RIKEN Center for Developmental Biology

2002 Annual Report

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

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

On The CoverDistribution of the ERM proteinsezrin (green) and moesin (red) in theforelimb bud of the mouse embryo.
Photo: Shigenobu Yonemura

All contents copyright (c) RIKEN Center for Developmental Biology, 2003.

Printed in Japan
Printed on recycled paper

RIKEN Center for Developmental Biology

2002 Annual ReportContents■ Organization

RIKEN Center for Developmental Biology………………………………………………

1■ Message from the Director/Introduction ……………… 2/3■ 2002 Highlights …………………………………………… 4/5

Deputy Directors
Shin-ichi Nishikawa
Shinichi Aizawa

Executive Advisor
Michio Seki

Director
Masatoshi Takeichi
Institutional Review Board

Core Program■ Cell Adhesion and Tissue Patterning (Masatoshi Takeichi)

8/9■ Stem Cell Biology (Shin-ichi Nishikawa)

10/11■ Vertebrate Body Plan (Shinichi Aizawa)

12/13■ Morphogenetic Signaling (Shigeo Hayashi)

14/15■ Cell Asymmetry (Fumio Matsuzaki)

16/17■ Organogenesis and Neurogenesis (Yoshiki Sasai)

18/19■ Evolutionary Regeneration Biology (Kiyokazu Agata)

20/21

Zentaro Kitagawa
Vice-Director, International Institute for Advanced Studies

Creative Research Promoting Program

Hiroko Ueno
Representative, Laboratory for Media and Communication

Mako Tanaka■ Neural Network Development (Chihiro Hama)

24/25■ Vertebrate Axis Formation (Masahiko Hibi)

26/27■ Positional Information (Shigeru Kondo)

28/29

IEXAS, Osaka University Medical School■ Evolutionary Morphology (Shigeru Kuratani)

30/31

Yasushi Yukinari■ Cell Migration (Kiyoji Nishiwaki)

32/33■ Pluripotent Cell Studies (Hitoshi Niwa)

34/35■ Mammalian Epigenetic Studies (Masaki Okano)

36/37■ Cell Fate Decision (Hitoshi Sawa)

38/39■ Developmental Genomics (Asako Sugimoto)

40/41■ Body Patterning (Yoshiko Takahashi)

42/43■ Genomic Reprogramming (Teruhiko Wakayama)

44/45■ Embryonic Induction (Hiroshi Sasaki)

46■ Germline Development (Akira Nakamura)47■ Chromatin Dynamics (Jun-ichi Nakayama)

48■ Stem Cell Translational Research (Takayuki Asahara)49■ Mammalian Molecular Embryology (Tony Perry)50■ Neuronal Differentiation and Regeneration (Hideki Enomoto)51■ Sensory Development (Raj Ladher)

52

Director, Kobe Film Office

Kazuto Kato
Associate Professor, Institute for Research in Humanities, Kyoto University

Tsutomu Miki Kurosawa

Staff Writer, Science News Department, The Yomiuri Shinbun

Shin-ichi Nishikawa
Deputy Director, RIKEN Center for Developmental Biology
Group Director, Laboratory for Stem Cell Biology,
RIKEN Center for Developmental Biology

Kiyokazu Agata
Group Director, Laboratory for Evolutionary Regeneration Biology,
RIKEN Center for Developmental Biology

Advisory Council
Igor Dawid, Chair

William Chia
MRC Center for Developmental Neurobiology (UK)Elaine Fuchs
Howard Hughes Medical InstituteDepartment of Molecular Genetics and Cell Biology
University of Chicago (USA)

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

2002 New Faces

Laboratory of Molecular Genetics
National Institute of Child Health and Human Development, NIH (USA)Peter Gruss
Department of Molecular Cell Biology
Max Planck Institute for Biophysical Chemistry (Germany)

Supporting Laboratories

Yoshiki Hotta
National Institute of Genetics (Japan)■ Cellular Morphogenesis (Shigenobu Yonemura)

Yoichi Nabeshima■ Animal Resources and Genetic Engineering (Shinichi Aizawa) / Sequencing Lab

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

54/55
56

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

Austin Smith■ Administration/Educational Activities ……………… 60/61

Center for Genome Research
University of Edinburgh (UK)Yoshimi Takai
Department of Molecular Biology and Biochemistry
Osaka University Graduate School/Faculty of Medicine

RIKEN CDB 2002 Annual Report■ 2002 Seminars………………………………… 57/58………………………………… 62/63■ City of Kobe/Medical Industry Development Project… 64/65

RIKEN CDB 2002 Annual Report1Development
Regeneration

Center for Developmental Biology

Regenerative Medicine

Masatoshi Takeichi
Director, CDB

Message from the Director
2002 represented a landmark year in the history of the RIKEN
Center for Developmental Biology, one which saw the completion of its main physical facilities, the addition of seven outstanding new laboratories, and the official opening of the Center itself in July. It has been a true pleasure to be able to playthe dual roles of center director and head of the laboratory for
Cell Adhesion and Tissue Patterning and to watch as the CDB
grew from no more than an inspired idea in 2000 to become
one of the largest developmental biology research centers in
the world today.
The Center for Developmental Biology was launched in April
2000 under the auspices of the Millennium Project research
initiative launched by former Prime Minister Keizo Obuchi.
The Millennium Projects were established to drive research in
the fields of information technology, environmental scienceand the study of aging, areas of vital importance to both
Japan and the world in the 21th century. The drafters of thisplan recognized the great potential for contributions by developmental and regeneration biologists in addressing the healthchallenges confronting an aging society, and so the conceptof a national center for developmental biology was born.
A vast array of details needed to be worked out in order tomake this vision a reality, and the RIKEN research organization provided the organizational structure and support environment needed, giving initial form to what is now the CDB andhelping to coordinate all aspects of the center's launch fromconstruction planning to human resources to facility management.
During the process of its establishment, the decision wasmade to locate the CDB within a new and dynamically growing biomedical research park in Kobe, one of Japan's most
attractive cities. This location situates the CDB within a world-

2 RIKEN CDB 2002 Annual Reportclass research setting, with the active support of local andnational governments and participation by public, academic
and corporate research organizations. A spirit of collegiality
and international cooperation pervades the atmosphere here,
and I look forward to seeing the results of collaborations between labs within the CDB with their colleagues at the Center,as well as their counterparts in the region, throughout Japan,
and around the world.
It has been my goal as director of the Center for Developmental Biology to develop a new, open model for research organizations within Japan, with an emphasis on providing the freedom and independence to envision new directions ofresearch, and the organizational and material support to makethose visions real. Nearly three years have passed since thebirth of the CDB as a concept, and the investment of time andmoney has already begun to bear fruit in the form of solid,
innovative research into the mechanisms of development,
regeneration and the scientific bases for regenerative medicine. The years ahead hold the promise of ushering in new
conceptual insights regarding the biological processes ofdevelopment, and of translating those insights into applications with the potential to revolutionize the way we think about
aging, disease and medical therapy. I invite you to explore
this report for a first glimpse into some of the challenges andmysteries that we have encountered, and a few of those thatstill await.

Why Study Development?
The study of developmental biology traces its roots back to Aristotle and the observations
he made on the changes that take place in hen's eggs as the embryos grow from a formless mass into a highly organized and recognizable chick. Scientists today continue to ask
many of the same questions about development that intrigued natural philosophers in thepast, but at dramatically increased levels of sophistication and complexity. How do theorgans of the body arise? How do cells differentiate? What enables animals to reproduce
and transmit genetic traits to their offspring? What are the biological bases underlying evolution? The science of development now extends to and impacts upon nearly every field of
biology in its quest to determine how the information stored in a fertilized egg can trigger
and orchestrate the processes leading to the establishment of unique individuals and theendlessly diverse species they comprise.
Questions of how the body maintains and heals itself also drive research into regeneration.
For centuries it has been known that different species have different self-healing powers,
but the reasons for these differences and the mechanisms by which our own bodies replenish cells lost to aging, injury or disease are now coming into much sharper focus. With the
emergence of improved techniques for studying regeneration at the cellular and molecularlevels, the roles of biological systems governing the maintenance and restoration of the
body's cells have grown clearer, spurring further interest into progenitor cell types known tobe capable of generating appropriate new cells in response to damage and loss.
These progenitor (or 'stem') cells play fascinating and crucial roles in development andregeneration, and science now looks to harness their potential for medical application bydiscovering ways to induce and guide their differentiation to supply natural replacement
cells to damaged organs and tissues. The emerging field of regenerative medicine looks totranslate research findings from the laboratory bench to the hospital bed and to develop
alternatives to the traditional medical approaches of pharmacological and mechanical intervention. Such therapies may someday make the physician's dream of using the body's
own cells to heal itself a reality.
Laboratories at the RIKEN Center for Developmental Biology pursue research into themechanisms of development and regeneration with a twofold mission: To make contributions to the betterment of human health and well-being by providing an academic foundation for regenerative medicine, and, equally important, to illuminate the laws of nature in
the grand panoply of the living world. To shed light and offer hope - these are the goals of
responsible scientists everywhere, goals that we share and hope will direct our work
toward deciphering some of life's greatest mysteries.

RIKEN CDB 2002 Annual Report3Center for Developmental Biology

2002 Highlights

3
4

7

March 22

July 3Publication of "Cadherin Regulates Dendritic Spine Morphogenesis"
The July issue of Neuron featured an article from the Takeichi research groupdetailing a heretofore unknown function of cadherin cell-adhesion molecules in
the formation of synapses between mammalian neurons.

July 8Completion of Research Building A and
CDB Experimental Animal Facility

Opening Ceremony and Public Science ForumThe CDB marked the completion of construction of Building A, adding 7 floors and 10,199 m2 of laboratory space tothe facility. The experimental animal facility (4,311 m2) was
officially opened on the same day, representing the launchof one of the world's most advanced facilities for the production and handling of experimental mice.

The CDB was officially inaugurated in an opening ceremony attended by members of the local and national government, including Kobe mayor Tatsuo Yadaand MEXT Senior Vice-Minister Takashi Aoyama. A lecture session open to
the public was held the same day at a nearby hall, with talks given by CDB
group directors and team leaders to an audience of more than 500 people.

July 9Visit by MEXT Minister Toyama

April 22-24

Atsuko Toyama, the Minister of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), visited the Center on its first full day of operation,
chatting with scientists and observing experiments at a number of labs. RIKEN
research is primarily funded through MEXT budget allocations.

Opening Symposium and Meeting of
the Advisory Council
The CDB held its first international symposium on "A New Paradigm in Developmental Biology," attended by developmental biology and regenerative medical
researchers from around the world. Speakers included members of the CDB Advisory Council, which convened its second session in
the two days immediately following the symposium.

April 259September 27
Completion of CDB Research Aquarium
The CDB's 455 m2 aquarium for housing and breeding experimental aquatic
species such as zebrafish and African clawed frogs officially opened on thisday.

Publication of "Dominant role of the nichein melanocyte stem-cell fate determination"
The work of the Nishikawa research group on the role of the microenvironment(or niche) in determining a stem cell's fate was featured on the cover of the April
25, 2002 issue of Nature.
Reprinted by permission from Nature.
Vol. 416 Publication Date 2002/4/25
Copyright: Macmillan Magazines Ltd.

5

May 17
Publication of "Heterotopic Shift ofEpithelial-Mesenchymal Interactions
in Vertebrate Jaw Evolution"
Research results from the Kuratani lab providing a concrete demonstration of the evo-devo concept of exaptation, in which a structure that originally evolved for one adaptive reason is converted to a different adaptive rolein another species, were published in Science magazine.

4 RIKEN CDB 2002 Annual Report

10

October 10
Publication of"FGFR-related gene nou-darake restricts
brain tissues to the head region of planarians"
The publication of the Agata group's work in determining the gene responsible for restricting brain neuraldevelopment to the head in planarians attracted news coverage in Japan and abroad.

11

November 25-26

12

December 6First CDB Retreat
The CDB held its first annual retreat on the beautiful island of Awaji, located ashort distance from Kobe in Japan's Inland Sea. The retreat gave CDB
research staff the chance to relax, mingle and exchange research findings andopinions in a series of oral presentation and poster sessions. As with all CDB
events, the retreat was held in English, allowing Japanese and non-Japanesescientists to communicate unimpeded by barriers of language or culture.

Completion of Research Building C
The opening of Building C (6 floors, 8,455 m2) represented the completion of
construction of the Center's physical facilities. A number of laboratories had
relocated to their permanent locations in Building C by the end of 2002, and all
current labs are scheduled to be in place by early spring of 2003.

RIKEN CDB 2002 Annual Report5Development

Regeneration

Core Program
The Core Program aims to promote highly innovative
developmental and regeneration studies in a strategic andmulti-disciplinary manner. This program constitutes thecore research framework to achieve the aims of the Millennium Project, and focuses on the three main themes of
the CDB:- Mechanisms of Development- Mechanisms of Regeneration- Scientific Bases of Regenerative Medicine
The Core Program consists of seven research groups,
each lead by an eminent scientist in these fields. In addition to the group director, each group includes a numberof research fellows and technical staff and in some casesa senior research fellow.

Partial view of ragworm (Perinereis nuntia) reproductive appendage
Photo: Nao Niwa

Regenerative
MedicineDevelopment

Regeneration

Core Program

Laboratory for Cell Adhesion and Tissue Patterning

Cell Recognition
Masatoshi Takeichi
Ph. D.

Masatoshi Takeichi is director of the
RIKEN Center for Developmental Biology (CDB; Kobe, Japan) as well as director of 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., Dr. Takeichi took a research fellowship at the Carnegie Institution
Department of Embryology under Dr.
Richard Pagano. He then returned to
Kyoto University, attaining a full professorship in the Department of Biophysics(1986-1999), before becoming professor in the Department of Cell and Developmental Biology in the Graduate
School of Biostudies at the same university. He assumed his current positions at the CDB in 2000. Dr. Takeichi
is best known for his discovery of cadherins, which are fundamental in themechanisms of cell-cell adhesion.

divisions. The innermost domain, to which theβ- and α-catenin complexes bind, is known to be
essential to cadherin function and mutations in thesecatenins have been implicated in some forms of cancer. One of Dr. Takeichi’s recent areas of interest has
been the role of the juxtamembrane (JM) domain,which is the binding site for another catenin, known
as p120. He found that cells lacking the JM domain
cannot undergo normal morphogenetic movement
during the development of chicken somites, suggesting that this domain plays a critical role in allowing
cells to relocate. These findings led Takeichi to propose the model that the JM domain is a signaling center involved in regulating cadherin activity, and may
be important not only in embryonic morphogenesis,
but in the processes of cancer invasion and metastasis as well.

The neural retina provides
the Takeichi lab with another systemuseful for the study ofdevelopmental processes

Cell adhesion, recognition andmorphogenesis
The formation of complex systems such as tissues
and organs requires a high degree of communicationand cooperation between their constituent cells.
Since the discovery of cadherins, the role of the cadherin superfamily of molecules in regulating cell-celladhesion has been studied extensively. These molecules form complexes with other intracellular factors
to create bonds spanning membranes and intercellular spaces to join cells with other cells expressing
similar cadherins. Such bonds are dynamic and regulable, acting more like Velcro tape than permanentglue. But recent work is revealing that the cadherins
may serve a wider range of functions than as simple
adhesives. Dr. Takeichi’s research group is engaged
in demonstrating expanded roles forthese molecules in the formation of complex structures such as neural synapses.

Staff
Group Director

Masatoshi Takeichi
Research Scientist

Shinichi Nakagawa
Visiting Scientist

Shinji Hirano
Special Postdoctoral Researcher

Takuji Tanoue
Research Specialist

Structure and function of
cadherin complexes

Midori Maekawa
Research Associate

Sachihiro Suzuki
Technical Staff

Cadherins are the main regulators ofcalcium-dependent cell-cell adhesion, butthey do not work alone. It has beenshown that the function of cadherins
relies on the formation of complexes witha group of molecules called catenins,which associate with the cytoplasmic tailof the cadherin molecule. The tail of thecadherin molecule comprises two main

Hitomi Ishigami
Miwako Harata
Masato Uemura
Junior Research Associate

Tetsuo Ichii
Student Trainee

Tetsuhisa Otani / Hideru Togashi
Koji Tanabe / Kanna Takaoka
Yoshiko Kametani / Kentaro Abe
Masakazu Kadowaki / Fumi Kubo
Shinsuke Nakao / Masamitsu Sone
AssistantMutsuko Aiso

Synapse formation
The synapses formed between neurons are of central
importance in neural signal transmission, but the processes by which synapses are formed remain poorly
understood. The Takeichi research group previously
discovered that cadherin complexes localize in interneuronal cell junctions in such a way that they do notinterfere with signal transmission as carried out bysynaptic vesicles and receptors on either side of thesynaptic complex. The group has now reported that
cadherins help to regulate the adhesion of dendritic
branches known as ‘spines’ to axons, which is a crucial step in synapse formation. During neural network
development, dendrites (protoplasmic processes that
conduct impulses toward the nerve cell body) extend
spines into the intercellular space. When a spine
makes contact with an axon (which conducts impulses away from the neural cell body), a mushroomshaped ‘head’ is formed, laying the foundation for a
new synaptic site. The Takeichi lab conducted lossof-function studies in hippocampal neurons with dominant negative mutations for N-cadherin, and in thoseobtained from αN-cadherin knockout mice, andfound that both groups of neurons formed synapses
with altered dendritic spines and synaptic organization. These findings indicate that cadherin/catenincomplexes may be critical regulators of the morphogenetic processes underlying synaptic plasticity.

Organization
anddevelopment of theneural retina
The retina, the innermost coatof the eyeball, is composed ofneural and non-neural elements. The neural retina, which
receives optical informationfrom the lens and interfaces
with the optic nerve to convey that information to thebrain, provides the Takeichi lab with another systemuseful for the study of cellular organization andstructure-specific developmental processes. The
group’s studies in this field have focused on the roleof the gene Wnt-2b in retinal development in chickembryos. The developing retina gradually organizes
into sheet-like laminated structures. Retinal cells in
isolation, however, fail to form these structures andinstead clump together in ‘rosettes.’ The molecularmechanism by which the laminated structures are
generated was unknown. Members of the Takeichigroup have now demonstrated that Wnt-2b producedin the ciliary margin, a small region located at the border of the retina with the iris, guides retinal cells toform laminar sheets. Using dissociated retinal cells in
culture, the team found that cells exposed to Wnt-2b
in culture organized into sheets, similar to thearrangement of cells in the normal retina. The same
cells, when exposed to Frizzled, a Wnt-antagonist,clumped together in rosettes.
A second study of Wnt-2b function revealed its importance in maintaining undifferentiated progenitor cells
in the ciliary marginal zone. Throughout embryonicdevelopment, the ciliary margin contains multipotent
cells that serve as progenitors for the specialized, differentiated cell types that ultimately form the complex
architecture of the retina.
When Wnt-2b is overexpressed in developing retinas, normal cell differentiation is inhibited. Additional
studies revealed that when
the Wnt signaling pathway
was blocked downstream,
neuronal cells differentiatedearlier than they normally
would have. All of thesefindings suggest a role for
Wnt-2b as a stem cell factor in the retina.

Blockade of cadherins in hippocampal neurons alters dendritic
spine shape. Red, a control
dendrite; green, a dendrite
expressing a dominantnegative cadherin.

Publications
Kubo F, Takeichi M, and Nakagawa
S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone.
Development (2003 in press).
Togashi H, Abe K, Mizoguchi A,
Chisaka O, and Takeichi M.
Cadherin regulates dendritic spine
morphogenesis. Neuron 35:77-89(2002).
Horikawa K, and Takeichi M. Requirement of juxtamembrane domain of
the cadherin cytoplasmic tail formorphogenetic cell rearrangement
during myotome development. J Cell
Biol 155:1297-306 (2001).

Wnt2b, expressed in the ciliary
margin (left), controls the undifferentiated state of retinal cells. Overexpression of Wnt2b, coinjected with
GFP (green), in retina induces thefolded expansion of an undifferentiated cell layer (right). Middle,
control, in which only GFP wasinjected.

Myotome patterning in chicken embryos is perturbed by the overexpression of mutant cadherins or p120-catenin in different fashions.

8 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report9Development

Regeneration

Regenerative
MedicineCore Program

Laboratory for Stem Cell Biology

Stem Cells
Shin-ichi Nishikawa
M. D. , Ph. D.
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 ResearchInstitute, before taking an assistant professorship in the same department in
1979. He spent the period from 198082 at the University of Cologne Institutefor Genetics (Germany) before returning to the Chest Disease Research Institute, where he was appointed associate
professor in the Department of Microbiology in 1983. He moved to the Kumamoto University Medical School in 1987
to take a professorship in the Department of Immunology, and returned to
Kyoto in 1993, as professor in the
Department of Molecular Genetics at
the Kyoto Graduate School of Medicine.
He was appointed CDB group director
in 2000.

Staff

Stem cell biology
Stem cells are undifferentiated cells characterized bytheir capacity to self-renew and to generate specialized cells indefinitely. These cells provide the source
for all of the myriad somatic cell types in the adultorganism. The study of stem cells stands at the heartof many of the central problems in developmental biology, but many questions regarding the nature ofthese cells, such as the extent to which they can be
reprogrammed to produce progeny of different fates
and the identities of the intrinsic and extrinsic molecular factors that regulate a cell’s ‘stemness,’ remainunanswered.

Group Director

Shin-ichi Nishikawa
Research Scientist

Takumi Era
Masatake Osawa
Yusuke Yoshida
Visiting Scientist

Igor M Samokhvalov
Timm T Schroeder
Collaborative Scientist

Mitsuhiro Okada
Eri Nishioka
Lars M Jakt
Muneaki Miyata
Masahiro Yasunaga
Tamie Morisawa
Satomi Nishikawa
Shin Kawamata
Special Postdoctoral Researcher

Chikara Furusawa
Technical Staff

Satomi Nishikawa
Mariko Moriyama
Kotomi Kawasaki
Chiga Okita
Natalia I Samokhvalova

Shin-ichi Nishikawa
believes that location isimportant in defining
a stem cell’s function

Student Trainee

Hidetoshi Sakurai
Atsushi Takebe
Shinsuke Tada
Kanako Yoshikawa
Ai Kotani
Mak Siu Shan
Masanori Takenaga
Yasuhiro Takashima
Masaki Kinoshita

Shin-ichi Nishikawa believes that location is importantin defining a stem cell’s function. Recent findingsthat stem cells can arise in one tissue type but later
be directed to give rise to another strongly suggestthat a stem cell’s role is at least partly determined bysignals from its surrounding environment. By studying
this microenvironment, known as the stem cell‘niche,’ the Nishikawa research group seeks to gain
insight into the identity and function of molecules
involved in stem cell activities, and the potential ofstem cell-based therapeutic applications.

