RIKEN Center for Developmental Biology

2009 Annual ReportContents

Message from the Center Director

2

Organizational Chart

4

2009 at the CDB

6News Highlights 1

10

Labs
Vertebrate Body Plan

18

Shinichi AIZAWA
Neuronal Differentiation
and Regeneration

19

Hideki ENOMOTO
Neocortical Development

20

Carina HANASHIMA
Morphogenetic Signaling

21

Shigeo HAYASHI
Vertebrate Axis Formation

22

Masahiko HIBI
Cell Lineage Modulation

23

Toru KONDO
Evolutionary Morphology

24

Shigeru KURATANI
Sensory Development

25

Raj LADHER
Cell Asymmetry

26

Fumio MATSUZAKIRIKEN Center for
Developmental Biology

2009
AnnualReport

The Center for Developmental Biology was launched in
April 2000 under the auspices of the Millennium Project
research initiative that was established to drive researchin 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. Locatedin Kobe, Japan, the Center’s research program isdedicated to developing a better understanding offundamental processes of animal development at themolecular and cell biological level, the even more
complex phenomena involved in organogenesis, and theNews Highlights 2

28

Labs
Germline Development

38
39
40
41
42
43
44

Developmental Genomics

59

Retinal Regeneration

60

Cell Adhesion and Tissue Patterning

61

Systems Biology

62

Hiroki R. UEDA
45

Mitinori SAITOU
Organogenesis and Neurogenesis

58

Masatoshi TAKEICHI

Tony PERRY
Mammalian Germ Cell Biology

Early Embryogenesis

Masayo TAKAHASHI

Masaki OKANO
Mammalian Molecular Embryology

57

Asako SUGIMOTO

Hitoshi NIWA
Mammalian Epigenetic Studies

Cell Fate Decision

Guojun SHENG

Takashi NISHIMURA
Pluripotent Cell Studies

56

Hitoshi SAWA

Shin-Ichi NISHIKAWA
Growth Control Signaling

Embryonic Induction
Hiroshi SASAKI

Jun-ichi NAKAYAMA
Stem Cell Biology

48

Labs

Akira NAKAMURA
Chromatin Dynamics

News Highlights 3

Genomic Reprogramming

63

Teruhiko WAKAYAMA
46

Yoshiki SASAI

biology of stem cells and regeneration. Scientists at the
CDB conduct world-class studies in a truly international
research environment, with ample core facilities andtechnical support and a critical mass of scientific talentand expertise. The CDB also benefits from belonging toJapan’s largest basic scientific research organization,
RIKEN, as well as its close ties to graduate and medical
schools and research institutes in Japan and othercountries, giving our scientists access to a broad networkof talent and technology across the country and aroundthe world.

News Highlights 466

Supporting Laboratories

72

RIKEN Kobe Institute80

CDB Symposium

82

CDB Seminars

84

About RIKEN

86

RIKEN Campuses

87

4

Message from the Center Director
The past year has seen the RIKEN Center for Developmental Biology continue in its tradition of advanced research into themechanisms of development, regeneration, and stem cell biology, as well as take the first steps in the second decade since
its establishment in 2000. The pace of change in these areas over the past ten years has truly been breathtaking, and we areproud as an institution to have been able to play at least some small part in pushing back the frontiers of our knowledge inthese exciting fields of inquiry.

Research labs at the CDB made important strides on a number of fronts in 2009, publishing important new findings in areas
ranging from the origins of the germline in mammals, to the genetic regulation of pluripotent stem cells, to the evolutionary
advent of the turtle carapace. Studies of developmental phenomena in the nervous system were a particular highlight, generating novel insights into the formation of the olfactory system and the inner ear, as well as the molecular underpinnings ofneuronal connectivity. Other studies focused on regulatory mechanisms that govern the circadian clock, and methods formeasuring the state of these internal timekeepers.

Our mission continues to emphasize an unfettered, creative approach to fundamental research into the mechanisms of development, organogenesis and regeneration, but within that broad remit we also recognize the importance of concentrated
focus on specific areas. To this end, we have established a pair of Strategic Programs within the CDB to allow for dedicatedand sustained support for research into stem cells and systems biology. Research projects in these programs are expected
not only to make significant contributions to individual scientific questions, but also to serve as platforms for spurring research advances and technology development across the entire field.

2009 was also a year of transitions, a welcome development as the Center grows and our scientists, many of whom enteredas fledgling investigators, move on to the next phases of their careers. By the end of the 2009 fiscal year, nearly one-thirdof the original 30 CDB laboratories will have made the transition to a new home, and I am pleased to be able to say that allof our research leader alumni have gone on to head laboratories at major universities in Japan and abroad. Our younger research scientists have also done extremely well in their post-CDB appointments, gaining fellowships and faculty positions at
research institutions throughout Europe, North America, and Asia. For our part, we welcomed one new research team andtwo new support units to the CDB in the past twelve months, and look forward to a new generation of research leaders andscientists to join us in the years to come.

Our commitment to outreach, dialogue, and international collaboration remains as strong as ever, and we conducted or
participated in numerous programs designed to foster mutual understanding of scientific issues and developments between
researchers and the public. We made special efforts to engage with the educational community, continuing in our summer
school programs for local high school students and workshops for educators. We similarly continue to support the researchcommunity in Japan by offering practical courses in techniques for pluripotent stem cell culture and differentiation several
times per year.

The excitement and anticipation surrounding the fields of development, organogenesis, stem cells and regenerative medicine
remains undiminished as we enter the second decade of the new century, and we will continue to dedicate ourselves to bettering the understanding of how bodies come to be, how they maintain themselves, and how those that are damaged might
one day be healed.

Masatoshi Takeichi
Director, RIKEN Center for Developmental Biology

5

RIKEN Kobe InstituteCore
Program
The Core Program aims to promote highly
innovative developmental and regeneration
studies in a strategic and multi-disciplinarymanner. This program focuses on the main
themes of the CDB: the mechanisms ofdevelopment, organogenesis, and strengthening the scientific basis of regenerative
medicine through the study of stem cells andregeneration.

Center for
Developmental
Biology
The Center for Developmental Biology (CDB) is a research
center within the RIKEN Kobe Institute, which also comprises
the Center for Molecular Imaging Science (CMIS), the Kobe
Research Promotion Division, which provides administrative
services and support, and the institutional Safety Center. The
CDB is home to a total of 34 laboratories in its Core Research
Program (7 groups), Center’s Director Strategic Programs (2projects), Creative Research Promoting Program (19 teams)
and Supporting Laboratories (6 labs). The CDB Director is
assisted by two Deputy Directors and is advised by the Advisory Council and the Institutional Review Board.

Vertebrate Body Plan
Shinichi AIZAWA Ph.D.

Morphogenetic Signaling
Shigeo HAYASHI Ph.D.

Evolutionary Morphology
Shigeru KURATANI Ph.D.

Cell Asymmetry
Fumio MATSUZAKI Ph.D.

Stem Cell Biology
Shin-Ichi NISHIKAWA M.D., Ph.D.

Organogenesis and Neurogenesis
Yoshiki SASAI M.D., Ph.D.

Cell Adhesion and Tissue Patterning

Center for Molecular Imaging Science

Masatoshi TAKEICHI Ph.D.

Research Promotion Division

Center Director’sStrategic Program

Safety Center

Institutional Review Board
The Institutional Review Board includes representatives from local academic,
research, medical, and lay organizations, as well as CDB research leaders, andmeets regularly to review and discuss programs and investigations with potential ethical, social, legal or public health and safety implications prior to theirimplementation.

This program allows for concentrated focus onpriority areas of research determined by the
CDB Center Director. The initial 10-year projects
will focus on stem cell and systems biology.

Pluripotent stem Cell Studies
Hitoshi NIWA M.D., Ph.D.

Systems Biology
Hiroki R. UEDA M.D., Ph.D.

Advisory Council
The CDB Advisory Council (DBAC) convenes regularly toreview the per formance and direction of the Center ’s
research and governance, and submits reports of its
findings and recommendations to aid in guiding future
activities and decision-making. The ten-member council
comprises top international scientists working in developmental biology, stem cells, and related fields.

6

Austin Smith, DBAC Chair

Haifan Lin

Margaret Buckingham

Toshio Suda

Stephen Cohen

Yoshimi Takai

University of Cambridge, UK
Institut Pasteur, France

Temasek Life Science Laboratories,
Singapore

Hiroshi Hamada

Osaka University, Japan

Ryoichiro Kageyama
Kyoto University, Japan

Yale University, USA
Keio University, Japan
Kobe University, Japan

Patrick Tam

University of Sydney, Australia

Christopher Wylie

Cincinnati Children's
Hospital Medical Center, USA

Director
Deputy Directors

Creative Research
Promoting Program

Supporting
Laboratories

The Creative Research Promoting Program provides
solid support to encourage young researchers tocarry out innovative and independent research plans.
The teams are afforded a great deal of flexibility andcontrol in regard to projects, budget use, and lab size.
The program also places great emphasis oncooperation and international collaboration.

The Supporting Labs provide a range of technical support functions available to all researchers working at
the CDB. They also conduct research and developmentof new lab equipment and analytic software, and provide training on the use of research technologies.

Neuronal Differentiation and Regeneration

Animal Resources and Genetic Engineering

Hideki ENOMOTO M.D., Ph.D.

Shinichi AIZAWA Ph.D.

Neocortical Development

Genetic Engineering Unit

Carina HANASHIMA Ph.D.

Shinichi AIZAWA Ph.D.

Vertebrate Axis Formation

Animal Resource Unit

Masahiko HIBI M.D., Ph.D.

Kazuki NAKAO

Cell Lineage Modulation

Electron Microscope

Toru KONDO Ph.D.

Shigenobu YONEMURA Ph.D.

Sensory Development

Bio-imaging Laboratory

Raj LADHER Ph.D.

Shigeo HAYASHI Ph.D.

Germline Development

Optical Image Analysis Unit

Akira NAKAMURA Ph.D.

Yuko MIMORI-KIYOSUE Ph.D.

Chromatin Dynamics

Genomics

Jun-ichi NAKAYAMA Ph.D.

Fumio MATSUZAKI Ph.D.

Growth Control Signaling

Genome Resource and Analysis Unit

Takashi NISHIMURA Ph.D.

Hiroshi TARUI Ph.D.

Pluripotent Cell Studies

Functional Genomics Unit

Hitoshi NIWA M.D., Ph.D.

Hiroki R. UEDA M.D., Ph.D.

Mammalian Epigenetic Studies

Proteomics

Masaki OKANO Ph.D.

Shigeo HAYASHI Ph.D.

Mammalian Molecular Embryology

Mass Spectrometry Analysis Unit

Tony PERRY Ph.D.

Akira NAKAMURA Ph.D.

Mammalian Germ Cell Biology

Division of Human Stem Cell Technology

Mitinori SAITOU M.D., Ph.D.

Yoshiki SASAI M.D., Ph.D.

Embryonic Induction

Human Stem Cell Technology Unit

Hiroshi SASAKI Ph.D.

Yoshiki SASAI M.D., Ph.D.

Cell Fate Decision

Science Policy and Ethics Studies Unit

Hitoshi SAWA Ph.D.

Douglas SIPP

Early Embryogenesis
Guojun SHENG Ph.D.

Developmental Genomics
Asako SUGIMOTO Ph.D.

Retinal Regeneration
Masayo TAKAHASHI M.D., Ph.D.

Systems Biology
Hiroki R. UEDA M.D., Ph.D.

Genomic Reprogramming
Teruhiko WAKAYAMA Ph.D.

7

2009 Highlights

Stem cell researchers gather in Kobe
February 4
The CDB hosted a meeting on “Stem Cell Research in the Asia-Pacific Region”
in collaboration with the Stem Cell Network: Asia-Pacific (SNAP) and the Japan
Society for the Promotion of Science (JSPS), with additional financial support fromNature Publishing Group. Around 30 scientists from around the region gatheredin Kobe for three days of talks on recent scientific advances and ethical, legal andsocial aspects of stem cell research.

Teru Wakayama wins JSPS Prize
March 9Team Leader Teruhiko Wakayama (Laboratory for Genomic Reprogramming) wasawarded the 5th JSPS Prize in recognition of his contributions to the study of animal reproduction through biotechnology. In 2008, Wakayama famously succeededin generating a clone from a dead mouse that had been frozen for more than 15
years. A list of all awards won by CDB scientists appears on p. 54.

New Programs and Appointments
April 1On the first day of the Japanese fiscal year, the CDB inaugurated its new Center
Director’s Strategic Program, to provide focused support for designated research
fields; the first two fields selected were stem cell and developmental systems biology. Also on this day, Yuko Mimori-Kiyosue joined the Center as head of the new
Optical Image Analysis Unit, which will provide support to CDB labs in microscopy
techniques and technology management.

CDB Meeting on Sensory Development
April 13-15
At the height of the cherry blossom season, the CDB hosted a three-day meetingon “Common Themes and New Concepts in Sensory Formation.” More than 30
international speakers gathered to present and discuss their latest findings andenjoy Kobe’s springtime clime. See a full list of CDB meetings from 2009 on p. 66.

8

CDB scientists win MEXT awards
April 14Group Director Yoshiki Sasai was honored with a Science and Technology
Prize from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for his work in neural differentiation from pluripotent stem
cells. That same day, Team Leader Mitinori Saitou was awarded with a
MEXT Young Investigators Award in recognition of his research into mammalian germline specification.

CDB holds its 7th Open House
April 25
The CDB opened its doors to the public for a day of learning,interaction and fun, with activities, exhibits, games, talks and ascience café event. Nearly 1,500 visitors came to find out more
about the CDB’s research and chat with scientists.

Research article featured on Cell cover
May 1
A major article on mammalian germline development published by the Laboratory
for Mammalian Germ Cell Biology was featured on the cover the journal Cell. To
learn more about this research, see the news highlight on p. 30.
Image copyright © 2009 Cell Press

Takashi Nishimura joins
as new Team Leader
July 1
Takashi Nishimura joined the CDB as Team Leader of the Laboratory for Growth
Control Signaling. Nishimura will focus on the regulation of tissue and organ
growth by genetically encoded and extrinsic signals, using the fruit fly Drosophilamelanogaster as a model system. Read more about his work on p. 41.

9

2009 Highlights

CDB research makes cover of Science
July 15
The latest work on evolutionary innovation of the turtle carapace by the Laboratory
for Evolutionary Morphology appeared on the cover of Science magazine on thisday. See p. 50 for a full summary of this noteworthy research.

Image copyright © 2009 AAAS

Summer school forhigh school students
August 4-6
The Center held two one-day summer school courses for local
high school students, giving them a chance to learn more about
the CDB’s research and to try their hand at Drosophila experiments. For a complete list of courses held in 2009, turn to p. 36.

ISDB Meeting in Edinburgh
September 6-10
Many CDB scientists traveled to Edinburgh, UK to attend the 2009 Congress of
the International Society of Developmental Biologists (ISDB). CDB Director Masatoshi Takeichi served as ISDB President from 2006 to 2009. In addition to providing administrative support to the society, the Center organized a space in the
exhibitor’s hall (shown).

Project Leaders appointed
October 1
Hitoshi Niwa and Hiroki R. Ueda became the first Project
Leaders to head Center Director’s Strategic Programsin Pluripotent Stem Cell Studies and Systems Biology.
These projects will concentrate on priority areas of research determined by the CDB Center Director.

10Annual retreat held in Sasayama
November 5-6
To promote interaction and exchange among research staff and students, the
CDB holds a yearly getaway every fall. This year, the retreat was held for the firsttime in a meeting center in the mountains of western Japan. Lab heads, scientists, students and administrators convened for a day and a half of talks, posters, discussion and collegial exchange.

IBM Japan Science Prize
November 10
Hiroki R. Ueda, Project Leader of the Laboratory for Systems Biology, was awarded the IBM Japan 23rd Annual Science Prize, for his contributions to the field ofsystems biology through the large-scale analysis of biological data.

Excellence in imaging
November 18
Renowned microscope manufacturer Leica Microsystems awarded Shigeo Hayashi of the Laboratory for Morphogenetic Signaling its 2009 Grand Prize in scientific imaging. The image, showing a terminal branch of the Drosophila trachea, waspreviously featured on the cover of the 2004 CDB Annual Report.

11Notch has a dHand in smooth muscle
differentiation
Not all development takes places inside the embryo.
Many important functions, such as the formation ofblood and blood vessels, are relegated to extraembryonic tissues in early developmental stages and reintroduced to the embryo proper subsequently. In the chicken embryo, the extraembryonic mesoderm gives rise toboth blood and endothelial cells, but it has been neverbeen shown whether this represents its full differentiative
gamut.
A study by Masahiro Shin and Hiroki Nagai in the Laboratory for Early Embryogenesis (Guojun Sheng; Team
Leader) revealed that this same extraembryonic tissue
is the earliest source of smooth muscle cells as well. In
an article published in Development, the team identified
the transcription factor dHand as a genetic marker ofsmooth muscle and showed that the segregation of thispopulation from blood and endothelial progenitors ismediated by Notch signaling.
Notch has been shown to be involved in vascular remodeling and specification, but little was known about
its role in ventral mesodermal differentiation (which gives
rise to the population of cells that populate the extraembryonic mesoderm). To address this, Shin made a truncated Notch construct engineered such that the pathway was continuously switched on and introduced it into
primitive streak-stage embryos to study the effects oncell fate. He found that a preponderance of the Notchexpressing cells took on a smooth muscle fate, with very
few differentiating into blood/endothelial progenitors.
In situ hybridization revealed that Notch is active in theposterior primitive streak, where ventral mesoderm isgenerated.
Conjecturing that Notch might act as a sorting mechanism to separate smooth muscle from blood and vascular progenitors, the team next sought a means oflabeling smooth muscle, as no genetic markers for thesecells had been identified. An in situ screen turned up thetranscriptional factor dHand, which they found to be expressed in early smooth muscle cells as well as vascularsmooth muscle layer and extraembryonic somatic mesoderm (also known to consist of smooth muscle cells) atlater stages of development.

Masahiro SHIN

12

Blood and endothelial progenitors have their own specific marker in the gene Scl. When the team went back andlooked at expression patterns in the constitutively Notchactive posterior streak, they found that cells expressing Notch also tended to express dHand, and not Scl.

©2009 The Company of Biologists
Shin M, et al. Notch mediates Wnt and BMP signals in the early separation of smooth muscle
progenitors and blood/endothelial common progenitors. Development 136. 595-603 (2009)

Figure 1
dHAND marks the smoothmuscle cells, which are tightly
wrapped around vessel cellswith developing blood cells
inside the vessel lumen (bothvessel and blood cells aremarked by Lmo2).

Inhibiting the Notch pathway abolished this exclusion. Interestingly, they found that although Notch
activation is associated with nearly exclusive smooth muscle cell contribution to the extraembryonicmesoderm, the absence of Notch has no effect on the induction or differentiation of these cells.
They next asked whether other signaling pathways known to work in the same vicinity might also be
involved in smooth muscle specification. While BMP appeared to function primarily by ventralizing themesoderm (leading to the induction of both smooth muscle and blood/endothelium), ectopic expression of Wnt strongly induced the smooth muscle marker dHand. Intriguingly, the signal responsible forinducing the blood/endothelial common progenitor has yet to be identified.

Figure 2
The circulatory system of earlychicken embr yos (E5 shownhere) is complex and contains
embryonic, allantoic, and yolk
sac sub-systems. The diversityof blood cells during this period
is revealed by the transcriptomic analysis of non-red blood
cells in circulation.

"Perhaps many researchers aren't so interested in extraembryonic tissues, but a similar tug-of-war
between muscles and vessels happens in cardiac development and in mesoderm stem cell differentiation in vitro," says Sheng. "So in that sense the molecular mechanisms uncovered in our studies
may be of more general interest."

13

Endothelial cells give birth to blood

An imaging technique developed through a collaboration between the CDB Laboratory for Stem Cell Biology (Shin-Ichi Nishikawa; Group Director) and Timm
Schroeder, a former CDB research scientist who now
heads the Independent Research Group for Hematopoiesis at the Helmholtz Zentrum München, German
Research Center for Environmental Health, Institute forStem Cell Research, yielded a compelling new insights
into the origins of blood in the mammalian embryo. This
represents the first joint publication between the two institutes, which established a research affiliation in 2005.
The debate over the ultimate source of embryonic hematopoiesis is fraught and longstanding, with competing hypotheses proposing that blood is generated frommesoderm, mesenchymal progenitors, endothelial precursors, or cells in the endothelium itself. But limits in thetechnologies used to visualize blood differentiation at thecellular level by observing only single genetic markers
at single points in time have meant that the existence ofhemogenic endothelial cells has remained in dispute.
Schroeder set out to address these shortcomings bydeveloping a bio-imaging approach that would enable
him to watch well-characterized individual endothelial
cells continuously during the differentiation process.
Starting with mesodermal precursors (the cells fromwhich endothelial cells originate) cultured under conditions known to induce endothelium, the team observed
their morphology and behavior using computer-assisted
time-lapse microscopy. From the total population, endothelial cells were isolated by selecting for the full set of
their known characteristics, including sheet morphology,
the ability to take up low-density lipoprotein, and theexpression of VE-cadherin and the tight junction marker,
claudin.
Similarly, blood cells were identified by their non-adherence, proliferation, expression of blood-specific marker
proteins, and the absence of endothelial characteristics.
Looking at more than 10,000 mesodermal colonies,
the team finally narrowed their focus down to 144 that
had endothelial sheet morphology and generated blood
cells. In all of these, they found that adherent endothelial
cells gave rise to blood cells that gradually detachedcompletely from the colony. The free-floating blood cells
expressed blood lineage markers in a similar sequence
to that of emerging hematopoietic cells in vivo.

Timm SCHROEDER

14©2009 Nature Publishing Group
Schroeder T, et al. Continuous single-cell imaging of blood generationfrom haemogenic endothelium. Nature 457. 896-900 (2009)

Figure 1
Hemogenic endothelial colony
with blood cells generated byit, above. (Left: phase contrast;
Right: Histone2B-VENUS labeled nuclei)

Importantly, the hemogenic endothelial cells failed to differentiate into either smooth muscle or cardiomyocytes, suggesting that they were not multipotent mesodermal cells. And they lacked the expression of CD45, a blood marker, further ruling out the possibility that they were bipotent blood-endothelial precursors. Indeed, the hemogenic cells gave rise solely to blood cells, while their neighboring
sister cells generated only endothelial progeny. Mesodermal cells taken from mouse embryos showedsimilar hemogenic capacity, indicating that the findings were not merely an artifact of cell culture.

Figure 2
Molecular dynamics during
endothelial-hematopoietic celltransition. Multiple molecularmarkers were simultaneously
detected at the single cell level
over days. Part of a haemogenic
endothelial cell transition isshown. Haemogenic endothelial
cells first express the endothelial marker VE-cadherin, but
not the hematopoietic markers
CD41 or CD45. Later, cells withendothelial morphology start
expressing CD41, and develop
into non-adherent blood cells
as identified by CD45 expression. Hours after start of themovie and cells of pedigree (arrowheads) are indicated. Scalebars: 50 um.

15

Ins and outs of the early embryo

Noriyuki NISHIOKAIn the first few days of mammalian development, the embryo transforms from a clump of undifferentiated cells toa fluid-filled sphere called the blastocyst, with distinct inner and outer cell types. The interior cells, known as theinner cell mass (ICM), go on to form the embryo proper, while the external layer of cells, or trophectoderm, contributes to extraembryonic tissues. Previous work has shown that the ICM-trophectoderm balance is maintainedby interdependent mutual inhibition between a pair of factors: Cdx2, which drives trophectodermal differentiation,
and Oct3/4, which maintains the ICM in an undifferentiated state. But the mechanisms involved in tipping thisbalance to either side in even earlier embryonic regions have never been worked out.
A study by Noriyuki Nishioka and colleagues in the Laboratory for Embryonic Induction (Hiroshi Sasaki; Team
Leader) has revealed that Hippo signaling controls the expression of both Cdx2 and Oct3/4 in the early embryo.
The report, published in Developmental Cell, describes an intricate interplay between factors in the embryo’s
inner and outer cells, in which the activation of the transcription factor Tead4 in outer cells switches on Cdx2
expression, while its suppression by the Hippo pathway in interior cells allows Oct3/4 to dominate. The authors
speculate that this may be triggered by differences in the cell adhesion environments in these two zones of theembryo.
Prior to the formation of the blastocyst, the cells in the embryo can only be distinguished from one another by
their position in the exterior (surface) or interior of the embryo. It has been thought that positional information fromthe inner and/or outer cells might influence the contest between Cdx2 and Oct3/4 that defines the initial differentiation events in the blastocyst, but the mechanism to account for this remained elusive. There was, however,
one tantalizing clue from previous work by the lab, which had showed that Tead4 is required for Cdx2 expression16©2009 Cell Press
Nishioka N, et al. The Hippo Signaling Pathway Components Lats and
Yap Pattern Tead4 Activity to Distinguish Mouse Trophectoderm
from Inner Cell Mass. Dev Cell 16. 398-410 (2009)

and the formation of the trophectoderm. The mystery was that Tead4 is expressed in every cell of theembryo, not just the cells that differentiate into trophectoderm. Another member of the Sasaki team,
Mitsunori Ota, added an important piece to the puzzle when, working on a separate line of inquiry, hefound that in cultured cells and later-stage, post-implantation embryos the activity of Tead4 relied on a
co-factor, Yap.
This prompted Nishioka to look for a possible role for Yap in the early embryo as well. Looking at the
distribution of the Yap protein, they were interested to find that while Yap localizes to the nucleus inouter cells, it is cytoplasmic in the inner ones. Importantly, this nuclear localization in outer cells precedes the expression of Cdx2, which suggested that, in these cells, Yap might trigger Tead4, whichthen switches on Cdx2. Overexpression of Yap or activated Tead4 caused Cdx2 to be expressed
aberrantly in inner cells, while the expression of a Yap protein engineered to lack its Tead-binding domain had no effect.
The question remained: What causes Yap to move to the nucleus only in outer cells? An experimentshowed that a Hippo pathway member, Lats, phosphorylates Yap in cultured cells, causing it to remain in the cytoplasm. In the embryo, they found that phosphorylated Yap is only found in the inner
cells, and that overexpression of Lats throughout the entire embryo causes a marked reduction of
nuclear Yap and failure to form trophectoderm.
Thinking about what might account for the inner cell-specific activity of Lats, Ota showed that theactivation of this factor causes Yap to stay in the cytoplasm. Armed with this knowledge of what happens in vitro, Nishioka and colleagues did a series of experiments in 8-cell embryos in which he either
interfered with cell-cell adhesion or dissociated and rearranged the cells of the embryo, and foundthat cellular adhesion indeed does activate Hippo signaling, and that the adhesive environment determined by a cell’s position affects the localization of Yap within the cell.
Other recent work from the Sasaki team had shown a role for Hippo signaling in the regulation ofTead, in a phenomenon known as contact inhibition of proliferation. In this latest work, they have demonstrated a similar link between cell contact and differentiation in the early mammalian embryo, thefirst evidence of how a cell’s
positional information determines its fate in this context.
“These new findings are
an important step towards
a better understanding of
the mechanisms underlying differentiation along theinner-outer axis of the early
embryo,” says Sasaki. “Butthere have also been reports that cell polarity is important in pre-implantationdifferentiation, and there ismuch we still do not know,
so we’re looking forward tolearning more about how
such events are controlledduring these critical earlystages of development.”

