RIKEN Center for Developmental Biology

2008 Annual Report

Contents
Greetings from the CDB Director ··········· 2
Organizational Chart ······························· 4April-May
Highlights ··········································· 34

January-March
Highlights ············································ 6

Labs

Labs

Cell Asymmetry ·································· 58

Fumio MATSUZAKI

Germline Development ······················ 59

Vertebrate Body Plan ························· 26

Akira NAKAMURA

Neuronal Differentiation
and Regeneration ······························· 27

Jun-ichi NAKAYAMA

Shinichi AIZAWA

Hideki ENOMOTO

Neocortical Development ·················· 28
Carina HANASHIMA

Morphogenetic Signaling ·················· 29
Shigeo HAYASHI

Vertebrate Axis Formation ················ 30
Masahiko HIBI

Cell Lineage Modulation ···················· 31
Toru KONDO

Evolutionary Morphology ·················· 32
Shigeru KURATANI

Sensory Development ························ 33
Raj LADHER

Chromatin Dynamics ························· 60
Stem Cell Biology ······························· 61

Shin-Ichi NISHIKAWA

Cell Migration ····································· 62

Kiyoji NISHIWAKI

Pluripotent Cell Studies ····················· 63

Hitoshi NIWA

Mammalian Epigenetic Studies ········ 64

Masaki OKANO

Mammalian Molecular Embryology ···· 65

Tony PERRY

Mammalian Germ Cell Biology ········· 66

Mitinori SAITOU

Organogenesis and Neurogenesis ···· 67

Yoshiki SASAIJune-AugustHighlights ··········································· 68
Labs
Embryonic Induction ·························· 86
Hiroshi SASAKI

Cell Fate Decision ······························ 87
Hitoshi SAWA

Early Embryogenesis ························· 88
Guojun SHENG

The Center for Developmental Biology was launched in
April 2000 under the auspices of the Millennium Project
research initiative that was established to drive
research in the fields of information technology, environmental science and the study of aging, areas of vital
importance to both Japan and the world in the 21st
century. Located in Kobe, Japan, the Center’s research
program is dedicated to developing a better understanding of fundamental processes of animal development at the molecular and cell biological level, the evenmore complex phenomena involved in organogenesis,
as well as the biology of stem cells and regeneration.
Scientists at the CDB conduct world-class studies in atruly international research environment, with ample
core facilities and technical support and a critical massof scientific talent and expertise. The CDB also benefits
from belonging to Japan’s largest basic scientificresearch organization, RIKEN, as well as its close ties tograduate and medical schools and research institutes
in Japan and other countries, giving our scientists
access to a broad network of talent and technology
across the country and around the world.

Developmental Genomics ················· 89
Asako SUGIMOTO

Retinal Regeneration ························· 90
Masayo TAKAHASHI

Cell Adhesion andTissue Patterning ······························· 91
Masatoshi TAKEICHI

Systems Biology ································· 92
Hiroki R. UEDA

Genomic Reprogramming ················· 93
Teruhiko WAKAYAMA

Supporting Laboratories ···················· 94

September-December
Highlights ············································· 96
RIKEN Kobe InstituteBudget and Staff ··································· 114
CDB Symposium ·································· 116
CDB Seminars ······································ 118
About RIKEN ········································· 120
RIKEN Campuses ································· 1211

2

Greetings from the CDB Director
2008 marks a turning point for the RIKEN Center for Developmental Biology, with the reformulation of our research mission
to keep pace with the rapid advances in the study of animal development and related fields and in concert with new five-year
mid-term plan of the Japanese Ministry of Education, Sports, Culture, Science and Technology (MEXT). As in previous years,
many of our labs will continue to focus on genetic, molecular and cell biological mechanisms of development, but we now
also seek to broaden that scope to include greater research into organogenesis, defined as studies that explore the formationof higher-order, three-dimensional functional structures involving multiple cell types. And even as we pursue the understandingof development and organogenesis at a fundamental level, many CDB labs will also work to strengthen the scientific foundations
for regenerative medicine through research into stem cells and regenerative processes.
With the CDB now in full operation for more than six years, many of the research programs have also entered into a second
five-year term, and the past year has seen more publications than ever before. But even more than quantity, what has
impressed is the breadth and diversity of CDB research across the board. Investigator-driven research has always been thehallmark of our organization, freeing our scientists to follow wherever nature and their curiosity may lead. In 2008, CDB labs
took advantage of that freedom to develop new insights into transcriptional repression in the germline, the migration ofneuronal progenitors, the embryonic origins of the blood system, and the genetic underpinnings of circennial biologicalrhythms. Several reports even drew international media attention, including the cloning of a mouse that had been dead andfrozen for 16 years, and the in vitro differentiation of organized cortical tissue from human embryonic stem cells. Our labs
also contributed to their fields through the development of research technologies from a method for purifying monoclonal
antibodies to low-abundance antigens, to software for bisulfite sequencing analysis, to an improved mouse model ofHirschsprung’s disease.
The CDB continues to contribute to the international research community as well, maintaining close involvement with andadministrative support for the International Society of Developmental Biologists (ISDB), the Asia-Pacific Developmental Biology
Network (APDBN), the Asia Reproductive Biotechnology Society (ARBS) and the Stem Cell Network: Asia-Pacific (SNAP).
Our support for Asia-Pacific regional initiatives further underscores our commitment to building and fostering relationships
with researchers throughout the region, such as through the sharing of transgenic and knockout mouse resources under the
auspices of an APDBN partnering program.
In Japan, we also collaborate widely with scientists in national universities and research centers, and host many graduate
students doing the bench work for their theses. We support students and the educational community as well through workshops
and courses targeting participant groups from high school students and teachers, to graduate students, to working scientists.
Two new highlights for 2008 were the launch of a workshop series for Japanese scientists wishing to learn basic laboratory
techniques and ethical procedures for the research use of human pluripotent cells, and a short course organized in conjunction
with the Japanese Society for Developmental Biology (JSDB) to provide local high school teachers with instruction in developmental biology concepts and techniques. We continued in our public outreach activities as well with an “Evening of Scienceand Music” hosted by the Japan Science and Technology Agency (JST) in Kyoto last summer.
A sampling of the full spectrum of CDB activities over the past twelve months is collected in the Annual Report you hold inyour hands. Every year we strive to make this report as informative and lively as possible, and to convey through it the senseof excitement we have at confronting the new challenges and opportunities of the years ahead. I hope you find it to be auseful reference and wish you all the best for 2009.

Masatoshi TAKEICHI

Director, RIKEN Center for Developmental Biology

3Neurorientation: Finding direction
in mammalian neuroepithelial cell divisions

When a cell divides, mitotic spindles form tosegregate the replicated chromosomes andallocate the contents of the parent cell to thetwo daughters. The orientation of thesespindles is therefore important, as it impacts
directly on the cellular components that eachdaughter will inherit, and can influence ordetermine whether the division will besymmetric (producing daughters of identicalcell fate) or asymmetric. Asymmetric cell
division is particularly key to stem cell biology,
as many types of stem and progenitor cell
self-renew by generating one self-copy alongwith a daughter of another fate. This is true ofthe cells of the neuroepithelium in mammals,
which serve as progenitors that both selfrenew and give rise to neurons, suggestingthat the orientation of the spindle apparatus
may play a significant role in ensuring asymmetric mitosis, but its contribution, if any,remains unclear.
Daijiro Konno, Go Shioi and colleagues in the
Laboratory for Cell Asymmetry (Fumio Matsuzaki; Group Director) shed light on the controlof spindle orientation in neuroepithelial cells,
and its function in maintaining the neuroprogenitor population. This work, published in
Nature Cell Biology, identified the gene LGN
as a critical component for orienting themitotic spindle so as to allow neuroepithelial
cell division to proceed asymmetrically.
Mitotic spindle orientation has been studied
extensively in the fruit fly Drosophila melanogaster, revealing an elaborate network ofgenes that steers the spindles and links themto the cell cortex. Recent work from a numberof labs has suggested that similar genetics
may underlie this process in mouse neuroepithelium as well. Shioi and Konno began bycreating a mutant mouse lacking the gene
Atsunori SHITAMUKAI, Daijiro KONNO, Go SHIOILGN, a murine counterpart to the Drosophila
gene, Pins (Partner of Inscuteable), which
participates in the cortical complex responsible for spindle orientation in the fly. Looking
at dividing cells in the brain preparations from the LGN knockouts, they found that their orientations had
become randomized, rather than occurring parallel to the epithelial surface as they do in wildtype animals.
A similar spindle misorientation phenotype could also be produced by overexpression of mInsc, the mouseortholog of Inscuteable, a Drosophila gene that functions to rotate the mitotic spindle perpendicular to theepithelial surface.

6©2008 Nature Publishing Group

Konno D, et al. Neuroepithelial progenitors undergo LGNdependent planar divisions to maintain self-renewability during
mammalian neurogenesis. Nat Cell Biol 10. 93-101 (2008)

To determine whether such misorientation had an effect on daughter cell fate, the Matsuzaki group next
examined the distribution of LGN mutant daughter cells expressing the gene Pax6, which normally marks
apical progenitors. They found that, rather than remaining in the ventricular zone (the region nearest theapical aspect of the neuroepithelium, where cell division generally occurs), Pax6 cells were scattered morebasally. This was also the case for cells overexpressing mInsc, suggesting linkage between the failure of
the spindles to orient properly and the basal scattering of progenitors.
Apical progenitor cells are normally polarized, extending processes called “feet” from both their apical andbasal surfaces. In wildtype, most progenitors divide in such a way that both daughters inherit part of themother cell’s apical foot. But a third member of the group, Atsunori Shitamukai, found that in the orientation
mutants, the apical foot was segregated to only one of the daughters at a much higher frequency. In both
cultured brain slice preparations and in vivo, the progenitors produced fewer apical and more non-surface
progenitors on division, and as it is the non-surface progenitors that primarily generate neurons, the rateof neurogenesis was unaffected despite this change in composition.
The larger picture that emerged from these findings was intriguing for its subtlety, as it suggests that theinheritance of the basal or apical compartment alone is not sufficient to determine cell fate of the apicalprogenitors; the failure to acquire an apical foot also seems to play a role in the generation of non-surface
progenitors. The group’s observations also call into question some widely held views on what constitutes
the normal orientation of neurogenic cell divisions, suggesting that such divisions take place within, rather
than perpendicular or oblique to, the epithelial plane.
“It has been predicted that randomizing the orientation of cell divisions should affect epithelial morphogenesis and other phenomena,” notes Matsuzaki. “So the finding that LGN knockout mice, in which the orientation is indeed random, are viable came as something of a surprise to us, and highlights the robustness inthe face of perturbations of mammalian development.”

Apical view of neural progenitor cell division. Bothdaughter receive apicalcomponents in the majorityof divisions. Boundary between apical cells shown by
ZO1:EGFP fusion protein(green). Centrosomes labeled with PACT domainKO1 (red).

7

The difference a domain makes

Shin-ichiro (with Rin and Juntaro) TAKEBAYASHIDNA methylation is an important form of epigenetic marking that generally leads to the formation of heterochromatin, in which DNA is packed securely away from the transcription machinery. The vertebrate genome
is stripped of nearly all of its methylation immediately following fertilization, after which different cells reacquire the distinct patterns of methyl residues appropriate to their differentiated state. The establishment
and maintenance of DNA methylation is therefore a critical function in the regulation of gene expression.
Three DNA methyltransferases have been identified in mammals: DNMT3A and -3B, which work in theestablishment of DNA methylation patterns, and DNMT1, which functions in their maintenance during
replication. DNMT1 is recruited to the site of DNA replication during the synthesis stage of cell division.
Interestingly, although mice carrying a null mutation for Dnmt1 exhibit very early embryonic lethality, indicatingthat this methyltransferase is required for development to proceed, the protein’s large number of transcription factor-binding domains suggests that it may have multiple functions, which have remained unclear.
Recent work by Shin-ichiro Takebayashi in the Laboratory for Mammalian Epigenetic Studies (Masaki
Okano; Team Leader) has brought an additional level of detail to our understanding of just how the DNMT1
enzyme achieves its developmentally important maintenance function. His work, published in Molecular8©2007 American Society for Microbiology

Takebayashi S, et al. Major and essential role for the DNA
methylation mark in mouse embryogenesis and stableassociation of DNMT1 with newly replicated regions. Mol Cell
Biol 27. 8243-58 (2007)

and Cellular Biology, reveals an essential role for the DNMT1 catalytic activity in mouse development, as
well as the requirement for preexisting DNA methylation for the localization of DNMT1 to replicating DNA.
Takebayashi began exploring the possibilities by making a subtle, single amino acid substitution in the
catalytic center of the protein encoded by the Dnmt1 gene, and used this to generate mutant embryonicstem cells and mouse embryos. He confirmed that in both the cells and the embryos DNA methylationlevels were greatly reduced, similar to the case in Dnmt1 null mutants. Even more tellingly, the catalytic defective mutant embryos were arrested shortly after gastrulation, failing to form somites and to undergo closureof the neural tube, phenotypes reminiscent of the null mutation. These findings point to a pivotal role forthe catalytic domain in enabling embryonic development.

DAPI

PCNA

DNMT1

Merge

Wildtype

Nearly total loss of methylation of genomic DNA in
nuclei Dnmt1 ps mutant
cells, in which DNMT1 fails
to localize to replicationsites. Immunofluorescenceshows DNMT1 and PCNA(a replication foci marker;
scale bar 10 µm) at E9.5.

The team examined the expression of multiple genes in their mutant mice and discovered a range of defects
in the regulation of multiple genes, including imprinted genes and retrotransposons, as well as cell growtharrest. Looking at the localization of heterochromatin marks, they found that these too were globally affected;
heterochromatin recruitment to pericentromeric regions was lower in all somatic cells. They also saw asmall percentage of cells that exhibited hyperacetylation (a marker of transcriptionally active chromatin), a
phenomenon that was never observed in wildtype cells. The picture that emerged was one of chromatin
structural and gene regulatory abnormalities on the loss of DNMT1 catalytic function, leading to early
embryonic lethality.
Curious about how their mutant allele might affect DNMT1 recruitment to replication foci (where it is foundin wildtype), they used immunofluorescence to track the mutated DNMT1 enzyme and discovered that it
was instead diffusely present throughout the nucleus, suggesting that this defect was specifically attributable
to the loss of the catalytic activity, rather than to some overarching functional change to the protein’s
structure.
Using a replication labeling with 5-methyldeoxy-CTP in embryonic stem cells without the all three active
DNA methyltransferases and endogenous DNA methylation, they found that preexisting DNA methylation
in a hemi-methylated state is a major determinant for the localization of DNMT1 to the replication foci.

9Tead4 triggers trophectoderm

The first few steps of mammalian development see a single fertilized egg divide several
times to form a compact sphere of cells
known as a morula, which subsequently
develops into a fluid-filled ball called a blastocyst. It is in the period surrounding the
emergence of the blastocyst that the first
cellular differentiation processes take place,notably the segregation of the inner cell mass(ICM), which gives rise to the embryo proper,from the trophectoderm, a tissue that contributes to extraembryonic tissues, such asplacenta. Recent studies have indicated an
important role for the mutual inhibition
between a pair of genes, Oct4 and Cdx2,
which regulate the ICM and trophectoderm,
respectively. The question of potential
upstream regulation of these genes, however,remains open.
Noriyuki Nishioka, Shinji Yamamoto andothers in the Laboratory for Embryonic Induction (Hiroshi Sasaki; Team Leader) showedthat the transcription factor Tead4 is required
for trophectoderm development, in a mannerthat suggests it functions upstream of Cdx2.
Their findings, published in the journal Mechanisms of Development, shed new light on thegenetic regulation of the earliest cell specialization event in mammalian embryogenesis.

Noriyuki NISHIOKAThe Sasaki lab’s study began with the generation of mice carrying a homozygous deletionof the Tead4 gene, following on their previous
work that showed roles for Tead-family genes
in the regulation of Foxa2, a transcription
factor known to regulate the development of
the midline signaling centers controlling
post-implantation development. They were
surprised to find that the knockout embryos
died at extremely early stages in development, prior to the implantation of the blastocyst into the uterine wall.
This unexpected early lethality prompted Nishioka and Yamamoto to re-examine the expression of Tead4,
and the related genes, Tead1, -2, and -3, in pre-implantation mouse embryos. Using RT-PCR to detect
Tead transcripts, they found that Tead4 expression switched on by the 4-cell stage; two other Tead genes,
Tead1 and Tead2, were also expressed. They next used immunohistochemistry to attempt to detect thevarious Tead proteins, and found both Tead1 and Tead4 in all blastomeres (as the individual cells of theearly embryo are called), as well as in the cells of trophectoderm and the inner cell mass slightly later indevelopment.

10©2007 Elsevier B.V.

Nishioka N, et al. Tead4 is required for specification oftrophectoderm in pre-implantation mouse embryos. Mech Dev
125. 270-83 (2008)

Using time-lapse videos of developing embryos, the team determined that, in Tead4-/- mutants, although
the embryos developed apparently normally up to the morula stage, they failed to form a blastocoel (thecavity of the blastocyst) even as cell proliferation proceeded apace. The blastocoel forms when fluid seeps
between gaps in the surface of the embryo and fills the interior, and it is known that this process can be
disturbed by defects in cell adhesion. But on studying pathways involved in the formation of adherens andtight junctions, cell polarity and various forms of signaling, they found no abnormalities that could accountfor the blastocoel failure.
They turned next to the trophectoderm, as this tissue is also a critical requirement for normal blastocyst
development. Testing for the expression of Cdx2, which is upregulated in the trophectoderm in wildtype
embryos and required for TE lineage specification, they found that the gene was only faintly expressed in
the Tead4 mutant embryo after the first several rounds of cell division, and not at all in subsequent stages.

Tead4 is essential for
trophectoderm development. (Left) Wildtype blastocyst showing trophectoderm (Cdx2, red) and innercell mass (Oct3/4, green),
respectively. (Right) A
Tead4 homozygous mutant
embryo at the comparable
developmental stage. The
blastocoel fails to form, andall cells assume inner cellmass fate, as revealed by
the expression of Oct3/4(green).

This was true of other TE-specific genes, such as Eomes and Fgfr2, as well. Cdx2 functions in the preimplantation embryo by repressing the expression of Oct4. When Nishioka and Yamamoto checked Oct4
expression, they found that it was expressed throughout the late blastocyst, indicating that the entire
embryo had adopted an inner cell mass fate.
Given that the ICM can serve as a source for embryonic stem (ES) cells, the team tried to establish an ES
cell line using Tead4 mutants, and succeeded in establishing three ES-like cell lines, one of which wascapable of forming colonies in culture and differentiating into all three germ layer lineages in vitro, suggestingthat Tead4 is not required for development of embryo proper.
“The finding that trophectoderm completely fails to form in Tead4 mutants provides an important clue for
us to better understand early development in mammals,” says Sasaki. “But the important question of how
this gene, which is expressed in every cell in the embryo, can prompt trophectodermal differentiation in
only a subset of those cells still needs to be addressed.”11

Keeping germline transcription under wraps

Kazuko Hanyu-NAKAMURA, Hiroko Sonobe-NOJIMAThe cells of the germline represent the future of a species, as they are the only cells capable of conveying
genetic information from one generation to the next. Given this critical function, it is vital that they beprotected from the influence of factors that might cause them to differentiate into somatic lineages. In manytaxa, this protection is achieved by the transient and global repression of RNA polymerase II (RNAPII)dependent transcription in germ cell progenitors, which keeps them from deviating from their purpose.
The RNAPII enzyme is encoded in all eukaryotic genomes, and catalyzes the transcription of DNA intomessenger RNAs (mRNAs). In some invertebrate species, such as the roundworm C. elegans and the fruit
fly Drosophila melanogaster, the repression of transcription in fledgling germline cells has been linked tothe absence of phosphorylation (a form of protein modification) of Ser2 residues in a specific domain in
the RNAPII protein, but the means by which this is achieved has been obscure.
Kazuko Hanyu-Nakamura, Hiroko Sonobe-Nojima and colleagues in the Laboratory for Germline Development (Akira Nakamura; Team Leader) have identified the factor responsible for such transcriptional repression in Drosophila germ cell precursors. Their work, published in Nature, reveals that the product of thepolar granule component (pgc) gene represses Ser2 phosphorylation of the RNAPII carboxy-terminal
domain (CTD) in germline progenitors known as pole cells by interfering with the recruitment of a second
factor, P-TEFb, which is known to play a role in CTD Ser2 phosphorylation in vivo.

12©2008 Nature Publishing Group

Hanyu-Nakamura K, et al. Drosophila Pgc protein inhibits P-TEFb
recruitment to chromatin in primordial germ cells. Nature 451.
730-3 (2008)

The project began somewhat serendipitously when the Nakamura team noted that pgc RNA, which was
assumed to be non-coding, in fact contains a sequence that suggests a translation start site. Going backto the completed Drosophila genome, they found that pgc should indeed be capable of encoding a small
protein, which they verified by immunostaining; antibodies against the Pgc peptide showed immunoreactivity in early-stage pole cells, before tapering off later in embryonic development.
They next generated flies with various mutations causing losses of pgc-function and discovered that, whilepole cells did form, there was no repression of Ser2 phosphorylation in the RNAPII carboxy-terminal domain,
causing the cells to degenerate by mid-stage embryogenesis. This defect could be rescued by the expression of intact Pgc. Moreover, misexpression of pgc in somatic cells resulted in the repression of Ser2
phosphorylation, strongly suggesting that the newfound protein plays a central role in transcriptional repression.

Posterior pole of the blastoderm - stage embryos immunostained for Pgc (green)
and CTD phospho-Ser2(magenta). Pgc is expressedin newly formed pole cells
and CTD Ser2 phosphorylation is repressed in thesecells (wild-type, left). In theabsence of Pgc, pole cells
fail to repress CTD Ser2
phosphorylation( pgc-,right).

The team next turned their attention to a second factor, P-TEFb (Positive transcription elongation factorb), which had previously been implicated in the phosphorylation of CTD Ser2 and promotion of productive
transcription elongation. Suspecting that this might be a target of Pgc, they used co-immunoprecipitationand pull-down assays to establish a specific interaction between the two factors, both in vitro and in vivo,in which Pgc interacts with Cdk9, a catalytic subunit of P-TEFb. Overexpression of P-TEFb in pole cells
produced a similar effect to that of loss of pgc function, suggesting that Pgc represses Ser2 phosphorylationin pole cells by interfering with the function of P-TEFb. But the finding that an MBP-Pgc construct, whichinteracted with P-TEFb, did not affect CTD phosphorylation by that factor in vitro indicated that Pgc’s
function was unlikely to regulate the catalytic activity of P-TEFb. They compared localization of P-TEFb in
untreated somatic tissue and tissue in which Pgc was misexpressed, and found a dramatic reduction of
their normal localization to active promoter regions on chromosomes. This suggested that Pgc may function
by sequestering P-TEFb and preventing its recruitment to promoter sites, a view that was shored up by
the finding that Pgc expression inhibited normal P-TEFb recruitment to heat shock genes following heatshock.
“Studies in C. elegans have shown that another germline protein, PIE-1, is also involved in regulating thephosphorylation of CTD Ser2 and that it appears to bind to the CyclinT subunit of P-TEFb,” says HanyuNakamura. “What’s interesting is that Pgc and PIE-1 are non-homologous, which means these germline
transcriptional repression systems may well have evolved independently.”

13

Critical role for maternal nucleolus innuclear transfer

Somatic cell nuclear transfer (SCNT), in which
the nucleus from one cell (usually an unfertilized oocyte) is removed and replaced by one
from another somatic cell, has been touted
as a potentially powerful tool to enable
personalized regenerative medicine, which
has led to the process of human SCNT being
called (somewhat prematurely), “therapeuticcloning.” Despite its promise, enucleation
imposes considerable stress on the egg, andthe great majority of embryos created by
nuclear transplantation fail to develop.
Numerous hypotheses have been proposed
to explain the high fail rate of cloning attempts,
but the cause has remained tantalizingly outof reach after more than a decade of intense
research.
New work by Sugako Ogushi in the Laboratory for Mammalian Germ Cell Biology(Mitinori Saitou; Team Leader) took a major
step toward answering that question with thedemonstration that the oocyte nucleolus isessential to early embryonic development inmammals. This study, done in collaboration
with scientists from Kobe University, Italy, andthe Czech Republic and published in thejournal Science, shows for the first time theindispensable role played by the maternal
nucleolus, possibly as the source of materials
needed to form the zygotic nucleolus in thefirst stages of embryogenesis.

Sugako OGUSHI

14

The nucleolus, which forms a part of thenucleus, is mainly responsible for assembling
ribosomes, the protein factory organelles inwhich peptides are translated from mRNA.
However, a fully-grown oocyte completely
ceases transcription including ribosomal RNA
synthesis. Its nucleolar structure apparently
contains no DNA and shows highly compacted, monotonous morphology of uncertain composition and function. Although
nuclear transfer generally involves the removalof the entire nucleus, Ogushi used a micromanipulation system to selectively pluck out only the nucleolus
from oocytes taken from pigs and mice, a procedure called enucleolation. She found that, although these
enucleolated oocytes were capable of normal maturation and could be activated parthenogenetically or
by fertilization, the activated cells failed to develop nucleoli of their own. Interestingly, this defect could be
rescued by reinjecting the maternal nucleolus into an enucleolated egg, but not by injection of nucleoli fromsomatic or ES cells.

©2008 the American Association for the Advancement of Science

Ogushi S, et al. The maternal nucleolus is essential for early
embryonic development in mammals. Science 319. 613-6(2008)

Following the development of enucleolated embryos, Ogushi found that pig embryos produced fromoocytes lacking nucleoli failed to develop past the first few cleavages, although their viability was restored
by reinjection of the missing maternal nucleolar material. Oocytes that were sham-operated by aspiratinga small amount of nucleoplasm but leaving the nucleoli intact developed zygotic nucleoli, and did not suffer
the same embryonic arrest, suggesting that the enucleolated embryos’ failure was not simply due to thetrauma of micromanipulation.
Suspecting that the maternal nucleolus might be the missing ingredient preventing SCNT embryos fromdeveloping normally, the team next tried injecting maternal nucleoli along with somatic cell nuclei into
oocytes that had had their own nuclei removed. This nucleolar coinjection alone, however, was insufficient
to rescue development, indicating that the maternal nucleolus is a necessary, but not sufficient, component
for mammalian embryogenesis. It is likely that additional factors in the germinal vesicle (as the oocyte
nucleus is called) are also required, as oocytes in which the germinal vesicle has disintegrated, releasing
its contents into the cytoplasm, support development after nuclear transfer, while those in which thegerminal vesicle has not broken down, do not.
The discovery that nucleoli are inherited solely from the mother adds these structures to the list of organelles,
such as mitochondria, that are passed on from only one of the parental lineages. It has been known foryears that the early embryo relies on a stock of materials from the oocyte to drive development over the
first few rounds of cell division. These new findings indicate that the nucleolus may be an important maternal
contribution, needed by the embryo to build nucleoli of its own.
“In this study, we showed that the nucleolar structure in a fully-grown oocyte is essential for early embryonicdevelopment, and that it contributes to the nucleolar structure of the paternal pronucleus, demonstratingthat the nucleolus of a zygote is exclusively of maternal origin” says Ogushi. “Although we still don’t know
its precise function and molecular components, we are now working to answer those questions. We hopethese studies will lead to a better understanding of how a totipotent zygote is constructed.”

Removal of the nucleolus
from the oocyte. A mouse
oocyte (upper panel) has aprominent nucleolus inside
the nucleus. The nucleolus
is gently aspirated by a verythin pipette (bottom panel).

15

Breakthrough in photoreceptor
differentiation from ES cells

Visual impairment can be caused by loss offunction in various parts of the eye, but the
majority of currently incurable causes of vision
loss, such as retinitis pigmentosa and agerelated macular degeneration, involve theretina. Although retinal neurons regenerate
only minimally in mammals under normal
conditions, this region at the back of the eye
represents one of the most promising targets
for regenerative medicine in the nervous
system. Such therapies may one day involve
stimulating endogenous stem cells to regenerate damaged neural tissue or, alternatively,
generating functional retinal cells in vitro andtransplanting them into patients.
A report by Fumitaka Osakada, Hanako Ikeda
and colleagues in the Laboratory for Retinal
Regeneration (Masayo Takahashi; Team
Leader) made an important stride towardrealizing that dream. Their study, conductedin collaboration with clinicians and scientists
from Kyoto University, and the CDB Laboratory for Organogenesis and Neurogenesis(Yoshiki Sasai; Group Director) and publishedin Nature Biotechnology, reports the highefficiency generation of rod and cone photoreceptors and pigmented epithelium frommouse, primate and human embryonic stem
cells under fully characterized culture conditions.
The Takahashi lab and others had previously
developed methods for inducing the differentiation of retinal neural progenitors, but these
relied on co-culture with embryonic retinal
tissue or other confounding factors, andgenerally produced retinal neural cells at
unacceptably low efficiencies. Working firstwith mouse embryonic stem (ES) cells, they
sought to identify factors capable of spurring
photoreceptor differentiation in a morereliable and efficient manner. After isolatingprogenitor cells and confirming their competence for further differentiation to retinal
precursors (as shown by their expression of
the marker gene Crx), the Takahashi teamfound that the γ-secretase inhibitor DAPT
steered the mitotic progenitors to adopt a
postmitotic precursor fate.
Fumitaka OSAKADA

16

©2008 Nature Publishing Group

Osakada F, et al. Toward the generation of rod and cone
photoreceptors from mouse, monkey and human embryonicstem cells. Nat Biotechnol 26. 215-24 (2008)

They next tested a panel of factors implicated in photoreceptor differentiation on the Crx+ precursor cells,
and found that a combination of aFGF, bFGF, taurine, Shh, and retinoic acid in retinal culture medium
prompted nearly 30% of the precursors to begin expressing the light-sensitive opsins characteristic ofeither rod or cone photoreceptors.
One drawback of the method used for inducing differentiation from mouse ES cells is that it involves theuse of fetal bovine serum, which problematizes its use for clinical applications. Seeking a workaround,
Osakada turned next to a chemically defined medium for use with ES cells from cynomolgus monkeys andfound that the serum free floating culture of embryoid body-like aggregates (SFEB) system worked forprimate cells as well, without requiring the use of bovine serum. They next tested a pair of factors that had
been shown to promote retinal-lineage differentiation – the Wnt-antagonist Dkk1 and the nodal-antagonist
Lefty-AS – and found that in combination, these were highly effective in boosting the differentiation of retinal
pigmented epithelium.
They further found that by treating retinal progenitors derived from monkey ES cells with retinoic acid andtaurine, two of the factors used in the mouse ES study, they could strongly promote differentiation of Crxpositive postmitotic precursors, and subsequently of rod and cone-like cells expressing opsins appropriate
to their putative cell types.
In a final set of experiments, the Takahashi lab assessed whether their findings using mouse and primate
ES cells could be replicated using human embryonic stem cells as well. Using the SFEB culture (originallydeveloped by the Sasai group) optimized for use with human ES cells, they found that as in the monkey
ES cell tests, the combination of Dkk1 and Lefty-A caused a significant number of the undifferentiated cells
to assume a retinal pigmented epithelial fate, as determined by morphology and gene expression. Differentiation of Crx+ precursors into photoreceptor-like cells using taurine and retinoic acid also proved successful,
although at slightly lower efficiency than in monkey cells, establishing this new technique as a potentially
important tool for generating human photoreceptors for use in drug screening and future regenerative
medical applications.
“Now we have the cells that might one
day be useful for retinal cell transplantation therapy, but it needs to be remembered that these are only some of the
raw materials. We will still need to establish methods for purifying them, andgetting them to function in vivo after
transplantation,” cautions Takahashi.
“We also need to recognize the differences in closeness to clinical application
between retinal pigmented epithelial
cells and photoreceptor cells. We now
need to think practically about how totranslate this basic research, and thehurdles to that lie ahead.”