Assistant

Sakura Yuoka
Maya Iwase

Melanocyte stem cell niches
One of the major challenges confronting stem cellresearchers is the host of technical and biologicalproblems associated with in vivo studies. Multipotent
stem cells are responsible for the normal development of their progeny cells, which means that experimental manipulations of these cells can have farreaching, even lethal, consequences. Nishikawa haschosen melanocyte stem cells as a research modelas these cells share some common characteristics
with the stem cells responsible for generating bloodand sperm, but offer several experimental advantages over those stem cell types. Melanocytes, which
produce skin and hair pigments, can be modified
extensively, even lost, without causing any more serious effect than alterations in skin and hair color, andsuch alterations can themselves serve as visual confirmation of an experiment’s result. Increased animal
survival due to the absence of lethal phenotypes also
makes the melanocyte system an ideal model fromboth the experimental and ethical perspectives.
In work published in 2002, Nishikawa and colleagues
established a system for profiling melanocyte stem
cells and tracking their locations in living hair follicles.
Follicles are known to contain melanocyte stem cells,
but the follicular microenvironment that sustains suchcells is still poorly understood. In this study, the Nishikawa group identified cells with high expression levels of melanocyte stem cell markers, and thenblocked the function of one of these molecules using
marker-specific antibody. The results of these experiments indicated that melanocyte stem cells localize
at two intrafollicular sites, the hair matrix and thebulge, each of which hosts a specific sub-populationof stem cells. The matrix, where hair growth and pigmentation take place, provides an environment inwhich amplifying progeny cells become committed toa stem cell fate, while the bulge serves as a reservoir
for stem cells at rest. Intermediate stem cells canmigrate from the bulge to the matrix and eventhrough the epidermis to neighboring follicles toreplenish the actively proliferating population. Nishikawa hopes to characterize these niches in more detailto achieve a better understanding of the extrinsic factors responsible for maintaining stem cells in different
states of activity.

Hematopoietic lineage maturation

stem cells at least, the process may follow a different
route. Blood-forming cells
are traditionally thought tooriginate in the lateral mesoderm, one of the three maindevelopmental regions of the mesodermal germ layer. The Nishikawa group developed tools to distinguish cell types generated during early embryogenesis
and identified several intermediate stages during thedifferentiation of blood cells from mesoderm. The
group discovered two points of blood cell line divergence – either directly from lateral mesoderm cells orfrom vascular endothelial cells – a finding that contrasts with the widely-held view that blood cells andendothelial cells diverge from common precursors
called hemangioblasts, and surprisingly indicates thatan increase in potency occurs at the endothelial
stage at which it would normally be expected to show
a decline.

Distribution of melanocytes in mouse follicle

Regulating vascular architecture
Other recent work by the group has revealed a factor
capable of maintaining the integrity of blood vessels
derived in isolation from their natural environment. In
the normal course of development, endothelial cells
and perivascular mural cells work together to form adiverse vascular architecture from a relatively simple
network. While the role played by mural cells in the
structuring of vascular tissue remains unclear, theirabsence is a hallmark of diseases such as diabetic
retinal disorders. Two proteins, angiopoietin-1 and
PDGF-B, have been implicated in mediating the interaction between endothelial and mural cells. The Nishikawa group introduced a PDGF-B antagonist into neonatal mice, preventing mural cells from participatingin the vascular formation process, and found that theresultant vascular system in the retina was poorly
organized and leaky. However, when they introduced
recombinant angiopoietin-1 to the eyes, they foundthat normal vascular architecture was restored, whichsuggests a potential application for angiopoietin in
the treatment of mural cell disorders.

Publications
Nishimura E, Siobhan J, Oshima H,
Yoshida H, Osawa M, Moriyama M,
Jackson IA, Barrandon Y, Miyachi Y,
Nishikawa SI. Dominant role of theniche in melanocyte stem cell fate
determination. Nature 416:854-60(2002).
Yoshida H, Naito A, Inoue JI, Satoh
M, Santee-Cooper SM, Ware CF,
Togawa A, Nishikawa S, Nishikawa
SI. Different Cytokines Induce Surface Lymphotoxin-alphabeta on IL-7
Receptor-alpha Cells that Differentially Engender Lymph Nodes and Peyer's Patches. Immunity 17(6):823-33(2002).
Uemura A, Ogawa M, Hirashima M,
Fujiwara T, Koyama S, Takagi H, Honda Y, Wiegand SJ, Yancopoulos GD,
Nishikawa SI. Recombinantangiopoietin-1 restores higher-orderarchitecture of growing blood vessels
in mice in the absence of mural cells.
J Clin Invest 110(11):1619-28 (2002).
Endoh M, Ogawa M, Orkin S, Nishikawa SI. SCL/tal-1-dependent process
determines a competence to selectthe definitive hematopoietic lineageprior to endothelial differentiation.
EMBO J 16;21(24):6700-8 (2002).

In the classical view of lineage maturation, cell lines
always develop toward increased specialization andaway from differentiative potency. However, evidence
exists to suggest that, in the case of hematopoietic

HoxB1 expression pattern in 9.5 d.p.c. mouse embryo

10 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report11Development

Regeneration

Core Program

Laboratory for Vertebrate Body Plan

Body Plan
Shinichi Aizawa
Ph. D.
Shinichi Aizawa received his Ph. D. inbiochemistry from 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 in the
Laboratory of Genetic Pathology at the
University of Washington (US). He
returned to the Tokyo Metropolitan Institute of Gerontology in 1982, where heremained until 1986 when he moved to
the RIKEN Tsukuba Life Science Center as a senior research associate. He
was appointed professor in the Kumamoto University School of Medicine
Department of Morphogenesis in 1994,
and served in that position until 2002.
Since 2000 he has served as CDB
deputy director and group director of
the Vertebrate Body Plan, as well asteam leader of the Laboratory for Animal Resources and genetic Engineering.

StaffGroup Director

Shinichi Aizawa
Research Scientist

Akihito Yamamoto
Yoko Suda
Takuya Murata
Nobuyoshi Takasaki
Daisuke Kurokawa
Takashi Nagano
Jun Kimura
Kohei Hatta
Collaborative Scientist

Akihiko Shimono
Technical Staff

Shoko Takehara
Tomomi Ohmura
Miwa Nakamura
Ai Inoue
Saori Nagayoshi
Student Trainee

Wataru Sato
Mariko Hirano
Kyoko Hashimoto

The vertebrate head-body boundary
The evolutionary development of the vertebrate headis the subject of a debate older than the concept ofevolution itself. Beginning with Goethe’s observationthat the cranium of a sheep seemed to be formed
from fused vertebrae, rival theories have been proposed in which it is asserted that the head is either
the evolution of some preexisting structure, or thatthe emergence of the head was an evolutionarily novel event. What is certain is that the body plan of modern vertebrates is composed of three distinct regions
characterized by the expression of different sets ofregulatory genes – the trunk, the head, and the intervening hindbrain/pharyngeal region.

Part-time Staff

Setsuko Date
Kazuki Suganuma
Assistant

Yukari Hata
Sayo Saito

Shinichi Aizawa’s research concentrates on the genetic and molecular characteristics of head development, particularly in the border between the fore-andhindbrain, which in vertebrates originates in segmented embryonic units known as ‘rhombomeres.’ Rhombomeres exhibit distinct gene expression and cell
behavior profiles, and are situated at the developmentally interesting boundary between the trunk region,
whose development is primarily regulated by homeotic Hox-family genes, and the rostral (anterior) head,
which is governed by a different set of genes, predominantly the homologs of Drosophila head gap
genes. Studying mutations in genes known to be
responsible for body patterning in these regions, theAizawa research group hopes to establish the factorsthat delimit and characterize the foremost rhombomeres, and thereby contribute to the understanding of the developmental criteria for the constitution of
the anterior head as conserved across phyla.

Head gap genes

Head Organizer ProjectTeam

Homologs of the Drosophila orthodenticle gene,which collectively form the Otx gene family, havebeen established as essential to the induction of theanterior head and the anterior-posterior body axis
itself in both invertebrate and vertebrate species. In
the mouse, Otx2 homozygous mutants entirely fail todevelop the fore- and midbrain. However, themechanisms by which this gene induces normal
anterior head development remain elusive. With theknowledge that signals from an embryonic neurode-

Senior Scientist

Isao Matsuo
Special Postdoctoral Researcher

Chiharu Yoshida
Technical Staff

Kuniko Kitajima
Hiroshi Nakano
Izumi Ishii
Part-time Staff

Nana Fujimoto

velopmental region called the anterior neural ridge
are also essential in the induction and patterning of
the forebrain, Aizawa set out to determine the preciserole played by Otx2 in mediating signals from thisorganizing center. Generating a series of allelic Otx2
mutants resulted in the unexpected discovery of ahypomorphic allele characterized by arrested forebrain and anterior head development. Unlike homozygous Otx2 mutants, however, these allelic embryos
were able to form apparently normal anteriorposterior axes and progenitor fields. Explant assays
were made by removing anterior neural plate sections from mutant embryos and transplanting theminto wild type embryos, and vice-versa. It was foundthat while neural plates from Otx frt-neo/- mutants wereable to respond normally to signals from the anterior
neural ridge in wild type embryos, the reverse was
not true, indicating that Otx2 may be involved in thedownstream mediation of forebrain-inducing signals,
such as Fgf8, emanating from this region.

Locus mapping provides
a means for identifying candidate
genes of potential
biomedical importance

Genetic determinants of craniofacial anomalies
In mice, embryos lacking a single copy of the Otx2
gene develop a range of phenotypes in which craniofacial development is disturbed. These phenotypes,
termed otocephaly or agnathia-holoprosencephaly,
are also observed as human congenital anomalies
and represent a failure of the head patterning program. However, in mice, the severity of the phenotype was linked to the genetic background of thestrain in which the mutation was induced. By crossing
strains exhibiting different phenotypes and performing genotypic analysis of the offspring, the Aizawa
group was able to identify two loci linked to abnormalities in the lower jaw in these mutants. One of these
loci, named Otmf2, may also be implicated in thedevelopment of holoprosencephaly, the most common congenital forebrain defect in humans, with anincidence of up to 1 in 250 early embryos. This study
revealed the power of the mapping of loci responsiblefor determining phenotypic severity in naturally occurring variations in different strains of experimental animals, both as a tool for genotypic analysis and as ameans of identifying candidate genes of potential biomedical importance.

Two enhancers of Otx2 expression in the forebrain and midbrain

Body patterning in primitive species
Amphioxus, commonly referred to as the lancelet, isa small, marine organism thought to represent one of
the earliest evolutionary ancestors of vertebrates as
the lancelet embryo develops a number of structurescommon to chordate development, such as thenotochord, during gastrulation. The polarized accumulation of β-catenin in the nuclei of specific cells is a
crucial step in the early development of multicellularanimals, playing roles in the formation of germ layers
and signaling centers. The Aizawa lab studied theactivity of β-catenin in the lancelet to investigate how
its localization impacted on morphogenesis in theseorganisms. These studies, in which the asymmetric
distribution of this protein was experimentally altered,indicated that embryos with non-typical β-catenin distributions are nonetheless able to establish dorsalventral axes, suggesting that dorsal morphogenesis
is not dependent on early embryonic polarization in
Amphioxus.
In experiments such as these, the Aizawa lab
endeavors to characterize the evolutionary, genetic,
and molecular bases of the vertebrate body plan, witha particular emphasis on the factors at work in inducing that crowning achievement of animal evolution,
the forebrain.

Publications
Hide T, Hatakeyama J, Kimura C, Tian
E, Takeda Na, Ushio Y, Shiroishi T,
Aizawa S, and Matsuo I. Genetic modifiers of otocephalic phenotypes in Otx2
heterozygous mutant mice. Development 129:4347-57 (2002).
Shinozaki K, Miyagi T, Yoshida M,
Miyata T, Ogawa M, Aizawa S, andSuda Y. Absence of Cajal-Retzius
cells and subplate neurons associated with defect of tangential cell migration from ganglionic eminence in
Emx1/2 double mutant cerebral cortex. Development 129:3479-92(2002).
Tian E, Kimura C, Takeda N, Aizawa
S, and Matsuo I. Otx2 is Required to
Respond to Signals from Anterior
Neural Ridge for Forebrain Specification. Dev Biol 242:204-23 (2001).
Kimura C, Shen M, Takeda N, Aizawa S, and Matsuo I. Complementary
Functions of Otx2 and Cripto in Initial
Patterning of Mouse Epiblast. Dev
Biol 235:12-32 (2001).
Suda Y, Houssain ZM, Kobayashi C,
Hatano O, Yoshida M, Matsuo I, andAizawa S. Emx2 directs the development of diencephalon in cooperation
with Otx2. Development 128:243350 (2001).

Enhancer of Otx2 expression in the anterior neuroectoderm

12 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report13Development

Regeneration

Core Program

Laboratory for Morphgenetic Signaling

Signaling
Shigeo Hayashi
Ph. D.
Shigeo Hayashi received his B. Sc. in
Biology from Kyoto University in 1982,
and his Ph. D. in Biophysics from thesame institution in 1987 for his work on
lens-specific regulation of the chicken
delta crystallin gene. Inspired by the discovery 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 toJapan to work in the National Instituteof Genetics. He became an associate
professor at the same Institute in 1994,
and professor in 1999. Also in 1999, hereceived the incentive prize of the
Genetics Society of Japan for Genetic
Studies on Drosophila Development.
He was named group director of theMorphogenetic Signaling researchgroup at the RIKEN CDB in May 2000.

A society of cells
The metaphor in which cells are likened to individuals
within a society is a common one in biology, andplays a particularly instructive role in understanding
many of the mechanisms of early development. Just
as students of social behavior seek to identify the factors responsible for human actions, developmental
biologists ask similar questions about what drives acell to divide, differentiate, migrate, or self-destruct.
The answers to both sets of questions generally
involve multiple factors. In animal development, thespecification of cell fates and their behavior as individuals and in groups is determined by interactions between molecular factors intrinsic and extrinsic to themicroscopic world of the cell – genes and their protein products, and signaling and transcription factors
received from the world beyond the cell’s membraneborders.

Group Director

Shigeo Hayashi

Staff

Research Scientist

Leo Tsuda
Atsushi Wada
Yoshiko Inoue
Kenji Oshima
Special Postdoctoral Researcher

Nao Niwa
Junior Research Associate

Kagayaki Kato
Technical Staff

Ai Akimoto
Masako Kaido
Michiko Takeda
Chiaki Kose
Hiromi Sakaguchi
Part-time Staff

Tadashi Sakata
Masayo Shindo
Kayoko Sakurai
Housei Wada
Sono Hashimoto
Hiromi Niwa
AssistantChisa Iimuro

Group Director

Shigeo Hayashi
Research Scientist

Leo Tsuda
Atsushi Wada
Yoshiko Inoue
Kenji Oshima
Special Postdoctoral Researcher

Nao Niwa

Questions of interand intracellular communication
inform Shigeo Hayashi’s
research

Junior Research Associate

Kagayaki Kato
Technical Staff

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

Tadashi Sakata
Masayo Shindo
Kayoko Sakurai
AssistantChisa Iimuro

14 RIKEN CDB 2002 Annual Report

The tracheal system stained for nuclei (green)
and luminal cavities (purple)

Questions of inter- and intracellular communication
inform Shigeo Hayashi’s research. His work seeks touncover genes and molecular factors that allow differentiated cells to communicate and coordinate witheach other to form ‘communities’ of tissue, organ andanatomical structure within the ‘society’ of the organ-

ism as a whole. Hayashi has chosen to focus his studies on the systems of limb and tracheal development
within the fruit fly Drosophila to outline the mechanisms of cellular behavior and interaction in organogenesis.

Development of appendages
Like many insects, Drosophila is a highly competentflier, evolutionarily adapted to a life spent on thewing. Embryological studies in Drosophila haveshown that both wings and legs share a common origin in developmental bodies known as limb primordia.
In their studies of limb formation, Hayashi’s grouptracks the cellular growth, differentiation and migration characteristic of cells destined to become wings
in the adult fly. Using time-lapse confocal microscopyof fluorescent-tagged cells, the group tracks threestages of the process of wing development: allocation, specification, and separation. In allocation, cells
in the primordium undergo preliminary specializationfrom cells in the surrounding area. Specification involves further differentiation between cells that willremain in place and contribute to leg formation, andthose that are marked for specification into wing tissue. The separation phase involves the migration of
wing-specified cells to dorsal locations, from which
the final wing structure arises. Non-migrating cells
remain to form the leg primordium, which consists ofproximal and distal domains. This simple organization
is further elaborated by a series of segmentation processes.
Now, armed with the ability to make a clear visual
record of the movements of wing and leg-specified
cells to their ultimate destinations within the body,
Hayashi and colleagues intend to pin down the molecular machinery that determines these outcomes using
genome screening and molecular biology techniques.
As the genetic factors involved in limb development
tend to be highly conserved across species, findings
from studies on the relatively simple fruit fly should offer insights into the fundamental mechanisms of organ
and limb development in humans and other species.

Tracheal development
The mechanisms of tracheal development in Drosophila are another focal area for research in
Hayashi’s lab. In fruit flies, the trachea is functionallyanalogous to the mammalian lung, and its development shares many characteristics with the development of many branched tubular systems including thelungs, kidneys, and blood vessels, which are all
essential to the distribution and regulation of liquids
and gases throughout the body in higher animals.

The adult Drosophila melanogaster fruit fly

The development of the trachea in Drosophila originates with ten pairs of tracheal primordia, located in
the contiguous thoracic and abdominal segments, T2
to A8. Cells from the primordium migrate, extend
branches decorated with fine cellular extensions
called filopodia, and respond to environmental cues
to seek out and establish connections with other tubular cells. The trachea ultimately extends to every segment in the adult individual. Hence, the process oftracheal tubulogenesis involves both intercellular
cooperation within individual segments, and coordination between cells throughout the entire body.
The migration of tracheal branches is controlled by acomplex guidance mechanism involving several extracellular factors such as Dpp, Wingless and Fgf.
Hayashi’s group is investigating this guidancemechanism and the role of the Rac signaling pathway, which is known to participate in the mechanismsof cell adhesion, migration and guidance. The group,in collaboration with researchers from Tokyo Metropolitan University, uses selective overexpression oftarget genes to produce gain-of-function mutants as apreliminary screen for new gene functions required
for tracheal patterning.
Gain-of-function studies serve to provide an initial
glimpse of the roles of such genes. By regulating theexpression of target genes and observing the phenotypes, it becomes possible to identify candidates
for further investigation. To date, the Hayashi grouphas completed screening studies of approximately
one-fourth of the Drosophila genome using randomly
inserted UAS transposons, which serve to activate
genes located downstream. Phenotypes of interest
include mutants displaying absent tracheal branches,branch misrouting, changes in cell and tubule shape,
and abnormal cell adhesion. The genes
implicated in these mutations become
candidates for follow-up studies to elucidate their involvement in tracheal development.

Publications
Kubota K, Goto S, Hayashi S. The
role of Wg signaling in the patterningof embryonic leg primordium in Drosophila. Dev Biol (in press 2003).
Chihara T, Kato K, Taniguchi M, Ng
J, Hayashi S. Rac promotes epithelial cell rearrangement during tracheal
tubulogenesisi in Drosophila. Development 130:1419-28 (2003).
Shiga Y, Yasumoto R, Yamagata H,
and Hayashi S. Evolving role ofAntennapedia protein in arthropod
limb
patterning.
Development
129:3555-61 (2002).
Hayashi S, Ito K, Sado Y, Taniguchi
M, Akimoto A, Takeuchi H, Aigaki T,
Matsuzaki F, Nakagoshi H, Tanimura
T, Ueda R, Uemura T, Yoshihara M,
and Goto S. GETDB,a database compiling expression patterns and molecular locations of a collection of Gal4
enhancer traps. Genesis 34:58-61(2002).
Goto S, Taniguchi M, Muraoka M,
Toyoda H, Sado Y, Kawakita M, andHayashi S. UDP–sugar transporter
implicated in glycosylation and processing of Notch. Nat Cell Biol
9:816-22 (2001).

Drosophila embryo expressing tau-GFP
fusion protein in the limb primordium
and sense organs of the head

RIKEN CDB 2002 Annual Report15

Development

Regeneration

Core Program

Laboratory for Cell Asymmetry

Asymmetry
Fumio Matsuzaki
Ph. D.
Fumio Matsuzaki received his B. Sc.
from the Department of Biochemistry
and Biophysics at the University ofTokyo in 1979, and his doctorate fromthe same institution in 1984, for his
work on the characterization of theerythrocyte cytoskeletal structure. He
spent the period from 1984 to 1988 as
a postdoctoral fellow, first in the Department of Cell Biology at the Tokyo Metropolitan Institute of Medical Science,then in the laboratory of Gerard Edelman at Rockefeller University. He
returned to Japan in 1988 to take a position as a section chief in the Department of Molecular Genetics in the
National Institute of Neuroscience. In
1998, he was appointed professor in
the Institute of Development, Aging andCancer at Tohoku University andremained there until taking his current
position as group director at the RIKEN
CDB.

ment of cell diversity, as the nervous system features
more cell types than any other organ system, and thefruit fly is highly amenable to genetic manipulation.
With the advent of techniques for molecular analysis,
neurodevelopmental studies have shown that neuralcells’ fates are determined at a very early stage in thedevelopment of the organism. In previous research,
Matsuzaki and others demonstrated that the fates ofdaughter neural cells are in large part determined byunequal distribution of fate-determining factors within
the mother cell during the process of cell division.
Two factors in particular, known as Prospero and
Numb, have been shown to localize to the basal sideof the neuroblast (the neural progenitor cell) prior toits division. This polar distribution results in two
daughter cells exhibiting distinct gene expression
patterns – a new neuroblast, and a smaller-sizedganglion mother cell (GMC) – in which Prospero andNumb are segregated in the GMC. In this process ofasymmetric division, the neuroblast remains undifferentiated and retains its multipotency, while the GMC
becomes committed to generating neurons and glial
cells.

