2002 Annual Report 2002 Annual Report

RIKEN Center for Developmental Biology

RIKEN Center for Developmental Biology 2002 Annual Report 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: [email protected]

Production

Text Douglas Sipp

Content Development Naoki Namba Douglas Sipp

Design TRAIS K. K.

Photography Yutaka Doi

On The Cover Distribution of the ERM proteins ezrin (green) and moesin (red) in the forelimb 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 Contents

■ Organization ……………………………………………… 1 RIKEN Center for Developmental Biology ■ Message from the ……………… Director/Introduction 2/3 ■ 2002 Highlights …………………………………………… 4/5

Deputy Directors Core Program Shin-ichi Nishikawa Shinichi Aizawa ■ Cell Adhesion and Tissue Patterning (Masatoshi Takeichi) 8/9 ■ Stem Cell Biology (Shin-ichi Nishikawa) 10/11 Executive Advisor ■ Vertebrate Body Plan (Shinichi Aizawa) 12/13 Michio Seki ■ Morphogenetic Signaling (Shigeo Hayashi) 14/15 Director ■ Cell Asymmetry (Fumio Matsuzaki) 16/17 Masatoshi Takeichi ■ Organogenesis and Neurogenesis (Yoshiki Sasai) 18/19

Institutional Review Board ■ Evolutionary Regeneration Biology (Kiyokazu Agata) 20/21

Zentaro Kitagawa Vice-Director, International Institute for Advanced Studies

Hiroko Ueno Creative Research Promoting Program Representative, Laboratory for Media and Communication

Mako Tanaka ■ Neural Network Development (Chihiro Hama) 24/25 Director, Kobe Film Office

Kazuto Kato ■ Vertebrate Axis Formation (Masahiko Hibi) 26/27 Associate Professor, Institute for Research in Humanities, Kyoto University ■ Positional Information (Shigeru Kondo) 28/29 Tsutomu Miki Kurosawa IEXAS, Osaka University Medical School ■ Evolutionary Morphology (Shigeru Kuratani) 30/31 Yasushi Yukinari ■ Cell Migration (Kiyoji Nishiwaki) 32/33 Staff Writer, Science News Department, The Yomiuri Shinbun ■ Pluripotent Cell Studies (Hitoshi Niwa) 34/35 Shin-ichi Nishikawa Deputy Director, RIKEN Center for Developmental Biology Group Director, Laboratory for Stem Cell Biology, ■ Mammalian Epigenetic Studies (Masaki Okano) 36/37 RIKEN Center for Developmental Biology ■ Cell Fate Decision (Hitoshi Sawa) 38/39 Kiyokazu Agata Group Director, Laboratory for Evolutionary Regeneration Biology, ■ Developmental Genomics (Asako Sugimoto) 40/41 RIKEN Center for Developmental Biology ■ Body Patterning (Yoshiko Takahashi) 42/43

Advisory Council ■ Genomic Reprogramming (Teruhiko Wakayama) 44/45 ■ Embryonic Induction (Hiroshi Sasaki) 46 Igor Dawid, Chair Laboratory of Molecular Genetics National Institute of Child Health and Human Development, NIH (USA) ■ Germline Development (Akira Nakamura) 47

William Chia ■ Chromatin Dynamics (Jun-ichi Nakayama) 48 MRC Center for Developmental Neurobiology (UK) ■ Stem Cell Translational Research (Takayuki Asahara) 49 Elaine Fuchs Howard Hughes Medical Institute ■ Mammalian Molecular Embryology (Tony Perry) 50 Department of Molecular Genetics and Cell Biology University of Chicago (USA) ■2002 New Faces Neuronal Differentiation and Regeneration (Hideki Enomoto) 51 Hajime Fujisawa Division of Biological Science ■ Sensory Development (Raj Ladher) 52 Nagoya University Graduate School of Science (Japan)

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) 54/55 Yoichi Nabeshima Animal Resources and Genetic Engineering (Shinichi Aizawa) / Sequencing Lab 56 Department of Pathology and Tumor Biology ■ Kyoto University Graduate School of Medicine (Japan)

Tatsutoshi Nakahata Department of Pediatrics Kyoto University Graduate School of Medicine (Japan) ■ Model Organisms ………………………………… 57/58

Austin Smith ■ Administration/Educational ……………… Activities 60/61 Center for Genome Research University of Edinburgh (UK) ■ 2002 Seminars ………………………………… 62/63 Yoshimi Takai ■ City of Kobe/Medical Industry Development Project … 64/65 Department of Molecular Biology and Biochemistry Osaka University Graduate School/Faculty of Medicine

RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 1 Development

Regeneration

Center for Developmental Biology Regenerative Medicine

Masatoshi Takeichi Director, CDB

Message from the Director Why Study Development?

2002 represented a landmark year in the history of the RIKEN class research setting, with the active support of local and The study of developmental biology traces its roots back to Aristotle and the observations Center for Developmental Biology, one which saw the comple- national governments and participation by public, academic he made on the changes that take place in hen's eggs as the embryos grow from a form- tion of its main physical facilities, the addition of seven out- and corporate research organizations. A spirit of collegiality less mass into a highly organized and recognizable chick. Scientists today continue to ask standing new laboratories, and the official opening of the Cen- and international cooperation pervades the atmosphere here, many of the same questions about development that intrigued natural philosophers in the ter itself in July. It has been a true pleasure to be able to play and I look forward to seeing the results of collaborations bet- past, but at dramatically increased levels of sophistication and complexity. How do the the dual roles of center director and head of the laboratory for ween labs within the CDB with their colleagues at the Center, organs of the body arise? How do cells differentiate? What enables animals to reproduce Cell Adhesion and Tissue Patterning and to watch as the CDB as well as their counterparts in the region, throughout Japan, and transmit genetic traits to their offspring? What are the biological bases underlying evo- grew from no more than an inspired idea in 2000 to become and around the world. lution? The science of development now extends to and impacts upon nearly every field of one of the largest developmental biology research centers in biology in its quest to determine how the information stored in a fertilized egg can trigger the world today. It has been my goal as director of the Center for Developmen- and orchestrate the processes leading to the establishment of unique individuals and the tal Biology to develop a new, open model for research organi- endlessly diverse species they comprise. The Center for Developmental Biology was launched in April zations within Japan, with an emphasis on providing the free- 2000 under the auspices of the Millennium Project research dom and independence to envision new directions of Questions of how the body maintains and heals itself also drive research into regeneration. initiative launched by former Prime Minister Keizo Obuchi. research, and the organizational and material support to make For centuries it has been known that different species have different self-healing powers, The Millennium Projects were established to drive research in those visions real. Nearly three years have passed since the but the reasons for these differences and the mechanisms by which our own bodies replen- the fields of information technology, environmental science birth of the CDB as a concept, and the investment of time and ish cells lost to aging, injury or disease are now coming into much sharper focus. With the and the study of aging, areas of vital importance to both money has already begun to bear fruit in the form of solid, emergence of improved techniques for studying regeneration at the cellular and molecular Japan and the world in the 21th century. The drafters of this innovative research into the mechanisms of development, levels, the roles of biological systems governing the maintenance and restoration of the plan recognized the great potential for contributions by devel- regeneration and the scientific bases for regenerative medi- body's cells have grown clearer, spurring further interest into progenitor cell types known to opmental and regeneration biologists in addressing the health cine. The years ahead hold the promise of ushering in new be capable of generating appropriate new cells in response to damage and loss. challenges confronting an aging society, and so the concept conceptual insights regarding the biological processes of of a national center for developmental biology was born. development, and of translating those insights into applica- These progenitor (or 'stem') cells play fascinating and crucial roles in development and A vast array of details needed to be worked out in order to tions with the potential to revolutionize the way we think about regeneration, and science now looks to harness their potential for medical application by make this vision a reality, and the RIKEN research organiza- aging, disease and medical therapy. I invite you to explore discovering ways to induce and guide their differentiation to supply natural replacement tion provided the organizational structure and support environ- this report for a first glimpse into some of the challenges and cells to damaged organs and tissues. The emerging field of regenerative medicine looks to ment needed, giving initial form to what is now the CDB and mysteries that we have encountered, and a few of those that translate research findings from the laboratory bench to the hospital bed and to develop helping to coordinate all aspects of the center's launch from still await. alternatives to the traditional medical approaches of pharmacological and mechanical inter- construction planning to human resources to facility manage- vention. Such therapies may someday make the physician's dream of using the body's ment. own cells to heal itself a reality.

During the process of its establishment, the decision was Laboratories at the RIKEN Center for Developmental Biology pursue research into the made to locate the CDB within a new and dynamically grow- mechanisms of development and regeneration with a twofold mission: To make contribu- ing biomedical research park in Kobe, one of Japan's most tions to the betterment of human health and well-being by providing an academic founda- attractive cities. This location situates the CDB within a world- tion 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.

2 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 3 Center for Developmental Biology July 3 2002 Highlights Publication of "Cadherin Regulates Dendritic Spine Morphogenesis" 7 The July issue of Neuron featured an article from the Takeichi research group detailing a heretofore unknown function of cadherin cell-adhesion molecules in the formation of synapses between mammalian neurons. March 22 Completion of Research Building A and July 8 3 CDB Experimental Animal Facility Opening Ceremony and Public Science Forum The CDB marked the completion of construction of Build- The CDB was officially inaugurated in an opening ceremony attended by mem- ing A, adding 7 floors and 10,199 m2 of laboratory space to bers of the local and national government, including Kobe mayor Tatsuo Yada the facility. The experimental animal facility (4,311 m2) was and MEXT Senior Vice-Minister Takashi Aoyama. A lecture session open to officially opened on the same day, representing the launch the public was held the same day at a nearby hall, with talks given by CDB of one of the world's most advanced facilities for the pro- group directors and team leaders to an audience of more than 500 people. duction and handling of experimental mice. July 9 Visit by MEXT Minister Toyama April 22-24 Atsuko Toyama, the Minister of the Ministry of Education, Culture, Sports, Sci- Opening Symposium and Meeting of ence and Technology (MEXT), visited the Center on its first full day of operation, 4 the Advisory Council chatting with scientists and observing experiments at a number of labs. RIKEN The CDB held its first international symposium on "A New Paradigm in Develop- research is primarily funded through MEXT budget allocations. mental Biology," attended by developmental biology and regenerative medical researchers from around the world. Speakers included members of the CDB Advi- sory Council, which convened its second session in September 27 the two days immediately following the symposium. Completion of CDB Research Aquarium 9 The CDB's 455 m2 aquarium for housing and breeding experimental aquatic April 25 species such as zebrafish and African clawed frogs officially opened on this Publication of "Dominant role of the niche day. in 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 October 10 25, 2002 issue of Nature. Publication of 10 "FGFR-related gene nou-darake restricts Reprinted by permission from Nature. brain tissues to the head region of planarians" Vol. 416 Publication Date 2002/4/25 The publication of the Agata group's work in determining the gene responsible for restricting brain neural Copyright: Macmillan Magazines Ltd. development to the head in planarians attracted news coverage in Japan and abroad. May 17 Publication of "Heterotopic Shift of November 25-26 5 Epithelial-Mesenchymal Interactions First CDB Retreat in Vertebrate Jaw Evolution" 11 The CDB held its first annual retreat on the beautiful island of Awaji, located a Research results from the Kuratani lab providing a concrete demonstration of the evo-devo concept of exapta- short distance from Kobe in Japan's Inland Sea. The retreat gave CDB tion, in which a structure that originally evolved for one adaptive reason is converted to a different adaptive role research staff the chance to relax, mingle and exchange research findings and in another species, were published in Science magazine. opinions in a series of oral presentation and poster sessions. As with all CDB events, the retreat was held in English, allowing Japanese and non-Japanese scientists to communicate unimpeded by barriers of language or culture.

December 6 Completion of Research Building C 12 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.

4 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 5 Regenerative Development Regeneration Medicine

Core Program

The Core Program aims to promote highly innovative developmental and regeneration studies in a strategic and multi-disciplinary manner. This program constitutes the core research framework to achieve the aims of the Mil- lennium 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 number of research fellows and technical staff and in some cases a senior research fellow.

Partial view of ragworm (Perinereis nuntia) reproductive appendage Photo: Nao Niwa 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 Biolo- gy (CDB; Kobe, Japan) as well as director of the Cell Adhesion and Tissue Pat- terning research group. He completed the B. Sc. and M. S. programs in biolo- divisions. The innermost domain, to which the Organization and gy at Nagoya University before receiv- β- and α-catenin complexes bind, is known to be development of the ing a doctorate in biophysics from Kyo- essential to cadherin function and mutations in these neural retina to University in 1973. After attaining his catenins have been implicated in some forms of canc- The retina, the innermost coat Ph. D., Dr. Takeichi took a research fel- er. One of Dr. Takeichi’s recent areas of interest has of the eyeball, is composed of lowship at the Carnegie Institution Department of Embryology under Dr. been the role of the juxtamembrane (JM) domain, neural and non-neural ele- Richard Pagano. He then returned to which is the binding site for another catenin, known ments. The neural retina, which Kyoto University, attaining a full profes- as p120. He found that cells lacking the JM domain receives optical information Blockade of cadherins in hippo- sorship in the Department of Biophysics cannot undergo normal morphogenetic movement from the lens and interfaces campal neurons alters dendritic (1986-1999), before becoming profes- spine shape. Red, a control sor in the Department of Cell and Devel- during the development of chicken somites, suggest- with the optic nerve to convey that information to the dendrite; green, a dendrite opmental Biology in the Graduate ing that this domain plays a critical role in allowing brain, provides the Takeichi lab with another system expressing a dominant- School of Biostudies at the same uni- cells to relocate. These findings led Takeichi to pro- useful for the study of cellular organization and negative cadherin. versity. He assumed his current posi- pose the model that the JM domain is a signaling cen- structure-specific developmental processes. The tions at the CDB in 2000. Dr. Takeichi ter involved in regulating cadherin activity, and may group’s studies in this field have focused on the role is best known for his discovery of cadherins, which are fundamental in the be important not only in embryonic morphogenesis, of the gene Wnt-2b in retinal development in chick mechanisms of cell-cell adhesion. but in the processes of cancer invasion and metasta- embryos. The developing retina gradually organizes sis as well. into sheet-like laminated structures. Retinal cells in isolation, however, fail to form these structures and instead clump together in ‘rosettes.’ The molecular Publications The neural retina provides mechanism by which the laminated structures are the Takeichi lab with another system generated was unknown. Members of the Takeichi Kubo F, Takeichi M, and Nakagawa useful for the study of group have now demonstrated that Wnt-2b produced S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. in the ciliary margin, a small region located at the bor- developmental processes Development (2003 in press). Cell adhesion, recognition and der of the retina with the iris, guides retinal cells to morphogenesis form laminar sheets. Using dissociated retinal cells in Togashi H, Abe K, Mizoguchi A, The formation of complex systems such as tissues Synapse formation culture, the team found that cells exposed to Wnt-2b Chisaka O, and Takeichi M. and organs requires a high degree of communication The synapses formed between neurons are of central in culture organized into sheets, similar to the Cadherin regulates dendritic spine morphogenesis. Neuron 35:77-89 and cooperation between their constituent cells. importance in neural signal transmission, but the pro- arrangement of cells in the normal retina. The same (2002). Since the discovery of cadherins, the role of the cad- cesses by which synapses are formed remain poorly cells, when exposed to Frizzled, a Wnt-antagonist, herin superfamily of molecules in regulating cell-cell understood. The Takeichi research group previously clumped together in rosettes. Horikawa K, and Takeichi M. Require- adhesion has been studied extensively. These mol- discovered that cadherin complexes localize in inter- ment of juxtamembrane domain of the cadherin cytoplasmic tail for ecules form complexes with other intracellular factors neuronal cell junctions in such a way that they do not A second study of Wnt-2b function revealed its impor- morphogenetic cell rearrangement to create bonds spanning membranes and intercellu- interfere with signal transmission as carried out by tance in maintaining undifferentiated progenitor cells during myotome development. J Cell lar spaces to join cells with other cells expressing synaptic vesicles and receptors on either side of the in the ciliary marginal zone. Throughout embryonic Biol 155:1297-306 (2001). similar cadherins. Such bonds are dynamic and regu- synaptic complex. The group has now reported that development, the ciliary margin contains multipotent lable, acting more like Velcro tape than permanent cadherins help to regulate the adhesion of dendritic cells that serve as progenitors for the specialized, dif- Staff glue. But recent work is revealing that the cadherins branches known as ‘spines’ to axons, which is a cru- ferentiated cell types that ultimately form the complex

Group Director may serve a wider range of functions than as simple cial step in synapse formation. During neural network architecture of the retina. Masatoshi Takeichi adhesives. Dr. Takeichi’s research group is engaged development, dendrites (protoplasmic processes that When Wnt-2b is overex- Research Scientist Shinichi Nakagawa in demonstrating expanded roles for conduct impulses toward the nerve cell body) extend pressed in developing reti- Visiting Scientist Shinji Hirano these molecules in the formation of com- spines into the intercellular space. When a spine nas, normal cell differentia- Special Postdoctoral Researcher plex structures such as neural synapses. makes contact with an axon (which conducts impuls- tion is inhibited. Additional Takuji Tanoue Research Specialist es away from the neural cell body), a mushroom- studies revealed that when Midori Maekawa Structure and function of shaped ‘head’ is formed, laying the foundation for a the Wnt signaling pathway Research Associate Sachihiro Suzuki cadherin complexes new synaptic site. The Takeichi lab conducted loss- was blocked downstream, Technical Staff Cadherins are the main regulators of of-function studies in hippocampal neurons with domi- neuronal cells differentiated Hitomi Ishigami Miwako Harata calcium-dependent cell-cell adhesion, but nant negative mutations for N-cadherin, and in those earlier than they normally Masato Uemura they do not work alone. It has been obtained from αN-cadherin knockout mice, and would have. All of these Wnt2b, expressed in the ciliary Junior Research Associate Tetsuo Ichii shown that the function of cadherins found that both groups of neurons formed synapses findings suggest a role for margin (left), controls the undifferen- Student Trainee tiated state of retinal cells. Overex- Tetsuhisa Otani / Hideru Togashi relies on the formation of complexes with with altered dendritic spines and synaptic organiza- Wnt-2b as a stem cell fac- pression of Wnt2b, coinjected with Koji Tanabe / Kanna Takaoka a group of molecules called catenins, tion. These findings indicate that cadherin/catenin tor in the retina. GFP (green), in retina induces the Yoshiko Kametani / Kentaro Abe folded expansion of an undifferen- Masakazu Kadowaki / Fumi Kubo which associate with the cytoplasmic tail complexes may be critical regulators of the morphoge- tiated cell layer (right). Middle, Shinsuke Nakao / Masamitsu Sone of the cadherin molecule. The tail of the netic processes underlying synaptic plasticity. control, in which only GFP was Assistant Mutsuko Aiso cadherin molecule comprises two main injected. 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 Report 9 Regenerative Development Regeneration Medicine Core 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 Depart- ment of Internal Medicine at the Kyoto University Chest Disease Research stem cells at least, the pro- Institute, before taking an assistant pro- Melanocyte stem cell niches cess may follow a different fessorship in the same department in One of the major challenges confronting stem cell route. Blood-forming cells 1979. He spent the period from 1980- researchers is the host of technical and biological are traditionally thought to 82 at the University of Cologne Institute problems associated with in vivo studies. Multipotent originate in the lateral meso- for Genetics (Germany) before return- Distribution of melanocytes in mouse follicle ing to the Chest Disease Research Insti- stem cells are responsible for the normal develop- derm, one of the three main tute, where he was appointed associate ment of their progeny cells, which means that experi- developmental regions of the mesodermal germ lay- professor in the Department of Micro- mental manipulations of these cells can have far- er. The Nishikawa group developed tools to distingu- biology in 1983. He moved to the Kuma- reaching, even lethal, consequences. Nishikawa has ish cell types generated during early embryogenesis moto University Medical School in 1987 to take a professorship in the Depart- chosen melanocyte stem cells as a research model and identified several intermediate stages during the ment of Immunology, and returned to as these cells share some common characteristics differentiation of blood cells from mesoderm. The Kyoto in 1993, as professor in the with the stem cells responsible for generating blood group discovered two points of blood cell line diver- Department of Molecular Genetics at and sperm, but offer several experimental advantag- gence Ð either directly from lateral mesoderm cells or the Kyoto Graduate School of Medicine. es over those stem cell types. Melanocytes, which from vascular endothelial cells Ð a finding that con- He was appointed CDB group director in 2000. produce skin and hair pigments, can be modified trasts with the widely-held view that blood cells and extensively, even lost, without causing any more seri- endothelial cells diverge from common precursors ous effect than alterations in skin and hair color, and called hemangioblasts, and surprisingly indicates that such alterations can themselves serve as visual con- an increase in potency occurs at the endothelial firmation of an experiment’s result. Increased animal stage at which it would normally be expected to show survival due to the absence of lethal phenotypes also a decline. makes the melanocyte system an ideal model from both the experimental and ethical perspectives. Regulating vascular architecture Publications Other recent work by the group has revealed a factor Staff Stem cell biology In work published in 2002, Nishikawa and colleagues capable of maintaining the integrity of blood vessels Nishimura E, Siobhan J, Oshima H, Yoshida H, Osawa M, Moriyama M, Group Director Stem cells are undifferentiated cells characterized by established a system for profiling melanocyte stem derived in isolation from their natural environment. In Jackson IA, Barrandon Y, Miyachi Y, Shin-ichi Nishikawa their capacity to self-renew and to generate special- cells and tracking their locations in living hair follicles. the normal course of development, endothelial cells Research Scientist Nishikawa SI. Dominant role of the Takumi Era ized cells indefinitely. These cells provide the source Follicles are known to contain melanocyte stem cells, and perivascular mural cells work together to form a niche in melanocyte stem cell fate Masatake Osawa Yusuke Yoshida for all of the myriad somatic cell types in the adult but the follicular microenvironment that sustains such diverse vascular architecture from a relatively simple determination. Nature 416:854-60 Visiting Scientist organism. The study of stem cells stands at the heart cells is still poorly understood. In this study, the Nishi- network. While the role played by mural cells in the (2002). Igor M Samokhvalov Timm T Schroeder of many of the central problems in developmental biol- kawa group identified cells with high expression lev- structuring of vascular tissue remains unclear, their Yoshida H, Naito A, Inoue JI, Satoh Collaborative Scientist ogy, but many questions regarding the nature of els of melanocyte stem cell markers, and then absence is a hallmark of diseases such as diabetic Mitsuhiro Okada M, Santee-Cooper SM, Ware CF, Eri Nishioka these cells, such as the extent to which they can be blocked the function of one of these molecules using retinal disorders. Two proteins, angiopoietin-1 and Togawa A, Nishikawa S, Nishikawa Lars M Jakt SI. Different Cytokines Induce Sur- Muneaki Miyata reprogrammed to produce progeny of different fates marker-specific antibody. The results of these experi- PDGF-B, have been implicated in mediating the inter- Masahiro Yasunaga and the identities of the intrinsic and extrinsic molecu- ments indicated that melanocyte stem cells localize action between endothelial and mural cells. The Nishi- face Lymphotoxin-alphabeta on IL-7 Tamie Morisawa Receptor-alpha Cells that Differential- lar factors that regulate a cell’s ‘stemness,’ remain at two intrafollicular sites, the hair matrix and the kawa group introduced a PDGF-B antagonist into neo- Satomi Nishikawa ly Engender Lymph Nodes and Pey- Shin Kawamata unanswered. bulge, each of which hosts a specific sub-population natal mice, preventing mural cells from participating er's Patches. Immunity 17(6):823-33 Special Postdoctoral Researcher Chikara Furusawa of stem cells. The matrix, where hair growth and pig- in the vascular formation process, and found that the (2002). Technical Staff mentation take place, provides an environment in resultant vascular system in the retina was poorly Satomi Nishikawa Shin-ichi Nishikawa Uemura A, Ogawa M, Hirashima M, Mariko Moriyama which amplifying progeny cells become committed to organized and leaky. However, when they introduced Fujiwara T, Koyama S, Takagi H, Hon- Kotomi Kawasaki believes that location is a stem cell fate, while the bulge serves as a reservoir recombinant angiopoietin-1 to the eyes, they found Chiga Okita important in defining da Y, Wiegand SJ, Yancopoulos GD, Natalia I Samokhvalova for stem cells at rest. Intermediate stem cells can that normal vascular architecture was restored, which Nishikawa SI. Recombinant Student Trainee a stem cell’s function Hidetoshi Sakurai migrate from the bulge to the matrix and even suggests a potential application for angiopoietin in angiopoietin-1 restores higher-order Atsushi Takebe through the epidermis to neighboring follicles to the treatment of mural cell disorders. architecture of growing blood vessels Shinsuke Tada in mice in the absence of mural cells. Kanako Yoshikawa Shin-ichi Nishikawa believes that location is important replenish the actively proliferating population. Nishika- J Clin Invest 110(11):1619-28 (2002). Ai Kotani in defining a stem cell’s function. Recent findings wa hopes to characterize these niches in more detail Mak Siu Shan Masanori Takenaga that stem cells can arise in one tissue type but later to achieve a better understanding of the extrinsic fac- Endoh M, Ogawa M, Orkin S, Nishika- Yasuhiro Takashima wa SI. SCL/tal-1-dependent process Masaki Kinoshita be directed to give rise to another strongly suggest tors responsible for maintaining stem cells in different determines a competence to select Assistant that a stem cell’s role is at least partly determined by states of activity. Sakura Yuoka the definitive hematopoietic lineage Maya Iwase signals from its surrounding environment. By studying prior to endothelial differentiation. this microenvironment, known as the stem cell Hematopoietic lineage maturation EMBO J 16;21(24):6700-8 (2002). ‘niche,’ the Nishikawa research group seeks to gain In the classical view of lineage maturation, cell lines insight into the identity and function of molecules always develop toward increased specialization and involved in stem cell activities, and the potential of away from differentiative potency. However, evidence stem cell-based therapeutic applications. 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 Report 11 Development Regeneration Core Program

