RIKEN Center for Developmental
ental Biology

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

2010 Annual Report

On The CoverSpontaneously formed conjoined twin chicken
embryos, stained for axial (Brachyury) and paraxial(Paraxis) mesoderm markers

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

RIKEN Center for Developmental Biology

2010 Annual ReportSendai Facility
519-1399 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-0845
Tel: +81-22-228-2111 Fax: +81-22-228-2122

Tsukuba InstituteContents

Message from the Director

2

Organizational Chart

4

2010 at the CDB

6News Highlights 1

10Itabashi Branch
1-7-13 Kaga, Itabashi, Tokyo 173-0003
Tel: +81-3-3963-1611 Fax: +81-3-3579-5940

Carina HANASHIMA
Morphogenetic Signaling

25

Shigeo HAYASHI
Vertebrate Axis Formation

26

Masahiko HIBI
Sensory Circuit Formation

Wako Institute24

Hideki ENOMOTO
Neocortical Development

Center for Intellectual Property Strategies
RIKEN Next-Generation Supercomputer R&D Center
XFEL Project Head OfficePublic Relations Office23

22

Shinichi AIZAWA
Neuronal Differentiation
and Regeneration

RIKEN Headquarters

Advanced Science Institute
Brain Science InstituteNishina Center for Accelerator-Based Science
RIKEN Innovation Center
RIKEN Biomass Engineering Program
Computational Science Research Program
2-1, Hirosawa, Wako, Saitama 351-0198
Tel: +81-48-462-1111 Fax: +81-48-462-1554

LabsVertebrate Body Plan

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

27

Tokyo Liaison Office2311, 23th floor, Fukoku Seimei Building,
2-2, Uchisaiwaicho 2-chome, Chiyoda-ku, Tokyo 100-0011
Tel: +81-3-3580-1981 Fax: +81-3-3580-1980

RIKEN Advanced Institute for Computational Science
7-1-26 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-940-5555 Fax: +81-78-304-4956

Takeshi IMAI
Cell Lineage Modulation

28

Toru KONDO
Evolutionary Morphology

29

Shigeru KURATANI
Sensory Development

30

Raj LADHER
Cell Asymmetry

31

Fumio MATSUZAKI
Yokohama Institute

RIKEN Center for
Developmental Biology

2010
AnnualReport

RIKEN
http://www.riken.jp/

The RIKEN Center for Developmental Biology waslaunched in April 2000 under the auspices of theMillennium Project research initiative, which wasestablished to drive research in areas of vital importanceto both Japan and the world in the 21st century. The
challenges of aging society were among the issues
addressed, and developmental biology was included
within this remit as a scientific field with the potential tocontribute to the development of regenerative medicine.
Located in Kobe, Japan, the Center’s research programis dedicated to developing a better understanding offundamental processes of animal development at the

RIKEN is a publicly funded researchorganization established to conduct
comprehensive research in science andtechnology, and to disseminate the resultsof its scientific research and technologicaldevelopments. RIKEN carries out basic
and applied research in a wide range offields, including physics, chemistry,
biology, medical science, and engineering.
RIKEN was founded in 1917 as a private
research organization, Rikagaku
kenkyuusho (The Institute of Physical andChemical Research). In 2003, the Institutewas reorganized as an Independent
Administrative Institution under the Ministryof Education, Culture, Sports, Science andTechnology (MEXT), and has continued toengage in wide-ranging research activities
spanning the basic and applied sciences.

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

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

Kobe InstituteCenter for Developmental Biology
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-306-0111 Fax: +81-78-306-0101
Center for Molecular Imaging Science
MI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku,
Kobe, Hyogo 650-0047
Tel: +81-78-304-7111 Fax: +81-78-304-7112

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

News Highlights 2

32

Labs
Germline Development

44
45
46
47
48
49
50

Physical Biology

69

Developmental Genomics

70

Retinal Regeneration

71

Cell Adhesion and Tissue Patterning

72

Masatoshi TAKEICHI
51

Mitinori SAITOU
Organogenesis and Neurogenesis

68

Masayo TAKAHASHI

Tony PERRY
Mammalian Germ Cell Biology

Early Embryogenesis

Asako SUGIMOTO

Masaki OKANO
Mammalian Molecular Embryology

67

Tatsuo SHIBATA

Hitoshi NIWA
Mammalian Epigenetic Studies

Cell Fate Decision

Guojun SHENG

Takashi NISHIMURA
Pluripotent Stem Cell Studies

66

Hitoshi SAWA

Shin-Ichi NISHIKAWA
Growth Control Signaling

Embryonic Induction
Hiroshi SASAKI

Jun-ichi NAKAYAMA
Stem Cell Biology

54

Labs

Akira NAKAMURA
Chromatin Dynamics

News Highlights 3

Systems Biology

73

Hiroki R. UEDA
52

Yoshiki SASAI

Genomic Reprogramming

74

Teruhiko WAKAYAMANews Highlights 476

Supporting Laboratories

86

RIKEN Kobe Institute

9
4

CDB Symposium

9
6

CDB Seminars

98

About RIKEN

100

RIKEN Campuses

101

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

12Message from the Director
The end of 2010 brings us to the end of our first decade of research at the RIKEN Center for Developmental Biology, a decade thathas seen well over 1,000 publications from nearly 50 laboratories and over a thousand research staff from dozens of countries thathave made the CDB their home. Our work has spanned an exciting and broad range of fields, from fundamental aspects of developmental cell and molecular biology, to research into stem cell maintenance and differentiation, to systematic approaches to genetic
regulatory networks, to fresh new insights into evolutionary phenomena. It has been an exciting time for developmental biology andassociated fields, and we have been fortunate to play a part on the global scientific stage.

This home is not, however, intended to be a permanent one for the majority of our research programs, but rather an incubator andaccelerator for talented young scientists to work in a collegial environment with a strong concentration of peers in related disciplines.
Our policy has always been one of encouraging positive turnover, in which older labs continually transition outward to make way forthe fresh ideas and approaches that new generations bring. In real terms, this means that the CDB is now in the process of rebirthand renewal, mirroring in our own structure one of the central themes of our research, as new labs focusing on mechanisms ofgrowth control, sensory circuitry, and physical biology joined the institute in the past year.

Despite the transitional phase we have entered, research at the CDB remains highly productive, with numerous high profile scientificarticles and awards attained by our staff in 2010. Highlights include new findings in the molecular machinery that harnesses forcesat the cell-cell junction, techniques for steering pluripotent stem cells toward specific neuronal fates, studies of asymmetric cell division, and insights into the evolution of the insect wing. Our record for conducting leading research has won recognition for many of
our investigators from organizations devoted to the advancement of the sciences in Japan and abroad.

The CDB was also honored to receive visits by then Japanese Prime Minister Yukio Hatoyama and Her Royal Highness, Princess
Srindhorn of Thailand in 2010, highlighting the high level of recognition our institute has begun to attract both in Japan and overseas. We strive to remain in the public eye at ground level as well, hosting numerous programs and events intended to promote a
better understanding of science and more opportunities for communication between our research staff and the general public. Ourannual Open House event received more visitors in 2010 than in any previous year, and our educational programs for students andhigh school teachers remain popular science learning events in the Kansai region of Japan.

Indeed, our location in the city of Kobe, in the heart of western Japan continue to provide opportunities for interaction and collaboration at the laboratory and institutional level. The RIKEN Kobe Institute, of which the CDB is the founding Center, is now home to
the Center for Molecular Imaging Science (CMIS), which focuses on positron emissions tomography technology and applications,and the newly established Quantitative Biology Center (QBiC), which will work on computational and systematic approaches tofundamental biological questions of complexity and dynamics. In addition, RIKEN is readying the launch of its Next-Generation Supercomputer facility only a few minutes from the CDB, bringing a new dimension of computational power and expertise to the area.
Kobe also boasts numerous biomedical and biotechnology companies, research institutes, and foundations, and the new Kobe City
Medical Center is set to open in spring of next year, which will enhance the possibilities for translational research, very much in linewith our original mission of helping to build a foundation in science for the field of regenerative medicine.

We can look back proudly on 2010 as another successful year for the institute, which could only be achieved through the dedicatedand collaborative efforts of our research and administrative staff, and the support and cooperation of our colleagues around theworld. As we enter our second decade, I would like to thank all those who have helped make the RIKEN CDB what it is today, andask for your continuing support as we work to open up new frontiers in development.

Masatoshi Takeichi
Director, RIKEN Center for Developmental Biology

3

RIKEN Kobe InstituteCore Program

Center for
Developmental
Biology
The Center for Developmental Biology (CDB) is a
research center within the RIKEN Kobe Institute, which
also comprises the Center for Molecular Imaging Science (CMIS), the Kobe Research Promotion Division,
which provides administrative services and support, andthe institutional Safety Center. The CDB is home to a
total of 33 laboratories in its Core Research Program (7groups), Center’s Director Strategic Programs (2 projects), Creative Research Promoting Program (19 teams)
and Supporting Laboratories (6 labs). The CDB Director
is assisted by two Deputy Directors and is advised by
the Advisory Council and the Institutional Review Board.

Center for Molecular Imaging Science
Research Promotion Division
Safety Center

Institutional Review Board
The RIKEN CDB’s Institutional Review Board (IRB) includes representatives from local academic, research, medical and legal organizations, aswell as CDB research leaders, and meets regularly to review anddiscuss investigations with potential ethical, legal, social or public healthand safety implications prior to their implementation. The IRB is coordinated by the Kobe Institute Safety Center.

The Core Program aims to promote highly innovative
developmental and regeneration studies in a strategic
and multi-disciplinary manner. This program focuseson the main themes of the CDB: the mechanisms ofdevelopment, organogenesis, and strengthening thescientific basis of regenerative medicine through thestudy of stem cells and regeneration.

Vertebrate Body Plan
Shinichi AIZAWA Ph.D.

Morphogenetic Signaling
Shigeo HAYASHI Ph.D.

Evolutionary Morphology
Shigeru KURATANI Ph.D.

Cell Asymmetry
Fumio MATSUZAKI Ph.D.

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

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

Cell Adhesion and Tissue Patterning
Masatoshi TAKEICHI Ph.D.

Center Director’sStrategic ProgramThis program allows for concentrated focus on priority
areas of research determined by the CDB Center Director.
The initial 10-year projects will focus on stem cell andsystems biology.

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

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

Physical Biology
Tatsuo SHIBATA Ph.D.

Advisory Council
The CDB Advisory Council (DBAC) convenes regularly to review theCenter’s performance and make recommendations on the direction of
its programs and governance. The DBAC reports its findings to aid in
guiding future activities and decision-making. The full texts of DBAC
reports can be viewed on the CDB website (www.cdb.riken.jp).

Margaret Buckingham

Austin Smith

Steve Cohen

Toshio Suda

Hiroshi Hamada

Yoshimi Takai

Haifan Lin

Patrick Tam

Institut Pasteur, France
EMBL, Germany

Osaka University, Japan
Yale University, USA

Yo-ichi Nabeshima
The ten-member Council comprises top international scientists working in developmental biology, stem cells and related fields.

4

Kyoto University, Japan

University of Cambridge, UK
Keio University, Japan
Osaka University, Japan
Children's Medical
Research Institute, Australia

Christopher Wylie

Cincinnati Children's Hospital
Research Foundation, USA

Director
Deputy Directors

Creative Research
Promoting Program

Supporting
Laboratories

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

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

Neuronal Differentiation and Regeneration

Animal Resources and Genetic Engineering

Hideki ENOMOTO M.D., Ph.D.

Shinichi AIZAWA Ph.D.

Neocortical Development

Genetic Engineering Unit

Carina HANASHIMA Ph.D.

Shinichi AIZAWA Ph.D.

Vertebrate Axis Formation

Animal Resource Unit

Masahiko HIBI M.D., Ph.D.

Kazuki NAKAO Ph.D.

Cell Lineage Modulation

Electron Microscope

Toru KONDO Ph.D.

Shigenobu YONEMURA Ph.D.

Sensory Development

Bio-imaging Laboratory

Raj LADHER Ph.D.

Shigeo HAYASHI Ph.D.

Germline Development
Akira NAKAMURA Ph.D.

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

Chromatin Dynamics

Genomics

Jun-ichi NAKAYAMA Ph.D.

Fumio MATSUZAKI Ph.D.

Growth Control Signaling

Genome Resource and Analysis Unit

Takashi NISHIMURA Ph.D.

Hiroshi TARUI Ph.D.

Mammalian Epigenetic Studies

Functional Genomics Unit

Masaki OKANO Ph.D.

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

Mammalian Molecular Embryology

Proteomics

Tony PERRY Ph.D.

Shigeo HAYASHI Ph.D.

Mammalian Germ Cell Biology
Mitinori SAITOU M.D., Ph.D.

Mass Spectrometry Analysis Unit
Akira NAKAMURA Ph.D.

Embryonic Induction

Division of Human Stem Cell Technology

Hiroshi SASAKI Ph.D.

Yoshiki SASAI M.D., Ph.D.

Cell Fate Decision

Human Stem Cell Technology Unit

Hitoshi SAWA Ph.D.

Yoshiki SASAI M.D., Ph.D.

Early Embryogenesis

Four-dimensional Tissue Analysis Unit

Guojun SHENG Ph.D.

Yoshiki SASAI M.D., Ph.D.

Developmental Genomics

Science Policy and Ethics Studies Unit

Asako SUGIMOTO Ph.D.

Douglas SIPP

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

Genomic Reprogramming
Teruhiko WAKAYAMA Ph.D.

5

2010 Highlights

Visit by Prime Minister Hatoyama
January 17
Japan’s then Prime Minister, Yukio Hatoyama, visited the RIKEN CDB
as part of a visit to Kobe’s biomedical research park. He was greetedby CDB Director Masatoshi Takeichi, Group Director Yoshiki Sasai, andTeam Leader Masayo Takahashi, and introduced to the Center’s work
using embryonic and induced pluripotent stem cells, as well as fundamental developmental and regenerative phenomena.

RIKEN Kids’ Lab
March 23
The CDB announced the opening of its “RIKEN Kids
Lab: The wonders of development and regeneration”
in the Kobe Science Museum. This child-friendly
space creates an environment for discovery and fun,with graphic and hands-on activities relating to topics
such as the development of the body from a single
cell, and the exciting future promise of stem cells in
medicine.

Frontiers in Organogenesis
March 23 – 25
The RIKEN CDB hosted its eighth annual symposium, “Frontiers in
Organogenesis,” bringing together nearly 200 participants from 16
countries to discuss recent advances in the study of the formation offunctional, complex biological structures. The meeting featured a wide
range of themed sessions, including patterning, imaging, evolution,and the dynamics of organogenic phenomena.

6Team Leaders win Ministry Prizes
April 5The Ministry of Education, Culture, Sports, Science and Technology(MEXT) announced that CDB Team Leaders Teruhiko Wakayama and
Jun-ichi Nakayama has been awarded Commendations for Scienceand Technology for their contributions to the advancement of theirfields. Wakayama received the Prize for Science and Technology for
Research for his work in somatic cell nuclear transfer, and Nakayama
received the Young Scientist’s Prize for his research on how dynamic
structural changes to chromatin regulate gene expression.

Takeshi Imai joins
as Team Leader
July 1The Center welcomed its newest Team Leader,Takeshi Imai, who heads the Laboratory for Sensory Circuit Formation. His research will focus on
the organization of the olfactory system in mouse.

CDB postcards a hit with
developmental biologists
July 2The RIKEN CDB debuted a set of decorative postcards
incorporating motifs from Japanese art and fashion into
depictions of 16 organisms used in developmental biology. The printed cards were distributed at scientific meetings and are no longer available, but the data can be
downloaded from the CDB website: www.cdb.riken.jp.

7

2010 Highlights

Intensive Lecture Programfor Grad School Affiliates
August 19 – 20
Every summer, the Center holds a two-day program for interested students enrolled in affiliated graduate and medical schools in the Kansai
region. Nearly 150 students gathered for talks and demonstrations ofsome of the advanced methods and technologies used by CDB labs.

Summer school for HS students
August 24, 26
The CDB held its annual summer school program for area high school students. The pair of oneday programs, consisting of lectures and demonstration experiments, focused on C. elegans research. More than 20 young learners joined in practicing techniques for observing, collecting andworking with these tiny organisms.

New Research Unit Leader,
Tatsuo Shibata
October 1
The Laboratory for Physical Biology was launched under the leadership of Tatsuo
Shibata, and as part of the Center Director’s Project on Systems Biology. The lab
will use concepts and methodology from mathematical in the study and elucidation of emerging questions in biology.

Lessons in development forhigh school teachers
October 2 – 3
The CDB held its third recurrent course in developmental biology for teachersof high school biology in conjunction with the Japanese Society of Developmental Biologists. This program is intended to give teachers an opportunity tolearn hands-on techniques in embryology that can be shared with their classes.

8Visit by HRH Princess
Sirindhorn of Thailand
October 11
Princess Maha Chakri Sirindhorn of Thailand paid a visit to the CDB during atour of the city of Kobe. After a brief introduction to the Center’s history, research mission, and organization, Her Royal Highness heard presentations on
the work being done in the Laboratory for Retinal Regeneration.

RIKEN Kobe Open House
November 20
This year saw more visitors to the Center’s annual Open House than ever before, withmore than 1,700 people turning out to enjoy talks, demonstrations and exhibits ondevelopment, regeneration, evolution and stem cells, as well as tours of the nearby
Center for Molecular Imaging Science and Next Generation Supercomputer.

Group Director Shigeru Kuratani
wins science prizes
November 10
Shigeru Kuratani was awarded the Hyogo Prefectural Science Prize in recognitionof his work on the evolutionary mechanisms that led to the innovation of the turtle
carapace, and the Alexander Kowalevsky Medal, granted annually by the St. Petersburg Society of Naturalists (Russia) for contributions in the fields of evolutionary
developmental biology and comparative zoology.

Annual Retreat in Sasayama
November 29 – 30
Scientific and administrative staff gathered for an overnight stay in the mountains of
Sasayama for active discussion of the latest developments in CDB research. This
once-a-year event serves as a forum for unfettered exchange among the Center’s
many labs.

9

A pair of paths to the insect wing

Ai AKIMOTO-KATO, Nao NIWA

The emergence of the wing is a critical innovation that underlies the evolutionary success and diverse speciation of insects. Two rival hypotheses have been proposed to explain the emergence of the first insect wings, one
suggesting that it arose de novo, the other that it represents a modification of a pre-existing structure. Given the
scarcity of early and intermediate examples in the fossil record, the resolution of this question will require a forensic examination of wing-related genes in basal insects and their relatives.
Work led by Nao Niwa and colleagues in the Laboratory for Morphogenetic Signaling (Shigeo Hayashi, Group Director) now suggests a possible solution based on genetic evidence that collates the two competing propositions
into a single theory of insect wing origins. Working with scientists from Nagoya and Shinshu Universities and the
University of Tsukuba, Niwa proposes that the expression of wing-related genes in limb branch primordial regions
drifted over time in such a way as to integrate into the dorsal body wall, which already showed potential for flat
sheet-like outgrowth.
Niwa’s study was born from a comparison of the advantages and limits of two previous models of wing evolution.
The first, referred to as the paranotal hypothesis, posits that the wing emerged as a novel extension of the thoracic tergum (the dorsal body wall), while its alternate, the limb branch hypothesis, suggests that wings are actually modifications of dorsal limb branches that were already in place. However, neither of these models can fully
explain the morphological features of the insect wing on its own.
The Hayashi group used knowledge of the anatomy and developmental genetics of a winged insect to draw
comparisons with two key insect species: a wingless basal insect (the bristletail Pedetontus unimaculatus) and

10©2010 John Wiley & Sons, Inc.
Niwa N, et al. Evolutionary origin of the insect wing via integration of two developmental modules.
Evolution & Development 12.168-76 (2010)

a primitive winged insect (the mayfly Ephoron eophilum). These models for comparison have specificmorphological traits that have previously been suggested to represent forerunners to the early wing
via adaptations to dorsal limb branches. The bristletail develops a rod-like outgrowth known as astylus from thoracic and abdominal appendages, while the mayfly nymph shows articulated tracheal
gill structures associated with abdomen. However, little is known about the genetic aspects of their
specification and growth.
The fruit fly Drosophila melanogaster, in contrast, is one of the most widely studied organisms in biology, and we have a detailed understanding of its wing growth at the genetic level. The gene wingless(wg) plays key roles inducing the wing disc primordium in the embryo and later promoting growthof the wing pouch. The wing does not form in the absence of wg activity, as indicated by the gene’s
name. vestigial (vg) also plays a role in growth control and specification of wing tissue. apterous (ap)specifies the flat shape of the wing by controlling subdivision of the wing into dorsal and ventral compartments.

Scanning electron micrographof the bristletail, Pedetontus unimaculatus, showing stick-likestylus attached to the base oflimbs, and tergal plates covering the lateral side of the body.
Expression analysis of wingrelated genes revealed the two
structures share key propertiesof wing development, suggesting a dual origin of the insectwing.

Niwa et al. analyzed gene expression patterns in the primordia of bristletail stylus and mayfly tracheal
gill, and found that wg and vg are expressed, results that are consistent with the limb branch hypothesis. However the expression of wg and vg is less regionalized than it is in the Drosophila wing disc,suggesting that positional information required for dorsal-ventral subdivision is missing. Interestingly,
wg and vg expression was also detected in the tergum of mayfly and bristletail, accompanied by ap
expression. In bristletail, the ap expression domain formed a sharp border, marking the margin of thetergal extension that forms the prominent flat lateral extensions. The expression of wg, vg, and ap in
the tergum supports the paranotal hypothesis. Given these new insights into the paranotal and limb
branch hypotheses, the authors propose a dual role for wg-vg signaling; an inductive role in the dorsal
limb branch and growth-promoting role in the tergal extension, where ap specifies the tergal margin in
the dorsal body wall. They speculate that the inductive signal may have drifted dorsally over evolutionary time toward the outgrowth of tergal extension, and that a synergistic effect may account for the
rapid advent of the wing once the two intersected. If this is correct, it would indicate that the two current theories of wing origins are both partially correct, but incomplete, and that the early wing may infact have arisen from a combination of paranotal and limb branch components.
“The evolutionary origin of the insect wing has fascinated many biologists,” says Hayashi. “I am very
pleased that we can now propose a model that provides a plausible explanation for previously unanswered questions and serves as a basis for future molecular studies.”

11

Epigenetic factors hold cell fate to the script

Yukimasa SHIBATA

DNA famously plays a central role in determining the body’s form and function, typically through the transcriptionof genes and the translation of these transcripts to proteins. Controlling this expression of genes such that they
are only “switched on” at the right times and in the right places requires reliable and precise regulatory mechanisms. The results of such regulatory activities result in the establishment and maintenance of cells with specificfunctional identities, or fates, but how is this spatiotemporal transcriptional control achieved?
Yukimasa Shibata and others in the Laboratory for Cell Fate Decision (Hitoshi Sawa, Team Leader) were interested in possible roles for epigenetic mechanisms in the upkeep of cell fate in C. elegans, as a body of previous
work had pointed to functions for such phenomena as DNA methylation and histone modifications in cell typespecific transcriptional regulation in various model systems. In a study published in the journal Development, they
showed that a pair of protein factors implicated in histone acetylation plays crucial roles in setting up and stably
maintaining cell fates. Acetylation is one of a number of modifications known to modify histone protein activity
and configuration, and to affect the expression of genes in associated regions of DNA within chromatin complexes.
The team identified the first of their molecules of interest in a mutation screen, which gave the first hint that thehistone-associated bromodomain factor BET-1 might play a role in cell fate stability. A mutant allele yielded phenotypes in which cell-fate maintenance is disrupted. The result of these aberrations was seen, for example, in the
mistaken adoption of inappropriate cell fates by progeny of tail neural precursor cells lacking bet-1 function, withthe result that neuroblasts gave rise to hypodermal cells (in wildtype animals, these neuroblasts produce neuralcells).

12

©2010 The Company of Biologists
Shibata Y, et al. Double bromodomain protein BET-1 and MYST HATs establish andmaintain stable cell fates in C. elegans. Development 137.1045-53 (2010)

In an extremely wide range of cell lineages, loss of BET-1 resulted in the ectopic expression of cell
type-specific markers and more than usual numbers of specific type of cells. In at least one case,they traced this effect to the regulation of the expression of a cell-fate determinant by BET-1, a generalmechanism that they suspect may be common to other lineages as well.
Armed with evidence of the ability of bet-1
mutation to cause cell fate transformationsin the somatic, germ, and neural lineages,
the Sawa team suspected that this mightalso be true in other cell populations. To
test this possibility, they observed the expression of marker genes over time in thehopes of gleaning insights into the effectsof loss of bet-1 function on cell fate stability. Again, they found that differentiatedcells switch between fates in mutants, but
not in wildtype. Further tests using heatshock treatment to conditionally rescue
the phenotype suggested that BET-1 establishes and maintains stable cell fates.
Pursuing the role of histone acetylationin cell fate establishment and maintenance, Shibata next looked at a family ofacetyltransferases known as MYST HATs,
and found that blocking their function
produced phenotypes closely resembling
those of the bet-1 mutants. Distribution
analyses showed that the localizationof bet-1 within the nucleus depends on
MYST factors, highlighting a possiblefunctional relationship between the two.
These findings shed new light into roles
for epigenetic processes in establishingand maintaining cellular identity, possibly
through the regulation of the expressionof cell fate determinant genes. As analogsof BET-1 and MYST proteins have been implicated in expression of developmental genes in other organisms, such as the fruit fly Drosophila melanogaster, these factors may have a more generally conserved function in cell fate maintenance and the prevention of wayward differentiation.

