RIKEN Center for Developmental Biology

2015 Annual Report

2015
Annual Report
RIKEN
Center for
Developmental Biology

Contents
2

Message from the Director

4

Organizational Chart

62015 at the CDB

Research Highlights
10

Oocyte maturation directed by PLK1Lab. for Chromosome Segregation

12

Generating 3D cerebellar structure andfunctional cerebellar neurons
Lab. for Organogenesis and Neurogenesis

14

Induction of retinal tissue with ciliary margin
from hESCs
Lab. for Organogenesis and Neurogenesis; In Vitro
Histogenesis

16

Chick embryo reveals conserved developmental
mechanisms among vertebrates
Lab. for Early Embryogenesis

18

Improved donor organ preservation method fortransplantation
Lab. for Organ Regeneration

20

Surprising instability of meiotic cell division
Lab. for Chromosome Segregation

22

Self-organized actin pattern in the tracheal tube
Lab. for Morphogenetic Signaling

24

Anchoring the signaling complex needed for cell
elongation
Lab. for Morphogenetic Signaling

26

Leading cause of age-related aneuploidy inoocytes revealed
Lab. for Chromosome Segregation

28

Just being naive? Early pluripotency in a birdmodel of development
Lab. for Sensory Development; Early Embryogenesis

30

Nutrient-dependent regulation of growth control
hormone revealed
Lab. Growth Control Signaling

The RIKEN Center for Developmental Biology was launchedin April 2000 under the auspices of the Millennium Project
research initiative, which was established to drive researchin areas of vital importance to both Japan and the world in
the 21st century. The challenges of an aging society wereamong the issues addressed, and developmental biology
was included within this remit as a scientific field with thepotential to contribute to the development of regenerative
medicine.
Located in Kobe, Japan, the Center’s research programs
are dedicated to developing a better understanding offundamental processes of animal development at the32

Hippocampal neurons from hES cells
Lab. for Organogenesis and Neurogenesis; In Vitro
Histogenesis

34

Putting a new spin on autonomously organizedepithelial movement
Lab. for Histogenetic Dynamics

36

Pulmonary neuroendocrine cell clusters at
airway branches
Lab. for Lung Development

38

Doubling teeth from single tooth germ
Lab. for Organ Regeneration

40

Transplantation of hESC-derived retinal tissue inprimate models of retinitis pigmentosa
Lab. for Retinal Regeneration

Laboratories

54

Sensory Development
Raj LADHER

55

Cell Asymmetry
Fumio MATSUZAKI

56

Lung Development
Mitsuru MORIMOTO

57

Growth Control Signaling
Takashi NISHIMURA

58

Pluripotent Stem Cell Studies
Hitoshi NIWA

59

Early Embryogenesis
Guojun SHENG

60

Retinal Regeneration
Masayo TAKAHASHI

61

Cell Adhesion and Tissue Patterning
Masatoshi TAKEICHI

62

44

In Vitro Histogenesis
Mototsugu EIRAKU

Organogenesis and Neurogenesis
Masatoshi TAKEICHI

63

45

Tissue Microenvironment
Hironobu FUJIWARA

Organ Regeneration
Takashi TSUJI

64

46

Organismal Patterning
Hiroshi HAMADA

Epithelial Morphogenesis
Yu-Chiun WANG

47

Neocortical Development
Carina HANASHIMA
Morphogenetic Signaling
Shigeo HAYASHI

65

2015 Events

66

2015 Courses

672015 Awards

70

RIKEN in Kobe

72

CDB Symposium

48
49

Developmental Epigenetics
Ichiro HIRATANI

50

Sensory Circuit Formation
Takeshi IMAI

51

Axial Pattern Dynamics
Hidehiko INOMATA

74

Seminars

76

About RIKEN

52

Chromosome Segregation
Tomoya KITAJIMA77

RIKEN Campuses

53

Histogenetic Dynamics
Erina KURANAGA

molecular and cell biological level, the more complex
phenomena involved in organogenesis, as well as the
biology of stem cells and regeneration. Scientists at the
CDB conduct world-class studies in a truly international
research environment, with ample core facilities andtechnical support and a critical mass of scientific talent andexpertise. The CDB also benefits from belonging to Japan’slargest basic scientific research organization, RIKEN, aswell as its close ties to graduate and medical schools andresearch institutes in Japan and other countries, giving ourscientists access to a broad network of talent and technology across the country and around the world.

Message from the Center Director

It was a great sense of pride and responsibility that I joined the RIKEN Center for Developmental Biology (CDB) as Director in April of
this year. My predecessor, Dr. Masatoshi Takeichi has set an incredibly high bar of achievement through his great leadership and hislong career of scientific achievements, and the success of the Center over the first 15 years of its history is a testament to his vision.
My task now is to guide the CDB into a new stage of its own development, one in which we continue to bring to light the mechanisms
by which the body is built, and find new ways of harnessing that knowledge in rebuilding bodies that have been damaged by illness,injury, or age.
Following a reorganization of the research system last year, the CDB of 2015 is a slimmer organization, with multidisciplinary programs
focused on cellular environment and response, organogenesis, stem cells and organ regeneration, and developmental biology andmathematical science, as well as its ongoing commitment to a world-renowned project in retinal regeneration. The CDB is also committed to working even more closely with other RIKEN Centers, including the Center for Life Science Technologies (CLST) and Quantitative Biology Center (QBiC), as well as the RIKEN-wide Chief Scientists program.
Our tighter research focus, however, has not slowed the pace of scientific achievement. This past year CDB labs have continued to
publish outstanding work in high-impact journals, ranging from fundamental insights in cell adhesion, evolutionary developmental biology, and morphogenesis, to more application-oriented studies in stem cell differentiation and transplantation methods. The work ofour research scientists continues to be recognized by awards from government agencies and private foundations, but more importantly through collaboration and interaction with their international peers.
This international focus continues to be a key feature of scientific life at the CDB, as exemplified by our annual symposium, whichconvened in March under the thought-provoking theme of “Time in Development.” As in previous years, this event drew a capacity
audience of 140+ speakers and attendees from around the world and across the spectrum of related fields. We look forward to welcoming another great assembly of leading scientists and thinkers to the 2016 Symposium, “Size in Development: Growth, Shape andAllometry,” on March 28–30.
While maintaining our focus on conducting world-class science, the CDB will also redouble its efforts to contribute to benefiting society through translational efforts as well. Given Japan’s national emphasis on developing new medical technologies, including regenerative medicine applications, to address the challenges of an aging society, the Center is well positioned to work with national, regional and local government and private sector organizations to make this dream a reality. The city of Kobe continues to promote theincredible growth and ongoing success of its biomedical research park here on Port Island, which is now home to over 300 medical
centers, research institutions, and biotechnology industry sites. Western Japan also boasts some of the world’s leading institutes inthe study of stem cell technology, including the Center for iPS cell Research and Application (CiRA) at Kyoto University, with which the
CDB continues to work closely in exploring the therapeutic potential of pluripotent stem cell-based approaches.
In closing, it is a new and exciting era for the CDB and for the study of development and regeneration, two interlinked processes thathave been at the core of our scientific focus since the Center was first established at turn of the millennium. The potential of thesefields remains enormous, but its realization will require the dedicated support and hard work of the global life science and biomedical
research community, and it is to that community that I pledge we will work even harder to contribute new innovations and insights on
the long road to discovery.

Hiroshi Hamada
Director, RIKEN Center for Developmental Biology

3

RIKEN Kobe Branch

CDB Organization 2015

Center for
Developmental Biology
The Center for Developmental Biology (CDB) is a
research center within the RIKEN Kobe Branch, which
also includes the Center for Life Science Technologies(CLST), the Quantitative Biology Center (QBiC), the HPCI
Program for Computational Life Sciences, the Kobe Administrative Division, which provides administrative services, and the institutional Safety Center. The CDB
underwent an organizational restructuring in 2014, resulting in major changes to laboratory designations anddepartmental affiliations. The majority of laboratories are
now designated as teams, and assigned to one of five

Deputy Director

Cellular Environment and Response
Research ProgramThis program includes labs focused on developing
platforms for gaining a better understanding of fundamental control principles of animal development, aimed
at learning new ways of overcoming developmental
disorders.

Morphogenetic Signaling
Shigeo HAYASHI Ph.D.

Developmental Epigenetics
Ichiro HIRATANI Ph.D.

programs, which are described in detail to the right. The

Chromosome Segregation

CDB Director is assisted by a Deputy Director, andTomoya KITAJIMA Ph.D.

advised by the Advisory Council, while governanceissues are discussed by the Management Committee.

Center for Life Science Technologies
Quantitative Biology Center
HPCI Program for Computational Life Sciences
Developmental Biology Planning Office

Growth Control Signaling
Takashi NISHIMURA Ph.D.

Organogenesis Research Program

Labs in this program study mechanisms underlying theformation of complex model systems, in the hopes ofgaining insight into the bases of the higher order structure of organs and functional development.

Neocortical Development
Carina HANASHIMA Ph.D.

Life Science Technologies Planning OfficeSensory Circuit Formation
Takeshi IMAI Ph.D.

Quantitative Biology Planning Office

Cell Asymmetry

Kobe Administrative Division

Cell Adhesion and Tissue Patterning

Kobe Safety Center

Epithelial Morphogenesis

Fumio MATSUZAKI Ph.D.
Masatoshi TAKEICHI Ph.D.
Yu-Chiun WANG Ph.D.

Advisory Council

Austin Smith

Haifan Lin

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

Christopher Wylie

Toshio Suda

Margaret Buckingham

Ryoichiro Kageyama

Patrick Tam

Hiroshi Hamada

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

University of Sydney, Australia

The nine-member Council comprises top international scientists
working in developmental biology, stem cells, and related fields.

4

Stephen Cohen

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

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

Osaka University, Japan(Until March 31, 2015)

Center Director Hiroshi Hamada
Office of the Director

Research Ethics Education Officer

Stem Cells and Organ Regeneration
Research Program

Research and Development Project

Labs in this program work on identifying methods forcontrolling organ formation and regeneration through thestudy of stem cells and organogenesis.

In Vitro Histogenesis
Mototsugu EIRAKU Ph.D.

Tissue Microenvironment
Hironobu FUJIWARA Ph.D.

Lung Development

This program conducts research using induced
pluripotent (iPS) and other types of stem cells to develop
approaches to regenerate retinal function, in active
collaboration with the Kyoto University Center for
Induced Pluripotent Stem Cell Research and Innovation(CiRA) and various Kobe-based research initiatives.

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

Mitsuru MORIMOTO Ph.D.

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

Organogenesis and Neurogenesis
Masatoshi TAKEICHI Ph.D.

Organ Regeneration
Takashi TSUJI Ph.D.

Developmental Biology andMathematical Science ProgramThis program aims to develop a systematic understanding of the complex cellular interactions underlying tissuemorphogenesis through quantitative mathematical
approaches and the development of computationalmodels of gene expression, signal modulation, anddynamic processes.

Senior Investigator Program

Sensory Development
Raj LADHER Ph.D.

Early Embryogenesis
Guojun SHENG Ph.D.

Axial Pattern Dynamics
Hidehiko INOMATA Ph.D.

Organismal Patterning
Hiroshi HAMADA M.D. Ph.D.

Histogenetic Dynamics
Erina KURANAGA Ph.D.

Management Committee

Hiroshi Hamada

Tomoya Kitajima

The Management Committee was established in November 2014
following the restructuring of the CDB. The Committee comprises
the CDB Director, Deputy Director and several laboratory heads inaddition to scientists outside the CDB, and convenes monthly to
discuss and make administrative decisions on key issues such as
recruitment of new laboratory heads and personnel, and budgetary allocations.

Toshihiko Oguru

Erina Kuranaga

Osamu Kamigaito

Tatsuo Shibata

Makoto Taiji

Masayo Takahashi

Hisato Kondo

Takashi Tsuji

Takeshi Imai

Katsutoshi Nukui

Director, CDB

Director, RIKEN Kobe Branch; Deputy Director, CDB
RIKEN Nishina Center for Accelerator Based Science
RIKEN Quantitative Biology Center
Kyoto Sangyo University
Lab. Sensory Circuit Formation, CDB

Lab. Chromosome Segregation, CDB
Lab. Histogenetic Dynamics, CDB
Coordinator, CDB

Lab. Retinal Regeneration, CDB
Lab. Organ Regeneration, CDB
Developmental Biology Planning Office5

2015 Highlights
The 13th CDB Symposium:Time in Development
The CDB held its 13th annual symposium on March 23–25 on thetheme, “Time in Development.” Close to 150 researchers fromaround the globe convened at the Center to share their work andengage in discussions on the different contexts of time in relation todevelopment.

New center director takes office
On April 1, the CDB welcomed Hiroshi Hamada, a professor in the Graduate School of
Frontier Biosciences, Osaka University and respected scientist known for his research on
the establishment of body axes in mammals, as the new center director.
On his appointment, he stated, “On the management side, I hope that we can work toincrease the transparency of the CDB. As for research, I would like to ensure that we takeheed of the balance between basic and applied research and also create an environmentthat fosters, especially for younger scientists, the pursuit of challenging research aimed
at discovering the true essence of biological phenomena.”
He established his own lab, the Laboratory for Organismal Patterning, at the CDB in October.

CDB scientists awarded
MEXT prizes
In a ceremony in April, Project Leader Masayo Takahashi and her researchteam, and Team Leader Takeshi Imai were recognized for their scientificachievements by Japan’s Minister of Education, Culture, Sports, Science,
and Technology (MEXT), receiving the Commendation for Science andTechnology. Takahashi, along with lab members Michiko Mandai, Chikako
Morinaga, Noriko Sakai and Chikako Yamada, received the Prize for Science and Technology for their work on the clinical development of iPSCderived RPE for transplantation. Imai was awarded the Young Scientist’s
Prize for his work on neural circuit formation using the olfactory system as amodel.

6

Exploring the interface between
developmental biology
and mechanics
The 26th CDB Meeting, “Mechanical Perspectives of Multicellular Organization,” was held September 8–9. The multidisciplinary meeting
attracted over 130 scientists with varied backgrounds including biology, medicine, physics, and engineering to share and engage in discussions on how biological fields can incorporate perspectives andtechnologies from physics and mechanics to understand biologicalprocesses.

Masayo Takahashi awarded
Ogawa-Yamanaka Stem Cell Prize
The Gladstone Institutes named Masayo Takahashi, project leader ofthe Laboratory for Retinal Regeneration, as the inaugural winner of the
Ogawa-Yamanaka Stem Cell Prize for her “trailblazing work leading thefirst clinical trial to use induced pluripotent stem cells (iPSCs) in humans.”

Update on the first transplantation of
iPSC-derived RPE cell sheet
RIKEN, the FBRI, and the Kobe Medical Center General Hospital held ajoint press conference in October to report on the progress of the recipient of the first iPSC-derived RPE cell transplantation in the pilot safety study of iPSC-based intervention for wet-type age-related maculardegeneration. The research team reported that the results of periodic
examinations during the monitoring period appear to be good, and thatno signs of tumorigenesis, abnormalities, and recurring neovascularization have been detected. The patient will continue to be monitored forthe next three years.

7

Research Highlights

Pituitary tissue generated from human ES cells.
Green, growth hormone; red, PITX1; blue, nucleus.
Image: Laboratory for Organ Regeneration

Oocyte maturation directed by PLK1

Tomoya KITAJIMA

C

hromosome segregation is one of the most important events during cell division, both in somatic cells (mitosis) and in germ cells (meiosis).
Improper segregation of chromosomes leads to a range
of defects in the daughter cells, and nowhere is correct
chromosome segregation more crucial than in the division of germ cells as they pass on genetic information to
the next generation of offspring. But surprisingly, errors
in chromosome segregation are known to occur withhigh frequencies in mammalian oocytes. Meiosis in oocytes is also unique, as the cell division cycle is arrested
for an extended period in prophase I before resuming
meiotic division. One factor that plays a major role in cell
division is Polo-like kinase 1 (PLK1), a serine/threonine
kinase, which has been implicated in multiple events during mitosis. The functions of PLK1 in oocytes undergoing
maturation (meiosis), however, are not well understood.
Now, a new joint study by team leader Tomoya Kitajima
and colleagues in the Laborator y for Chromosome
Segregation and collaborators at the Institute of Animal
Physiology and Genetics (Czech Republic) and European
Molecular Biology Laboratory (EMBL; Germany) looks at
the role of PLK1 during meiosis, using mouse oocytes asa model. Their findings, published in PLoS ONE, demonstrate that PLK1 controls meiotic cell division in a manner

10

similar to mitosis, such as promoting microtubule elongation and stabilizing microtubule-kinetochore binding.
They also find that PLK1 has functions specific to meiosis such as activating the anaphase promoting complex/
cyclosome (APC/C) for entry into anaphase I, for proper
chromosome segregation and maintaining chromosome
condensation during meiosis I-meiosis II transition.
Kitajima and colleagues first examined the normal localization patterns of PLK1 during meiosis of oocytes using
immunostaining and 4D live imaging techniques. PLK1
was initially found dispersed in the cytoplasm during prophase I, and was activated and localized to microtubuleorganizing centers (MTOCs) before nuclear envelope
breakdown (NEBD). When oocytes were treated with an
inhibitor to specifically block PLK1 function, there was amarked delay in the timing of NEBD and of chromosome
condensation, two events that signal the resumption ofmeiosis. The resumption of meiosis requires anotherkinase, CDK1, which is known to be activated by PLK1.
Analyses by the researchers indicated that in addition toactivating CDK1, PLK1 also regulates NEBD through analternative route. PLK1 was found to contribute to MTOC
formation, and to be required for proper meiotic spindle
assembly after NEBD as well.

Solc P, et al. Multiple Requirements of PLK1 during Mouse Oocyte
Maturation. PLoS One 10.e0116783 (2015)

Inhibiting PLK1 function also caused chromosomal misalignment in during metaphase I. When they investigatedthe cause of this misalignment, the researchers discovered a reduction in phosphorylation levels of BUBR1,
which is essential for kinetochore function, and an increase in the number of microtubules that were not attached to kinetochores (a region of the chromosome thatbinds to microtubules). As the PLK1 was seen to localize
to the kinetochores after NEBD under normal conditions,
their findings suggest that PLK1 stabilizes the binding
between kinetochores and microtubules.
Normally, when problems occur in chromosomal alignment, the spindle assembly checkpoint (SAC), a mechanism preventing the onset of anaphase I, is activatedthereby preventing oocytes from undergoing transition
from metaphase to anaphase. In PLK1-inhibited oocytes,
meiosis was arrested at metaphase. Upon closer examination, PLK1 was found to be essential for the degradation of EMI1, a molecule that prevents the APC/C frombeing activated to push oocytes to enter anaphase. In
contrast to what is seen in somatic cell division, inhibiting SAC alone in oocytes does not resume cell division,
thus APC/C activation via PLK1 is necessary for progression to anaphase. In addition, their experiments showthat even after APC/C is activated and oocytes enter
anaphase PLK1 contributes to chromosome segregation,
maintaining chromosome condensation, and also cytokinesis.
“The present study shows that PLK1 is an important factor that directs multiple events during oocyte maturation,
similar to PLK1 functions in mitosis; but there are alsoslight differences in its roles and contributions,” says
Kitajima. “Our joint research recently uncovered a binding partner of PLK1 called, Meikin, found in the kinetochores of oocytes which regulates meiosis-specific kinetochore function. Thus, the functions and contributionsof PLK1 during meiosis appear to be regulated by factors
uniquely assembled by the oocyte.”

Timelapse images of oocyte undergoing first meiotic division. PLK1 (green) localizes to MTOCs and kinetochores (red). By anaphase I (far right panel), PLK1
accumulates at the spindle midzone.