Expression of Yap (green) andCdx2 (red) in outer cells of theblastocyst.Blue shows labelingof nuclei with DAPI. Merged image at bottom right.

17

2009 Events
In addition to its research activities, the CDB conducts events every year to engage more closely with the public, as well as to foster closer
connections between labs and individual scientists. In 2009, the CDB held a number of successful public and closed events.

Open HouseThe RIKEN CDB held its annual Open House on April
25, welcoming more than 1,400 visitors from Kobe
and the surrounding areas of Japan to learn about theCenter’s research and enjoy exhibits and activities forthe curious of all ages. This event featured a talk by
Shigeru Kuratani on the evolutionary developmental
biology of the turtle shell, a special exhibit on green
fluorescent protein to mark the 2008 Nobel Prize, andthemed exhibits on germ cells and systems biology.
A science café was held to allow people to meet andexchange views on questions from the leading edgeof science, and a crafts corner, laboratory photo studio, and games for kids were all part of the fun.

CDB Retreat
The CDB held its institutional retreat at a new location in 2009, moving to the natural setting of a nature
center in the mountains of Sasayama. At this yearlytwo-day meeting, CDB lab leaders, research scientists and student trainees gather in a closed meeting
to encourage free and open discussion of preliminary
findings and unpublished results, and to get to knoweach other better through conversation and timespent together away from the lab. This year’s highlights included talks by two CDB alumni who left theCenter to start their own labs, and musical and visualentertainment provided by members of the CDB staff.

18

A zebra finch embryo at approximately stage HH23 of development,with inset showing it intact with extraembryonic tissues and yolk.

Vertebrate Body Plan
All vertebrate species share certain features in the development of the brain, inthat all vertebrate brains comprise four regions – telencephalon, diencephalon,
mesencephalon and metencephalon (or cerebellum) – an organizational trait thatis thought to have appeared with the advent of the vertebrate lineage. However,dramatic changes in this organization took place during the divergence of jawed(gnathostome) fishes from their jawless (agnathan) ancestors. Evolutionary modifications of the telencephalon first manifested in the reptiles, becoming evenmore pronounced with the development of a neocortex stratified into six layers
regionalized by distinct physiological functions. However, the mechanisms that
instruct the brain’s laminar regions to conform to anterior-posterior and dorsalventral axis patterning programs, and the means by which each individual region
is formed, remain unknown.

Shinichi AIZAWA Ph.D.
http://www.cdb.riken.jp/en/aizawa
Shinichi Aizawa received his Ph.D. in biochemistryfrom the Tokyo Kyoiku University Department of
Zoology in 1973. 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 at the University of Washington.
He returned to the Tokyo Metropolitan Instituteof Gerontology in 1982, where he remained until
1986, when he moved to the RIKEN Tsukuba Life
Science Center. He was appointed professor in
the Kumamoto University School of Medicine in
1994, and served in that position until 2002. Since
2000 he has served as CDB deputy director andgroup director of the Vertebrate Body Plan lab, as
well as head of the Animal Resources and Genetic
Engineering Laboratory. In January 2007, he wasappointed President of the Japanese Society ofDevelopmental Biologists. He also serves as editor for the journal, Mechanisms of Development.

The head is the most highly layered of the body’s structures and its development
begins with anterior-posterior axis formation. This A-P axis patterning is one of
the most primary phenomena in animal development, and the emergence of thevertebrate lineage has been accompanied by widespread adaptations to thisfundamental rule, as evidenced by the diversity of mechanisms by which vertebrate taxa achieve the common ends of axis formation and jaw induction.
A long road lies ahead in the search for the origins of the Bauplan of the vertebrate head, but application of powerful new molecular biological techniques
to the study of multiple vertebrate model species has now made it possible tostudy the question more comprehensively and in greater detail than ever before.
Our laboratory is focusing on the ontogenetic and phylogenetic roles played by
the Otx/Emx family of head gap genes as a route toward a better understandingof brain development. The question of what molecular mechanisms underlie thedevelopment of neocortical regions is central to that pursuit.

Staff
Group Director

Visiting Scientist

Research Specialist

Research Associate

Shinichi AIZAWA
Yoko SUDA
Masaki TAKEUCHI
Research Scientist

Mariko HIRANO
Hideharu HOSHINO
Fumitaka INOUE
Miyuki NORO
Mikihito SHIBATA
Michio YOSHIDA

Daisuke KUROKAWA
Yusuke SAKURAI
Technical Staff

Eriko KAJIKAWA
Hiromi NAKAO
Tomomi OMURA
Maiko TAKAHASHI
Assistant

Sayo SAITO

Publications
Suda Y, et al. Evolution of Otx paralogue usages in earlypatterning of the vertebrate head. Dev Biol 325. 282-95(2009)
Takeuchi M et al. Germ layer patterning in bichir and lamprey; an insight into its evolution in vertebrates. Dev Biol
332. 90-102 (2009)
Hirano M, et al. EPB41L5 functions to post-transcriptionallyregulate cadherin and integrin during epithelial-mesenchymal transition. J Cell Biol 182. 1217-30 (2008)
Shibata M, et al. MicroRNA-9 modulates Cajal-Retzius celldifferentiation by suppressing Foxg1 expression in mouse
medial pallium. J Neurosci 28. 10415-21 (2008)
Takasaki N, et al. Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart.
Embo J 26. 1649-59 (2007)
Kimura-Yoshida C, et al. Crucial roles of Foxa2 in mouseanterior-posterior axis polarization via regulation of anterior
visceral endoderm-specific genes. Proc Natl Acad Sci U S
A 104. 5919-24 (2007)

20

EPB41L5 regulates epithelial/mesenchymal transition post-transcriptionally; it binds to p120 catenin
through its N-terminal FERM domain, thereby destabilizing E-cadherin toward endocytosis and to
paxillin through its C-terminal, thereby activating integrin expression in cell surfaces or focal adhesion
formation.

Neuronal Differentiation and Regeneration
The architecture of the nervous system, which underlies much of the dynamismof animal life, is frequently described as complex, but closer examination reveals
great beauty as well. The construction of this exquisite and elaborate structure
involves the birth, growth and death of countless individual cells and is regulated
by intercellular communication and cooperative activity.
Our laboratory is interested in questions fundamental to the development of
the nervous system: determining the specific extracellular signals and cell autonomous routines that participate in the processes of neural development, andresolving the means by which diverse families of signaling molecules coordinate
and interact at the molecular and cellular levels. To address these questions, we
focus on a class of signaling molecules known as neurotrophic factors, mostparticularly, the GDNF Family Ligands (the GFLs). This family of neurotrophic factors includes four known members – GDNF (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 four co-receptors,
GRFα1-4. In vitro, these four receptors show differential affinities for specific
GFLs. GFL signaling has been shown to affect neuronal growth, survival and migration as well as axon guidance, and defects in this signaling system have been
implicated in a number of congenital health problems.
Our efforts will focus on the analysis of the physiological roles of neurotrophic
factors, which we hope will lead to an improved understanding of the molecularbases of neural development, differentiation and pathology, and ultimately topotential clinical applications in the treatment of nervous system trauma and disease.

Hideki Enomoto M.D., Ph.D.
http://www.cdb.riken.jp/en/enomoto
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.
After serving as the Chief of the Department ofPediatric Surgery, Matsudo Municipal Hospitalin 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 a position as ateam leader at the CDB in 2002.

Staff
Team Leader

Junior Research Associate

Research Scientist

Student Trainee

Technical Staff

Part-Time Staff

Hideki ENOMOTO
Pilaiwanwadee
HUTAMEKALIN
Toshihiro UESAKA
Takae HIRAI
Chihiro NISHIYAMA
Hiroko SONOBE

Mayumi NAGASHIMADA
Mitsuhiro IWASAKI
Katsuki YASUDA
Yohei YONEKURA
Toko KONDO
AssistantKaori HAMADA

Publications
Burzynski G, et al. Genetic model system studies of thedevelopment of the enteric nervous system, gut motility and
Hirschsprung’s disease. Neurogastroenterology & Motility,
21. 113-27 (2009) Review.
Gould TW, et al. Neurotrophic modulation of motor neuron
development. The Neuroscientist 15. 105-16 (2009) Review.
Uesaka T, et al. Diminished Ret expression compromises
neuronal survival in the colon and causes intestinal aganglionosis in mice. J Clin Invest 118. 1890-8 (2008)
Gould T W, et al. The neurotrophic effects of glial cell linederived neurotrophic factor on spinal motoneurons are
restricted to fusimotor subtypes. J Neurosci 28. 2131-46(2008)
Uesaka T, et al. Conditional ablation of GFRalpha1 in postmigratory enteric neurons triggers unconventional neuronal
death in the colon and causes a Hirschsprung's diseasephenotype. Development 134. 2171-81 (2007)

Developing enteric nervous system (ENS) in which GDNF receptor RET was conditionally inactivatedin a small population of ENS cells (mouse gut: embryonic day 14.5, Green: Ret-deficient cells, Red:
enteric neurons).

Enomoto H, et al. GFRalpha1 expression in cells lacking
RET is dispensable for organogenesis and nerve regeneration. Neuron 44. 623-36 (2004)21

Neocortical Development

Carina HANASHIMA Ph.D.
http://www.cdb.riken.jp/en/hanashima
Carina Hanashima received her Ph.D. fromWaseda University School of Science andEngineering in 1999. She worked as a postdoctoral fellow in the laboratory of Eseng Lai at
Memorial Sloan-Kettering Cancer Center, Cell
Biology Program from 1999 to 2002, where she
was engaged in developmental neuroscience.
She moved to Skirball Institute Developmental
Genetics Program in 2002 in Gord Fishell’slaboratory where she extended her research tocell specification in the neocortex. She was appointed team leader at the CDB in September
2007.

Staff
Team Leader

Technical Staff

Research Scientist

Part-time Staff

Carina HANASHIMA
Takuma KUMAMOTO
Ken-ichi MIZUTANI
Wataru OCHIAI

Yuko WADA
GunadiStudent Trainee

Youko YAMAMOTO

Publications
Fishell G and Hanashima C. Pyramidal neurons grow up
and change their mind. Neuron 57. 333-8 (2008)

The neocortex, by far the most complex structure of all vertebrate brain systems,
is organized into cytoarchitectonic groups of neurons that process unique sensory inputs such as pressure, light and sound. Nonetheless this highly orderedstructure is generated from a relatively simple sheet of neuroepithelium earlier
during development. Research in our laboratory aims to understand how diverse
arrays of cortical neurons are specified and coordinated into high-functional territories. Despite its well-defined anatomical character and functional significance,
the mechanisms underlying the precise assembly of distinct functional areas of
the cerebral cortex remains largely unknown. Progress has been impeded by ourpresent lack of understanding of the molecular mechanisms by which neuronalsubtypes within cortical layers and across function domains are specified. In ourlaboratory, we address important questions concerning neocortical development: 1) What are the mechanisms by which diverse cell fate is determined in the
neocortex? 2) How are neurons precisely arranged into distinct cortical areas
and establish cytoarchitectonic boundaries? 3) To what extent does the refinement of functional areas rely on environmental inputs? To investigate these questions we utilize the mouse as a model to understand the assembly of functional
subdivisions in the mammalian neocortex.
Previous work has shown that the fate of neocortical neurons is at least in partpre-determined within the progenitor cells. We have found that specification of
deep-layer projection neurons is dependent on transcriptional repressors thatfunction cell-autonomously to prevent neurons from acquiring an earlier neuronal phenotype. These results imply cortical intrinsic programs in which neuron
fate is established by temporal changes in gene expression may be co-opted.
However, in the mature cortex, cortical areas differ in their types and numbersof specific layer neurons along both the anteriorposterior (AP) and medial-lateral(ML) axes. We are exploring the extent to which intrinsic determinants control
the specification of neuronal subtypes within discrete regions of the neocortex,
as well as the extrinsic influences that refine the boundaries between functional
areas of neocortex. To further these studies, we employ genetic manipulations inmice that will enable conditional loss of gene and cellular functions, recombination mediated cell-lineage tracing, and systematic approaches to identify novel
molecules responsible for precise areal specification. Through these studies wewish to understand the mechanistic basis by which unique sensory perceptions
and functional circuitries develop in the human neocortex.

Hanashima C, et al. The role of Foxg1 and dorsal midline
signaling in the generation of Cajal-Retzius subtypes. J
Neurosci 27. 11103-11 (2007)
Hanashima C, et al. Foxg1 suppresses early cortical cellfate. Science 303. 56-9 (2004)

Recruitment of late-born neocortical neurons expressing membranetargeted GFP (green) and nuclear lacZ (red) to the motor cortex.

22

Morphogenetic Signaling
The main research interest in my lab focuses on the mechanisms by which cellcell and tissue-tissue interactions are modulated during embryonic morphogenesis. Our strategy is to identify intercellular signaling systems and intracellulartransducers that control cell-cell and tissue-tissue interactions. A range of vital
developmental processes, including the ability to build complex structures
through selective cell adhesion, to move through the body, and to form epithelial
tissues, rely on interactions between cells, and between cells and the extracellular matrix. The means by which cells are able to communicate, congregate andwork together to build a body is a central question in the study of morphogenesis.
The Drosophila tracheal system is a respiratory organ that delivers air directly
to tissues through a network of finely branched epithelial tubes. Tracheal development begins by invagination of ectodermal epithelium that subsequently
forms branches with specific types of cells migrating in stereotyped patterns.
The branching patterns and cell fate are instructed by external cues, including
FGF, WG, and Dpp. We are studying the roles of these signaling molecules in thespecification and migration of tracheal branches, as well as the mechanisms thatcoordinate cell movement and cell adhesion. We additionally use 4D confocal
imaging of GFP-labeled embryos to study the dynamism of cell and organelle
movement in living organisms. Using combinations of GFP markers and transcriptional enhancers of cell-specific expression, we have been able to capture
movements of tracheal cells at resolutions sufficient to image cytoskeletal organization and cell adhesion structures in single cells.
The development of appendages in Drosophila from primordial regions called
imaginal discs is a second area of interest. During this process, subpopulationsof cells in the imaginal discs segregate into distinct domains by coupling cell
growth and differentiation to cell sorting, which provides us with an opportunity
to study the regulation of cell affinity by positional information. Each limb primordium also coordinates its specific developmental pattern with other tissues, suchas muscles, motor nerves and trachea, which are specified independently inother parts of the embryo. This understanding of mechanisms of limb specification and proximal-distal axis formation gained from work on Drosophila, however,must also be validated by comparative analyses in other species with simpler
appendage structures. We focus on three species for these comparative studies:
the bristletail, Pedetontus unimaculatus, the mayfly, Ephemera japonica, and the
ragworm, Perinereis nuntia.

Shigeo HAYASHI Ph.D.
http://www.cdb.riken.jp/en/hayashi
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 thechicken delta crystallin gene. Inspired by thediscovery of the homeobox, he changed 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 to work in the National Institute of
Genetics. He became an associate professor
at the same Institute in 1994, and professor
in 1999. Also in 1999, he received the incentive prize of the Genetics Society of Japan for
Genetic Studies on Drosophila Development.
He was named group director of the Morphogenetic Signaling research group at the
RIKEN CDB in May 2000. His current researchinterests are dynamic aspects of cell adhesion,
cell migration and cell morphogenesis in Drosophila.

StaffGroup Director

Shigeo HAYASHI
Research Scientist

Kagayaki KATO
Takefumi KONDO
Nao NIWA
Tatsuhiko NOGUCHI
Tetsuhisa OTANIReiko TAJIRI
Kazunaga TAKIZAWA

Technical Staff

Ai AKIMOTO
Michiko KOIZUMIHousei WADA
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
AssistantTomoe DEGUCHI
Hiromi WANAKA

Publications
Kato K and Hayashi S. Practical guide of live imaging fordevelopmental biologists. Dev Growth Differ 50. 381-90(2008)
Kakihara K,et al. Conversion of plasma membrane topology
during epithelial tube connection requires Arf-like 3 small
GTPase in Drosophila. Mech Dev (2008)
Shindo M, et al. Dual function of Src in the maintenance ofadherens junctions during tracheal epithelial morphogenesis. Development 135. 1355-64 (2008)
Sakurai K T, et al. Differential control of cell affinity requiredfor progression and refinement of cell boundary during Drosophila leg segmentation. Dev Biol 309. 126-36 (2007)

Homophilic cell adhesion molecules Capricious and Tartan are required for proper placement ofsegmental boundary in the leg. The boundary of pretarsas and tarsal 5 segment is marked with red
signal, which normally appears as a ring. When the boundary is flanked by a group of cells lacking
Capricious and Tartan ( indicated by a loss of green signal ) the position of the boundary was shiftedinteriorly and the boundary appeared uneven.

Nishimura M, et al. A wave of EGFR signaling determines
cell alignment and intercalation in the Drosophila tracheal
placode. Development 134. 4273-82 (2007)
Kondo T, et al. Small peptide regulators of actin-based cellmorphogenesis encoded by a polycistronic mRNA. Nat Cell
Biol 9. 660-5 (2007)23

Vertebrate Axis Formation
During early vertebrate development, the embryonic dorso-ventral (DV) and anterior-posterior (AP) axes are established through a series of inductive signals thatprefigure and determine the concerted movements and differentiation of a groupof cells. The dorsal organizer plays a major role in the process of axis formation.
In amphibian and teleost fish embryogenesis, the program for dorsal organizer
formation begins soon after fertilization. In these species, dorsal determinants
are believed to be initially localized to the vegetal pole and later transported tothe dorsal side of the embryo.

Masahiko HIBI M.D., Ph.D.
http://www.cdb.riken.jp/en/hibi
Masahiko Hibi received his M.D. from Hiroshima
University School of Medicine in 1988, and his
Ph.D. from Osaka University in 1992. From 1992
to 1995, he worked as a postdoctoral fellow in
Michael Karin's lab in the University of California,
San Diego Department of Pharmacology, andthen returned to Japan as a research associate
in the Division of Molecular Oncology at Osaka
University Medical School. He was appointed
associate professor in the same division in 1999,
where he remained until he assumed his positionas team leader at the RIKEN CDB.

Staff
Team Leader

Masahiko HIBITechnical Staff

Special Postdoctoral
Researcher

Koji TANABE

Setsuko FUJII
Kana KADOWAKIBANDOAya KATSUYAMA

Research Scientist

Part-Time Staff

Shuichi KANI
Hideaki NOJIMA
Takashi SHIMIZU
Takeshi SHIMIZU

Isao HARIMOTO
Yuko HIROSE
Shigemi SHIBUYAKazuko YAMAMOTO

Publications
Nojima H, et al. Syntabulin, a motor protein linker, controls
dorsal determination. Development 137. 923-33 (2010)

Although the molecular nature of the dorsal determinants has not been elucidated, they are known to activate the canonical Wnt pathway and thereby leadto the expression of genes involved in the induction of the dorsal organizer. The
dorsal organizer generates secreted signaling molecules that participate in thegeneration of the DV axis in the mesoderm and endoderm, and the formation of
the neuroectoderm. We are working to identify the molecules that are involved in
the formation and function of the dorsal organizer. This search led us to identify
the protein Sizzled as a negative feedback regulator of BMP signaling that cooperates with the dorsal organizer protein Chordin to regulate DV axis formation.
We also found that Syntabulin, a linker of the kinesin I motor protein, is involved
in microtubule-dependent transport of dorsal determinants.
Neural patterning and neurogenesis as a model of cell fate determination, aprocess that is linked to axis formation, is also a question of interest to our team.
Neuronal tissues are generated in a stepwise manner in vertebrates; these steps
include neural induction, AP patterning and neurogenesis. In amphibian andteleost embryos, neuroectoderm is initially induced by the dorsal organizer proteins. The induced neuroectoderm is anterior in character and is subsequently
subjected to posteriorization and regionalization. Accordingly, the central nervous system becomes highly ordered along the AP axis, which is regionalized toforebrain, midbrain, hindbrain and spinal cord compartments in a head-to-tail direction. Our studies have revealed that two groups of genes play important rolesin the AP patterning of neural tissue. The zinc-finger genes Fezf1 and Fezf2 areexpressed in the anterior forebrain and control the AP patterning of forebrain byrepressing the caudal forebrain fate, while the caudal-related genes cdx1a andcdx4 are expressed in the posterior neural tissue and control the formation ofposterior spinal cord by repressing anterior fate. We are also extensively studying
the mechanisms that establish the complex structure of the cerebellum.

Bae Y-K, et al. Anatomy of zebrafish cerebellum and screen
for mutations affecting its development Dev Biol 330. 406426 (2009)
Hirata T, et al. Zinc-finger genes Fez and Fez-like function in
the establishment of diencephalon subdivisions. Development 133. 3993-4004 (2006)
Hirata T, et al. Zinc-finger gene Fez in the olfactory sensory
neurons regulates development of the olfactory bulb noncell-autonomously. Development 133. 1433-43 (2006)
Muraoka O, et al. Sizzled controls dorso-ventral polarity byrepressing cleavage of the Chordin protein. Nat Cell Biol 8.
329-38 (2006)
Shimizu T, et al. Cdx-Hox code controls competence for responding to Fgfs and retinoic acid in zebrafish neural tissue.
Development 133. 4709-19 (2006)

Development of cerebellar neurons in zebrafish larvae at 5 days post fertilization. Purkinje neurons(green) receive axons of granule cells (magenta) in wild type. Loss and abnormal extension of granulecell axons are observed in gazami and shiomaneki mutants, respectively.

24

Cell Lineage Modulation
Mankind has long wondered whether it might be possible to regenerate bodytissues and structures that have been lost. Recent research has shown that eventhe adult body contains a diverse range of tissue-specific somatic stem cells thatserve to maintain the function and integrity of tissues, opening a promising avenue toward possible applications in regenerative medicine. But the limits on thenumber of somatic stem cells present in any individual, coupled with the limited
availability of donors, have heightened interest in the development of new, alternative means of generating stem cells.
Research in our lab seeks to develop a better understanding of the molecularmechanisms involved in the reacquisition of “stemness” (stem cell characteristics) using the dedifferentiation of oligodendrocyte progenitor cells (OPCs), which
are abundantly present in the central nervous system, into neural stem-like cells,
knowledge which it is hoped may lead to new techniques for the generation ofneural stem cells. We are also interested in two other related subjects: one is thecharacterization of stem cell-like cancer cells (sometimes called “cancer stem
cells” or “cancer initiating cells”), found in malignant glioma, and to find markers
and targets for them, potentially leading to the development of novel anti-cancer
therapies. The other is to develop a better understanding of the molecular mechanisms of cellular senescence, which are involved in both the defects of tissuespecific stem cells and the suppression of tumorigenesis.

Toru KONDO Ph.D.
http://www.cdb.riken.jp/en/kondo
Toru Kondo received his B.Sc. from Waseda
University and his M.S. and Ph.D. from the
Osaka University Institute for Molecular andCellular Biology. He worked as a postdoctoral
fellow in Shigekazu Nagata's lab at Osaka
Bioscience Institute from 1994 to 1998 and in
Martin Raff's lab at University College London
MRC Laboratory for Molecular Cell Biology
from 1998 to 2001. He returned to Japan totake a position as an associate professor at
Kumamoto University in 2001 and moved toCambridge University Centre for Brain Repair
in 2002 to take a group leader position. He wasappointed team leader in 2005.

Staff
Team Leader

Toru KONDO
Research Scientist

Hiromi TAKANAGA
Nobuko TSUCHIDASTRAETEN
Visiting Researcher

Norishige YAMADA

Student Trainee

Yuki KUJURO
Kenji NISHIDE
Tatsuya TAKEZAKI
Agency Staff

Makiko NAKAHARA
Assistant

Yukie NAKAMURA

Technical Staff

Keiko MIWA
Yuka NAKATANI

Publications
Hide T, et al. Sox11 prevents tumorigenesis of gliomainitiating cells by inducing neuronal differentiation. Cancer
Res. 69. 7953-7959 (2009)
Murase M, et al. Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bonesarcomas. Br J Cancer 101. 1425-1432 (2009)
Nishide K, et al. Gliomagenesis from transformed neuralstem cells depleted of prominin1-expressing cells. PLoS
ONE 4. E6869 (2009)
Takanaga H, et al. Gli2 Is A Novel Regulator of Sox2 Expression In Telencephalic Neuroepithelial Cells. Stem Cells
27. 165-174 (2009)
Kondo T. Making of glioma-initiating cells. Inflamation andRegeneration 28. 537-542 (2008)
Kondo T. Stem cell-like glioma cells. Current Cancer Therapy Review 4. 201-205 (2008)

Morphology of rat C6 glioma cells in different culture conditions, FCS(A), bFGF(B), PDGF(C) or
PDGF+bFGF(D).

