RIKEN Center for Developmental Biology

2011 Annual ReportRIKEN Center for
Developmental Biology

2011
Annual Report

Contents
Message from the Director

2

Organizational Chart

4

2011 at the CDB

6News Highlights 1

10

Labs
Vertebrate Body Plan

24

Shinichi AIZAWA
Neuronal Differentiation
and Regeneration

25

Hideki ENOMOTO
Neocortical Development

26

Carina HANASHIMA
Morphogenetic Signaling

27

Shigeo HAYASHI
Sensory Circuit Formation

28

Takeshi IMAI
Histogenetic Dynamics

29

Erina KURANAGA
Evolutionary Morphology

30

Shigeru KURATANI
Sensory Development

31

Raj LADHERThe 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News Highlights 2

32

Labs
Cell Asymmetry

47
48
49
50
51

70

Physical Biology

71

Developmental Genomics

72

Retinal Regeneration

73

Masayo TAKAHASHI
52

Masaki OKANO
Organogenesis and Neurogenesis

Early Embryogenesis

Asako SUGIMOTO

Hitoshi NIWA
Mammalian Epigenetic Studies

69

Tatsuo SHIBATA

Takashi NISHIMURA
Pluripotent Stem Cell Studies

Cell Fate Decision

Guojun SHENG

Shin-Ichi NISHIKAWA
Growth Control Signaling

68

Hitoshi SAWA

Jun-ichi NAKAYAMA
Stem Cell Biology

Embryonic Induction
Hiroshi SASAKI

Akira NAKAMURA
Chromatin Dynamics

54

Labs
46

Fumio MATSUZAKI
Germline Development

News Highlights 3

Cell Adhesion and Tissue Patterning

74

Masatoshi TAKEICHI
52

Yoshiki SASAI

Systems Biology

75

Hiroki R. UEDA
Genomic Reprogramming

76

Teruhiko WAKAYAMA

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.

News Highlights 478

Supporting Laboratories

92

RIKEN Kobe Institute100

CDB Symposium

102

CDB Seminars

104

About RIKEN

106

RIKEN Campuses

107

2

Message from the Director
History will remember 2011 as a year in which Japan was confronted with an unprecedented series of disasters, natural andmanmade. Our research center was fortunately spared any physical damage, but like all of Japan, we are still coming to termswith the impact of these events. I would like to being this year’s end reflection by expressing our deepest gratitude to all of themany people who reached out to Japan in its moment of need, and particularly to the many scientists and research organizations that made such generous offers of help and support in the days following the Great East Japan Earthquake.
Our own achievements and challenges over the past year here at the RIKEN Center for Developmental Biology pale in significance against this backdrop, but it seems clear that the most effective way for us to contribute to the nation’s rebuilding efforts
is to continue in our scientific endeavors and support the greater community. Our research programs have certainly continued
to open new insights into a range of developmental phenomena, with novel findings into cell signaling, migration, asymmetry,and morphological change that have honed our appreciation for the complexity of the embryo. Stem cell research was a particular highlight in 2011, with CDB labs revealing cell culture techniques capable of inducing the self-organization of embryonicstem cells into tissue-like structures closely resembling the optic cup and the anterior pituitary, as well an assay using patientspecific induced pluripotent stem cells as a platform for drug validation and disease modeling in retinitis pigmentosa.
We continue to recruit capable young PIs into the center as part of our commitment to supporting the careers of talented scientists in a research-intensive environment; in 2011, we welcomed a new lab led by Erina Kuranaga that will focus on histogenetic
dynamics to the CDB. We also saw a number of our earliest team leaders move on to new positions in academia and government research institutes, a sign of the continuing success of our emphasis on fixed-term commitments to promising young
scientists in the field. With two new labs scheduled to open in early 2012, we remain on a course of continuous self-renewal,
chasing the horizons in these rapidly evolving fields of science.
We have also maintained our commitment to outreach and interaction, both with the international scientific community and thelay public. This fall, we hosted our largest Open House event, in conjunction with the other RIKEN Kobe research centers, giving over 1,000 visitors the opportunity to speak with our scientists and learn more about the CDB’s work. We were delighted
to host a delegation led by the Japanese Minister of Education, Culture, Sports, Science and Technology in December, during which he visited labs and spoke with a number of our research leaders. As in previous years, we also organized a numberof popular hands-on programs for local high school students and teachers, designed to stimulate and reward the curiosity oflearners and educators alike.
Our location in the city of Kobe remains a key factor in our ability to contribute to and conduct translational and cross-disciplinary studies. Not only has RIKEN’s presence in the city expanded to include four life sciences research institutions and theworld’s fastest supercomputer, but in July Kobe’s new municipal medical center opened just a short walk from the CDB, joining
the more than 200 public and private biomedical organizations that make their home on Port Island.
And so, we look back proudly on 2011 as a year of successes for the CDB, but with the recognition that these were overshadowed by events of greater historical magnitude. We could not have sustained this record of achievement without the dedicatedwork of our many investigators, student trainees, and administrative staff, or without the encouragement and well wishes of ourcolleagues around the world. And so I close, as I opened, with a message of gratitude to all who have supported us in our efforts over the past year, and wish you all the best for the one to come.

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.

Advisory Council
The CDB Advisory Council (DBAC) convenes regularly to review theCenter’s performance and make recommendations on the directionof 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).
The ten-member Council comprises top international scientists working in developmental biology, stem cells and related fields.

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.

Austin Smith

Haifan Lin

Christopher Wylie

Toshio Suda

University of Cambridge, UK
Cincinnati Children's Hospital
Medical Center, USA

Margaret Buckingham
Institut Pasteur, France

Patrick Tam

University of Sydney, Australia

Stephen Cohen

Temasek Life Sciences
Laboratory, Singapore

Yale university, USA
Keio University, Japan

Yoshimi Takai

Kobe University, Japan

Ryoichiro Kageyama
Kyoto University, Japan

Hiroshi Hamada

Osaka University, Japan

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
Hideki ENOMOTO M.D., Ph.D.

Neocortical Development
Carina HANASHIMA Ph.D.

Sensory Circuit Formation
Takeshi IMAI Ph.D.

Histogenetic Dynamics
Erina KURANAGA Ph.D.

Sensory Development
Raj LADHER Ph.D.

Germline Development
Akira NAKAMURA Ph.D.

Chromatin Dynamics
Jun-ichi NAKAYAMA Ph.D.

Growth Control Signaling
Takashi NISHIMURA Ph.D.

Mammalian Epigenetic Studies
Masaki OKANO Ph.D.

Embryonic Induction
Hiroshi SASAKI Ph.D.

Cell Fate Decision
Hitoshi SAWA Ph.D.

Early Embryogenesis
Guojun SHENG Ph.D.

Developmental Genomics
Asako SUGIMOTO Ph.D.

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

Genomic Reprogramming
Teruhiko WAKAYAMA Ph.D.

Animal Resources and Genetic Engineering
Shinichi AIZAWA Ph.D.

Genetic Engineering Unit
Shinichi AIZAWA Ph.D.

Animal Resource Unit
Kazuki NAKAO Ph.D.

Electron Microscope
Shigenobu YONEMURA Ph.D.

Bio-imaging Laboratory
Shigeo HAYASHI Ph.D.

Optical Image Analysis Unit
Yuko KIYOSUE

Genomics
Fumio MATSUZAKI Ph.D.

Genome Resource and Analysis Unit
Fumio MATSUZAKI Ph.D.

Functional Genomics Unit
Hiroki R. UEDA M.D., Ph.D.

Proteomics
Shigeo HAYASHI Ph.D.

Mass Spectrometry Analysis Unit
Akira NAKAMURA Ph.D.

Division of Human Stem Cell Technology
Yoshiki SASAI M.D., Ph.D.

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

Four-dimensional Tissue Analysis Unit
Yoshiki SASAI M.D., Ph.D.

Science Policy and Ethics Studies Unit
Douglas Sipp

2011 Highlights

New Team Leader Erina Kuranaga
joins the CDB
Erina Kuranaga, the new head of the Laboratory for Histogenetic
Dynamics, studies the roles played signaling pathways that underlie
programmed cell death in tissue development. The Kuranaga lab
joined the Center in January.

Examining emu embryology
Scientists from the Labs of Early Embryology andSensory Development created a stage series of emu
embryogenesis, showing parallels to that of the more
widely studied chick in that while these two very different
bird species proceed through similar embryonic stages, anumber of differences in timing and pace set them on the
widely diverging courses manifested in their adult forms.

Project leader Hiroki R. Ueda wins
JSPS Prize
Hiroki R. Ueda, leader of the CDB Center Director’s Project on Systems Biology,
was awarded the prestigious JSPS Prize by the Japan Society for the Promotionof Science for his studies of the design principles underlying the mammalian
circadian clock system. Ueda takes a systems biology and quantitative approachto developing a better understanding of the mechanisms at work in regulating theapproximately 24-hour circadian cycle operating in diverse forms of life.

6

Course in multicolor fluorescent insitu hybridization
In February, the CDB organized a course on multicolor fluorescencein situ hybridization (FISH) for the nuclear analysis of human stem
cells. This program was the latest in a series of training workshops
coordinated by the Division of Human Stem Cell Technology.

CDB Symposium:
Epigenetic Landscape in
Development and Disease
The RIKEN CDB hosted its ninth annual symposium,“Epigenetic landscapes in development and disease” onMarch 14–15. More than 180 people gathered to share anddiscuss the latest developments in the study of epigenetics
as it relates to ontogeny and pathology.

Self-organized optic cup fromES cells
Researchers from the Four-dimensional Tissue Analysis Unit andthe Laboratory for Neurogenesis and Organogenesis described how
mouse embryonic stem cells (ESCs) are able to differentiate and selfassemble assemble into an optic cup-like structure.

7

CDB scientists awarded
MEXT Prizes
Group Director Shigeru Kuratani and Yasuhide Ohinata,a research scientist in the Pluripotent Stem Cell Studies
lab, were honored with awards from the MEXT Ministerfor their accomplishments in scientific research. Kuratani
was noted for his career of achievements in comparative
vertebrate evolutionary developmental biology, while
Ohinata was recognized for his contributions to theunderstanding of germline formation in the early embryoand its recapitulation in vitro.

Hagfish vertebra-likestructures found
The Laboratory for Evolutionary Morphogenesis
demonstrated the presence of cartilaginous vertebralike elements and analyzed their anatomical features as
genuine vertebrae in a hagfish species, solidifying thephylogenetic position of hagfish as true cyclostomes.

Summer school for highschool students
The CDB held its annual summer school for local high school students in earlyAugust. The pair of one-day courses featured lectures, lab visits, and handson experiments on fluorescent labeling. After staining the cells, the students
watched their creations under a fluorescence microscope, enabling them toobserve phenomena, such as cell division.

Biology at the Interface
The CDB was delighted to co-organize and host themeeting “Cellular Development: Biology at the Interface”
from September 29 to October 1 to commemorate thetenth anniversary of the publication of Developmental
Cell, in cooperation with Cell Press, and Fondation Ipsen,with support from the Days of Molecular Medicine Global
Foundation.

8

2011 Highlights

Hands-on course for highschool teachers
A course in development for teachers of high school biology,
organized by the Office for Research Communications (ORC),
was held at the CDB in October. This course is held annuallyin conjunction with the Japanese Society of Developmental
Biologists, and gives groups of teachers an opportunity tolearn hands-on techniques in embryology, using methods andmaterials which they can adapt to their own classrooms.

Open HouseThe Center held its annual visitors’ day activities in November as part of the RIKEN
Kobe Institute Open House event, which featured talks, science-themed crafts andgames, demonstrations and open laboratories highlighting the research being doneat the Institute’s research centers. More than 1,200 visitors from around the Kobe
area participated in the event.

ES cells give rise to organized pituitary
tissue in vitro
A collaboration between the Division of Human Stem Cell Technology and the Laboratory
for Organogenesis and Neurogenesis made a new breakthrough in self-organizedtissue differentiation, steering mouse ESCs to give rise to tissue closely resembling thehormone-secreting component of the pituitary, known as the adenohypophysis, in vitro.

MEXT Minister visits Center
Masaharu Nakagawa, Japan’s Minister of Education, Sports,
Culture, Science and Technology (MEXT) paid a visit to the RIKEN
Center for Developmental Biology in December to hear about
research at a number of CDB labs. He showed especial interest in
the leading work being done by stem cell researchers at the Center,and their potential for applications in regenerative medicine.

9Key factor combines day andnight to hold back morning
As in manmade timepieces, the movements ofthe genetic clockworks that lie behind circadiancycles involve a remarkable amount of complexity. The mammalian circadian clock, for example, is thought to arise from the interactionsof around 20 transcription factors with specific
DNA sequences associated with morning, day,and night expression. Existing models of thisgenetic network can readily explain the basis
for the day and night activities, but the mechanism underlying morning expression remains
incompletely understood. It is thought that delayed negative feedback exerted by the morning (E/E’ box) inhibitor Cryptochrome 1 (Cry1),
which is itself expressed in evening, plays animportant role in keeping the biological clockon time. But just how it achieves this effect is
unknown.
Maki Ukai-Tadenuma and Rikuhiro G. Yamadaof the Laboratory for Systems Biology (Hiroki R.
Ueda, Project Leader), along with colleagues in
the Universities of Memphis (USA) and Fribourg(Switzerland), now report how delayed feedback repression is a key factor in mammalianclock function. Published in Cell, this workshows the role of Cry1 as mediator of delayed
negative feedback repression and fleshes out
the current understanding of the circadian circuitry.

Fumio
MATSUZAKI,Atsushi KITAJIMA
Rikuhiro
G. YAMADA,
Maki UKAI-TADENUMA

10

The team began by looking into the basis forthe evening expression of Cry1 using reporter
genes coding for the luciferase protein to detect transcriptional activity, and found that theCry1 promoter region induces the expressionof genes carrying the daytime expression motif.
A closer look at Cry1’s DNA revealed that its
intronic region contains a separate sequencethat induces nighttime clock genes. They next
stitched together a construct including thesepromoter and intron regions, ran another reporter assay to observe its behavior, and foundthat its expression switched on in circadianevening, suggesting that this in-between expression time is a result of the combination of
day and night regulatory elements. To test thismodel, the team tried to rescue clock functionin cells with homozygous deletions of both
Cry1 and Cry2 by inducing the evening expression of exogenous Cry1. They found that while©2011 Elsevier B.V.
Ukai-Tadenuma M, et al. Delay in feedback repression by cryptochrome 1 isrequired for circadian clock function. Cell 144.268-81 (2011)

the Cry1 promoter region alone was ineffective, when the promoter and intron regions were used inconjunction, the gene’s circadian rhythmicity was restored.
Using this same set-up, Ukai-Tadenuma and Yamada next tried changing the onset time of Cry1
expression, and found that as expression neared midday, meaning that the normal phase delay was
reduced, the amplitude of circadian oscillations grew smaller, in line with predictions. Similarly, prolonging the delay of exogenous Cry1 expression caused an increase in the length of the restored cycle.
The team’s findings were recapitulated by a relatively simple phase vector model, which not only successfully reproduces the findings from the current study, but numerous other aspects of the circadianclock network as well.
“In 1990, Paul Hardin at Texas A&M pointed out the importance of delayed feedback repression in
biological clocks, but it has taken 21 years to work out the mechanism behind it,” says Ueda. “We willcontinue exploring whether the current minimal transcriptional network model is complete, or whether
new regulatory systems remain to be discovered.”A minimal circuit model for the
mammalian circadian transcriptional
network. Morning (E/E’box), day (Dbox) and night (RRE) DNA elements
lie at the heart of the circadianclock. Solid lines indicate activating(green) and inhibitory (pink) interactions between clock gene types.
The dotted lines indicate how Cry1
represses mor ning expression
through the combined action of day
and night sequences.

11

Emus emulate chickdevelopmental stages

Hiroki NAGAI, Siu Shan MAK, Guojun SHENG, Raj LADHER
Birds are useful models in developmental biology, given their large, external eggs and an array ofclassic embryology techniques, but the study of avian development has been dominated by a single
species, the chicken Gallus gallus. While a few other bird varieties have been studied for purposesof comparison, these have all been from the “modern” species (neognaths), such as quail, duck andpheasant. A number of more basal bird species (palaeognaths), including ostrich, rhea, kiwi, and emu
survive, but their development has remained largely unstudied.
In a report published in Developmental Dynamics, Hiroki Nagai of the Laboratory of Early Embryogenesis (Guojun Sheng, Team Leader) and colleagues from the same lab as well as the Laboratory for
Sensory Development (Raj Ladher, Team Leader) provided a comparative description of the development of the emu, Dromaius novaehollandiae, with that of the better-known chick. The team found thatwhile the two birds proceed through similar embryonic stages, a number of differences in timing andpace set them on the widely diverging courses manifested in their adult forms.
Nagai’s approach centered on identifying emu equivalents for Hamilton-Hamburger stages in the chick.
The Hamburger-Hamilton (HH) system uses definitive morphological characteristics to determine thestate of development, and is the gold standard in staging chick embryos. As a general rule, emus take
2–3 times longer than chicks to reach the same HH stage.
The early development of the emu resembles that of the chick until HH7. During subsequent stages
in which somites form, however, the emu forms a greater number of these structures than the chick.
The emu somitogenic period was calculated to be 100–110 min, slightly longer than the 90 minutes inchick embryos.

12©2011 John Wiley & Sons, Inc.
Nagai H, et al. Embryonic development of the emu, Dromaius novaehollandiae.
Dev Dyn 240.162-75 (2011)

The forelimbs in adult emus are diminutive, which is reflected in development as well. The forelimbbuds form and undergo initial patterning, but these appendages fail to grow apace with the rest of theembryo body, including the hindlimbs. Interestingly, adult emus also have fewer forelimb digits thando other birds, a difference that is also observable at the stage of the limb’s patterning. Once the limb
buds have formed, the emu embryo begins a growth spurt that results in the enormous size differential
with the chick.

The emu, Dromaius novaehollandiae (left), and a comparison of emu
and chicken eggs (right)

In addition to their comprehensive morphological observations, the team looked at the expression of
a number of genes, such as Sonic hedgehog (Shh), Brachyury, and Chordin, known to be importantin early development. Expression patterns were similar to those in chick up to stage 7, when the firstsomite appears, with the single exception that Brachyury expression begins slightly later in emu.
“Since a brief description by Haswell in 1887, there have been no published studies of emu development,” says Sheng. “In staging these embryos, we learned of the high level of conservation of developmental routines across bird orders, which suggests that findings from the chick may well apply
generally to birds. That said, we also saw heterochrony in the development of specific tissues andstructures between chick and emu, so we look forward to studying these embryos in more detailthrough cell labeling, transplants, and imaging techniques.”

E m u e m b r y o s a t H H 3 +, H H 7 ,6-somite, HH18 and HH36 stagesof development

13

Modeling retinitis pigmentosa
with iPS cells

Fumio Zi-Bing
MATSUZAKI,AtsushiOKAMOTOKITAJIMA
JIN, Satoshi
Retinitis pigmentosa (RP) is a cluster of genetically determined eye disorders that cause visual defects,
such as night blindness and narrowing of the field of vision, due to progressive loss of rod photoreceptors. As many as 45 different genes have been linked to the inheritance of this disease, which suggests a diverse etiology and makes development of a standardized animal model problematic. Thus,
despite a range of clinical trials of nutritional and drug-based interventions for RP, the disease remains
untreatable. Better platforms for modeling the disease and testing drug candidates in vitro are urgently
needed.
New work by Zi-Bing Jin and colleagues in the Laboratory for Retinal Regeneration (Masayo Takahashi, Team Leader) looks to add a set of powerful new tools for those searching for treatments for
RP. In an article published in PLoS One, the team reports the generation of induced pluripotent stem
cells (iPSCs) from patients carrying mutations in several RP-associated genes, and the subsequent differentiation and characterization of rod photoreceptors from these genetically distinct, patient-derived
pluripotent cells.
After obtaining informed consent from five RP patients with distinct mutations in the RP1, RP9,PRPH2, or RHO gene, the team took samples of skin cells and used the fibroblasts as a starting point
for generating iPSCs. Using the classic reprogramming cocktail of Oct4, Sox2, Klf4, and c-Myc delivered via a retroviral vector, Jin and colleagues generated cell lines from each patient and verified theirconversion by tests for appropriate morphology, genetic and karyotypic integrity, and teratoma formation.
Using these iPSCs, the team next generated photoreceptors carrying the genetic signatures of each of
the five patient donors using a previously established stepwise protocol that steered the cells over four14©2011 Public Library of Science
Jin Z B, et al. Modeling retinal degeneration using patient-specific induced
pluripotent stem cells. PLoS One 6.e17084 (2011)

months in culture from an undifferentiated ES cell-like state through retinal progenitor, and photoreceptor precursor stages to the desired rod photoreceptor phenotype. The differentiated cells were shown
to express the rod photoreceptor marker rhodopsin at high levels, and to have similar electrophysiological function.
Interestingly, rod photoreceptor cells generated from iPSC colonies carrying RP-linked mutations
showed a tendency to degenerate, while cone photoreceptors and bipolar cells derived from the same
iPSCs were stable. The mechanisms underlying this instability turned out to be dependent on the affected gene. Rod photoreceptors generated from iPSCs with a mutation in RP9 showed evidence of
DNA oxidation, while those from iPSCs with a mutation in the gene encoding rhodopsin showed signsof stress on the endoplasmic reticulum, the site of protein synthesis.
As an initial proof-of-concept test of their suite of RPspecific rod photoreceptors in drug validation, Jin andcolleagues examined the effects of antioxidant vitamins inpreventing degeneration of these cells in vitro. Ascorbic
acid, α-tocopherol, and β–carotene have all been tested
in clinical trials as anti-oxidant therapies for RP, but none
had proved very effective. When the team tested these on
individual cells lines by treating them with one of the three
antioxidants for seven days at around the stage at which
rod photoreceptor degeneration occurs, they found that
α-tocopherol increased cell survival in the lines generated
from two patients both carrying mutations in RP9. The
same treatment was ineffective in cells from other patients, and ascorbic acid and β–carotene had no effect in any of the lines. These results, which show
the efficacy of α-tocopherol in promoting survival in RP9 rod photoreceptors, highlights the potential ofpatient-derived induced pluripotent stem cells in the study of disease mechanisms and in vitro testingof treatment approaches.

Rod cells induced from patientderived iPS cells.
The cells express rod-specific transmembrane protein, rhodopsin (red).

“Using iPSCs from cells donated by RP patients with different underlying genetic mutations, we wereable to show that rod photoreceptors generated from these cells underwent apoptosis in vitro, andshowed differing responses in a genetically determined manner to drug treatment,” says Takahashi.
“This is one of the first reports to demonstrate that patient-derived iPSCs may be useful in personalized medicine, as differential responses within a genetically diverse study group will tend to be lost in
the crowd. Future improvements in differentiation protocols, screening techniques, cost and efficiency
and the establishment of methods for isolating photoreceptors may open up new possibilities for theuse of these cells in drug screening.”

Patient-derived rod photoreceptors
undergo degeneration in vitro. The
bars show differentiation efficienciesof the iPS cells derived from fiveretinitis pigmentosa patients.