11

Generating 3D cerebellar structure andfunctional cerebellar neurons

Keiko MUGURUMAT

he brain’s cerebellar cortex, which is associatedwith motor control, consists of multiple cell types
arranged in a layered structure; from top to bottom, the molecular layer, the Purkinje cell layer and the
granule cell layer. Purkinje cells, found in the thin Purkinje
cell layer, have large cell bodies with highly arborized
dendrites fanning out toward the molecular layer above
and a long axon extending down to the deep cerebellar
nuclei; they play a central role in processing information
received from outside the cerebellum. Granule cells in
the granule cell layer are much smaller and transmit information received from around the body to the Purkinjecells. These two cell types arise from two distinct regionsof the developing cerebellum and later mesh together toform the layered structure of the cerebellum. While successful induction of certain cerebellar neurons in vitro
have been reported, recapitulating the developmental
processes to generate three-dimensional (3D) cerebellar
structure has proven to be a challenge.
New work led by research specialist Keiko Mugurumaof the Laboratory for Organogenesis and Neurogenesis(Masatoshi Takeichi, Team Leader), former group director
Yoshiki Sasai (dec.), and other colleagues details a method to generate functional cerebellar neurons from human

12

embryonic stem cells (hESCs) in vitro. They also showthat these induced neurons can self-organize to forma 3D structure resembling the human cerebellar cortex
seen in the early stages of development. Published inCell Reports, their work demonstrates how the additionof essential growth factors at key time points can produce cerebellum-like structure in a culture system.
In a previous study led by Muguruma, the group successfully generated cerebellar neurons from mouse
ESCs (mESCs) at relatively high efficiencies. There, using the SFEBq (serum-free culture of embryoid body-likeaggregate) method developed by the lab, they induced
the formation of Purkinje neurons by adding FGF2 andinsulin to the medium. Further addition of BMP4 to thesystem produced granule cells; however, they were notable to generate Purkinje neurons and granule cells simultaneously.
In the current study, they initially attempted to induce
cerebellar neurons from hESCs with the mESC method
but met with difficulties; whether or not the ROCK inhibitor which promotes cell survival and reaggregation of
hESCs was added to the 3D culture system, the resulting
floating aggregates were extremely fragile and difficult

Muguruma K, et al. Self-Organization of Polarized Cerebellar Tissue
in 3D Culture of Human Pluripotent Stem Cells. Cell Rep 10(4).537550 (2015)

to culture long-term. This problem was solved by adding
TGF-β-receptor blocker, a known neuroectoderm differentiation promoter. As with mESCs, Muguruma et al.
then confirmed that adding FGF2 leads to the inductionof isthmus organizer-like tissue in the hESC aggregateswithin 14 days in culture, and that by day 35, these aggregates also formed hollowed rosette structures within,which expressed markers in a pattern typically seen in
cerebellar plate neural epithelium (CPNE) that later gives
rise to cerebellum-specific neurons.
They next examined whether these CPNE cells havethe potential to generate mature cerebellar neurons. As
synaptogenesis between granule cells and Purkinje cells
promotes Purkinje cell maturation, FGF2-treated aggregates were dissociated and cocultured with granulecells isolated from mouse tissues. Cells expressing an
early differentiation marker for Purkinje cell progenitors
differentiated into mature Purkinje cells, identified bytheir characteristic morphology and expression of late
Purkinje-cell markers. Electrophysiological experimentson these hESC-derived Purkinje cells also demonstrated
they function similar to naturally born Purkinje cells.
A closer look at the hollowed rosettes formed in the aggregates at day 35 revealed that while many were small
and round, a few were relatively large and elliptical inshape. The elliptical rosettes expressed dorsal-specificmarkers on the outside and ventral-specific markers on
the inside, resembling the dorsal-ventral axis seen in

the neural tube. The team tested a range of conditions
and found that adding FGF19 from day 14 promoted theformation of these elliptical structures. The further addition of SDF1, a factor linked to cerebellar development,
at day 28 led to the formation of a large continuous neuroepithelial structure resembling the CPNE, rather than
the formation of multiple elliptical structures inside theaggregate. SDF1 also promoted differentiation of thecontinuous neuroepithelium to generate a three-layered
structure—the ventricular zone, Purkinje cell precursor
zone and rhombic lip-derived zone—similar to normal
cerebellar development. The periphery of one end of thehESC-derived neuroepithelium was also curled, and exhibited gene expression patterns seen in the rhombic lip
which normally generates the granule cells in vivo.
Muguruma and her colleagues are also attempting toestablish cerebellar disease models from patient-derived
hiPS cells by adapting their current protocol for human
iPS cells. These attempts to generate cerebellar tissue invitro could lead to understanding disease pathology or
to applications in drug screening.
“Cerebellar development involves very dynamic changes
in tissue morphology. So far we have only been able
to generate embryonic cerebellum, roughly equivalent
to the first trimester,” says Muguruma. “But, if we canproduce a more mature cerebellum tissue in vitro, it may
reveal the mechanisms underlying the dramatic morphological changes taking place during development.”

Left: hESC-derived Purkinje cell. Middle: Elliptical structures formed inside hESC aggregate, with D-V axis resembling the neural tube. Green, dorsal marker; magenta, ventral marker. Right: hESC-derived cerebellar cortex-like structure. Curled rhombic lip-like structure (blue arrow) generates granule cells,
which migrate (pink arrow) to align with Purkinje cells (green).

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Induction of retinal tissue withciliary margin from hESCs

Chikafumi OZONE, Atsushi KUWAHARA, Tokushige NAKANO

D

uring retinal development, a section of the neuroepithelium balloons outwards to form a saclike optic vesicle, which then folds inwards to
produce a two-layered optic cup. Cells in the outer layerof the optic cup differentiate into retinal pigment epithelium (RPE), while those in the inner layer differentiate into
neural retina which includes the light-sensing photoreceptor cells. At the peripheral margin of the embryonicretina lies the ciliary margin (CM), the junctional region
where the neural retina and the RPE converge. In birds,
fishes, and some reptiles, the CM is known to maintain astem cell population that contributes to retinal development and regeneration. Whether or not a similar stem
cell population exists in the CM of the mammalian eye isunclear, and in humans, where access to the embryonicretina is difficult, even less is known about this region.
A new study carried out by Atsushi Kuwahara in the
L aborator y for Organogenesis and Neurogenesis(Masatoshi Takeichi, Team Leader; laboratory closed
March 2015), Mototsugu Eiraku, team leader of the Laboratory for In Vitro Histogenesis, and others shows evidence that strongly suggests the CM of the embryonichuman retina also fosters a stem cell population whichplays a key role in the growth of the retina. The team de-14veloped a protocol allowing them to drive human embryonic stem cells (hESCs) to spontaneously self-organize
to form a three-dimensional (3D) retinal structure that
includes CM-like regions at high efficiencies. They thenfound that the CM-like region is a niche for retinal stem
cells and serves as a source of progenitors to produce
new neural retinal cells. This work, published in Nature
Communications, was a joint project with the Environmental Health Science Laboratory, Sumitomo Chemical
Company Ltd.
The Laboratory for Organogenesis and Neurogenesis,
under the direction of former group director Yoshiki
Sasai, previously developed the SFEBq method, a 3D
cell culturing method that can efficiently induce differentiation of pluripotent stem cells to form a neuroepithelial
sheet through the addition of a cocktail of signaling factors. The signaling factors stimulate cells to spontaneous
self-organize, further differentiate and produce 3D neuraltissues, partially recapitulating in vivo early embryonicdevelopment processes. To date, they have generated
tissues resembling cerebrum, hypothalamus, pituitary
gland, and cerebellum. They also successfully generated
3D stratified retinal tissue from both mouse and human
ESCs, which included a region at the neural retina–RPE

Kuwahara A, et al. Generation of a ciliary margin-like stem cell nichefrom self-organizing human retinal tissue. Nat Commun 6.6286 (2015)

boundary containing cells distinct from both cell types.
In the current study, the team tried to determine whether
CM could indeed be generated in hESC-derived retinal
tissue, and to uncover the role of the CM.
Kuwahara et al. first focused on improving the induction
efficiency of the initial differentiation step from ESCs toretinal tissues. They found that the transient addition of
BMP4 to the culture system when differentiation begins
could drive ESCs to differentiate into retinal structuresat markedly higher efficiencies and with greater stability,
naming this improved differentiation method, the BMP
method. This method also eliminated the need to add
extracellular matrix proteins to the culture media, the addition of which poses difficulties when considering large
scale production and future clinical applications. Analyses of retinal tissues generated with this method showedthat most had gene expression profiles characteristic ofneural retina, and not RPE.
They next speculated that if both neural retina and RPE
could be produced inside the cultured cell aggregates,
it might be possible to generate a CM, which in normal
development, forms at the junction between neural retina
and RPE. Past reports have indicated the importanceof Wnt signals for RPE differentiation as well as maintenance, and FGF signals for neural retina differentiationand maintenance. Focusing on these two signaling pathways, the team attempted to create a two-domain retinal
tissue containing both neural retina and RPE. They succeeded in establishing a protocol which could generate
the said retinal tissue with high efficiency and reproducibility; BMP-method derived retinal tissue is treated forsix days with Wnt agonists and FGF inhibitors to tip the
differentiation bias toward RPE cell fate, and then placed
again under conditions favorable for inducing neuralretina. Interestingly, analyses of the derived tissue indicated that it was initially biased toward a neural retina
fate, but then became transiently biased to an RPE fateafter treatment with Wnt agonists and FGF inhibitors
before resuming a neural retina fate. They called this the‘induction-reversal’ method because of the swinging ofcell fates between neural retina and RPE in the aggregates.
When retinal tissue generated via induction-reversal
method was cultured over a two-month period, both theneural retina and RPE continued to grow, with the tissue
eventually taking on a turnip-shaped form. The junctional
region between the neural retina and the RPE of thederived retinal structure was distinctly thin and tapered,and expressed genes specific to the CM. Following threemonths of culture, the neural retina comprised numerous
photoreceptor progenitors, and by five months, differentiated photoreceptor cells were present. The tapered
CM-like region was also found to contain a large numberof stem cells with high sphere-forming abilities, serving
as a source of progenitors for the growing retinal tissues.
“Our results are consistent with the current view that
RPE and neural retina are capable of fate transition. We
hope to further unveil the mechanisms involved humanretinal development through examining retinal formationin culture,” says Eiraku.
Lead author Kuwahara adds, “The protocol developed
here allows us to generate retinal tissue that more closely resembles the biological retina at higher efficiencies
and with greater stability. It is a step closer to realizing
regenerative medicine for retinal disorders, and we hope
to continue advancing our research in this regard.”

Left: Turnip-shaped hESC-derived retinal tissue using induction-reversal method at differentiation day 60. (Green, neuralretina; Dark region, RPE) Middle and right: Formation of CM-like zone (white brackets) at border region between neuralretina and RPE via spontaneous self-organization (differentiation day 63).

15

Chick embryo reveals conserved developmental
mechanisms among vertebratesT

he chick has long been a cherished model forstudying development, owing to the ease withwhich the embryo can be accessed and manipulated inside the eggshell. However, much of its first day
of development following fertilization, which occurs inside the hen before the egg is laid (oviposition), remains
shrouded in mystery. Studies of fish and mouse embryos
show that initial cleavage processes immediately after
fertilization heavily influence cell fate decisions and morphogenesis at later stages, but whether similar developmental processes unfold in avian embryos in the earlystages before egg-laying has not been closely examined
before.

Research Team at the RIKEN Center for Life Science
Technologies (CLST), closes in on the early avian developmental processes that take place after fertilization
but before egg-laying. Using both light and scanningelectron microscopy (SEM), they carried out detailed
observations of the developmental patterning of pre-oviposition chick embryos, providing the first look at early
embryonic development that occurs before egg-laying.
Their findings, published in Development, reveal several
surprising similarities in early developmental patterning
among birds and other vertebrate species.

In the 1970s, scientists Eyal-Giladi and Kochav created a developmental staging system for chick embryos
A new study by technical staff Hiroki Nagai of the Labo(EGK stages), which classifies early embryonic stages
ratory for Early Embryogenesis (Guojun Sheng, Seniorbased on morphological characteristics observed under
Investigator) along with other collaborators, includthe light microscope; the staging system divides develing Shigenobu Yonemura, head of the Ultrastructuralopmental processes between post-fertilization to preprimitive streak formation
into 14 stages, from EGKI to -XIV, with EGK-I to -X
covering early embr yonicstages before oviposition. In
the 1990s, with the help ofelectron microscopy, Etches
and colleagues were able toobserve ultrafine structuresof the later pre-ovipositional
EGK stage s (EGK- IV onward) in chick embryo. For
the current study by Nagai
and colleagues, they examined the morphologicalcharacteristics of early preovipositional EGK stages(EGK-I to -V: post-fertilization to 8 hours) in detail using both light and electron
microscopy, retrieving thefertilized eggs from the hens
Left: Images captured by SEM of chick embryos between EGK-I to -V. Cellularization of blastoderm as developwith a non-invasive, abdomment progresses. Right: Schematic view of cellularization process. Subgerminal cavity beneath the blastoderm
inal massage method.
gradually expands as blastomeres become completely cellularized (blue).

Zebra finch embryo at EGK-VIII/IX. Syncytial nuclei forms near the boundary between the yolk membrane and subgerminal cavity.

16

Nagai H, et al. Cellular analysis of cleavage-stage chick embryos
reveals hidden conservation in vertebrate early development.
Development 142.1279-86 (2015)

During EGK-I, several cleavage furrows appear on thesurface of the yolk on the animal pole side, forming
multinucleate cells. At EGK-II, cells of varying sizes thathave completed cellularization (compartmentation) arefound near the center, and by EGK-III, these cells begin
a series of rapid divisions and a space emerges between
cellularized cells and yolk cells, called the subgerminal
cavity.
The team then focused on two important events that
occur in early developmental stages to look for possible similarities with other vertebrate species. One was
zygotic gene activation (ZGA), when the embryo, whichis initially dependent on maternal transcription products
accumulated in the egg for development, switches over
to its own developmental regulatory system by activating transcription of its own genes. ZGA is thought to beclosely linked to epigenetic reprogramming mechanisms
in cells, but when and how this occurs has not been wellstudied in avian species. Upon examining the chick embryos, they discovered that ZGA takes place between
late EGK-II and early EGK-III (64- to 128-cell stages),similar to zebrafish embryos in which ZGA occurs at a
the 128-cell stage. That ZGA occurs at similar developmental timing in chick and zebrafish, which both haveyolk-rich eggs, suggests there is a common embryonicmolecular mechanism at work.
The second event was one specific to unevenly distributed yolk-rich eggs such as chick and zebrafish, theformation of the yolk syncytial layer (YSL). Research inzebrafish have revealed that the YSL forms during thecleavage stage near the boundary where the embryo(blastoderm) contacts the yolk and contributes to the dynamic collective migration of blastoderm cells; whether
a similar YSL forms in avian embryos has remained
unclear. Nagai et al. carefully examined pre- and postoviposition chick embryos and found YSL was present
between stages EGK-V to -XI. Analyses of other avian
species, such as finch and quail, also showed YSL formation at similar developmental timings as in the chick.
These findings indicate that YSL formation is a common
developmental phenomenon among birds and fishes.
“Several features of early embryonic development inchick closely resemble what is seen in zebrafish embryos,” says Sheng. “It will be interesting to determine
whether the features conserved across species are a
result of convergent evolution or of evolutionary constraints.”

Hiroki NAGAI

17

Improved donor organ preservation
method for transplantation

Takashi TSUJI, Jun ISHIKAWA

O

rgan transplantation is an approach consideredby medical specialists to replace an organ thatcan no longer carry out essential physiological
functions. Despite many advances in transplantation
research, there are still several hurdles that need to be
overcome to improve success rates and access to organtransplantation. Static cold preservation systems are
currently used to preserve donor organs prior to transplantation, but they can only maintain organs in healthy
states for relatively short periods—from a few hours toless than a day depending on the organ. New methods
to store organs in healthy conditions for longer periods
are thus needed as this will improve transplant successrates. There is also a worldwide shortage of organs available for transplantation, as the number of patients waitlisted for transplants continues to grow with no corresponding rise in the number of suitable donors or donororgans, necessitating the development of methods toexpand the use of organs from marginal donors, whoseorgan health may not be considered ideal for transplantation, to increase the donor organ pools.
Now, research associate Jun Ishikawa of the Laboratory
for Organ Regeneration (Takashi Tsuji, Team Leader)
together with researchers at the Tokyo University of Sci-

18

ence, Keio University, and Organ Technologies Inc. havedeveloped a new technique that can preserve donororgans for longer periods and can resuscitate organs
retrieved from donors after cardiac arrest. Using the rat
liver as a model organ for transplantation, they reveal a
set of conditions that can preserve donor livers for up to
two days while ensuring that liver physiological functions
are recovered after transplantation. Their findings, published in Scientific Reports, imparts important insights
toward establishing an efficient perfusion culture system
to extend the storage period of donor organs, as well as
to increase the donor organ pool by resuscitating organs
donated from marginal donors, such as those who diedfrom cardiac arrest.
The group first designed a perfusion culture system,
which simulates the circulatory system of the body, andplaced a liver extracted from a rat into the system’s organ chamber filled with culture medium, ensuring thatthe entire tissue was sufficiently perfused with medium.
The liver was connected via its portal vein and inferior
vena cava to their perfusion culture system. They tested
a range of experimental conditions and found that a
combination of cooling the culture to 22°C and adding
red blood cells (RBCs), which serves as oxygen trans-

Ishikawa J, et al. Hypothermic temperature effects on organ survivaland restoration. Sci Rep 5.9563 (2015)

porters, to the culture media maintained liver functions
for up to two days as confirmed by measuring concentrations of byproducts of physiological liver functions,such as albumin synthesis, urea, and bile. Under thiscondition, the levels of alanine aminotransferase, a protein marker indicative of hepatic disorders when high,were low, and tissue structures also remained intact,
indicating that liver damage was suppressed. The teamalso determined that liver cells could survive and recoverproliferative abilities after being cooled down to 22°C
and then warmed back to body-temperature (37°C) conditions, whereas under other colder temperatures, thecells did not recover proliferative activities or died whenreturned to 37°C conditions.
Next the group examined whether transplanting a livercultured in their 22°C perfusion system for 24 hours
could replace the function of the recipient’s natural liver.
They carried out an auxiliary liver transplantation, andcompared effectiveness of using their system with orwithout addition of RBCs and of using conventional cold
storage at 4°C on liver preservation period. Survival rates
of rats transplanted with livers stored at 4°C or cultured
in perfusion system without RBCs dropped dramaticallyafter transplantation, whereas recipients of livers cultured in RBC-added perfusion system all survived. One

week after the transplant, they removed most of the recipient’s natural liver to confirm its regenerative abilities.
Most recipients of livers stored at 4°C or in the perfusion
system without RBCs did not survive, while those transplanted with livers cultured in RBC-added perfusion system showed 100% survival rates for an additional week.
Normal metabolic functions were also maintained in thelivers cultured in their RBC-added system.
Ishikawa et al. also tested whether their perfusion system could be used to resuscitate livers obtained fromdonors after cardiac arrest. Organs from individuals
who have died from cardiac arrest are often not used fortransplantation as oxygen and energy (ATP) needed forphysiological functions are rapidly depleted due to lackof blood circulation to organs (ischemia), leading to tissue damage. They isolated the liver from a rat that had
been in cardiac arrest for 90 minutes and placed it intheir RBC-added perfusion culture system for 100 minutes. ATP levels in the liver dropped dramatically after
cardiac arrest as expected, but after it was placed in theperfusion culture system, there was a marked increasein ATP levels. Survival rates of recipients of the perfusion
culture-treated livers was 100% after two weeks, andeven showed regeneration of tissue mass after partial
removal of the recipient’s natural liver. Thus, their findings indicate livers extracted
from cardiac arrest donors
can be resuscitated by theaddition of RBCs to their
per fusion culture system
maintained at 22°C.

Setup of perfusion culture system. Image on right shows rat liver placed in organ culturing chamber.

“This study demonstrates
that longer organ preservation periods and resuscitation of ischemia-damaged
organs is possible using our
perfusion culture system,”says Tsuji. “Our next step is
to modify the system for pig
models, which are genetically closer to humans, withthe final goal of making thisavailable for human clinicalapplications.”

ATP levels in liver drop after cardiac arrest, but recover when placed in perfusion culture system.