Laboratory for Vertebrate Body Plan Body Plan

Shinichi Aizawa Ph. D. Shinichi Aizawa received his Ph. D. in biochemistry from the Tokyo Kyoiku Uni- versity Department of Zoology in 1973. He spent the period from 1974 to 1979 as an investigator at the Tokyo Metro- velopmental region called the anterior neural ridge politan Institute of Gerontology, then are also essential in the induction and patterning of two years as a research fellow in the the forebrain, Aizawa set out to determine the precise Laboratory of Genetic Pathology at the University of Washington (US). He role played by Otx2 in mediating signals from this returned to the Tokyo Metropolitan Insti- organizing center. Generating a series of allelic Otx2 tute of Gerontology in 1982, where he mutants resulted in the unexpected discovery of a remained until 1986 when he moved to hypomorphic allele characterized by arrested fore- the RIKEN Tsukuba Life Science Cen- ter as a senior research associate. He brain and anterior head development. Unlike homo- was appointed professor in the Kuma- zygous Otx2 mutants, however, these allelic embryos Two enhancers of Otx2 expression in the forebrain and midbrain moto University School of Medicine were able to form apparently normal anterior- Department of Morphogenesis in 1994, posterior axes and progenitor fields. Explant assays and served in that position until 2002. were made by removing anterior neural plate sec- Since 2000 he has served as CDB deputy director and group director of tions from mutant embryos and transplanting them Body patterning in primitive spe- the Vertebrate Body Plan, as well as into wild type embryos, and vice-versa. It was found cies team leader of the Laboratory for Ani- that while neural plates from Otx frt-neo/- mutants were Amphioxus, commonly referred to as the lancelet, is mal Resources and genetic Engineer- able to respond normally to signals from the anterior a small, marine organism thought to represent one of ing. neural ridge in wild type embryos, the reverse was the earliest evolutionary ancestors of vertebrates as not true, indicating that Otx2 may be involved in the the lancelet embryo develops a number of structures downstream mediation of forebrain-inducing signals, common to chordate development, such as the such as Fgf8, emanating from this region. notochord, during gastrulation. The polarized accumulation of β-catenin in the nuclei of specific cells is a Locus mapping provides crucial step in the early development of multicellular a means for identifying candidate animals, playing roles in the formation of germ layers The vertebrate head-body bound- Shinichi Aizawa’s research concentrates on the genet- Staff genes of potential and signaling centers. The Aizawa lab studied the Publications ary ic and molecular characteristics of head develop- activity of β-catenin in the lancelet to investigate how Group Director biomedical importance Shinichi Aizawa The evolutionary development of the vertebrate head ment, particularly in the border between the fore-and its localization impacted on morphogenesis in these Hide T, Hatakeyama J, Kimura C, Tian E, Takeda Na, Ushio Y, Shiroishi T, Research Scientist is the subject of a debate older than the concept of hindbrain, which in vertebrates originates in segment- organisms. These studies, in which the asymmetric Akihito Yamamoto Aizawa S, and Matsuo I. Genetic modi- Yoko Suda evolution itself. Beginning with Goethe’s observation ed embryonic units known as ‘rhombomeres.’ Rhom- Genetic determinants of cranio- distribution of this protein was experimentally altered, fiers of otocephalic phenotypes in Otx2 Takuya Murata Nobuyoshi Takasaki that the cranium of a sheep seemed to be formed bomeres exhibit distinct gene expression and cell facial anomalies indicated that embryos with non-typical β-catenin dis- heterozygous mutant mice. Develop- Daisuke Kurokawa from fused vertebrae, rival theories have been pro- behavior profiles, and are situated at the developmen- In mice, embryos lacking a single copy of the Otx2 tributions are nonetheless able to establish dorsal- ment 129:4347-57 (2002). Takashi Nagano Jun Kimura posed in which it is asserted that the head is either tally interesting boundary between the trunk region, gene develop a range of phenotypes in which cranio- ventral axes, suggesting that dorsal morphogenesis Shinozaki K, Miyagi T, Yoshida M, Kohei Hatta the evolution of some preexisting structure, or that whose development is primarily regulated by homeo- facial development is disturbed. These phenotypes, is not dependent on early embryonic polarization in Collaborative Scientist Miyata T, Ogawa M, Aizawa S, and Akihiko Shimono the emergence of the head was an evolutionarily nov- tic Hox-family genes, and the rostral (anterior) head, termed otocephaly or agnathia-holoprosencephaly, Amphioxus. Suda Y. Absence of Cajal-Retzius Technical Staff are also observed as human congenital anomalies cells and subplate neurons associat- Shoko Takehara el event. What is certain is that the body plan of mod- which is governed by a different set of genes, pre- Tomomi Ohmura ern vertebrates is composed of three distinct regions dominantly the homologs of Drosophila head gap and represent a failure of the head patterning pro- In experiments such as these, the Aizawa lab ed with defect of tangential cell migra- Miwa Nakamura tion from ganglionic eminence in characterized by the expression of different sets of genes. Studying mutations in genes known to be gram. However, in mice, the severity of the pheno- endeavors to characterize the evolutionary, genetic, Ai Inoue Emx1/2 double mutant cerebral cor- Saori Nagayoshi regulatory genes Ð the trunk, the head, and the inter- responsible for body patterning in these regions, the type was linked to the genetic background of the and molecular bases of the vertebrate body plan, with tex. Development 129:3479-92 Student Trainee Wataru Sato vening hindbrain/pharyngeal region. Aizawa research group hopes to establish the factors strain in which the mutation was induced. By crossing a particular emphasis on the factors at work in induc- (2002). Mariko Hirano that delimit and characterize the foremost rhom- strains exhibiting different phenotypes and performing that crowning achievement of animal evolution, Kyoko Hashimoto Tian E, Kimura C, Takeda N, Aizawa Part-time Staff bomeres, and thereby contribute to the understanding genotypic analysis of the offspring, the Aizawa the forebrain. S, and Matsuo I. Otx2 is Required to Setsuko Date ing of the developmental criteria for the constitution of group was able to identify two loci linked to abnormali- Kazuki Suganuma Respond to Signals from Anterior Assistant the anterior head as conserved across phyla. ties in the lower jaw in these mutants. One of these Neural Ridge for Forebrain Specifica- Yukari Hata Sayo Saito loci, named Otmf2, may also be implicated in the tion. Dev Biol 242:204-23 (2001). Head gap genes development of holoprosencephaly, the most com- Head Organizer Project Kimura C, Shen M, Takeda N, Aiza- Team Homologs of the Drosophila orthodenticle gene, mon congenital forebrain defect in humans, with an wa S, and Matsuo I. Complementary Senior Scientist which collectively form the Otx gene family, have incidence of up to 1 in 250 early embryos. This study Isao Matsuo Functions of Otx2 and Cripto in Initial Special Postdoctoral Researcher been established as essential to the induction of the revealed the power of the mapping of loci responsible Patterning of Mouse Epiblast. Dev Chiharu Yoshida anterior head and the anterior-posterior body axis for determining phenotypic severity in naturally occur- Biol 235:12-32 (2001). Technical Staff Kuniko Kitajima itself in both invertebrate and vertebrate species. In ring variations in different strains of experimental ani- Hiroshi Nakano Suda Y, Houssain ZM, Kobayashi C, the mouse, Otx2 homozygous mutants entirely fail to mals, both as a tool for genotypic analysis and as a Izumi Ishii Hatano O, Yoshida M, Matsuo I, and Part-time Staff develop the fore- and midbrain. However, the means of identifying candidate genes of potential bio- Aizawa S. Emx2 directs the develop- Nana Fujimoto mechanisms by which this gene induces normal medical importance. ment of diencephalon in cooperation anterior head development remain elusive. With the with Otx2. Development 128:2433- knowledge that signals from an embryonic neurode- 50 (2001).

Enhancer of Otx2 expression in the anterior neuroectoderm

12 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 13 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 the same institution in 1987 for his work on lens-specific regulation of the chicken delta crystallin gene. Inspired by the dis- ism as a whole. Hayashi has chosen to focus his stud- covery of the homeobox, he changed ies on the systems of limb and tracheal development his research focus to the developmental within the fruit fly Drosophila to outline the mechan- The adult Drosophila melanogaster fruit fly genetics of Drosophila and spent three isms of cellular behavior and interaction in organoge- years as a postdoctoral research asso- nesis. ciate in Matthew Scott’s lab at the Uni- The development of the trachea in Drosophila origi- versity of Colorado before returning to Publications Japan to work in the National Institute Development of appendages nates with ten pairs of tracheal primordia, located in of Genetics. He became an associate Like many insects, Drosophila is a highly competent the contiguous thoracic and abdominal segments, T2 Kubota K, Goto S, Hayashi S. The professor at the same Institute in 1994, flier, evolutionarily adapted to a life spent on the to A8. Cells from the primordium migrate, extend role of Wg signaling in the patterning and professor in 1999. Also in 1999, he of embryonic leg primordium in Dro- received the incentive prize of the wing. Embryological studies in Drosophila have branches decorated with fine cellular extensions sophila. Dev Biol (in press 2003). Genetics Society of Japan for Genetic shown that both wings and legs share a common ori- called filopodia, and respond to environmental cues Studies on Drosophila Development. gin in developmental bodies known as limb primordia. to seek out and establish connections with other tubu- Chihara T, Kato K, Taniguchi M, Ng He was named group director of the In their studies of limb formation, Hayashi’s group lar cells. The trachea ultimately extends to every seg- J, Hayashi S. Rac promotes epitheli- Morphogenetic Signaling research tracks the cellular growth, differentiation and migra- al cell rearrangement during tracheal group at the RIKEN CDB in May 2000. ment in the adult individual. Hence, the process of tubulogenesisi in Drosophila. Devel- tion characteristic of cells destined to become wings tracheal tubulogenesis involves both intercellular opment 130:1419-28 (2003). in the adult fly. Using time-lapse confocal microscopy cooperation within individual segments, and coordina- of fluorescent-tagged cells, the group tracks three tion between cells throughout the entire body. Shiga Y, Yasumoto R, Yamagata H, stages of the process of wing development: alloca- and Hayashi S. Evolving role of tion, specification, and separation. In allocation, cells The migration of tracheal branches is controlled by a Antennapedia protein in arthropod limb patterning. Development in the primordium undergo preliminary specialization complex guidance mechanism involving several extra- 129:3555-61 (2002). from cells in the surrounding area. Specification invol- cellular factors such as Dpp, Wingless and Fgf. ves further differentiation between cells that will Hayashi’s group is investigating this guidance Hayashi S, Ito K, Sado Y, Taniguchi remain in place and contribute to leg formation, and mechanism and the role of the Rac signaling path- M, Akimoto A, Takeuchi H, Aigaki T, Matsuzaki F, Nakagoshi H, Tanimura A society of cells those that are marked for specification into wing tis- way, which is known to participate in the mechanisms T, Ueda R, Uemura T, Yoshihara M, The metaphor in which cells are likened to individuals sue. The separation phase involves the migration of of cell adhesion, migration and guidance. The group, and Goto S. GETDB,a database com- within a society is a common one in biology, and wing-specified cells to dorsal locations, from which in collaboration with researchers from Tokyo Metro- piling expression patterns and molec- plays a particularly instructive role in understanding the final wing structure arises. Non-migrating cells politan University, uses selective overexpression of ular locations of a collection of Gal4 many of the mechanisms of early development. Just remain to form the leg primordium, which consists of target genes to produce gain-of-function mutants as a enhancer traps. Genesis 34:58-61 (2002). as students of social behavior seek to identify the fac- proximal and distal domains. This simple organization preliminary screen for new gene functions required tors responsible for human actions, developmental is further elaborated by a series of segmentation pro- for tracheal patterning. Goto S, Taniguchi M, Muraoka M, biologists ask similar questions about what drives a cesses. Toyoda H, Sado Y, Kawakita M, and cell to divide, differentiate, migrate, or self-destruct. Gain-of-function studies serve to provide an initial Hayashi S. UDPÐsugar transporter implicated in glycosylation and pro- The answers to both sets of questions generally Now, armed with the ability to make a clear visual glimpse of the roles of such genes. By regulating the cessing of Notch. Nat Cell Biol involve multiple factors. In animal development, the record of the movements of wing and leg-specified expression of target genes and observing the phe- 9:816-22 (2001). specification of cell fates and their behavior as individ- cells to their ultimate destinations within the body, notypes, it becomes possible to identify candidates

Group Director uals and in groups is determined by interactions bet- Hayashi and colleagues intend to pin down the molec- for further investigation. To date, the Hayashi group Shigeo Hayashi Staff ween molecular factors intrinsic and extrinsic to the ular machinery that determines these outcomes using has completed screening studies of approximately Research Scientist Leo Tsuda Group Director microscopic world of the cell Ð genes and their pro- genome screening and molecular biology techniques. one-fourth of the Drosophila genome using randomly Atsushi Wada Shigeo Hayashi tein products, and signaling and transcription factors As the genetic factors involved in limb development inserted UAS transposons, which serve to activate Yoshiko Inoue Research Scientist Kenji Oshima Leo Tsuda received from the world beyond the cell’s membrane tend to be highly conserved across species, findings genes located downstream. Phenotypes of interest Special Postdoctoral Researcher Atsushi Wada from studies on the relatively simple fruit fly should off- Nao Niwa Yoshiko Inoue borders. include mutants displaying absent tracheal branches, Junior Research Associate Kenji Oshima er insights into the fundamental mechanisms of organ branch misrouting, changes in cell and tubule shape, Kagayaki Kato Special Postdoctoral Researcher and limb development in humans and other species. and abnormal cell adhesion. The genes Technical Staff Nao Niwa Questions of inter- Ai Akimoto Junior Research Associate implicated in these mutations become Masako Kaido Kagayaki Kato and intracellular communication Michiko Takeda Technical Staff Tracheal development candidates for follow-up studies to elu- Chiaki Kose Ai Akimoto inform Shigeo Hayashi’s The mechanisms of tracheal development in Dro- cidate their involvement in tracheal devel- Hiromi Sakaguchi Masako Kaido research Part-time Staff Michiko Takeda sophila are another focal area for research in opment. Tadashi Sakata Chiaki Kose Hayashi’s lab. In fruit flies, the trachea is functionally Masayo Shindo Hiromi Sakaguchi Kayoko Sakurai Housei Wada Questions of inter- and intracellular communication analogous to the mammalian lung, and its develop- Housei Wada Student Trainee Sono Hashimoto Tadashi Sakata inform Shigeo Hayashi’s research. His work seeks to ment shares many characteristics with the develop- Hiromi Niwa Masayo Shindo uncover genes and molecular factors that allow differ- ment of many branched tubular systems including the Assistant Kayoko Sakurai Chisa Iimuro Assistant entiated cells to communicate and coordinate with lungs, kidneys, and blood vessels, which are all Chisa Iimuro Drosophila embryo expressing tau-GFP The tracheal system stained for nuclei (green) each other to form ‘communities’ of tissue, organ and essential to the distribution and regulation of liquids fusion protein in the limb primordium and luminal cavities (purple) anatomical structure within the ‘society’ of the organ- and gases throughout the body in higher animals. and sense organs of the head