Comparison of wildtype (A) andbet-1 mutants (B–D) reveals altered numbers of cells expressing cell type-specific markers
genes.

13

Anchors down: New roles for Wnt and
Src in nuclear localization
When cells divide, they often produce daughters withdifferent identities, or “fates.” This asymmetric division isan intricate process involving the sorting of intracellularcomponents to different sides of the mitotic cell so as toensure that they are allocated differentially to its progeny.
A growing body of work has revealed that such polarization relies on signals both from within and outside of
the cell, and typically involves mechanical action by elements of the mitotic machinery, such as centrosomes.
The roundworm C. elegans has served as one of themost powerful systems for studying these phenomena,
shedding light on the mechanisms that underlie asymmetric cell division.
Research by Kenji Sugioka in the Laboratory for Cell Fate
Decision (Hitoshi Sawa, Team Leader) examined how
the positioning of daughter cell nuclei, a critical event inasymmetric division, is regulated by signaling proteins.
Published in the journal Genes to Cells, this work reveals
roles for the Wnt and Src signaling pathways in ensuringthat cells in the early embryo split up their bequests appropriately.
Very early in development, when the roundworm embryo is made up of only four cells, many fundamentally
important decisions are being made that will determine
whether the embryo develops in the normal fashion.
One of these four, known as the EMS cell, is the sourceof the lineages that give rise to tissues such as muscle,gut, and the nervous system. Given its importance, theasymmetric division of EMS has been intensively studied
at the structural and molecular levels, and it is known
that this activity is directed by cues from its neighboring
P2 cell in the form of MOM-2 (Wnt) and other signals.
Sugioka used 4D live imaging to watch as embryoniccells divided, and made an intriguing observation. First,
the centrosomes of the anterior and posterior daughtersof the EMS cell behaved differently, with the posterior
centrosomes generally moving, along with the nucleus,
to anchor in the posterior cortex, while their anteriorcounterparts rarely did so. Looking at other asymmetrically dividing cells, he found that this posterior nuclear
anchoring was widely conserved.

Kenji SUGIOKA

14

Moving back to the EMS cell, Sugioka began to search
for a mechanism. Previous work had shown that the
EMS cell receives signals from its posterior neighbor, theP2 cell. These signals are mediated by at least two distinct pathways: MOM-2/Wnt through the Wnt/ß-catenin
pathway, and MES-1 through the tyrosine kinase SRC-1.

©2010 John Wiley & Sons, Inc.
Sugioka K and Sawa H. Regulation of asymmetric positioning of nuclei by Wnt and Src signaling and
its roles in POP-1/TCF nuclear asymmetry in Caenorhabditis elegans. Genes Cells 15.397-407 (2010)

To determine whether these signals play a role in nuclear anchoring, Sugioka looked for abnormal nuclear localization in posterior daughters of mutant or RNAi-treated cells, and found that loss of Wnt or
SRC function was associated with anchoring defects.
But these factors are also involved in the orientation of mitotic spindles, meaning that the effect on nuclear anchoring could be secondary. To rule this out, he quantitatively monitored spindle orientation inthree dimensions, and found that even when these structures were oriented within the normal rangein MOM-2 or SRC-1 RNAi embryos, nuclear cortical anchoring remained abnormal.

Nuclear localization in asymmetrically dividing cells of theearly C. elegans embryos (yellow). The cor tical anchoringof the nucleus is dependent
on the activity of centrosomes(white) regulated by Wnt andSrc signaling.

What then is the function of anchoring the nucleus to the posterior cortex? The team speculated it
might have a relationship with asymmetric localization of another factor, POP-1, which is linked with
distinct transcriptional activity and cell fate determination. By measuring the proximity of the nucleus
to the cell cortex in mutants lacking a protein required for centrosomes attachment to the cortex, theyfound that POP-1 asymmetry could be maintained even in the absence of nuclear anchoring. On looking more closely, using mutants in which asymmetric cell fate determination and POP-1 localization
were only partially defective, he was able to show a strong correlation between the proximity of thenucleus to the cortex with POP-1 asymmetry, suggesting that while nuclear cortical attachment itself
is not essential, it may support normal asymmetry by holding the nucleus close to the cell cortex.
“We know that, in many organisms, the nucleus is often found off-center, but the underlying mechanisms and significance are for the most part still unknown,” says Sugioka. “By showing how an extracellular signal can drive nuclear localization, and how the positioning of the nucleus near the cortex
makes it more accessible to signals, we have added what I think will be an important piece to thispuzzle.”15

New chromatin factor in DNA repair pathway

Noriyo HAYAKAWA, Tomohiro HAYAKAWA, Yasuko OHTANIMaintaining the integrity of the genome is a mission-critical role for the cell’s many DNA repair mechanisms. Errors of varying severity can creep into the code in the form of substitutions, deletions, reversals and extra copies,
but ruptures of the double helix itself can be even more catastrophic. Such double-stand breaks set off alarms,
as they can lead to massive rearrangements of long stretches of the genome, and eukaryotes have evolved anarsenal of detection, repair, and abort mechanisms to deal with this contingency. Homologous recombination is
one tactic used to salvage broken DNA, by making a template for the damaged sequence from a sister chromatid; a very similar process takes place during meiosis.
Studies of homologous recombination have revealed a number of the molecular factors that underlie the process,with the genes BRCA1 and BRCA2 (whose loss of function is a well-known predictor of breast cancer) playing
central roles. The proteins encoded by these genes are bound together in a complex with other factors known as
RAD51 and PALB2, which respectively form single-strand filaments of DNA and protein that are used in initiating
the recombination event, and help recruit BRCA2 to chromatin containing damaged DNA. The chromatin-sidetarget of the BRCA complex, however, remains unclear.
A study by Tomohiro Hayakawa of the Laboratory for Chromatin Dynamics (Jun-ichi Nakayama, Team Leader),Fun Zhang in Paul Andreassen’s lab at Cincinnati Children’s Research Foundation, and others added a new linkto that chain, showing that the chromodomain protein MRG15 binds directly to PALB2, and is required for homologous recombination. Published in The Journal of Cell Science, this work provides new insight into how the DNA
damage response network interacts with the genome through its nucleoprotein packaging.

16©2010 The Rockefeller University Press
Hayakawa T, et al.MRG15 binds directly to PALB2 andstimulates homology-directed repair of chromosomal breaks.
J Cell Sci 123.1124-30 (2010)

Knowing that a molecular relative of MRG15 complexes with PALB2, Hayakawa and Zhang started by
testing for a possible functional link using immunoprecipitation, and found that the two proteins show
affinity for each other. Further experiments using Western blotting revealed that MRG15 indeed interacts with the entire BRCA complex.
Testing next for function, the team found that MRG15 plays a vital role in homologous recombination; depletion of the protein caused a significant drop in the efficiency of this DNA repair mechanism.
Interestingly, MRG15 also appeared to be tied to cellular resistance to mitomycin C (MMC), a potent
DNA crosslinker that activates not only homologous recombination, but multiple DNA repair pathways. MMC resistance is mediated by the BRCA complex, and Hayakawa and Zhang found that lossof MRG15 function caused hypersensitivity to the crosslinking agent, similar to the phenotype wheneither BRCA2 or PALB2 are knocked down.
The recruitment of the BRCA complex to the site of DNA damage has been shown to rely on PALB2,
so the researchers looked for a possible role for MRG15 in this targeting. Using immunoblotting tolook at BRCA complex binding to chromatin in wild type and irradiated cells in which MRG15 was expressed normally or silenced by siRNA, they found that the DNA repair complex was associated withchromatin at much higher frequencies in untreated cells (see Fig.). They speculate that once PALB2
has attached to MRG15 in the chromatin, it then recruits the other elements of the BRCA complex,
enabling homologous recombination and possibly, other DNA repair mechanisms to proceed.
“Several lines of study have shown how dramatically chromatin structure can change over the courseof development,” says Hayakawa, “but we still know very little about what roles MRG15, PALB2, andthe BRCA complex might play in such rearrangements. We’re hopeful that by studying these interactions we will gain a better understanding of developmental processes.”

PA L B 2 (r e d ) l o c a l i z e s w i t hdamaged DNA (green) in cells
expressing MRG15 (top), but
not when its function is blocked(bottom).

17Genetic controller of cell senescence foundOne of the main functions of somatic (or adult) stem cells
is to keep the body’s systems in working order by generating replacements for cells that have been damaged
or aged. As the body grows older, however, its stem
cells gradually lose their ability to self-renew throughcell division, a phenomenon known as cell senescence.
A number of cell cycle mechanisms have been linked
to this form of cellular aging, but its molecular basis remains poorly understood.
A recent report by Yuki Kujuro and others in the Laboratory for Cell Lineage Modulation (Toru Kondo, Team
Leader) identifies a new genetic inducer of cell senescence in the nervous system. The gene, Ecrg4, encodes
a secreted protein that triggers the degradation of cell
cycle factors in oligodendrocyte progenitor and neuralprecursor cells, marking it as an important link in the aging of the brain.
Kujuro began by observing the aging process in oligodendrocyte progenitor cells (OPCs), and found anumber of irreversible changes in their morphology andinternal regulation that take place when they are cultured
in a high concentration of serum, which serves as anexperimental surrogate for in vivo aging. Looking next
at changes in gene expression, she found more than athousand were up- or down-regulated on high serum
culture, more than three hundred of which remained inthat state even after the culture medium was switchedback to a normal serum level.
Among the genes caught in this screen, Ecrg4, caught
the team’s interest for several reasons. The difference
between its expression levels in senescent and nonsenescent cells was greater than for any other gene, andits expression was also activated in senescing mouseembryonic fibroblasts, shown for the first time in thisreport. In addition, its expression is down-regulated intumors, which are notorious for their age-defying ability
to self-renew indefinitely.
To test for function, the team transfected rat OPCs with
Ecrg4 and watched for signs of senescence. Compared
to control populations, the transfected cells showed significantly higher activation of senescence markers. Subsequent experiments showed that the gene’s effect islikely achieved through a combination of the degradationof cell cycle factors, cyclin D1 and D3, and dephosphorylation of Rb.

Yuki KUJURO

18

©2010 National Academy of Sciences
Kujuro Y, et al. Esophageal cancer-related gene 4 is a secreted
inducer of cell senescence expressed by aged CNS precursor cells.
Proceedings of the National Academy of Sciences. 107.8259-64 (2010)

Having established a solid case for the involvement of Ecrg4 in OPC senescence in vitro, Kujuro next
looked at the mouse brain to see if it had similar effects in vivo. She found that while the gene showed
only weak expression in the brains of young mice, it was much higher in aged brain, specifically in theregions where OPCs and neural precursor cells reside. Interestingly, Ecrg4 was upregulated even interminally differentiated neurons, which have ceased dividing and should therefore be immune to senescence.

E xpression of Ecrg4 (red) is
induced in oligodendrocy tes(green, top panels) and neuralstem cells (green, bottom panels) as aging progresses from
2 months (left) to 20 months(right). Nuclei are stained blue.

“The question of ‘Why do we age?’ is a longstanding mystery, and since the discovery of stem cells,
people have speculated that it may be due in part to natural wear and tear affecting their ability tofunction,” says Kondo. “In this study, we identified one mechanism relating to the aging of neural stemand progenitor cells, and we’re hopeful that this will lead us to a better understanding to diseases of
the nervous system.”19

2010 Events
The RIKEN CDB strives to engage with the public through a variety of media, including its website, media coverage and direct interactions. This
final form of outreach takes the form of frequent site visits by groups and delegations from around the Japan, and in our annual Open House.

In addition to these public engagement activities, the Center also organizes events for bringing scientists together outside of the laboratoryenvironment, such as its annual Retreat.

Kagaku Ichiba and RIKEN Kids’ Lab
In February, the RIKEN CDB jointly organized a science exchange event, “Kagaku
Ichiba,” with the Kobe Science Museum, where it was held. This event, supported bya grant from the Japan Science and Technology Agency (JST), was held in an effort
to promote science to the general public and attended by people of all ages, and featured lectures and hands on exhibits for kids of all ages.

In March, the CDB opened the RIKEN Kids Lab in the Kobe Science Museum. This
space provides an environment for discovery and fun, with graphic and hands-on activities relating to topics such as the development of the body from a single cell, andthe exciting future promise of stem cells in medicine. Additional exhibits include an
introduction to green fluorescent protein (GFP) and the fantastic regenerative ability offlatworms known as planarians.

2010 Open House
The 2010 CDB Open House, held in conjunction with the RIKEN Kobe Institute’s Center for Molecular Imaging Science and the RIKEN Next Generation Supercomputer R&D
Center, was the most well-attended event in the CDB’s history, with over 1700 visitors
on a single day in November. The event featured a commemorative talk by Kobe Institute and CDB Director, Masatoshi Takeichi, marking ten full years of the Center’s history, and numerous open laboratories, where PIs, postdocs, technicians and students
welcomed the curious and explained their work. Themes for this year’s special exhibits
included stem cells and regenerative medicine, and cell and body morphology. For
younger visitors, a number of games and arts and crafts activities were available.

2010 Retreat
The CDB held its annual retreat for its research staff in the mountain setting of Sasayama. CDB lab leaders, research scientists and student trainees gathered in a closed
meeting to share and discuss the latest developments from their labs, and to get better acquainted through social activities and casual conversation away from the bench.
This year, CDB Advisory Council member Stephen Cohen of the Institute of Molecular
and Cell Biology in Singapore joined the Retreat as well, adding a voice of broad international scientific experience.

20

The first cell division of a C. elegans embryo

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

Shinichi AIZAWA Ph.D.
http://www.cdb.riken.jp/en/aizawa
Shinichi Aizawa received his Ph.D. in biochemistry from the Tokyo Kyoiku University Department of Zoology in 1973. He spent the period
from 1974 to 1979 as an investigator at the Tokyo Metropolitan Institute of Gerontology, then
two years as a research fellow at the University of Washington. He returned to the Tokyo
Metropolitan Institute of Gerontology in 1982,
where he remained until 1986, when he moved
to the RIKEN Tsukuba Life Science Center. He
was appointed professor in the Kumamoto University School of Medicine in 1994, and servedin that position until 2002. Since 2000 he hasserved as CDB deputy director and group director of the Vertebrate Body Plan lab, as wellas head of the Animal Resources and Genetic
Engineering Laboratory. In January 2007, he
was appointed President of the JapaneseSociety of Developmental Biologists. He also
serves as editor for the journal, Mechanisms ofDevelopment.

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

Staff
Group Director

Visiting Scientist

Research Specialist

Research Associate

Shinichi AIZAWA
Yoko SUDA
Masaki TAKEUCHI
Research Scientist

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

Daisuke KUROKAWA
Yusuke SAKURAI
Technical Staff

Eriko KAJIKAWA
Hiromi NAKAO
Tomomi OMURA
Maiko TAKAHASHI
Assistant

Sayo SAITO

Publications
Shibata M, et al. MicroRNA-9 regulates neurogenesis inmouse telencephalon by targeting multiple transcription factors. J Neurosci (2011) In press
Kurokawa D, et al. Evolutionary origin of the Otx2 enhancer
for its expression in visceral endoderm. Dev. Biol. 342, 11020 (2010).
Suda Y, et al. The same enhancer regulates the earliest
Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone. Development 137.
2939-49 (2010)
Suda Y, et al. Evolution of Otx paralogue usages in earlypatterning of the vertebrate head. Dev Biol 325. 282-95(2009)
Takeuchi M et al. Germ layer patterning in bichir and lamprey; an insight into its evolution in vertebrates. Dev Biol
332. 90-102 (2009)
Hirano M, et al. EPB41L5 functions to post-transcriptionallyregulate cadherin and integrin during epithelial-mesenchymal transition. J Cell Biol 182. 1217-30 (2008)22

microRNA-9 is required for neural progenitor proliferation and upper cortical plate formation: Together
with Elavl1, microRNA-9 targets the Nr2e1 mRNA 3’UTR to enhance its expression. microRNA-9 also
indirectly inhibits Pax6 expression by suppressing Meis2 expression. Both Nr2e1 and Pax6 are essential for upper cortical plate formation.

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

Hideki ENOMOTO M.D., Ph.D.
http://www.cdb.riken.jp/en/enomoto
Hideki Enomoto received his M.D. from the
Chiba University School of Medicine in 1988,
and his Ph.D. from the same institution in 1996.
After serving as the Chief of the Department ofPediatric Surgery, Matsudo Municipal Hospitalin Chiba, Japan, he moved to the Washington
University School of Medicine, St. Louis in
1997, to work as a research associate studying
the physiological roles of the GDNF Family of
Ligands in neural development and function.
He returned to Japan to take a position as ateam leader at the CDB in 2002.

Staff
Team Leader

Hideki ENOMOTO
Research Scientist

Junior Research Associate

Mayumi NAGASHIMADA
Yohei YONEKURA

Pilaiwanwadee
HUTAMEKALIN
Keisuke ITOH
Toshihiro UESAKA

Student Trainee

Technical Staff

Assistant

Chihiro NISHIYAMA
Hiroko SONOBE

Mitsuhiro IWASAKI
Part-Time Staff

Toko KONDO

Kaori HAMADA

Publications
Uesaka T, et al. Neural precursor death is central to thepathogenesis of intestinal aganglionosis in Ret hypomorphic
mice. J Neurosci 30. 5211-8 (2010)
Burzynski G, et al. Genetic model system studies of thedevelopment of the enteric nervous system, gut motility and
Hirschsprung’s disease. Neurogastroenterology & Motility,
21. 113-27 (2009) Review.
Uesaka T, et al. Diminished Ret expression compromises
neuronal survival in the colon and causes intestinal aganglionosis in mice. J Clin Invest 118. 1890-8 (2008)
Gould T W, et al. The neurotrophic effects of glial cell linederived neurotrophic factor on spinal motoneurons are
restricted to fusimotor subtypes. J Neurosci 28. 2131-46(2008)
Uesaka T, et al. Conditional ablation of GFRalpha1 in postmigratory enteric neurons triggers unconventional neuronal
death in the colon and causes a Hirschsprung's diseasephenotype. Development 134. 2171-81 (2007)

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

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

23

Neocortical Development

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

Staff
Team Leader

Technical Staff

Research Scientist

Part-time Staff

Carina HANASHIMA
Yuko GONDA
Takuma KUMAMOTO
Ken-ichi MIZUTANIVisiting Researcher

Torsten BULLMANN

Yuko WADA

Chika KUMAMOTO
Reiko ODA
Assistant

Yukie NAKAMURA

Student Trainee

Ken-ichi TOMA

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

Publications
Fishell G and Hanashima C. Pyramidal neurons grow up
and change their mind. Neuron 57. 333-8 (2008)
Hanashima C, et al. The role of Foxg1 and dorsal midline
signaling in the generation of Cajal-Retzius subtypes. J
Neurosci 27. 11103-11 (2007)
Hanashima C, et al. Foxg1 suppresses early cortical cellfate. Science 303. 56-9 (2004)

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

24

Morphogenetic Signaling
The main research interest in my lab is focused on the mechanisms by whichcell-cell and tissue-tissue interactions are modulated during embryonic morphogenesis. A range of vital developmental processes, including the ability to build
complex structures through selective cell adhesion, to move through the body,and to form epithelial tissues, rely on interactions between cells, and between
cells and the extracellular matrix. The means by which cells are able to communicate, congregate, and work together to build a body is a central question in
the study of morphogenesis. To tackle this problem, we use the model organism
Drosophila which is suited for genetic dissection of biological processes and forhigh-resolution imaging, and study the problem at three levels of cellular organization: single-cell, multiple cells, and organ.
Our study is centered on the tracheal system, a respiratory organ that delivers air
directly to tissues through a network of finely branched epithelial tubes. Tracheal
development begins by invagination of ectodermal epithelium that subsequently
forms branches with specific types of cells migrating in stereotyped patterns.
Cellular forces produce mechanical strain in the epithelium, and alleviation of thatstrain is essential for smooth tissue movement. As a consequence of epithelial
cells behaving as elastic bodies, their shape changes and movement proceedwith local fluctuations. We aim at elucidating (1) mechanisms that coordinate cell
movement, (2) mechanisms for alleviating tissue strain, and (3) mechanisms ofcross-talk between these two mechanisms. To accomplish these goals, the mechanical state of cells will be measured by combining techniques such as quantitative cell imaging, and various cell perturbation techniques are being used toassess the mechanical states of cells. The results of these analyses will be used
to construct epithelial cell models and simulations.
In addition, we study intracellular mechanisms of cell polarization and elongation
using model systems of mechanosensory bristles and spermatids, and higher
level organization in insect limbs.

Shigeo HAYASHI Ph.D.
http://www.cdb.riken.jp/en/hayashi
Shigeo Hayashi received his B. Sc. in Biology
from Kyoto University in 1982, and his Ph.D. in
Biophysics from the same institution in 1987,
for his work on lens-specific regulation of thechicken delta crystallin gene. Inspired by thediscovery of the homeobox, he changed his
research focus to the developmental genetics
of Drosophila and spent three years as a postdoctoral research associate in Matthew Scott's
lab at the University of Colorado before returning to Japan to work in the National Institute of
Genetics. He became an associate professor
at the same Institute in 1994, and professor
in 1999. Also in 1999, he received the incentive prize of the Genetics Society of Japan for
Genetic Studies on Drosophila Development.
He was named group director of the Morphogenetic Signaling research group at the
RIKEN CDB in May 2000. His current researchinterests are dynamic aspects of cell adhesion,
cell migration and cell morphogenesis in Drosophila.

StaffGroup Director

Shigeo HAYASHI
Research Scientist

Bo DONG
Kagayaki KATO
Takefumi KONDO
Nao NIWA
Tatsuhiko NOGUCHI
Tetsuhisa OTANIReiko TAJIRI

Technical Staff

Ai AKIMOTO
Michiko KOIZUMIHousei WADA
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
Assistant

Hiromi WANAKA

Publications
Otani T, et al. IKKε regulates cell elongation through recycling endosome shuttling. Developmental Cell 20. 219-32(2011)
Niwa N, et al. Evolutionary origin of the insect wing via integration of two developmental modules. Evol Dev 12. 16876 (2010)
Kawahashi K and Hayashi S. Dynamic intracellular distribution of Notch during activation and asymmetric cell division
revealed by functional fluorescent fusion proteins. Genes
Cells 15. 749-59 (2010)

Formation of new organ primodia often involves segregation from the epithelial placode by invagination. This picture shows a cross section of the Drosophila tracheal placode. At the center of theplacode, tracheal primordial cells (green) constrict apical region facing outside of the epithelia andinvaginate inwardly. This process involves complex interplay of cell boundary tension in the plane ofepithelia orchestrated by EGF receptor signaling and inward (basal) movement of cells driving invagination. Cell boundaries are marked with magenta.

Tajiri R, et al. Dynamic shape changes of ECM-producing
cells drive morphogenesis of ball-and-socket joints in the flyleg. Development 137. 2055-63 (2010)
Kaido M, et al. Essential requirement for RING finger E3
ubiquitin ligase Hakai in early embryonic development ofDrosophila. Genes Cells 14. 1067-77 (2009)25

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

Masahiko HIBI M.D., Ph.D.
Masahiko Hibi received his M.D. from Hiroshima
University School of Medicine in 1988, and his
Ph.D. from Osaka University in 1992. From 1992
to 1995, he worked as a postdoctoral fellow in
Michael Karin's lab in the University of California,
San Diego Department of Pharmacology, andthen returned to Japan as a research associate
in the Division of Molecular Oncology at Osaka
University Medical School. He was appointed
associate professor in the same division in 1999,
where he remained until he assumed his positionas team leader at the RIKEN CDB. He took aprofessorship at Nagoya University in 2009.

