CDB 2003Annual Report

ISSN 1349-0494 RIKEN Center for Developmental Biology

RIKEN Center for Developmental Biology 2003 Annual Report 2003 Annual Report

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

Production

Text Douglas Sipp

Content Development Naoki Namba Douglas Sipp

Design TRAIS K. K.

Photography Yutaka Doi

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

All contents copyright (c) RIKEN Center for Developmental Biology, 2004. Printed in Japan Printed on 100% Recycled Paper Contents

■ Organization ……………………………………………………… 1 ■ Message from the ……………………………………… Director 2/3 ■ 2003 Highlights …………………………………………………… 4/5 RIKEN Core Program ■ Evolutionary Regeneration Biology (Kiyokazu Agata) 8/9 Center for Developmental Biology ■ Vertebrate Body Plan (Shinichi Aizawa) 10/11 ■ Morphogenetic Signaling (Shigeo Hayashi) 12/13 ■ Cell Asymmetry (Fumio Matsuzaki) 14/15 ■ Stem Cell Biology (Shin-ichi Nishikawa) 16/17 Deputy Directors ■ Organogenesis and Neurogenesis (Yoshiki Sasai) 18/19 Shin-ichi Nishikawa Shinichi Aizawa ■ Cell Adhesion and Tissue Patterning (Masatoshi Takeichi) 20/21 Director

Masatoshi Takeichi Creative Research Promoting Program Institutional Review Board

■ Stem Cell Translational Research (Takayuki Asahara) 24/25 ■ Neuronal Differentiation and Regeneration (Hideki Enomoto) 26/27 Advisory Council ■ Neural Network Development (Chihiro Hama) 28/29

Igor Dawid, Chair ■ Vertebrate Axis Formation (Masahiko Hibi) 30/31 Laboratory of Molecular Genetics National Institute of Child Health and Human Development, NIH (USA) ■ Positional Information (Shigeru Kondo) 32/33 William Chia Evolutionary Morphology (Shigeru Kuratani) 34/35 MRC Center for Developmental Neurobiology (UK) ■

Elaine Fuchs ■ Sensory Development (Raj Ladher) 36/37 Howard Hughes Medical Institute Rockefeller University (USA) ■ Germline Development (Akira Nakamura) 38/39 Hajime Fujisawa ■ Chromatin Dynamics (Jun-ichi Nakayama) 40/41 Division of Biological Science Nagoya University Graduate School of Science (Japan) ■ Cell Migration (Kiyoji Nishiwaki) 42/43 Peter Gruss Department of Molecular Cell Biology ■ Pluripotent Cell Studies (Hitoshi Niwa) 44/45 Max Planck Institute for Biophysical Chemistry (Germany) ■ Mammalian Epigenetic Studies (Masaki Okano) 46/47 Yoshiki Hotta National Institute of Genetics (Japan) ■ Mammalian Molecular Embryology (Tony Perry) 48/49 Yoichi Nabeshima ■ Embryonic Induction (Hiroshi Sasaki) 50/51 Department of Pathology and Tumor Biology Kyoto University Graduate School of Medicine (Japan) ■ Cell Fate Decision (Hitoshi Sawa) 52/53 Tatsutoshi Nakahata Department of Pediatrics ■ Developmental Genomics (Asako Sugimoto) 54/55 Kyoto University Graduate School of Medicine (Japan) ■ Body Patterning (Yoshiko Takahashi) 56/57 Austin Smith Center for Genome Research 58/59 University of Edinburgh (UK) ■ Genomic Reprogramming (Teruhiko Wakayama)

Yoshimi Takai 2003 New Faces Department of Molecular Biology and Biochemistry Osaka University Graduate School/Faculty of Medicine ■ Mammalian Germ Cell Biology (Mitinori Saitou) 60 ■ Systems Biology (Hiroki R. Ueda) 61

Supporting Laboratories

■ Cellular Morphogenesis (Shigenobu Yonemura) 64/65 ■ Animal Resources and Genetic Engineering (Shinichi Aizawa) / Sequencing Lab 66/67

r e ■ Animal Facility & Research Aquarium 68 h d a L j a ■ Budget & Staff ……………………………………………………… 70 R : O ■ Collaborations & Affiliations ……………………………………… 71 T O H ■ Leading Project in ……………………… Regenerative Medicine 72/73 P . k ic ■ Seminars ……………………………………………………… 74/75 h c e th ■ CDB Symposium ……………………………………………………… 76 f o r a e le d id M RIKEN CDB 2003 Annual Report 1 Contents

Masatoshi Takeichi Creative Research Promoting Program Institutional Review Board

■ Stem Cell Translational Research (Takayuki Asahara) 24/25 ■ Neuronal Differentiation and Regeneration (Hideki Enomoto) 26/27 Advisory Council ■ Neural Network Development (Chihiro Hama) 28/29

Supporting Laboratories

■ Cellular Morphogenesis (Shigenobu Yonemura) 64/65 ■ Animal Resources and Genetic Engineering (Shinichi Aizawa) / Sequencing Lab 66/67

Masatoshi Takeichi Director, CDB

The days leading up to the year's end tend to be chaotic, but they also provide one with a valuable Domestically, Japan saw increased stimulus to look back on milestones passed and ahead to new objectives. As 2003 comes to a attention to a number of related issues and close, it's good to be able to take stock of the achievements made by researchers at the RIKEN Cen- research fields. The national government ter for Developmental Biology, the challenges we face in the years ahead, and the state of our rapid- unveiled plans for the Leading Projects, initiatives set ly evolving science. This year has seen the emergence of news and civic issues that have placed up to fund mid-term research into a number of key areas the fields of developmental biology, regeneration and regenerative medicine frequently in the identified as having the potential to improve public health, wel- spotlight and sometimes at the center of controversy on the world stage, a position accompanied by fare and economic well-being. As the largest center for the study of all the advantages and drawbacks that public prominence entails. developmental biology in Japan, the CDB received funding to carry out Leading Project research into potential applications for stem cells in regen- For us at the CDB, the year began in March with the unqualified (and uncontroversial) success of erative medicine. Embryonic stem cells also featured in the national news our first annual symposium on The Origin and Formation of Multicellular Systems, a three-day meet- this year, which saw the first establishment of a new line of human ES cells by ing of more than 200 distinguished speakers, poster presenters and attendees who gathered to dis- a Japanese laboratory in Kyoto University. ES cells represent a biological cuss metazoan biology, from its single-celled forebears to its greatest evolutionary achievement in resource of potentially revolutionary importance, but whose study demands careful the formation of neural networks comprising billions of cells. We look forward to hosting more such consideration and ethical restraint. The use of human ES cells is for biomedical meetings on a yearly basis, beginning with our next symposium, on Developmental Remodeling, in research is permitted in Japan, and in 2003 a number of CDB labs applied for permis- March of 2004, which will be held on the CDB campus in an auditorium designed to increase the sion to use the new Kyoto cell line. Approval of these research plans will allow scientists Center's capacity for hosting scientific meetings and expand its ability to provide a venue for inter- here to explore the secrets of cell differentiation and pluripotency, and perhaps one day to national exchange across fields. contribute to the development of cell-replacement therapies for intractable illnesses such as diabetes and Parkinson's disease. Research advances from CDB labs have also provided a string of highlights this year. As you will see from the pages of this report, scientists at the CDB have made strides forward on many As a basic research institute, the CDB also appreciates the fundamental importance of main- fronts, from uncovering new molecular mechanisms at work in the asymmetric division, signal- taining ties with the world of academia, and we have pursued active affiliations with some of ing and guided migration of cells, to developing new technologies that may one day enable Japan's top graduate school programs in the biosciences and regenerative medicine, allowing the targeted differentiation of embryonic stem cells toward clinically relevant ends. Studies in students from affiliated programs to conduct their academic research using the Center's facilities. evolutionary development have also provided broadened contexts and perspectives, finding This year, we were proud to formalize relationships with the Kyoto University Graduate School of possible roots for the central nervous system in the genome of a tiny flatworm and chal- Medicine, the Nara Advanced Institute of Science and Technology Graduate School of Biological lenging conventional wisdom about the regulatory origins of the vertebrate hindbrain. Sciences, and the Kwansei Gakuin University Bioscience Department, bringing the total number of such affiliations to six. Of course, this science did not take place in a vacuum. The central pillars of the Cen- ter's research mission — developmental biology, regeneration and the scientific Public communications and outreach also featured prominently on the Center's calendar this year. bases for regenerative medicine — are all vibrantly dynamic, truly global fields of In May, the CDB opened its doors to the public for its first Open House, which was attended by endeavor, and the CDB is pledged to remain fully engaged with international more than 1,000 visitors who took advantage of the opportunity to take in talks on stem cells and pro- colleagues at the organizational, laboratory and institutional levels. In 2003, grammed cell death, join in mock experiments, and sneak a peek at a day in the life of a develop- this commitment was characterized by the establishment of scientific mental biology lab. The Center also joined in the public educational activities at the Kobe BioWeek exchange relationships with the University of Texas Graduate School event in August, a biotechnology convention that saw more than 8,000 attendees, including many of Biomedical Sciences and the Temasek Life Sciences Laboratory who visited the CDB in a virtual sense via two-way video lectures and realistic software simulations Limited in Singapore, paving the way for new and better oppor- of laboratory equipment. tunities to visit and host researchers from these institu- tions. We look forward to increasing the number and In retrospect, the year has been a full one, and it has been gratifying to participate in the Center's range of our inter-organizational exchange activities both as a scientist and an administrator. So it is a great pleasure for me to introduce our programs in the years ahead. 2003 Annual Report, which illustrates the range and depth of research at the CDB and serves both as a spark for happy memories and as a source of inspiration as we enter a new year, with its promise of new discoveries and change.

2 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 3 Center for Developmental Biology Message from the Director

Masatoshi Takeichi Director, CDB

2 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 3 2003 HIGHLIGHTS August 27 - 9 Kobe BioWeek May 13 The city of Kobe played host to more than 8,000 visitors December 21 Publication of "Generation participating in Kobe Publication of "An NDPase of neural crest-derived BioWeek, a series of meetings links ADAM protease April 1 peripheral neurons and and events focusing on the glycosylation with organ New Graduate School floor plate cells from mouse August 19 growth and future directions of morphogenesis in Affiliations and primate embryonic Publication of "Traveling the biotechnology sector in the C. elegans" The CDB formalized its stem cells" stripes on the skin of a region and around Japan. The In work published in the online affiliations with the Kyoto In a study published in the mutant mouse" CDB joined in the public edition of Nature Cell Biology, University Graduate School of May 13 issue of the The August 19 issue of PNAS education activities, with two- the Laboratory for Cell Medicine and the NAIST Proceedings of the National featured a cover image taken way videoconference lectures Migration, under team leader Graduate School of Biological Academy of Sciences (PNAS), from a research article from and Q&A sessions, hands-on Kiyoji Nishiwaki, reported the Sciences, allowing doctoral the Sasai research group Shigeru Kondo's lab simulations of lab equipment role of a protein modification students from those schools to demonstrated the application describing the mathematical and a virtual lab tour program known as glycosylation in conduct their thesis research of stromal cell derived properties of a strikingly using web cameras that guiding the migration of in CDB laboratories under the inducing activity (SDIA) in the patterned mutant mouse allowed the public to watch gonadal cells in the nematode, guidance of lab heads high-efficiency induction of strain. CDB labs at work in real time. C. elegans. designated as visiting peripheral neurons from professors. embryonic stem cells.

July 2 October 1 Scientific exchange RIKEN Becomes agreement with Temasek Independent Administrative March 24 -26 Life Sciences Laboratory Institution June 24 Limited As part of a restructuring of First CDB Symposium: Publication of "Origin and The CDB established formal N Suzuki, M Hirata and S Kondo. Traveling Japanese public corporations, Origin and Formation of Stripes on the Skin of a Mutant Mouse. Proc evolutionary process of the ties with the Temasek Life RIKEN was officially Multicellular Systems Natl Acad Sci U S A 100:9680-5 (2003). Copy- CNS elucidated by Sciences Laboratory in right (2003) National Academy of Sciences, re-designated as an The CDB held its first annual comparative genomics Singapore, a non-profit U.S.A. independent administrative symposium at the nearby analysis of planarian ESTs" organization with affiliations to institution, a move intended to Portopia Hotel. The meeting On June 24, PNAS published the National University of simultaneously increase the drew more than 200 speakers this study by the Laboratory Singapore and Nanyang Institute's autonomy and and attendees from around for Evolutionary Regeneration Technological University accountability. As part of this the world, with lectures, poster May 2 Biology providing evidence of established to conduct transition, the Nobel Prize- presentations and lively Scientific exchange genetic roots for the central molecular biology and winning chemist Ryoji Noyori discussion on the origins of agreement with University nervous system in a primitive genetics research. was named the new president, metazoan biology, the of Texas Graduate School of bilaterian, the flatworm ushering in a new era for fundamental processes of Biomedical Sciences Dugesia japonica. RIKEN, which was first development and regeneration As part of its ongoing efforts to established in 1917. and the formation of higher- develop organizational ties with May 31 order structures, such as the international developmental CDB Open House neural networks. biology research community, The CDB opened its doors to October 20 -1 the CDB formalized an the public for a one-day event 2nd CDB Retreat agreement for active scientific featuring walk-through The CDB held its second exchange with the University of experiments, lab visits, talks annual retreat on the island of Texas Graduate School of on C. elegans and ES cell Awaji, located a short distance Biomedical Sciences at biology, quizzes, games and from Kobe in Japan's Inland Houston. The agreement video and poster Sea. The retreat gave CDB facilitates the hosting and presentations. research staff the chance to exchange of research staff and relax, mingle and exchange the joint sponsorship of scientific research findings and opinions meetings with one of the in two days of oral foremost academic centers for presentation and poster developmental biology research sessions. in the United States.

4 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 5 2003 HIGHLIGHTS August 27 - 9 Kobe BioWeek May 13 The city of Kobe played host to more than 8,000 visitors December 21 Publication of "Generation participating in Kobe Publication of "An NDPase of neural crest-derived BioWeek, a series of meetings links ADAM protease April 1 peripheral neurons and and events focusing on the glycosylation with organ New Graduate School floor plate cells from mouse August 19 growth and future directions of morphogenesis in Affiliations and primate embryonic Publication of "Traveling the biotechnology sector in the C. elegans" The CDB formalized its stem cells" stripes on the skin of a region and around Japan. The In work published in the online affiliations with the Kyoto In a study published in the mutant mouse" CDB joined in the public edition of Nature Cell Biology, University Graduate School of May 13 issue of the The August 19 issue of PNAS education activities, with two- the Laboratory for Cell Medicine and the NAIST Proceedings of the National featured a cover image taken way videoconference lectures Migration, under team leader Graduate School of Biological Academy of Sciences (PNAS), from a research article from and Q&A sessions, hands-on Kiyoji Nishiwaki, reported the Sciences, allowing doctoral the Sasai research group Shigeru Kondo's lab simulations of lab equipment role of a protein modification students from those schools to demonstrated the application describing the mathematical and a virtual lab tour program known as glycosylation in conduct their thesis research of stromal cell derived properties of a strikingly using web cameras that guiding the migration of in CDB laboratories under the inducing activity (SDIA) in the patterned mutant mouse allowed the public to watch gonadal cells in the nematode, guidance of lab heads high-efficiency induction of strain. CDB labs at work in real time. C. elegans. designated as visiting peripheral neurons from professors. embryonic stem cells.

4 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 5 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 Millennium Project, and focuses on the three main themes of the CDB: - Mechanisms of Development - Mechanisms of Regeneration - Scientific Bases of Regenerative Medicine The Core Program consists of seven research groups, each led by an eminent scientist. 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.

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i 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 Millennium Project, and focuses on the three main themes of the CDB: - Mechanisms of Development - Mechanisms of Regeneration - Scientific Bases of Regenerative Medicine The Core Program consists of seven research groups, each led by an eminent scientist. 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.

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i Functional regionalization of the planarian brain

Staff

Group Director Technical Staff Kouji Matsuzaki team was able to identify 116 clones Kiyokazu Agata Midori Nakayama Hideaki Ito with significant similarities to genes Research Scientist Tetsutaro Hayashi Sayaka Higuchi Noriko Funayama Tomomi Takamatsu Tomomi Takano closely linked to the nervous system in Yoshihiko Umesono Yumi Saito Kaneyasu Nishimura other species, including genes involved Hiroshi Imokawa Osamu Nishimura Part-time Staff Chiyoko Kobayashi Mikiko Nakatsukasa Machiko Teramoto in neurotransmission, the neural net- Nobuyasu Maki Chiharu Tanegashima Yuko Hirofuji work, brain morphogenesis and neural Shuichi Shigeno Student Trainee Keiko Momozu Shigenobu Yazawa Takeshi Inoue Assistant differentiation. These results point Takahiro Murakami Keiji Okamoto Shozou Sano Visiting Scientist Takaaki Karasawa toward a shared evolutionary origin for Norito Shibata Tomoko Shibata many nervous system genes, indicating that the nervous system is likely to have arisen in a single common ancestor of bilaterian animals. Intriguingly, nearly Publications Core Program 30% of the genes identified also have Hayasi S, Itho M, Agata K homologous sequences in yeast and the and Taira M. Conserved plant species, Arabidopsis thaliensis, Expression Patterns of FGF Receptor-like 1/nou-darake in both of which entirely lack neural development, cess. In the fourth step, which occurs by day four of Xenopus and Planarian. Dev regeneration, a homolog of the axon-guidance gene which suggests that a significant number of genes Dyn (2004). netrin is triggered. At this stage the discrete compon- that predate the advent of the nervous system have Its position on an important but scientifically under- ents of the regenerating brain begin to form connec- been co-opted to perform functions specific to neuro- Inoue T, Kumamoto H, Okamoto K, Umesono Y, explored branch of the evolutionary tree and its Brain regeneration by stages tions and higher-order organizations; chiasmata biology. Sakai M, Aiejandro SA and remarkable biology combine to make the planarian A planarian can re-grow a fully functional brain with- cross-linking eyes and brain develop, and connec- Agata K. Morphological and flatworm a fascinating and invaluable model for in five days following the amputation of its entire tions are set up between the brain and the ventral A planarian can re-grow Functional Recovery of the basic research in fields from evolutionary develop- head, an extraordinary feat of self-healing that Planarian Photosensing nerve cords, which serve as a rudimentary peripher- a fully functional brain within ment to regenerative medicine. Kiyokazu Agata has involves recapitulating the development of the al nervous system. Thus, the basic components and System During Head Regen- five days following the eration. Zoolog Sci (2004). adopted the freshwater planarian, Dugesia japonica, worm's entire nervous system. Researchers in the connections of the planarian neural network are in amputation of its head as a model species in his study of the origins and Agata group studied this process and identified pat- place by the fourth day, but the functional recovery Agata K. Regeneration and properties of stem cells, which are prevalent and terns of gene expression that indicate the regenera- of the brain is not completed until a final stage in Gene Regulation in Planari- experimentally accessible in these worms and which tion of the brain comprises five distinct stages. In the which two newly-identified genes are expressed is The Agata lab is also now participating in an interna- ans. Curr Opin Genet Dev 13:492-6 (2003). Kiyokazu Agata he believes hold the key to understanding the devel- first stage, at about eight hours after wound closure, entered. tional collaboration established to sequence and Ph. D. opment, organization and maintenance of the cellu- a noggin-like gene (Djnlg) is activated in the stump annotate a set of more than 10,000 planarian ESTs, Agata K, Tanaka T, Kiyokazu Agata received lar diversity that characterizes metazoan life. prior to the formation of a blastema, a mass of prolif- A parallel study revealed that these two genes, which promises to provide an invaluable resource Kobayashi C, Kato K and his doctorate from Kyoto Saitoh Y. Intercalary Regener- University in 1985 for his erating undifferentiated cells that serves as the fron- 1020HH and eye53, are necessary for planarians to for the study of evolutionary biology, comparative work on molecular cloning Isolating planarian stem cells tline of regeneration. Soon thereafter, the brain- regain their normal light avoidance behavior, known genomics, and the genetics underlying the unique ation in Planarians. Dev Dyn and gene expression of 226:308-16 (2003). crystallin genes in chicken. Agata's laboratory has developed a method for iden- specifying gene nou-darake is switched on in the as negative phototaxis. Knockdown of these genes characteristics of these organisms. The availability From 1983 to 1991, he tifying subsets of planarian stem cells, also called anterior fringe of the blastema, allowing the brain by RNAi had no discernible effects on brain or eye of an annotated database of planarian cDNAs may Mineta K, Nakazawa M, worked at the National Insti- Cebria F, Ikeo K, Agata K tute for Basic Biology, neoblasts. These somatic stem cells are the only rudiment to be formed. These first two steps occur morphology, but the worms failed to respond to light provide keys to the understanding of stem cell biolo- where he studied the molec- mitotically active cells in the planarian body, making within 24 hours of decapitation, and set the stage for stimuli in the normal manner, even after five days of gy, tissue plasticity and maintenance and other fun- and Gojobori T. Origin and ular characteristics of dedif- Evolutionary Process of the them susceptible to X-ray irradiation. Examination of the third phase in which brain patterning is estab- regeneration, when the structure of the brain has damentally important processes. ferentiated cells in transdif- Cns Elucidated by Compara- ferentiation. He took an cell populations shown to be vulnerable to elimina- lished by the expression of a set of three been fully reinstated. tive Genomics Analysis of associate professorship at tion by X-rays, revealed that planarian neoblasts, otd/Otx-related genes (relatives of Planarian Ests. Proc Natl the Himeji Institute of Tech- nology in 1991, and started like stem cells in other species, seem to exist in pro- which also function to pattern brain Evolution of the Acad Sci U S A 100:7666-71 his study of planarian regen- liferating and resting states. Real-time PCR analysis development in many other taxa, (2003). eration with Kenji Wata- central nervous indicates that each of these sub-populations including vertebrates) followed by nabe. He remained at the system Nakazawa M, Cebria F, Institute until 2000, when expresses discrete sets of genes. One fraction, X1, that of the planarian homolog of The planarian is one of the low- Mineta K, Ikeo K, Agata K he left to assume a professorship at Okayama Uni- expresses genes specific to actively cycling cells, Wnt at about 48 hours into est forms of animal known to and Gojobori T. Search for versity. He joined the while a second fraction, X2, lacks this expression the regenerative pro- possess a central nervous sys- the Evolutionary Origin of a RIKEN Center for Develop- Brain: Planarian Brain Char- profile and is thought to comprise stem cells tem, making it an apt model for mental Biology as a group acterized by Microarray. Mol director in 2000. in a state of quiescence. Interestingly, the study of the evolution of Biol Evol 20:784-91 (2003). many X1 cells also express signal recep- this most complex and elabo- tor molecules which are switched off in rately organized biological sys- Saito Y, Koinuma S, Wata- the X2 fraction, while the correspond- tem. Using clones of over nabe K and Agata K. Medio- lateral Intercalation in ing ligands are expressed in a variety 3,000 expressed sequence Planarians Revealed by Graft- of differentiated cells resistant to X-ray tags (ESTs) isolated from the ing Experiments. Dev Dyn irradiation. planarian head region, the 226:334-40 (2003). Expression of various neural-related genes Evolutionarily conserved expression patterns of planarian (right) and Xeno- pus nou-darake/FGFRL1 (left)

8 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 9 Functional regionalization of the planarian brain

Staff

8 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 9 Emx genes in early cortical development In the mouse, the development of the cerebrum is immediately preced- ed by the closure of the anterior neural plate at around E8.5 in the pre- sumptive forebrain-midbrain junction. In the earliest stages of cortico- genesis, the structures of the archipallium, the non-neuronal components of the choroid plexus, the hippocampal complex and the fimbria, are Staff generated. It has been suggested Group Director Technical Staff Student Trainee that the last of these structures, the Shinichi Aizawa Shoko Takehara Mariko Hirano Senior Scientist Miwa Nakamura Wataru Satoh fimbria, which are located at the bor- Isao Matsuo Tomomi Omura Yusuke Sakurai der of the cerebral cortex and the Research Scientist Saori Nagayoshi Assistant Yoko Suda Ai Inoue Sayo Saito Kohei Hatta Kuniko Kitajima Akihito Yamamoto Hiroshi Nakano Akihiko Shimono Hitomi Tsujii Nobuyoshi Takasaki Maiko Takahashi Daisuke Kurokawa Junior Research Associate Otx2 expression in fore- and Publications Takuya Murata Takashi Nagano midbrain at E10.5 Jun Kimura Izumi Oda-Ishii Kurokawa D, Takasaki N, Kiyo- Special Postdoctoral Researcher Chiharu Kimura-Yoshida nari H, Nakayama R, Kimura C, Matsuo I and Aizawa S. Regulation of Otx2 expression and its functions in mouse epi- Core Program blast and anterior neuroectoderm. Development (2004).

Kurokawa D, Kiyonari H, specific sites and stages of development. In the Nakayama R, Kimura C, Mat- past, the lab found enhancers responsible for pro- suo I and Aizawa S. Regula- moting Otx2 expression in the visceral endoderm, tion of Otx2 expression and its Despite their many differences, taxa as diverse as head development, and the molecular bases for the definitive anterior mesendoderm and the cephalic functions in mouse forebrain and midbrain. Development fish, amphibians, reptiles, birds and mammals share processes of anterior-posterior axis formation, head neural crest cells. These cis-regulatory elements (2004). a common body plan comprising three regions: the induction, brain regionalization and cortical develop- were all located relatively near the transcriptional trunk, the hindbrain/pharyngeal region, and the ros- ment. start site for the gene. More recently, the group has Kimura-Yoshida C, Kitajima K, tral head. Shin Aizawa is interested in the molecular identified and analyzed enhancers that guide Otx2 Oda-Ishii I, Tian E, Suzuki M, Yamamoto M, Suzuki T, bases for and phylogenetic origins of this regionaliza- Roles of Otx2 in head expression in epiblast, anterior neuroectoderm and Kobayashi M, Aizawa S and tion, concentrating primarily on the genetic activity development fore- and midbrain, which they have named the EP, choroid plexus, function as a local signaling center Isao Matsuo. Characterization and molecular attributes of head development. Otx2 plays central roles in each step of head devel- AN and FM enhancers, respectively. All of these ele- in cortical development. One model of archipallial of the pufferfish Otx2 cis- opment in vertebrate, but the regulatory mechan- ments are located more remotely from the coding patterning involves the expression of ligands, recep- regulators reveals evolutionari- All animals develop from the head. The formation of isms by which this gene’s activity is mediated have region (more than 80 kb upstream) than are the pre- tors, transcriptional factors and inhibitor molecules ly conserved genetic mechanisms for vertebrate head speci- inductive head organizer precedes that of the trunk remained largely unknown. The development of the to form morphogenetic gradients that direct the differ- Shinichi Aizawa viously identified Otx2 enhancers. fication. Development 131:57- Ph. D. organizer, as was first demonstrated by Spemann head traces back to before gastrulation, and is insep- entiation of areas on either side of the cortical hem, 71 (2004). Shinichi Aizawa received his and Mangold in their studies of amphibian embryoge- arably linked to the formation of the anterior- but the actions of specific players in this model Ph. D. in Zoology from the The development of Nishida A, Furukawa A, Koike Tokyo Kyoiku University, nesis. Gene knockout studies in mice, have demon- posterior axis. In mice, this process begins prior to the head is inseparably linked remain to be worked out. The Aizawa group’s stud- Department of Zoology in strated that the rhombomere r1/2 is the ground state gastrulation when the cells of the distal ventral endo- ies of Emx1 and Emx2, mouse homologs of the Dro- C, Tano Y, Aizawa S, Matsuo I 1973. He spent the period to the formation of and Furukawa T. Otx2 homeo- in the mammalian body plan. The head organizer derm migrate to the region that will become the ani- sophila head gap gene ems, have shown that these from 1974 to 1979 as an inves- the anterior-posterior axis box gene controls retinal photo- tigator at the Tokyo Metropoli- induces the rostral head anteriorly to this rhombo- mal’s anterior, forming the anterior visceral endo- two genes cooperate in two phases of cortical devel- receptor cell fate and pineal tan Institute of Gerontology, gland development. Nature then two years as a research mere, while the trunk organizer guides trunk develop- derm (AVE), which suppresses posteriorizing sign- opment. Previous work demonstrated that Emx1 fellow in the Laboratory of ment caudally to the same r1/2 landmark. Studying als in the adjacent epiblast. In previous work, mem- The activity of the AN enhancer is independent of and Emx2 work together to generate Cajal-Retzius Neuroscience 6:1255-63 (2003). Genetic Pathology at the Uni- mutations in genes responsible for body patterning in bers of the Aizawa lab demonstrated that Otx2 plays cells and subplate neurons. Recent work now indi- versity of Washington (US). that of the EP enhancer, and plays an essential role He returned to the Tokyo Met- this region, the Aizawa research group hopes to an essential role in triggering this anterior migration. in maintaining the anterior neuroectoderm through cates that the two genes also play a combinatory Shinozaki K, Miyagi T, Yoshi- ropolitan Institute of Gerontolo- reveal the genetic cascades for the constitution of the Following gastrulation, organizing centers in the epi- role in establishing the archipallium as distinct from da M, Miyata T, Ogawa M, gy in 1982, where he Otx2 expression, once that region has been remained until 1986 when he anterior head as conserved across vertebrate phyla. blast induce the formation of the anterior neuroecto- induced. The Aizawa group’s studies have also indi- the roof plate, immediately following the closure of Aizawa S and Suda Y. Absence of Cajal-Retzius cells moved to the RIKEN Tsukuba The groupﾕs research focuses on identifying and stu- derm, which subsequently regionalizes into multiple cated a phylogenetic relationship between these ele- the neural tube. Life Science Center as a sen- and subplate neurons associat- ior research associate. He was dying the functions of factors acting upstream and primitive structures destined to form the areas of the ments, in which FM is the most deeply conserved in ed with defects of tangential appointed professor in the downstream of Otx2, a master gene in vertebrate brain. In this process, the isthmus and anterior neu- the gnathostome (jawed vertebrate) lineage, while cell migration from ganglionic Kumamoto University School of Medicine Department of ral ridge act as local organizing the epiblast enhancer appears to have been eminence in Emx1/2 double Morphogenesis in 1994, and centers for mid- and forebrain acquired later, perhaps after the ascent of amphibi- mutant cerebral cortex. Devel- served in that position until opment 129:3479-92 (2002). 2002. Since 2000 he has development. ans, and to include the anterior neuroectoderm served as CDB deputy director enhancer as an essential component. Using these Hide T, Hatakeyama J, Kimura and group director of the Verte- brate Body Plan, as well as Otx2 functions as a master gene results as a springboard, Aizawa and colleagues C, Tian E, Takeda N, Ushio Y, team leader of the Laboratory in each phase of head ontogeny. next plan to investigate the significance of this phylo- Shiroishi T, Aizawa S and Mat- for Animal Resources and suo I. Genetic modifiers of oto- Aizawa and colleagues have genetic specialization of enhancers in terms of the genetic Engineering. He also cephalic phenotypes in Otx2 serves as a Managing Editor been working to identify and char- evolution of the vertebrate head, and to continue the heterozygous mutant mice. for the journal, Mechanisms of acterize regulatory factors that Development. search for upstream factors at work in the regulation Development 129:4347-57 control the gene’s expression in of Otx2. (2002). Fore- and midbrain structure at E12.5 in wildtype (left) and loss of archicortex and diencephalon in Emx2/Otx2 double mutants (right) Forebrain/midbrain at E10.5

10 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 11 Emx genes in early cortical development In the mouse, the development of the cerebrum is immediately preced- ed by the closure of the anterior neural plate at around E8.5 in the pre- sumptive forebrain-midbrain junction. In the earliest stages of cortico- genesis, the structures of the archipallium, the non-neuronal components of the choroid plexus, the hippocampal complex and the fimbria, are Staff generated. It has been suggested Group Director Technical Staff Student Trainee that the last of these structures, the Shinichi Aizawa Shoko Takehara Mariko Hirano Senior Scientist Miwa Nakamura Wataru Satoh fimbria, which are located at the bor- Isao Matsuo Tomomi Omura Yusuke Sakurai der of the cerebral cortex and the Research Scientist Saori Nagayoshi Assistant Yoko Suda Ai Inoue Sayo Saito Kohei Hatta Kuniko Kitajima Akihito Yamamoto Hiroshi Nakano Akihiko Shimono Hitomi Tsujii Nobuyoshi Takasaki Maiko Takahashi Daisuke Kurokawa Junior Research Associate Otx2 expression in fore- and Publications Takuya Murata Takashi Nagano midbrain at E10.5 Jun Kimura Izumi Oda-Ishii Kurokawa D, Takasaki N, Kiyo- Special Postdoctoral Researcher Chiharu Kimura-Yoshida nari H, Nakayama R, Kimura C, Matsuo I and Aizawa S. Regulation of Otx2 expression and its functions in mouse epi- Core Program blast and anterior neuroectoderm. Development (2004).

10 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 11 junction regions, resulting in the loss of tracheal cell Staff

adhesion and the detachment of these cells from the Group Director Special Postdoctoral Researcher epithelium. These complementary findings strongly Shigeo Hayashi Nao Niwa Research Scientist Kagayaki Kato suggest that a strictly maintained balance of Rac Leo Tsuda Technical Staff Atsushi Wada Ai Akimoto activity is essential in determining the proper junc- Yoshiko Inoue Masako Kaido tional assembly and disassembly of cadherins in the Kenji Oshima Michiko Takeda Housei Wada Drosophila tracheal epithelium, a role it is thought to Chiaki Kose fulfill by regulating cell adhesion and motility to Hiromi Sakaguchi Student Trainee ensure the appropriate remodeling of epithelial Tadashi Sakata Kayoko Sakurai sheets into tubules. Mayuko Nishimura Masayo Shindo Part-time Staff Kumi Ohnishi Hayashi seeks to develop an Chizuyo Itou improved understanding of the Ikuko Fukuzyou Assistant genetic control of Chisa Iimuro epithelial morphogenesis

A number of other signaling molecules that function in the tracheal system are known to cause gain-of- function phenotypes when overexpressed. In work related to the Rac study, the Hayashi lab has conducted a gain-of-function screen for genes that disrupt the tracheal epithelium on overexpression. The lab has identified several candidate genes whose hyperactivation leads to disruptions in epithelial inte- In a study published this year, the Hayashi group Core Program grity, including genes encoding regulators of actin determined that Wg signaling is required for Publications dynamics, cell adhesion and cell motility. The func- proximal-distal patterning during embryonic leg development, a role it performs by helping to recruit Chihara T, Kato K, Taniguchi tions of the individual candidate genes are now M, Ng J and Hayashi S. Rac being analyzed, with the goal of developing an cell types specific to the proximal and distal Promotes Epithelial Cell Rear- improved understanding of the genetic control of epi- rangement During Tracheal thelial morphogenesis. Tubulogenesis in Drosophila. Development 130:1419-28 Epithelial tissues are The regulation of epithelial (2003). constructed from adhesion Leg and wing specification sheets of cells with The ability of epithelial cells to attach to and disen- The insect wing is an evolutionary innovation that is Kubota K, Goto S and Hayashi distinct apical-basal gage from their neighbors is closely linked to the thought to have arisen as a derivative of the leg dur- S. The Role of Wg Signaling in the Patterning of Embryonic (top-bottom) polari- activity of cadherin family molecules, which span the ing an early stage in the evolution of the insect body Leg Primordium in Drosophila. ties, which form junc- cell membrane to regulate this dynamic cell-cell plan. The close relationship between the wing and Dev Biol 257:117-26 (2003). tions with each other adhesion. A number of studies have also indicated the leg can be observed in the embryogenesis of and interact with the roles for the small GTPase Rac as another key regu- Drosophila, which is a highly derived winged insect. Sugimura K, Yamamoto M, extracellular matrix, lator of cell adhesion, but these roles have yet to be It has been shown that wing and leg both derive Niwa R, Satoh D, Goto S, Tani- guchi M, Hayashi S and the macromolecular confirmed in the context of embryonic development. from a common precursive structure called the limb Ventral view of the thoracic region of a Drosophila embryo. Stripes of Wingless (purple) expression overlap the leg primordia (green) and Uemura T. Distinct Develop- Shigeo Hayashi environment in the In work published in 2003, members of the Hayashi primordium. This structure gives rise to both the limb specify the proximal leg identity. mental Modes and Lesion- Ph. D. spaces between cells. research group reported the results of their investiga- and wing imaginal discs, pouches of undifferentiated Induced Reactions of Den- drites of Two Classes of Dro- Shigeo Hayashi received A range of vital devel- tions into Rac's role in epithelial cell rearrangement cells in the larva that form the leg and wing in the domains. This finding counters previous work that his B. Sc. in Biology from sophila Sensory Neurons. J opmental processes, in the development of the Drosophila tracheal sys- adult. Kyoto University in 1982, suggested that Wg plays no role in late embryonic Neurosci 23:3752-60 (2003). and his Ph. D. in Biophys- including the abilities tem, a tubular network formed from epithelium. leg disc development, and led Hayashi to propose a ics from the same institution in 1987 for his work on to build complex struc- The Hayashi lab has investigated the role of Wing- new model for leg specification in which dorsal- Shiga Y, Yasumoto R, Yama- gata H and Hayashi S. Evolv- lens-specific regulation of Transmission electron micrograph tures through selec- The group found that reductions in Rac activity led less (Wg) signaling in the wing and leg primordia. proximal, ventral-proximal and distal cells are distin- the chicken delta crystallin of dorsal trunk of the trachea, which tive cell adhesion, to to increases in the amount and range of cadherin The Wg protein, which mediates a wide range of ing Role of Antennapedia Pro- gene. Inspired by the dis- consists of multiple epithelial cells guished by differences in the expression domains of tein in Arthropod Limb covery of the homeobox, he joined tightly by adherens junctions. move through the cell adhesion molecules in both embryonic epider- inductive interactions in Drosophila, is known to be specific genes, including Wg. The group also found Patterning. Development changed his research focus One of the tracheal cells is colored body, and to form epi- mis and tracheal epithelia, which they attribute to a required for the induction of imaginal discs during to the developmental genet- red and the tracheal lumen is that ectopic Wg signaling inhibits wing disc develop- 129:3555-61 (2002). ics of Drosophila and spent colored green. thelial tissues rely on post-transcriptional mechanism and changes in epi- embryonic development. In the leg imaginal disc, ment; ectopic activation of the Wg signal in the wing three years as a postdoctor- Hayashi S, Ito K, Sado Y, Tani- interactions between thelial structural organization. This strengthening of Wg serves as an axis determinant, triggering dorsal- disc causes a reduction in the number of wing cells al research associate in guchi M, Akimoto A, Takeuchi Matthew Scott’s lab at the cells and other cells, and with the extracellular intercellular adhesion was accompanied by a recipro- ventral pattern formation by specifying the ventral that develop. These results indicate that Wg deter- H, Aigaki T, Matsuzaki F, University of Colorado matrix. The means by which cells are able to cal decrease in the ability of epithelial cells to region of the leg, and by activating the Distal-less before returning to Japan to mines leg cell specification while inhibiting that of Nakagoshi H, Tanimura T, work in the National Insti- achieve these feats as they communicate, con- rearrange dynamically. Conversely, when they over- (Dll) gene in the distal region. However, Wingless's the wing, thereby playing the role of a crucial binary Ueda R, Uemura T, Yoshihara tute of Genetics. He gregate and work together to build a body are cen- expressed the constitutively active form of Rac in roles in leg development have not been fully switch in the establishment of wing and leg identi- M and Goto S. GETDB, a Data- became an associate pro- base Compiling Expression fessor at the same Institute tral to the study of morphogenesis, the focus of tracheal cells, the researchers found that cadherins explored. ties. Patterns and Molecular Loca- in 1994, and professor in research in the Hayashi laboratory. decreased and eventually disappeared from cell 1999. Also in 1999, he tions of a Collection of Gal4 received the incentive prize Enhancer Traps. Genesis of the Genetics Society of 34:58-61 (2002). Japan for Genetic Studies 0:00 1:00 2:00 3:00 4:00 5:00 6:00 7:00 on Drosophila Develop- Goto S, Taniguchi M, Muraoka ment. He was named group director of the Morphogenet- M, Toyoda H, Sado Y, Kawaki- ic Signaling research group ta M and Hayashi S. UDP- at the RIKEN CDB in May Sugar Transporter Implicated 2000. in Glycosylation and Process- ing of Notch. Nat Cell Biol Time course of tracheal development. Time lapse pictures of an 3:816-22 (2001). embryo expressing GFP in the tracheal system.