14 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 15 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 of Tokyo in 1979, and his doctorate from the same institution in 1984, for his work on the characterization of the ment of cell diversity, as the nervous system features of both extrinsic and intrin- erythrocyte cytoskeletal structure. He more cell types than any other organ system, and the sic signals, and the mechan- spent the period from 1984 to 1988 as fruit fly is highly amenable to genetic manipulation. ism by which cells of differ- a postdoctoral fellow, first in the Depart- With the advent of techniques for molecular analysis, ent sizes are generated in ment of Cell Biology at the Tokyo Metro- neurodevelopmental studies have shown that neural the process of mitotic divi- politan Institute of Medical Science, then in the laboratory of Gerard Edel- cells’ fates are determined at a very early stage in the sion. man at Rockefeller University. He development of the organism. In previous research, returned to Japan in 1988 to take a posi- Matsuzaki and others demonstrated that the fates of Asymmetric cell sizes In Drosophila, dividing neuro- tion as a section chief in the Depart- daughter neural cells are in large part determined by This last question prompted investigations that led to blasts localize the Miranda ment of Molecular Genetics in the (green) / Prospero complex National Institute of Neuroscience. In unequal distribution of fate-determining factors within the identification of a new regulatory function in a to be segregated into the daughter GMC. 1998, he was appointed professor in the mother cell during the process of cell division. family of proteins, known as G proteins, in Drosophila the Institute of Development, Aging and Two factors in particular, known as Prospero and neuroblast cell division, a process in which the result- Cancer at Tohoku University and Numb, have been shown to localize to the basal side ant neuroblast is much larger than the GMC. During remained there until taking his current of the neuroblast (the neural progenitor cell) prior to mitotic cell division, chromosomes replicate within the position as group director at the RIKEN CDB. its division. This polar distribution results in two mother cell and attach to spindles that draw them in daughter cells exhibiting distinct gene expression opposite directions to ensure that a full complement patterns Ð a new neuroblast, and a smaller-sized of chromosomes is available to each daughter cell. ganglion mother cell (GMC) Ð in which Prospero and The chromatids are drawn to the spindle poles Numb are segregated in the GMC. In this process of through the action of the cytoskeleton. In work asymmetric division, the neuroblast remains undiffer- carried out at the CDB, Matsuzaki’s lab has found entiated and retains its multipotency, while the GMC becomes committed to generating neurons and glial cells. Cells lacking the Gβ protein Intracellular asymmetry in question form All the cells in the body originate from a single proge- Further studies in the Matsuzaki and other labs a symmetrical mitotic spindle and nitor, and nearly all share an identical DNA code. showed that Prospero and Numb are tethered to the produce daughter cells of How then do the vast numbers of distinct cell types in basal cortex of the neuroblast by the molecules equal size with the complex organisms differentiate from one another? Miranda and PON (for Partner Of Numb), respec- determinants being normally The process of asymmetric division, in which regula- tively, and subsequently released into the newly- segregated. tory factors in a single mother cell are distributed formed GMC. The molecular mechanisms involved in Publications unequally to daughter cells produced by cell division, asymmetric distribution during cell polarization have is a key to answering that fundamental question. This further been shown to have homologous counterparts that Gβ protein signals are distributed unequally in Hayashi S, Ito K, Sado Y, Taniguchi asymmetric distribution of regulatory factors allows in vertebrates, providing strong evidence for the neuroblasts and seem to restrict the development of M, Akimoto A, Takeuchi H, Aigaki T, Matsuzaki F, Nakagoshi H, Tanimura resultant cells to assume different roles from each evolutionary conservation of intrinsic signaling in the microtubules, resulting in a shortening of the mitotic T, Ueda R, Uemura T, Yoshihara M, other in a process called fate determination. This process of asymmetric cell division. The Matsuzaki spindle on one side of the cell. These unequal spin- Goto S. GETDB, a database compil- process relies on the establishment of intracellular lab is now confronting questions regarding the roles dle lengths cause the neuroblast to divide at a clea- ing expression patterns and molecu- gradients of factors regulating the expression of vage site that is off-center, and the sizes of the daugh- lar locations of a collection of Gal4 enhancer traps. Genesis 34:58-61 genes, which results in differential patterns of gene ter cells reflect this imbalance, with the daughter (2002). expression in daughter cells when the cell undergoes neuroblast being more than twice the size of the mitosis. As it is the expression of genes, and not their GMC. Cells lacking the Gβ protein in question form a Nakagoshi H, Shirai T, Nabeshima simple presence or absence, which is primarily symmetrical mitotic spindle and produce daughter Y, and Matsuzaki F. Refinement of Staff responsible for determining cell identity, controlled cells of equal size with the determinants being wingless expression by a Wingless- and Notch-Responsive Homeodo- Group Director asymmetric division is critical to ensuring the develop- normally segregated. Normal gene expression and main protein, Defective Proventricu- Fumio Matsuzaki ment of appropriate numbers and types of daughter mitotic activity are gradually altered in neuroblasts lus. Dev Biol 249:44-56 (2002). Research Scientist Noyuki Fuse cells, and so, to the development of the organism as undergoing such divisions, suggesting that Yasushi Izumi a whole. asymmetrically-sized division is a mechanism that Ohshiro T, Yagami T, Zhang C, and Ayano Kawaguchi Matsuzaki F. Role of cortical tumor Go Shioi ensures the ‘stemness’ of neuroblasts by minimizing suppressor proteins in asymmetric Visiting Scientist Takako Isshiki Neural cell fate determination in the reduction of cell volume during consecutive divi- division of Drosophila neuroblast. Technical Staff Drosophila sions. This is the first demonstration of a molecular Nature 408:593-6 (2000). Kanako Hisata Nao Ohta Fumio Matsuzaki has dedicated his research to expli- mechanism responsible for asymmetric mitotic spin- Mai Saito cating the role of asymmetric division in determining dle formation, and provides a valuable platform for Matsuzaki F. Asymmetric division of Taeko Suetsugu Drosophila neural stem cells:a basis Student Trainee cell identity, using the Drosophila melanogaster further studies in fields ranging from microtubule for neural diversity. Curr Opin Neuro- Maki Maeda nervous system as a model. This system provides an dynamics to animal morphogenesis. biol 10:38-44 (2000). Assistant Chika Lee attractive research platform for studying the develop- In the developing mouse spinal cord, Prospero (green) is tran- siently expressed in neurons immediately after their birth from mitotic neural progenitors (red). 16 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 17 Regenerative Development Medicine Core Program

Laboratory for Organogenesis and Neurogenesis Neurogenesis

Yoshiki Sasai M. D. , Ph. D. Yoshiki Sasai received his M. D. from the Kyoto University School of Medicine in 1986, subsequently performing intern- ships in general practice and emergency medicine. He completed the Ph. D. course at the same institution in 1992, extent of the signaling networks involved in setting up for work on neural specific transcription- the dorsal-ventral axis and determining neural fate in Inducing neural al regulators. In 1993, he took a post- the ectoderm. Insights gained from studies in Xeno- differentiation in doctoral fellowship in the De Robertis pus are further tested in other organisms, such as mammals lab at the UCLA School of Medicine, zebrafish, chick and mouse, allowing for comparisons Over the past decade, the remaining there until 1996 when he was appointed associate professor at the across species and evidence-based speculation into understanding of early Kyoto University School of Medicine. the evolutionary conservation of these signaling neural differentiation in He assumed a professorship at the Kyo- mechanisms. The group is also actively developing Xenopus has advanced to University Institute for Frontier Medi- effective methods of inducing neuralization in mam- rapidly, but that knowl- cal Sciences in 1998, and was appointed group director at the CDB in 2000. mals, work which has potential for application in the edge is not immediately Expression of Tiarin (purple) in Dr Sasai serves on the editorial boards treatment of nerurodegenerative disorders, such as transferable to the study of neural induction in other Xenopus neurula; Neural plate is of Neuron, Development, Genesis and Parkinson’s disease. organisms. While the effects of anti-BMP signals are stained with Sox2 cRNA probe (light blue). Developmental Dynamics. sufficient to induce neuralization in the frog, addition- The Sasai group’s work has al signals seem to be necessary in the mouse. Until potential for application in quite recently, one of the main obstacles to the the treatment of nerurodegenerative detailed study of mammalian neural differentiation in disorders, vitro has been the lack of an experimental system such as Parkinson’s disease that 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 Tiarin, a novel dorsalization factor response to appropriate signals, making it a valuable In previous work, Sasai identified a number of neural tool in the embryologist’s experimental kit. Mouse differentiation factors that operate by blocking the embryonic stem (ES) cells promise similar differentia- Staff Induction of the dorsal nervous derm to maintain a neural fate. These molecules, effects of BMP4 (BMP for Bone Morphogenetic Pro- tive potential, but difficulties in their guided induction Group Director system which include Noggin, Chordin, Follistatin and their tein), a molecule which instructs undifferentiated cells have meant they have not seen widespread use in Yoshiki Sasai Research Scientist The vertebrate body plan can be construed as the dis- homologs, participate in elaborate signaling networks in the ectoderm to take up an epidermal fate. The con- studies of early patterning. Kenji Mizuseki tribution of three germ layers relative to three body in which factors collaborate and compete to initiate clusion of these studies was that anti-BMP factors, Noriaki Sasai Makoto Ikeya axes. These interacting sets of patterns are estab- the embryonic nervous system. which include Chordin and its immediate and indirect The Sasai lab has developed a technique for induc- Mami Takasaki lished in early embryogenesis, and serve to deter- downstream targets, actually serve to protect the ‘de- ing the differentiation of neural cells by culturing Junior Research Associate Kiichi Watanabe mine the position and specification of cells as they dif- Beginning with the identification of the neural induc- fault’ neural status of ectodermal cells. Sasai has mouse ES cells on plates of connective cells, called Technical Staff Publications Ayaka Nishiyama ferentiate and aggregate into tissues and organs. The ing factor Chordin in the early 1990s, Yoshiki Sasai’s recently added a new member to the growing family stromal cells. These cells have a strong neuralizing Yoko Nakazawa specification of the dorsal-ventral (or back-belly) axis research has focused on explicating the molecular of central nervous system dorsalizing factors with the effect, termed SDIA (for Stromal cell-Derived Induc- Tsuda H, Sasai N, Matsuo-Takasaki Tomoko Katayama M, Sakuragi M, Murakami Y, and Masako Kawada is significant in neural development: the central signaling mechanisms that allow the early embryo to identification of Tiarin (so named because its expres- ing Activity), and induce ES cells to generate dopami- Tomoko Haraguchi nervous system forms on the dorsal side of the body organize itself into a complex, mature organism. sion pattern in the Xenopus neurula resembles a tiara nergic neurons and their precursors at high rates of Sasai Y. Dorsalization of the Neural Michiru Matumura Tube by Xenopus Tiarin, a Novel Pat- worn on the head). efficiency. Such controlled neural induction repre- Assistant in all vertebrate species. This process is dictated by Using the African clawed frog, Xenopus laevis, as a terning factor Secreted by the Flank- Ukiko Miyake the effects of a number of signaling factors that dif- model in molecular embryological studies, Sasai and sents an important step toward the development of Ayumi Tanaka ing Non-Neural Head Ectoderm. Neu- Yuri Arizono fuse from organizing centers and direct dorsal ecto- his group are engaged in clarifying the structure and With the goal of isolating genes that encode factors an experimental platform amenable to the study of ron 33:515-28 (2002). involved in the patterning of the anterior central early neural differentiation in mammals. And, in work nervous system, the Sasai group performed a cDNA published in early 2002, Sasai and colleagues further Kawasaki H, Suemori H, Mizuseki Ke, Watanabe K, Urano F, Ichinose library screen, yielding nine candidate genes, includ- demonstrated that the SDIA effect is preserved in the H, Haruta M, Takahashi M, Yoshika- Dopaminergic neu- ing Tiarin, which exhibited neural-specific expression neural induction of primate ES cells as well, with wa K, Nishikawa S, Nakatsuji N, and rons derived from + primate ES cells by patterns. Subsequent profiling of the Tiarin gene, dopamine-producing cells being generated at a fre- Sasai Y. Generation of TH Dopami- + the SDIA method. which has homologs in chick and mouse, revealed quency of about 35%. This method provides an imme- nergic Neurons and Pax6 Pigment Epithelia from Primate ES cells by that it is expressed in the non-neural ectoderm flank- diate unlimited source for primate cells useful in SDIA. Proc Natl Acad Sci USA ing the anterior neural plate, a developmental region experimental studies of neurodegenerative disease, 99:1580-5 (2002). which gives rise to the vertebrate forebrain. Misex- and opens up avenues for developing stem cell- pression of Tiarin induces dorsal neural markers and based therapies for such diseases in the future. Sasai N, Mizuseki K, and Sasai Y. suppresses ventral markers, indicating its importance Requirement of FoxD3-class Signal- ing for Neural Crest Determination in in establishing dorsal and neural identity. Although Xenopus. Development 128:2525-36 the molecular mechanisms by which this is achieved (2001). 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.

18 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 19 Regenerative Development Medicine Core Program

Laboratory for Evolutionary Regeneration Biology Totipotent Stem Cells

Kiyokazu Agata Ph. D. Anti-synaptotagmin antibody staining showing axon bundles 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 trans- Whole-mount in situ RNA staining with differentiation. He took an associate pro- DjPC2 probe, showing neural cell bodies fessorship at the Himeji Institute of Technology in 1991, and started his study of planarian regeneration with Prof. Kenji Watanabe. He remained at the ency, Agata’s research group concentrated on cells knocked down by RNA interference resulted in ani- Publications Institute until 2000, when he left to containing ‘chromatoid bodies’, structures within the mals that developed brain cells throughout their bod- assume a professorship at Okayama cell marked by the presence of the gene DjvlgA (Du- ies. This gene seems to function by binding a second Agata K, Tanaka T, Kobayashi C, University. He joined the RIKEN Center Kato K, Saito Y. Intercalary Regener- for Developmental Biology as a group gesia japonica Vasa-Like Gene A, D. japonica being protein, FGF, to cells in the head region, causing ation in planarians. Dev Dyn director in 2000. the name of one species of planaria). Chromatoid them to adopt a brain neuron fate. Previous to this dis- 226(2):308-16 (2003). covery it had been thought that the planarian brain Planarians, flatworms produced some factor that inhibited brain growth in Asami M, Nakatsuka T, Hayashi T, other parts of the body, but the ndk findings strongly Kou K, Kagawa H, and Agata K. only a few millimeters in length, Cultivation and characterization of indicate that the reverse is true; cells in the head (and are known for their remarkable planarian neuronal cells isolated by those migrating to the wound site in decapitated ani- regenerative abilities fluorescence activated cell sorting mals) recruit a brain-inducing factor. (FACS). Zool Sci 19:1257-65 (2002). bodies are morphologically and compositionally simi- Evolutionary role of stem cells F Cebria, Kobayashi C, Nakazawa lar to germ plasm, which is found in totipotent germ In addition to the valuable insights they provide on M, Mineta K, Ikeo K, Gojobori T, Ito cells in Drosophila and C. elegans and is involved in the mechanisms of regeneration, stem cells can also M, Taira M, A Sánchez Alvarado, germline specification in those animals. shed light on the function of developmental process- and Agata K. FGFR-related gene es in evolution. Indeed, in Agata’s words, “Stem cells nou-darake restricts brain tissues to the head region of planarians. Evolutionary regeneration By comparing the compositions of chromatoid bodies are key to understanding evolution.” This is due to Nature 419:620-4 (2002). The process by which animals replace cells that have in undifferentiated neoblasts and cells committed to, their unique ability to give rise to cells of different been lost due to aging, injury or disease is known as for example, neural or muscle cell fates, Agata’s character than the antecedent cell. On the time scale Ogawa K, Ishihara S, Mineta K, regeneration. All animals have some ability to regen- group discovered that the content of chromatoid bod- of a single organism’s life span, this leads to the Nakazawa M, Ikeo K, Gojobori T, erate, but during the course of evolution, the degree ies is heterogeneous, and dependent on the cell’s development of complex cell systems within the Watanabe K, and Agata K. Induction of a noggin-like gene by ectopic D-V of that capacity has been distributed widely in differ- designated fate. This finding runs counter to the clas- organism. On the evolutionary time scale, however, it interaction during planarian regenera- ent species. While some animals, such as humans, sical view that only uncommitted, totipotent cells are may be one of the primary factors leading to the tion. Dev Biol 250:59-70 (2002). are quite limited in their regenerative capabilities, oth- capable of division in planaria, and suggests that cer- divergence of species. For example, investigations ers demonstrate much greater potential to replenish tain stem cells migrate through the planarian body to into how planarian stem cells differentiate into the ani- lost cells. Research at Kiyokazu Agata’s lab is the regenerating wound site, called the ‘blastema.’ mal’s simple eye could provide insights into how focused at the intersection of two fields of study: the These ‘committed’ stem cells exist in a state interme- more complex eyes devel- mechanisms of regeneration itself, and the evolution- diate between the totipotency of uncommitted stem oped in more highly evolved ary processes by which organisms have developed cells, and terminally committed cells that have lost animals. Agata’s group seeks Staff such diverse regenerative approaches. Stem cells the ability to divide. The group is now investigating to trace diversification within

Group Director are centrally important to both of these fields, and the factors responsible for maintaining the ‘stemness’ families of conserved genes Kiyokazu Agata Agata’s work seeks to identify molecules that main- of these cells prior to their arrival at the blastema. that are known to be linked to Research Scientist Noriko Funayama tain pluripotency in and present positional cues to stem cell identity and func- Yoshihiko Umesono these cells, as well to clarify their role in the evolution Stem cell regulation tion, in the hopes of clarifying Chiyoko Kobayashi Nobuyasu Maki of complex cell systems. Although totipotent stem cells are distributed through- the evolutionary contribution Maki Kashikawa (Yoshida) Katuaki Takechi out the planarian body, these cells are able to gener- of stem cells to the develop- Shuichi Shigeno Maintaining totipotency ate specialized cell types appropriate to the site of ment of more and more com- Visiting Scientist Norito Shibata Planarians, tiny flatworms only a few millimeters in injury when the animal is cut. This suggests that infor- plex cellular systems. Technical Staff length, are known for their remarkable regenerative mation regarding the position of the wound is con- Midori Nakayama Tetsutaro Hayashi abilities. A single planarian can be cut into dozens, veyed to the stem cell, allowing it to make the neces- Tomomi Takamatsu even hundreds, of pieces, and each segment will sary changes in gene expression to differentiate into Yumi Saito Mikako Dohi regenerate into a complete individual organism. This the cell type that needs to be regenerated. This year, Hiroshi Wake is due to the presence throughout the planarian body the Agata group announced the discovery of one Student Trainee Maki Asami of totipotent stem cells called neoblasts, which, given gene involved in such positional cueing. The gene, Takeshi Inoue Keiji Okamoto the proper signals, are capable of differentiating into named nou-darake (ndk; Japanese for ‘brains every- Takaaki Karasawa any cell type in the animal’s body. To study the where’), ensures that brain neurons only develop in Kouji Matsuzaki Brain expansion in ndk knock- Kazuya Ishizawa mechanism by which neoblasts maintain their totipot- the head region; experiments in which the gene was down planarian Regeneration in planarians

20 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 21 Regenerative Development Regeneration Medicine

Creative Research Promoting Program

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

Undifferentiated mouse ES cells Photo: Yayoi Toyooka Development Creative Research Promoting Program

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 Biochem- istry and was awarded a Ph. D from the same institution in 1985 for his work on the regulation of plasmid ColIb DNA rep- mutant strains in previous screening studies, Hama genes encoding proteins lication by inc and repY. He spent the and colleagues identified the gene still life (sif), which known to be involved in the period from 1985 to 1988 as a post-doc participates in the maintenance of motor function in regulation of vesicular traffic in the laboratory of Thomas Kornberg at Drosophila. This gene, which codes for a GEF in budding yeast. Hama the University of California, San Francis- (Guanine-nucleotide Exchange Factor) that activates hopes to elucidate the roles co before returning to Japan to continue his post-doctoral work at the National Rho family GTPases, has been shown to be involved of these proteins in neurite Institute of Neuroscience, NCNP, in the processes of synaptic formation. Later, Trio, extension, branching and Tokyo. He advanced to section chief in identified as a second GEF expressed in the nervous guidance, and synapse for- the Department of Molecular Genetics system, was revealed to be a pivotal factor for the reg- mation as well, by conducting Trio (purple) in the mushroom body in 1991, and remained at the NCNP of Drosophila brain until 2001 when he was appointed to ulation of axon extension. The Hama lab now plans conditional RNAi studies his current position at the CDB. to use RNAi gene silencing to perform functional using a DNA-based transgen- analyses of the approximately 20 known GEF-family ic approach. genes with the goal of identifying genes and molecular mechanisms contributing to neural development. Development of brain structure Publications A fourth prospective direction for research in the Hama hopes to take advantage Hama team lies in the functional analysis of a gene Saito M, Awasaki T, and Hama C. of the Drosophila olfactory identified in a large-scale mutant screen as involved Genetic analyses of essential genes in the structural development of the brain. Aberra- in cytological region 61D1-2 to 61F1- system as an experimental model 2 of Drosophila melanogaster. Mol to shed light on the genetic tions in the brain structures can be caused by defects Genet Genomics 268:446-54 (2002). bases of olfactory axon targeting of many developmental events including neuronal proliferation, axon guidance and synapse formation. Mutations in the gene in question exhibit smaller than Olfactory network guidance ordinary axon bundles and reduced numbers of cells Neural networks There are approximately one thousand different types in the brain tissue involved in olfactory learning and The formation of neural networks involves the orches- of odorant receptors in the mammalian olfactory sys- memory, which suggests that the gene regulates neu- tration of complex processes from neuronal migra- tem, but it is believed that any given olfactory neuron ronal proliferation. The brain contains a variety of neu- tion, to axon growth to synaptic formation. In the expresses only a single receptor type. By mechan- ronal types, which can be generated serially from sin- human brain, which comprises on the order of 100 bil- isms that remain largely unknown, the axons of olfac- gle neuronal precursors. Further analyses of the lion neurons, the mechanisms by which neuronal tory neurons expressing a receptor of the same type mutant may provide a hint about how switching of the cells are able to identify and form synapses with converge into one of a thousand glomeruli, which are neuronal types is regulated during brain develop- appropriate partners, and the ways that circuits discrete structures composed of the axon terminals ment. formed from interconnecting neurons are able to dem- and the dendrites of the target neurons. Using a onstrate adaptive plasticity are questions of stagger- mutant screening approach incorporating gene knock- By adopting a broad-based, multi-focus approach, ing intricacy and depth. Chihiro Hama seeks to out and GFP cell visualization techniques, Hama Hama seeks to illuminate the mechanisms by which address these complex problems by investigations hopes to take advantage of the simpler Drosophila cells that have followed the developmental route lead- into the neural network of the Drosophila fly, a com- olfactory system as an experimental model to shed ing to neuronal differentiation are further able to paratively simply structured system which nonethe- light on the genetic bases of olfactory axon targeting. organize into neural circuits, and ultimately to partici- less demonstrates the capacity for both instinctive pate in the function of the brain. behaviors and learning. While there are pronounced Vesicle transport in neural devel- differences in degrees of network sophistication and opment cell type diversity between Drosophila and more high- All cells contain cargo-bearing compartments known ly evolved organisms, they nonetheless share a large as vesicles. These vesicles function to deliver the number of homologous genes important in neural macromolecules that serve the cells as membrane development. Using a research strategy of large- components or secreted signals, and the metabolite scale mutant screening in the fly, the Hama research products of the cellular digestive process to the rest Staff team seeks to open new windows into the genetic of the cell, where they are stored or used to fuel activi-

Team Leader bases of neural circuitry. ty. Microtubules serve as ‘railways’ for vesicle trans- Chihiro Hama port; their ends anchor to sites on the cell cortex Research Scientist Minako Orihara GEF family genes where the vesicles fuse. It is likely that the extrusion Keita Endo of cell membrane at given sites, such as takes place Kazunaga Takizawa Screening for mutant phenotypes is an exacting pro- Hiroko Saito cess, involving methodical surveillance for anatomical in axon extension, requires vesicles to be transported Technical Staff Yuka Yoda or behavioral aberrations and requiring a thorough along microtubule in a specific orientation. To clarify Kyoko Ishikawa familiarity with the ‘normal’ characteristics of the how vesicle transport is involved in neural develop- Assistant Kanako Moriwaki experimental model. On observing reduced activity in ment, Hama plans to analyze Drosophila orthologs of SIF (green) in the periactive zones of neuromuscular Specific axonal targeting in the Drosophila synapses olfactory system

24 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 25 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 Medi- cine in 1988, and his Ph. D. from the Osaka University Institute for Molecular and Cellular Biology in 1992. From 1992 to 1995, he worked as a postdoc- its amenability to genetic studies, its rapid toral fellow in Michael Karin’s lab in the embryological development, and extensive University of California, San Diego data regarding mutant phenotypes, makes Department of Pharmacology, then the zebrafish an attractive platform for returned to Japan to take an assistant investigations into the genetic and molecu- professorship in the Division of Molecu- lar Oncology at Osaka University Medi- lar bases of organizer formation. Hibi focus- cal School. He was appointed associate es on the dynamic processes that take professor in the same division in 1999, place in the first 24 hours of zebrafish devel- where he remained until he assumed opment, a period in which the embryo his position as team leader at the RIKEN CDB. grows and differentiates from a single cell into a recognizably vertebrate body organ- Morpholino (MO)-mediated loss of Pnx, which contains ized along distinct axes. an Eh1 repressor motif and a homeodomain, leads to reduction in primary neurons expressing elavl3.

