CONTENTS
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・ 1
Organization of the National Institute for Basic Biology ・
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・ 2
Goals of the National Institute for Basic Biology ・
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・ 5
Personnel Changes and Awardees in 2012 ・
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・ 7
Cell Biology
Division of Cell Mechanisms・
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・ 8
Division of Intercellular Signaling Biology ・
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・ 11
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Laboratory of Neuronal Cell Biology ・
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・ 14
Laboratory of Cell Sociology・
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・ 16
Developmental Biology
Division of Morphogenesis ・
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・ 17
Division of Developmental Genetics ・
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・ 20
Division of Molecular and Developmental Biology ・
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・ 23
Division of Embryology ・
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・ 26
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Division of Germ Cell Biology ・
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・ 29
Laboratory of Molecular Genetics for Reproduction ・
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・ 32
Laboratory of Plant Organ Development ・
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・ 34
Neurobiology
Division of Molecular Neurobiology ・
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・ 36
Division of Brain Biology・
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Division of Brain Circuits ・
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・ 42
Laboratory of Neurophysiology・
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・ 44
Evolutionary Biology and Biodiversity
Division of Evolutionary Biology ・
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Division of Symbiotic Systems・
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Laboratory of Morphodiversity ・
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Laboratory of Bioresources ・
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Laboratory of Biological Diversity・
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・ 54
Environmental Biology
Division of Molecular Environmental Endocrinology・
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Division of Environmental Photobiology ・
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・ 63
Theoretical Biology
Laboratory of Genome Informatics・
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・ 65
Imaging Science
Laboratory for Spatiotemporal Regulations ・
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・ 67
Research Support
NIBB Core Research Facilities ・
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・ 68
NIBB BioResource Center・
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・ 73
NIBB Center of the IBBP・
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・ 76
Center for Radioisotope Facilities (Okazaki Research Facilities) ・
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・ 77
Strategic Planning Department・
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・ 78
Office of Public Relations・
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Office of International Cooperation・
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・ 79
Technical Division ・
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・ Inside back cover

The cover photographs are related to a study showing the role of the middle domain-specific WOX genes in leaf
development of Arabidopsis thaliana (Nakata et al., Plant Cell, 2012). This study led a proposal that a new domain,
named the middle domain, located between the adaxial and abaxial domains, has a distinct role from that of thepreviously identified two domains above: namely, the middle domain-specific WOX genes organize blade outgrowth
together with adaxial and abaxial regulators and prevent the mixing of the adaxial and abaxial domains to maintainproper patterns during leaf development. See page 34 of this report for details.

INTRODUCTION
n order to understand the survival strategies of avariety of organisms NIBB uses model organisms incollaboration with Japanese and international researchers touncover both the basic mechanisms that are common to allliving things, as well as the varied and unique systems
organisms have evolved to allow them to adapt to theirenvironments. It is necessary to continue to promote toplevel research in this field, therefore it is important that weboldly pioneer new areas of study, and exert our energies tofoster the young researchers who will lead us in the future.

I

In addition, as an inter-university research institute it isessential that the National Institute for Basic Biology sparesno effort in promoting cooperative use and collaborative
research. In answer to the desires of the research community,
NIBB has been strengthening our cooperative use andcollaborative research structure by setting up the DSLM
(Digital Scanned Light-sheet Microscope) cooperative
research program, the next Generation DNA sequencer
cooperative research program, and we have established the“NIBB BioResource Center” and the “NIBB Core Research
Facilities”. In addition, in order to protect biologicalresources that are indispensable to the advancement of thebiological sciences we have begun operating as the corefacility of the Interuniversity Bio-Backup Project (IBBP),
together with implementing plans in this fiscal year to begin
joint-use research aimed at the development of new
cryopreservation technology of biological resources. With
the current climate of emphasis being placed on the practicalapplication of research NIBB is expected to play a central
role in promotion of the importance of the study of basic
biology. To this end I believe it is necessary for NIBB tocome together and strive to propel research, as well as
expanding cooperative use and collaborative research with
universities and institutes throughout the world.
In 2012 we welcomed several new colleagues, including 4
assistant professors and 5 NIBB research fellows, while 2
colleagues transferred to other institutes.
Finally I would like to congratulate Prof. Yoshinori Ohsumi
for winning the Kyoto Prize, which is widely regarded as oneof the most prestigious international awards for lifetime
achievement in the arts and sciences, and Assistant Prof.
Shinichi Miyagawa for winning the NINS young ResearcherAward. I would also like to congratulate Dr. Kazuya
Kuboyama, Dr. Michitaro Shibata, and Dr. Kenji Fukushima
for receiving recognition for their research achievements asdetailed on page 7.
We always welcome any questions, comments andsuggestions concerning research activities and administrationof NIBB.

Mikio Nishimura, D. Agric.
Deputy Director General
June 11, 2013

1

ORGANIZATION OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) was
founded in 1977 on a hill overlooking the old town ofOkazaki in Aichi prefecture as one of the Inter-University
Research Institutes to promote and stimulate studies of
biology. NIBB conducts first-rate research on site as well as
in cooperation with national, public and private universities
and research organizations. NIBB attempts to elucidate thegeneral and fundamental mechanisms underlying various
biological phenomena throughout a wide variety of
biological fields, such as cell biology, developmental
biology, neurobiology, evolutionary biology, environmental
biology, and theoretical biology.
NIBB and four other national institutes, the National
Astronomical Observatory (NAO), the National Institute for
Fusion Science (NIFS), the National Institute forPhysiological Sciences (NIPS), and the Institute for
Molecular Science (IMS), constitute the National Institutesof Natural Sciences (NINS), which is one of the four InterUniversity Research Institute Corporations. NIBB’s activities
are supported by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan. The Okazaki
Institute for Integrative Bioscience was established as acommon facility for the three institutes (NIBB, NIPS, andIMS) of the Okazaki campus.

■ Organization

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Policy, Decision Making, and Administration
The Director General oversees the operation of NIBB,
assisted by the Advisory Committee for Programming andManagement. The Advisory Committee, comprised ofprofessors within NIBB and an equal number of leading
biologists outside NIBB, advises the Director General on
important matters such as planning joint research programs
as well as on the scientific activities of NIBB. The Advisory
Committee makes recommendations on the appointments of
new Directors General, faculty appointments, NIBB’s annual
budget and future prospects.
The Strategic Planning Department assists the directorgeneral with NIBB’s evaluation procedures and in planning along-range strategy for the institute. The Office of Public
Relations and International Cooperation is a central office forpublic relations and the management of conferences andother extramural activities.
Administration of NIBB is undertaken by the Okazaki
Administration Office under the direct auspices of the
Administration Office of NINS.

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3

Research and Research Support■ Members in NIBBNIBB’s research programs are conducted in research units
and research support units. Each research unit is called either
a division, led by a professor, or a laboratory, led by anassociate professor or an assistant professor. Each research
unit forms an independent project team. The NIBB Core
Research Facilities and NIBB BioResource Center werelaunched in 2010 to support research in NIBB and also to act
as an intellectual hub to promote collaboration among the
researchers of NIBB and other institutions. The NIBB
Center of the Inter-University Bio-Backup Project (IBBP
Center) was founded in 2012 to prevent loss of invaluablebiological resources. The Technical Division manages theactivities of the technical staff and helps to promote theactivities of each research unit and to maintain the common
resources of both the research support units of NIBB and theresearch facilities of the Okazaki campus. Center forRadioisotope Facilities are one of the latter and ran by thetechnical staff of NIBB.
The Okazaki Institute for Integrative Bioscience wasfounded to conduct interdisciplinary molecular research on
various biological phenomena and is shared by all three
institutes in Okazaki. Some of the divisions also function as
NIBB divisions.

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NIBB members

HAKOSHIMA, Toshio*
HIGASHIYAMA, Tetsuya
KONDO, Takao
KURUMIZAKA, Hitoshi*
MIZUSHIMA, Noboru
MORI, Ikue
OHTA, Kunihiro*
TAKABAYASHI, Junji
TANAKA, Ayumi
TSUKITA, Sachiko*
FUJIMORI, Toshihiko
HASEBE, Mitsuyasu
IGUCHI, Taisen
KAWAGUCHI, Masayoshi
KOBAYASHI, Satoru
NISHIMURA, Mikio
NODA, Masaharu
TAKADA, Shinji
UENO, Naoto
YAMAMORI, Tetsuo
YOSHIDA, ShoseiPLOOLRQ\HQ
LQWRWDO(as of April, 2013)

Professor, Nara Institute of Science and Technology
Professor, Nagoya University
Professor, Nagoya University
Professor, Waseda University
Professor, The University of Tokyo
Professor, Nagoya University
Professor, The University of Tokyo
Professor, Kyoto University
Professor, Hokkaido University
Professor, Osaka University
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology* new member from April 2013
# Chairperson and vice-chair will be nominated at meeting scheduled for the end of June, 2013.

4

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Non-NIBB members

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DVRIVW$SULO*UDGXDWH6WXGHQWVLQFXGLQJRQHXQGHUJUDGXDWHGOALS OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) sets five goals
for its activities in pursuing the progress of biology. We contributeto the world-wide community of biologists through our efforts toaccomplish these goals. This chapter briefly explains four of thesegoals. The last goal, the promotion of academic research, isaccomplished through our research activities, which are introduced
throughout this brochure.

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Promotion of Collaborative Research'HYHORSPHQW
RIQHZILHOGV&XOWLYDWLRQRIIXWXUHUHVHDUFKHUV■ Collaborative Research Support
Research activities in collaboration with NIBB’s divisions/laboratories using NIBB’s facilities are solicited from external
researchers. “Individual collaborative research projects” are thebasic form of collaboration support which provide external
researchers with travel and lodging expenses to visit NIBB’s
laboratories for collaborative research. For the use of NIBB’s
unique and excellent research instruments, such as the large
spectrograph, the DSLM, and next generation DNA sequencers
experimental projects are solicited and reviewed to provide
machine time and travel expenses. “Priority collaborative research
projects” are carried out in one to three years as group research byinternal and external researchers with the purpose of developing
pioneering research fields in biology and the “collaborative
research projects for model organism/technology development” arefor developing and establishing new model organisms and new
research technology. For these projects, research expenses in
addition to travel expenses are provided
Shortly after the Great East Japan Earthquake in March 2011 webegan to solicit special emergency collaborative research projects
to allow researchers whose labs sufferred damage to stay at our
institute to continue their work. Six projects from Tohoku
University, Chiba University, and the University of Tokyo etc. were
accepted.

■ NIBB Core Research Facilities
year
Priority collaborative research projects
Collaborative research projects for model organisms/
technology development
Individual collaborative research projects
NIBB workshops
Collaborative experiments using the large spectrograph
Collaborative experiments using the DSLM
Collaborative experiments using
the next generation DNA sequencer
Facility Use (Training Course Facility)
total
total

2009 2010 2011

2012

1

4

6

3

2

2

5
3

54
3
10
−

68
3
8
7

88
6
9
8

89
6
14
5

−

11

45

47

−
71

1
94

0
164

2
171

Collaborative Research Projects by Year

The NIBB Core Research Facilities were launched in 2010 tosupport research in NIBB and also to act as an intellectual hub topromote collaboration among the researchers of NIBB and otheracademic institutions. They consist of three facilities that aredeveloping and providing state-of-the-art technologies throughfunctional genomics, bioimaging and bioinformatics (p. 68).
The Functional Genomics Facility maintains a wide array of coreresearch equipment, including cutting edge tools such as next
generation DNA sequencers. The facility is dedicated to fostering
NIBB’s collaborative research by providing these tools as well as
expertise. The current focus is supporting functional genomics
works that utilize mass spectrometers and DNA sequencers,
holding training courses as one of these undertakings (p. 86). The
Spectrography and Bioimaging Facility manages research tools,
such as confocal microscopes, DSLM and the large spectrograph,and provides technical support and scientific advice to researchers.
These two facilities hold specially-appointed associate professors,
an expert in each field, with a mission to manage each facility as
well as conducting his own academic research. The Data
Integration and Analysis Facility supports large-scale biological
data analysis, such as genomic sequence analysis, expression data
analysis, and imaging data analysis. For this purpose, the facility
maintains high-performance computers with large-capacity storage
systems.

■ NIBB Center of the Inter-University Bio-Backup Project(IBBP Center)
The 2011 Tohoku Earthquake and Tsunami in March, 2011,
caused massive damage to important biological resources. To
prevent such loss, NIBB established the IBBP Center in 2012 incollaboration with seven national universities for multiplicate
preservation of genetic libraries and other invaluable bioresources(p.76).

International Cooperation and Outreach■ Collaborative Programs with Overseas Institutes
NIBB takes a leading role in the collaborative research programs
between the European Molecular Biology Laboratory (EMBL), a
research institute established in 1974 and funded by 21 mostly
European countries, and the National Institutes of Natural Sciences(NINS) and promotes personal and technological exchange throughjoint meetings, exchange between researchers and graduate
students, and the introduction of experimental equipment. The 10th
NIBB-EMBL symposium “Quantitative Bioimaging” was held inMarch, 2013, and will appear in the 2013 Annual Report.
NIBB formed an agreement with the Max Planck Institute for
Plant Breeding Research (MPIPZ) in April 2009 to start a new
initiative aimed at stimulating academic and scholarly exchange inthe field of plant sciences. NIBB and MPIPZ work together to planand promote joint research projects, collaborative symposia,
training courses and student exchange programs.
Collaborative programs have also been started with the Temasek
Life Sciences Laboratory (TLL), of Singapore and Princeton
University. The 4 th NIBB-MPIPZ-TLL Joint Symposium“Arabidopsis and Emerging Model Systems” was held in
November, 2012 at NIBB (p. 85).

■ NIBB Conference
The NIBB Conferences are international conferences on hot
topics in biology organized by NIBB’s professors. Since the first
conference in 1977 (the year of NIBB’s foundation), NIBB
Conferences have provided researchers in basic biology withvaluable opportunities for international exchange. The 58 th/60th
conference “Germline -Speciation, Sex, and Stem Cells-” was held5

in July, 2012 (p.84) and the 59 th conference “Neocortical
Organization” was held in March, 2012 (p. 83).

■ International Practical Course

A frame from a 3D movie ofliving Amoeba proteus firstvisualized by DSLM owing
to its excellent time resolution.

With the cooperation of researchers from Japan and abroad the
NIBB international practical course is given at a laboratory
specifically prepared for its use. The 7 th course “Genetics,
Genomics and Imaging in Medaka and Zebrafish” was held jointlywith TLL and the National University of Singapore (NUS) in July,2012 at NUS and TLL (p. 86). Graduate students and young
researchers from various areas including Germany, China, India,
and Italy, were provided with training in state-of-the-art researchtechniques. International conferences and courses are managed by
the Office of International Cooperation (p. 79).

living samples, and has developed an improved model (p. 67). The
Advisory Committee on Bioimaging, comprised of leading
researchers in the bioimaging field in Japan, is organized toformulate advice on NIBB’s imaging research. The Bioimaging
Forum provides an opportunity for researchers and company
engineers to frankly discuss practical difficulties and needs
regarding imaging (p. 87).

■ Bio-Resources■ Okazaki Biology Conferences

The National BioResource Project (NBRP) is a national project
for the systematic accumulation, storage, and supply of nationallyrecognized bio-resources (experimental animals and plants, cells,DNA, and other genetic resources), which are widely used asmaterials in life science research. To promote this national project,
NIBB has been appointed as a center for research on Medaka(Oryzia latipes) whose usefulness as a vertebrate model was firstshown by Japanese researchers. The usability of Medaka as a
research material in biology has drawn increasing attention since
its full genome sequence recently became available. NIBB is also a
sub-center for the NBRP’s work with Japanese morning glory. The
NIBB BioResource Center has equipment, facilities, and staff tomaintain Medaka and Japanese morning glory safely, efficiently,
and appropriately. The center also maintains other modelorganisms, such as mice, zebrafish, Arabidopsis, Lotus japonicus,
and Physcomitrella patens, and provides technical support andadvice for the appropriate use of these organisms (p. 73).

NIBB holds Okazaki Biology Conferences (OBC) that, with theendorsement of the Union of Japanese Societies for Biological
Science, aim to explore new research fields in biology and support
the formation of international communities in these fields. Dozens
of top-level researchers from Japan and abroad spend nearly one
week together for intensive discussions seeking strategies foraddressing future critical issues in biology. Past Conferences havepromoted the formation of international researcher communities.
The OBC8 “Speciation and Adaptation II: Environment andEpigenetics” was held in March, 2012 (p. 81), and the OBC9“Marine Biology II” was held in October, 2012 (p. 82).

Strains of Japanese morning glory maintained in the center■ Outreach
NIBB’s outreach activities aim to present cutting edge research
results to the public via mass media through press releases ordirectly through the internet. We made a major revision of ourEnglish web page and opened a video gallery containing an
English language introduction of NIBB in 2011. NIBB also
cooperates in the education of undergraduate and younger students
through lectures and workshops. Outreach activities are mostly
managed by the Office of Public Relations (p. 79).