19

Surprising instability of meiotic cell division

Shuhei YOSHIDA

O

ocytes display a surprisingly high frequency oferrors in cell division despite being a vital means
for animals, including humans, to pass on theirgenes to the next generation. During meiosis in oocytes,
sister kinetochores (KTs), one located on each chromosome making up a homologous chromosome pair (bivalents), must correctly form stable attachments to themicrotubules (MTs) and spindle apparatus for the bivalents to be properly segregated into two future daughter
cells. Errors in the chromosomal segregation process inoocytes are known to cause congenital diseases suchas Down syndrome or lead to miscarriages in humans.
The mechanisms and reasons for the high rate of errors
in chromosome segregation in oocytes remain largely
unknown.
A new study by Shuhei Yoshida and colleagues in the
Laborator y for Chromosome Segregation ( Tomoya
Kitajima, Team Leader) has now uncovered one of thecauses of the high rate of errors seen in the first meiotic
division (MI) in oocytes. They carried out a detailed analysis of the events during MI in mouse oocytes, specifically around the KT-MT attachment sites, and discoveredthat correct KT-MT attachments are initially relatively unstable due to the prolonged presence of Aurora B and C

20(B/C) kinase, an attachment destabilizer, around the attachment sites, contrary to what is observed in mitosis.
Their findings, published in Developmental Cell, suggestthat the modified features of KTs and chromosomes in
MI make it difficult for the oocyte to pry the KTs spatiallyaway from the influence of Aurora B/C, thus contributing
to errors in chromosome segregation.
Both mitosis and meiosis require the formation of correctand stable KT-MT attachments for proper segregation ofchromosomes. Past studies of mitosis suggest that stability of KT-MT attachments are linked to the regulationof KT phosphorylation, such as by Aurora B/C kinase
which localizes to the inner centromere. In mitosis, MTs
extending from opposite spindle poles form an attachment to KTs on the sister chromatids (bipolar attachments). The resulting tension draws the sister KTs apart,causing Aurora B/C kinase to become spatially separated from the KTs, leading to a drop in KT phosphorylationlevels and stabilizing the attachment site. For MI of meiosis, MTs extending from the same pole form attachmentswith KTs on the bivalents (monopolar attachments), but
the mechanisms at work to ensure correct, stable KT-MT
attachments has not been closely examined. Despite theinherent differences in KT-MT attachments, a stabilizing

Yoshida S, et al. Inherent Instability of Correct Kinetochore-Microtubule Attachments during Meiosis I in Oocytes. Dev Cell 33(5).589602 (2015)

mechanism similar to that seen in mitosis has been proposed for KT-MT attachments in MI.
Yoshida et al. first turned their attention to the localization of KT-MT attachment destabilizer Aurora B/C kinaseduring meiosis. During early MI, MTs emanating from anapolar spindle attach to sister KTs on the bivalents andthe spindle then begins to bipolarize causing the bivalents to be pulled and stretched in opposite directions,
eventually forming a belt-like spatial arrangement. But, at
this point, the KT-MT attachment remains unstable. Aurora B/C remained localized in the vicinity of the KT-MT
attachments even after the KTs were pulled apart by theMTs. In contrast, during meiosis II, which unfolds similarly to mitosis, Aurora B/C kinase was localized at somedistance from the KT-MT attachment sites when KTs
were being pulled apart. When the oocytes were treatedwith an Aurora B/C kinase inhibitor as bivalents are
stretched in MI, there was a marked rise in the number ofcorrect KT-MT attachments formed, suggesting that thelingering Aurora B/C around the KT-MT attachment sites
after bivalent stretching, as seen in MI, is a direct causeof the instability of KT-MT attachments.
How then are KT-MT attachments stabilized in the later
MI stages? Close examination of changes in phosphorylation levels of KT-MT attachments during MI revealed
high phosphorylation levels for some period after bivalent stretching, which then dropped markedly four to six
hours later. These results suggested the workings of amolecular mechanism in MI that decreases phosphoryla-tion levels at KT-MT attachment sites over a prolonged
period leading to a gradual stabilization of these attachments without having to physically distance Aurora B/Cfrom the KT-MT attachment sites.
To search for the factor blocking the phosphorylating activity of Aurora B/C around KT-MT attachments, Yoshida
et al. closed in on PP2A-B56, a phosphatase known toantagonize Aurora B/C activity by dephosphorylating
KTs during mitosis. When localization patterns of PP2AB56 during MI were analyzed, they found its concentration levels gradually increased over several hours aroundthe KT-MT attachment sites. They next searched for themechanism underlying the regulation of PP2A-B56 levels and found that Cdk1-dependent phosphorylation of
BubR1, known in mitosis as a checkpoint protein, assistsin the recruitment of the phosphatase to the vicinity ofKTs. Gradual PP2A-B56 accumulation at KTs counters
Aurora B/C activity, resulting in dephosphorylation of
KTs and progressive stabilization of KT-MT attachment
sites.
“Our study reveals the surprising instability of chromosome segregation in oocytes, and also a mechanism forstabilizing KT-MT attachments that is different from mitosis,” says Kitajima. “As the oocyte is the starting pointof development, it has evolved many unique features.
But we may be looking at an example of a biological paradox, if these features have unwittingly created a weak
spot in basic intracellular activity.”

Left: Aurora B/C remains localized close to KT-MT attachment sites in MI than compared with MII, after chromosomes begin being pulled in opposite poles.
(red, KTs; green, Aurora B/C complex; blue, chromosomes) Right: Inhibiting Aurora B/C activity results in an increase in correct KT-MT attachments. (red,KTs; green, MTs; blue, chromosomes).

21

Self-organized actin patternin the tracheal tube

Edouard HANNEZO, Bo DONG

P

eriodic structural patterns, as seen in centipedes
or woodlice, are fundamental body pattern forms
that are also commonly observed in humans
and other animals. How these periodic structural patterns arise is one enduring fundamental question ofdevelopmental phenomena. In the 1950s, Alan Turing, abrilliant mathematician and founder of the concept formodern computing, proposed that a reaction between
two molecules diffusing at different speeds could produce a striped pattern autonomously without preexisting
positional information, and that this molecular level selforganization was the basis of morphogenesis in living organisms. Many groups have since attempted to validate
his theory through biological experiments, and to date it
has been demonstrated at the cellular level to produce
striped patterns in fish. However, little progress has been
made to validate the theory in vivo at the molecular level.
New work by former research scientist Bo Dong in
the Laboratory for Morphogenetic Signaling (Shigeo
Hayashi, Team Leader), now a professor at the Ocean
University of China, in collaboration with Edouard
Hannezo at the Institut Curie in France reveals a selforganizing mechanism of actomyosin in the cell cortex
that produces the actin ring patterns in the tracheal tube

22of Drosophila. They demonstrate through mathematicalmodeling and experimental observations that actin ringpatterns are self-organized via actin flows created bymyosin contractility-induced forces and that these actin
rings are stabilized by extracellular mechanical factors.
Their study, published in Proceedings of National Academy of Sciences, establishes the relevance of Turing’s
theory in understanding biological phenomena.
In Drosophila tracheal tube formation, the epithelial cells
are aligned with the apical side facing the lumen (interior)of the tube. As lumen formation begins, evenly spaced
actin rings are generated along the tracheal tube, straddling the apical cell cortex (just below the cell membrane)of epithelial cells. A hard cuticular structure is then assembled around these actin rings after the larva hatchesfrom the egg. Similar periodic structures are also seen in
mammalian trachea and C. elegans embryos, which suggests that this is a fundamental mechanism for maintaining tubular or cylindrical structures.
How then is the actin ring pattern formed? To maintainthe evenly spaced patterns beyond cell boundaries, theteam speculated that there must be a mechanism functioning at the tissue level. Hannezo and Recho adopted

Hannezo E, et al. Cortical instability drives periodic supracellularactin pattern formation in epithelial tubes. Proc Natl Acad Sci U S A
112(28).8620-8625 (2015)a theoretical approach using mathematical modeling topredict that in a viscous cytoplasmic environment, therepeated contractility-driven forces of the myosin motor
protein causes actin flows, eventually giving rise to a periodic actin ring pattern.
Dong then set out to validate the model experimentally.
They first analyzed Drosophila tracheal tube development using fluorescence live imaging, which revealed agradual increase in actin concentration and appearanceof circumferential supracellular actin rings, each cell with
15 to 20 actin rings. Because myosin II colocalizes withactin, the rings were speculated to arise from the bindingof actin and myosin II to form the contractile actomyosin
complex. Fluorescence recovery after photobleaching (FRAP) experiments confirmed that actin flow andturnover did take place in the cortex, and when developing embryos were treated with a drug inhibiting myosin
contractility, the actin rings disappeared, indicating thatmyosin activity is required for tracheal actin ring formation. Thus, together these experiments validated that thebasic assumptions of the mathematical model were supported in vivo.
In their model, friction within the cell cortex was postulated as a hypothetical factor influencing actin flows,
playing a large role in orienting or positioning actin within
the cortex. It predicted that actin would be assembled
in a circumferential ring pattern when circumferential
friction was slightly higher than axial friction, and thata decrease in friction would lead to wider spacing between the actin rings. Thus, Dong et al. proposed thatthe source of friction was the binding between the actin
filaments, intercellular adhesion complex and cell cortex.
They examined flies with mutations in a gene encoding
a factor important for stabilizing the adhesion complex,

and found that intervals between actin rings were wider
in mutants than seen in the wildtype. In another experiment where actin-cell cortex binding was inhibited, theyalso found wider actin ring intervals. These results wereconsistent with the predictions made by their model, anddemonstrate that cell cortical friction contributes to ringpattern formation in the tracheal tube.
Another potential source of friction was the binding between the cell cortex and solid extracellular matrix (ECM)
that initially fills the tracheal tube. Chitin fibers, the main
ECM component, are aligned in the tube in an anteroposterior direction, distributed in a manner enabling directional friction. They examined a fly mutant strain thatcannot synthesize chitin and consequently has no chitin
fibers in the developing tube, and observed that contractile forces exerted by actin rings were sufficient to cause
localized constrictions of the tube. Intervals between
actin rings were uneven and irregular, resulting in a tube
with non-uniform circumference and several kinks along
its length. These results suggest that under normal conditions where ECM fills the lumen, friction between the
ECM and the cell cortex contributes to the stabilizationof actin rings and uniformity of the tracheal tube. Thus,
the actin dynamics observed in the tracheal tube areconsistent with the behavioral predictions of their model
when values for factors such as friction are manipulated.
“It was surprising to discover that molecular level selforganization, as proposed by Turing over 50 years ago, is
one mechanism maintaining the uniform circumferenceof the tracheal tube throughout its entire length,” says
Hayashi. “We hope to further investigate the physicallaws associated with tissue level morphogenesis whensupracellular structures that span the cells, such as actin
rings, are controlled by extracellular factors.”

Left: Model of actin ring formation. Right: Actin rings formed along the cortex of tracheal tube. In mutants for a factor involved in stabilizing adhesion complex (Src42 mutant) and those unable to produce ECM (kkv mutant), cortical friction decreased and intervals between actin rings were
wider than seen in wild type. This is consistent with predictions of the mathematical model.

23

Anchoring the signaling complex
needed for cell elongation

Tetsuhisa OTANI

A

lmost all cell types display some sort of polarity, resulting in a diversity of cell shapes andfunctional differentiation. Extensive and robustchanges in morphology can arise from polarization ofcellular components during development, which requires
tightly regulated intracellular transport systems capableof shuttling and localizing proteins and other molecularcargo to specific destinations within the cell. One widely
observed cell morphogenetic phenomenon is cell elongation, in which a cell elongates in one direction during its development. The distal tip of an elongating cell
serves as a hub for the signaling complexes that regulate
molecular cargo trafficking as well as cytoskeletal organization. While motor proteins are known to shuttle
molecular cargo to the distal tip along the polarized cytoskeleton, how the signaling molecules that coordinate
elongation are localized and maintained at the tip duringelongation remain poorly understood.
A new study published in Development by Tetsuhisa
Otani in the Laboratory for Morphogenetic Signaling(Shigeo Hayashi, Team Leader) and his colleagues offers
new insights into how distal tips of elongating cells areorganized and maintained. Using the mechanosensory
bristle of the Drosophila fruit fly as a model system, they24

demonstrate a mechanism regulating the correct transport and positioning of I K B kinase ε (IKKε), a protein
kinase that plays a central role sorting molecular signals
needed for elongation, near the distal tip of the elongating bristle.
In a previous work also led by Otani using the samemodel, they reported that once IKKε was localized andactivated at the distal tip, it acts as a signaling center tosort the various molecular cargos transported to the tip.
To solve the mystery of how IKKε is robustly maintainedat the distal tip during elongation, the group combed
through past publications for molecules that reportedlybind to IKKε.
Their search led them to the Spindle-F (Spn-F) protein,which has been shown to interact with IKKε as well as
contribute to the localization of active IKKε. These two
proteins were speculated to function as a pair due tosimilar polarity defects observed in oocytes of spn-F andof ikkε mutants. Upon closer examination of the respective mutants, Otani et al. discovered bristle morphology
defects in both mutants; bristles were shorter, with somedisplaying multiple branching near the tip or bulges along
the bristle. In normal bristles, activated IKKε and Spn-F

Otani T, et al. A transport and retention mechanism for the sustained
distal localization of Spn-F-IKKepsilon during Drosophila bristle
elongation. Development 142.2338-51 (2015)

are found co-localized at the tip of the bristle, but Spn-F
is also found dispersed in the cytoplasm. Time-lapse imaging experiments tracking cytoplasmic Spn-F behavior
revealed that the protein moved along microtubules tothe tip, and fluorescence recovery after photobleaching(FRAP) experiments also showed that Spn-F was stably
localized to the distal tip once transported there.
How then are IKKε and Spn-F transported to the tip?
Cytoplasmic dynein, a minus-end motor protein, was aprime candidate because microtubules in the elongating tip are oriented with the minus-ends facing the distal
tip. Indeed, when dynein activity was inhibited in thebristle, both IKKε and Spn-F were unable to localize
to the distal tip. When the domain regions of the SpnF were analyzed, they found that cytoplasmic dynein
and IKKε could bind simultaneously to Spn-F throughdistinct binding regions and that this simultaneous binding is crucial for cell elongation. Thus, Spn-F appears toserve as an adaptor protein linking IKKε to dynein, whichtransports them both along the microtubules to the distal
tip.
Next, Otani et al. turned their attention to the mechanism
underlying the sustained localization of IKKε at the tip after being transported there by cytoplasmic dynein. They
examined the possible role of a protein called Javelin-like(Jvl) at the distal tip as it is known to interact with Spn-F and also contributes to the polarization of active IKKεin oocytes. In jvl mutants, IKKε and Spn-F colocalized at
the distal tip during early stages of elongation, but in thelater stages, they were no longer localized at the tip, andbristle tips showed morphological defects. Time-lapse
imaging tracking the molecular dynamics of Jvl and SpnF revealed that each molecule actively moves along the
microtubules independently of the other, but when bothproteins colocalize together, they stop moving. The interaction of Jvl and Spn-F thus results in their immobilization, and consequently immobilizes IKKε, which is boundto Spn-F.
Based on their findings, they proposed that the IKKεSpn-F complex and Jvl are transported independently
to the distal tip in a unidirectional manner, and when Jvl
interacts with Spn-F at the tip, the IKKε-Spn-F complex
becomes anchored there.
“Spn-F is not just an adaptor molecule linking the motor protein to IKKε. It also serves as a signal to recruit
factors that regulate dynamics of IKKε after being transported to the tip,” says Hayashi. “Examining different
adaptor molecules may help us understand the elaborate
intracellular transport system underlying the morphogenesis of more complex cell shapes such as mammalian
neurons.”

Left: Electron microscopy image of Drosophila bristle. Right: Pupal stage bristle cell. IKKε (blue) and Spn-F (red) are colocalized near elongating tip (whitearrow).

25

Leading cause of age-related aneuploidy
in oocytes revealed

Yogo SAKAKIBARAT

he germ cell, more commonly referred to as oocyte or sperm, is the only cell type in the animal
body that contributes to the next generation bypassing on its genetic information. Despite its importance in reproduction, oocytes display a surprisingly
high rate of chromosome segregation errors as maternal
age increases. When an oocyte with abnormal chromosomal numbers (aneuploid) is fertilized, it often results ina miscarriage or in congenital disorders, such as Down
Syndrome, if it develops to term. While chromosome
segregation errors are known to occur during the firstmeiotic division (MI) of oocytes, the exact causes andmechanisms remain unclear.
A new study published in Nature Communications by
Yogo Sakakibara, a special postdoctoral researcher inthe Laboratory for Chromosome Segregation (Tomoya
Kitajima, Team Leader) and collaborators at the IVF
Namba Clinic, Japan, and the Karolinska Institute, Sweden, takes a closer look at the chromosome dynamics
during MI in both mouse and human oocytes. Using highresolution 3D imaging microscopy to track chromosome
movement, they demonstrate that premature separation ofbivalent chromosomes during MI is a major cause of chromosome segregation errors observed in aging oocytes.

26

Germ cells arise from meiosis, a specialized type of cell
division which reduces the chromosome number by halfthrough two rounds of cell division, meiosis I (MI) andmeiosis II (MII). During MI, maternal and paternal chromosomes pair and undergo synapsis to form homologous chromosome sets (bivalents); these bivalents thenseparate into univalents and are evenly divided into two
daughter cells. In MII, sister chromatids of univalents are
separated and are again evenly divided into two cells, resulting in haploid cells. A study published by Henderson
and Edwards in 1968 reported the observation of a highfrequency of univalents in chromosome spreads takenfrom aged oocytes undergoing MI, and proposed that
this was likely the cause of chromosome segregation
errors. Many studies following their work appeared tosupport this hypothesis; however, it remained difficult toprove directly due to small sample sizes and limitations
in experimental methods.
In the present study, Sakakibara and his collaborators
successfully used high-resolution 3D imaging technology to examine and analyze chromosome dynamics inlive oocytes during MI. They began by analyzing 275
oocytes obtained from naturally aged mice (16 months),
and found that 20 of those oocytes exhibited errors inSakakibara Y, et al. Bivalent separation into univalents precedes
age-related meiosis I errors in oocytes. Nat Commun 6.7550 (2015)

chromosome segregation. They identified three distinct
patterns of segregation errors. The first was balanced
predivision (45%), in which both pairs of sister chromatids making up a bivalent are separated prior to MII. The
second was unbalanced predivision (35%), in which one
pair of the sister chromatids of a bivalent is segregatedprematurely. In the third pattern, both pairs of sister
chromatids fail to separate, resulting in nondisjunction(20%). In cases of balanced predivision, the oocyte appears as though normal chromosomal segregation has
taken place after MI; however, errors will arise during
MII. In contrast, aneuploidy can be detected after MI in
oocytes that undergo unbalanced predivision or nondisjunction. These patterns of segregation errors in themouse, are consistent with reports from human genetic
studies and analysis of aneuploidy in human oocytes.
Chromosome segregation errors were not observed in
the 167 oocytes obtained from young mice (two months
old).
Next, the group re-examined the chromosome dynamics to determine the underlying cause of these segregation errors. Univalents were found in 80% of oocytes
exhibiting segregation errors during a period of MI whenbivalents should be present. They discovered that whilebivalents need to maintain their structure until segregation occurs in MI, some are unable to withstand thebipolar forces generated by the microtubules and consequently become hyperstretched until they eventually
separate prematurely into univalents. The same bipolar
forces bioriented the univalents, and in many cases, thesister chromatids of the univalents were mistakenly segregated in MI. These findings suggest that the glue holding the bivalent together until segregation is weakened,
corresponding with recent reports of reduced levels ofcohesin, a protein complex mediating cohesion between
sister chromatids of bivalents, in aged oocytes.
Sakakibara and his colleagues also analyzed chromosome segregation dynamics in human oocytes. They obtained oocytes that would otherwise be disposed of froma fertility clinic, with the donors’ consent, and found that,similar to the results seen in mice, oocytes from relatively
aged donors exhibited premature bivalent chromosome
separation during MI. The resulting univalents were thenpulled to opposite poles, strongly suggesting that premature segregation of bivalents is also a cause of chromosomal segregation errors seen in humans.
“While our study clearly demonstrated the major causeof age-related chromosomal
segregation errors observedin the mouse, we need to becareful when trying to interpret the data from humanoocytes,” explains Kitajima.
“We are planning to examine
the process of prematurebivalent separation from amolecular perspective. In
particular, we hope to reveal
the mechanism underlying
the reduction of cohesin inaged oocytes.”

Chromosome dynamics in aged mouse oocyte during MI. Chromosome (red) localization was traced by tracking
movements of kinetochores (green). One of the bivalents was hyperstretched by microtubule-generated bipolar
forces and eventually separated prematurely into univalents (arrowheads). Univalents then underwent balanced
predivision of sister chromatids.

Three patterns of segregation errors observed in oocytes from naturally aged mouse (16 months) during MI.