15

New role for phosphorylation inheterochromatin

Fumio MATSUZAKI,Atsushi KITAJIMA
Kyoko HIRAGAMI-HAMADA
A great many cellular processes are switched on or off by the modification of a given enzyme or otherprotein by addition of a phosphate molecule, known as phosphorylation. This regulatory activity occurs
widely in the cytoplasm, but can take place in the nucleus as well. Recent work has shown the HP1α,a protein that guides the formation of heterochromatin, a form of the DNA-protein structure know as
chromatin, is also subject to this post-translational modification, but the biological meaning of thisevent has remained unresolved.
A new study by Kyoko Hiragami-Hamada and colleagues in the Laboratory for Chromatin Dynamics (Jun-ichi Nakayama, Team Leader), working in collaboration with labs in Kobe University, Kwansei
Gakuin University, and AIST, have shown that phosphorylation of HP1α boosts its ability to bind to heterochromatin, resulting in stabilization of chromosomes. Published in Molecular and Cellular Biology,
this work opens up new insights into the interplay between protein modification and chromatin dynamics.
HP1 was first identified in Drosophila, and is now recognized as a highly conserved regulator of transcriptional repression in heterochromatin in eukaryotes. The protein has two similar binding domains:
a chromodomain (CD) region in its N-terminal region, and a chromo shadow domain (CSD) at the
carboxyl end. The CD binds to a methylated site on histone H3 (H3K9me3), and importantly the CSD
promotes binding to other HP1 proteins, which is essential to the formation and maintenance of heterochromatin. It has been suspected that HP1 may also be regulated by phosphorylation as well, but
this has never clearly been shown.
Hiragami-Hamada began by testing for binding activity between mammalian HP1 variants HP1α,
HP1β and HP1γ and H3K9me3, and found that while phosphorylation had no evident effect on the16©2011 American Society for Microbiology
Hiragami-Hamada K, et al. N-terminal phosphorylation of HP1α promotes its
chromatin binding. Mol Cell Biol (2011)

strength of this association in HP1β or HP1γ, binding of HP1α to methylated histone H3 appeared
to be markedly enhanced by the modification. She next used an electrophoresis technique known as
Phos-tag-PAGE to examine HP1α in its phosphorylated state, and found not only that multiple sites on
the protein were thus modified, but that phosphorylation increased during cell division as well.
To resolve the specific phosphorylation sites, she next used Phos-tag-PAGE in combination with targeted amino acid substitutions and identified serine 14 in the N-terminal and serine 93 in the protein’s
hinge region as major phospho-targets. Mass spectrometry revealed additional sites in serine 11–13 in
the N-terminal as well. Tests of binding affinity showed that it was the N-terminal sites, but not S93 in
the hinge, that are responsible for binding to H3K9me3 in a phosphorylation-dependent manner. Interestingly, while this modification was important for establishing the interaction, it was not required for its
stabilization.
With a better understanding of the biochemistry behind this interaction, Hiragami-Hamada next turned
to phosphorylated HP1α’s role within the cell. She found that, when phosphorylation of S14 was prevented by an amino acid substitution, HP1α accumulation at heterochromatic regions was reduced.
Similar experiments on N-terminal serines 11 to 13 showed that their phosphorylation also plays arole in targeting HP1α to heterochromatin. When the team replaced all four serines 11 through 14 with
substitute amino acids in cultured mouse cells, they observed an increase in chromosomal abnormalities. Serine 93 in the hinge region in contrast had no such effects.
“This work suggests that the chromodomain alone is not sufficient for binding the methylated histone;
the connections needs to be strengthened by phosphorylation of sites on the HP1α N-terminus, whichinterestingly contributes to chromosomal stabilization as well” says Nakayama. “We’re now curious
about how this phosphorylation is regulated, and what role, if any, it plays in serine 93.”HP1α accumulates in heterochromatic regions in wildtype cells (left),but not in cells engineered to lack
phosphorylation of the protein’s Nterminal region (right).

17

Conserved kinase protects cells
from endosomal traffic jam
Some molecules have a single, highly specificfunction in physiology, while others have amuch broader remit. Kinases in the IKK (inhibitor of nuclear factor κB (NF-κB) kinase) family
are of the latter sort, playing a variety of roles in
the immune system, cancer, and differentiation.
Previous work with a related kinase, IKKε, inDrosophila has shown that this protein regulates the cytoskeleton and cell elongation in a
number of contexts. It has yet to be shown,however, how it accomplishes this task.
A report by Tetsuhisa Otani in the Laboratoryof Morphogenetic Signaling (Shigeo Hayashi,Group Director) and colleagues, working in collaboration with the CDB Electron Microscope
and Proteomics labs, has done just that. In an
article published in Developmental Cell, thegroups show that this factor regulates shuttlingof endosomes in the tips of growing mechanosensory bristles, converting the directionof their trafficking from incoming to outward
bound.
Bristles form from single cells during the fly’s
pupal stage. A previous report had indicatedthat IKKε is expressed at the tips of thesestructures. Otani looked at the active, phosphorylated form of the molecule and foundthat it indeed accumulated at the tips of growing bristles. He next focused more closely atcytoskeletal organization in these structures inwildtype and IKKε mutants, and found that in
the mutant bristles, actin bundles were poorly
organized and frequently failed to attach totheir anchor points in the cell cortex. Transmission electron microscopy further showed thatwhile microtubules in mutants maintained theirusual orientation with the respect to the bristle,
an abnormal number of vesicles accumulated
in this space, suggesting that vesicle transport
was somehow affected.

Tetsuhisa OTANI

18

An examination of vesicle markers turned up a
promising candidate in Rab11, a recycling endosome protein known to be essential in bristle
morphogenesis. In contrast to its tendency tocluster around the tip in control animals, Rab11
was clumped in the bristle shaft in IKKε mutants. Other endosome markers also showedaberrant localization. The effect was replicatedin cultured cells, in which mutant IKKε caused
Rab11 to aggregate.

©2010©2011 Elsevier B.V.
Otani T, et al. IKKε Regulates Cell Elongation through Recycling Endosome
Shuttling. Dev Cell 20.219-32 (2011)

What then was the link between the defects in actin
bundles and endosome distribution in IKKε mutants? Looking at the timing of these events, Otani
found that Rab11 accumulation precedes the misrouting of actin, and was unaffected by various mutations that disturb the bundling of actin filaments,suggesting that IKKε’s roles in Rab11 distribution
and actin bundling are independent.
He next used photobleaching and time-lapse imaging to track the localization of Rab11 over time.
Rab11 normally moves along the bristle, shuttling
between shaft and tip in a state of dynamic equilibrium. The rate and overall directionality of this backand-forth varied, and was at its highest during bristle elongation. Looking for a mechanism, the groupexamined functional relationships between IKKε,Rab11, and the Rab11 effector, Nuf, which binds
to Rab11 and the Dynein light intermediate chain (acomponent in microtubule trafficking). What they found pointed to opposing roles for IKKε and Nuf inthe trafficking of Rab11. This antagonistic effect appears to be due to the phosphorylation of Nuf at a
specific amino acid (serine 225) by IKKε. Preliminary experiments using IKKε homologs in mammaliancells suggested that the function of this factor in the trafficking of recycling endosomes is conserved.

Activated IKKε (magenta) accumulates at the tips of growing bristles,
and actin bundles (green) run along
the long axis of growing bristles.

“In addition to their role as vehicles for cellular materials, endosomes also serve as a platform for various signaling activities in cell proliferation and immunity,” says Hayashi. “The identification of new
downstream targets of IKKε sheds light on the roles of endosomal trafficking in morphogenesis, innate
immunity, and cancer.”Electron microscopy image ofbristles19Novel factor behind ES cells’
neural default

Daisuke KAMIYA
Embryonic stem cells (ESCs) are highly regarded for their ability to give rise to the full range of cellularlineages found in the adult body, a capacity known as pluripotency. In most cases, this differentiationneeds to be steered by molecular cues that drive the stem cells’ progeny to adopt a specific fate, suchas blood, muscle or bone. But left to their own devices, ESCs tend to differentiate into neural lineages.
Indeed, in the absence of an inhibitory signal, such as BMP4, the preferred destination for ESC differentiation is neural ectoderm. This has been known for years, following demonstrations of the phenomenon in undifferentiated tissue in amphibians, but the mechanism behind this cell-intrinsic propensity
has never been explained.
Daisuke Kamiya and colleagues in the Laboratory for Organogenesis and Neurogenesis (Yoshiki Sasai,Group Director), working in collaboration with the Laboratories for Stem Cell Biology and Animal Resources and Genetic Engineering, have revealed how the nuclear protein Zfp521 is key to the default
neural fate. In an article published in Nature, the group reports that this factor is both necessary andsufficient in driving the intrinsic neural differentiation of mouse ESCs.
The study started from a GeneChip survey of mouse ES cells that had taken their first step down
the road to neural differentiation, as revealed by GFP knocked into Sox1, a neural progenitor-specificmarker. This turned up 104 genes of interest, 29 of which were even more interesting for their lack of
expression in mesodermal tissues. Of these, Zfp521 was particularly attractive as it was found to bea potent promoter of neural differentiation when added to ESCs. Forced expression of Zfp521 led toincreased expression of early neural markers such as Sox1, Sox3, and Ncad. The last of these, which
encodes N-cadherin, is normally suppressed by the neural inhibitor BMP4, but Kamiya found thatoverexpression of Zfp521 overrode this effect. In situ hybridization showed that Zfp521 expression

20©2011 Nature Publishing Group
Kamiya D, et al. Intrinsic transition of embryonic stem-cell differentiation into neuralprogenitors. Nature 470.503-09 (2011)

switches by around day 6.5 of development, and grows more pronounced in the neuroectodermal region of the epiblast, while it is absent in non-neural tissues.
To determine whether Zfp521 is truly necessary for neural differentiation of ESCs, Kamiya next
knocked down its function by short hairpin RNA, resulting in a dramatic reduction of cells expressing
neural markers. Importantly, this was not rescued by the addition of control cells, indicating that that
action was cell autonomous. When the Zfp521-knockdown ESCs were injected into blastocysts, theyfailed to contribute significantly to the rostral neural tube, indicating a failure in primary neurulation.
Wanting to know the specific window of time in which Zfp521 exerts its function, the group examined
its effects on a range of stage- and lineage-specific markers, from the earliest stages of undifferentiated ES cells, which reflects the developmental status of the inner cell mass (ICM), to various primarylineages differentiating from epiblast tissues. They found that while ICM development to epiblast was
unaffected by loss of Zfp521 function, epiblast cells themselves failed to make the transition to neuroectoderm. Chromatin immunoprecipitation (ChIP) assays revealed that Zfp521 associates with anumber of neuroectoderm-specific genes, but not with genes lined to the maintenance of pluripotency
or differentiation into other lineages, suggesting that its effect is achieved by activating neural genes
rather than inhibiting non-neural ones. This may be attributable to an interaction with a second factor,
the histone acetylase p300, with which Zfp521 also associates. The model of default neural differentiation that emerges from these findings suggests that Zfp521 functions as a transactivator of neuralspecification, autonomously steering undifferentiated ESCs to adopt a neuroectodermal fate.
“This set of findings has given us insight into the mechanisms behind how a colony of undifferentiatedcells such as ESCs can begin neural differentiation prompted early in development by Zfp521 in theabsence of countering signals, such as BMP4,” says Sasai. “What we would like to work out next iswhether this factor has similarly intrinsic effects in the epiblast as well.”Mouse ESCs overexpressing
Zfp521 in SFEB culture express
neural markers Sox1 (green) andN-cad (red) by day 5 of culture (rightpanel). Wildtype ESCs shown onleft (blue shows DAPI staining of
nuclei).

21

2011 Events
The RIKEN CDB strives to engage with the public through a variety of media, including its website, media coverage and direct interactions, such as guided tours, visitors’ day events, and other outreach activities. In addition to this work in public
engagement, the Center also organizes events for bringing scientists together outside of the laboratory environment.

Transcripts newsletter launched
The Office for Research Communications began publication of a quarterly bulletin, Transcripts, to keep staff informed about recent news and upcoming events. The newsletter contains information on new hires, important calendar events, RIKEN-wide programs, and eveninteresting spots in and around the city of Kobe.

Junior high school student internships
RIKEN Kobe Institute welcomed four students from local junior high schools affiliated with Kobe University as part ofa weeklong program to introduce participants to work in various settings. The young trainees spent three days at the
CDB, learning basic scientific techniques in the demo lab maintained by the Office for Research Communications,and visiting the Laboratory for Retinal Regeneration.

2011 Open HouseThe RIKEN Center for Developmental Biology held its annual visitors’ day activities on November 5 as part of the
RIKEN Kobe Institute Open House event, which featured talks, science-themed crafts and games, demonstrations
and open laboratories highlighting the research being done at the Institute’s research centers. More than 1,200 visitorsfrom around the Kobe area participated in the event. Group Director Yoshiki Sasai gave a morning talk on the organforming potential of pluripotent stem cells in vitro, explaining how ES and iPS
cells can be induced to give rise to complex tissue structures in a self-organized fashion. Multiple CDB labs also opened their doors to visitors, including
the Laboratories for Cell Asymmetry, Early Embryogenesis, and Neocortical
Development, and the Division for Human Stem Cell Technology, encouraging
interaction with the Center’s student trainees and scientists. Other features
included a an exhibition of model organisms used in the CDB’s research, ascience café event, and a craft-making corner co-organized by the Kobe Science Museum

Awaji Retreat
The CDB held its annual retreat for its research staff on the nearby island of
Awaji. CDB lab leaders, research scientists and student trainees gathered in aclosed 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, a number of former research scientists andleaders joined the Retreat, adding a voice of broad international scientific experience and insights into the career paths followed by CDB alumni.

22

Transgenic mouse embryo at E14.5 expressing KikGr, a fluorescent protein under the
regulation of Blimp1 photoconverted fromgreen to red by UV irradiation.

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
that is thought to have appeared with the advent of the vertebrate lineage. However, dramatic changes in this organization took place during the divergence ofjawed (gnathostome) fishes from their jawless (agnathan) ancestors. Evolutionary modifications of the telencephalon first manifested in the reptiles, becoming
even more 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

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 theTokyo 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.

and dorsal-ventral axis patterning programs, and the means by which each individual region is formed, remain unknown.
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 emergenceof the vertebrate lineage has been accompanied by widespread adaptations tothis fundamental rule, as evidenced by the diversity of mechanisms by whichvertebrate 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

Shinichi AIZAWA
Research Specialist

Yoko SUDA
Masaki TAKEUCHI
Research Scientist

Hideharu HOSHINO
Fumitaka INOUE
Miyuki NORO
Mikihito SHIBATA
Michio YOSHIDA

Research Associate

Yusuke SAKURAI
Technical Staff

Eriko KAJIKAWA
Hiromi NAKAO
Tomomi OHMURA
Maiko TAKAHASHI
Assistant

Sayo SAITO

Visiting Scientist

Daisuke KUROKAWA

Publications
Kurokawa D, et al. A lineage specific enhancer drives Otx2
expression in teleost organizer tissues. Mech Dev (2011)
Shibata M, et al. MicroRNA-9 regulates neurogenesis inmouse telencephalon by targeting multiple transcription factors. J Neurosci. 31, 3407-22. (2011)
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)24(A) gives descendants of distal visceral endoderm cells that expressed a head organizer gene (hog), (B)the descendants keeping the hog expression, and (C) their merged view.

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 coreceptors, 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 systemhave 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 anddisease.

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

Pilaiwanwadee
HUTAMEKALIN
Keisuke ITOH
Toshihiro UESAKA
Technical Staff

Student Trainee

Sachie ONO
Part-Time Staff

Toko KONDO
AssistantKaori HAMADA
Yuka NODA

Chihiro NISHIYAMA
Junior Research Associate

Mitsuhiro IWASAKI
Yohei YONEKURA

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. GFRα1 expression in cells lacking RET
is dispensable for organogenesis and nerve regeneration.
Neuron 44. 623-36 (2004)

25

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

Carina HANASHIMA
Research Scientist

Yuko GONDA
Takuma KUMAMOTO

Part-Time Staff

Chika KUMAMOTO
Reiko ODA
Assistant

Yukie NAKAMURA

Foreign Postdoctoral
Researcher

Torsten BULLMANN
Student Trainee

Ken-ichi TOMA
Technical Staff

Yuko WADA

The neocortex, by far the most complex structure of all vertebrate brain systems, is organized into cytoarchitectonic groups of neurons that process uniquesensory inputs such as pressure, light, and sound. Nonetheless this highly ordered structure 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 functionalsignificance, the mechanisms underlying the precise assembly of distinct functional areas of the cerebral cortex remains largely unknown. Progress has been
impeded by our present lack of understanding of the molecular mechanisms
by which neuronal subtypes within cortical layers and across function domains
are specified. In our laboratory, we address important questions concerning
neocortical development: 1) What are the mechanisms by which diverse cellfate 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 theassembly 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 regionsof 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.
Through these studies we wish to understand the mechanistic basis by which
unique sensory perceptions and functional circuitries develop in the human neocortex.

Publications
Kasukawa T, et al. Quantitative expression profile of distinct
functional regions in the adult mouse brain. PLoS One
6.e23228 (2011)
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.

26

Morphogenetic Signaling
The main research interest in my lab focuses on the mechanisms by which cellcell and tissue-tissue interactions are modulated during embryonic morphogenesis. 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 alleviationof that strain is essential for smooth-tissue movement. As a consequence ofepithelial cells behaving as elastic bodies, their shape changes and movement
proceed with local fluctuations. We aim at elucidating (1) mechanisms that coordinate cell movement, (2) mechanisms for alleviating tissue strain, and (3) mechanisms of cross-talk between these two mechanisms. To accomplish thesegoals, the mechanical state of cells will be measured by combining techniquessuch as quantitative cell imaging, and various cell perturbation techniques arebeing used to assess 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 research interests are
dynamic aspects of cell adhesion, cell migration and cell morphogenesis in Drosophila.

StaffGroup Director

Shigeo HAYASHI
Research Scientist

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

Technical Staff

Ai AKIMOTO
Michiko KOIZUMIHousei WADA
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
Assistant

Yayoi HAMAZAKI
Hiromi WANAKA

Publications
Otani T, et al. IKKε regulates cell elongation through recycling endosome shuttling. Dev Cell 20. 219-32 (2011)
Tajiri R, et al. Joint morphology in the insect leg: evolutionary
history inferred from Notch loss-of-function phenotypes in
Drosophila. Development 138.4621-6 (2011)
Noguchi T, et al. Sustained elongation of sperm tail promoted by local remodeling of giant mitochondria in Drosophila.
Curr Biol 21.805-14(2011)
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)

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.

Niwa N, et al. Evolutionary origin of the insect wing via integration of two developmental modules. Evol Dev 12.168-76(2010)

27

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 arraythemselves during development? In recent years, it has become clear that themouse olfactory system provides an excellent platform for addressing thesequestions experimentally. In this system, there are around 1,000 types of odorant receptors that are capable of detecting and discriminating between odorantmolecules. Each olfactory sensory neuron expresses a single type of odorant
receptor, and the axons of neurons expressing the same receptor type converge
on the same site 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 from a single specific type of olfactory sensory neuron.

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 University ofTokyo Graduate School of Science in 2006. He
did consecutive postdoctoral fellowships under
the CREST Program and the University of Tokyo in 2006 and 2007, before his appointment
as Assistant Professor at the same university in2009. 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.

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.

Staff
Team Leader

Takeshi IMAI
Research Scientist

Naho ATSUMI
Satoshi FUJIMOTO
Ryo IWATA

Student Trainee

Meng-Tsen KE
Assistant

Aya KATSUYAMA

Publications
Imai T. and Sakano H. Axon-axon interactions in neuronal
circuit assembly: lessons from olfactory map formation. Eur
J Neurosci 34.1647-54 (2011)
Imai T, et al. Topographic Mapping - The Olfactory System.
Cold Spring Harb Perspect Biol 2. A001776 (2010)
Imai T, 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 T, 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.

28

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).

Histogenetic Dynamics
The development of multicellular organisms involves the collective effect of multiple events at the level of the individual cell, such as proliferation, differentiation,adhesion, and migration. Programmed cell death, for example, is a process bywhich cells are selected for death at set times in development, allowing for the
sculpting of tissue, and is used in the adult organism to maintain homeostasis byeliminating cells that have developed abnormalities. Perturbations in cell deathsignaling can thus affect an organism’s physiological stability, and result in developmental defects, tumorigenesis, or neurodegenerative disease. Cell deathplays an important role in maintaining the cellular society not only by eliminating
unneeded cells at given sites and stages, but in other functions, such as regulating the proliferation and migration of neighboring cells, as well. Such cellularbehaviors give rise to cell networks capable of organizing into tissues, the studyof which requires a experimental approach to spatiotemporal information in living
systems, such as can be obtained through the real-time live imaging of biologicalphenomena.
We have chosen the fruit fly Drosophila melanogaster as our primary researchmodel, seeking to take advantage of its utility in developmental studies andwealth of genetic data in studying the coordination of histogenesis through live
imaging and genetic screens. To elucidate the role of cell death in histogenetic
processes, we will analyze caspase mutant phenotypes in which the exteriormale genitalia (terminalia) develops abnormally. In normal Drosophiladevelopment,
the terminalia rotates 360° as it forms, but in caspase mutants, this revolution isincomplete. Image analysis reveals that in wildtype, the speed of this rotation isvariable, with distinct initiation, acceleration, deceleration, and termination stages;
caspase inhibition results in loss of the acceleration phase, and failure in terminalia development. We will seek to identify how caspase function and cell deathcontrol acceleration of the rotation through searching for associated genes andlive imaging analysis. It has further been predicted that cell death alone cannot
account for rotation that maintains tissue area, suggesting other mechanisms arealso at work. We will conduct single-cell analyses to determine whether other behaviors such as proliferation or migration are also altered. Through the use of theextensive Drosophila genetics toolset and live imaging technologies, we hope tobe able to address questions that have proven technically challenging in the past,and by visualizing the activities of individual cells, develop a better understandingof how cellular network systems work in histogenesis.

Erina KURANAGA Ph.D.
http://www.cdb.riken.jp/en/kuranaga
Erina Kuranaga received her doctorate in medical science from the Osaka University Department of Cell Biology and Neuroscience in 2004,
after which she moved to the University of Tokyo
Graduate School of Medicine as assistant professor in the Department of Genetics. In 2006,
she was promoted to associate professor in thesame department, where she remained until she
joined the RIKEN Center for Developmental Biology as Team Leader in 2011. She was awarded
the Molecular Biology Society of Japan Young
Scientist Award in 2010.

Staff
Team Leader

Erina KURANAGA
Research Scientist

Emi MAEKAWA

Technical Staff

Ayako ISOMURA
Kayo NAGATA
Aimi TSUKIOKA
Part-Time Staff

Arata KURANAGA

Publications
Kuranaga E, et al. Apoptosis controls the speed of looping
morphogenesis in Drosophila male terminalia. Development
138.1493-9 (2011)
Koto A, et al. Apoptosis ensures spacing pattern formationof Drosophila sensory organs. Curr Biol 21.278-87 (2011)
Nakajima Y, et al. Nonautonomous apoptosis is triggered by
local cell cycle progression during epithelial replacement in
Drosophila. Mol Cell Biol 31.2499-512 (2011)
Koto A, et al. Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development.
J Cell Biol 187.219-31 (2009)
Kuranaga E, et al. Drosophila IKK-related kinase regulates
nonapoptotic function of caspases via degradation of IAPs.
Cell 126.583-96 (2006)
Kuranaga E, et al. Reaper-mediated inhibition of DIAP1induced DTRAF1 degradation results in activation of JNK in
Drosophila. Nat Cell Biol 4.705-10 (2002)

Dorsal (left) and ventral (right) views of Drosophila
pupae that express fluorescent protein in cells
located posterior component of each segment.
Location of male genitalia is pointed in yellow
square.