25Evolutionary Morphology
By studying the evolutionary designs of diverse animal species, I hope to gain adeeper insight into the secrets behind the fabrication of morphological designs.
Integrating the fields of evolutionary morphology and molecular genetics, ourlab seeks to expand the understanding of the relationship between genome andmorphology (or body plan) through investigating the evolutionary changes in developmental processes, and also the process of evolution in which phenotypic
selection shapes developmental programs. Our recent studies have focusedon novel traits found in the vertebrates, such as the jaw, the turtle shell, and the
mammalian middle ear. By analyzing the history of developmental patterns, I
hope to open new avenues toward answering as-yet unresolved questions about
phenotypic evolution in vertebrates at the level of body plans.

Shigeru KURATANI Ph.D.
http://www.cdb.riken.jp/en/kuratani
Shigeru Kuratani received his masters and Ph.D.
from the Kyoto University Department of Zoology. He spent the period from 1985 to 1988
studying comparative embryology in the Department of Anatomy, University of the Ryukyus,
and 1988 to 1991 working in experimental embryology in the Department of Anatomy at the
Medical College of Georgia before moving to the
Biochemistry Department at the Baylor Collegeof Medicine, where he was engaged in molecular
embryological research. He returned to Japan
in 1994 to take a position as associate professor at the Institute of Medical Embryology andGenetics 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. He was appointed group director in 2005.

Staff
Group Director

Junior Research Associate

Research Scientist

Student Trainee

Shigeru KURATANI
Shinichi AOTA
Naoki IRIE
Hiroshi NAGASHIMA
Kinya OTA
Takao SUZUKI
Motoki TADA
Masaki TAKECHITechnical Staff

Satoko FUJIMOTO
Tamami HIRAI

Through the study of evolutionarily novel structures, our lab has identified
changes in developmental mechanisms that have obliterated the structural homologies between organisms as evidenced in such novel structures as the jaw ingnathostomes (jawed vertebrates) and the turtle shell. Developmental studies of
the cranial region of the lamprey are intended to shed light on the true origins of
the vertebrate head and neck, as lampreys lack a number of important features,
including jaws, true tongues, and trapezius muscles, that are possessed only
by gnathostomes. We aim to resolve the question of what primary factors havechanged during evolution by comparing the developmental patterns that yieldsuch innovations, and by the experimental construction of phenocopies in one
animal that mimic structures in another.
The turtle’s shelled body pattern appears suddenly in the fossil record. Our lab’s
research into turtle carapace development addresses the developmental changes that resulted in this abrupt and dramatic morphological change, and is aimed
at identifying genes that function differently in turtle and other amniotes, which itis hoped will provide a key to discovering the true targets of natural selection in
the acquisition of a shell.

Fumiaki SUGAHARA

Noritaka ADACHI
Hiroki HIGASHIYAMA
Yasuhiro OISIPart-Time Staff

Maki MIYAUCHI
AssistantMika HIKAWA
Yuko HIROFUJI

Publications
Nagashima H, et al. Evolution of the turtle body plan by thefolding and creation of new muscle connections. Science
325. 193-6 (2009)
Kuraku S, et al. Noncanonical role of Hox14 revealed by its
expression patterns in lamprey and shark. Proc Natl Acad
Sci USA 105. 6679-83 (2008)
Ota K G, et al. Hagfish embryology with reference to theevolution of the neural crest. Nature 446. 672-5 (2007)
Nagashima H, et al. On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian
body plan. Development 134. 2219-26 (2007)
Kuraku S, et al. Comprehensive survey of carapacial ridgespecific genes in turtle implies co-option of some regulatory
genes in carapace evolution. Evol Dev 7. 3-17 (2005)
Takio Y, et al. Evolutionary biology: lamprey Hox genes andthe evolution of jaws. Nature 429. 1 p following 262 (2004)

26

Dorsal (left) and ventral (right) views of the carapace (dorsal half of the turtle shell) of
Chinese soft-shelled turtle. The carapace is made of laterally expanded ribs.

Sensory Development
Organogenesis is a monumental undertaking. From siting a given organ to its
correct location, to confirming that it is correctly engineered and constructed, toensuring the necessary functional connections with other organs, the embryo
has a daunting task before it; indeed, perhaps we should be surprised that theresult is so frequently successful. Taking sensory organogenesis as our model
system, we hope to be able to follow the route an organ takes in its development,
and seek to understand the steps that allow it to reach maturity. One aim is to beable to recapitulate these steps in vitro, with the ultimate goal of providing potentially clinically relevant applications.
The ear provides a particularly rich system for study. The inner ear, which converts mechanical stimulation to electrical impulses, is formed from a piece of tissue originally destined to become skin; a fate that is altered by instructive signals
in a process known as induction. In our research to date, we have identified themechanism and the signals that set this early tissue on its path toward an inner ear fate as well as those that determine its position. We next hope to learn
how these signals are themselves established. We are also investigating the rolethese signals play in the regulated differentiation of the otic placode, and in better understanding the cellular, molecular and embryological process responsible
for this elegantly engineered organ of hearing.
The inner ear cannot function alone. In higher vertebrates, it requires the middle
ear to transduce sound that has been channeled through the external ear, as
well as specialized regions within the brain to interpret the information it sends.
But how is this intricate integration achieved? Using a combination of molecularand embryological techniques, we are characterizing the systems the embryo
uses in the coordinated construction of the auditory apparatus.
We believe that an understanding of the embryological systems that control thedevelopment of the inner ear can be applied to the development of other organs
as well, sensory organs in particular. By working to comprehend the induction
and development of other sense organs, such as the olfactory system and theeye, we are now taking steps toward achieving insights into common mechanisms underpinning the development of organ systems.

Raj LADHER Ph.D.
http://www.cdb.riken.jp/en/ladher
Raj Ladher received his B. Sc. in biochemistry from Imperial College, London in 1992
and his Ph.D. in developmental biology fromthe National Institute for Medical Research in
1996, for his thesis on the cloning of the Xenopus homeobox gene, Xom. 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 the Tokyo Medical
and Dental University. In 2000, he moved to
the University of Utah as a postdoctoral fellow
in Gary Schoenwolf's laboratory, and was appointed team leader at the CDB in 2002.

Staff
Team Leader

Visiting Scientist

Research Scientist

Student Trainee

Rajesh LADHER
Xiao Rei CAI
Sabine FRETER
Akira HONDA
Siu Shan MAK
Yuko MUTA
Marie PASCHAKI
Vassil VASSILEV

Foreign Postdoctoral
Researcher

Tomoko KITA
Kazuya ONO

Part-Time Staff

Yoshiko KONDO
Yoko MATSUOKA
Kazuya ONO
Assistant

Noriko HIROIPaul O'NEILL

Publications
Teraoka M E, et al. Rostral paraxial mesoderm regulates
refinement of the eye field through the bone morphogenetic
protein (BMP) pathway. Dev Biol (2009)
Freter S, et al. Progressive restriction of otic fate: the role of
FGF and Wnt in resolving inner ear potential. Development
135. 3415-24 (2008)
Sai X and Ladher R K. FGF signaling regulates cytoskeletal
remodeling during epithelial morphogenesis. Curr Biol 18.
976-81 (2008)
Manley G A, and Ladher R K. Phylogeny and Evolutionof Ciliated mechanoreceptors Springer, Handbook of theSenses (Elsevier) ed. P. Dallos (2007)
Sakamoto T, et al. Hair cell differentiation becomes tissue
specific by E9.5 in mouse inner ear. Neuroreport 18. 841-4(2007)
Ladher R K, et al. FGF8 initiates inner ear induction in chickand mouse. Genes Dev 19. 603-13 (2005)

The chick middle ear is made of one bone, the columella. Alcian blue highlights the cartilage of thecolumella. On the left is a control columella, the right columella is from an anotic embryo.

Ladher R K and Schoenwolf G C. Neural Induction. Developmental Neurobiology (Plenum Press), eds. M. Rao and M.
Jacobson. (2005)27

Cell Asymmetry
Our group seeks to explore the molecular mechanisms underlying the organization of cells into highly ordered structures in the developing brain, where neuralstem cells generate a large number of neurons of different fates at appropriate
points in time. Asymmetric cell division plays an essential role in this process. We
have focused our study on the control of asymmetric division and cell polarity inneural precursor cells, and their roles in brain development in invertebrate (Drosophila) and vertebrate (mouse) systems.

Fumio MATSUZAKI Ph.D.
http://www.cdb.riken.jp/en/matsuzaki
Fumio Matsuzaki received his B. Sc. from the
Department of Biochemistry and Biophysics at
the University of 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 from1984 to 1988 as a postdoctoral fellow, first in the
Department of Cell Biology at the Tokyo Metropolitan Institute of Medical Science, then in thelaboratory of Gerald Edelman at the Rockefeller
University. He returned to Japan in 1988 to takea position as a section chief in the Departmentof Molecular Genetics in the National Instituteof Neuroscience. In 1998, he was appointed
professor in the Department of Developmental
Neurobiology at Tohoku University and remained
there until taking his current position as groupdirector at the RIKEN CDB.

Staff
Group Director

Visiting Scientist

Special Postdoctoral
Researcher

Technical Staff

Fumio MATSUZAKI
Daijiro KONNO

Research Scientist

Tomohiko IWANO
Atsushi KITAJIMA
Yoichi KOSODO
Hironori OGAWA
Atsunori SHITAMUKAI
Chieko TAKIZAWA
Takuya TSUJI
Shigeki YOSHIURA

Ayano KAWAGUCHI
Tomoko AOKIMisato IWASHITA
Aki MASUDANao OTA
Taeko SUETSUGU
Student Trainee

Masahiko INOUE
Tomoaki KATO
Yoshiaki SATO

Asymmetric cell division, in which daughter cells of different types are produced,
is a process fundamental to the generation of cells of divergent types during
proliferation. This type of division requires the polarized organization of mitotic
cells when it occurs cell-autonomously, and depends on asymmetric microenvironments when the process is non-cell autonomous. Drosophila neural stem
cells, called neuroblasts, provide an excellent model system for the investigationof fundamental aspects of asymmetric division, including the cell polarity that
promotes it. Neuroblasts typically undergo asymmetric divisions to produce two
daughter cells: another neuroblast, and a smaller ganglion mother cell (GMC)
to which neural fate determinants such as Numb and the Prospero transcription factor are asymmetrically partitioned. However, we do not yet understand anumber of fundamental aspects of the asymmetric division of neuroblasts. We
are addressing these issues using genetic screens for mutations affecting theasymmetric division of neuroblasts.
The vertebrate brain comprises a considerably larger number of neurons arranged in a vastly more complex functional network than that in Drosophila. This
complex organ develops from the two dimensional epithelial sheet that forms theneural tube, in which neuroepithelial cells initially proliferate and subsequently
yield neurons from neural stem cells. Hence the transition from the proliferative
to neurogenic phase is thought to be critical for determining brain size. Althoughprevious studies have proposed that neural stem cells divide symmetrically to
proliferate and asymmetrically to produce neurons, the mechanism by which
neural stem cells switch their mode of division from symmetric to asymmetricand the manner in which they simultaneously generate self-renewable and differentiating daughter cells are not well understood. We are investigating these
problems to understand the principles that organize the cellular architecture of
the vertebrate brain.

AssistantJunko ISHIGAI

Publications
Ogawa H, et al. Protein phosphatase 2A negatively regulates aPKC signaling by modulating phosphorylation of Par6 in Drosophila neuroblast asymmetric divisions. J. Cell
Science 122. 3242-3249 (2009)
Shioi G, et al. Structural Basis for Self-Renewal of Neural
Progenitors in Cortical Neurogenesis. Cereb Cortex 19(suppl_1). i55-i61 (2009)
Yamamoto Y, et al. The GC kinase Fray and Mo25 regulate
Drosophila asymmetric divisions. Biochem Biophys Res
Commun 366. 212-218 (2008)
Konno D, et al. Neuroepithelial progenitors undergo LGNdependent planar divisions to maintain self-renewability
during mammalian neurogenesis. Nat Cell Biol 10. 93-101(2008)
Kawaguchi A, et al. Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis.
Development 135. 3113-24 (2008)
Matsuzaki F and Sampath K Wiring the nervous system:from form to function. Development 134. 1819-22 (2007)28

In the developing mouse spinal cord, Prospero (green) is transiently expressed in newborn neurons
immediately after birth from mitotic neural progenitors (red).

Dorsal view of computerized axial tomography scanned
juvenile of Chinese soft-shelled turtle, Pelodiscus sinensis.

Eye field refined by BMPs

In vertebrates, the eye develops from a single patch at
the headward end of the neural plate, which later splits
into two, allowing a pair of eyes to form from optical vesicles that bulge outward from the sides of the developing
brain. The initial bisection of the eye field is achieved bysignaling from the neighboring prechordal mesoderm,
but how the subsequent shaping and fine-tuning of theresulting halved regions is directed is not well understood.
In a report published in Developmental Biology, Michael
Teraoka and colleagues in the Laboratory for Sensory
Development (Raj Ladher; Team Leader) described how
a region of paraxial mesoderm works latterly to refine
and restrict the eye field after its bisection in chick. The
team found that signals from the rostral cephalic paraxial
mesoderm help to limit the expression of eye field transcription factors through the action of the BMP signaling
pathway.
Teraoka began with experiments in which he removed
various tissue regions at developmental stages corresponding to eye formation. When he extirpated paraxial
mesoderm near the head, he saw the optical vesicles
subsequently became enlarged, due to an increase in
the total numbers of cells in these structures. As no effect was seen on cell proliferation, he surmised that theincrease in the numbers of optical vesicle cells might bedue to a misassignment of cell fate instead.
Following this classic embryological approach, he next
swapped rostral paraxial mesoderm from quails into regions of the embryonic chick head that do not normally
feature mesoderm. He found that this affected eye development in the converse manner: the optic cups werereduced in size and the lens vesicles were small or absent. The transplantation also affected the expression ofgenes associated with eye development, such as Pax6
and crystallin. Significantly, there was no effect on neuralpatterning in general, suggesting that the rostral paraxial
mesoderm has an eye-specific role. This role, in turn, is
specific to the rostral paraxial mesoderm as well; transplantation of other regions of quail mesoderm to thesame part of the embryonic chick head had no effect on
the development of the eye.

Michael TERAOKA

30

©2009 Elsevier B.V.
Teraoka M, et al. Rostral paraxial mesoderm regulates refinement of the eyefield through the bone morphogenetic protein (BMP) pathway.Dev Biol (2009)

Seeking a possible molecular mechanism be hind the rostral pa ra x ial
mesoderm’s eye-inhibiting function,
Teraoka focused on bone morphogenetic protein (BMP) signaling, which
he found to be expressed in theparaxial mesoderm and, importantly,
repressed in the axial mesoderm,which has no optic-inhibitory activity.
He next implanted tiny beads soaked
in BMP factors to test for effects on
optic gene expression, and found that
BMP4 caused reductions in several
eye-related genes without affecting
genes more generally involved in neural patterning. Knocking the BMP level
down in vivo by shRNA had the opposite, pro-optic effect of increasing
the size of the optic vesicles, which
was mirrored by the antagonism offactors downstream in the BMP pathway as well. The team speculates
that BMP’s role may be mediated by
its interplay with both canonical andnon-canonical Wnt signaling, anotherpathway known to be important in eye
development.

Figure 1
BMP4 soaked into a microgel
bead (blue) inhibits the formation of the eye, by acting on adownstream optic determinant,wnt8b.

“I think that the most important aspect of this work is the finding that organ-generating fields areconstantly being told what to do by their neighbors,” says Ladher. “In this way the final shape andposition is formed so that the organ can carry out its function.”

Figure 2
BMP4 soaked into a microgel
bead (blue) inhibits the formation of the eye, by acting on adownstream optic determinant,
Pax6.

31

A complicated birth for the germ cell lineage

Yasuhide OHINATA

Segregating the germ cells used in reproduction from the rest of the body’s (somatic) cells is a mission-criticaltask for the embryo for, more than any organ system, the germline is the body’s raison d’être. In many species,
the initial commitment of the germ lineage cells takes place early in development, and sets them off on a journey
through somatic regions before they reach their final home in the gonads. This is true for mammals as well; in themouse, the roots of the germline have been traced back to as early as the sixth day of development. But what
triggers the expression of the first germline-specific genes has been a mystery.
A study by Yasuhide Ohinata and colleagues in the Laboratory for Germ Cell Biology (Mitinori Saitou; Team
Leader) revealed signals even further upstream in the germ cell specification cascade, originating from the extraembryonic ectoderm. Reported in Cell, the newly unearthed mechanism, which involves both the Bmp and Wnt
pathways, was further shown to drive the high-efficiency differentiation of this tissue to the germ lineage in vitro,
yielding viable germ cells (sperm) capable of fertilization following transplantation into testes.
The importance of Bmp signaling, including the Smad proteins downstream, from the extraembryonic ectoderm
had been pointed at by earlier work from other labs, while Saitou’s lab had previously identified Blimp1, Prdm14
and stella as the earliest genetic factors in germline specification. Taking that as their starting point, Ohinata andcolleagues asked how these two known factors might be connected. In the embryo, the germline specifying factors Blimp1 and Prdm14 are expressed in only a limited region of the epiblast proximal to the extraembryonic ectoderm, while Bmp4 and Bmp8 are uniformly expressed throughout that tissue. Loss-of-function studies revealed
that germ cell specification is the result of an interaction between two signaling axes – one related to proximity to
the extraembryonic ectoderm (involving the Bmps) and a second that represented posterior epiblast properties,
which could be inhibited by an antagonistic signal from the anterior visceral endoderm (via a pathway mediatedby Smad2 and FoxH1).

32

©2009 Cell Press
Ohinata Y, et al. A signaling principle for the specificationof the germ cell lineage in mice. Cell 137. 571-84 (2009)

They next tried culturing these embryonic regions to see if their insights into the specification
mechanism could be replicated in vitro. Using Day 6 epiblast and extraembryonic ectoderm, they
showed that the addition of Bmp4 to the culture medium was sufficient to induce the expressionof the germline-specifying factor Blimp1 throughout the entire explanted region. Further studies
using mutants for the Wnt factor Wnt3, which is involved in early embryonic patterning, showedthat Bmp4’s instructive function is Wnt-dependent.
Interestingly, a second Bmp factor, Bmp8b, which is known to be important for the generationof primordial germ cells, had no direct effect on germ cells themselves. Using a mouse model inwhich the Bmp8b gene had been knocked out, and further in vitro studies, Ohinata et al. determined that the role of this protein is to inhibit development of the anterior visceral endoderm, atissue that has an inhibitory effect on Bmp4.
With this solider understanding of germ cell differentiation, Ohinata used the culture system thelab had developed to steer epiblasts to adopt this fate. The induced cells showed expression of
Blimp1 and other genetic and epigenetic hallmarks of germ cells. The real test of their character,though, came with their injection into the testes of sterile mice, which revealed that they were fully
capable of further differentiation
into viable sperm.
“In this study, we looked at theepiblast, which is composed ofpluripotent cells that give rise to
the embryo, so in that sense what
we learned may also be applicableto other pluripotent cells, such asembryonic or induced pluripotent
stem cells,” says Saitou. “Althoughsome ethical questions remain, it
may be possible to apply this technique for germ cell differentiationin the future, which may have applications in the treatment of infertility in addition to its fundamental
scientific interest.”

Sperm dif ferentiated frominduced primordial germ cells
engineered to express GFP (top)are fertilization-competent andcan be used to generate normal
offspring (bottom; green shows
GFP fluorescence)33

A metabolomics approach to measuring
circadian time
Our bodies, and those of all animals, have intrinsic mechanisms that ensure that their many internal systems, such as sleep and hormone secretion, follow the rhythms of the earthly day closely.
Many diseases, such as those of the heart and vasculature, also show changes in the severityof symptoms at different times of day, and disturbances of the circadian rhythm can create great
difficulties for individuals in their waking and sleeping lives. A technique for measuring the body’s
own time would open the prospects of a new “chronotherapy” approach to medicine. But wehave yet to decipher the code of the body’s clock.
A study by Yoichi Minami, Takeya Kasukawa and others in the Laboratory for Systems Biology(Hiroki R. Ueda, Team Leader) working in collaboration with colleagues at Keio University (Tsuruoka, Japan) has revealed a method for measuring bodily time by analyzing metabolic productsin the blood. This new approach to reading circadian status is published in the Proceedings of
the National Academy of Sciences USA.

Body time can be represented
by a profile of “time-indicating
metabolites” and can be accurately detected by “metabolitetimetable method”. In the day(lower right) ‘day-indicating
metabolites’ (orange) are atpresent at high levels, whereas‘night-indicating metabolites’(blue) are low. In the evening(upper left), the opposite is true.

The inspiration for the study actually came from the 18th century Swedish botanist Karl vonLinne (more commonly known as Linnaeus), who discovered that one could estimate the time of
day simply by watching which types of flowers had opened their blooms. Minami and Kasukawa
thought that they might be able to do the same thing by watching the changes in levels of metabolites and other factors instead. The Ueda lab had made a first important step toward that goalby developing a molecular timetable for the mammalian biological clock in 2004, but the method
involved analyzing gene expression in the liver, making it unfeasible for simple clinical uses.

34©2009 The National Academy of Sciences
Minami Y, et al. Measurement of internal body timeby blood metabolomics.Proc Natl Acad Sci U S A (2009)

The team turned next to a metabolomics approach, which entails samp-ling a comprehensive set of metabolites
in the bloodstream to get a snapshot of the body's state at any given time. However, such an approach had neverbeen applied to substances with oscillating levels in the blood. Minami, Kasukawa and colleagues drew blood at
4-hour intervals from mice for two days and analyzed the samples using a combination of liquid chromatography
and mass spectrometry at Keio University. Mathematical analysis of the samples revealed a set of 142 positiveion and 176 negative ion metabolic products that showed clear rises and falls over the course of a 24-hour day.
Importantly, mice with known circadian defects showed aberrant metabolic cycles as well.
The team tested this newly developed metabolite timetable by sampling mice at arbitrary times of day and comparing their results with the predictions from the new technique, and found that it provided an admirably accurate
picture of the body’s circadian state. Additional tests using mice of different genetic backgrounds, genders, ages,
and feeding conditions further confirmed the general applicability of the method. They next entrained mice to analtered daily rhythm and looked at whether the timetable would hold up in these “jet-lagged” animals. They foundthat on the first day of jet-lag, the circadian metabolites remained at their previous levels, but by day 5, they wereat an intermediate state between real time of day and the entrained time, and by day 14, the switchover to the
new cycle as measured by metabolomics was complete.
“People show much greater diversity than mice in their eating habits and daily schedules, so it would be important to take that into consideration in developing any clinical applications,” says Kasukawa. Minami also adds,“The technique will need to be simplified before it can enter widespread use. But I think this metabolite timetable
represents a significant step forward in the drive to realizing chronotherapy.”

Takeya KASUKAWA

Yoichi MINAMI

35

Opposing roles for Wnt receptors
in the olfactory system

Masao SAKURAI

Olfaction is one of the primary sensory modes by which many species, including insects, survey their environments. The olfactory system is therefore highly specialized and exquisitely sensitive to a very broad spectrum ofodorant signals. The fruit fly Drosophila melanogaster, for example, has around 1300 olfactory receptor neurons
each carrying one of around 60 odorant receptors. Adding to the complexity, these neurons bundle together into
50 distinct glomeruli, which can be found arrayed in a consistent pattern in the brain of any given fly. The control
mechanisms responsible for ensuring this olfactory patterning are known to involve the signaling factor Wnt5, butotherwise remain largely a mystery.
Work by Masao Sakurai and colleagues in the Laboratory of Neural Network Development (Chihiro Hama; Team
Leader) revealed that a pair of receptors – Drl and Drl-2 –are expressed in different olfactory cell types and play
opposing roles in Wnt signaling. These findings, published in The Journal of Neuroscience, shed new light on thecooperative mechanisms at work in establishing the olfactory circuitry.
The study began by looking at Wnt5 mutants, which Sakurai found resulted in a phenotype in which peripheral
glomeruli in the antennal lobes rotated clockwise of their wildtype positions. Knowing the role of Drl as a Wnt5
receptor, he next checked the drl mutant phenotype, but found it to be substantially different from that of Wnt5,

36

©2009 Society for Neuroscience
Sakurai M, et al. Differentially expressed Drl and Drl-2 play opposing roles in Wnt5 signaling
during Drosophila olfactory system development. J Neurosci 29. 4972-80 (2009)

suggesting that other receptors might also be involved. To test this possibility, the team engineered
flies to lack function of both drl and a related gene drl-2, and found that the double mutant phenotype
much more closely resembled that of the Wnt5 mutant, which suggests that both Drl and Drl-2 function as Wnt5 receptors.

A. Flies perceive odors througho l f a c to r y r e c e pto r n e u r o n s(ORNs) on the a nte nna a nd
maxillary palp (MP).
B. ORNs expressing the sameodorant receptor (as shown by
the same color) project axons
to one of 50 glomeruli in theantennal lobe (AL).
C. A stereotyped organizationof glomeruli in the wild-type
AL. The glomeruli DA1, VL1, V
and VA1d,v (dotted circles) are
shown in the right AL.
D. The glomeruli in the right AL
shift in a clockwise manner in
the Wnt5 mutant compared tothose in the wild type.

One reason for the differences in the Wnt5 and drl phenotypes is traceable to Drl’s antagonistic effecton Wnt5 signaling. The Drl protein is expressed in only a subset of second-order olfactory neurons
and glial cells, and it is in the latter that the repressive function manifests. Drl-2 in contrast is normally
expressed in the axons of olfactory receptor neurons. But when the Hama team misexpressed Drl-2
in the glial cells of drl mutants, they found it was capable of compensating for the loss of drl function,
which antagonizes Wnt5 signaling. This led to the somewhat enigmatic conclusion that these two
structurally related molecules play contrasting roles with respect to Wnt5, and that further, the choiceof role is determined by the cells in which they are expressed.
“The antagonistic function of Drl and Drl-2 is likely to arise from titration of Wnt5 ligand by receptor
binding,” notes Hama. “This simple physical mechanism invokes the general idea that many other receptors potentially have an antagonistic role in their respective signaling.”

