RIKEN Center for Developmental Biology

2016 Annual Report

2016
AnnualReport
RIKEN
Center for
Developmental Biology

Contents
2

Message from the Director

4

Organizational Chart

6

2016 at the CDB

Research Highlights
12

Mechanism orienting microtubules in epithelial
cellsLab. for Cell Adhesion and Tissue Patterning

14

Functional pituitary tissue generated from human
ESCs
Lab. for Organ Regeneration

16

Origins of complex vertebrate brain older thanpreviously thought
Evolutionary Morphology Laboratory (RIKEN)

18

Improved tissue clearing solution forsuper-resolution imaging
Lab. for Sensory Circuit Formation20

New insights into telomere maintenancemechanisms in embryonic stem cells
Lab. for Pluripotent Stem Cell Studies

22

Making skin from iPS cells
Lab. for Organ Regeneration

24

How cells can turn into donuts
Lab. for Morphogenetic Signaling

26

There’s no stopping time in apical progenitors
Lab. for Cell Asymmetry

28

Notch signaling critical for postnatal alveolar
morphogenesis
Lab. for Lung Development

30

Trehalose important for maintaining body waterhomeostasis
Lab. for Growth Control Signaling

32

Keeping actin filaments in order in fly bristles
Lab. for Morphogenetic Signaling

34

New tool for introducing genes into developing
mammalian brain
Lab. for Cell Asymmetry

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 facing an aging society
were among the issues addressed, and developmental biology was included within this remit as a scientific field withthe potential to contribute to the development of regenerative medicine.
Located in Kobe, Japan, the Center’ s research program isdedicated to developing a better understanding of fundamental processes of animal development at the molecular

60

Chromosome Segregation
Tomoya KITAJIMA

61

Histogenetic Dynamics
Erina KURANAGA

62

Cell Asymmetry
Fumio MATSUZAKI

One signal, two cell fates
Lab. for Morphogenetic Signaling

63

Lung Development
Mitsuru MORIMOTO

42

In vitro model for spinocerebellar ataxia
developed using patient iPSCs
Lab. for Cell Asymmetry

64

Growth Control Signaling
Takashi NISHIMURA

44

DAAM1 stabilizes adhesion between lateral
membranes of epithelial cells
Lab. for Cell Adhesion and Tissue Patterning

65

Pluripotent Stem Cell Studies
Hitoshi NIWA

66

Vascular Morphogenesis
Li-Kun PHNG

67

Retinal Regeneration
Masayo TAKAHASHI

68

Human Organogenesis
Minoru TAKASATO

69

Cell Adhesion and Tissue Patterning
Masatoshi TAKEICHI

70

Organ Regeneration
Takashi TSUJI

71

Epithelial Morphogenesis
Yu-Chiun WANG

36

Transplanted iPSC-derived retinal cells not
rejected if MHC-matched with recipient
Lab. for Retinal Regeneration

38

Disoriented axons lead to olfactory disorder after
traumaLab. for Sensory Circuit Formation

40

46

Lulling mice into a short hibernation-like stateLab. for Retinal Regeneration

48

HITI: An innovative in vivo genome editing
technology
Lab. for Cell Asymmetry

Laboratories
52

in vitro Histogenesis
Mototsugu EIRAKU

53

Tissue Microenvironment
Hironobu FUJIWARA

54

Organismal Patterning
Hiroshi HAMADA

55

Neocortical Development
Carina HANASHIMA
Morphogenetic Signaling
Shigeo HAYASHI

56
57

Developmental Epigenetics
Ichiro HIRATANI

58

Sensory Circuit Formation
Takeshi IMAI

59

Axial Pattern Dynamics
Hidehiko INOMATA

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 access to core facilities and technicalsupport and a critical mass of scientific talent and expertise.
The CDB also benefits from belonging to RIKEN, Japan’ slargest basic scientific research organization, as well as its
close ties to graduate and medical schools and research
institutions in Japan and other countries, giving our scientists access to a broad network of talent and technology
across the country and around the world.

Activities
74

2016 CDB Symposium

76

2016 Seminars

78

2016 Events

80

2016 Educational Programs

81

2016 Awards

84

RIKEN in Kobe

86

About RIKEN

87

RIKEN Campuses

Message from the Director

It has been a whirlwind, but rewarding second year serving as director of the RIKEN Center for Developmental Biology (CDB) as we
prepare for impending changes laid out by our parent organization RIKEN, following its designation as one of the three DesignatedNational Research and Developmental Institutes by the Japanese government. But rest assured that we have been working hard toensure that the high quality world class research for which CDB is known for will not be interrupted or lost by the imminent changes.
The laboratories at the CDB continued to have a productive year in 2016, publishing new findings that extend our knowledge on thefundamental principles of organismal development, how to harness these principles to recapitulate developmental phenomena invitro, as well as reveal insights into disease mechanisms. These include findings on mechanisms of cell specification and differentiation, those controlling morphology, to reports of in vitro generation of pituitary tissue, generation of complex skin tissue, and revealing
underlying pathogenesis of the olfactory disorder dysosmia. There were major developments on technological fronts as well, withseveral cutting-edge publications on new tools and methods that not only assist scientists working in the basic sciences but also havepotential applications in medicine, such as protocols for turning tissues transparent, and novel genome-editing technologies.
We were also delighted to welcome two new talented team leaders, Minoru Takasato and Li-Kun Phng, to our Center this year, whowill no doubt in due course make great contributions to our scientific programs. As part of our commitment to support the careers ofyoung capable researchers, we will continue to search for and recruit young talent to head new laboratories in a research-intensive
setting, and strive to strengthen educational programs geared toward undergraduate and graduate students, including medical students, to foster the next generation of scientists.
In July, the CDB’s research and management activities underwent an intensive evaluation and review process by members of the CDB
Advisory Council, a panel of nine prominent scientists from around the world working in fields related to developmental and stem cellbiology. I am pleased to say that, overall, the report submitted by the Council was positive and encouraging, and helped to renew myresolve to lead the Center to achieve higher levels of excellence.
One of the key initiatives implemented this year to this end was the launch of the RIKEN CDB–Otsuka Pharmaceutical Collaborative
Center (COCC), a partnership with Otsuka Pharmaceutical Co. Ltd. (Otsuka) that will draw on CDB’s strength in basic research indevelopmental and regenerative biology and Otsuka’s expertise in developing medicines and health-related products, to develop
seeds that can be translated into practical applications, focusing on areas related to neurodegenerative and kidney diseases. Other
new joint research announced with major industry partners this year include Kyocera Corporation for hair follicle regeneration, andSanten Pharmaceutical Co. Ltd, for retinal diseases.
Keeping pace with changing tides of science, we recognize that, now more than ever, the government and society have high expectations for basic science to produce advances to ameliorate our daily lives, and in this regard the CDB is committed to developing astronger pipeline from basic to translational science. Taking advantage of our location in the Kobe Biomedical Innovation Cluster, wehave been working to forge stronger ties with clinicians at nearby medical institutions, with the aim of identifying findings with translational potential. We have also established a clinical translational program for physician scientists that would allow them to work alongside CDB scientists to exchange insights and expertise to promote translation of basic science. This will complement the pioneeringresearch by the Laboratory for Retinal Regeneration that is working to establish an innovative therapy to treat retinal diseases using
pluripotent stem cells.
Looking to the future, we pledge to continue laying down a strong foundation of basic science in understanding how bodies arise andare maintained, but at the same time, keep our eyes open to spot translational opportunities through closer collaboration with industry and clinical counterparts. I ask for your continued support on our quest to open new frontiers in development and regeneration.

Hiroshi Hamada
Director, RIKEN Center for Developmental Biology

3

RIKEN Kobe Branch

CDB Organization 2016

Center for
Developmental Biology

Deputy Director

The Center for Developmental Biology (CDB) is a
research center within the RIKEN Kobe Branch, which
also includes the Center for Life Science Technologies

Cellular Environment and Response
Research Program(CLST), the Quantitative Biology Center (QBiC), the Advanced Institute for Computational Science (AICS) andthe Kobe Administrative Division, which provides administrative services. The CDB underwent an organizational
restructuring in 2014, resulting in major changes to labo-

This 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.

ratory designations and departmental affiliations. The

Morphogenetic Signaling

majority of laboratories are now designated as teams,

Shigeo HAYASHI Ph.D.

and assigned to one of five programs, described in detailto the right. In September 2016, the CDB partnered with
Otsuka Pharmaceutical Co. Ltd to launch the RIKEN

Developmental Epigenetics
Ichiro HIRATANI Ph.D.

Chromosome Segregation
Tomoya KITAJIMA Ph.D.

CDB–Otsuka Pharmaceutical Collaborative Center,

Growth Control Signaling

intended to facilitate transfer of scientific findings to com-

Takashi NISHIMURA Ph.D.

mercial applications. The CDB Director is assisted by a

Vascular Morphogenesis

Deputy Director and advised by the Advisory Council,

Li-Kun PHNG Ph.D.

while governance issues are discussed by the Management Committee.

Center for Life Science Technologies
Quantitative Biology Center
Advanced Institute for Computational Science

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.

Developmental Biology Planning OfficeLife Science Technologies Planning Office
Quantitative Biology Planning Office

Takeshi IMAI Ph.D.

Cell Asymmetry
Fumio MATSUZAKI Ph.D.

Cell Adhesion and Tissue Patterning

Computational Science Planning Office

Masatoshi TAKEICHI Ph.D.

Kobe Administrative Division

Yu-Chiun WANG Ph.D.

Epithelial Morphogenesis

Advisory Council

Janet Rossant

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(www.cdb.riken.jp).

Anne Ephrussi

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

4

Sensory Circuit Formation

The Hospital for Sick Children, Canada
European Molecular Biology Laboratory, Germany

Ryoichiro Kageyama
Kyoto University, Japan

Gordon Keller

Princess Margaret Cancer Centre, University
Health Network, Canada

Hiromitsu Nakauchi

Institute for Stem Cell Biology and Regenerative
Medicine, Stanford University School of Medicine, USA

Daniel St Johnston

Gurdon Institute, University ofCambridge, UK

Clifford Tabin

Harvard Medical School, USA

Patrick Tam

Children’ s Medical ResearchInstitute, University of Sydney,
Australia

Shosei Yoshida

National Institute for Basic
Biology, Japan

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 iPS
Cell Research and Application (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.

Human Organogenesis
Minoru TAKASATO 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.

Collaboration Centers ProgramThis program aims to facilitate collaborations with industry by pursuing longer term projects in areas related toneurodegenerative and kidney diseases, with a view todevelop seeds of innovation that can be translated into
practical applications.

Organismal Patterning

RIKEN CDB–Otsuka Pharmaceutical
Collaboration Center

Hiroshi HAMADA M.D. Ph.D.

Hiroshi HAMADA M.D. Ph.D.

Axial Pattern Dynamics
Hidehiko INOMATA Ph.D.

Histogenetic Dynamics
Erina KURANAGA Ph.D.

Management Committee
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 decisions on governance issues such as
recruitment of new laboratory heads and personnel, and budgetary allocations.

Hiroshi Hamada

Tatsuo Shibata

Toshihiko Oguru

Masayo Takahashi

Akihiko Koseki

Takashi Tsuji

Yoichi Shinkai

Takashi Nishimura

Hisato Kondoh

Carina Hanashima

Tomoya Kitajima

Yasuhide Furuta

Director, CDB

Director, RIKEN Kobe Branch; Deputy Director, CDB
RIKEN Center for Integrative Medical Sciences
RIKEN Cellular Memory Laboratory
Kyoto Sangyo University
Lab. Chromosome Segregation, CDB

Coordinator, CDB

Lab. Retinal Regeneration, CDB
Lab. Organ Regeneration, CDB
Lab. Growth Control Signaling, CDB
Lab. Neocortical Development, CDB
Coordinator, CDB

Katsutoshi Nukui

Developmental Biology Planning Office5

2016 Highlights
Team Leader Tomoya Kitajima
receives MEXT Prize
Tomoya Kitajima, team leader of the Laboratory for Chromosome
Segregation was honored with a commendation for Science andTechnology from the Minister of MEXT at a ceremony held in Tokyoon April 20. He received the Young Scientist’s Prize for his study ofthe mechanisms of chromosome segregation in mammalian oocytes
using high-resolution live-imaging in combination with micromanipulation and genetic engineering techniques.

CDB Director elected associate
member of EMBO
Director Hiroshi Hamada was elected as an Associate Member of theEuropean Molecular Biology Organization (EMBO) following thisyear’s membership elections, an honor reserved for a small numberof leading scientists residing outside Europe in recognition of theircontributions to research in the life sciences.

CDB signs MOU with
Chulalongkorn University
The CDB established an MOU with the Faculty of Pharmaceutical
Sciences of Chulalongkorn University, in Thailand. The signing ceremony was held at Chulalongkorn University in May, and a joint symposium was also held after the ceremony.

6New organizational framework
for iPSC clinical research
RIKEN CDB concluded an agreement with the Kobe City Medical Center General Hospital, Osaka University Hospital andKyoto University on May 30, to launch a new clinical research
project aimed at establishing an iPSC-based treatment for wettype age-related macular degeneration. Under the new partnership, Kyoto University will establish autologous and allogeneic iPSC lines, CDB will generate RPE cells that will be used
for transplantation from iPSCs, and Kobe General Hospital andOsaka University Hospital will perform the transplantation.

CDB Advisory Council convenes
The biennially organized CDB Advisory Council (AC) was held on July
14–16 at the CDB. AC members convened to hear about scientificprogress of each of the laboratories, as well as discuss and makesuggestions on the research activities at CDB, and its directions forthe future.

New faces at the CDB
The RIKEN CDB welcomed two new team leaders to its research programs. Minoru Takasato was appointed Team
Leader of the Laboratory for Human Organogenesis in August,and Li-Kun Phng joined the CDB in October as Team Leader ofthe Laboratory for Vascular Morphogenesis.

72016 Highlights
Strengthening researchcollaborations with industry
CDB announced the launch of several new collaborative research
projects with industry. The Laboratory for Organ Regeneration has
teamed with Kyocera Corporation and Organ Technologies Inc. withthe aim of developing technologies and products for treating alopecia(hair loss) through the regeneration of hair follicles, and the Laboratory for Retinal Regeneration has joined hands with Santen Pharmaceutical Co. Ltd. and FBRI to identify new drug candidates for treating
retinal degenerative diseases using retinal cells derived from iPSCs.

Team leader wins AustralianMuseum Eureka Prize
Minoru Takasato, team leader of the Laboratory for Human Organogenesis,
was announced as one of the winners of the 2016 Australian Museum Eureka
Prizes. At a gala event held in Sydney, Australia on August 31, he along withhis colleague, Professor Melissa Little from the Murdoch Childrens Institute,received the 2016 UNSW Eureka Prize for Scientific Research for their work
generating human kidney organoids from stem cells. This work was carriedout by Takasato during his previous post at the Murdoch Childrens Institute.

RIKEN CDB and Otsuka
Pharmaceutical launch newcollaborative research center
CDB signed an agreement with Otsuka Pharmaceutical Co. Ltd toestablish the RIKEN CDB-Otsuka Pharmaceutical Collaborative
Center (COCC) starting September 1. The new collaborative research
center is headed by Hiroshi Hamada, and will initially focus on theareas of kidney and neurodegenerative diseases.

8G7 Ministers visit RIKEN CDB
Health ministers of countries in the Group of Seven (G7) visited the
CDB on September 12 to learn about RIKEN, and hear a talk by
Masayo Takahashi on her research to establish iPSC-based treatment for age-related macular degeneration. The ministers also had
the opportunity to look at samples of iPSCs and retinal pigment epithelial cell sheets generated from iPSCs.

Going beyond skin-deepThe 27th CDB Meeting

Body Surface Tactics
Cellular crosstalk for the generation ofsuper-biointerfaces

November 14-15, 2016

The 27th CDB Meeting, “Body Surface Tactics: Cellular crosstalk forthe generation of super-biointerfaces,” was held November 14–15.
Scientists and students with an interest in some aspect of the skinassembled at the CDB to take part in the two-day meeting that focused on themes such as epithelial formation, maintenance and regeneration, new technologies, and skin dysfunction and therapeutics.

RIKEN Center for Developmental Biology
Kobe, Japan
Keynote speakers:
Yann Barrandon
École Polytechnique Fédérale de Lausanne, Switzerland

Cheng-Ming Chuong
University of Southern California, USA

Confirmed speakers:
Masayuki Amagai
Keio University/RIKEN Center for Integrative Medical Sciences, Japan

Hironobu Fujiwara
RIKEN Center for Developmental Biology, Japan

Hisao Honda
Kobe University, Japan

Mayumi Ito
New York University, USA

Kenji Kabashima
Kyoto University, Japan

Hiroyasu Kidoya
Osaka University, Japan

Akiharu Kubo
Keio University, Japan

Yoshiaki Kubota
Keio University, Japan

Michiyuki Matsuda
Kyoto University, Japan

Masaharu Nagayama
Hokkaido University, Japan

Daisuke Nanba
Tokyo Medical & Dental University, Japan

Emi Nishimura
Tokyo Medical & Dental University, Japan

Yoshiko Takahashi
Kyoto University, Japan

Takashi Tsuji
RIKEN Center for Developmental Biology, Japan

Registration deadline:
Friday, September 9, 2016
http://www.cdb.riken.jp/skin2016/

Organizers:
Akiharu Kubo, Daisuke Nanba,
Emi Nishimura, Hironobu Fujiwara

Seeking new frontiers in cilia andcentrosome research

Invited Speakers

Renata Basto
Institut Curie, France

Takanari Inoue

Johns Hopkins University, USA

Daniela Nicastro

University of Texas Southwestern Medical Center, USA

Karen Oegema

University of California San Diego, USA

Heymut Omran

University of Münster, Germany

Junmin Pan

Tsinghua University, P. R. China

The 28th CDB Meeting, “Cilia and Centrosomes: Current Advancesand Future Directions,” was organized November 27–29. Over 100
participants were in attendance to discuss roles of cilia and centrosomes in cell cycle, embryogenesis, and disease pathogenesis.
The 28th CDB Meeting

Cilia
and Centrosomes
Current Advances and Future Directions
November 27‒29, 2016

RIKEN Center for Developmental Biology (CDB), Kobe, Japan
Organizers
Masa Kikkawa, The University of Tokyo
Fumio Matsuzaki, RIKEN CDBRegistration deadline

Co-HostCilia Club
Grant-in-Aid for Scientiﬁc Research on Innovative Areas, MEXT
“Cilium-centrosome system regulating biosignal ﬂows”

Friday, September 30, 2016

http://www.cdb.riken.jp/cilia/2016

9Research Highlights

Super-resolution microscopy for microtubules in small intestinal epithelial cells. In wild
type mouse, microtubules (green) align along the apico-basal axis of cells. CAMSAP3(red) localizes to the apical region, capturing the minus-ends of microtubules. Red
signals in the lower middle region are noise.
Image: Mika Toya, Laboratory for Cell Adhesion and Tissue Patterning

Mechanism orienting microtubulesin epithelial cells

Mika TOYAE

pithelial cells are found covering flat surfaces andlining tubular organs of the body. They exhibit anapicobasal polarity, a characteristic that is essential for carrying out their numerous functions suchas secretion, absorption, and protection. One feature
contributing to epithelial cell polarization is the distinct intracellular organization of microtubules, a key componentof the cytoskeleton. In polarized epithelial cells, microtubules are arranged in longitudinal rows parallel to theapicobasal axis, appearing like a string curtain, but themechanisms at work to properly orient the microtubules
remained unclear.
In a study led by research scientist Mika Toya in the
Laboratory for Cell Adhesion and Tissue Patterning(Masatoshi Takeichi, Team Leader), the team demonstrated, using mouse intestine epithelial cells and humancolorectal adenocarcinoma-derived cell line (Caco-2)
as models, that microtubule-binding protein CAMSAP3(calmodulin-regulated–spectrin-associated protein 3) isimportant for microtubule orientation in polarized epithelial cells. They also showed that microtubule orientationis critical for correctly positioning organelles within thecell. Their work was published in the Proceedings of the
National Academy of Sciences USA.