Cultivation of Future Researchers
NIBB constitutes the Department of Basic Biology in the Schoolof Life Science of the Graduate University for Advanced Studies(SOKENDAI). The department provides a five-year course foruniversity graduates and a three-year doctoral course for graduate
students with a master’s degree.
Graduate students enrolled in other universities and institutions
can apply to be special research students eligible to conduct
research for fixed periods of time under the supervision of NIBB
professors.
In both cases above, graduate students can live an academic lifeand receive financial support from NIBB based on the research
assistant (RA) system from the beginning of the five-year course.
Due to the international collaboration with EMBL, graduate
students are encouraged to attend PhD student symposia held at
EMBL and provided an opportunity to give oral and poster
presentations, at least once during their master’s and doctoral
program.
Students from Japan and abroad can also come to NIBB throughour Internship Program. Internships give students an excellent way
to build international connections while experiencing hands on
research in a world class research institute (p. 87).

Development of New Fields of Biology■ Bioimaging
NIBB aims to maximize the application of modern lightmicroscopes and biophotonic probes for real time visualization of
biological phenomena and to develop new imaging techniques. As
part of our collaborative work with EMBL, NIBB introduced a
DSLM, which is effective for the three-dimensional observation of

6

Graduate students educated by NIBB■ Personnel changes in 2012*

Newly assigned in NIBB
Name

Position

Research Unit

Date

MAEDA, TaroNIBB Research Fellow

Functional Genomics Facility

March 16

MII, Yusuke

Assistant Professor

Division of Molecular and Developmental Biology

April 1ISHIKAWA, Masaki

Assistant Professor

Division of Evolutionary Biology

April 1

WAKE, Hiroaki

Assistant Professor

Division of Brain Circuits

April 1

GOYO-YAMADA, ShinoNIBB Research Fellow

Division of Cell Mechanisms

April 1MIYAKAWA, Hitoshi

NIBB Research Fellow

Division of Molecular Environmental Endocrinology

April 1TANAKA, Yasuhiro

NIBB Research Fellow

Division of Brain Circuits

April 1SOYANO, Takashi

NIBB Research Fellow

Division of Symbiotic Systems

July 1

TAKEHANA, Yusuke

Assistant Professor

Laboratory of Bioresources

November 1

Newly affiliated in other universities and institutes
Name

New Affiliation

Position

Date

HIWATASHI, Yuji

Aberystwyth University

Marie Curie Fellow

April 1WATANABE, Takaaki

Lerner Research Institute, Cleveland Clinic

Postdoctoral Research Fellow

April 1

* Changes in professors, associate and assistant professors, and NIBB research fellows are shown.

■ Awardees in 2012

Name

PositionAward

KUBOYAMA, Kazuya

NIBB Research Fellow

FUKUSHIMA, Kenji

Graduate Student

Young Investigator Award of Japanese Association
for Protein phosphatase Research
Japanese Society for Plant Systematics
Meeting Presentation Award (Oral Presentation Section)

SHIBATA, Michitaro

Graduate Student

Sokendai President’s AwardMIYAGAWA, Shinichi

Assistant Professor

NINS Young Researcher Award

OHSUMI, Yoshinori

Professor Emeritus

The Kyoto Prize

Note: On the unit member lists from P. 8 all members who belonged to the unit during 2012 are listed irrespective of the length of the period they were members.
Those appearing twice in the same list under different titles are those whose title was changed during 2012. The former title is indicated by an asterisk (*).

7

National Institute for Basic Biology

Cell Biology

DIVISION OF CELL MECHANISMS

Professor

Associate Professor

NISHIMURA, Mikio

HAYASHI, Makoto

Assistant Professor:

MANO, Shoji
YAMADA, Kenji
KONDO, Maki
GOTO-YAMADA, Shino
OIKAWA, Kazusato
KANAI, Masatake
WATANABE, Estuko
TANAKA, Mina
KAMIGAKI, Akane
NITO, Kazumasa
GOTO-YAMADA, Shino*
NAKAI, Atsushi*
CUI, Songkui*
SHIBATA, Michitaro
SAITO, Miyuki
NAKAYAMA, Tomomi
HIKINO, Kazumi
YOSHINORI, Yumi
YAMAGUCHI, Chinami
NAKAI, Atsushi
CUI, Songkui
UEDA, Chizuru

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:

Graduate Student:
Technical Assistant:

Secretary:

Since plants spread their roots in the ground, they must
survive in a given environment. To adapt to the environment,
they positively utilize environmental changes in the life cycle
as important signals that are necessary for their survival.
Plant cells can induce, degenerate and differentiate theirorganelles to adapt to environmental changes. The flexibilityof plant organelles is the basis of the strategy forenvironmental adaptation in plants.
The aim of this division is to clarify the molecularmechanisms underlying the induction, differentiation, andinteraction of organelles, and to understand the integratedfunction of individual plants through organelle dynamics.
The Scientific Research on Innovative Areas “Environmental
sensing of plants: Signal perception, processing and cellularresponses” was started to clarify the molecular mechanisms
underlying organelle differentiation and interaction.

pumpkin cotyledons, the reverse transition of leaf
peroxisomes to glyoxysomes occurs during senescence. Gene
expression, alternative splicing, protein translocation andprotein degradation control the functional transformation
between glyoxysomes and leaf peroxisomes.

II. Transcriptomics, proteomics and phenomicsof plant peroxisomes
Enzymes localized in plant peroxisomes are synthesized in
the cytosol and function after their post-translationaltransport into peroxisomes. Almost all of the peroxisomal
matrix proteins contain one of two targeting signals (PTS1and PTS2) within the molecules. PTS1 is a unique tripeptide
sequence found in the carboxyl terminus of the mature
proteins. In contrast, PTS2 is involved in a cleavable amino
terminal presequence of peroxisomal proteins that aresynthesized as a precursor protein with larger molecularmass.
We identified 256 gene candidates of PTS1- and PTS2containing proteins and another 30 genes of non-PTScontaining proteins from the Arabidopsis genome. Custommade DNA microarrays covering all these genes were used
to investigate expression profiles of the peroxisomal genes invarious organs. They revealed that peroxisomes in root cells
play a pivotal role in polyamine catabolism. We also made a
two-dimensional protein map of glyoxysomes and leaf
peroxisomes isolated from Arabidopsis and soybean. Peptide
MS fingerprinting analyses allowed us to identify novel
peroxisomal membrane proteins, i.e. voltage-dependentanion-selective channel and adenine nucleotide carrier 1(PNC1). We also found that peroxisomal membrane ATPbinding cassette transporter promotes seed germination by
inducing pectin degradation under the control of abscisicacid signaling. The overall results provide us with new
insights into plant peroxisomal functions.
Bioinformatic analysis of the Arabidopsis genome predicted
the presence of 15 kinds of genes, called PEX genes, forperoxisomal biogenesis factors. We demonstrated that PEX5
and PEX7 form a cytosolic receptor complex and recognize
PTS1- and PTS2-containing proteins, respectively. PEX14 isa peroxisomal membrane docking protein that captures the

I. R e v e r s i b l e t r a n s f o r m a t i o n o f p l a n t
peroxisomes
Dramatic metabolic changes that underlie the shift fromheterotrophic to autotrophic growth occur in the greening ofseed germination. Accompanying these metabolic changes
are the functional transformations of many constitutive
organelles. Etioplasts differentiate into chloroplasts while
mitochondria acquire the ability to oxidize glycine.
Glyoxysomes, which are peroxisomes engaged in the
degradation of reserve oil stored in oil body (Figure 1) via
β-oxidation and the glyoxylate cycle, are transformed intoleaf peroxisomes that function in several crucial steps ofphotorespiration. After the functional transition ofglyoxysomes to leaf peroxisomes during the greening of

8

Figure 1. Oil body visualized by oleosin-GFP. (A) Five-day-old darkgrown seedlings expressing oleosin-GFP. (B) Numerous oil bodies in thecell of dry seed. (C) High-resolution image of aggregated oil bodies. Bar= 5 μm.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

receptor-cargo complex. We also comprehensively
investigated whether or not these predicted PEX genes
function in peroxisome biogenesis by generating knockdown mutants that suppress PEX gene expression by RNAinterference. Phenotypes of these mutants allowed us to
identify the functional PEX genes, which can be classified
into two groups: PEX genes regulating for peroxisomal
protein import and PEX genes regulating for peroxisomal
morphology. We continue to investigate the detailed
molecular functions of other PEX genes. Of these, we
recently proposed that function of PEX7 is maintained by aquality control mechanism involving RabE1c.

III. Identification of novel components
essential for peroxisome biogenesis
To better understand peroxisome biogenesis and functions,
we isolated a number of Arabidopsis mutants having aberrant
peroxisome morphology (apem mutants) based on themhaving a different pattern of GFP fluorescence from theparent plant, GFP-PTS1, in which peroxisomes with normal
sizes and numbers can be visualized with GFP.
Of these apem mutants, APEM1 gene (whose defect causes
the elongation of peroxisomes and mitochondria) encodes
dynamin-related protein 3A, which is involved in division ofboth organelles. APEM2 and APEM4 (whose defects cause adecrease in the efficiency of protein transport) were revealed
to encode proteins homologous to PEROXIN 13 (PEX13)and PEX12, respectively, and both proteins are responsible
for protein translocation on peroxisomal membranes.
APEM9 is the plant-specific PEX that has a role in tethering
the PEX1-PEX6 complex on peroxisomal membranes. In
addition, we found that APEM3 encodes Peroxisomal
membrane protein 38, and that its defect causes enlargementof peroxisomes (Figure 2).

We are currently characterizing other apem mutants. Fromthese analyses, we will be able to identify the components
responsible for peroxisome biogenesis and functions, and toaddress the mechanism at the molecular level.

IV. ER derived organelles for protein storingand defense strategy
Plant cells develop various types of endoplasmic reticulum(ER)-derived structures with specific functions. ER bodies
are ER-derived compartments observed in Arabidopsis. They
are rod-shaped structures surrounded by ribosomes, and arewidely distributed in the epidermal cells of whole seedlings.
Undamaged rosette leaves have no ER bodies, but
accumulate ER bodies after wounding or jasmonic acidtreatment. This suggests that ER bodies function in the
defense against herbivores. ER bodies in seedlings include
β-glucosidase PYK10. When plant cells are damaged,
PYK10 forms large protein aggregates. The aggregate
formation increases glucosidase activity, possibly producing
toxic products. Arabidopsis nai1 mutants have no ER bodies
in the entire plant and do not accumulate PYK10. NAI1
encodes a transcription factor and regulates the expressionof PYK10 and NAI2. The Arabidopsis nai2 mutant has no ER
bodies and reduced accumulation of PYK10. NAI2 encodes aunique protein that localizes to the ER body. We found thatthe membrane protein of ER body 1 (MEB1) and MEB2 areintegral membrane proteins of the ER body. MEB1 andMEB2 have homology to yeast iron/manganese transporter
CCC1. Heterologous expression of MEB1 and MEB2 inyeast ccc1 mutant suppresses the iron toxicity (Figure 3),indicating that MEB1 and MEB2 are iron/manganesetransporters. These results suggest that the ER body has
specific membrane proteins that are involved in defense
against metal stress as well as pathogens and herbivores.
NAI2 deficiency relocates MEB1 and MEB2 to the ER
network. These findings indicate that NAI2 is a key factor
that enables ER body formation. We are now investigating
the function of NAI2 on ER body formation byheterologously expressing it in onion and tobacco cells.

V. Roles of molecular chaperones on celldifferentiation
Molecular chaperones are cellular proteins that function in
the folding and assembly of certain other polypeptides into
oligomeric structures. We have found that HSP90 inhibitor
induces genes with heat shock response element (HSE)

Figure 2. PMP38/APEM3 has a role as a transporter on the peroxisomal
membrane. (A, B) Compared to the parent plant (A), peroxisomes areenlarged in the apem3 mutant (B). (C) Subcellular localization andfunction of PMP38. PMP38-GFP (green) and RFP-PTS1 (magenta) weretransiently expressed in Arabidopsis leaf cells (Inset). PMP38 is present
as a dimer, and has a role in transport of NAD+, CoA and ADP. Scalebars, 10 μm (A, B) and (B) 1 μm (C).

Figure 3. MEB1 and MEB2 suppress the toxicity of iron. Yeast ironsensitive ccc1 mutants harboring empty vector, vector for MEB1, or
MEB2 were grown on synthetic minimal medium with (upper) or without(lower) 2 mM iron.

9National Institute for Basic Biology

Cell Biology

motifs in their promoters, suggesting that heat shocktranscription factor (HSF) is involved in the response.
Arabidopsis HSFs interacted with HSP90.2. Thus, it appears
that in the absence of heat shock, HSP90 actively suppresses
HSF function. During heat shock, HSP90 is transiently
inactivated, which leads to HSF activation. These data
indicate that HSP90 regulates correct gene expression forheat acclimatization in plants. We also observed that HSP90
is involved in hormone responses in Arabidopsis. The
evolutional and functional characterizations are now being
investigated.

ultrastructures of organelles by electron microscopy, soon. It
is expected that PODB2 will contribute to systems biology
through the combination of the included data with other‘omics’ data and computational analyses. In addition, we
updated the website, Plant Organelles World, which is basedon PODB2 as an educational tool to engage members of thenon-scientific community. We expect that PODB2 and Plant
Organelles World will enhance the understanding of plantorganelles among researchers and the general public whowant to explore plant biology.

VI. Update of The Plant Organelles Database 2(PODB2) and release of Plant Organelles
World

Publication List

The Plant Organelles Database 2 (PODB2) was built topromote a comprehensive understanding of organelle
dynamics. This public database is open to all researchers.
PODB2 consists of five individual units: the perceptive
organelles database, the organelles movie database, theorganellome database, the functional analysis database, andexternal links. The perceptive organelles database, which
was added as new content, shows organelles dynamics in
response to environmental stimuli (Figure 4). The organelles
movie database contains time-lapse images and 3D structure
rotations. The organellome database is a compilation of static
image data of various tissues of several plant species atdifferent developmental stages. The functional analysis
database is a collection of protocols for plant organelle
research. The amount of included data is increasing day byday. We will add new content, which is dedicated to〔Original papers〕
M., Nanba, C., Saito, M., Kondo, M., Takeda, A., Watanabe,• Hayashi,Y., and Nishimura, M. (2012). Loss of XRN4 function can trigger

•
•
•
•

cosuppression in a sequence-dependent manner. Plant Cell Physiol. 53,
1310-1321.
Nakano, R.T., Matsushima, R., Nagano, A.J., Fukao, Y., Fujiwara, M.,
Kondo, M., Nishimura, M., and Hara-Nishimura, I. (2012). ERMO3/
MVP1/GOLD36 is involved in a cell type-specific mechanism for
maintaining ER morphology in Arabidopsis thaliana. PLoS ONE 7,
e49103.
Nakayama, M., Kaneko, Y., Miyazawa, Y., Fujii, N., Higashitani, N.,
Wada, S., Ishida, H., Yoshimoto, K., Yamada, K., Nishimura, M., andTakahashi, H. (2012). A possible involvement of autophagy in
amyloplast degradation in columella cells during hydrotropic responseof Arabidopsis roots. Planta 236, 999-1012.
Negishi, T., Oshima, K., Hattori, M., Kanai, M., Mano, S., Nishimura,M., and Yoshida, K. (2012). Tonoplast- and plasma membrane-localized
aquaporin-family transporters in blue hydrangea sepals of aluminum
hyperaccumulating plant. PLoS ONE 7, e43189.
Toyokura, K., Hayashi, M., Nishimura, M., and Okada, K. (2012).
Adaxial-abaxial patterning: A novel function of the GABA shunt. Plant
Signal. Behav. 7, 705-707.

〔Original papers (E-publication ahead of print)〕
K., Narise, T., Sonoike, K., Hashimoto, H., Sato, N., Kondo,• Kobayashi,M., Nishimura, M., Sato, M., Toyooka, K., Sugimoto, K., Wada, H.

•

Masuda, T., and Ohta, H. Role of galactolipid biosynthesis incoordinated development of photosynthetic complexes and thylakoid
membranes during chloroplast biogenesis in Arabidopsis. Plant J. 2012
Nov. 26.
Yamada, K., Nagano, A.J., Nishina, M., Hara-Nishimura, I., andNishimura, M. Identification of two novel endoplasmic reticulum bodyspecific integral membrane proteins. Plant Physiol. 2012 Nov. 19.

〔Review articles〕
S., Kimori, Y., Takeda, T., Miwa, T., Nishikawa, S., Mimura, T.,• Mano,Nagatani, A., and Nishimura, M. (2012). Using image-based resources:•

Figure 4. The graphical user interface of the perceptive organelles
database in PODB2 (http://podb.nibb.ac.jp/Orgenellome).