Hibi focuses on the dynamic processes that take place in the first 24 hours of zebrafish Posterior neurogenesis in the Publications development zebrafish Bae Y-K, Shimizu T, Yabe T, Kim The nervous system is known to arise on the dorsal C-H, Hirata T, Nojima H, Muraoka O, side of the vertebrate gastrula, but this system dis- Hirano T, and Hibi M. A homeobox gene, pnx, is involved in the forma- Establishing the dorsal organizer plays anterior-posterior (A-P) asymmetry as well. The tion of posterior neurons in zebrafish. There is extensive evidence that an as-yet unknown fore-, mid- and hindbrain regions, and the spinal cord Development (in press 2003). Staff dorsal determinant molecule in the vegetal pole of the all form from precursive embryonic structures Shimizu T, Yamanaka Y, Nojima H, Team Leader Body axis determination in zebra- questions that have challenged and intrigued develop- very early zebrafish embryo is transported via the arranged along the A-P axis of the gastrula. In the Yabe T, Hibi M, and Hirano T. A nov- Masahiko Hibi fish mental biologists ever since. The problems of how intracellular microtubule network to a dorsally located Nieuwkoop model of neurogenesis, after a population Research Scientist el repressor-type homeobox gene, Osamu Muraoka In 1924, when Spemann and Mangold discovered the such organizing centers are formed, and how they signaling center known as the Nieuwkoop center, of ectodermal cells is committed to a neural fate (the ved, is involved in dharma/bozozok- Takashi Shimizu which in turn produces signals that induce the dorsal neuroectoderm), the population undergoes a pattern- mediated dorsal organizer formation Visiting Researcher axis-organizing properties of a small section of the function to steer the development of the body axes Young-Ki Bae newt embryo located at the dorsal lip, it was the first remain at the forefront of modern embryological organizer via interactions with the Wnt signaling path- ing process in which the cells are further differentiat- in zebrafish. Mech Dev 118:125-38 Technical Staff (2002). Chiho Fukae demonstration that one group of cells can determine research. way. Using both gain- and loss-of-function studies, ed to form spatially distinct sub-populations that will Yukiko Kuga the developmental fates of neighboring uncommitted Hibi has discovered a number of genes that partici- develop into the various regions of the central Student Trainee Ryu S-L, Fujii R, Yamanaka Y, Shimi- Taijiro Yabe cells. This group of fate-determining cells, which The research in Masahiko Hibi’s lab seeks to address pate in the induction of signaling centers and body nervous system, and, ultimately give rise to the prim- zu T, Yabe T, Hirata T, Hibi M, and Tsutomu Hirata axes, including dharma/bozozok and ved. His recent ary and secondary neurons that comprise the Hirano T. Regulation of dharma/bozo- Hideaki Nojima came to be known as the Spemann organizer, is these problems, using the zebrafish as a model sys- zok by the Wnt pathway. Dev Biol Assistant responsible for establishing the dorsal-ventral and tem for understanding dorsal organizer origins and work has focused on determining the exact roles nervous system proper. The Hibi research team has Shoko Iwaki 231:397-409 (2001). anterior-posterior body axes in amphibians, and corre- function. A region of the zebrafish embryo called the played by these genes in the signaling networks identified one gene, pnx, that is expressed in the pos-

sponding embryonic signaling centers have been ‘embryonic shield’ closely corresponds to the amphibi- responsible for axis formation. terior neuroectoderm, and regulates the formation of Hashimoto H, Yabe T, Hirata T, Shi- shown to play similar roles in fish, birds and mam- an organizer. The high level of evolutionary conserva- posterior neurons, such as those of the hindbrain and mizu T, Bae Y-K, Yamanaka Y, Hira- mals. The discovery of the Spemann organizer raised tion seen in the vertebrate organizer, combined with The dharma/bozozok (dha/boz) gene is known to spinal cord. pnx encodes a homeodomain protein no T, and Hibi M. Expression of the play a central role in the establishment of the dorsal that mediates posteriorizing signals as part of a larg- zinc finger gene fez-like in zebrafish forebrain. Mech Dev 97:191-5 organizer in zebrafish, and is capable of inducing the er signaling network that functions in the patterning (2000). organizer in a non-cell-autonomous manner. Hibi and later stages of neurogenesis. found that dha/boz is activated prior to gastrulation as part of a complex network of axis-determining factors The Hibi lab is also interested in the analysis of that includes both the Wnt and BMP signaling path- genes involved in ventralized zebrafish phenotypes. ways. However, several molecular steps in this pro- While there are many known dorsalized mutants in posed network remain unidentified, and the Hibi team zebrafish, ventralized phenotypes are comparatively is working to characterize the immediate and second- few. Hibi and colleagues are conducting analyses of ary downstream targets of the dha/boz gene product. one ventralizing gene, ogon, in the hopes of identify- One aspect that is becoming clear is that dha/boz ing its function in the dorsal-ventral axis signaling networks in concert with a group of ventrally-expressed work. Hibi plans to continue to explore the formation Mutations in ogon lead to ventralization of proteins that inhibit other dorsalizing factors further and function of signaling centers in the zebrafish the embryo. downstream. The interplay between these dorsalizing embryo, continuing on the genetic and molecular lev- and ventralizing factors is central to the formation of el the revolutionary work started by Spemann and the anterior neuroectoderm, the developmental Mangold nearly eighty years ago. region that gives rise to the forebrain.

26 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 27 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 an the skin corresponded to the overlying skin pattern. overseas fellowship at the Basel Uni- The existence of a spotted zebrafish leopard pheno- versity Biocenter under Walter Gehring. type indicates that genetic factors are at work in deter- He returned to Kyoto University in 1994 mining the spatial distributions of the pigmented bod- as assistant professor, where he ies; the reaction-diffusion model predicts that one or remained until 1998, when he was appointed professor in the Tokushima more activating and inhibiting factors interact to University Faculty of Integrated Arts influence the formation of these pattern variants. His and Sciences. Kondo was appointed team is now engaged in a collaborative mutant Regularly periodic formation of additional somites in extended chick CDB team leader in 2001. screening search to identify the gene or genes presomitic mesoderm responsible for maintaining regular stripes in these Kondo team sought to demonstrate patterning consist- fish. Publications ent with the Turing model. In its embryonic develop- Stripe directionality ment, the chick develops a regularly spaced series of Kondo S. The reaction-diffusion Angelfish present an unusual problem for the bodies adjacent to the neural tube, called somites, system:a mechanism for autonomous pattern formation in the reaction-diffusion model, in that related species dis- which eventually give rise to the vertebrae, ribs and animal skin. Genes Cells 7:535-41 play patterns with similar striping, but in which the other structures. As regular periodicity is one of the (2002). stripes are arranged in different directions. These features of Turing wave patterns, the Kondo team fish, which are also unusual for the ability of females sought to determine whether somitogenesis could be Shoji H, et.al. Directionality of stripe in male-less populations to change sex, develop explained by the reaction-diffusion model. Working formed by anisotropic diffusion. J Theor Biol 214:549-61 (2002). stripes only in the process of acquiring male gender. with gastrulating embryos, the team stretched the In one species, the stripes are vertically arrayed, region that somites arise from beyond its normal while in another closely related species, they form length, and found that somites in the elongated Staff horizontally. While the stripes themselves conform to region divided to maintain their spacing, just as the

Team Leader Reaction-diffusion wave pattern- Shigeru Kondo is interested in demonstrating the the predictions of the Turing equation, there is noth- model predicts. This experimental confirmation indi- Shigeru Kondo ing mathematical basis of pattern formation in developing inherent to the reaction diffusion model that would cates that a balance between activating and inhibiting Research Scientist Masashi Hirata The natural world is filled with patterns that can be ment, and using mathematical models as predictive account for such directionality. While several factors provides the basis for the pattern periodicity of Jun Ozaki somites, and the team is now working to identify the Motoomi Yamaguchi described by mathematical functions, such as the tools to aid in the identification of genes and mol- mathematical explanations, such as boundary condi- Technical Staff Fibonacci spiral of a nautilus shell, or the fractal ecules involved in the generation of spatial struc- tions or spatial gradients in reaction rate could con- specific genes and proteins involved. Kana Bando Miwako Okamura arrangement of branches on some trees. In 1952, the tures. Research in the Kondo lab focuses on skin sur- ceivably produce differences in stripe direction, Kon- Part-time Staff British mathematician Alan Turing proposed a simple face and morphogenetic patterning, both of which fea- do concluded that the developmental pattern suggest- Motoko Yoshizawa Student Trainee mathematical equation capable of generating a wide ture prominent examples of periodic structures that ed an inequality in distribution (anisotropy) was at Takeshi Nakagawa range of commonly observed patterns, such as can be described in terms of standing and moving work in the reaction-diffusion model. Comparisons of Assistant Sumire Hino stripes, spots and networks. This model, known as waves. skin patterning in scaled and scaleless fish indicate the reaction-diffusion model, demonstrates that the that scale characteristics can cause marked changes Female Female interaction between a local activator and a long-range Zebrafish stripes and spots in pattern formation, and computational modeling con- inhibitor can give rise to various periodic structures in As its name implies, the zebrafish is normally a firmed that even very small differences in a variable response to differences in their individual diffusion striped animal. Similar stripes can be generated such as scale structure or arrangement could pro- rates. When the activator’s diffusion rate exceeds using the reaction-diffusion model, which inspired duce the changes in stripe direction observed in that of the inhibitor, a ‘moving wave’ is formed, wher- Kondo to seek the underlying biological mechanisms these fish. This work underscores the important point eas a higher rate for the inhibitor results in a ‘stand- capable of fulfilling the model’s mathematical condi- that minute perturbations in diffusion are capable of ing wave’. Both forms of wave can create periodic tions. Histological analysis of the zebrafish’s pigment- generating widespread and visible morphological geometrical structures which exhibit the property of ed regions revealed that the relative distributions of effects. maintaining the size of the interval between struc- three types of pigmented bodies Ð melanophores, tures as the overall pattern grows. xanthophores and iridophores Ð near the surface of Minute perturbations in diffusion are capable of generating widespread and 3 weeks 3 weeks visible morphological effects

male male Periodicity in somitogenesis The reaction-diffusion model is not only applicable to Genicanthus melanospilos Genicanthus watanabei skin surface patterning, but to the formation of periodically repetitive internal structures as well. Using somitogenesis in the chick embryo as a model system, the 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 Report 29 Development Creative Research Promoting Program

Laboratory for Evolutionary Morphology Evolution of Structures

Shigeru Kuratani Ph. D. Shigeru Kuratani received his M. S. and Ph. D. from the Kyoto University Depart- ment of Zoology. He spent the period from 1988 to 1991 working in experimental embryology in the Department of Anatomy at the Medical College of the carapace at other points in evolutionary history, Georgia before moving to the Biochem- as the underlying genetic elements are presumed to istry Department, Baylor College of be conserved, making carapace formation one of the Medicine, where he was engaged in trajectories available to the process of morphological molecular embryological research. He development. returned to Japan in 1994 to take a position as associate professor at the Insti- tute of Medical Embryology and Genet- The Kuratani lab is undertaking a systematic search ics in the Kumamoto University School to identify the genes and molecular mechanisms relat- of Medicine. He moved to Okayama Uni- ed to carapace development. This search involves dif- versity to assume a professorship in the Department of Biology in 1997, where ferential screening of gene expression in chick and he remained until he was appointed turtle. The chick is used as a basis for comparison, team leader at the CDB. 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 jaw conferred an evolutionary advantage the rapid emergence of the turtle’s shell will become clear, and that the case for an evo-devo approach to Publications morphological divergence will be strengthened. molecular mechanisms common to oral development Shigetani Y, Sugahara F, Kawakami Y, in these groups have been mobilized to form mouth Murakami Y, Hirano S, and Kuratani S. Evolution of the jaw structures at several discrete evolutionary moments, Heterotopic shift of epithelial- mesenchymal interactions for verte- The emergence of the jaw was an evolutionarily and not as part of an unbroken continuum. This nice- brate jaw evolution. Science 296:1319- ly illustrates the evo-devo principle of exaptation, in important step for gnathostomes (jawed vertebrates), 21 (2002). Staff as it conferred, among other things, the ability to which a structure or mechanism that originally evolved for one adaptive reason is co-opted as an Kuratani S, Kuraku S, and Murakami Team Leader Evo-devo developmental mechanisms mediate the translation grasp prey securely in the mouth, which was impossi- Shigeru Kuratani Evolutionary developmental biology, sometimes of changes in the genome to changes in morphology. ble for agnathan (jawless) species. However, the autonomous adaptation in a different context. In Y. Lamprey as an Evo-Devo mod- Research Scientist el:lessons from comparative embryol- Yoshie Ohya experiments such as this, Kuratani hopes to bolster referred to as ‘evo-devo,’ looks at the ways that mouths of some agnathans, such as lampreys, fea- ogy and molecular phylogenetics. Yasunori Murakami the already strong argument for the central role of Katsuhisa Uchida changes in developmental genes and gene expres- ture upper and lower lips, which it was long believed Genesis 34:175-95 (2002). Masumi Nakazawa sion drive both the evolution of species and differenc- Kuratani seeks to deepen were the evolutionary forerunners of the two-part, molecular developmental biology in evolutionary pro- Rie Kusakabe cesses. Kuratani S, Nobusada Y, Horigome Yuichi Narita es in morphology. Evolution has frequently been the understanding of developmental bony jaw. Kuratani tested this hypothesis by observ- N, and Shigetani Y. Embryology of Technical Staff described as the result of the interplay between muta- biological mechanisms ing the expression patterns of the lamprey homologs Ryo Usuda the lamprey and evolution of the ver- Student Trainee tion and selection, but the advent of molecular devel- in the evolution of species of four genes known to be responsible for oral devel- tebrate jaw: insights from molecular Shigehiro Kuraku opment in chicks. He found that, in the lamprey and developmental perspectives. Hiroshi Nagashima opmental biology in the 1980s led to the increasingly Yoko Takio widely accepted view that developmental constraints embryo, these genes, and the molecular cascades Phil Trans Roy Soc 356:15-32 Keiko Yamamoto that their protein pro- (2001). Assistant have a hand in shaping the directions, or ‘trajecto- The turtle carapace Chiaki Nakayama ries,’ that evolutionary processes are most likely to fol- Evolution is generally considered to be a gradual products participate in, do low. This concept is perhaps best illustrated by the cess involving the accumulation of subtle changes indeed serve to regu- homeobox (Hox) genes, a developmentally important over long spans of time. But marked changes occa- late the development of set of genes that shares a highly conserved domain sionally do appear abruptly in the fossil record. This the mouth. However, (the 180-base-pair homeodomain) and plays a cen- is the case for the advent of the turtle’s shell, or ‘cara- interestingly, analysis tral role in regulating the morphological development pace,’ which appeared suddenly and with few recog- showed that the lam- of diverse organisms. nizable precursors (although two now-extinct distant prey mouth arises from phylogenetic groups also evolved carapaces). Such a different regions of the Combining experimental and analytic techniques phenomenon would be difficult to explain by mutation developing embryo from molecular biology, phylogenetics and compara- and selection alone, as it is improbable that the geno- than it does in the tive morphology, Shigeru Kuratani seeks to deepen mic alterations necessary to produce such a dramati- gnathostome chick. the understanding of the part played by developmen- cally new bodily structure would be achievable by the Thus, the vertebrate tal biological mechanisms, such as the Hox genes, in introduction of new genes in the short timeframe of jaw and the lamprey the evolutionary divergence of species. His approach the turtle’s emergence. Evo-devo theory predicts mouth are morphologi- involves examining related genes in morphologically instead that the genes responsible for carapace devel- cally somewhat similar and phylogenetically distinct animals to uncover the opment actually belong to a set of genes shared by and share certain gene ways in which context affects gene expression, and groups related to the turtle, such as lizards, dinosaurs expression patterns, thereby influences body development. By comparing and birds, but which function distinctly in the unique but derive from distinct the molecular bases of evolutionarily novel struc- context of the turtle’s molecular-genetic network. This mesenchymal domains, tures, Kuratani hopes to illustrate the means by which would also explain the independent appearance of which suggests that the Ammocoete larvae of Lampetra japonica

30 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 31 Development Creative Research Promoting Program

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 he worked on the molecular biology of the yeast S. cerevisiae. He joined NEC Corp. in 1986 as a researcher in the body cavity to form symmetrical gonad arms originat- Fundamental Research Laboratory stu- ing in a primordium on the ventral side of the body dying the molecular genetics of C. ele- and terminating dorsally. The migratory path of the gans. He left NEC in 1992 to work as a emerging gonad is determined by a pair of distal tip visiting researcher at Johns Hopkins cells, one located at each end of the gonad primordi- University, then returned to the company in 1993 to continue his work on um, but the mechanisms by which these cells initiate, Development of C. elegans gonad nematode development. He was award- steer and end migration at the correct stages in larval ed a Ph. D. by Osaka City University for development are largely unknown. work on the molecular analysis of C. elegans embryogenesis in 1994. He Nishiwaki sees the interactions between the base- and restoring normal gonadogenesis, even in the remained at NEC until receiving an Publications appointment as team leader at the ment membrane of the migrating gonad and that of complete absence of MIG-17. The biochemical analy- RIKEN CDB. the body wall as a key to the process of cell move- sis of these candidates is still ongoing, and it is Suzuki N, Buechner M, Nishiwaki K, ment, and his team has conducted a large-scale hoped that the results of these efforts will provide bet- Hall DH, Nakanishi H, Takai Y, Hisa- screen for phenotypes that demonstrate distal tip cell ter knowledge of the MIG-17 metalloprotease and the moto N, and Matsumoto K. A puta- migratory aberrance. One gene uncovered in this tive GDP-GTP exchange factor is identification of molecules that directly interact with required for development of the mutant hunt, mig-17 (the mig prefix indicates a MIG-17. excretory cell in C.elegans. EMBO mutant phenotype with a migratory defect), has been Rep 21:530-5 (2001). found to encode a member of the ADAM (A Disinte- Nishiwaki is interested grin And Metalloprotease) family of secreted proteins. Nishiwaki K, Hisamoto N, and Matsu- moto K. A metalloprotease distinte- Tracking studies using molecules genetically tagged in clarifying the network of proteins grin that controls cell migration in with green fluorescent protein revealed that the mig- Caenorhabditis elegans. Science 17 product, MIG-17, is secreted into the body cavity that interact with MIG-17 288:2205-8 (2000). by muscle cells in the body wall and binds to the Cell migration and organogenesis gonadal basement membrane at the developmental Nishiwaki K. Mutations affecting Nishiwaki’s work to date has revealed that the base- symmetrical migration of distal tip In animal development, cell division, cell migration stage when the distal tip cells make their initial turn ment membrane plays a far larger role in organogene- cells in Caenorhabditis elegans. and changes in cell shape are coordinated to gener- dorsal-ward. The research team is now pursuing stud- sis than that of simple structural support. As his func- Genetics 152:985-97 (1999). ate complex systems of tissue and organs. One ies of other genes associated with migratory defects, tional genetic studies of C. elegans have shown, aspect of this highly orchestrated process is the with a particular focus on phenotypes in which the basement membranes are integral in the control of movement of cells in sheets, an activity orchestrated migratory mechanism is disturbed, but not abolished. cell migration as well. Judging from the high degree by mechanisms which remain poorly understood. Kiy- These mutations allow for the tracking of molecular of homology with higher organisms, such as the oji Nishiwaki’s research team studies gonadogenesis action and cell behavior throughout the migratory pro- mouse, these migratory mechanisms seem to be evo- (the formation of reproductive organs) in the nema- cess, and to identify developmental checkpoints at lutionarily well-conserved. By focusing on the move- tode C. elegans as a model system to improve the which the distal tip cell’s guidance mechanisms may ments of a small group of cells in a tiny worm, Nishi- understanding of the molecular mechanisms that go awry. Preliminary studies indicate that proteins waki seeks to push back the frontiers of underlie cell migration during organogenesis. By associated with alterations in the glycosylation of knowledge of how cells in the bodies of ani- developing a clearer picture of the mechanisms held MIG-17 or in its secretion may form a matrix that mals across species are able to get to Staff in common across species, it is hoped that this directs the localization of MIG-17 to the migrating where they need to go.