27

Just being naive? Early pluripotency
in a bird model of development

Siu-Shan MAK, Anna WRABEL

E

mbryonic stem cells (ESCs) are perhaps the mostrecognized type of pluripotent stem cell derived
from mammalian embryos, but they have a lesser
known cousin as well, called epiblast stem cells (EpiSCs).
Whereas mouse ESCs are obtained from the inner cellmass (ICM) at the blastocyst stage of development, andcan give rise to all three germ layers as well as reproductive germ cells, their epiblast counterparts are derived
from a later stage, after the embryo has implanted into
the uterine lining, and no longer have the capacity for
germ cell differentiation. In recent years, these variant
states of pluripotency have come to be described asnaive and primed, respectively, and the transition to aprimed state in mouse is now known to be controlled by
the activin and FGF pathways. Human ESCs in contrast
are already in a primed state, and efforts to obtain naive
ESCs from the ICM have not borne fruit, highlighting interspecies differences in pluripotency.
In a report published in eLife, Siu-Shan Mak (lead author)and Anna Wrabel (technical assistant), in a joint study by
the Laboratories for Sensory Development (Raj Ladher)and Early Embryogenesis (Guojun Sheng) identified naive pluripotency in cells obtained from zebra finch eggs
immediately after laying. This lays the groundwork for ex-

28

panded studies into the establishment and maintenanceof pluripotency across amniote species, not only mammals. Ladher and Sheng have respectively since moved
to the National Centre for Biological Sciences (Bangalore)
and Kumamoto University.
Chicken and quail are the most commonly used avian
species in developmental research, but their embryos
have already developed substantially by the time theegg is oviposited. In this study, however, the Ladher andSheng labs recognized that the comparatively less advanced development of zebra finch embryos at the timeof oviposition afforded an opportunity to look for naive
pluripotent stem cells in bird.
The team first observed embryos at this stage and determined that they represented a state of development(stages 6–8) roughly comparable to the mouse blastocyst prior to epithelialization. Chicken embryos from laid
eggs in contrast have already undergone this transitionand correspond more closely to mouse epiblast.
Looking at the molecular profiles of zebra finch embryos
confirmed what their morphology suggested; not only
did they express canonical markers of pluripotency such

Mak S-S, et al. Characterisation of the finch embryo supports
evolutionary conservation of the naive stage of development inamniotes. eLIFE 4.e07178 (2015)
Mak S-S, et al. Zebra finch as a developmental model. genesis
53.669-667 (2015)

as Nanog, PouV and DNMT3b, but numerous markers ofnaive pluripotency as well. The expression of the lattermarkers dropped precipitously after stage 10, consistent with a clearly timed transition. Importantly, stage
6–8 embryos also expressed germline markers, whichlines up well with another feature of naive pluripotency inmouse ESCs. Interestingly, markers of naive pluripotency
were also observed, although at generally lower levels,in cells from chicken embryos at a later stage (~10) of development.
These findings point to the prospect that naive andprimed pluripotency may be conserved across amniote
taxa, not only in mammals. “We’re hoping to one day

identify bona fide pluripotent stem cells from zebra finch
embryos, which would enable the study of mechanisms
behind the establishment and maintenance of pluripotency in a much wider range of model systems,” says
Ladher.
This new work involved not only performing cell andmolecular biology experiments in zebra finch embryos,
but establishing the bases for rearing and housing zebra
finches under laboratory conditions. The same joint teampublished their methods for zebra finch breeding in the
October issue of genesis, in the interests of adding newexperimental options for avian embryological research.

Left: Expression of PouV in stage 8–10 zebra finch egg. Right: Expression levels of naive pluripotency markers in cells from zebra finch(green) and chicken (red) eggs.

29

Nutrient-dependent regulation of growth
control hormone revealed

Naoki OKAMOTO and Takashi NISHIMURA (second row, fourth and third from the right)Insulin/insulin-like growth factor (IGF) signaling plays
an important role in the regulation of biological processes such as growth, metabolism, reproduction
and longevity, and is known to be widely conserved
across species. In mammals, major players of thispathway are insulin and IGF, which have distinct roles
in metabolism and growth, respectively, whereas in the
Drosophila fruit fly, there are eight insulin-like peptides(Dilps) that fulfill these roles. The function and regulationof these endocrine hormones must be capable of readily
adapting to environmental changes, particularly to nutrient availability, but the mechanisms by which organisms
can sense these changes to regulate hormone levels remain poorly understood.
In new work published in the journal Developmental Cellby former research scientist Naoki Okamoto and teamleader Takashi Nishimura in the Laboratory for Growth
Control Signaling, they examine the function of nutrientdependent growth control hormone, Dilp5, and themechanisms regulating its production in Drosophila.
They pieced together their findings to reveal a complex
signaling relay and positive feedback mechanism at work
to control the expression of dilp5 in the insulin-producing
cells of the fly brain. Okamoto has since moved to the30

University of California, Riverside, to continue his research.
In Drosophila, Dilps are primarily secreted by insulinproducing cells (IPCs) in the brain in response to a signal
from the fat body (insect equivalent of vertebrate liver)which acts as a sensor of nutrient availability. The secreted Dilps then circulate throughout the body to act ontarget tissues. One of the main Dilps produced by IPCs
is Dilp5, an important regulator of body growth. Expression levels of dilp5 are initially low immediately after larval hatching, but rise sharply as larvae begin to feed, andexpression levels are also known to fluctuate depending
on availability of nutrients in the surrounding environment. A past study by Okamoto and his colleagues demonstrated that dilp5 expression was stimulated throughthe synergistic action of two transcription factors, Eyeless (Ey) and Dachshund (Dac), in IPCs. However, itremained unclear how Ey and Dac were tied to nutrientdependent changes in dilp5 expression.
In this study, the team first tested different nutrient mixtures to determine the critical nutrient source regulating
dilp5 expression was amino acids. Target of rapamycin(Tor) signaling, a well-known amino acid sensing path-Okamoto N, Nishimura T. Signaling from glia and cholinergic neurons
controls nutrient-dependent production of an insulin-like peptide for
Drosophila body growth. Dev Cell 35.295-310 (2015)

way, was analyzed as a potential upstream signal regulating IPCs, but the disruption of various factors in thispathway showed no effects on dilp5 expression. They
shifted their focus to search for a nutrient-dependent
molecule that could regulate the Ey-Dac protein complex, and transcription factor FoxO surfaced as a prime
candidate because its activity is known to be regulated
by nutrient availability, and can both activate and downregulate expression of a diverse array genes. Starvation
experiments revealed FoxO localization moved fromcytoplasm to nuclei of IPCs, and that nuclear FoxO caninteract directly with Ey, thereby competing with Dac forbinding to Ey. Thus, localization of FoxO within the cell
can switch expression of dilp5 on and off.
How then is FoxO localization regulated? They screened
for potential signaling factors in the IPC to uncover theactivation of anaplastic lymphoma kinase (Alk) leads toFoxO localization in the cytoplasm and promotes dilp5
expression. Alk was in turn found to be activated by asecreted ligand called Jelly belly (Jeb), discovered to be
produced by cholinergic neurons. Thus, Jeb secreted bycholinergic neurons surrounding the IPC regulated FoxO
localization indirectly. Further experiments showed that

Dilp6 produced by surface glial cell layer of the Drosophila central nervous system, which functions similarly tothe blood brain barrier of mammalian brain, was responsible for regulating Jeb. Surface glial cells are exposedto the hemolymph, and IPC-derived Dilps (Dilp2, 3, 5) released and circulating in the hemolymph stimulate dilp6
expression in glial cells, initiating the indirect signaling
relay to enhance dilp5 expression in the IPC through apositive feedback loop.
“It’s puzzling why Drosophila use such a complicatedmechanism. Under poor food conditions, dilp5 mutants
showed reduced growth rates and smaller adult bodysizes than wildtype, and required longer time for larval
development, suggesting that the multistep dilp5 regulatory mechanism is important to maintain insulin signals
needed for promote growth, even under nutrient-restricted conditions,” says Nishimura. “The fact that FoxO andcholinergic neurons are also involved in insulin regulationin mammals suggests the mechanism revealed in thisstudy has been conserved throughout evolution. We plan
to further explore the elaborate survival strategies thatorganisms have developed to adapt to environmental
changes.”

Left: FoxO in IPCs is shuttled from cytoplasm to the nucleus under starved conditions (green, IPC (dilp2);
pink, FoxO). Right: Model of the regulatory mechanisms of nutrient-dependent dilp5 expression.

31

Hippocampal neurons from hES cellsT

he hippocampus is the seat of memory formationand learning, and disturbances of hippocampal
function are known to lead to cognitive and psychiatric disorders, making this a brain region of fundamental importance in a range of biomedical research
fields. In developmental terms, the hippocampus emerges through the activity of an organizing center on neighboring tissues, but its location deep in the embryonicbrain makes it difficult to study experimentally, and themechanisms by which hippocampal cells are induced
remain unclear.
Now, junior research associate Hideya Sakaguchi andothers from the Laboratory for In Vitro Histogenesis(Mototsugu Eiraku, Team Leader) and the former Lab for
Organogenesis and Neurogenesis (Yoshiki Sasai, dec.)
report the induction of self-organizing hippocampus-liketissue from human embryonic stem cells (hESCs). Published in Nature Communications, this work provides a
new experimental resource for studies ranging from hippocampal development to Alzheimer’s disease.
This new report represents the most recent in a series ofexperiments aimed at inducing neuronal differentiationfrom pluripotent stem cells stretching back more than adecade, and makes use of the SFEBq (serum-free floating culture of embryoid body-like aggregates with quick
reaggregation) method developed by the same group.
In this study, the group sought to adjust SFEBq conditions to steer hESCs toward the primordial tissue in theembryonic brain (called medial pallium) from which the
hippocampus arises.
Using a previously reported SFEBq technique for inducing self-organized cerebral cortex-like tissue, Sakaguchi
sought to develop a cocktail of factors delivered at theright times and dosages to trigger hippocampal differentiation from hESCs. By tweaking the levels of Wnt
and BMP4, dorsalizing factors that strongly influence
neuronal differentiation, the group was able to induce
the expression of genetic markers of choroidal plexus,
the neighboring cortical hem, and finally medial pallium,
suggesting that a self-organized form of differentiation
was occurring in the same chronological order seen in
normal development.
They next attempted to generate hippocampus-like tissue from the self-organized medial pallium. By experimenting with different culture media and conditions,they gradually discovered a reliable method for directing
the medial pallium into hippocampus, as evidenced bymarker gene expression. As these cell colonies begin todestabilize by about 100 days in vitro, they dissociatedthem into individual neuronal cells between 73 and 84
days of culture and followed their developmental course.
After isolation, these individual cells re-aggregated, andby day 197 around 75% expressed markers, indicating
hippocampal differentiation. Moreover, markers of two
distinct neuronal cell types—pyramidal and granular—
were each observed in about one-third of the total popu-

Neuroepithelium at day 42 in vitro. Cortical hem (negative for TTR and Lmx1a) and medial pallium (green, Foxg1::Venus; magenta,
Lef1) form sequentially adjacent to the choroid plexus (red, TTR; white, Lmx1a) (left). At day 179 of culture, granule (Prox1, white) andpyramidal (KA1, red) neurons are seen (right).

32

Sakaguchi H, et al. Generation of functional hippocampal neurons
from self-organizing human embryonic stem cell-derived dorsomedial
telencephalic tissue. Nat Commun 6.8896 (2015)

lation, and glial- and astrocyte-like cells were present as
well.
Tests of neuronal function by patch-clamping confirmedthat the differentiated neuronal cells possessed working
synapses, and calcium imaging at day 143 showed synchronized depolarization, suggesting that the hippocampal tissue was exhibiting network-like activity.
“The ability to recapitulate hippocampal development
in vitro is a significant step forward,” says Eiraku. “Welook forward to the use of this system not only to study
biological phenomena such as patterning and regionalization, but as a resource in biomedical research intodiseases like Alzheimer’s and schizophrenia as well.”Hideya SAKAGUCHI, Mototsugu EIRAKU

33

Putting a new spin on autonomously
organized epithelial movementThe bending and stretching of sheets of epithelial
cells into channels, tubes, and other topologies
is a central process throughout much of development. In the pupa of Drosophila fruit flies, an interesting
phenomenon occurs in which the cells of the male reproductive organ (terminalia) rotate consistently in the same
direction with respect to the anterior-posterior axis,
resulting in the wrapping of the spermiduct around thehindgut. In order for the terminalia to make its circuit, thesurrounding epithelial cells must also move as a group in
the same direction. But how they do this without a clear‘conductor’ to coordinate their movements has been amystery.
A new report in Nature Communications from the Laboratory for Histogenetic Dynamics (Erina Kuranaga, Team

Leader) working with the Laboratory for Physical Biology(led by Tatsuo Shibata) in the RIKEN Quantitative Biology
Center (QBiC) shows how epithelial left-right asymmetry
can give rise to such autonomous cellular movements.
The pupal genitalia are located in the tail region, andtheir rotation is made possible by the coordinated movement of epithelial cells in abdominal segment eight (A8)that connect the terminalia with the abdomen. This A8
segment is divided into anterior (A8a) and posterior (A8p)components. Over 38 hours following the first 24 hours
after puparium formation, first A8p makes a 180° turn,after which A8a completes the rotation by turning theremaining 180°. In this new study, the team began byisolating these tissues and observing them in culture to
see whether they would retain their ability to rotate. They

Cells in adjacent epithelium show rightward-leaning positioning, as revealed by changes in the positions of cells marked by colored dots.

A model of unidirectional movement by left-right asymmetrical repositioning of cells. Accumulation of Myo-II at right-leaning cell boundaries results in directional contraction and rearrangement of cell positions.

34

Sato K, et al. Left–right asymmetric cell intercalation drives directional
collective cell movement in epithelial morphogenesis. Nat Commun
6.10074 (2015)

found that, even isolated from the rest of the abdomen,
A8 epithelium still rotated autonomously.
They next sought to follow the changes in the positionsof individual cells by labeling cell boundaries. Whilethese cells maintained their cell-cell junctions, what theteam found was that they constantly changed their borders and positions relative to each other throughout therotation process. And intriguingly, the rearrangement ofapical cell-cell boundaries showed a distinct rightward
slant with respect to the A-P axis.
When the team examined the localization of the motor
protein Myo-II, known to play a role in apical contraction,
they found that while its distribution was not polarizedasymmetrically within a cell prior to the start of rotation,
its polarization showed a clear right-handed preference
before and during the rotation of A8a. Higher concentrations of Myo-II were also associated with higher levelsof left-right asymmetry, suggesting that the lopsided
distribution of this factor might play a part in driving epithelium to move en masse.
They looked at Myo-II in a fly mutant for Myo ID, in which
the terminalia rotates opposite to the ordinary direction.
In these cells, Myo-II tended to be more highly accumulated in left-leaning cell boundaries, again suggesting arole for this factor in coordinating epithelial movement
at the group level. Finally, a mathematical simulation of
the effects of left-right asymmetry gave further computational support to the notion that such rearrangements
alone can give rise to autonomous movement by groupsof cells, which can be made to be unidirectionally rightward when the cells are neighbored to the anterior byother immobile cells.

Ayako ISOMURA, Emi MAEKAWA, Erina KURANAGA“These new results were made possible by combining
advanced live imaging and quantitative modeling techniques,” says Kuranaga. “This finding of how left-rightasymmetry can drive autonomous epithelial movementsshows promise beyond the fruit fly, and may be of fundamental importance in a number of organogenetic processes in other species as well.”

Katsuhiko SATO, Tetsuya HIRAIWA

35

Pulmonary neuroendocrine cell clusters at
airway branches(From left) Keishi KISHIMOTO, Akira YAMAOKA, Mitsuru MORIMOTO,
Masafumi NOGUCHI, Yuka NODA, Chisa MATSUOKA, Yuki KIKUCHI

T

he cellular composition of respiratory epithelium
varies in different sections of this highly branching organ system. The airway, for example, contains numerous ciliary cells capable of sweeping out
foreign material, while Club cells that secrete surfactant
protecting the airway surface dominate. A third type,neuroendocrine (NE) cells, has been found to aggregate
at branch points in the airway in many different species,
but the developmental process responsible for this localization has remained unknown.
A new study by research scientist Masafumi Noguchi
and others in the Laboratory for Lung Development(Mitsuru Morimoto, Team Leader) uses single cell resolution 3D time-lapse imaging of an entire lung lobe to show
how NE cells cluster at airway bifurcation points in themouse lung. They also find that Notch signaling plays acritical role in inhibiting NE cell differentiation, thus limiting the numbers of such cells. This work was publishedin the December issue of Cell Reports.
In order to overcome the historical challenge of observing minute changes in the developing lung over time,36

the team first generated a mouse in which epithelial
cell nuclei and NE cells were labeled with different genetic fluorescent reporters. They next used two-photon
microscopy to visualize whole-mount preparations ofcleared fetal lung at high precision and tissue depth, andused these images to generate a 3D model of the entire
airway, making it possible to analyze the sizes and spatial relationships of NE cell clusters, which they foundindeed form following a consistent pattern.
Previous works by the Morimoto team and other groups
have shown that Notch-Hes1 signaling is involved in
NE cell cluster formation, so in the new study they useda Hes1 knockout mouse to investigate how this would
affect the differentiation of emerging NE cells. In wild
type embryos, NE cells appear as solitary cells throughout the airway, not just at branch points, in a salt-andpepper distribution from about day 13.5, but in the Hes1
knockout the number of NE cells clearly increased, withgreater numbers of neighboring cells undergoing NE
cell differentiation, and much larger clusters of NE cells
at bifurcation points by day 16.5. This suggests that the
Notch signaling pathway plays a role in suppressing theNoguchi M. et al. Directed Migration of Pulmonary Neuroendocrine
Cells toward Airway Branches Organizes the Stereotypic Location of
Neuroepithelial Bodies. Cell Rep 13.2679–2686 (2015)

NE cell differentiation of adjacent cells as a means ofrestricting the NE cell numbers, and with the ultimate effect of limiting NE cell clusters to appropriate sizes.
What then leads differentiated NE cells to aggregate at
airway branches? Using a 4D live imaging setup, Noguchi visualized changes in cultured fetal lung lobes for
up to 15 hours, and was surprised to find that the NE
cells appear to be moving autonomously toward branch
points, crawling through the epithelium and taking a direct path to a distal bifurcation, an unusual example ofindependent epithelial cell migration in morphogenesis.
“So we know that NE cells cluster at branches, but thenext question is, what controls their migration to such
points. I think this would require at least three factors – achemoattractant to draw cells from distant sites, a second to anchor them at branch points, and a third to bindsimilar cells to each other,” says Morimoto. “If we cangain a better understanding of this process, I think it may
also shed light on the mechanisms at work in the metastasis of small cell lung cancer as well.”

Localization of NE cell clusters in 3D airway. Clusters at branch points shown in green, those at other sites in red.

NE cell (yellow) migration in developing airway epithelium (red). Initially isolated cell (arrowhead) converges toward bifurcation
point (arrow).

37

Doubling teeth from single tooth germF

ollowing tooth loss, dental treatments using artificial materials such as dentures, dental bridges
and implants are used to replace the tooth, however, these methods do not restore full physiological
function of the teeth. Other tooth replacement therapies
such tooth transplantation of wisdom tooth and transplantation of autologous or bioengineered tooth germs, agroup of cells in early development that give rise to teeth,
into regions of tooth loss, have recently been developedand shown to fully restore physiological tooth function.
As humans have a limited number of tooth germs, securing biological sources that do not cause immunologicalrejection remains a critical issue, and thus the development of a technique to increase tooth germ numbers isheavily anticipated.
A recent research collaboration between CDB’s Takashi
Tsuji, team leader of the Laboratory for Organ Regeneration, and graduate student Naomi Yamamoto andothers in Prof. Keiji Moriyama’s laboratory at the Tokyo
Medical and Dental University has led to the development of a unique approach for tooth regeneration, using
a mouse model. They split a single tooth germ into two
using mechanical force, which could subsequently betransplanted into the oral cavity and develop into two
fully functional teeth. Their approach and findings are
published in the online journal, Scientific Reports.
The tooth germ is derived from the embryonic ectoderm
through epithelial-mesenchymal interactions. The teamfirst extracted molar tooth germ from the jaw of an embryonic day 14.5 mouse, when the tooth germ is generated, and tied a knot (ligation) around the tooth germusing a thin nylon thread, specifically through the signaling center in an attempt to split the tooth germ. After
culturing the ligated tooth germ for six days, histologicalstaining revealed the formation of two tooth germs, eachsurrounded by epithelial tissue. The ligated tooth germwere then transplanted into the mouse subrenal capsule,and after 30 days of maturation, it gave rise to two teeth
that contained hard tissues such as enamel and dentin, which was surrounded by periodontal ligament andalveolar bone, as seen in natural teeth. The split teeth
however were half the size of natural molar teeth, andalso had half the number of dental cusps on the crown of
the tooth.
Next, the team carefully analyzed the germ splitting
process using time-lapse imaging, and found that after
ligation, the tooth germ signaling center, enamel knot,
appeared in each split germ. Epithelial tissues surrounding each split germ continued to proliferate, eventually
invaginating along the ligated surface resulting in two
separate germs. Gene expression analyses for genes
important in early tooth development showed split germs
displayed similar expression patterns as those in natural
tooth germ.
Past studies have shown that tooth morphology is regulated by reaction-diffusion waves of gene expression involving an activator, Lef1, and an inhibitor, Ectodin. Analyses of the expression patterns of these two genes were
performed in natural and split tooth germs. In natural
tooth germ, Lef1 was expressed in the enamel knot andneighboring mesenchyme, and Ectodin was expressedaround the region of Lef1 expression. The split tooth
germ showed smaller yet comparable expression patterns within each germ. Thus, ligation of the tooth germ
leads to re-regionalization of the tooth-forming field ineach split germ through reaction-diffusion waves of gene
expression.
The group then implanted the split tooth germ into thejaw bone of the mouse to test whether teeth would de-Left: Tooth germ split into two after ligation. Middle: Micro-CT of split tooth germ 50 days after transplantation into oral cavity of mouse. Right: Nerve fibers
in the tooth pulp and periodontal ligament of split tooth.