Staff
Team Leader

Masahiko HIBITechnical Staff

Special Postdoctoral
Researcher

Koji TANABE

Setsuko FUJII
Kana KADOWAKIBANDOAya KATSUYAMA

Research Scientist

Part-Time Staff

Shuichi KANI
Hideaki NOJIMA
Takashi SHIMIZU
Takeshi SHIMIZU

Isao HARIMOTO
Yuko HIROSE
Shigemi SHIBUYAKazuko YAMAMOTO

Publications

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

Tanabe K, et al. Atypical PKC regulates primary dendrite
specification of cerebellar Purkinje cells by localizing Golgi
apparatus. J Neurosc.30. 16983-92 (2010)
Shimizu T, et al. Zinc finger genes Fezf1 and Fezf2 control
neuronal differentiation by repressing Hes5 expression inforebrain. Development 137. 1875-85 (2010)
Kani S,et al. Proneural gene-linked neurogenesis in zebrafish cerebellum. Dev Biol 343. 1-17 (2010)
Nojima H, et al. Syntabulin, a motor protein linker, controls
dorsal determination. Development 137. 923-33 (2010)
Nagao Y, et al. Dynein axonemal intermediate chain 2 isrequired for formation of the left-right body axis and kidney
in medaka. Dev Biol 347. 53-61 (2010)

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

26

Sensory Circuit Formation
The mammalian nervous system is composed of enormous numbers of neurons,
but how do these cells take on diverse fates and organize and array themselves
during development? In recent years, it has become clear that the mouse olfactory system provides an excellent platform for addressing these questions experimentally. In this system, there are around 1,000 types of odorant receptors
that are capable of detecting and discriminating between odorant molecules.
Each olfactory sensory neuron expresses a single type of odorant receptor, andthe axons of neurons expressing the same receptor type converge on the samesite in the olfactory bulb. Olfactory sensory neurons connect axons to the dendrites of mitral and tufted (M/T) cells in the bulb, where each receives inputs froma single specific type of olfactory sensory neuron.
It has generally been thought that neuronal identities are genetically programmed, and that neuronal connectivity is maintained by molecular “lock andkey” mechanisms. The mouse olfactory system, however, is highly adaptive;
olfactory neuronal identities are dependent on peripheral inputs, and form thebasis for a self-organizing olfactory map. A better understanding of this flexibility
may provide new insights into the diversification of function that took place during the evolution of the human brain.
Our lab will seek to develop a better understanding of odorant receptor-dependent axon projection of olfactory sensory neurons, and the formation of neuronal
circuitry in the olfactory bulb dependent on inputs from these neurons. We will
also seek to develop next-generation genetic tools to aid in our developmental
and functional analyses of specific neuronal inputs in the brain.

Takeshi IMAI Ph.D.
http://www.cdb.riken.jp/en/imai
Takeshi Imai completed his Ph. D. in the laboratory of Prof. Hitoshi Sakano at the Universityof Tokyo Graduate School of Science in 2006.
He did consecutive postdoctoral fellowships
under the CREST Program and the Universityof Tokyo in 2006 and 2007, before his appointment as Assistant Professor at the same university in 2009. He received independent funding under the JST PRESTO program that year,
before joining the RIKEN CDB as Team Leader
in 2010. He also holds an adjunct position as
Associate Professor at the Kyoto University
Graduate School of Biostudies.

Staff
Team Leader

Takeshi IMAITechnical Staff

Momo FUJII

Student Trainee

Yuhei ASHIDA
Assistant

Aya KATSUYAMA

Publications
Imai T, et al. Topographic Mapping - The Olfactory System.
Cold Spring Harb Perspect Biol 2. A001776 (2010)
Imai et al., Pre-target axon sorting establishes the neuralmap topography. Science 325.585-90 (2009)
Imai T and Sakano H. Odorant receptor-mediated signaling
in the mouse. Curr Opin Neurobiol 18. 251-60 (2008)
Imai T and Sakano H. Roles of odorant receptors in projecting axons in the mouse olfactory system. Curr Opin Neurobiol 17. 507-15 (2007)
Imai et al. Odorant receptor-derived cAMP signals direct
axonal targeting. Science 314. 657-61 (2006)

Axonal projection of olfactory sensory neurons
to the olfactory bulb. Cyan, olfactory sensory
neurons expressing I7-CFP; Yellow, olfactory
sensory neurons expressing I7-dnPKA-YFP.

A graded olfactory map forms in the absence of the olfactory bulb. Fibrocellularmass of a bulb-less mutant mouse, stainedwith anti-gap43 (green) and anti-Neuropilin-1(red).

27

Cell Lineage Modulation
Mankind has long wondered whether it might be possible to regenerate bodytissues and structures that have been lost. Recent research has shown that eventhe adult body contains a diverse range of tissue-specific somatic stem cells thatserve to maintain the function and integrity of tissues, opening a promising avenue toward possible applications in regenerative medicine. But the limits on thenumber of somatic stem cells present in any individual, coupled with the limited
availability of donors, have heightened interest in the development of new, alternative means of generating stem cells.

Toru KONDO Ph.D.
Toru Kondo received his B.Sc. from Waseda
University and his M.S. and Ph.D. from the
Osaka University Institute for Molecular andCellular Biology. He worked as a postdoctoral
fellow in Shigekazu Nagata's lab at Osaka
Bioscience Institute from 1994 to 1998 and in
Martin Raff's lab at University College London
MRC Laboratory for Molecular Cell Biology
from 1998 to 2001. He returned to Japan totake a position as an associate professor at
Kumamoto University in 2001 and moved toCambridge University Centre for Brain Repair
in 2002 to take a group leader position. He wasappointed team leader in 2005. He was appointed professor at Ehime University in 2009.

Research in our lab seeks to develop a better understanding of the molecularmechanisms involved in the reacquisition of “stemness” (stem cell characteristics) using the dedifferentiation of oligodendrocyte progenitor cells (OPCs), which
are abundantly present in the central nervous system, into neural stem-like cells,
knowledge which it is hoped may lead to new techniques for the generation ofneural stem cells. We are also interested in two other related subjects: one is thecharacterization of stem cell-like cancer cells (sometimes called “cancer stem
cells” or “cancer initiating cells”), found in malignant glioma, and to find markers
and targets for them, potentially leading to the development of novel anti-cancer
therapies. The other is to develop a better understanding of the molecular mechanisms of cellular senescence, which are involved in both the defects of tissuespecific stem cells and the suppression of tumorigenesis.

Staff
Team Leader

Toru KONDO
Research Scientist

Hiromi TAKANAGA
Nobuko TSUCHIDASTRAETEN
Visiting Researcher

Norishige YAMADA

Student Trainee

Yuki KUJURO
Kenji NISHIDE
Tatsuya TAKEZAKI
Agency Staff

Makiko NAKAHARA
Assistant

Yukie NAKAMURA

Technical Staff

Keiko MIWA
Yuka NAKATANI

Publications
Hide T, et al. Combination of a Ptgs2 inhibitor and an EGFR
signaling inhibitor prevents tumorigenesis of oligodendrocytelineage derived glioma-initiating cells. Stem Cells (2011) In
press
Sumitomo A, et al. Transcriptional Mediator Subunit MED1/
TRAP220 in Stromal 1 Cells Is Involved in Hematopoietic
Stem/Progenitor Cell Support through Osteopontin Expression. Mol Cell Biol 30. 4808-17 (2010)

C

D

Kujuro Y, et al. Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed in aged CNS
precursor cells. Proc Natl Acad Sci USA 107. 8259-64(2010)
Hide T, et al. Sox11 prevents tumorigenesis of gliomainitiating cells by inducing neuronal differentiation. Cancer
Res 69. 7953-59 (2009)
Nishide K, et al. Gliomagenesis from transformed neuralstem cells depleted of prominin1-expressing cells. PLoS
ONE 4. e6869 (2009)
Takanaga H, et al. Gli2 is a novel regulator of sox2 expression in telencephalic neuroepithelial cells. Stem Cells 27.
165-74 (2009)28

Neural stem/precursor cells may be cells-of-origin for brain disorders. Brain tumor formed by induced
mouse glioma-initiating cell, NSCL61, shows a phenocopy of human GBM (A and B). Neural stem
cells (C, green: Musashi1) and oligodendrocyte precursor cells (D, green: NG2) in the aged mousebrain are positive for a new senescence marker, Ecrg4 (red).

Evolutionary Morphology
By studying the evolutionary designs of diverse animal species, I hope to gain adeeper insight into the secrets behind the fabrication of morphological designs.
Integrating the fields of evolutionary morphology and molecular genetics, ourlab seeks to expand the understanding of the relationship between genome andmorphology (or body plan) through investigating the evolutionary changes in developmental processes, and also the process of evolution in which phenotypic
selection shapes developmental programs. Our recent studies have focusedon novel traits found in the vertebrates, such as the jaw, the turtle shell, and the
mammalian middle ear. By analyzing the history of developmental patterns, I
hope to open new avenues toward answering as-yet unresolved questions about
phenotypic evolution in vertebrates at the level of body plans.
Through the study of evolutionarily novel structures, our lab has identified
changes in developmental mechanisms that have obliterated the structural homologies between organisms as evidenced in such novel structures as the jaw ingnathostomes (jawed vertebrates) and the turtle shell. Developmental studies of
the cranial region of the lamprey are intended to shed light on the true origins of
the vertebrate head and neck, as lampreys lack a number of important features,
including jaws, true tongues, and trapezius muscles, that are possessed only
by gnathostomes. We aim to resolve the question of what primary factors havechanged during evolution by comparing the developmental patterns that yieldsuch innovations, and by the experimental construction of phenocopies in one
animal that mimic structures in another.
The turtle’s shelled body pattern appears suddenly in the fossil record. Our lab’s
research into turtle carapace development addresses the developmental changes that resulted in this abrupt and dramatic morphological change, and is aimed
at identifying genes that function differently in turtle and other amniotes, which itis hoped will provide a key to discovering the true targets of natural selection in
the acquisition of a shell.

Shigeru KURATANI Ph.D.
http://www.cdb.riken.jp/en/kuratani
Shigeru Kuratani received his masters and Ph.D.
from the Kyoto University Department of Zoology. He spent the period from 1985 to 1988
studying comparative embryology in the Department of Anatomy, University of the Ryukyus,
and 1988 to 1991 working in experimental embryology in the Department of Anatomy at the
Medical College of Georgia before moving to the
Biochemistry Department at the Baylor Collegeof Medicine, where he was engaged in molecular
embryological research. He returned to Japan
in 1994 to take a position as associate professor at the Institute of Medical Embryology andGenetics in the Kumamoto University School of
Medicine. He moved to Okayama University toassume a professorship in the Department of
Biology in 1997, where he remained until he wasappointed team leader at the CDB. He was appointed group director in 2005.

Staff
Group Director

Shigeru KURATANI
Research Scientist

Shinichi AOTA
Naoki IRIE
Hiroshi NAGASHIMA
Kinya OTA
Juan PASCUAL ANAYA
Takao SUZUKI
Motoki TADA
Masaki TAKECHISpecial Postdoctoral
Researcher

Tatsuya HIRASAWA

Technical Staff

Satoko FUJIMOTO
Tamami HIRAI
Junior Research Associate

Noritaka ADACHI
Yasuhiro OISI
Fumiaki SUGAHARA
Student Trainee

Hiroki HIGASHIYAMA
Part-Time Staff

Maki MIYAUCHI
Kazuko YAMAMOTO
AssistantMika HIKAWA
Yuko HIROFUJI

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

CT-scanned skeleton of Pelodiscus sinensis juvenile

Takio Y, et al. Evolutionary biology: lamprey Hox genes andthe evolution of jaws. Nature 429. 1 p following 262 (2004)29

Sensory Development
Organogenesis is a monumental undertaking. From siting a given organ in its
correct location, to confirming that it is correctly engineered and constructed, toensuring the necessary functional connections with other organs, the embryo
has a daunting task before it; indeed, perhaps we should be surprised that theresult is so frequently successful. Taking sensory organogenesis as our model
system, we hope to be able to follow the route an organ takes in its development,
and seek to understand the steps that allow it to reach maturity. One aim is to beable to recapitulate these steps in vitro, with the ultimate goal of providing potentially clinically relevant applications.

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

Staff
Team Leader

Student Trainee

Research Scientist

Part-Time Staff

Rajesh LADHER
Xiao Rei CAIAkira HONDA
Tomoko KITA
Siu Shan MAK
Yuko MUTA

Kazuya ONO

Yoshiko KONDO
Yoko MATSUOKA
Anna WRABELAssistant

Noriko HIROI

Foreign Postdoctoral
Researcher

The ear provides a particularly rich system for study. The inner ear, which converts mechanical stimulation to electrical impulses, is formed from a piece of tissue originally destined to become skin; a fate that is altered by instructive signals
in a process known as induction. In our research to date, we have identified themechanism and the signals that set this early tissue on its path toward an inner ear fate as well as those that determine its position. We next hope to learn
how these signals are themselves established. We are also investigating the rolethese signals play in the regulated differentiation of the otic placode, and in better understanding the cellular, molecular and embryological process responsible
for this elegantly engineered organ of hearing.
The inner ear cannot function alone. In higher vertebrates, it requires the middle
ear to transduce sound that has been channeled through the external ear, as
well as specialized regions within the brain to interpret the information it sends.
But how is this intricate integration achieved? Using a combination of molecularand embryological techniques, we are characterizing the systems the embryo
uses in the coordinated construction of the auditory apparatus.
We believe that an understanding of the embryological systems that control thedevelopment of the inner ear can be applied to the development of other organs
as well, sensory organs in particular. By working to comprehend the induction
and development of other sense organs, such as the olfactory system and theeye, we are now taking steps toward achieving insights into common mechanisms underpinning the development of organ systems.

Paul O'NEILL

Publications
Ladher R K, et al. From shared lineage to distinct functions:the development of the inner ear and epibranchial placodes.
Development 137, 1777-1785 (2010)
Teraoka M E, et al. Rostral paraxial mesoderm regulates
refinement of the eye field through the bone morphogenetic
protein (BMP) pathway. Dev Biol (2009)
Freter S, et al. Progressive restriction of otic fate: the role of
FGF and Wnt in resolving inner ear potential. Development
135. 3415-24 (2008)
Sai X and Ladher R K. FGF signaling regulates cytoskeletal
remodeling during epithelial morphogenesis. Curr Biol 18.
976-81 (2008)
Manley G A, and Ladher R K. Phylogeny and evolutionof ciliated mechanoreceptors Springer, Handbook of theSenses (Elsevier) ed. P. Dallos (2007)
Sakamoto T, et al. Hair cell differentiation becomes tissue
specific by E9.5 in mouse inner ear. Neuroreport 18. 841-4(2007)
Ladher R K, et al. FGF8 initiates inner ear induction in chickand mouse. Genes Dev 19. 603-13 (2005)

30

Ciliated mechanoreceptors, or inner ear hair cells, transduce sound information in the organ of corti,
part of the mammalian cochlea. Shown are the 4 rows of inner ear hair cells that make up the organof corti, stained with alpha-tubulin (to show kinocilia in green) and phalloidin to show the actin basedstereocilia (in red).

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

Fumio MATSUZAKI Ph.D.
http://www.cdb.riken.jp/en/matsuzaki
Fumio Matsuzaki received his B. Sc. from the
Department of Biochemistry and Biophysics at
the University of Tokyo in 1979, and his doctorate from the same institution in 1984, for his
work on the characterization of the erythrocyte
cytoskeletal structure. He spent the period from1984 to 1988 as a postdoctoral fellow, first in the
Department of Cell Biology at the Tokyo Metropolitan Institute of Medical Science, then in thelaboratory of Gerald Edelman at the Rockefeller
University. He returned to Japan in 1988 to takea position as a section chief in the Departmentof Molecular Genetics in the National Instituteof Neuroscience. In 1998, he was appointed
professor in the Department of Developmental
Neurobiology at Tohoku University and remained
there until taking his current position as groupdirector at the RIKEN CDB.

Staff
Group Director

Visiting Scientist

Special Postdoctoral
Researcher

Technical Staff

Fumio MATSUZAKI
Daijiro KONNO

Research Scientist

Tomohiko IWANO
Atsushi KITAJIMA
Atsunori SHITAMUKAI
Shigeki YOSHIURA
Foreign Postdoctoral
Researcher

Jeremy Nicholas
PULVERS

Ayano KAWAGUCHI
Yoichi KOSODO
Tomoko AOKI
Aki MASUDANao OTA
Taeko SUETSUGU
Student Trainee

Masahiko INOUE
Tomoaki KATO
Yoshiaki SATO
Takuya YOKOMATSU
AssistantJunko ISHIGAI

Publications
Shitamukai et al. Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors
outside the germinal zone that resemble primate outersubventricular zone progenitors. J Neurosci in press
Kitajima A, et al. Progenitor properties of symmetricallydividing Drosophila neuroblasts during embryonic and larval
development. Dev Biol 347. 9-23 (2010)
Ogawa H, et al. Protein phosphatase 2A negatively regulates aPKC signaling by modulating phosphorylation of Par6 in Drosophila neuroblast asymmetric divisions. J. Cell
Science 122. 3242-3249 (2009)
Shioi G, et al. Structural Basis for Self-Renewal of Neural
Progenitors in Cortical Neurogenesis. Cereb Cortex 19(suppl_1). i55-i61 (2009)

In the developing mouse brain, neural stem cells normally maintain epithelial structure during asymmetric division. Oblique cleavages occur stochastically and generate a different type of self-renewing
neurogenic progenitors (white arrows) outside of the germinal zone. Divisions occur at 1h:46min,
21:39, and 41:18 in these consecutive images.

Konno D, et al. Neuroepithelial progenitors undergo LGNdependent planar divisions to maintain self-renewability
during mammalian neurogenesis. Nat Cell Biol 10. 93-101(2008)
Kawaguchi A, et al. Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis.
Development 135. 3113-24 (2008)31

α-catenin relies on force

Shigenobu YONEMURA

Near their apical surfaces, epithelial cells are bound to their neighbors through molecular interactions that collectively give rise to an active space known as the adherens junction. In addition to holding cells of the same typein close proximity, these junctions are believed to act as sensors for mechanical forces pushing and tugging on
the cellular membrane, allowing it to respond and maintain its shape and integrity through rearrangements of thecytoskeleton. It is suspected that cytoplasmic elements that attach to cadherins, the primary cell-cell adhesion
molecules, are responsible for this regulatory activity, but recent research has cast doubt on the most popular
contender, a protein called α-catenin, as the elusive molecule behind this mechanism.
Now, however, a study by Shigenobu Yonemura, head of the Electron Microscope Laboratory, and colleagues
has reinstated α-catenin as the leading candidate for the link between the adhesion machinery and the cytoskeleton. This latest work, published in Nature Cell Biology, reveals that the protein recruits a second factor, vinculin,known to interact with the cytoskeletal molecule actin in a manner determined by mechanical forces.
The key molecular complex in the adherens junction is known as the cadherin-catenin complex, which forms
when a group of catenin molecules (ß, α, and p120) attaches to the cytoplasmic tail of any one of the many classic cadherin adhesion molecules. α-catenin is also known to bind with actin filaments and multiple actin-binding
elements (including vinculin), which seemingly made it a natural candidate as the linker between cell adhesion
and the cytoskeleton. But recent evidence suggesting that α-catenin does not simultaneously bind both the cadherin complex and actin filaments in vivo put its role in doubt.

32©2010 Nature Publishing Group
Yonemura S, et al. α-Catenin as a tension transducer that induces
adherens junction development. . Nat Cell Biol 12, 533 - 542 (2010)

Intrigued by this problem, Yonemura and colleagues looked at a number of epithelial cell types in vitro
for clues into the mechanism. Observing that vinculin (but not α-catenin) accumulates at adherens
junctions only in the presence of the force-generating protein myosin II, the team conjectured thatα-catenin’s binding to vinculin might be dependent on the activity of myosin II. Dissection of the protein’s molecular structure revealed three active regions: the amino terminus, which binds ß-catenin;
the carboxy end, which binds various actin-binding factors; and a central region where vinculinbinding was thought to occur. Experimenting with various combinations of the presence and absenceof α-catenin and force generation, Yonemura found that, contrary to the original speculation, vinculin
molecules do not bind α-catenin in a force-dependent manner, but instead that a central region of theprotein inhibits vinculin binding and that the carboxy terminus is required for the force-dependent alleviation of this inhibition.

When α-catenin molecules (blue)
distributing evenly along the cellcell interface are stretched bymyosin II (green)-derived forces
applied at adherens junctions,they become bound to a monoclonal antibody to α-catenin (red),suggesting force-dependentchanges in α-catenin conformation.

Seeking to pinpoint its functional regions, the team generated a series of α-catenin mutants lacking
various regions, and identified the vinculin-binding region (residues 325–360) and the region responsible for releasing the inhibition of vinculin binding (residues 697–906). From this structural understanding, Yonemura developed a model for force-dependent binding between α-catenin and vinculin, in
which the application of mechanical force exposes the region of the α-catenin protein that releases
the inhibition of vinculin, allowing its recruitment. Further examination of α-catenin dynamics in theabsence and presence of cytoskeleton-generated forces revealed that its turnover was lower in presence of myosin II activity (which generates force), in line with predictions from the functional model.
And, importantly, the force-dependent mechanism was shown to be in effect in membrane-boundα-catenin as well, indicating that even when it functions as part of the cadherin-catenin complex,
α-catenin is still capable of responding to forces.

Model of force-dependent vinculin binding to α-catenin. N
and C represent the N and C
termini of α-catenin, respectively. A possible unknown linker isindicated with a question mark.

33

Morphogenesis of ball-and-socket
joint revealed
Insects wear their skeletons on the outside, but despite
their external location, these chitinous cuticles must perform many of the same functions as vertebrate bones. In
addition to their protective functions, exoskeletons also
provide structural support and defined range of motion
for limbs. This last role is the duty of the joints, which,as in vertebrate limbs, come in various configurations
and play central roles in such behaviors as mobility andfeeding. Indeed, an entire phylum of invertebrates, theArthropoda, was originally named after its members’“jointed feet.” But while the physiology of vertebrate
joints has been extensively studied, much less is know
about their development.
Reiko Tajiri in the Laboratory for Morphogenetic Signaling (Shigeo Hayashi, Group Director) in collaboration
with the Electron Microscopy Laboratory (Shigenobu
Yonemura, Laboratory Head) chose the leg of the fruit fly
Drosophila melanogaster as a model to study the formation of ball-and-socket joint skeletons. Using a combination of cell labeling and imaging technologies, the group
showed that this structure forms through a sequential
process involving extensive cell migration and tissue remodeling.
The study began with some basic observations, as thedevelopment of joint cuticles is largely unexplored. Aftercharacterizing the morphology and cell arrangementsin the mature joint by optical and electron microscopy,
Tajiri looked at the same region earlier in development
to watch how these come to be. The leg primordium
begins as grooved epithelium that gradually lengthens,with points of constriction at the future joints. The joints
themselves form when cells on the dorsal side constrict
more tightly and fold inward in a proximal direction,
deepening the furrow into an empty cavity. Once thiscavity has formed, the cells that line it begin to deposit
cuticle, forming the ball at the deepest part of the cavity,
and the socket on the dorsal side. The socket cuticle
later expands ventrally toward the bottom of the cavity,and eventually covers the entire surface.

Reiko TAJIRI

34

The group next turned to a structural feature known asplasma membrane plaques (PMPs), which were believed
to be the sites of chitin release and of the adhesion
between the cuticle and underlying cells. The question
Tajiri sought to answer was, do the cuticle-forming activity move like a wave over a bed of stationary cells, or do
the cells themselves move? The group used a genetic
labeling method to monitor the locations of cells within
the joint region at various stages, and identified three©2010 The Company of Biologists
Tajiri R, et al. Dynamic shape changes of
ECM-producing cells drive morphogenesis of ball-and-socket
joints in the fly leg. Development 137.2055-63 (2010)

drivers of reporter gene expression in distinct, but partially overlapping, subpopulations. By tracking
the behavior of the labeled cells, they determined that the apical surfaces of cells making up the balland socket components of the joint followed the distribution of PMPs.

( To p) D i s t a l p a r t of t h e l e g(tarsus) in Drosophila melanogaster, where segments areconnected through joints (arrows). (Middle). The joint cuticle
has the ball-and-socket architecture (cuticle in magenta andcell cortices in green). (Bottom)
Developing ball cuticle it is surrounded by plasma membrane
plaques (yellow arrows in theelectron micrograph).

The next step was to see which parts of the cuticle were produced by which of the genetically marked
regions. Tajiri and colleagues found that one subpopulation (expressing fng) was specific to the dorsal
aspect of the socket, while the other two, neur- and bib-, overlapped, with neur-expressing cells giving rise to ventral socket while bib-expressing cells formed the cuticle of the ball. In contrast to the
cuticle, the surfactant that lubricates the joint seemed to be manufactured more diffusely. Perhaps
unsurprisingly, blockade of chitin synthesis disrupted the entire joint cuticle, but interestingly had noeffect on the cuticle-secreting cells, which changed shape as usual even when the cuticle was chemically disrupted, indicating that the cuticle is unlikely to play a guidance role.
“Extracellular matrices occupy a major portion of skeletal elements of animal body, and while the rolesof extracellular matrices in cell signaling have been studied extensively, much less is known about
how cells instruct shaping of extracellular environment,” says Hayashi. “This study opens a new direction of research into the morphogenesis of non-cellular parts of insect exoskeletons, which vary enormously in shape, color, and function.”