Team Leader research will lead to insights into cell migration and gonad. Kiyoji Nishiwaki organ development in humans, and contribute to the Research Scientist Norio Suzuki understanding of human diseases involving distur- Protein interac- Katsuyuki Tamai bances in coordinated cellular movement. tions with MIG-17 Special Postdoctoral Researcher Yukihiko Kubota To gain deeper insight into Technical Staff Mitsue Sano Genetic control of gonad develop- the role of cell migration in Assistant ment nematode gonadogenesis, Rie Kuroki C. elegans has been a favorite model for geneticists Nishiwaki is interested in clari- and developmental biologists since its introduction by fying the network of proteins Sydney Brenner in the 1970s, as the lineage of each that interact with MIG-17. of the 959 cells can be traced precisely, its short The team searches for muta- reproductive cycle makes it amenable to breeding for tions in mapping experiments genetic experiments, and its transparent body allows designed to locate candidate for direct observation of individual cells under a light genes. These mutations microscope. In the nematode, the gonad develops have been found to be cap- through four larval stages (L1-L4) during which time able of rescuing the meander- MIG-17 is produced in and secreted from muscle cells and localized on the gonad surface after the first turn of the DTC Distal tip cell migration. The path of the migrating DTC is shown by the dotted line. the gonad-forming cells divide and move through the ing distal tip cell phenotype

32 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 33 Regenerative Creative Research Development Regeneration Medicine Promoting Program

Laboratory for Pluripotent Cell Studies Pluripotency

Hitoshi Niwa M. D. , Ph. D. Hitoshi Niwa received his M. D. from Nara Medical University in 1989, and his Ph. D. in medical physiology in 1993 from the Kumamoto University Gradu- ate School of Medicine for his work in gene trap methods. From 1993 to 1994, Inducing differentiation he worked as a research associate in A second field of central importance to the stem cell the Department of Developmental research community is the development of methods Genetics at the same university, before by which undifferentiated ES cells can be prompted taking a postdoctoral fellowship with to commit to a specific cell lineage. Studying factors Austin Smith at the University of Edin- burgh Centre for Genome Research. identified in published work on knockout mice, the He returned to Japan in 1996 as a Niwa research team analyzed transcription factors research associate in the Department with potential roles in the development of extraem- of Nutrition and Physiological Chemistry bryonic and primitive endoderm lineages. (Primitive at the Osaka University Graduate School of Medicine, where he remained endoderm derivatives include the parietal and viscer- until taking his current position at the al endoderm, the extraembryonic regions that give RIKEN CDB. rise to the yolk sac endoderm.) Two factors in particular, Gata-4 and Gata-6, known to be expressed in cells 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 undifferentiated ES cells into endodermal cells, and it was found that the 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 of Staff tissue-specific transcription factors, an important step

Team Leader Self-renewal and pluripotency the first factor found to elicit multiple differentiative toward the experimentally and clinically significant Hitoshi Niwa With their ability to self-renew indefinitely and to differ- outcomes dependent on its expression level. ES cells goal of controlling the differentiation of ES cells in cul- Research Scientist Shinji Masui entiate into cells of all three germ layer types (a differ- expressing normal levels of Oct-3/4 maintained their ture. Parietal endoderm cells induced by Gata-6 overexpression in ES cells Kazuya Ogawa Publications Satoshi Ohtsuka entiative capacity called pluripotency), embryonic pluripotency, while its overexpression resulted in dif- Yayoi Toyooka stem (ES) cells represent one of the great prospects ferentiation into primitive endoderm and mesoderm, ES cell growth in culture Niwa H, Masui S, Chambers I, Smith Itsuro Sugimura for regenerative medical applications, as well as an and its inhibition caused the cells to take up a trophec- This goal of controlling culture conditions to achieve AG, and Miyazaki J-I. Phenotypic Technical Staff complementation establishes require- Kadue Takahashi attractive platform for the study of a diverse spectrum todermal (the layer of extraembryonic cells that specific outcomes is important to the growth of ES Rika Yagi ments for specific POU domain and Hiroyuki Kitajima of developmental processes. However, the mechan- serves as the source of the placenta) fate. This estab- cells in vitro as well. The Niwa lab is working to devel- generic transactivation function of Student Trainee op a serum- and feeder-cellÐfree system for culturing Oct-3/4 in embryonic stem cells. Mol Yoko Sekita isms by which ES cells are able to maintain these lished Oct-3/4 as a primary regulator of pluripotency Ken-ichi Tominaga functions are incompletely known, and a better under- in ES cells, but gave rise to new questions regarding mouse ES cells, which necessitates developing a Cell Biol 22:1526-36 (2002). Yuko Iwamatsu standing of the ‘stemness’ of these cells will be neces- the biochemically active domain in the Oct-3/4 pro- detailed picture of both the extrinsic factors and the Sayako Matsui Fujikura F, Yamato E, Yonemura S, Assistant sary in order to take optimal advantage of their tein. In work published in the spring of 2002, the Niwa intrinsic networks that function in these cells in cul- Hosoda K, Masui S, Nakao K, Miya- Miho Ohtsuka remarkable properties. Two of the biggest challenges lab showed that the function of Oct-3/4 in ES cell ture. It is known that a single ES cell in isolation will zaki J-I, and Niwa H. Differentiation of facing stem cell research are determining the factors propagation depends on the combined presence of fail to proliferate, while colonies of such cells grow embryonic stem cells is induced by that allow ES cells to generate limitlessly self- specific POU and transactivation domains. By refin- normally. It is also known that even isolated single GATA factors. Genes Dev 16:784-9 (2002). renewable progeny, and identifying molecules that ing the understanding of the molecular characteristics cells can be induced to proliferate if the culture medi- direct the dividing ES cell to produce daughter cells involved in the Oct-3/4-based ES cell maintenance in um from a larger ES cell colony is transferred to the Tomioka M, Nishimoto M, Miyagi S, with specific fates. Beginning with his work in identify- this way, it may be possible to pinpoint important tar- single cell’s plate, suggesting that ES cells produce a Katayanagi T, Fukui N, Niwa H, Mura- ing Oct-3/4 as a transcription factor important in ES get genes and thereby elucidate the regulatory path- growth-stimulating factor that acts by community matsu M, and Okuda A. Identification of Sox-2 regulatory region which is cell maintenance, Hitoshi Niwa’s research has ways at work in the preservation of pluripotency. effect. Niwa has developed an assay system to under the control of Oct-3/4-Sox-2 addressed both of these challenges. isolate candidate molecules for this hypothetical complex. Nucleic Acids Res 30:3202- One aspect of this network that holds Niwa’s interest ‘stem cell autocrine factor’ (SAF), and is actively pur- 13 (2002). A better understanding of is the identification of factors that interact with Oct-3/4 suing the characterization of the most promising can- the ‘stemness’ of pluripotent cells to govern ES cell fate. The tight regulation of Oct-3/4 didates. The results of these analyses should Tanaka TT, Kunath T, Kimber WL, will be necessary Jaradat SA, Stagg CA, Usuda M, is required to maintain pluripotency in ES cells, sug- improve the ability of researchers to grow ES cells in Yokota T, Niwa H, Rossant J, Ko in order to take optimal advantage of gesting that a finely tuned feedback mechanism is at culture, facilitating the study of these fascinating and MSH. Gene expression profiling of their remarkable properties work in keeping Oct-3/4 transcription levels within the potentially revolutionarily important cells. embryo-derived stem cells reveals appropriate range. The resolution of the elements of candidate genes associated with pluripotency and lineage specificity. this hypothetical feedback loops remains the subject Genome Res 12:1921-8 (2002). Maintenance of pluripotency of ongoing research and several candidate compon- The POU-family transcriptional regulator Oct-3/4 was ents have been identified.

34 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 35 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 once established. This is important for the main- 1995 as a research fellow in cell biology tenance of consistent cellular function, but it is equal- Expression pattern of DNA at the Nagoya University BioScience ly important for new individuals to have the ability to methyltransferase Dnmt3b in mouse embryo at 8.5 d.p.c. Center before moving to Massachu- reset their methylation patterns. To use the corporate setts, where he worked as a research analogy, there needs to be a way for new companies fellow in medicine at Harvard Medical School and Massachusetts General spun off from a parent to remove old labels and bind- Hospital from 1995 to 2002, serving as ings and to establish their own distinct sets of instruc- an instructor in medicine at Harvard for tions. This process of establishing new instructions is the last two years of that period. He called de novo methylation, and it seems to play a was appointed team leader at the RIKEN Center for Developmental Biolo- crucial role in the early development of many ani- gy in 2001, and returned to work in mals, including humans. Japan full-time in 2002. This process of establishing new epigenetic instructions is known as de novo methylation

In previous work, Okano showed that de novo methylation in mice required the expression of a pair of methyltransferase genes, Dnmt3a and Dnmt3b, at a seem to function in a number of developmental pro- very early stage of embryonic development, as well cesses. It is thought that DNA methylation is the epi- Publications during gametogenesis. Prior to this finding, it was genetic mechanism that governs genomic imprinting, Hata K, Okano M, Lei H, and Li E. Epigenetic instructions believed that a related gene, Dnmt1, was solely and nearly all imprinted genes contain regions that Dnmt3L cooperates with the Dnmt3 One of the great questions remaining in the life sci- responsible for the methylation of DNA. Using lacZ are methylated differentially in maternal and paternal family de novo DNA methyltransferases to establish maternal imprints in ences concerns how it is possible for the body's reporter gene studies, Okano showed that these new- alleles, although it remains a mystery as to how meth- mice. Development 129(8):1983-93 many cell types to express only the limited set of ly identified genes are highly expressed in germline yltransferases such as the Dnmt3 family recognize (2002). genes required for the performance of their individual (Dnmt3a), ES (Dnmt3a and Dnmt3b) and progenitor the sequences of imprinted genes. In a series of functions when nearly every one of these cells is (Dnmt3b) cells, suggesting that they function in the ovary transplantation studies, Okano showed that Okano M, Bell DW, Haber DA, Li E. genetically identical. While a complete answer epigenetic reprogramming of DNA methylation states. Dnmt3a and Dnmt3b function in the methylation of DNA methyltransferases Dnmt3a and Dnmt3b are essential for de remains to be found, it is already evident that addi- Based on these findings, it is now believed that maternally imprinted genes, while colleagues demon- novo methylation and mammalian tions need to be made to the classical model of gene Dnmt1 functions primarily in methylation main- strated that a third gene, Dnmt3L, also participates in development. Cell 99(3):247-57 expression to account for a number of 'epigenetic' pro- tenance, which is necessary to the stable inheritance the same process. Although Dnmt3L shows no direct (1999). cesses, such as X chromosome inactivation, parental of tissue-specific methylation patterns, but that methyltransferase activity, it is thought that it may Okano M, Shaoping X, Li E. Cloning imprinting, position effect variegation and gene silenc- Dnmt3a and Dnmt3b are the principal determinants in help to regulate maternal imprinting by interacting and characterization of a family of ing, which do not depend on DNA sequence. The con- initiating DNA methylation. Knockout mutations of with the known methyltransferases Dnmt3a and novel mammalian DNA (cytosine-5) Staff cept of epigenetically determined differential gene Dnmt3a and Dnmt3b result in a variety of organic, Dnmt3b, with which it binds and colocalizes. methyltransferases. Nat Genet Team Leader expression can be likened to a corporation with a sin- neurological and spermatic defects, which indicates 19(7):219-20 (1998). Masaki Okano Research Scientist gle comprehensive manual that provides instructions that the establishment of the DNA methylation state In the future, the Okano lab will Shin-ichiro Takebayashi to every department, but which features a system plays an important part in many aspects of develop- study how epigenetic reprogram- Masaaki Oda Technical Staff whereby locally irrelevant sections of the manual can ment. ming works at a molecular level. Chisa Matsuoka Akiko Yamagiwa be labeled, bound up, and filed away unread. In this By identifying the genes Student Trainee analogy, DNA methylation can be seen as one of the DNA methylation in maternal involved in de novo DNA methy- Takao Nakayama Akiko Tsumura means by which unnecessary sets of DNA instruc- imprinting lation, they hope to discover the tions are labeled and bound. Methylation targets cyto- In more recent work published in 2002, Okano and means by which it is determined sine bases in the genetic code, and genes methylat- colleagues at the Cardiovascular Research Center at what genes will be methylated. ed in this way are generally rendered inactive, or 'si- Massachusetts General Hospital demonstrated a new Comparisons of the expression lenced.' Masaki Okano's research concentrates on role for the Dnmt3 family of genes, which they found patterns of such genes across the mechanisms by which DNA methylation is estab- to be essential to the process of maternal imprinting. germ layers, or in normal mice lished and maintained throughout development. In the mouse, the paternal and maternal genomes against clones created from both contain small numbers of differentially somatically derived cells should

De novo methylation expressed genes known as imprinted genes. The lead to new insights in the Gross morphology of Dnmt3a(-/-) Methylation lays down developmentally significant expression of such genes is determined by whether context-specificity of the DNA Dnmt3b(-/-) mouse embryo at 9.5 d.p.c. gene expression patterns that are difficult to alter they are inherited from the mother or father, and they methylation process. Distribution of methylcytosine in metaphase chromosomes of ES cells

36 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 37 Development Creative Research Promoting Program

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 from 1982 to 1991. He worked as a postdoctoral fellow at the California Institute of Regulation of cell polarity Integration of transcription- Technology on a Human Frontier Sci- Publications ence Program grant during the period The nematode offers a number of advantages for use regulating networks from 1991 to 1994, then at the Mas- as an experimental model, including its rapid repro- A second area of focus in Sawa’s work is unraveling Zhao X, Sawa H, Herman MA. Tcl-2 sachusetts Institute of Technology from ductive cycle, the complete accounting of its cell line- the ways in which complex intracellular signaling encodes a novel protein that acts 1994 to 1997 under support from the ages, its amenability to growth in culture and genetic networks are integrated in the determination of cell synergistically with Wnt signaling Howard Hughes Medical Institute. He pathways in C.elegans. Dev Biol (in returned to Japan in 1997 to continue manipulation, and the ease with which individual cells fate, using vulva development in C. elegans as a press 2003). his work at Osaka University, first as a in its transparent body can be observed in vivo under model system. The nematode exists either as a male postdoc, then as researcher funded by a low-power microscope. The Sawa research team or a hermaphrodite animal. The hermaphrodite vulva Sawa H, Kouike H, and Okano H. the Japan Science and Technology Cor- studies the asymmetric division of C. elegans T cells, is a small channel located near the midpoint of the Components of the SWI/SNF com- poration PRESTO program. He took his plex is required for asymmetric cell which give rise to pairs of daughter cells, one of ventral side, and functions both to receive sperm and current position at the RIKEN CDB in division in C.elegans. Mol Cell 6:617- 2001. which generates epithelial cells, while the other release fertilized eggs during reproduction. This 24 (2000). produces neural cell types. The Sawa research team extensively-studied organ comprises only 22 cells, uses molecular genetic techniques to probe the and its development is known to be mediated by both Rocheleau CE, Yasuda J, Shin TH, molecular mechanisms involved in cell fate specifica- the Ras and Notch signal transduction pathways. Lin R, Sawa H, Okano H, Priess JR, Davis RJ, Mello CC. WRM-1 acti- tion. By cloning the gene lin-17 (lin signifying lineage- Sawa is analyzing vulval mutants to gain a more vates the LIT-1 protein kinase to defective) from a mutant strain exhibiting aberrant detailed understanding of how these pathways func- transduce anterior/posterior polarity asymmetric cell division and analyzing its sequence, tion in the formation of this organ, a process that invol- signals in C.elegans. Cell 97:717-26 Sawa determined that lin-17 is homologous to the friz- ves a rigidly stereotyped pattern of mitotic divisions. (1999). zled gene in the fruit fly Drosophila, which is believed Such stereotyped cell division patterns are character- to serve as a receptor of Wnt-family ligands. Other istic of nematode development, and are one of its experiments in which the Wnt-family protein LIN-44 most attractive features as a model system, allowing was misexpressed ectopically resulted in the reversal researchers to pinpoint phenotypic deviations at the of T cell lineage polarity, indicating that the Wnt/Friz- level of the single cell. One result of this analysis of Asymmetric cell division and cell zled signaling pathway is important in regulating the vulval mutants has been the identification of diversity direction of polarization as occurs in the process of LET-19/TRAP240, components of a transcription- Our bodies are made up of the myriad and multiform asymmetric mitosis and adding to its wide and well- regulating complex Mediator, which operates down- descendants of a single fertilized egg. The process of characterized array of functions in such fundamental stream of Notch. The Mediator complex also incorpo- asymmetric cell division, in which a single mother cell processes as cell proliferation and differentiation. rates another component, SUR-2, which functions splits to generate daughter cells with discrete charac- downstream in the Ras pathway, strongly suggesting ters, is widely accepted as the basis of cellular divers- that that this complex plays a ity but due to the difficulties associated with tracking role in integrating the Notch LIN-17/Frizzled the lineages produced by any single cell, the mechan- The Sawa team has and Ras pathways in vulva isms by which this is achieved remain imperfectly also identified nearly forty development. LIN-44/Wnt understood. Utilizing the nematode C. elegans as a genes in a C. elegans model, Hitoshi Sawa is engaged in the study of the mutant screen Looking ahead, the Sawa molecular interactions that underlie this process. He research team intends to iden- Staff seeks also to resolve the ways in which transcription- tify SWI/SNF activators and regulating complexes integrate the intracellular signal- The Sawa team has also identified nearly forty genes target genes, and to deepen cell polarity Team Leader Hitoshi Sawa ing networks that collaborate to help determine cell from a screen of mutants in which both daughters of further the understanding of Research Scientist Yukinobu Arata fate. the T cells were found to produce only epithelial cells, the molecular mechanisms at Kumiko Oishi rather than epithelial and neural cells. Of that group, work in asymmetric cells in Masahiro Uchida Masaki Fujita the two genes psa-1 and psa-4 (psa for Phasmid the nematode, in the hopes of Technical Staff Socket Absent) were found to encode proteins homol- developing insights with appli- SWI/SNF Hisako Takeshita Noriko Sasakawa LIN-12/Notch ogous to yeast SWI3 and SWI2/SNF2, respectively. cation to research in mamma- Assistant Tomoko Nakashima DPY-22 These proteins are components of the SWI/SNF tran- lian cell asymmetry. He also 1° Cell Fate scription regulatory complex, which is known to be plans to continue his research LET-19 involved in asymmetric cell division in yeast, a single- into the integration of signals 2° Cell Fate celled organism that nonetheless exhibits asymmetric in the process of cell fate LET-425 mitosis. These results provide compelling evidence determination. that conserved mechanisms may be at work in the SUR-2 asymmetric division of both one- and multi-celled 3° Cell Fate inactive chromatin active chromatin organisms. Polar distribution of LIN-44/Wnt and LIN-17/Frizzled induces SWI/SNF LET-60/Ras Mediator complex function in a single neurally-fated daughter cell 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 Report 39 Development Creative Research Promoting Program

Laboratory for Developmental Genomics Functional Genomics

Asako Sugimoto C. elegans (adult hermaphrodite) Ph. D. Asako Sugimoto received her B. Sc. degree from the Department of Biophys- ics and Biochemistry in the University of Tokyo School of Science in 1987, and her doctorate from the same institution the role of networked genes in guiding development determined that the most functionally important in 1992. She worked as a postdoctoral Publications fellow in Joel Rothman’s laboratory in in a simple worm, new light will be shed on universal genes are located on chromosomes where the chanc- the University of Wisconsin Ð Madison mechanisms in the genetic regulation of the develop- es of a complete loss of function are reduced. Kodama Y, Rothman JH, Sugimoto from 1992 to 1996, before returning to mental program. A, Yamamoto M. The stem-loop bind- Japan to assume an assistant professor- In addition to further phenotypic characterization and ing protein CDL-1 is required for chro- ship at the University of Tokyo. She mosome condensation, progression remained in that position until 2002, pur- Sugimoto hopes to shed categorization of the embryonic lethal genes, Sugimo- of cell death and morphogenesis in suing concurrent work as a Japan Sci- new light on universal mechanisms to next studied their post-embryonic functions using a Caenorhabditis elegans. Develop- ence and Technology Corporation in the genetic regulation of conditional knockdown strategy. Approximately 60% ment 129:187-96 (2002). PRESTO researcher from 1997 to of the genes studied played post-embryonic roles as 2000. She was appointed team leader the developmental program. Sumiyoshi E, Sugimoto A, Yamamo- well, indicating that many genes play multiple roles at at the RIKEN CDB in 2001. to M. Protein phosphatase 4 is different stages in development. It is anticipated that required for centrosome maturation this comprehensive phenotype profiling will lead to in mitosis and sperm meiosis in C.ele- RNAi-based profiling of gene func- the identification of gene networks that play crucial gans. J Cell Sci 115:1403-10 (2002). tion roles in developmental processes. Maeda I, Kohara Y, Yamamoto M, The wild-type nematode is made up of less than Sugimoto A. Large-scale analysis of 1,000 cells, yet this simple organism exhibits a wide Programmed cell death gene function in Caenorhabditis ele- range of the specialized cell types, such as muscles In addition to their work on system- gans by high- throughput RNAi. Curr and nerves, that characterize more highly evolved level analysis of gene networks, the Biol 11:171-6 (2001). species. And, thanks to the complete knowledge of Sugimoto team is also engaged in Sugimoto A, Kusano A, Hozak RR, the lineage of every cell in the C. elegans body, the detailed analysis of specific cellular Derry WB, Zhu J, Rothman JH. Many differentiative fate of every one of those cells can be functions, such as programmed cell genomic regions are required for nor- traced from its origin in the fertilized egg to its role in death, in which cells self-destruct as mal embryonic programmed cell the fully-grown adult. The amenability of this worm’s part of the normal developmental death in Caenorhabditis elegans. Genetics 158:237-52 (2001). The gene network sequenced genome to reverse-genetic techniques process. Programmed cell death As recent findings of the limited numbers of genes in has also served to make it one of the preferred model has been extensively studied in C. many genomes, including our own, have indicated, organisms in the world of genetics research. The dis- elegans, and it is known that exactly the interactions between genes at the network level covery in the late 1990s that the introduction of dou- 131 cells undergo this programmed seem increasingly significant in determining the ble stranded RNA (dsRNA) could be used to ‘knock cell death in normal nematode intricate development and function of multicellular down’ the expression of specific genes in the nema- development. It has been shown organisms. While the characterization of genomes at tode has only added to its appeal, giving scientists that a single genetic cascade is the level of the individual gene remains an important the ability to inhibit gene function without disturbing responsible for inducing cell death challenge, an analysis of how genes function in net- its underlying DNA. Sugimoto has refined this tech- in C. elegans, and that this cascade works is now in equal demand. This represents a nique of RNA interference (RNAi) by developing a has been evolutionarily conserved. daunting task in highly evolved and complex species method in which nematodes directly uptake dsRNA in By seeking to identify the molecular such as Homo sapiens, prompting scientists to seek solution. This process of ‘RNAi by soaking’ offers events that control the death of cells more primitive and simpler models to gain fundamen- greater efficiency and ease-of-use than other RNAi during the development of the nema- Staff tal insights in the hopes that a significant number of methods and has made it possible to conduct system- tode, Sugimoto hopes to uncover