12 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 13 junction regions, resulting in the loss of tracheal cell Staff

12 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 13 entiated and retains its multipotency, while the GMC becomes committed to generating neurons and glial cells. Further studies in Matsuzaki lab showed that Prospero is tethered to the basal cortex of the neuroblast by the molecule Miranda, and subsequently released into the newly-formed GMC. Mechanisms that polarize cells — underlying the asymmetric distribution of those molecules — have further been shown to have homologous counterparts in vertebrates, providing strong evidence for the evolutionary conservation of intrinsic signaling in the process of asymmetric cell division. Coordinating asymmetric division The basal localization of determinants is by itself not sufficient for the parental neuroblast to segregate cell fate determinants to the daughter GMC. It is Asymmetric cell sizes Staff also necessary for the division to occur along the This last question prompted investigations that led Group Director Fumio Matsuzaki axis of polar distribution of the Miranda-Prospero to the identification of a new regulatory function for Research Scientist Naoyuki Fuse complex. This is achieved by the coordinated align- G proteins, in Drosophila neuroblast cell division, a Yasushi Izumi ment of the mitotic spindle along the apicobasal process in which the resultant neuroblast is much Ayano Kawaguchi Go Shioi axis. In the neuroblast, both the asymmetric localiza- larger than the GMC. During mitotic cell division, Tohru Yamamoto tion of cell fate determinants and spindle orientation chromosomes replicate within the mother cell and Technical Staff Kanako Hisata are governed by a multi-protein complex that attach to spindles that draw them in opposite direc- Taeko Suetsugu Core Program tions to ensure that a full complement of chro- Mai Saito Nao Ota The fates of mosomes is available to each daughter cell. The Tomoko Ikawa chromatids are drawn to the spindle poles through Junior Research Associate daughter neural cells are in Maki Maeda the action of molecular motors known as micro- Assistant large part determined by Chika Lee tubules. In work carried out at the CDB, Matsuzaki's unequal distribution of Part-time Staff lab has found that Gβ protein signals are distributed Chie Ueda Intracellular asymmetry tion. This process relies on the establishment of factors within the unequally in neuroblasts and seem to restrict the All the cells in the body originate from a single proge- intracellular gradients of factors regulating the mother cell during the development of microtubules, resulting in a shorten- nitor, and nearly all share an identical DNA code. expression of genes, which results in differential pat- process of cell division ing of the mitotic spindle on one side of the cell. How then do the vast numbers of distinct cell types terns of gene expression in daughter cells when the These unequal spindle lengths cause the neuroblast in complex organisms differentiate from one anoth- cell undergoes mitosis. As it is the expression of creates intracellular asymmetry. This protein com- to divide at a cleavage site that is off-center, and the er? The process of asymmetric division, in which reg- genes, and not their simple presence or absence, plex, named the "apical complex" for the site of its sizes of the daughter cells reflect this imbalance, ulatory factors in a single mother cell are distributed which is primarily responsible for determining cell localization in the neuroblast, includes two different with the daughter neuroblast being more than twice unequally to daughter cells produced by cell divi- identity, controlled asymmetric division is critical to signaling complexes: atypical protein kinase C the size of the GMC. Cells lacking the Gβ protein in sion, is a key to answering that fundamental ques- ensuring the development of appropriate numbers (aPKC) and heterotrimeric G protein. The Matsuzaki question form a symmetrical mitotic spindle and pro- tion. This asymmetric distribution of regulatory fac- and types of daughter cells, and so, to the develop- lab recently demonstrated that the two signaling duce daughter cells of equal size with the determi- Fumio Matsuzaki tors allows resultant cells to assume different roles ment of the organism as a whole. pathways included in the apical complex play differ- nants being normally segregated. This provides a Ph. D. from each other in a process called fate determina- ential roles in the asymmetric segregation of the valuable platform for further studies in fields ranging Fumio Matsuzaki received Neural cell fate determination determinants. aPKC signaling is essential for the from microtubule dynamics to animal morphogene- his B. Sc. from the Depart- ment of Biochemistry and in Drosophila localization of the basal Miranda-Prospero complex, sis. Biophysics at the University Fumio Matsuzaki has dedicated his research to expli- while the Pins molecule, known to be Publications of Tokyo in 1979, and his cating the role of asymmetric division in determining associated with an alpha subunit of the G doctorate from the same Izumi Y, Ohta N, Itoh-Furuya institution in 1984, for his cell identity, using the Drosophila melanogaster protein in the apical complex, plays a criti- A, Fuse N and Matsuzaki F. work on the characteriza- Differential Function of G Pro- tion of the erythrocyte cytos- nervous system as a model. This system provides cal role in orienting the mitotic spindle. keletal structure. He spent an attractive research platform for studying the Thus multiple intracellular signals operate tein and Baz/aPKC Signaling the period from 1984 to development of cell diversity, as the nervous system coordinately and differentially to create Pathways in Drosophia Neuro- 1988 as a postdoctoral fel- blast Asymmetric Division. J low, first in the Department features more cell types than any other organ sys- intracellular asymmetry, and promote the Cell Biol (2004). of Cell Biology at the Tokyo tem, and the fruit fly is highly amenable to genetic asymmetric division of neuroblasts. The Metropolitan Institute of Medical Science, then in manipulation. With the advent of techniques for Matsuzaki lab is now confronting ques- Fuse N, Hisata K, Katzen A L the laboratory of Gerard molecular analysis, Matsuzaki and others demon- tions regarding the roles of extrinsic sign- and Matsuzaki F. Heterotrimer- Edelman at Rockefeller Uni- ic G Proteins Regulate Daugh- strated that the fates of daughter neural cells are in als and intrinsic players, and the mechan- versity. He returned to ter Cell Size Asymmetry in Dro- Japan in 1988 to take a large part determined by unequal distribution of fac- ism by which cells of different sizes are sophila Neuroblast Divisions. position as a section chief in the Department of Molec- tors within the mother cell during the process of cell generated in the process of mitotic divi- Curr Biol 13:947-54 (2003). ular Genetics in the Nation- division. Two factors in particular, known as Pros- sion. al Institute of Neuroscience. pero and Numb, have been shown to localize to the Ohshiro T, Yagami T, Zhang C In 1998, he was appointed and Matsuzaki F. Role of Corti- professor in the Institute of basal side of the neuroblast (the neural progenitor cal Tumour-Suppressor Pro- Development, Aging and Cancer at Tohoku Universi- cell) prior to its division. This polar distribution teins in Asymmetric Division of ty and remained there until results in two daughter cells of distinct gene expres- Drosophila Neuroblast. Nature taking his current position 408:593-6 (2000). as group director at the sion — a new neuroblast, and a smaller-sized gan- RIKEN CDB. glion mother cell (GMC) — in which Prospero and In the developing mouse spinal cord, Pros- In Drosophila, dividing neuroblasts localize the Numb are segregated in the GMC. In this process of pero (green) is transiently expressed in Miranda (green) / Prospero complex to be segre- newborn neurons immediately after birth gated into the daughter GMC. asymmetric division, the neuroblast remains undiffer- from the mitotic neural progenitors (red).

14 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 15 entiated and retains its multipotency, while the GMC becomes committed to generating neurons and glial cells. Further studies in Matsuzaki lab showed that Prospero is tethered to the basal cortex of the neuroblast by the molecule Miranda, and subsequently released into the newly-formed GMC. Mechanisms that polarize cells — underlying the asymmetric distribution of those molecules — have further been shown to have homologous counterparts in vertebrates, providing strong evidence for the evolutionary conservation of intrinsic signaling in the process of asymmetric cell division. Coordinating asymmetric division The basal localization of determinants is by itself not sufficient for the parental neuroblast to segregate cell fate determinants to the daughter GMC. It is Asymmetric cell sizes Staff also necessary for the division to occur along the This last question prompted investigations that led Group Director Fumio Matsuzaki axis of polar distribution of the Miranda-Prospero to the identification of a new regulatory function for Research Scientist Naoyuki Fuse complex. This is achieved by the coordinated align- G proteins, in Drosophila neuroblast cell division, a Yasushi Izumi ment of the mitotic spindle along the apicobasal process in which the resultant neuroblast is much Ayano Kawaguchi Go Shioi axis. In the neuroblast, both the asymmetric localiza- larger than the GMC. During mitotic cell division, Tohru Yamamoto tion of cell fate determinants and spindle orientation chromosomes replicate within the mother cell and Technical Staff Kanako Hisata are governed by a multi-protein complex that attach to spindles that draw them in opposite direc- Taeko Suetsugu Core Program tions to ensure that a full complement of chro- Mai Saito Nao Ota The fates of mosomes is available to each daughter cell. The Tomoko Ikawa chromatids are drawn to the spindle poles through Junior Research Associate daughter neural cells are in Maki Maeda the action of molecular motors known as micro- Assistant large part determined by Chika Lee tubules. In work carried out at the CDB, Matsuzaki's unequal distribution of Part-time Staff lab has found that Gβ protein signals are distributed Chie Ueda Intracellular asymmetry tion. This process relies on the establishment of factors within the unequally in neuroblasts and seem to restrict the All the cells in the body originate from a single proge- intracellular gradients of factors regulating the mother cell during the development of microtubules, resulting in a shorten- nitor, and nearly all share an identical DNA code. expression of genes, which results in differential pat- process of cell division ing of the mitotic spindle on one side of the cell. How then do the vast numbers of distinct cell types terns of gene expression in daughter cells when the These unequal spindle lengths cause the neuroblast in complex organisms differentiate from one anoth- cell undergoes mitosis. As it is the expression of creates intracellular asymmetry. This protein com- to divide at a cleavage site that is off-center, and the er? The process of asymmetric division, in which reg- genes, and not their simple presence or absence, plex, named the "apical complex" for the site of its sizes of the daughter cells reflect this imbalance, ulatory factors in a single mother cell are distributed which is primarily responsible for determining cell localization in the neuroblast, includes two different with the daughter neuroblast being more than twice unequally to daughter cells produced by cell divi- identity, controlled asymmetric division is critical to signaling complexes: atypical protein kinase C the size of the GMC. Cells lacking the Gβ protein in sion, is a key to answering that fundamental ques- ensuring the development of appropriate numbers (aPKC) and heterotrimeric G protein. The Matsuzaki question form a symmetrical mitotic spindle and pro- tion. This asymmetric distribution of regulatory fac- and types of daughter cells, and so, to the develop- lab recently demonstrated that the two signaling duce daughter cells of equal size with the determi- Fumio Matsuzaki tors allows resultant cells to assume different roles ment of the organism as a whole. pathways included in the apical complex play differ- nants being normally segregated. This provides a Ph. D. from each other in a process called fate determina- ential roles in the asymmetric segregation of the valuable platform for further studies in fields ranging Fumio Matsuzaki received Neural cell fate determination determinants. aPKC signaling is essential for the from microtubule dynamics to animal morphogene- his B. Sc. from the Depart- ment of Biochemistry and in Drosophila localization of the basal Miranda-Prospero complex, sis. Biophysics at the University Fumio Matsuzaki has dedicated his research to expli- while the Pins molecule, known to be Publications of Tokyo in 1979, and his cating the role of asymmetric division in determining associated with an alpha subunit of the G doctorate from the same Izumi Y, Ohta N, Itoh-Furuya institution in 1984, for his cell identity, using the Drosophila melanogaster protein in the apical complex, plays a criti- A, Fuse N and Matsuzaki F. work on the characteriza- Differential Function of G Pro- tion of the erythrocyte cytos- nervous system as a model. This system provides cal role in orienting the mitotic spindle. keletal structure. He spent an attractive research platform for studying the Thus multiple intracellular signals operate tein and Baz/aPKC Signaling the period from 1984 to development of cell diversity, as the nervous system coordinately and differentially to create Pathways in Drosophia Neuro- 1988 as a postdoctoral fel- blast Asymmetric Division. J low, first in the Department features more cell types than any other organ sys- intracellular asymmetry, and promote the Cell Biol (2004). of Cell Biology at the Tokyo tem, and the fruit fly is highly amenable to genetic asymmetric division of neuroblasts. The Metropolitan Institute of Medical Science, then in manipulation. With the advent of techniques for Matsuzaki lab is now confronting ques- Fuse N, Hisata K, Katzen A L the laboratory of Gerard molecular analysis, Matsuzaki and others demon- tions regarding the roles of extrinsic sign- and Matsuzaki F. Heterotrimer- Edelman at Rockefeller Uni- ic G Proteins Regulate Daugh- strated that the fates of daughter neural cells are in als and intrinsic players, and the mechan- versity. He returned to ter Cell Size Asymmetry in Dro- Japan in 1988 to take a large part determined by unequal distribution of fac- ism by which cells of different sizes are sophila Neuroblast Divisions. position as a section chief in the Department of Molec- tors within the mother cell during the process of cell generated in the process of mitotic divi- Curr Biol 13:947-54 (2003). ular Genetics in the Nation- division. Two factors in particular, known as Pros- sion. al Institute of Neuroscience. pero and Numb, have been shown to localize to the Ohshiro T, Yagami T, Zhang C In 1998, he was appointed and Matsuzaki F. Role of Corti- professor in the Institute of basal side of the neuroblast (the neural progenitor cal Tumour-Suppressor Pro- Development, Aging and Cancer at Tohoku Universi- cell) prior to its division. This polar distribution teins in Asymmetric Division of ty and remained there until results in two daughter cells of distinct gene expres- Drosophila Neuroblast. Nature taking his current position 408:593-6 (2000). as group director at the sion — a new neuroblast, and a smaller-sized gan- RIKEN CDB. glion mother cell (GMC) — in which Prospero and In the developing mouse spinal cord, Pros- In Drosophila, dividing neuroblasts localize the Numb are segregated in the GMC. In this process of pero (green) is transiently expressed in Miranda (green) / Prospero complex to be segre- newborn neurons immediately after birth gated into the daughter GMC. asymmetric division, the neuroblast remains undiffer- from the mitotic neural progenitors (red).

14 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 15 Differentiation of mesoderm Staff and endoderm Publications Group Director Masaki Kinoshita Shin-ichi Nishikawa Yasuhiro Takashima In another ongoing project, members of the Nishika- Nishikawa SI, Honda K, Vieira Research Scientist Nobuyuki Kondo wa group are developing software to allow for P and Yoshida H. Organogene- Takumi Era Yasushi Kubota sis of Peripheral Lymphoid Masatake Osawa EPS Chandana improved DNA chip analyses and using it to build a Eri Nishioka Rasmus Freter Organs. Immunol Rev 195:72- Akiyoshi Uemura Masato Okuda database of the progressive intermediate states that 80 (2003). Atsushi Togawa Saori Yonetani endothelial cells pass through in the journey from Sentaro Kusuhara Visiting Scientist Sone M, Itoh H, Yamashita J, Igor M Samokhvalov Assistant ES (embryonic stem) to terminally differentiated cell. Timm T Schroeder Maya Iwase A number of mesoderm and endoderm cell lineages Yurugi-Kobayashi T, Suzuki Y, Collaborative Scientist Sakura Yuoka Kondo Y, Nonoguchi A, Sawa- and their intermediates are being used, as these are Mitsuhiro Okada Kaori Kawahara da N, Yamahara K, Miyashita Lars M Jakt available in pure cultures and their in vitro differentia- Muneaki Miyata K, Park K, Shibuya M, Nito S, Mari Kohno tion can be steered by the addition of the appro- Nishikawa S, Nakao K and Shigenobu Oshima Nishikawa SI. Different Differ- Naoko Yoshimura priate factors. entiation Kinetics of Vascular Masahiro Yasunaga Yoko Nakano Progenitor Cells in Primate Technical Staff The mesendodermal lineage, which has been clear- and Mouse Embryonic Stem Satomi Nishikawa ly defined at cell level by the Nishikawa group, Cells. Circulation 107:2085-8 Mariko Moriyama Masae Sato includes a number of important cell types, including (2003). Natalia I Samokhvalova endothelial cells, the primary constituent cells in Keiko Gion Hirashima M, Ogawa M, Kanako Yamaguchi many important organs including the lungs, liver and Nishikawa S, Matsumura K, Student Trainee Atsushi Takebe pancreas. Mesendodermal differentiation is charac- Kawasaki K, Shibuya M, and Hidetoshi Sakurai terized by the activity of specific marker genes Nishikawa SI. A Chemically Kanako Yoshikawa Defined Culture of VEGFr2+ Shinsuke Tada whose expression is switched on or off, depending Siushan Mak on the pathway a cell has followed and the stage it Cells Derived from Embryonic Stem Cells Reveals the Role has reached. Analysis of the stages in this stepwise of Vegfr1 in Tuning the process has also made it possible to develop a sys- Threshold for VEGF in Devel- Core Program tem for guiding differentiation from ES cell to endo- oping Endothelial Cells. Blood dermal, mesenchymal or mesodermal (paraxial and 101:2261-7 (2003). lateral) fates in vitro using combinations of extrinsic Hirai H, Ogawa M, Suzuki N, the follicle, as well as all of the GFP-negative cells factors. By identifying the characteristic markers of Yamamoto M, Breier G, Maz- from the bulge and the subset of GFP-negative cells cells at each step on each branch of the lineage tree da O, Imanishi J and Nishika- found specifically to adhere to bulge melanocytes. and collecting samples to produce DNA arrays, the wa SI. Hemogenic and Nonhe- Stem cell biology directed to give rise to another strongly substantiate Nishikawa lab is working to build a database of mogenic Endothelium Can Be Distinguished by the Activity of Stem cells of various types, characterized by their the model in which a stem cell's behavior and proper- These follicular regions were selected based on the cDNA profiles of the intermediate stages in ES cell Fetal Liver Kinase (Flk)-1 Pro- abilities to self-renew and to generate more highly ties are influenced by external signals. By studying results of a previous study that showed that the differentiation in culture, which can be queried and moter/Enhancer During Mouse specialized cells indefinitely, are the key to replenish- the microenvironments, known as 'niches,' in which bulge and matrix are the two main sites of localiza- visualized using software developed in the lab. Embryogenesis. Blood ing the body's cells. The study of these cells stands stem cells originate, abide and proliferate, the Nishi- Analyses of this cDNA chip database promise to 101:886-93 (2003). at the heart of some of the central questions in devel- kawa research group seeks to gain insight into the lead to a better understanding of the process of Profiling gene expression in Matsumura K, Hirashima M, opment, but many aspects of stem cell biology, identity and function of molecules involved in stem melanocytes and endothelial differentiation, and of stem cell specifica- Ogawa M, Kubo H, Hisatsune such as the limits (if any) of the ability to cell activity, and the potential of stem cell-based ther- resting stem cells promises to tion in general. This powerful new DNA analysis soft- H, Kondo N, Nishikawa S, Chi- ba T and Nishikawa SI. Modu- reprogram them to produce progeny cells apeutic applications. provide a new resource for ware application, which promises to make a great of diverse lineages, and the identities contribute to the harmonization of multiple DNA lation of VEGFR-2-Mediated scientists investigating Endothelial-Cell Activity by of the intrinsic and extrinsic molecu- Characterizing the stem cell array databases, is being developed in collaboration Shin-ichi Nishikawa stem cells' genetic signatures VEGF-C/VEGFR-3. Blood M. D. , Ph. D. lar factors that regulate a cell's deci- niche with the Institute of Biomedical Research and Inno- 101:1367-74 (2003). Shin-ichi Nishikawa sions whether to maintain stem- Working to isolate the defining characteristics of spe- vation, located adjacent to the CDB in the Kobe Bio- received his M. D. from the tion of melanocyte stem cells, making them excel- Kyoto University School of ness or to commit to differentia- cific stem cell niches, the Nishikawa group has cho- medical Industry Project park. Medicine in 1973. He per- tion have yet to be resolved. sen melanocyte stem cells as one of the main model lent sites for studies of niche properties. Each of formed his internship and these areas hosts a specific sub-population of stem residency in the Depart- systems for their research, as melanocytes are readi- ment of Internal Medicine at A stem cell's location and ly identified by the presence of a melanocyte- cells. The matrix niche, the site of active hair growth the Kyoto University Chest and pigmentation, provides an environment in which Disease Research Institute, immediate environment specific promoter, Dct, and are amenable to experi- before taking an assistant can be important factors mental manipulations that allow specific cell types to amplifying progeny become committed to a stem professorship in the same in defining its role. be isolated from mixed populations. As the exist- cell fate, while the bulge serves as a reservoir for department in 1979. He spent the period from 1980- Recent findings that ence of niche cells and the identification of their char- stem cells in their resting, or quiescent, state. Sub- 82 at the University of Colo- stem cells may acteristics can only be determined by first isolating tractive comparisons of cDNAs from stem cell, differ- gne Institute for Genetics (Germany) before returning arise from one the stem cells they support, Nishikawa's lab has entiated cell and 'niche-like' (keratinocyte) libraries to the Chest Disease tissue type developed an approach in which stem cells are dis- revealed approximately 200 non-redundant genes, Research Institute, where he was appointed asso- and then sected from their surroundings, dissociated and providing the lab with targets for further experiments ciate professor in the later be used to construct single cell libraries, a process in which the individual genes will be conditionally Department of Microbiology knocked out and the resulting phenotypes examined in 1983. He moved to the involving the technically challenging initial step of iso- Kumamoto University Medi- lating single intact hair follicles from the skin. Target- and analyzed. Other related studies, for which single cal School in 1987 to take a Melanocytes cell libraries of other resting stem cells are now professorship in the Depart- ed GFP expression is next used to label the follic- (marked red) found ment of Immunology, and being constructed, will entail profiling gene expres- in each hair follicle of ular melanocytes, making it possible to dis- returned to Kyoto in 1993, the mouse form a stem sion in melanocytes and resting stem cells, which as professor in the Depart- tinguish them from other cells. ment of Molecular Genetics cell system within these These cells are used to con- promises to provide a new resource for scientists minute organs. In this Representation of the relationships between different cell types based on the at the Kyoto Graduate investigating stem cells' genetic signatures. expression of approximately 700 probe sets School of Medicine. He was system, the most immature struct cDNA libraries of appointed CDB group direc- stem cell compartment is located (GFP-positive) melano- tor in 2000. in the upper region and the actively cycling compartment is located in the cytes from the bulge bottom of the follicle. and matrix regions of

16 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 17 Differentiation of mesoderm Staff and endoderm Publications Group Director Masaki Kinoshita Shin-ichi Nishikawa Yasuhiro Takashima In another ongoing project, members of the Nishika- Nishikawa SI, Honda K, Vieira Research Scientist Nobuyuki Kondo wa group are developing software to allow for P and Yoshida H. Organogene- Takumi Era Yasushi Kubota sis of Peripheral Lymphoid Masatake Osawa EPS Chandana improved DNA chip analyses and using it to build a Eri Nishioka Rasmus Freter Organs. Immunol Rev 195:72- Akiyoshi Uemura Masato Okuda database of the progressive intermediate states that 80 (2003). Atsushi Togawa Saori Yonetani endothelial cells pass through in the journey from Sentaro Kusuhara Visiting Scientist Sone M, Itoh H, Yamashita J, Igor M Samokhvalov Assistant ES (embryonic stem) to terminally differentiated cell. Timm T Schroeder Maya Iwase A number of mesoderm and endoderm cell lineages Yurugi-Kobayashi T, Suzuki Y, Collaborative Scientist Sakura Yuoka Kondo Y, Nonoguchi A, Sawa- and their intermediates are being used, as these are Mitsuhiro Okada Kaori Kawahara da N, Yamahara K, Miyashita Lars M Jakt available in pure cultures and their in vitro differentia- Muneaki Miyata K, Park K, Shibuya M, Nito S, Mari Kohno tion can be steered by the addition of the appro- Nishikawa S, Nakao K and Shigenobu Oshima Nishikawa SI. Different Differ- Naoko Yoshimura priate factors. entiation Kinetics of Vascular Masahiro Yasunaga Yoko Nakano Progenitor Cells in Primate Technical Staff The mesendodermal lineage, which has been clear- and Mouse Embryonic Stem Satomi Nishikawa ly defined at cell level by the Nishikawa group, Cells. Circulation 107:2085-8 Mariko Moriyama Masae Sato includes a number of important cell types, including (2003). Natalia I Samokhvalova endothelial cells, the primary constituent cells in Keiko Gion Hirashima M, Ogawa M, Kanako Yamaguchi many important organs including the lungs, liver and Nishikawa S, Matsumura K, Student Trainee Atsushi Takebe pancreas. Mesendodermal differentiation is charac- Kawasaki K, Shibuya M, and Hidetoshi Sakurai terized by the activity of specific marker genes Nishikawa SI. A Chemically Kanako Yoshikawa Defined Culture of VEGFr2+ Shinsuke Tada whose expression is switched on or off, depending Siushan Mak on the pathway a cell has followed and the stage it Cells Derived from Embryonic Stem Cells Reveals the Role has reached. Analysis of the stages in this stepwise of Vegfr1 in Tuning the process has also made it possible to develop a sys- Threshold for VEGF in Devel- Core Program tem for guiding differentiation from ES cell to endo- oping Endothelial Cells. Blood dermal, mesenchymal or mesodermal (paraxial and 101:2261-7 (2003). lateral) fates in vitro using combinations of extrinsic Hirai H, Ogawa M, Suzuki N, the follicle, as well as all of the GFP-negative cells factors. By identifying the characteristic markers of Yamamoto M, Breier G, Maz- from the bulge and the subset of GFP-negative cells cells at each step on each branch of the lineage tree da O, Imanishi J and Nishika- found specifically to adhere to bulge melanocytes. and collecting samples to produce DNA arrays, the wa SI. Hemogenic and Nonhe- Stem cell biology directed to give rise to another strongly substantiate Nishikawa lab is working to build a database of mogenic Endothelium Can Be Distinguished by the Activity of Stem cells of various types, characterized by their the model in which a stem cell's behavior and proper- These follicular regions were selected based on the cDNA profiles of the intermediate stages in ES cell Fetal Liver Kinase (Flk)-1 Pro- abilities to self-renew and to generate more highly ties are influenced by external signals. By studying results of a previous study that showed that the differentiation in culture, which can be queried and moter/Enhancer During Mouse specialized cells indefinitely, are the key to replenish- the microenvironments, known as 'niches,' in which bulge and matrix are the two main sites of localiza- visualized using software developed in the lab. Embryogenesis. Blood ing the body's cells. The study of these cells stands stem cells originate, abide and proliferate, the Nishi- Analyses of this cDNA chip database promise to 101:886-93 (2003). at the heart of some of the central questions in devel- kawa research group seeks to gain insight into the lead to a better understanding of the process of Profiling gene expression in Matsumura K, Hirashima M, opment, but many aspects of stem cell biology, identity and function of molecules involved in stem melanocytes and endothelial differentiation, and of stem cell specifica- Ogawa M, Kubo H, Hisatsune such as the limits (if any) of the ability to cell activity, and the potential of stem cell-based ther- resting stem cells promises to tion in general. This powerful new DNA analysis soft- H, Kondo N, Nishikawa S, Chi- ba T and Nishikawa SI. Modu- reprogram them to produce progeny cells apeutic applications. provide a new resource for ware application, which promises to make a great of diverse lineages, and the identities contribute to the harmonization of multiple DNA lation of VEGFR-2-Mediated scientists investigating Endothelial-Cell Activity by of the intrinsic and extrinsic molecu- Characterizing the stem cell array databases, is being developed in collaboration Shin-ichi Nishikawa stem cells' genetic signatures VEGF-C/VEGFR-3. Blood M. D. , Ph. D. lar factors that regulate a cell's deci- niche with the Institute of Biomedical Research and Inno- 101:1367-74 (2003). Shin-ichi Nishikawa sions whether to maintain stem- Working to isolate the defining characteristics of spe- vation, located adjacent to the CDB in the Kobe Bio- received his M. D. from the tion of melanocyte stem cells, making them excel- Kyoto University School of ness or to commit to differentia- cific stem cell niches, the Nishikawa group has cho- medical Industry Project park. Medicine in 1973. He per- tion have yet to be resolved. sen melanocyte stem cells as one of the main model lent sites for studies of niche properties. Each of formed his internship and these areas hosts a specific sub-population of stem residency in the Depart- systems for their research, as melanocytes are readi- ment of Internal Medicine at A stem cell's location and ly identified by the presence of a melanocyte- cells. The matrix niche, the site of active hair growth the Kyoto University Chest and pigmentation, provides an environment in which Disease Research Institute, immediate environment specific promoter, Dct, and are amenable to experi- before taking an assistant can be important factors mental manipulations that allow specific cell types to amplifying progeny become committed to a stem professorship in the same in defining its role. be isolated from mixed populations. As the exist- cell fate, while the bulge serves as a reservoir for department in 1979. He spent the period from 1980- Recent findings that ence of niche cells and the identification of their char- stem cells in their resting, or quiescent, state. Sub- 82 at the University of Colo- stem cells may acteristics can only be determined by first isolating tractive comparisons of cDNAs from stem cell, differ- gne Institute for Genetics (Germany) before returning arise from one the stem cells they support, Nishikawa's lab has entiated cell and 'niche-like' (keratinocyte) libraries to the Chest Disease tissue type developed an approach in which stem cells are dis- revealed approximately 200 non-redundant genes, Research Institute, where he was appointed asso- and then sected from their surroundings, dissociated and providing the lab with targets for further experiments ciate professor in the later be used to construct single cell libraries, a process in which the individual genes will be conditionally Department of Microbiology knocked out and the resulting phenotypes examined in 1983. He moved to the involving the technically challenging initial step of iso- Kumamoto University Medi- lating single intact hair follicles from the skin. Target- and analyzed. Other related studies, for which single cal School in 1987 to take a Melanocytes cell libraries of other resting stem cells are now professorship in the Depart- ed GFP expression is next used to label the follic- (marked red) found ment of Immunology, and being constructed, will entail profiling gene expres- in each hair follicle of ular melanocytes, making it possible to dis- returned to Kyoto in 1993, the mouse form a stem sion in melanocytes and resting stem cells, which as professor in the Depart- tinguish them from other cells. ment of Molecular Genetics cell system within these These cells are used to con- promises to provide a new resource for scientists minute organs. In this Representation of the relationships between different cell types based on the at the Kyoto Graduate investigating stem cells' genetic signatures. expression of approximately 700 probe sets School of Medicine. He was system, the most immature struct cDNA libraries of appointed CDB group direc- stem cell compartment is located (GFP-positive) melano- tor in 2000. in the upper region and the actively cycling compartment is located in the cytes from the bulge bottom of the follicle. and matrix regions of

16 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 17 Staff

Group Director Technical Staff Visiting Students Yoshiki Sasai Ayaka Nishiyama Tatsunori Sakamoto Research Scientist Yoko Nakazawa Akiko Arakawa Kenji Mizuseki Masako Kawada Morio Ueno Noriaki Sasai Tomoko Haraguchi Keiji Tsuji Makoto Ikeya Michiru Matsumura Takashi Irioka Mami Takasaki Tomoko Katayama Assistant Collaborative Scientist Student Trainee Ukiko Miyake Primate motoneurons Keiko Muguruma Makoto Sakuragi Ayumi Tanaka Hidehiko Inomata Takahiko Sato Yuri Arizono Hong-Lin Su Daisuke Kamiya Junko Yamada Hanako Ikeda Junior Research Associate Kiichi Watanabe Takayuki Onai an antagonistic effect to BMP4, and cause the cells they act upon to take on a ventral character. Periph- Core Program eral neurons are generally dorsal in their origins, while much of the central nervous system derives Publications from more ventral developmental regions. Normal Mizuseki K, Sakamoto T, Wata- nerve cells and precursors at high rates of efficien- development involves the constant, dynamic inter- nabe K, Muguruma K, Ikeya cy. In 2003, the lab built on that previous work, set- play between patterning factors that steer neural pre- M, Nishiyama A, Arakawa A, ting out to address the question of whether the neu- cursors toward either a ventral or dorsal fate. Suemori H, Nakatsuji N, Kawa- saki H, Murakami F and Sasai Induction and patterning of works involved in setting up the dorsal-ventral axis ral differentiation of ES cells could be guided with Y. Generation of neural crest- the nervous system and determining neural fate in the ectoderm, work even greater precision and diversity by adding pat- Sasai's group also investigated the effects of Shh derived peripheral neurons The interactions between cells from different germ which led to the recent identification of Tiarin, a nov- terning factors — soluble molecules that work to (Sonic hedgehog), a factor that suppresses dorsal and floor plate cells from layers and their distributions along body axes are el dorsalization factor. The group is now also active- determine the identities of cells in a developmental and promotes ventral development in vivo. As with mouse and primate embryonic established in early embryogenesis and serve to ly developing effective methods of inducing neurali- region — to the SDIA-cultured cells. BMP4, Shh provokes concentration-dependent differ- stem cells. Proc Natl Acad Sci U S A 100:5828-33 (2003). determine the spatial disposition and normal devel- zation in mammals, work which has potential for entiation in SDIA-cultured cells, analogous to its opment of tissues and organs. The specification of application in the treatment of neurodegenerative Although BMP signals have an function in the body. A low concentration guides Tsuda H, Sasai N, Matsuo- the dorsal-ventral (back-belly) axis is significant in disorders, such as Parkinson's disease. inhibitory effect on SDIA-derived neural precursors to develop into Takasaki M, Sakuragi M, Mura- neural development in that the central nervous sys- motor neurons, while higher concentrations bring kami Y and Sasai Y. Dorsaliza- neural development in tion of the neural tube by Xeno- tem forms on the dorsal side of the body in all verte- Inducing neural differentiation about differentiation into neural types that in normal earlier embryonic stages, pus tiarin, a novel patterning Yoshiki Sasai brate orders. This process is dictated by the effects in mammals development would derive from the floor plate, the factor secreted by the flanking M. D. , Ph. D. of a number of signaling factors that diffuse from The Sasai lab previously reported the development they work to promote the ventralmost region of neural development. Tests nonneural head ectoderm. Yoshiki Sasai gained his M. organizing centers and direct dorsal ectoderm to of a technique for inducing the differentiation of neu- neural crest at a later stage in revealed that these SDIA and Shh-treated neurons Neuron 33:515-28 (2002). D. from the Kyoto Universi- ty School of Medicine in maintain a neural fate. These molecules, which ral cells by culturing mouse ES cells on plates of development display both the molecular markers and physiologi- Kawasaki H, Suemori H, Mizu- 1986, subsequently per- include Noggin, Chordin, Follistatin and their homol- connective cells, called stromal cells. These cells cal attributes (such as directional axon guidance) forming internships in gener- seki K, Watanabe K, Urano F, al practice and emergency ogs, participate in elaborate signaling networks in have a strong neuralizing effect, termed SDIA (for Using BMP4, Sasai's group succeeded in causing characteristic of the same types of neurons in vivo, Ichinose H, Haruta M, Taka- medicine. He received his which factors collaborate and compete to initiate the Stromal cell-Derived Inducing Activity), inducing the SDIA-treated ES cells to differentiate into cell demonstrating that the cells produced by this meth- hashi M, Yoshikawa K, Nishika- Ph. D. from the same institu- wa S, Nakatsuji N and Sasai tion in 1992, for work on embryonic nervous system. types that normally derive from the neural crest, a od are structurally and functionally similar to natural Y. Generation of dopaminergic neural specific transcription- developmental region that gives rise to nerve cells in floor plate cells. neurons and pigmented epithe- al regulators. In 1993, he Beginning with the identifica- took a postdoctoral fellow- the peripheral nervous system, including sensory, lia from primate ES cells by ship in the De Robertis lab tion of the neural inducing autonomic and enteric (gut) neurons. Although BMP Taken together, these findings show that SDIA- stromal cell-derived inducing at the UCLA School of Medi- factor Chordin in the early generated neural precursors respond to patterning activity. Proc Natl Acad Sci U cine, remaining there until signals have an inhibitory effect on neural develop- S A 99:1580-5 (2002). 1996 when he was appoint- 1990s, Yoshiki Sasai's ment in earlier embryonic stages, the same signals factors with a versatility similar to that of their in vivo ed associate professor at research has focused on work to promote the neural crest at a later stage in counterparts. Higher concentrations of the dorsaliz- the Kyoto University School Sasai N, Mizuseki K and Sasai of Medicine. He assumed a explicating the molecular sig- development. The group found that they were able ing factor BMP4 induce increasingly dorsal neural Y. Requirement of FoxD3- professorship at the Kyoto naling mechanisms that to induce peripheral neural types that are naturally types, while greater doses of Shh have the same class signaling for neural crest University Institute for Fron- determination in Xenopus. tier Medical Sciences in enable the early embryo to derived from the neural crest, including autonomic effect in ventralizing neural precursors. This work Development 128:2525-36 1998, and was appointed organize itself into a com- and sensory neurons and smooth muscle cells, represents the first efficient generation of such a group director at the CDB (2001). in 2000. He serves on the plex, mature organism. when they treated the SDIA-treated cells with medi- broad spectrum of neuronal types from ES cells in editorial boards of Neuron, Using the African clawed an concentrations of BMP4. A lower concentration culture, and the fact that the process has been dem- Kawasaki H, Mizuseki K, Nishi- Development, Genesis and kawa S, Kaneko S, Kuwana Y, Developmental Dynamics. frog, Xenopus laevis, as a prompts the same cells to differentiate into dorsal onstrated effective not only in mouse, but also in model in molecular embryo- central nervous system neurons. primate cells, marks a significant step toward the Nakanishi S, Nishikawa SI and Sasai Y. Induction of midbrain logical studies, Sasai and ability to induce specific types of neuron from ES dopaminergic neurons from his group are engaged in The neural crest arises from the dorsalmost region cell-derived precursors that is one of the foremost ES cells by stromal cell- clarifying the structure and of the developing nervous system, and BMP4 is challenges confronting researchers in regenerative derived inducing activity. Neu- extent of the signaling net- regarded as a 'dorsalizing' factor. Other factors have medicine. ron 28:31-40 (2000).

