RIKEN Center for Developmental Biology

2012 Annual Repor
ReportRepo
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RIKEN Center for
Developmental
Biology

2012
AnnualReportContents
Message from the Director

2

Organizational Chart

4

2012 at the CDB

6News Highlights 1

10

Labs
Vertebrate Body Plan

24

Shinichi AIZAWA
Neuronal Differentiation
and Regeneration

25

Hideki ENOMOTO
Tissue Microenvironment

26

Hironobu FUJIWARA
Neocortical Development

27

Carina HANASHIMA
Morphogenetic Signaling

28

Shigeo HAYASHI
Sensory Circuit Formation

29

Takeshi IMAI
Chromosome Segregation

30

Tomoya KITAJIMA
Histogenetic Dynamics

31

Erina KURANAGA
Evolutionary Morphology

32

Shigeru KURATANI
Sensory Development

33

Raj LADHERThe RIKEN Center for Developmental Biology was launchedin April 2000 under the auspices of the Millennium Project
research initiative, which was established to drive researchin areas of vital importance to both Japan and the world in
the 21st century. The challenges of aging society wereamong the issues addressed, and developmental biology
was included within this remit as a scientific field with thepotential to contribute to the development of regenerative
medicine.
Located in Kobe, Japan, the Center’s research program isdedicated to developing a better understanding offundamental processes of animal development at theNews Highlights 2

34

Labs
Cell Asymmetry

48
49
50
51
52
53

70

Physical Biology

71

Retinal Regeneration

72

Cell Adhesion and Tissue Patterning

73

Masatoshi TAKEICHI
54

Takashi NISHIMURA
Pluripotent Stem Cell Studies

Early Embryogenesis

Masayo TAKAHASHI

Shin-Ichi NISHIKAWA
Growth Control Signaling

69

Tatsuo SHIBATA

Jun-ichi NAKAYAMA
Stem Cell Biology

Organogenesis and Neurogenesis

Guojun SHENG

Akira NAKAMURA
Chromatin Dynamics

68

Yoshiki SASAI

Yoshihiro MORISHITA
Germline Development

Mammalian Epigenetic Studies
Masaki OKANO

Mitsuru MORIMOTO
Developmental Morphogeometry

56

Labs

Fumio MATSUZAKI
Lung Development

News Highlights 3

Systems Biology

74

Hiroki R. UEDA
55

Hitoshi NIWA

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 talent andexpertise. The CDB also benefits from belonging to Japan’slargest basic scientific research organization, RIKEN, aswell as its close ties to graduate and medical schools andresearch institutes in Japan and other countries, giving ourscientists access to a broad network of talent andtechnology across the country and around the world.

Genomic Reprogramming

75

Teruhiko WAKAYAMANews Highlights 476

Supporting Laboratories

86

RIKEN Kobe Institute94

CDB Symposium

96

CDB Seminars

98

About RIKEN

100

RIKEN Campuses

101Message from the Director
December 2012 marked the tenth anniversary of the completion of our second main research building at the RIKEN Center for Developmental Biology, a date which also in a way represents the unofficial start of full-scale research activities at the Center. Over thecourse of the past decade, the CDB has grown and matured as an institute committed to a deeper understanding of fundamental
processes and mechanisms that underlie animal development, organogenesis, regeneration, and stem cell biology, with an eye tosupporting the development of regenerative medicine by providing a solid scientific foundation for translational studies.

The past year has seen a great many changes in the CDB’s laboratory programs, with several labs moving on after two successful
five-year terms at the Center, and six new teams and units joining our institute since January. These many new faces bring a new
injection of talent, energy, and ideas to the research staff, in line with our philosophy of balancing continuity with change, and diversity of perspectives and approaches with a common cause of pushing back the frontiers of knowledge and contributing to thegreater good.

Even as these transitions have played out in recent years, our scientists have remained consistently innovative and productive. In
2012, our labs published studies in such diverse areas as somatic cell nuclear transfer, the control of cell polarity, orientation and differentiation, cytoskeleton dynamics, and the phylogenetic origins of vertebrates. In addition to these advances in fundamental biology,
we are proud to support efforts to translate stem cell biology into clinical applications in full compliance with the national regulatory
framework for stem cell clinical research. The award of the 2012 Nobel Prize in Physiology or Medicine to Sir John Gurdon for his work
on cloning by SCNT, and Shinya Yamanaka for his development of induced pluripotent stem cells lent further impetus to the field, and
it is gratifying that the Japanese government has redoubled its support for the development of regenerative medicine and other applications of stem cells.

This past year, the Center expanded its programs for engaging with students at all levels of higher education. We now hold summer
and winter courses for local high school students, as well as practical workshops for high school science teachers in the fall. In August,we established a lab internship program for undergrads, complementing our annual intensive lecture program for graduate andmedical school students.

The upcoming year will be a transformative one for RIKEN, as it reorganizes its research system at the start of a new 5-year plan withgreater focus on multidisciplinary collaboration and the development of applications to address challenges in energy, environment,
health, and human welfare. The Center for Developmental Biology will continue to focus on its core strengths of developmental biology, regeneration and stem cell research, but with a keener eye to develop new quantitative approaches, and closer cooperation withlabs throughout the RIKEN system, enabling us to take the fullest advantage of the extraordinary resources and facilities this institution
provides.

We will also look to strengthen even further our ties with colleagues around the world, as evidenced by the new cooperative research
agreements the CDN entered into with three institutes in Barcelona, Spain in the past year. Our research staff is increasingly diverse,with more than one in ten of our scientists coming from overseas, and our very successful annual symposium marked its tenth anniversary as well. We look forward to the continued collaboration of the international community in our shared endeavors, and we thankour colleagues from around the world for their continued support.

The upcoming year promises to be an exciting one, for the CDB, for Japan, and for the world. We hope to welcome many more newfaces here in Kobe, and to see the start of important new undertakings in line with our research mission and the nation’s plan to contribute to human society through scientific progress and technology development. It is with a combined sense of profound responsibility and wonderful opportunity that we have undertaken these challenges, and I would like to thank all of our many colleagues andsupporters for their cooperation and encouragement in these efforts.

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 Quantitative Biology Center (QBiC) the Kobe Research
Promotion Division, which provides administrative services and support, and the institutional Safety Center. The
CDB is home to a total of 32 laboratories in its Core Research Program (7 groups), Center Director’s Strategic
Programs (4 projects), Creative Research Promoting Program (14 teams), Regenerative Medicine Development
Program (1 project) and Supporting Laboratories (6 labs).
The CDB Director is assisted by two Deputy Directors andis advised by the Advisory Council and the Institutional
Review Board.

Center for Molecular Imaging Science
Quantitative Biology Center
HPCI Program for Computational Life Sciences
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, as well as CDB research leaders, and meets regularly toreview and discuss investigations with potential ethical, legal, social
or public health and 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
the Center’s performance and make recommendations on the
direction of its programs and governance. The DBAC reports its
findings to aid in guiding future activities and decision-making.
The full texts of DBAC reports can be viewed on the CDB website(www.cdb.riken.jp).
The nine-member Council comprises top international scientists
working in developmental biology, stem cells and related fields.

4

The Core Program aims to promote highly innovative developmental and regeneration studies in a
strategic and multi-disciplinary manner. This program focuses on the main themes of the CDB: themechanisms of development, organogenesis, andstrengthening the scientific basis of regenerative
medicine through the study of stem cells andregeneration.

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 provides for concentrated focus onpriority areas of research determined by the CDB
Center Director. The initial 10-year projects will
focus on stem cell and systems 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.

Developmental Morphogeometry
Yoshihiro MORISHITA Ph.D.

Austin Smith

Haifan Lin

Christopher Wylie

Toshio Suda

Margaret Buckingham

Ryoichiro Kageyama

Patrick Tam

Hiroshi Hamada

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

University of Sydney, Australia

Stephen Cohen

Institute of Molecular
and Cell Biology - A*STAR, Singapore

Yale university, USA
Keio University, Japan
Kyoto University, Japan

Osaka University, Japan

Director Masatoshi TAKEICHI
Deputy Directors Shin-Ichi NISHIKAWA, Shinichi AIZAWA

Creative Research
Promoting Program

Supporting
Laboratories

The Creative Research Promoting Program provides
solid support to encourage young researchers to carryout innovative and independent research plans. The
teams are afforded a great deal of flexibility and control
in regard to projects, budget use, and lab size. The
program also emphasizes 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 development of
new lab equipment and analytic software, and provide
training on the use of research technologies.

Neuronal Differentiation and Regeneration

Animal Resources and Genetic Engineering

Hideki ENOMOTO M.D., Ph.D.

Yasuhide FURUTA Ph.D.

Tissue Microenvironment

Genetic Engineering Unit

Hironobu FUJIWARA Ph.D.

Shinichi AIZAWA Ph.D.

Neocortical Development

Animal Resource Unit

Carina HANASHIMA Ph.D.

Kazuki NAKAO Ph.D.

Sensory Circuit Formation

Electron Microscope

Takeshi IMAI Ph.D.

Shigenobu YONEMURA Ph.D.

Chromosome Segregation

Bio-imaging

Tomoya KITAJIMA Ph.D.

Shigeo HAYASHI Ph.D.

Histogentic Dynamics
Erina KURANAGA Ph.D.

Optical Image Analysis Unit
Yuko KIYOSUE Ph.D.

Sensory Development

Genomics

Raj LADHER Ph.D.

Fumio MATSUZAKI Ph.D.

Lung Development

Genome Resource and Analysis Unit

Mitsuru MORIMOTO Ph.D.

Shigehiro KURAKU Ph.D.

Germline Development

Functional Genomics Unit

Akira NAKAMURA Ph.D.

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

Chromatin Dynamics

Proteomics

Jun-ichi NAKAYAMA Ph.D.

Shigeo HAYASHI Ph.D.

Growth Control Signaling
Takashi NISHIMURA Ph.D.

Mass Spectrometry Analysis Unit
Akira NAKAMURA Ph.D.

Mammalian Epigenetic Studies

Division of Human Stem Cell Technology

Masaki OKANO Ph.D.

Yoshiki SASAI M.D., Ph.D.

Early Embryogenesis

Human Stem Cell Technology Unit

Guojun SHENG Ph.D.

Yoshiki SASAI M.D., Ph.D.

Genomic Reprogramming

Four-dimensional Tissue Analysis Unit

Teruhiko WAKAYAMA Ph.D.

Yoshiki SASAI M.D., Ph.D.

Science Policy and Ethics Studies Unit
Douglas SIPP

Regenerative Medicine
Development Program
The Regenerative Medicine Development Program provides support for labs engaged in the clinical translationof research relating to regenerative medicine, such ascell transplantation and tissue engineering.

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

5

2012 Highlights

Secrets of cloning
efficiency unlocked

The cloning of mammals by somatic cell nuclear transfer has beenhaunted by remarkably low efficiency – only a few percent of SCNT
attempts are successful, even after more than a decade of researchin the field. The Lab for Genomic reprogramming found that early
defects in chromosome segregation may be the reason behind cloning failure.

Three new research leaders

The RIKEN CDB appointed three new leaders to its research programs in the first three months of 2012: Yoshihiro Morishita was named Unit Leader of the Laboratory
for Developmental Morphogeometry, Tomoya Kitajima
was appointed Team Leader of the Laboratory for Chromosomal Segregation, and Shigehiro Kuraku returned to
the CDB as head of the Genome Resource and Analysis
Unit.

Tre1 orients stem cell alignment

The Laboratory for Cell Asymmetry reported how the G Protein-coup­
led receptor Tre1 controls the orientation of neuroblast in the fruit fly.
This stem cell alignment is critical to the establishment of cell polarity
and tissue patterning in the fly brain, and may represent a more widelyconserved feature of asymmetric cell division in general.

6

CDB Symposium on Quantitative
Developmental Biology

The CDB held its tenth annual symposium, “Quantitative Developmental Biology” on March 26-28. Around 160 participants fromaround the world gathered to discuss computational, and theoretical
approaches to the study of development.

Human ES cells giverise to retinal tissue

Building on technqiues first developed in mouse embryonic stem
cells, the Laboratory for Organogenesis and Neurogenesis showedthat human ES cells can also be induced to self-organize into complex, three-dimensional tissue-like structures in vitro. In this study,
the Sasai group derived an hESC-derived optic cup, which is theembryonic tissue that gives rise to the retina.

Summer lectures forgrad school affiliates

As part of its commitment to contribute to the development of future
research leaders, the CDB held a two-day lecture program for graduate and medical school students. More than 150 aspiring scientists
joined the event, which included talks by CDB scientists and visits to
many of the Center’s labs.

7

2012 Highlights

Summer school at the CDB

The Office for Research Communications organized two one-day
courses developed in cooperation with the Laboratory for Early Embryogenesis (Guojun Sheng, Team Leader). The curriculum focusedon basic facts about blood biology, and the development of theblood system in chick, as well as an introduction to globin switching,and how defects in this mechanism are linked to disease.

Enteric neural crest
cells cut corners

New work by the Lab for Neuronal Differentiation and Regeneration
revealed a surprising feature of the development of the enteric nervous system, showing how subset of enteric neural crest cells inmouse takes a short cut across the mesentery on the way to innervating the large intestine.

Roots of the paratympanic
organ resolved

Birds possess a little-studied sensory structure called the paratympanic organ, which is thought to be used in monitoring air pressureor altitude. The Laboratory for Sensory Development has now shownthat this organ traces its origin back to a related structure in fish.

8

High school teachers come to learn

The CDB’s annual course in developmental biology for high school teachers welcomed over
20 biology instructors to the center. Instruction focused on how transcription factors establish cell identity, and included a practical component on the culture and observation of chickembryos.

More new faces

The RIKEN CDB welcomed three new laboratory heads inthe closing months of 2012: Yasuhide Furuta was appointed head of the Laboratory for Animal Resources andGenetic Engineering, Hironobu Fujiwara was named
Team Leader of the Laboratory for Tissue Microenvironment, and Mitsuru Morimoto joined as Team Leader of theLaboratory for Lung Development.

CDB Group Director Yoshiki Sasai
awarded science prizes

In 2012, Yoshiki Sasai, group director of the Lab for Organogenesis
and Neurogenesis, was honored with a trio of prestigious science
prizes – the Inoue Science Prize, the Yamazaki-Teiichi Prize, and the
Takeda Medical Prize – in recognition of his work in the self-organization of embryonic stem cells (ESCs) into various tissue types.

9Chromosome segregation underlies
cloning efficiency
The announcement of the birth of Dolly the sheep in1997 represented the first cloning by somatic cell nuclear
transfer (SCNT) of a mammal, and sparked a global fascination with the possibilities of creating genetic copies
of individual organisms. This technology has been adopted for use in animal husbandry and conservation, but inthe 15 years since that first achievement, SCNT remains
plagued by an extraordinarily low efficiency in giving rise
to viable cloned offspring, which has never risen above
a few percent of attempts. Many groups have tackled
this challenge through studies of gene expression andepigenetic signatures in cloned embryos, but to date theproblem of low efficiency remains unsolved.
A new report by Eiji Mizutani, Kazuo Yamagata, andothers in the Laboratory for Genomic Reprogramming(Teruhiko Wakayama, Team Leader) may shed new lighton this murky question, showing that defects in chromosome segregation during early developmental stages
may lie at its root. Published in Developmental Biology,
this study takes advantage of live imaging technologies that enable the microscopic observation of living
embryos. Mizutani has since moved to the RIKEN BioResources Center in Tsukuba, and Yamagata to Osaka
University.
Yamagata and Mizutani began by focusing on the possibility that chromosomal abnormalities, rather thantranscriptional or epigenetic differences, might accountfor the lack of progress in SCNT efficiency. To do so inlive embryos, however, meant that they needed to find
non-invasive approaches as an alternative to other traditional technologies such as immunolabeling and in situ
hybridization, which require embryos to be fixed in advance. This led them to begin to develop a live imaging
system used in this study, which allows users to observe
chromosome distribution in very early embryos without
disturbing their development.
They used mRNAs to introduce fluorescent probes forhistone H2B and α-tubulin to label the nucleus and mitotic spindle, and tweaked the doses to optimal levels
using their microscopy setup to minimize stress to theembryos. They next watched cloned embryos develop
for around 60 hours to the morula-blastocyst stage using
their 3D fluorescence imaging system, before returning
the embryos to surrogate mothers, and confirming theirdevelopmental viability.

Eiji MIZUTANI, Kazuo YAMAGATA

10This meticulous observation yielded a surprising finding: of 330 embryos studied, around 80% showed some©2012 Elsevier B.V.
Mizutani E, et al. Abnormal chromosome segregation at early cleavage is a major causeof the full-term developmental failure of mouse clones. Dev Biol 364.56-65 (2012)aberrant chromosomal segregation by the 8-cell stage. All such embryos failed to develop to term.
Interestingly, however, the rate of chromosomal abnormality between the 8- and 16-cell stages was
only 2.5%, and by the end of this early embryonic period, only 7.1% of the cloned embryos had notexperienced a chromosome segregation error. Clearly, defects in chromosomal segregation in thefirst few embryonic cell divisions set the stage for developmental failure.

Video showing chromosome
segregation (image on rightlabeled for H2B (red) and tubulin (green).

Mizutani and Yamagata next selected 72 cloned embryos at random and implanted them in surrogate mothers one-by-one to check their developmental potential. Of these, only three resulted in live
births, and on reviewing the fluorescence imaging of their early development in vitro, they found that
all three embryos were free of chromosomal defects by the 8-cell stage.
“We believe that the technology described in this study can be used to improve the screening ofcloned embryos for the absence or presence of chromosomal segregation errors, which appear to
be strongly connected to their developmental viability,” says Wakayama. “We also recognize however that there are likely to be other factors than epigenetics and chromosomal segregation behind
SCNT efficiency rates, and we hope to continue in our search for those in future work.”

11

New conserved mechanism for insulin
regulation

Kanako HIGUCHI, Junko SHINNO, Yuka NISHIMORI, Naoki OKAMOTO

Peptides of the insulin family are found across a broad range of taxa spanning both vertebrates andinvertebrates, in which they play roles in the regulation of processes such as metabolism, growth,
reproduction, and longevity. The genome of the fruit fly Drosophila melanogaster includes sevengenes encoding such peptides, known as dilps (Drosophila insulin-like peptides), which are primarily
expressed in secretory insulin-producing cells (IPCs) within the brain. The expression of each dilp is
independently regulated, but just how this level of coordination is achieved remains poorly understood.
Now, Naoki Okamoto and others in the Laboratory for Growth Control Signaling (Takashi Nishimura,Team Leader) have identified an important clue to this puzzle, showing that a highly conserved nuclear protein Dachshund (Dac) acts as a transcriptional regulator specific to dilp5. Published in theProceedings of the National Academy of Science, these findings may open new avenues of understanding into the means by which diverse insulin-family peptides are kept working in harmony.
The IPCs of the fruit fly brain express several genes known to be involved in the development of theeye. The team knocked down the function of several of these genes in a tissue-specific manner tocheck for possible effects on dilp expression. They found that the knockdown of dac was accompanied by a dramatic down-regulation of dilp5, suggesting that Dac might function in Dilp5 regulation
in IPCs.

12©2012 National Academy of Sciences
Okamoto N, et al. Conserved role for the Dachshund protein with Drosophila Pax6
homolog Eyeless in insulin expression. Proc Natl Acad Sci U S A 109.2406-11 (2012)

An examination of Dac expression in IPCs
showed the protein is expressed continuously throughout development. When Okamoto next analyzed homozygous mutantsfor dac, he found that while IPCs formed
normally, the expression of dilp5 in thesecells was markedly lower in young larvae,
but intriguingly its expression was normal
in later larval stages, suggesting stagedependent differential mechanisms for its
regulation. A subsequent analysis using
heterozygous dac mutants revealed that
expression levels of that gene correlated
closely with the expression of dilp5. A second protein called Eyeless (Ey) is known tocontrol dilp5 expression, and he found thatey mutants revealed similar phenotypes as found in the dac studies.

E xpression of Dac (red) in
the IPCs (green) of larval (left)
and adult (right) flies. Upper
panels show IPCs and axons(green) with Dac labeled (red).
Lower panels show the areas
indic ate d by white dot te dlines above at higher magnification.

The team next focused on possible interactions between Dac and Ey, but found that the loss offunction of one had no discernible effect on the other, suggesting that their expressions are independently regulated. Their effects on dilp5 were synergistic, however, as shown by tests in which theexpression of both was perturbed. Looking next for an interaction between Dac, Ey and the dilp5
promoter, they found that while Dac alone did not interact, the binding of Ey to the dilp5 promoter
was accelerated in the presence of Dac. Further experiments revealed Dac forms a physical complex with Ey, and with itself, through specific protein domains.
Knowing that Dac is evolutionarily conserved in mammals, Okamoto next turned to the insulinsecreting β-cells in the islets of Langerhans of the pancreas, in which Dach and Pax6, homologs ofthe Drosophila Dac and Ey, play important developmental roles. Importantly, Pax6 is also involvedin the transcription of genes encoding critical pancreatic hormones, such as insulin and glucagon.
Using cultured rat β-cells-derived cell line, they examined the functions of these two regulatory
proteins and found that, as in the fruit fly, Pax6 and Dach had similar combinatorial effects on theactivation of insulin expression.
“The expression of dilp5 can change in
response to nutritional status, and we still
don’t know how this gene is regulated inlater stage larvae,” says Nishimura, “whichsuggests that the regulatory situation iseven more complex than we expected.
We are looking forward to tackling the link
between nutrition and dilp5 expression in
future work.”

In wildtype young larvae (left)
IPCs (green) express Dilp5(red), while in dac mutants(right) Dilp5 expression isdramatically reduced.

13

New, lower-cost approach tofluorescence imaging

Kazuo YAMAGATA

Fluorescence imaging is a foundational technology in modern cell and molecular biology, making it
possible to label specific molecules and analyze their structural and functional properties. This imaging technique is not, however, without its disadvantages. The fluorescent light used to obtain images, for example, can cause significant damage to living cells, and many of the dyes used lose theircolor quickly after exposure. Cost can also be an issue, as expensive equipment such as mercury
lamps and lasers are need to generate the light needed to excite fluorescent molecules into theirlight-emitting state.
Now, Kazuo Yamagata and others in the Laboratory for Genomic Reprogramming (Teruhiko Wakayama, Team Leader) have showed that halogen lamps can serve as a cheaper, lower-intensity lightsource for use in fluorescence imaging studies in living cells. The report of this new technologicaldevelopment is published in PLoS ONE. Yamagata has since moved to take a position at Osaka University.
The demand for an alternative light source was first brought home to the team through their frequentinteractions with labs in developing countries in Asia, for whom mercury lamps and lasers can beprohibitively expensive. “We knew that the motivation levels are very high in these labs, but their limited financial resources make it difficult to acquire advanced microscopes, which inevitably deepensthe ‘research divide’ between developed and developing nations,” says Wakayama. “Fluorescenceimaging traditionally requires powerful light sources, but with the advent of newer molecules, wethought it might be possible to achieve similar results even with weaker lamps.”

14©2012 Public Library of Science
Yamagata K, et al. Fluorescence cell imaging and manipulation using
conventional halogen lamp microscopy. PLoS One 7.e31638 (2012)

Imaging of this sort requires the use of excitation filters, which exclude light of wavelengths otherthan those needed to charge target fluorescent molecules into an excited, light-emitting state. In
collaboration with Olympus, Yamagata et al. developed an adaptor to allow ordinary upright andinverted microscopes using only halogen lamps to be equipped with such filters, as well as filters toabsorb light other than that emitted from a fluorescing sample. When they tested this new equipment on fluorescence-labeled mouse embryos, they found the results to be remarkably vivid, comparable to those typical of a traditional fluorescence microscope.
Fluorescence imaging normally produces images on a dark field, due to the filtering of light at visible
wavelengths, but this can prove disadvantageous when scientists desire to observe cellular features in a brighter context. By modifying their adaptors, however, the team was able to create filters
smaller than the optical path with “leaky” diaphragms that can be opened to allow light around the
periphery of the image area to escape filtration, thereby making it possible to obtain bright field andfluorescence images simultaneously.