Team Leader basic mechanisms are shared by many branches of atic high-throughput studies of gene suppression the regulatory foundations of this Asako Sugimoto the phylogenetic tree. more rapidly than ever before, achieving knock down important mechanism. Research Scientist Naoko Sakumoto analysis rates of up to 800 genes per month. Special Postdoctoral Researcher The Sugimoto team is also engaged Fumio Motegi Asako Sugimoto has adopted as a model the nema- Technical Staff tode C. elegans for its tractability to the systematic Starting with a cDNA library of approximately 10,000 in investigating the molecular Nobuko Uodome Chie Hayakawa functional analysis of its genome using unique high- genes expressed in developmental processes, the mechanisms underlying the forma- Yumi Iida throughput screening techniques. By studying the Sugimoto lab used RNAi to knock down each gene’s tion and movement of mitotic spin- Student Trainee Miwa Furuya functional interaction of genes within C. elegans, function and built a database in which the resulting dles, and has identified a gene Rika Maruyama whose genome has been completely sequenced, the phenotypes were sorted by developmental outcome. involved in spindle formation. Assistant Fumi Amimori Sugimoto research team seeks to identify the means To date, more than 4,000 phenotypes have been cat- Future research will employ live by which genes working in combination help to estab- egorized, and it was found that loss of function in imaging of microtubule and cen- lish and direct developmental processes. The lab more than 25% of these genes resulted in lethal, mor- trosomes using GFP-tagged pro- also looks to take the findings from these studies as a phologically altered or sterile phenotypes. Such devel- teins in combination of RNAi knock- base for advancing the understanding of developmen- opmentally important genes also seem to appear less down analysis to provide a clearer tally important mechanisms such as programmed cell frequently on the sex chromosome. The C. elegans picture of the role of specific genes death (apoptosis) and microtubule dynamics in mito- male has only a single such chromosome, and selec- in this process. sis. Sugimoto hopes that by opening windows into tive pressure in the process of evolution may have One-cell stage embryo. (Blue: chromosomes, Green: microtubules, Embryogenesis of Red: Protein Phosphatase 4) C. elegans 40 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 41 Development Creative Research Promoting Program

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 Depart- ment of Biophysics where she received her master’s and doctoral degrees in developmental biology. She pursued process, in which epithelial cells assume a mesenchy- consecutive postdoctoral fellowships in mal fate, is also observed during development. The developmental biology at the Institut Takahashi lab would like to clarify the molecular d’Embryologie du CNRS (1988 to mechanisms that regulate this mesenchymal- 1991), the University of Oregon Institute epithelial transition, and are concentrating in on the of Neuroscience (1991 to 1993) and Columbia University (1994). She roles played by members of the Rho family of pro- returned to Japan as an associate proteins in this process. Both the inhibition of Rac1 and fessor at Kitasato University in 1994, the overexpression of Cdc42 were found to induce where she worked until 1998, when she the conversion of epithelial cells to mesenchyme, took an associate professorship at the Nara Institute of Science and Technolo- while interference with Cdc42 function caused gy Graduate School of Biological Sci- mesenchymal cells to take on epithelial characteris- ences. She was appointed team leader tics. These suggestive findings led Takahashi to pro- at the RIKEN CDB in 2001. pose that the mesenchymal-epithelial transition is determined by relative Rac1 and Cdc42 activity levels, with higher levels of Rac1 inducing an epithelial fate, while higher levels of Cdc42 tip the balance in the mesenchymal direction. The simple egg develops into a marvelously complex organism

Publications You can’t tell an embryo Suetsugu R, Sato Y, Takahashi Y. what to do, Pax2 expression in mesodermal Staff only listen to what it can tell you segmentation and its relationship with EphA4 and Lunatic fringe during Team Leader Pattern formation in the early anterior-posterior axis. Somites arise one by one from chicken somitogenesis. Gene Expres- Yoshiko Takahashi embryo this segmental plate in a head-down direction. In this Special Postdoctoral Researcher sion Patterns 2:157-61 (2002). Yuki Sato Pattern formation, the process by which the bounda- sense, if the segmentation of invertebrates such as the Periodicity Technical Staff Rinako Suetsugu ries that define germ layers and tissue types are fruit fly can be described as an ‘egg-slicer’ effect in The fact that, despite the marked differences in the teach her about pattern formation. And pattern forma- Sato Y, Yasuda K, and Takahashi Y. Toshiharu Kasai established, occurs at highly specific sites and devel- which all cuts are made simultaneously, then segmenta- developmental mechanisms at work in invertebrates tion, of course, has many lessons for those who wish Morphological boundary forms by a Akemi Uchiyama novel inductive event mediated by opmental stages in the early embryo. This closely tion in vertebrates might be likened to a ‘meat-slicer,’ in and vertebrates, organisms as diverse as the Dro- to understand the bases of development itself. Student Trainee Lunatic-Fringe and Notch during Yukiko Nakaya coordinated regulation prevents cells from differentiat- which cuts are made sequentially, one after another. sophila fly and the chick exhibit patterns with similarly Takashi Yoshino somitic segmentation. Development Tadayoshi Watanabe ing inappropriately, and enables the establishment of regular temporal and spatial inter- 129:3633-44 (2002). Emi Ohata complex and specialized tissues and organs in the In experiments designed to elucidate the molecular vals intrigues Takahashi. Her lab Assistant Naomi Akira adult body. One type of pattern formation involves the mechanisms that drive this vertebrate meat-slicer, Taka- has been participating in collabora- Shiroi A, Yoshikawa M, Yokota H, Fukui H, Ishizaka S, Tatsumi K, and tive research to determine whether regimented formation of periodically alternating hashi made sectional transplantations and locally target- GFP Takahashi Y. Identification of insulin bands of differentiated cells along the anterior- ed expression of various genes, the results of which led the establishment and maintenance producing cells derived from embryo- posterior body axis. The results of this process of seg- her to propose the existence of a region responsible for of such intervals can be explained Electroporation nic stem cells by zinc-chelating dithi- mentation can be observed in the orderly structures inducing segmentation, which she has named the ‘seg- by the reaction-diffusion model (see zone. Stem Cells 20:284-92 (2002). of the vertebrae and spinal ganglia. In vertebrates, menter.’ Transplantation of this segmenter into the undif- pp. 28-9). Takahashi Y, Osumi N, and Patel N. segmentation takes place in the embryonic region ferentiated segmental plate triggers ectopic boundary Body Patterning. Proc Natl Acad Sci known as the ‘somitic mesoderm,’ and involves the formation, or segmentation. The segmenter region also The details of her current work 98:12338-9 (2001). transient formation of segment organizing bodies specifically expresses a factor that is believed to regu- aside, Takahashi emphasizes that called somites. Yoshiko Takahashi studies segmenta- late Notch signaling, a pathway which has been exten- what drew her to science and main- Sudo H, Takahashi Y, Tonegawa A, Arase Y, Aoyama H, Mizutani-koseki tains her interest is the opportuni- tion and somitogenesis in the chick embryo, and has sively studied for its diverse roles in the determination of Y, Moriya H, Wiling J, Christ B, Koseki made inroads into achieving a better knowledge of cell fate and differentiation. Moreover, Takahashi found ties it offers to those willing and H. Inductive signals from the somato- how groups of cells are able to cleave at specific it was possible to mimic the effects of segmenter trans- able to learn from natural phenome- pleure mediated by bone morphoge- boundary lines and re-organize into periodically seg- plantation by modulating the expression of activated na. “You can’t tell an embryo what netic proteins are essential for the formation of the sternal component of mented patterns. Investigations at the Takahashi lab Notch in specific unsegmented mesodermal regions, to do, only listen to what it can tell avian ribs. Dev Biol 232(2):284-300 seek to explain at the molecular level the mechan- suggesting a new function for the Notch pathway in you.” But her range of interests is (2001). isms of pattern formation in development. boundary formation during mesodermal segmentation. not limited to any single field; she prefers to pursue avenues that oth- The segmenter and Notch signaling Epithelialization ers may have overlooked. She stud- In the early chick embryo, two bands of unsegmented After the segmenter has established the boundaries ies molecules and genes for what mesoderm, collectively referred to as the ‘segmental of a somite, the mesenchymal cells in the border they can teach her about somites, plate,’ flank the neural tube, which runs along the region change to become epithelial cells. The reverse and somites for what they can 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 Report 43 Regenerative Creative Research Development Regeneration Medicine 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 of Tokyo Department of Veterinary Anato- Although he is perhaps best known for his pioneering ized, and found that these cells, too, are capable of my in 1996. He received a postdoctoral Publications fellowship from the Japanese Society work in the cloning of mice, Teruhiko Wakayama has developing into live-born, normal mice. Previously, it for the Promotion of Science in 1996 dedicated his research to exploring the mysteries of had been thought that polar bodies might be reposi- Tamashiro KL, Wakayama T, Akutsu and spent the next three years at the fertilization. By observing the effects of varying experi- tories for damaged DNA and, so, unlikely to develop H, Yamazaki Y, Lachey JL, Wortman Yanagimachi lab in the University of MD, Seeley RJ, D'Alessio DA, mental conditions on the interaction between the egg normally if fertilized. The viability of polar bodies as Hawaii Medical School, where he suc- Woods SC, Yanagimachi R, Sakai ceeded in producing the world’s first (or oocyte) and extrinsic genetic matter, Wakayama’s surrogate oocytes has implications for the field of RR. Cloned mice have an obese phe- cloned mouse. He was appointed to an research team seeks to understand the fundamental human in vitro fertilization, in which the supply of notype not transmitted to their off- assistant professorship at the same basis by which the fertilized egg is ‘reprogrammed’ eggs is frequently a limiting factor. spring. Nat Med 8:262-7 (2002). institution in 1998, and moved to Rocke- from a highly differentiated state to become the toti- feller University as a research assistant Wakayama T, Tabar V, Rodriguez I, professor in 1999. He spent a year as a potent progenitor of the myriad cell types in the adult Cloning Perry ACF, Studer L, Mombaerts P. researcher at Advanced Cell Technolo- body. Wakayama also strives to develop new labora- For many people, the concept of cloning conjures Differentiation of embryonic stem cell gy before returning to Japan to take his tory techniques to make the difficult processes of images of indistinguishably identical replicas of an lines generated from adult somatic current position at the RIKEN CDB. manipulating eggs simpler and more efficient. individual. But Wakayama notes, “Clones are by no cells by nuclear transfer. Science 292:740-3 (2001). means perfect copies of the original.” Experimentally cloned animals exhibit a range of embryonic and The fertilization of the egg is adult epigenetic defects. The process of cloning the initiation of does, however, provide an excellent system for study- the entire developmental ing the mechanisms of fertilization, as it allows the cascade specific control of factors such as nuclear source. In cloning experiments using nuclei from different sourc- es, such as cumulus cells, somatic cells and embryo- Freeze-dried sperm nic stem cells, Wakayama has observed variations in Staff Sperm preserved for use in experiments and in vitro patterns of live-birth rates and developmental abnor-

Team Leader Fertilization that allow it to dock with the outer surface of the unfer- fertilization is traditionally frozen in liquid nitrogen at malities, which he hopes will fuel further investiga- Teruhiko Wakayama In most animals, the sperm and the egg (collectively tilized egg, disintegrate, and deliver its genetic cont- extremely low temperatures (around -190ûC). This tions at the molecular level. In the future, he plans to Research Scientist Satoshi Kishigami known as gametes) are highly specialized cells that ents. Penetration by the sperm also induces dramatic cryopreservation allows the sperm to be maintained continue to explore the secrets of the fertilized egg at Nguyen Van Thuan viably for very long periods, but requires expensive the cellular level. In his own words, “Eggs are very Technical Staff enable the organism to reproduce in a way that com- effects in the egg, including biochemical and structur- Sayaka Wakayama mingles the genetic information from both partners in al reactions that prevent other sperm from entering, facilities and some degree of technical skill in beautiful and mysterious. I never get tired of watching a sexual pairing. This can only be achieved by the the transition from meiotic to mitotic cell division, and handling. In the 1990s, Wakayama developed a them.” fusion of gametes in the process known as fertiliza- a series of intracellular changes that represents the method by which sperm could be freeze-dried and tion. Under natural conditions, fertilization involves a first steps toward the construction of a new individual. remain viable under low-cost, low-maintenance condi- complex chain of events and interactions, which in Seen from this perspective, the fertilization of the egg tions. Sperm freeze-dried in such a way were later mammals begins with the entry of the sperm into the is not merely the event by which two haploid gen- used in ICSI fertilization experiments, and although female reproductive tract. The environment within the omes merge, but also the initiation of the entire devel- they were technically ‘dead,’ they demonstrated an uterus and oviduct produces changes in the sperm opmental cascade. effective ‘fertilization shelf-life’ of around thirty days when stored at room temperature and considerably longer when refrigerated at 4ûC. Wakayama continues to be interested in developing more efficient and economical techniques for sperm storage, and hopes to improve the storable duration and viability rates for sperm preserved in this manner.

Egg alterations 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 factors of two and attempted sperm injection, he found that Nuclear transfer by microinjection eggs of one-half normal size were competent to produce normal individuals, indicating a certain degree of functional redundancy in oocyte composition. In Wakame, the first mouse cloned at the RIKEN CDB 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-

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Development Creative Research Promoting Program Development Regeneration Creative Research Promoting Program Induction Germ Cells

Laboratory for Embryonic Induction Laboratory for Germline Development

Signaling centers in mammalian the node and notochord. Using a marker gene to Germ cells development trace gene expression, his team identified the Germ cells are the only cell types capable of transmit- HNF3β enhancer, and has found that the key ting genetic information from generation to genera- The early mouse embryo is characterized by its dyna- mechanism of its regulation is activation by a pair of tion, and these cells are characterized by equally mic transformation from a simple, nearly featureless transcription factors, functioning downstream of a unique developmental processes as well. In many cylinder of cells to a recognizable embryonic body in known node-inducing signal, Wnt. The team will stu- types of animals, the formation and differentiation of the space of a few days during gastrulation. This dy the effects of the loss of function of these mol- germ cells is controlled by mRNAs and proteins local- remarkably rapid transition is achieved by the highly ecules in knockout mice, and search for potential ized in the specific cytoplasmic region within eggs, coordinated differentiation of cells toward specific co-factors using a yeast interaction-screening sys- called germ plasm. Germ plasm mRNAs are translat- fates, a feat orchestrated by a number of cellular loci, tem. ed in a spatio-temporally regulated manner, but the collectively known as ‘signaling centers.’ These cen- means by which the germ plasm is formed and ters produce diffusible proteins that serve as regula- The serendipitous discovery of a previously unknown achieves this controlled translation remain obscure. tors, guiding neighboring cell communities to take up mouse phenotype in which the head fails to develop Akira Nakamura studies the establishment of the Dro- Hiroshi Sasaki Akira Nakamura the roles they will play in the adult animal, or steering has also spurred research in Sasaki’s lab. It is rare sophila germ line as a model of the processes of Ph. D. them away from inappropriate fates. for such ‘headless’ phenotypes to survive to later Ph. D. germ plasm formation and differentiation, as well as Hiroshi Sasaki received his B. Sc. in Akira Nakamura received both his stages of embryonic development, and the known for the insights this system can provide into the gener- zoology from the University of Tokyo, baccalaureate and his Ph. D. from and his master’s and Ph. D. in devel- Hiroshi Sasaki’s research team investigates the net- cases that do have all been linked to mutations in the University of Tsukuba. He spent al mechanisms of mRNA localization and translation. opmental biology from the same insti- work of signaling centers that work to induce normal node-related genes. The phenotype under investiga- a year as a post-doctoral fellow at tution. He worked as a research development in mammals. The node, the center tion develops normally in early embryogenesis, but the same institution before moving to The Drosophila egg shares cytoplasm with neighbor- associate in Atsushi Kuroiwa’s lab at responsible for induction of body parts in the mouse, exhibits a loss of anterior markers in late gastrulation, the Department of Biology at McGill ing nurse cells via an incomplete cell membrane, Tohoku University from 1990 to University in Montreal in 1995 to Maternal RNP particles shown by green fluorescent 1992, and in Brigid Hogan’s lab at is of particular interest, as it plays a role correspond- when the node normally induces the head. Sasaki is work as a post-doc under Paul Las- allowing mRNAs and proteins from the nurse cells to protein imaging Vanderbilt University from 1992 to ing to the amphibian Spemann organizer, which first now performing a functional analysis of how one can- ko. He returned to Japan in 1997 as be transported to the egg in the form of ribonucleopro- 1994. He returned to Japan to take a sparked Sasaki’s fascination with signaling centers. didate gene interacts with other signaling molecules. a research associate at the Universi- teins (RNPs). In order to ensure the proper function position as assistant professor at Sasaki has identified a number of transcription fac- In these and other studies, Sasaki seeks to identify ty of Tsukuba. He was appointed of these mRNAs, their translation must be prevented Osaka University beginning in 1995, assistant professor in the university’s tors involved in the formation and the function of the molecules involved in signal center formation and during their transport. Screening proteins with locali- where he remained until his appoint- Gene Research Center and Institute the function of Pgc is inhibited, germ cells fail to ment as team leader of the Labora- node in previous studies, and he continues to be function in the early embryonic development of the of Biological Sciences in 2000, and zation patterns similar to those of transported migrate to the embryonic gonads. Another maternal tory for Embryonic Induction at the acutely involved with questions of how these centers mouse. began a three-year term as a PRES- mRNAs, the Nakamura team identified Me31B, which effect mutant, named N14, results in a similar pheno- RIKEN Center for Developmental are formed, and how they regulate development. TO researcher in the Japan Science is involved in the translational silencing of maternal type, suggesting that these genes play roles in germ Biology. and Technology Corporation (JST) in mRNAs. The Me31B complex also includes a protein cell maintenance and migration, and possibly their December 2001. He was appointed Node-specific tran- CDB team leader in March 2002. involved in directing the cytoplasmic transport of programmed cell death. Their loss of function might scription factor net- mRNAs, suggesting that RNP complex proteins coop- correspond to a loss of ‘germness’ in these cells, a works eratively regulate both the translation and transport of finding that provides a new potential link between Hepatocyte Nuclear Factor 3β mRNAs. The lab is now working on the functional germ cells and undifferentiated stem cells. (HNF3β) is a highly conserved characterization of other RNP proteins they have iden- transcription factor expressed tified through several approaches. Future work in the Nakamura lab will focus on taking specifically in embryonic signal- what has been learned from the study of germ line ing centers. Sasaki is engaged In previous work, the Nakamura team identified a nov- establishment in Drosophila and searching for pat- in an analysis of molecules that el non-translatable RNA, Pgc (for Polar Granule Com- terns of conservation and divergence in chordates, activate HNF3β expression in ponent), which is localized in the germ plasm. When using the sea squirt as a model.

Staff Staff

Team Leader Team Leader Hiroshi Sasaki Akira Nakamura Research Scientist Enhancer of HNF3β drives gene expression in the signal- Research scientist Atsushi Sawada Publications ing centers, node and notochord (blue). Kazuko Hanyu-Nakamura Publications Visiting Scientist Keiji Sato Noriyuki Nishioka Maki Shirae Nishizaki Y, Shimazu K, Kondoh H, Sasaki H, Nishizaki Y, Hui C.-c, Naka- Styhler S, Nakamura A, and Lasko Carrera P, Johnstone O, Nakamura Technical Staff Technical Staff Hiroko Sato Sasaki H. Identification of essential fuku M, and Kondoh H. Regulation of Chiaki Nakamoto P. VASA localization requires the A, Casanova J, Jäckle H, and Las- Yayoi Minami sequence motifs in the node/no- Gli2 and Gli3 activities by an amino- Hiroko Sonobe SPRY-domain and SOCS-box con- ko P. VASA mediates translation Assistant tochord enhancer of Foxa2 (Hnf3β) terminal repression domain:implica- taining protein,GUSTAVUS. Dev through interaction with a Drosophi- Misaki Harano gene that are conserved across verte- tion of Gli2 and Gli3 as primary media- Cell 3:65-876 (2002). la yIF2 homolog. Mol Cell 5:181-7 brate species. Mech Dev 102:57-66 tors of Shh signaling. Development (2000). (2001). 126:3915-24 (1999). Nakamura A, Amikura R, Hanyu K, and Kobayashi S. Me31B silences Nakamura A, Amikura R, Mukai M, translation of oocyte-localizing Kobayashi S, and Lasko PF. RNAs through the formation of cyto- Requirement for a non-coding RNA plasmic RNP complex during Dro- in Drosophila polar granules for sophila oogenesis. Development germ cell establishment. Science 128:3233-42 (2001). 274:2075-9 (1996).

