2

3

4

5

6

7

Remembering Dr. Yoshiki Sasai

On August 5, all of us at the CDB were shocked and deeply
saddened to learn of the sudden, tragic death of Yoshiki
Sasai. Dr. Sasai was one of the CDB’ s founding groupdirectors, and an internationally regarded developmental andstem cell biologist. His laboratory was known for its innovative
research into dorsoventral axis patterning in the embryo andthe induced differentiation of pluripotent stem cells,
particularly into neuronal lineages.

Dr. Sasai joined the RIKEN Center for Developmental Biology

in 2000, leaving a professorship at Kyoto University, which hehad attained at the age of only 36. In addition to his research
efforts, Dr. Sasai played a central role in guiding theestablishment of the CDB facilities and research infrastructure,
as well as in helping to develop an international work
environment.

His earliest work at the CDB focused on two complementary areas: gaining a better understanding of themolecular interactions underlying dorsoventral specification in the Xenopus embryo, and learning ways to steer thedifferentiation of mouse embryonic stem cells down specific neural lineages, such as dopaminergic or sensory
neurons. This work produced a series of high-profile publications that made the Sasai laboratory a world leader inboth fields.

A subsequent study on the problem of maintaining human embryonic stem cells in culture yielded an unexpected
breakthrough when members of the lab discovered that inhibition of the Rho-associated protein kinase (ROCK)
pathway resulted in markedly improved survival of ESCs in vitro. ROCK inhibition has gone on to become astandard technique for the culture of human ESCs around the world.

Perhaps the most noteworthy accomplishments, however, were yet to come. The following year, the Sasai lab
made the first of its reports of the self-organized formation of three-dimensional neural tissue-like structures from

ESCs in vitro. By minimizing external stimuli during ESC culture, the lab was able to show that mouse ESCs areable to spontaneously give rise to highly organized tissue closely resembling the embryonic cerebral cortex invitro.

A string of related work followed soon thereafter, with demonstrations of similar activity giving rise to optic cup
and pituitary (adeno-hypophysis) tissue from mouse ESCs, and recapitulations of the cortical and retinal
self-organization phenomena using human ESCs as well.

Dr. Sasai’ s untimely death represents a loss that the international scientific community is still coming to terms

with. For all of us at the CDB, he will be remembered as an enormously capable and highly driven colleague,
mentor, and friend.

8First patient undergoes novel
iPSC-based procedure
for age-related maculardegeneration

In September 2014, a female patient sufferingfrom exudative age-related maculardegeneration (AMD) became the world’ s firstrecipient of an experimental transplant of a cellsheet derived from induced pluripotent stem

cell (iPSCs). The patient is part of a pilot study
led by Masayo Takahashi (Project Leader,
Laboratory for Retinal Regeneration) incollaboration with Yasuo Kurimoto (Head,
Ophthalmology Department, Institute for

Biomedical Research and Innovation Hospital),
who performed the procedure. This study isdesigned to make a preliminary evaluation of
the safety of the transplantation of

iPSC-derived sheets of retinal pigment

Restructuring of CDB in theaftermath of the STAP scandal

epithelium (RPE) cells. There will be a one-year
intensive monitoring period to evaluate thetransplant for adverse events and possible earlysignals of clinical benefit, after which the patient
will undergo periodic follow-up for a total ofthree years post-transplant. The plan for thecurrent pilot study is to enroll six volunteer

participants in total. Additional information
about the study is available at:

http://www.riken-ibri.jp/AMD/english/index.html.

In response to recommendations from anexternal committee investing the STAP

misconduct incident, RIKEN undertook a major

reorganization of the Center for Developmental
Biology. From November 2014, the CDB labs
have been reorganized into four fundamental

research programs: the Cellular Environment
and Response Research Program, theOrganogenesis Research Program, the StemCells and Organ Regeneration Research

Program, the Mathematical Developmental
Biology Program as well as a Research andDevelopment Program, which will seek todevelop new biomedical technologies based on
CDB research results. Many laboratories werereassigned to other RIKEN Centers, including
the Center for Life Science Technologies (CLST)
and the Quantitative Biology Center (QBiC),
which is expected to promote greaterinteraction between life science research

centers in Kobe. The new CDB also features aflatter research organization, in which alllaboratories share the same designation ofTeam, as distinct from the multi-tiered system
employed from 2000 to 2014. A detailed

overview of the post-reform organization of the
CDB can be found on the CDB website, and on
RPE cells generated from iPSCs

p.5 of this report.

9Preclinical testing of iPSC-derived RPEshows efficacy and safety in animal models

Hiroyuki KAMAO

Age-related macular degeneration (AMD) is a diseaseof the eye that affects visual acuity in the elderly.
Degeneration of the retinal pigment epithelium (RPE), a
pigmented cell layer in the eye that supports and supplies
nutrients to the photoreceptors, is a major pathogenic
factor in AMD. Of the major forms of this disease,neovascular AMD is more prevalent in Asian populations.
There is currently no cure for AMD, but several groups are
exploring the possibility that pluripotent stem cells may be
a useful source for generating functional RPE for use intransplantation. The efficacy and safety of such stem cellderived tissue, however, needs to be tested extensively
before studies in human can begin.
A new preclinical study by Hiroyuki Kamao and colleagues
in the Laboratory for Retinal Regeneration reports that
RPE cell sheets generated from induced pluripotent
stem cells (iPSCs) show an excellent efficacy and safety
profile in animal transplant models. Published in Stem Cell

10

Reports, this work lays the groundwork for the world’s firstpilot clinical study on transplanting hiPSC-RPE cell sheets
to AMD patients.
Kamao et al. produced RPE cell sheets that do not havean integrated artificial scaffold, but can nonethelesswithstand the manipulations required for transplantation
procedures. They were able to do so by growing the stem
cell-derived RPE cells on a degradable temporary collagenscaffold. Several groups have previously reported using
artificial scaffolds to generate RPE cell sheets; however,these scaffolds run the risk of causing inflammation or
creating physical barriers that limit nutrient flow fromchoroid to photoreceptor cells. The group confirmed
that, in vitro, the cell sheets expressed RPE markers
and basement membrane components and had formed
functional tight junctions exhibiting polarized secretion of
growth factors similar to the activity of native RPE.

©2014 Elsevier B.V.
Kamao H, et al. Characterization of Human Induced Pluripotent Stem Cell-Derived
Retinal Pigment Epithelium Cell Sheets Aiming for Clinical Application.
Stem Cell Reports 2 1–14 (2014)

Kamao and colleagues next tested the function of thesehiPSC-RPE cell sheets in vivo by transplanting them into
the subretinal space in immunosuppressed three-weekold RCS rats, a model of inherited retinal degeneration.
Using electroretinogram (ERG), they watched for changes
in photoreceptor activity at six weeks post-transplant, andthree weeks later measured the thickness of the outer
nuclear layer (ONL), where the photoreceptors reside.
Compared to control and sham-surgery animals, rats thatreceived transplants of either hiPSC-RPE cell sheets or cellsuspensions maintained the ONL and showed significant
restoration of ERG responses, suggesting that the hiPSCgenerated RPE cell sheets are functionally similar to native
RPE.
To confirm the feasibility of using the iPSC-derived grafts
in clinical studies, the group tested and compared theimmunogenicity of autologous and allogeneic grafts incynomolgus monkeys. To verify whether immunogenic
responses are triggered by allogeneic or autologous
grafts in vivo, Kamao transplanted cell sheets derived
from a single donor monkey into the subretinal space offour non-immunosuppressed monkeys—the donor andthree MHC-incompatible animals—and observed them

Grafts are cut from hiPSC-RPE cell sheet using a laser micro dissection
system.

for one year. The group found that tissues surrounding
the allografts showed typical signs of graft rejection,
while those surrounding the autograft showed no signsof rejection. The transplanted cells also did not contributeto tumor formation, which has been one of the major
concerns for pluripotent stem cell-derived cell products.
A final comparison between the transplantation of cellsuspensions and cell sheets indicated that cell sheets
offer several advantages; they can be transplanted directlyat the lesion site and monitored easily, as they remainanchored in place.
“Safety is of the utmost importance, and these animal
experiments show that autologous grafts are notrejected and non-tumorigenic. Tumor formation in theeye is relatively rare to begin with, and at least for AMD,transplantation does not require a large number of cells,
so quality control is relatively simpler,” says Takahashi. “We
can also detect subtle changes in the eye, so in the eventthat any abnormalities develop, we can catch them at an
early stage. All of these factors make AMD a good target
for the world’s first clinical study of iPS cells.”

Monkey iPSC-RPE cell sheet (arrow) transplanted into subretinal space of a
non-human primate.

11New recipe for hPSC cryopreservation

Teruo AKUTA, Shin-Ichi NISHIKAWA

Human pluripotent stem cells (hPSCs) remain in the
spotlight for the possibilities they hold out for uses inregenerative medicine. In the lab, hPSCs are famously
difficult to handle, as they are extremely sensitive toexternal stresses such as those caused by experimental
manipulations or changes in culture conditions, as thesecan lead to rapid changes in cell properties or cell death.
One especially stressful technique is cryopreservation,
which is characterized by low recovery rates of
hPSCs after the thawing processes. Further technicaldevelopments to cryopreservation are needed to improvecell recovery rates, but any such method must also be
simple, efficient, and inexpensive.
In a new study focused on hPSC preservation, Teruo
Akuta, Keitaro Imaizumi, and colleagues in the Laboratory
for Stem Cell Biology (Shinichi Nishikawa, Group Director;
the Nishikawa lab closed in March 2013) have developed a
new slow-freezing method that can be used to store large

12

quantities of hPSCs easily and cost-effectively. Publishedin PLoS One, they took a commercially available medium
used for the cryopreservation of cells from cord bloodand bone marrow, and optimized it specifically for hPSCs.
The group also determined the most effective dissociation
solution to be used in combination with the modified
medium. The study was carried out in collaboration withthe laboratory of Shin Kawamata (Vice Director, Foundationfor Biomedical and Innovation).
The two methods currently used for hPSC cryopreservation
are vitrification and slow-freezing. Vitrification, which
requires special skills, uses liquid nitrogen to quickly freeze
the cells placed in a medium with high concentrations ofcryoprotectants and is not suited for the cryopreservationof large quantities of cells. The slow-freezing method,where cells are suspended in cryopreservation media andthen slowly allowed to freeze in a deep freezer overnight, issimpler and can be used to cryopreserve large quantities©2014 Public Library of Science
Imaizumi K, et al. A simple and highly effective method forslow-freezing human pluripotent stem cells using dimethyl sulfoxide,hydroxyethyl starch and ethylene glycol. PLOS ONE 9. e88696 (2014)of cells. The efficacy of any form of cryopreservation
depends on the dissociation step carried out just prior tofreezing, in which the colonies are broken up into small
clusters, as the surface area of clusters bathed directly bycryoprotectant varies by the size of cell clusters following
dissociation.
The group first examined five known dissociating solutions
to determine which is most suited for the cryopreservationof hPSCs in combination with a popular commerciallyavailable freezing medium. Recovery rates of cells
treated with different solutions were then examined
post-cryopreservation by staining them with alkaline
phosphatase (ALP). They found that treatment with
Pronase/EDTA resulted in relatively small, uniform cellcluster sizes, and yielded the highest recovery frequency(44%). Trypsin/EDTA also generated small cell clusters,
but the cell recovery frequency was less than half of thatseen for Pronase/EDTA; other dissociation solutions tested
produced large variations in cell cluster size and cell death
after thawing.
Given its favorable properties, Imaizumi et.al. next
looked to develop a hPSC-specific cryopreservation
medium compatible with Pronase/EDTA. They prepareda panel of new cryopreservation formulas by changing
the concentration of reagents in the freezing medium,and adding other known cryoprotectants. When they
compared the efficacy of cell recovery after thawing, theyfound that the addition of ethylene glycol (EG) yielded
the highest post-thaw recovery rates. They tweaked theformula further to identify the optimal concentration ofreagents plus EG. The most efficient formula, CP-5E,
consisted of a mix of 6% hydroxyethyl starch, a natural
cryoprotectant derived from plants, 5% dimethyl sulfoxide
and 5% EG.
The group next tested the optimized cryopreservation
medium in combination with the Pronase/EDTA solution
on different hiPSC and hESC lines, and found to yieldrecovery frequencies of over 80%. When the group
evaluated the cells before and after cryopreservation, theyfound no significant differences in cell properties. hPSCs
subjected to the new cryopreservation method maintained
their proliferative capacity and pluripotency on thawing,
and showed no structural chromosomal abnormalities.
A major advantage of the hPSC cryopreservation medium
developed by Imaizumi et. al. is the absence of animaland protein components. By simplifying the composition,
they were able to avoid such risks as lot-to-lot variationand infections from pathogenic agents, which can makebovine serum albumin or serum solutions problematic.
The coupling of this new method with a novel dissociation
agent has also made cryopreservation of hPSCs more
efficient and straightforward.
“The increasing demand for use of hPSCs in drug
discovery and regenerative medicine has made it essential(for scientists) to establish safe, simple, efficient, andinexpensive methods for handling these cells,” says
Nishikawa. “We hope that this study will lower some of thetechnical barriers to starting stem cell research, and help
accelerate both basic and applied studies in the field.”Comparison of ALP stainings of hiPSC line 201B7 before and after cryopreservation.
Colony numbers are comparable.

Cell growth curve. Proliferative capacity is maintained
before freezing (blue) and after thawing (red).

13

Mechanisms of chemotaxis andrandom cell movement explained

Masatoshi NISHIKAWA

For centuries, compasses have been used as navigational
instruments enabling explorers to use directional
information to guide them to their destinations. Likewise,
during animal development, some cells follow a
compass-like mechanism that guides their movements
in response to exter nal cues in a process calledchemotaxis. Chemotactic cells sense chemical gradientsin the surrounding environment that induce intracellular
asymmetry of the signaling pathway biased in the directionof the gradient and direct the cells either toward oraway from the stimulus. However, even in the absenceof external gradients, intracellular asymmetry can beproduced spontaneously by the cell, a process that may
be linked to random cell movements. The Laboratory for
Physical Biology (Tatsuo Shibata, Unit Leader) previously
published a quantitative model of a gradient sensing
system that reconstructs the phenomenon of spontaneous
intracellular polarization and describes how this increases

14cell sensitivity and robustness, allowing them to respondto even shallow gradients.
Now, in a report published in Biophysical Journal,
Masatoshi Nishikawa and colleagues in the same labvalidate these models, specifically showing that excitabilityof the signaling pathway leads to spontaneous intracellularpolarization, which can trigger both random movementin the absence of a chemoattractant gradient and biased
movement in the direction of the gradient. Using theameba, Dictyostelium discoideum, they show that the
dynamics of spontaneous intracellular asymmetry isimportant for directional cell movement, and that this
response is driven by an excitability-based mechanism.
The spatiotemporal dynamics of the phosphatidylinositol
3,4,5-triphosphate (PIP3) pathway plays an importantrole in chemotaxis in Dictyostelium cells, where a PIP3enriched membrane domain is produced in response©2014 Cell Press
Nishikawa M, et al. Excitable signal transduction induces both spontaneous and directional cell
asymmetries in the phosphatidylinositol lipid signaling system for eukaryotic chemotaxis.
Biophys J 106.723-34 (2014)

to external stimulation (cAMP) driving changes tocytoskeleton to form pseudopods used for chemotaxis.
Nishikawa et al. observed that cells exhibited random
spontaneous PIP3-domain formation both in absence
and presence of cAMP, with this spontaneous response
appearing more frequently in presence of cAMP. They
also tracked and quantified the cells’ transient response
levels and times when stimulated with various cAMP
concentrations, and found that the average cell responses
were similar, irrespective of cAMP concentration. The sizeof the PIP3 domain observed at peak response levels was
proportional to cAMP concentration, while the overall PIP3
signal levels remained constant, suggesting that because
the intensity of the responses was not influenced by thestrength of the external stimulus but determined by the
intracellular intrinsic activity, this is an excitable system.
If the chemotactic response is indeed driven by an
excitability-based system, when the external stimulus
exceeds a certain threshold to trigger a response, any
responses unfolding even after the stimulus is removed
can be considered to be maintained by the cell itself.
When the group applied a short impulse stimulus that was
shorter than the average response time, the cells showed
intracellular polarization of PIP3 domains similar to that
produced spontaneously, and as the length of impulses
were increased, larger PIP3-domains were produced,
consistent with their expectations. They also confirmedthat the PIP3 signaling system has a refractory period,
another indicator of an excitable system, by applying aseries of successive stimuli to the cells at various timeintervals.

The researchers next looked at how external gradientsaffected spontaneous intracellular polarization. Cells
placed in conditions with a chemical gradient are
known to induce localization of PIP3 in the membrane
domain facing the highest cAMP concentration, driving
chemotaxis in the same direction. From a theoreticalperspective, spontaneous polarization of PIP3 domains,which appears in random directions in the absence of a
gradient, becomes biased in the presence of an external
gradient. The group found that, when presented with agradient, the PIP3 domains appearing at random in theabsence of a gradient exhibit a bias in the direction of thegradient. But, there were no significant changes in theintensity of the PIP3 response before or after the gradient
was applied. These experiments demonstrate that spatialcAMP gradients orient the localization of the PIP3 domain,
without changing the characteristics of the PIP3 domain.
“Our current study clearly shows that the signaling pathway
for chemotaxis has excitability-based characteristics,” says
Shibata. “Cells do not act only in response to external
stimuli. They have a set response pattern, which, whenaltered slightly, produces sensitive and robust responses
for diverse situations. In this study, we found that the
localization of the set response pattern changes according
to the gradient, which may also explain how multicellularaxis and polarity formation occur. The same system mayfunction in complex multicellular morphogenesis as well.”Model of the biased-excitability system. In the absence of external stimulus (left), PIP3 domains areproduced in random manner. In the presence of external stimulus (right), PIP3 domains are oriented to
localize in the direction of the stimulus.

15

Express line to erythropoiesis

Primitive erythropoiesis during animal development
requires stepwise differentiation from pluripotent epiblast
cells, which undergo gastrulation to form the three germlayers: endoderm, ectoderm, and mesoderm. Newly
formed mesoderm generates hemangioblast cells, theembryonic origins of the blood and vascular system, byinitiating expression of early hematopoietic factors. Thesehemangioblasts then give rise to primitive erythrocytes.
A number of studies have noted a delay between theappearance of early hematopoietic markers in mesoderm
and that of erythroid lineage-specific globin genes inhemangioblasts. The purpose and the mechanisms at
work during this delay, however, remain unclear.
New work by Wei Weng and colleagues in the Laboratory
for Early Embryogenesis (Guojun Sheng, Team Leader)
now shows that epiblast cells can be made to differentiate
directly into the erythroid lineage in vivo through forced
expression of five erythropoiesis-related transcription
factors and inhibition of the FGF pathway. Published in
Stem Cell Reports, these findings suggest a new modeof induced differentiation that bypasses the step-by-steproutine seen during development.
Sheng’s group previously reported that FGF signaling
has an inhibitory effect on primitive blood differentiation inchicken. Using an FGF inhibitor to block FGF expression
levels in the embryo, this time, Weng and Sheng looked at
the effect of FGF activity on early erythropoiesis and on theexpression of early hematopoietic factors, SCL and LMO2.
A comparison of the effects of FGF inhibition in embryos
before or after endogenous expression of SCL and
LMO2, suggested that FGF is needed to switch on bothof these genes in mesoderm. In FGF-inhibited embryos,
they found that exogenous expression of SCL and LMO2
in mesoderm is sufficient to induce robust erythroid
differentiation.

Wei WENG (with Yun-chan)

16

Steering pluripotent epiblast cells to differentiate directly
into the erythroid lineage proved to be trickier than justinhibiting FGF and turning on two genes. Weng and Shengsearched transcriptomic data for candidate genes withsimilar expression profiles to SCL and LMO2 and putative
regulatory roles during the transition to erythropoiesis.
They identified LDB1 and E2A, both of which have been
implicated in mammalian hematopoiesis, and are thought
to form a complex with SCL, LMO2 and GATA2 to regulate
erythropoiesis in vitro.

©2014 Elsevier B.V.
Weng W and Sheng G. Five transcription factors and FGF pathway
inhibition efficiently induce erythroid differentiation in the epiblast.
Stem Cell Reports 262-70 (2014)

They made expression constructs of these five genes (SCL,
LMO2, GATA2, LDB1, and E2A) and found that when theyelectroporated the five constructs in combination with
FGF inhibition before the endogenous expression of early
hematopoietic regulatory genes, they were able to induce
strong expression of rho, an erythroid marker, in epiblast.
Interestingly, the combination of these five gene constructs
and FGF inhibition also induced expression of a gene
involved in heme biosynthesis in epiblast, suggesting that
this directed differentiation also initiates expression of otheressential factors required for primitive erythropoiesis.

“ M a n y g ro u p s h a v e t r i e d u s i n g o n e o r t w o k e yhematopoietic genes to induce erythroid differentiationfrom mesoderm. What we have done here is to look at
inducing erythroid differentiation in mesoderm as wellas other lineages,” says Weng. “We have been able toget high efficiency in inducing differentiation not only of
mesoderm into a specific lineage, but also in pluripotent
epiblast and in ectoderm derivatives that are alreadyless pluripotent than epiblast. But it remains to be seen
whether this same efficiency can be achieved in vitro.”

Primitive erythropoiesis during embryonic development (left). Direct
induction of erythroid differentiation in the epiblast (right).

Globin-positive cells induced in the forebrain and head surface ectoderm
by five transcription factors (SCL, LMO2, LDB1, E2A, and GATA2).

17

2014 Events
The RIKEN Center for Developmental Biology strives to engage with the public through a variety of media, including its website andprinted materials, media coverage, and direct interactions such as guided tours, Open House events, and other public outreachactivities. In addition to its open to the public activities, the CDB also organizes events intended to bring the Center’s scientists
together for discussion outside of the laboratory environment.

Start of CDB Lecture Series
The CDB Lecture Series is the Center’s new flagship forum for talks byleading international scientists, who are invited to introduce their latest
research and visit individual laboratories over the course of two days
for additional discussion. The annual schedule is intended to featureapproximately eight CDB Lectures by eminent researchers. The program wasinitiated in fall of this year, and the inaugural CDB Lecture was delivered by
Ben Simons of the University of Cambridge on September 8.

Open House 2014
The RIKEN CDB held its annual Open House on Saturday, October 25, 2014.
Around 1,500 people visited the Center to see and learn firsthand about thelatest research in development and regenerative biology. This year’s OpenHouse featured public lectures by Tomoya Kitajima (Team Leader, Laboratory
for Chromosomal Segregation) who talked about the causes of oocyte aging
and by Takashi Tsuji (Group Director, Laboratory for Organ Regeneration)on the latest developments of research carried out in his laboratory related
to teeth and hair regeneration. Twenty-four laboratories also took part in theevent by organizing booths that introduced their research through posters
and alluring research images. Many laboratories set up microscopes at theirbooths to allow the visitors to observe actual cell and tissue samples as well
as some model organisms used in their research. Scientists and graduate
students were on hand to explain and answer questions about their researchfrom the visitors who dropped by their booths. In a special lecture program
co-organized with Kobe City, Masayo Takahashi (Project Leader, Laboratory
for Retinal Regeneration) gave a talk to a full crowd on the current state of theher clinical research using iPSC-derived retinal pigment epithelial cells at theneighboring Institute for Biomedical Research and Innovation.