Primate sensory neurons

18 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 19 Staff

Primate sensory neurons

18 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 19 Staff ered that Fat1 is required for the stable maintenance of tight associations between cells and the normal Group Director Masatoshi Takeichi organization of actin. This inhibition of Fat1 also Research Scientist suppressed the formation of cell polarity, which is a Shinichi Nakagawa Shinji Hirano general phenomenon induced in epithelial cell Publications Research Associate sheets during wound healing. Molecular studies Sachihiro Suzuki Tanabe K, Takeichi M, and Research Specialist uncovered an apparent partnership between Fat1 Midori Maekawa Nakagawa S. Identification of Special Postdoctoral Researcher and Ena/VASP (vasodilator stimulated phosphopro- a nonchordate-type classic Takuji Tanoue tein) proteins; Fat1 colocalizes and binds with cadherin in vertebrates: Chick- Technical Staff en Hz cadherin is expressed in Hitomi Ishigami Ena/VASP proteins in filopodia, lamellipodia and cell Miwako Harata junctions and the group's recent studies indicate horizontal cells of the neural Makoto Uemura retina and contains a Junior Research Associate that the Ena/VASP proteins may act as links in the nonchordate-specific domain Tetsuo Ichii molecular regulatory chain leading from Fat1 to complex. Dev Dyn (2004). Student Trainee Tetsuhisa Otani actin, although the presence of other linkages was Hideru Togashi also suggested. The study's findings also suggest Abe K, Chisaka O, van Roy F, Yoshiko Kametani and Takeichi M. Stability of Koji Tanabe that Fat1 co-acts with the classic cadherins to rein- Kentaro Abe dendritic spines and synaptic Masakazu Kadowaki force or maintain cell-cell junctions, which would contacts is controlled by Fumi Kubo make this molecule a significant player on both αN-catenin. Nat Neurosci Shinsuke Nakao Masamitsu Sone sides of the cell membrane. (2004). Assistant Mutsuko Aiso S Nakagawa, S Takada, R Contacts between neurons Takada and M Takeichi. Identi- Cadherin complexes are known to play diverse roles fication of the Laminar- in a cell type-specific manner. In an ongoing study Inducing Factor: Wnt-Signal of cadherin-catenin function in interneuronal junc- from the Anterior Rim Induces Correct Laminar Formation of tions, Takeichi's lab has found that N-catenin, a α the Neural Retina in Vitro. Dev Core Program form of α-catenin specific to the nervous system, is Biol 260:414-25 (2003). essential to the stabilization of dendritic spines. These projections are important for establishing and Kubo F, Takeichi M and Naka- maintaining contacts between axons and dendrites, gawa S. Wnt2b controls retinal Characterizing Fat1 function cell differentiation at the ciliary Cadherins do not act alone. Spanning both sides of the two principal types of extensions from the marginal zone. Development the cell membrane, cadherin molecules bind with neuronal cell body. Previous work showed that 130:587-98 (2003). The development of multicellular systems and organ- molecules in regulating cell-cell adhesion has been other cadherins of like type in the space between cadherins help to regulate the adhesion of dendritic isms depends on the ability of cells to congregate studied extensively. These molecules form complex- cells, while in the cytoplasm they form anchorage Nakagawa S, Takada S, Taka- Cadherins do not da R and Takeichi M. Identifi- and cooperate in diverse ways. Individual cells must es with other intracellular factors to create bonds complexes with other proteins, such as various act alone cation of the laminar-inducing be able to identify each other and distinguish spanning membranes and intercellular spaces to members of the catenin family, which in turn bind factor: Wnt-signal from the between diverse cell types. They need to be able to join cells with other cells expressing similar cadher- with molecules present within the cell and partici- spines to axons, which is a crucial step in synapse anterior rim induces correct form bonds discriminatively, such that cells that ins. Cadherin bonds are dynamically stable — pate in important cell signaling networks. In 2003, formation, a finding which was complemented by laminar formation of the neural retina in vitro. Dev Biol must aggregate in order to form a tissue do so, although the intercellular junctions they form persist the Takeichi lab investigated the function of a this year's elucidation of αN-catenin's role. The func- 260:414-25 (2003). while cells with different missions remain detached in terms of the whole cell, the individual bonds are mammalian cadherin known as Fat1, which is found tional loss of this molecule resulted in dramatic from one another. These functions of cell recogni- regulable and can respond to changes in cell state. in the peripheries of cells. The group found that Fat1 increases in spine motility and shape alterations, Togashi H, Abe K, Mizoguchi tion and bonding are achieved by several types of Masatoshi Takeichi has been engaged in exploring acts to regulate the function of actin, a primary while αN-catenin overexpression caused spines, A, Takaoka K, Chisaka O and Takeichi M. Cadherin regu- Masatoshi Takeichi molecules known as CAMs (for cellular adhesion the structure, function and biological implications of component of the cytoskeleton that functions in the which normally extend and retract intermittently, to lates dendritic spine morphoge- Ph. D. molecules), which variously bind with partner the cadherin superfamily since his discovery of the turn over at a much lower rate, resulting in abnormal control of cellular motility and changes in cell nesis. Neuron 35:77-89 Masatoshi Takeichi is direc- molecules present on the surfaces of substrates, first cadherin molecules more than twenty years morphology. Among members of the cadherin fami- accumulations of mature spines over time. Taken (2002). tor of the RIKEN Kobe Insti- tute and the Center for matrices and other cells. Since the discovery of ago. ly, Fat cadherins are distinguished by their unusually together, these results suggest that αN-catenin is a Developmental Biology, as cadherins, the role of the cadherin superfamily of large number of extracellular repeat domains — critical agent in maintaining the appropriate balance well as the Cell Adhesion and Tissue Patterning nearly seven times the number found in classic between stability and turnover in synaptic contacts. research group at the same cadherins — and by the institute. He completed the B. Sc. and M. S. programs uniqueness of the primary in biology at Nagoya Uni- sequences of their cytoplas- versity before receiving a doctorate in biophysics mic domains. Three Fat from Kyoto University in cadherins have been identi- 1973. After attaining his Ph. D., he took a research fel- fied in mammals — Fat1, 2 lowship at the Carnegie and 3. It has been shown Institution Department of that the Drosophila homolog Embryology under Richard Pagano. He then returned of these molecules plays to Kyoto University, attain- roles in cell proliferation or in ing a full professorship in the Department of Biophys- the determination of planar ics (1986-1999), before polarity, but the function of becoming professor in the Department of Cell and the vertebrate Fats has Developmental Biology in remained unknown. the Graduate School of Bio- studies at the same university. He assumed his current Using RNA interference positions at the CDB in 2000. (RNAi) to knock down Fat1 Overexpression of αN-catenin in hippocampal neurons causes overproduction of dendritic spines function, researchers in the (right). Left, control. Laboratory for Cell Adhesion Fat1 cadherin (green) localizes at cell-cell contact sites (left), colocalizing with F-actin (red/yellow, right) in epithelial cells. and Tissue Patterning discov-

20 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 21 Staff ered that Fat1 is required for the stable maintenance of tight associations between cells and the normal Group Director Masatoshi Takeichi organization of actin. This inhibition of Fat1 also Research Scientist suppressed the formation of cell polarity, which is a Shinichi Nakagawa Shinji Hirano general phenomenon induced in epithelial cell Publications Research Associate sheets during wound healing. Molecular studies Sachihiro Suzuki Tanabe K, Takeichi M, and Research Specialist uncovered an apparent partnership between Fat1 Midori Maekawa Nakagawa S. Identification of Special Postdoctoral Researcher and Ena/VASP (vasodilator stimulated phosphopro- a nonchordate-type classic Takuji Tanoue tein) proteins; Fat1 colocalizes and binds with cadherin in vertebrates: Chick- Technical Staff en Hz cadherin is expressed in Hitomi Ishigami Ena/VASP proteins in filopodia, lamellipodia and cell Miwako Harata junctions and the group's recent studies indicate horizontal cells of the neural Makoto Uemura retina and contains a Junior Research Associate that the Ena/VASP proteins may act as links in the nonchordate-specific domain Tetsuo Ichii molecular regulatory chain leading from Fat1 to complex. Dev Dyn (2004). Student Trainee Tetsuhisa Otani actin, although the presence of other linkages was Hideru Togashi also suggested. The study's findings also suggest Abe K, Chisaka O, van Roy F, Yoshiko Kametani and Takeichi M. Stability of Koji Tanabe that Fat1 co-acts with the classic cadherins to rein- Kentaro Abe dendritic spines and synaptic Masakazu Kadowaki force or maintain cell-cell junctions, which would contacts is controlled by Fumi Kubo make this molecule a significant player on both αN-catenin. Nat Neurosci Shinsuke Nakao Masamitsu Sone sides of the cell membrane. (2004). Assistant Mutsuko Aiso S Nakagawa, S Takada, R Contacts between neurons Takada and M Takeichi. Identi- Cadherin complexes are known to play diverse roles fication of the Laminar- in a cell type-specific manner. In an ongoing study Inducing Factor: Wnt-Signal of cadherin-catenin function in interneuronal junc- from the Anterior Rim Induces Correct Laminar Formation of tions, Takeichi's lab has found that N-catenin, a α the Neural Retina in Vitro. Dev Core Program form of α-catenin specific to the nervous system, is Biol 260:414-25 (2003). essential to the stabilization of dendritic spines. These projections are important for establishing and Kubo F, Takeichi M and Naka- maintaining contacts between axons and dendrites, gawa S. Wnt2b controls retinal Characterizing Fat1 function cell differentiation at the ciliary Cadherins do not act alone. Spanning both sides of the two principal types of extensions from the marginal zone. Development the cell membrane, cadherin molecules bind with neuronal cell body. Previous work showed that 130:587-98 (2003). The development of multicellular systems and organ- molecules in regulating cell-cell adhesion has been other cadherins of like type in the space between cadherins help to regulate the adhesion of dendritic isms depends on the ability of cells to congregate studied extensively. These molecules form complex- cells, while in the cytoplasm they form anchorage Nakagawa S, Takada S, Taka- Cadherins do not da R and Takeichi M. Identifi- and cooperate in diverse ways. Individual cells must es with other intracellular factors to create bonds complexes with other proteins, such as various act alone cation of the laminar-inducing be able to identify each other and distinguish spanning membranes and intercellular spaces to members of the catenin family, which in turn bind factor: Wnt-signal from the between diverse cell types. They need to be able to join cells with other cells expressing similar cadher- with molecules present within the cell and partici- spines to axons, which is a crucial step in synapse anterior rim induces correct form bonds discriminatively, such that cells that ins. Cadherin bonds are dynamically stable — pate in important cell signaling networks. In 2003, formation, a finding which was complemented by laminar formation of the neural retina in vitro. Dev Biol must aggregate in order to form a tissue do so, although the intercellular junctions they form persist the Takeichi lab investigated the function of a this year's elucidation of αN-catenin's role. The func- 260:414-25 (2003). while cells with different missions remain detached in terms of the whole cell, the individual bonds are mammalian cadherin known as Fat1, which is found tional loss of this molecule resulted in dramatic from one another. These functions of cell recogni- regulable and can respond to changes in cell state. in the peripheries of cells. The group found that Fat1 increases in spine motility and shape alterations, Togashi H, Abe K, Mizoguchi tion and bonding are achieved by several types of Masatoshi Takeichi has been engaged in exploring acts to regulate the function of actin, a primary while αN-catenin overexpression caused spines, A, Takaoka K, Chisaka O and Takeichi M. Cadherin regu- Masatoshi Takeichi molecules known as CAMs (for cellular adhesion the structure, function and biological implications of component of the cytoskeleton that functions in the which normally extend and retract intermittently, to lates dendritic spine morphoge- Ph. D. molecules), which variously bind with partner the cadherin superfamily since his discovery of the turn over at a much lower rate, resulting in abnormal control of cellular motility and changes in cell nesis. Neuron 35:77-89 Masatoshi Takeichi is direc- molecules present on the surfaces of substrates, first cadherin molecules more than twenty years morphology. Among members of the cadherin fami- accumulations of mature spines over time. Taken (2002). tor of the RIKEN Kobe Insti- tute and the Center for matrices and other cells. Since the discovery of ago. ly, Fat cadherins are distinguished by their unusually together, these results suggest that αN-catenin is a Developmental Biology, as cadherins, the role of the cadherin superfamily of large number of extracellular repeat domains — critical agent in maintaining the appropriate balance well as the Cell Adhesion and Tissue Patterning nearly seven times the number found in classic between stability and turnover in synaptic contacts. research group at the same cadherins — and by the institute. He completed the B. Sc. and M. S. programs uniqueness of the primary in biology at Nagoya Uni- sequences of their cytoplas- versity before receiving a doctorate in biophysics mic domains. Three Fat from Kyoto University in cadherins have been identi- 1973. After attaining his Ph. D., he took a research fel- fied in mammals — Fat1, 2 lowship at the Carnegie and 3. It has been shown Institution Department of that the Drosophila homolog Embryology under Richard Pagano. He then returned of these molecules plays to Kyoto University, attain- roles in cell proliferation or in ing a full professorship in the Department of Biophys- the determination of planar ics (1986-1999), before polarity, but the function of becoming professor in the Department of Cell and the vertebrate Fats has Developmental Biology in remained unknown. the Graduate School of Bio- studies at the same university. He assumed his current Using RNA interference positions at the CDB in 2000. (RNAi) to knock down Fat1 Overexpression of αN-catenin in hippocampal neurons causes overproduction of dendritic spines function, researchers in the (right). Left, control. Laboratory for Cell Adhesion Fat1 cadherin (green) localizes at cell-cell contact sites (left), colocalizing with F-actin (red/yellow, right) in epithelial cells. and Tissue Patterning discov-

20 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 21 N

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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 use, 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. N

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Staff Goukassian D A, Kishore R, Team Leader Student Trainee Krasinski K, Dolan C, Luede- Takayuki Asahara Masakazu Kuriwaka mann C, Yoon Y S, Kearney Senior Scientist Assistant Satoshi Murasawa Kanako Ota M, Hanley A, Ma H, Asahara Hiromi Nishimura T, Isner J M and Losordo D Research Scientist W. Engineering the Satoshi Hasegawa Response to Vascular Injury. Tsuyoshi Hamada Technical Staff Divergent Effects of Deregu- Akira Oyamada lated E2f1 Expression on Kazuyo Sadamoto Vascular Smooth Muscle Cells and Endothelial Cells A colony of endothelial progenitor cells on the surface of an injured Results in Endothelial Recov- blood vessel ery and Inhibition of Neointi- Creative Research mal Growth. Circ Res (2003). Promoting Program Iwakura A, Luedemann C, Shastry S, Hanley A, Kear- progenitor cells in tissue sections from the SDF-1 sites in the hearts of pigs, to test the effects of the ney M, Aikawa R, Isner J M, and uninjected groups, which showed that the EPCs concentrated delivery of autologous EPCs. The Asahara T and Losordo D W. accumulated at nearly two times the rate in the results showed that the targeted transplantation of Estrogen-Mediated,Endothel- ial Nitric Oxide Synthase- The emerging field of stem cell-based therapy contin- tions. The vascularization of regenerating tissue is a limbs which had been injected with SDF-1 com- CD31+ hematopoietic cells reduced ischemic dam- Dependent Mobilization of ues to receive attention as one of the most promis- critical component of the natural healing process as pared to that in the control group. This represents age and promoted neovascularization, resulting in Bone Marrow-Derived ing frontiers of medical science. Building on previ- well as fundamental to the recovery of blood vessels an important first proof-of-principle demonstration improved cardiac function, while pigs receiving Endothelial Progenitor Cells ous work in which he identified bone marrow- that have been damaged, blocked or lost, and the that the homing of EPCs to a damaged area can be CD31- or control solutions experienced no such ben- Contributes to Reendotheliali- zation after Arterial Injury. Cir- derived endothelial progenitor cells (EPCs) and dem- ability to promote and regulate the growth of new augmented by an extrinsic factor. efits. A complementary study involving the targeted culation (2003). onstrated their role in the generation of new blood blood vessels using EPCs will provide impetus and transplantation of human CD34+ cells into ischemic vessels, Takayuki Asahara seeks to characterize new areas to explore for researchers and clinicians Targeted and autologous rat hearts showed that non-autologous cells can Kawamoto A, Tkebuchava T, adult stem and progenitor cells with even greater dif- working to develop treatments for disorders of the transplantation also incorporate into damage sites and differentiate Yamaguchi J, Nishimura H, ferentiative potential, and simultaneously to trans- cardiovascular and other systems. EPCs represent an extremely promising new mode into functioning endothelial cells, even when the Yoon Y S, Milliken C, Uchida S, Masuo O, Iwaguro H, Ma late that research into clinically relevant advances. of promoting the therapeutic growth of blood ves- transplanted quantities are one-twentieth the H, Hanley A, Silver M, Kear- Takayuki Asahara Stimulating EPC accumulation sels, but significant obstacles must be overcome amounts used in similar previous experiments. ney M, Losordo DW, Isner M. D. , Ph. D. Researchers in the Asahara lab found that EPCs tak- before that promise can be realized. Previous stud- JM and Asahara T. Intramyo- Takayuki Asahara received en from human subjects and expanded ex vivo con- ies in rats have used quantities of EPCs that would Clinical work cardial Transplantation of his M. D. from Tokyo Medi- Autologous Endothelial Pro- tributed to the vascularization of the damaged limbs require impracticably large supplies of peripheral In addition to his role as team leader of the CDB Lab- cal College in 1984, and genitor Cells for Therapeutic performed residencies in of mice at higher efficiencies if the limbs were first blood to derive equivalent amounts of EPCs for use oratory for Stem Cell Translational Research, Asa- Neovascularization of Myo- cardiology and emergency injected with a factor known as SDF-1 (stromal cell- in humans, and the possibility of rejection of blood hara also serves as director of the regenerative med- cardial Ischemia. Circulation medicine. He worked as a 107:461-8 (2003). research fellow in cardiolo- derived factor-1), a chemokine originally identified in from non-self sources by the immune system icine program at the neighboring Institute for Biomed- gy at the Tokyo Medical mouse bone marrow and whose only known recep- ical Research and Innovation. In its role as a transla- Yamaguchi J, Kusano KF, College Hospital from 1989 tor is expressed in hematopoietic stem cells. The tional research lab, Asahara's team is beginning clin- to 1993, before moving to a The vascularization of Masuo O, Kawamoto A, Sil- CXCR4 transmembrane receptor, present in CD34+ fellowship in cardiovascular regenerating tissue is a ical tests of the therapeutic uses of EPCs in the treat- ver M, Murasawa S, Bosch- research at St. Elizabeth’s hematopoietic cells, shows a strong attraction to ment of Buerger Disease (thromboangiitis obliter- Marce M, Masuda H, Losor- Hospital in Boston (USA). critical component of the SDF-1 and is believed to help recruit stem cells to ans), an inflammatory condition involving the do DW, Isner JM and Asa- He was appointed assistant hara T. Stromal Cell-Derived professor at Tufts School of sites in need of vascular replacement or repair. natural healing process obstruction of small and medium-sized blood ves- Factor-1 Effects on Ex Vivo Medicine in 1995, and asso- sels in the limbs, and linked with heavy tobacco use. Expanded Endothelial Proge- ciate professor at the Tokai Vaculogenesis achieved by endothelial progenitor cells University Institute of Medi- This belief was borne out by the EPC transplanta- remains another important potential barrier. Mem- Plans are in place to expand the patient population nitor Cell Recruitment for cal Sciences in 2000. In tion study, in which the researchers first isolated bers of the Asahara team sought to address these of this preliminary study into the therapeutic trans- Ischemic Neovascularization. addition to his current posi- Blood vessels are formed by two distinct physiologi- large numbers of human EPCs for transplantation questions by developing a system for the local, rath- plantation of EPCs, which may offer a new mode of Circulation 107:1322-8 tion as CDB team leader, (2003). Dr. Asahara serves as direc- cal processes in the adult body. In angiogenesis, into mouse models of limb ischemia — localized oxy- er than systemic, transplantation of EPCs freshly iso- treatment for this disease which at present frequent- tor of Regenerative Medi- new blood vessels are generated from pre-existing, gen starvation of tissue resulting in damage to blood lated from the host. These studies were conducted ly necessitates the surgical amputation of ischemic Yamamoto K, Takahashi T, cine and Research at the differentiated endothelial cells. Vasculogenesis, on vessels. The transplanted human EPCs were found using a swine model of myocardial (heart muscle) Asahara T, Ohura N, Sokabe Kobe Institute of Biomedi- digits or limbs. T, Kamiya A and Ando J. Pro- cal Research and Innova- the other hand, involves the recruitment and differen- to improve neovascularization in the ischemic ischemia, from which small quantities of blood were tion, and Professor of Physi- tiation of previously undifferentiated EPCs at the site mouse limbs, as well as to reduce the secondary drawn to allow the isolation and expansion of EPCs, liferation, Differentiation, and ology at the Tokai Universi- Tube Formation by Endotheli- ty School of Medicine. of new blood vessel growth. These EPCs are them- effects of prolonged ischemia, which include necro- identified by the presence of the marker molecule al Progenitor Cells in selves the progeny of adult stem cells known as sis and autoamputation. The incorporation of the CD31. Catheters were used to inject similar quanti- Response to Shear Stress. J hemangioblasts, which can be induced to demon- transplanted EPCs at ischemic sites was deter- ties of either CD31+ or CD31- cells, or of a control Appl Physiol 95:2081-8 strate true pluripotency under the right culture condi- mined by measuring the uptake of labeled human solution containing no cells, directly into ischemic (2003).

24 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 25 Publications

24 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 25 expressed in blood vessels and attracts growing axons emanating from sympathetic ganglion that expresses RET. This interaction is crucial for sympathetic axons to follow vascular pathways and to Publications reach the final target tissues. The fourth GFL- Gianino S, Grider J R, Cress- receptor pairing of Persephin and GFR 4 is known α well J, Enomoto H and to be expressed ubiquitously at low levels, but its Heuckeroth R O. GDNF avail- developmental activity remains obscure. ability determines enteric neuron number by controlling In related work, members of the Enomoto lab precursor proliferation. Devel- opment 130:2187-98 (2003). studied the roles of RET and TRKA (neurotrophin receptor) in sympathetic neurogenesis and main- Enomoto H, Crawford P A, tenance. Both RET and TRKA are expressed in Gorodinsky A, Heuckeroth R developing sympathetic neurons, but the balance O, Johnson E M, Jr. and Mil- brandt J. RET signaling is between these two receptor types shifts over time, Staff essential for migration, axon- with RET predominating during earlier phases of al growth and axon guidance Team Leader Hideki Enomoto axon growth and migration, while TRK assumes a of developing sympathetic Research Scientist more important role in ensuring the perinatal sur- neurons. Development Shiho Ohmori 128:3963-74 (2001). Yukinao Shibukawa vival and maintenance of these cells. Technical Staff Kayoko Inoue Enomoto H, Heuckeroth R Mayumi Nagashimada Studies of RET signaling are of O, Golden J P, Johnson E M Mayumi Akatsuka and Milbrandt J. Develop- Assistant particular interest for Kaori Hamada their value in biomedical research ment of cranial parasympa- thetic ganglia requires sequential actions of GDNF Studies of RET signaling are of particular interest for and neurturin. Development Creative Research their value in biomedical research. Hirschsprung's 127:4877-89 (2000). Promoting Program disease, a congenital disorder afflicting about 1 in 5,000 newborn children in which enteric neuron pre- Baloh R H, Enomoto H, John- son E M, Jr. and Milbrandt J. cursors fail to colonize the distal part of the gut The GDNF family ligands resulting in a loss of bowel motility, chronic constipa- and receptors - implications tion and bowel obstruction, is caused by a loss-of- for neural development. Curr Neurotrophic factors rotrophic factors includes four known members — function mutation in the Ret gene. Hyperactivation of Opin Neurobiol 10:103-10 (2000). The early stages of neural network development see GDNF (for Glial cell line Derived Neurotrophic Fac- RET results in abnormal development of the neural the growth of a number of neurons in excess of the tor), Neurturin, Artemin and Persephin. The GFLs crest, a developmentally transient structure that nor- number that will ultimately populate any given signal via receptor complexes formed by the RET mally gives rise to neurons, glia, endocrine cells and region. Neurons that have formed synaptic connec- receptor tyrosine kinase and one of four mesenchymal cells, and has been linked to the onco- tions establish what is known as a trophic interaction co-receptors, GRFα1-4. In vitro, these four recep- genesis of endocrine tumors, such as thyroid carci- in which the target tissue provides the neuron with tors show differential affinities for specific GFLs, with noma and pheochromocytoma. chemical signals necessary for its survival and con- GFR?1 showing the greatest ability to interact with tinued function. The supply of such signals is limit- the range of GFL family members. In the future, the Enomoto research team plans to GFP staining of RET-expressing motor and sensory neurons in the ed, and neurons which fail brainstem of RET-GFP mice conduct systematic analyses of RET-bearing neu- Hideki Enomoto to compete successfully GDNF ligand-receptor pairings rons to achieve a better understanding of the molec- M. D. , Ph. D. for these factors Enomoto studied phenotypes turin or GFRα2 deficiency, but the phenotypes are ular mechanisms involved in RET and GFL-receptor Hideki Enomoto received undergo apoptosis, of mice in which individual less severe, with kidneys forming normally and indef- signaling. One immediate focus will be on investigat- his M. D. from the Chiba a controlled die-off GFL, GFR or Ret genes inite effects on sympathetic and motor neural devel- ing roles for RET, GDNF and GFR 1 in the postna- University School of medi- α α cine in 1988, and his Ph. D. that establishes are disrupted and found opment. Analysis of the consequences of these tal maintenance of motor, sensory and dopaminer- from the same institution in an appropriate that, in receptor-ligand mutations in developing enteric and parasympathet- gic neurons, 1996 for his work in the molecular cloning of the balance in the interactions in vivo, ic neurons suggests that GDNF-GFRα1 functions in using a Cre-Lox human DAN gene. After neural population. there are affinitive pair- neural precursors to regulate fundamental neurode- conditional GFL-GFRα INTERACTIONS serving as the Chief of the ings between GFRα1- velopmental processes such as migration and prolif- knockout strate- Department of Pediatric Sur- gery, Matsudo Municipal The target-derived GDNF, GFRα2-NRTN eration, while the later expression of Neurturin- gy. By providing GDNF NRTN ARTN PSPN Hospital in Chiba, Japan, chemical signals in and GFRα3-ARTN. GFRα2 in target tissues serves to maintain previous- insights into the he moved to the Washing- the trophic interaction The loss of GDNF- ly established neurons. functions of ton University School of Medicine, St. Louis in 1997, are known as neuro- GFRα1 system had GFL-specific to work as a research asso- trophic factors. The study the strongest develop- Artemin-GFRα3 signaling seems to operate in a neurotrophic ciate studying the physiological roles of the GDNF fami- of these signaling proteins mental effects, caus- more specific subset of cells, sympathetic neurons, factor signaling ly of ligands in neural devel- dates back to the discovery of ing defects in enteric, with no apparent function in enteric or central neural pathways, Eno- opment and function. He nerve growth factor (NGF) by autonomic, sensory development. The sympathetic nervous system aris- moto hopes to returned to Japan to take a position as a team leader at Levi-Montalcini and colleagues in and motor neurons as es from the neural crest and follows a stepwise contribute to the CDB in 2002. the 1950s. Subsequent research has well as failures in the developmental pathway that directs its neurons to the develop- revealed the diversity of neurotrophic fac- development of the establish their appropriate functional roles and posi- ment of stem tors, which are now thought to include neuro- kidney, and resulting tions throughout the body. In a series of signal- cell-basedthera- trophins (of which NGF is a member), neuropoie- in lethality by the P0 directed decisions, sympathetic precursor cells pies for nervous tins, fibroblast growth factors, and the transforming X-gal staining of a mouse stage of develop- migrate and differentiate into neurons soon after system disor- growth factor-β superfamily. Hideki Enomoto is pri- embryo expressing tau-lacZ ment, birth. Similar they undergo lineage commitment and restriction to ders. under the GFRα1 promoter GFR 1 GFR 2 GFR 3 GFR 4 marily interested in this last group, the GDNF Family neuronal populations a catecholaminergic (sympathetic neurotransmitter- α α α α RET RET RET Ligands (the GFLs) in particular. This family of neu- are affected by Neur- producing) fate. Enomoto found that Artemin is

26 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 27 expressed in blood vessels and attracts growing axons emanating from sympathetic ganglion that expresses RET. This interaction is crucial for sympathetic axons to follow vascular pathways and to Publications reach the final target tissues. The fourth GFL- Gianino S, Grider J R, Cress- receptor pairing of Persephin and GFR 4 is known α well J, Enomoto H and to be expressed ubiquitously at low levels, but its Heuckeroth R O. GDNF avail- developmental activity remains obscure. ability determines enteric neuron number by controlling In related work, members of the Enomoto lab precursor proliferation. Devel- opment 130:2187-98 (2003). studied the roles of RET and TRKA (neurotrophin receptor) in sympathetic neurogenesis and main- Enomoto H, Crawford P A, tenance. Both RET and TRKA are expressed in Gorodinsky A, Heuckeroth R developing sympathetic neurons, but the balance O, Johnson E M, Jr. and Mil- brandt J. RET signaling is between these two receptor types shifts over time, Staff essential for migration, axon- with RET predominating during earlier phases of al growth and axon guidance Team Leader Hideki Enomoto axon growth and migration, while TRK assumes a of developing sympathetic Research Scientist more important role in ensuring the perinatal sur- neurons. Development Shiho Ohmori 128:3963-74 (2001). Yukinao Shibukawa vival and maintenance of these cells. Technical Staff Kayoko Inoue Enomoto H, Heuckeroth R Mayumi Nagashimada Studies of RET signaling are of O, Golden J P, Johnson E M Mayumi Akatsuka and Milbrandt J. Develop- Assistant particular interest for Kaori Hamada their value in biomedical research ment of cranial parasympa- thetic ganglia requires sequential actions of GDNF Studies of RET signaling are of particular interest for and neurturin. Development Creative Research their value in biomedical research. Hirschsprung's 127:4877-89 (2000). Promoting Program disease, a congenital disorder afflicting about 1 in 5,000 newborn children in which enteric neuron pre- Baloh R H, Enomoto H, John- son E M, Jr. and Milbrandt J. cursors fail to colonize the distal part of the gut The GDNF family ligands resulting in a loss of bowel motility, chronic constipa- and receptors - implications tion and bowel obstruction, is caused by a loss-of- for neural development. Curr Neurotrophic factors rotrophic factors includes four known members — function mutation in the Ret gene. Hyperactivation of Opin Neurobiol 10:103-10 (2000). The early stages of neural network development see GDNF (for Glial cell line Derived Neurotrophic Fac- RET results in abnormal development of the neural the growth of a number of neurons in excess of the tor), Neurturin, Artemin and Persephin. The GFLs crest, a developmentally transient structure that nor- number that will ultimately populate any given signal via receptor complexes formed by the RET mally gives rise to neurons, glia, endocrine cells and region. Neurons that have formed synaptic connec- receptor tyrosine kinase and one of four mesenchymal cells, and has been linked to the onco- tions establish what is known as a trophic interaction co-receptors, GRFα1-4. In vitro, these four recep- genesis of endocrine tumors, such as thyroid carci- in which the target tissue provides the neuron with tors show differential affinities for specific GFLs, with noma and pheochromocytoma. chemical signals necessary for its survival and con- GFR?1 showing the greatest ability to interact with tinued function. The supply of such signals is limit- the range of GFL family members. In the future, the Enomoto research team plans to GFP staining of RET-expressing motor and sensory neurons in the ed, and neurons which fail brainstem of RET-GFP mice conduct systematic analyses of RET-bearing neu- Hideki Enomoto to compete successfully GDNF ligand-receptor pairings rons to achieve a better understanding of the molec- M. D. , Ph. D. for these factors Enomoto studied phenotypes turin or GFRα2 deficiency, but the phenotypes are ular mechanisms involved in RET and GFL-receptor Hideki Enomoto received undergo apoptosis, of mice in which individual less severe, with kidneys forming normally and indef- signaling. One immediate focus will be on investigat- his M. D. from the Chiba a controlled die-off GFL, GFR or Ret genes inite effects on sympathetic and motor neural devel- ing roles for RET, GDNF and GFR 1 in the postna- University School of medi- α α cine in 1988, and his Ph. D. that establishes are disrupted and found opment. Analysis of the consequences of these tal maintenance of motor, sensory and dopaminer- from the same institution in an appropriate that, in receptor-ligand mutations in developing enteric and parasympathet- gic neurons, 1996 for his work in the molecular cloning of the balance in the interactions in vivo, ic neurons suggests that GDNF-GFRα1 functions in using a Cre-Lox human DAN gene. After neural population. there are affinitive pair- neural precursors to regulate fundamental neurode- conditional GFL-GFRα INTERACTIONS serving as the Chief of the ings between GFRα1- velopmental processes such as migration and prolif- knockout strate- Department of Pediatric Sur- gery, Matsudo Municipal The target-derived GDNF, GFRα2-NRTN eration, while the later expression of Neurturin- gy. By providing GDNF NRTN ARTN PSPN Hospital in Chiba, Japan, chemical signals in and GFRα3-ARTN. GFRα2 in target tissues serves to maintain previous- insights into the he moved to the Washing- the trophic interaction The loss of GDNF- ly established neurons. functions of ton University School of Medicine, St. Louis in 1997, are known as neuro- GFRα1 system had GFL-specific to work as a research asso- trophic factors. The study the strongest develop- Artemin-GFRα3 signaling seems to operate in a neurotrophic ciate studying the physiological roles of the GDNF fami- of these signaling proteins mental effects, caus- more specific subset of cells, sympathetic neurons, factor signaling ly of ligands in neural devel- dates back to the discovery of ing defects in enteric, with no apparent function in enteric or central neural pathways, Eno- opment and function. He nerve growth factor (NGF) by autonomic, sensory development. The sympathetic nervous system aris- moto hopes to returned to Japan to take a position as a team leader at Levi-Montalcini and colleagues in and motor neurons as es from the neural crest and follows a stepwise contribute to the CDB in 2002. the 1950s. Subsequent research has well as failures in the developmental pathway that directs its neurons to the develop- revealed the diversity of neurotrophic fac- development of the establish their appropriate functional roles and posi- ment of stem tors, which are now thought to include neuro- kidney, and resulting tions throughout the body. In a series of signal- cell-basedthera- trophins (of which NGF is a member), neuropoie- in lethality by the P0 directed decisions, sympathetic precursor cells pies for nervous tins, fibroblast growth factors, and the transforming X-gal staining of a mouse stage of develop- migrate and differentiate into neurons soon after system disor- growth factor-β superfamily. Hideki Enomoto is pri- embryo expressing tau-lacZ ment, birth. Similar they undergo lineage commitment and restriction to ders. under the GFRα1 promoter GFR 1 GFR 2 GFR 3 GFR 4 marily interested in this last group, the GDNF Family neuronal populations a catecholaminergic (sympathetic neurotransmitter- α α α α RET RET RET Ligands (the GFLs) in particular. This family of neu- are affected by Neur- producing) fate. Enomoto found that Artemin is

26 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 27 the production of genetically mosaic flies in which specific olfactory neurons are homozygous for mutations in otherwise heterozygous animals. This screening approach has already yielded approximately 70 mutant lines exhibiting phenotypes in which axons mistar- get, overshoot, arborize improp- erly or fail to project entirely. Some of these mutations are Targeting of olfactory sensory neurons into specific glomeruli in the Staff now being mapped on the chro- antenna lobes mosome by SNP analyses and Team Leader Chihiro Hama the use of chromosomal defi- Research Scientist ciencies. Minako Orihara Keita Endo Kazunaga Takizawa Vesicle transport in neural Hiroko Saito Technical Staff development Yuka Yoda Kyoko Ishikawa All cells contain cargo-bearing compartments known Assistant as vesicles. These vesicles function to deliver the Kanako Moriwaki macromolecules that serve the cells as membrane Publications components or secreted signals, and the metabolite Saito M, Awasaki T and products of the cellular digestive process to the rest Hama C. Genetic analyses of the cell, where they are stored or used to fuel of essential genes in cytologi- Creative Research cal region 61D1-2 to 61F1-2 of Drosophila melanogaster. Promoting Program The Hama lab is interested in Mol Genet Genomics resolving the mechanisms by 268:446-54 (2002). synthesis. The Hama lab's studies indicate that Sle, which olfactory neurons identify Sone M, Suzuki E, Hoshino a novel perinucleolar protein, may modulate nucleo- and home to specific targets M, Hou D, Kuromi H, Fukata lar assembly and consequently promotes the prolifer- M, Kuroda S, Kaibuchi K, Neural networks ral development. Using a research strategy of large- ation of neuroblasts. Nabeshima Y and Hama C. Neural network development involves the orchestra- scale mutant screening in the fly, the Hama activity. Microtubules serve as 'railways' for vesicle Synaptic development is con- tion of complex processes from neuronal differentia- research team seeks to open new windows into the transport; their ends anchor to sites on the cell cor- trolled in the periactive zones Olfactory axon guidance of Drosophila synapses. tion to axon growth to the formation of synapses. The genetic bases of neural circuitry. The Drosophila olfactory sensory system comprises tex where the vesicles fuse. It is likely that the extru- Development 127:4157-68 mechanisms by which neuronal cells are able to iden- around 1,300 neurons that are positioned in the sion of cell membrane at given sites, such as takes (2000). tify and form synapses with appropriate partners, and Slender lobes head appendages and project into the antennal lobe place in axon extension, requires vesicles to be the ways that circuits formed from interconnecting The mushroom body, containing on the order of 2000 of the brain, which contains 43 glomerular synaptic transported along microtubule in a specific orienta- Awasaki T, Saito M, Sone M, Suzuki E, Sakai R, Ito K and neurons are able to demonstrate adaptive plasticity neurons, is the functional center for olfactory leaning tion. To clarify how vesicle transport is involved in clusters of sensory neurons and interneurons. Each Hama C. The Drosophila trio are questions of bewildering intricacy and depth. and memory in Drosophila. Minako Orihara in the individual sensory neuron expresses only one or two neural development, members of the Hama lab are plays an essential role in pat- Hama lab has identified the gene slender lobes (sle), types of the approximately 60 known types of odor- now analyzing Drosophila orthologs of genes encod- terning of axons by regulat- Chihiro Hama Chihiro Hama is working to address these questions whose homozygous mutation results in sterile flies ant receptors, and all neurons expressing the same ing proteins known to be involved in the regulation ing their directional extension. Neuron 26:119-31 Ph. D. through investigations into the neural network of the with thinner axonal bundles and reduced neuron odorant receptor project to the same glomerulus. of vesicular traffic in budding yeast. Hama hopes to (2000). Chihiro Hama received his fruit fly Drosophila, a system which, in spite of its counts in the mushroom body, a phenotype attribut- The Hama lab is interested in resolving the mechan- elucidate the roles of these proteins in neurite exten- B. Sc. and M. Sc. from the comparatively simple structure, demonstrates the able to the failure of flies lacking the sle gene to sus- sion, branching and guidance, and synapse forma- University of Tokyo Depart- isms by which these specialized neurons are able to ment of Biophysics and Bio- capacity for instinctive behaviors, learning and mem- tain the normally high rate of cellular proliferation by identify and home to specific targets over consider- tion as well, by conducting conditional RNAi studies chemistry and was award- ory. While there are profound differences in degrees mushroom body neuroblasts (neural progenitor cells). able distances with such great accuracy during the using a DNA-based transgenic approach. ed a Ph. D from the same institution in 1985 for his of network sophistication and cell type diversity bet- organization of the olfactory network. work on the regulation of ween Drosophila and more highly evolved organ- The protein encoded by the sle gene is found in the This research seeks both to clarify the plasmid ColIb DNA replica- isms such as mammals, they nonetheless share a nuclei of a variety of cells, including neuroblasts, roles of known genetic elements and to tion by inc and repY. He spent the period from 1985 large number of homologous genes important in neu- and is abundant in the perinucleolar region during uncover new pieces needed to solve to 1988 as a post-doc in interphase. Genetic analyses this intricate puzzle through screening the laboratory of Thomas have revealed that a majority of Kornberg at the University for new mutations that affect the projec- of California, San Francisco interphase nucleoli in the mutant tion of olfactory neurons. before returning to Japan to are shifted to a particular state of continue his post-doctoral work at the National Insti- assembly that transiently or rare- Keita Endo in the Hama lab has devel- tute of Neuroscience, ly occurs in the wild type. Elec- oped a system to generate mutant flies NCNP, Tokyo. He tron microscopy has showed that and screen them for olfactory neuron advanced to section chief in the Department of Molec- the nucleoli in mutant neuro- projection phenotypes by visualizing ular Genetics in 1991, and blasts are unusually densely specific projection patterns. The first remained at the NCNP until packed, and are separated from 2001 when he was appoint- step of this approach takes advantage ed to his current position at the perinucleolar heterochroma- of an enhancer trap line that enables the CDB. tins that surround or tightly asso- the forced expression of marker genes ciate with nucleoli in wild type. in specific subsets of olfactory sensory Similar phenotypes were also neurons, making it possible to visualize observed in neurons that are qui- the extension of their axons toward the Mushroom body in the Drosophila brain escent during mitosis and DNA glomeruli. The second phase entails Antenna segment (top arrow) and maxillary palp (bottom arrow); olfactory sensory apparatuses in Drosophila