Photoreceptor cells differentiated from human ES
cells.

17

ES cells show diversity

The first few days of embryonic developmentin the mouse witness the zygote undergo atransformation from a single cell, to a
raspberry-like cluster (morula), to a hollow ball(blastocyst) before implanting in the uterine
wall. Cell differentiation begins by about thethird day of embryogenesis, with exterior cells
entering the trophectodermal lineage, a tissue
that forms the placenta and other extraembryonic tissues, and interior cells, which takethe first step of the path leading to various
somatic fates. The inner cell mass (ICM) of
the blastocyst is famous as the source ofembryonic stem cells, although it has beenshown that ES-like pluripotent cells can also
be obtained from the epiblast, a transient
structure that arises slightly later in development, after implantation. The epiblast in turngives rise to primitive ectoderm, which isconsidered to be the source of the somatic
lineages that yield the many differentiated cell
types in the adult body. ES cells are of greatinterest because they show similar ability togive rise to all somatic lineages in culture, but
it remains unclear whether all ES cells are truly
created equal, or whether they show diversity
among themselves.

Yayoi TOYOOKA

A study by Yayoi Toyooka and colleagues inthe Laboratory for Pluripotent Cell Studies(Hitoshi Niwa; Team Leader) came down
clearly in favor of the heterogeneity theory,
showing that subsets of ES cells exhibit gene
expression variations corresponding to theICM, epiblast and primitive ectoderm stagesof development. In a report published in
Development, Toyooka, revealed that while
all ES cells express the pluripotency marker
Oct3/4, they show fluctuations in the expression of another gene previously taken as an
ES hallmark, Rex1.
The study began with the establishment of asystem for visualizing the expression of these
two genes using a pair of fluorescent proteins. When they cultured the cells using a method to select for
Oct3/4-expressing cells, they found that some of these expressed Rex1 while others did not, with predominantly Rex1+ colonies showing compacted morphologies, and Rex1- colonies tending to be flatter.
They used quantitative PCR to examine gene expression in the two subpopulations in more detail, andfound that the overall expression patterns in Rex1+ cells was similar to that seen in inner mass cells, while
Rex1 - cells expressed genes in patterns reminiscent of primitive ectoderm. Interestingly, when they18

©2008 Company of Biologists

Toyooka Y, et al. Identification and characterization of subpopulations in undifferentiated ES cell culture. Development 135.
909-18 (2008)

separated these fractions into purified populations, they found that each was able to give rise to the otherspontaneously; that is, Rex1+ cells showed up in Rex1- colonies, and vice versa. This worked even incolonies derived from a single Rex1-positive or negative cell.
It has long been known that cells from the inner cell mass or epiblast can contribute to chimera formation
when injected into blastocyst-stage embryos, but cells taken from primitive ectoderm cannot. Investigating
whether this might be true of ES cells in which Rex1 expression is switched on or off, Toyooka et al. injected
either Rex1+ or Rex1- cells into mouse blastocysts and found that, similar to ICM and epiblast cells, theRex1 + cells contributed strongly to chimeric embryonic tissues, but that, as is the case for primitive
ectoderm, the Rex1-cells did not.
Given this apparent correspondence of Rex1+ and Rex1- cells to the inner cell mass and primitive ectoderm
stage of development, respectively, the Niwa team next checked whether their differentiation would follow
a similar pattern. When differentiation was induced in colonies enriched for Rex1- cells by the withdrawalof LIF (a factor that keeps mouse ES cells in an undifferentiated state) they showed a lower tendency togive rise to extraembryonic tissue than did a control group, indicating another similarity with primitive
ectoderm cells in vivo. Conversely, Rex1- cells were comparatively easier to steer down somatic pathways,
such as mesoderm or neuroectoderm, than were their Rex1+ counterparts.
“I suppose that many people who have worked with ES cells already suspected that they were not entirely homogeneous,” says Toyooka, “but now, by looking at Rex1 expression, we’ve been able to show that there
are definite subpopulations in terms of both gene expression and differentiative potential. Perhaps thiswork will serve as a model for studying the control of fluctuations in gene expression.”

Upper panels show Rex1 positive cells labeled by
GFP. Both Rex1+ and Rex1cells can be seen. The lower
panel shows a cartoon of
the two ES cell subpopulations.

19

Rbsn-5, a new factor in pole
plasm assembly

Tsubasa TANAKAThe Drosophila oocyte is a polarized structure, with clearly demarcated anterior and posterior ends showing
distinct patterns of RNA and protein localization. The posterior pole is home to the pole plasm, from which
the cells of the germline originate, and whose assembly is mediated by the protein Oskar (Osk). Interestingly,
osk RNA yields two isoforms, long and short Osk, each with distinct functions – short Osk serves as arecruiter of pole plasm components, while long Osk tethers them to the posterior cell cortex through interactions with the actin cytoskeleton governed by some as-yet unknown mechanism.
A study by Tsubasa Tanaka of the Laboratory for Germline Development (Akira Nakamura; Team Leader)has pointed to a hitherto unsuspected link between Oskar and the endocytic pathway that may help to
explain how this protein fulfills its function. The report, published in Development, reveals that the endosomal
protein Rabenosyn-5 is required, apparently downstream of Osk, for pole plasm assembly.
The study arose from a mutagenesis screen to identify mutations that affected the formation of pole plasm,a special form of cytoplasm found in the posterior end of the Drosophila oocyte. The screen netted one
promising candidate factor that was identified as a homolog of the protein Rabenosyn-5 (Rbsn-5), a Rabeffector protein in mouse that functions in endocytosis, a process in which materials are absorbed fromthe extracellular environment and sorted for processing.
Given the failure of pole plasm assembly in the rbsn-5 mutant, Tanaka checked for a possible link to Oskar,
and found that although the initial recruitment of osk RNA to the posterior pole was unaffected, its accumulation was unstable and deteriorated at later stages. Knowing that osk RNA is carried to the posterior via apolarized network of microtubules, he investigated a the effects of loss of rbsn-5 function on this process,and again found that while microtubule polarity was established, it failed to be maintained.
Although this opened up a plausible explanation for the pole plasm assembly defects in rbsn-5 mutants,
the close co-localization of Rbsn-5 and Osk in oocytes suggested that there might be something more to
the story. Knowing the association of Rbsn-5 with endosomes in other species, Tanaka looked for20©2008 Company of Biologists

Tanaka T and Nakamura A. The endocytic pathway acts
downstream of Oskar in Drosophila germ plasm assembly.
Development 135. 1107-17 (2008)

correspondences with other endosomal markers, and found that all showed polarized (posterior) distributions similar to both Rbsn-5 and Osk. This posterior localization of endosomes appeared briefly but was
subsequently lost in an osk-null mutant, suggesting that the maintenance of endocytic polarization is dependent on Osk. (The initial establishment of endocytic polarity appears rather to rely on the even earlier oocyte
A-P polarization factor, Gurken.)Experiments in which osk was misexpressed at the anterior pole further showed the involvement of Osk
in the endocytic pathway. Anteriorly misexpressed Osk recruits endosomal proteins and stimulates
endocytic activity. Wondering whether this might be a function specific to one of the two Osk isoforms,
Tanaka tried expressing short and long Osk in the anterior and found that, while the ectopic expression ofshort Osk alone had no noticeable effect, misexpression of long Osk was sufficient to recruit endosomes
to the anterior pole of the oocyte.
It has long been suspected that long Osk works by anchoring pole plasm components to the posterior
cortex, but the involvement of Rbsn-5 in microtubule polarity in normal oocytes made it impossible to disentangle its known role in this process from a second possible role in Osk-mediated tethering. But as rbsn-5oocytes maintain their competence to localize RNA to the anterior pole, they provide a unique environment
for focusing on this question. Looking at Osk recruitment and maintenance in an rbsn-5 mutant in which
Osk was misexpressed at the anterior pole, Tanaka found that the anchoring of Osk and other pole plasm
components to the cortex was weak, resulting in their diffusion into the cytoplasm. Although Osk promotes
long F-actin projections from the cortex at both anterior and posterior ends of wildtype oocytes, the anterior
misexpression of Osk in rbsn-5 oocytes induced large aberrant F-actin aggregates, which diffuse alongwith pole plasm components into the cytoplasm, suggesting that long Osk stimulates endocytic activity,
which in turn promotes proper F-actin reorganization to anchor pole plasm components to the oocyte
cortex.
“Although the genetic approach used in this study reveals that the endocytic pathway acts downstream oflong Osk to anchor pole plasm components,” says Nakamura, “further study of the molecular basis of long
Osk function in endocytic activity is still needed to develop a better understanding of pole plasm assembly.”

F-actin

Wildtype

Oskar misexpression

F-actin at the anterior pole inwildtype or rbsn-5 oocytes
without or with misexpression of Oskar. Anterior misexpression of Oskar promotes long F-actin projections emanating fromcortical actin bundles inwild type oocytes (middle
image), but induces largeaberrant F-actin aggregates in rbsn-5 oocytes(right ).

21

Cross purposes: Dual role for Src in control
of cell-cell adhesion

Tissues in the embryo need to be capable ofboth stability and plasticity, which means thatthe connections between their component
cells must be subject to dynamic controls.
This regulation of intercellular adhesion at
adherens junction is achieved through theaction of multiple genetic pathways working
on a range of adhesion and cytoskeletal
molecules. Interestingly, the same regulatory
pathways can be hijacked in cancer cells,
allowing them to metastasize and form
tumors. The Src family of oncogenes, forexample, is known to downregulate theexpression of E-cadherin, a major cell-celladhesion molecule, but the wide range ofrelated molecules with overlapping functions
has made this relationship difficult to study inmammals.
A study by Masayo Shindo and colleagues
from the Laboratory for Morphogenetic
Signaling (Shigeo Hayashi; Group Director)
took a new tack by looking at the function of
Src in epithelial morphogenesis in the fruit fly
Drosophila melanogaster. Their report,
published in Development, suggests that Src
has the somewhat paradoxical dual functionof suppressing cell adhesion by E-cadherin
while simultaneously upregulating its expression.

Masayo SHINDO

The study began with a gain of function
screen for mutations producing alteredepithelial morphology during tracheal
development in the fly, which yielded the Src
family members Src42A and Src64B, both ofwhich caused defects in tracheal integrity on overexpression. Using antibodies to monitor the activity ofthese genes in vivo, the authors determined that these genes were upregulated in a range of epithelial
tissues undergoing cell rearrangement.
Focusing back on the embryonic tracheal system, the group next studied the effects of loss of Src42A
function and found multiple defects involving a ramified structure known as the dorsal branch (a group ofcells that gives rise to the trachea), including reductions in number of cells, and delays in branch extension
and cell intercalation.
Knowing of the link between Src and E-cadherin in other contexts, Shindo et al. re-examined the functionof E-cadherin in tracheal development using the cadherin gene, shotgun (shg). Loss-of-function shg
mutants showed disruptions in cellular attachments in tracheal branches, while overexpression of the genecaused delays in dorsal branch elongation and cell rearrangements. Overactivation of Src42A caused
reduced tracheal cell adhesion phenotype, which was partially suppressed by co-expression of E-cadherin,suggesting that E-cadherin is a rate-limiting component of the tracheal cell adhesion under control of Src.

22

©2008 The Company of Biologists

Shindo M, et al. Dual function of Src in the maintenance ofadherens junctions during tracheal epithelial morphogenesis.
Development 135. 1355-64 (2008)

They next used a technology known as fluorescence recovery after photobleaching (FRAP), in which afluorescent-tagged sample is zapped with high intensity light causing it to lose its fluorescence (known as“bleaching”) and then watched to determine the rate of recovery, to study the effects of Src on adherens
junctions, where cadherins normally accumulate. Looking at GFP-labeled α-catenin, an E-cadherin-binding
molecule, as a measure of adherens junction dynamics, they made an interesting discovery. Entry of
α-catenin-GFP into adherens junction was slowed down upon loss of Src function and increased upongain of Src function. “We suspected that Src enhances turnover of α-catenin in adherens junctions,” says
Hayashi, who heads the lab in which the study was done.
One puzzling question remained. Although reduction of E-cadherin explains inhibition of cell adhesion by
Src, it does not explain why activated Src permitted a larger amount of α-catenin to enter adherens junction.
The hint was obtained when they investigated the expression of Armadillo (the Drosophila version of
β-catenin, another binding partner of cadherin). They discovered that Src promoted Armadillo expression
and its downstream target, E-cadherin. This led to the remarkable conclusion that, even as Src downregulates the E-cadherin protein at the adherens junction, it upregulates E-cadherin transcription. This dual
function explains the increased turnover rate of cell adhesion molecules upon elevation of Src, making adherens junctions more dynamic.
“The dual function of Src we’ve discovered provides an answer to the long-standing question of why embryonic epithelial tissues are plastic enough to be able to adopt a variety of morphologies,” says Hayashi.
“The mechanism also explains why Src-transformed cells not only detach from original tissues, but also
re-settle frequently in a new position, an essential feature of malignant cancer metastasis.”

Figure 1

Figure 1
Activation of Src in adherens junctions in Drosophila
embryos. Src42A is distributed broadly in cell membranes (right). A phosphorylated, active, Src42A (pSrc)
was preferentially localized
to adherens junctions of tracheal epithelium (center).
Merged picture is shown at
the left.
Figure 2

Figure 2(Movie)
Metastatic behavior of tracheal cells expressing activated Src42A. Two cells
on one of tracheal branches detached from the stalkand settled on the adjacentbranch.

23

Spring comes to the field

The circadian rhythm that runs on an
approximately 24-hour cycle is perhaps thebest-known biological clock, but many
animals such as hibernating bears and migratory birds also follow a longer-term rhythm
that regulates seasonal behaviors fromfeeding to reproduction. The molecularunderpinnings of this “biological calendar,”however, have largely remained a mystery.
A new collaborative study between Takeya
Kasukawa of the CDB Functional Genomics
Unit (Hiroki R. Ueda, Unit Leader) and Takashi
Yoshimura’s group at Nagoya University andothers now reveals a “spring hormone” thatworks to trigger seasonal breeding in theJapanese quail, Coturnix japonica. In work
published in Nature, the group found that thehormone thyrotrophin (TSH), which issecreted from the anterior lobe of the pituitary
gland in response to light exposure mimicking
that of a long day in spring, triggers seasonal
breeding in this domestic fowl.
The work was enabled by the application of
DNA microarray technology to the quail,which serves as a useful model for the studyof seasonal breeding due to its rapid anddramatic responsiveness to changes in day
length. As with many birds, the Japanesequail maintains gonads of small size in nonbreeding periods, reducing its burden,
developing them to functional size only at thestart of mating season. Yoshimura’s group
had previously shown that thyrotrophin secretion was one of the earliest events in the long
day-induced pathway.
The joint project began with a comprehensive
analysis by DNA microarray of changes ingene expression when birds raised in short
day length conditions were shifted to a longer
light exposure environment, similar to thechange in day length from winter to spring. Of
the nearly 30,000 genes analyzed, roughly
300 showed changes in expression levelassociated with the day length shift, with TSH
being the earliest responder. Further study
revealed that this hormone is triggered in thepars tuberalis, a section of the anterior
pituitary lobe, and binds to its receptor, TSHR,
Takeya KASUKAWA

24

©2008 Nature Publishing Group

Nakao N, et al. Thyrotrophin in the pars tuberalis triggers
photoperiodic response. Nature 452. 317-22 (2008)

in the overlying hypothalamus, enabling it to bypass the blood-brain barrier and enter the brain.The groupnext looked for a possible interaction between thyrotrophin and a second factor known as type-2 deiodinase,or DIO2, which Yoshimura’s lab had previously shown to be important in gonad activation, and found that
TSH indeed upregulates DIO2. They confirmed this in vivo by injecting TSH into cerebral ventricles, which
stimulated both DIO2 expression and gonadal growth.
In addition to the identification of this molecular pathway involved in seasonal breeding, the discovery of
the role of the pars tuberalis put paid to a longstanding question of the function of this hitherto mysterious
structure. The new link between TSH, which is known for its roles in thermoregulation and metabolism,
and reproduction also represents an important discovery, that may lead to new insights into seasonal biological cycles with implications in fields ranging from animal husbandry to seasonal affective disorders.

A) Many animals adjust their
daily rhythms to the lengthof day in a given season.
B) The quail used in this research is a common domestic fowl in Japan, and exhibits highly sensitive responses to seasonal change.
C) On being shifted into along-day light exposure
regime, TSH levels are elevated by 14 H after midnightof the first day of exposure.

25

Vertebrate Body Plan
http://www.cdb.riken.jp/en/aizawa

Shinichi AIZAWA Ph. D.

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 Universityof 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 has served as CDB deputy director and group director of the Vertebrate Body Plan lab, as well ashead of the Animal Resources and Genetic Engineering Laboratory. In January 2007, he was appointed President of the Japanese Societyof Developmental Biologists. He also serves as managing editor for the journal, Mechanisms of Development.

All vertebrate species share certain features in the development of the brain, in that all vertebrate brains comprise four regions –telencephalon, diencephalon, mesencephalon and metencephalon (or cerebellum) – an organizational trait that is thought tohave appeared with the advent of the vertebrate lineage. However, dramatic changes in this organization took place during thedivergence of jawed (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. The mechanisms that instruct the brain’s laminar regions to conform toanterior-posterior and dorsal-ventral axis patterning programs, and the means by which each individual region is formed however,remain unknown.
The head is the most layered of the body’s structures and its development begins with anterior-posterior axis formation. ThisA-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 bywhich 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 Bauplanof the vertebrate head, but application of powerful molecular biological techniques to the study of multiple vertebrate model species
has now made it possible to study 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 understanding of brain development. The question of what molecularmechanisms underlie the development of neocortical regions iscentral to that pursuit.

EPB41L5 regulates epithelial/mesenchyme transition post-transcriptionally; it binds
to p120 catenin through its N-terminal FERM domain, thereby destabilizing E-cadherin
toward endocytosis and to paxillin through its C-terminal, thereby activating integrin
expression in cell surfaces or focal adhesion formation.

Staff
Group Director

Shinichi AIZAWA
Research Specialist

Yoko SUDA

Research Scientist

Hideharu HOSHINO
Fumitaka INOUE
Mikihito SHIBATA
Masaki TAKEUCHISpecial Postdoctoral
Researcher

Mariko HIRANO
Visiting Scientist

Daisuke KUROKAWA
Research Associate

Yusuke SAKURAI

26

Publications
Technical Staff

Akiko FUKUHARA
Eriko KAJIKAWA
Hiromi NAKAO
Tomomi OMURA
Maiko TAKAHASHI
Assistant

Sayo SAITO

Suda Y, et al. Evolution of Otx paralogue
usages in early patterning of the vertebrate head. Dev Biol 325. 282-95(2009)

Takasaki N, et al. Acetylated YY1 regulates Otx2 expression in anterior neuroectoderm at two cis-sites 90 kb apart.
Embo J 26. 1649-59 (2007)

Hirano M, et al. EPB41L5 functions topost-transcriptionally regulate cadherin
and integrin during epithelial-mesenchymal transition. J Cell Biol 182.
1217-30 (2008)

Kimura-Yoshida C, et al. Crucial rolesof Foxa2 in mouse anterior-posterior
axis polarization via regulation of anterior visceral endoderm-specific genes.
Proc Natl Acad Sci U S A 104. 5919-24(2007)

Shibata M, et al. MicroRNA-9 modulates Cajal-Retzius cell differentiation by
suppressing Foxg1 expression inmouse medial pallium. J Neurosci 28.
10415-21 (2008)

Furushima K, et al. Mouse homologuesof Shisa antagonistic to Wnt and Fgf signalings. Dev Biol 306. 480-92 (2007)

Kurokawa D, et al. Evolutionary constraint on Otx2 neuroectoderm enhancers-deep conservation from skate tomouse and unique divergence in teleost. Proc Natl Acad Sci U S A 103.
19350-5 (2006)

Neuronal Differentiation andRegeneration
http://www.cdb.riken.jp/en/enomoto

Hideki ENOMOTO M.D., Ph. D.

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 of Pediatric Surgery, Matsudo Municipal Hospital in 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 GDNFFamily of
Ligands in neural development and function. He returned to Japan to take a position as a team leader at the CDB in 2002.

The architecture of the neural system, which underlies much of the dynamism of 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 specificextracellular signals and cell autonomous routines that participate in the processes of neural development, and resolving themeans by which diverse families of signaling molecules coordinate and interact at the molecular and cellular levels. To addressthese 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 (for Glial cell line Derived Neurotrophic Factor), Neurturin, Artemin and Persephin. The GFLs signal via receptor complexes formed by the RET receptor tyrosine
kinase and one of four co-receptors, GRFα1-4. In vitro, these four receptors show differential affinities for specific GFLs. GFL
signaling has been shown to affect neuronal growth, survival and migration as well as axon guidance, and defects in this signaling
system have been implicated in a number of congenital health problems.
Our efforts will focus on the analysis of the physiological roles of neurotrophic factors, which we hope will lead to an improved
understanding of the molecular bases of neural development, differentiation and pathology, and ultimately to potential clinicalapplications in the treatment of nervous system trauma and disease.

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

Staff

Publications

Team Leader

Part-Time Staff

Research Scientist

Assistant

Hideki ENOMOTO
Toshihiro UESAKA

Technical Staff

Takae HIRAI
Chihiro NISHIYAMA
Hiroko SONOBE

Junior Research
Associate

Mayumi NAGASHIMADA
Student Trainee

Mitsuhiro IWASAKI
Katsuki YASUDA
Yohei YONEKURA

Toko KONDO

Kaori HAMADA

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 line-derived neurotrophic factor on spinal motoneurons
are restricted to fusimotor subtypes. J
Neurosci 28. 2131-46 (2008)
Uesaka T, et al. Conditional ablation of
GFRalpha1 in postmigratory entericneurons triggers unconventional neuronal death in the colon and causes a
Hirschsprung's disease phenotype.
Development 134. 2171-81 (2007)

Vohra B P, et al. Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration andneuritogenesis. Dev Biol 298. 259-71(2006)
Enomoto H, et al. GFRalpha1 expression in cells lacking RET is dispensablefor organogenesis and nerve regeneration. Neuron 44. 623-36 (2004)
Enomoto H, et al. RET signaling is essential for migration, axonal growth andaxon guidance of developing sympathetic neurons. Development 128.
3963-74 (2001)27

Neocortical Development
http://www.cdb.riken.jp/en/hanashima

Carina HANASHIMA Ph. D.

Carina Hanashima received her Ph. D. from Waseda University School of Science and Engineering 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 wasengaged in developmental neuroscience. She moved to Skirball Institute Developmental Genetics Program in 2002 in Gord Fishell’s laboratory
where she extended her research to cell specification in the neocortex. She was appointed team leader at the CDB in September 2007.

The neocortex, by far the most complex structure of all vertebrate brain systems, is organized into cytoarchitectonic groups ofneurons that process unique sensory inputs such as pressure, light and sound. Nonetheless this highly ordered structure is
generated from a relatively simple sheet of neuroepithelium earlier during development. Research in our laboratory aims to understand how diverse arrays of cortical neurons are specified and coordinated into high-functional territories. Despite its well-defined
anatomical character and functional significance, the mechanisms underlying the precise assembly of distinct functional areasof the cerebral cortex remains largely unknown. Progress has been impeded by our present lack of understanding of themolecular mechanisms by which neuronal subtypes both 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 cell fate is determined in the neocortex? 2) How are neurons precisely arranged into distinct cortical areas and establish
cytoarchitectonic boundaries? 3) To what extent does the refinement of functional areas rely on environmental inputs? To investigate these questions we utilize 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 part predetermined within the progensitor cells. We
have found that specification of deep-layer projection neurons is dependent on transcriptional repressors that function cellautonomously to prevent neurons from acquiring an earlier neuronal phenotype. These results imply cortical intrinsic programs
may be co-opted in which neuron fate is established by temporal changes in gene expression. However, in the mature cortex,
cortical areas differ in their types and numbers of 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 cellularfunctions, recombination mediated cell-lineage tracing, and systematic approaches to identify novel molecules responsible for
precise areal specification. Through these studies we wish to understandthe mechanistic basis by which unique sensory perceptions and functional
circuitries develop in the human neocortex.

Recruitment of late-born neocortical neurons expressing membrane-targeted GFP (green) andnuclear lacZ (red) to the motor cortex.

Staff
Team Leader

Carina HANASHIMA
Research Scientist

Ken-ichi MIZUTANI
Wataru OCHIAI
Technical Staff

Yuko WADA

Student Trainee

Youko YAMAMOTO

28

Publications
Hanashima C, et al. The role of Foxg1and dorsal midline signaling in the generation of Cajal-Retzius subtypes. J
Neurosci 27. 11103-11 (2007)
Hanashima C, et al. Foxg1 suppresses
early cortical cell fate. Science 303.
56-9 (2004)
Hanashima C, et al. Brain factor-1 controls the proliferation and differentiationof neocortical progenitor cells throughindependent mechanisms. J Neurosci
22. 6526-36 (2002)

Morphogenetic Signaling
http://www.cdb.riken.jp/en/hayashi

Shigeo HAYASHI Ph. D.

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

The main research interest in my lab focuses on the mechanisms by which cell-cell and tissue-to-tissue interactions are modulated
during embryonic morphogenesis. Our strategy is to identify intercellular signaling systems and intracellular transducers that
control cell-cell and tissue-tissue interactions. 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.
The Drosophila tracheal system is a respiratory organ that delivers air directly to tissues through a network of finely branchedepithelial tubes. Tracheal development begins by invagination of ectodermal epithelium that subsequently forms branches withspecific types of cells migrating in stereotyped patterns. The branching patterns and cell fate are instructed by external cues,
including FGF, WG and Dpp. We are studying the roles of these signaling molecules in the specification and migration of tracheal
branches, as well as the mechanisms that coordinate cell movement and cell adhesion. We additionally use 4D confocal imaging
of GFP-labeled embryos to study the dynamism of cell and organelle movement in living organisms. Using combinations of
GFP markers and transcriptional enhancers of cell-specific expression, we have been able to capture movements of tracheal
cells at resolutions sufficient to image cytoskeletal organization and cell adhesion structures in single cells.
The development of appendages in Drosophila from primordial regions called imaginal discs is a second area of interest. During
this process, subpopulations of cells in the imaginal discs segregate into distinct domains by coupling cell growth and differentiation to cell sorting, which provides us with an opportunity to study the regulationof cell affinity by positional information. Each limb primordium also coordinates
its specific developmental pattern with other tissues, such as muscles, motor
nerves and trachea, which are specified independently in other parts of theembryo. This understanding of mechanisms of limb specification andproximal-distal axis formation gained from work on Drosophila, however, mustalso be validated by comparative analyses in other species with simpler
appendage structures. We focus on three species for these comparative
studies: the bristletail, Pedetontus unimaculatus, the mayfly, Ephemera
japonica, and the ragworm, Perinereis nuntia.

Homophilic cell adhesion molecules Capricious and Tartan are required for proper placement ofsegmental boundaries in the leg. The boundary of pretarsas and tarsal 5 segment is marked by the
red signal, which normally appears as a ring. When the boundary is flanked by a group of cells
lacking Capricious and Tartan marked by a loss of green signal, the position of the boundary shifts
to the interior and appears staggered.

Staff
Group Director

Shigeo HAYASHI

Research Scientist

Kagayaki KATO
Mayuko NISHIMURA
Nao NIWA
Tatsuhiko NOGUCHI
Tetsuhisa OTANIReiko TAJIRI
Kazunaga TAKIZAWA
Minoru TATENO

Technical Staff

Ai AKIMOTO
Masako KAIDO
Michiko KOIZUMIHousei WADA

Publications
Student Trainee

Ken KAKIHARA
Koji KAWAHASHI
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
AssistantTomoe DEGUCHI

Kato K, et al. Practical guide of live
imagining for developmental biologists.
Develop. Growth Differ. 50:381-390(2008)
Kakihara K, et al. Conversion of plasma
membrane topology during epithelial
tube connection requires Arf-like 3
small GTPase in Drosophila. Mech Dev(2008)

Nishimura M, et al. A wave of EGFR signaling determines cell alignment and intercalation in the Drosophila tracheal
placode. Development 134. 4273-82(2007)
Kondo T, et al. Small peptide regulatorsof actin-based cell morphogenesis encoded by a polycistronic mRNA. Nat
Cell Biol 9. 660-5 (2007)

Shindo M, et al. Dual function of Src in
the maintenance of adherens junctions
during tracheal epithelial morphogenesis. Development 135. 1355-64 (2008)
Sakurai K T, et al. Differential control ofcell affinity required for progression andrefinement of cell boundary during Drosophila leg segmentation. Dev Biol 309.
126-36 (2007)29

Vertebrate Axis Formation
http://www.cdb.riken.jp/en/hibi

Masahiko HIBI M.D., Ph. D.

Masahiko Hibi received his M.D. from Hiroshima University School of Medicine in 1988, and his Ph. D. from Osaka University in 1992. From1992 to 1995, he worked as a postdoctoral fellow in Michael Karin's lab in the University of California, San Diego Department of Pharmacology,
and then returned to Japan as a research associate in the Division of Molecular Oncology at Osaka University Medical School. He wasappointed associate professor in the same division in 1999, where he remained until he assumed his position as team leader at the RIKEN
CDB.

During early vertebrate development, the embryonic dorso-ventral (DV) and anterior-posterior (AP) axes are established througha series of inductive signals that prefigure and determine the concerted movements and differentiation of a group of cells. The
dorsal organizer plays a major role in the process of axis formation. In amphibian and teleost fish embryogenesis, the programfor dorsal organizer formation begins soon after fertilization. In these species, dorsal determinants are believed to be initiallylocalized to the vegetal pole and later transported to the dorsal side of the embryo.
Although the molecular nature of the dorsal determinants has not been elucidated, they are known to activate the canonicalWnt pathway and thereby lead to the expression of genes involved in the induction of the dorsal organizer. The dorsal organizer
generates secreted signaling molecules that participate in the generation of the DV axis in the mesoderm and endoderm, andthe formation of the neuroectoderm. We are working to identify the molecules that are involved in the formation and functionof the dorsal organizer. This search led us to identify the protein Sizzled as a negative feedback regulator of BMP signaling thatcooperates with the dorsal organizer protein Chordin to regulate DV axis formation. We also remain actively interested in determining the molecular identities of dorsal determinants.
Neural patterning and neurogenesis as a model of cell fate determination, a process that is linked to axis formation, is alsoa
question of interest to our team. Neuronal tissues are generated in a stepwise manner in vertebrates; these steps include neural induction, AP patterning and neurogenesis. In amphibian and teleost embryos, neuroectoderm is initially induced by the dorsalorganizer proteins. The induced neuroectoderm is anterior in character and is subsequently subjected to posteriorization andregionalization. Accordingly, the central nervous system becomes highly ordered along the AP axis – regionalized to forebrain,
midbrain, hindbrain and spinal cord compartments in a head-to-tail direction. Our studies have revealed that two groups ofgenes play important roles in the AP patterning of neural tissue. The zinc-finger genes Fezf1 and Fezf2 are expressed in theanterior forebrain and control the AP patterning of forebrain by repressing the caudal forebrain fate, while the caudal-related
genes cdx1a and cdx4 are expressed in the posterior neural tissue and control the formation of posterior spinal cord by repressing
anterior fate. We are also extensively studying the mechanisms that establish the complex structure of the cerebellum.

Development of cerebellar neurons in zebrafish larvae at 5 days post fertilization.
Purkinje neurons (green) receive axons of granule cells (magenta) in wildtype
but do not in one of the mutants isolated by our team.