Caudal view of a Drosophila male terminalia
showing cells in posterior compartment(green).

29

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, andthe mammalian middle ear. By analyzing the history of developmental patterns,
I hope to open new avenues toward answering as-yet unresolved questions
about phenotypic evolution in vertebrates at the level of body plans.

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

Staff
Group Director

Shigeru KURATANI
Research Scientist

Shinichi AOTA
Naoki IRIE
Christian MITGUTSCH
Hiroshi NAGASHIMA
Takayuki ONAI
Kinya OTA
Juan PASCUAL ANAYA
Fumiaki SUGAHARA
Takao SUZUKI
Motoki TADA
Masaki TAKECHISpecial Postdoctoral
Researcher

Tatsuya HIRASAWA

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
in gnathostomes (jawed vertebrates) and the turtle shell. Developmental studiesof the cranial region of the lamprey are intended to shed light on the true originsof 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
have changed during evolution by comparing the developmental patterns thatyield such 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 it is hoped will provide a key to discovering the true targets of natural selection in the acquisition of a shell.

Visiting Scientist

Ingmar WERNEBURG
Technical Staff

Satoko FUJIMOTO
Tamami HIRAI
Junior Research Associate

Noritaka ADACHI
Yasuhiro OISI
Student Trainee

Hiroki HIGASHIYAMA
Part-Time Staff

Maki MIYAUCHI
Kazuko YAMAMOTO
AssistantMika HIKAWA
Yuko HIROFUJI
Tomoko OYANAGI

Publications
Ota K. G., et al. Identification of vertebra-like elements andtheir possible differentiation from sclerotomes in the hagfish.
Nat Commun 2.373 (2011)
Irie N. and Kuratani S. Comparative transcriptome analysis
reveals vertebrate phylotypic period during organogenesis.
Nat Commun 2.248 (2011)
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)
Takio Y, et al. Evolutionary biology: lamprey Hox genes andthe evolution of jaws. Nature 429. 1 p following 262 (2004)30

CT-scanned skeleton of Pelodiscus sinensis juvenile

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

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

Staff
Team Leader

Rajesh LADHER
Research Scientist

Xiao Rei CAIAkira HONDA
Tomoko KITA
Siu Shan MAK
Yuko MUTA
Foreign Postdoctoral
Researcher

Student Trainee

Kazuya ONO
Part-Time Staff

Yoshiko KONDO
Yoko MATSUOKA
Anna WRABELAssistant

Noriko HIROIPaul O'NEILL
Visiting Researcher

Stephen FREEMAN

Publications
Ladher R. K. Squeezing into differentiation. Dev Cell 21.6078 (2011)
Nagai H, et al. Embryonic development of the emu, Dromaius novaehollandiae. Dev Dyn 240.162-75 (2011)
Ladher R. K., et al. From shared lineage to distinct functions: the development of the inner ear and epibranchialplacodes. Development 137.1777-85 (2010)
Teraoka M. E, et al. Rostral paraxial mesoderm regulates
refinement of the eye field through the bone morphogenetic
protein (BMP) pathway. Dev Biol 330.389-98 (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)

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).

31

The self-made eye: Formationof optic cup from ES cells

Nozomu TAKATA, Eriko SAKAKURA, Mototsugu EIRAKU, Masako KAWADA
Fumio MATSUZAKI,Atsushi
Developmental processes are increasingly well-characterizedat the molecularand cellKITAJIMA
biological levels, but how more complex tissues and organs involving the coordinated action of multiple cell types
in three dimensions is achieved remains something of a black box. One question of particular interestand importance is whether signaling interactions between neighboring tissues are essential to guidingorganogenesis, or whether these can arise autonomously from developmental routines inherent to agiven primordial tissue. Finding answers to these questions will be critical both to a better understanding of embryonic phenomena and to the ability to control the differentiation of cell populations into desired configurations.
A breakthrough new report by Mototsugu Eiraku, deputy leader of the Four-dimensional Tissue Analysis Unit and colleagues in the Laboratory for Neurogenesis and Organogenesis (Group Director, Yoshiki
Sasai), as well as the RIKEN VCAD Program, and Kyoto and Osaka Universities, describes how mouseembryonic stem cells (ESCs) are able to differentiate and assemble into an optic cup, capable of giving
rise to a tissue exhibiting the stratified structure characteristic of the retina in vivo. Published in Nature,
the study used a cutting-edge three-dimensional tissue culture system not only to demonstrate thisself-organizing capacity of pluripotent stem cells, but the underlying cell dynamics as well.
The mechanistic basis for the formation of the optic cup, with its complex two-walled structure, hasbeen a longstanding question in embryology. The retina, with its origins in the lateral midbrain, is partof the central nervous system. Its development begins with the formation of the optic vesicle, a pocketof epithelium that deepens and pinches to form the optic cup, which develops a double layer of cells,with pigment epithelium on the outer, and neural retina on the inner wall. It has generally been thoughtthat this transformation is triggered by chemical and physical influences from other tissues, such aslens or cornea, but some, including the father of experimental embryology, Hans Spemann, have suggested that perhaps external induction or force is not necessary.

32©2011 Nature Publishing Group
Eiraku M, et al. Self-organizing optic-cup morphogenesis in three-dimensional
culture. Nature 472 51-56 (2011)

To resolve this question, Eiraku et al. built on a series of techniques and findings emerging from theuse of the SFEBq (serum-free culture of embryoid body–like aggregates) ES cell culture system developed by the Sasai lab, which had previously been used to differentiate these pluripotent stem cells
into a wide range of neuronal cell types, including, recently, structurally organized cerebral cortical neurons. By adding extracellular matrix proteins to the SFEBq medium, the group was able to epitheliallyorganized retinal precursors at high efficiencies by day 7 of culture. One day later, optic vesicle-likestructure began to form, followed by bi-layered optic cup-like structures by day 10. The pigmented
and neuronal character of the outer and inner layers of cells in these spontaneously formed tissues
were confirmed by gene expression, indicating that optic cup development had been recapitulated invitro, and importantly, in the absence of any external signaling sources, such as lens, demonstrating
the capacity for self-organization.
They next used multi-photon microscopy to explore the mechanisms behind this process of self-assembly in 3D. They found that after the ES cell-derived retinal precursors differentiated into pigmented
epithelial and neuronal layers, the tissue underwent a four step morphological rearrangement on its
way to assuming the optic cup structure. When they examined cytoskeletal behaviors in this process,they noted that myosin activity dropped in the region of the epithelium that bend inward to form thecup, giving the flexibility needed to form a pocket driven by expansion of the epithelium through cell
division.
Computer simulation of the mechanics behind this revealed that threeprincipal forces can explain the optic cup-forming event. First, the aregion of the epithelium must lose rigidity, allowing it to buckle inward,
after which cells at the hinge points (defined by the border between
presumptive pigment epithelium and neuronal regions) must undergo
apical constriction, giving them a wedge-like shape. Once these conditions are met, expansion of the tissue surface by cell division results
further involution of the cup, all of which are very much in line with theexperimental findings.

Optic cup derived from mouse ES
cells. GFP shows expression of Rx.

As a final test of the in vitro structure’s ability to mirror its embryonic counterpart, Eiraku excised theneuronal layer from the ES cell-derived optic cup and allowed it to develop in 3D cell culture underconditions optimized for spurring neuronal maturation. He found that the retinal neurons underwent
active mitosis and ultimately organized into a six-layer stratified and synapse-forming neuronal structure closely resembling that of the post-natal retina.
“What we’ve been able to do in this study is resolve a nearly century-old problem in embryology, byshowing that retinal precursors have the inherent ability to give rise to the complex structure of the
optic cup,” says Sasai. “It’s exciting to think that we are now well on the way to becoming able to generate not only differentiated cell types, but organized tissues from ES and iPS cells, which may open
new avenues toward applications in regenerative medicine.” Potential applications include regenerative
medicine approaches to the treatment of retinal degenerative disorders, such as retinitis pigmentosa.

Four-step morphological transformation in formation of optic cup
in vitro revealed by two-photon
microscopy over three-period.

33

Transcriptomic insights into thevertebrate phylotypic stage

Fumio MATSUZAKI,AtsushiNaoki
KITAJIMA
Shigeru KURATANI,
IRIE
April 10, 2011 –The concept of the phylotypic stage traces its roots back to early comparative observations of embryos from different vertebrate taxa, in which it was noted that embryonic morphologies
appeared to converge on a shared body plan before veering off in specialized directions. This gave rise
to a profound debate over the evolutionary basis for this phenomenon; specifically, whether it could
best be explained by a “funnel” model, in which the commonality of traits is highest at the earliest
stages of embryogenesis, and gradually but unilaterally narrows over time, or an “hourglass” model,
where homology is highest at a point later in development as the body plan is being established, anddiffers more widely before and after.
A new comparative transcriptomic analysis of four vertebrate species conducted by Naoki Irie in the
Laboratory for Evolutionary Morphology (Shigeru Kuratani, Group Director) has now revealed that genetic expression is most highly conserved across taxa at the pharyngula stage of development. Published in Nature Communications, these latest findings strongly suggest that the hourglass model isthe more accurate description of how the vertebrate phylotype manifests.
Irie’s decision to study this question using a gene expression approach broke with the long history ofmorphological comparisons. He sampled tissue from mouse, chicken, and frog embryos across multiple developmental stages to allow for comparisons of changes in gene expression, and further supplemented this data set with information from previously published transcriptomic studies in a fourth taxa,
zebrafish, thus providing representative samples from mammal, bird, amphibian and fish species. He
took advantage of the supercomputing capabilities at the RIKEN integrated Cluster of Clusters (RICC)
for the processing power needed for comparison of this enormous set of data points.

34

©2011 Nature Publishing Group
Irie N. and Kuratani S. Comparative transcriptome analysis reveals vertebrate
phylotypic period during organogenesis. Nat Commun 2.248 (2011)

As development proceeds at different paces in different species, and organs likewise emerge at different points, making straightforward comparisons can be extremely problematic. To overcome thischallenge, Irie compared gene expression similarities from all combinations of developmental stages
among four species using various statistical models. For two species comparisons, he found that thehighest similarity was seen in intermediate stages of embryogenesis (from neural to late pharyngula
stages). More sophisticated computational analysis with four species pairing analysis revealed thatpharyngular embryos had the highest transcriptomic similarity of any stage.

Funnel (left) and hourglass (right)
models of changes in commonalityand diversity in vertebrate ontogeny.

To ascertain the detailed molecular characteristics of this phylotypic stage, the group went on to identify genes showing conserved expression during the pharyngula stage, but that were not constitutively
expressed throughout embryogenesis, and identified 109 gene sets, including Hox genes, transcription factors, cell-cell signaling genes, and morphogens. Interestingly, within these sets, developmental
genes were more highly represented than in sets with different expression profiles. The data generated
in this study has been deposited in the ArrayExpress and Gene Expression Omnibus repositories.
“It seems that the notion that genetic programs underlying early development are resistant to change
needs to be reconsidered in light of this data,” says Irie. “We’ll be interested in working out how early
genetic flexibility is achieved while maintaining the robustness of gene expression at the phylotypicpharyngula stage.”

Embryos at stages of greatest similarity in gene expression. Clockwise,from top right: mouse (E9.5), chicken (HH16), zebrafish (24 hours), andAfrican clawed frog (stages 28 and
31)

35

PGL proteins work in germ
granule assembly

Masafumi YONETANI, Momoyo (and Shiyori) HANAZAWA-MATSUYAMA
The separation of the germ cells responsible for the transmission of genetic information across generations through reproduction from the somatic tissues that form the rest of the body is a critical eventin early development. In many organisms, germ cells are characterized by the inclusion of organelles
known generally as germ granules, whose function appears to be linked to germ cell specification anddifferentiation. In the nematode C. elegans, these organelles are historically known as P granules, andare made up of a combination of messenger RNAs and protein components, only some of which havebeen identified.
Momoyo Hanazawa and Masafumi Yonetani in the Laboratory for Developmental Genomics (Team
Leader, Asako Sugimoto; now at Tohoku University) have now sorted out a crucial role for a pair ofproteins in germ granule formation. The study, published in the Journal of Cell Biology, shows how
PGL-1 and -3 self-assemble into scaffolds that recruit ribonucleoprotein components to developing
granules.
The team began by developing an assay system to test individual components of P granules for theability to form granules independent of other granule-associated proteins in a mammalian cell-based
system. Of the fourteen proteins tested, only two – PGL-1 and PGL-3 – assembled into granular clusters. To test this capacity in C. elegans cells, Hanazawa induced the expression of these PGL proteinsin embryonic and adult somatic cells and confirmed the formation of granules in these cellular environments as well, suggesting that these factors are able to self-organize into structure that may help to
recruit other P granule components.
To test this notion, they next checked whether RNAs and proteins co-localized with the PGL complexes. They found that the granules stained positive by a ribonucleic acid-specific dye, and that eight of36

©2011 The Rockerfeller University Press
Hanazawa M, et al. PGL proteins self associate and bind RNPs to mediate germ
granule assembly in C. elegans. J Cell Biol 192.929-37 (2011)

12 granule proteins tested also co-expressed with PGL-3. They next used a loss-of-function approachin which they depleted the PGLs from C. elegans embryos using mutations or RNAi, and determinedthat granule formation was greatly reduced in the PGL-deficient animals. This was true for membersof the GLH family (homologs of VASA in fruit fly), which typically co-localize with PGL granules in germ
cells as well. Analysis of cells in which both GLH-1 and -4 were knocked down below the detection
threshold showed that while PGL proteins were still able to self-aggregate, their GLH partners wererequired for maintaining the multicomponent granular structures.
Yonetani and Hanazawa next performed a structural analysis of PGL-3 to identify possible functional
domains. They found two domains are essential to form germ granules: one is an RNA binding domain
in the protein’s C-terminal, which appeared to be essential for the capture and incorporation of RNAs
to granules, and the other a self-association domain in the N-terminal, which is essential for forming
globular granules. These findings suggest that the self-association of RNA binding proteins, such as
PGL proteins, is crucial for forming germ granules containing diverse RNA and proteins.
Sugimoto notes, “There are many RNA-and-protein containing granules (ribonucleoprotein granules,
or RNP granules) in a cell, which play important roles in gene expressions. Our finding may provide ageneral mechanism for the formation of RNP granules.”4-cell C. elegans embryos. Green:
PGL-3, Red: GLH-1. Top: normal
PGL-3 proteins form granules with
GLH-1 in the germ lineage cell(rightmost cell). Bottom: PGL-3 protein that lacks the self-association
domain is dispersed in the cytoplasm of both somatic and germ
lineage cells.

37

Apoptosis accelerates
morphogenesis

Ayako ISOMURA, Erina KURANAGA, Aimi TSUKIOKA, Emi MAEKAWA
The process of programmed cell death, known as apoptosis, plays a critical role in many developmental events, such as the sculpting of digits in vertebrate limbs, in which the death of the cells that would
otherwise form inter-digital webbing enables individual fingers and toes to be formed. The importanceof the removal of cells in such contexts is well known, but in recent years, we have begun to learn
more about additional roles for apoptosis in the formation of tissues and organs.
A report by Erina Kuranaga, Team Leader of the Laboratory for Histogenetic Dynamics, and colleagues
revealed one such novel function for apoptosis in speeding the rotation of ring-like structures in the developing male reproductive system in the fruit fly Drosophila, a process that is necessary for its formation. This work, which was initiated at the University of Tokyo, is published in the journal Development.
Kuranaga began by analyzing the part played by apoptosis in morphogenetic processes using the
Drosophila system with its powerful genetics and decades-old store of research findings. In 1930,
it was noted that in this fly the male reproductive structure, known as the terminalia, makes a full
360-degree rotation during development. More than half a century later, researchers observed that defects in apoptosis result in incomplete rotation, and consequently reduced reproductive function andshortening of the structural links with the terminalia. In her recent study, Kuranaga brought live imaging
technology to bear on observation of the rotational kinetics of this system.
The terminalia originate in the rostral section of the embryo, and comprise a ring-shaped region known
as A8 that encircles the two inner regions A9 and A10. Kuranaga used fluorescent proteins to visualize the nuclei of cells in these domains and observe their movements in living embryos. They foundthat rotation begins approximately 24 hours after the formation of the puparium, and takes around12 hours to complete a full turn. But when the team blocked the activity of caspases responsible for38©2011 The Company of Biologists
Kuranaga E, et al. Apoptosis controls the speed of looping morphogenesis in
Drosophila male terminalia.Development 138.1493-9 (2011)

inducing apoptosis, they found that while this rotation began at the same developmental time pointand continued over the same 12-hour period, it failed to complete a 360° spin. To gain a better understanding of how this happened, they quantitatively analyzed the terminalia rotation in developing
flies, and determined that in wildtype the process involves four distinct steps: initiation, acceleration,
deceleration, and termination. In apoptosis-defective flies, however, the acceleration stage was not
maintained, leading to incomplete rotation.

A8
A9
A10
His2Av-mRFP+

Caudalview of a Drosophia maleterminalia(See CDB website for video)

The team next conducted more sophisticated live imaging observations, allowing them to track the behaviors of individual cells. They saw that in the ring-like A8 domain the inner and outer populations ofcells behaved differently – the interior cells rotated 360° in synchrony with the cells of the A9 domain,
but the outer cells only rotated half that distance, or 180°. Kuranaga also noted that the outer ringcells began their rotation just as the inner cells were entering the acceleration phase, suggesting a link
between these events. She also observed for the first time that apoptosis occurs in the A8 domain.
In embryos in which apoptosis was blocked, the inner cells in A8 moved as in wildtype, but the rotation of the outer cells failed to begin. By analyzing the frequency of apoptotic events and speed of rotation of outer cells in wildtype embryos as well, the team was able to show clear correlation between
the two. Taken together, the findings suggest that apoptosis is required for the rotation of the outer
ring, which in turn drives the acceleration of the inner ring of the A8 domain. And indeed, upregulationof the pro-apoptotic pathway resulted in faster rotation of the terminalia.
“The outer ring seems to function as a kind of moving walkway,” says Kuranaga. “When the inner ring
steps onto it, its speed increases, allowing it to complete its rotation within the normal period. We arestill working out how might apoptosis drive this, but suspect it may be related to the relief of some mechanical stress generated by cellular movements.”

Comparison of the speed of genitalia rotation between normal flies(black) and flies-inhibiting apoptosis
(red)

39

Mitochondria shape cellmorphology
Animals employ a surprising array of tricks toensure their reproductive chances, and successful innovations, such as the feathers ina male peacock’s tail, can drive evolutionarychange. In another remarkable example, somespecies of Drosophila develop sperm as longas 6 cm, around 30 times the length of the fly
itself. Such elongated sperm are more successful in reaching eggs, resulting in the genesof their possessors being evolved throughnatural selection. But how are such dramatic
cellular morphologies achieved?
Tatsuhiko Noguchi and colleagues in the Laboratory for Morphogenetic Signaling (Shigeo
Hayashi, Group Director) have now shown in
the fruit fly, Drosophila melanogaster, how mitochondria play a critical role in sperm elongationin this species. The report, published in Current
Biology, reveals that interactions between giant
mitochondria and microtubules underlie andmaintain the sperm’s extraordinary stretch.
Drosophila sperm cells undergo changes inshape during their maturation that leave them
200 times longer without any increase in volume. Four primary structures run along thesperm’s longitudinal axis: the flagellar axoneme,
cytoplasmic microtubules, giant mitochondria,
and cables of F-actin. It has previously beenshown that the axoneme is not required forsperm elongation, but the role of the othercomponents has remained unknown. In theirrecent work, Noguchi et al. extracted intact
sperm cells and maintained them in culture, allowing them to apply live imaging techniques to
get to the bottom of these questions.

Fumio MATSUZAKI,Atsushi NOGUCHIKITAJIMA
Michiko
KOIZUMI, Tatsuhiko40

The group began by chemically interfering withactin and microtubule function, which revealed
that while actin was dispensable, microtubules
were necessary for sperm tail elongation. They
followed up by inhibiting microtubule growth ina localized fashion, and found indications thatthe sperm tail end is the focus of elongation
activity. They looked next at the role of mitochondria. Giant mitochondria form from thefusion of multiple smaller mitochondria, but Noguchi found that when he inhibited this fusion,
sperm elongation was markedly reduced. Even
more interestingly, the crucial property of theseorganelles was shown to be their length andshape, rather than their better-known energygenerating respiratory activity.

©2011 Elsevier B.V.
Noguchi T, et al. Sustained Elongation of Sperm Tail Promoted by Local
Remodeling of Giant Mitochondria in Drosophila. Curr Biol 21,805-814 (2011)

The discovery that both microtubules and mitochondria are essential for sperm elongation naturally
suggested the next experiment, in which the group asked if they act on each other. When microtubules were ablated in the absence of axoneme, giant mitochondria reverted from their elongated form
to a rounded shape. When mitochondrial fusion was inhibited, microtubules assembled preferentiallyon fragmented mitochondria. Electron microscopic imaging further revealed structures cross-linking
both between microtubules and between microtubules and mitochondria.

Sperm cells undergoing elongation.
Microtubule (green) and mitochondria (red) are labeled. N: nucleus,
bb: basal body, axo: axoneme, CM:
cytoplasmic microtubule.

In loss-of-function mutants for Milton, a protein cross-linking mitochondria to microtubule motor kinesin, this sliding action, and consequently sperm elongation, were lost, suggesting a role for this motor
protein in the process. Further experiments in which microtubules were transiently depolymerized andallowed to regrow indicated that these cytoskeletal structures use the mitochondrial surface as a substrate for growth.
Given these findings, the group proposes that sperm elongation plays out as follows. First, microtubules form at the surface of giant mitochondria in the sperm tail, after which microtubules pair up by
cross-linking and slide along the mitochondrial surface, causing them to lengthen and thin, creating afoothold for more microtubules to accumulate and continue the elongation process, as well as to stabilize the structure.
These results represent the first finding that mitochondria can contribute to cell morphogenesis. “Thediscovery that the mitochondria that occupy our cells can act not only as cellular power plants, but
in germ line development as well is intriguing,” says Hayashi. “Mitochondria have a strong bi-layered
membrane, and can additionally provide the energy needed for flagellar motion, so the fact that they
are used as a scaffold in this context makes perfect sense.”