12

Microtubules are involved in many cellular functions
such as chromosome segregation, intracellular transport, cell motility and cell structure maintenance. The
tubular structure of the microtubule has polarity, a minus- and plus-end, with the plus-end being associatedwith rapid elongation. In epithelial cells, microtubules
are aligned along the apicobasal axis, with minus-ends
oriented near the apical side and plus-ends toward thebasal side. Past studies from the same laboratory using
cultured cells identified CAMSAP3 (also called Nezha) as
a protein binding specifically to microtubule minus-ends.
In the current study, the team focused on revealing themechanism underlying microtubule orientation in polarized epithelial cells.
CAMSAP3 is known to bind to microtubule minus-ends
through its CKK-domain at the C-terminus, so the teamfirst generated a Camsap3 mutant mouse line, which
expresses a CAMPSAP3 lacking the CKK-domain. Homozygous mutants displayed growth impairments, andabout 15% of these mice died before postnatal day 30.
When epithelial cells of the small intestine were examined, they noticed those from Camsap3 mutants lacked
the normally punctate distribution of CAMSAP3 on theapical side. Comparisons of microtubule distribution incells using stimulated-emission depletion (STED) super-Toya M, et al. CAMSAP3 orients the apical-to-basal polarity of
microtubule arrays in epithelial cells. Proc Natl Acad Sci U S A 113(2):332–7 (2016)

resolution microscopy revealed that microtubules in cells
from wildtype were bound to the apically distributed
CAMSAP3 and aligned longitudinally along the apicobasal axis, whereas in mutant cells, microtubules were disorganized, displaying no specific orientation. Thus, theseexperiments suggested that apically distributed CAMSAP3 steers proper microtubule orientation by binding to
the microtubule minus-ends, anchoring them in place.

Modified from article © 2016 Toya M, et al.

They next examined arrangements of organelles in cells
from Camsap3 mutant mice. In wildtype mice, cell nuclei
were found in near the basal side at relatively similar distances from the basal membrane, whereas in Camsap3
mutants, nuclei positions were disorganized, with someeven positioned close to the apical side. Position of theGolgi apparatus, generally found just above (apical to)
the nucleus, was also abnormal in Camsap3 mutants,
scattered to different areas of the cytoplasm. Cells frommutants also appeared to have problems maintaining
consistent cell height along apicobasal axis. Electron microscopy revealed mitochondria elongation, and in somecells, the normally apically positioned adapter protein
ezrin was mislocalized. Thus, it appears that organelles
cannot be correctly localized when microtubules are
misoriented as seen in Camsap3 mutant cells.

Toya also analyzed CAMSAP3 function in Caco-2 cells, ahuman colorectal adenocarcinoma-derived cell line, culturing them in a media that allowed the cells to maintainepithelial-like polarity. When CAMSAP3 was depleted in
Caco-2 cells, they found microtubule misalignment andmislocalization of organelles. Analyses of the CAMSAP3
domains revealed that the CC1 domain contained anamino acid sequence that was critical for CAMSAP3’s
apical localization. CAMSAP3 also requires binding tomicrotubules through its microtubule-binding CKK domain for apical localization.
“Our combined experiments using STED super-resolution microscopy and generating Camsap3 gene knockouts has allowed us to analyze microtubule alignment
in epithelial cells in greater detail than ever before,” explains Takeichi. “We hope to look at CAMSAP3 function
in other cell types, and understand how microtubules
bound to CAMSAP3 control intracellular structure. It will
also be interesting to determine the underlying causes of
the growth defects observed in Camsap3 mutant mice.”

Left: Distribution of CAMSAP3 (red) and microtubules (green) in mouse intestinal epithelial cells. Upper right row: Position of nuclei (blue, nucleus; green,
apical protein ezrin; red, basal membrane) Lower right row: Position of Golgi apparatus (blue, nucleus; green, apical cortex; red, Golgi). Scale bar for bothrows, 10 μm. All panel sets, wildtype is on left and Camsap3 mutant is on right.

13

Functional pituitary tissue generated fromhuman ESCs

Chikafumi OZONET

he pituitary, located at the base of the brain, isthe central regulator of the endocrine system,
controlling hormone secretions within the body. It
secretes different hormones in response to signals fromthe hypothalamus, and also controls hormone secretionlevels in response to feedback signals from the body.
Disruption of hormone production in the pituitary canlead to problems with blood pressure, electrolyte balance, growth, and fertility, highlighting the importance of
this tissue in maintaining homeostasis. While hormone
replacement therapies are currently used to treat some ofthese conditions, they need to be administered throughout the patient’s life and cannot emulate the precise
regulatory control of the natural endocrine system, which
responds to the ever changing needs of the body. Thus,a method to produce pituitary tissue that is capable ofresponding to regulatory signals would be a step forward
developing an effective therapy for pituitary diseases.
A research collaboration between research associate
Chikafumi Ozone of the Laboratory for Organ Regeneration (Takashi Tsuji, Team Leader), associate professor
Hidetaka Suga of Nagoya University and others has culminated in the successful generation of anterior pituitary
tissue from human embryonic stem cells (ESCs). Furthermore, they were able to steer the induction of various

14functional hormone-producing cells of the anterior pituitary. Their achievements were published in the online
journal, Nature Communications.
The current work builds on previous work by Suga andformer group director Yoshiki Sasai (dec.) of the Laboratory for Organogenesis and Neurogenesis, in which theygenerated self-organizing pituitary tissue in vitro frommouse ESCs, using a 3D floating cell culture method
called SFEBq which was developed by the same lab.
The pituitar y primordium, also known as Rathke’s
pouch, emerges from the oral ectoderm upon receiving
inductive signals from the neighboring hypothalamicneural epithelia (NE). The team began by determining the optimal conditions for inducing both hypothalamic NE and oral ectoderm in the same culture. Aftermany attempts, they discovered that the addition ofa hedgehog pathway agonist and BMP4 to cultured
human ESC-aggregates led to the efficient inductionof oral ectoderm-like tissue around hypothalamic NE.
After three weeks, they noticed a thickening of some
sections of the oral ectoderm that expressed an early
pituitary progenitor marker, LHX3, which eventually invaginated to form a Rathke’s pouch-like structure, andthe addition of FGF, important in early pituitary forma-Ozone C, et al. Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells. Nat Commun 7. 10351(2016)

Modified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 Ozone C, et al. / © 2016 RIKEN CDB, Lab. for Organ Regeneration

tion, produced Rathke’s pouch-like structure at higher
frequencies.
The group then examined whether their derived pituitary primordium could differentiate into mature pituitaryhormone-producing cells. By culture day 67 to 70, they
identified differentiation of adrenocorticotropic hormone(ACTH)-producing cells (corticotrophs) through immunostaining analyses and the observation of secretory
granules in the cell cytoplasm. They also found that,consistent with reports from past studies, treating theirderived pituitary tissue with glucocorticoids induced differentiation of somatotrophs, which produce growth hormone, and at much lower frequencies, of cells producing
prolactin and thyroid stimulating hormone. When a Notch
signaling inhibitor was added, they saw induction of gonadotrophs. Thus, their results show human ESC-derived
pituitary primordium could differentiate into different
hormone-producing cells of the anterior pituitary.
They also examined the regulatory responses of theirderived corticotrophs and found that, similar to in vivo
responses, ACTH release was stimulated with corticotropin-releasing hormones whereas glucocorticoids
inhibited ACTH release. Likewise, they found their human
ESC-derived somatotrophs responded similarly to natural ones in vivo.

Finally, they analyzed whether their hESC-derived pituitary could function in vivo by transplanting their pituitary tissue into the subrenal capsule of a hypopituitary
mouse model. Extraction of the pituitary in mouse islethal, causing death within a matter of weeks due toglucocorticoid deficiency arising from lack of ACTH. Tendays after transplantation, they observed engraftment ofcorticotrophs as well as a rise in ACTH levels stimulating
glucocorticoid release. Comparisons between mice thatreceived transplants and those that did not, revealed
improvements to some hypopituitary symptoms, suchas activity levels, which had dropped due to glucocorticoid deficiency, weight stability and length of survival.
Three to four months after transplantation, the tissue
graft showed signs of a vascular system and retained its
hormone-secreting function.
“While there have been previous reports of pituitary induction from human ESCs, our study differs in that wehave recapitulated the in vivo pituitary development invitro, and are the first to demonstrate that our derived
pituitary tissue can respond to regulatory signals, andshow some therapeutic effects when transplanted intohypopituitary mouse model,” says Suga. “Our work
provides a platform for understanding pathogenesis of
pituitary diseases and for developing future therapeutic
applications.”

Co-induction of ventral neural epithelium (green) and non-neural ectoderm (white) from hESCs. A Rathke’s pouch-like structure(LHX3+, red) began to form between culture days 26 (left) to 27 (middle and right).

Corticotrophs differentiated from human ESC(green, ACTH-positive)

15

Origins of complex vertebrate brain older thanpreviously thought(From left) Wataru TAKAGI, Tamami HIRAI, Noritaka ADACHI, Shigeru KURATANI, Juan PASCUAL ANAYA, Fumiaki SUGAHARA

C

yclostomes are jawless vertebrate fish that lackpaired fins and have only one nostril. They are
regarded as being more primitive forms of vertebrates, and there are presently only two surviving groupsof cyclostomes—hagfish and lampreys. The latest common ancestor they shared with gnathostomes, the jawedvertebrate species including humans, was thought to be
500 million years ago. The acquisition of a highly regionalized complex brain is considered the hallmark of vertebrates, and the brain of cyclostomes have been characterized as being ancestral forms of the vertebrate brain
due its more simple regionalization. The actual evolutionary origins of the vertebrate, however, remain unclear.
RIKEN visiting scientist Fumiaki Sugahara and researchscientist Juan Pascual Anaya of the Evolutionary Morphology Laboratory (Shigeru Kuratani, Chief Scientist)and others performed detailed analyses of the embryonic brain of cyclostomes and discovered the existence of two key brain domains—the medial ganglioniceminence (MGE) and the rhombic lip—common acrossall gnathostomes but previously thought to be absent incyclostomes. Their discovery provides strong evidence
that the fundamental plan for vertebrate brain development was already established before the divergence of
cyclostomes and gnathostomes over 500 million years

16

ago. This work, published in Nature, was carried out
when the laboratory was part of the CDB. Sugahara hassince moved to and continues his research at the Hyogo
College of Medicine, Japan.
The blueprint for brain development is remarkably wellconserved across gnathostome species. In the embryonic brain, the MGE emerges from the ventral region of
the subpallium (future cerebrum), from which GABAergic
interneurons migrate to the cerebral cortex. The rhombic lip, which gives rise to the cerebellum importantfor motor control, appears along the dorsal hindbrain.
The apparent absence of the MGE and rhombic lip indeveloping lamprey brains suggested that these domains emerged in gnathostomes after divergence of
cyclostomes and gnathostomes. However, GABAergic
interneurons, presumed to be of MGE origin, have beenfound in adult lampreys, which alludes to the existenceof an MGE region in embryonic lamprey brains, conflicting with conventional evolutionary models.
Sugahara and his collaborators began by making a detailed examination of brain development in hagfish, theonly other extant cyclostome groups. They made serial
sections of the embryonic head region at several different developmental stages, and used immunostaining andSugahara F, et al. Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain. Nature 531. 97–100 (2016)

Adapted by permission from Macmillan Publishers Ltd: Nature 531. 97-100, copyright 2016 / © 2016 RIKEN Evolutionary Morphology Laboratory

in situ hybridization methods to stain for specific geneexpressing regions and tissues, and then reconstructed
the brain in 3D using computer software. These results
were then compared to those obtained from brains oflamprey and cloudy cat sharks, an example of a gnathostome offshoot to cyclostomes, at similar developmental
stages. Hagfish had many common developmental brain
features such as the positioning of key structures andnerve fibers as lamprey and shark. However, unlike inlamprey, the team identified expression patterns of MGE
marker genes, Nkx2.1 and Hedgehog, in the subpallium region, and of rhombic lip marker genes, Pax6 andAtoh1, along the dorsal hindbrain region, confirming thathagfish form an MGE and rhombic lip domain during
brain development.
Then how can the absence of the MGE and rhombic lip
in lamprey, which exist in the hagfish and gnathostomes,
be explained? One possibility is that common brain features between cyclostomes and gnathostomes were lost
secondarily in lamprey, and another is that the gnathostomes and hagfish evolved similar features independently through convergent evolution. The team revisited theembryonic lamprey brain armed with newly publishedgenomic data for lamprey, and uncovered additional
orthologues of MGE and rhombic lip marker genes thatwere previously undetected. They confirmed that orthologous genes for Nkx2.1, Pax6, and Atoh1 in the lamprey
were expressed in patterns comparable to those seen in
hagfish and gnathostomes, indicating that an MGE andrhombic lip-like domain exist in the embryonic lamprey
brain.
“The brain architecture of cyclostomes were presumed
to be less complex than those of gnathostomes because
two key brain domains common across all vertebrate
brains could not be found. But our current study showsthat these brain domains are indeed present in cyclostomes, indicating that the fundamental vertebrate brain
plan was established well over 500 million years ago,”says Kuratani. “Most gnathostomes subsequently developed more elaborate brain regions leading to the forms
seen today, so our next step is to understand how eachbrain region was acquired and evolved. Also of interestis the origins and evolution of cerebral neocortex andcerebellum.”

Embryonic brain regionalization in cyclostomes (hagfish, lamprey) and gnathostomes (mouse)

Hagfish (behind) and lamprey (front)

17

Improved tissue clearing solution forsuper-resolution imaging

Meng-Tsen KE (with Ying-Chu)As biologists seek to gain further insights on subcellular structures deeply embedded in various
tissues, high-resolution fluorescence microscopyusing various fluorescent markers has emerged as a powerful tool to ‘see’ inside tissues in three-dimension (3D).
High-resolution imaging remains challenging, however,as it is sensitive to light scattering and spherical aberrations, which in turn creates blurred or inaccurate images.
To facilitate 3D imaging, especially of thicker tissue samples, several protocols for turning tissues transparent ina process called optical clearing have been developed toreduce light scattering and spherical aberrations. Whilesome optical clearing agents achieve very high tissuetransparency, many include use of harsh chemicals which
can cause tissue damage or swelling resulting in morphological changes to more delicate structures. Thus, theseclearing agents are unsuited for visualizing subcellularfeatures at high resolution.
In a study by Foreign Postdoctoral Researcher MengTsen Ke in the Laboratory for Sensory Circuit Formation(Takeshi Imai, Team Leader) and other collaborators,
they report the development of a tissue-clearing solution
SeeDB2 that can be used in combination with different
types of high-resolution fluorescence microscopes, allowing them to analyze tiny fine structures lodged deep

18

within thick tissue samples at super-resolutions. Published in Cell Reports, their study also demonstrated theapplications of their tissue clearing solution for largescale detailed 3D imaging of neuronal circuits in themouse and fruit fly brain at synaptic-scale resolutions.
The Laboratory for Sensory Circuit Formation previously
developed SeeDB (see deep brain), a non-hazardous,
fructose-based clearing agent that can rapidly clear tissues within three days with minimal damage to tissue
structures simply by soaking the tissue. Despite the wellpreserved state of structures in samples treated with
SeeDB, obtaining high resolution images of these embedded structures have been hampered by the resolution limit of the objective lenses.
To increase resolution power of fluorescence microscopy, immersion oil, which has a high refractive index (RI),
is normally placed between the glass coverslip and objective lens. RIs of clearing agents, including SeeDB, arelower than that of the coverslip and immersion oil, thus
when the lens focus shifts deeper in a tissue, the light
passing through tissue can no longer converge resulting in spherical aberrations. Spherical aberrations canbe minimized when RI of cleared tissues matches that of
immersion oil.

Ke M T, et al. Super-resolution mapping of neuronal circuitry with anindex optimized clearing agent. Cell Rep 14. 2718–32 (2016)

Modified from article and licensed under CC BY-NC-ND (creativecommons.org/licenses/by-nc-nd/4.0/legalcode) © 2016 The Authors.

The team searched for aqueous solutions with high RIs
and came across iohexol, an iodine compound used as acontrasting medium for X-ray imaging. After testing various formulas, they found that the combination of iohexol,
small amounts of saponin (weak detergent) and TrisEDTA buffer improved its permeation into tissues as wellas the transparency of tissues to match the RI of immersion oil and the glass coverslip. This improved formula
was named SeeDB2, and tissues could easily be cleared
within two days, in some cases as short as a few hours,
depending on the thickness of the sample by simple
soaking. Like its predecessor, SeeDB2 also minimized
tissue swelling during the clearing process, and could
stably maintain bright fluorescence signals emitted byseveral different fluorescent proteins and fluorophores
used for high-resolution fluorescence microscopy.
The performance of SeeDB2 was tested using different
biological samples in combination with several commercial super-resolution microscopes. When imaging
SeeDB2-treated mouse oocytes, which are relatively
larger than the average cell, they were able to see individual microtubule strands in fine detail, where previously it had been difficult to do so. In mouse brain tissues
cleared with SeeDB2, they were able to visualize neurons
located at depths approximately 10 times deeper thanpreviously possible as well as resolve fine neuronal features including spines on the dendritic processes whichhave diameters less than 1μm.
The team also demonstrated applications of SeeDB2
and super-resolution microscopy for imaging neuronal
circuitry in brain tissues of mouse. They focused on theneuronal synapses, structures where electrochemicalsignals between neurons are exchanged, and which dueto their small size (˂ 1μm) have been difficult to observe
directly. Many cognitive disorders have been linked toabnormalities of neuronal synapses, thus, understanding
synaptic structure will also add to our comprehensionof how these disorders arise. They compared the dendrites of normal neurons with those in which an essential
subunit of a glutamate receptor NMDAR was knocked
out. In normal neurons, dendritic spines displayed different morphologies, some with pointed spines and others
that were mushroom-shaped. Whereas excitatory synapses are known to localize at tips of dendritic spines,
examination of inhibitory synapses clearly revealed they
localize to the shaft of dendritic spines as well as the tips.
In knockout neurons, while the spine density was comparable to normal neurons, the team observed that sizesof some spines were larger than those of normal neurons
and that inhibitory synapses were densely localized attips of spines. They also successfully imaged the neuralcircuitry in brain of Drosophila, revealing unique synaptic
structures in 3D that could not be distinguished before.
“SeeDB2 is a simple and highly versatile clearing agent,
particularly useful for imaging fine structures in tissues
at high resolutions. The other clearing agents each havetheir own advantages, therefore scientists should choose
the one that best suits their purpose," says Imai. “Electron
microscopy has been the only means available until now
to study neural synaptic structures, a time-consuming
and laborious method. Combining the use of SeeDB2
with super-resolution microscopy will facilitate the studyof connectomics of neural networks by revealing a manifold of detailed information on these delicate subcellular
structures at synaptic level.”

Reconstructed image of mouse cerebral cortex cleared with SeeDB2 using super-resolution
microscopy (Airyscan). Fine structures of the dendritic spines and synapses can be clearly seen.

19

New insights into telomere maintenancemechanism in embryonic stem cellsEmbryonic stem cells (ESCs) are derived from cells
extracted from the inner cell mass of early stageembryos. When cultured under the right conditions,they are capable of dividing and proliferating indefinitely,
all the while retaining the potential to become any somatic
cell type in the body. In most somatic cells, the terminal
ends of DNA called telomeres become shorter after eachsuccessive round of cell division. Once telomere shortening reaches a critical limit, the cell stops dividing, eventually progresses into senescence (aging) and finally cell
death. The exceptions to this are germ cells, stem cells
and cancer cells, which can repair their telomeres throughtelomere elongation and continue to proliferate. In ESCs,
a DNA-binding protein called ZSCAN4 is known to play
an important role in regulating telomere elongation andgenomic stability; however, its gene is not expressed in all
ESCs, and it was unclear what triggered its expression.
In a study by research scientist Yoko Futatsugi andTeam Leader Hitoshi Niwa in the Laboratory for Pluripotent Stem Cell Studies, they investigated the conditions
leading to the activation of Zscan4 expression in mouse
ESCs by monitoring its activity at the single-cell level andfound that it is activated in response to telomere shortening. They also discovered that the cell-cycle lengths
were longer when Zscan4 was expressed, presumablyreflecting the time necessary for telomere maintenance.
Their findings were published in the journal Stem Cell
Reports.
Futatsugi and Niwa first transfected mouse ESCs with
two transgenes, each expressing different colored fluorescence when activated. They then established an experimental system where a nuclear marker was used to
identify and track individual cells as well as analyze theZscan4 activity quantitatively over time. Individual mouse
ESCs were tracked over a period of 120 hours, and a celllineage tree was constructed based on the tracking data.
The team also analyzed the cell-cycle length, and telomere length in these cells, and looked for correlations
between these variables and Zscan4 expression levels.

Yoko FUTATSUGI

20

They were surprised to find that the cell-cycle length ofmouse ESCs, which were thought to be approximately
12 hours, in fact displayed considerable variation, ranging between 10–30 hours. When they examined whether
a correlation existed between cell-cycle length and Zscan4 expression levels, cells with longer cell cycles werediscovered to show higher expression levels of Zscan4.
Those cells with high Zscan4 expression levels also had
shorter cell-cycle lengths in the following round of cell
division. Searching for a link between cell-cycle lengthand telomere length, they found that cells with longer
cell cycles had shorter telomeres. Thus, these results

Nakai-Futatsugi Y and Niwa H. Zscan4 is activated after telomere
shortening in mouse embryonic stem cells. Stem Cell Reports 6(4):483–495 (2016)strongly suggested that as the ESCs undergo successive
rounds of cell division, their telomeres become shorter,
which triggers activation of Zscan4 expression. Zscan4
activation, in turn, functions to elongate the shortened
telomeres, resulting in extended cell-cycle lengths.