CDB Retreat in Sasayama
The CDB held its annual research retreat in the mountains of Sasayama
on September 29 and 30. Laboratory heads, research scientists, student
trainees, technical staff and members of the administration took part in thisclosed meeting to share the latest developments in their work, as well as
to get better acquainted with colleagues from other parts of the Center.
The 2-day program featured talks by recent appointees, as well as special
lectures by a number of scientists from other institutes, including CDB alumni
Masahiko Hibi (Nagoya University) and Yuki Sato (Kyushu University).

18

Drosophila embryo at the beginning of tracheal
placed invagination (magenta). Cell outline islabeled green.

Aizawa Laboratory
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 ofGerontology, then two years as a research fellow at the University of Washington. He returned to the Tokyo
Metropolitan Institute of Gerontology in 1982, where he remained until 1986, when he moved to the RIKEN
Tsukuba Life Science Center. He was appointed professor in the Kumamoto University School of Medicine
in 1994, and served in that position until 2002. Since 2000 he has served as CDB deputy director and groupdirector of the Vertebrate Body Plan lab, as well as head of the Animal Resources and Genetic Engineering
Laboratory.
The Aizawa lab closed as part of the reorganization of the CDB in November 2014.
Dr. Aizawa is now affiliated with RIKEN CLST.

Pre-gastrular developmental routines have changed dramatically over the course ofvertebrate evolution. The yolk, for example, has been stored in various amounts, andcleavage development has changed from the holoblastic cleavage seen in amphibian, bichir,lamprey, and mammal to the meroblastic form in teleost, reptile, and avian. Extraembryonicstructures in amniotes, especially those in mammal, were acquired later in vertebrate
evolution, and were formed by altering post-cleavage development. In light of thesealterations, it is obscure how the mechanisms of anterior-posterior (A-P) axis formationand head development are related and have diverged in each family of vertebrates; these
are among the most fundamental events in vertebrate development. Our aim is to assess
A-P axis formation in an ancestral amniote and its divergence in each amniote lineage bycomparative studies in extant amniote animals: mouse, rabbit, pig, Suncus, chick, quail, softshelled turtle, gecko, and gray short-tailed opossum.

Staff
Research Specialist

Technical Staff

Yoko SUDA
Michio YOSHIDA

Michiko INOUE
Eriko KAJIKAWA
Tomomi OHMURA
Daisuke YAMAMOTO

Research Scientist

Hideharu HOSHINO
Visiting Scientist

Daisuke KUROKAWA
Masaki TAKEUCHI

Publications
Courchet J, et al. Terminal axon branching is regulatedby the LKB1-NUAK1 kinase pathway via presynaptic
mitochondrial capture. Cell 153.1510-25 (2013)
Kurokawa D, et al. Otx2 expression in anterior
neuroectoderm and forebrain/midbrain is directed by morethan six enhancers. Dev Biol 387.203-13 (2014)
Huilgol D, et al. Dual origins of the mammalian accessory
olfactory bulb revealed by an evolutionarily conserved
migratory stream. Nat Neurosci 16.157-65 (2013)
Abe T, et al. Visualization of cell cycle in mouse embryoswith Fucci2 reporter directed by Rosa26 promoter.
Development 140.237-46 (2013)
Inoue F, et al. Gbx2 directly restricts Otx2 expression
to forebrain and midbrain, competing with class III POU
factors. Mol Cell Biol 32.2618-27 (2012)

Suncussoft-shelled turtle

20

gray short-tailed opossum

gecko

Reconstitutive Developmental Biology
Miki EBISUYA Ph.D.

Miki Ebisuya received her Ph.D. from the Graduate School of Biostudies, Kyoto University under thesupervision of Prof. Eisuke Nishida in 2008. She worked briefly as a postdoctoral fellow in the samelaboratory before being appointed a group leader of the Career-Path Promotion Unit at Kyoto University in
2009, where she began research in synthetic biology. In 2013, she was selected as a PRESTO researcher
in the area of design and control of cellular functions, and was also appointed to her current position asunit leader of the Laboratory for Reconstitutive Developmental Biology at the CDB.
As part of the reorganization of the CDB in November 2014, the Reconstitutive Developmental Biology lab
was transferred to the RIKEN Quantitative Biology Center.

Staff
Research Scientist

Mitsuhiro MATSUDA
Ryoji SEKINE
Technical Staff

Yuko SAKAIDE

Publications
Matsuda M, et al. Synthetic lateral inhibition governs celltype bifurcation with robust ratios. Nat Commun DOI:
10.1038/ncomms7195, in press
Imajo M, et al. Dual role of YAP and TAZ in renewal of theintestinal epithelium. Nat Cell Biol 17.7-19 (2015)
Koga M, et al. Foxd1 is a mediator and indicator of the cell
reprogramming process. Nat Commun 5.3197 (2014)
Matsuda M, et al. Synthetic signal propagation throughdirect cell-cell interaction. Sci Signal 5.ra31 (2012)

In our lab, we strive to create or reconstitute biological mechanisms. Our aim ofreconstitution is to test the sufficiency of current understandings of mechanisms of interest,
as well as to discover unexplained or unexpected elements through observation. We areparticularly focused on reconstituting mechanisms of intercellular communications. In
metazoan development, for instance, intercellular communications induce the spontaneous
differentiation of groups of cells underlying the cellular patterns seen in various tissues (known
as self-organized cell differentiation patterns). We use simplified, artificial networks of the
Delta-Notch pathway, which is responsible for signaling between adjacent cells, in an effort
to reconstitute the essence of self-organized cell differentiation patterns in mammalian cells
in vitro. Our ongoing projects also include the reconstitution of asymmetric pattern formation
using artificial Nodal-Lefty networks, and the reconstitution of an intercellular synchronization
mechanism. Artificially reconstituted systems also have the advantage of facilitating
measurements and the modification of parameters, which we hope will contribute to thequantitative understanding of intercellular communications.

Examples of gene networks for cell-cellcommunication.
Mutual activation between adjacent cells
results in signal propagation (upper),while mutual inhibition results in cellpattern formation (lower).

Reconstitution of signal propagation.
The gene expression signal (green) propagated to theneighboring cells along a line.

21

In Vitro Histogenesis
Mototsugu EIRAKU Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/eiraku.html
Mototsugu Eiraku received his Ph. D. from The University of Kyoto in 2005, and joined the RIKEN Brain
Science Institute as a research scientist that same year. In 2006, he joined the RIKEN CDB Laboratory
for Organogenesis and Neurogenesis, where he served as a research scientist until 2010. That year, he
was promoted to Deputy Unit Leader of the Four-dimensional Tissue Analysis Unit within the Division for
Human Stem Cell Technology, and in 2013 was appointed Unit Leader. From November 2014, he hasserved as leader of the In Vitro Histogenesis Unit.

Recent advances in stem cell technology have enabled the generation of various potentially
medically useful cell types from ES and iPS cells, including neurons, photoreceptors,
and cardiac muscle. However, the extent to which such cells mimic their in vivo function
when plated on culture dishes is limited. In vitro histogenesis unit seeks to develop new
approached to cell technology that will allow for more realistic in vitro recapitulation throughthree-dimensional tissue formation from stem cells. To this end, we establish efficient threedimensional culture of ES cell-derived brain and retinal tissues, and also develop new
cutting-edge live imaging technology and related optic devices for four-dimensional analysisof relatively large tissues. We also support and collaborate with CDB and non-CDB users ofthese optics technologies.

Staff
Research Scientist

Technical Staff

Shunsuke MORI
Nozomu TAKATA

Eriko SAKAKURA

Visiting Scientist

Yuiko HASEGAWA

Hidetaka SUGA

Junior Research Associate

Publications
Kadoshima T. et al. Self-organization of axial polarity, insideout layer pattern, and species-specific progenitor dynamics
in human E cell-derived neocortex. Proc Natl Acad Sci U S
A 110. 20284-9 (2013)
Nakano T et al. Self-formation of optic cup and storable
stratified neural retina from human ESCs. Cell Stem Cell 10.
771-85 (2012)
Eiraku M et al. Relaxation-expansion model for self-driven
retinal morphogenesis: a hypothesis from the perspective of
biosystems. Bioessays 34. 17-25 (2012)
Eiraku M. and Sasai Y. Mouse embryonic stem cell culture
for generation of three-dimensional retinal and corticaltissues. Nature Protocols 7. 69-79 (2011)
Eiraku M, et al. Self-organizing optic-cup morphogenesis inthree-dimensional culture. Nature 472. 51-56 (2011)
Eiraku M. et al. Self-organized formation of polarized corticaltissues from ESCs and its active manipulatin by extrinsic
signals. Cell Stem Cell 3. 512-32 (2008)

Photoreceptor cell (red) and bipolarcells (green) in ES cell-derived neuralretinaSelf-organized optic cup formationfrom ES cell

22

ES cell-derived neuroepithelium (lifeact-GFP)Neuronal Differentiation and Regeneration
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 GDNF Family 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 Neuronal Differentiation and Regeneration lab closed in April 2014.
Dr. Enomoto is now affiliated with Kobe University.

Staff
Research Scientist

Student Trainee

Pilaiwanwadee HUTAMEKALIN
Keisuke ITOH
Toshihiro UESAKA

Sachie ONO
Part-Time Staff

Chihiro NISHIYAMA

Azusa FUKUDA
Toko KONDO
Akira YAMAOKA

Junior Research Associate

Assistant

Mitsuhiro IWASAKI
Yohei YONEKURA

Yuka NODA

Technical Staff

Publications
Uesaka T et al. GDNF signaling levels control migration andneuronal differentiation of enteric ganglion precursors. J
Neurosci 33. 16372-82 (2013)
Nishiyama C, et al. Trans-mesenteric neural crest cells are
the principal source for the colonic enteric nervous system.
Nat Neurosci 15.1211-18 (2012)
Nagashimada M, et al. Autonomic neurocristopathyassociated mutations in PHOX2B dysregulate Sox10
expression. J Clin Invest 122.3145-58 (2012)
Uesaka T and Enomoto H. Neural precursor death iscentral to the pathogenesis of intestinal aganglionosis in Ret
hypomorphic mice. J Neurosci 30.5211-8 (2010)
Uesaka T, et al. Diminished Ret expression compromises
neuronal survival in the colon and causes intestinal
aganglionosis in mice. J Clin Invest 118.1890-8 (2008)
Gould T W, et al. The neurotrophic effects of glial cell linederived neurotrophic factor on spinal motoneurons are
restricted to fusimotor subtypes. J Neurosci 28.2131-46(2008)

The architecture of the nervous 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 deathof countless individual cells and is regulated by intercellular communication and cooperative
activity.
Our laboratory is interested in questions fundamental to the development of the nervous
system: determining the specific extracellular signals and cell autonomous routines thatparticipate in the processes of neural development, and resolving the means by which diverse
families of signaling molecules coordinate and interact at the molecular and cellular levels. To
address these questions, we focus on a class of signaling molecules known as neurotrophic
factors, most particularly the GDNF Family Ligands (the GFLs). This family of neurotrophic
factors includes four known members – GDNF (Glial cell line Derived Neurotrophic Factor),
Neurturin, Artemin, and Persephin. The GFLs signal via receptor complexes formed by the
RET receptor tyrosine kinase and one of four co-receptors, GRFα1-4. In vitro, these four
receptors show differential affinities for specific GFLs. GFL signaling has been shown toaffect neuronal growth, survival and migration as well as axon guidance, and defects in thissignaling 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 neuraldevelopment, differentiation and pathology, and ultimately to potential clinical applications in
the treatment of nervous system trauma and disease.

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

23

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

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

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

24

Staff
Research Scientist

Assistant

Chun-Chun CHENG
Ritsuko MORITA
Ko TSUTSUI

Asako NAKAGAWA
Part-Time Staff

Sonoko TOCHITANITechnical Staff

Noriko BAN-SANZEN

Publications
Donati G, et al. Epidermal Wnt/beta-catenin signaling
regulates adipocyte differentiation via secretion of
adipogenic factors. Proc Natl Acad Sci U S A 111.E1501-9(2014)
Fujiwara H, et al. The basement membrane of hair follicle
stem cells is a muscle cell niche. Cell 144.577-89 (2011)
Watt F. M. and Fujiwara H. Cell-extracellular matrix
interactions in normal and diseased skin. Cold Spring Harb
Perspect Biol 3. (2011)
Ferreira M, et al. An activating ß1 integrin mutation increases
the conversion of benign to malignant skin tumors. Cancer
Res 69.1334-42 (2009)
Fujiwara H, et al. Regulation of mesodermal differentiationof mouse embryonic stem cells by basement membranes. J
Biol Chem 282.29701-11 (2007)
Fujiwara H, et al. Rac regulates integrin-mediatedendothelial cell adhesion and migration on laminin-8. Exp
Cell Res 292.67-77 (2004)

Neocortical Development
Carina HANASHIMA Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/hanashima.html
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 was engaged 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.

Staff
Research Scientist

Technical Staff

Yuko GONDA
Takuma KUMAMOTO
Ken-ichi TOMA

Chihiro NISHIYAMA

Foreign Postdoctoral Researcher

Torsten BULLMANN
Visiting Researcher

Tien-Cheng WANG

Part-Time Staff

Chika KUMAMOTO
Reiko ODA
Assistant

Eri KOJIMA

Publications
Toma K, et al. The timing of upper-layer neurogenesis isconferred by sequential derepression and negative feedback
from deep-layer neurons. J Neurosci 34. 13259-76 (2014)
Kumamoto T and Hanashima C. Neuronal subtype
specification in establishing mammalian neocortical circuits.
Neurosci Res (2014)
Yeh M, et al. Robo1 modulates proliferation andneurogenesis in the developing neocortex. J Neurosci 34.
5717-31 (2014)
Kumamoto T, et al. Foxg1 coordinates the switch fromnonradially to radially migrating glutamatergic subtypes in
the neocortex through spatiotemporal repression. Cell Rep
3. 931-45 (2013)
Gonda Y, et al. Robo1 regulates the migration and laminar
distribution of upper-layer pyramidal neurons of the cerebral
cortex. Cereb Cortex 23. 1495-508 (2013)
Kasukawa T, et al. Quantitative expression profile of distinct
functional regions in the adult mouse brain. PLoS One
6.e23228 (2011)

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

The neocortex is composed of distinct
neuronal subtypes that establish 6 layers.
Coronal section of a one-week-old mouseneocortex showing Reelin (layer I, cyan),
Brn2 (layer II/III, red), RORß (layer IV, green),
Ctip2 (layer V/VI, blue) expressing neurons.

Robo1 receptor is necessary for inside-out layer formation.Upon Robo1-suppression, laterborn neurons labeled with DsRed at embryonic day (E) 16 (magenta) cannot migrate pastearlier-born cells (E15 GFP-labeled cells, green).

25

Morphogenetic Signaling
Shigeo HAYASHI Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/hayashi.html
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 andcell morphogenesis in Drosophila.

Our research aim is to understand fundamental mechanisms of animal morphogenesis
with particular interest in the mechanical basis of tissue movement and its interaction withextracellular environment. Our main research focus is the tracheal system in the Drosophila
embryo, a network of tubular epithelium used as a respiratory organ. Trachea is formed
through invagination, tube formation, elongation, fusion and final maturation as a respiratoryorgan. We are particularly interested in the mechanical control of epithelial archetechtures.
Epithelium is stabilized by cell-cell adhesion and cell-matrix adhesion. Breaking this stability
is essential for initiating morphogenetic movement. We found that prospective tracheal
primordium is under negative tension (pressurized). Anisotropic redistribution of tissue
tension and timely mitosis initiates local mechanical instability that leads to tissue invagination
movement (Kondo and Hayashi, 2013). Once tracheal network is formed, tube diameterand length are enlarged to reach the final size. Tracheal size change involves increase in cellsize, espacially the increase of apical cell area facing the luminal side. Key question is how
indivisually controlled cellular growth is coordinated to form coherent tissue archetecture.
We found that extracellular matrix in the luminal space plays a central role by providing
mechanical stability to the tubules (Dong et al., 2013, 2014). Defects in extracellular matrix
components leads to destabilization of tube shape and malformation, resembling tuble
shapes found in organs in pathological conditions.
Another area of research is the mechanism of cell morphogenesis. We ask the question of towhat extent single cells can autonomously organize nanometer scale cellular patterns. Ourstudies have uncovered the role of cellular trafficking center as an organizer of cell elongation(Otani et al., 2011).

Staff
Research Scientist

Technical Staff

Toshiya ANDO
Xiaorui CAI
Bo DONG
Aki HAYASHI
Takuya MAEDA
Yosuke OGURA
Tetsuhisa OTANIAkiyo KIMPARA
Housei WADA

Special Postdoctoral Researcher

Takefumi KONDOInternational Program Associate

Guangxia MIAO
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
Assistant

Ryoko ARAKI

Publications
Dong B, et al. A fat body-derived apical extracellular matrix
enzyme is transported to the tracheal lumen and is required
for tube morphogenesis in Drosophila. Development
141.4104-9 (2014)
Miao G. and Hayashi S. Manipulation of gene expression by
infrared laser heat shock and its application to the study oftracheal development in Drosophila. Dev Dyn (2014)
Dong B, et al. Balance between apical membrane growthand luminal matrix resistance determines epithelial tubule
shape. Cell Rep 7. 941-50 (2014)
Niwa N, et al. Homeogenetic inductive mechanism ofsegmentation in polychaete tail regeneration. Dev Biol 381.
460-70 (2013)
Dong B, et al. Rab9 and retromer regulate retrograde
trafficking of luminal protein required for epithelial tube
length control. Nat Commun 4.1358 (2013)
Kondo T. and Hayashi S. Mitotic cell rounding accelerates
epithelial invagination. Nature 494.125-9 (2013)
Otani T, et al. IKKepsilon regulates cell elongation throughrecycling endosome shuttling. Dev Cell 20.219-32 (2011)

Drosophila embryo at the beginning of tracheal placed invagination (magenta). Celloutline is labeled green.

26

Developmental Epigenetics
Ichiro HIRATANI Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/hiratani.html
Ichiro Hiratani received his B. Sc. in Biological Sciences from the University of Tokyo in 1998, and his
Ph.D. from the same institute in 2003 for his work on Xenopus embryonic development with Masanori
Taira. In 2003, he moved to the United States for his postdoctoral training with David Gilbert at the State
University of New York Upstate Medical University and studied the developmental regulation of DNA
replication timing and nuclear genome organization. He later moved with Gilbert to Florida State University
in 2006 and returned to Japan in 2010 to take a position as Assistant Professor at the National Instituteof Genetics. In 2011, he was awarded a grant from the JST PRESTO program in Epigenetics. He wasappointed to his current position as Team Leader at the RIKEN CDB in October 2013.

Staff
Research Scientist

Student Trainee

Hisashi MIURA
Naoyuki SARAI
Tsuneyuki TATSUKEDavid MEYER
Technical Staff

Akie TANIGAWA

Publications
Ryba T, et al. Genome-scale analysis of replication timing:from bench to bioinformatics. Nat Protoc 6.870-95 (2011)
Hiratani I. and Gilbert D. M. Autosomal lyonization of
replication domains during early Mammalian development.
Adv Exp Med Biol 695.41-58 (2010)
Ryba T, et al. Evolutionarily conserved replication timing
profiles predict long-range chromatin interactions anddistinguish closely related cell types. Genome Res 20.76170 (2010)
Hiratani I, et al. Genome-wide dynamics of replication
timing revealed by in vitro models of mouse embryogenesis.
Genome Res 20.155-69 (2010)
Hiratani I, et al. Replication timing and transcriptional
control: beyond cause and effect--part II. Curr Opin Genet
Dev 19.142-9 (2009)
Hiratani I, et al. Global reorganization of replication domains
during embryonic stem cell differentiation. PLoS Biol 6.e245(2008)
Hiratani I, et al. Differentiation-induced replication-timingchanges are restricted to AT-rich/long interspersed nuclear
element (LINE)-rich isochores. Proc Natl Acad Sci U S A
101.16861-6 (2004)

Early- and late-replicating DNA localize tothe interior (green) and periphery (red) of
the nucleus, respectively. Because of thisrelationship, genome-wide DNA replication
profiling (graphs) can be used to deduce the 3D
genome organization at the sequence level.

We wish to clarify the molecular mechanisms underlying global facultative heterochromatin
formation during early mouse embryogenesis, with the belief that understanding thedevelopmental regulation of higher-order chromosome organization will lead to a deeper
understanding of cell differentiation.
The term facultative heterochromatin refers to chromosomal regions that condense, become
inactivated, and are stably maintained in this manner after a certain developmental stage.
A classic example is the inactive X chromosome in mammals, which becomes detectable
immediately prior to the formation of germ layers and is stably maintained thereafter in all
downstream lineages. Intriguingly, we recently discovered that many autosomal domains
also undergo a similar process of facultative heterochromatin formation at the samedevelopmental stage, which accounts for more than 6% of the genome. This suggests thatfacultative heterochromatin formation at this stage is not specific to the inactive X, but rather
a more widespread phenomenon affecting the entire genome. Recent studies have also
revealed low reprogramming efficiency of cells immediately after this developmental stage,already as low as downstream somatic cell types. Thus, this facultative heterochromatin is acommon epigenetic feature of all somatic cells beyond the germ layer formation stage, andthe reprogramming experiments imply a potential link to the cell's differentiated state.
For these reasons, we combine genome-wide approaches with molecular and cell biology
and imaging techniques to elucidate the molecular mechanisms underlying the facultative
heterochromatin formation process. In the future, we will address the biological significanceof this phenomenon and eventually wish to understand the fundamental implications ofhigher-order chromosome organization.

Genome-wide DNA replication profiling
during ES cell differentiation can reveal
domains that show large-scale changes innuclear organization.

Electron microscopy reveals a large-scale genome
reorganization during pre- (left) to post-epiblast (right)
transition, consistent with predictions made by DNA
replication profiling.

27

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

The mammalian central nervous system is composed of enormous numbers of neurons.
How do these neurons establish their identity and form functional neuronal circuitries? To
address this question, we are studying the mouse olfactory system as a model system. In
the mouse olfactory system, odorants are detected by ~1,000 types of olfactory sensory
neurons, each expressing a single type of odorant receptor. The olfactory bulb, the primaryolfactory area of the brain, receives inputs from olfactory sensory neurons through 1,000
sets of glomeruli. These inputs are then processed in the olfactory bulb circuits and are thensent out to the olfactory cortex. These complex neuronal circuits emerge in an autonomous
fashion based on cell-cell interactions and neuronal activity after birth, rather than by strict
genetic programs. Using the olfactory bulb as a model system, we are trying to understandsimple rules behind the formation of complex neuronal circuitry.