37

2009 Courses
As part of its commitment to promoting awareness and understanding of its field of research, and maintaining close ties with the academic
and research communities, the RIKEN CDB dedicates itself to conducting training and public educational programs for a wide range of audiences. Although not a teaching institution, the Center works with students, educators, professional scientists and other organizations todevelop, implement and promote a variety of instructional courses throughout the year.

Pluripotent stem cell courses
The CDB organizes a series of beginners’ workshops on the use of humanstem cells in collaboration with the Kyoto University Center for iPS Research and Application and the RIKEN Bioresource Center. In 2009, thesetwo-day learning programs were held in February, April, and November,and provided instruction for dozens of participants from academia and industry in the basics of human pluripotent stem cell research.

Summer school forhigh school students
The RIKEN CDB held two summer school days for Kansai area high school
students on August 4th and 6th. Now in its third year, this year’s program
was organized with cooperation from the Laboratory for Morphogenetic
Signaling. Both days featured a talk in the morning by Shigeo Hayashi(Group Director, Laboratory for Morphogenetic Signaling), followed by avisit to his lab, and hands-on lab work in the afternoon using Drosophila (fruitfly) larvae.

Science lesson for teachers
The RIKEN CDB held its recurring course for high school science teachers in conjunction with the Japanese Society of Developmental Biologists
on October 3 and 4. This is the second time the course, which is aimed atmaking a contribution to high school education in the biological sciences,
has been held since its launch last year. This year’s main themes were theuse of chicken embryos as a classroom material, and organizer transplant
experiments, which were introduced through lectures and lab work.

38

Expression of Gfrα 1GFP (green) and E-cadherin
protein (red) in embryonic day 13.5 mouse kidney

Germline Development

Akira NAKAMURA Ph.D.
http://www.cdb.riken.jp/en/nakamura
Akira Nakamura received both his baccalaureateand his Ph.D. from the University of Tsukuba.
He spent a year as a post-doctoral fellow at thesame institution before moving to the Department of Biology at McGill University in Montreal
in 1995 to work as a postdoc under Paul Lasko.
He returned to Japan in 1997 as a research associate at the University of Tsukuba. He wasappointed assistant professor in the university's
Gene Research Center and Institute of Biological
Sciences in 2000, and began a three-year term
as a PRESTO researcher in the Japan Scienceand Technology Corporation (JST) in December
2001. He was appointed CDB team leader inMarch 2002.

Staff
Team Leader

Technical Staff

Research Scientist

Student Trainee

Akira NAKAMURA
Kazuko HANYUNAKAMURA
Yasuko KATO
Maki SHIRAEKURABAYASHI
Tsubasa TANAKA

Kazuki MATSUDA
Kenji MAEKUBO

Germ cells are the only cell types capable of transmitting genetic information
across generations, and their formation is characterized by unique developmental processes as well. In many types of animals, including the Drosophila fruit fly,the formation and differentiation of germ cells is controlled by mRNAs and proteins localized in a specific cytoplasmic region within eggs, called germ plasm.
Germ plasm mRNAs are translated in a spatiotemporally regulated manner, but
the means by which the germ plasm is formed and regulates germ cell development remain largely unknown. Our research team studies the establishment of
the Drosophila germ line as a model of the processes of germ plasm assemblyand germ cell differentiation. We expect that our study will also provide insights
into the general mechanisms of mRNA localization and translation, which areimportant to a number of other developmental processes including asymmetriccell division, cell migration and axis formation, and postnatal synaptic plasticity
as well.
One area of interest is the mechanism of translational repression in germline
development. In one example of this critically important form of regulation, thetranslation of the RNA for the maternal gene oskar, which functions in embryonicpatterning and the formation of germline cells in Drosophila, is repressed during
its transport to the posterior pole of the oocyte. We are now studying the function of proteins, such as the recently identified factor, Cup, that regulates therepression of oskar translation during its localization to the oocyte posterior. In
another concurrent project, we are focusing on the roles of wunen2 and polar
granule component (pgc), which are known to function in lipid signaling and global transcriptional regulation in germline cells during embryogenesis.
In addition to the study of fruit fly germline development, we are also beginningto undertake investigations using the ascidian, Ciona intestinalis. Our team will
explore the genetic regulation of ascidian germline development by characterizing promoter regions of germline specific genes and trans-acting factors that
regulate germline specific gene expression.

Assistant

Eri YAMASHITA

Publications
Tanaka T and Nakamura A. The endocytic pathway acts
downstream of Oskar in Drosophila germ plasm assembly.
Development 135. 1107-17 (2008)
Hanyu-Nakamura K, et al. Drosophila Pgc protein inhibits
P-TEFb recruitment to chromatin in primordial germ cells.
Nature 451. 730-3 (2008)
Shirae-Kurabayashi M, et al. Dynamic redistribution of vasa
homolog and exclusion of somatic cell determinants during
germ cell specification in Ciona intestinalis. Development
133. 2683-93 (2006)
Barbee S A, et al. Staufen- and FMRP-containing neuronal
RNPs are structurally and functionally related to somatic P
bodies. Neuron 52. 997-1009 (2006)
Nakamura A, et al. Drosophila cup is an eIF4E binding protein that associates with Bruno and regulates oskar mRNAtranslation in oogenesis. Dev Cell 6. 69-78 (2004)
Hanyu-Nakamura K, et al. Germ cell-autonomous Wunen2
is required for germline development in Drosophila embryos.
Development 131. 4545-53 (2004)

Migrating gem cells (blue) in stage 10 Drosophila embryo.

40

Chromatin Dynamics
Multicellular organisms are made up of diverse populations of many different
types of cells, each of which contains an identical set of genetic information coded in its DNA. Cell differentiation and the process of development itself dependon the ability of individual cells to maintain the expression of different genes, andfor their progeny to do so through multiple cycles of cell division. In recent years,
we have begun to understand that the maintenance of specific patterns of gene
expression does not rely on direct modifications to the DNA sequence encoding
the organism’s genome, but rather takes place in a heritable, “epigenetic” manner. DNA methylation, chromatin modifications, and RNA silencing are some of
the best known epigenetic phenomena. Recent studies have begun to show thatthese different mechanisms are closely interrelated, but a detailed understandingof these systems has yet to be developed.
Our team investigates how modifications to the structure and configuration ofchromatin (complexes of nuclear DNA and proteins that provide the structural
basis of chromosomes) contribute to epigenetic gene regulation and how suchmodifications are transmitted over generations of cellular division by studying
events at the molecular scale in the excellent model organism, fission yeast(Schizosaccharomyces pombe), and in cultured mammalian cells.
Histones are a set of DNA packing proteins present in nucleosomes, the fundamental building blocks of chromatins. In our current studies, we are particularlyinterested in determining the specific histone modifications and the molecular
recognition processes that enable modified histones to work together to establish and maintain higher-order chromatin structures. We also seek to clarify the
picture of how dynamic rearrangements of chromatin structure are triggeredby examining the structure and function of protein complexes that bind to andmodify histones. Through these approaches we aim to understand the molecular mechanisms that underlie complex epigenetic phenomena in developmental
processes.

Jun-ichi NAKAYAMA Ph.D.
http://www.cdb.riken.jp/en/nakayama
Jun-ichi Nakayama received his bachelor's,
master's and Ph.D. degrees in bioscience fromthe Tokyo Institute of Technology, the last in
1999 for his work on the cloning and characterization of mammalian telomerase components.
He spent the period from 1999 to 2001 as a
postdoctoral researcher at Cold Spring Harbor
Laboratory in Shiv Grewal's lab, before returning to Japan in December 2001 as a PRESTO
researcher in the Japan Science and Technology Corporation (JST). He was appointed teamleader of the Laboratory for Chromatin Dynamics in 2002.

Staff
Team Leader

Jun-ichi NAKAYAMA
Research Scientist

Kyoko HAMADA
Tomohiro HAYAKAWA
Aki HAYASHIAtsuko SHIRAI

Student Trainee

Mayumi ISHIDA
Erina KITANO
Gohei NISHIBUCHI
Assistant

Sayaka SENOTechnical Staff

Noriyo HAYAKAWA
Rika KAWAGUCHI
Yasuko OHTANI

Publications
Sadaie M, et al. Balance between distinct HP1 proteins
controls heterochromatin assembly in fission yeast. Mol Cell
Biol 28. 6973-88 (2008)
Iida T, et al. siRNA-mediated heterochromatin establishment
requires HP1 and is associated with antisense transcription.
Mol Cell 31. 178-89 (2008)
Hayakawa T, et al. RBP2 is an MRG15 complex component
and down-regulates intragenic histone H3 lysine 4 methylation. Genes Cells 12. 811-26 (2007)
Buker S M, et al. Two different Argonaute complexes are required for siRNA generation and heterochromatin assemblyin fission yeast. Nat Struct Mol Biol 14. 200-7 (2007)
Iida T, et al. Conserved ribonuclease, Eri1, negatively regulates heterochromatin assembly in fission yeast. Curr Biol
16. 1459-64 (2006)
Sadaie M, et al. A chromodomain protein, Chp1, is required
for the establishment of heterochromatin in fission yeast.
Embo J 23. 3825-35 (2004)

Model for RNAi-mediated heterochromatin assembly in fission yeast

41

Stem Cell Biology
The stem cell system can be thought of as a means by which organisms maintain cell renewal. All forms of life require the endless recycling of both materials
and energy, making it impossible for either organisms or individual cells to live
independent of their environments. In bodies made up of many cells working incooperation, such as our own, the need to replace and renew is not limited tosimple materials and energy supply; the constant generation of new cells is alsoessential to the maintenance of the individual, a process that has developedin the evolution of multicellular life. In this sense, the process of cell renewal inmaintaining the body’s integrity and function provides a meaningful example of
the relationship of the cell to the organism, the individual to the whole, which wehope to explore more fully using the biology of stem cells as our model.

Shin-Ichi NISHIKAWA M.D., Ph.D.
http://www.cdb.riken.jp/en/nishikawa
Shin-Ichi Nishikawa received his M.D. from the
Kyoto University School of Medicine in 1973. He
performed his internship and residency in the Department of Internal Medicine at the Kyoto University Chest Disease Research Institute, before taking
an assistant professorship in the same department
in 1979. He spent the period from 1980-82 at the
University of Cologne Institute for Genetics 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 Faculty of Medicine in
1987 to take a professorship in the Department of
Immunopathology, and returned to Kyoto in 1993,
as professor in the Department of Molecular Genetics at the Kyoto University Faculty of Medicine. He
was appointed CDB group director in 2000.

We are especially interested in the question of how cells maintain the never-ending self-renewal that sustains the organism as a whole. This will require, at thevery least, solving the riddles of two essential processes. The first of these is theneed for old cells destined for replacement to be able to disengage or otherwise
be dislocated from their environmental milieux. The second is the requirement for
preparing new cells in replacement. To investigate the first of these mechanisms,
our group uses a system for labeling cells with special dyes, which allows us tomonitor their location and behavior. We have also developed a system for differentiating embryonic stem (ES) cells in culture to study the second question of
new cell production. Currently, we are focusing on developing different stem cellsystems, particularly hematopoietic stem cells. This ongoing research project in
our lab will allow us to explore how the self-renewing stem cell system is formed
and will lead to the development of methods to generate bona fide hematopoietic stem cells from ES cells.

Staff
Group Director

Shin-Ichi NISHIKAWA
Research Scientist

Visiting Scientist

Herve Le MOUELLIC
Yosuke TANAKA

Julien BOUISSAC
Lars M JAKT
Hiroshi KATAOKA
Igor M SAMOKHVALOVYoshiteru SASAKI
Yan-Fung WONG

Technical Staff

Research Specialist

Junior Research Associate

Research Associate

Part-Time Staff

Reiko NAKAGAWA
Rasmus FRETER
Yasushi KUBOTA

Collaborative Scientist

Shin KAWAMATA

Misato HAYASHI
Satoko MORIWAKI
Satomi NISHIKAWA
Yuri SHIMIZU
Naoko YOSHIOKA
Kenichiro KOBAYASHI
Miho HOSHIJIMA
Yoko OTSUKA
Kazumi YANAIAssistantChisato NISHIMURA
Satomi SHINOHARA

Publications
Kubota Y, et al. Necdin restricts proliferation of hematopoietic stem cells during hematopoietic regeneration. Blood
114. 4383-92 (2009)
Kondo N, et al. Thrombin induces rapid disassembly ofclaudin-5 from the tight junction of endothelial cells. Exp Cell
Res 315. 2879-87 (2009)
Kinoshita M, et al. The novel protein kinase Vlk is essential
for stromal function of mesenchymal cells. Development
136. 2069-79 (2009)
Eilken H M, et al. Continuous single-cell imaging of blood
generation from haemogenic endothelium. Nature 457. 896900 (2009)
Egawa G, et al. Transient expression of ephrin b2 in perinatal skin is required for maintenance of keratinocyte homeostasis. J Invest Dermatol 129. 2386-95 (2009)42

PDGFRα+ mesenchymal stem cells derived from neuroepithelial cells in 14.5 embryo.

Growth Control Signaling
The processes of animal development, including organ size and body size, aregenetically predetermined, but these processes are also influenced by environmental factors such as nutrition and temperature. The close link between celland tissue growth control and environmental cues ensures that developmental
transitions occur at the appropriate time during animal development.
Cell proliferation and differentiation in each tissue and organ are kept under strict
regulation both spatially and temporally. Research has revealed the nature of
spatial signals such as growth factors and morphogens, but the way in whichthese signals direct cell and tissue growth over time remains poorly understood.
For example, how do signals sent to quiescent cells direct them to enter the cell
cycle and begin proliferating at appropriate developmental stages; and how dothey know when to exit the cell cycle and/or undergo differentiation? In addition
to the intrinsic gene expression programs, growth and developmental timing arealso governed by nutrient availability. Most species have a standard body size,
but developing organisms are also capable of adapting their growth to fluctuating nutritional states through metabolic regulation. Therefore, linking the nutrient
sensory system to an endocrine signaling network allows organisms to control
the timing of cell proliferation and differentiation.
Our team’s research aims to shed light on the molecular basis for growth control
and developmental timing at the cellular and tissue/organ level using Drosophila
as a model system. In particular, we are interested in addressing the following
questions: 1) how do organisms adapt their growth program to changes in energy needs and states; 2) what are the molecular mechanisms that sense nutrient
availability and regulate cell/tissue size; and 3) how do endocrine signals interact
with metabolic and growth regulators? We will combine biochemical and genetic
approaches, along with quantitative and qualitative imaging and cell-biologicalanalysis, to identify and characterize the relevant signal transduction pathways.

Takashi NISHIMURA Ph.D.
http://www.cdb.riken.jp/en/nishimura
Takashi Nishimura obtained his Ph.D. in Kozo
Kaibuchi’s lab at the Nagoya University Graduate School of Medicine for his work on theregulation of neuronal polarization. He did
postdoctoral research in the same laboratory
before moving to the Institute of Molecular Biotechnology in Vienna, Austria in 2006 to pursue
further postdoctoral work in Jürgen Knoblich’s
group. He returned to Japan in 2009, working briefly as a visiting scientist at the RIKEN
Center for Developmental Biology prior to his
appointment as Team Leader in July of thesame year.

Staff
Team Leader

Takashi NISHIMURA
Technical Staff

Yukako HIRAO
Yuka NISHIMORI
Chiaki OOKURA

Publications
Wirtz-Peitz F, et al. Linking cell cycle to asymmetric division:
Aurora-A phosphorylates the Par complex to regulate Numb
localization. Cell 135. 161-73 (2008)
Nishimura T and Kaibuchi K. Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3.
Dev Cell 13. 15-28 (2007)
Nishimura T, et al. Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2. Mol Biol
Cell 17. 1273-85 (2006)
Nishimura T, et al. PAR-6-PAR-3 mediates Cdc42-induced
Rac activation through the Rac GEFs STEF/Tiam1. Nat Cell
Biol 7. 270-7 (2005)
Nishimura T, et al. Role of the PAR-3-KIF3 complex in theestablishment of neuronal polarity. Nat Cell Biol 6. 328-34(2004)
Nishimura T, et al. CRMP-2 regulates polarized Numbmediated endocytosis for axon growth. Nat Cell Biol 5. 81926 (2003)

Drosophila late larval brain showing proliferating cells (green)

43Pluripotent Cell Studies
Pluripotency is a term used to describe the ability of certain types of stem cells thatare capable of giving rise to at least one type of cell from all three germ layers –endoderm, mesoderm and ectoderm. Stem cells are also characterized by theirability to produce copies of themselves with similar differentiative potential, as well
as more specialized cells with different properties through differentiation. Embryonic stem (ES) cells are the best known example of a type of stem cell possessing these properties of pluripotency and self-renewal. In our lab, we use ES cells
as a model system for studying the molecular mechanisms that determine andenable the mysterious properties of pluripotent cells.

Hitoshi NIWA M.D., Ph.D.
http://www.cdb.riken.jp/en/niwa
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 workin gene trap methods. From 1993 to 1994, heworked as a research associate in the Department of Developmental Genetics at the same
university, before taking a postdoctoral fellowship with Austin Smith at the University of Edinburgh Centre for Genome Research. He returned
to Japan in 1996 as a research associate in
the Department of Nutrition and Physiological
Chemistry at the Osaka University Graduate
School of Medicine, where he remained until
taking his current position as Team Leader at the
RIKEN CDB. He was appointed Project Leaderof the Laboratory for Pluripotent Stem Cell Studies in October 2009.

Pluripotency can be primarily determined by a particular set of transcription
factors, for as we now know, pluripotency can be induced by 4 transcription
factors. These transcription factors should form a self-organizing network able
to stabilize their expression and maintain pluripotency. At the same time, the network should have the sensitivity to signals that induce differentiation. How can
the transcription factor network satisfy these contradictory requirements? Whatis the principle behind the structure of the transcription factor networks governing developmental processes? We are now trying to answer these questions bystudying ES cells and the changes they undergo in their differentiation toward
trophoblast stem cells, extraembryonic endoderm cells, and primitive ectoderm
cells.
In October 2009, the Laboratory for Pluripotent Cell Studies was re-designatedas a Project Lab in the Center Director’s Strategic Program for Stem Cell research, and renamed the Laboratory for Pluripotent Stem Cell Studies.

Staff
Team Leader

Student Trainee

Research Scientist

Part-Time Staff

Hitoshi NIWA
Kenjiro ADACHI
Jitsutaro KAWAGUCHI
Masaki KINOSHITA
Kazuhiro MURAKAMI
Satomi NISHIKAWATORIKAI
Kazuya OGAWA
Satoshi OHTSUKA
Itsuro SUGIMURA

Toshimi SUGIMOTO
Sachiko HASHIMOTO
Yayoi NAKAI
AssistantMiho SAKURAI

Publications
Niwa H, et al. A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460. 11822 (2009)
Niwa H, et al. Platypus Pou5f1 reveals the first steps in theevolution of trophectoderm differentiation and pluripotency
in mammals. Evol Dev 10. 671-82 (2008)
Toyooka Y, et al. Identification and characterization of subpopulations in undifferentiated ES cell culture. Development
135. 909-18 (2008)
Niwa H. How is pluripotency determined and maintained?
Development 134. 635-46 (2007)
Masui S, et al. Pluripotency governed by Sox2 via regulationof Oct3/4 expression in mouse embryonic stem cells. Nat
Cell Biol 9. 625-35 (2007)

44

Figure 1

Chimeric embryos generated by injectionof Gata6GR ES cells into blastocysts.
These ES cells carry the constitutivelyactive Egfp transgene and contribute toprimitive ectoderm (upper panels), whereas
they contribute to parietal endoderm after
induction of Gata6 activity with dexamethasone (lower panels).

Figure 2

Schematic model of transcription factor
networks for pluripotent stem cells, trophectoderm and primitive endoderm.

Mammalian Epigenetic Studies
The development and physiological function of multicellular organisms relies on
the ability of their component individual cells, which as a rule contain identicalgenetic information, to express subsets of genes in a selective manner. In orderto maintain lineages of cells of different types, the gene expression programs
established in ancestral precursor cells must be transmitted stably to many
generations of progeny. The fundamental mechanisms by which patterns ofgene expression are preserved across cycles of cell division without a change
to the underlying genome sequence are commonly referred to as “epigenetic”
processes. These processes produce chemical modifications to and structural
remodeling of chromatin, nuclear structures that store the cell’s DNA, thereby
allowing individual cells to regulate the switching on and off of the expression of
specific genes with great precision and flexibility.
Our team studies epigenetics by focusing on a particular set of molecules that
act in the methylation of DNA, with the goal of identifying their roles in the regulation of chromatin function in embryonic development and the processes oflineage commitment and plasticity in cell differentiation. We conduct phenotype
analyses of mice in which various genes involved in DNA methylation have beenknocked out, as well as biochemical and cell biological studies of embryonicstem (ES) cells to explore how dynamic changes in DNA methylation function as
regulatory factors in mammalian embryogenesis and cell differentiation, and themeans by which DNA methylation works to maintain cell proliferation and chromosome function. We anticipate that a deeper understanding of these questions
will lead to new insights in the context-specificity of the DNA methylation process
as well as the roles of methylation in development, health, and regeneration.

Masaki OKANO Ph.D.
http://www.cdb.riken.jp/en/okano
Masaki Okano received his baccalaureate
and master's degrees from Kyoto University,and his doctorate in biochemistry from the
Research Division in Agriculture at the same
institution in 1994. He spent the period from1994 to 1995 as a research fellow in cell biology at the Nagoya University BioScience
Center before moving to Massachusetts, where
he worked as a 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 thelast two years of that period. He was appointed
team leader at the RIKEN Center for Developmental Biology in 2001, and returned to work inJapan full-time in 2002.

Staff
Team Leader

Masaki OKANO
Research Scientist

Yuichi KUMAKI
Morito SAKAUE
Takashi TAMURA

Technical Staff

Chisa MATSUOKA
Akiko YAMAGIWA
Assistant

Fumika NAKAYAMA

Publications
Kumaki Y, et al. QUMA: quantification tool for methylation
analysis. Nucleic Acids Res 36. W170-5 (2008)
Takebayashi S, et al. Major and essential role for the DNA
methylation mark in mouse embryogenesis and stable association of DNMT1 with newly replicated regions. Mol Cell
Biol 27. 8243-58 (2007)
Sharif J, et al. The SRA protein Np95 mediates epigenetic
inheritance by recruiting Dnmt1 to methylated DNA. Nature450. 908-12 (2007)
Oda M, et al. DNA methylation regulates long-range genesilencing of an X-linked homeobox gene cluster in a lineagespecific manner. Genes Dev 20. 3382-94 (2006)
Tsumura A, et al. Maintenance of self-renewal ability ofmouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11.
805-14 (2006)

Immunofluorescence showing Np95 localization to methylated DNA during DNA replication in ES cells.

45

Mammalian Molecular Embryology
There can be few, if any, cells as specialized as sperm and egg; they appear
unique (you can even see a mammalian oocyte with the naked eye) and don't
typically divide by themselves. Yet when they combine at fertilization, the single
cell they generate is transformed within hours to produce a totipotent cell: one
which is completely unspecialized, in that from it all cell types develop to produce an entire individual.

Tony PERRY Ph.D.
http://www.cdb.riken.jp/en/perry
Tony Perry received his B.Sc. in Microbiology
from the Department of Bacteriology at the
University of Bristol, England and his doctorate
from the University of Liverpool four years later.
From 1989 he became a postdoctoral fellow andtemporary lecturer working on epididymal sperm
maturation at the University of Bristol and in1996 won a European Molecular Biology Travel
Fellowship to study oocyte activation. Dr. Perrydeveloped a novel method of transgenesis which
he primarily worked on at the Rockefeller University. In June 2002 he took his present position
as Team Leader of the Laboratory of Mammalian
Molecular Embryology at the RIKEN CDB, where
he started to work on the mechanisms of key
cytoplasmic events at fertilization in mammals,
including chromatin remodeling and meiotic exit.

Staff
Team Leader

Anthony C.F. PERRYTechnical Staff

Research Scientist

Toru SUZUKI
Matthew D. VERMILYEA

Atsuko KUBO
Erina OKUDA
Heide OLLER
Emi SUZUKI

Collaborative Researcher

Assistant

Naoko YOSHIDA

Eriko NOMURA

Publications
Perry A C and Verlhac M H. Second meiotic arrest and exit
in frogs and mice. EMBO Rep 9. 246-51 (2008)
Yoshida N, et al. Broad, ectopic expression of the sperm
protein PLCZ1 induces parthenogenesis and ovarian tumours in mice. Development 134. 3941-52 (2007)

Our laboratory combines molecular and cell biology with micromanipulation ofmouse gametes and embryos to study the nucleo-cytoplasmic events that occur
immediately after sperm-egg union (oocyte activation) and their developmental
consequences. One long-standing question concerns why oocytes don't begin
to divide by themselves in the absence of a sperm. To address this, our groupdeveloped a novel approach that revealed that the removal of the protein Emi2
from oocytes caused them to resume the cell cycle as if the oocytes had been
activated by a sperm. We then extended this to show that Emi2 works throughCdc20 and that both parthenogenetic activation and fertilization require Cdc20.
This may be the first formal demonstration that the signaling required for parthenogenesis and fertilization has molecular components common to both; this issignificant given the application of parthenogenetic activation in nuclear transfer
and other current research.
During meiotic resumption, sperm chromatin undergoes almost complete remodeling; its nucleoproteins, which are mostly protamines, are removed andsupplanted by maternal histones which are subsequently modified. We study
and manipulate this overall process and have found that chromatin remodelingvaries depending on the provenance of the nucleus; the fate of somatic cell chromatin is different from chromatin associated with a sperm-derived genome, evenwhen both are in the same oocyte. Chromatin remodeling is not necessarily ofcritical importance; gross hyper-acetylation during fertilization has only a limited
effect, if any, on development. We are analyzing the developmental significanceof different modes of epigenetic remodeling and how they are regulated.
We are also interested in additional interactions between sperm head components and the oocyte cytoplasm, in a bid to discover what happens during
fertilization and the earliest moments of the new embryo. It would be useful toattribute molecular identities to the proteins involved in these interactions andcharacterize them functionally. This task is a daunting one, as the sperm contributes >500 distinct nuclear and cytoplasmic protein species at fertilization, andyet detecting them in newly fertilized oocytes and embryos requires exquisitely
sensitive methods. With a greater understanding of any sperm contribution todevelopment, our lab hopes to gain insights into the processes by which embryonic stem cells are formed and carcinogenesis is initiated.