10

Databases for plant organelle dynamics and applications based onimage information. In Introduction to Sequence and Genome Analysis.
iConcept Press Ltd. ed. (Brisbane, iConcept Press Ltd.), chapter 16, pp.
1-25.
Tanaka, Y., Kimura, T., Hikino, K., Goto, S., Nishimura, M., Mano, S.,
and Nakagawa, T. (2012). Gateway vectors for plant genetic
engineering: Overview of plant vectors, application for bimolecularfluorescence complementation (BiFC) and multigene construction. In
Genetic Engineering – Basics, New Applications and Responsiblities.
Barrera-Saldaña, H. A ed. (Rijeka, INTECH), pp. 35-58.

DIVISION OF INTERCELLULAR SIGNALING BIOLOGY

Professor

MATSUBAYASHI, Yoshikatsu

Assistant Professor:
Postdoctoral Fellow:
Visiting Scientist:
Technical Assistant:
Secretary:

SHINOHARA, Hidefumi
KAKITA, Mitsuru
OKAMOTO, Satoru
MORI, Ayaka
YOSHII, Tomoaki
OGAWA-OHNISHI, MariYASUE, Naoko
OKUBO, Masayo

Cell-to-cell signaling mediated by secreted signals andmembrane-localized receptors is one of the criticalmechanisms by which growth and development ofmulticellular organisms are cooperatively regulated. Signal
molecules that specifically bind receptors are generally
referred to as ligands. Because membrane-localized receptors
act as master switches of complex intracellular signaling,
identification of the ligand-receptor pair is one of the central
issues of post-genome research. We are working to clarify
the mechanisms by which plant development is regulated
through identification of novel ligands such as small peptides
and their specific receptors using Arabidopsis genome
information, biochemical analysis and phenotypicobservation.

tyrosine sulfation of secreted peptides and proteins. Thisapproach is based on the assumption that the severe shortroot phenotype of the tpst-1 mutant reflects deficiencies inthe biosynthesis of all the functional tyrosine-sulfatedpeptides, including undiscovered peptide signals. RGFs areproduced from ≈100-amino-acid precursor peptides via posttranslational sulfation and proteolytic processing. RGF
family peptides are expressed mainly in the stem cell area
and the innermost layer of central columella cells, anddiffuse into the meristematic region. RGF peptides regulate
root development by stabilizing PLETHORA transcription
factor proteins which are specifically expressed in root
meristem and mediate patterning of the root stem cell niche(Figure 1).
To gain more insight into RGF signaling, we havedeveloped a positive screening system to identify
Arabidopsis mutants with altered response to RGF peptides.
Several mutants that are less sensitive to RGF have been
identified and are currently being further analyzed.

I. Secreted peptide signals
Following complete sequencing of the Arabidopsis genome,
a number of genes encoding small secreted peptides havebeen identified by in silico database analysis. Based on ourown analysis, we identified 979 putative secreted peptide
genes with an open reading frame (ORF) size between 50
and 150 amino acids in the Arabidopsis genome. These 979
ORFs include many functionally uncharacterized peptides.
Although estimation of the total number of biologically
relevant secreted peptide signals is difficult at present, thepresence of many “orphan receptors” among receptor-likekinases in Arabidopsis suggest that a substantial number ofintercellular signals remain to be identified.
One structurally characteristic group of peptide signals is“small post-translationally modified peptides”. These
peptides are characterized by the small size of mature
peptides (less than 20 amino acids) and the presence of posttranslational modifications. In these peptide signals, peptide
chain length and post-translational modifications are
generally very important for their receptor binding activity
and physiological functions.

1-1 Root meristem growth factor (RGF)
Root meristem growth factor (RGF) is a 13-amino-acid
tyrosine-sulfated peptide involved in maintenance of the root
stem cell niche in Arabidopsis identified by our group in
2010. RGF was identified in a search for sulfated peptides
that recover root meristem defects of the loss-of-function
mutant of tyrosylprotein sulfotransferase (tpst-1). TPST is apost-translational modification enzyme that catalyzes

Figure 1. Mode of action of RGF peptide. (A) Structure of RGF1. (B)Expression domain of RGF genes. (C) Localization of RGF peptides
visualized by anti-RGF1 antibody. (D) Expression pattern of
PLETHORA (PLT2-GFP) transcription factor, a downstream target of
RGF signaling.

1-2 Other novel peptide signal candidates
The common feature of known small post-translationallymodified peptide signals is that they are encoded by multiple
paralogous genes whose primary products are approximately
70- to 110-amino-acid cysteine-poor secreted polypeptides
that share short conserved domains near the C-terminus. We
have identified several novel polypeptide families that fulfill
the above criteria by in silico screening and determined theirmature structures by analyzing apoplastic peptide fractions
by nano LC-MS/MS. Functional analysis of these peptides is
now going on.

II. Post-translational modification mechanisms
Post-translational modifications are known to affect peptide
conformation through steric interactions with the peptide
backbone, thereby modulating the binding ability andspecificity of peptides for target receptor proteins. To date,11

National Institute for Basic Biology

Cell Biology

the following types of post-translational modification havebeen identified in secreted peptide signals in plants: tyrosine
sulfation and hydroxyproline arabinosylation (Figure 2).

signals in plants. We have succeeded in the stereoselective
total synthesis of β-1,2-linked tri-arabinosylated CLV3
peptide ([Ara 3]CLV3) (Figure 3 and 4). Comparison of
mono-, di- and tri-arabinosylated CLV3 glycopeptides
revealed that the biological activity increased progressively
as arabinose chain length increased. Thus, arabinose chain
length of CLV3 is important for its biological activity.

Figure 3. Synthesis of triarabinosylated Hyp building block.

Figure 2. Post-translational modifications in secreted peptide signals inplants.

2-1 Tyrosine sulfation
Arabidopsis tyrosylprotein sulfotransferase (AtTPST) is a
Golgi-localized 62-kDa transmembrane protein identified byour group in 2009. AtTPST is expressed throughout the plantbody, and the highest levels of expression are observed in theroot apical meristem. A loss-of-function mutant of AtTPST(tpst-1) displayed a marked dwarf phenotype accompaniedby stunted roots, loss of root stem cells, pale green leaves
and early senescence, indicating the important roles of
sulfated peptides in plant growth and development. Threeknown sulfated peptide signals, PSK, PSY and RGF, canalmost fully restore root defects of tpst-1 when added to theculture medium, but can not fully restore phenotypes in theabove-ground parts of the plant. This observation suggeststhat as yet undiscovered sulfated peptides may regulate plantdevelopment. A search for novel sulfated peptide signals is
now in progress.

2-2 Hydroxyproline arabinosylation
Hyp residues in several secreted peptide signals, such as
CLV3 and CLE2 are further modified with an O-linked
L -arabinose chain. This modification is physiologicallyimportant for these peptide signals. Biosynthesis of Hypbound β-1,2-linked triarabinoside involves two distinct
arabinosyltransferases. The first is responsible for theformation of a β-linkage with the hydroxyproline(hydroxyproline arabinosyltransferase), and the second forms
a β-1,2-linkage between arabinofuranose residues(arabinosyltransferase). Arabinosyltransferase has alreadybeen reported, but there have been no reports onhydroxyproline arabinosyltransferase (HPAT). We haveestablished an in vitro assay system to detect HPAT activity
and are currently attempting to purify this enzyme by affinity
chromatography.

12

Figure 4. Shoot apical meristem of wild-type, clv3-1 seedlings and clv3-1
seedlings treated with chemically synthesized [Ara3]CLV3 at 30 nM.

2-4 Conformation of arabinosylated peptides
NMR spectroscopy and NOE-based structure calculations
revealed the structural impact of the arabinose chain on
peptide conformation. The arabinose chain of [Ara3]CLV3
extends toward the C-terminal end of the peptide, and its
non-reducing end is positioned proximal to the peptide
backbone (Figure 5). Consequently, the arabinose chaincauses distinct distortion in the C-terminal half of the peptide
in a highly directional manner. The established syntheticroute of [Ara 3 ]CLV3 will greatly contribute to ourunderstanding of the biology and biochemistry ofarabinosylated peptide signals in plants.

Figure 5. Energy-minimized structure of [Ara3]CLV3 resulting from asimulated annealing protocol that incorporated NOE-derived distance
restraints.

2-3 Chemical synthesis of arabinosylated peptides

III. Receptors for secreted peptide signals

Ar a b i n o sy l a t io n o f h y d ro x y p ro lin e (H y p) is aposttranslational modification often found in secreted peptide

The receptors or putative receptors for peptide signals
identified to date belong to the receptor kinase (RK) orreceptor-like protein (RLP) families. Among RKs, the largest
subfamily is the leucine-rich repeat RK (LRR-RK) family,
which consists of 216 members in Arabidopsis. The majorityof receptors for small post-translationally modified peptide
signals belong to this family. Especially, an increasing
number of LRR X and LRR XI members are now being
confirmed as receptors for several endogenous small peptide
ligands, suggesting that these subgroups are an attractive
target for binding analysis with novel peptide signals.

3- 1 Receptor expression library
Although both genetic and biochemical methods have beenused to identify ligand-receptor pairs in plants, genetic
redundancy often interferes with the former approach, andthe low levels at which ligand and receptor molecules areoften present in tissues can make the latter approach very
difficult. As described in the above section, an increasing
number of ligand candidates are being identified. If
individual receptor kinases could be functionallyoverexpressed in certain cells at sufficiently high levels andsufficiently high quality for biochemical binding analysis,
such a receptor library would facilitate identification ofligand-receptor pairs in plants. To this end, we established afunctional and efficient expression system of plant receptor
kinases in tobacco BY-2 cells and prepared an expression
library of all the potential receptor candidates.

LRR-RKs is slightly deviatory from the LRR consensus. Our
results indicate that ligand recognition mechanisms of plant
LRR-RKs are more complex and diversified than anticipated.

Publication List
〔Original paper〕
H., Moriyama, Y., Ohyama, K., and Matsubayashi, Y.
• Shinohara,(2012). Biochemical mapping of a ligand-binding domain within
Arabidopsis BAM1 reveals diversified ligand recognition mechanismsof plant LRR-RKs. Plant J. 70, 845-854.

〔Original paper (E-publication ahead of print)〕
H., and Matsubayashi, Y. Chemical synthesis of Arabidopsis• Shinohara,
CLV3 glycopeptide reveals the impact of Hyp arabinosylation on
peptide conformation and activity. Plant Cell Physiol. 2012 Dec 19.

〔Review article〕
Y. (2012). Recent progress in research on small post• Matsubayashi,translationally modified peptide signals in plants. Genes Cells. 17, 1-10.

3- 2 Structural basis for ligand recognition
Leucine-rich repeat receptor kinases (LRR-RKs) comprisethe largest subfamily of the transmembrane receptor kinases
in plants. In several LRR-RKs, a loop-out region called an“island domain” that intercepts the extracellular tandem
LRRs at a position near the transmembrane domain
constitutes the ligand-binding pocket, but the absence of theisland domain in numerous LRR-RKs raises questions about
which domain specifically recognizes the corresponding
ligands in non-island domain-carrying LRR-RKs. We
determined, by photoaffinity labeling followed by chemicaland enzymatic digestion, that BAM1, a CLV1/BAM family
LRR-RK whose extracellular domain is comprised of 22
consecutive LRRs, directly interacts with the small peptide
ligand CLE9 at the LRR6-8 region that is relatively distalfrom the transmembrane domain (Figure 6). Multiple
sequence alignment and homology modeling revealed thatthe inner concave side of LRR6-8 of the CLV1/BAM family

Figure 6. Deduced structure of BAM1 extracellular LRR domain byhomology modeling and comparison with the BRI1 crystal structure.
Ligand-binding domains are highlighted in red.

13

National Institute for Basic Biology

Cell Biology

LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:
Technical Assistant:

NAKAYAMA, Kei
MATSUDA, Chisato

The transport of specific mRNAs and local control oftranslation in neuronal dendrites represent an important gene
expression system that provides localized protein synthesis indendrites at just the right time and place. It is believed that
this system controls the location at which neurites willconnect to each other, thereby forming neural networks. Our
main interest is to understand the mechanisms and roles ofmRNA transport and local translation in neuronal dendrites.
It is known that specific mRNAs are recruited into “RNAgranules” in neuronal dendrites. RNA granules aremacromolecular complexes composed mainly of mRNAs,
ribosomes and RNA-binding proteins, and mediate thetransport and local translation of their mRNA cargoes in
response to synaptic stimulation (Figure 1). We areresearching RNA granule factors regulating mRNA transportand local translation, their target mRNAs, and themechanisms of localized protein synthesis using mice inorder to better understand its relation to the formation ofsynapses and neural networks, memory, learning, andbehavior.

is associated with defects in fetal brainstem development. To
investigate the role of RNG105 in higher brain functions,e.g., memory and learning, in adult mice, we generated
RNG105 conditional knockout (cKO) mice using the Cre/
loxP system. Alpha-CaMKII promoter was used to drive Cre
recombinase since its promoter activity was low during
embryonic stages but elevated after birth in the brain. We
successfully obtained RNG105 cKO mice that grew into
adults (Figure 2A). Expression of RNG105 was markedly
reduced in the cerebrum, especially in the hippocampal
pyramidal neurons of adult cKO mice (Figure 2B). Open
field behavior testing revealed that exploratory activity of
RNG105 cKO mice in a novel environment was not changed
between the first and later trials, although exploratory
activity of wild-type mice was reduced with increasing
number of trials, suggesting that the knockout mice had someproblems in being acclimated to a new environment. We are
currently analyzing learning and memory in the cKO mice.
RNG105 has one paralog, RNG140, which has RNAbinding domains highly conserved with RNG105. RNG105
and RNG140 are localized to different kinds of RNA
granules and their timing of expression is also different:
RNG105 is highly expressed in embryos, but RNG140 ishighly expressed in adults (Shiina and Tokunaga, J. Biol.
Chem. 285, 24260-24269, 2010). We have obtained RNG140
knockout mice and are going to investigate the role of
RNG140 in higher brain functions in adult mice.

Figure 1. A model for local translation in neuronal dendrites. SpecificmRNAs are recruited into RNA granules and transported to dendrites.
Translation of the mRNAs is induced locally upon synaptic stimulation,
which modifies local postsynapses to regulate synaptic connection andnetwork formation.

I. Knockout mice for RNA granule proteins
We previously identified RNA granule protein 105(RNG105), an RNA-binding protein, as a component ofRNA granules. RNG105 is responsible for mRNA transport
to dendrites, which is required for the encoded proteins to betranslated and function in dendrites for proper networking of
neurons (Shiina et al., J. Neurosci. 30, 12816-12830, 2010).
RNG105 knockout mice exhibit reduced dendritic synapse
formation and reduced dendritic arborization, which resultsin poor development of neuronal networks. The knockout
neonates die soon after birth due to respiratory failure, which

14

Figure 2. RNG105 conditional knockout (cKO) mice. A, 10-week-old
adult wild-type and RNG105 cKO mice. B, Brain hippocampal slices
from wild-type and RNG105 cKO mice were immunostained with ananti-RNG105 antibody. S, somatic layer; D, dendritic layer ofhippocampal pyramidal neurons. RNG105 was reduced in pyramidal
neurons of RNG105 cKO mice.

II. Molecular characterization of the RNA
granule complex
RNA granules are abundantly formed in neurons, but notobserved in many other types of cells. However, theformation of RNA granules is induced by stress such asoxidation and virus infection in the cells. They are called“stress granules” and have common features with neuronal
RNA granules, e.g., they are macromolecular complexes
containing ribosomes and mRNAs, and repress translation ofcargo mRNAs. We previously found that RNG105 was acomponent of not only neuronal RNA granules but also stressgranules, and further that overexpression of RNG105 infibroblastic cells induced the formation of stress granules
which contain mRNAs. Because knowledge about stressgranules helps us in studying neuronal RNA granules, ourresearch includes identification and characterization ofmolecular components of stress granules.
We have identified a novel component of stress granules,
NFAR2. NFAR2 was recruited into RNG105-induced stressgranules and enhanced the assembly of the stress granules(Figure 3). It is known that cellular stress activates masterkinases including PKR, which induces translation repression
and stress granule formation. We found that NFAR2
phosphorylation by PKR enhanced its localization to, and theformation of, stress granules. Furthermore, we found that
NFAR2 was bound by its partner NF45 and antagonized its
effect on stress granules in basal condition, but it overcame
the antagonistic regulation by NF45 when phosphorylated by
PKR. Because PKR is reportedly implicated in learning andmemory, these results will provide insights into theregulatory mechanism of neuronal RNA granule formation in
the brain.

Figure 3. NFAR2 is a component of stress granules. A6 cells wereco-transfected with RNG105-monomeric red fluorescent protein 1(mRFP1) and NFAR2-green fluorescent protein (GFP). Expression of
RNG105 induced the formation of cytoplasmic stress granules. NFAR2
was predominantly localized to the nucleus, and it was also recruited intoand enlarged the stress granules (arrows). Scale bar, 10 μm.