38Yamamoto N. et al. Functional tooth restoration utilising split germs
through re-regionalisation of the tooth-forming field. Sci Rep 5.18393(2015)

velop and erupt similar to natural tooth. The transplanted
germs became engrafted into the surrounding tissue,
and two months post-transplantation, micro-CT analysis revealed that the newly erupted teeth were aligned
and in contact with the teeth on the opposing jaw. Whenorthodontic methods similar to braces were applied tothe split teeth, the bone supporting the teeth underwent
remodeling to accommodate teeth movement, indicating
the presence of a functional periodontal ligament. Nerve
fibers were also detected in the tooth pulp and periodontal ligament of the split teeth, as seen in natural teeth.
Analyses of response to noxious stimuli, such as orthodontic force or pulp exposure, revealed both engrafted
and natural teeth showed comparable responses, indicating that split teeth were properly connected to thecentral nervous system.
“Not only does tooth loss affect physiological tooth functions such as teeth alignment, chewing and speech, it
can also affect overall health,” explains Tsuji. “In the future, our method could be used as a new form of regenerative medicine therapy for patients with tooth loss bygenerating multiple tooth germs from the patient’s own
tooth germs and later implanting them.”

Keiji MORIYAMA, Naomi YAMAMOTO

Takashi TSUJI

39

Transplantation of hESC-derived retinal tissue
in primate models of retinitis pigmentosa

R

etinitis pigmentosa (RP) is a degenerative condition that ultimately damages the photoreceptors
at the back of the eye, one of the many layers ofspecialized cells in the complex retina. Patients afflictedwith RP suffer from loss of visual acuity, poor night vision and a narrowed visual field. Despite ongoing efforts
to develop effective treatments using gene transfer andbioengineering approaches, to date RP remains resistant to therapy. The emerging paradigm of cell therapy
represents another potential alternative, and the 4D cell
culture techniques developed by CDB Team Leader
Mototsugu Eiraku and former Group Director Yoshiki
Sasai (dec.), in which embryonic stem cells (ESCs) can
be steered to give rise to highly organized, three-dimensional tissue-like structures in vitro provides a promising
starting point for the development of potential cellular
resources for transplantation.

stem cells (iPSCs) is capable of maturation and integration into host tissue in mouse models, which promptedto them to move one step closer toward clinical testingby attempting a similar approach using macaque modelsof RP, which have the advantage of closer similarity tohumans.
Before attempting transplants in macaques, the group
first used a rat model to identify culture conditions forgenerating retina-like tissue with the ability to survive,
mature, and function in the host environment. After experimenting with a range of differentiation times and culture conditions, they determined that slightly less mature
cells, at 50–60 days of in vitro differentiation, produced
superior grafts in terms of thickness and ability to integrate.

Now, a new study by Hiroshi Shirai, Michiko Mandai
and others in the Laboratory for Retinal Regeneration(Masayo Takahashi, Project Leader) reports the successful transplantation of human ESC-derived retinal tissue
into a non-human primate model of retinitis pigmentosa.
This work, published in the Proceedings of the National
Academy of Sciences, represents an exciting new step
toward the development of cell-based treatments for RP.

Next, the group needed to develop a monkey model ofRP, a disease in which degeneration progresses fromthe peripheral part of the eye inwards, resulting in visualacuity losses in the center of the field of vision. The scientists used two different approaches to simulate thisradiating degeneration, chemical injections and photocoagulation by laser light, producing selective foci ofdamage suitable for use in transplantation experiments
and subsequent monitoring.

The same group had previously shown that retinal tissue
generated from mouse ESCs and induced pluripotent

They next transplanted hESC-derived retina tissue after~60 days of differentiation into the subretinal space ofTransplantation of hESC-derived retinal precursor tissue into immunocompromised rat yields mature photoreceptor cells withboth inner and outer segments (left). Transplantation of hESC-derived retinal precursor tissue into subretinal space in a primate
model of RP produces mature photoreceptors (green) proximate to host bipolar cells (white) suggestive of synapse formation(right).

40

Shirai H. et al. Transplantation of human embryonic stem cell-derived
retinal tissue in two primate models of retinal degeneration. PNASPlus 113.E81-E90 (2016)

immune suppressed macaque RP models. They observed proliferative growth of the grafted tissue for up to
120 days, and ongoing differentiation as evidenced by
the expression of early photoreceptor markers by day 90
and markers of rod and cone cells by day 150 of differentiation. Additionally, they observed growth patterns suggesting the formation of synaptic connections between
host bipolar cells and graft photoreceptors, although at
low frequencies.
In establishing primate models for preclinical studies ofcell transplantation in RP, this study opens up new possibilities for testing of different culture and surgical delivery techniques. “In the future, we hope to test whether
iPSC-derived tissue will show similar ability to integrate
and function in the host retina,” says Mandai.

Hiroshi SHIRAI, Michiko MANDAI

41

Laboratories

Fluorescence image of zebrafish embryo that was
injected with fluorescent dye at the one-cell stage.
Image: Laboratory for Axial Pattern Dynamics

In Vitro Histogenesis

http://www.cdb.riken.jp/en/research/laboratory/eiraku.html

Team Leader

Mototsugu EIRAKU Ph.D.
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 worked as a researchscientist 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 wasappointed Unit Leader. From November 2014, he has served as leader of the In Vitro Histogenesis Team.

Staff
Research Scientist

Shunsuke MORI
Satoru OKUDA
Nozomu TAKATA

Foreign Postdoctoral Researcher

Nicholas Robert LOVE
Research Associate

Yuiko HASEGAWA

In vitro generation of a functional organ with complex structures is a major challenge of cell biology. Toward this goal, it is a reasonable strategy to recapitulate the ontogeny that is the mostefficient and robust process for organogenesis acquired through evolution. Our laboratory aims
to clarify molecular and cellular dynamics underlying organogenesis, and to develop new technologies for in vitro recapitulation, that is, 3D functional organ formation from stem cells. These
researches aim to make important contributions to the field of developmental biology, stem cellbiology and regenerative medicine.

Visiting Scientist

Atsushi KUWAHARA
Daiki NUKAYA
Hidetaka SUGA
Morio UENO
Technical Staff

Tomoko HARAGUCHI
Masako KAWADA
Eriko SAKAKURA
Junior Research Associate

Hideya SAKAGUCHI
Student Trainee

Rika YAMADA
AssistantFumi WAGAI

Recent Publications
Sakaguchi H, et al. Generation of functional hippocampal neurons from self-organizing humanembryonic stem cell-derived dorsomedial telencephalic tissue. Nat Commun 6. 8896 (2015)
Kuwahara A, et al. Generation of a ciliary
margin-like stem cell niche from self-organizing
human retinal tissue. Nat Commun 6. 6286
(2015)
Okuda S, et al. Vertex dynamics simulations ofviscosity-dependent deformation during tissuemorphogenesis. Biomech Model Mechanobiol
14.413-25 (2015)
Kadoshima T, et al. Self-organization of axial
polarity, inside-out layer pattern, and speciesspecific progenitor dynamics in human E cellderived 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)

Self-organized optic cup formation from ES cell

Eiraku M, et al. Relaxation-expansion model for
self-driven retinal morphogenesis: a hypothesis
from the perspective of biosystems. Bioessays
34. 17-25 (2012)

ES cell-derived neuroepithelium (lifeact-GFP)

44

Photoreceptor cell (red) and bipolar cells (green) in ES cellderived neural retina

Tissue Microenvironment

http://www.cdb.riken.jp/en/research/laboratory/fujiwara.html

Team Leader

Hironobu FUJIWARA Ph.D.
Hironobu Fujiwara attained his baccalaureate from Kyoto Pharmaceutical University, and went
on to receive his M.S. and Ph.D. from Osaka University, the latter in 2003 for his work on thecharacterization of the human laminin-8 protein. From 2003 to 2007, he worked 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 that these specialized
tissue microenvironments instruct the fate and behaviors of cells. The aim of our lab is to gain abetter 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 of organs. We are particularly interested in the roleof regional specialization of the extracellular matrix (ECM) in the formation of the stem cell microenvironment, or niche, in the hair follicle. A deeper knowledge of this will provide a molecular basis
to an improved understanding of the niche regulation of stem cells and organ formation, and thedevelopment of tailor-made microenvironments for different lineages of stem cells in the skin.

Staff
Research Scientist

Ritsuko MORITA
Ko TSUTSUIVisiting Scientist

Natsumi SAITO
Norio UEMATSU
Technical Staff

Noriko BAN-SANZENIntern Student

Eloi Franco TREPAT
Part-Time Staff

Sonoko TOCHITANIAssistant

Asako NAKAGAWA

Recent Publications
Donati G, et al. Epidermal Wnt/beta-cateninsignaling 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 ofhair follicle stem cells is a muscle cell niche. Cell
144.577-89 (2011)
Watt F. M. and Fujiwara H. Cell-extracellularmatrix interactions in normal and diseased skin.
Cold Spring Harb Perspect Biol 3. a005124(2011)
Ferreira M, et al. An activating beta1 integrin
mutation increases the conversion of benign tomalignant skin tumors. Cancer Res 69.1334-42(2009)
Fujiwara H, et al. Regulation of mesodermal
differentiation of mouse embryonic stem
cells by basement membranes. J Biol Chem
282.29701-11 (2007)
Fujiwara H, et al. Rac regulates integrin-mediated endothelial cell adhesion and migration onlaminin-8. Exp Cell Res 292.67-77 (2004)

The image shows arrector pili muscles anchored to the bulge of hair follicles in a whole-mount
preparation of 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 anuclear counter-stain (blue).

45

Organismal Patterning
http://www.cdb.riken.jp/en/research/laboratory/hamada.html

Team Leader

Hiroshi HAMADA M.D., Ph.D.
Hiroshi Hamada was appointed director of the RIKEN Center for Developmental Biology in
April 2015, and also established the Laboratory for Organismal Patterning at the Center in
October of the same year. He currently holds a joint appointment as Professor in the Graduate School of Frontier Biosciences at Osaka University. He received his M.D. and Ph.D. from
Okayama University in 1979, and worked at the National Institutes of Health (USA) and Memorial University (Canada) for nine years before returning to Japan. His interest in development
stems from earlier work on embryonal carcinoma cells, which he performed in Canada. His
current interests are the mechanisms underlying symmetry-breaking and the origins of body
axes.

Staff
Visiting Scientist

Katsuyoshi TAKAOKA
AssistantKaori SONEMy lab studies how left-right asymmetries develop in the mouse embryo. In particular, we focus
on two types of cilia that are required for left-right symmetry breaking: rotating cilia that generate
leftward fluid flow, and immotile cilia that sense the fluid flow. We also study the role of maternal
epigenetic regulators in pre-implantation development. We address these questions by integrating
live imaging, structural biology, fluid dynamics and mathematical modeling.

Recent Publications
Shinohara K., et al. Absence of radial spokes inmouse node cilia is required for rotational movement but confers ultrastructural instability as atrade-off. Dev Cell 35.236-246 (2015)
Nakamura T., et al. Fluid flow and interlinked
feedback loops establish left-right asymmetricdecay of Cerl2 mRNA in the mouse embryo.
Nat Commun. 3.1322 (2012)
Yoshiba S., et al. Cilia at the node of mouseembryos sense fluid flow for left-right determination via Pkd2. Science 338.226-231 (2012)
Shinohara K., et al. Two rotating cilia in thenode cavity are sufficient to break left-rightsymmetry in the mouse embryo. Nat Commun.
3.622 (2012)
Takaoka K., et al. Origin and role of distal
visceral endoderm, a group of cells that determines anterior-posterior polarity of the mouseembryo. Nat Cell Biol 13. 743-752 (2011)
Hashimoto M., et al. Planar polarization of thenode cells determines the rotation axis of thenode cilia. Nat Cell Biol 12.170-176 (2010)

Role of motile and immotile cilia in left-right symmetry breaking46

Neocortical Development

http://www.cdb.riken.jp/en/research/laboratory/hanashima.html

Team Leader

Carina HANASHIMA Ph.D.
Carina Hanashima received her Ph.D. from Waseda University School of Science and Engineering in 1999. She worked as a postdoctoral fellow in the laboratory of Eseng Lai at Memorial Sloan-Kettering Cancer Center, Cell Biology Program from 1999 to 2002, where she
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 tocell specification in the neocortex. She was appointed Team Leader at the CDB in September
2007.

The neocortex, the most complex structure of all vertebrate brain systems, is organized into cytoarchitectonic groups of neurons that process unique sensory inputs, such as pressure, light andsound. This highly ordered structure is nonetheless generated from a relatively simple sheet ofneuroepithelium during development. Research in our laboratory aims to understand how thesediverse arrays of cortical neurons are specified and coordinated into high-functional territories:
1) What is the mechanism by which diverse cell fate is determined in the neocortex? 2) How areneurons precisely arranged into distinct cortical layers and areas? 3) To what extent does the establishment of functional areas rely on environmental inputs?

Staff
Research Scientist

Yuko GONDA
Takuma KUMAMOTO
Kenichi TOMA
Tien-Cheng WANG
Visiting Researcher

Pei-Shan HOU
Technical Staff

Chihiro NISHIYAMA
Part-Time Staff

Reiko ODA

Recent work from the laboratory has shown that the fate of neocortical neurons is controlled
through cortical intrinsic programs in which neuron fate is established by temporal changes intranscriptional regulatory network. However, in the mature cortex, cortical areas differ in theirtypes and numbers of specific layer neurons along both the anteriorposterior (AP) and medial-lateral (ML) axes. We are exploring the extent to which intrinsic determinants control the specificationof neuronal subtypes within discrete regions of the neocortex, as well as the extrinsic influencesthat refine the boundaries between functional areas of neocortex. To further these studies, we employ genetic manipulations in mice that will enable conditional loss of gene and cellular functions,recombination mediated cell-lineage tracing, and systematic approaches to identify novel molecules responsible for precise areal specification. Through these studies we wish to understandthe mechanistic basis by which unique sensory perceptions and functional circuitries develop in
the human neocortex.

Assistant

Eri KOJIMA

Recent Publications
Toma, K, et al. Encoding and decoding time inneural development. Dev Growth Differ 58. 5972 (2016)
Toma, K. and Hanashima, C. Switching modes
in corticogenesis: mechanisms of neuronal subtype transitions and integration in the cerebral
cortex. Frontiers in Neuroscience 9.274 eCollection (2015)
Bullmann, T, et al. A transportable, inexpensive
electroporator for in utero electroporation. Dev
Growth Differ 57(5): 369-377 (2015)
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)
Kumamoto T. and Hanashima C. Neuronalsubtype specification in establishing mammalian
neocortical circuits. Neurosci Res 86.37-49(2014)Yeh M. L, et al. Robo1 modulates proliferationand neurogenesis in the developing neocortex.
J Neurosci 34.5717-31 (2014)

The neocortex is composed of distinct neuronal subtypes that establish 6 layers.
Coronal section of a one-week-old mouse neocortex 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,later-born neurons labeled with DsRed at embryonic day (E) 16 (magenta) cannot migrate
past earlier-born cells (E15 GFP-labeled cells, green).

47

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

Team Leader

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

Staff
Research Scientist

Toshiya ANDO
Wei-Chen CHU
Takefumi KONDO
Takuya MAEDA
Yosuke OGURA
Technical Staff

Xiaorui CAIAkiyo KIMPARA
Tetsuhisa OTANIHousei WADA
International Program Associate

Guangxia MIAO
Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
Assistant

Ryoko ARAKIMai SHIBATA

Our research aim is to understand fundamental mechanisms of animal morphogenesis with particular interest in the mechanical basis of tissue movement and its interaction with extracellularenvironment. Our main research focus is the tracheal system in the Drosophila embryo, a networkof tubular epithelium used as a respiratory organ. Trachea is formed through invagination, tube
formation, elongation, fusion and final maturation as a respiratory organ. We are particularly interested in the mechanical control of epithelial architectures. Epithelium is stabilized by cell-celladhesion 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 the tracheal network
is formed, tube diameter and length are enlarged to reach the final size. Tracheal size change involves increase in cell size, especially the increase of apical cell area facing the luminal side. Keyquestion is how individually controlled cellular growth is coordinated to form coherent tissue architecture. 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 tubule shapes found inorgans in pathological conditions.

Recent Publications
Dong B, et al. Cortical instability drives periodic
supracellular actin pattern formation in epithelial
tubes. Proc Natl Acad Sci 112. 8620-25 (2015)
O Tani, et al. A transport and retention mechanism for the sustained distal localization of SpnF-IKKε during Drosophila bristle elongation.
Development 142.2338-51 (2015)

Another area of research is the mechanism of cell morphogenesis. We ask the question to what
extent single cells can autonomously organize nanometer scale cellular patterns. Our studies haveuncovered the role of the cellular trafficking center as an organizer of cell elongation (Otani et al.,2011).

Miao G and Hayashi S. Manipulation of gene
expression by infrared laser heat shock and its
application to the study of tracheal development
in Drosophila. Dev Dyn 244.479-87 (2015)
Dong B, et al. Balance between apical membrane growth and luminal matrix resistancedetermines epithelial tubule shape. Cell Rep
7.941-50 (2014)
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)

Drosophila embryo at the beginning of tracheal placed invagination (magenta). Cell outline is labeledgreen.

48

Developmental Epigenetics

http://www.cdb.riken.jp/en/research/laboratory/hiratani.html

Team Leader

Ichiro HIRATANI Ph.D.
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 developmentwith Masanori Taira. In 2003, he moved to the United States for his postdoctoral training withDavid Gilbert at the State University of New York Upstate Medical University and studied thedevelopmental 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 aposition as Assistant Professor at the National Institute of Genetics. In 2011, he was awarded a
grant from the JST PRESTO program in Epigenetics. He was appointed to his current position
as Team Leader at the RIKEN CDB in October 2013.

We wish to clarify the molecular mechanisms underlying global facultative heterochromatin formation during early mouse embryogenesis, with the belief that understanding the developmental
regulation of higher-order chromosome organization will lead to a deeper understanding of celldifferentiation.

Staff
Research Scientist

Hisashi MIURA
Rawin POONPERM
Naoyuki SARAI
Student Trainee

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 offacultative heterochromatin formation at the same developmental stage, which accounts for morethan 6% of the genome. This suggests that facultative 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 a common epigenetic feature of all somatic cells beyond the germ layer formation stage, and the reprogramming experiments imply a potential link to the cell’s differentiatedstate.
For these reasons, we combine genome-wide approaches with molecular and cell biology andimaging techniques to elucidate the molecular mechanisms underlying the facultative heterochromatin formation process. In the future, we will address the biological significance of this phenomenon and eventually wish to understand the fundamental implications of higher-order chromosome
organization.

David MEYER

Technical Staff

Akie TANIGAWA

Recent Publications
Shang W. H. et al, Chromosome engineering
allows the efficient isolation of vertebrate neocentromeres. Dev Cell 24.635-48 (2013)
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 695.4158 (2010)
Ryba T, et al. Evolutionarily conserved replication timing profiles predict long-range chromatininteractions and distinguish closely related cell
types. Genome Res 20.761-70 (2010)
Hiratani I, et al. Genome-wide dynamics ofreplication timing revealed by in vitro models ofmouse embryogenesis. Genome Res 20.15569 (2010)
Hiratani I, et al. Replication timing and transcriptional control: beyond cause and effect--part II.
Curr Opin Genet Dev 19.142-9 (2009)

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

Genome-wide DNA replication profiling during ES celldifferentiation can reveal domains that show largescale changes in nuclear 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.