Yoshida N, et al. Epigenetic discrimination by mouse metaphase II oocytes mediates asymmetric chromatin remodeling independently of meiotic exit. Dev Biol 301. 464-77(2007)
Yoshida N and Perry A C. Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nat Protoc 2. 296-304 (2007)
Shoji S, et al. Mammalian Emi2 mediates cytostatic arrest
and transduces the signal for meiotic exit via Cdc20. Embo
J 25. 834-45 (2006)
Amanai M, et al. A restricted role for sperm-borne microRNAs in mammalian fertilization. Biol Reprod 75. 877-84(2006)
Amanai M, et al. Injection of mammalian metaphase II
oocytes with short interfering RNAs to dissect meiotic andearly mitotic events. Biol Reprod 75. 891-8 (2006)

46

Cytoplasm from a mouse egg expressing a mitochondrially-targeted green fluorescent protein wastransplanted into one cell of a two-cell embryo; the paired images show the same embryo after cell
division, on the left with laser illumination only, and merged with DIC illumination on the right. For afuller explanation, see Shoji et al., EMBO J. 25, 834-845 (2006).

Mammalian Germ Cell Biology
All of the diverse cell types in the body can be broadly classed as either somatic or germline cells. In contrast to somatic cells, which work to maintain theconstant, stable form and function of an individual organism’s body, germ cells
provide the faithfully-replicated information needed to establish subsequent
generations of individuals. In order to fulfill this role, these cells need to exhibit
certain unique properties, including the ability to revert (generally through fusion
with another germline cell) to a state of developmental totipotency and maintainthat totipotent state until the start of ontogeny, the ability to undergo epigenetic
reprogramming, and to divide meiotically.
Research in our laboratory is geared to elucidating the developmental bases ofgermline function at the molecular levels from the very earliest stages of germline
development. Specifically, our subjects of interest are the molecular mechanisms(a signaling as well as transcriptional principle) involved in the formation of mouseprimordial germ cells (PGCs), the cellular progenitors of sperm and oocytes, thegenome-wide epigenetic reprogramming that takes place immediately following
PGC establishment, the development and application of technologies to enable
the analysis of transcriptional regulatory networks at the single-cell level, and ultimately, reconstitution of germ cell development in vitro.
Our studies have so far shown that germ cell specification in mice integrates
three key events: repression of the somatic program, reacquisition of potential
pluripotency, and genome-wide epigenetic reprogramming. We have identified aPR-domain-containing protein, Blimp1 (also known as Prdm1), as a critical factor
for PGC specification. Using a highly representative single-cell microarray technology that we developed, we identified complex but highly ordered genomewide transcription dynamics associated with PGC specification. This analysis
not only demonstrated a dominant role of Blimp1 for the repression of the genes
normally down-regulated in PGCs relative to their somatic neighbors, but also
revealed the presence of gene expression programs initiating independently
from Blimp1. Among such programs, we identified Prdm14, another PR-domaincontaining protein, as a key regulator for the reacquisition of potential pluripotency and genome-wide epigenetic reprogramming. The launch of the germ celllineage in mice, therefore, is orchestrated by two independently acquired PRdomain-containing transcriptional regulators, Blimp1 and Prdm14. Furthermore,
we have uncovered a signaling principle for germ cell fate specification. Suchstudies may provide fundamental information on the reconstruction of the germ
cell lineage from pluripotent stem cells in vitro.

Mitinori SAITOU M.D., Ph.D.
http://www.cdb.riken.jp/en/saitou
Mitinori Saitou received his M.D. from the Kyoto
University Faculty of Medicine in 1995, andreceived his Ph.D. in 1999 for his study of thestructure and function of tight junctions under
Shoichiro Tsukita in the Kyoto University Graduate School of Medicine. He then moved to theWellcome Trust/Cancer Research Campaign
Institute (present name: Wellcome Trust/Cancer Research UK Gurdon Institute), where heworked as a postdoctoral research associate in
Azim Surani's laboratory, focusing on the originof the germ line in the mouse. He was appointed
team leader at the CDB in 2003, and received
a three-year grant from the Japan Science andTechnology Corporation (JST) PRESTO programthat same year, and was affiliated to the Kyoto
University Graduate School of Biostudies asassociate professor in 2004. He assumed a
professorship at the Kyoto University Graduate
School of Medicine in 2009.

Staff
Team Leader

Research Fellow

Special Postdoctoral
Researcher

Technical Staff

Mitinori SAITOU
Kazuki KURIMOTO
Sugako OGUSHI
Yoshiyuki SEKI
Research Scientist

Yasuhide OHINATA
Jun UEDA
Yukihiro YABUTA
Masashi YAMAJI

Mitsue SANO

Mayo SHIGETA
Kaori YAMANAKA
Student Trainee

Takayuki HIROTA
Satomi KIKUOKA
Part-time staff

Miho MIURA
AssistantKazuyo NAKATANI

Publications
Yamaji M, et al. Functional reconstruction of NANOS3
expression in the germ cell lineage by a novel transgenic reporter reveals distinct subcellular localizations of NANOS3.
Reproduction 139. 381-93(2010)
Saitou, M. Germ cell specification in mice. Curr Opin Genet
Dev 19. 386-395 (2009)
Ohinata Y, et al. A signaling principle for the specification of
the germ cell lineage in mice. Cell 137. 571-84 (2009)
Ohinata Y, et al. A comprehensive, non-invasive visualizationof primordial germ cell development in mice by the Prdm1mVenus and Dppa3-ECFP double transgenic reporter.
Reproduction 136. 503-14 (2008)
Yamaji M, et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet 40. 10161022 (2008)

Sperm differentiated from induced primordial germ cells from the epiblasts engineered to express GFP(left) are fertilization-competent and can be used to generate normal offspring (right; green shows
GFP fluorescence)

Kurimoto K, et al. Complex genome-wide transcription
dynamics orchestrated by Blimp1 for the specification of thegerm cell lineage in mice. Genes Dev 22. 1617-35 (2008)47

Organogenesis and Neurogenesis
The complexity of the fully formed brain defies description, yet this organ arises
from a nondescript clump of cells in the embryo. The specification of the dorsalventral axis is significant in neural 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 to maintain a neural fate. Thesemolecules, which include Noggin, Chordin, Follistatin and their homologs, participate in elaborate signaling networks in which factors collaborate and compete
to initiate the embryonic nervous system.

Yoshiki SASAI M.D., Ph.D.
http://www.cdb.riken.jp/en/sasai
Yoshiki Sasai received his M.D. from the Kyoto
University School of Medicine in 1986, subsequently performing internships in general practiceand emergency medicine. He completed the Ph.D.
course at the same institution in 1992, for work onneural specific transcriptional regulators. In 1993,
he took 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 School of Medicine. He assumed a professorship at the Kyoto
University Institute for Frontier Medical Sciencesin 1998, and was appointed group director at the
CDB in 2000. He serves on the editorial boards of
Neuron, Genesis, and Developmental Dynamics.

Staff
Group Director

Yoshiki SASAI
Collaborative Scientist

Satoshi ANDO
Makoto IKEYA
Morio UENO

Research Specialist

Keiko MUGURUMATAMADA
Research Scientist

Toshihiro ARAMAKI
Mototsugu EIRAKU
Hidehiko INOMATA
Daisuke KAMIYA
Research Associate

Akira TAKAI
Nozomu TAKATA

Technical Staff

Satoe BANNO-NISHIDE
Tomoko HARAGUCHI
Masako KAWADA
Michiru MATSUMURAIMOTO
Ayaka NISHIYAMA
Rieko YAKURANISHIZAWA

Using the African clawed frog, Xenopus laevis, as a model in molecular embryological studies, our group is engaged in clarifying the structure and extent of thesignaling networks involved in setting up the dorsal-ventral axis and determining
neural fate in the ectoderm. These studies focus on molecules that operate at
early stages in embryonic development to lay down a complex of permissive, instructive and inhibitory patterns that determines the growth and differentiation of
the brain in its earliest stages of development.
Our group is now also actively developing effective methods of inducing neuralization in mammals, work which it is hoped will contribute to basic researchby providing in vitro experimental systems for use in the analysis of mammalian
neurogenesis. In addition, this approach has potential for application in the treatment of neurodegenerative disorders, such as Parkinson’s disease. Using asystem developed in our lab, we have succeeded in inducing mouse and primate
embryonic stem (ES) cells to differentiate into a range of specialized neuronal
types. The application of similar techniques to human ES cells represents a fieldof study that, although still at an early stage, shows immense clinical promise.
By studying very early neurogenesis, and the mechanisms of neuronal differentiation, our lab aims to understand the molecular basis underpinning the formationof so intricate a system as the mammalian neural network, in the hopes that, bylooking at the elegant simplicity of the embryonic brain, it may one day be possible to understand the cellular bases of the mind's activity.

Junior Research Associate

Teruko DANJOUInternational Program
Associate

Anne H. B. FOSBY
Part-Time Staff

Masako SUZUKI
Assistant

Mako MIYAGI
Ayumi TANAKA-IKEYA
Fumi WAGAI

Publications
Sasai N, et al. Ectodermal factor restricts mesoderm differentiation by inhibiting p53. Cell 133. 878-90 (2008)
Wataya T, et al. Minimization of exogenous signals in ES cell
culture induces rostral hypothalamic differentiation. Proc
Natl Acad Sci U S A 105. 11796-801 (2008)
Inomata H, et al. Robust stability of the embryonic axial pattern
requires a secreted scaffold for chordin degradation. Cell 134.
854-65 (2008)
Eiraku M, et al. Self-organized formation of polarized corticaltissues from ESCs and its active manipulation by extrinsic
signals. Cell Stem Cell 3. 519-32 (2008)
Watanabe K, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25.
681-6 (2007)
Onai T, et al. XTsh3 is an essential enhancing factor of canonical Wnt signaling in Xenopus axial determination. Embo
J 26. 2350-60 (2007)

48

Telencephalic precursors cells induced by SFEB culture (Bf1 shown inred)Time-lapse images of spindle (Tubulin, green) and nuclear (histoneH2B, red) dynamics during mouse pre-implantation development.

LIF follows two paths to pluripotency

Mouse embryonic stem (ES) cells, distinct from their human counterparts, rely on a cytokine named leukemia
inhibitory factor (LIF) to keep them in their pluripotent, self-renewing state; deprived of LIF, they begin to differentiate. A number of transcription factors, namely Oct3/4, Sox2 and Nanog, form the core circuitry of pluripotency
and serve as the molecular hallmarks of undifferentiated ES cells, but how LIF integrates with this triad has
remained something of a mystery to ES cell biologists. A better understanding of this mechanism could yield
important insights into how embryonic cells maintain their pluripotency in the face of internal dynamics and environmental change.
Findings from the Laboratory of Pluripotent Cell Studies (Hitoshi Niwa; Team Leader) show that LIF sits at thehead of a pair of pathways that interact with separate elements in the pluripotency genetic core. These parallel
circuits may help to stabilize the transcription factor network that sustains stem cells in culture. This work was
published in the journal Nature.
It has been known for some time that a complex, interlocking network of transcriptional pathways is involved in
ES cell maintenance, but not all of the pathway components have been identified. LIF is essential for keeping
mouse ES cells in an undifferentiated state, but previous work has shown that this requirement can be bypassed

Hitoshi NIWA

50

©2009 Nature Publishing GroupNiwa H, et al. A parallel circuit of LIF signalling pathways maintains
pluripotency of mouse ES cells. Nature 460. 118-22 (2009)by the overexpression of Nanog or the activation of Stat3, a critical factor in a signaling cascade
known as the Jak-Stat3 pathway. Other studies involving the analysis of possible targets of the ES
cell core circuitry revealed that the transcription factor-encoding genes Klf4 and Tbx3 are also able tocompensate for the absence of LIF.
Niwa began by trying to piece together the relationships between these multiple factors in maintaining
pluripotency. He first looked at the expression levels of pluripotency circuitry elements in mouse ES
cells in which the absence of LIF was compensated for by transgenic Nanog, Klf4, or Tbx3. In all threelines, Oct3/4 remained stable, but interestingly in the Nanog and Tbx3 lines, Klf4 expression dropped
to levels similar to that of wildtype ES cells cultured without LIF, which was coupled with reduced expression of Sox2. This jibes well with a previous suggestion that Sox2 and Nanog work independently
to activate Oct3/4.
Staining of ES cells with antibodies to Tbx3, Klf4 and Nanog revealed that even as Oct3/4 levels
remained homogeneous, the levels of these three factors vary from cell to cell in an apparently uncoordinated fashion. Somehow these fluctuating expression levels must work together to produce asingle, uniform output. Returning to the LIF linkage, Niwa next tried reintroducing LIF to the transgenic
ES cells, and found that while neither Tbx3 nor Nanog responded, Klf4 levels changed after LIF was
added to the medium.

Schematic model of transcription factor networks for pluripotent stem cells, trophectoderm
and primitive endoderm.

Previous studies of LIF have shown it to work via three separate signaling routes, known as the JakStat3, PI(3)K-Akt, and MAP kinase pathways; in mouse ES cells, LIF is the only factor in control of the
Jak-Stat3 pathway, while the other two are multiply regulated. Wanting to identify the roles of eachpathway in ES cell maintenance, Niwa used specific inhibitors and activators of each of the downstream pathways before stimulating the cells with LIF. The team found that while Klf4 is downregulatedby the Jak-Stat3 blockade, Tbx3 and Nanog were not. In contrast, when the PI(3)K-Akt was activated,
Tbx3 and Nanog remained switched on, while Klf4 was unaffected. MAP kinase appeared to work as
a negative control on Tbx3; when the MAPK pathway was inhibited, Tbx3 levels rose. The larger picture that arises from this work is one of parallel LIF-activated pathways, which engage in crosstalk toensure that Oct3/4 remains unperturbed in ES cells.
“In this study we showed that the pluripotency-associated transcription factors have a hierarchicalnetwork structure defined by pathways that integrate external signals," says Niwa. “In this scheme,
Klf4 and Tbx3 work as signal transmitters while Oct3/4 sits at the core, and Sox2 and Nanog are intermediates. This new model may help to improve our understanding of how pluripotency is robustly
maintained under various environmental conditions."

51

Something fold, something new:
Evolutionary morphology of the turtle shell

Shigeru KURATANI, Hiroshi NAGASHIMA, Fumiaki SUGAHARA, Masaki TAKECHI

The elephant’s trunk, the giraffe’s neck, the bat’s wing all provide examples of how evolution tinkers with preexisting body parts, giving rise to adaptations that are simultaneously new and familiar. But there are also cases
in which a novelty enters the body plan without evident precursors, defying explanation by such bricolage. The
turtle’s shell, or carapace, is one such invention; it appears to have burst into the fossil record with almost no
prior inkling of its emergence.
Work published in Science by Hiroshi Nagashima and coworkers in the Laboratory for Evolutionary Morphology(Shigeru Kuratani; Group Director) showed that the musculoskeletal changes that led to abrupt appearance of theturtle shell involved a combination of altered tissue folding and new connectivity. And, by analyzing the structureof a proto-turtle fossil recently unearthed in China, the group revealed an intermediate body plan between that ofancient uncarapaced reptiles in the same lineage and the turtles, tortoises, and terrapins of today.
Nagashima began by making anatomic comparisons of mouse, chicken and turtle embryos at the stages in development at which structural changes that underlie the formation of the carapace first become apparent. Specifically, the group focused on how connections between muscles associated with the ribs and scapulae might
differ in turtle and other vertebrate embryos, for although it is known that the shell itself forms through the fusionof ribs, the developmental steps that lead up to this novel structure have been unclear.

52©2009 the American Association for the Advancement of Science
Nagashima H, et al. Evolution of the turtle body plan by the folding
and creation of new muscle connections. Science 325. 193-6 (2009)

What Nagashima found was that, despite the radically different modified appearance of the adultturtle, the changes to the embryo are in fact quite modest. In contrast to the bird and mammal, theembryonic turtle’s scapula forms anterior to the ribs, and the muscles that attach to these bones
maintain their connectivities. The ribs themselves are shorter, confined to the axial region, meaningthat they can only grow to the sides and anterior of the embryo, eventually covering the scapula. The
cause of this “axial arrest” of the ribs remains uncertain, but it is this change in rib growth coupledwith altered folding of tissue at the boundary between the axis and body wall that fundamentally distinguishes the turtle from other vertebrates.

Comparison of skeletal andmuscular structure in humanand turtle.

A second innovation the group identified was a number of new attachment points among what are
known as “in-and-out muscles,” which primarily connect limbs to the trunk. They found that musclesin the turtle’s upper thoracic region, such as the pectorals and the latissimus dorsi, arose more anteriorly and established different connectivities to bones than those seen in chicken and mouse.
The insights into turtle musculoskeletal development gained from comparisons with extant animals
mesh well with the perspective provided by fossils of extinct species from the lineage that led to theturtles. The skeleton of the most ancient known turtle, Odontochelys, which was first described in
2008, shows that axial arrest of the ribs had already begun 220 million years ago. The ribs, however,
did not show the splayed arrangement seen in the ribs of modern turtles, nor did they fuse to form afull carapace.
“In this study, we used fossil evidence to develop comparisons in the skeletal architecture of modern
turtles and their ancestors, but the question of muscle homologies remains unanswered, due to thelack of soft tissue," says Kuratani. "But we feel that a general knowledge of the vertebrate developmental plan will still allow us to draw reasonable conclusions about how at least some of the muscles
might have connected to the skeleton in the most ancient turtles, via an evolutionary developmental
biology approach that we feel may find broader application among paleontologists."

53

Synapses stay true:
Selective neural connectivity in vitro
The brain is a bramble, with thorny branches of neuronal projections intertwining into a dense network. But a
closer look reveals an order to its seemingly thicket-like structure, in which axons from one neuron connect withdendrites from specific partners. In cultured neurons, however, these relationships become blurred, and synapses have been shown to form between mismatched neurons, raising questions about the mechanisms that control
neuronal recognition in vitro.
Shoko Ito of the Laboratory for Cell Adhesion and Tissue Patterning (Masatoshi Takeichi; Group Director) reexamined this notion of in vitro neuronal promiscuity and found that, in contrast to previous allegations, at least somedendrites remain true to their partners. In studies of synapse formation using cerebellar granule cell dendrites,
Ito showed that while these may come into contact with axons of all types in cell culture, they synapse only withmossy fiber axons, just as they do in vivo. This work was published in the Proceedings of the National Academyof Sciences.

Shoko ITO

54

©2009 The National Academy of Sciences
Ito S, et al. Dendrites of cerebellar granule cells correctly recognize their target
axons for synaptogenesis in vitro. Proc Natl Acad Sci U S A 106. 12782-7 (2009)

Figure 1
Synapse formation bet ween
granule ce ll de ndr ite s (blueor light blue) and mossy fibera xons (green). Synapses aredetected by staining for PSD95(red), a postsynaptic marker.

The cerebellum features two characteristic neuronal cells types: the huge and hugely intricate Purkinjecells, which form synapses with climbing fibers from a medullary region called the inferior olivary nucleus, and granule cells which synapse with mossy fiber axons from a different region known as the
pontine nuclei. Ito began with a neuronal co-culture system in which granule cells were grown in thepresence of climbing fibers and hippocampal neurons, neither of which would they ordinarily form
synapses within the brain. These explants projected axons as normal, and the granule cells extended
dendrites, but although they in some cases the neurites came into close contact with each other, synapses of the type seen in vivo failed to form.
Only when granule cells were cultured with their typical binding partners, mossy fiber axons from thepontine, did they form synapses which are functionally normal and morphologically identical to thoseseen in vivo, as indicated by the expression of synaptic markers. Axons from the inferior olivary andhippocampal cultures also apparently formed synapses, but on closer examination these showed
their arrangement was abnormal, and they showed impaired release of synaptic vesicles, which areresponsible for the cycling of neurotransmitters.
This new work clearly shows that the selective synapse formation known to occur in vivo is replicablein cultured neurons as well. The mechanisms by which such selection takes place, however, remain
unknown and must await further studies now that this fundamental principle has been shown to beconserved in vitro.

Figure 2
FM staining of co-cultured
granule cells and mossy fibers
shows recycling of synaptic
vesicles. Differential inter ference contrast microscopy on
left; center and left panels shownormal uptake and release ofsynaptic vesicles in these cultured cells.

55

2009 Awards
As one of the world’s leading institutes in the fields of developmental biology and stem cell research, the RIKEN CDB strives to maintain thehighest levels of creativity, impact and quality in its scientific output. The individual achievements of many of its scientists, laboratory heads,
junior scientists and student trainees alike, have been recognized for numerous awards from government agencies, private foundations, andindustry.

The Center takes great pride in the recognition garnered by its scientists. A list of all prizes awarded in 2009 to CDB scientists follows:

Date

Name

PositionLaboratory

Award

Organization

3/9/2009

Teruhiko Wakayama

Team Leader

Genomic
Reprogramming

Japanese Academy Medal

The Japan Academy

3/9/2009

Teruhiko Wakayama

Team Leader

Genomic
Reprogramming

5th annual JSPS PrizeJapan Society for the
Promotion of Science

3/16/2009

Sayaka Wakayama

Research Scientist

Genomic
Reprogramming

2nd Annual Shiseido
Research Grant for Female
Scientists

Shiseido Co.,Ltd.

4/2/2009

Hiroshi Nagashima

Research Scientist

Evolutionary Morphology

147th Meeting of the Japanese Society of Veterinary
Science Outstanding
Poster Award

The Japanese Society of
Veterinary Science

4/14/2009

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Minister of Education,
Culture, Sports,Science,
and Technology Prize for
Science and Technology

Japanese Ministry of
Education, Culture, Sports,
Science, and Technology(MEXT)

4/14/2009

Mitinori Saitou

Team Leader

Mammalian Germ Cell
Biology

Minister of Education, Culture, Sports,Science, andTechnology Prize for Young
Investigators

Japanese Ministry of
Education, Culture, Sports,
Science, and Technology(MEXT)

4/17/2009

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Tokyo Techno Forum 21
Gold Medal Prize

Tokyo Techno Forum 21

5/26/2009

Teruhiko Wakayama

Team Leader

Genomic
Reprogramming

NISTEP’s Nice Step
Researcher Prize

National Institute of Scienceand Technology Policy

10/27/2009

Yoichi Minami

Research Scientist

Systems Biology

16th Annual Meeting for
the Japanese Society for
Chronobiology Outstanding Poster Award

Japanese Society for
Chronobiology

11/10/2009

Hiroko Ueda

Project Leader

Systems Biology

IBM Japan Science Prize(Computer science category)Japan IBM

11/18/2009

Chong Li

Junior Research
Associate

Genomic
Reprogramming

Certificate of Award for
Excellent Poster Presentation

The Asia Reproductive
Biotechnology Society

12/5/2009

Motoki Tada

Research Scientist

Evolutionary Morphology

Leica Microscope PhotoContest 2009 Outstanding
Photo Award

Leica Microsystems

12/9/2009

Shigeo Hayashi

Group Director

Morphogenetic Signaling

Leica Microscope PhotoContest 2009 Grand Prize

Leica Microsystems

56

Parvalbumin7 and Vglut1 are expressed in Purkinje cells (magenta) andparallel fibers of granule cells (green) in zebrafish larval cerebellum.

Embryonic Induction
During vertebrate embryogenesis, simply structured regions of undifferentiatedcells organize and cooperate to give rise to the anterior-posterior and dorsalventral body axes, the neural tube and other embryonic structures deriving fromthe three germ layers, following developmental patterns that can be collectively
summarized as the adult body plan. The embryo is a hive of activity, including
cell differentiation, proliferation and migration, strictly regulated in both space
and time by molecular signals emitted by special regions of the embryo referred
to as “signaling centers.” These centers produce diffusible proteins that serve toregulate embryonic development, guiding neighboring cell communities to takeup the roles they will play in the adult animal, or steering them away from inappropriate fates.

Hiroshi SASAKI Ph.D.
http://www.cdb.riken.go.jp/en/sasaki
Hiroshi Sasaki received his B. Sc. in zoology
from the University of Tokyo, and his master's
and Ph.D. in developmental biology from thesame institution. He worked as a research associate in Atsushi Kuroiwa's group at Tohoku University from 1990 to 1992, and in Brigid Hogan's
lab at Vanderbilt University from 1992 to 1994.
He returned to Japan to take a position as assistant professor at Osaka University beginningin 1995, where he remained until his appointment as team leader at the RIKEN CDB.

Research in our laboratory focuses on these signaling centers, especially thoseof the mouse, as a model for studying the regulation of embryogenetic processes. We focus particularly on the node and notochord, which are of central
importance in the formation of the early embryo. We focus on the control of theexpression of the Foxa2 transcription factor in the formation and maintenance of
signaling centers, as well as search for new factors involved in the control of embryonic development by such centers. We have recently revealed that membersof the Tead family of transcription factors are important not only for signaling
center formation, but also in a broad range of processes in mouse development,
including cell proliferation and differentiation in pre- and post-implantation embryos. Our lab is now analyzing the roles of Tead family proteins and their regulation by cell-cell contact information as a new approach to the study of earlymouse development.

