ISSN 1349-0494

RIKEN Center for Developmental Biology

2003 Annual Report

http://www.cdb.riken.jp
CENTER FOR DEVELOPMENTAL BIOLOGY (CDB)
2-2-3 MINATOJIMA-MINAMIMACHI, CHUO-KU
KOBE, 650-0047
JAPAN
PHONE: +81-78-306-0111 FAX: +81-78-306-0101
EMAIL: contact@cdb.riken.jp

Production
Text
Douglas Sipp
Content Development
Naoki Namba
Douglas Sipp
DesignTRAIS K. K.
Photography
Yutaka Doi

On The Cover
Terminal branch of the Drosophila
trachea. A single terminal celllabeled with GFP (green) with a large
polyploid nucleus (purple) spreads
numerous cytoplasmic tubules withintracellular lumen (tracheole) overthe surface of the larval gut.
PHOTO: Shigeo Hayashi

All contents copyright (c) RIKEN Center for Developmental Biology, 2004.

Printed in Japan
Printed on 100%Recycled Paper

RIKEN Center for Developmental Biology

2003 Annual ReportContents■ Organization………………………………………………………

■ Message from the Director■ 2003 Highlights

1………………………………………

2/3……………………………………………………

4/5

Core Program

RIKEN
Center for Developmental Biology■ Evolutionary Regeneration Biology (Kiyokazu Agata)

Deputy Directors
Shin-ichi Nishikawa
Shinichi Aizawa

8/9■ Vertebrate Body Plan (Shinichi Aizawa)

10/11■ Morphogenetic Signaling (Shigeo Hayashi)

12/13■ Cell Asymmetry (Fumio Matsuzaki)

14/15■ Stem Cell Biology (Shin-ichi Nishikawa)

16/17■ Organogenesis and Neurogenesis (Yoshiki Sasai)

18/19■ Cell Adhesion and Tissue Patterning (Masatoshi Takeichi)

20/21

Director
Masatoshi Takeichi
Creative Research Promoting Program

Institutional Review Board■ Stem Cell Translational Research (Takayuki Asahara)

24/25■ Neuronal Differentiation and Regeneration (Hideki Enomoto)

26/27■ Neural Network Development (Chihiro Hama)

28/29■ Vertebrate Axis Formation (Masahiko Hibi)

30/31■ Positional Information (Shigeru Kondo)

32/33

MRC Center for Developmental Neurobiology (UK)■ Evolutionary Morphology (Shigeru Kuratani)

34/35

Elaine Fuchs■ Sensory Development (Raj Ladher)

36/37

Howard Hughes Medical InstituteRockefeller University (USA)■ Germline Development (Akira Nakamura)

38/39

Hajime Fujisawa■ Chromatin Dynamics (Jun-ichi Nakayama)

40/41■ Cell Migration (Kiyoji Nishiwaki)

42/43■ Pluripotent Cell Studies (Hitoshi Niwa)

44/45

Advisory Council
Igor Dawid, Chair
Laboratory of Molecular Genetics
National Institute of Child Health and Human Development, NIH (USA)William Chia

Division of Biological Science
Nagoya University Graduate School of Science (Japan)

Peter Gruss
Department of Molecular Cell Biology
Max Planck Institute for Biophysical Chemistry (Germany)■ Mammalian Epigenetic Studies (Masaki Okano)

46/47

National Institute of Genetics (Japan)■ Mammalian Molecular Embryology (Tony Perry)

48/49

Yoichi Nabeshima■ Embryonic Induction (Hiroshi Sasaki)

50/51■ Cell Fate Decision (Hitoshi Sawa)

52/53■ Developmental Genomics (Asako Sugimoto)54/55■ Body Patterning (Yoshiko Takahashi)

56/57■ Genomic Reprogramming (Teruhiko Wakayama)

58/59

Yoshiki Hotta

Department of Pathology and Tumor Biology
Kyoto University Graduate School of Medicine (Japan)

Tatsutoshi Nakahata
Department of Pediatrics
Kyoto University Graduate School of Medicine (Japan)

Austin Smith
Center for Genome Research
University of Edinburgh (UK)Yoshimi Takai

2003 New Faces

Department of Molecular Biology and Biochemistry
Osaka University Graduate School/Faculty of Medicine■ Mammalian Germ Cell Biology (Mitinori Saitou)

60■ Systems Biology (Hiroki R. Ueda)

61■ Cellular Morphogenesis (Shigenobu Yonemura)

64/65■ Animal Resources and Genetic Engineering (Shinichi Aizawa) / Sequencing Lab

66/67■ Animal Facility & Research Aquarium

68■ Budget & Staff………………………………………………………

70■ Collaborations & Affiliations ………………………………………

71■ Leading Project in Regenerative Medicine ……………………… 72/73■ Seminars……………………………………………………… 74/75■ CDB Symposium ………………………………………………………

76

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Supporting Laboratories

RIKEN CDB 2003 Annual Report1Contents■ Organization………………………………………………………

■ Message from the Director■ 2003 Highlights

1………………………………………

2/3……………………………………………………

4/5

Core Program

RIKEN
Center for Developmental Biology■ Evolutionary Regeneration Biology (Kiyokazu Agata)

Deputy Directors
Shin-ichi Nishikawa
Shinichi Aizawa

8/9■ Vertebrate Body Plan (Shinichi Aizawa)

10/11■ Morphogenetic Signaling (Shigeo Hayashi)

12/13■ Cell Asymmetry (Fumio Matsuzaki)

14/15■ Stem Cell Biology (Shin-ichi Nishikawa)

16/17■ Organogenesis and Neurogenesis (Yoshiki Sasai)

18/19■ Cell Adhesion and Tissue Patterning (Masatoshi Takeichi)

20/21

Director
Masatoshi Takeichi
Creative Research Promoting Program

Institutional Review Board■ Stem Cell Translational Research (Takayuki Asahara)

24/25■ Neuronal Differentiation and Regeneration (Hideki Enomoto)

26/27■ Neural Network Development (Chihiro Hama)

28/29■ Vertebrate Axis Formation (Masahiko Hibi)

30/31■ Positional Information (Shigeru Kondo)

32/33

MRC Center for Developmental Neurobiology (UK)■ Evolutionary Morphology (Shigeru Kuratani)

34/35

Elaine Fuchs■ Sensory Development (Raj Ladher)

36/37

Howard Hughes Medical InstituteRockefeller University (USA)■ Germline Development (Akira Nakamura)

38/39

Hajime Fujisawa■ Chromatin Dynamics (Jun-ichi Nakayama)

40/41■ Cell Migration (Kiyoji Nishiwaki)

42/43■ Pluripotent Cell Studies (Hitoshi Niwa)

44/45

Advisory Council
Igor Dawid, Chair
Laboratory of Molecular Genetics
National Institute of Child Health and Human Development, NIH (USA)William Chia

Division of Biological Science
Nagoya University Graduate School of Science (Japan)

Peter Gruss
Department of Molecular Cell Biology
Max Planck Institute for Biophysical Chemistry (Germany)■ Mammalian Epigenetic Studies (Masaki Okano)

46/47

National Institute of Genetics (Japan)■ Mammalian Molecular Embryology (Tony Perry)

48/49

Yoichi Nabeshima■ Embryonic Induction (Hiroshi Sasaki)

50/51■ Cell Fate Decision (Hitoshi Sawa)

52/53■ Developmental Genomics (Asako Sugimoto)54/55■ Body Patterning (Yoshiko Takahashi)

56/57■ Genomic Reprogramming (Teruhiko Wakayama)

58/59

Yoshiki Hotta

Department of Pathology and Tumor Biology
Kyoto University Graduate School of Medicine (Japan)

Tatsutoshi Nakahata
Department of Pediatrics
Kyoto University Graduate School of Medicine (Japan)

Austin Smith
Center for Genome Research
University of Edinburgh (UK)Yoshimi Takai

2003 New Faces

Department of Molecular Biology and Biochemistry
Osaka University Graduate School/Faculty of Medicine■ Mammalian Germ Cell Biology (Mitinori Saitou)

60■ Systems Biology (Hiroki R. Ueda)

61■ Cellular Morphogenesis (Shigenobu Yonemura)

64/65■ Animal Resources and Genetic Engineering (Shinichi Aizawa) / Sequencing Lab

66/67■ Animal Facility & Research Aquarium

68■ Budget & Staff………………………………………………………

70■ Collaborations & Affiliations ………………………………………

71■ Leading Project in Regenerative Medicine ……………………… 72/73■ Seminars……………………………………………………… 74/75■ CDB Symposium ………………………………………………………

76

M

id

dl

e

ea

ro

ft

he

ch

ic k

.P

HO

TO
:R
aj
La
dh
er

Supporting Laboratories

RIKEN CDB 2003 Annual Report1Center for Developmental Biology

Message from the Director
Masatoshi Takeichi
Director, CDB

The days leading up to the year's end tend to be chaotic, but they also provide one with a valuablestimulus to look back on milestones passed and ahead to new objectives. As 2003 comes to aclose, it's good to be able to take stock of the achievements made by researchers at the RIKEN Center for Developmental Biology, the challenges we face in the years ahead, and the state of our rapidly evolving science. This year has seen the emergence of news and civic issues that have placed
the fields of developmental biology, regeneration and regenerative medicine frequently in the
spotlight and sometimes at the center of controversy on the world stage, a position accompanied by
all the advantages and drawbacks that public prominence entails.
For us at the CDB, the year began in March with the unqualified (and uncontroversial) success of
our first annual symposium on The Origin and Formation of Multicellular Systems, a three-day meeting of more than 200 distinguished speakers, poster presenters and attendees who gathered to discuss metazoan biology, from its single-celled forebears to its greatest evolutionary achievement in
the formation of neural networks comprising billions of cells. We look forward to hosting more suchmeetings on a yearly basis, beginning with our next symposium, on Developmental Remodeling, inMarch of 2004, which will be held on the CDB campus in an auditorium designed to increase theCenter's capacity for hosting scientific meetings and expand its ability to provide a venue for international exchange across fields.
Research advances from CDB labs have also provided a string of highlights this year. As you willsee from the pages of this report, scientists at the CDB have made strides forward on many
fronts, from uncovering new molecular mechanisms at work in the asymmetric division, signaling and guided migration of cells, to developing new technologies that may one day enable
the targeted differentiation of embryonic stem cells toward clinically relevant ends. Studies in
evolutionary development have also provided broadened contexts and perspectives, finding
possible roots for the central nervous system in the genome of a tiny flatworm and challenging conventional wisdom about the regulatory origins of the vertebrate hindbrain.
Of course, this science did not take place in a vacuum. The central pillars of the Center's research mission — developmental biology, regeneration and the scientificbases for regenerative medicine — are all vibrantly dynamic, truly global fields ofendeavor, and the CDB is pledged to remain fully engaged with international
colleagues at the organizational, laboratory and institutional levels. In 2003,
this commitment was characterized by the establishment of scientificexchange relationships with the University of Texas Graduate Schoolof Biomedical Sciences and the Temasek Life Sciences Laboratory
Limited in Singapore, paving the way for new and better opportunities to visit and host researchers from these institutions. We look forward to increasing the number andrange of our inter-organizational exchange
programs in the years ahead.

2 RIKEN CDB 2003 Annual Report

Domestically, Japan saw increased
attention to a number of related issues andresearch fields. The national government
unveiled plans for the Leading Projects, initiatives set
up to fund mid-term research into a number of key areas
identified as having the potential to improve public health, welfare and economic well-being. As the largest center for the study ofdevelopmental biology in Japan, the CDB received funding to carry out
Leading Project research into potential applications for stem cells in regenerative medicine. Embryonic stem cells also featured in the national news
this year, which saw the first establishment of a new line of human ES cells bya Japanese laboratory in Kyoto University. ES cells represent a biologicalresource of potentially revolutionary importance, but whose study demands carefulconsideration and ethical restraint. The use of human ES cells is for biomedical
research is permitted in Japan, and in 2003 a number of CDB labs applied for permission to use the new Kyoto cell line. Approval of these research plans will allow scientists
here to explore the secrets of cell differentiation and pluripotency, and perhaps one day tocontribute to the development of cell-replacement therapies for intractable illnesses such as
diabetes and Parkinson's disease.
As a basic research institute, the CDB also appreciates the fundamental importance of maintaining ties with the world of academia, and we have pursued active affiliations with some ofJapan's top graduate school programs in the biosciences and regenerative medicine, allowing
students from affiliated programs to conduct their academic research using the Center's facilities.
This year, we were proud to formalize relationships with the Kyoto University Graduate School of
Medicine, the Nara Advanced Institute of Science and Technology Graduate School of Biological
Sciences, and the Kwansei Gakuin University Bioscience Department, bringing the total number ofsuch affiliations to six.
Public communications and outreach also featured prominently on the Center's calendar this year.
In May, the CDB opened its doors to the public for its first Open House, which was attended bymore than 1,000 visitors who took advantage of the opportunity to take in talks on stem cells and programmed cell death, join in mock experiments, and sneak a peek at a day in the life of a developmental biology lab. The Center also joined in the public educational activities at the Kobe BioWeek
event in August, a biotechnology convention that saw more than 8,000 attendees, including many
who visited the CDB in a virtual sense via two-way video lectures and realistic software simulationsof laboratory equipment.
In retrospect, the year has been a full one, and it has been gratifying to participate in the Center's
activities both as a scientist and an administrator. So it is a great pleasure for me to introduce our2003 Annual Report, which illustrates the range and depth of research at the CDB and serves bothas a spark for happy memories and as a source of inspiration as we enter a new year, with its promise of new discoveries and change.

RIKEN CDB 2003 Annual Report3Center for Developmental Biology

Message from the Director
Masatoshi Takeichi
Director, CDB

The days leading up to the year's end tend to be chaotic, but they also provide one with a valuablestimulus to look back on milestones passed and ahead to new objectives. As 2003 comes to aclose, it's good to be able to take stock of the achievements made by researchers at the RIKEN Center for Developmental Biology, the challenges we face in the years ahead, and the state of our rapidly evolving science. This year has seen the emergence of news and civic issues that have placed
the fields of developmental biology, regeneration and regenerative medicine frequently in the
spotlight and sometimes at the center of controversy on the world stage, a position accompanied by
all the advantages and drawbacks that public prominence entails.
For us at the CDB, the year began in March with the unqualified (and uncontroversial) success of
our first annual symposium on The Origin and Formation of Multicellular Systems, a three-day meeting of more than 200 distinguished speakers, poster presenters and attendees who gathered to discuss metazoan biology, from its single-celled forebears to its greatest evolutionary achievement in
the formation of neural networks comprising billions of cells. We look forward to hosting more suchmeetings on a yearly basis, beginning with our next symposium, on Developmental Remodeling, inMarch of 2004, which will be held on the CDB campus in an auditorium designed to increase theCenter's capacity for hosting scientific meetings and expand its ability to provide a venue for international exchange across fields.
Research advances from CDB labs have also provided a string of highlights this year. As you willsee from the pages of this report, scientists at the CDB have made strides forward on many
fronts, from uncovering new molecular mechanisms at work in the asymmetric division, signaling and guided migration of cells, to developing new technologies that may one day enable
the targeted differentiation of embryonic stem cells toward clinically relevant ends. Studies in
evolutionary development have also provided broadened contexts and perspectives, finding
possible roots for the central nervous system in the genome of a tiny flatworm and challenging conventional wisdom about the regulatory origins of the vertebrate hindbrain.
Of course, this science did not take place in a vacuum. The central pillars of the Center's research mission — developmental biology, regeneration and the scientificbases for regenerative medicine — are all vibrantly dynamic, truly global fields ofendeavor, and the CDB is pledged to remain fully engaged with international
colleagues at the organizational, laboratory and institutional levels. In 2003,
this commitment was characterized by the establishment of scientificexchange relationships with the University of Texas Graduate Schoolof Biomedical Sciences and the Temasek Life Sciences Laboratory
Limited in Singapore, paving the way for new and better opportunities to visit and host researchers from these institutions. We look forward to increasing the number andrange of our inter-organizational exchange
programs in the years ahead.

2 RIKEN CDB 2003 Annual Report

Domestically, Japan saw increased
attention to a number of related issues andresearch fields. The national government
unveiled plans for the Leading Projects, initiatives set
up to fund mid-term research into a number of key areas
identified as having the potential to improve public health, welfare and economic well-being. As the largest center for the study ofdevelopmental biology in Japan, the CDB received funding to carry out
Leading Project research into potential applications for stem cells in regenerative medicine. Embryonic stem cells also featured in the national news
this year, which saw the first establishment of a new line of human ES cells bya Japanese laboratory in Kyoto University. ES cells represent a biologicalresource of potentially revolutionary importance, but whose study demands carefulconsideration and ethical restraint. The use of human ES cells is for biomedical
research is permitted in Japan, and in 2003 a number of CDB labs applied for permission to use the new Kyoto cell line. Approval of these research plans will allow scientists
here to explore the secrets of cell differentiation and pluripotency, and perhaps one day tocontribute to the development of cell-replacement therapies for intractable illnesses such as
diabetes and Parkinson's disease.
As a basic research institute, the CDB also appreciates the fundamental importance of maintaining ties with the world of academia, and we have pursued active affiliations with some ofJapan's top graduate school programs in the biosciences and regenerative medicine, allowing
students from affiliated programs to conduct their academic research using the Center's facilities.
This year, we were proud to formalize relationships with the Kyoto University Graduate School of
Medicine, the Nara Advanced Institute of Science and Technology Graduate School of Biological
Sciences, and the Kwansei Gakuin University Bioscience Department, bringing the total number ofsuch affiliations to six.
Public communications and outreach also featured prominently on the Center's calendar this year.
In May, the CDB opened its doors to the public for its first Open House, which was attended bymore than 1,000 visitors who took advantage of the opportunity to take in talks on stem cells and programmed cell death, join in mock experiments, and sneak a peek at a day in the life of a developmental biology lab. The Center also joined in the public educational activities at the Kobe BioWeek
event in August, a biotechnology convention that saw more than 8,000 attendees, including many
who visited the CDB in a virtual sense via two-way video lectures and realistic software simulationsof laboratory equipment.
In retrospect, the year has been a full one, and it has been gratifying to participate in the Center's
activities both as a scientist and an administrator. So it is a great pleasure for me to introduce our2003 Annual Report, which illustrates the range and depth of research at the CDB and serves bothas a spark for happy memories and as a source of inspiration as we enter a new year, with its promise of new discoveries and change.

RIKEN CDB 2003 Annual Report

3

2003

H I G H L I G H T S
August 27 - 9
May 13
April 1
New Graduate School
Affiliations
The CDB formalized its
affiliations with the Kyoto
University Graduate School of
Medicine and the NAIST
Graduate School of Biological
Sciences, allowing doctoral
students from those schools to
conduct their thesis researchin CDB laboratories under theguidance of lab heads
designated as visiting
professors.

Publication of "Generationof neural crest-derived
peripheral neurons andfloor plate cells from mouseand primate embryonicstem cells"
In a study published in the
May 13 issue of theProceedings of the National
Academy of Sciences (PNAS),
the Sasai research groupdemonstrated the applicationof stromal cell derived
inducing activity (SDIA) in thehigh-efficiency induction of
peripheral neurons fromembryonic stem cells.

August 19
Publication of "Travelingstripes on the skin of amutant mouse"
The August 19 issue of PNAS
featured a cover image takenfrom a research article from
Shigeru Kondo's lab
describing the mathematicalproperties of a strikingly
patterned mutant mousestrain.

July 2

March 24 -26
First CDB Symposium:
Origin and Formation ofMulticellular Systems
The CDB held its first annual
symposium at the nearby
Portopia Hotel. The meetingdrew more than 200 speakers
and attendees from aroundthe world, with lectures, poster
presentations and lively
discussion on the origins ofmetazoan biology, thefundamental processes ofdevelopment and regenerationand the formation of higherorder structures, such asneural networks.

4 RIKEN CDB 2003 Annual Report

June 24
Publication of "Origin andevolutionary process of the
CNS elucidated bycomparative genomics
analysis of planarian ESTs"On June 24, PNAS publishedthis study by the Laboratory
for Evolutionary Regeneration
Biology providing evidence ofgenetic roots for the central
nervous system in a primitive
bilaterian, the flatworm
Dugesia japonica.

May 2
Scientific exchange
agreement with Universityof Texas Graduate School of
Biomedical Sciences
As part of its ongoing efforts todevelop organizational ties with
the international developmental
biology research community,
the CDB formalized anagreement for active scientificexchange with the University ofTexas Graduate School of
Biomedical Sciences at
Houston. The agreement
facilitates the hosting andexchange of research staff andthe joint sponsorship of scientificmeetings with one of theforemost academic centers fordevelopmental biology researchin the United States.

May 31
CDB Open HouseThe CDB opened its doors to
the public for a one-day event
featuring walk-throughexperiments, lab visits, talks
on C. elegans and ES cellbiology, quizzes, games andvideo and poster
presentations.

Scientific exchange
agreement with Temasek
Life Sciences Laboratory
LimitedThe CDB established formalties with the Temasek Life
Sciences Laboratory in
Singapore, a non-profit
organization with affiliations to
the National University ofSingapore and Nanyang
Technological University
established to conductmolecular biology andgenetics research.

Kobe BioWeek
The city of Kobe played host
to more than 8,000 visitors
participating in Kobe
BioWeek, a series of meetingsand events focusing on thegrowth and future directions of
the biotechnology sector in theregion and around Japan. The
CDB joined in the public
education activities, with twoway videoconference lectures
and Q&A sessions, hands-on
simulations of lab equipment
and a virtual lab tour programusing web cameras thatallowed the public to watch
CDB labs at work in real time.

December 21
Publication of "An NDPaselinks ADAM proteaseglycosylation with organ
morphogenesis in
C. elegans"
In work published in the online
edition of Nature Cell Biology,
the Laboratory for Cell
Migration, under team leader
Kiyoji Nishiwaki, reported therole of a protein modification
known as glycosylation in
guiding the migration of
gonadal cells in the nematode,
C. elegans.

October 1

N Suzuki, M Hirata and S Kondo. Traveling
Stripes on the Skin of a Mutant Mouse. Proc
Natl Acad Sci U S A 100:9680-5 (2003). Copyright (2003) National Academy of Sciences,
U.S.A.

RIKEN BecomesIndependent Administrative
Institution
As part of a restructuring ofJapanese public corporations,
RIKEN was officiallyre-designated as anindependent administrative
institution, a move intended tosimultaneously increase theInstitute's autonomy andaccountability. As part of thistransition, the Nobel Prizewinning chemist Ryoji Noyori
was named the new president,
ushering in a new era for
RIKEN, which was firstestablished in 1917.

October 20 -1
2nd CDB Retreat
The CDB held its second
annual retreat on the island of
Awaji, located a short distancefrom Kobe in Japan's Inland
Sea. The retreat gave CDB
research staff the chance torelax, mingle and exchange
research findings and opinions
in two days of oralpresentation and poster
sessions.

RIKEN CDB 2003 Annual Report

5

2003

H I G H L I G H T S
August 27 - 9
May 13
April 1
New Graduate School
Affiliations
The CDB formalized its
affiliations with the Kyoto
University Graduate School of
Medicine and the NAIST
Graduate School of Biological
Sciences, allowing doctoral
students from those schools to
conduct their thesis researchin CDB laboratories under theguidance of lab heads
designated as visiting
professors.

Publication of "Generationof neural crest-derived
peripheral neurons andfloor plate cells from mouseand primate embryonicstem cells"
In a study published in the
May 13 issue of theProceedings of the National
Academy of Sciences (PNAS),
the Sasai research groupdemonstrated the applicationof stromal cell derived
inducing activity (SDIA) in thehigh-efficiency induction of
peripheral neurons fromembryonic stem cells.

August 19
Publication of "Travelingstripes on the skin of amutant mouse"
The August 19 issue of PNAS
featured a cover image takenfrom a research article from
Shigeru Kondo's lab
describing the mathematicalproperties of a strikingly
patterned mutant mousestrain.

July 2

March 24 -26
First CDB Symposium:
Origin and Formation ofMulticellular Systems
The CDB held its first annual
symposium at the nearby
Portopia Hotel. The meetingdrew more than 200 speakers
and attendees from aroundthe world, with lectures, poster
presentations and lively
discussion on the origins ofmetazoan biology, thefundamental processes ofdevelopment and regenerationand the formation of higherorder structures, such asneural networks.

4 RIKEN CDB 2003 Annual Report

June 24
Publication of "Origin andevolutionary process of the
CNS elucidated bycomparative genomics
analysis of planarian ESTs"On June 24, PNAS publishedthis study by the Laboratory
for Evolutionary Regeneration
Biology providing evidence ofgenetic roots for the central
nervous system in a primitive
bilaterian, the flatworm
Dugesia japonica.

May 2
Scientific exchange
agreement with Universityof Texas Graduate School of
Biomedical Sciences
As part of its ongoing efforts todevelop organizational ties with
the international developmental
biology research community,
the CDB formalized anagreement for active scientificexchange with the University ofTexas Graduate School of
Biomedical Sciences at
Houston. The agreement
facilitates the hosting andexchange of research staff andthe joint sponsorship of scientificmeetings with one of theforemost academic centers fordevelopmental biology researchin the United States.

May 31
CDB Open HouseThe CDB opened its doors to
the public for a one-day event
featuring walk-throughexperiments, lab visits, talks
on C. elegans and ES cellbiology, quizzes, games andvideo and poster
presentations.

Scientific exchange
agreement with Temasek
Life Sciences Laboratory
LimitedThe CDB established formalties with the Temasek Life
Sciences Laboratory in
Singapore, a non-profit
organization with affiliations to
the National University ofSingapore and Nanyang
Technological University
established to conductmolecular biology andgenetics research.

Kobe BioWeek
The city of Kobe played host
to more than 8,000 visitors
participating in Kobe
BioWeek, a series of meetingsand events focusing on thegrowth and future directions of
the biotechnology sector in theregion and around Japan. The
CDB joined in the public
education activities, with twoway videoconference lectures
and Q&A sessions, hands-on
simulations of lab equipment
and a virtual lab tour programusing web cameras thatallowed the public to watch
CDB labs at work in real time.

December 21
Publication of "An NDPaselinks ADAM proteaseglycosylation with organ
morphogenesis in
C. elegans"
In work published in the online
edition of Nature Cell Biology,
the Laboratory for Cell
Migration, under team leader
Kiyoji Nishiwaki, reported therole of a protein modification
known as glycosylation in
guiding the migration of
gonadal cells in the nematode,
C. elegans.

October 1

N Suzuki, M Hirata and S Kondo. Traveling
Stripes on the Skin of a Mutant Mouse. Proc
Natl Acad Sci U S A 100:9680-5 (2003). Copyright (2003) National Academy of Sciences,
U.S.A.

RIKEN BecomesIndependent Administrative
Institution
As part of a restructuring ofJapanese public corporations,
RIKEN was officiallyre-designated as anindependent administrative
institution, a move intended tosimultaneously increase theInstitute's autonomy andaccountability. As part of thistransition, the Nobel Prizewinning chemist Ryoji Noyori
was named the new president,
ushering in a new era for
RIKEN, which was firstestablished in 1917.

October 20 -1
2nd CDB Retreat
The CDB held its second
annual retreat on the island of
Awaji, located a short distancefrom Kobe in Japan's Inland
Sea. The retreat gave CDB
research staff the chance torelax, mingle and exchange
research findings and opinions
in two days of oralpresentation and poster
sessions.

RIKEN CDB 2003 Annual Report5The Core Program aims to promote highly innovative developmental and regeneration
studies in a strategic and multi-disciplinary manner. This program constitutes the coreresearch framework to achieve the aims of the Millennium Project, and focuses on thethree main themes of the CDB:
- Mechanisms of Development- Mechanisms of Regeneration- Scientific Bases of Regenerative Medicine
The Core Program consists of seven research groups, each led by an eminent scientist.
In addition to the group director, each group includes a number of research fellows andtechnical staff and in some cases a senior research fellow.

ahiko Hibi
O: Mas
PHOT
bryo
h em
rafis
Zeb

The Core Program aims to promote highly innovative developmental and regeneration
studies in a strategic and multi-disciplinary manner. This program constitutes the coreresearch framework to achieve the aims of the Millennium Project, and focuses on thethree main themes of the CDB:
- Mechanisms of Development- Mechanisms of Regeneration- Scientific Bases of Regenerative Medicine
The Core Program consists of seven research groups, each led by an eminent scientist.
In addition to the group director, each group includes a number of research fellows andtechnical staff and in some cases a senior research fellow.

ahiko Hibi
O: Mas
PHOT
bryo
h em
rafis
Zeb

Functional regionalization of
the planarian brain

Staff
Group Director

Technical Staff

Kiyokazu Agata

Midori Nakayama
Tetsutaro Hayashi
Tomomi Takamatsu
Yumi Saito
Osamu Nishimura
Mikiko Nakatsukasa
Chiharu Tanegashima

Research Scientist

Noriko Funayama
Yoshihiko Umesono
Hiroshi Imokawa
Chiyoko Kobayashi
Nobuyasu Maki
Shuichi Shigeno
Shigenobu Yazawa
Takahiro Murakami
Visiting Scientist

Norito Shibata

Student Trainee

Takeshi Inoue
Keiji Okamoto
Takaaki Karasawa
Tomoko Shibata

Kouji Matsuzaki
Hideaki Ito
Sayaka Higuchi
Tomomi Takano
Kaneyasu NishimuraPart-time Staff

Machiko Teramoto
Yuko Hirofuji
Keiko Momozu
AssistantShozou Sano

Core Program

Kiyokazu Agata
Ph. D.
Kiyokazu Agata received
his doctorate from Kyoto
University in 1985 for his
work on molecular cloning
and gene expression of
crystallin genes in chicken.
From 1983 to 1991, heworked at the National Institute for Basic Biology,
where he studied the molecular characteristics of dedifferentiated cells in transdifferentiation. He took an
associate professorship at
the Himeji Institute of Technology in 1991, and started
his study of planarian regeneration with Kenji Watanabe. He remained at theInstitute until 2000, when
he left to assume a professorship at Okayama University. He joined the
RIKEN Center for Developmental Biology as a groupdirector in 2000.

Its position on an important but scientifically underexplored branch of the evolutionary tree and its
remarkable biology combine to make the planarian
flatworm a fascinating and invaluable model forbasic research in fields from evolutionary development to regenerative medicine. Kiyokazu Agata has
adopted the freshwater planarian, Dugesia japonica,
as a model species in his study of the origins andproperties of stem cells, which are prevalent andexperimentally accessible in these worms and which
he believes hold the key to understanding the development, organization and maintenance of the cellular diversity that characterizes metazoan life.

Isolating planarian stem cells
Agata's laboratory has developed a method for identifying subsets of planarian stem cells, also called
neoblasts. These somatic stem cells are the only
mitotically active cells in the planarian body, making
them susceptible to X-ray irradiation. Examination ofcell populations shown to be vulnerable to elimination by X-rays, revealed that planarian neoblasts,
like stem cells in other species, seem to exist in proliferating and resting states. Real-time PCR analysis
indicates that each of these sub-populations
expresses discrete sets of genes. One fraction, X1,
expresses genes specific to actively cycling cells,
while a second fraction, X2, lacks this expression
profile and is thought to comprise stem cells
in a state of quiescence. Interestingly,
many X1 cells also express signal receptor molecules which are switched off in
the X2 fraction, while the corresponding ligands are expressed in a variety
of differentiated cells resistant to X-ray
irradiation.

Brain regeneration by stages
A planarian can re-grow a fully functional brain within five days following the amputation of its entire
head, an extraordinary feat of self-healing thatinvolves recapitulating the development of theworm's entire nervous system. Researchers in theAgata group studied this process and identified patterns of gene expression that indicate the regeneration of the brain comprises five distinct stages. In thefirst stage, at about eight hours after wound closure,a noggin-like gene (Djnlg) is activated in the stumpprior to the formation of a blastema, a mass of proliferating undifferentiated cells that serves as the frontline of regeneration. Soon thereafter, the brainspecifying gene nou-darake is switched on in theanterior fringe of the blastema, allowing the brain
rudiment to be formed. These first two steps occur
within 24 hours of decapitation, and set the stage forthe third phase in which brain patterning is established by the expression of a set of threeotd/Otx-related genes (relatives ofwhich also function to pattern brain
development in many other taxa,
including vertebrates) followed bythat of the planarian homolog of
Wnt at about 48 hours into
the regenerative pro-cess. In the fourth step, which occurs by day four ofregeneration, a homolog of the axon-guidance gene
netrin is triggered. At this stage the discrete components of the regenerating brain begin to form connections and higher-order organizations; chiasmata
cross-linking eyes and brain develop, and connections are set up between the brain and the ventral
nerve cords, which serve as a rudimentary peripheral nervous system. Thus, the basic components andconnections of the planarian neural network are inplace by the fourth day, but the functional recoveryof the brain is not completed until a final stage inwhich two newly-identified genes are expressed isentered.
A parallel study revealed that these two genes,
1020HH and eye53, are necessary for planarians toregain their normal light avoidance behavior, known
as negative phototaxis. Knockdown of these genes
by RNAi had no discernible effects on brain or eyemorphology, but the worms failed to respond to lightstimuli in the normal manner, even after five days ofregeneration, when the structure of the brain has
been fully reinstated.

team was able to identify 116 clones
with significant similarities to genes
closely linked to the nervous system inother species, including genes involvedin neurotransmission, the neural network, brain morphogenesis and neuraldifferentiation. These results point
toward a shared evolutionary origin formany nervous system genes, indicatingthat the nervous system is likely to havearisen in a single common ancestor ofbilaterian animals. Intriguingly, nearly
30% of the genes identified also havehomologous sequences in yeast and theplant species, Arabidopsis thaliensis,
both of which entirely lack neural development,
which suggests that a significant number of genesthat predate the advent of the nervous system havebeen co-opted to perform functions specific to neurobiology.

Publications
Hayasi S, Itho M, Agata K
and Taira M. Conserved
Expression Patterns of FGF
Receptor-like 1/nou-darake in
Xenopus and Planarian. Dev
Dyn (2004).
Inoue T, Kumamoto H,
Okamoto K, Umesono Y,
Sakai M, Aiejandro SA andAgata K. Morphological andFunctional Recovery of the
Planarian
Photosensing
System During Head Regeneration. Zoolog Sci (2004).

A planarian can re-growa fully functional brain within
five days following theamputation of its head

Agata K. Regeneration andGene Regulation in Planarians. Curr Opin Genet Dev
13:492-6 (2003).

The Agata lab is also now participating in an international collaboration established to sequence andannotate a set of more than 10,000 planarian ESTs,
which promises to provide an invaluable resource
for the study of evolutionary biology, comparative
genomics, and the genetics underlying the uniquecharacteristics of these organisms. The availabilityof an annotated database of planarian cDNAs may
provide keys to the understanding of stem cell biology, tissue plasticity and maintenance and other fundamentally important processes.

Agata
K,
TanakaT,
Kobayashi C, Kato K and
Saitoh Y. Intercalary Regeneration in Planarians. Dev Dyn
226:308-16 (2003).
Mineta K, Nakazawa M,
Cebria F, Ikeo K, Agata K
and Gojobori T. Origin and
Evolutionary Process of theCns Elucidated by Comparative Genomics Analysis of
Planarian Ests. Proc Natl
Acad Sci U S A 100:7666-71(2003).

Evolution of thecentral nervous
systemThe planarian is one of the lowest forms of animal known topossess a central nervous system, making it an apt model forthe study of the evolution ofthis most complex and elaborately organized biological system. Using clones of over
3,000 expressed sequencetags (ESTs) isolated from theplanarian head region, theNakazawa M, Cebria F,
Mineta K, Ikeo K, Agata K
and Gojobori T. Search for
the Evolutionary Origin of aBrain: Planarian Brain Characterized by Microarray. Mol
Biol Evol 20:784-91 (2003).
Saito Y, Koinuma S, Watanabe K and Agata K. MediolateralIntercalationin
Planarians Revealed by Grafting Experiments. Dev Dyn
226:334-40 (2003).

Expression of various neural-related genes

Evolutionarily conserved expression patterns of planarian (right) and Xenopus nou-darake/FGFRL1 (left)

8 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report9

Functional regionalization of
the planarian brain

Staff
Group Director

Technical Staff

Kiyokazu Agata

Midori Nakayama
Tetsutaro Hayashi
Tomomi Takamatsu
Yumi Saito
Osamu Nishimura
Mikiko Nakatsukasa
Chiharu Tanegashima

Research Scientist

Noriko Funayama
Yoshihiko Umesono
Hiroshi Imokawa
Chiyoko Kobayashi
Nobuyasu Maki
Shuichi Shigeno
Shigenobu Yazawa
Takahiro Murakami
Visiting Scientist

Norito Shibata

Student Trainee

Takeshi Inoue
Keiji Okamoto
Takaaki Karasawa
Tomoko Shibata

Kouji Matsuzaki
Hideaki Ito
Sayaka Higuchi
Tomomi Takano
Kaneyasu NishimuraPart-time Staff

Machiko Teramoto
Yuko Hirofuji
Keiko Momozu
AssistantShozou Sano

Core Program

Kiyokazu Agata
Ph. D.
Kiyokazu Agata received
his doctorate from Kyoto
University in 1985 for his
work on molecular cloning
and gene expression of
crystallin genes in chicken.
From 1983 to 1991, heworked at the National Institute for Basic Biology,
where he studied the molecular characteristics of dedifferentiated cells in transdifferentiation. He took an
associate professorship at
the Himeji Institute of Technology in 1991, and started
his study of planarian regeneration with Kenji Watanabe. He remained at theInstitute until 2000, when
he left to assume a professorship at Okayama University. He joined the
RIKEN Center for Developmental Biology as a groupdirector in 2000.

Its position on an important but scientifically underexplored branch of the evolutionary tree and its
remarkable biology combine to make the planarian
flatworm a fascinating and invaluable model forbasic research in fields from evolutionary development to regenerative medicine. Kiyokazu Agata has
adopted the freshwater planarian, Dugesia japonica,
as a model species in his study of the origins andproperties of stem cells, which are prevalent andexperimentally accessible in these worms and which
he believes hold the key to understanding the development, organization and maintenance of the cellular diversity that characterizes metazoan life.

Isolating planarian stem cells
Agata's laboratory has developed a method for identifying subsets of planarian stem cells, also called
neoblasts. These somatic stem cells are the only
mitotically active cells in the planarian body, making
them susceptible to X-ray irradiation. Examination ofcell populations shown to be vulnerable to elimination by X-rays, revealed that planarian neoblasts,
like stem cells in other species, seem to exist in proliferating and resting states. Real-time PCR analysis
indicates that each of these sub-populations
expresses discrete sets of genes. One fraction, X1,
expresses genes specific to actively cycling cells,
while a second fraction, X2, lacks this expression
profile and is thought to comprise stem cells
in a state of quiescence. Interestingly,
many X1 cells also express signal receptor molecules which are switched off in
the X2 fraction, while the corresponding ligands are expressed in a variety
of differentiated cells resistant to X-ray
irradiation.

Brain regeneration by stages
A planarian can re-grow a fully functional brain within five days following the amputation of its entire
head, an extraordinary feat of self-healing thatinvolves recapitulating the development of theworm's entire nervous system. Researchers in theAgata group studied this process and identified patterns of gene expression that indicate the regeneration of the brain comprises five distinct stages. In thefirst stage, at about eight hours after wound closure,a noggin-like gene (Djnlg) is activated in the stumpprior to the formation of a blastema, a mass of proliferating undifferentiated cells that serves as the frontline of regeneration. Soon thereafter, the brainspecifying gene nou-darake is switched on in theanterior fringe of the blastema, allowing the brain
rudiment to be formed. These first two steps occur
within 24 hours of decapitation, and set the stage forthe third phase in which brain patterning is established by the expression of a set of threeotd/Otx-related genes (relatives ofwhich also function to pattern brain
development in many other taxa,
including vertebrates) followed bythat of the planarian homolog of
Wnt at about 48 hours into
the regenerative pro-cess. In the fourth step, which occurs by day four ofregeneration, a homolog of the axon-guidance gene
netrin is triggered. At this stage the discrete components of the regenerating brain begin to form connections and higher-order organizations; chiasmata
cross-linking eyes and brain develop, and connections are set up between the brain and the ventral
nerve cords, which serve as a rudimentary peripheral nervous system. Thus, the basic components andconnections of the planarian neural network are inplace by the fourth day, but the functional recoveryof the brain is not completed until a final stage inwhich two newly-identified genes are expressed isentered.
A parallel study revealed that these two genes,
1020HH and eye53, are necessary for planarians toregain their normal light avoidance behavior, known
as negative phototaxis. Knockdown of these genes
by RNAi had no discernible effects on brain or eyemorphology, but the worms failed to respond to lightstimuli in the normal manner, even after five days ofregeneration, when the structure of the brain has
been fully reinstated.

team was able to identify 116 clones
with significant similarities to genes
closely linked to the nervous system inother species, including genes involvedin neurotransmission, the neural network, brain morphogenesis and neuraldifferentiation. These results point
toward a shared evolutionary origin formany nervous system genes, indicatingthat the nervous system is likely to havearisen in a single common ancestor ofbilaterian animals. Intriguingly, nearly
30% of the genes identified also havehomologous sequences in yeast and theplant species, Arabidopsis thaliensis,
both of which entirely lack neural development,
which suggests that a significant number of genesthat predate the advent of the nervous system havebeen co-opted to perform functions specific to neurobiology.

Publications
Hayasi S, Itho M, Agata K
and Taira M. Conserved
Expression Patterns of FGF
Receptor-like 1/nou-darake in
Xenopus and Planarian. Dev
Dyn (2004).
Inoue T, Kumamoto H,
Okamoto K, Umesono Y,
Sakai M, Aiejandro SA andAgata K. Morphological andFunctional Recovery of the
Planarian
Photosensing
System During Head Regeneration. Zoolog Sci (2004).

A planarian can re-growa fully functional brain within
five days following theamputation of its head

Agata K. Regeneration andGene Regulation in Planarians. Curr Opin Genet Dev
13:492-6 (2003).

The Agata lab is also now participating in an international collaboration established to sequence andannotate a set of more than 10,000 planarian ESTs,
which promises to provide an invaluable resource
for the study of evolutionary biology, comparative
genomics, and the genetics underlying the uniquecharacteristics of these organisms. The availabilityof an annotated database of planarian cDNAs may
provide keys to the understanding of stem cell biology, tissue plasticity and maintenance and other fundamentally important processes.

Agata
K,
TanakaT,
Kobayashi C, Kato K and
Saitoh Y. Intercalary Regeneration in Planarians. Dev Dyn
226:308-16 (2003).
Mineta K, Nakazawa M,
Cebria F, Ikeo K, Agata K
and Gojobori T. Origin and
Evolutionary Process of theCns Elucidated by Comparative Genomics Analysis of
Planarian Ests. Proc Natl
Acad Sci U S A 100:7666-71(2003).

Evolution of thecentral nervous
systemThe planarian is one of the lowest forms of animal known topossess a central nervous system, making it an apt model forthe study of the evolution ofthis most complex and elaborately organized biological system. Using clones of over
3,000 expressed sequencetags (ESTs) isolated from theplanarian head region, theNakazawa M, Cebria F,
Mineta K, Ikeo K, Agata K
and Gojobori T. Search for
the Evolutionary Origin of aBrain: Planarian Brain Characterized by Microarray. Mol
Biol Evol 20:784-91 (2003).
Saito Y, Koinuma S, Watanabe K and Agata K. MediolateralIntercalationin
Planarians Revealed by Grafting Experiments. Dev Dyn
226:334-40 (2003).

Expression of various neural-related genes

Evolutionarily conserved expression patterns of planarian (right) and Xenopus nou-darake/FGFRL1 (left)

8 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report9

Emx genes in earlycortical development
In the mouse, the development of
the cerebrum is immediately preceded by the closure of the anterior neural plate at around E8.5 in the presumptive forebrain-midbrain junction. In the earliest stages of corticogenesis, the structures of the archipallium, the non-neuronal components of the choroid plexus, the hippocampal complex and the fimbria, aregenerated. It has been suggested
that the last of these structures, thefimbria, which are located at the border of the cerebral cortex and the

StaffGroup Director

Technical Staff

Student Trainee

Shinichi Aizawa

Shoko Takehara
Miwa Nakamura
Tomomi Omura
Saori Nagayoshi
Ai Inoue
Kuniko Kitajima
Hiroshi Nakano
Hitomi Tsujii
Maiko Takahashi

Mariko Hirano
Wataru Satoh
Yusuke Sakurai

Senior Scientist

Isao Matsuo
Research Scientist

Yoko Suda
Kohei Hatta
Akihito Yamamoto
Akihiko Shimono
Nobuyoshi Takasaki
Daisuke Kurokawa
Takuya Murata
Jun Kimura

Assistant

Sayo Saito

Otx2 expression in fore- andmidbrain at E10.5

Junior Research Associate

Takashi Nagano
Izumi Oda-Ishii

Kurokawa D, Takasaki N, Kiyonari H, Nakayama R, Kimura
C, Matsuo I and Aizawa S.
Regulation of Otx2 expression
and its functions in mouse epiblast and anterior neuroectoderm. Development (2004).

Special Postdoctoral Researcher

Chiharu Kimura-Yoshida

Core Program

Despite their many differences, taxa as diverse as
fish, amphibians, reptiles, birds and mammals share
a common body plan comprising three regions: thetrunk, the hindbrain/pharyngeal region, and the rostral head. Shin Aizawa is interested in the molecularbases for and phylogenetic origins of this regionalization, concentrating primarily on the genetic activity
and molecular attributes of head development.

Shinichi Aizawa
Ph. D.
Shinichi Aizawa received his
Ph. D. in Zoology from theTokyo Kyoiku University,
Department of Zoology in1973. He spent the period
from 1974 to 1979 as an investigator at the Tokyo Metropolitan Institute of Gerontology,
then two years as a research
fellow in the Laboratory of
Genetic Pathology at the University of Washington (US).
He returned to the Tokyo Metropolitan Institute of Gerontology in 1982, where heremained until 1986 when hemoved to the RIKEN Tsukuba
Life Science Center as a senior research associate. He wasappointed professor in the
Kumamoto University Schoolof Medicine Department ofMorphogenesis in 1994, andserved in that position until
2002. Since 2000 he hasserved as CDB deputy director
and group director of the Vertebrate Body Plan, as well asteam leader of the Laboratory
for Animal Resources andgenetic Engineering. He also
serves as a Managing Editor
for the journal, Mechanisms ofDevelopment.

All animals develop from the head. The formation of
inductive head organizer precedes that of the trunkorganizer, as was first demonstrated by Spemannand Mangold in their studies of amphibian embryogenesis. Gene knockout studies in mice, have demonstrated that the rhombomere r1/2 is the ground statein the mammalian body plan. The head organizer
induces the rostral head anteriorly to this rhombomere, while the trunk organizer guides trunk development caudally to the same r1/2 landmark. Studying
mutations in genes responsible for body patterning inthis region, the Aizawa research group hopes toreveal the genetic cascades for the constitution of theanterior head as conserved across vertebrate phyla.
The groupﾕs research focuses on identifying and studying the functions of factors acting upstream anddownstream of Otx2, a master gene in vertebratehead development, and the molecular bases for theprocesses of anterior-posterior axis formation, head
induction, brain regionalization and cortical development.

Roles of Otx2 in headdevelopment
Otx2 plays central roles in each step of head development in vertebrate, but the regulatory mechanisms by which this gene’s activity is mediated haveremained largely unknown. The development of thehead traces back to before gastrulation, and is inseparably linked to the formation of the anteriorposterior axis. In mice, this process begins prior togastrulation when the cells of the distal ventral endoderm migrate to the region that will become the animal’s anterior, forming the anterior visceral endoderm (AVE), which suppresses posteriorizing signals in the adjacent epiblast. In previous work, members of the Aizawa lab demonstrated that Otx2 plays
an essential role in triggering this anterior migration.
Following gastrulation, organizing centers in the epiblast induce the formation of the anterior neuroectoderm, which subsequently regionalizes into multiple
primitive structures destined to form the areas of thebrain. In this process, the isthmus and anterior neural ridge act as local organizing
centers for mid- and forebrain
development.
Otx2 functions as a master genein each phase of head ontogeny.
Aizawa and colleagues havebeen working to identify and characterize regulatory factors that
control the gene’s expression in

Fore- and midbrain structure at E12.5 in wildtype (left) and loss of archicortex and diencephalon in
Emx2/Otx2 double mutants (right)

specific sites and stages of development. In thepast, the lab found enhancers responsible for promoting Otx2 expression in the visceral endoderm,
definitive anterior mesendoderm and the cephalicneural crest cells. These cis-regulatory elements
were all located relatively near the transcriptional
start site for the gene. More recently, the group has
identified and analyzed enhancers that guide Otx2
expression in epiblast, anterior neuroectoderm andfore- and midbrain, which they have named the EP,
AN and FM enhancers, respectively. All of these elements are located more remotely from the coding
region (more than 80 kb upstream) than are the previously identified Otx2 enhancers.

The development of
the head is inseparably linked
to the formation of
the anterior-posterior axis
The activity of the AN enhancer is independent ofthat of the EP enhancer, and plays an essential rolein maintaining the anterior neuroectoderm throughOtx2 expression, once that region has been
induced. The Aizawa group’s studies have also indicated a phylogenetic relationship between these elements, in which FM is the most deeply conserved in
the gnathostome (jawed vertebrate) lineage, while
the epiblast enhancer appears to have beenacquired later, perhaps after the ascent of amphibians, and to include the anterior neuroectoderm
enhancer as an essential component. Using theseresults as a springboard, Aizawa and colleagues
next plan to investigate the significance of this phylogenetic specialization of enhancers in terms of theevolution of the vertebrate head, and to continue thesearch for upstream factors at work in the regulationof Otx2.

Kurokawa D, Kiyonari H,
Nakayama R, Kimura C, Matsuo I and Aizawa S. Regulation of Otx2 expression and its
functions in mouse forebrain
and midbrain. Development(2004).
Kimura-Yoshida C, Kitajima K,
Oda-Ishii I, Tian E, Suzuki M,
Yamamoto M, Suzuki T,
Kobayashi M, Aizawa S andIsao Matsuo. Characterizationof the pufferfish Otx2 cisregulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification. Development 131:5771 (2004).

choroid plexus, function as a local signaling centerin cortical development. One model of archipallial
patterning involves the expression of ligands, receptors, transcriptional factors and inhibitor molecules
to form morphogenetic gradients that direct the differentiation of areas on either side of the cortical hem,
but the actions of specific players in this model
remain to be worked out. The Aizawa group’s studies of Emx1 and Emx2, mouse homologs of the Drosophila head gap gene ems, have shown that these
two genes cooperate in two phases of cortical development. Previous work demonstrated that Emx1and Emx2 work together to generate Cajal-Retzius
cells and subplate neurons. Recent work now indicates that the two genes also play a combinatoryrole in establishing the archipallium as distinct fromthe roof plate, immediately following the closure of
the neural tube.

Nishida A, Furukawa A, Koike
C, Tano Y, Aizawa S, Matsuo I
and Furukawa T. Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal
gland development. Nature
Neuroscience
6:1255-63(2003).
Shinozaki K, Miyagi T, Yoshida M, Miyata T, Ogawa M,
Aizawa S and Suda Y.
Absence of Cajal-Retzius cells
and subplate neurons associated with defects of tangential
cell migration from ganglioniceminence in Emx1/2 double
mutant cerebral cortex. Development 129:3479-92 (2002).
Hide T, Hatakeyama J, KimuraC, Tian E, Takeda N, Ushio Y,
Shiroishi T, Aizawa S and Matsuo I. Genetic modifiers of otocephalic phenotypes in Otx2
heterozygous mutant mice.
Development 129:4347-57(2002).

Forebrain/midbrain at E10.5

10 RIKEN CDB 2003 Annual Report

Publications

RIKEN CDB 2003 Annual Report11

Emx genes in earlycortical development
In the mouse, the development of
the cerebrum is immediately preceded by the closure of the anterior neural plate at around E8.5 in the presumptive forebrain-midbrain junction. In the earliest stages of corticogenesis, the structures of the archipallium, the non-neuronal components of the choroid plexus, the hippocampal complex and the fimbria, aregenerated. It has been suggested
that the last of these structures, thefimbria, which are located at the border of the cerebral cortex and the

StaffGroup Director

Technical Staff

Student Trainee

Shinichi Aizawa

Shoko Takehara
Miwa Nakamura
Tomomi Omura
Saori Nagayoshi
Ai Inoue
Kuniko Kitajima
Hiroshi Nakano
Hitomi Tsujii
Maiko Takahashi

Mariko Hirano
Wataru Satoh
Yusuke Sakurai

Senior Scientist

Isao Matsuo
Research Scientist

Yoko Suda
Kohei Hatta
Akihito Yamamoto
Akihiko Shimono
Nobuyoshi Takasaki
Daisuke Kurokawa
Takuya Murata
Jun Kimura

Assistant

Sayo Saito

Otx2 expression in fore- andmidbrain at E10.5

Junior Research Associate

Takashi Nagano
Izumi Oda-Ishii

Kurokawa D, Takasaki N, Kiyonari H, Nakayama R, Kimura
C, Matsuo I and Aizawa S.
Regulation of Otx2 expression
and its functions in mouse epiblast and anterior neuroectoderm. Development (2004).

Special Postdoctoral Researcher

Chiharu Kimura-Yoshida

Core Program

Despite their many differences, taxa as diverse as
fish, amphibians, reptiles, birds and mammals share
a common body plan comprising three regions: thetrunk, the hindbrain/pharyngeal region, and the rostral head. Shin Aizawa is interested in the molecularbases for and phylogenetic origins of this regionalization, concentrating primarily on the genetic activity
and molecular attributes of head development.

Shinichi Aizawa
Ph. D.
Shinichi Aizawa received his
Ph. D. in Zoology from theTokyo Kyoiku University,
Department of Zoology in1973. He spent the period
from 1974 to 1979 as an investigator at the Tokyo Metropolitan Institute of Gerontology,
then two years as a research
fellow in the Laboratory of
Genetic Pathology at the University of Washington (US).
He returned to the Tokyo Metropolitan Institute of Gerontology in 1982, where heremained until 1986 when hemoved to the RIKEN Tsukuba
Life Science Center as a senior research associate. He wasappointed professor in the
Kumamoto University Schoolof Medicine Department ofMorphogenesis in 1994, andserved in that position until
2002. Since 2000 he hasserved as CDB deputy director
and group director of the Vertebrate Body Plan, as well asteam leader of the Laboratory
for Animal Resources andgenetic Engineering. He also
serves as a Managing Editor
for the journal, Mechanisms ofDevelopment.

All animals develop from the head. The formation of
inductive head organizer precedes that of the trunkorganizer, as was first demonstrated by Spemannand Mangold in their studies of amphibian embryogenesis. Gene knockout studies in mice, have demonstrated that the rhombomere r1/2 is the ground statein the mammalian body plan. The head organizer
induces the rostral head anteriorly to this rhombomere, while the trunk organizer guides trunk development caudally to the same r1/2 landmark. Studying
mutations in genes responsible for body patterning inthis region, the Aizawa research group hopes toreveal the genetic cascades for the constitution of theanterior head as conserved across vertebrate phyla.
The groupﾕs research focuses on identifying and studying the functions of factors acting upstream anddownstream of Otx2, a master gene in vertebratehead development, and the molecular bases for theprocesses of anterior-posterior axis formation, head
induction, brain regionalization and cortical development.

Roles of Otx2 in headdevelopment
Otx2 plays central roles in each step of head development in vertebrate, but the regulatory mechanisms by which this gene’s activity is mediated haveremained largely unknown. The development of thehead traces back to before gastrulation, and is inseparably linked to the formation of the anteriorposterior axis. In mice, this process begins prior togastrulation when the cells of the distal ventral endoderm migrate to the region that will become the animal’s anterior, forming the anterior visceral endoderm (AVE), which suppresses posteriorizing signals in the adjacent epiblast. In previous work, members of the Aizawa lab demonstrated that Otx2 plays
an essential role in triggering this anterior migration.
Following gastrulation, organizing centers in the epiblast induce the formation of the anterior neuroectoderm, which subsequently regionalizes into multiple
primitive structures destined to form the areas of thebrain. In this process, the isthmus and anterior neural ridge act as local organizing
centers for mid- and forebrain
development.
Otx2 functions as a master genein each phase of head ontogeny.
Aizawa and colleagues havebeen working to identify and characterize regulatory factors that
control the gene’s expression in

Fore- and midbrain structure at E12.5 in wildtype (left) and loss of archicortex and diencephalon in
Emx2/Otx2 double mutants (right)

specific sites and stages of development. In thepast, the lab found enhancers responsible for promoting Otx2 expression in the visceral endoderm,
definitive anterior mesendoderm and the cephalicneural crest cells. These cis-regulatory elements
were all located relatively near the transcriptional
start site for the gene. More recently, the group has
identified and analyzed enhancers that guide Otx2
expression in epiblast, anterior neuroectoderm andfore- and midbrain, which they have named the EP,
AN and FM enhancers, respectively. All of these elements are located more remotely from the coding
region (more than 80 kb upstream) than are the previously identified Otx2 enhancers.

The development of
the head is inseparably linked
to the formation of
the anterior-posterior axis
The activity of the AN enhancer is independent ofthat of the EP enhancer, and plays an essential rolein maintaining the anterior neuroectoderm throughOtx2 expression, once that region has been
induced. The Aizawa group’s studies have also indicated a phylogenetic relationship between these elements, in which FM is the most deeply conserved in
the gnathostome (jawed vertebrate) lineage, while
the epiblast enhancer appears to have beenacquired later, perhaps after the ascent of amphibians, and to include the anterior neuroectoderm
enhancer as an essential component. Using theseresults as a springboard, Aizawa and colleagues
next plan to investigate the significance of this phylogenetic specialization of enhancers in terms of theevolution of the vertebrate head, and to continue thesearch for upstream factors at work in the regulationof Otx2.

Kurokawa D, Kiyonari H,
Nakayama R, Kimura C, Matsuo I and Aizawa S. Regulation of Otx2 expression and its
functions in mouse forebrain
and midbrain. Development(2004).
Kimura-Yoshida C, Kitajima K,
Oda-Ishii I, Tian E, Suzuki M,
Yamamoto M, Suzuki T,
Kobayashi M, Aizawa S andIsao Matsuo. Characterizationof the pufferfish Otx2 cisregulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification. Development 131:5771 (2004).

choroid plexus, function as a local signaling centerin cortical development. One model of archipallial
patterning involves the expression of ligands, receptors, transcriptional factors and inhibitor molecules
to form morphogenetic gradients that direct the differentiation of areas on either side of the cortical hem,
but the actions of specific players in this model
remain to be worked out. The Aizawa group’s studies of Emx1 and Emx2, mouse homologs of the Drosophila head gap gene ems, have shown that these
two genes cooperate in two phases of cortical development. Previous work demonstrated that Emx1and Emx2 work together to generate Cajal-Retzius
cells and subplate neurons. Recent work now indicates that the two genes also play a combinatoryrole in establishing the archipallium as distinct fromthe roof plate, immediately following the closure of
the neural tube.