35

Bird’s eye view of mesodermal gene expression

The mesoderm, one of the three embryonic germ layers, is the source of a broad range of cell types, from muscle, bone and cartilage, to the reproductive system. In two major amniote groups (birds and mammals) the mesoderm arises from the ectoderm in the form of a structure known as the primitive streak. The cells in this transitory strip of mesodermal precursors are guided by positional cues that roughly translate starting points along
the anterior-posterior axis of the streak to tissue destinations along the dorsal-ventral axis of the embryo. Cells
from anterior sites in the primitive streak tend to give rise to dorsal structures, while more posterior streak cells
contribute to ventral tissue types. The molecular underpinnings of this guidance system, however, have yet to becomprehensively studied.
Cantas Alev and colleagues in the Laboratory for Early Embryogenesis (Guojun Sheng, Team Leader), in collaboration with the members of the Laboratories for Systems Biology and Stem Cell Biology, surveyed the transcriptomic landscape of the primitive streak using genomic arrays, developing a finer understanding of the regulationof the dorsal-ventral gradient in the developing mesoderm. Published in the journal Development, their study ofevents in the early chick embryogenesis provides a valuable resource for the study of tissue patterning in amniotes.
Using more than 250 embryos collected at the full primitive streak stage (HH4), Alev et al. began by dissecting out
a rectangular section containing the streak from each, and subdividing it into four equal parts along its A-P axis.
Next, using a commercially available genome array of around 22,000 unique genes, they tested the segments fordifferences in the expression of individual genes. While they found that close to 40% of the chicken genome isexpressed across the entire streak, they did identify a number of genes whose expression levels varied from seg-

Yuping WU, Cantas ALEV, Takeya KASUKAWA

36

©2010 The Company of Biologists
Alev C, et al. Transcriptomic landscape of the primitive streak. Development 137.2863-74 (2010)

ment to segment. The team analyzed all genes that could be assigned a Gene Ontology classification,
which is a method for categorizing genetic functional activity.
Alev found that around 14% of the expressed transcripts could be grouped into six clusters definedby the expression in various combinations of segments, such as [1,0,0,0] or [0,0,0,1], representing
either dorsal-to-ventral (Group A) or ventral-to-dorsal (Group D) asymmetric expression. While many
Gene Ontology categories were evenly represented across both groups, the team did find that genes
involved in transcriptional regulation and receptor-mediated signaling showed biased expression, withtranscriptional regulators expressed more in Group D, and signaling genes more in Group A.

Mesoderm precursors, marked
by the pan-mesodermal marker
Brachyury (left panel), received i f fe re nti a l p at te r n i n g c u e salong the length of the primitive
streak (e.g., Chordin in the middle panel and Cdx1 in the rightpanel).

In their analysis, the authors focused particularly on three major developmental pathways—Wnt,
TGFß/BMP, and FGF—but found that for the most part their core components were not differentiallyexpressed. However, regulators of these pathways frequently did show either dorsal or ventral distributions. The picture that emerges from this set of findings is of a complex, multi-tiered network ofgenes interacting with and regulating the activity of key pathways involved in setting up the D-V axis.
"To embryologists and stem cell biologists alike, the primitive streak is a fascinating structure that defies simplification," says Sheng. "Our study offers a resource for achieving a better understanding of
its molecular complexity in vivo. We're hopeful that this understanding can also help achieve efficientand targeted mesoderm lineage differentiation in translational research."

37

ROCK’s role in hESC suicide prevention

Toshihiro ARAMAKI, Masatoshi OHGUSHI, Michiru MATSUMURA-IMOTO

Embryonic stem cells (ESCs) are known for their ability to be grown indefinitely in self-renewing culture, and fortheir pluripotency—the differentiative potential to give rise to all the cell types in the body. These characteristics
are common to such cells in both mouse and human, but ESCs from these species also exhibit important differences, one of the most prominent of which is the greater susceptibility to cell death of human ESCs on dissociation; mouse ESCs are made of sturdier stuff, and grow happily even when isolated from their cell-mates. Althoughmethods have been developed to prevent hESCs from self-destructing, including a breakthrough report from theLaboratory for Organogenesis and Neurogenesis (Yoshiki Sasai, Group Director) in 2007, the mechanisms behind
its occurrence have never been explained.
A new study by led by Masatoshi Ohgushi in the same group, in collaboration with the CDB Laboratory for
Pluripotent Stem Cell Studies and scientists at Kyoto University, now provides an answer to that puzzle. Published in Cell Stem Cell, the authors report that hESC cells die due to a hyperactivation of myosin that is triggeredby loss of cell-cell adhesion and mediated by the Rho signaling pathway. These changes in molecular activity areaccompanied, but not dependent on, changes in cells morphology known as “blebbing,” in which the cells perform a “dance of death” as an early signal of their demise.
Ohgushi began by building on previous work from the same lab, which had shown that inhibition of a componentof the Rho signaling pathway known as ROCK can prevent human ESCs from dying on separation from theircolony. He used a combination of cell sorting and live imaging to identify features of dying hESCs, and found thatquickly after dissociation the cells became motile and began to form distinct outpockets in their surface membranes, called blebs. This symptom is known to be the result of the actomyosin system, which regulates cytoskel-

38©2010 Elsevier B.V.
Ohgushi M, et al. Molecular pathway and cell state responsible for dissociation-induced
apoptosis in human pluripotent stem cells. Cell Stem Cell 7.225-39 (2010)

etal behavior, but it is ordinarily a short-lived phenomenon, a kind of convulsive last gasp in a dying
cell’s final minutes; in hESCs, however, it lasts for as long as a day. And interestingly, while blebbing is
usually under the control of the caspase pathway that regulates canonical forms of programmed cell
death, its onset in hESCs is caspase-independent, but can be prevented by ROCK inhibition.
It turns out that the reason for ROCK’s role is its effect on non-muscle myosin light chain 2 (MLC2).
ROCK is upregulated following dissociation of hESCs, which triggers an elevation of MLC2, hyperactivating myosin and generating the intracellular contractive forces that underlie blebbing. Interestingly,
however, Ohgushi found that inhibition of blebbing did not prevent apoptosis.
The group’s first supposed the true cause of this programmed cell death in hESCs on dissociation
would be a phenomenon known as anoikis (from the Greek word for “homeless”), in which cells detached from their basement membrane spontaneously undergo apoptosis. But live imaging showedthat even when dissociated hESCs remained attached to the basement membrane, they blebbed
and died. It appeared that the loss of lateral, not basal, connections was to blame, suggesting a rolefor cadherin-mediated cell-cell adhesion. They tested this notion by depleting calcium ions (cadherin
function is calcium-dependent) and cadherin knockdown by RNAi in hESCs, and in both cases observed blebbing and apoptosis, suggesting that theloss of intercellular adhesion
plays a triggering role in theapoptotic cascade. This jibes
well with known differences
between the cellular statesof mouse and human ESCs;
specifically, the similarity ofhESCs to mouse e piblast
cells.
Loss of cadherin function also
induced the upregulation of
the Rho signaling pathway, of
which ROCK is a component.
Previous work has shown that this pathway is subject to multiple upstream activators, so Ohgushi etal. performed an shRNA knockdown screen to identify responsible factors, and turned up a heretofore
poorly characterized Rho-GEF family member known as Abr.

Extensive blebbing in dissociated hESC. Nucleus, green; cell
membrane, red.

The involvement of Abr cements the importance of Rho signaling in the demise of dissociated hESCs.
Importantly, Rho is frequently found to interact with another small G protein, Rac. Using a pulldown
assay, Ohgushi looked at the control of this factor in dissociated hESCs, and found that, in contrast
to Rho, Rac was significantly downregulated, strongly suggesting that Rho and Rac have reciprocal
roles in this process. The take-home message appears to be that loss of cadherin-mediated cell-celladhesion in dissociated hESCs upregulates Abr, leading to a Rho-high/Rac-low state, consequentelevation of ROCK, and the hyperactivation of myosin, causing blebbing and apoptotic death.
The developmental and evolutionary significance of this mechanism remains a stimulating open question. “In mouse, the cells of the ICM show no polarity, while those of the epiblast do, enabling themto adhere and form a sheet-like structure, and we have seen that dissociated mouse epiblast cells
undergo the same apoptosis as do dissociated hESCs,” says Sasai. “This dance of death inducedby the hyperactivation of myosin is an intriguing cellular phenomenon in the development of a multicelled ‘society’ during early embryogenesis.”39

DNA methylation not requiredin extraembryonic tissues

Chisa MATSUOKA, Morito SAKAUE, Akiko YAMAGIWA

Genomic techniques have not only shown us the diversity of genetic mechanisms for establishing, maintaining
and regulating the body’s functions, they have also brought into clear focus the fact that life is not ruled by genetics alone. The study of heritable changes in phenotype independent of the underlying DNA is collectively referred
to as epigenetics, and has begun to reveal the intricacy and fundamental importance of such processes as chromatin remodeling and the modification of genomic sequences by various proteins. DNA methylation is one suchmodification, in which methyl residues are added to and maintained at specific sites on the genome through theaction of methyltransferases. These have been shown to be essential to the growth and survival of somatic cells
but, interestingly, not to pluripotent embryonic stem cells (ESCs). The question of whether DNA methylation functions in any type of differentiated cells comes down to their role in extraembryonic tissues, which has never been
fully determined.
To address this question, Morito Sakaue, Hiroshi Ohta, and colleagues in the Laboratories of Mammalian Epigenetic Studies (Masaki Okano, Team Leader), Genomic Reprogramming, and Pluripotent Stem Cell Studies studied extraembryonic development in mice lacking all three DNA methyltransferases known to have physiological
function. In a report published in Current Biology, they showed that in these triple knockout (TKO) embryos, while
differentiation into the embryo proper was affected, the development of extraembryonic stem cells and tissue
was normal.
Sakaue and Ohta examined developmental abilities of mouse embryos deficient for active DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, (TKO) by using nuclear transfer (NT). No abnormalities were seen in thedevelopment of NT embryos up to the blastocyst stage, even in the absence of all three Dnmts. To examine subsequent stages, the team made aggregates of TKO-NT and wildtype embryos (DNMT mutations are known tobe lethal to embryos), and found that few mutant cells were detected in the embryo proper, but they contributed40©2010 Elsevier B.V.
Sakaue M, et al. DNA methylation is dispensable for the growth andsurvival of the extraembryonic lineages. Curr Biol 20.1452-7 (2010)

to extraembryonic tissues in the chimeras. So, while DNA methylation is not necessary for the earliest
differentiation event that separates the extraembryonic tissue from the embryo itself or for further differentiation in the extraembryonic tissues, it is required for cell survival within the embryo.
Their next question was, Why? On observing the TKO embryos, they saw signs that somatic differentiation had begun to take place, but that the cells did not survive. Sakaue and Ohta used retinoic acidto steer mutant ESCs toward a neuroectodermal fate, and found that, just as in wildtype, the cells expressed the appropriate genetic markers but that they were subject to extensive apoptotic cell death,possibly linked to the DNA damage response. When they turned to the trophectoderm, however, they
did not find unusual levels of apoptosis. When they used Gata4 to induce a second extraembryoniclineage, known as primitive endoderm, these too showed no significant differences from wildtype,suggesting that DNA methylation is not required in extraembryonic tissues in the mouse.
As a final test of the role of DNA methyltransferases, Ohta and Sakaue made trophectoderm stem (TS)cells from triple knockout blastocysts made by nuclear transfer. They found that, like the differentiated extraembryonic cells, the TS cells showed normal behavior and viability, and could proliferate
for dozens of passages in culture. Using genetic inducers, they further showed that these stem cells
were capable of differentiating into all of the expected cell types, and even contributing to placenta invivo. Despite these similarities, however, the mutant TS cells showed differences in gene expression
revealed by microarray, suggesting that DNA methylation does have a regulatory function in TS cells.

D n m t 1 -/- D n m t 3 a -/- D n m t 3 b -/(TKO) cells (green) contribute toplacenta in a chimeric TKO NT/
wildtype embryo. Left, a brightfield image; right, a dark field
image with green fluorescence.

“In somatic cells, epigenetic marks on chromosomes support the stable maintenance of cellular properties over the long term, while cellular properties are reprogrammed during embryogenesis, and areassociated with the reorganization of epigenetic marks; that is, cells have ‘stable’ and ‘flexible’ modesin their epigenetic regulation” says Okano. “Mechanisms that tolerate growth without DNA methylationin ES and TS cells may underlie the epigenetic flexibility of these cell types and early embryos.”41

2010 Courses
As part of its commitment to promoting awareness and understanding of development, regeneration, evolution, stem cells, regenerative medicine and related fields, and its interests in maintaining close ties with the academic and research communities, the RIKEN CDB dedicates itself
to conducting training and educational programs for a wide range of audiences. Although not a teaching institution, the Center works with students, educators, and scientists, as well as other organizations, to develop, implement, and promote a variety of instructional courses throughout the year.

Beginners’ course in human ES cell culture
The Division for Human Stem cell Technology held a number of courses for first-time research users of human embryonic stem cells. The instruction focuses on freezing and thawing, generation of feeder cells, and techniques for confirming pluripotency. All steps were
taught using human induced pluripotent stem cells (iPSCs), kindly provided by the Kyoto
University Center for iPS Cell Research and Application (CiRA). Students who complete thiscourse are eligible to apply for permission to use human ESCs under the guidelines established by the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

Summer school for area high school students
The CDB held its annual summer school program for area high school students on August
24 and 26. The one-day program of lectures and demonstration experiments focusedon C. elegans research. The lecture, by Team Leader Hitoshi Sawa, explained why theseroundworms are useful in developmental biology, what types of research they can be used
in, and how they have contributed to Nobel Prize-winning science. The students next visited the Sawa lab before trying their hand at simple biology techniques, such as observing
roundworms under a microscope and collecting them for further study. The next comparedmutant phenotypes against wildtype animals, and collected eggs and watched the first few
rounds of zygotic cell division.

Developmental methods for high school biology teachers
The RIKEN Center for Developmental Biology held its third recurrent course in developmental biology for teachers of high school biology on October 2 and 3. Held in conjunction
with the Japanese Society of Developmental Biologists every year in the fall, the course isintended to give teachers an opportunity to learn hands-on techniques in embryology. This
year’s program, which focused on chicken development, featured a lecture by Prof. Sadao
Yasugi of Kyoto Sangyo University on the history of embryology studies in chicken, andan overview of the current state of the science in areas such as body axis formation andorganogenesis. Folowing the talk, Yasuo Ishii, a lecturer at the same university, led a twoday training course in which teachers learned how to extract, culture, and observe chickembryos, and to prepare them for microscopic examination. This was complemented by ademonstration of similar techniques in the Japanese quail by Yoshina Usui, a teacher at Higashi Suma High School. All aspects of the training course were designed to enable teachers to apply the subject matter and techniques in their own classrooms.

42

Egg of hagfish, Epatretus burgeri, containing pharyngula-stage embryoGermline Development

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

Staff
Team Leader

Technical Staff

Research Scientist

Student Trainee

Akira NAKAMURA
Takako AWAI
Kazuko HANYUNAKAMURA
Yasuko KATO
Maki SHIRAEKURABAYASHI

Kazuki MATSUDA
Kenji MAEKUBO

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

Assistant

Eri YAMASHITA

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

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

44

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

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

Staff
Team Leader

Jun-ichi NAKAYAMA
Research Scientist

Kyoko HAMADA
Tomohiro HAYAKAWA
Aki HAYASHIAtsuko SHIRAI

Student Trainee

Mayumi ISHIDA
Erina KITANO
Gohei NISHIBUCHI
Assistant

Sayaka SENOTechnical Staff

Noriyo HAYAKAWA
Rika KAWAGUCHI
Yasuko OHTANI

Publications
Hayakawa T, et al. MRG15 directly binds to PALB2 andstimulates homology-directed repair of chromosomal
breaks. J Cell Sci 123. 1124-30 (2010)
Shimada A, et al. Phosphorylation of Swi6/HP1 regulates
transcriptional gene silencing at heterochromatin. Genes
Dev 23. 18-23 (2009)
Hayashi M T, et al. The heterochromatin protein Swi6/HP1
activates replication origins at pericentromeres and silent
mating-type locus. Nat Cell Biol 11. 357-62 (2009)
Sadaie M, et al. Balance between distinct HP1 proteins
controls heterochromatin assembly in fission yeast. Mol Cell
Biol 28. 6973-88 (2008)
Iida T, et al. siRNA-mediated heterochromatin establishment
requires HP1 and is associated with antisense transcription.
Mol Cell 31. 178-89 (2008)
Buker S M, et al. Two different Argonaute complexes are required for siRNA generation and heterochromatin assemblyin fission yeast. Nat Struct Mol Biol 14. 200-7 (2007)

Disruption of HP1α N-terminal phosphorylation leads to defects in its heterochromatic localization(upper panels) and increased chromosomal instability (lower panels).

45

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

Shin-Ichi NISHIKAWA M.D., Ph.D.
http://www.cdb.riken.jp/en/nishikawa
Shin-Ichi Nishikawa received his M.D. from the
Kyoto University School of Medicine in 1973. He
performed his internship and residency in the
Department of Internal Medicine at the Kyoto
University Chest Disease Research Institute,
before taking an assistant professorship in thesame department in 1979. He spent the period
from 1980-82 at the University of Cologne Institute for Genetics before returning to the Chest
Disease Research Institute, where he was appointed associate professor in the Departmentof Microbiology in 1983. He moved to the Kumamoto University Faculty of Medicine in 1987,
and returned to Kyoto in 1993, as professor at
the Kyoto University Faculty of Medicine. He
was appointed CDB group director in 2000.

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

Staff
Group Director

Visiting Researcher

Research Scientist

Technical Staff

Shin-Ichi NISHIKAWA
Julien BOUISSAC
Kenichiro KOBAYASHI
Igor M SAMOKHVALOVYoshiteru SASAKIYosuke TANAKA
Yan-Fung WONG
Research Specialist

Lars M JAKTReiko NAKAGAWA
Research Associate

Rasmus FRETER
Yasushi KUBOTA

Takao YAMADA

Misato HAYASHI
Satoko MORIWAKI
Satomi NISHIKAWA
Nataliya
SAMOKHVALOVA
Yuri SHIMIZU
Naoko YOSHIOKA
Student Trainee

Leo BLONDEL
Matteo PLUCHINOTTA
Part-Time Staff

Collaborative Scientist

Shin KAWAMATA

Miho HOSHIJIMA
Yoko OTSUKA
Kazumi YANAIVisiting Scientist

AssistantTeruo AKUTA
Keitaro IMAIZUMIHiroshi KATAOKA
Masao MATSUO
Herve Le MOUELLIC

Naeko MINAMI
Chisato NISHIMURA
Satomi SHINOHARA

Publications
Freter R, et al. Adult stem cells exhibit global suppressionof RNA polymerase II serine-2 phosphorylation. Stem Cells
28.1571-80 (2010)
Kubota Y, et al. Necdin restricts proliferation of hematopoietic stem cells during hematopoietic regeneration. Blood
114. 4383-92 (2009)
Kondo N, et al. Thrombin induces rapid disassembly ofclaudin-5 from the tight junction of endothelial cells. Exp Cell
Res 315. 2879-87 (2009)
Kinoshita M, et al. The novel protein kinase Vlk is essential
for stromal function of mesenchymal cells. Development
136. 2069-79 (2009)
Eilken H M, et al. Continuous single-cell imaging of blood
generation from haemogenic endothelium. Nature 457. 896900 (2009)
Egawa G, et al. Transient expression of ephrin b2 in perinatal skin is required for maintenance of keratinocyte homeostasis. J Invest Dermatol 129. 2386-95 (2009)46

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

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

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

Staff
Team Leader

Agency Staff

Research Scientist

Part-Time Staff

Takashi NISHIMURA
Yoshiko HASHIMOTO
Naoki OKAMOTO

Chieko HIJIRIYAMA
Hiroko KOBAYASHITechnical Staff

Yuka NISHIMORI

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

Drosophila larval brain showing proliferating cells (green) and insulin-producing cells (red)47

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

Hitoshi NIWA M.D., Ph.D.
http://www.cdb.riken.jp/en/niwa
Hitoshi Niwa received his M.D. from Nara Medical University in 1989, and his Ph.D. in medical
physiology in 1993 from the Kumamoto University Graduate School of Medicine for his workin gene trap methods. From 1993 to 1994, heworked as a research associate in the Department of Developmental Genetics at the same
university, before taking a postdoctoral fellowship with Austin Smith at the University of Edinburgh Centre for Genome Research. He returned
to Japan in 1996 as a research associate in
the Department of Nutrition and Physiological
Chemistry at the Osaka University Graduate
School of Medicine, where he remained until
taking his current position as Team Leader at the
RIKEN CDB. He was appointed Project Leaderof the Laboratory for Pluripotent Stem Cell Studies in October 2009.

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

Staff
Project Leader

Technical Staff

Research Scientist

Student Trainee

Hitoshi NIWA

Kenjiro ADACHI
Jitsutaro KAWAGUCHI
Masaki KINOSHITA
Kazuhiro MURAKAMI
Yoko NAKAI-FUTATSUGISatomi NISHIKAWATORIKAI
Satoshi OHTSUKA

Setsuko FUJII
Shunsuke ITO
Toshimi SUGIMOTO
Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI
AssistantMiho SAKURAI

Visiting Scientist

Yasuhide OHINATA

Publications
Murakami K., et al. Choice of random rather than imprinted
X inactivation in female embryonic stem cell-derived extraembryonic cells. Development 138.197-202 (2011)
Niwa H, et al. A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460. 11822 (2009)
Niwa H, et al. Platypus Pou5f1 reveals the first steps in theevolution of trophectoderm differentiation and pluripotency
in mammals. Evol Dev 10. 671-82 (2008)
Toyooka Y, et al. Identification and characterization of subpopulations in undifferentiated ES cell culture. Development
135. 909-18 (2008)
Niwa H. How is pluripotency determined and maintained?
Development 134. 635-46 (2007)
Masui S, et al. Pluripotency governed by Sox2 via regulationof Oct3/4 expression in mouse embryonic stem cells. Nat
Cell Biol 9. 625-35 (2007)

48

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

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

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

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

Staff
Team Leader

Masaki OKANO
Research Scientist

Yuichi KUMAKI
Morito SAKAUE
Takashi TAMURA

Technical Staff

Chisa MATSUOKA
Akiko YAMAGIWA
Assistant

Fumika NAKAYAMA

Publications
Sakaue M, et al. DNA methylation is dispensable for thegrowth and survival of the extraembryonic lineages. Curr
Biol 20. 1452-7 (2010)
Kumaki Y, et al. QUMA: quantification tool for methylation
analysis. Nucleic Acids Res 36. W170-5 (2008)
Takebayashi S, et al. Major and essential role for the DNA
methylation mark in mouse embryogenesis and stable association of DNMT1 with newly replicated regions. Mol Cell
Biol 27. 8243-58 (2007)
Sharif J, et al. The SRA protein Np95 mediates epigenetic
inheritance by recruiting Dnmt1 to methylated DNA. Nature450. 908-12 (2007)
Oda M, et al. DNA methylation regulates long-range genesilencing of an X-linked homeobox gene cluster in a lineagespecific manner. Genes Dev 20. 3382-94 (2006)
Tsumura A, et al. Maintenance of self-renewal ability ofmouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11.
805-14 (2006)

Placenta tissue in a chimera between a nuclear transfer embryo using a Dnmt1-/-Dnmt3a-/-Dnmt3b-/ES cell nucleus (green) and a wildtype embryo. Cells without DNA methylation contribute to extraembryonic lineages.

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

Tony PERRY Ph.D.
Tony Perry received his B.Sc. in Microbiology
from the Department of Bacteriology at the
University of Bristol, England and his doctorate
from the University of Liverpool four years later.
From 1989 he became a postdoctoral fellow andtemporary lecturer working on epididymal sperm
maturation at the University of Bristol and in1996 won a European Molecular Biology Travel
Fellowship to study oocyte activation. Dr. Perrydeveloped a novel method of transgenesis which
he primarily worked on at the Rockefeller University. In June 2002 he took his present position
as Team Leader of the Laboratory of Mammalian
Molecular Embryology at the RIKEN CDB, where
he started to work on the mechanisms of key
cytoplasmic events at fertilization in mammals,
including chromatin remodeling and meiotic exit.
He was appointed Reader at the University ofBath in 2009.

Staff
Team Leader

Anthony C.F. PERRY
Research Scientist

Toru SUZUKI

Collaborative Researcher

Technical Staff

Heide OLLER
Emi SUZUKI
Assistant

Eriko NOMURA

Naoko YOSHIDA

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

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

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

50

Cytoplasm from a mouse egg expressing a mitochondrially-targeted green fluorescent protein wastransplanted into one cell of a two-cell embryo; the paired images show the same embryo after cell
division, on the left with laser illumination only, and merged with DIC illumination on the right.

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

Mitinori SAITOU M.D., Ph.D.
Mitinori Saitou received his M.D. from the
Kyoto University Faculty of Medicine in 1995,
and received his Ph.D. in 1999 for his study ofthe structure and function of tight junctions
under Shoichiro Tsukita in the Kyoto University
Graduate School of Medicine. He then moved
to the Wellcome Trust/Cancer Research Campaign Institute (present name: Wellcome Trust/Cancer Research UK Gurdon Institute), where
he worked as a postdoctoral research associate in Azim Surani's laboratory, focusing on theorigin of the germ line in the mouse. He wasappointed team leader at the CDB in 2003, andreceived a three-year grant from the Japan
Science and Technology Corporation (JST )PRESTO program that same year, and was affiliated to the Kyoto University Graduate Schoolof Biostudies as associate professor in 2004.
He moved to the Kyoto University Graduate
School of Medicine as professor in 2010.