Snap shot view of spermatid elongation

41

Willin and Par3 work together inapical constriction

Fumio MATSUZAKI,AtsushiTakashi (andYuri) KITAJIMAISHIUCHI
Epithelial cells often take the shape of truncated wedges, with an apical surface narrower than thebasal face. This form of cellular polarization plays a central role in morphogenetic processes in which
sheets of homogeneous tissue bend, fold over, or roll up due to minute changes in the geometry of
their component cells. The primary connections between cells in such epithelial sheets are known asapical junctional complexes (AJCs), which both bind cells of like type to their neighbors and engage
with the cytoskeleton elements called actomyosin cables to modify and reinforce cell shapes andbehaviors. The contractility of such cables is under the control of a family of kinases known as ROCK
proteins, which form complexes near AJCs and enhance actomyosin contraction through phosphorylation of myosin light chain. But how ROCK interacts with factors involved in cell polarity remains
something of a mystery.
Takashi Ishiuchi in the Laboratory for Cell Adhesion and Tissue Patterning (Masatoshi Takeichi, Group
Director) has begun to tie the threads together with the discovery that a previously enigmatic protein
called Willin works cooperatively with the known polarity protein Par3 to phosphorylate ROCK, a critical trigger of apical constriction. Published in Nature Cell Biology, this new work shed light on thecomplex regulation of this important cellular morphogenetic process.
The study began with an examination of Willin’s localization, which revealed that it clusters near the
apical junction. A subsequent immunoprecipitation assay turned up an interaction with Par3, catching
the group’s attention, due to Par proteins’ involvement in cell polarization. They tested whether expression of Willin would affect Par3 localization, and found that while this had no effect, it did increase theaccumulation of Par3 partner aPKC at the cell membrane. A second round of immunoprecipitation
showed that Willin associates with aPKC as well, which deletion mutant studies revealed to be dependent on a region in the Willin protein known as JFR.

42

©2011 Nature Publishing Group
Ishiuchi T. and Takeichi M. Willin and Par3 cooperatively regulate epithelial apicalconstriction through aPKC-mediated ROCK phosphorylation.
Nat Cell Biol 13.860-6 (2011)

As Willin expression had no measurable effect on Par3 levels, Ishiuchi tried the converse experimentand showed that Par3 is not required for Willin’s upregulation of aPKC. Willin was also not required for
the Par3-aPKC association, indicating that while Willin could interact with both of these proteins, it appeared to do so by independent mechanisms.
Curious about Willin’s true role, the group knocked down the function of both Willin and Par3 in cells,
and found that this resulted in tightening of the apical junctional complex, secondary to a dramatic reduction in the localization of aPKC near the membrane. It appeared that aPKC was the critical downstream factor for limiting apical constriction. This was confirmed by experiments using a membranetargeted aPKC construct, which was able to rescue excessive actomyosin contractility.
But how does aPKC achieve this effect? Ishiuchi had observed that ROCK inhibition abrogated apical constriction, so he turned his focus to possible interactions between the two factors, with an
intuition that this activity might rely on phosphorylating activity by aPKC. On treating ROCK1 with aphosphatase, he found that this caused a change in its molecular mass, suggesting that the protein isindeed phosphorylated in vivo, which the group was then able to verify using a system to detect phosphorylation by band-shift. Analysis of the distribution of wildtype and phosphorylated ROCK proteins
revealed that while wildtype ROCKs tended to aggregate at the apical junction, when phosphorylated
they remained in the cytoplasm.
“The Par3-aPKC complex is known to regulate the apico-basal polarity of cells, and Willin is thought
to be a relative of the Drosophila protein Expanded, a component of the Hippo pathway for growth
control,” says Takeichi. “It will be interesting to work out how the ability of these proteins to regulate
actomyosin contraction at AJCs is related to their other physiological functions in future studies.”

Apical constriction is lost on knockdown of ROCK in cells lacking Par3
and Willin(red: Z0-1, green: cells
expressing ROCK shRNA)43

2011 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.

Training in multicolor FISH technqiues for scientists
The Center organized a course on multicolor fluorescence in situ hybridization(FISH) for the nuclear analysis of human stem cells on February 3 and 4, providing instruction in techniques for the detection of chromosomal abnormalities
that can appear in ES and iPS cells when cultured for long periods. Twenty
participants from academia, industry and other research institutes joined thecourse, which was organized under the auspices of the MEXT Project for the
Realization of Regenerative Medicine and support from Carl Zeiss MicroImaging and Abbot Japan.

Developmental biology over summer break
The CDB held its annual summer school for local high school students on August 2
and 4. The one-day courses featured lectures, lab visits, and hands-on experimentson fluorescent labeling. Unit Leader Shigenobu Yonemura (Electron Microscope Laboratory) opened the day with a lecture on how cells respond to tissue damage, andhis work on studying the mechanisms of wound healing in vitro. The lab practicum
began with a demonstration of how to use a micropipette, followed by the generationof fluorescent staining solutions, which the class used to label tubulin red and DNA
blue. Once the cells had been stained, the students watched their creations undera fluorescence microscope, enabling them to observe phenomena such as cell divisions.

Course in development for local HS teachers
The recurrent course in developmental biology for teachers of highschool biology, organized by the Office for Research Communications(ORC), was held at the CDB on October 1 and 2, 2011. This course
is held annually in the fall in conjunction with the Japanese Societyof Developmental Biologists, and gives the teachers an opportunity
to learn hands-on techniques in embryology. The program featured
a lecture by Prof. Sadao Yasugi of Kyoto Sangyo University on thehistory of embryology studies in chicken and on the molecular biological mechanism of cell differentiation, followed by a two-day training
course in which the teachers learned how to extract, culture, and observe chick embryos under the microscope.

44

Chick embryo at day E6, showing extensive
extra-embryonic vascular network.

Cell Asymmetry
Our group explores the mechanisms underlying the organization of cells into
highly ordered structures in the developing brain. During brain development,
neural stem cells generate a large number of neurons and glia of different fates
at appropriate points in time; the framework and size of the brain depend on
the spatiotemporal behavior of neural stem cells, which are highly dynamic in
their modes of division and gene expression. We focus our study on the programs by which behaviors of neural stem cells are controlled, using invertebrate(Drosophila) and vertebrate (mouse) model systems.

Fumio MATSUZAKI Ph.D.
http://www.cdb.riken.jp/en/matsuzaki
Fumio Matsuzaki received his B. Sc. from the
Department of Biochemistry and Biophysics at
the University of Tokyo in 1979, and his doctorate from the same institution in 1984. He spent
the period from 1984 to 1988 as a postdoctoral
fellow, first in the Department of Cell Biology at
the Tokyo Metropolitan Institute of Medical Science, then in the laboratory of Gerald Edelmanat the Rockefeller University. He returned to Japan in 1988 to take a position as a section chiefin the Department of Molecular Genetics in the
National Institute of Neuroscience. In 1998, he
was appointed professor in the Department ofDevelopmental Neurobiology at Tohoku University and remained there until taking his current
position as group director at the RIKEN CDB.

Staff
Group Director

Fumio MATSUZAKI
Special Postdoctoral
Researcher

Daijiro KONNO
Research Scientist

Tomohiko IWANO
Atsushi KITAJIMA
Tomokazu OSHIROAtsunori SHITAMUKAI
Shigeki YOSHIURA
Foreign Postdoctoral
Researcher

Jeremy Nicholas
PULVERS

Visiting Scientist

Ayano KAWAGUCHI
Yoichi KOSODO
Technical Staff

Tomoko AOKI
Aki MASUDANao OTA
Taeko SUETSUGU
Student Trainee

Masahiko INOUE
Yoshiaki SATO
Takuya YOKOMATSU

Drosophila neural stem cells, called neuroblasts, provide an excellent model
system for the investigation of fundamental aspects of asymmetric division, aprocess essential to the generation of cells of divergent type during proliferation. We have been investigating mechanisms controlling asymmetric divisions,
including the cell polarity and spindle orientation. We also extend our researchscope to understand how neurogenesis is controlled in tissue space dependingon the environments that surround the nervous system. We recently identified
an extrinsic mechanism that controls the orientation of division (cell polarity) inneuroblasts relative to the overlying ectoderm (Yoshiura et al., 2011), which determines the orientation of neural tissue growth.
The vertebrate brain evolved rapidly resulting in an expansion of the size of thebrain, which comprises a larger number of neurons arranged in a vastly more
complex functional network than that in invertebrate. Neural stem cells typicallyadopt three states – proliferative (symmetrically dividing), neurogenic (asymmetrically dividing), and resting – and undergo transitions among the states,on which the size, complexity and basic organization of the brain depend. We
investigate mechanisms that determine the individual states of neural stem cells,
and control transitions between states in mouse. We recently discovered a noveltransition in the developing mouse cortex from radial glia (typical neural stem
cells with the epithelial structure) to another type of neural stem cell, basal radial
glia (Shitamukai et al., 2011 and see figure), which is known as a major population of neural stem cells in mammals with large brains, such as primates. We areinvestigating the mechanisms that underlie the formation, maintenance, and expansion of this type of stem cells, by developing model mice that produce large
numbers of basal radial glia. We have also revealed a principle underlying the
dynamic behavior (interkinetic nuclear migration) of populations of neural stem
cells (Kosodo et al., 2011).

Part-Time Staff

Megumi SHIBATA
AssistantJunko ISHIGAI

Publications
Yoshiura S, et al. Tre1 GPCR signaling orients stem cell divisions in the Drosophila central nervous system. Dev Cell 22.
1-13 (2012)
Kosodo Y, et al. Regulation of interkinetic nuclear migration
by cell cycle-coupled active and passive mechanisms in thedeveloping brain. EMBO J 30.1690-704 (2011)
Wang C, et al. An ana2/ctp/mud complex regulates spindleorientation in Drosophila neuroblasts. Dev Cell 21.520-33(2011)
Shitamukai A, 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 31.3683-95(2011)
Kitajima A, et al. Progenitor properties of symmetricallydividing Drosophila neuroblasts during embryonic and larval
development. Dev Biol 347. 9-23 (2010)
Kato T. M, et al. Lunatic fringe potentiates Notch signaling in
the developing brain. Mol Cell Neurosci 45.12-25 (2010)46

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.
(http://www.cdb.riken.jp/en/04_news/articles/11/110704_progenitors.html)

Germline Development
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 embryonic patterning and the formation of germline cells in Drosophila, is repressed
during its transport to the posterior pole of the oocyte. We are now studying thefunction of proteins, such as the recently identified factor, Cup, that regulates
the repression 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 andglobal 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.

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

Akira NAKAMURA
Research Scientist

Takako AWAI
Kazuko HANYUNAKAMURA
Yasuko KATO
Maki SHIRAEKURABAYASHITechnical Staff

Kazuki MATSUDA
Assistant

Eri YAMASHITA

Publications
Shirae-Kurabayashi M, et al. Ci-Pem-1 localizes to the
nucleus and represses somatic gene transcription in the
germline of Ciona intestinalis embryos. Development 138.
2871-81 (2011)
Tanaka T, et al. Drosophila Mon2 couples Oskar-induced
endocytosis with actin remodeling for cortical anchorage ofthe germ plasm. Development 138. 2523-32 (2011)
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)

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

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).

47

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
depend on the ability of individual cells to maintain the expression of different
genes, and for their progeny to do so through multiple cycles of cell division.
In recent years, we have begun to understand that the maintenance of specificpatterns 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 that these different mechanisms are closely interrelated,but a detailed understanding of these systems has yet to be developed.

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

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.

Assistant

Sayaka SENOTechnical Staff

Noriyo HAYAKAWA
Rika KAWAGUCHI
Yasuko OHTANI

Publications
Hiragami-Hamada K, et al. N-terminal phosphorylation of
HP1 promotes its chromatin binding. Mol Cell Biol 31.
1186-200 (2011)
Kitano E, et al. Roles of fission yeast Grc3 protein in ribosomal RNA processing and heterochromatic gene silencing.
J Biol Chem 286. 15391-402 (2011)
Shirai A, et al. Methylation of ribosomal protein L42 regulates ribosomal function and stress adapted cell growth.
J Biol Chem 285. 22448-60 (2010)
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)48

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

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.
We are especially interested in the question of how cells maintain the neverending self-renewal that sustains the organism as a whole. This will require, atthe very least, solving the riddles of two essential processes. The first of theseis the need for old cells destined for replacement to be able to disengage orotherwise be dislocated from their environmental milieux. The second is therequirement for preparing new cells in replacement. To investigate the first ofthese mechanisms, our group uses a system for labeling cells with special dyes,
which allows us to monitor their location and behavior. We have also developeda 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 cell systems, 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 generatebona fide hematopoietic stem cells from ES cells.

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.

Staff
Group Director

Shin-Ichi NISHIKAWA

Technical Staff

Lars Martin JACT
Kenichiro KOBAYASHI
Igor M SAMOKHVALOVYoshiteru SASAKIYosuke TANAKA
Yan-Fung WONG

Misato HAYASHI
Satoko MORIWAKI
Satomi NISHIKAWA
Nataliya
SAMOKHVALOVA
Iori SATO
Yuri SHIMIZU
Naoko YOSHIOKA

Research Specialist

Student Trainee

Research Scientist

Reiko NAKAGAWA
Collaborative Scientist

Shin KAWAMATA
Visiting Scientist

Teruo AKUTA
Keitaro IMAIZUMIHiroshi KATAOKA
Shigeo MASUDA
Masao MATSUO
Herve Le MOUELLIC
Taro MUTO
Ruttachuk
RUNGSIWIWUT
Visiting Researcher

German Dario
GAMARGO ORTEGA
Stephan PETTITTE
Matteo PLUCHINOTTA
Anna STRATMANN
Part-Time Staff

Miho HOSHIJIMA
Satoshi NAKAI
Yoko OTSUKA
Kazumi YANAIAssistantJunko KOYA
Naeko MINAMI
Satomi SHINOHARA

Takao YAMADA

Publications
Ban H, et al. Efficient generation of transgene-free humaninduced pluripotent stem cells (iPSCs) by temperaturesensitive Sendai virus vectors. Proc Natl Acad Sci U S A
108.14234-9 (2011)
Fukushima Y, et al. Sema3E-PlexinD1 signaling selectively
suppresses disoriented angiogenesis in ischemic retinopathy in mice. J Clin Invest 121.1974-85 (2011)
Ishitobi H, et al. Molecular basis for Flk1 expression
in hemato-cardiovascular progenitors in the mouse.
Development 138.5357-68 (2011)
Kamiya D, et al. Intrinsic transition of embryonic stem-celldifferentiation into neural progenitors. Nature 470.503-9(2011)
Kataoka H, et al. Etv2/ER71 induces vascular mesoderm
from Flk1+PDGFRα+ primitive mesoderm. Blood (2011)
Nishikawa-Torikai S, et al. Functional characterization ofmelanocyte stem cells in hair follicles. J Invest Dermatol
131.2358-67 (2011)49

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.

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

Staff
Team Leader

Takashi NISHIMURA

Technical Staff

Special Postdoctoral
Researcher

Rinna NAKAMORIYuka NISHIMORI
Yuki YAMAUCHI

Naoki OKAMOTO

Part-Time Staff

Research Scientist

Kanako HIGUCHI
Junko SHINNO

Tomoka MURAI

Cell proliferation and differentiation in each tissue and organ are kept understrict regulation both spatially and temporally. Research has revealed the natureof 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 andgenetic approaches, along with quantitative and qualitative imaging and cellbiological analysis, to identify and characterize the relevant signal transduction
pathways.

Publications
Okamoto N, et al. Conserved role for the Dachshund proteinwith Drosophila Pax6 homolog Eyeless in insulin expression.
PNAS 108. 2406-2411 (2012)
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)

50

Cluster of neurosecretory cells in the larval brain called insulin-producing cells of the
fruit fly Drosophila melanogaster, showing expression of Drosophila insulin-like peptide(Green) and nuclear protein Dachshund (Red).

Pluripotent Stem Cell Studies
Pluripotency is a term used to describe the ability of certain types of stem cells
that are 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 possessingthese properties of pluripotency and self-renewal. In our lab, we use ES cells as amodel system for studying the molecular mechanisms that determine and enable
the mysterious properties of pluripotent cells.
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 shouldhave the sensitivity to signals that induce differentiation. How can the transcription factor network satisfy these contradictory requirements? What is the principle
behind the structure of the transcription factor networks governing developmental
processes? We are now trying to answer these questions by studying ES cells andthe 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-designated asa Project Lab in the Center Director’s Strategic Program for Stem Cell Research,and renamed the Laboratory for Pluripotent Stem Cell Studies.

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.

Staff
Project Leader

Hitoshi NIWA
Research Scientist

Kenjiro ADACHI
Jitsutaro KAWAGUCHI
Masaki KINOSHITA
Kazuhiro MURAKAMI
Satomi NISHIKAWATORIKAI
Satoshi OHTSUKA
Masaki SHIGETA
Tomoyuki TSUKIYAMA
Hiroki URA

Technical Staff

Setsuko FUJII
Student Trainee

Shunsuke ITO
Toshimi SUGIMOTO
Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI
Yoko NAKAI-FUTATSUGIAssistantMiho 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)

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.

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)

51

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.

Masaki OKANO Ph.D.
http://www.cdb.riken.jp/en/okano
Masaki Okano received his baccalaureate andmaster’s degrees from Kyoto University, and his
doctorate in biochemistry from the Research
Division in Agriculture at the same institution in
1994. He spent the period from 1994 to 1995 asa 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 Hospitalfrom 1995 to 2002, serving as an instructor in
medicine at Harvard for the last two years ofthat period. He was appointed team leader at
the RIKEN Center for Developmental Biology in
2001, and returned to work in Japan full-time in2002.

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.

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)

52

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.

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 isdictated by the effects of a number of signaling factors that diffuse from organizing centers and direct dorsal ectoderm to maintain a neural fate. These molecules, which include Noggin, Chordin, Follistatin and their homologs, participate
in elaborate signaling networks in which factors collaborate and compete toinitiate the embryonic nervous system.
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 differentiationof 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 thetreatment of neurodegenerative disorders, such as Parkinson’s disease. Using asystem developed in our lab, we have succeeded in inducing mouse and humanembryonic stem (ES) cells to differentiate into a range of specialized neuronal
types, including those of the cerebral cortex, cerebellar cortex and retina.
By studying very early neurogenesis, and the mechanisms of neuronal differentiation, our lab aims to understand the molecular basis underpinning the formation of so intricate a system as the mammalian neural network. In addition, we
seek to understand the principles underlying the shapes of organs, and are currently studying self-organization of cortex, retina, and adenohypophysis using
three-dimensional ES cell culture.

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
Research Specialist

Keiko MUGURUMA
Research Scientist

Toshihiro ARAMAKI
Hidehiko INOMATA
Makoto NASU
Nozomu TAKATA
Research Associate

Akira TAKAI
Visiting Scientist

Satoshi ANDO
Makoto IKEYA
Daisuke KAMIYA
Morio UENO

Technical Staff

Tomoko HARAGUCHI
Masako KAWADA
Ayaka NISHIYAMA
Mika SOEN
Rieko YAKURANISHIZAWA
Junior Research Associate

Shogo YAMADA
Student Trainee

Taisuke KADOSHIMA
Atsushi SHIRAISHI
Part-Time Staff

Masako SUZUKI
Assistant

Mako MIYAGI
Ayumi TANAKA-IKEYA
Fumi WAGAI

Publications
Suga H, et al. Self-formation of functional adeno-hypophysis
in three-dimensional culture Nature 480. 57–62 (2011)
Eiraku M, et al. Self-organizing optic-cup morphogenesis inthree-dimensional culture. Nature 472. 51-56 (2011)
Kamiya D, et al. Intrinsic transition of ES cell differentiation
into neural progenitors. Nature 470. 503-509 (2011)
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 generationand tissue integration of ES cell-derived Purkinje cells.
Nat Neurosci 13. 1171-80 (2010)

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

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)53

Planarian Islet homolog works
in posterior differentiation
Planarians are a group of small flatworms withsignificant capacity for regeneration, whichenables them to reform entire bodies from individual amputated fragments. To do so, however, each fragment needs to have a sense ofwhere it should be in the body, so that missing
head and tail regions form as needed and in
the appropriate location with respect to the restof the regenerating body. This positional information is thought to be conferred by gradientsin the expression of various genes that activate,
modulate or inhibit cell differentiation. The Wnt
signaling pathway, for example, is known to benecessary for the specification of posterior regions. The molecular mechanisms that underlie
this function, however, are poorly understood.
A new study by Tetsutaro Hayashi and others in
the Genome Resource and Analysis Unit (Fumio
Matsuzaki, Unit Leader) now reveals an additional factor, encoded by a planarian LIM- homeobox gene known as Djislet, is required forthe posterior differentiation of Wnt-expressing
cells. Published in Development, this reports
yields new insights into the coordination that
occurs between stem and differentiated cells in
an important model of animal regeneration.
The study began with the isolation of an apparent homolog of the vertebrate gene Islet
from an EST (expressed sequence tag) libraryfor the planarian species Dugesia japonica,
which is indigenous to Japan. In vertebrates,
the Islet family of LIM-homeobox transcription
factors is known to be involved in the proliferation, maintenance and migration of stem andprogenitor cells in a range of tissues. When
Hayashi knocked down the function of thisgene, dubbed Djislet, using RNA interference(RNAi), he found that its loss of function resulted in a tailless regeneration phenotype infragments from any part of the body (head or
trunk), suggesting that the gene might functionin posterior differentiation and regeneration.

Tetsutaro
HAYASHI
Fumio MATSUZAKI,Atsushi KITAJIMA

54

This suggested in turn the possibility of afunctional link to the Wnt signaling pathway,
which had previously been shown to be required for posterior specification and regeneration in another flatworm species, Schmidtea
mediterranea. Analysis of the expression of©2011 The Company of Biologists
Hayashi T, et al. A LIM-homeobox gene is required for differentiationof Wnt-expressing cells at the posterior end of the planarian body.
Development 138.3679-88 (2011)

these genes, Djislet and Djwnt1/P-1, showed co-localization at the midline of the blastema, the locus
of regeneration in planarians, at day 3 of regeneration. Moreover, in Djislet(RNAi) worms, the expression of Djwnt1/P-1 disappeared from the posterior blastema, suggesting that this gene functions
downstream of the Islet function.
Interestingly, the loss of Isletfunction had a more limited effect than that of Wnt, in that
Djwnt1/P-1(RNAi) worms show
both tailless regeneration andoccasional formation of twoheaded regenerants (known as
the Janus-heads phenotype),
while Djislet(RNAi) animals regenerated without tails, but never with two heads. Examining
gene expression in the processof Dugesia regeneration moreclosely, the lab found Wnt is expressed in two phases, at days 1 and 3, with the Islet homolog required
only for the second. Importantly, this second phase is senstitive to γ-ray irradiation, suggesting that it
involves stem cell-derived cells.

Djislet(RNAi) animals show tail-lessregeneration phenotype. Djwnt1/
P-1(RNAi) animals show twoheaded regenerants (Janus-heads
phenotype). Arrowhead, pharyngeal
neuron. Dashed lines indicate theamputation site.

Hayashi next checked the effect of Djislet(RNAi) on a panel of genes known to function in the posterior
blastema, and was able to groups these into three classes: Class I, which includes genes expressedin γ-ray-insensitive cells not regulated by Djislet; Class II genes which are partly downregulated by lossof Djislet function; and Class III, which are mainly expressed in γ-ray-sensitive cells and strongly downregulated by Djislet(RNAi) at day 3 of regeneration. These findings collectively point to a role for Islet via
Wnt signaling in the maintenance and activation of posterior genes in differentiating blastema cells for
tail formation.