Modified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 The Authors.

Futatsugi and Niwa also examined the relationship between pluripotency marker gene Rex1 and expression of
Zscan4, but they did not find any correlations between
these two factors. This suggested that telomere elongation by Zscan4 functions independent to the mechanism
maintaining ESC pluripotency.

“ESCs are known to be a heterogeneous population ofcells. In this study, our single-cell tracking method uncovered surprising revelations about the telomere elongation mechanism that could not be identified simply
through examinations of ESC colonies,” explains Futatsugi. “As Zscan4 appears to be activated through somemechanism sensing telomere length, our next step is totry understanding the details of this regulatory mechanism. We hope that further unveiling of innate characteristics of ESCs will lead to the development of more stable culturing methods of pluripotent stem cells, including
iPS cells.”

Expression of Zscan4 in cultured mouse ESCs. Using nuclear marker H2B (middle, red) as a reference marking individual cells, expression levels of Zscan4(right panel, green) were analyzed temporally and quantitatively in each individual cell. Notice only one portion of cells expresses Zscan4.

Left: Example of cell lineage tree. Cells were sequentially numbered in the order they emerged(small black numbers). Numbers in green indicate
cell-cycle length (hours). Zscan4 activity is indicatedon a scale of red intensity. Cells expressed Zscan4transiently. Top, right: Zscan4 activity is higher
when cell-cycle length is longer. Bottom, right: When
Zscan4 expression levels are high, the next round ofcell division tends to be shorter.

21

Making skin from iPS cells(From left) Ryohei MINAMIDE, Koh-ei TOYOSHIMA, Takashi TSUJI, Miho OGAWA, Kyousuke ASAKAWA

T

he skin is a complex organ system comprisingmultiple organs. In addition to the epithelium, dermis and subcutaneous fat—the three tissue layers
making up the bulk of the skin—it includes appendage
organs such as sweat glands, sebaceous glands and hairfollicles. These organs are all connected to structures
such as sensory nerves or muscles, and work together
to produce physiological responses. Many scientists
have been working to develop artificial skin that closely
resembles the function and structure of natural skin fortherapeutic use to restore skin function in patients suffering from different skin-related diseases or injuries and foruse in testing the safety or effects of cosmetics or drugs.
Thus far, it has been possible to induce pluripotent stem
cells to differentiate into specific cell types of the skin, but
generating a three-dimensional (3D) skin structure complete with appendage organs has proven to be difficult.
A research team headed by Takashi Tsuji, Team Leaderof the Laboratory for Organ Regeneration, reported thesuccessful generation of skin tissue complete with appendage organs such as hair follicles and sebaceous
glands from mouse induced pluripotent stem cells(iPSCs). They developed a new approach to efficiently
induce generation of 3D skin tissues from iPSCs bytransplanting them into an in vivo system. Furthermore,

22

they demonstrated that their derived skin tissue was fully
functional when implanted into the skin of a nude mouse,
with the transplanted skin engrafting with the host tissue
and its hair follicles exhibiting regular hair eruption cycles
similar to that of natural skin. This work was published in
the online journal Science Advances.
Generally, the transplantation of iPSCs or embryonicstem cells into an in vivo system leads to the formationof teratomas, a tumor consisting of a mix of tissue types
derived from the three embryonic germ layers. Tsuji’sgroup began by searching for conditions favorable for inducing the formation of epithelial tissues, which is essential for organogenesis, from iPSCs at high efficiencies.
They developed a novel method to transplant numerous
embryoid bodies (EBs) formed from culturing iPSCs
clustered in drop of collagen gel into a live mouse, calling this the clustering-dependent embryoid body (CDB)
transplantation method. The team found that transplantation of clustered EBs in collagen drop produced fourtimes as many cysts containing epithelial tissues thanobserved from transplantation of single iPSCs or EBs.
These cystic epithelia possessed the three epithelial tissue layers as well as hair follicles and sebaceous glands
that make up whole skin tissue all arranged akin to thatof natural skin. They also discovered that stimulating EBs

Modified from article and licensed under CC BY-NC 4.0 (creativecommons.org/licenses/by-nc/4.0/legalcode) ©2016 The Authors. / © 2016 RIKEN CDB, Lab. for Organ Regeneration.

Takagi R, et al. Bioengineering a 3D integumentary organ systemfrom iPS cells using an in vivo transplantation model. Science
Advances 2(4): e1500887 (2016)

with Wnt10b, a signaling factor important for hair follicle
formation, markedly increased the number of hair follicles formed within iPSC-derived skin.

skin. The color, shape, type, pattern and density of theerupted hair was also confirmed to be similar to that ofnormal mice skin.

Next, the team cut out small units of skin tissue withhair follicles from the cystic epithelia, implanted themsubcutaneously into the back skin of a mouse. To traceengraftment of the transplanted skin unit, skin tissue
made from male mouse-derived iPSCs was transplanted
into a female mouse, and using Y-FISH analysis, whichlabels Y-chromosome positive cells, they verified that
their iPSC-derived skin engrafted with the surrounding
host tissue and showed no signs of tumorigenesis. Close
analysis of the transplanted skin tissue revealed that it
formed proper connections to nerves, muscles, and other structures of the host, and the hair follicles themselves
underwent repeated hair cycles consistent with natural“Our CDB method appears to induce complex skin tissue through the interactions between outer epithelial
surfaces of neighboring EBs, which may be creating
epithelial niches with increased surface areas that undergo epithelial-mesenchymal interactions needed forskin organogenesis,” says Tsuji. “The finding that Wnt10
signaling increases induction frequency of hair follicles
provides an important hint for future studies attempting
to replicate skin organogenesis in a culture system. We
aim to understand the basic principles at work duringorgan induction as well as strive to develop artificial skinthat can be used for human clinical applications and inindustry.”EBs transplanted into adult mouse using CDB method (upper left, view from top; lower left, sideview) resulted in formation of various structures making up the skin tissue inside the explants (left).

Skin tissue units including hair follicles (left) from iPSC-derived cystic epithelia were excised and transplanted subcutaneously in a nudemouse. Transplanted tissue unit became engrafted in host tissue (middle: explant-derived Y-chromosome-positive cells (green) and nucleus(blue)) and displayed normal repeating hair cycles (right).

23

How cells can turn into donuts

Kagayaki KATOF

rom a topological perspective, a ball and a fork
are considered equivalent forms as one can be
deformed into other by stretching or shrinking
the form of the outer surface. They are inequivalent to adonut (torus), as neither forms can be continuously deformed into a torus. In the natural world, however, where
cells making up the animal body are found in diverse
shapes, there appears to be way to turn a sphere-likecell into a torus. This extreme example is seen in tip cellsof the developing Drosophila tracheal system. Tracheal
primordia arise in each segment of the Drosophila embryo to form an epithelial tube unit, and tubular branchesof each unit must connect to those in neighboring segments to form a continuous network circulating the body.
Tip cells are located at the tips of migrating tubules andlead the branches to pair and connect with a neighboring
branch. As tip cell pairs begin adhering to one another,
they also undergo dramatic shape changes to facilitate
luminal fusion, but how this shape change occurs was notwell understood.

In a paper published in Nature Communications, research scientists Kagayaki Kato (now at National Institutes of Natural Sciences, Okazaki, Japan) and Bo Dong
(now at Ocean University, China) of the Laboratory forMorphogenetic Signaling (Shigeo Hayashi, Team Leader)24

reveal a mechanism underlying topological changes of
the tip cells observed in the developing branches of the
Drosophila tracheal system. Their study demonstrates
that microtubules within the tip cells play an importantrole in bringing about the dramatic shape changes.
To change the cell topology from sphere-like into a torus
requires opposing sides of the cell to compress inwards,
followed by fusion of the cell membrane. The team employed live imaging technology to track the behavior of
tip cells destined to pair and fuse, from migration, tocell-cell contact, and finally fusion to connect the tracheal branches. During migration, the leading edge of
tip cells extended a radial array of filopodia, with microtubules actively elongating in the direction of the target
cell and punctate distribution of the cell adhesion molecule E-cadherin. When tip cells came into contact with
their partner, they saw E-cadherin accumulating near thetip cell interface and forming new cell adhesion sites.
Furthermore, dense central bundles of actomyosin andmicrotubules were found to span across the pairing tip
cells. Morphological changes in tip cells were compromised when myosin activity was blocked, indicating that
intrinsic actomyosin forces were driving the contractionof tip cells.

Kato K, et al. Microtubule-dependent balanced cell contraction andluminal-matrix modification accelerate epithelial tube fusion. Nat
Commun 7. 11141 (2016)

The team also investigated the effects of disrupting
microtubule organization by treating the tip cells withdifferent microtubule severing factors. They found that
contraction of interfacing tip cells showed a range ofproblems, from an imbalance of contractile forces between tip cell leading to asynchronous contraction, todelays in or prolonged contraction phase, or even failed
tip cell fusion. Thus, these observations suggested thata microtubule mechanism underlies the synchronizationof cell fusion of the cells.

Modified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 Kato K, et al.

Microtubules also serve as transport routes to shuttle
molecular cargo within the cell, and the lab previously
reported that in fly tracheal formation, the chitin deacetylase Serpentine (Serp) is shuttled by vesicular transport
to be secreted in the tracheal lumen to promote luminal

formation. When microtubules were disrupted in the tip
cells, the Serp transport machinery was also disrupted
and tracheal branch fusion was arrested before luminal
fusion was complete.
“A cell changing its topology to assume a torus-shapeis an extremely rare occurrence, but our current study
shows that even these radical morphological changes
can be realized by skillful manipulation of cellular components found in any cell. Studying these extreme examples may shed light on the universal principles guiding
morphological changes in cells,” explains Hayashi. “It is
still unclear how microtubules can sense and balance thecontractile and tensile forces. We would like to understand the mechanism linking the sensing of these forcesby microtubules to cell morphogenesis.”

Tubular arrangement of epithelial tissues in the fly tracheal system. The tracheal network extending to all regions of the body is generated by connections formed between tracheal branches (left,lateral view; right, dorsal view). Bottom left and right panels were taken at stage 14 of development.

Microtubules (middle panel and magenta in right panel) formed bundles in the contact area of the paired tip cells. In this area, the actin filaments (left panel
and green in right panel) overlapped with microtubules and formed dense bundles used for cell contraction.

25

There’s no stopping time in apical progenitors

The most striking feature of the mammalian brain
is the presence of a large, complex cerebral cortex, a six-layered structure comprising different
neuronal types and each layer being derived from selfrenewing neuroepithelial cells, called apical progenitors(APs), found in the ventricular zone. APs initially proliferate through symmetric cell division, but later switch todifferentiation mode, undergoing asymmetric cell division
to produce one AP that will continue to self-renew andanother daughter cell that becomes an intermediate progenitor (IP) which will differentiate to give rise to neurons.
As the type of neurons born from IPs changes according
to progression of time, from deep-layer neurons to more
upper-layer neurons, the precursor APs are thought to“sense” temporal progression of development to change
fate of their differentiating progeny, but the underlying
mechanism regulating time in the cell remains unknown.
A collaborative study between research scientist Daijiro Konno of the Laboratory for Cell Asymmetry (Fumio
Matsuzaki, Team Leader), Ayano Kawaguchi, an alumnusof the laboratory and currently associate professor at
Nagoya University, and others has identified a group of
so-called “temporal-axis genes” in neural progenitors—
genes that show fluctuations in expression levels onlywith respect to progression of developmental time—using the mouse model. They used single cell transcriptomics to analyze changes in gene expression at several
different developmental stages, and also revealed that
temporal expression patterns of the temporal-axis genes
were unaffected by cell-cycle arrest, thus functioning independent to cell cycle. Their findings were published in
the online journal Nature Communications.

Fumio MATSUZAKI, Ayano KAWAGUCHI, Daijiro KONNO

26

Identifying the molecular factors that regulate the timing
when APs temporally change their properties, including switch from proliferation to neuronal production, hasbeen a challenge as this process is not synchronized.
Thus it is difficult to make a distinction between genes
involved in temporal change of degree of differentiationand those involved only in shift of temporal pattern of
AP identity. In this study, the research group decided
to apply single cell transcriptomics to comprehensively
analyze and compare all genes transcribed within acell at several different developmental stages (E11, E14
and E16). From their transcriptome data, they used astatistical method called principal component analysis(PCA) to identify a set of genes that showed changes ingene expression only in correlation to temporal progression. These changes in gene expression patterns wereindependent to differential fate of progenitors (whether
the cell is an AP or an IP), and most of the temporalaxis genes displayed the largest changes in expression
around E12.

Okamoto M, et al. Cell-cycle-independent transitions in temporal
identity of mammalian neural progenitor cells. Nat Commun 7. 11349(2016)

Modified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 Okamoto M, et al.

The cell cycle would appear as the most likely mechanism involved in regulating time within a cell since it isa fundamental mechanism found in all cells, with genes
being activated in turn to prompt cells to steadily advance to the next stage of the cell cycle. The groupdevised an experimental system to transiently arrest
the cell cycle of APs in the embryonic mouse brain by
activating and terminating expression of genes involvedin cell-cycle progression via electroporation, and thentracked the effects of transient cell-cycle arrest on expression patterns of temporal-axis genes. They foundthat, despite the temporary cell-cycle arrest, expression
patterns of the temporal-axis genes, which displayedconsiderable changes in expression around E12, remained unchanged. Furthermore, when neuronal types
that are born after transient cell-cycle arrest were examined, the team noticed that these APs failed to produce
deep-layer neurons, and instead differentiated into thesame type of upper-layer neurons that would be produced under normal conditions if cell cycle had not beenarrested. Together, these findings suggest that the abilityof APs to produce sequentially diverse neurons is independent to the cell cycle.
“Our experiments also suggested the existence of two
types of temporal-axis genes, those that can be controlled cell intrinsically and those that require an extrinsic
signal to trigger gene expression changes,” explains
Matsuzaki. “Whereas the question of how living organisms keep time has remained largely a mystery, ourpresent study presents evidence refuting the widelyheld hypothesis of the involvement of the cell cycle, atleast in the context of brain development. Our next stepis to identify the key temporal-axis genes, and eventually reveal the complete mechanism controlling temporalprogression in cells.”

Schematic of mammalian cerebral development. Self-renewing APs gradually switch to differentiation mode, producing IPs which will differentiate into different types of neurons. The fates ofdifferentiating neurons transitions over time, from deep-layer neurons to upper-layer neurons.

Left: PCA was used to identify temporal-axis genes (those closely aligned to pink arrow), which exhibit changes in expressionin correlation with temporal progression and independent from differentiation state. Right: Temporal-axis gene profile. Temporal change of gene expression occurs mainly around E12 (top panels). Transient cell-cycle arrest does not affect the temporalchange of gene expression.

27

Notch signaling critical for postnatal alveolar
morphogenesis(From left) Keishi KISHIMOTO, Akira YAMAOKA, Yuki KIKUCHI, Mitsuru MORIMOTO, Chisa MATSUOKA, Hirofumi KIYOKAWA, Yuka NODA

Po-Nien TSAO (Taiwan University)P

Wellington CARDOSO, Munemasa MORI Koichi HASEGAWA, Atsuyasu SATO, Susumu SATO
(Columbia University Medical Center)(Kyoto University)

ulmonar y alveoli are small, spongy, sac-likestructures found at the terminal ends of the
branching respiratory system and is the regionof the lung where gas exchange takes place. They comprise the planar type I alveolar epithelial cells involved
in gas exchange, the round type II alveolar epithelial
cells that secrete surfactants to regulate humidity inside alveolar sacs, and the myofibroblasts that form thesepta between alveolar ducts and help maintain the lungstructure, which undergoes significant expansion andcontraction during respiration. Alveolar development (alveologenesis) is unique in that it spans both embryonicand postnatal stages of development, and the distinctive
morphology of the alveoli arises postnatally following theinitial contact with air inhaled through respiration. The
molecular mechanisms underlying alveologenesis, especially those involved postnatally, however, remain largely
unknown.

28

A study by technical staff Chisa Matsuoka and Team
Leader Mitsuru Morimoto of CDB’s Laboratory for Lung
Development, published in the Proceedings of the National Academy of Sciences USA, demonstrated thecritical role of Notch signaling in postnatal alveolar development using the mouse as a model. They showedthat disruption of Notch signaling, Notch2 in particular,
in epithelial cells lining the airways leads to abnormal
alveolar morphology reminiscent of the phenotypes ofbronchopulmonary dysplasia, with enlarged alveolar
airspaces and decreased cellularity, reducing efficiencyof gas exchange. This study was carried out in collaboration with National Taiwan University and Columbia
University.
The Notch signaling pathway is important in lung morphogenesis, particularly in the differentiation of bronchial
epithelial cells, and mice lacking epithelial Notch die
shortly after birth, making it difficult to study the roleTsao P, et al. Epithelial Notch signaling regulates lung alveolar morphogenesis and airway epithelial integrity. Proc Natl Acad Sci U S A
113. 8242–8247 (2016)of Notch in postnatal alveolar development. Thus, tocarry out a more detailed analysis of alveolar development, the research team examined a Notch-deficientmouse line which shows moderate lethality, as well asa few knockout mouse lines for Notch signaling-related
genes that partially impair Notch function. Analysis of a
Pofut1 knockout mouse line which leads to inactivationof epithelial Notch signals revealed that progression ofprenatal pulmonary development is relatively normal,
but abnormalities arise postnatally during alveologenesis. The lungs of Pofut1 mutants were reminiscent of
the emphysema-like phenotype with enlarged airspacesin the alveolar structure. Notch2 was discovered to be
the critical signal required for postnatal alveologenesis,
with the lungs of Notch2 mutants displaying significantly
reduced numbers of type II cells in the alveoli. The teamdetermined that a drop in proliferation of type II cells in
Notch2 mutants that may lead to overall reduction numbers of type II cells seen in alveoli, as type II cells serve
as progenitors to generate both type I and type II cells.

Modified from article © 2016 Tsao P, et al. / © 2016 Lab. for Lung Development

Notch-deficient mice also exhibited abnormalities in themesenchymal cells of alveoli in addition to the epithelial
cells. During postnatal alveolar development, the alveolar septa begin to form from the mesenchymal myofibroblast cells lining the pulmonary epithelium, increasing

pulmonary surface area for gas exchange. In the Notch2deficient mouse, the number of myofibroblast cells foundin the distal respiratory branches were reduced which
in turn resulted in fewer septa formation. These myofibroblast cells are known to be activated to proliferate
through PDGF-A, a growth factor secreted by the epithelial cells and known as a downstream factor of Notch
signaling. PDGF-A expression was indeed reduced in
Notch2-defective epithelium. Thus, loss of Notch signals
disrupted epithelial-mesenchymal interactions, causing
severe abnormalities in alveolar morphogenesis.
“Our present study focused on developmental eventsoccurring postnatally, rather than those seen prenatally,and has opened up new avenues of research. Emphysema is caused not only by impaired alveolar development, but can arise during adulthood triggered by external factors or from aging. Thus, understanding postnatallung morphogenesis may help us to discover ways toregenerate damaged lung tissues,” explained Morimoto.
“There are many factors at work in an in vivo system thatmake it difficult to gain clear insights into normal developmental processes. We are now working to develop an
in vitro system to recapitulate pulmonary development,
which would allow us to clue in on the key mechanisms
required for this process.”

Histological comparison of the lungs in wildtype and in Notch-deficient (Pofut1 mutant) mice. Whereas embryonic lungs were indistinguishable, after birth, the mutant lungs displayed abnormal alveologenesis, withairspaces within tissue becoming enlarged and tissue becoming less dense (left). Mutant lungs had fewer
numbers of proliferating type II cells (light blue and arrowheads) (right).

Role of Notch2 in postnatal alveologenesis

29

Trehalose important for maintaining body waterhomeostasis

Miki YOSHIDA

T

rehalose is a disaccharide sugar synthesized andused as a source of energy in many plants andinsects. In insects, trehalose is the primary sugarcompound circulating in the hemolymph (insect circulatory system), and in addition to being an energy source,is thought to play an important role in preventing desiccation due to its stability and chemical inertness. There
have been some reports of insects in dry environments
having higher levels of trehalose in the hemolymph thantheir counterparts in other environments. However, many
aspects of the physiological role of trehalose and its metabolism remain largely unknown.

In insects, trehalose is synthesized in the fat body, theinsect-equivalent of the mammalian liver, by Tps1, andthen released into the hemolymph. Using a Drosophila
Tps1 mutant strain, the lab previously demonstrated thatlack of trehalose under starvation or nutrient-deficientconditions has significant effects on growth and viabilityof the fly. The Treh enzyme, in contrast to Tps1, functions to breakdown trehalose into two glucose molecules
through hydrolysis. Treh is found in two different forms inthe body, a secreted form and a cytoplasmic form, due
to alternative splicing, but their respective physiologicalroles remained unclear.