Intracellular asymmetry

Staff
Group Director

Fumio Matsuzaki
Research Scientist

Noyuki Fuse
Yasushi Izumi
Ayano Kawaguchi
Go Shioi
Visiting Scientist

Takako Isshiki
Technical Staff

Kanako Hisata
Nao Ohta
Mai Saito
Taeko Suetsugu
Student Trainee

Maki Maeda
AssistantChika Lee

16 RIKEN CDB 2002 Annual Report

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

Further studies in the Matsuzaki and other labs
showed that Prospero and Numb are tethered to thebasal cortex of the neuroblast by the molecules
Miranda and PON (for Partner Of Numb), respectively, and subsequently released into the newlyformed GMC. The molecular mechanisms involved inasymmetric distribution during cell polarization havefurther been shown to have homologous counterpartsin vertebrates, providing strong evidence for theevolutionary conservation of intrinsic signaling in theprocess of asymmetric cell division. The Matsuzaki
lab is now confronting questions regarding the roles

Neural cell fate determination in
Drosophila
Fumio Matsuzaki has dedicated his research to explicating the role of asymmetric division in determining
cell identity, using the Drosophila melanogasternervous system as a model. This system provides anattractive research platform for studying the developIn the developing mouse spinal cord, Prospero (green) is transiently expressed in neurons immediately after their birth frommitotic neural progenitors (red).

of both extrinsic and intrinsic signals, and the mechanism by which cells of different sizes are generated in
the process of mitotic division.

Asymmetric cell sizes
This last question prompted investigations that led tothe identification of a new regulatory function in a
family of proteins, known as G proteins, in Drosophila
neuroblast cell division, a process in which the resultant neuroblast is much larger than the GMC. Duringmitotic cell division, chromosomes replicate within themother cell and attach to spindles that draw them in
opposite directions to ensure that a full complementof chromosomes is available to each daughter cell.
The chromatids are drawn to the spindle poles
through the action of the cytoskeleton. In work
carried out at the CDB, Matsuzaki’s lab has found

In Drosophila, dividing neuroblasts localize the Miranda(green) / Prospero complex
to be segregated into thedaughter GMC.

Cells lacking the Gβ protein
in question forma symmetrical mitotic spindle andproduce daughter cells of
equal size with thedeterminants being normallysegregated.

Publications
that Gβ protein signals are distributed unequally inneuroblasts and seem to restrict the development of
microtubules, resulting in a shortening of the mitotic
spindle on one side of the cell. These unequal spindle lengths cause the neuroblast to divide at a cleavage site that is off-center, and the sizes of the daughter cells reflect this imbalance, with the daughter
neuroblast being more than twice the size of the
GMC. Cells lacking the Gβ protein in question form asymmetrical mitotic spindle and produce daughter
cells of equal size with the determinants being
normally segregated. Normal gene expression andmitotic activity are gradually altered in neuroblasts
undergoing such divisions, suggesting thatasymmetrically-sized division is a mechanism that
ensures the ‘stemness’ of neuroblasts by minimizing
the reduction of cell volume during consecutive divisions. This is the first demonstration of a molecularmechanism responsible for asymmetric mitotic spindle formation, and provides a valuable platform forfurther studies in fields ranging from microtubule
dynamics to animal morphogenesis.

Hayashi S, Ito K, Sado Y, Taniguchi
M, Akimoto A, Takeuchi H, Aigaki T,
Matsuzaki F, Nakagoshi H, Tanimura
T, Ueda R, Uemura T, Yoshihara M,
Goto S. GETDB, a database compiling expression patterns and molecular locations of a collection of Gal4
enhancer traps. Genesis 34:58-61(2002).
Nakagoshi H, Shirai T, NabeshimaY, and Matsuzaki F. Refinement of
wingless expression by a Winglessand Notch-Responsive Homeodomain protein, Defective Proventriculus. Dev Biol 249:44-56 (2002).
Ohshiro T, Yagami T, Zhang C, andMatsuzaki F. Role of cortical tumor
suppressor proteins in asymmetric
division of Drosophila neuroblast.
Nature 408:593-6 (2000).
Matsuzaki F. Asymmetric division of
Drosophila neural stem cells:a basis
for neural diversity. Curr Opin Neurobiol 10:38-44 (2000).

RIKEN CDB 2002 Annual Report17Regenerative
Medicine

Development

Core Program

Laboratory for Organogenesis and Neurogenesis

Neurogenesis
Yoshiki Sasai
M. D. , Ph. D.

Yoshiki Sasai received his M. D. fromthe Kyoto University School of Medicine
in 1986, subsequently performing internships in general practice and emergency medicine. He completed the Ph. D.
course at the same institution in 1992,
for work on neural 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 wasappointed associate professor at the
Kyoto University School of Medicine.
He assumed a professorship at the Kyoto University Institute for Frontier Medical Sciences in 1998, and was appointed group director at the CDB in 2000.
Dr Sasai serves on the editorial boardsof Neuron, Development, Genesis andextent of the signaling networks involved in setting up
the dorsal-ventral axis and determining neural fate in
the ectoderm. Insights gained from studies in Xenopus are further tested in other organisms, such aszebrafish, chick and mouse, allowing for comparisonsacross species and evidence-based speculation into
the evolutionary conservation of these signalingmechanisms. The group is also actively developing
effective methods of inducing neuralization in mammals, work which has potential for application in thetreatment of nerurodegenerative disorders, such as
Parkinson’s disease.

Developmental Dynamics.

The Sasai group’s work has
potential for application in
the treatment of nerurodegenerative
disorders,such as Parkinson’s disease

Tiarin, a novel dorsalization factor
Staff
Group Director

Yoshiki Sasai
Research Scientist

Kenji Mizuseki
Noriaki Sasai
Makoto Ikeya
Mami Takasaki
Junior Research Associate

Kiichi Watanabe
Technical Staff

Ayaka Nishiyama
Yoko Nakazawa
Tomoko Katayama
Masako Kawada
Tomoko Haraguchi
Michiru Matumura
Assistant

Ukiko Miyake
Ayumi Tanaka
Yuri Arizono

Induction of the dorsal nervous
systemThe vertebrate body plan can be construed as the distribution of three germ layers relative to three bodyaxes. These interacting sets of patterns are established in early embryogenesis, and serve to determine the position and specification of cells as they differentiate and aggregate into tissues and organs. The
specification of the dorsal-ventral (or back-belly) axis
is significant in neural development: the central
nervous system forms on the dorsal side of the bodyin all vertebrate species. This process is dictated by
the effects of a number of signaling factors that diffuse from organizing centers and direct dorsal ecto-derm to maintain a neural fate. These molecules,
which include Noggin, Chordin, Follistatin and theirhomologs, participate in elaborate signaling networks
in which factors collaborate and compete to initiate
the embryonic nervous system.
Beginning with the identification of the neural inducing factor Chordin in the early 1990s, Yoshiki Sasai’s
research has focused on explicating the molecularsignaling mechanisms that allow the early embryo toorganize itself into a complex, mature organism.
Using the African clawed frog, Xenopus laevis, as amodel in molecular embryological studies, Sasai andhis group are engaged in clarifying the structure andDopaminergic neurons derived fromprimate ES cells by
the SDIA method.

18 RIKEN CDB 2002 Annual Report

In previous work, Sasai identified a number of neuraldifferentiation factors that operate by blocking theeffects of BMP4 (BMP for Bone Morphogenetic Protein), a molecule which instructs undifferentiated cells
in the ectoderm to take up an epidermal fate. The conclusion of these studies was that anti-BMP factors,
which include Chordin and its immediate and indirect
downstream targets, actually serve to protect the ‘default’ neural status of ectodermal cells. Sasai has
recently added a new member to the growing familyof central nervous system dorsalizing factors with theidentification of Tiarin (so named because its expression pattern in the Xenopus neurula resembles a tiara
worn on the head).
With the goal of isolating genes that encode factors
involved in the patterning of the anterior central
nervous system, the Sasai group performed a cDNA
library screen, yielding nine candidate genes, including Tiarin, which exhibited neural-specific expression
patterns. Subsequent profiling of the Tiarin gene,which has homologs in chick and mouse, revealed
that it is expressed in the non-neural ectoderm flanking the anterior neural plate, a developmental region
which gives rise to the vertebrate forebrain. Misexpression of Tiarin induces dorsal neural markers andsuppresses ventral markers, indicating its importancein establishing dorsal and neural identity. Although
the molecular mechanisms by which this is achieved
remain obscure, it is known that Tiarin operates independently of other major signaling networks known to
influence body patterning, making it an important new
factor in the patterning of the neural tube.

Inducing neuraldifferentiation in
mammals
Over the past decade, theunderstanding of earlyneural differentiation in
Xenopus has advanced
rapidly, but that knowledge is not immediatelytransferable to the study of neural induction in otherorganisms. While the effects of anti-BMP signals are
sufficient to induce neuralization in the frog, additional signals seem to be necessary in the mouse. Until
quite recently, one of the main obstacles to thedetailed study of mammalian neural differentiation invitro has been the lack of an experimental systemthat offers the relative advantages of the animal cap
assay used in Xenopus. The animal cap in the frog
blastula is capable of producing a variety of tissues in
response to appropriate signals, making it a valuable
tool in the embryologist’s experimental kit. Mouseembryonic stem (ES) cells promise similar differentiative potential, but difficulties in their guided induction
have meant they have not seen widespread use instudies of early patterning.
The Sasai lab has developed a technique for inducing the differentiation of neural cells by culturing
mouse ES cells on plates of connective cells, called
stromal cells. These cells have a strong neuralizing
effect, termed SDIA (for Stromal cell-Derived Inducing Activity), and induce ES cells to generate dopaminergic neurons and their precursors at high rates of
efficiency. Such controlled neural induction represents an important step toward the development ofan experimental platform amenable to the study ofearly neural differentiation in mammals. And, in work
published in early 2002, Sasai and colleagues further
demonstrated that the SDIA effect is preserved in theneural induction of primate ES cells as well, withdopamine-producing cells being generated at a frequency of about 35%. This method provides an immediate unlimited source for primate cells useful inexperimental studies of neurodegenerative disease,and opens up avenues for developing stem cellbased therapies for such diseases in the future.

Expression of Tiarin (purple) in
Xenopus neurula; Neural plate isstained with Sox2 cRNA probe(light blue).

Publications
Tsuda H, Sasai N, Matsuo-Takasaki
M, Sakuragi M, Murakami Y, andSasai Y. Dorsalization of the Neural
Tube by Xenopus Tiarin, a Novel Patterning factor Secreted by the Flanking Non-Neural Head Ectoderm. Neuron 33:515-28 (2002).
Kawasaki H, Suemori H, Mizuseki
Ke, Watanabe K, Urano F, Ichinose
H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S, Nakatsuji N, andSasai Y. Generation of TH+ Dopaminergic Neurons and Pax6+ Pigment
Epithelia from Primate ES cells by
SDIA. Proc Natl Acad Sci USA
99:1580-5 (2002).
Sasai N, Mizuseki K, and Sasai Y.
Requirement of FoxD3-class Signaling for Neural Crest Determination in
Xenopus. Development 128:2525-36(2001).

RIKEN CDB 2002 Annual Report19Regenerative
Medicine

Development

Core Program

Laboratory for Evolutionary Regeneration Biology

Totipotent Stem Cells

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

Anti-synaptotagmin antibody staining
showing axon bundles

Whole-mount in situ RNA staining with
DjPC2 probe, showing neural cell bodies

ency, Agata’s research group concentrated on cells
containing ‘chromatoid bodies’, structures within thecell marked by the presence of the gene DjvlgA (Dugesia japonica Vasa-Like Gene A, D. japonica being
the name of one species of planaria). Chromatoid

Planarians, flatworms
only a few millimeters in length,
are known for their remarkable
regenerative abilities

knocked down by RNA interference resulted in animals that developed brain cells throughout their bodies. This gene seems to function by binding a second
protein, FGF, to cells in the head region, causing
them to adopt a brain neuron fate. Previous to this discovery it had been thought that the planarian brain
produced some factor that inhibited brain growth inother parts of the body, but the ndk findings strongly
indicate that the reverse is true; cells in the head (andthose migrating to the wound site in decapitated animals) recruit a brain-inducing factor.

Publications
Agata K, Tanaka T, Kobayashi C,
Kato K, Saito Y. Intercalary Regeneration in planarians. Dev Dyn
226(2):308-16 (2003).
Asami M, Nakatsuka T, Hayashi T,
Kou K, Kagawa H, and Agata K.
Cultivation and characterization ofplanarian neuronal cells isolated byfluorescence activated cell sorting(FACS). Zool Sci 19:1257-65(2002).

bodies are morphologically and compositionally similar to germ plasm, which is found in totipotent germ
cells in Drosophila and C. elegans and is involved in
germline specification in those animals.

Evolutionary regeneration
The process by which animals replace cells that havebeen lost due to aging, injury or disease is known asregeneration. All animals have some ability to regenerate, but during the course of evolution, the degreeof that capacity has been distributed widely in different species. While some animals, such as humans,
are quite limited in their regenerative capabilities, others demonstrate much greater potential to replenish
lost cells. Research at Kiyokazu Agata’s lab is
focused at the intersection of two fields of study: themechanisms of regeneration itself, and the evolutionary processes by which organisms have developed
such diverse regenerative approaches. Stem cells
are centrally important to both of these fields, andAgata’s work seeks to identify molecules that maintain pluripotency in and present positional cues tothese cells, as well to clarify their role in the evolutionof complex cell systems.

Staff
Group Director

Kiyokazu Agata
Research Scientist

Noriko Funayama
Yoshihiko Umesono
Chiyoko Kobayashi
Nobuyasu Maki
Maki Kashikawa (Yoshida)
Katuaki Takechi
Shuichi Shigeno

Maintaining totipotency

Visiting Scientist

Planarians, tiny flatworms only a few millimeters inlength, are known for their remarkable regenerative
abilities. A single planarian can be cut into dozens,even hundreds, of pieces, and each segment will
regenerate into a complete individual organism. Thisis due to the presence throughout the planarian bodyof totipotent stem cells called neoblasts, which, given
the proper signals, are capable of differentiating into
any cell type in the animal’s body. To study themechanism by which neoblasts maintain their totipot-

Norito Shibata
Technical Staff

Midori Nakayama
Tetsutaro Hayashi
Tomomi Takamatsu
Yumi Saito
Mikako Dohi
Hiroshi Wake
Student Trainee

Maki Asami
Takeshi Inoue
Keiji Okamoto
Takaaki Karasawa
Kouji Matsuzaki
Kazuya Ishizawa

By comparing the compositions of chromatoid bodies
in undifferentiated neoblasts and cells committed to,for example, neural or muscle cell fates, Agata’sgroup discovered that the content of chromatoid bodies is heterogeneous, and dependent on the cell’s
designated fate. This finding runs counter to the classical view that only uncommitted, totipotent cells arecapable of division in planaria, and suggests that certain stem cells migrate through the planarian body to
the regenerating wound site, called the ‘blastema.’
These ‘committed’ stem cells exist in a state intermediate between the totipotency of uncommitted stem
cells, and terminally committed cells that have lostthe ability to divide. The group is now investigating
the factors responsible for maintaining the ‘stemness’of these cells prior to their arrival at the blastema.

Stem cell regulation
Although totipotent stem cells are distributed throughout the planarian body, these cells are able to generate specialized cell types appropriate to the site of
injury when the animal is cut. This suggests that information regarding the position of the wound is conveyed to the stem cell, allowing it to make the necessary changes in gene expression to differentiate into
the cell type that needs to be regenerated. This year,
the Agata group announced the discovery of one
gene involved in such positional cueing. The gene,
named nou-darake (ndk; Japanese for ‘brains everywhere’), ensures that brain neurons only develop in
the head region; experiments in which the gene was

Evolutionary role of stem cells
In addition to the valuable insights they provide on
the mechanisms of regeneration, stem cells can also
shed light on the function of developmental processes in evolution. Indeed, in Agata’s words, “Stem cells
are key to understanding evolution.” This is due totheir unique ability to give rise to cells of different
character than the antecedent cell. On the time scaleof a single organism’s life span, this leads to thedevelopment of complex cell systems within theorganism. On the evolutionary time scale, however, it
may be one of the primary factors leading to thedivergence of species. For example, investigations
into how planarian stem cells differentiate into the animal’s simple eye could provide insights into how
more complex eyes developed in more highly evolved
animals. Agata’s group seeks
to trace diversification withinfamilies of conserved genesthat are known to be linked tostem cell identity and function, in the hopes of clarifying
the evolutionary contributionof stem cells to the development of more and more complex cellular systems.

F Cebria, Kobayashi C, Nakazawa
M, Mineta K, Ikeo K, Gojobori T, Ito
M, Taira M, A Sánchez Alvarado,and Agata K. FGFR-related genenou-darake restricts brain tissues tothe head region of planarians.
Nature 419:620-4 (2002).
Ogawa K, Ishihara S, Mineta K,
Nakazawa M, Ikeo K, Gojobori T,
Watanabe K, and Agata K. Inductionof a noggin-like gene by ectopic D-Vinteraction during planarian regeneration. Dev Biol 250:59-70 (2002).

Brain expansion in ndk knockdown planarian

Regeneration in planarians

20 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report21

Development

Regeneration

Regenerative
Medicine

Creative Research
Promoting Program
The Creative Research Promoting Programprovides solid support to encourage relatively
young researchers to carry out innovative andindependent research plans. The teams areallowed a great deal of flexibility in regard toprojects, budget, and lab size. The program also
places great emphasis on cooperation and international participation. It is hoped that thisunique system will help to cultivate a new generation of leading researchers by fostering thecreativity and originality of investigators in abottom-up fashion.

Undifferentiated mouse ES cells
Photo: Yayoi Toyooka

Creative Research Promoting Program

Development

Laboratory for Neural Network Development

Neural Networks
Chihiro Hama
Ph. D.

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

mutant strains in previous screening studies, Hamaand colleagues identified the gene still life (sif), which
participates in the maintenance of motor function in
Drosophila. This gene, which codes for a GEF(Guanine-nucleotide Exchange Factor) that activates
Rho family GTPases, has been shown to be involvedin the processes of synaptic formation. Later, Trio,
identified as a second GEF expressed in the nervous
system, was revealed to be a pivotal factor for the regulation of axon extension. The Hama lab now plans
to use RNAi gene silencing to perform functional
analyses of the approximately 20 known GEF-family
genes with the goal of identifying genes and molecular mechanisms contributing to neural development.

Hama hopes to take advantageof the Drosophila olfactory
system as an experimental model
to shed light on the genetic
bases of olfactory axon targeting

Olfactory network guidance
Neural networks
The formation of neural networks involves the orchestration of complex processes from neuronal migration, to axon growth to synaptic formation. In thehuman brain, which comprises on the order of 100 billion neurons, the mechanisms by which neuronalcells are able to identify and form synapses withappropriate partners, and the ways that circuits
formed from interconnecting neurons are able to demonstrate adaptive plasticity are questions of staggering intricacy and depth. Chihiro Hama seeks toaddress these complex problems by investigations
into the neural network of the Drosophila fly, a comparatively simply structured system which nonetheless demonstrates the capacity for both instinctive
behaviors and learning. While there are pronounced
differences in degrees of network sophistication andcell type diversity between Drosophila and more highly evolved organisms, they nonetheless share a large
number of homologous genes important in neuraldevelopment. Using a research strategy of largescale mutant screening in the fly, the Hama researchteam seeks to open new windows into the genetic
bases of neural circuitry.

Staff
Team Leader

Chihiro Hama
Research Scientist

GEF family genes

Minako Orihara
Keita Endo
Kazunaga Takizawa
Hiroko Saito

Screening for mutant phenotypes is an exacting process, involving methodical surveillance for anatomicalor behavioral aberrations and requiring a thoroughfamiliarity with the ‘normal’ characteristics of theexperimental model. On observing reduced activity inTechnical Staff

Yuka Yoda
Kyoko Ishikawa
Assistant

Kanako Moriwaki
SIF (green) in the periactive zones of neuromuscularsynapses

24 RIKEN CDB 2002 Annual Report

There are approximately one thousand different typesof odorant receptors in the mammalian olfactory system, but it is believed that any given olfactory neuron
expresses only a single receptor type. By mechanisms that remain largely unknown, the axons of olfactory neurons expressing a receptor of the same typeconverge into one of a thousand glomeruli, which are
discrete structures composed of the axon terminals
and the dendrites of the target neurons. Using amutant screening approach incorporating gene knockout and GFP cell visualization techniques, Hama
hopes to take advantage of the simpler Drosophila
olfactory system as an experimental model to shedlight on the genetic bases of olfactory axon targeting.

genes encoding proteins
known to be involved in theregulation of vesicular traffic
in budding yeast. Hama
hopes to elucidate the rolesof these proteins in neurite
extension, branching andguidance, and synapse formation as well, by conducting
conditional RNAi studies
using a DNA-based transgenic approach.

Development of brain structure
A fourth prospective direction for research in theHama team lies in the functional analysis of a gene
identified in a large-scale mutant screen as involvedin the structural development of the brain. Aberrations in the brain structures can be caused by defects
of many developmental events including neuronal proliferation, axon guidance and synapse formation.
Mutations in the gene in question exhibit smaller thanordinary axon bundles and reduced numbers of cells
in the brain tissue involved in olfactory learning andmemory, which suggests that the gene regulates neuronal proliferation. The brain contains a variety of neuronal types, which can be generated serially from single neuronal precursors. Further analyses of themutant may provide a hint about how switching of theneuronal types is regulated during brain development.

Trio (purple) in the mushroom bodyof Drosophila brain

Publications
Saito M, Awasaki T, and Hama C.
Genetic analyses of essential genes
in cytological region 61D1-2 to 61F12 of Drosophila melanogaster. Mol
Genet Genomics 268:446-54 (2002).

By adopting a broad-based, multi-focus approach,
Hama seeks to illuminate the mechanisms by whichcells that have followed the developmental route leading to neuronal differentiation are further able toorganize into neural circuits, and ultimately to participate in the function of the brain.

Vesicle transport in neural development
All cells contain cargo-bearing compartments known
as vesicles. These vesicles function to deliver themacromolecules that serve the cells as membranecomponents or secreted signals, and the metabolite
products of the cellular digestive process to the restof the cell, where they are stored or used to fuel activity. Microtubules serve as ‘railways’ for vesicle transport; their ends anchor to sites on the cell cortex
where the vesicles fuse. It is likely that the extrusionof cell membrane at given sites, such as takes placein axon extension, requires vesicles to be transported
along microtubule in a specific orientation. To clarify
how vesicle transport is involved in neural development, Hama plans to analyze Drosophila orthologs ofSpecific axonal targeting in the Drosophila
olfactory system
RIKEN CDB 2002 Annual Report25

Development

Creative Research Promoting Program

Laboratory for Vertebrate Axis Formation

Axis Formation
Masahiko Hibi
M. D. , Ph. D.
Masahiko Hibi received his M. D. from
Hiroshima University, School of Medicine in 1988, and his Ph. D. from the
Osaka University Institute for Molecular
and Cellular Biology in 1992. From1992 to 1995, he worked as a postdoctoral fellow in Michael Karin’s lab in the
University of California, San Diego
Department of Pharmacology, thenreturned to Japan to take an assistantprofessorship 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 position as team leader at the
RIKEN CDB.

its amenability to genetic studies, its rapid
embryological development, and extensive
data regarding mutant phenotypes, makes
the zebrafish an attractive platform forinvestigations into the genetic and molecular bases of organizer formation. Hibi focuses on the dynamic processes that takeplace in the first 24 hours of zebrafish development, a period in which the embryo
grows and differentiates from a single cell
into a recognizably vertebrate body organized along distinct axes.