Staff
Research Scientist

Student Trainee

Satoshi FUJIMOTO
Ryo IWATA
Meng-Tsen KE
Marcus LEIWE

Shuhei AIHARA
Aya MURAI
Richi SAKAGUCHITechnical Staff

Grandjean HARISH

Miwako NOMURA
Yoichi SAKASHITAInternAssistant

Eri YAMASHITA

Publications
Imai T. Construction of functional neuronal circuitry in theolfactory bulb. Semin Cell Dev Biol 35. 180-188 (2014)
Nakashima A, et al. Agonist-Independent GPCR Activity
Regulates Anterior-Posterior Targeting of Olfactory Sensory
Neurons. Cell 154. 1314-25 (2013)
Ke M. T, et al. SeeDB: a simple and morphology-preserving
optical clearing agent for neuronal circuit reconstruction.
Nat Neurosci 16. 1154-61(2013)
Imai T, et al. Topographic Mapping - The Olfactory System.
Cold Spring Harb Perspect Biol 2. a001776 (2010)
Imai et al., Pre-target axon sorting establishes the neuralmap topography. Science 325. 585-90 (2009)
Imai et al. Odorant receptor-derived cAMP signals direct
axonal targeting. Science 314. 657-61 (2006)

Adult Thy1-YFP-H mouse brain was cleared with an
optical clearing agent SeeDB and imaged using twophoton microscopy. 3D rendering image of a volume of 4mm x 5 mm x 2 mm, encompassing cerebral cortex andhippocampus, is shown.

28

Tracing of sister mitral cells associated with a common glomerulus in the olfactorybulb. Neurons connecting to a single glomerulus were labelled by neuronal tracer andthe sample was cleared with SeeDB.

Axial Pattern Dynamics
Hidehiko INOMATA Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/inomata.html
Hidehiko Inomata gained his doctorate from the Tokyo Institute of Technology in 2003, and moved to takea research scientist position at the Foundation for Biomedical Research and Innovation in Kobe, Japanthat same year. From 2004 to 2012, he worked as a research scientist in the RIKEN CDB Laboratory for
Organogenesis and Neurogenesis, and in 2012, he was appointed a Senior Scientist in the same lab.
In 2011, he was also named a researcher in the Japan Science and Technology Agency (JST) PRESTO
program. He was appointed Team Leader of the Laboratory for Axial Pattern Dynamics in 2014.

Developmental processes take place through the exchange of information by cells within theconstrained spatial environment of the embryo. Such intercellular communication is essential
for the formation of a well-ordered body; in its absence, our individual cells would behave
in an uncoordinated fashion, and fail to follow the patterns needed for the development ofa head, limbs, or other body parts. Factors that play central roles in such developmental
signaling are known as morphogens.

Staff
Research Associate

Taichi MATSUNAGATechnical Staff

Setsuko KANAMURA
Kaori NIIMI

Publications
Inomata H, et al. Scaling of dorsal-ventral patterningby embryo size-dependent degradation of Spemann'sorganizer signals. Cell 153.1296-311 (2013)
Takai A, et al. Anterior neural development requires Del1,a matrix-associated protein that attenuates canonical Wnt
signaling via the Ror2 pathway. Development 137.3293-302(2010)
Inomata H, et al. Robust stability of the embryonic axial
pattern requires a secreted scaffold for chordin degradation.
Cell 134.854-65 (2008)
Arakawa A, et al. The secreted EGF-Discoidin factor xDel1
is essential for dorsal development of the Xenopus embryo.
Dev Biol 306.160-9 (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. A scaffold protein JIP-1b enhancesamyloid precursor protein phosphorylation by JNK and
its association with kinesin light chain 1. J Biol Chem
278.22946-55 (2003)

In our research we will seek to gain a deeper understanding into processes informed bypositional information in a spatial context (developmental fields), using vertebrate (mainly frogand zebrafish) axis formation as a model. The establishment of the frog dorsoventral axis
depends on gradients of morphogens secreted by the organizer region. In order to ensurethat development based on simple concentration gradients is stably reproducible, cell-cellcommunications mediate by morphogens need to be robust against perturbations. One
example of such robustness can be seen in the response of a frog embryo when bisected:
such embryos follow normal developmental patterns, despite being half the ordinary size, aphenomenon known as ‘scaling.’ Our team has previously shown how scaling is maintained
through morphogen-mediated intercellular communication when the spatial size of theembryo is perturbed. In our lab, we address visualization of morphogen gradients and invivo imaging along with biochemical approaches to study how developmental robustness ismaintained.
We are also working to develop methods for controlling the shape of morphogen gradients.
Gradients are primarily regulated by production, diffusion, and degradation, which indicatesthat by controlling these factors, it should be possible to arbitrarily design gradients thatreconstruct tissue patterns in the embryo. By using such methods, we hope to gain a deeper
understanding of developmental systems.

FRAP assays of mEGFP-tagged Sizzled shown by snapshots. From left to rightcolumns; before bleaching, at the end of bleaching, 50 s after bleaching, and 230 s
after bleaching.

Graded DV patterning by the reconstructed organizer. Substantial pSmad accumulation
was observed on the side opposite to the reconstructed organizer, indicating that the
influence of chordin was distance-sensitive.

29

FGF makes a noise in maintaining
hair cell progenitors

Sound is generated by vibrations of air molecules,
which are detected and interpreted by the ear. These
vibrations are transduced, in turn, from the ear’s tympanic
membrane, to the ossicular bones in the inner ear, to theperilymph in the spiral-shaped cochlea, and finally to themechanosensory stereocilia of the hair cells (HCs). HCs
then send electrical signals to the brain to be interpreted
into what we perceive as sound. HCs in the cochlea arehighly organized, with the mammalian cochlea comprising
three rows of outer hair cells (OHCs), a single row of innerhair cells (IHCs), and populations of support cells thatsurround the HCs. Damage to the HCs by extrinsic or
intrinsic factors can lead to hearing impairments, and oncedamaged, the hair cells cannot regenerate themselves.
The mechanisms of HC development have long beenstudied as deciphering this process may lead to inroads ingenerating new HCs to replace damaged ones.
In a recent study, Kazuya Ono and colleagues in the
Laboratory for Sensory Development (Raj K. Ladher,
Laboratory head) shed new light on the role of one
signaling pathway, the fibroblast growth factor (FGF)
pathway, in cochlear HC development. Published in PLOS
Genetics, they demonstrate that this pathway plays acrucial role in the maintenance of pre-hair cell progenitors
and in the commitment of progenitors to differentiate into
hair cells.
HCs form from an area of the inner ear epithelia which isinduced to express Sox2, a transcription factor involvedin maintaining pluripotency, by Notch-Jagged (Jag) 1
signaling. BMP signaling then specifies a region of cells
within the Sox2-positive patch, also called sensory patch,
to form the prosensory domain. These are the immediate
precursors of HCs and support cells. The prosensory
domain cells subsequently stop proliferating and a second
wave of Notch signaling selectively induces the prosensory
domain to differentiate into HCs or support cells. In
addition to these pathways, studies by others have shownthat the FGFR1 pathway is also important in cochlear
HC development, as deleting the Fgfr1 gene resulted in
HC loss. However, the question remained: how does thissignaling pathway function in HC development.

Kazuya ONO, Tomoko KITA

30

Ono and his colleagues decided to focus on addressing
this question by first determining when this signal isneeded by reducing Fgfr1 levels at different stages in miceand noting its effects. They used two Fgfr1 conditional©2014 Public Library of Science
Ono K, et al. FGFR1-Frs2/3 signalling maintains sensory progenitors during
inner ear hair cell formation. PLoS Genet 10.e1004118 (2014)

mutant mouse lines, which differed in the timing of Fgfr1
deletion; in the first line, Fgfr1 deletion occurred just prior
to E10.5, before the induction of the sensory patch, andin the second line, the gene was deleted at E12.5. HC
loss was observed in both lines; however, the loss wasmore dramatic when Fgfr1 levels were reduced at E10.5,
suggesting that FGFR1 signaling was needed before
E12.5.

signaling, Sox2 was not maintained. Thus, the FGFR1
signaling pathway appears to be important for maintaining
Sox2 expression during sensory patch formation, but is
not involved in initiating the exiting of the cell cycle. Further
analysis of the FGFR1 signaling pathway also revealed that
the MAP kinase signaling cascade, acting through Frs2/3,
is turned on downstream of FGFR1 and regulates Sox2
expression.

Having established that FGFR1 signaling is needed before
E12.5, the group next turned their attention on determining
which step was disrupted. The defect was traced backto the earliest step in HC development, the formation of
the Sox2 positive sensory patch. They found that while
Sox2 was switched on normally, in the absence of FGF“In this study, we answer the longstanding question of how
FGF signals regulate the hair cell numbers,” says Ladher.
“It may be possible in the future to produce hair cells in
culture, which can then be transplanted to replace thedamaged inner ear hair cells, by modulating FGF signals.”

Cross sections of cochleae. Left: Three outer hair cells (OHCs) and one inner hair cell (IHC) surrounded by Sox2-positive support cells (pink). Middle:
Cochlea from mutant with relatively weak phenotype. Only 2 OHCs can be seen. Right: Cochlea from mutant with severe phenotype. No HCs detected.

31

Eye on ESC- and iPSC-derived retinal
tissue transplantation

Michiko MANDAI, Juthaporn ASSAWACHANANONT

Retinitis pigmentosa (RP) is a group of genetic diseasesof the eye that are characterized by the degeneration ofphotoreceptors, a type of neural cell which detects light.
Symptoms of RP include night blindness and progressive
narrowing of peripheral vision, and in some advanced
cases, may lead to blindness. The pathogenesis of thisdisease is as varied as the large number of known causal
genes or mutations, and there are currently no effective
treatments available. In recent years, cell transplantation
using cells derived from embryonic stem cells (ESCs) or
induced pluripotent stem cells (iPSCs) has emerged asa potential treatment method for RP, but progress has
been hindered by technical issues to obtain sufficientnumbers of photoreceptor cells at an ideal developmental
stage for transplantation, which can survive and form
functional synapses with neighboring neural networks
within the complex retinal tissue. A groundbreaking reportpublished in 2011 by Mototsugu Eiraku (Unit Leader, Four-32

dimensional Tissue Analysis Unit) and the Laboratory for
Organogenesis and Neurogenesis of RIKEN CDB cleared
this barrier, showing that ESCs could be induced to form
photoreceptors as well as three-dimensional optic cups
similar to retinal tissues in a 3D culture system, paving theway to consider tissue level transplantation.
Now, a new study carried out by International Program
Associate, Juthaporn Assawachananont, Deputy Project
Leader, Michiko Mandai, and others in the Laboratory for
Retinal Regeneration (Project Leader, Masayo Takahashi),shows in that ESC- or iPSC-derived retina sheetstransplanted in the eye can survive and integrate with thehost retina over an extended period. Published in Stem
Cell Reports, their study suggests that, at least in mouse,transplanted retina sheets derived from ESCs or iPSCs arecapable of developing structures similar to normal retinal
tissues and possibly neural networks, especially whengrafts are prepared from younger stage cultures.

©2014 Elsevier B.V.
Assawachananont J, et al. Transplantation of embryonic and induced
pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice.
Stem Cell Reports 2. 662-74 (2014)

The team first modified the 3D culturing method
developed by Eiraku et al. (2011) to generate larger
quantities of retinal tissue from mouse ESCs and iPSCs.
They tweaked the culture medium by adding a retinoic
acid antagonist (RRA) and increasing the concentration of
extracellular matrix, which led to a more efficient inductionof mESCs and miPSCs into retinal progenitors. Using
this modified medium, they examined the optic vesiclelike structures that eventually developed into retinalike structures at differentiation day (DD) 24 composedof an assembly of typical cell types found in the retina
such as photoreceptors, amacrine cells, horizontal cells,
and glial cells. The differentiation patterns of these cells
resembled the development of the mouse retina, and cell
cultures younger than DD20 were considered equivalent
to embryonic retinal tissues, whereas cultures DD21 andolder were similar to postnatal retinas.
The group then examined the feasibility of transplanting
grafts of retina-like sheets prepared from ESC- or iPSCderived optic cup structures induced in 3D culture. Retinalike sheets from various stages between DD11 to 24
were prepared and then transplanted in the subretinal
space of mouse models for retinal degeneration, whichhave lost most of their rod photoreceptors, and the posttransplantation effects were tracked over the course of six
months. When retina sheets from younger cultures (DD1117) were transplanted into host retina, close to 90% of

iPSC-derived retina-like sheet (DD14) transplanted to mouseforms retina-like structure, including outer nuclear layer(ONL), inner nuclear layer (INL), and inner plexiform layer.

the grafts went on to form a structured outer nuclear layer(ONL) made up of the cell body of photoreceptors. In
contrast, in transplants using retina sheets prepared from
cultures DD18 and older, most of the grafts (80%) failed tomaintain an ONL structure.
Closer examination of the transplanted area revealed
that mature photoreceptors in the grafts which formed
ONL also possessed the typical outer segment andinner segment. The outer segment had stacked disc-likearrangement when they were nestled adjacent to hostretinal pigment epithelial cells, while the inner segment
contained mitochondria. Immunostaining experiments
indicated that photoreceptors in the grafts showedassembly of their synaptic ends with dendrites of hostbipolar cells, resembling normal retinal development. The
grafts showed a range of patterns, but those preparedfrom cultures DD17 and younger showed the mostpotential to form synapses and maintain an ONL structure.
“Our study is the first to demonstrate that retinal
tissue derived from ESCs or iPSCs can be used fortransplantation, and the transplanted tissue can survive
and integrate with the host retina, even in advanced
stages of degeneration,” explains Mandai. “We still need tocarry out more experiments to determine the functionalityof the transplanted retinal tissue, but we hope to explore
the possibility of translating our work into clinical studies
for humans, using human derived tissues.”

3D reconstruction of immunostaining of transplanted
area. Photoreceptors (green) from graft form
synaptic connections (red) with host bipolar cells.

33

Balancing act by ECM regulates lengthof tracheal tube

A complex network of luminal structures plays an importantrole in bodily functions such as transporting gases andfluids associated with metabolism or homeostasis. The
tube length and diameter of these tubular networks vary
according to size of the animal, the location inside thebody, and of the molecules that will circulate through thetube to ensure efficient transport. The Drosophila embryo,for example, prior to hatching, develops a tracheal
tube that fits its tiny body. Past studies of this tracheal
system indicate that apical membrane synthesis, lumen
components, and cell-cell adhesion between epithelial
cells have an important role in tracheal tube formation;
however, the detailed mechanism has remained unclear.
In a new study published in Cell Reports, Foreign
Postdoctoral Researcher, Bo Dong, from the Laboratory
for Morphogenetic Signaling (Shigeo Hayashi, Group
Director) in collaboration with Edouard Hannezo of theInstitut Curie in France reveal that the extracellular matrix(ECM) plays a regulatory role in determining tube length.
Using the Drosophila tracheal system, they demonstratethat elastic ECM and its interaction with the encircling
apical membrane of the tracheal epithelium are key factors
in regulating tube length.
The Drosophila tracheal system consists of a thick tube
of epithelial cells running through the center of the bodywith smaller and narrower tubes branching off to thedorsal and ventral sides. During development, the tracheal
lumen is filled with ECM, which is broken down prior
to hatching and replaced by gases allowing it to fill its
physiological role. The tracheal epithelial cells are arranged
with their apical membrane facing the lumen and the basal
membrane facing the outside.

Edouard HANNEZO, Shigeo HAYASHI, Bo DONG

34

D o n g e t a l . f i r s t s e a rc h e d f o r m u t a n t s s h o w i n gabnormalities in tracheal tube length, and came acrossa fly mutant that had an abnormally long and sinusoidalshaped tracheal tube, but with a diameter similar to thatin wild-type flies. This fly had a mutation in the shrub
gene, which encodes part of a protein complex involved
in membrane remodeling for vesicular transport. They
examined the cell membrane on both apical and basalsides of trachea epithelial cells by immunostaining eachside with different colored markers and found that,compared with wild-type, only the apical membrane of
shrub mutants had elongated along the anterior-posterior(AP) axis. The basal membrane was nearly unchanged andthere were no significant differences in cell number.

©2014 Cell Press
Dong B, et al. Balance between Apical Membrane Growth andLuminal Matrix Resistance Determines Epithelial Tubule Shape. Cell Rep 7. 941-50 (2014)

The defective transport machinery in shrub mutants resultsin many membrane proteins accumulating in the cytoplasm
rather than at the membrane; the apical membrane protein
Crumbs (Crb), which is involved in apical membranebiogenesis, is one such protein. Crb is normally localized
to the apical membrane, but in the shrub mutant, Crb
was found amassed in the endosome, stimulating apicalmembrane overgrowth.
As the orderly wave-like pattern of the trachea tube in
shrub mutants suggests a mechanical effect, such as aforce antagonistic to the elongational force of the apicalmembrane, the group shifted to focus on the ECM
filling the tracheal lumen, which is made up of chitin, apolysaccharide that uniformly fills the lumen, and of protein
components such as Dumpy (Dp) a giant protein involvedin maintaining epithelial integrity. They used GFP-tagged
luminal proteins and FRAP (fluorescence recovery after
photobleaching) experiments to investigate the physicalproperties of Dp and found that while it barely moves
within the lumen, the protein itself is stretched as the tube
elongates. Thus, the resistance from the elastic ECM
is likely the force that counteracts the elongating force
exerted by the apical membrane.

Dong et al. used these findings to establish a physicalmodel of tube length that recreates the mechanism
through computer simulation. Their model assumes thatthe apical membrane encircles and binds to the elastic
cylindrical matrix; when apical membrane growth isstimulated, it creates an elongating force along the AP
axis, which will in turn stretch the ECM. The trachea willstop elongating when the apical membrane’s elongating
force and the ECM’s elastic force are balanced. The
group was able to verify the relevance of their model by
confirming that it gave close predictions of the actual
phenotypes observed in shrub and other mutants withvarying lengths and shapes of elongated trachea.
“As one of the major components of the body, the ECM
is often seen as having a supportive role in the body, but
our current study reveals that it has a prominent role inregulating morphogenesis as well,” says Hayashi. “Wehope to continue closing in on the mechanisms underlying
how shapes of organisms, which are closely associatedwith function, are determined.”

Left: Drosophila tracheal tube. The shrub mutant has an abnormally elongated and wavy tracheal tube running through the embryo. Right: Stainingof tracheal lumen (pink), and of apical membrane (light blue), basement membrane (green) and nucleus (dark blue) of tracheal epithelial cells shows
excess apical membrane growth in shrub mutant, but with no major differences in cell number.

35

Epidermal Wnt/ß-catenin signal regulates
adipocyte differentiation in underlying hypodermis

Hironobu FUJIWARA

The mammalian skin is an important multifunctional organwith roles in sensation, thermoregulation and metabolism,
in addition to protecting the body from the external
environment. It is organized into a complex layered
structure of epidermis, dermis and hypodermis, with eachlayer made up of a mix of cell and tissue types. Thesecells and tissues use intercellular communication to guidemorphogenesis, as well as maintenance (or regeneration)of the skin. The synchronicity between the growth cycleof hair follicles (HFs) and the fluctuation in the thickness ofthe dermal adipocyte layer (hypodermis) is one model ofintercellular communication in the skin. The hypodermis
increases in thickness as HFs enter the growth phase toextend deeper into the dermal layer, and becomes thinnerwhen HFs regress and enter the quiescent phase. Whilethe hypodermis has been reported to secrete factors that
control the HF growth cycle, it remained unknown whether,conversely, the HFs regulate adipogenesis or thickness ofthe hypodermis.

36

Now, a new study by Hironobu Fujiwara (Team Leader,
Laboratory for Tissue Microenvironment) and his former
colleagues at the Cancer Research UK Cambridge
Research Institute, in collaboration with scientists at Kings
College London, reveals that activation of the canonical
Wnt/ß-catenin signaling in the epidermis, which is known
to promote HF growth, induces adipocyte differentiationin the hypodermis. Published in the Proceedings of the
National Academy of Sciences, they show that epidermal
Wnt/ß-catenin signal regulates morphogenesis of thehypodermis by triggering the secretion of adipogenic
factors, synchronizing the HF growth cycle with adipocyte
differentiation.
Because activation of the epidermal Wnt/ß-cateninsignaling pathway is known to induce HFs to enter thegrowth phase, the research group turned their attention tothis signal to determine its effects on the hypodermis. They
first generated a transgenic mouse, in which the epidermal©2014 National Academy of Sciences
Donati G, et al. Epidermal Wnt/beta-catenin signaling regulates adipocyte
differentiation via secretion of adipogenic factors.
Proc Natl Acad Sci U S A 111.E1501-9 (2014)

Wnt/ß-catenin signaling was blocked. During embryonicskin development, HF growth appeared normal in thismouse, but the hypodermis was thinner overall than that ofwildtype. There was, however, an increase in the thicknessof the reticular dermis, the lower layer of the dermis, whichappeared to compensate for the thinner hypodermis. In
the adult mutant mouse, adipocyte progenitor numbers
were normal, but there was a significant decrease in the
thickness of the differentiated adipocyte layer. Therefore,
epidermal Wnt/ß-catenin signaling is important for
hypodermal adipocyte differentiation, and when blocked,
limits the differentiation of adipocyte progenitors to matureadipocytes.
The group next analyzed the effects of activating theepidermal Wnt/ß-catenin signal in the hair growth cycle,
and found that HFs in the rest phase were induced toenter the growth phase and the hypodermis showed
simultaneous expansion when Wnt/ß-catenin was
activated. While there were no changes in the number ofadipocyte progenitors, they did see a rise in the thicknessof the differentiated adipocyte layer. And when epidermal
Wnt/ß-catenin signal was activated in a transgenic mousewith impaired HF development, they observed that
adipogenesis could be induced even in the absence of
HFs, indicating that Wnt/ß-catenin signal acts directly on
the dermal layer to induce adipogenesis, not indirectly via
HF formation.
Because the epidermis and dermis are separated by abasement membrane, it was thought that the epidermal
Wnt/ß-catenin signal is conveyed to the subdermal layer
via a factor secreted by the epidermis. After reanalyzing
gene expression profile data of previously reported
transgenic mice with induced activation of epidermal Wnt/
ß-catenin signal, the group discovered that genes related

to extracellular regions were upregulated in the epidermis,and genes related to fat metabolism, differentiation, andproliferation were upregulated in the underlying dermis.
Thus, the analyses suggest that a secreted factor is
produced from the epidermis, and that responses such as
adipocyte proliferation and differentiation are seen in thedermis when epidermal Wnt/ß-catenin is activated.
To confirm whether the epidermis secretes adipogenic
factor(s), the group cultured adipocyte progenitors
in medium conditioned with epidermis from different
transgenic mice. In medium conditioned with epidermis
in which Wnt/ß-catenin activation was induced, moreprogenitors entered adipogenesis prematurely thanseen under WT conditions, and in medium conditioned
with Wnt/ß-catenin–inhibited epidermis, very few
adipocytes were generated. These results indicated thatepidermal Wnt/ß-catenin signal triggers the secretion ofan adipogenic factor from the epidermis, which in turninduces adipocyte differentiation. They tested possible
candidate factors gleaned from the gene expression profile
data, and identified three factors (BMP2, BMP6, andIgf2) secreted by the epidermis that triggered adipocyte
differentiation.
“Our study demonstrates that epidermal Wnt signal
re g u l a t e s a d i p o g e n e s i s i n t h e h y p o d e r m i s , a n dsynchronizes adipocyte differentiation with HF growth
cycle,” says Fujiwara. “For growing HFs to extend below
the skin, additional space needs to be created in thedermal layer to accommodate the downgrowth. It is
possible that inducing adipocyte differentiation, which has
high volume expansion rates, is an efficient way for HFs toaccomplish this.”