Mouse embr yo imaged by
fl uorescence microscopy using an upright optical microscope equipped with halogen
lamps and an excitation filter.
Inner cell mass (red) and trophectoderm cells (green) are
shown.

Length of exposure to intense light is a critical factor in determining cellular damage during imaging.
To compare the efficiency of their halogen lamp system with that of traditional mercury lamps, theteam injected oocytes with phycoerythrin-labeled antibody against histone (or fluorescent dye for the
nucleus), and observed them using each approach. Those observed using a conventional fluorescence microscope faded within around 30 seconds, but the same sample tested using the halogen
light system remained continuously visible for over 10 min. This lower-power approach may havereal biological consequences - early embryos imaged using conventional technology show reduced
developmental viability on implant in surrogate mothers, but those visualized using the new systemshowed no such effect.
“One of the biggest advantages of this approach is its low cost, which should put it within reach formany labs in economically developing parts of the world,” says Wakayama. “Of course, there aremany different types of halogen lamp, so we will need to keep studying the optimum intensity, but
we are hopeful that the adaptors we have developed will make it possible to attach fluorescence filters to ordinary optic microscopes at much lower cost.”

15

Triple-jointed: Notch signaling a key toinsect joint diversity

ReikoTAJIRI

Insects show a remarkable variety of joint structures in their limbs, which despite their tiny size andsimplicity and the rigid, external nature of their skeletons, rival our own in diversity of function. The
basis for this articular range, however, remains something of a mystery.
Now, Reiko Tajiri and others in the Laboratory for Morphogenetic Signaling (Shigeo Hayashi, Group
Director) have shown that, diversity of appearance notwithstanding, insect limb joint structures can
be classed into one of three general forms, and that the developmental decisions that guide theirmorphogenesis are under the control of Notch signaling. Published in Development, this work goesa long way to building a framework for understanding how insect joints are formed.
This new study built in previous work from the Hayashi lab, which showed that cell differentiationand movement are key processes in the sculpting of a specific type of joint – the ball-and-socket
form - in the adult Drosophila leg, known as the tarsus. Taking this as a starting point, Tajiri begancomparing tarsal joint morphologies in other insect species to gain a clearer understanding of theextent to which this model is conserved. The ball-and-socket type joint is not found in the tarsal
limbs of primitive insects (Apterygota, Paleoptera), in which the joint cavity is covered entirely by cuticle with no underlying ball-socket distinction, which suggests that the cell differentiation needed toform this form of joint does not take place.
In other joints in the tarsus of several primitive species, however, the joint is formed by two cuticularcomponents positioned side-by-side, without the encirclement seen in a true ball-and-socket joint.

16©2012 The Company of Biologists
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)

In other, more highly diverged insect lineages, such as Polyneoptera, Paraneoptera, and Holometabola, ball-and socket-joints are common, in contrast to their basal relations. From these observations, the group realized that only three main morphologies account for all tarsal limb joint forms in
insects: uniform, side-by-side, and ball-and-socket.
These findings additionally suggested that the formation of the most complex ball-and-socket joint
relies generally on the two essential modular processes of cell differentiation and cell movement,
and that gains or losses in either of these results in the failure of ball-and-socket formation. The evident diversity of joint forms in insect limbs presumably has evolutionary significance; each joint typemay confer advantages under specific environmental conditions, and all may have evolved multiple
times independently. But what Tajiri wished to know was, how might such diversity arise at the genetic level?
Previous reports have demonstrated the importance of the Notch signaling pathway in Drosophilalimb development, so the group conditionally knocked down Notch at various stages in pupal development to watch for possible changes in joint structure. By altering the absence, presence and timing of Notch expression using a temperature-sensitive mutant, Tajiri was able to shift ordinarily balland-socket joints to take on the simpler uniform conformation, or a range of incomplete variations
on its usual form. Labeling of a subset of cells in the developing joint showed that Notch knockdown
affected cell motility, resulting in the failure of the cell movement step of joint formation.

Ball-and-socket, uniform, andside-by-side joint types in insect limb (scale bar = 10 µm)

But what of differentiation? Using a constitutively active form of its gene and an RNAi construct tointerfere with its expression, Tajiri tested the role of differing levels of Notch expression role in determining the fate of cuticle cells. The model that developed was clear: higher levels of Notch signaling
drive “ball” differentiation, while lower levels promote “socket.”“What was fascinating about this study was that it showed that Notch signaling is not simply aswitch driving differentiation, but a subtle regulator of the balance between differentiation and cell
movement,” says Tajiri, who has since moved to the University of Tokyo. “It will be interesting to findout what specific factors Notch interacts with at each step in joint morphogenesis, which may leadto a better understanding of the links between development and evolution in this system.”

17

Stem cells show sense of direction

Shigeki YOSHIURA

Brains, whether they belong to flies, mice, or humans, are built of numerous types of neurons and supporting
cells. This great diversity is the result of asymmetric cell divisions, in which daughter cells often inherit different
sets of protein components from their progenitors, and consequently develop different properties. Such activity
is highly coordinated, to ensure that the appropriate number and type of cells are produced at just the right times
and locations. The Drosophila neuroblast, a type of stem cell, provides a good example of this, dividing perpendicular to its overlying epithelium to give rise to a pair of daughter cells of which one is a more highly differentiatedintermediate neural progenitor and the other is a neuroblast like its parent cell. The means by which this division isoriented, however, are not well understood. Previous work has indicated that centrosomes and microtubules playa crucial role in larval brains, but other studies suggest that other factors may be important as well in embryoniccentral nervous systems.
Shigeki Yoshiura and others in the Laboratory for Cell Asymmetry (Fumio Matsuzaki, Group Director) have now
found that the key may lie in molecular signals across the cell membrane that tell neuroblasts how to align. Published in Developmental Cell, the group shows that the G protein-coupled receptor (GPCR) Tre1 is a key factor ina molecular interaction that orients these cells with respect to the epithelium.
The group’s study began with a genetic screen for non-cell-autonomous factors involved in orientation, by monitoring the orientation of neuroblasts in embryos lacking genes coding for candidate membrane proteins. They
found that mutation of the rhodopsin family orphan GPCR gene trapped in endoderm 1 (tre1) was associatedwith orientation defects, raising the possibility of a causal relationship. Deletion mutants for tre1 showed significant perturbations of neuroblast orientation with respect to the epithelial ectoderm, resulting in disorganized18©2012 Elsevier B.V.
Yoshiura S, et al. Tre1 GPCR signaling orients stem cell divisions in the
Drosophila central nervous system. Dev Cell 22.79-91 (2012)

stratification of the developing tissue in central
nervous system (CNS). This phenotype could
be rescued by introducing a genomic fragmentof the gene, strongly suggesting that it was
responsible for the neuroblasts’ disorientation.

EP
NB
GMC

Interestingly, however, cell polarity was unaffected by tre1 deletion, and the early-stage
Miranda pH3 DAPI
orientation defects in such mutants were rectified over the course of development by someEP
unknown compensatory mechanism. Yoshiura
NB
et al. speculated that this correction might beGMC
due to interactions between neurons and otherNR
neighboring cells, such as glia. And indeed
when they tested flies in which both tre1 andElav Ase DAPI
repo, a critical glial cell gene, were deleted,even later-stage neuronal organization wasaberrant, suggesting that neuron-glia interactions are required for the development of the CNS.

In wild type embr yos (left),neuroblasts (NB) asymmetrically divide perpendicular
to the epithelial layer (EP) to
bud off the daughter ganglion
mother cell (GMC) inward,
giving rise to a layered organization from the epithelium toneurons (NR). In contrast, intre1 mutants, the orientationof the polarity and division ofneuroblasts is randomized(right upper), and the developing nervous tissue is disarranged (bottom right).

Using flies in which the distribution of Tre1 could be visualized, the group next sought to determine its mode offunction. Yoshiura found that this protein was distributed widely in neuroblasts across the cell cortex, but to amuch weaker degree in ganglion mother cells and neurons, indicating that it might function in a neuroblast-autonomous fashion. Disruption of microtubule activity had no effect on orientation in neuroblasts in the embryonic
CNS in which Tre1 was normally expressed, suggesting that this protein functions independent of microtubules atembryonic stages.
To exclude the possibility that Tre1 helps orient neuroblasts via signals from a tissue other than epithelium (suchas mesoderm), the group examined a brain region in which only epithelium, neuroblasts and their progeny arepresent, and found that the cells remained oriented perpendicular to the epithelium, as expected.
They next used co-immunoprecipitation to find binding partners of Tre1, and found that it interacts with Goα, aneuroblast protein of unknown function. Using a toxin that specifically interferes with Goα, they found that loss of
this protein’s function results in a phenotype mirroring that of tre1 deletion, suggesting that Tre1 may orient neuroblasts in response to signals from the epithelium, by facilitating GDP-GTP hydrolysis by Goα.
These findings brought another factor into the picture, as the protein Pins is known to interact with Goα in vitro.
Pins is known for its role in mitotic spindle orientation, via binding with another Gα protein, Gαi. In vivo tests using
a different assay, however, indicated that only the Goα-Pins interaction depends on Tre1, suggesting that only the
GTP form (but not the GDP form) of Goα binds to Pins. Additional testing using Gαi, in addition to Goα and Pins,in tissue cultures, showed that to be the case - the GTP form of Goα showed much higher interactivity with Pins
than the GDP form of Goα or Gαi. Constructing a cell culture assay system, the group also found that Tre1 could
recruit Pins in the presence of Goα.
The scenario that emerges is a sort of “handoff” mechanism; binding with the GDP form of Gαi is necessary andsufficient for Pins to be recruited to the cell membrane. At the membrane, the GTP form of Goα, in turn, is generated from its GDP form only on the apical side proximal to the adjacent epithelium, from which an unidentified
signal presumably activates Tre1. Gαi is then replaced by the GTP form of Goα, due to its more stable binding toPins, which serves to recruit Pins to the apical membrane. In neuroblasts, Pins associates with the polarity-organizing aPKC-Par complex via interaction with Inscuteable, and consequently recruits this complex to the apicalside of neuroblasts. As the aPKC-Par complex determines the polarity and orientation of division in neuroblasts,
the Tre1-Pins mechanism ultimately directs the orientation of neuroblasts with respect to the epithelium.

19

Pair of factors directs heterochromatin
assembly via RNAi
The enormous information set encoded in a cell’s genome is bundled into tightly wound coils of DNA and proteins known as chromatin. The prevailing view has beenthat this structure takes two main forms: loosely packed
stretches of euchromatin which tend to permit active
transcription, and more securely locked down heterochromatin, where gene expression is typically silenced,
and which provides the structural basis for regions suchas the centromere and telomeres. Heterochromatin is theend product a cascade of events involving multiple molecular complexes that interact to form this critical chromosomal component, which is characterized in part byits coating of the protein HP1. Some studies have suggested that HP1 plays a role upstream in the cascade as
well, but the details of this have resisted explication.
Now, Aki Hayashi and others in the Laboratory for Chromatin Dynamics (Jun-ichi Nakayama, Team Leader) haveshown that the co-factors Ers1 and the HP1 homolog
Swi6 work together to recruit the RNAi machinery to sitesof heterochromatin assembly in the fission yeast, Schizosaccharomyces pombe. Published in the Proceedings of
the National Academy of Sciences, this work refines ourunderstanding of how this crucial basis of chromosome
structure is formed.
Heterochromatin assembly is regulated by Histone H3
Lysine 9 (H3K9me) methylation. In fission yeast, the histone methyltransferase Clr4 marks specific sites on chromosomes, serving as a binding site for chromodomain
proteins such as Swi6, which in turn induces heterochromatin compaction. Interestingly, the molecular machinery
behind RNAi, best known as an epigenetic form of posttranscriptional regulation, also plays a part. In S. pombe,
the RNA-induced transcriptional silencing (RITS) complex
and RNA-dependent RNA polymerase complex (RDRC)
both function in the RNAi process, but recent work has
shown that these are, surprisingly, also essential to heterochromatin assembly in the centromeres, as loss offunction of either results in aberrant centromeres anddefective gene silencing. This growing body of work has
shown that both RITS and RDRC localize at centromere
heterochromatic regions, highlighting the possibility that
this RNAi machinery is involved in heterochromatin assembly.

Aki HAYASHI

20

Hayashi approached this question using a genetic
screen for mutations affecting gene silencing at the centromeres, which yielded a number of hits associated with
the RNAi machinery, including a factor called Ers1, which
had previously been necessary to RNAi, but remained©2012 National Academy of Sciences
Hayashi A, et al. Heterochromatin protein 1 homologue Swi6 acts in concert with Ers1 to regulate
RNAi-directed heterochromatin assembly. Proc Natl Acad Sci U S A 109.6159-64 (2012)poorly understood. The team found that ers1 mutants showed not only defective silencing, but also
low production of siRNAs (short interfering RNAs) and reduced H3K9 methylation. On testing theability of various genes involved in heterochromatin assembly or RNAi to rescue these phenotypes,
they found that overexpression of the RNA helicase Hrr1 and the methyltransferase Clr4 had a partially compensatory effect, pointing to the possibility of a functional connection. Yeast two-hybrid
assays showed that Ers1 and Hrr1 bind directly, but did not show any physical association between
either Ers1 or Hrr1 with Clr4.

Functional relationships between Ers1 and other factors
linking RNAi and heterochromatin assembly

Given these findings, questions remained regarding the molecular mechanisms behind the functional
association. Hayashi et al. next used EGFP to label Ers1 in wildtype cells, allowing them to observe
its punctate localization in the nucleus, consistent with co-localization with heterochromatin. In Clr4
and Swi6 mutants, however, the localization was more diffuse, indicating their importance to the localization of Ers1 to heterochromatic regions. A subsequent yeast two-hybrid screen confirmed thatErs1 binds directly with Swi6, fitting a piece into a puzzle that had begun clarifying into a scenario in
which the molecular complex of Ers1 and Swi6 is recruited to heterochromatin regions methylatedby Clr4.
The bigger picture reveals that Ers1 interacts with both Hrr1 and Swi6. Hrr1 is a necessary component of RDRC, which the team found was recruited to centromeres by Ers1 and Swi6. Thus, Swi6
acts as a kind of platform to localize this complex to these highly methylated heterochromatic regions in order to proliferate further siRNA generation.
“Recent work has revealed the direct association between the RITS and RDRC complexes,” says
Nakayama. “It seems that Ers1 is playing a critical role in bridging the RNAi machinery with heterochromatin assembly. It is will be very interesting to see whether this bridging mechanism, as well as
the mechanism behind RNAi mediated heterochromatin formation, are conserved in other organisms. ”

21

2012 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.

Kobe-Barcelona joint meeting
The Center hosted a joint meeting with colleagues from research centers and graduate school programs in Barcelona, including theCenter for Genomic Regulation, Universitat Pompeu-Fabra, and the Institute for Research in Biomedicine. The CDB subsequently entered into programs of cooperation with all three organizations.

RNA Sciences in Cell andDevelopmental Biology II
This two-day program in June brought together scientists from around the world
to discuss new developments and insights into the diverse function of RNAs inthe developing embryo. This represents the second CDB meeting with this focus,
following a successful start in 2010.

Kobe Institute Open HouseThe RIKEN Kobe Institute held its annual Open House on October 20, welcoming
more than 1,500 visitors to learn more about the research being done at its research centers, including the Center for Developmental Biology.

2012 Retreat
The CDB held its annual retreat in the mountains of Sasayama on September 25
to 26. This overnight stay is designed to encourage research scientists and students share their most recent work in an relaxed, informal setting. This year’s
retreat additionally featured talks by CDB alumnus Kinya G. Ota and Advisory
Council member Christopher Wylie.

22Wholemount staining of gut from a Ret EGFP/+ embryo (E11.0) using anti-GFP and anti-PECAM antibodies, which detect enteric neural crest cells and vascular endothelial cells, respectively. A
population of enteric neural crest cells (green) in the midgut are observed to cross the mesentery
during a gut hairpin bending period. Note that midgut and hidngut are tightly apposed, sandwiching mesentery.
Image: Chihiro NISHIYAMA, Laboratory for Neuronal Differentiation and Regeneration

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
and dorsal-ventral axis patterning programs, and the means by which each individual region is formed, remain unknown.

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

Staff
Group Director

Technical Staff

Shinichi AIZAWA

Eriko KAJIKAWA
Tomomi OHMURA

Research Specialist

Yoko SUDA
Michio YOSHIDA

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

Assistant

Sayo KIYONARI

Research Scientist

Hideharu HOSHINO
Miyuki NORO
Visiting Scientist

Daisuke KUROKAWA
Masaki TAKEUCHI

Recent Publications
Inoue F, al at. Gbx2 expression to forebrain and midbrain,competing with class Ⅲ POU factors. Mol Cel Biol 32.
2618-27 (2012)
Kurokawa D, et al. A lineage specific enhancer drives Otx2
expression in teleost organizer tissues. Mech Dev 128. 65361 (2012)
Shibata M, et al. MicroRNA-9 regulates neurogenesis inmouse telencephalon by targeting multiple transcription factors. J Neurosci. 31. 3407-22 (2011)

A

B

C

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 andlater expression in the cortical ventricular zone.
Development 137. 2939-49 (2010)
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) shows descendants of distal visceral endoderm cells that expressed a head organizer gene (hog), (B)the descendants retaining 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,
most particularly 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 fourco-receptors, GRFα1-4. In vitro, these four receptors show differential affinities
for specific GFLs. GFL signaling has been shown to affect neuronal growth,survival and migration as well as axon guidance, and defects in this signaling
system have been implicated in a number of congenital health problems.
Our efforts will focus on the analysis of the physiological roles of neurotrophic
factors, which we hope will lead to an improved understanding of the molecularbases of neural development, differentiation and pathology, and ultimately topotential clinical applications in the treatment of nervous system trauma 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

Student Trainee

Hideki ENOMOTO

Sachie ONO

Research Scientist

Part-Time Staff

Pilaiwanwadee
HUTAMEKALIN
Keisuke ITOH
Toshihiro UESAKA

Azusa FUKUDA
Toko KONDO
Akira YAMAOKA

Technical Staff

Kaori HAMADA
Yuka NODA

Chihiro NISHIYAMA

AssistantJunior Research Associate

Mitsuhiro IWASAKI
Yohei YONEKURA

Recent Publications
Nishiyama et al. Trans-mesenteric neural crest cells are theprincipal source for the colonic enteric nervous system. Nat
Neurosci 15.1211-18 (2012)
Nagashimada et al. Autonomic neurocristopathy-associatedmutations in PHOX2B dysregulate Sox10 expression. J Clin
Invest 122.3145-58 (2012)
Uesaka T and Enomoto H. Neural precursor death is central
to the pathogenesis of intestinal aganglionosis in Ret hypomorphic mice. J Neurosci 30.5211-8 (2010)
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 et al. Conditional ablation of GFRalpha1 in postmigratory enteric neurons triggers unconventional death in
the colon and causes a Hirschsprung’s disease phenotype.
Development 134.2171-81 (2007)

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

25

Tissue Microenvironment
There is a wide diversity of environments on earth, from the North Pole to the
Sahara, in which well-adapted organisms live. The traits and behaviors of all
such organisms have been developed and modified through exposure to their
specific environments.

Hironobu FUJIWARA Ph.D.
http://www.cdb.riken.jp/en/fujiwara
Hironobu Fujiwara attained his baccalaureate
from Kyoto Pharmaceutical University, andwent on to receive his M.S. and Ph. D. from
Osaka University, the latter in 2003 for his workon the characterization of the human laminin-8
protein. From 2003 to 2007, he served as a
postdoctoral researcher at the Osaka University Institute for Protein research, with support
from the ERATO Sekiguchi Biomatrix Signaling
Project (2003 – 2006). He then moved to theCancer Research UK Cambridge Research Institute, where he completed a second postdoctoral fellowship, before returning to Japan totake a position as a Team Leader at the RIKEN
CDB in 2012.

Staff
Team Leader

Hironobu FUJIWARA
Technical Staff

Noriko BAN-SANZEN
Assistant

Asako NAKAGAWA

Recent Publications
Fujiwara H, et al. The basement membrane of hair follicle
stem cells is a muscle cell niche. Cell 144.577-89 (2011)
Watt F. M. and Fujiwara H. Cell-extracellular matrix interactions in normal and diseased skin. Cold Spring Harb Perspect Biol 3.(2011)

In our own bodies, we also have millions of different environments in whichcells reside, which are known as cellular or tissue microenvironments. A seriesof recent studies has shown that these specialized tissue microenvironments
instruct the fate and behaviors of cells. The aim of our lab is to gain a better
understanding of the mechanisms underlying the ways in which tissue microenvironments are regionally specialized, and how these specialized microenvironments then instruct cellular behavior and communication, and the formation oforgans. We are particularly interested in the role of regional specialization of theextracellular matrix (ECM) in the formation of the stem cell microenvironment, or
niche. A deeper knowledge of this will provide a molecular basis to an improved
understanding of the niche regulation of stem cells, and the development of
tailor-made microenvironments for different lineages of stem cells.
The ECM is divided in two major groups – the fibrillar interstitial matrix, whichfills the interstitial connective tissues, and the basement membrane (BM), a thinsheet-like ECM located at the borders of tissues. Stem cells in most tissues reside at this border, adhere to the BM and interact with neighboring niche cells.
By virtue of its remarkable heterogeneity in composition, the BM contributes
to the spatial organization of niches, and modulates the local concentration ofadhesive and soluble signalling molecules that are available to stem cells. A recent study by our team has shown that the molecular composition of the BM in
the mouse hair follicle stem cell niche, the bulge, is highly specialized. One stem
cell-derived component, nephronectin, is important for the development andpositioning of the bulge-residing arrector pili muscles, which, among other functions, are responsible for goosebumps. This was the first report to show thatstem cells regulate the fate and positioning of surrounding niche cells throughthe specialization of the BM. To gain further insight into fundamental aspects ofthe microenvironmental regulation of stem cells, we use mouse skin as a modeland seek to better understand 1) the molecular landscape of BM specializationin the stem cell niche, 2) mechanisms by which the BM in the stem cell niche isregionally specialized, and 3) how the specialized BM controls stem cell nicheformation, stem cell behavior and the conversation between stem cells and theirneighboring cells.

Ferreira M, et al. An activating beta1 integrin mutation increases the conversion of benign to malignant skin tumors.
Cancer Res 69.1334-42 (2009)
Fujiwara H, et al. Regulation of mesodermal differentiationof mouse embryonic stem cells by basement membranes. J
Biol Chem 282.29701-11 (2007)
Fujiwara H, et al. Rac regulates integrin-mediated endothelial cell adhesion and migration on laminin-8. Exp Cell Res
292.67-77 (2004)

The image shows arrector pili muscles anchored to the bulge of hair follicles in a whole-mount preparation of dorsal skin in mouse, viewed from the dermal side. Arrector pili muscles are visualized by staining
for α-smooth muscle actin (green) and SM22α (red). The whole-mount is labelled with a nuclear counterstain (blue).