Nishida A, Furukawa A, Koike
C, Tano Y, Aizawa S, Matsuo I
and Furukawa T. Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal
gland development. Nature
Neuroscience
6:1255-63(2003).
Shinozaki K, Miyagi T, Yoshida M, Miyata T, Ogawa M,
Aizawa S and Suda Y.
Absence of Cajal-Retzius cells
and subplate neurons associated with defects of tangential
cell migration from ganglioniceminence in Emx1/2 double
mutant cerebral cortex. Development 129:3479-92 (2002).
Hide T, Hatakeyama J, KimuraC, Tian E, Takeda N, Ushio Y,
Shiroishi T, Aizawa S and Matsuo I. Genetic modifiers of otocephalic phenotypes in Otx2
heterozygous mutant mice.
Development 129:4347-57(2002).

Forebrain/midbrain at E10.5

10 RIKEN CDB 2003 Annual Report

Publications

RIKEN CDB 2003 Annual Report11Staffjunction regions, resulting in the loss of tracheal celladhesion and the detachment of these cells from theepithelium. These complementary findings strongly
suggest that a strictly maintained balance of Rac
activity is essential in determining the proper junctional assembly and disassembly of cadherins in the
Drosophila tracheal epithelium, a role it is thought tofulfill by regulating cell adhesion and motility toensure the appropriate remodeling of epithelial
sheets into tubules.

Group Director

Special Postdoctoral Researcher

Shigeo Hayashi

Nao Niwa
Kagayaki Kato

Research Scientist

Leo Tsuda
Atsushi Wada
Yoshiko Inoue
Kenji Oshima

Technical Staff

Ai Akimoto
Masako Kaido
Michiko Takeda
Housei Wada
Chiaki Kose
Hiromi Sakaguchi
Student Trainee

Tadashi Sakata
Kayoko Sakurai
Mayuko Nishimura
Masayo ShindoPart-time Staff

Kumi Ohnishi
Chizuyo Itou
Ikuko Fukuzyou

Hayashi seeks to develop an
improved understanding of thegenetic control ofepithelial morphogenesis

Ph. D.
Shigeo Hayashi received
his B. Sc. in Biology from
Kyoto University in 1982,
and his Ph. D. in Biophysics from the same institution in 1987 for his work on
lens-specific regulation of
the chicken delta crystallin
gene. Inspired by the discovery of the homeobox, hechanged his research focus
to the developmental genetics of Drosophila and spent
three years as a postdoctoral research associate in
Matthew Scott’s lab at the
University of Colorado
before returning to Japan towork in the National Institute of Genetics. He
became an associate professor at the same Institutein 1994, and professor in
1999. Also in 1999, hereceived the incentive prizeof the Genetics Society ofJapan for Genetic Studies
on Drosophila Development. He was named groupdirector of the Morphogenetic Signaling research groupat the RIKEN CDB in May
2000.

Chisa Iimuro

A number of other signaling molecules that functionin the tracheal system are known to cause gain-offunction phenotypes when overexpressed. In work
related to the Rac study, the Hayashi lab has conducted a gain-of-function screen for genes that disrupt the tracheal epithelium on overexpression. The
lab has identified several candidate genes whose
hyperactivation leads to disruptions in epithelial integrity, including genes encoding regulators of actin
dynamics, cell adhesion and cell motility. The functions of the individual candidate genes are now
being analyzed, with the goal of developing an
improved understanding of the genetic control of epithelial morphogenesis.

Core Program

Shigeo Hayashi

Assistant

Epithelial tissues areconstructedfromsheets of cells with
distinct apical-basal(top-bottom) polarities, which form junctions with each otherand interact with theextracellular matrix,
the macromolecularenvironment in thespaces between cells.
A range of vital developmental processes,
including the abilities
to build complex structures through selecTransmission electron micrographof dorsal trunk of the trachea, whichtive cell adhesion, toconsists of multiple epithelial cells
move through thejoined tightly by adherens junctions.
One of the tracheal cells is coloredbody, and to form epired and the tracheal lumen iscolored green.
thelial tissues rely oninteractions between
cells and other cells, and with the extracellularmatrix. The means by which cells are able toachieve these feats as they communicate, congregate and work together to build a body are central to the study of morphogenesis, the focus ofresearch in the Hayashi laboratory.

0:00

1:00

The regulation of epithelial
adhesion

The insect wing is an evolutionary innovation that isthought to have arisen as a derivative of the leg during an early stage in the evolution of the insect bodyplan. The close relationship between the wing andthe leg can be observed in the embryogenesis of
Drosophila, which is a highly derived winged insect.
It has been shown that wing and leg both derivefrom a common precursive structure called the limb
primordium. This structure gives rise to both the limb
and wing imaginal discs, pouches of undifferentiatedcells in the larva that form the leg and wing in theadult.

Chihara T, Kato K, Taniguchi
M, Ng J and Hayashi S. Rac
Promotes Epithelial Cell Rearrangement During Tracheal
Tubulogenesis in Drosophila.
Development 130:1419-28(2003).
Kubota K, Goto S and Hayashi
S. The Role of Wg Signaling in
the Patterning of Embryonic
Leg Primordium in Drosophila.
Dev Biol 257:117-26 (2003).

4:00

5:00

Sugimura K, Yamamoto M,
Niwa R, Satoh D, Goto S, Taniguchi M, Hayashi S andUemura T. Distinct Developmental Modes and LesionInduced Reactions of Dendrites of Two Classes of Drosophila Sensory Neurons. J
Neurosci 23:3752-60 (2003).

Ventral view of the thoracic region of a Drosophila embryo. Stripes ofWingless (purple) expression overlap the leg primordia (green) andspecify the proximal leg identity.

domains. This finding counters previous work thatsuggested that Wg plays no role in late embryonicleg disc development, and led Hayashi to propose a
new model for leg specification in which dorsalproximal, ventral-proximal and distal cells are distinguished by differences in the expression domains ofspecific genes, including Wg. The group also foundthat ectopic Wg signaling inhibits wing disc development; ectopic activation of the Wg signal in the wing
disc causes a reduction in the number of wing cells
that develop. These results indicate that Wg determines leg cell specification while inhibiting that of
the wing, thereby playing the role of a crucial binary
switch in the establishment of wing and leg identities.

The Hayashi lab has investigated the role of Wingless (Wg) signaling in the wing and leg primordia.
The Wg protein, which mediates a wide range of
inductive interactions in Drosophila, is known to berequired for the induction of imaginal discs during
embryonic development. In the leg imaginal disc,Wg serves as an axis determinant, triggering dorsalventral pattern formation by specifying the ventral
region of the leg, and by activating the Distal-less(Dll) gene in the distal region. However, Wingless'sroles in leg development have not been fully
explored.

The group found that reductions in Rac activity led
to increases in the amount and range of cadherincell adhesion molecules in both embryonic epidermis and tracheal epithelia, which they attribute to a
post-transcriptional mechanism and changes in epithelial structural organization. This strengthening ofintercellular adhesion was accompanied by a reciprocal decrease in the ability of epithelial cells torearrange dynamically. Conversely, when they overexpressed the constitutively active form of Rac intracheal cells, the researchers found that cadherins
decreased and eventually disappeared from cell

3:00

Publications

Leg and wing specification

The ability of epithelial cells to attach to and disengage from their neighbors is closely linked to theactivity of cadherin family molecules, which span thecell membrane to regulate this dynamic cell-celladhesion. A number of studies have also indicatedroles for the small GTPase Rac as another key regulator of cell adhesion, but these roles have yet to be
confirmed in the context of embryonic development.
In work published in 2003, members of the Hayashi
research group reported the results of their investigations into Rac's role in epithelial cell rearrangementin the development of the Drosophila tracheal system, a tubular network formed from epithelium.

2:00

In a study published this year, the Hayashi group
determined that Wg signaling is required forproximal-distal patterning during embryonic leg
development, a role it performs by helping to recruit
cell types specific to the proximal and distal

6:00

Shiga Y, Yasumoto R, Yamagata H and Hayashi S. Evolving Role of Antennapedia Protein in Arthropod Limb
Patterning.
Development
129:3555-61 (2002).
Hayashi S, Ito K, Sado Y, Taniguchi M, Akimoto A, Takeuchi
H, Aigaki T, Matsuzaki F,
Nakagoshi H, Tanimura T,
Ueda R, Uemura T, Yoshihara
M and Goto S. GETDB, a Database Compiling Expression
Patterns and Molecular Locations of a Collection of Gal4
Enhancer Traps. Genesis
34:58-61 (2002).

7:00
Goto S, Taniguchi M, Muraoka
M, Toyoda H, Sado Y, Kawakita M and Hayashi S. UDPSugar Transporter Implicatedin Glycosylation and Processing of Notch. Nat Cell Biol
3:816-22 (2001).

Time course of tracheal development. Time lapse pictures of anembryo expressing GFP in the tracheal system.

12 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report13

Staffjunction regions, resulting in the loss of tracheal celladhesion and the detachment of these cells from theepithelium. These complementary findings strongly
suggest that a strictly maintained balance of Rac
activity is essential in determining the proper junctional assembly and disassembly of cadherins in the
Drosophila tracheal epithelium, a role it is thought tofulfill by regulating cell adhesion and motility toensure the appropriate remodeling of epithelial
sheets into tubules.

Group Director

Special Postdoctoral Researcher

Shigeo Hayashi

Nao Niwa
Kagayaki Kato

Research Scientist

Leo Tsuda
Atsushi Wada
Yoshiko Inoue
Kenji Oshima

Technical Staff

Ai Akimoto
Masako Kaido
Michiko Takeda
Housei Wada
Chiaki Kose
Hiromi Sakaguchi
Student Trainee

Tadashi Sakata
Kayoko Sakurai
Mayuko Nishimura
Masayo ShindoPart-time Staff

Kumi Ohnishi
Chizuyo Itou
Ikuko Fukuzyou

Hayashi seeks to develop an
improved understanding of thegenetic control ofepithelial morphogenesis

Ph. D.
Shigeo Hayashi received
his B. Sc. in Biology from
Kyoto University in 1982,
and his Ph. D. in Biophysics from the same institution in 1987 for his work on
lens-specific regulation of
the chicken delta crystallin
gene. Inspired by the discovery of the homeobox, hechanged his research focus
to the developmental genetics of Drosophila and spent
three years as a postdoctoral research associate in
Matthew Scott’s lab at the
University of Colorado
before returning to Japan towork in the National Institute of Genetics. He
became an associate professor at the same Institutein 1994, and professor in
1999. Also in 1999, hereceived the incentive prizeof the Genetics Society ofJapan for Genetic Studies
on Drosophila Development. He was named groupdirector of the Morphogenetic Signaling research groupat the RIKEN CDB in May
2000.

Chisa Iimuro

A number of other signaling molecules that functionin the tracheal system are known to cause gain-offunction phenotypes when overexpressed. In work
related to the Rac study, the Hayashi lab has conducted a gain-of-function screen for genes that disrupt the tracheal epithelium on overexpression. The
lab has identified several candidate genes whose
hyperactivation leads to disruptions in epithelial integrity, including genes encoding regulators of actin
dynamics, cell adhesion and cell motility. The functions of the individual candidate genes are now
being analyzed, with the goal of developing an
improved understanding of the genetic control of epithelial morphogenesis.

Core Program

Shigeo Hayashi

Assistant

Epithelial tissues areconstructedfromsheets of cells with
distinct apical-basal(top-bottom) polarities, which form junctions with each otherand interact with theextracellular matrix,
the macromolecularenvironment in thespaces between cells.
A range of vital developmental processes,
including the abilities
to build complex structures through selecTransmission electron micrographof dorsal trunk of the trachea, whichtive cell adhesion, toconsists of multiple epithelial cells
move through thejoined tightly by adherens junctions.
One of the tracheal cells is coloredbody, and to form epired and the tracheal lumen iscolored green.
thelial tissues rely oninteractions between
cells and other cells, and with the extracellularmatrix. The means by which cells are able toachieve these feats as they communicate, congregate and work together to build a body are central to the study of morphogenesis, the focus ofresearch in the Hayashi laboratory.

0:00

1:00

The regulation of epithelial
adhesion

The insect wing is an evolutionary innovation that isthought to have arisen as a derivative of the leg during an early stage in the evolution of the insect bodyplan. The close relationship between the wing andthe leg can be observed in the embryogenesis of
Drosophila, which is a highly derived winged insect.
It has been shown that wing and leg both derivefrom a common precursive structure called the limb
primordium. This structure gives rise to both the limb
and wing imaginal discs, pouches of undifferentiatedcells in the larva that form the leg and wing in theadult.

Chihara T, Kato K, Taniguchi
M, Ng J and Hayashi S. Rac
Promotes Epithelial Cell Rearrangement During Tracheal
Tubulogenesis in Drosophila.
Development 130:1419-28(2003).
Kubota K, Goto S and Hayashi
S. The Role of Wg Signaling in
the Patterning of Embryonic
Leg Primordium in Drosophila.
Dev Biol 257:117-26 (2003).

4:00

5:00

Sugimura K, Yamamoto M,
Niwa R, Satoh D, Goto S, Taniguchi M, Hayashi S andUemura T. Distinct Developmental Modes and LesionInduced Reactions of Dendrites of Two Classes of Drosophila Sensory Neurons. J
Neurosci 23:3752-60 (2003).

Ventral view of the thoracic region of a Drosophila embryo. Stripes ofWingless (purple) expression overlap the leg primordia (green) andspecify the proximal leg identity.

domains. This finding counters previous work thatsuggested that Wg plays no role in late embryonicleg disc development, and led Hayashi to propose a
new model for leg specification in which dorsalproximal, ventral-proximal and distal cells are distinguished by differences in the expression domains ofspecific genes, including Wg. The group also foundthat ectopic Wg signaling inhibits wing disc development; ectopic activation of the Wg signal in the wing
disc causes a reduction in the number of wing cells
that develop. These results indicate that Wg determines leg cell specification while inhibiting that of
the wing, thereby playing the role of a crucial binary
switch in the establishment of wing and leg identities.

The Hayashi lab has investigated the role of Wingless (Wg) signaling in the wing and leg primordia.
The Wg protein, which mediates a wide range of
inductive interactions in Drosophila, is known to berequired for the induction of imaginal discs during
embryonic development. In the leg imaginal disc,Wg serves as an axis determinant, triggering dorsalventral pattern formation by specifying the ventral
region of the leg, and by activating the Distal-less(Dll) gene in the distal region. However, Wingless'sroles in leg development have not been fully
explored.

The group found that reductions in Rac activity led
to increases in the amount and range of cadherincell adhesion molecules in both embryonic epidermis and tracheal epithelia, which they attribute to a
post-transcriptional mechanism and changes in epithelial structural organization. This strengthening ofintercellular adhesion was accompanied by a reciprocal decrease in the ability of epithelial cells torearrange dynamically. Conversely, when they overexpressed the constitutively active form of Rac intracheal cells, the researchers found that cadherins
decreased and eventually disappeared from cell

3:00

Publications

Leg and wing specification

The ability of epithelial cells to attach to and disengage from their neighbors is closely linked to theactivity of cadherin family molecules, which span thecell membrane to regulate this dynamic cell-celladhesion. A number of studies have also indicatedroles for the small GTPase Rac as another key regulator of cell adhesion, but these roles have yet to be
confirmed in the context of embryonic development.
In work published in 2003, members of the Hayashi
research group reported the results of their investigations into Rac's role in epithelial cell rearrangementin the development of the Drosophila tracheal system, a tubular network formed from epithelium.

2:00

In a study published this year, the Hayashi group
determined that Wg signaling is required forproximal-distal patterning during embryonic leg
development, a role it performs by helping to recruit
cell types specific to the proximal and distal

6:00

Shiga Y, Yasumoto R, Yamagata H and Hayashi S. Evolving Role of Antennapedia Protein in Arthropod Limb
Patterning.
Development
129:3555-61 (2002).
Hayashi S, Ito K, Sado Y, Taniguchi M, Akimoto A, Takeuchi
H, Aigaki T, Matsuzaki F,
Nakagoshi H, Tanimura T,
Ueda R, Uemura T, Yoshihara
M and Goto S. GETDB, a Database Compiling Expression
Patterns and Molecular Locations of a Collection of Gal4
Enhancer Traps. Genesis
34:58-61 (2002).

7:00
Goto S, Taniguchi M, Muraoka
M, Toyoda H, Sado Y, Kawakita M and Hayashi S. UDPSugar Transporter Implicatedin Glycosylation and Processing of Notch. Nat Cell Biol
3:816-22 (2001).

Time course of tracheal development. Time lapse pictures of anembryo expressing GFP in the tracheal system.

12 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report13entiated and retains its multipotency, while the GMC
becomes committed to generating neurons and glial
cells. Further studies in Matsuzaki lab showed that
Prospero is tethered to the basal cortex of the neuroblast by the molecule Miranda, and subsequently
released into the newly-formed GMC. Mechanismsthat polarize cells — underlying the asymmetric distribution of those molecules — have further beenshown to have homologous counterparts in vertebrates, providing strong evidence for the evolutionary conservation of intrinsic signaling in the processof asymmetric cell division.

Coordinating asymmetric
division
The basal localization of determinants is by itself not
sufficient for the parental neuroblast to segregate
cell fate determinants to the daughter GMC. It isalso necessary for the division to occur along theaxis of polar distribution of the Miranda-Prosperocomplex. This is achieved by the coordinated alignment of the mitotic spindle along the apicobasal
axis. In the neuroblast, both the asymmetric localization of cell fate determinants and spindle orientation
are governed by a multi-protein complex that

Core Program

Intracellular asymmetry

Fumio Matsuzaki
Ph. D.

All the cells in the body originate from a single progenitor, and nearly all share an identical DNA code.
How then do the vast numbers of distinct cell types
in complex organisms differentiate from one another? The process of asymmetric division, in which regulatory factors in a single mother cell are distributed
unequally to daughter cells produced by cell division, is a key to answering that fundamental question. This asymmetric distribution of regulatory factors allows resultant cells to assume different roles
from each other in a process called fate determina-tion. This process relies on the establishment of
intracellular gradients of factors regulating theexpression of genes, which results in differential patterns of gene expression in daughter cells when thecell undergoes mitosis. As it is the expression ofgenes, and not their simple presence or absence,
which is primarily responsible for determining cell
identity, controlled asymmetric division is critical toensuring the development of appropriate numbers
and types of daughter cells, and so, to the development of the organism as a whole.

Neural cell fate determination
in Drosophila

Fumio Matsuzaki received
his B. Sc. from the Department of Biochemistry andBiophysics at the Universityof Tokyo in 1979, and his
doctorate from the same
institution in 1984, for his
work on the characterization of the erythrocyte cytoskeletal structure. He spent
the period from 1984 to
1988 as a postdoctoral fellow, first in the Department
of Cell Biology at the Tokyo
Metropolitan Institute ofMedical Science, then in
the laboratory of Gerard
Edelman at Rockefeller University. He returned toJapan in 1988 to take aposition as a section chiefin the Department of Molecular Genetics in the National Institute of Neuroscience.
In 1998, he was appointed
professor in the Institute ofDevelopment, Aging andCancer at Tohoku University and remained there until
taking his current position
as group director at the
RIKEN CDB.
In Drosophila , dividing neuroblasts localize theMiranda (green) / Prospero complex to be segregated into the daughter GMC.

14 RIKEN CDB 2003 Annual Report

Fumio Matsuzaki has dedicated his research to explicating the role of asymmetric division in determining
cell identity, using the Drosophila melanogasternervous system as a model. This system provides
an attractive research platform for studying thedevelopment of cell diversity, as the nervous system
features more cell types than any other organ system, and the fruit fly is highly amenable to genetic
manipulation. With the advent of techniques formolecular analysis, Matsuzaki and others demonstrated that the fates of daughter neural cells are inlarge part determined by unequal distribution of factors within the mother cell during the process of cell
division. Two factors in particular, known as Prospero and Numb, have been shown to localize to thebasal side of the neuroblast (the neural progenitorcell) prior to its division. This polar distribution
results in two daughter cells of distinct gene expression — a new neuroblast, and a smaller-sized ganglion mother cell (GMC) — in which Prospero andNumb are segregated in the GMC. In this process ofasymmetric division, the neuroblast remains undiffer-

The fates ofdaughter neural cells are inlarge part determined byunequal distribution offactors within themother cell during theprocess of cell division
creates intracellular asymmetry. This protein complex, named the "apical complex" for the site of its
localization in the neuroblast, includes two different
signaling complexes: atypical protein kinase C(aPKC) and heterotrimeric G protein. The Matsuzaki
lab recently demonstrated that the two signaling
pathways included in the apical complex play differential roles in the asymmetric segregation of thedeterminants. aPKC signaling is essential for the
localization of the basal Miranda-Prospero complex,
while the Pins molecule, known to be
associated with an alpha subunit of the Gprotein in the apical complex, plays a critical role in orienting the mitotic spindle.
Thus multiple intracellular signals operate
coordinately and differentially to create
intracellular asymmetry, and promote theasymmetric division of neuroblasts. The
Matsuzaki lab is now confronting questions regarding the roles of extrinsic signals and intrinsic players, and the mechanism by which cells of different sizes are
generated in the process of mitotic division.

Staff

Asymmetric cell sizes

Group Director

This last question prompted investigations that led
to the identification of a new regulatory function for
G proteins, in Drosophila neuroblast cell division, aprocess in which the resultant neuroblast is muchlarger than the GMC. During mitotic cell division,
chromosomes replicate within the mother cell andattach to spindles that draw them in opposite directions to ensure that a full complement of chromosomes is available to each daughter cell. The
chromatids are drawn to the spindle poles throughthe action of molecular motors known as microtubules. In work carried out at the CDB, Matsuzaki's
lab has found that Gβ protein signals are distributed
unequally in neuroblasts and seem to restrict thedevelopment of microtubules, resulting in a shortening of the mitotic spindle on one side of the cell.
These unequal spindle lengths cause the neuroblast
to divide at a cleavage site that is off-center, and thesizes of the daughter cells reflect this imbalance,with the daughter neuroblast being more than twice
the size of the GMC. Cells lacking the Gβ protein inquestion form a symmetrical mitotic spindle and produce daughter cells of equal size with the determinants being normally segregated. This provides avaluable platform for further studies in fields ranging
from microtubule dynamics to animal morphogenesis.

Fumio Matsuzaki
Research Scientist

Naoyuki Fuse
Yasushi Izumi
Ayano Kawaguchi
Go Shioi
Tohru Yamamoto
Technical Staff

Kanako Hisata
Taeko Suetsugu
Mai Saito
Nao Ota
Tomoko Ikawa
Junior Research Associate

Maki Maeda
AssistantChika Lee
Part-time Staff

Chie Ueda

Publications
Izumi Y, Ohta N, Itoh-Furuya
A, Fuse N and Matsuzaki F.
Differential Function of G Protein and Baz/aPKC Signaling
Pathways in Drosophia Neuroblast Asymmetric Division. J
Cell Biol (2004).
Fuse N, Hisata K, Katzen A L
and Matsuzaki F. Heterotrimeric G Proteins Regulate Daughter Cell Size Asymmetry in Drosophila Neuroblast Divisions.
Curr Biol 13:947-54 (2003).
Ohshiro T, Yagami T, Zhang C
and Matsuzaki F. Role of Cortical Tumour-Suppressor Proteins in Asymmetric Division of
Drosophila Neuroblast. Nature
408:593-6 (2000).

In the developing mouse spinal cord, Prospero (green) is transiently expressed innewborn neurons immediately after birthfrom the mitotic neural progenitors (red).

RIKEN CDB 2003 Annual Report15

entiated and retains its multipotency, while the GMC
becomes committed to generating neurons and glial
cells. Further studies in Matsuzaki lab showed that
Prospero is tethered to the basal cortex of the neuroblast by the molecule Miranda, and subsequently
released into the newly-formed GMC. Mechanismsthat polarize cells — underlying the asymmetric distribution of those molecules — have further beenshown to have homologous counterparts in vertebrates, providing strong evidence for the evolutionary conservation of intrinsic signaling in the processof asymmetric cell division.

Coordinating asymmetric
division
The basal localization of determinants is by itself not
sufficient for the parental neuroblast to segregate
cell fate determinants to the daughter GMC. It isalso necessary for the division to occur along theaxis of polar distribution of the Miranda-Prosperocomplex. This is achieved by the coordinated alignment of the mitotic spindle along the apicobasal
axis. In the neuroblast, both the asymmetric localization of cell fate determinants and spindle orientation
are governed by a multi-protein complex that

Core Program

Intracellular asymmetry

Fumio Matsuzaki
Ph. D.

All the cells in the body originate from a single progenitor, and nearly all share an identical DNA code.
How then do the vast numbers of distinct cell types
in complex organisms differentiate from one another? The process of asymmetric division, in which regulatory factors in a single mother cell are distributed
unequally to daughter cells produced by cell division, is a key to answering that fundamental question. This asymmetric distribution of regulatory factors allows resultant cells to assume different roles
from each other in a process called fate determina-tion. This process relies on the establishment of
intracellular gradients of factors regulating theexpression of genes, which results in differential patterns of gene expression in daughter cells when thecell undergoes mitosis. As it is the expression ofgenes, and not their simple presence or absence,
which is primarily responsible for determining cell
identity, controlled asymmetric division is critical toensuring the development of appropriate numbers
and types of daughter cells, and so, to the development of the organism as a whole.

Neural cell fate determination
in Drosophila

Fumio Matsuzaki received
his B. Sc. from the Department of Biochemistry andBiophysics at the Universityof Tokyo in 1979, and his
doctorate from the same
institution in 1984, for his
work on the characterization of the erythrocyte cytoskeletal structure. He spent
the period from 1984 to
1988 as a postdoctoral fellow, first in the Department
of Cell Biology at the Tokyo
Metropolitan Institute ofMedical Science, then in
the laboratory of Gerard
Edelman at Rockefeller University. He returned toJapan in 1988 to take aposition as a section chiefin the Department of Molecular Genetics in the National Institute of Neuroscience.
In 1998, he was appointed
professor in the Institute ofDevelopment, Aging andCancer at Tohoku University and remained there until
taking his current position
as group director at the
RIKEN CDB.
In Drosophila , dividing neuroblasts localize theMiranda (green) / Prospero complex to be segregated into the daughter GMC.

14 RIKEN CDB 2003 Annual Report

Fumio Matsuzaki has dedicated his research to explicating the role of asymmetric division in determining
cell identity, using the Drosophila melanogasternervous system as a model. This system provides
an attractive research platform for studying thedevelopment of cell diversity, as the nervous system
features more cell types than any other organ system, and the fruit fly is highly amenable to genetic
manipulation. With the advent of techniques formolecular analysis, Matsuzaki and others demonstrated that the fates of daughter neural cells are inlarge part determined by unequal distribution of factors within the mother cell during the process of cell
division. Two factors in particular, known as Prospero and Numb, have been shown to localize to thebasal side of the neuroblast (the neural progenitorcell) prior to its division. This polar distribution
results in two daughter cells of distinct gene expression — a new neuroblast, and a smaller-sized ganglion mother cell (GMC) — in which Prospero andNumb are segregated in the GMC. In this process ofasymmetric division, the neuroblast remains undiffer-

The fates ofdaughter neural cells are inlarge part determined byunequal distribution offactors within themother cell during theprocess of cell division
creates intracellular asymmetry. This protein complex, named the "apical complex" for the site of its
localization in the neuroblast, includes two different
signaling complexes: atypical protein kinase C(aPKC) and heterotrimeric G protein. The Matsuzaki
lab recently demonstrated that the two signaling
pathways included in the apical complex play differential roles in the asymmetric segregation of thedeterminants. aPKC signaling is essential for the
localization of the basal Miranda-Prospero complex,
while the Pins molecule, known to be
associated with an alpha subunit of the Gprotein in the apical complex, plays a critical role in orienting the mitotic spindle.
Thus multiple intracellular signals operate
coordinately and differentially to create
intracellular asymmetry, and promote theasymmetric division of neuroblasts. The
Matsuzaki lab is now confronting questions regarding the roles of extrinsic signals and intrinsic players, and the mechanism by which cells of different sizes are
generated in the process of mitotic division.

Staff

Asymmetric cell sizes

Group Director

This last question prompted investigations that led
to the identification of a new regulatory function for
G proteins, in Drosophila neuroblast cell division, aprocess in which the resultant neuroblast is muchlarger than the GMC. During mitotic cell division,
chromosomes replicate within the mother cell andattach to spindles that draw them in opposite directions to ensure that a full complement of chromosomes is available to each daughter cell. The
chromatids are drawn to the spindle poles throughthe action of molecular motors known as microtubules. In work carried out at the CDB, Matsuzaki's
lab has found that Gβ protein signals are distributed
unequally in neuroblasts and seem to restrict thedevelopment of microtubules, resulting in a shortening of the mitotic spindle on one side of the cell.
These unequal spindle lengths cause the neuroblast
to divide at a cleavage site that is off-center, and thesizes of the daughter cells reflect this imbalance,with the daughter neuroblast being more than twice
the size of the GMC. Cells lacking the Gβ protein inquestion form a symmetrical mitotic spindle and produce daughter cells of equal size with the determinants being normally segregated. This provides avaluable platform for further studies in fields ranging
from microtubule dynamics to animal morphogenesis.

Fumio Matsuzaki
Research Scientist

Naoyuki Fuse
Yasushi Izumi
Ayano Kawaguchi
Go Shioi
Tohru Yamamoto
Technical Staff

Kanako Hisata
Taeko Suetsugu
Mai Saito
Nao Ota
Tomoko Ikawa
Junior Research Associate

Maki Maeda
AssistantChika Lee
Part-time Staff

Chie Ueda

Publications
Izumi Y, Ohta N, Itoh-Furuya
A, Fuse N and Matsuzaki F.
Differential Function of G Protein and Baz/aPKC Signaling
Pathways in Drosophia Neuroblast Asymmetric Division. J
Cell Biol (2004).
Fuse N, Hisata K, Katzen A L
and Matsuzaki F. Heterotrimeric G Proteins Regulate Daughter Cell Size Asymmetry in Drosophila Neuroblast Divisions.
Curr Biol 13:947-54 (2003).
Ohshiro T, Yagami T, Zhang C
and Matsuzaki F. Role of Cortical Tumour-Suppressor Proteins in Asymmetric Division of
Drosophila Neuroblast. Nature
408:593-6 (2000).

In the developing mouse spinal cord, Prospero (green) is transiently expressed innewborn neurons immediately after birthfrom the mitotic neural progenitors (red).

RIKEN CDB 2003 Annual Report15

StaffGroup Director

Shin-ichi Nishikawa
Research Scientist

Takumi Era
Masatake Osawa
Eri Nishioka
Akiyoshi Uemura
Atsushi Togawa
Visiting Scientist

Igor M Samokhvalov
Timm T Schroeder
Collaborative Scientist

Mitsuhiro Okada
Lars M Jakt
Muneaki Miyata
Mari Kohno
Shigenobu Oshima
Naoko Yoshimura
Masahiro Yasunaga
Yoko Nakano

Masaki Kinoshita
Yasuhiro Takashima
Nobuyuki Kondo
Yasushi Kubota
EPS Chandana
Rasmus Freter
Masato Okuda
Saori Yonetani
Sentaro Kusuhara
Assistant

Maya Iwase
Sakura Yuoka
Kaori Kawahara

Technical Staff

Satomi Nishikawa
Mariko Moriyama
Masae Sato
Natalia I Samokhvalova
Keiko Gion
Kanako Yamaguchi
Student Trainee

Atsushi Takebe
Hidetoshi Sakurai
Kanako Yoshikawa
Shinsuke Tada
Siushan Mak

Core Program

Stem cell biology

Shin-ichi Nishikawa
M. D. , Ph. D.
Shin-ichi
Nishikawareceived his M. D. from the
Kyoto University School of
Medicine in 1973. He performed his internship andresidency in the Department of Internal Medicine at
the Kyoto University Chest
Disease Research Institute,
before taking an assistantprofessorship in the samedepartment in 1979. He
spent the period from 198082 at the University of Cologne Institute for Genetics(Germany) before returning
to the Chest Disease
Research Institute, where
he was appointed associate professor in the
Department of Microbiology
in 1983. He moved to the
Kumamoto University Medical School in 1987 to take aprofessorship in the Department of Immunology, andreturned to Kyoto in 1993,
as professor in the Department of Molecular Genetics
at the Kyoto Graduate
School of Medicine. He wasappointed CDB group director in 2000.

Stem cells of various types, characterized by theirabilities to self-renew and to generate more highly
specialized cells indefinitely, are the key to replenishing the body's cells. The study of these cells stands
at the heart of some of the central questions in development, but many aspects of stem cell biology,
such as the limits (if any) of the ability toreprogram them to produce progeny cellsof diverse lineages, and the identitiesof the intrinsic and extrinsic molecular factors that regulate a cell's decisions whether to maintain stemness or to commit to differentiation have yet to be resolved.
A stem cell's location and
immediate environment
can be important factors
in defining its role.
Recent findings thatstem cells may
arise from one
tissue type
and thenlater beMelanocytes(marked red) foundin each hair follicle ofthe mouse form a stem
cell system within theseminute organs. In thissystem, the most immature
stem cell compartment is locatedin the upper region and the actively
cycling compartment is located in thebottom of the follicle.

16 RIKEN CDB 2003 Annual Report

the follicle, as well as all of the GFP-negative cells
from the bulge and the subset of GFP-negative cells
found specifically to adhere to bulge melanocytes.
directed to give rise to another strongly substantiate
the model in which a stem cell's behavior and properties are influenced by external signals. By studying
the microenvironments, known as 'niches,' in which
stem cells originate, abide and proliferate, the Nishikawa research group seeks to gain insight into theidentity and function of molecules involved in stem
cell activity, and the potential of stem cell-based therapeutic applications.

Characterizing the stem cellnicheWorking to isolate the defining characteristics of specific stem cell niches, the Nishikawa group has chosen melanocyte stem cells as one of the main model
systems for their research, as melanocytes are readily identified by the presence of a melanocytespecific promoter, Dct, and are amenable to experimental manipulations that allow specific cell types tobe isolated from mixed populations. As the existence of niche cells and the identification of their characteristics can only be determined by first isolating
the stem cells they support, Nishikawa's lab has
developed an approach in which stem cells are dissected from their surroundings, dissociated andused to construct single cell libraries, a process
involving the technically challenging initial step of isolating single intact hair follicles from the skin. Targeted GFP expression is next used to label the follicular melanocytes, making it possible to distinguish them from other cells.
These cells are used to construct cDNA libraries of(GFP-positive) melanocytes from the bulge
and matrix regions ofThese follicular regions were selected based on the
results of a previous study that showed that thebulge and matrix are the two main sites of localiza-

Profiling gene expression inmelanocytes andresting stem cells promises toprovide a new resource forscientists investigatingstem cells' genetic signatures
tion of melanocyte stem cells, making them excellent sites for studies of niche properties. Each ofthese areas hosts a specific sub-population of stem
cells. The matrix niche, the site of active hair growthand pigmentation, provides an environment in which
amplifying progeny become committed to a stem
cell fate, while the bulge serves as a reservoir forstem cells in their resting, or quiescent, state. Subtractive comparisons of cDNAs from stem cell, differentiated cell and 'niche-like' (keratinocyte) libraries
revealed approximately 200 non-redundant genes,
providing the lab with targets for further experiments
in which the individual genes will be conditionallyknocked out and the resulting phenotypes examined
and analyzed. Other related studies, for which single
cell libraries of other resting stem cells are now
being constructed, will entail profiling gene expression in melanocytes and resting stem cells, which
promises to provide a new resource for scientists
investigating stem cells' genetic signatures.

Differentiation of mesoderm
and endoderm

Publications

In another ongoing project, members of the Nishikawa group are developing software to allow for
improved DNA chip analyses and using it to build a
database of the progressive intermediate states that
endothelial cells pass through in the journey fromES (embryonic stem) to terminally differentiated cell.
A number of mesoderm and endoderm cell lineages
and their intermediates are being used, as these areavailable in pure cultures and their in vitro differentiation can be steered by the addition of the appropriate factors.

Nishikawa SI, Honda K, Vieira
P and Yoshida H. Organogenesis of Peripheral Lymphoid
Organs. Immunol Rev 195:7280 (2003).
Sone M, Itoh H, Yamashita J,
Yurugi-Kobayashi T, Suzuki Y,
Kondo Y, Nonoguchi A, Sawada N, Yamahara K, Miyashita
K, Park K, Shibuya M, Nito S,
Nishikawa S, Nakao K and
Nishikawa SI. Different Differentiation Kinetics of Vascular
Progenitor Cells in Primate
and Mouse Embryonic StemCells. Circulation 107:2085-8(2003).

The mesendodermal lineage, which has been clearly defined at cell level by the Nishikawa group,
includes a number of important cell types, including
endothelial cells, the primary constituent cells inmany important organs including the lungs, liver andpancreas. Mesendodermal differentiation is characterized by the activity of specific marker genes
whose expression is switched on or off, dependingon the pathway a cell has followed and the stage it
has reached. Analysis of the stages in this stepwise
process has also made it possible to develop a system for guiding differentiation from ES cell to endodermal, mesenchymal or mesodermal (paraxial andlateral) fates in vitro using combinations of extrinsic
factors. By identifying the characteristic markers ofcells at each step on each branch of the lineage tree
and collecting samples to produce DNA arrays, theNishikawa lab is working to build a database of
cDNA profiles of the intermediate stages in ES celldifferentiation in culture, which can be queried andvisualized using software developed in the lab.
Analyses of this cDNA chip database promise tolead to a better understanding of the process ofendothelial differentiation, and of stem cell specification in general. This powerful new DNA analysis software application, which promises to make a great
contribute to the harmonization of multiple DNA
array databases, is being developed in collaboration
with the Institute of Biomedical Research and Innovation, located adjacent to the CDB in the Kobe Biomedical Industry Project park.

Hirashima M, Ogawa M,
Nishikawa S, Matsumura K,
Kawasaki K, Shibuya M, andNishikawa SI. A ChemicallyDefined Culture of VEGFr2+
Cells Derived from Embryonic
Stem Cells Reveals the Roleof Vegfr1 in Tuning theThreshold for VEGF in Developing Endothelial Cells. Blood
101:2261-7 (2003).
Hirai H, Ogawa M, Suzuki N,
Yamamoto M, Breier G, Mazda O, Imanishi J and Nishikawa SI. Hemogenic and Nonhemogenic Endothelium Can Be
Distinguished by the Activity of
Fetal Liver Kinase (Flk)-1 Promoter/Enhancer During Mouse
Embryogenesis.
Blood
101:886-93 (2003).
Matsumura K, Hirashima M,
Ogawa M, Kubo H, Hisatsune
H, Kondo N, Nishikawa S, Chiba T and Nishikawa SI. Modulation of VEGFR-2-MediatedEndothelial-Cell Activity by
VEGF-C/VEGFR-3.
Blood
101:1367-74 (2003).

Representation of the relationships between different cell types based on theexpression of approximately 700 probe sets

RIKEN CDB 2003 Annual Report17

StaffGroup Director

Shin-ichi Nishikawa
Research Scientist

Takumi Era
Masatake Osawa
Eri Nishioka
Akiyoshi Uemura
Atsushi Togawa
Visiting Scientist

Igor M Samokhvalov
Timm T Schroeder
Collaborative Scientist

Mitsuhiro Okada
Lars M Jakt
Muneaki Miyata
Mari Kohno
Shigenobu Oshima
Naoko Yoshimura
Masahiro Yasunaga
Yoko Nakano

Masaki Kinoshita
Yasuhiro Takashima
Nobuyuki Kondo
Yasushi Kubota
EPS Chandana
Rasmus Freter
Masato Okuda
Saori Yonetani
Sentaro Kusuhara
Assistant

Maya Iwase
Sakura Yuoka
Kaori Kawahara

Technical Staff

Satomi Nishikawa
Mariko Moriyama
Masae Sato
Natalia I Samokhvalova
Keiko Gion
Kanako Yamaguchi
Student Trainee

Atsushi Takebe
Hidetoshi Sakurai
Kanako Yoshikawa
Shinsuke Tada
Siushan Mak

Core Program

Stem cell biology

Shin-ichi Nishikawa
M. D. , Ph. D.
Shin-ichi
Nishikawareceived his M. D. from the
Kyoto University School of
Medicine in 1973. He performed his internship andresidency in the Department of Internal Medicine at
the Kyoto University Chest
Disease Research Institute,
before taking an assistantprofessorship in the samedepartment in 1979. He
spent the period from 198082 at the University of Cologne Institute for Genetics(Germany) before returning
to the Chest Disease
Research Institute, where
he was appointed associate professor in the
Department of Microbiology
in 1983. He moved to the
Kumamoto University Medical School in 1987 to take aprofessorship in the Department of Immunology, andreturned to Kyoto in 1993,
as professor in the Department of Molecular Genetics
at the Kyoto Graduate
School of Medicine. He wasappointed CDB group director in 2000.

Stem cells of various types, characterized by theirabilities to self-renew and to generate more highly
specialized cells indefinitely, are the key to replenishing the body's cells. The study of these cells stands
at the heart of some of the central questions in development, but many aspects of stem cell biology,
such as the limits (if any) of the ability toreprogram them to produce progeny cellsof diverse lineages, and the identitiesof the intrinsic and extrinsic molecular factors that regulate a cell's decisions whether to maintain stemness or to commit to differentiation have yet to be resolved.
A stem cell's location and
immediate environment
can be important factors
in defining its role.
Recent findings thatstem cells may
arise from one
tissue type
and thenlater beMelanocytes(marked red) foundin each hair follicle ofthe mouse form a stem
cell system within theseminute organs. In thissystem, the most immature
stem cell compartment is locatedin the upper region and the actively
cycling compartment is located in thebottom of the follicle.

16 RIKEN CDB 2003 Annual Report

the follicle, as well as all of the GFP-negative cells
from the bulge and the subset of GFP-negative cells
found specifically to adhere to bulge melanocytes.
directed to give rise to another strongly substantiate
the model in which a stem cell's behavior and properties are influenced by external signals. By studying
the microenvironments, known as 'niches,' in which
stem cells originate, abide and proliferate, the Nishikawa research group seeks to gain insight into theidentity and function of molecules involved in stem
cell activity, and the potential of stem cell-based therapeutic applications.

Characterizing the stem cellnicheWorking to isolate the defining characteristics of specific stem cell niches, the Nishikawa group has chosen melanocyte stem cells as one of the main model
systems for their research, as melanocytes are readily identified by the presence of a melanocytespecific promoter, Dct, and are amenable to experimental manipulations that allow specific cell types tobe isolated from mixed populations. As the existence of niche cells and the identification of their characteristics can only be determined by first isolating
the stem cells they support, Nishikawa's lab has
developed an approach in which stem cells are dissected from their surroundings, dissociated andused to construct single cell libraries, a process
involving the technically challenging initial step of isolating single intact hair follicles from the skin. Targeted GFP expression is next used to label the follicular melanocytes, making it possible to distinguish them from other cells.
These cells are used to construct cDNA libraries of(GFP-positive) melanocytes from the bulge
and matrix regions ofThese follicular regions were selected based on the
results of a previous study that showed that thebulge and matrix are the two main sites of localiza-

Profiling gene expression inmelanocytes andresting stem cells promises toprovide a new resource forscientists investigatingstem cells' genetic signatures
tion of melanocyte stem cells, making them excellent sites for studies of niche properties. Each ofthese areas hosts a specific sub-population of stem
cells. The matrix niche, the site of active hair growthand pigmentation, provides an environment in which
amplifying progeny become committed to a stem
cell fate, while the bulge serves as a reservoir forstem cells in their resting, or quiescent, state. Subtractive comparisons of cDNAs from stem cell, differentiated cell and 'niche-like' (keratinocyte) libraries
revealed approximately 200 non-redundant genes,
providing the lab with targets for further experiments
in which the individual genes will be conditionallyknocked out and the resulting phenotypes examined
and analyzed. Other related studies, for which single
cell libraries of other resting stem cells are now
being constructed, will entail profiling gene expression in melanocytes and resting stem cells, which
promises to provide a new resource for scientists
investigating stem cells' genetic signatures.

Differentiation of mesoderm
and endoderm

Publications

In another ongoing project, members of the Nishikawa group are developing software to allow for
improved DNA chip analyses and using it to build a
database of the progressive intermediate states that
endothelial cells pass through in the journey fromES (embryonic stem) to terminally differentiated cell.
A number of mesoderm and endoderm cell lineages
and their intermediates are being used, as these areavailable in pure cultures and their in vitro differentiation can be steered by the addition of the appropriate factors.

Nishikawa SI, Honda K, Vieira
P and Yoshida H. Organogenesis of Peripheral Lymphoid
Organs. Immunol Rev 195:7280 (2003).
Sone M, Itoh H, Yamashita J,
Yurugi-Kobayashi T, Suzuki Y,
Kondo Y, Nonoguchi A, Sawada N, Yamahara K, Miyashita
K, Park K, Shibuya M, Nito S,
Nishikawa S, Nakao K and
Nishikawa SI. Different Differentiation Kinetics of Vascular
Progenitor Cells in Primate
and Mouse Embryonic StemCells. Circulation 107:2085-8(2003).

The mesendodermal lineage, which has been clearly defined at cell level by the Nishikawa group,
includes a number of important cell types, including
endothelial cells, the primary constituent cells inmany important organs including the lungs, liver andpancreas. Mesendodermal differentiation is characterized by the activity of specific marker genes
whose expression is switched on or off, dependingon the pathway a cell has followed and the stage it
has reached. Analysis of the stages in this stepwise
process has also made it possible to develop a system for guiding differentiation from ES cell to endodermal, mesenchymal or mesodermal (paraxial andlateral) fates in vitro using combinations of extrinsic
factors. By identifying the characteristic markers ofcells at each step on each branch of the lineage tree
and collecting samples to produce DNA arrays, theNishikawa lab is working to build a database of
cDNA profiles of the intermediate stages in ES celldifferentiation in culture, which can be queried andvisualized using software developed in the lab.
Analyses of this cDNA chip database promise tolead to a better understanding of the process ofendothelial differentiation, and of stem cell specification in general. This powerful new DNA analysis software application, which promises to make a great
contribute to the harmonization of multiple DNA
array databases, is being developed in collaboration
with the Institute of Biomedical Research and Innovation, located adjacent to the CDB in the Kobe Biomedical Industry Project park.

Hirashima M, Ogawa M,
Nishikawa S, Matsumura K,
Kawasaki K, Shibuya M, andNishikawa SI. A ChemicallyDefined Culture of VEGFr2+
Cells Derived from Embryonic
Stem Cells Reveals the Roleof Vegfr1 in Tuning theThreshold for VEGF in Developing Endothelial Cells. Blood
101:2261-7 (2003).
Hirai H, Ogawa M, Suzuki N,
Yamamoto M, Breier G, Mazda O, Imanishi J and Nishikawa SI. Hemogenic and Nonhemogenic Endothelium Can Be
Distinguished by the Activity of
Fetal Liver Kinase (Flk)-1 Promoter/Enhancer During Mouse
Embryogenesis.
Blood
101:886-93 (2003).
Matsumura K, Hirashima M,
Ogawa M, Kubo H, Hisatsune
H, Kondo N, Nishikawa S, Chiba T and Nishikawa SI. Modulation of VEGFR-2-MediatedEndothelial-Cell Activity by
VEGF-C/VEGFR-3.
Blood
101:1367-74 (2003).

Representation of the relationships between different cell types based on theexpression of approximately 700 probe sets

RIKEN CDB 2003 Annual Report17

StaffGroup Director

Technical Staff

Visiting Students

Yoshiki Sasai
Kenji Mizuseki
Noriaki Sasai
Makoto Ikeya
Mami Takasaki

Ayaka Nishiyama
Yoko Nakazawa
Masako Kawada
Tomoko Haraguchi
Michiru Matsumura
Tomoko Katayama

Tatsunori Sakamoto
Akiko Arakawa
Morio Ueno
Keiji Tsuji
Takashi Irioka

Collaborative Scientist

Student Trainee

Keiko Muguruma
Hidehiko Inomata
Hong-Lin Su
Hanako Ikeda

Makoto Sakuragi
Takahiko Sato
Daisuke Kamiya

Ukiko Miyake
Ayumi Tanaka
Yuri Arizono
Junko Yamada

Research Scientist

AssistantPrimate motoneurons

Junior Research Associate

Kiichi Watanabe
Takayuki Onai

Core Program

Induction and patterning of
the nervous system

Yoshiki Sasai
M. D. , Ph. D.
Yoshiki Sasai gained his M.
D. from the Kyoto University School of Medicine in1986, subsequently performing internships in general practice and emergency
medicine. He received his
Ph. D. from the same institution in 1992, for work onneural specific transcriptional regulators. In 1993, hetook a postdoctoral fellowship in the De Robertis lab
at the UCLA School of Medicine, remaining there until
1996 when he was appointed associate professor at
the Kyoto University Schoolof Medicine. He assumed a
professorship at the Kyoto
University Institute for Frontier Medical Sciences in
1998, and was appointed
group director at the CDB
in 2000. He serves on theeditorial boards of Neuron,Development, Genesis andDevelopmental Dynamics.

The interactions between cells from different germlayers and their distributions along body axes are
established in early embryogenesis and serve todetermine the spatial disposition and normal development of tissues and organs. The specification of
the dorsal-ventral (back-belly) axis is significant inneural development in that the central nervous system forms on the dorsal side of the body in all vertebrate orders. This process is dictated by the effects
of a number of signaling factors that diffuse fromorganizing centers and direct dorsal ectoderm tomaintain a neural fate. These molecules, which
include Noggin, Chordin, Follistatin and their homologs, participate in elaborate signaling networks inwhich factors collaborate and compete to initiate theembryonic nervous system.

works involved in setting up the dorsal-ventral axis
and determining neural fate in the ectoderm, work
which led to the recent identification of Tiarin, a novel dorsalization factor. The group is now also actively developing effective methods of inducing neuralization in mammals, work which has potential for
application in the treatment of neurodegenerative
disorders, such as Parkinson's disease.

Inducing neural differentiationin mammals
The Sasai lab previously reported the developmentof a technique for inducing the differentiation of neural cells by culturing mouse ES cells on plates ofconnective cells, called stromal cells. These cells
have a strong neuralizing effect, termed SDIA (forStromal cell-Derived Inducing Activity), inducing

Beginning with the identification of the neural inducing
factor Chordin in the early
1990s, Yoshiki Sasai's
research has focused on
explicating the molecular signaling mechanisms thatenable the early embryo toorganize itself into a complex, mature organism.
Using the African clawedfrog, Xenopus laevis, as amodel in molecular embryological studies, Sasai andhis group are engaged inclarifying the structure andextent of the signaling net-

nerve cells and precursors at high rates of efficiency. In 2003, the lab built on that previous work, setting out to address the question of whether the neural differentiation of ES cells could be guided witheven greater precision and diversity by adding patterning factors — soluble molecules that work to
determine the identities of cells in a developmental
region — to the SDIA-cultured cells.

Although BMP signals have an
inhibitory effect onneural development inearlier embryonic stages,
they work to promote theneural crest at a later stage indevelopment
Using BMP4, Sasai's group succeeded in causing
the SDIA-treated ES cells to differentiate into cell
types that normally derive from the neural crest, adevelopmental region that gives rise to nerve cells in
the peripheral nervous system, including sensory,
autonomic and enteric (gut) neurons. Although BMP
signals have an inhibitory effect on neural development in earlier embryonic stages, the same signals
work to promote the neural crest at a later stage indevelopment. The group found that they were able
to induce peripheral neural types that are naturally
derived from the neural crest, including autonomic
and sensory neurons and smooth muscle cells,
when they treated the SDIA-treated cells with median concentrations of BMP4. A lower concentration
prompts the same cells to differentiate into dorsal
central nervous system neurons.
The neural crest arises from the dorsalmost regionof the developing nervous system, and BMP4 isregarded as a 'dorsalizing' factor. Other factors havean antagonistic effect to BMP4, and cause the cells
they act upon to take on a ventral character. Peripheral neurons are generally dorsal in their origins,
while much of the central nervous system derives
from more ventral developmental regions. Normal
development involves the constant, dynamic interplay between patterning factors that steer neural precursors toward either a ventral or dorsal fate.

Publications
Mizuseki K, Sakamoto T, Watanabe K, Muguruma K, Ikeya
M, Nishiyama A, Arakawa A,
Suemori H, Nakatsuji N, Kawasaki H, Murakami F and Sasai
Y. Generation of neural crestderived peripheral neurons
and floor plate cells frommouse and primate embryonicstem cells. Proc Natl Acad Sci
U S A 100:5828-33 (2003).

Sasai's group also investigated the effects of Shh(Sonic hedgehog), a factor that suppresses dorsal
and promotes ventral development in vivo. As with
BMP4, Shh provokes concentration-dependent differentiation in SDIA-cultured cells, analogous to its
function in the body. A low concentration guides
SDIA-derived neural precursors to develop into
motor neurons, while higher concentrations bringabout differentiation into neural types that in normal
development would derive from the floor plate, theventralmost region of neural development. Tests
revealed that these SDIA and Shh-treated neurons
display both the molecular markers and physiological attributes (such as directional axon guidance)
characteristic of the same types of neurons in vivo,
demonstrating that the cells produced by this method are structurally and functionally similar to natural
floor plate cells.

Tsuda H, Sasai N, MatsuoTakasaki M, Sakuragi M, Murakami Y and Sasai Y. Dorsalization of the neural tube by Xenopus tiarin, a novel patterning
factor secreted by the flanking
nonneural head ectoderm.
Neuron 33:515-28 (2002).
Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F,
Ichinose H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S, Nakatsuji N and Sasai
Y. Generation of dopaminergic
neurons and pigmented epithelia from primate ES cells bystromal cell-derived inducing
activity. Proc Natl Acad Sci U
S A 99:1580-5 (2002).

Taken together, these findings show that SDIAgenerated neural precursors respond to patterning
factors with a versatility similar to that of their in vivocounterparts. Higher concentrations of the dorsalizing factor BMP4 induce increasingly dorsal neural
types, while greater doses of Shh have the same
effect in ventralizing neural precursors. This work
represents the first efficient generation of such a
broad spectrum of neuronal types from ES cells in
culture, and the fact that the process has been demonstrated effective not only in mouse, but also inprimate cells, marks a significant step toward theability to induce specific types of neuron from ES
cell-derived precursors that is one of the foremost
challenges confronting researchers in regenerative
medicine.

Sasai N, Mizuseki K and Sasai
Y. Requirement of FoxD3class signaling for neural crest
determination in Xenopus.
Development 128:2525-36(2001).
Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y,
Nakanishi S, Nishikawa SI and
Sasai Y. Induction of midbrain
dopaminergic neurons fromES cells by stromal cellderived inducing activity. Neuron 28:31-40 (2000).

Primate sensory neurons

18 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report19

StaffGroup Director

Technical Staff

Visiting Students

Yoshiki Sasai
Kenji Mizuseki
Noriaki Sasai
Makoto Ikeya
Mami Takasaki

Ayaka Nishiyama
Yoko Nakazawa
Masako Kawada
Tomoko Haraguchi
Michiru Matsumura
Tomoko Katayama

Tatsunori Sakamoto
Akiko Arakawa
Morio Ueno
Keiji Tsuji
Takashi Irioka

Collaborative Scientist

Student Trainee

Keiko Muguruma
Hidehiko Inomata
Hong-Lin Su
Hanako Ikeda

Makoto Sakuragi
Takahiko Sato
Daisuke Kamiya

Ukiko Miyake
Ayumi Tanaka
Yuri Arizono
Junko Yamada

Research Scientist

AssistantPrimate motoneurons

Junior Research Associate

Kiichi Watanabe
Takayuki Onai

Core Program

Induction and patterning of
the nervous system

Yoshiki Sasai
M. D. , Ph. D.
Yoshiki Sasai gained his M.
D. from the Kyoto University School of Medicine in1986, subsequently performing internships in general practice and emergency
medicine. He received his
Ph. D. from the same institution in 1992, for work onneural specific transcriptional regulators. In 1993, hetook a postdoctoral fellowship in the De Robertis lab
at the UCLA School of Medicine, remaining there until
1996 when he was appointed associate professor at
the Kyoto University Schoolof Medicine. He assumed a
professorship at the Kyoto
University Institute for Frontier Medical Sciences in
1998, and was appointed
group director at the CDB
in 2000. He serves on theeditorial boards of Neuron,Development, Genesis andDevelopmental Dynamics.

The interactions between cells from different germlayers and their distributions along body axes are
established in early embryogenesis and serve todetermine the spatial disposition and normal development of tissues and organs. The specification of
the dorsal-ventral (back-belly) axis is significant inneural development in that the central nervous system forms on the dorsal side of the body in all vertebrate orders. This process is dictated by the effects
of a number of signaling factors that diffuse fromorganizing centers and direct dorsal ectoderm tomaintain a neural fate. These molecules, which
include Noggin, Chordin, Follistatin and their homologs, participate in elaborate signaling networks inwhich factors collaborate and compete to initiate theembryonic nervous system.

works involved in setting up the dorsal-ventral axis
and determining neural fate in the ectoderm, work
which led to the recent identification of Tiarin, a novel dorsalization factor. The group is now also actively developing effective methods of inducing neuralization in mammals, work which has potential for
application in the treatment of neurodegenerative
disorders, such as Parkinson's disease.

Inducing neural differentiationin mammals
The Sasai lab previously reported the developmentof a technique for inducing the differentiation of neural cells by culturing mouse ES cells on plates ofconnective cells, called stromal cells. These cells
have a strong neuralizing effect, termed SDIA (forStromal cell-Derived Inducing Activity), inducing

Beginning with the identification of the neural inducing
factor Chordin in the early
1990s, Yoshiki Sasai's
research has focused on
explicating the molecular signaling mechanisms thatenable the early embryo toorganize itself into a complex, mature organism.
Using the African clawedfrog, Xenopus laevis, as amodel in molecular embryological studies, Sasai andhis group are engaged inclarifying the structure andextent of the signaling net-

nerve cells and precursors at high rates of efficiency. In 2003, the lab built on that previous work, setting out to address the question of whether the neural differentiation of ES cells could be guided witheven greater precision and diversity by adding patterning factors — soluble molecules that work to
determine the identities of cells in a developmental
region — to the SDIA-cultured cells.