Hibi focuses on the dynamic
processes that take placein the first 24 hours of zebrafish
development

Establishing the dorsal organizer
Staff
Team Leader

Masahiko Hibi
Research Scientist

Osamu Muraoka
Takashi Shimizu
Visiting Researcher

Young-Ki Bae
Technical Staff

Chiho Fukae
Yukiko Kuga
Student Trainee

Taijiro Yabe
Tsutomu Hirata
Hideaki Nojima
Assistant

Shoko Iwaki

Body axis determination in zebrafish
In 1924, when Spemann and Mangold discovered theaxis-organizing properties of a small section of thenewt embryo located at the dorsal lip, it was the first
demonstration that one group of cells can determine
the developmental fates of neighboring uncommitted
cells. This group of fate-determining cells, whichcame to be known as the Spemann organizer, is
responsible for establishing the dorsal-ventral andanterior-posterior body axes in amphibians, and corresponding embryonic signaling centers have beenshown to play similar roles in fish, birds and mammals. The discovery of the Spemann organizer raisedquestions that have challenged and intrigued developmental biologists ever since. The problems of how
such organizing centers are formed, and how theyfunction to steer the development of the body axes
remain at the forefront of modern embryologicalresearch.
The research in Masahiko Hibi’s lab seeks to addressthese problems, using the zebrafish as a model system for understanding dorsal organizer origins andfunction. A region of the zebrafish embryo called the‘embryonic shield’ closely corresponds to the amphibian organizer. The high level of evolutionary conservation seen in the vertebrate organizer, combined with

Mutations in ogon lead to ventralization of
the embryo.

26 RIKEN CDB 2002 Annual Report

There is extensive evidence that an as-yet unknown
dorsal determinant molecule in the vegetal pole of thevery early zebrafish embryo is transported via theintracellular microtubule network to a dorsally locatedsignaling center known as the Nieuwkoop center,
which in turn produces signals that induce the dorsalorganizer via interactions with the Wnt signaling pathway. Using both gain- and loss-of-function studies,
Hibi has discovered a number of genes that participate in the induction of signaling centers and bodyaxes, including dharma/bozozok and ved. His recent
work has focused on determining the exact roles
played by these genes in the signaling networks
responsible for axis formation.
The dharma/bozozok (dha/boz) gene is known toplay a central role in the establishment of the dorsalorganizer in zebrafish, and is capable of inducing theorganizer in a non-cell-autonomous manner. Hibi
found that dha/boz is activated prior to gastrulation aspart of a complex network of axis-determining factorsthat includes both the Wnt and BMP signaling pathways. However, several molecular steps in this proposed network remain unidentified, and the Hibi teamis working to characterize the immediate and secondary downstream targets of the dha/boz gene product.
One aspect that is becoming clear is that dha/boz
works in concert with a group of ventrally-expressed
proteins that inhibit other dorsalizing factors further
downstream. The interplay between these dorsalizing
and ventralizing factors is central to the formation of
the anterior neuroectoderm, the developmental
region that gives rise to the forebrain.

Morpholino (MO)-mediated loss of Pnx, which contains
an Eh1 repressor motif and a homeodomain, leads toreduction in primary neurons expressing elavl3.

Posteriorzebrafishneurogenesis

in

the

The nervous system is known to arise on the dorsalside of the vertebrate gastrula, but this system displays anterior-posterior (A-P) asymmetry as well. The
fore-, mid- and hindbrain regions, and the spinal cord
all form from precursive embryonic structures
arranged along the A-P axis of the gastrula. In the
Nieuwkoop model of neurogenesis, after a populationof ectodermal cells is committed to a neural fate (theneuroectoderm), the population undergoes a patterning process in which the cells are further differentiated to form spatially distinct sub-populations that willdevelop into the various regions of the central
nervous system, and, ultimately give rise to the primary and secondary neurons that comprise thenervous system proper. The Hibi research team has
identified one gene, pnx, that is expressed in the posterior neuroectoderm, and regulates the formation ofposterior neurons, such as those of the hindbrain andspinal cord. pnx encodes a homeodomain proteinthat mediates posteriorizing signals as part of a larger signaling network that functions in the patterningand later stages of neurogenesis.

Publications
Bae Y-K, Shimizu T, Yabe T, Kim
C-H, Hirata T, Nojima H, Muraoka O,
Hirano T, and Hibi M. A homeobox
gene, pnx , is involved in the formation of posterior neurons in zebrafish.
Development (in press 2003).
Shimizu T, Yamanaka Y, Nojima H,
Yabe T, Hibi M, and Hirano T. A novel repressor-type homeobox gene,ved, is involved in dharma/bozozokmediated dorsal organizer formationin zebrafish. Mech Dev 118:125-38(2002).
Ryu S-L, Fujii R, Yamanaka Y, Shimizu T, Yabe T, Hirata T, Hibi M, andHirano T. Regulation of dharma/bozozok by the Wnt pathway. Dev Biol
231:397-409 (2001).
Hashimoto H, Yabe T, Hirata T, Shimizu T, Bae Y-K, Yamanaka Y, Hirano T, and Hibi M. Expression of thezinc finger gene fez-like in zebrafish
forebrain. Mech Dev 97:191-5(2000).

The Hibi lab is also interested in the analysis ofgenes involved in ventralized zebrafish phenotypes.
While there are many known dorsalized mutants inzebrafish, ventralized phenotypes are comparatively
few. Hibi and colleagues are conducting analyses of
one ventralizing gene, ogon, in the hopes of identifying its function in the dorsal-ventral axis signaling network. Hibi plans to continue to explore the formationand function of signaling centers in the zebrafish
embryo, continuing on the genetic and molecular level the revolutionary work started by Spemann and
Mangold nearly eighty years ago.

RIKEN CDB 2002 Annual Report27

Development

Regeneration

Creative Research Promoting Program

Laboratory for Positional Information

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

the skin corresponded to the overlying skin pattern.
The existence of a spotted zebrafish leopard phenotype indicates that genetic factors are at work in determining the spatial distributions of the pigmented bodies; the reaction-diffusion model predicts that one ormore activating and inhibiting factors interact toinfluence the formation of these pattern variants. His
team is now engaged in a collaborative mutant
screening search to identify the gene or genes
responsible for maintaining regular stripes in thesefish.

Stripe directionality

Staff
Team Leader

Shigeru Kondo
Research Scientist

Masashi Hirata
Jun Ozaki
Motoomi Yamaguchi
Technical Staff

Kana Bando
Miwako Okamura
Part-time Staff

Motoko Yoshizawa
Student Trainee

Takeshi Nakagawa
AssistantSumire Hino

Reaction-diffusion wave patterning
The natural world is filled with patterns that can be
described by mathematical functions, such as the
Fibonacci spiral of a nautilus shell, or the fractal
arrangement of branches on some trees. In 1952, theBritish mathematician Alan Turing proposed a simple
mathematical equation capable of generating a wide
range of commonly observed patterns, such as
stripes, spots and networks. This model, known as
the reaction-diffusion model, demonstrates that theinteraction between a local activator and a long-range
inhibitor can give rise to various periodic structures in
response to differences in their individual diffusion
rates. When the activator’s diffusion rate exceedsthat of the inhibitor, a ‘moving wave’ is formed, whereas a higher rate for the inhibitor results in a ‘standing wave’. Both forms of wave can create periodic
geometrical structures which exhibit the property ofmaintaining the size of the interval between structures as the overall pattern grows.

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

Zebrafish stripes and spots
As its name implies, the zebrafish is normally astriped animal. Similar stripes can be generatedusing the reaction-diffusion model, which inspired
Kondo to seek the underlying biological mechanisms
capable of fulfilling the model’s mathematical conditions. Histological analysis of the zebrafish’s pigmented regions revealed that the relative distributions of
three types of pigmented bodies – melanophores,
xanthophores and iridophores – near the surface ofAngelfish present an unusual problem for thereaction-diffusion model, in that related species display patterns with similar striping, but in which thestripes are arranged in different directions. Thesefish, which are also unusual for the ability of females
in male-less populations to change sex, develop
stripes only in the process of acquiring male gender.
In one species, the stripes are vertically arrayed,while in another closely related species, they formhorizontally. While the stripes themselves conform to
the predictions of the Turing equation, there is nothing inherent to the reaction diffusion model that would
account for such directionality. While several
mathematical explanations, such as boundary conditions or spatial gradients in reaction rate could conceivably produce differences in stripe direction, Kondo concluded that the developmental pattern suggested an inequality in distribution (anisotropy) was atwork in the reaction-diffusion model. Comparisons ofskin patterning in scaled and scaleless fish indicatethat scale characteristics can cause marked changes
in pattern formation, and computational modeling confirmed that even very small differences in a variable
such as scale structure or arrangement could produce the changes in stripe direction observed inthese fish. This work underscores the important pointthat minute perturbations in diffusion are capable ofgenerating widespread and visible morphologicaleffects.

Minute perturbations in
diffusion are capable of generatingwidespread andvisible morphological effects

Regularly periodic formation of additional somites in extended chickpresomitic mesoderm

Kondo team sought to demonstrate patterning consistent with the Turing model. In its embryonic development, the chick develops a regularly spaced series ofbodies adjacent to the neural tube, called somites,
which eventually give rise to the vertebrae, ribs andother structures. As regular periodicity is one of thefeatures of Turing wave patterns, the Kondo teamsought to determine whether somitogenesis could be
explained by the reaction-diffusion model. Working
with gastrulating embryos, the team stretched theregion that somites arise from beyond its normal
length, and found that somites in the elongatedregion divided to maintain their spacing, just as themodel predicts. This experimental confirmation indicates that a balance between activating and inhibiting
factors provides the basis for the pattern periodicity ofsomites, and the team is now working to identify the
specific genes and proteins involved.

Publications
Kondo S. The reaction-diffusion
system:a mechanism for autonomous pattern formation in the
animal skin. Genes Cells 7:535-41(2002).
Shoji H, et.al. Directionality of stripe
formed by anisotropic diffusion. J
Theor Biol 214:549-61 (2002).

FemaleFemale3 weeks

3 weeks

male

male

Periodicity in somitogenesis
The reaction-diffusion model is not only applicable toskin surface patterning, but to the formation of periodically repetitive internal structures as well. Using somitogenesis in the chick embryo as a model system, theGenicanthus melanospilos

Genicanthus watanabei

Pattern alterations during sexual change in Genicanthus

Autonomous formation of 2-D periodic pattern by Turing mechanism

28 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report29Creative Research Promoting Program

Development

Laboratory for Evolutionary Morphology

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

the carapace at other points in evolutionary history,
as the underlying genetic elements are presumed tobe conserved, making carapace formation one of the
trajectories available to the process of morphologicaldevelopment.
The Kuratani lab is undertaking a systematic search
to identify the genes and molecular mechanisms related to carapace development. This search involves differential screening of gene expression in chick andturtle. The chick is used as a basis for comparison,as birds are close evolutionary relatives of turtles,
and share extremely similar genomes. By observing
changes in gene expression in transgenic embryos, it
is hoped that the developmental context that allowed
the rapid emergence of the turtle’s shell will become
clear, and that the case for an evo-devo approach tomorphological divergence will be strengthened.

Evolution of the jaw
Staff
Team Leader

Shigeru Kuratani
Research Scientist

Yoshie Ohya
Yasunori Murakami
Katsuhisa Uchida
Masumi Nakazawa
Rie Kusakabe
Yuichi Narita
Technical Staff

Ryo Usuda
Student Trainee

Shigehiro Kuraku
Hiroshi Nagashima
Yoko Takio
Keiko Yamamoto
Assistant

Chiaki Nakayama

Evo-devo
Evolutionary developmental biology, sometimes
referred to as ‘evo-devo,’ looks at the ways thatchanges in developmental genes and gene expression drive both the evolution of species and differences in morphology. Evolution has frequently been
described as the result of the interplay between mutation and selection, but the advent of molecular developmental biology in the 1980s led to the increasingly
widely accepted view that developmental constraints
have a hand in shaping the directions, or ‘trajectories,’ that evolutionary processes are most likely to follow. This concept is perhaps best illustrated by the
homeobox (Hox) genes, a developmentally important
set of genes that shares a highly conserved domain(the 180-base-pair homeodomain) and plays a central role in regulating the morphological development
of diverse organisms.
Combining experimental and analytic techniquesfrom molecular biology, phylogenetics and comparative morphology, Shigeru Kuratani seeks to deepen
the understanding of the part played by developmental biological mechanisms, such as the Hox genes, inthe evolutionary divergence of species. His approach
involves examining related genes in morphologicallyand phylogenetically distinct animals to uncover theways in which context affects gene expression, andthereby influences body development. By comparing
the molecular bases of evolutionarily novel structures, Kuratani hopes to illustrate the means by whichdevelopmental mechanisms mediate the translationof changes in the genome to changes in morphology.

Kuratani seeks to deepen
the understanding of developmental
biological mechanisms
in the evolution of species

The turtle carapace
Evolution is generally considered to be a gradual process involving the accumulation of subtle changes
over long spans of time. But marked changes occasionally do appear abruptly in the fossil record. Thisis the case for the advent of the turtle’s shell, or ‘carapace,’ which appeared suddenly and with few recognizable precursors (although two now-extinct distantphylogenetic groups also evolved carapaces). Such aphenomenon would be difficult to explain by mutationand selection alone, as it is improbable that the genomic alterations necessary to produce such a dramatically new bodily structure would be achievable by the
introduction of new genes in the short timeframe of
the turtle’s emergence. Evo-devo theory predicts
instead that the genes responsible for carapace development actually belong to a set of genes shared bygroups related to the turtle, such as lizards, dinosaurs
and birds, but which function distinctly in the uniquecontext of the turtle’s molecular-genetic network. This
would also explain the independent appearance ofThe emergence of the jaw was an evolutionarily
important step for gnathostomes (jawed vertebrates),
as it conferred, among other things, the ability to
grasp prey securely in the mouth, which was impossible for agnathan (jawless) species. However, themouths of some agnathans, such as lampreys, feature upper and lower lips, which it was long believed
were the evolutionary forerunners of the two-part,bony jaw. Kuratani tested this hypothesis by observing the expression patterns of the lamprey homologs
of four genes known to be responsible for oral development in chicks. He found that, in the lamprey
embryo, these genes, and the molecular cascadesthat their protein products participate in, do
indeed serve to regulate the development of
the mouth. However,interestingly, analysis
showed that the lamprey mouth arises fromdifferent regions of thedeveloping
embryothan it does in thegnathostome
chick.
Thus, the vertebrate
jaw and the lamprey
mouth are morphologically somewhat similarand share certain gene
expression patterns,
but derive from distinct
mesenchymal domains,which suggests that the

The jaw conferred an evolutionary advantage

Publications
molecular mechanisms common to oral developmentin these groups have been mobilized to form mouth
structures at several discrete evolutionary moments,
and not as part of an unbroken continuum. This nicely illustrates the evo-devo principle of exaptation, in
which a structure or mechanism that originally
evolved for one adaptive reason is co-opted as anautonomous adaptation in a different context. In
experiments such as this, Kuratani hopes to bolster
the already strong argument for the central role ofmolecular developmental biology in evolutionary processes.

Shigetani Y, Sugahara F, Kawakami Y,
Murakami Y, Hirano S, and Kuratani S.
Heterotopic shift of epithelialmesenchymal interactions for vertebrate jaw evolution. Science 296:131921 (2002).
Kuratani S, Kuraku S, and Murakami
Y. Lamprey as an Evo-Devo model:lessons from comparative embryology and molecular phylogenetics.
Genesis 34:175-95 (2002).
Kuratani S, Nobusada Y, Horigome
N, and Shigetani Y. Embryology ofthe lamprey and evolution of the vertebrate jaw: insights from molecularand developmental perspectives.
Phil Trans Roy Soc 356:15-32(2001).

Ammocoete larvae of Lampetra japonica

30 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report31Creative Research Promoting Program

Development

Laboratory for Cell Migration

Cell Migration
Kiyoji Nishiwaki
Ph. D.
Kiyoji Nishiwaki received his B. Sc.
from Osaka City University and M. Sc.
from Osaka University where heworked on the molecular biology of theyeast S. cerevisiae. He joined NEC
Corp. in 1986 as a researcher in theFundamental Research Laboratory studying the molecular genetics of C. elegans. He left NEC in 1992 to work as avisiting researcher at Johns Hopkins
University, then returned to the company in 1993 to continue his work on
nematode development. He was awarded a Ph. D. by Osaka City University forwork on the molecular analysis of C. elegans embryogenesis in 1994. He
remained at NEC until receiving an
appointment as team leader at the
RIKEN CDB.

Cell migration and organogenesis
In animal development, cell division, cell migrationand changes in cell shape are coordinated to generate complex systems of tissue and organs. One
aspect of this highly orchestrated process is the
movement of cells in sheets, an activity orchestrated
by mechanisms which remain poorly understood. Kiyoji Nishiwaki’s research team studies gonadogenesis(the formation of reproductive organs) in the nematode C. elegans as a model system to improve theunderstanding of the molecular mechanisms that
underlie cell migration during organogenesis. By
developing a clearer picture of the mechanisms heldin common across species, it is hoped that thisresearch will lead to insights into cell migration andorgan development in humans, and contribute to theunderstanding of human diseases involving disturbances in coordinated cellular movement.

Staff
Team Leader

Kiyoji Nishiwaki
Research Scientist

Norio Suzuki
Katsuyuki Tamai
Special Postdoctoral Researcher

Yukihiko Kubota
Technical Staff

Genetic control of gonad development

Mitsue Sano
Assistant

Rie KurokiDistal tip cell migration. The path of the migrating DTC is shown by the dotted line.

32 RIKEN CDB 2002 Annual Report

C. elegans has been a favorite model for geneticists
and developmental biologists since its introduction by
Sydney Brenner in the 1970s, as the lineage of eachof the 959 cells can be traced precisely, its short
reproductive cycle makes it amenable to breeding for
genetic experiments, and its transparent body allows
for direct observation of individual cells under a lightmicroscope. In the nematode, the gonad develops
through four larval stages (L1-L4) during which time
the gonad-forming cells divide and move through the

body cavity to form symmetrical gonad arms originating in a primordium on the ventral side of the bodyand terminating dorsally. The migratory path of theemerging gonad is determined by a pair of distal tip
cells, one located at each end of the gonad primordium, but the mechanisms by which these cells initiate,steer and end migration at the correct stages in larval
development are largely unknown.
Nishiwaki sees the interactions between the basement membrane of the migrating gonad and that of
the body wall as a key to the process of cell movement, and his team has conducted a large-scale
screen for phenotypes that demonstrate distal tip cell
migratory aberrance. One gene uncovered in thismutant hunt, mig-17 (the mig prefix indicates amutant phenotype with a migratory defect), has beenfound to encode a member of the ADAM (A Disintegrin And Metalloprotease) family of secreted proteins.
Tracking studies using molecules genetically taggedwith green fluorescent protein revealed that the mig17 product, MIG-17, is secreted into the body cavity
by muscle cells in the body wall and binds to the
gonadal basement membrane at the developmental
stage when the distal tip cells make their initial turndorsal-ward. The research team is now pursuing studies of other genes associated with migratory defects,
with a particular focus on phenotypes in which the
migratory mechanism is disturbed, but not abolished.
These mutations allow for the tracking of molecularaction and cell behavior throughout the migratory process, and to identify developmental checkpoints at
which the distal tip cell’s guidance mechanisms may
go awry. Preliminary studies indicate that proteinsassociated with alterations in the glycosylation of
MIG-17 or in its secretion may form a matrix that
directs the localization of MIG-17 to the migrating
gonad.

Development of C. elegans gonad

and restoring normal gonadogenesis, even in thecomplete absence of MIG-17. The biochemical analysis of these candidates is still ongoing, and it ishoped that the results of these efforts will provide better knowledge of the MIG-17 metalloprotease and theidentification of molecules that directly interact with
MIG-17.

Nishiwaki is interested
in clarifying
the network of proteinsthat interact with MIG-17
Nishiwaki’s work to date has revealed that the basement membrane plays a far larger role in organogenesis than that of simple structural support. As his functional genetic studies of C. elegans have shown,basement membranes are integral in the control ofcell migration as well. Judging from the high degreeof homology with higher organisms, such as themouse, these migratory mechanisms seem to be evolutionarily well-conserved. By focusing on the movements of a small group of cells in a tiny worm, Nishiwaki seeks to push back the frontiers ofknowledge of how cells in the bodies of animals across species are able to get towhere they need to go.

Publications
Suzuki N, Buechner M, Nishiwaki K,
Hall DH, Nakanishi H, Takai Y, Hisamoto N, and Matsumoto K. A putative GDP-GTP exchange factor isrequired for development of theexcretory cell in C.elegans. EMBO
Rep 21:530-5 (2001).
Nishiwaki K, Hisamoto N, and Matsumoto K. A metalloprotease distintegrin that controls cell migration in
Caenorhabditis elegans . Science
288:2205-8 (2000).
Nishiwaki K. Mutations affecting
symmetrical migration of distal tip
cells in Caenorhabditis elegans .
Genetics 152:985-97 (1999).

Proteininteractions with MIG-17
To gain deeper insight into
the role of cell migration innematode gonadogenesis,
Nishiwaki is interested in clarifying the network of proteinsthat interact with MIG-17.
The team searches for mutations in mapping experiments
designed to locate candidate
genes. These mutations
have been found to be capable of rescuing the meandering distal tip cell phenotype

MIG-17 is produced in and secreted from muscle cells and localized on
the gonad surface after the first turn of the DTC

RIKEN CDB 2002 Annual Report33

Development

Regeneration

Regenerative
Medicine

Creative Research
Promoting Program

Laboratory for Pluripotent Cell Studies

Pluripotency
Hitoshi Niwa
M. D. , Ph. D.
Hitoshi Niwa received his M. D. fromNara Medical University in 1989, and
his Ph. D. in medical physiology in 1993
from the Kumamoto University Graduate School of Medicine for his work ingene trap methods. From 1993 to 1994,
he worked 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 Departmentof Nutrition and Physiological Chemistryat the Osaka University Graduate
School of Medicine, where he remained
until taking his current position at the
RIKEN CDB.