26

Neocortical Development
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. This highly ordered structure
nonetheless 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 how do they establish cytoarchitectonic boundaries?
3) To what extent does the refinement of functional areas rely on environmental
inputs? To investigate these questions we utilize mouse as a model to understand the assembly of functional subdivisions in the mammalian neocortex.
Previous work has shown that the fate of neocortical neurons is at least in partpre-determined within the progenitor cells. We have found that specification of
deep-layer projection neurons is dependent on transcriptional repressor thatfunction cell-autonomously to prevent neurons from acquiring an earlier neuronal phenotype. These results imply cortical intrinsic programs in which neuron
fate is established by temporal changes in gene expression may be co-opted.
However, in the mature cortex, cortical areas differ in their types and numbersof specific layer neurons along both the anteriorposterior (AP) and medial-lateral(ML) axes. We are exploring the extent to which intrinsic determinants control
the specification of neuronal subtypes within discrete regions of the neocortex,
as well as the extrinsic influences that refine the boundaries between functional
areas of neocortex. To further these studies, we employ genetic manipulations
in mice that will enable conditional loss of gene and cellular functions, recombination mediated cell-lineage tracing, and systematic approaches to identify
novel molecules responsible for precise areal specification. Through thesestudies we wish to understand the mechanistic basis by which unique sensory
perceptions and functional circuitries develop in the human neocortex.

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

Junior Research Associate

Carina HANASHIMA

Ken-ichi TOMA

Research Scientist

Technical Staff

Yuko GONDA
Takuma KUMAMOTO

Yuko WADA

Foreign Postdoctoral
Researcher

Chika KUMAMOTO
Reiko ODA

Torsten BULLMANN
Research Associate

Tien-Cheng WANG

Part-Time Staff

Assistant

Miyuki MIURA

Recent Publications
Gonda Y, et al. Robo1 Regulates the Migration and Laminar
Distribution of Upper-Layer Pyramidal Neurons of the Cerebral Cortex. Cereb Cortex (2012)
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 membrane-targeted
GFP (green) and nuclear lacZ (red) to the motor cortex.

27

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 for high-resolution imaging, and study the problem at three levels of cellularorganization: single-cell, multiple cells, and organ.

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

Our study is centered on the tracheal system, a respiratory organ that delivers
air directly to tissues through a network of finely branched epithelial tubes. Tracheal development begins by invagination of ectodermal epithelium that subsequently forms branches with specific types of cells migrating in stereotyped
patterns. Cellular forces produce mechanical strain in the epithelium, and alleviation of that strain is essential for smooth-tissue movement. As a consequenceof epithelial cells behaving as elastic bodies, their shape changes and movement proceed with local fluctuations. We aim at elucidating (1) mechanismsthat coordinate cell movement, (2) mechanisms for alleviating tissue strain, and(3) mechanisms of cross-talk between these two mechanisms. To accomplishthese goals, the mechanical state of cells will be measured by combining techniques such as quantitative cell imaging, and various cell perturbation techniques are being used to assess the mechanical states of cells. The results ofthese 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.

Staff
Group Director

Technical Staff

Shigeo HAYASHIAi AKIMOTO
Akiyo KIMPARA
Housei WADA

Research Scientist

Kagayaki KATO
Takuya MAEDA
Tetsuhisa OTANI
Foreign Postdoctoral
Researcher

Bo DONG
Special Postdoctoral
Researcher

Takefumi KONDOInternational Program
Associate

Guangxia MIAO
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
Assistant

Ryoko ARAKI

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

28

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 the placode,
tracheal primordial cells (green) constrict apical region facing outside of the epithelia and invaginate
inwardly. This process involves a complex interplay of cell boundary tension in the plane of epithelia
orchestrated by EGF receptor signaling and inward (basal) movement of cells driving invagination. Cellboundaries are marked with magenta.

Sensory Circuit Formation
The mammalian nervous system is composed of enormous numbers of neurons, but how do these cells take on diverse fates and organize and array themselves during development? In recent years, it has become clear that the mouseolfactory system provides an excellent platform for addressing these questions
experimentally. In this system, there are 1,000 types of odorant receptors thatare capable of detecting and discriminating between odorant molecules. Eacholfactory sensory neuron expresses a single type of odorant receptor, and theaxons of neurons expressing the same receptor type converge on the same site
in the olfactory bulb. Olfactory sensory neurons connect axons to the dendritesof mitral and tufted (M/T) cells in the bulb, where each receives inputs from asingle specific type of olfactory sensory neurons.
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 receptordependent axon projection of olfactory sensory neurons, and the formation ofneuronal circuitry in the olfactory bulb dependent on inputs from these neurons.
We will also seek to develop next-generation genetic tools and imaging techniques to aid in our developmental and functional analyses of specific neuronal
circuits in the brain.

Takeshi Imai Ph.D.
http://www.cdb.riken.jp/en/imai
Takeshi Imai completed his Ph.D. in the laboratory of Prof. Hitoshi Sakano at the Universityof Tokyo Graduate School of Science in 2006.
He did consecutive postdoctoral fellowships in
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.

Staff
Team Leader

Student Trainee

Takeshi IMAI

Meng-Tsen KE

Research Scientist

AssistantSatoshi FUJIMOTO
Ryo IWATA

Eri YAMASHITA

Technical Staff

Miwako NOMURA

Recent Publications
Imai T. Positional information in neural map development:
Lessons from the olfactory system. Dev Growth Differ
54.358-365 (2012)
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, et al. Odorant receptor-derived cAMP signals direct
axonal targeting. Science 314. 657-61 (2006)

Mitral and tufted cells in the mouse olfactory bulb labeled with TMR-dextran. Each mitral/tufted cell extends a single primary dendrite to a single glomerulus, where it receives inputs from a single specific typeof olfactory sensory neurons.

29Chromosome Segregation
In order to maintain genetic information across generations, cells must allocate
chromosomes equally to daughter cells during mitosis. Meiotic divisions of the
mammalian oocyte, however, are known to exhibit a higher frequency of errors in chromosomal segregation than in other cell types. Oocytes formed fromsuch divisions are aneuploid, meaning they have incorrect numbers of chromosomes; if these are fertilized and develop to term, the resulting individual may
exhibit congenital anomalies, such as trisomy 21 (Down syndrome). Such errors
in chromosomal segregation are also known to increase with the age of themother, and this risk may be a contributing factor to the low birth rates seen in
many developed nations.

Tomoya Kitajima Ph.D.
http://www.cdb.riken.jp/en/kitajima
Tomoya Kitajima received his Master’s anddoctoral degrees from the University of Tokyo,
for his thesis on genetic screens for meiosisspecific proteins regulating chromosome
segregation in fission yeast. After receiving his
Ph.D. in 2004, he served as research associate
at the Institute of Molecular and Cellular Biosciences at the same university, before moving
to the European Molecular Biology Laboratory
in Heidelberg, Germany as a postdoctoral
researcher. He was appointed Team Leader at
the CDB in 2012.

Using the mouse as a model, we will seek to conduct detailed and comprehensive analyses of the dynamics of chromosomes and the molecular machinerythat underlies chromosome segregation during cell division. We plan to takeadvantage of the latest live imaging technologies to study the chromosome dynamics of the mouse oocyte at a level detail unprecedented in other cell types.
Oocyte chromosomes behave in ways distinct from those in other cells, andthese unique dynamics may provide insights into novel mechanisms for chromosome allocation. By combining live imaging with genetics techniques such
as RNAi and gene knockouts, we hope to study the mechanisms underlying
chromosomal segregation in oocyte meiosis, and identify the causes behind
age-related increases in ploidy errors.

Staff
Team Leader

Tomoya KITAJIMA
Research Scientist

Yogo SAKAKIBARA
Shuhei YOSHIDATechnical Staff

Masako KAIDO
AssistantKaori HAMADA

Recent Publications
Kitajima T. S, et al. Complete kinetochore tracking reveals
error-prone homologous chromosome biorientation in mammalian oocytes. Cell 146.568-81 (2011)
Lee J, et al. Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells. Nat Cell
Biol 10.42-52 (2008)
Kitajima T. S, et al. Shugoshin collaborates with protein
phosphatase 2A to protect cohesin. Nature 441.46-52(2006)
Kitajima T. S, et al. Human Bub1 defines the persistentcohesion site along the mitotic chromosome by affecting
Shugoshin localization. Curr Biol 15.353-9 (2005)
Kitajima T. S, et al. The conserved kinetochore protein
shugoshin protects centromeric cohesion during meiosis.
Nature 427.510-7 (2004)
Kitajima T. S, et al. Distinct cohesin complexes organize
meiotic chromosome domains. Science 300.1152-5 (2003)

Tracking of kinetochores (green)and chromosomes

30

Prometaphase of meiosis I. Microtubules (green), kinetochores(red), chromosomes (blue).

Kinetochore tracking during anaphase of meiosis I.

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
by eliminating cells that have developed abnormalities. Perturbations in cell
death signaling can thus affect an organism’s physiological stability, and result
in developmental defects, tumorigenesis, or neurodegenerative disease. Cell
death plays 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 cellular behaviors give rise to cell networks capable of organizing into tissues, thestudy of which requires a experimental approach to spatiotemporal informationin living systems, such as can be obtained through the real-time live imaging of
biological phenomena.
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 Drosophila development, the terminalia rotates 360° as it forms, but in caspase mutants, thisrevolution is incomplete. Image analysis reveals that in wildtype, the speed ofthis rotation is variable, with distinct initiation, acceleration, deceleration, andtermination stages; caspase inhibition results in loss of the acceleration phase,and failure in terminalia development. We will seek to identify how caspasefunction and cell death control acceleration of the rotation through searching for
associated genes and live imaging analysis. It has further been predicted thatcell death alone cannot account for rotation that maintains tissue area, suggesting other mechanisms are also at work. We will conduct single-cell analyses todetermine whether other behaviors such as proliferation or migration are also altered. Through the use of the extensive Drosophila genetics toolset and live imaging technologies, we hope to be able to address questions that have proventechnically challenging in the past, and by visualizing the activities of individual
cells, develop a better understanding of how cellular network systems work inhistogenesis.

Erina KURANAGA Ph.D.
http://www.cdb.riken.jp/en/kuranaga
Erina Kuranaga received her doctorate in medical science from the Osaka University Graduate School of Medicine in 2004, after which
she moved to the University of Tokyo Graduate
School of Pharmaceutical Sciences as assistant professor in the Department of Genetics. In 2006, she was promoted to associate
professor in the same department, where sheremained until she joined the RIKEN Center for
Developmental Biology as team leader in 2011.
She was awarded the Wiley-Blackwell Prize
2012.

Staff
Team Leader

Student Trainee

Erina KURANAGA

Shuji SHIMAMURA

Research Scientist

Part-Time Staff

Emi MAEKAWA

Arata KURANAGATechnical Staff

Ayako ISOMURA
Aimi TSUKIOKA

Recent Publications
Sekine Y, et al. The Kelch repeat protein KLHDC10 regulates oxidative stress-induced ASK1 activation by suppressing PP5. Mol Cell 48.692-704 (2012)
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 triggeredby local cell cycle progression during epithelial replacementin Drosophila. Mol Cell Biol 31.2499-512 (2011)
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 DE-Cadherin:GFP-expressing
Drosophila. This image was taken before rotation.

31

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 seek 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 andPh.D. from the Kyoto University Department
of Zoology. He spent the period from 1985
to 1988 studying comparative embr yology
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 College of Medicine, where he wasengaged in molecular embryological research.
He returned to Japan in 1994 to take a positionas associate professor at the Institute of Medical Embryology and Genetics in the Kumamoto
University School of Medicine. He moved to
Okayama University to assume a professorshipin the Department of Biology in 1997, where heremained until he was appointed team leader
at the CDB. He was appointed group director
in 2005.

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.

Staff
Group Director

Technical Staff

Shigeru KURATANI

Satoko FUJIMOTO
Tamami HIRAI

Research Scientist

Noritaka ADACHI
Naoki IRIE
Christian MITGUTSCH
Takayuki ONAI
Juan PASCUAL ANAYA
Fumiaki SUGAHARA
Motoki TADA
Special Postdoctoral
Researcher

Tatsuya HIRASAWA

Junior Research Associate

Yasuhiro OISI
Student Trainee

Hiroki HIGASHIYAMA
Part-Time Staff

Kazuko YAMAMOTO
Assistant

Yuko HIROFUJI
Naeko MINAMI
Tomoko OYANAGI

Recent Publications
Oisi Y, et al. Craniofacial development of hagfishes and theevolution of vertebrates. Nature 493.175-80 (2013)
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. Science325.193-6 (2009)
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)32

CT-scanned skeleton of Pelodiscus sinensis juvenile

Sensory Development
Organogenesis is a monumental undertaking. From siting a given organ to 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 tolearn how these signals are themselves established. We are also investigating
the role these 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
the development of the inner ear can be applied to the development of otherorgans, 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 biochemistryfrom Imperial College, London in 1992 and his
Ph. D. in developmental biology from the National Institute for Medical Research in 1996,
for his thesis on the cloning of the Xenopus homeobox gene, Xom. He worked as a research
associate at King's College, London from 1996
to 2000 in the lab of Pip Francis-West, and firstcame to Japan during that period as a visiting scientist at the Tokyo Medical and Dental
University. In 2000, he moved to the Universityof Utah as a postdoctoral fellow in Gary Schoenwolf's laboratory. He was appointed teamleader at the CDB in 2002.

Staff
Team Leader

Student Trainee

Rajesh LADHER

Kazuya ONO

Research Scientist

Part-Time Staff

Akira HONDA
Tomoko KITA
Siu Shan MAK
Yuko MUTAPaul O'NEILL
Xiao Rei SAI

Yoshiko KONDO
Yoko MATSUOKA
Anna WRABELAssistant

Noriko HIROIVisiting Researcher

Stephen FREEMAN

Recent Publications
Freter S, et al. Pax2 modulates proliferation during thespecification of the otic and epibranchial placodes. Dev Dyn
241.1716-1728 (2012)
O’Neill P, et al. The amniote paratympanic organ develops
from a previously undiscovered sensory placode. Nat Commun 3.1041(2012)
Zou Y, et al. Induction of the chick columella and its integration with the inner ear. Dev Dyn 241.1104-10 (2012)
Ladher R. K. Squeezing into differentiation. Dev Cell 21.6078 (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)
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)

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

Sai X. and Ladher R. K. FGF signaling regulates cytoskeletal remodeling during epithelial morphogenesis. Curr Biol
18.976-81 (2008)33

Tissue interactions behind the middle ear

YiHui ZOU, Siu Shan MAK, Raj LADHER

The vertebrate ear forms in three parts – inner, middle, and outer – that are derived independently
from different embryonic tissues. Despite these distinct origins, however, the adult ear manifests asa functionally integrated whole, suggesting that these regions must be closely coordinated during
embryonic development. The precise manner in which this transpires, however, remains somethingof a mystery. In the case of the inner and middle ear, for example, the inner ear forms from a neuroectodermal primordium called the otic placode, followed by the middle ear, which arises from a migratory cell population derived from the neural crest. While the induction of the inner ear is fairly wellunderstood, that of the middle ear, as well the integration between these neighboring tissue regions,is less clear.
Now, YiHui Zou, Siu-Shan Mak, and colleagues in the Laboratory for Sensory Development (Raj
Ladher, Team Leader) have shed new light on this process, showing that an underlying strip of mesoderm is needed to induce the bony middle ear in chick. Published in Developmental Dynamics, thisreport opens up avenues toward an improved understanding of the ways in which organ systems
involving multiple tissue types can be integrated during development.
In mammals, the middle ear is famously made up of three bones, known familiarly as the hammer,
anvil, and stirrup, while in birds it comprises only a single bone, the columella. This structure arises
from a neural crest-derived population of precursor cells, which at the relevant stage of development are surrounded by the inner ear, the pharyngeal endoderm, and the cephalic paraxial mesoderm (CPM). The first contact between the nascent columella and these tissues is with the CPM,

34

©2012 John Wiley & Sons, Inc.
Zou Y, et al. Induction of the chick columella and its integration with the inner ear.
Dev Dyn 241.1104-10 (2012)at Hamburger-Hamilton stages 10–11, around 40 hours
after fertilization. Zou and Mak decided to test whether
this tissue would show inductive activity by transplanting
mesoderm taken from the same site and developmental
stage of quail embryos. Quail CPM from HH10 had noeffect on chick columella, but when they did the same
experiment using CPM from quail HH8 embryos, theyfound morphological changes in the recipient columella,
which was interesting as this stage precedes that at
which direct contact between these tissues occurs innormal development.
The team speculated that the cephalic paraxial mesoderm might work indirectly by exerting an effect on another neighboring tissue that subsequently induces middle ear. Previous work had shown that CPM induces theotic placode, the tissue that gives rise to the inner ear,so Mak and Zou first tried ablating the inner ear and watching for effects on columella development.
Although some anomalies were detected in the fine structure of the ossicle, the effects were modest
and suggested that the tissue mediating the CPM’s inductive action is not inner ear.

Chick middle ear: the middle
ear ossicle of the chicken,
the columella, is the crossshaped bone in this skeletal
stain of the 14-day-old chickhead. Mineralized bone (red) ;cartilage (blue).

They turned next to the pharyngeal endoderm (PE), which is known to act in the formation of otherskeletal elements. Again using a classical explant approach, Zou and Mak transferred corresponding quail tissue into chick embryos and watched for changes in columella formation. When tissue
from the second branchial arch (the site of origin for the middle ear) was transplanted from a HH10
quail embryos into chick embryos at the same stage, they observed a range of severe defects in
middle ear development; transplants of other endodermal tissue had no such effect, suggesting thatpharyngeal endoderm works specifically in this context.
Given that both the CPM and thepharyngeal endoderm affect columella tissue patterning, the teamconjectured that CPM might be
triggering inductive activity in thePE, which is known to express
Fgf19, the signaling molecule primarily responsible for inner ear induction. Chick endoderm cultured
in isolation did not show expressionof this gene, but when this tissue
was recombined with quail CPM, it
was switched on, suggesting that
endodermal Fgf19 is regulated by
one or more signals from the cephalic paraxial mesoderm.

A pair of dissected columellae,
stained for cartilage, from theleft and right side of a chick at
day 12 of development.

“How organ systems with components derived from different lineages develop in a coordinated andintegrated fashion has not really been investigated,” says Ladher. “The auditory system gives us
some insight into one way that integration could happen.”35

Celsr1 bridges key processes information of neural tube

Masatoshi TAKEICHI, Tamako (and Tomoki) NISHIMURA

Topology plays a central role in development. Sheets of cells held together by adhesion molecules
fold or curl into functional barriers separating regions of the body or tubes that serve as tightly
sealed channels. The neural tube is one such structure, which forms from an epithelial sheet called
the neural plate that bends in, rolls up, and pinches off to form an enclosed cylinder that subsequently serves as the source of an extremely wide range of cells types; indeed, this transient structure is sometimes referred to as the “fourth germ layer.” The early steps of neural tube formation
involve a process of cell rearrangement known as convergent extension, in which sheets of cells
spread toward each other and merge by intercalation, as well as a signaling pathway known as planar cell polarity (PCP) that reorients the alignment of intracellular components in the merging cells.
How PCP and convergent extension work together to form the neural tube, however, is unknown.
Using embryonic chick as a model, Tamako Nishimura and others in the Laboratory for Cell Adhesion and Tissue Patterning (Masatoshi Takeichi, Group Director) have shown that the PCP signaling molecule Celsr1 is concentrated in the apical junctions of polarized cells in the merging neuralplates. Published in Cell, this work shows how this single molecule coordinates both apical constriction and convergence of neuroepithelial sheets in the nascent neural tube.
The inward bending of the neural plate relies on the purse string-like constriction of the apical surfaces of certain key cells in the region to generate a “hinge” effect, a process that is known to involve the activation of actomyosin in the region by the Rho kinase, ROCK. The resulting depression
resembles a furrow, rather than a bowl, suggesting that this apical constriction is oriented along theanterior-posterior (A-P) axis of the neural plate.

36©2012 Elsevier B.V.
Nishimura T, et al. Planar cell polarity links axes of spatial dynamics in neural-tube closure.
Cell 149.1084-97 (2012)

To gain a better understanding of how this occurs, Nishimura watched adherens junctions in theneural plate in chick embryos as the component cells remodeled the tissue by apical constriction.
She found that activated actomyosin (as identified by the molecule pMLC) accumulated at junctions
perpendicular to the A-P axis, and that cells tended to contract uniformly in the direction of the midline. Live imaging analyses of the region further revealed changes in both the cell morphology of theapically constricted cells and rearrangements of spatial relationships between cells along this axis,
pointing to the possibility that this oriented constriction is a driver of convergent extension.

Celsr1 co-localizes with contracting actin filaments (F-actin) in cell junctions perpendicular to the A-P axis (top).
Interference with Celsr1 function results in defects in neuraltube closure (bottom).

This next raised the question of how this orientation is generated. Previous work from the Takeichi
lab identified the Drosophila protein Flamingo as a member of the cadherin molecular superfamily
involved in the regulation of planar cell polarity. Interestingly, mice lacking Celsr1, a vertebrate homolog of Flamingo, exhibit defects in neural tube closure – a common form of congenital defect inhumans. The group speculated that Celsr1 might play a role in determining cell polarity and thus theorientation of apical constriction. Similar to pMLC, Celsr1 localized in junctions perpendicular to theneural plate A-P axis, and knockdown of the gene function by siRNA produced neural tube defects
reminiscent of those seen in Celsr1 knockout mice, and while pMLC activity was maintained, its
localization to the cell-cell junctions was reduced. Cellular rearrangements underlying convergent
extension were also diminished, strongly suggesting that Celsr1 plays a critical role in orienting apical constriction in this context.
“Studies in Drosophila have highlighted the potential for local constriction of cell adhesion sites toexert significant effects on tissue morphology,” says Takeichi. “In this study, we have shown how
this can occur in vertebrates as well. We next hope to focus on what drives the localization of Celsr1
and, more generally, whether this system is conserved in other tissues, and other taxa.”37

Role for Robo in cortical neuron
distribution

Yuko GONDA

The cerebral cortex is the thin, but structurally complex, surface of the mammalian forebrain andserves as the locus for important functions of the nervous such as the processing of sensory inputs, decision-making, and memory. This “gray matter” shows a neatly stratified organization into
six layers, each characterized by a distinct type of neuron, all of which connect and interact withother parts of the cortex and the rest of the brain. Positioning of cells in their appropriate locations
is therefore key to brain function. The mechanisms by which cortical neurons arrive at and establish
themselves in their specific destinations, however, remain poorly understood.
Yuko Gonda in the Laboratory for Neocortical Development (Carina Hanashima, Team Leader),working with colleagues at the National Institute of Neuroscience, have now shown how the receptor
protein Robo1 helps determine the positioning of certain neurons in the neocortex. Published in thejournal Cerebral Cortex, this work reveals an important new molecular basis underlying neuronal arrangement in the cortex.
The Robo proteins were first identified as receptors for chemorepulsive signals in Drosophila, andwere later found to operate in axon guidance in the mammalian central nervous system as well. In ascreen to identify genes expressed in subset of neocortical neurons, Gonda first found that Robo1
mRNA exhibit layer-specific expression. Further analyses revealed the Robo1+ cells to be pyramidal
neurons, and that the protein localized mainly in dendrites and axons.

38©2012 Oxford University Press
Gonda Y, et al. Robo1 regulates the migration and laminar distribution of
upper-layer pyramidal neurons of the cerebral cortex. Cereb Cortex (2012)

To better understand its function, the team first examined mice in which Robo1 had been deleted
from the genome. They found higher densities of pyramidal cells in cortical layers II/III in theseknockout mice, resulting in a thinning of these layers and consequent shift of layer IV toward thepial surface of the cortex. To further determine the cell-autonomous requirement for Robo1 in thesecells, Gonda introduced shRNA constructs against Robo1 in discrete population of layers II/III pyramidal neurons by electroporation, and found that greater numbers of these cells were retained in
the intermediate zone as opposed to entering the cortical plate as in controls, suggesting that lossof Robo1 function affects early neuronal migration in the embryonic cortex.