Hypodermis in the skin is thinner in Wnt/ß-catenin blocked transgenic mouse than in WT. Left: Changes in volume of hypodermis over 223 days after birthin WT and transgenic mouse. Two center panels: Skin sections. Transgenic mouse has thinner hypodermis and thicker dermis. Right: Immunostaining ofsections. Differentiated adipocytes marked by Fabp4 antibody (green).

37

2014 Courses
As part of its commitment to promoting awareness and understanding of development, regeneration, evolution, stem cells,
regenerative medicine and related fields, and its interests in maintaining close ties with the academic and research communities, the
RIKEN CDB dedicates itself to conducting training and educational programs for a wide range of audiences. Although not a degreegranting institution, the Center works with students, educators, and scientists, as well as other organizations, to develop, implement,
and promote a variety of instructional courses throughout the year.

Summer School for high school students
The CDB’s eighth annual one-day summer workshops for high school students were
held this year on August 5 and 28. The theme of this year’s workshop was “Development
and Differentiation,” and included a talk and laboratory tour by a CDB scientist and lab
experiments. Taking C. elegans as a model, the students learned how molecular factors
function during cell division, and how different types of cells could be produced throughasymmetrical cell division by showing movies and images. In the hands-on component,
students used SDS-PAGE electrophoresis to analyze proteins from brain, liver, andmuscle tissues.

Undergrad internships
The CDB invited 34 undergraduate students from universities around Japan to spend
one week in August as an intern working alongside research scientists in various labs at
the CDB. In addition to carrying out small-scale projects in the host labs, the students
had the chance to hear about the research being done at the CDB through lectures,and lab visits.

Graduate student lectures
The RIKEN CDB maintains close ties with a number of major graduate and medical
schools in the Kansai area, and hosts a two-day lecture program every year to
introduce the students to the labs at the center. Nearly 200 students convened at the
CDB this year to take part in this year’s program, which was held on August 27 and 28.
The event also included exhibitions of model organisms and tours of the lab facilities,
such as the research aquarium and electron microscopy room.

High school teacher workshop
The CDB, with support from the Japanese Society for Developmental Biologists (JSDB),
organized a weekend workshop for high school teachers held on October 4 and 5.
This annual workshop gives the teachers the opportunity to learn actual experimental
techniques used in developmental biology, and learn how they can adapt thesetechniques for a classroom setting. This year’s course focused on microscopic analysisof tissue morphology.

38

Turning the page (scanning EM view of acleavage furrow in an early stage chickembryo, resembling an open book).

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

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

Staff

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

Publications

Research Scientist

Student Trainee

Aurélien COURTOIS
Yogo SAKAKIBARA
Shuhei YOSHIDAYi DING
AssistantKaori HAMADA

Visiting Scientist

Hirohisa KYOGOKUTechnical Staff

Masako KAIDO

Kyogoku H, et al. Nucleolus Precursor Body (NPB): A
Distinct Structure in Mammalian Oocytes and Zygotes.
Nucleus 5.493-8 (2014)
Kitajima T. S, et al. Complete kinetochore tracking reveals
error-prone homologous chromosome biorientation in
mammalian oocytes. Cell 146.568-81 (2011)
Lee J, et al. Unified mode of centromeric protection byshugoshin in mammalian oocytes and somatic cells. Nat
Cell Biol 10.42-52 (2008)
Kitajima T. S, et al. Shugoshin collaborates with protein
phosphatase 2A to protect cohesin. Nature 441.46-52(2006)
Kitajima T. S, et al. Human Bub1 defines the persistentcohesion site along the mitotic chromosome by affecting
Shugoshin localization. Curr Biol 15.353-9 (2005)
Kitajima T. S, et al. The conserved kinetochore protein
shugoshin protects centromeric cohesion during meiosis.
Nature 427.510-7 (2004)

Chromosomes form the belt-like structure during
prometaphase.

40

Kinetochore-microtubule attachment

Histogenetic Dynamics
Erina KURANAGA Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/kuranaga.html
Erina Kuranaga received her doctorate in medical science from the Osaka University Graduate School of
Medicine in 2004, after which she moved to the University of Tokyo Graduate School of Pharmaceutical
Sciences as assistant professor in the Department of Genetics. In 2006, she was promoted toassociate professor in the same department, where she remained until she joined the RIKEN Center for
Developmental Biology as team leader in 2011. She was awarded the Wiley-Blackwell Prize 2012 andEditor-in-Chief Prize from Development, Growth and Differentiation in 2014.

Staff
Research Scientist

Student Trainee

Emi MAEKAWA
Daiki UMETSU

Yuka HAYASHI
Yuhei KAWAMOTO
Shuji SHIMAMURA

Technical Staff

Ayako ISOMURA
Aimi TSUKIOKA

Part-Time Staff

Arata KURANAGA
Yoko UMEGAKI

Publications
Obata F, Kuranaga E, et al. Necrosis-driven systemic
immune response alters SAM metabolism through the
FOXO-GNMT axis. Cell Rep 7.821-833 (2014)
Takeishi A, Kuranaga E, et al. Homeostatic epithelial renewal
in the gut is required for dampening a fatal systemic woundresponse in Drosophila. Cell Rep 3.919-930 (2013)
Kuranaga E, et al. Apoptosis controls the speed of looping
morphogenesis in Drosophila male terminalia. Development
138.1493-9 (2011)
Koto A, Kuranaga E, et al. Apoptosis ensures spacing
pattern formation of Drosophila sensory organs. Curr Biol
21.278-87 (2011)
Nakajima Y, et al. Nonautonomous apoptosis is triggeredby local cell cycle progression during epithelial replacementin Drosophila. Mol Cell Biol 31.2499-512 (2011)
Kuranaga E, et al. Drosophila IKK-related kinase regulates
nonapoptotic function of caspases via degradation of IAPs.
Cell 126.583-96 (2006)

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

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

Caudal view of DE-Cadherin:GFPexpressing Drosophila. This
image was taken before rotation.

41

Evolutionary Morphology
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 professorshipin 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.
As part of the reorganization of the CDB in November 2014, the Evolutional Morphology lab wastransferred to the RIKEN Chief Scientist Laboratories program.

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 thefields of evolutionary morphology and molecular genetics, our lab seeks to expand theunderstanding of the relationship between genome and morphology (or body plan) throughinvestigating the evolutionary changes in developmental processes, and also the processof 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
seek to open new avenues toward answering as-yet unresolved questions about phenotypicevolution in vertebrates at the level of body plans.
Through the study of evolutionarily novel structures, our lab has identified changes indevelopmental mechanisms that have obliterated the structural homologies between
organisms as evidenced in such novel structures as the jaw in gnathostomes (jawedvertebrates) 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 lampreyslack 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 primaryfactors have changed during evolution by comparing the developmental patterns that yieldsuch innovations, and by the experimental construction of phenocopies in one animal thatmimic structures in another.
The turtle’s shelled body pattern appears at the end of a graded series of changes in
the fossil record. Our lab’s research into turtle carapace development addresses thedevelopmental changes that resulted in this abrupt and dramatic morphological change, andis aimed at identifying genes that function differently in turtle and other amniotes, which it ishoped will provide a key to discovering the true targets of natural selection in the acquisitionof a shell.

CT-scanned skeleton of Pelodiscus sinensis juvenile

42

Staff
Research Scientist

Student Trainee

Tatsuya HIRASAWA
Dutel HUGO
Rie KUSAKABE
Takayuki ONAI
Juan PASCUAL ANAYA

Hiroki HIGASHIYAMA
Shinnosuke HIGUCHI
Masashi UMEDA

Visiting Researcher

Part-Time Staff

Christine BÖHMER
Technical Staff

Satoko FUJIMOTO
Tamami HIRAI
Yasuhiro OISI
Iori SATO

InternHülya ŞENShigemi SHIBUYAKazuko YAMAMOTO
Assistant

Naeko MINAMI
Tomoko OYANAGI

Publications
Hirasawa T, et al. The endoskeletal origin of the turtle
carapace. Nat Commun 4.2107 (2013)
Wang Z, et al. The draft genomes of soft-shell turtle andgreen sea turtle yield insights into the development andevolution of the turtle-specific body plan. Nat Genet 45.
701-6 (2013)
Oisi Y, et al. Craniofacial development of hagfishes and theevolution of vertebrates. Nature 493.175-80 (2013)
Ota K. G, et al. Identification of vertebra-like elements andtheir possible differentiation from sclerotomes in the hagfish.
Nat Commun 2.373 (2011)
Nagashima H, et al. Evolution of the turtle body plan by thefolding and creation of new muscle connections. Science
325. 193-6 (2009)
Ota K G, et al. Hagfish embryology with reference to theevolution of the neural crest. Nature 446. 672-5 (2007)

Sensory Development
Raj LADHER Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/ladher.html
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 thecloning 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 avisiting scientist at the Tokyo Medical and Dental University. In 2000, he moved to the University of Utah as
a postdoctoral fellow in Gary Schoenwolf's laboratory. He was appointed team leader at the CDB in 2002.

Staff
Research Scientist

Part-Time Staff

Akira HONDA
Xiao Rei SAI

Yoshiko KONDO

Visiting Researcher

Noriko HIROI

Stephen FREEMAN
Tomoko KITA

Assistant

Research Associate

Kazuya ONO

Publications
Ono K, et al. FGFR1-Frs2/3 signalling maintains sensory
progenitors during inner ear hair cell formation. PLoS Genet
10.e1004118 (2014)
Sai X, et al. Junctionally restricted RhoA activity is necessary
for apical constriction during phase 2 inner ear placodeinvagination Dev. Biol. 394. 206-16 (2014)
Paschaki M, et al. Retinoic acid regulates olfactory
progenitor cell fate and differentiation. Neural Dev 8.13
(2013)
Honda A, et al. From placode to labyrinth: Culture of thechicken inner ear. Methods 66. 447-53 (2013)
O'Neill P, et al. The amniote paratympanic organ develops
from a previously undiscovered sensory placode. Nat
Commun 3. 1041 (2012)

Hearing loss is the most prevalent sensory disorder not only in Japan, but also the world,with over 5% of the global population showing disabling hearing loss, rising to 50% in theaged population. Hearing occurs in the inner ear, a complex structure that houses thesensory receptors important for sound detection, as well as other non-sensory cell types
that are equally important in the sensitive and precise detection of sound. This complex
organ forms from a relatively simple structure known as the otic placode, found in thesurface ectoderm of the early embryo. It is clear that the otic placode results from dynamic
epigenetic processes that convert ectoderm into otic placode and with it establish changes
in the morphology of the placode, driving the internalization of the otic placode to make thefirst rudiments of the inner ear.
Developmental mechanisms also subdivide the otic placode into different cellular territories
that include both sensory and non-sensory progenitors. These form as a result of balancing
specification and patterning with proliferation, and then subsequently the maturation ofthese cell-types. We want to understand the blueprint for making an inner ear, with particularemphasis on integrating extrinsic signals, the genes that they control, with the cellular andsub-cellular changes that drive phenotypic changes that adapt the cell to its function. Our
ultimate aim is to use our developmental knowledge to make better cell differentiation
protocols that can ultimately used to design screening protocols, diagnostic tools and cellulartherapies that help in alleviating hearing loss.

Ladher R K, et al. From shared lineage to distinct functions:the development of the inner ear and epibranchial placodes.
Development 137. 1777-1785 (2010)

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

43

Cell Asymmetry
Fumio MATSUZAKI Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/matsuzaki.html
Fumio Matsuzaki is engaged in the study of the genetic programs underlying neural development,with a focus on the asymmetric division of neural stem cells. His laboratory has discovered several key
mechanisms controlling asymmetric divisions in neural progenitor cells using Drosophila and mouse asmodel systems. He has also recently found a novel type of self-renewing progenitor in the developing
cerebral cortex in rodents, providing new insights into the enormous increase in brain size during
mammalian evolution.

Our group explores the mechanisms underlying the organization of cells into highly orderedstructures in the developing brain. During brain development, neural stem cells generatea large number of neurons and glia of different fates at appropriate points in time; theframework and size of the brain depend on the spatiotemporal behavior of neural stem cells,
which are highly dynamic in their modes of division and gene expression. Using invertebrate(Drosophila) and vertebrate (mouse) model systems, we focus our study on genetic andepigenetic programs, by which behaviors of neural stem cells are controled and brain
development is governed.
Drosophila neural stem cells, called neuroblasts, provide an excellent model system forthe investigation of fundamental aspects of asymmetric division, a process essential to
the generation of cells of divergent type during proliferation. We have been investigating
mechanisms controlling asymmetric divisions, including the cell polarity and spindleorientation. We also extend our research scope to understand how neurogenesis iscontrolled in tissue space depending on the environments that surround the nervous system.
We recently identified an extrinsic mechanism that controls the orientation of division (cellpolarity) in neuroblasts relative to the overriding ectoderm (Yoshiura et al., 2012), which
determines the orientation of neural tissue growth.
The vertebrate brain evolved rapidly, resulting in an expansion of the size of the brain,
which comprises a larger number of neurons arranged in a vastly more complex functional
network than that in invertebrate. Neural stem cells typically adopt three states - proliferative(symmetrically dividing), neurogenic (asymmetrically dividing), and resting - and undergotransitions among the states, on which the basic organization of the brain depend. We areinvestigating mechanisms that determine the individual states of neural stem cells, andcontrol transitions between states in mouse as well as mechanisms for generating neuralprogenitor cell diversity (see figure). We recently discovered a novel transition in the divisionmode in the developing mouse cortex from radial glia (typical neural stem cells with theepithelial structure) to translocating neural stem cells, basal radial glia (Shitamukai et al.,2011), which become a major population of neural stem cells in mammals with gyrencephalicbrains, such as primates and ferrets. We are investigating the mechanisms that underlie theformation, maintenance, and expansion of these neural stem cells, by using model mice that
produce large numbers of basal radial glia as well as ferrets.

Staff
Research Scientist

Student Trainee

Ikumi FUJITA
Daijiro KONNO
Atsunori SHITAMUKAI
Yuji TSUNEKAWA
Shigeki YOSHIURA

Yuki KOBAYASHI
Shun MASE
Kaga SAKAI
Yoko WATANABE

Visiting Scientist

Yoko OTSUKA
Megumi SHIBATA

Ayano KAWAGUCHI
Yoichi KOSODO
Technical Staff

Chiaki KISHIDA
Raymond TERHUNE-KUNIKANE
Taeko SUETSUGU
Nobuko TANIYAMA

Part-Time Staff

AssistantJunko ISHIGAI

Publications
Kasahara K, et al. Ubiquitin-proteasome system controls
ciliogenesis at the initial step of axoneme extension. Nat
Commun 5.5081 (2014)
Pilz G. A, et al. Amplification of progenitors in the
mammalian telencephalon includes a new radial glial celltype. Nat Commun 4.2125 (2013)
Matsuzaki F, and Shitamukai A. Cell division modes andcleavage planes of neural progenitors during mammaliancortical development. Cold Spring Harb Perspect Biol.
Mammalian Develop. doi 10.1101/cshperspect.a015719(2013)
Konno D, et al. The mammalian DM domain transcription
factor Dmrta2 is required for early embryonic developmentof the cerebral cortex. PLoS One 7.e46577 (2012)
Yoshiura S, et al. Tre1 GPCR signaling orients stem cell
divisions in the Drosophila central nervous system. Dev Cell
22.79-91 (2012)
Shitamukai A, et al. Oblique radial glial divisions in thedeveloping mouse neocortex induce self-renewing
progenitors outside the germinal zone that resemble primate
outer subventricular zone progenitors. J Neurosci 31.368395 (2011)

During brain development, the ganglionic eminence in the ventral telencephalon
generates a large number of diverse types of neurons including GABAergic
interneurons. We have revealed that the ganglionic eminence generate a variety
of progenitors that eventually produce a range of different cell lineages. RG, radial
glia; SAP, subapical progenitor; BP, basal progenitor.

44

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

Staff
Research Scientist

Part-Time Staff

Keishi KISHIMOTO
Masafumi NOGUCHI

Junko SAKAI
Akira YAMAOKA

Technical Staff

AssistantChisa MATSUOKA

Yuka NODA

Publications
Morimoto M, et al. Different assemblies of Notch receptors
coordinate the distribution of the major bronchial Clara,
ciliated and neuroendocrine cells. Development 139.436573 (2012)
Morimoto M, et al. Canonical Notch signaling in thedeveloping lung is required for determination of arterialsmooth muscle cells and selection of Clara versus ciliatedcell fate. J Cell Sci 123.213-24 (2010)
Demehri S, et al. Skin-derived TSLP triggers progression
from epidermal-barrier defects to asthma. PLoS Biol
7.e1000067 (2009)
Morimoto M. and Kopan R. rtTA toxicity limits the usefulnessof the SP-C-rtTA transgenic mouse. Dev Biol 325.171-8(2009)
Morimoto M, et al. The negative regulation of Mesp2 bymouse Ripply2 is required to establish the rostro-caudal
patterning within a somite. Development 134.1561-9 (2007)
Morimoto M, et al. Cooperative Mesp activity is required fornormal somitogenesis along the anterior-posterior axis. Dev
Biol 300.687-98 (2006)
Morimoto M, et al. The Mesp2 transcription factor
establishes segmental borders by suppressing Notch
activity. Nature 435.354-9 (2005)

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

Distribution of Clara (green), neuroendocrine (red) and SPNC (blue) cells

Three dimensional image of embryonicbronchi reconstructed from multi-photon
images45

Developmental Morphogeometry
Yoshihiro MORISHITA Ph.D.

Yoshihiro Morishita received his M.Sc. and Ph.D. from the University of Tokyo Graduate School of
Frontier Sciences. From 2005 to 2007, he served as Project Assistant Professor in the Kyushu University
Department of Biology. From 2007 to 2011, he served both as assistant professor in the Theoretical
Biological Laboratory at Kyushu University and as a researcher funded under several JST programs. He
was named Research Unit Leader at the RIKEN CDB in 2012.
As part of the reorganization of the CDB in November 2014, the Developmental Morphogeometry lab
was transferred to the RIKEN Quantitative Biology Center.

Developmental phenomena comprise a multiscalar system extending across a rangeof spatial scales, from molecular to cellular to histological. Such phenomena are also
multiphysical, in that they involve the transmission and reception of positional information
through diffusion and reactions of chemicals, and the generation of forces within tissues
and concomitant geometrical deformation through the proliferation and migration of cells.
The Laboratory for Developmental Morphogeometry takes theoretical and experimental
approaches to the study of phenomena comprising multiple scales and properties, andthe quantitative measurement of dynamic and coordinated interactions between suchphenomena.
An example of this is seen in organogenesis, a process in which the various tissue regionsthat give rise to the organ exhibit changes in volume at different rates, or anisoptropic
expansion and contraction. Such deformations can be quantified as tensor quantities(geometrical characteristics). The morphological differences between various organs, or
between homologous organs in different species can thus be explained as spatio-temporal
patterns in the tensor quantities of each object. We are now able to extract such patterns
using organ-level quantitative imaging and statistical analysis. By combining and comparing
such data with the accumulated body of molecular and cellular evidence, we hope todevelop clearer insights into the relationships between macro-scale organ morphogenesis
and micro-scale phenomena.
The ability of individual cells to recognize and respond to (for example, through proliferationand differentiation) their positions within a tissue is also essential to tissue growth andpatterning. This necessitates accurate “spatial recognition” on the part of cells, which receive
environmental cues (such as gradients of growth factors or interactions with neighboring
cells), but this is complicated by uncertainty arising from perturbations within the organism(such as inter-individual variations in morphogen expression levels). Questions of how to
maximize the accuracy of the transmission and reading of information against a backgroundof uncertainty is formalized as problems in information coding. Analysis of such problems hasrevealed optimal sites of the expression of information sources (morphogens) and optimal
designs for the form and parameter values of response functions implemented by intracellularbiochemical reactions. By comparing the results of these theoretical analyses withexperimental observations, we can begin to assess the extent to which actual developmental
systems are designed to optimal criteria.

Staff
Research Scientist

Student Trainee

Ken-ichi HIRONAKA
Yukitaka ISHIMOTO
Toru KAWANISHI
Aiko KAWASUMI
Sangwoo LEE
Daisuke OHTSUKA

Naofumi KAWAHIRA
Technical Staff

Kaori TSUJINO
You WATAOKA
Part-Time Staff

Megumi MORIOKA

Publications
Morishita Y. and Suzuki T. Bayesian inference of wholeorgan deformation dynamics from limited space-time point
data. J Theor Biol 357.74-85 (2014)
Lee S. W, et al. Canalization-based vein formation in a
growing leaf. J Theor Biol 353.104-20 (2014)
Hironaka K. and Morishita Y. Cellular sensory mechanisms
for detecting specific fold-changes in extracellular cues.
Biophys J 106.279-88 (2014)
Hironaka K. I. and Morishita Y. Encoding and decoding of
positional information in morphogen-dependent patterning.
Curr Opin Genet Dev (2012)
Morishita Y. and Iwasa Y. Coding design of positional
information for robust morphogenesis. Biophys J 101.232435 (2011)
Morishita Y. and Iwasa Y. Optimal placement of multiple
morphogen sources. Phys Rev E Stat Nonlin Soft Matter
Phys 77.041909 (2008)

Our lab will seek to use 1) analysis of measured data and 2) study system designs throughtheoretical formulation and computer simulations with an eye to developing a better
understanding of these phenomena.

Information coding process in computer science (A) andin developmental biology (B). In former, information iscoded through binary electronic signals, while in latter,the information, spatial coordinate, is coded throughchemicals. (C) Optimal arrangement of morphogen source(Shh-source) to maximize the precision of positional
information in vertebrate limb bud.

46

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

The processes of animal development, including organ size and body size, are geneticallypredetermined, but these processes are also influenced by environmental factors suchas nutrition and temperature. The close link between cell and tissue growth control andenvironmental cues ensures that developmental transitions occur at the appropriate time
during animal development.