A study carried out by CDB student trainee Miki Yoshidaof the Laboratory for Growth Control Signaling (Takashi
Nishimura, Team Leader) and others revealed the physiological functions of two enzymes involved in trehalosemetabolism in Drosophila—the trehalose-synthesizing
enzyme, trehalose-6-phosphate synthase (Tps1), andthe trehalose-hydrolyzing enzyme, trehalase ( Treh).
They also show the importance of trehalose metabolism
for normal growth and development, maintaining systemic water homeostasis, and tolerating desiccation.
Their work was published in the online journal, Scientific
Reports.

The group began by taking a close look at Drosophila
carrying mutations in the gene encoding Treh. Using the
CRISPER/Cas9 system, they generated different Treh mutant strains—for the secreted form, for the cytoplasmic
form, and for both forms of Treh. Treh mutants for bothforms did not survive past pupal stages. Cytoplasmic
Treh mutants also displayed lethality during pupal stages,
whereas secreted Treh mutants produced viable adults,
indicating that cytoplasmic Treh plays a more critical rolein development, especially beyond the pupal stages.

30

The team next examined the Tps1 mutant strain and Trehmutant strain to determine the function of trehalose inModified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 Yoshida M, et al. / © 2016 RIKEN CDB, Lab. for Growth Control Signaling

Yoshida M, et al. Molecular characterization of Tps1 and Treh genes
in Drosophila and their role in body water homeostasis. Sci Rep 6.
30582 (2016)

Drosophila. Tps1 mutants are unable to synthesize trehalose, and consequently have lower levels of trehalosein the body than found in wildtype. Treh mutants on theother hand are unable to breakdown trehalose, and thus
have higher levels of trehalose than wildtype. They foundthat both mutants showed markedly reduced levels of
glucose, and had lower tolerance for starvation andnutrient-deficient conditions. The group also analyzed
the relative water content distribution within the body,and found that when trehalose levels were high, watercontent increased in the hemolymph but decreased intissues. Thus, the hemolymph of Tps1 mutants appeared
more viscous than seen in Treh mutants. Interestingly,
when fly mutants were analyzed for tolerance to desiccation conditions, Tps1 and Treh mutants both showed
higher lethality than wildtype, indicating that normal tre-

halose metabolism as well as high levels of trehalose isessential for protecting the fly from desiccation.
“The hemolymph does not function solely as the circulatory system to transport factors within the body. It also
serves as the medium where catabolic reactions takeplace to produce physiologically important molecules,“says Nishimura. “Here, we revealed conditions when
impairment of Tps1 and Treh, two enzymes that have opposing functions, produce similar phenotypes. Our next
step will be carry out a closer analysis of Treh mutants,
focusing on features specific to this mutant. Mutants
with high trehalose levels may be useful as a diseasemodel for diabetes, which is characterized by high sugar
levels in the body.”

Different Treh mutants (secretory, cytoplasmic, null) all exhibit higher trehalose levels than wildtype.
However, levels of glucose, which is the product of trehalose metabolism, is markedly reduced.

Water content distribution in wildtype, Tps1 mutant and Treh mutant. Relative water content in hemolymph and tissues changes in correlation withfluctuations in trehalose levels.

31

Keeping actin filaments in order in fly bristles

Tetsuhisa OTANIT

he bristles on the external surface of the Drosophila fruit fly are mechanosensory organs that form
from the robust elongation of single cells during
pupal stages of development. On the inside of the bristle,
parallel bundles of actin filaments are aligned in an orderly fashion along the long axis of the bristle shaft, nestledclose to the cell cortex. A further magnified view of thebristle reveals that the actin filaments making up the actin
bundles are aligned in hexagonal paracrystalline lattice,
but how this distinct and orderly arrangement, which isessential for maintaining the robust yet supple structureof the bristle, is formed remained relatively unknown.
In a study published in Development by former CDB
scientist Tetsuhisa Otani and colleagues in the Laboratory for Morphogenetic Signaling (Shigeo Hayashi, Team
Leader), working in collaboration with the RIKEN Center
for Life Science Technology (CLST)’s Ultrastructural Research Team (Shigenobu Yonemura, Team Leader), they
reveal that protein kinase IKKε, important for distal tip
elongation in Drosophila bristles, is also involved in promoting actin bundling by protecting the actin crosslinking protein Fascin from becoming phosphorylated byanother protein kinase called, Protein Kinase C (PKC).
Fascin, when phosphorylated by PKC is unable to crosslink actin filaments into hexagonal arrangement. IKKε32

uses a double inhibition mechanism to promote actin
bundling by inhibiting PKC activity, which in turn prevents PKC from phosphorylating Fascin and facilitatesbundling activity.
Otani previously reported that protein kinase IKKε islocalized and activated at the distal tip of the elongating bristle, serving as a signaling center regulating the
shuttling of molecular cargo required for elongation.
They reported that fly mutants for ikkε showed kinked orbranching bristles as opposed to straight smooth bristles
because the signaling center could not be maintained at
the elongating tip. These mutants were also seen to display morphological abnormalities along the bristle shaft,and the group turned their attention to uncovering otherroles of IKKε in bristle morphogenesis.
The alignment of actin filaments is known to be mediatedby two types of actin-binding proteins, Forked which
bundles newly formed actin filaments at the bristle tip
and Fascin which packages actin filaments into hexagonal arrangement. They found that of the two proteins,which normally distribute along actin filaments, in theikkε mutants, only the localization of Fascin was disrupted, in addition to displaying disorganized actin filament
bundles. These results led them to speculate that Fascin

Otani T, et al. IKKε inhibits PKC to promote Fascin-dependent actin
bundling. Development 143. 3806–3816 (2016)

functions to bind actin filaments together, and that IKKεregulates Fascin-mediated actin bundling.

Modified from article and licensed under CC BY 3.0 (creativecommons.org/licenses/by/3.0/) © 2016 Otani T, et al.

How exactly does IKKε regulate Fascin? Phosphorylationof Fascin by PKC was known to disrupt actin bundling
activity in bristles. Thus, the team examined the different
mutant fly strains to determine the relationship between
IKKε, PKC, and Fascin. They determined that Fascin localizes along actin bundles only in the unphosphorylated
state, and that phosphorylation by PKC disrupts Fascin
localization along actin bundles. IKKε was also foundto play a role in inhibiting phosphorylation of Fascin by
PKC. As PKC is known to be activated when it translocates and binds to the cell cortex, the team showed,
using cultured cells, that IKKε could prevent PKC acti-

vation. These results indicate that IKKε could indirectly
maintain Fascin-mediated acting bundling through the
inhibition of PKC activity.
“PKC activation is not required for normal bristle formation as pkc mutants show no visible abnormalities inbristle morphology. What then is the merit of adopting adouble inhibitory mechanism to regulate bristle formation?” says Hayashi. “Having a factor like IKKε keeping
PKC activity in check may be the key to maintaining
safe and stable cell and body functions, as PKC plays acentral role in many intracellular signaling pathways anderrors in regulation of PKC activity can lead to cancer orlifestyle diseases, such as diabetes.”

Electron-microscopic image of bristle cross section. Actin bundles spaced at regular intervals
along the cell cortex (A). Magnified view of actin bundles reveal individual actin filaments arearranged in hexagonal paracrystalline arrangement (B,C).

In wildtype flies (top row), Fascin and Forked localize along actin filaments in wildtype flies. In ikkε mutant strains (middle and bottom rows), morphology ofbristle is abnormal and Fascin localization was disrupted (E, F).

33

New tool for introducing genes into developing
mammalian brain(From left) Ikumi FUJITA, Raymond TERHUNE-KUNIKANE, Fumio MATSUZAKI, Yuji TSUNEKAWA, Atsunori SHITAMUKAII

n utero electroporation is a technique that is widelyused in the study of brain development to introduce
DNA or RNA into the brain of the developing embryo
inside the uterus by applying electrical pulses. Thismethod can be used to label cells by inducing fluorescentprotein expression vectors, or to modify gene function by
overexpression, mis-expression or knocking down expression of a specific gene and examining phenotypes attissue level. It remained difficult, however, to modify thegene itself using in utero electroporation. If gene knock-in
was made possible via in utero electroporation, it would
be possible to alter the genome of a specific cell population in the brain during development, facilitating labelingof cells, lineage tracing, or analyses of the localizationand dynamics of a protein at the cellular level in vivo.

Research scientist Yuji Tsunekawa, student trainee
Raymond Terhune and others in the Laboratory for Cell
Asymmetry (Fumio Matsuzaki, Team Leader) have now
developed a new tool for introducing genes into the developing mammalian brain by combining the use of the
CRISPR/Cas9 system and in utero electroporation. In a
paper published in Development, they demonstrate thehigh efficiency of their gene knock-in method to insert
transgenes into a target site in neural progenitors in theembryonic mouse brain. Furthermore, the team refined

34

their protocol to insert two different colored fluorescentmarkers into a target gene in each homologous chromosome, which enabled them to visualize by color the cellswith homozygous knocked-in alleles.
The success of a gene knock-in protocol is dependent
on the efficacy of gene targeting events, which is usually very low. Invention of the CRISPR/Cas9 system hasenabled the genome to be edited with ease and highprecision. Tsunekawa and his collaborators have beenworking to develop CRISPR/Cas9-based gene-editing
techniques that can efficiently modify the genome, andhave more universal applications, one of which culminated in the development of HITI, which can efficiently
knock-in genes in differentiated cells in vivo (Suzuki K,
et al., Nature 2016). Concurrently with the developmentof HITI, the team tried developing a simple gene knockin method targeting neural progenitor cells in embryonicbrain in utero by combining the CRISPR/Cas9 methodwith in utero electroporation.
CRISPR/Cas9 system-based genome-editing technology
exploits the two intrinsic DNA repair pathways of cells:
homology-directed repair (HDR) and non-homologous
end joining (NHEJ). Whereas the HITI approach used the
NHEJ pathway to insert genes into both proliferating andTsunekawa Y, et al. Developing a de novo targeted knock-in method
based on in utero electroporation into the mammalian brain. Development 143. 3216–3222 (2016)

non-proliferating cells, to specifically target proliferatingneural progenitors in the brain, the team elected to use
the HDR pathway, which functions only in actively dividing cells but is more accurate and efficient. They first
designed the vector such that the EGFP sequence to beinserted was flanked by homology arms and tested their
method to knock-in EGFP (green) fluorescent marker
targeted to the Tubb3 gene, a neural marker. They found
EGFP was knocked-in in approximately 20% of the electroporated cells. Proving that their concept did work, theteam then determined the best homology arm lengths for
efficient gene insertion and optimizing the vector template to minimize leaky expression.

Modified from article and licensed under CC BY 3.0 (creativecommons.org/licenses/by/3.0/) © 2016 Tsunekawa Y, et al.

Genes knocked-in to the cell are inserted at random
into one target loci of homologous chromosomes, thus
resulting in cells carrying gene insertions in either one of
the two loci (heterozygous) or in both loci (homozygous)of homologous chromosomes. The team wondered
whether it was possible to distinguish between heterozygous and homozygous targeted cells, and designed a
protocol in which two targeting vectors, each with a different colored fluorescent marker, were electroporated

simultaneously in utero into the mouse brain. The theory
was that cells in which both loci had been knocked-in
would fluoresce both colored signals to produce a different colored signal, thus indicating a cell carried homozygous knocked-in alleles. When the team tested their
protocol targeting Tubb3 gene with EGFP (green) andmCherry (red), they found cells expressing both EGFPand mCherry produced a yellow signal, indicating that
donor sequence had been knocked-in to both Tubb3
gene loci of those cells (approximately 5% of the electroporated cells).
“Efficient observation of simultaneous fluorescenceof two different colored signals is made possible by
the CRISPR/Cas9 system’s efficiency for introducing
transgenes. Using this method, we can now identify cellswith homozygous knocked-in alleles, and also trace celllineages with live-imaging,” explains Matsuzaki. “Wealso showed that our new protocol can be used in ferretmodels, demonstrating that this can be used for nonrodent animals for which generating genetically modified animals is difficult, and spur research advances inneurosciences.”

Left: Schematic of gene knock-in using in utero electroporation. Right: Efficient knock-in demonstrated using CRISPR/Cas9 system. Scale bar, 100 μm.

When two different colored fluorescent markers (EGFP, green; mCherry, red) were electroporated
simultaneously, cells with homozygous knocked-in alleles can be identified by yellow fluorescencein the Merge panel. BFP (blue) indicates all electroporated cells. Scale bar, 50 μm.

35Transplanted iPSC-derived retinal cells not
rejected if MHC-matched with recipient

Sunao SUGITA

Cells of an animal’s immune system are capable of differentiating between self and non-selfcells by recognizing proteins expressed on thecell surface called, major histocompatibility complexes(MHC). In humans, these cell surface proteins are called
human leukocyte antigens (HLA). When immune cells
encounter MHCs they do not recognize, an immune response is triggered to rid the body of the foreign entity.
This mechanism is important for protecting the body from
pathogens, but can be a hindrance when considering cellor organ transplantation from one individual to another.
A research team led by Sunao Sugita, deputy project leader of the Laboratory for Retinal Regeneration (Masayo Takahashi, Project Leader) have now
demonstrated experimentally that immune responses
can be avoided when retinal pigment epithelial (RPE)
cells generated from induced pluripotent cells (iPSCs)
derived from a MHC/HL A homoz ygous donor are
matched with MHC/HL A of recipient. The researchteam developed an in vivo experimental method in a
primate model, in which they transplanted RPE cells
derived from iPSCs from a MHC homozygote monkey
into a different monkey, and an in vitro experimental
system using RPE cells derived from human iPSCs andco-cultured them with human immune cells, including

36

T cells. Their study was published in two separate papers in Stem Cell Reports.
The laboratory has been working to establish treatments for retinal diseases using iPSC-derived cells.
They developed a method to generate RPE cells fromhuman iPSCs, and in 2014, performed the first autologous RPE transplantation into a human patient with agerelated macular degeneration (AMD), a disease thatdamages the RPE cell layer of the retina. As the original
cell source for autologous transplantation is the patientsthemselves—iPSCs derived from patient’s own cells
are differentiated into RPE cells, and later transplantedback into the patient—the potential for rejection wasconsidered minimal. However, as this method is costly
and time-consuming, the group has been considering
allogeneic transplantation (iPSC-derived cells generated
from one person transplanted into another person) as
an approach to overcome these issues. While allogeneictransplantation raises concerns for risk of immune rejection and need for use of immunosuppressant drugs, intheory, matching MHC/HLA of graft and recipient should
reduce potential for immune rejection, similar to organtransplantation, but this had not been tested before for
iPSC-derived cells.

Sugita S, et al. Lack of T-cell response to iPS cell-derived retinal
pigment epithelial cells from HLA homozygous donors. Stem Cell
Reports 7(4). 619–634 (2016)
Sugita S, et al. Successful Transplantation of Retinal Pigment Epithelial Cells from MHC Homozygote iPSCs in MHC-Matched Models.
Stem Cell Reports 7(4). 635–648 (2016)

The team generated RPE cells and sheets from monkey
iPSC lines with an MHC homozygous profile, and thentransplanted these RPE cells or sheets into the sub-retinal space of the eyes of MHC-matched or mismatchedmonkeys. In MHC-mismatched monkeys, the teamobserved retinal tissue damage and signs of immune rejection, such as infiltration of inflammatory cells into thesubretinal space, following transplantation. On the other
hand, no signs of immune rejection were observed inrecipients of MHC-matched grafts, and the sheet grafts
survived for at least six months post-transplantation
without the need to use immunosuppressant drugs.

Modified from article and licensed under CC BY-NC-ND 4.0 (creativecommons.org/licenses/by-nc-nd/4.0/) © 2016 The Authors.

Sugita and his colleagues also examined immunogenicity using an in vitro experimental system with human
iPSC-derived RPE cells. They generated iPSC lines from
HLA homozygous donors, and then differentiated theminto RPE cells, which were then co-cultured with lymphocytes isolated from a different donor. The researchers found that when HLA types were mismatched, there
was an increase in inflammatory cell numbers as well as
a rise in levels of IFNɣ inflammatory cytokine, similar towhat is observed in early stages of an immune response.
In contrast, the matching of at least three HLA gene loci

(HLA-A, HLA-B, and HLA-DRBI) between iPSC-derived
RPE cells and the donor immune cells was sufficient for
preventing the trigger of an immune response. The HLA
matching of these three loci have been known to be critical when considering organ transplantation or bone marrow transplants, and the present study demonstrated forthe first time that the same is true for allogeneic transplantation of iPSC-derived RPE cells.
The laboratory announced the start of a new clinicalresearch study in June 2016, which includes plans to
use the iPSC bank established at the Center for iPS Cell
Research and Application (CiRA) at Kyoto University toderive RPE cells for allogeneic transplantations.
“The potential risks posed to the whole body in using immunosuppressant drugs is a high price to pay when considering the size of the eye. But our current study showsthat these drugs may not be needed if at least three HLAloci are matched,” explains Sugita. “While RPE cells
were the focus of our studies, the in vivo and in vitro experimental systems that we developed may be adaptableand prove useful for other groups working towards iPSCbased clinical applications in other tissues or organs.”

Schematic of in vivo experimental model using
monkeys. When RPE cells generated from monkey MHC homozygote iPSCs were transplanted
into MHC-matched recipient monkey (illustrated
as blue gene) no immune rejection was observed.

Left: When MHC-mismatched, an inflammatory nodule with many infiltrating inflammatory cells was observed around the graft.
Right: When MHC-matched, no signs of rejection were observed and graft survived in subretinal space.

37

Disoriented axons lead to olfactory disorder after
traumaAya MURAI

O

lfaction or sense of smell, while not critical for
life, can greatly influence the quality of life. The
olfactory system is capable of detecting anddiscriminating a wide range of airborne odor moleculesin the air; odorants inspired into the nasal cavity contact
odorant receptors (ORs) expressed by olfactory sensory
neurons (OSNs) of the olfactory epithelium, and the OSNs
then relay the information via the glomeruli in the olfactory bulb (OB) to the mitral and tufted cells, the second
order neurons in the olfactory system. While the olfactory
neural circuits are stably maintained throughout life, damage to OSN axons due to head trauma in adults can leadto an olfactory disorder called dysosmia, where patients
experience reduced odor sensitivity as well as unpleasant perception of different odors. It has yet to be shownhow the neural circuitry is affected after OSN axon injury,
which may yield hints for ways to treat dysosmia.
Junior research associate Aya Murai and others in the
Laboratory for Sensory Circuit Formation (Takeshi Imai,Team Leader), in collaboration with Okayama University
Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, carried out a detailed analysis of amouse model for dysosmia, and discovered that during
the recovery process following axonal injury, OSN axonal
targeting was impaired and the connectivity between ax-

38

ons of OSNs and dendrites of mitral cells was also markedly reduced. Their study, published in the online journal
eNeuro, unveiled the mechanism underlying dysosmia
pathogenesis.
The neural circuitry of the olfactory system is elaborate
and complex. Each OSN expresses a single OR type,
and OSNs expressing the same ORs all converge theiraxons to one glomerulus in the OB. Olfactory information is interpreted from the activation patterns of over
1,000 sets of glomeruli, stimulated by over 1,000 typesof OSNs. Unlike other neurons, OSNs are unique in thatthey can be regenerated, and undergo continuous turnover throughout life, while maintaining the original topographical circuitry. After damage to OSN axons by headtrauma, newly generated OSNs often show impaired
axonal connections, but why this occurred was not wellunderstood.
The team surgically transected a portion of OSN axons
projecting to the olfactory bulb to generate a mousemodel simulating axonal injury following head trauma,
and then examined the recovery process of the OSN,
searching for the mechanism underlying dysosmia. Normal OSN turnover takes place in two phases; first, OSN
roughly determine the anteroposterior course to projectMurai A, et al. Distorted Coarse Axon Targeting and Reduced
Dendrite Connectivity Underlie Dysosmia after Olfactory Axon Injury.
eNeuro 3. ENEURO.0242–16 (2016)

their axons, and then as axonal projection progresses,
will refine their path as needed to their glomerulus target.
In contrast, in the axonal injury mouse model, the initial
anteroposterior course targeting of axons was often impaired, with axons extending a direction opposite to thatseen in normal OSN turnover. Their experiments also
revealed a concentration of mistargeted axons in moreanterior region of olfactory bulb and formation of small
glomerulus-like structures innervated by heterogeneous
mix of OSN axons.

Modified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 Murai A, et al.

They also analyzed the mitral cells, which relay signals
received from the glomerulus to the olfactory cortex.
Mitral cells are normally connected to the glomerulus
through a single primary dendrite, but after OSN axonal
injury, only half of the mitral cells were found to maintainconnections with OSNs, and the arborized tips of the

primary dendrites showed signs of atrophy contributing
to reduction in connectivity. Live imaging in vivo of mitral cell activity also confirmed that axonal injury model
showed reduced olfactory sensitivity than normal mouse.
“When OSN axons are severed due to severe headtrauma, we saw impaired targeting of axons as well as
reduced connectivity to mitral cells. This suggests thatin normal OSN turnover, the existing OSN axons act aspart guide and part scaffold to direct new OSN axons tothe correct target,” explains Imai. “Thus, degenerationand eventual loss of ‘scaffold’ axons after injury appears
to be the underlying cause of dysosmia. It may be possible to prevent dysosmia if OSN axon degeneration after
injury can be delayed enough to allow newly generating
OSNs axons to be guided to their correct targets. We
would like to continue seeking hints to treat dysosmia.”