Suppression of Robo1 affects
inside-out layer formation of
neocortical layers II/III neurons. In control shRNA transfe cte d ne urons, late -bor npyramidal cells electroporated with DsRed at E16.5(magenta) are able to migrate
past earlier-born cells (green),
resulting in an inside-out neuronal distribution. In Robo1
suppressed cells, neurons
fa il to u n d e rg o b i r th d ate dependent segregation ofthese neurons.

The laminar organization of cortical neurons follows an inside-out pattern, in which the early-bornneurons migrate to deeper layers of the cortex, while later-born generations make their way successively to more superficial layers. In Robo1 shRNA transfected layers II/III pyramidal neurons, although
the cell movement into the cortical plate was initially delayed, these neurons eventually migrated tothe surface of the cortical plate. However, after arrival, these neurons remained in the most superficial part of layers II/III. As the molecular mechanism by which the relative inside-out positioningof cortical neurons is established remains largely unknown, Gonda next assessed the distribution
pattern of temporal cohorts of Robo1 suppressed neurons by performing sequential electroporation
analysis. The experiment revealed that Robo1 suppression leads to abnormal laminar distribution ofneurons in which the inside-out pattern was compromised. Interference with Robo1 function also
caused defects in the growth and branching of neurites, while leaving neuronal identity intact. The
picture that emerges is one in which Robo1 is required for determining the layer-specific final positioning of pyramidal neurons, and fine-tuning of axon projections in distinct cortical layers.
“The laminar organization of the mammalian cortex is its most distinguishing feature, but we only
have a limited understanding of the molecular factors that give rise to this structure,” says Hanashima. “What we have shown here is how a layer-specific axonal guidance receptor regulates thearrangement of neurons in upper layers of the cortex, which suggests that other molecular control
systems may be at work in determining neuronal location in other layers as well.”39

Self-organized retinal tissue from humanembryonic stem cells

Tokushige NAKANO, Satoshi ANDO

Pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)
are valued for their ability to give rise to all the many cell types in the adult body. Since the first derivation of human ESCs in 1998, scientists have developed a variety of techniques for inducing thesecells to differentiate into specific cell populations at reasonably high efficiencies. More recently, anumber of reports have shown that mouse ESC can be prompted not only to give rise to formlesscell aggregates, but remarkably well-organized tissue-like structures in vitro, a feat that has been
likened to the recapitulation of developmental phenomena in a culture dish.
Now, Tokushige Nakano and colleagues in the Laboratory for Organogenesis and Neurogenesis(Yoshiki Sasai, Group Director) in collaboration with Sumitomo Chemical, have shown that human
ESCs exhibit that same capacity for generating self-organized, complex tissue structures with theirreport of an hESC-derived optic cup. Published in Cell Stem Cell, the report also describes thegroup’s development of a cryopreservation technology capable of freezing these retinal tissues intact.
This represents the latest in a series of achievement in induced differentiation from ESCs by the
Sasai group. Using a cell culture system (SFEBq) developed in the same lab, Sasai has previously
shown controlled differentiation of a range of neuronal cell types including dopaminergic neurons,
cerebellar Purkinje cells, as well as cortical, sensory and motor neurons. In recent years, the grouphas shown that mouse ESCs can be steered to self-organize into complex tissue-like structures involving multiple cell types, including cerebral cortex, optic cup, and pituitary hypophysis, revealing40©2012 Elsevier B.V.
Nakano T, et al. Self-formation of optic cups and storable
stratified neural retina from human ESCs. Cell Stem Cell 10.771-85 (2012)

an intrinsic developmental capacity in these pluripotent cells independent of signals from the surrounding microenvironment.
Their most recent achievement built on the lab’s previous demonstration of optic cup formationfrom mouse ESCs, but required modifications to that protocol to account for difference in tissue
size, number of cells, culture media, and the time points at which the growth factors that steer theprocess needed to be added. The optimized 22–26 day process began with a population of around
9000 hESCs, which formed retinal precursors after about two weeks in culture, after which thestructure gradually remodeled to form the optic cup. Interestingly, the resulting structure was around
twice the diameter of that derived from mouse ESCs, apparently reflecting the size differences in theembryos of the two species.

Formation of optic cyst- (top)and optic cup-like structuresfrom human ESCs (bottom
left). Staining reveals neuraldifferentiation in inner layer(bottom center), and distinct
laminar structure of retina(bottom right).

When the neural retina was cut away from induced optic cups and cultured, it showed even further
differentiative capacity, giving rise to ganglion cells, photoreceptors, and other cells types by day 40,
and forming a laminar retinal structure resembling that of the adult retina by day 126. Intriguingly, Nakano also observed cone photoreceptors, a cell type not seen in mouse ESC-derived tissue. Recognizing that the long culture times required to give rise to these tissues could represent a pitfall forclinical use, the group also developed a system for pre-treating the optic cups prior to cryopreservation, allowing them to be frozen, stored, and thawed with minimal damage.
“The question of just how complex a structure we can obtain from embryonic stem cells is a fascinating one from the developmental perspective,” says Sasai. “We are hopeful that these findings
may help to lay a foundation for a regenerative medicine in which intact organized tissues, not justgroups of cells, can be developed for use in cell transplantation.”

41

Chromodomain binding to nucleic acids key
to heterochromatin assembly and function

Mayumi ISHIDA, Yasuko OHTANIChromosomes in eukaryotic cells contain regions of compactly bundled DNA and protein called heterochromatin, which serve as loci for the epigenetic regulation of gene expression, frequently in theform of gene silencing. In a sense, genes in heterochromatic regions are “locked away” from transcription factors that might trigger their expression. Recent studies in the fission yeast, Schizosaccharomyces pombe, have shown that the RNAi machinery is involved in heterochromatin assemblyin centromeres, but direct mechanistic links between these two processes have remained elusive.
New work by Mayumi Ishida and colleagues in the Laboratory for Chromatin Dynamics (Jun-ichi
Nakayama, Team Leader) has now shown how the RNAi factor Chp1 contributes to heterochromatin
assembly and gene silencing through direct binding not only to methylated histones but to DNA andRNA as well. Published in Molecular Cell, this new report provides new mechanistic insights into thiscrucial genetic regulatory system.
The construction of heterochromatin regions relies on a process called histone methylation, in which
DNA-associated histones are decorated with methyl residues as a kind of identifying label. In fission
yeast, a methyltransferase called Clr4 methylates the ninth lysine of histone H3 (called H3K9me),
which then binds HP1 proteins (such as Swi6 and Chp2) resulting in the formation of a transcriptionally silent chromatin region. RNAi, in contrast, is a mechanism by which targeted RNAs are degraded post-transcriptionally. The centromeric regions of fission yeast chromosomes express noncoding RNAs at low levels; after their transcription, these are cleaved into double strands by RNAi
machineries and enzymatically diced to short sequence fragments known as siRNAs. These ribonu-42©2012 Elsevier B.V.
Ishida M, et al. Intrinsic nucleic acid-binding activity of Chp1
chromodomain is required for heterochromatic gene silencing. Mol Cell 47.228-41 (2012)

clear snippets form part of a larger complex called RITS, which itself binds to chromatin by targetingof these non-coding RNAs. This elaborate process is known to recruit Clr4 to the centromere andinduce histone methylation.
With this as a background, the Nakayama lab’s most recent study focused on Chp1, a component of
the RITS complex, which plays a critical role in tethering RITS to the centromere. Notably, the Chp1
protein structure features a region known as a chromodomain (CD), which it shares with other CD
proteins, such as Clr4, Swi6, and Chp2. Chp1 also includes a different domain called RRM, which is
known as an RNA binding site in other proteins, leading them to ask whether it would have the samefunction here. On conducting an electrophoretic binding assay to answer this question, they were
surprised to find not only that RNA was bound not only by RRM, but by the chromodomain as well.
This effect was intensified nearly ten-fold by histone methylation, which also enabled DNA binding
by Chp1, suggesting that the binding of this protein to chromatin is stabilized via a CD-mediated interaction with H3K9me.

Four chromodomain proteinsaf fe cting hete rochromatin
formation in the fission yeast.
These domains (labeled CD)
in Chp1 and Clr4 bind to DNA
and RNA, stabilizing chromatin and working to establish
heterochromatic structure.

Interesting, but was this discovery important in vivo as well? Ishida generated Chp1 mutants to testfor effects on chromatin structure and found that defects in either the methylated histone- or the
nucleic acid-binding domains resulted in a dramatic reduction in localization of Chp1 to the centromere, and a concomitant drop in heterochromatin function. The team will be focusing next onwhether this activity is unique to Chp1, or more widely shared by its chromodomain protein kin.
“Our next step will be to look into whether the nucleic acid-binding function of Chp1 is at work inother heterochromatic regions, and in other eukaryotes,” says Nakayama. “This will be an importantpart of our broader focus on unraveling the roles of CD proteins in general in heterochromatin assembly and RNAi.”

43

The body’s clock from a blood sample

Our bodies have a daily activity cycle that tracks closelywith the natural day-night rhythm of the rotating earth,and helps regulate such physiological functions as hormone secretion, blood pressure, and sleep. The importance of these biological clocks can be seen from themany diseases, such as asthma, myocardial infraction,and even some allergies, that show clear tendencies toworsen or occur at specific times of day. Misalignment
between internal time and the outside environment isalso tied to other problems, such as jetlag, hormone dysregulation, cognitive problems, and affective disorders,
all of which make the ability to make clear and accurate
measurements of the body’s clock an important researchgoal.
Now, Takeya Kasukawa and others in the Functional
Genomics Unit and Laboratory for Systems Biology (bothheaded by Hiroki R. Ueda) reveal a convenient, noninvasive method for calculating body time in humans.
This work, conducted in collaboration with researchers
from Keio University, the National Center of Neurology
and Psychiatry, and Hokkaido University, and publishedin the Proceedings of the National Academy of Sciences,shows how a timetable of oscillating metabolites provides a readout of the internal clock of potentially great
clinical value.
Previously published methods for telling internal time
have been based on measurements of known cycling
molecules, such as melatonin or cortisol, but these necessitated the isolation and monitoring of the subject andtaking multiple samples over an extended period, making
them labor-intensive to researchers and burdensome topatients. Taking a hint from an observation made by Carlvon Linné (better known as Linnaeus), who noted that, asdifferent flowers bloom at different times of day, it should
be possible to tell the time simply by seeing which flowers are in bloom, Kasukawa et al looked for a way toread a molecular version of this “flower clock” in humans.
The Ueda lab had previously developed a method for
doing just that in mouse, by developing and referring toa 24-hour timetable showing the cycling levels of a large
number of hundreds of metabolites.

Takeya KASUKAWA

44

In this latest study, Kasukawa sought to apply thosesame techniques in humans, beginning by measuring
the rise and fall of metabolite levels over the course of a
24-hour day in three human subjects following predetermined activity routines, in which the subjects stayed inlight- and temperature-controlled environments, receiving specific calorie amount every two hours and giving

©2012 National Academy of Sciences
Kasukawa T, et al. Human blood metabolite timetable indicates internal body time. PNAS 109.15036-41 (2012)

blood samples on the hour over a day and a half period. This so-called constant routine minimizes
the effects of external influences on internal state making it possible to extract readouts of the unperturbed endogenous levels of metabolites in the blood. These fluctuating abundances were measured by liquid chromatography and mass spectrometry, making it possible to construct a timetableplotting metabolite levels against body time.

Changes in metabolite levelsin blood samples from threehuman subjects over a 24h o u r p e r i o d. Po s i t i v e i o nmetabolites shown at top,
negative ion metabolites at
b ot to m. (G r e e n i n d i c a te s
lower and magenta higher
abundances)

The next step was validation. Blood samples were taken at arbitrary times from three different subjects, analyzed for the same metabolites, and compared to the molecular circadian schedule. Samples were also taken from the six subjects subjected to shift routines, in which external and internal
time signals are purposely uncoordinated. Comparing the results from all these groups, Kasukawa
et al. found that the readouts matched neatly with the subjects’ actual internal time, with a maximum
margin of error of only three hours. The success of this method provides an accurate new way toevaluate biological time in humans without requiring multiple blood draws over extended periods.
The Ueda lab’s technique has great potential for uses in the study of rhythm disorders and development of effective treatments. “We hope to improve the accuracy and ease of use of this metabolite
timetable approach in the future,” says Kasukawa, “and to show its potential for clinical applicationby using it to analyze actual patient blood samples as well.”45

2012 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 dedicatesitself 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.

Learning programs forhigh school students
The CDB holds regular courses and workshops for area high school students tolearn more about embryogenesis and get a feel for research in the lab. This year,
the Office for Research Communications organized courses in August and December, giving dozens of local students a chance to study concepts and experimental technqiues in cell and developmental biology.

Internships for undergrads
In August, the Center welcomed undergraduate students from local universities
to perform one-week internships in various CDB laboratories. Interns selectedfrom among eight research areas, and worked side-by-side with research scientists in host labs.

Graduate school lecture program
Also in August, the CDB hosted its annual intensive lecture program for graduate
students potentially interested in pursuing research in developmental biology or
related fields. More than 100 students joined the program, which included talks
by CDB research leaders and visits to labs.

Training workshop for high school teachers
The CDB’s annual course in developmental biology for high school teachers was held on
October 6–7, with over 20 biology instructors in attendance. The course began with aseminar on how transcription factors establish cell identity followed by a practical coursein techniques for culturing and studying chick and quail embryos.

46

Fluorescent microscopic images of mouse embryos expressing the
Fucci2 cell cycle reporter transgene, from early cleavage (10 o’clock),post-implantation (3 o’clock), through mid-gestation (center) stages. Individual cells at specific phases of the cell cycle (Red, Yellow, Green)
can be readily distinguished in live embryos and tissues.
Image: Takaya ABE, Laboratory for Animal Resources and Genetic Engineering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 thespatiotemporal behavior of neural stem cells, which are highly dynamic in theirmodes of division and gene expression. We focus our study on the programsby 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 is engaged in the study of thegenetic programs underlying neural development, with a focus on the asymmetric divisionof neural stem cells. His laboratory has discovered several key mechanisms controlling asymmetric divisions in neural progenitor cells using
Drosophila and mouse as model systems. He
has also recently found a novel type of selfrenewing progenitor in the developing cerebral
cortex in rodents, providing new insights into
the enormous increase in brain size during
mammalian evolution.

Staff
Group Director

Technical Staff

Fumio MATSUZAKI

Tomoko AOKI
Aki MASUDA
Asuka MOMIYAMA
Taeko SUETSUGU
Raymond TerhuneKUNIKANE

Research Scientist

Hiroshi ISHIMOTO
Tomohiko IWANO
Daijiro KONNO
Atsunori SHITAMUKAI
Shigeki YOSHIURA

Student Trainee

Research Associate

Takuya YOKOMATSU

Noriko YUTSUDO

Part-Time Staff

Visiting Scientist

Ayano KAWAGUCHI
Yoichi KOSODO

Hiromi ISHIMOTO
Erina MATSUMOTO
Megumi SHIBATA
AssistantJunko ISHIGAI

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 overriding ectoderm (Yoshiura et al., 2012), 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 novel transition in the division mode in the developing mouse cortex from radial glia (typical neural stem cells with the epithelial structure) to another type ofneural 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 gyrencephalic brains, such as primates and ferrets. We are investigating the mechanisms that underlie the formation, maintenance, and expansion of this typeof stem cells, by developing model mice that produce large numbers of basal
radial glia.

Recent Publications
Matsuzaki F, and Shitamukai A. Cell division modes andcleavage planes of neural progenitors during mammaliancortical development. Cold Spring Harb Perspect Biol.
Mammalian Develop. in press.
Iwano T, et al. Prox1 postmitotically defines dentate gyrus
cells by specifying granule cell identity over CA3 pyramidal
cell fate in the hippocampus. Development 139.3051-62(2012)
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)
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)

48

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, and41:18 in these consecutive images.
(http://www.cdb.riken.jp/en/04_news/articles/11/110704_progenitors.html)

Lung Development
The organs of the body all play critical functional roles, which are made possible by the arrangement of differentiated cells into the structures specific tothat organ. Such structures are formed throughout development, with the lateembryonic and immediate postnatal periods being particularly important for thefunctional maturation of organ systems. Defects that arise during these organogenetic processes are closely linked to a wide range of diseases, while after
birth the body is constantly exposed to potentially damaging environmental
stresses. The adult body does manifest a certain degree of regenerative ability,
although this is by no means complete. To study organ formation, repair and regeneration, we have focused our research on the respiratory system in mouse.
Respiratory organs in higher mammals are characterized by their efficient gas
exchange, enabled by the functions of specialized cells. The development ofsuch organs relies on the coordinated activities of both epithelial and mesenchymal tissue types, which arise from tissue-specific populations of stem cells
in the developing embryo. The epithelial tissues of the conducting airways serveas the channel for the intake and exhalation of gases in the respiratory cycle,
and are composed mainly of Clara, ciliated, and neuroendocrine cells. In ourwork to date, we have shown how these various cells interact and exchange
information to maintain the appropriate balance in their respective cell numbers
and distributions.
Despite its location in the body’s interior, the respiratory tract is constantly
exposed to environmental factors, such as infection by viruses and bacteria,smoke, and chemical toxins, that may damage the airway epithelium. This damage is rapidly repaired by regenerating epithelial cells supplied by somatic stem
cells in the adult tissue, and as in development, the numbers and distribution ofcells in the epithelium must be maintained at levels appropriate to each regionof the airway.

Mitsuru MORIMOTO Ph.D.
http://www.cdb.riken.jp/en/morimoto
Mitsuru Morimoto received his Ph.D. in life
sciences in 2003 from Tokyo University of
Pharmacy and Life Sciences. From 2003 to
2006, he studied the molecular mechanismsof somitogenesis using mouse genetics at
the National Institute of Genetics. He thenmoved to the Washington University School of
Medicine in St. Louis to work with Dr. Raphael
Kopan, where he extended his research to lungorganogenesis. He returned to the National Institute of Genetics in 2010, and was appointed
Team Leader at the RIKEN CDB in 2012.

Staff
Team Leader

Mitsuru MORIMOTO
Part-Time Staff

Junko SAKAI
Assistant

We focus on issues of how tissue morphology influences the formation of stem
cell niches in the development, repair and regeneration of respiratory organs, aswell as mechanisms regulating cell proliferation and differentiation in developmental and regenerative processes.

Yuka NODA

Recent Publications
Morimoto M, et al. Different assemblies of Notch receptors
coordinate the distribution of the major bronchial Clara, ciliated and neuroendocrine cells. Development 139.4365-73(2012)
Morimoto M, et al. Canonical Notch signaling in the developing lung is required for determination of arterial smoothmuscle cells and selection of Clara versus ciliated cell fate. J
Cell Sci 123.213-24 (2010)
Demehri S, et al. Skin-derived TSLP triggers progression from epidermal-barrier defects to asthma. PLoS Biol
7.e1000067 (2009)
Morimoto M. and Kopan R. rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse. Dev Biol 325.1718 (2009)
Morimoto M, et al. The negative regulation of Mesp2 bymouse Ripply2 is required to establish the rostro-caudal
patterning within a somite. Development 134.1561-9 (2007)
Morimoto M, et al. The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity.
Nature 435.354-9 (2005)

Airway epithelium is composed of various specialized cell types that benefit functional airways. Immunofluorescence staining of E18.5 lung with anti-CC10 (green), anti-CGRP (red) and anti-SSEA-1 (blue)shows localizations of Clara, neuroendocrine and SPNC cells.

49

Developmental Morphogeometry
Developmental phenomena comprise a multiscalar system extending across a
range of spatial scales, from molecular to cellular to histological. Such phenomena are also multiphysical, in that they involve the transmission and receptionof positional information through diffusion and reactions of chemicals, and thegeneration of forces within tissues and concomitant geometrical deformation
through the proliferation and migration of cells. The Laboratory for Developmental Morphogeometry takes theoretical and experimental approaches to thestudy of phenomena comprising multiple scales and properties, and the quantitative measurement of dynamic and coordinated interactions between suchphenomena.

Yoshihiro MORISHITA Ph.D.
http://www.cdb.riken.jp/en/morishita
Yoshihiro Morishita received his M.Sc. and
Ph.D. from the University of Tokyo Graduate
School of Frontier Sciences. From 2005 to
2007, he served as Project Assistant Professor
in the Kyushu University Department of Biology. From 2007 to 2011, he served both as assistant professor in the Theoretical Biological
Laboratory at Kyushu University and as a researcher funded under several JST programs.
He was named Research Unit Leader at the
RIKEN CDB in 2012.

Staff
Unit Leader

Technical Staff

Yoshihiro MORISHITA

Yu WATAOKA

Research Scientist

Part-Time Staff

Yukitaka ISHIMOTO
Daisuke OHTSUKA

Yuko FUJIYAMA

Student Trainee

Ken-ichi HIRONAKA
Sangwoo LEE

Recent Publications
Hironaka K. I. and Morishita Y. Encoding and decoding of
positional information in morphogen-dependent patterning.
Curr Opin Genet Dev (2012)
Morishita Y. and Iwasa Y. Coding design of positional information for robust morphogenesis. Biophys J 101.2324-35(2011)
Morishita Y. and Iwasa Y. Estimating the spatiotemporal
pattern of volumetric growth rate from fate maps in chicklimb development. Dev Dyn 238.415-22 (2009)
Morishita Y. and Iwasa Y. Growth based morphogenesis ofvertebrate limb bud. Bull Math Biol 70.1957-78 (2008)
Morishita Y. and Iwasa Y. Optimal placement of multiple
morphogen sources. Phys Rev E Stat Nonlin Soft Matter
Phys 77.041909 (2008)
Morishita Y, et al. An optimal number of molecules for signal
amplification and discrimination in a chemical cascade. Biophys J 91.2072-81 (2006)

An example of this is seen in organogenesis, a process in which the various
tissue regions that give rise to the organ exhibit changes in volume at different
rates, or anisoptropic expansion and contraction. Such deformations can bequantified as tensor quantities (geometrical characteristics). The morphologicaldifferences between various organs, or between homologous organs in different species can thus be explained as spatio-temporal patterns in the tensor
quantities of each object. We are now able to extract such patterns using organ-level quantitative imaging and statistical analysis. By combining and comparing such data with the accumulated body of molecular and cellular evidence,
we hope to develop clearer insights into the relationships between macro-scaleorgan morphogenesis and micro-scale phenomena.
The ability of individual cells to recognize and respond to (for example, throughproliferation and differentiation) their positions within a tissue is also essential
to tissue growth and patterning. This necessitates accurate “spatial recognition” on the part of cells, which receive environmental cues (such as gradientsof growth factors or interactions with neighboring cells), but this is complicatedby uncertainty arising from perturbations within the organism (such as interindividual variations in morphogen expression levels). Questions of how to
maximize the accuracy of the transmission and reading of information against
a background of uncertainty is formalized as problems in information coding.
Analysis of such problems has revealed optimal sites of the expression of information sources (morphogens) and optimal designs for the form and parametervalues of response functions implemented by intracellular biochemical reactions. By comparing the results of these theoretical analyses with experimental
observations, we can begin to assess the extent to which actual developmental
systems are designed to optimal criteria.
Our lab will seek to use
1) analysis of measured
data and 2) study systemdesigns through theoreticalformulation and computer
simulations with an eye todeveloping a better understanding of these phenomena.

Optimal arrangement of morphogen source (Shh expression
region) to maximize the precision of positional information in vertebrate limb bud.

50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 baccalaureate and his Ph.D. from the University ofTsukuba. He spent a year as a post-doctoral
fellow at the same 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 ofTsukuba. He was appointed assistant professor in the university's Gene Research Centerand Institute of Biological Sciences in 2000,
and began a three-year term as a PRESTO researcher in the Japan Science and Technology
Corporation (JST) in December 2001. He wasappointed CDB team leader in March 2002.