15National Institute for Basic Biology

Cell Biology

LABORATORY OF CELL SOCIOLOGY

Assistant Professor

HAMADA, Yoshio

Technical Assistant:

GONDA, Naoko

Mammalian eggs implant onto the maternal uterine wallafter hatching at the blastula stage, due to a lack of enoughnutrients to support development until birth. In order totransfer nutrients and oxygen into the fetus, and metabolic
wastes into the mother, the placenta has evolved to establish
close connections between the maternal and fetal
vasculature. There is amazing diversity from one animal toanother in the features, such as gross structures, microscopicarrangements and developmental processes, but the functionof this organ is invariable.
Primates and rodents have the ancestral type of placenta,
classified as heomochorial, in which maternal blood is notcontained within endothelial cell lined vessels but rather is in
direct contact with epithelial cells of the fetal placenta thatare derived from the trophoblast cell lineage. In mice,maternal blood coming out from the spiral arteries invaded
by trophoblast cells that replace the endothelium passes
across the fetal layers of the placenta and then enters into thelabyrinth where fetal blood vessels form and feto-maternal
exchange occurs. Maternal and fetal vascular circuits in theestablished mouse placenta are shown schematically in
Figure 1.
Notch2 null mutation induces lethality around embryonicday 11 because of impaired maternal vasculature formation.
However, both cellular and molecular events in the processof vasculature formation are little known, especially as to
how the maternal blood bed expands among the tightly
packed trophoblast mass. Our histological studies showedthat a stream of maternal blood along the interface between
labyrinthine and spongiotrophoblast was slightly opened in
the mutant placenta. Expression of the Notch2 gene isobserved in spongiotrophoblast and Giant cells, but in neither
the labyrinthine trophoblast or multi-nuclear
Syncytiotrophoblast that is derived by cell fusion of themono-nuclear labyrinthine trophoblast (Figure 2). Thus, weconclude that cellular and molecular events to form thematernal blood bed occur in spongiotrophoblast and Giantcell layers.
We have assumed that the cellular event that expands thematernal blood bed is the deletion of spongiotrophoblast in asignificant number from the developing placenta. Thus, weare trying to detect trophoblast cell death or detachment withvarious methods.

Figure 1. Schematic maternal and fetal vascular circuits in mouseplacenta. Maternal blood flows in streams drawn in yellow in the
direction shown by small red arrows. Fetal blood running in the artery(blue line) reach at the border spongio- and labyrinth layers and return tofetal vein (red line).

Figure 2. Expression of Notch2 gene in developing mouse placenta. Notch2 gene is expressed in the ectoplacental cone at E8.0, but not in the chorion. At
E9.0, the ectoplacental cone and chorion differentiate into spongiotrophoblast (Sp) and labyrinthine trophoblast (Lb), respectively. The expression isdetected in Sp. At E10.0, differentiation of syncytiotrophoblast (SynT) and vasculogenesis of fetal blood are obvious in the placenta. Notch2 expression
occurs in Sp, Ginat cell layer (Gc), and mesenchymal cells derived from allantois. However, it is not detected in SynT and Lb far away from maternal
blood or separated by SynT.

16

National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

UENO, NaotoKINOSHITA, Noriyuki

Assistant Professor:

TAKAHASHI, Hiroki
SUZUKI, Makoto
TAKAGI, Chiyo
HASHIMOTO, Masakazu
SUZUKI, Miho
HARA, Yusuke
MIYAGI, Asuka
HAYASHI, Kentaro
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
SUZUKI, Atsuko
MIYAKE, Satoko
TSUGE, Toyoko

Technical Staff:
Postdoctoral Fellow:
PRESTO Researcher:
Graduate Student:
Technical Assistant:
Secretary:

The complex morphogenesis of organisms is achieved bydynamic rearrangements of tissues during embryogenesis, inwhich change in cellular morphology as well as orchestrated
cell movements are involved. For cells to know how theyshould change their shape and where they should move,
information called “cell polarity” is essential. How then is
the cell polarity established within cells? Is it intrinsicallyformed within the cells or triggered by extracellular cues?
Furthermore, little is known as to how coordinated andcomplex cell movements are controlled in time and space.
We attempt to understand the mechanisms underlying these
events using several model animals, including frogs, fish,
mice and ascidians, taking physical parameters such as force
in consideration, in addition to conventional molecular andcellular biology.

precedent tissue Leading Edge Mesoderm (LEM). Whensurgically isolated the LEM migrates fairly rapidly toward
the predetermined anterior side, while the following axial
mesoderm shows little directed tissue migration. We
hypothesized that the LEM generates traction force on thefollowing axial mesoderm. To prove the biologicalsignificance of the force generated by the LEM, we haveremoved the LEM, cultured to the end of gastrulation, andfound that the movement of the LEM is required for theproper narrowing and elongation of the axial mesoderm(Figure 1).

Figure 1. (A, A’) External appearance of early neurula embryos. Shamoperated embryo (A) in which the LEM was remained but not the LEMremoved (A’) embryo closed the blastopore and underwent convergent
extension (B, B’, respectively)

I. Biological sign ific ance of force fo rmorphogenesis

II. The roles of PCP core components inmouse development

Physical forces are a non-negligible environmental factor
that can guide the morphogenesis of organisms. Such forces
are generated by tissue-tissue interactions during early
development where drastic tissue remodeling occurs. One
good example is neural tube formation. In vertebrates, theneural tube that is the primordial organ of the central nervous
system and is formed by the bending of the neural plate thatis a flat sheet of neuroepithelial cells. The tissue remodelingis driven by cellular morphogenesis in which selected cells in
the neural plate change their shapes from cuboidal to an
elongated wedge-like shape. Recent studies have revealed
that this cell shape change is controlled by cytoskeletal
dynamics, namely the remodeling of F-actin andmicrotubules (Suzuki, M. et al. Dev. Growth Differ., 2012) .
In addition, we also found that non-neural deep layer cells
that underlie the non-neural ectoderm generate force to bring
the two neural folds to the dorsal midline to close the tube
(Morita, H. et al. Development, 2012).
Another example is the axial mesoderm, which elongatesalong the anterior-posterior axis during gastrulation cell
movements by which rearrangement of the three germ layers
is driven. The axial mesoderm is led by the anteriorly

In epithelia, the roles of planar cell polarity (PCP) havebeen extensively studied, whereas in non-epithelia, they have
yet to be fully understood. We are exploring the roles of PCP
in the mesenchymal and neural tissues by using mouse
genetics and RNAi knock-down technology.
Recently, we generated a hypomorphic allele of mouse
Prickle1, one of the core PCP factors. As we have reported(Tao, H. et al. Proc. Natl. Acad. Sci., USA, 2009) Prickle1
null/null mice die around E6.0 in gestation due to the failureof gastrulation. In contrast, Prickle1 hypomorphic mutant
mice survive to P0 in a Mendelian manner but eventually diea day after birth. Interestingly, the mutant mice also
displayed abnormal morphology in some tissues where
Prickle1 is expressed. Now, we are investigating the cellularand molecular mechanisms underlying the abnormal
morphology. We hope that in combination with comparative
genomic analysis, these PCP mutant mice will serve as goodmodels that can explain morphological variations ofmammals.

III. Regulation of cell adhesion by theubiquitin system during gastrulation
17

National Institute for Basic Biology

Developmental Biology

During gastrulation, dorsal mesoderm cells migrate toward
the midline and align along the anteroposterior axis to form
the notochord. In this process, cells change their shape andmigrate in a highly coordinated manner. To achieve thissystematic cell movement, cell-to-cell interaction must be
tightly regulated. The cell adhesion molecule cadherin plays
a crucial role in this regulatory system. It is known that of thecadherin family members, paraxial protocadherin (PAPC) is
an important molecule that regulates cell adhesion in
mesoderm cells. We have found that the localization and thestability of PAPC protein is regulated by the ubiquitin
system. The inhibition of E3 ubiquitin ligase SCF/β-TrCP
blocks its plasma membrane localization. The inhibition of
PAPC ubiquitination by the dominant-negative ubiquitin
protein also impaired the plasma membrane localization andweakened cell adhesion of mesoderm cells (Figure 2).
Furthermore, this ubiquitin system regulates the localizationof another cell adhesion molecule, C-cadherin. Our findings
uncovered a novel mechanism of regulation of cell adhesion
proteins by the ubiquitin system, which plays a crucial role in
the actively-migrating mesoderm cells.

extra- and intracellular cues. The level of cytoplasmic
calcium is increased by its influx from either extracellularspace or intracellular storage areas such as the endoplasmic
reticulum (ER). We found that inhibition of calcium influx
delayed neural tube closure (NTC), suggesting that calcium
signaling plays an important role(s) in AC. Long-term timelapse imaging with calcium indicators revealed the dynamic
calcium transients throughout NTC. Occurrence of thetransients correlated with AC at the cellular level and withthe speed of NTC at tissue level. Furthermore, the forced
increase of cytoplasmic calcium by caged-compounds caused
cell shape change similar to AC. These suggest that calcium
is a positive regulator of AC and accelerates NTC to achieve
the correct formation of the hollow structure of the primitive
CNS.

Figure 3. By injecting in vitro transcribed mRNA, we observed theintracellular calcium patterns during Xenopus neural tube closure. Duringneural cells undergoing apical constriction, calcium transients at single
cell level dynamically occurred, sometimes resulting in multicellular
propagation. (Left) Frequency of calcium transients visualized bypseudocolor, (right) Time-projection of calcium patterns by colored circle
representing area of the transients.

V. Notochord and evolution of chordates
Figure 2. The ubiquitin system regulates PAPC localization and celladhesion. In the normal mesoderm cells, PAPC localizes at the plasma
membrane and cytoplasmic vesicles. Dominant-negative ubiquitin(DN-Ubi), which inhibits polyubiquitination, impairs PAPC localization
to the plasma membrane and weakens cell adhesion.

IV. Cellular morphogenesis during neural tube
closureFor the morphogenesis of organs, cellular morphogenesis as
well as cellular behaviors plays critical roles. During early
development of the central nervous system (CNS) invertebrates, the neuroepithelial cells undergo a typical shapechange, called apical constriction (AC), the cumulative
action of which cause the neural plate to bend to form theneural tube. In AC, cell apices are contracted and stabilized,
causing cells to adopt wedge-like shapes from columnar
ones. Recent studies have revealed that AC is controlled bycytoskeletal dynamics, namely the remodeling of F-actin,
and non-muscle myosin II activity, yet how AC isdynamically controlled in time and space is not fully
understood.
Calcium ions act as second messengers, triggered by both18

Gastrulation is a morphogenetic movement that is essential
for the formation of two- or three-germ-layered embryos.
Brachyury is transiently expressed in the blastopore region,
where it confers on cells the ability to undergo invagination.
This process is involved in the formation of the archenteronin all metazoans. This is a “primary” function of Brachyury.
During the evolution of chordates, they gained an additional
expression domain at the dorsal midline region of theblastopore. In the new expression domain, Brachyury served
its “secondary” function, recruiting another set of target
genes to form a dorsal axial organ, the notochord (Figure 4).
In order to better understand the molecular mechanisms
underlying the origin of the notochord during chordate
evolution, we are currently investigating to compare theBrachyury gene regulatory networks of hemichordates (anonchordate deuterostome closest to chordates) andcephalochordates (the most basal chordate).

VI. Epigenetic modification in the invertebrate
genomeEpigenetic modifications, such as DNA methylation andhistone modification, alter DNA accessibility and chromatin
structure, thereby regulating patterns of gene expression.
These processes are crucial to normal development and•••Figure 4. A schematic representation of the innovation of secondary
Brachyury function that leads the notochord formation during chordate
evolution. (A) Brachyury appears to be expressed continuously in theblastopore, but engaged cells never continue expressing the gene. (B) The
expression of Brachyury in the cells fated to form the notochord is nottransient, but instead continues till the process of notochorddifferentiation progresses to some extent.

Chiba, K., Kominami, K., Jung, S.K., Okawa, K., Nozaki, M., Kubota,
H.Y., and Ueno, N. (2012). Multiple functions of FADD in apoptosis,
NF-κB-related signaling, and heart development in Xenopus embryos.
Genes Cells 17, 875-896.
Tao, H., Inoue, K., Kiyonari, H., Bassuk, A.G., Axelrod, J.D., Sasaki,
H., Aizawa, S., and Ueno, N. (2012). Nuclear localization of Prickle2 isrequired to establish cell polarity during early mouse embryogenesis.
Dev. Biol. 364, 138-148.
Tran, L.D., Hino, H., Quach, H., Lim, S., Shindo, A., Mimori-Kiyosue,
Y., Mione, M., Ueno, N., Winkler, C., Hibi, M., and Sampath, K. (2012).
Dynamic microtubules at the vegetal cortex predict the embryonic axis
in zebrafish. Development 139, 3644-3652.
Uno, Y., Nishida, C., Tarui, H., Ishishita, S., Takagi, C., Nishimura, O.,
Ishijima, J., Ota, H., Kosaka, A., Matsubara, K., Murakami, Y.,
Kuratani, S., Ueno N., Agata, K., and Matsuda, Y. (2012). Inference ofthe protokaryotypes of amniotes and terapods and the evolutionary
processes of microchromosomes from comparative gene mapping.
PLoS ONE 7, e53027.

〔Review articles〕
N., Tagawa, K., and Takahashi, H. (2012). How was the• Satoh,notochord born? Evol. Dev. 14, 56-75.
M., Morita, H., and Ueno, N. (2012). Molecular mechanisms• Suzuki,of cell shape changes that contribute to vertebrate neural tube closure.
Dev. Growth Differ. 54, 266-276.

differentiation of distinct cell lineages in mammals. In
invertebrates, gene coding regions are the primary targets of
DNA methylation, but the role of DNA methylation in
actively transcribed genes is unknown. We investigated thetissue variability of both the global levels and distribution of
5mC in the seasquirt Ciona intestinalis. We found that global
5mC content of early developmental embryos is high, but isstrikingly reduced in body wall tissues. We chose sperm andadult muscle cells, with high and reduced levels of global
5mC respectively, for genome-wide analysis of 5mC targets.
By means of CXXC-affinity purification followed by deep
sequencing (CAP-seq), and genome-wide bisulfite
sequencing (BS-seq), we designated body-methylated andunmethylated genes in each tissue. Surprisingly, bodymethylated and unmethylated gene groups are identical in the
sperm and muscle cells. We conclude that gene body
methylation is not a direct regulator of tissue specific gene
expression in C. intestinalis. Instead, methylated genes areoften stably or maternally expressed. Moreover, wedemonstrate that transgenes can be modified by gene body
methylation, when their expression is driven by promoters ofendogenously body-methylated genes. Our findings reveal
constant targeting of gene body methylation irrespective ofcell types, and they emphasize a correlation between gene
body methylation and ubiquitously expressed genes.

Publication List
〔Original papers〕
G.G., Sarazin, H., Li, R., Suzuki, M., Ueno, N., and Liu. X. J.
• Leblond,(2012). Translation of incenp during oocyte maturation is required for••embryonic development in Xenopus laevis. Biol. Reprod. 86, 161, 1-8.
Morita, H., Kajiura-Kobayashi, H., Takagi, C., Yamamoto, T.S.,
Nonaka, S., and Ueno, N. (2012). Cell movements of the deep layer of
non-neural ectoderm underlie complete neural tube closure in Xenopus.
Development 139, 1417-1426.
Sakamaki, K., Takagi, C., Kitayama, A., Kurata, T., Yamamoto, T.S.,

19

National Institute for Basic Biology

Developmental Biology

DIVISION OF DEVELOPMENTAL GENETICS

Professor

KOBAYASHI, Satoru

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:
Visiting Scientist:
Technical Assistant:
Secretary:

HAYASHI, Yoshiki
SATO, Masanao
NODA, Chiyo
OTA, Ryoma
FUJISAWA, Chiemi
SUGIYAMA, Arisa
SHINOZUKA, Yuko
OHARA, Yuya
GOTO, Ayako
SATO, Kaori
YAMAMOTO, Manami
WASHIO, Midori
HONDA, SatokoGerm cells are specialized cells that can transmit genetic
materials from one generation to the next in sexual
reproduction. All of the other cells of the body are somatic
cells. This separation of germ and somatic cells is one of theoldest problems in developmental biology. In many animalgroups, a specialized portion of egg cytoplasm, or germ
plasm, is inherited by the cell lineage which gives rise togerm cells. This cell lineage is called germline. The germline
progenitors eventually migrate into the gonads, where theydifferentiate as germline stem cells (GSC) to form eggs andsperm when the organisms are physically matured. Ourlaboratory aims to find the molecular mechanisms regulating
germline segregation, germline sex determination and GSC
niche function in Drosophila.