Staff
Research Scientist

Part-Time Staff

Naoki OKAMOTO
Tetsuo YASUGIOkiko HABARA
Kanako HIGUCHI
Hitomi KUBO
Noriko NISHIMURA
Junko SHINNOTechnical Staff

Takayuki YAMADA
Student Trainee

Miki YOSHIDA

Publications
Matsuda H., et al. Flies without Trehalose. J Biol Chem
290.1244-55 (2015)
Okamoto N, et al. A secreted decoy of InR antagonizes
insulin/IGF signaling to restrict body growth in Drosophila.
Genes Dev 27.87-97 (2013)
Okamoto N, et al. Conserved role for the Dachshund proteinwith Drosophila Pax6 homolog Eyeless in insulin expression.
Proc Natl Acad Sci U S A 109.2406-11 (2012)
Wirtz-Peitz F, et al. Linking cell cycle to asymmetric division:
Aurora-A phosphorylates the Par complex to regulate Numb
localization. Cell 135. 161-73 (2008)
Nishimura T and Kaibuchi K. Numb controls integrin
endocytosis for directional cell migration with aPKC andPAR-3. Dev Cell 13. 15-28 (2007)

Cell proliferation and differentiation in each tissue and organ are kept under strict regulation
both spatially and temporally. Research has revealed the nature of spatial signals, such as
growth factors and morphogens, but the way in which these signals direct cell and tissue
growth over time remains understood. In addition, growth and developmental timing are also
governed by nutrient availability. Most species have a standard body size, but developing
organisms are also capable of adapting their growth to fluctuating nutritional states throughmetabolic regulation. Therefore, linking the nutrient sensing system to an endocrine signaling
network allows organisms to control the timing of cell proliferation and differentiation.
Our team’s research aims to shed light on the molecular basis for growth control anddevelopmental timing at the cellular and tissue/organ level using Drosophila as a model
system. In particular, we are interested in addressing the following questions: 1) how doorganisms adapt their growth program to changes in energy needs and states; 2) what are
the molecular mechanisms that sense nutrient availability and regulate body size; and 3) how
do endocrine signals interact with metabolic and growth regulators?
To better understand the interface between nutrient availability and growth regulation,
we are focusing on how nutrition controls systemic growth through Drosophila insulinlike peptides (Dilps). Members of the insulin family of peptides have conserved roles in theregulation of growth and metabolism in a wide variety of metazoans. We are now analyzing
the molecular mechanism underlying the nutrient-dependent expression of Dilp genes. We
have also conducted in vivo RNAi screening to identify new players regulating growth anddevelopmental timing at the organismal level. We described the first demonstration of theglia-derived endocrine factor regulating systemic body growth. The identification of SDR
protein in Drosophila provides a new concept for the regulation of insulin/IGF signaling.

A wild-type female fly (right) and an insulin-like receptor mutant female (left)

47Strategy of bending by giant cadherins

loses its elongated morphology, and consequently, cell-celladhesion is also lost. The ectodomains of “non-classical”
cadherins such as protocadherins, Fat, and Dachsous
have varying numbers of EC domains, ranging from five
up to 34. However, these larger cadherins have also beenfound to occupy intracellular spaces with spans smaller
than the linear length of its ectodomain. Thus, this raises
the question of how larger cadherin molecules are able tofit into relatively narrow intercellular spaces.
Now, a new study by research scientist Yoshikazu
Tsukasaki of the Laboratory for Cell Adhesion and Tissue
Patterning (Masatoshi Takeichi, Team Leader) and othercolleagues demonstrates a strategy of bending adopted
by the giant cadherins, Fat and Dachsous, which allows
them to squeeze into narrow intercellular spaces despite
their large size. Published in the Proceedings of the
National Academy of Sciences (PNAS), their findings
indicate that some segments of the large ectodomain of
Fat and Dachsous have been modified to prevent Ca2+binding, and facilitate the folding of these domains toassume a more compact form. Tsukasaki is now at the
University of Texas Health Science Center.

Yoshikazu TSUKASAKIThe cadherin superfamily of transmembrane proteins play
an important role in mediating cell-cell adhesion, which isfundamental for maintaining organization of multicellularorganisms. They require calcium ions (Ca2+) to function,
and also regulate many developmental processes such as
tissue and neural circuit formation through their intercellularinteractions. Cell-cell adhesion is achieved throughhomophilic or heterophilic binding between cadherinsof adjacent cells through their filamentous extracellulardomains (ectodomains). The so-called “classical”
cadherins have ectodomains consisting of five repetitive
units called, extracellular cadherin (EC) domains, which arejoined together with a segment containing a Ca2+-binding
motif (CBM), an amino acid sequence that can bind Ca2+.
When Ca2+ binds to the CBM, it activates the cadherin
by causing the ectodomain to elongate into a filamentous
morphology and interacts with its partner molecule in celladhesion. But when Ca 2+ is depleted, the ectodomain

48

The mammalian Fat4 and Dachsous1 form a heterophilicbinding that regulates planar cell polarity and proliferation.
Fat4 is the largest molecule within the cadherin
superfamily with 34 EC domains, and Dachsous1 is also
large with 27 EC domains. Tsukasaki et al. first examined
purified ectodomain structures of both cadherins undertransmission electron microscopy (TEM), and found thatwhile the N-terminal end of ectodomains, the region
furthest from the cell membrane, displayed a relatively
linear morphology, the C-terminal end which is close to
the membrane had multiple locations where the filament
was sharply bent. When the amino acid sequences of the
CBMs were analyzed, they found modifications of aminoacid residues in several of the CBMs near the C-terminal
end, suggesting the possibility that these modifications
prevent Ca2+-binding.
The group generated mutants of the classical cadherin,
E-cadherin, by replacing one of its CBMs with a modified
CBM from Fat4. TEM revealed that these E-cadherin
mutants displayed a bent arrangement in the location
where the CBM was switched. Gel filtration experiments
measuring elution speed showed that both wildtype
E-cadherin run in Ca 2+-free conditions and the CBMreplaced E-cadherin mutants moved slower than the©2014 National Academy of Sciences
Tsukasaki Y, et al. Giant cadherins Fat and Dachsous self-bendto organize properly spaced intercellular junctions. Proc Natl Acad Sci U S A (2014)

wildtype E-cadherin run in presence of Ca2+; these results
supported their hypothesis that the modified CBM found in
Fat and Dachsous is unable to bind Ca2+, thus leading to anonlinear ectodomain.
To further understand the morphological changes that
occur in CBM-replaced E-cadherin, the group carried out
computer simulations using the elastic network model
based on the known X-ray crystal structure of E-cadherin.
The simulations showed that, in CBM-replaced E-cadherin,
the segments with modified CBM exhibited a wide rangeof bent configurations, consistent with TEM observations.
They applied the same method for Fat4 and Dachsous1,
and successfully created atomic models that matchedmorphologies seen under TEM. In a separate series ofexperiments, they also found that the four most N-terminal
ectodomains appeared to be sufficient for Ca2+-mediatedheterophilic binding between Fat4 and Dachsous1, andthat this binding is unaffected even when the C-terminal
end is bent.
Tsukasaki et al. next analyzed the morphology of Fat4 andDachsous1 in vivo, by transfecting a cell line with Fat4 orDachsous1 cDNAs. When these separately transfected
cells were co-cultured, they observed the formation of
Fat4-Dachsous1 bindings at the cell boundaries between
the two cell types just above the tight junctions. Closer
analysis with TEM revealed that the intercellular spaces inwhich the Fat4-Dachsous1 binding complexes were foundwere approximately 47 nm wide, matching the dimensionsof the molecules calculated from the elastic networkmodel. Observations of Fat4-Dachsous1 binding in themouse embryonic cortex also showed the span between
cell boundaries were similar to those observed in the coculture of transfected cells.
“It is a mystery as to why giant cadherins have gone as
far as losing their Ca 2+-binding ability to squeeze into
intracellular spaces by folding,” says Takeichi. “What isinteresting is that the size of Fat and Dachsous, and thepositions of modified CBMs within the ectodomain havebeen conserved across species. No doubt the bending
strategy revealed in our study is closely linked to thephysiological function of these molecules.”TEM images of Fat4 (left) and Dachsous1 (middle). In both molecules, the N-terminal end had an elongated configuration, while the C-terminal end had abent configuration. The right panel shows a simulated 3D structure of Fat4 (red) and Dachsous1 obtained from the elastic network model.

49

Protocadherin mediates collective axon
extension of neurons

Shuichi HAYASHI

Formation of the complex neural network involves neurons
extending a single axon to target and synapse with adendrite sprouting from another neuron, which will in turn
synapse with the next neuron. Some axons can extend
up to one hundred times the length of the cell body toreach their target. The axons of neurons making up the
amygdala, which is located deep in the middle of thebrain mass, migrate along the hippocampus, making along journey to reach the hypothalamus. These extending
axons move collectively by forming fascicles (or bundles)consisting of several hundred to several thousand axons
derived from the same neuronal group, but the underlying
mechanism regulating fascicle formation and collective
axonal migration remained unclear.
In a new study published in Developmental Cell, researchscientist Shuichi Hayashi of the Laboratory for Cell
Adhesion and Tissue Patterning (Masatoshi Takeichi, Team

50

Leader) and colleagues identify one factor that plays anessential role in the collective axon extension of amygdala
neurons. Using the developing mouse brain as a model,
their findings indicate that protocadherin-17 (Pcdh17),
a member of the cadherin superfamily of cell adhesion
molecules, mediates axon-axon interactions and recruits
actin regulators to the interaxonal contact sites which
modify the surrounding actin dynamics to promote cell
migration.
Protocadherins are broadly classified as being either
clustered or non-clustered. Past studies suggest thatclustered protocadherins have some function in the selfrepelling mechanism of dendrites to prevent binding
between dendrites from the same neuron, while nonclustered protocadherins have been implicated as a
crucial player in axon elongation during neural network
formation in the brain. In this study, Hayashi et al.

©2014 Elsevier B.V.
Hayashi S, et al. Protocadherin-17 mediates collective axon
extension by recruiting actin regulator complexes to interaxonal contacts.
Dev Cell 30.673-87 (2014)

focused their attention on a non-clustered protocadherin,
Pcdh17, and its expression in amygdala neurons. Pcdh17
expression was detected in several subsets of amygdala
neurons. Upon close examination, they found that Pcdh17
expression in these neuronal subsets was localized aroundthe interaxonal adhesion sites, leading them to consider
the role of Pcdh17 in the collective elongation of axons in
amygdala neurons.
They next generated a Pcdh17 knockout mouse line, andan analysis of its phenotype revealed that loss of Pcdh17
results in abnormal axonal elongation; the axon fascicle
extending from the amygdala to the hypothalamus wasthinner. The thinner fascicle was due to a collapse of
the growth cones, the motor center located at the tipof growing axons. Labeling the Pcdh17 expressing andnon-Pcdh17 expressing neurons with different coloredfluorescent proteins, the group observed growth conemigration using live-imaging and found that, in wild-typeconditions, when the elongating axons came into contact
with an axon from other neurons of the same subtype, it
continued to elongate along the other axon, whereas in
the Pcdh17 mutant, the axon stopped elongating when
it came into contact with another neuron of the same
subtype.
So then, how is the migration of axons regulated? Hayashi
et al. confirmed that, similar to other cadherins whichmediate intercellular interactions, the extracellular domainof Pcdh17 facilitates the binding of axons of similar
neuronal types. Using a pull-down assay, they next looked
into which molecules bind to the intracellular domain of
Pcdh17 and identified several factors that make up theWAVE complex, which is involved in actin polymerization.
They also determined that Pcdh17 works together with
the WAVE complex to recruit other downstream factorsthat plays a role in cell motility of the growth cone as well
as at the interaxonal adhesion sites. The role of Pcdh17
mediating cell migration was further verified by observing
the behavior of Pcdh17-transfected cultured cells thatshowed similar directional movements to neuronal growthcones. Thus, the results showed that Pcdh17 promotes
cell migration by localizing at interaxonal adhesion sites
and recruiting the WAVE complex and further downstream
factors, which enhance the efficiency of axon elongation.
“The function of cadherins largely differs depending on
the molecules it coordinates with,” explains Takeichi.
“Neuronal axons can extend on their own, but when they
form a fascicle, the contact enhances axon migration.
Recent research shows that malfunction of protocadherins
can lead to autism and schizophrenia. As the amygdala
is a part of the brain that functions in emotion andmemory, future studies looking at the relationship between
protocadherins and amygdala will be very interesting.”

Still frames taken from live-imaging movie of axon elongation of amygdala neurons. Under normal conditions, whenan axon comes into contact with another axon, it will continue elongating along the other neuron (top row). In Pcdh17
mutants, an axon stops elongating when it comes into contact with another axon (bottom row). Neurons express Pcdh17
are labeled green, non-Pcdh17 expressing neurons are labeled red.

51

Fat body protein shapes tracheal system

Bo DONGThe Drosophila tracheal system is a branched network ofepithelial tubes extending throughout the body to transportoxygen to the tissues. During tracheal development, thetracheal lumen is filled with apical extracellular matrix(ECM), which is later cleared out and replaced by gases
prior to larval hatching. Components of the apical ECM,
such as proteins and chitin, which is also an importantcomponent of the exoskeleton protecting the fly’s
exterior, play important roles in tracheal morphogenesis
by regulating the length and diameter of the tubes. One
ECM protein required for tracheal development is a chitin
deacetylase, serpentine (Serp). Past studies have reported
that overexpression or loss of serp leads to overelongatedand convoluted tracheal tubes. While tracheal cells
are the major source of ECM proteins, including Serp,secreted into the lumen, it remains unclear whether andto what extent other tissues contribute to tracheal tube
development.

52

Now, new work by Bo Dong and colleagues in the
Laboratory for Morphogenetic Signaling (Shigeo
Hayashi, Team Leader) reveals the Drosophila fat body,a mesoderm-derived tissue that plays physiological roles
similar to the vertebrate liver, as a surprising new source ofluminal Serp during tracheal morphogenesis. Published in
the journal Development, their findings demonstrate that a
non-epithelial tissue plays a role in tracheal morphogenesis
and provides additional evidence of the fat bodysupporting the developmental process of various organs.
The insect fat body functions as a site of synthesis andstorage of sugars, lipids and proteins, which are secreted
into the hemolymph to be delivered to tissues andorgans of the body as needed to maintain homeostasis.
Dong et al. first confirmed the presence Serp in the fat
body as well as in the tracheal system of fly embryos,
consistent with previous reports. Then, using two different©2014 The Company of Biologists
Dong B, et al. A fat body-derived apical extracellular matrix enzyme istransported to the tracheal lumen and is required for tube morphogenesis
in Drosophila. Development 141.4104-9 (2014)

mutant fly strains (rab9 and shrub) in which glitches invesicular mechanisms affect Serp localization resulting inphenotypes similar to serp mutants (i.e. overelongatedtracheal tubes), they found that both mutants showed
higher levels of Serp in the fat bodies and lower Serp levelsin the tracheal lumen than seen in wildtype, a patternthat was not observed with other apical ECM proteins.
Additional experiments specifically blocking endocytic or
exocytic pathways of fat bodies indicated that the sourceof Serp accumulation in the fat bodies of the mutants wascell-intrinsic synthesis, ruling out the possibility that Serp
was being translocated there from an external source.
Under normal conditions, Serp does not remain localized
in the fat body, so the group next examined whether
Serp synthesized by the fat body can be delivered to thelumen of the developing tracheal tube. They ectopically
expressed a secreted luminal marker protein containing
the chitin-binding domain of Serp (Serp-CBD-GFP) in
the fat body and found that the marker accumulated in
the tracheal lumen. In contrast, when a functionally inert
secreted protein marker with a molecular size similar toSerp-CBD-GFP was expressed, it was absent from thelumen. Further, the authors used the technique of IR-laser
induced local heat shock (Miao and Hayashi, 2014) todemonstrate that Serp-GFP could be translocated to thelumen even after the developing tracheal tube acquired
epithelial barrier function, suggesting that Serp is taken up
by tracheal cells from the hemolymph and deposited in thelumen via transcytosis.
Finally, the group looked at whether fat body-derived Serpplays a functional role in tracheal development. When Serp
was ectopically expressed in the fat body of serp mutants,
they observed that the overelongated tube phenotype
could be rescued, suggesting that Serp secreted from thefat body plays a role in tracheal morphogenesis. Closer
examination also revealed that the function of fat bodyderived Serp appears to overlap with that of tracheal cellderived Serp and supports tracheal tube development
when the tracheal source is limited.
“To find that the fat body influences tracheal tube
morphogenesis was unexpected,” says Hayashi. “We
speculate that the fat body plays a role in balancing the
supply of a key enzyme to the trachea and epidermis sothat differentiation of those epidermal organs is controlled
under the same temporal schedule.”

Tracheal cells secrete Serp protein into the lumen (magenta). Fat body cells (green) surround the trachea
and serve as a secondary source of Serp.

53

2014 Awards
As one of the world’s leading institutes in the fields of developmental biology and stem cell research, the RIKEN CDB strives tomaintain the highest levels of originality, impact and quality in its scientific contributions. The individual achievements and careersof many of its scientific staff have been recognized with awards from numerous organizations, including government ministries andagencies, private foundations and academic societies.
The CDB takes great pride in the achievements of its researchers, as individuals and as indispensable members of their laboratories.

Awardee

54

PositionLaboratory

Award

Organization

Mitsuru Morimoto

Team Leader

Lung Development

The Young Scientists’Prize, The Commendationfor Science and Technology

The Minister of Education, Culture,
Sports, Science and Technology

Mitsuru Morimoto

Team Leader

Lung Development

International Session
Award

The 55th Annual Meeting of theJapanese Respiratory Society

Erina Kuranaga

Team Leader

Histogenetic
Dynamics

Editor-in-Chief Prize, Most
Cited Paper

Development, Growth &Differentiation (Wiley Blackwell)

Masatoshi
TakeichiTeam Leader

Cell Adhesion andTissue Patterning

AAAS Fellow

American Association for the
Advancement of Science (AAAS)Fluorescence microscopy image showing
a pair of U251 cells in contact with one
another. Protocadherin-17 (green) recruits
actin-polymerization regulators including theWAVE-complex (magenta) to peripheral cellcell contacts and convert them into a motile
structure.

55

Pluripotent Stem Cell Studies
Hitoshi NIWA M.D., Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/niwa.html
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. He was appointed
project leader of the Laboratory for Pluripotent Stem Cell Studies in October 2009.

Pluripotency is a term used to describe the ability of certain types of stem cells that arecapable of giving rise to at least one type of cell from all three germ layers - endoderm,
mesoderm and ectoderm. Stem cells are also characterized by their ability to produce copiesof themselves with similar differentiative potential, as well as more specialized cells withdifferent properties through differentiation. Embryonic stem (ES) cells are the best known
example of a type of stem cell possessing these properties of pluripotency and self - renewal.
In our lab, we use ES cells as a model system for studying the molecular mechanisms that
determine and enable the mysterious properties of pluripotent cells.
Pluripotency can be primarily determined by a particular set of transcription factors, for as
we now know, pluripotency can be induced by four transcription factors. These transcription
factors should form a self-organizing network able to stabilize their expression and maintainpluripotency. At the same time, the network should have the sensitivity to signals thatinduce differentiation. How can the transcription factor network satisfy these contradictory
requirements? What is the principle behind the structure of the transcription factor networks
governing developmental processes? We are now trying to answer these questions bystudying ES cells and the changes they undergo in their differentiation toward trophoblaststem cells, extraembryonic endoderm cells, and primitive ectoderm cells.

Staff
Research Scientist

Yoko NAKAI-FUTATSUGISatomi NISHIKAWA-TORIKAI
Satoshi OHTSUKA
Robert Odell STEPHENSON
Tomoyuki TSUKIYAMA
Research Associate

Mariko YAMANE
Visiting Scientist

Hiroki URA
Technical Staff

Setsuko FUJII

Kumi MATSUURA
Azusa NOMA
Junior Research Associate

Shunsuke ITOStudent Trainee

Marius KLANGWART
Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI
AssistantMiho SAKURAI

Publications
Adachi K, et al. Context-Dependent Wiring of Sox2
Regulatory Networks for Self-Renewal of Embryonic andTrophoblast Stem Cells. Mol Cell 52.380-92 (2013)
Ohtsuka S, et al. E-cadherin promotes incorporation ofmouse epiblast stem cells into normal development. PLoS
One 7.e45220 (2012)
Martello G, et al. Esrrb is a pivotal target of the gsk3/tcf3
axis regulating embryonic stem cell self-renewal. Cell Stem
Cell 11.491-504 (2012)
Niwa H, et al. A parallel circuit of LIF signalling pathways
maintains pluripotency of mouse ES cells. Nature 460. 11822 (2009)
Niwa H. How is pluripotency determined and maintained?
Development 134. 635-46 (2007)

Chimeric embryos generated by injection of Gata6GR
ES cells into blastocysts. These ES cells carry theconstitutively-active Egfp transgene and contribute toprimitive ectoderm (upper panels), whereas they contributeto parietal endoderm after induction of Gata6 activity withdexamethasone (lower panels).

56

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

Organogenesis and Neurogenesis
Yoshiki SASAI M.D., Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/takeichi2.html
Yoshiki Sasai received his M.D. from the Kyoto University School of Medicine in 1986, subsequently
performing internships in general practice and emergency medicine. He completed the Ph.D. course
at the same institution in 1992, for work on neural specific transcriptional regulators. In 1993, he took a
postdoctoral fellowship in the De Robertis lab at the UCLA School of Medicine, remaining there until 1996
when he was appointed associate professor at the Kyoto University School of Medicine. He assumed a
professorship at the Kyoto University Institute for Frontier Medical Sciences in 1998, and was appointed
group director at the CDB in 2000. He serves on the editorial boards of Cell, Neuron, Developmental Cell,
Genesis, and Developmental Dynamics.
In memoriam Yoshiki Sasai (1962–2014)

Staff
Research Specialist

Technical Staff

Keiko MUGURUMA
Satoru OKUDA
Douglas SIPP
Nozomu TAKATA
Momoko WATANABE

Tomoko HARAGUCHI
Tomoko HORIKIRI
Masako KAWADA
Ayaka NISHIYAMA
Mika SOEN
Rieko YAKURA-NISHIZAWA

Foreign Postdoctoral Researcher

Junior Research Associate

Research Scientist

Nicholas Robert LOVE
Research Associate

Chikafumi OZONE
Visiting Scientist

Satoshi ANDO
Yoshihito ISHIDA
Atsushi KUWAHARA
Tokushige NAKANOMorio UENO

Hideya SAKAGUCHI
Atsushi SHIRAISHI

The complexity of the fully formed brain defies description, yet this organ arises from anondescript clump of cells in the embryo. The specification of the dorsal-ventral (back-belly)
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 anumber of signaling factors that diffuse from organizing centers and direct dorsal ectoderm
to maintain a neural fate. These molecules, which include Noggin, Chordin, Follistatin andtheir homologs, participate in elaborate signaling networks in which factors collaborate andcompete to initiate the embryonic nervous system.