The expression patterns of Djwnt1/
P-1 mRNA in the head regenerants
during posterior regeneration (from1 to 4 days after posterior amputation). The expression of Djwnt1/P-1
is eliminated by Djislet(RNAi) and
γ-ray irradiation at day 3, but not
day 1.

55

Hagfish show some backbone

The earliest roots of the phylum Vertebrata
trace back more than 500 million years, to theappearance of the body plan characterized by
the vertebrae for which it is named. Hagfish,
cartilaginous jawless fishes that lack vertebrae,
have thus sometimes been excluded fromthe vertebrate ranks, despite sharing many
characters with other members of the taxon.
In such schemes, the hagfish are technicallyinvertebrates, but are classed together withvertebrates as ‘craniates,’ referring to their welldeveloped crania. The debate about hagfish’strue taxonomic place has raged for more thana century, but is their lack of vertebral elements
enough to leave them an evolutionary odd manout?
Vertebra-like cartilaginous nodules had in fact
been found in hagfish over a century ago, but
the existence of these has been neglected in
the decades since that discovery. Kinya G. Ota
and colleagues in the Laboratory for Evolutionary Morphogenesis (Shigeru Kuratani, Group
Director) have now conclusively shown thepresence of these elements and analyzed theiranatomical features as genuine vertebrae in theJapanese in-shore hagfish, Eptatreus burgeri.
They further identified true vertebra-like traits inembryonic hagfish developmental patterns andprocesses, although these are for the most partlost in the adult. Reporting in Nature Communications, the group suggests that the hagfish
are, along with the lampreys, indeed true members of the vertebrate cyclostomes.
Ota’s work was enabled by the lab’s access tohagfish embryos for anatomical and histologicalanalyses, something that only became possible
when the Kuratani lab succeeded in breeding the animals in captivity several years ago.
Building on this achievement by developing along-term incubation system, Ota gained access to a sufficiently large number of fertilized
eggs to investigate their development at multiple stages.

Fumio MATSUZAKI, Atsushi
KITAJIMAKinya
G. OTA56

The early pharyngula-stage hagfish was foundto develop just under 80 somites, the repeating structures along the anterior-posterior axisof the embryo that in most vertebrates serveas the primordia for many of the bones and©2011 Nature Publishing GroupOta K. G, et al. Identification of vertebra-like elements and their possible
differentiation from sclerotomes in the hagfish. Nat Commun 2.373 (2011)

skeletal muscles in the trunk. Looking at gene expression in the somatic region of the trunk, Ota noted
similarities in the expression of characteristic genes in patterns corresponding to those of jawed (gnathostome) vertebrates. Specifically, hagfish Twist and Pax1/9 were expressed in segmentally arranged
ventral mesenchyme derived from the medial part of epithelial somites, while Pax3/7 and MyoD wererespectively switched on dermomyotome and myotome, respectively, indicating that this stage of hagfish somitogenesis follows a similar routine for the formation of somite-derived muscle and skin to thatin later vertebrates.

Histological view of the cartilaginous
vertebra of the hagfish

By the late pharyngula stage in hagfish, an additional dozen or so somites have formed, including
those that give rise to vertebra-like elements at around somite 90. Ota found that the Pax1/9-expressing cells at this stage had become more broadly distributed, suggesting that some had migrated
further ventrally, and might thus correspond to the sclerotome in later vertebrates, which is the somitic
component that gives rise to the vertebrae.
Given these similarities, the group surmised that hagfish somites indeed form both dermomyotome
and sclerotome, indicating homology with other vertebrates. And indeed close examination of theadult anatomy yielded cartilaginous axial elements to either side of the dorsal aorta that may represent
the hagfish’s vertebra-like equivalent. Although these cartilages lack the metameric quality of true vertebrae, their location and arrangement within the body are reminiscent of ventral components of gnathostome vertebrae known as the neural spine and neural arches.

Whole-mount stained adult hagfish
showing tail section with cartilaginous nodules beneath the notochord57

Out of the ventricular zone:
OVZ cells arise from basal inheritance

Fumio MATSUZAKI,Atsushi
KITAJIMA
Daijiro
KONNO, Atsunori
SHITAMUKAIThe developing mammalian brain is characterized by rapid proliferation and differentiation, needed togenerate the diversity of neural cells and tissues that make up the adult organ. During this stage, one
cellular population known as radial glia cells plays the part of neural stem cells, capable of both selfrenewal and the generation of differentiated progeny. These cells feature processes at both their apicaland basal ends, which are allocated asymmetrically to daughter cells in a context-dependent manner,
but the relationship between such segregation and the ability to self-renew remains poorly understood.
New research by Atsunori Shitamukai and others in the Laboratory for Cell Asymmetry (Fumio Matsuzaki, Group Director) shed new light on the angle of cell division as a crucial determinant of daughter
cell fate, with progeny produced by oblique divisions assuming a new combination of differential fates
depending on whether they inherit the apical or basal process. Published in The Journal of Neuroscience, these findings are in strong agreement with previous work in the primate nervous system, suggesting that the mouse can serve as a useful model for more detailed studies of this neurodevelopmental process.
The group began by observing the fates of daughter cells from apical and basal progenitors, using anelectroporation system previously developed by the lab, which enabled them to distinguish between
daughter cell types they called apical and other progenitors, after the location at which their subsequent cell division occurs. Apical progenitors, or radial glia cells, divide at the apical surface after interkinetic nuclear migration (INM) and form the apical junction, while basal progenitors divide a single
time in the subventricular zone (SVZ), giving rise to a pair of neurons. The group tested whether thefates of daughter cells in such divisions were dependent on the size of the inherited apical membrane,
as had been suggested by some previous studies, but found that in this context the question of selfrenewal vs. differentiation was unaffected by differences in inherited domain size.

58©2011 Society for Neuroscience
Shitamukai A., et al. Oblique radial glial divisions in the developing mouse neocortex
induce self-renewing progenitors outside the germinal zone that resemble primate
outer subventricular zone progenitors. J Neurosci 31.3683-95 (2011)

Shitamukai next examined whether the inheritance of the basal process played a role. All apical progenitors divided asymmetrically with regard to this structure, meaning only a single daughter inherits it.
By monitoring subsequent changes in the morphology and behavior of daughter cells that had or had
not inherited basal process, they were able to determine that those cells that inherited a process often
maintained their ability to self-renew, becoming either apical or nonsurface progenitors; daughter cells
that did not inherit the basal process showed no such proliferation.
Expression of genetic indicators of Notch activation, a hallmark of self-renewal ability in these cells,
again showed that inheritance of the basal process is associated with the generation of self-renewing
progenitor cells.
Shitamukai et al. then employed a strategy previously developed by the
Matsuzaki lab for genetically perturbing the orientation of the cleavage
plane, to examine the respective roles of the apical and basal domains ondaughter cell fate. They found that such misorientation at apical divisions
induces progenitors outside the ventricular zone (outer VZ progenitors)
that retain the basal process and, unlike basal progenitors, are capable ofself-renewal. Analysis of marker genes for differentiation and self-renewal
further supported these findings.

In the developing mouse brain,neural stem cells normally maintainepithelial 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.
(See CDB website for video)

Previous work by the lab had shown that even in normal mouse brain slices, there are occasional
progenitor cell divisions in which only a single daughter inherits the apical domain. To determine
whether these basal-only daughters in wildtype slices become outer VZ progenitors as well, the groupobserved unmodified both live brain slices and fixed brain sections and found that, in this context as
well, there is a small population of outer VZ progenitors that retain the basal process and proliferative
capacity. Interestingly, the survival of these progenitor cells depends on Notch signaling with their own
progeny cells.
Neural progenitor cells that are equivalent to outer
VZ progenitors, known as either outer subventricular zone (OSVZ) progenitor or outer glial cells, havebeen identified in the developing brain of gyrencephalic mammals, including ferrets and primates.
In primates in particular, these OSVZ progenitors
predominate among self-renewing neural progenitor cells from mid-neurogenic stages onwards, andgenerate most of the neurons in the upper layers of
the neocortex. Therefore, the appearance of OSVZ progenitor cells has been thought to be importantin the evolution of the brain, as a cause of the expansion in brain size and the consequent emergenceof brain gyration in these species. Some have speculated that rodents, which are agyrencephalic,
might lack OSVZ progenitors as well. Shitamukai et al., however, have shown that the OSVZ type ofprogenitor cells had already evolved in the rodent taxa, although their population is smaller than that ofradial glial cells. The group also proposes how OSVZ progenitor cells are generated in primates, suggesting that they may be born from radial gilal cells by oblique apical divisions in which one daughter
cell loses the apical domain, as happens in mice.

Scheme showing transitions in neural stem cell modes. The first transition is from symmetric to asymmetric division, while the second is fromradial glia (apical progenitor) to OVZ
progenitor.

Matsuzaki notes, “A number of human hereditary diseases are thought to be caused by abnormal
behaviors of neural progenitors. Some of these may be due to abnormalities in OSVZ progenitor cells,
which would have made them difficult to study experimentally, as this is a minor population in mice.
Our LGN mutant mice generate a number of the OSVZ progenitors comparable to that of radial glial
cells, which makes it possible to analyze some properties and genetic defects of the OSVZ progenitors
using this mouse strain as a model.”59

Dual role for AIR-1 in mitotic
spindle assembly

Fumio MATSUZAKI, AtsushiMika
KITAJIMA
TOYA
Mitotic cell division involves a critical process in which replicated chromosomes separated by spindle
fibers are pulled apart into separate daughter cells. In the roundworm C. elegans, such mitotic
spindles, comprising microtubules and associated proteins, are assembled through both γ-tubulindependent and -independent processes. Previous studies have shown that γ-tubulin-independent microtubule assembly relies on the function of AIR-1, the C. elegans homolog of Aurora A kinase, a key
regulator of numerous mitotic events. But just how AIR-1 works in this aspect of microtubule assembly
remains unclear.
Mika Toya and others in the Laboratory for Developmental Genomics (Asako Sugimoto, Team Leader)have succeeded in deciphering AIR-1’s role. In a report published in Nature Cell Biology, the teamused live cell imaging and RNAi approaches to show that AIR-1 stabilizes the spindle in a non-kinasedependent fashion in addition to its kinase-dependent role at the centrosomes.
Toya, who has since joined the CDB Laboratory for Cellular Adhesion and Tissue Patterning, began
by observing microtubules in mitotic cells in which the expression of air-1 or tbg-1 (the C. elegans
γ-tubulin gene) were knocked down by RNA interference in living embryos. They found that while microtubule numbers and lengths remained roughly constant in the air-1(RNAi) embryos, these increased
significantly during chromosome condensation in the tbg-1(RNAi) cells, suggesting the two mechanisms are different.
In other organisms, in addition to centrosomes (the major microtubule organizing centers), condensed
chromatin can also trigger microtubule assembly. To determine whether this is the case in C. elegans
as well, they tested air-1 and tbg-1 knockdown in embryos in which chromatin-stimulated microtubules were made readily visible, and found that while γ-tubulin appeared to play no role, loss of AIR-1

60

©2011 Nature Publishing Group
Toya M, et al. A kinase-independent role for Aurora A in the assembly of mitotic
spindle microtubules in Caenorhabditis elegans embryos.
Nat Cell Biol 13.708-14 (2011)

function caused microtubules to grow to shorter lengths and in fewer numbers. Immunofluorescencealso showed that AIR-1 localized around condensed chromatin, but γ-tubulin was not seen in theseregions.

AIR-1(P)-AIR-1

DNA

MergedKinase-active form of AIR-1, (P)AIR-1, detected at the centrosomes
as a subpopulation of the panAIR-1 signal

The kinase activity of AIR-1 depends on phosphorylation of the protein at a specific site. To monitor
such activity, the team developed a phospho-specific AIR-1 antibody, allowing them to specificallyvisualize the subpopulation of AIR-1 proteins showing kinase activity. Interestingly, the phospho-AIR-1
signal was not detected in proximity to condensed chromatin, suggesting that its kinase activity was
not involved in this aspect of its function.
The team tested this idea by generating transgenic embryos that expressed the kinase-inactive formof AIR-1. Toya et al. found that while AIR-1 association with microtubules was unaffected by the absence of kinase activity, its kinase-inactive form appears to be critical for stabilizing microtubules once
assembled. Kinase activity was likewise found to be dispensable for the chromatin-stimulated microtubule assembly. The association of the kinase-inactive form of AIR-1 with microtubules was regulated
by levels of the wildtype protein – in the presence of wildtype AIR-1, microtubule localization of thekinase-inactive construct was reduced. Given these findings, the team speculates that the balance between active and inactive forms of AIR-1 may be important in controlling mitotic spindle assembly andbehaviors.
“The next step will be to find out whether Aurora A in other organisms, including mammals, has similar
kinase-independent functions,” says Sugimoto, now at Tohoku University. “We speculate that kinaseindependent roles of protein kinases may be more widespread, although these have been overlookedin previous studies.”

tbg-1(RNAi);
spd-5(RNAi)

air-1(RNAi);
spd-5(RNAi)
Assembly of chromatin-stimulated
microtubules. Chromatin-stimulated
microtubules were assembled in theabsence of γ-tubulin (left); this was
dramatically reduced in the absenceof AIR-1 (right).
(See CDB website for video)61

Mon2 tethers plasm to pole

The uneven distribution of cellular components
is a fundamental process in development anddifferentiation, as can be seen in the sequestration of maternal RNAs and proteins in a formof cytoplasm known as germ plasm at theposterior pole of the Drosophila oocyte, whichis allocated only to daughter cells that go on toform the germ lineage. Failure to maintain thisgerm plasm at the egg’s polar region results in
defects in germ cell formation, highlighting its
critical importance to the fly’s reproductive viability. The means by which the germ plasm isanchored to the oocyte pole, however, remain
incompletely understood.
Tsubasa Tanaka and colleagues in the Laboratory for Germline Development (Akira Nakamura, Team Leader) have now worked out at least
part of this mystery, finding that the protein
Mon2 works with a second factor, Oskar (Osk),
to remodel actin and hold the germ plasm in its
proper place. Published in Development, thiswork may shed light on more general principlesof the generation of cellular polarity.
The role of Osk in germ plasm assembly iswell established – osk RNA is carried to the
oocyte’s posterior pole where it is translated,
forms complexes with other polar components,
and triggers the remodeling of F-actin at thecell cortex. The resulting actin fibers serve as
anchor points in the polar region. The Nakamura team previously found that Osk appears
to accomplish this feat through the activation of
the endocytic pathway, a mechanism by whichcells absorb and recycle molecules on the cell
membrane, but the mechanistic link between
this process and actin remodeling remained
uncertain.

Fumio MATSUZAKI, Atsushi
TsubasaKITAJIMATANAKA62

To investigate more closely, Tanaka began byidentifying six mutants in which polar factors
such as Osk and Vasa failed to stay localized
at the poles. Analysis of the underlying genetic
changes revealed that all six were caused bymutations in the gene encoding Mon2. Whenthey looked at the patterns of Mon2 expression
in the oocyte, they found that while it began asa punctate expression throughout the egg, it
gradually shifted to the cortex, resembling the
localization of Golgi and some endosomes.

©2011 The Company of Biologists
Tanaka T, et al. Drosophila Mon2 couples Oskar-induced endocytosis with actin
remodeling for cortical anchorage of the germ plasm.
Development 138.2523-32 (2011)

The team next asked what function Mon2 might have in maintaining the germ plasm at the pole. In the
mutants, they found no changes in microtubule assembly, needed to convey pole factors to their destinations, or in the ability of Osk to activate endocytosis. But when they looked at its effect on actin,
they found that Mon2 loss of function interfered with the cytoskeletal component’s remodeling. Analysis of genetic interactions between Mon2, Osk and the endocytic regulator Rab5 revealed that Mon2
operates downstream of both Osk and the endocytosis it triggers.
When Tanaka tested for protein binding partners for Mon2, he found that it physically associated with apair of actin nucleators, Cappuccino (Capu) and Spire (Spir). Interestingly, Spir includes a Rab-binding
domain, pointing to a possible interaction with endosomes. Loss of Capu and Spir function resulted in
the loss of actin projections at the polar cortex; the actin fibers instead accumulated in fuzzy ball-likeaccretions, indicating a failure of the actin remodeling requisite for germ plasm tethering. Knowing thatboth Spir and Capu interact with Rho1, a protein known for its roles in actin regulation, the team next
tried to copy the loss of Capu and Spir phenotype by interfering with Rho expression, which resulted
in the expected failure of actin remodeling. They further found that in Mon2 mutants, Rho’s normal localization at the oocyte posterior pole was lost. The picture that develops is one in which the activationof endocytosis by Osk spurs actin remodeling by Mon2 downstream of Spir, Capu and Rho, thereby
providing an anchorage for the germ plasm.
“There have been a number of recent indications that the endosome serves as a platform for the assembly of molecular complexes, as well as other lines of evidence that show that intracellular vesicles,
such as endosomes, play a role in ferrying molecules through the cell,” says Nakamura. “Given theimportance of these functions, what we have learned about Mon2 in this study may have implications
for the generation of polarity in other contexts as well.”Loss of Mon2 function results in a
failure to maintain pole plasm at the
pole

63

Pushing in, crowding out

The surface, or cortex, of the mammalian brain
is formed from highly organized layers comprising distinct types of neural cells. This cellular
diversity, and the underlying order that informs
the overall cortical structure, are largely the
result of routines that play out during the brainsdevelopment in embryonic stages. Importantamong these is a process called asymmetriccell division, in which progenitor cells give riseto daughter cells of different fates. Interestingly,
neural progenitors remain on the move during
this process, shuttling their nuclei back andforth between the apical and basal sides of thedeveloping cortex in an action known as interkinetic nuclear migration (INM). These movements are timed in sync with the cell cycle, butjust how this is accomplished has remained
enigmatic.
Yoichi Kosodo and others in the Laboratory
for Cell Asymmetry (Fumio Matsuzaki, Group
Director) have identified a pair of mechanismsthat couple INM to the mitotic cycle. In an article published in The EMBO Journal, the groupshows that cell-autonomous basal-to-apical
nuclear migration proceeds under the control ofthe microtubule-associated protein Tpx2, which
drives migration of the nucleus in the opposite
direction via a displacement, or “crowding out,”
effect.
The group began by developing a system toallow them to track nuclear movement during
INM in living tissue, enabling them to identify
a number of novel features of this behavior
and its coordination with various stages of thecell cycle. To test whether interkinetic nuclear
migration in fact depends on cell cycle progression, they used an inhibitor to arrest neuralprogenitors at the G1 phase, and found that
this prevented the apical migration of nuclei atthe start of INM. Previous studies had raised
the possibility that this linkage might rely on thefunction of Tpx2, a microtubule nucleating protein, the expression of which is regulated by thecell cycle. Monitoring of a fluorescent-tagged
version of the protein revealed that it associateswith microtubules in the apical processes ofneural progenitor cells.

Fumio MATSUZAKI, Atsushi
Yoichi KITAJIMAKOSODO

64

Kosodo next interfered with Tpx2 function using RNAi to examine whether it is involved inapical nuclear migration, and found that its

©2011 European Molecular Biology Organization
Kosodo Y, et al. Regulation of interkinetic nuclear migration by cell cycle-coupled active andpassive mechanisms in the developing brain. EMBO J 30.1690-1704 (2011)

knockdown resulted in a significant decrease in this aspect of INM; specifically, that loss of Tpx2 function caused a slowdown in the speed of nuclear movement toward the apical region. Observations of
microtubule distribution in wildtype and Tpx2-inhibited cells indicated that the factor works to localize
microtubules in the apical process of neural progenitor cells. Taken together, these results suggest thatbasal-to-apical nuclear migration during the G2 phase is dependent on the reorganization of microtubules by Tpx2.

Neural progenitors undergo dynamic oscillations of nuclei, calledinterkinetic nuclear migration, anddivide facing the ventricular zone
(apical side). In this movie, cellswith nuclear GFP migrate basally at
G1 phase while those in G2 phase(cytoplasmic GFP) migrate apically.
(See CDB website for video)

What then of nuclear migration in the opposite direction? In G1-arrested neural progenitor cells, thegroup observed that nuclei accumulated basally, raising the possibility of a separate mechanism behind this. Using a magnetic fluorescent microbead assay, they tracked the motion of apically-locatedbeads in cultured brain slices, and found that this drew them to the basal side, from which they did
not subsequently return to the apical region, even when nuclei continued to migrate in that direction. It
appeared that their translocation was therefore non-cell-autonomous, but the mechanism remained in
doubt.
One possibility was that the active migration in the basal-to-apical direction increases nuclear density
in the apical region, which might lead to a “crowding out” effect. Kosodo et al. tested this by arresting
the cell cycle at S phase by drug treatment and observing the effects on nuclear migration over time.
Cell cycle arrest causes a drop in the number of apically migrating nuclei. They found that this consequently reduced the rate at which nuclei moved in the basal direction as well. The same phenomenon
was observed in observations of microbeads, suggesting that it was not due to some unknown bioactive property of the mitotic inhibitor, but that basal movements of nuclei or beads are tightly linked
with apically directed nuclear movements. To further test their hypothesis that apical-to-basal nuclear
migration is the result of displacement, the group, in collaboration with Akatsuki Kimura at National
Institute for Genetics, constructed a computational simulation of INM to model conditions that might
influence nuclear migration in the apical-to-basal direction, and found that their in silico predictions
jibed well with their in vivo observations, strongly suggesting that this aspect of INM is driven by physical displacement of nuclei from the basal region.
“In the developing brain, a huge number of neural progenitors must engage in highly dynamic
movements in order to give rise to its myriad
neurons,” says Matsuzaki. “What we havediscovered is that the independent but linked
mechanisms that drive movements in opposing
directions help to avoid extreme deviations or
collisions of progenitors and maintain the structural order of the brain’s morphology. Indeed, thismay represent a fundamental strategy for maintaining order in dynamic cell populations.”

A computer modeled simulation
faithfully mimics the behavior of
apical to basal nuclear migrationof neural progenitors. The model
assumes that G1 nuclei move suchthat the local nuclear density is
minimized, whereas G1 nuclei apically migrate at a constant speed(See CDB website for video)

65

2011 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 the highest levels of originality, impact and quality in its scientific contributions. The individual achievements andcareers of many of its scientific staff have been recognized with awards from numerous organizations, including government
ministries and agencies, private foundations and academic societies.
The CDB takes great pride in the achievements of its researchers, as individuals and as indispensable members of their laboratories.
Awardee

66

PositionLaboratory

Award

Organization

Shigeru KurataniGroup Director

Evolutionary
Morphogenesis

Alexander Kowalevsky
MedalSaint-Petersburg Society of
Naturalists

Hiroshi Nagashima

Research scientist

Evolutionary
Morphogenesis

2011 Achievement in
AnatomyThe Japanese Association of
Anatomists

Yukari Terashita

Junior Research
Associate

Genomic
Reprogramming

Prize for Academic
Excellence

Japanese Society of
Mammalian Ova Research

Sayaka Wakayama

Research scientist

Genomic
Reprogramming

Prize for Academic
Excellence

Japanese Society of
Mammalian Ova Research

Chong Li

Research scientist

Genomic
Reprogramming

Prize for Academic
Excellence

Japanese Society of
Mammalian Ova Research

Kazuo Yamagata

Visiting scientist

Genomic
Reprogramming

Prize for Academic
Excellence

Japanese Society of
Mammalian Ova Research

Teruhiko Wakayama

Team Leader

Genomic
Reprogramming

Prize for Academic
Excellence

Japanese Society of
Mammalian Ova Research

Hiroki R. Ueda

Project Leader

Systems Biology

JSPS PrizeJapan Society for the
Promotion of Science

Shigeru KurataniGroup Director

Evolutionary
Morphogenesis

MEXT Prize

Ministry for Education,
Culture, Sports, Science andTechnology

Yosuke Tanaka

Research scientist

Stem Cell Biology

Poster Prize

Max Delbrück Centrum
Berlin-Buch

Yasuhide Ohinata

Visiting scientist

Pluripotent Stem cell
Studies

MEXT Prize

Ministry for Education,
Culture, Sports, Science andTechnology

Yukari Terashita

Junior Research
Associate

Genomic
ReprogrammingAward for excellent
poster presentation

Asian Reproductive
Biotechnology Society

Mototsugu Eiraku

Deputy Unit Leader

Four-dimensional
Tissue Analysis

Mitsubishi Kagaku Prize

Molecular Biology Society ofJapan

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Inoue Prize for Science

Inoue Foundation for ScienceE14.5 mouse inner ear bone labyrinth, containing the cochlea andvestibular system, as visualized by injection of ink (paint filling).