I. Role of maternal Ovo protein in the germlineof Drosophila embryos
It has been proposed that germline-specific gene expression
is initiated by the function of maternal factors that areenriched in the germ plasm. However, such factors haveremained elusive. We have done a genome-wide survey ofmaternal transcripts that are enriched in the germ plasm andencode transcription factors for germline-specific gene
expression of vasa and/or nanos. We finally identified 6transcripts required for germline-specific gene expression byknockdown experiments using RNA interference (RNAi).
Among the 6 transcripts, we focused on ovo. The ovo geneencodes for a DNA-binding, C2H2 Zn-finger protein that is
involved in oogenesis and in epidermal development. The
ovo gene produces at least three alternate isoforms. Ovo-A
and Ovo-B function as a negative and a positivetranscriptional regulator in the germline, respectively. OvoSvb is expressed in the epidermal cells and is required fortheir differentiation. We found that Ovo-B is the major
isoform expressed in PGCs during embryogenesis. To
understand its function, we over-expressed the Ovo-A
repressor only in PGCs, and examined their developmental
fate. Our data shows that the reduction in maternal Ovo-B
activity results in a decrease in the number of primordial
germ cells during post-embryonic stages. Thus, maternal

20

Ovo-B has an essential role in germline development in both
sexes. Experiments for identifying the downstream genes
regulated by Ovo-B in germline are now on-going.

II. Mechanism regulating sex determination of
PGCs
It is widely accepted in mammals and Drosophila that malesexual development is imposed in PGCs by the sex of the
gonadal soma, and that PGCs assume a female fate in theabsence of a masculinizing environment. How PGCs initiate
female development, however, is a long-standing question in
reproductive and developmental biology.
We have reported that Sex lethal (Sxl) was expressed in XX
female, but not XY male PGCs, during their migration to the
gonads. To determine whether Sxl induces female
development in XY PGCs, we induced Sxl expression in XY
PGCs using nanos-Gal4 and UAS-Sxl, and transplanted these
PGCs into XX females. We found that these XY PGCs
entered the oogenic pathway and produced mature oocytes in
XX females. These oocytes contributed to progeny
production. In contrast, XY PGCs did not enter the oogenic
pathway. These observations demonstrate that Sxl expression
in XY PGCs during embryogenesis induces functional egg
differentiation in the female soma.
Our findings provide powerful evidence for Sxl as a mastergene that directs a female germline fate. Experiments for
identifying the downstream genes regulated by Sxl and the
upstream genes inducing Sxl only in XX female PGCs are in
progress.

III. The role of heparan sulfate proteoglycans
in JAK/ STAT signaling and distribution ofits ligand, Unpaired.
The JAK/STAT pathway plays vital roles in development
and homeostasis in animals. Drosophila, with its complete
set of JAK/STAT components, provides a powerful genetic
system to analyze the molecular functions of this essential
pleiotropic pathway. In addition to its functions inembryonic, larval, and imaginal development, JAK/STAT
signaling plays a critical role during several steps ofDrosophila oogenesis. First, JAK activity is necessary in thesomatic cells of the GSC niche to regulate production of the
BMP signal that maintains GSCs. Furthermore, in the
germarium, JAK signaling is required for maintenance andfunction of the supporting somatic escort cells. As cysts exit
the germarium, JAK activity regulates the formation of stalkcells that separate developing egg chambers. Later inoogenesis, constant JAK activity is necessary for proper
migration of the border cell cluster towards the posterior of
the egg chamber. In ovaries with reduced JAK activity, there
is a reduction in the number of border cells and stretchedcells (terminal fates), and a concomitant increase in thenumber of main body cells. Furthermore, increased JAK
activity from overexpression of the Janus kinase, Hop, or the
Upd ligand causes cells to adopt terminal fates within thefollicular epithelium. These results are consistent with theobserved graded activation of JAK in the follicularepithelium and suggest that Upd acts as a morphogen during
oogenesis. However, it has not been shown whether thisshown to control BMP, Wnt, and Hh signaling. A previous
study has shown that Upd protein expressed in cultured cells
is tightly associated with the extracellular matrix and the
addition of free heparin releases Upd into the medium. Theseobservations suggested that Upd normally associates with
HSPGs and thus may be regulated in a mechanism analogous
to other morphogens.
We demonstrated that Upd indeed forms an extracellular
gradient that activates JAK in a concentration dependent
manner. As is the case for other secreted morphogens, Upd
signaling was regulated by glypicans. Mutations in dally anddlp or in the HS biosynthetic enzymes, sulfateless (sfl) andHS 2-O sulfotransferase (Hs2st) led to aberrant JAK/STAT
pathway activation and disruption of stalk cell specification.
These alterations in JAK/STAT signaling and celldifferentiation can be attributed to effects on the normal
extracellular gradient of Upd by loss or changes inmodification of the glypicans. Biochemical andhistochemical studies showed that Dally and Upd physicallybind to each other and co-localize on the surface ofDrosophila S2 cells. In vivo, Upd accumulation on cells
lacking glypicans was dramatically reduced, and reciprocallywas enhanced upon ectopic expression of Dally (Figure 1).
These results suggest that Drosophila glypicans serve tostabilize a novel morphogen, Upd, at the cell surface during
oogenesis.

IV. The role of HSPGs in germline stem cellniche of Drosophila.

Figure 1. Dally regulates distribution of Upd.
(A-C) Distribution of Upd protein in dally-over expressed ovary. (A)dally-overexpressed clone (Green). (B) Upd protein distribution (Red).
Upd is normally distributed at the apical surface of posterior follicle cells(Arrow). Upd accumulate on the cell surface of dally-over expressedclone (Brackets). (C) Merged image of A and B.

putative morphogen forms a concentration gradient thatreflects the pattern of JAK activation.
Morphogens are important molecules in development andare defined by their ability to direct different cell fates over a
distance in a concentration-dependent manner. It has been
well established that extracellular signaling molecules of the
Wingless/Wnt, Hedgehog (Hh), and Bone Morphogenetic
Protein (BMP) families act as morphogens during
Drosophila development. Despite extensive studies on theactivities of these morphogens, it is not fully understood how
these molecules generate and maintain their gradients in atissue. A class of molecules that affect the gradient formationof all these morphogens is heparan sulfate proteoglycans(HSPGs). HSPGs are a family of carbohydrate-modified
proteins abundantly found in the extracellular matrix and on
the cell surface. Three families of HSPGs are widelyconserved during animal evolution: syndecans, glypicans,and perlecans. In particular, two Drosophila HSPGs of theglypican family, dally and dally-like protein (dlp), have been

Stem cells posses the remarkable capacity to generate
daughter cells that retain a stem-cell identity and others thatdifferentiate. Stem cells reside in dedicated cellular
microenvironments termed stem-cell niches. These niches
dictate stem-cell identity, maintain the stem cell population,and coordinate proper homeostatic production ofdifferentiated cells. The GSC niche in Drosophila gonads is a
useful model system for studying the stem-cell niche,
because the cellular components of this niche have beencharacterized and the signaling pathways, such as BMPs and
JAK/STAT which are essential for GSC maintenance, are
known. Ligands for these signaling pathways (niche signals)
are secreted from the niche cells, and are received by GSCs
to activate the pathway responsible for GSC maintenance.
Thus, the GSC niche is defined as the specialized region
retaining a sufficient amount of niche signals for GSC
maintenance. However, it is not well understood how the
distribution of the niche signals is precisely controlled in
GSC niche.
We identified dally and dlp as important components of the
GSC niche in both sexes. In the female GSC niche, dally was
expressed in niche cells. On the other hand, both of theglypicans were expressed in male GSC niche cells.
Mutations for these glypicans caused a significant reduction
in GSC number in both ovary and testis. In the dally mutant
ovary, GSC lost appropriate activation of the signaling
pathway by Dpp (a BMP homologue acting as a niche signal
in female GSC niche). Conversely, ectopic expression of
dally in female gonads caused an increase in GSC number
with ectopic activation of the Dpp signaling pathway. These

21

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Developmental Biology

results strongly suggest that dally defines the female GSC
niche by regulating distribution of Dpp.
To address this, we have been trying hard to visualize Dpp
distribution in the female GSC niche. By modifying
protocols for antibody staining and generating new
antibodies, we succeeded in visualization of Dpp
distribution. We found that Dpp distribution was significantly
expanded when dally was ectopically expressed in female
gonads, while Dpp-producing cells were unaffected (Figure2). The above results support our model that glypicans define
the GSC niche by regulating extracellular distribution ofniche signals.

Figure 2. Distribution of GSC niche signals.
(A,B) Dpp distribution in distal tip region (germarium) of normal ovary(A) and of ovary expressing dally in the somatic cells throughout
germarium (B). Green signal indicates Dpp protein (Brackets). Dpp
distribution is expanded in dally-expressing ovary, compared to thatobserved in normal ovary. Arrows show niche cells, which are the sourceof Dpp. Asterisks indicate GSCs. (C, D) Distribution of male GSC niche
signals, Upd (Green, C) and Gbb (Green, D). Distribution of both niche
signal was limited within male GSC niche. Asterisks indicate GSCs.

We also investigated molecular function of glypicans in themale GSC niche. We found that dlp is required for signaling
pathway by Gbb (the other BMP homologue acting as aniche signal in male GSC niche). Upd is also known as male
GSC niche signal. Since our data showed that dally regulates
the morphogen gradient of Upd (see section III), we
speculate that dally and dlp separately regulate Upd and Gbb
distribution in the male GSC niche, respectively. For the firststep to address this possibility, we tried to visualize the Upd
and Gbb distribution in the male GSC niche. We found that
Upd and Gbb were both enriched in the male GSC niche(Figure 2). By utilizing this system, we are now investigating
whether these glypicans define the male GSC niche via
regulating distribution of niche signals.

Publication List
〔Original papers〕
Y., Sexton, T.R., Dejima, K., Perry, D.W., Takemura, M.,• Hayashi,
Kobayashi, S., Nakato, H., and Harrison, D.A. (2012). Glypicans
regulate JAK/ STAT signaling and distribution of the Unpaired
morphogen. Development 139, 4162-4171.

22

C., and Kobayashi, S. (2012). Germline stem cells• Nishimiya-Fujisawa,
and sex determination in Hydra. Int. J. Dev. Biol. 56, 499-508.
Y., Kayashima, Y., Hayashi, Y., Kobayashi, S., and Yamakawa• Ohhara,
Kobayashi, K. (2012). Expression of beta-adrenergic-like octopamine
receptors during Drosophila development. Zool. Sci., 29, 83-89.

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:

Technical Assistant:

Secretary:

YABE, Taijiro
MII, Yusuke
UTSUMI, Hideko
CHEN, Qiuhong
KAMETANI, Yoshiko
TAKADA, Ritsuko
WANGLAR, Chimwar
TSUNOKUNI, Hiroyuki
WANGLAR, Chimwar*
SHINOZUKA, Takuma
KAMAGATA, Takanori
TAKASHIRO, Kayoko
NISHIGUCHI, Kimiko
ITO, Yukiko
TOMIDA, SanaiOHHARA, Kaori
UKAI, Sakie

The body and tissues of the developing embryo arerepeatedly divided into sub-regions specified bycharacteristic gene expression and morphology. The processthat gives rise to these sub-regions, according to a definedpattern, is called “pattern formation” or “patterning.” Ourlaboratories aim to understand the molecular mechanism
underlying pattern formation by several different approaches.
The most popular model to explain the patterning process isthe “morphogen gradient and threshold” theory. Many
genetic results indicate that secreted signal proteins such as
Wnt, BMP, and Hedgehog function as morphogens in many
aspects of the patterning process. In spite of theaccumulation of genetic evidence, however, the biochemical
characteristics of morphogens, including modification andhigher order structure, remain to be elucidated. Thus, one ofour major goals is to reveal the real image of morphogens
and the molecular mechanism underlying the formation ofmorphogen gradients, including the secretion andextracellular transport of these morphogens.
The segmental sub-regions of several specific tissues,
including the paraxial mesoderm (or somites) and thepharyngeal pouches, by contrast, appear not to be simply
directed by the morphogen gradient and threshold, but by aunique mechanism proceeding periodically. For instance,
somites are sequentially generated in an anterior-to-posteriororder by converting oscillatory gene expression into
periodical structures. The molecular mechanism underlying
this conversion and morphological segmentation, however, is
not yet fully understood. Thus, another goal of our current
studies is to reveal the molecular mechanism of this otherand unique mode of patterning that underlies the periodical
and sequential sub-division in the development of somites
and pharyngeal arches.

I. Lipid modification and extracellulartrafficking of Wnt proteins
The Wnt family of secreted signal proteins plays a key rolein numerous aspects of embryogenesis. Most Wnt proteinstransmit their signals locally in most of the cases where theyfunction, presumably since their secretion and transport areunder tight control. One important step regulating theextracellular transport of various secreted signal proteins
involves post-translational modification with lipid moieties.
We found that murine Wnt-3a is modified with a monounsaturated fatty acid, palmitoleic acid, at a conserved Ser
residue. Wnt-3a defective in this modification is not secreted
from cells in culture or in Xenopus embryos, but is retainedin the endoplasmic reticulum (ER). Thus, the Wnt protein
appears to require a particular lipid modification for proper
intracellular transport during the secretory process. We
expect that the discovery of this unexpected lipid
modification might provide a clue regarding the higher-order
structure of secreted Wnt proteins and their gradientformation.
The palmitoleoylation requires an enzyme, Porcupine(Porcn), a member of the family of membrane-boundO-acyltransferases (MBOAT). The Porcn gene was first
identified as a segment polarity gene, like wingless, inDrosophila and is evolutionally conserved from worms tomammals. Porcn proteins localize to the endoplasmic
reticulum (ER) and are required for Wnt trafficking from the
ER in mammalian cells and probably in Drosophila embryos.
Porcn overexpression promotes lipid-modification of Wnt1
and Wnt3a and causes a steepened Wnt gradient in the chickneural tube, suggesting its important function in vertebrate
embryogenesis.
To reveal the biological significance of palmitoleoylation
by Porcn, we used zebrafish as a model system and examinedthe effects of defects in Porcn function on Wnt signals
mainly in early embryonic stages because the roles of Wnt
signals have been precisely characterized in early zebrafish
embryos. We identified two zebrafish homologs of
porcupine, porcn and porcupine-like (porcn-l). Zebrafish
porcn, but not porcn-l, restores secretion of Wnt proteins inFigure 1. Porcn deficiency causes abnormalities in the convergence andextension. Zebrafish embryos injected with control (A, C) and Porcnspecific (B, D) MOs were shown, respectively. The notochord was
expanded and shortened due to defect in the convergence and extension
movement during gastrulation. The brackets indicate the notochords.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

23

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Developmental Biology
porcn-deficient mouse L cells. Morpholino-mediatedknockdown of porcn in zebrafish embryos impairs
convergence and extension (CE) during gastrulation withoutchanging embryonic patterning (Figure 1). Moreover, porcn
interacts genetically with wnt5b and wnt11 in regulating CE.
In contrast, porcn-deficient embryos do not exhibit
phenotypes caused by failure in canonical Wnt signaling,which is activated by several Wnt ligands, including Wnt3a.
Furthermore, expression of genes regulated by the canonical
Wnt signaling pathway is not perturbed in knockdown
embryos relative to that in the controls. While the trafficking
and lipidation of ectopically expressed zebrafish Wnt5b andmouse Wnt5a are impaired in porcn-deficient embryos, thoseof ectopically expressed Wnt3a are less or not affected(Figure 2). In addition, the secretion of Wnt5a is inhibited byless porcn inhibitor than that of Wnt3a in 293 cells. Thus,
decrease of Porcn activity does not equivalently affecttrafficking and lipidation of different Wnt proteins inzebrafish embryos and in mammalian culture cells. These
results suggest that the mechanism of trafficking andmodification of Wnt proteins appear to be inconsistent
between different types of Wnts. We are currently examining
the molecular mechanism underlying the variability of Wnt
modification.
In addition to the study of the secretory process of Wnt
proteins, we are also examining the extracellular transport ofWnt proteins during embryogenesis using frog and mouseembryos.

II. M o l e c u l a r m e c h a n i s m o f s o m i t edevelopment.
The molecular mechanism underlying the periodical
formation of somites is coupled to an internal oscillator,
referred to as the “segmentation clock,” which has been
evidenced by the cyclic expression of genes in the presomitic
mesoderm (PSM). For example, hairy/Enhancer of split(Espl)-related bHLH genes, including her1 and her7 inzebrafish, are expressed in a dynamic pattern of stripes
across the PSM in a posterior to anterior direction. The
oscillating and anteriorly propagating wave of gene
expression, which is maintained in the posterior PSM,
becomes fixed to cause segmentation in the anterior PSM.
Prior to morphological segmentation, a segmental pattern,characterized by the periodical borders between neighboring
somites and by the rostro-caudal within a somite, isestablished in the anterior PSM. We have already shown that
two Ripply genes, Ripply1 and Ripply2 play essential rolesin this patterning. To gain insight into the mechanism of
somite segmentation, we are examining the segmental
patterning in zebrafish embryos by focusing on Ripply andrelated molecules.

24

Figure 2. Secretion of Wnt proteins in zebrafish embryos. Confocal
images of localization of EGFP-tagged zebrafish Wnt5b (A, B), mouse
Wnt5a (C, D), and mouse Wnt3a (E, F) with membrane markers, inepiblast cells of embryos injected with 5mis control MO (A, C, E,) or
porcn MO (B, D, F) are shown. Merged images of Wnts and membranemarkers are also presented. Secretion of Wnt5a, but not Wnt3a, isdefective in Porcn-deficient embryos.