Staff
Team Leader

Masahiko HIBI
Special Postdoctoral
Researcher

Koji TANABE
Research Scientist

Shuichi KANI
Hideaki NOJIMA
Takashi SHIMIZU
Takeshi SHIMIZU
Technical Staff

Kana BANDO
Setsuko FUJII
Aya KATSUYAMA

30

Publications
Part-Time Staff

Isao HARIMOTO
Yuko HIROSE
Shigemi SHIBUYA
Yoko TAKADO
Kazuko YAMAMOTO

Hirata T, et al. Zinc-finger genes Fez andFez-like function in the establishment of
diencephalon subdivisions. Development 133. 3993-4004 (2006)
Hirata T, et al. Zinc-finger gene Fez inthe olfactory sensory neurons regulates
development of the olfactory bulb
non-cell-autonomously. Development
133. 1433-43 (2006)
Muraoka O, et al. Sizzled controls dorso-ventral polarity by repressing cleavage of the Chordin protein. Nat Cell Biol
8. 329-38 (2006)

Shimizu T, et al. Cdx-Hox code controls
competence for responding to Fgfs andretinoic acid in zebrafish neural tissue.
Development 133. 4709-19 (2006)

Cell Lineage Modulation
http://www.cdb.riken.jp/en/kondo

Toru KONDO Ph. D.

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

Mankind has long wondered whether it might be possible to regenerate body tissues and structures that have been lost. Recent research has shown that even the adult body contains a diverse range of tissue-specific somatic stem cells that serve to maintainthe function and integrity of tissues, opening a promising avenue toward possible applications in regenerative medicine. But
the limits on the number of somatic stem cells present in any individual, coupled with the limited availability of donors, have heightened interest in the development of new, alternative means of generating stem cells.
Research in our lab seeks to develop a better understanding of the molecular mechanisms involved in the reacquisition of “stemness” (stem cell characteristics) using the dedifferentiation of oligodendrocyte progenitor cells (OPCs), which are abundantly
present in the central nervous system, into neural stem-like cells, knowledge which it is hoped may lead to new techniques forthe generation of neural stem cells. We are also interested in two other related subjects: one is the characterization of stem
cell-like cancer cells (sometimes called “cancer stem cells” or “cancer initiating cells”), found in malignant glioma, and to findmarkers and targets for them, potentially leading to the development of novel anti-cancer therapies. The other is to develop abetter understanding of the molecular mechanisms of cellular senescence, which are involved in both the defects of tissue-specificstem cells and the suppression of tumorigenesis.

Morphology of rat C6 glioma cells in different culture conditions, FCS(A), bFGF(B),PDGF(C) or PDGF+bFGF(D).

Staff
Team Leader

Toru KONDO

Research Scientist

Hiromi TAKANAGA
Nobuko TSUCHIDA-STRAETEN

Technical Staff

Keiko MIWA
Yuka NAKATANI

Student Trainee

Yuki KUJURO
Kenji NISHIDE
Tatsuya TAKEZAKI

Assistant

Yukie NAKAMURA

Publications
Takanaga H, et al. Gli2 is a novel regulator of sox2 expression in telencephalic neuroepithelial cells. Stem Cells(In press)
Kondo T. Stem cell-like glioma cells.
Current cancer therapy Review 4. 201205 (2008)
Hide T, et al. Brain tumor stem cells as
research and treatment targets. Brain
Tumor Pathol 25. 67-72 (2008)

Lyssiotis CA, et al. Inhibition of histone
deacetylase activity induces developmental plasticity in oligodendrocyte
progenitor cells. Proc Natl Acad Sci
USA 104. 14982-7 (2007)
Kondo T. Stem cell-like cancer cells incancer cell lines. Cancer Biomarkers 3.
245-50 (2007)

Hampton DW, et al. A potential role forbone morphogenetic protein signallingin glial cell fate determination following
adult CNS injury in vivo. Eur J Neurosci
26. 3024-35 (2007)31

Evolutionary Morphology
http://www.cdb.riken.jp/en/kuratani

Shigeru KURATANI Ph. D.

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

By studying the evolutionary designs of diverse animal species, I hope to gain a deeper insight into the secrets behind the fabrication of morphological designs. Integrating the fields of evolutionary morphology and molecular genetics, our lab seeks to expand
the understanding of the relationship between genome and morphology (or body plan) through investigating the evolutionarychanges in developmental processes, and also the process of evolution in which phenotypic selection shapes developmental
programs. Our recent studies have focused on novel traits found in the vertebrates, such as the jaw, the turtle shell, and the
mammalian middle ear. By analyzing the history of developmental patterns, I hope to open new avenues toward answering
as-yet unresolved questions about phenotypic evolution in vertebrates at the level of body plans.
Through the study of evolutionarily novel structures, our lab has identified changes in developmental mechanisms that haveobliterated the structural homologies between organisms as evidenced in such novel structures as the jaw in gnathostomes(jawed vertebrates) and the turtle shell. Developmental studies of the cranial region of the lamprey are intended to shed light on
the true origins of the vertebrate head and neck, as lampreys lack a number of important features, including jaws, true tongues
and trapezius muscles, that are possessed only by gnathostomes. We aim to resolve the question of what primary factors havechanged during evolution by comparing the developmental patterns that yield 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 theacquisition of a shell.
We have also recently started to study wing pattern evolution in moths in aneffort to understand the relationship between phenotype and development.
By employing morphometric strategies, we seek to identify relationships
between developmental and functional modules in wing patterns, which wehope will lead to molecular developmental searches for genes favored bynatural selection.

Dorsal (left) and ventral (right) views of the carapace (dorsal half of the turtle shell) of Chinese softshelled turtle. The carapace forms from laterally expanded ribs.

Staff
Group Director

Part-Time Staff

Research Scientist

Assistant

Shigeru KURATANI
Shinichi AOTA
Hiroshi NAGASHIMA
Kinya OTA
Takao SUZUKI
Motoki TADA
Masaki TAKECHI
Motoomi YAMAGUCHITechnical Staff

Satoko FUJIMOTO
Yoko TAKIO
Junior Research
Associate

Fumiaki SUGAHARA

32

Publications

Student Trainee

Yasuhiro OISIMaki MIYAUCHI
Mika HIKAWA
Yuko HIROFUJI

Ota K G, et al. Hagfish embryology withreference to the evolution of the neuralcrest. Nature 446. 672-5 (2007)
Nagashima H, et al. On the carapacial
ridge in turtle embryos: its developmental origin, function and the chelonian
body plan. Development 134. 2219-26(2007)
Kuraku S, et al. Comprehensive surveyof carapacial ridge-specific genes inturtle implies co-option of some regulatory genes in carapace evolution. EvolDev 7. 3-17 (2005)

Takio Y, et al. Evolutionary biology: lamprey Hox genes and the evolution ofjaws. Nature 429. 1 p following 262(2004)
Shigetani Y, et al. Heterotopic shift ofepithelial-mesenchymal interactions invertebrate jaw evolution. Science 296.
1316-9 (2002)

Sensory Development
http://www.cdb.riken.jp/en/ladher

Raj LADHER Ph. D.

Raj Ladher received his B. Sc. in biochemistry from 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 first came to Japan during that period as a visiting
scientist at the Tokyo Medical and Dental University. In 2000, he moved to the University of Utah as a postdoctoral fellow in Gary Schoenwolf'slaboratory, and was appointed team leader at the CDB in 2002.

Organogenesis is a monumental undertaking. From siting a given organ to its correct location, to confirming that it is correctly
engineered and constructed, to ensuring the necessary functional connections with other organs, the embryo has a daunting
task before it; indeed, perhaps we should be surprised that the result 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 thesteps 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 of tissue originally destined to become skin; a fate that is altered by instructive signals in a process known
as induction. In our research to date, we have identified the mechanism and the signals that set this early tissue on its path
toward an inner ear fate as well as those that determine its position. We next hope to learn how these signals are themselves
established. We are also investigating the 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 channeledthrough the external ear, as well as specialized regions within the brain to interpret the information it sends. But how is thisintricate integration achieved? Using a combination of molecular and embryological techniques, we are characterizing thesystems 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 other organs, sensory organs in particular. By working to comprehend the induction and developmentof other sense organs, such as the olfactory system andthe eye, we are now taking steps toward achieving insights
into common mechanisms underpinning the developmentof organ systems.

The chick middle ear is made of one bone, the columella. Alcian blue highlights the cartilage of the columella. On the left is a control columella, theright columella is from an anotic embryo.

Staff
Team Leader

Rajesh LADHER

Research Scientist

XiaoRei CAIAkira HONDA
Tomoko KITA
Yuko MUTA
Marie PASCHAKI
Vassil VASSILEV

Visiting Scientist

Sabine FRETER
Siu Shan MAKPaul O'NEILL

Part-Time Staff

Yoshiko KONDO
Yoko MATSUOKA

Assistant

Noriko HIROI

Publications
Freter S, et al. Progressive restriction ofotic fate: the role of FGF and Wnt in resolving inner ear potential. Development 135. 3415-24 (2008)
Sai X and Ladher R K. FGF signaling
regulates cytoskeletal remodeling
during epithelial morphogenesis. Curr
Biol 18. 976-81 (2008)
Manley G A and Ladher R K. Phylogeny
and Evolution of Ciliated Mechanoreceptors. The Senses: A Comprehensive Reference; Audition, vol. 3 (ed. P.
Dallos and D. Oertel), pp. 1-34. San
Diego: Academic Press.(2008)
Sakamoto T, et al. Hair cell differentiation becomes tissue specific by E9.5 inmouse inner ear. Neuroreport 18.
841-4 (2007)

Ladher R K, et al. FGF8 initiates inner
ear induction in chick and mouse.
Genes Dev 19. 603-13 (2005)
Ladher R K and Schoenwolf G C.
Making a Neural Tube: neural induction
and neurulation. Developmental Neurobiology, (ed. M. H. Rao): Kluwer.
(2005).
Wright T J, Ladher R K, et al. Mouse
FGF15 is the ortholog of human andchick FGF19, but is not uniquely required for otic induction. Dev Biol 269.
264-75 (2004)

33

Shroom3 recruits ROCKs
to the apical junction

Tamako NISHIMURA

Epithelial tissues are made up of highly ordered sheets of cells, that can stretch, squeeze, bend and roll
into all manner of bulges, concavities and tubes. But for essentially two-dimensional sheets to take onthese three-dimensional appearances requires changes in shape at the level of individual cells. This requires
not only the regulation of morphological events within a given cell, but precise coordination across themany cells that make up the tissue, in order to assure that the process plays out true to the developmental
plan. The invagination and closure of the neural tube, in which a sheet of cells destined to become thecentral nervous system forms a depression whose edges fold over to form a hollow cylinder, is one of thebest-studied instances of such structural remodeling, but the molecular mechanisms that underlie thisprocess remain incompletely understood.
A study this year conducted by Tamako Nishimura of the Laboratory for Cell Adhesion and Tissue Patterning(Masatoshi Takeichi; Group Director) identifies the role of one player in neural tube morphogenesis, theactin-binding protein Shroom3. In a report published in Development, Nishimura showed that this protein
binds certain Rho kinases, recruiting them to the apical cell junction, a site at which uppermost lateralsurfaces of epithelial cells connect, and which serves as a hinge and anchor point for much tissue remodelingactivity.
Shroom3 was originally identified as the culprit in a mouse mutant whose phenotype was characterized
by incomplete neural tube closure (the name deriving from the mushroom-like appearance of the mutant
embryo’s head). Gene expression studies had shown that it is expressed strongly in the neural epithelium,34

©2008 The Company of Biologists

Nishimura T and Takeichi M. Shroom3-mediated recruitment of
Rho kinases to the apical cell junctions regulates epithelial andneuroepithelial planar remodeling. Development 135. 1493-502(2008)

but its mechanism of action remained a mystery. To address this question, Nishimura first searched forbinding partners, and found that a number of Rho kinases (known as ROCKs) associated with Shroom3
in immunoprecipitation assays. In vitro tests further revealed that Shroom3 and ROCKs co-localized at theapical cell junction, an effect that could be blocked by interfering with their binding activity.
Looking next in vivo, Nishimura studied the Shroom3-ROCK association in embryonic chicken, andconfirmed that the two were concentrated in the apical junctions of neuroepithelial cells lining the neuraltube. Blocking Shroom3 by RNAi resulted in the failure of ROCKs to localize to the apical junction, and consequent defects in neural tube closure. When she watched the interactions between cells in the closing neuraltube, Nishimura observed assemblies of 5 or 6 cells into rosette patterns, suggesting coordinated contraction of the cell shapes, presumably dependent on actomyosin, which is known to be regulated by ROCKs.
Such rosettes, again, were lost on ROCK inhibition.
The picture that emerges from Nishimura’s findings suggests that Shroom3 recruits ROCKs to the apicaljunction, where they interact with myosin and actin cytoskeletal components to give shape to the forming
neural tube. “There are still many unknowns in terms of the mechanisms behind changes in cell shape,such as those that occur in the neural tube,” says Nishimura. “I think the Shroom/ROCK complex is particularly interesting in this context, for the way it localizes in a polarized way to only one aspect of the cells andthus contributes to the sculpting of the entire tissue.”ROCK-lMergedShrm-△ASD2

Shrm-Full

Shrm

Co-localization of Shroom3
and ROCK at apical junctions (middle row) andloss of ROCK localizationin Shroom3 mutant cells(bottom row). Note the lossof tension in the mutant cell
membranes.

35

Zeroing in on beta globins in
the early embryo

Our red blood cells, or erythrocytes, act as vehicle for conveying oxygen from the respiratory system to the cells of the body, a feat they
achieve thanks to the ornate structure of hemoglobin, a molecule comprising alpha andbeta globin subunits and an iron-based heme.
There are multiple types of both alpha andbeta globin, and different combinations ofthese types result in differing degrees of affinity
for oxygen, which has implications in both embryonic development and adult physiology. In
the chicken, the three subtypes of alphaglobin (?, αD and αA) and the four beta globins(ρ, βH, βA and ε) show different spatiotemporal patterns of expression during the formationof the red blood system during embryogenesis, a process that plays out in two waves –an initial phase of primitive extraembryonicerythropoiesis, followed by a second wavethat takes place within the embryo. This transition is characterized by a shift from the embryonic beta globins (ρ and ε) to βA, the major
adult beta globin. But due to technical limitations, attempts to study the details of this sequence of events have yielded somewhat
conflicting results.
In a study published in Developmental Dynamics, Cantas Alev and colleagues in the
Laboratory for Early Embryogenesis (Guojun
Sheng; Team Leader) and other CDB labs
have shown through protein analyses that β
A is expressed even in the early stage of primitive erythropoiesis in the chicken.
Previous reports using classical embryology
and molecular biology had suggested that
there was a clear separation between primitive (embryonic) and definitive (adult) beta globins, and that the transition between these
two stages occurred as early as embryonicday 4 (E4) or as late as day 13. Analysis ofmRNA transcripts, in particular, had indicateda switching on and rapid increase of βA expression in the period between days 5 and 7of embryogenesis.
Alev et al., however, took a new approach tothe question, first using mass spectrometry
to sort out and quantify each of the globinsfrom samples of embryonic blood. In contrast
Cantas ALEV

36

©2008 Wiley-Liss,Inc

Alev C, et al. BetaA, the major beta globin in definitive red blood
cells, is present from the onset of primitive erythropoiesis inchicken. Dev Dyn 237. 1193-7 (2008)

to previous reports indicating that βA expression is first detectable at around E2(stage HH13 in the Hamburger-Hamilton
system for evaluating chicken development), the team found that it is present as
early as two days earlier, coincident with theprimitive wave of blood development.
Looking even earlier, they found signs of β
A expression as early as HH8, with significant transcription by HH9. Interestingly,
even at this early stage of development, thelevel of the purportedly “adult” beta globin
βA was seen concurrent with that of the“embryonic” ρ subtype (although at a much
lower level). In situ hybridization further
showed that, by HH10~11, βA beta globin
is expressed in the extraembryonic mesoderm as well.

Figure 1

Figure 1
Each of the four beta globins
has unique tryptic digestion
pattern that can be used formass-spectrometry based
semi-quantitative assessment of its relative abundance.

Figure 2

The findings from this multi-technique strategy are in general agreement with thescheme in which the transition from primitive (ρ and ε) to definitive (βA) beta globins
occurs in the period of E5~7, but pushes
back the start point for βA expression to theearliest stage of blood formation, suggesting that the distribution of the various subtypes may be a function of relative abundances rather than strict transcriptional
control. “We still don’t know what βA is
doing in the primitive stage of hematopoiesis,” admits Alev. “But it’s interesting thatthe other adult beta globin, βH, is completely undetectable at that stage, so we’dlike to try to work out how to account forthese differences in the regulation of transcription.”

Figure 2
Expression of betaA in primitive erythrocytes revealed
by intron-specific in situ hybridization.

37

Subtraction a plus for monoclonal
antibody research

Antibodies have become an indispensable
component of the experimental repertory inmany life sciences laboratories, used insuch techniques as immunohistochemistry,
Western blotting and immunoprecipitation. It
remains a challenge, however, to isolate
antibodies of precisely the right affinity for
antigens called for under a given experimental
design. A method developed by Kazumasa
Takeda and others in the Laboratory for
Developmental Genomics (Asako Sugimoto;Team Leader) now looks set to lower thebarrier to the creation of monoclonal antibodies to low-abundance antigens. In a proofof-concept demonstration published in
Genes to Cells, of this “antigen subtraction”
approach, the team was able to isolate morethan 30 monoclonal antibodies specific tostructures in the C. elegans embryo, including
P granules (specifically present in cells of thegermline), muscle, pharynx and hypodermis,
as well as previously unreported cellular
structures. Importantly, this method can beapplied to other species as well, which willallow researchers in other labs to develop new,highly specific antibody probes for cells andtissues at specific developmental stages.
The project traces its roots back more than
20 years, to when Takeda was using monoclonal antibodies as part of a virus research
project at Kyoto University. On reading areview on apoptosis and roundworm germline
development, he wondered whether he might
be able to apply the techniques he’d beenusing in virology to develop antibodies for
specific C. elegans structures as well, andquietly began to experiment. In the late1980s, he generated and screened morethan 1,300 hybridomas (cancer cellengineered to produce a single species ofantibody). “I had to be a bit careful when I
was working on these,” admits Takeda, “asthey weren’t really part of the research
program of the lab I was working in at thetime.”
Despite his intensive initial efforts, Takeda
was frustrated to find that many of his
antibodies were not specific to any single
structure in immunostaining, and tended toChie WATANABE, Kazumasa TAKEDA

38

©2008 John Wiley & Sons, Inc.

Takeda K, et al. Efficient production of monoclonal antibodies
recognizing specific structures in Caenorhabditis elegans
embryos using an antigen subtraction method. Genes Cells
13, 653-665 (2008)

stain the entire C. elegans embryo. Reasoning that the solution would lie in finding a way to sift out nonspecific antibodies, he devised a clever two-stage system in which first antibodies against abundant andnon-specific antigens are pooled and then used to subtract out the corresponding antigens from the embryonic extracts. The mixture that remained after this subtraction should then be enriched with less abundant
proteins, which could then be used for a second round of immunization, hopefully yielding targets capableof marking specific cell or tissue types. Takeda found that in the first round of immunization, only about
3% of monoclonal antibodies showed a specific pattern in immunostaining tests; the rest generally stained
the entire embryo. But after subtraction, almost 50% produced signals in specific structures or cell types.
Takeda had to discontinue this project for various reasons, but in 2003, after retiring from Kyoto University,
he emailed Sugimoto to ask her if she would be interested in his collection of hybridomas and monoclonal antibodies, which led to an offer to join her lab and resume his work on an official basis. Takeda and Chie
Watanabe, a technician in the lab, then developed a collection of 35 monoclonal antibodies for a range ofstructures including P granules, body wall muscle, hypodermal cells, and pharynx. This antibody libraryhas already been used to produce images by immunostaining of previously unknown histological andcellular structures, which can be viewed on the Sugimoto lab homepage: http://cdb.riken.jp/dge. The lab
has already received more than 30 requests for antibodies from labs around the world, and is pleased tomake them available to the global C. elegans research community. Within the lab, the antibodies haveopened up new direction for research and are now being used in analyses of the structure and assembly
mechanism of P granules.
“The C. elegans research community has always been based on the open sharing of information andfindings,” says Sugimoto. “So we have made the entire database accessible to everyone in the field, in thehopes of distributing these antibodies as widely as possible.”

Figure 1

Figure 2

Figure 1
Images showing immunostaining using antibodies
generated by antigen subtraction.
Figure 2
A schematic of the antigen
subtraction method.

39

Novel ectodermal factor blocks
mesodermal fate

Rieko YAKURA-NISHIZAWA, Noriaki SASAI

The development of the embryo is a tale of either/or gateways and roads not taken; decisions made at an
early stage in development can put constraints on all those made subsequently. The same is often true ofthe induction of differentiation of pluripotent cells in culture, which requires step-by-step instructions to guidecells to adopt a terminally differentiated fate. For this reason, the identification of those early signals thatsteer undifferentiated cells toward a specific germ layer is of fundamental interest to scientists working bothin vivo and in vitro. The African clawed frog, Xenopus laevis, provides an excellent model for studying
questions of early vertebrate development through analyses of a pluripotent region of the nascent embryo
known as the animal cap, which is the site of in vivo germ layer specification and is amenable to tissue
culture as well. That notwithstanding, there have been no reports of a zygotic factor able to regulate one of
the earliest lineage decisions during gastrulation, the choice between ectodermal and mesodermal fates.
That changed this year, when Noriaki Sasai and colleagues in the Laboratory for Organogenesis and Neurogenesis (Yoshiki Sasai; Group Director) identified a novel ectodermal factor that prevents mesodermal
differentiation in both Xenopus animal cap and mouse embryonic stem cells in a report published in Cell.
This factor acts by an unsuspected molecular mechanism, the inhibition of p53, a gene best known for its
role in genomic remodeling and cancer.
Having observed that mRNAs from stage 11.5 animal caps (about a half-day into development) suppressed
mesodermal differentiation, the group screened a library of cDNAs to try to pinpoint this inhibitory function.
“There were already a number of reports of maternal lineage determinants,” says Sasai, “but the fact thatthese were residual proteins that had been expressed in the egg, rather than newly in the embryo, madeit unlikely that they could account for fate specification during gastrulation.” On completing the screen, five
molecules showed mesoderm-inhibiting activity, and as four were already relatively well-characterized, they
narrowed their focus to the fifth, a gene encoding zinc-finger protein known as XFDL156.

40

©2008 Cell Press

Sasai N, et al. Ectodermal factor restricts mesoderm differentiation by inhibiting p53. Cell 133. 878-90 (2008)

Sasai first tried overexpression experiments in animal caps that had been induced toward the mesodermal
lineage, and found that XFDL suppressed mesodermal, but not neural or endodermal, markers. The
converse experiment, in which XFDL function was blocked by antisense morpholinos, resulted in mesodermal expansion, suggesting that this factor suppresses mesodermal differentiation in the ectoderm. The
question was, how?
As recent studies have shown a role for the transcription factor p53 in mesodermal differentiation, Sasai
looked for a possible functional relationship between these two proteins in animal cap assays. After confirming by a double knockdown that its function was dependent on p53, he next tested the effect of XFDL on
p53 (and its co-factor p300) and determined that it effectively inhibited p53’s ability both to activate the mesoderm marker gene, Mix.2 and to promote apoptosis in the animal cap. Immunoprecipitation showed thatXFDL and p53 co-precipitated, indicating a direct interaction between the two. Further analyses, by gel
shift assay and chromatin immunoprecipitation, revealed that XFDL-bound p53 fails to bind to its target
genes.

An
An

control

Veg

VegT MO

Veg

VegT

Expression of mesodermal marker (VegT, shownby arrowheads) limited topresumptive mesoderm inwildtype embryo (left) andexpanded throughout presumptive ectoderm region
on XFDL156 knockdown(right).

Investigating the structural underpinnings of this interaction, Sasai et al. next made a number of p53
constructs engineered to lack various protein domains, allowing them to identify a regulatory domain in
the carboxyl-terminal region as the sequence necessary for XFDL binding. Interestingly, a search in othergenomes for related genes turned up a pair of zinc-finger protein encoding genes in the mouse that shared
the ability to inhibit mesodermal differentiation and to interfere with p53, suggesting that mammalian ES
cells may use a mechanism for ectodermal differentiation similar to that in the frog.
“With these findings. I think we have managed to add a piece that has long been missing from the puzzleof early development from undifferentiated, pluripotent cells,” says Yoshiki Sasai. “We’re hopeful that thiswill take us one step closer to the day when we will be able to control every major step in the process ofdifferentiation from ES cell to neuron.”41

Improved model yields new insights into
Hirschsprung’s disease

The long tract of the gut is innervated by a
network of peripheral neurons that control,
among other functions, the peristaltic actionthat moves food through the lower half of thealimentary canal. In Hirschsprung’s disease,a congenital disorder, neurons fail to develop
in the large intestine, which can lead to symptoms ranging in severity from chronic constipation to total blockage and enlargement of
the colon, which can be life-threatening. The
primary genetic cause of Hirschsprung’s has
been linked to mutations in the Ret gene.
Recent evidence has also shown that thebinding of the protein GDNF (glial cell linederived neurotrophic factor) to its receptor
GFRα1 activates RET, which is necessary forthe survival of enteric neurons, leading to thequestion of precisely how Ret regulates
neuronal survival and death in the developing
colon, particularly with reference toHirschsprung’s disease.
Toshihiro Uesaka and colleagues in the
Laboratory for Neuronal Differentiation andRegeneration (Hideki Enomoto; Team Leader)have developed a mouse model ofHirschsprung’s in which Ret gene expression
levels can be reduced conditionally, allowing
them to study the gene’s involvement in an
animal model with greater precision than everbefore. This work was published in The Journalof Clinical Investigation.
Uesaka had previously shown that loss of
GFRα1 function at a specific stage late inmouse development caused a near-total lossof innervation in the large intestine. “In theconditional GFRα1 mouse, we found analmost complete loss of the neuronal networkin the lower intestine due to apoptotic cell
death, a phenotype that closely resembles
Hirschsprung’s disease,” says Uesaka. “Asthis disease is characterized by mutations in
the Ret gene, we suspected that apoptosismight be involved there as well.”In mice in which GDNF, GFRα1 or Ret are
ablated unconditionally, the enteric nervous
system fails to develop beyond the stomach.
In the sense that it produces a loss of theenteric neural network, this phenotype could
Toshihiro UESAKA

42

©2008, The American Society for Clinical Investigation

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)

be said to resemble Hirschsprung’s disease, but in fact such extreme cases are rare, and many patientsshow only a loss of neurons in the colon. Previous reports have also shown that Ret-mutant mice exhibit
abnormal kidney and motor neuron development as well, which is not seen in Hirschsprung’s, indicating
the need for a more sophisticated experimental model.
“By designing a system for conditionally inactivating Ret at a specific developmental stage in the mouse
fetus, we were able to confirm that, just as with GFRα1, the loss of Ret function at a specific developmental
stage results in a reduction in the innervation of the colon,” comments Uesaka. “We knew that in humanHirschsprung’s patients there’s a mutation in the Ret gene of a region that regulates its expression level,
so we next looked at a series of mutant mice in which Ret expression levels were reduced to various
extents.”
Mice in which Ret expression was reduced to 50% of the wildtype level were asymptomatic, but in adifferent mutant in which levels were 30% of wildtype, colonization of the gut by enteric neural crest-derived
cells initially appeared to play out as normal, but these cells subsequently disappeared. This phenotype
occurred in about half of all the animals, in a gender distribution reminiscent of that in Hirschsprung’s. The
phenotype was limited to gut innervation without any of the other defects, such as kidney and motoneuron
anomalies, that are seen in unconditional Ret knockouts. And, interestingly, the gender distribution showeda slight male bias, consistent with the tendency in the Hirschsprung’s patient population.
Uesaka next looked into the mechanism by which low levels of Ret expression might bring about such neuronal loss, and found that in the mutants neuronal migration into the colon is delayed. Reductions in Ret
induced at later developmental stages also caused a loss of colonic innervation, suggesting that lower Ret
levels may both interfere with the survival and maintenance of enteric neurons and trigger the programmed
cell death that underlies Hirschsprung’s pathogenesis.
“It had previously been thought that Hirschsprung’s disease was caused by a defect in cell migration, notby cell death,” says Uesaka. “But our findings suggest that insufficient supply of a survival signal is at fault.
I am hoping to look more closely at the underlying mechanisms in the future to contribute to a better understanding of this disease.”

Reduced expression of Ret
causes aganglionosis in the
distal colon in vivo.

43

Unsuspected neuronal specificity in
GDNF function

Neurons are born with an appetite for molecular factors that regulate multiple aspects of
their development and function, and in manycases are necessary for their very survival.
One such neurotrophic factor, GDNF (glial
cell-line derived neurotrophic factor), is
known to play important roles in gene expression, targeting, survival, and synapse pruningin spinal motor neurons, which innervate
skeletal muscles and convey efferent signals
from the brain. Skeletal muscle comprises
different kinds of muscle fibers – the extrafusal
fibers that contract and extend to generatetension, and the intrafusal fibers, which aresheathed in sensory receptors that detect the
speed and length of a muscle as it isstretched. Mice lacking GDNF or its receptors
are known to suffer motor neuronal deficits,
but it has never been determined whether thisis a general effect, or specific to a given
subpopulation.

Thomas (and Margot ) GOULD

Research by Thomas Gould of the Laboratory
for Neuronal Differentiation and Regeneration(Hideki Enomoto; Team Leader) reveals that,somewhat surprisingly, loss of the function of
GDNF or its receptors affects only the gamma
motor neurons, which innervate intrafusal
fibers. The study, published in The Journal of
Neuroscience, further highlighted the exquisite specificity of GDNF signaling function by
showing that the period in which GDNF isneeded for motor neuron survival is limited to
the first few days after birth.
In starting their work, Gould et al. were confronted by a number of technical challenges in teasing out thedetails of GDNF function, the thorniest of which is that mice entirely lacking GNDF function die at aroundthe time of birth due to dysfunctions in kidney and gut. “The enormous repertoire of mice in the lab madethis sort of thorough analysis possible,” says Gould. “”Our conditional mutant mice permit the deletion of
GDNF receptors in specific tissue types such as motor neurons, which avoids the lethal phenotype causedby constitutive deletion and thus facilitates the investigation of the role of GDNF signaling in specific regions
or cell types.”
The difficulty of visualizing and counting motor neurons presented a second hurdle to the team, which it
met by developing a new labeling method which they used first to verify the normal number of lumbar
motor neurons in wildtype and GDNF-knockout newborn mice. When they looked at different hindlimb
muscles, they found that in most muscles innervation was normal in both wildtype and mutant. Only threemuscle types – peroneal, gluteus maximus, and iliopsoas – showed deficits, consistent with previous
findings showing a role for GDNF in peroneal axon guidance. Intriguingly, however, in the occasional GDNF
ligand-receptor knockouts in which peroneal innervation remains normal, the mice still lacked greaternumbers of neurons that could be accounted for by deficits in the other two groups alone.