67

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

Research in our laboratory focuses on these signaling centers, especially thoseof the mouse, as a model for studying the regulation of embryogenetic processes. We focus particularly on the node and notochord, which are of central
importance in the formation of the early embryo. We focus on the control of theexpression of the Foxa2 transcription factor in the formation and maintenanceof signaling centers, as well as search for new factors involved in the control ofembryonic development by such centers. We have recently revealed that members of 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-implantationembryos. Our lab is now analyzing the roles of Tead family proteins and theirregulation by cell-cell contact information as a new approach to the study ofearly mouse development.

Staff
Team Leader

Hiroshi SASAKI
Research Scientist

Yoshikazu HIRATE
Keiichiro KAMURA
Hiroshi MAMADA
Ken-ichi WADA

Student Trainee

Yuu IMUTA
Part-Time Staff

Megumi SHIBATA

Technical Staff

Shino HIRAHARA
Aya KATSUYAMA
Akiko MORII

Publications
Wada K, et al. Hippo pathway regulation by cell morphology
and stress fibers. Development 138.3907-14 (2011)
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)68

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.

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 fatedto undergo 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.jp/en/sawa
Hitoshi Sawa obtained his baccalaureate,master’s and doctoral degrees from Kyoto
University in the period from 1982 to 1991.
He worked as a postdoctoral fellow at the
California Institute of Technology on a Human
Frontier Science Program grant during theperiod from 1991 to 1994, then at the Massachusetts Institute of Technology from 1994 to
1997 under support from the Howard Hughes
Medical Institute. He returned to Japan in 1997
to continue his work at Osaka University, firstas a postdoc, then as researcher funded by theJapan Science and Technology Corporation
PRESTO program. He took his current positionat the RIKEN CDB in 2001.

Staff
Team Leader

Hitoshi SAWA
Research Scientist

Yuji HOSOIRI
Kenji SUGIOKA
Technical Staff

Hatsumi OKADA
Tomomi TAKANO

Junior Research Associate

Yuko YAMAMOTO
Part-Time Staff

Tomoko SUGIMOTO
Assistant

Tomoko NAKASHIMA

Publications
Shibata Y, et al. Multiple functions of PBRM-1/Polybromoand LET-526/Osa-containing chromatin remodeling complexes in C. elegans development. Dev Biol 361.349-57(2012)
Yamamoto Y, et al. Multiple Wnts redundantly control polarity orientation in Caenorhabditis elegans epithelial stem
cells. PLoS Genet 7.e1002308 (2011)
Sugioka K, et al. Wnt regulates spindle asymmetry to generate asymmetric nuclear beta-catenin in C. elegans. Cell
146.942-54 (2011)
Arata Y, et al. Extracellular control of PAR protein localization
during asymmetric cell division in the C. elegans embryo.
Development 137. 3337-45 (2010)

Asymmetric cortical localization of the Wnt pathway components

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)69

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

Guojun SHENG Ph.D.
http://www.cdb.riken.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
Research Scientist

Meng-Chi LIN
Yukiko NAKAYA
Fumie NAKAZAWA
Wei WENG

Technical Staff

Cantas ALEV
Hiroki NAGAI
Kanako OTA
Erike Widyasari
SUKOWATI
YuPing WU

Publications
Shin M, et al. Activin/TGF-beta signaling regulates Nanog
expression in the epiblast during gastrulation. Mech Dev
128.268-78 (2011)
Nagai H, et al. Embryonic development of the emu, Dromaius novaehollandiae. Dev Dyn 240. 162-175 (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. Nature Cell Biol 10. 765-775 (2008)
Nakazawa F., et al. Negative regulation of primitive
hematopoiesis by the FGF signaling pathway. Blood
108.3335-43 (2006)

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.

70

Physical Biology
Advances in measurement technologies have afforded us glimpses into dynamic functional processes, such as morphogenesis and information processing,in cells 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 of such 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 andelucidation of these emerging questions in biology.
One example of such a phenomenon is seen in cellular chemotaxis, in whichcells recognize concentration gradients of attractant molecule and respondby directional 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 ofranging 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 that guides the direction of its movement, raising the question of how
cells are able to detect and follow such weak and noisy signals. We now knowthat within their tiny intracellular spaces, cells comprise many interacting molecules that work in 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 fascinatingproblems in biology through concepts and methods adapted from the mathematical sciences.

Tatsuo SHIBATA Ph.D.
http://www.cdb.riken.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
Tetsuya HIRAIWA
Naotoshi NAKAMURA
Masatoshi NISHIKAWA
Katsuhiko SATO

Student Trainee

Naohiro AKUZAWA
Toshinori NAMBA
Junior Research Associate

Prabhat SHANKAR
Assistant

Satoko KINOSHITA

Visiting Researcher

Seirin LEE

Publications
Ooyama S. and Shibata T. Hierarchical organization of noise
generates spontaneous signal in Paramecium cell. J Theor
Biol 283.1-9 (2011)
Arai Y., et al. Self-organization of the phosphatidylinositol
lipids signaling system for random cell migration. Proc Natl
Acad Sci U S A 107.12399-404 (2010)
Nishikawa M. and Shibata T. Nonadaptive fluctuation in an
adaptive sensory system: bacterial chemoreceptor. PLoS
One 5.e11224 (2010)
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)

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.

Shibata T. and Fujimoto K. Noisy signal amplification in
ultrasensitive signal transduction. Proc Natl Acad Sci U S A
102.331-6 (2005)

71

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 “asymmetric cell division”), the mother cell is polarized before dividing and cytoplasmic
components are asymmetrically segregated. A mitotic spindle is formed andpositioned 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.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
School of Life Sciences, Tohoku University.

Staff
Team Leader

Asako SUGIMOTO
Special Postdoctoral
Researcher

Mika TOYA
Research Scientist

Eisuke SUMIYOSHI

Part-Time Staff

Kanako KAWAI
Student Trainee

Masafumi YONETANIAssistant

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.

Taeko IMURA

Technical Staff

Kayo NAGATA

Publications
Toya M, et al. A kinase-independent role for Aurora A in theassembly of mitotic spindle microtubules in C. elegans embryos. Nat Cell Biol 13, 708-14 (2011)
Hanazawa M, et al. PGL proteins self associate and bind
RNPs to mediate germ granule assembly in C. elegans. J
Cell Biol 192.929-37(2011)
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)
Toya M, et al. Imaging of mitotic spindle dynamics inCaenorhabditis elegans embryos. Methods Cell Biol 97,
359-72 (2010)
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.

72

Retinal Regeneration
The retina has been called the “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 new hope that the ability to regenerate neurons and even to reconstitute theneural network may be retained in the adult retina. We are now exploring theexciting prospect that, by transplanting cells from outside of the retina or by regeneration from 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 viable grafts upon transplantation and induce the reconstitution of functional neural networks. We also hope to develop means of promoting true regeneration by
activating endogenous stem cells to replace cells lost to trauma or disease 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.jp/en/takahashi
Masayo Takahashi received her M.D. from Kyoto
University in 1986, and her Ph.D. from the same
institution in 1992. After serving as assistantprofessor in the Department of Ophthalmology,
Kyoto University Hospital, she moved to the Salk
Institute in 1996, where she discovered the potential of stem cells as a tool for retinal therapy.
She came back to Kyoto University Hospital in1998, and since 2001 served as an associate
professor at the Translational Research Centerin the Kyoto University Hospital. She joined the
CDB as a team leader of the retinal regeneration
research team in 2006. Her clinical specialty isretinal disease, macular diseases and retinal hereditary diseases in particular. Her aim is to understand these diseases at a fundamental leveland develop retinal regeneration therapies.

Staff
Team Leader

Masayo TAKAHASHI
Research Scientist

Jun KANEKO
Michiko MANDAI
Akiko SUGA
Research Associate

Chie ISHIGAMI
Hiroyuki KAMAO
Satoshi OKAMOTO
Visiting Scientist

Yasuhiko HIRAMI
Zi-Bing JIN
Yuko MIBU
Chikako MORINAGA
Visiting Technical Staff

Yumiko SHIBATA
Motoki TERADA

1
Human iPS cells

2
Retinal pigment epithelial cells differentiatedfrom iPS cells

Technical Staff

Momo FUJII
Kyoko ISEKI
Kanako KAWAI
Akane NOMORI
Wataru OHASHI
Kazuyo SADAMOTO
Noriko SAKAI
Akihiro TACHIBANA
Yu WATAOKA
Chikako YAMADA
Student Trainee

Juthaporn
ASSAWACHANANONTSun JIANAN
Hirofumi KINOSHITA
Part-Time Staff

Yuuki ARAI
Tomoko KATAYAMA
Yuumi TADA
Tomoko YOKOTA

Publications
Jin ZB., et al. Modeling retinal degeneration using patientspecific induced pluripotent stem cells. PLoS One 6.e17084(2011)
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)
Osakada F, et al. Wnt signaling promotes regeneration in
the retina of adult mammals. J Neurosci 27. 4210-9 (2007)
Ooto S, et al. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina. Proc Natl Acad
Sci USA 101. 13654-9 (2004)

3
Purified retinal pigment epithelial cells

4
Photoreceptor cells differentiated from iPS cells

Haruta M, et al. In vitro and in vivo characterization of pigment epithelial cells differentiated from primate embryonicstem cells. Invest Ophthalmol Vis Sci 45. 1020-5 (2004)
Haruta M., et al. Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue. Nat Neurosci 4.1163-4(2001)

73

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 tissuespecific multicellular structure, such as epithelial sheets and neural networks.
We are also interested in the molecular basis of how the tissue architecture is
disrupted during carcinogenesis, a process that is thought to accelerate the metastasis of cancer cells. For these studies, we are focusing on the roles playedby cell-cell adhesion and recognition molecules, the cadherin family of adhesion
molecules in particular, as these are known to be indispensable for tissue construction.

Masatoshi TAKEICHI Ph.D.
http://www.cdb.riken.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

Shuichi HAYASHI
Takashi ISHIUCHI
Shoko ITO
Kyoko KUBOTA
Wenxiang MENG
Shoko NAKAMURA
Tamako NISHIMURA
Anna PLATEK
Katsutoshi TAGUCHI
Mika TOYA
Yoshikazu TSUKASAKI
Vassil VASSILEV

Research Associate

Shigenori NAGAE
Nobutoshi TANAKA
Technical Staff

Sylvain HIVER
Yoko INOUE
Saeko KOBAYASHI
Miwako NOMURA
Hiroko SAITOAssistant

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 filamentsor microtubules, in modulating cell assembly. We are therefore studying themechanisms underlying the crosstalk between cadherins and such cytoskeletal
systems. These studies include analysis of the functions of microtubule minus
end-associated proteins and kinesin motors, as well as actin dynamics regulators, in cell-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 thecadherin superfamily, deficiencies of which have been implicated in neuronal
defects in brain development. Through these studies, we expect to gain deeper
mechanistic insights into the ways by which cells build elaborate structures of
the animal body.

Mutsuko AISOWATANABE

Publications
Ishiuchi T. and Takeichi M. Willin and Par3 cooperatively regulate epithelial apical constriction through aPKC-mediatedROCK phosphorylation. Nat Cell Biol 13.860-6 (2011)
Taguchi K., et al. Mechanosensitive EPLIN-dependent remodeling of adherens junctions regulates epithelial reshaping. J Cell Biol 194.643-56 (2011)
Ito S and Takeichi M. Dendrites of cerebellar granule cells
correctly recognize their target axons for synaptogenesis invitro. Proc Natl Acad Sci USA 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)
Nakao S, et al. Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction. J Cell Biol
182. 395-410 (2008)74

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-cellularlevel and apply them to system-level questions of complex and dynamic biological systems, such as the mammalian circadian clock. In October 2009, ourlaboratory was re-designated as a Project Lab in the Center Director’s Strategic
Program for Systems Biology research to promote the challenging tasks. Basedon the achievements over the last eight years, we strongly feel that it is now thetime 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 numberof the generated animals (ideally with a single animal). Full utilization of thesetechnologies 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, seasonalaffective disorder and sleep disorder.

Hiroki R. UEDA M.D., Ph.D.
http://www.cdb.riken.jp/en/ueda
Hiroki R. Ueda received his M.D. from the Facultyof Medicine of the University of Tokyo in 2000, andreceived his Ph. D. from the University of Tokyo
Graduate School of Medicine in March 2004. Whilean undergraduate, he worked as a research assistant on a biological simulation system project at
Sony Computer Science Laboratories. As a graduate student he next went on to work, first as a
researcher (2000) and then as a group leader (2002),
at Yamanouchi Pharmaceuticals, on a project
studying biological clock mechanisms in fly andmouse. 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 was promoted
project leader of the Laboratory for System Biology
under CDB’s Center Director’s Strategic Program.

Staff
Project Leader

Hiroki R. UEDA
Research Scientist

Yohei M. KOYAMA
Arthur MILLIUS
Tomoko MORIYAMA
Masato NAKAJIMA
Yasutaka NIWA
Hideki UKAI
Rikuhiro G. YAMADASpecial Postdoctoral
Researcher

Hiroki DANNO
Etsuo A. SUSAKI
Foreign Postdoctoral
Researcher

Craig C JOLLEY
Technical Staff

Hiroshi FUJISHIMA
Junko HARA
Tomoyo HASHIGUCHI
Masahiro HAYASHI
Shino HIRAHARA
Mayumi ISHIDAMarie MURAMATSU
Yuta SHINOHARA
Maki UKAI-TADENUMA
Research Fellow

Genshiro SUNAGAWA
Student Trainee

Ryokichi TANAKA
Takashi YASUKAWA
Junior Research Associate

Hiroyuki TAMIYA
Visiting Scientist

Tetsuya KOBAYASHI
Kohei MASUMOTO
Atsushi WADA
Part-Time Staff

Yuko SASAGAWA
Kaori TSUJINO
Agency Staff

Chikako IMAI
AssistantSumire HINO
Maki NOMOTO

Publications
Ukai-Tadenuma M, et al. Delay in feedback repression by
cryptochrome 1 is required for circadian clock function. Cell
144.268-81 (2011)

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.

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)
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 U S A 105. 14946-51 (2008)
Ukai H, et al. Melanopsin-dependent photo-perturbation
reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat Cell Biol 9. 1327-34 (2007)75

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.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 Promotionof Science in 1996 and spent the next two
years at the Yanagimachi lab in the Universityof 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
Mikiko TOKORO
Sayaka WAKAYAMA
Visiting Scientist

Fumiaki ITOI
Satoshi KISHIGAMI
Haruko OBOKATA
Kazuo YAMAGATA

Junior Research Associate

Yukari TERASHITA
Student Trainee

Hirohisa KYOGOKUTechnical 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
they lead to reprogramming failure, and possible solutions to the problem all
remain completely unknown. Despite these challenges, cloning continues tobe a biotechnology of great promise, given its potential applications in the fieldof regenerative medicine, such as the generation of embryonic stem (ES) cellsby 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 of rejection.
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 efficiencyof the 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 forpreserving embryonic lethal and infertile strains of laboratory mice, and continue
to explore the development of new and better techniques for sperm and oocytepreservation and other reproductive biological research technologies.

Yuko SAKAIDE
Kaori YAMANAKA
Assistant

Tomoko OYANAGI

Publications
Li C, et al. Intracytoplasmic sperm injection with mouse
spermatozoa preserved without freezing for six months
can lead to full-term development. Biol Reprod 85.1183-90(2011)
Ono T, et al. Offspring from intracytoplasmic sperm injectionof aged mouse oocytes treated with caffeine or MG132.
Genesis 49.460-71 (2011)
Hikichi T, et al. Functional full-term placentas formed fromparthenogenetic embryos using serial nuclear transfer.
Development 137.2841-7 (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)
Wakayama S, et al. Production of healthy cloned mice frombodies frozen at -20 degrees C for 16 years. Proc Natl Acad
Sci USA 105. 17318–17322 (2008)76

Transfer of a somatic nucleus into an enucleated egg

The submucosa of the small intestine contains numerous blood andlymphatic vessels (blue and magenta, respectively), as well as an
intrinsic network of neurons (green). In mouse, these submucosal
neuronal precursors proliferate until two weeks after birth.

First germline transcriptional
repressor in ascidian foundMaki SHIRAE-KURABAYASHIIn most animal embryos, molecular measures are in place to prevent nascent germline cells from mistakenly differentiating into a somatic lineage. This is typically achieved by the repression of somaticgene expression by specific genetic factors in the germ plasm, such as Pgc in Drosophila and PIE-1
in C. elegans. It has been suspected that transcriptional repression is also at work in embryos of theascidian Ciona intestinalis, which also has a germ plasm-like cytoplasmic compartment known as the
postplasm, but the factors and mechanisms that might lie behind this process remain unknown.
New work by Maki Shirae-Kurabayashi and colleagues in the Laboratory for Germline Development(Akira Nakamura, Team Leader) has identified what appears to be a key transcriptional repressor in the
ascidian, a protein product of Ci-pem-1 RNA, which is an ascidian specific component of the postplasm. This study, published in the journal Development, shows that Ci-Pem-1 interacts with anothergeneral co-repressor of mRNA transcription, lending further support to the knockdown findings.
Transcriptional repressors in other widely studied species, such as fruit fly and nematode, work by
inhibiting the phosphorylation of the C-terminal domain in RNA polymerase II, a critical enzyme inmRNA transcription. Shirae-Kurabayashi began this study by examining such RNAPII phosphorylationin C. intestinalis blastomeres, at a stage of development in which the germline begins to emerge as a
distinct lineage, as signaled by the inheritance of the postplasm. The team analyzed RNAPII phosphorylation in individual blastomeres using anti-phospho-CTD antibodies, and found that germline blastomeres showed lower levels of phosphorylation than did somatic cells.

78©2011 The Company of Biologists
Shirae-Kurabayashi M, et al. Ci-Pem-1 localizes to the nucleus andrepresses somatic gene transcription in the germline of Ciona intestinalis
embryos. Development 138.2871-81 (2011)

Familiar with the expression of Ci-pem-1 RNA from a previous study, Shirae-Kurabayashi used immunostaining to visualize the distribution of its protein product. They previously reported that the
postplasmic Ci-pem-1 RNA is ejected from the germline during gastrulation by an asymmetric cell
division (Shirae-Kurabayashi et al., 2006). In the present study, the team found that the signal of its
protein product also dropped in the germline by the tailbud stage. More intriguingly, they found that
this protein localizes to the nuclei in the germline blastomeres in the most posterior region of thecleavage-stage embryo. This nuclear accumulation, however, fluctuated over the course of the cell
cycle, appearing only in late telophase when the nuclear envelope reassembles following replication.
To eliminate the possibility that this reaction was an artifact of cross-reactivity, they re-tested in blastomeres injected with a morpholino against Ci-pem-1, and found, in line with expectations, that MO
knockdown prevented its nuclear localization.
Thinking that this protein might play a role in transcriptional control in germline cells, the team examined the effects of Ci-pem-1 knockdown on embryonic gene expression. When they knocked down
this factor, they detected ectopic expression of a number of genes ordinarily repressed in germline
blastomeres. The expression of both beta-catenin and GATA-dependent genes was de-repressed in
the knocked-down germline blastomeres, indicating that Ci-pem-1 exerts a broadly repressive effecton somatic gene expression.

Ciona intestinalis embryos at 1, 4,
8 and 16-cell stage probed for Cipem-1 mRNA (green) and Ci-Pem-1
protein (magenta). In the pair ofgermline blastomeres, Ci-pem-1
mRNA is highly concentrated in the
postplasm at the posterior cortex,
while Ci-Pem-1 protein is enrichedin the nuclei.

The structure of the Ci-Pem-1 protein includes a motif reminiscent of binding domains in other species that associate with the transcriptional co-repressor Groucho. Shirae-Kurabayashi performed a coimmunoprecipitation assay using tagged constructs for these two proteins, and found that Groucho
associated with GFP-labeled Ci-Pem-1, but not with GFP alone, pointing to a physical interaction ofpotential functional significance.
“Unlike Pgc in Drosophila and PIE-1 in C. elegans, our findings suggest that Ci-Pem-1 appears to
repress somatic gene expression without directly inhibiting RNAP II phosphorylation, indicating that
distinct mechanisms and factors are responsible for transcriptional quiescence in different organisms,”
says Nakamura. “Interestingly, recent work by Mitinori Saitou, a former CDB team leader, has shownthat the silencing of somatic transcriptional programs also occurs during the establishment of mousegerm cells, which are induced through cell-cell signaling without the germ plasm, meaning the active
repression of somatic transcriptional programs during the establishment of the germline is a conserved
hallmark in animal development, regardless of the mode of germ cell formation.”