III. Molecular mechanism of mesoderm
development from stem cell-likeprogenitors.
The posterior body of the vertebrate is generated fromprogenitor cells residing in the tailbud. The mesodermal
progenitor cells (MPCs), which appear to have stem cell-likecharacteristics, continuously produce the presomitic
mesoderm (PSM) cells, which further differentiate into
somites. Accumulating evidence has revealed the molecularmechanism underlying the maintenance of theundifferentiated state of the MPCs. For instance, in zebrafish
embryos, wnt and zebrafish orthologues of brachyury, no tail(ntl) and bra, mutually activate their expressions in the
MPCs and this autoregulatory loop is essential for
maintenance of the undifferentiated state of the MPC.
On the other hand, the molecular mechanism promoting
MPC differentiation into PSM cells should also be elucidated
for a better understanding of the MPC-based development of
the paraxial mesoderm. Of note, genetic studies with thezebrafish have indicated that the molecular mechanisms
underlying the development of somites are not the same
between trunk and tail. For instance, spadetail (spt)/tbx16

mutant embryos are impaired in the development of their
trunk somites, but generate relatively normal tail ones.
However, in contrast to this phenotype restricted to the trunk
somites, other evidence suggests that spt is required for PSM
differentiation in both trunk and tail somites and that some
additional factors compensate the loss of Spt function during
tail development. Therefore, for understanding the molecularmechanism that controls the maintenance and subsequentdifferentiation of the MPCs, it is important to reveal thefunction of Spt and these additional factors during thedevelopment of tail somites, especially in terms of theirinteraction with the Wnt/Brachyury autoregulatory loop.
One candidate as an additional factor seems to be
Mesogenin1 (Msgn1), which is a bHLH transcription factor
expressed in the PSM. Interestingly, mouse embryos
deficient in functional Msgn1 have impaired development of
their posterior somites, in spite of having normal formationof the first 7 somites, as well as show an abnormal
accumulation of an undifferentiated cell mass at the tip of
their tail. Thus, Msgn1 seems to be involved in PSM
differentiation during the development of posterior, or tail,somites. However, it is still uncertain as to how thedifferentiation from the MPCs to PSM cells is controlled by
msgn1 during somite development. Furthermore, it hasremained to be elucidated whether zebrafish msgn1 interacts
with spt during PSM differentiation during tail development.
We assessed the functions of msgn1 in zebrafish
development by injecting msgn1 specific MO into wild-type
and spt mutant eggs. Zebrafish embryos defective in msgn1
and spt failed to differentiate into PSM cells in taildevelopment and show increased expression of wnt8 and ntl(Figure 3). Msgn1 acted in a cell-autonomous manner and as
a transcriptional activator in PSM differentiation. The
expression of msgn1 initially overlapped with that of ntl inthe ventral tail bud, as previously reported; and its misexpression caused ectopic expression of tbx24, a PSM
marker gene, only in the tail bud and posterior notochord,both of which expressed ntl in zebrafish embryos.
Furthermore, the PSM-inducing activity of misexpressed
msgn1 was enhanced by co-expression with ntl. Thus, Msgn1
exercised its PSM-inducing activity in cells expressing ntl.
Based on these results, we speculate that msgn1 expressionin association with that of ntl may allow the differentiation ofprogenitor cells to proceed during development of somites inthe tail.

Publication List
〔Original papers〕
Q., Takada, R., and Takada S. (2012). Loss of Porcupine impairs• Chen,convergent extension during gastrulation in zebrafish. J. Cell Sci. 125,,

2224-2234.

C.H., Chou, C.W., Takada, S., and Liu, Y.W. (2012). Development• Chiu,and fibronectin signaling requirements of the zebrafish interrenal
vessel. PLoS ONE 7, e43040.

Yabe, T., and Takada, S. (2012). Mesogenin causes embryonic• mesoderm
progenitors to differentiate during development of zebrafish,

tail somites. Dev. Biol. 370, 213-222.

Figure 3. msgn1 and spt are required for thedifferentiation of posterior mesoderm cells inzebrafish. Wild-type embryos injected with
control (5mis) MO (A, E, I, and M) or msgn1
MO (B, F, J, and N) and spt homozygous
embryos injected with 5mis MO (C, G, K, andO) or msgn1 MO (D, H, L, and P) at the
24-somite stage were hybridized with tbx24(A-D), papc (E-H), ntl (I-L) or wnt8 (M-P)
probes. The expression of paraxial mesoderm
markers, tbx24 and papc in the PSM isseverely reduced in the msgn1 MO-injected spt
homozygous embryo (D, H). Injection of
msgn1 MO into wild-type embryos increased
the expression area of ntl and wnt8, both ofwhich are expressed in the mesoderm
progenitor cells in the tailbud, in the tailbud (J,N) compared with that for the control (5mis)
MO (I, M). In addition, ntl and wnt8expressing cells are also increased by msgn1
MO in spt mutant embryos (K, L, O, and P).

25

National Institute for Basic Biology

Developmental Biology

DIVISION OF EMBRYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Visiting Scientist:
Technical Assistant:
Secretary:

TOYOOKA, Yayoi
KOYAMA, Hiroshi
OKA, Sanae
SATO, Yasufumi
KOBAYAKAWA, Satoru
SHI, Dongbo
HIGUCHI, Yoko
KATO, Azusa

The aim of our research is to understand the events
underlying early mammalian development during the period
from pre-implantation to establishment of the body axes.
Early events during embryogenesis in mammals are
relatively less understood, as compared to in other animals.
This is mainly due to difficulties in approaching thedeveloping embryo in the uterus of the mother. Anothercharacteristic of mammalian embryos is their highly
regulative potential. The pattern of cell division andallocation of cells within an embryo during the early stages
vary between embryos. The timing of the earliest
specification events that control the future body axes is still
under investigation. Functional proteins or other cellularc o m p o n e n t s h av e n o t b ee n fo u n d th a t loc a liz e
asymmetrically in the fertilized egg. We want to provide
basic and fundamental information about the specification ofembryonic axes, differentiation of specific cell lineages,
behaviors of cells, and the regulation of body shape in earlymammalian development. We are also interested in therelationship between embryos and their environment, theoviducts and the uterus.

I. Establishment of live imaging system forobservation of early mouse development
To understand the mechanisms underlying embryonicdevelopment and morphogenesis, it has become common toobserve the behavior of cells and gene expression in living
embryos in a variety of animal species. Progress in GFP
technologies, genetic engineering and embryonic
manipulation concomitantly with the progress in optics andmicroscope technologies, including development of highly
sensitive photo-detectors, has allowed us to observe
developing embryos live, even in mammals. It is useful tovisualize nuclei, cell shapes, cytoskeleton or other organelles
to observe cells and cell behaviors in living mouse embryos.
We have established a series of transgenic mouse lines forlive imaging, which is a part of a collaborative project taking
place in the Laboratory for Animal Resources and Genetic
Engineering, Riken CDB. In each mouse, cDNA encoding
fusion protein with fluorescent protein and a localization
sequence were inserted into Rosa26 locus. The sequence forthe fusion protein was following stop sequences that are
surrounded by loxP sites on both sides. These loxP sites canbe recognized by an enzyme, Cre recombinase, that catalyzes

26

Figure 1. Examples of 7.5 day embryos expressing reporter fluorescentfusion proteins ubiquitously. These embryos express fusion proteinsshown on the bottom.

recombination between two loxP sites to remove the stop
sequences. Thus, when Cre recombinase is activated in a
spatial-temporally specific manner, the following reporter
fluorescent proteins are expressed in a specific way. And
once this irreversible reaction is induced in the germ line, thederived offspring possess the transgene without the stop
sequence and express the reporter fusion protein
ubiquitously.
We are also establishing several reporter mouse lines in thelab to study gene expression patterns during periimplantation stage of mouse development. In these mice,
cDNA encoding fluorescent proteins are connected to themouse genomic DNA sequences of enhancer/promoter
region of important gene encoding factors regulating celldifferentiation in these stages. Combining these two types ofreporter transgenic mouse lines, we have started analysis ofbehaviors of cells comparing gene expression properties atthe single cell level.
For the live imaging of early mammalian embryos, acombined microscope and incubation system is an important
tool. Conventional CO2 incubators provide better conditions
compared with microscope top incubation chambers,
including stability of temperature and humidity, to support
embryonic development in vitro. Incubation microscopes
have also recently become commercially available, however,these are expensive for personal use. We have modified an
incubation microscope with wide field fluorescentillumination, which is relatively inexpensive. We added aspinning disc confocal system and sensitive EM-CCD
camera for observation of developing mouse embryos withless photo-toxicity and higher spatial resolution.
We are trying to observe and reveal aspects of cell shape,morphogenesis, cell lineage, gene expression and celldifferentiation by combining these techniques.

II. Histological observation of mouse embryos
developing in the uterus
One of the characteristics of mammalian development isthat embryos develop in the uterus after implantation. Directinteraction between embryos and the cells of the uterus isimportant. In studies on developmental biology of
mammalian embryos, embryos are usually removed from theuterus, and isolated embryos are analyzed. We have been
analyzing early embryonic development of mice comparing
the changes in the uterine epithelium. To observe themorphogenesis of embryos within the uterus, serial sectionsof implanted uteruses were made, stained with hematoxylin
and eosin, and the images of the embryos within the uteruses
are captured to make high resolution three-dimensional
re-constructions. Figure 2 shows an example of a section. We
are preparing samples obtained from various developmental
stages. Precise positions of embryonic implantation and

Figure 2. A pregnant uterus 3.5 days after fertilization. Developing
embryos and the shape of uterus epithelium can be observed.

changes in the uterine epithelium correlating with embryonicdevelopment will be examined using these images.

III. Studying early development of rabbits as a newmodel of mammalian embryogenesis
Mice have been the main experimental animal used for the
study of mammalian developmental biology because genetics
and genetic engineering including transgenesis and targetedmutagenesis are applicable for mice. We also have been
mainly studying mouse embryogenesis. However, rodent
embryos do not necessarily provide ideas common in
mammalian embryogenesis because the styles of early
embryonic development differ between species, and rodent
embryos do not provide a typical style when compared withother animals. We searched for animals that are suitable forthe study of early embryogenesis comparing possible
candidates, and decided to use rabbits. We are studying
morphological changes and gene expression during earlystages of development until the peri-implantation stage. We
are focusing especially on the formation of germ layers andbody axes, and compare with corresponding stages in mice.

IV. Formation, maintenance of ell polarity and tissuemorphogenesis in the mouse oviduct
The oviducts (fallopian tubes) are tubes connecting theperiovarian spaces and the uterine horns. The ova released
from the ovary are transported through the oviduct, where
fertilization occurs with the spermatozoon moving from theuterus. We are focusing on the region of the oviduct close to
the opening to the ovary. In this region many multi-ciliatedcells exist. These cilia move in one direction along the ovaryuterus axis. This directional movement of multi-cilia mightplay a major role in the transportation of ovum fromperiovarian space, although muscle contractions also playroles in the region close to the uterus. This suggested that theovum was transported by the directionally beating cilia. This
directional beating of cilia was based on the polarity of the
microtubule assembly in the cilia. Skeletal microtubules in
the cilia are arranged in a “9 +2”array, and the central twobundles are aligned facing the same direction in each cell.
This suggests that oviduct epithelial cells possess polarity
along the cell surface parallel to the longitudinal axis of theoviduct; this type of cellular polarity is called “Planner Cell
Polarity (PCP)”. We have been studying how PCP isestablished during development, and how this polarity is
maintained over a long period in later stages.
We are now focusing on several genes involved in PCP
formation. Molecular basis of PCP formation has been
genetically studied, particularly using fruit fly, and several
genes have been shown to be involved in the regulation of
PCP formation. Homologues of these genes are also known
in mammalian species, and some of them have been shown
to be playing similar roles. Disruption of functions of someof these genes resulted in abnormal PCP formation in skin,
inner ear and early embryos of mice. We are also focusing onsome of the PCP regulators, and are trying to understand how
oviduct epithelial cells establish and maintain polarity. We
would like to reveal mechanisms including the molecularfunctions, cellar shape, tissue morphology and involvementof mechanical forces in this system.

IV. Analysis of mechanical properties of cells
during embryonic development
Mechanics is one of the essential components for biologicalprocesses including cell shape transformation, tissuemorphogenesis etc. However, how mechanical properties
such as force and material stiffness regulate these processes
is poorly understood. To approach this problem, measuringcellular geometric information and mechanical properties arenecessary. We developed an image processing based
technique to measure cellular geometric information fromfluorescent microscopic images and a framework totheoretically estimate the mechanical properties. The image
processing technique enabled us to robustly detect cell
contour from images with high noise and non-uniform
illumination, although the algorithm of the technique is quite
simple. By employing the image processing technique, we
successfully extracted geometric information of early
embryonic cells during cytokinesis in C. elegans and ofmouse cells in cell sheets. In the framework for estimating
mechanical properties, geometric information was combinedwith a mechanical simulation, which was technically basedon the data assimilation (Figure 3). We spatio-temporally
estimated cell surface stiffness during cytokinesis bysystematically fitting the in vivo cell shape to the mechanicalsimulation. We found that cell polar and equatorial regions
were stiffer and softer, respectively. Further theoretical27

National Institute for Basic Biology

Developmental Biology

modeling showed that the relative difference of stiffness
between the two regions could be a primary determinant for
cleavage furrow ingression during cytokinesis. We
speculated that the relative contributions of cell surface
stiffness and the contractile ring could explain the contractile
ring dependent and independent cytokinesis.

Figure 3. Theoretical estimation of cell surface stiffness. Schematic
illustration of estimation.

Publication List
〔Original papers〕
K., Komatsu, K., Fujimori, T., and Kobayashi, T.J. (2012).
• Bashar,Automatic extraction of nuclei centroids of mouse embryonic cells
from fluorescence microscopy images. PLoS ONE, 7, e35550.

L., Kobayakawa, S., Yoshiki, A., and Abe, K. (2012). High• Cao,
resolution intravital imaging of subcellular structures of mouseabdominal organs using a microstage device. PLoS ONE, 7, e33876.

H., Umeda, T., Nakamura, K., Higuchi,T., and Kimura, A.
• Koyama,(2012). A high-resolution shape fitting and simulation demonstrated
equatorial cell surface softening during cytokinesis and its promotive
role in cytokinesis. PLoS ONE, 7, e31607.

28

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:
NIBB Research Fellow:
Technical Staff:
Graduate Student:
Technical Assistant:
Secretary:

HARA, Kenshiro
KITADATE, Yu
NAKAMURA, Yoshiaki
MIZUGUCHI, Hiroko
IKAMI, Kanako
TOKUE, Moe
NONAMI, Yuta
INADA, KanaMARUYAMA, Ayumi
SUGIMOTO, Ryo
KUBOKI, Yuko

Mammalian testes produce numerous sperm for a long
period. This property that is fundamental for continuity oflife across generations is supported by the robust stem cellsystem, which is defined by the ability of proliferation that is
associated with self-renewal (maintenance of their own
undifferentiated population) and differentiation (productionof different cell type(s)),
The Division of Germ Cell Biology, which was launched in
2008, aims to fully understand the mouse sperm stem cellsystem in vivo under the context of tissue architecture of
testis. So far, we have revealed some characteristics of thispotent and interesting stem cell system. First, differentiating
germ cells that had been believed to be irreversiblycommitted to differentiation still retain the self-renewing
potential and can contribute to stem cell pool maintenance(“potential stem cells”). Secondly, “reversion” from potential
stem cells occurs at a higher frequency when testicular tissue
is damaged and regeneration is induced. Thirdly, theundifferentiated spermatogonia population that includes both
“actual” and “potential” stem cells localized to thevasculature (vascular- associated niche). We also discoveredthat stem cells turn over in an unexpectedly frequent andmathematically stochastic manner under steady-state
situations that continuously produce sperm. (Nakagawa etal., Dev. Cell 2007, Yoshida et al., Science 2007; Nakagawa
et al., Science 2010; Klein et al., Cell Stem Cell 2010).
In 2012, we have extended these investigations for the
deeper understanding of this stem cell system. Firstly, we
have investigated the nature and the behavior of the stem
cells at a single cell resolution. Secondly, we have challenged
the cellular and molecular nature of the vascular-associatedniche. Thirdly, a mechanism underlying the coordinatedspatio-temporal regulation of stem cells has been proposed.

I. Stem cell behavior at a single cell resolutionin undisturbed testicular tissue
For the purpose of function-based investigation of stem
cells, it is essential to analyze the behavior of cells over time.
We have previously established experimental systems tostudy stem cell behavior in mouse spermatogenesis overtime, without affecting the tissue architecture, namely liveimaging and pulse-labeling experiments. For live-imaging,taking advantage of the fluorescent protein-labeling, we haveachieved continual filming of particular subsets ofspermatogonia in the testes (Figure 1: Yoshida et al., Science2007). For pulse-labeling, we have also establishedirreversible labeling of a particular spermatogonial
population using tamoxifen-dependent activation of cre
recombinase fused with a domain of the mutated humanestrogen receptor (Figure 2: Yoshdia et al., Development
2006, Nakagawa et al., Dev. Cell 2007, Nakagawa Science2010).