Staff
Team Leader

Technical Staff

Special Postdoctoral
Researcher

Student Trainee

Mitinori SAITOU
Kazuki KURIMOTO
Sugako OGUSHI
Research Scientist

Yukihiro YABUTA
Masashi YAMAJI

Visiting Researcher

Mayo SHIGETA
Kaori YAMANAKA
Takayuki HIROTA
Part-time staff

Miho MIURA
AssistantKazuyo NAKATANI

Yasuhide OHINATA

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

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

Kurimoto K, et al. Complex genome-wide transcription
dynamics orchestrated by Blimp1 for the specification of thegerm cell lineage in mice. Genes Dev 22. 1617-35 (2008)51

Organogenesis and Neurogenesis
The complexity of the fully formed brain defies description, yet this organ arises
from a nondescript clump of cells in the embryo. The specification of the dorsalventral axis is significant in neural development in that the central nervous
system forms on the dorsal side of the body in all vertebrate orders. This process is dictated by the effects of a number of signaling factors that diffuse fromorganizing centers and direct dorsal ectoderm to maintain a neural fate. Thesemolecules, which include Noggin, Chordin, Follistatin and their homologs, participate in elaborate signaling networks in which factors collaborate and compete
to initiate the embryonic nervous system.

Yoshiki SASAI M.D., Ph.D.
http://www.cdb.riken.jp/en/sasai
Yoshiki Sasai received his M.D. from the Kyoto
University School of Medicine in 1986, subsequently performing internships in general practice and emergency medicine. He completed the
Ph.D. course at the same institution in 1992, forwork on neural specific transcriptional regulators. In 1993, he took a postdoctoral fellowshipin the De Robertis lab at the UCLA School of
Medicine, remaining there until 1996 when he
was appointed associate professor at the Kyoto
University School of Medicine. He assumed a
professorship at the Kyoto University Institutefor Frontier Medical Sciences in 1998, and was
appointed group director at the CDB in 2000. He
serves on the editorial boards of Neuron, Genesis, and Developmental Dynamics.

Staff
Group Director

Yoshiki SASAI
Collaborative Scientist

Satoshi ANDO
Makoto IKEYA
Morio UENO

Research Specialist

Keiko MUGURUMATAMADA
Research Scientist

Toshihiro ARAMAKI
Mototsugu EIRAKU
Hidehiko INOMATA
Daisuke KAMIYA
Makoto NASU
Nozomu TAKATA
Research Associate

Akira TAKAITechnical Staff

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

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

Junior Research Associate

Teruko DANJOUInternational Program
Associate

Anne H. B. FOSBY
Part-Time Staff

Masako SUZUKI
Assistant

Mako MIYAGI
Ayumi TANAKA-IKEYA
Fumi WAGAI

Publications
Aramaki T, et al. Jiraiya attenuates BMP signaling by interfering with type II BMP receptors in neuroectodermal patterning. Dev Cell 19. 547-61 (2010)
Muguruma K, et al. Ontogeny-recapitulating generation andtissue integration of ES cell-derived Purkinje cells. Nat Neurosci 13. 1171-80 (2010)
Ohgushi M, et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7.225-39 (2010)
Sasai N, et al. Ectodermal factor restricts mesoderm differentiation by inhibiting p53. Cell 133. 878-90 (2008)
Inomata H, et al. Robust stability of the embryonic axial pattern
requires a secreted scaffold for chordin degradation. Cell 134.
854-65 (2008)
Eiraku M, et al. Self-organized formation of polarized corticaltissues from ESCs and its active manipulation by extrinsic
signals. Cell Stem Cell 3. 519-32 (2008)52

Self-formation of layered cortical tissue in three-dimensional
culture of human ES cells (red: the telencephalic marker Bf1)

Scanning electron microscope (SEM) image of
Drosophila mechanosensory bristle

Orientation of asymmetry by external factors

Yukinobu ARATA

The diversity of cell types in the animal body is the result processes such as asymmetric cell division, in which
a mother cell divides mitotically to give rise to two daughters of different character. The axis of asymmetric cell
division is critical for determining the future positions of the differentiated progeny in developing embryos. These
processes have been intensively studied in models such as the developing nervous system in the fruit fly and in
the earliest stages of roundworm embryogenesis. Despite the importance of these phenomena and the scientificattention paid to them, a number of fundamental questions remain. Particularly controversial among these is the
problem of whether various events that occur during asymmetric division are regulated by extrinsic or intrinsic
mechanisms.
Yukinobu Arata and others in the Laboratory for Cell Fate Decision (Hitoshi Sawa, Team Leader), working in collaboration with scientists from the University of North Carolina at Chapel Hill, addressed this question in fine detail withrespect to some of the earliest cell divisions in the C. elegans embryo. In a report published in the journal Development, they showed that while the asymmetric distribution of the protein PAR-2 occurs in a cell-autonomous manner, its localization to the point of cell-cell contact relies on external signals mediated by the transmembrane protein
MES-1 and tyrosine kinase SRC-1.
Arata and colleagues focused on a set of germline precursor cells (P0 through P4) that arise in successive roundsof asymmetric cell division in the very early C. elegans embryo, and their interactions with neighboring cells. One
important characteristic of the P cell lineage is that the axis of the P3 and P4 polarity is in reversed orientation tothat of the P0 and P1 cells. This polarity reversal allows the P2 cell to give rise to a P3 daughter in contact withthe endodermal precursor E, and for P3 to generate a P4 cell that is also in contact with an endodermal precur-54©2010 The Company of Biologists
Arata Y, et al. Extracellular control of PAR protein localization during
asymmetric cell division in the C. elegans embryo. Development 137.3337-45 (2010)

sor, E.x. PAR polarity proteins are known to localize to the points of contact between the germline andendodermal precursor cells, suggesting extracellular signals are at work. However, some previous
studies had suggested that the regulation of the polarity reversal that takes place between the P2 andP3 divisions is cell-intrinsic.
The team sought to gain a better understanding of
this process by isolating individual P-lineage cells fromdeveloping embryos in vitro using glass micropipettes.
They were surprised to find that the P4 cells derivingfrom isolates were frequently in inverted position, suggesting that the P3 division axis might rely on extracellular cues. To test this possibility, Arata altered thecontact site by isolating cells and re-attaching germline
precursors with either of the endodermal precursor
cells and watched for effects on the division axis. The results showed that endodermal precursor cells
exercise a clear influence on P cell disposition, indicating the axis of division is determined by extracellular signals. The daughters of P2 and 3 cells attached to control cells, however, showed highlyvariable division axes, highlighting the necessity of external inputs in axis orientation.

16-cell stage of C. elegans embryo

They used a similar strategy to look at protein distribution and centrosome positioning. In P cells
exposed to both endodermal precursor and control cells, asymmetric distribution of PAR proteins occurred, but interestingly the localization of these proteins to the point of cell-cell contact failed to takeplace in P cells attached to control cells. Simultaneously, the centrosome in P cells in contact withendodermal lineage cells consistently located adjacent to the site of contact between cells, but whencontrol cells were used, the centrosome location was variable, suggesting that external cues from theendodermal cells determine the orientation of asymmetric cell division by regulating both PAR protein
distribution and centrosome orientation.
The molecules responsible remained unknown but, based on previous reports of the role of two
factors—MES-1 and SRC-1—in establishing differential daughter cell sizes, the team investigated thepossibility that they might be involved in axis determination as well.
In embryos with losses of function in mes-1 and src-1 they found that the PAR-2 protein was asymmetrically distributed as usual, but that it often failed to accumulate at the cell-cell contact site indicating that these genes shows clear defects in axis orientation. And lastly, to determine whether
MES-1 and SRC-1 were required in the P lineage cell or its endodermal neighbor, they tested various
combinations of chimeric pairs of P and endodermal cells using cells isolated from mutants and wild
type embryos, and found that while the transmembrane protein MES-1 plays an essential part on
both sides of the cell-cell junction, the tyrosine kinase SRC-1 is needed only in the signal-receiving P
cell. This is the first experimental demonstration in which cell positions were manipulated to directly
test the role of cell signaling on directional control of PAR protein localization during asymmetric cell
division, work that may shed new light on the directional
control of cell polarization in many other developmental
processes.
“I observed cell divisions of cells isolated by micropipettefrom developing embryos in vitro, and I have to say that I
never got tired of watching them; they’re quite beautiful,”says Arata. “This study began when I noticed that theprogeny of P2 and P3 were sometimes in positions opposite to their normal ones. I wanted to find out why.”

Cells in the embryo were isolated and manipulated using a
glass micropipette.

55

Purkinje neurons from ES cells

Keiko MUGURUMA

The cerebellum is one of the largest regions of the mammalian brain, and is responsible for controlling motor andother bodily functions. These functions are enabled by the activity of a distinct group of large and highly arborizedcells known as Purkinje neurons, which integrate multiple inputs from other parts of the brain through their many
dendrites and send inhibitory signals to cerebellar nuclei involved in motor coordination. The loss of Purkinje cellfunction is associated with a severe form of ataxia (loss of motor function), making this cell type one of tremendous potential both for the study of disease and the development of future clinical applications. Attempts to generate Purkinje neurons from stem cell sources at high efficiencies in vitro, however, have proven difficult.
Keiko Muguruma and colleagues in the Laboratory for Organogenesis and Neurogenesis (Yoshiki Sasai, Group
Director), working in collaboration with scientists from the KAN Institute and Kyoto University, achieved a breakthrough in Purkinje cell induction, selectively deriving these neurons from mouse embryonic stem (ES) cells. In areport published in Nature Neuroscience, the group showed that, by recapitulating aspects of the cerebellar developmental environment, they were able to generate functional Purkinje cells in quantity.
The work began using a technique for neuronal induction using serum-free culture of embryoid body-like aggregates (SFEBq) previously developed by the group and applied to the differentiation of numerous neural cell types.
In order to adapt this protocol to Purkinje induction, they looked to how these neurons are triggered to develop in
the embryo itself. Purkinje cell precursors are induced by signals from a region in the posterior midbrain known
as the isthmic organizer. By simulating this structure in cultured cells, Muguruma sought to give rise secondarily
to Purkinje neurons.

56©2010 Nature Publishing GroupK. Muguruma, et al. Ontogeny-recapitulating generation and tissue
integration of ES cell-derived Purkinje cells. Nat Neurosci 13 1171-80 (2010)

Using a trial and error approach, she tested various transcription factors at different timepoints and indifferent orders, before finally arriving at a method for inducing cerebellar tissue. This involves SFEBqculturing mouse ES cells for 1–2 days before adding the caudalizing factors insulin and FGF2, yielding
an isthmic organizer-like tissue after four days. This region secretes inductive factors, which over timelead to the differentiation of cerebellar precursors at a very high efficiency of around 80%. Subsequent
addition of an inhibitor of the ventralizing factor hedgehog to these precursors at day 13 gives rise to
Purkinje neurons at a rate of around 30% of all cells by day 19. Subjecting this population to cell sorting by flow cytometry allows for even greater purities, of up to about 90%.

ES cell-derived Purkinje cells(yellow) integrate into mouse
cerebellum

The Purkinje neurons derived through this approach were similar in morphology and surface marker
expression, but would they function in the same way as their embryonic equivalents? Muguruma introduced ES-derived Purkinje cells into the cerebella of day 16 mouse embryos to find out. Allowing
the transplanted embryos to develop to term, she found that their cerebellar tissues contained manysuch cells, testifying to the power of the new technique. The transplanted Purkinje neurons not only
integrated into the tissue, but also formed functional neural circuits, and exhibited normal electrophysiology and neuronal inputs.
“It is still early days,” says Sasai, “but if we are able to derive populations of human cerebellar neurons
in a similar fashion, these might be of great benefit to the study and treatment of diseases such asspinocerebellar ataxia in the future.”57

Del1 quells Wnt in head development

In amphibians, the anterior-posterior axis develops
headfirst, with forebrain representing the default fate
and more caudal tissues emerging secondarily throughthe action of multiple posteriorizing factors. These factors play an instructive role in specifying the fates thattargeted regions adopt, but also work to suppress thedevelopment of anterior structures. It is this combinationof inhibition and induction that sets up the gradient oftissue identities along the A-P axis. Of the many known
factors of this kind, Wnt stands out for its potent caudalizing effect, but what prevents such posterior factors
from transforming anterior structures as well?
That question has been answered in part through astudy by Akira Takai and others in the Laboratory for
Organogenesis and Neurogenesis (Yoshiki Sasai, Group
Director), which showed that the secreted protein Del1
inhibits Wnt signaling in embryonic head regions in the
African clawed frog, Xenopus laevis. Published in thejournal Development, the report describes how this extracellular matrix protein staves off its posteriorizing effects through interaction with a downstream componentof the canonical Wnt pathway.
Del1 was already well known to the group from previous
work in which they had shown that the protein inhibited
BMP, a major ventralizing factor that works in the establishment of the dorsal-ventral axis. So they were intrigued when they found that injection of Del1 mRNA intoa different part of the early embryo resulted in a phenotype with an oversized head and eyes. To validate thatfinding, they tested for Del1 effect in artificially induced
secondary axes, which can be triggered by injection ofanti-BMP factors, such as Chd. Such axes rarely develop anterior structures, but when Del1 was co-injectedwith Chd, eye tissue formed, indicating that misexpression of Del1 promotes anteriorization. The converse wasalso true—loss of Del1 function (induced by morpholino)caused embryos to develop smaller than normal heads.
The group next looked at the structure of the Del1 protein in an effort to identify the domain responsible for
its anteriorizing activity. Wildtype Del1 has two EGF-likeand two Discoidin domains. By selectively deleting individual domains and testing their effects on animal cap
embryos (a useful model of amphibian development),
they pinpointed the anteriorizing function to the Discoidin domains.

Akira TAKAI58

Now that they had the responsible domain, Takai et al.
sought to identify where it fit into the larger picture in©2010 The Company of Biologists
Takai A, et al. Anterior neural development requires Del1, a matrix-associated proteinthat attenuates canonical Wnt signaling via the Ror2 pathway. Development 137. 3293-302 (2010)

molecular terms. Knowing that Wnt is an important inducer of posterior fates, they tested for Del1
effects in Wnt-posteriorized embryos. They found that co-injection of Del1 was sufficient to rescue
embryos from this phenotype, while co-injection of other anti-BMP factors did not, excluding the possibility that Del1’s effect was due to its established role as a BMP antagonist. And, importantly, Del1
did not affect phenotypes induced by other posteriorizing factors, such as FGF or Nodal, indicatingthat its effect in this context is specific to the Wnt pathway.

Del1 function is required forhead development (wildtype at
top; Del1 morphant at bottom).

The group performed a TOPflash assay, which uses luciferase to visualize ß-catenin-mediated transcriptional activation, to watch how Del1 might be interacting with the canonical Wnt pathway (inwhich ß-catenin is a key component). Their initial results suggested that Del1 works downstream of ßcatenin, which was reconfirmed in immunostaining experiments that showed that ß-catenin localized
to the nucleus even in the presence of Del1. The mechanism, however, remained unclear.
The non-canonical Wnt signaling pathway protein Ror2 seemed a promising candidate, as it had previously been shown to have a downstream inhibitory effect on Wnt. A preliminary test of this notion,in which Ror2 was inhibited in an embryo in which Del1 was overexpressed, showed a weakening of
the Del1+ phenotype, suggesting possible interactivity. In a clever test of this relationship, the group
added a morpholino that inhibited Ror2 to embryos that had been induced by Del1, and found thatthe depletion of Ror2 significantly reduced the usual anteriorization.
“We found it intriguing that Del1, a matrix protein secreted from the anterior neural region, functions as
a barrier to protect the region from caudalizing Wnt signals,” says Sasai. “You might say that Del1 acts
as a kind of shield in the maintenance of regional identity.”

Model schematic of how Del1i n h i b i t s Wn t /ß - c a te n i n i n a
Ror2-dependent manner.

59

New anti-BMP mechanism unveiled

Rieko YAKURA, Toshihiro ARAMAKIPowerful molecular players frequently require major powerful inhibitors to keep them in check. Bone morphogenetic protein, or BMP, is one such factor, playing central roles in multiple developmental processes; its suppression in appropriate contexts is likewise a critical component of many embryogenetic programs. The induction
and patterning of neural tissues provides a good example of this to-and-fro. BMP, which is highly expressed on
the embryo’s ventral side, is suppressed by inhibitors secreted from the Spemann organizer, leading to neuralinduction. BMP signaling is also involved in the subsequent pattern formation in this neural tissue. But, while it isclear that the suppression of the BMP pathway at given sites and time-points is crucial to the patterning of theearly embryo, just how this is accomplished is imperfectly understood.
Toshihiro Aramaki and colleagues in the Laboratory for Organogenesis and Neurogenesis (Yoshiki Sasai, Group
Director) shed light on this process through the lens of a new controller of BMP signaling. In a report publishedin Developmental Cell, the group identified the membrane protein Jiraiya as a specific inhibitor of a subunit of the
BMP receptor in the African clawed frog, Xenopus laevis.
Jiraiya first caught the researchers’ eye when it was captured in a screen for genes downstream of the BMP inhibitor Chordin. Its dorsal expression pattern recalled a character, named Jiraiya, who appears mounted on anamphibian in a famous kaubuki play, thus earning it its colorful name. The protein is first expressed in the dorsal
ectoderm of the early gastrula, shifting subsequently to the dorsal aspect of the neural tube. When Aramaki
knocked down its function using a morpholino antisense oligonucleotide, he saw an increase in the scope of expression of lateral neural plate marker genes in the treated embryos, accompanied by upregulation of activated60©2010 Elsevier B.V.
Aramaki T, et al. Jiraiya attenuates BMP signaling by interfering with type II
BMP receptors in neuroectodermal patterning. Dev Cell 19.547-61 (2010)

Smad1, a factor downstream in the BMP pathway, suggesting that this newly identified protein worked
to limit dorsal neuralization.
To get a clearer sense of the mechanism, the group used an animal cap assay of embryonic cells totest the effect of Jiraiya overexpression. They found that Jiraiya interferes with the function of BMPRII,
one of two subunits of the BMP receptor. The suppressor effect was specific to BMP; no inhibition
was seen, for example, of a second member of the TGF-ß family, activin. The question was: how does
Jiraiya achieve this? Immunostaining revealed that Jiraiya tended to accumulate more in the cytoplasm than at the plasma membrane, suggesting it might work intracellularly to block the function of
the membrane-bound BMPRII, which was supported by the failure of this receptor component to integrate into the cell membrane in cells overexpressing Jiraiya.

Expansion of lateral neural plate
marker gene expression (right)
following Jiraiya knockdown

They next looked for the site of interaction between these proteins by creating a series of mutant proteins from which various domains were deleted. They found that when BMPRII lost its C-terminal, it
became much less sensitive to Jiraiya’s quelling effect. Compellingly, the BMPRII tail domain contains
a highly conserved 6-amino snippet not found in any other TGF-ß receptor family member, suggestinga locus for the specificity of the BMPRII-Jiraiya interaction. The finding represents the first identification of a receptor-level inhibitor of BMP.
“We hope to be able to tease out Jiraiya’s mechanism of action in vivo,” says Sasai. “It will be especially interesting to work out how Jiraiya functions in mammals, and specifically whether it inhibits the
BMP pathway there as well.”

BMPRII (green) accumulates at
plasma membrane in control
cells (left), but is sequesteredcy toplasmically in Jiraiyaoverexpressing cells (right),
resulting in much lower expression of this receptor subunit in
the membrane.

61

Stage-dependent outcomes ofsymmetric stem cell division
Asymmetric cell division, in which a single mother cell
divides to give rise daughters with different properties
such as gene expression and morphology, is a fundamental mechanism in the generation of the many typesof cells in animal bodies. This phenomenon has been
extensively studied in the nervous system of the fruit fly
Drosophila melanogaster, in which specific protein factors are allocated to the basal side of dividing neuronalprogenitors, steering the cell that inherits these basal
determinants to a ganglion mother cell (GMC) fate, while
its mitotic sibling becomes a larger neuroblast. Past
studies have shown that daughter cell characteristics
such as cell size and protein localization are under thecontrol of independent molecular mechanisms. But thedownstream consequences of the loss of asymmetry inthese features remain something of an open question.
Working with mutants for a pair of genes linked to themaintenance of asymmetric mitosis, Atsushi Kitajima
and others in the Laboratory for Cell Asymmetry (Fumio
Matsuzaki, Group Director), in collaboration with the
National Institute of Genetics, observed how the loss ofthese functions leads to abnormal progeny. The study,
published in Developmental Biology, revealed that, inembryos of these mutants, the daughter cells that would
normally form neuroblasts instead differentiate into neurons.
Kitajima began by generating flies carrying mutations for
Gß13 (which is associated with maintaining differential
sizes in the daughters of neural progenitors) and dlg (anoncogene linked with controlling the asymmetric intracellular localization of fate determinants). As expected,
neuroblast progeny in these cells showed abnormal
symmetry in cell size and equal distribution of determinant proteins. The group next monitored the fate ofcells born from the mutant neuroblasts in embryos, anddetermined that while the newborn daughters bore thehallmarks of neuroblasts and GMCs, the stem cell natureof the progeny neuroblasts was not maintained, as mostultimately differentiated into neurons.

Fumio MATSUZAKI, Atsushi KITAJIMA

62

Asymmetric cell division is seen not only at embryonicstages, but in larval flies as well. Using a conditional
knockout to interfere with Gß13 and dlg function in specific tissues and observe the lineage effects. Interestingly, Kitajima et al. found that unlike in the embryo, thedouble-mutant cells failed to differentiate, and proliferated abnormally instead in a manner similar to that seen
in mutants of the basal determinant Prospero (itself an
oncogene). The authors speculate that the larger size©2010 Elsevier B.V.
Kitajima A, et al. Progenitor properties of symmetrically dividing Drosophila
neuroblasts during embryonic and larval development. Dev Biol 347. 9-23 (2010)of the mutant progeny may prevent a sufficient amount of Prospero from being allocated to a single
daughter (the presumptive GMC), leading to misregulation of cell proliferation in larval neural tissue, asurmise that was borne out by the finding that overexpression of Prospero in the double-mutants rescued the phenotype.

In asymmetrically dividing
wildtype cells (right), Miranda(green) is sequestered to only
one of the daughter cells. In
dlg-Gß mutants, Miranda is distributed to both daughters.

“It is intriguing how symmetrically dividing stem cells behave so differently in embryonic and larval
contexts,” comments Matsuzaki. “We see how the same combination of mutations can lead to terminal differentiation in one milieu, but tumor-like over-proliferation in another. In the future, we hope todetermine whether this holds true in other species as well.”Cartoon scheme showing differences in the differentiationand proliferation of progeny
cells born from wildtype, as well
as dlg-Gß embryonic and adultstage neuronal progenitor cells.

632010 Awards
As one of the world’s leading institutes in the fields of developmental biology and stem cell research, the RIKEN CDB strives to maintain thehighest levels of originality, impact and quality in its scientific contributions. The individual achievements and careers of many of its scientificstaff have been recognized with awards from numerous organizations, including government ministries and agencies, private foundations andacademic societies.

The CDB takes great pride in the achievements of its researchers, as individuals and as indispensable members of their laboratories.

AwardeePositionLaboratory

Award

Organization

Teruhiko Wakayama

Team Leader

Genomic Reprogramming

MEXT Prize

Ministry of Education, Culture, Sports,
Science and Technology

Jun-ichi Nakayama

Team Leader

Chromatin Dynamics

MEXT Prize

Ministry of Education, Culture, Sports,
Science and Technology

Naoki Irie

Research Scientist

Evolutionary
Morphogenesis

2010 Poster Prize

Society of Evolutionary Studies,Japan

Takao Suzuki

Research Scientist

Evolutionary
Morphogenesis

2010 Poster Prize

Society of Evolutionary Studies,Japan

Chong Li

Junior Research Associate

Genomic Reprogramming

Best Poster Prize

International Society for Transgenic
Technologies

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Osaka Science Prize

Osaka Science and Technology
Foundation

Shigeru KurataniGroup Director

Evolutionary
Morphogenesis

Hyogo Science Prize

Hyogo Prefecture

Yukari Terashita

Student Trainee

Genomic ReprogrammingAward for excellent poster
presentation

Asia Reproductive Biotechnology
Society

Teruhiko Wakayama

Team Leader

Genomic Reprogramming

Yamazaki Teiichi Prize

Foundation for Promotion of Material
Science and Technology of Japan64

CDK9 is expressed at the day of birth in melanocyte stem cells (melanocyte (Trp2) in red, CDK9 ingreen, note the red cells in the upper part of the hair follicle are positive for green signal)Embryonic Induction
During vertebrate embryogenesis, simply structured regions of undifferentiatedcells organize and cooperate to give rise to the anterior-posterior and dorsalventral body axes, the neural tube and other embryonic structures deriving fromthe three germ layers, following developmental patterns that can be collectively
summarized as the adult body plan. The embryo is a hive of activity, including
cell differentiation, proliferation and migration, strictly regulated in both space
and time by molecular signals emitted by special regions of the embryo referred
to as “signaling centers.” These centers produce diffusible proteins that serve toregulate embryonic development, guiding neighboring cell communities to takeup the roles they will play in the adult animal, or steering them away from inappropriate fates.