28 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 29 the production of genetically mosaic flies in which specific olfactory neurons are homozygous for mutations in otherwise heterozygous animals. This screening approach has already yielded approximately 70 mutant lines exhibiting phenotypes in which axons mistar- get, overshoot, arborize improp- erly or fail to project entirely. Some of these mutations are Targeting of olfactory sensory neurons into specific glomeruli in the Staff now being mapped on the chro- antenna lobes mosome by SNP analyses and Team Leader Chihiro Hama the use of chromosomal defi- Research Scientist ciencies. Minako Orihara Keita Endo Kazunaga Takizawa Vesicle transport in neural Hiroko Saito Technical Staff development Yuka Yoda Kyoko Ishikawa All cells contain cargo-bearing compartments known Assistant as vesicles. These vesicles function to deliver the Kanako Moriwaki macromolecules that serve the cells as membrane Publications components or secreted signals, and the metabolite Saito M, Awasaki T and products of the cellular digestive process to the rest Hama C. Genetic analyses of the cell, where they are stored or used to fuel of essential genes in cytologi- Creative Research cal region 61D1-2 to 61F1-2 of Drosophila melanogaster. Promoting Program The Hama lab is interested in Mol Genet Genomics resolving the mechanisms by 268:446-54 (2002). synthesis. The Hama lab's studies indicate that Sle, which olfactory neurons identify Sone M, Suzuki E, Hoshino a novel perinucleolar protein, may modulate nucleo- and home to specific targets M, Hou D, Kuromi H, Fukata lar assembly and consequently promotes the prolifer- M, Kuroda S, Kaibuchi K, Neural networks ral development. Using a research strategy of large- ation of neuroblasts. Nabeshima Y and Hama C. Neural network development involves the orchestra- scale mutant screening in the fly, the Hama activity. Microtubules serve as 'railways' for vesicle Synaptic development is con- tion of complex processes from neuronal differentia- research team seeks to open new windows into the transport; their ends anchor to sites on the cell cor- trolled in the periactive zones Olfactory axon guidance of Drosophila synapses. tion to axon growth to the formation of synapses. The genetic bases of neural circuitry. The Drosophila olfactory sensory system comprises tex where the vesicles fuse. It is likely that the extru- Development 127:4157-68 mechanisms by which neuronal cells are able to iden- around 1,300 neurons that are positioned in the sion of cell membrane at given sites, such as takes (2000). tify and form synapses with appropriate partners, and Slender lobes head appendages and project into the antennal lobe place in axon extension, requires vesicles to be the ways that circuits formed from interconnecting The mushroom body, containing on the order of 2000 of the brain, which contains 43 glomerular synaptic transported along microtubule in a specific orienta- Awasaki T, Saito M, Sone M, Suzuki E, Sakai R, Ito K and neurons are able to demonstrate adaptive plasticity neurons, is the functional center for olfactory leaning tion. To clarify how vesicle transport is involved in clusters of sensory neurons and interneurons. Each Hama C. The Drosophila trio are questions of bewildering intricacy and depth. and memory in Drosophila. Minako Orihara in the individual sensory neuron expresses only one or two neural development, members of the Hama lab are plays an essential role in pat- Hama lab has identified the gene slender lobes (sle), types of the approximately 60 known types of odor- now analyzing Drosophila orthologs of genes encod- terning of axons by regulat- Chihiro Hama Chihiro Hama is working to address these questions whose homozygous mutation results in sterile flies ant receptors, and all neurons expressing the same ing proteins known to be involved in the regulation ing their directional extension. Neuron 26:119-31 Ph. D. through investigations into the neural network of the with thinner axonal bundles and reduced neuron odorant receptor project to the same glomerulus. of vesicular traffic in budding yeast. Hama hopes to (2000). Chihiro Hama received his fruit fly Drosophila, a system which, in spite of its counts in the mushroom body, a phenotype attribut- The Hama lab is interested in resolving the mechan- elucidate the roles of these proteins in neurite exten- B. Sc. and M. Sc. from the comparatively simple structure, demonstrates the able to the failure of flies lacking the sle gene to sus- sion, branching and guidance, and synapse forma- University of Tokyo Depart- isms by which these specialized neurons are able to ment of Biophysics and Bio- capacity for instinctive behaviors, learning and mem- tain the normally high rate of cellular proliferation by identify and home to specific targets over consider- tion as well, by conducting conditional RNAi studies chemistry and was award- ory. While there are profound differences in degrees mushroom body neuroblasts (neural progenitor cells). able distances with such great accuracy during the using a DNA-based transgenic approach. ed a Ph. D from the same institution in 1985 for his of network sophistication and cell type diversity bet- organization of the olfactory network. work on the regulation of ween Drosophila and more highly evolved organ- The protein encoded by the sle gene is found in the This research seeks both to clarify the plasmid ColIb DNA replica- isms such as mammals, they nonetheless share a nuclei of a variety of cells, including neuroblasts, roles of known genetic elements and to tion by inc and repY. He spent the period from 1985 large number of homologous genes important in neu- and is abundant in the perinucleolar region during uncover new pieces needed to solve to 1988 as a post-doc in interphase. Genetic analyses this intricate puzzle through screening the laboratory of Thomas have revealed that a majority of Kornberg at the University for new mutations that affect the projec- of California, San Francisco interphase nucleoli in the mutant tion of olfactory neurons. before returning to Japan to are shifted to a particular state of continue his post-doctoral work at the National Insti- assembly that transiently or rare- Keita Endo in the Hama lab has devel- tute of Neuroscience, ly occurs in the wild type. Elec- oped a system to generate mutant flies NCNP, Tokyo. He tron microscopy has showed that and screen them for olfactory neuron advanced to section chief in the Department of Molec- the nucleoli in mutant neuro- projection phenotypes by visualizing ular Genetics in 1991, and blasts are unusually densely specific projection patterns. The first remained at the NCNP until packed, and are separated from 2001 when he was appoint- step of this approach takes advantage ed to his current position at the perinucleolar heterochroma- of an enhancer trap line that enables the CDB. tins that surround or tightly asso- the forced expression of marker genes ciate with nucleoli in wild type. in specific subsets of olfactory sensory Similar phenotypes were also neurons, making it possible to visualize observed in neurons that are qui- the extension of their axons toward the Mushroom body in the Drosophila brain escent during mitosis and DNA glomeruli. The second phase entails Antenna segment (top arrow) and maxillary palp (bottom arrow); olfactory sensory apparatuses in Drosophila

28 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 29 Hibi and colleagues set out to clone the ogon gene Neurogenesis (which is also known as mercedes and short tail), in Vertebrate neuronal tissues are order to determine its precise relationship with Chor- generated in a stepwise fashion. din, and the molecular bases for its action in regulat- These steps include neural inducing the establishment of the dorsal-ventral axis. The tion, antero-posterior patterning, results of that positional cloning showed that ogon and neurogenesis. In amphibian encodes a homolog of the Secreted Frizzled (Siz- and teleost (bony fish) embryos, zled) protein, which has similarities to the Wnt recep- neuroectoderm is induced by BMP inhibitors derived from organizing Organizing centers, centers. Induced neuroectoderm is by default anterior in character, but which emerge soon after a subset in some tissues is subse- fertilization, play pivotal roles in quently subject to posteriorizing setting up positional axes transformations. The hindbrain and in the zebrafish embryo spinal cord, which are posteriorized regions, are specified by a signal from a region known as non- Staff tor, Frizzled. The canonical Wnt pathway is known axial mesendoderm, while anterior neuroectoderm Team Leader Masahiko Hibi to play a role in axis formation, but it has been sug- that does not receive this posteriorizing signal devel- Research Scientist gested that ogon/sizzled influences the dorsal- ops into fore- and midbrain. After these initial pro- Osamu Muraoka Takashi Shimizu ventral axis independently of its effect cesses of neural induction and patterning, neurogen- Taijiro Yabe on Wnt. To test this idea, members of ic regions, the domains in which neurogenesis takes Visiting Scientist Young-Ki Bae the Hibi lab compared the activity of place, are established. Tsutomu Hirata Technical Staff Ogon/Sizzled with that of Wnt inhibi- Chiho Fukae tors in ectopic expression studies, The Hibi lab has been working to identify genes Yukiko Kamimura Hanae Akiyama and found that the effects of involved in the control of this patterning and neuro- Student Trainee Creative Research Ogon/Sizzled misexpression genesis, focusing on two genes in particular: the Hideaki Nojima Assistant Promoting Program resembled those of BMP, zinc finger gene fez-like expressed in the fore- Shoko Muraoka but not Wnt, inhibitors, brain, and the posterior neuron-specific suggesting an excep- homeobox gene pnx. In collaborative tional mode of work, they showed that fez-like is action. required for the formation of hypotha- Vertebrate embryogenesis begins with a seemingly In work published in 2003, the Hibi lab analyzed and lamic monoaminergic neurons in formless egg that, through processes of division, isolated the responsible gene for a mutant named They next looked zebrafish. These neurons produce growth, differentiation, migration and rearrange- ogon, which displays ventralized phenotypes, and at the possible neurotransmitters such as dopamine ment, rapidly gives rise to a highly organized struc- found that the ogon locus encodes a zebrafish involvement of and serotonin, which are centrally ture characterized by a number of definitive axes. homologue of the protein Secreted Frizzled (Siz- Chordin (which is important to the regulation of mood, This transformation is particularly striking and rapid zled), which functions as negative feedback regula- known to be essen- behavior, endocrine and cognitive Publications in the zebrafish (Danio rerio), which in the space of tor of BMP signaling. While numerous mutations are tial in the process of functions in humans. Levkowitz G, Zeller J, Sirot- a single day develops from an ovum to a recogniz- known to result in dorsalized phenotypes, ogon is dorsalization) in kin H I, French D, Schilbach ably vertebrate body laid down in respect to multiple only the second zebrafish mutant reported to show Ogo/Szl’s effects by A separate study identified a homeo- S, Hashimoto H, Hibi M, Tal- polar vectors. The speed of its growth and reproduc- clearly ventralized phenotypes, such as expanded injecting ogon/sizzled box gene, pnx, involved in the devel- bot W S and Rosenthal A. tion, coupled with its amenability to genetic studies ventral tail fins, blood, pronephron, and other posteri- RNA into mutants (chordi- opment of posterior neurons. This Zinc finger protein too few Masahiko Hibi controls the development of M. D. , Ph. D. and the revealing pellucidity of its embryo, make this or structures. These patterns are similar to the pheno) lacking a functional gene, which is regulated by a signal monoaminergic neurons. Nat Masahiko Hibi received his organism an ideal system for the study of axis forma- notypes observed in mutations of the gene for the chordin gene. This from the posteriorizing non-axial Neurosci 6:28-33 (2003). M. D. from Hiroshima Uni- tion in early vertebrate development. BMP antagonist, Chordin, which suggested that failed to rescue the ven- mesendoderm and Notch signaling, versity, School of Medicine Bae Y K, Shimizu T, Yabe T, in 1988, and his Ph. D. Ogon might also be involved in BMP inhibition. tralized phenotype of The maternal-effect mutant acts as a transcriptional repressor. Kim CH, Hirata T, Nojima H, from the Osaka University Regulating the homozygous chordino tokkaebi displays a completely Misexpression of pnx results in an Institute for Molecular and ventralized phenotype. Muraoka O, Hirano T and Cellular Biology in 1992. dorsal-ventral axis mutants, indicating that increase in neural precursor cells Hibi M. A homeobox gene, From 1992 to 1995, he Masahiko Hibi’s laboratory focuses on the molecular Ogon/Sizzled activity and neurons, while its repression pnx, is involved in the forma- worked as a postdoctoral genetic cascade of events stemming from organiz- depends on Chordin. caused reductions in certain population of posterior neurons in fellow in Michael Karin’s lab ing centers in the zebrafish embryo. These centers, The detailed functional tions of posterior neurons, indicating zebrafish. Development in the University of Califor- 130:1853-65 (2003). nia, San Diego Department which emerge soon after fertilization, play pivotal relationship between that the gene participates in posteri- of Pharmacology, then roles in setting up positional axes; Hibi’s lab is par- these two dorsalizing fac- or neurogenesis. Yabe T, Shimizu T, Muraoka returned to Japan to take an assistant professorship ticularly interested in the formation of the dorsal- tors remains to be O, Bae Y K, Hirata T, Nojima in the Division of Molecular ventral (back-belly) axis, an essential step in the worked out, but these ini- H, Kawakami A, Hirano T Oncology at Osaka Univers- and Hibi M. Ogon/Secreted establishment of the body plan. An embryonic tial findings suggest that ity Medical School. He was Frizzled functions as a nega- appointed associate profes- region known in zebrafish as the dorsal organizer Ogon/Sizzled augments tive feedback regulator of sor in the same division in guides dorsoventral patterning by generating induc- Chordin activity either by Bmp signaling. Development 1999, where he remained 130:2705-16 (2003). until he assumed his positive signals, including inhibitors of BMPs and Wnts inhibiting a Chordin tion as team leader at the (molecules which ultimately determine dorsal and inhibitor, by upregulating RIKEN CDB. Shimizu T, Yamanaka Y, ventral fates). These molecules play roles in a com- Chordin activity, or by Nojima H, Yabe T, Hibi M plex network of axis-determining factors, and the making BMP more sensi- and Hirano T. A novel interplay between dorsalizing and ventralizing fac- tive to Chordin’s inhibi- repressor-type homeobox tors is central to the formation of the dorsal meso- tory effects. gene, ved, is involved in dharma/bozozok-mediated derm and endoderm, and the neuroectoderm. dorsal organizer formation in zebrafish. Mech Dev 118:125-38 (2002). Pnx is required for the formation of motoneurons. ogon mutant and ogon-overexpressing embryos. Ogon functions as a negative feedback regulator of BMP signaling

30 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 31 Hibi and colleagues set out to clone the ogon gene Neurogenesis (which is also known as mercedes and short tail), in Vertebrate neuronal tissues are order to determine its precise relationship with Chor- generated in a stepwise fashion. din, and the molecular bases for its action in regulat- These steps include neural inducing the establishment of the dorsal-ventral axis. The tion, antero-posterior patterning, results of that positional cloning showed that ogon and neurogenesis. In amphibian encodes a homolog of the Secreted Frizzled (Siz- and teleost (bony fish) embryos, zled) protein, which has similarities to the Wnt recep- neuroectoderm is induced by BMP inhibitors derived from organizing Organizing centers, centers. Induced neuroectoderm is by default anterior in character, but which emerge soon after a subset in some tissues is subse- fertilization, play pivotal roles in quently subject to posteriorizing setting up positional axes transformations. The hindbrain and in the zebrafish embryo spinal cord, which are posteriorized regions, are specified by a signal from a region known as non- Staff tor, Frizzled. The canonical Wnt pathway is known axial mesendoderm, while anterior neuroectoderm Team Leader Masahiko Hibi to play a role in axis formation, but it has been sug- that does not receive this posteriorizing signal devel- Research Scientist gested that ogon/sizzled influences the dorsal- ops into fore- and midbrain. After these initial pro- Osamu Muraoka Takashi Shimizu ventral axis independently of its effect cesses of neural induction and patterning, neurogen- Taijiro Yabe on Wnt. To test this idea, members of ic regions, the domains in which neurogenesis takes Visiting Scientist Young-Ki Bae the Hibi lab compared the activity of place, are established. Tsutomu Hirata Technical Staff Ogon/Sizzled with that of Wnt inhibi- Chiho Fukae tors in ectopic expression studies, The Hibi lab has been working to identify genes Yukiko Kamimura Hanae Akiyama and found that the effects of involved in the control of this patterning and neuro- Student Trainee Creative Research Ogon/Sizzled misexpression genesis, focusing on two genes in particular: the Hideaki Nojima Assistant Promoting Program resembled those of BMP, zinc finger gene fez-like expressed in the fore- Shoko Muraoka but not Wnt, inhibitors, brain, and the posterior neuron-specific suggesting an excep- homeobox gene pnx. In collaborative tional mode of work, they showed that fez-like is action. required for the formation of hypotha- Vertebrate embryogenesis begins with a seemingly In work published in 2003, the Hibi lab analyzed and lamic monoaminergic neurons in formless egg that, through processes of division, isolated the responsible gene for a mutant named They next looked zebrafish. These neurons produce growth, differentiation, migration and rearrange- ogon, which displays ventralized phenotypes, and at the possible neurotransmitters such as dopamine ment, rapidly gives rise to a highly organized struc- found that the ogon locus encodes a zebrafish involvement of and serotonin, which are centrally ture characterized by a number of definitive axes. homologue of the protein Secreted Frizzled (Siz- Chordin (which is important to the regulation of mood, This transformation is particularly striking and rapid zled), which functions as negative feedback regula- known to be essen- behavior, endocrine and cognitive Publications in the zebrafish (Danio rerio), which in the space of tor of BMP signaling. While numerous mutations are tial in the process of functions in humans. Levkowitz G, Zeller J, Sirot- a single day develops from an ovum to a recogniz- known to result in dorsalized phenotypes, ogon is dorsalization) in kin H I, French D, Schilbach ably vertebrate body laid down in respect to multiple only the second zebrafish mutant reported to show Ogo/Szl’s effects by A separate study identified a homeo- S, Hashimoto H, Hibi M, Tal- polar vectors. The speed of its growth and reproduc- clearly ventralized phenotypes, such as expanded injecting ogon/sizzled box gene, pnx, involved in the devel- bot W S and Rosenthal A. tion, coupled with its amenability to genetic studies ventral tail fins, blood, pronephron, and other posteri- RNA into mutants (chordi- opment of posterior neurons. This Zinc finger protein too few Masahiko Hibi controls the development of M. D. , Ph. D. and the revealing pellucidity of its embryo, make this or structures. These patterns are similar to the pheno) lacking a functional gene, which is regulated by a signal monoaminergic neurons. Nat Masahiko Hibi received his organism an ideal system for the study of axis forma- notypes observed in mutations of the gene for the chordin gene. This from the posteriorizing non-axial Neurosci 6:28-33 (2003). M. D. from Hiroshima Uni- tion in early vertebrate development. BMP antagonist, Chordin, which suggested that failed to rescue the ven- mesendoderm and Notch signaling, versity, School of Medicine Bae Y K, Shimizu T, Yabe T, in 1988, and his Ph. D. Ogon might also be involved in BMP inhibition. tralized phenotype of The maternal-effect mutant acts as a transcriptional repressor. Kim CH, Hirata T, Nojima H, from the Osaka University Regulating the homozygous chordino tokkaebi displays a completely Misexpression of pnx results in an Institute for Molecular and ventralized phenotype. Muraoka O, Hirano T and Cellular Biology in 1992. dorsal-ventral axis mutants, indicating that increase in neural precursor cells Hibi M. A homeobox gene, From 1992 to 1995, he Masahiko Hibi’s laboratory focuses on the molecular Ogon/Sizzled activity and neurons, while its repression pnx, is involved in the forma- worked as a postdoctoral genetic cascade of events stemming from organiz- depends on Chordin. caused reductions in certain population of posterior neurons in fellow in Michael Karin’s lab ing centers in the zebrafish embryo. These centers, The detailed functional tions of posterior neurons, indicating zebrafish. Development in the University of Califor- 130:1853-65 (2003). nia, San Diego Department which emerge soon after fertilization, play pivotal relationship between that the gene participates in posteri- of Pharmacology, then roles in setting up positional axes; Hibi’s lab is par- these two dorsalizing fac- or neurogenesis. Yabe T, Shimizu T, Muraoka returned to Japan to take an assistant professorship ticularly interested in the formation of the dorsal- tors remains to be O, Bae Y K, Hirata T, Nojima in the Division of Molecular ventral (back-belly) axis, an essential step in the worked out, but these ini- H, Kawakami A, Hirano T Oncology at Osaka Univers- and Hibi M. Ogon/Secreted establishment of the body plan. An embryonic tial findings suggest that ity Medical School. He was Frizzled functions as a nega- appointed associate profes- region known in zebrafish as the dorsal organizer Ogon/Sizzled augments tive feedback regulator of sor in the same division in guides dorsoventral patterning by generating induc- Chordin activity either by Bmp signaling. Development 1999, where he remained 130:2705-16 (2003). until he assumed his positive signals, including inhibitors of BMPs and Wnts inhibiting a Chordin tion as team leader at the (molecules which ultimately determine dorsal and inhibitor, by upregulating RIKEN CDB. Shimizu T, Yamanaka Y, ventral fates). These molecules play roles in a com- Chordin activity, or by Nojima H, Yabe T, Hibi M plex network of axis-determining factors, and the making BMP more sensi- and Hirano T. A novel interplay between dorsalizing and ventralizing fac- tive to Chordin’s inhibi- repressor-type homeobox tors is central to the formation of the dorsal meso- tory effects. gene, ved, is involved in dharma/bozozok-mediated derm and endoderm, and the neuroectoderm. dorsal organizer formation in zebrafish. Mech Dev 118:125-38 (2002). Pnx is required for the formation of motoneurons. ogon mutant and ogon-overexpressing embryos. Ogon functions as a negative feedback regulator of BMP signaling

30 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 31 Turing mechanism in zebrafish stripes Publications The zebrafish (Danio rerio) is strikingly patterned in Suzuki N, Hirata M and Kon- bands of light and dark, lending it the zebra-like do S. Traveling stripes on the skin of a mutant mouse. appearance for which it is named. This striped effect Proc Natl Acad Sci U S A is achieved by the tightly regulated distribution of 100:9680-5 (2003). three types of pigment cells (chromatophores) in the hypodermis — xanthophores, which produce yellow Shoji H, Mochizuki A, Iwasa pigment, melanophores, which produce black, and Y, Hirata M, Watanabe T, Hioki S and Kondo S. Origin iridophores, which have a glittering appearance. Iri- of directionality in the fish dophores are evenly distributed, but xanthophores stripe pattern. Dev Dyn and melanophores appear in mutually exclusive 226:627-33 (2003). bands, which appear as stripes in the adult fish. Not- Staff Hirata M, Nakamura K, Kane- ing resemblances between zebrafish stripe develop- maru T, Shibata Y and Kon- Team Leader Shigeru Kondo ment and mathematical patterns generated by the do S. Pigment cell Research Scientist Turing mechanism, Kondo speculated that a organization in the hypoder- Masashi Hirata mis of zebrafish. Dev Dyn Jun Ozaki reaction-diffusion effect might be at work. To test Motoomi Yamaguchi this hypothesis, he used lasers to ablate specific 227:497-503 (2003). Technical Staff Miwako Okamura populations of pigment cells and observed their Kana Bando re-growth. When a band of xanthophores or mela- Student Trainee Takeshi Nakagawa nophores is ablated, small precursor cells are gener- Motoko Yoshizawa (Iwashita) Assistant ated to replace the cells that have been lost. The ini- Sumire Hino tial number of replacement cells that arise is in excess of the final number, indicating that some of Creative Research Promoting Program

this follicular attrition and the subsequent re-activation by neigh- The patterning of the Traveling wave mice boring follicles produces natural world In research published this year, Kondo identified a a phenotype in which the The question of how complex patterns arise from mathematical similarity between a well- mouse's skin color at first uniform- seemingly disorganized or formless initial structures characterized reaction that produces wave-like pat- ly oscillates between dark and light colora- represents an intriguing challenge to mathemati- terns in chemical media and a pattern-forming phe- tion, then begins to take on a remarkable striped The zebrafish skin pattern is generated cians, physicists, chemists and biologists alike. The- nomenon in the skin of a mutant mouse. These by combinations of cells producing vari- appearance, with bands of pigmentation that origi- ous pigments oretical work indicates that the mechanisms underly- mice, which have defects in a gene responsible for nate from the region under the forearms and travel ing pattern formation are similar in both biological hair follicle development, develop bands of dark- across the body surface in all directions. These the precursors die off during the process of regener- and non-biological systems, and a number of ened skin that traverse the body surface in waves. waves first appear at about three months after birth, ation. And while in principle any type of chroma- mathematical models capable of describing pattern Kondo, working in collaboration with researchers and continue to arise and propagate throughout the tophore can grow at any location in the skin, generation in chemical media have been proposed, from the Medical School of Mie University, showed life of the animal. xanthophore bands regenerate only xanthophores Shigeru Kondo but the greater complexity of living systems has that these traveling waves of skin coloration are strik- and melanophores only melanophores, suggesting Ph. D. made it much more difficult to demonstrate a ingly similar to nonlinear waves produced by the Kondo identified similarities that cells of both types inhibit the growth of their Shigeru Kondo received his mathematical basis for biological patterns. In 1952, Belousov-Zhabotinskii (BZ) reaction in chemical sys- counterparts. Using mathematical simulations, Kon- doctorate from the Kyoto between wave-like patterns the British mathematician Alan Turing proposed a tems, suggesting a shared underlying principle. The do developed a model to account for this banded University Faculty of Medi- observed in chemical media and cine in 1988. He spent the simple mathematical equation capable of generating research began when Kondo learned of a mutant skin patterning, in which chromatophores of each period from 1989 to 1990 a pattern-forming phenomenon in as a postdoctoral fellow in a wide range of patterns commonly found in the nat- strain of mouse with an unusual striped phenotype. type produce both short-range inhibitors of pigment Masami Muramatsu’s lab at ural world, such as stripes, spots and reticulations. The mutation, a splicing defect in the Foxn1 (Whn or the skin of a mutant mouse cells of the same type and long-range inhibitors of the University of Tokyo, This model, known as the reaction-diffusion model, nude) gene, causes hair follicle development to ter- the complementary cell type. When mathematical before taking an overseas fellowship at the Basel Uni- demonstrates that the interaction between a local minate just after skin pigments begin to accumulate. By tracking the movements of traveling waves in the simulations using versity Biocenter under activator and a long-range inhibitor can give rise to The immature follicles die off and are quickly Foxn1 mouse skin pattern, Kondo ascertained that inhibitors of varying Walter Gehring. He various periodic structures in response to differenc- replaced by a new hair cycle. The cyclical nature of their formation was fundamentally similar to that of strengths of diffusion returned to Kyoto University in 1994 as assistant profes- es in their individual diffusion rates. waves generated by the BZ reaction. Although this rates are performed, sor, where he remained finding does not directly identify the specific molecu- this double negative until 1998, when he was appointed professor in the Shigeru Kondo is interested in demonstrating the lar mechanism at work in the Foxn1 phenotype, feedback relationship Tokushima University Facul- mathematical basis of pattern formation in develop- mathematical analysis suggests a candidate mol- gives rise to a range ty of Integrated Arts and Sci- ment, and using mathematical models as predic- ecule, Shh, which satisfies the necessary criteria for of Turing patterns simi- ences. Kondo was appointed CDB team leader in tive tools to aid in the identification of genes a molecule key to traveling wave pattern formation. lar to skin patterns 2001. and molecules involved in the generation of And, as many other species demonstrate similar seen in nature, a find- spatial structures. Research in the Kondo striping mechanisms, this report may help to provide ing that provides new lab focuses on skin surface and morpho- biologists with a mathematical model to explain this avenues of inquiry for genetic patterning, both of which feature skin patterning phenomenon. investigators studying prominent examples of periodic struc- the molecular mechan- tures that can be described in terms of isms of pattern forma- Altered striping in zebrafish skin three weeks after chromatopohore ablation standing and moving waves. tion in biological systems. Traveling wave skin pattern of the newly isolated mutant traveling wave (tw) mouse. Each wave arises at the base of the fore- limb, and moves like a wave spreading on the surface of water.

32 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 33 Turing mechanism in zebrafish stripes Publications The zebrafish (Danio rerio) is strikingly patterned in Suzuki N, Hirata M and Kon- bands of light and dark, lending it the zebra-like do S. Traveling stripes on the skin of a mutant mouse. appearance for which it is named. This striped effect Proc Natl Acad Sci U S A is achieved by the tightly regulated distribution of 100:9680-5 (2003). three types of pigment cells (chromatophores) in the hypodermis — xanthophores, which produce yellow Shoji H, Mochizuki A, Iwasa pigment, melanophores, which produce black, and Y, Hirata M, Watanabe T, Hioki S and Kondo S. Origin iridophores, which have a glittering appearance. Iri- of directionality in the fish dophores are evenly distributed, but xanthophores stripe pattern. Dev Dyn and melanophores appear in mutually exclusive 226:627-33 (2003). bands, which appear as stripes in the adult fish. Not- Staff Hirata M, Nakamura K, Kane- ing resemblances between zebrafish stripe develop- maru T, Shibata Y and Kon- Team Leader Shigeru Kondo ment and mathematical patterns generated by the do S. Pigment cell Research Scientist Turing mechanism, Kondo speculated that a organization in the hypoder- Masashi Hirata mis of zebrafish. Dev Dyn Jun Ozaki reaction-diffusion effect might be at work. To test Motoomi Yamaguchi this hypothesis, he used lasers to ablate specific 227:497-503 (2003). Technical Staff Miwako Okamura populations of pigment cells and observed their Kana Bando re-growth. When a band of xanthophores or mela- Student Trainee Takeshi Nakagawa nophores is ablated, small precursor cells are gener- Motoko Yoshizawa (Iwashita) Assistant ated to replace the cells that have been lost. The ini- Sumire Hino tial number of replacement cells that arise is in excess of the final number, indicating that some of Creative Research Promoting Program

32 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 33 Embryonic developmental stages in Chinese soft-shelled turtle

neurons develop in conserved rhombomere-specific positions, similar to those observed in the gnathostome zebrafish. Interestingly, in lamprey the positions of other sets of hindbrain neurons — the trige- Staff minal and facial motor nuclei — do not map neatly to rhombomeric borderlines, as they do in gnathos- Team Leader Student Trainee Shigeru Kuratani Yoko Takio tomes. Rather, the trigeminal-facial nerve originates Research Scientist Hiroshi Nagashima in the middle of rhombomere 4, in the region of Yoshie Kawashima Shigehiro Kuraku Katsuhisa Uchida Assistant expression of the lamprey Hox gene LjHox3. When Masumi Nakazawa Chiaki Nakayama retinoic acid (which is known to alter Hox gene Yuuji Narita Rie Kusakabe expression and associated developmental pro- Yasunori Murakami grams) was introduced to the developing hindbrain Technical Staff Ryo Usuda region, it caused positional shifts of both LjHox3 expression and branchiomotor nuclei, but no apparent changes in segmentation or the positions of retic- Creative Research ular neurons. Promoting Program Nature prefers to repurpose or tinker with Publications tionary history, as the underlying genetic elements existing genes rather than to Murakami Y, Pasqualett M, are presumed to be conserved, making carapace for- introduce novelties ex nihilo Takio Y, Hirano S, Riiji F and mation one of the trajectories available to the pro- Kuratani S. Segmental devel- Evo-devo of gradual evolution in incremental steps. Such a cess of morphological development. opment of reticulospinal and Darwin conceived evolution as a process driven by phenomenon is difficult to explain by mutation and These findings indicate that, in the lamprey, hind- branchiomotor neuros in the lamprey:insights into evolu- the interplay between random mutation and natural selection alone, as it is improbable that the genomic brain neural identities and rhombomeric segmenta- In other closely related species, ribs are entirely tion of the vertebrate hind- selection, but the advent of molecular developmen- alterations necessary to produce such a dramatical- internal and reside interior to the scapulae (corre- tion are governed by independent mechanisms, pro- brain. Development (2004). tal biology in the 1980s led to the recognition that ly new bodily structure would be achievable by the sponding to the shoulder blades in humans). In the viding strong counter-evidence to one prevailing inherent genetic constraints also have a role in defin- introduction of new genes in the short timeframe of turtle, however, the rib bones grow exterior to the model that suggests that the establishment of neu- Kusakabe R, Tochinai S and Kuratani S. Expression of for- ing the trajectories that evolutionary processes are the turtle's emergence. Evo-devo (evolutionary scapulae to form the external carapace. Using ronal identity is a Hox-dependent process. Based on eign genes in lamprey most likely to follow. The homeobox (Hox) genes, a developmental biology) theory predicts instead that cross-species explantation, cDNA library and cell their discoveries, embryos: an approach to stu- developmentally important set of genes provide one the genes responsible for carapace development sorting studies, researchers in the Kuratani lab have Kuratani et al. off- dy evolutionary changes in instantiation of this concept; they play central roles actually belong to a set of genes shared by groups identified a number of developmental switching er an alternate gene regulation. J Exp Zool- Shigeru Kuratani in regulating the morphological development of related to the turtle, but which function distinctly in points that enable this exteriorization of this portion model in which og Part B Mol Dev Evol 296:87-97 (2003). Ph. D. organisms ranging from yeast and plants to animals the unique context of the turtle's molecular-genetic of the turtle endoskeleton. Their findings indicate the positional Shigeru Kuratani received from every branch of the phylogenetic tree and network. This would also explain the independent that alterations in a number of essentially conserved concurrence bet- Kuratani S. Evolutionary his M. S. and Ph. D. from share a highly conserved domain (the 180-base-pair appearance of the carapace at other points in evolu- ween Hox deveropmental biology and the Kyoto University Depart- genes, rather than genetic novelty, have resulted in ment of Zoology. He spent homeodomain) that indicates a non-random mode of the turtle's unique anatomical phenotype. expression, vertebrate head segmenta- the period from 1988 to evolutionary selection. rhombomere tion:a perspective from dever- 1991 working in experimen- opmental constraint. Theory tal embryology in the Hindbrain evolution identity and spe- Biosciences 122:230-51 Department of Anatomy at Combining experimental and analytic techniques In vertebrates, the hindbrain is a segmented struc- cific subsets of (2003). the Medical College of from molecular biology, phylogenetics and compara- ture, subdivided into clearly demarcated units called hindbrain neu- Georgia before moving to Kuratani S. Evolution of the the Biochemistry Depart- tive morphology, Shigeru Kuratani seeks to deepen rhombomeres, which generate specific sets of neurons is the result vertebrate jaw - homology ment, Baylor College of the understanding of the part played by developmen- of a convergent rons. The lancelet Amphioxus, a more primitive chor- and developmental con- Medicine, where he was tal biological mechanisms, such as the Hox genes, process in which engaged in molecular date, however, lacks this hindbrain segmentation. straints. Paleontol. Res 7:89- embryological research. He in the divergence of species. His approach involves The lamprey, a jawless fish that arose in the interval originally inde- 102(2003). returned to Japan in 1994 examining related genes in phylogenetically distinct between non-vertebrate chordates (such as pendent mechan- to take a position as asso- Kuratani S. The heterotopic animals to uncover the ways in which context affects Amphioxus) and gnathostomes (jawed animals), isms became ciate professor at the Insti- shift in developmental pat- tute of Medical Embryology The rib grows laterally towards the edge gene expression, and thereby influences body devel- provides a relevant model for studying the emer- linked over evolu- terns and evolution of the and Genetics in the Kuma- of the body in the turtle embryo. opment. By comparing the molecular bases of the gence of the hindbrain developmental plan. tionary time. jaw in vertebrates. Springer moto University School of Medicine. He moved to emergence of discrete structures, Kuratani hopes to Verlag (2003). Okayama University to illustrate the means by which developmental In work published in 2004, investigators in the Kura- Studies such as these underscore a pair of evo- Uchida K, Murakami Y, Kura- assume a professorship in mechanisms mediate the translation of changes in devo precepts: that molecular designs capable of the Department of Biology tani lab labeled reticulospinal and branchial motor ku S, Hirano S and Kuratani in 1997, where he the genome to changes in morphology. neurons (which derive from rhombomeres) to reveal supporting viable ontogenies tend to act as magnets S. Development of the adeno- remained until he was the neuronal organization of the hindbrain of the for convergent evolution, and that Nature is parsimo- hypophysis in the lamprey: appointed team leader at evolution of the epigenetic the CDB. The turtle's shell Japanese lamprey, Lethenteron japonicum, and nious with her creations, preferring to repurpose or patterning programs in organ- The abrupt advent of the turtle's shell, or 'carapace,' studies the expression patterns of rhombomere- tinker with existing genes rather than to introduce ogenesis. J Exp Zool (Mol which appeared with few recognizable precursors, specific genes. They found that lamprey reticular novelties ex nihilo. Dev Evol) 300B:32-47(2003). provides a remarkable exception to the general rule Removal of carapacial ridge results in a characteristic fan-shape splaying of turtle ribs.