79

Hippo signaling follows form

Fumio MATSUZAKI, Atsushi
KITAJIMA
Ken-IchiWADA
Cells have a well-developed sense of their surroundings, identifying and interacting with many diverse
components of their environment. This is clearly seen in the proliferation of cells in culture – a population will grow steady until the cell density crosses a given threshold, triggering a shutdown. Thisphenomenon, known as “cell contact inhibition of proliferation,” has been tied to the Hippo signaling
pathway, but the molecular mechanism remains mysterious. Specifically, it is unknown whether the key
cue behind this process is cell morphology or cell-cell adhesion, or some combination of the two.
A study by Ken-Ichi Wada and others in the Laboratory for Embryonic Induction (Hiroshi Sasaki, Team
Leader) looked at individual cells to examine the effects of cell morphology on Hippo signaling in isolation from other influences. Published in Development, the report of this work describes how cell shapeand underlying cytoskeletal proteins, such as F-actin, regulate the Hippo pathway in vitro. Wada now
works at the RIKEN Advanced Science Institute, while Sasaki has moved to the Kumamoto University
Institute of Molecular Embryology and Genetics.
In a previous study, the Sasaki team had shown how changes in cell density control Hippo activity;
Hippo is down-regulated at lower densities, resulting in the nuclear accumulation of a coactivator
protein, Yap, increasing activity of Tead, a transcription factor that promotes proliferation. Conversely,at higher densities, Hippo is activated, causing the phosphorylation and cytoplasmic retention of Yap,
preventing Tead activity and, as a result, stoppage of proliferation. This report, however, left open thequestion of how Hippo signaling is able to detect the density of cells in an environment.
Numerous studies have shown links between cell-cell adhesion and regulation of the Hippo pathway,
and other reports have described the effect of cell morphology on proliferation, which led Wada to
speculate that cell shape might also play be a missing piece in the Hippo–proliferation puzzle. In order80©2011 The Company of Biologists
Wada K, et al. Hippo pathway regulation by cell morphology and stress fibers.
Development 138.3907-14 (2011)

to study this, however, he first needed anexperimental system that would allow him to
examine cells in isolation from contact withother cells. By culturing cells in microdomainsof different sizes, he was able to grow single
cells in spatial constraints that affected theirmorphology. In smaller 20 × 20 μm domains,the cells assumed a compact, rounded form,
while in roomier 70 × 70 μm quarters, they
became flattened and spread out. Looking
next at the expression of the Hippo pathway
factor Yap, he noted that in cells grown in thesmaller domains, Yap remained outside thenucleus, while in those cultured in the larger
domains it was localized in the nucleus, suggesting that morphology can act as a regulator of the distribution of this protein.
But how is this regulatory control achieved?
The key may lie in a form of F-actin known asstress fibers. In cells cultured in the 50 × 50
μm domains, as a model of lower cell density, stress fibers were abundant and present
throughout the cells, while in the 20 × 20 μm
domains their distribution was punctate and only faintly detectable. To determine whether the apparent
correlation between stress fiber quantity and cell density works in Hippo regulation, the team next disrupted stress fiber formation using an actin-inhibiting agent. As expected, this caused Yap to localizein the cytoplasm, as occurs in high-cell-density environments.

In smaller, rounded cells (left), Yap(red) accumulates outside the nucleus (blue), while in flattened cells(right) with extensive F-actin (green),
Yap localize within the nucleus.

This latest work form the Sasaki team cements a role for cell morphology in the regulation of proliferation mediated by the Hippo pathway. “It will be interesting to tease out how Hippo signaling integrates
diverse information inputs relating to a cell’s shape and its adhesive contacts with neighboring cells,”
says Sasaki. “We know that actin is also involved in cell-cell adhesion, so that may be one key to understanding the link to Hippo. We are also very interested in the role that Hippo plays in controlling differentiation in the preimplantation mammalian embryo, and whether these latest findings will prove tobe important to our understanding of early development as well.”Stress fibers (green) are foundthroughout untreated cells, in which
Yap (red) accumulates in the nucleus, while in cells treated with anactin-inhibitor (CytoD, LatA), there
are fewer stress fibers and Yap iscytoplasmic.

81

BrainStars project maps
regional gene expression in brain

Fumio
MATSUZAKI,Atsushi
KITAJIMA
TakeyaKASUKAWA,Koh-hei
MASUMOTO
The mammalian brain is one of the pinnacles of evolutionary achievement; myriad interconnecting neurons in discrete functional domains give rise to an organ of astonishing complexity. Numerous efforts
have been made to profile the molecular signatures of the brain’s many regions, typically using a technology known as in situ hybridization to visualize gene transcripts at specific sites. Other more comprehensive techniques for analyzing gene expression, such as DNA microarrays, also exist, but thesehave yet to be used in measuring the central nervous system’s genetic activity across a wide range ofdifferent regions.
Takeya Kasukawa and Itoshi Nikaido of the Functional Genomics Unit, Koh-hei Masumoto of the Laboratory for Systems Biology (both headed by Hiroki R. Ueda) and others have done just that, profilinggenome-wide expression across nearly 50 brain regions in the mouse. Published in PLoS ONE, thiswork identified a large number of genes showing interesting characteristics such as multi-state expression, control properties, and putative ligand-receptor interactions. The results have been compiled into
an open, searchable database at http://brainstars.org.
The group sampled 51 brain regions associated with distinct functions, such as various sensory modalities, internal clocks, memory, fear, and feeding, at 4-hour intervals over the course of a day. They
next used quantitative PCR (qPCR) to analyze these samples for the expression of region-specificgenes, and used global clustering to identify developmental, evolutionary and anatomical groupings,
noting a high degree of consistency with expectations, but a few notable exceptions as well. Retina,
pituitary and pineal tissue, for example, were markedly distinct from the other 48 regions in this analysis, which may be due to differences in anatomy or cell-type composition.
Their next step was to identify and analyze multi-state patterns of gene expression, finding more than
8,000 genes showing multimodal expression across the sampled regions. Such multiple states may82©2011 Public Library of Science
Kasukawa T, et al. Quantitative expression profile of distinct functional regions in
the adult mouse brain. PLoS One 6.e23228 (2011)reflect either differential regulation in different cell types,
or heterogeneity in the balance of various cell types indifferent brain regions. A closer examination revealed
a number of interesting genes with from two to four
distinct states, including various G-protein-coupled receptors (GPCRs) and a large proportion of homeobox
and nuclear receptor genes.
Such multi-state expression data was then used to
identify candidate marker genes, defined as those
whose highest or lowest expression state occurs only
in a single region of the central nervous system. Morethan 2,000 such genes were found, including severaltranscriptional regulator/target gene pairs. Not all
genes are multimodal, however; the study also founda class of genes with single-state expression acrossmultiple CNS regions, suggesting that they may function as stable internal controls.
To test the utility of their dataset, Kasukawa, Nikaido
and Masumoto focused on neurotransmitter (NT) andneurohormone (NH) ligands and receptors as a meansof measuring the interconnectivity between CNS regions, counting the number of NH/NT ligand-receptor
expressions for every pairing of CNS regions, andplotted these by density. In examples of the revelatory
power of this approach, they identified a high level ofconnectivity between the hypothalamic and olfactory
bulb regions, and large number of inbound connections to the suprachiasmatic nucleus, the circadianclock center, suggesting the requirement for multiple
inputs in keeping the body’s internal clock runningon time. A number of other intrinsic connections, described in detail in the article and the public database,were also uncovered.
The final validation of the group’s findings came froma comparison to several previous studies of regional
gene expression in the mouse brain. Despite thesmaller sampling sizes of the BrainStars study, it
showed good levels of correlation with existing in situ
hybridization data sets, with some differences howeverin findings for smaller and more complex regions, suchas the hypothalamus.
The BrainStars database provides arrange of information on gene expression in the mouse brain, including
gene expression profiles the results of various analyses,” says Kasukawa. “We are hopeful that this resource will make a contribution to CNS research, andwould be delighted to see it used widely by others.

Map of 44 of the brain regions
sampled in this study.

(A) Color-coded table of CNS
regions expressing genes coding
for NH/NT ligands in one region
and cognate receptor genes in theother. (B) Map of putative strong
connections among CNS regions.

83

EPLIN responds to force

Epithelial cells stay connected in a numberof ways, relying on combinations of adhesion
molecules to recognize and maintain contact
with their neighbors. One form of epithelial cellcell adhesion involves the zonula adherens,a belt-like region at the cell border at which
complexes of cadherins, catenins, and othermolecules, including actin filaments in the cytoskeleton, accumulate. Sheets of epithelial
tissue undergo rearrangements in the courseof numerous developmental and disease processes, ranging from convergent extension to
wound closure. In the latter, purse-string-likeactomyosin cables linked to adherens junctions(AJs) contract around the leading edges of cell
margins, closing the space between cells. It
is not known, however, how actin cooperates
with the cadherin machinery in the remodelingof the AJ.
New work by Katsutoshi Taguchi and othersin the Laboratory for Cell Adhesion and Tissue
Patterning (Masatoshi Takeichi, Group Director)
shows that the protein EPLIN plays a key rolein the process, as a mechanosensitive regulatorof junctional remodeling. Published in the Journal of Cell Biology, this research sheds newlight on how physical forces, such as stretching, help determine cellular properties and behaviors.
Taguchi began by immunostaining a line ofcultured epithelial cells for E-cadherin and Factin, showing their accumulation at adherens
junctions. Interestingly, this labeling revealed
that these junctions took two different forms. In
the interior of a cell colony, the proteins aligned
linearly, whereas at the fringes, the junctions
were punctate in form. These junction types
appeared to be interconvertible, as both forms
could be identified within single colonies.

Fumio MATSUZAKI,Atsushi KITAJIMA
Katsutoshi
TAGUCHI84

In both the linear and the punctate adherens
junctions, E-cadherin, β- and αE-catenins,and actin fibers play dynamic structural roles.
Several proteins, including EPLIN and vinculin, have been identified as linkers between
the cadherin-catenin complex and the actin
cytoskeleton to date, prompting Taguchi to examine their possible roles. Through additional
immunostaining, he found that while vinculin is©2011 The Rockerfeller University Press
Taguchi K, et al. Mechanosensitive EPLIN-dependent remodeling of adherens
junctions regulates epithelial reshaping. J Cell Biol 194.643-56 (2011)

present in both linear and punctate AJs, EPLIN was only detectable in the linear type. Knockdown of
the two proteins by RNAi further supported a role for EPLIN; loss of the function of this protein allowed
the formation of punctate AJs only, suggesting that it is required in the formation of the linear adherens
junction.
What then is the reason for the lackof EPLIN at punctate AJs? The
group focused on F-actin as a candidate. In linear AJs, actin fibers run
parallel to the cell membrane, whilein the punctate they are perpendicular, suggesting a possible influence
by mechanical forces, such as tension, in realigning the cytoskeleton.
Taguchi tested this hypothesis bylaser ablating anchor points targeted by F-actin in punctate AJs. Lossof this tension-generating structure
resulted in immediate upregulation of both EPLIN and E-cadherin, and rearrangement of the adherens
junctions to the linear form. In the converse experiment, when parallel actin fibers were ablated fromlinear AJs, the EPLIN signal was lost, suggesting that this molecule responds to “pulling” forces oncells by accumulating at linear junctions. A subsequent test of this idea using a stretching apparatus toexert tension on epithelial colonies showed that this form of stress stimulates the localization of EPLIN
at the cell-cell junction.

In interior cells, E-cadherin and
EPLIN align to form robust linear
adherens junctions (top), while incells at the periphery, cadherin is
distributed in a punctate fashion(bottom).

The current study provides new insight into how EPLIN plays a mechanosensory role in the remodeling of adherens junctions in response to mechanical forces. The punctate adherens junction may playa stabilizing role by giving flexibility to otherwise rigid sheets of epithelium under stress. “Many types
of epithelial tissue are subject to external mechanical stimuli, but they tend to be quite resilient and remain intact even when pulled and stretched,” says Takeichi. “It seems that these sheets of cells musthave some mechanism for detecting and responding to these kinds of stresses. It will be interesting to
explore whether the function of EPLIN observed in vitro in this study plays roles in vivo as well.”

EPLIN accumulates at linear AJs
where the cell membrane is subject
to tension, whereas it is absent
from the punctate AJS that form incells not exposed to such mechanical stress.

85Self-organized pituitary-liketissue from mouse ES cells

Taisuke KADOSHIMA, Hidetaka SUGA, Mika SOEN, Maki MINAGUCHI
Fumio
MATSUZAKI,Atsushi
KITAJIMA
The possibility that functional, three-dimensional tissues
and organs
may be derived
from pluripotent
cells,
such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), represents one of the grandchallenges of stem cell research, but is also one of the fundamental goals of the emerging field of regenerative medicine. Developmental biology has played a central role in informing such efforts, as it has beenshown that stem cell differentiation can be directed to follow a given lineage pathway by culturing stem cells
in conditions that recapitulate the specific cellular and molecular environment from which such cells normally
emerge during embryogenesis. Intriguingly, recent work has shown that when ES cells are cultured under theappropriate conditions, they can be driven to self-organize into complex, three-dimensional tissue-like structures that closely resemble their physiological counterparts, a remarkable advance for the field.
New work by Hidetaka Suga of the Division of Human Stem Cell Technology, Yoshiki Sasai, Group Directorof the Laboratory for Organogenesis and Neurogenesis, and others has unlocked the most recent achievement in self-organized tissue differentiation, steering mouse ESCs to give rise to tissue closely resembling thehormone-secreting component of the pituitary, known as the adenohypophysis, in vitro. Conducted in collaboration with Yutaka Oiso at the Nagoya University Graduate School of Medicine, this work was publishedin Nature.
The Sasai group has complied an impressive list of achievements in induced differentiation using an ES cell
culture technique dubbed SFEBq (shorthand for “serum-free floating culture of embryoid body-like aggregates with quick re-aggregation”), including high-efficiency methods for the differentiation of dopaminergic,
cerebral cortex, cerebellar Purkinje, and other neuronal cell types. In recent years, refinements of this approach have enabled the first tantalizing insights into the developmental capacity of pluripotent stem cells,
showing their ability to give rise to multi-cell-type populations of cortical and retinal neurons that spontaneously self-organize into stratified tissue nearly identical to that of the developing embryo.
In the group’s most recent work, Suga sought to use SFEBq to derive the secretory component of the pituitary (hypophysis) from mouse ES cells. In embryonic development, the pituitary emerges from a region of the
non-neural (rostral) head ectoderm, adjacent to the anterior neural plate. Guided by molecular interactions,86©2011 Nature Publishing GroupSuga H, et al. Self-formation of functional adenohypophysis in three-dimensional
culture. Nature 480.57-62 (2011)

this placodal region forms an indentation, known as Rathke’s pouch, in the developmental predecessor of
the roof of the mouth, and eventually gives rise to the anterior section of the pituitary, the source of hormones
involved in growth, reproduction and modulating the physiological response to stress.
The Sasai lab had previously reported how a modified version of the SFEBq approach lacking extrinsic
growth factors could spur ES cells to give rise to hypothalamic neurons. Building on this finding, Suga foundby further tweaking the conditions he could steer populations of such stem cells to differentiate simultaneously into the neighboring rostral head ectoderm and hypothalamic neuroectoderm, both of which arerequired to development of the adenohypophysis. Interestingly, the cells in these clusters spontaneously organized into distinct layers resembling those of the corresponding embryonic tissues. This effect appears to
be attributable to the increase of BMP signaling in larger cell aggregates, leading to differentiation into nonneural ectoderm. To test whether co-culture of this ectoderm with hypothalamic tissue would lead to the formation of Rathke’s pouch-like structures, Suga observed the ESC-derived aggregates for nearly two weeks,
and found that by adding the signaling factor Sonic hedgehog, he was able to induce the self-directed formation of this vesicular tissue.
The next important test was whether
this in vitro anterior pituitary anlage
would show similar functionality tothe physiological activity of the adenohypophysis. Of the many criticallyimportant pituitary hormones, theychose adenocorticotropic hormone(ACTH) for their first assay. Previous
research had suggested that Notch
signaling interferes with the development of ACTH-secreting cells, so the group added a Notch blocker to the culture medium and found that
this triggered the generation of such cells at high efficiencies. Following a similar principle, they showed thatby adding Wnt, glucocorticoid and insulin to the culture at appropriate doses and stages, they could obtain
growth hormone-secreting cells in quantity. By varying the recipe of the growth factor cocktail, they were able
to induce other pituitary hormones as well. And, critically, the group was able to show that in vitro hormone
secretion could respond to requisite signals and engage in regulatory feedback just as in the body.

Rathke’s pouch forms from ES
cells cultured using the SFEBq approach (left: bright field image; right:
Lim3::Venus expression)(See CDB website for video)

In a final series of experiments, Suga et al. transplanted the ESC-derived ACTH-secreting tissue into thekidneys of adult mice whose own pituitary glands had been ablated, to see whether it would be capableof compensating for pituitary function in this model system. Within a week of transplantation, these mice
showed strong overall survival, a marked rise in ACTH levels and a concomitant increase in corticosterone (aglucocorticoid hormone stimulated by ACTH) over untransplanted controls, which uniformly weakened anddied within eight weeks of hypophysectomy.
Yoshiki Sasai, leader of the study, commented on this most recent demonstration of the remarkable self-organizing capabilities
of embryonic stem cells in vitro. “We have previously shown how
ES cells can give rise to self-organized, three-dimensional neuronal and sensory tissues, and in this report we describe for the
first time how this principle can be used to generate to an endocrine tissue, suggesting our approach is of general applicability.
Suga, himself an endocrinologist, remarks, “We currently treat
pituitary deficiencies by hormone replacement, but achieving
the correct dosage is not a straightforward problem, given thenaturally fluctuating levels secreted within the body. I am hopefulthat this new finding will lead to further advances in regenerative
medicine in the endocrine system.”

ACTH-secreting cells (red) generated from ESC-derived Rathke’s
pouch

87

New mutant mice for live imaging

Fumio MATSUZAKI, Atsushi
KITAJIMA
TakayaABE
The study of developmental processes frequently demands the ability to study how individual cells
look and act. This has been bolstered in recent years by the development of technologies for fluorescence live imaging, which have made it possible to visually examine and monitor cells in living embryosof various species. Applications of live imaging techniques have been introduced in the mouse as well,
but these are complicated the inability to simultaneously label multiple tissues using discrete markers,
and the random integration of fluorescence genes, which makes it difficult to constrain sites and dosages of expression.
In a step forward for the field, the Laboratory for Animal Resources and Genetic Engineering (LARGE;
Shinichi Aizawa, Laboratory Head) has developed 12 new mutant strains of mice engineered to conditionally express fluorescent markers at seven different cellular sites, such as nucleus or cell membrane.
These new mice, engineered using conditional Cre/loxP recombination, express fluorescent proteins at
levels sufficient for strong visualization, and enable dual labeling by different fluorescent signals at userdetermined developmental sites and time-points. Published in Genesis, these novel mouse resources
are now available for distribution from LARGE.
In tackling the challenges posed by mouse biology for live imaging techniques, the lab focused on the
ROSA26 locus, which enables introduced genes to be expressed nearly ubiquitously in mouse tissues, and can be mutated on both sites in a pair of chromosomes without causing abnormal phenotypes. They took advantage of these properties by introducing genes for fluorescent proteins into the
ROSA26 locus and expressing them conditionally using the Cre/loxP system, which made it possible
to engineer a series of mice specially adapted for use in live imaging studies.

88©2011 John Wiley & Sons, Inc.
Shioi G, et al. A mouse reporter line to conditionally mark nuclei and cell
membranes for in vivo live-imaging. Genesis 49.570-8 (2011)
Abe T, et al. Establishment of conditional reporter mouse lines at ROSA26 locus
for live cell imaging. Genesis 49.579-90 (2011)

They kicked off the effort by fusing genes for any of six fluorescence signals, such as EGFP (green) andmCherry (red), with genetic markers of subcellular sites, such as nucleus and mitochondria, yielding a
total of 28 reporter genes. Testing these for specificity and strength of expression narrowed the field
to 16 candidates, which were then introduced by homologous recombination into the ROSA26 locus
in mouse embryonic stem cells (ESCs) accompanied by a stop sequence flanked by loxP sequences,making it possible to conditionally switch on their expression only in cells expressing Cre.
They next crossed these 16 reportermouse lines with mice designed to express Cre constitutively, and examined
their patterns of expression in E7.5 embryos. Twelve of the 16 expressed thereporter genes at appropriate sites andsufficient quantities to enable their use inimaging applications. For their next step,
the team crossed mice that heterozygously expressed Venus (red) and mCherry (green) in the cell membrane and nucleus, respectively,
thereby generating dual-labeled embryos, and making it possible to study the detailed movements ofnuclei in from stage E6.5 to E7.5.
As a final test of the specificity of expression, they crossed mice expressing Venus (green) in the cell membranewith other designed to express Cre
only in the notochord. Observing theresulting embryos at E8.5 of development showed clear membrane expression of Venus in a tissue-specific manner.

Conditional expression of Venus(green) at the notochord (left);the image at right shows the samesite at higher magnification, nuclei
stained blue by DAPI

List of 12 fluorescent reportermouse lines

The group published a second report in the same issue of Genesis, describing a proof-of-concept
application of a dual-labeled line expressing mCherry in nucleus and EGPF in the plasma membrane.
Again using the Cre/loxP system, they were able to achieve the specific expression of the genetic
reporters for these two organelles only in fetal Sertoli cells, indicating the usefulness of these new resources in live imaging studies.

Expression patterns in E7.5 embryo
in sagittal sections (top) and visceralendoderm (bottom)

89

Spindle mechanism behind
nuclear asymmetry
A host of factors, both internal and external, areinvolved in determining whether a mitotic cell
division will give rise to daughter cells of like ordifferent types. Extrinsic signals can exert a variety of effects within the cell, from directly regulating the transcription of genes to remodelingthe cytoskeleton. But how structural changes
such as the latter translate to downstream effects on cell symmetry remains only poorly understood.
Recent work by Kenji Sugioka and others in
the Lab for Cell Fate Decision (Hitoshi Sawa,
Team Leader) has revealed a new mechanism
by which external Wnt signaling generates mitotic spindle asymmetry, and thereby promotes
asymmetric localization of ß-catenin within the
nucleus. Published in Cell, this work reveals
a novel means by which external signals canaffect cellular properties in a cytoskeletonmediated fashion in the roundworm C. elegans.
Both Sawa and Sugioka have since moved to
the National Institute of Genetics in Mishima,
Japan.
Early in roundworm development, a cell referred
to as EMS receives external Wnt signals from aneighboring cell located posterior to it. Previous
work has showed that this asymmetric signal
causes WRM-1 (a C. elegans homolog of ßcatenin) to accumulate selectively at the anterior cortex of the EMS cell, which subsequently
sets up an asymmetry during mitosis resulting
in the accumulation of WRM-1 in the posterior
daughter cell’s nucleus, and a second factor,
POP-1, in the nucleus of the anterior daughter.
The upshot of these allocations is that the anterior and posterior progeny take on different
development fates, with the anterior daughter
giving rise to muscle, and the posterior, gut.

Fumio MATSUZAKI, Atsushi
KITAJIMA
Kenji SUGIOKA

90

Suspecting that the cytoskeletal component
known as microtubules might play a role inestablishing this nuclear asymmetry, Sugioka
treated EMS cells with a microtubule-disrupting
chemical, and found that this indeed appeared to affect asymmetric WRM-1 and POP1 localization in treated nuclei. Looking moreclosely at the behavior of microtubules during
EMS cell division, the team found that astral
microtubules increased in the cells’ anterior as©2011 Elsevier B.V.
Sugioka K, et al. Wnt regulates spindle asymmetry to generate asymmetric nuclear
beta-catenin in C. elegans. Cell 146.942-54 (2011)

mitosis progressed, with the greatest anterior-posterior differential occurring at the end stages of division. Inhibiting Wnt downstream factors WRM-1 and APR-1 by RNAi resulted in a decrease in anterior
microtubules, indicating that their asymmetric localization is regulated by Wnt signaling.
APR-1 co-localizes with WRM-1 at the anterior cell cortex, and it has been suggested that its mammalian counterpart (APC) has a stabilizing effect on microtubules. Sugioka used a GFP fusion protein
to examine the distribution of APR-1 in dividing EMS cells. Early in mitosis there was no apparent
asymmetry in its localization, but it gradually accumulated in the anterior side of the cell at later stages,
strongly reminiscent of the changes in microtubule distribution observed the previous experiment. Inhibition of Wnt also reduced or eliminated this asymmetry, strongly suggesting that this signaling pathway controls the development of asymmetry by inducing unilateral localization of APR-1, leading to theasymmetric accumulation of microtubules. A subsequent experiment showed that APR-1 binds to andstabilizes the plus-ends of microtubules at the anterior cortex, suggesting a mechanistic basis for thiseffect.
The team next used an optical (laser) disruption system to directly disrupt microtubule formation andobserve the effects on the asymmetric nuclear localization of WRM-1 and POP-1. Perturbing anterior
microtubule formation resulted in loss of asymmetric accumulation of these factors in nuclei, while
dsirupting posterior microtubules conversely increased this asymmetry. Interestingly, when they used
the same technique to induce microtubule asymmetry in Wnt mutants in which microtubules do notform asymmetrically, they found that the nuclear asymmetry of WRM-1 and POP-1 was restored. Thisset of findings makes a strong case for the role of microtubules in establishing the asymmetric nuclear
accumulation of cell fate determinants in dividing C. elegans cells.
“It will be interesting to find out exactly what role microtubules are playing in conveying nuclear factors
and establishing asymmetry,” says Sawa. “We will also be studying the question of how general thisregulatory mechanism is to other biological contexts.”