Staff
Team Leader

Hitoshi Niwa
Research Scientist

Shinji Masui
Kazuya Ogawa
Satoshi Ohtsuka
Yayoi Toyooka
Itsuro Sugimura
Technical Staff

Kadue Takahashi
Rika Yagi
Hiroyuki Kitajima
Student Trainee

Yoko Sekita
Ken-ichi Tominaga
Yuko Iwamatsu
Sayako Matsui
AssistantMiho Ohtsuka

Self-renewal and pluripotency
With their ability to self-renew indefinitely and to differentiate into cells of all three germ layer types (a differentiative capacity called pluripotency), embryonicstem (ES) cells represent one of the great prospects
for regenerative medical applications, as well as an
attractive platform for the study of a diverse spectrumof developmental processes. However, the mechanisms by which ES cells are able to maintain thesefunctions are incompletely known, and a better understanding of the ‘stemness’ of these cells will be necessary in order to take optimal advantage of theirremarkable properties. Two of the biggest challenges
facing stem cell research are determining the factorsthat allow ES cells to generate limitlessly selfrenewable progeny, and identifying molecules that
direct the dividing ES cell to produce daughter cellswith specific fates. Beginning with his work in identifying Oct-3/4 as a transcription factor important in ES
cell maintenance, Hitoshi Niwa’s research hasaddressed both of these challenges.

A better understanding ofthe ‘stemness’ of pluripotent cells
will be necessary
in order to take optimal advantage of
their remarkable properties

Maintenance of pluripotency
The POU-family transcriptional regulator Oct-3/4 was

34 RIKEN CDB 2002 Annual Report

the first factor found to elicit multiple differentiative
outcomes dependent on its expression level. ES cells
expressing normal levels of Oct-3/4 maintained theirpluripotency, while its overexpression resulted in differentiation into primitive endoderm and mesoderm,
and its inhibition caused the cells to take up a trophectodermal (the layer of extraembryonic cells thatserves as the source of the placenta) fate. This established Oct-3/4 as a primary regulator of pluripotency
in ES cells, but gave rise to new questions regarding
the biochemically active domain in the Oct-3/4 protein. In work published in the spring of 2002, the Niwa
lab showed that the function of Oct-3/4 in ES cell
propagation depends on the combined presence ofspecific POU and transactivation domains. By refining the understanding of the molecular characteristics
involved in the Oct-3/4-based ES cell maintenance inthis way, it may be possible to pinpoint important target genes and thereby elucidate the regulatory pathways at work in the preservation of pluripotency.
One aspect of this network that holds Niwa’s interestis the identification of factors that interact with Oct-3/4
to govern ES cell fate. The tight regulation of Oct-3/4
is required to maintain pluripotency in ES cells, suggesting that a finely tuned feedback mechanism is atwork in keeping Oct-3/4 transcription levels within theappropriate range. The resolution of the elements ofthis hypothetical feedback loops remains the subjectof ongoing research and several candidate components have been identified.

Inducing differentiation
A second field of central importance to the stem cellresearch community is the development of methodsby which undifferentiated ES cells can be prompted
to commit to a specific cell lineage. Studying factors
identified in published work on knockout mice, theNiwa research team analyzed transcription factors
with potential roles in the development of extraembryonic and primitive endoderm lineages. (Primitive
endoderm derivatives include the parietal and visceral endoderm, the extraembryonic regions that giverise to the yolk sac endoderm.) Two factors in particular, Gata-4 and Gata-6, known to be expressed incells from these lineages, were studied to test whether their expression was sufficient to promote the differentiation of ES cells. Overexpression of the factors
resulted in the specific conversion of undifferentiatedES cells into endodermal cells, and it was found thatthe induced expression of either of these GATA factors resulted in the expression of the other as well.
This result was the first successful attempt to induce
differentiation of ES cells by the misexpression oftissue-specific transcription factors, an important step
toward the experimentally and clinically significant
goal of controlling the differentiation of ES cells in culture.

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

Parietal endoderm cells induced by Gata-6 overexpression in ES cells

Publications
Niwa H, Masui S, Chambers I, Smith
AG, and Miyazaki J-I. Phenotypiccomplementation establishes requirements for specific POU domain andgeneric transactivation function of
Oct-3/4 in embryonic stem cells. Mol
Cell Biol 22:1526-36 (2002).
Fujikura F, Yamato E, Yonemura S,
Hosoda K, Masui S, Nakao K, Miyazaki J-I, and Niwa H. Differentiation ofembryonic stem cells is induced by
GATA factors. Genes Dev 16:784-9(2002).
Tomioka M, Nishimoto M, Miyagi S,
Katayanagi T, Fukui N, Niwa H, Muramatsu M, and Okuda A. Identificationof Sox-2 regulatory region which isunder the control of Oct-3/4-Sox-2
complex. Nucleic Acids Res 30:320213 (2002).
Tanaka TT, Kunath T, Kimber WL,
Jaradat SA, Stagg CA, Usuda M,
Yokota T, Niwa H, Rossant J, Ko
MSH. Gene expression profiling ofembryo-derived stem cells reveals
candidate genes associated with pluripotency and lineage specificity.
Genome Res 12:1921-8 (2002).

RIKEN CDB 2002 Annual Report35

Development

Regeneration

Creative Research Promoting Program

Laboratory for Mammalian Epigenetic Studies

Epigenetics

Masaki Okano
Ph. D.
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 from 1994 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 forthe last 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.

once established. This is important for the maintenance of consistent cellular function, but it is equally important for new individuals to have the ability toreset their methylation patterns. To use the corporate
analogy, there needs to be a way for new companies
spun off from a parent to remove old labels and bindings and to establish their own distinct sets of instructions. This process of establishing new instructions iscalled de novo methylation, and it seems to play a
crucial role in the early development of many animals, including humans.

Expression pattern of DNA
methyltransferase Dnmt3b inmouse embryo at 8.5 d.p.c.

This process ofestablishing new epigenetic
instructions is known as
de novo methylation

Epigenetic instructions
One of the great questions remaining in the life sciences concerns how it is possible for the body's
many cell types to express only the limited set ofgenes required for the performance of their individualfunctions when nearly every one of these cells is
genetically identical. While a complete answer
remains to be found, it is already evident that additions need to be made to the classical model of gene
expression to account for a number of 'epigenetic' processes, such as X chromosome inactivation, parental
imprinting, position effect variegation and gene silencing, which do not depend on DNA sequence. The concept of epigenetically determined differential gene
expression can be likened to a corporation with a single comprehensive manual that provides instructions
to every department, but which features a system
whereby locally irrelevant sections of the manual canbe labeled, bound up, and filed away unread. In thisanalogy, DNA methylation can be seen as one of themeans by which unnecessary sets of DNA instructions are labeled and bound. Methylation targets cytosine bases in the genetic code, and genes methylated in this way are generally rendered inactive, or 'silenced.' Masaki Okano's research concentrates on
the mechanisms by which DNA methylation is established and maintained throughout development.

Staff
Team Leader

Masaki Okano
Research Scientist

Shin-ichiro Takebayashi
Masaaki Oda
Technical Staff

Chisa Matsuoka
Akiko Yamagiwa
Student Trainee

Takao Nakayama
Akiko Tsumura

De novo methylation
Methylation lays down developmentally significant
gene expression patterns that are difficult to alter

In previous work, Okano showed that de novo methylation in mice required the expression of a pair ofmethyltransferase genes, Dnmt3a and Dnmt3b, at avery early stage of embryonic development, as wellduring gametogenesis. Prior to this finding, it was
believed that a related gene, Dnmt1, was solely
responsible for the methylation of DNA. Using lacZ
reporter gene studies, Okano showed that these newly identified genes are highly expressed in germline(Dnmt3a), ES (Dnmt3a and Dnmt3b) and progenitor(Dnmt3b) cells, suggesting that they function in theepigenetic reprogramming of DNA methylation states.
Based on these findings, it is now believed thatDnmt1 functions primarily in methylation maintenance, which is necessary to the stable inheritanceof tissue-specific methylation patterns, but that
Dnmt3a and Dnmt3b are the principal determinants ininitiating DNA methylation. Knockout mutations ofDnmt3a and Dnmt3b result in a variety of organic,
neurological and spermatic defects, which indicatesthat the establishment of the DNA methylation stateplays an important part in many aspects of development.

DNA methylation
imprintinginmaternal

In more recent work published in 2002, Okano andcolleagues at the Cardiovascular Research Center at
Massachusetts General Hospital demonstrated a newrole for the Dnmt3 family of genes, which they foundto be essential to the process of maternal imprinting.
In the mouse, the paternal and maternal genomes
both contain small numbers of differentiallyexpressed genes known as imprinted genes. The
expression of such genes is determined by whether
they are inherited from the mother or father, and they

seem to function in a number of developmental processes. It is thought that DNA methylation is the epigenetic mechanism that governs genomic imprinting,and nearly all imprinted genes contain regions thatare methylated differentially in maternal and paternalalleles, although it remains a mystery as to how methyltransferases such as the Dnmt3 family recognize
the sequences of imprinted genes. In a series ofovary transplantation studies, Okano showed that
Dnmt3a and Dnmt3b function in the methylation ofmaternally imprinted genes, while colleagues demonstrated that a third gene, Dnmt3L, also participates inthe same process. Although Dnmt3L shows no direct
methyltransferase activity, it is thought that it may
help to regulate maternal imprinting by interactingwith the known methyltransferases Dnmt3a andDnmt3b, with which it binds and colocalizes.
In the future, the Okano lab will
study how epigenetic reprogramming works at a molecular level.
By identifying the genes
involved in de novo DNA methylation, they hope to discover themeans by which it is determined
what genes will be methylated.
Comparisons of the expression
patterns of such genes acrossgerm layers, or in normal miceagainst clones created fromsomatically derived cells should
lead to new insights in thecontext-specificity of the DNA
methylation process.

Publications
Hata K, Okano M, Lei H, and Li E.
Dnmt3L cooperates with the Dnmt3
family de novo DNA methyltransferases to establish maternal imprints inmice. Development 129(8):1983-93(2002).
Okano M, Bell DW, Haber DA, Li E.
DNA methyltransferases Dnmt3a
and Dnmt3b are essential for denovo methylation and mammaliandevelopment. Cell 99(3):247-57(1999).
Okano M, Shaoping X, Li E. Cloning
and characterization of a family ofnovel mammalian DNA (cytosine-5)
methyltransferases. Nat Genet
19(7):219-20 (1998).

Gross morphology of Dnmt3a(-/-)
Dnmt3b (-/-) mouse embryo at
9.5 d.p.c.

Distribution of methylcytosine in metaphase chromosomes of ES cells

36 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report37Creative Research Promoting Program

Development

Laboratory for Cell Fate Decision

Asymmetric Cell Division
Hitoshi Sawa
Ph. D.
Hitoshi Sawa obtained his baccalaureate, master’s and doctoral degrees
from Kyoto University in the period from1982 to 1991. He worked as a postdoctoral fellow at the California Institute ofTechnology on a Human Frontier Science Program grant during the period
from 1991 to 1994, then at the Massachusetts Institute of Technology from1994 to 1997 under support from theHoward Hughes Medical Institute. He
returned to Japan in 1997 to continue
his work at Osaka University, first as a
postdoc, then as researcher funded by
the Japan Science and Technology Corporation PRESTO program. He took his
current position at the RIKEN CDB in
2001.

Asymmetric cell division and cell
diversity
Our bodies are made up of the myriad and multiform
descendants of a single fertilized egg. The process ofasymmetric cell division, in which a single mother cell
splits to generate daughter cells with discrete characters, is widely accepted as the basis of cellular diversity but due to the difficulties associated with tracking
the lineages produced by any single cell, the mechanisms by which this is achieved remain imperfectly
understood. Utilizing the nematode C. elegans as amodel, Hitoshi Sawa is engaged in the study of themolecular interactions that underlie this process. He
seeks also to resolve the ways in which transcriptionregulating complexes integrate the intracellular signaling networks that collaborate to help determine cell
fate.

Staff
Team Leader

Hitoshi Sawa
Research Scientist

Yukinobu Arata
Kumiko Oishi
Masahiro Uchida
Masaki Fujita
Technical Staff

Hisako Takeshita
Noriko Sasakawa

LIN-12/Notch

Assistant

DPY-22

Tomoko Nakashima

1° Cell Fate

LET-192° Cell Fate
LET-425
SUR-2

LET-60/Ras

3° Cell Fate

Mediator complex

Regulation of cell polarity
The nematode offers a number of advantages for use
as an experimental model, including its rapid reproductive cycle, the complete accounting of its cell lineages, its amenability to growth in culture and genetic
manipulation, and the ease with which individual cells
in its transparent body can be observed in vivo undera low-power microscope. The Sawa research teamstudies the asymmetric division of C. elegans T cells,
which give rise to pairs of daughter cells, one ofwhich generates epithelial cells, while the otherproduces neural cell types. The Sawa research teamuses molecular genetic techniques to probe themolecular mechanisms involved in cell fate specification. By cloning the gene lin-17 (lin signifying lineagedefective) from a mutant strain exhibiting aberrant
asymmetric cell division and analyzing its sequence,
Sawa determined that lin-17 is homologous to the frizzled gene in the fruit fly Drosophila, which is believedto serve as a receptor of Wnt-family ligands. Other
experiments in which the Wnt-family protein LIN-44
was misexpressed ectopically resulted in the reversalof T cell lineage polarity, indicating that the Wnt/Frizzled signaling pathway is important in regulating thedirection of polarization as occurs in the process ofasymmetric mitosis and adding to its wide and wellcharacterized array of functions in such fundamental
processes as cell proliferation and differentiation.

The Sawa team has
also identified nearly forty
genes in a C. elegans
mutant screen

The Sawa team has also identified nearly forty genes
from a screen of mutants in which both daughters of
the T cells were found to produce only epithelial cells,
rather than epithelial and neural cells. Of that group,
the two genes psa-1 and psa-4 (psa for Phasmid
Socket Absent) were found to encode proteins homologous to yeast SWI3 and SWI2/SNF2, respectively.
These proteins are components of the SWI/SNF transcription regulatory complex, which is known to be
involved in asymmetric cell division in yeast, a singlecelled organism that nonetheless exhibits asymmetricmitosis. These results provide compelling evidence
that conserved mechanisms may be at work in theasymmetric division of both one- and multi-celledorganisms.

Integrationoftranscriptionregulating networks

Publications

A second area of focus in Sawa’s work is unravelingthe ways in which complex intracellular signaling
networks are integrated in the determination of cellfate, using vulva development in C. elegans as amodel system. The nematode exists either as a male
Sawa H, Kouike H, and Okano H.
or a hermaphrodite animal. The hermaphrodite vulva
Components of the SWI/SNF comis a small channel located near the midpoint of theplex is required for asymmetric cell
ventral side, and functions both to receive sperm anddivision in C.elegans. Mol Cell 6:617release fertilized eggs during reproduction. This
24 (2000).
extensively-studied organ comprises only 22 cells,
Rocheleau CE, Yasuda J, Shin TH,
and its development is known to be mediated by both
Lin R, Sawa H, Okano H, Priess JR,
the Ras and Notch signal transduction pathways.
Davis RJ, Mello CC. WRM-1 actiSawa is analyzing vulval mutants to gain a morevates the LIT-1 protein kinase todetailed understanding of how these pathways functransduce anterior/posterior polarity
signals in C.elegans. Cell 97:717-26
tion in the formation of this organ, a process that invol(1999).
ves a rigidly stereotyped pattern of mitotic divisions.
Such stereotyped cell division patterns are characteristic of nematode development, and are one of its
most attractive features as a model system, allowing
researchers to pinpoint phenotypic deviations at thelevel of the single cell. One result of this analysis ofvulval mutants has been the identification of
LET-19/TRAP240, components of a transcriptionregulating complex Mediator, which operates downstream of Notch. The Mediator complex also incorporates another component, SUR-2, which functions
downstream in the Ras pathway, strongly suggestingthat that this complex plays arole in integrating the Notch
LIN-17/Frizzled
and Ras pathways in vulva
development.
LIN-44/Wnt
Zhao X, Sawa H, Herman MA. Tcl-2
encodes a novel protein that acts
synergistically with Wnt signaling
pathways in C.elegans. Dev Biol (inpress 2003).

Looking ahead, the Sawa
research team intends to identify SWI/SNF activators andtarget genes, and to deepen
further the understanding of
the molecular mechanisms at
work in asymmetric cells in
the nematode, in the hopes ofdeveloping insights with application to research in mammalian cell asymmetry. He alsoplans to continue his research
into the integration of signals
in the process of cell fate
determination.

Polar distribution of LIN-44/Wnt and
LIN-17/Frizzled induces SWI/SNF
function in a single neurally-fateddaughter cell

cell polarity

SWI/SNF

inactive chromatin

active chromatin

Ras and Notch signals set primary to tertiary cell fates via the Mediator
complex in vulval development

38 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report39Creative Research Promoting Program

Development

Laboratory for Developmental Genomics

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

C. elegans (adult hermaphrodite)the role of networked genes in guiding development
in a simple worm, new light will be shed on universal
mechanisms in the genetic regulation of the developmental program.

Sugimoto hopes to shed
new light on universal mechanisms
in the genetic regulation of
the developmental program.

RNAi-based profiling of gene function

The gene network
As recent findings of the limited numbers of genes inmany genomes, including our own, have indicated,
the interactions between genes at the network level
seem increasingly significant in determining the
intricate development and function of multicellularorganisms. While the characterization of genomes atthe level of the individual gene remains an important
challenge, an analysis of how genes function in networks is now in equal demand. This represents adaunting task in highly evolved and complex species
such as Homo sapiens, prompting scientists to seek
more primitive and simpler models to gain fundamental insights in the hopes that a significant number ofbasic mechanisms are shared by many branches ofthe phylogenetic tree.

Staff
Team Leader

Asako Sugimoto
Research Scientist

Naoko Sakumoto
Special Postdoctoral Researcher

Asako Sugimoto has adopted as a model the nematode C. elegans for its tractability to the systematicfunctional analysis of its genome using unique highthroughput screening techniques. By studying thefunctional interaction of genes within C. elegans,whose genome has been completely sequenced, theSugimoto research team seeks to identify the meansby which genes working in combination help to establish and direct developmental processes. The lab
also looks to take the findings from these studies as abase for advancing the understanding of developmentally important mechanisms such as programmed cell
death (apoptosis) and microtubule dynamics in mitosis. Sugimoto hopes that by opening windows into

Fumio Motegi
Technical Staff

Nobuko Uodome
Chie Hayakawa
Yumi Iida
Student Trainee

Miwa Furuya
Rika Maruyama
AssistantFumi Amimori

One-cell stage embryo. (Blue: chromosomes, Green: microtubules,
Red: Protein Phosphatase 4)

40 RIKEN CDB 2002 Annual Report

The wild-type nematode is made up of less than
1,000 cells, yet this simple organism exhibits a wide
range of the specialized cell types, such as muscles
and nerves, that characterize more highly evolved
species. And, thanks to the complete knowledge ofthe lineage of every cell in the C. elegans body, thedifferentiative fate of every one of those cells can betraced from its origin in the fertilized egg to its role in
the fully-grown adult. The amenability of this worm’s
sequenced genome to reverse-genetic techniques
has also served to make it one of the preferred modelorganisms in the world of genetics research. The discovery in the late 1990s that the introduction of double stranded RNA (dsRNA) could be used to ‘knock
down’ the expression of specific genes in the nematode has only added to its appeal, giving scientists
the ability to inhibit gene function without disturbing
its underlying DNA. Sugimoto has refined this technique of RNA interference (RNAi) by developing amethod in which nematodes directly uptake dsRNA in
solution. This process of ‘RNAi by soaking’ offers
greater efficiency and ease-of-use than other RNAi
methods and has made it possible to conduct systematic high-throughput studies of gene suppression
more rapidly than ever before, achieving knock down
analysis rates of up to 800 genes per month.
Starting with a cDNA library of approximately 10,000
genes expressed in developmental processes, theSugimoto lab used RNAi to knock down each gene’sfunction and built a database in which the resulting
phenotypes were sorted by developmental outcome.
To date, more than 4,000 phenotypes have been categorized, and it was found that loss of function in
more than 25% of these genes resulted in lethal, morphologically altered or sterile phenotypes. Such developmentally important genes also seem to appear lessfrequently on the sex chromosome. The C. elegans
male has only a single such chromosome, and selective pressure in the process of evolution may havedetermined that the most functionally important
genes are located on chromosomes where the chances of a complete loss of function are reduced.
In addition to further phenotypic characterization andcategorization of the embryonic lethal genes, Sugimoto next studied their post-embryonic functions using a
conditional knockdown strategy. Approximately 60%of the genes studied played post-embryonic roles as
well, indicating that many genes play multiple roles atdifferent stages in development. It is anticipated that
this comprehensive phenotype profiling will lead tothe identification of gene networks that play crucial
roles in developmental processes.

Programmed cell death
In addition to their work on systemlevel analysis of gene networks, theSugimoto team is also engaged indetailed analysis of specific cellularfunctions, such as programmed cell
death, in which cells self-destruct aspart of the normal developmental
process. Programmed cell deathhas been extensively studied in C.
elegans, and it is known that exactly
131 cells undergo this programmed
cell death in normal nematodedevelopment. It has been shownthat a single genetic cascade is
responsible for inducing cell deathin C. elegans, and that this cascadehas been evolutionarily conserved.
By seeking to identify the molecular
events that control the death of cells
during the development of the nematode, Sugimoto hopes to uncover
the regulatory foundations of thisimportant mechanism.

Publications
Kodama Y, Rothman JH, Sugimoto
A, Yamamoto M. The stem-loop binding protein CDL-1 is required for chromosome condensation, progressionof cell death and morphogenesis in
Caenorhabditis elegans. Development 129:187-96 (2002).
Sumiyoshi E, Sugimoto A, Yamamoto M. Protein phosphatase 4 isrequired for centrosome maturationin mitosis and sperm meiosis in C.elegans. J Cell Sci 115:1403-10 (2002).
Maeda I, Kohara Y, Yamamoto M,
Sugimoto A. Large-scale analysis ofgene function in Caenorhabditis elegans by high- throughput RNAi. Curr
Biol 11:171-6 (2001).
Sugimoto A, Kusano A, Hozak RR,
Derry WB, Zhu J, Rothman JH. Many
genomic regions are required for normal embryonic programmed cell
death in Caenorhabditis elegans.
Genetics 158:237-52 (2001).