Staff
Team Leader

Student Trainee

Research Scientist

Part-Time Staff

Hiroshi SASAKI
Yoshikazu HIRATE
Hiroshi MAMADANoriyuki NISHIOKA
Mitsunori OTA
Ken-ichi WADA

Yuu IMUTA

Megumi SHIBATA
Assistant

Misaki HARANO

Technical Staff

Shino HIRAHARA
Akiko MORII

Publications
Nishioka N, et al. The Hippo signaling pathway components
Lats and Yap pattern Tead4 activity to distinguish mouse
trophectoderm from inner cell mass. Dev Cell 16. 398-410(2009)
Yamamoto S, et al. Cthrc1 selectively activates the planarcell polarity pathway of Wnt signaling by stabilizing the Wntreceptor complex. Dev Cell 15. 23-36 (2008)
Ota M and Sasaki H. Mammalian Tead proteins regulate cellproliferation and contact inhibition as transcriptional mediators of Hippo signaling. Development 135. 4059-69 (2008)
Sawada A, et al. Redundant roles of Tead1 and Tead2 innotochord development and the regulation of cell proliferation and survival. Mol Cell Biol 28. 3177-89 (2008)
Nishioka N, et al. Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech Dev
125. 270-83 (2008)
Sawada A, et al. Tead proteins activate the Foxa2 enhancer
in the node in cooperation with a second factor. Development 132. 4719-29 (2005)

Day 8.5 mouse embryos showing that the notochord and node (signaling centers regulating trunk/taildevelopment; stained purple on the left) is absent in the Tead1;Tead2 double mutant (right; remaining
staining is in the primitive streak). Tead1;Tead2 double mutants also have defects in cell proliferation
and apoptosis.

58

Cell Fate Decision
The development of multicellular organisms involves a single cell (such as a fertilized egg) which gives rise to diverse progeny through successive cell divisions.
The process of cell division in which the resulting daughter cells are of different
characters or “fates” is known as asymmetric cell division. One example of thisform of cell division is seen in stem cells, which can produce one copy of themselves and one more specialized (differentiated) cell in a single division. Asymmetric cell division is central to the generation of cellular diversity, but because
in higher organisms a given cell’s “family relations” (known as cell lineage) arefrequently unclear, it can be difficult to study the process in detail.
In the nematode, C. elegans, however, thanks to the transparency of the worm’s
body it is possible to track the division and other activity of individual cells in theliving animal using only a low-magnification light microscope. In our laboratory,
we take advantage of this ability to monitor developmental processes in realtime, a method which we use in combination with genetic studies to investigate
the mechanisms that underlie asymmetric division. We have determined thatnearly every asymmetric cell division in C. elegans is mediated by β-catenins
acting in the Wnt signaling pathway. This pathway works to polarize cells fated toundergo asymmetric division; we seek to gain a better understanding of how it
achieves this by studying the intracellular localization of the Wnt pathway’s molecular components.
We have also discovered numerous mutant C. elegans phenotypes in which theasymmetric division of specific cells is disturbed, allowing us to identify morethan 50 genes involved in this process. By investigating the function of thesegenes, we hope to clarify the biological mechanisms of asymmetric cell divisioneven further.

Hitoshi SAWA Ph.D.
http://www.cdb.riken.go.jp/en/sawa
Hitoshi Sawa obtained his baccalaureate,master's and doctoral degrees from Kyoto
University in the period from 1982 to 1991.
He worked 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 from 1994 to
1997 under support from the Howard Hughes
Medical Institute. He returned to Japan in 1997
to continue his work at Osaka University, firstas a postdoc, then as researcher funded by theJapan Science and Technology Corporation
PRESTO program. He took his current positionat the RIKEN CDB in 2001.

Staff
Team Leader

Junior Research Associate

Research Scientist

Student Trainee

Hitoshi SAWA
Yukinobu ARATA
Yuji HOSOIRI
Yukimasa SHIBATA
Technical Staff

Noriko SASAKAWA
Tomomi TAKANO

Yuko YAMAMOTO
Kenji SUGIOKA
Part-Time Staff

Tomoko SUGIMOTO
Kenji SUGIOKA
Assistant

Tomoko NAKASHIMA

Publications
Zinovyeva A Y, et al. Complex network of Wnt signaling regulates neuronal migrations during Caenorhabditis elegans
development. Genetics 179. 1357-71 (2008)
Kanamori T, et al. β-catenin asymmetry is regulated by
PLA1 and retrograde traffic in C. elegans stem cell divisions.
Embo J 27. 1647-57 (2008)
Mizumoto K and Sawa H. Cortical β-catenin and APC regulate asymmetric nuclear β-catenin localization during asymmetric cell division in C. elegans. Dev Cell 12. 287-99 (2007)
Goldstein B, et al. Wnt signals can function as positionalcues in establishing cell polarity. Dev Cell 10. 391-6 (2006)
Arata Y, et al. Wnt signaling and a Hox protein cooperatively
regulate psa-3/Meis to determine daughter cell fate after
asymmetric cell division in C. elegans. Dev Cell 11. 105-15(2006)
Takeshita H and Sawa H. Asymmetric cortical and nuclear
localizations of WRM-1/β-catenin during asymmetric cell
division in C. elegans. Genes Dev 19. 1743-8 (2005)

Asymmetric cortical localization of the Wnt pathway components.

59

Early Embryogenesis
The lab studies how mesoderm cells form and differentiate during early vertebrate development, using the chick as the main model organism. Our research
currently focuses on two aspects: 1) the formation and early regionalization of
mesoderm cells during gastrulation; 2) differentiation of ventral-most mesoderm
cell types.

Guojun SHENG Ph.D.
http://www.cdb.riken.go.jp/en/sheng
Guojun Sheng received his B.S. in microbiology from Fudan University in Shanghai, China
in 1990. He entered the Rockefeller University
the next year, attending the summer course inembryology at the Woods Hole Marine Biological
Laboratory in 1996 and receiving his Ph.D. under
Claude Desplan in 1997. He spent the period
from 1998 to 2000 in Claudio Stern's laboratory
in Columbia University, moving with Dr. Stern toUniversity College London in 2001. He was appointed Team Leader at the RIKEN CDB in 2004.

Mesoderm cells are derived from ectoderm cells during gastrulation through aprocess called epithelial to mesenchymal transition (EMT). During EMT, mesoderm cells leave epithelial-shaped ectoderm sheet and become migratory. We
are interested in understanding the molecular and cellular mechanisms governing this EMT. We are also interested in understanding how this process is linked
to mesoderm fate specification in the ectoderm and to mesoderm regionalization
along the dorsal-ventral axis during gastrulation.
After their formation, mesoderm cells give rise to several well-defined lineages:
axial, paraxial, intermediate, lateral plate and extraembryonic. The extraembryonic lineage contains three cell types: blood, endothelial and smooth muscle.
We are investigating how these three cell types are specified and how they areorganized to form functional hematopoietic and vascular systems.

Staff
Team Leader

Collaborative Scientist

Special Postdoctoral
Researcher

Technical Staff

Guojun SHENG
Yukiko NAKAYA

Research Scientist

Fumie NAKAZAWA
Masahiro SHIN
Wei WENG

Cantas ALEV

Hiroki NAGAI
Kanako OTA
Erike Widyasari
SUKOWATI
YuPing WU

Publications
Alev C, et al. Dynamic expression of Endoglin, a TGF-betaco-receptor, during pre-circulation vascular development inchick Int J Dev Biol 54. 737-42 (2010)
Alev C, et al. Genomic organization of zebra finch alphaand beta globin genes and their expression in primitive anddefinitive blood in comparison with globins in chicken. Dev
Genes Evol 219. 353-60 (2009)
Alev C, et al. NFATc2 Ser170 phosphorylation marks mitosis
in development and ES cells. Cell Cycle 8. 1803-5 (2009)
Shin M, et al. Notch mediates Wnt and BMP signals in theearly separation of smooth muscle progenitors and blood/
endothelial common progenitors. Development 136. 595603 (2009)
Nagai H and Sheng G Definitive erythropoiesis in chicken
yolk sac. Dev Dyn 237. 3332-41 (2008)
Nakaya Y, et al. RhoA and microtubule dynamics control
cell-basement membrane interaction in EMT during gastrulation. Nat Cell Biol 10. 765-775 (2008)

The circulatory system of early chicken embryos (E5 shown here) is complex and contains embryonic,
allantoic, and yolk sac sub-systems. The diversity of blood cells during this period is revealed by thetranscriptomic analysis of non-red blood cells in circulation.

60

Developmental Genomics
Fertilized eggs give rise to complex animal structures through strict coordination of cell divisions, cell fate determination and differentiation. To generate two
daughter cells with distinct cell fates from a single cell division (called “asymmetriccell division”), the mother cell is polarized before dividing and cytoplasmic components are asymmetrically segregated. A mitotic spindle is formed and positioned along the polarized axis, and the cell division produces two daughter cells
that contain distinct cell contents.
Our laboratory aims to understand the gene/protein networks controlling these
dynamic cellular processes using the nematode Caenorhabditis elegans embryos as an experimental model. This organism provides an extremely usefulsystem for studying such processes at the whole genome level, as its genome
has been fully sequenced and the lineage of each cell in its body is known. Using
high-resolution live microscopy to trace dynamic behaviors of proteins in combination with gene knockdown by RNAi, we are investigating the gene networks
that control these processes.
Our current research focuses on understanding the spatio-temporal regulatory
mechanisms of mitotic spindle formation. We revealed that two genetically separate pathways for microtubule assembly, one involving γ-tubulin and the otherrequiring aurora-A kinase, are responsible for the formation of mitotic spindles
in early C. elegans embryos. We are investigating how these pathways spatiallyand temporally coordinate to form mitotic spindles.
In addition to studying mitotic spindle formation, we are also studying two otherphenomena which are crucial for cell fate determination in early embryogenesisof C. elegans. The first is how mitotic spindles are positioned within the cell according to the established cell polarity. The second is how germ granules (large
complexes of mRNAs and proteins, believed to be the “germ cell determinants”)
are assembled and segregated specifically into the germ lineage. Through thesestudies, we hope to develop a more detailed picture of the regulation of dynamic
cellular processes by gene networks.

Asako SUGIMOTO Ph.D.
http://www.cdb.riken.go.jp/en/sugimoto
Asako Sugimoto received her B. Sc. 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's laboratory in the University of Wisconsin-Madison from 1992 to 1996,
before returning to Japan to assume an assistant professorship at the University of Tokyo.
She remained in that position until 2002, pursuing concurrent work as a Japan Science andTechnology Corporation PRESTO researcher
from 1997 to 2000. She was appointed teamleader at the RIKEN CDB in 2001.

Staff
Team Leader

Asako SUGIMOTO
Special Postdoctoral
Researcher

Technical Staff

Yumi IIDA
Kayo NAGATA
Chie WATANABE

Momoyo HANAZAWA
Mika TOYA

Part-Time Staff

Research Scientist

Student Trainee

Yohei SASAGAWA
Masahiro TERASAWA
Takehito URUNO
Research Consultant

Kanako KAWAI
Masafumi YONETANIAssistant

Taeko IMURA

Kazumasa TAKEDA

Publications
Konishi T, et al. The Caenorhabditis elegans DDX-23, a
homolog of yeast splicing factor PRP28, is required forthe sperm-oocyte switch and differentiation of various cell
types. Dev Dyn 237. 2367-77 (2008)
Takeda K, et al. Efficient production of monoclonal antibodies recognizing specific structures in Caenorhabditis elegans
embryos using an antigen subtraction method. Genes Cells(2008)
Maruyama R, et al. EGG-3 regulates cell-surface and cortex
rearrangements during egg activation in Caenorhabditis
elegans. Curr Biol 17. 1555-60 (2007)
Motegi F, et al. Two phases of astral microtubule activity
during cytokinesis in C. elegans embryos. Dev Cell 10. 50920 (2006)
Motegi F and Sugimoto A. Sequential functioning of the
ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos. Nat Cell Biol 8. 97885 (2006)

The first two rounds of cell divisions in C. elegans embryos. Centrosomes and cell membrane arelabeled with GFP. Top: wildtype. Bottom: par-3 mutant, showing a defect in the cell division axis.

61

Retinal Regeneration
The retina has been called the “most approachable part of the brain,” owing to
its relatively simple structure and its location near the body surface, and for thesereasons it serves as a useful and experimentally amenable model of the central
nervous system. Until very recently, it was thought that in adult mammals theretina was entirely incapable of regenerating, but we now know that at least new
retinal neurons can be generated after being damaged. This has opened up newhope that the ability to regenerate neurons and even to reconstitute the neuralnetwork may be retained in the adult retina. We are now exploring the excitingprospect that, by transplanting cells from outside of the retina or by regenerationfrom intrinsic progenitor cells, it may one day be possible to restore lost function
to damaged retinas.

Masayo TAKAHASHI M.D., Ph.D.
http://www.cdb.riken.go.jp/en/takahashi
Masayo Takahashi received her M.D. from Kyoto
University in 1986, and her Ph.D. from the same
institution in 1992. After serving as assistantprofessor in the Department of Ophthalmology,
Kyoto University Hospital, she moved to the Salk
Institute in 1996, where she discovered the potential of stem cells as a tool for retinal therapy.
She came back to Kyoto University Hospital in1998, and since 2001 served as an associate
professor at the Translational Research Centerin the Kyoto University Hospital. She joined the
CDB as a team leader of the retinal regeneration
research team in 2006. Her clinical specialty isretinal disease, macular diseases and retinal hereditary diseases in particular. Her aim is to understand these diseases at a fundamental leveland develop retinal regeneration therapies.

Staff
Team Leader

Masayo TAKAHASHI
Research Scientist

Kohei HOMMA
Michiko MANDAI
Fumitaka OSAKADA
Akiko SUGA
Shintaro YAMAMOTO
Research Associate

Satoshi OKAMOTO
Visiting Scientist

Yasuhiko HIRAMI

Foreign Postdoctoral
Researcher

Zi-Bing JIN

Our research into retinal regeneration seeks to achieve clinical applications by
developing methods for inducing stem cells to differentiate into retinal neurons
and pigmented epithelial cells in sufficient quantities for use in the treatment ofpatients suffering from conditions in which such cells have been damaged or
lost. We must also ensure that such cells establish viable grafts upon transplantation and induce the reconstitution of functional neural networks. We also hope
to develop means of promoting true regeneration by activating endogenous stem
cells to replace cells lost to trauma or disease and thus repair damaged tissues.
Access to a broad spectrum of developmental biological research information
will be key to the achievement of these goals, and we appreciate the opportunities for exchange that working in the environment provided by the RIKEN CDB.
Therapeutic applications cannot be developed from basic research alone; theclinical approach – a thorough understanding of the medical condition to betreated is equally important. For conditions such as retinitis pigmentosa, eventhe successful transplantation of cells in animal models may not necessarily betranslatable to a human clinical therapy without an understanding of the underlying genetics and possible immunological involvement. Our goal is to study retinal
regeneration based on both a strong foundation in basic research and solid clinical evidence.

Technical Staff

Kyoko ISEKI
Chie ISHIGAMI
Akane NOMORI
Kazuyo SADAMOTO
Noriko SAKAI
Yu WATAOKA
Chikako YAMADA
Student Trainee

Hiroyuki KAMAO
Takayuki NAGAI

Publications
Osakada F, et al. Toward the generation of rod and cone
photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol 26. 215-24 (2008)
Jin Z B, et al. Identifying pathogenic genetic background of
simplex or multiplex retinitis pigmentosa patients: a large scale
mutation screening study. J Med Genet 45. 465-72 (2008)
Osakada F, et al. Wnt signaling promotes regeneration in
the retina of adult mammals. J Neurosci 27. 4210-9 (2007)
Ooto S, et al. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina. Proc Natl Acad
Sci U S A 101. 13654-9 (2004)
Haruta M, et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonicstem cells. Invest Ophthalmol Vis Sci 45. 1020-5 (2004)
Haruta M, et al. Induction of photoreceptor-specific phenotypes
in adult mammalian iris tissue. Nat Neurosci 4. 1163-4 (2001)

62

Immunostaining of bipolar cells in the adult mouse retina

Cell Adhesion and Tissue Patterning
Animal cells organize into tissues with complex architecture. Our lab is exploring
the molecular mechanisms by which individual cells assemble into a tissue-specific multicellular structure, such as epithelial sheets and neural networks. We arealso interested in the molecular basis of how the tissue architecture is disrupted
during carcinogenesis, a process that is thought to accelerate the metastasis ofcancer cells. For these studies, we are focusing on the roles played by cell-celladhesion and recognition molecules, the cadherin family of adhesion moleculesin particular, as these are known to be indispensable for tissue construction.
Cell-cell adhesion is an active process, which can be affected by a number ofcell-internal and external factors. This dynamic nature of cell-cell adhesion is implicated in various processes of cell assembly, such as the tightening or loosening of cell contacts and changing of cell assembly patterns. We are conducting
cell biological analyses to unravel the principal mechanisms that can regulate
cell-cell adhesion. A growing body of evidence suggests that cadherins cooperate with the cytoskeletal and/or motile machineries, such as actin filaments or
microtubules, in modulating cell assembly. We are therefore studying the mechanisms underlying the crosstalk between cadherins and such cytoskeletal systems. These studies include analysis of the functions of microtubule minus endassociated proteins and kinesin motors, as well as actin dynamics regulators, incell-cell contact regulation.
A second area of interest to our lab is to learn how the cell-cell adhesion machinery contributes to morphogenetic cell behavior. We are analyzing the rolesof cadherin in cell movement, in order to explore the mechanisms underlying thecontact-dependent regulation of cell locomotion. At the tissue level, using theembryonic brain as a model system, our team is attempting to determine how
cadherin and associated molecules control the remodeling of neuroepithelial layers, such as neural tube closure and the migration of neuronal progenitor cells.
We are also investigating the roles of protocadherins, members of the cadherin
superfamily, deficiencies of which have been implicated in neuronal defects inbrain development. Through these studies, we expect to gain deeper mechanistic insights into the ways by which cells build elaborate structures of the animal
body.

Masatoshi TAKEICHI Ph.D.
http://www.cdb.riken.go.jp/en/takeichi
Masatoshi Takeichi is director of the RIKEN Center
for Developmental Biology as well as directorof the Cell Adhesion and Tissue Patterning research group. He completed the B. Sc. and M. S.
programs in biology at Nagoya University before
receiving a doctorate in biophysics from Kyoto
University in 1973. After attaining his Ph.D., he took
a research fellowship at the Carnegie Institution
Department of Embryology under Richard Pagano.
He then returned to Kyoto University, attaining aprofessorship in the Department of Biophysics(1986-1999), before becoming professor in the
Department of Cell and Developmental Biology in
the Graduate School of Biostudies at the same university. He assumed the directorship of the CDB in
2000.

Staff
Group Director

Masatoshi TAKEICHI
Research Scientist

Syuichi HAYASHI
Wenxiang MENG
Shoko NAKAMURA
Tamako NISHIMURA
Katsutoshi TAGUCHI
Yoshikazu TSUKASAKI
Masato UEMURA
Visiting Scientist

Anna PLATEKTechnical Staff

Miwako NOMURA
Hiroko SAITO
Chika YOSHII
Junior Research Associate

Takashi ISHIUCHI
Shigenori NAGAE
Student Trainee

Shoko ITO
Nobutoshi TANAKA
Agency Staff

Yoko INOUE
AssistantMutsukoAISO-WATANABE

Publications
Ito S and Takeichi M Dendrites of cerebellar granule cells
correctly recognize their target axons for synaptogenesis invitro. Proc Natl Acad Sci U S A 106. 12782-7 (2009)
Ishiuchi T, et al. Mammalian Fat and Dachsous cadherins
regulate apical membrane organization in the embryonic
cerebral cortex. J Cell Biol 185. 959-67 (2009)
Meng W, et al. Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell
135. 948-59 (2008)
Nishimura T and Takeichi M. Shroom3-mediated recruitmentof Rho kinases to the apical cell junctions regulates epithelial
and neuroepithelial planar remodeling. Development 135.
1493-502 (2008)

Identification of a novel protein, termed Nezha, which binds non-centrosomal microtubules at theminus ends. Triple immunofluorescence staining for microtubules (green), Nezha (blue), and EB1 (red),a plus-end binding protein.

Nakao S, et al. Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction. J Cell Biol
182. 395-410 (2008)
Abe K and Takeichi M EPLIN mediates linkage of thecadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc Natl Acad Sci U S A 105. 13-9
(2008)

63

Systems Biology

http://www.cdb.riken.go.jp/en/ueda
Hiroki R. Ueda received his M.D. from the Facultyof Medicine of the University of Tokyo in 2000, andreceived his Ph.D. from the University of Tokyo Graduate School of Medicine in March 2004. While an undergraduate, he worked as a research assistant on abiological simulation system project at Sony Computer
Science Laboratories. As a graduate student he next
went on to work, first as a researcher (2000) and thenas a group leader (2002), at Yamanouchi Pharmaceuticals, on a project studying biological clock mechanisms in fly and mouse. He was appointed team leaderof Laboratory for Systems Biology in 2003, and Leaderof the Functional Genomics Unit at the CDB in 2004.
He was also appointed as Professor (Invited) of Osaka
University in 2006. In 2009, he was promoted project
leader of the Laboratory for Systems Biology under
CDB’s Center Director’s Strategic Program.

Staff
Team Leader

Hiroki R. UEDA
Research Scientist

Junior Research Associate

Genshiro SUNAGAWA
Kaori TSUJINO

Yohei KOYAMA
Kohei MASUMOTO
Yoichi MINAMI
Masato NAKAJIMA
Hideki UKAI

Special Postdoctoral Researcher

Technical Staff

Yoko SAKAKIDA
Yuko SASAGAWA

Hiroshi FUJISHIMA
Masahiro HAYASHI
Mayumi ISHIDA
Maki UKAI-TADENUMA

Rikuhiro YAMADA
Student Trainee

Ryokichi TANAKA
Part-Time Staff

AssistantSumire HINO

Visiting Scientist

Tetsuya KOBAYASHIAtsushi WADA

Publications
Isojima Y, et al. CKIepsilon/delta-dependent phosphorylation
is a temperature-insensitive, period-determining process in the
mammalian circadian clock. Proc Natl Acad Sci U S A 106.
15744-9 (2009)
Minami Y, et al. Measurement of internal body time by blood
metabolomics. Proc Natl Acad Sci U S A (2009)
Ukai-Tadenuma M, et al. Proof-by-synthesis of the transcriptional
logic of mammalian circadian clocks. Nat Cell Biol 10. 1154-63(2008)
Kumaki Y, et al. Analysis and synthesis of high-amplitude Ciselements in the mammalian circadian clock. Proc Natl Acad Sci
U S A 105. 14946-51 (2008)
Ukai H, et al. Melanopsin-dependent photo-perturbation reveals
desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell Biol 9. 1327-34 (2007)
Matsumoto A, et al. A functional genomics strategy reveals
clockwork orange as a transcriptional regulator in the Drosophila
circadian clock. Genes Dev 21. 1687-700 (2007)

64

The Laboratory for Systems Biology (LSB) has specific aims to investigate development of systems-biological approaches and their application to systemlevel questions of complex and dynamic biological systems, such as mammalian
circadian clocks.
In pursuing the research aims outlined above, we have been mainly focusing on
the development of a systems-biological approach. We have successfully developed new strategies and technologies for genome-wide profiling, bioinformatics,
quantitative measurement, perturbation of cellular state, and implementationof artificial circuits in cells. We have also applied these systems-biological approaches to specific system-level questions, which has led to a number of new
discoveries and inventions. Over the next five years, we plan on fully integratingthese approaches in an attempt to realize a system-level understanding of the
mammalian circadian clock. In order to facilitate these processes, we will also
commit to the development of key technologies such as functional genomics
used for complete identification of the mammalian circadian clock and Micro
Electro Mechanical Systems (MEMS, also known as microfluidics) for quantitative perturbation of the mammalian circadian clock. These key technologies also
have the potential to be applied to the study of developmental problems.
In October 2009, the Laboratory for Systems Biology was re-designated as a
Project Lab in the Center Director’s Strategic Program for Systems Biology research.

Substrate phosphoylation(mol/mol CKI)

Hiroki R. UEDA M.D., Ph.D.

Recent large-scale efforts in genome-sequencing, expression profiling andfunctional screening have produced an embarrassment of riches for life science researchers and biological data can now be accessed in quantities thatare orders of magnitude greater than were available even a few years ago. The
growing need for interpretation of data sets, as well as the accelerating demand
for their integration to a higher level understanding of life, has set the stage forthe advent of systems biology, in which biological processes and phenomena
are approached as complex and dynamic systems. Systems Biology is a naturalextension of molecular biology, and can be defined as "biology after the identification of key genes." We see systems-biological research as a multi-stage process, beginning with the comprehensive identification and quantitative analysis ofindividual system components and their networked interactions, and leading to
the ability to drive existing systems toward a desired state and design new ones
based on an understanding of structure and underlying principles.

ΔCKIε
100

50

0

0

50

100 150 200 250

Time (min)Temperature-insensitive activity of CKIεTemperature dependency of the CKIε phosphorylation activity for the PER2-peptide substrate wasmeasured at 25°C (blue) and 35°C (red). CKIε phosphorylated the peptide substrate at similar rates
whether at 25°C or 35°C, indicating a strong temperature-insensitivity

Genomic Reprogramming
A limitless number of clones of an animal can be generated from its somatic
cells. Only a few years ago, such a statement would have belonged to the realmof science fiction, but now thanks to advances in the technology known as micromanipulation, which allows researchers to work with individual cells and their
nuclei, that fiction has become reality. The efficiency of the cloning procedure(measured as the ratio of live offspring to cloning attempts), however, remains
quite low, at only a few percent, and even in those attempts that do lead to live
births, the cloned animals consistently exhibit a range of severe developmental
abnormalities and pathologies.
The cause of these defects is thought to be due to imperfections in the nuclear
transfer technique used to remove the nucleus from a host cell and replace
it with another from a donor; imperfections which presumably lead to total orpartial failure of reprogramming, the process in which a cell’s developmental
potential is reset to a totipotent state (such as is exhibited by a naturally fertilizedegg). However, the specific details of the technical flaws, the ways in which theylead to reprogramming failure, and possible solutions to the problem all remaincompletely unknown. Despite these challenges, cloning continues to be a biotechnology of great promise, given its potential applications in the field of regenerative medicine, such as the generation of embryonic stem (ES) cells by nuclear
transfer, which may one day allow us to grow replacement cells that perfectly
match a patient’s own genetic and immune profile, thereby eliminating the risk ofrejection.
We use the mouse as a model system to study cloning under a range of experimental conditions with the goals of achieving improvements in the efficiency ofthe cloning procedure, gaining a better understanding of reprogramming mechanisms and analyzing the properties of ES cells derived via somatic nuclear transfer. We also use nuclear transfer technology to develop methods for preserving
embryonic lethal and infertile strains of laboratory mice, and continue to explore
the development of new and better techniques for sperm and oocyte preservation and other reproductive biological research technologies.