Staff
Team Leader

Assistant

Akira NAKAMURA

Yoko UMEGAKIEri YAMASHITA

Research Scientist

Takako AWAI
Kazuko HANYUNAKAMURA
Yasuko KATO
Maki SHIRAEKURABAYASHI
Yuko SUKEGAWA

Recent 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)
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).

Migrating germ cells (blue) in stage 10 Drosophila embryo

51

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 inter-related,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

Student Trainee

Jun-ichi NAKAYAMA

Gohei NISHIBUCHI

Research Scientist

Assistant

Aki HAYASHIAtsuko SHIRAI

Sayaka SENO
Shinobu SHIMAHARA

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.

Research Associate

Mayumi ISHIDATechnical Staff

Rika KAWAGUCHI
Yasuko OHTANI

Recent Publications
Ishida M, et al. Intrinsic nucleic acid-binding activity of Chp1
chromodomain is required for heterochromatic gene silencing. Mol Cell 47.228-41 (2012)
Kawakami K, et al. A novel RNAi protein, Dsh1, assembles
RNAi machinery on chromatin to amplify heterochromatic
siRNA. Genes Dev 26.1811-24 (2012)
Hayashi A, et al. Heterochromatin protein 1 homologue Swi6 acts in concert with Ers1 to regulate RNAidirected heterochromatin assembly. Proc Natl Acad Sci
USA 109.6159-64 (2012)
Shiomi Y et al. Two different RFC proteins, Ctf18 and RFC1
separately control PCNA-CRL4Cdt2-mediated Cdt1 proteolysis during S phase and following UV-irradiation. Mol Cell
Biol 32.2279-88 (2012)
Hiragami-Hamada K, et al. N-terminal phosphorylationof HP1a 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)

Swi6/HP1 recruits RDRC to heterochromatin through Ers1, an RNAi factor intermediate. Heterochromatic
localization of Ers1 is dependent on Swi6/HP1 (upper, left). Yeast two-hybrid assay testing interaction
between Ers1 and other RNAi factors (upper, right). Model for Swi6-mediated siRNA generation at heterochromatin (lower).

52

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 professorshipin the same department in 1979. He spent theperiod 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 Department of Microbiology in 1983. He moved to
the Kumamoto University Faculty of Medicine
in 1987 to take a professorship in the Department of Immunopathology, and returned to
Kyoto in 1993, as professor in the Departmentof Molecular Genetics at the Kyoto University
Faculty of Medicine. He was appointed CDB
group director in 2000.

Staff
Group Director

Technical Staff

Shin-Ichi NISHIKAWA

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

Research Scientist

Igor M SAMOKHVALOVYoshiteru SASAKIYosuke TANAKA
Yan-Fung WONG
Research Specialist

Lars Martin JACT
Reiko NAKAGAWA
Collaborative Scientist

Shin KAWAMATA
Visiting Scientist

Teruo AKUTA
Guo DING
Keitaro IMAIZUMIHiroshi KATAOKA
Shigeo MASUDA
Herve Le MOUELLIC
Taro MUTO
Visiting Researcher

Takao YAMADA

Student Trainee

German Dario
CAMARGO ORTEGA
Lars HANSSEN
Matteo PLUCHINOTTA
Mehrnoosh Fathi
ROUDSARI
Part-Time Staff

Miho HOSHIJIMA
Yoko OTSUKA
Noriaki SATO
Kazumi YANAIAssistantJunko KOYA
Naeko MINAMI

Recent Publications
Jakt L. M, et al. A continuum of transcriptional identities
visualized by combinatorial fluorescent in situ hybridization.
Development 140.216-25 (2013)
Tanaka Y, et al. The transcriptional programme controlled
by Runx1 during early embryonic blood development. Dev
Biol 366.404-19 (2012)
Tanaka Y, et al. Early ontogenic origin of the hematopoietic
stem cell lineage. Proc Natl Acad Sci USA 109.4515-20(2012)The Last Supper

Kobayashi K, et al. Epigenetic regulation of the neuroblastoma genes, Arid3b and Mycn. Oncogene (2012)
Hayashi M, et al. Endothelialization and altered hematopoiesis by persistent Etv2 expression in mice. Exp Hematol
40.738-50 e11 (2012)53

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 the same
year.

Staff
Team Leader

Agency Staff

Takashi NISHIMURA

Aya MASUDA

Special Postdoctoral Researcher

Part-Time Staff

Naoki OKAMOTO

Kanako HIGUCHI
Noriko NISHIMURA
Junko SHINNO

Research Scientist

Tomoka MURAITechnical Staff

Cell proliferation and differentiation in each tissue and organ are kept understrict regulation both spatially and temporally. Research has revealed the nature of spatial signals, such as growth factors and morphogens, but the way inwhich these signals direct cell and tissue growth over time remains understood.
In addition, growth and developmental timing are also 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 throughmetabolic regulation. Therefore, linking the nutrient sensing 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 body size; and 3) how do endocrine signals interact with metabolic and growth regulators?
To better understand the interface between nutrient availability and growth
regulation, we are now focusing on how nutrition controls systemic growththrough Drosophila insulin-like peptides (Dilps). Members of the insulin family of
peptides have conserved roles in the regulation of growth and metabolism in awide variety of metazoans. We have also conducted in vivo RNAi screening to
identify new players regulating growth and developmental timing at the organismal level.

Rinna NAKAMORI
Takayuki YAMADA
Yuki YAMAUCHI

Recent Publications
Okamoto N, et al. A secreted decoy of InR antagonizes
insulin/IGF signaling to restrict body growth in Drosophila.
Genes Dev 27. 87-97 (2013)
Okamoto N, et al. Conserved role for the Dachshund proteinwith Drosophila Pax6 homolog Eyeless in insulin expression.
PNAS 109. 2406-11 (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)

Wild-type female fly (right) and insulin-like receptor mutant female (left)

54

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 bytheir ability to produce copies of themselves with similar differentiative potential,
as well as more specialized cells with different properties through differentiation. Embryonic stem (ES) cells are the best known example of a type of stem
cell possessing these properties of pluripotency and self-renewal. In our lab,
we use ES cells as a model system for studying the molecular mechanisms that
determine 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 four transcription factors. These transcription factors should form a self-organizing network able tostabilize their expression and maintain pluripotency. At the same time, the network should have the sensitivity to signals that induce differentiation. How can
the transcription factor network satisfy these contradictory requirements? Whatis the principle behind the structure of the transcription factor networks governing developmental processes? We are now trying to answer these questions bystudying ES cells and the changes they undergo in their differentiation toward
trophoblast stem cells, extraembryonic endoderm cells, and primitive ectoderm
cells.

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 forhis work in gene trap methods. From 1993 to
1994, he worked as a research associate in the
Department of Developmental Genetics at thesame university, before taking a postdoctoral
fellowship with Austin Smith at the Universityof Edinburgh Centre for Genome Research.
He returned to Japan in 1996 as a research
associate in the Department of Nutrition andPhysiological Chemistry at the Osaka University Graduate School of Medicine, where he remained until taking his current position as teamleader at the RIKEN CDB. He was appointed
project leader of the Laboratory for Pluripotent
Stem Cell Studies in October 2009.

Staff
Project Leader

Technical Staff

Hitoshi NIWA

Setsuko FUJII

Research Scientist

Junior Research Associate

Jitsutaro KAWAGUCHI
Satomi NISHIKAWATORIKAI
Satoshi OHTSUKA
Masaki SHIGETA
Robert Odell
STEPHENSON
Tomoyuki TSUKIYAMA

Shunsuke ITO

Research Associate

Mariko YAMANE

Student Trainee

Toshimi SUGIMOTO
Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI
Yoko NAKAI-FUTATSUGIAssistantMiho SAKURAI

Visiting Scientist

Yasuhide OHINATA
Hiroki URA

Recent Publications
Ohtsuka S, et al. E-cadherin promotes incorporation ofmouse epiblast stem cells into normal development. PLoS
One 7.e45220 (2012)
Martello G, et al. Esrrb is a pivotal target of the gsk3/tcf3
axis regulating embryonic stem cell self-renewal. Cell Stem
Cell 11.491-504 (2012)
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)

Chimeric embryos generated by injection ofGata6GR ES cells into blastocysts. These
ES cells carry the constitutively-active Egfp
transgene and contribute to primitive 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 andprimitive endoderm.

Kitajima H. and Niwa H. Clonal expansion of human pluripotent stem cells on gelatin-coated surface. Biochem Biophys
Res Commun 396.933-8 (2010)
Niwa H. Mouse ES cell culture system as a model of development. Dev Growth Differ 52.275-83 (2010)
Niwa H, et al. A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460.11822 (2009)55

A fast track to gut innervation

Chihiro NISHIYAMA

The gut is richly innervated with enteric neurons that autonomically control such behaviors as peristaltic movement, secretion and blood flow, earning this long stretch of the nervous system the distinction of being called “the second brain.” During development, the enteric nervous system begins
as a migratory population of neural crest cells, wending their way down to populate the entire lengthof the intestines, which can be several times as long as the entire body. This long journey has always
been thought to closely follow the winding course of the intestinal wall – when these neurons fail tocomplete the trip, it can lead to a complete lack of lower gut innervation, as seen in the congenitalcondition called Hirschprung’s disease. In some patients, however, aganglionic sections, known asskip areas, in the large intestine are found alternating with normally innervated gut, a phenomenon
that the conventional model of enteric innervation cannot explain.
New work by Chihiro Nishiyama and others in the Laboratory for Neuronal Differentiation and Regeneration (Hideki Enomoto, Team Leader) shows how a subset of enteric neural crest cells in mousetakes a short cut across the mesentery on their way to innervate the large intestine. Published in Nature Neuroscience, these findings open up new and unexpected insights into the longest migrationof neurons in the embryo.
In the mouse, the population of the gut with enteric neurons takes place over the period of embryonic day 9.5 to 14, during which the guts itself undergoes extensive growth and morphologicalchanges. At E10, the gut is still a nearly straight tube, but by the following day, it develops a hairpin
loop that gives rise to the mid- and hindgut sections (which form the embryonic bases for the small

56

©2012 Nature Publishing Group
Nishiyama C, et al. Trans-mesenteric neural crest cells are the principal
source of the colonic enteric nervous system. Nat Neurosci 15.1211-8 (2012)and large intestines). By E11.5, these gut segments begin to pull away from each other. Nishiyama et
al. used live imaging to observe the migration of cells in tissue sections from the gut over time, using
embryos engineered to express one of two fluorescent proteins – GFP and KikumeGR. Both canbe used to label enteric neurons, but KikumeGR has the added advantage of changing color fromgreen to red on exposure to ultraviolet light, making it possible for the team to track the movementsof select populations of cells through living gut tissue. Using this technique, they found that in theE12.5–13.5 embryo, precursor cells form bundles that spread in a network throughout the hindgut,
with the wavefront region at the distal edge contributing more than 80% of the hindgut neural network.
But how did these precursors arrive at their destination? Looking more closely at the fluorescent-labeled E11.5 gut, Nishiyama found that the population arose around the time that the mid- and hindgut regions are brought into close proximity by the folding action. What the team saw next surprised
them; they found that these wavefront cells are derived from a cell population that traveled straightacross the mesentery from the mid to the hind region, not from cells taking the long way around thebend. This corner cutting by migrating cells plays a crucial role; when the team blocked this transmesenteric movement, they found a dramatic delay in hindgut colonization by precursors.

Cells (KikumeGR, red) alignedwith mesentery in E11.5 gut
proliferate and migrate, giving
rise to the neural network of
the hindgut. A subpopulationof cells traverses the mesentery to reach the hindgut(right).

Next, using a mouse model of Hirschprung’s disease developed by the lab in 2008, they looked forconnections between this novel form of migration and disease. They found that in Hirschsprung’s
mice, the trans-mesenteric migration was markedly reduced, suggesting that a failure in this mechanism leads to the defects in gut innervation as well as the skip areas observed in some patients.
Enteric neurons in Hirschsprung’s disease are known to show reduced expression of GDNF, an important factor in neuronal development and differentiation, suggesting the intriguing possibility thatthe defects in trans-mesenteric migration may be tied to aberrant GDNF signaling.
“In mouse, the enteric neurons only have a window of a single day in which the mid- and hindgut areclose enough to enable neurons to take this path across the mesentery, so it is really wonderful tosee how tightly the timing of cell migration and morphological changes in the tissue are coordinated,” says Enomoto. The lab plans to study differences in cell populations that take the trans-mesenteric shortcut and those that take the long way round, as well as to investigate possible applicationsof these findings in the development of cell therapies for Hirschsprung’s disease.

57

Evolutionary and developmental roots ofthe paratympanic organ resolved

Birds, many species of which experience rapid changes
in air pressure during flight, possess a specialized structure called the paratympanic organ (PTO) in the middle
ear that appears to function as a kind of barometer or
altimeter. This fluid-filled sac is connected to the tympanum (ear drum) by a ligament-like tissue, by which
minute pressure-induced changes in the membrane arecommunicated to the PTO, triggering deformations that
stimulate mechanosensory hair cells lining its inner walls.
The PTO is for the most part a birds-only structure, andis not present in mammals, amphibians, or most reptiles.
The developmental origins of this organ, however, remain
poorly understood.
Using the chick embryo as a model, Paul O’Neill andothers in the Laboratory for Sensory Development (Raj
Ladher, Team Leader) have now found that the PTO
arises from a newly discovered tissue primordium known
as a placode, and reconfirm its homology to a sensory
organ in fish known as the spiracular organ. This work,
published in Nature Communications, was done in collaboration with Clare Baker from Cambridge University,and resolves a long-standing question over the embryonic and evolutionary roots of this sense organ.
Previous studies have suggested that the PTO andspiracular organ (a sensory pouch found in fish thatis thought to detect jaw movements) are homologous
structures, based on similarities in tissue morphology,
anatomical position, innervation, and the presence of
mechanosensory hair cells. The case for this homology
was called into question, however, by fate mapping experiments in chick embryos conducted in the 1980s thatshowed that the geniculate placode, which gives rise tofacial neurons innervating the tongue, is the embryonicsource tissue for the PTO. As the spiracular organ arises
from a different placode, this made the homology between the two organs seem a less likely prospect, andfor years the evolutionary relationship between them has
been disputed. But O’Neill doubted that a placode thatin other species gives rise solely to neurons would also
generate the hair cells of the PTO, and so decided to
study its development in greater detail to get to the bottom of this question.

Paul O’NEILL58

The gene Sox2 is known to play a critical role in hair cell
development, so he first examined its expression pattern
at embryonic stages when the PTO begins to form, andobserved a small patch of Sox2 expression immediately
adjacent to the geniculate placode. Interestingly, the©2012 Nature Publishing GroupO’Neill P, et al. The amniote paratympanic organ develops from apreviously undiscovered sensory placode. Nat Commun 3.1041 (2012)

Sox2+ region did not express geniculate placode markers, suggesting that it represented a distinct
entity. O’Neill next traced the fate of cells derived from the region and found that it gave rise to boththe PTO and a population of PTO neurons which migrate to the geniculate ganglion. These findings
pointed to a dramatic conclusion – that the PTO is not derived from the geniculate placode, but rather a previously undiscovered separate placode directly alongside it, which the team has christened
the PTO placode. Interestingly, although neurons from the geniculate and this newly described placode both reside within the geniculate ganglion, they remain segregated by gene expression and theregions of the brain to which they project.

A: Schematic showing thepositions of the PTO placode(red) and epibranchial placodes (yellow).
B: The PTO placode expresses the transcription factor
Sox2.
C-E: Expression of epibranchial placode markers. The PTO
pl a c o d e a nd e pibr a nc hi a lplacode are separate entities
that are located adjacent toeach other.
F: PTO neurons (red) migratefrom the PTO to join the geniculate ganglion.

To confirm that the PTO placode and geniculate placode are separate entities, the team next performed experiments in which different regions of head ectoderm were transplanted from quail embryos to the PTO precursor region of chick embryos to evaluate the developmental effects. Whenequivalent quail tissue was grafted into chick, both the PTO and the geniculate ganglion formed,
but when quail tissue from different regions of the head or trunk was transplanted, the PTO failed
to form, while the geniculate ganglion developed normally, suggesting that the two structures areformed by different developmental mechanisms.
The discovery of a novel placode is an exciting finding by any standards, and in this case one thathelps put a decades-old controversy over the embryonic and evolutionary origins of the PTO to rest.
“The next step is to identity the key molecular signals underpinning PTO and spiracular organ formation, and hopefully to understand the evolutionary mechanisms responsible for PTO loss or retention
in particular species” commented O’Neill.

59

Mutations in Phox2b tied to common
neural crest disorders

Mayumi NAGASHIMADAThe neural crest is a transient, mobile population of cells that gives rise to a wide array of cell types,
including skeletal, neural, endocrine, and pigment cells during development, leading some to refer
to it as the fourth germ layer. Given its importance, it is not surprising that alterations in neural crest
cell migration and differentiation can lead to serious congenital defects, a group of developmental
disorders collectively known as neurocristopathies. Most such diseases, which include such common disorders as neuroblastoma and Hirschsprung’s disease, clearly have a genetic basis, but thedetails of the underlying defect are not well understood.
Scientists in the Laboratory for Neuronal Differentiation and Regeneration (Hideki Enomoto, Team
Leader), led by Mayumi Nagashimada, have now uncovered defects in a specific gene are associated with a group of neurocristopathies of the autonomic nervous system. Published in the Journal of
Clinical Investigation, this report shows how mutations in Phox2b perturb the expression of anothergene, Sox10, which plays a central role in neural crest development. Nagashimada has since taken aposition at the Brain/Liver Interface Medicine Research Center in Kanazawa University.
Interestingly, despite the fact that neuroblastoma is a cancer of the sympathetic nervous system
while Hirschsprung’s is a defect in gut innervation, these diseases sometime occur in the same patient. This is particularly the case in patients with a rare condition called central hypoventilation syndrome (CCHS), a respiratory disorder in which breathing often stops during sleep – CCHS patientsshow a 500 to 1000 times higher incidence of neuroblastoma and Hirschsprung’s disease. Previous
genetic studies have shown that a specific mutation in the gene Paired-like homeobox 2b (PHOX2B)

60

©2012 American Society for Clinical Investigation
Nagashimada M, et al. Autonomic neurocristopathy-associated mutations in
PHOX2B dysregulate Sox10 expression. J Clin Invest 122.3145-58 (2012)is associated with the CCHS-neuroblastoma-Hirschsprung’s triad (CCHS-HSCR-NB). This gene encodes a homeodomain-bearing transcription factor, which itself has been implicated in neural crest
development. Of the various mutations known to occur in PHOX2B, many CCHS-HSCR-NB patients
exhibit frameshifts affecting the gene’s open reading frame. To study the pathogenic mechanism ofsuch mutations more closely, the team generated mice engineered to carry the same frameshift mutation in Phox2b.
The resultant mice proved to be viable for much of development, but showed 100% lethality in theperinatal period. Analyses of heterozygous Phox2b mutant offspring showed they suffered fromhypoxia due to failure of spontaneous respiration, and defects in colon innervation and sympathetic
ganglion formation – an extraordinarily close match to the symptoms of the CCHS-HSCR-NB association in humans.

Glia
Glia

Model of how defect in Phox2b leads to Sox10 dysregulation and perturbed neuronglia balance in neural crestderived nervous system (right;
wildtype shown on left)

In mouse, the cells of the autonomic nervous system characteristically express a pair of genes,
Phox2b and Sox10; the former in differentiated neurons and the latter in glia. Nagashimada lookedat the expression of these genes in two autonomic populations, enteric and sympathetic ganglia, inthe mutant animals, and found that Phox2b-expressing cells were markedly reduced in both, while
the ratio of Sox10+ cells within the population was dramatically increased. Evidently, the frameshift
resulted in a dysregulation of Sox10, freeing it from transcriptional control by Phox2b and resulting in
disturbed self-renewal and proliferation of neural progenitors and a bias in differentiation toward glial
fate. These imbalances in gene regulation subsequently manifest as the diverse and widespread
symptoms observed in the mouse model, and presumably in human CCHS-HSCR-NB patients as
well.
“This is a very important finding, as we have been able to identify the molecular basis for a developmental pathology of the neural crest,” says Enomoto. “But this is only one of many mutations associated with neurocristopathies such as Hirschprung’s and neuroblastoma, so we hope to continue
developing additional mouse models in order to develop a better understanding of and, one day,treatments for these diseases.”61

Talin links cytoskeleton and cell
membrane in migrating and dividing cells

Masatsune TSUJIOKA

Cells can show a remarkable range of motility, creeping over substrates using a variety of pushes, pulls,
stretches, and drags to get from A to B. This range of motion is achieved through the concerted efforts ofmotor proteins and structural complexes collectively known as the cytoskeleton. In addition to propulsion,
however, cells also need to find footholds on surfaces in order to get the traction needed to advance or withdraw. Mobile single-celled organisms, such as the amoeba Dictyostelium, provide excellent living models forstudying the molecular basis of such mechanisms, as they spend much of their lives solitary and on the crawl.
New work by Masatsune Tsujioka and colleagues in the Electron Microscope Laboratory (Shigenobu Yonemura, Laboratory Head) reveals how a protein called talin A links the cytoskeletal complex actomyosin to
the plasma membrane of Dictyostelium cells as they move and divide. Published in the Proceedings of the
National Academy of Sciences, this work was conducted in collaboration with colleagues at Yamaguchi and
Kyoto Universities, the University of Edinburgh, Osaka University, Core Research for Evolutional Science andTechnology Agency (CREST), and the RIKEN Quantitative Biology Center (QBiC).
Cortical actomyosin is a contractile fiber-like complex of filamentous actin and the motor protein myosin II, andis the driving force behind many forms of changes in cell morphology and movement. This activity requires
connections between this cytoskeletal molecular complex and the membrane of the cell, but how this is accomplished has remained something of a mystery. Talin, a protein known to function in integrin-mediated cellsubstrate adhesions, has been implicated as a candidate for this role, as loss of talin function has been shownin previous studies to affect bond between cells and their substrate. Knowing this, Tsujioka opted to study
the question more closely in Dictyostelium as it possesses only a single myosin II gene, simplifying the task of62©2012 National Academy of Sciences
Tsujioka M, et al. Talin couples the actomyosin cortex to the plasma membrane
during rear retraction and cytokinesis. Proc Natl Acad Sci U S A 109.12992-7 (2012)

studying it in isolation, and it shows aremarkable range of motile behaviors.
He began by examining the subcellular
localization of talin A (the Dictyostelium
homolog of mammalian talins). Usinga GFP-tagged construct and immunostaining techniques, Tsujioka foundthat this protein co-localized withmyosin II at the back end of migrating
amoebae. He next used a sophisticated piece of kit called a total internalreflection fluorescence microscope,
which enables very thin sections of asample to be visualized, to check fordirect binding between the two, but
was intrigued to find that while talin A
and myosin II remained close to one
another, they were not directly connected. Both did co-precipitate with actin, raising the possibility that thisfibrous protein served as the link.

(A) Co-localization of talin
A and myosin II during cell
division. (B) Defects in cell
division in cells lacking talin
A (bot tom) compared towildt ype (top); the mutant
cells show separation ofcontractile ring and plasmam e m b r a n e (f ro m 16 m i n),
leading to mitotic failure.