34 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 35 Embryonic developmental stages in Chinese soft-shelled turtle

34 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 35 Sensory cellular differentiation The conversion of uncommitted precursor cells in Publications the otic placode to differentiated, functional otic sub- Ladher R K & Schoenwolf G types is very precisely timed. If it occurs premature- C in Developmental Neuro- ly, the resulting organ is underdeveloped, with too biology (ed. Rao, M. H.) few cells forming the adult structure. If it happens Kluwer (2003). too late, there is a danger that these cells will still be immature and unable to fulfill their needed roles in Francis-West P H, Ladher R K and Schoenwolf G C. subsequent developmental processes. An example Development of the sensory of this temporal regulation is seen in the wiring of organs. Sci Prog 85:151-73 the inner ear to the brain, which requires the forma- (2002). tion of neurons from the otic placode. If these cells Ladher R K, Anakwe K U, are not formed when this process occurs, the func- Gurney A L, Schoenwolf G C tion of the auditory system is compromised. But Staff and Francis-West P H. Identi- what is the nature of this control? By using embryo- fication of synergistic signals Team Leader logical studies, combined with gene manipulation initiating inner ear develop- Raj Ladher ment. Science 290:1965-7 Research Scientist and ex-ovo culture, Raj's lab is addressing this ques- (2000). Michael F Teraoka Escaño tion, finding the basis for this precise regulation in Technical Staff Pabel Antonio Delgado the functional characteristics of specific signaling Susan Boerner Assistant molecules. Their control and effects on the develop- Lio Hiroi ment of the inner ear represents one of the most exciting aspects of research in Raj's lab, with implications in regenerative technologies and tissue Creative Research engineering-based therapeutics. Promoting Program

duration of early ear development. Signaling interactions, acting upon the cell surface, trigger a series of transduction events that are interpreted in the Developing sense organs opmental program initiation, cell differentiation and nucleus of the responding cell as transcription fac- The embryo provides many examples of elegant the integration of discrete cellular motifs in structural- tors. Raj's lab is characterizing the transcriptional micro-engineering, wedding intricate organic struc- ly and neuronally specialized organs. response to the different signals that act to collec- tures with specialized cellular function. The sensory tively specify the inner ear. By understanding their organs provide a particularly good example of this Signals in inner ear induction function, Raj hopes to be able to determine a sophistication. By initiating in the correct place, differ- Sense organs originate in placodes, thickened mechanism of molecular synergy. entiating into the correct subtypes and finally integrat- regions within the early ectoderm. The inner ear arising with structures of diverse origin to form a sensory es from the otic placode, a cluster of cells which Morphogenesis of the complex, these structures permit the animal to appre- ultimately gives rise to the mature inner ear. Raj is otic placode hend its environment by processing a range of physi- studying the inductive factors that dictate the loca- Although the adult inner ear is located in the interior Raj Ladher tion and timing of these developmental processes. of the animal it is nonetheless an ectodermal struc- Ph. D. In a series of tissue manipulation experiments con- ture, originating from a placode embedded within Raj Ladher received his B. ducted previously, he identified a trio of signaling fac- the outermost layer of the embryo. Through a pro- Sc. in biochemistry from tors — FGF-8, FGF-19, and Wnt-8c — each of Imperial College, London in cess known as invagination, the otic placode pinch- 1992 and his Ph. D. in which localizes in a separate germ layer, and which es off from the ectoderm. This represents one of the developmental biology from cooperate to induce inner ear development. In this the National Institute for Medical Research in 1996, network, it appears that FGF-8, expressed in the Raj hopes to reconcile for his thesis on the cloning endodermal layer, induces the expression of FGF- the events that occur Nuclei marked using DAPI (blue), of the Xenopus homeobox 19 in the immediately overlying mesoderm. FGF-19 Development of microtubules marked using phalloi- gene, Xom, under the outside the cell with in turn induces Wnt-8c in the neuroectoderm, trigger- other sensory organs din (green). A section through a supervsion of Jim Smith. the biological changes chick embryo at stage 17 shows the He worked as a research ing a complex regulatory loop in which FGF-19 and As well as the inner ear, Raj's lab is also investigating morphogenesis of the otic placode associate at King’s College, Wnt-8c maintain each other's expression for the occurring within the development of other sensory organs, in particu- during invagination. This results London from 1996 to 2000 lar the nose and the eye. Using embryological manip- from a dynamic rearrangement of in the lab of Pip Francis- subcellular components. West, and first came to Expression of Fgf-19 is in purple. Shown is a section through a stage first cellular consequences of inner ear induction, 22 embryo. Fgf-19 is expressed in the condensing acoustic ganglia at ulations, the Lab for Sensory Development is map- Japan during that period as and Raj has focused on this process to understand a visiting scientist at the these stages, suggesting a role in the later development of the inner ping the tissues that are responsible for the formation Tokyo Medical and Dental ear as well as in its induction. the functional significance of signaling factors and and differentiation of these structures. Though still in University. In 2000, he their transcriptional responses. Working from the their infancy, these research projects do indicate that moved to the University of hypothesis that extracellular cues choreograph this Utah as a postdoctoral fel- cal and chemical stimuli. The development of these tissue interactions distinct from each other and from low in Gary Schoenwolf’s systems involves an extraordinary level of coordina- complex dance of cells, modifying their shape, motili- those operating to induce the inner ear are responsi- laboratory, and was appoint- tion between cells and emergent tissues at very early ty, growth and the location of subcellular compon- ble for the control of their development. ed team leader at the ents, Raj hopes to be able to reconcile the events RIKEN CDB in 2002. stages of embryogenesis. Research in Raj Ladher's lab focuses on the induction of the inner ear and the that occur outside the cell with the biological chang- In the future, the Ladher lab looks to extend its peripheral olfactory system. The chick embryo is es occurring within. research scope to other model systems, with the used as a model system, chosen for its tractability to goal of determining whether some as-yet unidenti- classical embryological techniques and the opportuni- fied general mechanisms are at work in sensory ties it affords for investigating the processes of devel- organogenesis. Fgf-19 is expressed in the mesoderm of the chicken embryo from early stages.

36 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 37 Sensory cellular differentiation The conversion of uncommitted precursor cells in Publications the otic placode to differentiated, functional otic sub- Ladher R K & Schoenwolf G types is very precisely timed. If it occurs premature- C in Developmental Neuro- ly, the resulting organ is underdeveloped, with too biology (ed. Rao, M. H.) few cells forming the adult structure. If it happens Kluwer (2003). too late, there is a danger that these cells will still be immature and unable to fulfill their needed roles in Francis-West P H, Ladher R K and Schoenwolf G C. subsequent developmental processes. An example Development of the sensory of this temporal regulation is seen in the wiring of organs. Sci Prog 85:151-73 the inner ear to the brain, which requires the forma- (2002). tion of neurons from the otic placode. If these cells Ladher R K, Anakwe K U, are not formed when this process occurs, the func- Gurney A L, Schoenwolf G C tion of the auditory system is compromised. But Staff and Francis-West P H. Identi- what is the nature of this control? By using embryo- fication of synergistic signals Team Leader logical studies, combined with gene manipulation initiating inner ear develop- Raj Ladher ment. Science 290:1965-7 Research Scientist and ex-ovo culture, Raj's lab is addressing this ques- (2000). Michael F Teraoka Escaño tion, finding the basis for this precise regulation in Technical Staff Pabel Antonio Delgado the functional characteristics of specific signaling Susan Boerner Assistant molecules. Their control and effects on the develop- Lio Hiroi ment of the inner ear represents one of the most exciting aspects of research in Raj's lab, with implications in regenerative technologies and tissue Creative Research engineering-based therapeutics. Promoting Program

36 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 37 eIF4G, and thereby inhibits the initiation of transla- previously unexpected roles for a mater- tion. Findings that a mutant form of Cup lacking the nally supplied factor involved in lipid sig- sequence with which it binds eIF4E failed to repress naling, which the team is now working to oskar translation in vivo, that Cup interacts with Bru- characterize in more detail. Mutation of no in a yeast two-hybrid assay, and that the Cup- the underlying gene results in a pheno- eIF4E complex associates with Bruno in vivo sug- type similar to that of the Pgc mutant, in gest that these three proteins form a complex that which germ cells fail to migrate to the achieves translational repression by interactions embryonic gonads. Nakamura is now with both the 3’ and 5’ ends of the oskar RNA. A investigating the roles of both genes in similar model of protein interactions is observed in the maintenance and migration of germ the translational repression of the cyclin-B1 RNA in cells. the Xenopus African clawed frog, indicating that this paradigm of translational repression through the 5’ Development of the Staff /3’ interactions is conserved across species. Ciona intestinalis germline Team Leader Akira Nakamura In addition to their investigation of fruit fly germline Research Scientist Nakamura next intends to look into the means by development, scientists in the Nakamura lab are Kazuko Hanyu-Nakamura Maki Shirae which the repressor effects of the eIF4E-Cup-Bruno also beginning investigations using the ascidian, Cio- Keiji Sato complex are alleviated at the appropriate develop- na intestinalis, a member of the chordate lineage Tsubasa Tanaka Technical Staff mental stage, after the oskar ribonucleoprotein com- from which all vertebrates arose. Commonly Hiroko Sonobe plex has reached and anchored to its appropriate referred to as sea squirts, these animals provide a Chiaki Nakamoto destination at the pole of the oocyte. good model for studying vertebrate evolution. Soon

Model of the translational repression of Publications Creative Research oskar RNA in oogenesis. The mechan- Nakamura A, Sato K and Promoting Program isms by which translational repression is coupled with RNA localization remain to Hanyu-Nakamura K. Dro- be solved. sophila Cup Is an eIF4E Bind- ing Protein that Associates with Bruno and Regulates oskar mRNA Translation in Oogenesis. Dev Cell 6:69-78 Germ cells are the only cell types capable of trans- Translational repression (2004). mitting genetic information across generations, and RNA activity during Drosophila oogenesis involves a their formation is characterized by unique develop- number of sequential processes. The Drosophila Kawashima T, Nakamura A, Yasuda K and Kageyama Y. mental processes as well. In many types of animals, oocyte shares cytoplasm with neighboring nurse Dmaf, a novel member of Maf including the Drosophila fruit fly, the formation and cells via an incomplete cell membrane, allowing transcription factor family is differentiation of germ mRNAs and proteins from the nurse cells to be trans- expressed in somatic gonadal cells is controlled by ported to the oocyte in the form of ribonucleopro- cells during embryonic devel- mRNAs and proteins teins. Following their export from the nurse cell opment and gametogenesis in Drosophila. Gene Expr Pat- localized in a specific nuclei, mRNAs are translationally repressed, or terns 3:663-7 (2003). cytoplasmic region within ‘masked,’ and transported to specified regions of the Akira Nakamura eggs, called germ plasm. oocyte, where they establish fixed and precise locali- Styhler S, Nakamura A and Ph. D. Germ plasm mRNAs are zations and regain their ability to undergo transla- Lasko P. VASA localization requires the SPRY-domain Akira Nakamura received translated in a spatiotem- tion. In one example of this critically important regu- and SOCS-box containing both his baccalaureate and porally regulated manner, lation, the translation of the RNA for the maternal after fertilization, the ascidian egg develops into a his Ph. D. from the Universi- protein, GUSTAVUS. Dev ty of Tsukuba. He spent a but the means by which gene oskar, which has critical functions in embryo- Polar granule maintenance small free-swimming tadpole possessing a Cell 3:865-76 (2002). year as a post-doctoral fel- the germ plasm is formed nic patterning and the formation of germline cells, is Polar granules are large complexes of ribonucleopro- notochord, an evolutionary forebear of the spinal low at the same institution and achieves this con- repressed during its transport to the posterior pole of teins that store RNAs and proteins required for the cord, and a rudimentary nervous system, both of Sano H, Nakamura A and before moving to the Kobayashi S. Identification of Department of Biology at trolled translation remain the oocyte. This transcript-specific repression is formation of germ cells in the fruit fly. A non-coding which are lost when it enters its immobile adult a transcriptional regulatory McGill University in Montre- largely unknown. Akira known to be mediated by the protein Bruno, which Polar granule component (Pgc) RNA has been stage. region for germline-specific al in 1995 to work as a expression of vasa gene in post-doc under Paul Lasko. Nakamura studies the binds to the 3’ UTR of oskar mRNA, but the underly- shown to be important in the maintenance of these He returned to Japan in establishment of the Dro- ing mechanisms have remained obscure. germ plasm organelles; in Pgc antisense knock- Nakamura seeks to analyze the regulatory mechan- Drosophila melanogaster. Mech Dev 112:129-39 1997 as a research asso- sophila germ line as a down embryos, germ cells are formed but subse- isms of germline development in Ciona, which are ciate at the University of (2002). model of the processes quently degenerate, which has led to the hypothesis interesting in that while several lines of evidence Tsukuba. He was appoint- Following their export from the ed assistant professor in of germ plasm formation that Pgc serves as a form of supportive scaffold indicate that germ cells are formed from maternally Nakamura A, Amikura R, Han- the university’s Gene nurse cell nuclei, mRNAs are yu K and Kobayashi S. Research Center and Insti- and differentiation, as maintaining the structural integrity of polar granules. derived germ plasm, it also appears that germ cells Me31B silences translation of tute of Biological Sciences well as for the insights translationally repressed, or The Nakamura lab is now exploring Pgc function in can be regenerated after metamorphosis, suggest- oocyte-localizing RNAs in 2000, and began a three- this system can provide ‘masked,’ and transported to more detail by isolating a complete loss-of-function ing that two independent mechanisms may be at year term as a PRESTO through the formation of cyto- researcher in the Japan Sci- into the general mechan- specified regions of the oocyte mutant, allowing for more specific analyses of the work. In research conducted in collaboration with plasmic RNP complex during ence and Technology Cor- isms of mRNA localiza- gene’s role. They also plan to look for the gene’s Suma Aqualife Park, a local municipal aquarium, Drosophila oogenesis. Devel- poration (JST) in December tion and translation, In recent work, the Nakamura lab demonstrated that functional domains, using Pgc orthologs from other Nakamura’s team will explore the genetic regulation opment 128:3233-42 (2001). 2001. He was appointed which are important to a an ovarian protein, Cup, is another protein required species of Drosophila as a basis for comparison. of ascidian germline development by selecting can- CDB team leader in March Carrera P, Johnstone O, 2002. number of other develop- to inhibit the premature translation of oskar mRNA, didate genes from EST and genome databases (a Nakamura A, Casanova J, mental processes includ- and that Cup achieves this by binding to a second In other ongoing research, members of the Naka- draft sequence of the Ciona genome is available), Jackle H and Lasko P. VASA ing asymmetric cell divi- protein, eIF4E, a 5’ cap-binding general translation mura lab are using forward-genetics techniques to confirm the spatial and temporal patterns of their mediates translation through Drosophila germ plasm and sion, cell migration and initiation factor. The binding with Cup prevents eIF- search for novel genes involved in germ cell develop- expression and characterize promoter regions and interaction with a Drosophila germ cells visualized using yIF2 homolog. Mol Cell GFP-Vasa fusion protein. axis formation and likely 4E from binding with a different partnering molecule, ment. Early results of those studies have revealed trans-acting factors. 5:181-7 (2000). synaptic plasticity.

38 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 39 eIF4G, and thereby inhibits the initiation of transla- previously unexpected roles for a mater- tion. Findings that a mutant form of Cup lacking the nally supplied factor involved in lipid sig- sequence with which it binds eIF4E failed to repress naling, which the team is now working to oskar translation in vivo, that Cup interacts with Bru- characterize in more detail. Mutation of no in a yeast two-hybrid assay, and that the Cup- the underlying gene results in a pheno- eIF4E complex associates with Bruno in vivo sug- type similar to that of the Pgc mutant, in gest that these three proteins form a complex that which germ cells fail to migrate to the achieves translational repression by interactions embryonic gonads. Nakamura is now with both the 3’ and 5’ ends of the oskar RNA. A investigating the roles of both genes in similar model of protein interactions is observed in the maintenance and migration of germ the translational repression of the cyclin-B1 RNA in cells. the Xenopus African clawed frog, indicating that this paradigm of translational repression through the 5’ Development of the Staff /3’ interactions is conserved across species. Ciona intestinalis germline Team Leader Akira Nakamura In addition to their investigation of fruit fly germline Research Scientist Nakamura next intends to look into the means by development, scientists in the Nakamura lab are Kazuko Hanyu-Nakamura Maki Shirae which the repressor effects of the eIF4E-Cup-Bruno also beginning investigations using the ascidian, Cio- Keiji Sato complex are alleviated at the appropriate develop- na intestinalis, a member of the chordate lineage Tsubasa Tanaka Technical Staff mental stage, after the oskar ribonucleoprotein com- from which all vertebrates arose. Commonly Hiroko Sonobe plex has reached and anchored to its appropriate referred to as sea squirts, these animals provide a Chiaki Nakamoto destination at the pole of the oocyte. good model for studying vertebrate evolution. Soon

38 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 39 chromosomes, while Chp2 mutations result in weak silencing defects in three heterochromatic regions: centromeres, telomeres and the mat locus.

The group has demonstrated that Swi6, Chp1 and Chp2 localize at three heterochromatic regions (centromere, telomere and mat locus), and that this localization is clearly dependent on H3-lys9 methylation mediated by Clr4. Following on a Staff

series of experiments in which the gene for Team Leader each protein was disrupted, Nakayama has Jun-ichi Nakayama Research Scientist developed a model in which Chp1 function is Tetsushi Iida specific to the establishment and spreading Tomohiro Hayakawa Visiting Scientist of heterochromatin, while Chp2 and Swi6 Mahito Sadaie Technical Staff form a dimer that supports the stable main- Yasuko Ohtani tenance of heterochromatin. They also found Rika Kawaguchi that, among three heterochromatic regions, centromeres are more dynamic and require establishment steps. A model of heterochromatin formation in fission yeast

Nakayama is also interested in how chromo- Creative Research heterochromatin assembly. The number of species domain proteins function in higher eukaryotic cells. Promoting Program of these proteins, which possess characteristic SET In previous study, Nakayama found an evolutionally- Publications or chromodomain sequence motifs, is much smaller conserved chromodomain protein is a stable com- Nakayama J, Xiao G, Noma K, in yeast than it is in human, making S. pombe an apt ponent of histone deacetylase complex in fission Malikzay A, Bjerling P, Ekwall model for studying the basic means by which these yeast. Using mammalian cells, he is now investigat- K, Kobayashi R and Grewal SI. Alp13, an MRG family pro- molecules respond to the methyl modifications to his- ing the function of a chromodomain protein which tein, is a component of fission Modifications to the nuclear DNA-protein chromatin encoding DNA. The amino-terminal tails of histones tone residues. The methylation of specific sites on has been linked to cell senescence and develop- yeast Clr6 histone deacetylase complex are central to the epigenetic regulation of protrude from the nucleosome and are subject to the histone H3 catalyzed by a SET domain- ment. required for genomic integrity. gene transcription, an activity that must be main- covalent modifications including phosphorylation, containing methyltransferase provides epigenetic Embo J 22:2776-87 (2003). tained and propagated across mitotic cycles and acetylation, and methylation. These histone modifica- markers that allow chromodomain proteins to bind Through his work on heterochromatin histone modifica- Tamaru H, Zhang X, McMillen throughout the development of the organism proper tions affect higher-order chromatin structure and the histone and direct it toward eu- or heterochroma- tions, Nakayama has uncovered potentially important D, Singh PB, Nakayama J, in order for cells to establish and maintain their iden- influence gene expression. tin assembly or to initiate developmentally regulated new roles for proteins in the establishment, main- Grewal SI, Allis CD, Cheng X tities. Chromatin occurs in highly-condensed and gene silencing. tenance and transmission of epigenetic information. and Selker EU. Trimethylated less spatially concentrated states, known as hetero- These findings show that the definition of a gene as a lysine 9 of histone H3 is a Heterochromatin also functions in mark for DNA methylation in chromatin and euchromatin, respectively. Hetero- The Nakayama lab has shown the histone methyl- simple string of DNA nucleotides needs to be expand- telomeres, which play key roles in Neurospora crassa. Nat Genet chromatin regions have fewer genes overall than transferase Clr4 to be essential for heterochromatin ed to include the action of proteins in the functional replicative senescence and cancer, 34:75-9 (2003). euchromatic stretches of the genome, and many of assembly as an upstream element that helps Swi6 genetic unit. In the future, Nakayama plans to perform Jun-ichi Nakayama the genes that are found there remain unexpressed. and centromeres, the to localize correctly. Two other chromodomain pro- more detailed analyses of the molecular mechanisms Nakagawa H, Lee JK, Hurwitz J, Allshire RC, Nakayama J, Ph. D. However, heterochromatin functions as more than a linchpins of mitosis teins, Chp1 and Chp2, have also been implicated in that underlie epigenetic function, as well as studies in Grewal SI, Tanaka K and Mur- Jun-ichi Nakayama locked closet to store stretches of unused DNA; in heterochromatin formation and function. Disruption higher organisms and epigenetic gene expression in received his bachelor’s, akami Y. Fission yeast CENP- master’s and doctoral fact, there appear to be a number of functionally dis- In previous studies, Nakayama demonstrated that of the gene encoding Chp1causes defects in centro- developmental processes. B homologs nucleate centro- degrees in bioscience from tinct types of heterochromatin serving in a spectrum heterochromatin protein binding states play a role in meric silencing and higher mitotic loss rates in mini- meric heterochromatin by pro- the Tokyo Institute of Tech- of developmentally important capacities, from the the regulation of gene silencing. Nakayama per- moting heterochromatin- nology, the last in 1999 for specific histone tail modifica- his work on the cloning and transcriptional regulation of cell-type specific genes formed detailed analyses of the binding states of tions. Genes Dev 16:1766-78 characterization of mamma- to genomic self-defense by compartmentalizing and Swi6, a homolog of the mammalian HP-1 heterochro- (2002). lian telomerase components. He spent the period neutralizing foreign mobile genetic elements that matin protein at the silent mating-type (mat) locus of from 1999 to 2001 as a might otherwise interfere with proper gene function. the fission yeast. The results of that study showed Nakayama J, Allshire RC, Klar AJS and Grewal SIS. Implicat- postdoctoral researcher at Heterochromatin also functions in two genetically sil- that Swi6 protein is a dosage-critical component the Cold Spring Harbor Lab- ing DNA polymerase a in epi- oratory in Shiv Grewal’s ent chromosomal regions: telomeres, which play key involved in imprinting the mat locus. This binding of genetic control of silencing in lab, before returning to roles in replicative senescence and cancer, and cen- Swi6 is maintained both across mitotic cell cycles fission yeast. EMBO J Japan in December 2001 20:2857-66(2001). as a PRESTO researcher tromeres, the linchpins of mitosis. and intergenerationally, as it is propagated through in the Japan Science and meiosis as well. Nakayama's study also showed that Nakayama J, Rice JC, Strahl Technology Corporation Using the fission yeast Saccharomyces pombe as a the deacetylation and subsequent methylation of a (JST). He was appointed BD, Allis CD and Grewal SI. team leader in the RIKEN model system, Jun-ichi Nakayama focuses on inves- specific histone H3 lysine residue are essential to Role of histone H3 lysine 9 CDB Laboratory for Chro- tigations of heterochromatin dynamics, modifications this process. Chemical modifications to histones methylation in epigenetic con- matin Dynamics in 2002. to the DNA-packing proteins called 'histones' in par- are fundamental epigenetic processes that can act trol of heterochromatin assembly. Science 292:110-3 (2001). ticular, and the molecular mechanisms that allow to switch the expression of a target gene on or off.

such chemical states to be heritably transmitted. His- Nakayama J, Klar AJ and Gre- tones are the primary protein constituents of the Nakayama has now linked Swi6 function to a wal SI. A chromodomain pro- nucleosome, the most basic unit of chromosomal number of other chromodomain proteins in fission AB tein, Swi6, performs imprinting organization, which provides highly compact but yeast that seem to act in a stepwise and context- functions in fission yeast dur- Localization of a chromodomain protein (green) and phosphorylated Localization of a chromodomain protein (green) and SC-35 (magenta) ing mitosis and meiosis. Cell readily accessible packaging for a cell's gene- sensitive fashion to silence genes in the process of form of RNA polymerase II (magenta) in HeLa cell. under alpha-amanitin treatment in HeLa cell. 101:307-17 (2000).

40 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 41 chromosomes, while Chp2 mutations result in weak silencing defects in three heterochromatic regions: centromeres, telomeres and the mat locus.

40 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 41 demonstrated that MIG-17 Publications molecules in MIG-23 mutants had lower than nor- Nishiwaki K, Kubota Y, Chi- mal molecular weights con- gira Y, Roy SK, Suzuki M, Schvarzstein M, Jigami Y, sistent with the loss of glyco- Hisamoto N and Matsumoto sylation, while immunoblot- K. An NDPase links ADAM ting studies confirmed that in protease glycosylation with these mutants MIG-17 failed organ morphogenesis in C. to bind sugars at the normal elegans. Nat Cell Biol 6:31-7 (2004). levels. MIG-17 molecules designed with deficient glyco- Gumienny TL, Brugnera E, sylation sites also failed to Tosello-Trampont AC, localize at the gonad and Kinchen JM, Haney LB, Nishi- waki K, Walk SF, Nemergut showed no ability to direct its ME, Macara IG, Francis R, migration. Genetic analyses Staff Schedl T, Qin Y, Van Aelst L, revealed that MIG-23 is Hengartner MO and Team Leader Student Trainee Ravichandran KS. CED- Kiyoji Nishiwaki Ryoko Murakami expressed in muscle cells, 12/ELMO, a novel member Research Scientist Assistant which reconciles well with Norio Suzuki Rie Kuroki of the CrkII/Dock180/Rac Yukihiko Kubota Part-time Staff MIG-17's muscle cell origins. pathway, is required for pha- Katsuyuki Tamai Midori Tatsumoto Taken together, these gocytosis and cell migration. Shinji Ihara Asami Sumitani Kiyotaka Ohkura results indicate that MIG-17 Cell 107:27-41 (2001). Technical Staff molecules are secreted from Mitsue Sano Suzuki N, Buechner M, Nishi- muscle cells after glycosyla- waki K, Hall DH, Nakanishi tion by MIG-23, a modifica- H, Takai Y, Hisamoto N and tion which enables their Matsumoto K. A putative Creative Research recruitment to the migrating GDP-GTP exchange factor is Promoting Program gonad. required for development of the excretory cell in Caenor- habditis elegans. EMBO Rep anti-coagulatory effects. ADAM family proteases Questions and implications 2:530-5 (2001). have been implicated in a number of human physio- Although Golgi body NDPases such as MIG-23 are logical and pathological processes, including blood capable of modifying a number of other secreted pro- Nishiwaki K, Hisamoto N and Matsumoto K. A metallopro- way to destinations in the body, following molecular teins, the Nishiwaki study indicates that the effects Cell migration and coagulation disorders, rheumatoid arthritis and asth- tease disintegrin that con- organogenesis guidance cues generated by mechanisms that ma. These proteins act to proteolytically release of the NDPase dysfunction are especially prominent trols cell migration in Cell division, cell migration and changes in cell remain enigmatic. Kiyoji Nishiwaki studies gonadoge- membrane-bound growth factors or to disrupt com- in the abnormal glycosylation and function of an Caenorhabditis elegans. Sci- shape are coordinated to generate complex tissue nesis in the nematode C. elegans as a model sys- ponents of extracellular matrices such as collagen ADAM family protease, suggesting that glycosyla- ence 288:2205-8 (2000). and organ systems during animal development and tem to improve the understanding of the molecular and proteoglycans (which help to build skin and soft tion may play a more important role in the function regeneration. One important aspect of this minutely bases of cell migration during organ development. tissue). of proteins in this family than it does in that of other regulated process is the movement of cells in By developing a clearer picture of evolutionarily con- secreted proteins. ADAM family proteases are sheets, an activity orchestrated by mechanisms served mechanisms, this research promises to pro- This research promises to known to be involved in a number of human diseas- which remain poorly understood. During organoge- vide insights into cell migration and organ develop- provide insights into cell es, and the question of whether glycosylation nesis, sheets of cells migrate and spread over the ment in humans, and contribute to the understand- defects play a role in their pathogenesis merits fur- migration and organ development underlying tissues while maintaining selective adhe- ing of human diseases involving disturbances in ther investigation. Research such as this provides Kiyoji Nishiwaki sive bonds with cells of similar type, and make their coordinated cellular movement. in humans insights into basic biological mechanisms that may Ph. D. one day make it possible to treat a range of health Kiyoji Nishiwaki received Guidance of the gonad conditions by restoring normal ADAM protease gly- his B. Sc. from Osaka City Nishiwaki's team has analyzed a number of muta- University and M. Sc. from Interactions between the basement membrane of tions that result in aberrant gonadal phenotypes simi- cosylation in cases in which it is aberrant. Osaka University where he the migrating gonad and that of the body wall are lar to those seen in mig-17 mutants, including the worked on the molecular biology of the yeast S. cere- keystone processes in gonadal development. The mutation of the gene that encodes MIG-23. This pro- visiae. He joined NEC C. elegans gonad is U-shaped, the result of the tein was found to be structurally related to nucleo- Corp. in 1986 as a research- directed migration of cells known as distal tip cells side diphosphatases (NDPases), glycosylating pro- er in the Fundamental Research Laboratory study- present at the leading ends of the developing gonad teins found in the Golgi apparatus, the intracellular ing the molecular genetics in larvae. The gene mig-17, which Nishiwaki pre- processing center where proteins are modified, sort- of C. elegans. He left NEC viously showed to be essential to this guided move- in 1992 to work as a visiting ed and released from the cell. Glycosylation invol- researcher at Johns Hop- ment, is expressed in muscle cells and secreted into ves the addition of a sugar to a site on a protein kins University, then the body cavity where it binds to the migrating gona- chain, a post-translational modification whose roles returned to the company in 1993 to continue his work dal tip cells. Distal tip cells in worms engineered to are incompletely understood, but which is thought to on nematode development. lack the MIG-17 protein fail to steer cell migration serve a regulatory function in some signaling mol- He was awarded a Ph. D. properly, resulting in abnormal development and a ecules. On further investigation, MIG-23 mutants by Osaka City University for work on the molecular anal- 'meandering' gonad phenotype. However, the showed reductions in NDPase activity. The wild-type ysis of C. elegans embryo- means by which MIG-17 coordinates the timing and MIG-23 protein successfully rescued yeast mutants genesis in 1994. He direction of the developing gonad's progress though remained at NEC until for NDPase, lending solid support to the case for the receiving an appointment the larval body has remained unknown. NDPase nature of MIG-23. as team leader at the RIKEN CDB. MIG-17 is an ADAM (a disintegrin and metallopro- The similarity between the MIG-23 and MIG-17 phe- tease) protease, a family of secreted or membrane notypes suggested that the mutation of MIG-23 proteins first identified as components of various MIG-17 is glycosylated by MIG-23 and secreted from the muscle cells to localize on the The U-shaped gonad in the wild type becomes abnormal in mig-23 might result in abnormal MIG-17 glycosylation. gonad surface where it is required for the migration of distal tip cells. and mig-17 mutants due to the misdirected migration of distal tip cells. snake venoms, where they have hemorrhagic and Using gel electrophoresis, Nishiwaki and colleagues

42 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 43 demonstrated that MIG-17 Publications molecules in MIG-23 mutants had lower than nor- Nishiwaki K, Kubota Y, Chi- mal molecular weights con- gira Y, Roy SK, Suzuki M, Schvarzstein M, Jigami Y, sistent with the loss of glyco- Hisamoto N and Matsumoto sylation, while immunoblot- K. An NDPase links ADAM ting studies confirmed that in protease glycosylation with these mutants MIG-17 failed organ morphogenesis in C. to bind sugars at the normal elegans. Nat Cell Biol 6:31-7 (2004). levels. MIG-17 molecules designed with deficient glyco- Gumienny TL, Brugnera E, sylation sites also failed to Tosello-Trampont AC, localize at the gonad and Kinchen JM, Haney LB, Nishi- waki K, Walk SF, Nemergut showed no ability to direct its ME, Macara IG, Francis R, migration. Genetic analyses Staff Schedl T, Qin Y, Van Aelst L, revealed that MIG-23 is Hengartner MO and Team Leader Student Trainee Ravichandran KS. CED- Kiyoji Nishiwaki Ryoko Murakami expressed in muscle cells, 12/ELMO, a novel member Research Scientist Assistant which reconciles well with Norio Suzuki Rie Kuroki of the CrkII/Dock180/Rac Yukihiko Kubota Part-time Staff MIG-17's muscle cell origins. pathway, is required for pha- Katsuyuki Tamai Midori Tatsumoto Taken together, these gocytosis and cell migration. Shinji Ihara Asami Sumitani Kiyotaka Ohkura results indicate that MIG-17 Cell 107:27-41 (2001). Technical Staff molecules are secreted from Mitsue Sano Suzuki N, Buechner M, Nishi- muscle cells after glycosyla- waki K, Hall DH, Nakanishi tion by MIG-23, a modifica- H, Takai Y, Hisamoto N and tion which enables their Matsumoto K. A putative Creative Research recruitment to the migrating GDP-GTP exchange factor is Promoting Program gonad. required for development of the excretory cell in Caenor- habditis elegans. EMBO Rep anti-coagulatory effects. ADAM family proteases Questions and implications 2:530-5 (2001). have been implicated in a number of human physio- Although Golgi body NDPases such as MIG-23 are logical and pathological processes, including blood capable of modifying a number of other secreted pro- Nishiwaki K, Hisamoto N and Matsumoto K. A metallopro- way to destinations in the body, following molecular teins, the Nishiwaki study indicates that the effects Cell migration and coagulation disorders, rheumatoid arthritis and asth- tease disintegrin that con- organogenesis guidance cues generated by mechanisms that ma. These proteins act to proteolytically release of the NDPase dysfunction are especially prominent trols cell migration in Cell division, cell migration and changes in cell remain enigmatic. Kiyoji Nishiwaki studies gonadoge- membrane-bound growth factors or to disrupt com- in the abnormal glycosylation and function of an Caenorhabditis elegans. Sci- shape are coordinated to generate complex tissue nesis in the nematode C. elegans as a model sys- ponents of extracellular matrices such as collagen ADAM family protease, suggesting that glycosyla- ence 288:2205-8 (2000). and organ systems during animal development and tem to improve the understanding of the molecular and proteoglycans (which help to build skin and soft tion may play a more important role in the function regeneration. One important aspect of this minutely bases of cell migration during organ development. tissue). of proteins in this family than it does in that of other regulated process is the movement of cells in By developing a clearer picture of evolutionarily con- secreted proteins. ADAM family proteases are sheets, an activity orchestrated by mechanisms served mechanisms, this research promises to pro- This research promises to known to be involved in a number of human diseas- which remain poorly understood. During organoge- vide insights into cell migration and organ develop- provide insights into cell es, and the question of whether glycosylation nesis, sheets of cells migrate and spread over the ment in humans, and contribute to the understand- defects play a role in their pathogenesis merits fur- migration and organ development underlying tissues while maintaining selective adhe- ing of human diseases involving disturbances in ther investigation. Research such as this provides Kiyoji Nishiwaki sive bonds with cells of similar type, and make their coordinated cellular movement. in humans insights into basic biological mechanisms that may Ph. D. one day make it possible to treat a range of health Kiyoji Nishiwaki received Guidance of the gonad conditions by restoring normal ADAM protease gly- his B. Sc. from Osaka City Nishiwaki's team has analyzed a number of muta- University and M. Sc. from Interactions between the basement membrane of tions that result in aberrant gonadal phenotypes simi- cosylation in cases in which it is aberrant. Osaka University where he the migrating gonad and that of the body wall are lar to those seen in mig-17 mutants, including the worked on the molecular biology of the yeast S. cere- keystone processes in gonadal development. The mutation of the gene that encodes MIG-23. This pro- visiae. He joined NEC C. elegans gonad is U-shaped, the result of the tein was found to be structurally related to nucleo- Corp. in 1986 as a research- directed migration of cells known as distal tip cells side diphosphatases (NDPases), glycosylating pro- er in the Fundamental Research Laboratory study- present at the leading ends of the developing gonad teins found in the Golgi apparatus, the intracellular ing the molecular genetics in larvae. The gene mig-17, which Nishiwaki pre- processing center where proteins are modified, sort- of C. elegans. He left NEC viously showed to be essential to this guided move- in 1992 to work as a visiting ed and released from the cell. Glycosylation invol- researcher at Johns Hop- ment, is expressed in muscle cells and secreted into ves the addition of a sugar to a site on a protein kins University, then the body cavity where it binds to the migrating gona- chain, a post-translational modification whose roles returned to the company in 1993 to continue his work dal tip cells. Distal tip cells in worms engineered to are incompletely understood, but which is thought to on nematode development. lack the MIG-17 protein fail to steer cell migration serve a regulatory function in some signaling mol- He was awarded a Ph. D. properly, resulting in abnormal development and a ecules. On further investigation, MIG-23 mutants by Osaka City University for work on the molecular anal- 'meandering' gonad phenotype. However, the showed reductions in NDPase activity. The wild-type ysis of C. elegans embryo- means by which MIG-17 coordinates the timing and MIG-23 protein successfully rescued yeast mutants genesis in 1994. He direction of the developing gonad's progress though remained at NEC until for NDPase, lending solid support to the case for the receiving an appointment the larval body has remained unknown. NDPase nature of MIG-23. as team leader at the RIKEN CDB. MIG-17 is an ADAM (a disintegrin and metallopro- The similarity between the MIG-23 and MIG-17 phe- tease) protease, a family of secreted or membrane notypes suggested that the mutation of MIG-23 proteins first identified as components of various MIG-17 is glycosylated by MIG-23 and secreted from the muscle cells to localize on the The U-shaped gonad in the wild type becomes abnormal in mig-23 might result in abnormal MIG-17 glycosylation. gonad surface where it is required for the migration of distal tip cells. and mig-17 mutants due to the misdirected migration of distal tip cells. snake venoms, where they have hemorrhagic and Using gel electrophoresis, Nishiwaki and colleagues

42 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 43 Staff

Team Leader Hitoshi Niwa Research Scientist Shinji Masui Kazuya Ogawa Satoshi Ohtsuka Yayoi Toyooka Visiting Scientist Itsuro Sugimura Hiroyuki Kitajima Technical Staff Rika Yagi Kadue Takahashi Student Trainee Ken-ichi Tominaga Yoko Sekita Yuko Iwamatsu Sayako Matsui Daisuke Shimosato Assistant Rie Kuroki

Trophoblast stem (TS) cells, derived from ES cells, grown on feeder cells in the presence of recombinant FGF4 Creative Research Promoting Program expression of either of these factors simultaneously enon). The researchers found that SDIA acts to induced the expression of the other from the endog- maintain Oct 3/4 expression in ES cells, which enous gene. New work suggests that the homeobox appears to be important as a promoter of the differ- gene Cdx-2 can serve as a similar trigger for the entiation of ES cells into neural lineages. ES cells induction of trophectoderm from ES cells expressing from which Oct 3/4 had been deleted lost their ability low levels of the pluripotency maintaining gene, Oct to differentiate into neurons, while heightened levels Self-renewal and renewable progeny, and the identification of mol- 3/4. Using regulable activation of Cdx-2 in vitro, of Oct 3/4 intensified the neurogenic effects of SDIA. pluripotency ecules that direct the dividing ES cell to produce Niwa successfully induced ES cells to differentiate This finding raises the possibility that, in addition to Their ability to self-renew indefinitely and to differen- daughter cells of specific types. Hitoshi Niwa's into trophoblast stem cells. These results, in combi- its function as a maintainer of pluripotency, Oct 3/4 tiate into cells of all three germ layer types (a differ- research addresses both of these challenges, with nation with the GATA study, have led to the develop- also plays a second key role by regulating neuronal entiative capacity termed 'pluripotency'), makes the aims of developing solid scientific foundations ment of a model in which Oct 3/4, when maintained differentiation in a concentration-dependent manner. embryonic stem (ES) cells one of the most promis- and reliable technologies to support this exciting at an appropriate mid-range level, inhibits the differ- ing subjects of study in regenerative medicine, as field of biomedicine. entiation of ES cells into either the trophectodermal ES cell growth in well as an attractive model system for research into or primitive endodermal lineages by suppressing culture a spectrum of developmental processes. However, Inducing differentiation Cdx-2 and Gata-6, respectively. This goal of controlling culture conditions to achieve the mechanisms by which ES cells are able to main- The development of methods by which undifferentiat- specific outcomes is important to the growth of ES tain these capabilities are incompletely understood, ed ES cells can be prompted to commit to a specific Hitoshi Niwa Maintaining pluripotency cells in vitro as well. The Niwa lab is working to M. D. , Ph. D. and a better understanding of the stemness of these cell lineage is a field of central importance to the The POU-family transcriptional regulator Oct 3/4 develop a serum- and feeder-cell-free system for cul- Hitoshi Niwa received his cells will be necessary in order to be able to take stem cell research community. Studying factors iden- was the first factor found to elicit multiple differentia- turing mouse ES cells, which necessitates develop- M. D. from Nara Medical best advantage of their remarkable properties. tified in work on knockout mice, the Niwa research Publications University in 1989, and his tive outcomes dependent on its expression level. ES ing a detailed picture of both the extrinsic factors team has been engaged in the analysis of transcrip- Ph. D. in medical physiolo- cells expressing median levels of Oct 3/4 maintain and the intrinsic networks that function in these cells Tokuzawa Y, Kaiho E, Mar- Two of the biggest challenges that now face stem tion factors with potential roles in the development gy in 1993 from the Kuma- their pluripotency, while its overexpression results in in culture. It is known that a single ES cell in isola- uyama M, Takahashi K, Mit- moto University Graduate cell research are the determination of the factors of extraembryonic cell lineages, extraembryonic differentiation into primitive endoderm and meso- tion will fail to proliferate, while colonies of such cells sui K, Maeda M, Niwa H and School of Medicine for his that allow ES cells to generate limitlessly self- endoderm and trophectoderm. Extraembryonic Yamanaka S. Fbx15 is a nov- derm and its inhibition causes the cells to take up a grow normally. It is also known that even isolated work in gene trap methods. endoderm derivatives include el target of Oct3/4 but is dis- From 1993 to 1994, he trophectodermal fate. These findings established single cells can be induced to proliferate if the cul- pensable for embryonic stem the parietal and visceral endo- worked as a research asso- Oct 3/4 as a primary regulator of pluripotency in ES ture medium from a larger ES cell colony is trans- cell self-renewal and mouse derm, which give rise to the yolk ciate in the Department of cells, but the function of this regulator in the commit- ferred to the single cell's plate, suggesting that ES development. Mol Cell Biol Developmental Genetics at sac covering embryos in utero 23:2699-708 (2003). ment of more specific lineages remains an open cells produce a growth-stimulating factor that acts the same university, before during development, and taking a postdoctoral fellow- issue. by community effect. Niwa has developed an assay trophectoderm derivatives, the Shimozaki K, Nakashima K, ship with Austin Smith at system to isolate candidate molecules for this puta- Niwa H and Taga T. Involve- source of the placenta that sus- the University of Edinburgh tive 'stem cell autocrine factor' (SAF), and is actively ment of Oct3/4 in the Centre for Genome tains the developing mammalian In addition to its function as a pursuing the characterization of the most promising enhancement of neuronal dif- Research. He returned to embryos. maintainer of pluripotency, ferentiation of ES cells in candidates. The results of these analyses should Japan in 1996 as a Oct 3/4 may neurogenesis-inducing cul- research associate in the improve the ability of researchers to grow ES cells in tures. Development In previous research, the Niwa Department of Nutrition and also play a second key role by culture, facilitating the study of these fascinating and 130:2505-12 (2003). lab showed that the overexpres- Physiological Chemistry at regulating neuronal differentiation potentially revolutionarily important cells. the Osaka University Gradu- sion of GATA transcription fac- Ishida C, Ura K, Hirao A, ate School of Medicine, tors such as Gata-4 or Gata-6 Sasaki H, Toyoda A, Sakaki where he remained until tak- resulted in the specific conver- In 2003, the Niwa lab contributed to a study that Y, Niwa H, Li E and Kaneda ing his current position at demonstrated a role for Oct 3/4 in neurogenesis pro- Y. Genomic organization and the RIKEN CDB. sion of undifferentiated ES cells promoter analysis of the into extraembryonic endodermal moted by stromal cell derived inducing activity Dnmt3b gene. Gene cells, and that the exogenous (SDIA, see p. 18-19 for a description of this phenom- 310:151-9 (2003). Terminally differentiated trophoblast giant cells derived from ES cells, showing large, flat morphologies and polyploidy