49

Sensory Circuit Formation
http://www.cdb.riken.jp/en/research/laboratory/imai.html

Team Leader

Takeshi IMAI Ph.D.
Takeshi Imai completed his Ph.D. in the laboratory of Prof. Hitoshi Sakano at the University ofTokyo Graduate School of Science in 2006. He did consecutive postdoctoral fellowships 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 Schoolof Biostudies.

Staff
Research Scientist

Satoshi FUJIMOTO
Ryo IWATA
Meng-Tsen KE
Marcus LEIWE
Technical Staff

Miwako NOMURA
Yoichi SAKASHITA
Student Trainee

Shuhei AIHARA
Aya MURAI
Richi SAKAGUCHI
Assistant

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 thisquestion, 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 asingle type of odorant receptor. The olfactory bulb, the primary olfactory 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 then sent 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 understand simple rules behind the formation of complex neuronal circuitry.

Eri YAMASHITA

Recent Publications
Ke M. T., et al. Super-resolution mapping of
neuronal circuitry with an index-optimized
clearing agent. Cell Rep doi:10.1016/jcelrep.2016.02.057 (2016)
Imai T. Construction of functional neuronal circuitry in the olfactory bulb. Semin Cell Dev Biol
35C.180-88 (2014)
Nakashima A, et al. Agonist-independent GPCR
activity regulates anterior-posterior targeting ofolfactory 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 T, et al. Pre-target axon sorting establishes
the neural map topography. Science 325.58590 (2009)

Adult Thy1-YFP-H mouse brain was cleared with an opticalclearing agent SeeDB and imaged using two-photon microscopy. 3D rendering image of a volume of 4 mm x 5 mm x 2mm, encompassing cerebral cortex and hippocampus, isshown.

Tracing of sister mitral cells associated with a common glomerulus in the olfactory bulb.
Neurons connecting to a single glomerulus was labelled by neuronal tracer and thesample was cleared with SeeDB.

50

Axial Pattern Dynamics

http://www.cdb.riken.jp/en/research/laboratory/inomata.html

Team Leader

Hidehiko INOMATA Ph.D.
Hidehiko Inomata received his doctorate from the Tokyo Institute of Technology in 2003, andmoved to take a research scientist position at the Foundation for Biomedical Research and Innovation in Kobe, Japan that 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 named a researcher in the Japan
Science and Technology Agency (JST) PRESTO program. He was appointed Team Leader ofthe Laboratory for Axial Pattern Dynamics in 2014. In 2015, he was also named a researcher in
the Japan Science and Technology Agency (JST) PRESTO program.

Developmental processes take place through the exchange of information by cells within the constrained spatial environment of the embryo. Such intercellular communication is essential for theformation 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 of a head, limbs, or otherbody parts. Factors that play central roles in such developmental signaling are known as morphogens.
In our research, we will seek to gain a deeper understanding into processes informed by positional information in a spatial context (developmental fields), using vertebrate (mainly frog andzebrafish) 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 ensure that development
based on simple concentration gradients is stably reproducible, cell-cell communications mediated 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, a phenomenon known as ‘scaling.’ Our
team has previously shown how scaling is maintained through morphogen-mediated intercellularcommunication when the spatial size of the embryo is perturbed. In our lab, we address visualization of morphogen gradients and in vivo imaging along with biochemical approaches to study how
developmental robustness is maintained.
We are also working to develop methods for controlling the shape of morphogen gradients. Gradients are primarily regulated by production, diffusion, and degradation, which indicates that bycontrolling these factors, it should be possible to arbitrarily design gradients that reconstruct tissue patterns in the embryo. By using such methods, we hope to gain a deeper understanding ofdevelopmental systems.

Staff
Research Scientist

Takehiko ICHIKAWA
Shinya MATSUKAWA
Technical Staff

Setsuko KANAMURA
Kaori NIIMI
Part-Time Staff

Mako MIYAGIMasako SUZUKI

Recent Publications
Inomata H, et al. Scaling of dorsal-ventral patterning by embryo size-dependent degradationof Spemann’s organizer signals. Cell 153.1296311 (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 developmentof 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 enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin lightchain 1. J Biol Chem 278.22946-55 (2003)

FRAP assays of mEGFP-tagged Sizzled shown by snapshots. From left to right columns;
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.

51

Chromosome Segregation
http://www.cdb.riken.jp/en/research/laboratory/kitajima.html

Team Leader

Tomoya KITAJIMA Ph.D.
Tomoya Kitajima received his Master’s and doctoral degrees from the University of Tokyo, for
his thesis on identification of Shugoshin as a conserved protector of chromosome cohesion
at centromeres. After receiving his Ph.D. in 2004, he worked as a research associate at theInstitute of Molecular and Cellular Biosciences at the same university, before moving to theEuropean Molecular Biology Laboratory in Heidelberg, Germany, as a postdoctoral researcher.
He was appointed Team Leader at the CDB in 2012.

Staff
Research Scientist

Aurélien COURTOIS
Yogo SAKAKIBARA
Shuhei YOSHIDA
Visiting Scientist

Hirohisa KYOGOKU
Student Trainee

Takeshi ASAKAWA
Yi DING
Namine TABATA
AssistantKaori HAMADA

Recent Publications
Sakakibara Y et al. Bivalent separation into univalents precedes age-related meiosis I errors inoocytes. Nat Commun 6.7550 (2015)

The oocyte becomes an egg through meiosis. The egg is fertilized by a sperm and undergoes
repeated cell divisions to give rise to an entire body. We study chromosome segregation duringmeiosis in oocytes and during mitosis in fertilized eggs, taking advantage of techniques for highthroughput and high-resolution live imaging of mouse oocytes combined with micromanipulationand genetic engineering methods. The first cell division that oocytes undergo is meiosis I. Chromosome segregation in this division is error-prone and the rate of errors increases with maternalage. Subsequently, chromosomes are segregated in meiosis II upon fertilization, and then segregated again in mitosis after DNA replication. We will reveal distinct mechanisms for chromosome
segregation during these subsequent but fundamentally different cell divisions. By uncovering themechanism of chromosome segregation during meiosis I in oocytes, we understand why oocyte
meiosis I is error-prone and related to age. Comparing the mechanisms in meiosis I with thosefound in meiosis II and mitosis may provide insights into the capacity of cells to flexibly use different strategies for chromosome segregation. The findings will be exploited to collaborative studies
with reproductive medicine.

Yoshida S. et al. Inherent instability of correct
kinetochore-microtubule attachments during meiosis I in oocytes. Developmental Cell
33(5).589–602 (2015)
Kim J et al. Meikin is a conserved regulator ofmeiosis-I-specific kinetochore function. Nature
517(7535).466-471 (2015).
Solc P et al. Multiple requirements of PLK1 during mouse oocyte maturation. PLOS ONE 10(2):e0116783 (2015).
Kyogoku H et al. Nucleolus Precursor Body
(NPB): A Distinct Structure in Mammalian
Oocytes and Zygotes. Nucleus 5(6).493-498(2014).
Kitajima TS et al. Complete kinetochore tracking reveals error-prone homologous chromosome biorientation in mammalian oocytes. Cell
146.568-81 (2011)

Kinetochore-microtubule attachments

52

Chromosome segregation error

Histogenetic Dynamics

http://www.cdb.riken.jp/en/research/laboratory/kuranaga.html

Team Leader

Erina KURANAGA Ph.D.
Erina Kuranaga received her doctorate in medical science from the Osaka University Graduate School of Medicine in 2004, after which she moved to the University of Tokyo Graduate
School of Pharmaceutical Sciences as assistant professor in the Department of Genetics. In
2006, she was promoted to associate professor in the same department, where she remained
until she joined the RIKEN Center for Developmental Biology as Team Leader in 2011. She wasawarded the Wiley-Blackwell Prize 2012 and Editor-in-Chief Prize from Development, Growthand Differentiation in 2014.

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 death at set times
in development, allowing for the sculpting of tissue, and is used in the adult organism to maintainhomeostasis by eliminating cells that have developed abnormalities. Perturbations in cell deathsignaling can thus affect an organism’s physiological stability, and result in developmental defects,
tumorigenesis, or neurodegenerative diseases. Cell death plays an important role in maintaining
the cellular society not only by eliminating unneeded cells at given sites and stages, but in otherfunctions, such as regulating the proliferation and migration of neighboring cells, as well. Suchcellular behaviors give rise to cell networks capable of organizing into tissues, the study of which
requires an experimental approach for obtaining spatiotemporal information in living systems, forexample, through real-time live imaging of biological phenomena.
We have chosen the fruit fly Drosophila melanogaster as our primary research model, seeking totake advantage of its utility in developmental studies and wealth of genetic data in studying thecoordination of histogenesis through live imaging and genetic screens. To elucidate the role of cell
death in histogenetic processes, we will analyze caspase mutant phenotypes in which the exteriormale genitalia (terminalia) develops abnormally. In normal Drosophila development, the terminalia
rotates 360° as it forms, but in caspase mutants, 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 deathcontrol acceleration of the rotation by searching for associated genes and live imaging analysis.
It has further been predicted that cell death alone cannot account for rotation that maintains tissue area, suggesting other mechanisms are also at work. We will conduct single-cell analyses todetermine whether other behaviors such as proliferation or migration are also altered. Throughthe use of the extensive Drosophila genetics toolset and live imaging technologies, we hope to beable to address questions that have proven technically challenging in the past, and by visualizing
the activities of individual cells, develop a better understanding of how cellular network systems
work in histogenesis.

Dorsal (left) and ventral (right) viewsof Drosophila pupae that express
fluorescent protein in cells locatedposterior component of each segment. Yellow square indicates locationof male genitalia.

Staff
Research Scientist

Emi MAEKAWA
Hiroyuki UECHI
Daiki UMETSU
Technical Staff

Ayako ISOMURA
Student Trainee

Yuka HAYASHI
Yuhei KAWAMOTO
Part-Time Staff

Arata KURANAGA
Yoko UMEGAKI

Recent Publications
Sato K, et al. Left-right asymmetric cell intercalation drives directional collective cell movement
in epithelial morphogenesis. Nat Commun
6.10074 (2015)
Obata F, et al. Necrosis-driven systemic immune response alters SAM metabolism throughthe FOXO-GNMT axis. Cell Rep 7.821-833(2014)
Takeishi A, et al. Homeostatic epithelial renewal
in the gut is required for dampening a fatal systemic wound response in Drosophila. Cell Rep
3.919-930 (2013)
Kuranaga E, et al. Apoptosis controls the speedof looping morphogenesis in Drosophila maleterminalia. Development 138.1493-9 (2011)
Nakajima Y, et al. Nonautonomous apoptosis is
triggered by local cell cycle progression during
epithelial replacement in Drosophila. Mol Cell
Biol 31.2499-512 (2011)
Kuranaga E, et al. Drosophila IKK-related kinase
regulates nonapoptotic function of caspases via
degradation of IAPs. Cell 126.583-96 (2006)

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

53

Sensory Development
Senior Investigator

Raj LADHER Ph.D.
Raj Ladher received his B.Sc. in biochemistry from Imperial College, London in 1992 and his
Ph.D. in developmental biology from the National Institute for Medical Research in 1996, for histhesis on the cloning of the Xenopus homeobox gene, Xom. He worked as a research associate at King’s College, London from 1996 to 2000 in the lab of Pip Francis-West, and first cameto Japan during that period as a visiting scientist at the Tokyo Medical and Dental University.
In 2000, he moved to the University of Utah as a postdoctoral fellow in Gary Schoenwolf’s laboratory. He was appointed Team Leader at the CDB in 2002. He is now a member of the facultyat the National Centre for Biogocial Sciences (NCBS) in Bangalore, India.
The Sensory Development lab closed in March 2015.
Dr. Ladher in now affiliated with NCBS in Bangalore, India.

Staff
Research Scientist

Akira HONDA

Visiting Researcher

Tomoko KITA

Part-Time Staff

Yoshiko KONDO
Assistant

Noriko HIROI

Recent Publications
Mak S-S, et al. Characterization of the finch
embryo supports evolutionary conservation of
the naive stage of development in amniotes.
Elife. doi:10.7554/eLife.07178 (2015)
Freeman S., et al. Expression of the heparansulfate 6-O-endosulfatases, Sulf1 and Sulf2, in
the avian and mammalian inner ear suggests arole for sulfation in inner ear development. Dev
Dyn. 244.168-180 (2015)
Ono K, et al. FGFR1-Frs2/3 signalling maintains
sensory progenitors during inner ear hair cell
formation. PLoS Genet 10.e1004118 (2014)

Hearing loss is the most prevalent sensory disorder not only in Japan, but also the world, withover 5% of the global population showing disabling hearing loss, rising to 50% in the aged population. Hearing occurs in the inner ear, a complex structure that houses the sensory receptors
important for sound detection, as well as other non-sensory cell types that are equally importantin the sensitive and precise detection of sound. This complex organ forms from a relatively simple
structure known as the otic placode, found in the surface ectoderm of the early embryo. It is clearthat 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 internalizationof the otic placode to make the first 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 of these cell-types.
We want to understand the blueprint for making an inner ear, with particular emphasis on integrating extrinsic signals, the genes that they control, with the cellular and sub-cellular changes thatdrive 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 be used todesign screening protocols, diagnostic tools and cellular therapies that help in alleviating hearingloss.

Sai X, et al. Junctionally restricted RhoA activity
is necessary for apical constriction during phase2 inner ear placode invagination Dev. Biol.
394.206-16 (2014)
O’Neill P, et al. The amniote paratympanic
organ develops from a previously undiscovered
sensory placode. Nat Commun 3.1041 (2012)
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 mammaliancochlea. Shown are the four rows of inner ear hair cells that make up the organ of corti, stained with alpha-tubulin (to show kinocilia in green) and phalloidin to show the actin-based stereocilia (in red).

54

Cell Asymmetry

http://www.cdb.riken.jp/en/research/laboratory/matsuzaki.html

Team Leader

Fumio MATSUZAKI Ph.D.
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 as model systems. He has also recently found a novel type of selfrenewing progenitor in the developing cerebral cortex in rodents, providing new insights into
the enormous increase in brain size during mammalian evolution.

Our group explores the mechanisms underlying the organization of cells into highly ordered structures in the developing brain. During brain development, neural stem cells generate a large number of neurons and glia of different fates at appropriate points in time; the framework and size ofthe 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 and epigenetic programs, by which behaviors ofneural stem cells are controlled and brain development is governed.
Drosophila neural stem cells, called neuroblasts, provide an excellent model system for the investigation of fundamental aspects of asymmetric division, a process essential to the generation ofcells of divergent type during proliferation. We have been investigating mechanisms controlling
asymmetric divisions, including the cell polarity and spindle orientation. We also extend our research scope to understand how neurogenesis is controlled in tissue space depending on the environments that surround the nervous system. We recently identified an extrinsic mechanism that
controls the orientation of division (cell polarity) 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 thatin invertebrates. Neural stem cells typically adopt three states—proliferative (symmetrically dividing), neurogenic (asymmetrically dividing), and resting—and undergo transitions among the states,on which the basic organization of the brain depend. We are investigating mechanisms that determine the individual states of neural stem cells, and control transitions between states in mouse as
well as mechanisms for generating neural progenitor cell diversity (see figure). We recently discovered a novel transition in the division mode in the developing mouse cortex from radial glia (typicalneural stem cells with the epithelial structure) to translocating neural stem cells, basal radial glia
(Shitamukai et al., 2011), which become a major population of neural stem cells in mammals withgyrencephalic brains, such as primates and ferrets. We are investigating the mechanisms that underlie the formation, maintenance, and expansion of these neural stem cells, by using model micethat produce large numbers of basal radial glia as well as ferrets.

Staff
Research Specialist

Keiko MUGURUMA
Atsunori SHITAMUKAI
Special Postdoctoral Researcher

Shigeki YOSHIURA
Research Scientist

Ikumi FUJITA
Daijiro KONNO
Yuji TSUNEKAWA

Visiting Researcher

Wu QUAN

Visiting Scientist

Yoshihito ISHIDA
Taisuke KADOSHIMA
Ayano KAWAGUCHI
Tomoko KITA
Yoichi KOSODO
Technical Staff

Chiaki KISHIDA
Ayaka NISHIYAMA
Taeko SUETSUGU
Yuko TAKEUCHIRyo YOSHIDA
Junior Research Associate

Shun MASE
Atsushi SHIRAISHI
Student Trainee

Kalyn KAWAMOTO
Yuki KOBAYASHI
Raymond TERHUNE-KUNIKANE
Fumiya KUSUMOTO
Yoko WATANABE
Part-Time Staff

Misato IWASHITA
Yoko OTSUKA
AssistantJunko ISHIGAI

Recent Publications
Okamoto et al. Cell cycle–independent transitions in temporal identity of mammalian neuralprogenitor cells. Nat Commun. In press.
Matsuzaki F, and Shitamukai A. Cell divisionmodes and cleavage planes of neural progenitors during mammalian cortical development.
Cold Spring Harb Perspect Biol. 7.a015719(2015)
Klotz L, et al. Cardiac lymphatics are heterogeneous in origin and respond to injury. Nature.
522. 62-67 (2015)
Kasahara K, et al. Ubiquitin-proteasome system
controls ciliogenesis at the initial step of axoneme extension. Nat Commun 5.5081 (2014)

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.

Mora-Bermúdez F, et al. Specific polar subpopulations of astral microtubules control spindleorientation and symmetric neural stem cell division. Elife. doi: 10.7554/eLife.02875 (2014)
Pilz GA, et al. Amplification of progenitors in the
mammalian telencephalon includes a new radial
glial cell type. Nat Commun 4.2125 (2013)55

Lung Development

http://www.cdb.riken.jp/en/research/laboratory/morimoto.html

Team Leader

Mitsuru MORIMOTO Ph.D.
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 School of Medicine in St. Louis to work with Dr. Raphael Kopan, where
he extended his research to lung organogenesis. He returned to the National Institute of Genetics in 2010, and was appointed Team Leader at the RIKEN CDB in 2012.

Staff
Research Scientist

Keishi KISHIMOTO
Masafumi NOGUCHI
Student Trainee

Yuki KIKUCHI

Technical Staff

Chisa MATSUOKA
Part-Time Staff

Akira YAMAOKA
Assistant

Yuka NODA

Recent Publications
Noguchi M, et al. Directed migration of pulmonary neuroendocrine cells toward airway
branches organizes the stereotypic location ofneuroepithelial bodies. Cell Rep 13.2679-2686(2015)
Morimoto M, et al. Different assemblies ofNotch receptors coordinate the distribution of
the major bronchial Clara, ciliated and neuroendocrine cells. Development 139.4365-73 (2012)
Morimoto M, et al. Canonical Notch signaling in
the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate. J Cell Sci
123.213-24 (2010)
Demehri S, et al. Skin-derived TSLP triggers
progression from epidermal-barrier defects toasthma. PLoS Biol 7.e1000067 (2009)
Morimoto M. and Kopan R. rtTA toxicity limits
the usefulness of the SP-C-rtTA transgenic
mouse. Dev Biol 325.171-8 (2009)
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 the arrangement 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 theseorganogenetic 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, enabledby the functions of specialized cells. The development of such organs relies on the coordinatedactivities of both epithelial and mesenchymal tissue types, which arise from tissue-specific populations of stem cells in the developing embryo. The epithelial tissues of the conducting airways
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 theirrespective cell numbers and distributions.
Despite its location in the body’s interior, the respiratory tract is constantly exposed to environmental factors, such as infection by viruses and bacteria, smoke, and chemical toxins, that may
damage the airway epithelium. This damage is rapidly repaired by regenerating epithelial cells
supplied by somatic stem cells in the adult tissue, and as in development, the numbers and distribution 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/niche tissuecomplexes 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

56

Reconstructed 3D-branching bronchiole on PC

Position of neuroendocrine cells within the entire geometricarchitecture of the airways.

Growth Control Signaling
http://www.cdb.riken.jp/en/research/laboratory/nishimura.html

Team Leader

Takashi NISHIMURA Ph.D.
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 same laboratory before moving to the Institute of Molecular Biotechnology
in Vienna, Austria in 2006 to pursue further postdoctoral work in Jürgen Knoblich’s group. He
returned to Japan in 2009, working briefly as a visiting scientist at the RIKEN Center for Developmental Biology prior to his appointment as Team Leader in July of the same year.