44

©2008 The Society for Neuroscience

Gould T W, et al. The neurotrophic effects of glial cell line-derived
neurotrophic factor on spinal motoneurons are restricted tofusimotor subtypes. J Neurosci 28. 2131-46 (2008)

This prompted Gould to look at the possibility that rather than those innervating a specific muscle group,
motor neurons projecting to a specific muscle fiber type might be affected by loss of GDNF signal. Indeed,
while extrafusal skeletomotor neurons (also known as alpha motor neurons) were completely unaffectedin knockouts of Ret (a vital component of the GDNF receptor complex), this same mutation caused near-total
reduction in the number of fusimotor (or gamma) neurons. Similar fusimotor deficits were seen in knockouts
of GDNF and GFRα1 (the other half of the Ret receptor complex). Observation of myelinated axons in motor
neuron-specific Ret and GFRα1 conditional knockouts confirmed that this deficit was confined to smalldiameter, gamma motor neurons. Finally, the analysis of mice expressing the fluorescent reporter CFP in
one or both Ret loci (Ret-CFP knockin mice) revealed that the reductions in gamma motor neuron counts
result at least partially from programmed cell death during initial innervation of intrafusal muscle fibers.
The team also crossed mice with conditional mutations in either Ret or GFRα1 to those expressing atamoxifen-inducible Cre to study the requirement for GDNF at different points in fetal and postnatal development, and found that gamma motor neurons depend on the GDNF pathway only during the late developmental period during which cell death occurs.
“In addition to the biology, what we found has some interesting implications for medicine,” says Gould. “In
ALS (amyotrophic lateral sclerosis), a fatal degenerative disease characterized by the selective loss of alphamotor neurons, for example, it had been thought that the loss of these neurons might be an instance ofdysregulation of GDNF signaling. But our findings show that it is the gamma neurons, rather than the alphaneurons, that appear most sensitive to the trophic actions of GDNF, which raises questions about theclinical relevance of this pathway in the pathogenesis of ALS.”

Top Row, Sections of the
fourth lumbar ventral root
stained with toluidine blue
from P42 mice reveal a lossof small- but not large-diameter motor axons in motor
neuron-specific Ret and
GFRa1knockouts. BottomRow, In contrast to those of
WT mice (left panel), intrafusal muscle fibers detected by parvalbumin-immunoreactive sensory nerve
terminals (red) fail to receive
motor innervation from g
MNs (GFP immunoreactivity, green) in P0 Ret knockout mice (right panel). In
contrast, extrafusal muscle
fibers (detected by the presence of nAChR clusters,blue) receive normal motor
innervation from a MNs.

45

Untangling the roots of the germline

Kazuki KURIMOTO

Evolution has devised two ways for the germline to arise during development: through preformation, in
which germ cells are determined by maternal factors segregated into a few cells from the very beginningof development, and epigenesis, in which signals delivered by the embryo itself instruct undifferentiatedcells to adopt germline fate. Several popular model organisms, including C. elegans and Drosophila, takethe preformation route, but most species, including mammals such as human and mouse, feature epigenetic
germline development. So the question of how genetic signals induce the first few germ cells is one of fundamental importance and interest.
A genome-wide study of transcriptional dynamics conducted by Kazuki Kurimoto and others in the Laboratory for Mammalian Germ Cell Biology (Mitinori Saitou; Team Leader) has revealed how a complex networkorchestrated by the regulatory factor Blimp1 guides the specification of primordial germ cells (PGCs) in themouse. This work, which brought a sophisticated single-cell microarray and PCR analytical approach tobear on the question of what sets these cells apart from their somatic neighbors early in mammalian development. The article was published in the journal Genes and Development.
“The entire mouse germline arises from a small group of about 40 cells quite early in the embryo’s development,” says Kurimoto, “which meant we had to be able to analyze very small samples. The single-cell
technology developed in our lab a few years ago was essential to making that possible.” The study, whichlooked at transcriptional dynamics and gene interactions across the entire genome in both wildtype embryos
and those carrying mutations in the definitive PGC marker Blimp1 (also known as Prdm1), represents thefirst such comprehensive study in any mammalian cell lineage.
The team began by comparing gene expression patterns in cells taken from embryos at 6 different stagesof development between day E6.25, when the germline is first specified, and 8.25, when the somites begin
to appear and germline commitment is complete. One mainstream theory of germline specification has itthat Blimp1 acts to repress “somatic” genetic programs, such as those enacted by Hox genes (represented46©2008, Cold Spring Harbor Laboratory Press

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

here by Hoxb1). The picture that emerged from this close analysis, however, was a bit more complex:
undifferentiated cells (marked by the gene Sox2) that become specified by Blimp1 for germline fate firsttransiently express Hoxb1 at levels similar to those in the surrounding soma before ultimately repressing
this and other somatic genes.
Comparison of genome-wide expression patterns in wildtype and Blimp1-mutant embryos also shed lighton the dynamics and functions of specification and somatic genes in the germline anlage. Among thehighlights of the spectrum of new insights were the findings that nearly all genes relating to the establishment
or maintenance of DNA methylation are repressed in PGCs, and that germline specification occurs withouta requirement for epithelial-mesenchymal transitions. This comprehensive trove of gene expression profilesof cells undergoing PGC specification now allows a more detailed understanding of what distinguishes thenascent germ lineage from its somatic neighbors at the genome-wide scale.
Kurimoto’s analysis of loss-of-Blimp1-function embryos yielded even more intriguing gold, with the revelation that while this gene certainly plays a master role, it apparently does not work alone. While Blimp1 wasessential for the repression of almost all of the genes normally downregulated in PGCs, the transcriptionof many genes that had been presumed to be under the sole control of Blimp1 was unaffected in the
mutants, suggesting that other factors must also play a part. “What we saw was that some genes specificto the germline would switch on to some extent even if Blimp1 wasn’t working,” says Kurimoto. “That said,in the mutant, the individual cells still couldn’t coordinate all the many genes required. It seems that Blimp1
functions something like the conductor of an enormous orchestra, making sure that everything plays according to the score.”

Differentiation of PGCs atdifferent stages of early development.

47

Breaking away: A new role for RhoA in EMT

Yukiko NAKAYA

The cells of the embryo can be classed roughly by their structures into epithelial and mesenchymal types;
epithelium is characterized by its more rigid borders, clear apical-basal polarity, and well-defined physicaljunctions with other cells and the extracellular basement membrane, while mesenchymal cells show noneof these interactions and have more free-form morphologies. These cell types, however distinct, are notpermanent, and switches from epithelium to mesenchyme or vice-versa occur in many developmental
contexts, a phenomenon known generally as epithelial-mesenchymal transition (EMT) or mesenchymalepithelial transition (MET). Given the frequency and importance of such events, they have been widely
studied in cultured cells, but due to the technical challenges it presents, the regulation of EMT in vivo has
yet to be adequately investigated.
Working with chicken embryos, Yukiko Nakaya and others in the Laboratory for Early Embryogenesis(Guojun Sheng; Team Leader) have shown that EMT during gastrulation plays out as a serial, stepwise
process, beginning with the breakdown of the basement membrane. In an article published in Nature Cell
Biology, Nakaya reports that this initial stage in the epithelial-mesenchymal transition is controlled by thedownregulation of the factor RhoA, a small Rho GTPase known to regulate cytoskeletal rearrangements.
Gastrulation is a process that takes place during early development in which, through a series of tissue movements, the early mammalian embryo transforms itself from an almost featureless cylinder to a rudimentary
but recognizable body composed of three germ layers: ectoderm, mesoderm and endoderm. In the chicken,
epithelial cells from a structure known as the epiblast ingress to form mesoderm (the germ layer that gives
rise to tissues including heart, blood, bone and muscle), requiring them to undergo EMT in which theydissolve their attachments to neighboring cells and the underlying extracellular substrate.

48

©2008 Nature Publishing Group

Nakaya Y, et al. RhoA and microtubule dynamics control cellbasement membrane interaction in EMT during gastrulation.
Nat Cell Biol (2008)

Cross –section of a gastrulation-stage chicken embryowith cell nuclei stained blue,
RhoA in red and the basement membrane (marked
by laminin) in green. Loss of
RhoA coincides with breakdown of the basement membrane.

49

How cells go pear-shaped

The organs of the body are born from simple
sheets of cells that later take on their elaborate and complex forms during development,a process known as morphogenesis. It is
known that such developmental processes
involve changes at the level of individual cells,
but what influences groups of cells to collectively change shape in a coordinated fashion
remains largely a mystery.
The inner ear, which originates as a flat patchof epithelium that subsequently invaginatesand pinches off to form a hollow cyst,
provides an excellent system for the study ofsuch organogenesis. In a study published in
Current Biology, XiaoRei Sai and Raj Ladherof the Laboratory for Sensory Development(Raj Ladher; Team Leader) have revealed therole of extrinsic signaling in the morphogenesis of the inner ear in the chicken embryo.
Through a series of experiments involving
chemical inhibition of signaling pathway
components, they showed that, following apreliminary induction phase, FGF signalingfrom the underlying mesoderm instructs thecells of the ectoderm that are destined to
become the inner ear to expand basally,
contributing to the transformation of columnar epithelial cells with parallel lateral sides
into more trapezoidal forms, like voussoirs in
an arch. This allows the cells to invaginate
and form a concavity that ultimately pinches
off to form the otocyst.
“We knew that morphogenesis was one of thefirst events after inner ear induction, which iscontrolled by a pair of FGF molecules, so wewere curious as to whether FGFs played a rolein this as well,” says Ladher.
Sai and Ladher first confirmed that FGF was
required for the morphogenetic stage of innerear development by removing ectoderm that
had been induced to become inner ear fromembryos at different developmental
timepoints. They found that even after induction, the tissue needed to remain in contact
with its normal surroundings in order formorphogenesis to occur, suggesting that one
or more external signals is required. Using
microscopic beads soaked in the FGF factors
XiaoRei SAI

50

©2008 Cell Press

Sai X and Ladher R K. FGF signaling regulates cytoskeletal
remodeling during epithelial morphogenesis. Curr Biol 18.
976-81 (2008)

known to induce the inner ear, they determined that FGF signaling to the basal aspect of the prospective
inner ear was necessary to its subsequent morphogenesis.
Knowing that changes in cell shape are often the result of cytoskeletal rearrangement, the team next lookedat microtubules and actin during the morphogenetic phase, and noted that while microtubules appeared
not to change, actin filaments gradually became depleted from the basal aspect of the ectoderm in a mannersuggesting that a local change in actin polymerization (rather than its localization) was responsible.
In looking to connect FGF signaling with the change in actin dynamics, they homed in on one of two known
FGF signaling pathways, involving the phosphorylation of phospholipase Cγ (PLCγ), which they had linked
to basal FGF signaling through inhibitionof PLCγ and another downstream factor,
protein kinase C (PKC). Suspecting thatthe mechanism responsible for the basal
expansion of ectodermal cells was actin
depolymerization, they first surveyed forwell-known actin depolymerization
factors, but were unable to detect them inthe basal otic ectoderm. They turned next
to myosin II, a factor that typically mediates
the contraction of actin filaments, butwhich had recently been shown to play apart in actin clearance and turnover as
well.
Activation of non-muscle myosin II relieson the phosphorylation of a regulatory
region known as the myosin light chain(MLC), and when the team used an
antibody specific to phosphorylated lightchain, they found it localized to the basalside of the otic tissue. Inhibition of myosin
II activity also prevented invagination,suggesting that its activity is needed formorphogenesis of the inner ear.
With strong evidence that the FGF–PLCγ
cascade and activated myosin light chain
are both involved in the basal expansionof otic ectoderm during its morphogenesis, they sought to link them by studying the effects of inhibitionof upstream factors on MLC phosphorylation, and found that it could be blocked by the inhibition of either
FGF or PLCγ. Using a general inhibitor of protein synthesis, they further showed that this activation of
myosin II was a direct effect of FGF signaling, as even in the absence of translation, the activation of the
FGF-PLCγ-myosin II pathway caused the depolymerization of F-actin in the basal aspect of otic ectodermal
cells, necessary for the apical constriction and basal expansion that gives them their wedge-shaped
morphology.

Scanning electron micrographs of the chick inner
ear at 30 and 36 hours ofdevelopment. The inner ear
is a flat epithelial sheet at 30
hours, by 36 hours the inner
ear has invaginated. In thisreport, the Ladher teamshows that FGF controls
the cytoskeletal remodelling events that lead to thismorphological change.

“This is the first time to our knowledge that the shaping of cells involves something more than cell-intrinsic
mechanisms,” says Ladher. “And as we see very similar processes in other aspects of development, likethe formation of the neural tube from an originally flat plate of cells, it will be interesting to see how general
the use of extrinsic signaling is in other such morphogenetic events.”

51

QUMA tool for bisulfite sequencing analysis

Yuichi KUMAKI

Bisulfite sequencing is a technology for determining the methylation status of stretches of genomic DNA,
one of the most widely studied forms of epigenetic marking. The technique relies on the interesting factthat when DNA is treated with the ion HSO3-, known as bisulfite, it causes all cytosine residues in thesequence to convert into the related nucleobase, uracil. This occurs in all cases but for one important exception: methylated cytosines remain unchanged. This makes it possible to determine the methylation statusof specific sites in the treated sequence with high confidence. One of the main limiting factors in the application of bisulfite sequencing to basic and medical research, however, remains the analysis of the data generated by the DNA sequencer, which requires multiple steps of processing and refinement, including subjective
quality checks that may introduce researcher bias.
That all may change with the release of QUMA (quantification tool for methylation analysis), a new softwaretool for bisulfite sequencing analysis developed by Yuichi Kumaki and colleagues in the Laboratory for
Mammalian Epigenetic Studies (Masaki Okano; Team Leader), which addresses some of the shortcomings
of previous such applications. The software, which is described in an article in Nucleic Acids Research, isavailable for download under a Creative Commons License from: http://quma.cdb.riken.jp/“I started developing QUMA because I had seen and experienced myself how much time was required fortasks like aligning sequences by hand and generating output for figures, ” says Kumaki. “I think that people
studying DNA methylation will find that this tool will reduce the amount of time they spend on these routine
tasks.”

52

©2008 Oxford University Press

Kumaki Y, et al. QUMA: quantification tool for methylation
analysis. Nucleic Acids Res 36. W170-5 (2008)

QUMA (pronounced koo-ma), boasts a number of features geared toward making bisulfite sequencing
studies simpler. The user interface is easy to use and modify, and nearly all of the output data can be downloaded and configured to user specs. It provides a one-stop service for virtually all of the data-processing
functions needed to analysis the methylation of sequences, such as sequence alignment, trimming, quality
checks, visualization and statistical analysis. This represents a significant improvement over previous
software that required, for example, manual alignment and trimming of sequences prior to analysis. QUMA
puts quality control in the hands of the user with adjustable cutoff parameters, which can be shared withanalysis data, increasingly their reproducibility. And, for users who wish to work with sensitive data, theentire software package can be run over a local network from a CD-ROM.

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“We’re looking forward to receiving feedback from the DNA methylation community on ways to improve
the program, and suggestions for new features, such as the ability to analyze methylation status of individual
alleles,” says Kumaki. “The code is entirely open source, and distributed free via our website, so we hopethat this finds a broad user base in the basic and medical research communities.”

11

Sample output automatically generated by QUMA’s
user-configurable image
generator.

53

Blood cell type diversity in the early embryo

Brendan MCINTYRE

In chickens, as in other vertebrates, the emergence of the blood system takes place in two waves, a firstthat originates in extraembryonic tissue, and a second within the developing embryo itself. The nature ofthis early hematopoiesis is of great interest to scientists and clinicians alike, but the small size of the embryo
in most popular model organisms makes it difficult to obtain enough material to conduct comprehensive
studies. The comparatively large size of the chicken embryo at the transition from primitive (extraembryonic)
to definitive hematopoiesis in the embryo proper makes it a good platform for investigating the changes
that take place during this critical stage in the development of the blood system.

54©2008 BioMed Central Ltd.

McIntyre B A, et al. Expression profiling of circulating non-red
blood cells in embryonic blood. BMC Dev Biol 8. 21 (2008)

Brendan McIntyre and others in the Laboratory for Early Embryogenesis (Guojun Sheng; Team Leader)
took advantage of this plentiful supply to examine gene expression profiles of blood during the period
between embryonic days 4 and 6, when the specification of definitive hematopoietic lineages occurs.
Through microarray data analysis, he found that circulating non-red blood cells include a full complementof blood progenitors and stem cells, meaning that this population may serve as a useful alternative to embryonic stem cells and perinatal blood for scientists interested in embryonic hematopoiesis.
The team began by taking samples from chicken embryos at days E4 and E6, and centrifuging them tosort the heavier red cells from the other lighter components of the blood system. Additional cell sorting by
FACS and classical staining techniques enabled them to isolate a population of cells from which all red
blood had been excluded. RT-PCR, a technique for amplifying gene transcripts, showed that the non-red
blood population expressed the hematopoietic stem cell (HSC) marker CD34, while the red blood fraction
did not, indicating that the non-red sample represented the sole source of blood stem cells in the embryo.
McIntyre et al. next used Affymetrix gene chips to compare levels of gene expression, and identified anumber of candidates whose expression was higher in the non-red blood cells (but, significantly, was notelevated in the heart). Their analysis revealed that numerous genes associated with hematopoietic stem
cells, including those used in cell signaling, cell-matrix adhesion and communication between cells at gap
junctions. In addition to these HSC genes, the team found markers of the myeloid and lymphoid lineages,
providing the first evidence in chick of lymphoid differentiation at this early stage of development. The
search also yielded six functionally uncharacterized genes, which it is hoped will provide new insights into
HSC function and differentiation on further study.

The circulatory system of
early chicken embryos (E5shown here) is complex andcontains 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.

55

Science communications activities
The CDB takes engagement, outreach and public understanding of science very seriously, and organizes various
events and programs every year to build relationships of mutual understanding and trust with the community atlarge.

2008 Open HouseThe CDB held its fifth annual Open House in May, welcoming more than 1,000 visitors who came to learn about theCenter’s research ongoing projects and recent achievements through a variety of exhibitions and activities. In themorning, CDB Group Director Shigeo Hayashi (Laboratory for Morphogenetic Signaling) gave a talk on “Learning
about development and regeneration from insects.” Two themed exhibits on “Development and Disease” and “Headand Brain Patterning” gave guests the opportunity to speak with researchers about their work and how CDB research
connects to “big picture” academic and healthcare issues. This year’s Open House was held in conjunction with the
RIKEN Molecular Imaging Research Program and the city of Kobe’s Medical Industry Research Project, and drew
people from around the Kansai region of western Japan.

NIHITI ©Takagi Masakatsu Photo:Kazuhiro Tanda

An Evening of Science and Music
Over 800 people filled the Kyoto Kaikan Hall on August 22, 2008 to take in “An Evening of Science and Music”, sponsored by the Japan Society for Science and Technology (JST) and co-organized by RIKEN. This event was part of an
initiative by JST to combine art and science with the goal of conveying the wonders of science to a wider audience.
This collaborative event featured a talk by Masatoshi Takeichi (Director of the RIKEN Center for Developmental Biology)
followed by a performance by Masakatsu Takagi (a popular video artist and musician). One of the highlights of theevening was the world premiere of Takagi’s newest piece “NIHITI”, the concept of which originated from his visit to
the CDB earlier this year to learn more about cells and biological phenomena. A dialogue between Takeichi and Takagi
followed the lecture, where they exchanged their views on common ground between art and science. Asked about
what inspired him in his work, Takeichi said, “Regardless of whether you are looking at flowers or insects, all life phenomena are beautiful. I carry out research with the hope that by attempting to reveal the essence of life, we will stumble
upon an ultimately beautiful phenomenon. Art and science are alike in that they both seek ultimate beauty.”

56

The posterior pole of a Drosophila blastoderm embryo stained for Vasa (blue) andtranscription-engaged RNA polymerase II (red). Nuclear membrane is visualized with WGA lectin.

Cell Asymmetry
http://www.cdb.riken.jp/en/matsuzaki

Fumio MATSUZAKI Ph. D.

Fumio Matsuzaki received his B. Sc. from the Department of Biochemistry and Biophysics at the University of Tokyo in 1979, and his
doctorate from the same institution in 1984, for his work on the characterization of the erythrocyte cytoskeletal structure. He spent theperiod from 1984 to 1988 as a postdoctoral fellow, first in the Department of Cell Biology at the Tokyo Metropolitan Institute of Medical
Science, then in the laboratory of Gerald Edelman at the Rockefeller University. He returned to Japan in 1988 to take a position as a section
chief in the Department of Molecular Genetics in the National Institute of Neuroscience. In 1998, he was appointed professor in the Departmentof Developmental Neurobiology at Tohoku University and remained there until taking his current position as group director at the RIKEN
CDB.

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

In the developing mouse spinal cord, Prospero (green) is transiently expressed in new born neurons immediately after birth from mitotic neural progenitors (red).

Staff
Group Director

Fumio MATSUZAKI
Special Postdoctoral
Researcher

Daijiro KONNO
Research Scientist

Tomohiko IWANO
Ayano KAWAGUCHI
Atsushi KITAJIMA
Yoichi KOSODO
Hironori OGAWA
Atsunori SHITAMUKAI
Chieko TAKIZAWA
Shigeki YOSHIURA
Research Associate

Yoshihiro YAMAMOTO

58

Publications
Technical Staff

Tomoko AOKIMisato IWASHITA
Aki MASUDANao OTA
Taeko SUETSUGU
Student Trainee

Tomoaki KATO
AssistantJunko ISHIGAI

Yamamoto Y, et al. The GC kinase Frayand Mo25 regulate Drosophila asymmetric divisions. Biochem Biophys Res
Commun 366. 212-218 (2008)

Izumi Y, et al. Drosophila Pins-binding
protein Mud regulates spindle-polarity
coupling and centrosome organization.
Nat Cell Biol 8. 586-93 (2006)

Konno D, et al. Neuroepithelial progenitors undergo LGN-dependent planar
divisions to maintain self-renewability
during mammalian neurogenesis. Nat
Cell Biol 10. 93-101 (2008)

Fujimura Y, et al. MDGA1, an IgSF molecule containing a MAM domain, heterophilically associates with axon- andmuscle-associated binding partners
through distinct structural domains.
Brain Res 1101. 12-9 (2006)

Kawaguchi A, et al. Single-cell geneprofiling defines differential progenitor
subclasses in mammalian neurogenesis. Development 135. 3113-24 (2008)
Matsuzaki F and Sampath K. Wiring thenervous system: from form to function.
Development 134. 1819-22 (2007)

Matsuzaki F. Drosophila G-protein signalling: intricate roles for Ric-8? Nat Cell
Biol 7. 1047-9 (2005)

Germline Development
http://www.cdb.riken.jp/en/nakamura

Akira NAKAMURA Ph. D.

Akira Nakamura received both his baccalaureate and his Ph. D. from the University of Tsukuba. 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 of Tsukuba. He was appointed assistant professor in theuniversity's Gene Research Center and 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 was appointed CDB team leader in March 2002.

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 anddifferentiation 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 establishmentof the Drosophila germ line as a model of the processes of germ plasm assembly and germ cell differentiation. We expect that
our study will also provide insights into the general mechanisms of mRNA localization and translation, which are important toa number of other developmental processes including asymmetric cell 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 criticallyimportant form of regulation, the translation of the RNA for the maternal gene oskar, which functions in embryonic patterningand the formation of germline cells in Drosophila, is repressed during its transport to the posterior pole of the oocyte. We are
now studying the function of proteins, such as the recently identified factor, Cup, that regulates 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 and global transcriptional regulation in germline cells
during embryogenesis.
In addition to the study of fruit fly germline development, we are also beginning to 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.

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

Staff
Team Leader

Akira NAKAMURA

Research Scientist

Kazuko HANYU-NAKAMURA
Maki SHIRAE-KURABAYASHI
Tsubasa TANAKA

Technical Staff

Kazuki MATSUDA

Assistant

Eri YAMASHITA

Publications
Tanaka T and Nakamura A. The endocytic pathway acts downstream of
Oskar in Drosophila germ plasm assembly. Development 135. 1107-17(2008)
Hanyu-Nakamura K, et al. Drosophila
Pgc protein inhibits P-TEFb recruitment
to chromatin in primordial germ cells.
Nature 451. 730-3 (2008)
Shirae-Kurabayashi M, et al. Dynamic
redistribution of vasa homolog and exclusion of somatic cell determinants
during germ cell specification in Ciona
intestinalis. Development 133. 2683-93(2006)

Barbee S A, et al. Staufen- and FMRPcontaining neuronal RNPs are structurally and functionally related to somatic
P bodies. Neuron 52. 997-1009 (2006)
Nakamura A, et al. Drosophila Cup is
an eIF4E binding protein that associates with Bruno and regulates oskar
mRNA translation in oogenesis. Dev
Cell 6. 69-78 (2004)
Hanyu-Nakamura K, et al. Germ cellautonomous Wunen2 is required for
germline development in Drosophila
embryos. Development 131. 4545-53(2004)

59

Chromatin Dynamics
http://www.cdb.riken.jp/en/nakayama

Jun-ichi NAKAYAMA Ph. D.

Jun-ichi Nakayama received his bachelor's, master's and Ph. D. degrees in bioscience from the 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 team leader of the Laboratory for Chromatin Dynamics
in 2002.

Multicellular organisms are made up of diverse populations of many different types of cells, each of which contains an identicalset 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 specific patterns of gene expression does not rely on
direct modifications to the DNA sequence encoding the organism’s genome, but rather takes place in a heritable, “epigenetic”
manner. DNA methylation, chromatin modifications and post-transcriptional gene silencing by double-stranded RNA molecules
are some of the best known epigenetic phenomena. Recent studies have begun to show that these different mechanisms areclosely inter-related, but a detailed understanding of these systems has yet to be developed.
Our team investigates how modifications to the structure and configuration of chromatin (complexes of nuclear DNA and proteinsthat provide the structural basis of chromosomes) contribute to epigenetic gene regulation and how such modifications aretransmitted over generations of cellular division by studying events at the molecular scale in the 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 ourcurrent studies, we are particularly interested in determining the specific histone modifications and the molecular recognition
processes that enable modified histones to work together to construct and maintain higher-order chromatin structures. We
also seek to clarify the picture of how dynamic rearrangementsof chromatin structure are triggered by examining the structureand function of protein complexes that bind to and modify
histones. Through these approaches we aim to understand themolecular mechanisms that underlie complex epigenetic
phenomena in developmental processes.

Model for the RNAi-mediated heterochromatin assembly in fission yeast.

Staff
Team Leader

Jun-ichi NAKAYAMA
Research Scientist

Kyoko HAMADA
Tomohiro HAYAKAWA
Aki HAYASHI
Mahito SADAIE
Atsuko SHIRAITechnical Staff

Noriyo HAYAKAWA
Rika KAWAGUCHI
Yasuko OHTANI
Student Trainee

Mayumi ISHIDA
Erina KITANO
Assistant

Sayaka SENO

60

Publications
Sadaie M, et al. Balance between distinct HP1 proteins controls heterochromatin assembly in fission yeast. Mol Cell
Biol 28, 6973-88 (2008)
Iida, T, et al. siRNA-mediated heterochromatin establishment requires HP1and Is associated with antisense transcription. Mol Cell 31, 178-89 (2008)
Hayakawa T, et al. RBP2 is an MRG15
complex component and down-regulates intragenic histone H3 lysine 4
methylation. Genes Cells 12. 811-26(2007)

Buker S M, et al. Two different Argonaute complexes are required for siRNA
generation and heterochromatin assembly in fission yeast. Nat Struct Mol
Biol 14. 200-7 (2007)
Iida T, et al. Conserved ribonuclease,
Eri1, negatively regulates heterochromatin assembly in fission yeast. Curr
Biol 16. 1459-64 (2006)
Sadaie M, et al. A chromodomain protein, Chp1, is required for the establishment of heterochromatin in fission
yeast. EMBO J 23. 3825-35 (2004)

Stem Cell Biology
http://www.cdb.riken.jp/en/nishikawa

Shin-Ichi NISHIKAWA M.D., Ph. D.

Shin-Ichi Nishikawa received his M.D. from the Kyoto University School of Medicine in 1973. He performed his internship and residency in the
Department of Internal Medicine at the Kyoto University Chest Disease Research Institute, before taking an assistant professorship in the samedepartment in 1979. He spent the period from 1980-82 at the University of Cologne Institute for Genetics before returning to the Chest Disease
Research Institute, where he was appointed associate professor in the Department of Microbiology in 1983. He moved to the Kumamoto
University Medical School in 1987 to take a professorship in the Department of Immunology, and returned to Kyoto in 1993, as professor in the
Department of Molecular Genetics at the Kyoto Graduate School of Medicine. He was appointed CDB group director in 2000.

The stem cell system can be thought of as a means by which organisms maintain cell renewal. All forms of life require the endlessrecycling of both materials and energy, making it impossible for either organisms or individual cells to live independent of theirenvironments. In bodies made up of many cells working in cooperation, such as our own, the need to replace and renew is not
limited to simple materials and energy supply; the constant generation of new cells is also essential to the maintenance of theindividual, a process that has developed in the evolution of multicellular life. In this sense, the process of cell renewal in maintaining
the body’s integrity and function provides a meaningful example of the relationship of the cell to the organism, the individual to thewhole, which we hope 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 never-ending self-renewal that sustains the organism as awhole. This will require, at the very least, solving the riddles of two essential processes. The first of these is the need for old cells
destined for replacement to be able to disengage or otherwise be dislocated from their environmental milieux. The second is therequirement for preparing new cells in replacement. To investigate the first of these mechanisms, our group uses a system forlabeling cells with special dyes, which allows us to monitor their location and behavior. We have also developed a system fordifferentiating embryonic stem (ES) cells in culture to study the second question of new cell production. Our current interest in thisproject is the development of hematopoietic stem cells, endothelial cells and pigment cells. These three ongoing research projects
within our lab allow us to explore the problem of cell renewal in self-maintenance from multiple angles, an approach that we hope
will provide new insights into this fundamental process.

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

Staff
Group Director

Shin-Ichi NISHIKAWA

Research Scientist

Julien BOUISSAC
Lars M JAKT
Hiroshi KATAOKA
Igor M SAMOKHVALOVYoshiteru SASAKI
Yan-Fung WONG

Research Specialist

Reiko NAKAGAWA

Research Associate

Rasmus FRETER
Yasushi KUBOTA

Publications
Collaborative Scientist

Gyohei EGAWA
Takumi ERA
Shin KAWAMATA
Sentaro KUSUHARA
Masatake OSAWA
Atsushi TOGAWA
Akiyoshi UEMURA
Visiting Scientist

Naoki IZUMIHerve LE MOUELLIC
Yosuke TANAKA
Technical Staff

Misato HAYASHI
Satoko MORIWAKI
Satomi NISHIKAWA
Yuri SHIMIZU
Naoko YOSHIOKA

Junior Research
Associate

Kenichiro KOBAYASHI
Student Trainee

Yoko FUKUSHIMA
Masaki KINOSHITA
Satomi TORIKAI-NISHIKAWA
Part-Time Staff

Miho HOSHIJIMA
Yoko OTSUKA
Kazumi YANAIAssistantKaori KAWAHARA
Satomi SHINOHARA

Era T, et al. Multiple mesoderm subsets
give rise to endothelial cells, whereas
hematopoietic cells are differentiatedonly from a restricted subset in embryonic stem cell differentiation culture.
Stem Cells 26. 401-11 (2008)

Sasaki Y, et al. NIK overexpression amplifies, whereas ablation of its TRAF3binding domain replaces BAFF:BAFFR-mediated survival signals in B cells.
Proc Natl Acad Sci U S A 105. 10883-8(2008)Moriyama M, et al. Multiple roles ofNotch signaling in the regulation of epidermal development. Dev Cell 14.
594-604 (2008)

Takashima Y, et al. Neuroepithelial cells
supply an initial transient wave of MSC
differentiation. Cell 129. 1377-88(2007)

Yonetani S, et al. In vitro expansion of
immature melanoblasts and their ability
to repopulate melanocyte stem cells in
the hair follicle. J Invest Dermatol 128.
408-20 (2008)

Samokhvalov I M, et al. Cell tracingshows the contribution of the yolk sac
to adult haematopoiesis. Nature 446.
1056-61 (2007)

61

Cell Migration
http://www.cdb.riken.jp/en/nishiwaki

Kiyoji NISHIWAKI Ph. D.