Figure 1. Live-imaging of fluorescence-labeled cells. An example of liveimaging observation of spermatogonia in a living testis. Spermatogonia
labeled with GFP under Ngn3 promoter (white) were continuously
filmed, shown cell division (30h and 76h) and active migration. A part of
selected frames are shown. Numerals indicate the elapsed time in hours.
(From Yoshida et al. Science 2007)

Figure 2. Pulse-labeling of subset spermatogonia with marker expression.
(A) Principle of pulse-label of a stem cell. When a self-renewing stem
cell is pulse-labeled, its descendants (shown in blue here) include bothstem and differentiating cells. Then, the clone will persist in the tissue, as
shown in the actual experiments in the following panels. (B)Spermatogonia labeled with LacZ expression (colored in blue) observed
2 days after induction of labeling with Tamoxifen administration. Asingle(1), Apair (2), Aaligned-4 (4), Aaligned-8 (8) are shown. (C) Three months after
the pulse, labeled cells that are descendant of the induced spermatogonia
form patches occupying a continual segment of the tubules includingstem and differentiating cells. This reflects the stem cell function of theoriginally labeled cell. (From Yoshida Reproduction 2012 and Nakagawa
et al., Dev. Cell 2007)29

National Institute for Basic Biology

Developmental Biology

In addition, our results strongly suggest that within theundifferentiated group of spermatogonia consisting of Asingle,
Apaired and Aaligned cells, a small subset expressing GFRα1(glial cell line-derived neurotropic factor receptor alpha 1) is
responsible for the stem cell functions, while thoseexpressing Ngn3 (neurogenin 3, a bHLH transcription factor)
are destined for differentiation while maintaining theirpotential to get back into being GFRα1-positive and selfrenew (Nakagawa et al., 2010).
In addition, in collaboration with Professor Benjamin
Simons, a theoretical physicist of Cambridge University, we
have shown that the spermatogenic stem cells show astochastic and continuous turnover between neighbors(Nakagawa et al., Dev. Cell 2007, Klein et al., Cell Stem Cell
2010): Instead of the ‘text book’ type of asymmetric
divisions that always give rise to one self-renewing and one
differentiating daughter, fate selection for stem cells either toremain undifferentiated or to differentiate appears to bestochastic. As a result, fates of stem cell-derived cohorts arehighly variable and show neutral competition between stem
cell clones.
Based on these preceding findings, we began investigating
the behavior of GFRα1+ spermatogonia in undisturbed testis
using live-imaging and pulse-labeling at a single cellresolution. These have allowed us to directly demonstrate forthe first time that this tiny subset of spermatogonia indeed
acts as the stem cell pool supporting the steady-state turnoverof spermatogenic cells. We are now intensively challenging
the problem of how to link the actual single-cell-level
behavior (such as cell division, fragmentation of
spermatogonial syncytia, fate selection, whether todifferentiate or remain undifferentiated, and death) of the
GFRα1+ spermatogonia and the overall robustness of thestem cell system at a level of population, with the aid ofmathematical modeling.

II. N a t u r e o f t h e s t e m c e l l n i c h e
microenvironment in the mouse testis
It is generally believed that some specialized
microenvironment within tissue is involved in the stem cellsystem as an indispensable element that regulates the stem
cell behavior, designated as the “stem cell niche”. In contrast
to well-defined examples of the stem cell systems
accompanying anatomically defined niches where stem cells
always settle and differentiation accompanies their exit fromthe niche region, such as Drosophila gonads, mammalian
testis do not exhibit such a defined substructure. Therefore it
has long been a mystery how stem cell behavior is regulated
in the context of this tissue, namely the seminiferous tubules(Figure 3). Regarding this issue, it has been shown that,within the ‘basal compartment’ of seminiferous tubules, theundifferentiated spermatogonia show a preferential
localization to the area adjacent to blood vessels that make a
network running through the interstitial space between theseminiferous tubules (Chiarini-Garcia, Biology of
Reproduction 2001, Yoshida et al., Science 2007). However,again, lacking a discrete niche structure, this example of astem cell niche has been considered to stand on a facultative
nature.

30

Figure 3. Architecture of seminiferous tubules, the spermatogenic centerin the testis. Schema of seminiferous tubules. In basal compartment
between the basement membrane and tight junction between Sertoli cells,
undifferentiated spermatogonia (the population responsible for the stem
cell pool) show a preferential localization to blood vessels running
between the tubules. The differentiating progeny of the stem cells stratify
into four layers, each of which is comprised of mitotic spermatogonia,
meiotic spermatocytes, and haploid round and elongating spermatids.
(Modified from Spradling et al, 2011)

Based on these preceding observations of our own andothers, we hypothesize that the blood vessel-adjacent area is
specialized and optimal for harboring and regulating the stem
spermatogonia, and that this feature is reflected by the
specialized gene expression in this area. We therefore havebeen challenging this issue by searching for genes showing
localized expression to such a presumptive niche area. We
first collected the area of interest using microdissection
techniques on sectioned testis specimens, followed by cDNA
microarray and in situ hybridization analyses to search forgenes showing preferential expression. We have been
studying these genes with regard to the spatial relationshipbetween the sites of expression of these genes with
undifferentiated (and stem) spermatogonia, as well as thepossible function of these genes in stem cell regulation.
These findings will provide basic and fundamental
information on stem cell regulation in tissues harboring
facultative niche.

III. Spatio-temporal stem cell regulation by
retinoic acid metabolism
In seminiferous tubules, differentiation of Ngn3-positive
undifferentiated spermatogonia into Kit-positive stage occurs
periodically with an interval of 8.6 days, which is followedby programmed differentiation process toward spermatozoa
that takes 35 days. This temporal regulation is essential forthe integrity of the tissue and continuity of spermatogenesis
in that this process supports the regular stratification of germ
cells in the wall of seminiferous tubules from stem cells tomature testis (Figure 3).
Based on the classic finding that retinoids (whose
prominent active form is retinoic acid) are essential for the
timed differentiation of undifferentiated spermatogonia, weinvestigated how the differentiation of spermatogonia isregulated by retinoic acid signaling, taking advantage ofexperimental modulation of retinoid supply and examinationof retinoid metabolism-related enzyme expression patterns(Sugimoto et al., Mech. Dev. 2012). It has been demonstrated
that a series of enzymes involved in retinoic acid metabolism
at different steps are expressed separately but in a beautifully

orchestrated manner among multiple differentiating germ cell
types (i.e., meiotic and haploid cells) and somatic Sertoli
cells (which nourish all the steps of germ cells in
seminiferous tubules), suggesting strongly that RA
concentration changes in a 8.6 day cyclic manner, and that it
increases at the same timing with that of differentiation of
spermatogonia into Kit-positive. In addition, expression of
RA metabolism-related genes in Sertoli cells are underregulation of RA signaling, forming positive and negative
regulatory loops, leading us to propose that it is RA itself thatcoordinates the orchestration of metabolism between
neighboring cell types that constitute the seminiferous
tubules (Figure 4).

identified. As well, mathematical analyses and modeling will
be quite powerful to link the observations obtained indifferent scales.

Publication List
〔Original papers〕
S., Hamasaki, H., Sekiguchi, S., Hara, K., Ishii, Y., Kyuwa,• Koyanagi,S., and Yoshikawa, Y. (2012). Effects of ubiquitin C-terminal hydrolase
L1 deficiency on mouse ova. Reproduction 143, 271-279.

Y., Usui, F., Miyahara, D., Mori, T., Ono, T., Kagami, H.,• Nakamura,
Takeda, K., Nirasawa, K., and Tagami, T. (2012). X-irradiation removes••

endogenous primordial germ cells (PGCs) and increases germline
transmission of donor PGCs in chimeric chickens. J. Reprod. Dev. 58,
432-437.
Sato, T., Yokonishi, T., Komeya, M., Katagiri, K., Kubota, Y., Matoba,
S., Ogonuki, N., Ogura, A., Yoshida, S., and Ogawa, T. (2012). Testis
tissue explantation cures spermatogenic failure in c-Kit ligand mutant
mice. Proc. Natl. Acad. Sci. USA 109, 16934-16938.
Sugimoto, R., Nabeshima, Y., and Yoshida, S. (2012). Retinoic acidmetabolism links the periodical differentiation of germ cells with thecycle of Sertoli cells in mouse seminiferous epithelium. Mech. Dev.
128, 610-624.

〔Review article〕
S. (2012). Elucidating the identity and behavior of• Yoshida,spermatogenic stem cells in the mouse testis. Reproduction 144, 293302.

Figure 4. Proposed model for coordination of periodic differentiation ofgerm cells linked by retinoid metabolism. (A) Particular stages of meiotic
cells (LP) and haploid cells (RT) send the ‘go’ and ‘wait’ signals to Aundff
and Sertoli cells, by regulating the local RA metabolism to increase anddecrease the RA concentration, respectively. (B) These signals will occur
reciprocally and periodically to maintain the seminiferous epithelial
cycle. (From Sugimoto et al., Mech. Dev. 2012)

IV. Perspectives
As described above, we have been tackling the robust stem
cell system that supports mouse spermatogenesis fromdifferent aspects and scales. Once this research is achieved itwill be quite intriguing to ask how these events are linked
with each other and integrated into the robustness of thewhole stem cell system. For that purpose, it is clear that westill need to know about molecules (and genes that encode
these factors) that play essential roles but have not been

31

National Institute for Basic Biology

Developmental Biology

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION

Associate Professor

TANAKA, Minoru

NIBB Research Fellow:
Graduate Student:
Technical Assistant:
Secretary:

hypothesis, we tried recovering the number of germ cells
using sox9b and amhrII heterozygous compound mutant. In
the compound mutant, germ cells come back to the normal
number and no sex reversal was observed, supporting ouridea that germ cells are important for the proper
manifestation of sex

YAMAMOTO, Yasuhiro
NISHIMURA, Toshiya
SAKAE, Yuta
KINOSHITA, Chie
WATAKABE, Ikuko
YONEMITSU, Masako

Laboratory scope
Our laboratory aims to reveal the molecular mechanisms of
the formation of the gonads and sex differentiation. We use
medaka fish (Oryzias latipes) for these purposes and havebeen generating transgenic medaka (Figure 1) enabling us to
identify different cell lineages by fluorescence and to analyze
the process of gonad formation and sex differentiation in
vivo. Additionally, in order to identify the genes essential forgonadogenesis, we carried out a mutational screening of
medaka with defective gonads and are performing a
positional cloning. With these two unique analytical methods(visualizing cells and mutants), we are attempting to unveil
the fundamental mechanisms of sex differentiation andplasticity common to many organisms.

I. Cellular biphasic process critical for
manifestation of the sex
In gonochoristic vertebrates such as medaka and humans, agene on the sex chromosome is responsible for thedetermination of sex. Once the process of sex determinationis triggered by the gene, the animal begins to develop intoeither female or male and does not change the direction
during its life cycle. The sex differentiation is unidirectional.
On the other hand, it has been described that sex is aconsequence of balancing between female and male process(biphasic process) because sex reversal is often reported evenin gonochoristic vertebrates.
As the results of our previous studies, we have revealed thatgerm cells are critical for the biphasic process. In the absenceof germ cells, we found that medaka exhibit complete malesecondary characteristics at both endocrine and gene levels(Kurokawa et al., 2007 PNAS). This indicates that germ cells
are essential for formation of ovaries. In addition, in theabsence of germ cells, somatic cells are predisposed to maledevelopment.
This view was also supported by our recent mutant analysis(Nakamura et al., 2012 PLoS ONE). Medaka with amutation in the sox9b gene show a female to male sex
reversal. In the mutant, an initial pathway to determine thesex functions and the gonad normally is formed. But wefound that sox9b mutant does not maintain the germ cells.
The extracellular matrix produced by supporting cells
surrounding germ cells is largely disorganized in the mutant(Figure 1). Therefore it was expected that loss of germ cells
but not impairment of the initial male pathway might cause afemale to male sex reversal. In order to prove this32

Figure 1. Extracellular matrix (laminin: red) in the sox9b mutant gonad(bottom panels) and in the wild-type gonad (upper panels). Distribution
is impaired in the mutant. Green; sox9b-expressing supporting cells.
Blue; germ cells (from PLoS ONE 2012).

II. How the number of germ cells are regulated
during the course of early sex determination
process
As mentioned above we can say that regulation of germ cellnumber is an important component of proper sex
differentiation. We have previously identified the gene
responsible for this regulation, amhrII (anti-Mullerian
hormone receptor). The ligand for this receptor is AMH(anti-Müllerian hormone), which is known to be secreted
from male supporting cells (Sertoli cells) during mammalian
sex differentiation, and is critical for Müllerian duct (female
reproductive organ) regression in males. But in teleosts, there
are no organs found that are equivalent to the Müllerian duct.
In addition, AMH belongs to a phylogenetically old andconserved type of TGFβ superfamily. These collectively
suggest some conserved function other than Müllerian duct
regression.
The mutant, called hotei, has a mutation in amhrII and
exhibits a hypertrophic phenotype of germ cells and a maleto female sex reversal. This suggested that an AMH systemregulates germ cell number and sex.

Figure 2. Hetrozygous sox9b-mutant medaka have a normal appearance
but some exhibit female to male sex reversal.

First we isolated other alleles of amhrII, which causes astop codon at the very last N terminal region and lacks
important domains. Therefore it is very likely that thesealleles are a functional null. Consistent with this, the mutated
AMHRIIs fail to transmit an intercellular signal in thecultured cells. These allelic mutants displayed the same
phenotype as the hotei mutant, excessive number of germ
cells and sex reversal, indicating that a amhrII mutation in
hotei also causes a functional null phenotype.
Next we investigated the expression of both ligand andreceptor. As expected from the expression in mammals,
medaka amh is expressed in supporting cells that directly
surround germ cells. Interestingly amhrII is also detected insupporting cells. This suggests that the AMH system
functions in supporting cells in an autocrine manner. To
further confirm this, chimeric gonads were generated withsomatic cells from the hotei mutant and wild-type germ cells.
These chimeric gonads showed an increasing number ofgerm cells. On the other hand, gonads with normal somatic
cells, and germ cells from the hotei mutant did not exhibit
any hotei phenotype. The result of chimeric analyses
supports the expression analysis, whereby hyperactive germ
cell proliferation is a result of loss of the AMH signal in
supporting cells but is not due to a direct effect of AMH on
germ cells.

III. The AMH system regulates mitotically
active type I germ cells.
During sex differentiation in medaka gonads, there arefound two different types of germ cells. In males, germ cells
divide intermittently and the daughter germ cells are readily
enclosed with surrounded supporting cells (type I division).
In females, however, there is an additional type of germ cell,
dividing synchronously and successively to form germ cell
cysts (type II division). Therefore females have more germ
cells than males. Our previous results using the zenzai
mutant indicated that type I is a self-renewal type of division
while type II division occurs in germ cells that are committed
to gametogenesis (Saito et al., 2008 Dev. Biol.). In addition,
we proved the presence of germline stem cells in mature
medaka ovaries (Nakamura et al., 2010 Science).
A BrdU incorporation experiment revealed the presence of
mitotically quiescent germ cells, a good indication of thestem character, in the developing gonads (Figure 3). In thehotei mutants, the number of the quiescent type I germ cells
did not change, compared to that in wild-type. But the
mutant has more type I germ cells totally than the wild type.
This suggests that the AMH system does not regulatetransition from a quiescent type of type I germ cells to
mitotically active type I germ cells but promotes mitosis of
typeI germ cells that had already been committed to themitotic process (Nakamura et al., 2012 Development).
Collectively these analyses demonstrate that the AMH
system acts in supporting cells autonomously and that thisactivity consequently regulates mitotically active type I germ
cells. In the mutant, loss of AMH activity results in more
germ cells, which then causes a male to female sex reversal.

Figure 3. Presence of mitotically quiescent type I germ cells (arrows).
Blue; germ cells, Red; BrdU, Green; supporting cells..

Publication List
〔Original papers〕
K., Bubenshchikova, E., Powell, R., Fukuyo, Y., Nakamura,• Ichimura,T., Tran, U., Oda, S., Tanaka, M., Wessely, O., Kurihara, H., Sakai, T.,••

and Obara, T. (2012). A comparative analysis of glomerulus
development in the pronephros of medaka and zebrafish. PLoS ONE 7,
e45286.
Nakamura, S., Watanabe, I., Nishimura, T., Picard, J-Y., Toyoda, A.,
Taniguchi, Y., di Clemente, N., and Tanaka, M. (2012).
Hyperproliferation of mitotically active germ cells dues to defective
anti-Müllerian hormone signaling mediates sex reversal in medaka.
Development 139, 2283-2287.
Nakamura, S., Watanabe, I., Nishimura, T., Toyoda, A., Taniguchi, Y.,
and Tanaka, M. (2012). Analysis of medaka sox9 orthologue reveals a
conserved role in germ cell maintenance. PLoS ONE 7, e29982.