When Tsujioka expressed talin A in Dictyostelium cells lacking both myosin II and endogenous talin, he foundthat it spread throughout the entirety of polarized migrating cells, rather than just in the rear, indicating that its
distribution relies on myosin. To test whether the talin A protein plays a functional role in cell motility, the team
set up a system for observing Dictyostelium cells as they moved, taking advantage of this amoeba’s wellknown taste for cAMP. When talin A-null cells were presented with a chemical gradient generated by a microcapillary, they would set off to follow it, but their posterior ends often remained stuck behind them, resemblinga long tail that failed to detach from the substrate. Suspecting that this sticky predicament might be mediatedby a cell-substrate adhesion molecule called SibA, they next generated double knockouts for both proteinsand found that the cellular tails vanished.
Actomyosin also plays a role in mitotic cell division, so Tsujioka next asked whether talin might be at work inthis process as well. As in migrating cells, talin co-localized with myosin II, in this case mid-cell near the cleavage furrow, and again talin’s accumulation appeared to rely on myosin II. In dividing wildtype cells, this furrow
gradually folds inward, a form of movement called ingression, eventually splitting the cell into a pair of daughter
cells. In cells in which talin A was deleted, however, the furrow was wider and longer than in wildtype, and responded differently to mechanical stresses (in this case, being sandwiched tightly under a sheet of agarose),which appeared to be due to a disconnect between the actomyosin contractile ring and the plasma membrane.
As a last step, the lab conducted a structural analysis of the talin A protein to look for possible binding sitesthat might account for its apparent coupling role. They found that while the talin C-terminal contains an actin
binding site, explaining its bond with actomyosin, a locus on its N-terminal domain bound with a phospholipid
called PtdIns(4,5)P2. PtdIns(4,5)P2 is a known talin activator in mammals and is also enriched in the tail ends
of Dictyostelium migrating cells, suggesting that this might be the molecule that activates talin A, which conveys contractile force in migrating and dividing cells.
“The fact that a similar separation between the plasma membrane and the underlying actin has been seen to
occur in talin mutant cells in mammals as well suggests that that this discovery of a novel function for the molecular in Dictyostelium may be evolutionarily conserved,” says Tsujioka. “We hope to dig deeper in future analyses of this new function and to identify other molecules involved in constructing and regulating these links.”

63

Prox1 controls differentiation ofhippocampal granule cells
As is true of many brain structures, the hippocampus,
which plays a central role in memory and learning, ismade up of multiple cell types organized into domains
controlling different functions. Distinct from many otherbrain regions, however, the hippocampus is a site ofactive neuronal generation in the adult. Its distinctive
pyramidal neurons are contained mainly in the cornu
ammonis (CA), while another hippocampal region called
the dentate gyrus (DG) is characterized by granule cells.
While it is known that the survival of these granule cells
relies on the activity of specific transcription factors, how
they arise during the differentiation of the hippocampus
has remained unknown.
In a recent study, Tomohiko Iwano and others in the
Laboratory for Cell Asymmetry (Fumio Matsuzaki, Group
Director) have now shown that the transcription factor
Prox1 specifies the identity of postmitotic granule cells inmouse, while steering them away from a pyramidal fate.
Published in Development, this work reveals how thissingle factor contributes to generating the cellular diversity of the hippocampus.
For neurons, granule cells are tiny, but they play a majorrole in diverse brain regions, including memory formationin the dentate gyrus. Taking a lead from previous studies
that showed Prox1 to be involved in maintaining granulecell survival, Iwano sought to gain a better understanding of whether this transcription factor might play a rolein the differentiation of these neurons as well. As theProx1 gene is constantly switched on in granule cells,
the group first generated conditional knockouts allowing
them to selectively switch it off at any stage in development. When they looked at DG neurons in which Prox1
was homozygously deleted in the postmitotic stage, theprospective granule cells lost the expression of all markers of both mature and immature neurons of that type.
Even more interestingly, however, these conditional Prox1
mutant cells in the DG began to exhibit gene expression patterns and morphology reminiscent of pyramidalcells, the typical neuronal subtype found in a specific CA
subregion of the hippocampus – a remarkable transformation for cells which might otherwise be thought of as
terminally differentiated. Iwano found that granule cells
exhibited similar expression levels of at least 18 marker
genes characteristic of CA3 pyramidal neurons after
postmitotic knockout of Prox1, and began to show typical pyramidal dendrite morphology as well.

Tomohiko IWANO, Aki MASUDA

64

©2012 The Company of Biologists
Iwano T, et al. Prox1 postmitotically defines dentate gyrus cells by specifying granule cell
identity over CA3 pyramidal cell fate in the hippocampus. Development 139.3051-62 (2012)

By looking at the effects of loss of Prox1 function at various stages in hippocampal development(which occurs mainly from late embryonic to neonatal stages of the animal, although granule cells
continue to be generated throughout life in the DG), the group was able to show that at an advanced
stage of hippocampal development and even in adulthood, Prox1 knockout was able to convert incipient granule cells to pyramidal-like neurons.
A key aspect of a neuron’s function is its connectivity with other parts of the brain, both by accepting inputs and by projecting its axon to a specific site. Granule cells and pyramidal neurons ordinarily project to different destinations, so Iwano next checked whether Prox1-deficient DG cells would
mimic not only pyramidal cells’ gene expression and morphology, but their circuit-forming behavior
as well. He found that while some of the Prox1-homozygous granule cells retained their characteristic axonal destinations, the range of projection was greatly expanded to include sites typical of thoseof CA3 pyramidal neurons.

Prox1 steers mature neuralprecursor cells toward a granule cell fate.

Having determined the effects of Prox1 depletion, the group next tried the converse experiment toobserve how its overexpression would affect hippocampal cell fate. Iwano watched primary culturesof cells electroporated with a Prox1 plasmid, they exhibited a much stronger tendency to gene expression and morphology typical of granule cells, as opposed to control cultures in which the pyramidal fate predominated, suggesting that Prox1 overexpression promotes granule cell fate at theexpense of pyramidal. Electroporation of the same plasmid in vivo had equally pronounced effects in
inducing the granule cell phenotype. Thus, the group indicated that both depletion and acquisitionof Prox1 expression at postmitotic hippocampal cells could induce a conversion of these cells’ neuronal fate (from granule cell to pyramidal, and from pyramidal to granule, respectively), demonstratingthat Prox1 is a kind of on-off switch for pyramidal vs. granule neurons, even in at advanced stages ofneuronal maturation.
“It has been thought that pyramidal neurons and granule cells in the hippocampus are of totally different origin, but our finding reveals that their destinies are not so securely fixed, at least not untilright before their maturation by the integration into the circuit, which takes about three weeks after
their birth,” says Matsuzaki. “This may be a property common to other neurons as well, which ensures the flexibility and tunability of neural circuit formation during both brain development and adultneurogenesis.”65

2012 Awards
As one of the world’s leading institutes in the fields of developmental biology and stem cell research, the RIKEN CDB strives to maintain thehighest levels of originality, impact and quality in its scientific contributions. The individual achievements and careers of many of its scientificstaff have been recognized with awards from numerous organizations, including government ministries and agencies, private foundations andacademic societies.

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

AwardeePositionLaboratory

Award

Organization

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Inoue Prize for Science

Inoue Foundation for Science

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Tsukahara Nakaakira
Memorial Award

Brain Science Foundation

Hiroki R. Ueda

Project Leader

Systems Biology

Tsukahara Nakaakira
Memorial Award

Brain Science Foundation

Mototsugu Eiraku

Deputy Unit Leader

Four-dimensional
Tissue Analysis

MEXT Prize

Ministry for Education, Culture,
Sports, Science and Technology

Erina Kuranaga

Team Leader

Histogenetic Dynamics

Development, Growth& Differentiation
Wiley-Blackwell Prize 2012

Wiley-Blackwell

Masatoshi Takeichi

Director of the RIKEN
Center for Developmental
Biology2012 Citation Laureate

Thomson ISI

Teruhiko Wakayama

Team Leader

Genomic ReprogrammingAward for Excellent Poster
Presentation

Asian Reproductive Biotechnology
Society

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Yamazaki-Teiichi Prize

Foundation for Promotion of Material
Science and Technology of Japan

Yoshiki Sasai

Group Director

Organogenesis and
Neurogenesis

Takeda Medical Prize

Takeda Science Foundation

66

Z-Projection through one of the 3 cristae in the inner ear of an E14 developing chick embryo. The
cristae are mechanosensory organs responsible for detecting acceleration and gravity. This image
shows expression of a heparan sulfate modifier in the mechanosensory hair cells (green), nuclei
labelled with DAPI (blue), and F-actin labelled with phalloidin (red). We believe the modification ofcell surface heparan sulfate plays an important role in patterning the mechanosensory organs of
the inner ear.
Image: Stephen FREEMAN, Laboratory for Sensory Development

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
and master's degrees from Kyoto University,and his doctorate from the Research Divisionin Agriculture at the same institution in 1994.
He spent the period from 1994 to 1995 as a
research fellow in cell biology at the Nagoya
University BioScience Center before moving
to Massachusetts, where he worked as a research fellow in medicine at Harvard Medical
School and Massachusetts General Hospitalfrom 1995 to 2002. He was appointed teamleader at the RIKEN Center for Developmental
Biology in 2001, and returned to work in Japan
full-time in 2002. In 2012, he served as a seniorinvestigator at the CDB.

Our laboratory studies epigenetics by focusing on a particular set of moleculesthat act in the methylation of DNA, with the goal of identifying their roles in theregulation of chromatin function in embryonic development and the processesof lineage commitment and plasticity in cell differentiation. We conduct phenotype analyses of mice in which various genes involved in DNA methylation
have been knocked out, as well as biochemical and cell biological studies ofembryonic stem (ES) cells to explore how dynamic changes in DNA methylation
function as regulatory factors in mammalian embryogenesis and cell differentiation, and the means by which DNA methylation works to maintain cell proliferation and chromosome function. We anticipate that a deeper understanding ofthese 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
Senior Investigator

Masaki OKANO
Research Scientist

Takashi TAMURA
Technical Staff

Chisa MATSUOKA

Recent Publications
Ehara T, et al. Role of DNA methylation in the regulation of
lipogenic glycerol-3-phosphate acyltransferase 1 gene expression in the mouse neonatal liver. Diabetes 61.2442-50(2012)
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)

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 extraembryoniclineages.

68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. Usinga system developed in our lab, we have succeeded in inducing mouse and human embryonic 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, for work on neural specific transcriptional
regulators. In 1993, he took a postdoctoral
fellowship in the De Robertis lab at the UCLA
School of Medicine, remaining there until 1996
when he was appointed associate professor
at the Kyoto University School of Medicine. He
assumed a professorship at the Kyoto University Institute for Frontier Medical Sciences in
1998, and was appointed group director at the
CDB in 2000. He serves on the editorial boardsof Neuron, Developmental Cell, Genesis, andDevelopmental Dynamics.

Staff
Group Director

Junior Research Associate

Yoshiki SASAI

Hideya SAKAGUCHI

Research Specialist

Student Trainee

Keiko MUGURUMA

Taisuke KADOSHIMA
Chikafumi OZONEAtsushi SHIRAISHI
Kazuhiro TAKASHIMA

Senior Scientist

Hidehiko INOMATA
Research Scientist

Nozomu TAKATA
Visiting Scientist

Satoshi ANDO
Atsushi KUWAHARA
Makoto NASU
Morio UENO

Part-Time Staff

Masako SUZUKI
Assistant

Mako MIYAGI-INOMATA
Ayumi TANAKA-IKEYA
Fumi WAGAI

Technical Staff

Masayo FUJIWARA
Tomoko HARAGUCHI
Masako KAWADA
Ayaka NISHIYAMA
Mika SOEN
Rieko YAKURANISHIZAWA

Recent Publications
Nakano T, et al. Self-Formation of Optic Cups and Storable Stratified Neural Retina from Human ESCs. Cell Stem
Cell 10.771-85 (2012)
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)

Self-organized formation of optic cup from human ES cells

Muguruma K, et al. Ontogeny-recapitulating generation andtissue integration of ES cell-derived Purkinje cells. Nat Neurosci 13. 1171-80 (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 the 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 theperiod from 1998 to 2000 in Claudio Stern'slaboratory in Columbia University, moving with
Dr. Stern to University 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

Technical Staff

Guojun SHENG

Hiroki NAGAI
Kanako OTA
Erike Widyasari
SUKOWATI
YuPing WU

Research Scientist

Cantas ALEV
Meng-Chi LIN
Yukiko NAKAYA
Wei WENG

Part-Time Staff

Maiko SEZAKI

Recent 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 the early chicken embryo (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 the transcriptomic 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 in groups, emerges from the coordinated interaction of numerous molecular and genetic factors, and the need for integrated, systems-based
approaches to the study 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 willseek to use concepts and methodology from mathematical sciences such as
physics in the study and elucidation of these emerging questions in biology.
One example of such a phenomenon is seen in cellular chemotaxis, in whichcells recognize concentration gradients of attractant molecules and respondby directional movements necessary for functions such as the exploration of the environment by single-celled organisms, and morphogenesis inmetazoa. Chemotactically responsive cells are able to detect differentials
in the concentration of an attractant molecule of only a few percent, which,given cell sizes of ranging in the tens of micrometers, translates to a real difference of just a few dozen molecules. Cells are capable of interpreting thisminute difference as a gradient that guides the direction of its movement,
raising the question of how cells are able to detect and follow such weakand noisy signals. We now know that 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.
A[PIP3][PIP3][PIP2]
2= VPTEN [PTEN]+VPI3K[PIP3]PIP 3 [PIP3]+ D
K PTEN +[PIP3]
K PI3K +[PIP2]t[PIP3][PIP2][PIP2]= VPTEN [PTEN]
VPI3K+ k PIP 2 [PIP2]+ D 2 [PIP2]K PTEN +[PIP3]
K PI3K +[PIP2]t[PTEN]=Vass [PTEN]totalt

1
L[PTEN]K PIP3[PIP2]K PIP3 +[PIP3] K PIP2 +[PIP2]PTEN[PTEN]+ DPTEN

20

30

2

0

10

20

20

30

30

10

20

F

Visiting Researcher

Tatsuo SHIBATA

Seirin LEE

Research Scientist

Technical Staff

Akinori BABA
Tetsuya HIRAIWA
Naotoshi NAKAMURA
Masatoshi NISHIKAWA
Katsuhiko SATO
Foreign Postdoctoral Researcher

Marcel HOERNING

Satoru IWATANI
Junior Research Associate

Toshinori NAMBA
Prabhat SHANKAR
Assistant

Satoko KINOSHITA[PTEN]

Angle θ (π)
00 1 2Time (min)10Angle θ (π)
00 1 2Time (min)

2

θ (π)
D 0AngleE
1 2Time (min)101Time (min)Time (min)00

Angle θ (π)
00 1 2

Staff
Unit Leader

Baba A, et al. Directional sensing of deformed cells underfaint gradients, Phys. Rev. E 86.060901 (R) (2012)

PtdIns(3,4,5)P3

B Angle θ (π) C

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.

Recent Publications

PTEN

θ

Tatsuo SHIBATA Ph.D.

10

20

30

G

Shibata T, et al. Modeling the self-organized phosphatidylinositol lipid signaling system in chemotactic cells using
quantitative image analysis. J Cell Sci 125. 5138-50 (2012)
Namba T, et al. The relation of signal transduction to thesensitivity and dynamic range of bacterial chemotaxis. Biophys J 103.1390-9 (2012)
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)

Time (AU)(A) Spontaneous asymmetry formation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns (3,4,5) P3,
green) and PTEN (red), and an explanatory theoretical model.(B, C) Spatiotemporal diagrams of the membrane distributions.(D-F) Stochastic numerical simulations of the theoretical model. (G) Temporal evolutionof the distributions on the cell surface calculated from the theoretical model. (Shibata, et al. 2012).

Nishikawa M. and Shibata T. Nonadaptive fluctuation in an
adaptive sensory system: bacterial chemoreceptor. PLoS
One 5.e11224 (2010)71

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.

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. fromthe same institution in 1992. After ser vingas assistant professor in the Department ofOphthalmology, 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 returned to Kyoto University Hospital in 1998, and since 2001 served
as an associate professor at the Translational
Research Center in the Kyoto University Hospital. She joined the CDB as a team leader ofthe retinal regeneration research team in 2006.
Her clinical specialty is retinal disease - macular diseases and retinal hereditary diseases
in particular. Her aim is to understand these
diseases at a fundamental level and develop
retinal regeneration therapies.

Staff
Project Leader

Technical Staff

Masayo TAKAHASHI

Momo FUJIITomoyo HASHIGUCHI
Kyoko ISEKI
Kanako KAWAI
Noriko SAKAI
Akihiro TACHIBANA
Chikako YAMADA

Deputy Project Leader

Michiko MANDAI
Sunao SUGITA
Research Scientist

Jun KANEKO
Chikako MORINAGA
Akishi ONISHI
Akiko SUGA
Research Associate

Chie ISHIGAMI
Yuko MIBU
Satoshi OKAMOTO
Visiting Scientist

Yasuhiko HIRAMI
Zi-Bing JIN
Visiting Technical Staff

Yumiko SHIBATA
Motoki TERADA

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,
and we 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 andsolid clinical evidence.

International Program
Associate

Juthaporn
ASSAWACHANANONT
Student Trainee

Shinichiro ITO
Sun JIANAN
Hiroyuki KAMAO
Hirofumi KINOSHITA
Part-Time Staff

Tomoko KATAYAMA
Yuumi TADA
Tomoko YOKOTA

Recent Publications

1
Human iPS cells

2
Retinal pigment epithelial cells differentiatedfrom iPS cells

Jin ZB., et al. Modeling retinal degeneration using patient-specific 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)
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)

72

3
Purified retinal pigment epithelial cells

4
Photoreceptor cells differentiated from iPS cells

Cell Adhesion and Tissue Patterning
Animal cells organize into tissues with complex architecture. Our lab is exploring
the molecular mechanisms by which individual cells assemble into a tissue-specific multicellular structure, such as epithelial sheets and neural networks. We
are also interested in the molecular basis of how tissue architecture is disrupted
during carcinogenesis, a process that is thought to accelerate the metastasis ofcancer cells. For these studies, we are focusing on the roles played by cell-celladhesion and recognition molecules, the cadherin family of adhesion moleculesin particular, as these are known to be indispensable for tissue construction.
Our current studies are divided into three categories:
1) Cell-cell adhesion is a dynamic process, and this nature of cell-cell adhesion
is implicated in various cell behaviors, such as contact-dependent regulation ofcell movement and cancer metastasis. A growing body of evidence suggeststhat cadherins cooperate with cytoskeletal and/or motility machineries, suchas actin regulators, non-muscle myosins, and Rho GTPases, in modulating cell
assembly. We are therefore studying the molecular mechanisms underlying thecrosstalk between cadherins and such cytoskeletal systems.
2) A second area of interest to our lab is to gain a better understanding of how
the cell-cell adhesion machinery contributes to animal morphogenesis. Usingmouse and chicken embryos, we are analyzing the roles of cadherins and associated proteins in various morphogenetic processes, including neural tube closure and neural crest migration. We are also investigating the roles of membersof the cadherin superfamily known as protocadherins, deficiencies of whichhave been implicated in brain disorders. Through these studies, we expect togain deeper mechanistic insights into the ways by which cells build the elaborate structures of the animal body.
3) In addition, we have recently begun analyzing the functions of microtubule
minus end-associated proteins, Nezha/CAMSAPs. These proteins regulate microtubule assembly patterns, centrosomal function, and organelle positioning.
We are exploring molecular mechanisms underlying such regulatory activity,
as well as the roles of these molecules in cellular morphogenesis, such as polarized epithelial formation and axon growth, with the aim of uncovering novel
functions of non-centrosomal microtubules.

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

Collaborative Scientist

Masatoshi TAKEICHI

Wenxiang MENG
Shoko NAKAMURA
Tamako NISHIMURA
Katsutoshi TAGUCHI

Research Scientist

Shuichi HAYASHI
Takashi ISHIUCHI
Shoko ITO
Kyoko KUBOTA
Anna PLATEKMika TOYA
Vassil VASSILEV
Research Associate

Shigenori NAGAE
Nobutoshi TANAKA

Technical Staff

Sylvain HIVER
Yoko INOUE
Saeko KOBAYASHI
Miwako NOMURA
Hiroko SAITOAssistantMutsukoAISO-WATANABE

Recent Publications
Tanaka N, et al. Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specific organization of non-centrosomal
microtubules. Proc. Natl. Acad. Sci. USA 109.20029-34(2012)
Nishimura T, et al. Planar cell polarity links axes of spatial
dynamics in neural-tube closure. Cell 149.1084-97 (2012)
Ishiuchi T. and Takeichi M. Willin and Par3 cooperatively
regulate epithelial apical constriction through aPKC-mediated ROCK 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)
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)

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 theunique distributions shown here.

Meng W, et al. Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell
135. 948-59 (2008)73

Systems Biology

Hiroki R. UEDA M.D., Ph.D.
http://www.cdb.riken.jp/en/ueda
Hiroki R. Ueda received his M.D. from theFaculty of Medicine of the University of Tokyoin 2000, and received his Ph.D. from the University of Tokyo Graduate School of Medicine
in March 2004. While an undergraduate, heworked as a research assistant on a biologicalsimulation 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
and mouse. He was appointed team leader ofLaboratory for Systems Biology in 2003, andLeader 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 theLaboratory for System Biology under CDB’s
Center Director’s Strategic Program.

Staff
Project Leader

Hiroki R. UEDA
Research Scientist

Yohei M. KOYAMA
Masato NAKAJIMA
Yasutaka NIWA
Hideki UKAI
Rikuhiro G. YAMADASpecial Postdoctoral Researcher

Hiroki DANNO
Etsuo A. SUSAKI
Foreign Postdoctoral Researcher

Craig C. JOLLEY
Dimitri PERRIN

Naoshi KOIDE
Marie MURAMATSU
Yuta SHINOHARA
Kaori TSUJINO
Maki UKAI-TADENUMA
Junko YOSHIDA
Research Fellow

Genshiro SUNAGAWA

Over the next several years, we intend to develop a efficient experimental platform to identify, monitor, and perturb cellular networks within an organism. To
this aim, we will attempt to invent and combine several key technologies ranging from (i) rapid engineering of the genome of ES cells, (ii) generation of “100%chimera” animals for F0 phenotyping, and (iii) phenotype analysis of a small
number of the generated animals (ideally with a single animal). Full utilizationof these technologies will formulate cellular-to-organism-level systems biology,
which will provide new strategies and concepts for the diagnosis, treatment,
and prevention of biological-time-related disorders, including rhythm disorder,
seasonal affective disorder, and sleep disorder.

Hiroyuki TAMIYA
Junior Research Associate

Genki KANDA
Visiting Scientist

Tetsuya KOBAYASHI
Kohei MASUMOTO
Atsushi WADA

Technical Staff

Agency Staff

Hiroshi FUJISHIMA
Junko HARA
Shino HIRAHARAOver 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 challenging research end­eavors. Based on the achievements over the past eight years, we strongly feelthat it is now the time for us to take the next step forward toward experimental
systems biology at the cellular-to-organism level.

Student Trainee

Arthur MILLIUS

Visiting Researcher

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
of individual system components and their networked interactions, and leading
to the ability to drive existing systems toward a desired state and design newones based on an understanding of structure and underlying principles.