Kiyoji Nishiwaki received his B. Sc. and M. Sc. from Osaka City University and was awarded a Ph. D. by the same institution for work on the molecular biology of C.elegans in 1994. He joined NEC Corporation in 1986 as a researcher in the Fundamental Research Laboratory, studying
the molecular genetics of C. elegans. He left NEC in 1992 to work as a visiting researcher at Johns Hopkins University, then returned to the company in 1993 to continue his work on nematode molecular genetics. He remained at NEC until receiving an appointment as team leader at
the RIKEN CDB.

In many forms of organogenesis, we can observe the coordinated movement of epithelial cells in sheets. For example, in thedevelopment of the lung, tubular epithelial sheets repeatedly extend and subdivide in branching patterns and thereby give riseto the minutely ramified and intricate structure of the airway. Epithelium is characterized by the presence of a basement
membrane, an extracellular substrate in the form of a protein matrix, which plays important roles in regulating the direction anddistance of epithelial cell migration. The main component of the basement membrane is collagen, but it comprises hundredsof other proteins as well, and abnormalities in the function of these proteins can lead to a range of developmental anomalies
and adult pathologies.
We study the function of basement membranes in migration of epithelial sheets using a model organism called C. elegans,which is a tiny roundworm species about 1 mm in length. In this worm, the gonads develop following a stereotyped path inwhich cells at the leading end of the migrating gonad (known as distal tip cells, or DTCs) travel along a U-shaped route in thelarval body, thereby giving rise to an organ of that shape. The proper migration of the developing gonad relies on surface interactions mediated by the basement membranes of the gonad and the body wall. We study various mutant worms in which thedirection of gonadal cell migration is abnormal to search for clues to the genetic and molecular bases of DTC guidance. Oneof the genes we have been focusing on encodes a metalloprotease named MIG-17, which localizes in the gonadal cell basement
membrane and plays an important part in the determination of the DTC’s migratory route by breaking down or modifying othermembrane proteins. We have also discovered that FBL-1, a member of the fibulin family of secreted proteins, is localized tothe basement membrane in response to MIG-17 activity and also plays a role in directing cell migration. It is our hope that
research such as this will provide insights into basic biological mechanisms that may one day make it possible to treat a rangeof health conditions in which cell migration is aberrant.

Expression of fbl-1::Venus in the head region.

Staff
Team Leader

Kiyoji NISHIWAKI
Research Scientist

Shinji IHARA
Hon-Song KIM
Yukihiko KUBOTA
Toshihiro SASSA
Katsuyuki K. TAMAI
Technical Staff

Kayo NAGATA
Asami SUMITANI
Student Trainee

Yuko KITANOKae MIZUTANI
Keigo NAKAYA

62

Publications
Kubota Y, et al. MIG-17/ADAMTS controls cell migration by recruiting nidogento the basement membrane in C. elegans. Proc Natl Acad Sci U S A 105.
20804-9 (2008)

Ihara S and Nishiwaki K. Prodomaindependent tissue targeting of an
ADAMTS protease controls cell migration in Caenorhabditis elegans. Embo J
26. 2607-20 (2007)

Ihara S and Nishiwaki K. Stage-specific activation of MIG-17/ADAMTS
controls cell migration in Caenorhabditis elegans. FEBS J. 275(17):4296305. (2008)

Suzuki N, et al. Chondroitin acts in theguidance of gonadal distal tip cells in C.
elegans. Dev Biol 300. 635-46 (2006)

Tamai K K and Nishiwaki K. bHLH transcription factors regulate organ morphogenesis via activation of an
ADAMTS protease in C. elegans. Dev
Biol 308. 562-71 (2007)

Kubota Y, et al. The conserved oligomeric Golgi complex acts in organ morphogenesis via glycosylation of an
ADAM protease in C. elegans. Development 133. 263-73 (2006)

Pluripotent Cell Studies
http://www.cdb.riken.jp/en/niwa

Hitoshi NIWA M.D., Ph. D.

Hitoshi Niwa received his M.D. from Nara Medical University in 1989, and his Ph. D. in medical physiology in 1993 from the Kumamoto
University Graduate School of Medicine for his work in gene trap methods. From 1993 to 1994, he worked as a research associate in the Department of Developmental Genetics at the same university, before taking a postdoctoral fellowship with Austin Smith at the University of
Edinburgh Centre for Genome Research. He returned to Japan in 1996 as a research associate in the Department of Nutrition and Physiological
Chemistry at the Osaka University Graduate School of Medicine, where he remained until taking his current position as Team Leader at the
RIKEN CDB.

Pluripotency is a term used to describe the ability of certain types of stem cells that are capable of giving rise to at leastone
type of cell from all three germ layers – endoderm, mesoderm and ectoderm. Stem cells are also characterized by their 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 propertiesof 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 4 transcription factors. These transcription factors should form a self-organizing network able to stabilize their expression and maintain pluripotency. At the same time, the network should have the sensitivity to signals that induce differentiation.
How can the transcription factor network satisfy these contradictory requirements? What is the principle behind the structureof 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.

Chimeric embryos generated by injection of Gata6GR ES cells into blastocysts. These ES cells carry the constitutively active Egfp transgene and contribute to primitive ectoderm (upper panels), whereas they contributeto parietal endoderm after induction of Gata6 activity when treated with dexamethasone (lower panels).

Staff
Team Leader

Hitoshi NIWA

Research Scientist

Kenjiro ADACHI
Kazuhiro MURAKAMI
Yuhki NAKATAKE
Kazuya OGAWA
Itsuro SUGIMURA

Publications
AssistantMiho SAKURAI

Toyooka Y, et al. Identification andcharacterization of subpopulations in
undifferentiated ES cell culture. Development 135. 909-18 (2008)

Technical Staff

Niwa H, et al. Platypus Pou5f1 reveals
the first steps in the evolution of trophectoderm differentiation and pluripotency in mammals. Evol Dev 10. 671-82(2008)

Student Trainee

Masui S, et al. Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells.
BMC Dev Biol 8. 45 (2008)

Part-Time Staff

Niwa H. How is pluripotency determined and maintained? Development
134. 635-46 (2007)

Kadue TAKAHASHI
Rika YAGI
Makoto SHIKI
Daisuke SHIMOSATO
Toshimi SUGIMOTO
Sachiko HASHIMOTO
Yayoi NAKAI

Masui S, et al. Pluripotency governedby Sox2 via regulation of Oct3/4 expression in mouse embryonic stem
cells. Nat Cell Biol 9. 625-35 (2007)

63

Mammalian Epigenetic
Studies
http://www.cdb.riken.jp/en/okano

Masaki OKANO Ph. D.

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

The development and physiological function of multicellular organisms relies on the ability of their component individual cells,
which as a rule contain identical genetic information, to express subsets of genes in a selective manner. In order to maintainlineages 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 of gene expression are preserved
across cycles of cell division without a change to the underlying genome sequence are commonly referred to as “epigenetic”
processes. These processes produce chemical modifications to and structural remodeling of chromatin, nuclear structures that
store the cell’s DNA, thereby allowing individual cells to regulate the switching on and off of the expression of specific genes
with great precision and flexibility.
Our team studies epigenetics by focusing on a particular set of molecules that act in the methylation of DNA, with the goal ofidentifying their roles in the regulation of chromatin function in embryonic development and the processes of 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 of embryonic stem (ES) cells to explore how dynamicchanges in DNA methylation function as regulatory factors in mammalian embryogenesis and cell differentiation, and the meansby which DNA methylation works to maintain cell proliferation and chromosome function. We anticipate that a deeper understanding of these questions will lead to new insights in the context-specificity of the DNA methylation process as well as the roles of methylation in development, health and regeneration.

Immunofluorescence showing Np95 localization to methylated
DNA during DNA replication in ES cells.

Staff
Team Leader

Masaki OKANO
Research Scientist

Morito SAKAUE
Takashi TAMURA
Research Associate

Yuichi KUMAKI
Technical Staff

Chisa MATSUOKA
Akiko YAMAGIWA
Assistant

Fumika NAKAYAMA

64

Publications
Kumaki Y, et al. QUMA: quantification
tool for methylation analysis. Nucleic
Acids Res 36. W170-5 (2008)
Takebayashi S, et al. Major andessential role for the DNA methylation
mark in mouse embryogenesis andstable 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 byrecruiting Dnmt1 to methylated DNA.
Nature 450. 908-12 (2007)
Oda M, et al. DNA methylation regulates
long-range gene silencing of an Xlinked homeobox gene cluster in a
lineage-specific manner. Genes Dev
20. 3382-94 (2006)

Tsumura A, et al. Maintenance of selfrenewal ability of mouse embryonicstem cells in the absence of DNA
methyltransferases Dnmt1, Dnmt3a
and Dnmt3b. Genes Cells 11. 805-14(2006)

Mammalian Molecular
Embryology
http://www.cdb.riken.jp/en/perry

Tony PERRY Ph. D.

Tony Perry received his B.Sc. in Microbiology from the Department of Bacteriology at the University of Bristol, England and his doctorate
from the University of Liverpool four years later. From 1989 he became a postdoctoral fellow and temporary lecturer working on epididymal
sperm maturation at the University of Bristol and in 1996 won a European Molecular Biology Travel Fellowship to study oocyte activation.
Dr. Perry developed a novel method of transgenesis which he primarily worked on at the Rockefeller University. In June 2002 he took his
present position as Team Leader of the Laboratory of Mammalian Molecular Embryology at the RIKEN CDB, where he started to work on
the mechanisms of key cytoplasmic events at fertilization in mammals, including chromatin remodeling and meiotic exit.

There can be few, if any, cells as specialized as sperm and egg; they appear unique (you can even see a mammalian oocyte
with the naked eye) and don't typically divide by themselves. Yet when they combine at fertilization, the single cell they generate
is transformed within hours to produce a totipotent cell: one which is completely unspecialized in that from it all cell types
develop to produce an entire individual.
Our laboratory combines molecular and cell biology with micromanipulation of mouse gametes and embryos to study the
nucleo-cytoplasmic events that occur immediately after sperm-egg union (oocyte activation) and their developmental consequences. One long-standing question concerns why oocytes don't begin to divide by themselves in the absence of a sperm.
To address this, our group developed a novel approach that revealed that the removal of the protein Emi2 from oocytes caused
them to resume the cell cycle as if the oocytes had been activated by a sperm. We then extended this to show that Emi2 works
through Cdc20 and that both parthenogenetic activation and fertilization require Cdc20. This may be the first formal demonstration that the signaling required for parthenogenesis and fertilization has molecular components common to both; this is significant
given the application of parthenogenetic activation in nuclear transfer and other current research.
During meiotic resumption, sperm chromatin undergoes almost complete remodeling; its nucleoproteins, which are mostly
protamines, are removed and supplanted by maternal histones which are subsequently modified. We study and manipulate
this overall process and have found that chromatin remodeling varies depending on the provenance of the nucleus; the fate ofsomatic cell chromatin is different from chromatin associated with a sperm-derived genome, even when both are in the same
oocyte. Chromatin remodeling is not necessarily of critical importance; gross hyper-acetylation during fertilization has only alimited effect, if any, on development. We are analyzing the developmental significance of different modes of epigenetic remodelingand how they are regulated.
We are also interested in additional interactions between sperm head components and the oocyte cytoplasm, in a bid to discoverwhat happens during fertilization and the earliest moments of the new embryo. It would be useful to attribute molecular identities
to the proteins involved in these interactions and characterize them functionally. This task is adaunting one, as the sperm contributes >500 distinct nuclear and cytoplasmic protein species
at fertilization, and yet detecting them in newly fertilized oocytes and embryos requires
exquisitely sensitive methods. With a greater understanding of any sperm contribution todevelopment, our lab hopes to gain insights into the processes by which embryonic stem cells
are formed and carcinogenesis is initiated.
Twelve hours earlier, this 4-cell mouse embryo had just two cells. One of these had been injected with oocyte cytoplasm(containing fluorescent mitochondria so that it's easier to see). In the frog, this classic experiment causes the injected
cell to stop dividing due to a long-sought cytostatic factor (CSF). The result: a 3-cell embryo. But the embryo above has
4-cells - the experiment doesn't work very well in the mouse, requiring a novel approach to identifying mouse CSF.

Staff
Team Leader

Anthony C.F. PERRY

Research Scientist

Hongmei LI
Toru SUZUKI
Matthew D. VERMILYEA

Collaborative Researcher

Naoko YOSHIDATechnical Staff

Atsuko KUBO
Erina OKUDA
Heide OLLER
Emi SUZUKI

Assistant

Eriko NOMURA

Publications
Perry A C and Verlhac M H. Second
meiotic arrest and exit in frogs and mice.
EMBO Rep 9. 246-51 (2008)
Yoshida N, et al. Broad, ectopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice. Development 134.
3941-52 (2007)
Yoshida N, et al. Epigenetic discrimination by mouse metaphase II oocytes
mediates asymmetric chromatin remodeling independently of meiotic exit.
Dev Biol 301. 464-77 (2007)
Yoshida N and Perry A C. Piezo-actuated mouse intracytoplasmic sperm injection (ICSI). Nat Protoc 2. 296-304(2007)

Shoji S, et al. Mammalian Emi2 mediates cytostatic arrest and transduces
the signal for meiotic exit via Cdc20.
Embo J 25. 834-45 (2006)
Amanai M, et al. A restricted role forsperm-borne microRNAs in mammalian fertilization. Biol Reprod 75. 877-84(2006)
Amanai M, et al. Injection of mammalian
metaphase II oocytes with short interfering RNAs to dissect meiotic and early
mitotic events. Biol Reprod 75. 891-8(2006)

65

Mammalian Germ Cell Biology
http://www.cdb.riken.jp/en/saitou

Mitinori SAITOU M.D., Ph. D.

Mitinori Saitou received his M.D. from the Kyoto University Faculty of Medicine in 1995, and received his Ph. D. in 1999 for his study of the structure and function of tight junctions under Shoichiro Tsukita in the Kyoto University Graduate School of Medicine. He then moved to theWellcome Trust/Cancer Research Campaign Institute (present name: Wellcome Trust/Cancer Research UK Gurdon Institute), where worked
as a postdoctoral research associate in Azim Surani's laboratory, focusing on the origin of the germ line in the mouse. He was appointed
team leader at the CDB in 2003, and received a three-year grant from the Japan Science and Technology Corporation (JST) PRESTO
program that same year, and was affiliated to the Kyoto University Graduate School of Biostudies as associate professor in 2004. He willassume a professorship at the Kyoto University Graduate School of Medicine in 2009.

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

Heat map representation of the copy numbers of the indicated genes in single ICM cells at E3.5
(upper) and E4.5 (lower). PE, primitive endoderm. EPI, epiblast.

Staff
Team Leader

Technical Staff

Special Postdoctoral
Researcher

Student Trainee

Mitinori SAITOU
Kazuki KURIMOTO
Sugako OGUSHI
Yoshiyuki SEKI
Research Scientist

Yasuhide OHINATA
Jun UEDA
Yukihiro YABUTA
Masashi YAMAJI
Research Fellow /Student Trainee

Mitsue SANO

66

Publications
Mayo SHIGETA
Kaori YAMANAKA
Takayuki HIROTA
Satomi KIKUOKA
AssistantKazuyo NAKATANI

Kurimoto K, et al. Specification of thegerm cell lineage in mice: a process orchestrated by the PR-domain proteins,
Blimp1 and Prdm14. Cell Cycle 7.
3514-8 (2008)

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

Ohinata Y, et al. A comprehensive,
non-invasive visualization of primordial
germ cell development in mice by thePrdm1-mVenus and Dppa3-ECFP
double transgenic reporter. Reproduction 136. 503-14 (2008)

Ogushi S, et al. The maternal nucleolus
is essential for early embryonic development in mammals. Science 319.
613-6 (2008)

Yamaji M, et al. Critical function ofPrdm14 for the establishment of thegerm cell lineage in mice. Nat Genet(2008)

Saitou M, et al. Single-cell cDNA highdensity oligonucleotide microarray
analysis: detection of individual cell
types and properties in complex biological processes. Reprod Biomed Online
16. 26-40 (2008)

Organogenesis and
Neurogenesis
http://www.cdb.riken.jp/en/sasai

Yoshiki SASAI M.D. , Ph. D.

Yoshiki Sasai received his M. D. from the Kyoto University School of Medicine in 1986, subsequently performing internships in generalpractice 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 thereuntil 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 boards of
Neuron, Genesis, and Developmental Dynamics.

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 dorsal-ventral axis is significant in neural development in that the central nervous system forms on thedorsal side of the body in all vertebrate orders. This process is dictated 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
to initiate 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 the signaling 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 complexof permissive, instructive and inhibitory patterns that determines the growth and differentiation of the brain in its earliest stagesof development.
Our group is now also actively developing effective methods of inducing neuralization in mammals, work which it is hoped willcontribute to basic research by providing in vitro experimental systems for use in the analysis of mammalian neurogenesis. In
addition, this approach has potential for application in the treatment of neurodegenerative disorders, such as Parkinson disease.
Using a system developed in our lab, we have succeeded in inducing mouse and primate embryonic stem (ES) cells to differentiate
into a range of specialized neuronal types. The application of similar techniques to human ES cells represents a field of study
that, although still at an early stage, shows immense clinical promise.
By studying very early neurogenesis, and the mechanisms of neuronal differentiation, our lab aims to understand the molecular basis underpinning the formation of
so intricate a system as the mammalian neural network, in the hopes that, by looking
at the elegant simplicity of the embryonic brain, it may one day be possible to understand the cellular bases of the mind's activity.

Telencephalic precursor cells induced by SFEB culture (Bf1 shown in red).

Staff
Group Director

Yoshiki SASAI
Collaborative Scientist

Tomoko NAGASE
Morio UENO

Research Specialist

Keiko MUGURUMA-TAMADASpecial Postdoctoral
Researcher

Makoto IKEYA
Research Scientist

Toshihiro ARAMAKI
Mototsugu EIRAKU
Hidehiko INOMATA
Daisuke KAMIYA
Noriaki SASAI
Research Associate

Takafumi WATAYA

Publications
Technical Staff

Satoe BANNO-NISHIDE
Kumi FUKUSHIMA
Tomoko HARAGUCHI
Masako KAWADA
Michiru MATSUMURA-IMOTO
Ayaka NISHIYAMA
Rieko YAKURA-NISHIZAWA

Junior Research
Associate

Teruko DANJOU
Akira TAKAI
Part-Time Staff

Masako SUZUKI

Assistant

Mako MIYAGI
Ayumi TANAKA-IKEYA
Fumi WAGAI

Sasai N, et al. Ectodermal factor restricts mesoderm differentiation by inhibiting p53. Cell 133. 878-90 (2008)
Wataya T, et al. Minimization of exogenous signals in ES cell culture induces
rostral hypothalamic differentiation.
Proc Natl Acad Sci U S A. 105. 11797801 (2008)

Watanabe K, et al. A ROCK inhibitor
permits survival of dissociated humanembryonic stem cells. Nat Biotechnol
25. 681-6 (2007)
Onai T, et al. XTsh3 is an essential enhancing factor of canonical Wnt signaling in Xenopus axial determination.
Embo J 26. 2350-60 (2007)

Inomata H, et al. Robust stability of theembryonic axial pattern requires a secreted scaffold for chordin degradation.
Cell 134. 854-65 (2008)
Eiraku M, et al. Self-organized formation of polarized cortical tissues from
ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3. 519-32(2008)

67

Cthrc1 ties Wnt PCP factors together

Shinji YAMAMOTO

Wnt activate pathways that play central roles in a wide range of developmental processes, from body axis
specification to stem cell proliferation to morphogenetic cell movements. Two of the main programs are
the canonical pathway, in which Wnt stabilizes β-catenin allowing it to interact with various transcription
factors in the nucleus, and the non-canonical, planar cell polarity (PCP) pathway, which features the activation of numerous small GTPases and downstream factors important in cell morphology and motility. Different
Wnt ligands are associated with each of these pathways (Wnt 1, 3a and 8 are known as canonical, while
Wnt5a and 11 are non-canonical), but the means by which pathways are selectively activated have remained
unclear.
Work by Shinji Yamamoto and others in the Laboratory for Embryonic Induction (Hiroshi Sasaki; Team
Leader) shows for the first time how an extracellular cofactor enables the selective activation of a Wnt
pathway. The report, published in Developmental Cell, reveals how a secreted glycoprotein, Cthrc1, helps
switch on the PCP molecular cascade by stabilizing the interaction between Wnt ligands and their receptors.

68©2008 Elsevier Inc.

Yamamoto S, et al. Cthrc1 selectively activates the planar cell
polarity pathway of Wnt signaling by stabilizing the Wntreceptor complex. Dev Cell 15. 23-36 (2008)

In the PCP pathway (as in canonical Wnt signaling) the primary receptor is the surface protein Frizzled, but importantly, the co-receptors in the two pathways differ; canonical signaling uses LRP5 and LRP6, while PCP
uses a molecule named Ror2.
Yamamoto’s first inkling of a role for Cthrc1 came from a screen for genes expressed in the notochord (atransient axial structure found in all chordate embryos). Drawn by the fact that it codes for a secreted
protein expressed in both the notochord and inner ear, suggesting the possible involvement of the Wnt/PCP
pathway, he first created a null mutant, but found no phenotype when Cthrc1 alone was ablated. Whenhe studied its loss of function in mice carrying a heterozygous mutation in another PCP pathway element(Vangl2), however, he found defects in several developmental processes, including neural tube closure andorientation of the cochlear hair cells in the inner ear.
Using co-culture immunoprecipitation, a method newly developed by the Sasaki lab to detect interactions
between proteins, Yamamoto et al. next asked whether Cthrc1 binds with the co-receptor Ror2. Intriguingly,
they found that it not only binds this PCP-specific co-receptor, but to Wnt ligands and the Frizzled receptor
as well, opening up the possibility that Cthrc1 strengthens the interaction between components, some ofwhich are general to Wnt signaling and others that are specific to the PCP pathway. Conversely, Cthrc1
appears to suppress canonical Wnt signaling by some unknown mechanism.
“The fact that the Cthrc1 solo knockout had no phenotype suggests that there are other genes able tocompensate for its loss of function,” says Sasaki, “so one of the next challenges will be to identify othergenes that work in the selective activation of either the canonical or non-canonical Wnt pathways.”

Figure 1

Figure 1
Misorientation of inner ear
hair cells in a Cthrc1 homozygous / Vangl2 heterozygous mutant (right), a phenotype typical of defects inthe planar cell polarity (PCP)
pathway.

Figure 2

Figure 2
Model of the proposed
function of Cthrc1 in stabilizing the interaction between Wnt/PCP pathway
components (left). The canonical Wnt ligand-receptor combination is shown atright.

69

New germline specification gene foundGerm cells diverge from their somatic counterparts fairly early during mammalian development, undergoing at least three processes:
the repression of somatic genes, the reacquisition of the potential for pluripotency, andsubsequent epigenetic reprogramming to acommitted germline fate. The genetic factors
involved in germline specification have been
traced as far back as day 6.25 of embryonicdevelopment, when the gene Prdm1 (alsoknown as Blimp1) is switched on in a handfulof cells in the epiblast, in what is believed tobe the first critical step in the pathway todetermining germline fate. A recent genomewide study of transcriptional dynamics in early
germline progenitors by the Laboratory for
Mammalian Germ Cell Biology (Mitinori
Saitou; Team Leader) has revealed, however,that the network is more diverse than previously expected, with Prdm1 acting as a sortof conductor keeping this genetic orchestra
in harmony.
Masashi Yamaji and others from the Saitou
lab discovered that a gene identified in theirprevious analysis, Prdm14, plays a critical rolein the establishment of the germ cell lineage.
In a study published in Nature Genetics, theyreported that this gene, which encodes atranscription factor expressed only in the
germline, is necessary for two of the three hallmark events in the acquisition of germ cell
fate.
Prdm14 is first expressed transiently in the
inner cell mass, a cluster of cells in the interiorof the 3.5 day-old embryo that give rise to theembryo proper, but fades out by day 5.5,
before reappearing at around E6.5 at a stage
called the early streak, in which the germline
master gene Prdm1 is also just beginning to
be expressed. This early germline-specificexpression prompted Yamaji et al. to look
more closely for a role in the specification ofprimordial germ cells (PGCs). To do so, they
established mutant mouse lines in which
Prdm14 was deleted. The homozygous
Prdm14-/- mutants were of normal appearance, but both the males and females weresterile, their gonads being entirely devoid ofgerm cells.
Yoshiyuki SEKI, Masashi YAMAJI, Kazuki KURIMOTO

70

©2008 Nature Publishing Group

Yamaji M, et al. Critical function of Prdm14 for the establishmentof the germ cell lineage in mice. Nat Genet (2008)

The team examined germline development in the knockout embryos to determine the stage at which
Prdm14 functions, and found that while somatic genetic programs (as represented by Hoxb1) wererepressed, the cells failed to regain pluripotency (indicated by expression of Sox2) or to undergo thewidespread epigenetic reprogramming indicated by the erasure of histone H3 lysine(K)9 di-methylation
and upregulation of H3K27tri-metylation). Further in vitro experiments using primordial germ cells from thePrdm14 mutants showed that, as suggested by their inability to reinitiate Sox2 expression, they were also
unable to de-differentiate to pluripotent embryonic germ cell-like cultures, which is another feature of
wildtype PGCs.
Interestingly, when Yamaji and colleagues analyzed the position of Prdm14 in the genetic network associatedwith PGC specification they found that although its initial activation does not require Prdm1, its subsequent
maintenance and/or upregulation do. The trigger for Prdm14’s activation appears rather to be dependent
on the Bmp4-Smad pathway, a separate signaling routine at work in the early mammalian embryo.
“We now know that the specification of the germ cell lineage is orchestrated by the two transcriptional regulators, Prdm1 and Prdm14,” says Saitou. “Our next challenges will be to clarify the biochemical mechanism
by which these two key factors function and to reconstruct germ cell specification in vitro. We hope that
this work will provide a useful foundation for reproductive and regenerative medicine.”

Expression of Prdm1(Blimp1)
and Prdm14 in the emerging
PGCs visualized by Prdm1and Prdm14-mVenus, respectively.

71

Hypothalamic neurons from ES cells

Takafumi WATAYA

The hypothalamus is a small ventral region of the brain that sits atop the pituitary gland and serves as a
critical interface between the nervous and endocrine systems. Although it only accounts for about 1/300of the total brain mass, the hypothalamus plays important roles in the regulation of the autonomic nervous
system and a set of behaviors that include feeding, sexuality, and the fight-or-flight response. Despite its
importance, however, the development of the hypothalamus remains imperfectly understood, due in partto a lack of effective means for studying its differentiation in vitro.
A series of experiments by Takafumi Wataya and colleagues in the Laboratory for Organogenesis and Neurogenesis (Yoshiki Sasai; Group Director) demonstrating a technique for inducing hypothalamic differentiationfrom mouse embryonic stem (ES) cells in culture stands to add significantly to our understanding of thedevelopment of this tiny but complex brain region. In an article published in the Proceedings of the National
Academy of Sciences USA, Wataya showed that, by modifying a technique used for inducing neuronaldifferentiation in pluripotent cells, it is possible to generate populations of cells expressing specific hypothalamic markers at high efficiency.
The study began when scientists in the Sasai lab noticed that mouse ES cells cultured using a method
known as SFEB (for serum-free culture of embryoid body-like aggregates) very rarely expressed hypothalamic marker genes, although this technique had been used with great success in giving rise to other72©2008 The National Academy of Sciences

Wataya T, et al. Minimization of exogenous signals in ES cell
culture induces rostral hypothalamic differentiation. Proc Natl
Acad Sci U S A. 105. 11797-801 (2008)

specific neuronal populations, including telencephalic (forebrain) precursors. “We knew that the medium
typically used in SFEB culture contained a number of bioactive factors, such as insulin,” says Wataya, “sowe tested whether these might somehow be interfering with hypothalamic differentiation.”
Assays in which ES cells were SFEB-cultured in the presence or absence of insulin at different stages
revealed that this hormone had a strong inhibitory effect on the differentiation of precursor cells expressing
the hypothalamic marker gene, Rax. Interestingly, SFEB culture in the absence of insulin and otherexogenous growth factors yielded populations of cells in which 60~70% expressed Rax and other genes
indicative of hypothalamic neuronal fate. When the Rax-positive precursors were treated with the growth
factor Sonic hedgehog, they found that cells went on to differentiate into neurons specific to different
hypothalamic nuclei involved in hunger and satiety, which are of great potential value for studies of thecellular bases of metabolic regulation.
“Hypothalamus function is implicated in a wide range of medical conditions, including appetite and metabolic
problems such as anorexia and overeating, but its small size has made it difficult to study in vivo,” says
Wataya. “So we’re hopeful that the ability to grow large populations of homogeneous hypothalamic neurons
through ES cell culture will make a contribution to the study of pathogenesis as well as the screening of
new drug candidates.”
The discovery that exogenous factors work to inhibit hypothalamic differentiation also intriguingly suggeststhat this rostral-ventral region may be thought of as a kind of zero-point in the Cartesian positional patterningof the early neuroectoderm. Taken together with the enormous biological importance and high degree of
evolutionary conservation of this region, Wataya’s findings raise the interesting possibility that the hypothalamic precursor fate stands as a default outcome in brain development, opening new avenues of understanding of the evolution of nervous systems as well.

Hypothalamic vasopressinergic neurons derived frommouse ES cell.

73

Pairing up and settling down

Shinsuke NAKAO

Individual cells are capable of moving over surfaces by using a variety of mechanisms, such as the formationof ruffling regions of cell membrane known as lamellipodia that allow the cell to creep across a substrate.
But when two cells encounter each other, the interaction causes them to settle down, this loss of movement
being one aspect of a phenomenon known as contact inhibition. A number of signaling and adhesion
factors, including members of the classic cadherin family of molecules, have been implicated in this contactmediated inhibition of cell movement, but a detailed understanding of the process has remained elusive.
Shinsuke Nakao and others in the Laboratory for Cell Adhesion and Tissue Patterning (Masatoshi Takeichi;
Group Director) have added another piece to this puzzle with the revelation that another cadherin known
as OL-protocadherin is involved in recruiting cytoskeletal regulators to cell-cell contact sites, a previously
unknown means of modulating cell migration.
OL-protocadherin (OL-pc) shows a similar like-binds-like mode of interaction to that seen in other cadherins,
although its homotypic bonds are comparatively weaker. Previous work from the Takeichi lab had shownthat rather than acting as a straightforward adhesion molecule, OL-pc might instead function in othercellular processes – neurons in mice lacking OL-pc, for example, showed defects in axon extension.
This novel role prompted the group to look for interactions with other molecules, using a technique known
as a pull-down assay, in which the OL-pc protein was used as “bait” to survey for other proteins that bindwith it. Their search for binding partners turned up the protein named Nap1 that is known to associatewith a second factor called WAVE1, which functions in the assembly of the actin cytoskeleton protein into
forms necessary for cell migration.