The Sugimoto team is also engaged
in investigating the molecularmechanisms underlying the formation and movement of mitotic spindles, and has identified a gene
involved in spindle formation.
Future research will employ live
imaging of microtubule and centrosomes using GFP-tagged proteins in combination of RNAi knockdown analysis to provide a clearer
picture of the role of specific genes
in this process.
Embryogenesis of
C. elegans
RIKEN CDB 2002 Annual Report41Creative Research Promoting Program

Development

Laboratory for Body Patterning

Body Patterning
Yoshiko Takahashi

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

process, in which epithelial cells assume a mesenchymal fate, is also observed during development. The
Takahashi lab would like to clarify the molecularmechanisms that regulate this mesenchymalepithelial transition, and are concentrating in on theroles played by members of the Rho family of proteins in this process. Both the inhibition of Rac1 andthe overexpression of Cdc42 were found to induce
the conversion of epithelial cells to mesenchyme,
while interference with Cdc42 function caused
mesenchymal cells to take on epithelial characteristics. These suggestive findings led Takahashi to propose that the mesenchymal-epithelial transition isdetermined by relative Rac1 and Cdc42 activity levels, with higher levels of Rac1 inducing an epithelial
fate, while higher levels of Cdc42 tip the balance inthe mesenchymal direction.
The simple egg develops into a marvelously complex organism

Publications
You can’t tell an embryo
what to do,
only listen to what it can tell you

Staff
Team Leader

Yoshiko Takahashi
Special Postdoctoral Researcher

Yuki Sato
Technical Staff

Rinako Suetsugu
Toshiharu Kasai
Akemi Uchiyama
Student Trainee

Yukiko Nakaya
Takashi Yoshino
Tadayoshi Watanabe
Emi Ohata
Assistant

Naomi Akira

Pattern formation in the early
embryoPattern formation, the process by which the boundaries that define germ layers and tissue types are
established, occurs at highly specific sites and developmental stages in the early embryo. This closely
coordinated regulation prevents cells from differentiating inappropriately, and enables the establishment ofcomplex and specialized tissues and organs in theadult body. One type of pattern formation involves theregimented formation of periodically alternating
bands of differentiated cells along the anteriorposterior body axis. The results of this process of segmentation can be observed in the orderly structuresof the vertebrae and spinal ganglia. In vertebrates,
segmentation takes place in the embryonic region
known as the ‘somitic mesoderm,’ and involves thetransient formation of segment organizing bodies
called somites. Yoshiko Takahashi studies segmentation and somitogenesis in the chick embryo, and has
made inroads into achieving a better knowledge ofhow groups of cells are able to cleave at specificboundary lines and re-organize into periodically segmented patterns. Investigations at the Takahashi lab
seek to explain at the molecular level the mechanisms of pattern formation in development.

anterior-posterior axis. Somites arise one by one fromthis segmental plate in a head-down direction. In thissense, if the segmentation of invertebrates such as thefruit fly can be described as an ‘egg-slicer’ effect inwhich all cuts are made simultaneously, then segmentation in vertebrates might be likened to a ‘meat-slicer,’ inwhich cuts are made sequentially, one after another.
In experiments designed to elucidate the molecularmechanisms that drive this vertebrate meat-slicer, Takahashi made sectional transplantations and locally targeted expression of various genes, the results of which led
her to propose the existence of a region responsible forinducing segmentation, which she has named the ‘segmenter.’ Transplantation of this segmenter into the undifferentiated segmental plate triggers ectopic boundary
formation, or segmentation. The segmenter region also
specifically expresses a factor that is believed to regulate Notch signaling, a pathway which has been extensively studied for its diverse roles in the determination ofcell fate and differentiation. Moreover, Takahashi found
it was possible to mimic the effects of segmenter transplantation by modulating the expression of activatedNotch in specific unsegmented mesodermal regions,suggesting a new function for the Notch pathway inboundary formation during mesodermal segmentation.

The segmenter and Notch signaling

Epithelialization

In the early chick embryo, two bands of unsegmented
mesoderm, collectively referred to as the ‘segmental
plate,’ flank the neural tube, which runs along theAfter the segmenter has established the boundaries
of a somite, the mesenchymal cells in the borderregion change to become epithelial cells. The reverse

Suetsugu R, Sato Y, Takahashi Y.
Pax2 expression in mesodermal
segmentation and its relationship with
EphA4 and Lunatic fringe during
chicken somitogenesis. Gene Expression Patterns 2:157-61 (2002).

Periodicity
The fact that, despite the marked differences in thedevelopmental mechanisms at work in invertebrates
and vertebrates, organisms as diverse as the Drosophila fly and the chick exhibit patterns with similarly
regular temporal and spatial intervals intrigues Takahashi. Her lab
has been participating in collaborative research to determine whether
the establishment and maintenanceof such intervals can be explainedby the reaction-diffusion model (seepp. 28-9).

teach her about pattern formation. And pattern formation, of course, has many lessons for those who wish
to understand the bases of development itself.

GFP
Electroporation

The details of her current work
aside, Takahashi emphasizes thatwhat drew her to science and maintains her interest is the opportunities it offers to those willing andable to learn from natural phenomena. “You can’t tell an embryo what
to do, only listen to what it can tellyou.” But her range of interests is
not limited to any single field; she
prefers to pursue avenues that others may have overlooked. She studies molecules and genes for what
they can teach her about somites,and somites for what they canSato Y, Yasuda K, and Takahashi Y.
Morphological boundary forms by anovel inductive event mediated by
Lunatic-Fringe and Notch duringsomitic segmentation. Development
129:3633-44 (2002).
Shiroi A, Yoshikawa M, Yokota H,
Fukui H, Ishizaka S, Tatsumi K, andTakahashi Y. Identification of insulin
producing cells derived from embryonic stem cells by zinc-chelating dithizone. Stem Cells 20:284-92 (2002).
Takahashi Y, Osumi N, and Patel N.
Body Patterning. Proc Natl Acad Sci
98:12338-9 (2001).
Sudo H, Takahashi Y, Tonegawa A,
Arase Y, Aoyama H, Mizutani-koseki
Y, Moriya H, Wiling J, Christ B, Koseki
H. Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for theformation of the sternal component ofavian ribs. Dev Biol 232(2):284-300(2001).

Electroporation used in combination with classical explant techniques allows for pinpoint gene
expression in target tissues

42 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report43Development

Regeneration

Regenerative
Medicine

Creative Research
Promoting Program

Laboratory for Genomic Reprogramming

Reprogramming
Teruhiko Wakayama
Ph. D.
Teruhiko Wakayama received his B.
Sc. and M. Sc. from Ibaraki University,and was awarded a Ph. D. in reproductive biology from the University ofTokyo Department of Veterinary Anatomy in 1996. He received a postdoctoral
fellowship from the Japanese Society
for the Promotion of Science in 1996
and spent the next three years at the
Yanagimachi lab in the University of
Hawaii Medical School, where he succeeded in producing the world’s firstcloned mouse. He was appointed to an
assistant professorship at the same
institution in 1998, and moved to Rockefeller University as a research assistantprofessor in 1999. He spent a year as a
researcher at Advanced Cell Technology before returning to Japan to take hiscurrent position at the RIKEN CDB.

Although he is perhaps best known for his pioneering
work in the cloning of mice, Teruhiko Wakayama has
dedicated his research to exploring the mysteries offertilization. By observing the effects of varying experimental conditions on the interaction between the egg(or oocyte) and extrinsic genetic matter, Wakayama’s
research team seeks to understand the fundamental
basis by which the fertilized egg is ‘reprogrammed’
from a highly differentiated state to become the totipotent progenitor of the myriad cell types in the adultbody. Wakayama also strives to develop new laboratory techniques to make the difficult processes ofmanipulating eggs simpler and more efficient.

The fertilization of the egg is
the initiation of
the entire developmental
cascadeFreeze-dried sperm
Staff
Team Leader

Teruhiko Wakayama
Research Scientist

Satoshi Kishigami
Nguyen Van Thuan
Technical Staff

Sayaka Wakayama

Fertilization
In most animals, the sperm and the egg (collectively
known as gametes) are highly specialized cells thatenable the organism to reproduce in a way that commingles the genetic information from both partners ina sexual pairing. This can only be achieved by thefusion of gametes in the process known as fertilization. Under natural conditions, fertilization involves acomplex chain of events and interactions, which inmammals begins with the entry of the sperm into thefemale reproductive tract. The environment within theuterus and oviduct produces changes in the spermthat allow it to dock with the outer surface of the unfertilized egg, disintegrate, and deliver its genetic contents. Penetration by the sperm also induces dramaticeffects in the egg, including biochemical and structural reactions that prevent other sperm from entering,
the transition from meiotic to mitotic cell division, anda series of intracellular changes that represents thefirst steps toward the construction of a new individual.
Seen from this perspective, the fertilization of the egg
is not merely the event by which two haploid genomes merge, but also the initiation of the entire developmental cascade.

Sperm preserved for use in experiments and in vitro
fertilization is traditionally frozen in liquid nitrogen at
extremely low temperatures (around -190˚C). This
cryopreservation allows the sperm to be maintained
viably for very long periods, but requires expensive
facilities and some degree of technical skill in
handling. In the 1990s, Wakayama developed amethod by which sperm could be freeze-dried andremain viable under low-cost, low-maintenance conditions. Sperm freeze-dried in such a way were later
used in ICSI fertilization experiments, and although
they were technically ‘dead,’ they demonstrated aneffective ‘fertilization shelf-life’ of around thirty days
when stored at room temperature and considerably
longer when refrigerated at 4˚C. Wakayama continues to be interested in developing more efficient andeconomical techniques for sperm storage, and hopes
to improve the storable duration and viability rates forsperm preserved in this manner.

ized, and found that these cells, too, are capable ofdeveloping into live-born, normal mice. Previously, it
had been thought that polar bodies might be repositories for damaged DNA and, so, unlikely to develop
normally if fertilized. The viability of polar bodies assurrogate oocytes has implications for the field of
human in vitro fertilization, in which the supply ofeggs is frequently a limiting factor.

Cloning
For many people, the concept of cloning conjures
images of indistinguishably identical replicas of anindividual. But Wakayama notes, “Clones are by nomeans perfect copies of the original.” Experimentallycloned animals exhibit a range of embryonic andadult epigenetic defects. The process of cloning
does, however, provide an excellent system for studying the mechanisms of fertilization, as it allows the
specific control of factors such as nuclear source. In
cloning experiments using nuclei from different sources, such as cumulus cells, somatic cells and embryonic stem cells, Wakayama has observed variations inpatterns of live-birth rates and developmental abnormalities, which he hopes will fuel further investigations at the molecular level. In the future, he plans tocontinue to explore the secrets of the fertilized egg at
the cellular level. In his own words, “Eggs are very
beautiful and mysterious. I never get tired of watchingthem.”

Publications
Tamashiro KL, Wakayama T, Akutsu
H, Yamazaki Y, Lachey JL, Wortman
MD, Seeley RJ, D'Alessio DA,
Woods SC, Yanagimachi R, Sakai
RR. Cloned mice have an obese phenotype not transmitted to their offspring. Nat Med 8:262-7 (2002).
Wakayama T, Tabar V, Rodriguez I,
Perry ACF, Studer L, Mombaerts P.
Differentiation of embryonic stem celllines generated from adult somatic
cells by nuclear transfer. Science
292:740-3 (2001).

Egg alterations

Nuclear transfer by microinjection

44 RIKEN CDB 2002 Annual Report

Wakayama is also interested in whether a partial egg
is sufficient to produce a normally developed individual when fertilized. In a series of experiments in which
he sequentially reduced the oocyte mass by factorsof two and attempted sperm injection, he found thateggs of one-half normal size were competent to produce normal individuals, indicating a certain degreeof functional redundancy in oocyte composition. In
similar work, he investigated the competency of polar
bodies (small satellite cells produced during the meiotic division of the egg) to produce offspring when fertil-

Wakame, the first mouse cloned at the RIKEN CDB

RIKEN CDB 2002 Annual Report45

2002 New Faces

2002 New Faces
Development

Creative Research Promoting Program

Development

Regeneration

Creative Research Promoting Program

Induction

Germ Cells

Laboratory for Embryonic Induction

Signaling centers in mammaliandevelopment

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

The early mouse embryo is characterized by its dynamic transformation from a simple, nearly featurelesscylinder of cells to a recognizable embryonic body inthe space of a few days during gastrulation. This
remarkably rapid transition is achieved by the highly
coordinated differentiation of cells toward specificfates, a feat orchestrated by a number of cellular loci,collectively known as ‘signaling centers.’ These centers produce diffusible proteins that serve as regulators, guiding neighboring cell communities to take up
the roles they will play in the adult animal, or steering
them away from inappropriate fates.
Hiroshi Sasaki’s research team investigates the network of signaling centers that work to induce normal
development in mammals. The node, the center
responsible for induction of body parts in the mouse,
is of particular interest, as it plays a role corresponding to the amphibian Spemann organizer, which firstsparked Sasaki’s fascination with signaling centers.
Sasaki has identified a number of transcription factors involved in the formation and the function of thenode in previous studies, and he continues to beacutely involved with questions of how these centers
are formed, and how they regulate development.

Laboratory for Germline Development

Germ cells

the node and notochord. Using a marker gene totrace gene expression, his team identified the
HNF3β enhancer, and has found that the key
mechanism of its regulation is activation by a pair oftranscription factors, functioning downstream of aknown node-inducing signal, Wnt. The team will study the effects of the loss of function of these molecules in knockout mice, and search for potential
co-factors using a yeast interaction-screening system.
The serendipitous discovery of a previously unknown
mouse phenotype in which the head fails to develop
has also spurred research in Sasaki’s lab. It is rare
for such ‘headless’ phenotypes to survive to laterstages of embryonic development, and the known
cases that do have all been linked to mutations in
node-related genes. The phenotype under investigation develops normally in early embryogenesis, but
exhibits a loss of anterior markers in late gastrulation,
when the node normally induces the head. Sasaki is
now performing a functional analysis of how one candidate gene interacts with other signaling molecules.
In these and other studies, Sasaki seeks to identify
molecules involved in signal center formation andfunction in the early embryonic development of themouse.

Node-specific transcription factor networks

Akira Nakamura
Ph. D.
Akira Nakamura received both his
baccalaureate and his Ph. D. fromthe University of Tsukuba. He spenta year as a post-doctoral fellow at
the same institution before moving to
the Department of Biology at McGill
University in Montreal in 1995 towork as a post-doc under Paul Lasko. He returned to Japan in 1997 asa research associate at the University of Tsukuba. He was appointed
assistant professor in the university’s
Gene Research Center and Instituteof Biological Sciences in 2000, andbegan a three-year term as a PRESTO researcher in the Japan Scienceand Technology Corporation (JST) in
December 2001. He was appointed
CDB team leader in March 2002.

Hepatocyte Nuclear Factor 3β(HNF3β) is a highly conserved
transcription factor expressed
specifically in embryonic signaling centers. Sasaki is engaged
in an analysis of molecules that
activate HNF3β expression in

Hiroko Sato
Yayoi Minami
Assistant

Misaki Harano

46 RIKEN CDB 2002 Annual Report

Future work in the Nakamura lab will focus on taking
what has been learned from the study of germ line
establishment in Drosophila and searching for patterns of conservation and divergence in chordates,
using the sea squirt as a model.

Akira Nakamura
Enhancer of HNF3β drives gene expression in the signaling centers, node and notochord (blue).

Research Scientist

Technical Staff

the function of Pgc is inhibited, germ cells fail tomigrate to the embryonic gonads. Another maternal
effect mutant, named N14, results in a similar phenotype, suggesting that these genes play roles in germ
cell maintenance and migration, and possibly theirprogrammed cell death. Their loss of function might
correspond to a loss of ‘germness’ in these cells, afinding that provides a new potential link between
germ cells and undifferentiated stem cells.

Staff

Hiroshi Sasaki

Noriyuki Nishioka

Maternal RNP particles shown by green fluorescentprotein imaging

Team Leader

Team Leader

Visiting Scientist

The Drosophila egg shares cytoplasm with neighboring nurse cells via an incomplete cell membrane,
allowing mRNAs and proteins from the nurse cells tobe transported to the egg in the form of ribonucleoproteins (RNPs). In order to ensure the proper functionof these mRNAs, their translation must be prevented
during their transport. Screening proteins with localization patterns similar to those of transported
mRNAs, the Nakamura team identified Me31B, whichis involved in the translational silencing of maternal
mRNAs. The Me31B complex also includes a protein
involved in directing the cytoplasmic transport ofmRNAs, suggesting that RNP complex proteins cooperatively regulate both the translation and transport ofmRNAs. The lab is now working on the functional
characterization of other RNP proteins they have identified through several approaches.
In previous work, the Nakamura team identified a novel non-translatable RNA, Pgc (for Polar Granule Component), which is localized in the germ plasm. When

Staff

Atsushi Sawada

Germ cells are the only cell types capable of transmitting genetic information from generation to generation, and these cells are characterized by equallyunique developmental processes as well. In many
types of animals, the formation and differentiation ofgerm cells is controlled by mRNAs and proteins localized in the specific cytoplasmic region within eggs,
called germ plasm. Germ plasm mRNAs are translated in a spatio-temporally regulated manner, but themeans by which the germ plasm is formed andachieves this controlled translation remain obscure.
Akira Nakamura studies the establishment of the Drosophila germ line as a model of the processes ofgerm plasm formation and differentiation, as well as
for the insights this system can provide into the general mechanisms of mRNA localization and translation.

Publications
Nishizaki Y, Shimazu K, Kondoh H,
Sasaki H. Identification of essential
sequence motifs in the node/notochord enhancer of Foxa2 (Hnf3β)
gene that are conserved across vertebrate species. Mech Dev 102:57-66(2001).

Sasaki H, Nishizaki Y, Hui C.-c, Nakafuku M, and Kondoh H. Regulation ofGli2 and Gli3 activities by an aminoterminal repression domain:implication of Gli2 and Gli3 as primary mediators of Shh signaling. Development
126:3915-24 (1999).

Research scientist

Kazuko Hanyu-Nakamura
Keiji Sato
Maki Shirae
Technical Staff

Chiaki Nakamoto
Hiroko Sonobe

Publications
Styhler S, Nakamura A, and Lasko
P. VASA localization requires the
SPRY-domain and SOCS-box containing protein,GUSTAVUS. Dev
Cell 3:65-876 (2002).
Nakamura A, Amikura R, Hanyu K,
and Kobayashi S. Me31B silencestranslation of oocyte-localizing
RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development
128:3233-42 (2001).

Carrera P, Johnstone O, Nakamura
A, Casanova J, Jäckle H, and Lasko P. VASA mediates translation
through interaction with a Drosophila yIF2 homolog. Mol Cell 5:181-7(2000).
Nakamura A, Amikura R, Mukai M,
Kobayashi S, and Lasko PF.
Requirement for a non-coding RNA
in Drosophila polar granules for
germ cell establishment. Science
274:2075-9 (1996).
RIKEN CDB 2002 Annual Report47

2002 New Faces
Development

Creative Research Promoting Program

2002 New Faces
Development

Chromatin

Jun-ichi Nakayama
Ph. D.
Jun-ichi Nakayama received hisbachelor’s, master’s and doctoral
degrees in bioscience from theTokyo Institute of Technology, thelast 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 theCold Spring Harbor Laboratory in
Shiv Grewal’s lab, before returning toJapan in December 2001 as a PRESTO researcher in the Japan Scienceand Technology Corporation (JST).
He was appointed team leader in the
RIKEN CDB Laboratory for Chromatin Dynamics in 2002.

In recent years, it has become increasingly evident
that the controlled expression of genes cannot be
accounted for by DNA sequences alone. Epigenetic(meaning ‘heritable, but occurring independently ofgene sequence’) regulation of gene transcription
must be maintained and propagated across cell division cycles and throughout the development of theorganism proper in order for cells to establish andmaintain their identities as specialized cell types.
While many of the mechanisms by which such regulation is achieved remain unknown, it is clear that modifications to the nuclear DNA-protein chromatin complex are central to epigenetics. Chromatin occurs inhighly-condensed and less condensed forms, which
are known as heterochromatin and euchromatin,
respectively. Jun-ichi Nakayama’s research will concentrate on heterochromatin dynamics, modifications
to the DNA-packing proteins called ‘histones’ in particular, and the molecular mechanisms that allow suchchemical states to be heritably transmitted.

Heterochromatin
gene silencing

proteins

and

The Nakayama research team uses the fission yeast
S. pombe as a model system, as its chromatin structure is closely similar to that of higher organisms. In
previous
studies,Nakayama demonstrated that heterochromatinprotein binding statesplay a role in the regulation of transcriptional
gene silencing. Nakayama performed detailed
analyses of the binding
states of Swi6, a homolog of the mammalian

A new basis for regenerative medicine

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

Staff
Fission yeasts stained by DAPI(left) or detected by GFP-Swi6(right)

Visiting Scientist

Yasuko Ohtani
Chikako Nishimoto
Junko Toga

48 RIKEN CDB 2002 Annual Report

Takayuki Asahara brings a background in clinical cardiology to the CDB, and plans to use his experienceat both the bedside and the research bench to investigate the potential for applications of post-natal pluripotent stem cells in regenerative therapies. Building
on previous work in which he identified bone marrowderived endothelial progenitor cells (EPCs) and demonstrated their role in the generation of new bloodvessels, Asahara seeks to characterize stem cells in
the adult body with even greater differentiative potential, and one day to translate that research into clinically relevant advances.

In addition to their capacity for facultative tissuespecific differentiation, the ability of stem cells andtheir progenitor cell descendants to home to sites ofactive regeneration makes them attractive for use asvectors in targeted gene therapy. Adult stem cells
also preferentially maintain a quiescent, nondifferentiating state, and so they are believed to represent less of a tumorigenic risk than embryonic stem
cells when transplanted into human patients, as,indeed, experience with hematopoietic stem cells has
shown. With the goal of realizing truly translational
research, Asahara also plans to study optimal methods for delivering stem cells to localized and systemwide targets in regenerative and gene therapy.

Blood vessel formation and organogenesis
One result of Asahara’s and others’ work in the
1990s was the realization that blood vessels areformed by two distinct physiological processes in theadult body. In angiogenesis, new blood vessels are
generated from pre-existing, differentiated endothelial
cells. Conversely, vasculogenesis involves the recruitment and differentiation of previously undifferentiatedEPCs at the site of new blood vessel growth. These
EPCs are themselves the progeny of more differentia-

Team Leader

Jun-ichi Nakayama

Technical Staff

The use of transplanted adult stem cells to regenerate blood cells represents one of the first and, todate, most successful applications of regenerative
medicine. But the developing field of stem cell-based
therapy continues to receive attention as one of themost promising frontiers of medical science. Pluripotent cells, with their ability to produce both pluripotent
and differentiated progeny on cell division, have enormous potential for clinical application, and are thesubject of intense research in medical fields fromautoimmune and neurodegenerative disease to disorders of the metabolism.

tively potent adult stem cells known as hemangioblasts, which can be induced to demonstrate true pluripotency under appropriate culture conditions. Asahara’s research at the CDB will hinge on developing
gene expression profiles of post-natal pluripotent
stem cells and performing functional assays of suchcells at various stages of differentiation to elucidate
the genetic and molecular mechanisms that underlie
vasculo- and organogenesis.