Although BMP signals have an
inhibitory effect onneural development inearlier embryonic stages,
they work to promote theneural crest at a later stage indevelopment
Using BMP4, Sasai's group succeeded in causing
the SDIA-treated ES cells to differentiate into cell
types that normally derive from the neural crest, adevelopmental region that gives rise to nerve cells in
the peripheral nervous system, including sensory,
autonomic and enteric (gut) neurons. Although BMP
signals have an inhibitory effect on neural development in earlier embryonic stages, the same signals
work to promote the neural crest at a later stage indevelopment. The group found that they were able
to induce peripheral neural types that are naturally
derived from the neural crest, including autonomic
and sensory neurons and smooth muscle cells,
when they treated the SDIA-treated cells with median concentrations of BMP4. A lower concentration
prompts the same cells to differentiate into dorsal
central nervous system neurons.
The neural crest arises from the dorsalmost regionof the developing nervous system, and BMP4 isregarded as a 'dorsalizing' factor. Other factors havean antagonistic effect to BMP4, and cause the cells
they act upon to take on a ventral character. Peripheral neurons are generally dorsal in their origins,
while much of the central nervous system derives
from more ventral developmental regions. Normal
development involves the constant, dynamic interplay between patterning factors that steer neural precursors toward either a ventral or dorsal fate.

Publications
Mizuseki K, Sakamoto T, Watanabe K, Muguruma K, Ikeya
M, Nishiyama A, Arakawa A,
Suemori H, Nakatsuji N, Kawasaki H, Murakami F and Sasai
Y. Generation of neural crestderived peripheral neurons
and floor plate cells frommouse and primate embryonicstem cells. Proc Natl Acad Sci
U S A 100:5828-33 (2003).

Sasai's group also investigated the effects of Shh(Sonic hedgehog), a factor that suppresses dorsal
and promotes ventral development in vivo. As with
BMP4, Shh provokes concentration-dependent differentiation in SDIA-cultured cells, analogous to its
function in the body. A low concentration guides
SDIA-derived neural precursors to develop into
motor neurons, while higher concentrations bringabout differentiation into neural types that in normal
development would derive from the floor plate, theventralmost region of neural development. Tests
revealed that these SDIA and Shh-treated neurons
display both the molecular markers and physiological attributes (such as directional axon guidance)
characteristic of the same types of neurons in vivo,
demonstrating that the cells produced by this method are structurally and functionally similar to natural
floor plate cells.

Tsuda H, Sasai N, MatsuoTakasaki M, Sakuragi M, Murakami Y and Sasai Y. Dorsalization of the neural tube by Xenopus tiarin, a novel patterning
factor secreted by the flanking
nonneural head ectoderm.
Neuron 33:515-28 (2002).
Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F,
Ichinose H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S, Nakatsuji N and Sasai
Y. Generation of dopaminergic
neurons and pigmented epithelia from primate ES cells bystromal cell-derived inducing
activity. Proc Natl Acad Sci U
S A 99:1580-5 (2002).

Taken together, these findings show that SDIAgenerated neural precursors respond to patterning
factors with a versatility similar to that of their in vivocounterparts. Higher concentrations of the dorsalizing factor BMP4 induce increasingly dorsal neural
types, while greater doses of Shh have the same
effect in ventralizing neural precursors. This work
represents the first efficient generation of such a
broad spectrum of neuronal types from ES cells in
culture, and the fact that the process has been demonstrated effective not only in mouse, but also inprimate cells, marks a significant step toward theability to induce specific types of neuron from ES
cell-derived precursors that is one of the foremost
challenges confronting researchers in regenerative
medicine.

Sasai N, Mizuseki K and Sasai
Y. Requirement of FoxD3class signaling for neural crest
determination in Xenopus.
Development 128:2525-36(2001).
Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y,
Nakanishi S, Nishikawa SI and
Sasai Y. Induction of midbrain
dopaminergic neurons fromES cells by stromal cellderived inducing activity. Neuron 28:31-40 (2000).

Primate sensory neurons

18 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report19ered that Fat1 is required for the stable maintenanceof tight associations between cells and the normalorganization of actin. This inhibition of Fat1 also
suppressed the formation of cell polarity, which is a
general phenomenon induced in epithelial cellsheets during wound healing. Molecular studies
uncovered an apparent partnership between Fat1and Ena/VASP (vasodilator stimulated phosphoprotein) proteins; Fat1 colocalizes and binds with
Ena/VASP proteins in filopodia, lamellipodia and celljunctions and the group's recent studies indicatethat the Ena/VASP proteins may act as links in themolecular regulatory chain leading from Fat1 toactin, although the presence of other linkages was
also suggested. The study's findings also suggestthat Fat1 co-acts with the classic cadherins to reinforce or maintain cell-cell junctions, which would
make this molecule a significant player on bothsides of the cell membrane.

Staff
Group Director

Masatoshi Takeichi
Research Scientist

Shinichi Nakagawa
Shinji Hirano
Research Associate

Sachihiro Suzuki
Research Specialist

Midori Maekawa
Special Postdoctoral Researcher

Takuji TanoueTechnical Staff

Hitomi Ishigami
Miwako Harata
Makoto Uemura
Junior Research Associate

Tetsuo Ichii
Student Trainee

Tetsuhisa Otani
Hideru Togashi
Yoshiko Kametani
Koji Tanabe
Kentaro Abe
Masakazu Kadowaki
Fumi Kubo
Shinsuke Nakao
Masamitsu Sone

Publications
Tanabe K, Takeichi M, andNakagawa S. Identification ofa nonchordate-type classic
cadherin in vertebrates: Chicken Hz cadherin is expressed inhorizontal cells of the neuralretina and contains anonchordate-specific domain
complex. Dev Dyn (2004).
Abe K, Chisaka O, van Roy F,
and Takeichi M. Stability ofdendritic spines and synaptic
contacts is controlled by
αN-catenin. Nat Neurosci(2004).

AssistantMutsuko Aiso

Core Program
Characterizing Fat1 function

Masatoshi Takeichi
Ph. D.
Masatoshi Takeichi is director of the RIKEN Kobe Institute and the Center for
Developmental Biology, as
well as the Cell Adhesion
and Tissue Patterning
research group at the same
institute. He completed theB. Sc. and M. S. programsin biology at Nagoya University before receiving adoctorate in biophysics
from Kyoto University in
1973. After attaining his Ph.
D., he took a research fellowship at the Carnegie
Institution Department ofEmbryology under Richard
Pagano. He then returned
to Kyoto University, attaining a full professorship in
the Department of Biophysics (1986-1999), before
becoming professor in the
Department of Cell andDevelopmental Biology in
the Graduate School of Biostudies at the same university. He assumed his current
positions at the CDB in
2000.

The development of multicellular systems and organisms depends on the ability of cells to congregate
and cooperate in diverse ways. Individual cells must
be able to identify each other and distinguishbetween diverse cell types. They need to be able toform bonds discriminatively, such that cells thatmust aggregate in order to form a tissue do so,while cells with different missions remain detachedfrom one another. These functions of cell recognition and bonding are achieved by several types ofmolecules known as CAMs (for cellular adhesion
molecules), which variously bind with partner
molecules present on the surfaces of substrates,
matrices and other cells. Since the discovery of
cadherins, the role of the cadherin superfamily ofmolecules in regulating cell-cell adhesion has beenstudied extensively. These molecules form complexes with other intracellular factors to create bonds
spanning membranes and intercellular spaces tojoin cells with other cells expressing similar cadherins. Cadherin bonds are dynamically stable —although the intercellular junctions they form persist
in terms of the whole cell, the individual bonds are
regulable and can respond to changes in cell state.
Masatoshi Takeichi has been engaged in exploring
the structure, function and biological implications of
the cadherin superfamily since his discovery of thefirst cadherin molecules more than twenty years
ago.

Fat1 cadherin (green) localizes at cell-cell contact sites (left), colocalizing with F-actin (red/yellow, right) in epithelial cells.

20 RIKEN CDB 2003 Annual Report

Cadherins do not act alone. Spanning both sides of
the cell membrane, cadherin molecules bind withother cadherins of like type in the space between
cells, while in the cytoplasm they form anchorage
complexes with other proteins, such as various
members of the catenin family, which in turn bindwith molecules present within the cell and participate in important cell signaling networks. In 2003,
the Takeichi lab investigated the function of a
mammalian cadherin known as Fat1, which is foundin the peripheries of cells. The group found that Fat1
acts to regulate the function of actin, a primary
component of the cytoskeleton that functions in thecontrol of cellular motility and changes in cellmorphology. Among members of the cadherin family, Fat cadherins are distinguished by their unusuallylarge number of extracellular repeat domains —nearly seven times the number found in classic
cadherins — and by theuniqueness of the primary
sequences of their cytoplasmic domains. Three Fat
cadherins have been identified in mammals — Fat1, 2
and 3. It has been shownthat the Drosophila homologof these molecules plays
roles in cell proliferation or in
the determination of planar
polarity, but the function of
the vertebrate Fats hasremained unknown.
Using RNA interference(RNAi) to knock down Fat1
function, researchers in the
Laboratory for Cell Adhesion
and Tissue Patterning discov-

S Nakagawa, S Takada, R
Takada and M Takeichi. Identification of the LaminarInducing Factor: Wnt-Signal
from the Anterior Rim Induces
Correct Laminar Formation of
the Neural Retina in Vitro. Dev
Biol 260:414-25 (2003).

Contacts between neurons
Cadherin complexes are known to play diverse roles
in a cell type-specific manner. In an ongoing studyof cadherin-catenin function in interneuronal junctions, Takeichi's lab has found that αN-catenin, aform of α-catenin specific to the nervous system, isessential to the stabilization of dendritic spines.
These projections are important for establishing andmaintaining contacts between axons and dendrites,
the two principal types of extensions from theneuronal cell body. Previous work showed that
cadherins help to regulate the adhesion of dendritic

Kubo F, Takeichi M and Nakagawa S. Wnt2b controls retinal
cell differentiation at the ciliary
marginal zone. Development
130:587-98 (2003).
Nakagawa S, Takada S, Takada R and Takeichi M. Identification of the laminar-inducing
factor: Wnt-signal from theanterior rim induces correct
laminar formation of the neuralretina in vitro. Dev Biol
260:414-25 (2003).

Cadherins do not
act alonespines to axons, which is a crucial step in synapse
formation, a finding which was complemented bythis year's elucidation of αN-catenin's role. The functional loss of this molecule resulted in dramaticincreases in spine motility and shape alterations,while αN-catenin overexpression caused spines,
which normally extend and retract intermittently, toturn over at a much lower rate, resulting in abnormal
accumulations of mature spines over time. Taken
together, these results suggest that αN-catenin is a
critical agent in maintaining the appropriate balance
between stability and turnover in synaptic contacts.

Togashi H, Abe K, Mizoguchi
A, Takaoka K, Chisaka O andTakeichi M. Cadherin regulates dendritic spine morphogenesis.
Neuron 35:77-89(2002).

Overexpression of αN-catenin in hippocampal neurons causes overproduction of dendritic spines(right). Left, control.

RIKEN CDB 2003 Annual Report21ered that Fat1 is required for the stable maintenanceof tight associations between cells and the normalorganization of actin. This inhibition of Fat1 also
suppressed the formation of cell polarity, which is a
general phenomenon induced in epithelial cellsheets during wound healing. Molecular studies
uncovered an apparent partnership between Fat1and Ena/VASP (vasodilator stimulated phosphoprotein) proteins; Fat1 colocalizes and binds with
Ena/VASP proteins in filopodia, lamellipodia and celljunctions and the group's recent studies indicatethat the Ena/VASP proteins may act as links in themolecular regulatory chain leading from Fat1 toactin, although the presence of other linkages was
also suggested. The study's findings also suggestthat Fat1 co-acts with the classic cadherins to reinforce or maintain cell-cell junctions, which would
make this molecule a significant player on bothsides of the cell membrane.

Staff
Group Director

Masatoshi Takeichi
Research Scientist

Shinichi Nakagawa
Shinji Hirano
Research Associate

Sachihiro Suzuki
Research Specialist

Midori Maekawa
Special Postdoctoral Researcher

Takuji TanoueTechnical Staff

Hitomi Ishigami
Miwako Harata
Makoto Uemura
Junior Research Associate

Tetsuo Ichii
Student Trainee

Tetsuhisa Otani
Hideru Togashi
Yoshiko Kametani
Koji Tanabe
Kentaro Abe
Masakazu Kadowaki
Fumi Kubo
Shinsuke Nakao
Masamitsu Sone

Publications
Tanabe K, Takeichi M, andNakagawa S. Identification ofa nonchordate-type classic
cadherin in vertebrates: Chicken Hz cadherin is expressed inhorizontal cells of the neuralretina and contains anonchordate-specific domain
complex. Dev Dyn (2004).
Abe K, Chisaka O, van Roy F,
and Takeichi M. Stability ofdendritic spines and synaptic
contacts is controlled by
αN-catenin. Nat Neurosci(2004).

AssistantMutsuko Aiso

Core Program
Characterizing Fat1 function

Masatoshi Takeichi
Ph. D.
Masatoshi Takeichi is director of the RIKEN Kobe Institute and the Center for
Developmental Biology, as
well as the Cell Adhesion
and Tissue Patterning
research group at the same
institute. He completed theB. Sc. and M. S. programsin biology at Nagoya University before receiving adoctorate in biophysics
from Kyoto University in
1973. After attaining his Ph.
D., he took a research fellowship at the Carnegie
Institution Department ofEmbryology under Richard
Pagano. He then returned
to Kyoto University, attaining a full professorship in
the Department of Biophysics (1986-1999), before
becoming professor in the
Department of Cell andDevelopmental Biology in
the Graduate School of Biostudies at the same university. He assumed his current
positions at the CDB in
2000.

The development of multicellular systems and organisms depends on the ability of cells to congregate
and cooperate in diverse ways. Individual cells must
be able to identify each other and distinguishbetween diverse cell types. They need to be able toform bonds discriminatively, such that cells thatmust aggregate in order to form a tissue do so,while cells with different missions remain detachedfrom one another. These functions of cell recognition and bonding are achieved by several types ofmolecules known as CAMs (for cellular adhesion
molecules), which variously bind with partner
molecules present on the surfaces of substrates,
matrices and other cells. Since the discovery of
cadherins, the role of the cadherin superfamily ofmolecules in regulating cell-cell adhesion has beenstudied extensively. These molecules form complexes with other intracellular factors to create bonds
spanning membranes and intercellular spaces tojoin cells with other cells expressing similar cadherins. Cadherin bonds are dynamically stable —although the intercellular junctions they form persist
in terms of the whole cell, the individual bonds are
regulable and can respond to changes in cell state.
Masatoshi Takeichi has been engaged in exploring
the structure, function and biological implications of
the cadherin superfamily since his discovery of thefirst cadherin molecules more than twenty years
ago.

Fat1 cadherin (green) localizes at cell-cell contact sites (left), colocalizing with F-actin (red/yellow, right) in epithelial cells.

20 RIKEN CDB 2003 Annual Report

Cadherins do not act alone. Spanning both sides of
the cell membrane, cadherin molecules bind withother cadherins of like type in the space between
cells, while in the cytoplasm they form anchorage
complexes with other proteins, such as various
members of the catenin family, which in turn bindwith molecules present within the cell and participate in important cell signaling networks. In 2003,
the Takeichi lab investigated the function of a
mammalian cadherin known as Fat1, which is foundin the peripheries of cells. The group found that Fat1
acts to regulate the function of actin, a primary
component of the cytoskeleton that functions in thecontrol of cellular motility and changes in cellmorphology. Among members of the cadherin family, Fat cadherins are distinguished by their unusuallylarge number of extracellular repeat domains —nearly seven times the number found in classic
cadherins — and by theuniqueness of the primary
sequences of their cytoplasmic domains. Three Fat
cadherins have been identified in mammals — Fat1, 2
and 3. It has been shownthat the Drosophila homologof these molecules plays
roles in cell proliferation or in
the determination of planar
polarity, but the function of
the vertebrate Fats hasremained unknown.
Using RNA interference(RNAi) to knock down Fat1
function, researchers in the
Laboratory for Cell Adhesion
and Tissue Patterning discov-

S Nakagawa, S Takada, R
Takada and M Takeichi. Identification of the LaminarInducing Factor: Wnt-Signal
from the Anterior Rim Induces
Correct Laminar Formation of
the Neural Retina in Vitro. Dev
Biol 260:414-25 (2003).

Contacts between neurons
Cadherin complexes are known to play diverse roles
in a cell type-specific manner. In an ongoing studyof cadherin-catenin function in interneuronal junctions, Takeichi's lab has found that αN-catenin, aform of α-catenin specific to the nervous system, isessential to the stabilization of dendritic spines.
These projections are important for establishing andmaintaining contacts between axons and dendrites,
the two principal types of extensions from theneuronal cell body. Previous work showed that
cadherins help to regulate the adhesion of dendritic

Kubo F, Takeichi M and Nakagawa S. Wnt2b controls retinal
cell differentiation at the ciliary
marginal zone. Development
130:587-98 (2003).
Nakagawa S, Takada S, Takada R and Takeichi M. Identification of the laminar-inducing
factor: Wnt-signal from theanterior rim induces correct
laminar formation of the neuralretina in vitro. Dev Biol
260:414-25 (2003).

Cadherins do not
act alonespines to axons, which is a crucial step in synapse
formation, a finding which was complemented bythis year's elucidation of αN-catenin's role. The functional loss of this molecule resulted in dramaticincreases in spine motility and shape alterations,while αN-catenin overexpression caused spines,
which normally extend and retract intermittently, toturn over at a much lower rate, resulting in abnormal
accumulations of mature spines over time. Taken
together, these results suggest that αN-catenin is a
critical agent in maintaining the appropriate balance
between stability and turnover in synaptic contacts.

Togashi H, Abe K, Mizoguchi
A, Takaoka K, Chisaka O andTakeichi M. Cadherin regulates dendritic spine morphogenesis.
Neuron 35:77-89(2002).

Overexpression of αN-catenin in hippocampal neurons causes overproduction of dendritic spines(right). Left, control.

RIKEN CDB 2003 Annual Report21eity of th
Nasal cav

m ous
e PH
OTO

igen
: Sh
obu

Yo

ne m
ura

The Creative Research Promoting Program provides solid support to encourage relatively young researchers
to carry out innovative and independent research plans. The teams are allowed a great deal of flexibility inregard to projects, budget use, and lab size. The program also places great emphasis on cooperation andinternational participation. It is hoped that this unique system will help to cultivate a new generation ofleading researchers by fostering the creativity and originality of investigators in a bottom-up fashion.

e
ity of th
Nasal cav

m ous
e PH
OTO

igen
: Sh
obu

Yo

ne m
ura

The Creative Research Promoting Program provides solid support to encourage relatively young researchers
to carry out innovative and independent research plans. The teams are allowed a great deal of flexibility inregard to projects, budget use, and lab size. The program also places great emphasis on cooperation andinternational participation. It is hoped that this unique system will help to cultivate a new generation ofleading researchers by fostering the creativity and originality of investigators in a bottom-up fashion.

Publications
Staff
Team Leader

Student Trainee

Takayuki Asahara

Masakazu Kuriwaka

Senior Scientist

AssistantSatoshi Murasawa
Hiromi Nishimura

Kanako Ota

Goukassian D A, Kishore R,
Krasinski K, Dolan C, Luedemann C, Yoon Y S, Kearney
M, Hanley A, Ma H, Asahara
T, Isner J M and Losordo D
W.
EngineeringtheResponse to Vascular Injury.
Divergent Effects of Deregulated E2f1 Expression on
Vascular Smooth Muscle
Cells and Endothelial Cells
Results in Endothelial Recovery and Inhibition of Neointimal Growth. Circ Res (2003).

Research Scientist

Satoshi Hasegawa
Tsuyoshi Hamada
Technical Staff

Akira Oyamada
Kazuyo Sadamoto
A colony of endothelial progenitor cells on the surface of an injured
blood vessel

Creative Research
Promoting Program

The emerging field of stem cell-based therapy continues to receive attention as one of the most promising frontiers of medical science. Building on previous work in which he identified bone marrowderived endothelial progenitor cells (EPCs) and demonstrated their role in the generation of new bloodvessels, Takayuki Asahara seeks to characterize
adult stem and progenitor cells with even greater differentiative potential, and simultaneously to translate that research into clinically relevant advances.

Stimulating EPC accumulation

Takayuki Asahara
M. D. , Ph. D.
Takayuki Asahara received
his M. D. from Tokyo Medical College in 1984, andperformed residencies in
cardiology and emergency
medicine. He worked as a
research fellow in cardiology at the Tokyo Medical
College Hospital from 1989
to 1993, before moving to afellowship in cardiovascularresearch at St. Elizabeth’s
Hospital in Boston (USA).
He was appointed assistantprofessor at Tufts School of
Medicine in 1995, and associate professor at the Tokai
University Institute of Medical Sciences in 2000. In
addition to his current position as CDB team leader,
Dr. Asahara serves as director of Regenerative Medicine and Research at the
Kobe Institute of Biomedical Research and Innovation, and Professor of Physiology at the Tokai University School of Medicine.

tions. The vascularization of regenerating tissue is a
critical component of the natural healing process aswell as fundamental to the recovery of blood vesselsthat have been damaged, blocked or lost, and theability to promote and regulate the growth of new
blood vessels using EPCs will provide impetus andnew areas to explore for researchers and cliniciansworking to develop treatments for disorders of the
cardiovascular and other systems.

Researchers in the Asahara lab found that EPCs taken from human subjects and expanded ex vivo contributed to the vascularization of the damaged limbs
of mice at higher efficiencies if the limbs were first
injected with a factor known as SDF-1 (stromal cellderived factor-1), a chemokine originally identified inmouse bone marrow and whose only known receptor is expressed in hematopoietic stem cells. The
CXCR4 transmembrane receptor, present in CD34+
hematopoietic cells, shows a strong attraction to
SDF-1 and is believed to help recruit stem cells tosites in need of vascular replacement or repair.
Vaculogenesis achieved by endothelial progenitor cells

Blood vessels are formed by two distinct physiological processes in the adult body. In angiogenesis,
new blood vessels are generated from pre-existing,differentiated endothelial cells. Vasculogenesis, on
the other hand, involves the recruitment and differentiation of previously undifferentiated EPCs at the siteof new blood vessel growth. These EPCs are themselves the progeny of adult stem cells known ashemangioblasts, which can be induced to demonstrate true pluripotency under the right culture condi-

24 RIKEN CDB 2003 Annual Report

This belief was borne out by the EPC transplantation study, in which the researchers first isolated
large numbers of human EPCs for transplantation
into mouse models of limb ischemia — localized oxygen starvation of tissue resulting in damage to bloodvessels. The transplanted human EPCs were foundto improve neovascularization in the ischemic
mouse limbs, as well as to reduce the secondary
effects of prolonged ischemia, which include necrosis and autoamputation. The incorporation of thetransplanted EPCs at ischemic sites was determined by measuring the uptake of labeled humanprogenitor cells in tissue sections from the SDF-1
and uninjected groups, which showed that the EPCs
accumulated at nearly two times the rate in thelimbs which had been injected with SDF-1 compared to that in the control group. This represents
an important first proof-of-principle demonstrationthat the homing of EPCs to a damaged area can beaugmented by an extrinsic factor.

Targeted and autologoustransplantation
EPCs represent an extremely promising new modeof promoting the therapeutic growth of blood vessels, but significant obstacles must be overcome
before that promise can be realized. Previous studies in rats have used quantities of EPCs that would
require impracticably large supplies of peripheral
blood to derive equivalent amounts of EPCs for use
in humans, and the possibility of rejection of bloodfrom non-self sources by the immune system

The vascularization ofregenerating tissue is a
critical component of thenatural healing process
remains another important potential barrier. Members of the Asahara team sought to address thesequestions by developing a system for the local, rather than systemic, transplantation of EPCs freshly isolated from the host. These studies were conductedusing a swine model of myocardial (heart muscle)
ischemia, from which small quantities of blood weredrawn to allow the isolation and expansion of EPCs,
identified by the presence of the marker moleculeCD31. Catheters were used to inject similar quantities of either CD31+ or CD31- cells, or of a control
solution containing no cells, directly into ischemic

Iwakura A, Luedemann C,
Shastry S, Hanley A, Kearney M, Aikawa R, Isner J M,
Asahara T and Losordo D W.
Estrogen-Mediated,Endothelial Nitric Oxide SynthaseDependent Mobilization ofBone Marrow-Derived
Endothelial Progenitor Cells
Contributes to Reendothelialization after Arterial Injury. Circulation (2003).

sites in the hearts of pigs, to test the effects of theconcentrated delivery of autologous EPCs. The
results showed that the targeted transplantation of
CD31+ hematopoietic cells reduced ischemic damage and promoted neovascularization, resulting in
improved cardiac function, while pigs receiving
CD31- or control solutions experienced no such benefits. A complementary study involving the targeted
transplantation of human CD34+ cells into ischemic
rat hearts showed that non-autologous cells canalso incorporate into damage sites and differentiate
into functioning endothelial cells, even when thetransplanted quantities are one-twentieth theamounts used in similar previous experiments.

Kawamoto A, Tkebuchava T,
Yamaguchi J, Nishimura H,
Yoon Y S, Milliken C, Uchida
S, Masuo O, Iwaguro H, Ma
H, Hanley A, Silver M, Kearney M, Losordo DW, Isner
JM and Asahara T. Intramyocardial Transplantation ofAutologous Endothelial Progenitor Cells for Therapeutic
Neovascularization of Myocardial Ischemia. Circulation
107:461-8 (2003).

Clinical work
In addition to his role as team leader of the CDB Laboratory for Stem Cell Translational Research, Asahara also serves as director of the regenerative medicine program at the neighboring Institute for Biomedical Research and Innovation. In its role as a translational research lab, Asahara's team is beginning clinical tests of the therapeutic uses of EPCs in the treatment of Buerger Disease (thromboangiitis obliterans), an inflammatory condition involving theobstruction of small and medium-sized blood vessels in the limbs, and linked with heavy tobacco use.
Plans are in place to expand the patient populationof this preliminary study into the therapeutic transplantation of EPCs, which may offer a new mode oftreatment for this disease which at present frequently necessitates the surgical amputation of ischemic
digits or limbs.

RIKEN CDB 2003 Annual Report

Yamaguchi J, Kusano KF,
Masuo O, Kawamoto A, Silver M, Murasawa S, BoschMarce M, Masuda H, Losordo DW, Isner JM and Asahara T. Stromal Cell-Derived
Factor-1 Effects on Ex Vivo
Expanded Endothelial Progenitor Cell Recruitment for
Ischemic Neovascularization.
Circulation 107:1322-8(2003).
Yamamoto K, Takahashi T,
Asahara T, Ohura N, Sokabe
T, Kamiya A and Ando J. Proliferation, Differentiation, andTube Formation by Endothelial Progenitor Cells in
Response to Shear Stress. J
Appl Physiol 95:2081-8(2003).

25

Publications
Staff
Team Leader

Student Trainee

Takayuki Asahara

Masakazu Kuriwaka

Senior Scientist

AssistantSatoshi Murasawa
Hiromi Nishimura

Kanako Ota

Goukassian D A, Kishore R,
Krasinski K, Dolan C, Luedemann C, Yoon Y S, Kearney
M, Hanley A, Ma H, Asahara
T, Isner J M and Losordo D
W.
EngineeringtheResponse to Vascular Injury.
Divergent Effects of Deregulated E2f1 Expression on
Vascular Smooth Muscle
Cells and Endothelial Cells
Results in Endothelial Recovery and Inhibition of Neointimal Growth. Circ Res (2003).

Research Scientist

Satoshi Hasegawa
Tsuyoshi Hamada
Technical Staff

Akira Oyamada
Kazuyo Sadamoto
A colony of endothelial progenitor cells on the surface of an injured
blood vessel

Creative Research
Promoting Program

The emerging field of stem cell-based therapy continues to receive attention as one of the most promising frontiers of medical science. Building on previous work in which he identified bone marrowderived endothelial progenitor cells (EPCs) and demonstrated their role in the generation of new bloodvessels, Takayuki Asahara seeks to characterize
adult stem and progenitor cells with even greater differentiative potential, and simultaneously to translate that research into clinically relevant advances.

Stimulating EPC accumulation

Takayuki Asahara
M. D. , Ph. D.
Takayuki Asahara received
his M. D. from Tokyo Medical College in 1984, andperformed residencies in
cardiology and emergency
medicine. He worked as a
research fellow in cardiology at the Tokyo Medical
College Hospital from 1989
to 1993, before moving to afellowship in cardiovascularresearch at St. Elizabeth’s
Hospital in Boston (USA).
He was appointed assistantprofessor at Tufts School of
Medicine in 1995, and associate professor at the Tokai
University Institute of Medical Sciences in 2000. In
addition to his current position as CDB team leader,
Dr. Asahara serves as director of Regenerative Medicine and Research at the
Kobe Institute of Biomedical Research and Innovation, and Professor of Physiology at the Tokai University School of Medicine.

tions. The vascularization of regenerating tissue is a
critical component of the natural healing process aswell as fundamental to the recovery of blood vesselsthat have been damaged, blocked or lost, and theability to promote and regulate the growth of new
blood vessels using EPCs will provide impetus andnew areas to explore for researchers and cliniciansworking to develop treatments for disorders of the
cardiovascular and other systems.

Researchers in the Asahara lab found that EPCs taken from human subjects and expanded ex vivo contributed to the vascularization of the damaged limbs
of mice at higher efficiencies if the limbs were first
injected with a factor known as SDF-1 (stromal cellderived factor-1), a chemokine originally identified inmouse bone marrow and whose only known receptor is expressed in hematopoietic stem cells. The
CXCR4 transmembrane receptor, present in CD34+
hematopoietic cells, shows a strong attraction to
SDF-1 and is believed to help recruit stem cells tosites in need of vascular replacement or repair.
Vaculogenesis achieved by endothelial progenitor cells

Blood vessels are formed by two distinct physiological processes in the adult body. In angiogenesis,
new blood vessels are generated from pre-existing,differentiated endothelial cells. Vasculogenesis, on
the other hand, involves the recruitment and differentiation of previously undifferentiated EPCs at the siteof new blood vessel growth. These EPCs are themselves the progeny of adult stem cells known ashemangioblasts, which can be induced to demonstrate true pluripotency under the right culture condi-

24 RIKEN CDB 2003 Annual Report

This belief was borne out by the EPC transplantation study, in which the researchers first isolated
large numbers of human EPCs for transplantation
into mouse models of limb ischemia — localized oxygen starvation of tissue resulting in damage to bloodvessels. The transplanted human EPCs were foundto improve neovascularization in the ischemic
mouse limbs, as well as to reduce the secondary
effects of prolonged ischemia, which include necrosis and autoamputation. The incorporation of thetransplanted EPCs at ischemic sites was determined by measuring the uptake of labeled humanprogenitor cells in tissue sections from the SDF-1
and uninjected groups, which showed that the EPCs
accumulated at nearly two times the rate in thelimbs which had been injected with SDF-1 compared to that in the control group. This represents
an important first proof-of-principle demonstrationthat the homing of EPCs to a damaged area can beaugmented by an extrinsic factor.

Targeted and autologoustransplantation
EPCs represent an extremely promising new modeof promoting the therapeutic growth of blood vessels, but significant obstacles must be overcome
before that promise can be realized. Previous studies in rats have used quantities of EPCs that would
require impracticably large supplies of peripheral
blood to derive equivalent amounts of EPCs for use
in humans, and the possibility of rejection of bloodfrom non-self sources by the immune system

The vascularization ofregenerating tissue is a
critical component of thenatural healing process
remains another important potential barrier. Members of the Asahara team sought to address thesequestions by developing a system for the local, rather than systemic, transplantation of EPCs freshly isolated from the host. These studies were conductedusing a swine model of myocardial (heart muscle)
ischemia, from which small quantities of blood weredrawn to allow the isolation and expansion of EPCs,
identified by the presence of the marker moleculeCD31. Catheters were used to inject similar quantities of either CD31+ or CD31- cells, or of a control
solution containing no cells, directly into ischemic

Iwakura A, Luedemann C,
Shastry S, Hanley A, Kearney M, Aikawa R, Isner J M,
Asahara T and Losordo D W.
Estrogen-Mediated,Endothelial Nitric Oxide SynthaseDependent Mobilization ofBone Marrow-Derived
Endothelial Progenitor Cells
Contributes to Reendothelialization after Arterial Injury. Circulation (2003).

sites in the hearts of pigs, to test the effects of theconcentrated delivery of autologous EPCs. The
results showed that the targeted transplantation of
CD31+ hematopoietic cells reduced ischemic damage and promoted neovascularization, resulting in
improved cardiac function, while pigs receiving
CD31- or control solutions experienced no such benefits. A complementary study involving the targeted
transplantation of human CD34+ cells into ischemic
rat hearts showed that non-autologous cells canalso incorporate into damage sites and differentiate
into functioning endothelial cells, even when thetransplanted quantities are one-twentieth theamounts used in similar previous experiments.

Kawamoto A, Tkebuchava T,
Yamaguchi J, Nishimura H,
Yoon Y S, Milliken C, Uchida
S, Masuo O, Iwaguro H, Ma
H, Hanley A, Silver M, Kearney M, Losordo DW, Isner
JM and Asahara T. Intramyocardial Transplantation ofAutologous Endothelial Progenitor Cells for Therapeutic
Neovascularization of Myocardial Ischemia. Circulation
107:461-8 (2003).

Clinical work
In addition to his role as team leader of the CDB Laboratory for Stem Cell Translational Research, Asahara also serves as director of the regenerative medicine program at the neighboring Institute for Biomedical Research and Innovation. In its role as a translational research lab, Asahara's team is beginning clinical tests of the therapeutic uses of EPCs in the treatment of Buerger Disease (thromboangiitis obliterans), an inflammatory condition involving theobstruction of small and medium-sized blood vessels in the limbs, and linked with heavy tobacco use.
Plans are in place to expand the patient populationof this preliminary study into the therapeutic transplantation of EPCs, which may offer a new mode oftreatment for this disease which at present frequently necessitates the surgical amputation of ischemic
digits or limbs.

RIKEN CDB 2003 Annual Report

Yamaguchi J, Kusano KF,
Masuo O, Kawamoto A, Silver M, Murasawa S, BoschMarce M, Masuda H, Losordo DW, Isner JM and Asahara T. Stromal Cell-Derived
Factor-1 Effects on Ex Vivo
Expanded Endothelial Progenitor Cell Recruitment for
Ischemic Neovascularization.
Circulation 107:1322-8(2003).
Yamamoto K, Takahashi T,
Asahara T, Ohura N, Sokabe
T, Kamiya A and Ando J. Proliferation, Differentiation, andTube Formation by Endothelial Progenitor Cells in
Response to Shear Stress. J
Appl Physiol 95:2081-8(2003).

25

expressed in blood vessels and attracts growing
axons emanating from sympathetic ganglion that
expresses RET. This interaction is crucial for sympathetic axons to follow vascular pathways and toreach the final target tissues. The fourth GFLreceptor pairing of Persephin and GFRα4 is known
to be expressed ubiquitously at low levels, but its
developmental activity remains obscure.

Staff
Team Leader

Hideki Enomoto
Research Scientist

Shiho Ohmori
Yukinao Shibukawa

Publications
Gianino S, Grider J R, Cresswell J, Enomoto H and
Heuckeroth R O. GDNF availability determines entericneuron number by controllingprecursor proliferation. Development 130:2187-98 (2003).

In related work, members of the Enomoto lab
studied the roles of RET and TRKA (neurotrophin
receptor) in sympathetic neurogenesis and maintenance. Both RET and TRKA are expressed indeveloping sympathetic neurons, but the balance
between these two receptor types shifts over time,with RET predominating during earlier phases ofaxon growth and migration, while TRK assumes amore important role in ensuring the perinatal survival and maintenance of these cells.

Enomoto H, Crawford P A,
Gorodinsky A, Heuckeroth R
O, Johnson E M, Jr. and Milbrandt J. RET signaling isessential for migration, axonal growth and axon guidanceof developing sympathetic
neurons. Development
128:3963-74 (2001).

Technical Staff

Kayoko Inoue
Mayumi Nagashimada
Mayumi Akatsuka
AssistantKaori Hamada

Studies of RET signaling are of particular interest for
their value in biomedical research. Hirschsprung's
disease, a congenital disorder afflicting about 1 in
5,000 newborn children in which enteric neuron precursors fail to colonize the distal part of the gut
resulting in a loss of bowel motility, chronic constipation and bowel obstruction, is caused by a loss-offunction mutation in the Ret gene. Hyperactivation of
RET results in abnormal development of the neuralcrest, a developmentally transient structure that normally gives rise to neurons, glia, endocrine cells andmesenchymal cells, and has been linked to the oncogenesis of endocrine tumors, such as thyroid carcinoma and pheochromocytoma.

Creative Research
Promoting Program

Neurotrophic factors

Hideki Enomoto
M. D. , Ph. D.
Hideki Enomoto received
his M. D. from the Chiba
University School of medicine in 1988, and his Ph. D.
from the same institution in
1996 for his work in themolecular cloning of thehuman DAN gene. After
serving as the Chief of the
Department of Pediatric Surgery, Matsudo Municipal
Hospital in Chiba, Japan,
he moved to the Washington University School of
Medicine, St. Louis in 1997,
to work as a research associate studying the physiological roles of the GDNF family of ligands in neural development and function. He
returned to Japan to take aposition as a team leader at
the CDB in 2002.

The early stages of neural network development see
the growth of a number of neurons in excess of thenumber that will ultimately populate any given
region. Neurons that have formed synaptic connections establish what is known as a trophic interactionin which the target tissue provides the neuron withchemical signals necessary for its survival and continued function. The supply of such signals is limited, and neurons which fail
to compete successfully
for these factors
undergo apoptosis,
a controlled die-off
that establishes
an appropriatebalance in theneural population.
The target-derived
chemical signals in
the trophic interaction
are known as neurotrophic factors. The studyof these signaling proteinsdates back to the discovery ofnerve growth factor (NGF) by
Levi-Montalcini and colleagues in
the 1950s. Subsequent research has
revealed the diversity of neurotrophic factors, which are now thought to include neurotrophins (of which NGF is a member), neuropoietins, fibroblast growth factors, and the transforming
growth factor-β superfamily. Hideki Enomoto is primarily interested in this last group, the GDNF Family
Ligands (the GFLs) in particular. This family of neu-

26 RIKEN CDB 2003 Annual Reportrotrophic factors includes four known members —GDNF (for Glial cell line Derived Neurotrophic Factor), Neurturin, Artemin and Persephin. The GFLs
signal via receptor complexes formed by the RET
receptor tyrosine kinase and one of fourco-receptors, GRFα1-4. In vitro, these four receptors show differential affinities for specific GFLs, with
GFR?1 showing the greatest ability to interact withthe range of GFL family members.
GDNF ligand-receptor pairings
Enomoto studied phenotypesof mice in which individual
GFL, GFRα or Ret genes
are disrupted and foundthat, in receptor-ligand
interactions in vivo,
there are affinitive pairings between GFRα1GDNF, GFRα2-NRTN
and GFRα3-ARTN.
The loss of GDNFGFRα1 system had
the strongest developmental effects, causing defects in enteric,autonomic, sensory
and motor neurons aswell as failures in thedevelopment of thekidney, and resulting
in lethality by the P0
stage of developX-gal staining of a mouseembryo expressing tau-lacZment, birth. Similar
under the GFRα1 promoter
neuronal populations
are affected by Neur-

Enomoto H, Heuckeroth R
O, Golden J P, Johnson E M
and Milbrandt J. Development of cranial parasympathetic ganglia requires
sequential actions of GDNF
and neurturin. Development
127:4877-89 (2000).

Studies of RET signaling are ofparticular interest for
their value in biomedical research

GFP staining of RET-expressing motor and sensory neurons in thebrainstem of RET-GFP miceturin or GFRα2 deficiency, but the phenotypes areless severe, with kidneys forming normally and indefinite effects on sympathetic and motor neural development. Analysis of the consequences of thesemutations in developing enteric and parasympathetic neurons suggests that GDNF-GFRα1 functions inneural precursors to regulate fundamental neurodevelopmental processes such as migration and proliferation, while the later expression of NeurturinGFRα2 in target tissues serves to maintain previously established neurons.
Artemin-GFRα3 signaling seems to operate in amore specific subset of cells, sympathetic neurons,with no apparent function in enteric or central neuraldevelopment. The sympathetic nervous system arises from the neural crest and follows a stepwise
developmental pathway that directs its neurons toestablish their appropriate functional roles and positions throughout the body. In a series of signaldirected decisions, sympathetic precursor cells
migrate and differentiate into neurons soon after
they undergo lineage commitment and restriction toa catecholaminergic (sympathetic neurotransmitterproducing) fate. Enomoto found that Artemin is

Baloh R H, Enomoto H, Johnson E M, Jr. and Milbrandt J.
The GDNF family ligands
and receptors - implications
for neural development. Curr
Opin Neurobiol 10:103-10(2000).

In the future, the Enomoto research team plans to
conduct systematic analyses of RET-bearing neurons to achieve a better understanding of the molecular mechanisms involved in RET and GFL-receptor
signaling. One immediate focus will be on investigating roles for RET, GDNF and GFRα1 in the postnatal maintenance of motor, sensory and dopaminergic
neurons,
using a Cre-Lox
conditional
GFL-GFRα INTERACTIONS
knockout strategy. By providing
GDNF
NRTN
ARTN
PSPN
insights into thefunctionsof
GFL-specificneurotrophic
factor signaling
pathways, Enomoto hopes tocontributetothedevelopment of stem
cell-basedtherapies for nervous
system disorders.
GFRα1
GFRα2
GFRα3
GFRα4
RET
RET
RET

RIKEN CDB 2003 Annual Report27

expressed in blood vessels and attracts growing
axons emanating from sympathetic ganglion that
expresses RET. This interaction is crucial for sympathetic axons to follow vascular pathways and toreach the final target tissues. The fourth GFLreceptor pairing of Persephin and GFRα4 is known
to be expressed ubiquitously at low levels, but its
developmental activity remains obscure.

Staff
Team Leader

Hideki Enomoto
Research Scientist

Shiho Ohmori
Yukinao Shibukawa

Publications
Gianino S, Grider J R, Cresswell J, Enomoto H and
Heuckeroth R O. GDNF availability determines entericneuron number by controllingprecursor proliferation. Development 130:2187-98 (2003).

In related work, members of the Enomoto lab
studied the roles of RET and TRKA (neurotrophin
receptor) in sympathetic neurogenesis and maintenance. Both RET and TRKA are expressed indeveloping sympathetic neurons, but the balance
between these two receptor types shifts over time,with RET predominating during earlier phases ofaxon growth and migration, while TRK assumes amore important role in ensuring the perinatal survival and maintenance of these cells.

Enomoto H, Crawford P A,
Gorodinsky A, Heuckeroth R
O, Johnson E M, Jr. and Milbrandt J. RET signaling isessential for migration, axonal growth and axon guidanceof developing sympathetic
neurons. Development
128:3963-74 (2001).

Technical Staff

Kayoko Inoue
Mayumi Nagashimada
Mayumi Akatsuka
AssistantKaori Hamada

Studies of RET signaling are of particular interest for
their value in biomedical research. Hirschsprung's
disease, a congenital disorder afflicting about 1 in
5,000 newborn children in which enteric neuron precursors fail to colonize the distal part of the gut
resulting in a loss of bowel motility, chronic constipation and bowel obstruction, is caused by a loss-offunction mutation in the Ret gene. Hyperactivation of
RET results in abnormal development of the neuralcrest, a developmentally transient structure that normally gives rise to neurons, glia, endocrine cells andmesenchymal cells, and has been linked to the oncogenesis of endocrine tumors, such as thyroid carcinoma and pheochromocytoma.

Creative Research
Promoting Program

Neurotrophic factors

Hideki Enomoto
M. D. , Ph. D.
Hideki Enomoto received
his M. D. from the Chiba
University School of medicine in 1988, and his Ph. D.
from the same institution in
1996 for his work in themolecular cloning of thehuman DAN gene. After
serving as the Chief of the
Department of Pediatric Surgery, Matsudo Municipal
Hospital in Chiba, Japan,
he moved to the Washington University School of
Medicine, St. Louis in 1997,
to work as a research associate studying the physiological roles of the GDNF family of ligands in neural development and function. He
returned to Japan to take aposition as a team leader at
the CDB in 2002.

The early stages of neural network development see
the growth of a number of neurons in excess of thenumber that will ultimately populate any given
region. Neurons that have formed synaptic connections establish what is known as a trophic interactionin which the target tissue provides the neuron withchemical signals necessary for its survival and continued function. The supply of such signals is limited, and neurons which fail
to compete successfully
for these factors
undergo apoptosis,
a controlled die-off
that establishes
an appropriatebalance in theneural population.
The target-derived
chemical signals in
the trophic interaction
are known as neurotrophic factors. The studyof these signaling proteinsdates back to the discovery ofnerve growth factor (NGF) by
Levi-Montalcini and colleagues in
the 1950s. Subsequent research has
revealed the diversity of neurotrophic factors, which are now thought to include neurotrophins (of which NGF is a member), neuropoietins, fibroblast growth factors, and the transforming
growth factor-β superfamily. Hideki Enomoto is primarily interested in this last group, the GDNF Family
Ligands (the GFLs) in particular. This family of neu-

26 RIKEN CDB 2003 Annual Reportrotrophic factors includes four known members —GDNF (for Glial cell line Derived Neurotrophic Factor), Neurturin, Artemin and Persephin. The GFLs
signal via receptor complexes formed by the RET
receptor tyrosine kinase and one of fourco-receptors, GRFα1-4. In vitro, these four receptors show differential affinities for specific GFLs, with
GFR?1 showing the greatest ability to interact withthe range of GFL family members.
GDNF ligand-receptor pairings
Enomoto studied phenotypesof mice in which individual
GFL, GFRα or Ret genes
are disrupted and foundthat, in receptor-ligand
interactions in vivo,
there are affinitive pairings between GFRα1GDNF, GFRα2-NRTN
and GFRα3-ARTN.
The loss of GDNFGFRα1 system had
the strongest developmental effects, causing defects in enteric,autonomic, sensory
and motor neurons aswell as failures in thedevelopment of thekidney, and resulting
in lethality by the P0
stage of developX-gal staining of a mouseembryo expressing tau-lacZment, birth. Similar
under the GFRα1 promoter
neuronal populations
are affected by Neur-

Enomoto H, Heuckeroth R
O, Golden J P, Johnson E M
and Milbrandt J. Development of cranial parasympathetic ganglia requires
sequential actions of GDNF
and neurturin. Development
127:4877-89 (2000).

Studies of RET signaling are ofparticular interest for
their value in biomedical research

GFP staining of RET-expressing motor and sensory neurons in thebrainstem of RET-GFP miceturin or GFRα2 deficiency, but the phenotypes areless severe, with kidneys forming normally and indefinite effects on sympathetic and motor neural development. Analysis of the consequences of thesemutations in developing enteric and parasympathetic neurons suggests that GDNF-GFRα1 functions inneural precursors to regulate fundamental neurodevelopmental processes such as migration and proliferation, while the later expression of NeurturinGFRα2 in target tissues serves to maintain previously established neurons.
Artemin-GFRα3 signaling seems to operate in amore specific subset of cells, sympathetic neurons,with no apparent function in enteric or central neuraldevelopment. The sympathetic nervous system arises from the neural crest and follows a stepwise
developmental pathway that directs its neurons toestablish their appropriate functional roles and positions throughout the body. In a series of signaldirected decisions, sympathetic precursor cells
migrate and differentiate into neurons soon after
they undergo lineage commitment and restriction toa catecholaminergic (sympathetic neurotransmitterproducing) fate. Enomoto found that Artemin is

Baloh R H, Enomoto H, Johnson E M, Jr. and Milbrandt J.
The GDNF family ligands
and receptors - implications
for neural development. Curr
Opin Neurobiol 10:103-10(2000).

In the future, the Enomoto research team plans to
conduct systematic analyses of RET-bearing neurons to achieve a better understanding of the molecular mechanisms involved in RET and GFL-receptor
signaling. One immediate focus will be on investigating roles for RET, GDNF and GFRα1 in the postnatal maintenance of motor, sensory and dopaminergic
neurons,
using a Cre-Lox
conditional
GFL-GFRα INTERACTIONS
knockout strategy. By providing
GDNF
NRTN
ARTN
PSPN
insights into thefunctionsof
GFL-specificneurotrophic
factor signaling
pathways, Enomoto hopes tocontributetothedevelopment of stem
cell-basedtherapies for nervous
system disorders.
GFRα1
GFRα2
GFRα3
GFRα4
RET
RET
RET

RIKEN CDB 2003 Annual Report27

the production of geneticallymosaic flies in which specificolfactory neurons are homozygous for mutations in otherwise heterozygous animals.
This screening approach has
already yielded approximately
70 mutant lines exhibiting phenotypes in which axons mistarget, overshoot, arborize improperly or fail to project entirely.
Some of these mutations are
now being mapped on the chromosome by SNP analyses andthe use of chromosomal deficiencies.

Staff
Team Leader

Chihiro Hama
Research Scientist

Minako Orihara
Keita Endo
Kazunaga Takizawa
Hiroko Saito

Vesicle transport in neuraldevelopment

Technical Staff

Yuka Yoda
Kyoko Ishikawa

All cells contain cargo-bearing compartments known
as vesicles. These vesicles function to deliver themacromolecules that serve the cells as membranecomponents or secreted signals, and the metabolite
products of the cellular digestive process to the restof the cell, where they are stored or used to fuelAssistant

Kanako Moriwaki

Creative Research
Promoting Program

Neural networks
Neural network development involves the orchestration of complex processes from neuronal differentiation to axon growth to the formation of synapses. The
mechanisms by which neuronal cells are able to identify and form synapses with appropriate partners, andthe ways that circuits formed from interconnecting
neurons are able to demonstrate adaptive plasticity
are questions of bewildering intricacy and depth.

Chihiro Hama
Ph. D.
Chihiro Hama received his
B. Sc. and M. Sc. from the
University of Tokyo Department of Biophysics and Biochemistry and was awarded a Ph. D from the same
institution in 1985 for his
work on the regulation ofplasmid ColIb DNA replication by inc and repY. He
spent the period from 1985
to 1988 as a post-doc in
the laboratory of Thomas
Kornberg at the Universityof California, San Francisco
before returning to Japan tocontinue his post-doctoral
work at the National Institute of Neuroscience,
NCNP,
Tokyo.
He
advanced to section chiefin the Department of Molecular Genetics in 1991, andremained at the NCNP until
2001 when he was appointed to his current position at
the CDB.

Chihiro Hama is working to address these questions
through investigations into the neural network of the
fruit fly Drosophila, a system which, in spite of its
comparatively simple structure, demonstrates thecapacity for instinctive behaviors, learning and memory. While there are profound differences in degrees
of network sophistication and cell type diversity between Drosophila and more highly evolved organisms such as mammals, they nonetheless share alarge number of homologous genes important in neu-

Mushroom body in the Drosophila brain

ral development. Using a research strategy of largescale mutant screening in the fly, the Hama
research team seeks to open new windows into thegenetic bases of neural circuitry.

Slender lobes
The mushroom body, containing on the order of 2000
neurons, is the functional center for olfactory leaning
and memory in Drosophila. Minako Orihara in theHama lab has identified the gene slender lobes (sle),
whose homozygous mutation results in sterile flies
with thinner axonal bundles and reduced neuron
counts in the mushroom body, a phenotype attributable to the failure of flies lacking the sle gene to sustain the normally high rate of cellular proliferation by
mushroom body neuroblasts (neural progenitor cells).
The protein encoded by the sle gene is found in the
nuclei of a variety of cells, including neuroblasts,
and is abundant in the perinucleolar region during
interphase. Genetic analyses
have revealed that a majority ofinterphase nucleoli in the mutant
are shifted to a particular state ofassembly that transiently or rarely occurs in the wild type. Electron microscopy has showed thatthe nucleoli in mutant neuroblasts are unusually densely
packed, and are separated fromthe perinucleolar heterochromatins that surround or tightly associate with nucleoli in wild type.
Similar phenotypes were also
observed in neurons that are quiescent during mitosis and DNA

Targeting of olfactory sensory neurons into specific glomeruli in theantenna lobes

synthesis. The Hama lab's studies indicate that Sle,a novel perinucleolar protein, may modulate nucleolar assembly and consequently promotes the proliferation of neuroblasts.

Olfactory axon guidance
The Drosophila olfactory sensory system comprises
around 1,300 neurons that are positioned in thehead appendages and project into the antennal lobeof the brain, which contains 43 glomerular synaptic
clusters of sensory neurons and interneurons. Eachindividual sensory neuron expresses only one or two
types of the approximately 60 known types of odorant receptors, and all neurons expressing the sameodorant receptor project to the same glomerulus.
The Hama lab is interested in resolving the mechanisms by which these specialized neurons are able toidentify and home to specific targets over considerable distances with such great accuracy during theorganization of the olfactory network.
This research seeks both to clarify theroles of known genetic elements and to
uncover new pieces needed to solve
this intricate puzzle through screening
for new mutations that affect the projection of olfactory neurons.

Publications
Saito M, Awasaki T and
Hama C. Genetic analysesof essential genes in cytological region 61D1-2 to 61F1-2
of Drosophila melanogaster.
Mol Genet Genomics
268:446-54 (2002).

The Hama lab is interested inresolving the mechanisms bywhich olfactory neurons identify
and home to specific targets

Sone M, Suzuki E, Hoshino
M, Hou D, Kuromi H, Fukata
M, Kuroda S, Kaibuchi K,
Nabeshima Y and Hama C.
Synaptic development is controlled in the periactive zones
of Drosophila synapses.
Development 127:4157-68(2000).

activity. Microtubules serve as 'railways' for vesicletransport; their ends anchor to sites on the cell cortex where the vesicles fuse. It is likely that the extrusion of cell membrane at given sites, such as takes
place in axon extension, requires vesicles to betransported along microtubule in a specific orientation. To clarify how vesicle transport is involved inneural development, members of the Hama lab are
now analyzing Drosophila orthologs of genes encoding proteins known to be involved in the regulationof vesicular traffic in budding yeast. Hama hopes to
elucidate the roles of these proteins in neurite extension, branching and guidance, and synapse formation as well, by conducting conditional RNAi studies
using a DNA-based transgenic approach.

Awasaki T, Saito M, Sone M,
Suzuki E, Sakai R, Ito K andHama C. The Drosophila trio
plays an essential role in patterning of axons by regulating their directional
extension. Neuron 26:119-31(2000).

Keita Endo in the Hama lab has developed a system to generate mutant flies
and screen them for olfactory neuron
projection phenotypes by visualizing
specific projection patterns. The firststep of this approach takes advantage
of an enhancer trap line that enables
the forced expression of marker genes
in specific subsets of olfactory sensory
neurons, making it possible to visualize
the extension of their axons toward theglomeruli. The second phase entails
Antenna segment (top arrow) and maxillary palp (bottom arrow); olfactory sensory
apparatuses in Drosophila

28 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report29the production of geneticallymosaic flies in which specificolfactory neurons are homozygous for mutations in otherwise heterozygous animals.
This screening approach has
already yielded approximately
70 mutant lines exhibiting phenotypes in which axons mistarget, overshoot, arborize improperly or fail to project entirely.
Some of these mutations are
now being mapped on the chromosome by SNP analyses andthe use of chromosomal deficiencies.

Staff
Team Leader

Chihiro Hama
Research Scientist

Minako Orihara
Keita Endo
Kazunaga Takizawa
Hiroko Saito

Vesicle transport in neuraldevelopment

Technical Staff

Yuka Yoda
Kyoko Ishikawa

All cells contain cargo-bearing compartments known
as vesicles. These vesicles function to deliver themacromolecules that serve the cells as membranecomponents or secreted signals, and the metabolite
products of the cellular digestive process to the restof the cell, where they are stored or used to fuelAssistant

Kanako Moriwaki

Creative Research
Promoting Program

Neural networks
Neural network development involves the orchestration of complex processes from neuronal differentiation to axon growth to the formation of synapses. The
mechanisms by which neuronal cells are able to identify and form synapses with appropriate partners, andthe ways that circuits formed from interconnecting
neurons are able to demonstrate adaptive plasticity
are questions of bewildering intricacy and depth.

Chihiro Hama
Ph. D.
Chihiro Hama received his
B. Sc. and M. Sc. from the
University of Tokyo Department of Biophysics and Biochemistry and was awarded a Ph. D from the same
institution in 1985 for his
work on the regulation ofplasmid ColIb DNA replication by inc and repY. He
spent the period from 1985
to 1988 as a post-doc in
the laboratory of Thomas
Kornberg at the Universityof California, San Francisco
before returning to Japan tocontinue his post-doctoral
work at the National Institute of Neuroscience,
NCNP,
Tokyo.
He
advanced to section chiefin the Department of Molecular Genetics in 1991, andremained at the NCNP until
2001 when he was appointed to his current position at
the CDB.

Chihiro Hama is working to address these questions
through investigations into the neural network of the
fruit fly Drosophila, a system which, in spite of its
comparatively simple structure, demonstrates thecapacity for instinctive behaviors, learning and memory. While there are profound differences in degrees
of network sophistication and cell type diversity between Drosophila and more highly evolved organisms such as mammals, they nonetheless share alarge number of homologous genes important in neu-

Mushroom body in the Drosophila brain

ral development. Using a research strategy of largescale mutant screening in the fly, the Hama
research team seeks to open new windows into thegenetic bases of neural circuitry.

Slender lobes
The mushroom body, containing on the order of 2000
neurons, is the functional center for olfactory leaning
and memory in Drosophila. Minako Orihara in theHama lab has identified the gene slender lobes (sle),
whose homozygous mutation results in sterile flies
with thinner axonal bundles and reduced neuron
counts in the mushroom body, a phenotype attributable to the failure of flies lacking the sle gene to sustain the normally high rate of cellular proliferation by
mushroom body neuroblasts (neural progenitor cells).
The protein encoded by the sle gene is found in the
nuclei of a variety of cells, including neuroblasts,
and is abundant in the perinucleolar region during
interphase. Genetic analyses
have revealed that a majority ofinterphase nucleoli in the mutant
are shifted to a particular state ofassembly that transiently or rarely occurs in the wild type. Electron microscopy has showed thatthe nucleoli in mutant neuroblasts are unusually densely
packed, and are separated fromthe perinucleolar heterochromatins that surround or tightly associate with nucleoli in wild type.
Similar phenotypes were also
observed in neurons that are quiescent during mitosis and DNA

Targeting of olfactory sensory neurons into specific glomeruli in theantenna lobes

synthesis. The Hama lab's studies indicate that Sle,a novel perinucleolar protein, may modulate nucleolar assembly and consequently promotes the proliferation of neuroblasts.