Hiroshi SASAKI Ph.D.
http://www.cdb.riken.go.jp/en/sasaki
Hiroshi Sasaki received his B. Sc. in zoology
from the University of Tokyo, and his master's
and Ph.D. in developmental biology from thesame institution. He worked as a research associate in Atsushi Kuroiwa's group at Tohoku University from 1990 to 1992, and in Brigid Hogan's
lab at Vanderbilt University from 1992 to 1994.
He returned to Japan to take a position as assistant professor at Osaka University beginningin 1995, where he remained until his appointment as team leader at the RIKEN in 2002.

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

Staff
Team Leader

Student Trainee

Research Scientist

Part-Time Staff

Hiroshi SASAKI
Yoshikazu HIRATE
Hiroshi MAMADA
Ken-ichi WADA

Yuu IMUTA

Megumi SHIBATA
Assistant

Misaki HARANO

Technical Staff

Shino HIRAHARA
Aya KATSUYAMA
Akiko MORII

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

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

66

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

Hitoshi SAWA Ph.D.
http://www.cdb.riken.go.jp/en/sawa
Hitoshi Sawa obtained his baccalaureate,master's and doctoral degrees from Kyoto
University in the period from 1982 to 1991.
He worked as a postdoctoral fellow at the
California Institute of Technology on a Human
Frontier Science Program grant during theperiod from 1991 to 1994, then at the Massachusetts Institute of Technology from 1994 to
1997 under support from the Howard Hughes
Medical Institute. He returned to Japan in 1997
to continue his work at Osaka University, firstas a postdoc, then as researcher funded by theJapan Science and Technology Corporation
PRESTO program. He took his current positionat the RIKEN CDB in 2001. He was appointed
team leader at the RIKEN CDB in 2001. He wasappointed professor in the National Institute of
Genetics in 2010.

Staff
Team Leader

Junior Research Associate

Research Scientist

Part-Time Staff

Hitoshi SAWA
Yukinobu ARATA
Yuji HOSOIRI
Yukimasa SHIBATA
Kenji SUGIOKA

Yuko YAMAMOTO

Tomoko SUGIMOTO
Assistant

Tomoko NAKASHIMA

Technical Staff

Hatsumi OKADA
Noriko SASAKAWA
Tomomi TAKANO

Publications
Arata Y, et al. Extracellular control of PAR protein localization
during asymmetric cell division in the C. elegans embryo.
Development 137. 3337-45 (2010)
Shibata Y, et al. Double bromodomain protein BET-1 andMYST HATs establish and maintain stable cell fates in C.
elegans. Development 137. 1045-53 (2010)
Sugioka K and Sawa H. Regulation of asymmetric positioning of nuclei by Wnt and Src signaling and its roles in POP1/TCF nuclear asymmetry in C. elegans. Genes Cells 15.
397-407 (2010)
Mizumoto K and Sawa H. Cortical β-catenin and APC regulate asymmetric nuclear β-catenin localization during asymmetric cell division in C. elegans. Dev Cell 12. 287-99 (2007)
Arata Y, et al. Wnt signaling and a Hox protein cooperatively
regulate psa-3/Meis to determine daughter cell fate after
asymmetric cell division in C. elegans. Dev Cell 11. 105-15(2006)

Asymmetric cortical localization of the Wnt pathway components

Goldstein B, et al. Wnt signals can function as positionalcues in establishing cell polarity. Dev Cell 10. 391-6 (2006)67

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

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

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

Staff
Team Leader

Guojun SHENG
Special Postdoctoral
Researcher

Yukiko NAKAYA

Research Scientist

Meng-Chi LIN
Fumie NAKAZAWA
Wei WENG

Technical Staff

Hiroki NAGAI
Kanako OTA
Erike Widyasari
SUKOWATI
YuPing WU
Part-Time Staff

Manjula
BRAHNAJOSYULA

Visiting Scientist

Cantas ALEV

Publications
Nagai H, et al. Embryonic development of the emu, Dromaius novaehollandiae. Developmental Dynamics 240. 162175 (2011)
Nagai H., et al. A modified cornish pasty method for ex ovo
culture of the chick embryo. Genesis 49. 46-52 (2011)
Nakaya Y, et al. Involvement of dystroglycan in epithelialmesenchymal transition during chick gastrulation. Cells Tissues Organs 193. 64-73 (2011)
Alev C, et al. Transcriptomic landscape of the primitive
streak. Development 137. 2863-74 (2010)
Shin M, et al. Notch mediates Wnt and BMP signals in theearly separation of smooth muscle progenitors and blood/
endothelial common progenitors. Development 136. 595603 (2009)
Nakaya Y, et al. RhoA and microtubule dynamics control
cell-basement membrane interaction in EMT during gastrulation. Nat Cell Biol 10. 765-775 (2008)

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

68

Physical Biology
Advances in measurement technologies have afforded us glimpses into dynamic
functional processes, such as morphogenesis and information processing, incells and tissues involved in development and regeneration. The truly organic
dynamism of the biological phenomena exhibited by living cells, individually or ingroups, emerges from the coordinated interaction of numerous molecular andgenetic factors, and the need for integrated, systems-based approaches to thestudy of design and operating principles in such “living” phenomena is becoming
increasingly clear. This will require not only technologies for the measurement ofsuch elements but the development of applicable mathematical methods as well.
In Laboratory for Physical Biology, we will seek to use concepts and methodology from mathematical sciences such as physics in the study and elucidation ofthese emerging questions in biology.
One example of such a phenomenon is seen in cellular chemotaxis, in whichcells recognize concentration gradients of attractant molecule and respond bydirectional movements necessary for functions such as the exploration of the environment by single-celled organisms, and morphogenesis in metazoa. Chemotactically responsive cells are able to detect differentials in the concentration of
an attractant molecule of only a few percent, which, given cell sizes of ranging in
the tens of micrometers, translates to a real difference of just a few dozen molecules. Cells are capable of interpreting this minute difference as a gradient thatguides the direction of its movement, raising the question of how cells are able
to detect and follow such weak and noisy signals. We now know that within theirtiny intracellular spaces, cells comprise many interacting molecules that workin a highly orchestrated fashion, and thus give rise to emergent order enabling
their orientation. Using quantitative fluorescence imaging data and the analysis
of mathematical models, we seek to gain a better understanding of such mechanisms.
Mathematical modeling of the essential aspects of observed phenomena of interest is a useful approach to evaluating whether we have sufficient knowledgeof associated molecules, reactions, and cellular interactions to explain them. The
abstracted mathematical idea of particular phenomena may further reveal general principles that underlie the living systems more broadly across diverse taxa.
We seek to contribute to the thorough exploration of these fascinating problems
in biology through concepts and methods adapted from the mathematical sciences.

Tatsuo SHIBATA Ph.D.
http://www.cdb.riken.go.jp/en/shibata
Tatsuo Shibata received his B.Sc. in Physics from Kyoto University, and Ph.D. from the
Graduate School of Arts and Sciences, University of Tokyo in 1999, after which he worked
as a postdoctoral research fellow at the Kyoto
University Research Institute for Mathematical
Sciences for two years. In 2001, he moved toGermany to do a second postdoc at the FritzHaber-Institut. He returned to Japan in 2002,
taking an associate professorship at Hiroshima
University. In 2007, he was additionally appointed as a researcher under the PRESTO
program coordinated by the Japan Scienceand Technology Agency (JST). In October of2010, he took his current position of Research
Unit Leader at the RIKEN CDB.

Staff
Unit Leader

Tatsuo SHIBATA
Research Scientist

Akinori BABA
Masatoshi NISHIKAWA

Student Trainee

Naohiro AKUZAWA
Toshinori NAMBA
Shunsuke OOYAMA
Ayumi TAKEMOTO
Assistant

Satoko KINOSHITA

Publications
Arai Y, et al. Self-organization of the phosphatidylinositol
lipids signaling system for random cell migration. Proc Natl
Acad Sci USA 107. 12399-404 (2010)
Nishikawa M and Shibata T. Nonadaptive fluctuation in an
adaptive sensory system: bacterial chemoreceptor. PLoS
One 5. e11224 (2010)
Shibata T and Ueda M. Noise generation, amplification andpropagation in chemotactic signaling systems of living cells.
Biosystems 93. 126-32 (2008)
Ueda M and Shibata T. Stochastic signal processing andtransduction in chemotactic response of eukaryotic cells.
Biophys J 93. 11-20 (2007)
Ishihara S,et al. Cross talking of network motifs in gene regulation that generates temporal pulses and spatial stripes.
Genes Cells 10. 1025-38 (2005)
Shibata T and Fujimoto K. Noisy signal amplification in ultrasensitive signal transduction. Proc Natl Acad Sci USA 102.
331-6 (2005)

Simulated stochastic behavior ofsignal transduction system that is
responsible for response and adaptation in bacterial chemotaxis.

Spontaneous formation of localized signal of Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and the averaged dynamics of PIP3 and PTEN.

69

Developmental Genomics
Fertilized eggs give rise to complex animal structures through strict coordination of cell divisions, cell fate determination and differentiation. To generate two
daughter cells with distinct cell fates from a single cell division (called “asymmetriccell division”), the mother cell is polarized before dividing and cytoplasmic components are asymmetrically segregated. A mitotic spindle is formed and positioned along the polarized axis, and the cell division produces two daughter cells
that contain distinct cell contents.

Asako SUGIMOTO Ph.D.
http://www.cdb.riken.go.jp/en/sugimoto
Asako Sugimoto received her B. Sc. from the
Department of Biophysics and Biochemistry in
the University of Tokyo, School of Science in
1987, and her doctorate from the same institution in 1992. She worked as a postdoctoral fellow in Joel Rothman's laboratory in the University of Wisconsin-Madison from 1992 to 1996,
before returning to Japan to assume an assistant professorship at the University of Tokyo.
She remained in that position until 2002, pursuing concurrent work as a Japan Science andTechnology Corporation PRESTO researcher
from 1997 to 2000. She was appointed teamleader at the RIKEN CDB in 2001. In April 2010,
she was appointed professor at Graduate Schoolof Life Sciences, Tohoku University.

Staff
Team Leader

Technical Staff

Special Postdoctoral
Researcher

Part-Time Staff

Asako SUGIMOTO
Momoyo HANAZAWA
Mika TOYA
Research Scientist

Yohei SASAGAWA
Eisuke SUMIYOSHI
Masahiro TERASAWA

Yumi IIDA
Kayo NAGATA

Our laboratory aims to understand the gene/protein networks controlling these
dynamic cellular processes using the nematode Caenorhabditis elegans embryos as an experimental model. This organism provides an extremely usefulsystem for studying such processes at the whole genome level, as its genome
has been fully sequenced and the lineage of each cell in its body is known. Using
high-resolution live microscopy to trace dynamic behaviors of proteins in combination with gene knockdown by RNAi, we are investigating the gene networks
that control these processes.
Our current research focuses on understanding the spatio-temporal regulatory
mechanisms of mitotic spindle formation. We revealed that two genetically separate pathways for microtubule assembly, one involving γ-tubulin and the otherrequiring aurora-A kinase, are responsible for the formation of mitotic spindles
in early C. elegans embryos. We are investigating how these pathways spatiallyand temporally coordinate to form mitotic spindles.
In addition to studying mitotic spindle formation, we are also studying two otherphenomena which are crucial for cell fate determination in early embryogenesisof C. elegans. The first is how mitotic spindles are positioned within the cell according to the established cell polarity. The second is how germ granules (large
complexes of mRNAs and proteins, believed to be the “germ cell determinants”)
are assembled and segregated specifically into the germ lineage. Through thesestudies, we hope to develop a more detailed picture of the regulation of dynamic
cellular processes by gene networks.

Kanako KAWAI
Student Trainee

Masafumi YONETANIAssistant

Taeko IMURA

Publications
Hanazawa M, et al. PGL proteins self associate and bind
RNPs to mediate germ granule assembly in C. elegans. J
Cell Biol (2011) in press
Terasawa M., et al. Caenorhabditis elegans ortholog of the
p24/p22 subunit, DNC-3, is essential for the formation of
the dynactin complex by bridging DNC-1/p150 (Glued) and
DNC-2/dynamitin. Genes Cells 15.1145-57 (2010)
Maruyama R, et al. EGG-3 regulates cell-surface and cortex
rearrangements during egg activation in Caenorhabditis
elegans. Curr Biol 17. 1555-60 (2007)
Motegi F, et al. Two phases of astral microtubule activity
during cytokinesis in C. elegans embryos. Dev Cell 10. 50920 (2006)
Motegi F and Sugimoto A. Sequential functioning of the
ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos. Nat Cell Biol 8. 97885 (2006)

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

70Retinal Regeneration
The retina has been called the “most approachable part of the brain,” owing to
its relatively simple structure and its location near the body surface, and for thesereasons it serves as a useful and experimentally amenable model of the central
nervous system. Until very recently, it was thought that in adult mammals theretina was entirely incapable of regenerating, but we now know that at least new
retinal neurons can be generated after being damaged. This has opened up newhope that the ability to regenerate neurons and even to reconstitute the neuralnetwork may be retained in the adult retina. We are now exploring the excitingprospect that, by transplanting cells from outside of the retina or by regenerationfrom intrinsic progenitor cells, it may one day be possible to restore lost function
to damaged retinas.
Our research into retinal regeneration seeks to achieve clinical applications by
developing methods for inducing stem cells or embryonic stem cells to differentiate into retinal neurons and pigment epithelial cells in sufficient quantities
for use in the treatment of patients suffering from conditions in which such cells
have been damaged or lost. We must also ensure that such cells establish viablegrafts upon transplantation and induce the reconstitution of functional neuralnetworks. We also hope to develop means of promoting true regeneration by
activating endogenous stem cells to replace cells lost to trauma or disease andthus repair damaged tissues. Access to a broad spectrum of developmental biological research information will be key to the achievement of these goals, andwe appreciate the opportunities for exchange that working in the environment
provided by the RIKEN CDB.
Therapeutic applications cannot be developed from basic research alone; theclinical approach—a thorough understanding of the medical condition to betreated—is equally important. For conditions such as retinitis pigmentosa, eventhe successful transplantation of cells in animal models may not necessarily betranslatable to a human clinical therapy without an understanding of the underlying genetics and possible immunological involvement. Our goal is to study retinal
regeneration based on both a strong foundation in basic research and solid clinical evidence.

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

Staff
Team Leader

Visiting Technical Staff

Research Scientist

Technical Staff

Masayo TAKAHASHI
Jun KANEKO
Michiko MANDAI
Akiko SUGA

Research Associate

Chie ISHIGAMI
Hiroyuki KAMAO
Satoshi OKAMOTO

Kyoko ISEKI
Akane NOMORI
Kazuyo SADAMOTO
Noriko SAKAI
Akihiro TACHIBANA
Yu WATAOKA
Chikako YAMADA

Visiting Scientist

Student Trainee

Yasuhiko HIRAMI
Zi-Bing JIN

1
Human iPS cells

2
Retinal pigment epithelial cells differentiatedfrom iPS cells

Motoki TERADA

Juthaporn
ASSAWACHANANONT

Publications
Jin ZB, Okamoto S, Mandai M and Takahashi M. Induced
Pluripotent Stem Cells for Retinal Degenerative Diseases: A
New Perspective on the Challenges, J Genetics 88:4, 417424 (2009)
Osakada F, et al. Toward the generation of rod and cone
photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol 26. 215-24 (2008)
Jin Z B, et al. Identifying pathogenic genetic background of
simplex or multiplex retinitis pigmentosa patients: a large scale
mutation screening study. J Med Genet 45. 465-72 (2008)
Osakada F, et al. Wnt signaling promotes regeneration in
the retina of adult mammals. J Neurosci 27. 4210-9 (2007)3
Purified retinal pigment epithelial cells

4
Photoreceptor cells differentiated from iPS cells

Ooto S, et al. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina. Proc Natl Acad
Sci U S A 101. 13654-9 (2004)
Haruta M, et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonicstem cells. Invest Ophthalmol Vis Sci 45. 1020-5 (2004)71

Cell Adhesion and Tissue Patterning
Animal cells organize into tissues with complex architecture. Our lab is exploring
the molecular mechanisms by which individual cells assemble into a tissue-specific multicellular structure, such as epithelial sheets and neural networks. We arealso interested in the molecular basis of how the tissue architecture is disrupted
during carcinogenesis, a process that is thought to accelerate the metastasis ofcancer cells. For these studies, we are focusing on the roles played by cell-celladhesion and recognition molecules, the cadherin family of adhesion moleculesin particular, as these are known to be indispensable for tissue construction.

Masatoshi TAKEICHI Ph.D.
http://www.cdb.riken.go.jp/en/takeichi
Masatoshi Takeichi is director of the RIKEN
Center for Developmental Biology as well as
director of the Cell Adhesion and Tissue Patterning research group. He completed the B.
Sc. and M. S. programs in biology at Nagoya
University before receiving a doctorate in biophysics from Kyoto University in 1973. Afterattaining his Ph.D., he took a research fellowship at the Carnegie Institution Department ofEmbryology under Richard Pagano. He thenreturned to Kyoto University, attaining a professorship in the Department of Biophysics (19861999), before becoming professor in the Department of Cell and Developmental Biology in
the Graduate School of Biostudies at the same
university. He assumed the directorship of the
CDB in 2000.

Staff
Group Director

Masatoshi TAKEICHI
Research Scientist

Syuichi HAYASHI
Shoko ITO
Wenxiang MENG
Shoko NAKAMURA
Tamako NISHIMURA
Anna PLATEK
Katsutoshi TAGUCHI
Yoshikazu TSUKASAKI
Vassil VASSILEV
Research Associate

Shigenori NAGAE
Nobutoshi TANAKA

Technical Staff

Sylvain HIVER
Saeko KOBAYASHI
Miwako NOMURA
Hiroko SAITO
Chika YOSHII
Junior Research Associate

Takashi ISHIUCHI

Cell-cell adhesion is an active process, which can be affected by a number ofcell-internal and external factors. This dynamic nature of cell-cell adhesion is implicated in various processes of cell assembly, such as the tightening or loosening of cell contacts and changing of cell assembly patterns. We are conducting
cell biological analyses to unravel the principal mechanisms that can regulate
cell-cell adhesion. A growing body of evidence suggests that cadherins cooperate with the cytoskeletal and/or motile machineries, such as actin filaments or
microtubules, in modulating cell assembly. We are therefore studying the mechanisms underlying the crosstalk between cadherins and such cytoskeletal systems. These studies include analysis of the functions of microtubule minus endassociated proteins and kinesin motors, as well as actin dynamics regulators, incell-cell contact regulation.
A second area of interest to our lab is to learn how the cell-cell adhesion machinery contributes to morphogenetic cell behavior. We are analyzing the rolesof cadherin in cell movement, in order to explore the mechanisms underlying thecontact-dependent regulation of cell locomotion. At the tissue level, using theembryonic brain as a model system, our team is attempting to determine how
cadherin and associated molecules control the remodeling of neuroepithelial layers, such as neural tube closure and the migration of neuronal progenitor cells.
We are also investigating the roles of protocadherins, members of the cadherin
superfamily, deficiencies of which have been implicated in neuronal defects inbrain development. Through these studies, we expect to gain deeper mechanistic insights into the ways by which cells build elaborate structures of the animal
body.

Agency Staff

Yoko INOUE
AssistantMutsukoAISO-WATANABE

Publications
Ito S and Takeichi M Dendrites of cerebellar granule cells
correctly recognize their target axons for synaptogenesis invitro. Proc Natl Acad Sci U S A 106. 12782-7 (2009)
Ishiuchi T, et al. Mammalian Fat and Dachsous cadherins
regulate apical membrane organization in the embryonic
cerebral cortex. J Cell Biol 185. 959-67 (2009)
Meng W, et al. Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell
135. 948-59 (2008)
Nishimura T and Takeichi M. Shroom3-mediated recruitmentof Rho kinases to the apical cell junctions regulates epithelial
and neuroepithelial planar remodeling. Development 135.
1493-502 (2008)
Nakao S, et al. Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction. J Cell Biol
182. 395-410 (2008)
Abe K and Takeichi M EPLIN mediates linkage of thecadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc Natl Acad Sci U S A 105. 13-9
(2008)

72

Double-immunostaining for F-actin (green) and Kusabira Orange-tagged E-cadherin (red) introduced
into A431D cells. In these cells, E-cadherin dynamically moves along cortical actin filaments, resulting
in the unique distributions shown here.

Systems Biology
Recent large-scale efforts in genome-sequencing, expression profiling andfunctional screening have produced an embarrassment of riches for life science researchers and biological data can now be accessed in quantities thatare orders of magnitude greater than were available even a few years ago. The
growing need for interpretation of data sets, as well as the accelerating demand
for their integration to a higher level understanding of life, has set the stage forthe advent of systems biology, in which biological processes and phenomena
are approached as complex and dynamic systems. Systems Biology is a naturalextension of molecular biology, and can be defined as "biology after the identification of key genes." We see systems-biological research as a multi-stage process, beginning with the comprehensive identification and quantitative analysis ofindividual system components and their networked interactions, and leading to
the ability to drive existing systems toward a desired state and design new ones
based on an understanding of structure and underlying principles.
Over the last several years, the Laboratory for Systems Biology (LSB) has worked
to establish Experimental Systems Biology at the Molecular-to-Cellular level andapply them to system-level questions of complex and dynamic biological systems, such as the mammalian circadian clock. In October 2009, our laboratory
was re-designated as a Project Lab in the Center Director’s Strategic Programfor Systems Biology research to promote the challenging tasks. Based on theachievements over the last eight years, we strongly feel that it is now the time
for us to take the next step forward toward Experimental Systems Biology at the
Cellular-to-Organism level.
Over the next several years, we intend to develop a efficient experimental platform to identify, monitor and perturb cellular networks within a organism. To thisaim, we attempt to invent and combine several key technologies ranging from (i)a rapid engineering of the genome of ES cells, (ii) generation of “100% chimera”
animals for F0 phenotyping, and (iii) phenotype analysis of the a small number ofthe generated animals (ideally with a single animal). Full utilization of these technologies will formulate cellular-to-organism-level Systems Biology, which will provide new strategies and concepts for the diagnosis, treatment, and prevention of
biological-time-related disorders, including rhythm disorder, seasonal affective
disorder, and sleep disorders.

Hiroki R. UEDA M.D., Ph.D.
http://www.cdb.riken.go.jp/en/ueda
Hiroki R. Ueda received his M.D. from the Faculty of Medicine of the University of Tokyo in
2000, and received his Ph.D. from the Universityof Tokyo Graduate School of Medicine in March
2004. While an undergraduate, he worked asa research assistant on a biological simulation
system project at Sony Computer Science Laboratories. As a graduate student he went on towork as a researcher (2000) and then as a groupleader (2002), at Yamanouchi Pharmaceuticals,
He was appointed team leader of Laboratory
for Systems Biology in 2003, and Leader of the
Functional Genomics Unit at the CDB in 2004.
He was also appointed as Professor (Invited)of Osaka University in 2006. In 2009, he waspromoted project leader of the Laboratory for
Systems Biology under CDB’s Center Director’sStrategic Program.

Staff
Project Leader

Visiting Researcher

Research Scientist

Junior Research Associate

Hiroki R. UEDA
Yohei KOYAMA
Tomoko MORIYAMA
Masato NAKAJIMA
Yasutaka NIWA
Hideki UKAI
Technical Staff

Hiroshi FUJISHIMA
Masahiro HAYASHI
Mayumi ISHIDA
Maki UKAI-TADENUMA
Visiting Scientist

Tetsuya KOBAYASHI
Kohei MASUMOTO
Atsushi WADA

Etsuo SUSAKI

Genshiro SUNAGAWA
Kaori TSUJINO

Special Postdoctoral Researcher

Rikuhiro YAMADA
Student Trainee

Ryokichi TANAKA
Takashi YASUKAWA
Part-Time Staff

Yoko SAKAKIDA
Yuko SASAGAWA
AssistantSumire HINO

Publications
Ukai-Tadenuma M, et al. Delay in Feedback Repression by
Cryptochrome 1 Is Required for Circadian Clock Function.
Cell 144.268-281 (2011)
Masumoto K. H, et al. Acute induction of Eya3 by late-night
light stimulation triggers TSHbeta expression in photoperiodism. Curr Biol 20.2199-206 (2010)

Delayed Feedback repression: The essence of the clock
Cryptochrome1 (Cry1), an essential mammalian clock component, serves as a strong repressor ofmorning-time transcription and displays a delayed expression in the evening. Ukai-Tadenuma and
Yamada et al. revealed that the delayed expression of Cry1 is realized by a combinatorial transcriptional regulation via day-time and night-time cis-elements, which can be simply recapitulated by aphase-vector model. They also revealed that delay in feedback repression is required for mammalianclock function, and that the prolonged delay in feedback repression slows circadian oscillation. These
results suggest that the delayed-feedback repression is a design principle of the transcriptional circuitin mammalian circadian clocks.