Left: Olfactory bulb viewed from above. After axonal transection, axonal projections show a large anterior
shift (green: OSNs expressing MOR29B, one type of OR; scale bar 500 μm). Right: Cross-section of olfactory
bulb viewed from front. Nrp1-positive axons normally targeted to the posterior were mistargeted to anterior.
Nrp1-positive and -negative axons were jumbled and formed small glomerular-like structures (blue, nucleus;
green, ONS; red, Nrp1; scale bar 100 μm).

Left: Arborization of mitral cell dendrites is greatly reduced after axonal transection (blue, nucleus; green,
mitral cell). Right: When the mitral cell dendrites were traced, they were found to rarely innervate the glomerular layer, and showed less arborization. Scale bar, 100μm.

39One signal, two cell fates

Guangxia MIAO

T

ubular networks in the body are essential for transporting fluids such as blood through blood vessels,
or promoting exchange of gases in the trachea andbronchiolar structures of the respiratory system. Formation of tubular networks are triggered by signaling factors secreted during early stages of development, which
directs some cells to become so-called ‘leading’ tip cells
to begin protruding outward from an initially sphericalepithelial cyst and lead ‘follower’ stalk cells to specific locations to form the branching structures. In tracheal tube
formation, FGF signaling is known as the major branching
signal for specifying tip cells. Whereas in most animal
species only one tip cell is specified, in Drosophila, two
tip cells are in fact specified by FGF. Interestingly, these
two tip cells will later differentiate into two distinct cell
types. One becomes a fusion cell (FC) that connects
branches of tracheal primordia from adjacent body segments together by undergoing topological change into adonut shape, and the other differentiates into a terminal
cell (TC), which acquires a complex bifurcating and lumenal structure, and functions similar to bronchioles. The
mechanism underlying the divergent cell fates of tip cells
remained poorly understood.
A study led by research scientist Guangxia Miao of
the Laboratory for Morphogenetic Signaling (Shigeo

40

Hayashi, Team Leader) zoomed in on the Drosophila tracheal primordia and the mechanisms involved in sequential specification of two tip cell fates by a single inductive FGF signal. Their work, published in Development,
revealed that whereas FGF initially specifies FC fate anddirects branch migration to their destination, once there,
the expression of transcription factor Escargot (Esg) suppresses FGF signaling in the FC and permits activationof FGF signaling in an adjacent cell to differentiate into
a terminal cell (TC), resulting in two cells with distinct
properties.
FGF signaling has been known to play multiple roles
during tracheal tube formation. First, it specifies the FCtype of tip cells that will lead branch migration. Following specification, FCs will in turn inhibit FGF signaling inneighboring cells through activation of Notch-Delta, converting them into stalk cells that will trail behind tip cells.
FGF signaling is also involved in guiding FC migrationand differentiation of tip cells into TCs. How this single
FGF signal can control multiple aspects of cell differentiation and cell motility was unclear.
The team began by analyzing the distribution of thecells in which FGF signaling was activated. During earlystages of tracheal development, downstream factors ofModified from article and licensed under CC BY 3.0 (creativecommons.org/licenses/by/3.0/legalcode) © 2016 Miao G, et al. / © 2016 RIKEN CDB, Lab. for Morphogenetic Signaling

Miao G, et al. Escargot controls the sequential specification of two
tracheal tip cell types by suppressing FGF signaling in Drosophila.
Development 143. 4261–4271 (2016)

FGF signaling were activated in a small group of cells
in the primordium that includes the future FC and TC,
but by stages 13 to 15, FGF signaling in these cells, withthe exception of the TC, was lost. The team then turned
their attention to Esg, whose expression appeared to belinked with FC specification. Analysis of Esg mutant flies
revealed that FCs in these mutants failed to fuse with its
partner FC, continued to migrate and elongate, and as
seen in TCs, formed bifurcations. Further, Esg-mutant
FCs were confirmed to show TC-like properties such asactivated FGF signaling and expression of TC-specificmarker genes. These findings indicated that Esg is expressed specifically in FCs, functioning to suppress FGF
signaling and prevent FCs from differentiating into TCs.
When the team used live-imaging to track FC and TC
positions, they found that between stages 14 to 15, FCs
and TCs always undergo an anterior-to-posterior positional shift. This positional shift appeared to be criticalfor establishing the tracheal network, but it was not clear

what signal was controlling this event. Using the technique previously developed by the lab, in which ectopic
FGF expression could be induced at any place and timeby laser irradiation, they examined the behavior of FCs
and TCs to FGF expressed ectopically in different areas
around the nascent tube. FCs moved away from FGF
signals, whereas TCs moved toward FGF signals andcreated new bifurcations. In Esg mutants, FCs lost its
repulsive response to ectopic FGF. Thus, the positional
shift is brought about by the difference in responses between FCs and TCs to FGF signals.
“Analogous to train track switching devices, Esg coordinates time-lagged cell responses of tip cells to FGF,
which results in two cells with distinct properties,” explains Hayashi. “There are more cell types in the bodythan there are signaling factors. Thus the cells must adopt
strategies allowing it to reuse a single signal to generate
cell type diversity, and this study gives a glimpse into one
such mechanism that evolved in organisms.”

Left: Diagram and microscope image of branching morphogenesis of Drosophila trachea system. Tip cells and stalkcells are specified at stage 14, and the tip cells differentiate into FC and TC at stage 15. Anterior is to the left, andposterior is to the right. Right: The TC is a single cell, but is capable of acquiring a complex bifurcating and lumenal
formations, and functions similarly to mammalian bronchioles.

Left: In Esg mutants, FCs fail to fuse with their partner FCs, resulting in multiple branching. Right: Prospective
FCs in Esg mutants become TC-like, resulting in two TCs. Scale bar, 10μm.

41

In vitro model for spinocerebellar ataxia
developed using patient-derived iPSCs

Keiko MUGURUMA, Yoshihito ISHIDAT

he cerebellum is a vital region of the brain intimately involved in the regulation of motor coordinationand some aspects of cognition. Disorders affecting the cerebellum can be debilitating, leading to lossof speech articulation and uncoordinated and unsteady
gait. One group of hereditary genetic diseases linked tothe cerebellum is spinocerebellar ataxia (SCA), an intractable disorder characterized by progressive loss of
motor coordination, while the patients retain full mental
capacity. There are numerous types of SCA and many
causative genes have been identified, but much remains
unknown about underlying molecular mechanisms of
SCA pathogenesis and no effective treatments are currently available for this disease. Thus, there is a need fora suitable model system to study SCA pathogenesis andto screen for drug compounds that may be effective fortreating SCA.

Now, a study by CDB visiting scientist Yoshihito Ishida*1
and research specialist Keiko Muguruma of the Laboratory for Cell Asymmetry has reported the successful
generation of mature Purkinje cells from induced pluripotent stem cells (iPSCs) derived from SCA type 6 (SCA6)
patients. They further revealed that when placed underconditions of stress, the patient-derived Purkinje cells
exhibit some vulnerabilities, and demonstrated that this42

stress culturing method can be used as an in vitro model
system for evaluating drug candidates for treating SCA.
Their work, published in the journal Cell Reports, wascarried out in collaboration with Hiroshima University
and Kyoto University.
SCA6 is characterized by selective degeneration andloss of Purkinje cells in the cerebellum, and cerebellar
atrophy. It is caused by a mutation in the CACNA1A geneencoding the Cav2.1, the α1-subunit of a calcium channel. The mutated gene has an expanded CAG trinucleotide repeat in the region encoding the Cav2.1 C-terminal
domain, resulting in an abnormally long section of glutamine residues (or polyQ). Muguruma, who previously
reported the successful generation of mature Purkinjecells from human embryonic stem cells and iPSCs, andher collaborators wondered whether it would be possible
to simulate pathology of SCA6 in vitro by applying their
protocol for Purkinje cell differentiation using patientderived iPSCs.
The team generated iPSCs from skin or blood cells of
healthy donors and of SCA6 patients with heterozygous
alleles or homozygous alleles for CAG repeats, and confirmed that their established iPSC lines have the same
number of CAG repeats as their respective donors.

Ishida Y, et al. Vulnerability of Purkinje Cells Generated from Spinocerebellar Ataxia Type 6 Patient-Derived iPSCs. Cell Rep 17(6):1482–1490 (2016)

Modified from article and licensed under CC BY-NC-ND 4.0 (creativecommons.org/licenses/by-nc-nd/4.0/legalcode) © 2016 The Authors.

The iPSCs were then differentiated into Purkinje cells,
and similar to known SCA pathology, patient-derived
Purkinje cells showed high cytoplasmic levels of Cav2.1.
A fragment of the C-terminal domain of Cav2.1 containing the CAG repeat, called α1ACT, also functions as atranscription factor targeting TAF1 in the nucleus, whichis involved in cell growth and development. Expression
levels of α1ACT and TAF1 were found be to markedly
reduced in patient-derived Purkinje cells than in control
Purkinje cells, which was consistent with reports fromother studies indicating that α1ACT containing CAG repeat expansion loses ability to activate transcription of
TAF1 and consequently inhibits neural cell growth anddevelopment.
Recapitulating in vivo disease pathology in a cell culture
system within a relatively short period presented a challenge as SCA6 is a late-onset disease, meaning symptoms begin appearing later in life. Thyroid hormone T3 isknown to be an essential factor for Purkinje cell maturation and maintenance, the group temporarily depleted
T3 from their culture system to create conditions ofstress and analyzed its effect on Purkinje cells. Whereas
Purkinje cells from healthy donors showed no significant
changes, many of the patient-derived Purkinje cells diedor if they did survive, had significantly reduced dendritic
arborization, indicating that T3 depletion caused vulnerability in patient-derived Purkinje cells.

Left: Cav2.1 accumulation levels were higher in SCA6 Purkinje cells than healthy
ones, with homozygous SCA6 Purkinje cells showing highest accumulation. Right:
In contrast, α1ACT and TAF1 expression levels were lower in SCA6 Purkinje cells
than control Purkinje cells, with homozygous SCA6 Purkinje cells showing lowest
expression levels.

The group also explored the possibility of using theirpatient-derived Purkinje cells following stress for drug
assays, testing different compounds on their effectiveness in reducing the vulnerabilities observed in patientderived Purkinje cells. They found that thyroid releasing
hormone (TRH), which has been used in treatments for
SCA6, and a compound called Riluzole, which has beenused in treatments for another neurodegenerative disorder called ALS, both displayed positive effects in improving survival and maintenance of patient-derived Purkinjecells.
“The strength of our in vitro model comes from combining the techniques for generating human iPSCs frompatients with those for inducing self-organizing cerebellar tissues and Purkinje cells to recapitulate ontogenesis,” explains Muguruma. “Other types of SCA involve
degeneration of other neural cells in the cerebellum aswell as in the cerebrum. With our expertise in generating
cerebellar and cerebral tissues from disease-specific
iPSCs, we hope to contribute to unveiling the pathology
of different SCA types and other neurodegenerative
diseases.”*1 V isiting scientist Yoshihito Ishida is employed by Shionogi &Co. Ltd., however the company was not directly involved inany aspect of this study.

Depletion of T3 from culture media created stress conditions,which led to degeneration of patient-derived Purkinje cells.
Addition of TRH or Riluzole could stave off the vulnerabilities
caused by T3 depletion. Scale bar, 20 μm.

43DAAM1 stabilizes adhesion between lateral
membranes of epithelial cells

Tamako NISHIMURA

E

pithelial cells are arranged as polarized monolayer
sheets and are often found lining the surface or lumen of organs. The epithelial cell edge that comes
into contact with fluids or the external environment isreferred to as the apical side, while the opposite side that
attaches to connective tissues is referred to as the basalside. To maintain the sheet-like configuration, epithelial
cells attach to each other along their lateral membranes
through a number of special junctional structures. Near
the apical side is a complex of ‘tight junction’ and ‘adherens junction,’ known as the apical junctional complex(AJC), which is lined with a bundle of actin filaments (Factin) that lies beneath the apical membrane forming acircumferential actin belt, and has been well studied todate. Below the AJC is a junctional structure called lateral
membrane contacts (LCs), which spans most of the lateral
membrane region and whose functions are not as well
documented.
A study led by visiting researcher Tamako Nishimura in
the Laboratory for Cell Adhesion and Tissue Patterning (Masatoshi Takeichi, Team Leader), published in
the Journal of Cell Biology, took a close look at LCs ofepithelial cells and uncovered a role played by DAAM1,
a formin family protein associated with actin polymerization, in stabilizing the adhesion of lateral membranes

44of adjacent epithelial cells. They also revealed the molecular mechanism underpinning instability of LCs in theabsence of DAAM1.
In a past study from the same laboratory, also led by Nishimura, in which they reported the mechanism involved
in apical constriction of neural epithelial cells duringneural tube formation, DAAM1 was identified as one
factor recruited to the cell-cell adhesion sites to mediate constriction. Studies by other groups have reported
observations of abnormal sarcomere formation andcell-cell adhesion between cardiomyocytes in DAAM1
mutant mouse, and of reduced DAAM1 expression possibly contributing to invasiveness of astrocytoma cells,
one type of brain tumor cells; thus, suggesting DAAM1
playing another role in regulation of cell-cell adhesion(junctions).
Nishimura’s team used a mouse mammary gland epithelial cell line to first examine the localization of DAAM1,
and found that it accumulated at epithelial junctions
along the lateral membrane. F-actin was also distributed along the lateral membrane, overlapping with thatof DAAM1, and differing from F-actin distribution seenin the apical actin belt. When DAAM1 was knocked
down, they observed reduced F-actin localization along

Nishimura T, et al. DAAM1 stabilizes epithelial junctions by restraining WAVE complex-dependent lateral membrane motility. J Cell Biol
215(4). 559–573 (2016)

the lateral membrane, as well as larger tilt angles of LC
membranes. Time-lapse imaging revealed more dynamic
lateral membrane movements in DAAM1-depleted cells
compared to control cells. When cells were cultured in agel matrix for several days, control cells formed spherical cysts, with cells arranged in radial manner, whereas
DAAM1-depleted cells formed disorganized aggregates,with some cells appearing to be extruded out from theaggregates. Interestingly, in co-culture of control and
DAAM1-depleted cells, DAAM1-depleted cells extended
long protrusions into nearby cell layers, reminiscent of
behavior seen in invasive cells, when flanked by control
cells. Hence, these results suggest that DAAM1 mediates stability of cell junctions through F-actin, which inturn contributes to maintenance of epithelial integrity.

Modified from article and licensed under CC BY-NC-SA 4.0 (creativecommons.org/licenses/by-nc-sa/4.0/) © 2016 Nishimura T, et al.

The team next investigated why the LCs become unstable after DAAM1 depletion. During normal cell migration,
cell edges in the direction of migration move dynamicallyforming lamellipodium, and its motility is known to be
mediated by a molecular complex called the WAVE com-

plex. The team thus speculated that the WAVE complex
also plays a role in LC instability following DAAM1 depletion. They analyzed the WAVE complex components
in DAAM1-depleted cells and identified WAVE2 and its
upstream regulator Rac as effectors of LC instability, resulting in acquired membrane motility.
“Our current study demonstrated that DAAM1 maintains stability of epithelial cell junctions by suppressing
WAVE complex-mediated membrane mobility,” explains
Nishimura. “It was surprising to discover that epithelial
cells, which are considered stable when packed together, in fact harbor the potential to acquire motility.”
Takeichi adds, “The WAVE complex–mediated motilityof LC membranes is likely critical for repair mechanisms
involving cell migration, such as wound healing. At thesame time, errors in DAAM1 regulation of the WAVE
complex may lead to cancer cell-like invasive behavior.
Therefore, detailed studies on DAAM1 functions in vivo
may reveal clues into mechanisms of cancer invasion.”

Left: DAAM1 was localized at LCs of epithelial cells. When cells viewed from a direction perpendicular to the cell layer, DAAM1 localization was seen to overlap with F-actin distributed in lateral membrane (arrowheads) as opposed to F-actin localized in the apical actin
belt (arrows). Right: Tracings of relative signal intensity of DAAM1 and F-actin distributed along dotted line ‘a’ in the image to the left.

Left: DAAM1 knockdown results in abnormal tilting of lateral membrane faces, and exhibit more dynamic motility of the lateral membrane than control cells. Right: When cells were cultured in Matrigel for several days, control cells formed cysts in which cells were arranged radially, whereas DAAM1-depleted cells showed disorganized aggregation with some cells being extruded from the cell mass.

45

Lulling mice into a short hibernation-like state

Masayo TAKAHASHI, Genshiro SUNAGAWA

T

he approach of winter signals the start of the hibernation period for some animals, such as squirrels and bears. Hibernation, a period of days or
weeks of inactivity, can be considered an extreme formof energy conservation as some animals are known tolower their metabolic rates to just a few percent of normal
awake levels, thereby reducing demand for oxygen during this period. A shorter hibernation-like state (minutes
to hours) called torpor is also induced in some animals,
and is marked similarly by active reduction in metabolism(hypometabolism). The underlying mechanisms regulatinghibernation and torpor remain largely unknown as hibernating and torpor species are few and far between, making them difficult subjects to use in research. Recently,
however, there have been reports that mice, which arecommonly used in research, can undergo torpor undercertain conditions, hinting at possibilities of studying thisphenomena in the laboratory. Understanding how active
hypometabolism can be induced may benefit humans aswell in clinical applications, where reducing oxygen consumption by inducing hypometabolism could slow down
progress of brain damage after stroke, or help prolong
storage periods for organs or tissues for transplantation.
In a study led by research scientist Genshiro Sunagawa
in the Laboratory for Retinal Regeneration (Masayo Taka-46

hashi, Project Leader), published in the online journal
Scientific Reports, they created an algorithm for detecting torpor based on actual measured values of baseline
metabolism of individual mice, which can be used tostably observe and evaluate torpor states of mice. The
team further analyzed the mouse thermoregulatory system between normal (awake) and torpid states to reveal
a common feature between torpor and hibernation.
Warm-blooded animals have an internal thermoregulatory system to maintain a relatively stable body temperature. When temperatures in the outside environment
drop, the body is more prone to losing heat, and consequently begins to actively generate heat to compensate
for the heat loss. Normally, if this mechanism stops
functioning and the body temperature falls below a certain threshold or reference temperature, the tissues andcells incur damages and eventually die, but this does
not occur in hibernating or torpid animals. Instead, theseanimals appear to possess a mechanism that signals
to the body to abandon or suppress its normally active
thermoregulatory system.
Torpor takes place over a shorter period and is more unstable compared with hibernation, and even shows great
variation between individual animals. This has made itSunagawa G A and Takahashi M. Hypometabolism during Daily
Torpor in Mice is Dominated by Reduction in the Sensitivity of the
Thermoregulatory System. Sci Rep 6. 37011 (2016)

difficult to clearly define and evaluate states of torpor.
The team prepared a chamber fitted with sensors tomeasure and record body temperature and oxygen consumption of mice under controlled ambient temperature
conditions. Baseline metabolism of the individual mice
were calculated from the data collected over 24 hoursof observation during which time they had no physicalcontact with researchers, and values falling outside therange of the baseline metabolism (outliers) were definedas ‘torpor.’ Using their model to evaluate torpor states,
they found that a constant ambient temperature between
12°C to 24°C and restricting access to food for 24 hours
could stably and reliably induce torpor in mice.

Modified from article and licensed under CC BY 4.0 (creativecommons.org/licenses/by/4.0/legalcode) © 2016 Sunagawa G A, et al.

The team then created a mathematical model for the
body’s thermoregulatory system in response to ambient
temperature changes, and using the data collected fromthe above experiments, estimated the parameters thatchanged significantly. Parameters included how easily
the body loses heat, the reference temperature the bodyis trying to maintain, the actual body temperature, andthe sensitivity of the thermoregulatory system. During hibernation, both the reference temperature and sensitivityof the thermoregulatory system show a dramatic reduction. In torpid mice, they find that reference temperatureshows only minimal changes, while the sensitivity of thethermoregulatory system falls to levels similar to thatseen in hibernation.
“Our study demonstrates that a common feature of hibernation and torpor is that sensitivity of the thermoregulatory system is significantly reduced during these states.
This suggests that to induce active hypometabolism
there is either a signal relayed to the body to suppress or
reduce thermogenesis, or there is a mechanism signaling
to the body to ignore the default thermogenesis system.
Our goal is to unveil the complete mechanism regulating
this phenomena,” explains Sunagawa. “This work also
paves the way to use mice as a model to understand active hypometabolism, as well as seek possibilities for applying hypometabolism in clinical applications.”