Student Trainee

Taisuke KADOSHIMA
Kazuhiro TAKASHIMA
Part-Time Staff

Masako SUZUKI
Assistant

Mako MIYAGI-INOMATA
Masayo OKUGAWA
Fumi WAGAI

Publications
Kadoshima T, et al. Self-organization of axial polarity,
inside-out layer pattern, and species-specific progenitor
dynamics in human ES cell-derived neocortex.
Proc Natl Acad Sci U S A 110.20284-9 (2013)
Inomata H, et al. Scaling of Dorsal-Ventral Patterning
by Embryo Size-Dependent Degradation of Spemann’s
Organizer Signals. Cell 153 1296-1311(2013)
Suga H, et al. Self-formation of functional adenohypophysis
in three-dimensional culture. Nature 480. 57-62 (2011)
Eiraku M, et al. Self-organizing optic-cup morphogenesis inthree-dimensional culture. Nature 472 51-56(2011)
Kamiya D, et al. Intrinsic transition of embryonic stem-celldifferentiation into neural progenitors. Nature 470. 503-509(2011)
Muguruma K, et al. Ontogeny-recapitulating generationand tissue integration of ES cell-derived Purkinje cells. Nat
Neurosci 13 1171-80 (2010)

Using the African clawed frog, Xenopus laevis , as a model in molecular embryologicalstudies, 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 complex of permissive, instructive and inhibitory patterns that determines thegrowth and differentiation of the brain in its earliest stages of development.
The group is now also actively developing effective methods of inducing neuralization in
mammals, work which it is hoped will contribute to basic research by providing in vitro
experimental systems for use in the analysis of mammalian neurogenesis. In addition, thisapproach has potential for applications in the treatment of neurodegenerative disorders, suchas 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 specializedneuronal types. The application of similar techniques to human ES cells represents a field ofstudy that, although it remains at quite 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 underpinningthe formation of so intricate a system as
the mammalian neural network, in the hopes that, by looking at the elegant simplicity of theembryonic brain, it may one day be possible to understand the cellular bases of the mind's
activity.

Self-formation of layered cortical tissue fromhuman ES cells

Self-organized formation of optic cup from human
ES cells

57

Early Embryogenesis
Guojun SHENG Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/sheng.html
Guojun Sheng received his B.S. in microbiology from Fudan University in Shanghai, China in 1990. He
entered the Rockefeller University the next year, attending the summer course in embryology at the Woods
Hole Marine Biological Laboratory in 1996 and receiving his Ph.D. under Claude Desplan in 1997. Hespent the period from 1998 to 2000 in Claudio Stern's laboratory in Columbia University, moving with Dr.
Stern to University 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 main model organism. Our research currently focuseson two aspects: 1) the formation and early regionalization of mesoderm cells during
gastrulation; 2) differentiation of the ventral-most mesoderm cell types.
Mesoderm cells are derived from the epiblast during gastrulation through a process called
epithelial to mesenchymal transition (EMT). During EMT, mesoderm cells leave epithelialshaped epiblast sheet and become migratory. We are interested in understanding
the molecular and cellular mechanisms governing this EMT. We are also interested inunderstanding how this process is linked to mesoderm fate specification in the epiblast andto mesoderm regionalization along the dorsal-ventral axis during gastrulation.
After their formation, mesoderm cells give rise to several well-defined lineages: axial, paraxial,
intermediate, lateral plate and extraembryonic. The extraembryonic lineage contains threecell types: blood, endothelial and smooth muscle. We are investigating how these threecell types are specified and how they are organized to form functional hematopoietic andvascular systems.

Staff
Research Scientist

Part-Time Staff

Cantas ALEV
Yukiko NAKAYA
Wei WENG

Anna Barbara WRABELAssistant

Yumi KATO

Technical Staff

Hiroki NAGAI
Fumie NAKAZAWA
Erike Widyasari SUKOWATIYuPing WU

Publications
Weng W and Sheng G.Five transcription factors and FGF
pathway inhibition efficiently induce erythroid differentiationin the epiblast. Stem Cell Reports 262-70 (2014)
Nakaya Y, et al. Epiblast integrity requires CLASP and
Dystroglycan-mediated microtubule anchoring to the basal
cortex. J Cell Biol 202. 637-51 (2013)
Alev C, et al. Decoupling of amniote gastrulation and streak
formation reveals a morphogenetic unity in vertebrate
mesoderm induction. Development 140.2691-6 (2013)
Alev C, et al. Transcriptomic landscape of the primitive
streak. Development 137.2863-74 (2010)
Shin M, et al. Notch mediates Wnt and BMP signals in theearly separation of smooth muscle progenitors and blood/
endothelial common progenitors. Development 136. 595603 (2009)
Nakaya Y, et al. RhoA and microtubule dynamics control
cell-basement membrane interaction in EMT during
gastrulation. Nat Cell Biol 10.765-75 (2008)

An anamniote-like, circumblastoporal ring of
mesoderm precursors (blue: brachyury) can be
induced in the chick embryo by subgerminal cavity
injection of FGF growth factor, suggesting that theprimitive streak is not an essential component of
mesoderm formation in amniotes.

A cross-section of the chick primitive streak with epiblast cells in the middle undergoing EMT. Red: betaDystroglycan (basolaterally localized in lateral epiblast cells and downregulated in medial epiblast cells); Green:
nuclei.

58

Physical Biology
Tatsuo SHIBATA Ph.D.

Tatsuo Shibata received his B.Sc. in Physics from Kyoto University, and Ph.D. from the Graduate Schoolof Arts and Sciences, University of Tokyo in 1999, after which he worked as a postdoctoral research fellow
at the Kyoto University Research Institute for Mathematical Sciences for two years. In 2001, he moved
to Germany to do a second postdoc at the Fritz-Haber-Institut. He returned to Japan in 2002, taking an
associate professorship at Hiroshima University. In 2007, he was additionally appointed as a researcher
under the PRESTO program coordinated by the Japan Science and Technology Agency (JST). In Octoberof 2010, he took his current position of research unit leader at the RIKEN CDB.
As part of the reorganization of the CDB on November 21, 2014, the Physical Biology lab wastransferred to the RIKEN Quantitative Biology Center.

Staff
Research Scientist

Student Trainee

Katsuyoshi MATSUSHITA
Naotoshi NAKAMURA
Katsuhiko SATO

Prabhat SHANKAR
Assistant

Satoko KINOSHITA

Technical Staff

Yuko WADA

Publications
Hiraiwa T, et al. Relevance of intracellular polarity toaccuracy of eukaryotic chemotaxis. Phys Biol 11.056002(2014)
Nishikawa M, et al. Excitable signal transduction induces
both spontaneous and directional cell asymmetries in thephosphatidylinositol lipid signaling system for eukaryotic
chemotaxis. Biophys J 106.723-34 (2014)
Shibata T, et al. Intracellular encoding of spatiotemporal
guidance cues in a self-organizing signaling system forchemotaxis in dictyostelium cells. Biophys J 105.2199-209(2013)
Hiraiwa T, et al. Theoretical model for cell migration withgradient sensing and shape deformation. Eur Phys J E Soft
Matter 36.9846 (2013)
Baba A., et al. Directional sensing of deformed cells underfaint gradients. Phys Rev E Stat Nonlin Soft Matter Phys
86.060901 (2012)
Shibata T, et al. Modeling the self-organizedphosphatidylinositol lipid signaling system in chemotactic
cells using quantitative image analysis. J Cell Sci 125.513850 (2012)

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

A

C

B

(A) 300msec cAMP stimulus induce asymmetric activation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) indicating an excitable
reaction. (B) Spontaneously formed asymmetry of PtdIns(3,4,5)P3 (left panels) in a random direction, which is biased to the external gradientdirection when it is applied (right panels). (C) Biased excaitability is the principle of gradient sensing. (Nishikawa, et al. 2014).

59

Retinal Regeneration
Masayo TAKAHASHI M.D., Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/takahashi.html
Masayo Takahashi received her M.D. from Kyoto University in 1986, and her Ph.D. from the same
institution in 1992. After serving as an assistant professor in the Department of Ophthalmology, Kyoto
University Hospital, she moved to the Salk Institute in 1996, where she discovered the potential of stem
cells as a tool for retinal therapy. She returned to Kyoto University Hospital in 1998, and since 2001 served
as an associate professor at the Translational Research Center in the same 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 to understand these diseases at afundamental 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 usefuland 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 know that at least new retinal neurons can be generated after being damaged. Thishas opened up new hope that the ability to regenerate neurons and even to reconstitutethe neural network may be retained in the adult retina. We are now exploring the excitingprospect that, by transplanting cells from outside of the retina or by regeneration from
intrinsic progenitor cells, it may one day be possible to restore lost function to damaged
retinas.
Our research into retinal regeneration seeks to achieve clinical applications by developing
methods for inducing stem cells or embryonic stem cells to differentiate into retinal neurons
and pigment epithelial cells in sufficient quantities for use in the treatment of patients sufferingfrom conditions in which such cells have been damaged or lost. We must also ensure thatsuch cells establish viable grafts upon transplantation and induce the reconstitution offunctional neural networks. We also hope to develop means of promoting true regenerationby activating endogenous stem cells to replace cells lost to trauma or disease andthus repair damaged tissues. Access to a broad spectrum of developmental biologicalresearch information will be key to the achievement of these goals, and we appreciate theopportunities for exchange that working in the environment provided by the RIKEN CDB.
Therapeutic applications cannot be developed from basic research alone; the clinicalapproach—a thorough understanding of the medical condition to be treated is equallyimportant. For conditions such as retinitis pigmentosa, even the successful transplantation ofcells in animal models may not necessarily be translatable to a human clinical therapy without
an understanding of the underlying genetics and possible immunological involvement. Our
goal is to study retinal regeneration based on both a strong foundation in basic research andsolid clinical evidence.

Staff
Deputy Project Leader

Michiko MANDAI
Sunao SUGITA
Research Scientist

Hiroshi AKIMARU
Jun KANEKO
Hiroyuki KITAJIMA
Naoshi KOIDE
Chikako MORINAGA
Akishi ONISHI
Akiko SUGA
Research Associate

Yuuki ARAI
Akihiro TACHIBANATechnical Staff

Momo FUJII
Kanako KAWAI
Tomoyo HASHIGUCHI
Naoko HAYASHI

Shino HIRAHARA
Yukako HIRAO
Ayumi HONO
Kyoko ISEKI
Hiromi ITO
Miwa OKADA
Noriko SAKAI
Yumiko SHIBATA
Junki SHO
Hitomi TAKAKUBO
Motoki TERADA
Kazuko TSUJIMOTO
Chikako YAMADARyo YOSHIDA
Part-Time Staff

Tomoko KATAYAMA
Ryoko TAKEDA
Tomoko YOKOTA

Publications
Assawachananont J, et al. Transplantation of embryonic andinduced pluripotent stem cell-derived 3D retinal sheets into
retinal degenerative mice. Stem Cell Reports 2.662-74 (2014)
Kamao H, et al. Characterization of Human Induced
Pluripotent Stem Cell-Derived Retinal Pigment Epithelium
Cell Sheets Aiming for Clinical Application. Stem Cell
Reports 2.205-18 (2014)
Jin Z. B, et al. Modeling retinal degeneration using patient-specificinduced pluripotent stem cells. PLoS One 6.e17084 (2011)
Osakada F, et al. Toward the generation of rod and cone
photoreceptors from mouse, monkey and human embryonicstem cells. Nat Biotechnol 26. 215-24 (2008)
Osakada F, et al. Wnt signaling promotes regeneration in
the retina of adult mammals. J Neurosci 27. 4210-9 (2007)
Ooto S, et al. Potential for neural regeneration after
neurotoxic injury in the adult mammalian retina. Proc Natl
Acad Sci U S A 101. 13654-9 (2004)
Haruta M, et al. In vitro and in vivo characterization of pigment
epithelial cells differentiated from primate embryonic stem cells.
Invest Ophthalmol Vis Sci 45. 1020-5 (2004)

Mouse iPSC-derived photoreceptors are morphologicallyable to form synapses after transplantation into host animals.

60

iPSC-derived retinal pigment epithelium cells and cellsheet (generated for clinical use)

Human iPSCs (immunostained red:SSEA-4, blue:
DAPI)

Cell Adhesion and Tissue Patterning
Masatoshi TAKEICHI Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/takeichi.html
Masatoshi Takeichi is director of the RIKEN Center for Developmental Biology as well as director of the Cell
Adhesion and Tissue Patterning research group. He completed the B. Sc. and M. S. programs in biology
at Nagoya University before receiving a doctorate in biophysics from Kyoto University in 1973. Afterattaining his Ph.D., he took a research fellowship at the Carnegie Institution Department of Embryology
under Richard Pagano. He then returned to Kyoto University, attaining a professorship in the Departmentof Biophysics (1986-1999), before becoming professor in the Department of Cell and Developmental
Biology in the Graduate School of Biostudies at the same university. Dr. Takeichi served as CDB Director
from 2000 to 2014.

Staff
Research Scientist

Collaborative Scientist

Shuichi HAYASHI
Shoko ITO
Toshiya KIMURA
Shigenori NAGAE
Anna PLATEK
Varisa PONGRAKHANANON
Nobutoshi TANAKA
Takuji TANOUE
Mika TOYA
Vassil VASSILEV

Shoko NAKAMURA

Visiting Scientist

Technical Staff

Sylvain HIVER
Yoko INOUE
Miwa KAWASAKI
Hiroko SAITO
Part-Time Staff

Haruka MORISHITA
AssistantMutsuko AISO-WATANABE

Tamako NISHIMURA

Publications
Tsukasaki Y, et al. Giant cadherins Fat and Dachsous selfbend to organize properly spaced intercellular junctions.
Proc. Natl. Acad. Sci. USA 45. 16011-16016 (2014)
Hayashi S, et al. Protocadherin-17 mediates collective
axon extension by recruiting actin regulator complexes tointeraxonal contacts. Dev Cell 30.673-87 (2014)
Takeichi M. Dynamic contacts: rearranging adherens
junctions to drive epithelial remodelling. Nat Rev Mol Cell
Biol 15.397-410 (2014)
Tanaka N, et al. Nezha/CAMSAP3 and CAMSAP2
cooperate in epithelial-specific organization ofnoncentrosomal microtubules. Proc Natl Acad Sci U S A
109.20029-34 (2012)
Nishimura T, et al. Planar cell polarity links axes of spatial
dynamics in neural-tube closure. Cell 149.1084-97 (2012)
Ishiuchi T. and Takeichi M. Willin and Par3 cooperatively
regulate epithelial apical constriction through aPKCmediated ROCK phosphorylation. Nat Cell Biol 13.860-6(2011)
Meng W, et al. Anchorage of microtubule minus ends toadherens junctions regulates epithelial cell-cell contacts.
Cell 135. 948-59 (2008)

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

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

61

Organ Regeneration
Takashi TSUJI Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/tsuji.html
Takashi Tsuji received his Masters degree from Niigata University in 1986, and after working in thepharmaceuticals industry for three years, returned to complete his doctorate at Kyushu University, andhe received his doctorate in 1992 from Niigata University. He conducted research at Niigata University
from 1992 to 1994 before moving to serve as researcher and then senior scientist at JT Inc. From
2001, he moved to Tokyo University of Science, and in 2007 he was appointed a full professor in thesame university. During his academic career, he received numerous prestigious grants and awards, andparticipated in numerous industry collaborations. He joined the CDB as a Group Director in 2014, and has
served as Team Leader since the restructuring of the Center in November 2014.

Organogenesis begins with the formation of patterned developmental fields during early
embryogenesis, which provide environments appropriate for the induction of specificorgans. Most organs emerge from primordia induced by interactions between epithelial andmesenchymal tissue and, following organ-specific morphological changes, develop intofunctional structures.
Our group is working to gain a more complete understanding of the roles of epithelialmesenchymal interactions in organ induction, development, and morphogenesis. Using
technologies developed in our group for the three dimensional (3D) control of epithelial stem
cells and mesenchymal stem cells, we have generated regenerative primordia for teeth, hairfollicles and endocrine tissue, such as salivary glands, and shown that these functionallyintegrate with surrounding tissue following transplantation into adult mice. By recapitulatingorganogenetic fields as seen in the early embryo to steer the self-organized formation of
three-dimensional tissue-like structures from pluripotent stem cells, such as embryonicstem cells (ESCs) or induced pluripotent stem cells (iPSCs), we seek both to elucidate themechanisms by which such fields induce organogenesis and to develop new technologies
for use in regenerative medicine. We are also interested in using 4D cell dynamics imaging
techniques to quantitatively analyze organ-specific morphogenesis, and to analyze, model,
and simulate molecular pathways involved in their control, in the hopes of gaining new
insights into the fundamental principles underlying organogenetic processes. Building onthese fundamental studies, we further seek to conduct applications research towards thedevelopment of technologies for use in therapeutic organ regeneration.

Staff
Collaborative Scientist

Student

Miho OGAWA
Katsunari TEZUKA
Koh-ei TOYOSHIMA

Jun ISHIKWA
Kei SAKAKIBARA

Research Scientist

Mayumi MUROFUSHI

Kyosuke ASAKAWA
Chikafumi OZONEAssistant

Technical Staff

Yuma FUKUTANI
Ryohei MINAMIDE
Yukiko MORIOKA
Hisae TSUJI

Publications
Oshima M, et al. Functional tooth restoration by nextgeneration bio-hybrid implant as a bio-hybrid artificial organ
replacement therapy. Sci Rep 4.6044 (2014)
Ogawa M, et al. Functional salivary gland regeneration
by transplantation of a bioengineered organ germ. Nat
Commun 4.2498 (2013)
Hirayama M, et al. Functional lacrimal gland regeneration
by transplantation of a bioengineered organ germ. Nat
Commun 4.2497 (2013)
Toyoshima K. E, et al. Fully functional hair follicle
regeneration through the rearrangement of stem cells andtheir niches. Nat Commun 3.784 (2012)
Ikeda E, et al. Fully functional bioengineered tooth
replacement as an organ replacement therapy. Proc Natl
Acad Sci U S A 106.13475-80 (2009)
Nakao K, et al. The development of a bioengineered organ
germ method. Nat Methods 4.227-30 (2007)

Bioengineered organ germ

62

Bioengineered hair follicle

Bioengineered toothSystems Biology
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, andreceived his Ph.D. from the University of Tokyo Graduate School of Medicine in March 2004. While anundergraduate, he worked as a research assistant on a biological simulation system project at Sony
Computer Science Laboratories. As a graduate student he next went on to work, first as a researcher (2000)
and then as a group leader (2002), at Yamanouchi Pharmaceuticals, on a project studying biological clock
mechanisms in fly 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. In 2009, he was promoted project leader of the Laboratory for
System Biology under CDB’s Center Director’s Strategic Program.
The Systems Biology lab closed in September 2014. Dr. Ueda is now affiliated with RIKEN QBiC
and the University of Tokyo.

Staff
Special Postdoctoral Researcher

Technical Staff

Yasutaka NIWA

Hiroshi FUJISHIMA
Junko HARA
Maki UKAI-TADENUMA
Junko YOSHIDA

Research Scientist

Genshiro SUNAGAWA
Senior Research Scientist

Hideki UKAI
Rikuhiro G. YAMADA

Student Trainee

Foreign Postdoctoral Researcher

Visiting Scientist

Craig C. JOLLEY
Dimitri PERRIN

Assistant

Visiting Researcher

Genki KANDA
Kohei MASUMOTO
Noriko OKAMOTO

Arthur MILLIUS

Publications
Susaki E. A, et al. Whole-brain imaging with single-cellresolution using chemical cocktails and computational
analysis. Cell 157.726-39 (2014)
Sunagawa G. A, et al. FASTER: an unsupervised fully
automated sleep staging method for mice. Genes Cells
18.502-18 (2013)
Jolley C. C, et al. A design principle for a posttranslational
biochemical oscillator. Cell Rep 2.938-50 (2012)
Ukai-Tadenuma M, et al. Delay in feedback repression by
cryptochrome 1 is required for circadian clock function. Cell
144.268-81 (2011)
Kasukawa T, et al. Human blood metabolite timetableindicates internal body time. Proc Natl Acad Sci U S A
109.15036-41 (2012)
Isojima Y, et al. CKIepsilon/delta-dependent phosphorylation
is a temperature-insensitive, period-determining process inthe mammalian circadian clock. Proc Natl Acad Sci U S A
106.15744-9 (2009)

Recent large-scale efforts in genome-sequencing, expression profiling and functional
screening have produced an embarrassment of riches for life science researchers andbiological data can now be accessed in quantities that are orders of magnitude greater thanwere available even a few years ago. The growing need for interpretation of data sets, as wellas 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 extension ofmolecular biology, and can be defined as "biology after the identification of key genes." Wesee systems-biological research as a multi-stage process, beginning with the comprehensive
identification and quantitative analysis of individual system components and their networked
interactions, and leading to the ability to drive existing systems toward a desired state anddesign new ones based on an understanding of structure and underlying principles.
Over the last several years, the Laboratory for Systems Biology (LSB) has worked toestablish experimental systems biology at the molecular-to-cellular level and apply them tosystem-level questions of complex and dynamic biological systems, such as the mammalian
circadian clock. In October 2009, our laboratory was re-designated as a Project Lab in theCenter Director’s Strategic Program for Systems Biology research to promote challenging
research endeavors. Based on the achievements over the past years, we strongly feel that it
is now the time for us to take the next step forward toward experimental systems biology at
the cellular-to-organism level.
Over the next several years, we intend to develop an efficient experimental platform to
identify, monitor, and perturb cellular networks within organism. To this aim, we will attempt
to invent and combine several key technologies ranging from (i) rapid engineering of the
genome of ES cells, (ii) generation of “100% chimera” animals for F0 phenotyping, and (iii)
phenotype analysis of a small number of the generated animals (ideally with a single animal).
Full utilization of these technologies will formulate cellular-to-organism-level systems biology,
which will provide new strategies and concepts for the diagnosis, treatment, and preventionof biological-time-related disorders, including rhythm disorder, seasonal affective disorder,and sleep disorder.

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

63

Epithelial Morphogenesis
Yu-Chiun WANG Ph.D.
http://www.cdb.riken.jp/en/research/laboratory/wang.html
Yu-Chiun Wang completed his B.Sc. (1996) and M.S. (1998) from the National Taiwan University, Taiwan,
and received his doctorate in 2006 from the University of Chicago, U.S.A. under the supervision of Prof.
Edwin Ferguson for his work on the regulation of BMP signaling during dorsal-ventral patterning in the
Drosophila for which he was awarded the 2007 Larry Sandler Memorial Award for best dissertation of
Drosophila research. In 2007, he moved to Princeton University to work as a postdoctoral fellow in thelaboratory of Eric Wieschaus and was also awarded a postdoctoral research fellowship from the Helen
Hay Whitney Foundation for his work on understanding the mechanisms of the formation of the cephalicfurrow and dorsal transverse folds during Drosophila gastrulation. He was appointed Team Leader of theLaboratory for Epithelial Morphogenesis at the CDB in October 2013.