Olfactory axon guidance
The Drosophila olfactory sensory system comprises
around 1,300 neurons that are positioned in thehead appendages and project into the antennal lobeof the brain, which contains 43 glomerular synaptic
clusters of sensory neurons and interneurons. Eachindividual sensory neuron expresses only one or two
types of the approximately 60 known types of odorant receptors, and all neurons expressing the sameodorant receptor project to the same glomerulus.
The Hama lab is interested in resolving the mechanisms by which these specialized neurons are able toidentify and home to specific targets over considerable distances with such great accuracy during theorganization of the olfactory network.
This research seeks both to clarify theroles of known genetic elements and to
uncover new pieces needed to solve
this intricate puzzle through screening
for new mutations that affect the projection of olfactory neurons.

Publications
Saito M, Awasaki T and
Hama C. Genetic analysesof essential genes in cytological region 61D1-2 to 61F1-2
of Drosophila melanogaster.
Mol Genet Genomics
268:446-54 (2002).

The Hama lab is interested inresolving the mechanisms bywhich olfactory neurons identify
and home to specific targets

Sone M, Suzuki E, Hoshino
M, Hou D, Kuromi H, Fukata
M, Kuroda S, Kaibuchi K,
Nabeshima Y and Hama C.
Synaptic development is controlled in the periactive zones
of Drosophila synapses.
Development 127:4157-68(2000).

activity. Microtubules serve as 'railways' for vesicletransport; their ends anchor to sites on the cell cortex where the vesicles fuse. It is likely that the extrusion of cell membrane at given sites, such as takes
place in axon extension, requires vesicles to betransported along microtubule in a specific orientation. To clarify how vesicle transport is involved inneural development, members of the Hama lab are
now analyzing Drosophila orthologs of genes encoding proteins known to be involved in the regulationof vesicular traffic in budding yeast. Hama hopes to
elucidate the roles of these proteins in neurite extension, branching and guidance, and synapse formation as well, by conducting conditional RNAi studies
using a DNA-based transgenic approach.

Awasaki T, Saito M, Sone M,
Suzuki E, Sakai R, Ito K andHama C. The Drosophila trio
plays an essential role in patterning of axons by regulating their directional
extension. Neuron 26:119-31(2000).

Keita Endo in the Hama lab has developed a system to generate mutant flies
and screen them for olfactory neuron
projection phenotypes by visualizing
specific projection patterns. The firststep of this approach takes advantage
of an enhancer trap line that enables
the forced expression of marker genes
in specific subsets of olfactory sensory
neurons, making it possible to visualize
the extension of their axons toward theglomeruli. The second phase entails
Antenna segment (top arrow) and maxillary palp (bottom arrow); olfactory sensory
apparatuses in Drosophila

28 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report29Hibi and colleagues set out to clone the ogon gene(which is also known as mercedes and short tail), in
order to determine its precise relationship with Chordin, and the molecular bases for its action in regulating the establishment of the dorsal-ventral axis. The
results of that positional cloning showed that ogon
encodes a homolog of the Secreted Frizzled (Sizzled) protein, which has similarities to the Wnt recep-

Organizing centers,
which emerge soon after
fertilization, play pivotal roles insetting up positional axesin the zebrafish embryo
tor, Frizzled. The canonical Wnt pathway is known
to play a role in axis formation, but it has been suggested that ogon/sizzled influences the dorsalventral axis independently of its effecton Wnt. To test this idea, members ofthe Hibi lab compared the activity of
Ogon/Sizzled with that of Wnt inhibitors in ectopic expression studies,and found that the effects of
Ogon/Sizzled misexpression
resembled those of BMP,
but not Wnt, inhibitors,
suggesting an exceptional mode ofaction.

Creative Research
Promoting Program

Masahiko Hibi
M. D. , Ph. D.
Masahiko Hibi received his
M. D. from Hiroshima University, School of Medicine
in 1988, and his Ph. D.
from the Osaka University
Institute for Molecular andCellular Biology in 1992.
From 1992 to 1995, heworked as a postdoctoral
fellow in Michael Karin’s lab
in the University of California, San Diego Departmentof Pharmacology, thenreturned to Japan to takean assistant professorshipin the Division of Molecular
Oncology at Osaka University Medical School. He wasappointed associate professor in the same division in
1999, where he remained
until he assumed his position as team leader at the
RIKEN CDB.

Vertebrate embryogenesis begins with a seemingly
formless egg that, through processes of division,
growth, differentiation, migration and rearrangement, rapidly gives rise to a highly organized structure characterized by a number of definitive axes.
This transformation is particularly striking and rapid
in the zebrafish (Danio rerio), which in the space ofa single day develops from an ovum to a recognizably vertebrate body laid down in respect to multiple
polar vectors. The speed of its growth and reproduction, coupled with its amenability to genetic studies
and the revealing pellucidity of its embryo, make thisorganism an ideal system for the study of axis formation in early vertebrate development.

In work published in 2003, the Hibi lab analyzed andisolated the responsible gene for a mutant named
ogon, which displays ventralized phenotypes, andfound that the ogon locus encodes a zebrafish
homologue of the protein Secreted Frizzled (Sizzled), which functions as negative feedback regulator of BMP signaling. While numerous mutations are
known to result in dorsalized phenotypes, ogon is
only the second zebrafish mutant reported to show
clearly ventralized phenotypes, such as expanded
ventral tail fins, blood, pronephron, and other posterior structures. These patterns are similar to the phenotypes observed in mutations of the gene for the
BMP antagonist, Chordin, which suggested that
Ogon might also be involved in BMP inhibition.

Regulating thedorsal-ventral axis
Masahiko Hibi’s laboratory focuses on the molecular
genetic cascade of events stemming from organizing centers in the zebrafish embryo. These centers,
which emerge soon after fertilization, play pivotal
roles in setting up positional axes; Hibi’s lab is particularly interested in the formation of the dorsalventral (back-belly) axis, an essential step in theestablishment of the body plan. An embryonicregion known in zebrafish as the dorsal organizer
guides dorsoventral patterning by generating inductive signals, including inhibitors of BMPs and Wnts
(molecules which ultimately determine dorsal andventral fates). These molecules play roles in a complex network of axis-determining factors, and theinterplay between dorsalizing and ventralizing factors is central to the formation of the dorsal mesoderm and endoderm, and the neuroectoderm.

ogon mutant and ogon-overexpressing embryos. Ogon functions as a negative feedback regulator of BMP signaling

30 RIKEN CDB 2003 Annual Report

They next lookedat the possible
involvement of
Chordin (which is
known to be essential in the process of
dorsalization)
in
Ogo/Szl’s effects byinjecting ogon/sizzled
RNA into mutants (chordino) lacking a functional
chordin gene. This
failed to rescue the ventralized phenotype ofhomozygous chordino
mutants, indicating that
Ogon/Sizzled activity
depends on Chordin.
The detailed functional
relationshipbetweenthese two dorsalizing factors remains to beworked out, but these initial findings suggest that
Ogon/Sizzled augments
Chordin activity either byinhibiting a Chordin
inhibitor, by upregulating
Chordin activity, or bymaking BMP more sensitive to Chordin’s inhibitory effects.

Neurogenesis
Vertebrate neuronal tissues are
generated in a stepwise fashion.
These steps include neural induction, antero-posterior patterning,
and neurogenesis. In amphibianand teleost (bony fish) embryos,
neuroectoderm is induced by BMP
inhibitors derived from organizing
centers. Induced neuroectoderm isby default anterior in character, but
a subset in some tissues is subsequently subject to posteriorizing
transformations. The hindbrain andspinal cord, which are posteriorized regions, arespecified by a signal from a region known as nonaxial mesendoderm, while anterior neuroectoderm
that does not receive this posteriorizing signal develops into fore- and midbrain. After these initial processes of neural induction and patterning, neurogenic regions, the domains in which neurogenesis takes
place, are established.

Staff
Team Leader

Masahiko Hibi
Research Scientist

Osamu Muraoka
Takashi Shimizu
Taijiro Yabe
Visiting Scientist

Young-Ki Bae
Tsutomu Hirata
Technical Staff

Chiho Fukae
Yukiko Kamimura
Hanae Akiyama

The Hibi lab has been working to identify genes
involved in the control of this patterning and neurogenesis, focusing on two genes in particular: thezinc finger gene fez-like expressed in the forebrain, and the posterior neuron-specifichomeobox gene pnx. In collaborative
work, they showed that fez-like isrequired for the formation of hypothalamic monoaminergic neurons inzebrafish. These neurons produce
neurotransmitters such as dopamineand serotonin, which are centrally
important to the regulation of mood,
behavior, endocrine and cognitive
functions in humans.

The maternal-effect mutant
tokkaebi displays a completely
ventralized phenotype.

Student Trainee

Hideaki Nojima
Assistant

Shoko Muraoka

Publications
Levkowitz G, Zeller J, Sirotkin H I, French D, Schilbach
S, Hashimoto H, Hibi M, Talbot W S and Rosenthal A.
Zinc finger protein too few
controls the development of
monoaminergic neurons. Nat
Neurosci 6:28-33 (2003).

A separate study identified a homeobox gene, pnx, involved in the development of posterior neurons. This
gene, which is regulated by a signal
from the posteriorizing non-axial
mesendoderm and Notch signaling,acts as a transcriptional repressor.
Misexpression of pnx results in an
increase in neural precursor cells
and neurons, while its repression
caused reductions in certain populations of posterior neurons, indicatingthat the gene participates in posterior neurogenesis.

Bae Y K, Shimizu T, Yabe T,
Kim CH, Hirata T, Nojima H,
Muraoka O, Hirano T and
Hibi M. A homeobox gene,pnx, is involved in the formation of posterior neurons inzebrafish. Development
130:1853-65 (2003).
Yabe T, Shimizu T, Muraoka
O, Bae Y K, Hirata T, Nojima
H, Kawakami A, Hirano T
and Hibi M. Ogon/Secreted
Frizzled functions as a negative feedback regulator of
Bmp signaling. Development
130:2705-16 (2003).
Shimizu T, Yamanaka Y,
Nojima H, Yabe T, Hibi M
and Hirano T. A novel
repressor-type homeobox
gene, ved, is involved indharma/bozozok-mediateddorsal organizer formation inzebrafish. Mech Dev
118:125-38 (2002).

Pnx is required for the formation of motoneurons.

RIKEN CDB 2003 Annual Report31Hibi and colleagues set out to clone the ogon gene(which is also known as mercedes and short tail), in
order to determine its precise relationship with Chordin, and the molecular bases for its action in regulating the establishment of the dorsal-ventral axis. The
results of that positional cloning showed that ogon
encodes a homolog of the Secreted Frizzled (Sizzled) protein, which has similarities to the Wnt recep-

Organizing centers,
which emerge soon after
fertilization, play pivotal roles insetting up positional axesin the zebrafish embryo
tor, Frizzled. The canonical Wnt pathway is known
to play a role in axis formation, but it has been suggested that ogon/sizzled influences the dorsalventral axis independently of its effecton Wnt. To test this idea, members ofthe Hibi lab compared the activity of
Ogon/Sizzled with that of Wnt inhibitors in ectopic expression studies,and found that the effects of
Ogon/Sizzled misexpression
resembled those of BMP,
but not Wnt, inhibitors,
suggesting an exceptional mode ofaction.

Creative Research
Promoting Program

Masahiko Hibi
M. D. , Ph. D.
Masahiko Hibi received his
M. D. from Hiroshima University, School of Medicine
in 1988, and his Ph. D.
from the Osaka University
Institute for Molecular andCellular Biology in 1992.
From 1992 to 1995, heworked as a postdoctoral
fellow in Michael Karin’s lab
in the University of California, San Diego Departmentof Pharmacology, thenreturned to Japan to takean assistant professorshipin the Division of Molecular
Oncology at Osaka University Medical School. He wasappointed associate professor in the same division in
1999, where he remained
until he assumed his position as team leader at the
RIKEN CDB.

Vertebrate embryogenesis begins with a seemingly
formless egg that, through processes of division,
growth, differentiation, migration and rearrangement, rapidly gives rise to a highly organized structure characterized by a number of definitive axes.
This transformation is particularly striking and rapid
in the zebrafish (Danio rerio), which in the space ofa single day develops from an ovum to a recognizably vertebrate body laid down in respect to multiple
polar vectors. The speed of its growth and reproduction, coupled with its amenability to genetic studies
and the revealing pellucidity of its embryo, make thisorganism an ideal system for the study of axis formation in early vertebrate development.

In work published in 2003, the Hibi lab analyzed andisolated the responsible gene for a mutant named
ogon, which displays ventralized phenotypes, andfound that the ogon locus encodes a zebrafish
homologue of the protein Secreted Frizzled (Sizzled), which functions as negative feedback regulator of BMP signaling. While numerous mutations are
known to result in dorsalized phenotypes, ogon is
only the second zebrafish mutant reported to show
clearly ventralized phenotypes, such as expanded
ventral tail fins, blood, pronephron, and other posterior structures. These patterns are similar to the phenotypes observed in mutations of the gene for the
BMP antagonist, Chordin, which suggested that
Ogon might also be involved in BMP inhibition.

Regulating thedorsal-ventral axis
Masahiko Hibi’s laboratory focuses on the molecular
genetic cascade of events stemming from organizing centers in the zebrafish embryo. These centers,
which emerge soon after fertilization, play pivotal
roles in setting up positional axes; Hibi’s lab is particularly interested in the formation of the dorsalventral (back-belly) axis, an essential step in theestablishment of the body plan. An embryonicregion known in zebrafish as the dorsal organizer
guides dorsoventral patterning by generating inductive signals, including inhibitors of BMPs and Wnts
(molecules which ultimately determine dorsal andventral fates). These molecules play roles in a complex network of axis-determining factors, and theinterplay between dorsalizing and ventralizing factors is central to the formation of the dorsal mesoderm and endoderm, and the neuroectoderm.

ogon mutant and ogon-overexpressing embryos. Ogon functions as a negative feedback regulator of BMP signaling

30 RIKEN CDB 2003 Annual Report

They next lookedat the possible
involvement of
Chordin (which is
known to be essential in the process of
dorsalization)
in
Ogo/Szl’s effects byinjecting ogon/sizzled
RNA into mutants (chordino) lacking a functional
chordin gene. This
failed to rescue the ventralized phenotype ofhomozygous chordino
mutants, indicating that
Ogon/Sizzled activity
depends on Chordin.
The detailed functional
relationshipbetweenthese two dorsalizing factors remains to beworked out, but these initial findings suggest that
Ogon/Sizzled augments
Chordin activity either byinhibiting a Chordin
inhibitor, by upregulating
Chordin activity, or bymaking BMP more sensitive to Chordin’s inhibitory effects.

Neurogenesis
Vertebrate neuronal tissues are
generated in a stepwise fashion.
These steps include neural induction, antero-posterior patterning,
and neurogenesis. In amphibianand teleost (bony fish) embryos,
neuroectoderm is induced by BMP
inhibitors derived from organizing
centers. Induced neuroectoderm isby default anterior in character, but
a subset in some tissues is subsequently subject to posteriorizing
transformations. The hindbrain andspinal cord, which are posteriorized regions, arespecified by a signal from a region known as nonaxial mesendoderm, while anterior neuroectoderm
that does not receive this posteriorizing signal develops into fore- and midbrain. After these initial processes of neural induction and patterning, neurogenic regions, the domains in which neurogenesis takes
place, are established.

Staff
Team Leader

Masahiko Hibi
Research Scientist

Osamu Muraoka
Takashi Shimizu
Taijiro Yabe
Visiting Scientist

Young-Ki Bae
Tsutomu Hirata
Technical Staff

Chiho Fukae
Yukiko Kamimura
Hanae Akiyama

The Hibi lab has been working to identify genes
involved in the control of this patterning and neurogenesis, focusing on two genes in particular: thezinc finger gene fez-like expressed in the forebrain, and the posterior neuron-specifichomeobox gene pnx. In collaborative
work, they showed that fez-like isrequired for the formation of hypothalamic monoaminergic neurons inzebrafish. These neurons produce
neurotransmitters such as dopamineand serotonin, which are centrally
important to the regulation of mood,
behavior, endocrine and cognitive
functions in humans.

The maternal-effect mutant
tokkaebi displays a completely
ventralized phenotype.

Student Trainee

Hideaki Nojima
Assistant

Shoko Muraoka

Publications
Levkowitz G, Zeller J, Sirotkin H I, French D, Schilbach
S, Hashimoto H, Hibi M, Talbot W S and Rosenthal A.
Zinc finger protein too few
controls the development of
monoaminergic neurons. Nat
Neurosci 6:28-33 (2003).

A separate study identified a homeobox gene, pnx, involved in the development of posterior neurons. This
gene, which is regulated by a signal
from the posteriorizing non-axial
mesendoderm and Notch signaling,acts as a transcriptional repressor.
Misexpression of pnx results in an
increase in neural precursor cells
and neurons, while its repression
caused reductions in certain populations of posterior neurons, indicatingthat the gene participates in posterior neurogenesis.

Bae Y K, Shimizu T, Yabe T,
Kim CH, Hirata T, Nojima H,
Muraoka O, Hirano T and
Hibi M. A homeobox gene,pnx, is involved in the formation of posterior neurons inzebrafish. Development
130:1853-65 (2003).
Yabe T, Shimizu T, Muraoka
O, Bae Y K, Hirata T, Nojima
H, Kawakami A, Hirano T
and Hibi M. Ogon/Secreted
Frizzled functions as a negative feedback regulator of
Bmp signaling. Development
130:2705-16 (2003).
Shimizu T, Yamanaka Y,
Nojima H, Yabe T, Hibi M
and Hirano T. A novel
repressor-type homeobox
gene, ved, is involved indharma/bozozok-mediateddorsal organizer formation inzebrafish. Mech Dev
118:125-38 (2002).

Pnx is required for the formation of motoneurons.

RIKEN CDB 2003 Annual Report31Turing mechanism in zebrafish stripes

Staff
Team Leader

Shigeru Kondo
Research Scientist

Masashi Hirata
Jun Ozaki
Motoomi Yamaguchi
Technical Staff

Miwako Okamura
Kana Bando
Student Trainee

Takeshi Nakagawa
Motoko Yoshizawa (Iwashita)
AssistantSumire Hino

Publications

The zebrafish (Danio rerio) is strikingly patterned inbands of light and dark, lending it the zebra-likeappearance for which it is named. This striped effectis achieved by the tightly regulated distribution of
three types of pigment cells (chromatophores) in the
hypodermis — xanthophores, which produce yellow
pigment, melanophores, which produce black, andiridophores, which have a glittering appearance. Iridophores are evenly distributed, but xanthophores
and melanophores appear in mutually exclusive
bands, which appear as stripes in the adult fish. Noting resemblances between zebrafish stripe development and mathematical patterns generated by theTuring mechanism, Kondo speculated that areaction-diffusion effect might be at work. To test
this hypothesis, he used lasers to ablate specificpopulations of pigment cells and observed theirre-growth. When a band of xanthophores or melanophores is ablated, small precursor cells are generated to replace the cells that have been lost. The initial number of replacement cells that arise is inexcess of the final number, indicating that some of

Suzuki N, Hirata M and Kondo S. Traveling stripes on
the skin of a mutant mouse.
Proc Natl Acad Sci U S A
100:9680-5 (2003).
Shoji H, Mochizuki A, Iwasa
Y, Hirata M, Watanabe T,
Hioki S and Kondo S. Originof directionality in the fish
stripe pattern. Dev Dyn
226:627-33 (2003).
Hirata M, Nakamura K, Kanemaru T, Shibata Y and Kondo S. Pigment cellorganization in the hypodermis of zebrafish. Dev Dyn
227:497-503 (2003).

Creative Research
Promoting Program

The patterning of thenatural world

Shigeru Kondo
Ph. D.
Shigeru Kondo received his
doctorate from the Kyoto
University Faculty of Medicine in 1988. He spent theperiod from 1989 to 1990
as a postdoctoral fellow in
Masami Muramatsu’s lab at
the University of Tokyo,
before taking an overseas
fellowship at the Basel University Biocenter under
WalterGehring.
He
returned to Kyoto University
in 1994 as assistant professor, where he remained
until 1998, when he wasappointed professor in theTokushima University Faculty of Integrated Arts and Sciences. Kondo was appointed CDB team leader in
2001.

The question of how complex patterns arise fromseemingly disorganized or formless initial structures
represents an intriguing challenge to mathematicians, physicists, chemists and biologists alike. Theoretical work indicates that the mechanisms underlying pattern formation are similar in both biologicaland non-biological systems, and a number ofmathematical models capable of describing patterngeneration in chemical media have been proposed,
but the greater complexity of living systems has
made it much more difficult to demonstrate amathematical basis for biological patterns. In 1952,
the British mathematician Alan Turing proposed a
simple mathematical equation capable of generatinga wide range of patterns commonly found in the natural world, such as stripes, spots and reticulations.
This model, known as the reaction-diffusion model,
demonstrates that the interaction between a local
activator and a long-range inhibitor can give rise tovarious periodic structures in response to differences in their individual diffusion rates.

Traveling wave mice
In research published this year, Kondo identified amathematical similarity between a wellcharacterized reaction that produces wave-like patterns in chemical media and a pattern-forming phenomenon in the skin of a mutant mouse. These
mice, which have defects in a gene responsible forhair follicle development, develop bands of darkened skin that traverse the body surface in waves.
Kondo, working in collaboration with researchers
from the Medical School of Mie University, showedthat these traveling waves of skin coloration are strikingly similar to nonlinear waves produced by theBelousov-Zhabotinskii (BZ) reaction in chemical systems, suggesting a shared underlying principle. The
research began when Kondo learned of a mutant
strain of mouse with an unusual striped phenotype.
The mutation, a splicing defect in the Foxn1 (Whn ornude) gene, causes hair follicle development to terminate just after skin pigments begin to accumulate.
The immature follicles die off and are quickly
replaced by a new hair cycle. The cyclical nature of

Shigeru Kondo is interested in demonstrating themathematical basis of pattern formation in development, and using mathematical models as predictive tools to aid in the identification of genes
and molecules involved in the generation of
spatial structures. Research in the Kondo
lab focuses on skin surface and morphogenetic patterning, both of which featureprominent examples of periodic structures that can be described in terms ofstanding and moving waves.

this follicular attrition
and the subsequentre-activation by neighboring follicles producesa phenotype in which themouse's skin color at first uniformly oscillates between dark and light coloration, then begins to take on a remarkable striped
appearance, with bands of pigmentation that originate from the region under the forearms and travel
across the body surface in all directions. Thesewaves first appear at about three months after birth,and continue to arise and propagate throughout thelife of the animal.

Kondo identified similarities
between wave-like patterns
observed in chemical media anda pattern-forming phenomenon in
the skin of a mutant mouse
By tracking the movements of traveling waves in the
Foxn1 mouse skin pattern, Kondo ascertained that
their formation was fundamentally similar to that ofwaves generated by the BZ reaction. Although thisfinding does not directly identify the specific molecular mechanism at work in the Foxn1 phenotype,mathematical analysis suggests a candidate molecule, Shh, which satisfies the necessary criteria fora molecule key to traveling wave pattern formation.
And, as many other species demonstrate similar
striping mechanisms, this report may help to provide
biologists with a mathematical model to explain thisskin patterning phenomenon.

The zebrafish skin pattern is generated
by combinations of cells producing various pigments

the precursors die off during the process of regeneration. And while in principle any type of chromatophore can grow at any location in the skin,xanthophore bands regenerate only xanthophores
and melanophores only melanophores, suggestingthat cells of both types inhibit the growth of theircounterparts. Using mathematical simulations, Kondo developed a model to account for this bandedskin patterning, in which chromatophores of eachtype produce both short-range inhibitors of pigment
cells of the same type and long-range inhibitors of
the complementary cell type. When mathematicalsimulations
using
inhibitors of varying
strengths of diffusion
rates are performed,
this double negative
feedback relationshipgives rise to a rangeof Turing patterns similar to skin patterns
seen in nature, a finding that provides new
avenues of inquiry forinvestigators studying
the molecular mechanisms of pattern formaAltered striping in zebrafish skin three weeks after chromatopohore ablation
tion in biological systems.

Traveling wave skin pattern of the newly isolated mutant traveling wave (tw) mouse. Each wave arises at the base of the forelimb, and moves like a wave spreading on the surface of water.

32 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report33Turing mechanism in zebrafish stripes

Staff
Team Leader

Shigeru Kondo
Research Scientist

Masashi Hirata
Jun Ozaki
Motoomi Yamaguchi
Technical Staff

Miwako Okamura
Kana Bando
Student Trainee

Takeshi Nakagawa
Motoko Yoshizawa (Iwashita)
AssistantSumire Hino

Publications

The zebrafish (Danio rerio) is strikingly patterned inbands of light and dark, lending it the zebra-likeappearance for which it is named. This striped effectis achieved by the tightly regulated distribution of
three types of pigment cells (chromatophores) in the
hypodermis — xanthophores, which produce yellow
pigment, melanophores, which produce black, andiridophores, which have a glittering appearance. Iridophores are evenly distributed, but xanthophores
and melanophores appear in mutually exclusive
bands, which appear as stripes in the adult fish. Noting resemblances between zebrafish stripe development and mathematical patterns generated by theTuring mechanism, Kondo speculated that areaction-diffusion effect might be at work. To test
this hypothesis, he used lasers to ablate specificpopulations of pigment cells and observed theirre-growth. When a band of xanthophores or melanophores is ablated, small precursor cells are generated to replace the cells that have been lost. The initial number of replacement cells that arise is inexcess of the final number, indicating that some of

Suzuki N, Hirata M and Kondo S. Traveling stripes on
the skin of a mutant mouse.
Proc Natl Acad Sci U S A
100:9680-5 (2003).
Shoji H, Mochizuki A, Iwasa
Y, Hirata M, Watanabe T,
Hioki S and Kondo S. Originof directionality in the fish
stripe pattern. Dev Dyn
226:627-33 (2003).
Hirata M, Nakamura K, Kanemaru T, Shibata Y and Kondo S. Pigment cellorganization in the hypodermis of zebrafish. Dev Dyn
227:497-503 (2003).

Creative Research
Promoting Program

The patterning of thenatural world

Shigeru Kondo
Ph. D.
Shigeru Kondo received his
doctorate from the Kyoto
University Faculty of Medicine in 1988. He spent theperiod from 1989 to 1990
as a postdoctoral fellow in
Masami Muramatsu’s lab at
the University of Tokyo,
before taking an overseas
fellowship at the Basel University Biocenter under
WalterGehring.
He
returned to Kyoto University
in 1994 as assistant professor, where he remained
until 1998, when he wasappointed professor in theTokushima University Faculty of Integrated Arts and Sciences. Kondo was appointed CDB team leader in
2001.

The question of how complex patterns arise fromseemingly disorganized or formless initial structures
represents an intriguing challenge to mathematicians, physicists, chemists and biologists alike. Theoretical work indicates that the mechanisms underlying pattern formation are similar in both biologicaland non-biological systems, and a number ofmathematical models capable of describing patterngeneration in chemical media have been proposed,
but the greater complexity of living systems has
made it much more difficult to demonstrate amathematical basis for biological patterns. In 1952,
the British mathematician Alan Turing proposed a
simple mathematical equation capable of generatinga wide range of patterns commonly found in the natural world, such as stripes, spots and reticulations.
This model, known as the reaction-diffusion model,
demonstrates that the interaction between a local
activator and a long-range inhibitor can give rise tovarious periodic structures in response to differences in their individual diffusion rates.

Traveling wave mice
In research published this year, Kondo identified amathematical similarity between a wellcharacterized reaction that produces wave-like patterns in chemical media and a pattern-forming phenomenon in the skin of a mutant mouse. These
mice, which have defects in a gene responsible forhair follicle development, develop bands of darkened skin that traverse the body surface in waves.
Kondo, working in collaboration with researchers
from the Medical School of Mie University, showedthat these traveling waves of skin coloration are strikingly similar to nonlinear waves produced by theBelousov-Zhabotinskii (BZ) reaction in chemical systems, suggesting a shared underlying principle. The
research began when Kondo learned of a mutant
strain of mouse with an unusual striped phenotype.
The mutation, a splicing defect in the Foxn1 (Whn ornude) gene, causes hair follicle development to terminate just after skin pigments begin to accumulate.
The immature follicles die off and are quickly
replaced by a new hair cycle. The cyclical nature of

Shigeru Kondo is interested in demonstrating themathematical basis of pattern formation in development, and using mathematical models as predictive tools to aid in the identification of genes
and molecules involved in the generation of
spatial structures. Research in the Kondo
lab focuses on skin surface and morphogenetic patterning, both of which featureprominent examples of periodic structures that can be described in terms ofstanding and moving waves.

this follicular attrition
and the subsequentre-activation by neighboring follicles producesa phenotype in which themouse's skin color at first uniformly oscillates between dark and light coloration, then begins to take on a remarkable striped
appearance, with bands of pigmentation that originate from the region under the forearms and travel
across the body surface in all directions. Thesewaves first appear at about three months after birth,and continue to arise and propagate throughout thelife of the animal.

Kondo identified similarities
between wave-like patterns
observed in chemical media anda pattern-forming phenomenon in
the skin of a mutant mouse
By tracking the movements of traveling waves in the
Foxn1 mouse skin pattern, Kondo ascertained that
their formation was fundamentally similar to that ofwaves generated by the BZ reaction. Although thisfinding does not directly identify the specific molecular mechanism at work in the Foxn1 phenotype,mathematical analysis suggests a candidate molecule, Shh, which satisfies the necessary criteria fora molecule key to traveling wave pattern formation.
And, as many other species demonstrate similar
striping mechanisms, this report may help to provide
biologists with a mathematical model to explain thisskin patterning phenomenon.

The zebrafish skin pattern is generated
by combinations of cells producing various pigments

the precursors die off during the process of regeneration. And while in principle any type of chromatophore can grow at any location in the skin,xanthophore bands regenerate only xanthophores
and melanophores only melanophores, suggestingthat cells of both types inhibit the growth of theircounterparts. Using mathematical simulations, Kondo developed a model to account for this bandedskin patterning, in which chromatophores of eachtype produce both short-range inhibitors of pigment
cells of the same type and long-range inhibitors of
the complementary cell type. When mathematicalsimulations
using
inhibitors of varying
strengths of diffusion
rates are performed,
this double negative
feedback relationshipgives rise to a rangeof Turing patterns similar to skin patterns
seen in nature, a finding that provides new
avenues of inquiry forinvestigators studying
the molecular mechanisms of pattern formaAltered striping in zebrafish skin three weeks after chromatopohore ablation
tion in biological systems.

Traveling wave skin pattern of the newly isolated mutant traveling wave (tw) mouse. Each wave arises at the base of the forelimb, and moves like a wave spreading on the surface of water.

32 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report33

Embryonic developmental stages in Chinese soft-shelled turtle

Staff
Team Leader

Student Trainee

Shigeru Kuratani

Yoko Takio
Hiroshi Nagashima
Shigehiro Kuraku

Research Scientist

Yoshie Kawashima
Katsuhisa Uchida
Masumi Nakazawa
Yuuji Narita
Rie Kusakabe
Yasunori Murakami

Assistant

Chiaki Nakayama

Technical Staff

Ryo Usuda

Creative Research
Promoting Program

Evo-devo

Shigeru Kuratani
Ph. D.
Shigeru Kuratani received
his M. S. and Ph. D. fromthe Kyoto University Department of Zoology. He spent
the period from 1988 to
1991 working in experimental embryology in the
Department of Anatomy at
the Medical College ofGeorgia before moving to
the Biochemistry Department, Baylor College ofMedicine, where he wasengaged in molecular
embryological research. He
returned to Japan in 1994
to take a position as associate professor at the Institute of Medical Embryology
and Genetics in the Kumamoto University School of
Medicine. He moved to
Okayama University toassume a professorship in
the Department of Biology
in
1997,
where
he
remained until he wasappointed team leader at
the CDB.

Darwin conceived evolution as a process driven by
the interplay between random mutation and natural
selection, but the advent of molecular developmental biology in the 1980s led to the recognition that
inherent genetic constraints also have a role in defining the trajectories that evolutionary processes aremost likely to follow. The homeobox (Hox) genes, adevelopmentally important set of genes provide one
instantiation of this concept; they play central roles
in regulating the morphological development oforganisms ranging from yeast and plants to animals
from every branch of the phylogenetic tree andshare a highly conserved domain (the 180-base-pairhomeodomain) that indicates a non-random mode ofevolutionary selection.

of gradual evolution in incremental steps. Such aphenomenon is difficult to explain by mutation andselection alone, as it is improbable that the genomic
alterations necessary to produce such a dramatically new bodily structure would be achievable by the
introduction of new genes in the short timeframe of
the turtle's emergence. Evo-devo (evolutionary
developmental biology) theory predicts instead thatthe genes responsible for carapace development
actually belong to a set of genes shared by groups
related to the turtle, but which function distinctly inthe unique context of the turtle's molecular-genetic
network. This would also explain the independent
appearance of the carapace at other points in evolu-

tionary history, as the underlying genetic elements
are presumed to be conserved, making carapace formation one of the trajectories available to the process of morphological development.

The turtle's shell
The abrupt advent of the turtle's shell, or 'carapace,'
which appeared with few recognizable precursors,
provides a remarkable exception to the general rule

34 RIKEN CDB 2003 Annual Report

Nature prefers torepurpose or tinker withexisting genes rather than to
introduce novelties ex nihilo

In vertebrates, the hindbrain is a segmented structure, subdivided into clearly demarcated units calledrhombomeres, which generate specific sets of neurons. The lancelet Amphioxus, a more primitive chordate, however, lacks this hindbrain segmentation.
The lamprey, a jawless fish that arose in the interval
between non-vertebrate chordates (such as
Amphioxus) and gnathostomes (jawed animals),
provides a relevant model for studying the emergence of the hindbrain developmental plan.

These findings indicate that, in the lamprey, hindbrain neural identities and rhombomeric segmentation are governed by independent mechanisms, providing strong counter-evidence to one prevailingmodel that suggests that the establishment of neuronal identity is a Hox-dependent process. Based on
their discoveries,
Kuratani et al. offer an alternate
model in which
thepositional
concurrence between
Hox
expression,rhombomere
identity and specific subsets ofhindbrain neurons is the resultof a convergent
process in which
originally independent mechanisms
becameThe rib grows laterally towards the edge
linked over evoluof the body in the turtle embryo.
tionary time.

In work published in 2004, investigators in the Kuratani lab labeled reticulospinal and branchial motor
neurons (which derive from rhombomeres) to reveal
the neuronal organization of the hindbrain of theJapanese lamprey, Lethenteron japonicum, andstudies the expression patterns of rhombomerespecific genes. They found that lamprey reticularStudies such as these underscore a pair of evodevo precepts: that molecular designs capable ofsupporting viable ontogenies tend to act as magnets
for convergent evolution, and that Nature is parsimonious with her creations, preferring to repurpose ortinker with existing genes rather than to introduce
novelties ex nihilo.

In other closely related species, ribs are entirelyinternal and reside interior to the scapulae (corresponding to the shoulder blades in humans). In theturtle, however, the rib bones grow exterior to thescapulae to form the external carapace. Using
cross-species explantation, cDNA library and cellsorting studies, researchers in the Kuratani lab haveidentified a number of developmental switching
points that enable this exteriorization of this portionof the turtle endoskeleton. Their findings indicatethat alterations in a number of essentially conserved
genes, rather than genetic novelty, have resulted in
the turtle's unique anatomical phenotype.

Hindbrain evolution
Combining experimental and analytic techniquesfrom molecular biology, phylogenetics and comparative morphology, Shigeru Kuratani seeks to deepen
the understanding of the part played by developmental biological mechanisms, such as the Hox genes,in the divergence of species. His approach involves
examining related genes in phylogenetically distinct
animals to uncover the ways in which context affects
gene expression, and thereby influences body development. By comparing the molecular bases of theemergence of discrete structures, Kuratani hopes toillustrate the means by which developmental
mechanisms mediate the translation of changes in
the genome to changes in morphology.

neurons develop in conserved rhombomere-specificpositions, similar to those observed in the gnathostome zebrafish. Interestingly, in lamprey the positions of other sets of hindbrain neurons — the trigeminal and facial motor nuclei — do not map neatly
to rhombomeric borderlines, as they do in gnathostomes. Rather, the trigeminal-facial nerve originatesin the middle of rhombomere 4, in the region of
expression of the lamprey Hox gene LjHox3. When
retinoic acid (which is known to alter Hox gene
expression and associated developmental programs) was introduced to the developing hindbrain
region, it caused positional shifts of both LjHox3
expression and branchiomotor nuclei, but no apparent changes in segmentation or the positions of reticular neurons.

Publications
Murakami Y, Pasqualett M,
Takio Y, Hirano S, Riiji F and
Kuratani S. Segmental development of reticulospinal andbranchiomotor neuros in thelamprey:insights into evolution of the vertebrate hindbrain. Development (2004).
Kusakabe R, Tochinai S and
Kuratani S. Expression of foreign genes in lamprey
embryos: an approach to study evolutionary changes ingene regulation. J Exp Zoolog Part B Mol Dev Evol
296:87-97 (2003).
Kuratani S. Evolutionary
deveropmental biology andvertebrate head segmentation:a perspective from deveropmental constraint. Theory
Biosciences 122:230-51(2003).
Kuratani S. Evolution of thevertebrate jaw - homology
and developmental constraints. Paleontol. Res 7:89102(2003).
Kuratani S. The heterotopic
shift in developmental patterns and evolution of thejaw in vertebrates. Springer
Verlag (2003).
Uchida K, Murakami Y, Kuraku S, Hirano S and Kuratani
S. Development of the adenohypophysis in the lamprey:
evolution of the epigenetic
patterning programs in organogenesis. J Exp Zool (MolDev Evol) 300B:32-47(2003).

Removal of carapacial ridge results in a characteristic fan-shape
splaying of turtle ribs.

RIKEN CDB 2003 Annual Report35

Embryonic developmental stages in Chinese soft-shelled turtle

Staff
Team Leader

Student Trainee

Shigeru Kuratani

Yoko Takio
Hiroshi Nagashima
Shigehiro Kuraku

Research Scientist

Yoshie Kawashima
Katsuhisa Uchida
Masumi Nakazawa
Yuuji Narita
Rie Kusakabe
Yasunori Murakami

Assistant

Chiaki Nakayama

Technical Staff

Ryo Usuda

Creative Research
Promoting Program

Evo-devo

Shigeru Kuratani
Ph. D.
Shigeru Kuratani received
his M. S. and Ph. D. fromthe Kyoto University Department of Zoology. He spent
the period from 1988 to
1991 working in experimental embryology in the
Department of Anatomy at
the Medical College ofGeorgia before moving to
the Biochemistry Department, Baylor College ofMedicine, where he wasengaged in molecular
embryological research. He
returned to Japan in 1994
to take a position as associate professor at the Institute of Medical Embryology
and Genetics in the Kumamoto University School of
Medicine. He moved to
Okayama University toassume a professorship in
the Department of Biology
in
1997,
where
he
remained until he wasappointed team leader at
the CDB.

Darwin conceived evolution as a process driven by
the interplay between random mutation and natural
selection, but the advent of molecular developmental biology in the 1980s led to the recognition that
inherent genetic constraints also have a role in defining the trajectories that evolutionary processes aremost likely to follow. The homeobox (Hox) genes, adevelopmentally important set of genes provide one
instantiation of this concept; they play central roles
in regulating the morphological development oforganisms ranging from yeast and plants to animals
from every branch of the phylogenetic tree andshare a highly conserved domain (the 180-base-pairhomeodomain) that indicates a non-random mode ofevolutionary selection.

of gradual evolution in incremental steps. Such aphenomenon is difficult to explain by mutation andselection alone, as it is improbable that the genomic
alterations necessary to produce such a dramatically new bodily structure would be achievable by the
introduction of new genes in the short timeframe of
the turtle's emergence. Evo-devo (evolutionary
developmental biology) theory predicts instead thatthe genes responsible for carapace development
actually belong to a set of genes shared by groups
related to the turtle, but which function distinctly inthe unique context of the turtle's molecular-genetic
network. This would also explain the independent
appearance of the carapace at other points in evolu-

tionary history, as the underlying genetic elements
are presumed to be conserved, making carapace formation one of the trajectories available to the process of morphological development.

The turtle's shell
The abrupt advent of the turtle's shell, or 'carapace,'
which appeared with few recognizable precursors,
provides a remarkable exception to the general rule

34 RIKEN CDB 2003 Annual Report

Nature prefers torepurpose or tinker withexisting genes rather than to
introduce novelties ex nihilo

In vertebrates, the hindbrain is a segmented structure, subdivided into clearly demarcated units calledrhombomeres, which generate specific sets of neurons. The lancelet Amphioxus, a more primitive chordate, however, lacks this hindbrain segmentation.
The lamprey, a jawless fish that arose in the interval
between non-vertebrate chordates (such as
Amphioxus) and gnathostomes (jawed animals),
provides a relevant model for studying the emergence of the hindbrain developmental plan.

These findings indicate that, in the lamprey, hindbrain neural identities and rhombomeric segmentation are governed by independent mechanisms, providing strong counter-evidence to one prevailingmodel that suggests that the establishment of neuronal identity is a Hox-dependent process. Based on
their discoveries,
Kuratani et al. offer an alternate
model in which
thepositional
concurrence between
Hox
expression,rhombomere
identity and specific subsets ofhindbrain neurons is the resultof a convergent
process in which
originally independent mechanisms
becameThe rib grows laterally towards the edge
linked over evoluof the body in the turtle embryo.
tionary time.

In work published in 2004, investigators in the Kuratani lab labeled reticulospinal and branchial motor
neurons (which derive from rhombomeres) to reveal
the neuronal organization of the hindbrain of theJapanese lamprey, Lethenteron japonicum, andstudies the expression patterns of rhombomerespecific genes. They found that lamprey reticularStudies such as these underscore a pair of evodevo precepts: that molecular designs capable ofsupporting viable ontogenies tend to act as magnets
for convergent evolution, and that Nature is parsimonious with her creations, preferring to repurpose ortinker with existing genes rather than to introduce
novelties ex nihilo.

In other closely related species, ribs are entirelyinternal and reside interior to the scapulae (corresponding to the shoulder blades in humans). In theturtle, however, the rib bones grow exterior to thescapulae to form the external carapace. Using
cross-species explantation, cDNA library and cellsorting studies, researchers in the Kuratani lab haveidentified a number of developmental switching
points that enable this exteriorization of this portionof the turtle endoskeleton. Their findings indicatethat alterations in a number of essentially conserved
genes, rather than genetic novelty, have resulted in
the turtle's unique anatomical phenotype.

Hindbrain evolution
Combining experimental and analytic techniquesfrom molecular biology, phylogenetics and comparative morphology, Shigeru Kuratani seeks to deepen
the understanding of the part played by developmental biological mechanisms, such as the Hox genes,in the divergence of species. His approach involves
examining related genes in phylogenetically distinct
animals to uncover the ways in which context affects
gene expression, and thereby influences body development. By comparing the molecular bases of theemergence of discrete structures, Kuratani hopes toillustrate the means by which developmental
mechanisms mediate the translation of changes in
the genome to changes in morphology.

neurons develop in conserved rhombomere-specificpositions, similar to those observed in the gnathostome zebrafish. Interestingly, in lamprey the positions of other sets of hindbrain neurons — the trigeminal and facial motor nuclei — do not map neatly
to rhombomeric borderlines, as they do in gnathostomes. Rather, the trigeminal-facial nerve originatesin the middle of rhombomere 4, in the region of
expression of the lamprey Hox gene LjHox3. When
retinoic acid (which is known to alter Hox gene
expression and associated developmental programs) was introduced to the developing hindbrain
region, it caused positional shifts of both LjHox3
expression and branchiomotor nuclei, but no apparent changes in segmentation or the positions of reticular neurons.

Publications
Murakami Y, Pasqualett M,
Takio Y, Hirano S, Riiji F and
Kuratani S. Segmental development of reticulospinal andbranchiomotor neuros in thelamprey:insights into evolution of the vertebrate hindbrain. Development (2004).
Kusakabe R, Tochinai S and
Kuratani S. Expression of foreign genes in lamprey
embryos: an approach to study evolutionary changes ingene regulation. J Exp Zoolog Part B Mol Dev Evol
296:87-97 (2003).
Kuratani S. Evolutionary
deveropmental biology andvertebrate head segmentation:a perspective from deveropmental constraint. Theory
Biosciences 122:230-51(2003).
Kuratani S. Evolution of thevertebrate jaw - homology
and developmental constraints. Paleontol. Res 7:89102(2003).
Kuratani S. The heterotopic
shift in developmental patterns and evolution of thejaw in vertebrates. Springer
Verlag (2003).
Uchida K, Murakami Y, Kuraku S, Hirano S and Kuratani
S. Development of the adenohypophysis in the lamprey:
evolution of the epigenetic
patterning programs in organogenesis. J Exp Zool (MolDev Evol) 300B:32-47(2003).

Removal of carapacial ridge results in a characteristic fan-shape
splaying of turtle ribs.

RIKEN CDB 2003 Annual Report35Sensory cellulardifferentiation

Staff
Team Leader

Raj Ladher
Research Scientist

Michael F Teraoka Escaño
Technical Staff

Pabel Antonio Delgado
Susan Boerner
Assistant

Lio Hiroi

Creative Research
Promoting Program

Developing sense organs
The embryo provides many examples of elegant
micro-engineering, wedding intricate organic structures with specialized cellular function. The sensory
organs provide a particularly good example of thissophistication. By initiating in the correct place, differentiating into the correct subtypes and finally integrating with structures of diverse origin to form a sensory
complex, these structures permit the animal to apprehend its environment by processing a range of physi-

Raj Ladher
Ph. D.
Raj Ladher received his B.
Sc. in biochemistry from
Imperial College, London in
1992 and his Ph. D. indevelopmental biology fromthe National Institute for
Medical Research in 1996,
for his thesis on the cloningof the Xenopus homeobox
gene, Xom, under thesupervsion of Jim Smith.
He worked as a research
associate at King’s College,
London from 1996 to 2000
in the lab of Pip FrancisWest, and first came to
Japan during that period as
a visiting scientist at theTokyo Medical and Dental
University. In 2000, hemoved to the University of
Utah as a postdoctoral fellow in Gary Schoenwolf’slaboratory, and was appointed team leader at the
RIKEN CDB in 2002.

opmental program initiation, cell differentiation andthe integration of discrete cellular motifs in structurally and neuronally specialized organs.

Signals in inner ear induction
Sense organs originate in placodes, thickened
regions within the early ectoderm. The inner ear arises from the otic placode, a cluster of cells which
ultimately gives rise to the mature inner ear. Raj is
studying the inductive factors that dictate the location and timing of these developmental processes.
In a series of tissue manipulation experiments conducted previously, he identified a trio of signaling factors — FGF-8, FGF-19, and Wnt-8c — each ofwhich localizes in a separate germ layer, and which
cooperate to induce inner ear development. In thisnetwork, it appears that FGF-8, expressed in theendodermal layer, induces the expression of FGF19 in the immediately overlying mesoderm. FGF-19
in turn induces Wnt-8c in the neuroectoderm, triggering a complex regulatory loop in which FGF-19 and
Wnt-8c maintain each other's expression for the

Expression of Fgf-19 is in purple. Shown is a section through a stage
22 embryo. Fgf-19 is expressed in the condensing acoustic ganglia atthese stages, suggesting a role in the later development of the inner
ear as well as in its induction.

Publications
Ladher R K & Schoenwolf G
C in Developmental Neurobiology (ed. Rao, M. H.)
Kluwer (2003).
Francis-West P H, Ladher R
K and Schoenwolf G C.
Development of the sensory
organs. Sci Prog 85:151-73(2002).
Ladher R K, Anakwe K U,
Gurney A L, Schoenwolf G C
and Francis-West P H. Identification of synergistic signals
initiating inner ear development. Science 290:1965-7(2000).

duration of early ear development. Signaling interactions, acting upon the cell surface, trigger a series oftransduction events that are interpreted in the
nucleus of the responding cell as transcription factors. Raj's lab is characterizing the transcriptional
response to the different signals that act to collectively specify the inner ear. By understanding theirfunction, Raj hopes to be able to determine amechanism of molecular synergy.

Morphogenesis of theotic placode
Although the adult inner ear is located in the interiorof the animal it is nonetheless an ectodermal structure, originating from a placode embedded within
the outermost layer of the embryo. Through a process known as invagination, the otic placode pinches off from the ectoderm. This represents one of the

Raj hopes to reconcile
the events that occur
outside the cell withthe biological changes
occurring withinfirst cellular consequences of inner ear induction,
and Raj has focused on this process to understandthe functional significance of signaling factors andtheir transcriptional responses. Working from the
hypothesis that extracellular cues choreograph thiscomplex dance of cells, modifying their shape, motility, growth and the location of subcellular components, Raj hopes to be able to reconcile the events
that occur outside the cell with the biological changes occurring within.

cal and chemical stimuli. The development of thesesystems involves an extraordinary level of coordination between cells and emergent tissues at very earlystages of embryogenesis. Research in Raj Ladher's
lab focuses on the induction of the inner ear and the
peripheral olfactory system. The chick embryo isused as a model system, chosen for its tractability toclassical embryological techniques and the opportunities it affords for investigating the processes of devel-

The conversion of uncommitted precursor cells in
the otic placode to differentiated, functional otic subtypes is very precisely timed. If it occurs prematurely, the resulting organ is underdeveloped, with too
few cells forming the adult structure. If it happens
too late, there is a danger that these cells will still be
immature and unable to fulfill their needed roles insubsequent developmental processes. An exampleof this temporal regulation is seen in the wiring of
the inner ear to the brain, which requires the formation of neurons from the otic placode. If these cells
are not formed when this process occurs, the function of the auditory system is compromised. But
what is the nature of this control? By using embryological studies, combined with gene manipulationand ex-ovo culture, Raj's lab is addressing this question, finding the basis for this precise regulation in
the functional characteristics of specific signaling
molecules. Their control and effects on the development of the inner ear represents one of the most
exciting aspects of research in Raj's lab, with implications in regenerative technologies and tissue
engineering-based therapeutics.

Nuclei marked using DAPI (blue),
microtubules marked using phalloidin (green). A section through achick embryo at stage 17 shows themorphogenesis of the otic placodeduring invagination. This resultsfrom a dynamic rearrangement ofsubcellular components.

Development ofother sensory organs
As well as the inner ear, Raj's lab is also investigating
the development of other sensory organs, in particular the nose and the eye. Using embryological manipulations, the Lab for Sensory Development is mapping the tissues that are responsible for the formationand differentiation of these structures. Though still in
their infancy, these research projects do indicate thattissue interactions distinct from each other and fromthose operating to induce the inner ear are responsible for the control of their development.
In the future, the Ladher lab looks to extend its
research scope to other model systems, with thegoal of determining whether some as-yet unidentified general mechanisms are at work in sensory
organogenesis.

Fgf-19 is expressed in the mesoderm of the chicken embryo
from early stages.

36 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report37Sensory cellulardifferentiation

Staff
Team Leader

Raj Ladher
Research Scientist

Michael F Teraoka Escaño
Technical Staff

Pabel Antonio Delgado
Susan Boerner
Assistant

Lio Hiroi

Creative Research
Promoting Program

Developing sense organs
The embryo provides many examples of elegant
micro-engineering, wedding intricate organic structures with specialized cellular function. The sensory
organs provide a particularly good example of thissophistication. By initiating in the correct place, differentiating into the correct subtypes and finally integrating with structures of diverse origin to form a sensory
complex, these structures permit the animal to apprehend its environment by processing a range of physi-

Raj Ladher
Ph. D.
Raj Ladher received his B.
Sc. in biochemistry from
Imperial College, London in
1992 and his Ph. D. indevelopmental biology fromthe National Institute for
Medical Research in 1996,
for his thesis on the cloningof the Xenopus homeobox
gene, Xom, under thesupervsion of Jim Smith.
He worked as a research
associate at King’s College,
London from 1996 to 2000
in the lab of Pip FrancisWest, and first came to
Japan during that period as
a visiting scientist at theTokyo Medical and Dental
University. In 2000, hemoved to the University of
Utah as a postdoctoral fellow in Gary Schoenwolf’slaboratory, and was appointed team leader at the
RIKEN CDB in 2002.

opmental program initiation, cell differentiation andthe integration of discrete cellular motifs in structurally and neuronally specialized organs.

Signals in inner ear induction
Sense organs originate in placodes, thickened
regions within the early ectoderm. The inner ear arises from the otic placode, a cluster of cells which
ultimately gives rise to the mature inner ear. Raj is
studying the inductive factors that dictate the location and timing of these developmental processes.
In a series of tissue manipulation experiments conducted previously, he identified a trio of signaling factors — FGF-8, FGF-19, and Wnt-8c — each ofwhich localizes in a separate germ layer, and which
cooperate to induce inner ear development. In thisnetwork, it appears that FGF-8, expressed in theendodermal layer, induces the expression of FGF19 in the immediately overlying mesoderm. FGF-19
in turn induces Wnt-8c in the neuroectoderm, triggering a complex regulatory loop in which FGF-19 and
Wnt-8c maintain each other's expression for the

Expression of Fgf-19 is in purple. Shown is a section through a stage
22 embryo. Fgf-19 is expressed in the condensing acoustic ganglia atthese stages, suggesting a role in the later development of the inner
ear as well as in its induction.

Publications
Ladher R K & Schoenwolf G
C in Developmental Neurobiology (ed. Rao, M. H.)
Kluwer (2003).
Francis-West P H, Ladher R
K and Schoenwolf G C.
Development of the sensory
organs. Sci Prog 85:151-73(2002).
Ladher R K, Anakwe K U,
Gurney A L, Schoenwolf G C
and Francis-West P H. Identification of synergistic signals
initiating inner ear development. Science 290:1965-7(2000).

duration of early ear development. Signaling interactions, acting upon the cell surface, trigger a series oftransduction events that are interpreted in the
nucleus of the responding cell as transcription factors. Raj's lab is characterizing the transcriptional
response to the different signals that act to collectively specify the inner ear. By understanding theirfunction, Raj hopes to be able to determine amechanism of molecular synergy.

Morphogenesis of theotic placode
Although the adult inner ear is located in the interiorof the animal it is nonetheless an ectodermal structure, originating from a placode embedded within
the outermost layer of the embryo. Through a process known as invagination, the otic placode pinches off from the ectoderm. This represents one of the

Raj hopes to reconcile
the events that occur
outside the cell withthe biological changes
occurring withinfirst cellular consequences of inner ear induction,
and Raj has focused on this process to understandthe functional significance of signaling factors andtheir transcriptional responses. Working from the
hypothesis that extracellular cues choreograph thiscomplex dance of cells, modifying their shape, motility, growth and the location of subcellular components, Raj hopes to be able to reconcile the events
that occur outside the cell with the biological changes occurring within.

cal and chemical stimuli. The development of thesesystems involves an extraordinary level of coordination between cells and emergent tissues at very earlystages of embryogenesis. Research in Raj Ladher's
lab focuses on the induction of the inner ear and the
peripheral olfactory system. The chick embryo isused as a model system, chosen for its tractability toclassical embryological techniques and the opportunities it affords for investigating the processes of devel-

The conversion of uncommitted precursor cells in
the otic placode to differentiated, functional otic subtypes is very precisely timed. If it occurs prematurely, the resulting organ is underdeveloped, with too
few cells forming the adult structure. If it happens
too late, there is a danger that these cells will still be
immature and unable to fulfill their needed roles insubsequent developmental processes. An exampleof this temporal regulation is seen in the wiring of
the inner ear to the brain, which requires the formation of neurons from the otic placode. If these cells
are not formed when this process occurs, the function of the auditory system is compromised. But
what is the nature of this control? By using embryological studies, combined with gene manipulationand ex-ovo culture, Raj's lab is addressing this question, finding the basis for this precise regulation in
the functional characteristics of specific signaling
molecules. Their control and effects on the development of the inner ear represents one of the most
exciting aspects of research in Raj's lab, with implications in regenerative technologies and tissue
engineering-based therapeutics.

Nuclei marked using DAPI (blue),
microtubules marked using phalloidin (green). A section through achick embryo at stage 17 shows themorphogenesis of the otic placodeduring invagination. This resultsfrom a dynamic rearrangement ofsubcellular components.

Development ofother sensory organs
As well as the inner ear, Raj's lab is also investigating
the development of other sensory organs, in particular the nose and the eye. Using embryological manipulations, the Lab for Sensory Development is mapping the tissues that are responsible for the formationand differentiation of these structures. Though still in
their infancy, these research projects do indicate thattissue interactions distinct from each other and fromthose operating to induce the inner ear are responsible for the control of their development.
In the future, the Ladher lab looks to extend its
research scope to other model systems, with thegoal of determining whether some as-yet unidentified general mechanisms are at work in sensory
organogenesis.

Fgf-19 is expressed in the mesoderm of the chicken embryo
from early stages.

36 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report37eIF4G, and thereby inhibits the initiation of translation. Findings that a mutant form of Cup lacking thesequence with which it binds eIF4E failed to repress
oskar translation in vivo, that Cup interacts with Bruno in a yeast two-hybrid assay, and that the CupeIF4E complex associates with Bruno in vivo suggest that these three proteins form a complex that
achieves translational repression by interactions
with both the 3’ and 5’ ends of the oskar RNA. A
similar model of protein interactions is observed in
the translational repression of the cyclin-B1 RNA in
the Xenopus African clawed frog, indicating that thisparadigm of translational repression through the 5’/3’ interactions is conserved across species.
Nakamura next intends to look into the means bywhich the repressor effects of the eIF4E-Cup-Bruno
complex are alleviated at the appropriate developmental stage, after the oskar ribonucleoprotein complex has reached and anchored to its appropriate
destination at the pole of the oocyte.

Ph. D.
Akira Nakamura received
both his baccalaureate andhis Ph. D. from the University of Tsukuba. He spent a
year as a post-doctoral fellow at the same institution
before moving to the
Department of Biology at
McGill University in Montreal in 1995 to work as a
post-doc under Paul Lasko.
He returned to Japan in
1997 as a research associate at the University ofTsukuba. He was appointed assistant professor in
the university’s Gene
Research Center and Institute of Biological Sciencesin 2000, and began a threeyear term as a PRESTO
researcher in the Japan Science and Technology Corporation (JST) in December
2001. He was appointed
CDB team leader in March
2002.

Development of the
Ciona intestinalis germline

Staff
Team Leader

Akira Nakamura

In addition to their investigation of fruit fly germline
development, scientists in the Nakamura lab are
also beginning investigations using the ascidian, Ciona intestinalis, a member of the chordate lineage
from which all vertebrates arose. Commonlyreferred to as sea squirts, these animals provide agood model for studying vertebrate evolution. Soon

Research Scientist

Kazuko Hanyu-Nakamura
Maki Shirae
Keiji Sato
Tsubasa Tanaka
Technical Staff

Hiroko Sonobe
Chiaki Nakamoto

Publications

Model of the translational repression ofoskar RNA in oogenesis. The mechanisms by which translational repression iscoupled with RNA localization remain tobe solved.