74©2008 The Rockefeller University Press

Nakao S, et al. Contact-dependent promotion of cell migrationby the OL-protocadherin-Nap1 interaction. J Cell Biol 182.
395-410 (2008)

After identifying the specific molecular region at which Nap1 binds to OL-pc, Nakao et al. next investigatedits cell biological function by transfecting OL-pc into a line of cells that shows high motility when cultured
at low densities. Typical of other cadherins, OL-pc tends to accumulate in regions of contact between
cells, while Nap1 and WAVE1 are concentrated in lamellipodia. In cells transfected with exogenous OL-pc,
both Nap1 and WAVE1 became detectable at cell-cell contacts as well, phenotypes that were not seen
when OL-pc lacking the Nap1-binding region was used.
As Nap1 and WAVE1 are known to regulate cell movement, Nakao was curious about a possible role for
OL-pc in this process as well. In cells moving freely at low culture densities, OL-pc misexpression had noapparent effect when observed using time-lapse microscopy. But when the cells were grown in more
crowded quarters, enabling the formation of stabler cell-cell contacts, OL-pc-expressing cultures showed
higher motility than their untransfected counterparts, with the cells moving in a jumpy, uncoordinatedfashion. Tests in which Nap1 and WAVE1 were knocked down using RNAi revealed that OL-pc’s function
relied at least in part on the function of these actin regulators.
Interestingly, cells lacking N-cadherin, a classic cadherin expressed in neurons, mimicked the OL-pc misexpression phenotype, which, combined with observations of altered organization of adherens junctions intransfected cells, suggests that the OL-pc-Nap1-WAVE complex acts by remodeling actin assembly atcell-cell contact sites, causing changes in the structure of the cell-cell junction. The group speculates that
this may be the basis for the increase in cell motility in contact inhibition mediated by cadherin adhesion
is weakened due to the alteration of cell-cell junction structure.

Co-localization of OL-pc(green) and Nap1 (red) at
cell-cell contacts in cells
transfected with OL-pc.

75

Knowing when to stop: New stability factor
controls robustness of the dorsal-ventral axis

One of the key questions in development is
how tissues know when to stop growing.
Early experiments on the head organizer byHans Spemann and colleagues famously
showed that this signaling region can prompt
the growth of a second dorsal axis whentransplanted into the ventral region of an amphibian embryo. Interestingly, the features,such as heads, that develop in the embryowith the organizer largely removed maintainsizes similar to those found in wildtype embryos, suggesting that dorsal-ventral developmental routines are robust against massive perturbations in their regulatory environment. But while the interplay of factors thatassigns back and belly fates is well characterized, it has never clearly been shown how thisrobustness, in the face of change, is maintained.
A study by Hidehiko Inomata and colleagues
in the Laboratory for Organogenesis andTomoko HARAGUCHI, Hidehiko INOMATA
Neurogenesis (Yoshiki Sasai; Group Director)
identified a new factor responsible for stabilizing the dorsal-ventral axial pattern in Xenopus
laevis, a popular system for the study of signaling in early development. This work, published in the journal
Cell, shows that a secreted scaffold protein, ONT1, and the neural inducer Chordin (a BMP antagonist) in
contact with a degradatory enzyme, allowing it to fine-tune BMP signaling in the axial tissue.
The study emerged in part from a theoretical conundrum surrounding the network of dorsal-ventral regulatory factors: the mutually inhibitory relationship between dorsalizing (such as Chordin) and ventralizing
proteins (BMP4) appeared to be vulnerable to instabilities capable of triggering catastrophic changes in
the patterning of the embryo. The observation that this rarely occurs under either natural or experimental
conditions opened up the possibility that some unknown factor might be at work restoring the axial balance
when things begin to go astray. One such factor, ADMP, was shown in a study by a different lab to workin this manner, but its loss-of-function phenotype suggested that it alone could not account for the sturdy
robustness of this system.
Inomata’s work began with the identification of the ONT1 protein in chicken by others in the Sasai lab. A
member of the olfactomedin family and related to other neurodevelopmental regulatory proteins such as
Noelin and Tiarin, ONT1 was interesting also for the expression of its homolog in the dorsal side of theembryo, from which the nervous system arises in frog. Using morpholinos to study its knockdown phenotype, the group found that loss of ONT1 function resulted in dorsalization as evidenced both by morphology
and gene expression.
Wanting to know more about how it might relate to other components of the BMP regulatory network that
controls dorsal-ventral patterning, they used tagged ONT1 in immunoprecipitation assays against a range
of BMPs, their receptors and modulators, as well as other candidates such as Wnt and Nodal. Strong
affinity was shown for the BMP antagonist Chordin, but also intriguingly with a group of proteinases(collectively referred to as B1TP proteins) known to degrade Chordin. Analysis of deletion mutations showedthat the interactions with Chordin and the B1TP enzymes depend on different ONT1 domains.

76©2008 Cell Press

Inomata H, et al. Robust stability of the embryonic axial pattern
requires a secreted scaffold for chordin degradation. Cell 134.
854-65 (2008)

This dual role of ONT1 yoking Chordin to enzymes brought up the possibility that this protein works to titrate
Chordin levels in dorsal-ventral patterning. Despite the apparently primary role of Chordin as a BMP antagonist, this inhibitory system is quite robust in response to changes in Chordin availability. Knowing that evena significant gain of Chordin function usually has only moderate dorsalization, Inomata tested whether the
combination of Chordin protein injection and ONT1 deletion would disrupt the system’s stability, and foundthat it did result in a hyperdorsalized phenotype as measured by gene expression and morphology.
In vivo tests of ONT1 at different concentrations showed that its role as a scaffold linking Chordin and B1TP
enzymes is dose-dependent; normal levels of ONT1 reduce Chordin by promoting its degradation but,seemingly paradoxically, its overexpression actually leaves Chordin protected by binding to individual
proteins and preventing the enzymes from coming into contact with and digesting their target. This scaffold
model was further supported (at the molecular level by) immunoprecipitation assays that revealed increased
Chordin-B1TP association in the presence of ONT1.
Interestingly, the group found that ONT1 is negatively regulated by ventralizing BMP signals, just as is
Chordin. Given that its mode of action as a facilitator of Chordin degradation differs from ADMP, a previously
reported pro-BMP (or to be more accurate, anti-anti-BMP) factor also shown to have only a moderate
loss-of-function phenotype, they performed a double knockdown to see if the two work in concert. The
ONT1-ADMP morphant indeed showed much stronger dorsalization, suggesting the factors play complementary roles, a kind of double safeguard that underlies the dorsal-ventral system’s robustness.
“Past studies have developed a pretty clear picture of deterministic events in development,” says Inomata,“but we have a poorer of understanding of how developmental programs recover when confronted withpotentially catastrophic perturbations. This work shows that there is a kind of emergency manual for dealing
with crisis situations, and maintaining this system’s robustness.”

Figure 1

Figure 1
ONT1 binds to B1P and Chd
as a scaffold protein.

Figure 2

Figure 2
ONT1 is required for robustness resistance to increased Chordin in DV patterning of the early Xenopus
embryo.

77

Molecular basis of neural progenitor diversity

Mammalian brains feature dozens of neuronalsubtypes generated principally by the divisionand differentiation of progenitor cells during
embryonic development. In the neocortex, forexample, elongated progenitor cells straddle
and shuttle across the ventricular zone along
this apical-basal axis, undergoing mitosis at
the apical surface to give rise to the multiple
strata of cortical neurons. But while molecularmarkers have been identified that distinguishapical progenitors from their basal counterparts, the mechanism that underlies theirdifferentiation has remained largely a mystery.
A thoroughgoing statistical analysis of gene
expression profiles of individual neocorticalprogenitor cells by Ayano Kawaguchi andcolleagues in the Laboratory for Cell Asymmetry (Fumio Matsuzaki; Group Director), as wellas the Laboratories for Mammalian Germ Cell
Biology (Mitinori Saitou; Team Leader) and
Systems Biology (Hiroki R. Ueda; Team
Leader), yielded new insights into the molecular basis of their heterogeneity. Using a combination of single-cell cDNA amplification, cluster analysis, and in situ hybridization, thegroup identified a set of genes that distinguishes the stem cell-like apical progenitorsfrom their more differentiated basal counterparts, as well as a possible role for Notch
signaling in maintaining these two progenitor
populations. Their findings were published in
the journal Development.
Ayano KAWAGUCHI

Kawaguchi began by manually selecting arandom assortment of individual cells(including several mature neurons used ascontrols) from the ventricular/subventricular zone of embryonic mouse forebrains, and used a high-efficiency
single-cell PCR technique to analyze their expression of a set of 20 marker genes known to be associatedwith various populations of progenitors and neurons. This profiling work enabled them to class the cells
into five groups: undifferentiated and neuronally-biased apical progenitors from the ventricular zone, subventricular (basal) progenitors, young neurons, and mature neurons.
To validate these initial findings, the group next performed hierarchical cluster analyses of the cells using
microarray data. In both supervised and unsupervised analyses, the gene expression profiles of the individual
cells allowed them to be grouped into four clusters closely corresponding to the results of the cDNA
analysis. Cluster I cells included the full set of undifferentiated progenitors, and Cluster IV cells all the differentiated neurons. Clusters II and III showed highly similar gene expression and were most closely matchedwith the neuronally-biased ventricular zone progenitors and the basal progenitors, respectively. Clustering
revealed that these two groups occupy the same branch of the hierarchical tree as the neurons, setting
them apart from the undifferentiated progenitors in Cluster I.

78©2008 The Company of Biologists

Kawaguchi A, et al. Single-cell gene profiling defines differential
progenitor subclasses in mammalian neurogenesis. Development 135. 3113-24 (2008)

Equipped with a clear in vitro picture of neuroprogenitor taxonomy, Kawaguchi next looked in vivo, to see
if gene expression patterns would match the predictions of the cluster analysis. In situ hybridization revealed
distinct patterns of expression along the apical-basal axis, consistent with the expectation that the Cluster
II cells represented progenitors that have begun to differentiate, and Cluster III basal progenitors in thesubventricular zone. The high degree of similarity in gene expression between Clusters II and III raised thepossibility that the Cluster II cells were young progenitors in the ventricular zone en route to more basal
destinations in the subventricular zone, where they would mature and divide to produce a pair of terminallydifferentiated neurons.
Cluster I cells, in contrast, appeared more likely to be undifferentiated apical progenitors, whose nuclei undergo
elevator-like shuttling in vivo. Looking at levels of molecules associated with neuroprogenitor fate determination, Kawaguchi et al. found a remarkable heterogeneity in factors in the Notch signaling pathway, although
it was unclear whether this was due to transient contact or loss of contact by individual cells with sourcesof Notch ligands in their microenvironments, or an inherent diversity within the Cluster I population.
In any case, Notch signaling provided the strongest distinction between the Cluster I and Cluster II/III
groups. Expression of the Notch ligand Delta-like 1 was seen most strongly in the Cluster II cells, suggestingthat these young basal progenitors are an important Notch signaling source. Inhibition of this pathway incultures including Cluster I (apical progenitor) cells resulted in dramatic increases in the number of cells
that converted from an apical to a basal progenitor fate. The picture that emerges is one in which young
basal progenitors express a ligand capable of inhibiting the differentiation of their neighbors as they maketheir way to the subventricular zone, thereby maintaining the balance between undifferentiated and committed progenitors.
“This is the first work to provide a genome-wide basis for the molecular properties and variety of neuralprogenitors in mammalian developing brains,” says Matsuzaki. “This allows us to clearly delineate one
dynamic feedback system controlling the balance between self-renewal and differentiation among progenitors.”

Clustering of neural progenitor cells from a mid embryonic stage based on single
cell gene profiling.

79New-found control principles forbiological clocks

Maki UKAI-TADENUMA, Yuichi KUMAKIThe advent of high-throughput genomics about a decade ago has yielded an extraordinary wealth of
information on genome sequences, cDNAs, transcriptional start sites and binding sites for a growing
spectrum of species. This burgeoning trove has simultaneously created the demand for new analytical
tools, using computer algorithms to sift through the data, sometimes across genomes, in search of patternsof interest, identify new genes and regulatory factors and potentially synthesize new ones. As with any
attempt to make sense of dizzyingly complex systems, such tools tend to work best when set to work on
problems with at least some well-defined parameters. But once this combination of high-quality data sets
and appropriate tools is in place, they represent a powerful new means of interrogating the molecular codesthat underlie the machineries of life.
Using the mammalian circadian clock as a model system, Yuichi Kumaki and Maki Ukai-Tadenuma of theLaboratory for Systems Biology (Hiroki R. Ueda; Team Leader) have done just that. In a study conductedin collaboration with scientists at the University of Pennsylvania, Kinki University and INTEC Systems
Institute, the Ueda team constructed a database of DNA regulatory regions in mammalian genomes,
surveyed it with statistical methods to predict new targets, and finally validated their model with synthetic
regulatory elements. Their work, published in the Proceedings of the National Academy of Sciences USA,demonstrates the power of the systems biology approach in uncovering new biological principles.
The oscillatory nature of circadian gene expression in mammals is maintained by the cyclical interaction oftranscription factors, which produce periodic transcriptional read-out through three sets of elements whose
activity peaks in morning (E-box), daytime (D-box) and night (RRE). While dozens of such clock-control
elements are known, as an estimated 5-10% of mammalian genes show circadian expression, muchremains to be learned. To address this question, the Ueda team assembled a database of human andmouse enhancers and promoters (two types of DNA regulatory regions), and customized a statisticalmethod known as a Hidden Markov Model (HMM) to compare data and search probabilistically for new
clock-controlled elements.

80©2008 The National Academy of Sciences

Kumaki Y, et al. Analysis and synthesis of high-amplitude Ciselements in the mammalian circadian clock. Proc Natl Acad Sci
U S A 105. 14946-51 (2008)

They found that the Hidden Markov Model performed best in non-coding regions conserved between
mouse and human, and that while D-box and RRE elements showed no bias in their distribution, E-box
sequences tended to be grouped around transcriptional start sites. Their search turned up more than
6,500 predicted E-box, D-box and RRE elements. After winnowing out false positives, tests for clock-like
temporal expression patterns using the top 100 putative clock-controlled elements for each of the three
categories showed highly consistent peak expression times in approximate phase with other circadianelements.
The team next validated their candidates
in vitro, picking the ten highest-confidence
E-box, D-box, and RRE elements, andtransfecting them fused to a luminescentreporter into cells to observe their transcription. After stimulation to synchronize
the cells’ circadian rhythms, 4 of thetransfected E-box candidates, 7 of the
D-box, and 6 RREs showed strong daily
oscillations. They next checked theexpression of these 17 genes in vivo,
examining endogenous transcripts fromseven different mouse tissues and foundcircadian patterns of expression for 13.
As systems biology seeks not only to findwhat is in a system, but also conceptuallywhat might be, the Ueda team lookedback to the HMM. The Hidden Markov
Model is essentially a means of inferring
probabilities about unknown states in asystem by looking at known propertiesof the same system, such as probabilitiesof the state generating a given outcome(“emission”) and of transition from state
to state. Kumaki and Ukai-Tadenuma
emitted sequences from the E-box,
D-box and RRE models, and filtered out the naturally occurring ones, leaving only those that appeared not
to be present in the genome (but which nonetheless are predicted to have similar activity to their naturalcounterparts). Taking one “high-scoring” and one “low-scoring” element for each of the models, both ofwhich contained the same consensus sequence but different flanking sequences, they tested them in asynthetic reporter system and found that all of the high-scoring elements showed high-amplitude circadiantranscriptional activity. This synthetic reporter assay clearly provided the evidence that the flanking
sequences around the consensus sequence can function to titer the amplitude and oscillating rhythm of
their transcriptional output.

Cartoon of experimental
design of the PNAS study.

すこしThinking that such flanking sequences might play a role in changing the binding affinity (and thereby alter
the amplitude) of clock gene regulators, the team analyzed the affinities of activators and repressors incompetitive binding assays. Interestingly, among the E-box elements (but no D-box or RRE), those which
had previously been classed as low-scoring had higher affinity for activators and approximately normal
affinity for repressors, while the affinities for activators and repressors in high-scoring E-box elements was
closer, suggesting that a balance between direct activators and repressors is important for generating
high-amplitude E-box output.

81Principles proven: Transcriptional logicof the mammalian biological clock

Much like their mechanical counterparts,
mammalian biological clocks rely on complex
inner workings. In mouse, the circadian clockis established by a system of interlinked
genetic regulatory loops consisting of at least
three sets of clock-controlled DNA elementsthat mediate transcriptional output at different
times of day: morning, daytime and night.
With three such elements already identified,
the genetic regulation of the biological clock
already rivals the most sophisticated clockworks in its complexity, but the dynamic
principles that govern this system have neverbeen clearly defined.

Maki UKAI-TADENUMA, Takeya KASUKAWA

A study by Maki Ukai-Tadenuma of theLaboratory for Systems Biology and Takeya
Kasukawa of the Functional Genomics Unit
(both of which are led by Hiroki R. Ueda; Team
Leader/Unit Leader) provided new insights
into the transcriptional logic of the mammalian
circadian clock. In an article published in
Nature Cell Biology, Ukai-Tadenuma andKasukawa describe how they simulated thiscircuitry in cellulo using synthetic factors tomanipulate and observe the system dynamics of the biological clock.

The work began with the development of aclock-cell culture system in which the expression of a synthetic transcriptional activator and repressor were put under the control of morning (E’-box),
daytime (D-box) or night (RRE) clock-control DNA elements. By transiently transfecting the host cells withthe activator and repressor, along with a reporter construct used to monitor output, the team was able towatch their transcriptional activity in real-time over the course of several days per transfection.
Testing a hypothesis that a morning activator combined with a nighttime repressor would be sufficient toproduce daytime transcriptional output (which was based on experimental results from previous work in
the Ueda lab), they put their synthetic activator and repressor under the control of E’-box and RRE elements,and observed the resulting output oscillations as indicated by the luminescent reporter. The transcriptional
output tracked very closely (within 1 hour) of the rhythm seen in the output of the natural daytime phase
controlled by D-box elements.
A related principle for nighttime output, determined by a daytime activator and morning repressor, yielded
similar confirmation of theory; the transcriptional output of the synthetic circuitry oscillated in near-lockstep
with the natural daytime clock, providing an important proof-by-synthesis validation of the logics underlying
the regulation of the mammalian biological clock in vivo.
Confident that their synthetic system could faithfully mimic at least two natural daily rhythms, Ukai-Tadenuma
and Kasukawa next attempted to generate other circadian phases. The pairing of a night activator and a
daytime repressor, for example, generated high-amplitude transcriptional output in the pre-dawn period,
while other combinations of morning, daytime and night activators and repressors likewise resulted in oscilla-82©2008 Nature Publishing GroupUkai-Tadenuma M, et al. Proof-by-synthesis of the transcriptional logic of mammalian circadian clocks. Nat Cell Biol 10.
1154-63 (2008)

tions with regular circadian phases. Given these results, they reasoned that in principle, it should be possible
to generate transcriptional output that would peak at times corresponding to (subjective) midday, dawn,
dusk or late at night.
A previous report from the Ueda lab had proposed an even more general model for circadian oscillations
under genetic control. In this model, the expression of a repressor in advance of an activator element(“repressor-precedes-activator”) results in the delay of transcriptional output, while in the opposite case(“activator-precedes-repressor”), the phase is advanced. A corollary to this is that larger discrepancies
between the repressor and activator phases (“repressor antiphasic to activator”) create higher amplitude
oscillations. Using this as a starting point, they searched for theoretical sets of parameters likely to generate
specific outputs, and created an input-output plot against which they could test the theory using theirsynthetic activator and repressor. As with the morning, day and night rhythms, their experimental data
matched nicely with the predictions of the general activator-repressor theory, showing that, in this system
at least, the virtual can provide a window into the real, just as manmade clocks keep us informed of thenatural passage of time.
“Physical simulations allow us to mirror and manipulate actual biological systems with increasing precisionand ease, making it possible to perform all kinds of analyses,” says Ueda. “This approach should be equallyvalid in the study of dynamic, complex biological systems other than biological clocks as well, so we are
hopeful that it will find a broad range of applications.”

Figure 1

Figure 1
Scheme of complex circadian transcriptional network identified previously
by Ueda lab.

Figure 2
すこしFigure 2
Cartoon of daytime transcriptional output generated by morning activator andnighttime repressor (left),and nighttime output generated by daytime activator and morning repressor(right).

83

Societies and Networks
The CDB strives to contribute to the advancement of developmental biology and related fields in Asia andaround the world by supporting the work of numerous scientific societies and networks.

International Society of Developmental Biologists (ISDB)
http://www.developmental-biology.org/

The ISDB serves the global developmental biology community
through its support for international meetings, the establishmentof regional networks, its website listing developmental biology societies and meetings around the world, and its official journal,
Mechanisms of Development. In 2006, CDB Director Masatoshi
Takeichi was elected president of the ISDB.

Asia-Pacific Developmental Biology Network (APDBN)
http://www.apdbn.org/

The APDBN was established in 2004 to promote and support the
study of development and related fields in the Asia-Pacific region.
The APDBN provides multiple travel awards every year to subsidize regional travel by students and young investigators, hosts
meetings and satellite sessions, and administers a mutant mousecollaborative program in conjunction with the CDB Laboratories
for Animal Resources and Genetic Engineering.

Asia Reproductive Biotechnology Society (ARBS)
http://www.cdb.riken.jp/arb/

Established in 2004, the ARBS has held major annual meetings
throughout Southeast Asia, as a forum for discussing the latest
advances in reproductive biology with applications in veterinary
and conservation biology, and with additional implications in theunderstanding of stem cells and regenerative medicine. The 2009
annual meeting of the ARBS will be held in Cambodia. The CDB
hosts the ARBS website, and has served as co-host for the first
four ARBS meetings.

Stem Cell Network, Asia-Pacific (SNAP)
http://www.asiapacificstemcells.org

This network was launched in 2007 to help develop the field ofstem cell research in the Asia-Pacific region. Scientific and government representatives from Australia, China, India, Japan,
Korea, Singapore, Taiwan and Thailand participated in the launchmeeting in Bangkok, and the organization held its first scientificmeetings in Beijing and Kyoto, and launched its website in 2008.
CDB Deputy Director Shin-Ichi Nishikawa serves as chair of the
SNP steering committee.

84

Whole-mount X-gal staining of Rettlac/+ mouse embryo (E12.5)

Embryonic Induction
http://www.cdb.riken.jp/en/sasaki

Hiroshi SASAKI Ph. D.

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

During vertebrate embryogenesis, simply structured regions of undifferentiated cells organize and cooperate to give rise to theanterior-posterior and dorsal-ventral body axes, the neural tube and other embryonic structures deriving from the three germlayers, following developmental patterns that can be collectively summarized as the adult body plan. The embryo is a hive ofactivity, including cell differentiation, proliferation and migration, strictly regulated in both space and time by molecular signals
emitted by special regions of the embryo referred to as “signaling centers.” These centers produce diffusible proteins that serve
to regulate embryonic development, guiding neighboring cell communities to take up the roles they will play in the adult animal,or steering them away from inappropriate fates.
Research in our laboratory focuses on these signaling centers, especially those of the mouse, as a model for studying the regulation of embryogenetic processes. We focus particularly on the node and notochord, which are of central importance in the formation of the early embryo. We focus on the control of the expression of the Foxa2 transcription factor in the formation and maintenance of signaling centers, as well as search for new factors involved in the control of embryonic development by such centers.
We have recently revealed that members of the Tead family of transcription factors are important not only for signaling center
formation, but also in a broad range of processes in mouse development, including cell proliferation and differentiation in preand post-implantation embryos. Our lab is now analyzing the roles of Tead family proteins and their regulation by cell-cell contact
information as a new approach to the study of early mouse development.

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

Staff
Team Leader

Research Scientist

Yamamoto S, et al. Cthrc1 selectively
activates the planar cell polarity pathway of Wnt signaling by stabilizing the
Wnt-receptor complex. Dev Cell 15.
23-36 (2008)

Technical Staff

Ota M and Sasaki H. Mammalian Teadproteins regulate cell proliferation andcontact inhibition as transcriptional
mediators of Hippo signaling. Development 135. 4059-69 (2008)

Student Trainee

Sawada A, et al. Redundant roles ofTead1 and Tead2 in notochord development and the regulation of cell proliferation and survival. Mol Cell Biol 28.
3177-89 (2008)

Hiroshi SASAKI
Noriyuki NISHIOKA
Mitsunori OTA
Ken-ichi WADA
Shinji YAMAMOTO
Shino HIRAHARA
Akiko MORII
Hiroko SATO
Kanako UKITA
Yuu IMUTA

Part-Time Staff

Megumi SHIBATA

86

Publications

Assistant

Misaki HARANO

Nishioka N, et al. Tead4 is required
for specification of trophectoderm in
pre-implantation mouse embryos.
Mech Dev 125. 270-83 (2008)
Sawada A, et al. Tead proteins activate
the Foxa2 enhancer in the node in cooperation with a second factor. Development 132. 4719-29 (2005)
Nishioka N, et al. Ssdp1 regulates headmorphogenesis of mouse embryos byactivating the Lim1-Ldb1 complex.
Development 132. 2535-46 (2005)

Cell Fate Decision
http://www.cdb.riken.jp/en/sawa

Hitoshi SAWA Ph. D.

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

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

Asymmetric cortical localization of the Wnt pathway components.

Staff

Publications

Team Leader

Part-Time Staff

Research Scientist

Assistant

Hitoshi SAWA
Yukinobu ARATA
Kota MIZUMOTO
Yukimasa SHIBATA
Takashi YANOTechnical Staff

Noriko SASAKAWA
Mayumi SHIBUYATomomi TAKANO

Junior Research
AssociateKenji SUGIOKA
Yuko YAMAMOTO

Tomoko SUGIMOTO
Tomoko NAKASHIMA

Zinovyeva A Y, et al. Complex networkof Wnt signaling regulates neuronalmigrations during Caenorhabditis elegans development. Genetics 179.
1357-71 (2008)

Arata Y, et al. Wnt signaling and a Hox
protein cooperatively regulate psa3/Meis to determine daughter cellfate after asymmetric cell division in
C. elegans. Dev Cell 11. 105-15 (2006)

Kanamori T, et al. Beta-catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell
divisions. Embo J 27. 1647-57 (2008)

Takeshita H and Sawa H. Asymmetriccortical and nuclear localizations of
WRM-1/beta-catenin during asymmetric cell division in C. elegans. Genes
Dev 19. 1743-8 (2005)

Mizumoto K and Sawa H. Cortical beta-catenin and APC regulate asymmetric nuclear beta-catenin localization during asymmetric cell division in
C. elegans. Dev Cell 12. 287-99 (2007)
Goldstein B, et al. Wnt signals can function as positional cues in establishingcell polarity. Dev Cell 10. 391-6 (2006)87

Early Embryogenesis
http://www.cdb.riken.jp/en/sheng

Guojun SHENG Ph. D.

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

The lab studies how mesoderm cells form and differentiate during early vertebrate development, using the chick as the mainmodel organism. Our research currently focuses on two aspects: 1) the formation and early regionalization of mesoderm cells
during gastrulation; 2) differentiation of ventral-most mesoderm cell types.
Mesoderm cells are derived from ectoderm cells during gastrulation through a process 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 plateand
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 are organized to form functional hematopoietic and vascularsystems.

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

Staff
Team Leader

Guojun SHENG
Special Postdoctoral
Researcher

Yukiko NAKAYA
Research Scientist

Brendan McINTYRE
Fumie NAKAZAWA
Masahiro SHIN
Wei WENG
Collaborative Scientist

Cantas ALEVTechnical Staff

Hiroki NAGAI
Kanako OTA
Erike Widyasari SUKOWATIYuPing WU

88

Publications
Shin M, et al. Notch mediates Wnt and
BMP signals in the early separation ofsmooth muscle progenitors andblood/endothelial common progenitors. Dev 136. 595-603 (2009)
Nakazawa F, et al. PBRL, a putative
peripheral benzodiazepine receptor,
in primitive erythropoiesis. Gene Expr
Patterns (2008)
Nagai H and Sheng G. Definitive erythropoiesis in chicken yolk sac. Dev Dyn
237. 3332-41 (2008)
Nakaya Y, et al. RhoA and microtubule
dynamics control cell-basement membrane interaction in EMT during gastrulation. Nat Cell Biol (2008)

McIntyre B A, et al. Expression profilingof circulating non-red blood cells inembryonic blood. BMC Dev Biol 8. 21(2008)
Alev C, et al. BetaA, the major beta
globin in definitive red blood cells, ispresent from the onset of primitive
erythropoiesis in chicken. Dev Dyn 237.
1193-7 (2008)

Developmental Genomics
http://www.cdb.riken.jp/en/sugimoto

Asako SUGIMOTO Ph. D.

Asako Sugimoto received her B. Sc. from the Department of Biophysics and Biochemistry in the University of Tokyo, School of Science in 1987,
and her doctorate from the same institution in 1992. She worked as a postdoctoral fellow in Joel Rothman's laboratory in the University of
Wisconsin-Madison from 1992 to 1996, before returning to Japan to assume an assistant professorship at the University of Tokyo. She
remained in that position until 2002, pursuing concurrent work as a Japan Science and Technology Corporation PRESTO researcher from 1997
to 2000. She was appointed team leader at the RIKEN CDB in 2001.

Fertilized eggs give rise to complex animal structures through strict coordination of cell divisions, cell fate determination anddifferentiation. To generate two daughter cells with distinct cell fates from a single cell division (called “asymmetric cell division”),
the mother cell is polarized before dividing and cytoplasmic components are asymmetrically segregated. A mitotic spindle isformed and positioned along the polarized axis, and the cell division produces two daughter cells that contain distinct cell
contents.
Our laboratory aims to understand the gene/protein networks controlling these dynamic cellular processes using the nematode
Caenorhabditis elegans embryos as an experimental model. This organism provides an extremely useful system for studying
such processes at the whole genome level, as its genome has been fully sequenced, and the lineage of each cell in its body is
known. Using high-resolution live microscopy to trace dynamic behaviors of proteins in combination with gene knockdown by RNAi, we are investigating the gene networks that control theseprocesses.
Our current research focuses on understanding the spatio-temporal regulatory mechanisms of
mitotic spindles formation. We revealed that two genetically separate pathways for microtubule
assembly, one involving γ-tubulin and the other requiring aurora-A kinase, are responsible forthe formation of mitotic spindles in early C. elegans embryos. We are investigating how thesepathways spatially and temporally coordinate to form mitotic spindles.
In addition to studying mitotic spindle formation, we are also studying two other phenomena
which are crucial for cell fate determination in early embryogenesis of C. elegans. The first is
how mitotic spindles are positioned within the cell according to the established cell polarity.
The second is how germ granules (large complexes of mRNAs and proteins, believed to be the“germ cell determinants”) are assembled and segregated specifically into the germ lineage.
Through these studies, we hope to develop a more detailed picture of the regulation of dynamic
cellular processes by gene networks.

P granules (magenta) at 2-cell, 4-cell, ~100-cell and pretzel-stage C. elegans embryos. P granules are segregated onlyin the germ lineage. P granules were visualized by the KT2 monoclonal antibody. DNA (green) are stained by DAPI.

Staff

Publications

Team Leader

Part-Time Staff

Special Postdoctoral
Researcher

Student Trainee

Momoyo HANAZAWA
Mika TOYA

Assistant

Asako SUGIMOTO

Research Scientist

Takafumi KONISHI
Masahiro TERASAWA
Takehito URUNO

Research Consultant

Kazumasa TAKEDA

Technical Staff

Yumi IIDA
Nobuko UODOME
Chie WATANABE

Kanako KAWAI
Masafumi YONETANI
Taeko IMURA

Konishi T, et al. The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the
sperm-oocyte switch and differentiation of various cell types. Dev Dyn 237.
2367-77 (2008)
Takeda K, et al. Efficient production of
monoclonal antibodies recognizing
specific structures in Caenorhabditis
elegans embryos using an antigen
subtraction method. Genes Cells 13,
653-665 (2008)

Motegi F, et al. Two phases of astral
microtubule activity during cytokinesis
in C. elegans embryos. Dev Cell 10.
509-20 (2006)
Motegi F and Sugimoto A. Sequentialfunctioning of the ECT-2 RhoGEF,
RHO-1 and CDC-42 establishes cell
polarity in Caenorhabditis elegans
embryos. Nat Cell Biol 8. 978-85 (2006)

Maruyama R, et al. EGG-3 regulates
cell-surface and cortex rearrangements
during egg activation in Caenorhabditis
elegans. Curr Biol 17. 1555-60 (2007)89

Retinal Regeneration
http://www.cdb.riken.jp/en/takahashi

Masayo TAKAHASHI M.D., Ph. D.