Isojima Y, et al. CKIepsilon/delta-dependent phosphorylation
is a temperature-insensitive, period-determining process inthe mammalian circadian clock. Proc Natl Acad Sci USA
106. 15744-9 (2009)
Ukai-Tadenuma M, et al. Proof-by-synthesis of the transcriptional logic of mammalian circadian clocks. Nat Cell
Biol 10. 1154-63 (2008)
Kumaki Y, et al. Analysis and synthesis of high-amplitude
Cis-elements in the mammalian circadian clock. Proc Natl
Acad Sci USA 105. 14946-51 (2008)
Ukai H, et al. Melanopsin-dependent photo-perturbation
reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell Biol 9. 1327-34 (2007)73

Genomic Reprogramming
A limitless number of clones of an animal can be generated from its somatic
cells. Only a few years ago, such a statement would have belonged to the realmof science fiction, but now thanks to advances in the technology known as micromanipulation, which allows researchers to work with individual cells and their
nuclei, that fiction has become reality. The efficiency of the cloning procedure(measured as the ratio of live offspring to cloning attempts), however, remains
quite low, at only a few percent, and even in those attempts that do lead to live
births, the cloned animals consistently exhibit a range of severe developmental
abnormalities and pathologies.

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

Staff
Team Leader

Teruhiko WAKAYAMA
Research Scientist

Chong LI
Hiroshi OHTA
Mikiko TOKORO
Sayaka WAKAYAMA
Kazuo YAMAGATA

Technical Staff

Yuko SAKAIDE
Kaori YAMANAKA
Part-Time Staff

The cause of these defects is thought to be due to imperfections in the nuclear
transfer technique used to remove the nucleus from a host cell and replace
it with another from a donor; imperfections which presumably lead to total orpartial failure of reprogramming, the process in which a cell’s developmental
potential is reset to a totipotent state (such as is exhibited by a naturally fertilizedegg). However, the specific details of the technical flaws, the ways in which theylead to reprogramming failure, and possible solutions to the problem all remaincompletely unknown. Despite these challenges, cloning continues to be a biotechnology of great promise, given its potential applications in the field of regenerative medicine, such as the generation of embryonic stem (ES) cells by nuclear
transfer, which may one day allow us to grow replacement cells that perfectly
match a patient’s own genetic and immune profile, thereby eliminating the risk ofrejection.
We use the mouse as a model system to study cloning under a range of experimental conditions with the goals of achieving improvements in the efficiency ofthe cloning procedure, gaining a better understanding of reprogramming mechanisms and analyzing the properties of ES cells derived via somatic nuclear transfer. We also use nuclear transfer technology to develop methods for preserving
embryonic lethal and infertile strains of laboratory mice, and continue to explore
the development of new and better techniques for sperm and oocyte preservation and other reproductive biological research technologies.

Tetsuo ONO
Yukari TERASHITA
Assistant

Tomoko OYANAGI

Visiting Scientist

Satoshi KISHIGAMI

Publications
Ono T., et al. Inhibition of class IIb histone deacetylase
significantly improves cloning efficiency in mice. Biol Reprod
83.929-37 (2010)
Li C, et al. An efficient method for generating transgenic
mice using NaOH-treated spermatozoa. Biol Reprod 82.
331-40 (2010)
Wakayama S, et al. Detrimental effects of microgravity on
mouse preimplantation development in vitro. PLoS One 4.
e6753 (2009)
Yamagata K, et al., Assessment of chromosomal integrity
using a novel live-cell imaging technique in mouse embryos
produced by intracytoplasmic sperm injection. Hum. Reprod. 24. 2490-9 (2009)
Wakayama S, et al. Production of healthy cloned mice frombodies frozen at -20 degrees C for 16 years. Proc Natl Acad
Sci U S A 105. 17318-22 (2008)
Bui H T, et al. The cytoplasm of mouse germinal vesicle
stage oocytes can enhance somatic cell nuclear reprogramming. Development 135. 3935-45 (2008)
Tabar V, et al. Therapeutic cloning in individual parkinsonian
mice. Nat Med 14. 379-81 (2008)

74

Transfer of a somatic nucleus into an enucleated egg

Emu embryo at HH-equivalent stage 36

Transcriptional off-switch in stem cells

Rasmus FRETER

Stem cells are known mainly for their ability to self-renew and to give rise to cells of different types, but many
have a third important characteristic as well: they can shut down, becoming “quiescent.” This is particularly crucial for stem cells that function in the adult body, as they need to be able to maintain tissue homeostasis by dividing to produce progeny cells in precisely the right numbers to maintain integrity without over-proliferating. Thisdormant state has been studied mainly in term of cell cycle regulation, cell metabolism, and interactions with thestem cell niche. More recent evidence suggests that transcriptional activity is also suppressed in quiescent stem
cells, but the mechanisms by which this slowdown is achieved have yet to be identified.
A study by Rasmus Freter and others in the Laboratory for Stem Cell Biology (Shin-Ichi Nishikawa, Group Director) revealed the mechanistic basis for stem cells’ transcriptional slumber. Published in the journal Stem Cells, thisnew work reveals that adult stem cells globally suppress the phosphorylation of a specific amino acid in the tailend of the crucial enzyme, RNA polymerase II (RNApII), in mouse.
Freter began the study knowing that mRNA transcription is markedly reduced in quiescent melanocyte stem
cells. Looking for the underlying mechanism, he tested for the phosphorylation of serine 2 in the C-terminal domain of RNApII, which is known to trigger elongation, a necessary step in the synthesis of mRNA. While melanocytes and migrating progenitors stained positive for phosphorylated ser2, this was downregulated in stem cells
that had settled into their niche in the hair follicle.
Quiescence is typically induced when a stem cell is deprived of survival factors, which in the case of melanocyte stem cells include SCF/c-Kit. To determine whether a relationship existed between this stem cell factor and76©2010 AlphaMed Press
Freter R, et al. Adult stem cells exhibit global suppression of RNApolymerase II serine-2 phosphorylation. Stem Cells 28.1571-80 (2010)

phosphorylation of this amino acid, the group used a transgenic mouse in which SCF is constitutively
overexpressed. They found that the melanocyte stem cells in adult animals lacked phosphorylated
ser2, but interestingly, that this was present at the time of birth. Apparently a short-lived signal caused
dephosphorylation in the immediate postnatal period, but was later overridden by the constitutive effect of SCF in the mutant animals.
Looking for clues behind the dephosphorylation in the transgenic adults, Freter analyzed the expression of the cyclin-dependent kinase complex p-TEFb, which is composed of CDK9 and a cyclin partner, and is known to trigger the phosphorylation of ser2. He found that while CDK9 was unperturbed
in differentiated cells, it was specifically downregulated in melanocyte stem cells, presumably as aresult of the global transcriptional suppression that takes place in these cells. This supposition wasbolstered by the finding that conditional knock-in of CDK9 did not induce phosphorylation in thesemelanoblasts. The group further found that the inhibition of CDK9, used here as a readout of thephosphorylation status of ser2, increased the survival of cultured cells during growth factor starvation
by suppression of cell death pathways.

Absence of mRNA transcription
elongation in adult melanocyte
stem cells (melanocytes (Trp2)
in red, and mRNA transcription(CTD-Ser2-p) in green).

To determine whether the role of ser2 phosphorylation in the C-terminal domain was common toquiescent stem cells in other tissues, the group examined a range of other stem cell types, including
satellite cells in muscle, spermatogonia stem cells and blood-producing hematopoietic stem cells,in addition to the melanocyte and keratinocyte stem cells from the hair system. They found that in allcases, the inactive stem cells showed lower ser2 phosphorylation than that seen in actively cycling
cells, suggesting that global transcriptional suppression may be a general feature of stem cell quiescence.
“Downregulation of CTD Ser2 phosphorylation is beneficial for cell survival when cells are confronted
by stress, and thus maintains the stem cell compartment throughout the life of the organism,” says
Freter. “The detection of transcriptional quiescent cells in organs, or even cancer tissues, may therefore be useful in the identification of cells with stem cell-like properties."

77

New twist on ex ovo culture in bird

Hiroki NAGAI, Meng-Chi LIN

Birds, such as the chicken, provide an excellent model for the study of many developmental processes, andremain one of the most commonly studied species in classical embryology. The earliest stages of avian embryonic development are not amenable, however, to manipulation within the egg, necessitating methods for embryo
culture. A number of techniques for sustaining the growth of chicken embryos ex ovo have been developed, but
are limited by cumbersome set-up and relatively short survival times. Methodological advances might therefore
expand the utility of ex ovo approaches and enable studies of later occurring developmental phenomena.
Hiroki Nagai and colleagues in the Laboratory for Early Embryogenesis (Guojun Sheng, Team Leader) have done
just that with a new twist on ex ovo chick embryo culture, which they have christened the modified Cornish pasty(MC) method, as it is based on a previous technique that involves folding the explanted embryo in two, similar
to the preparation of the well-known British folded pastry. The new method, described in the journal Genesis, issimpler to perform than its predecessors and sustains embryos to later stages of development.
The protocol involves removing the embryo from the egg during the primitive streak phase of development,
between stages 3 and 4 on the Hamburger-Hamilton (HH) scale, folding it along an axis parallel to the primitive
streak so that it takes on a half-moon shape, and transferring it to culture medium. Interestingly, within 24 hours
most of the embryos “inflate,” apparently due to active transport of fluid, and rise to the surface of the medium.
This fluid transport is important, as it maintains epiblast tension, which is crucial for early development. The
majority of embryos maintained under these conditions grew at normal rates for the first day of culture, but later
slowed slightly relative to normal development.

78©2010 John Wiley & Sons, Inc.
Nagai H, et al. A modified cornish pasty method forex ovo culture of the chick embryo. Genesis (2010)

Importantly, about half of all MC cultured embryos survived with normal morphology until HH18, an
advance over previous methods, which could only sustain growth until HH13–15. This survival gain issignificant in that a number of features of vascular, head, and limb formation only begin to appear ataround stage 18. In a number of the MC embryos, growth of the head region continued beyond HH18,
although limb development was arrested, meaning that the new method might prove useful in ex ovo
studies of even later stages of head development.

The MC culture is prepared byfolding the streak stage embryoand trimming the margins (left).
Af ter one day in culture, theembryo inflates into a balloon
shape, with the embryo proper
positioned on top and growing
healthily.

To confirm that their new method could be used in complement with other manipulations, Nagai et al.
tested several embryological techniques in the folded explants. They found that embryos electroporated with GFP grew normally and were stained as expected with fluorescent signals. They next trieda trickier experiment, known as parabiosis, in which a pair of embryos is co-cultured in close contact
with each other, resulting in twin embryos with shared blood circulation. Using a slightly modified version of their new method, they found that they could generate parabiotic pairs of chicks, quails, andeven combinatorial parabiosis involving a chick-quail pairing. Such experimental systems are useful inthe study of blood development.
“The chick is a wonderful model organism for developmental biology studies, but the modified Cornish pasty culture, or the dumpling culture as I like to call it, shows that we still don’t understand theembryo well, as no one would have predicted that twinning and parabiosis can be created this way,”says Sheng. “Our next goal is to use this technique to test some of the hypotheses regarding the origin of definitive hematopoietic stem cells.”

A pair of chick /chick twins, after
2-day culture with the MC method. The lef t-side embr yo has
been injected intracardially witha fluorescent dye, and vascularfusion of the twins is highlighted
by fluorescent signals in the vasculature of the right-side embryo
shortly after injection.

79

X inactivation a toss-up in ES cells

Cells have evolved a variety of strategies for ensuringthat they get just the right doses of gene products. X
inactivation, in which one of the two X chromosomes infemale mammals is epigenetically silenced, is one of thebest known and most intensively studied of these mechanisms. One of this process’s more interesting features
is that cell lineage determines whether inactivation occurs in a randomized or deterministic fashion. In somatic
lineages differentiated in post-implantation embryo, X
inactivation can take place in either the paternally or maternally supplied chromosome, while in extraembryonictissues segregated from pluripotent stem cells in pre-implantation embryo, the paternal X is always inactivated.
But has this transition already been made in the pluripotent stem cells of inner cell mass, which represents boththe first fruit of differentiation and the starting point for all
somatic lineages?
Kazuhiro Murakami in the Laboratory for Pluripotent
Stem Cell Studies (Hitoshi Niwa, Project Leader) andcolleagues used a pair of modified mouse embryonicstem (ES) cell lines to answer that question, and foundthat when the pluripotent cells were forced to differentiate into extraembryonic lineages, X inactivation in theirprogeny cells took place randomly. Their findings, published in Development, help to better constrain the timing of this crucial epigenetic event.
The team selected two lines of female mouse ES celllines in which the paternal and maternal X chromosomes
could be readily distinguished, due to genomic polymorphism or fluorescent labeling. ES cells such as these do
not normally give rise to extraembryonic lineages, suchas trophectoderm or primitive endoderm, but previous
work by the Niwa lab had shown that forced expression of Cdx2 or Gata6 could steer ES cells down those
respective pathways. Murakami used this combinationof cells and transcription factors to generate ES-derived
extraembryonic lineages in which the pattern of X inactivation could be visualized.
X inactivation is associated with the epigenetic silencing
by trimethylation of histone 2 lysine 27 (H3K27) as wellas the coating with Xist RNA, so the team first confirmedthat their induced trophectoderm and primitive ectoderm
cells had indeed undergone silencing, and found that,in contrast to undifferentiated ES cells in which inactivation has not occurred, the extraembryonic cells showed
clear Xist-H3K27 association at a single locus.

Kazuhiro MURAKAMI80©2010 The Company of Biologists
Murakami K, et al. Choice of random rather than imprinted X inactivation infemale embryonic stem cell-derived extra-embryonic cells. Development 138.197-202 (2011)

The question was: did this happen only in the maternal X, or randomly to either the paternally or maternally donated chromosome?
Murakami et al. first used a PCR-restriction
fragment length polymorphism (RFLP) assay to detect transcripts specific to eachgenomic donor. While both sets of transcripts are expected to appear in undifferentiated cells, only the maternal transcripts
appear in normal extraembryonic tissues,
due to imprinted X inactivation. However, in
the induced trophectoderm and primitive
endoderm (in which X inactivation clearlyoccurs), both paternal and maternal X chromosome contributions remained, suggesting that the process occurred randomly.
This finding was supported by results ofparallel experiments using a second ES cellline in which the paternal X chromosome
was labeled by EGFP. If imprinted X inactivation took place in the induced extraembryonic cells, thefluorescent signal would be lost, but in both primitive endoderm and trophectoderm, the cells glowed,
testifying to the stochastic nature of the chromosomal silencing.

Xist RNA/H3K27me3 immunoFISH in induced extra-embryonic cells. The spreading of
X ist RNAs was dete cte d by
Xist exon probes (green). The
H3K27me3 signal was detectedby Anti-H3K 27me3 antibody(red).

To make a final confirmation that the X inactivation imprint is already lost in the inner cell mass, theteam, in collaboration with the Laboratory for Genomic Reprogramming (Teruhiko Wakayama, Team
Leader) used the ES cells expressing EGFP on its paternal X chromosome to generate cloned blastocysts. As anticipated by the results from their in vitro differentiation studies, X inactivation in the trophectoderm of these blastocysts occurred randomly, in contrast to the usual imprinting seen in the same
tissue in control embryos. The imprinting that leads to selective X inactivation, it seems, is completely
lost in ES cells.
“The precise developmental time-point of the transition in the X chromsome’s epigenetic state from
imprinted to random X inactivation was unknown, but we were able to show that the early epiblastfrom which ES cells are derived completes the transition event by erasing all epigenetic modifications
for imprinted X inactivation,” says Niwa. “The epigenetic state of ES cells may be a sort of tabula rasa.”

Phase-contrast and GFP fluore scence image s of B142bgeoEG Cdx2ER and Gata6GR
ES cell lines. The active state of
the paternal X chromosome was
detected by GFP fluorescence.
Nuclei were counterstained by
Hoechst 33342 (blue).

81

Better cloning through chemistry

Tetsuo ONO

Cloning of animals by nuclear transfer remains an art as much as it is a science. Mouse cloning efficiency has lingered in the low single digits since the first success in 1998, at which time only 1–2% of all attempts was capableof generating cloned offspring. In the intervening years, that number has only risen by a few percentage points,
and even when using the best available techniques, a cloning success rate of above 5% is considered high. A
number of recent advances in cloning technology have involved the use of chemicals that interfere with the actionof epigenetic regulators known as histone deacetylases (HDACs). But the reason why these inhibitors promotecloning success is unknown.
Tetsuo Ono and others in the Laboratory for Genomic Reprogramming (Teruhiko Wakayama, Team Leader) have
now identified a pair of new HDAC inhibitors that improve the rate of full-term development in cloned mice. These
new cloning boosters share a subset of characteristics with previously identified molecules of similar function,
which may point the way to a better mechanistic understanding of how these new ingredients work.
There are multiple types of HDAC, which are categorized into five classes (I, IIa, IIb, III and IV), and are expressed
nearly ubiquitously, functioning as regulators of gene expression. Their inhibitors play an equally important role infine-tuning their regulatory effects, but can also have cytotoxic effects, suggesting that the selection of inhibitor
can have a major effect on whether it helps or harms the cloning process.
To determine which works best in raising the efficiency of cloning and genomic reprogramming, Ono tested
a number of HDACi proteins, with specificities for different classes of HDAC. These included well-established
HDAC inhibitors, such as trichostatin A and scriptaid, as well as two new molecules, suberoylanilide hydroxamic82

©2010 Biology of Reproduction
Ono T, et al. Inhibition of class IIb histone deacetylase significantly
improves cloning efficiency in mice. Biol Reprod 83.929-37 (2010)acid (SAHA) and oxamflatin—all of which inhibit HDAC classes I, IIa and IIb. The effects of valproic
acid, an inhibitor of HDAC I and IIa, but importantly not of IIb, were also examined.
The team treated cloned embryos with varying concentrations of these HDAC and followed their development through hallmark stages such as blastocyst formation and full-term development. They
found that at all stages, the embryos treated with trichostatin A or the new factors SAHA and oxamflatin outperformed those treated with valproic acid and untreated controls. The new factors yielded
particularly strong improvements in cloning efficiency, with 9.4% of SAHA treated embryos and up to
7.5% of the oxamflatin-treated group reaching full term.
Immunostaining of the cloned blastocysts revealed that the SAHA and oxamflatin-treated embryos
had significantly higher numbers of cells in the inner cell mass (the population of cells that gives riseto the embryo proper), suggesting that these HDAC inhibitors might enhance the reprogramming ofsomatic cell nuclei. TUNEL assays, which reveal apoptotic activity, showed that the SAHA and oxamflatin blastocysts had fewer cells undergoing programmed cell death, while those treated with valproic
acid were more apoptotic.
As a final functional test, Ono et al. generated embryonic stem cell lines from the cloned blastocysts(ntES cell lines) treated with the various factors, and found that HDACi-treated clones were two to
three time more likely to be able to give rise to a pluripotent stem cell line.
“We wanted to know why only cloned embryos require HDACi treatment to encourage genomic reprogramming,” says Wakayama. “What we found was that perhaps the oocyte cytoplasm reprograms
the somatic cell nucleus too strongly. By inhibiting a particular oocyte HDAC, we could achieve more
appropriate reprogramming of donor nuclei, resulting in a higher cloning success rate.”

Incidence of apoptosis (TUNEL
assay) in cloned blastocysts
treated with SAHA (right) andVPA (left). The apoptotic cells(green spots) were significantly
decreased in SAHA treated embryos.

83The first light of day heralds spring’s coming

Many organisms in both the plant and animal kingdoms
rely on length-of-day cues to detect seasonal change,
and modulate their physiological activities to suit thetime of year. This photoperiodism is involved in sexual
maturation as well as hibernation and migratory cycles inanimals, and has been implicated in seasonal affective
disorder in humans. Previous research has shown that,in quail, an increase in day length induced the expression of the thyroid stimulating hormone, ß subunit (TSHß),which is a crucial regulator of the bird’s physiological
function. But just how information about the length of
daylight is transduced to TSHß has remained a mystery.
Now, Koh-hei Masumoto and colleagues in the Laboratory for Systems Biology (Hiroki R. Ueda, Project Leader)show in a unique mouse model that the transcription
factor Eya3 not only induces TSHß expression but does
so in response to light stimuli delivered early in the daily
cycle. Their results, which appear in Current Biology,
reveal an interesting mechanism behind the seasonal
control of TSHß expression.
Most of the mouse strains commonly used in research
do not exhibit photoperiodism, but, it was recently reported that melatonin-proficient CBA/N mice show increased expression of TSHß in the pars tuberalis regionof the brain in response to longer daylight, just as in thequail. Identification of this mouse model was an important first step, as it meant that the lab could bring thewealth of accumulated knowledge of mouse physiology
and advanced genetic techniques, to bear on the problem.
Masumoto started by conducting a whole-genome
microarray analysis on pars tuberalis samples from micethat had been raised in either long-day (16 hours of light,
8 hours dark), or short-day (8h light, 16h dark) conditions. He found 246 genes (including TSHß) strongly
expressed in the long-day samples, and 57 that wereupregulated in the short-day mouse brain. Wanting todetermine what would happen to the induction of TSHßwhen mice were shifted between short-day and longday regimes, Masumoto found that while switching thelights off eight hours later (in a sense, delaying night) had
no effect on TSHß, the opposite adjustment, speeding
the arrival of morning’s light by eight hours, caused a
rapid increase in the mRNA’s expression.

Koh-hei MASUMOTO, Maki UKAI-TADENUMA

84

But it still wasn’t clear what linked its expression tochanges in the onset of day, so the team did a second
round of DNA microarray testing, looking for genes that©2010 Elsevier B.V.
Masumoto KH, et al. Acute induction of Eya3 by late-night light stimulation
triggers TSHß expression in photoperiodism. Curr Biol 20.2199-206 (2010)

began to be expressed in advance of the switched-on TSHß. They found a total of 34, and homed inon a favorite candidate, Eya3, by virtue of, among other things, its known role as a transcriptional factor. Eya family factors work by forming complexes with Six family proteins, enabling them to regulate
target genes. Looking back to the pars tuberalis from the brains of long-day mice, Masumoto et al.
found that both Eya3 and Six1 were strongly expressed. Testing this association in an in vitro reporter
assay, they confirmed that the co-expression of these two factors led to activation of the TSHß promoter. Further, this activation is enhanced by Tef and Hlf. Although Eya3’s expression under long-day
conditions has been known previously from work in birds and sheep, this current work from the Ueda
lab points both to its broad conservation and its mechanism of action.

Shifting the start of the light period forward by 8 hours for miceraised in short-day conditions
results in upregulation of Eya3,
which in turn induces TSHß
expression.

“In natural settings, of course, the progression toward a longer day is more gradual, so we think thatthe induction of TSHß by Eya3 should also play out over a longer time,” says Ueda. “But in this study
we have shown that it is possible to induce Eya3 in a short period by manipulating exposure to light,
which should be useful for future research into photoperiodism. We will also be very interested inworking out how information about day length is relayed to this transcription factor, and why it is thatmorning light plays such a key role in determining photoperiod.”85

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

Shinichi AIZAWA Ph.D.

Genetic Engineering Unit
Shinichi AIZAWA Ph.D.

Staff
Unit Leader

Shinichi AIZAWA
Research Scientist

Shuichi KANI
Go SHIOI
Hirotaka TAO

Visiting Scientist

Toshihiko FUJIMORITechnical Staff

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

CS7 BL/6 Es(HK4)-derived offspring

Animal Resource Unit
Kazuki NAKAO Ph.D.

Staff
Unit Leader

Kazuki NAKAO
Attending
Veterinarian

Naoko KAGIYAMATechnical Staff

Kenichi INOUE
Yuki KANEKO
Takuya KAWADA
Hiroshi KIYONARI
Miho SATO
Mayo SHIGETA
Aki SHIRAISHI
Norie TANAKA
TomokoTOKUNAGA
Sachi YAKAWA
SayakaYOSHIMURA

Assistant

Yuki TSUJI

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

Publications
Kiyonari H, Kaneko M, Abe S and Aizawa S. Threeinhibitors of FGF receptor, ERK, and GSK3 establishes
germline-competent embryonic stem cells of C57BL/6N
mouse strain with high efficiency and stability. Genesis
48.317-27 (2010)
Nakao K. and Inoue K. Cryopreservation for broader production of transgenic mice by DNA injection into zygotes.
Exp Anim 59.225-9 (2010)86

Mice maintained by the Animal Resource Unit

Uchiyama Y, Sakaguchi M,Terabayashi T, Inenaga T, Inoue
S, Kobayashi C, Oshima N, Kiyonari H, Nakagata N, Sato
Y, Sekiguchi K, Miki H, Araki E, Fujimura S, Tanaka S S,
Nishinakamura R. Kif26b, a kinesin family gene, regulates
adhesion of the embryonic kidney mesenchyme. Proc
Natl Acad Sci USA 107.9240-5 (2010)
Oboki K, Ohno T, Kajiwara N, Arae K, Morita H, Ishii A,
Nambu A, Abe T, Kiyonari H, Sudo K, Okumura K, Saito H,
Nakae S. IL-33 is a crucial amplifier of innate rather thanacquired immunity. Proc Natl Acad Sci USA 107.18581-6(2010)Electron Microscope
Detailed knowledge of the shapes of individual cells and fine subcellular structures is vital to the understanding of biologicalstructures and organization. In developmental processes in particular, changes in cell morphology and behavior play fundamental roles. The Electron Microscope Laboratory provides technical assistance and expert guidance in the use of transmission and scanning electron microscopy (TEM and SEM) and related technologies in morphological analyses.