Microtubules increase in anteriorside of asymmetrically dividing EMS
cell (top left); asymmetry is lost in
Wnt mutant (top right). Only theposterior daughter in EMS cell division expresses gut marker end-1(red), but again this asymmetry is
lost in the Wnt mutant.

91

Animal Resource andGenetic Engineering
Mutant mice are an important resource used in biological and medical research. The quality and efficacy of research isgreatly dependent on how easily mutant mice can be generated, propagated, and housed, but researchers today tend not toengage in routine generation of mutant mice. This laboratory’s major function is to develop mutant mice for research in thefields 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

Technical Staff

Shinichi AIZAWA

Takaya ABE
Kana BANDO
Naoko HATAMOTO
MichikoHIGASHIKAWA
Mari KANEKO
Taketsugu MITSU
Megumi WATASE

Research Specialist

Hiroshi KIYONARI
Go SHIOI
Research Scientist

Shuichi KANI
Hirotaka TAO
Visiting Scientist

Toshihiko FUJIMORIThe 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.

A mouse line expressing H2B-EGFP ubiquitously

Animal Resource Unit
Kazuki NAKAO Ph.D.

Staff
Unit Leader

Technical Staff

Kazuki NAKAO

Kenichi INOUE
Yuki KANEKO
Takuya KAWADA
Miho SATO
Mayo SHIGETA
Aki SHIRAISHI
Norie TANAKA
TomokoTOKUNAGA
Sachi YAKAWA
Junko YOSHIDA
Sayaka YOSHIMURA

Attending Veterinarian

Naoko KAGIYAMA
Research Specialist

Hiroshi KIYONARIThe 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 andreceiving 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 forcolony expansion and strain cryopreservation.
We also develop technologies for the study of
reproductive biology.

Assistant

Yuki TSUJI

Publications
Abe T, et al. Establishment of conditional reporter mouselines at ROSA26 locus for live cell imaging. Genesis
49.579-90 (2011)

Kuroda K, et al. Behavioral alterations associated withtargeted disruption of exons 2 and 3 of the Disc1 gene in
the mouse. Hum Mol Genet 20.4666-83 (2011)

Shioi G, et al. A mouse reporter line to conditionallymark nuclei and cell membranes for in vivo live-imaging.
Genesis 49.570-8 (2011)

Doi M, et al. Circadian regulation of intracellular G-protein
signalling mediates intercellular synchrony and rhythmicity in the suprachiasmatic nucleus. Nat Commun 2.327(2011)

Sato Y, et al. Cartilage acidic protein-1B (LOTUS), an
endogenous Nogo receptor antagonist for axon tract
formation. Science 333.769-73 (2011)
Kobayashi M, et al. Decrease in topoisomerase I is
responsible for activation-induced cytidine deaminase(AID)-dependent somatic hypermutation. Proc Natl Acad
Sci U S A 108.19305-10 (2011)92

Injection of C57BL/6 ES(HK3i) cells into 8 cellstage embryo for chimeric mouse productionElectron Microscope
Detailed knowledge of the shapes of individual cells and fine subcellular structures is vital to the understanding of biological structures and organization. In developmental processes in particular, changes in cell morphology and behavior playfundamental roles. The Electron Microscope Laboratory provides technical assistance and expert guidance in the use oftransmission 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, assistancein the use of 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 withspecific advice as to the 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, andthe biophysical aspects of morphological rearrangements in epithelial
cells.

Staff
Laboratory Head

Research Fellow

Shigenobu
YONEMURA

Akira ONODERA
Ayuko SAKANETechnical Staff

Assistant

Hanako HAYASHI
Kisa KAKIGUCHI
Kazuyo MISAKIMai SHIBATA

Visiting Scientist

Masatsune
TSUJIOKA

Publications
Suga H, et al. Self-formation of functional adenohypophysis in three-dimensional culture. Nature
480.57-62 (2011)
Yonemura S. A mechanism of mechanotransduction at the cell-cell interface: emergence ofalpha-catenin as the center of a force-balancing
mechanism for morphogenesis in multicellularorganisms. Bioessays 33.732-6 (2011)
Yonemura S. Cadherin-actin interactions at adherens junctions. Curr Opin Cell Biol 23.515-22(2011)
Otani T, et al. IKKepsilon regulates cell elongation
through recycling endosome shuttling. Dev Cell
20.219-32 (2011)
Yonemura S, et al. alpha-Catenin as a tension
transducer that induces adherens junction development. Nat Cell Biol 12.533-42 (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).

93

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 a secure environment for the effective use of these resources.

Shigeo HAYASHI Ph.D.

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

Staff
Unit Leader

Part-Time Staff

Yuko KIYOSUE

Satoko NAKAMURA

Research scientist

Agency Staff

Togo SHIMOZAWA

Emiko MAEKAWA

Technical Staff

Tomoko HAMAJI

Publications
Hotta A, et al. Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cellbasal 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

94

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

Fumio MATSUZAKI Ph.D.

Genome Resource and Analysis Unit
Fumio MATSUZAKI 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
perform gene expression analysis and high-throughput gene screening
using a DNA sequencing system that combines traditional DNA sequencers with the next generation sequencers, making it possible to apply it toa wide range of genomic and epigenomic studies. We can also custommake DNA microarray systems. Gene resources derived from a varietyof 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, as well as providing solid support, we
aim to respond flexibly to the needs of each research request.

Staff
Unit Leader

Visiting Scientist

Fumio MATSUZAKI

Osamu NISHIMURA

Hiroshi TARUI(~ MARCH 2011)

Assistant

Technical Staff

Chiharu
TANEGASHIMA

Tetsutaro HAYASHI
Kazu ITOMI
Asuka MOMIYAMA
Kaori TATSUMI

Publications
Yazawa S, et al. Planarian Hedgehog/Patchedestablishes anterior-posterior polarity by regulating Wnt signaling. Proc Natl Acad Sci U S A 106.
22329-34 (2009)
Maki N, et al. Expression of stem cell pluripotency factors during regeneration in newts. Dev
Dyn 238. 1613-6 (2009)
Egger B, et al. To be or not to be a flatworm: theacoel controversy. PLoS One 4. 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
284. 23260-71 (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 to accelerate the Center’s research. We are striving to implement
two types of technologies: 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, and developing threedimensional expression analysis in organs.

Staff
Unit Leader

Technical Staff

Hiroki R. UEDA

Junko SAKAI
Kenichiro UNO

Special Postdoctoral
Researcher

Yohei SASAGAWA

Assistant

Yuka TOKUMINE

Research Specialist

Takeya KASUKAWA
Itoshi NIKAIDO

Publications
Kasukawa T, et al. Quantitative expression profile
of distinct functional regions in the adult mousebrain. PLoS One 6.e23228 (2011)
Masumoto K.H, et al. Acute induction of Eya3 bylate-night light stimulation triggers TSHβ expression in photoperiodism. Curr Biol 20. 2199-206(2010)
Isojima Y, et al. CKIε/δ-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock.
Proc Natl Acad Sci U S A 106. 15744-9 (2009)
Kawaguchi A, et al. Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis. Development 135. 311324 (2008)95

Proteomics
The identification of proteins from trace amounts of biologically important protein complexes is a powerful technique andhas 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
Zou P, et al. p57(Kip2) and p27(Kip1) cooperate
to maintain hematopoietic stem cell quiescencethrough interactions with Hsc70. Cell Stem Cell
9.247-61 (2011)
Kitano E., et al. Roles of fission yeast Grc3
protein in ribosomal RNA processing andheterochromatic gene silencing. J Biol Chem
286.15391-402 (2011)

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.

Hiragami-Hamada K, et al. N-terminal phosphorylation of HP1α promotes its chromatin binding.
Mol Cell Biol 31. 1186-200 (2011)
Otani T, et al. IKKe regulates cell elongation
through recycling endosome shuttling Dev. Cell
20.219-32 (2011)
Hayakawa T, et al. MRG15 binds directly to
PALB2 and stimulates homology-directed repairof 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)

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

96

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 andanalysis 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 a full spectrum of support services tolabs within the CDB and throughout Japan with an interest 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 to establish a solid scientific foundation for regenerative medicine.

Staff
Unit Leader

Technical Staff

Yoshiki SASAI

Michiru
MATSUMURAIMOTO
Maki MINAGUCHI

Deputy Unit Leader

Masatoshi OHGUSHI
Research Specialist

Hiroyuki KITAJIMA
Visiting Scientist

Part-Time Staff

Yoshinori NAKAI

Tokushige NAKANO

Publications
Ohgushi M. and Sasai Y. Lonely death dance ofhuman pluripotent stem cells: ROCKing between
metastable cell states. Trends Cell Biol 21.27482 (2011)
Ohgushi M, et al. Molecular pathway and cellstate responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem
Cell 7.225-39 (2010)

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

Staff
Unit Leader

Technical Staff

Yoshiki SASAI

Eriko SAKAKURA

Deputy Unit Leader

Junior Research
Associate

Mototsugu EIRAKU
Research Scientist

Yuiko HASEGAWA

Hidetaka SUGA

Publications
Eiraku M, et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature
472, 51-56. (2011)
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)

Science Policy and Ethics Studies Unit
Douglas SIPPThe field of stem cell research has been subject to legal, social, andethical tensions across a broad range of issues, from the research useof human embryos to the optimization of pathways for the translation ofbasic 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 ofhuman stem cell research.

Staff
Unit Leader

Douglas SIPP

Publications
Cheng T. et al. Stem cell research on the rise in
China. Cell Stem Cell (2012)
Sipp D. Global challenges in stem cell researchand the many roads ahead. Neuron 70.573-6(2011)
Sipp D. The unregulated commercialization ofstem cell treatments: a global perspective. Front
Med 5.348-55 (2011)97

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 range of seasonal activities, all withina short train ride or drive from the center of the city. Japan’s
renowned public transport system allows rapid and convenient access to both local and national destinations, and themany area airports, including the Kobe Airport located lessthan 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 andapartments are available to suit all budgets.

The bustling heart of Kobe includes a variety of distinct
neighborhoods. The downtown area of Sannomiya sits
square in the city’s center, and its range of international
restaurants and late-night 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 centerlies 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 andseas, with easy access to many natural spots. A short driveor bus ride takes you to the island of Awaji, with its beaches
and first-rate seafood, and hiking trails crisscross the forested mountains that span the entire length of the city,
beckoning hikers and picnickers to enjoy a day in the fresh
air. The city is also dotted with parks that come into bloomat 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 ofmany of the country’s most popular destinations. Kyoto, theancient capital, is only an hour away by train, making it possible to enjoy some of Japan’s most popular historic sites
and cultural assets. Nara, another early city, features herdsof tame deer in the precincts of some of the world’s oldest
wooden buildings. Complementing the old-world style ofthese two cities is Japan’s second city of Osaka, which offers a hip and modern take on urban living.

RIKEN Kobe Institute
The RIKEN Kobe Institute was established in April 2000 as an organizational 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 to regenerative medicine. In April 2007, the Kobe Institute welcomed a new institution, the Molecular Imaging Research Program, which carries out research into bioimaging technologies such as positron emission tomography. In autumn 2008, this program was redesignated as the Center for Molecular
Imaging Science (CMIS). In April 2011, the Kobe Institute welcomed its newest research center, the Quantitative Biology
Center, which will focus on systems, simulation, and computational approaches to the life sciences phenomena.
The Kobe Institute seeks both to help develop a foundation of knowledge into biological phenomena and, through translational research efforts conducted with the Institute for Biomedical Research and Innovation and other nearby institutions, tohelp bridge basic science to novel applications in medical therapy and drug discovery, thereby contributing to the health andwelfare of the people of Japan.
The Kobe Institute administrative structure comprises the Research Promotion Division and the Safety Center.

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

Institutional Review Board

100

Center for
Molecular Imaging Science
The Center for Molecular Imaging Science (CMIS)
was established in October 2008 as an expansion of
the Molecular Imaging Research Program, which waslaunched by RIKEN in July 2005. Molecular imaging
is the only non-invasive technique for quantitative
monitoring of changes in concentration or distributionof target molecules in living organisms. This is madepossible 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 ontranslational projects that span basic research, suchas 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.

Quantitative Biology Center
The RIKEN Quantitative Biology Center (QBiC) focuses on the complex spatiotemporal relationships
between components of biological systems throughinnovative measurement, analysis, and modelingtechnologies and techniques. These will be used to
predict, design, simulate, and manipulate cellular
phenomena, which may help to revolutionize research and applications in the life sciences, including
such fields as regenerative medicine and diagnostics.

2011 CDB Budget
Overhead andadministration

Personnel

1,501

1,412

Total: 3,818(million yen)

Research expenses

905
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 signif icant component of theCenter’ s total funding every year, and were particularly significant in the fiscal year ending March 2011, representing an additional 955 million yen in research, facilities, and operating funds.

Private industry

66

RIKEN internal grants

34

Foundations, etc.

23
Other government
agencies

126

Total: 955

Research Promotion Division
The Kobe Institute Research Promotion Division(KRPD) provides a full range of administrative services required by CDB labs, with sections 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 disaster prevention measures,and educates staff on and ensures complianceswith 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

706(million yen)

2011 CDB Staff
Laboratory heads
Deputy unit leaders
Research scientists
Research associates
Technical staff
Assistants
Visiting scientists
Student trainees
Part-time staff
Other

25
2
105
6
97
21
113
39
34
22

Total

464

Research Promotion Division**Administrative staff for Kobe Institute

83

101

2011 CDB Symposium

Epigenetic Landscapein Development and Disease
March 14 - 15, 2011
The RIKEN CDB hosted its ninth annual symposium, “Epigenetic landscape in development and disease” on March
14–15. This year’s symposium was held on an abbreviatedtwo-day schedule in the aftermath of the Great Tohoku
Earthquake and tsunami that struck northeastern Japan on
March 11, and opened with a moment of silence for thoseaffected by the disaster.
Although a number of speakers and attendees were forced
to cancel their participation due to travel disruptions andother concerns, more than 180 people were able to gather
to share and discuss the latest developments in the study of
epigenetics as it relates to ontogeny and pathology. In addition to the 21 talks, each day included a poster session inwhich nearly 70 poster presenters discussed their work.
The CDB symposium series, launched in 2003, was established as a forum for addressing diverse aspects of developmental biology and the mechanisms of regeneration andaims to promote the free, timely and borderless exchange of
research achievements.

Session 1

Jun-ichi Nakayama (RIKEN CDB, Japan)
Klaus Hansen (University of Copenhagen, Denmark)Session 2

Tetsuji Kakutani (National Institute of Genetics, Japan)
Rudolf Jaenisch(Whitehead Institute for Biomedical Research & MIT, USA)
Session 3

Ichiro Hiratani(National Institute of Genetics, Japan)

Kazuo Yamagata(RIKEN Center for Developmental Biology, Japan)

Tetsuya J. Kobayashi (The University of Tokyo, Japan)
Session 4

Mark L. Siegal (New York University, USA)
Richard A. Young(Whitehead Institute for Biomedical Research & MIT, USA)

Hiroki R. Ueda (RIKEN CDB, Japan)
Session 5

Minoru S.H. Ko (National Institutes of Health, USA)
Emma Whitelaw(Queensland Institute of Medical Research, Australia)

Shahragim Tajbakhsh (Institut Pasteur, France)1022012 CDB Symposium

Quantitative Developmental
Biology
March 26 – 28, 2012
The tenth annual symposium “Quantitative Developmental Biology”
will be held on March 26 – 28, 2012 in the CDB Auditorium. We plan
to discuss experimental, computational, and theoretical approaches toward understanding principles of development. By facilitating
communication between the leaders in these fields and promising
younger scientists at a collegial forum, we hope to create a uniqueatmosphere that provides a comprehensive perspective on how we
can apply quautitative approaches to elucidate principles of development, which we hope will generate fresh perspectives and newinteractions.

Invited Speakers

Buzz Baum

MRC Laboratory for Molecular Cell Biology, UK

Christopher Chen
University of Pennsylvania, USA

Cheng Ming Chuong

University of Southern California, USA

Lance Davidson

University of Pittsburgh, USA

Suzanne Eaton

Max Planck Institute of Molecular Cell Biology and Genetics, Germany

Darren Gilmour

European Molecular Biology Laboratory, Germany

Shigeo Hayashi

RIKEN Center for Developmental Biology, Japan

Frank Jülicher

Max Planck Institute for the Physics of Complex Systems, Germany

Shigeru Kondo

Osaka University, Japan

Edwin M. Munro

The University of Chicago, USA

Eugene W. Myers

HHMI Janelia Farm Research Campus, USA

Stuart A. Newman
Session 6

Hitoshi Niwa (RIKEN CDB, Japan)
Berthold Göttgens (University of Cambridge, UK)
Session 7

Rudolf Jaenisch(Whitehead Institute for Biomedical Research & MIT, USA)

Michael Kagey(Whitehead Institute for Biomedical Research, USA)

Yanina Tsenkina (University of Edinburgh, UK)
Yan Fung Wong (RIKEN CDB, Japan)
Session 8

New York Medical College, USA

Shuichi Onami

RIKEN Quantitative Biology Center , Japan

Przemyslaw Prusinkiewicz
The University of Calgary, Canada

Satoshi Sawai

The University of Tokyo, Japan

Ralf Schnabel
TU Braunschweig, Germany

James Sharpe

Centre for Genomic Regulation, Spain

Kiyoe Ura (Osaka University, Japan)
Kristian Helin (University of Copenhagen, Denmark)Tatsuo Shibata

Session 9
Austin Smith

Tadashi Uemura(Wellcome Trust Centre for Stem Cell Research, UK)

RIKEN Center for Developmental Biology, Japan

Kyoto University, Japan

Cornelis Weijer

University of Dundee, UK

Orion Weiner

University of California, San Francisco, USA

103

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 600 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 2011.
Date

104

Title

Speaker

01-11

Evolutionary design of robust oscillatory genetic networks

Yasuaki KOBAYASHI

01-24

Physical approach to the origin of life: How nature separates molecules

Yusuke T. MAEDA

01-25

Building the vertebrate body: molecular mechanisms of progenitor cellmaintenance and lineage specification

Benjamin L. MARTIN

01-26

Transcriptional control of midbrain dopaminergic neuron development

Siew-Lan ANG

02-01

Towards a gene regulatory network for sense organ progenitors

Andrea STREIT

02-02

Molecular insights into the mammalian circadian clock: A link to nuclear receptormediated transcription and flexibility of its transcriptional networks

Jürgen A. RIPPERGER

02-08

Mathematical approaches to developmental biology; geometrical analysis oforgan deformation and coding design of positional information

Yoshihiro MORISHITA

02-08

Hydroxylation regulates chromatin demethylation

Yuichi TSUKADA

03-09

Using patient-derived iPS cells to investigate cellular phenotypes in Timothy
syndrome

Masayuki YAZAWA

04-11

Analysis of germ cell immortality

Shawn AHMED

04-14

Estimating the dynamics of forces during morphogenesis

Shuji ISHIHARA

04-21

How do cell-surface molecules specify synaptic-layer targeting in the visualsystem?

Takashi SUZUKI

05-16

Mechanical forces due to cell destruction: Biophysical investigation of the role ofapoptosis in tissue dynamics during embryogenesis

Yusuke TOYAMA

05-17

Polarity is destiny

Henrik SEMB

06-01

The role of chromatin orchestra in circadian rhythms

Satchidananda PANDA

06-09

Stem cells and the origin of prostate cancer

Michael M. SHEN

06-09

Complete 3D kinetochore tracking reveals error-prone homologous chromosomebiorientation during mouse oocyte meiosis

Tomoya KITAJIMA

06-10

Genome-wide RNAi screen identifies novel genes required for the response tohypoxia

Pablo WAPPNER

06-13

Molecular mechanisms of small RNA pathways in Drosophila

Katsutomo OKAMURA

06-16

Introduction to CSH Asia/CSHL and the importance of effective communication inscience

Maoyen CHI

Date

Title

Speaker

06-23

Synthetic cell biology: Visualizing and manipulating cell signaling

Takanari INOUE

07-11

Sleep/wakefulness control using optogenetics in mice

Akihiro YAMANAKA

07-20

Dissecting microRNA function using mouse genetics and genomics

Stefan A. MULJO

07-26

The contributions of palaeontology to the study of development in a molecular
world

Marcelo SÁNCHEZ

08-01

What happened to the genome of vertebrate closest living relatives?

Daniel CHOURROUT

08-17

A matter of commitment: TGFβ signaling in the regulation of neural stem/
progenitor cells

Yasuhide FURUTA

09-13

Multifaceted analysis of the Drosophila epithelial barrier junction, the septate
junction

Kenzi OSHIMA

09-13

The retina as a model to explore different pools of neuronal progenitors

Caren NORDEN

09-14

Immunoglobulin gene conversion and somatic hypermutation: regulation,
evolution and biotechnology

Hiroshi ARAKAWA

09-15

Dynamics of cell cycle transitions

Attila CSIKASZ-NAGY

10-11

Inter-axonal communication defines presynaptic tiling in C. elegans

Kota MIZUMOTO

10-17

Non-conservation of developmental ‘toolkit’ genes

Shigehiro KURAKU

10-19

Breaking symmetry: Polarization of the C. elegans zygote

Fumio MOTEGI

10-24

Establishment of intestinal stem cell culture system: from mouse to humanToshiro SATO

11-21

Modeling ablation experiments at the cell and tissue level

Philippe MARCQ

11-22

The zebrafish lateral line as a model to study cell migration, morphogenesis andregeneration

Tatjana PIOTROWSKI

12-09

The functional interplay of chromatin insulators and transcription

Olivier CUVIER

12-12

Neuronal mechanisms that induce memories in the fly brain

Hiromu TANIMOTO

12-28

Neuronal cilia : Regulatory mechanism of ciliary length and function in feeding
behavior

Yoshihiro OMORI

105

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 practical application.
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 an interest in learning more about RIKEN’s organization, activities andhistory 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/

106Sendai
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RIKEN
http://www.riken.jp/

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

On The CoverElongation of giant mitochondria showing increasein length while volume remains constant.

Printed in Japan using soy inks, waterless printing
methods, and paper from sustainable resources.
RIKEN 2012-033