44 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 45 Staff

44 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 45 around E8.0, prior to somitogenesis, and early embryonic lethality by E9.5. These loss-of-function defects indicate that the establishment of the DNA methylation state plays an important part in many Publications aspects of mouse development, an area the Okano Dodge JE, Ramsahoye BH, lab will continue to explore. Wo ZG, Okano M and Li E. De novo methylation of The establishment of the MMLV provirus in embryonic DNA methylation state plays an stem cells: CpG versus non- CpG methylation. Gene important part in many aspects 289:41-8 (2002). of mouse development Okano M and Li E. Genetic Staff Abnormal methylation has been implicated in a analyses of DNA methyltransferase genes in mouse mod- number of human congenital anomalies as well. ICF Team Leader el system. J Nutr 132:2462S- Masaki Okano (Immunodeficiency-Centromeric instability-Facial 5S (2002). Research Scientist Shin-ichiro Takebayashi anomalies) syndrome, which is characterized by Masaaki Oda immune deficiency, heterochromatin instability and Hata K, Okano M, Lei H and Technical Staff Li E. Dnmt3L cooperates Chisa Matsuoka defects in facial development, results from a muta- Akiko Yamagiwa with the Dnmt3 family of de Makiko Okano tion in the gene encoding Dnmt3b. A mutation in the novo DNA methyltransferas- Student Trainee gene encoding the methyl-cytosine binding protein, es to establish maternal Takao Nakayama imprints in mice. Develop- Akiko Tsumura MeCP2, is also known to cause Rett syndrome, a ment 129:1983-93 (2002). Assistant neurological disorder that occurs almost entirely in Fumika Nakayama females. In this form of mental retardation, develop- Okano M, Bell DW, Haber ment appears normal until the child is 6-18 months DA and Li E. DNA methyl- old, after which the child loses communication skills transferases Dnmt3a and Creative Research and develops disturbances in gait and use of the Dnmt3b are essential for de Promoting Program hands and slowed growth of the head. novo methylation and mammalian development. Cell 99:247-57 (1999). As de novo methylation activity takes place mainly In the future, the Okano lab will study how epigenet- in embryonic stem (ES) cells, early postimplantation ic reprogramming works at a molecular level. By embryos and gametogenesis, ES cells provide a identifying the genes involved in de novo DNA meth- The methylation of mammalian DNA plays an import- Dnmt3a and Dnmt3b highly suitable experimental model system for inves- ylation, they hope to discover the means by which it ant role in determining gene expression. The Methylation lays down developmentally significant tigating the molecular mechanisms underlying the is determined what genes will be methylated. Com- number and locations of methyl tags provide molecu- gene expression patterns that tend not to alter once establishment of new methylation patterns. Okano is parisons of the expression patterns of such genes lar icons marking genes as inactive, or in some established. In previous work, Okano showed that studying the consequences of conditional knockout across germ layers, or in normal mice against cases, active. Certain types of protein recognize and de novo methylation in mice required the expression and overexpression of methyltransferase genes on clones created from somatically derived cells should bind to the tagged DNA, affecting the expression pat- of a pair of methyltransferase genes, Dnmt3a and mouse development using ES cells and transgenic lead to new insights in the context-specificity of the terns of the genes it encodes. During embryonic Dnmt3b, at a very early stage of embryonic develop- mice, in the hopes of uncovering links with tumorige- DNA methylation process as well as the roles of development, DNA methylation patterns are estab- ment, as well during gametogenesis. Prior to this nesis, congenital nervous system defects and abnor- methylation in development, health, and regenera- lished in a sequence of steps involving both the finding, it was believed that a related gene, Dnmt1, mal cell differentiation. tion. removal of methyl tags at the preim- was solely responsible for Masaki Okano plantation stage and the establish- the methylation of DNA. Developmental Ph. D. ment of new sets of tags, a pro- Using lacZ reporter roles of Masaki Okano received his cess known as de novo methyla- gene studies, Okano methylation baccalaureate and master’s tion, which occurs after implan- showed that these Knockout mutations of degrees from Kyoto Uni- tation and again during game- genes are expressed versity, and his doctorate in Dnmt3a and Dnmt3b biochemistry from the togenesis. These methyla- in early embryonic result in a spectrum of Research Division in Agri- tion patterns can subse- and ES cells, suggest- organic, neurological culture at the same institu- quently be maintained ing that they function and spermatic defects. tion in 1994. He spent the and transmitted across in the epigenetic Dnmt3b mutants gener- period from 1994 to 1995 as a research fellow in cell generations by main- reprogramming of ally die by embryonic biology at the Nagoya Uni- tenance methylation. DNA methylation stage 16.5 due to wide- versity BioScience Center states. Based on spread defects affecting before moving to Massachu- Masaki Okano's research these findings, it is organ development and setts, where he worked as concentrates on the now known that Dnmt1 a research fellow in medi- general growth. Loss of cine at Harvard Medical mechanisms by which DNA [Dnmt3a-/-,Dnmt3b-/-] mouse embryo functions primarily in Dnmt3a function produc- School and Massachusetts methylation is established failed to turn and form somites. methylation maintenance, es less pronounced General Hospital from 1995 and maintained throughout Mouse embryos at 9.5 d.p.c. Left; wild which is necessary for the effects; embryogenesis to 2002, serving as an type, Right; mutant development, focusing on the stable inheritance of tissue- appears normal, but instructor in medicine at Harvard for the last two properties and activity of the Dnmt family of methyl- specific methylation patterns, but that Dnmt3a and most mutants die by the years of that period. He transferases, which includes Dnmt1, Dnmt3a and Dnmt3b are the principal determinants in initiating fourth week after birth was appointed team leader Dnmt3b. He particularly focuses on working out and DNA methylation. Recent work has also shown that and show abnormal at the RIKEN Center for clarifying the roles of de novo methyltransferases in isoforms of Dnmt3a and Dnmt3b function as main- growth, intestinal motili- Developmental Biology in embryogenesis and cell differentiation, seeing these tenance methyltransferases in mouse embryonic 2001, and returned to work ty and spermatogene- enzymes as necessary agents in maintaining the stem cells, indicating a broader role for these pro- Dnmt3b expression in the embryonic ectoderm and the chorion shown by IRES-βgeo marker driven by the in Japan full-time in 2002. sis. The deletion of both endogenous Dnmt3b promoter. proper balance between stability and plasticity in the teins. genes results in devel- expression of specific genes. opmental arrest at

46 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 47 around E8.0, prior to somitogenesis, and early embryonic lethality by E9.5. These loss-of-function defects indicate that the establishment of the DNA methylation state plays an important part in many Publications aspects of mouse development, an area the Okano Dodge JE, Ramsahoye BH, lab will continue to explore. Wo ZG, Okano M and Li E. De novo methylation of The establishment of the MMLV provirus in embryonic DNA methylation state plays an stem cells: CpG versus non- CpG methylation. Gene important part in many aspects 289:41-8 (2002). of mouse development Okano M and Li E. Genetic Staff Abnormal methylation has been implicated in a analyses of DNA methyltransferase genes in mouse mod- number of human congenital anomalies as well. ICF Team Leader el system. J Nutr 132:2462S- Masaki Okano (Immunodeficiency-Centromeric instability-Facial 5S (2002). Research Scientist Shin-ichiro Takebayashi anomalies) syndrome, which is characterized by Masaaki Oda immune deficiency, heterochromatin instability and Hata K, Okano M, Lei H and Technical Staff Li E. Dnmt3L cooperates Chisa Matsuoka defects in facial development, results from a muta- Akiko Yamagiwa with the Dnmt3 family of de Makiko Okano tion in the gene encoding Dnmt3b. A mutation in the novo DNA methyltransferas- Student Trainee gene encoding the methyl-cytosine binding protein, es to establish maternal Takao Nakayama imprints in mice. Develop- Akiko Tsumura MeCP2, is also known to cause Rett syndrome, a ment 129:1983-93 (2002). Assistant neurological disorder that occurs almost entirely in Fumika Nakayama females. In this form of mental retardation, develop- Okano M, Bell DW, Haber ment appears normal until the child is 6-18 months DA and Li E. DNA methyl- old, after which the child loses communication skills transferases Dnmt3a and Creative Research and develops disturbances in gait and use of the Dnmt3b are essential for de Promoting Program hands and slowed growth of the head. novo methylation and mammalian development. Cell 99:247-57 (1999). As de novo methylation activity takes place mainly In the future, the Okano lab will study how epigenet- in embryonic stem (ES) cells, early postimplantation ic reprogramming works at a molecular level. By embryos and gametogenesis, ES cells provide a identifying the genes involved in de novo DNA meth- The methylation of mammalian DNA plays an import- Dnmt3a and Dnmt3b highly suitable experimental model system for inves- ylation, they hope to discover the means by which it ant role in determining gene expression. The Methylation lays down developmentally significant tigating the molecular mechanisms underlying the is determined what genes will be methylated. Com- number and locations of methyl tags provide molecu- gene expression patterns that tend not to alter once establishment of new methylation patterns. Okano is parisons of the expression patterns of such genes lar icons marking genes as inactive, or in some established. In previous work, Okano showed that studying the consequences of conditional knockout across germ layers, or in normal mice against cases, active. Certain types of protein recognize and de novo methylation in mice required the expression and overexpression of methyltransferase genes on clones created from somatically derived cells should bind to the tagged DNA, affecting the expression pat- of a pair of methyltransferase genes, Dnmt3a and mouse development using ES cells and transgenic lead to new insights in the context-specificity of the terns of the genes it encodes. During embryonic Dnmt3b, at a very early stage of embryonic develop- mice, in the hopes of uncovering links with tumorige- DNA methylation process as well as the roles of development, DNA methylation patterns are estab- ment, as well during gametogenesis. Prior to this nesis, congenital nervous system defects and abnor- methylation in development, health, and regenera- lished in a sequence of steps involving both the finding, it was believed that a related gene, Dnmt1, mal cell differentiation. tion. removal of methyl tags at the preim- was solely responsible for Masaki Okano plantation stage and the establish- the methylation of DNA. Developmental Ph. D. ment of new sets of tags, a pro- Using lacZ reporter roles of Masaki Okano received his cess known as de novo methyla- gene studies, Okano methylation baccalaureate and master’s tion, which occurs after implan- showed that these Knockout mutations of degrees from Kyoto Uni- tation and again during game- genes are expressed versity, and his doctorate in Dnmt3a and Dnmt3b biochemistry from the togenesis. These methyla- in early embryonic result in a spectrum of Research Division in Agri- tion patterns can subse- and ES cells, suggest- organic, neurological culture at the same institu- quently be maintained ing that they function and spermatic defects. tion in 1994. He spent the and transmitted across in the epigenetic Dnmt3b mutants gener- period from 1994 to 1995 as a research fellow in cell generations by main- reprogramming of ally die by embryonic biology at the Nagoya Uni- tenance methylation. DNA methylation stage 16.5 due to wide- versity BioScience Center states. Based on spread defects affecting before moving to Massachu- Masaki Okano's research these findings, it is organ development and setts, where he worked as concentrates on the now known that Dnmt1 a research fellow in medi- general growth. Loss of cine at Harvard Medical mechanisms by which DNA [Dnmt3a-/-,Dnmt3b-/-] mouse embryo functions primarily in Dnmt3a function produc- School and Massachusetts methylation is established failed to turn and form somites. methylation maintenance, es less pronounced General Hospital from 1995 and maintained throughout Mouse embryos at 9.5 d.p.c. Left; wild which is necessary for the effects; embryogenesis to 2002, serving as an type, Right; mutant development, focusing on the stable inheritance of tissue- appears normal, but instructor in medicine at Harvard for the last two properties and activity of the Dnmt family of methyl- specific methylation patterns, but that Dnmt3a and most mutants die by the years of that period. He transferases, which includes Dnmt1, Dnmt3a and Dnmt3b are the principal determinants in initiating fourth week after birth was appointed team leader Dnmt3b. He particularly focuses on working out and DNA methylation. Recent work has also shown that and show abnormal at the RIKEN Center for clarifying the roles of de novo methyltransferases in isoforms of Dnmt3a and Dnmt3b function as main- growth, intestinal motili- Developmental Biology in embryogenesis and cell differentiation, seeing these tenance methyltransferases in mouse embryonic 2001, and returned to work ty and spermatogene- enzymes as necessary agents in maintaining the stem cells, indicating a broader role for these pro- Dnmt3b expression in the embryonic ectoderm and the chorion shown by IRES-βgeo marker driven by the in Japan full-time in 2002. sis. The deletion of both endogenous Dnmt3b promoter. proper balance between stability and plasticity in the teins. genes results in devel- expression of specific genes. opmental arrest at

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Team Leader Technical Staff Assistant Tony Perry Satoko Fujimoto Yayoi Ikeda Research Scientist Shaoyun Wang Hisashi Kishi Manami Amanai Naoko Yoshida Shisako Shoji A schematic of RNAi by transgenesis Creative Research Promoting Program

Molecular identities of PNM proteins tion and establish what roles they play in subse- Protein id Role Reference quent development. Such roles may not be restrict- Calicin (PT60) F-actin-interacting; provides rigidity Longo et al., 1987 ed to short-term development, as a growing body of By mechanisms still unknown, fertili- ry lab's interests center on attributing Cylicin I Role unknown Hess et al., 1993 evidence indicates that they have far-reaching con- Publications zation achieves the transformation molecular identities to the proteins Cylicin II Role unknown Hess et al., 1995 sequences, even after the resulting adult is in old Perry AC and Wakayama T. of two highly specialized cells — involved in these interactions and Arp-T1 Actin-related; role unknown Heid et al., 2002 age. Untimely ends and new a sperm and an unfertilized egg characterizing them functionally. Arp-T2 Actin-related; role unknown Heid et al., 2002 beginnings in mouse cloning. Nat Genet 30:243-4 (2002). (oocyte) — to a state of totipoten- SubH2Bv Histone H2B variant Aul and Oko, 2002 Novel methods of genetic cy: a single-cell embryo that can This task is large, because PERF15 Lipophilic transport protein-related; Oko and Morales, 1994 modulation using gametes Perry AC, Rothman A, de las role unknown give rise to an entire individual. beneath the membrane of a Utilizing the integrated molecular, cellular, and Heras JI, Feinstein P, Mom- Stat4 Jak-Stat signaling; role unknown Herrada & Wolgemuth, 1997 The first moments of this remark- sperm head reside macromolecu- embryological approach from the work in his labora- baerts P, Cooke HJ and c-Yes Protein tyrosine kinase ; Leclerc and Goupil, 2002 able process are known as oocyte lar complexes that include a tory, Perry expects to gain insights that will enable Wakayama T. Efficient meta- role unknown phase II transgenesis with dif- activation, an intricate orchestration nucleus (containing the paternal gen- Tony Perry PT32 WBP2, a c-Yes BP; Gong et al., 1997 us to genetically alter embryos in new ways. He has ferent transgene archetypes. Ph. D. of sub-cellular events that include all ome and associated proteins) and a role unknown developed a novel method of genome manipulation Nat Biotechnol 19:1071-3 Tony Perry received his B.Sc. the checks and balances that presage surrounding cytoplasmic matrix, the peri- p30 Calmodulin BP; role unknown Leclerc and Goupil, 2000 known as metaphase II (mII) transgenesis. This effi- (2001). in Microbiology from the healthy growth of a new embryo. Oocyte nuclear matrix (PNM). It has been estimat- cient method of transgenesis works when oocytes Perry AC. Hijacking oocyte Department of Bacteriology at activation can be observed at the light ed that the PNM comprises some 230 pro- The Perry team's analysis of sperm-oocyte interac- (normally arrested at mII) are co-injected with a the University of Bristol, Eng- DNA repair machinery in land and his doctorate from microscope level as dramatic change in the tein species. Because it is juxtaposed to the tions at fertilization begins with demembranated nucleus and transgene (tg) DNA. Typically, sperm transgenesis? Mol Reprod the University of Liverpool morphology of the newly fertilized egg and inner leaflet of enshrouding sperm head mem- sperm that are subsequently exposed to standard- Dev 56:319-24 (2000). four years later. In 1989 he provides an incredible read-out of the underly- branes, the PNM rapidly comes into contact ized conditions that recapitulate key aspects of the To date, few PNM proteins became a postdoctoral fellow ing molecular mechanisms. The Laboratory of with the oocyte cytoplasm at fertilization and is oocyte cytoplasm. Proteins solubilized in this way Perry AC, Wakayama T, working on epididymal sperm have been described, and the Cooke IM and Yanagimachi Mammalian Molecular Embryology combines an immediate source of paternally-contributed can be injected into eggs, typically by piezo- maturation at Bristol Universi- function of almost none of R. Mammalian oocyte activa- ty and in 1996 won a Euro- molecular and cellular biology with piezo- molecules that potentially modulate develop- actuated microinjection, to probe their function. tion by the synergistic action pean Molecular Biology Trav- actuated micromanipulation of mouse (Mus mus- ment. However, owing in part to its size and com- Such functional analysis is coupled to molecular them is known of discrete sperm head com- el Fellowship to work in the culus) gametes and embryos to study oocyte acti- plexity, its excoriation and disassembly of the analyses that span several disciplines. The proteins ponents: induction of calcium laboratory of Ryuzo Yanagi- transients and involvement vation and its developmental consequences. PNM takes hours to complete within the oocyte can be purified and identified; Perry's team employs heads are depleted of their membranes by deter- machi on the mechanism of of proteolysis. Dev Biol oocyte activation, which activation period. During this time, it is likely to chromatographic and state-of-the art 2D electrophor- gent extraction prior to mixing with tg DNA and 217:386-93 (2000). remains one of his research Interactions between release molecules that may be placed in one of etic methods to this end. Antibodies raised against co-injection. This has several advantages over len- interests. From there, Dr. Per- gamete cytoplasms several categories, according to whether they (i) peptides identified in this way are used to localize tiviral methods of tg introduction; cloning and propa- Perry AC, Wakayama T, ry moved first to the Rockefell- Microinjection of an isolated sperm head into an have an immediate function in neoembryogene- the proteins before, during and after fertilization; anti- gation in viral vectors is not required and the method Kishikawa H, Kasai T, Okabe er University and then to the M, Toyoda Y and Yanagi- oocyte can result in the birth of normal young. sis, (ii) are stored for later use, or (iii) are target- lends itself to tgs in the megabase range, which is company Advanced Cell Tech- body function can also be assayed by microinjec- machi R. Mammalian trans- nology, working primarily on Thus, the sperm head — the part of the head ed for rapid proteolytic degradation. Each cate- tion. Owing to dissimilarities between frog and mam- too large for lentiviral delivery. Perry and members genesis by intracytoplasmic novel methods of genome that lies beneath its membranes — apparently gory implies an intimate series of interactions malian oocytes and the paucity of material obtain- of his lab are adapting the method to facilitate high sperm injection. Science manipulation. In 2002 Dr. Per- 284:1180-3 (1999). ry took his present position as contains all of the paternal information sufficient with the surrounding cytoplasm. However, to able from the latter, genetic methods are used to throughput targeted knock-out and -down phe- Team Leader of the Labora- for full development. Tony Perry is systematically date, few proteins of the PNM have been identify mouse oocyte proteins that interact with the notypes. Developing these approaches is an import- tory of Mammalian Molecular elucidating interactions between sub-membrane described, and the function of almost none of sperm proteins that have been characterized. Collec- ant supplementary part of their work to facilitate the Embryology at the RIKEN sperm head components and the oocyte at fertili- them is known. This represents an extraordinary tively, these studies hold forth the promise of ena- molecular dissection of sperm protein function in the CDB, where he works on mechanisms in mammalian zation, particularly during the moments soon gap in the understanding of a conserved biologi- bling the identification of oocyte signaling pathways context of embryos and whole animals. preimplantation embryos. after the sperm has entered the oocyte. The Per- cal structure. and processes that become operational at fertiliza-

An early model of the sperm-borne oocyte activating factor

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An early model of the sperm-borne oocyte activating factor

48 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 49 term. However, a limited number of such headless birth phenotypes have been reported, all of which have been linked to mutations in node-related genes, such as Lim1 and Otx2. The fortuitous discovery of a previously unknown headless phenotype in the mouse prompted Sasaki to investigate the genetic cause of this mutation, which he named headshrinker (hsk), and to study the gene's role in the induction of the head. Gene expression analysis revealed losses in the expression of important head- organizing genes in hsk mutants, resulting in the impaired function of the late-stage head organizer or Staff the prechordal plate, a fore/midbrain-inducing Team Leader Hiroshi Sasaki mesenchymal mass that forms at the anterior end of Research Scientist the developing embryo. Atsushi Sawada Shinji Yamamoto Yukari Yada (2004) Northern blotting showed reduced levels in the Visiting Scientist Noriyuki Nishioka expression of the mRNA for the gene Ssdp1 (a Technical Staff sequence-specific, single-stranded-DNA-binding pro- Hiroko Sato Yayoi Minami tein) in headshrinker mutants, and the researchers Yuuri Tsuboi found that they could rescue the headless pheno- Student Trainee Norifumi Namekawa type by using Ssdp1cDNA. Ssdp1/hsk encodes a Assistant Misaki Harano protein that binds with Ldb1, which itself binds LIM- homeodomain proteins and other developmentally important transcription factors. Sasaki proposes a Creative Research model for the interaction of these three co-factors in Promoting Program which Sspd1 enhances the activation of target genes by the Lim1-Ldb1 complex. In an analysis sponding increases or decreases in the area of Fox- using a labeled reporter gene, Sspd1 alone showed a2 expression. The Foxa2 enhancer element CS3 almost no activity, but boosted the activity of both (which binds with TEAD) functions downstream of Lim1 and Ldb1 on co-expression, and had the stron- Wnt, a known node-inducing signal and the synergis- gest effect when all three factors were present. In Publications Signaling centers in Activation of nodal tic activation of Foxa2 by TEAD and Wnt has also addition to the lethal loss of the head, hsk mutants Sekimizu K, Nishioka N, mammalian development differentiation Sasaki H, Takeda H, Karl- The earliest stages of mouse embryogenesis play Foxa2 (also known as Hepatocyte Nuclear Factor strom R and Kawakami A. out over the course of a few days as a series of 3β, HNF3β) is a highly-conserved transcription fac- The zebrafish iguana locus dynamic transformations from a simple, nearly fea- tor required for the formation of embryonic signaling encodes Dzip1, a novel zinc finger protein required for tureless blastocyst to a recognizable vertebrate centers in the mouse. The Sasaki lab is engaged in proper regulation of hedge- body by the end of gastrulation. This rapid and strik- an analysis of molecules that activate Foxa2 expres- hog signaling.Development ing transition is achieved by the highly coordinated sion in both node and notochord. Members of the (2004). differentiation of cells toward specific fates, a feat Sasaki team previously identified the key regulatory orchestrated by a number of organizing loci, collec- element of the Foxa2 enhancer, CS3, and have now Karlstrom RO, Tyurina OV, Kawakami A, Nishioka N, Tal- Hiroshi Sasaki tively known as ‘signaling centers.’ These centers determined that this element binds to a member of bot WS, Sasaki H and Schier Ph. D. produce diffusible proteins that serve to regulate the TEAD/TEF family of proteins, four transcription AF. Genetic analysis of Hiroshi Sasaki received his embryonic development, guiding neighboring cell factors found in both mice and humans, which share zebrafish gli1 and gli2 B. Sc. in zoology from the communities to take up the roles they will play in the a common DNA-binding domain. TEAD proteins are reveals divergent require- University of Tokyo, and his ments for gli genes in verte- adult animal, or steering them away from inappro- widely expressed in the mouse embryo and their master’s and Ph. D. in brate development. developmental biology from priate fates. activity is regulated by interaction with multiple tran- Development 130:1549-64 the same institution. He scription factors and co-activators. Complementary (2003). worked as a research asso- studies of TEAD activation showed that increases or ciate in Atsushi Kuroiwa’s Foxa2 (HNF3§) is a key regulator of the signaling centers, node These recent findings suggest a Sheng H, Goich S, Wang A, lab at Tohoku University decreases in TEAD activation resulted in corre- and notochord in mouse embryos. Grachtchouk M, Lowe L, Mo from 1990 to 1992, and in new model to R, Lin K, de Sauvage FJ, Brigid Hogan’s lab at explain node induction been demonstrated in zebrafish, indicating that this exhibit a range of other developmental abnormali- Sasaki H, Hui CC and Dlu- Vanderbilt University from gosz AA. Dissecting the 1992 to 1994. He returned pathway has been conserved in at least some other ties, implicating Ssdp1 as a co-activator molecule oncogenic potential of Gli2: to Japan to take a position vertebrate taxa as well. A second signaling factor with multiple and diverse embryogenetic roles. deletion of an NH(2)-terminal as assistant professor at Hiroshi Sasaki’s research team investigates the net- known to work in node induction, Nodal, has also fragment alters skin tumor Osaka University beginning work of signaling centers that work to induce normal been linked to TEAD activity. These recent findings Sasaki continues to pursue a deeper understanding phenotype. Cancer Res in 1995, where he development in mammals. The node, a center 62:5308-16 (2002). remained until his appoint- suggest a new model to explain node induction, in of the structure and function of signaling centers ment as team leader of the responsible for induction of body parts in the mouse, which TEAD complements the function of multiple using an array of approaches and techniques. Other Dai P, Shinagawa T, Nomura Laboratory for Embryonic is a particular focus of Sasaki’s research. He has activating co-factors from primary signaling centers ongoing work in the lab is aimed at isolating cells T, Harada J, Kaul SC, Wadh- Induction at the RIKEN Cen- identified a number of transcription factors involved by activating the enhancer element CS3 to induce from transgenic mice engineered to express LacZ in wa R, Khan MM, Akimaru H, ter for Developmental Biolo- in the formation and the function of the node in previ- gy. Foxa2 expression. the node and notochord, which promises to speed Sasaki H, Colmenares C and ous studies, and he remains intent on solving ques- the identification of new genes specifically Ishii S. Ski is involved in tran- tions of how these centers are formed, and how they headshrinker expressed in those regions. scriptional regulation by the repressor and full-length set courses for the developing embryo to follow. Although a variety of mutations can cause the head forms of Gli3. Genes Dev to fail to develop properly, it is rare for embryos in 16:2843-8 (2002). headshrinker mutant lacks head induction by the signaling center, and shows headless phenotype at birth (left). which the head is entirely lacking to develop to full

50 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 51 term. However, a limited number of such headless birth phenotypes have been reported, all of which have been linked to mutations in node-related genes, such as Lim1 and Otx2. The fortuitous discovery of a previously unknown headless phenotype in the mouse prompted Sasaki to investigate the genetic cause of this mutation, which he named headshrinker (hsk), and to study the gene's role in the induction of the head. Gene expression analysis revealed losses in the expression of important head- organizing genes in hsk mutants, resulting in the impaired function of the late-stage head organizer or Staff the prechordal plate, a fore/midbrain-inducing Team Leader Hiroshi Sasaki mesenchymal mass that forms at the anterior end of Research Scientist the developing embryo. Atsushi Sawada Shinji Yamamoto Yukari Yada (2004) Northern blotting showed reduced levels in the Visiting Scientist Noriyuki Nishioka expression of the mRNA for the gene Ssdp1 (a Technical Staff sequence-specific, single-stranded-DNA-binding pro- Hiroko Sato Yayoi Minami tein) in headshrinker mutants, and the researchers Yuuri Tsuboi found that they could rescue the headless pheno- Student Trainee Norifumi Namekawa type by using Ssdp1cDNA. Ssdp1/hsk encodes a Assistant Misaki Harano protein that binds with Ldb1, which itself binds LIM- homeodomain proteins and other developmentally important transcription factors. Sasaki proposes a Creative Research model for the interaction of these three co-factors in Promoting Program which Sspd1 enhances the activation of target genes by the Lim1-Ldb1 complex. In an analysis sponding increases or decreases in the area of Fox- using a labeled reporter gene, Sspd1 alone showed a2 expression. The Foxa2 enhancer element CS3 almost no activity, but boosted the activity of both (which binds with TEAD) functions downstream of Lim1 and Ldb1 on co-expression, and had the stron- Wnt, a known node-inducing signal and the synergis- gest effect when all three factors were present. In Publications Signaling centers in Activation of nodal tic activation of Foxa2 by TEAD and Wnt has also addition to the lethal loss of the head, hsk mutants Sekimizu K, Nishioka N, mammalian development differentiation Sasaki H, Takeda H, Karl- The earliest stages of mouse embryogenesis play Foxa2 (also known as Hepatocyte Nuclear Factor strom R and Kawakami A. out over the course of a few days as a series of 3β, HNF3β) is a highly-conserved transcription fac- The zebrafish iguana locus dynamic transformations from a simple, nearly fea- tor required for the formation of embryonic signaling encodes Dzip1, a novel zinc finger protein required for tureless blastocyst to a recognizable vertebrate centers in the mouse. The Sasaki lab is engaged in proper regulation of hedge- body by the end of gastrulation. This rapid and strik- an analysis of molecules that activate Foxa2 expres- hog signaling.Development ing transition is achieved by the highly coordinated sion in both node and notochord. Members of the (2004). differentiation of cells toward specific fates, a feat Sasaki team previously identified the key regulatory orchestrated by a number of organizing loci, collec- element of the Foxa2 enhancer, CS3, and have now Karlstrom RO, Tyurina OV, Kawakami A, Nishioka N, Tal- Hiroshi Sasaki tively known as ‘signaling centers.’ These centers determined that this element binds to a member of bot WS, Sasaki H and Schier Ph. D. produce diffusible proteins that serve to regulate the TEAD/TEF family of proteins, four transcription AF. Genetic analysis of Hiroshi Sasaki received his embryonic development, guiding neighboring cell factors found in both mice and humans, which share zebrafish gli1 and gli2 B. Sc. in zoology from the communities to take up the roles they will play in the a common DNA-binding domain. TEAD proteins are reveals divergent require- University of Tokyo, and his ments for gli genes in verte- adult animal, or steering them away from inappro- widely expressed in the mouse embryo and their master’s and Ph. D. in brate development. developmental biology from priate fates. activity is regulated by interaction with multiple tran- Development 130:1549-64 the same institution. He scription factors and co-activators. Complementary (2003). worked as a research asso- studies of TEAD activation showed that increases or ciate in Atsushi Kuroiwa’s Foxa2 (HNF3§) is a key regulator of the signaling centers, node These recent findings suggest a Sheng H, Goich S, Wang A, lab at Tohoku University decreases in TEAD activation resulted in corre- and notochord in mouse embryos. Grachtchouk M, Lowe L, Mo from 1990 to 1992, and in new model to R, Lin K, de Sauvage FJ, Brigid Hogan’s lab at explain node induction been demonstrated in zebrafish, indicating that this exhibit a range of other developmental abnormali- Sasaki H, Hui CC and Dlu- Vanderbilt University from gosz AA. Dissecting the 1992 to 1994. He returned pathway has been conserved in at least some other ties, implicating Ssdp1 as a co-activator molecule oncogenic potential of Gli2: to Japan to take a position vertebrate taxa as well. A second signaling factor with multiple and diverse embryogenetic roles. deletion of an NH(2)-terminal as assistant professor at Hiroshi Sasaki’s research team investigates the net- known to work in node induction, Nodal, has also fragment alters skin tumor Osaka University beginning work of signaling centers that work to induce normal been linked to TEAD activity. These recent findings Sasaki continues to pursue a deeper understanding phenotype. Cancer Res in 1995, where he development in mammals. The node, a center 62:5308-16 (2002). remained until his appoint- suggest a new model to explain node induction, in of the structure and function of signaling centers ment as team leader of the responsible for induction of body parts in the mouse, which TEAD complements the function of multiple using an array of approaches and techniques. Other Dai P, Shinagawa T, Nomura Laboratory for Embryonic is a particular focus of Sasaki’s research. He has activating co-factors from primary signaling centers ongoing work in the lab is aimed at isolating cells T, Harada J, Kaul SC, Wadh- Induction at the RIKEN Cen- identified a number of transcription factors involved by activating the enhancer element CS3 to induce from transgenic mice engineered to express LacZ in wa R, Khan MM, Akimaru H, ter for Developmental Biolo- in the formation and the function of the node in previ- gy. Foxa2 expression. the node and notochord, which promises to speed Sasaki H, Colmenares C and ous studies, and he remains intent on solving ques- the identification of new genes specifically Ishii S. Ski is involved in tran- tions of how these centers are formed, and how they headshrinker expressed in those regions. scriptional regulation by the repressor and full-length set courses for the developing embryo to follow. Although a variety of mutations can cause the head forms of Gli3. Genes Dev to fail to develop properly, it is rare for embryos in 16:2843-8 (2002). headshrinker mutant lacks head induction by the signaling center, and shows headless phenotype at birth (left). which the head is entirely lacking to develop to full

50 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 51 Factors in asymmetric T cell division Publications Additional work has also focused on T hypodermal Zhao X, Sawa H and Her- cells, which in normal development divide asymmet- man MA. tcl-2 encodes a novel protein that acts rically to produce non-identical daughter cells of neu- synergistically with Wnt sig- ral or hypodermal fate. Recent work by the Sawa naling pathways in C. ele- team has shown that three genes, psa-3, ceh-20 gans. Dev Biol 256:276-89 and nob-1 (homologs of Meis, Pbx and Hox, respec- (2003). tively) are involved in this asymmetric T mitosis, as Sawa H, Kouike H and Oka- mutations in any of these genes result in the abnor- no H. Components of the mal loss of asymmetry in cell divisions, in which the SWI/SNF complex are posterior daughters of the T cell are hypodermal required for asymmetric cell rather than neural in character. division in C. elegans. Mol Staff Cell 6:617-24 (2000).

Team Leader Hitoshi Sawa Investigations into the role of PSA-3 uncovered that Rocheleau CE, Yasuda J, Research Scientist expression of PSA-3 is stronger in the posterior T Shin TH, Lin R, Sawa H, Oka- Masaki Fujita no H, Priess JR, Davis RJ Masahiro Uchida cell daughter. psa-3 expression is dependent on a Yukimasa Shibata consensus binding site within its promoter region for and Mello CC. WRM-1 acti- Special Postdoctoral Researcher vates the LIT-1 protein Yukinobu Arata the transcriptional factor POP-1/TCF, which has pre- kinase to transduce anteri- Kumiko Oishi viously been reported to be required for asymmetric or/posterior polarity signals Technical Staff Hisako Takeshita T cell division, suggesting that the psa-3 gene is a in C. elegans. Cell 97:717-26 Noriko Sasakawa (1999). Assistant direct target of Wnt signaling and functions as a cell Tomoko Nakashima fate determinant in asymmetric cell division. The asymmetry of PSA-3/Meis expression is also per- turbed in lin-44/Wnt and lin-17/Frizzled mutants, cor- Creative Research roborating the argument for a downstream role for Promoting Program psa-3 in the Wnt pathway.

ter produces neural cells. In lin-44 mutants, however, Expression of psa-3/Meis in the T cell lineage is also the opposite is often the case, with anterior daughters markedly reduced in nob-1/Hox and ceh-20/Pbx frequently giving rise to neural cells, and posterior mutants, indicating that, NOB-1/Hox and CEH- Asymmetric cell division T cell polarity daughters generating cells in the hypodermal lineage. 20/Pbx also act in the regulation of psa-3/Meis and cell diversity The Wnt pathway represents one of the most highly This phenotype is attributable to a reversal in parent expression. Taken together, these data indicate that The animal body is constructed from the myriad and conserved and widely adapted signaling systems in cell polarity, which occurs in about 70% of lin-44 the expression of psa-3/Meis is controlled by binary multiform progeny cells from a single fertilized egg. biology, playing developmentally critical roles in the reg- mutants. Misexpression of lin-44 at the anterior of the T mechanisms; Wnt signaling establishes cellular Asymmetric cell division, in which a single mother ulation of cell fate, proliferation and polarity in species cell in lin-44 mutants caused a significant enhancement polarity, while the Hox-Pbx complex regulates posi- cell splits to generate daughter cells with discrete from worm to human. Wnt signaling, in fact, transpires of the reversal phenotype; 97% of such mutants tional identity. In other metazoans, both Meis and characters, is the fundamental process by which cell- along multiple divergent routes. In the canonical Wnt showed switching of fates in T cell daughters. These Pbx are known to bind directly as co-factors to Hox, ular diversity is achieved, but remains imperfectly pathway in Drosophila (the species in which it was first results demonstrate that the orientation of T cell polarity raising the possibility that 20/Pbx and NOB-1/Hox understood at the molecular level. Using the nema- described), Wnt binds to the transmembrane receptor is regulated by the LIN-44 signal. proteins may form a ternary complex with PSA- tode C. elegans as a model system, Hitoshi Sawa is Frizzled, which activates a series of molecules and 3/Meis in C. elegans as well. In vitro studies show Hitoshi Sawa engaged in the study of the genetic and molecular molecular complexes within the cell, thereby prevent- that PSA-3/Meis interacts directly with CEH-20/Pbx Ph. D. interactions that underlie asymmetric division, and ing the degradation of β-catenin and allowing it to accu- Sawa is engaged and NOB-1/Hox, lending support to the model in Hitoshi Sawa obtained his the related phenomenon of cellular polarity, in which mulate and signal transcription factors that act on the in the study of the genetic and which PSA-3/Meis functions as a cell fate determi- baccalaureate, master’s the contents of individual cells are distributed une- pathway's ultimate downstream gene targets. Studying molecular interactions nant by forming a complex with CEH-20/Pbx and and doctoral degrees from venly, giving distinct characters to daughter cells on the effects of lin-44 (a Wnt homolog) on asymmetric divi- NOB-1/Hox in asymmetric cell division of the T cell. Kyoto University in the peri- that underlie asymmetric od from 1982 to 1991. He division. He seeks also to resolve the ways in which sion in the nematode, the Sawa team ectopically division and cellular polarity worked as a postdoctoral transcription-regulating complexes integrate the expressed and examined its effects on the division of T fellow at the California Insti- intracellular signaling networks that collaborate to cells, which form a diverse lineage of hypodermal and tute of Technology on a help determine cell fate. neural cells arising from a single progenitor. In normal A pathway homologous to the Wnt cascade exists in Human Frontier Science Program grant during the development, the anterior daughter of the parent T cell C. elegans, and it has been suggested that a period from 1991 to 1994, produces hypodermal cells, while the posterior daugh- number of downstream factors corresponding to Expression of PSA-3/Meis is directly induced by then at the Massachusetts many of the most prominent Wnt signaling compon- POP-1/TCF. PSA-3 forms Institute of Technology from ents also play roles in roundworm asymmetric cell complex with CEH-20/Pbx and 1994 to 1997 under support NOB-1/Hox to determine from the Howard Hughes division. POP-1, the C. elegans homolog of asymmetric cell fates. Medical Institute. He TCF/LEF-1, a transcription factor acting downstream returned to Japan in 1997 of Wnt, has recently been shown to be required for to continue his work at Osa- asymmetric T cell division. In the canonical Wnt path- ka University, first as a post- doc, then as researcher way, TCF/LEF-1 is a target of β-catenin, but the funded by the Japan Sci- direct involvement of β-catenin in C. elegans asym- ence and Technology Cor- metric cell division has never clearly been demon- poration PRESTO program. strated. Researchers in the Sawa lab found that He took his current position Ectopic expression of at the RIKEN CDB in 2001. LIN-44/Wnt anterior to the T wrm-1/β-catenin mutants show defects in asymmet- cell strongly reverses the ric T cell division, similar to those observed in lin-17 polarity of T cell division. mutants, suggesting that that the asymmetric cell division is controlled by β -catenin.