46 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 47 2002 New Faces 2002 New Faces

Regenerative Creative Research Development Creative Research Promoting Program Development Regeneration Medicine Promoting Program Chromatin Stem Cell Organogenesis

Laboratory for Chromatin Dynamics Laboratory for Stem Cell Translational Research

Epigenetic control of gene expres- HP-1 heterochromatin protein at silent mating type A new basis for regenerative medi- tively potent adult stem cells known as hemangio- sion (mat) locus of the fission yeast. The results of that stu- cine blasts, which can be induced to demonstrate true plur- In recent years, it has become increasingly evident dy showed that Swi6 protein is a dosage-critical com- The use of transplanted adult stem cells to regener- ipotency under appropriate culture conditions. Asa- that the controlled expression of genes cannot be ponent involved in imprinting the mat locus. This bind- ate blood cells represents one of the first and, to hara’s research at the CDB will hinge on developing accounted for by DNA sequences alone. Epigenetic ing of Swi6 is maintained both across mitotic cell date, most successful applications of regenerative gene expression profiles of post-natal pluripotent (meaning ‘heritable, but occurring independently of cycles and intergenerationally, as it is propagated medicine. But the developing field of stem cell-based stem cells and performing functional assays of such gene sequence’) regulation of gene transcription through meiosis as well. Nakayama’s study also therapy continues to receive attention as one of the cells at various stages of differentiation to elucidate must be maintained and propagated across cell divi- showed that the deacetylation and subsequent methy- most promising frontiers of medical science. Pluripot- the genetic and molecular mechanisms that underlie sion cycles and throughout the development of the lation of a specific histone H3 lysine residue are ent cells, with their ability to produce both pluripotent vasculo- and organogenesis. organism proper in order for cells to establish and essential to this process. Chemical modifications and differentiated progeny on cell division, have enor- maintain their identities as specialized cell types. such as deacetylation, methylation, phosphorylation mous potential for clinical application, and are the In addition to their capacity for facultative tissue- While many of the mechanisms by which such regula- and ubiquitination are fundamental epigenetic pro- subject of intense research in medical fields from specific differentiation, the ability of stem cells and tion is achieved remain unknown, it is clear that modi- cesses that can act to switch the expression of a tar- autoimmune and neurodegenerative disease to disor- their progenitor cell descendants to home to sites of Jun-ichi Nakayama Takayuki Asahara fications to the nuclear DNA-protein chromatin com- get gene on or off. ders of the metabolism. active regeneration makes them attractive for use as Ph. D. M. D. , Ph. D. plex are central to epigenetics. Chromatin occurs in vectors in targeted gene therapy. Adult stem cells Jun-ichi Nakayama received his Takayuki Asahara received his M. D. highly-condensed and less condensed forms, which Through his work on this evolutionarily conserved het- Takayuki Asahara brings a background in clinical car- also preferentially maintain a quiescent, non- bachelor’s, master’s and doctoral from Tokyo Medical College in 1984, degrees in bioscience from the are known as heterochromatin and euchromatin, erochromatin protein, Nakayama has uncovered and performed residencies in cardiol- diology to the CDB, and plans to use his experience differentiating state, and so they are believed to repre- Tokyo Institute of Technology, the respectively. Jun-ichi Nakayama’s research will con- potentially important new roles for the molecule in the ogy and emergency medicine. He at both the bedside and the research bench to inves- sent less of a tumorigenic risk than embryonic stem last in 1999 for his work on the clon- centrate on heterochromatin dynamics, modifications establishment, maintenance and transmission of epi- worked as a research fellow in car- tigate the potential for applications of post-natal pluri- cells when transplanted into human patients, as, ing and characterization of mamma- to the DNA-packing proteins called ‘histones’ in partic- genetic information. His findings suggest that the con- diology at the Tokyo Medical College potent stem cells in regenerative therapies. Building indeed, experience with hematopoietic stem cells has lian telomerase components. He Hospital from 1989 to 1993, before spent the period from 1999 to 2001 ular, and the molecular mechanisms that allow such cept of a gene as a simple string of DNA nucleotides moving to a fellowship in cardiovasc- on previous work in which he identified bone marrow- shown. With the goal of realizing truly translational as a postdoctoral researcher at the chemical states to be heritably transmitted. needs to be expanded to include the action of pro- ular research at St. Elizabeth’s Hospi- derived endothelial progenitor cells (EPCs) and dem- research, Asahara also plans to study optimal meth- Cold Spring Harbor Laboratory in teins in the functional genetic unit. In the future, tal in Boston (USA). He was appoint- onstrated their role in the generation of new blood ods for delivering stem cells to localized and system- Shiv Grewal’s lab, before returning to Heterochromatin proteins and Nakayama plans to perform more detailed analyses ed assistant professor at Tufts vessels, Asahara seeks to characterize stem cells in wide targets in regenerative and gene therapy. Japan in December 2001 as a PRES- School of Medicine in 1995, and gene silencing of the molecular mechanisms that underlie epigenetic the adult body with even greater differentiative poten- TO researcher in the Japan Science associate professor at the Tokai Uni- and Technology Corporation (JST). The Nakayama research team uses the fission yeast function, as well as studies in higher organisms and versity Institute of Medical Sciences tial, and one day to translate that research into clini- He was appointed team leader in the S. pombe as a model system, as its chromatin struc- epigenetic gene expression in developmental pro- in 2000. In addition to his current cally relevant advances. RIKEN CDB Laboratory for Chroma- ture is closely similar to that of higher organisms. In cesses. position as CDB team leader, Dr. tin Dynamics in 2002. previous studies, Asahara serves as director of Regen- Blood vessel formation and organo- erative Medicine and Research at Nakayama demonstrat- the Kobe Institute of Biomedical genesis ed that heterochromatin Research and Innovation, and Pro- One result of Asahara’s and others’ work in the protein binding states fessor of Physiology at the Tokai Uni- 1990s was the realization that blood vessels are play a role in the regula- versity School of Medicine. formed by two distinct physiological processes in the tion of transcriptional adult body. In angiogenesis, new blood vessels are gene silencing. Nakaya- generated from pre-existing, differentiated endothelial ma performed detailed cells. Conversely, vasculogenesis involves the recruit- analyses of the binding ment and differentiation of previously undifferentiated states of Swi6, a homol- EPCs at the site of new blood vessel growth. These og of the mammalian EPCs are themselves the progeny of more differentia-

Staff Staff

Team Leader Team Leader Jun-ichi Nakayama Fission yeasts stained by DAPI(left) or detected by GFP-Swi6(right) Takayuki Asahara Vasculogenesis achieved by endothelial progenitor cells Visiting Scientist Senior Scientist Mahito Sadaie Satoshi Murasawa Technical Staff Publications Hiromi Nishimura Publications Yasuko Ohtani Research Scientist Chikako Nishimoto Nakagawa H, Lee J-K, Hurwitz J, All- Nakayama J, Rice JC, Strahl BD, Allis Satoshi Hasegawa Asahara T, Kawamoto A, Kalka C, Murasawa S, Llevadot J, Silver M, Asa- Walter DH, Rittig K, Bahlmann FH, Junko Toga shire RC, Nakayama J, Grewal SIS, CD, and Grewal SIS. Role of Histone Tsuyoshi Hamada Masuda H. Therapeutic potential of hara T, et.al. Constitutive human telo- Kirchmair R, Silver M, Murayama T, Tanaka K, and Murakami Y. Fission H3 Lysine 9 Methylation in Epigenetic Technical Staff bone marrow-derived endothelial proge- merase reverse transcriptase expres- Nishimura H, Losordo DW, Asahara T, Akira Oyamada yeast CENP-B homologs nucleate Control of Heterochromatin Assem- Kazuyo Sadamoto nitor cells for cardiovascular ischemic sion enhances regenerative properties Isner JM. Statin therapy accelerates centromeric heterochromatin by bly. Science 292:110-3 (2001). Student Trainee diseases Gene Ther Reg 1 (4):361-75 of endothelial progenitor cells. Circula- reendothelialization: a novel effect prompting heterochromatin-specific Masakazu Kuriwaka (2003) tion 106(9):1133-9 (2002). involving mobilization and incorporation histone tail modifications. Genes Dev Nakayama J, Klar AJS, and Grewal Assistant of bone marrow-derived endothelial pro- Kanako Ota 16:1766-78 (2002). SIS. A Chromodomain Protein, Swi6, Asahara T, Isner JM. Vascular cells and Iwaguro H, Yamaguchi J, Kalka C, Mura- genitor cells. Circulation 105(25):3017- Performs Imprinting Functions in Fis- Angiogenesis; Endothelial progenitor sawa S, Asahara T, et.al. Endothelial 24 (2002). Nakayama J, Allshire RC, Klar AJS, sion Yeast during Mitosis and miosis. cells for vascular regeneration. J Hema- progenitor cell VEGF gene transfer for and Grewal SIS. Implicating DNA poly- Cell 101:307-17 (2000). totherapy Stem Cell Res 11(4):579-82 vascular regeneration. Circulation merase a in epigenetic control of (2002). 105(6):732-8 (2002). silencing in fission yeast. EMBO J 20:2857-66 (2001). 48 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 49 2002 New Faces 2002 New Faces

Regenerative Creative Research Regenerative Creative Research Development Regeneration Medicine Promoting Program Development Regeneration Medicine Promoting Program Embryology Neurotrophic Factors

Laboratory of Mammalian Molecular Embryology Laboratory for Neuronal Differentiation and Regeneration

The developmental program is initiated Neurotrophic factors moments after the penetration of an Early neural network development is characterized by egg by a fertilizing sperm. Although the growth of neurons in excess of the number that these initiation events are quintessen- will ultimately populate any given region. Neurons tial to the ontogeny of all mammals, that have formed synaptic connections with a target they remain one of nature's most close- tissue need to establish what is known as a ‘trophic ly guarded secrets. Somehow, within interaction’ in which the target provides the neuron hours of coming together, the two high- with chemical signals necessary for its survival and ly specialized cells that begin this pro- continued function. The supply of such signals is limit- cess, produce a single cell that is utter- ed and neurons which fail to compete successfully for ly unspecialized: it can give rise to all these factors undergo the programmed cell death rou- cell types - in fact, it produces an indi- tine, apoptosis, a controlled die-off that establishes vidual. The single cell is, of course, a an appropriate balance in the neural population. The Tony Perry Hideki Enomoto 1-cell embryo, and the two cells that target-derived chemical signals in the trophic interac- Ph. D. must unite to produce the embryo are M. D. , Ph. D. tion are known as neurotrophic factors. The study of Explant culture of primitive sympathetic ganglia in mouse treated with Tony Perry received his B.Sc. in Hideki Enomoto received his M. D. gametes - an egg (or oocyte) from the these signaling proteins dates back to the discovery Artemin, showing robust neurite outgrowth Microbiology from the Department of Transgenic mouse and offspring expressing green fluorescent protein from the Chiba University School of Bacteriology at the University of Bris- maternal side and a sperm from the medicine in 1988, and his Ph. D. of nerve growth factor (NGF) by Levi-Montalcini and tol, England and his doctorate from paternal side. from the same institution in 1996 for colleagues in the 1950s. Subsequent research has trophins. Enomoto’s work has shown, however, that the University of Liverpool four years his work in the molecular cloning of revealed the diversity of neurotrophic factors, which the role of RET signaling is distinct from that of the later. In 1989 he became a postdoc- the human DAN gene. After serving Adopting the mouse as a model system, the labora- consequences. The characterization of these activat- are now thought to include neurotrophins (of which neurotrophins in that it is involved in the migration, toral fellow working on epididymal as the Chief of the Department of sperm maturation at Bristol Univers- tory of Tony Perry is trying to bring molecular and cell- ing factors at the molecular level is fundamental to an Pediatric Surgery, Matsudo Munici- NGF is a member), neuropoietins, fibroblast growth axonal growth and axon guidance of developing sym- ity and in 1996 won a European ular techniques to dissect the magical events occur- improved understanding of the process of fertilization, pal Hospital in Chiba, Japan, he factors, and transforming growth factors. Hideki Eno- pathetic nerve cells, not the survival of established Molecular Biology Travel Fellowship ring within the earliest embryos. What are the key and knowledge of the mechanisms involved has the moved to the Washington University moto is primarily interested in this last group, and the neurons. This suggests that RET and TRK (the neuro- to work in the laboratory of Ryuzo molecules involved, how do they relate to each other, potential for application in novel methods of contra- School of Medicine, St. Louis in GDNF (for Glial cell line Derived Neurotrophic Factor) trophin receptor) activate independent signaling path- Yanagimachi on the mechanism of 1997, to work as a research asso- and what influence do they have on developmental Family Ligands (the GFLs) in particular. By pinpoint- oocyte activation, which remains one ception and could open up new avenues in the treat- ciate studying the physiological roles ways to perform their complementary functions. of his research interests. From outcome? Central to these analyses is a novel meth- ment of some forms of infertility. of the GDNF family of ligands in neu- ing the extrinsic signals responsible for inducing nor- there, Dr. Perry moved first to the od termed piezo-actuated micromanipulation. This ral development and function. He mal neural development, he hopes to open up new The Enomoto research team plans to conduct system- Rockefeller University and then to allows scientists for the first time to manipulate In addition to describing the earliest events of a new returned to Japan to take a position avenues for ES cell-based therapies in the treatment atic DNA chip analyses of enteric and autonomic neu- the company Advanced Cell Technol- as a team leader at the CDB in 2002. mouse eggs and embryos, which are exquisitely sen- embryonic life, the Perry laboratory has an interest in of human neurological disorders. rons to achieve a better understanding of the molecu- ogy, working primarily on novel methods of genome manipulation. In sitive to mechanical trauma. exploiting these findings to develop novel methods of lar mechanisms involved in RET and TRK signaling. 2002 Dr. Perry took his present posi- genome manipulation. This work includes the devel- Staff The GFLs signal through a receptor complex compris- And, by gaining insights into the roles played by spe- tion as Team Leader of the Labora- Based on his work in this area, Perry has developed ing the RET receptor tyrosine kinase and the opment of a new technique for introducing transgene Team Leader cific neurotrophic factor signaling pathways, Enomoto tory of Mammalian Molecular Embry- a model that seeks to explain how a sperm signals to DNA via co-injection with a nucleus into an egg. Hideki Enomoto co-receptor GRFα. In genetic knockout studies, Eno- hopes to contribute to the development of stem cell- ology at the RIKEN CDB, where he the egg to initiate its dedifferentiation to a totipotent Research Scientist moto discovered that the GFLs play a critical role in will work on mechanisms in mamma- Since such eggs are typically arrested at the second Yukinao Shibukawa based neuroregenerative therapies. Shiho Ohmori lian preimplantation embryos. state. In this model, one or more sperm-borne oocyte meiotic metaphase, the method is called metaphase the development of enteric, sensory and autonomic Technical Staff activating factors act to trigger signaling networks in II (or 'mII') transgenesis. The mII transgenesis meth- Kayoko Inoue neurons, establishing the GFLs as a neurotrophic the moments that immediately follow fertilization. A od enables the efficient introduction of large DNA seg- Mayumi Nagashimada family of equal importance to the better-known neuro- Assistant growing body of evidence indicates that such net- ments to produce transgenic mice. Kaori Hamada works have direct and far-reaching developmental

Staff

Team Leader Tony Perry Research Scientist Hisashi Kishi Noriko Yoshida Publications Publications Technical Staff Satoko Fujimoto Perry ACF, Rothman A, de las ponents: induction of calcium tran- Enomoto H, Crawford PA, Gorodinsky thetic ganglia requires sequential parasympathetic neurons. Neuron Assistant Heras JI, Feinstein P, Mombaerts sients and involvement of proteoly- A, Heuckeroth RO, Johnson EM Jr, actions of GDNF and neurturin. Devel- 22:253-63 (1999). Lio Hiroi P, Cooke HJ, Wakayama T. Effi- sis. Dev Biol 217:386-93 (2000). and Milbrandt J. RET signaling is opment 127:4877-89 (2000). cient metaphase II transgenesis essential for migration, axonal growth Enomoto H, Araki T, Jackman A, with different transgene archetypes. Perry ACF, Wakayama T, Kishika- and axon guidance of developing sym- Heuckeroth RO, Enomoto H, Grider Heuckeroth RO, Snider WD, Johnson Nat Biotechnol 19:1071-3 (2001). wa H, Kasai T, Okabe M, Toyoda Y, pathetic neurons. Development JR, Golden JP, Hanke JA, Jackman EM Jr, and Milbrandt J. GFRa1- Yanagimachi R. Mammalian trans- 128:3963-74 (2001). A, Molliver DC, Bardgett ME, Snider deficient mice have deficits in the Perry ACF, Wakayama T, Cooke genesis by intracytoplasmic sperm WD, Johnson EM, and Milbrandt J. enteric nervous system and kidneys. IM, Yanagimachi R. Mammalian injection. Science 284:1180-3 Enomoto H, Heuckeroth RO, Golden Gene targeting reveals a critical role Neuron 21:317-24 (1998). oocyte activation by the synergistic (1999). JP, Johnson EM Jr, and Milbrandt J. for neurturin in the development and action of discrete sperm head com- Development of cranial parasympa- maintenance of enteric, sensory and

50 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 51 Regenerative 2002 New Faces Development Regeneration Medicine

Development Regeneration Creative Research Promoting Program Sensory Organogenesis

Laboratory for Sensory Development Supporting Laboratories

Sensory organogenesis a complex regulatory loop in which FGF-19 and Wnt- The supporting labo- Sensory systems in vertebrates represent one of the 8c maintain each other’s expression for the duration ratories offer tech- pinnacles of evolutionary achievement, wedding of early ear development. nical support, devel- intricate organic structures with specialized cellular op new technology, function to permit the animal to apprehend its environ- Sensory cellular differentiation and conduct inde- ment by processing a range of physical and chemical The operation of factors from non-ectodermal layers pendent research stimuli. The development of these systems involves seems to be critical to cell differentiation in the inner projects. Their serv- an extraordinary level of coordination between cells ear as well. In a series of experiments in which he ices are available and emergent tissues at very early stages of embryo- tested various tissue recombinations of sections from to all CDB research genesis, and provides an ideal model for the study of germ layers neighboring the site of inner ear develop- groups and teams. such inductive interactions in general. Research in ment, Raj found that even though the mesodermal Raj Ladher’s lab focuses on the induction of the inner inducer FGF-19 could initiate inner ear formation, dif- ear in the chick embryo, a model system chosen for ferentiation did not occur. He is currently testing the Raj Ladher the chick’s tractability to classical embryological tech- hypotheses that FGF-19 may have an inhibitory Ph. D. niques and the opportunities it affords for investigat- effect on differentiation at later stages, and that it Raj Ladher received his B. Sc. in bio- ing the processes of developmental program initia- may play a role in recruiting cells to the developing chemistry from Imperial College, Lon- don in 1992 and his Ph. D. in devel- tion, cell differentiation and the integration of discrete inner ear. opmental biology from the National cellular motifs in a structurally and neuronally special- Institute for Medical Research in ized organ. In the future, the Ladher lab looks to extend its 1996, for his thesis on the cloning of research scope to other model systems, with the goal the Xenopus homeobox gene, Xom, under the supervsion of Jim Smith. Inner ear induction of determining whether some as-yet unidentified gen- He worked as a research associate Sense organs originate in placodes, thickened eral mechanisms are at work in sensory organogene- at King’s College, London from 1996 regions within the early ectoderm. The inner ear aris- sis. to 2000 in the lab of Pip Francis- es from the otic placode, a cluster of cells which, in a West, and first came to Japan during process known as ‘invagination,’ recedes from the that period as a visiting scientist at the Tokyo Medical and Dental Uni- ectodermal surface and is folded over, resulting in the versity. In 2000, he moved to the Uni- formation of a segregated hollow called the otocyst versity of Utah as a postdoctoral fel- which ultimately gives rise to the mature inner ear. low in Gary Schoenwolf’s laboratory, Raj is studying the inductive factors that dictate the and was appointed team leader at the RIKEN CDB in 2002. location and timing of these developmental processes. In a series of tissue manipulation experiments conducted previously, he identified a trio of signaling Staff factors Ð FGF-8, FGF-19, and Wnt-8c Ð each of

Team Leader which localizes in a separate germ layer, and which Raj Ladher cooperate to induce inner ear development. In this Technical Staff Susan Boerner network, it appears that FGF-8, expressed in the Assistant Noriko Hiroi endodermal layer, induces the expression of FGF-19 in the immediately overlying mesoderm. FGF-19 in turn induces Wnt-8c in the neuroectoderm, triggering

Expression patterns of fgf-19 and Publications wnt-8c. The gene products co-operate to induce the inner ear in non-neural Karabagli H, Karabagli P, Ladher sion during Chick Organogenesis. ectoderm, adjacent to the wnt-8c RK, Schoenwolf GC. Comparison of Anat Rec 268:1-6 (2002). neural domain and above the fgf-19 the Expression Patterns of Several mesodermal domain. Fibroblast Growth Factors during Ladher R, Anakwe K, Gurney AL, Chick Gastrulation and Neurulation. Schoenwolf GC, Francis-West PH. Anat Embryol 205:365-70 (2002). Identification of synergistic signals initiating inner ear development. Sci- Karabagli H, Karabagli P, Ladher ence 290:1965-8 (2000). RK, Schoenwolf GC. A Survey of Fibroblast Growth Factor Expres- Late-stage embryo of C. elegans ced-5 mutant Photo: Asako Sugimoto