Creative Research
Promoting Program

Akira Nakamura

previously unexpected roles for a maternally supplied factor involved in lipid signaling, which the team is now working tocharacterize in more detail. Mutation of
the underlying gene results in a phenotype similar to that of the Pgc mutant, inwhich germ cells fail to migrate to theembryonic gonads. Nakamura is now
investigating the roles of both genes in
the maintenance and migration of germ
cells.

Germ cells are the only cell types capable of transmitting genetic information across generations, andtheir formation is characterized by unique developmental processes as well. In many types of animals,
including the Drosophila fruit fly, the formation anddifferentiation of germ
cells is controlled by
mRNAs and proteins
localized in a specificcytoplasmic region within
eggs, called germ plasm.
Germ plasm mRNAs aretranslated in a spatiotemporally regulated manner,
but the means by which
the germ plasm is formed
and achieves this controlled translation remain
largely unknown. Akira
Nakamura studies theestablishment of the Drosophila germ line as amodel of the processesof germ plasm formationand differentiation, as
well as for the insights
this system can provide
into the general mechanisms of mRNA localization and translation,
which are important to anumber of other developmental processes including asymmetric cell diviDrosophila germ plasm andsion, cell migration andgerm cells visualized using
axis formation and likely
GFP-Vasa fusion protein.
synaptic plasticity.

38 RIKEN CDB 2003 Annual Report

Nakamura A, Sato K and
Hanyu-Nakamura K. Drosophila Cup Is an eIF4E Binding Protein that Associateswith Bruno and Regulates
oskar mRNA Translation in
Oogenesis. Dev Cell 6:69-78(2004).

Translational repression
RNA activity during Drosophila oogenesis involves anumber of sequential processes. The Drosophila
oocyte shares cytoplasm with neighboring nurse
cells via an incomplete cell membrane, allowing
mRNAs and proteins from the nurse cells to be transported to the oocyte in the form of ribonucleoproteins. Following their export from the nurse cell
nuclei, mRNAs are translationally repressed, or‘masked,’ and transported to specified regions of theoocyte, where they establish fixed and precise localizations and regain their ability to undergo translation. In one example of this critically important regulation, the translation of the RNA for the maternal
gene oskar, which has critical functions in embryonic patterning and the formation of germline cells, isrepressed during its transport to the posterior pole ofthe oocyte. This transcript-specific repression is
known to be mediated by the protein Bruno, whichbinds to the 3’ UTR of oskar mRNA, but the underlying mechanisms have remained obscure.

Following their export from thenurse cell nuclei, mRNAs aretranslationally repressed, or‘masked,’ and transported to
specified regions of the oocyte
In recent work, the Nakamura lab demonstrated thatan ovarian protein, Cup, is another protein required
to inhibit the premature translation of oskar mRNA,and that Cup achieves this by binding to a second
protein, eIF4E, a 5’ cap-binding general translation
initiation factor. The binding with Cup prevents eIF4E from binding with a different partnering molecule,Kawashima T, Nakamura A,
Yasuda K and Kageyama Y.
Dmaf, a novel member of Maftranscription factor family isexpressed in somatic gonadal
cells during embryonic development and gametogenesis
in Drosophila. Gene Expr Patterns 3:663-7 (2003).

Polar granule maintenance
Polar granules are large complexes of ribonucleoproteins that store RNAs and proteins required for theformation of germ cells in the fruit fly. A non-coding
Polar granule component (Pgc) RNA has beenshown to be important in the maintenance of thesegerm plasm organelles; in Pgc antisense knockdown embryos, germ cells are formed but subsequently degenerate, which has led to the hypothesisthat Pgc serves as a form of supportive scaffold
maintaining the structural integrity of polar granules.
The Nakamura lab is now exploring Pgc function in
more detail by isolating a complete loss-of-function
mutant, allowing for more specific analyses of thegene’s role. They also plan to look for the gene’sfunctional domains, using Pgc orthologs from otherspecies of Drosophila as a basis for comparison.
In other ongoing research, members of the Nakamura lab are using forward-genetics techniques tosearch for novel genes involved in germ cell development. Early results of those studies have revealed

Styhler S, Nakamura A and
Lasko P. VASA localization
requires the SPRY-domain
and SOCS-box containing
protein, GUSTAVUS. Dev
Cell 3:865-76 (2002).

after fertilization, the ascidian egg develops into asmall free-swimming tadpole possessing a
notochord, an evolutionary forebear of the spinal
cord, and a rudimentary nervous system, both ofwhich are lost when it enters its immobile adultstage.

Sano H, Nakamura A andKobayashi S. Identification ofa transcriptional regulatory
region for germline-specificexpression of vasa gene in
Drosophila melanogaster .
Mech Dev 112:129-39(2002).

Nakamura seeks to analyze the regulatory mechanisms of germline development in Ciona, which areinteresting in that while several lines of evidence
indicate that germ cells are formed from maternallyderived germ plasm, it also appears that germ cells
can be regenerated after metamorphosis, suggesting that two independent mechanisms may be atwork. In research conducted in collaboration with
Suma Aqualife Park, a local municipal aquarium,
Nakamura’s team will explore the genetic regulationof ascidian germline development by selecting candidate genes from EST and genome databases (adraft sequence of the Ciona genome is available),
confirm the spatial and temporal patterns of theirexpression and characterize promoter regions andtrans-acting factors.

RIKEN CDB 2003 Annual Report

Nakamura A, Amikura R, Hanyu K and Kobayashi S.
Me31B silences translation ofoocyte-localizing RNAs
through the formation of cytoplasmic RNP complex during
Drosophila oogenesis. Development 128:3233-42 (2001).
Carrera P, Johnstone O,
Nakamura A, Casanova J,
Jackle H and Lasko P. VASA
mediates translation throughinteraction with a DrosophilayIF2 homolog. Mol Cell
5:181-7 (2000).

39

eIF4G, and thereby inhibits the initiation of translation. Findings that a mutant form of Cup lacking thesequence with which it binds eIF4E failed to repress
oskar translation in vivo, that Cup interacts with Bruno in a yeast two-hybrid assay, and that the CupeIF4E complex associates with Bruno in vivo suggest that these three proteins form a complex that
achieves translational repression by interactions
with both the 3’ and 5’ ends of the oskar RNA. A
similar model of protein interactions is observed in
the translational repression of the cyclin-B1 RNA in
the Xenopus African clawed frog, indicating that thisparadigm of translational repression through the 5’/3’ interactions is conserved across species.
Nakamura next intends to look into the means bywhich the repressor effects of the eIF4E-Cup-Bruno
complex are alleviated at the appropriate developmental stage, after the oskar ribonucleoprotein complex has reached and anchored to its appropriate
destination at the pole of the oocyte.

Ph. D.
Akira Nakamura received
both his baccalaureate andhis Ph. D. from the University of Tsukuba. He spent a
year as a post-doctoral fellow at the same institution
before moving to the
Department of Biology at
McGill University in Montreal in 1995 to work as a
post-doc under Paul Lasko.
He returned to Japan in
1997 as a research associate at the University ofTsukuba. He was appointed assistant professor in
the university’s Gene
Research Center and Institute of Biological Sciencesin 2000, and began a threeyear term as a PRESTO
researcher in the Japan Science and Technology Corporation (JST) in December
2001. He was appointed
CDB team leader in March
2002.

Development of the
Ciona intestinalis germline

Staff
Team Leader

Akira Nakamura

In addition to their investigation of fruit fly germline
development, scientists in the Nakamura lab are
also beginning investigations using the ascidian, Ciona intestinalis, a member of the chordate lineage
from which all vertebrates arose. Commonlyreferred to as sea squirts, these animals provide agood model for studying vertebrate evolution. Soon

Research Scientist

Kazuko Hanyu-Nakamura
Maki Shirae
Keiji Sato
Tsubasa Tanaka
Technical Staff

Hiroko Sonobe
Chiaki Nakamoto

Publications

Model of the translational repression ofoskar RNA in oogenesis. The mechanisms by which translational repression iscoupled with RNA localization remain tobe solved.

Creative Research
Promoting Program

Akira Nakamura

previously unexpected roles for a maternally supplied factor involved in lipid signaling, which the team is now working tocharacterize in more detail. Mutation of
the underlying gene results in a phenotype similar to that of the Pgc mutant, inwhich germ cells fail to migrate to theembryonic gonads. Nakamura is now
investigating the roles of both genes in
the maintenance and migration of germ
cells.

Germ cells are the only cell types capable of transmitting genetic information across generations, andtheir formation is characterized by unique developmental processes as well. In many types of animals,
including the Drosophila fruit fly, the formation anddifferentiation of germ
cells is controlled by
mRNAs and proteins
localized in a specificcytoplasmic region within
eggs, called germ plasm.
Germ plasm mRNAs aretranslated in a spatiotemporally regulated manner,
but the means by which
the germ plasm is formed
and achieves this controlled translation remain
largely unknown. Akira
Nakamura studies theestablishment of the Drosophila germ line as amodel of the processesof germ plasm formationand differentiation, as
well as for the insights
this system can provide
into the general mechanisms of mRNA localization and translation,
which are important to anumber of other developmental processes including asymmetric cell diviDrosophila germ plasm andsion, cell migration andgerm cells visualized using
axis formation and likely
GFP-Vasa fusion protein.
synaptic plasticity.

38 RIKEN CDB 2003 Annual Report

Nakamura A, Sato K and
Hanyu-Nakamura K. Drosophila Cup Is an eIF4E Binding Protein that Associateswith Bruno and Regulates
oskar mRNA Translation in
Oogenesis. Dev Cell 6:69-78(2004).

Translational repression
RNA activity during Drosophila oogenesis involves anumber of sequential processes. The Drosophila
oocyte shares cytoplasm with neighboring nurse
cells via an incomplete cell membrane, allowing
mRNAs and proteins from the nurse cells to be transported to the oocyte in the form of ribonucleoproteins. Following their export from the nurse cell
nuclei, mRNAs are translationally repressed, or‘masked,’ and transported to specified regions of theoocyte, where they establish fixed and precise localizations and regain their ability to undergo translation. In one example of this critically important regulation, the translation of the RNA for the maternal
gene oskar, which has critical functions in embryonic patterning and the formation of germline cells, isrepressed during its transport to the posterior pole ofthe oocyte. This transcript-specific repression is
known to be mediated by the protein Bruno, whichbinds to the 3’ UTR of oskar mRNA, but the underlying mechanisms have remained obscure.

Following their export from thenurse cell nuclei, mRNAs aretranslationally repressed, or‘masked,’ and transported to
specified regions of the oocyte
In recent work, the Nakamura lab demonstrated thatan ovarian protein, Cup, is another protein required
to inhibit the premature translation of oskar mRNA,and that Cup achieves this by binding to a second
protein, eIF4E, a 5’ cap-binding general translation
initiation factor. The binding with Cup prevents eIF4E from binding with a different partnering molecule,Kawashima T, Nakamura A,
Yasuda K and Kageyama Y.
Dmaf, a novel member of Maftranscription factor family isexpressed in somatic gonadal
cells during embryonic development and gametogenesis
in Drosophila. Gene Expr Patterns 3:663-7 (2003).

Polar granule maintenance
Polar granules are large complexes of ribonucleoproteins that store RNAs and proteins required for theformation of germ cells in the fruit fly. A non-coding
Polar granule component (Pgc) RNA has beenshown to be important in the maintenance of thesegerm plasm organelles; in Pgc antisense knockdown embryos, germ cells are formed but subsequently degenerate, which has led to the hypothesisthat Pgc serves as a form of supportive scaffold
maintaining the structural integrity of polar granules.
The Nakamura lab is now exploring Pgc function in
more detail by isolating a complete loss-of-function
mutant, allowing for more specific analyses of thegene’s role. They also plan to look for the gene’sfunctional domains, using Pgc orthologs from otherspecies of Drosophila as a basis for comparison.
In other ongoing research, members of the Nakamura lab are using forward-genetics techniques tosearch for novel genes involved in germ cell development. Early results of those studies have revealed

Styhler S, Nakamura A and
Lasko P. VASA localization
requires the SPRY-domain
and SOCS-box containing
protein, GUSTAVUS. Dev
Cell 3:865-76 (2002).

after fertilization, the ascidian egg develops into asmall free-swimming tadpole possessing a
notochord, an evolutionary forebear of the spinal
cord, and a rudimentary nervous system, both ofwhich are lost when it enters its immobile adultstage.

Sano H, Nakamura A andKobayashi S. Identification ofa transcriptional regulatory
region for germline-specificexpression of vasa gene in
Drosophila melanogaster .
Mech Dev 112:129-39(2002).

Nakamura seeks to analyze the regulatory mechanisms of germline development in Ciona, which areinteresting in that while several lines of evidence
indicate that germ cells are formed from maternallyderived germ plasm, it also appears that germ cells
can be regenerated after metamorphosis, suggesting that two independent mechanisms may be atwork. In research conducted in collaboration with
Suma Aqualife Park, a local municipal aquarium,
Nakamura’s team will explore the genetic regulationof ascidian germline development by selecting candidate genes from EST and genome databases (adraft sequence of the Ciona genome is available),
confirm the spatial and temporal patterns of theirexpression and characterize promoter regions andtrans-acting factors.

RIKEN CDB 2003 Annual Report

Nakamura A, Amikura R, Hanyu K and Kobayashi S.
Me31B silences translation ofoocyte-localizing RNAs
through the formation of cytoplasmic RNP complex during
Drosophila oogenesis. Development 128:3233-42 (2001).
Carrera P, Johnstone O,
Nakamura A, Casanova J,
Jackle H and Lasko P. VASA
mediates translation throughinteraction with a DrosophilayIF2 homolog. Mol Cell
5:181-7 (2000).

39

chromosomes, while Chp2 mutations result in weak silencing
defects in three heterochromatic
regions: centromeres, telomeres
and the mat locus.
The group has demonstrated that
Swi6, Chp1 and Chp2 localize atthree heterochromatic regions(centromere, telomere and mat
locus), and that this localization isclearly dependent on H3-lys9
methylation mediated by Clr4. Following on aseries of experiments in which the gene foreach protein was disrupted, Nakayama hasdeveloped a model in which Chp1 function is
specific to the establishment and spreadingof heterochromatin, while Chp2 and Swi6
form a dimer that supports the stable maintenance of heterochromatin. They also foundthat, among three heterochromatic regions,centromeres are more dynamic and requireestablishment steps.

A model of heterochromatin formation in fission yeast

Creative Research
Promoting Program

Jun-ichi Nakayama
Ph. D.
Jun-ichiNakayama
received his bachelor’s,
master’s and doctoral
degrees in bioscience fromthe Tokyo Institute of Technology, the last in 1999 forhis work on the cloning andcharacterization of mammalian telomerase components. He spent the period
from 1999 to 2001 as a
postdoctoral researcher at
the Cold Spring Harbor Laboratory in Shiv Grewal’slab, before returning toJapan in December 2001
as a PRESTO researcher
in the Japan Science andTechnology Corporation(JST). He was appointed
team leader in the RIKEN
CDB Laboratory for Chromatin Dynamics in 2002.

Modifications to the nuclear DNA-protein chromatincomplex are central to the epigenetic regulation ofgene transcription, an activity that must be maintained and propagated across mitotic cycles andthroughout the development of the organism properin order for cells to establish and maintain their identities. Chromatin occurs in highly-condensed andless spatially concentrated states, known as heterochromatin and euchromatin, respectively. Heterochromatin regions have fewer genes overall thaneuchromatic stretches of the genome, and many of
the genes that are found there remain unexpressed.
However, heterochromatin functions as more than alocked closet to store stretches of unused DNA; infact, there appear to be a number of functionally distinct types of heterochromatin serving in a spectrumof developmentally important capacities, from thetranscriptional regulation of cell-type specific genes
to genomic self-defense by compartmentalizing andneutralizing foreign mobile genetic elements thatmight otherwise interfere with proper gene function.
Heterochromatin also functions in two genetically silent chromosomal regions: telomeres, which play key
roles in replicative senescence and cancer, and centromeres, the linchpins of mitosis.
Using the fission yeast Saccharomyces pombe as amodel system, Jun-ichi Nakayama focuses on investigations of heterochromatin dynamics, modifications
to the DNA-packing proteins called 'histones' in particular, and the molecular mechanisms that allow
such chemical states to be heritably transmitted. Histones are the primary protein constituents of thenucleosome, the most basic unit of chromosomalorganization, which provides highly compact but
readily accessible packaging for a cell's gene-

40 RIKEN CDB 2003 Annual Reportencoding DNA. The amino-terminal tails of histones
protrude from the nucleosome and are subject tocovalent modifications including phosphorylation,acetylation, and methylation. These histone modifications affect higher-order chromatin structure andinfluence gene expression.

Heterochromatin also functions intelomeres, which play key roles in
replicative senescence and cancer,
and centromeres, thelinchpins of mitosis
In previous studies, Nakayama demonstrated thatheterochromatin protein binding states play a role in
the regulation of gene silencing. Nakayama performed detailed analyses of the binding states of
Swi6, a homolog of the mammalian HP-1 heterochromatin protein at the silent mating-type (mat) locus of
the fission yeast. The results of that study showedthat Swi6 protein is a dosage-critical component
involved in imprinting the mat locus. This binding of
Swi6 is maintained both across mitotic cell cycles
and intergenerationally, as it is propagated throughmeiosis as well. Nakayama's study also showed thatthe deacetylation and subsequent methylation of a
specific histone H3 lysine residue are essential tothis process. Chemical modifications to histones
are fundamental epigenetic processes that can act
to switch the expression of a target gene on or off.
Nakayama has now linked Swi6 function to anumber of other chromodomain proteins in fission
yeast that seem to act in a stepwise and contextsensitive fashion to silence genes in the process ofheterochromatin assembly. The number of speciesof these proteins, which possess characteristic SETor chromodomain sequence motifs, is much smaller
in yeast than it is in human, making S. pombe an apt
model for studying the basic means by which thesemolecules respond to the methyl modifications to histone residues. The methylation of specific sites on
the histone H3 catalyzed by a SET domaincontaining methyltransferase provides epigenetic
markers that allow chromodomain proteins to bindthe histone and direct it toward eu- or heterochromatin assembly or to initiate developmentally regulated
gene silencing.
The Nakayama lab has shown the histone methyltransferase Clr4 to be essential for heterochromatin
assembly as an upstream element that helps Swi6
to localize correctly. Two other chromodomain proteins, Chp1 and Chp2, have also been implicated inheterochromatin formation and function. Disruptionof the gene encoding Chp1causes defects in centromeric silencing and higher mitotic loss rates in mini-

Staff
Team Leader

Jun-ichi Nakayama
Research Scientist

Tetsushi Iida
Tomohiro Hayakawa
Visiting Scientist

Mahito SadaieTechnical Staff

Yasuko Ohtani
Rika Kawaguchi

Nakayama is also interested in how chromodomain proteins function in higher eukaryotic cells.
In previous study, Nakayama found an evolutionallyconserved chromodomain protein is a stable component of histone deacetylase complex in fission
yeast. Using mammalian cells, he is now investigating the function of a chromodomain protein which
has been linked to cell senescence and development.

Publications
Nakayama J, Xiao G, Noma K,
Malikzay A, Bjerling P, Ekwall
K, Kobayashi R and Grewal
SI. Alp13, an MRG family protein, is a component of fission
yeast Clr6 histone deacetylase
required for genomic integrity.
Embo J 22:2776-87 (2003).

Through his work on heterochromatin histone modifications, Nakayama has uncovered potentially importantnew roles for proteins in the establishment, maintenance and transmission of epigenetic information.
These findings show that the definition of a gene as asimple string of DNA nucleotides needs to be expanded to include the action of proteins in the functional
genetic unit. In the future, Nakayama plans to perform
more detailed analyses of the molecular mechanismsthat underlie epigenetic function, as well as studies inhigher organisms and epigenetic gene expression indevelopmental processes.

Tamaru H, Zhang X, McMillen
D, Singh PB, Nakayama J,
Grewal SI, Allis CD, Cheng X
and Selker EU. Trimethylated
lysine 9 of histone H3 is amark for DNA methylation in
Neurospora crassa. Nat Genet
34:75-9 (2003).
Nakagawa H, Lee JK, Hurwitz
J, Allshire RC, Nakayama J,
Grewal SI, Tanaka K and Murakami Y. Fission yeast CENPB homologs nucleate centromeric heterochromatin by promoting heterochromatinspecific histone tail modifications. Genes Dev 16:1766-78(2002).
Nakayama J, Allshire RC, Klar
AJS and Grewal SIS. Implicating DNA polymerase a in epigenetic control of silencing infission yeast. EMBO J
20:2857-66(2001).
Nakayama J, Rice JC, Strahl
BD, Allis CD and Grewal SI.
Role of histone H3 lysine 9
methylation in epigenetic control of heterochromatin assembly. Science 292:110-3 (2001).

A
Localization of a chromodomain protein (green) and phosphorylated
form of RNA polymerase II (magenta) in HeLa cell.

Nakayama J, Klar AJ and Grewal SI. A chromodomain protein, Swi6, performs imprinting
functions in fission yeast during mitosis and meiosis. Cell
101:307-17 (2000).

B
Localization of a chromodomain protein (green) and SC-35 (magenta)
under alpha-amanitin treatment in HeLa cell.

RIKEN CDB 2003 Annual Report41

chromosomes, while Chp2 mutations result in weak silencing
defects in three heterochromatic
regions: centromeres, telomeres
and the mat locus.
The group has demonstrated that
Swi6, Chp1 and Chp2 localize atthree heterochromatic regions(centromere, telomere and mat
locus), and that this localization isclearly dependent on H3-lys9
methylation mediated by Clr4. Following on aseries of experiments in which the gene foreach protein was disrupted, Nakayama hasdeveloped a model in which Chp1 function is
specific to the establishment and spreadingof heterochromatin, while Chp2 and Swi6
form a dimer that supports the stable maintenance of heterochromatin. They also foundthat, among three heterochromatic regions,centromeres are more dynamic and requireestablishment steps.

A model of heterochromatin formation in fission yeast

Creative Research
Promoting Program

Jun-ichi Nakayama
Ph. D.
Jun-ichiNakayama
received his bachelor’s,
master’s and doctoral
degrees in bioscience fromthe Tokyo Institute of Technology, the last in 1999 forhis work on the cloning andcharacterization of mammalian telomerase components. He spent the period
from 1999 to 2001 as a
postdoctoral researcher at
the Cold Spring Harbor Laboratory in Shiv Grewal’slab, before returning toJapan in December 2001
as a PRESTO researcher
in the Japan Science andTechnology Corporation(JST). He was appointed
team leader in the RIKEN
CDB Laboratory for Chromatin Dynamics in 2002.

Modifications to the nuclear DNA-protein chromatincomplex are central to the epigenetic regulation ofgene transcription, an activity that must be maintained and propagated across mitotic cycles andthroughout the development of the organism properin order for cells to establish and maintain their identities. Chromatin occurs in highly-condensed andless spatially concentrated states, known as heterochromatin and euchromatin, respectively. Heterochromatin regions have fewer genes overall thaneuchromatic stretches of the genome, and many of
the genes that are found there remain unexpressed.
However, heterochromatin functions as more than alocked closet to store stretches of unused DNA; infact, there appear to be a number of functionally distinct types of heterochromatin serving in a spectrumof developmentally important capacities, from thetranscriptional regulation of cell-type specific genes
to genomic self-defense by compartmentalizing andneutralizing foreign mobile genetic elements thatmight otherwise interfere with proper gene function.
Heterochromatin also functions in two genetically silent chromosomal regions: telomeres, which play key
roles in replicative senescence and cancer, and centromeres, the linchpins of mitosis.
Using the fission yeast Saccharomyces pombe as amodel system, Jun-ichi Nakayama focuses on investigations of heterochromatin dynamics, modifications
to the DNA-packing proteins called 'histones' in particular, and the molecular mechanisms that allow
such chemical states to be heritably transmitted. Histones are the primary protein constituents of thenucleosome, the most basic unit of chromosomalorganization, which provides highly compact but
readily accessible packaging for a cell's gene-

40 RIKEN CDB 2003 Annual Reportencoding DNA. The amino-terminal tails of histones
protrude from the nucleosome and are subject tocovalent modifications including phosphorylation,acetylation, and methylation. These histone modifications affect higher-order chromatin structure andinfluence gene expression.

Heterochromatin also functions intelomeres, which play key roles in
replicative senescence and cancer,
and centromeres, thelinchpins of mitosis
In previous studies, Nakayama demonstrated thatheterochromatin protein binding states play a role in
the regulation of gene silencing. Nakayama performed detailed analyses of the binding states of
Swi6, a homolog of the mammalian HP-1 heterochromatin protein at the silent mating-type (mat) locus of
the fission yeast. The results of that study showedthat Swi6 protein is a dosage-critical component
involved in imprinting the mat locus. This binding of
Swi6 is maintained both across mitotic cell cycles
and intergenerationally, as it is propagated throughmeiosis as well. Nakayama's study also showed thatthe deacetylation and subsequent methylation of a
specific histone H3 lysine residue are essential tothis process. Chemical modifications to histones
are fundamental epigenetic processes that can act
to switch the expression of a target gene on or off.
Nakayama has now linked Swi6 function to anumber of other chromodomain proteins in fission
yeast that seem to act in a stepwise and contextsensitive fashion to silence genes in the process ofheterochromatin assembly. The number of speciesof these proteins, which possess characteristic SETor chromodomain sequence motifs, is much smaller
in yeast than it is in human, making S. pombe an apt
model for studying the basic means by which thesemolecules respond to the methyl modifications to histone residues. The methylation of specific sites on
the histone H3 catalyzed by a SET domaincontaining methyltransferase provides epigenetic
markers that allow chromodomain proteins to bindthe histone and direct it toward eu- or heterochromatin assembly or to initiate developmentally regulated
gene silencing.
The Nakayama lab has shown the histone methyltransferase Clr4 to be essential for heterochromatin
assembly as an upstream element that helps Swi6
to localize correctly. Two other chromodomain proteins, Chp1 and Chp2, have also been implicated inheterochromatin formation and function. Disruptionof the gene encoding Chp1causes defects in centromeric silencing and higher mitotic loss rates in mini-

Staff
Team Leader

Jun-ichi Nakayama
Research Scientist

Tetsushi Iida
Tomohiro Hayakawa
Visiting Scientist

Mahito SadaieTechnical Staff

Yasuko Ohtani
Rika Kawaguchi

Nakayama is also interested in how chromodomain proteins function in higher eukaryotic cells.
In previous study, Nakayama found an evolutionallyconserved chromodomain protein is a stable component of histone deacetylase complex in fission
yeast. Using mammalian cells, he is now investigating the function of a chromodomain protein which
has been linked to cell senescence and development.

Publications
Nakayama J, Xiao G, Noma K,
Malikzay A, Bjerling P, Ekwall
K, Kobayashi R and Grewal
SI. Alp13, an MRG family protein, is a component of fission
yeast Clr6 histone deacetylase
required for genomic integrity.
Embo J 22:2776-87 (2003).

Through his work on heterochromatin histone modifications, Nakayama has uncovered potentially importantnew roles for proteins in the establishment, maintenance and transmission of epigenetic information.
These findings show that the definition of a gene as asimple string of DNA nucleotides needs to be expanded to include the action of proteins in the functional
genetic unit. In the future, Nakayama plans to perform
more detailed analyses of the molecular mechanismsthat underlie epigenetic function, as well as studies inhigher organisms and epigenetic gene expression indevelopmental processes.

Tamaru H, Zhang X, McMillen
D, Singh PB, Nakayama J,
Grewal SI, Allis CD, Cheng X
and Selker EU. Trimethylated
lysine 9 of histone H3 is amark for DNA methylation in
Neurospora crassa. Nat Genet
34:75-9 (2003).
Nakagawa H, Lee JK, Hurwitz
J, Allshire RC, Nakayama J,
Grewal SI, Tanaka K and Murakami Y. Fission yeast CENPB homologs nucleate centromeric heterochromatin by promoting heterochromatinspecific histone tail modifications. Genes Dev 16:1766-78(2002).
Nakayama J, Allshire RC, Klar
AJS and Grewal SIS. Implicating DNA polymerase a in epigenetic control of silencing infission yeast. EMBO J
20:2857-66(2001).
Nakayama J, Rice JC, Strahl
BD, Allis CD and Grewal SI.
Role of histone H3 lysine 9
methylation in epigenetic control of heterochromatin assembly. Science 292:110-3 (2001).

A
Localization of a chromodomain protein (green) and phosphorylated
form of RNA polymerase II (magenta) in HeLa cell.

Nakayama J, Klar AJ and Grewal SI. A chromodomain protein, Swi6, performs imprinting
functions in fission yeast during mitosis and meiosis. Cell
101:307-17 (2000).

B
Localization of a chromodomain protein (green) and SC-35 (magenta)
under alpha-amanitin treatment in HeLa cell.

RIKEN CDB 2003 Annual Report41

StaffTeam Leader

Student Trainee

Kiyoji Nishiwaki

Ryoko Murakami

Research Scientist

Assistant

Norio Suzuki
Yukihiko Kubota
Katsuyuki Tamai
Shinji Ihara
Kiyotaka Ohkura

Rie Kuroki
Part-time Staff

Midori Tatsumoto
Asami Sumitani

Technical Staff

Mitsue Sano

Creative Research
Promoting Program

Cell migration andorganogenesis

Kiyoji Nishiwaki

Cell division, cell migration and changes in cell
shape are coordinated to generate complex tissue
and organ systems during animal development andregeneration. One important aspect of this minutely
regulated process is the movement of cells in
sheets, an activity orchestrated by mechanisms
which remain poorly understood. During organogenesis, sheets of cells migrate and spread over theunderlying tissues while maintaining selective adhesive bonds with cells of similar type, and make their

way to destinations in the body, following molecular
guidance cues generated by mechanisms thatremain enigmatic. Kiyoji Nishiwaki studies gonadogenesis in the nematode C. elegans as a model system to improve the understanding of the molecularbases of cell migration during organ development.
By developing a clearer picture of evolutionarily conserved mechanisms, this research promises to provide insights into cell migration and organ development in humans, and contribute to the understanding of human diseases involving disturbances incoordinated cellular movement.

anti-coagulatory effects. ADAM family proteases
have been implicated in a number of human physiological and pathological processes, including blood
coagulation disorders, rheumatoid arthritis and asthma. These proteins act to proteolytically releasemembrane-bound growth factors or to disrupt components of extracellular matrices such as collagenand proteoglycans (which help to build skin and soft
tissue).

This research promises toprovide insights into cell
migration and organ developmentin humans

Ph. D.

Guidance of the gonad

Kiyoji Nishiwaki received
his B. Sc. from Osaka City
University and M. Sc. from
Osaka University where heworked on the molecularbiology of the yeast S. cerevisiae. He joined NEC
Corp. in 1986 as a researcher in the Fundamental
Research Laboratory studying the molecular genetics
of C. elegans. He left NEC
in 1992 to work as a visiting
researcher at Johns Hopkins
University,
thenreturned to the company in1993 to continue his workon nematode development.
He was awarded a Ph. D.
by Osaka City University forwork on the molecular analysis of C. elegans embryogenesis in 1994. He
remained at NEC until
receiving an appointment
as team leader at the
RIKEN CDB.

The U-shaped gonad in the wild type becomes abnormal in mig-23
and mig-17 mutants due to the misdirected migration of distal tip cells.

42 RIKEN CDB 2003 Annual Report

Interactions between the basement membrane of
the migrating gonad and that of the body wall are
keystone processes in gonadal development. The
C. elegans gonad is U-shaped, the result of thedirected migration of cells known as distal tip cells
present at the leading ends of the developing gonadin larvae. The gene mig-17, which Nishiwaki previously showed to be essential to this guided movement, is expressed in muscle cells and secreted into
the body cavity where it binds to the migrating gonadal tip cells. Distal tip cells in worms engineered tolack the MIG-17 protein fail to steer cell migration
properly, resulting in abnormal development and a'meandering' gonad phenotype. However, themeans by which MIG-17 coordinates the timing anddirection of the developing gonad's progress though
the larval body has remained unknown.

Nishiwaki's team has analyzed a number of mutations that result in aberrant gonadal phenotypes similar to those seen in mig-17 mutants, including themutation of the gene that encodes MIG-23. This protein was found to be structurally related to nucleoside diphosphatases (NDPases), glycosylating proteins found in the Golgi apparatus, the intracellular
processing center where proteins are modified, sorted and released from the cell. Glycosylation involves the addition of a sugar to a site on a protein
chain, a post-translational modification whose roles
are incompletely understood, but which is thought toserve a regulatory function in some signaling molecules. On further investigation, MIG-23 mutants
showed reductions in NDPase activity. The wild-type
MIG-23 protein successfully rescued yeast mutantsfor NDPase, lending solid support to the case for the
NDPase nature of MIG-23.

MIG-17 is an ADAM (a disintegrin and metalloprotease) protease, a family of secreted or membraneproteins first identified as components of various
snake venoms, where they have hemorrhagic and

The similarity between the MIG-23 and MIG-17 phenotypes suggested that the mutation of MIG-23
might result in abnormal MIG-17 glycosylation.
Using gel electrophoresis, Nishiwaki and colleagues

demonstrated that MIG-17
moleculesin
MIG-23mutants had lower than normal molecular weights consistent with the loss of glycosylation, while immunoblotting studies confirmed that inthese mutants MIG-17 failed
to bind sugars at the normal
levels. MIG-17 molecules
designed with deficient glycosylation sites also failed to
localize at the gonad andshowed no ability to direct its
migration. Genetic analyses
revealed that MIG-23 isexpressed in muscle cells,
which reconciles well with
MIG-17's muscle cell origins.
Taken
together,these
results indicate that MIG-17
molecules are secreted frommuscle cells after glycosylation by MIG-23, a modification which enables their
recruitment to the migrating
gonad.

Publications
Nishiwaki K, Kubota Y, Chigira Y, Roy SK, Suzuki M,
Schvarzstein M, Jigami Y,
Hisamoto N and Matsumoto
K. An NDPase links ADAM
protease glycosylation withorgan morphogenesis in C.
elegans. Nat Cell Biol 6:31-7(2004).
Gumienny TL, Brugnera E,
Tosello-Trampont AC,
Kinchen JM, Haney LB, Nishiwaki K, Walk SF, Nemergut
ME, Macara IG, Francis R,
Schedl T, Qin Y, Van Aelst L,
HengartnerMOandRavichandran KS. CED12/ELMO, a novel memberof the CrkII/Dock180/Rac
pathway, is required for phagocytosis and cell migration.
Cell 107:27-41 (2001).
Suzuki N, Buechner M, Nishiwaki K, Hall DH, Nakanishi
H, Takai Y, Hisamoto N and
Matsumoto K. A putative
GDP-GTP exchange factor isrequired for development of
the excretory cell in Caenorhabditis elegans. EMBO Rep
2:530-5 (2001).

Questions and implications
Although Golgi body NDPases such as MIG-23 arecapable of modifying a number of other secreted proteins, the Nishiwaki study indicates that the effectsof the NDPase dysfunction are especially prominent
in the abnormal glycosylation and function of an
ADAM family protease, suggesting that glycosylation may play a more important role in the functionof proteins in this family than it does in that of othersecreted proteins. ADAM family proteases are
known to be involved in a number of human diseases, and the question of whether glycosylation
defects play a role in their pathogenesis merits further investigation. Research such as this provides
insights into basic biological mechanisms that may
one day make it possible to treat a range of healthconditions by restoring normal ADAM protease glycosylation in cases in which it is aberrant.

Nishiwaki K, Hisamoto N and
Matsumoto K. A metalloprotease disintegrin that controls cell migration in
Caenorhabditis elegans. Science 288:2205-8 (2000).

MIG-17 is glycosylated by MIG-23 and secreted from the muscle cells to localize on the
gonad surface where it is required for the migration of distal tip cells.

RIKEN CDB 2003 Annual Report43StaffTeam Leader

Student Trainee

Kiyoji Nishiwaki

Ryoko Murakami

Research Scientist

Assistant

Norio Suzuki
Yukihiko Kubota
Katsuyuki Tamai
Shinji Ihara
Kiyotaka Ohkura

Rie Kuroki
Part-time Staff

Midori Tatsumoto
Asami Sumitani

Technical Staff

Mitsue Sano

Creative Research
Promoting Program

Cell migration andorganogenesis

Kiyoji Nishiwaki

Cell division, cell migration and changes in cell
shape are coordinated to generate complex tissue
and organ systems during animal development andregeneration. One important aspect of this minutely
regulated process is the movement of cells in
sheets, an activity orchestrated by mechanisms
which remain poorly understood. During organogenesis, sheets of cells migrate and spread over theunderlying tissues while maintaining selective adhesive bonds with cells of similar type, and make their

way to destinations in the body, following molecular
guidance cues generated by mechanisms thatremain enigmatic. Kiyoji Nishiwaki studies gonadogenesis in the nematode C. elegans as a model system to improve the understanding of the molecularbases of cell migration during organ development.
By developing a clearer picture of evolutionarily conserved mechanisms, this research promises to provide insights into cell migration and organ development in humans, and contribute to the understanding of human diseases involving disturbances incoordinated cellular movement.

anti-coagulatory effects. ADAM family proteases
have been implicated in a number of human physiological and pathological processes, including blood
coagulation disorders, rheumatoid arthritis and asthma. These proteins act to proteolytically releasemembrane-bound growth factors or to disrupt components of extracellular matrices such as collagenand proteoglycans (which help to build skin and soft
tissue).

This research promises toprovide insights into cell
migration and organ developmentin humans

Ph. D.

Guidance of the gonad

Kiyoji Nishiwaki received
his B. Sc. from Osaka City
University and M. Sc. from
Osaka University where heworked on the molecularbiology of the yeast S. cerevisiae. He joined NEC
Corp. in 1986 as a researcher in the Fundamental
Research Laboratory studying the molecular genetics
of C. elegans. He left NEC
in 1992 to work as a visiting
researcher at Johns Hopkins
University,
thenreturned to the company in1993 to continue his workon nematode development.
He was awarded a Ph. D.
by Osaka City University forwork on the molecular analysis of C. elegans embryogenesis in 1994. He
remained at NEC until
receiving an appointment
as team leader at the
RIKEN CDB.

The U-shaped gonad in the wild type becomes abnormal in mig-23
and mig-17 mutants due to the misdirected migration of distal tip cells.

42 RIKEN CDB 2003 Annual Report

Interactions between the basement membrane of
the migrating gonad and that of the body wall are
keystone processes in gonadal development. The
C. elegans gonad is U-shaped, the result of thedirected migration of cells known as distal tip cells
present at the leading ends of the developing gonadin larvae. The gene mig-17, which Nishiwaki previously showed to be essential to this guided movement, is expressed in muscle cells and secreted into
the body cavity where it binds to the migrating gonadal tip cells. Distal tip cells in worms engineered tolack the MIG-17 protein fail to steer cell migration
properly, resulting in abnormal development and a'meandering' gonad phenotype. However, themeans by which MIG-17 coordinates the timing anddirection of the developing gonad's progress though
the larval body has remained unknown.

Nishiwaki's team has analyzed a number of mutations that result in aberrant gonadal phenotypes similar to those seen in mig-17 mutants, including themutation of the gene that encodes MIG-23. This protein was found to be structurally related to nucleoside diphosphatases (NDPases), glycosylating proteins found in the Golgi apparatus, the intracellular
processing center where proteins are modified, sorted and released from the cell. Glycosylation involves the addition of a sugar to a site on a protein
chain, a post-translational modification whose roles
are incompletely understood, but which is thought toserve a regulatory function in some signaling molecules. On further investigation, MIG-23 mutants
showed reductions in NDPase activity. The wild-type
MIG-23 protein successfully rescued yeast mutantsfor NDPase, lending solid support to the case for the
NDPase nature of MIG-23.

MIG-17 is an ADAM (a disintegrin and metalloprotease) protease, a family of secreted or membraneproteins first identified as components of various
snake venoms, where they have hemorrhagic and

The similarity between the MIG-23 and MIG-17 phenotypes suggested that the mutation of MIG-23
might result in abnormal MIG-17 glycosylation.
Using gel electrophoresis, Nishiwaki and colleagues

demonstrated that MIG-17
moleculesin
MIG-23mutants had lower than normal molecular weights consistent with the loss of glycosylation, while immunoblotting studies confirmed that inthese mutants MIG-17 failed
to bind sugars at the normal
levels. MIG-17 molecules
designed with deficient glycosylation sites also failed to
localize at the gonad andshowed no ability to direct its
migration. Genetic analyses
revealed that MIG-23 isexpressed in muscle cells,
which reconciles well with
MIG-17's muscle cell origins.
Taken
together,these
results indicate that MIG-17
molecules are secreted frommuscle cells after glycosylation by MIG-23, a modification which enables their
recruitment to the migrating
gonad.

Publications
Nishiwaki K, Kubota Y, Chigira Y, Roy SK, Suzuki M,
Schvarzstein M, Jigami Y,
Hisamoto N and Matsumoto
K. An NDPase links ADAM
protease glycosylation withorgan morphogenesis in C.
elegans. Nat Cell Biol 6:31-7(2004).
Gumienny TL, Brugnera E,
Tosello-Trampont AC,
Kinchen JM, Haney LB, Nishiwaki K, Walk SF, Nemergut
ME, Macara IG, Francis R,
Schedl T, Qin Y, Van Aelst L,
HengartnerMOandRavichandran KS. CED12/ELMO, a novel memberof the CrkII/Dock180/Rac
pathway, is required for phagocytosis and cell migration.
Cell 107:27-41 (2001).
Suzuki N, Buechner M, Nishiwaki K, Hall DH, Nakanishi
H, Takai Y, Hisamoto N and
Matsumoto K. A putative
GDP-GTP exchange factor isrequired for development of
the excretory cell in Caenorhabditis elegans. EMBO Rep
2:530-5 (2001).

Questions and implications
Although Golgi body NDPases such as MIG-23 arecapable of modifying a number of other secreted proteins, the Nishiwaki study indicates that the effectsof the NDPase dysfunction are especially prominent
in the abnormal glycosylation and function of an
ADAM family protease, suggesting that glycosylation may play a more important role in the functionof proteins in this family than it does in that of othersecreted proteins. ADAM family proteases are
known to be involved in a number of human diseases, and the question of whether glycosylation
defects play a role in their pathogenesis merits further investigation. Research such as this provides
insights into basic biological mechanisms that may
one day make it possible to treat a range of healthconditions by restoring normal ADAM protease glycosylation in cases in which it is aberrant.

Nishiwaki K, Hisamoto N and
Matsumoto K. A metalloprotease disintegrin that controls cell migration in
Caenorhabditis elegans. Science 288:2205-8 (2000).

MIG-17 is glycosylated by MIG-23 and secreted from the muscle cells to localize on the
gonad surface where it is required for the migration of distal tip cells.

RIKEN CDB 2003 Annual Report43StaffTeam Leader

Hitoshi Niwa
Research Scientist

Shinji Masui
Kazuya Ogawa
Satoshi Ohtsuka
Yayoi Toyooka
Visiting Scientist

Itsuro Sugimura
Hiroyuki Kitajima
Technical Staff

Rika Yagi
Kadue Takahashi
Student Trainee

Ken-ichi Tominaga
Yoko Sekita
Yuko Iwamatsu
Sayako Matsui
Daisuke Shimosato
Assistant

Rie Kuroki

Trophoblast stem (TS) cells, derived from ES cells, grown on feeder cells in the presence of recombinant FGF4

Creative Research
Promoting Program

Self-renewal andpluripotency

Hitoshi Niwa
M. D. , Ph. D.
Hitoshi Niwa received his
M. D. from Nara Medical
University in 1989, and his
Ph. D. in medical physiology in 1993 from the Kumamoto University Graduate
School of Medicine for his
work in gene trap methods.
From 1993 to 1994, heworked as a research associate in the Department ofDevelopmental Genetics atthe same university, before
taking a postdoctoral fellowship with Austin Smith at
the University of Edinburgh
Centrefor
GenomeResearch. He returned toJapan in 1996 as a
research associate in the
Department of Nutrition andPhysiological Chemistry at
the Osaka University Graduate School of Medicine,where he remained until taking his current position at
the RIKEN CDB.

Their ability to self-renew indefinitely and to differentiate into cells of all three germ layer types (a differentiative capacity termed 'pluripotency'), makes
embryonic stem (ES) cells one of the most promising subjects of study in regenerative medicine, as
well as an attractive model system for research intoa spectrum of developmental processes. However,
the mechanisms by which ES cells are able to maintain these capabilities are incompletely understood,
and a better understanding of the stemness of thesecells will be necessary in order to be able to takebest advantage of their remarkable properties.
Two of the biggest challenges that now face stem
cell research are the determination of the factorsthat allow ES cells to generate limitlessly self-

renewable progeny, and the identification of molecules that direct the dividing ES cell to produce
daughter cells of specific types. Hitoshi Niwa's
research addresses both of these challenges, withthe aims of developing solid scientific foundations
and reliable technologies to support this exciting
field of biomedicine.

Inducing differentiation
The development of methods by which undifferentiated ES cells can be prompted to commit to a specificcell lineage is a field of central importance to thestem cell research community. Studying factors identified in work on knockout mice, the Niwa researchteam has been engaged in the analysis of transcription factors with potential roles in the developmentof extraembryonic cell lineages, extraembryonicendoderm and trophectoderm. Extraembryonicendoderm derivatives include
the parietal and visceral endoderm, which give rise to the yolk
sac covering embryos in utero
duringdevelopment,
andtrophectoderm derivatives, thesource of the placenta that sustains the developing mammalianembryos.
In previous research, the Niwa
lab showed that the overexpression of GATA transcription factors such as Gata-4 or Gata-6
resulted in the specific conversion of undifferentiated ES cells
into extraembryonic endodermal
cells, and that the exogenous

expression of either of these factors simultaneously
induced the expression of the other from the endogenous gene. New work suggests that the homeobox
gene Cdx-2 can serve as a similar trigger for the
induction of trophectoderm from ES cells expressing
low levels of the pluripotency maintaining gene, Oct
3/4. Using regulable activation of Cdx-2 in vitro,
Niwa successfully induced ES cells to differentiate
into trophoblast stem cells. These results, in combination with the GATA study, have led to the development of a model in which Oct 3/4, when maintainedat an appropriate mid-range level, inhibits the differentiation of ES cells into either the trophectodermalor primitive endodermal lineages by suppressing
Cdx-2 and Gata-6, respectively.

Maintaining pluripotency
The POU-family transcriptional regulator Oct 3/4
was the first factor found to elicit multiple differentiative outcomes dependent on its expression level. ES
cells expressing median levels of Oct 3/4 maintaintheir pluripotency, while its overexpression results indifferentiation into primitive endoderm and mesoderm and its inhibition causes the cells to take up atrophectodermal fate. These findings establishedOct 3/4 as a primary regulator of pluripotency in ES
cells, but the function of this regulator in the commitment of more specific lineages remains an open
issue.

In addition to its function as amaintainer of pluripotency,
Oct 3/4 may
also play a second key role byregulating neuronal differentiationenon). The researchers found that SDIA acts tomaintain Oct 3/4 expression in ES cells, whichappears to be important as a promoter of the differentiation of ES cells into neural lineages. ES cells
from which Oct 3/4 had been deleted lost their ability
to differentiate into neurons, while heightened levelsof Oct 3/4 intensified the neurogenic effects of SDIA.
This finding raises the possibility that, in addition toits function as a maintainer of pluripotency, Oct 3/4
also plays a second key role by regulating neuronaldifferentiation in a concentration-dependent manner.

ES cell growth incultureThis goal of controlling culture conditions to achieve
specific outcomes is important to the growth of ES
cells in vitro as well. The Niwa lab is working todevelop a serum- and feeder-cell-free system for culturing mouse ES cells, which necessitates developing a detailed picture of both the extrinsic factors
and the intrinsic networks that function in these cells
in culture. It is known that a single ES cell in isolation will fail to proliferate, while colonies of such cells
grow normally. It is also known that even isolated
single cells can be induced to proliferate if the culture medium from a larger ES cell colony is transferred to the single cell's plate, suggesting that ES
cells produce a growth-stimulating factor that acts
by community effect. Niwa has developed an assay
system to isolate candidate molecules for this putative 'stem cell autocrine factor' (SAF), and is actively
pursuing the characterization of the most promising
candidates. The results of these analyses should
improve the ability of researchers to grow ES cells in
culture, facilitating the study of these fascinating andpotentially revolutionarily important cells.

Publications
Tokuzawa Y, Kaiho E, Maruyama M, Takahashi K, Mitsui K, Maeda M, Niwa H and
Yamanaka S. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem
cell self-renewal and mouse
development. Mol Cell Biol
23:2699-708 (2003).
Shimozaki K, Nakashima K,
Niwa H and Taga T. Involvement of Oct3/4 in theenhancement of neuronal differentiation of ES cells inneurogenesis-inducing cultures.
Development
130:2505-12 (2003).
Ishida C, Ura K, Hirao A,
Sasaki H, Toyoda A, Sakaki
Y, Niwa H, Li E and Kaneda
Y. Genomic organization andpromoter analysis of theDnmt3b gene. Gene
310:151-9 (2003).

In 2003, the Niwa lab contributed to a study that
demonstrated a role for Oct 3/4 in neurogenesis promoted by stromal cell derived inducing activity(SDIA, see p. 18-19 for a description of this phenom-

Terminally differentiated trophoblast giant cells derived from ES cells, showing large, flat
morphologies and polyploidy

44 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report45

StaffTeam Leader

Hitoshi Niwa
Research Scientist

Shinji Masui
Kazuya Ogawa
Satoshi Ohtsuka
Yayoi Toyooka
Visiting Scientist

Itsuro Sugimura
Hiroyuki Kitajima
Technical Staff

Rika Yagi
Kadue Takahashi
Student Trainee

Ken-ichi Tominaga
Yoko Sekita
Yuko Iwamatsu
Sayako Matsui
Daisuke Shimosato
Assistant

Rie Kuroki

Trophoblast stem (TS) cells, derived from ES cells, grown on feeder cells in the presence of recombinant FGF4

Creative Research
Promoting Program

Self-renewal andpluripotency

Hitoshi Niwa
M. D. , Ph. D.
Hitoshi Niwa received his
M. D. from Nara Medical
University in 1989, and his
Ph. D. in medical physiology in 1993 from the Kumamoto University Graduate
School of Medicine for his
work in gene trap methods.
From 1993 to 1994, heworked as a research associate in the Department ofDevelopmental Genetics atthe same university, before
taking a postdoctoral fellowship with Austin Smith at
the University of Edinburgh
Centrefor
GenomeResearch. He returned toJapan in 1996 as a
research associate in the
Department of Nutrition andPhysiological Chemistry at
the Osaka University Graduate School of Medicine,where he remained until taking his current position at
the RIKEN CDB.

Their ability to self-renew indefinitely and to differentiate into cells of all three germ layer types (a differentiative capacity termed 'pluripotency'), makes
embryonic stem (ES) cells one of the most promising subjects of study in regenerative medicine, as
well as an attractive model system for research intoa spectrum of developmental processes. However,
the mechanisms by which ES cells are able to maintain these capabilities are incompletely understood,
and a better understanding of the stemness of thesecells will be necessary in order to be able to takebest advantage of their remarkable properties.
Two of the biggest challenges that now face stem
cell research are the determination of the factorsthat allow ES cells to generate limitlessly self-

renewable progeny, and the identification of molecules that direct the dividing ES cell to produce
daughter cells of specific types. Hitoshi Niwa's
research addresses both of these challenges, withthe aims of developing solid scientific foundations
and reliable technologies to support this exciting
field of biomedicine.

Inducing differentiation
The development of methods by which undifferentiated ES cells can be prompted to commit to a specificcell lineage is a field of central importance to thestem cell research community. Studying factors identified in work on knockout mice, the Niwa researchteam has been engaged in the analysis of transcription factors with potential roles in the developmentof extraembryonic cell lineages, extraembryonicendoderm and trophectoderm. Extraembryonicendoderm derivatives include
the parietal and visceral endoderm, which give rise to the yolk
sac covering embryos in utero
duringdevelopment,
andtrophectoderm derivatives, thesource of the placenta that sustains the developing mammalianembryos.
In previous research, the Niwa
lab showed that the overexpression of GATA transcription factors such as Gata-4 or Gata-6
resulted in the specific conversion of undifferentiated ES cells
into extraembryonic endodermal
cells, and that the exogenous

expression of either of these factors simultaneously
induced the expression of the other from the endogenous gene. New work suggests that the homeobox
gene Cdx-2 can serve as a similar trigger for the
induction of trophectoderm from ES cells expressing
low levels of the pluripotency maintaining gene, Oct
3/4. Using regulable activation of Cdx-2 in vitro,
Niwa successfully induced ES cells to differentiate
into trophoblast stem cells. These results, in combination with the GATA study, have led to the development of a model in which Oct 3/4, when maintainedat an appropriate mid-range level, inhibits the differentiation of ES cells into either the trophectodermalor primitive endodermal lineages by suppressing
Cdx-2 and Gata-6, respectively.

Maintaining pluripotency
The POU-family transcriptional regulator Oct 3/4
was the first factor found to elicit multiple differentiative outcomes dependent on its expression level. ES
cells expressing median levels of Oct 3/4 maintaintheir pluripotency, while its overexpression results indifferentiation into primitive endoderm and mesoderm and its inhibition causes the cells to take up atrophectodermal fate. These findings establishedOct 3/4 as a primary regulator of pluripotency in ES
cells, but the function of this regulator in the commitment of more specific lineages remains an open
issue.

In addition to its function as amaintainer of pluripotency,
Oct 3/4 may
also play a second key role byregulating neuronal differentiationenon). The researchers found that SDIA acts tomaintain Oct 3/4 expression in ES cells, whichappears to be important as a promoter of the differentiation of ES cells into neural lineages. ES cells
from which Oct 3/4 had been deleted lost their ability
to differentiate into neurons, while heightened levelsof Oct 3/4 intensified the neurogenic effects of SDIA.
This finding raises the possibility that, in addition toits function as a maintainer of pluripotency, Oct 3/4
also plays a second key role by regulating neuronaldifferentiation in a concentration-dependent manner.

ES cell growth incultureThis goal of controlling culture conditions to achieve
specific outcomes is important to the growth of ES
cells in vitro as well. The Niwa lab is working todevelop a serum- and feeder-cell-free system for culturing mouse ES cells, which necessitates developing a detailed picture of both the extrinsic factors
and the intrinsic networks that function in these cells
in culture. It is known that a single ES cell in isolation will fail to proliferate, while colonies of such cells
grow normally. It is also known that even isolated
single cells can be induced to proliferate if the culture medium from a larger ES cell colony is transferred to the single cell's plate, suggesting that ES
cells produce a growth-stimulating factor that acts
by community effect. Niwa has developed an assay
system to isolate candidate molecules for this putative 'stem cell autocrine factor' (SAF), and is actively
pursuing the characterization of the most promising
candidates. The results of these analyses should
improve the ability of researchers to grow ES cells in
culture, facilitating the study of these fascinating andpotentially revolutionarily important cells.

Publications
Tokuzawa Y, Kaiho E, Maruyama M, Takahashi K, Mitsui K, Maeda M, Niwa H and
Yamanaka S. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem
cell self-renewal and mouse
development. Mol Cell Biol
23:2699-708 (2003).
Shimozaki K, Nakashima K,
Niwa H and Taga T. Involvement of Oct3/4 in theenhancement of neuronal differentiation of ES cells inneurogenesis-inducing cultures.
Development
130:2505-12 (2003).
Ishida C, Ura K, Hirao A,
Sasaki H, Toyoda A, Sakaki
Y, Niwa H, Li E and Kaneda
Y. Genomic organization andpromoter analysis of theDnmt3b gene. Gene
310:151-9 (2003).

In 2003, the Niwa lab contributed to a study that
demonstrated a role for Oct 3/4 in neurogenesis promoted by stromal cell derived inducing activity(SDIA, see p. 18-19 for a description of this phenom-

Terminally differentiated trophoblast giant cells derived from ES cells, showing large, flat
morphologies and polyploidy

44 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report45around E8.0, prior to somitogenesis, and early
embryonic lethality by E9.5. These loss-of-function
defects indicate that the establishment of the DNA
methylation state plays an important part in many
aspects of mouse development, an area the Okano
lab will continue to explore.

Publications
Dodge JE, Ramsahoye BH,
Wo ZG, Okano M and Li E.
De novo methylation of
MMLV provirus in embryonicstem cells: CpG versus nonCpG methylation. Gene
289:41-8 (2002).

The establishment of the
DNA methylation state plays animportant part in many aspectsof mouse development
Staff
Team Leader

Masaki Okano
Research Scientist

Shin-ichiro Takebayashi
Masaaki Oda
Technical Staff

Chisa Matsuoka
Akiko Yamagiwa
Makiko Okano
Student Trainee

Takao Nakayama
Akiko Tsumura
Assistant

Fumika Nakayama

Creative Research
Promoting Program

Masaki Okano
Ph. D.
Masaki Okano received his
baccalaureate and master’s
degrees from Kyoto University, and his doctorate inbiochemistry from the
Research Division in Agriculture at the same institution in 1994. He spent theperiod from 1994 to 1995
as a research fellow in cellbiology at the Nagoya University BioScience Center
before moving to Massachusetts, where he worked asa research fellow in medicine at Harvard Medical
School and Massachusetts
General Hospital from 1995
to 2002, serving as an
instructor in medicine at
Harvard for the last two
years of that period. He
was appointed team leader
at the RIKEN Center for
Developmental Biology in
2001, and returned to workin Japan full-time in 2002.

The methylation of mammalian DNA plays an important role in determining gene expression. The
number and locations of methyl tags provide molecular icons marking genes as inactive, or in somecases, active. Certain types of protein recognize andbind to the tagged DNA, affecting the expression patterns of the genes it encodes. During embryonicdevelopment, DNA methylation patterns are established in a sequence of steps involving both theremoval of methyl tags at the preimplantation stage and the establishment of new sets of tags, a process known as de novo methylation, which occurs after implantation and again during gametogenesis. These methylation patterns can subsequently be maintained
and transmitted acrossgenerations by maintenance methylation.