52 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 53 Factors in asymmetric T cell division Publications Additional work has also focused on T hypodermal Zhao X, Sawa H and Her- cells, which in normal development divide asymmet- man MA. tcl-2 encodes a novel protein that acts rically to produce non-identical daughter cells of neu- synergistically with Wnt sig- ral or hypodermal fate. Recent work by the Sawa naling pathways in C. ele- team has shown that three genes, psa-3, ceh-20 gans. Dev Biol 256:276-89 and nob-1 (homologs of Meis, Pbx and Hox, respec- (2003). tively) are involved in this asymmetric T mitosis, as Sawa H, Kouike H and Oka- mutations in any of these genes result in the abnor- no H. Components of the mal loss of asymmetry in cell divisions, in which the SWI/SNF complex are posterior daughters of the T cell are hypodermal required for asymmetric cell rather than neural in character. division in C. elegans. Mol Staff Cell 6:617-24 (2000).

52 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 53 knock down the expression of Phenotype analysis specific genes in the nema- The data sets produced by high- tode has only added to its throughput phenotype analyses can appeal, giving scientists the be formidably large and unwieldy, and ability to inhibit gene function the lack of standardized descriptive without disturbing its underly- terms and categories can limit the ing DNA. Sugimoto has accessibility to the wealth of informa- refined this technique of RNA tion they contain. To improve the val- interference (RNAi) by devel- ue, ease-of-use and distributability of oping a method in which her RNAi phenotyping results, Sugimo- nematodes directly uptake to has developed a taxonomic system dsRNA in solution. This pro- and nomenclature to enable the pre- cess of 'RNAi by soaking' off- cise and consistent description of ers greater efficiency and embryonic and post-embryonic phe- ease-of-use than other RNAi notypes using a checklist of more than 50 identifying Staff methods and has enabled sys- traits, such as abnormalities in cell division, cellular Team Leader tematic high-throughput stud- differentiation, organogenesis, morphogenesis, Asako Sugimoto ies of gene suppression to be growth, and movement. She plans to make these Research Scientist Naoko Iida conducted more rapidly than RNAi phenotype profiles available to the C. elegans Special Postdoctoral Researcher ever before possible. research community, in the hopes of establishing a Fumio Motegi Technical Staff universal taxonomy for phenotype analysis. Phe- Nobuko Uodome Chie Hayakawa Starting with a cDNA library of notypes described using this system can be subject- Yumi Iida approximately 10,000 genes ed to hierarchical clustering analysis, which makes it Student Trainee Miwa Furuya expressed in developmental Rika Maruyama processes, the Sugimoto lab Assistant Creative Research Fumi Amimori used RNAi to knock down Sugimoto has developed Part-time Staff Promoting Program each gene's function and has a taxonomic system and Kazumasa Takeda started to construct a data- nomenclature to enable base in which the resulting the precise and consistent phenotypes are sorted by description of embryonic and Interactions between genes at the network level are these studies as a base for advancing the under- developmental outcome. To post-embryonic phenotypes of fundamental importance in instructing the develop- standing of developmentally important mechanisms. date, nearly 6,000 phenotypes ment and function of multicellular organisms. While Sugimoto hopes that by opening windows into the have been categorized, and it possible to categorize genes based on relatedness the characterization of genomes at the level of the role of networked genes in guiding development in a was found that loss of function between phenotypes. The genes clustered by this solitary gene remains an important challenge in simple worm, new light will be shed on universal in more than 25% of these Publications method are likely to be involved in the same develop- many yet unsequenced organisms, the analysis of mechanisms in the genetic regulation of the develop- genes resulted in lethal, mor- mental process, thus this analysis will provide piv- Mito Y, Sugimoto A and how genes function in networks is now in increasing mental program. phologically altered or sterile otal information to uncover genetic networks Yamamoto M. Distinct devel- demand for those genomes that are already avail- phenotypes. Many lethal phe- opmental function of two Cae- involved in the regulation of development. Members able. In the post-genome era, scientists seek to RNAi-based profiling of notypes result in developmen- norhabditis elegans of the Sugimoto team are now utilizing these RNAi understand the roles of genes in evolution through gene function tal arrest and death at very homologs of the cohesin sub- analysis data to investigate developmental process- unit Scc1/Rad21. Mol Biol comparative genomics studies as well as to charac- The wild-type nematode is built from precisely 959 early stages, which typically es such as microtubule dynamics in mitosis, morpho- Cell 14:2399-409 (2003). Asako Sugimoto terize genes in isolation and understand their func- somatic cells, yet this simple organism exhibits a prevents the study of the genesis, and programmed cell death. Ph. D. tions in the context of interactive networks, a field of wide range of the specialized cell types, such as underlying gene's function in Sawa M, Suetsugu S, Sugi- Asako Sugimoto received investigation known as functional genomics. muscles and neurons, that characterize more highly later development. By expos- moto A, Miki H, Yamamoto her B. Sc. degree from the derived species. And, thanks to the complete knowl- ing worms to RNAi-inducing M and Takenawa T. Essen- Department of Biophysics tial role of the C. elegans Asako Sugimoto has adopted the nematode edge of the lineage of every cell in the C. elegans dsRNA at the L1 stage of lar- and Biochemistry in the Uni- Arp2/3 complex in cell migra- versity of Tokyo School of Caenorhabditis elegans as an experimental model body, the differentiative pathway of val development, Sugimoto is tion during ventral enclosure. Science in 1987, and her to take advantage every one of those cells can be fol- able to study the post- J Cell Sci 116:1505-18 doctorate from the same of its tractability lowed from its origin in the fertil- embryonic function of genes (2003). institution in 1992. She to the ized egg to its role in the that are lethal when knocked worked as a postdoctoral Sumiyoshi E, Sugimoto A fellow in Joel Rothman’s fully-grown down in embryos. This is criti- and Yamamoto M. Protein laboratory in the University cally important to the under- phosphatase 4 is required for of Wisconsin Ð Madison standing of genetic networks centrosome maturation in from 1992 to 1996, before in post-embryonic develop- mitosis and sperm meiosis in returning to Japan to C. elegans. J Cell Sci assume an assistant profes- ment, as more than one out of 115:1403-10 (2002). sorship at the University of every ten genes analyzed by Tokyo. She remained in Kodama Y, Rothman JH, Caenorhabditis elegans the lab so far is essential for that position until 2002, pur- (adult hermaphrodite) embryogenesis. Sugimoto's Sugimoto A and Yamamoto suing concurrent work as a Embryogenesis of M. The stem-loop binding Japan Science and Tech- systematic functional anal- studies to date show that C. elegans Germ cells in meiotic prophase I. protein CDL-1 is required for nology Corporation PRES- ysis of its genome using unique approximately 50% of all such (Blue: DNA, Red: A specific locus on Chromosome V) chromosome condensation, TO researcher from 1997 to high-throughput screening techniques. adult. The amen- genes play post-embryonic progression of cell death and 2000. She was appointed By studying the interactions between the approx- ability of this worm's roles as well, supporting the morphogenesis in Caenor- team leader at the RIKEN habditis elegans. Develop- CDB in 2001. imately 19,000 genes predicted for C. elegans, the sequenced genome to reverse-genetic techniques concept that a great number ment 129:187-96 (2002). Sugimoto research team seeks to identify the has also served to make it one of the standard mod- of genes play multiple roles at means by which sets of genes working in combina- el organisms in the world of genetics research. The different stages in develop- tion help to establish and direct developmental pro- discovery in the late 1990s that the introduction of ment. cesses. The lab also looks to take the findings from double stranded RNA (dsRNA) could be used to

54 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 55 knock down the expression of Phenotype analysis specific genes in the nema- The data sets produced by high- tode has only added to its throughput phenotype analyses can appeal, giving scientists the be formidably large and unwieldy, and ability to inhibit gene function the lack of standardized descriptive without disturbing its underly- terms and categories can limit the ing DNA. Sugimoto has accessibility to the wealth of informa- refined this technique of RNA tion they contain. To improve the val- interference (RNAi) by devel- ue, ease-of-use and distributability of oping a method in which her RNAi phenotyping results, Sugimo- nematodes directly uptake to has developed a taxonomic system dsRNA in solution. This pro- and nomenclature to enable the pre- cess of 'RNAi by soaking' off- cise and consistent description of ers greater efficiency and embryonic and post-embryonic phe- ease-of-use than other RNAi notypes using a checklist of more than 50 identifying Staff methods and has enabled sys- traits, such as abnormalities in cell division, cellular Team Leader tematic high-throughput stud- differentiation, organogenesis, morphogenesis, Asako Sugimoto ies of gene suppression to be growth, and movement. She plans to make these Research Scientist Naoko Iida conducted more rapidly than RNAi phenotype profiles available to the C. elegans Special Postdoctoral Researcher ever before possible. research community, in the hopes of establishing a Fumio Motegi Technical Staff universal taxonomy for phenotype analysis. Phe- Nobuko Uodome Chie Hayakawa Starting with a cDNA library of notypes described using this system can be subject- Yumi Iida approximately 10,000 genes ed to hierarchical clustering analysis, which makes it Student Trainee Miwa Furuya expressed in developmental Rika Maruyama processes, the Sugimoto lab Assistant Creative Research Fumi Amimori used RNAi to knock down Sugimoto has developed Part-time Staff Promoting Program each gene's function and has a taxonomic system and Kazumasa Takeda started to construct a data- nomenclature to enable base in which the resulting the precise and consistent phenotypes are sorted by description of embryonic and Interactions between genes at the network level are these studies as a base for advancing the under- developmental outcome. To post-embryonic phenotypes of fundamental importance in instructing the develop- standing of developmentally important mechanisms. date, nearly 6,000 phenotypes ment and function of multicellular organisms. While Sugimoto hopes that by opening windows into the have been categorized, and it possible to categorize genes based on relatedness the characterization of genomes at the level of the role of networked genes in guiding development in a was found that loss of function between phenotypes. The genes clustered by this solitary gene remains an important challenge in simple worm, new light will be shed on universal in more than 25% of these Publications method are likely to be involved in the same develop- many yet unsequenced organisms, the analysis of mechanisms in the genetic regulation of the develop- genes resulted in lethal, mor- mental process, thus this analysis will provide piv- Mito Y, Sugimoto A and how genes function in networks is now in increasing mental program. phologically altered or sterile otal information to uncover genetic networks Yamamoto M. Distinct devel- demand for those genomes that are already avail- phenotypes. Many lethal phe- opmental function of two Cae- involved in the regulation of development. Members able. In the post-genome era, scientists seek to RNAi-based profiling of notypes result in developmen- norhabditis elegans of the Sugimoto team are now utilizing these RNAi understand the roles of genes in evolution through gene function tal arrest and death at very homologs of the cohesin sub- analysis data to investigate developmental process- unit Scc1/Rad21. Mol Biol comparative genomics studies as well as to charac- The wild-type nematode is built from precisely 959 early stages, which typically es such as microtubule dynamics in mitosis, morpho- Cell 14:2399-409 (2003). Asako Sugimoto terize genes in isolation and understand their func- somatic cells, yet this simple organism exhibits a prevents the study of the genesis, and programmed cell death. Ph. D. tions in the context of interactive networks, a field of wide range of the specialized cell types, such as underlying gene's function in Sawa M, Suetsugu S, Sugi- Asako Sugimoto received investigation known as functional genomics. muscles and neurons, that characterize more highly later development. By expos- moto A, Miki H, Yamamoto her B. Sc. degree from the derived species. And, thanks to the complete knowl- ing worms to RNAi-inducing M and Takenawa T. Essen- Department of Biophysics tial role of the C. elegans Asako Sugimoto has adopted the nematode edge of the lineage of every cell in the C. elegans dsRNA at the L1 stage of lar- and Biochemistry in the Uni- Arp2/3 complex in cell migra- versity of Tokyo School of Caenorhabditis elegans as an experimental model body, the differentiative pathway of val development, Sugimoto is tion during ventral enclosure. Science in 1987, and her to take advantage every one of those cells can be fol- able to study the post- J Cell Sci 116:1505-18 doctorate from the same of its tractability lowed from its origin in the fertil- embryonic function of genes (2003). institution in 1992. She to the ized egg to its role in the that are lethal when knocked worked as a postdoctoral Sumiyoshi E, Sugimoto A fellow in Joel Rothman’s fully-grown down in embryos. This is criti- and Yamamoto M. Protein laboratory in the University cally important to the under- phosphatase 4 is required for of Wisconsin Ð Madison standing of genetic networks centrosome maturation in from 1992 to 1996, before in post-embryonic develop- mitosis and sperm meiosis in returning to Japan to C. elegans. J Cell Sci assume an assistant profes- ment, as more than one out of 115:1403-10 (2002). sorship at the University of every ten genes analyzed by Tokyo. She remained in Kodama Y, Rothman JH, Caenorhabditis elegans the lab so far is essential for that position until 2002, pur- (adult hermaphrodite) embryogenesis. Sugimoto's Sugimoto A and Yamamoto suing concurrent work as a Embryogenesis of M. The stem-loop binding Japan Science and Tech- systematic functional anal- studies to date show that C. elegans Germ cells in meiotic prophase I. protein CDL-1 is required for nology Corporation PRES- ysis of its genome using unique approximately 50% of all such (Blue: DNA, Red: A specific locus on Chromosome V) chromosome condensation, TO researcher from 1997 to high-throughput screening techniques. adult. The amen- genes play post-embryonic progression of cell death and 2000. She was appointed By studying the interactions between the approx- ability of this worm's roles as well, supporting the morphogenesis in Caenor- team leader at the RIKEN habditis elegans. Develop- CDB in 2001. imately 19,000 genes predicted for C. elegans, the sequenced genome to reverse-genetic techniques concept that a great number ment 129:187-96 (2002). Sugimoto research team seeks to identify the has also served to make it one of the standard mod- of genes play multiple roles at means by which sets of genes working in combina- el organisms in the world of genetics research. The different stages in develop- tion help to establish and direct developmental pro- discovery in the late 1990s that the introduction of ment. cesses. The lab also looks to take the findings from double stranded RNA (dsRNA) could be used to

54 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 55 Epithelial-mesenchymal teins, notably Rho, Rac1 and Cdc42, are known to Staff transitions in somites play roles in cytoskeletal rearrangement and cell Team Leader Somites comprise both epithelial and mesenchymal locomotion in vitro, cycling between GDP-bound Yoshiko Takahashi cells, two contrasting cell types that vividly demon- inactive and GTP-bound active states, and trigger- Research Scientist Daisuke Saito strate the range of possibilities in cellular function ing downstream effectors linked to the cytoskeleton Special Postdoctoral Researcher and morphology. Epithelial cells, which in adults when activated. Yuki Sato Technical Staff form the skin and the linings of many organs, Rinako Suetsugu Toshiharu Kasai adhere to each other tightly and have polarized Now, studies in the Takahashi lab are beginning to Yuji Katagiri structures with distinct basal and apical sides. By reveal additional functions for Rho family members Akemi Uchiyama Junior Research Associate comparison, mesenchymal cells are less rigidly in the context of somitic development. Both the inhi- Ryosuke Tadokoro structured and form looser aggregations. However, bition of Rac1 and the overexpression of Cdc42 Student Trainee Yukiko Nakaya these two cell types are by no means isolated or were found to induce the conversion of epithelial Tadayoshi Watanabe Emi Ohata independent from one another; indeed, a great cells to mesenchyme, while interference with Cdc42 Itsuki Kashin many developmental function caused Assistant Tomoko Oyanagi and physiological phe- mesenchymal cells to Part-time Staff nomena rely on interac- take on epithelial char- Naomi Akira tions between epitheli- acteristics. These roles um and mesenchyme. seem to be at least part- An extreme example of ly specific to somites; their inter-relatedness is neither Cdc42 nor Rac1 the ability of cells of one was found to be type to convert into the involved in epithelial- other, in a process mesenchymal transi- known as epithelial- tions in epiblast cells. mesenchymal transi- These findings suggest Creative Research tion. Such transitions that the mesenchymal- Promoting Program (which can proceed in epithelial transition that either an epithelial-to- occurs during somitoge-

mesenchymal or the Cdc42, a Rho family GTPase, plays an important role in nesis is determined by reverse direction) are mesenchymal to epithelial transitions in somitogenesis. relative Rac1 and not only essential to nor- Cdc42 activity levels, mal embryonic development and organogenesis, with higher levels of Rac1 inducing an epithelial fate, Pattern formation, the process by which the border referred to as the 'segmental plate,' flank the neural they also play roles in wound healing and the patho- while higher levels of Cdc42 tip the balance in the zones that define germ layers and tissue types are tube, which runs along the anterior-posterior axis. genesis of cancer. mesenchymal direction. She now seeks to further Publications established, is strictly regulated to occur at specific Somites arise one by one from this segmental plate characterize the induction of the activity of these Takahashi Y. Somitogenesis sites and stages in the developing embryo. This in a head-down direction, directed by the inductive After the boundaries of a somite have been estab- Rho family members in the earliest stages of the in vertebrate development, in close coordination prevents cells from differentiating activity of a putative regulatory region Takahashi lished, the mesenchymal cells in the border region establishment of the somite by segmenter activity. "Encyclopedia of Life Scienc- es", Nature Publishing Group inappropriately, and enables the establishment of has termed the 'segmenter.' Recent work in her lab undergo a transition, acquiring the polarized struc- (2004). complex and specialized tissues and organs in the is aimed at explicating transitions in cell structure ture and properties of epithelial cells. This results in Takahashi emphasizes that what drew her to sci- embryo and the adult body. One type of pattern for- and type that take place in nascent somites once a somite body in which a core of mesenchymal cells ence and maintains her interest are the opportuni- Takahashi Y. Shaping up of mation involves the regimented establishment of their initial boundaries have been established. is circumscribed by an outer layer of cells of epitheli- ties and challenges it offers to those willing and able the chic chick: boundary for- periodically alternating bands, or segments, of differ- al character. The Takahashi lab seeks to clarify the to learn from natural phenomena. Her interests are mation shared between somitogenesis and brain Yoshiko Takahashi entiated cells along the anterior-posterior body axis. molecular mechanisms that regulate this diverse and far-ranging; she chooses to pursue development. Review arti- Ph. D. The results of this process of segmentation can be mesenchymal-epithelial transition, and is concentrat- avenues that others may have overlooked, and cle, International Journal of Yoshiko Takahashi received observed in repeated orderly structures such as ver- ing on the roles played by members of the Rho fami- encourages her students and colleagues to do the Developmental Biology her B. Sc. from the Universi- tebrae, ribs and spinal ganglia. In vertebrates, one ly of GTPase proteins in this process. These pro- same. (2004). ty of Hiroshima, before mov- form of segmentation takes place in the embryonic ing to the Kyoto University Tonegawa A, Kasai T and Department of Biophysics region known as the 'somitic mesoderm,' and invol- Takahashi Y. Systematic where she received her mas- ves the formation of transient segment-organizing screening for signaling mol- ter’s and doctoral degrees in bodies called somites. ecules expressed during developmental biology. She somitogenesis by the signal pursued consecutive postdoc- sequence trap method. Dev toral fellowships in develop- Somites comprise both Biol 262:32-50 (2003). mental biology at the Institut d’Embryologie du CNRS epithelial and mesenchymal cells, Suetsugu R, Sato Y and (1988 to 1991), the Universi- two cell types that Takahashi Y. Pax 2 expres- ty of Oregon Institute of Neu- sion in mesodermal segmen- roscience (1991 to 1993) vividly demonstrate the range of tation and its relationship and Columbia University possibilities in with EphA4 and Lunatic- (1994). She returned to cellular function and morphology fringe during chicken somito- Japan as an associate pro- genesis. Mech Dev 119 fessor at Kitasato University Suppl 1:S155-9 (2002). in 1994, where she worked Yoshiko Takahashi's research involves molecular until 1998, when she took an and cellular analyses of segmentation and associate professorship at Sato Y, Yasuda K and Taka- the Nara Institute of Science somitogenesis in the chick embryo, which are invalu- hashi Y. Morphological and Technology Graduate able for the insights they provide into how popula- boundary forms by a novel School of Biological Scienc- tions of cells are able to cleave at specific boundary Transitions between epithelial and mesenchymal states are inductive event mediated by pivotal to early morphogenetic processes. Lunatic fringe and Notch dur- es. She was appointed team lines and reorganize into highly ordered and reitera- leader at the RIKEN CDB in ing somitic segmentation. 2001. tive patterns. In the early chick embryo, two strips of Development 129:3633-44 unsegmented paraxial mesoderm, collectively (2002). 2-day embryo showing somites beautifully arrayed in pairs along the anterior-posterior axis

56 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 57 Epithelial-mesenchymal teins, notably Rho, Rac1 and Cdc42, are known to Staff transitions in somites play roles in cytoskeletal rearrangement and cell Team Leader Somites comprise both epithelial and mesenchymal locomotion in vitro, cycling between GDP-bound Yoshiko Takahashi cells, two contrasting cell types that vividly demon- inactive and GTP-bound active states, and trigger- Research Scientist Daisuke Saito strate the range of possibilities in cellular function ing downstream effectors linked to the cytoskeleton Special Postdoctoral Researcher and morphology. Epithelial cells, which in adults when activated. Yuki Sato Technical Staff form the skin and the linings of many organs, Rinako Suetsugu Toshiharu Kasai adhere to each other tightly and have polarized Now, studies in the Takahashi lab are beginning to Yuji Katagiri structures with distinct basal and apical sides. By reveal additional functions for Rho family members Akemi Uchiyama Junior Research Associate comparison, mesenchymal cells are less rigidly in the context of somitic development. Both the inhi- Ryosuke Tadokoro structured and form looser aggregations. However, bition of Rac1 and the overexpression of Cdc42 Student Trainee Yukiko Nakaya these two cell types are by no means isolated or were found to induce the conversion of epithelial Tadayoshi Watanabe Emi Ohata independent from one another; indeed, a great cells to mesenchyme, while interference with Cdc42 Itsuki Kashin many developmental function caused Assistant Tomoko Oyanagi and physiological phe- mesenchymal cells to Part-time Staff nomena rely on interac- take on epithelial char- Naomi Akira tions between epitheli- acteristics. These roles um and mesenchyme. seem to be at least part- An extreme example of ly specific to somites; their inter-relatedness is neither Cdc42 nor Rac1 the ability of cells of one was found to be type to convert into the involved in epithelial- other, in a process mesenchymal transi- known as epithelial- tions in epiblast cells. mesenchymal transi- These findings suggest Creative Research tion. Such transitions that the mesenchymal- Promoting Program (which can proceed in epithelial transition that either an epithelial-to- occurs during somitoge-

56 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 57 Staff

Team Leader Teruhiko Wakayama Research Scientist Satoshi Kishigami Nguyen Van Thuan Hiroshi Ohta Takafusa Hikichi Technical Staff Sayaka Wakayama Student Trainee Eiji Mizutani Assistant Kana Tachibana Oocyte (large, upper left) and somatic (small, bottom center) spindles following nuclear transfer Creative Research Promoting Program

posed to explain the inefficiency of this procedure, cient than fertilization using mature spermatozoa, but while accounting for the fact that cloning is not alto- the reason for this lower developmental competency gether impossible. It has been suggested that the remains unknown. The Laboratory for Genomic Repro- The experimental cloning of animals has a history code in a specialized cell's nucleus is sufficient to process of removing the nucleus from an oocyte (an gramming is now comparing in vitro fertilization using that extends back more than fifty years, to when instruct an egg into which it is transplanted to give unfertilized egg) or the absence of chromosomal spermatozoa and spermatids under controlled condi- Briggs and King successfully produced tadpoles by rise to a normal individual. information during the several hours in which the tions in an effort to identify the differences between transplanting the nuclei from embryonic cells into egg is missing a nucleus may somehow damage or these stages in the developing sperm cell. Future frogs' eggs whose own nuclei had been removed. At Although questions of whether genetic information is cause the loss of factors that would normally act to research will look at changes in epigenetic modifica- the time, cloning was used not as a technique to be lost or irreversibly altered during development were reprogram the genetic information in the fertilizing tions to the sperm genome over time as a possible studied for its immediate application, but as a laid to rest, new questions arose to take their place. (or transplanted) cell's nucleus. To test this possibili- explanation for their disparate potentials. means of testing a fundamental question of repro- For Teruhiko Wakayama, the most intriguing issue ty, Wakayama re-ordered conventional cloning meth- ductive biology: whether the processes of fertiliza- raised by the ability of animals to be cloned using odology by first transferring nuclei from cumulus New technologies Publications tion and later development, in which the body's cells the nuclei of specialized somatic cells (which form cells into oocytes whose own nuclei were still pres- Sperm preserved for use in experiments and in vitro Singh U, Fohn L, Wakayama become more specialized and functionally distinct, the body and, unlike sperm and eggs, cannot nor- Teruhiko Wakayama ent, and only then removing the mitotic spindle fertilization is traditionally frozen in liquid nitrogen at T, Ohgane J, Steinhoff C, Lip- Ph. D. involves a loss of genetic information, or whether all mally produce a new individual), is that of reprogram- derived from the native nucleus. These experiments extremely low temperatures (around -190ûC). This kowitz B, Schulz R, Orth A, Teruhiko Wakayama cells retain the full set of genetic code, even after dif- ming, the processes by which the genome receives resulted in the generation of live offspring at a rate cryopreservation allows the sperm to be maintained Ropers HH, Behringer R, received his B. Sc. and M. ferentiation has proceeded. The success of these ini- new sets of coding instructions enabling it to order of efficiency similar to that of standard cloning by viably for very long periods, but requires expensive Tanaka A, Shiota K, Yanagi- Sc. from Ibaraki University, machi R, Nuber U and Fun- tial experiments in cloning by nuclear transfer dem- the development of all the cells in a new individual nuclear transfer, providing evidence that tends to facilities and some degree of technical skill in han- and was awarded a Ph. D. dele R. Abnormal gene in reproductive biology from onstrated conclusively that cells do maintain intact while remaining intact and fundamentally counter the hypothesis that the temporary absence dling. Oocytes and fertilized eggs are also extremely expression in mouse placen- the University of Tokyo genomes even after differentiation, as the genetic unchanged in each of those cells. The Wakayama of a nucleus is responsible for the poor developmen- labile, and must likewise be stored cryogenically. topathies caused by interspe- Department of Veterinary lab studies mammalian cloning tal prospects of NT oocytes. But these requirements tend to limit the access of cies hybridization, cloning, Anatomy in 1996. He and fertilization with the same germ cells to researchers unequipped with liquid and Esx1 mutation. Develop- received a postdoctoral fel- mental Dynamics (2004). lowship from the Japanese basic goal as drove Briggs and Many hypotheses nitrogen facilities, preventing the spread of the tech- Society for the Promotion of King in their nuclear transfer nology and the development of research using these Wakayama S, Cibelli JB and Science in 1996 and spent have been proposed to explain studies a half century ago: to cells. Wakayama hopes to develop new, less expen- Wakayama T. Effect of tim- the next three years at the cloning's inefficiency, answer central questions in the ing of the removal of oocyte Yanagimachi lab in the Uni- sive and less technically demanding methods for the while accounting for the fact chromosomes before or after versity of Hawaii Medical biology of animal reproduction. storage and maintenance of germ cells for experi- injection of somatic nucleus School, where he succeed- that it is not altogether impossible mental use. Recent tests using a modified commer- on development of NT ed in producing the world’s Cloning cially available culture medium showed that under embryos. Cloning Stem Cells first cloned mouse. He was Scientists in the Wakayama lab have adopted similar 5:181-9 (2003). appointed to an assistant efficiencies the right conditions spermatozoa can be preserved professorship at the same In all species and in all experi- approaches to the study of fertilization — experiments for long periods, up to 70 days, at 4 degrees Cel- Tamashiro KL, Wakayama T, institution in 1998, and mental methods tested to date, that involve the substitution of components or the reor- sius, a temperature that can be maintained using Akutsu H, Yamazaki Y, moved to Rockefeller Uni- cloning by nuclear transfer has dering of natural sequences of events to test for specif- ordinary and inexpensive refrigeration equipment. Lachey JL, Wortman MD, versity as a research assis- consistently low efficiency rates ic biological function. In nature, fertilization occurs after Seeley RJ, D'Alessio DA, tant professor in 1999. He This new preservation method opens up opportuni- Woods SC, Yanagimachi R spent a year as a research- — generally below 5% of enu- a mature spermatozoon fuses with and activates an ties in animal breeding and reproductive biology and Sakai RR. Cloned mice er at Advanced Cell Tech- cleated eggs fertilized by oocyte, but some laboratory techniques achieve fertili- research to scientists working under limited budgets, have an obese phenotype nology before returning to nuclear transfer go on to devel- zation by artificially activating the egg and then inject- which Wakayama hopes may help to make these Japan to take his current not transmitted to their off- op into live-born offspring. Many ing a spermatid, which is an incompletely maturated position at the RIKEN CDB. fields of science more accessible to developing spring. Nat Med 8:262-7 hypotheses have been pro- sperm cell. Such techniques are known to be less effi- countries and smaller labs. (2002). Wakame, the first mouse cloned at the RIKEN CDB

58 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 59 Staff

58 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 59 Publications Publications

Payer B, Saitou M, Barton SC, Thresher R, Dixon JP, Zahn D, Ueda HR, Hayashi S, Matsuyama S, Yomo T, Hashimoto S, Kay S A, Hogenesch Colledge WH, Carlton MB, Nakano T and Surani MA. stella is a J B and Iino M. Universality and Flexibility in Gene Expression from Bacteria to maternal effect gene required for normal early development in Human. Proc Nat Sci USA (2004). mice. Curr Biol 13:2110-7 (2003). Ueda H R, Chen W, Adachi A, Wakamatsu H, Hayashi S, Takasugi T, Nagano M, Saitou M, Payer B, Lange UC, Erhardt S, Barton SC and Sura- Nakahama K, Suzuki Y, Sugano S, Iino M, Shigeyoshi Y and Hashimoto S. A tran- ni MA. Specification of germ cell fate in mice. Philos Trans R scription factor response element for gene expression during circadian night. Soc Lond B Biol Sci 358:1363-70 (2003). Nature 418:534-9 (2002).

Lange UC, Saitou M, Western PS, Barton SC and Surani MA. Ueda H R, Hirose K and Iino M. Intercellular coupling mechanism for synchronized The fragilis interferon-inducible gene family of transmembrane and noise-resistant circadian oscillators. J Theor Biol 216:501-12 (2002). proteins is associated with germ cell specification in mice. BMC Staff Dev Biol 3:1 (2003). Ueda H R, Matsumoto A, Kawamura M, Iino M, Tanimura T and Hashimoto S. Staff Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J Team Leader Team Leader Mitinori Saitou Saitou M, Barton SC and Surani MA. A molecular programme Biol Chem 277:14048-52 (2002). Hiroki R. Ueda Research Scientist for the specification of germ cell fate in mice. Nature 418:293- Research Scientist Kazuki Kurimoto 300 (2002). Ueda H R, Hagiwara M and Kitano H. Robust oscillations within the interlocked Hideki Ukai Yasuhide Ohinata feedback model of Drosophila circadian rhythm. J Theor Biol 210:401-6 (2001). Collaborative Scientist Yukihiro Yabuta Yuichi Kumaki Collaborative Scientist Technical Staff Yukiko Ono Maki Ukai-Tadenuma Technical Staff Hiroshi Fujishima Mihoko Yuasa Kenichiro Uno Mayo Shigeta Student Trainee Yoichi Minami Rikuhiro Yamada Ryotaku Kito Assistant Ikuko Tada

A case could be made for the statement that the cells, research that ultimately yielded a number of Recent large-scale efforts in genome-sequencing tral (suprachiasmatic nucleus; SCN) and peripheral ultimate role of all of the other types of cells in the molecules that act to distinguish cells on the path- and expression analysis have produced an embar- (liver) circadian clocks. This phase of the study body is to ensure that germline cells — sperm and way to differentiation into germ cells. The first of rassment of riches for life science researchers - bio- required the development of a genome-wide promot- 2003 eggs — are able to fulfill their task of conveying these molecules, fragilis, is the product of a gene logical data can now be accessed in quantities that er database, which the Ueda lab will make available 2003 New Faces genetic information from one generation to the next. found to be strongly and specifically expressed in are orders of magnitude greater than were available to all researchers at the CDB. Subsequent phases New Faces But despite this centrality, many aspects of germ cells en route to assuming a germ cell fate, the even a few years ago. This burgeoning set of raw involved determining gene transcription start sites cell biology have been, and remain to this day, poor- result of inductive signaling from cells lying outside data has not, however, necessarily led to equally across the entire genome, predicting the regulatory ly understood. There has been, however, progress the embryo proper. A second such protein, stella, explosive advances in the understanding of the rela- sequences involved in time-specific transcription, in deepening this understanding, particularly in acts even more specifically, and is expressed only in tionships between its component parts. The need for and studying their actual functions in vitro using a regard to the mechanisms by which germ cells are a core subset of fragilis-expressing primordial germ integration has set the stage for the advent of sys- high-throughput real-time monitoring system for specified and segregated from the larger population cells. The co-expression of fragilis and stella serves tems biology, in which discrete biological processes luciferase-tagged transgenes. Analysis of the tran- of somatic cells early in development. This is to distinguish nascent germ cells from the somatic and phenomena are approached as complex, inter- scriptional regulation of gene expression in the morn- achieved by different means in different species. In cell community in the developing embryo, and their active systems. Hiroki Ueda sees systems biology ing, daytime, evening and night periods revealed a some animals, germ cell fate is predetermined from activity is thought to play a part in the gene network research as a multi-stage process, beginning with gene network comprising sixteen inter-regulating the very onset of development, in others, including that maintains germ cell characteristics and sup- the identification and analysis of individual system activators and inhibitors of time-linked gene expres- all mammals, all cells in the early embryo have the presses those of the somatic lineage. Single cell components and their networked interactions, and sion. potential to take on a germline fate, but only a small analysis has also revealed that repression of Hox leading to the ability to control existing systems and percentage of them actually do. genes and maintenance of genes linked to pluripot- design new ones based on an understanding of The initial success of the systems approach to the ency, such as Oct4, are the two major events that structure and underlying principles. mammalian circadian clock has been encouraging, Mitinori Saitou Mitinori Saitou studies the ways in which germ cell occur during germ cell lineage-restriction. and the Ueda lab now seeks to apply similar Hiroki R. Ueda M. D. , Ph. D. fate is specified in mammalian embryonic develop- The Ueda lab has taken the mammalian circadian genome-wide, high-throughput technologies to more Ph. D. Mitinori Saitou received his ment, using the mouse as a representative model. Although progress has been made, much about the clock as a relatively simple and self-contained initial involved and elaborate developmental processes. Hiroki R. Ueda received his MD from the Kyoto He joins the CDB from Azim Surani's lab at the Well- specification of the germline has yet to be explained. model for the study of a biological system. In addi- Guiding that research will be Einstein's (and the sys- MD from Faculty of Medi- University Faculty of cine, the University of Medicine in 1995, and was come Trust/Cancer Research UK Institute, where he The identification of fragilis and stella now offers a tion to its advantages as a basic research model, tem biologist's) dictum to "Make everything as sim- Tokyo in 2000, and will awarded a Ph D in 1999 for worked on the single cell analysis of primordial germ means to identify incipient germ cells, but the initial the function of the circadian clock is intimately ple as possible, but not simpler." receive PhD from Graduate his study of the structure molecular trigger for germ cell involved in the control of metabolic and hormonal School of Medicine, the Uni- and function of mammalian versity of Tokyo in March tight junctions under Prof. determination and key (epi-) cycles, and its dysregulation is linked to the onset 2004. While an undergradu- Shoichiro Tsukita in the genetic properties that determined and symptomatology of numerous human diseases, ate student, he worked as a Kyoto University Graduate research assistant on a bio- germ cells acquire are still including sleep disorders. An improved understand- Analysis of the transcrip- School of Medicine. After a tional regulation of gene logical simulation system brief fellowship in the same unknown. The Saitou lab plans to ing at the system level promises to provide biomedi- expression in the morn- project at Sony Computer department, he moved to develop comprehensive gene cal and clinical investigators with a powerful new ing, daytime, evening and Science Laboratories. the Wellcome Trust/Cancer night periods has While a graduate student, Research UK Institute in expression analysis at the single arsenal to attack these conditions. revealed a gene network he next went on to work, 2000, where worked as a cell level to search for the molecu- comprising more than first as a researcher (2000) postdoctoral research lar mechanisms that underlie this To address complex and dynamic sixteen component and then as a group leader associate in Azim Surani's factors. (2002), at Yamanouchi laboratory, focusing on the process, and to identify the exter- biological systems such as the circa- Pharmaceutical, on a pro- origin and properties of the ject studying biological nal factors that instruct a pluripot- dian clock, it is necessary to make E/E'box D-box germ line in the mouse. He clock mechanisms in fly remained there until 2003, ent cell to take on a germ cell comprehensive and precise meas- and mouse. He was when he returned to Japan fate. The lab also hopes to devel- urements of the system's dynamics appointed team leader at to take up his current op technologies to maintain imma- and to work out the organization of the CDB in 2003, and will position as head of the assume this position full CDB Laboratory for ture oocytes (egg cells), and to its underlying gene network. The time in April 2004. Mammalian Germ Cell induce their maturation in vitro, Ueda lab has conducted a genome- Biology. He received a thereby enabling oocyte-mediated wide screen and statistical analysis three-year grant from the Japan Science and gene transfer techniques. of gene expression to work out the Technology Corporation clock-controlled genes that are (JST) PRESTO program RRE that same year. rhythmically expressed in the cen- Gene expression profiles of BMP4 negative single PGCs and neighbouring somatic cells

60 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 61 Publications Publications

60 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 61 The supporting laboratories offer technical services, develop new technology, and conduct independent research projects. Their services are available to all CDB research groups and teams.