Teruhiko WAKAYAMA Ph.D.
http://www.cdb.riken.go.jp/en/wakayama
Teruhiko Wakayama received his B. Sc. and M.
Sc. from Ibaraki University, and was awarded a
Ph.D. in reproductive biology from the University of Tokyo Department of Veterinary Anatomy
in 1996. He received a postdoctoral fellowshipfrom the Japanese Society for the Promotion of
Science in 1996 and spent the next two years
at the Yanagimachi lab in the University of
Hawaii Medical School, where he succeededin creating the world's first cloned mouse. He
was appointed to an assistant professorship at
the same institution in 1998, and moved to theRockefeller University as a research assistantprofessor in 1999. He spent a year as a researcher at Advanced Cell Technology before
returning to Japan to take his current positionat the RIKEN CDB.

Staff
Team Leader

Technical Staff

Research Scientist

Junior Research Associate

Teruhiko WAKAYAMA
Eiji MIZUTANIHiroshi OHTA
Sayaka WAKAYAMA
Kazuo YAMAGATA
Visiting Scientist

In-Jeoung BAEK
Satoshi KISHIGAMI

Yuko SAKAIDE
Chong LI

Part-Time Staff

Tetsuo ONO
Assistant

Rie MAEDA
Tomoko OYANAGI

Publications
Li C, et al. An efficient method for generating transgenic
mice using NaOH-treated spermatozoa. Biol Reprod 82.
331-40 (2010)
Wakayama S, et al. Detrimental effects of microgravity on
mouse preimplantation development in vitro. PLoS One 4.
e6753 (2009)
Yamagata K, et al., Assessment of chromosomal integrity
using a novel live-cell imaging technique in mouse embryos
produced by intracytoplasmic sperm injection. Hum. Reprod. 24. 2490-9 (2009)
Wakayama S, et al. Production of healthy cloned mice frombodies frozen at -20 degrees C for 16 years. Proc Natl Acad
Sci U S A 105. 17318-22 (2008)
Bui H T, et al. The cytoplasm of mouse germinal vesicle
stage oocytes can enhance somatic cell nuclear reprogramming. Development 135. 3935-45 (2008)

Transfer of a somatic nucleus into an enucleated egg

Tabar V, et al. Therapeutic cloning in individual parkinsonian
mice. Nat Med 14. 379-81 (2008)

65

2009 Meetings
Scientific gatherings are at the core of international exchanges among researchers, and the RIKEN CDB has devoted a tremendous amountof effort into ensuring that every aspect of the meetings it hosts or sponsors meet the highest standards of quality in their programs, venues,
and logistics. In addition to its annual symposium and many CDB Seminars, the CDB hosts or supports numerous meetings every year.

Stem Cell Research in the
Asia-Pacific Region
February 2 – 4 at Kobe Chamber of Commerce and RIKEN CDB

In this three-day event, co-organized by the JSPS and the Stem Cell Network: AsiaPacific, around 40 scientists and experts from other fields gathered to discuss the
ethical, legal, commercial, and social aspects of stem cell research and applicationsin the Asia-Pacific region, and to present their latest findings in the study of a rangeof stem cells including ES, iPS, mesenchymal, and hematopoietic.

Common Themes and New Concepts
in Sensory Formation
April 13 – 15 at RIKEN CDB

Hosted by the Laboratory for Sensory Development and colleagues from the RIKEN
BSI and the U.K., this meeting drew leading scientists from around the world toaddress common themes in sensory development and novel approaches to thisresearch, with particular focus on the specification of sensory cells, mechanisms of
sensory cell organization, and the use of non-model systems.

Building the Body Plan:

How Cell Adhesion,Signaling andCytoskeletal Regulation Shape Morphogenesis
September 21 – 23 at Kyoto International Conference Center

The CDB joined with the American and Japanese Societies for Cell Biology to sponsor this meeting, designed to allow grad students and postdocs to interact withleaders in the field from around the world in an intimate setting. Three days of talks
focused on topics such as cell junction formation and dynamics, signaling via cell
contacts, epithelial remodeling, cell migration for morphogenesis, and neural morphogenesis and axon recognition.

Frontiers in Stem Cell Biology andEmbryonic Manipulation
November 9 at RIKEN CDB

In this one-day meeting, leaders in the fields of stem cells and contemporary embryology
gathered to discuss their recent work and to participate in a lively discussion of outstanding
problems and future prospects in their fields.

66Section view of paratympanic organ, stained to show nuclei and cytoplasm;
the organ appears as an oval structure, lying next to the future middle ear.

Fat and Daschous cadherins work at the apicalmembrane in cerebral cortical cells
A study by Takashi Ishiuchi, Takuji Tanoue and others in
the Laboratory for Cell Adhesion and Tissue Patterning(Masatoshi Takeichi; Group Director) shed new light onan unexpected role for a pair of cadherin molecules inthe embryonic brain. The group revealed that, in mammals, the little-studied Fat and Daschous cadherins help
to organize the apical membrane structure in neural progenitor cells in the cerebral cortex.
The cadherins are a superfamily of transmembrane molecules best known for the roles of the classic cadherins
in cell-cell junctions, where they help cells recognize other cells of similar type and stick together by a like-bindslike mechanism. But there are other cadherin types as
well, some of which are of indeterminate function andmuch greater size than their classic counterparts. The
Fat cadherins, for example, have nearly 7 times thenumber of extracellular “cadherin repeat” segments thandoes the typical classic cadherin, E-cadherin; the atypical cadherin, Daschous, is similarly oversized. A numberof gross phenotypes have recently been reported inmice lacking Fat4 cadherin (one of the four mammalian Fat cadherins), but the molecular and cellular levelmechanisms behind these have yet to be worked out.
Ishiuchi began by making antibodies against Fat4 andDaschous1 and using these to map distribution of these
proteins by immunostaining, which revealed high concentrations in the cerebral cortex. The two molecules
were co-localized in the vicinity of the adherens junction,
but unlike the junction proteins beta-catenin and ZO-1,
their expression was discontinuous and apically shifted,features that had previously been reported for the distribution of these proteins in Drosophila.
Given the known properties of Fat and Daschous in flies,
the group next considered they might bind heterophilically (binding between unlike pairs), in contrast to thehomophilic binding characteristic of classic cadherins.
They transfected Fat4 and Daschous1 into fibroblasts
lacking classic cadherins, and found that the molecules
became concentrated only at the boundaries between
the Fat and Daschous transfectants, where threadlike
filopodia made contact with neighboring cells, suggesting a heterophilic mechanism. This balance between
Fat and Daschous was dynamically regulated as well –when Fat4 was depleted, Daschous1 levels dropped.
When Daschous1 was removed, however, Fat4 was upregulated.

Takashi ISHIUCHI

68

©2009 The Rockefeller University Press
Ishiuchi T, et al. Mammalian Fat and Dachsous cadherins regulate apical membraneorganization in the embryonic cerebral cortex. J Cell Biol 185. 959-67 (2009)

Looking to the inner side of the junction, Ishiuchi sought after cytoplasmic binding partners of Fat4
and Daschous1, and using immunoprecipitation found that it associated with members of the Pals
protein complex. Fat4 co-localized with Pals1 and a second member of the complex, MUPP1, suggesting a functional interaction apical of the cell-cell junction. Interestingly, Pals1 and MUPP1 localized
remained even in the absence of Fat4 and Daschous1, indicating that their recruitment to the apicalregion relies on a distinct mechanism.

The apical gap between cells
is lost in Fat4 loss-of-function
cells (right). Wildtype cell-celljunction (with gap indicated in
the boxed area) shown to left.

To investigate the cellular roles of the complex comprising Fat4, Daschous1, Pals1 and MUPP1,
Ishiuchi and co-workers used RNAi to block the functions of each molecule and observed the effects.
A minute gap apical to the adherens junction is normally seen between plasma membranes of adjacent cells in the intercellular regional, but when the function of either Fat or Pals was interfered with,this gap disappeared. As this form of intercellular gaps appear to be prominent only in certain cell
types, including neural progenitor cells, Fat and Daschous may exert cell type-specific effects on theorganization of the apical membrane.
The current work, published in The Journal of Cell Biology, showed both the conserved nature of
the molecular properties of the Fat and Daschous cadherins and a new function for these proteins
and their binding partners in the mammalian cortex. “We still don’t know how Fat4 and Daschous1
regulate junction structure in the apical region,” says Ishiuchi, “so we are going to need to look at possible functional relationships with other factors that are known to work in that part of the cell, such as
Crumbs.”69

Embryos feel gravity’s pull

Were lengthy space flights to become a reality, thequestion of whether humans and other animals could
reproduce normally when living in low gravity would
need to be addressed. Previous studies have focusedon species of fish, amphibians and birds, but little work
has been done to date on mammalian reproduction in
microgravity due to the limitations imposed by technology and the stress responses in rats and mice.
Sayaka Wakayama and colleagues in the Laboratory for
Genomic Reprogramming (Teruhiko Wakayama; Team
Leader) studied reproduction and early development inmice in simulated microgravity conditions, and madesome unexpected findings. In an article published in
PLoS One, they showed that while fertilization is possible
in low gravity, subsequent development is perturbed.
To achieve the most realistic simulation of microgravity,
the team used a newly developed device known as athree-dimensional clinostat, which uses rotation on twoaxes to evenly distribute gravitational pull in all directions
over time (rather than the unidirectional downward pull
we normally experience). Previous studies on mammalian development had been performed using a onedimensional clinostat, but the results were equivocal.
Wakayama first tested whether in vitro fertilization would
be possible in microgravity, and found that the successrate for fertilization was no different from that at 1G,
which suggests that mouse gametes are unaffected by
low gravity.
As a next step, they allowed zygotes created by IVF continue to develop in the clinostat before implanting theminto recipient females. But while the embryos could
develop to the 2-cell and even the blastocyst stage,
they did much less well when transferred into a surrogate mother – the rate of generation of offspring fromthe microgravity embryos was little more than half thatwhen using embryos derived in normal gravity. Fertilityof the mice born from microgravity embryos, however,
was normal, as shown by their ability to mate and haveoffspring of their own.

Sayaka (and Ryuta) WAKAYAMA

70

©2009 Public Library of Science
Wakayama S, et al. Detrimental effects of microgravity on mouse
preimplantation development in vitro. PLoS One 4. e6753 (2009)

Figure 1
Preimplantation embryos developed in simulated microgravity(right) and earth-surface gravity(left) conditions.

To determine the reason behind the developmental problems, Wakayama took a closer look at the microgravity embryos’ cell differentiation and polarization. In the early embryo, there is normally a clear
demarcation between regions expressing Cdx2 (which contribute to the extraembryonic trophectoderm) and those expressing Oct4 (which give rise to the embryo proper). But in the microgravity embryos, an unusual number of cells expressed both. Surprisingly, the blastocysts remained polarized,with the inner cell mass located in the expected position.
These results have important implications for the prospects of reproduction in space, but remain preliminary in their scope. “What we have shown using this new technology will still need to be replicatedin actual microgravity conditions,” cautions Wakayama. “So we are looking forward to being able toperform those experiments once the Japanese experiment module Kibō in the International Space
Station is complete.”

Figure 2
3-D clinostat used in experiments

71New basis for the temperature-insensitive period
regulation of the biological clock

Hideki UKAI, Masato NAKAJIMA
Although the circadian clock is exquisitely tuned to changes in light intensity and many chemical compounds, it
is robust in the face of temperature variations. Although this temperature-insensitivity is seen in a wide range oftaxa from bacteria to mammals, its fundamental basis has eluded description. One possibility is that the clock
mechanism includes at least one biochemical component that is unaffected by temperature fluctuations, but at
first blush this seems counterintuitive, given the highly temperature-sensitive nature of most such reactions.
A collaboration of researchers including Yasushi Isojima from the Advanced Computational Sciences Department, RIKEN, Masato Nakajima, Hideki Ukai and others from the Laboratory for Systems Biology (Hiroki R. Ueda;
Team Leader), and Joseph S. Takahashi’s group from Northwestern University, which was formed to elucidate
underlying mechanisms of the circadian clocks by the chemical-biological approach, has now identified an essential regulatory component that shows the selective environmental sensitivity that would be required to explain
the flexible-yet-robust workings of the biological clock.
The study began with a screen of more than 1,200 pharmacological compounds to test their effect on clock periodicity, which yielded 10 that had the strongest ability to lengthen clock period in mouse and human cells. The
majority of these had never been identified as affecting the length of the circadian period, but two were known to
inhibit a family of protein kinases known as CKI (casein kinase I). Taking this lead, the team knocked down various proposed target genes of the 10 compounds to check for possible roles in clock regulation, and found that
two – CKIε/δ and CKIδ – had the greatest period-lengthening effects. Going back to their compounds of interest, they found that 9 of the 10 inhibited the catalytic domain of CKI.

72

©2009 The National Academy of Sciences
Nakajima M, et al. CKIε/δ-dependent phosphorylation is a temperature-insensitive,
period-determining process in the mammalian circadian clock. Proc Natl Acad Sci U S A 106. 15744-9 (2009)

CKIε is known to regulate the degradation of an important clock factor known as PER2 (which CKIδis thought to regulate as well), so Isojima, Nakajima and Ukai next checked whether the two test compounds would inhibit this as well. They found that compounds that putatively inhibit CKI had a significant positive effect on PER2 stability and slowed its degradation, suggesting that these compounds
indeed operate via their effects on CKIε/δ.
Using the same two compounds, they next tested the extent to which they could lengthen the normal
circadian period, and found that both increased the duration of a single cycle by more than double.
Using cells expressing a construct of the circadian gene Per2 fused with the gene encoding luciferase
to assist in the visualization of its expression, they found that its cyclical expression reflected thechanges of PER2 stability seen on administration of the potent compounds and, importantly, that therate of degradation of the PER2 protein was temperature-insensitive.
To confirm whether the underlying CKIε/δ kinase activity is also robust in the face of temperaturechanges, the collaborators created a peptide substrate derived from the CKIε/δ binding domainthought to be phosphorylated by CKIs, and tested it against the catalytic domain of CKIε at temperatures between 25º and 35º C. Kinase activity remained stable against changes across this range. Interestingly, this temperature-insensitivity was reduced in an autophosphorylating version of CKIε, andwhen they tested the activity of the kinase with commercially available and general substrates.

Model of the screening systemused to test chemical comp o u n d s w i th ef fe c ts o n th ecircadian clock

As a final confirmatory step, they used the Per2-luciferase construct to compare degradation rates inclock cells to those of luciferase alone (which shows temperature-sensitive degradation). As expected,
the Per2 constructs significantly outlasted luciferase alone against a range of temperatures, and thiseffect was intensified by enhancing the reaction by Per2 and CKI by forced expression of the CKIεcatalytic domain.
These results clearly point to casein kinase I ε/δ factors as the enzymatic drivers behind temperaturecompensation in the circadian clock. This new knowledge of the basis for period-determination andtemperature-insensitivity may additionally prove to be of value in the development of circadian regulatory drugs.

73Animal Resource andGenetic Engineering
Mutant mice are an important resource used in biological and medical research. The quality and efficacy of research is greatly
dependent on how easily mutant mice can be generated, propagated, and housed, but researchers today tend not to engage inroutine generation of mutant mice. This laboratory’s major function is to develop mutant mice for research in the fields of developmental biology and regenerative science, and to maintain the CDB’s experimental rodent resources.

Shinichi AIZAWA Ph.D.

Genetic Engineering Unit
Shinichi AIZAWA Ph.D.

Staff
Unit Leader

Shinichi AIZAWA
Research Scientist

Go SHIOI
Hirotaka TAO

Visiting Scientist

Fardin FATHI
Toshihiko FUJIMORITechnical Staff

Takaya ABE
Naoko HATAMOTO
MichikoHIGASHIKAWA
Mari KANEKO
Hiroshi KIYONARI
Makiko OGAWA
Naoko OHSHIMA
Yoshihiro TAKAMIYA
Megumi WATASEThe Genetic Engineering Unit works with research labs within the CDB, as well as otherlabs in Japan and throughout the Asia-Pacificregion to develop mutant mice useful to thestudy of development and regeneration. In
these joint development projects, we receive
sequence information for genes of interest
from our collaborators, and perform all subsequent stages of the development from construction of the targeting vector to generationof chimeras, making about 100 new knockout
mutants every year. We also develop transgenic mice for the CDB and Kansai biologicalresearch communities. In addition, we develop new bioimaging technologies to aid in
the visualization of mutant mouse embryos at
the tissue, cell and organelle level.

CS7 BL/6 Es(HK4)-derived offspring.

Animal Resource Unit
Kazuki NAKAO

Staff
Unit Leader

Kazuki NAKAO
Attending
Veterinarian

Naoko KAGIYAMATechnical Staff

Kenichi INOUE
Masahiro OHNO

Assistant

Mayumi
MIZUSHIRO
Yuki TSUJI

The Animal Resource Unit maintains andcares for CDB’s laboratory mouse and rat
resources in a Specific Pathogen Free (SPF)
environment. We also handle the shipping
and receiving of mutant mice both within the
CDB and with other domestic and overseas
institutions. In addition, we provide pregnant
females, fertilized mouse eggs, and services
for colony expansion and strain cryopreservation. We also develop technologies for the
study of reproductive biology.

Mice maintained by the Animal Resource
Unit.

Publications
Islam S M, Shinmyo Y, Okafuji T, Su Y, Bin Nase I, Ahmed
G, Zhang S, Chen S, Ohta K, Kiyonari H, Abe T, Tanaka S,
Nishinakamura R, Terashima T, Kitamura T and Tanaka H.
Draxin, a Repulsive Guidanc Protein for Spinal Cord andForebrain Commissures. Science 323 388-393 (2009)
Kinoshita M, Era T, Jakt L M and Nishikawa S. The novel
protein kinase Vlk is essential for stromal function of mesenchymal cells. Development 136 2069-2079 (2009)74

Tao H, Suzuki M, Kiyonari H, Abe T, Sasaoka T and Ueno
N. Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical-basal polarity. Proc Natl Acad Sci
USA 106 14426-14431 (2009)
Ima T, Yamazaki T, Kobayakawa R, Abe T, Suzuki M and
Sakano H. Pre-target axon sorting establishes the neuralmap topography. Science 325 585-590 (2009)Electron Microscope
Detailed knowledge of the shapes of individual cells and fine subcellular structures is vital to the understanding of biologicalstructures and organization. In developmental processes in particular, changes in cell morphology and behavior play fundamental roles. The Electron Microscope Laboratory provides technical assistance and expert guidance in the use of transmission and scanning electron microscopy (TEM and SEM) and related technologies in morphological analyses.

Shigenobu YONEMURA Ph.D.
Our technical support activities aim to assist scientists with all procedures
related to conventional transmission and scanning electron microscopy,
including the preparation of specimens of any type, assistance in the useof equipment, and the recording and printing of images. We also provide
instructions equipment use, specimen preparation, and interpretation of images. In all cases, we seek to provide researchers with specific advice as tothe appropriate electron micrograph analysis before and during the observation, in order to facilitate the efficient use of electron microscopy in the CDB’s
research activities.
Our lab additionally conducts research into cytoskeletal elements, and thebiophysical aspects of morphological rearrangements in epithelial cells.

Staff
Team Leader

Shigenobu
YONEMURA
Special Postdoctoral
Researcher

Masatsune
TSUJIOKA

Technical Staff

Kazuyo MISAKI

Sachiko ONISHI
Makiko F. UWOStudent Trainee

Tomoki FUJITA
Research Fellow

Ayuko SAKANEAssistant

Mai SHIBATA

Publications
Takata N, Itoh B, Misaki K, Hirose T, Yonemura
S and Okada M. Non-receptor tyrosine kinase
CSK-1 controls pharyngeal muscle organizationin Caenorhabditis elegans. Genes Cells 14: 381393 (2009)
Takeuchi K, Nakano Y, Kato U, Kaneda M, Aizu
M, Awano W, Yonemura S, Kiyonaka S, MoriY, Yamamoto and Umeda M. Changes in temperature preferences and energy homeostasis in
dystroglycan mutants. Science 323 1740-1743(2009)
Horikoshi Y, Suzuki A, Yamanaka T, Sasaki K,
Mizuno K, Sawada H, Yonemura S, and Ohno S.
Interaction between PAR-3 and the aPKC-PAR-6
complex is indispensable for apical domain development of epithelial cells. J Cell Sci 122 1595606 (2009)
Ishiuchi T, Misaki K, Yonemura S, Takeichi M and
Tanoue T. Mammalian Fat and Dachsous cadherins regulate apical membrane organization in theembryonic cerebral cortex. J. Cell Biol 185:959967 (2009)

Figure 1
Cells organizing the notochord of zebrafish embryo.

Figure 2
α-catenin molecules stretched by forces produced by Myosin
II (green) are selectively labeled with α18 antibody (red).

75

Bio-imaging
The goal of the Bioimaging Laboratory is to assist in the operation and maintenance of the Center’s core imaging facility andsupport scientists through the provision of superior optical imaging technologies, analysis and presentation of results. The lab
will manage central optical microscopy systems, image analysis software and the associated IT environment, and promote asecure environment for the effective use of these resources.

Shigeo HAYASHI Ph.D.

Optical Image Analysis Unit
Yuko MIMORI-KIYOSUE Ph.D.

Staff
Unit Leader

Yuko MIMORI-KIYOSUE
Technical Staff

Tomoko HAMAJI
Agency Staff

Tatsunori HAGIHARA
Kayoko NAGAHAMA

Bio-imaging is an interdisciplinary process that integrates molecular cellbiology and biochemistry, as well as technology from optics, engineering,and computer sciences. It has enabled scientists to visualize biologicalprocesses at the cellular and molecular levels and today, has become an
indispensable field for research on biological systems. We aim to design
an imaging environment that can adapt to changing scientific demands
and can contribute to the progress of biology. We hope to accelerate biological research by matching research concepts with the latest technologies from the different specialized fields.
In addition, we aim to shed light on the molecular mechanisms controllingthe microtubule cytoskeleton in the cell/tissue morphogenesis by making
full use of the available technology.

Inverted microscope maintained by the Optical Image Analysis Unit.

76

Genomics
The Genomics Laboratory works to support a wide range of gene analysis and functional genomics research, providing genome-scale, high-throughput services in sequencing gene expression analysis to all CDB labs. All projects can be initiated andfollowed using an internal website designed to ensure a smooth workflow and timely reporting of results.

Fumio MATSUZAKI Ph.D.

Genome Resource and Analysis Unit
Hiroshi TARUI Ph.D.
The Genome Resource and Analysis Unit aims to support a wide gamut ofgene analysis research, ranging from DNA sequencing and gene expression
analysis to storage and distribution of gene resources. Our facility can performgene expression analysis and high-throughput gene screening using a DNA
sequencing system that combines traditional DNA sequencers with the nextgeneration sequencer 454 sequencer, making it possible to apply it to a wider
range of genomic experiments, We can also custom-make DNA microarraysystems. Gene resources derived from a variety of species can also be stored
and distributed to researchers according to their requests. By building upon
existing technologies with the goal of creating new techniques and ideas, aswell as providing solid support, we aim to respond flexibly to the needs ofeach research request.

Staff
Unit Leader

Visiting Scientist

Technical Staff

Part-Time Staff

Hiroshi TARUITetsutaro HAYASHI
Kazu ITOMIYu LIN
Asuka MOMIYAMA
Kaori TATSUMI

Osamu NISHIMURA
Minako MOTOISHI
Assistant

Chiharu
TANEGASHIMA

Publications
Maki N, Suetsugu-Maki R, Tarui H, Agata K, Del
Rio-Tsonis K and Tsonis PA. Expression of stem
cell pluripotency factors during regeneration innewts. Dev Dyn 238(6), 1613-6 (2009)
Egger B, et al. To be or not to be a flatworm: theacoel controversy. PLoS One 4(5), e5502 (2009)
Eto K, Eda K, Hayano M, Goto S, Nagao K,
Kawasaki T, Kashimura H, Tarui H, Nishimura O,
Agata K, Abe SI. Reduced expression of a RNAbinding protein by prolactin leads to translationalsilencing of programmed cell death protein 4 andapoptosis in newt spermatogonia. J Biol Chem(2009)

Functional Genomics Unit
Hiroki R. UEDA M.D., Ph.D.
The Functional Genomics Unit (FGU) provides functional genomics services to
CDB labs and introduces and develops cutting-edge technologies to accelerate research. The FGU has been working to develop GeneChip and cell-based
screening technologies. For the past 3 years, FGU has provided GeneChip
service and informatics for more than 1,250 experiments in a range of research projects conducted using diverse model organisms and protocols. The
FGU has constructed protocols including several quality checks of samples toensure reliable GeneChip service, launched an intranet website, held technicalseminars and project meetings for first-time users, and held a service meeting every week for sharing important information and discussing problems our
GeneChip service. We have also established several informatics methods tosupport this service in the future.
The FGU has additionally developed cell-based screening system using a fulllength cDNA library, and conducted several genome-wide assays in a pilot
study to identify regulators for transcription processes.

Staff
Unit Leader

Hiroki R. UEDA
Research Scientist

Technical Staff

Junko NISHIO
Kenichiro UNO

Takeya KASUKAWA
Itoshi NIKAIDO

Agency Staff

Visiting Scientist

Assistant

Hiroki DANNO

Chikako IMAI
Ikuko TADA

Publications
Nakazawa F et al.: PBRL, a putative peripheral
benzodiazepine receptor, in primitive erythropoiesis, Gene Expr Patterns. 9, 114-21 (2009)
Sakurai M et al.: Differentially expressed Drl andDrl-2 play opposing roles in Wnt5 signaling during Drosophila olfactory system development, J
Neurosci 29, 4972-80 (2009)77

Proteomics
The identification of proteins from trace amounts of biologically important protein complexes is a powerful technique and has
become an indispensable approach to the study of development and regeneration. A better understanding of the protein components of cells and tissues may yield new insights into the molecular structure and function that underlies the spectrum of
biological phenomena and improve our ability to manipulate and recapitulate them.