Dnmt3a and Dnmt3b

Methylation lays down developmentally significant
gene expression patterns that tend not to alter once
established. In previous work, Okano showed thatde novo methylation in mice required the expressionof a pair of methyltransferase genes, Dnmt3a andDnmt3b, at a very early stage of embryonic development, as well during gametogenesis. Prior to thisfinding, it was believed that a related gene, Dnmt1,
was solely responsible forthe methylation of DNA.
Using lacZ reporter
gene studies, Okano
showed that thesegenes are expressedin early embryonicand ES cells, suggesting that they functionin the epigenetic
reprogramming ofDNAmethylation
states. Based on
Masaki Okano's researchthese findings, it isconcentrates on the
now known that Dnmt1
mechanisms by which DNA
functions primarily in[Dnmt3a-/-,Dnmt3b-/-] mouse embryo
failed to turn and form somites.
methylation is establishedmethylation maintenance,
Mouse embryos at 9.5 d.p.c. Left; wild
and maintained throughout
which is necessary for thetype, Right; mutant
development, focusing on thestable inheritance of tissueproperties and activity of the Dnmt family of methylspecific methylation patterns, but that Dnmt3a andtransferases, which includes Dnmt1, Dnmt3a andDnmt3b are the principal determinants in initiating
Dnmt3b. He particularly focuses on working out andDNA methylation. Recent work has also shown thatclarifying the roles of de novo methyltransferases in
isoforms of Dnmt3a and Dnmt3b function as mainembryogenesis and cell differentiation, seeing thesetenance methyltransferases in mouse embryonic
enzymes as necessary agents in maintaining thestem cells, indicating a broader role for these proproper balance between stability and plasticity in theteins.
expression of specific genes.

46 RIKEN CDB 2003 Annual Report

Okano M and Li E. Genetic
analyses of DNA methyltransferase genes in mouse model system. J Nutr 132:2462S5S (2002).

Abnormal methylation has been implicated in a
number of human congenital anomalies as well. ICF(Immunodeficiency-Centromeric instability-Facial
anomalies) syndrome, which is characterized byimmune deficiency, heterochromatin instability anddefects in facial development, results from a mutation in the gene encoding Dnmt3b. A mutation in thegene encoding the methyl-cytosine binding protein,
MeCP2, is also known to cause Rett syndrome, aneurological disorder that occurs almost entirely infemales. In this form of mental retardation, development appears normal until the child is 6-18 months
old, after which the child loses communication skills
and develops disturbances in gait and use of thehands and slowed growth of the head.

As de novo methylation activity takes place mainlyin embryonic stem (ES) cells, early postimplantationembryos and gametogenesis, ES cells provide ahighly suitable experimental model system for investigating the molecular mechanisms underlying theestablishment of new methylation patterns. Okano is
studying the consequences of conditional knockout
and overexpression of methyltransferase genes onmouse development using ES cells and transgenic
mice, in the hopes of uncovering links with tumorigenesis, congenital nervous system defects and abnormal cell differentiation.

Hata K, Okano M, Lei H and
Li E. Dnmt3L cooperates
with the Dnmt3 family of denovo DNA methyltransferases to establish maternal
imprints in mice. Development 129:1983-93 (2002).
Okano M, Bell DW, Haber
DA and Li E. DNA methyltransferases Dnmt3a andDnmt3b are essential for denovo methylation and mammalian development. Cell
99:247-57 (1999).

In the future, the Okano lab will study how epigenetic reprogramming works at a molecular level. By
identifying the genes involved in de novo DNA methylation, they hope to discover the means by which itis determined what genes will be methylated. Comparisons of the expression patterns of such genes
across germ layers, or in normal mice against
clones created from somatically derived cells should
lead to new insights in the context-specificity of the
DNA methylation process as well as the roles ofmethylation in development, health, and regeneration.

Developmentalroles ofmethylation
Knockout mutations ofDnmt3a and Dnmt3b
result in a spectrum oforganic, neurologicaland spermatic defects.
Dnmt3b mutants generally die by embryonicstage 16.5 due to widespread defects affecting
organ development andgeneral growth. Loss of
Dnmt3a function produces less pronounced
effects; embryogenesis
appears normal, but
most mutants die by the
fourth week after birthand show abnormal
growth, intestinal motility and spermatogenesis. The deletion of bothgenes results in developmental arrest at

Dnmt3b expression in the embryonic ectoderm and the chorion shown by IRES-βgeo marker driven by theendogenous Dnmt3b promoter.

RIKEN CDB 2003 Annual Report47

around E8.0, prior to somitogenesis, and early
embryonic lethality by E9.5. These loss-of-function
defects indicate that the establishment of the DNA
methylation state plays an important part in many
aspects of mouse development, an area the Okano
lab will continue to explore.

Publications
Dodge JE, Ramsahoye BH,
Wo ZG, Okano M and Li E.
De novo methylation of
MMLV provirus in embryonicstem cells: CpG versus nonCpG methylation. Gene
289:41-8 (2002).

The establishment of the
DNA methylation state plays animportant part in many aspectsof mouse development
Staff
Team Leader

Masaki Okano
Research Scientist

Shin-ichiro Takebayashi
Masaaki Oda
Technical Staff

Chisa Matsuoka
Akiko Yamagiwa
Makiko Okano
Student Trainee

Takao Nakayama
Akiko Tsumura
Assistant

Fumika Nakayama

Creative Research
Promoting Program

Masaki Okano
Ph. D.
Masaki Okano received his
baccalaureate and master’s
degrees from Kyoto University, and his doctorate inbiochemistry from the
Research Division in Agriculture at the same institution in 1994. He spent theperiod from 1994 to 1995
as a research fellow in cellbiology at the Nagoya University BioScience Center
before moving to Massachusetts, where he worked asa research fellow in medicine at Harvard Medical
School and Massachusetts
General Hospital from 1995
to 2002, serving as an
instructor in medicine at
Harvard for the last two
years of that period. He
was appointed team leader
at the RIKEN Center for
Developmental Biology in
2001, and returned to workin Japan full-time in 2002.

The methylation of mammalian DNA plays an important role in determining gene expression. The
number and locations of methyl tags provide molecular icons marking genes as inactive, or in somecases, active. Certain types of protein recognize andbind to the tagged DNA, affecting the expression patterns of the genes it encodes. During embryonicdevelopment, DNA methylation patterns are established in a sequence of steps involving both theremoval of methyl tags at the preimplantation stage and the establishment of new sets of tags, a process known as de novo methylation, which occurs after implantation and again during gametogenesis. These methylation patterns can subsequently be maintained
and transmitted acrossgenerations by maintenance methylation.

Dnmt3a and Dnmt3b

Methylation lays down developmentally significant
gene expression patterns that tend not to alter once
established. In previous work, Okano showed thatde novo methylation in mice required the expressionof a pair of methyltransferase genes, Dnmt3a andDnmt3b, at a very early stage of embryonic development, as well during gametogenesis. Prior to thisfinding, it was believed that a related gene, Dnmt1,
was solely responsible forthe methylation of DNA.
Using lacZ reporter
gene studies, Okano
showed that thesegenes are expressedin early embryonicand ES cells, suggesting that they functionin the epigenetic
reprogramming ofDNAmethylation
states. Based on
Masaki Okano's researchthese findings, it isconcentrates on the
now known that Dnmt1
mechanisms by which DNA
functions primarily in[Dnmt3a-/-,Dnmt3b-/-] mouse embryo
failed to turn and form somites.
methylation is establishedmethylation maintenance,
Mouse embryos at 9.5 d.p.c. Left; wild
and maintained throughout
which is necessary for thetype, Right; mutant
development, focusing on thestable inheritance of tissueproperties and activity of the Dnmt family of methylspecific methylation patterns, but that Dnmt3a andtransferases, which includes Dnmt1, Dnmt3a andDnmt3b are the principal determinants in initiating
Dnmt3b. He particularly focuses on working out andDNA methylation. Recent work has also shown thatclarifying the roles of de novo methyltransferases in
isoforms of Dnmt3a and Dnmt3b function as mainembryogenesis and cell differentiation, seeing thesetenance methyltransferases in mouse embryonic
enzymes as necessary agents in maintaining thestem cells, indicating a broader role for these proproper balance between stability and plasticity in theteins.
expression of specific genes.

46 RIKEN CDB 2003 Annual Report

Okano M and Li E. Genetic
analyses of DNA methyltransferase genes in mouse model system. J Nutr 132:2462S5S (2002).

Abnormal methylation has been implicated in a
number of human congenital anomalies as well. ICF(Immunodeficiency-Centromeric instability-Facial
anomalies) syndrome, which is characterized byimmune deficiency, heterochromatin instability anddefects in facial development, results from a mutation in the gene encoding Dnmt3b. A mutation in thegene encoding the methyl-cytosine binding protein,
MeCP2, is also known to cause Rett syndrome, aneurological disorder that occurs almost entirely infemales. In this form of mental retardation, development appears normal until the child is 6-18 months
old, after which the child loses communication skills
and develops disturbances in gait and use of thehands and slowed growth of the head.

As de novo methylation activity takes place mainlyin embryonic stem (ES) cells, early postimplantationembryos and gametogenesis, ES cells provide ahighly suitable experimental model system for investigating the molecular mechanisms underlying theestablishment of new methylation patterns. Okano is
studying the consequences of conditional knockout
and overexpression of methyltransferase genes onmouse development using ES cells and transgenic
mice, in the hopes of uncovering links with tumorigenesis, congenital nervous system defects and abnormal cell differentiation.

Hata K, Okano M, Lei H and
Li E. Dnmt3L cooperates
with the Dnmt3 family of denovo DNA methyltransferases to establish maternal
imprints in mice. Development 129:1983-93 (2002).
Okano M, Bell DW, Haber
DA and Li E. DNA methyltransferases Dnmt3a andDnmt3b are essential for denovo methylation and mammalian development. Cell
99:247-57 (1999).

In the future, the Okano lab will study how epigenetic reprogramming works at a molecular level. By
identifying the genes involved in de novo DNA methylation, they hope to discover the means by which itis determined what genes will be methylated. Comparisons of the expression patterns of such genes
across germ layers, or in normal mice against
clones created from somatically derived cells should
lead to new insights in the context-specificity of the
DNA methylation process as well as the roles ofmethylation in development, health, and regeneration.

Developmentalroles ofmethylation
Knockout mutations ofDnmt3a and Dnmt3b
result in a spectrum oforganic, neurologicaland spermatic defects.
Dnmt3b mutants generally die by embryonicstage 16.5 due to widespread defects affecting
organ development andgeneral growth. Loss of
Dnmt3a function produces less pronounced
effects; embryogenesis
appears normal, but
most mutants die by the
fourth week after birthand show abnormal
growth, intestinal motility and spermatogenesis. The deletion of bothgenes results in developmental arrest at

Dnmt3b expression in the embryonic ectoderm and the chorion shown by IRES-βgeo marker driven by theendogenous Dnmt3b promoter.

RIKEN CDB 2003 Annual Report

47

pA

A

intron

(a)

1E Cβ

(b)

1E Cβ

1 kb

EGFP

pA

B

pA

intron

(c)

EGFP

EGFP

EGFP

(e)

(d)

pA

intronintron

mRNA

pA

pA

pA

EGFP

C

intronintron
1E Cβ

cRNA1

(f)

EGFP

cRNA2

dsRNA

mRNA

intronintronintron
EGFP

EGFP

EGFP

cRNA

EGFP

pA

(g)
dsRNA

Staff
Team Leader

Technical Staff

AssistantTony Perry

Satoko Fujimoto
Shaoyun Wang
Manami Amanai

Yayoi Ikeda

Research Scientist

Hisashi Kishi
Naoko Yoshida
Shisako Shoji

A schematic of RNAi by transgenesis

Creative Research
Promoting Program
Molecular identities of PNM proteins

Tony Perry
Ph. D.
Tony Perry received his B.Sc.
in Microbiology from the
Department of Bacteriology at
the University of Bristol, England and his doctorate fromthe University of Liverpool
four years later. In 1989 hebecame a postdoctoral fellow
working on epididymal sperm
maturation at Bristol University and in 1996 won a European Molecular Biology Travel Fellowship to work in thelaboratory of Ryuzo Yanagimachi on the mechanism ofoocyte activation, which
remains one of his researchinterests. From there, Dr. Perry moved first to the Rockefeller University and then to thecompany Advanced Cell Technology, working primarily on
novel methods of genome
manipulation. In 2002 Dr. Perry took his present position as
Team Leader of the Laboratory of Mammalian Molecular
Embryology at the RIKEN
CDB, where he works onmechanisms in mammalianpreimplantation embryos.

By mechanisms still unknown, fertilization achieves the transformationof two highly specialized cells —a sperm and an unfertilized egg(oocyte) — to a state of totipotency: a single-cell embryo that cangive rise to an entire individual.
The first moments of this remarkable process are known as oocyte
activation, an intricate orchestrationof sub-cellular events that include all
the checks and balances that presage
healthy growth of a new embryo. Oocyte
activation can be observed at the lightmicroscope level as dramatic change in themorphology of the newly fertilized egg andprovides an incredible read-out of the underlying molecular mechanisms. The Laboratory ofMammalian Molecular Embryology combines
molecular and cellular biology with piezoactuated micromanipulation of mouse (Mus musculus) gametes and embryos to study oocyte activation and its developmental consequences.

Interactions between
gamete cytoplasms
Microinjection of an isolated sperm head into an
oocyte can result in the birth of normal young.
Thus, the sperm head — the part of the headthat lies beneath its membranes — apparently
contains all of the paternal information sufficientfor full development. Tony Perry is systematicallyelucidating interactions between sub-membrane
sperm head components and the oocyte at fertilization, particularly during the moments soonafter the sperm has entered the oocyte. The Per-ry lab's interests center on attributing
molecular identities to the proteins
involved in these interactions andcharacterizing them functionally.
This task is large, because
beneath the membrane of a
sperm head reside macromolecular complexes that include anucleus (containing the paternal genome and associated proteins) and asurrounding cytoplasmic matrix, the perinuclear matrix (PNM). It has been estimated that the PNM comprises some 230 protein species. Because it is juxtaposed to theinner leaflet of enshrouding sperm head membranes, the PNM rapidly comes into contact
with the oocyte cytoplasm at fertilization and is
an immediate source of paternally-contributed
molecules that potentially modulate development. However, owing in part to its size and complexity, its excoriation and disassembly of thePNM takes hours to complete within the oocyte
activation period. During this time, it is likely torelease molecules that may be placed in one ofseveral categories, according to whether they (i)have an immediate function in neoembryogenesis, (ii) are stored for later use, or (iii) are targeted for rapid proteolytic degradation. Each category implies an intimate series of interactions
with the surrounding cytoplasm. However, todate, few proteins of the PNM have been
described, and the function of almost none ofthem is known. This represents an extraordinary
gap in the understanding of a conserved biological structure.

Protein id

Role

Reference

Calicin (PT60)

F-actin-interacting; provides rigidity Longo et al., 1987

Cylicin I

Role unknown

Hess et al., 1993

Cylicin II

Role unknown

Hess et al., 1995

Arp-T1

Actin-related; role unknown

Heid et al., 2002

Arp-T2

Actin-related; role unknown

Heid et al., 2002

SubH2Bv

Histone H2B variant

Aul and Oko, 2002

PERF15

Lipophilic transport protein-related;role unknown

Oko and Morales, 1994

Stat4

Jak-Stat signaling; role unknown

Herrada & Wolgemuth, 1997

c-Yes

Protein tyrosine kinase ;role unknown

Leclerc and Goupil, 2002

PT32

WBP2, a c-Yes BP;
role unknown

Gong et al., 1997

p30

Calmodulin BP; role unknown

Leclerc and Goupil, 2000

The Perry team's analysis of sperm-oocyte interactions at fertilization begins with demembranatedsperm that are subsequently exposed to standardized conditions that recapitulate key aspects of theoocyte cytoplasm. Proteins solubilized in this way
can be injected into eggs, typically by piezoactuated microinjection, to probe their function.
Such functional analysis is coupled to molecularanalyses that span several disciplines. The proteinscan be purified and identified; Perry's team employs
chromatographic and state-of-the art 2D electrophoretic methods to this end. Antibodies raised against
peptides identified in this way are used to localize
the proteins before, during and after fertilization; antibody function can also be assayed by microinjection. Owing to dissimilarities between frog and mammalian oocytes and the paucity of material obtainable from the latter, genetic methods are used to
identify mouse oocyte proteins that interact with thesperm proteins that have been characterized. Collectively, these studies hold forth the promise of enabling the identification of oocyte signaling pathways
and processes that become operational at fertiliza-tion and establish what roles they play in subsequent development. Such roles may not be restricted to short-term development, as a growing body ofevidence indicates that they have far-reaching consequences, even after the resulting adult is in oldage.

Publications
Perry AC and Wakayama T.
Untimely ends and new
beginnings in mouse cloning.
Nat Genet 30:243-4 (2002).

Novel methods of genetic
modulation using gametes

Perry AC, Rothman A, de las
Heras JI, Feinstein P, Mombaerts P, Cooke HJ and
Wakayama T. Efficient metaphase II transgenesis with different transgene archetypes.
Nat Biotechnol 19:1071-3(2001).

Utilizing the integrated molecular, cellular, andembryological approach from the work in his laboratory, Perry expects to gain insights that will enable
us to genetically alter embryos in new ways. He hasdeveloped a novel method of genome manipulation
known as metaphase II (mII) transgenesis. This efficient method of transgenesis works when oocytes(normally arrested at mII) are co-injected with anucleus and transgene (tg) DNA. Typically, sperm

Perry AC. Hijacking oocyte
DNA repair machinery intransgenesis? Mol Reprod
Dev 56:319-24 (2000).

To date, few PNM proteins
have been described, and thefunction of almost none ofthem is known

Perry AC, Wakayama T,
Cooke IM and Yanagimachi
R. Mammalian oocyte activation by the synergistic actionof discrete sperm head components: induction of calcium
transients and involvementof proteolysis. Dev Biol
217:386-93 (2000).

heads are depleted of their membranes by detergent extraction prior to mixing with tg DNA andco-injection. This has several advantages over lentiviral methods of tg introduction; cloning and propagation in viral vectors is not required and the method
lends itself to tgs in the megabase range, which is
too large for lentiviral delivery. Perry and membersof his lab are adapting the method to facilitate highthroughput targeted knock-out and -down phenotypes. Developing these approaches is an important supplementary part of their work to facilitate themolecular dissection of sperm protein function in thecontext of embryos and whole animals.

Perry AC, Wakayama T,
Kishikawa H, Kasai T, Okabe
M, Toyoda Y and Yanagimachi R. Mammalian transgenesis by intracytoplasmic
sperm injection. Science
284:1180-3 (1999).

An early model of the sperm-borne oocyte activating factor

48 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report

49

pA

A

intron

(a)

1E Cβ

(b)

1E Cβ

1 kb

EGFP

pA

B

pA

intron

(c)

EGFP

EGFP

EGFP

(e)

(d)

pA

intronintron

mRNA

pA

pA

pA

EGFP

C

intronintron
1E Cβ

cRNA1

(f)

EGFP

cRNA2

dsRNA

mRNA

intronintronintron
EGFP

EGFP

EGFP

cRNA

EGFP

pA

(g)
dsRNA

Staff
Team Leader

Technical Staff

AssistantTony Perry

Satoko Fujimoto
Shaoyun Wang
Manami Amanai

Yayoi Ikeda

Research Scientist

Hisashi Kishi
Naoko Yoshida
Shisako Shoji

A schematic of RNAi by transgenesis

Creative Research
Promoting Program
Molecular identities of PNM proteins

Tony Perry
Ph. D.
Tony Perry received his B.Sc.
in Microbiology from the
Department of Bacteriology at
the University of Bristol, England and his doctorate fromthe University of Liverpool
four years later. In 1989 hebecame a postdoctoral fellow
working on epididymal sperm
maturation at Bristol University and in 1996 won a European Molecular Biology Travel Fellowship to work in thelaboratory of Ryuzo Yanagimachi on the mechanism ofoocyte activation, which
remains one of his researchinterests. From there, Dr. Perry moved first to the Rockefeller University and then to thecompany Advanced Cell Technology, working primarily on
novel methods of genome
manipulation. In 2002 Dr. Perry took his present position as
Team Leader of the Laboratory of Mammalian Molecular
Embryology at the RIKEN
CDB, where he works onmechanisms in mammalianpreimplantation embryos.

By mechanisms still unknown, fertilization achieves the transformationof two highly specialized cells —a sperm and an unfertilized egg(oocyte) — to a state of totipotency: a single-cell embryo that cangive rise to an entire individual.
The first moments of this remarkable process are known as oocyte
activation, an intricate orchestrationof sub-cellular events that include all
the checks and balances that presage
healthy growth of a new embryo. Oocyte
activation can be observed at the lightmicroscope level as dramatic change in themorphology of the newly fertilized egg andprovides an incredible read-out of the underlying molecular mechanisms. The Laboratory ofMammalian Molecular Embryology combines
molecular and cellular biology with piezoactuated micromanipulation of mouse (Mus musculus) gametes and embryos to study oocyte activation and its developmental consequences.

Interactions between
gamete cytoplasms
Microinjection of an isolated sperm head into an
oocyte can result in the birth of normal young.
Thus, the sperm head — the part of the headthat lies beneath its membranes — apparently
contains all of the paternal information sufficientfor full development. Tony Perry is systematicallyelucidating interactions between sub-membrane
sperm head components and the oocyte at fertilization, particularly during the moments soonafter the sperm has entered the oocyte. The Per-ry lab's interests center on attributing
molecular identities to the proteins
involved in these interactions andcharacterizing them functionally.
This task is large, because
beneath the membrane of a
sperm head reside macromolecular complexes that include anucleus (containing the paternal genome and associated proteins) and asurrounding cytoplasmic matrix, the perinuclear matrix (PNM). It has been estimated that the PNM comprises some 230 protein species. Because it is juxtaposed to theinner leaflet of enshrouding sperm head membranes, the PNM rapidly comes into contact
with the oocyte cytoplasm at fertilization and is
an immediate source of paternally-contributed
molecules that potentially modulate development. However, owing in part to its size and complexity, its excoriation and disassembly of thePNM takes hours to complete within the oocyte
activation period. During this time, it is likely torelease molecules that may be placed in one ofseveral categories, according to whether they (i)have an immediate function in neoembryogenesis, (ii) are stored for later use, or (iii) are targeted for rapid proteolytic degradation. Each category implies an intimate series of interactions
with the surrounding cytoplasm. However, todate, few proteins of the PNM have been
described, and the function of almost none ofthem is known. This represents an extraordinary
gap in the understanding of a conserved biological structure.

Protein id

Role

Reference

Calicin (PT60)

F-actin-interacting; provides rigidity Longo et al., 1987

Cylicin I

Role unknown

Hess et al., 1993

Cylicin II

Role unknown

Hess et al., 1995

Arp-T1

Actin-related; role unknown

Heid et al., 2002

Arp-T2

Actin-related; role unknown

Heid et al., 2002

SubH2Bv

Histone H2B variant

Aul and Oko, 2002

PERF15

Lipophilic transport protein-related;role unknown

Oko and Morales, 1994

Stat4

Jak-Stat signaling; role unknown

Herrada & Wolgemuth, 1997

c-Yes

Protein tyrosine kinase ;role unknown

Leclerc and Goupil, 2002

PT32

WBP2, a c-Yes BP;
role unknown

Gong et al., 1997

p30

Calmodulin BP; role unknown

Leclerc and Goupil, 2000

The Perry team's analysis of sperm-oocyte interactions at fertilization begins with demembranatedsperm that are subsequently exposed to standardized conditions that recapitulate key aspects of theoocyte cytoplasm. Proteins solubilized in this way
can be injected into eggs, typically by piezoactuated microinjection, to probe their function.
Such functional analysis is coupled to molecularanalyses that span several disciplines. The proteinscan be purified and identified; Perry's team employs
chromatographic and state-of-the art 2D electrophoretic methods to this end. Antibodies raised against
peptides identified in this way are used to localize
the proteins before, during and after fertilization; antibody function can also be assayed by microinjection. Owing to dissimilarities between frog and mammalian oocytes and the paucity of material obtainable from the latter, genetic methods are used to
identify mouse oocyte proteins that interact with thesperm proteins that have been characterized. Collectively, these studies hold forth the promise of enabling the identification of oocyte signaling pathways
and processes that become operational at fertiliza-tion and establish what roles they play in subsequent development. Such roles may not be restricted to short-term development, as a growing body ofevidence indicates that they have far-reaching consequences, even after the resulting adult is in oldage.

Publications
Perry AC and Wakayama T.
Untimely ends and new
beginnings in mouse cloning.
Nat Genet 30:243-4 (2002).

Novel methods of genetic
modulation using gametes

Perry AC, Rothman A, de las
Heras JI, Feinstein P, Mombaerts P, Cooke HJ and
Wakayama T. Efficient metaphase II transgenesis with different transgene archetypes.
Nat Biotechnol 19:1071-3(2001).

Utilizing the integrated molecular, cellular, andembryological approach from the work in his laboratory, Perry expects to gain insights that will enable
us to genetically alter embryos in new ways. He hasdeveloped a novel method of genome manipulation
known as metaphase II (mII) transgenesis. This efficient method of transgenesis works when oocytes(normally arrested at mII) are co-injected with anucleus and transgene (tg) DNA. Typically, sperm

Perry AC. Hijacking oocyte
DNA repair machinery intransgenesis? Mol Reprod
Dev 56:319-24 (2000).

To date, few PNM proteins
have been described, and thefunction of almost none ofthem is known

Perry AC, Wakayama T,
Cooke IM and Yanagimachi
R. Mammalian oocyte activation by the synergistic actionof discrete sperm head components: induction of calcium
transients and involvementof proteolysis. Dev Biol
217:386-93 (2000).

heads are depleted of their membranes by detergent extraction prior to mixing with tg DNA andco-injection. This has several advantages over lentiviral methods of tg introduction; cloning and propagation in viral vectors is not required and the method
lends itself to tgs in the megabase range, which is
too large for lentiviral delivery. Perry and membersof his lab are adapting the method to facilitate highthroughput targeted knock-out and -down phenotypes. Developing these approaches is an important supplementary part of their work to facilitate themolecular dissection of sperm protein function in thecontext of embryos and whole animals.

Perry AC, Wakayama T,
Kishikawa H, Kasai T, Okabe
M, Toyoda Y and Yanagimachi R. Mammalian transgenesis by intracytoplasmic
sperm injection. Science
284:1180-3 (1999).

An early model of the sperm-borne oocyte activating factor

48 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report49term. However, a limited number of such headlessbirth phenotypes have been reported, all of whichhave been linked to mutations in node-related
genes, such as Lim1 and Otx2. The fortuitous discovery of a previously unknown headless phenotype
in the mouse prompted Sasaki to investigate thegenetic cause of this mutation, which he named
headshrinker (hsk), and to study the gene's role in
the induction of the head. Gene expression analysis
revealed losses in the expression of important headorganizing genes in hsk mutants, resulting in the
impaired function of the late-stage head organizer or
the prechordal plate, a fore/midbrain-inducing
mesenchymal mass that forms at the anterior end of
the developing embryo.

Staff
Team Leader

Hiroshi Sasaki
Research Scientist

Atsushi Sawada
Shinji Yamamoto
Yukari Yada (2004)
Visiting Scientist

Noriyuki Nishioka
Technical Staff

Hiroko Sato
Yayoi Minami
Yuuri Tsuboi
Student Trainee

Norifumi Namekawa
Assistant

Misaki Harano

Creative Research
Promoting Program

Hiroshi Sasaki
Ph. D.
Hiroshi Sasaki received his
B. Sc. in zoology from the
University of Tokyo, and his
master’s and Ph. D. indevelopmental biology fromthe same institution. He
worked as a research associate in Atsushi Kuroiwa’s
lab at Tohoku University
from 1990 to 1992, and in
Brigid Hogan’s lab at
Vanderbilt University from1992 to 1994. He returned
to Japan to take a positionas assistant professor at
Osaka University beginning
in
1995,
where
he
remained until his appointment as team leader of theLaboratory for Embryonic
Induction at the RIKEN Center for Developmental Biology.

Signaling centers inmammalian development

Activation of nodal
differentiation

The earliest stages of mouse embryogenesis playout over the course of a few days as a series ofdynamic transformations from a simple, nearly featureless blastocyst to a recognizable vertebratebody by the end of gastrulation. This rapid and striking transition is achieved by the highly coordinateddifferentiation of cells toward specific fates, a featorchestrated by a number of organizing loci, collectively known as ‘signaling centers.’ These centersproduce diffusible proteins that serve to regulate
embryonic development, guiding neighboring cellcommunities to take up the roles they will play in theadult animal, or steering them away from inappropriate fates.

Foxa2 (also known as Hepatocyte Nuclear Factor
3β, HNF3β) is a highly-conserved transcription factor required for the formation of embryonic signaling
centers in the mouse. The Sasaki lab is engaged in
an analysis of molecules that activate Foxa2 expression in both node and notochord. Members of the
Sasaki team previously identified the key regulatory
element of the Foxa2 enhancer, CS3, and have now
determined that this element binds to a member of
the TEAD/TEF family of proteins, four transcription
factors found in both mice and humans, which share
a common DNA-binding domain. TEAD proteins arewidely expressed in the mouse embryo and theiractivity is regulated by interaction with multiple transcription factors and co-activators. Complementarystudies of TEAD activation showed that increases ordecreases in TEAD activation resulted in corre-

These recent findings suggest a
new model toexplain node induction

Foxa2 (HNF3ß) is a key regulator of the signaling centers, nodeand notochord in mouse embryos.

headshrinker
headshrinker mutant lacks head induction by the signaling center, andshows headless phenotype at birth (left).

Publications
Sekimizu K, Nishioka N,
Sasaki H, Takeda H, Karlstrom R and Kawakami A.
The zebrafish iguana locus
encodes Dzip1, a novel zincfinger protein required forproper regulation of hedgehog signaling. Development(2004).
Karlstrom RO, Tyurina OV,
Kawakami A, Nishioka N, Talbot WS, Sasaki H and Schier
AF. Genetic analysis ofzebrafish gli1 and gli2
reveals divergent requirements for gli genes in vertebrate
development.
Development 130:1549-64(2003).

been demonstrated in zebrafish, indicating that thispathway has been conserved in at least some othervertebrate taxa as well. A second signaling factor
known to work in node induction, Nodal, has also
been linked to TEAD activity. These recent findings
suggest a new model to explain node induction, in
which TEAD complements the function of multiple
activating co-factors from primary signaling centersby activating the enhancer element CS3 to induce
Foxa2 expression.

Hiroshi Sasaki’s research team investigates the network of signaling centers that work to induce normal
development in mammals. The node, a center
responsible for induction of body parts in the mouse,
is a particular focus of Sasaki’s research. He has
identified a number of transcription factors involvedin the formation and the function of the node in previous studies, and he remains intent on solving questions of how these centers are formed, and how they
set courses for the developing embryo to follow.

50 RIKEN CDB 2003 Annual Reportsponding increases or decreases in the area of Foxa2 expression. The Foxa2 enhancer element CS3(which binds with TEAD) functions downstream ofWnt, a known node-inducing signal and the synergistic activation of Foxa2 by TEAD and Wnt has also

Northern blotting showed reduced levels in theexpression of the mRNA for the gene Ssdp1 (asequence-specific, single-stranded-DNA-binding protein) in headshrinker mutants, and the researchers
found that they could rescue the headless phenotype by using Ssdp1cDNA. Ssdp1/hsk encodes aprotein that binds with Ldb1, which itself binds LIMhomeodomain proteins and other developmentally
important transcription factors. Sasaki proposes amodel for the interaction of these three co-factors inwhich Sspd1 enhances the activation of target
genes by the Lim1-Ldb1 complex. In an analysis
using a labeled reporter gene, Sspd1 alone showedalmost no activity, but boosted the activity of both
Lim1 and Ldb1 on co-expression, and had the strongest effect when all three factors were present. In
addition to the lethal loss of the head, hsk mutants

Sheng H, Goich S, Wang A,
Grachtchouk M, Lowe L, Mo
R, Lin K, de Sauvage FJ,
Sasaki H, Hui CC and Dlugosz AA. Dissecting theoncogenic potential of Gli2:
deletion of an NH(2)-terminal
fragment alters skin tumor
phenotype. Cancer Res
62:5308-16 (2002).

exhibit a range of other developmental abnormalities, implicating Ssdp1 as a co-activator moleculewith multiple and diverse embryogenetic roles.
Sasaki continues to pursue a deeper understandingof the structure and function of signaling centers
using an array of approaches and techniques. Other
ongoing work in the lab is aimed at isolating cells
from transgenic mice engineered to express LacZ in
the node and notochord, which promises to speed
the identification of new genes specifically
expressed in those regions.

Dai P, Shinagawa T, Nomura
T, Harada J, Kaul SC, Wadhwa R, Khan MM, Akimaru H,
Sasaki H, Colmenares C and
Ishii S. Ski is involved in transcriptional regulation by the
repressor and full-length
forms of Gli3. Genes Dev
16:2843-8 (2002).

Although a variety of mutations can cause the headto fail to develop properly, it is rare for embryos inwhich the head is entirely lacking to develop to full

RIKEN CDB 2003 Annual Report51term. However, a limited number of such headlessbirth phenotypes have been reported, all of whichhave been linked to mutations in node-related
genes, such as Lim1 and Otx2. The fortuitous discovery of a previously unknown headless phenotype
in the mouse prompted Sasaki to investigate thegenetic cause of this mutation, which he named
headshrinker (hsk), and to study the gene's role in
the induction of the head. Gene expression analysis
revealed losses in the expression of important headorganizing genes in hsk mutants, resulting in the
impaired function of the late-stage head organizer or
the prechordal plate, a fore/midbrain-inducing
mesenchymal mass that forms at the anterior end of
the developing embryo.

Staff
Team Leader

Hiroshi Sasaki
Research Scientist

Atsushi Sawada
Shinji Yamamoto
Yukari Yada (2004)
Visiting Scientist

Noriyuki Nishioka
Technical Staff

Hiroko Sato
Yayoi Minami
Yuuri Tsuboi
Student Trainee

Norifumi Namekawa
Assistant

Misaki Harano

Creative Research
Promoting Program

Hiroshi Sasaki
Ph. D.
Hiroshi Sasaki received his
B. Sc. in zoology from the
University of Tokyo, and his
master’s and Ph. D. indevelopmental biology fromthe same institution. He
worked as a research associate in Atsushi Kuroiwa’s
lab at Tohoku University
from 1990 to 1992, and in
Brigid Hogan’s lab at
Vanderbilt University from1992 to 1994. He returned
to Japan to take a positionas assistant professor at
Osaka University beginning
in
1995,
where
he
remained until his appointment as team leader of theLaboratory for Embryonic
Induction at the RIKEN Center for Developmental Biology.

Signaling centers inmammalian development

Activation of nodal
differentiation

The earliest stages of mouse embryogenesis playout over the course of a few days as a series ofdynamic transformations from a simple, nearly featureless blastocyst to a recognizable vertebratebody by the end of gastrulation. This rapid and striking transition is achieved by the highly coordinateddifferentiation of cells toward specific fates, a featorchestrated by a number of organizing loci, collectively known as ‘signaling centers.’ These centersproduce diffusible proteins that serve to regulate
embryonic development, guiding neighboring cellcommunities to take up the roles they will play in theadult animal, or steering them away from inappropriate fates.

Foxa2 (also known as Hepatocyte Nuclear Factor
3β, HNF3β) is a highly-conserved transcription factor required for the formation of embryonic signaling
centers in the mouse. The Sasaki lab is engaged in
an analysis of molecules that activate Foxa2 expression in both node and notochord. Members of the
Sasaki team previously identified the key regulatory
element of the Foxa2 enhancer, CS3, and have now
determined that this element binds to a member of
the TEAD/TEF family of proteins, four transcription
factors found in both mice and humans, which share
a common DNA-binding domain. TEAD proteins arewidely expressed in the mouse embryo and theiractivity is regulated by interaction with multiple transcription factors and co-activators. Complementarystudies of TEAD activation showed that increases ordecreases in TEAD activation resulted in corre-

These recent findings suggest a
new model toexplain node induction

Foxa2 (HNF3ß) is a key regulator of the signaling centers, nodeand notochord in mouse embryos.

headshrinker
headshrinker mutant lacks head induction by the signaling center, andshows headless phenotype at birth (left).

Publications
Sekimizu K, Nishioka N,
Sasaki H, Takeda H, Karlstrom R and Kawakami A.
The zebrafish iguana locus
encodes Dzip1, a novel zincfinger protein required forproper regulation of hedgehog signaling. Development(2004).
Karlstrom RO, Tyurina OV,
Kawakami A, Nishioka N, Talbot WS, Sasaki H and Schier
AF. Genetic analysis ofzebrafish gli1 and gli2
reveals divergent requirements for gli genes in vertebrate
development.
Development 130:1549-64(2003).

been demonstrated in zebrafish, indicating that thispathway has been conserved in at least some othervertebrate taxa as well. A second signaling factor
known to work in node induction, Nodal, has also
been linked to TEAD activity. These recent findings
suggest a new model to explain node induction, in
which TEAD complements the function of multiple
activating co-factors from primary signaling centersby activating the enhancer element CS3 to induce
Foxa2 expression.

Hiroshi Sasaki’s research team investigates the network of signaling centers that work to induce normal
development in mammals. The node, a center
responsible for induction of body parts in the mouse,
is a particular focus of Sasaki’s research. He has
identified a number of transcription factors involvedin the formation and the function of the node in previous studies, and he remains intent on solving questions of how these centers are formed, and how they
set courses for the developing embryo to follow.

50 RIKEN CDB 2003 Annual Reportsponding increases or decreases in the area of Foxa2 expression. The Foxa2 enhancer element CS3(which binds with TEAD) functions downstream ofWnt, a known node-inducing signal and the synergistic activation of Foxa2 by TEAD and Wnt has also

Northern blotting showed reduced levels in theexpression of the mRNA for the gene Ssdp1 (asequence-specific, single-stranded-DNA-binding protein) in headshrinker mutants, and the researchers
found that they could rescue the headless phenotype by using Ssdp1cDNA. Ssdp1/hsk encodes aprotein that binds with Ldb1, which itself binds LIMhomeodomain proteins and other developmentally
important transcription factors. Sasaki proposes amodel for the interaction of these three co-factors inwhich Sspd1 enhances the activation of target
genes by the Lim1-Ldb1 complex. In an analysis
using a labeled reporter gene, Sspd1 alone showedalmost no activity, but boosted the activity of both
Lim1 and Ldb1 on co-expression, and had the strongest effect when all three factors were present. In
addition to the lethal loss of the head, hsk mutants

Sheng H, Goich S, Wang A,
Grachtchouk M, Lowe L, Mo
R, Lin K, de Sauvage FJ,
Sasaki H, Hui CC and Dlugosz AA. Dissecting theoncogenic potential of Gli2:
deletion of an NH(2)-terminal
fragment alters skin tumor
phenotype. Cancer Res
62:5308-16 (2002).

exhibit a range of other developmental abnormalities, implicating Ssdp1 as a co-activator moleculewith multiple and diverse embryogenetic roles.
Sasaki continues to pursue a deeper understandingof the structure and function of signaling centers
using an array of approaches and techniques. Other
ongoing work in the lab is aimed at isolating cells
from transgenic mice engineered to express LacZ in
the node and notochord, which promises to speed
the identification of new genes specifically
expressed in those regions.

Dai P, Shinagawa T, Nomura
T, Harada J, Kaul SC, Wadhwa R, Khan MM, Akimaru H,
Sasaki H, Colmenares C and
Ishii S. Ski is involved in transcriptional regulation by the
repressor and full-length
forms of Gli3. Genes Dev
16:2843-8 (2002).

Although a variety of mutations can cause the headto fail to develop properly, it is rare for embryos inwhich the head is entirely lacking to develop to full

RIKEN CDB 2003 Annual Report51Factors in asymmetric
T cell division

Staff
Team Leader

Hitoshi Sawa
Research Scientist

Masaki Fujita
Masahiro Uchida
Yukimasa Shibata
Special Postdoctoral Researcher

Yukinobu Arata
Kumiko Oishi
Technical Staff

Hisako Takeshita
Noriko Sasakawa
Assistant

Tomoko Nakashima

Creative Research
Promoting Program

Asymmetric cell divisionand cell diversity

Hitoshi Sawa
Ph. D.
Hitoshi Sawa obtained his
baccalaureate, master’sand doctoral degrees from
Kyoto University in the period from 1982 to 1991. Heworked as a postdoctoral
fellow at the California Institute of Technology on a
Human Frontier Science
Program grant during theperiod from 1991 to 1994,
then at the Massachusetts
Institute of Technology from1994 to 1997 under support
from the Howard Hughes
MedicalInstitute.
Hereturned to Japan in 1997
to continue his work at Osaka University, first as a postdoc, then as researcher
funded by the Japan Science and Technology Corporation PRESTO program.
He took his current positionat the RIKEN CDB in 2001.

The animal body is constructed from the myriad andmultiform progeny cells from a single fertilized egg.
Asymmetric cell division, in which a single mothercell splits to generate daughter cells with discrete
characters, is the fundamental process by which cellular diversity is achieved, but remains imperfectly
understood at the molecular level. Using the nematode C. elegans as a model system, Hitoshi Sawa is
engaged in the study of the genetic and molecularinteractions that underlie asymmetric division, andthe related phenomenon of cellular polarity, in which
the contents of individual cells are distributed unevenly, giving distinct characters to daughter cells on
division. He seeks also to resolve the ways in whichtranscription-regulating complexes integrate theintracellular signaling networks that collaborate tohelp determine cell fate.

Ectopic expression of
LIN-44/Wnt anterior to the Tcell strongly reverses thepolarity of T cell division.

52 RIKEN CDB 2003 Annual Report

T cell polarity
The Wnt pathway represents one of the most highly
conserved and widely adapted signaling systems inbiology, playing developmentally critical roles in the regulation of cell fate, proliferation and polarity in species
from worm to human. Wnt signaling, in fact, transpires
along multiple divergent routes. In the canonical Wnt
pathway in Drosophila (the species in which it was firstdescribed), Wnt binds to the transmembrane receptor
Frizzled, which activates a series of molecules andmolecular complexes within the cell, thereby preventing the degradation of β-catenin and allowing it to accumulate and signal transcription factors that act on thepathway's ultimate downstream gene targets. Studying
the effects of lin-44 (a Wnt homolog) on asymmetric division in the nematode, the Sawa team ectopically
expressed and examined its effects on the division of T
cells, which form a diverse lineage of hypodermal andneural cells arising from a single progenitor. In normal
development, the anterior daughter of the parent T cell
produces hypodermal cells, while the posterior daugh-ter produces neural cells. In lin-44 mutants, however,
the opposite is often the case, with anterior daughtersfrequently giving rise to neural cells, and posterior
daughters generating cells in the hypodermal lineage.
This phenotype is attributable to a reversal in parent
cell polarity, which occurs in about 70% of lin-44
mutants. Misexpression of lin-44 at the anterior of the Tcell in lin-44 mutants caused a significant enhancementof the reversal phenotype; 97% of such mutants
showed switching of fates in T cell daughters. These
results demonstrate that the orientation of T cell polarity
is regulated by the LIN-44 signal.

Sawa is engaged
in the study of the genetic andmolecular interactions
that underlie asymmetric
division and cellular polarity
A pathway homologous to the Wnt cascade exists in
C. elegans, and it has been suggested that a
number of downstream factors corresponding tomany of the most prominent Wnt signaling components also play roles in roundworm asymmetric cell
division. POP-1, the C. elegans homolog of
TCF/LEF-1, a transcription factor acting downstreamof Wnt, has recently been shown to be required forasymmetric T cell division. In the canonical Wnt pathway, TCF/LEF-1 is a target of β-catenin, but thedirect involvement of β-catenin in C. elegans asymmetric cell division has never clearly been demonstrated. Researchers in the Sawa lab found that
wrm-1/β-catenin mutants show defects in asymmetric T cell division, similar to those observed in lin-17
mutants, suggesting that that the asymmetric cell
division is controlled by β -catenin.

Publications

Additional work has also focused on T hypodermal
cells, which in normal development divide asymmetrically to produce non-identical daughter cells of neural or hypodermal fate. Recent work by the Sawa
team has shown that three genes, psa-3, ceh-20
and nob-1 (homologs of Meis, Pbx and Hox, respectively) are involved in this asymmetric T mitosis, as
mutations in any of these genes result in the abnormal loss of asymmetry in cell divisions, in which theposterior daughters of the T cell are hypodermalrather than neural in character.

Zhao X, Sawa H and Herman MA. tcl-2 encodes a novel protein that acts
synergistically with Wnt signaling pathways in C. elegans. Dev Biol 256:276-89(2003).

Investigations into the role of PSA-3 uncovered that
expression of PSA-3 is stronger in the posterior T
cell daughter. psa-3 expression is dependent on aconsensus binding site within its promoter region for
the transcriptional factor POP-1/TCF, which has previously been reported to be required for asymmetric
T cell division, suggesting that the psa-3 gene is adirect target of Wnt signaling and functions as a cellfate determinant in asymmetric cell division. The
asymmetry of PSA-3/Meis expression is also perturbed in lin-44/Wnt and lin-17/Frizzled mutants, corroborating the argument for a downstream role for
psa-3 in the Wnt pathway.

Rocheleau CE, Yasuda J,
Shin TH, Lin R, Sawa H, Okano H, Priess JR, Davis RJ
and Mello CC. WRM-1 activates the LIT-1 protein
kinase to transduce anterior/posterior polarity signals
in C. elegans. Cell 97:717-26(1999).

Sawa H, Kouike H and Okano H. Components of the
SWI/SNF complex are
required for asymmetric cell
division in C. elegans. Mol
Cell 6:617-24 (2000).

Expression of psa-3/Meis in the T cell lineage is also
markedly reduced in nob-1/Hox and ceh-20/Pbx
mutants, indicating that, NOB-1/Hox and CEH20/Pbx also act in the regulation of psa-3/Meis
expression. Taken together, these data indicate thatthe expression of psa-3/Meis is controlled by binary
mechanisms; Wnt signaling establishes cellularpolarity, while the Hox-Pbx complex regulates positional identity. In other metazoans, both Meis andPbx are known to bind directly as co-factors to Hox,
raising the possibility that 20/Pbx and NOB-1/Hox
proteins may form a ternary complex with PSA3/Meis in C. elegans as well. In vitro studies showthat PSA-3/Meis interacts directly with CEH-20/Pbx
and NOB-1/Hox, lending support to the model inwhich PSA-3/Meis functions as a cell fate determinant by forming a complex with CEH-20/Pbx and
NOB-1/Hox in asymmetric cell division of the T cell.

Expression of PSA-3/Meis isdirectly induced byPOP-1/TCF. PSA-3 forms
complex with CEH-20/Pbx and
NOB-1/Hox to determine
asymmetric cell fates.

RIKEN CDB 2003 Annual Report53Factors in asymmetric
T cell division

Staff
Team Leader

Hitoshi Sawa
Research Scientist

Masaki Fujita
Masahiro Uchida
Yukimasa Shibata
Special Postdoctoral Researcher

Yukinobu Arata
Kumiko Oishi
Technical Staff

Hisako Takeshita
Noriko Sasakawa
Assistant

Tomoko Nakashima

Creative Research
Promoting Program

Asymmetric cell divisionand cell diversity

Hitoshi Sawa
Ph. D.
Hitoshi Sawa obtained his
baccalaureate, master’sand doctoral degrees from
Kyoto University in the period from 1982 to 1991. Heworked as a postdoctoral
fellow at the California Institute of Technology on a
Human Frontier Science
Program grant during theperiod from 1991 to 1994,
then at the Massachusetts
Institute of Technology from1994 to 1997 under support
from the Howard Hughes
MedicalInstitute.
Hereturned to Japan in 1997
to continue his work at Osaka University, first as a postdoc, then as researcher
funded by the Japan Science and Technology Corporation PRESTO program.
He took his current positionat the RIKEN CDB in 2001.

The animal body is constructed from the myriad andmultiform progeny cells from a single fertilized egg.
Asymmetric cell division, in which a single mothercell splits to generate daughter cells with discrete
characters, is the fundamental process by which cellular diversity is achieved, but remains imperfectly
understood at the molecular level. Using the nematode C. elegans as a model system, Hitoshi Sawa is
engaged in the study of the genetic and molecularinteractions that underlie asymmetric division, andthe related phenomenon of cellular polarity, in which
the contents of individual cells are distributed unevenly, giving distinct characters to daughter cells on
division. He seeks also to resolve the ways in whichtranscription-regulating complexes integrate theintracellular signaling networks that collaborate tohelp determine cell fate.

Ectopic expression of
LIN-44/Wnt anterior to the Tcell strongly reverses thepolarity of T cell division.

52 RIKEN CDB 2003 Annual Report

T cell polarity
The Wnt pathway represents one of the most highly
conserved and widely adapted signaling systems inbiology, playing developmentally critical roles in the regulation of cell fate, proliferation and polarity in species
from worm to human. Wnt signaling, in fact, transpires
along multiple divergent routes. In the canonical Wnt
pathway in Drosophila (the species in which it was firstdescribed), Wnt binds to the transmembrane receptor
Frizzled, which activates a series of molecules andmolecular complexes within the cell, thereby preventing the degradation of β-catenin and allowing it to accumulate and signal transcription factors that act on thepathway's ultimate downstream gene targets. Studying
the effects of lin-44 (a Wnt homolog) on asymmetric division in the nematode, the Sawa team ectopically
expressed and examined its effects on the division of T
cells, which form a diverse lineage of hypodermal andneural cells arising from a single progenitor. In normal
development, the anterior daughter of the parent T cell
produces hypodermal cells, while the posterior daugh-ter produces neural cells. In lin-44 mutants, however,
the opposite is often the case, with anterior daughtersfrequently giving rise to neural cells, and posterior
daughters generating cells in the hypodermal lineage.
This phenotype is attributable to a reversal in parent
cell polarity, which occurs in about 70% of lin-44
mutants. Misexpression of lin-44 at the anterior of the Tcell in lin-44 mutants caused a significant enhancementof the reversal phenotype; 97% of such mutants
showed switching of fates in T cell daughters. These
results demonstrate that the orientation of T cell polarity
is regulated by the LIN-44 signal.

Sawa is engaged
in the study of the genetic andmolecular interactions
that underlie asymmetric
division and cellular polarity
A pathway homologous to the Wnt cascade exists in
C. elegans, and it has been suggested that a
number of downstream factors corresponding tomany of the most prominent Wnt signaling components also play roles in roundworm asymmetric cell
division. POP-1, the C. elegans homolog of
TCF/LEF-1, a transcription factor acting downstreamof Wnt, has recently been shown to be required forasymmetric T cell division. In the canonical Wnt pathway, TCF/LEF-1 is a target of β-catenin, but thedirect involvement of β-catenin in C. elegans asymmetric cell division has never clearly been demonstrated. Researchers in the Sawa lab found that
wrm-1/β-catenin mutants show defects in asymmetric T cell division, similar to those observed in lin-17
mutants, suggesting that that the asymmetric cell
division is controlled by β -catenin.

Publications

Additional work has also focused on T hypodermal
cells, which in normal development divide asymmetrically to produce non-identical daughter cells of neural or hypodermal fate. Recent work by the Sawa
team has shown that three genes, psa-3, ceh-20
and nob-1 (homologs of Meis, Pbx and Hox, respectively) are involved in this asymmetric T mitosis, as
mutations in any of these genes result in the abnormal loss of asymmetry in cell divisions, in which theposterior daughters of the T cell are hypodermalrather than neural in character.

Zhao X, Sawa H and Herman MA. tcl-2 encodes a novel protein that acts
synergistically with Wnt signaling pathways in C. elegans. Dev Biol 256:276-89(2003).

Investigations into the role of PSA-3 uncovered that
expression of PSA-3 is stronger in the posterior T
cell daughter. psa-3 expression is dependent on aconsensus binding site within its promoter region for
the transcriptional factor POP-1/TCF, which has previously been reported to be required for asymmetric
T cell division, suggesting that the psa-3 gene is adirect target of Wnt signaling and functions as a cellfate determinant in asymmetric cell division. The
asymmetry of PSA-3/Meis expression is also perturbed in lin-44/Wnt and lin-17/Frizzled mutants, corroborating the argument for a downstream role for
psa-3 in the Wnt pathway.

Rocheleau CE, Yasuda J,
Shin TH, Lin R, Sawa H, Okano H, Priess JR, Davis RJ
and Mello CC. WRM-1 activates the LIT-1 protein
kinase to transduce anterior/posterior polarity signals
in C. elegans. Cell 97:717-26(1999).

Sawa H, Kouike H and Okano H. Components of the
SWI/SNF complex are
required for asymmetric cell
division in C. elegans. Mol
Cell 6:617-24 (2000).

Expression of psa-3/Meis in the T cell lineage is also
markedly reduced in nob-1/Hox and ceh-20/Pbx
mutants, indicating that, NOB-1/Hox and CEH20/Pbx also act in the regulation of psa-3/Meis
expression. Taken together, these data indicate thatthe expression of psa-3/Meis is controlled by binary
mechanisms; Wnt signaling establishes cellularpolarity, while the Hox-Pbx complex regulates positional identity. In other metazoans, both Meis andPbx are known to bind directly as co-factors to Hox,
raising the possibility that 20/Pbx and NOB-1/Hox
proteins may form a ternary complex with PSA3/Meis in C. elegans as well. In vitro studies showthat PSA-3/Meis interacts directly with CEH-20/Pbx
and NOB-1/Hox, lending support to the model inwhich PSA-3/Meis functions as a cell fate determinant by forming a complex with CEH-20/Pbx and
NOB-1/Hox in asymmetric cell division of the T cell.

Expression of PSA-3/Meis isdirectly induced byPOP-1/TCF. PSA-3 forms
complex with CEH-20/Pbx and
NOB-1/Hox to determine
asymmetric cell fates.

RIKEN CDB 2003 Annual Report53knock down the expression of
specific genes in the nematode has only added to its
appeal, giving scientists theability to inhibit gene function
without disturbing its underlying DNA. Sugimoto has
refined this technique of RNAinterference (RNAi) by developing a method in which
nematodes directly uptake
dsRNA in solution. This process of 'RNAi by soaking' offers greater efficiency andease-of-use than other RNAi
methods and has enabled systematic high-throughput studies of gene suppression to be
conducted more rapidly thanever before possible.

Creative Research
Promoting Program

Asako Sugimoto
Ph. D.
Asako Sugimoto received
her B. Sc. degree from the
Department of Biophysics
and Biochemistry in the University of Tokyo School of
Science in 1987, and her
doctorate from the same
institution in 1992. She
worked as a postdoctoral
fellow in Joel Rothman’slaboratory in the Universityof Wisconsin – Madisonfrom 1992 to 1996, before
returning to Japan toassume an assistant professorship at the University ofTokyo. She remained inthat position until 2002, pursuing concurrent work as aJapan Science and Technology Corporation PRESTO researcher from 1997 to
2000. She was appointed
team leader at the RIKEN
CDB in 2001.

Interactions between genes at the network level areof fundamental importance in instructing the development and function of multicellular organisms. Whilethe characterization of genomes at the level of thesolitary gene remains an important challenge inmany yet unsequenced organisms, the analysis of
how genes function in networks is now in increasing
demand for those genomes that are already available. In the post-genome era, scientists seek tounderstand the roles of genes in evolution throughcomparative genomics studies as well as to characterize genes in isolation and understand their functions in the context of interactive networks, a field ofinvestigation known as functional genomics.
Asako Sugimoto has adopted the nematode
Caenorhabditis elegans as an experimental model
to take advantage
of its tractability
to thethese studies as a base for advancing the understanding of developmentally important mechanisms.
Sugimoto hopes that by opening windows into therole of networked genes in guiding development in a
simple worm, new light will be shed on universal
mechanisms in the genetic regulation of the developmental program.

RNAi-based profiling ofgene function
The wild-type nematode is built from precisely 959
somatic cells, yet this simple organism exhibits awide range of the specialized cell types, such asmuscles and neurons, that characterize more highly
derived species. And, thanks to the complete knowledge of the lineage of every cell in the C. elegans
body, the differentiative pathway of
every one of those cells can be followed from its origin in the fertilized egg to its role in the
fully-grown

Caenorhabditis elegans(adult hermaphrodite)

systematic functional analysis of its genome using uniquehigh-throughput screening techniques.
By studying the interactions between the approximately 19,000 genes predicted for C. elegans, theSugimoto research team seeks to identify themeans by which sets of genes working in combination help to establish and direct developmental processes. The lab also looks to take the findings from

54 RIKEN CDB 2003 Annual Report

Embryogenesis of
C. elegans

adult. The amenability of this worm's
sequenced genome to reverse-genetic techniques
has also served to make it one of the standard model organisms in the world of genetics research. The
discovery in the late 1990s that the introduction of
double stranded RNA (dsRNA) could be used toStarting with a cDNA library of
approximately 10,000 genes
expressed in developmental
processes, the Sugimoto lab
used RNAi to knock downeach gene's function and has
started to construct a database in which the resulting
phenotypes are sorted bydevelopmental outcome. To
date, nearly 6,000 phenotypes
have been categorized, and it
was found that loss of function
in more than 25% of thesegenes resulted in lethal, morphologically altered or sterile
phenotypes. Many lethal phenotypes result in developmental arrest and death at very
early stages, which typically
prevents the study of theunderlying gene's function inlater development. By exposing worms to RNAi-inducing
dsRNA at the L1 stage of larval development, Sugimoto isable to study the postembryonic function of genesthat are lethal when knocked
down in embryos. This is critically important to the understanding of genetic networks
in post-embryonic development, as more than one out of
every ten genes analyzed by
the lab so far is essential forembryogenesis. Sugimoto's
studies to date show thatapproximately 50% of all suchgenes play post-embryonicroles as well, supporting theconcept that a great numberof genes play multiple roles atdifferent stages in development.

Phenotype analysis
The data sets produced by highthroughput phenotype analyses canbe formidably large and unwieldy, andthe lack of standardized descriptive
terms and categories can limit theaccessibility to the wealth of information they contain. To improve the value, ease-of-use and distributability of
her RNAi phenotyping results, Sugimoto has developed a taxonomic system
and nomenclature to enable the precise and consistent description ofembryonic and post-embryonic phenotypes using a checklist of more than 50 identifying
traits, such as abnormalities in cell division, cellulardifferentiation, organogenesis, morphogenesis,
growth, and movement. She plans to make these
RNAi phenotype profiles available to the C. elegans
research community, in the hopes of establishing auniversal taxonomy for phenotype analysis. Phenotypes described using this system can be subjected to hierarchical clustering analysis, which makes it

Staff
Team Leader

Asako Sugimoto
Research Scientist

Naoko Iida
Special Postdoctoral Researcher

Fumio Motegi
Technical Staff

Nobuko Uodome
Chie Hayakawa
Yumi Iida
Student Trainee

Miwa Furuya
Rika Maruyama
AssistantFumi Amimori

Sugimoto has developed
a taxonomic system andnomenclature to enable
the precise and consistentdescription of embryonic andpost-embryonic phenotypes

Part-time Staff

Kazumasa Takeda

possible to categorize genes based on relatedness
between phenotypes. The genes clustered by thismethod are likely to be involved in the same developmental process, thus this analysis will provide pivotal information to uncover genetic networks
involved in the regulation of development. Membersof the Sugimoto team are now utilizing these RNAi
analysis data to investigate developmental processes such as microtubule dynamics in mitosis, morphogenesis, and programmed cell death.