Masayo Takahashi received her M.D. from Kyoto University in 1986, and her Ph. D. from the same institution in 1992. After serving as assistantprofessor in the Department of Ophthalmology, Kyoto University Hospital, she moved to the Salk Institute in 1996, where she discovered thepotential of stem cells as a tool for retinal therapy. She came back 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 of the retinal regeneration
research team in 2006. Her clinical specialty is retinal disease – macular diseases and retinal hereditary diseases in particular. Her aim is tounderstand these diseases at a fundamental level and develop retinal regeneration therapies.

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 these reasons it serves as a useful and experimentally amenable model of the central nervous system.
Until very recently, it was thought that, in adult mammals, the retina was entirely incapable of regenerating, but we now knowthat at least new retinal neurons can be generated after being damaged. This has opened up new hope that the ability toregenerate neurons and even to reconstitute the neural network may be retained in the adult retina. We are now exploring theexciting prospect that, by transplanting cells from outside of the retina or by regeneration from intrinsic progenitor cells, it may
one day be possible to restore lost function to damaged retinas.
Our research into retinal regeneration seeks to achieve clinical applications by developing methods for inducing stem cells todifferentiate into retinal neurons and pigmented epithelial cells in sufficient quantities for use in the treatment of patients sufferingfrom 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 and thus repair damaged
tissues. Access to a broad spectrum of developmental biological research information will be key to the achievement of thesegoals, and we appreciate the opportunities for exchange that working in the environment the RIKEN CDB provides.
Therapeutic applications cannot be developed from basic research alone; the clinical approach – a thorough understanding of
the medical condition to be treated – is equally important. For conditions such as retinitis pigmentosa, even the successfultransplantation of cells in animal models may not necessarily be translatable toa human clinical therapy without an understanding of the underlying genetics
and possible immunological involvement. Our goal is to study retinal regeneration
based on both a strong foundation in basic research and solid clinical evidence.

Immunostaining of bipolar cells in the adult mouse retina.

Staff
Team Leader

Masayo TAKAHASHI
Research Scientist

Kohei HOMMA
Michiko MANDAI
Fumitaka OSAKADA
Research Associate

Satoshi OKAMOTO
Visiting Scientist

Yasuhiko HIRAMI
JSPS Postdoctoral Fellow

Zi-Bing JIN

Technical Staff

Kyoko ISEKI
Chie ISHIGAMI
Akane NOMORI
Chikako YAMADA

90

Publications
Student Trainee

Takayuki NAGAI

Osakada F, et al. Toward the generationof rod and cone photoreceptors frommouse, monkey and human embryonicstem cells. Nat Biotechnol 26. 215-24(2008)Jin Z B, et al. Identifying pathogenic
genetic background of simplex or multiplex retinitis pigmentosa patients: alarge scale mutation screening study.
J Med Genet 45. 465-72 (2008)
Osakada F, et al. Wnt signaling promotes regeneration in the retina of
adult mammals. J Neurosci 27. 4210-9(2007)

Ooto S, et al. Potential for neural regeneration after neurotoxic injury in
the adult mammalian retina. Proc Natl
Acad Sci U S A 101. 13654-9 (2004)
Haruta M, et al. In vitro and in vivo characterization of pigment epithelial cells
differentiated from primate embryonicstem cells. Invest Ophthalmol Vis Sci
45. 1020-5 (2004)
Haruta M, et al. Induction of photoreceptor-specific phenotypes in adultmammalian iris tissue. Nat Neurosci 4.
1163-4 (2001)

Cell Adhesion andTissue Patterning
http://www.cdb.riken.jp/en/takeichi

Masatoshi TAKEICHI Ph. D.

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. After attaining his Ph. D., he took a research fellowship at the Carnegie Institution Department of Embryology under
Richard Pagano. He then returned to Kyoto University, attaining a professorship in the Department of Biophysics (1986-1999 ), 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.

The general research focus of our lab is to study the cellular and molecular basis of the mechanisms by which animal cells areorganized into precisely ordered multicellular structures, such as tissues and neural networks. We are particularly interested in
the roles played by cell-cell adhesion and recognition molecules in these processes. The cadherin family of adhesion molecules
and associated signaling systems are essential for the construction of tissues; this adhesion machinery is the main subject ofour research.
Mechanisms of cell-cell adhesion and its remodeling: Cell-cell adhesion is remodeled during morphogenesis or cancer metastasis, and this nature of cell-cell adhesion is important for regulating many types of morphogenetic cell behavior. Cadherin activity
is modulated by the interactions with actin filaments or microtubules, whose processes are mediated by catenins, a group of
cadherin-associated proteins. We are studying the mechanisms underlying the crosstalk between cadherins and such cytoskeletal systems, with the goal of gaining novel insights into the mechanisms of cell-cell adhesion remodeling.
Cell rearrangement and tissue patterning: A second area of interest to our lab is the mechanisms by which animal tissues areorganized through the processes of dynamic cell rearrangement, such as cell migration and relocation. At cellular levels, we are exploring the roles of cadherin molecules in cell contact-dependent regulation of cell migration. At tissue levels, using the embryonicbrains as model systems, our team is attempting to determine how cadherin and associated molecules control the morphogenesis
of neuroepithelial layers, as well as the migration of neural precursor cells. We are also investigating the roles of protocadherins
and Fat cadherins, members of the cadherin superfamily, in the regulation of cell-cell contacts and tissue organization.
Synapse formation: We are further interested in the mechanisms underlying theformation of neural networks, particularly interneuronal recognition during
synapse formation. The cadherin-catenin complex is localized in synaptic
contacts, and different cadherin subtypes are expressed by different neuronsof the brain. We are investigating ways to determine how synaptic contacts are
regulated by this adhesion system, as well as by other adhesion molecules,
such as Ig-superfamily members.

Identification of a novel protein, Nezha, which binds non-centrosomal microtubules at the minus ends.
Triple immunofluorescence staining for microtubules (green), Nezha (blue), and EB1(red), a plus-end
binding protein.

Staff

Publications

Group Director

Junior Research
Associate

Research Scientist

Takashi ISHIUCHI
Shigenori NAGAE

Masatoshi TAKEICHI
Syuichi HAYASHI
Wenxiang MENG
Shoko NAKAMURA
Tamako NISHIMURA
Katsutoshi TAGUCHI
Yoshikazu TSUKASAKI
Masato UEMURA

Visiting Scientist

Anna PLATEKTechnical Staff

Miwako NOMURA
Hiroko SAITO
Chika YOSHII

Student Trainee

Shoko ITO
Nobutoshi TANAKA

AssistantMutsukoAISO-WATANABE

Meng W, et al. Anchorage of microtubule minus ends to adherens junctions
regulates epithelial cell-cell contacts.
Cell 135. 948-59 (2008)
Nishimura T and Takeichi M.
Shroom3-mediated recruitment of Rho
kinases to the apical cell junctions regulates epithelial and neuroepithelial
planar remodeling. Development 135.
1493-502 (2008)
Nakao S, et al. Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction. J Cell Biol
182. 395-410 (2008)

Abe K and Takeichi M. EPLIN mediates
linkage of the cadherin catenin complex
to F-actin and stabilizes the circumferential actin belt. Proc Natl Acad Sci U S
A 105. 13-9 (2008)
Uemura M, et al. OL-Protocadherin isessential for growth of striatal axons
and thalamocortical projections. Nat
Neurosci 10. 1151-9 (2007)
Kametani Y and Takeichi M. Basal-toapical cadherin flow at cell junctions.
Nat Cell Biol 9. 92-8 (2007)91

Systems Biology
http://www.cdb.riken.jp/en/ueda

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

Hiroki R. Ueda received his M.D. from the Faculty of Medicine of the University of Tokyo in 2000, and received his Ph. D. from the University ofTokyo Graduate School of Medicine in March 2004. While an undergraduate, he worked as a research assistant on a biological simulation
system project at Sony Computer Science Laboratories. As a graduate student he next went on to work, first as a researcher (2000) and then asa group leader (2002), at Yamanouchi Pharmaceuticals, on a project studying biological clock mechanisms in fly and mouse. He was appointed
team leader of Laboratory for Systems Biology in 2003, and Leader of the Functional Genomics Unit at the CDB in 2004. He was also appointed
as Professor (Invited) of Osaka University in 2006.

Recent large-scale efforts in genome-sequencing, expression profiling and functional screening have produced an embarrassment of riches for life science researchers and biological data can now be accessed in quantities that are 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 for the advent of systems biology, in which biological processes and phenomena are approached as complex and dynamic systems. Systems Biology is a natural extensionof molecular biology, and can be defined as "biology after the identification of key gene(s)." 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 control existing systems toward the desired state and design
new ones based on an understanding of structure and underlying principles.
The Laboratory for Systems Biology (LSB) has specific aims to investigate development of systems-biological approaches andtheir application to system-level questions of complex and dynamic biological systems, such as mammalian circadian clocks.
In attempting to accomplish the research aims outlined above, we have been mainly focusing on the development of a systemsbiological approach. We have successfully developed new strategies and technologies for genome-wide profiling, bioinformatics,
quantitative measurement, perturbation of cellular state, and implementation of artificial circuits in cells. We have also appliedthese systems-biological approaches to specific system-level questions, which has led to a number of new discoveries andinventions. Over the next five years, we plan on fully integrating these approaches in an
attempt to realize a system-level understanding of the mammalian circadian clock. In
order to facilitate these processes, we will also commit to the development of key
technologies such as functional genomics used for complete identification of the mammalian circadian clock and Micro Electro Mechanical Systems (MEMS, also known as
microfluidics) for quantitative perturbation of the mammalian circadian clock. These key
technologies also have the potential to be applied to the study of developmental
problems.

Clock sculpture outside Gare St Lazare train station in Paris. The cellular clocks inside us can be also become
desynchronized on exposure to critical light pulse(s).

StaffTeam Leader

Junior Research
Associate

Research Scientist

Genshiro SUNAGAWA
Kaori TSUJINO

Hiroki R. UEDA
Kohei MASUMOTO
Yoichi MINAMI
Masato NAKAJIMA
Hideki UKAI
Technical Staff

Special Postdoctoral
Researcher

Rikuhiro YAMADA
Student Trainee

Ukai-Tadenuma M, et al. Proof-by-synthesis of the transcriptional logic of
mammalian circadian clocks. Nat Cell
Biol 10. 1154-63 (2008)
Nakao N, et al. Thyrotrophin in the pars
tuberalis triggers photoperiodic response. Nature 452. 317-22 (2008)

Hiroshi FUJISHIMA
Yohei KOYAMA
Tamami HIRAIAssistant
Mayumi ISHIDA
Sumire HINO
Maki UKAI-TADENUMA

Kumaki Y, et al. Analysis and synthesisof high-amplitude Cis-elements in the
mammalian circadian clock. Proc Natl
Acad Sci U S A 105. 14946-51 (2008)

Visiting Scientist

Koyama Y M, et al. Perturbational formulation of principal component analysis in molecular dynamics simulation.
Phys Rev E Stat Nonlin Soft Matter
Phys 78. 046702 (2008)

Tetsuya KOBAYASHIAtsushi WADA

92

Publications
Ukai H, et al. Melanopsin-dependent
photo-perturbation reveals desynchronization underlying the singularity of
mammalian circadian clocks. Nat Cell
Biol 9. 1327-34 (2007)
Matsumoto A, et al. A functional genomics strategy reveals clockwork
orange as a transcriptional regulator in
the Drosophila circadian clock. Genes
Dev 21. 1687-700 (2007)

Genomic Reprogramming
http://www.cdb.riken.jp/en/wakayama

Teruhiko WAKAYAMA Ph. D.

Teruhiko Wakayama received his B. Sc. and M. Sc. from Ibaraki University, and was awarded a Ph. D. in reproductive biology from the
University of Tokyo Department of Veterinary Anatomy in 1996. He received a postdoctoral fellowship from the Japanese Society for the
Promotion of Science in 1996 and spent the next two years at the Yanagimachi lab in the University of Hawaii Medical School, where hesucceeded in creating the world's first cloned mouse. He was appointed to an assistant professorship at the same institution in 1998, andmoved to the Rockefeller University as a research assistant professor in 1999. He spent a year as a researcher at Advanced Cell Technology
before returning to Japan to take his current position at the RIKEN CDB.

A limitless number of clones of an animal can be generated from its somatic cells. Only a few years ago, such a statement
would have belonged to the 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 thecloning procedure (measured as the ratio of live offspring to cloning attempts), however, remains quite low, at only a few percent,and even in those attempts that do lead to live births, the cloned animals consistently exhibit a range of severe developmental
abnormalities and pathologies.
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 or partial failure of reprogramming, the process in which a cell’s developmental potential is reset to a totipotent state (such as is exhibited by anaturally 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 to be a biotechnology of great promise, given its potential applications in the field of regenerative medicine, such as the generation of embryonic stem (ES) cells by nuclear transfer, which may one day allow us to grow replacement cells that perfectly match a patient’s
own genetic and immune profile, thereby eliminating the risk 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 of the cloning procedure, gaining a better understanding of reprogramming mechanisms andanalyzing the properties of ES cells derived via somatic nuclear transfer. We also use nuclear transfer technology to develop
methods for preserving embryonic lethal and infertile strains of laboratory mice, and continue to explore the development of
new and better techniques for sperm and oocyte preservation and other reproductive biological research technologies.

Transfer of a somatic nucleus into an enucleated egg

Staff

Publications

Team Leader

Junior Research
Associate

Research Scientist

Chong LI

Teruhiko WAKAYAMA
Thuy Hong BUITakafusa HIKICHI
Eiji MIZUTANIHiroshi OHTA
Sayaka WAKAYAMA
Kazuo YAMAGATA

Visiting Scientist

In-Jeoung BAEK
Satoshi KISHIGAMI

Technical Staff

Yuko SAKAIDE

Part-Time Staff

Manjula BRAHMAJOSYULA
Tetsuo ONO

Assistant

Tomoko OYANAGI

Bui H T, et al. The cytoplasm of mouse
germinal vesicle stage oocytes canenhance somatic cell nuclear reprogramming. Development 135. 3935-45(2008)
Mizutani E, et al. Propagation of senescent mice using nuclear transfer embryonic stem cell lines. Genesis 46. 478-83(2008)

Tabar V, et al. Therapeutic cloning inindividual parkinsonian mice. Nat Med
14. 379-81 (2008)
Wakayama S, et al. Production ofhealthy cloned mice from bodies frozen
at -20 degrees C for 16 years. Proc
Natl Acad Sci U S A 105. 17318-22(2008)

Ohta H, et al. The birth of mice fromtesticular spermatozoa retrieved fromfrozen testicular sections. Biol Reprod
78. 807-11 (2008)
Ono T, et al. Nuclear Transfer Preserves
the Nuclear Genome of Freeze-Dried
Mouse Cells. J Reprod Dev (2008)

93

Animal Resources andGenetic Engineering Laboratory
Shinichi AIZAWA Ph. D.

Genetic Engineering Unit
Shinichi AIZAWA Ph. D.

Animal Resource Unit
Kazuki NAKAO

The Genetic Engineering Unit works with research labs within the CDB, as
well as other labs in Japan and throughout the Asia-Pacific region to develop
mutant mice useful to the study of development and regeneration. In thesejoint development projects, we receive sequence information for genes ofinterest from our collaborators, and perform all subsequent stages of the development from construction of the targeting vector to generation of chimeras,
making about 100 new knockout mutants every year. We also develop transgenic mice for the CDB and Kansai biological research communities. In addition, we develop new bioimaging technologies to aid in the visualization ofmutant mouse embryos at the tissue, cell and organelle level.

The Animal Resource Unit maintains and cares for CDB’s laboratory mouseand rat resources in a specific pathogen free (SPF) environment. We also handle
the shipping and receiving of mutant mice both within the CDB and with otherinstitutions in Japan and overseas. In addition, we provide pregnant females,mouse embryos, and services for colony expansion and cryopreservation offertilized eggs of strains prepared by CDB for in vitro fertilization (IVF) breeding.
We also develop technologies for the study of reproductive biology.

Staff
Unit Leaders

Shinichi AIZAWA
Kazuki NAKAO
Attending Veterinarian

Naoko KAGIYAMA
Research Scientist

Go SHIOI

Visiting Scientist

Toshihiko FUJIMORITechnical Staff

Takaya ABE
Naoko HATAMOTO
Michiko HIGASHIKAWA
Kenichi INOUE
Mari KANEKO
Hiroshi KIYONARI
Masahiro OHNO
Naoko OHSHIMA
Yoshihiro TAKAMIYA
Megumi WATASE
Assistant

Mayumi MIZUSHIROGenomics Laboratory
Fumio MATSUZAKI Ph. D.

Genome Resource and Analysis Unit
Hiroshi TARUI Ph. D.

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

The Genome Resource and Analysis Unit aims to support a wide gamut ofgene analysis research, ranging from DNA sequencing and gene expression
analysis to storage and distribution of gene resources. Our facility can performgene expression analysis and high-throughput gene screening using a DNA
sequencing system that can also custom-make DNA microarray systems.
Gene resources derived from a variety of species can also be stored and distributed to researchers according to their requests. By building upon existing
technologies with the goal of creating new techniques and ideas, as well as
providing solid support, we aim to respond flexibly to the needs of eachresearch request.

The Functional Genomics Unit (FGU) provides functional genomics services
to CDB labs, and introduces and develops cutting-edge technologies to accelerate research. The FGU has been working to develop GeneChip and cellbased screening technologies. For the past 3 years, FGU has provided
GeneChip service and informatics for more than 1,250 experiments in a rangeof research projects conducted using diverse model organisms and protocols.
The FGU has constructed protocols including several quality checks ofsamples to ensure reliable GeneChip service, launched an intranet website,held technical seminars and project meetings for first-time users, and held aservice meeting every week for sharing important information and discussing
problems our GeneChip service. We have also established several informatics
methods to support this service in the future.
The FGU has additionally developed a cell-based screening system using afull-length cDNA library, and conducted several genome-wide assays in a pilot
study to identify regulators for transcription processes.

94

Staff
Unit Leader

Hiroshi TARUITechnical Staff

Tetsutaro HAYASHI
Kazu ITOMI
Asuka MOMIYAMA
Kaori TATSUMI
Visiting Technician

Osamu NISHIMURA
Part-Time Staff

Minako MOTOISHI
Assistant

Chiharu TANEGASHIMA

Staff
Unit Leader

Hiroki R. UEDA
Research Scientist

Takeya KASUKAWA
Itoshi NIKAIDOTechnical Staff

Junko NISHIO
Kenichiro UNO
Assistant

Ikuko TADAElectron Microscope Laboratory
Shigenobu YONEMURA Ph. D.
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, as wellas support for live imaging using optical microscopy.

Staff
Team Leader

Shigenobu YONEMURA

Special Postdoctoral
Researcher

Masatsune TSUJIOKA
Technical Staff

Kazuyo MISAKI
Sachiko ONISHI
Makiko F. UWOStudent Trainee

Tomoki FUJITA
Ayuko SAKANEAssistant

Mai SHIBATA

Proteomics Laboratory
Shigeo HAYASHI Ph. D.

Mass Spectrometry Analysis Unit
Akira NAKAMURA Ph. D.

The identification of the individual components of molecular complexes
and elucidation of their structures can provide fundamental insights into
the intricate mechanisms of development and regeneration. The Mass
Spectrometry Analysis subunit uses LC-MASS spectrometry, a technology that enables the high-speed analysis of protein components from evenminute biological samples. The subunit undertakes the analysis of protein
modifications as well.

Staff
Lab Head

Shigeo HAYASHI

Unit Leader

Akira NAKAMURA

Technical Staff

Kaori SHINMYOZUDivision of Human Stem Cell
Technology
Yoshiki SASAI M.D., Ph. D.
Despite their great promise and fundamental interest, stem cells – both embryonic and somatic –can be challenging to study and manipulate in vitro. The Division of Human Stem Cell Technology(DHSCT) 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 theirderivatives in their research. The DHSCT provides technological expertise, training and support
in hESC culture, maintenance, distribution and management, meeting the needs of both experienced and first-time users in the life sciences research community. Working with labs in academia
and industry, the DHSCT seeks to contribute to achieving the goals of translational research, inline with the CDB mission to establish a solid scientific foundation for regenerative medicine. In
parallel to hESCs, technical development for the use of human iPS cells is being planned.

Staff
Division Chief

Yoshiki SASAI

Deputy Chief

Hitoshi NIWA

Research Specialist

Hiroyuki KITAJIMA

Research Scientist

Masatoshi OHGUSHITechnical Staff

Michiru MATSUMURA-IMOTO

Company-SponsoredResearch Trainee

Satoshi ANDO95

On again and off again:
Fgf signaling in inner ear development

Sabine FRETER

The past decade has seen dramatic progress in our understanding of the molecular factors that direct theearly development of the ear. It is known that, in both chicken and mouse, FGF signaling from the mesoderm
initiates the induction of the inner ear, and that Wnt signals from the overlying neural plate also play a role.
But the order of events in this stepwise process, and the specific functions of these two signaling pathways
in inner ear induction have proven elusive.
New work by Sabine Freter and colleagues in the Laboratory for Sensory Organogenesis (Raj Ladher;
Team Leader) now provides a higher resolution view into this problem. In an article published in Development,
the team revealed how FGF and Wnt work in setting up and then progressively restricting the fate of theembryonic region that ultimately gives rise to the inner ear.
Findings from previous studies, in which it was found that explants from the same pre-otic region isolated
from embryos at closely spaced developmental stages (defined by number of somites) showed differing
expression of certain genes. To see if this would hold true for genes used in inner ear induction, Freter
removed presumptive explants at the 5-somite stage and tested for the otic marker Soho1, finding only
minimal expression. When she took explants from the same region at the 7~8 somite stage, however, they
showed high levels of Soho1 transcription.
Returning to previous findings from the Ladher team which showed that FGF signaling is needed to trigger
the first steps toward otic differentiation, but that FGF expression subsequently fades in the region, Freter
decided to test whether the downregulation of FGF was merely incidental, or in fact a prerequisite for subsequent stages in the process. Using a genetic trick to leave FGF permanently switched on, and she foundthat, while it worked to expand the field of tissue from which the inner ear ultimately should emerge (which96© 2008 The Company of Biologists

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)

they call the otic-epibranchial progenitor domain, or OEPD), genes needed to induce differentiation into
the inner ear proper failed to switch on. This came as something of a surprise, as the link between FGF
expression and otic development is well established. Indeed, when the team tried suppressing FGF signaling, they found again that the development of the inner ear failed. (Interestingly, the expression of non-otic
genes was unaffected by constitutive FGF.)
In chick, a second signaling pathway known as Wnt has also been implicated in otic development. Usinga mutant downstream of Wnt that results in the pathway being constantly activated, Freter looked for
effects in the otic and non-otic areas of the OEPD. In a near mirror image of the case for FGF in the second
stage of otic development, she found that Wnt was required for otic development, but not so for the epibranchial regions of the OEPD.
This sets up a delicate balance between the programs needed to drive otic and non-otic differentiationfrom a common progenitor region that is induced by FGF. Whereas otic differentiation relies on the combination of Wnt signaling and downregulation of FGF, epibranchial differentiation requires FGF to be maintained
and Wnt shut off.
“This idea that a factor required for one stage in a multistep developmental process can actually have an
inhibitory effect on subsequent differentiation of certain lineages was surprising to us at first, but now seems
to make a great deal of sense,” says Freter. “It will be interesting to see if this is a common feature in thegenetic control of other developmental routines.”

FGF3+19
over-expression

FGF3+19
suppression

Effects of FGF overexpression (left) and suppression(right) on Pax2 and Soho1

97

Division of duties in heterochromatin
assembly

Rika KAWAGUCHI, Mahito SADAIE

Gene expression is controlled not only by regulatory elements that bind to the DNA, but by the way the
DNA itself is packed away in chromosomes. These structures are composed of a combination of DNA andproteins known as chromatin, in which the genetic material is wrapped up into tight coils. In its “open” conformation (euchromatin), genes tend to be more transcriptionally active than in heterochromatin, where genesilencing is common. This transcriptional silencing in heterochromatic regions is controlled by various
factors, including members of the HP1 protein family, but whether and how these proteins interact toachieve this function remains unknown.
Mahito Sadaie (now at the Cambridge Research Institute Cancer-Research UK), Rika Kawaguchi andcolleagues in the Laboratory for Chromatin Dynamics (Jun-ichi Nakayama; Team Leader) demonstrated
how, in fission yeast, the two HP1 proteins Swi6 and Chp2 play distinct, but cooperative, roles in heterochromatin assembly. In an article published in Molecular and Cellular Biology, the team revealed that while Swi6
contributes to the repressive structure through the ability of these molecules to self-associate, Chp2
functions by recruiting other chromatin-modulating factors.
Both Swi6 and Chp2 had previously been linked to heterochromatin assembly, but it was not clear whether
their roles were discrete, or simply redundant. Taking advantage of yeast genetics, Sadaie first tested
whether there was functional overlap by complementarily expressing Chp2 in a swi6-negative mutant strain,
and Swi6 in a chp2 mutant. In neither case was the transcriptional silencing defect rescued, suggestingthat both were required in this mechanism, but each in a different way.

98© 2008 American Society for Microbiology

Sadaie M, et al. Balance between distinct HP1 proteins controls
heterochromatin assembly in fission yeast. Mol Cell Biol 28,
6973-88 (2008)

One clear difference between the two is their levels of expression ; Western blot analyses showed that the
Chp2 protein was only about 1/80 as abundant as Swi6, indicating differential regulation. The team foundthat this asymmetric balance was functionally important, for when they overexpressed Chp2 in cells expressing normal levels of Swi6 it disrupted heterochromatin assembly. Seeking possible molecular interactions,
Sadaie performed pull-down assays and found that while Swi6 proteins bind with other proteins of thesame, Chp2 showed no such self-association.
The team turned next to studying how Swi6 and Chp2 are involved in the recruitment of other chromatinregulating factors to heterochromatic regions. Swi6 was known from previous reports to influence thebinding of a pair of factors, Epe1 and Clr3, to heterochromatin; Sadaie et al. showed that Chp2 also
physically associates with Epe1, and that it has a higher affinity for Clr3 than does Swi6, meaning that it islikely to play the dominant role in recruiting this protein to heterochromatic regions. The case for this interaction was further strengthened by experiments showing that the overexpression of Clr3 rescued the silencing
defect in chp2 mutants.
The picture that emerged from these findings is that, in fission yeast at least, heterochromatin organization
relies on the proper balance between two members of the same protein family, each of which plays adistinct role. “It may not be possible to extrapolate these results directly to other species,” says Nakayama,“but the mechanisms we have found for the dynamic regulation of heterochromatin and gene silencing infission yeast may enable a deeper understanding of how similar functions are achieved in mammals.”

Figure 1

Δswi6 + Chp2

Δswi6

3
1

wild type

Derepressed2

Derepressed
Δchp2 + Swi6

Δchp2

4
Repressed

5

Partially derepressed

Partially derepressed

Chp2 dimer
Chp2 OP in wt

Swi6 dimer

Figure 1
Cartoon showing swi6 andchp2 mutant phenotypes.

Partially derepressed

Figure 2

Epe1

Dose-dependent
repression

Me

Clr3

Swi6

Chp2

CSDCSDCSDCSD

CD

CD

Me

CD
Me

Me

CD
Me

Specific nuclear
association

Me

Figure 2
Discrete functions of Swi6
and Chp2.

99

An atom-by-atom approach to molecularconformational change

Yohei M. KOYAMAFor proteins and other biological molecules, function follows on structure. Scientists have simulated themolecular dynamics by calculating interactions between atoms and determining their stability in different
conformations and the likelihood of transitions between states. One method for analyzing simulated data,
known as principal component analysis (PCA), which uses the individual atoms’ positions (atomic coordinates) works well at extracting conformational fluctuation and predicting changes in response to perturbations, but runs into difficulties when dealing with major transitions, such as those involving multiple stableconformations.
Yohei M. Koyama of the Laboratory for Systems Biology (Hiroki R. Ueda; Team Leader) introduced a versionof PCA that takes advantage of the potential energy in atom-atom interactions, an approach he has named
PEPCA (for potential energy PCA). The work, which allows for the analysis and prediction of major conformational changes, was published in Physical Review E.
PCA using atomic Cartesian coordinates works well in analyzing conformational fluctuations around asingle stable conformation. But since these coordinates depend on the overall molecular motion, theconfounding factors of translational and rotational motion must first be eliminated. And while this is possible
even in large molecules for translational motion, factoring out rotational motion has proven to be a knottier
problem.

100

© 2008 The American Physical Society

Koyama Y M, et al. Perturbational formulation of principal
component analysis in molecular dynamics simulation. Phys
Rev E Stat Nonlin Soft Matter Phys 78. 046702 (2008)

Koyama’s approach was first to look at the traditional method and make some generalizations, in which
he determined that PCA can be used as a means of finding perturbations that cause the largest conformational change. In order to avoid the problems with conventional PCA, he developed an alternative to atomic
coordinates, using the potential energy of interactions between atoms as a surrogate. And, as this energy
is determined solely by the relative positions of a pair of interacting atoms, it is unaffected by overallmolecular motion, meaning that the problematic removal of rotational motion does not come into play.
To show the utility of PEPCA method, Koyama focused on a conformational transition in the alanine dipeptide
as his first test bed. He discovered in his analysis that the perturbation that induced the largest conformational change involved a change in the distribution of a pair of stable states, and identified important interactions in determining the stability of and transitions between these two conformations, demonstrating thevalue of the new approach in both identifying and predicting molecular conformational change.
“With the development of high-speed computational facilities specializing in molecular dynamics simulations, we’re now increasingly able to simulate physiologically significant conformational changes,” says
Ueda. “And I think that the PEPCA method will help in determining the atomic interactions that are criticalto molecular function, which could be of value, for example, in rational drug design.”

Figure 1

Figure 1
Combination of interactions resulting in largest
conformational change in
PEPCA analysis.

Figure 2

Figure 2
Modeling of the effects ofdiffering strengths of positive and negative interactions affects probabilistic
outcomes.

101

Clones from dead, frozen mice

Sayaka WAKAYAMA

The cloning of animals by nuclear transfer has been shown in a wide range of species from laboratory
standards such as mice and rats, to agriculturally important sheep, pigs, horses and cows. The cloning
procedure typically involves extracting the nucleus from a somatic cell taken from one animal (the donor)
and injecting into a fertilizing egg from which the nucleus has been removed. The ability of the oocyte toreprogram the transferred nucleus from a somatic to a totipotent state remains one of the great mysteriesof cell and developmental biology. But the fact that cloning by somatic cell nuclear transfer remains a very
low efficiency process has led many to believe that the integrity of the donor cells providing the genome
may be critical to its success.
This wisdom has been turned on its head by a study by Sayaka Wakayama and colleagues in the Laboratory
for Genomic Reprogramming (Teruhiko Wakayama; Team Leader) in which they report the cloning using
nuclear material from mice that had been dead and frozen for 16 years. Their report, published in theProceedings for the National Academy of Sciences, showed the possibility of nuclear transfer using genomic
material from cells that had been frozen at a relatively high temperature and without cryoprotectants, which
has implications for species conservation and the possibility of “resurrecting” extinct species.
The team began by confirming that tissues frozen for a week could be used as donors in cloning experiments,and found that, interestingly, brain cells yielded the highest efficiencies as genomic donors; higher, in fact,
than seen in previous experiments that had used healthy, living cells. The researchers speculate that this102© 2008 The National Academy of Sciences

Wakayama S, et al. Production of healthy cloned mice frombodies frozen at -20 degrees C for 16 years. Proc Natl Acad Sci
U S A 105. 17318-22 (2008)

may be due to the high levels of sugars in brain tissue, or denaturing, in which the genomic tightly packed
genome comes slightly uncoiled. Wakayama next tried using samples from two male mice that had beenfrozen at -20° C for 16 years without any chemical protectants, but was unable to generate clones using
the standard SCNT procedure.
In addition to straight somatic cell nuclear transfer, a second strategy is available to cloners, in which SCNT
is used to generate early stage embryos and these embryos are allowed to develop to the blastocyst stage,by which point they have a small, internal clump of cells, known as the inner cell mass; this inner cell massis removed and cultured as embryonic stem (ES) cell colonies. These “ES by nuclear transfer” (ntES) cells
can then be injected into other blastocysts and, through a process known as “tetraploid complementation,”giving rise to an individual in which nearly 100% of its nuclei carry the donor genome.