The central question in developmental biology is how cells, tissues and organs acquire their
specific functions and shapes. A large body of work over the past several decades has
yielded a broad understanding of how functional specialization is achieved through differential
gene expression. In contrast, far less is known about how cell shapes and tissue structures
are controlled and remodeled. Although a general theme has emerged whereby cytoskeletal
elements control the cell shapes, while alteration of individual cell shapes collectively
organizes the tissue architecture, the underlying molecular and mechanical mechanisms
remain poorly understood. My lab aims at identifying novel mechanisms that orchestrate theformation of three-dimensional epithelial structures. Our long-term goal is to comprehensively
understand the mechanistic principles of tissue morphogenesis in order to conceptualize theorigin of morphological diversity both within an organism and among evolutionary lineages.
We are currently focusing on how modifications of epithelial cell polarity control cell shapes
using gastrulating Drosophila embryos as the model system. Our previous work identified
a novel mechanism for cell shape changes whereby cell shortening is induced upon abasal repositioning of the apical-basal polarity and cell-cell adhesive apparatus adherens
junctions. The cell shortening occurs in two narrow strips of cells, producing heterogeneities
in cell height within the tissue, thereby allowing it to bend. The polarity-based mechanism
represents the first instance wherein the initiation of epithelial folding does not involve thecanonical myosin-dependent apical constriction. Since cell-cell adhesion and apical-basal
polarization are two fundamental features of epithelial tissues, our work potentially heralds a
general mechanism for cell shape changes and epithelial folding. In addition, we found thatafter initiation, the depths of epithelial folds differ depending on the degrees of neighboring
cell invagination. Genetic evidence suggests that the strength of mechanical coupling
between adherens junctions and their underlying actin cytoskeleton determines the extent ofcell invagination. Our ongoing work promises to identify genes and forces that sculpt distinct
morphological features.

Staff
Research Scientist

AssistantNiraj K. NIRALA
Percival P. SANGEL

Yuko FUJIYAMATechnical Staff

Mustafa SAMI
Michiko TAKEDA

Publications
Khan, Z. et al. Quantitative 4D analyses of epithelial folding
during Drosophila gastrulation. Development 141.2895-900(2014)
Gavin-Smyth, J. et al. A genetic network conferring
canalization to a bistable patterning system in Drosophila.
Curr Biol 23.2296-302 (2013)
Wang Y.-C, et al. Distinct Rap1 activity states control theextent of epithelial invagination via alpha-catenin. Dev Cell
25.299-309 (2013)
Wang Y.-C. et al. Differential positioning of adherens
junctions is associated with initiation of epithelial folding.
Nature 484.390-3 (2012)
Wang Y.-C. and Ferguson E. L. Spatial bistability of Dppreceptor interactions during Drosophila dorsal-ventral
patterning. Nature 434.229-34 (2005)
Podos S. D. et al. The DSmurf ubiquitin-protein
ligase restricts BMP signaling spatially and temporally
during Drosophila embryogenesis. Dev Cell 1.567-78(2001)

We employ an integrated approach that combines genetic manipulation, two-photon deep
tissue live imaging and computational cell shape reconstruction. We are also in the processof designing novel imaging strategies that could be used to visualize mechanical forces andcomputational algorithms that reconstruct and quantify 4D cell shapes. Furthermore, we will
launch a multidisciplinary, international collaboration that combines genetics, computationaland evolutionary approaches to analyze the history and function of transiently formed
epithelial structures that do not eventually contribute to a body part or organ.

Dorsal fold formation during Drosophila gastrulation.
The dorsal epithelium of the Drosophila gastrula forms twoepithelial (anterior and posterior) folds, shown here with the
mid-sagittal optical sections at an early (A) and a late (B) stage.
Adherens junctions are labeled in green; plasma membrane inmagenta.

64

Reconstruction of 3D cell shape during dorsal foldformation.
EDGE4D software was developed in collaboration
with Dr. Zia Khan (University of Maryland). 3D
reconstruction of cell shape was based on cell
membrane labeling. Immunolabeling of adherens
junctions (orange in A and B) defines the boundaries
between the apical and basal-lateral compartments(A’ and B’).

Plastic cast of airway

65

Inklings of insight into inner ear development

Xiaorei SAI, Raj LADHER

The inner ear is located in the inside of the head, howeverat the earliest stages of embryonic development, it forms
as a thickening of the ectoderm (epithelia) on the outersurface of the embryo, referred to as the otic placode. This
placode invaginates into the mesenchyme of the future
head region, eventually budding off to form an otic vesicle,
the precursor of inner ear organs. Invagination of the otic
placode, similar to many epithelial invagination processes,
involves two phases: first, there is an expansion of thebasal membrane of otic cells, followed by a constrictionof the apical side of otic cells. Whereas the molecularmechanisms that drive basal membrane expansion in
the first phase of otic invagination are beginning to beunderstood, the mechanisms controlling constriction of theapical side in the second phase remain relatively unknown.
Now, new work published in Developmental Biology byresearch scientist Xiaorei Sai of the Laboratory for Sensory
Development (Raj K. Ladher, Team Leader) and colleagues
has uncovered a molecular pathway coordinating the66

apical constriction of otic cells during the second phase ofotic invagination. Using chicken embryos as a model, they
demonstrate that localized activation of Ras homolog genefamily member A (RhoA) protein at the apical cell junctions
triggers a signaling pathway leading to recruitment andactivation of non-muscle myosin-II, an actin-binding
protein, to initiate apical constriction of the otic placode.
A previous study from this laboratory (Sai and Ladher,2008) showed that basal activation of non-muscle myosinII instigated basal expansion of the otic placode in phase
1 of otic invagination; myosin-II is activated by basally
localized FGF signaling, which leads to depletion of actin
filaments on the basal side, and consequently, basal
expansion. Studies of similar invagination processes,
such as gastrulation and lens placode formation, by othergroups have indicated that activation of myosin-II is also
important for apical constriction, where activated myosin-II
triggers a signaling cascade leading to constriction of actin
filaments on the apical side. Thus, these findings suggest©2014 Elsevier B.V.
Sai X, et al. Junctionally restricted RhoA activity is necessary for apicalconstriction during phase 2 inner ear placode invagination. Dev Biol 394.206-16 (2014)

that activation of myosin-II at apical and basal sides of theotic placode involve different molecular cues, resulting incontrasting activities for myosin-II on actin filaments.
So what are the molecular cues at work at the apicalside? Sai et al. focused their sights on the RhoA protein,a member of the Rho family of small GTPases, which is
known to regulate actin cytoskeleton dynamics and has
also been implicated in promoting apical constrictionof epithelial cells during invagination processes in othermodel systems. In these systems, RhoA triggers Rhoassociated coiled-coil protein kinases (ROCKs) tophosphorylate myosin light chain (MLC) to activate
myosin-II, leading to constriction of actin filaments. In
the case of the otic placode, Sai et al. observed a rise in
RhoA expression during phase 2 of otic invagination, withhigh levels detected in the apical junction complex. RhoA
activation assays on otic placode extracted from embryos
during this same period also confirmed the presence
RhoA activity. When RhoA activity was knocked downusing small RNAi, they detected reduced levels of active
apical MLC as well as actin in the apical junctions, and alarger apical surface area than control embryos suggested
that minimal or no constriction had taken place. Thus,
these results highlight the importance of RhoA for apicalconstriction. They also looked into whether ROCKs,
potential RhoA downstream factors, also played a role inphase 2 constriction by inhibiting ROCK expression andfound that otic invagination was blocked in addition to
disruption of the localization of actin and activated MLC toapical junctions, indicating ROCKs were also needed forapical constriction.
The team next attempted to determine the molecule
triggering RhoA activity. Rho family small GTPases are
generally switched on by guanine exchange factors (GEFs),so they performed a screening for apically localized
GEFs with using antibodies. The ArhGEF11 protein
was deemed a putative RhoA GEF since its expression
became concentrated in the apical area as otic placodeinvagination progressed, overlapping with expressionof junctional marker ZO-1. Knocking down normal
ArhGEF11 activity levels led to reduced activity of myosinII and localization of actin to apical junctions. ArhGEF11
expression was not affected when RhoA was knocked
down, thereby indicating that ArhGEF11 was upstream of
RhoA and also involved in apical constriction.
Studies on neural tube formation had reported that aplanar cell polarity signal, Celsr1, was needed to activate
myosin-II through RhoA and its upstream GEF. Thus, theteam speculated that Celsr1 may be an upstream regulatorof RhoA and its GEF, ArhGEF11, to enhance apicalconstriction in the otic placode. Expression experiments
showed that while weak Celsr1 expression is detectedin the ectoderm just as the otic placode is beginningestablished, during the phase I of otic morphogenesis,
its expression is upregulated and localized to the apicaljunction, reaching a peak during the phase 2. When Celsr1
expression levels were reduced in otic placodes during
the phase 2, they found reduced activity and localizationof actomyosin at the apical cell junction, resembling what
was seen in ArhGEF11 knockdown experiments.
Based on their data, they proposed a model for apicalconstriction seen in phase 2 of otic invagination, where
the planar cell polarity protein Celsr1 is upregulated in
the apical otic placode as otic morphogenesis begins,
and accumulates at the apical cell junctions. Celsr1 thenrecruits ArhGEF11 to the apical junction, which in turnactivates RhoA, a key factor needed to trigger ROCKs toactivate myosin-II, leading to constriction of actin filaments
anchored to the apical junction.
“It is clear that epithelial remodeling in the neural tube
and during inner ear invagination share a common
morphogenetic toolbox,” says Ladher. “Thus findings in
one of these processes could be used and adapted tounderstand conditions with clinical significance, such asneural tube defects, body wall closure defects and woundhealing.“

Scanning electron micrograph showing the inner ear
after 50 hours of development. The inner ear, which
formed on the surface ectoderm, has now completed
its invagination, and is embedded within the headmesenchyme

67

Cellular communication is key for
upper-layer neurogenesis

Kenichi TOMA

The neocortex is a six-layered brain region unique tomammals and is made up of a complex network ofmultiple cell types. This region is particularly large inprimates, including humans, and is associated withhigher order brain functions such as perception, thinking,memory, and language. Neurons of the neocortex arederived from progenitor cells that lie in the ventricular zone,
and these progenitors divide and differentiate to generate
deep-layer (DL) projection neurons and then upper-layer(UL) projection neurons in a timely manner, with the laterborn neurons migrating to upper layers. The transition fromgenerating one neuronal subtype to another is critical for
creating the layered organization of the neocortex, but themechanisms involved in this regulation are still shrouded inmystery.
Now, a study by research scientist Kenichi Toma of
the Laboratory for Neocortical Development (Carina
Hanashima, Team Leader) and colleagues, published in68

the Journal of Neuroscience, identifies the cues involved intriggering the onset of UL neurogenesis in the developing
neocortex. Using a series of transgenic mouse lines,
they found that induction of UL neurogenesis requires
sequential derepression of transcription factors, triggered
by Foxg1, and also a feedback signal from earlier-born,
differentiated DL neurons. Thus, the neural progenitors
use a combination of intrinsic gene regulatory mechanisms
and extrinsic cues from surrounding cells to switch fromDL to UL neurogenesis.
In neocortical development, Cajal-Retzius (CR) cells,
which occupy the most superficial layer of the cortex,
are generated first, followed by DL neurons that are
generated from embryonic day (E)11.5, and UL neurons
born at E14.5. These neurons then undergo a processof maturation to form a six-layered structure. Previous
work from the laboratory has shown that expression of atranscription factor, Foxg1, triggers progenitors to enter©Society of Neuroscience
Toma K, et al. The timing of upper-layer neurogenesis is conferred by sequential
derepression and negative feedback from deep-layer neurons. J Neurosci 34.13259-76 (2014)

DL neurogenesis from CR cell production (see CDB News:
April 5, 2013). The regulatory mechanisms involved in
the switch from DL neurogenesis to UL neurogenesis,
however, remained unclear.
In the current study, the team examined developmentof UL neurons in detail using transgenic mouse lines
previously developed in the lab, in which timing of
Foxg1 expression could be manipulated. Under normal
conditions, the expression of this gene first appears
around E8.5. When they delayed the expression onset toa later developmental stage (at E14.5), the period of CR
neuron generation was prolonged concomitantly, until theexpression of Foxg1 rapidly induced DL neurogenesis.
Using different markers to label the cohort of neurons
produced at E14.5 and those produced the next day, theteam found that at E18.5 most of the earlier-born neurons
had differentiated into DL neurons, while the later-bornneurons had primarily differentiated into UL neurons.
The further 1-day delay of Foxg1 induction to E15.5 also
gave similar results, suggesting that irrespective of the
progression of development, the onset of UL neurogenesis
cannot bypass DL neurogenesis.
Maintaining the layered structure of the neocortex
also requires regulation of transcription factors. Eachdifferentiated layer subtype expresses specific transcription
factors, and is segregated from the other layer subtypes
through mutual repression of four transcription factors.
However, how this repression network is triggered toconfer fate specification in neurogenesis was not wellunderstood. Toma et al. speculated that Foxg1 was
the trigger to tip the balance, and looked into the gene
expression levels of the four transcription factors afterFoxg1 was induced. Tbr1 was the only transcription
factor out of the four that showed a marked decrease in
expression levels after Foxg1 induction. The cells in which

Tbr1 was suppressed after Foxg1 induction also showeda rise in expression of DL neuron marker, and went on
to differentiate into DL neurons, indicating that inductionof Foxg1 leads to suppression of Tbr1 in progenitor cells
signaling them to commit to DL neuronal fate.
So after induction of DL neurogenesis, how do theprogenitors time the switch over to UL neurogenesis?
Under culture conditions, progenitors need to be
maintained under relatively high density conditions to
induce differentiation of UL neurons. Fixing on this point,
the team examined whether onset of UL neurogenesis
could be regulated by an extrinsic signal originating fromDL neurons themselves. They generated a mouse line
in which DL neurons produced between E11.5 to E13.5
were specifically ablated, and found that, despite a large
drop in DL neuronal population immediately after ablation,
their numbers had recovered substantially by E18.5. UL
neurons were also generated, although slightly fewer
than seen under normal conditions. Closer examination
revealed a prolonged period of DL neurogenesis, which inturn led to a delay in the onset of UL neurogenesis. These
results indicate that the differentiated DL neurons senda signal to the progenitors to halt DL neurogenesis andinitiate UL neurogenesis.
“The mechanisms uncovered in this study are extremely
useful systems for generating the necessary neuronalsubtypes in sequential order, as well as maintaining therelative balance of their number regardless of the size
differences, for example, between mouse and humanbrains,” says Hanashima. “Our next step is to reveal whatmolecular signal from DL neurons is relayed to progenitors
to facilitate the switch to UL neurogenesis, bringing us
closer to understanding the mechanisms of neocorticalneurogenesis.”

Coelectroporation of Foxg1 and GFP constructs (shown in green)in brain cells of Foxg1 knockout mouse, represses Tbr1 expression(red) and triggers the expression of DL neuron marker Ctip2 (blue)69

When flies are low on sugar

Hiroko MATSUDA (Front:left), Takayuki YAMADA (Back:second from the right)

Sugars such as glucose circulating in the blood and storedin tissues are important sources of energy and carbon
necessary for living organisms to maintain metabolic
homeostasis. In many invertebrates, including insects,
trehalose is the predominant sugar found circulating in the
blood (or hemolymph). The unique chemical propertiesof trehalose are thought to protect organisms fromenvironmental stresses such as desiccation and changes
in temperature. Trehalose levels in the hemolymph are
known to be regulated by a pair of endocrine hormones,
similar to how insulin and glucagon regulate glucose levels
in humans, to control the glycometabolism of an organism
in response to availability of nutrients. However, the precise
function of trehalose during the process of development
remains largely unknown.
In a new study carried out by research scientist Hiroko
Matsuda and technical staff Takayuki Yamada in the
Laboratory for Growth Control Signaling (Takashi

70

Nishimura, Team Leader) and others, published in the
Journal of Biological Chemistry, they use the Drosophila
system to analyze the physiological role of trehalose. Theirfindings reveal that trehalose plays an important role inregulating glycometabolism and growth as well as on theviability of the fly, especially when there are changes inexternal factors such as nutrient availability.
In Drosophila, glycogen, the main storage form of
glucose, is stored in tissues, whereas trehalose is foundstored and circulating in relatively high concentrationsin the hemolymph. When needed, these storage sugars
are broken down into glucose, which is used by tissues
to produce energy in the form of ATP. In this study, tounderstand the role of trehalose during development,
Matsuda et al. generated Drosophila mutants that lacked
the trehalose-synthesizing enzyme, trehalose-6-phosphatesynthase (Tps1), and compared their developmental
physiology with wildtype flies.

©American Society for Biochemistry and Molecular Biology
Matsuda H, et al. Flies without Trehalose. J Biol Chem (2014)

The group first examined the levels of trehalose in wildtype
flies at different developmental stages. Trehalose wasundetected in early stage embryos, but as development
progressed from late embryonic to larval stages,trehalose levels began to rise significantly, and during thepupal period, saw a sharp decline. These observations
suggested that trehalose was synthesized during
embryonic and larval development, and then consumed
during pupal development. They also confirmed thatthe main site of trehalose synthesis was the fat body,an organ with functions similar to the mammalian liver.
When development of Tps1 mutants was examined, as
expected, trehalose production and trehalose was notdetected. When grown under normal nutrient conditions,the mutants displayed relatively normal development until
the pupal stage, albeit slightly smaller in size than wildtype;but, they died before eclosion. Thus, while trehalose isimportant for viability of the fly, their findings suggest that
it is not essential for larval development up to the pupal
stage, at least under normal nutrient conditions.
So Matsuda et al. next investigated how dietary stress(starvation) would affect the larval period of Tps1 mutants.
When the mutant larvae were placed on a water-only diet,most died after the first day of starvation, while wildtype
larvae survived at least three days on the same diet. The
starved Tps1 mutant larvae showed no major changes in
the levels of ATP and of hormones that regulate energy
metabolism, and also began producing energy by breaking

down the proteins and stored fat in the body, as is seen
in starved wildtype larvae. However, a closer examination
revealed a significant reduction in glycogen levels in thecentral nervous system of the Tps1 mutants. Many cells
in the brain also underwent apoptosis. Thus, the lack oftrehalose synthesis in Tps1 mutants results in a localized
depletion of energy sources in brain cells which use
glucose as its primary source of energy, causing defects inthe brain and likely lethality.
The group further tested the effects of nutrient availabilityon the development of Tps1 mutants, placing them undera number of different dietary conditions. Under a lowsugar diet, the mutant larvae died before the pupal stage.
In contrast, under a low-protein (normal sugar) diet, mostof the mutant larvae reached the pupal stage, but with adelay in the timing of puparium formation and a markedly
smaller pupa sizes. An analysis of the developmental
physiology revealed that Tps1 mutants had reduced
insulin/insulin-like growth factor signaling activity, which is
involved in growth control of the fly.
“Our study showed that Tps1 mutants can survive to thepupal stage if dietary sugar is available, but die before
eclosion, so we would like to close in on the exact roletrehalose plays during the pupal period,” says Nishimura.
“It would also be interesting to understand how the
Drosophila uses the two storage sugars, glycogen andtrehalose, for the same or different purposes.”Central nervous system of larval stage Drosophila. After starvation, glycogen levels of
the Tps1 mutant (right) are markedly reduced compared with wildtype (left).

Under low protein conditions, the pupa sizes Tps1 mutants(right) are smaller than wildtype (left).

71Animal Resources and Genetic Engineering
Yasuhide FURUTA Ph.D.

Genetically engineered mice are one essential tool in modern biomedical research. The quality and efficacy of research
are greatly dependent on how efficiently mutant mice can be generated, propagated, and housed. The major role of theLaboratory for Animal Resources and Genetic Engineering (LARGE) is to provide comprehensive services to develop
mutant mouse models that are useful for research in animal development and regeneration, and to maintain and enhancethe experimental rodent resources at the CDB. LARGE comprises two complementary units that work to accomplishthese missions.
As part of the reorganization of the CDB in November 2014, the LARGE Unit was transferredto the RIKEN Center for Life Science Technologies.

Genetic Engineering Unit

Yasuhide FURUTA Ph.D.
Staff
Senior Advisor

Shinichi AIZAWA
Research Specialist

Takaya ABE
Go SHIOI
Visiting Scientist

The Genetic Engineering Unit works with research labs within the
CDB, as well as others in Japan and throughout the Asia-Pacificregion to develop genetically engineered mice useful for the study ofdevelopment and regeneration. In these joint development projects,
we receive sequence information of the subject genes from ourcollaborators, and perform all subsequent stages of mutant mouseproduction from constructing the targeting vectors to generating
chimeras, making about one hundred new mutant mouse lines
every year. In addition, we develop new tools and technologies
for bioimaging to aid in the visualization of mouse development at
tissue, cell, and organelle levels.

Publications
Miyazaki H, et al. Singular localization of
sodium channel beta4 subunit in unmyelinatedfibres and its role in the striatum. Nat Commun
5.5525 (2014)
Lee M, et al. p120-catenin regulates REST and
CoREST, and modulates mouse embryonicstem cell differentiation. J Cell Sci 127.403751 (2014)
Ishiguro K, et al. Meiosis-specific cohesin
mediates homolog recognition in mouse
spermatocytes. Genes Dev 28.594-607 (2014)

Toshihiko FUJIMORITechnical Staff

Kana BANDO
Naoko HATAMOTO
Michiko HIGASHIKAWA
Yoshiko MUKUMOTO
Megumi WATASEYui YAMASHITA
Student Trainee

Atsumi DENDA

Chimeric mice exhibiting mixed coat color

An E9.5 embryo expressing theR26p-Fucci2 cell cycle probes

Animal Resource Unit

Hiroshi KIYONARI Ph.D.
Staff
Attending Veterinarian

Naoko KAGIYAMA
Research Consultant

Kazuki NAKAO
Technical Staff

Karin AKIYAMA
Kenichi INOUE
Mari KANEKO
Yuki KANEKO
Takuya KAWADA
Masako NODA
Miho SATO
Mayo SHIGETA
Aki SHIRAISHI
Tomoko TOKUNAGA
Sachi YAKAWA
Daisuke YAMAMOTO

The Animal Resource Unit maintains and cares for the CDB’s
laboratory mouse and rat resources in a Specific Pathogen Free (SPF)
environment. We also handle transfer of mutant mice both within
the CDB and to/from other domestic and overseas institutions. In
addition, we provide pregnant females, fertilized mouse eggs, andservices for colony expansion and strain cryopreservation. We also
carry out research projects aimed at developing new technologies
in reproductive biology. More recently, we have established colonies
of a metatherian species, the gray short-tail opossum (Monodelphis
domestica), and a reptilian species, the gecko (Paroedura picta),
establishing optimal environment and breeding conditions, withthe aim for distribution of these new model animals to the researchcommunity in the near future.

Assistant

Masayo OKUGAWA
Yuki TSUJI

Pronuclear injection of DNA for generationof transgenic mice

72

Publications
Hayashi S, et al. Protocadherin-17 mediates
collective axon extension by recruiting actin
regulator complexes to interaxonal contacts.
Dev Cell 30.673-87 (2014)
Susaki E. A, et al. Whole-brain imaging withsingle-cell resolution using chemical cocktails
and computational analysis. Cell 157.726-39(2014)
Goriki A, et al. A novel protein, CHRONO,
functions as a core component of the
mammalian circadian clock. PLoS Biol12.e1001839 (2014)

Electron Microscope
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 inthe use of transmission and scanning electron microscopy (TEM and SEM) and related technologies in morphologicalanalyses.
As part of the reorganization of the CDB in November 2014, the Electron Microscope Lab was transferredto the RIKEN Center for Life Science Technologies.