Publications
Mito Y, Sugimoto A andYamamoto M. Distinct developmental function of two Caenorhabditis
elegans
homologs of the cohesin subunit Scc1/Rad21. Mol Biol
Cell 14:2399-409 (2003).
Sawa M, Suetsugu S, Sugimoto A, Miki H, Yamamoto
M and Takenawa T. Essential role of the C. elegans
Arp2/3 complex in cell migration during ventral enclosure.
J Cell Sci 116:1505-18(2003).
Sumiyoshi E, Sugimoto A
and Yamamoto M. Protein
phosphatase 4 is required forcentrosome maturation inmitosis and sperm meiosis in
C. elegans. J Cell Sci
115:1403-10 (2002).
Kodama Y, Rothman JH,
Sugimoto A and Yamamoto
M. The stem-loop binding
protein CDL-1 is required forchromosome condensation,
progression of cell death andmorphogenesis in Caenorhabditis elegans. Development 129:187-96 (2002).

Germ cells in meiotic prophase I.
(Blue: DNA, Red: A specific locus on Chromosome V)

RIKEN CDB 2003 Annual Report55

knock down the expression of
specific genes in the nematode has only added to its
appeal, giving scientists theability to inhibit gene function
without disturbing its underlying DNA. Sugimoto has
refined this technique of RNAinterference (RNAi) by developing a method in which
nematodes directly uptake
dsRNA in solution. This process of 'RNAi by soaking' offers greater efficiency andease-of-use than other RNAi
methods and has enabled systematic high-throughput studies of gene suppression to be
conducted more rapidly thanever before possible.

Creative Research
Promoting Program

Asako Sugimoto
Ph. D.
Asako Sugimoto received
her B. Sc. degree from the
Department of Biophysics
and Biochemistry in the University of Tokyo School of
Science in 1987, and her
doctorate from the same
institution in 1992. She
worked as a postdoctoral
fellow in Joel Rothman’slaboratory in the Universityof Wisconsin – Madisonfrom 1992 to 1996, before
returning to Japan toassume an assistant professorship at the University ofTokyo. She remained inthat position until 2002, pursuing concurrent work as aJapan Science and Technology Corporation PRESTO researcher from 1997 to
2000. She was appointed
team leader at the RIKEN
CDB in 2001.

Interactions between genes at the network level areof fundamental importance in instructing the development and function of multicellular organisms. Whilethe characterization of genomes at the level of thesolitary gene remains an important challenge inmany yet unsequenced organisms, the analysis of
how genes function in networks is now in increasing
demand for those genomes that are already available. In the post-genome era, scientists seek tounderstand the roles of genes in evolution throughcomparative genomics studies as well as to characterize genes in isolation and understand their functions in the context of interactive networks, a field ofinvestigation known as functional genomics.
Asako Sugimoto has adopted the nematode
Caenorhabditis elegans as an experimental model
to take advantage
of its tractability
to thethese studies as a base for advancing the understanding of developmentally important mechanisms.
Sugimoto hopes that by opening windows into therole of networked genes in guiding development in a
simple worm, new light will be shed on universal
mechanisms in the genetic regulation of the developmental program.

RNAi-based profiling ofgene function
The wild-type nematode is built from precisely 959
somatic cells, yet this simple organism exhibits awide range of the specialized cell types, such asmuscles and neurons, that characterize more highly
derived species. And, thanks to the complete knowledge of the lineage of every cell in the C. elegans
body, the differentiative pathway of
every one of those cells can be followed from its origin in the fertilized egg to its role in the
fully-grown

Caenorhabditis elegans(adult hermaphrodite)

systematic functional analysis of its genome using uniquehigh-throughput screening techniques.
By studying the interactions between the approximately 19,000 genes predicted for C. elegans, theSugimoto research team seeks to identify themeans by which sets of genes working in combination help to establish and direct developmental processes. The lab also looks to take the findings from

54 RIKEN CDB 2003 Annual Report

Embryogenesis of
C. elegans

adult. The amenability of this worm's
sequenced genome to reverse-genetic techniques
has also served to make it one of the standard model organisms in the world of genetics research. The
discovery in the late 1990s that the introduction of
double stranded RNA (dsRNA) could be used toStarting with a cDNA library of
approximately 10,000 genes
expressed in developmental
processes, the Sugimoto lab
used RNAi to knock downeach gene's function and has
started to construct a database in which the resulting
phenotypes are sorted bydevelopmental outcome. To
date, nearly 6,000 phenotypes
have been categorized, and it
was found that loss of function
in more than 25% of thesegenes resulted in lethal, morphologically altered or sterile
phenotypes. Many lethal phenotypes result in developmental arrest and death at very
early stages, which typically
prevents the study of theunderlying gene's function inlater development. By exposing worms to RNAi-inducing
dsRNA at the L1 stage of larval development, Sugimoto isable to study the postembryonic function of genesthat are lethal when knocked
down in embryos. This is critically important to the understanding of genetic networks
in post-embryonic development, as more than one out of
every ten genes analyzed by
the lab so far is essential forembryogenesis. Sugimoto's
studies to date show thatapproximately 50% of all suchgenes play post-embryonicroles as well, supporting theconcept that a great numberof genes play multiple roles atdifferent stages in development.

Phenotype analysis
The data sets produced by highthroughput phenotype analyses canbe formidably large and unwieldy, andthe lack of standardized descriptive
terms and categories can limit theaccessibility to the wealth of information they contain. To improve the value, ease-of-use and distributability of
her RNAi phenotyping results, Sugimoto has developed a taxonomic system
and nomenclature to enable the precise and consistent description ofembryonic and post-embryonic phenotypes using a checklist of more than 50 identifying
traits, such as abnormalities in cell division, cellulardifferentiation, organogenesis, morphogenesis,
growth, and movement. She plans to make these
RNAi phenotype profiles available to the C. elegans
research community, in the hopes of establishing auniversal taxonomy for phenotype analysis. Phenotypes described using this system can be subjected to hierarchical clustering analysis, which makes it

Staff
Team Leader

Asako Sugimoto
Research Scientist

Naoko Iida
Special Postdoctoral Researcher

Fumio Motegi
Technical Staff

Nobuko Uodome
Chie Hayakawa
Yumi Iida
Student Trainee

Miwa Furuya
Rika Maruyama
AssistantFumi Amimori

Sugimoto has developed
a taxonomic system andnomenclature to enable
the precise and consistentdescription of embryonic andpost-embryonic phenotypes

Part-time Staff

Kazumasa Takeda

possible to categorize genes based on relatedness
between phenotypes. The genes clustered by thismethod are likely to be involved in the same developmental process, thus this analysis will provide pivotal information to uncover genetic networks
involved in the regulation of development. Membersof the Sugimoto team are now utilizing these RNAi
analysis data to investigate developmental processes such as microtubule dynamics in mitosis, morphogenesis, and programmed cell death.

Publications
Mito Y, Sugimoto A andYamamoto M. Distinct developmental function of two Caenorhabditis
elegans
homologs of the cohesin subunit Scc1/Rad21. Mol Biol
Cell 14:2399-409 (2003).
Sawa M, Suetsugu S, Sugimoto A, Miki H, Yamamoto
M and Takenawa T. Essential role of the C. elegans
Arp2/3 complex in cell migration during ventral enclosure.
J Cell Sci 116:1505-18(2003).
Sumiyoshi E, Sugimoto A
and Yamamoto M. Protein
phosphatase 4 is required forcentrosome maturation inmitosis and sperm meiosis in
C. elegans. J Cell Sci
115:1403-10 (2002).
Kodama Y, Rothman JH,
Sugimoto A and Yamamoto
M. The stem-loop binding
protein CDL-1 is required forchromosome condensation,
progression of cell death andmorphogenesis in Caenorhabditis elegans. Development 129:187-96 (2002).

Germ cells in meiotic prophase I.
(Blue: DNA, Red: A specific locus on Chromosome V)

RIKEN CDB 2003 Annual Report55

Epithelial-mesenchymaltransitions in somites

Creative Research
Promoting Program

Yoshiko Takahashi
Ph. D.
Yoshiko Takahashi received
her B. Sc. from the University of Hiroshima, before moving to the Kyoto University
Department of Biophysics
where she received her master’s and doctoral degrees indevelopmental biology. She
pursued consecutive postdoctoral fellowships in developmental biology at the Institutd’Embryologie du CNRS
(1988 to 1991), the University of Oregon Institute of Neuroscience (1991 to 1993)
and Columbia University(1994). She returned toJapan as an associate professor at Kitasato University
in 1994, where she worked
until 1998, when she took an
associate professorship at
the Nara Institute of Scienceand Technology Graduate
School of Biological Sciences. She was appointed teamleader at the RIKEN CDB in
2001.

Pattern formation, the process by which the borderzones that define germ layers and tissue types are
established, is strictly regulated to occur at specificsites and stages in the developing embryo. This
close coordination prevents cells from differentiating
inappropriately, and enables the establishment ofcomplex and specialized tissues and organs in theembryo and the adult body. One type of pattern formation involves the regimented establishment of
periodically alternating bands, or segments, of differentiated cells along the anterior-posterior body axis.
The results of this process of segmentation can beobserved in repeated orderly structures such as vertebrae, ribs and spinal ganglia. In vertebrates, oneform of segmentation takes place in the embryonicregion known as the 'somitic mesoderm,' and involves the formation of transient segment-organizing
bodies called somites.

referred to as the 'segmental plate,' flank the neuraltube, which runs along the anterior-posterior axis.
Somites arise one by one from this segmental platein a head-down direction, directed by the inductive
activity of a putative regulatory region Takahashi
has termed the 'segmenter.' Recent work in her lab
is aimed at explicating transitions in cell structure
and type that take place in nascent somites once
their initial boundaries have been established.

teins, notably Rho, Rac1 and Cdc42, are known toplay roles in cytoskeletal rearrangement and cell
locomotion in vitro, cycling between GDP-boundinactive and GTP-bound active states, and triggering downstream effectors linked to the cytoskeleton
when activated.

Staff
Team Leader

Somites comprise both epithelial and mesenchymal
cells, two contrasting cell types that vividly demonstrate the range of possibilities in cellular function
and morphology. Epithelial cells, which in adults
form the skin and the linings of many organs,adhere to each other tightly and have polarized
Now, studies in the Takahashi lab are beginning tostructures with distinct basal and apical sides. By
reveal additional functions for Rho family members
comparison, mesenchymal cells are less rigidly
in the context of somitic development. Both the inhistructured and form looser aggregations. However,bition of Rac1 and the overexpression of Cdc42
these two cell types are by no means isolated orwere found to induce the conversion of epithelial
independent from one another; indeed, a greatcells to mesenchyme, while interference with Cdc42
many developmental
functioncausedand physiological phemesenchymal cells tonomena rely on interactake on epithelial chartions between epitheliacteristics. These roles
um and mesenchyme.
seem to be at least partAn extreme example ofly specific to somites;
their inter-relatedness isneither Cdc42 nor Rac1
the ability of cells of one
was found to betype to convert into theinvolved in epithelialother, in a process
mesenchymal transiknown as epithelialtions in epiblast cells.
mesenchymal transiThese findings suggest
tion. Such transitions
that the mesenchymal(which can proceed inepithelial transition thateither an epithelial-tooccurs during somitogemesenchymal or thenesis is determined by
Cdc42, a Rho family GTPase, plays an important role inreverse direction) are
relative Rac1 andmesenchymal to epithelial transitions in somitogenesis.
not only essential to norCdc42 activity levels,
mal embryonic development and organogenesis,
with higher levels of Rac1 inducing an epithelial fate,they also play roles in wound healing and the pathowhile higher levels of Cdc42 tip the balance in thegenesis of cancer.
mesenchymal direction. She now seeks to further
characterize the induction of the activity of these
After the boundaries of a somite have been estabRho family members in the earliest stages of thelished, the mesenchymal cells in the border region
establishment of the somite by segmenter activity.
undergo a transition, acquiring the polarized structure and properties of epithelial cells. This results in
Takahashi emphasizes that what drew her to scia somite body in which a core of mesenchymal cells
ence and maintains her interest are the opportuniis circumscribed by an outer layer of cells of epithelities and challenges it offers to those willing and able
al character. The Takahashi lab seeks to clarify theto learn from natural phenomena. Her interests aremolecular mechanisms that regulate thisdiverse and far-ranging; she chooses to pursue
mesenchymal-epithelial transition, and is concentratavenues that others may have overlooked, anding on the roles played by members of the Rho famiencourages her students and colleagues to do thely of GTPase proteins in this process. These prosame.

Yoshiko Takahashi
Research Scientist

Daisuke Saito
Special Postdoctoral Researcher

Yuki Sato
Technical Staff

Rinako Suetsugu
Toshiharu Kasai
Yuji Katagiri
Akemi Uchiyama
Junior Research Associate

Ryosuke Tadokoro
Student Trainee

Yukiko Nakaya
Tadayoshi Watanabe
Emi Ohata
Itsuki Kashin
Assistant

Tomoko Oyanagi
Part-time Staff

Naomi Akira

Publications
Takahashi Y. Somitogenesis
in vertebrate development, in"Encyclopedia of Life Sciences", Nature Publishing Group(2004).
Takahashi Y. Shaping up of
the chic chick: boundary formation shared between somitogenesis and brain
development. Review article, International Journal of
Developmental Biology(2004).
Tonegawa A, Kasai T and
Takahashi Y. Systematic
screening for signaling molecules expressed duringsomitogenesis by the signal
sequence trap method. Dev
Biol 262:32-50 (2003).

Somites comprise bothepithelial and mesenchymal cells,
two cell types thatvividly demonstrate the range ofpossibilities incellular function and morphology
Yoshiko Takahashi's research involves molecularand cellular analyses of segmentation andsomitogenesis in the chick embryo, which are invaluable for the insights they provide into how populations of cells are able to cleave at specific boundarylines and reorganize into highly ordered and reiterative patterns. In the early chick embryo, two strips ofunsegmented paraxial mesoderm, collectively

56 RIKEN CDB 2003 Annual Report

Suetsugu R, Sato Y and
Takahashi Y. Pax 2 expression in mesodermal segmentation and its relationshipwith EphA4 and Lunaticfringe during chicken somitogenesis. Mech Dev 119
Suppl 1:S155-9 (2002).
Sato Y, Yasuda K and Takahashi Y. Morphologicalboundary forms by a novel
inductive event mediated by
Lunatic fringe and Notch during somitic segmentation.
Development 129:3633-44(2002).

Transitions between epithelial and mesenchymal states arepivotal to early morphogenetic processes.

2-day embryo showing somites beautifully arrayed in pairs along the anterior-posterior axis

RIKEN CDB 2003 Annual Report57

Epithelial-mesenchymaltransitions in somites

Creative Research
Promoting Program

Yoshiko Takahashi
Ph. D.
Yoshiko Takahashi received
her B. Sc. from the University of Hiroshima, before moving to the Kyoto University
Department of Biophysics
where she received her master’s and doctoral degrees indevelopmental biology. She
pursued consecutive postdoctoral fellowships in developmental biology at the Institutd’Embryologie du CNRS
(1988 to 1991), the University of Oregon Institute of Neuroscience (1991 to 1993)
and Columbia University(1994). She returned toJapan as an associate professor at Kitasato University
in 1994, where she worked
until 1998, when she took an
associate professorship at
the Nara Institute of Scienceand Technology Graduate
School of Biological Sciences. She was appointed teamleader at the RIKEN CDB in
2001.

Pattern formation, the process by which the borderzones that define germ layers and tissue types are
established, is strictly regulated to occur at specificsites and stages in the developing embryo. This
close coordination prevents cells from differentiating
inappropriately, and enables the establishment ofcomplex and specialized tissues and organs in theembryo and the adult body. One type of pattern formation involves the regimented establishment of
periodically alternating bands, or segments, of differentiated cells along the anterior-posterior body axis.
The results of this process of segmentation can beobserved in repeated orderly structures such as vertebrae, ribs and spinal ganglia. In vertebrates, oneform of segmentation takes place in the embryonicregion known as the 'somitic mesoderm,' and involves the formation of transient segment-organizing
bodies called somites.

referred to as the 'segmental plate,' flank the neuraltube, which runs along the anterior-posterior axis.
Somites arise one by one from this segmental platein a head-down direction, directed by the inductive
activity of a putative regulatory region Takahashi
has termed the 'segmenter.' Recent work in her lab
is aimed at explicating transitions in cell structure
and type that take place in nascent somites once
their initial boundaries have been established.

teins, notably Rho, Rac1 and Cdc42, are known toplay roles in cytoskeletal rearrangement and cell
locomotion in vitro, cycling between GDP-boundinactive and GTP-bound active states, and triggering downstream effectors linked to the cytoskeleton
when activated.

Staff
Team Leader

Somites comprise both epithelial and mesenchymal
cells, two contrasting cell types that vividly demonstrate the range of possibilities in cellular function
and morphology. Epithelial cells, which in adults
form the skin and the linings of many organs,adhere to each other tightly and have polarized
Now, studies in the Takahashi lab are beginning tostructures with distinct basal and apical sides. By
reveal additional functions for Rho family members
comparison, mesenchymal cells are less rigidly
in the context of somitic development. Both the inhistructured and form looser aggregations. However,bition of Rac1 and the overexpression of Cdc42
these two cell types are by no means isolated orwere found to induce the conversion of epithelial
independent from one another; indeed, a greatcells to mesenchyme, while interference with Cdc42
many developmental
functioncausedand physiological phemesenchymal cells tonomena rely on interactake on epithelial chartions between epitheliacteristics. These roles
um and mesenchyme.
seem to be at least partAn extreme example ofly specific to somites;
their inter-relatedness isneither Cdc42 nor Rac1
the ability of cells of one
was found to betype to convert into theinvolved in epithelialother, in a process
mesenchymal transiknown as epithelialtions in epiblast cells.
mesenchymal transiThese findings suggest
tion. Such transitions
that the mesenchymal(which can proceed inepithelial transition thateither an epithelial-tooccurs during somitogemesenchymal or thenesis is determined by
Cdc42, a Rho family GTPase, plays an important role inreverse direction) are
relative Rac1 andmesenchymal to epithelial transitions in somitogenesis.
not only essential to norCdc42 activity levels,
mal embryonic development and organogenesis,
with higher levels of Rac1 inducing an epithelial fate,they also play roles in wound healing and the pathowhile higher levels of Cdc42 tip the balance in thegenesis of cancer.
mesenchymal direction. She now seeks to further
characterize the induction of the activity of these
After the boundaries of a somite have been estabRho family members in the earliest stages of thelished, the mesenchymal cells in the border region
establishment of the somite by segmenter activity.
undergo a transition, acquiring the polarized structure and properties of epithelial cells. This results in
Takahashi emphasizes that what drew her to scia somite body in which a core of mesenchymal cells
ence and maintains her interest are the opportuniis circumscribed by an outer layer of cells of epithelities and challenges it offers to those willing and able
al character. The Takahashi lab seeks to clarify theto learn from natural phenomena. Her interests aremolecular mechanisms that regulate thisdiverse and far-ranging; she chooses to pursue
mesenchymal-epithelial transition, and is concentratavenues that others may have overlooked, anding on the roles played by members of the Rho famiencourages her students and colleagues to do thely of GTPase proteins in this process. These prosame.

Yoshiko Takahashi
Research Scientist

Daisuke Saito
Special Postdoctoral Researcher

Yuki Sato
Technical Staff

Rinako Suetsugu
Toshiharu Kasai
Yuji Katagiri
Akemi Uchiyama
Junior Research Associate

Ryosuke Tadokoro
Student Trainee

Yukiko Nakaya
Tadayoshi Watanabe
Emi Ohata
Itsuki Kashin
Assistant

Tomoko Oyanagi
Part-time Staff

Naomi Akira

Publications
Takahashi Y. Somitogenesis
in vertebrate development, in"Encyclopedia of Life Sciences", Nature Publishing Group(2004).
Takahashi Y. Shaping up of
the chic chick: boundary formation shared between somitogenesis and brain
development. Review article, International Journal of
Developmental Biology(2004).
Tonegawa A, Kasai T and
Takahashi Y. Systematic
screening for signaling molecules expressed duringsomitogenesis by the signal
sequence trap method. Dev
Biol 262:32-50 (2003).

Somites comprise bothepithelial and mesenchymal cells,
two cell types thatvividly demonstrate the range ofpossibilities incellular function and morphology
Yoshiko Takahashi's research involves molecularand cellular analyses of segmentation andsomitogenesis in the chick embryo, which are invaluable for the insights they provide into how populations of cells are able to cleave at specific boundarylines and reorganize into highly ordered and reiterative patterns. In the early chick embryo, two strips ofunsegmented paraxial mesoderm, collectively

56 RIKEN CDB 2003 Annual Report

Suetsugu R, Sato Y and
Takahashi Y. Pax 2 expression in mesodermal segmentation and its relationshipwith EphA4 and Lunaticfringe during chicken somitogenesis. Mech Dev 119
Suppl 1:S155-9 (2002).
Sato Y, Yasuda K and Takahashi Y. Morphologicalboundary forms by a novel
inductive event mediated by
Lunatic fringe and Notch during somitic segmentation.
Development 129:3633-44(2002).

Transitions between epithelial and mesenchymal states arepivotal to early morphogenetic processes.

2-day embryo showing somites beautifully arrayed in pairs along the anterior-posterior axis

RIKEN CDB 2003 Annual Report57

StaffTeam Leader

Teruhiko Wakayama
Research Scientist

Satoshi Kishigami
Nguyen Van Thuan
Hiroshi Ohta
Takafusa Hikichi
Technical Staff

Sayaka Wakayama
Student Trainee

Eiji Mizutani
AssistantKana Tachibana

Oocyte (large, upper left) and somatic(small, bottom center) spindles following nuclear transfer

Creative Research
Promoting Program

Teruhiko Wakayama
Ph. D.
Teruhiko
Wakayama
received his B. Sc. and M.
Sc. from Ibaraki University,and was awarded a Ph. D.
in reproductive biology fromthe University of Tokyo
Department of Veterinary
Anatomy in 1996. Hereceived a postdoctoral fellowship from the JapaneseSociety for the Promotion of
Science in 1996 and spent
the next three years at the
Yanagimachi lab in the University of Hawaii Medical
School, where he succeeded in producing the world’s
first cloned mouse. He wasappointed to an assistantprofessorship at the same
institution in 1998, andmoved to Rockefeller University as a research assistant professor in 1999. He
spent a year as a researcher at Advanced Cell Technology before returning toJapan to take his current
position at the RIKEN CDB.

The experimental cloning of animals has a history
that extends back more than fifty years, to when
Briggs and King successfully produced tadpoles bytransplanting the nuclei from embryonic cells into
frogs' eggs whose own nuclei had been removed. At
the time, cloning was used not as a technique to bestudied for its immediate application, but as ameans of testing a fundamental question of reproductive biology: whether the processes of fertilization and later development, in which the body's cells
become more specialized and functionally distinct,
involves a loss of genetic information, or whether allcells retain the full set of genetic code, even after differentiation has proceeded. The success of these initial experiments in cloning by nuclear transfer demonstrated conclusively that cells do maintain intact
genomes even after differentiation, as the genetic

code in a specialized cell's nucleus is sufficient to
instruct an egg into which it is transplanted to giverise to a normal individual.
Although questions of whether genetic information islost or irreversibly altered during development werelaid to rest, new questions arose to take their place.
For Teruhiko Wakayama, the most intriguing issue
raised by the ability of animals to be cloned using
the nuclei of specialized somatic cells (which form
the body and, unlike sperm and eggs, cannot normally produce a new individual), is that of reprogramming, the processes by which the genome receives
new sets of coding instructions enabling it to orderthe development of all the cells in a new individual
while remaining intact and fundamentally
unchanged in each of those cells. The Wakayama
lab studies mammalian cloning
and fertilization with the same
basic goal as drove Briggs andKing in their nuclear transfer
studies a half century ago: toanswer central questions in the
biology of animal reproduction.

Cloning
efficiencies
In all species and in all experimental methods tested to date,cloning by nuclear transfer has
consistently low efficiency rates— generally below 5% of enucleated eggs fertilized by
nuclear transfer go on to develop into live-born offspring. Many
hypotheses have been pro-

posed to explain the inefficiency of this procedure,
while accounting for the fact that cloning is not altogether impossible. It has been suggested that theprocess of removing the nucleus from an oocyte (anunfertilized egg) or the absence of chromosomal
information during the several hours in which theegg is missing a nucleus may somehow damage or
cause the loss of factors that would normally act toreprogram the genetic information in the fertilizing(or transplanted) cell's nucleus. To test this possibility, Wakayama re-ordered conventional cloning methodology by first transferring nuclei from cumulus
cells into oocytes whose own nuclei were still present, and only then removing the mitotic spindle
derived from the native nucleus. These experiments
resulted in the generation of live offspring at a rateof efficiency similar to that of standard cloning by
nuclear transfer, providing evidence that tends tocounter the hypothesis that the temporary absenceof a nucleus is responsible for the poor developmental prospects of NT oocytes.

Many hypotheses
have been proposed to explain
cloning's inefficiency,
while accounting for the factthat it is not altogether impossible
Scientists in the Wakayama lab have adopted similar
approaches to the study of fertilization — experimentsthat involve the substitution of components or the reordering of natural sequences of events to test for specific biological function. In nature, fertilization occurs after
a mature spermatozoon fuses with and activates anoocyte, but some laboratory techniques achieve fertilization by artificially activating the egg and then injecting a spermatid, which is an incompletely maturated
sperm cell. Such techniques are known to be less effi-

cient than fertilization using mature spermatozoa, but
the reason for this lower developmental competency
remains unknown. The Laboratory for Genomic Reprogramming is now comparing in vitro fertilization using
spermatozoa and spermatids under controlled conditions in an effort to identify the differences between
these stages in the developing sperm cell. Future
research will look at changes in epigenetic modifications to the sperm genome over time as a possible
explanation for their disparate potentials.

New technologies

Publications

Sperm preserved for use in experiments and in vitro
fertilization is traditionally frozen in liquid nitrogen at
extremely low temperatures (around -190˚C). This
cryopreservation allows the sperm to be maintained
viably for very long periods, but requires expensive
facilities and some degree of technical skill in handling. Oocytes and fertilized eggs are also extremely
labile, and must likewise be stored cryogenically.
But these requirements tend to limit the access ofgerm cells to researchers unequipped with liquid
nitrogen facilities, preventing the spread of the technology and the development of research using thesecells. Wakayama hopes to develop new, less expensive and less technically demanding methods for the
storage and maintenance of germ cells for experimental use. Recent tests using a modified commercially available culture medium showed that under
the right conditions spermatozoa can be preserved
for long periods, up to 70 days, at 4 degrees Celsius, a temperature that can be maintained using
ordinary and inexpensive refrigeration equipment.
This new preservation method opens up opportunities in animal breeding and reproductive biology
research to scientists working under limited budgets,
which Wakayama hopes may help to make thesefields of science more accessible to developing
countries and smaller labs.

Singh U, Fohn L, Wakayama
T, Ohgane J, Steinhoff C, Lipkowitz B, Schulz R, Orth A,
Ropers HH, Behringer R,
Tanaka A, Shiota K, Yanagimachi R, Nuber U and Fundele R. Abnormal gene
expression in mouse placentopathies caused by interspecies hybridization, cloning,and Esx1 mutation. Developmental Dynamics (2004).
Wakayama S, Cibelli JB and
Wakayama T. Effect of timing of the removal of oocyte
chromosomes before or after
injection of somatic nucleus
on development of NT
embryos. Cloning Stem Cells
5:181-9 (2003).
Tamashiro KL, Wakayama T,
Akutsu H, Yamazaki Y,
Lachey JL, Wortman MD,
Seeley RJ, D'Alessio DA,
Woods SC, Yanagimachi R
and Sakai RR. Cloned mice
have an obese phenotype
not transmitted to their offspring. Nat Med 8:262-7(2002).

Wakame, the first mouse cloned at the RIKEN CDB

58 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report59StaffTeam Leader

Teruhiko Wakayama
Research Scientist

Satoshi Kishigami
Nguyen Van Thuan
Hiroshi Ohta
Takafusa Hikichi
Technical Staff

Sayaka Wakayama
Student Trainee

Eiji Mizutani
AssistantKana Tachibana

Oocyte (large, upper left) and somatic(small, bottom center) spindles following nuclear transfer

Creative Research
Promoting Program

Teruhiko Wakayama
Ph. D.
Teruhiko
Wakayama
received his B. Sc. and M.
Sc. from Ibaraki University,and was awarded a Ph. D.
in reproductive biology fromthe University of Tokyo
Department of Veterinary
Anatomy in 1996. Hereceived a postdoctoral fellowship from the JapaneseSociety for the Promotion of
Science in 1996 and spent
the next three years at the
Yanagimachi lab in the University of Hawaii Medical
School, where he succeeded in producing the world’s
first cloned mouse. He wasappointed to an assistantprofessorship at the same
institution in 1998, andmoved to Rockefeller University as a research assistant professor in 1999. He
spent a year as a researcher at Advanced Cell Technology before returning toJapan to take his current
position at the RIKEN CDB.

The experimental cloning of animals has a history
that extends back more than fifty years, to when
Briggs and King successfully produced tadpoles bytransplanting the nuclei from embryonic cells into
frogs' eggs whose own nuclei had been removed. At
the time, cloning was used not as a technique to bestudied for its immediate application, but as ameans of testing a fundamental question of reproductive biology: whether the processes of fertilization and later development, in which the body's cells
become more specialized and functionally distinct,
involves a loss of genetic information, or whether allcells retain the full set of genetic code, even after differentiation has proceeded. The success of these initial experiments in cloning by nuclear transfer demonstrated conclusively that cells do maintain intact
genomes even after differentiation, as the genetic

code in a specialized cell's nucleus is sufficient to
instruct an egg into which it is transplanted to giverise to a normal individual.
Although questions of whether genetic information islost or irreversibly altered during development werelaid to rest, new questions arose to take their place.
For Teruhiko Wakayama, the most intriguing issue
raised by the ability of animals to be cloned using
the nuclei of specialized somatic cells (which form
the body and, unlike sperm and eggs, cannot normally produce a new individual), is that of reprogramming, the processes by which the genome receives
new sets of coding instructions enabling it to orderthe development of all the cells in a new individual
while remaining intact and fundamentally
unchanged in each of those cells. The Wakayama
lab studies mammalian cloning
and fertilization with the same
basic goal as drove Briggs andKing in their nuclear transfer
studies a half century ago: toanswer central questions in the
biology of animal reproduction.

Cloning
efficiencies
In all species and in all experimental methods tested to date,cloning by nuclear transfer has
consistently low efficiency rates— generally below 5% of enucleated eggs fertilized by
nuclear transfer go on to develop into live-born offspring. Many
hypotheses have been pro-

posed to explain the inefficiency of this procedure,
while accounting for the fact that cloning is not altogether impossible. It has been suggested that theprocess of removing the nucleus from an oocyte (anunfertilized egg) or the absence of chromosomal
information during the several hours in which theegg is missing a nucleus may somehow damage or
cause the loss of factors that would normally act toreprogram the genetic information in the fertilizing(or transplanted) cell's nucleus. To test this possibility, Wakayama re-ordered conventional cloning methodology by first transferring nuclei from cumulus
cells into oocytes whose own nuclei were still present, and only then removing the mitotic spindle
derived from the native nucleus. These experiments
resulted in the generation of live offspring at a rateof efficiency similar to that of standard cloning by
nuclear transfer, providing evidence that tends tocounter the hypothesis that the temporary absenceof a nucleus is responsible for the poor developmental prospects of NT oocytes.

Many hypotheses
have been proposed to explain
cloning's inefficiency,
while accounting for the factthat it is not altogether impossible
Scientists in the Wakayama lab have adopted similar
approaches to the study of fertilization — experimentsthat involve the substitution of components or the reordering of natural sequences of events to test for specific biological function. In nature, fertilization occurs after
a mature spermatozoon fuses with and activates anoocyte, but some laboratory techniques achieve fertilization by artificially activating the egg and then injecting a spermatid, which is an incompletely maturated
sperm cell. Such techniques are known to be less effi-

cient than fertilization using mature spermatozoa, but
the reason for this lower developmental competency
remains unknown. The Laboratory for Genomic Reprogramming is now comparing in vitro fertilization using
spermatozoa and spermatids under controlled conditions in an effort to identify the differences between
these stages in the developing sperm cell. Future
research will look at changes in epigenetic modifications to the sperm genome over time as a possible
explanation for their disparate potentials.

New technologies

Publications

Sperm preserved for use in experiments and in vitro
fertilization is traditionally frozen in liquid nitrogen at
extremely low temperatures (around -190˚C). This
cryopreservation allows the sperm to be maintained
viably for very long periods, but requires expensive
facilities and some degree of technical skill in handling. Oocytes and fertilized eggs are also extremely
labile, and must likewise be stored cryogenically.
But these requirements tend to limit the access ofgerm cells to researchers unequipped with liquid
nitrogen facilities, preventing the spread of the technology and the development of research using thesecells. Wakayama hopes to develop new, less expensive and less technically demanding methods for the
storage and maintenance of germ cells for experimental use. Recent tests using a modified commercially available culture medium showed that under
the right conditions spermatozoa can be preserved
for long periods, up to 70 days, at 4 degrees Celsius, a temperature that can be maintained using
ordinary and inexpensive refrigeration equipment.
This new preservation method opens up opportunities in animal breeding and reproductive biology
research to scientists working under limited budgets,
which Wakayama hopes may help to make thesefields of science more accessible to developing
countries and smaller labs.

Singh U, Fohn L, Wakayama
T, Ohgane J, Steinhoff C, Lipkowitz B, Schulz R, Orth A,
Ropers HH, Behringer R,
Tanaka A, Shiota K, Yanagimachi R, Nuber U and Fundele R. Abnormal gene
expression in mouse placentopathies caused by interspecies hybridization, cloning,and Esx1 mutation. Developmental Dynamics (2004).
Wakayama S, Cibelli JB and
Wakayama T. Effect of timing of the removal of oocyte
chromosomes before or after
injection of somatic nucleus
on development of NT
embryos. Cloning Stem Cells
5:181-9 (2003).
Tamashiro KL, Wakayama T,
Akutsu H, Yamazaki Y,
Lachey JL, Wortman MD,
Seeley RJ, D'Alessio DA,
Woods SC, Yanagimachi R
and Sakai RR. Cloned mice
have an obese phenotype
not transmitted to their offspring. Nat Med 8:262-7(2002).

Wakame, the first mouse cloned at the RIKEN CDB

58 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report59

Publications
Payer B, Saitou M, Barton SC, Thresher R, Dixon JP, Zahn D,
Colledge WH, Carlton MB, Nakano T and Surani MA. stella is amaternal effect gene required for normal early development inmice. Curr Biol 13:2110-7 (2003).
Saitou M, Payer B, Lange UC, Erhardt S, Barton SC and Surani MA. Specification of germ cell fate in mice. Philos Trans R
Soc Lond B Biol Sci 358:1363-70 (2003).
Lange UC, Saitou M, Western PS, Barton SC and Surani MA.
The fragilis interferon-inducible gene family of transmembraneproteins is associated with germ cell specification in mice. BMC
Dev Biol 3:1 (2003).

Staff
Team Leader

Saitou M, Barton SC and Surani MA. A molecular programme
for the specification of germ cell fate in mice. Nature 418:293300 (2002).

Mitinori Saitou
Research Scientist

Kazuki Kurimoto
Yasuhide Ohinata
Yukihiro Yabuta

Publications
Ueda HR, Hayashi S, Matsuyama S, Yomo T, Hashimoto S, Kay S A, HogeneschJ B and Iino M. Universality and Flexibility in Gene Expression from Bacteria to
Human. Proc Nat Sci USA (2004).
Ueda H R, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, Nagano M,
Nakahama K, Suzuki Y, Sugano S, Iino M, Shigeyoshi Y and Hashimoto S. A transcription factor response element for gene expression during circadian night.
Nature 418:534-9 (2002).
Ueda H R, Hirose K and Iino M. Intercellular coupling mechanism for synchronized
and noise-resistant circadian oscillators. J Theor Biol 216:501-12 (2002).

Staff

Ueda H R, Matsumoto A, Kawamura M, Iino M, Tanimura T and Hashimoto S.
Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J
Biol Chem 277:14048-52 (2002).

Team Leader

Hiroki R. Ueda
Research Scientist

Ueda H R, Hagiwara M and Kitano H. Robust oscillations within the interlocked
feedback model of Drosophila circadian rhythm. J Theor Biol 210:401-6 (2001).

Hideki Ukai
Collaborative Scientist

Yuichi Kumaki

Collaborative Scientist

Technical Staff

Yukiko Ono

Maki Ukai-Tadenuma
Hiroshi Fujishima
Kenichiro Uno

Technical Staff

Mihoko Yuasa
Mayo Shigeta

Student Trainee

Yoichi Minami
Rikuhiro Yamada
Ryotaku Kito
Assistant

Ikuko Tada

2003
New Faces

Mitinori Saitou
M. D. , Ph. D.
Mitinori Saitou received his
MD from the Kyoto
University
FacultyofMedicine in 1995, and wasawarded a Ph D in 1999 forhis study of the structureand function of mammaliantight junctions under Prof.
Shoichiro Tsukita in the
Kyoto University Graduate
School of Medicine. After abrief fellowship in the samedepartment, he moved to
the Wellcome Trust/Cancer
Research UK Institute in
2000, where worked as a
postdoctoral
research
associate in Azim Surani'slaboratory, focusing on theorigin and properties of thegerm line in the mouse. He
remained there until 2003,
when he returned to Japanto take up his current
position as head of the
CDB
LaboratoryforMammalian Germ Cell
Biology. He received athree-year grant from theJapan
ScienceandTechnology Corporation(JST) PRESTO programthat same year.

A case could be made for the statement that theultimate role of all of the other types of cells in the
body is to ensure that germline cells — sperm andeggs — are able to fulfill their task of conveying
genetic information from one generation to the next.
But despite this centrality, many aspects of germ
cell biology have been, and remain to this day, poorly understood. There has been, however, progressin deepening this understanding, particularly inregard to the mechanisms by which germ cells are
specified and segregated from the larger populationof somatic cells early in development. This isachieved by different means in different species. In
some animals, germ cell fate is predetermined fromthe very onset of development, in others, including
all mammals, all cells in the early embryo have thepotential to take on a germline fate, but only a small
percentage of them actually do.
Mitinori Saitou studies the ways in which germ cellfate is specified in mammalian embryonic development, using the mouse as a representative model.
He joins the CDB from Azim Surani's lab at the Wellcome Trust/Cancer Research UK Institute, where heworked on the single cell analysis of primordial germ

cells, research that ultimately yielded a number ofmolecules that act to distinguish cells on the pathway to differentiation into germ cells. The first ofthese molecules, fragilis, is the product of a genefound to be strongly and specifically expressed incells en route to assuming a germ cell fate, theresult of inductive signaling from cells lying outside
the embryo proper. A second such protein, stella,
acts even more specifically, and is expressed only ina core subset of fragilis-expressing primordial germ
cells. The co-expression of fragilis and stella serves
to distinguish nascent germ cells from the somatic
cell community in the developing embryo, and theiractivity is thought to play a part in the gene networkthat maintains germ cell characteristics and suppresses those of the somatic lineage. Single cell
analysis has also revealed that repression of Hox
genes and maintenance of genes linked to pluripotency, such as Oct4, are the two major events that
occur during germ cell lineage-restriction.
Although progress has been made, much about the
specification of the germline has yet to be explained.
The identification of fragilis and stella now offers ameans to identify incipient germ cells, but the initialmolecular trigger for germ cell
determination and key (epi-)
genetic properties that determined
germ cells acquire are still
unknown. The Saitou lab plans todevelop comprehensive gene
expression analysis at the single
cell level to search for the molecular mechanisms that underlie thisprocess, and to identify the external factors that instruct a pluripotent cell to take on a germ cellfate. The lab also hopes to develop technologies to maintain immature oocytes (egg cells), and to
induce their maturation in vitro,
thereby enabling oocyte-mediatedgene transfer techniques.

Recent large-scale efforts in genome-sequencing
and expression analysis have produced an embarrassment of riches for life science researchers - biological data can now be accessed in quantities thatare orders of magnitude greater than were available
even a few years ago. This burgeoning set of raw
data has not, however, necessarily led to equally
explosive advances in the understanding of the relationships between its component parts. The need for
integration has set the stage for the advent of systems biology, in which discrete biological processes
and phenomena are approached as complex, interactive systems. Hiroki Ueda sees systems biology
research as a multi-stage process, beginning with
the identification and analysis of individual systemcomponents and their networked interactions, andleading to the ability to control existing systems anddesign new ones based on an understanding ofstructure and underlying principles.
The Ueda lab has taken the mammalian circadianclock as a relatively simple and self-contained initialmodel for the study of a biological system. In addition to its advantages as a basic research model,
the function of the circadian clock is intimately
involved in the control of metabolic and hormonal
cycles, and its dysregulation is linked to the onset
and symptomatology of numerous human diseases,
including sleep disorders. An improved understanding at the system level promises to provide biomedical and clinical investigators with a powerful newarsenal to attack these conditions.
To address complex and dynamic
biological systems such as the circadian clock, it is necessary to makecomprehensive and precise measurements of the system's dynamics
and to work out the organization of
its underlying gene network. The
Ueda lab has conducted a genomewide screen and statistical analysis
of gene expression to work out theclock-controlled genes that arerhythmically expressed in the cen-tral (suprachiasmatic nucleus; SCN) and peripheral(liver) circadian clocks. This phase of the study
required the development of a genome-wide promoter database, which the Ueda lab will make available
to all researchers at the CDB. Subsequent phases
involved determining gene transcription start sites
across the entire genome, predicting the regulatory
sequences involved in time-specific transcription,and studying their actual functions in vitro using ahigh-throughput real-time monitoring system forluciferase-tagged transgenes. Analysis of the transcriptional regulation of gene expression in the morning, daytime, evening and night periods revealed agene network comprising sixteen inter-regulating
activators and inhibitors of time-linked gene expression.

2003
New Faces

The initial success of the systems approach to themammalian circadian clock has been encouraging,and the Ueda lab now seeks to apply similar
genome-wide, high-throughput technologies to more
involved and elaborate developmental processes.
Guiding that research will be Einstein's (and the system biologist's) dictum to "Make everything as simple as possible, but not simpler."

Hiroki R. Ueda
Ph. D.

Analysis of the transcriptional regulation of gene
expression in the morning, daytime, evening andnight periods hasrevealed a gene network
comprising more thansixteen component
factors.
E/E'box

D-box

RRE

Gene expression profiles of BMP4 negative single PGCs and neighbouring somatic cells

60 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report

61

Hiroki R. Ueda received his
MD from Faculty of Medicine, the University ofTokyo in 2000, and will
receive PhD from Graduate
School of Medicine, the University of Tokyo in March
2004. While an undergraduate student, he worked as a
research assistant on a biological simulation systemproject at Sony Computer
ScienceLaboratories.
While a graduate student,
he next went on to work,
first as a researcher (2000)
and then as a group leader(2002), at Yamanouchi
Pharmaceutical, on a project studying biologicalclock mechanisms in fly
and mouse. He wasappointed team leader at
the CDB in 2003, and willassume this position fulltime in April 2004.

Publications
Payer B, Saitou M, Barton SC, Thresher R, Dixon JP, Zahn D,
Colledge WH, Carlton MB, Nakano T and Surani MA. stella is amaternal effect gene required for normal early development inmice. Curr Biol 13:2110-7 (2003).
Saitou M, Payer B, Lange UC, Erhardt S, Barton SC and Surani MA. Specification of germ cell fate in mice. Philos Trans R
Soc Lond B Biol Sci 358:1363-70 (2003).
Lange UC, Saitou M, Western PS, Barton SC and Surani MA.
The fragilis interferon-inducible gene family of transmembraneproteins is associated with germ cell specification in mice. BMC
Dev Biol 3:1 (2003).

Staff
Team Leader

Saitou M, Barton SC and Surani MA. A molecular programme
for the specification of germ cell fate in mice. Nature 418:293300 (2002).

Mitinori Saitou
Research Scientist

Kazuki Kurimoto
Yasuhide Ohinata
Yukihiro Yabuta

Publications
Ueda HR, Hayashi S, Matsuyama S, Yomo T, Hashimoto S, Kay S A, HogeneschJ B and Iino M. Universality and Flexibility in Gene Expression from Bacteria to
Human. Proc Nat Sci USA (2004).
Ueda H R, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, Nagano M,
Nakahama K, Suzuki Y, Sugano S, Iino M, Shigeyoshi Y and Hashimoto S. A transcription factor response element for gene expression during circadian night.
Nature 418:534-9 (2002).
Ueda H R, Hirose K and Iino M. Intercellular coupling mechanism for synchronized
and noise-resistant circadian oscillators. J Theor Biol 216:501-12 (2002).

Staff

Ueda H R, Matsumoto A, Kawamura M, Iino M, Tanimura T and Hashimoto S.
Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J
Biol Chem 277:14048-52 (2002).

Team Leader

Hiroki R. Ueda
Research Scientist

Ueda H R, Hagiwara M and Kitano H. Robust oscillations within the interlocked
feedback model of Drosophila circadian rhythm. J Theor Biol 210:401-6 (2001).

Hideki Ukai
Collaborative Scientist

Yuichi Kumaki

Collaborative Scientist

Technical Staff

Yukiko Ono

Maki Ukai-Tadenuma
Hiroshi Fujishima
Kenichiro Uno

Technical Staff

Mihoko Yuasa
Mayo Shigeta

Student Trainee

Yoichi Minami
Rikuhiro Yamada
Ryotaku Kito
Assistant

Ikuko Tada

2003
New Faces

Mitinori Saitou
M. D. , Ph. D.
Mitinori Saitou received his
MD from the Kyoto
University
FacultyofMedicine in 1995, and wasawarded a Ph D in 1999 forhis study of the structureand function of mammaliantight junctions under Prof.
Shoichiro Tsukita in the
Kyoto University Graduate
School of Medicine. After abrief fellowship in the samedepartment, he moved to
the Wellcome Trust/Cancer
Research UK Institute in
2000, where worked as a
postdoctoral
research
associate in Azim Surani'slaboratory, focusing on theorigin and properties of thegerm line in the mouse. He
remained there until 2003,
when he returned to Japanto take up his current
position as head of the
CDB
LaboratoryforMammalian Germ Cell
Biology. He received athree-year grant from theJapan
ScienceandTechnology Corporation(JST) PRESTO programthat same year.

A case could be made for the statement that theultimate role of all of the other types of cells in the
body is to ensure that germline cells — sperm andeggs — are able to fulfill their task of conveying
genetic information from one generation to the next.
But despite this centrality, many aspects of germ
cell biology have been, and remain to this day, poorly understood. There has been, however, progressin deepening this understanding, particularly inregard to the mechanisms by which germ cells are
specified and segregated from the larger populationof somatic cells early in development. This isachieved by different means in different species. In
some animals, germ cell fate is predetermined fromthe very onset of development, in others, including
all mammals, all cells in the early embryo have thepotential to take on a germline fate, but only a small
percentage of them actually do.
Mitinori Saitou studies the ways in which germ cellfate is specified in mammalian embryonic development, using the mouse as a representative model.
He joins the CDB from Azim Surani's lab at the Wellcome Trust/Cancer Research UK Institute, where heworked on the single cell analysis of primordial germ

cells, research that ultimately yielded a number ofmolecules that act to distinguish cells on the pathway to differentiation into germ cells. The first ofthese molecules, fragilis, is the product of a genefound to be strongly and specifically expressed incells en route to assuming a germ cell fate, theresult of inductive signaling from cells lying outside
the embryo proper. A second such protein, stella,
acts even more specifically, and is expressed only ina core subset of fragilis-expressing primordial germ
cells. The co-expression of fragilis and stella serves
to distinguish nascent germ cells from the somatic
cell community in the developing embryo, and theiractivity is thought to play a part in the gene networkthat maintains germ cell characteristics and suppresses those of the somatic lineage. Single cell
analysis has also revealed that repression of Hox
genes and maintenance of genes linked to pluripotency, such as Oct4, are the two major events that
occur during germ cell lineage-restriction.
Although progress has been made, much about the
specification of the germline has yet to be explained.
The identification of fragilis and stella now offers ameans to identify incipient germ cells, but the initialmolecular trigger for germ cell
determination and key (epi-)
genetic properties that determined
germ cells acquire are still
unknown. The Saitou lab plans todevelop comprehensive gene
expression analysis at the single
cell level to search for the molecular mechanisms that underlie thisprocess, and to identify the external factors that instruct a pluripotent cell to take on a germ cellfate. The lab also hopes to develop technologies to maintain immature oocytes (egg cells), and to
induce their maturation in vitro,
thereby enabling oocyte-mediatedgene transfer techniques.

Recent large-scale efforts in genome-sequencing
and expression analysis have produced an embarrassment of riches for life science researchers - biological data can now be accessed in quantities thatare orders of magnitude greater than were available
even a few years ago. This burgeoning set of raw
data has not, however, necessarily led to equally
explosive advances in the understanding of the relationships between its component parts. The need for
integration has set the stage for the advent of systems biology, in which discrete biological processes
and phenomena are approached as complex, interactive systems. Hiroki Ueda sees systems biology
research as a multi-stage process, beginning with
the identification and analysis of individual systemcomponents and their networked interactions, andleading to the ability to control existing systems anddesign new ones based on an understanding ofstructure and underlying principles.
The Ueda lab has taken the mammalian circadianclock as a relatively simple and self-contained initialmodel for the study of a biological system. In addition to its advantages as a basic research model,
the function of the circadian clock is intimately
involved in the control of metabolic and hormonal
cycles, and its dysregulation is linked to the onset
and symptomatology of numerous human diseases,
including sleep disorders. An improved understanding at the system level promises to provide biomedical and clinical investigators with a powerful newarsenal to attack these conditions.
To address complex and dynamic
biological systems such as the circadian clock, it is necessary to makecomprehensive and precise measurements of the system's dynamics
and to work out the organization of
its underlying gene network. The
Ueda lab has conducted a genomewide screen and statistical analysis
of gene expression to work out theclock-controlled genes that arerhythmically expressed in the cen-tral (suprachiasmatic nucleus; SCN) and peripheral(liver) circadian clocks. This phase of the study
required the development of a genome-wide promoter database, which the Ueda lab will make available
to all researchers at the CDB. Subsequent phases
involved determining gene transcription start sites
across the entire genome, predicting the regulatory
sequences involved in time-specific transcription,and studying their actual functions in vitro using ahigh-throughput real-time monitoring system forluciferase-tagged transgenes. Analysis of the transcriptional regulation of gene expression in the morning, daytime, evening and night periods revealed agene network comprising sixteen inter-regulating
activators and inhibitors of time-linked gene expression.

2003
New Faces

The initial success of the systems approach to themammalian circadian clock has been encouraging,and the Ueda lab now seeks to apply similar
genome-wide, high-throughput technologies to more
involved and elaborate developmental processes.
Guiding that research will be Einstein's (and the system biologist's) dictum to "Make everything as simple as possible, but not simpler."

Hiroki R. Ueda
Ph. D.

Analysis of the transcriptional regulation of gene
expression in the morning, daytime, evening andnight periods hasrevealed a gene network
comprising more thansixteen component
factors.
E/E'box

D-box

RRE

Gene expression profiles of BMP4 negative single PGCs and neighbouring somatic cells

60 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report

61

Hiroki R. Ueda received his
MD from Faculty of Medicine, the University ofTokyo in 2000, and will
receive PhD from Graduate
School of Medicine, the University of Tokyo in March
2004. While an undergraduate student, he worked as a
research assistant on a biological simulation systemproject at Sony Computer
ScienceLaboratories.
While a graduate student,
he next went on to work,
first as a researcher (2000)
and then as a group leader(2002), at Yamanouchi
Pharmaceutical, on a project studying biologicalclock mechanisms in fly
and mouse. He wasappointed team leader at
the CDB in 2003, and willassume this position fulltime in April 2004.

Somitogenesis in
chick
en em
bryo
PHO
TO:
Yos
hik
oT
aka
ha
sh
i

The supporting laboratories offer technical services, develop newtechnology, and conduct independent research projects. Their services are available to all CDB research groups and teams.

Somitogenesis in
chick
en em
bryo
PHO
TO:
Yos
hik
oT
aka
ha
sh
i

The supporting laboratories offer technical services, develop newtechnology, and conduct independent research projects. Their services are available to all CDB research groups and teams.

tubules in the mitotic apparatus, and which is essential to ensuring that each progeny cell receives one
and only one set of chromosomes. However, thequestion of which complement of mitotic apparatus
microtubules — astral or central — spatially determines this cleavage remains unresolved. Yonemura
studied Rho accumulation at the putative furrow site,taking this as an index of cleavage plane determination signaling, and analyzed the roles of both microtubule organizations by selectively disrupting each.
The study showed that both astral and central microtubules are able to transmit determinant signals to
the cell cortex, a finding that helps explain how
astral microtubules that extend to the cell cortex canplay an important role in cleavage plane determination in larger cells, such as oocytes, in which central
microtubules are located more remotely from thecortex, while being dispensable in the same process
in smaller cells in which central microtubules arelocated more proximally to the cell's periphery.

Actin fibers andthe adherens junction

Supporting
Laboratories

Shigenobu
Yonemura
Ph. D.
Shigenobu
Yonemura
received his B. Sc., M. Sc. and
Ph. D. from the University ofTokyo, earning his doctorate in1988 for thesis work under
Prof. I. Mabuchi. He spent a
year as a postdoctoral fellow
at the same institution before
moving to pursue a fellowshipat Johns Hopkins University
from 1989 to 1990. He
returned to Japan as an assistant professor in the Department of Cell Biology at the
National Institute for Physiological Sciences, where heremained until 1995. He joined
the Kyoto University Faculty of
Medicine the same year, firstas an assistant professor, thenas a lecturer in the Department of Cell Biology, a position he retained until his
appointment as CDB teamleader in 2001.

Cellular morphology is defined by interactions involving mechanical and osmotic stresses from the cell's
external environment, its permeable membrane, its
viscous cytosolic content, and an internal frameworkcomprising pre-stressed tubular struts, tensile
cables and a lattice-like filamentous cortex. Microtubules are one important and extensively studiedcomponent of this cytoskeletal structure, acting bothas internal stress-bearing braces and pathways toguide intracellular transport. A second critical formgiving element, actin filaments, are most prevalent
in cellular peripheries, where they provide a stable
but regulable meshwork enabling the cell to maintain its structural integrity while responding to external and internal stimuli through shape alterations
and movements.

Actin filaments maintain cellstructural integrity while
responding to external andinternal stimuli
The behavior of actin is intimately linked to the activity of a group of related small GTPase proteins
known as the Rho family, which is characterized by
the actin-regulatory functions of its three primary
members, Rho, Rac and Cdc42. The spatial andtemporal localization of Rho-family proteins has
been shown to be important in the structural organization of cells in culture. Shigenobu Yonemura is
working to develop technologies to allow the precise
determination of the localization of these proteins in
cultured cells and tissues within specific biologicalcontexts.

64 RIKEN CDB 2003 Annual Report

Cleavage plane determination
The final stages of mitotic cell division involve a
process known as cytokinesis, in which the parent
cell cleaves in two, partitioning cytoplasmic
elements and genetic information into each of thedaughter cells. Cytokinesis begins with theappearance of a cleavage furrow at the cell surface,a process which is known to be specified by micro-

NRK cells at cytokinesis. Rho (magenta) accumulates at the equatorial cell cortex depending on microtubule organizations (green).

NRK cells contacting each other thorough adherens junctions (magenta) where actin filament bundles (green) are tightly connected.

tion and of actin bundles. The
study showed that disruption ofactin bundles affects the integrityof the adherens junction, indicating the importance of Rho-actin
signaling in this form of cell-celladhesion.

ERM protein
recognition-site
structure
ERM proteins, named for three
representative members of thefamily, ezrin, radixin and moesin, act as links
between actin filaments and the plasma membrane.
Yonemura and colleagues studied the crystal structure of a functional domain of radixin to identify the
structural basis for adhesion molecule recognition
by one ERM protein, radixin. Radixin plays a role in
the formation of microvilli, cellular extensions inwhich actin filaments are closely associated with theplasma membrane. The importance of radixin insites of bilirubin secretion is suggested by the finding that mice lacking radixin, which is the dominant
ERM protein in the liver, show severely impaired
hepatic microvilli formation, resulting in a phenotype
similar to human conjugated hyperbilirubinemia, arare autosomal recessive liver disorder. This
work provides the first
insights into the structural bases for the recognition between an ERM
protein and an adhesion
molecule, and may
further serve as anexperimental model forthe study of molecular
processes involved inbile secretion.

Staff
Team Leader

Shigenobu Yonemura
Research Scientist

Yukako Nishimura
Research Specialist

Kazuyo Minakuchi
Research Associate

Nagatoki Kinoshita
Technical Staff

Naoko Inoue
Makiko Uwo
Yuka Miyake
Student Trainee

The main zone of contact between a cell and its
neighbors is called the adherens junction, which isdefined by the selective binding of transmembraneadhesion molecules that are themselves anchoredto molecular complexes
located within each cell'sinterior. In fibroblastic
cells in culture, bundles ofactin filaments that run
perpendicular to theplasma membrane areconnected to the components of the adherens junction, a phenomenon thatis also observed in epithelial cell adhesion. In manycells, the mature adherens junction forms a
contiguous belt, although
In addition to its full-time
during the process of its
research efforts, theMouse neutrophils migrating on a substratum. Phase contrast (blue)
formationitpassesYonemura lab offers
and fluorescence image of ERM proteins (magenta) are merged.
through a stage charactersite-wide support forized by discrete, spot-like adhesions. The Yonemura
electron microscopy, services available to alllab used NRK cells, a type of non-polarized fibroresearch staff at the CDB. By providing fluorescence
blast which forms spot-like junction zones, to study
and electron microscopic visualizations as well as
the role of actin fibers in adherens junction formainstruction in techniques, the team hopes to help
tion. In this cell line, the adherens junction and its
ensure the quality, fidelity and ease-of-interpretation
associated actin bundles can be disrupted andof visualization data generated atreconstituted by removing or adding serum to thethe Center.
culture medium. Using this system, herevealed that the activity of thesmall GTPase, Rho, isrequired both for theformation of theadherens
j u n c-Yuko Shimada

Publications
Hamada K, Shimizu T, Yonemura S, Tsukita S and
Hakoshima T. Structural
basis of adhesion-molecule
recognition by ERM proteins
revealed by the crystal structure of the radixin-ICAM-2
complex. Embo J 22:502-14(2003).
Fujikura J, Yamato E, Yonemura S, Hosoda K, Masui S,
Nakao K, Miyazaki Ji J and
Niwa H. Differentiation ofembryonic stem cells is
induced by GATA factors.
Genes Dev 16:784-9 (2002).
Kikuchi S, Hata M, Fukumoto
K, Yamane Y, Matsui T,
Tamura A, Yonemura S,
Yamagishi H, Keppler D and
Tsukita S. Radixin deficiency
causes conjugated hyperbilirubinemia with loss of Mrp2
from bile canalicular membranes. Nat Genet 31:320-5(2002).
Yonemura S, Matsui T and
Tsukita S. Rho-dependentand -independent activation
mechanisms of ezrin/radixin/moesin proteins: anessential role for polyphosphoinositides in vivo. J
Cell Sci 115:2569-80 (2002).