Staff

Team Leader

Mahito Sadaie

Creative Research
Promoting Program

Laboratory for Stem Cell Translational ResearchHP-1 heterochromatin protein at silent mating type(mat) locus of the fission yeast. The results of that study showed that Swi6 protein is a dosage-critical component involved in imprinting the mat locus. This binding of Swi6 is maintained both across mitotic cell
cycles and intergenerationally, as it is propagatedthrough meiosis as well. Nakayama’s study also
showed that the deacetylation and subsequent methylation of a specific histone H3 lysine residue are
essential to this process. Chemical modifications
such as deacetylation, methylation, phosphorylation
and ubiquitination are fundamental epigenetic processes that can act to switch the expression of a target gene on or off.
Through his work on this evolutionarily conserved heterochromatin protein, Nakayama has uncovered
potentially important new roles for the molecule in theestablishment, maintenance and transmission of epigenetic information. His findings suggest that the concept of a gene as a simple string of DNA nucleotides
needs to be expanded to include the action of proteins in the functional genetic unit. In the future,
Nakayama plans to perform more detailed analysesof the molecular mechanisms that underlie epigenetic
function, as well as studies in higher organisms andepigenetic gene expression in developmental processes.

Regenerative
Medicine

Stem Cell Organogenesis

Laboratory for Chromatin Dynamics

Epigenetic control of gene expression

Regeneration

Takayuki Asahara

Vasculogenesis achieved by endothelial progenitor cells

Senior Scientist

Satoshi Murasawa
Hiromi Nishimura

Publications
Nakagawa H, Lee J-K, Hurwitz J, Allshire RC, Nakayama J, Grewal SIS,
Tanaka K, and Murakami Y. Fission
yeast CENP-B homologs nucleate
centromeric heterochromatin byprompting heterochromatin-specifichistone tail modifications. Genes Dev
16:1766-78 (2002).
Nakayama J, Allshire RC, Klar AJS,
and Grewal SIS. Implicating DNA polymerase a in epigenetic control ofsilencing in fission yeast. EMBO J
20:2857-66 (2001).

Nakayama J, Rice JC, Strahl BD, Allis
CD, and Grewal SIS. Role of Histone
H3 Lysine 9 Methylation in Epigenetic
Control of Heterochromatin Assembly. Science 292:110-3 (2001).

Research Scientist

Satoshi Hasegawa
Tsuyoshi Hamada
Technical Staff

Akira Oyamada
Kazuyo Sadamoto
Student Trainee

Masakazu Kuriwaka
Nakayama J, Klar AJS, and Grewal
SIS. A Chromodomain Protein, Swi6,
Performs Imprinting Functions in Fission Yeast during Mitosis and miosis.
Cell 101:307-17 (2000).

Publications
Asahara T, Kawamoto A, Kalka C,
Masuda H. Therapeutic potential ofbone marrow-derived endothelial progenitor cells for cardiovascular ischemic
diseases Gene Ther Reg 1 (4):361-75(2003)

Murasawa S, Llevadot J, Silver M, Asahara T, et.al. Constitutive human telomerase reverse transcriptase expression enhances regenerative propertiesof endothelial progenitor cells. Circulation 106(9):1133-9 (2002).

Asahara T, Isner JM. Vascular cells andAngiogenesis; Endothelial progenitor
cells for vascular regeneration. J Hematotherapy Stem Cell Res 11(4):579-82(2002).

Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Asahara T, et.al. Endothelial
progenitor cell VEGF gene transfer for
vascular regeneration. Circulation
105(6):732-8 (2002).

Assistant

Kanako Ota

Walter DH, Rittig K, Bahlmann FH,
Kirchmair R, Silver M, Murayama T,
Nishimura H, Losordo DW, Asahara T,
Isner JM. Statin therapy accelerates
reendothelialization: a novel effectinvolving mobilization and incorporationof bone marrow-derived endothelial progenitor cells. Circulation 105(25):301724 (2002).

RIKEN CDB 2002 Annual Report492002 New Faces
Development

Tony Perry
Ph. D.
Tony Perry received his B.Sc. in
Microbiology from the Department ofBacteriology at the University of Bristol, England and his doctorate fromthe University of Liverpool four years
later. In 1989 he became a postdoctoral fellow working on epididymal
sperm maturation at Bristol University and in 1996 won a European
Molecular Biology Travel Fellowship
to work in the laboratory of Ryuzo
Yanagimachi on the mechanism ofoocyte activation, which remains oneof his research interests. From
there, Dr. Perry moved first to theRockefeller University and then to
the company Advanced Cell Technology, working primarily on novel methods of genome manipulation. In
2002 Dr. Perry took his present position as Team Leader of the Laboratory of Mammalian Molecular Embryology at the RIKEN CDB, where he
will work on mechanisms in mammalian preimplantation embryos.

Creative Research
Promoting Program

Regenerative
Medicine

Regeneration

2002 New Faces
Development

Regenerative
Medicine

Regeneration

Creative Research
Promoting Program

Embryology

Neurotrophic Factors

Laboratory of Mammalian Molecular Embryology

Laboratory for Neuronal Differentiation and Regeneration

The developmental program is initiatedmoments after the penetration of anegg by a fertilizing sperm. Althoughthese initiation events are quintessential to the ontogeny of all mammals,
they remain one of nature's most closely guarded secrets. Somehow, within
hours of coming together, the two highly specialized cells that begin this process, produce a single cell that is utterly unspecialized: it can give rise to allcell types - in fact, it produces an individual. The single cell is, of course, a1-cell embryo, and the two cells thatmust unite to produce the embryo are
gametes - an egg (or oocyte) from thematernal side and a sperm from thepaternal side.
Adopting the mouse as a model system, the laboratory of Tony Perry is trying to bring molecular and cellular techniques to dissect the magical events occurring within the earliest embryos. What are the key
molecules involved, how do they relate to each other,and what influence do they have on developmental
outcome? Central to these analyses is a novel method termed piezo-actuated micromanipulation. This
allows scientists for the first time to manipulatemouse eggs and embryos, which are exquisitely sensitive to mechanical trauma.
Based on his work in this area, Perry has developed
a model that seeks to explain how a sperm signals tothe egg to initiate its dedifferentiation to a totipotent
state. In this model, one or more sperm-borne oocyte
activating factors act to trigger signaling networks inthe moments that immediately follow fertilization. A
growing body of evidence indicates that such networks have direct and far-reaching developmental

Neurotrophic factors

Hideki Enomoto
M. D. , Ph. D.
Transgenic mouse and offspring expressing green fluorescent protein

consequences. The characterization of these activating factors at the molecular level is fundamental to an
improved understanding of the process of fertilization,and knowledge of the mechanisms involved has thepotential for application in novel methods of contraception and could open up new avenues in the treatment of some forms of infertility.
In addition to describing the earliest events of a newembryonic life, the Perry laboratory has an interest in
exploiting these findings to develop novel methods ofgenome manipulation. This work includes the development of a new technique for introducing transgene
DNA via co-injection with a nucleus into an egg.
Since such eggs are typically arrested at the second
meiotic metaphase, the method is called metaphase
II (or 'mII') transgenesis. The mII transgenesis method enables the efficient introduction of large DNA segments to produce transgenic mice.

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 forhis work in the molecular cloning of
the human DAN gene. After serving
as the Chief of the Department ofPediatric Surgery, Matsudo Municipal Hospital in Chiba, Japan, hemoved to the Washington University
School of Medicine, St. Louis in
1997, to work as a research associate studying the physiological rolesof the GDNF family of ligands in neural development and function. He
returned to Japan to take a position
as a team leader at the CDB in 2002.

Staff
Team Leader

Hideki Enomoto
Research Scientist

Yukinao Shibukawa
Shiho Ohmori
Technical Staff

Kayoko Inoue
Mayumi Nagashimada
Assistant

Early neural network development is characterized by
the growth of neurons in excess of the number thatwill ultimately populate any given region. Neurons
that have formed synaptic connections with a target
tissue need to establish what is known as a ‘trophic
interaction’ in which the target provides the neuron
with chemical signals necessary for its survival andcontinued function. The supply of such signals is limited and neurons which fail to compete successfully forthese factors undergo the programmed cell death routine, apoptosis, a controlled die-off that establishes
an appropriate balance in the neural population. The
target-derived chemical signals in the trophic interaction are known as neurotrophic factors. The study ofthese signaling proteins dates back to the discoveryof nerve growth factor (NGF) by Levi-Montalcini andcolleagues in the 1950s. Subsequent research has
revealed the diversity of neurotrophic factors, which
are now thought to include neurotrophins (of which
NGF is a member), neuropoietins, fibroblast growth
factors, and transforming growth factors. Hideki Enomoto is primarily interested in this last group, and the
GDNF (for Glial cell line Derived Neurotrophic Factor)
Family Ligands (the GFLs) in particular. By pinpointing the extrinsic signals responsible for inducing normal neural development, he hopes to open up new
avenues for ES cell-based therapies in the treatmentof human neurological disorders.
The GFLs signal through a receptor complex comprising the RET receptor tyrosine kinase and theco-receptor GRFα. In genetic knockout studies, Enomoto discovered that the GFLs play a critical role inthe development of enteric, sensory and autonomic
neurons, establishing the GFLs as a neurotrophic
family of equal importance to the better-known neuro-

Explant culture of primitive sympathetic ganglia in mouse treated with
Artemin, showing robust neurite outgrowthtrophins. Enomoto’s work has shown, however, thatthe role of RET signaling is distinct from that of theneurotrophins in that it is involved in the migration,axonal growth and axon guidance of developing sympathetic nerve cells, not the survival of establishedneurons. This suggests that RET and TRK (the neurotrophin receptor) activate independent signaling pathways to perform their complementary functions.
The Enomoto research team plans to conduct systematic DNA chip analyses of enteric and autonomic neurons to achieve a better understanding of the molecular mechanisms involved in RET and TRK signaling.
And, by gaining insights into the roles played by specific neurotrophic factor signaling pathways, Enomoto
hopes to contribute to the development of stem cellbased neuroregenerative therapies.

Kaori Hamada

StaffTeam Leader

Tony Perry
Research Scientist

Hisashi Kishi
Noriko Yoshida

Publications

Publications

Technical Staff

Satoko Fujimoto
Assistant

Lio Hiroi

Perry ACF, Rothman A, de las
Heras JI, Feinstein P, Mombaerts
P, Cooke HJ, Wakayama T. Efficient metaphase II transgenesis
with different transgene archetypes.
Nat Biotechnol 19:1071-3 (2001).
Perry ACF, Wakayama T, Cooke
IM, Yanagimachi R. Mammalianoocyte activation by the synergistic
action of discrete sperm head com-

50 RIKEN CDB 2002 Annual Reportponents: induction of calcium transients and involvement of proteolysis. Dev Biol 217:386-93 (2000).
Perry ACF, Wakayama T, Kishikawa H, Kasai T, Okabe M, Toyoda Y,
Yanagimachi R. Mammalian transgenesis by intracytoplasmic sperm
injection. Science 284:1180-3(1999).

Enomoto H, Crawford PA, Gorodinsky
A, Heuckeroth RO, Johnson EM Jr,
and Milbrandt J. RET signaling isessential for migration, axonal growth
and axon guidance of developing sympathetic neurons. Development
128:3963-74 (2001).
Enomoto H, Heuckeroth RO, Golden
JP, Johnson EM Jr, and Milbrandt J.
Development of cranial parasympa-

thetic ganglia requires sequential
actions of GDNF and neurturin. Development 127:4877-89 (2000).
Heuckeroth RO, Enomoto H, Grider
JR, Golden JP, Hanke JA, Jackman
A, Molliver DC, Bardgett ME, Snider
WD, Johnson EM, and Milbrandt J.
Gene targeting reveals a critical rolefor neurturin in the development andmaintenance of enteric, sensory andparasympathetic neurons. Neuron
22:253-63 (1999).
Enomoto H, Araki T, Jackman A,
Heuckeroth RO, Snider WD, Johnson
EM Jr, and Milbrandt J. GFRa1deficient mice have deficits in theenteric nervous system and kidneys.
Neuron 21:317-24 (1998).

RIKEN CDB 2002 Annual Report512002 New Faces
Development

Regeneration

Development

Regeneration

Regenerative
Medicine

Creative Research Promoting Program

Sensory Organogenesis
Laboratory for Sensory Development

Supporting Laboratories
Sensory organogenesis

Raj Ladher
Ph. D.
Raj Ladher received his B. Sc. in biochemistry from Imperial College, London in 1992 and his Ph. D. in developmental biology from the National
Institute for Medical Research in
1996, for his thesis on the cloning of
the Xenopus homeobox gene, Xom,
under the supervsion of Jim Smith.
He worked as a research associate
at King’s College, London from 1996
to 2000 in the lab of Pip FrancisWest, and first came to Japan duringthat 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 RIKEN CDB in 2002.

Staff
Team Leader

Raj Ladher
Technical Staff

Susan Boerner
Assistant

Noriko Hiroi

Sensory systems in vertebrates represent one of the
pinnacles of evolutionary achievement, wedding
intricate organic structures with specialized cellularfunction to permit the animal to apprehend its environment by processing a range of physical and chemical
stimuli. The development of these systems involves
an extraordinary level of coordination between cells
and emergent tissues at very early stages of embryogenesis, and provides an ideal model for the study ofsuch inductive interactions in general. Research in
Raj Ladher’s lab focuses on the induction of the inner
ear in the chick embryo, a model system chosen forthe chick’s tractability to classical embryological techniques and the opportunities it affords for investigating the processes of developmental program initiation, cell differentiation and the integration of discrete
cellular motifs in a structurally and neuronally specialized organ.

Inner ear induction
Sense organs originate in placodes, thickened
regions within the early ectoderm. The inner ear arises from the otic placode, a cluster of cells which, in aprocess known as ‘invagination,’ recedes from theectodermal surface and is folded over, resulting in theformation of a segregated hollow called the otocyst
which ultimately gives rise to the mature inner ear.
Raj is studying the inductive factors that dictate thelocation and timing of these developmental processes. In a series of tissue manipulation experiments
conducted previously, he identified a trio of signaling
factors – FGF-8, FGF-19, and Wnt-8c – each ofwhich localizes in a separate germ layer, and which
cooperate to induce inner ear development. In thisnetwork, it appears that FGF-8, expressed in theendodermal layer, induces the expression of FGF-19
in the immediately overlying mesoderm. FGF-19 inturn induces Wnt-8c in the neuroectoderm, triggering

Sensory cellular differentiation
The operation of factors from non-ectodermal layers
seems to be critical to cell differentiation in the inner
ear as well. In a series of experiments in which hetested various tissue recombinations of sections fromgerm layers neighboring the site of inner ear development, Raj found that even though the mesodermal
inducer FGF-19 could initiate inner ear formation, differentiation did not occur. He is currently testing the
hypotheses that FGF-19 may have an inhibitory
effect on differentiation at later stages, and that it
may play a role in recruiting cells to the developing
inner ear.
In the future, the Ladher lab looks to extend its
research scope to other model systems, with the goalof determining whether some as-yet unidentified general mechanisms are at work in sensory organogenesis.

Publications
Karabagli H, Karabagli P, Ladher
RK, Schoenwolf GC. Comparison of
the Expression Patterns of Several
Fibroblast Growth Factors during
Chick Gastrulation and Neurulation.
Anat Embryol 205:365-70 (2002).
Karabagli H, Karabagli P, Ladher
RK, Schoenwolf GC. A Survey of
Fibroblast Growth Factor Expres-

The supporting laboratories offer technical support, develop new technology,
and conduct independent research
projects. Their services are available
to all CDB researchgroups and teams.

a complex regulatory loop in which FGF-19 and Wnt8c maintain each other’s expression for the durationof early ear development.

sion during Chick Organogenesis.
Anat Rec 268:1-6 (2002).

Expression patterns of fgf-19 andwnt-8c. The gene products co-operate
to induce the inner ear in non-neural
ectoderm, adjacent to the wnt-8c
neural domain and above the fgf-19
mesodermal domain.

Ladher R, Anakwe K, Gurney AL,
Schoenwolf GC, Francis-West PH.
Identification of synergistic signals
initiating inner ear development. Science 290:1965-8 (2000).

Late-stage embryo of C. elegans ced-5 mutant
Photo: Asako Sugimoto

52 RIKEN CDB 2002 Annual Report

Development

Regeneration

Regenerative
Medicine

Supporting Laboratories

Laboratory for Cellular Morphogenesis

Morphology
Shigenobu Yonemura
Ph. D.
Shigenobu Yonemura received his B.
Sc., M. Sc. and Ph. D. from the University of Tokyo, earning his doctorate in1988 for thesis work under Prof. I.
Mabuchi. He spent a year as a postdoctoral fellow at the same institution
before moving to pursue a fellowship at
Johns Hopkins University from 1989 to
1990. He returned to Japan as an assistant professor in the Department of Cell
Biology at the National Institute forPhysiological Sciences, where heremained until 1995. He joined the Kyoto University Faculty of Medicine thesame year, first as an assistant professor, then as a lecturer in the Department of Cell Biology, a position heretained until his appointment as CDB
team leader in 2001.

Staff
Team Leader

Shigenobu Yonemura
Research Specialist

Yukako Nishimura
Kazuyo Minakuchi
Research Associate

Nagatoki Kinoshita
Technical Staff

Makiko Uwo
Naoko Inoue
Yuka Miyake

Cell morphology and imaging
Since the advent of the microscope in the 17th century, the development of novel methods of delineating
and distinguishing biological structures at evergreater resolutions has stood at the heart of progressin the life sciences. The most powerful electron microscopes now provide scientists with the ability to visualize biological objects as small as 2 nanometers in
size, which has opened up new windows of insight
into the relationship between form and function at the
sub-cellular scale. In theory, even greater resolutions
could be achieved, but the limits imposed by thematerials under study – organic molecules withsometimes fragile structures – have kept such microscopes from realizing their full potential. One of themain goals in the field of microscopy is the development of new techniques in specimen preparation andimage enhancement that will allow scientists to overcome these limitations and probe the hidden world
even more deeply.
Shigenobu Yonemura brings a background in celland molecular biology to bear on visualization methods and technology with applications in the study ofanimal development. He seeks to explore the links
between cell shape, function and behavior by improving the means by which the cellular architecture canbe brought to light. His special emphasis is on developing ways to localize with greater precision themolecular components of the cytoskeleton, a complex structure that determines cell morphology and

54 RIKEN CDB 2002 Annual Report

participates in the movement and interactions of cellswith their surroundings.

Visualizing Rho-family proteins
The Rho (for Ras Homologous) family of proteins –principally Cdc42, Rac and Rho – has been extensively studied for its effects on the structural activity ofcells. These proteins serve as molecular switches
capable of interacting with the actin cytoskeleton andinducing cells to form a range of surface structures,such as cylindrical microvilli projections and stress fibers that serve to anchor cells to the substratum. The
Yonemura research team is studying the localizationof Rho family members with the goal of gaining
insight into how these molecules contribute to cellularmorphogenesis.

Specimen preparation is considered
to be as important as
microscope power in
the imaging of biological samples
One of the perennial technical challenges in the
microscopic study of cells has been assuring that the
specimen is not damaged or altered in the process of
preparing it for examination. Indeed, specimen preparation is considered to be just as important as microscope power in the imaging of biological samples. In
previous work on improving specimen preparation for
immunofluorescence imaging, Yonemura introduced

Publications

Rho is rapidly recruited to elongating microvilli on EGF-stimulated A431 cells.

the denaturing agent trichloroacetic acid (TCA) for its
fixative and preservative properties. TCA fixation produces good results in the visualization of Rho and
Rac with few of the drawbacks of other traditional
methods.
However, recent localization studies of the Rho family
have produced inconsistent, sometimes contradictory, results. In studies using immunochemical andgreen fluorescent protein (GFP) tagging of the Rho
protein, widely divergent distribution patterns werereported, calling into question the reliability of thesevisualization techniques. Yonemura has been developing monoclonal antibodies specific to each of thethree Rho isoforms – RhoA, RhoB and RhoC. To
ensure the validity of the staining of the endogenous
target protein, Yonemura expressed an epitopetagged target protein in cultured cells. These cells
were processed for immunofluorescence imaging
using antibodies against both endogenous andepitope-tagged target proteins. As antibodies to theendogenous protein should also bind to the epitopetagged proteins, the failure of an imaging method toshow a high degree of correspondence in the localization of the two indicates a flaw in the antibody-fixation
protocol. By introducing such improvements to fixation and evaluation methodologies, Yonemura hopes
to reinforce scientific rigor in the field of microscopic
visualization and to be able to apply these techniques
to the study of other members of the Rho family.

simple nor direct. Rather, Rho plays an integral partin an intracellular signaling network of great, but still
unfathomed, complexity. Yonemura is working on
one aspect of this network – the interactions between
Rho, protein kinase C, and ERM, a cross-linker protein that joins actin to the cell membrane. Using theyeast two-hybrid system to identify proteins in this signaling network, Yonemura seeks to address some of
the many as-yet unanswered questions of Rho function.

Kikuchi S, Hata M, Fukumoto K,
Yamane Y, Matsui T, Tamura A,
Yonemura S, Yamagishi H, D Keppler, Tsukita Sh, and Tsukita Sa.
Radixin deficiency causes conjugated hyperbilirubinemia with loss of
Mrp2 from bile canalicular membranes. Nature Genet 24:320-5(2002).
Yonemura S, Matsui T, Tsukita Sh,
and Tsukita Sa. Rho-dependent and–independent activation mechanismsof ezrin/radixin/moesin proteins: anessential role for polyphosphoinositides in vivo. J Cell Sci 115:2569-80(2002).

In addition to its full-time research efforts, the Yonemura team offers common-use microscopy services
to other CDB laboratories. By providing fluorescenceand electron microscopic imaging and instruction intechniques, the team hopes to help ensure the quality, fidelity and ease-of-interpretation of visualization
data generated at the Center.

The actin cytoskeleton
The skeletal framework of the cell is built up of an
intricate assembly of filaments and accessory proteins, cooperating to stabilize the cell and give it
shape. One of the main types of filaments is the actin
filament (or microfilament), which, in addition to determining surface morphology, also play a critical role indriving cellular movement. As described above, theRho-family proteins help to regulate the function of
the actin cytoskeleton and, thereby, many aspects ofcellular behavior as a whole. But the relationship between Rho expression and actin response is neither

In dividing animal cells, Rho (red)is concentrated in the furrow
region. Blue: nuclei, Green: microtubules.