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

Staff
Laboratory Head

Visiting Scientist

Technical Staff

Student Trainee

Shigenobu
YONEMURA

Kisa KAKIGUCHI
Kazuyo MISAKI
Sachiko ONISHI
Makiko F. UWO

Masatsune
TSUJIOKA

Tomoki FUJITA
Research Fellow

Ayuko SAKANEAssistant

Mai SHIBATA

Publications
Yonemura S, et al. α-Catenin as a tension transducer that induces adherens junction development. Nat Cell Biol 12. 533-42 (2010)
Tajiri R, et al. Dynamic shape changes of ECMproducing cells drive morphogenesis of ball-andsocket joints in the fly leg. Development 137.
2055-63 (2010)
Iida A, et al. Metalloprotease-dependent onset ofblood circulation in zebrafish. Curr Biol 20. 11106 (2010)
Yamamoto M, et al. Mib-Jag1-Notch signaling
regulates patterning and structural roles of thenotochord by controlling cell-fate decisions. Development 137. 2527-37 (2010)

Cells organizing the notochord of zebrafish embryo

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

87

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

Shigeo HAYASHI Ph.D.

Optical Image Analysis Unit
Yuko MIMORI-KIYOSUE Ph.D.
This unit runs the CDB’s common-use Imaging facility.

Staff
Unit Leader

Yuko
MIMORI-KIYOSUE
Technical Staff

Agency Staff

Nobuyuki
NAKANISHI
Yuka YAMAMOTO

Tomoko HAMAJI
Part-time Staff

Satoko NAKAMURA

Publications
Hotta A, Kawakatsu T, Nakatani T, Sato T, Matsui
C, Sukezane T, Akagi T, Hamaji T, Grigoriev I,
Akhmanova A, Takai Y and Mimori-Kiyosue Y.
Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex
through LL5alpha/beta. J Cell Biol 189.901-17(2010)

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

Inverted microscope maintained by the Optical Image Analysis Unit.

88

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

Fumio MATSUZAKI Ph.D.

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

Staff
Unit Leader

Visiting Scientist

Technical Staff

Part-Time Staff

Hiroshi TARUITetsutaro HAYASHI
Kazu ITOMIYu LIN
Asuka MOMIYAMA
Kaori TATSUMI

Osamu NISHIMURA
Minako MOTOISHI
Assistant

Chiharu
TANEGASHIMA

Publications
Yazawa S, et al. Planarian Hedgehog/Patchedestablishes anterior-posterior polarity by regulating Wnt signaling. Proc Natl Acad Sci USA 106.
22329-34 (2009)
Maki N, et al. Expression of stem cell pluripotency factors during regeneration in newts. Dev
Dyn 238(6), 1613-6 (2009)
Egger B, et al. To be or not to be a flatworm: theacoel controversy. PLoS One 4(5), e5502 (2009)
Eto K, et al. Reduced expression of a RNAbinding protein by prolactin leads to translationalsilencing of programmed cell death protein 4 andapoptosis in newt spermatogonia. J Biol Chem(2009)

Functional Genomics Unit
Hiroki R. UEDA M.D., Ph.D.
The Functional Genomics Unit (FGU) has two missions: to provide functional
genomics services to the laboratories within the CDB, and develop and introduce cutting-edge technologies related to functional genomics in order toaccelerate the Center’s research. We are striving to implement two types oftechnologies: 1) expression analysis, and 2) high-throughput measurement
and perturbation. For expression analysis, we first introduced GeneChip technology, which is mainly used to measure expression profiles of genes in cells
or tissues. For high-throughput measurement and perturbation, we introduced
cell-based screening technology, which examines gene functions in cells. We
will integrate technologies for expression analysis and high-throughput measurement and perturbation to develop new functional genomics methods. In
particular, we will focus on strengthening single-cell expression analysis, anddeveloping three-dimensional expression analysis in organs.

Staff
Unit Leader

Visiting Scientist

Special Postdoctoral
Researcher

Technical Staff

Yohei SASAGAWA

Junko NISHIO
Kenichiro UNO

Research Specialist

Agency Staff

Hiroki R. UEDA

Takeya KASUKAWA
Itoshi NIKAIDO

Hiroki DANNO

Chikako IMAI
Assistant

Ikuko TADA

Publications
Alev C, et al. Transcriptomic landscape of theprimitive streak. Development 137. 2863-74(2010)
Masumoto K.H, et al. Acute induction of Eya3 bylate-night light stimulation triggers TSHß expression in photoperiodism. Curr Biol 20. 2199-206(2010)
Isojima Y, et al. CKIε/δ-dependent phosphorylation is a temperature-insensitive, perioddetermining process in the mammalian circadianclock. Proc Natl Acad Sci USA 106. 15744-9(2009)
Kawaguchi A, et al. Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis. Development 135. 311324 (2008)89

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

Shigeo HAYASHI Ph.D.

Mass Spectrometry Analysis Unit
Akira NAKAMURA Ph.D.

Staff
Lab Head

Shigeo HAYASHI
Unit Leader

Akira NAKAMURA
Technical Staff

Kaori SHINMYOZU

Publications
Otani T, et al. IKKe regulates cell elongation
through recycling endosome shuttling Dev. Cell
20.1-14 (2011)
Hayakawa T, et al. MRG15 binds directly to
PALB2 and stimulates homology-directed repairof chromosomal breaks. J Cell Sci 123.1124-30(2010)The Mass Spectrometry Analysis Unit uses LC-MASS spectrometry, atechnology that enables the high-speed analysis of protein components
from even minute biological samples. Its support activity takes the forms
of protein identification services and analysis of protein modifications
such as phosphorylation, ubiquitination, etc. The unit supports laboratories within the CDB, and collaborations with a number of laboratories
at other institutions. The lab receives gel slices containing proteins fromresearchers and conducts all subsequent steps, including de-staining ofgels, in-gel digestion of proteins, peptide extraction, and operating the
LC-MS/MS.
In the most recent fiscal year, the unit received more than 30 spectrometry requests and nearly 900 samples for use in identification.

Shirai A, et al. Methylation of ribosomal protein
L42 regulates ribosomal function and stressadapted cell growth. J Biol Chem 285.22448-60(2010)
Terasawa M, et al. Caenorhabditis elegans
ortholog of the p24/p22 subunit, DNC-3, is essential for the formation of the dynactin complex
by bridging DNC-1/p150 (Glued) and DNC-2/
dynamitin. Genes Cells 15.1145-57 (2010)

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

90

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

Yoshiki SASAI M.D., Ph.D. ( Deputy Chief Hitoshi NIWA M.D., Ph.D.)

Human Stem Cell Technology Unit
Yoshiki SASAI M.D., Ph. D.
Stem cells – both embryonic and somatic-can be challenging to study and manipulate
in vitro. The Division of Human Stem Cell Technology Unit was established to provide afull spectrum of support services to labs within the CDB and throughout Japan with aninterest in using human embryonic stem cells (hESCs) and their derivatives in their research. The unit provides expertise, training, and support in hESC culture, maintenance,
distribution and management, for users in the life sciences community. We seek to contribute to achieving the goals of translational research, in line with the CDB mission toestablish a solid scientific foundation for regenerative medicine.

Staff
Unit Leader

Yoshiki SASAI
Deputy Unit Leader

Masatoshi
OHGUSHI

Research Specialist

Hiroyuki KITAJIMA
Research Scientist

Technical Staff

Michiru
MATSUMURAIMOTO
Maki MINAGUCHI
Eriko SAKAKURA
Company-SponsoredResearch Trainee

Hidetaka SUGA

Taisuke
KADOSHIMA

Collaborative
Scientist

Part-Time Staff

Tokushige NAKANO

Yoshinori NAKAI

Publications
Ohgushi, M., et al. Molecular Pathway and Cell
State Responsible for Dissociation-Induced
Apoptosis in Human Embryonic Stem Cells. Cell
Stem Cell 7, 225-239 (2010)
Nagase T, et al. Pericellular matrix of deciduaderived mesenchymal cells: a potent humanderived substrate for the maintenance culture ofhuman ES cells. Dev Dyn 238 1118-1130 (2009)

Four-dimensional Tissue Analysis Unit
Yoshiki SASAI M.D., Ph. D.
Recent advances in stem cell technology have enabled the generation of various potentially medically useful cell types from ES and iPS cells, but the extent to which suchcells mimic their in vivo function when plated on culture dishes is limited. The Four-dimensional Tissue Analysis Unit seeks to develop new approaches to cell culture that willallow for more realistic in vitro recapitulation through the formation of three-dimensional
tissue from stem cells. We will establish efficient 3D culture of ES cell-derived brain andretinal tissues, and develop cutting-edge live imaging technologies and optic devices forthe 4D analysis of large tissues. We also support and work with users of these technologies within and outside the CDB.

Staff
Unit Leader

Yoshiki SASAI

Deputy Unit Leader

Mototsugu EIRAKU

Publications
Danjo T, et al. Subregional specification of embryonic stem cell-derived ventral telencephalictissues by timed and combinatory treatment withextrinsic signals. J Neurosci 31.1919-33 (2011)
Eiraku M, et al. Self-organized formation of polarized cortical tissues from ESCs and its active
manipulation by extrinsic signals. Cell Stem Cell
3.519-32 (2008)

Science Policy and Ethics Studies Unit
Douglas SIPPThe field of stem cell research has been subject to legal, social, and ethical tensions
across a broad range of issues, from the research use of human embryos to the optimization of pathways for the translation of basic research into clinical applications. We
seek to compare different science policy approaches to these issues and identify regulatory frameworks best suited to the development and promulgation of stem cell applications. We will further explore approaches to the clinical translation of human stem cellresearch.

Staff
Unit Leader

Douglas SIPP

Publications
Gold standards in the Diamond Age: The commodification of pluripotency. Sipp, D. Cell Stem
Cell 5 360 – 363 (2009)
Sipp, D. Stem cells and regenerative medicine on
the Asian horizon. Regenerative Medicine 4 911–
918 (2009)91

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

92

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

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

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

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

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

Kobe Institute Administrative Structure
Kobe Research Promotion Division

Planning
Finance
General Affairs
Human Resources
Facilities
Computer Networks
Office for Research Communications
Library
Kobe Safety Center

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

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

94

Institutional Review Board

2010 CDB Budget
Overhead andadministration

Personnel

1,623

1,353

Total: 4,195(million yen)

Research expenses

Center for
Molecular Imaging Science
The Center for Molecular Imaging Science (CMIS) was establishedin October 2008 as an expansion of the Molecular Imaging Research Program, which was launched by RIKEN in July 2005. Molecular imaging is the only non-invasive technique for quantitative
monitoring of changes in concentration or distribution of target
molecules in living organisms. This is made possible through the
integration of multiple fields, including chemistry, physics, molecular biology, pharmaceutical science, medical science, engineering,and computer science. Our Center brings together researchers
from these diverse areas to work on translational projects thatspan basic research, such as compound design, and the development of instruments, animal research, and clinical research.
This collaborative strategy makes our institution very unique within
the Japanese research system.

1,219
In additional to the dedicated funds outlined above, individuallabs and investigators are encouraged to compete for externalfunding as well, from sources such as the Japan Society for the
Promotion of Science (JSPS), the Japan Science and Technology Agency, and other governmental and private sources. Theseex ternal funds represent a significant component of theCenter’ s total funding every year, and were particularly significant in the fiscal year ending March 2010, representing an additional 2.5 billion yen in research, facilities, and operating funds.

Private industry

RIKEN internal grants

115

6Foundations, etc.

Other government
agencies

16

122

Total: 2,500

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

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

MEXT direct
funding

2,241(million yen)2010 CDB Staff
Laboratory heads
Deputy unit leaders
Research scientists
Research associates
Technical staff
Assistants
Visiting scientists
Student trainees
Part-time staff
Research Promotion Division
Other

28
2
118
7
107
24
82
44
32
52
22

Total

518

95

2010 CDB Symposium

Frontiers in Organogenesis
March 23 – 25, 2010

The CDB hosted its eighth annual symposium, “Frontiers in Organogenesis” on March 23 – 25.
The three-day program featured a wide range of oral presentations organized into sessions focused on themes relating to the patterning,
imaging, evolution, and dynamics of organogenic phenomena. In addition to the 25 talks, each day included a poster session in whichmore than 90 poster presenters discussed their work.
The CDB symposium series, launched in 2003, was established as a forum for addressing diverse aspects of developmental biology andthe mechanisms of regeneration and aims to promote the free, timely and borderless exchange of research achievements.

Session 1

Session 3

Naoto Ueno (National Institute for Basic Biology, Japan)
Stefano Piccolo (University of Padova, Italy)
Masatoshi Takeichi (RIKEN CDB, Japan)

Jukka Jernvall (University of Helsinki, Finland)
Shigeru Kuratani (RIKEN CDB, Japan)
Neil H. Shubin (University of Chicago, USA)

Session 2

Session 4

Atsushi Miyawaki (RIKEN Brain Science Institute, Japan)
Scott E. Fraser (California Institute of Technology, USA)
Didier Stainier (University of California, San Francisco, USA)
Joachim Wittbrodt (University of Heidelberg, Germany)

Ryoichiro Kageyama (Kyoto University, Japan)
Oliver Pourquié

96(Howard Hughes Medical Institute,
Stowers Institute for Medical Research, USA)

Hiroyuki Takeda (The University of Tokyo, Japan)
Yukiko Gotoh (The University of Tokyo, Japan)
Yoshiki Sasai (RIKEN CDB, Japan)

2011 CDB Symposium

Epigenetic Landscape in
Development and Disease
March 14 – 16, 2011
The ninth annual symposium “Epigenetic Landscape in Development and Disease” will be held on March 14 – 16, 2011
in the CDB Auditorium. We plan to discuss the question of to
how extract an epigenetic landscape from the ever-growing
set of data on epigenetic states. By facilitating communication between the leaders in these fields and promising
younger scientists at a collegial forum, we hope to create aunique atmosphere that provides a comprehensive perspective on the current knowledge of the dynamics of organogenesis, leading to the generation of fresh perspectives andinteractions.

Invited Speakers
C. David Allis

The Rockefeller University, USA
Session 5

Benoit G. Bruneau(Gladstone Institute of Cardiovascular Disease, USA)

Toshihiko Ogura (Tohoku University, Japan)
Elazar Zelzer (Weizmann Institute of Science, Israel)
Andrew McMahon (Harvard University, USA)

Adrian Bird

Wellcome Trust Centre for Cell Biology, UK

Joseph Ecker

The Salk Institute, USA

Klaus H. Hansen

University of Copenhagen, Denmark

Kristian Helin

University of Copenhagen, Denmark

Session 6

Rudolf JaenischBrigid L.M. Hogan (Duke University Medical Center, USA)
Mark A. Krasnow

Thomas Peter Jenuwein(Duke University Medical Center, Stanford University School of Medicine
and Howard Hughes Medical Institute, USA)

Christopher V.E. Wright (Vanderbilt University, USA)
Hideki Enomoto (RIKEN CDB, Japan)
Cliff Tabin (Harvard Medical School, USA)Whitehead Institute for Biomedical Research and MIT, USA
Max-Planck Institute of Immunobiology, Germany

Tetsuji Kakutani

National Institute of Genetics, Japan

Minoru S.H. Ko

National Institute on Aging, NIH, USA

Avi Ma’ayan

Systems Biology Center New York, USA

Jun-ichi Nakayama
RIKEN CDB, Japan

Hitoshi Niwa
RIKEN CDB, Japan

Stuart H. OrkinDana-Farber Cancer Institute, USA

Nikolaus Rajewsky

Max-Delbrück-Center for Molecular Medicine, Germany

Mark Siegal

New York University, USA

Austin Smith

Wellcome Trust Centre for Stem Cell Research, UK

Hiroki R. Ueda
RIKEN CDB, Japan

Kiyoe Ura

Osaka University, Japan

Michiel Vermeulen

University Medical Centre Utrecht, The Netherlands

Emma Whitelaw

Queensland Institute of Medical Research, Australia

Kazuo Yamagata
RIKEN CDB, Japan

Richard A. YoungWhitehead Institute for Biomedical Research and MIT, USA

97

CDB Seminars
The RIKEN CDB makes special efforts to provide a full and diverse series of invited seminars by scientists from around theworld. To date, the Center has hosted more than 500 such talks, in addition to numerous meetings, distinguished lectures, internal forums, and colloquia. The following speakers presented CDB Seminars in the period from January to December 2010.

98

date

title

speaker

01-07

Stoichiometry of the integrin adhesion complex in Drosophila tissues

Yoshiko INOUE

01-13

Regulation of VEGF-induced angiogenesis by receptor endocytosis

Masanori NAKAYAMA

01-15

Gamete activation and interactions required for reproductive success in C. elegans.

Andrew SINGSON

01-20

Using stem cells and reprogramming to study ALS

Kevin EGGAN

01-21

Mechanisms of musculoskeletal patterning and integration during craniofacial development

Rich SCHNEIDER

01-25

Chicken embryonic stem cells (cESC) as a non-mammalian embryonic stem cell model

Bertrand PAIN

01-25

Role of cell death for completion of organogenesis within developmental time window

Erina KURANAGA

01-26

Molecular and cellular mechanisms of C. elegans Q neuroblast development

Guangshuo OU

01-28

Primary contribution to zebrafish heart regeneration by a subpopulation of cardiomyocytes

Kazu KIKUCHI

02-15

Targeted capture and next-generation sequencing identifies TPRN, encoding TAPERIN, as
the mutated gene in nonsyndromic human deafness DFNB79

Thomas FRIEDMAN

03-03

Primate pluripotent stem cells: Bridging gaps in scientific knowledge between mice andhumansGerald SCHATTEN

03-04

Regulation of Rap2A by the ubiquitin ligase Nedd4-1 controls neurite development

Hiroshi KAWABE

03-15

FGF regulation of cortical patterning

John RUBENSTEIN

03-18

Cell polarity regulator Pard6b is essential for trophectoderm formation in the preimplantation mouse embryo

Vernadeth B. ALARCON

03-18

Molecular analyses of mouse mesoderm formation and axial elongation morphogenesis
using embryonal carcinoma cells

Yusuke MARIKAWA

03-19

Translational control of the self-renewal versus differentiation decision in the germ line

Rafal CIOSK

03-26

Identification and analysis of novel Spemann/Mangold organizer genes

Heiko LICKERT

04-01

Therapeutic and research potential of human stem cells: How GE sees the future of stem
cells

Stephen MINGER

04-02

TRIOBP: a novel actin bundler that generates resilient rootlets of inner ear hair cell stereocilia

Shin-ichiro KITAJIRI

05-07

Behind the scenes of scientific publishing

Katherine BROWN

05-13

Differential metabolomics for assessment of N-Acetyl-L-Cysteine pretreatment in strenuous
exercise: A quantitative Model of oxidative stress inhibition for fatigue reduction

Philip Britz-McKIBBIN

05-17

Tumor cell dormancy and recurrence: a view from EMT

Hisataka SABE

05-18

Microtubules break symmetry in C. elegans zygotes by protecting PAR-2 from corticalexclusion by PKC-3 kinase

Fumio MOTEGI

date

title

speaker

06-17

Rules for enteric nervous system assembly: Biological and Mathematical Insights

Don NEWGREEN and
Kerry LANDMAN

06-24

FGF signaling during avian gastrulation and mesoderm lineage diversification

Parker B. ANTIN

06-25

Wnt signals during stem cell self-renewal and tissue repair

Roel NUSSE

06-28

Developmental roles of Dchs1-Fat4 signaling in mice

Philippa FRANCIS-WEST

07-07

Wnts and Fgfs, via AKT and MAPK, direct discrete aspects of trigeminal placode development

Claire Ann CANNING

07-20

Exploring the creation of a tissue engineered trachea: Keeping our eyes towards the clinicalworld

Koji KOJIMA

07-29

Basement membrane movement breaks tissue boundaries during uterine-vulval attachment in C.elegans

Shinji IHARA

07-30

Evolution of a novel behavior mediated by the lateral line system adapts blind cavefish tolife in darkness

Masato YOSHIZAWA

08-02

Chemotaxis in gradients: How a cell knows when to move and turn

Arthur MILLIUS

08-03

Dormancy in normal and malignant stem cells

Andreas TRUMPP

08-23

All paths lead to the thymus: A cross-species genetic analysis of Hoxa3 function.

Nancy MANLEY

08-23

Fate mapping studies reveal that adult microglia derive from primitive macrophages

Florent GINHOUX

08-23

Human artificial chromosome meets stem cells

Mitsuo OSHIMURA

09-01

Pattern formation in enteric nervous system development: Building a complex nervous
system and human disease mechanisms

Robert O. HEUCKEROTH

09-29

Tracking stem cells at the single cell level: New tools for old questions

Timm SCHROEDER

10-04

Derivation of scalable vasculogenic precursors from human pluripotent stem cells during
the onset of vasculogenesis

Ayelet DAR-OAKNIN

11-04

Integrin activation and cytoskeletal remodeling mediated by LIM domain proteinsFrieder SCHOECK

11-08

Cortical representation of olfactory bulb input revealed by retrograde mono-transsynaptic
labeling

Kazunari MIYAMICHI

11-11

Asymmetric cell division and tumorigenesis in Drosophila neural stem cell lineages

Juergen A. KNOBLICH

11-15

Stepwise assembly of a tissue-specific network in the vertebrate brain

Manuel IRIMIA

12-01

DnaA and the timing of chromosome replication in E. coli: A systems biology modeling approach.

Marco Cosentino
LAGOMARSINO

12-09

Regulation of endoplasmic reticulum stress in Drosophila

Hyung Don RYOO

12-13

Extensive regeneration ability in Amphioxus (Branchiostoma lanceolatum) provides insight
into the evolution of chordate regeneration processes.

Ildikó SOMORJAI

12-21

Nuage: Birth and beyond of germline piRNA

Toshie KAI

99

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

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

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

100

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

Tsukuba InstituteContents

Message from the Director

2

Organizational Chart

4

2010 at the CDB

6News Highlights 1

10Itabashi Branch
1-7-13 Kaga, Itabashi, Tokyo 173-0003
Tel: +81-3-3963-1611 Fax: +81-3-3579-5940

Carina HANASHIMA
Morphogenetic Signaling

25

Shigeo HAYASHI
Vertebrate Axis Formation

26

Masahiko HIBI
Sensory Circuit Formation

Wako Institute24

Hideki ENOMOTO
Neocortical Development

Center for Intellectual Property Strategies
RIKEN Next-Generation Supercomputer R&D Center
XFEL Project Head OfficePublic Relations Office23

22

Shinichi AIZAWA
Neuronal Differentiation
and Regeneration

RIKEN Headquarters

Advanced Science Institute
Brain Science InstituteNishina Center for Accelerator-Based Science
RIKEN Innovation Center
RIKEN Biomass Engineering Program
Computational Science Research Program
2-1, Hirosawa, Wako, Saitama 351-0198
Tel: +81-48-462-1111 Fax: +81-48-462-1554

LabsVertebrate Body Plan

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

27

Tokyo Liaison Office2311, 23th floor, Fukoku Seimei Building,
2-2, Uchisaiwaicho 2-chome, Chiyoda-ku, Tokyo 100-0011
Tel: +81-3-3580-1981 Fax: +81-3-3580-1980

RIKEN Advanced Institute for Computational Science
7-1-26 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-940-5555 Fax: +81-78-304-4956

Takeshi IMAI
Cell Lineage Modulation

28

Toru KONDO
Evolutionary Morphology

29

Shigeru KURATANI
Sensory Development

30

Raj LADHER
Cell Asymmetry

31

Fumio MATSUZAKI
Yokohama Institute

RIKEN Center for
Developmental Biology

2010
AnnualReport

RIKEN
http://www.riken.jp/

The RIKEN Center for Developmental Biology waslaunched in April 2000 under the auspices of theMillennium Project research initiative, which wasestablished to drive research in areas of vital importanceto both Japan and the world in the 21st century. The
challenges of aging society were among the issues
addressed, and developmental biology was included
within this remit as a scientific field with the potential tocontribute to the development of regenerative medicine.
Located in Kobe, Japan, the Center’s research programis dedicated to developing a better understanding offundamental processes of animal development at the

RIKEN is a publicly funded researchorganization established to conduct
comprehensive research in science andtechnology, and to disseminate the resultsof its scientific research and technologicaldevelopments. RIKEN carries out basic
and applied research in a wide range offields, including physics, chemistry,
biology, medical science, and engineering.
RIKEN was founded in 1917 as a private
research organization, Rikagaku
kenkyuusho (The Institute of Physical andChemical Research). In 2003, the Institutewas reorganized as an Independent
Administrative Institution under the Ministryof Education, Culture, Sports, Science andTechnology (MEXT), and has continued toengage in wide-ranging research activities
spanning the basic and applied sciences.

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

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

Kobe InstituteCenter for Developmental Biology
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-306-0111 Fax: +81-78-306-0101
Center for Molecular Imaging Science
MI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku,
Kobe, Hyogo 650-0047
Tel: +81-78-304-7111 Fax: +81-78-304-7112

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

RIKEN Center for Developmental
ental Biology

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

2010 Annual Report

On The CoverSpontaneously formed conjoined twin chicken
embryos, stained for axial (Brachyury) and paraxial(Paraxis) mesoderm markers

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

RIKEN Center for Developmental Biology

2010 Annual Report