Chikako IMAI
Assistant

Maki NOMOTO

Recent Publications
Jolley C. C, et al. A design principle for a posttranslational
biochemical oscillator. Cell Rep 2.938-50 (2012)
Ukai-Tadenuma M, et al. Delay in feedback repression by
cryptochrome 1 is required for circadian clock function. Cell
144.268-81 (2011)
Masumoto K. H, et al. Acute induction of Eya3 by late-night
light stimulation triggers TSHbeta expression in photoperiodism. Curr Biol 20.2199-206 (2010)
Isojima Y, et al. CKIepsilon/delta-dependent phosphorylation is a temperature-insensitive, period-determining process in the 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)

74

Post-translational processes such as protein phosphorylation are vital for circadian rhythms in manyorganisms. In cyanobacteria, circadian proteins can be incubated with ATP to form an in vitro post-translational oscillator (PTO) that operates in the absence of transcription and translation. It is still unknown
whether components of the mammalian clock may also be able to function as a PTO. In a recent paper,
Jolley, Ode, and Ueda developed a mathematical model to examine the possibility of oscillations in a simple system with only three components. They found that two essential design motifs are necessary forsustained post-translational oscillation: a preferred ordering of phosphorylation states ("single-molecule
oscillators") and synchronization of these autonomous oscillators by enzyme sequestration.

Genomic Reprogramming
A theoretically limitless number of clones of an animal can be generated fromits somatic cells. Only a few years ago, such a statement would have belonged
to the realm of 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 thatdo lead to live births, the cloned animals consistently exhibit a range of severe
developmental abnormalities and pathologies.
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 fertilized egg). 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, potentially 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 efficiency ofthe cloning procedure, gaining a better understanding of reprogramming mechanisms and analyzing the properties of ES cells derived via somatic nuclear
transfer. We also use nuclear transfer technology to develop methods for preserving embryonic lethal and infertile strains of laboratory mice, and continue toexplore the development of new and better techniques for sperm and oocytepreservation and other reproductive biological research technologies.

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
fellowship from the Japanese Society for the
Promotion of Science in 1996 and spent thenext two years at the Yanagimachi lab in the
University of Hawaii Medical School, where hesucceeded in creating the world's first cloned
mouse. He was appointed to an assistantprofessorship at the same institution in 1998,
and moved to the Rockefeller University as a
research assistant professor in 1999. He spenta year as a researcher at Advanced Cell Technology before returning to Japan to take hiscurrent position at the RIKEN CDB.

Staff
Team Leader

Student Trainee

Teruhiko WAKAYAMA

Hirohisa KYOGOKU
Yukari TERASHITA

Research Scientist

Chong LI
Sayaka WAKAYAMA
Visiting Scientist

Fumiaki ITOI
Satoshi KISHIGAMI
Koji KOJIMAHaruko OBOKATA
Mikiko TOKORO
Kazuo YAMAGATA

Technical Staff

Yuko SAKAIDE
Kaori YAMANAKA
Assistant

Sakura HIRAUCHI

Recent Publications
Itoi F, et al. Offspring from mouse embryos developed using
a simple incubator-free culture system with a deoxidizing
agent. PLoS One 7(10).e47512 (2012)
Terashita Y, et al. Latrunculin A can improve the birth rateof cloned mice and simplify the nuclear transfer protocol by
gently inhibiting actin polymerization. Biol Reprod 86.180
1-6 (2012)
Yamagata K, et al. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy. PLoS
One 7.e31638 (2012)
Mizutani E, et al. Abnormal chromosome segregation at
early cleavage is a major cause of the full-term developmental failure of mouse clones. Dev Biol 364.56-65 (2012)
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)

Transfer of a somatic nucleus into an enucleated egg

Ono T, et al. Offspring from intracytoplasmic sperm injectionof aged mouse oocytes treated with caffeine or MG132.
Genesis 49.460-71 (2011)75

N-cadherin keeps cornea pumping

The cornea of the eye serves a dual role, protecting thesensitive interior tissue from the environment and supporting the lens by focusing incoming light. With a thickness of only 0.5 mm in humans, the cornea nonethelesshas a complex stratified structure, including epithelial,
stromal, and endothelial layers. The innermost of these,
the endothelium, separates the corneal stroma from theaqueous humor of the eye’s interior, acts as a point ofentry for nutrients into the non-vascular overlying tissue,
and as a pump that siphons away excess fluids fromthe corneal interior. Defects in endothelial function canthus lead to accumulations of fluid that cloud the otherwise transparent cornea, leading to blurring of vision.
Although it is known that this selectively permeable barrier is created by junctions between endothelial cells, themolecular mechanisms behind it have remained unclear.
A new study by Vassil S. Vassilev in the Laboratory for
Cell Adhesion and Tissue Patterning (Masatoshi Takeichi,
Group Director) and others in CDB now show that Ncadherin plays an important role in maintaining corneal
integrity. In an article published in Investigative Ophthalmology and Visual Science, the group shows that loss of
N-cadherin in mouse leads to defects in endothelial celljunctions, leading to stromal edema and a range of otherdefects in the cornea.
Corneal endothelial cells are bound to their neighbors inmonolayers by two forms of cell-cell adhesion: tight junctions and adherens junctions, which together are known
as the apical junction complex. Tight junctions are established by multiple types of adhesion molecule and form aphysical barrier between cells, while adherens junctions
are formed by classic cadherin adhesion molecules,
which work to stabilize the links between adjacent cells
and to regulate cell morphology through interactions withthe cytoskeleton. In the cornea, the N-cadherin gene ishighly expressed in the fetal through adult stages, which
led the group to investigate the exact role of this molecule in corneal function.

Vassil S. VASSILEV

76

Vassilev began by generating a conditional knockout
mouse in which the N-cadherin gene was deleted specifically in neural crest cells, the main embryonic sourceof the corneal endothelium. After confirming that N-cadherin expression was lost in mutant corneal endothelial
junctions, the group next examined the tissue structureand found it to be much more opaque than wildtype cornea. Interestingly, although N-cadherin was selectively
ablated from the endothelium, the effects of its loss wereseen in the other corneal layers. In the stromal layer, col-©2012 Association for Research in Vision and Ophthalmology
Vassilev V. S, et al. Loss of N-cadherin from the endothelium causes stromal edema andepithelial dysgenesis in the mouse cornea. Invest Ophthalmol Vis Sci 53.7183-93 (2012)

lagen fibrils became unevenly spaced and fluid accumulated, resulting in edema, while the epithelium grew thinner and exhibited delamination and abnormal apoptosis.
But how does loss of an endothelial cadherin perturb the tissue architecture of other corneal layers?
Suspecting the answer might lie in (fine) structural changes of the endothelial layer, Vassilev et al.
compared the cytoskeleton of mutant and unmodified cells by staining for F-actin (which connectswith cadherins via catenin linker molecules). In wildtype endothelium, actin molecules ring the cells’
circumference, maintaining the structure of cell-cell junctions. In the conditional N-cadherin knockouts, however, the actin cytoskeleton was malformed, leading to defects in nuclear morphology andcellular adhesion. Additionally, the group discovered defects in endothelial tight junctions, findingthat expression of the tight junction marker ZO-1 was significantly downregulated and the junctions
disorganized. Tests of the ion pumping function of mutant endothelial cells further revealed that theirability to transport sodium and water across endothelium layer was disturbed. This suggested lossof the endothelial barrier function leading to abnormal distribution of electrolytes and the excessive
accumulation of fluids in the corneal tissue.

Loss of N-cadherin leads toturbidit y of corneal tissue(bottom left; wildtype shownto p l e f t). S tro m a l l aye r i s
edematous and epitheliumis thinner in mutant cornea(bottom right, compared towildtype (top right)“In vitro studies had suggested that cadherin-based adherens junctions can be important for the
maintenance of tight junctions, but here we were able to show that this is the case in vivo as well,”says Takeichi. “In this study, we showed how the adherens junction plays a critical role in the exquisite architecture that gives the cornea its transparency. We next hope to use this conditional knockout system to investigate the function of N-cadherin in other neural crest-derived tissues.”

77

New role for Pax2 in placodemaintenanceThickenings in ectodermal tissue in the early embryo,known as placodes, serve as the developmental starting
points for a great many neural and sensory tissues. The
region that gives rise to the inner ear, known as the otic
placode, and the epibranchial placode, which generates
craniofacial neurons, are induced by the FGF signaling
pathway acting on a shared domain called the posterior
placodal area (PPA). Studies in a variety of model organisms have suggested that the Pax family of genes may
also function in the formation of these placodes, or the
PPA as a whole, but the evidence has remained equivocal.
Now, Sabine Freter and colleagues in the Laboratory
for Sensory Development (Raj Ladher, Team Leader) incollaboration with the Genome Resource and Analysis
Unit have shown that Pax2 functions not as an inducer,
but as a regulator of cell proliferation, in the otic and epibranchial placodes. Published in Developmental Dynamics, the report adds a new twist to our understanding of
Pax family genes in tissue maintenance.
Early work in mouse, zebrafish, and frog indicated thatthe gene Pax8 is expressed earlier than Pax2 in thenascent inner ear, and it was thought that either orboth might play an inductive role in otic placode or PPA
formation. The understanding of the function of these
Pax genes was muddled somewhat when subsequentstudies revealed that Pax8 mutants showed no inner
ear phenotype, and to add to the confusion, analyses ofchick development revealed that loss of Pax2 function
had no effect on induction of the PPA. Seeking to get tothe bottom of the question, Freter sought to study therole of Pax2/8 in chicken otic development. Interestingly,
after attempts to clone the Pax8 gene and to identify it
through searches of the chick genome, the team was
unable to identify the orthologous sequence in chick,or in the genomes of other related species, suggesting
it had been evolutionarily lost. Pax2, however, is highly
conserved, indicating that it is likely the sole Pax genefunctioning in PPA development in birds.

Sabine FRETER

78

Freter next used RNAi to interfere with Pax2 function, but
found that this had no effect on PPA formation. Similarly,
overexpression of the gene also left the PPA apparently
unaffected. Suspecting that the true role of Pax2 might
be in inner ear differentiation rather than induction, theteam looked at a later stage in development, when inner
ear-specific genes normally begin to be expressed. Pax2
knockdown resulted in the complete loss of expressionof Soho1, an early marker of inner ear, but did not appre-©2012 John Wiley & Sons, Inc.
Freter S, et al. Pax2 modulates proliferation during specificationof the otic and epibranchial placodes. Dev Dyn 241.1716-28 (2012)ciably affect the patterning of the otocyst, although it did cause a reduction in size. Its overexpression, in contrast, inhibited inner ear differentiation.
The epibranchial placode, which contributes to various cranial ganglia, also derives from the PPA, so
Freter investigated whether Pax2 inhibition would affect its formation or differentiation as well. She
found that, as in the otic placode, repression of Pax2 function had no effect on the expression ofgenes associated with the induction of this placode, but dramatically reduced expression of genetic
markers of committed neuronal progenitors. And, as in the inner ear, overexpression of Pax2 interfered with epibranchial differentiation as well.

Pa x2 electroporation (bluecells) reduces uptake of thethymidine homologs, BrdU
(green) and EdU (red) in the
inner ear precursor.

Seeking to better understand the mechanism by which Pax2 affects the otocyst and epibranchial
tissue, the team next investigated the possibilities that its effects could be due to altered cell deathor differentiation. The absence of caspase upregulation following Pax2 knockdown suggested thatan effect on programmed cell death could be ruled out. The cell cycle, however, was slowed significantly by Pax2 inhibition, causing an overall reduction of mitotic activity in affected cells. Interestingly, overexpression of Pax2 had no appreciable effect on cell cycle.
“Given that Pax is known to work in the maintenance of precursor cells in other tissues, such as
skeletal muscle, these latest results point to the possibility that Pax plays a general role in cell division and the maintenance of an undifferentiated state,” says Ladher.

79

CAMSAPs regulate microtubule
dynamics in epithelial cells
Microtubules organize themselves differently in different
types of dividing cells. While they grow radially out fromthe centrosome during interphase in many cell types, thisis not the case in epithelial cells, in which their minusends are mainly anchored in the cytoplasm instead.
Despite its importance to mitotic dynamics, however, themeans by which the distribution of centrosomal and noncentrosomal microtubules is established and maintainedin different cells has a remained a mystery.
A study by Nobutoshi Tanaka and others in the Laboratory for Cell Adhesion and Tissue Patterning (Masatoshi
Takeichi, Group Director) has now shown that a pair ofmolecules, CAMSAP3 (also known as Nezha) and CAMSAP2, work together to regulate and maintain noncentrosomal microtubule organization in cultured human cells.
Published in the Proceedings of the National Academyof Sciences, this work reveals the mechanism by whichinterphase epithelial cells establish this distinct form ofcytoskeletal organization.
Microtubules are polarized molecular chains, in which
the terminal site of growth by polymerization is called theplus end; the minus end is found at the other pole. Minus
ends have been shown to anchor in the centrosome,from which microtubules extend radially, a process
mediated by numerous molecular factors. But some microtubules stabilize without tethering to the centrosome,by mechanisms that remain poorly understood. Seeking to gain more insight into this these noncentrosomal
microtubules, Tanaka focused on CAMSAP3, which theTakeichi group had previously shown to bind to microtubule minus ends, and a related molecule CAMSAP2.

Nobutoshi TANAKA, Wenxiang MENG80

The group first looked at the localization of the two molecules and found that both tended to clump together insmall clusters throughout the intracellular space. Whenthey simultaneously visualized the plus ends of microtubules by immunostaining, they noted that the CAMSAP
clusters tended to be located at the opposite poles, suggesting an affinity for minus-ends. Having established aphysical connection, Tanaka et al. next sought after thefunctional role. The plus-end protein, EB-1, which theyused in their immunostaining experiments, is known tobind to microtubule plus ends and contribute to theirpolymerization. Using specific siRNAs to knock down
the function of each of the CAMSAPs, the group foundthat loss of the function of either molecule resulted in a
reduction of EB1 radiating growth. This effect was amplified when both CAMSAPs were knocked down simultaneously, suggesting that these proteins promote stable
extension of microtubules.

©2012 National Academy of Sciences
Tanaka N, et al. Nezha/CAMSAP3 and CAMSAP2 cooperate in epithelial-specificorganization of noncentrosomal microtubules. Proc Natl Acad Sci U S A 49.20029-34 (2012)

In wildtype epithelial cells in low-density cultures, most microtubules are arrayed in a pattern surrounding the nucleus. The centrosomes locate at random sites, and only rarely nucleate radial microtubules. But the loss of function of either CAMSAP3 or -2 triggered a rearrangement in which themicrotubules densely covered the nucleus. Even more intriguingly, the double knockdown of both
CAMSAPs caused some epithelial cells to develop uncharacteristic centrosomal radiation of microtubules. Cells lacking either CAMSAP also showed post-translational changes in the tubulin protein,which may account for the altered microtubule dynamics.

In control cells cultured at
low density, microtubules are
distributed encircling the nucleus (left), and are not seenradiating from centrosomes(stained red, arrowheads).
Knockdown of either CAMSAP2 or CAMSAP3 (middle
panels) triggers accumulationof microtubules at centrosome sites, while simultaneous knockdown of both
induces the radial polymerization of microtubules from
centrosome sites.

The arrangement of microtubules growing from CAMSAP sites appears to have functional consequences as well. In CAMSAP-depleted cells, early endosomes accumulated around the centrosome,while in control cells these organelles are dispersed throughout the cytoplasm. The Golgi apparatus,
which is normally distributed in the perinuclear region, was more widely distributed in CAPSAMknockdown cells.
“While this series of experiments points to some very interesting possibilities, we will need to study
these phenomena more closely in epithelial tissue in vivo rather than low-density cell culture,” notes
Takeichi. “As epithelial cells show distinct microtubule orientation toward their apical surfaces, wemay be able to detect whether CAMSAPs play regulatory role in the establishment of this polarity,and how their depletion affects cell morphology and behavior in intact tissue.”81

Ruling the airways: Notch controls
bronchial cell fates and distributions

Mitsuru MORIMOTO, Junko SAKAI, Yuka NODA

Nestled deep within the body, the epithelial lining of the respiratory system is nonetheless seriously
exposed. Its direct contact with environmental air necessitates protective mechanisms that both seal
off the respiratory tract from other compartments of the body and neutralize microbial invaders. Thisis achieved by the coordinated action of the functionally specialized various cell types that make up
the lining of the airway. These respiratory cell populations include major ciliated cells, exocrine Claracells, and neuroendocrine (NE) cells, all of which are generated by a common epithelial progenitor
cell type during embryogenesis. One interesting feature of lung histology is that the relative distributions of these cell types varies along the proximal-distal axis, with ciliated cells capable of sweeping
away microscopic particles and organisms more common near the mouth, and secretory Clara cells
that help maintain humidity and bronchiole structure increasing in deeper regions of the lung. Neuroendocrine cells are less common than ciliated and Clara cells, and are found in clusters throughout the lung epithelium. Despite their importance, the mechanisms by which these spatial changes
in lung cell distribution are regulated remain largely a mystery.
Mitsuru Morimoto, Team Leader of the Laboratory for Lung Development, working with colleagues
at the National Institute of Genetics and Washington University of St. Louis, has now shown that
Notch signaling controls the differentiation and distribution of these three lung epithelial cell types
in mouse. Published in Development, this work sheds new light on how the diversity of lung cells isestablished and maintained.

82©2012 The Company of Biologists
Morimoto M, et al. Different assemblies of Notch receptors coordinate the distributionof the major bronchial Clara, ciliated and neuroendocrine cells. Development 139.4365-73 (2012)

The Notch signaling pathway is triggered by interactions between ligands and receptors bound within cell membranes, making it a much more local affair than signaling involving secreted moleculesthat diffuse away from cells. Morimoto had previously noted that when Notch signaling is reduced
during development, the number of Clara cells in the lung epithelium decreases, while ciliated cells
expand in number. Other work had also shown that the Notch pathway ligand Jag1 is expressed inciliated cells, which it was suggested might induce Clara cell differentiation in neighboring cells. To
work out the details of how Notch works in lung cell fate determination, Morimoto in this most recent
study tested how ablating the Notch receptors Notch1-3 affected the relative distributions of epithelial cell types in the airway. His first finding came as something of a surprise – Clara cell differentiation
relies on Notch2, not Notch1 as previously believed.

Clara cells (green), neuroepithelial cells (red) and SPNC
cells (blue) in fetal mouseairway epithelium. Clara cells
are distributed throughout
the airway, while NE cells areobserved in clusters. SPNC
cells are induced in a Notchd e p e n d e n t m a n n e r, a n d
appear to regulate NE cellcluster size.

Morimoto further observed that simultaneous deletion of all three receptors caused the size of neuroendocrine cell clusters to increase, an effect not seen when these factors were knocked out individually or in other combinations, suggesting that Notch1-3 have additive functions in the regulationof NE cell differentiation. Even more intriguingly, Notch activity was not observed in the neuroendocrine cells themselves, but rather in a distinct population of their neighbors, apparently representing a hitherto unidentified cell type, which the team dubbed SPNC cells, after a characteristic gene
expression profile (SSEA-1+ peri-pNEB N1ICD+ CC10−). Morimoto found that these SPNC cells aremaintained by juxtacrine Notch signaling from adjacent NE cells, suggesting that once neuroendocrine cells have been induced, they function to maintain local SPNC cells, which in turn provide signals that regulate the size of NE cell clusters.
“The combinatorial effects of Notch factors in the airway epithelium may be the mechanism underlying the distribution of ciliated, Clara and neuroepithelial cells. While the ciliated/Clara selection issensitive to dosage of Notch2, NE cells are regulated synergistically by the combination of Notch1-3,
which may account for various pattern of ciliated/Clara cells and the uniform distribution of NE cellcluster throughout the epithlieum,” says Morimoto. “In future studies, we will be interested in looking
more deeply into how the Notch signaling works in Clara cell differentiation, and inducers of NE cells
as well.”83

Craniofacial development holds key tohagfish phylogeny
The position occupied by cyclostomes, such as lampreys and hagfish, within the phylogenetic tree has
been a controversial question for decades. Distinct
from gnathostomes (vertebrates possessing a jaw), thecyclostomes are jawless, and have only a single nostrillike facial structure, as opposed to the gnathostome’s
two. This solitary nostril (an olfactory organ known as thenasohypophyseal duct) is itself the focus of a debate overthe evolutionary relationship between hagfish and lampreys; while the duct dead-ends in the lamprey, it opens
into the pharynx in hagfish. There are also disagreements
within the research community as to the developmental
source of the adenophophysis, which opens into thenasohypophyseal duct in hagfish - specifically whether it
is endodermal in origin, as suggested by some previous
studies, or ectodermal, as is the case for both lampreys
and the gnathostomes. Finding answers to these questions has long been frustrated by the difficulty in obtaining hagfish embryos for detailed examination.
New work by Yasuhiro Oisi and others in the Laboratory
for Evolutionary Morphology (Shigeru Kuratani, Group Director) in collaboration with colleagues in CDB and Academia Sinica (Taiwan), provides morphological evidence
that reveals that development of the olfactory organs
and adenohypophysis is roughly homologous in hagfish
and lampreys. Published in Nature, this study solidifies
our understanding of hagfish as close relatives of their
cyclostome kin, and sheds new light on the evolution ofvertebrate craniofacial structure.
Several years ago, the Kuratani lab develop a systemfor breeding the hagfish Eptatretus burgeri in captivity, abreakthrough that provided the lab with a much-needed
supply of embryos after a nearly century-long droughtthat forced hagfish biologists to rely on studies publishedin the 19th century. Oisi used this resource to conduct
a staged series examination of embryos to examine thedevelopment of the NHP and adenohypophysis, and toresolve their germ layer origin.

Yasuhiro OISHI, Shigeru KURATANI

84

Observation of embryos at the mid-neurula stage suggested that the adenohypophysis arise from a transient
ectodermal tissue, similar to other vertebrates including
lamprey, and not from the endoderm as had previously
been reported. The ectodermal origin of the adenohypophysis was further supported by gene expression
profiles consistent with an ectodermal source, and byfollowing changes in gene expression and morphology
across the relevant development stages, the group was
further able to exclude the possibility that the hagfish ad-©2012 Nature Publishing Group
Oisi Y, et al. Craniofacial development of hagfishes and the evolution of vertebrates.
Nature 493.175-80 (2013)enohypophysis differentiates from the prechordal plate (endodermal thickening). This suggests thatthe developmental differences between hagfish and lampreys are smaller than had been suspected,
as well as a commonality in important aspects of craniofacial development between both extant vertebrate groups: cyclostomes and gnathostomes.

Sagittal sections of hagfish
and lamprey heads. NHP is ofectodermal origin in both animals, and located anterior tothe oropharyngeal membrane(opm), which defines ecto/
endodermal boundary.

In lampreys, the nasohypophyseal duct forms from a median complex of placodes known as thenasohypophyseal plate (NHP), which corresponds to a pair of discrete placodes - hypophyseal andnasal – found in gnathostomes. The differences in the gross morphology of the adult hagfish had led
some to suspect that its development might differ significantly from that in lamprey. Oisi set out totest this hypothesis by studying changes in genetic markers of NHP and the gnathostome placodes.
He found that, as in lamprey, the hagfish NHP takes the form of a single median placode, suggesting
the structures are conserved across the cyclostomes. The distinct morphological features observedin the craniofacial structures in adults of the two taxa thus appear to be attributable not to differences in their developmental origins, but to hagfish-specific modifications at subsequent embryonicstages.

Comparison of ve r te brateheads. Hagfish and lamprey
share embryonic craniofacial
features. This pattern does
not appear during any developmental stage in other living
gnathostomes. ah, adenohypophysis; nhd, nasohypophyseal duct.

85

Animal Resources and Genetic Engineering
Genetically engineered mice are an important resource used in biomedical research. The progress and quality of research is greatly
dependent on how efficiently mutant mice can be generated, propagated, and housed. However, researchers today tend not to engage in routine generation of mutant mice. The major function of our laboratory is to develop mutant mice for research in the fields ofdevelopmental biology and regenerative science, and to maintain and enhance the experimental rodent resources in the CDB.