Left: Temperature-controlled mouse chamber. Mouse cages were placed in the temperature-controlled chamber, and oxygen consumption was recorded. A
transmitter to measure body temperature was implanted into the mouse. Right: Based on actual measured values, torpor was defined as outliers from thebaseline metabolism (black dots).

47

HITI: An innovative in vivo genome-editing
technology

Targeted genome-editing technology has generated great interest for biomedical research fields
for its potential in clinical applications to treat or
improve symptoms of some genetic diseases. In particular, the development of the CRISPR/Cas9 system has
revolutionized the biology field with the ease and precision in which the genome can be manipulated. This innovative system exploits the activity of CRISPR/Cas9, thebacterial immune system used to respond to pathogen
invasion, and the cell’s intrinsic DNA repair mechanism,
either via non-homologous end joining (NHEJ), which
is seen in both proliferating and non-proliferating cells,
or homology-directed repair (HDR), which is used only
in proliferating (dividing) cells. Using CRISPR/Cas9 in
combination with NHEJ-mediated or with HDR-mediatedmethods have proven useful for knocking out functionof genes through errors caused by small DNA sequenceinsertions or deletions at target sites during NHEJ repair
or specific sequence modifications in gene of interest. In
contrast, gene knock-ins have been more challenging, as
this usually utilizes the HDR-mediated mechanism, whichis unavailable in differentiated cells. Thus, if the NHEJ
mechanism, which is found in both proliferating and nonproliferating cells, can be exploited for efficient targeted
gene integration, it would pave the way for potential use
in vivo, where the majority of cells are non-proliferative.
Research scientist Yuji Tsunekawa in the Laboratory for
Cell Asymmetry (Fumio Matsuzaki, Team Leader), together with Keiichiro Suzuki and Juan Carlos Izpisua Belmonte at the Salk Institute of Biological Studies, USA, andother collaborators, developed a novel genome-editing
technology, which exploits the NHEJ repair mechanismwith the CRISPR/Cas9 system called, homology-independent targeted integration (HITI). This technology allows them to insert transgenes into both proliferating andnon-proliferating cells efficiently. Their work, publishedin Nature, further demonstrates that HITI can be used forgene editing in vivo in the mouse and rat models.
HITI donor vectors were constructed to ensure robust
gene integration only when inserted in the forward direction, and if inserted in the reverse direction or is unintegrated, the DNA would undergo further cleavage by Cas9
until inserted correctly or gRNA is no longer able to bindto target sequences due to errors during NHEJ repair(see scheme). When the HITI donor vector was tested inhuman cell lines, the team observed gene integration attarget sites at efficiencies approximately ten times higher
than conventional HDR-mediated methods. Effectiveness of HITI in non-proliferating cells was also examinedusing mouse primary neurons, in which they found gene
insertion in 60% of transfected cells.

Yuji TSUNEKAWA

48

Suzuki K, et al. In vivo genome editing via CRISPR/Cas9 mediatedhomology-independent targeted integration. Nature 540. 144–149(2016)

Adapted by permission from Macmillan Publishers Ltd: Nature 540. 144-149, copyright 2016/ © 2016 RIKEN CDB

The team next tested whether their method can beused in non-dividing cells in vivo. The adeno-associatedvirus (AAV) expressing Cas9 systems and HITI donor
was injected directly into different tissues of adult mice,such as the brain and muscles, and they confirmed thattransgenes were correctly knocked in some cells in thevicinity of injection sites. When HITI-AAV vectors weredelivered intravenously just after birth, gene insertion was
confirmed in a small percentage (3–10%) of cells withinvarious tissues of the body. The team also injected their
HITI-AAV vector in the eye of a rat model for retinitis pigmentosa (RP), after which they detected a rise in mRNA
expression levels of Merkt, a gene implicated in RP, aswell as thickening of degenerating photoreceptor layer.

“As efficiency of gene insertion in vivo is still low andsome mistargeting and mutations of target genes do
occur, there is still much work that need to be done toimprove the HITI method before it can be used in clinicalapplications. Nevertheless, our study clearly demonstrates that in vivo genome editing is possible,” explains
Tsunekawa. “The HITI technology is sure to become apowerful tool not only for clinical applications, but also
for basic research. It can be used to visualize and track
fates of specific cells by inserting fluorescent marker
genes, or modify genomes in animals, such as non-human primates, for which generation of transgenic models
has been difficult.”

Scheme of HITI method, which uses NHEJ-mediated targeted integration. If cleavage is repaired without gene integration or with integration in reverse direction, DNA undergoes additional cleavage until forward gene integration or gRNA can no longer bind to target cleavage sequence due to errors from NHEJ repair.

Targeted GFP knock-in using HITI in adult mouse neurons in vivo. HITI-AAV was constructed so GFP cassette would be inserted downstream of Tubb3 gene, which is expressed in neurons. When HITI-AAV was injected directly into adult mouse brain, manyneurons expressing GFP were observed, indicating GFP gene was correctly integrated(lower panels), whereas in the control, cell transfection was observed with very little integration (minimal GFP signals) was detected (top panels).

49

Laboratories

Cerebellar Purkinje cells generated from healthy donor-derived iPSCs. L7 (green) and
GRID2 (red) are Purkinje cell-specific markers, and DAPI (blue) marks cell nuclei.
Image: Keiko Muguruma, Laboratory for Cell Asymmetry

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 Kyoto University 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 research scientist
until 2010. That year, he was appointed as Deputy Unit Leader of the Four-dimensional Tissue
Analysis Unit within the Division for Human Stem Cell Technology, and in 2013, was promoted
to Unit Leader. From November 2014, he has served as Team Leader of the Laboratory for invitro Histogenesis.

Staff
Senior Researcher

Masatoshi OHGUSHI
Research Scientist

Shunsuke MORI
Yusuke SETO

Special Postdoctoral Researcher

Satoru OKUDA

Research Associate

In vitro generation of a functional organ with complex structures is one major goal for the field ofdevelopmental and cell biology. To achieve this goal, it is a reasonable strategy to recapitulate
the ontogeny, which is the most efficient and robust process for organogenesis that has beenacquired through evolution, in an in vitro system. Our laboratory aims to clarify the molecular andcellular dynamics underlying organogenesis, and to develop new technologies for in vitro recapitulation, that is, three-dimensional functional organ formation from stem cells. These researches
aim to make important contributions to the field of developmental biology, stem cell biology, andregenerative medicine.

Yuiko HASEGAWA
Visiting Scientist

Takao KURODA
Atsushi KUWAHARA
Daiki NUKAYA
Morio UENO
Technical Staff

Masako KAWADA
Eriko SAKAKURA
AssistantFumi WAGAI

Recent Publications
Hasegawa Y, et al. Emergence of dorsal-ventral
polarity in ESC-derived retinal tissue. Development 143 (21). 3895–3906 (2016)
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 EES
cell-derived neocortex. Proc Natl Acad Sci U S
A 110. 20284–9 (2013)
Nakano T, et al. Self-formation of optic cup
and storable stratified neural retina from human
ESCs. Cell Stem Cell 10. 771–85 (2012)

Self-organized optic cup formation from ES cells

Emergence of dorso-ventral polarity in ES cellderived optic cup

52

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 Institute, where he completed a second postdoctoral fellowship before
returning to Japan in 2012 to take a position as a Team Leader at the RIKEN CDB.

Staff
Research Scientist

Ritsuko MORITA
Ko TSUTSUIVisiting Scientist

Natsumi SAITO
Norio UEMATSU
Technical Staff

Noriko BAN-SANZEN
Student

Hiroki MACHIDA
Visiting Student

In our bodies, we have millions of different environments in which cells reside, which are known
as cellular or tissue microenvironments. These specialized tissue microenvironments instruct thefate and behaviors of cells. The aim of our lab is to gain a better understanding of the mechanisms
underlying the ways in which tissue microenvironments are regionally specialized, and how these
specialized microenvironments in turn instruct cell behavior, cell-cell communication, and organ
formation. Our projects are focused on understanding 1) the extrinsic regulation of stem cells and2) the role of extracellular matrix (ECM) heterogeneity in organogenesis, using mammalian skin as amodel. A more in depth knowledge of these mutually related research focuses will provide a molecular basis to further understand how microenvironments regulate stem cells and organ formation,and for developing tailor-made microenvironments for different lineages of stem cells in the skin.

Kei HASHIMOTO
Part-Time Staff

Takuya OKAWARA
Assistant

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(4). 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 cellsby 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 labeled with anuclear counterstain (blue).

53

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 received his M.D. and Ph.D. from Okayama University in 1979, andworked at the National Institutes of Health (USA) and Memorial University of Newfoundland(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
Research Scientist

Takahiro IDE
Katsura MINEGISHI
Katsutoshi MIZUNORyo NABESHIMA
Natsumi SHIMIZU

My 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.

Visiting Scientist

Katsuyoshi TAKAOKA
Technical Staff

Yayoi IKAWA
Eriko KAJIKAWA
Hiromi NISHIMURA
Visiting Technician

Kei SHIOZAWA

International Program Associate

Wang Kyaw TWAN
Student Trainee

e7.5

Symmetry Breaking

Lynda LAMRI
AssistantKaori SONE

e8.5

Recent Publications

e9.5

Ｒ

Ｌ

Nodal
Lefty

Patterning

Minegishi K, et al. A Wnt5 activity asymmetry
and intercellular signaling polarize node cells
for breaking left-right symmetry in the mouseembryo. Dev Cell (2017, in press)
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)

e10.5

Organogenesis
Lung

Stomach

Blood
Vesselse11.5

Three steps for generating left-right asymmetry

Yoshiba S, et al. Cilia at the node of mouse embryos sense fluid flow for left-right determination
via Pkd2. Science 338. 226–231 (2012)
Shinohara K, et al. Two rotating cilia in the nodecavity are sufficient to break left-right symmetry
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 mouse embryo.
Nat Cell Biol 13. 743–752 (2011)

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

54

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 wasengaged in research in developmental neuroscience. She moved to Skirball Institute Developmental Genetics Program in 2002, to Gord Fishell’s laboratory where she extended her
research to cell specification in the neocortex. She was appointed Team Leader at the CDB in
September 2007.

Staff
Research Scientist

Pei-Shan HOU
Kenichi TOMA
Tien-Cheng WANG
Technical Staff

Chihiro NISHIYAMA
Part-Time Staff

Reiko ODA

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 fates are 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?

AssistantRisa IMAMURA
Eri KOJIMA

Recent Publications
Toma K, et al. Encoding and decoding time inneural development. Dev Growth Differ 58(1):59–72 (2016)
Toma K and Hanashima C. Switching modes incorticogenesis: mechanisms of neuronal subtype transitions and integration in the cerebral
cortex. Front Neurosci 9. 274 (2015)
Bullmann T, et al. A transportable, inexpensive
electroporator for in utero electroporation. Dev
Growth Differ 57(5): 369–377 (2015)

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 anterior-posterior (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.

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)

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).

The neocortex is composed of distinct neuronal subtypes that establish six 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.

55

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 at 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, and Team Leader of the same group in 2014. 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
Yuki ITAKURA
Yosuke OGURA
Technical Staff

Housei WADA

Part-Time Staff

Ikuko FUKUZYOUNoriko MORIMITU
Assistant

Ryoko ARAKIMai SHIBATA

Recent Publications
Miao G and Hayashi S. Escargot controls thesequential specification of two tracheal tip cell
types by suppressing FGF signaling in Drosophila. Development 143. 4261–4271 (2016)
Otani T, et al. IKKepsilon inhibits PKC to promote Fascin-dependent actin bundling. Development 143. 3806–3816 (2016)
Kato K, et al. Microtubule-dependent balanced
cell contraction and luminal-matrix modification
accelerate epithelial tube fusion. Nat Commun 7.
11141 (2016)

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 the 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 into a respiratory organ. We are particularlyinterested 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 an increase in apical cell area facing the luminal side. A key
question 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 lead to destabilization of tube shape and malformation, resulting in tubule morphology seenin organs under pathological conditions.
Another research area of interest is the mechanism of cell morphogenesis. Here we ask the question to what extent single cells can autonomously organize nanometer scale cellular patterns. Ourstudies have uncovered the role of the cellular trafficking center as an organizer of cell elongation(Otani et al., 2011, 2015, 2016).

Hannezo E, et al. A cortical instability drives
periodic supracellular actin pattern formation inepithelial tubes. Proc Natl Acad Sci U S A 112.
8620–8625 (2015)
Dong B, et al. Rab9 and retromer regulate retrograde trafficking of luminal protein required forepithelial 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 boundaries aremarked green.

56

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 institution in 2003 for his work on Xenopus embryonic development with Masanori Taira. In 2003, he moved to the United States for his postdoctoral training
with David Gilbert at the State University of New York Upstate Medical University and studied
the developmental regulation of DNA replication timing and nuclear genome organization. He
later moved with Gilbert to Florida State University in 2006, and then returned to Japan in 2010
to take a position as Assistant Professor at the National Institute of Genetics. In 2011, he wasawarded 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.

Staff
Research Scientist

Hisashi MIURA
Rawin POONPERM
Saori TAKAHASHITechnical Staff

Akie TANIGAWA
Student Trainee

Marc ABELLADavid MEYER

Part-Time Staff

Yoshiko KONDO

Recent Publications
Shang WH, 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 lyonizationof replication domains during early Mammaliandevelopment. Adv Exp Med Biol 695. 41–58(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)

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.
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 is 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 differentiated state.
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.

Hiratani I, et al. Genome-wide dynamics ofreplication timing revealed by in vitro models ofmouse embryogenesis. Genome Res 20.155–
69 (2010)
Hiratani I, et al. Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol 6. e245 (2008)

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

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

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.

57

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 under
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

Junko HARA
Miwako NOMURA
Student Trainee

Shuhei AIHARA
Aya MURAI
Richi SAKAGUCHIThe 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.

Assistant

Eri YAMASHITA

Recent Publications
Murai A, et al. Distorted Coarse Axon Targeting
and Reduced Dendrite Connectivity Underlie
Dysosmia after Olfactory Axon Injury. eNeuro 3.
e0242-16 (2016)
Ke M T, et al. Super-Resolution Mapping of
Neuronal Circuitry with an Index-Optimized
Clearing Agent. Cell Rep 14. 2718–2732 (2016)
Imai T. Construction of functional neuronal circuitry in the olfactory bulb. Semin Cell Dev Biol
35.180–88 (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)

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.

Super-resolution mapping of neuronal circuitry.
Brain slices of a Thy1-YFP-H transgenic mousewere cleared with SeeDB2, imaged with superresolution microscopy, and reconstructed in 3D.

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

58

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 andInnovation 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, wasappointed 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.

Staff
Research Scientist

Takehiko ICHIKAWA
Shinya MATSUKAWA
Research Associate

Ayumi TAKEMOTO

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

Technical Staff

Setsuko KANAMURA
Kaori NIIMIInternship

Léo ADENIS
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 153.1296–
311 (2013)
Takai A, et al. Anterior neural development
requires Del1, a matrix-associated protein that
attenuates canonical Wnt signaling via the Ror2
pathway. Development 137. 3293–302 (2010)
Inomata H, et al. Robust stability of the embryonic axial pattern requires a secreted scaffold
for chordin degradation. Cell 134. 854–65(2008)
Arakawa A, et al. The secreted EGF-Discoidin
factor xDel1 is essential for dorsal developmentof the Xenopus embryo. Dev Biol 306. 160–9(2007)

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 and zebrafish)
axis formation as a model. The establishment of the frog dorsoventral axis depends on gradientsof 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 seenin 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 intercellular communication
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.

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; 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.

59

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 served as research associate at the Instituteof Molecular and Cellular Biosciences at the same university, before moving to the European
Molecular Biology Laboratory in Heidelberg, Germany as a postdoctoral researcher. He wasappointed Team Leader at the CDB in 2012.

Staff
Research Scientist

Aurélien COURTOIS
Shuhei YOSHIDA
Visiting Scientist

Shu HASHIMOTO
Hirohisa KYOGOKU
Student Trainee

Takeshi ASAKAWA
Yi DING
Namine TABATA
AssistantKaori HAMADA

Recent Publications

The oocyte becomes an egg through meiosis. The egg fertilizes with 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.

Sakakibara Y, et al. Bivalent separation into
univalents precedes age-related meiosis I errors
in oocytes. Nat Commun 6. 7550 (2015)
Yoshida S, et al. Inherent instability of correct
kinetochore-microtubule attachments duringmeiosis I in oocytes. Dev 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

60

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 Schoolof 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 was appointed as professor at Tohoku University in 2016.

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
Kawamoto Y, et al. Apoptosis in Cellular Society: Communication between Apoptotic Cells
and Their Neighbors. Int J Mol Sci 17(12): 2144.
(2016)
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)
Kuranaga E, et al. Drosophila IKK-related kinase
regulates nonapoptotic function of caspases via
degradation of IAPs. Cell 126. 583–96 (2006)

Dorsal (left) and ventral (right) views ofDrosophila pupae that express fluorescent protein in cells located posterior
component of each segment. Yellow
box indicates location of male genitalia.

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 to spatiotemporal information in living systems, such as canbe obtained through the 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 caspases function and how
cell death controls 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 the tissue area, suggesting other mechanisms are also at work. We will conduct single-cell
analyses to determine whether other behaviors such as proliferation or migration are also altered.
Through the use of the extensive Drosophila genetics toolset and live imaging technologies, wehope to be able 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.

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

61

Laboratory for 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.

Staff
Team Leader

Fumio MATSUZAKI
Research Specialist

Keiko MUGURUMA
Atsunori SHITAMUKAI
Special Postdoctoral Researcher

Ikumi FUJITA

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.

Research Scientist

Daijiro KONNO
Yuji TSUNEKAWA
Shigeki YOSHIURA
Visiting Researcher

Wu QUAN

Visiting Scientist

Noriomi EGUCHIMariko IKEDA
Yoshihito ISHIDA
Misato IWASHITA
Taisuke KADOSHIMA
Ayano KAWAGUCHI
Tomoko KITA
Technical Staff

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

Shun MASE
Atsushi SHIRAISHI
Student Trainee

Kalyn KAWAMOTO
Raymond TERHUNE-KUNIKANE
Fumiya KUSUMOTO
International Program Associate

Merve BILGIC

Part-Time Staff

Yoko OTSUKA
AssistantJunko ISHIGAI
Tomoko KAMATANI

Recent Publications
Suzuki K, et al. In vivo genome editing via CRISPR-Cas9 mediated homology-independent targeted integration. Nature 540.144–149 (2016)
Tsunekawa Y, et al. Developing a de novo
targeted knock-in method based on in utero
electroporation into the mammalian brain. Development 143. 3216–22 (2016)
Okamoto M, et al. Cell cycle–independent transitions in temporal identity of mammalian neuralprogenitor cells. Nat Commun 7. 11349 (2016)
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)
Pilz G. A, et al. Amplification of progenitors in
the mammalian telencephalon includes a newradial glial cell type. Nat Commun 4. 2125 (2013)
Yoshiura S, et al. Tre1 GPCR signaling orients
stem cell divisions in the Drosophila central
nervous system. Dev Cell 22. 79–91 (2012)62

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 expansionof the size of the brain, which comprises a larger number ofneurons arranged in a vastly more complex functional network
than that in 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 theindividual states of neural stem cells, and control transitions
between states in mouse as well as mechanisms for generating neural progenitor cell diversity. We recently discovered anovel transition in the division mode in the developing mousecortex from radial glia (typical neural stem cells with the epithelial structure) to translocating neural stem cells, basal radial glia
(Shitamukai et al., 2011), which become a major population ofneural stem cells in mammals with gyrencephalic brains, suchas primates and ferrets. We are investigating the mechanismsthat underlie the formation, maintenance, and expansion ofthese neural stem cells, by using model mice that produce large
numbers of basal radial glia as well as ferrets.

We developed a novel method based on the CRISPR/Cas9 tool andin utero electroporation to knock-in genes into the developing brain.
This method enables us to distinguish homozygous knock-in cells
as yellow-colored cells by using two different colored fluorescencegenes as donors (EGFP and mCherry). The image shows an embryonic brain where two colored donors are knocked-in in β-tubulin
genes to produce fusion proteins.

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
Hirofumi KIYOKAWA
Masafumi NOGUCHI
Student Trainee

Yuki KIKUCHI

Technical Staff

Chisa MATSUOKA
Part-Time Staff

Bryan ITO
Akira YAMAOKA
Assistant

Yuka NODA

Recent Publications
Tsao P, et al. Epithelial Notch signaling regulates lung alveolar morphogenesis and airway
epithelial integrity. Proc Natl Acad Sci U S A
113. 8242–7 (2016)
Noguchi M, et al. Directed migration of pulmonary neuroendocrine cells toward airway
branches organizes the stereotypic location ofneuroepithelial bodies. Cell Rep13. 2679–2686(2015)
Morimoto M, et al. Different assemblies of Notch
receptors coordinate the distribution of the major bronchial Clara, ciliated and neuroendocrine
cells. Development 139. 4365–73 (2012)
Morimoto M, et al. Canonical Notch signaling in
the developing lung is required for determinationof arterial smooth muscle cells and selectionof Clara versus ciliated cell fate. J Cell Sci 123.
213–24 (2010)
Morimoto M and Kopan R. rtTA toxicity limits
the usefulness of the SP-C-rtTA transgenic
mouse. Dev Biol 325. 171–8 (2009)

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, thebody 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 Club, 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 infections 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.