Electron Microscope

Shigenobu YONEMURA Ph.D.
Staff
Research Scientist

Hanako HAYASHI
Kumiko TORISAWA
Technical Staff

Kisa KAKIGUCHI
Kazuyo MISAKI
Suzuka TAKAHASHI
Visiting Scientist

Masatsune TSUJIOKA
Research Fellow

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

Akira ONODERA
Ayuko SAKANEStudent Trainee

Yu AMANO
Assistant

Mai SHIBATA

Cells organizing the notochord of zebrafish embryo

Publications
Y o n e m u r a S . D i f f e r e n t i a l s e nsitivity ofepithelial cells to extracellular matrix in polarity
establishment. PLoS One 9.e112922 (2014)
Ebrahim S, et al. NMII forms a contractile
transcellular sarcomeric network to regulate
apical cell junctions and tissue geometry. Curr
Biol 23.731-6 (2013)
Tsujioka M, et al. Talin couples the actomyosin
cortex to the plasma membrane during rear
retraction and cytokinesis. Proc Natl Acad Sci
U S A 109.12992-7 (2012)
Yonemura S. A mechanism of
mechanotransduction at the cell-cellinterface: emergence of alpha-catenin as thecenter of a force-balancing mechanism formorphogenesis in multicellular organisms.
Bioessays 33.732-6 (2011)
Yonemura S. Cadherin-actin interactions
at adherens junctions. Curr Opin Cell Biol
23.515-22 (2011)
Yonemura S, et al. alpha-Catenin as a tension
transducer that induces adherens junction
development. Nat Cell Biol 12.533-42 (2010)

Alpha-catenin molecules stretched by forces produced by Myosin II (green)
are selectively labeled with alpha18 antibody(red).

73

Bio-Imaging
Shigeo HAYASHI Ph.D.

The role of the Bioimaging Laboratory is to assist in the operation and maintenance of the Center’s core imaging facility
and support scientists through the provision of superior optical imaging technologies, analysis and presentation of results.
The lab will manage central optical microscopy systems, image analysis software and the associated IT environment, andpromote a secure environment for the effective use of these resources.
As part of the reorganization of the CDB in November 2014, the Bio-imaging lab was transferredto the RIKEN Center for Life Science Technologies.

Optical Image Analysis Unit

Yuko KIYOSUE Ph.D.
Staff
Technical Staff

Tomoko HAMAJI
Technical Staff

YuPing WUAssistant

Yuki TANOThis unit runs common use CDB Imaging Facility. Bio-imaging is aninterdisciplinary process that integrates molecular cell biology andbiochemistry, as well as technology from optics, engineering, andcomputer sciences. It has enabled scientists to visualize biologicalprocesses at the cellular and molecular levels and today, has
become an indispensable field for research on biological systems.
We aim to design an imaging environment that can adapt tochanging scientific demands and can contribute to the progressof biology. We hope to accelerate biological research by matchingresearch concepts with the latest technologies from the different
specialized fields.
Our laboratory studies mechanisms of life woven by cellular skeleton
molecules. We aslo aim to develop our basic reasearch to contributeto the development of treatments for disease and health promotion.

Publications
van der Vaart B, et al. CFEOM1-AssociatedKinesin KIF21A Is a Cortical Microtubule
Growth Inhibitor. Dev Cell 27. 145-60 (2013)
Shimozawa T, et al. Improving spinning disk
confocal microscopy by preventing pinholecross-talk for intravital imaging. Proc Natl
Acad Sci U S A 110.3399-404 (2013)
Nakamura S, et al. Dissecting the Nanoscale
Distributions and Functions of MicrotubuleEnd-Binding Proteins EB1 and ch-TOG in
Interphase HeLa Cells. PLoS One 7.e51442(2012)
Mimori-Kiyosue Y. Shaping microtubules into
diverse patterns: molecular connections forsetting up both ends. Cytoskeleton (Hoboken)
68.603-18 (2011)
Kosodo Y, et al. Regulation of interkinetic
nuclear migration by cell cycle-coupled active
and passive mechanisms in the developing
brain. EMBO J 30.1690-704 (2011)
Hotta A., et al. Laminin-based cell adhesion
anchors microtubule plus ends to the epithelial
cell basal cortex through LL5alpha/beta. J Cell
Biol 189.901-17 (2010)

Inverted microscope maintained by the Optical Image Analysis Unit

74

Genomics
Fumio MATSUZAKI Ph.D.

The Genomics Laboratory works to support a wide range of genomic and epigenomic research and functional genomics
research, providing genome-scale, high-throughput services in sequencing gene expression analysis to all CDB labs.
All projects can be initiated and followed using an internal website designed to ensure a smooth workflow and timely
reporting of results.
As part of the reorganization of the CDB in November 2014, the Genomics lab was transferredto the RIKEN Center for Life Science Technologies.

Genome Resource and Analysis Unit

Shigehiro KURAKU Ph.D.
Staff
Research Specialist

Mitsutaka KADOTA
Research Scientist

Munazah ANDRABI
Yuichiro HARA
Miyuki NORO
Technical Staff

Kazu ITOMI
Kana MIYAGISHI
Osamu NISHIMURA
Kaori TANAKA
Chiharu TANEGASHIMA
Kaori TATSUMI
Kenichiro UNO

The Genome Resource and Analysis Unit (GRAS) provides a broad
range of biologist-oriented support for Sanger and massively parallel
deep sequencing, sequence informatics and gene expression
profiling. In parallel, we conduct our original research projects
on vertebrate comparative genomics, focusing on cross-species
comparisons of developmental programs. Above all, GRAS aims
to create an integrative research support station with active
communication between laboratory staff and bioinformaticians,
and take full advantage of evolving massively parallel sequencing
technologies to apply them to transcriptomic, epigenetic andgenomic projects in developmental biology arising in the whole
institute.

Assistant

Sayo SAITO
Student Trainee

Publications
Kuraku S, et al. aLeaves facilitates on-demand
exploration of metazoan gene family trees
on MAFFT sequence alignment server withenhanced interactivity. Nucleic Acids Res
41.W22-8 (2013)
Smith J. J, et al. Sequencing of the sea
lamprey (Petromyzon marinus) genome
provides insights into vertebrate evolution. Nat
Genet 45.415-21, 21e1-2 (2013)
Kuraku S. Impact of asymmetric gene
repertoire between cyclostomes andgnathostomes. Semin Cell Dev Biol 24.119-27(2013)
Oisi Y, et al. Craniofacial development of
hagfishes and the evolution of vertebrates.
Nature 493.175-80 (2013)
Wang Z, et al. The draft genomes of soft-shellturtle and green sea turtle yield insights into
the development and evolution of the turtlespecific body plan. Nat Genet 45.701-6 (2013)
Adachi K, et al. Context-dependent wiring ofsox2 regulatory networks for self-renewal ofembryonic and trophoblast stem cells. Mol
Cell 52.380-92 (2013)

Fumio MOTONE

Our main sequencing platform, illumina HiSeq 1500, in operation since spring 2012

Microfluidics-based equipment for single cell gene
expression profiling75

Proteomics
Shigeo HAYASHI Ph.D.

The identification of proteins from trace amounts of biologically important protein complexes help researchers to discovernovel molecular pathways and has become an indispensable approach to the study of development and regeneration. A
better understanding of the protein components of cells and tissues may yield new insights into the molecular structureand function that underlies the spectrum of biological phenomena and improve our ability to manipulate and recapitulatethem.
The Proteomics lab was closed as part of the reorganization of the CDB in November 2014.

Mass Spectrometry Analysis Unit

Shigeo HAYASHI Ph.D.
Staff
Research Specialist

Reiko NAKAGAWA

The Mass Spectrometry Analysis Unit uses LC-MASS spectrometry,a technology that enables the high-speed analysis of protein
components from even minute biological samples. Its support
activity takes the forms of protein identification services and analysis
of protein modifications such as phosphorylation, ubiquitination,and proteolysis. The unit supports laboratories within the CDB, andcollaborations with a number of laboratories at other institutions.
The lab receives gel slices containing proteins from researchers andconducts all subsequent steps, including de-staining of gels, in-gel
digestion of proteins, peptide extraction, and operating the LC-MS/
MS.

Mass Spectrometer Orbitrap Velos Pro

PAL HTS-xt autosampler

76

MASCOT proteome analysis server

Publications
Nishibuchi G, et al. N-terminal phosphorylationof HP1alpha increases its nucleosome-binding
specificity. Nucleic Acids Res 42.12498-511(2014)
Kamata K, et al. The N-terminus and Tudor
domains of Sgf29 are important for its
heterochromatin boundary formation function.
J Biochem 155.159-71 (2014)
Nishibuchi G, et al. Physical and functional
interactions between the histone H3K4
demethylase KDM5A and the nucleosome
remodeling and deacetylase (NuRD) complex.
J Biol Chem 289.28956-70 (2014)
Izumi H, et al. p54nrb/NonO and PSF promote
U snRNA nuclear export by accelerating its
export complex assembly. Nucleic Acids Res
42.3998-4007 (2014)

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

The Division of Human Stem Cell Technology (DHSCT) was established to provide support services to any lab with an interest in using human embryonic stem
cells (hESCs) and their derivatives in their research. The DHSCT provides technological expertise, training and support in hESC culture, maintenance, distribution
and management, as well as monitoring and analysis of global trends in stem cell research and regulation.
The Human Stem Cell Technology Division was closed as part of the reorganization of the CDB in November 2014.

Human Stem Cell Technology Unit

Deputy Unit Leader

Masatoshi OHGUSHI Ph.D.
Staff
Research Specialist

Hiroyuki KITAJIMA
Visiting Scientist

Taisuke KADOSHIMA
Daiki NUKAYA

Stem cells - both embryonic and somatic - can be challenging tostudy and manipulate in vitro. The Division of Human Stem Cell
Technology Unit was established to provide a full spectrum of
support services to labs within the CDB and throughout Japan
with an interest in using human embryonic stem cells (hESCs)
and their derivatives in their research. The unit provides expertise,training, and support in hESC culture, maintenance, distribution andmanagement, for users in the life sciences community. We seek tocontribute to achieving the goals of translational research, in linewith the CDB mission to establish a solid scientific foundation forregenerative medicine.

Publications
Nakano T, et al. Self-formation of optic cups
and storable stratified neural retina fromhuman ESCs. Cell Stem Cell 10.771-85 (2012)
Ohgushi, M., et al. Molecular Pathway and Cell
State Responsible for Dissociation-Induced
Apoptosis in Human Embryonic Stem Cells.
Cell Stem Cell 7, 225-239 (2010)

Technical Staff

Masayo FUJIWARA
Michiru MATSUMURA-IMOTO
Maki MINAGUCHI
Junior Research Associate

Chikafumi OZONEPart-Time Staff

Yuko MARUYAMA

Science Policy and Ethics Studies Unit
Masatoshi TAKEICHI Ph.D.
(Acting Unit Leader)

The field of stem cell research has been subject to legal, social, andethical tensions across a broad range of issues, from the researchuse of human embryos to the optimization of pathways for thetranslation of basic research into clinical applications. We seek tocompare different science policy approaches to these issues andidentify regulatory frameworks best suited to the development
and promulgation of stem cell applications. We will further explore
approaches to the clinical translation of human stem cell research.

Publications
Sipp D. Direct-to-consumer stem cellmarketing and regulatory responses. StemCells Transl Med 2.638-40 (2013)
Sipp D. and Turner L. U.S. Regulation of StemCells as Medical Products. Science 338.12961297 (2012)77

Kobe sits at the heart of the Kansai region of westernJapan, close to both the bright lights of Osaka andKyoto’s tranquility. The local climate is temperate, making it possible to enjoy a wide range of seasonal activities, all within a short train ride or drive from the centerof the city. Japan’s renowned public transport systemallows rapid and convenient access to both local andnational destinations, and the many area airports, including the Kobe Airport located less than 10 minutes
from the CDB, and the Kansai International Airport,
provide immediate gateways to any destination in theworld. Costs of living are comparable to those in many
major Western cities, and comfortable modern homes
and apartments are available to suit all budgets.
The bustling heart of Kobe includes a variety of distinct
neighborhoods. The downtown area of Sannomiya sits
square in the city’s center, and its range of international
restaurants and latenight bars promise a great eveningout any night of the week. The neighboring Motomachi
district offers a mix of upscale department stores andfunky shopping arcades, standing in contrast to thecolorful Chinatown that’s right next door. A short walknorth from city center lies the old foreign settlement ofKitano, whose clapboard houses and well-kept parks
are a perfect retreat from the dynamism downtown.

Kobe occupies a splendid location between mountains
and seas, with easy access to many natural spots. A
short drive or bus ride takes you to the island of Awaji,
with its beaches and first-rate seafood, and hiking trails
crisscross the forested mountains that span the entire
length of the city, beckoning hikers and picnickers toenjoy a day in the fresh air. The city is also dotted withparks that come into bloom at the start of cherry blossom season, and its many rivers, beaches and scenic
areas make it easy to take enjoyable day trips, inside
the city or out.
Its central location in Japan puts Kobe within close
reach of many of the country’s most popular destinations. Kyoto, the ancient capital, is only an hour away bytrain, making it possible to enjoy some of Japan’s mostpopular historic sites and cultural assets. Nara, anotherearly city, features herds of tame deer in the precinctsof some of the world’s oldest wooden buildings. Complementing the old-world style of these two cities isJapan’s second city of Osaka, which offers a hip andmodern take on urban living.

RIKEN in Kobe
Budget and Staff
CDB Symposium
Seminars
About RIKEN
RIKEN Campuses

80

81

2014 CDB Symposium

Regeneration of Organs:
Programming and Self-organization
March 10­–12, 2014
The RIKEN CDB hosted its twelfth annual symposium on March
10 to 12 in the CDB auditorium. This year’s symposium wasthemed, “Regeneration of Organs: Programming and Selforganization,” and drew an audience of 180 scientists andstudents from 15 different countries to take part in a program oftalks and discussions exploring inroads into our understandingof the phenomena of regeneration and organogenetic
processes.
The three-day program featured 30 invited and selected talks
and over 60 poster presentations that covered a range of
topics related to regeneration including molecular mechanisms
underlying regeneration, self-organization in regeneration, selforganization from a mathematical perspective, and mechanisms
determining shape and size of organs and organisms. The
co-organizers this year were Yoshihiro Morishita and Hitoshi
Niwa from the CDB, Kiyokazu Agata of Kyoto University (Japan),and Elly Tanaka of Center for Regenerative Therapies Dresden(Germany).
The CDB symposium series was launched in 2003 as a forum
to encourage the timely exchange and discussion of recent
findings, progress and trends in developmental biology,
regeneration and other related fields.

Session 1

Yoshihiko Umesono (The University of Tokushima, Japan)
Elly M. Tanaka(Center for Regenerative Therapies Dresden, Germany)

Akira Satoh (Okayama University, Japan)
Kenneth Poss (Duke University Medical Center, USA)
Ashley W. Seifert (University of Kentucky, USA)
Feng Chen (Stanford University, USA)
Session 2

Andras Simon (Karolinska Institute, Sweden)
Daniel Goldman (University of Michigan, USA)
Koji Tamura (Tohoku University, Japan)
Alan Rodrigues (Harvard Medical School, USA)
Takashi Takeuchi (Tottori University, Japan)
Ryo Nakamura (The University of Tokyo, Japan)
Session 3

Hans Meinhardt(Max Planck Institute for Developmental Biology, Germany)

Naama Barkai (Weizmann Institute of Science, Israel)
Tatsuo Shibata (RIKEN CDB, Japan)
Yoshihiro Morishita (RIKEN CDB, Japan)
Taiji Adachi (Kyoto University, Japan)
Yanlan Mao(MRC Laboratory for Molecular Cell Biology, University College
London, UK)82

2015 CDB SymposiumTime in Development
March 23–25, 2015
The thirteenth annual symposium “Time in Development” will
be held on March 23–25, 2015 in the CDB Auditorium. Thissymposium will focus on ‘time’. There are various time scales
in developmental events, which are mutually related and tightly
coordinated; the timing of cells to divide and differentiate,
time as developmental stages and specific events (suchas metamorphosis), time as the lifespan of a species, timealong the course of evolution, or even the heterochronicallyshifted time that we perceive through comparison of thedevelopmental time table of different animals. We aim to
discuss and understand the various contexts of 'developmental
time,' while reflecting on the concept of time used in our own
research. Intrinsic genetic programs coordinate developmental
time through regulatory networks of transcription factors.
More complex timing programs involve systemic signalingthat coordinates the developmental processes among tissues
within an organism. The duration of developmental intervals
can be regulated in response to nutritional or environmental
cues. Developmental timing mechanisms ensure correct tissueorganization, patterning, and size in a variety of organisms.
How time is controlled during development, or alternatively,
how development employs temporal cues (clocks/oscillators)
to generate cell diversity and coordinated patterns are
therefore central themes in development.

Invited Speakers

Victor R. Ambros(University of Massachusetts Medical School, USA)
Session 4

Pierre Léopold (Institut de Biologie Valrose (iBV), France)
Richard Adams (University of Cambridge, UK)
Tsuyoshi Hirashima (Kyoto University, Japan)
Kenneth D. Irvine (Rutgers University and HHMI, USA)
Ginés Morata (Universidad Autónoma de Madrid, Spain)
Shinichi Hayashi (Tohoku University, Japan)
Session 5

Manuel Serrano(Spanish National Cancer Research Center (CNIO), Spain)

Kaoru Sugimoto (California Institute of Technology, USA)
Takashi Tsuji (Tokyo University of Science, Japan)
Karl R. Koehler (Indiana University School of Medicine, USA)
Atsuhiro Taguchi (Kumamoto University, Japan)
Yoshiki Sasai (RIKEN CDB, Japan)

Adam Antebi(Max Planck Institute for Biology of Ageing, Germany)

Constance Cepko (Harvard Medical School and HHMI, USA)
Claude Desplan (New York University, USA)
Yukiko Gotoh (The University of Tokyo, Japan)
Carina Hanashima (RIKEN CDB, Japan)
Naoki Irie (The University of Tokyo, Japan)
Ryoichiro Kageyama (Kyoto University, Japan)
Takao Kondo (Nagoya University, Japan)
Fred Nijhout (Duke University, USA)
Takashi Nishimura (RIKEN CDB, Japan)
Andrew Oates (MRC National Institute for Medical Research, UK)
Michael B. O’Connor (University of Minnesota, USA)
Patrick H. O’Farrell(University of California, San Francisco, USA)

Linda Partridge(Max Planck Institute for Biology of Ageing, Germany)

Olivier Pourquié(Institute of Genetics and Molecular and Cellular Biology (IGBMC),
France)Yun-Bo Shi (National Institutes of Health, USA)
Ralf J. Sommer(Max Planck Institute for Developmental Biology, Tübingen, Germany)

Claudio Stern (University College London, UK)
Stefan Thor (Linköping University, Sweden)
Pavel Tomancak(Max Planck Institute of Molecular Cell Biology and Genetics,
Germany)

83

Seminars
The RIKEN CDB makes special efforts to provide a full and diverse series of invited seminars by scientists from around theworld. To date, the Center has hosted more than 720 such talks, in addition to numerous meetings, internal forums, andcolloquia. The following speakers presented seminars in the period from January to December 2014.
Date

84

Title

Speaker

01-29

New therapies for retinal degenerative disorders

02-04

Regional allocation and origin of stem cells in the postnatal brain

02-06

Artificial induction and disease-related conversion of the hepatic fate

02-17

The cell biology and biophysics of Ciona notochord morphogenesis, from anactomyosin perspective

Di JIANG

03-05

Planar Cell Polarity and septins compartmentalize cortical actomyosin to directcollective cell movement

Asako SHINDO

03-13

The two faces of Hippo : Mammalian Hippo signaling in tissue homeostasis
and disease

03-13

The Hippo pathway in organ growth control

04-11

Patterning mechanisms responsible for generating neurovascular network
during organogenesis

04-17

Investigation of beta-Spectrin function in the Hippo signaling pathway

Yan YAN

05-22

Anisotropic stress orients remodelling of mammalian limb bud ectoderm

Hirotaka TAO

06-10

Coping with stress: Dynamic structural changes in αα-catenin regulate thecadherin-actin linkage

Noboru ISHIYAMA

07-22

Coordinated actomyosin kinetics in generating self-organized patternformation in the cell cortex

07-23

How to visualize published ChIP-seq raw data

Shinya OKI

07-24

Genetically speaking, BIG≠FAT

Akihiro MORI

09-08

Defining the strategies of stem cell self-renewal

Akiko MAEDA

Arturo ALVAREZ-BUYLLA

Atsushi SUZUKI

Randy L. JOHNSON

Georg HALDER

Yoh-suke MUKOUYAMA

Masatoshi NISHIKAWA

Benjamin SIMONS

Date

Title

Speaker

09-09

Cyclic conversion of 5-methylcytosine to 5-hydroxymethylcytosine during thecell cycle in mouse embryonic stem cells

Masako TADA

09-16

A quantum jump in acorn worm diversity: deep-sea technology meetsmolecular phylogenetics

09-24

The Drosophila antimicrobial response at the time of the Cas9/CRISPR genetargeting revolution

Bruno LEMAITRE

10-30

Roles of Myosin II and Myosin III in regulation of hair cell apical morphology

Seham EBRAHIM

10-31

Know thy neighbor: The role of contact area geometry on cell-cell signaling

David SPRINZAK

10-31

Shaping the mammalian inner ear sensory organs by the vertebrate planar cell
polarity pathway

11-12

The genomics of deafness: gene discovery and regulation

11-13

Wounds: to heal or not to heal, that is the question

11-20

Epithelial spreading in zebrafish gastrulation

12-02

Unique mechanisms of successful fertilization in birds

Tomohiro SASANAMI

12-04

Spatial-temporal-spatial transformation in visual map plasticity: A novel rule in
input dependent organization of circuit

Masateru HIRAMOTO

12-08

An evolving retinoic acid signaling pathway active during evolution

Vincent LAUDET

12-11

How birds create diverse feather colors — Avian melanocyte stem cells andregulation of feather pigment patterns

Sung-Jan LIN

12-17

Mammalian odorant receptors: deorphanization, accessory proteins andreceptor gene choice

Nicholas D. HOLLAND

Ping CHEN

Karen B. AVRAHAM

Sa Kan YOO

Carl-Philipp HEISENBERG

Hiroaki MATSUNAMI

85

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

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RIKEN Research
RIKEN publishes the monthly print and online newsletter RIKENRESEARCH to draw the world’s attention to some of the institute’s
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The core component of RIKEN RESEARCH is short, easy-to-understand‘Research Highlight’ articles explaining for a broad scientific audience asampling of the latest research articles published by RIKEN scientists.
http://www.riken.jp/en/research/rikenresearch/8687