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

Staff
Research Scientist

Naoki OKAMOTO
Tetsuo YASUGI

Research Associate

Kota BANZAI

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 timeremains 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 capableof adapting their growth to fluctuating nutritional states through metabolic regulation. Therefore,
linking the nutrient sensing system to an endocrine signaling network allows organisms to control
the timing of cell proliferation and differentiation.
Our team’s research aims to shed light on the molecular basis for growth control and developmental timing at the cellular and tissue/organ level using Drosophila as a model system. In particular, we are interested in addressing the following questions: 1) how do organisms adapt their
growth program to changes in energy needs and states; 2) what are the molecular mechanismsthat 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 insulin-like peptides (Dilps).
Members of the insulin family of peptides have conserved roles in the regulation of growth andmetabolism 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 and developmental timing at the organismallevel. We described the first demonstration of the glia-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.

Visiting Scientist

Ken-ichi HIRONAKA
Technical Staff

Takayuki YAMADA
Part-Time Staff

Okiko HABARA
Kanako HIGUCHI
Hitomi KUBO
Noriko NISHIMURA
Tomomi OKUGOUCHI
Junko SHINNO

Recent Publications
Okamoto N, et al. Signaling from glia and cholinergic neurons controls nutrient-dependent
production of an insulin-like peptide for Drosophila body growth. Dev Cell 35.295-310 (2015)
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 protein with 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 and PAR-3. Dev Cell 13.15-28 (2007)

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

57

Pluripotent Stem Cell Studies
http://www.cdb.riken.jp/en/research/laboratory/niwa.html

Team Leader

Hitoshi NIWA M.D., Ph.D.
Hitoshi Niwa received his M.D. from Nara Medical University in 1989, and his Ph.D. in medical
physiology in 1993 from the Kumamoto University Graduate School of Medicine for his work ingene trap methods. From 1993 to 1994, he worked as a research associate in the Departmentof Developmental Genetics at the same university, before taking a postdoctoral fellowshipwith Austin Smith at the University of Edinburgh Centre for Genome Research. He returned toJapan 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 theLaboratory for Pluripotent Stem Cell Studies in October 2009.

Staff
Research Scientist

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

Mariko YAMANE
Visiting Scientist

Hiroki URA

Technical Staff

Kumi MATSUURA
Azusa NOMA
Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI
AssistantMiho SAKURAI

Recent Publications
Yamane, M., Fujii, S., Ohtsuka, S. and Niwa,
H.: Zscan10 is dispensable for maintenance ofpluripotency in mouse embryonic stem cells.
Biochem Biophys Res Commun 468.26-831(2015)

Pluripotency is a term used to describe the ability of certain types of stem cells that are capableof giving rise to at least one type of cell from all three germ layers—endoderm, mesoderm andectoderm. Stem cells are also characterized by their ability to produce copies of themselves withsimilar differentiative potential, as well as more specialized cells with different properties throughdifferentiation. 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 maintain pluripotency.
At the same time, the network should have the sensitivity to signals that induce differentiation.
How can the transcription factor network satisfy these contradictory requirements? What is theprinciple behind the structure of the transcription factor networks governing developmental processes? We are now trying to answer these questions by studying ES cells and the changes they
undergo in their differentiation toward trophoblast stem cells, extraembryonic endoderm cells, andprimitive ectoderm cells.

Ohtsuka, S. and Niwa, H. The differential activation of intracellular signaling pathways confers
the permissiveness of embryonic stem cell
derivation from different mouse strains. Development 142.431-437 (2015)
Fujii S. et al. Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells. Sci Rep,
16;5;9146 (2015)
Adachi K, et al. Context-dependent wiring ofsox2 regulatory networks for self-renewal ofembryonic and trophoblast stem cells. Mol Cell
52.380-92 (2013)
Niwa H, et al. A parallel circuit of LIF signalling
pathways maintains pluripotency of mouse ES
cells. Nature 460.118-22 (2009)
Niwa H. How is pluripotency determined andmaintained? Development 134.635-46 (2007)

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

58

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

Early Embryogenesis
Senior Investigator

Guojun SHENG Ph.D.
Guojun Sheng received his B.S. in microbiology from Fudan University in Shanghai, China in
1990. He entered the Rockefeller University the next year, attending the summer course inembryology at the Woods Hole Marine Biological Laboratory in 1996 and receiving his Ph.D.
under Claude Desplan in 1997. He spent the period from 1998 to 2000 in Claudio Stern’s laboratory in Columbia University, moving with Dr. Stern to University College London in 2001. He
was appointed Team Leader at the RIKEN CDB in 2004.
The Early Embryogenesis lab closed in September 2015.
Dr. Sheng is now affiliated with Kumamoto University.

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

Staff
Research Scientist

Cantas ALEV
Yukiko NAKAYA
Wei WENG
Technical Staff

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

Hiroki NAGAI
Erike Widyasari SUKOWATIYuPing WU
Part-Time Staff

Anna Barbara WRABELAssistant

Noriko HIROI
Fumie NAKAZAWA

Recent Publications
Mak S-S, et al. Characterization of the finch
embryo supports evolutionary conservation of
the naive stage of development in amniotes.
Elife. doi: 10.7554/elife.07178 (2015)
Nagai H, et al. Cellular analysis of cleavagestage chick embryos reveals hidden conservation in vertebrate early development. Development (2015)
Weng W. and Sheng G. Five transcription
factors and FGF pathway inhibition efficiently
induce erythroid differentiation in the epiblast.
Stem Cell Reports 2.262-70 (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 gastrulationand streak formation reveals a morphogenetic
unity in vertebrate mesoderm induction. Development 140.2691-6 (2013)

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

Alev C, et al. Transcriptomic landscape of theprimitive streak. Development 137.2863-74(2010)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.

59

Retinal Regeneration

http://www.cdb.riken.jp/en/research/laboratory/takahashi.html

Project Leader

Masayo TAKAHASHI M.D., Ph.D.
Masayo Takahashi received her M.D. in 1986, and her Ph.D. in 1992 from Kyoto University. After anassistant professorship in the Department of Ophthalmology, Kyoto University Hospital, she moved
to the Salk Institute in 1995, where she discovered the potential of stem cells as a tool for retinal
therapy. She returned to Kyoto University Hospital in 1997, and was appointed associate professor
at the Translational Research Center in the same hospital in 2001. She joined the CDB as a teamleader of the Lab for Retinal Regeneration in 2006. In 2013, her team launched a pilot clinical studyof autologous iPS cell-derived RPE cell sheets for exudative aged-related macular degeneration(AMD), and performed the first RPE cell sheet graft transplantation in Sept. 2014. Her clinical specialty is retinal disease–specifically, macular diseases and retinal hereditary diseases. Her aim is tounderstand these diseases at a fundamental level and develop retinal regeneration therapies.

Staff
Deputy Project Leader

Michiko MANDAI
Sunao SUGITA

Research Scientist

Jun KANEKO
Hiroyuki KITAJIMA
Naoshi KOIDE
Takesi Hoyos MATSUYAMA
Chikako MORINAGA
Akishi ONISHIGenshiro SUNAGAWA

The retina has been called the “approachable part of the brain,” owing to its relatively simple
structure and its location near the body surface, and for these reasons it serves as a useful andexperimentally amenable model of the central nervous system. Until very recently, it was thoughtthat in adult mammals the retina was entirely incapable of regenerating, but we now know that atleast new retinal neurons can be generated after being damaged. This has opened up new hopethat the ability to regenerate neurons and even to reconstitute the neural network may be retainedin the adult retina. We are now exploring the exciting prospect that, by transplanting cells fromoutside of the retina or by regeneration from intrinsic progenitor cells, it may one day be possible
to restore lost function to damaged retinas.

Research Associate

Yuuki ARAI
Akihiro TACHIBANATechnical Staff

Momo FUJII
Kanako KAWAI
Tomoyo HASHIGUCHI
Naoko HAYASHI
Yukako HIRAO
Ayumi HONO
Kyoko ISEKI
Hiromi ITOMichiru MATSUMURA
Noriko SAKAI
Yumiko SHIBATA
Junki SHO
Motoki TERADA
Kazuko TSUJIMOTO
Chikako YAMADA
Part-Time Staff

Shoko FUJINO

Recent Publications

Our research into retinal regeneration seeks to achieve clinical applications by developing methods
for inducing stem cells or embryonic stem cells to differentiate into retinal neurons and pigment
epithelial cells in sufficient quantities for use in the treatment of patients suffering from conditions
in which such cells have been damaged or lost. We must also ensure that such cells establish viable grafts upon transplantation and induce the reconstitution of functional neural networks. We
also hope to develop means of promoting true regeneration by activating endogenous stem cells
to replace cells lost to trauma or disease, and thus repair damaged tissues. Access to a broad
spectrum of developmental biological research information will be key to achieving these goals,
and we appreciate the opportunities for exchange that are available working at the RIKEN CDB.
Therapeutic applications cannot be developed from basic research alone; the clinical approach athorough understanding of the medical condition to be treated is equally important. For conditions
such as retinitis pigmentosa, even the successful transplantation of cells in animal models may notnecessarily 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 basedon both a strong foundation in basic research and solid clinical evidence.

Shirai H, et al. Transplantation of human embryonic stem cell-derived retinal tissue in two
primate models of retinal degeneration. Proc
Natl Acad Sci U S A 113. E81-E90 (2016)
Sugita S, et al. Inhibition of T-cell activation by
retinal pigment epithelial cells derived from induced pluripotent stem cells. Invest Ophthalmol
Vis Sci. 56(2).1051-62 (2015)
Assawachananont J, et al. Transplantation of
Embryonic and Induced Pluripotent Stem CellDerived 3D Retinal Sheets into Retinal Degenerative Mice. Stem Cell Reports 2.662-74 (2014)
Kamao H, et al. Characterization of humaninduced pluripotent stem cell-derived retinal
pigment epithelium cell sheets aiming for clinicalapplication. Stem Cell Reports 2.205-18 (2014)
Jin Z. B, et al. Modeling retinal degeneration
using patient-specific induced pluripotent stem
cells. PLoS One 6.e17084 (2011)
Osakada F, et al. Toward the generation of rod
and cone photoreceptors from mouse, monkey
and human embryonic stem cells. Nat Biotechnol 26.215-24 (2008)

60

iPSC-derived retinal pigment epithelium cells and cell sheet (generated forclinical use)

Mouse iPSC-derived photoreceptors aremorphologically able to form synapses after
transplantation into host model animals.

Human iPSCs (immunostained red:
SSEA-4, blue:DAPI)Cell Adhesion and Tissue Patterning

http://www.cdb.riken.jp/en/research/laboratory/takeichi.html

Team Leader

Masatoshi TAKEICHI Ph.D.
Masatoshi Takeichi is Team Leader of the Laboratory for Cell Adhesion and Tissue Patterning.
He completed the B.Sc. and M.S. programs in biology at Nagoya University before receiving
a doctorate in biophysics from Kyoto University in 1973. After attaining his Ph.D., he took aresearch fellowship at the Carnegie Institution Department of Embryology under Richard Pagano. He then returned to Kyoto University, attaining a professorship in the Department of Biophysics (1986–1999), before becoming professor in the Department of Cell and Developmental
Biology in the Graduate School of Biostudies at the same university. He assumed the directorship of the CDB in 2000, and resigned from this position in 2014.

Animal cells organize into tissues with complex architecture. Our lab is exploring the molecularmechanisms by which individual cells assemble into a tissue-specific multicellular structure, suchas epithelial sheets and neural networks. We are also interested in the molecular basis of how
tissue architecture is disrupted during carcinogenesis, a process that is thought to accelerate
the metastasis of cancer cells. For these studies, we are focusing on the roles played by cell-celladhesion and recognition molecules, the cadherin family of adhesion molecules in particular, as
these are known to be indispensable for tissue construction. Our current studies are divided into
three categories:

Staff
Research Scientist

Shuichi HAYASHI
Shoko ITO
Toshiya KIMURA
Anna PLATEK
Varisa PONGRAKHANANON
Takuji TANOUE
Mika TOYA
Vassil VASSILEV
Visiting Scientist

Tamako NISHIMURA

1) Cell-cell adhesion is a dynamic process, and this nature of cell-cell adhesion is implicated invarious 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, inmodulating cell assembly. We are therefore studying the molecular mechanisms underlying thecrosstalk between cadherins and such cytoskeletal systems, and their roles in epithelial junction
formation.

Technical Staff

Sylvain HIVER
Yoko INOUE
Miwa KAWASAKI
Hiroko SAITOAssistantMutsuko AISO-WATANABE

Recent Publications
2) A second area of interest to our lab is to gain a better understanding of how the cell-cell adhesion machinery contributes to animal morphogenesis. Using mouse embryos, we are analyzing
the roles of cadherins and associated proteins in various morphogenetic processes, including
neural crest migration. We are also investigating the roles of members of the cadherin superfamily
known as protocadherins, deficiencies of which have been implicated in human brain disorders.
Through these studies, we expect to gain deeper mechanistic insights into the ways by whichcells build the elaborate structures of the animal body.
3) In addition, we have been analyzing the functions of microtubule minus end-associated proteins, Nezha/CAMSAPs. These proteins regulate microtubule assembly patterns, centrosomal
function, and organelle positioning. We are exploring the roles of these molecules in cellular morphogenesis, such as polarized epithelial formation and axon growth, with the aim of uncoveringnovel functions of non-centrosomal microtubules.

Toya M., et al. CAMSAP3 orients the apical-tobasal polarity of microtubule arrays in epithelial
cells. Proc Natl Acad Sci U S A 113.332-337(2016)
Tsukasaki Y, et al. Giant cadherins Fat andDachsous self-bend to organize properly
spaced intercellular junctions. Proc Natl Acad
Sci U S A 111.1601-6 (2014)
Hayashi S, et al. Protocadherin-17 mediates
collective axon extension by recruiting actin
regulator complexes to interaxonal 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 of noncentrosomal microtubules. Proc Natl
Acad Sci U S A 109.20029-34 (2012)
Nishimura T., et al. Planar cell polarity links axesof spatial dynamics in neural-tube closure. Cell
149.1084-97 (2012)

A pair of U251 cells in contact with one another, triple-immunostained for Protocadherin17-EGFP (green), the WAVE complex protein Abi-1 (magenta) and DNA (blue). Protocadherin17
recruits the WAVE complex to cell-cell contact sites, and convert them into a motile structure. Cell edges of this structure
actively move, contributing to collective cell migration.

Microtubules (green) and CAMSAP3 (red) in intestinal epithelial cells.
CAMSAP3 localizes at the apical cortex of the cells, and tether microtubules to this site through its binding to their minus ends (left). WhenCAMSAP3 is knocked out, the longitudinal arrays of microtubules are
disrupted (right).

61

Organogenesis and Neurogenesis
Team Leader

Masatoshi TAKEICHI Ph.D.
This laboratory was led by Yoshiki Sasai M.D. Ph.D. until August 2014, after which Masatoshi Takeichi served asinterim leader. The laboratory closed in March 2015.

Recent Publications
Sakaguchi H, et al. Generation of functional hippocampal neurons from self-organizing humanembryonic stem cell-derived dorsomedial telencephalic tissue. Nat Commun 6, 8896 (2015)
Ohgushi M, et al. Rho-signaling-directed YAP/TAZ activity underlies the long-term survival andexpansion of human embryonic stem cells. Cell
Stem Cell 17, 448-461 (2015)
Kuwahara A, et al. Generation of a ciliary
margin-like stem cell niche from self-organizing
human retinal tissue. Nat Commun 6, 6286
(2015)
Muguruma K, et al. Self-organization of polarized cerebellar tissue in 3D culture of humanpluripotent stem cells. Cell Rep 10. 537-550(2015)

The complexity of the fully formed brain defies description, yet this organ arises from a nondescript clump of cells in the embryo. The specification of the dorsal-ventral (back-belly) axis issignificant in neural development in that the central nervous system forms on the dorsal side of
the body in all vertebrate orders. This process is dictated by the effects of a number of signaling
factors that diffuse from organizing centers and direct dorsal ectoderm to maintain a neural fate.
These molecules, which include Noggin, Chordin, Follistatin and their homologs, participate inelaborate signaling networks in which factors collaborate and compete to initiate the embryonic
nervous system.
Using the African clawed frog, Xenopus laevis, as a model in molecular embryological studies, thegroup was 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 focusedon molecules that operate at early stages in embryonic development to lay down a complex ofpermissive, instructive and inhibitory patterns that determines the growth and differentiation of thebrain in its earliest stages of development.
The group was also active in 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, this approach has potential for
applications in the treatment of neurodegenerative disorders, such as Parkinson disease. Usinga system developed by the lab, they have succeeded in inducing mouse and primate embryonicstem (ES) cells to differentiate into a range of specialized neuronal types. The application of similar
techniques to human ES cells represents a field of study that, although it remains at quite an earlystage, shows immense clinical promise.
By studying very early neurogenesis and the mechanisms of neuronal differentiation, the lab
aimed 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 the embryonic 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

62

Self-organized formation of optic cup fromhuman ES cells

Organ Regeneration

http://www.cdb.riken.jp/en/research/laboratory/tsuji.html

Team Leader

Takashi TSUJI Ph.D.
Takashi Tsuji received his Master’s degree from Niigata University in 1986, and after working in
the pharmaceutical industry for three years, returned to complete his doctorate at Kyushu University, and he received his doctorate in 1992 from Niigata University. He conducted researchat Niigata University from 1992 to 1994 before moving to serve as researcher and then seniorscientist at JT Inc. From 2001, he moved to Tokyo University of Science, and in 2007 he wasappointed a full professor in the same university. During his academic career, he received numerous prestigious grants and awards, and participated 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 specific organs. Most organs
emerge from primordia induced by interactions between epithelial and mesenchymal tissue and,following organ-specific morphological changes, develop into functional structures.
Our group is working to gain a more complete understanding of the roles of epithelial-mesenchymal 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, hair follicles and endocrine tissue,
such as salivary glands, and shown that they can functionally integrate with surrounding tissue following transplantation into adult mice. By recapitulating organogenetic fields as seen in the early
embryo to steer the self-organized formation of 3D tissue-like structures from pluripotent stem
cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), we seek
both to elucidate the mechanisms by which such fields induce organogenesis and to develop newtechnologies 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 on these fundamental studies, we further seek to conduct applied research towards the development of technologies
for use in therapeutic organ regeneration.

Staff
Collaborative Scientist

Miho OGAWA
Koh-ei TOYOSHIMA
Research Scientist

Kyosuke ASAKAWA
Mamoru ISHII
Jun ISHIKWA
Ryohei MINAMIDE
Chikafumi OZONETechnical Staff

Yukiko MORIOKA
Yumi NISHIMURA
Azusa NOMA
Hiroko SASAKIAya SHIOKAWA
Miki TAKASEStudent

Kei SAKAKIBARA
Assistant

Asako ANDO
Mayumi MUROFUSHI

Recent Publications
Chikafumi Ozone, et al. Functional anterior
pituitary generated in self-organizing cultureof human embryonic stem cells, Nat Commun
7.10351 (2015)
Ogawa M, et al. Functional salivary gland regeneration by transplantation of a bioengineeredorgan 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 ofstem cells and their 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 106.1347580 (2009)
Nakao K, et al. The development of a bioengineered organ germ method. Nat Methods
4.227-30 (2007)

Bioengineered tooth

Bioengineered salivary gland

Bioengineered hair follicle

63

Epithelial Morphogenesis
http://www.cdb.riken.jp/en/research/laboratory/wang.html

Team Leader

Yu-Chiun WANG Ph.D.
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 the laboratory 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 cephalic furrow and dorsaltransverse folds during Drosophila gastrulation. He was appointed Team Leader of the Laboratory for Epithelial Morphogenesis at the CDB in October 2013.

Staff
Research Scientist

Niraj K. NIRALA
Technical Staff

Mustafa SAMI
Michiko TAKEDAInternational Program Associate

Anthony ERITANO
Assistant

Yuko FUJIYAMA

Recent 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 the extent 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 ofepithelial folding. Nature 484.390-3 (2012)
Wang Y. C. and Ferguson E. L. Spatial bistability of Dpp-receptor 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)

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 abroad 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 andremodeled. Although a general theme has emerged whereby cytoskeletal elements control thecell shapes and 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 the formation of three-dimensional epithelial structures. Our long-term goal is to comprehensively understand the mechanistic principles of tissuemorphogenesis in order to conceptualize the origin 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 a basal repositioningof 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 the canonical 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 that after 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 of cell invagination. Our ongoing work promises to identify genes and forces that sculpt
distinct morphological features.
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 process of designing novel imaging strategies that could be used to visualize mechanical forces and computationalalgorithms that reconstruct and quantify 4D cell shapes. Furthermore, we will launch a multidisciplinary, international collaboration that combines genetics, computational and 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 two epithelial(anterior and posterior) folds, shown herewith the mid-sagittal optical sections at
an early (A) and a late (B) stage (green,
adherensjunctions; magenta, plasmamembrane).