Morimoto M, et al. The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity. Nature 435. 354–9 (2005)

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

3D computer reconstruction of branching bronchiole

Position of neuroendocrine cells within the entire geometricarchitecture of the airways

63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.

Staff
Research Scientist

Kota BANZAIThe 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.

Research Associate

Masako MINO

Visiting Scientist

Ken-ichi HIRONAKA
Technical Staff

Takayuki YAMADA
Student Trainee

Ryota MATSUSHITA
Part-Time Staff

Okiko HABARA
Noriko NISHIMURA
Junko SHINNO

Recent Publications
Yoshida M, et al. Molecular characterization of
Tps1 and Treh genes in Drosophila and theirrole in body water homeostasis. Sci Rep 6.
30582 (2016)
Okamoto N and Nishimura T. Signaling fromglia and cholinergic neurons controls nutrientdependent 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 theDachshund 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)

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 poorly understood. In addition, growth and developmental timing are also governed bynutrient availability. Most species have a standard body size, but developing organisms are also
capable of adapting their growth to fluctuating nutritional states through metabolic regulation.
Therefore, linking the nutrient-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 peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. We have demonstrated the molecular mechanism underlying the nutrient-dependent expression of a Dilp gene. 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. Because Dilp regulates both growth and metabolism during development, we are
analyzing the physiological significance of the regulation of sugar metabolism by insulin/IGF signaling. Our work focusing on the blood sugar trehalose revealed that metabolism of hemolymph sugar
plays a critical role for body growth under poor dietary conditions. Dietary condition-specific phenotype in Drosophila provides new insights into the significance of gene-environment interactions.

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

64

Pluripotent Stem Cell Studies
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 in gene trap
methods. From 1993 to 1994, he worked as a research associate in the Department of Developmental
Genetics at the same university, before taking a postdoctoral fellowship with Austin Smith at the
University of Edinburgh Centre for Genome Research. He returned to Japan in 1996 as a research associate in the Department of Nutrition and Physiological Chemistry at the Osaka University Graduate
School of Medicine, where he remained until taking a position as Team Leader at the RIKEN CDB. He
was appointed Project Leader of the Laboratory for Pluripotent Stem Cell Studies in October 2009.
The Laboratory for Pluripotent Stem Cell Studies closed in March 2016.
Dr. Niwa is now at Kumamoto University.

Staff
Research Scientist

Yoko NAKAI-FUTATSUGISatoshi OHTSUKA
Robert Odell STEPHENSON
Research Associate

Mariko YAMANE
Technical Staff

Kumi MATSUURA

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.

Part-Time Staff

Sachiko HASHIMOTO
Yayoi NAKAI

Recent Publications
Niwa H, et al. The evolutionally-conserved function of group B1 Sox family members confers
the unique role of Sox2 in mouse ES cells. BMC
Evol Biol 16. 173 (2016)
Nakai-Futatsugi Y and Niwa H. Zscan4 is activated after telomere shortening in mouse embryonic stem cells. Stem Cell Reports 6. 1–13
(2016)

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 that is 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? Whatis the principle behind the structure of the transcription factor networks governing developmental
processes? We are now trying to answer these questions by studying ES cells and the changes
they undergo in their differentiation toward trophoblast stem cells, extraembryonic endoderm
cells, and primitive ectoderm cells.

Yamane M, et al. Zscan10 is dispensable formaintenance of pluripotency in mouse embryonic stem cells. Biochem Biophys Res Commun
468. 826–831 (2015)
Kinoshita M, et al. Sox7 is dispensable for primitive endoderm differentiation from mouse ES
cells BMC Dev Biol 15. 37 (2015)
Fujii S, et al. Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells. Sci Rep
5. 9146 (2015)
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)

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).

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

65

Vascular Morphogenesis
http://www.cdb.riken.jp/en/research/laboratory/phng.html

Team Leader

Li-Kun Phng Ph.D.
Li-Kun Phng received her B.Sc. in Pharmacology from the University of Bristol in 2002, her
M.Sc. for Research in Life Science from the University of Edinburgh in 2004, and her Ph.D.
from University College London in 2009, for her work on blood vessel development in thelaboratory of Holger Gerhardt at the Cancer Research UK London Research Institute. She
was awarded the EMBO and HFSP Long-term Fellowships to perform postdoctoral researchat the European Molecular Biology Laboratory (EMBL), Heidelberg in 2009, and in VIB/KU
Leuven, Belgium in 2011. She next moved to the National Cerebral and Cardiovascular Center
Research Institute, Osaka, in 2014, after receiving the JSPS Postdoctoral Fellowship for Foreign Researchers. She was appointed to her current position as Team Leader at RIKEN CDB in
October 2016.

Staff
Technical Staff

Akane NOMORIAssistant

Emi TANIGUCHI

Recent Publications
Gebala V, et al. Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo. Nat Cell Biol 18(4): 443–451(2016)
Phng LK, et al. Formin-mediated actin polymerization at endothelial junctions is required forvessel lumen formation and stabilization. Dev
Cell 32. 123–132 (2015)
Phng LK, et al. Filopodia are dispensable forendothelial tip cell guidance. Development 140.
4031–4040 (2013)
Phng LK, et al. Nrarp coordinates endothelial
Notch and Wnt signalling to control vessel density in angiogenesis. Dev Cell 16. 70–82 (2009)
Hellström M, et al. Dll4 signalling throughNotch1 regulates formation of tip cells during
angiogenesis. Nature 445. 776–770 (2007)

The establishment of an intricate network of interconnected blood vessels is essential for the development of many tissues and organs. Tissue vascularization frequently occurs through sprouting angiogenesis, where new blood vessels arise from pre-existing ones, and encompasses amultitude of cellular processes including polarized collective cell migration, proliferation, anastomosis and lumen formation. While many key molecules and signaling pathways have been identified to regulate endothelial tip/stalk cell specification, blood vessel guidance and arterial-venous
differentiation, there is still a poor understanding of how angiogenic signals are relayed to the cell’smachinery to drive changes in endothelial cell morphology, behavior and consequently, the final
pattern of the vasculature.
My laboratory aims to unravel fundamental mechanisms that regulate endothelial cell dynamics
and coordination during blood vessel morphogenesis. Previous studies on actin cytoskeleton
revealed that specialized F-actin of different dynamics and subcellular localization drive distinct
steps of vessel morphogenesis. For example, the transient polymerization of F-actin at the apicalmembrane controls lumen expansion while a stable pool of F-actin at endothelial cell junctions
stabilizes nascent lumens to produce a functional vascular network. In the future, we aim to investigate the role of force generation in regulating endothelial cell dynamics and vessel morphogenesis, the mechanisms controlling actomyosin activity in endothelial cells and the molecular regulation of vessel lumen formation. Our long-term goal is to understand how upstream angiogenic
signals as well as hemodynamic forces regulate force generation and endothelial cell behavior.
To achieve our research goals, we employ live imaging at high temporal and spatial resolution,
advanced fluorescent microscopy techniques, genetics and chemical biology to elucidate themechanical and molecular mechanisms of blood vessel morphogenesis using the zebrafish as ourmodel organism.

Endothelial cells are highly plastic and undergo extensive cell shapechanges during blood vessel morphogenesis. Green, F-actin. Magenta,
membrane.

66

F-actin and apical membrane dynamics during lumen invagination. Local and transient actin polymerisation (5’, 10’) and myosin activity at apical membranes retract inverse blebs and can cause lumen collapse (15’). Remnants of membranes eventually fuse and integrate with the apical membraneof invaginating lumen (25’ to 55’).

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 1996, where she discovered the potential of stem cells as a tool for retinal
therapy. She returned to Kyoto University Hospital in 1998, and was appointed associate professor
at the Translational Research Center in the same hospital in 2001. She joined the CDB as a Team
Leader 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 September 2014. Her clinicalspecialty is retinal disease–specifically, macular diseases and retinal hereditary diseases. Her aim
is to understand these diseases at a fundamental level and develop retinal regeneration therapies.

Staff
Deputy Project Leader

Michiko MANDAI
Sunao SUGITA

Research Scientist

Yuuki ARAI
Hiroyuki KITAJIMA
Naoshi KOIDE
Takesi Hoyos MATSUYAMA
Chikako MORINAGA
Satoshi NAKADOMARI
Akishi ONISHIGenshiro SUNAGAWA
Hung-Ya TU
Akiko YOSHIDA
Research Associate

Akihiro TACHIBANATechnical Staff

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

Shoko FUJINO

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.
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 fromconditions 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 toa broad spectrum of developmental biological research information will be key to the achievementof these goals, and we appreciate the opportunities for exchange that working in the environment
provided by the RIKEN CDB.
Therapeutic applications cannot be developed from basic research alone; the clinical approach—a thorough 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 not necessarily be translatable to a human clinical therapy without an understanding of theunderlying genetics and possible immunological involvement. Our goal is to study retinal regeneration based on both a strong foundation in basic research and solid clinical evidence.

Recent Publications
Mandai M, et al. iPSC-derived retinal transplants
improve vision in rd1 end-stage retinal degeneration mice. Stem Cell Reports 8. 69–83 (2017)
Sugita S, et al. Successful transplantation ofretinal pigment epithelial cells from MHC homozygote iPS cells in MHC-matched models.
Stem Cell Reports 7(4): 635–648 (2016)
Sugita S, et al. Lack of T-cell response to iPS
cell-derived retinal pigment epithelial cells from
HLA homozygous donors. Stem Cell Reports
7(4): 619–634 (2016)
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)

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)

67

Human Organogenesis
http://www.cdb.riken.jp/en/research/laboratory/takasato.html

Team Leader

Minoru TAKASATO Ph.D.
Minoru Takasato is Team Leader of the Laboratory for Human Organogenesis at the RIKEN
CDB, where he heads the kidney regeneration project. He received his B.Sc. in anthropology
from the University of Tokyo, Japan in 2002, and completed his Ph.D. focusing on the study ofmouse kidney development in the same university in 2008 before joining Professor Little’s laboratory as a postdoctoral fellow at Institute for Molecular Bioscience, University of Queensland,
Australia, in 2009. He is an expert in the directed differentiation of human pluripotent stem cells
to kidney with his most recent work being published in Nature Cell Biology (2014) and Nature(2015). His most recent studies describe the generation of mini-kidneys from induced pluripotent stem cells for use in drug screening and disease modelling. His work has drawn a great
deal of international interest in kidney development and stem cell research fields.

Staff
Research Scientist

Wei ZHAO

Visiting Researcher

Yoshiki SAHARATechnical Staff

Kisa KAKIGUCHI
Kazuhiro OFUJI

Recent Publications
Takasato M, et al. Generation of kidney organoids from human pluripotent stem cells. Nat
Protoc 11. 1681–1692 (2016)
Takasato M, et al. Kidney organoids from human iPS cells contain multiple lineages andmodel human nephrogenesis. Nature 526.
564–568 (2015)
Takasato M and Little M H. The origin of the
mammalian kidney: implications for recreating
the kidney in vitro. Development 142. 1937–
1947 (2015)

What is the ultimate goal of regenerative research using human pluripotent stem cells? We think
this is to recreate a whole replaceable organ in vitro via directed differentiation. Due to the continuous rise in the incidence of end-stage renal disease around the world (approximately 7% perannum), there is an urgent demand for regenerative strategies to compensate for the loss of renal
function in these patients. In our previous study, we developed a protocol by which human pluripotent stem cells can be differentiated into the intermediate mesoderm that can self-organize into
kidney organoids. While these kidney organoids comprise all anticipated renal tissues, including
nephrons, collecting duct, blood vessels and renal interstitium, they are still far from the real human kidney in terms of their size, tissue complexity, maturity and functionality. By precisely recapitulating the developmental processes of the human kidney in directed differentiation of humanpluripotent stem cells, we are trying to achieve the ultimate goal of generating a three-dimensional
kidney that is functional and that can also be transplanted into patients. We appreciate knowledge
from basic developmental biology that is essential for such regenerative studies; therefore, we arealso highly interested in studies of human embryology. Utilizing our unique technology that generates hPSC-derived kidney organoids from pluripotent stages in vitro, we are focusing particularly
on uncovering the developmental mechanisms of the human mesoderm and kidney.

Takasato M, et al. Directing human embryonicstem cell differentiation towards a renal lineage
generates a self-organizing kidney. Nat Cell Biol
16. 118–126 (2014)
Hendry C E, et al. Direct transcriptional reprogramming of adult cells to embryonic nephronprogenitors. J Am Soc Nephrol 24. 1424–1434(2013)
Takasato M, et al. Trb2, a mouse homolog oftribbles, is dispensable for kidney and mouse
development. Biochem Biophys Res Commun373. 648–652 (2008)

Kidney organoids derived from human iPS cells. The organoid contains
two kidney progenitors, the ureteric tree (yellow with cyan) and nephronprogenitor (red), as well as developing nephrons (yellow).

68

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.

Staff
Research Scientist

Shuichi HAYASHI
Shoko ITO
Toshiya KIMURA
Anna PLATEK
Varisa PONGRAKHANANON
Takuji TANOUE
Mika TOYA

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:

Visiting Scientist

Tamako NISHIMURA
Technical Staff

Sylvain HIVER
Yoko INOUE
Miwa KAWASAKI
Hiroko SAITO
Vassil VASSILEV
AssistantMutsuko AISO-WATANABE

Recent Publications
Nishimura T, et al. DAAM1 stabilizes epithelial junctions by restraining WAVE complexdependent lateral membrane motility J Cell Biol
215. 559–573 (2016)
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)

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.
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.

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)

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 with 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. WhenCAMSAP3 is knocked out, the longitudinal arrays of microtubules are
disrupted.

69

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 pharmaceuticals industry for three years, returned to complete his doctorate at Kyushu
University, and he received his doctorate in 1992 from Niigata University. He conducted research at Niigata University from 1992 to 1994 before moving to serve as researcher and thensenior scientist at JT Inc. From 2001, he moved to Tokyo University of Science, and in 2007 he
was appointed full professor at 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.

Staff
Research Scientist

Kyousuke ASAKAWA
Etsuko IKEDA
Mamoru ISHII
Jun ISHIKAWA
Ryohei MINAMIDE
Makoto TAKEO
Visiting Scientist

Miho OGAWA
Koh-ei TOYOSHIMA
Technical Staff

Tomoyo IGA
Ikumi KONISHI
Yukiko MORIOKA
Azusa NOMA
Hiroko SASAKIAya SHIOKAWA
Miki TAKASE
Yuko TAMAI
Ayako TSUCHIYA
Hisashi UTSUNOMIYAAssistant

Mayumi MUROFUSHI
Sayaka NAKAMURA

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, andfollowing 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 these 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
new technologies for use in regenerative medicine. We are also interested in using 4D cell dynamics imaging techniques to quantitatively analyze organ-specific morphogenesis, and to analyze,
model, and simulate molecular pathways involved in their control, in the hopes of gaining new
insights into the fundamental principles underlying organogenetic processes. Building on thesefundamental studies, we further seek to conduct applications research towards the developmentof technologies for use in therapeutic organ regeneration.

Recent Publications
Takagi R, et al. Bioengineering a 3D integumentary organ system from iPS cells using an in vivotransplantation model. Science Advances 2(4):e1500887 (2016)
Ozone C, et al. Functional anterior pituitarygenerated in self-organizing culture of humanembryonic stem cells. Nat Commun 7. 10351(2016)
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 bioengineeredorgan germ. Nat Commun 4. 2497 (2013)
Toyoshima K E, et al. Fully functional hair follicle regeneration through the rearrangementof stem cells and their niches. Nat Commun 3.
784 (2012)

Mouse iPS cell-derived hair

Ikeda E, et al. Fully functional bioengineered
tooth replacement as an organ replacement
therapy. Proc Natl Acad Sci U S A 106. 13475–
80 (2009)

Bioengineered hair follicle

70

Bioengineered tooth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
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 bistabilityof 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, while alteration of individual cell shapes collectively organizes the tissue architecture,
the underlying molecular and mechanical mechanisms remain poorly understood. My lab aims at
identifying novel mechanisms that orchestrate 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. Adherens
junctions are labeled in green; plasma
membrane in magenta.

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

71

Activities

3D image of skin from talk of adult mouse stained with antibodies for Integrin-α6(green), and Ki67 (red), and DAPI (blue). Quiescent skin stem cells of hair follicles arestained green. Proliferating stem cells are stained red.
Image: Hironobu Fujiwara, Laboratory for Tissue Microenvironment

2016 CDB Symposium

Size in Development: Growth, Shape and Allometry
March 28–30, 2016
The RIKEN CDB hosted its fourteenth annual symposium on
March 28 to 30, in the CDB auditorium. The theme of this year’s
symposium was entitled, “Size in Development: Growth, Shapeand Allometry,” and an audience of 176 scientists and students
from over 18 different countries convened at the Center to takepart in a program of talks and discussions exploring the question of organismal size control.
The three-day program featured over 30 invited and selected
talks and poster sessions in which more than 60 presenters discussed their research that covered a range of topics including
growth control, organ size and shape, scaling and morphogenesis, and evolution of allometry. The co-organizers of this year’s
symposium were Shigeo Hayashi, Hidehiko Inomata and Mitsuru
Morimoto from the CDB, and Stefano Piccolo from University of
Padua Medical School, Italy.
The CDB symposium series was launched in 2003 as a forum
to encourage the timely exchange and discussion of recent findings, progress and trends in developmental biology, regeneration and other related fields.

Session 1

Organ Size and Shape Control

Yingzi Yang
(National Human Genome Research Institute, NIH and Harvard School of Dental Medicine, USA)

Mary Baylies(Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, USA)

Alberto Rosello-Diez (Sloan Kettering Institute, USA)
Shigeo Hayashi (RIKEN Center for Developmental Biology, Japan)
Mitsuru Morimoto (RIKEN Center for Developmental Biology, Japan)
Justin Crest (University of California, Berkeley, USA)
Mototsugu Eiraku (RIKEN Center for Developmental Biology, Japan)
James Wells(Cincinnati Children’s Hospital Medical Center and University of Cincinnati, USA)

Richard A. Schneider (University of California, San Francisco, USA)
Session 2

Growth Control

Hiroshi Sasaki (Osaka University, Japan)
Kazuo Yamamoto (Nagasaki University School of Medicine, Japan)
Stefano Piccolo (University of Padua Medical School, Italy)
Makoto Furutani-Seiki(Yamaguchi University School of Medicine, Japan and University of Bath, UK)

Georg Halder(VIB Center for the Biology of Disease, and KU Leuven Center for Human Genetics, University of Leuven, Belgium)

Yusuke Mii (National Institute for Basic Biology, Japan)
Iswar K. Hariharan (University of California, Berkeley, USA)
Duojia Pan(Howard Hughes Medical Institute and Johns Hopkins University School of Medicine, USA)

Igor Adameyko (Karolinska Institutet, Sweden)

2017 CDB Symposium

Towards Understanding
Human Development,
Heredity, and Evolution
March 27–29, 2017
Session 3

Scaling and Morphogenetic Control

Hidehiko Inomata (RIKEN Center for Developmental Biology, Japan)
Kazutaka Hosoda (Kyoto University, Japan)
Anna Kicheva (IST Austria, Austria)
Rebecca Heald (University of California, Berkeley, USA)
Hirohisa Kyogoku (RIKEN Center for Developmental Biology, Japan)
Timothy E. Saunders(Mechanobiology Institute, National University of Singapore, Singapore)

Alexander Aulehla (EMBL Heidelberg, Germany)
Kana Ishimatsu (Harvard Medical School, USA)
Nadia Rosenthal(The Jackson Laboratory, USA and Imperial College London, UK)

Session 4

Allometry in Evolution

Ehab Abouheif (McGill University, Canada)
Toru Miura (Hokkaido University, Japan)
Alejandro Burga (University of California, Los Angeles, USA)
Mitsuyasu Hasebe (National Institute for Basic Biology, Japan)
David M. Kingsley(Howard Hughes Medical Institute and Stanford University, USA)

Elaine A. Ostrander(National Human Genome Research Institute, National Institutes of Health, USA)The fifteenth annual symposium, “Towards Understanding
Human Development, Heredity, and Evolution,” will be heldon March 27–29, 2017, at the RIKEN CDB.
Recent advances in technologies, including stem cell culture, live imaging, single-cell approaches, next-generation
sequencing, and genome editing, have led to an unprecedented expansion in our knowledge on human development
and heredity. Comparative studies between humans andother mammals have also shed light on evolutionary processes, and in turn provided critical insights into the mechanisms for key diseased states. Given this exciting progress
and promising prospects in the relevant fields, it is importantand also timely to discuss current understandings of humandevelopment, heredity, and evolution, in addition to exploring the possibilities that have opened up to reconstitutesuch processes in vitro using pluripotent and other stem
cells. The CDB Symposium 2017 will cover a broad rangeof topics, including human germline development, early
embryogenesis, organogenesis from stem cells and relevant
disease models, as well as human genetics and evolution.