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S tubules in the mitotic apparatus, and which is essen- tion and of actin bundles. The tial to ensuring that each progeny cell receives one study showed that disruption of and only one set of chromosomes. However, the actin bundles affects the integrity question of which complement of mitotic apparatus of the adherens junction, indicat- microtubules — astral or central — spatially deter- ing the importance of Rho-actin mines this cleavage remains unresolved. Yonemura signaling in this form of cell-cell studied Rho accumulation at the putative furrow site, adhesion. taking this as an index of cleavage plane determination signaling, and analyzed the roles of both micro- ERM protein tubule organizations by selectively disrupting each. recognition-site The study showed that both astral and central micro- structure tubules are able to transmit determinant signals to ERM proteins, named for three the cell cortex, a finding that helps explain how representative members of the astral microtubules that extend to the cell cortex can family, ezrin, radixin and moesin, act as links play an important role in cleavage plane determina- between actin filaments and the plasma membrane. Staff tion in larger cells, such as oocytes, in which central Yonemura and colleagues studied the crystal struc- Team Leader Shigenobu Yonemura microtubules are located more remotely from the ture of a functional domain of radixin to identify the Research Scientist cortex, while being dispensable in the same process structural basis for adhesion molecule recognition Yukako Nishimura Research Specialist in smaller cells in which central microtubules are by one ERM protein, radixin. Radixin plays a role in Kazuyo Minakuchi located more proximally to the cell's periphery. the formation of microvilli, cellular extensions in Research Associate Nagatoki Kinoshita which actin filaments are closely associated with the Technical Staff Naoko Inoue Actin fibers and plasma membrane. The importance of radixin in Makiko Uwo the adherens junction sites of bilirubin secretion is suggested by the find- Yuka Miyake Student Trainee The main zone of contact between a cell and its ing that mice lacking radixin, which is the dominant Yuko Shimada Supporting neighbors is called the adherens junction, which is ERM protein in the liver, show severely impaired defined by the selective binding of transmembrane hepatic microvilli formation, resulting in a phenotype Laboratories adhesion molecules that are themselves anchored similar to human conjugated hyperbilirubinemia, a to molecular complexes rare autosomal reces- located within each cell's sive liver disorder. This interior. In fibroblastic work provides the first cells in culture, bundles of insights into the struc- Cellular morphology is defined by interactions involv- Cleavage plane determination actin filaments that run tural bases for the recog- ing mechanical and osmotic stresses from the cell's The final stages of mitotic cell division involve a perpendicular to the nition between an ERM external environment, its permeable membrane, its process known as cytokinesis, in which the parent plasma membrane are protein and an adhesion viscous cytosolic content, and an internal framework cell cleaves in two, partitioning cytoplasmic connected to the compon- molecule, and may Publications comprising pre-stressed tubular struts, tensile elements and genetic information into each of the ents of the adherens junc- further serve as an cables and a lattice-like filamentous cortex. Micro- daughter cells. Cytokinesis begins with the tion, a phenomenon that experimental model for Hamada K, Shimizu T, Yone- mura S, Tsukita S and tubules are one important and extensively studied appearance of a cleavage furrow at the cell surface, is also observed in epithel- the study of molecular Hakoshima T. Structural component of this cytoskeletal structure, acting both a process which is known to be specified by micro- ial cell adhesion. In many processes involved in basis of adhesion-molecule as internal stress-bearing braces and pathways to cells, the mature adher- bile secretion. recognition by ERM proteins guide intracellular transport. A second critical form- ens junction forms a revealed by the crystal struc- giving element, actin filaments, are most prevalent contiguous belt, although In addition to its full-time ture of the radixin-ICAM-2 Shigenobu complex. Embo J 22:502-14 in cellular peripheries, where they provide a stable during the process of its research efforts, the Yonemura Mouse neutrophils migrating on a substratum. Phase contrast (blue) (2003). Ph. D. but regulable meshwork enabling the cell to main- formation it passes and fluorescence image of ERM proteins (magenta) are merged. Yonemura lab offers Shigenobu Yonemura tain its structural integrity while responding to exter- through a stage character- site-wide support for Fujikura J, Yamato E, Yone- received his B. Sc., M. Sc. and nal and internal stimuli through shape alterations ized by discrete, spot-like adhesions. The Yonemura electron microscopy, services available to all mura S, Hosoda K, Masui S, Ph. D. from the University of Nakao K, Miyazaki Ji J and Tokyo, earning his doctorate in and movements. lab used NRK cells, a type of non-polarized fibro- research staff at the CDB. By providing fluorescence Niwa H. Differentiation of 1988 for thesis work under blast which forms spot-like junction zones, to study and electron microscopic visualizations as well as embryonic stem cells is Prof. I. Mabuchi. He spent a year as a postdoctoral fellow Actin filaments maintain cell the role of actin fibers in adherens junction forma- instruction in techniques, the team hopes to help induced by GATA factors. at the same institution before tion. In this cell line, the adherens junction and its ensure the quality, fidelity and ease-of-interpretation Genes Dev 16:784-9 (2002). structural integrity while moving to pursue a fellowship associated actin bundles can be disrupted and of visualization data generated at at Johns Hopkins University responding to external and Kikuchi S, Hata M, Fukumoto from 1989 to 1990. He NRK cells at cytokinesis. Rho (magenta) accumulates at the equato- reconstituted by removing or adding serum to the the Center. K, Yamane Y, Matsui T, returned to Japan as an assis- internal stimuli rial cell cortex depending on microtubule organizations (green). culture medium. Using this system, he Tamura A, Yonemura S, tant professor in the Depart- Yamagishi H, Keppler D and ment of Cell Biology at the revealed that the activity of the Tsukita S. Radixin deficiency National Institute for Physiolog- The behavior of actin is intimately linked to the activ- small GTPase, Rho, is causes conjugated hyperbilir- ical Sciences, where he ity of a group of related small GTPase proteins required both for the remained until 1995. He joined ubinemia with loss of Mrp2 the Kyoto University Faculty of known as the Rho family, which is characterized by formation of the from bile canalicular mem- Medicine the same year, first the actin-regulatory functions of its three primary adherens branes. Nat Genet 31:320-5 as an assistant professor, then members, Rho, Rac and Cdc42. The spatial and junc- (2002). as a lecturer in the Depart- ment of Cell Biology, a posi- temporal localization of Rho-family proteins has Yonemura S, Matsui T and tion he retained until his been shown to be important in the structural organi- Tsukita S. Rho-dependent appointment as CDB team leader in 2001. zation of cells in culture. Shigenobu Yonemura is and -independent activation working to develop technologies to allow the precise mechanisms of ezrin/radix- determination of the localization of these proteins in in/moesin proteins: an The tip of a villus of the essential role for poly- cultured cells and tissues within specific biological small intestine of the rat. phosphoinositides in vivo. J Aged cells that are exfo- contexts. NRK cells contacting each other thorough adherens junctions (magen- Cell Sci 115:2569-80 (2002). liated highly express Rho ta) where actin filament bundles (green) are tightly connected. protein (green).

64 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 65 tubules in the mitotic apparatus, and which is essen- tion and of actin bundles. The tial to ensuring that each progeny cell receives one study showed that disruption of and only one set of chromosomes. However, the actin bundles affects the integrity question of which complement of mitotic apparatus of the adherens junction, indicat- microtubules — astral or central — spatially deter- ing the importance of Rho-actin mines this cleavage remains unresolved. Yonemura signaling in this form of cell-cell studied Rho accumulation at the putative furrow site, adhesion. taking this as an index of cleavage plane determination signaling, and analyzed the roles of both micro- ERM protein tubule organizations by selectively disrupting each. recognition-site The study showed that both astral and central micro- structure tubules are able to transmit determinant signals to ERM proteins, named for three the cell cortex, a finding that helps explain how representative members of the astral microtubules that extend to the cell cortex can family, ezrin, radixin and moesin, act as links play an important role in cleavage plane determina- between actin filaments and the plasma membrane. Staff tion in larger cells, such as oocytes, in which central Yonemura and colleagues studied the crystal struc- Team Leader Shigenobu Yonemura microtubules are located more remotely from the ture of a functional domain of radixin to identify the Research Scientist cortex, while being dispensable in the same process structural basis for adhesion molecule recognition Yukako Nishimura Research Specialist in smaller cells in which central microtubules are by one ERM protein, radixin. Radixin plays a role in Kazuyo Minakuchi located more proximally to the cell's periphery. the formation of microvilli, cellular extensions in Research Associate Nagatoki Kinoshita which actin filaments are closely associated with the Technical Staff Naoko Inoue Actin fibers and plasma membrane. The importance of radixin in Makiko Uwo the adherens junction sites of bilirubin secretion is suggested by the find- Yuka Miyake Student Trainee The main zone of contact between a cell and its ing that mice lacking radixin, which is the dominant Yuko Shimada Supporting neighbors is called the adherens junction, which is ERM protein in the liver, show severely impaired defined by the selective binding of transmembrane hepatic microvilli formation, resulting in a phenotype Laboratories adhesion molecules that are themselves anchored similar to human conjugated hyperbilirubinemia, a to molecular complexes rare autosomal reces- located within each cell's sive liver disorder. This interior. In fibroblastic work provides the first cells in culture, bundles of insights into the struc- Cellular morphology is defined by interactions involv- Cleavage plane determination actin filaments that run tural bases for the recog- ing mechanical and osmotic stresses from the cell's The final stages of mitotic cell division involve a perpendicular to the nition between an ERM external environment, its permeable membrane, its process known as cytokinesis, in which the parent plasma membrane are protein and an adhesion viscous cytosolic content, and an internal framework cell cleaves in two, partitioning cytoplasmic connected to the compon- molecule, and may Publications comprising pre-stressed tubular struts, tensile elements and genetic information into each of the ents of the adherens junc- further serve as an cables and a lattice-like filamentous cortex. Micro- daughter cells. Cytokinesis begins with the tion, a phenomenon that experimental model for Hamada K, Shimizu T, Yone- mura S, Tsukita S and tubules are one important and extensively studied appearance of a cleavage furrow at the cell surface, is also observed in epithel- the study of molecular Hakoshima T. Structural component of this cytoskeletal structure, acting both a process which is known to be specified by micro- ial cell adhesion. In many processes involved in basis of adhesion-molecule as internal stress-bearing braces and pathways to cells, the mature adher- bile secretion. recognition by ERM proteins guide intracellular transport. A second critical form- ens junction forms a revealed by the crystal struc- giving element, actin filaments, are most prevalent contiguous belt, although In addition to its full-time ture of the radixin-ICAM-2 Shigenobu complex. Embo J 22:502-14 in cellular peripheries, where they provide a stable during the process of its research efforts, the Yonemura Mouse neutrophils migrating on a substratum. Phase contrast (blue) (2003). Ph. D. but regulable meshwork enabling the cell to main- formation it passes and fluorescence image of ERM proteins (magenta) are merged. Yonemura lab offers Shigenobu Yonemura tain its structural integrity while responding to exter- through a stage character- site-wide support for Fujikura J, Yamato E, Yone- received his B. Sc., M. Sc. and nal and internal stimuli through shape alterations ized by discrete, spot-like adhesions. The Yonemura electron microscopy, services available to all mura S, Hosoda K, Masui S, Ph. D. from the University of Nakao K, Miyazaki Ji J and Tokyo, earning his doctorate in and movements. lab used NRK cells, a type of non-polarized fibro- research staff at the CDB. By providing fluorescence Niwa H. Differentiation of 1988 for thesis work under blast which forms spot-like junction zones, to study and electron microscopic visualizations as well as embryonic stem cells is Prof. I. Mabuchi. He spent a year as a postdoctoral fellow Actin filaments maintain cell the role of actin fibers in adherens junction forma- instruction in techniques, the team hopes to help induced by GATA factors. at the same institution before tion. In this cell line, the adherens junction and its ensure the quality, fidelity and ease-of-interpretation Genes Dev 16:784-9 (2002). structural integrity while moving to pursue a fellowship associated actin bundles can be disrupted and of visualization data generated at at Johns Hopkins University responding to external and Kikuchi S, Hata M, Fukumoto from 1989 to 1990. He NRK cells at cytokinesis. Rho (magenta) accumulates at the equato- reconstituted by removing or adding serum to the the Center. K, Yamane Y, Matsui T, returned to Japan as an assis- internal stimuli rial cell cortex depending on microtubule organizations (green). culture medium. Using this system, he Tamura A, Yonemura S, tant professor in the Depart- Yamagishi H, Keppler D and ment of Cell Biology at the revealed that the activity of the Tsukita S. Radixin deficiency National Institute for Physiolog- The behavior of actin is intimately linked to the activ- small GTPase, Rho, is causes conjugated hyperbilir- ical Sciences, where he ity of a group of related small GTPase proteins required both for the remained until 1995. He joined ubinemia with loss of Mrp2 the Kyoto University Faculty of known as the Rho family, which is characterized by formation of the from bile canalicular mem- Medicine the same year, first the actin-regulatory functions of its three primary adherens branes. Nat Genet 31:320-5 as an assistant professor, then members, Rho, Rac and Cdc42. The spatial and junc- (2002). as a lecturer in the Depart- ment of Cell Biology, a posi- temporal localization of Rho-family proteins has Yonemura S, Matsui T and tion he retained until his been shown to be important in the structural organi- Tsukita S. Rho-dependent appointment as CDB team leader in 2001. zation of cells in culture. Shigenobu Yonemura is and -independent activation working to develop technologies to allow the precise mechanisms of ezrin/radix- determination of the localization of these proteins in in/moesin proteins: an The tip of a villus of the essential role for poly- cultured cells and tissues within specific biological small intestine of the rat. phosphoinositides in vivo. J Aged cells that are exfo- contexts. NRK cells contacting each other thorough adherens junctions (magen- Cell Sci 115:2569-80 (2002). liated highly express Rho ta) where actin filament bundles (green) are tightly connected. protein (green).

64 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 65 Publications

K Chida, T Hara, T Hirai, C Konishi, K Nakamura, K Nakao, A Aiba, M Katsuki and T Kuroki. Disruption of Protein Kinase Ceta Results in Impairment of Wound Healing and Enhancement of Tumor For- mation in Mouse Skin Carcinogenesis. Cancer Res 63:2404-8 (2003).

J M Kim, K Nakao, K Nakamura, I Saito, M Katsuki, K Arai and H Masai. Inactiva- tion of Cdc7 Kinase in Mouse ES Cells Results in S-Phase Arrest and P53- Dependent Cell Death. Embo J 21:2168- 79 (2002).

K Kuwahara, S Fujimura, Y Takahashi, N Nakagata, T Takemori, S Aizawa and N Sakaguchi. Germinal Center-Associated Nuclear Protein Contributes to Affinity Mat- uration of B Cell Antigen Receptor in T Cell-Dependent Responses. Proc Natl Acad Sci U S A (2004).

S Muto, A Aiba, Y Saito, K Nakao, K Nakamura, K Tomita, T Kitamura, M Kura- bayashi, R Nagai, E Higashihara, P C Har- ris, M Katsuki and S Horie. Pioglitazone Improves the Phenotype and Molecular Supporting Defects of a Targeted Pkd1 Mutant. Hum Supporting Mol Genet 11:1731-42 (2002). Laboratories Laboratories S Takaki, Y Tezuka, K Sauer, C Kubo, S M Kwon, E Armstead, K Nakao, M Katsu- ki, R M Perlmutter and K Takatsu. Impaired Lymphopoiesis and Altered B Cell Subpopulations in Mice Overexpress- ing Lnk Adaptor Protein. J Immunol The study of model organisms and systems is inte- 170:703-10 (2003). The CDB Sequencing Laboratory provides DNA sequenc- gral to biological research. Such systems provide ing and analysis services for use by all laboratories at the scientists with the means to search for broadly Y Wakabayashi, H Watanabe, J Inoue, N Takeda, J Sakata, Y Mishima, J Hitomi, T Center. Equipment is available for small, medium and Staff shared mechanisms underlying developmental and Yamamoto, M Utsuyama, O Niwa, S Aiza- large-scale requests, with turnaround times ranging from Research Scientist regenerative processes across species, and wa and R Kominami. Bcl11b Is Required under one week for medium-scale projects to one month Hiroshi Tarui conversely to identify those traits that earn each for Differentiation and Survival of Alpha- Technical Staff beta T Lymphocytes. Nat Immunol 4:533- for larger jobs, at rates of up to 1,920 samples per day. Junko Ueda species its unique branch on the phylogenetic tree. 9 (2003). Kenichiro Uno Smaller request are fulfilled especially rapidly, and results Mikako Dohi The mouse is one of the most important and widely can be delivered as soon as the following day when Takashige Hakozaki Staff used model organisms in science today, prized for Yukako Hirao sequence-ready samples are submitted. Analysis services its amenability to genetic manipulation, its high level Team Leader for editing, homology search, and Shinichi Aizawa of homology with humans, and the trove of data Attending Veterinarian assembling are also available. Naoko Kagiyama regarding its physiology, genetics and development Research Associate that has accumulated over nearly one century of Kazuki Nakao In addition to DNA sequencing, the Special Postdoctoral Researcher intensive scientific research. Kenryo Furushima lab also offers a full range of DNA Senior Technical Staff Hitoshi Miyachi The Laboratory for Animal Resources and Genetic In 2003, the LARGE team started to microarray services, from the amplifi- Rika Nakayama cation of target DNA and the prepara- Hiroshi Kiyonari Engineering (LARGE) provides an important suite of expand its services and initiate new Technical Staff services related to the generation of experimental programs, notably the generation of tion of probe cDNA, to microarray Chiaki Tamamura Akemi Hara mice to labs within the CDB and around Japan. In its target vectors from sequence informa- image data analysis. All work Takaya Abe requests can be submitted and Naoko Ohshima role as a CDB support laboratory, the LARGE team tion alone, and the independent Hirotake Nishino produces transgenic and knockout mouse models to production of novel genetically- tracked online, and results are Assistant Kaori Nasu the specifications of modified constructs, a drive that gener- returned to the applicant's personal scientists working in ates on the order of 40-50 new mutant folder on the CDB intranet a wide range of strains per year. Such strains serve file server, making it possi- genetic, embryologi- as research platforms with the poten- ble for CDB research staff cal and biomedical tial to provide new insights into a to sequence samples of research projects, Chimera mice range of important research problems, interest without leaving maintaining the high- from the developmental mechanisms their desktop computers. est quality standards of organ development to the genetic The lab also stores and and rapid turnar- to these core functions, the LARGE staff provides a bases of human disease. The lab will distributes DNA resources, ound to ensure fast number of other services, such as cloning by also function as part of Japan's making clones available to and easy access to nuclear transfer and cryopreservation of mouse system of Mouse Embryo Banks, with all members of the CDB researchers working zygotes and sperm. The lab also performs a number a special emphasis on producing, stor- staff. within the Center of maintenance and logistical functions, such as the ing and cataloging embryos for use in and throughout the specific pathogen free (SPF) housing, cleaning, developmental biology and regenera- country. In addition processing and distribution of animals. tive medical research.

66 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 67 Publications

66 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 67 Administration and Animal Facility Activities

Mouse biology provides a valuable platform for investigations into many of the fundamental processes and mechanisms of mammalian physiology and metabolism, and is widely used as a model organism in developmental biology research. With the sequencing of the mouse genome, mice can now be modified for use in research into the developmental consequences of genetic alterations, and as models of human disease.

The CDB animal facility provides the technology, equipment and staff to enable the humane, hygienic, efficient and economical handling of large numbers of experimental mice. This 24,000 cage SPF facility has the capacity to handle and care for up to 100,000 mice. Cutting edge technology, such as semi- automated cage cleaning, makes it possible to maintain high standards of hygiene and steril- ity, minimizing the risk of outbreaks of conta- gion among the mouse populations. The facility also provides services for the production of transgenic and knockout mice to specifications and on demand.

Research Aquarium

The CDB research aquarium serves as home to a number of water-dwelling species used in research into development and regeneration, including the zebrafish (Danio rerio) and the African clawed frog (Xenopus laevis). These, and other aquatic species, provide useful models for studies ranging from developmental genomics to classical embryology.

The aquarium includes seven climate-controlled rooms geared to providing optimal environments for the handling and breeding of freshwater and marine species, and utilizes reverse osmotic technology to maintain consistent tank- water purity. In addition to the commonly used zebrafish and African clawed frog, the facility also houses specimens from more novel models used in evolutionary development research, such as the lamprey, Lampetra japonica.

Photo: Yutaka Doi 68 RIKEN CDB 2003 Annual Report Administration and Animal Facility Activities

Research Aquarium

Photo: Yutaka Doi 68 RIKEN CDB 2003 Annual Report Graduate School 2003 CDB Budget Affiliates The Center for Developmental Biology was launched in April 2000 under the auspices of the Millennium Project Encouraging young scientists to participate in advanced research pro- research initiative launched by former Prime Minister Keizo Obuchi. The Millennium Projects were established to drive K research in the fields of information technology, environmental science and the study of aging, areas of vital importance o jects as they prepare for careers in the life sciences is essential to b to both Japan and the world in the 21st century. The organization and operations of the CDB were consigned to RIKEN, e ensuring the development of future generations of researchers. The U CDB has collaborative educational programs with a number of gradu- which coordinates the construction management and administration of the Center. n i ate schools in Western Japan, providing students with opportunities v e r for hands-on laboratory benchwork and lectures on topics in develop- s i ment and regeneration. The center also has also accepted 50 stud- t y , ents from other universities as research associates or trainees. In addi- G r tion to regularly scheduled scientific seminars, students can attend FY2003 a d events such as the CDB Forum research progress series and the Cen- u 762 a ter's annual retreat. The Junior Research Associate program estab- t e lished by RIKEN also provides financial support to 8 graduate students S c h working in CDB labs. 1827 o o l o f S c ie n ce a 2732 n Gr N d aduat a Te e School of Biostudies, Kyoto University Facility Expenses ra c A hn (energy expenses, maintenance,etc.) d o v log Grad an y uate School of Medicine, Kyoto University c De Research Management Expenses ed par i tmen (personnel expenses, etc.) ns t of Li tit fe Scien (Million Yen) ute ce Department of Developmental and Regenerative medicine of Research Expenses Sci ence and T echnology Graduate School of Biological Sciences

CDB Staff (December 2002) Advisory Council Kwa nsei Gakuin University Bioscience Department GD/TL/UL 30 The CDB Advisory Council, chaired by Dr Igor Dawid (Nation- Research scientists 101 al Institute of Child Health and Human Development, NIH) sub- University of Texas Graduat Research associates 3 mits regular external reports regarding the scientific adminis- e School of Biome Technical staff 105 tration and the state of research progress at the Center. The dica l Sc Assistants 25 ten-member Council includes top scientists in related fields Temasek Life Sciences Laborato ien ry ces Visiting researchers 9 from Japan and around the world and serves as an unbiased a t H Collaborative scientists 30 review board for CDB research activities. ou st Student trainees 47 on Part time staff 53 Research promotion division 43 Institutional Review Board Other 65 The Institutional Review Board includes representatives from local academic, research and lay organizations as well as Scientific Exchange Total 511 CDB research staff, and meets regularly to review and dis- cuss the ethical and social implications of programs and inves- Programs tigations being conducted at the CDB. The results of the The CDB is also engaged in programs of cooperation with research Board's discussions are submitted to the Center Director and organizations in other parts of Japan and around the world. This taken into consideration when planning research activities. activity is based primarily on cooperation between individual research laboratories. These scientific exchange agreements facilitate processes such as the hosting and dispatch of research staff for international collaborative research, the joint sponsorship of scientific meetings and the sharing of intellectual property produced in collaborations.

70 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 71 Graduate School 2003 CDB Budget Affiliates The Center for Developmental Biology was launched in April 2000 under the auspices of the Millennium Project Encouraging young scientists to participate in advanced research pro- research initiative launched by former Prime Minister Keizo Obuchi. The Millennium Projects were established to drive K research in the fields of information technology, environmental science and the study of aging, areas of vital importance o jects as they prepare for careers in the life sciences is essential to b to both Japan and the world in the 21st century. The organization and operations of the CDB were consigned to RIKEN, e ensuring the development of future generations of researchers. The U CDB has collaborative educational programs with a number of gradu- which coordinates the construction management and administration of the Center. n i ate schools in Western Japan, providing students with opportunities v e r for hands-on laboratory benchwork and lectures on topics in develop- s i ment and regeneration. The center also has also accepted 50 stud- t y , ents from other universities as research associates or trainees. In addi- G r tion to regularly scheduled scientific seminars, students can attend FY2003 a d events such as the CDB Forum research progress series and the Cen- u 762 a ter's annual retreat. The Junior Research Associate program estab- t e lished by RIKEN also provides financial support to 8 graduate students S c h working in CDB labs. 1827 o o l o f S c ie n ce a 2732 n Gr N d aduat a Te e School of Biostudies, Kyoto University Facility Expenses ra c A hn (energy expenses, maintenance,etc.) d o v log Grad an y uate School of Medicine, Kyoto University c De Research Management Expenses ed par i tmen (personnel expenses, etc.) ns t of Li tit fe Scien (Million Yen) ute ce Department of Developmental and Regenerative medicine of Research Expenses Sci ence and T echnology Graduate School of Biological Sciences

70 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 71 Leading Project in Regenerative Medicine

In 2003, the Japanese Ministry of Education, Culture, The implementation of the Leading Project in Regenerative Two new Research Unit laboratories have been recruited to conduct Sports, Science and Technology (MEXT) inaugurated a Medicine has been consigned to universities and research goal-oriented, fixed-term research projects as part of this program. number of Leading Projects to drive research into areas organizations across Japan, and a number of CDB labs These Research Units are funded by Leading Project grants and are identified as having great potential to make contributions have received funding to conduct research into key areas financially independent of RIKEN, but operate, in whole or in part, in to the nation's economy, environment, social climate or including the experimental manipulation and maintenance laboratories located within the RIKEN CDB campus, enabling them to health and welfare. One such project was introduced to of stem cells, the reprogramming and targeted differentia- capitalize on the extensive shared-use equipment and facilities at the promote research leading to applications for stem cells tion of somatic stem cells, and the ex vivo reconstitution of Center. in regenerative medicine. physiological structures with potential clinical uses in regenerative medicine. Shin-ichi Nishikawa, Group Director of the CDB Laboratory for Stem Cell Biology, was appointed to head this Leading Project.

Organ Regeneration The Research Unit for Organ Regeneration, headed by Hideki Tani- guchi, focuses on studies of the differentiative and regulatory mechanisms underlying endodermal tissues and organs in the digestive system, which include liver, pancreas, intestine and sali- vary glands. By analyzing stem cells isolated from each of these Cell Plasticity tissue types, the Taniguchi lab seeks to confirm the existence of Mitsuko Kosaka heads the Research Unit for Cell Plasticity, which "endodermal stem cells" that enable the high levels of cellular plas- seeks to determine the limits and possibilities in the differentiative ticity seen in digestive organs. Using cell sorting technologies, Tani- potential of somatic stem cells. Their work focuses on differentia- guchi is working to characterize these putative endodermal stem tion in the iris of the eye, a population of pigmented cells that cells by sorting and analyzing cells from the livers and other diges- contains stem cells that show an intriguing plasticity, or ability to tive organs of fetal mice. The identification of molecular markers assume more than one cellular fate. The Kosaka lab will inves- specific to such cells will make it possible to isolate them by flow tigate the mechanisms by which stem cells in the iris can be cytometry and to pursue further investigations into their functional induced to give rise to cells of other types, such as photoreceptive roles in the development of the endoderm-derived digestive Hideki Taniguchi neural cells. This work is of great clinical promise in the treatment system. The ability to identify and isolate stem cells capable of M. D. , Ph. D. of vision loss due to nerve damage, as cells from the iris are read- giving rise to endodermal lineage tissue types would also be of Unit Leader enormous potential value in the development of regenerative medi- Hideki Taniguchi ily obtainable and may one day be used as an autologous source Collaborative Scientist of retinal replacement cells if they can be steered reliably to take cal applications for the treatment of diabetes. Takeshi Sugaya Technical Staff up a neural fate. Recent findings from the lab suggesting that iris Midori Yokura stem cells express genes characteristic of highly undifferentiated In a concurrent project, Taniguchi is working to develop a system Naoyo Kajitani (2004) Mitsuko Kosaka Student Trainee Ph. D. pluripotent stem cells also offer new avenues for exploration for for isolating and guiding the differentiation of stem cells from the Masato Sugano (2004)

Unit Leader researchers studying the genetic regulation of differentiative pancreases of adult mice, which is aimed at establishing the foun- Mitsuko Kosaka potency. dations for cell replacement therapies using insulin-producing Research Scientist Maki Asami pancreatic beta cells grown in vitro. Junji Tsuchida Visiting Scientist Guangwei Sun

72 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 73 Leading Project in Regenerative Medicine

72 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 73 2003 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 2003.1.15 Hox genes and brainstem patterning Filippo M Rijli 2003.8.26 Impaired spermatogenesis in mice lacking Caf1 Tadashi Yamamoto 2003.1.16 Neural patterning in vertebrate embryogenesis Nobue Itasaki 2003.9.11 Mechanisms controlling heart and blood vessel growth Kenneth Walsh 2003.1.17 Left-right asymmetry in the zebrafish forebrain Marnie Halpern 2003.9.12 Genomic approaches for functional annotation of the mammalian genome John Hogenesch 2003.1.27 Patterning formation in neocortical area by FGF8 Tomomi Fukuchi-Shimogori 2003.9.19 Genetic analysis of mouse forebrain and midbrain development Siew-Lan Ang

To branch or not to branch: HAMLET controls dendritic arbor branching and lineage 2003.9.29 Fibronectin requirement in branching morphogenesis Takayoshi Sakai 2003.1.29 Adrian Moore restriction in the Drosophila peripheral nervous system 2003.10.3 Chemical modulation of embryonic development and oncogenesis James K Chen 2003.2.21 Wnt signaling in development and disease Randall T. Moon 2003.10.10 Hox in leukemia and as avenues to hematopoietic stem cell expansion R Keith Humphries

2003.3.11 The new revolution in developmental biology: Expansion and reconciliation Scott Gilbert Characterisation of two modulators of the FGF signal transduction pathway,and 2003.10.14 Michael Tsang 2003.3.27 Imaging cell movements that pattern the vertebrate embryo Scott Fraser their role in axial polarity

2003.3.27 Induction and evolution of the neural crest Marianne Bronner Fraser 2003.10.14 Patterning neurogenesis in the zebrafish embryo Motoyuki Itoh

Functional expression cloning of Nanog, a gene conferring cytokine independent ES 2003.10.16 Endoderm as a source of patterning information for early chick embryogenesis Gary Schoenwolf 2003.3.27 Ian Chambers cell self-renewal Structure and function of Spen proteins: A conserved repressive function in 2003.10.23 Mariko Ariyoshi developmental signalling pathways Differentiation of embryonic stem cells into hepatocytes: biological functions and 2003.4.22 Takahiro Ochiya therapeutic applications 2003.10.24 Regulation of temporal and spatial patterns of gene expression in Dictyostelium William Loomis

2003.4.22 ES cells as a model system for pancreatic differentiation Shoen Kume Phylogenetic conservation of a cis-acting regulator that controls polarized 2003.10.27 Toshihiko Shiroishi expression of Sonic hedgehog (Shh) in limb buds 2003.5.13 Dynamics of cellular interactions during T cell recognition in 3-dimensional tissue Ellen Robey visualized by two-photon microscopy 2003.10.29 Sniffing out smell's code and map Naoshige Uchida

2003.5.16 BMP-3b and BMP-3 function as different dorsalizing factors in Xenopus embryos Shin-ichiro Nishimatsu Molecular mechanisms specifying muscle-identity and myotube number in the 2003.11.14 K.VijayRaghavan 2003.5.21 Building brains from embryonic stem cells: Naive cultures offer greatest potential Hidemasa Kato Drosophila adult 2003.5.22 Considerations in microarray experimental design and data analysis Ali R Zareh 2003.11.18 Genesis of the neural primordia and their regional diversities through regulation of Sox2 Hisato Kondoh 2003.6.18 The role of myosin VI in the spermatogenesis of Drosophila Noguchi Tatsuhiko 2003.11.20 Nuclear interpretation of the Dpp morphogen in Drosophila melanogaster Markus Affolter

Cellular functions and molecular mechanisms of signaling pathways mediated by 2003.11.26 Genes that regulate the formation and regeneration of skeletal muscle Margaret Buckingham 2003.6.23 Kazuyoshi Yonezawa target of rapamycin 2003.11.27 Patterning and growth in Drosophila limb development Konrad Basler

2003.7.3 Diversity of CNR/protocadherin family in the brain Takeshi Yagi Secondary rearrangement of TcR alpha chain in vivo: shaping and reshaping 2003.12.2 Osami Kanagawa 2003.7.4 Membrane traffic: Molecular mechanisms and real-time imaging Akihiko Nakano T cell repertoire

2003.7.8 Ontogeny of the hematopoietic system M Bruno Peault Proteolytic cleavage of meiotic cohesin REC8 by separase is required for 2003.12.3 Nobuaki Kubo 2003.7.17 PAX6 and SOX2 in disease, development and evolution Veronica van Heyningen murine spermatogenesis 2003.7.18 Quiescent hematopoietic stem cells in the niche Toshio Suda 2003.12.5 Function and regulation of centralspindlin, a kinesin/RhoGAP complex, in cytokinesis Masanori Mishima 2003.8.4 Manipulating mouse genome from single gene knockout to chromosome engineering Ichiko Nishijima 2003.12.9 Eph receptors and ephrins in neural map development Masaru Nakamoto 2003.8.4 Genetic regulation of retinal interneuron differentiation Akihira Otoshi 2003.12.15 Establishment of the vertebrate body plan in relation to limb formation Mikiko Tanaka 2003.8.6 Self-renewal of neural stem cells and CNS-repair Hideyuki Okano 2003.12.15 Genetic cascades that regulate gastrulation cell movements in zebrafish Masazimi Tada 2003.8.7 Regulation of spindle orientation by centrosome and APC tumor suppressor Yukiko M Yamashita 2003.12.16 Wnt-mediated axon guidance at the midline of Drosophila Shingo Yoshikawa 2003.8.8 A protein interaction matrix for an integrin adhesion complex within C. elegans muscle Hiroshi Qadota 2003.12.26 Msx genes function in neural crest development Mamoru Ishii

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2003.3.27 Induction and evolution of the neural crest Marianne Bronner Fraser 2003.10.14 Patterning neurogenesis in the zebrafish embryo Motoyuki Itoh

74 RIKEN CDB 2003 Annual Report RIKEN CDB 2003 Annual Report 75 RIKEN

Photodynamics Research Center

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

Tsukuba Insitute The First Annual BioResource Center 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074 Tel.+81-29-836-9111 Fax.+81-29-836-9109

CDB Symposium Wako Main CampusWako Institute The Origin and Formation of Multicellular Systems Discovery Research Institute Frontier Research System The CDB held its first annual symposium at the nearby Portopia Hotel. Brain Science Institute 2-1, Hirosawa, Wako, Saitama 351-0198 The meeting drew more than 200 speakers and attendees from Tel.+81-48-462-1111 Fax.+81-48-462-1554 around the world, with lectures, poster presentations and lively discussion on the origins of metazoan biology, the fundamental processes of Yokohama Institute development and regeneration and the formation of higher-order struc- Genomic Sciences Center tures, such as neural networks. Plant Science Center SNP Research Center Research Center for Allergy and Immunology The Symposium was organized into five sessions over three days, 1-7-22, Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045 Tel.+81-45-503-9111 Fax.+81-45-503-9113 with full and short talks by 28 distinguished speakers.

Bio-Mimetic Control Research Center Monday, March 24 Tuesday, March 25 (In Nagoya Science Park Research and Development Center) Session 1 Session 3 2271-130, Anagahora, Shidami, Moriyama-ku, The Origin of Multicellular Systems Cellular Polarization and Nagoya, Aichi 463-0003 Tel.+81-52-736-5850 Fax.+81-52-736-5854 9:00 am 9:40 am Peter W Holland, Oxford University Asymmetric Division 9:40 am 10:00 am Hitoshi Suga, Kyoto University 9:00 am 9:40 am David Ish-Horowicz, Cancer Research UK Tokyo 10:00 am 10:40 am Nicole King, University of Wisconsin 9:40 am 10:20 am Anthony A Hyman, Max Planck Institute Kyoto 10:50 am 11:30 am Yoko Watanabe, 10:40 am 11:20 am Yuh-Nung Jan, UCSF Osaka National Science Museum, Japan 11:00 am 11:40 am Fumio Matsuzaki, RIKEN CDB 11:30 am 12:10 pm Peter Devreotes, Johns Hopkins University 11:40 am 12:00 pm Tadashi Uemura, Kyoto University

Session 2 Session 4 Germ Cells, Stem Cells and Intercellular Signaling and Regeneration Morphogenetic Cell Behavior 1:30 pm 2:10 pm Judith C Kimble, University of 1:30 pm 2:10 pm Hiroshi Hamada, Osaka University Wisconsin-Madison 2:10 pm 2:50 pm Edward M DeRobertis, UCLA 2:10 pm 2:50 pm Allan Spradling, Carnegie Institution of 2:50 pm 3:30 pm Olivier Pourquie, Stowers Institute for Washington Medical Research RIKEN is a publicly funded research organization estab- 3:10 pm 3:30 pm Yuzo Niki, Ibaraki University 3:50 pm 4:10 pm Yasumasa Bessho, Kyoto University lished to conduct comprehensive research in science and tech- Harima Institute 3:30 pm 4:10 pm Christopher Wylie, 4:10 pm 4:30 pm Yukiko Gotoh, University of Tokyo nology and to disseminate the results of its scientific research and Cincinnati Children's Hospital 4:30 pm 5:10 pm Elaine Fuchs,The Rockefeller University 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148 Tel.+81-791-58-0808 Fax.+81-791-58-0800 technological developments. RIKEN carries out basic and applied 8:00 pm 8:40 pm Shin-ichi Nishikawa, RIKEN CDB 5:10 pm 5:50 pm Tom Curran, St Jude Childrenís Research research in a wide range of fields, including physics, chemistry, medical sci- 8:40 pm 9:00 pm Ian Chambers, University of Edinburgh Hospital ence, biology, and engineering. 9:00 pm 9:40 pm Kiyokazu Agata, RIKEN CDB Kobe Institute Wednesday, March 26 RIKEN was first founded in 1917 as a private research foundation, Rikagaku Ken- Session 5 Center for Developmental Biology kyuusho (The Institute of Physical and Chemical Research). In 2003, the Institute Cellular Basis for 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, was reorganized as an Independent Administrative Institution under the Ministry of Neuronal Circuit Formation Hyogo 650-0047 Tel.+81-78-306-0111 Fax.+81-78-306-0101 Education, Culture, Sports, Science and Technology (MEXT), since which time it has 9:00 am 9:40 am Hajime Fujisawa, Nagoya University engaged in wide-ranging research activities spanning the basic and applied sciences. 9:40 am 10:20 am Peter Mombaerts, The Rockefeller University 10:40 am 11:20 am Christine E Holt, Cambridge University 11:20 am 12:00 pm Mu-ming Poo, UC Berkeley

76 RIKEN CDB 2003 Annual Report RIKEN