Yasuhide FURUTA 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
YoshikoMUKUMOTO
Megumi WATASE

Research Specialist

Hiroshi KIYONARI
Go SHIOI
Visiting Scientist

Toshihiko
FUJIMORI

Student Trainee

Behnoush
KHALEDIAN
Prem Swaroop
YADAVThe 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 for the
study 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 fromconstruction of the targeting vector to generation of chimeras, making about 100 new
knockout mutants every year. In addition, wedevelop new bioimaging technologies to aid
in the visualization of mutant mouse embryos
at the tissue, cell and organelle levels.

An E9.5 embryo expressing the R26p-Fucci2
cell cycle probes

Animal Resource Unit
Kazuki NAKAO Ph.D.

Staff
Unit Leader

Technical Staff

Kazuki NAKAO

Kenichi INOUE
Yuki KANEKO
Takuya KAWADA
Masako NODA
Miho SATO
Mayo SHIGETA
Aki SHIRAISHI
Norie TANAKA
Tomoko TOKUNAGA
Sachi YAKAWA

Attending Veterinarian

Naoko KAGIYAMA
Research Specialist

Hiroshi KIYONARIAssistant

The Animal Resource Unit maintains andcares for CDB’s laboratory mouse and rat
resources in a Specific Pathogen Free (SPF)
environment. We also handle 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
for colony expansion and strain cryopreservation. We also develop technologies for the
study of reproductive biology.

Injection of C57BL/6 ES(HK3i) cells into 8 cellstage embryo for chimeric mouse production

Yuki TSUJI

Recent Publications
Abe T, et al. Visualization of cell cycle in mouse embryoswith Fucci2 reporter directed by Rosa26 promoter. Development 140.237-46 (2013)
Shinohara R, et al. A role for mDia, a Rho-regulated actin
nucleator, in tangential migration of interneuron precursors.
Nat Neurosci 15.373-80, S1-2 (2012)
Niisato E, et al. CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus.
Dev Neurobiol 72.1447-57 (2012)
Tao H, et al. Nuclear localization of Prickle2 is required toestablish cell polarity during early mouse embryogenesis.
Dev Biol 364.138-48 (2012)86

Nakae J, et al. Novel repressor regulates insulin sensitivity
through interaction with Foxo1. EMBO J 31.2275-95 (2012)
Kanemaru K, et al. Epidermal phospholipase Cdelta1 regulates granulocyte counts and systemic interleukin-17 levels
in mice. Nat Commun 3.963 (2012)
Nishimura R, et al. Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association withtranscription factor Runx2 during endochondral ossification.
J Biol Chem 287.33179-90 (2012)
Iwano T, et al. Prox1 postmitotically defines dentate gyrus
cells by specifying granule cell identity over CA3 pyramidal
cell fate in the hippocampus. Development 139.3051-62(2012)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 play fundamental roles.
The Electron Microscope Laboratory provides technical assistance and expert guidance in the use of transmission and scanningelectron 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 toconventional transmission and scanning electron microscopy, including the preparation of specimens of any type, assistance in 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, and the biophysicalaspects of morphological rearrangements in epithelial cells.

Staff
Laboratory Head

Research Fellow

Shigenobu
YONEMURA

Akira ONODERA
Ayuko SAKANE

Research Scientist

Student Trainee

Hanako HAYASHIYu AMANO

Technical Staff

Assistant

Kisa KAKIGUCHI
Kazuyo MISAKIMai SHIBATA

Visiting Scientist

Yasuhiro INOUE
Masatsune
TSUJIOKA

Recent Publications
Tsujioka M, et al. Talin couples the actomyosin
cortex to the plasma membrane during rear retraction and cytokinesis. Proc Natl Acad Sci U S
A 109.12992-7 (2012)
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)

Cells organizing the notochord of zebrafish embryo

Yonemura S, et al. alpha-Catenin as a tension
transducer that induces adherens junction development. Nat Cell Biol 12.533-42 (2010)

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

87

Bio-imaging
The role of the Bioimaging Laboratory is to assist in the operation and maintenance of the Center’s core imaging facility and supportscientists 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

Emiko MAEKAWA

Research Scientist

Agency Staff

Togo SHIMOZAWA

Taisaku NOGITechnical Staff

Tomoko HAMAJI

Bio-imaging is an interdisciplinary process that integrates molecular cell biology and biochemistr y, as well as technology from optics, engineering, andcomputer sciences. It has enabled scientists to visualize biological processes
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 theprogress of biology. We hope to accelerate biological research by matching research concepts with the latest technologies from the different specialized fields.

Recent Publications
Shimozawa T, et al. Improving spinning disc confocal microscopy by preventing pinhole crosstalk for intravital imaging. Proc Natl Acad Sci U S
A in press (2013)
Nakamura S, et al. Dissecting the Nanoscale
Distributions and Functions of Microtubule-EndBinding Proteins EB1 and ch-TOG in Interphase
HeLa Cells. PLoS One 7.e51442 (2012)

In addition, we aim to shed light on the molecular mechanisms controlling the microtubule cytoskeleton in the cell/tissue morphogenesis by making full use of the available
technology.

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)

Inverted microscope maintained by the Optical Image Analysis Unit

88

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 reporting of results.

Fumio MATSUZAKI Ph.D.

Genome Resource and Analysis Unit
Shigehiro KURAKU Ph.D.
The Genome Resource and Analysis Unit (GRAS) provides a broad range of biologistoriented support for Sanger and massively parallel deep sequencing, sequence informatics and gene expression profiling. In parallel, we conduct our original research
projects on vertebrate comparative genomics, focusing on evolution of developmental
programs. Above all, GRAS aims to create an integrative research support station withactive communication between laboratory staff and bioinformaticians, and take fulladvantage of evolving massively parallel sequencing technologies to apply them totranscriptomic, epigenetic and genomic projects in developmental biology arising in
the whole institute.

Staff
Unit Leader

InternShigehiro KURAKU

Nathalie FEINER

Research Scientist

Part-Time Staff

Mitsutaka KADOTA

Naomi KAWASHIMA
Ayako TANIGUCHITechnical Staff

Tetsutaro HAYASHI
Kazu ITOMI
Osamu NISHIMURA
Chiharu
TANEGASHIMA
Kaori TATSUMI

Recent Publications
Kuraku S. Impact of asymmetric gene repertoire
between cyclostomes and gnathostomes. Semin
Cell Dev Biol in press (2013)
Oisi Y, et al. Craniofacial development of hagfishes and the evolution of vertebrates. Nature
493.175-80 (2013)
Matsubara K, et al. Intra-genomic GC heterogeneity in sauropsids: evolutionary insights from
cDNA mapping and GC3 profiling in snake. BMC
Genomics 13.604 (2012)

Our main sequencing platform, illumina HiSeq 1000, in operation since spring 2012

Our long-standing solution, Roche 454 FLX+,in operation since 2007

Freter S, et al. Pax2 modulates proliferation
during specification of the otic and epibranchialplacodes. Dev Dyn 241.1716-28 (2012)

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 cuttingedge 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
three-dimensional 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

Recent Publications
Kasukawa T, et al. Human blood metabolite
timetable indicates internal body time. Proc Natl
Acad Sci U S A 109. 15036-41 (2012)
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 TSHßexpression in photoperiodism. Curr Biol 20. 2199-206(2010)
Isojima Y, et al. CKIε/δ-dependent phosphorylation is a temperature-insensitive, perioddetermining process in the mammalian circadianclock. Proc Natl Acad Sci U S A 106. 15744-9(2009)

89

Proteomics
The identification of proteins from trace amounts of biologically important protein complexes is a powerful technique and has become an indispensable approach to the study of development and regeneration. A better understanding of the protein componentsof 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.

Reiko NAKAGAWA

The Mass Spectrometry Analysis Unit uses LC-MASS spectrometry, a technologythat enables the high-speed analysis of protein components from even minute biological samples. Its support activity takes the forms of protein identification services andanalysis 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 from researchersand conducts all subsequent steps, including de-staining of gels, in-gel digestion ofproteins, peptide extraction, and operating the LC-MS/MS.

Recent Publications

In the most recent fiscal year, the unit received more than 30 spectrometry requests
and nearly 900 samples for use in identification.

Staff
Lab Head

Shigeo HAYASHI
Unit Leader

Akira NAKAMURA
Research Specialist

Terabayashi T, et al. Phosphorylation of Kif26b
promotes its polyubiquitination and subsequent
proteasomal degradation during kidney development. PLoS One 7.e39714 (2012)
Shiomi Y, et al. Two different replication factor
C proteins, Ctf18 and RFC1, separately control
PCNA-CRL4Cdt2-mediated Cdt1 proteolysis
during S phase and following UV irradiation. Mol
Cell Biol 32.2279-88 (2012)
McCloskey A, et al. hnRNP C tetramer measures
RNA length to classify RNA polymerase II transcripts for export. Science 335.1643-6 (2012)
Satoh R, et al. Role of the RNA-binding protein
Nrd1 in stress granule formation and its implication in the stress response in fission yeast. PLoS
One 7.e29683 (2012)
Hatanaka A, et al. Fub1p, a novel protein isolated
by boundary screening, binds the proteasome
complex. Genes Genet Syst. 86.305-14 (2011)
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)

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

90

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

Michiru MATSUMURAIMOTO
Maki MINAGUCHI

Research Specialist

Part-Time Staff

Deputy Unit Leader

Hiroyuki KITAJIMA

Yoshinori NAKAI

Visiting Scientist

Tokushige NAKANO

Recent 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 of various potentially medically useful cell types from ES and iPS cells, but the extent to which suchcells mimic their in vivo function when plated on culture dishes is limited. The Fourdimensional Tissue Analysis Unit seeks to develop new approaches to cell culture thatwill 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 cell-derived
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 ofthese technologies within and outside the CDB.

Staff
Unit Leader

Technical Staff

Yoshiki SASAI

Eriko SAKAKURA

Deputy Unit Leader

Junior Research
Associate

Mototsugu EIRAKU
Visiting Scientist

Yuiko HASEGAWA

Hidetaka SUGA

Recent Publications
Eiraku M, et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature
472. 51-56 (2011)
Eiraku M. and Sasai Y. Mouse embryonic stem
cell culture for generation of three-dimensional
retinal and cortical tissues. Nature Protocols 7.
69-79 (2011)

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

Staff
Unit Leader

Douglas SIPP

Recent Publications
Sipp D. and Turner L. U.S. Regulation of StemCells as Medical Products. Science 338.12961297 (2012)
Sipp D. Pay-to-participate funding schemes inhuman cell and tissue clinical studies. Regenerative Medicine 7.105-111 (2012)
Yuan W, et al. Stem cell science on the rise inchina. Cell Stem Cell 10.12-15 (2012)91

Kobe sits at the heart of the Kansai region of western Japan,
close to both the bright lights of Osaka and Kyoto’s tranquility.
The local climate is temperate, making it possible to enjoy a wide
range of seasonal activities, all within a short train ride or drivefrom the center of the city. Japan’s renowned public transportsystem allows rapid and convenient access to both local andnational destinations, and the many area airports, including theKobe Airport located less than 10 minutes from the CDB, and theKansai International Airport, provide immediate gateways to any
destination in the world. Costs of living are comparable to those inmany 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 thecity’s center, and its range of international restaurants and latenight bars promise a great evening out any night of the week. The
neighboring Motomachi district offers a mix of upscale department stores and funky shopping arcades stand in contrast to thecolorful Chinatown that’s right next door. A short walk north fromcity center lies the old foreign settlement of Kitano, whose clapboard houses and well-kept parks are a perfect retreat from the
dynamism downtown.

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

Kobe occupies a splendid location between mountains and seas,with easy access to many natural spots. A short drive or bus ridetakes you to the island of Awaji, with its beaches and first-rate
seafood, and hiking trails crisscross the forested mountains thatspan 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 bloom at the start of cherry blossom season, andits many rivers, beaches and scenic areas make it easy to takeenjoyable day trips, inside the city or out.

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

RIKEN Kobe Institute
The RIKEN 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, fromfundamental studies of development and stem cells, to cuttingedge 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 wasredesignated as the Center for Molecular Imaging Science (CMIS).
In April 2011, the Kobe Institute established its newest research
center, the Quantitative Biology Center, which will focus on systems, simulation, and computational approaches to life sciences
phenomena.

The RIKEN 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, to help
bridge basic science to novel applications in medical therapy anddrug discovery, thereby contributing to the health and welfare 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

94

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

Quantitative Biology Center
The RIKEN Quantitative Biology Center (QBiC) focuses on thecomplex spatiotemporal relationships between components ofbiological systems through innovative measurement, analysis,
and modeling technologies and techniques. These will be used
to predict, design, simulate, and manipulate cellular phenomena,
which may help to revolutionize research and applications in thelife sciences, including such fields as regenerative medicine anddiagnostics.

2012 CDB Budget
Overhead andadministration

Personnel

1,455

1,502

Total: 3,858(million yen)

Research expenses

901
In additional to the dedicated funds outlined above, individuallabs and investigators are encouraged to compete for external funding as well, from sources such as the Japan Society
for the Promotion of Science (JSPS), the Japan Science andTechnology Agency (JST ), and other governmental andprivate sources. These external funds represent a significant
component of the Center’s total funding every year.

RIKEN
internal grants

Private industry

87

45Foundations, etc.

1

Other
government
agencies

158

Total: 980

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

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

MEXT direct
funding689(million yen)2012 CDB Staff
Laboratory heads
Deputy leaders
Research scientists
Research associates
Technical staff
Assistants
Visiting scientists
Student trainees
Part-time staff
Other

31
4
94
8
91
20
112
32
46
30

Total

468

Research Promotion Division*

78*Administrative staff for Kobe Institute952012 CDB Symposium

Quantitative Developmental Biology
March 26-28, 2012
The RIKEN CDB held its tenth annual symposium, “Quantitative Developmental Biology” on March 26–28. Around 160 participants from
14 countries gathered to interact and review experimental, computational, and theoretical approaches to the study of principles of development. In addition to the 30 talks over the course of the three-day program, days 1 and 2 included poster sessions in which over 70
poster presenters discussed their work.
This year’s event was co-organized by the CDB’s Shigeo Hayashi and Tatsuo Shibata , as well as Suzanne Eaton of the Max Planck Institute of Molecular Cell Biology and Genetics (Germany), Shigeru Kondo of Osaka University, and Shuichi Onami of the RIKEN Quantitative
Biology Center. Additional support for the meeting was provided by 11 organizations.
The CDB symposium series, launched in 2003, was established as a forum for addressing diverse aspects of developmental and regenerative biology, and aims to promote the free, timely and borderless exchange of research achievements.

Session 1

Session 5

Suzanne Eaton (Max Planck Institute of Molecular Cell Biology
Edwin W. Munro (The Univeristy of Chicago, USA)

Shigeru Kondo (Osaka University, Japan)
Cheng Ming Chuong (University of Southern California, USA)
Philip J. Murray (University of Oxford, UK)

Session 2

Session 6

Shigeo Hayashi (RIKEN CDB, Japan)
Kaoru Sugimura (Kyoto University, Japan)

Stuart A. Newman (New York Medical College, USA)
Przemyslaw Prusinkiewicz (The University of Calgary, Canada)
Dina A. Faddah (MIT/Whitehead Institute for Biomedical Research, USA)and Genetics, Germany)

Session 3

Cornelis J. Weijer (University of Dundee, UK)
Darren Gilmour(European Molecular Biology Laboratory, Germany)

Martin Behrndt(Institute of Science and Technology Austria, Austria)
Session 4

Tadashi Uemura (Kyoto University, Japan)
Philipp Khuc Trong (University of Cambridge, UK)96

Session 7

Frank Jülicher (Max Planck Institute for Physics of Complex
Systems, Germany)

Hidehiko Inomata (RIKEN QBiC, Japan)
Eugene Myers (HHMI, Janelia Farm Research Campus, USA)
Session 8

Shuichi Onami (RIKEN QBiC, Japan)
Ralf Schnabel (TU Braunschweig, Germany)
James Sharpe (Center for Genomic Regulation, Spain)2013 CDB Symposium

The Making of a
Vertebrate
March 4–6, 2013
The eleventh annual symposium “The Making of a
Vertebrate” will be held on March 4–6, 2013 in the
CDB Auditorium. This symposium will focus on thecurrent understanding of developmental mechanisms
in vertebrates at the cell and molecular levels, focusing
not only on what we have learned, but what remains
unknown and avenues to future discovery. Discussion
will center on the latest developments in such topics as
the fundamental structures of the vertebrate body andevolutionary mechanisms leading to the emergence ofdiversity.

Invited Speakers

Shinichi Aizawa(RIKEN CDB, Japan)

Detlev Arendt(EMBL, Germany)
Session 9

Edward M. De Robertis

Tatsuo Shibata (RIKEN CDB, Japan)

Denis Duboule

Orion D. Weiner (University of California, San Francisco, USA)
Tetsuya Nakamura (Osaka University, Japan)(Univ. of Geneva & EPFL, Switzerland)

Session 10

Edith Heard

Jérôme Solon (Center for Genomic Regulation, Spain)
Christopher S. Chen (University of Pennsylvania, USA)
Satoshi Sawai (The University of Tokyo, Japan)(CNRS, France)

Session 11(Nagoya Univ., Japan)

Masako Tamada (Sloan-Kettering Institute, USA)
Buzz Baum (MRC Laboratory for Molecular Cell Biology, UK)
Lance Davidson (University of Pittsburgh, USA)(UC San Diego, USA)

(HHMI/UCLA, USA)

Hiroshi Hamada(Osaka Univ., Japan)

Andreas Hejnol(Sars Intl Centre for Marine Molecular Biol, Norway)

Masahiko Hibi
Nicholas D. Holland
Hidehiko Inomata(RIKEN CDB, Japan)

Naoki Irie
(RIKEN CDB, Japan)

Fumitoshi Ishino(Tokyo Medical and Dental Univ., Japan)

David Kingsley(Stanford Univ., USA)

Shigeru Kuratani(RIKEN CDB, Japan)

Chris Lowe
(Stanford Univ., USA)

Miguel Manzanares(CNIC, Spain)

Hitoshi Niwa
(RIKEN CDB, Japan)

Luis Puelles(Univ. of Murcia, Spain)

Toshihiko Shiroishi(NIG, Japan)

Billie J. Swalla(Univ. of Washington, USA)

Hiroyuki Takeda(The Univ. of Tokyo, Japan)

97

CDB Seminars
The RIKEN CDB makes special efforts to provide a full and diverse series of invited seminars by scientists from around theworld. To date, the Center has hosted more than 650 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 2012.

98

Date

Title

Speaker

01-23

Two types of Notch signaling cooperate in epithelial patterning during lung organogenesis

Mitsuru MORIMOTO

02-06

A gene network regulating temporal changes in neural stem cell function

Jinsuke NISHINO

02-10

The stem cell niche is giving me goosebumps, and more

Hironobu FUJIWARA

02-23

The role of genetic programs and activity in the development of cortical interneuron
diversity

Gordon FISHELL

03-02

Mitotic microtubules in mammalian neurogenesis

Felipe MORA-BERMÚDEZ

03-07

Dissecting the role of morphogenesis in the origins of the first two cell lineages in themouse embryo

Robert Odell
STEPHENSON

03-08

Secreted semaphorins from degenerating larval ORN axons direct adult projection neuron
dendrite targeting

Lora B. SWEENEY

04-05

Spatial regulation of VEGF receptor endocytosis in angiogenesis

Masanori NAKAYAMA

04-10

What songbirds can tell us

Kentaro ABE

05-11

PDGF signaling pathways in development and homeostasis

Phil SORIANO

06-01

Microfabricated substrates as a tool to study cell mechanotransduction & collective cell
migrationBenoît LADOUX

06-18

Cell culture engineering toward large scale chondroprogenitor production from humanpluripotent stem cells

Naoki NAKAYAMA

06-18

Non-canonical Wnt signaling maintains hematopoietic stem cell through flamingo andFrizzled8 in the niche

Ryohichi SUGIMURA

06-18

The power of cell autonomous growth in the absence of ERK signalling is gained justbefore implantation

Thorsten BOROVIAK

06-19

BMP signaling and spinal cord development

Naihe JING

06-29

Conserved and diversified roles of eIF5-mimic proteins in eukaryotes

Katsura ASANO

07-03

Symmetrical divisions dedicated to generating distinct cone photoreceptor types

Sachihiro SUZUKI

07-05

Investigation of the physical and functional links between the APC/C and the centrosome
in Drosophila

Yuu KIMATA

07-13

Dissecting the route of human cellular reprogramming

Kazutoshi TAKAHASHI

07-13

Chromosome and spindle pole-derived signals generate an intrinsic code for spindle
position and orientation

Tomomi KIYOMITSU

07-19

Systematic single-cell analysis of development: worm and beyond

Zhirong BAO

07-23

Molecular mechanisms of the initial kinetochore-microtubule interaction in early mitosis

Shinya KOMOTO

Date

Title

Speaker

08-27

Divergent tissue dynamics of neural and non-neural ectoderm in the zebrafish

Stephen YOUNG

09-03

Intersection of transcriptional and signalling activity for head morphogenesis in mouseembryos

Patrick TAM

09-18

How do misfolded proteins and altered RNA metabolism cause ALS?

Robert BALOH

10-03

The exit from ground state pluripotency

Austin SMITH

10-31

Identifying a novel small-molecule inhibitor of the giant AAA+ ATPase using fission yeast
chemical genetics

Shigehiro A. KAWASHIMA

10-31

How to publish in Science

L. Bryan RAY

11-12

Annotation of the chicken W sex chromosome using RNA-seq reveals candidate femaledetermining genes

Craig SMITH

11-12

Angiogenesis in the zebrafish embryo: From single cell imaging to single cell functional
analysis; a new approach

Markus AFFOLTER

11-13

Specification and patterning of the neural plate border - special focus on lens and olfactory
placodesLena GUNHAGA

11-19

Making fate decisions at the exit of pluripotency: what ES cells tell us about the embryo

Alfonso MARTINEZ-ARIAS

11-22

Cellular and molecular mechanisms of lineage segregation during mesoderm development

Chaya KALCHEIM

11-26

Phylogenomics in the light of ever-growing sequencing data

Toni GABALDON

11-27

Transposon technologies towards iPS cell gene therapy

Akitsu HOTTA

11-30

Migrating transient neurons: organizing activity in patterning of the cerebral cortex

Alessandra PIERANI

12-04

From mechanical instabilities of epithelial tissues to morphogenesis, stem cell dynamics
and cancerogenesis

Edouard HANNEZO

12-06

Systemic control of hematopoietic progenitors in Drosophila

Utpal BANERJEE

12-07

How can microtubules establish protein patterns at the cell wall?

Núria Taberner
CARRETERO

12-10

Haploid ES cells as a tool for forward genetic approaches in mammals

Martin LEEB

12-10

Actin-driven chromosome transport in starfish oocytes

Masashi MORI

12-17

Genetic screens in haploid mouse ES cells to determine mechanisms of drug toxicity andresistance

Stephen PETTITT

12-25

Cis-element evolution of the Dlx genes as an underlying mechanism in toolkit gene cooption in vertebrate appendages

Kenta SUMIYAMA

12-28

Wnt/Dkk negative feedback loop regulates sensory organ size in the lateral line system offish

Hironori WADA

12-28

Mechanisms of nuclear reprogramming by eggs and oocytes

Kei MIYAMOTO

99

About RIKEN
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100

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http://www.cdb.riken.jp/
CENTER FOR DEVELOPMENTAL BIOLOGY
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KOBE 650-0047
JAPAN
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On The Cover
Caudal view of Drosophila male terminalia at 24 hr
after puparium formation. All nuclei (magenta) andcells in posterior compartment of each segment(green) are shown.
Image: Ayako ISOMURA, Laboratory for Histogenetic
Dynamics

Printed in Japan using soy inks, waterless printing
methods, and paper from sustainable resources.
RIKEN 2013-030