52 RIKEN CDB 2002 Annual Report Regenerative Development Regeneration 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 Universi- ty of Tokyo, earning his doctorate in 1988 for thesis work under Prof. I. Mabuchi. He spent a year as a postdoctoral fellow at the same institution Publications before moving to pursue a fellowship at Johns Hopkins University from 1989 to Kikuchi S, Hata M, Fukumoto K, 1990. He returned to Japan as an assis- Yamane Y, Matsui T, Tamura A, tant professor in the Department of Cell Yonemura S, Yamagishi H, D Kep- Biology at the National Institute for pler, Tsukita Sh, and Tsukita Sa. Physiological Sciences, where he Radixin deficiency causes conjugat- remained until 1995. He joined the Kyo- ed hyperbilirubinemia with loss of to University Faculty of Medicine the Mrp2 from bile canalicular mem- same year, first as an assistant profes- branes. Nature Genet 24:320-5 (2002). sor, then as a lecturer in the Depart- Rho is rapidly recruited to elongating microvilli on EGF-stimulated A431 cells. ment of Cell Biology, a position he retained until his appointment as CDB Yonemura S, Matsui T, Tsukita Sh, team leader in 2001. the denaturing agent trichloroacetic acid (TCA) for its simple nor direct. Rather, Rho plays an integral part and Tsukita Sa. Rho-dependent and fixative and preservative properties. TCA fixation pro- in an intracellular signaling network of great, but still Ðindependent activation mechanisms duces good results in the visualization of Rho and unfathomed, complexity. Yonemura is working on of ezrin/radixin/moesin proteins: an essential role for polyphosphoinosi- Rac with few of the drawbacks of other traditional one aspect of this network Ð the interactions between tides in vivo. J Cell Sci 115:2569-80 methods. Rho, protein kinase C, and ERM, a cross-linker pro- (2002). tein that joins actin to the cell membrane. Using the However, recent localization studies of the Rho family yeast two-hybrid system to identify proteins in this sig- have produced inconsistent, sometimes contradic- naling network, Yonemura seeks to address some of tory, results. In studies using immunochemical and the many as-yet unanswered questions of Rho func- green fluorescent protein (GFP) tagging of the Rho tion. protein, widely divergent distribution patterns were Staff reported, calling into question the reliability of these In addition to its full-time research efforts, the Yone- visualization techniques. Yonemura has been devel- mura team offers common-use microscopy services Team Leader Cell morphology and imaging participates in the movement and interactions of cells Shigenobu Yonemura Since the advent of the microscope in the 17th cen- with their surroundings. oping monoclonal antibodies specific to each of the to other CDB laboratories. By providing fluorescence Research Specialist Yukako Nishimura tury, the development of novel methods of delineating three Rho isoforms Ð RhoA, RhoB and RhoC. To and electron microscopic imaging and instruction in Kazuyo Minakuchi ensure the validity of the staining of the endogenous techniques, the team hopes to help ensure the quali- Research Associate and distinguishing biological structures at ever- Visualizing Rho-family proteins Nagatoki Kinoshita greater resolutions has stood at the heart of progress The Rho (for Ras Homologous) family of proteins Ð target protein, Yonemura expressed an epitope- ty, fidelity and ease-of-interpretation of visualization Technical Staff tagged target protein in cultured cells. These cells data generated at the Center. Makiko Uwo in the life sciences. The most powerful electron micro- principally Cdc42, Rac and Rho Ð has been extensive- Naoko Inoue scopes now provide scientists with the ability to visual- ly studied for its effects on the structural activity of were processed for immunofluorescence imaging Yuka Miyake ize biological objects as small as 2 nanometers in cells. These proteins serve as molecular switches using antibodies against both endogenous and size, which has opened up new windows of insight capable of interacting with the actin cytoskeleton and epitope-tagged target proteins. As antibodies to the into the relationship between form and function at the inducing cells to form a range of surface structures, endogenous protein should also bind to the epitope- sub-cellular scale. In theory, even greater resolutions such as cylindrical microvilli projections and stress fib- tagged proteins, the failure of an imaging method to could be achieved, but the limits imposed by the ers that serve to anchor cells to the substratum. The show a high degree of correspondence in the localiza- materials under study Ð organic molecules with Yonemura research team is studying the localization tion of the two indicates a flaw in the antibody-fixation sometimes fragile structures Ð have kept such micro- of Rho family members with the goal of gaining protocol. By introducing such improvements to fixa- scopes from realizing their full potential. One of the insight into how these molecules contribute to cellular tion and evaluation methodologies, Yonemura hopes main goals in the field of microscopy is the develop- morphogenesis. to reinforce scientific rigor in the field of microscopic ment of new techniques in specimen preparation and visualization and to be able to apply these techniques to the study of other members of the Rho family. image enhancement that will allow scientists to over- Specimen preparation is considered come these limitations and probe the hidden world to be as important as even more deeply. The actin cytoskeleton microscope power in The skeletal framework of the cell is built up of an the imaging of biological samples Shigenobu Yonemura brings a background in cell intricate assembly of filaments and accessory pro- and molecular biology to bear on visualization meth- teins, cooperating to stabilize the cell and give it ods and technology with applications in the study of One of the perennial technical challenges in the shape. One of the main types of filaments is the actin animal development. He seeks to explore the links microscopic study of cells has been assuring that the filament (or microfilament), which, in addition to deter- between cell shape, function and behavior by improv- specimen is not damaged or altered in the process of mining surface morphology, also play a critical role in ing the means by which the cellular architecture can preparing it for examination. Indeed, specimen prepa- driving cellular movement. As described above, the be brought to light. His special emphasis is on devel- ration is considered to be just as important as micro- Rho-family proteins help to regulate the function of In dividing animal cells, Rho (red) oping ways to localize with greater precision the scope power in the imaging of biological samples. In the actin cytoskeleton and, thereby, many aspects of is concentrated in the furrow cellular behavior as a whole. But the relationship bet- region. Blue: nuclei, Green: micro- molecular components of the cytoskeleton, a com- previous work on improving specimen preparation for tubules. plex structure that determines cell morphology and immunofluorescence imaging, Yonemura introduced ween Rho expression and actin response is neither

54 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 55 Regenerative Development Regeneration Medicine Supporting Laboratories Experimental Mice

Laboratory for Animal Resources and Genetic Engineering Model Organisms

es transgenic and knockout mouse models to the specifications of scientists working in a wide range of genetic, embryological and biomedical research projects, maintaining the highest quality standards and YEAST ROUNDWORM rapid turnaround to ensure fast and easy access to Common name : fission yeast Common name : nematode researchers working within the Center and through- Scientific name : Schizosaccharomyces pombe Scientific name : Caenorhabditis elegans out the country. In addition to these core functions, Yeasts are known for their use as fermenting With exactly 959 somatic cells in the body of a nor- the LARGE staff provides a number of other services, agents in the production of wine and beer, but mal adult, the nematode offers perhaps the best- such as nuclear transfer cloning, isolating homolo- they are also found in life sciences laboratories characterized model of a multicellular organism gous recombinants and culturing embryonic stem around the world, performing a very different but possessing simple nervous, muscle and reproduc- cells, and works to develop improved methods of equally valuable service. The fis- tive systems. It is possible to track the fates of indi- sample handling and storage, such as higher- sion yeast has served as an exper- vidual cells in its transparent body on a micro- efficiency modes of cryopreservation. The lab also imental model for the past fifty scope slide glass, and the exact fate of every cell The study of model organisms and systems is inte- performs a number of maintenance and logistical years, thanks to it amenability to in every cell lineage is known. In 1998, geneticists molecular genetic studies and the added to that body of knowledge by sequencing Staff gral to biological research. Such systems provide sci- functions, such as the specific pathogen free (SPF) entists with the means to search for broadly shared housing, cleaning, processing and distribution of ani- ease which it can be grown and the nematode genome, which was found to con- Team Leader mechanisms underlying developmental and regenera- mals. manipulated in culture, and it is tain around 19,000 genes, many of which have Shinichi Aizawa perhaps best known for its contri- human homologs. The discovery of the RNA inter- Postdoctoral Researcher tive processes across species, and conversely to Kenryo Furushima In the coming year, the LARGE team plans to expand butions to our knowledge of chro- ference mechanism that allows genes to be func- Research Associate identify those traits that earn each species its unique Kazuki Nakao branch on the phylogenetic tree. The mouse is one of its services and initiate new programs, notably the matin structure. tionally 'knocked down' has further fueled interest Technical Staff in these tiny creatures as model organisms. Hitoshi Miyachi the most important and widely used model organisms generation of target vectors from sequence informa- Rika Nakayama in science today, prized for its amenability to genetic tion, and the independent production of novel Hiroshi Kiyonari Technical Assistant manipulation, its high level of homology with humans, genetically-modified constructs, a drive that is antici- FLATWORM Chiaki Tamamura pated to generate on the order of 80-100 new mutant Akemi Hara and the trove of data regarding its physiology, genet- Common name : planarian Takashi Nomura ics and development that has accumulated over near- strains per year. Such strains serve as research plat- Scientific name : Dugesia japonica Tsunehisa Fujita forms with the potential to provide new insights into a Assistant ly one century of intensive scientific research. Found in freshwater streams and ponds, planari- Kaori Nasu range of important research problems, from the ans are small worms only a few millimeters long The Laboratory for Animal Resources and Genetic mechanisms of organ development to the genetic which possess simple brains, nervous and diges- Engineering (LARGE) provides an important suite of bases of human disease. The lab will also function as tive systems, and a strangely endearing pair of services related to the generation of experimental part of Japan’s system of Mouse Embryo Banks, with rudimentary eyes. Their most striking characteris- mice to labs within the CDB and around Japan. In its a special emphasis on producing, storing and catalog- tic, however, is their regenerative ability; cut a sin- FLY role as a CDB support laboratory, the LARGE team, ing embryos for use in developmental biology and gle animal into dozens, even hundreds of pieces, Common name : fruit fly under CDB Deputy Director Shinichi Aizawa, produc- regenerative medical research. and each fragment can develop into a new individ- Scientific name : Drosophila melanogaster ual. This remarkable capacity has attracted the The fruit fly came into its own as a laboratory mod- attention of biologists interested in the mechan- el when scientists in the early twentieth century Publications isms of regeneration and much progress has discovered Drosophila was easy to keep, feed been made in recent years in understanding how and breed, and by the 1920s fruit flies were the Ino M, Yoshinaga T, Wakamori M, Miyamoto N, Taka- Fukami K, Nakao K, Inoue T, Kataoka Y, Kurokawa M, Nishinakamura R, Matsumoto Y, Nakao K, Nakamura K, subject of extensive experiments in heredity in the hashi E, Sonoda J, Kagaya T, Oki T, Nagasu T, Nishiza- Fissore RA, Nakamura K, Katsuki M, Mikoshiba K, Yoshi- Sato A, Copeland NG, Gilbert DJ, Jenkins NA, Scully S, the planarian achieves its death-defying multiplica- wa Y, Tanaka I, Imoto K, Aizawa S, Koch S, Schwartz A, da N, and Takenawa T. Requirement of phospholipase Lacey DL, Katsuki M, Asashima M, and Yokota T. Murine tion. The answer lies in the totipotent cells distrib- labs of Nobel Prize-winner T. H. Morgan and else- Niidome T, Sawada K, Mori Y. Functional disorders of the Cdelta4 for the zona pellucida-induced acrosome reac- homolog of SALL1 is essential for ureteric bud invasion in uted throughout its where. As its popularity as a model increased, sympathetic nervous system in mice lacking the sub- tion.Science 292:920-3 (2001). kidney development. Development 128:3105-15 (2001). α1B tiny body, cells it is data on all aspects of fly biology, from molecular unit (Cav2.2) of N-type calcium channels. Proc Natl Acad Sci 98:5323-8 (2002). Tsuzuki T, Egashira A, Igarashi H, Iwakuma T, Nakatsuru hoped will provide genetics to learned behaviors, burgeoned. The dis- Y, Tominaga Y, Kawate H, Nakao K, Nakamura K, Ide F, clues to the emerg- covery of homeotic genes in the fly, which deter- Kim JM, Nakao K, Nakamura K, Saito I, Katsuki M, Arai Kura S, Nakabeppu Y, Katsuki M, Ishikawa T, and Seki- ing field of regenera- mine segment identity and which were found to be K, and Masai H. Inactivation of Cdc7 kinase in mouse ES guchi M. Spontaneous tumorigenesis in mice defective in conserved in animals from worms to humans, cells results in S-phase arrest and p53-dependent cell the MTH1 gene encoding 8-oxo-dGTPase. Proc Natl tive medicine. death. EMBO J 21:2168-21679 (2002). Acad Sci 98:11456-61 (2001). made Drosophila a favorite of developmental biologists as well. Sequencing Lab Staff The CDB sequencing lab provides DNA sequencing and analysis services for use by all laboratories at the Center. Equipment is available for Sequencing Lab Research Scientist small, medium and large-scale requests, with turnaround times from less than one week for smaller scale projects to one month for longer jobs, Hiroshi Tarui 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 Technical Staff when sequence-ready samples are submitted. Analysis services for editing, homology search, and assembling are also available. All work Osamu Nishimura requests can be submitted and tracked online, and results are returned to the applicant's personal folder on the CDB intranet file server, making Yukako Hirao Takako Sano 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 Report 57 Model Organisms Administration and Activities

FISH BIRD Common name : zebrafish Common name : chick (chicken) Scientific name : Danio rerio Scientific name : Gallus gallus The zebrafish, with its short reproductive cycle The chick embryo has been in use as a model and high number of eggs per clutch, is one of the since the time of Aristotle, making it one of the most popular vertebrate systems used in genetic most exhaustively studied systems in biology. The analysis. Its rapid development has also made it egg's extrauterine development made it attractive popular in the developmental biology and embryol- in the days before surgical techniques. The chick- ogy communities, as has the fact that its eggs are en's regular laying cycle and the fact that fertilized externally fertilized and translucent, making it eggs can be cold-stored for several weeks, tempo- possible to follow its developmental progress rarily freezing their development, ensures con- easily using a dissect- stant access to embryos. Chick embryonic devel- ing microscope. The opment shows some similarity to that of mam- tractability of this fish mals, and the flat blastodisc of the chick is mor- to gene knockdown phologically similar to the human embryo at the methods and the exist- same stage of development. The introduction of ence of techniques for techniques for embryonic sectional explantation tracing the fates of and the production of chick-quail chimeras has individual cells have only served to add to the popularity of the chick also helped to estab- embryo as a model system. lish it as a favorite system in developmental genetics. MOUSE Common name : mouse Scientific name : Mus musculus For nearly one hundred years FROG the mouse has Common name : African clawed frog been the animal Scientific name : Xenopus laevis of choice for mod- The African clawed frog has a long history of use eling human in the study of vertebrate embryology and develop- genetics and dis- ment. As aquatic animals, they undergo external ease. This appli- development, making it relatively cation has been easy to manipulate the fertilized egg borne out by the recent completed sequencing of as compared to animals in which the the mouse genome, which revealed an extremely embryo grows within the uterus. high homology between the mouse and human Their eggs are also quite large, genomes. As fellow mammals, humans and mice about 1 mm in diameter, which is also share many physiological traits and suscepti- about ten times the size of a mouse bilities, making the mouse an attractive model in oocyte, allowing for various surgical biomedical studies. Its rapid breeding cycle and manipulations and biochemical small size further add to its appeal, and it is little analyses. Ovulation can be induced wonder that scientists have chosen this animal for relatively simply using luteinizing hormone, which developing or popularizing a wide range of genet- eliminates some of the problems associated with ic techniques from transgenesis to gene knock- extended reproductive cycles. And their rapid pro- outs to cloning by nuclear transfer. gress from fertilization to neurulation in less than 24 hours provides scientists with a dynamic picture of the first stages of life. 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 fields Educational Activities of physics, chemistry, basic life sciences, engineering and medicine at laboratories located across Japan. The Center for Devel- opmental Biology was established as the first research center within the RIKEN Kobe Institute in April 2000.

Encouraging young scientists to participate in advanced research projects as they prepare for careers in the life sciences is 2002 CDB Budget essential to ensuring the development of future generations of researchers. The CDB has collaborative educational programs with a number of graduate schools in Japan, providing students with opportunities for hands-on laboratory benchwork and lec- tures on topical issues in development and regeneration. The center also has also accepted 47 students from other universities Facility Expenses as research assistants or trainees. In addition to regularly scheduled scientific seminars, students can attend events such as the FY2002 (energy expenses,maintenance,etc.) 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 Management Expenses 570570 (personnel expenses,etc.)

Research Expenses

2,0002,000 CDB Staff (December 2002) Group …Directors/Team Leaders 26 Research …………… Scientists 71 Research …………… Associates 3 Technical …………………… Staff 82 Assistants ………………………… 23 3,1393,139 Visiting …………… Researchers 9 Collaborative ………… Scientists 19 Student ………………… Trainees 34 Part-time …………………… Staff 46 Research … Promotion Division 38 (Million Yen) Other ……………………………… 28

Total ……………………………… 379

Advisory Council Institutional Review Board

The CDB Advisory Council, chaired by Dr Igor Dawid (Na- The Institutional Review Board includes representatives tional Institute of Child Health and Human Development, from local academic, media and lay organizations as well NIH) submits regular external reports regarding the scientif- as CDB research staff, and meets regularly to review and ic administration and the state of research progress at the discuss the ethical and social implications of programs and Center. The ten-member Council includes top scientists in investigations being conducted at the CDB. The results of related fields from Japan and around the world and acts as the Board's discussions are submitted to the Center Direc- an unbiased review board for CDB research activities. tor 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 Report 61 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, is Joohong Ahnn 2002.5.27 Germline stem cells and the niche in Drosophila melanogaster Toshie Kai involved in movement, fertility, egg laying, and growth in C. elegans 2002.6.7 The evolution of homeobox gene cluster P. W. Holland 2002.11.5 Clonal identification of pluripotent stem cells in the developing liver and pan- Hideki Taniguchi 2002.7.2 How the cavefish lost its eyes: A problem in development and evolution Yoshiyuki Yamamoto creas using flow cytometric cell sorting 2002.7.23 Control of epithelial-mesenchymal transitions in the neural crest Don Newgreen 2002.11.14 Sensory signaling and neural modulation in the nematode Caenorhabditis Ikue Mori 2002.7.24 Cell polarity and asymmetric cell divisions in the Drosophila PNS Francois Schweisguth elegans 2002.8.8 The 1st CDB Meeting; Head development (I) (multiple speakers) 2002.11.20 Distinct Rac regulators in cell migration, axon outgrowth and cell-corpse Yi-Chun Wu 2002.8.13 The 2nd CDB Meeting: Left-right determination in the lancelet embryo Kinya Yasui engulfment in C. elegans 2002.8.13 The 2nd CDB Meeting: Molecular studies of hemichordate development: New Kumifumi Tagawa 2002.11.21 Regulation of expression and function of cell differentiation factor Nkx2.2 Hirotaka Watada insights into bilaterian evolution 2002.11.21 The tao of stem cells in the germline Haifan Lin 2002.8.13 The 2nd CDB Meeting: Body plan of the sea urchin embryo Koji Akasaka 2002.11.28 Transcriptional regulation of signalling competence in derivatives of the spe- Joshua M. Brickman 2002.8.19 Transposon-mediated transgenesis and insertional mutagenesis in zebrafish Kouichi Kawakami mann organizer 2002.9.5 Construction of human artificial chromosome and its possible application for Mitsuo Oshimura 2002.12.5 Nuclear organization of chromosomes in fission yeast Yasushi Hiraoka regenerative medicine 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 mic stability 2002.12.9 Evolution of complex form and function in the developing mammalian heart Richard Harvey 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 histogenesis 2002.12.17 Use of the Cre/LoxP system to study mouse development by a non-biased Werner Muller 2002.10.24 Analysis of the function of isthmin in zebrafish embryos Isato Araki approach 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 Uchida 2002.10.30 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 mouse 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 and Austin Smith (University of Edinburgh) Regenerative Medicine Yoshiki Sasai (CDB) Igor Dawid speaks at the Opening Symposium (April 22, 2002) Takayuki Asahara (St. Elizabeth's Medical Center)

Concluding Remarks Shin-ichi Nishikawa (CDB)

62 RIKEN CDB 2002 Annual Report RIKEN CDB 2002 Annual Report 63 City of Kobe Medical Industry Development Project

The CDB is located in the international supply. The information infrastructure is well-developed, city of Kobe. This port city has been and access to broadband internet connections, mobile the major entrance to western Japan phone service and In 1998, Kobe inaugurated its Medical Industry Development The Medical Industry Development Project seeks to draw par- for over a century, giving the city a satellite TV is more Project, marking the launch of one of Japan's first integrated ticipants from academia, government research institutions cosmopolitan atmosphere and unique charm. With 1.5 mil- prevalent than near- biomedical research parks. The CDB plays a central role as a and life sciences industries by providing world-class facilities lion residents, the city is the sixth-largest in Japan, offer- ly anywhere else in basic research facility in this growing international center of and infrastructure, and a range of support services for basic ing a perfect mix of urban sophistication and access to the world. excellence. The CDB facilities are connected by an elevated and corporate research and development. Plans for 2003 some of Japan's more beautiful natural and historical walkway to the adjacent Institute for Biomedical Research include the construction of a Translational Research Informat- attractions. Major national uni- and Innovation (IBRI) research labs, fostering communica- ics Center and a Biomedical Accelerator business incubation versities and cultural tions and collaborations between researchers at both loca- facility, and the continued recruitment of biomedical research Situated in the heart of the Kansai region of western attractions are locat- tions. And the completion of the IBRI clinical testing hospital firms from within Japan and abroad. CDB scientists are alrea- Japan, Kobe is a short drive ed both within the (scheduled for April 2003) will further expand the translational dy working in close collaboration with colleagues at the IBRI or train ride away from Osa- city and in the sur- research opportunities for CDB scientists and their collabora- and industry research labs in projects that promise to realize ka and Kyoto, and sits nes- Kyoto rounding Kansai region, tors. the potential of the bench-to-bedside translational research tled in the foothills of the providing a stimulating intel- approach. Rokko Mountains facing out lectual and educational Kawanishi onto the Inland Sea. The climate. And thanks to the Miki Takarazuka climate is pleasant and advent of global communi- Nishinomiya Osaka KIMEC Center features four distinct sea- Kobe Itami Airport cations and information CDB Bldg C Shin-Kobe Sta. 1F communal labs sons similar to those in oth- Ashiya technology, Kobe is secure- Akashi Sannomiya Amagasaki Osaka 2F~5F labs er temperate climes. An ly connected to the world- 6F library, seminar room Translational Research Informatics Center Nara Medical Informatics for Preclinical and Clinical Studies exceptional public transpor- CDB wide network of ideas. Kobe Airport tation system provides con- (2005) venient access to destina- Exciting. Comfortable. Beau- tions within Kobe and Awaji tiful. Fun. These are some around Japan, and the Kan- JR Shinkansen of the words our visitors JR CDB Research Aquarium sai International Airport Toll Road from overseas have used to Kansai International serves as the region's hub Airport describe their experience in for overseas travel. Kobe. The Center offers dedicated support to for- Costs of living are comparable to those found in many eign research staff and Western cities. Kobe's shopping district is considered guests, from assistance Sapporo second-to-none in Japan, and shops specializing in import- with accommodations and ed goods make it easy to purchase items from around the visas to short-term visitors, world. Houses and apartments are clean, comfortable and to renting apartments and modern, and hotels and convention space are in ample Sendai help with setting up laboratories for newly arrived Kanazawa staff as they acclimate them- Tokyo Kobe International KOBE Yokohama Okayama selves to life in Japan. Business Center 1 and 2 Hiroshima Nagoya Osaka Whether as a traveler or a rental labs and offices Fukuoka Takamatsu potential resident, we encourage you to visit the Kagoshima IBRI Lab Bldg CDB and the city of Kobe to 1F rental labs 2F CDB labs (stem cells) see for yourself how enjoy- CDB Animal Facility 3F CDB labs (stem cells)áFACScells)áFACS 1F mouse facility, transgenic/KO lab 4Fá5F4Fá5F Cell Processing Center able life in Japan can be. 2F mouse facility, operation/virus rooms IBRI Clinical Bldg 1F operation room, cafeteria CDB Bldg B 2F outpatient 1F mouse/rat facility 3F clinical lab 2F communal facility 4F office 5F hospital CDB Bldg A IBRI Medical Imaging 1F office, EM lab, lobby, radioactivity lab 1F MRI(3T), open MRI, CT-guided irradiation 2F communal facility, cold room, virus lab 2F PET 3F~6F labs 7F labs, library, seminar room, lounge

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