RIKEN CDB 2002 Annual Report55

Development

Regenerative
Medicine

Regeneration

Supporting Laboratories

Experimental MiceLaboratory for
Animal Resources andGenetic Engineering

The study of model organisms and systems is integral to biological research. Such systems provide scientists with the means to search for broadly sharedmechanisms underlying developmental and regenerative processes across species, and conversely toidentify those traits that earn each species its uniquebranch on the phylogenetic tree. The mouse is one of
the most important and widely used model organisms
in science today, prized for its amenability to genetic
manipulation, its high level of homology with humans,
and the trove of data regarding its physiology, genetics and development that has accumulated over nearly one century of intensive scientific research.

Staff
Team Leader

Shinichi Aizawa
Postdoctoral Researcher

Kenryo Furushima
Research Associate

Kazuki Nakao
Technical Staff

Hitoshi Miyachi
Rika Nakayama
Hiroshi Kiyonari
Technical Assistant

Chiaki Tamamura
Akemi Hara
Takashi Nomura
Tsunehisa Fujita
AssistantKaori Nasu

The Laboratory for Animal Resources and Genetic
Engineering (LARGE) provides an important suite ofservices related to the generation of experimental
mice to labs within the CDB and around Japan. In its
role as a CDB support laboratory, the LARGE team,under CDB Deputy Director Shinichi Aizawa, produc-es transgenic and knockout mouse models to thespecifications of scientists working in a wide range ofgenetic, embryological and biomedical research projects, maintaining the highest quality standards andrapid turnaround to ensure fast and easy access toresearchers working within the Center and throughout the country. In addition to these core functions,the LARGE staff provides a number of other services,such as nuclear transfer cloning, isolating homologous recombinants and culturing embryonic stem
cells, and works to develop improved methods ofsample handling and storage, such as higherefficiency modes of cryopreservation. The lab also
performs a number of maintenance and logistical
functions, such as the specific pathogen free (SPF)
housing, cleaning, processing and distribution of animals.
In the coming year, the LARGE team plans to expand
its services and initiate new programs, notably thegeneration of target vectors from sequence information, and the independent production of novel
genetically-modified constructs, a drive that is anticipated to generate on the order of 80-100 new mutant
strains per year. Such strains serve as research platforms with the potential to provide new insights into a
range of important research problems, from themechanisms of organ development to the genetic
bases of human disease. The lab will also function aspart of Japan’s system of Mouse Embryo Banks, witha special emphasis on producing, storing and cataloging embryos for use in developmental biology andregenerative medical research.

Publications
Ino M, Yoshinaga T, Wakamori M, Miyamoto N, Takahashi E, Sonoda J, Kagaya T, Oki T, Nagasu T, Nishizawa Y, Tanaka I, Imoto K, Aizawa S, Koch S, Schwartz A,
Niidome T, Sawada K, Mori Y. Functional disorders of thesympathetic nervous system in mice lacking the α1B subunit (Cav2.2) of N-type calcium channels. Proc Natl Acad
Sci 98:5323-8 (2002).
Kim JM, Nakao K, Nakamura K, Saito I, Katsuki M, AraiK, and Masai H. Inactivation of Cdc7 kinase in mouse ES
cells results in S-phase arrest and p53-dependent cell
death. EMBO J 21:2168-21679 (2002).

Staff
Sequencing Lab
Research Scientist

Hiroshi Tarui
Technical Staff

Osamu Nishimura
Yukako Hirao
Takako Sano

Fukami K, Nakao K, Inoue T, Kataoka Y, Kurokawa M,
Fissore RA, Nakamura K, Katsuki M, Mikoshiba K, Yoshida N, and Takenawa T. Requirement of phospholipase
Cdelta4 for the zona pellucida-induced acrosome reaction.Science 292:920-3 (2001).

Nishinakamura R, Matsumoto Y, Nakao K, Nakamura K,
Sato A, Copeland NG, Gilbert DJ, Jenkins NA, Scully S,
Lacey DL, Katsuki M, Asashima M, and Yokota T. Murine
homolog of SALL1 is essential for ureteric bud invasion in
kidney development. Development 128:3105-15 (2001).

Tsuzuki T, Egashira A, Igarashi H, Iwakuma T, Nakatsuru
Y, Tominaga Y, Kawate H, Nakao K, Nakamura K, Ide F,
Kura S, Nakabeppu Y, Katsuki M, Ishikawa T, and Sekiguchi M. Spontaneous tumorigenesis in mice defective in
the MTH1 gene encoding 8-oxo-dGTPase. Proc Natl
Acad Sci 98:11456-61 (2001).

Model Organisms
YEAST

ROUNDWORM

Common name : fission yeast
Scientific name : Schizosaccharomyces pombe
Yeasts are known for their use as fermenting
agents in the production of wine and beer, butthey are also found in life sciences laboratories
around the world, performing a very different but
equally valuable service. The fission yeast has served as an experimental model for the past fifty
years, thanks to it amenability tomolecular genetic studies and theease which it can be grown andmanipulated in culture, and it isperhaps best known for its contributions to our knowledge of chromatin structure.

Common name : nematode
Scientific name : Caenorhabditis elegans
With exactly 959 somatic cells in the body of a normal adult, the nematode offers perhaps the bestcharacterized model of a multicellular organism
possessing simple nervous, muscle and reproductive systems. It is possible to track the fates of individual cells in its transparent body on a microscope slide glass, and the exact fate of every cellin every cell lineage is known. In 1998, geneticists
added to that body of knowledge by sequencing
the nematode genome, which was found to contain around 19,000 genes, many of which havehuman homologs. The discovery of the RNA interference mechanism that allows genes to be functionally 'knocked down' has further fueled interestin these tiny creatures as model organisms.

FLATWORM
Common name : planarian
Scientific name : Dugesia japonica
Found in freshwater streams and ponds, planarians are small worms only a few millimeters longwhich possess simple brains, nervous and digestive systems, and a strangely endearing pair of
rudimentary eyes. Their most striking characteristic, however, is their regenerative ability; cut a single animal into dozens, even hundreds of pieces,and each fragment can develop into a new individual. This remarkable capacity has attracted theattention of biologists interested in the mechanisms of regeneration and much progress has
been made in recent years in understanding how
the planarian achieves its death-defying multiplication. The answer lies in the totipotent cells distributed throughout its
tiny body, cells it ishoped will provide
clues to the emerging field of regenerative medicine.

FLY
Common name : fruit fly
Scientific name : Drosophila melanogaster
The fruit fly came into its own as a laboratory model when scientists in the early twentieth century
discovered Drosophila was easy to keep, feed
and breed, and by the 1920s fruit flies were thesubject of extensive experiments in heredity in thelabs of Nobel Prize-winner T. H. Morgan and elsewhere. As its popularity as a model increased,data on all aspects of fly biology, from molecular
genetics to learned behaviors, burgeoned. The discovery of homeotic genes in the fly, which determine segment identity and which were found to beconserved in animals from worms to humans,
made Drosophila a favorite of developmental biologists as well.

Sequencing Lab
The CDB sequencing lab provides DNA sequencing and analysis services for use by all laboratories at the Center. Equipment is available forsmall, medium and large-scale requests, with turnaround times from less than one week for smaller scale projects to one month for longer jobs,
at rates of up to 1,920 samples per day. Smaller jobs are completed especially rapidly, and results can be delivered as soon as the following day
when sequence-ready samples are submitted. Analysis services for editing, homology search, and assembling are also available. All work
requests can be submitted and tracked online, and results are returned to the applicant's personal folder on the CDB intranet file server, making
it possible for CDB research staff to sequence samples of interest without leaving their desktop computers.

56 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report57Model Organisms

Administration and Activities

FISH

BIRD

Common name : zebrafish
Scientific name : Danio rerio
The zebrafish, with its short reproductive cycle
and high number of eggs per clutch, is one of themost popular vertebrate systems used in genetic
analysis. Its rapid development has also made it
popular in the developmental biology and embryology communities, as has the fact that its eggs are
externally fertilized and translucent, making it
possible to follow its developmental progresseasily using a dissecting microscope. The
tractability of this fish
to gene knockdown
methods and the existence of techniques for
tracing the fates ofindividual cells havealso helped to establish it as a favorite
system in developmental genetics.

Common name : chick (chicken)
Scientific name : Gallus gallus
The chick embryo has been in use as a model
since the time of Aristotle, making it one of themost exhaustively studied systems in biology. The
egg's extrauterine development made it attractivein the days before surgical techniques. The chicken's regular laying cycle and the fact that fertilized
eggs can be cold-stored for several weeks, temporarily freezing their development, ensures constant access to embryos. Chick embryonic development shows some similarity to that of mammals, and the flat blastodisc of the chick is morphologically similar to the human embryo at thesame stage of development. The introduction oftechniques for embryonic sectional explantationand the production of chick-quail chimeras has
only served to add to the popularity of the chickembryo as a model system.

FROG
Common name : African clawed frog
Scientific name : Xenopus laevis
The African clawed frog has a long history of use
in the study of vertebrate embryology and development. As aquatic animals, they undergo external
development, making it relatively
easy to manipulate the fertilized egg
as compared to animals in which theembryo grows within the uterus.
Their eggs are also quite large,
about 1 mm in diameter, which isabout ten times the size of a mouse
oocyte, allowing for various surgicalmanipulations and biochemical
analyses. Ovulation can be induced
relatively simply using luteinizing hormone, which
eliminates some of the problems associated withextended reproductive cycles. And their rapid progress from fertilization to neurulation in less than
24 hours provides scientists with a dynamic picture of the first stages of life.

MOUSE
Common name : mouse
Scientific name : Mus musculus
For nearly one
hundred years
the mouse has
been the animalof choice for modelinghuman
genetics and disease. This application has beenborne out by the recent completed sequencing of
the mouse genome, which revealed an extremely
high homology between the mouse and humangenomes. As fellow mammals, humans and micealso share many physiological traits and susceptibilities, making the mouse an attractive model inbiomedical studies. Its rapid breeding cycle andsmall size further add to its appeal, and it is little
wonder that scientists have chosen this animal fordeveloping or popularizing a wide range of genetic techniques from transgenesis to gene knockouts to cloning by nuclear transfer.

Kobe night skyline
Photo: Yutaka Doi

58 RIKEN CDB 2002 Annual ReportAdministration
RIKEN (The Institute of Physical and Chemical Research) was established in 1917 to conduct comprehensive research in science and technology and to disseminate the results of its research and development efforts. RIKEN is organized as a public corporation under the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and conducts research in the fieldsof physics, chemistry, basic life sciences, engineering and medicine at laboratories located across Japan. The Center for Developmental Biology was established as the first research center within the RIKEN Kobe Institute in April 2000.

2002 CDB Budget
Facility Expenses(energy expenses,maintenance,etc.)

FY2002

570
570

Research Management Expenses(personnel expenses,etc.)

Educational Activities
Encouraging young scientists to participate in advanced research projects as they prepare for careers in the life sciences isessential to ensuring the development of future generations of researchers. The CDB has collaborative educational programswith a number of graduate schools in Japan, providing students with opportunities for hands-on laboratory benchwork and lectures on topical issues in development and regeneration. The center also has also accepted 47 students from other universities
as research assistants or trainees. In addition to regularly scheduled scientific seminars, students can attend events such as the
CDB Forum research progress series and the Center's annual retreat. The Junior Research Associate program established by
RIKEN also provides financial support to 4 graduate students working in CDB labs.

Research Expenses

2,000
2,000

CDB Staff (December 2002)

3,139
3,139

(Million Yen)

Group Directors/Team Leaders …
Research Scientists ……………
Research Associates ……………
Technical Staff ……………………
Assistants …………………………
Visiting Researchers ……………
Collaborative Scientists …………
Student Trainees …………………
Part-time Staff ……………………
Research Promotion Division …
Other ………………………………

26
71
3
82
23
9
19
34
46
38
28

Total ……………………………… 379

Advisory Council
The CDB Advisory Council, chaired by Dr Igor Dawid (National Institute of Child Health and Human Development,
NIH) submits regular external reports regarding the scientific administration and the state of research progress at theCenter. The ten-member Council includes top scientists inrelated fields from Japan and around the world and acts as
an unbiased review board for CDB research activities.

Institutional Review Board
The Institutional Review Board includes representatives
from local academic, media and lay organizations as wellas CDB research staff, and meets regularly to review anddiscuss the ethical and social implications of programs andinvestigations being conducted at the CDB. The results of
the Board's discussions are submitted to the Center Director and taken into consideration when planning research
activities.

2002 Graduate School Affiliates
Graduate School of Biostudies, Kyoto University
CDB: Masatoshi Takeichi
Kobe University, Graduate School of Science and Technology
Department of Life Science
CDB: Kiyokazu Agata, Shigeo Hayashi, Hitoshi Sawa
Department of Developmental and Regenerative Medicine
CDB: Hitoshi Niwa, Masahiko Hibi, Asako Sugimoto

60 RIKEN CDB 2002 Annual Report

RIKEN CDB 2002 Annual Report612002 Seminars
The CDB hosts regular seminars by distinguished speakers from within Japan and abroad as part of its committed
effort to promote the borderless exchange of scientific information. All CDB seminars are held in English. In addition to these seminars, the CDB also holds monthly Forums, in which CDB researchers share findings with their colleagues, and a range of informal lecture offerings in English and Japanese.

Date

Title

Speaker

Date

Title

Speaker

2002.4.16

The rediscovery of the lymphatic system

Michael Detmar

2002.10.30

Calcineurin, a component of G-protein coupled phosphorylation pathways, isJoohong Ahnn

2002.5.27

Germline stem cells and the niche in Drosophila melanogasterToshie Kai

2002.6.7

The evolution of homeobox gene cluster

P. W. Holland

2002.7.2

How the cavefish lost its eyes: A problem in development and evolution

Yoshiyuki Yamamoto

2002.7.23

Control of epithelial-mesenchymal transitions in the neural crest

Don Newgreen

2002.7.24

Cell polarity and asymmetric cell divisions in the Drosophila PNS

Francois Schweisguth

2002.8.8

The 1st CDB Meeting; Head development (I)(multiple speakers)

2002.8.13

The 2nd CDB Meeting: Left-right determination in the lancelet embryo

Kinya Yasui

2002.8.13

The 2nd CDB Meeting: Molecular studies of hemichordate development: New

Kumifumi Tagawa

insights into bilaterian evolution
2002.8.13

The 2nd CDB Meeting: Body plan of the sea urchin embryo

Koji Akasaka

2002.8.19

Transposon-mediated transgenesis and insertional mutagenesis in zebrafish

Kouichi Kawakami

2002.9.5

Construction of human artificial chromosome and its possible application for

Mitsuo Oshimura

regenerative medicine

involved in movement, fertility, egg laying, and growth in C. elegans
2002.11.5

Clonal identification of pluripotent stem cells in the developing liver and pan-

Hideki Taniguchi

creas using flow cytometric cell sorting
2002.11.14

Sensory signaling and neural modulation in the nematode Caenorhabditis

Ikue Morielegans
2002.11.20

Distinct Rac regulators in cell migration, axon outgrowth and cell-corpse

Yi-Chun Wu

engulfment in C. elegans
2002.11.21

Regulation of expression and function of cell differentiation factor Nkx2.2

Hirotaka Watada

2002.11.21

The tao of stem cells in the germline

Haifan Lin

2002.11.28

Transcriptional regulation of signalling competence in derivatives of the spe-

Joshua M. Brickmanmann organizer
2002.12.5

Nuclear organization of chromosomes in fission yeast

Yasushi Hiraoka

2002.12.5

Dynamic organization of the nuclear envelope

Tokuko Haraguchi

2002.9.12

CDB Meeting: Head Development (II)(multiple speakers)

2002.12.6

Cell signaling mechanisms that establish left-right asymmetry

H. Joe Yost

2002.9.23

Models for the generation of the primary body axes in higher organisms

Hans Meinhardt

2002.12.6

Making and breaking the embryonic mouse heart: A transgenic approach

Simon J. Conway

2002.9.27

A 600 kDa microtubule-associated factor that contributes to maintaining geno-

Yoshihiro Nakatani

2002.12.9

New insights into gastrulation and neural induction in the mouse embryo

Jerome Collignon

2002.12.9

Evolution of complex form and function in the developing mammalian heart

Richard Harvey

mic stability
2002.10.7

Essential role of Sall1 in kidney development

Ryuichi Nishinakamura

2002.12.11

What do we know of hematopoietic stem cells?

Hideo Ema

2002.10.22

Morphological asymmetry in dividing neuroepithelial cells:Its impact on brain

Takaki Miyata

2002.12.16

Regulation of motor pool sorting by expression of Type II cadherins

Stephen Price

2002.12.17

Use of the Cre/LoxP system to study mouse development by a non-biased

Werner Muller

histogenesis
2002.10.24

Analysis of the function of isthmin in zebrafish embryos

Isato Araki

2002.10.24

Genetic regulation of mouse urogenital organogenesis

Richard R. Behringer

2002.12.24

Human central nervous system stem cells for cell transplantation

Nobuko Uchida2002.10.30

Systems-biological analysis of dynamic and complex biological systems:

Hiroki Ueda

2002.12.25

Identification of novel genes important for pluripotency and tumorigenicity of

Shinya Yamanaka

Application to mammalian circadian rhythms

approachmouse ES cells

The CDB Opening Symposium
A New Paradigm in Developmental Biology

2002.4.22

Introduction to CDB

Shun-ichi Kobayashi (RIKEN President)
Masatoshi Takeichi (CDB Director)

Novel Concepts of Development

Igor Dawid (NIH/NICHD)Peter Gruss (Max-Planck-Institute of Biophysical Chemistry)

Cellular Mechanisms

William Chia (MRC Centre for Developmental Neurobiology)
Elaine Fuchs (The University of Chicago)
Nobutaka Hirokawa (University of Tokyo)

Genetic Reprogramming andRegenerative Medicine

Austin Smith (University of Edinburgh)
Yoshiki Sasai (CDB)
Takayuki Asahara (St. Elizabeth's Medical Center)

Concluding Remarks

Shin-ichi Nishikawa (CDB)

62 RIKEN CDB 2002 Annual Report

Igor Dawid speaks at the Opening Symposium (April 22, 2002)

RIKEN CDB 2002 Annual Report63Medical Industry Development ProjectCity of Kobe
The CDB is located in the international
city of Kobe. This port city has been
the major entrance to western Japanfor over a century, giving the city acosmopolitan atmosphere and unique charm. With 1.5 million residents, the city is the sixth-largest in Japan, offering a perfect mix of urban sophistication and access tosome of Japan's more beautiful natural and historicalattractions.

supply. The information infrastructure is well-developed,
and access to broadband internet connections, mobile
phone service andsatellite TV is moreprevalent than nearly anywhere else in
the world.

Situated in the heart of the Kansai region of westernJapan, Kobe is a short driveor train ride away from OsaKyotoka and Kyoto, and sits nestled in the foothills of the
Rokko Mountains facing out
Kawanishi
onto the Inland Sea. The
Miki
Takarazuka
climate is pleasant andNishinomiya
Osaka
Kobe
features four distinct seaItami Airport
Shin-Kobe Sta.
sons similar to those in othAshiya
Osaka
Amagasaki
Sannomiya
Akashier temperate climes. An
Nara
CDB
exceptional public transporKobe Airport(2005)
tation system provides convenient access to destinations within Kobe andAwaji
JR Shinkansen
around Japan, and the KanJR
sai International Airport
Toll Road
Kansai International
Airportserves as the region's hub
for overseas travel.
Costs of living are comparable to those found in many
Western cities. Kobe's shopping district is consideredsecond-to-none in Japan, and shops specializing in imported goods make it easy to purchase items from around theworld. Houses and apartments are clean, comfortable andmodern, and hotels and convention space are in ample

SapporoSendaiKanazawa
Okayama
Hiroshima

KOBE
Osaka

FukuokaKagoshima

Takamatsu

Yokohama
NagoyaTokyo

Major national universities and cultural
attractions are located both within thecity and in the surrounding Kansai region,
providing a stimulating intellectual and educational
climate. And thanks to theadvent of global communications and information
technology, Kobe is securely connected to the worldwide network of ideas.
Exciting. Comfortable. Beautiful. Fun. These are someof the words our visitorsfrom overseas have used todescribe their experience inKobe. The Center offers
dedicated support to foreign research staff andguests, from assistancewith accommodations andvisas to short-term visitors,
to renting apartments andhelp with setting up laboratories for newly arrived
staff as they acclimate themselves to life in Japan.
Whether as a traveler or apotential
resident,
we
encourage you to visit the
CDB and the city of Kobe tosee for yourself how enjoyable life in Japan can be.

In 1998, Kobe inaugurated its Medical Industry Development
Project, marking the launch of one of Japan's first integrated
biomedical research parks. The CDB plays a central role as abasic research facility in this growing international center of
excellence. The CDB facilities are connected by an elevatedwalkway to the adjacent Institute for Biomedical Researchand Innovation (IBRI) research labs, fostering communications and collaborations between researchers at both locations. And the completion of the IBRI clinical testing hospital(scheduled for April 2003) will further expand the translational
research opportunities for CDB scientists and their collaborators.

The Medical Industry Development Project seeks to draw participants from academia, government research institutions
and life sciences industries by providing world-class facilities
and infrastructure, and a range of support services for basic
and corporate research and development. Plans for 2003
include the construction of a Translational Research Informatics Center and a Biomedical Accelerator business incubation
facility, and the continued recruitment of biomedical researchfirms from within Japan and abroad. CDB scientists are already working in close collaboration with colleagues at the IBRI
and industry research labs in projects that promise to realize
the potential of the bench-to-bedside translational research
approach.

KIMEC Center

CDB Bldg C
1F communal labs
2F~5F labs
6F library, seminar room

Translational Research Informatics Center
Medical Informatics for Preclinical and Clinical Studies

CDB Research Aquarium

Kobe International
Business Center 1 and 2
rental labs and offices

IBRI Lab Bldg

CDB Animal Facility
1F mouse facility, transgenic/KO lab
2F mouse facility, operation/virus rooms

1F rental labs
2F CDB labs (stem cells)
3F CDB labs (stem cells)·FACS
4F·5F Cell Processing Center

IBRI Clinical Bldg
1F operation room, cafeteria
2F outpatient
3F clinical lab
4F office
5F hospital

CDB Bldg B
1F mouse/rat facility
2F communal facility

64 RIKEN CDB 2002 Annual Report

CDB Bldg A

IBRI Medical Imaging

1F office, EM lab, lobby, radioactivity lab
2F communal facility, cold room, virus lab
3F~6F labs
7F labs, library, seminar room, lounge

1F MRI(3T), open MRI, CT-guided irradiation
2F PET

RIKEN CDB 2002 Annual Report65