Mouse cloned from dead,frozen donor.

The Wakayama team first determined that the 16-year old frozen tissue could serve as a donor source,
establishing 46 ntES lines from brain cell nuclear material. Using these pluripotent cells, they createdtetraploid embryos and allowed them to develop in surrogate mothers. This time, the process worked,
producing four true clones and nine chimeras, as determined by genotype and coat color.
Although one previous report had shown cloning from frozen material, those samples had been stored at
the much lower temperature of -80° C, and in the presence of agents that may have some cryoprotective
effect. “This is the first time a mammal has been cloned from a sample stored at conditions reasonably
close to what might be expected in permafrost, making which gives some hope for those who might seek
to clone extinct species from frozen carcasses” says Wakayama. “The higher temperature also puts thisapproach more closely in the reach of people working in the field to preserve samples from rare or endangered species.”

103

Cerebral cortical tissue from ES cells

Mototsugu EIRAKU , Masako KAWADA

The cortical regions of the brain are structurally complex and functionally important, providing the neuralsubstrate for many of the brain’s highest functions, such as language, memory, emotion and thought. The
cortex begins to form from the anterior end of the neural plate, and is characterized by its laminar structurein which neurons born at different developmental stages migrate to different cortical layers, each with its
own functions and circuitry. Given this highly stratified and interconnected three-dimensional architecture,
corticogenesis would at first blush seem to require involve myriad, complex processes difficult to reproduce
in vitro.
Work by Mototsugu Eiraku and others in the Laboratory for Organogenesis and Neurogenesis (Yoshiki
Sasai; Group Director) revealed a surprisingly straightforward method for generating three-dimensional
aggregates of multiple, spatiotemporally organized cortical neuron types from embryonic stem (ES) cells.
The report, published in Cell Stem Cell, opens new insights into the genetic control of cortical development,
but also provides a technique for generating different types of cortical neurons for use in the study of pathogenesis and drug discovery.
The Sasai group had previously developed an ES cell culture method, known as serum-free culture of embryoid body-like aggregates (SFEB), to induce the generation of forebrain neuronal precursors that expressed
cortical marker genes from mouse embryonic stem cells. By tweaking the SFEB protocol, causing the cells
to re-aggregate more quickly after dissociation, Eiraku found he could increase the efficiency of corticaldifferentiation. Naming the modified method, SFEBq (for quick-aggregation), he found that not only did thetechnique yield higher percentages of cortical precursors, but also that these reliably went on to form
polarized neuroepithelial structures.
The group next cultured the mouse ES cell-derived cortical cell masses within forebrain slices, and foundthat the explants preferentially migrated into pallial regions, as would be expected from the normal destination of such cells in vivo. When they transplanted the same cells into the brains of newborn mice, theyfound that within four weeks they had developed into pyramidal neuron-like cells with typical morphology
and patterns of axon extension.

104

© 2008 Cell Press

Eiraku M, et al. Self-organized formation of polarized corticaltissues from ESCs and its active manipulation by extrinsic
signals. Cell Stem Cell 3. 519-32 (2008)

To study the functionality of the cells, Eiraku looked at spontaneous neuronal activity (as measured bysurges of calcium ions) in the cells, and found that this was sensitive to known chemical activators andinhibitors of such activity, indicating that it depended on local synaptic connections. Neonatal cortex is
known to exhibit a unique form of coordinated activity, characterized by an oscillating wave of Ca2+ thatwashes across relatively large fields of neurons. Surprisingly, even this calcium wave is recapitulated in the
ES cell-derived cortical tissues, showing that they mimic the properties of the neonatal cortex to a remarkableextent.
During embryogenesis, the cortex undergoes regional specialization as the result of molecular signals
emitted by surrounding tissues, such as FGF, which induces rostral cortical regions, and Wnt and BMP,
which are required for the specification of the choroid plexus and cortical hem. When treated with thesefactors in culture, the SFEBq-differentiated cortical progenitor-like cells responded to these signals similarly
to their embryonic counterparts, giving rise to the apposite neuronal types.
Eiraku and colleagues next sought to find out whether the mouse ES-cell derived cells would follow thestereotypical “inside early, outside late” birthing pattern observed for cortical neurons in vivo. Watching thesequence of marker expression in their cultured cortical-like cells, they found that they closely mirrored thetemporal changes in gene expression seen in the embryonic cortex.
After examining the temporal aspects of birthday-dependent differentiation, the Sasai group turned to thestructure of the cells, which form hollow rosette-like clusters in suspension culture. Here again, they foundthat these rosettes comprised at least four distinct zones (as shown by gene expression), mimicking certain
structural aspects of the early cortex.
Having extensively studied the ability ofmouse ES cells to give rise to cells closely resembling cortical neurons, Eiraku et al. tested
whether the SFEBq approach would work forhuman ES cells as well. Although the cells did
not cluster into rosettes, forming larger,
mushroom-shaped aggregates instead, theresulting continuous neuroepithelia expressed genetic markers characteristic ofcortex and showed some of the regionalization
seen in the mouse ES-derived structures.
“The most surprising finding for us was thata patternless clump of cells can reorganize
itself into something that so closely resembles
cortical tissue, so we’re eager to find out more
about the role of such self-organization inbrain development,” says Sasai. “What’smore, the fact that we were able to derive notjust individual cell types, but functional tissue
from ES cells may have a real impact in termsof both its medical significance and its value
in drug discovery.”Immunostaining of crosssectioned cortical tissue
generated from human ES
cells in vitro.

105

Tracks to the junction: Microtubules tethered
to zonula adherens

Wenxiang MENG, Masatoshi TAKEICHI

Microtubules are long polymer strands with functionally distinct plus- and minus-ends that function in awide range of processes from morphogenesis to the shuttling of motor proteins through the cytoplasm.
While many microtubules are anchored by their minus-ends to the centrosome, in epithelial cells some microtubules have non-centrosomal minus-ends, whose termination site has remained a mystery.
Research by Wenxiang Meng and others in the Laboratory for Cell Adhesion and Tissue Patterning(Masatoshi Takeichi; Group Director) revealed an important role for microtubules in the formation and maintenance of the zonula adherens, a lateral region in epithelial cells in which cell-cell adhesion proteins, suchas cadherins, are highly concentrated. In an article published in Cell, Meng and colleagues identified a pairof novel proteins, PLEKHA7 and Nezha, which are involved in recruiting and dynamically anchoring the
microtubules to the cadherin machinery.
The study began with a search for proteins that associate with p120 catenin, one of the cytoplasmic factorsthat binds to the cadherin tail in the cell’s interior. PLEKHA7 emerged from a pulldown assay for p120
catenin binding partners, and on immunostaining, the group determined that the protein localized at cell-celljunctions in a p120 catenin-dependent manner. Testing for function, they found that overexpression of
PLEKHA7 caused greater amounts of cadherin to accumulate at the zonula adherens, while its depletionby siRNA had the opposite effect.
The group next looked for other proteins that might interact with PLEKHA7 in a new pulldown assay, andcame up with a second unknown protein, which they dubbed Nezha, after a god who appears in the
Chinese adventure “Journey to the West." Immunofluorescence-tagged Nezha showed that it co-localizes
with PLEKHA7 at the zonula adherens in a co-dependent fashion. Overexpression of Nezha in cultured106© 2008 Cell Press

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

cells highlighted a role in microtubule bundling, prompting Meng et al. to look more deeply into this interaction. Double-immunostaining of Nezha and microtubules revealed a tendency for Nezha to associate withthe minus-ends, a relative rarity in contrast to the many proteins that interact with microtubule plus-ends.
A capping experiment using chemically tagged Nezha confirmed the association between this protein andmicrotubule minus-ends.
Wanting to know more about the dynamics of the Nezha/minus-end interaction in living cells, Meng watched
the behavior of fluorescence-labeled Nezha and α-tubulin using time-lapse microscopy. He found that
Nezha formed concentrated points from which single microtubule strands emerged and retracted, suggesting it was serving as a kind of anchor. These Nezha-microtubule complexes were themselves situated in
PLEKHA7-positive regions in the zonula adherens, lending further weight to a functional relationship among
these players.
In addition to the roles in dynamically stabilizing cell structure, microtubules serve an important role as
tracks for motor proteins moving through the cytoplasm. Thinking that Nezha might tether microtubules
to provide a route for minus-end-directed motor proteins to the cell junction, Meng tested a number ofsuch candidates and found that the minus-end-directed protein KIFC3 was indeed localized at the zonula
adherens. Loss-of-function experiments showed that its role there was important, as KIFC3 knockdown
resulted in disorganization of E-cadherin in this zone. When the group interfered with microtubule polymerization in these cells using an inhibitor, the junctional localization of KIFC3 gradually faded before disappearing altogether, suggesting that PLEKHA7, Nezha, KIFC3 and microtubule minus-ends work together inmaintaining zonula adherens integrity, which is essential for preserving the typical epithelial morphology ofthese cells.
“The minus-end specificity of Nezha is an intriguing discovery,” says Meng. “It’s going to be exciting to learn more
about how this PLEKHA7/Nezha/KIFC3 system relates to the processes of embryogenesis and proliferation andinvasion in cancers.”

Nezha-Kusabira Orange
and EB1-GFP are doubly
expressed in a Caco2 cell.
Arrowheads indicate Nezha
signals, and arrows point toEB1 signals growing out of
the Nezha signals.

107

Tead plays a part in cell contact inhibitionof proliferation

Mitsunori OTA

108

© 2008 The Company of Biologists

Ota M and Sasaki H. Mammalian Tead proteins regulate cellproliferation and contact inhibition as transcriptional mediatorsof Hippo signaling. Development 135. 4059-69 (2008)

Knowing of the role of Hippo signaling in cell proliferation, Ota next examined whether it might be involvedin the regulation of Tead. Constitutive expression of Hippo pathway components caused the downregulationof both nuclear Yap1 and Tead activity, indicating that cell contact modulates the activity of Tead proteinsthrough the Hippo pathway.
The converse experiment, in which Tead was activated by misexpression of Yap1 or activated forms of the
Tead protein, resulted in upregulation of growth, suppression of apoptosis, and promotion of tumorigenesis,
supporting the case for roles for Yap1 and Tead in controlling cell proliferation. This prompted them to look
for the gene targets of these factors, using microarrays to analyze expression profiles. Their tests showed
considerable overlap in the genes controlled by the two factors, suggesting that both work on similartargets to regulate growth.
But on looking more closely in vivo, a more nuanced picture emerged. Using embryos with a homologous
deletion of the two major Tead genes, or of Yap1, they found that the expression of only a small fractionof the genes induced by Tead in their cultured cells was affected in the knockout embryos, suggesting thatthese factors regulate different targets in a cell type-specific manner. Protein distribution patterns showed
diversity as well. While Tead1 was expressed in all tissues in embryos between days 8.5 and 10.5, it wasparticularly strong in heart muscle. Similarly, Yap1 was widely expressed, with the highest levels in the nodeand notochord, as well as in myocardium, where it co-localized with Tead. These findings point to cell
type-dependent differences in the Hippo pathway, which regulates contact inhibition of proliferation by its
effects on Yap1 and, consequently, Tead.
“We knew from studies in fly that Hippo was linked to the control of organ size,” says Sasaki, “what we’ve
done here is show not only that it plays a role in cell contact inhibition of proliferation in mouse, but thatthe pathway involves Tead proteins. We have been studying other roles for Tead as well, so we’ll beinterested to see how the Hippo pathway and cell contact information as well fit into the greater picture of
mammalian embryogenesis.”Yap1 localizes in the nuclei
in cells proliferating at low
density (top left), but diffuses into the cytoplasm inhigh-density culture, inwhich growth is slowed(bottom left). Tead shows asimilar pattern of localization (right panels).

109

Protein network reshapes the ECM

Kayo NAGATA, Yukihiko KUBOTA (back), Asami SUMITANI, Kiyoji NISHIWAKI

Studies of how the body is put together tend to focus on cells as the building blocks of tissues and organs,but the extracellular matrix, the collective name for various meshworks of proteins and complex carbohydrates that play a vital role in holding the body’s diverse cells together. Basement membranes are one such
extracellular matrix, comprising layers of collagen and other fibrous proteins and proteoglycans, which
serve the important functions of anchoring epithelial tissue and providing a barrier between the body’s
compartments. The migration of distal tip cells (DTCs) during gonadogenesis in C. elegans has been used
as a relatively simple system for studying the part played by basement membranes in organ formation.
But though it has been shown that an ADAMTS family protease known as MIG-17 is required for controllingthe direction of the distal tip cells as they followed their U-shaped path through the larval body, possibly
by remodeling the basement membrane, but the picture downstream of MIG-17 remained unclear.
A new study by Yukihiko Kubota, Kiyotaka Ohkura and colleagues in the Laboratory for Cell Migration(Kiyoji Nishiwaki; Team Leader) has now revealed a new network of proteins which, in mutant form
suppressed the defects induced by mutation of MIG-17. Interestingly, these suppressor mutations (in thegenes let-2 and fbl-1) appear to work by distinct mechanisms.

110

© 2008 The National Academy of Sciences

Kubota Y, et al. MIG-17/ADAMTS controls cell migration by
recruiting nidogen to the basement membrane in C. elegans.
Proc Natl Acad Sci U S A 105. 20804-9 (2008)

The study began with a screen for genetic defects affecting gonadal migration, using mig-17 mutant worms
as a starting point. Previous work by the Nishiwaki lab had identified one such suppressor mutant, in which
a mutant form of the fibulin-like protein encoded by fbl-1 suppresses MIG-17 defects in a dominant gain-offunction manner. The mutagenesis screened turned up two new mutations in the gene let-2 (which encodes
a C. elegans version of collagen IV), both of which bypass the mig-17 migration defect.
Although neither let-2 nor fbl-1 mutants alone show significant migration phenotypes, when Kubota and
Ohkura introduced mutations into both genes, they found severe gonadal defects, suggesting a genetic
interaction. They next looked at another basement membrane protein, nidogen, which is known to bindto type IV collagen, in order to determine whether it played a part in the equation. But although nidogen
and MIG-17 appeared to be at least partially redundant, the combination of mutant nid-1 with either of
let-2 collagen mutants had no effect on their suppression of mig-17-induced migration defects.
The loss of nid-1 had a strong effect, however, on the ability of the fbl-1 mutation to suppress the mig-17
phenotype, indicating that the two suppressor mutations of let-2 and fbl-1 operate respectively by nidogenindependent and -dependent mechanisms. As nidogen normally binds to collagen in the basement
membrane, the team checked whether this was affected in the various migration-related mutations, alone
or in combination. Using immunohistochemistry, they found that mutations in mig-17 and let-2 had
weakened on nidogen localization, while loss of fbl-1 function caused a more dramatic deficit. Intriguingly,
the introduction of either of the suppressor mutations in a mig-17 mutant background amplified the accumulation of NID-1. Overexpression of NID-1 in mig-17 mutant worms significantly rescued the gonadal defect,suggesting that its accumulation in the basement membrane plays an important role in mig-17-dependent
control DTC migration.
The picture that emerges is complex, and still incomplete. Although the details of the relationship between
FBL-1 and NID-1, and the precise role for LET-2 in the regulation of migration along the basement membraneremain for further study, the findings of Kubota, Ohkura, et al. point to a much more elaborate role for the
extracellular matrix in organogenesis than previously suspected.
“We know of a number of genetic diseases involving ADAMTS proteases associated with changes in theextracellular matrix, but how these proteins affect the ECM is largely unknown,” says Nishiwaki. “We have
now found that in C. elegans, MIG-17/ADAMTS recruits factors to the basement membrane in a determinedorder, giving us one of the first insights into the molecular control of organogenesis by this system.”(A-C) Cross-sections ofworms. Weakened NID-1
localization (magenta) in
mig-17 mutants (A) is recovered to wild type levels
when combined with let-2(B) or fbl-1 (C) suppressor
mutants. (D) Model for theprotein cascade downstream of MIG-17. MIG17-dependent proteolysisof an unknown substrate
recruits and activates
FBL-1C, which then recruits NID-1 to the basement membrane to control
DTC migration. MIG-17dependent proteolysis also
activates LET-2 to induce
NID-1-dependent and -independent MIG-17 pathways.

111

Educational activities
While the CDB is not a teaching institution, we maintain close links with the local academic community and work
to make a contribution by supporting active educators and students with an interest in learning more about developmental biology and experimental approaches.

Intensive lecture program forgrad students
The RIKEN CDB hosted its fifth intensive lecture programfor graduate school affiliates on July 17 and 18. The program drew more than 150 participants from the Center’saffiliated graduate and medical schools. The two-day event
featured scientific talks by CDB lab leaders, exhibitions ofmodel organisms, and demonstrations of research tools
and techniques.

Summer school forhigh school students
The CDB held a pair of single day courses for local highstudents interested in the life sciences on August 5 and 7,
building on the success of last year’s summer school initiative. Each day’s program featured a talk by Shigenobu
Yonemura, head of the CDB’s Electron Microscopy Laboratory, followed by a visit to his lab, as well as a chance
to do some hands-on bench work (staining cultured cells
by immunofluorescence) and a tour of the Kobe Medical
Industry research park in which the CDB is located.

Workshop forhigh school teachers
The CDB hosted a two-day educational program for area
high school teachers on October 4 and 5, with support
from the Japanese Society for Developmental Biology(JSDB), the Kobe Educational Council and the Japan Science and Technology Agency (JST). Thirty teachers of biology from the Kansai region of Japan joined the course,which featured hands-on experiments in Xenopus (African
clawed frog), zebrafish and chick embryology, and lecturesby Masaki Taira (Associate Professor, University of Tokyo)
and Hitoshi Niwa (CDB Laboratory for Pluripotent Cell
Studies).

112

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

Kobe night view. Photography: Natsuhiko KUSUMOTO

RIKEN Kobe Institute
The RIKEN Kobe Institute was established in April 2002 as an organizational framework for the newly launched Center
for Developmental Biology (CDB), which conducts a wide range of research from fundamental studies of development
and stem cells to work with the potential to make a contribution to the field of regenerative medicine. In April 2007, theKobe Institute welcomed a new institution, the Molecular Imaging Research Program (MIRP). In autumn 2008, the MIRP
was re-designated as the Center for Molecular Imaging Science.
The Kobe Institute seeks both to help develop a foundation of knowledge into biological phenomena and, throughtranslational research efforts conducted with the Institute for Biomedical Research and Innovation (IBRI) and otherneighboring institutions, to help bridge basic science to applications in medical therapy and drug discovery and thereby
to contribute 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
Facilities
Human Resources
Library
Computer Networks
Science Communications
and International Affairs

Kobe Safety Center
Institutional Review Board

114

Research Promotion Division
The Kobe Institute Research Promotion Division (RPD)
provides a full range of administrative services requiredby CDB labs, with sections responsible for planning, finances, general affairs and facilities, as well as support
for scientific meeting logistics, computer and information
networks, science communications and international affairs, and the CDB library.

Overheadandadministrative

Personnel

1,554

1,493

Total: 4,467

Safety Center
The KI Safety Center provides training and supervision forlab and workplace safety, implements and monitors disaster prevention measures, and educates staff on andensures compliance with national and institutional regulations governing laboratory practice and technology. The
office also handles all administrative affairs of the Institutional Review Board, the CDB’s research ethics review
committee, and administers the Institute’s medical center.

Center for
Molecular Imaging Science
In 2005, the Molecular Imaging Research Program wasselected by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) as an important part ofits efforts to promote research and development in life
sciences that respond to real societal needs. Since then,
RIKEN has been striving to establish a core center for
drug development molecular imaging. In the fall of 2006,
the Kobe MI R&D Center Building was completed withthe support of the Kobe municipal government andothers, in which RIKEN’s facility for the Molecular Imaging
Research Program (MIRP) was now located. In October
2008, to further advance research and improve its research system, the MIRP was reorganized and inaugurated as the Center for Molecular Imaging Science (CMIS).
The CMIS has developed many novel probes for molecular imaging, contributing to drug discovery and the development of diagnostic and therapeutic indicators.

Research
expenses(million yen)

1,420
In addition to these intramural funds, individual labs and investigators areencouraged to compete for external funding as well, from sources such as theJapan Society for the Promotion of Science (JSPS), other government agencies, private foundations and industry. These external funds form an importantcomponent of the CDB’s overall funding mix, representing more than 1 billion
yen in external funding for CDB research programs in the fiscal year ending inMarch 2008.

Collaborative
research

Private sector
grants14
MEXT
direct funding

97

365

Total: 996
Othergovernment
agencies(million yen)

521

Laboratory heads ·························· 29
Research scientists ····················· 107
Research associates ······················· 6Technical staff ····························· 102
Assistants ······································ 27
Visiting scientists ··························· 63
Student trainees ···························· 43
Part-time staff ································ 30
Research Promotion Division ········ 51
Other ··············································· 52
Total ············································· 510

115

2008 CDB Symposium

Turning Neurons into a Nervous System
March 24-26, 2008
The RIKEN CDB held its sixth annual symposium on the theme “Turning Neurons into a Nervous System,” from March
24 to 26. The three-day program featured a broad spectrum of talks ranging from the genetic and molecular activities
that guide the morphological and migratory behaviors of individual neurons, to the complex networks that allow huge
numbers of neurons to work together in a coordinated fashion to achieve higher brain functions. In addition to the nearly
30 invited talks, each day included a poster session in which more than 80 presenters discussed their work. The annual
symposium series, which was launched in 2002, was established as a forum for addressing diverse aspects of developmental biology and the mechanisms of regeneration and aims to promote the free, timely and borderless exchangeof research achievements.

Session 1

Session 5

William H. KLEIN

Barbara HEMPSTEAD(The University of Texas,
M. D. Anderson Cancer Center, USA)
Matthew KELLEY (NIDCD/NIH, USA)

(Weill Medical College of Cornell University, USA)

Raj LADHER(RIKEN Center for Developmental Biology, Japan)
William SNIDER (UNC Neuroscience Center, USA)

Session 2

Elizabeth GROVE (University of Chicago, USA)
Yoshiki SASAI

Session 6(RIKEN Center for Developmental Biology, Japan)(RIKEN Brain Science Institute, Japan)
Silvia ARBER (University of Basel, Switzerland)

Hitoshi OKAMOTO

Session 3

Adrian W. MOORE(RIKEN Brain Science Institute, Japan)
Kozo KAIBUCHI (Nagoya University, Japan)
Vivian Yi Nuo POON (Stanford University, USA)

Session 7

Masatoshi TAKEICHI(RIKEN Center for Developmental Biology, Japan)
Takeshi IMAI (The University of Tokyo, Japan)
Mineko KENGAKU (RIKEN Brain Science Institute, Japan)

Session 4

Hideki ENOMOTO(RIKEN Center for Developmental Biology, Japan)

Takako MAKITA(Howard Hughes Medical Institute,
Johns Hopkins University, USA)
Hideaki TANAKA (Kumamoto University, Japan)

Session 8

Dietmar SCHMUCKER (Harvard Medical School, USA)
Larry ZIPURSKY(Howard Hughes Medical Institute/ UCLA, USA)
Session 9

Thomas C. SÜDHOF
(Howard Hughes Medical Institute, USA)Mei ZHEN (Samuel Lunenfeld Research Institute, Canada)

116

2009 CDB Symposium

Shape and Polarity
March 23-25, 2009
Session 10

Joshua R. SANES (Harvard University, USA)
Aaron DIANTONIO(Washington University School of Medicine, USA)
Session 11

Mario R. CAPECCHI (University of Utah, USA)
Session 12

Joseph G. GLEESONThe seventh CDB symposium on “Shape and Polarity” will be held on March 23 to 25 in the CDB Auditorium. The focus will be on questions such as how
cells become polarized and the roles of cell polarity,and how cell populations organize into complex
tissue and organs through rearrangement of polarized cells. The function of anisotropic organizationand distribution of components such as proteins at
the cellular level will also be addressed, along with
its function in asymmetric cell division and other developmental processes.

(University of California, San Diego, USA)

Christopher A. WALSH(Howard Hughes Medical Institute, BIDMC, USA)

Li-Huei TSAI
(Picower Institute for Learning and Memory, USA)
Session 13

Yi Eve SUN (UCLA Medical School, USA)
Hideyuki OKANO(Keio University School of Medicine, Japan)

Arturo Alvarez-BUYLLA(University of California, San Francisco, USA)
Session 14

Ikue MORI (Nagoya University, Japan)
Edwin W. RUBEL (University of Washington, USA)
Eiji MATSUNAGA (RIKEN Brain Science Institute, Japan)
Session 15

Herwig BAIER (University of California, San Francisco, USA)
Takao K. HENSCH (Harvard University, USA)

Invited speakers

Julie AHRINGER (University of Cambridge, UK)
Jeffrey D. AXELROD (Stanford University, USA)
Ping CHEN (Emory University, USA)Jon CLARKE (King's College London, UK)
Anne EPHRUSSI (EMBL, Germany)
Jiri FRIML (Ghent University, Belgium)
Bob GOLDSTEIN (University of North Carolina at Chapel Hill, USA)
Hiroshi HAMADA (Osaka University, Japan)
Shigeo HAYASHI (RIKEN CDB, Japan)
Carl-Philipp HEISENBERG (MPI-CBG, Germany)
Robert INSALL (Beatson Institute for Cancer Research, UK)
Christopher KINTNER (The Salk Institute, USA)
Juergen KNOBLICH (IMBA, Austria)
Thomas LECUIT (IBDML, France)
Fumio MATSUZAKI (RIKEN CDB, Japan)
Roberto MAYOR (University College London, UK)
Hiroki NISHIDA (Osaka University, Japan)
Shigeo OHNO (Yokohama City University, Japan)
Sarah RUSSELL (Peter MacCallum Cancer Centre, Australia)
Hitoshi SAWA (RIKEN CDB, Japan)
Rudolf WINKLBAUER (University of Toronto, Canada)
Zhenbiao YANG (University of California, Riverside, USA)

Session 16

Tobias BONHOEFFER(Max-Planck-Institut für Neurobiologie, Germany)

Barry J. DICKSON(IMP-Research Institute of Molecular Pathology, Austria)

117

CDB Seminars
The CDB has made special efforts to develop a full and diverse program of invited seminars by scientists from aroundthe world. To date, the Center has hosted more than 400 such talks, in addition to numerous meetings, internal forums
and colloquia. The following speakers presented CDB Seminars in the period from January to December 2008.

118

date

title

speaker

01-09

FANTOM and genome-wide technologies

Yoshihide HAYASHIZAKI

02-01

Drosophila as a model for investigating obstructive pulmonary diseases:
Suppression of NF-kB-mediated innate immune responses in flies and humans
by elevated CO2 levels (hypercapnia)

Greg BEITEL

02-01

Genome evolution and the origins of the innate immune repertoire: insightsfrom the coral Acropora and other "lower" animals

David J. MILLER

03-10

Kruppel-like factors: Key molecular regulators of cardiovascular biology

Mukesh K. JAIN

04-01

RNAi screens for novel candidate insulin receptor-like proteins turned up an
unexpected role of EGF signaling in aging

Hiroaki IWASA

04-01

Getting published in Nature Neuroscience

Annette MARKUS

04-15

From stem cells to neuronal networks, from mouse to man: Genetic
mechanisms of development and evolution of the cerebral cortex

Pierre VANDERHAEGHEN

04-17

Molecular motors, adaptor proteins and Rab GTPases control vesicle
movements along microtubules

Ilya GRIGORIEV

04-17

Live imaging analysis of morphogenesis and lineage formation in the earlymouse embryo

Yojiro YAMANAKA

04-23

Kid-mediated anaphase chromosome compaction safeguards mouse early
embryos against multinuclear formation

Miho OHSUGI

04-30

Mechanosensory Tip-link is an asymmetric filament with Cadherin 23 andProtocadherin 15 at opposite ends

Hirofumi SAKAGUCHI

05-08

Nuclear transport as a regulator of cell differentiation

Noriko YASUHARA

05-08

Analysis of axis formation by observing the cell behaviors during the earlymouse development

Toshihiko FUJIMORI

05-08

Spermatogenic-stem-cell System and its spatiotemporal regulation in themouse testis

Shosei YOSHIDA

05-12

Macro- and micro-environmental regulation of ectodermal organ stem cells

Cheng-Ming CHUONG

05-19

Physical forces generated by cells and sensed by cells

Toshihiko OGURA

06-02

Integrating positional information in Xenopus and Drosophila embryos

Eddy M. DE ROBERTIS

06-02

The role of transcription factor Six3 in forebrain development

Lila Solnica-KREZEL

06-06

Multipotency of early and late Isl1+ cardiac progenitors

Atsushi NAKANO

06-12

Characteristics of epithelial intercellular adhesion and cell proliferation

Sachiko TSUKITA

07-02

Sculpting cell membranes: Understanding pathways of endocytosis andexocytosis

Harvey T. MCMAHON

date

title

speaker

07-07

Trophic factor signaling in the nervous system

Carlos IBANEZ

07-15

Life and death in dopamine neurons - Insights from TGFbeta-HIPK2 signaling
pathway

Eric HUANG

07-16

Biosynthesis and processing of brain-derived neurotrophic factor (BDNF) in
CNS neurons

Tomoya MATSUMOTO

07-22

Regulation and function of Hox genes during chick paraxial mesoderm
development

Tadahiro IIMURA

09-05

Chemicals and neurons controlling nematode behavior

Paul STERNBERG

09-05

Scientific publishing, choices and challenges

Katrina KELNER

09-11

Sex determination and the role of SRY

Ryohei SEKIDO

09-22

Intrinsic factors controlling neural stem cell properties: a tale of two Sox genes

Robin LOVELL-BADGE

10-31

Nuclear regulation during newt lens regeneration

Nobuyasu MAKI

11-10

The role of lineage and cell interactions in the specification of blood stem cellfate during embryonic development of the zebrafish

Robert HO

11-10

Using zebrafish to study pancreas development

Victoria PRINCE

11-13

Translational control of cellular senescence

Joel D. RICHTER

11-14

Understanding Wnt/beta-catenin signaling in development and disease

Xi HE

11-19

Meganucleases with tailored specificities for genome engineering purposes

Julianne SMITH

12-02

Defining the spatiotemporal origins of GABAergic cortical interneuron subtypes

Goichi MIYOSHI

12-03

Population dynamics of human ES cell cultures: Self-renewal, adaptation andcancerPeter W. ANDREWS

12-03

Neural stem cells and neurogenesis in the adult central nervous system

Jonas FRISÉN

12-05

Molecular control of cell fate specification in the cerebral cortex

Victor TARABYKIN

12-08

Microtubules, microfluidics, and cell shape

Phong T. TRAN

12-08

Adenomatous Polyposis Coli (APC) protein directly nucleates actin assembly by
a novel mechanism

Kyoko OKADA

12-08

Biologists in the director's chair: Communicating with Maya animation software

Dan W. NOWAKOWSKI

12-09

Human embryonic stem cell Research at Royan Institute

Hossein BAHARVAND

12-24

Nematode dauer formation as a case study for evolution of developmental
plasticity

Akira OGAWA

119