64

Reconstruction of 3D cell shape during
dorsal fold formation. EDGE4D software
was developed in collaboration with Dr.
Zia Khan (Univ. of Maryland). 3D reconstruction of cell shape was based on cell
membrane labeling. Immunolabeling ofadherens junctions (orange in A, B) defines the boundaries between the apicaland basal-lateral compartments (A’, B’).

2015 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 this work in public engagement, the CDB also organizes events for bringing scientists together outside of thelaboratory environment.

The 26th CDB Meeting: Mechanistic Perspectives ofMulticellular Organization
The 26th CDB Meeting, “Mechanical Perspectives of Multicellular Organization,” washeld September 8 to 9. The meeting was organized by a group of young researchers
primarily from the CDB, focusing on the theme of physics and mechanics involved intissue formation of multicellular organisms. Over 130 scientists and students with diverse backgrounds ranging from biology, medicine, physics, and engineering convenedat the CDB to take part in the two days of talks and discussions. Talks were given on
the themes of morphological dynamics, quantitative measurements of active passive
cell behaviors, in vitro constructions and controls of multicellular structures, and computational simulations of multicellular systems. Key note lectures were given by PierreFrançois Lenne, Frank Jülicher and Hiroshi Hamada, three leading scientists in this interdisciplinary research field.

CDB Retreat in Sasayama
The annual CDB Retreat was held September 29 and 30 in the mountains of Sasayama,
located in northern Hyogo Prefecture. Laboratory heads, research scientists, student
trainees, technical staff and members of the administration participated in this closed
meeting to share and discuss the latest developments in their work in addition to getting
better acquainted with each other outside normal lab settings. The two-day program
featured talks by recent appointees, research scientists and students, as well as special
lectures by Chikara Furusawa from RIKEN QBiC and CDB alumnus Yoshiko Takahashi(Kyoto University).

Open House 2015
The CDB opened its doors to the public as part of the RIKEN Kobe Open House on
Saturday, October 24. Over 1,600 people visited the CDB to see and learn firsthandabout the latest research in development and regenerative biology. All of the laboratories within the CDB took part in the Open House in some capacity. Six CDB laboratories
welcomed visitors to take a peek inside their laboratories, and see sample specimens
related to the research carried out in the respective laboratories. Another popular event
was the model organism exhibit where a variety of organisms from chicken and frogs,
to flies and fish used in developmental biology research were on display. New this year
was a talk show featuring a panel of CDB and QBiC scientists, as well as a booth wherevisitors could try carrying out actual experimental techniques used in the lab. For thoselooking for a quiet spot, there was a book lounge showcasing books that were recommended by CDB scientists. This is the largest annual event held at the CDB that gives
an opportunity for scientists and the public to engage face-to-face.

65

2015 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.

CDB internship for undergraduate students
The CDB invited 29 undergraduate students from universities around Japan to spend
the first week of August as an intern working alongside scientists in various labs on the
CDB campus. The busy week included talks given by CDB scientists, and opportunities
to visit different labs, in addition to carrying out small-scale projects in the host labs. Theinternship wrapped up with a presentation session on the last day, where the interns
shared their findings and their interpretation of the results with the others. This is the
fourth year the internship has been organized.

Summer school for high school students
The ninth annual one-day summer school for high school students was held at the CDB
on August 4 and 6. This popular summer program includes a talk by a CDB scientist, alaboratory visit and scientific experiments. This year’s program was organized aroundthe topic of genes, with the students learning about how genes regulate development
and shape our bodies, as well as how individual differences arise from even smallchanges to a gene. The lab experiments were designed so the students could predict
their alcohol tolerance levels by analyzing their own DNA, extracted from cells collectedfrom their mouths.

Intensive lecture program for graduate students
The CDB maintains close ties with a number of graduate and medical schools in the
Kansai area, and hosts a two-day lecture program every year for graduate students
enrolled in partnering institutions. Lectures are given by laboratory heads and scientists
at the CDB, introducing the students to the research carried out at the Center. Close to
190 students took part in the event held on August 26 and 27. The program includes
exhibits of model organisms, and tours of labs and other facilities such as the researchaquarium, electron microscopy room, and gene sequencing unit.

High school teacher workshop
A weekend workshop for high school biology teachers, held on October 3 and 4, wasco-organized by the CDB and the Japanese Society for Developmental Biologists(JSDB). In its eighth year, the workshop aims to help enrich biology education in the highschool classroom by giving teachers a refresher course in developmental biology basics, as well as giving them an opportunity to learn actual experimental techniques usedin the field. The theme this year was the ‘organizer’, a region of early stage embryos that
influences development of other parts of the embryo, and the program included a talkby Hidehiko Inomata, head of Laboratory for Axial Pattern Dynamics, on the recent developments in organizer-related research, a tour of his laboratory and a practical session
with experiments using embryos of chicken and African clawed frogs (Xenopus). The
program also introduced a few experiments that could be adapted for a classroom setting.
As an extension of this workshop, several teachers who took part in this program had
the opportunity to bring their own students to a special tutorial organized at the CDB in
December and teach them to carry out similar experiments.

66

2015 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 careers ofmany 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.

AwardeePositionLaboratory

Award

Organization

Masayo Takahashi

Project Leader

RetinalRegeneration

The Johnson & Johnson
Innovation Award

The Japanese Society for
Regenerative Medicine

Masayo Takahashi
Michiko Mandai
Chikako Morinaga
Noriko Sakai
Chikako Yamada

Project Leader
Deputy Project Leader
Researcher
Technical Staff
Technical Staff

RetinalRegeneration

MEXT Prize (Science andTechnology Prize)

The Ministry of Education,
Culture, Sports, Scienceand Technology (MEXT)

Takeshi ImaiTeam Leader

Sensory Circuit
Formation

MEXT Prize (The Young
Scientist’s Prize)

The Ministry of Education,
Culture, Sports, Scienceand Technology (MEXT)

Masayo Takahashi

Project Leader

RetinalRegeneration

The Award for Academic
Startups 2015

Japan Science andTechnology Agency

Masayo Takahashi

Project Leader

RetinalRegeneration

The Ogawa-Yamanaka
Stem Cell Prize

Gladstone Institutes, USA

Hirohisa Kyogoku

Visiting Scientist

Chromosome
Segregation

The 108th SRD Meeting,
Outstanding Presentation
Award (Oral Presentation)

The Society for
Reproduction andDevelopment

Masayo Takahashi

Project Leader

RetinalRegeneration

Hyogo Prefecture Science
Prize

Hyogo Prefecture

Masayo Takahashi

Project Leader

RetinalRegeneration

The Soroptimist JapanFoundation’s DreamAward

The Soroptimist JapanFoundation

Masayo Takahashi

Project Leader

RetinalRegeneration

Kyoto Akebono Prize

Kyoto Prefecture

Hirohisa Kyogoku

Visiting Scientist

Chromosome
Segregation

ARBS 12th Annual
Conference Excellent
Poster Presentation Award

Asian Reproductive
Biotechnology Society

Masayo Takahashi

Project Leader

RetinalRegeneration

Nikkei Woman:
Woman of the Year 2016

Nikkei WOMAN,
Nikkei BP

Hirohisa Kyogoku

Visiting Scientist

Chromosome
Segregation

BMB2015 Award for
Outstanding Presentation
by Young Scientist

Biochemistry andMolecular Biology

67

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

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

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

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

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

Kobe at sunset. Photography: Shigekazu SHINYARIKEN in Kobe
The RIKEN Center for Developmental Biology was the first research center established by RIKEN in the city of Kobe, only thesecond in all of Western Japan. In 2013, RIKEN made sweepingchanges to its organizational structure, which eliminated regional
administrative organizations referred to as Institutes (including theformer RIKEN Kobe Institute, of which CDB was a member), andconferred greater autonomy to the individual research centers.
These are now grouped by proximity into a number of Branches
around the country.
The RIKEN Kobe Branch is now home to several other research
centers, including the RIKEN Center for Life Science Technologies(CLST), and the Osaka-based RIKEN Quantitative Biology Center(QBiC). The CLST was established in 2013 through the fusion ofresearch programs at the former RIKEN Yokohama and Kobe
Institutes, and focuses on technology research and development
to support the medical and pharmaceutical sectors, while QBiC
focuses on measurement, analysis, and modeling technologies
and techniques to model cell dynamics.
Kobe is also home to the RIKEN High-Performance Computer
Infrastructure Program for Computational Life Sciences (HPCI)and the RIKEN Advanced Institute for Computational Sciences

70(AICS). AICS and HPCI are both associated with the national K
Supercomputer project, working respectively to generate cuttingedge scientific results and technological breakthroughs throughcollaboration and integration of computational and computer sciences, and to promote computational science and technology inthe life sciences.

Administrative support at RIKEN Kobe and CDB
Kobe Branch

RIKEN Kobe Administrative Division
General Affairs
Human Resources
Finance
Information Networks Office – Kobe Branch
RIKEN Kobe Safety Center
Developmental Biology Planning Office
The Planning Office coordinates funding and external review activities, as well as providing support
for international communications, scientific meetings, and the Center’s non-Japanese staff.

Life Science Technologies Planning Office
Quantitative Biology Planning OfficeCenter forLife Science Technologies (CLST)
The RIKEN Center for Life Science Technologies (CLST) works todevelop key technologies for breakthroughs in medical and pharmaceutical applications, and to conduct groundbreaking R&D forthe next-generation of life sciences. The CLST aims to promoteresearch on biomolecules and into life science technologies,
focusing on designing molecular structures at the atomic level,
manipulating molecular function at the cellular level, and tracing
molecular dynamics at the whole-body level.

2015 CDB Budget
Research
expenses

Overhead andadministration

213

430

Total: 1,582
Personnel(million yen)

938

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

In addition to the dedicated funds outlined above, individual labs
and investigators are encourages to compete for external funding as well, from sources such as the Japan Society for the Promotion of Science (JSPS), the Japan Science and Technology
Agency (JST), and other governmental and private sources.
These external funds represent a significant component of theCenter’s total funding every year.
Grant-in-Aid for
Scientific Research(MEXT)

Others (Foundations,
Private Industry, etc. )

357

96

Kobe Administrative Services
Since the reform of the RIKEN organization in early 2013, theKobe Administrative Division has been dedicated to providing
core administrative services to the entire RIKEN Kobe Branch. Its
main areas of responsibility are general affairs, facilities management, human resources, contracts, finances and accounting. The
Information Networks Office maintains both network access andmultiple intranet services of the Kobe Branch. Within the CDB,
the Developmental Biology Planning Office coordinates important
activities including budget and funding management, and administrative support for laboratory performance reviews and contract
renewals. The Library Office manages the CDB research literaturecollections and interlibrary loans.

Safety Center
The Kobe Safety Center provides training and supervision for lab
and workplace safety, implements and monitors disaster prevention measures, and educates staff on and ensures complianceswith national and institutional regulations governing laboratory
practice and technology. The office also handles all administrative
affairs of the Institutional Review Board, which meets regularly
to review and discuss investigations with potential ethical, legal,social or public health and safety implications prior to their implementation.

Total: 1,273

Grants(Other
Ministries)

54

Grants (JST)766(million yen)2015 CDB Staff
Laboratory heads
Deputy leaders
Research scientists
Research associates
Technical staff
Assistants
Visiting scientists
Student trainees
Part-time staff
Other

18
2
49
7
59
14
100
22
33
7

Total

311

Kobe Branch Administration

71

71

2015 CDB SymposiumTime in Development
March 23-25, 2015
The RIKEN CDB hosted its thirteenth annual symposium on
March 23 to 25 in the CDB auditorium. This year’s symposium
was themed, “Time in Development,” and drew an audience of
144 scientists and students from over 10 different countries totake part in a program of talks and discussions exploring the different contexts of time in relation to development.
The three-day program featured over 30 invited and selected
talks and more than 50 poster presentations that covered a
range of topics including temporal cell fate, oscillation and patterning, developmental timing, life span, and evolutionary time.
The co-organizers of this year’s symposium were Carina Hanashima and Takashi Nishimura from the CDB, Shigeru Kuratanifrom RIKEN and Claudio Stern (University College London, UK).
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.

72

Session 1: Temporal Cell Fate

Yukiko Gotoh (The University of Tokyo, Japan)
Carina Hanashima(RIKEN Center for Developmental Biology, Japan)

Fengzhu Xiong (Harvard Medical School, USA)
Claude Desplan (New York University, USA)
Stefan Thor (Linköing University, Sweden)
Connie Cepko(Harvard Medical School and Howard Hughes Medical Institute, USA)

Session 2: Oscillation and Patterning

Olivier Pourquié(Harvard Medical School and Brigham and Woman’s Hospital, USA)

Erik Clark (University of Cambridge, UK)
Taijiro Yabe (National Institute for Basic Biology, Japan)
Ryoichiro Kageyama (Kyoto University, Japan)
Takao Kondo (Nagoya University, Japan)
Berta Verd (Centre for Genomic Regulation (CRG), Spain)
Andrew C. Oates (MRC National Institute for Medical Research, UK)
Koichiro Uriu (RIKEN, Japan)
Claudio D Stern (University College London, UK)2016 CDB Symposium

Size in Development:
Growth, Shape and Allometry
March 28-30, 2016

Session 3: Developmental Timing

Patrick H. O’Farrell (University of California, San Francisco, USA)
Takashi Saitou (Ehime University Hospital, Japan)
Chieh Chang (University of Illinois at Chicago, USA)
Victor Ambros (University of Massachusetts Medical School, USA)
Yun-Bo Shi(Eunice Kennedy Shriver National Institute of Child Health and Human
Development (NICHD), NIH, USA)

H. Fred Nijhout (Duke University, USA)
Michael B. O’Connor (University of Minnesota, USA)
Ryusuke Niwa (University of Tsukuba, Japan)
Takashi Nishimura (RIKEN Center for Developmental Biology, Japan)

The fourteenth annual symposium “Size in Development: Growth, Shape and Allometry” will be held on
March 28-30, 2016 in the CDB Auditorium.
This symposium will focus on the question of organismal size control. The size of animals and plants varies
greatly among species, and sometimes within species
as well. Despite this variability, individuals maintainremarkably constant shapes through the poorly understood process of scaling, and deviations in shape control often lead to pathological conditions. Furthermore,allometric conversion of organ shapes is a major driverof evolutionary diversification. Recent advances in the
study of growth control and morphogen functions, aswell as quantitative approaches of tissue shape control
have brought us a fresh opportunity to revisit theselong-standing questions.

Session 4: Life Span

Invited Speakers

Ayumi Nakamura (University of North Carolina at Chapel Hill, USA)
Shigeru Kuratani (RIKEN, Japan)

Ehab Abouheif(McGill University, Canada)

Alexander Aulehla
Session 5: Evolutionary Time

Ralf J. Sommer(Max Planck Institute for Developmental Biology, Germany)

Tatsuya Hirasawa (RIKEN, Japan)
Shigehiro Kuraku(RIKEN Center for Life Science Technologies (CLST), Japan)

Naoki Irie (The University of Tokyo, Japan)
Qi Zhou (University of California, Berkeley, USA)
Pavel Tomancak(Max Planck Institute of Molecular Cell Biology and Genetics, Germany)(EMBL Heidelberg, Germany)

Mary Baylies(Sloan-Kettering Institute for Cancer Research, USA)

Mototsugu Eiraku(RIKEN CDB, Japan)

Makoto Furutani-Seiki(University of Bath, UK)

Georg Halder(VIB Center for the Biology of Disease, Belgium)

Shigeo Hayashi(RIKEN CDB, Japan)

Iswar Hariharan(University of California, Berkeley, USA)

Mitsuyasu Hasebe(National Institute for Basic Biology, Japan)

Rebecca Heald(University of California, Berkeley, USA)

Hidehiko Inomata(RIKEN CDB, Japan)

David Kingsley(Stanford University, USA)

Mitsuru Morimoto(RIKEN CDB, Japan)

Elaine A. Ostrander(National Institutes of Health, USA)

Duojia Pan(Howard Hughes Medical Institute and Johns Hopkins
University School of Medicine, USA)

Stefano Piccolo(University of Padova, Italy)

Nadia Rosenthal(The Jackson Laboratory, USA)

Hiroshi Sasaki(Osaka University, Japan)

Richard Schneider(University of California, San Francisco, USA)James M. Wells(Cincinnati Children’s Hospital Medical Center, USA)

Yingzi Yang

(Harvard School of Dental Medicine, USA)73

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 750 such talks, in addition to numerous meetings, internal forums, andcolloquia. The following speakers presented seminars in the period from January to December 2015.

Date

74

Title

Speaker

01-13

Liquid like compartments in cells: Implications for polarity and disease

Anthony HYMAN

02-05

Mechanism and Reconstitution In Vitro of Mammalian Germ Cell Development

Mitinori SAITOU

02-23

Dynamic programming and reprogramming of the stomatal lineage of plants

Dominique BERGMANN

03-02

Autophagy: an intracellular degradation system

Noboru MIZUSHIMA

03-12

How to screen out liars

Yoshihiro TAGUCHI

04-13

Transition zone: The dramatic change of lung progenitors to terminallydifferentiated cells

Munemasa MORI

04-17

Practical application of Platanus genome assembler

04-20

Making and patterning the spinal cord: cells, morphogens and transcriptional
networks

James BRISCOE

05-25

Cell shape and morphogenesis: sub cellular and supracellular mechanisms

Maria LEPTIN

06-01

Symmetry breaking in mouse development

Takashi HIIRAGI

06-08

Reverse genetics in Xenopus tropicalis as a model for understanding human
genetic diseases involved in eye and brain formation

06-26

Modeling Electrodiffusion and Osmosis in Physiological Systems

07-22

Spatially asymmetric neuronal connectivity in motion-sensitive circuits

07-24

Dissecting alternative pathways and functions of the microRNA biogenesis
machinery in mammalian neurogenesis and neurodevelopmental disorders

07-29

Degeneration and regeneration of circuits in the vertebrate retina

08-18

Epigenetic reprogramming in cancer and energy metabolism

Mitsuyoshi NAKAO

08-20

In toto imaging reveals multi-scale mechanisms for robustness in patterningand morphogenesis

Sean MEGASON

08-21

Engineering hematopoietic stem cells from human pluripotent stem cells

Rei KAJITANI

Takuya NAKAYAMA

Yoichiro MORI

Keisuke YONEHARADavide DE PIETRI TONELLI

Rachel WONG

Ryohichi SUGIMURA

Date

Title

Speaker

08-25

Forming and transforming tubes in the mouse lung

10-19

Stem cell dynamics in the mouse testis

10-28

The Making of Blood Vessels through Sprouting Angiogenesis

Li-Kun PHNG

11-20

Gene and genome duplications, and vertebrate evolution

Yukuto SATO

11-30

Genome-wide remodeling of sperm epigenome upon fertilization

Azusa INOUE

11-30

Endogenous stem cells in lung repair and regeneration

Brigid HOGAN

12-03

Regulating mesoderm regionalization generates kidney organoids from humanpluripotent stem cells

Minoru TAKASATO

12-10

Noise, Regulation, and Cell-Cell Heterogeneity

Namiko MITARAI

12-15

Nail stem cell and its unique role in digit bone homeostasis and regeneration

Makoto TAKEO

12-16

Two-photon imaging of subthreshold membrane potential dynamics at cellular
resolution in vivo

Yuki BANDO

12-17

Extrinsic Regulation of Muscle Stem Cell Function

Jichao CHEN

Shosei YOSHIDAC. Florian BENTZINGER

75

About RIKEN
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The core component of RIKEN Research areshort, easy-to-understand ‘Research Highlight’articles explaining for a broad scientific audience a sampling of the latest research articles
published by RIKEN scientists.
http://www.riken.jp/en/research/rikenresearch/76

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RIKEN was founded in 1917 as a private
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http://www.cdb.riken.jp/
RIKEN CENTER FOR DEVELOPMENTAL BIOLOGY
2-2-3 MINATOJIMA-MINAMIMACHI, CHUO-KU
KOBE 650-0047 JAPAN
PHONE: +81-78-306-0111 FAX: +81-78-306-0101
EMAIL: cdb@cdb.riken.jp

On The Cover
3D reconstructed image of developing mouse lung at
E16.5 showing localization of epithelial and neuroendocrine cells.
Image: Laboratory for Lung Development
Printed in Japan using soy inks, waterless printing
methods, and paper from sustainable resources.
RIKEN 2016-017