The tip of a villus of thesmall intestine of the rat.
Aged cells that are exfoliated highly express Rho
protein (green).

RIKEN CDB 2003 Annual Report65

tubules in the mitotic apparatus, and which is essential to ensuring that each progeny cell receives one
and only one set of chromosomes. However, thequestion of which complement of mitotic apparatus
microtubules — astral or central — spatially determines this cleavage remains unresolved. Yonemura
studied Rho accumulation at the putative furrow site,taking this as an index of cleavage plane determination signaling, and analyzed the roles of both microtubule organizations by selectively disrupting each.
The study showed that both astral and central microtubules are able to transmit determinant signals to
the cell cortex, a finding that helps explain how
astral microtubules that extend to the cell cortex canplay an important role in cleavage plane determination in larger cells, such as oocytes, in which central
microtubules are located more remotely from thecortex, while being dispensable in the same process
in smaller cells in which central microtubules arelocated more proximally to the cell's periphery.

Actin fibers andthe adherens junction

Supporting
Laboratories

Shigenobu
Yonemura
Ph. D.
Shigenobu
Yonemura
received his B. Sc., M. Sc. and
Ph. D. from the University ofTokyo, earning his doctorate in1988 for thesis work under
Prof. I. Mabuchi. He spent a
year as a postdoctoral fellow
at the same institution before
moving to pursue a fellowshipat Johns Hopkins University
from 1989 to 1990. He
returned to Japan as an assistant professor in the Department of Cell Biology at the
National Institute for Physiological Sciences, where heremained until 1995. He joined
the Kyoto University Faculty of
Medicine the same year, firstas an assistant professor, thenas a lecturer in the Department of Cell Biology, a position he retained until his
appointment as CDB teamleader in 2001.

Cellular morphology is defined by interactions involving mechanical and osmotic stresses from the cell's
external environment, its permeable membrane, its
viscous cytosolic content, and an internal frameworkcomprising pre-stressed tubular struts, tensile
cables and a lattice-like filamentous cortex. Microtubules are one important and extensively studiedcomponent of this cytoskeletal structure, acting bothas internal stress-bearing braces and pathways toguide intracellular transport. A second critical formgiving element, actin filaments, are most prevalent
in cellular peripheries, where they provide a stable
but regulable meshwork enabling the cell to maintain its structural integrity while responding to external and internal stimuli through shape alterations
and movements.

Actin filaments maintain cellstructural integrity while
responding to external andinternal stimuli
The behavior of actin is intimately linked to the activity of a group of related small GTPase proteins
known as the Rho family, which is characterized by
the actin-regulatory functions of its three primary
members, Rho, Rac and Cdc42. The spatial andtemporal localization of Rho-family proteins has
been shown to be important in the structural organization of cells in culture. Shigenobu Yonemura is
working to develop technologies to allow the precise
determination of the localization of these proteins in
cultured cells and tissues within specific biologicalcontexts.

64 RIKEN CDB 2003 Annual Report

Cleavage plane determination
The final stages of mitotic cell division involve a
process known as cytokinesis, in which the parent
cell cleaves in two, partitioning cytoplasmic
elements and genetic information into each of thedaughter cells. Cytokinesis begins with theappearance of a cleavage furrow at the cell surface,a process which is known to be specified by micro-

NRK cells at cytokinesis. Rho (magenta) accumulates at the equatorial cell cortex depending on microtubule organizations (green).

NRK cells contacting each other thorough adherens junctions (magenta) where actin filament bundles (green) are tightly connected.

tion and of actin bundles. The
study showed that disruption ofactin bundles affects the integrityof the adherens junction, indicating the importance of Rho-actin
signaling in this form of cell-celladhesion.

ERM protein
recognition-site
structure
ERM proteins, named for three
representative members of thefamily, ezrin, radixin and moesin, act as links
between actin filaments and the plasma membrane.
Yonemura and colleagues studied the crystal structure of a functional domain of radixin to identify the
structural basis for adhesion molecule recognition
by one ERM protein, radixin. Radixin plays a role in
the formation of microvilli, cellular extensions inwhich actin filaments are closely associated with theplasma membrane. The importance of radixin insites of bilirubin secretion is suggested by the finding that mice lacking radixin, which is the dominant
ERM protein in the liver, show severely impaired
hepatic microvilli formation, resulting in a phenotype
similar to human conjugated hyperbilirubinemia, arare autosomal recessive liver disorder. This
work provides the first
insights into the structural bases for the recognition between an ERM
protein and an adhesion
molecule, and may
further serve as anexperimental model forthe study of molecular
processes involved inbile secretion.

Staff
Team Leader

Shigenobu Yonemura
Research Scientist

Yukako Nishimura
Research Specialist

Kazuyo Minakuchi
Research Associate

Nagatoki Kinoshita
Technical Staff

Naoko Inoue
Makiko Uwo
Yuka Miyake
Student Trainee

The main zone of contact between a cell and its
neighbors is called the adherens junction, which isdefined by the selective binding of transmembraneadhesion molecules that are themselves anchoredto molecular complexes
located within each cell'sinterior. In fibroblastic
cells in culture, bundles ofactin filaments that run
perpendicular to theplasma membrane areconnected to the components of the adherens junction, a phenomenon thatis also observed in epithelial cell adhesion. In manycells, the mature adherens junction forms a
contiguous belt, although
In addition to its full-time
during the process of its
research efforts, theMouse neutrophils migrating on a substratum. Phase contrast (blue)
formationitpassesYonemura lab offers
and fluorescence image of ERM proteins (magenta) are merged.
through a stage charactersite-wide support forized by discrete, spot-like adhesions. The Yonemura
electron microscopy, services available to alllab used NRK cells, a type of non-polarized fibroresearch staff at the CDB. By providing fluorescence
blast which forms spot-like junction zones, to study
and electron microscopic visualizations as well as
the role of actin fibers in adherens junction formainstruction in techniques, the team hopes to help
tion. In this cell line, the adherens junction and its
ensure the quality, fidelity and ease-of-interpretation
associated actin bundles can be disrupted andof visualization data generated atreconstituted by removing or adding serum to thethe Center.
culture medium. Using this system, herevealed that the activity of thesmall GTPase, Rho, isrequired both for theformation of theadherens
j u n c-Yuko Shimada

Publications
Hamada K, Shimizu T, Yonemura S, Tsukita S and
Hakoshima T. Structural
basis of adhesion-molecule
recognition by ERM proteins
revealed by the crystal structure of the radixin-ICAM-2
complex. Embo J 22:502-14(2003).
Fujikura J, Yamato E, Yonemura S, Hosoda K, Masui S,
Nakao K, Miyazaki Ji J and
Niwa H. Differentiation ofembryonic stem cells is
induced by GATA factors.
Genes Dev 16:784-9 (2002).
Kikuchi S, Hata M, Fukumoto
K, Yamane Y, Matsui T,
Tamura A, Yonemura S,
Yamagishi H, Keppler D and
Tsukita S. Radixin deficiency
causes conjugated hyperbilirubinemia with loss of Mrp2
from bile canalicular membranes. Nat Genet 31:320-5(2002).
Yonemura S, Matsui T and
Tsukita S. Rho-dependentand -independent activation
mechanisms of ezrin/radixin/moesin proteins: anessential role for polyphosphoinositides in vivo. J
Cell Sci 115:2569-80 (2002).

The tip of a villus of thesmall intestine of the rat.
Aged cells that are exfoliated highly express Rho
protein (green).

RIKEN CDB 2003 Annual Report65

Publications
K Chida, T Hara, T Hirai, C Konishi, K
Nakamura, K Nakao, A Aiba, M Katsuki
and T Kuroki. Disruption of Protein Kinase
Ceta Results in Impairment of Wound
Healing and Enhancement of Tumor Formation in Mouse Skin Carcinogenesis.
Cancer Res 63:2404-8 (2003).
J M Kim, K Nakao, K Nakamura, I Saito,
M Katsuki, K Arai and H Masai. Inactivation of Cdc7 Kinase in Mouse ES Cells
Results in S-Phase Arrest and P53Dependent Cell Death. Embo J 21:216879 (2002).
K Kuwahara, S Fujimura, Y Takahashi, N
Nakagata, T Takemori, S Aizawa and N
Sakaguchi. Germinal Center-AssociatedNuclear Protein Contributes to Affinity Maturation of B Cell Antigen Receptor in T
Cell-Dependent Responses. Proc Natl
Acad Sci U S A (2004).
S Muto, A Aiba, Y Saito, K Nakao, K
Nakamura, K Tomita, T Kitamura, M Kurabayashi, R Nagai, E Higashihara, P C Harris, M Katsuki and S Horie. Pioglitazone
Improves the Phenotype and Molecular
Defects of a Targeted Pkd1 Mutant. Hum
Mol Genet 11:1731-42 (2002).

Supporting
Laboratories

Staff
Team Leader

Shinichi Aizawa
Attending Veterinarian

Naoko Kagiyama
Research Associate

Kazuki Nakao
Special Postdoctoral Researcher

Kenryo Furushima

S Takaki, Y Tezuka, K Sauer, C Kubo, S
M Kwon, E Armstead, K Nakao, M Katsuki, R M Perlmutter and K Takatsu.
Impaired Lymphopoiesis and Altered B
Cell Subpopulations in Mice Overexpressing Lnk Adaptor Protein. J Immunol
170:703-10 (2003).

The study of model organisms and systems is integral to biological research. Such systems provide
scientists with the means to search for broadly
shared mechanisms underlying developmental andregenerative processes across species, andconversely to identify those traits that earn eachspecies its unique branch on the phylogenetic tree.
The mouse is one of the most important and widelyused model organisms in science today, prized for
its amenability to genetic manipulation, its high levelof homology with humans, and the trove of data
regarding its physiology, genetics and developmentthat has accumulated over nearly one century ofintensive scientific research.

Y Wakabayashi, H Watanabe, J Inoue, N
Takeda, J Sakata, Y Mishima, J Hitomi, T
Yamamoto, M Utsuyama, O Niwa, S Aizawa and R Kominami. Bcl11b Is Required
for Differentiation and Survival of Alphabeta T Lymphocytes. Nat Immunol 4:5339 (2003).

Senior Technical Staff

Hitoshi Miyachi
Rika Nakayama
Hiroshi Kiyonari
Technical Staff

Chiaki Tamamura
Akemi Hara
Takaya Abe
Naoko Ohshima
Hirotake Nishino
AssistantKaori Nasu

The Laboratory for Animal Resources and Genetic
Engineering (LARGE) provides an important suite ofservices related to the generation of experimental
mice to labs within the CDB and around Japan. In its
role as a CDB support laboratory, the LARGE teamproduces transgenic and knockout mouse models tothe specifications ofscientists working ina wide range ofgenetic, embryological and biomedical
research projects,
maintaining the highest quality standards
and rapid turnaround to ensure fastand easy access toresearchers working
within the Centerand throughout thecountry. In addition

66 RIKEN CDB 2003 Annual Report

Chimera miceto these core functions, the LARGE staff provides anumber of other services, such as cloning by
nuclear transfer and cryopreservation of mousezygotes and sperm. The lab also performs a numberof maintenance and logistical functions, such as the
specific pathogen free (SPF) housing, cleaning,processing and distribution of animals.

In 2003, the LARGE team started to
expand its services and initiate newprograms, notably the generation oftarget vectors from sequence information alone, and the independent
production of novel geneticallymodified constructs, a drive that generates on the order of 40-50 new mutant
strains per year. Such strains serve
as research platforms with the potential to provide new insights into a
range of important research problems,from the developmental mechanismsof organ development to the genetic
bases of human disease. The lab will
also function as part of Japan's
system of Mouse Embryo Banks, witha special emphasis on producing, storing and cataloging embryos for use indevelopmental biology and regenerative medical research.

Supporting
Laboratories

The CDB Sequencing Laboratory provides DNA sequencing and analysis services for use by all laboratories at theCenter. Equipment is available for small, medium andlarge-scale requests, with turnaround times ranging fromunder one week for medium-scale projects to one monthfor larger jobs, at rates of up to 1,920 samples per day.
Smaller request are fulfilled especially rapidly, and results
can be delivered as soon as the following day whensequence-ready samples are submitted. Analysis services
for editing, homology search, andassembling are also available.

Staff
Research Scientist

Hiroshi Tarui
Technical Staff

Junko Ueda
Kenichiro Uno
Mikako Dohi
Takashige Hakozaki
Yukako Hirao

In addition to DNA sequencing, thelab also offers a full range of DNA
microarray services, from the amplification of target DNA and the preparation of probe cDNA, to microarray
image data analysis. All work
requests can be submitted andtracked online, and results are
returned to the applicant's personal
folder on the CDB intranet
file server, making it possible for CDB research staff
to sequence samples ofinterest without leaving
their desktop computers.
The lab also stores anddistributes DNA resources,
making clones available toall members of the CDB
staff.

RIKEN CDB 2003 Annual Report67

Publications
K Chida, T Hara, T Hirai, C Konishi, K
Nakamura, K Nakao, A Aiba, M Katsuki
and T Kuroki. Disruption of Protein Kinase
Ceta Results in Impairment of Wound
Healing and Enhancement of Tumor Formation in Mouse Skin Carcinogenesis.
Cancer Res 63:2404-8 (2003).
J M Kim, K Nakao, K Nakamura, I Saito,
M Katsuki, K Arai and H Masai. Inactivation of Cdc7 Kinase in Mouse ES Cells
Results in S-Phase Arrest and P53Dependent Cell Death. Embo J 21:216879 (2002).
K Kuwahara, S Fujimura, Y Takahashi, N
Nakagata, T Takemori, S Aizawa and N
Sakaguchi. Germinal Center-AssociatedNuclear Protein Contributes to Affinity Maturation of B Cell Antigen Receptor in T
Cell-Dependent Responses. Proc Natl
Acad Sci U S A (2004).
S Muto, A Aiba, Y Saito, K Nakao, K
Nakamura, K Tomita, T Kitamura, M Kurabayashi, R Nagai, E Higashihara, P C Harris, M Katsuki and S Horie. Pioglitazone
Improves the Phenotype and Molecular
Defects of a Targeted Pkd1 Mutant. Hum
Mol Genet 11:1731-42 (2002).

Supporting
Laboratories

Staff
Team Leader

Shinichi Aizawa
Attending Veterinarian

Naoko Kagiyama
Research Associate

Kazuki Nakao
Special Postdoctoral Researcher

Kenryo Furushima

S Takaki, Y Tezuka, K Sauer, C Kubo, S
M Kwon, E Armstead, K Nakao, M Katsuki, R M Perlmutter and K Takatsu.
Impaired Lymphopoiesis and Altered B
Cell Subpopulations in Mice Overexpressing Lnk Adaptor Protein. J Immunol
170:703-10 (2003).

The study of model organisms and systems is integral to biological research. Such systems provide
scientists with the means to search for broadly
shared mechanisms underlying developmental andregenerative processes across species, andconversely to identify those traits that earn eachspecies its unique branch on the phylogenetic tree.
The mouse is one of the most important and widelyused model organisms in science today, prized for
its amenability to genetic manipulation, its high levelof homology with humans, and the trove of data
regarding its physiology, genetics and developmentthat has accumulated over nearly one century ofintensive scientific research.

Y Wakabayashi, H Watanabe, J Inoue, N
Takeda, J Sakata, Y Mishima, J Hitomi, T
Yamamoto, M Utsuyama, O Niwa, S Aizawa and R Kominami. Bcl11b Is Required
for Differentiation and Survival of Alphabeta T Lymphocytes. Nat Immunol 4:5339 (2003).

Senior Technical Staff

Hitoshi Miyachi
Rika Nakayama
Hiroshi Kiyonari
Technical Staff

Chiaki Tamamura
Akemi Hara
Takaya Abe
Naoko Ohshima
Hirotake Nishino
AssistantKaori Nasu

The Laboratory for Animal Resources and Genetic
Engineering (LARGE) provides an important suite ofservices related to the generation of experimental
mice to labs within the CDB and around Japan. In its
role as a CDB support laboratory, the LARGE teamproduces transgenic and knockout mouse models tothe specifications ofscientists working ina wide range ofgenetic, embryological and biomedical
research projects,
maintaining the highest quality standards
and rapid turnaround to ensure fastand easy access toresearchers working
within the Centerand throughout thecountry. In addition

66 RIKEN CDB 2003 Annual Report

Chimera miceto these core functions, the LARGE staff provides anumber of other services, such as cloning by
nuclear transfer and cryopreservation of mousezygotes and sperm. The lab also performs a numberof maintenance and logistical functions, such as the
specific pathogen free (SPF) housing, cleaning,processing and distribution of animals.

In 2003, the LARGE team started to
expand its services and initiate newprograms, notably the generation oftarget vectors from sequence information alone, and the independent
production of novel geneticallymodified constructs, a drive that generates on the order of 40-50 new mutant
strains per year. Such strains serve
as research platforms with the potential to provide new insights into a
range of important research problems,from the developmental mechanismsof organ development to the genetic
bases of human disease. The lab will
also function as part of Japan's
system of Mouse Embryo Banks, witha special emphasis on producing, storing and cataloging embryos for use indevelopmental biology and regenerative medical research.

Supporting
Laboratories

The CDB Sequencing Laboratory provides DNA sequencing and analysis services for use by all laboratories at theCenter. Equipment is available for small, medium andlarge-scale requests, with turnaround times ranging fromunder one week for medium-scale projects to one monthfor larger jobs, at rates of up to 1,920 samples per day.
Smaller request are fulfilled especially rapidly, and results
can be delivered as soon as the following day whensequence-ready samples are submitted. Analysis services
for editing, homology search, andassembling are also available.

Staff
Research Scientist

Hiroshi Tarui
Technical Staff

Junko Ueda
Kenichiro Uno
Mikako Dohi
Takashige Hakozaki
Yukako Hirao

In addition to DNA sequencing, thelab also offers a full range of DNA
microarray services, from the amplification of target DNA and the preparation of probe cDNA, to microarray
image data analysis. All work
requests can be submitted andtracked online, and results are
returned to the applicant's personal
folder on the CDB intranet
file server, making it possible for CDB research staff
to sequence samples ofinterest without leaving
their desktop computers.
The lab also stores anddistributes DNA resources,
making clones available toall members of the CDB
staff.

RIKEN CDB 2003 Annual Report67Animal Facility

Administration and
Activities

Mouse biology provides a valuable platform for investigations into many of the fundamental processes and mechanisms of mammalian physiology and metabolism, and is widelyused as a model organism in developmental biology research. With the sequencing of
the mouse genome, mice can now be modified for use in research into the developmental consequences of genetic alterations, and as models of human disease.
The CDB animal facility provides the technology, equipment and staff to enable thehumane, hygienic, efficient and economical handling of large numbers of experimental
mice. This 24,000 cage SPF facility has thecapacity to handle and care for up to 100,000
mice. Cutting edge technology, such as semiautomated cage cleaning, makes it possible tomaintain high standards of hygiene and sterility, minimizing the risk of outbreaks of contagion among the mouse populations. The
facility also provides services for the production of transgenic and knockout mice to specifications and on demand.

Research Aquarium
The CDB research aquarium serves as home to a number ofwater-dwelling species used in research into development
and regeneration, including the zebrafish (Danio rerio) andthe African clawed frog (Xenopus laevis). These, and otheraquatic species, provide useful models for studies ranging
from developmental genomics to classical embryology.
The aquarium includes seven climate-controlled rooms
geared to providing optimal environments for the handling and breeding of freshwaterand marine species, and utilizes reverse osmotic technology to maintain consistent tankwater purity. In addition to the commonlyused zebrafish and African clawed frog, thefacility also houses specimens from morenovel models used in evolutionary development research, such as the lamprey,
Lampetra japonica.

Photo: Yutaka Doi

68 RIKEN CDB 2003 Annual ReportAnimal Facility

Administration and
Activities

Mouse biology provides a valuable platform for investigations into many of the fundamental processes and mechanisms of mammalian physiology and metabolism, and is widelyused as a model organism in developmental biology research. With the sequencing of
the mouse genome, mice can now be modified for use in research into the developmental consequences of genetic alterations, and as models of human disease.
The CDB animal facility provides the technology, equipment and staff to enable thehumane, hygienic, efficient and economical handling of large numbers of experimental
mice. This 24,000 cage SPF facility has thecapacity to handle and care for up to 100,000
mice. Cutting edge technology, such as semiautomated cage cleaning, makes it possible tomaintain high standards of hygiene and sterility, minimizing the risk of outbreaks of contagion among the mouse populations. The
facility also provides services for the production of transgenic and knockout mice to specifications and on demand.

Research Aquarium
The CDB research aquarium serves as home to a number ofwater-dwelling species used in research into development
and regeneration, including the zebrafish (Danio rerio) andthe African clawed frog (Xenopus laevis). These, and otheraquatic species, provide useful models for studies ranging
from developmental genomics to classical embryology.
The aquarium includes seven climate-controlled rooms
geared to providing optimal environments for the handling and breeding of freshwaterand marine species, and utilizes reverse osmotic technology to maintain consistent tankwater purity. In addition to the commonlyused zebrafish and African clawed frog, thefacility also houses specimens from morenovel models used in evolutionary development research, such as the lamprey,
Lampetra japonica.

Photo: Yutaka Doi

68 RIKEN CDB 2003 Annual ReportGraduate School
Affiliates2003 CDB Budget

Encouraging young scientists to participate in advanced research projects as they prepare for careers in the life sciences is essential toensuring the development of future generations of researchers. The
CDB has collaborative educational programs with a number of graduate schools in Western Japan, providing students with opportunities
for hands-on laboratory benchwork and lectures on topics in development and regeneration. The center also has also accepted 50 students from other universities as research associates or trainees. In addition to regularly scheduled scientific seminars, students can attend
events such as the CDB Forum research progress series and the Center's annual retreat. The Junior Research Associate program established by RIKEN also provides financial support to 8 graduate students
working in CDB labs.

Ko b eUni v

The Center for Developmental Biology was launched in April 2000 under the auspices of the Millennium Project
research initiative launched by former Prime Minister Keizo Obuchi. The Millennium Projects were established to drive
research in the fields of information technology, environmental science and the study of aging, areas of vital importanceto both Japan and the world in the 21st century. The organization and operations of the CDB were consigned to RIKEN,
which coordinates the construction management and administration of the Center.

er si

t y,
Gra

FY2003

du

762

ate

Sc
ho

1827

ol
of
Sc

ie

nc

2732
Facility Expenses(energy expenses, maintenance,etc.)

Research Management Expenses(personnel expenses, etc.)(Million Yen)

Research Expenses

CDB Staff (December 2002)

Advisory Council

GD/TL/UL
Research scientists
Research associates
Technical staff
Assistants
Visiting researchers
Collaborative scientists
Student trainees
Part time staff
Research promotion division
Other

30
101
3
105
25
9
30
47
53
43
65

The CDB Advisory Council, chaired by Dr Igor Dawid (National Institute of Child Health and Human Development, NIH) submits regular external reports regarding the scientific administration and the state of research progress at the Center. The
ten-member Council includes top scientists in related fields
from Japan and around the world and serves as an unbiased
review board for CDB research activities.

Total

511

70 RIKEN CDB 2003 Annual Report

Na

ra

e

an

Ad

d

va

Te

nc

G rad

ch

ed

no

in s

lo g

y

titu

Dep

te o

ua t e School of Biostudies, Kyoto University

G radu
a rtm

f Sc

ent o

f L ife S

ience

ate School of Medicine, Kyoto University

cience De

and Tepartment of Developmental and Regenerative medicine

c h no logy

K w a n sei

University of Texas Graduate School of Biom
edica
l Sc
ien
e
Temasek Life Scienc s Laboratory
ces

Graduate School of Biological Sciences

Gakuin University

Bioscience Department

at
Ho
us
to
n

Institutional Review Board
The Institutional Review Board includes representatives fromlocal academic, research and lay organizations as well as
CDB research staff, and meets regularly to review and discuss the ethical and social implications of programs and investigations being conducted at the CDB. The results of theBoard's discussions are submitted to the Center Director andtaken into consideration when planning research activities.

Scientific Exchange
Programs
The CDB is also engaged in programs of cooperation with researchorganizations in other parts of Japan and around the world. This
activity is based primarily on cooperation between individual
research laboratories. These scientific exchange agreements
facilitate processes such as the hosting and dispatch of researchstaff for international collaborative research, the joint sponsorship ofscientific meetings and the sharing of intellectual property producedin collaborations.

RIKEN CDB 2003 Annual Report71Graduate School
Affiliates2003 CDB Budget

Encouraging young scientists to participate in advanced research projects as they prepare for careers in the life sciences is essential toensuring the development of future generations of researchers. The
CDB has collaborative educational programs with a number of graduate schools in Western Japan, providing students with opportunities
for hands-on laboratory benchwork and lectures on topics in development and regeneration. The center also has also accepted 50 students from other universities as research associates or trainees. In addition to regularly scheduled scientific seminars, students can attend
events such as the CDB Forum research progress series and the Center's annual retreat. The Junior Research Associate program established by RIKEN also provides financial support to 8 graduate students
working in CDB labs.

Ko b eUni v

The Center for Developmental Biology was launched in April 2000 under the auspices of the Millennium Project
research initiative launched by former Prime Minister Keizo Obuchi. The Millennium Projects were established to drive
research in the fields of information technology, environmental science and the study of aging, areas of vital importanceto both Japan and the world in the 21st century. The organization and operations of the CDB were consigned to RIKEN,
which coordinates the construction management and administration of the Center.

er si

t y,
Gra

FY2003

du

762

ate

Sc
ho

1827

ol
of
Sc

ie

nc

2732
Facility Expenses(energy expenses, maintenance,etc.)

Research Management Expenses(personnel expenses, etc.)(Million Yen)

Research Expenses

CDB Staff (December 2002)

Advisory Council

GD/TL/UL
Research scientists
Research associates
Technical staff
Assistants
Visiting researchers
Collaborative scientists
Student trainees
Part time staff
Research promotion division
Other

30
101
3
105
25
9
30
47
53
43
65

The CDB Advisory Council, chaired by Dr Igor Dawid (National Institute of Child Health and Human Development, NIH) submits regular external reports regarding the scientific administration and the state of research progress at the Center. The
ten-member Council includes top scientists in related fields
from Japan and around the world and serves as an unbiased
review board for CDB research activities.

Total

511

70 RIKEN CDB 2003 Annual Report

Na

ra

e

an

Ad

d

va

Te

nc

G rad

ch

ed

no

in s

lo g

y

titu

Dep

te o

ua t e School of Biostudies, Kyoto University

G radu
a rtm

f Sc

ent o

f L ife S

ience

ate School of Medicine, Kyoto University

cience De

and Tepartment of Developmental and Regenerative medicine

c h no logy

K w a n sei

University of Texas Graduate School of Biom
edica
l Sc
ien
e
Temasek Life Scienc s Laboratory
ces

Graduate School of Biological Sciences

Gakuin University

Bioscience Department

at
Ho
us
to
n

Institutional Review Board
The Institutional Review Board includes representatives fromlocal academic, research and lay organizations as well as
CDB research staff, and meets regularly to review and discuss the ethical and social implications of programs and investigations being conducted at the CDB. The results of theBoard's discussions are submitted to the Center Director andtaken into consideration when planning research activities.

Scientific Exchange
Programs
The CDB is also engaged in programs of cooperation with researchorganizations in other parts of Japan and around the world. This
activity is based primarily on cooperation between individual
research laboratories. These scientific exchange agreements
facilitate processes such as the hosting and dispatch of researchstaff for international collaborative research, the joint sponsorship ofscientific meetings and the sharing of intellectual property producedin collaborations.

RIKEN CDB 2003 Annual Report71Leading Project in
In 2003, the Japanese Ministry of Education, Culture,
Sports, Science and Technology (MEXT) inaugurated anumber of Leading Projects to drive research into areas
identified as having great potential to make contributions
to the nation's economy, environment, social climate or
health and welfare. One such project was introduced topromote research leading to applications for stem cells
in regenerative medicine.

The implementation of the Leading Project in Regenerative
Medicine has been consigned to universities and researchorganizations across Japan, and a number of CDB labs
have received funding to conduct research into key areas
including the experimental manipulation and maintenanceof stem cells, the reprogramming and targeted differentiation of somatic stem cells, and the ex vivo reconstitution ofphysiological structures with potential clinical uses in regenerative medicine. Shin-ichi Nishikawa, Group Director of the
CDB Laboratory for Stem Cell Biology, was appointed tohead this Leading Project.

Regenerative Medicine
Two new Research Unit laboratories have been recruited to conduct
goal-oriented, fixed-term research projects as part of this program.
These Research Units are funded by Leading Project grants and arefinancially independent of RIKEN, but operate, in whole or in part, inlaboratories located within the RIKEN CDB campus, enabling them tocapitalize on the extensive shared-use equipment and facilities at theCenter.

Organ Regeneration

Cell Plasticity

Mitsuko Kosaka
Ph. D.
Unit Leader

Mitsuko Kosaka
Research Scientist

Maki Asami
Junji Tsuchida

Mitsuko Kosaka heads the Research Unit for Cell Plasticity, whichseeks to determine the limits and possibilities in the differentiative
potential of somatic stem cells. Their work focuses on differentiation in the iris of the eye, a population of pigmented cells that
contains stem cells that show an intriguing plasticity, or ability toassume more than one cellular fate. The Kosaka lab will investigate the mechanisms by which stem cells in the iris can be
induced to give rise to cells of other types, such as photoreceptive
neural cells. This work is of great clinical promise in the treatmentof vision loss due to nerve damage, as cells from the iris are readily obtainable and may one day be used as an autologous sourceof retinal replacement cells if they can be steered reliably to takeup a neural fate. Recent findings from the lab suggesting that iris
stem cells express genes characteristic of highly undifferentiatedpluripotent stem cells also offer new avenues for exploration forresearchers studying the genetic regulation of differentiative
potency.

The Research Unit for Organ Regeneration, headed by Hideki Taniguchi, focuses on studies of the differentiative and regulatory
mechanisms underlying endodermal tissues and organs in thedigestive system, which include liver, pancreas, intestine and salivary glands. By analyzing stem cells isolated from each of thesetissue types, the Taniguchi lab seeks to confirm the existence of"endodermal stem cells" that enable the high levels of cellular plasticity seen in digestive organs. Using cell sorting technologies, Taniguchi is working to characterize these putative endodermal stem
cells by sorting and analyzing cells from the livers and other digestive organs of fetal mice. The identification of molecular markers
specific to such cells will make it possible to isolate them by flow
cytometry and to pursue further investigations into their functionalroles in the development of the endoderm-derived digestivesystem. The ability to identify and isolate stem cells capable ofgiving rise to endodermal lineage tissue types would also be ofenormous potential value in the development of regenerative medical applications for the treatment of diabetes.

Hideki Taniguchi
M. D. , Ph. D.
Unit Leader

Hideki Taniguchi
Collaborative Scientist

Takeshi Sugaya
Technical Staff

In a concurrent project, Taniguchi is working to develop a system
for isolating and guiding the differentiation of stem cells from thepancreases of adult mice, which is aimed at establishing the foundations for cell replacement therapies using insulin-producing
pancreatic beta cells grown in vitro.

Midori Yokura
Naoyo Kajitani (2004)
Student Trainee

Masato Sugano (2004)

Visiting Scientist

Guangwei Sun

72 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report73Leading Project in
In 2003, the Japanese Ministry of Education, Culture,
Sports, Science and Technology (MEXT) inaugurated anumber of Leading Projects to drive research into areas
identified as having great potential to make contributions
to the nation's economy, environment, social climate or
health and welfare. One such project was introduced topromote research leading to applications for stem cells
in regenerative medicine.

The implementation of the Leading Project in Regenerative
Medicine has been consigned to universities and researchorganizations across Japan, and a number of CDB labs
have received funding to conduct research into key areas
including the experimental manipulation and maintenanceof stem cells, the reprogramming and targeted differentiation of somatic stem cells, and the ex vivo reconstitution ofphysiological structures with potential clinical uses in regenerative medicine. Shin-ichi Nishikawa, Group Director of the
CDB Laboratory for Stem Cell Biology, was appointed tohead this Leading Project.

Regenerative Medicine
Two new Research Unit laboratories have been recruited to conduct
goal-oriented, fixed-term research projects as part of this program.
These Research Units are funded by Leading Project grants and arefinancially independent of RIKEN, but operate, in whole or in part, inlaboratories located within the RIKEN CDB campus, enabling them tocapitalize on the extensive shared-use equipment and facilities at theCenter.

Organ Regeneration

Cell Plasticity

Mitsuko Kosaka
Ph. D.
Unit Leader

Mitsuko Kosaka
Research Scientist

Maki Asami
Junji Tsuchida

Mitsuko Kosaka heads the Research Unit for Cell Plasticity, whichseeks to determine the limits and possibilities in the differentiative
potential of somatic stem cells. Their work focuses on differentiation in the iris of the eye, a population of pigmented cells that
contains stem cells that show an intriguing plasticity, or ability toassume more than one cellular fate. The Kosaka lab will investigate the mechanisms by which stem cells in the iris can be
induced to give rise to cells of other types, such as photoreceptive
neural cells. This work is of great clinical promise in the treatmentof vision loss due to nerve damage, as cells from the iris are readily obtainable and may one day be used as an autologous sourceof retinal replacement cells if they can be steered reliably to takeup a neural fate. Recent findings from the lab suggesting that iris
stem cells express genes characteristic of highly undifferentiatedpluripotent stem cells also offer new avenues for exploration forresearchers studying the genetic regulation of differentiative
potency.

The Research Unit for Organ Regeneration, headed by Hideki Taniguchi, focuses on studies of the differentiative and regulatory
mechanisms underlying endodermal tissues and organs in thedigestive system, which include liver, pancreas, intestine and salivary glands. By analyzing stem cells isolated from each of thesetissue types, the Taniguchi lab seeks to confirm the existence of"endodermal stem cells" that enable the high levels of cellular plasticity seen in digestive organs. Using cell sorting technologies, Taniguchi is working to characterize these putative endodermal stem
cells by sorting and analyzing cells from the livers and other digestive organs of fetal mice. The identification of molecular markers
specific to such cells will make it possible to isolate them by flow
cytometry and to pursue further investigations into their functionalroles in the development of the endoderm-derived digestivesystem. The ability to identify and isolate stem cells capable ofgiving rise to endodermal lineage tissue types would also be ofenormous potential value in the development of regenerative medical applications for the treatment of diabetes.

Hideki Taniguchi
M. D. , Ph. D.
Unit Leader

Hideki Taniguchi
Collaborative Scientist

Takeshi Sugaya
Technical Staff

In a concurrent project, Taniguchi is working to develop a system
for isolating and guiding the differentiation of stem cells from thepancreases of adult mice, which is aimed at establishing the foundations for cell replacement therapies using insulin-producing
pancreatic beta cells grown in vitro.

Midori Yokura
Naoyo Kajitani (2004)
Student Trainee

Masato Sugano (2004)

Visiting Scientist

Guangwei Sun

72 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report732003 Seminars
The CDB hosts regular seminars by distinguished speakers from within Japan and abroadas part of its committed effort to promote the borderless exchange of scientificinformation. All CDB seminars are held in English. In addition to these seminars, the CDB
also holds monthly Forums, in which CDB researchers share findings with theircolleagues, and a range of informal lecture offerings in English and Japanese.

Date

Title

Speaker

Date

Title

Speaker

2003.1.15

Hox genes and brainstem patterning

Filippo M Rijli

2003.8.26

Impaired spermatogenesis in mice lacking Caf1

Tadashi Yamamoto

2003.1.16

Neural patterning in vertebrate embryogenesis

Nobue Itasaki

2003.9.11

Mechanisms controlling heart and blood vessel growth

Kenneth Walsh

2003.1.17

Left-right asymmetry in the zebrafish forebrain

Marnie Halpern

2003.9.12

Genomic approaches for functional annotation of the mammalian genome

John Hogenesch2003.1.27

Patterning formation in neocortical area by FGF8

Tomomi Fukuchi-Shimogori

2003.9.19

Genetic analysis of mouse forebrain and midbrain development

Siew-Lan Ang

2003.9.29

Fibronectin requirement in branching morphogenesis

Takayoshi Sakai

2003.1.29

To branch or not to branch: HAMLET controls dendritic arbor branching and lineage
restriction in the Drosophila peripheral nervous system

Adrian Moore
2003.10.3

Chemical modulation of embryonic development and oncogenesis

James K Chen

2003.2.21

Wnt signaling in development and disease

Randall T. Moon

2003.10.10

Hox in leukemia and as avenues to hematopoietic stem cell expansion

R Keith Humphries

2003.3.11

The new revolution in developmental biology: Expansion and reconciliation

Scott Gilbert
2003.10.14

Imaging cell movements that pattern the vertebrate embryo

Scott Fraser

Characterisation of two modulators of the FGF signal transduction pathway,and
their role in axial polarity

Michael Tsang2003.3.27
2003.3.27

Induction and evolution of the neural crest

Marianne Bronner Fraser

2003.10.14

Patterning neurogenesis in the zebrafish embryoMotoyuki Itoh2003.10.16

Endoderm as a source of patterning information for early chick embryogenesis

Gary Schoenwolf

2003.3.27

Functional expression cloning of Nanog, a gene conferring cytokine independent ES
cell self-renewal

2003.10.23

Structure and function of Spen proteins: A conserved repressive function indevelopmental signalling pathways

Mariko Ariyoshi

2003.10.24

Regulation of temporal and spatial patterns of gene expression in Dictyostelium

William Loomis

2003.10.27

Phylogenetic conservation of a cis-acting regulator that controls polarized
expression of Sonic hedgehog (Shh) in limb buds

Toshihiko Shiroishi

2003.10.29

Sniffing out smell's code and map

Naoshige Uchida2003.11.14

Molecular mechanisms specifying muscle-identity and myotube number in the
Drosophila adult

K.VijayRaghavan

Ian Chambers

2003.4.22

Differentiation of embryonic stem cells into hepatocytes: biological functions andtherapeutic applications

Takahiro Ochiya

2003.4.22

ES cells as a model system for pancreatic differentiation

Shoen Kume

2003.5.13

Dynamics of cellular interactions during T cell recognition in 3-dimensional tissue
visualized by two-photon microscopy

Ellen Robey

2003.5.16

BMP-3b and BMP-3 function as different dorsalizing factors in Xenopus embryos

Shin-ichiro Nishimatsu

2003.5.21

Building brains from embryonic stem cells: Naive cultures offer greatest potential

Hidemasa Kato

2003.5.22

Considerations in microarray experimental design and data analysis

Ali R Zareh

2003.11.18

Genesis of the neural primordia and their regional diversities through regulation of Sox2

Hisato Kondoh

2003.6.18

The role of myosin VI in the spermatogenesis of Drosophila

Noguchi Tatsuhiko

2003.11.20

Nuclear interpretation of the Dpp morphogen in Drosophila melanogaster

Markus Affolter

Cellular functions and molecular mechanisms of signaling pathways mediated bytarget of rapamycin

2003.11.26

Genes that regulate the formation and regeneration of skeletal muscle

Margaret Buckingham

2003.6.23

Kazuyoshi Yonezawa
2003.11.27

Patterning and growth in Drosophila limb development

Konrad Basler

2003.7.3

Diversity of CNR/protocadherin family in the brain

Takeshi Yagi
2003.12.2

Membrane traffic: Molecular mechanisms and real-time imaging

Akihiko Nakano

Secondary rearrangement of TcR alpha chain in vivo: shaping and reshaping
T cell repertoire

Osami Kanagawa

2003.7.4
2003.7.8

Ontogeny of the hematopoietic system

M Bruno Peault
2003.12.3

PAX6 and SOX2 in disease, development and evolution

Veronica van Heyningen

Proteolytic cleavage of meiotic cohesin REC8 by separase is required formurine spermatogenesis

Nobuaki Kubo

2003.7.17
2003.7.18

Quiescent hematopoietic stem cells in the nicheToshio Suda

2003.12.5

Function and regulation of centralspindlin, a kinesin/RhoGAP complex, in cytokinesis

Masanori Mishima

2003.8.4

Manipulating mouse genome from single gene knockout to chromosome engineering

Ichiko Nishijima

2003.12.9

Eph receptors and ephrins in neural map development

Masaru Nakamoto

2003.8.4

Genetic regulation of retinal interneuron differentiation

Akihira Otoshi

2003.12.15

Establishment of the vertebrate body plan in relation to limb formation

Mikiko Tanaka

2003.8.6

Self-renewal of neural stem cells and CNS-repair

Hideyuki Okano

2003.12.15

Genetic cascades that regulate gastrulation cell movements in zebrafish

Masazimi Tada

2003.8.7

Regulation of spindle orientation by centrosome and APC tumor suppressor

Yukiko M Yamashita

2003.12.16

Wnt-mediated axon guidance at the midline of Drosophila

Shingo Yoshikawa

2003.8.8

A protein interaction matrix for an integrin adhesion complex within C. elegans muscle

Hiroshi Qadota

2003.12.26

Msx genes function in neural crest development

Mamoru Ishii

74 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report752003 Seminars
The CDB hosts regular seminars by distinguished speakers from within Japan and abroadas part of its committed effort to promote the borderless exchange of scientificinformation. All CDB seminars are held in English. In addition to these seminars, the CDB
also holds monthly Forums, in which CDB researchers share findings with theircolleagues, and a range of informal lecture offerings in English and Japanese.

Date

Title

Speaker

Date

Title

Speaker

2003.1.15

Hox genes and brainstem patterning

Filippo M Rijli

2003.8.26

Impaired spermatogenesis in mice lacking Caf1

Tadashi Yamamoto

2003.1.16

Neural patterning in vertebrate embryogenesis

Nobue Itasaki

2003.9.11

Mechanisms controlling heart and blood vessel growth

Kenneth Walsh

2003.1.17

Left-right asymmetry in the zebrafish forebrain

Marnie Halpern

2003.9.12

Genomic approaches for functional annotation of the mammalian genome

John Hogenesch2003.1.27

Patterning formation in neocortical area by FGF8

Tomomi Fukuchi-Shimogori

2003.9.19

Genetic analysis of mouse forebrain and midbrain development

Siew-Lan Ang

2003.9.29

Fibronectin requirement in branching morphogenesis

Takayoshi Sakai

2003.1.29

To branch or not to branch: HAMLET controls dendritic arbor branching and lineage
restriction in the Drosophila peripheral nervous system

Adrian Moore
2003.10.3

Chemical modulation of embryonic development and oncogenesis

James K Chen

2003.2.21

Wnt signaling in development and disease

Randall T. Moon

2003.10.10

Hox in leukemia and as avenues to hematopoietic stem cell expansion

R Keith Humphries

2003.3.11

The new revolution in developmental biology: Expansion and reconciliation

Scott Gilbert
2003.10.14

Imaging cell movements that pattern the vertebrate embryo

Scott Fraser

Characterisation of two modulators of the FGF signal transduction pathway,and
their role in axial polarity

Michael Tsang2003.3.27
2003.3.27

Induction and evolution of the neural crest

Marianne Bronner Fraser

2003.10.14

Patterning neurogenesis in the zebrafish embryoMotoyuki Itoh2003.10.16

Endoderm as a source of patterning information for early chick embryogenesis

Gary Schoenwolf

2003.3.27

Functional expression cloning of Nanog, a gene conferring cytokine independent ES
cell self-renewal

2003.10.23

Structure and function of Spen proteins: A conserved repressive function indevelopmental signalling pathways

Mariko Ariyoshi

2003.10.24

Regulation of temporal and spatial patterns of gene expression in Dictyostelium

William Loomis

2003.10.27

Phylogenetic conservation of a cis-acting regulator that controls polarized
expression of Sonic hedgehog (Shh) in limb buds

Toshihiko Shiroishi

2003.10.29

Sniffing out smell's code and map

Naoshige Uchida2003.11.14

Molecular mechanisms specifying muscle-identity and myotube number in the
Drosophila adult

K.VijayRaghavan

Ian Chambers

2003.4.22

Differentiation of embryonic stem cells into hepatocytes: biological functions andtherapeutic applications

Takahiro Ochiya

2003.4.22

ES cells as a model system for pancreatic differentiation

Shoen Kume

2003.5.13

Dynamics of cellular interactions during T cell recognition in 3-dimensional tissue
visualized by two-photon microscopy

Ellen Robey

2003.5.16

BMP-3b and BMP-3 function as different dorsalizing factors in Xenopus embryos

Shin-ichiro Nishimatsu

2003.5.21

Building brains from embryonic stem cells: Naive cultures offer greatest potential

Hidemasa Kato

2003.5.22

Considerations in microarray experimental design and data analysis

Ali R Zareh

2003.11.18

Genesis of the neural primordia and their regional diversities through regulation of Sox2

Hisato Kondoh

2003.6.18

The role of myosin VI in the spermatogenesis of Drosophila

Noguchi Tatsuhiko

2003.11.20

Nuclear interpretation of the Dpp morphogen in Drosophila melanogaster

Markus Affolter

Cellular functions and molecular mechanisms of signaling pathways mediated bytarget of rapamycin

2003.11.26

Genes that regulate the formation and regeneration of skeletal muscle

Margaret Buckingham

2003.6.23

Kazuyoshi Yonezawa
2003.11.27

Patterning and growth in Drosophila limb development

Konrad Basler

2003.7.3

Diversity of CNR/protocadherin family in the brain

Takeshi Yagi
2003.12.2

Membrane traffic: Molecular mechanisms and real-time imaging

Akihiko Nakano

Secondary rearrangement of TcR alpha chain in vivo: shaping and reshaping
T cell repertoire

Osami Kanagawa

2003.7.4
2003.7.8

Ontogeny of the hematopoietic system

M Bruno Peault
2003.12.3

PAX6 and SOX2 in disease, development and evolution

Veronica van Heyningen

Proteolytic cleavage of meiotic cohesin REC8 by separase is required formurine spermatogenesis

Nobuaki Kubo

2003.7.17
2003.7.18

Quiescent hematopoietic stem cells in the nicheToshio Suda

2003.12.5

Function and regulation of centralspindlin, a kinesin/RhoGAP complex, in cytokinesis

Masanori Mishima

2003.8.4

Manipulating mouse genome from single gene knockout to chromosome engineering

Ichiko Nishijima

2003.12.9

Eph receptors and ephrins in neural map development

Masaru Nakamoto

2003.8.4

Genetic regulation of retinal interneuron differentiation

Akihira Otoshi

2003.12.15

Establishment of the vertebrate body plan in relation to limb formation

Mikiko Tanaka

2003.8.6

Self-renewal of neural stem cells and CNS-repair

Hideyuki Okano

2003.12.15

Genetic cascades that regulate gastrulation cell movements in zebrafish

Masazimi Tada

2003.8.7

Regulation of spindle orientation by centrosome and APC tumor suppressor

Yukiko M Yamashita

2003.12.16

Wnt-mediated axon guidance at the midline of Drosophila

Shingo Yoshikawa

2003.8.8

A protein interaction matrix for an integrin adhesion complex within C. elegans muscle

Hiroshi Qadota

2003.12.26

Msx genes function in neural crest development

Mamoru Ishii

74 RIKEN CDB 2003 Annual Report

RIKEN CDB 2003 Annual Report75

RIKEN
Photodynamics Research Center
519-1399, Aoba, Aramaki, Aoba-ku, Sendai,
Miyagi 980-0845
Tel.+81-22-228-2111 Fax.+81-22-228-2122

Tsukuba Insitute

The First Annual

CDB Symposium
The Origin and Formation of Multicellular Systems
The CDB held its first annual symposium at the nearby Portopia Hotel.
The meeting drew more than 200 speakers and attendees fromaround the world, with lectures, poster presentations and lively discussion on the origins of metazoan biology, the fundamental processes ofdevelopment and regeneration and the formation of higher-order structures, such as neural networks.
The Symposium was organized into five sessions over three days,
with full and short talks by 28 distinguished speakers.

Tuesday, March 25

Session 1
The Origin of Multicellular Systems

Session 3
Cellular Polarization andAsymmetric Division

9:40 am

9:40 am

10:00 am Hitoshi Suga, Kyoto University

Peter W Holland, Oxford University

9:00 am

9:40 am

10:00 am 10:40 am Nicole King, University of Wisconsin

9:40 am

10:20 am Anthony A Hyman, Max Planck Institute

10:50 am 11:30 am Yoko Watanabe,

10:40 am 11:20 am Yuh-Nung Jan, UCSF

National Science Museum, Japan

Session 2
Germ Cells, Stem Cells andRegeneration

Session 4
Intercellular Signaling andMorphogenetic Cell Behavior

1:30 pm

Judith C Kimble, University of

1:30 pm

2:10 pm

Hiroshi Hamada, Osaka University

Wisconsin-Madison

2:10 pm

2:50 pm

Edward M DeRobertis, UCLA

Allan Spradling, Carnegie Institution of

2:50 pm

3:30 pm

Olivier Pourquie, Stowers Institute for

2:50 pm

Washington
3:30 pm

Yuzo Niki, Ibaraki University

3:50 pm

4:10 pm

Yasumasa Bessho, Kyoto University

3:30 pm

4:10 pm

Christopher Wylie,

4:10 pm

4:30 pm

Yukiko Gotoh, University of Tokyo

Cincinnati Children's Hospital

4:30 pm

5:10 pm

Elaine Fuchs,The Rockefeller University

5:10 pm

5:50 pm

Tom Curran, St Jude Childrenís Research

8:40 pm

Shin-ichi Nishikawa, RIKEN CDB

8:40 pm

9:00 pm

Ian Chambers, University of Edinburgh

9:00 pm

9:40 pm

Kiyokazu Agata, RIKEN CDB

Tokyo
Kyoto
Osaka

Harima Institute1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148
Tel.+81-791-58-0808 Fax.+81-791-58-0800

Hospital
Wednesday, March 26

Session 5
Cellular Basis for
Neuronal Circuit Formation
9:00 am

9:40 am

9:40 am

10:20 am Peter Mombaerts, The Rockefeller

Hajime Fujisawa, Nagoya University
University

10:40 am 11:20 am Christine E Holt, Cambridge University
11:20 am 12:00 pm Mu-ming Poo, UC Berkeley

76 RIKEN CDB 2003 Annual Report(In Nagoya Science Park Research and Development Center)
2271-130, Anagahora, Shidami, Moriyama-ku,
Nagoya, Aichi 463-0003
Tel.+81-52-736-5850 Fax.+81-52-736-5854

Medical Research

3:10 pm

8:00 pm

Genomic Sciences Center
Plant Science Center
SNP Research Center
Research Center for Allergy and Immunology
1-7-22, Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045
Tel.+81-45-503-9111 Fax.+81-45-503-9113

11:00 am 11:40 am Fumio Matsuzaki, RIKEN CDB
11:40 am 12:00 pm Tadashi Uemura, Kyoto University

2:10 pm

Yokohama InstituteDavid Ish-Horowicz, Cancer Research UK

11:30 am 12:10 pm Peter Devreotes, Johns Hopkins University

2:10 pm

Wako Main CampusWako InstituteDiscovery Research Institute
Frontier Research System
Brain Science Institute2-1, Hirosawa, Wako, Saitama 351-0198
Tel.+81-48-462-1111 Fax.+81-48-462-1554

Bio-Mimetic Control Research Center

Monday, March 24

9:00 am

BioResource Center
3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074
Tel.+81-29-836-9111 Fax.+81-29-836-9109

RIKEN is a publicly funded research organization established to conduct comprehensive research in science and technology and to disseminate the results of its scientific research andtechnological developments. RIKEN carries out basic and appliedresearch in a wide range of fields, including physics, chemistry, medical science, biology, and engineering.

Kobe InstituteCenter for Developmental Biology
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047
Tel.+81-78-306-0111 Fax.+81-78-306-0101

RIKEN was first founded in 1917 as a private research foundation, Rikagaku Kenkyuusho (The Institute of Physical and Chemical Research). In 2003, the Institutewas reorganized as an Independent Administrative Institution under the Ministry of
Education, Culture, Sports, Science and Technology (MEXT), since which time it has
engaged in wide-ranging research activities spanning the basic and applied sciences.

RIKEN
Photodynamics Research Center
519-1399, Aoba, Aramaki, Aoba-ku, Sendai,
Miyagi 980-0845
Tel.+81-22-228-2111 Fax.+81-22-228-2122

Tsukuba Insitute

The First Annual

CDB Symposium
The Origin and Formation of Multicellular Systems
The CDB held its first annual symposium at the nearby Portopia Hotel.
The meeting drew more than 200 speakers and attendees fromaround the world, with lectures, poster presentations and lively discussion on the origins of metazoan biology, the fundamental processes ofdevelopment and regeneration and the formation of higher-order structures, such as neural networks.
The Symposium was organized into five sessions over three days,
with full and short talks by 28 distinguished speakers.

Tuesday, March 25

Session 1
The Origin of Multicellular Systems

Session 3
Cellular Polarization andAsymmetric Division

9:40 am

9:40 am

10:00 am Hitoshi Suga, Kyoto University

Peter W Holland, Oxford University

9:00 am

9:40 am

10:00 am 10:40 am Nicole King, University of Wisconsin

9:40 am

10:20 am Anthony A Hyman, Max Planck Institute

10:50 am 11:30 am Yoko Watanabe,

10:40 am 11:20 am Yuh-Nung Jan, UCSF

National Science Museum, Japan

Session 2
Germ Cells, Stem Cells andRegeneration

Session 4
Intercellular Signaling andMorphogenetic Cell Behavior

1:30 pm

Judith C Kimble, University of

1:30 pm

2:10 pm

Hiroshi Hamada, Osaka University

Wisconsin-Madison

2:10 pm

2:50 pm

Edward M DeRobertis, UCLA

Allan Spradling, Carnegie Institution of

2:50 pm

3:30 pm

Olivier Pourquie, Stowers Institute for

2:50 pm

Washington
3:30 pm

Yuzo Niki, Ibaraki University

3:50 pm

4:10 pm

Yasumasa Bessho, Kyoto University

3:30 pm

4:10 pm

Christopher Wylie,

4:10 pm

4:30 pm

Yukiko Gotoh, University of Tokyo

Cincinnati Children's Hospital

4:30 pm

5:10 pm

Elaine Fuchs,The Rockefeller University

5:10 pm

5:50 pm

Tom Curran, St Jude Childrenís Research

8:40 pm

Shin-ichi Nishikawa, RIKEN CDB

8:40 pm

9:00 pm

Ian Chambers, University of Edinburgh

9:00 pm

9:40 pm

Kiyokazu Agata, RIKEN CDB

Tokyo
Kyoto
Osaka

Harima Institute1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148
Tel.+81-791-58-0808 Fax.+81-791-58-0800

Hospital
Wednesday, March 26

Session 5
Cellular Basis for
Neuronal Circuit Formation
9:00 am

9:40 am

9:40 am

10:20 am Peter Mombaerts, The Rockefeller

Hajime Fujisawa, Nagoya University
University

10:40 am 11:20 am Christine E Holt, Cambridge University
11:20 am 12:00 pm Mu-ming Poo, UC Berkeley

76 RIKEN CDB 2003 Annual Report(In Nagoya Science Park Research and Development Center)
2271-130, Anagahora, Shidami, Moriyama-ku,
Nagoya, Aichi 463-0003
Tel.+81-52-736-5850 Fax.+81-52-736-5854

Medical Research

3:10 pm

8:00 pm

Genomic Sciences Center
Plant Science Center
SNP Research Center
Research Center for Allergy and Immunology
1-7-22, Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045
Tel.+81-45-503-9111 Fax.+81-45-503-9113

11:00 am 11:40 am Fumio Matsuzaki, RIKEN CDB
11:40 am 12:00 pm Tadashi Uemura, Kyoto University

2:10 pm

Yokohama InstituteDavid Ish-Horowicz, Cancer Research UK

11:30 am 12:10 pm Peter Devreotes, Johns Hopkins University

2:10 pm

Wako Main CampusWako InstituteDiscovery Research Institute
Frontier Research System
Brain Science Institute2-1, Hirosawa, Wako, Saitama 351-0198
Tel.+81-48-462-1111 Fax.+81-48-462-1554

Bio-Mimetic Control Research Center

Monday, March 24

9:00 am

BioResource Center
3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074
Tel.+81-29-836-9111 Fax.+81-29-836-9109

RIKEN is a publicly funded research organization established to conduct comprehensive research in science and technology and to disseminate the results of its scientific research andtechnological developments. RIKEN carries out basic and appliedresearch in a wide range of fields, including physics, chemistry, medical science, biology, and engineering.

Kobe InstituteCenter for Developmental Biology
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047
Tel.+81-78-306-0111 Fax.+81-78-306-0101

RIKEN was first founded in 1917 as a private research foundation, Rikagaku Kenkyuusho (The Institute of Physical and Chemical Research). In 2003, the Institutewas reorganized as an Independent Administrative Institution under the Ministry of
Education, Culture, Sports, Science and Technology (MEXT), since which time it has
engaged in wide-ranging research activities spanning the basic and applied sciences.

ISSN 1349-0494

RIKEN Center for Developmental Biology

2003 Annual Report

http://www.cdb.riken.jp
CENTER FOR DEVELOPMENTAL BIOLOGY (CDB)
2-2-3 MINATOJIMA-MINAMIMACHI, CHUO-KU
KOBE, 650-0047
JAPAN
PHONE: +81-78-306-0111 FAX: +81-78-306-0101
EMAIL: contact@cdb.riken.jp

Production
Text
Douglas Sipp
Content Development
Naoki Namba
Douglas Sipp
DesignTRAIS K. K.
Photography
Yutaka Doi

On The Cover
Terminal branch of the Drosophila
trachea. A single terminal celllabeled with GFP (green) with a large
polyploid nucleus (purple) spreads
numerous cytoplasmic tubules withintracellular lumen (tracheole) overthe surface of the larval gut.
PHOTO: Shigeo Hayashi

All contents copyright (c) RIKEN Center for Developmental Biology, 2004.

Printed in Japan
Printed on 100%Recycled Paper

RIKEN Center for Developmental Biology

2003 Annual Report