Shigeo HAYASHI Ph.D.

Mass Spectrometry Analysis Unit
Akira NAKAMURA Ph.D.

Staff
Lab Head

Shigeo HAYASHI
Unit Leader

Akira NAKAMURA
Technical Staff

Kaori SHINMYOZU

Publications
Kakihara K, Shinmyozu K, Kato K, Wada H and
Hayashi S. Conversion of plasma membrane topology during epithelial tube connection requires
Arf-like 3 small GTPase in Drosophila. Mech Dev
125, 325-336 (2008)
Sadaie M, Shinmyozu K and Nakayama J. A
conserved SET-domain methyltransferase, Set11,
modifies ribosomal protein Rpl12 in fission yeast.
J Biol. Chem 283 7185-7195 (2008)

The Mass Spectrometry Analysis Unit uses LC-MASS spectrometry, atechnology that enables the high-speed analysis of protein components
from even minute biological samples. Its support activity takes the forms
of protein identification services and analysis of protein modifications
such as phosphorylation, ubiquitination, etc. The unit supports laboratories within the CDB, and collaborations with a number of laboratories
at other institutions. The lab receives gel slices containing proteins fromresearchers and conducts all subsequent steps, including de-staining ofgels, in-gel digestion of proteins, peptide extraction, and operating the
LC-MS/MS.
In the most recent fiscal year, the unit received more than 30 spectrometry requests and nearly 900 samples for use in identification.

Alev C, McIntyre B A, Nagai H, Shin M, Shinmyozu K, Jakt L M, and Sheng G. Beta A, themajor beta globin in definitive red blood cells, ispresent from the onset of primitive erythropoiesis
in chicken. Dev Dyn 237 1193-1197 (2008)
Alev C, Shinmyozu K, McIntyre B A, and Sheng
G. Genomic organization of zebra finch alphaand beta globin genes and their expression inprimitive and definitive blood in comparison withglobins in chicken. Dev Genes Evol 219, 353360 (2009)

LC-MASS spectrometry system used in the Mass Spectrometry Analysis Unit.

78

Human Stem Cell Technology
The Division of Human Stem Cell Technology (DHSCT) was established to provide support services to any lab with an interest in
using human embryonic stem cells (hESCs) and their derivatives in their research. The DHSCT provides technological expertise,training and support in hESC culture, maintenance, distribution and management, as well as monitoring and analysis of global
trends in stem cell research and regulation.

Yoshiki SASAI M.D., Ph.D.

Human Stem Cell Technology Unit
Yoshiki SASAI M.D., Ph.D.

Human embryonic stem cells cultured on a fibroblast feeder layer.

Despite their great promise and fundamental interest, stem cells – both embryonic and somatic – canbe challenging to study and manipulate in vitro. The
Human Stem Cell Technology Unit was establishedto provide a full spectrum of support services tolabs within the CDB and throughout Japan withan interest in using human embryonic stem cells(hESCs) and their derivatives in their research. The
unit provides technological expertise, training andsupport in hESC culture, maintenance, distribution and management, meeting the needs of bothexperienced and first-time users in the life sciences
research community. Working with labs in academia
and industry, we seek to contribute to achieving thegoals of translational research, in line with the CDB
mission to establish a solid scientific foundation forregenerative medicine. In parallel to hESCs, technical development for the use of human iPS cells is
being planned.

Staff
Division Chief

Yoshiki SASAI
Deputy Chief

Hitoshi NIWA
Research Specialist

Hiroyuki KITAJIMA
Research Scientist

Masatoshi
OHGUSHI

Research Associate

Hidetaka SUGATechnical Staff

Michiru
MATSUMURAIMOTO
Maki MINAGUCHI
Company-SponsoredResearch Trainee

Tokushige NAKANO

Publications
Nagase,T.,Ueno,M.,Matsumura,M.,Muguruma,K.,
Ohgushi,M.,Kondo,N.,Kanematsu, D., Kanemura, Y., Sasai, Y. Pericellular matrix of deciduaderived mesenchymal cells: a potent humanderived substrate for the maintenance culture ofhuman ES cells. Dev Dyn 238 1118-1130 (2009)
Osakada, F., Jin, ZB., Hirami, Y., Ikeda, H., Danjyo, T., Watanabe, K., Sasai, Y., Takahashi, M.
In vitro differentiation of retinal cells from humanpluripotent stem cells by small-molecule induction. J Cell Sci. 3, 169-179 (2009)

Science Policy andEthics Studies Unit
Douglas SIPP

Studying global phenomena in stem
cell research.

The field of stem cell research has received a great
deal of attention due to the combination of fundamental scientific interest, therapeutic promise, andcommercial potential it entails. But it has also beensurrounded by legal, social, and ethical tensions
across a broad range of issues, from the researchuse of human embryos to the optimization of pathways for the translation of basic research into clinicalapplications. We will seek to compare different science policy approaches to these issues and identify
regulatory frameworks best suited to the development and promulgation of stem cell applications. We
will further explore social and ethical perspectives
on the translation of human stem cell research, with
an emphasis on the Asia-Pacific region.

Staff
Unit Leader

Douglas SIPP

Publications
Kiatpongsan, S and Sipp, D. Monitoring andregulating offshore stem cell clinics. Science 323
1564 – 1565 (2009)
Sipp, D. Stem cell research in Asia: A criticalview. J of Cell Biochem 107 853 – 856 (2009)
Gold standards in the Diamond Age: The commodification of pluripotency. Sipp, D. Cell Stem
Cell 5 360 – 363 (2009)
Sipp, D. Stem cells and regenerative medicine on
the Asian horizon. Regenerative Medicine 4 911– 918 (2009)79

Kobe sits at the heart of the Kansai region of western Japan, close
to both the bright lights of Osaka and Kyoto’s tranquility. The local climate is temperate, making it possible to enjoy a wide rangeof seasonal activities, all within a short train ride or drive from thecenter of the city. Japan’s renowned public transport system allows rapid and convenient access to both local and national destinations, and the many area airports, including the Kobe Airport
located less than 10 minutes from the CDB, and the Kansai International Airport, provide immediate gateways to any destination in
the world. Costs of living are comparable to those in many major
Western cities, and comfortable modern homes and apartments
are available to suit all budgets.

80

The bustling heart of Kobe includes a variety of distinct neighborhoods. The downtown area of Sannomiya sits square in thecity’s center, and its range of international restaurants and latenight bars promise a great evening out any night of the week. The
neighboring Motomachi district offers a mix of upscale department
stores and funky shopping arcades stand in contrast to the colorful Chinatown that’s right next door. A short walk north from city
center lies the old foreign settlement of Kitano, whose clapboard
houses and well-kept parks are a perfect retreat from the dynamism downtown.

RIKEN Kobe InstituteBudget and Staff
CDB Symposium
CDB Seminars
About RIKEN
RIKEN Campuses

Kobe occupies a splendid location between mountains and seas,with easy access to many natural spots. A short drive or bus ridetakes you to the island of Awaji, with its beaches and first-rate seafood, and hiking trails crisscross the forested mountains that spanthe entire length of the city, beckoning hikers and picnickers toenjoy a day in the fresh air. The city is also dotted with parks thatcome into bloom at the start of cherry blossom season, and its
many rivers, beaches and scenic areas make it easy to take enjoyable day trips, inside the city or out.

Its central location in Japan puts Kobe within close reach of manyof the country’s most popular destinations. Kyoto, the ancient capital, is only an hour away by train, making it possible to enjoy someof Japan’s most popular historic sites and cultural assets. Nara,another early city, features herds of tame deer in the precincts ofsome of the world’s oldest wooden buildings. Complementing theold-world style of these two cities is Japan’s second city of Osaka,
which offers a hip and modern take on urban living.

Kobe night view. Photography: Shigekazu SHINYARIKEN Kobe Institute
The RIKEN Kobe Institute was established in April 2000 as anorganizational framework for the newly launched Center for Developmental Biology (CDB), which conducts a wide range of research, from fundamental studies of development and stem cells,
to cutting-edge work with the potential to make a contribution toregenerative medicine. In April 2007, the Kobe Institute welcomeda new institution, the Molecular Imaging Research Program, which
carries out research into bioimaging technologies such as positronemission tomography. In autumn 2008, this program was redesignated as the Center for Molecular Imaging Science (CMIS).

The Kobe Institute seeks both to help develop a foundation ofknowledge into biological phenomena and, through translational
research efforts conducted with the Institute for Biomedical Research and Innovation and other nearby institutions, to help bridge
basic science to novel applications in medical therapy and drug
discovery, thereby contributing to the health and welfare of thepeople of Japan.

The Kobe Institute administrative structure comprises the Research Promotion Division and the Safety Center.

82

Kobe Institute Administrative Structure
Kobe Research Promotion Division

Planning
Finance
General Affairs
Human Resources
Facilities
Computer Networks
Science Communications andInternational Affairs
Library
Kobe Safety Center

Institutional Review Board

Center for
Molecular Imaging Science
In 2005, the Molecular Imaging Research Program was selectedby Japan’s Ministry of Education, Culture, Sports, Science andTechnology (MEXT) as an important part of its efforts to promoteresearch and development in life sciences that respond to societal
needs. Since that time RIKEN strove to establish a core centerfor drug development molecular imaging. In the fall of 2006, theKobe MI R&D Center Building was completed with the supportof the Kobe municipal government, and served as home to the
RIKEN facility for the Molecular Imaging Research Program (MIRP).
In October 2008, MIRP was reorganized and inaugurated as theCenter for Molecular Imaging Science (CMIS) to further advance
this field of research, and in April 2009 the Center expanded to 5
teams and 2 units. The CMIS has developed many novel probes
for molecular imaging, contributing to drug discovery and the development of diagnostic and therapeutic indicators.

Research Promotion Division
The Kobe Institute Research Promotion Division (KRPD) provides
a full range of administrative services required by CDB labs, withsections responsible for planning, finance, general affairs, humanresources and facilities, as well as support for scientific meeting
logistics, computer and information networks, science communications and international affairs, and the CDB library.

2009 CDB Budget
Overhead andadministration

Personnel

1,655

1,354

Total: 4,417(million yen)

Research expenses

1,408
In addition to these intramural funds, individual labs and investigators are encouraged to compete for external funding as well,from sources such as the Japan Society for the Promotion of
Science (JSPS), other government agencies, private foundations, and industry. These external funds form an importantcomponent of the CDB’ s overall funding mix, representing
more than 1 billion yen in external funding for CDB research programs in the fiscal year ending in March 2009.

Private industry

RIKEN internal grants

51

14Foundations, etc.

Other government
agencies

11

80

Safety Center
The Kobe Institute Safety Center provides training and supervision
for lab and workplace safety, implements and monitors disasterprevention measures, and educates staff on and ensures compliances with national and institutional regulations governing laboratory practice and technology. The office also handles all administrative affairs of the Institutional Review Board, and administers
the institute’s nursing station.

Total: 1,133

MEXT direct
funding977(million yen)2009 CDB Staff
Laboratory heads
Research scientists
Research associates
Technical staff
Assistants
Visiting scientists
Student trainees
Part-time staff
Research Promotion Division
Other

30
116
8
108
27
72
49
34
56
29

Total

529

Center for Molecular Imaging Science832009 CDB Symposium

Shape and Polarity
March 23–25, 2009
The CDB held its seventh annual symposium on the theme, “Shape and Polarity” on March 23 – 25. The three-day program featured awide range of talks on aspects of how cells become polarized and the roles of cell polarity, and how cell populations organize into complex tissues and organs through rearrangement of polarized cells. In addition to more than 20 invited talks, each day included a poster
session, at which more than 80 presenters introduced and discussed their work. The annual symposium series was launched in 2002 to
establish a forum for addressing diverse aspects of developmental biology and related fields, and continues to promote the free, timely,and global exchange of research achievements.

Session 1

Session 5

Shigeo Ohno (Yokohama City University, Japan)
Julie Ahringer (University of Cambridge, UK)

Tadashi Uemura (Kyoto University, Japan)
Matias Simons (University Hospital Freiburg, Germany)

Session 2

Session 6

Anne Ephrussi (EMBL, Germany)

Ping Chen (Emory University, USA)
Christopher Kintner (The Salk Institute, USA)
Rudolf Winklbauer (University of Toronto, Canada)

Pankaj Dhonukshe (Utrecht University, The Netherlands)

Session 3

Session 7

Jürgen A. Knoblich (IMBA, Austria)
Sarah M. Russell (Peter MacCallum Cancer Centre, Australia)
Hitoshi Sawa (RIKEN CDB, Japan)

Carl-Philipp Heisenberg (MPI-CBG, Germany)
Thomas Lecuit (IBDML, France)
Mirjam Mayer (MPI-CBG, Germany)

Session 4

Session 8

Fumio Matsuzaki (RIKEN CDB, Japan)
Xavier Morin (IBDML, France)
Jon Clarke (King's College London, UK)

Christophe Marcelle (IBDML, France)
Shigeo Hayashi (RIKEN CDB, Japan)
Bob Goldstein (University of North Carolina at Chapel Hill, USA)84

2009 CDB Symposium

Frontiers in Organogenesis
March 23–25, 2010
The eighth CDB Symposium “Frontiers in Organogenesis” will beheld on March 23 – 25, 2010 in the CDB Auditorium. The focus
will be on questions such as spatiotemporal patterning, inductive
signals and cellular dynamics, mechanical interactions in histogenesis, growth control and shaping and evolutionary modulation of organogenesis. By facilitating communication between theleaders and promising younger scientists in these fields at a collegial forum, this meeting seeks to create a unique atmosphere thatprovides a comprehensive perspective into the current knowledgeof the dynamics or organogenesis.

Invited speakers
Arkhat Abzhanov
Harvard University, USA
Session 9

Robert Insall (Beatson Institute for Cancer Research, UK)
Hiroko Sano (Ochanomizu University, Japan)
Roberto Mayor (University College London, UK)

Benoit G. Bruneau

Gladstone Institute of Cardiovascular Disease, USA

Hideki Enomoto

RIKEN Center for Developmental Biology, Japan

Scott E. Fraser

California Institute of Technology, USA

Yukiko Gotoh
Session 10

Takuya F. Sakaguchi (UCSF, USA)
Patrick O. Humbert (Peter MacCallum Cancer Centre, Australia)
Zhenbiao Yang (University of California-Riverside, USA)
Kenji Matsuno (Tokyo University of Science, Japan)
Session 11

Jerome Gros (Harvard Medical School, USA)
Hiroki Nishida (Osaka University, Japan)
Hiroshi Hamada (Osaka University, Japan)

The University of Tokyo, Japan

Brigit Hogan

Duke University Medical Center, USA

Jukka Jernvall

University of Helsinki, Finland

Ryoichiro Kageyama
Kyoto University, JapanMark A. Krasnow

Stanford University School of Medicine andHoward Hughes Medical Institute, USA

Shigeru Kuratani

RIKEN Center for Developmental Biology, Japan

Gail R. Martin

University of California, San Francisco, USA

Andy McMahon
Harvard University, USA

Atsushi Miyawaki

RIKEN Brain Science Institute, Japan

Toshihiko Ogura
Tohoku University, Japan

Stefano Piccolo

University of Padova, Italy

Olivier Pourquie

Howard Hughes Medical Institute,
Stowers Institute for Medical Research, USA

Yoshiki Sasai

RIKEN Center for Developmental Biology, Japan

Neil Shubin

University of Chicago, USA

Didier Stainier

University of California, San Francisco, USA

Cliff Tabin

Harvard Medical School, USA

Hiroyuki Takeda

The University Tokyo, Japan

Masatoshi Takeichi

RIKEN Center for Developmental Biology, Japan

Naoto Ueno

National Institute for Basic Biology, Japan

Joachim Wittbrodt

University of Heidelberg, Germany

Christopher V. E. Wright
Vanderbilt University, USA

Elazar Zelzer

Weizmann Institute of Science, Israel

85

CDB Seminars
The CDB has made special efforts to develop a full and diverse program of invited seminars by scientists from around theworld. To date, the Center has hosted around 450 such talks, in addition to numerous meetings, internal forums and colloquia. The following speakers presented CDB Seminars in the period from January to December 2009.

86

date

title

speaker

01-06

Genetic regulation of renal progenitor cells

Akio KOBAYASHI

01-06

C. elegans: A simple approach to understanding stem cells

Myon-Hee LEE

01-19

Atypical cadherin Fat4 governs planar cell polarity (PCP) in vertebrates

Sakura SABURI

01-19

A molecular mechanism for balancing self-renewal with differentiation in
Drosophila neural stem cells

Takashi NISHIMURA

02-17

Transcriptional silencing and lineage commitment in mice

Brian D. HENDRICH

03-03

Signaling in the neural crest lineages during cardiovascular and peripheral
nervous system development

Yuka MORIKAWA

03-09

Stem Cells: Niche, competition, aging and applications

Ting XIE

03-12

Chemokine-mediated migration control of osteoclast precursors visualized by in vivo bone marrow imaging: A novel point of regulation for
osteoimmunology

Masaru ISHII

03-12

Regulation of Xist function in X chromosome inactivation

Hiroyuki KISHIMOTO

03-18

Regulation of planar cell polarity by Smurf ubiquitin ligases

Masahiro NARIMATSU

04-03

Wnt signaling in mammalian embryonic development

Yingzi YANG

04-09

Developmental regulation of avian stem cells and the application for organ
regenerations

Hiroshi KAGAMI

04-16

Novel functions of tumor suppressor RB protein and p53 in nucleus andnear plasma membrane

Yoichi TAYA

04-20

Embryonic stem cells and pluripotency

Austin SMITH

04-24

Notch signaling is essential for coordinating cell fate, morphogenesis andmigration in the lateral line primordium

Miho MATSUDA

05-27

Roles of Dpr2 in TGF-β signaling and in development

Anming MENG

05-27

Regulation of zebrafish embryonic cell migration and patterning by steroids

Bon-chu CHUNG

06-01

Extrinsic and intrinsic regulators of synapse formation in C. elegans

Kang SHEN

06-02

Epiblast stem cells

Paul TESAR

06-02

Towards generation of visceral endoderm from XEN cells

Tilo KUNATH

06-03

From precursor to product: nephron induction, patterning and repair

Andrew Paul McMAHON

06-04

Regulation of TCF3 phosphorylation and function by Wnt signaling

Hiroki HIKASA

06-05

Post-translational regulation in the Drosophila germ line

Paul LASKO

06-08

Induction and trans-generational inheritance of responses to a novel challenge presented to developing flies

Yoav SOEN

06-08

Prospective isolation of blastocyst- and epiblast-stage precursors fromhuman embryonic stem cells

Micha DRUKKER

07-29

Signalling at tight junctions and regulation of gene expression in epithelial
cellsKarl MATTER

07-29

Tight junction remodeling participates in cytoskeletally-mediated barrier
regulation: The unique role of zonula occludens-1

Jerrold R. TURNER

date

title

speaker

09-07

Developmental evolution of digit identity in birds

Günter P. WAGNER

09-10

Identification and characterization of molecules directing axonal growth
and target field innervations

Kenji MANDAI

09-11

Stemming vision loss with stem cells

Martin FRIEDLANDER

09-14

Novel family of neurotrophic factors – structure, biology and therapeuticpotential

Mart SAARMA

09-19

Tribal networks in cerebral cortex

Henry KENNEDY

09-19

Role of G1 phase regulation in mouse and primate corticogenesis

Colette DEHAY

09-19

Managing stem cells and modulating mitosis: Key roles for centrosomes
and their regulation

Mark PEIFER

09-25

Genetic mechanisms of axon regeneration after injury in C. elegans: rolesof second messengers and MAP kinase pathways

Andrew CHISHOLM

09-30

C. elegans as a model system to study autophagy

Hong ZHANG

10-06

Role of membrane-deforming proteins and endocytosis in neuronalmigration and morphogenesis.

Franck POLLEUX

10-14

Using zebrafish genomics to elucidate human disease

Thomas BECKER

10-14

Genome architecture and new strategies to analyze gene regulation andactivity

Silke RINKWITZ

11-02

Nuclear dynamics in fission yeast Schizosaccharomyces pombe

Pernilla BJERLING

11-11

Characterization of microtubule-end structure and γ-tubulin in plants

Yoshinobu MINEYUKI

11-13

Role of cell polarity genes for photoreceptor morphogenesis in Drosophila

Kwang-Wook CHOI

11-13

Effects of neuropeptide in early postnatal brain development

Ichiko NISHIJIMA

11-19

Niche-associated polarization of a stem cell in Drosophila

Chip FERGUSON

12-04

Control of frontal cortical size and neuronal number during development

Setsuko SAHARA

12-08

Delineating the molecular mechanisms of chromatin protein dynamics inembryonic stem cells

Eran MESHORER

12-11

Elucidating the mechanism underlying embryonic stem cell self-renewal

Qi-Long YING

12-14

"Hitchhiking" to the microtubule plus end: identification of a molecularmechanism

Anna AKHMANOVA

12-15

RNAi-dependent DNA methylation of repeat elements and gene expression homeostasis in Arabidopsis

Vincent COLOT

12-17

The Tetrahymena Argonaute-binding protein Giw1p directs a mature
Argonaute-siRNA complex to the nucleus

Kazufumi MOCHIZUKI

12-21

Understanding the epigenetical functions of histone H3-lysine 56 modification in DNA repair, transcription, and chromatin structural regulation.

Toshiaki TSUBOTA

12-24

Odorant receptors and neural map formation

Takeshi IMAI

12-24

Statistical analysis and mathematical modeling of spontaneous activities
of chemotactic cells

Tatsuo SHIBATA

12-25

How can a protein that protects the brain during injury be harmful during
brain development?

Seong-Seng TAN

87

About RIKEN
The mission of RIKEN is to conduct comprehensive research in science and technology (excluding only the humanities andsocial sciences) as provided for under the "RIKEN Law," and to publicly disseminate the results of its scientific research andtechnological developments. RIKEN carries out high level experimental and research work in a wide range of fields, including physics, chemistry, medical science, biology, and engineering, covering the entire range from basic research to practicalapplication.
RIKEN was first organized in 1917 as a private research foundation, and reorganized in 2003 as an independent administrative institution under the Ministry of Education, Culture, Sports, Science and Technology.

RIKEN Website
The RIKEN website provides a full, rich experience for online
visitors to the institute. The site contains important links to allmaterials, as well as databases and other electronic resources developed by RIKEN labs. We encourage those with aninterest in learning more about RIKEN's organization, activities
and history to visit : http://www.riken.jp/

RIKEN Research
RIKEN publishes the monthly print and online newsletter
RIKEN RESEARCH to draw the world's attention to some of
RIKEN's best research in a timely and easy to understandfashion. This magazine provides a central resource for up-todate information on key achievements of the numerous RIKEN
institutes and research centers, along with related news andretrospectives on the history of institute. The core componentof RIKEN RESEARCH is short, easy-to-understand 'ResearchHighlight' articles explaining for a broad scientific audience asampling of the latest research articles published by RIKEN
scientists. http://www.rikenresearch.riken.jp/88Sendai Facility

519-1399 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-0845
Tel: +81-22-228-2111 Fax: +81-22-228-2122

Tsukuba Institute

BioResource Center
3-1-1 Koyadai,Tsukuba, Ibaraki 305-0074
Tel: +81-29-836-9111 Fax: +81-29-836-9109

RIKEN
Headquarters

Center for Intellectual Property Strategies
RIKEN Next-Generation Supercomputer R&D Center
XFEL Project Head Office

Wako Institute

Advanced Science Institute
Brain Science InstituteNishina Center for Accelerator-Based Science
Computational Science Research Program
2-1, Hirosawa, Wako, Saitama 351-0198
Tel: +81-48-462-1111 Fax: +81-48-462-1554
Itabashi Branch
1-7-13 Kaga, Itabashi, Tokyo 173-0003, Japan
Tel: +81-(0)3-3963-1611 Fax: +81-(0)3-3579-5940
Komagome Branch
2-28-8 Honkomagome, Bunkyo, Tokyo 113-0021, Japan
Tel: +81-(0)3-5395-2818 Fax: +81-(0)3-3947-1752

Yokohama InstitutePlant Science Center
Plant Science Center
Research Center for Allergy and Immunology
Omics Science Center
Systems and Structural Biology Center
Bioinformatics And Systems Engineering division
Center of Research Network for Infectious Diseases
1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045
Tel: +81-45-503-9111 Fax: +81-45-503-9113

Nagoya Facility

2271-130, Anagahora, Shidami, Moriyama-ku,
Nagoya, Aichi 463-0003
Tel: +81-52-736-5850 Fax: 0+81-52-736-5854

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

Center for Molecular Imaging Science

MI R&D Center, Minatojima-minamimachi 6-7-3, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-304-7111 Fax: +81-78-304-7112

Harima Institute
RIKEN SPring-8 Center 1-1-1, Kouto, Mikazuki, Sayo, Hyogo 679-5148
Tel: +81-791-58-0808 Fax: +81-791-58-0800

RIKEN
http://www.riken.jp/

RIKEN is a publicly funded research organization established to conduct comprehensive research inscience and technology, and to disseminate the results of its scientific research and technologicaldevelopments. RIKEN carries out basic and applied research in a wide range of fields, including physics,
chemistry, biology, medical science, and engineering.
RIKEN was founded in 1917 as a private research organization, Rikagaku kenkyuusho (The Institute ofPhysical and Chemical Research). In 2003, the Institute was reorganized as an Independent Administrative
Institution under the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and has
continued to engage in wide-ranging research activities spanning the basic and applied sciences.

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

On The CoverOverexpression of alpha-catenin-ezrin fusion protein(green) in human R2/7 cells induces filopodia-likestructures. Actin filaments (magenta) are also shown.

Printed in Japan using soy inks, waterless printing
methods, and paper from sustainable resources.