Invited Speakers

Déborah Bourc’his(Institut Curie, France)

Guillaume Bourque(McGill University and Genome Quebec Innovation Center, Canada)

Patrick F. Chinnery(University of Cambridge and MRC Mitochondrial Biology Unit, UK)

Mototsugu Eiraku(RIKEN CDB, Japan)

Anne Ferguson-Smith(University of Cambridge, UK)

Zev J. Gartner(University of California, San Francisco, USA)

Edith Heard(Institut Curie, France)

Agnar Helgason(deCODE Genetics, Iceland)

Rudolf Jaenisch(MIT, Whitehead Institute, USA)

Yoshiya Kawaguchi(Center for iPS Cell Research and Application, Kyoto University, Japan)

Tomoya Kitajima(RIKEN CDB, Japan)

Arnold Kriegstein(University of California, San Francisco, USA)

Rasmus Nielsen(University of California, Berkeley, USA)

Ryuichi Nishinakamura(Kumamoto University, Japan)

Molly Przeworski(Columbia University, USA)

Lluis Quintana-Murci(Institut Pasteur-CNRS, France)

Mitinori Saitou(Kyoto University, JST ERATO, Japan)

Rickard Sandberg(Karolinska Institutet, Sweden)

Austin Smith(Cambridge Stem Cell Institute, UK)

Michael Snyder(Stanford University, USA)

Azim Surani(Wellcome Trust/Cancer Research UK Gurdon Institute, UK)

Gen Suwa(The University Museum, The University of Tokyo, Japan)

Takashi Tsuji(RIKEN CDB, Japan)

75

2016 Seminars
The RIKEN CDB makes special efforts to provide a full and diverse series of invited seminars by scientists fromaround the world. To date, the Center has hosted close to 800 such talks, in addition to numerous meetings, internal
forums, and colloquia. The following speakers were invited to the CDB to give a seminar in the period from January
to December 2016.

76

Date

Title

Speaker

01-14

Integrated genomics approaches for the identification of causative variants andgenes in GWAS-identified risk loci: Application to inflammatory bowel disease

Michel GEORGES

01-22

Evolution and development of the bifurcated caudal axial skeleton in the twin-tailgoldfishKinya G. OTA

02-26

Rocaglates convert DEAD-box protein eIF4A into a sequence-selective
translational repressor

Shintaro IWASAKI

03-09

Nature Publishing Group - Scientific data seminar

Varsha KHODIYAR

03-15

Biochemical analysis of genome functions using the locus-specific chromatin
immunoprecipitation technologies: Key tools to elucidate 4D Nucleome

03-24

Antisense therapy for Fukuyama type congenital muscular dystrophy

03-31

Dynamics of bilaterian genome architecture evolution: Insights from the octopus
and hemichordate genomes

04-20

The human ciliopathy protein JBTS17 is required for basal body docking andintraflagellar transport for ciliogenesis

Michinori TORIYAMA

04-22

Biomedical and Engineering Seminar

Shunji YAMANAKA

04-28

Development of new cell-free regeneration therapies based on the novel protein
complex inducing tissue-regenerating microenvironment

Akihito YAMAMOTO

05-10

Controlling contractile instabilities in the actomyosin cortex

Stephan GRILL

06-06

Warburg-like metabolism regulates vertebrate somitogenesis

Masayuki OGINUMA

06-07

Mechanism of the asymmetry of signaling endosomes during asymmetric
division

06-08

Dissecting the regulatory circuitry of microRNAs in neutrophils

06-13

Molecular mechanisms underlying faithful chromosome segregation-- Inner
centromere-shugoshin network

06-27

Defining the stem cell lineages in adult skin epidermis

06-30

Versatile protein tagging with split fluorescence protein: A scalable strategy for
GFP tagging of endogenous human proteins

Sayaka SEKINE

07-12

Formation of the vertebrate gut

Clifford J. TABIN

07-13

Cell lineages in the early mammalian blastocyst

Janet ROSSANT

08-02

Enhancer control of transcriptional bursting

Takashi FUKAYA

09-09

The evodevo & physics of skin appendage and skin colour patterning invertebrates

Hodaka FUJII

Mariko TANIGUCHI-IKEDA

Oleg SIMAKOV

Marcos GONZALEZ-GAITAN

Qing DENG

Yoshinori WATANABE

Aiko SADA

Michel C. MILINKOVITCH

Date

Title

Speaker

09-14

Developmental genetics of mammalian reproductive organs

09-26

From nerve growth to synapse formation and plasticity

Mu-ming POO

10-03

In for the long haul: Maintaining chromosome cohesion in mammalian oocytes

Mary HERBERT

10-03

The final cut: control of progression through meiosis II

Wolfgang ZACHARIAE

10-18

Human naïve pluripotent stem cells and early embryogenesis

Yasuhiro TAKASHIMA

10-19

Pervasive translational regulation of the cell signaling circuitry underlies
mammalian development

Kotaro FUJII

10-31

Stem cell heterogeneity and function in development and regeneration

Shahragim TAJBAKHSH

11-11

Developmental control of replication timing and chromosome architectureDavid M. GILBERT

11-21

The developmental basis for the recurrent evolution of deuterostomy andprotostomy

José M. MARTIN-DURAN

11-24

Mechanical forces influence three-dimensional cell behaviours during mousemandibular arch development

Hirotaka TAO

11-25

Order from chaos: Rules and self-organizing properties of division patterns inplantsAlexis MAIZEL

11-28

Shaping cells and tissues: From embryonic development to synthetic biology

Stefano DE RENZIS

11-30

Homology and diversity in animal genomesPeter W. H. HOLLAND

12-05

Investigating the interplay between obesity and cancer using Drosophila

Susumu HIRABAYASHI

12-07

The regulatory mechanism of gastric stem cell and its malignant transformation

Yoku HAYAKAWA

12-08

Decoding cellular immortality using genetically engineered human pluripotent
stem cells

Kunitoshi CHIBA

12-12

Assembly of neural circuits

12-14

An extensive HLH transcription factor network promoting longevity in response
to signals from the gonad

Shuhei NAKAMURA

12-19

Mouse embryo keeps paternal chromosomes away from maternal spindle tomaintain the ploidy during fertilization

Masashi MORI

12-20

X chromosome inactivation initiated by dysfunctional Xist RNA

Takashi SADO

Richard BEHRINGER

Liqun LUO

77

2016 Events
The RIKEN Center for Developmental Biology strives to engage with the public through a variety of media, including its website,
printed materials and media coverage, and through direct interactions such as guided tours, open house events, and other public
outreach activities. The CDB also organizes events for the scientific community to facilitate the exchange of information between
scientists from around the world in a setting outside the laboratory environment.

CDB Retreat in Awaji
The Center’s research staff gathered on Awaji Island for the annual CDB Retreat from
September 29–30. Held in a new location this year on the southern end of Awaji Island, the laboratory heads, research scientists, student trainees, and technical staff
took part in the closed meeting to share and openly discuss the latest developmentsin their respective projects outside normal lab settings. The program included shorttalks by students and young scientists, and lectures by Team Leaders Minoru Takasato and Erina Kuranaga, QBiC Unit Leader Yuchi Taniguchi, and CDB alumnus Hiroshi Sawa (National Institute of Genetics).

KAIST-CDB-CAS Joint Meeting
The 3rd joint meeting between RIKEN CDB, KAIST’s Department of Biological Sciences (Korea), and Chinese Academy of Sciences’ Institute of Genetics and Developmental Biology (China) was held at the KAIST Institute, in Daegu, Korea from October
27 to 28. Five team leaders from the CDB attended the meeting which is held every
two years, rotating locations amongst the three institutions with the aim to strengthen
institutional ties as well as share and discuss recent findings in the area of developmental biology.

Open House 2016
The CDB, along with the other RIKEN Kobe Campus research centers held its annual
Open House on Saturday, November 5. Over 1,700 people of all ages dropped by the
CDB to learn about the research being carried out at the Center. One of the highlightsof the event is the Open Labs where visitors are permitted to walk through the laboratories, see samples of cells and tissues under microscopes, and talk to scientists
about their research. Other events included an exhibit introducing different model organisms used in research, a DNA experiment corner where visitors could try out techniques used by scientists for DNA analysis, and a talk show and Q&A session with
scientists working at the CDB campus. There was also a book lounge featuring books
recommended by CDB scientists, and a handicraft corner for kids making animalmasks and color-your-own bags. This is the Center’s largest annual event geared forthe general public, allowing scientists to engage with the public face-to-face.

78

The 27th CDB Meeting “Body Surface Tactics: Cellularcrosstalk for the generation of super-biointerfaces”
The 27th CDB Meeting entitled, “Body Surface Tactics: Cellular crosstalk for the generation of super-biointerfaces,” co-hosted by the Japan Skin Research Club and the
RIKEN Symposium, was held November 14 and 15. Over 130 students and scientists
from both academia and industry studying the body surface in different disciplines,
from cell and developmental biology, regenerative biology, vascular and neurobiology,
to immunology, evolution, mathematics and medicine, were in attendance. The talks
and discussions covered a broad range of topics including epithelial formation, functional unit formation, maintenance and regeneration, evo-devo, new technologies, anddysfunction and therapeutics. Keynote lectures were given by Yann Barrandon of
EPFL in Switzerland and Singapore A-STAR, and Cheng-Ming Chuong of the University of Southern California, USA.

The 28th CDB Meeting “Cilia and Centrosomes: Current
Advances and Future Directions”
The 28th CDB Meeting was held from November 27 to 29. There were 101 participants from within Japan and abroad attended the meeting, which was co-hosted by
the Cilia Club and the MEXT Grant-in-Aid for Scientific Research on Innovative Areas“Cilium-centrosome system regulating biosignal flows.” Research on cilia and centrosomes have progressed along parallel paths, and only recently have researchers
begun to realize the importance of tying these two fields together to gain a morecomprehensive perspective of the common molecular mechanisms regulating thefunction of these organelles and their association with various diseases. Twenty-twotalks and over 30 posters were presented at the meeting, on a range of topics including roles of the cilia and centrosomes in cell cycle, embryogenesis, and disease pathogenesis.

79

2016 Educational Programs
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 an academic 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.

Intensive lecture program for graduate students
The CDB has formed partnerships with a number of graduate schools in the Kansai
region, and organizes a two-day lecture program every year for graduate students
enrolled in partnering institutions. This year’s lecture program was held August 3 to 4,
and included talks by selected laboratory heads and research scientists on their research and tours of labs and some of the technical facilities. Some graduate students
attending the lecture program can also receive credit towards their degree program.

Summer school for high school students
The tenth annual one-day summer school program for high school students was heldon August 18 and 23. This program features a talk by a CDB scientist, a tour inside a
working laboratory, and a hands-on scientific experiment. The theme of this year was
genes and their functions, and the students analyzed the DNA extracted from theirown cheek epithelial cells to determine whether or not they carry a single nucleotide
polymorphism in their gene encoding an enzyme involved in metabolizing alcohol. Twoscientists in the Laboratory for Organismal Patterning contributed to the program bygiving a talk on their research as well as showing the students actual specimen samples and some of the state-of-art equipment used in their lab.

Internship for undergraduate students
Thirty-four undergraduate students from universities around Japan were invited tospend a week, from August 22 to 26, as interns in the participating laboratories on the
CDB campus. The internship program offers the students the chance to work on
small-scale projects under the supervision of scientists in the hosting labs, as well as
listen to lectures by laboratory heads and visit different labs. The activity-filled week
concluded with a presentation session on the final day, where the interns presented
the findings from their projects to the other participants.

High school teachers’ workshop
A one-day practical workshop for high school biology teachers was held at the CDB
on October 2. The program was co-organized by the CDB, the Japanese Society for
Developmental Biologists (JSDB), and the Hyogo Prefectural High School Educational
Committee for Biology. Twenty-eight teachers (18 participants and ten observers),
mainly from around the Kansai area, took part in the workshop. This year’s theme was
genetics using the fruit fly, and the program was led by Team Leader Shigeo Hayashi,
who also oversaw its development. The aim was for the teachers to gain a firsthandunderstanding of how genes and environmental factors affect phenotypes throughexamining Drosophila mutants caused by sex-linked inheritance or epigenetic factors.
The program was designed to facilitate the teachers to take what they learned back to
the classroom. Several of the teachers who participated in this workshop also had thechance to teach their own students about what they learned by serving as TAs in atutorial program modified for high school students in December.

80

2016 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 of many of its scientific staff have been recognized with awards from numerous organizations, including government
ministries and agencies, private foundations and academic societies. The CDB takes great pride in the achievements of its researchers, as individuals and as indispensable members of their laboratories.

AwardeePositionLaboratory

Award

Organization

Tomoya Kitajima

Team Leader

Chromosome
Segregation

MEXT Prize(The Young Scientist’s
Prize)The Ministry of Education,
Culture, Sports, Scienceand Technology (MEXT)

Hiroshi Hamada

Director

Organismal
Patterning

EMBO Associate Member

The European Molecular
Biology Organization(EMBO)

Minoru Takasato

Team Leader

Human
Organogenesis

The 2016 AustralianMuseum Eureka Prize for
Scientific Research

The Australian Museum

Shuhei Yoshida

Research Scientist

Chromosome
Segregation

MBSJ 39th Annual
Meeting Excellent Poster
Presentation Award

The Molecular Biology
Society of Japan81

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
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.

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 life science
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 of research programs at the former RIKEN Yokohama and Kobe Institutes, and focuses on technology researchand development to support the medical and pharmaceuticalsectors, while QBiC focuses on measurement, analysis, and modeling technologies and techniques to model cell dynamics.
Kobe is also home to the RIKEN Advanced Institute for Computational Science (AICS), which is associated with the national K
Supercomputer project.

Administrative support at RIKEN Kobe and CDB
Kobe Branch

RIKEN Kobe Administrative Division
General Affairs
Human Resources
Finance
Osaka Administrative Section
Information Networks Office – Kobe Branch
RIKEN Kobe Safety Center
Planning Office for each Center

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.

Quantitative Biology Center (QBiC)
2016 CDB Budget

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.

212

Advanced Institute for
Computational Science (AICS)

934

The RIKEN Advanced Institute for Computational Science (AICS)
houses the K supercomputer, which is used to generate cuttingedge scientific results and technological breakthroughs throughcollaboration and integration of computational and computer sciences, to assist and advance the science of forecasting in areasthat are linked directly to our daily lives, from predicting weather
patterns and effects of natural disasters, to drug design and development of new devices and materials. More recently, they are
involved in the development of a supercomputer to succeed the K
computer.

414

Total: 1,560
Personnel(million yen)In addition to the dedicated funds outlined above, individual labs
and investigators are encouraged to compete for external funding 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 the Center’s total
funding every year.
Grant-in-Aid for
Scientific Research(MEXT)

Others (Foundations,
Private Industry, etc. )

212

194

Kobe Administrative Services
The Kobe Administrative Division dedicated to providing coreadministrative services to the entire RIKEN Kobe Branch. Its mainareas 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. The Safety Center
provides training and supervision for lab and workplace safety,
implements and monitors disaster prevention measures, andensures compliances with national and institutional regulations
governing laboratory practice and technology. Within the CDB,
the Developmental Biology Planning Office coordinates important
activities including budget and funding management, and administrative support for laboratory performance reviews, as well as
provides support for domestic and international communications
and for organizing scientific meetings. The Library Office manages
the CDB research literature collections and interlibrary loans.

Research
expenses

Overhead andadministration

Total: 1,156
Grants (JST, etc.)

750(million yen)2016 CDB Staff
Laboratory heads
Deputy leaders
Research scientists
Research associates
Technical staff
Assistants
Visiting scientists
Student trainees
Part-time staff
Other

19
2
54
4
61
16
121
27
25
10

Total

339

Kobe Branch Administration

84

85

About RIKEN
The mission of RIKEN is to conduct comprehensive research in science and technology (excluding only the humanities andsocial sciences) as provided for under the “RIKEN Law,” and to publicly disseminate the results of its scientific research andtechnological developments. RIKEN carries out high level experimental and research work in a wide range of fields, including physics, chemistry, medical science, biology, and engineering, covering the entire range from basic research to practicalapplication.
RIKEN was first organized in 1917 as a private research foundation, and reorganized in 2003 as an independent administrative institution under the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and designated in 2015 asa National Research and Development Institute. RIKEN will be celebrating its centennial in 2017.

RIKEN Website
The RIKEN website provides an in-depth resource for online visitors to the institute. The site
contains important links to the latest researchachievements, all materials as well as databases and other electronic resources developed by
RIKEN laboratories. For those with an interestin learning more about RIKEN’s organization,
activities, and history, visit:
http://www.riken.jp

RIKEN Research
RIKEN publishes the quarterly print and online
magazine RIKEN Research to draw the world’s
attention to some of RIKEN’s best research ina timely and easy-to-understand fashion. Thismagazine serves as a central resource for upto-date information on key achievements of thenumerous RIKEN research centers, along withinsights into the people, programs and facilities. The core component of RIKEN Researchis the short, easy-to-read ‘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/

86Sendai
Center for Advanced Photonics
519-1399 Aoba, Aramaki, Aoba-ku, Sendai 980-0845
Tel: +81-22-228-2111 Fax: +81-22-228-2122

Tsukuba
BioResource Center
3-1-1 Koyadai,Tsukuba, Ibaraki 305-0074
Tel: +81-29-836-9111 Fax: +81-29-836-9109

RIKEN Headquarters
Wako Administrative Headquarters
Center for Emergent Matter Science
Center for Advanced Photonics
Center for Sustainable Resource Science
Brain Science InstituteNishina Center for Accelerator-Based Science
Advanced Center for Computing and Communication
Cluster for Industry Partnerships
Cluster for Science and Technology Hub
Chief Scientist Laboratories
Associate Chief Scientist Laboratories
Distinguished Senior Scientist Laboratories
Initiative Research Units
Special Research Units
Research Groups
Global Research Cluster
2-1 Hirosawa, Wako, Saitama 351-0198
Tel: +81-48-462-1111 Fax: +81-48-462-1554

Tokyo Liaison Office
Fukoku Seimei Building, 23rd floor (Room2311)
2-2-2 Uchisaiwaicho, Chiyoda-ku, Tokyo 100-0011
Tel: +81-3-3580-1981 Fax: +81-3-3580-1980
Center for Advanced Intelligence Project
Nihonbashi 1-chome Mitsui Building, 15th floor,
1-4-1 Nihonbashi, Chuo-ku, Tokyo 103-0027
Tel: +81-48-467-3626

Yokohama
Center for Sustainable Resource Science
Center for Integrative Medical Sciences
Center for Life Science Technologies
1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045
Tel: +81-45-503-9111 Fax: +81-45-503-9113

RIKEN
http://www.riken.jp/

RIKEN is a publicly funded research organization established to conduct comprehensive research in science and technology, and to disseminate the results of itsscientific research and technological development. RIKEN carried out basic andapplied research in a wide range of fields,
including physics, chemistry, biology,
medical science, and engineering.
RIKEN was founded in 1917 as a private
research organization, Rikagaku kenkyusho(The Institute of Physical and Chemical
Research). In 2003, RIKEN was reorganized as an Independent Administrative
Institution under the Ministry of Education,
Culture, Sports, Science, and Technology(MEXT), and in 2015, was designated as a
National Research and Development
Institute. RIKEN continues to engage inwide-ranging research activities spanningthe basic and applied sciences.

Nagoya2271-130 Anagahora, Shimoshidami, Moriyama-ku, Nagoya, Aichi 463-0003
Tel: +81-48-462-1111 Fax: +81-48-462-1554

Osaka
Quantitative Biology Center
6-2-3 Furuedai, Suita, Osaka 565-0874
Tel: +81-6-6155-0111 Fax: +81-6-6155-0112

Kobe
Center for Developmental Biology
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-306-0111 Fax: +81-78-306-0101
Center for Life Science Technologies
6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-304-7111 Fax: +81-78-304-7112
Advanced Institute for Computational Science
7-1-26 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047
Tel: +81-78-940-5555 Fax: +81-78-304-4955

HarimaSPring-8 Center
1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148
Tel: +81-791-58-0808 Fax: +81-791-58-0800

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

On The Cover
3D rendering of super-resolution images of a transgenic
Thy1-YFP-H mouse brain cleared with SeeDB2.
Image: Meng-Tsen Ke, Takeshi ImaiLaboratory for Sensory Circuit Formation
Printed in Japan using soy inks, waterless printing
methods, and paper from sustainable resources.
RIKEN 2017-013