〔Original paper (E-publication ahead of print)〕
K., Kamei, K., Kinoshita, M., Czerny, T., and Tanaka, M. A
• Kobayashi,
heat-inducible cre/loxP gene induction system in medaka. Genesis 2012
Nov 3.

33

National Institute for Basic Biology

Developmental Biology

LABORATORY OF PLANT ORGAN DEVELOPMENT †Director General

OKADA, Kiyotaka

Assistant Professor:
Postdoctoral Fellow:

Visiting Scientist:

Technical Assistant:

Secretary:

TATEMATSU, Kiyoshi
IGARASHI, Hisako
NAKATA, Miyuki
TOYOKURA, Koichi
URAWA, Hiroko
YABE, Kimihiko
IKEUCHI, Momoko
IWASAKI, Akira
NAKATA, Miyuki*
TAMESHIGE, Toshiaki
HARA, Reiko
MATSUMOTO, Miwako
NAKAMORI, Chihiro
SUGIMOTO, Nagisa
TSUZUKI, Yumiko
SAKAGAMI, Mari

Plant organs, leaves, flowers, and roots show impressive,
symmetrical shapes, based on an ordered arrangement ofdifferentiated cells. The organs are formed from a group of
undifferentiated cells located at the tip of the stem or the
root. In the case of leaves, the process of organogenesis starts
with the formation of a leaf primordium in the peripheral
zone of the shoot apical meristem (SAM) at a fixed position,
following an order called phyllotaxis. Cells in theprimordium then proliferate and differentiate according tothree spatially fixed axes: the apical-basal axis, the lateral(central-marginal) axis, and the adaxial-abaxial (foresidebackside) axis. In the course of proliferation anddifferentiation, plant cells are believed to exchange
information with neighboring or separated cells in order toregulate organ architecture. We are trying to understand themechanisms of information exchange between plant cells
during the development of lateral organs, such as leaves,sepals, petals, stamens and carpels by using genetic, andbiochemical approaches.

another primary miRNA transcript backbone, pri-miR319a,in the abaxial epidermal cells, the activity of miR165 wasobserved in the whole region of leaf primordia, showing thatsequence and/or structure of MIR165A primary transcript isnecessary to restrict the miR165-active region to the abaxialside cells. Thus we concluded that the number of cells inwhich miR165 can move is determined depending on the
MIR165A primary transcript (Tatematsu et al., in
preparation).
To examine the mechanisms of establishment of the
adaxial-abaxial axis, we isolated novel mutants which show
altered patterns of FIL promoter:GFP expression, and named
them enlarged fil-expression domain (enf). We revealed that
one of them, enf1, has a mutation in a gene that encodes
SUCCINIC SEMALDEHYDE DEHYDROGENASE, whichcatalyzes the conversion of succinic semialdehyde (SSA) tosuccinate, and that SSA and/or its derivatives affect the axisdependent cell fate in leaf primordia (Toyokura et al., 2011,2012). We also isolated some suppressor mutants of enf1,
which show normal leaf shapes and expression domain of
FIL promoter:GFP. We determined the genes, which havethe mutation for the suppression of enf1 phenotype, using
next-generation sequence methods, and revealed that one ofthem has the mutation in a gene which encodes atransaminase enzyme.
Detailed analysis of temporal and spatial expression patternof FIL in a developing leaf primordium using reporter genecombined with the Cre-LoxP recombinant system showed
dynamic shift of the FIL-expression domain (Figure 1). This

I. Genetic approach
Recent studies of Arabidopsis mutants show several genes
are involved in the axes-dependent control of lateral organ
development. The adaxial- and abaxial-specific tissue
differentiation in the leaf primordium is determined by theprecise expression of the adaxial marker genes, HD-Zip III
including PHABULOSA (PHB), and the abaxial marker
genes, FILAMENTOUS FLOWER (FIL) and YABBY. Using
the reporter gene-system, we visualized the function domainof microRNA165/166 (miR165/166), which targeted theHD-Zip III messenger RNA, and showed that miR165/166
act in the epidermal and mesophyll cells locating in theabaxial side and determine the adaxial-specific expression of
PHB. One of the MIR165/166 genes, MIR165A, is expressedin the abaxial epidermal cells. We revealed that MIR165A is
enough to repress the PHB expression in the cells located in
the abaxial side, indicating that miR165 is likely to move tocells at the adaxial side. When miR165 was produced by

34

Figure 1. Cell lineages of FIL-expressing cells were visualized using atransgenic line having FIL promoter:Cre-GR and 35S:LoxP-ter-LoxPYFP. After dexamethasone (DEX) treatment, YFP is expressed
constitutively in the FIL-expressing cells by Cre-LoxP recombinantsystem. We applied DEX at various timing after seed germination andchecked YFP signal in the third leaves of 12-day-old seedlings. Photo
was taken from the adaxial side. Green and red colors indicate YFP and
autofluorescence of chlorophyll, respectively. Strong green colors
indicate YFP expressed in the adaxial epidermal- and adaxial mesophyll
cells, and weak colors indicate YFP expressed in the adaxial mesophyll
cells, but not in the adaxial epidermis. The day of DEX treatment after
seed germination is indicated at the bottom left of each panel. Bars
represent 1 mm.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).
†: This laboratory was closed on 31 March, 2013.

result indicates that the boundary between the expression
domain of FIL and that of PHB moves to the abaxial side
during leaf development. Another enlarged-FIL-expression
domain mutant, enf2, which has abaxialized leaves, showedslower moving of the boundary than that in wild type. We
also indicated that the ENF2 gene encoded a plastidlocalized unknown protein, and that the state of expression ofchloroplast genome-encoding genes might be a factor
determining the rate of the boundary shifting between the
PHB- and the FIL-expression domains. Using a simple
mathematical model, we also predicted that general cellularfunctions, such as production and/or degradation of thedetermining genes, regulate the rate of the boundary shifting
during leaf development (Tameshige et al., in press).
We examined the role of homeobox-related gene, PRESSEDFLOWER (PRS) and its homolog, WOX1, and showed thatthey are required for forming the margin-specific oblong
cells. Genetic analyses showed that PRS and WOX1 control
blade outgrowth and determine the expression domains of
the adaxial- and the abaxial-determining genes in themarginal region of leaf primordia. Based on the observations
we proposed the middle domain model (Nakata et al., 2012;
Nakata and Okada 2012). To identify the genes acting in thedownstream of WOX1, we carried out gene expression
profiling using Arabidopsis genome array in the WOX1
overexpressing transgenic plants. This analysis indicates thatWOX1 directly regulates the expression level of several
adaxial and abaxial determining genes, suggesting that threedomains, the adaxial-, the abaxial-, and the middle-domain,interact mutually during leaf developments. We also foundthat the expression of some auxin-inducible genes was
repressed in the WOX1 overexpression plants. To reveal the
relationships between WOX1 and auxin, we are analyzing theauxin responsibility and expression of auxin-related genes inthe shoot apex of wild type and prs wox1 seedlings (Nakata
et al., in preparation).
To reveal how floral organs fix their forms throughdevelopment processes, we analyzed a series of mutants
named folded petals (fop) with stacked and folded petals
when flowers open. We found that epidermal cells of themutant petal show traces of stacking with the epidermal cellsof sepals. FOP proteins are related to wax/cutin synthesis ortransport. Thus, we proposed that wax/cutin components
secreted by FOP proteins on the surface of the petal
epidermis might act as a lubricant between the floral organs
in a developing bud (Takeda et al., 2013).

II. Biochemical approach
We are taking a biochemical approach to study of theintercellular signaling system by analyzing small peptides,
which are present in the apoplastic region of the SAM. We
purified fractions containing small peptides from apoplast
fractions of the curds of cauliflower (Brassica oleracea L.
var. botrytis), and found a putative lipid transfer protein(LTP) has a role in increasing the number of SAMs in
Arabidopsis seedling when applied exogenously. The
cauliflower LTP gene was highly expressed in the curd, andthe expression of the Arabidopsis ortholog was observed in
the L1 cell layers of the SAM (Figure 2A, B). When LTP

expressed under constitutive active promoter in Arabidopsis,the transgenic plants showed increased number of SAMs
near the shoot apex (Figure 2C). Moreover, to analyze therole of LTP gene in planta, we made RNAi knockdown lines
of Arabidopsis LTP. In some seedlings of the knockdown
lines, multiple cotyledon and/or filamentous leaves wereobserved. Thus, we concluded that LTP proteins in apoplasts
are responsible for SAM maintenance and formation.

Figure 2. (A) Expression levels of cauliflower LTP genes in cauliflower
organs analyzed by qRT-PCR methods. (B) Pattern of in situ
hybridization of Arabidopsis LTP gene showing expression in epidermis
of a wild type seedling. A blue arrowhead indicates the SAM. (C) (Left)
10-day-old seedling of Arabidopsis LTP overexpression plants. (Right) A
longitudinal section of the seedling. Black arrowheads indicate theSAMs, and black dashed lines indicate shapes of the SAMs.

Publication List
〔Original papers〕
A.*, Tatematsu, K.*, Hanada, K.*, Duermeyer, L., Okamoto, M.,• Endo,Yonekura-Sakakibara, K., Saito, K., Toyoda, T., Kawakami, N.,

•
•
•
•

Kamiya, Y., Seki, M., and Nambara, E. (2012). Tissue-specifictranscriptome analysis reveals cell wall metabolism, flavonolbiosynthesis, and defense responses are activated in the endosperm ofgerminating Arabidopsis thaliana seeds. Plant Cell Physiol. 53, 16-27.
(*: Equally contributed)
Nakata, M., Matsumoto, N., Tsugeki, R., Rikirsch, E. Laux, T., andOkada, K. (2012). Roles of the middle domain-specific WUSCHELRELATED-HOMEOBOX genes in early development of leaves in
Arabidopsis. Plant Cell 24, 519-535.
Nakata, M., and Okada, K. (2012). The three-domain model: A newmodel for the early development of leaves in Arabidopsis thaliana.
Plant Signal. Behav. 7, 1423-1427.
Sakai, T., Mochizuki, S., Haga, K., Uehara, Y., Suzuki, A., Harada, A.,
Wada, T., Ishiguro, S., and Okada, K. (2012). The WAVY GROWTH 3
E3 ligase family controls the gravitropic responses in Arabidopsis root.
Plant J. 70, 303-314.
Toyokura, K., Hayashi, M., Nishimura, M., and Okada, K. (2012).
Adaxial-abaxial patterning: A novel function of the GABA shunt. Plant
Signal. Behav. 7, 705-707.

35

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

Associate Professor

NODA, Masaharu

SHINTANI, Takafumi

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
KUBOYAMA, Kazuya
FUJIKAWA, Akihiro
SUZUKI, Ryoko
MATSUMOTO, Masahito
SAKURABA, Jyuichi
MIYAZAKI, Yuriko
MATSUDA, Takashi
YU, Yang
HATTORI, Nobuko
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
WADA, Kotoe
KONISHI, Mie
KODAMA, Akiko

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:

Technical Assistant:

Secretary:

Recently, we generated Apc2-deficient mice by a genetargeting technique. In the homozygous mutant mice, robust
defects in neuronal lamination were observed in the cortex(Figure 1A), hippocampus, cerebellum (Figure 1B), andolfactory bulb. In vivo BrdU labeling andimmunohistochemical analyses with specific markers
revealed that the laminary abnormalities are a result ofdysregulated neuronal migration by a cell-autonomousmechanism. Dissociated Apc2-deficient cerebellar granulecells showed no obvious alterations in migration under nonstimulated conditions, however, Brain-derived neurotrophic
factor (BDNF)-stimulated directional migration was
perturbed.
Total internal reflection fluorescence (TIRF) microscopy
revealed that APC2 is distributed along actin fibers as well as
microtubules. BDNF-stimulated F-actin formation at theleading edge was impaired in migrating Apc2-deficientneurons, along with dysregulation of Rho GTPase activity.
Thus APC2 is an essential mediator of the cytoskeletal
regulation at leading edges in response to extracellularsignals. The phenotypes observed in Apc2-deficient micesuggest that mutations in APC2 in humans may cause aneurodevelopmental disorder.

We have been studying the molecular and cellularmechanisms underlying the development of the vertebrate
central nervous system (CNS), mainly using the visualsystems of chicks and mice. This research covers many
developmental events including the patterning of the retina,
neuronal terminal differentiation, axonal navigation,
branching and targeting, synapse formation, refinement andplasticity. The scope of our interests also encompasses themechanisms for various functions of the mature brain,including body-fluid regulation, behavior control, learning,and memory.

I. Mechanisms for neural circuit formation
Topographic maps are a fundamental feature of neuralnetworks in the nervous system. We have long studied themolecular mechanisms for regional specification in thedeveloping retina as the basis of the topographic retinotectal
projection. We are now focusing our attention on themolecular mechanisms underlying axon branching andarborization for synapse formation, along with elimination of
mistargeted axons and branches. Among the region-specificmolecules in the developing retina, we have already foundseveral molecules that induce abnormal branching orarborization when their expression was experimentallymanipulated in vivo.
One is adenomatous polyposis coli 2 (APC2), which ispreferentially expressed in the nervous system from early
developmental stages through to adulthood. APC2 isdistributed along microtubules in growth cones as well asaxon shafts of retinal axons. The knockdown of Apc2 inchick retinas reduced the stability of microtubules in retinal
axons and yielded abnormal behaviors of growth cones
including a reduced response to ephrin-A2 and misprojectionin the tectum without making clear target zones.

36

Figure 1. Laminary defects in the brain of Apc2-deficient mice. A, Sagital
sections of P30 cerebral cortex stained with anti-NeuN (green, a neuronspecific marker) and anti-FoxP2 (red, a polymorphous cell-specificmarker). The cerebral cortex of the wild-type (Apc2 +/+) mouse isorganized into six layers and FoxP2-positive cells are mainly distributedin layer VI. In the Apc2-deficient mouse (Apc2-/-), however, they areobserved broadly in layers II to V. Right panels are schematic drawings of
distribution patterns of neuronal subtypes in the wild-type and Apc2deficient mouse. In the Apc2-deficient mouse, cortical layers were poorly
organized and layer boundaries are blurred and indistinct. Scale bars, 50
μm. B, Sagital sections of P20 cerebellum stained with anti-NeuN (green,a granule cell-specific marker) and anti-calbindin D-28K (red, a Purkinjecell-specific marker). Arrows and arrowheads indicate ectopically
distributed Purkinje and granule cells, respectively. Right panels areschematic drawings of distribution patterns of neuronal subtypes in thewild-type and Apc2-deficient mouse. ML, molecular layer; PCL, Purkinje
cell layer; GCL, granule cell layer. Scale bars, 50 μm.

II. Regeneration of retinal axons
CNS neurons in fish can regrow their axons after nerve
transection, while CNS neurons in mammals lose theircapacity to regenerate. Thus, we have been investigating
genes involved in optic nerve regeneration using fish visualsystems. We found that expression of coagulation factor XIIIA subunit (FXIII-A), a protein cross-linking enzyme, isupregulated in the goldfish optic nerve and retina during
regeneration. The cells producing FXIII-A were astrocytes/
microglial cells and retinal ganglion cells (RGCs) in the optic
nerve and retina, respectively. Overexpression of FXIII-A in
RGCs and addition of extracts of optic nerves with injury toretinal explants induced significant neurite outgrowth fromthe retina. From these observations, we concluded that theincrease of FXIII in RGCs promotes neurite sprouting frominjured RGCs, whereas the increase of FXIII in optic nerves
facilitates elongation of regrowing axons.

III.Physiological roles of protein tyrosine
phosphatase receptor type Z
Protein-tyrosine phosphatase receptor type Z (Ptprz, also
known as PTPζ/RPTPβ) is a member of the R5 receptor-likeprotein tyrosine phosphatase (RPTP) subfamily. Ptprz ispredominantly expressed in glial and neuronal cells in thecentral nervous system (CNS) and its physiologicalimportance has been demonstrated through studies withPtprz-deficient mice. Ptprz modulates hippocampal synaptic
plasticity: adult Ptprz-deficient mice display impairments inspatial and contextual learning. Ptprz is expressed also in thestomach, where it is used as a receptor for VacA, a cytotoxin
secreted by Helicobacter pylori: Ptprz-deficient mice areresistant to gastric ulcer induction by VacA.
This year, we revealed that Ptprz plays a negative role inoligodendrocyte differentiation in early CNS development
and remyelination in demyelinating CNS diseases, throughthe dephosphorylation of its substrates such as p190RhoGAP.
We first found an early onset of the expression of myelin
basic protein (MBP), a major protein of the myelin sheath,and early initiation of myelination in vivo during
development of the Ptprz-deficient mouse, as compared withthe wild-type (Figure 2A). In addition, oligodendrocytes
appeared earlier in primary cultures from Ptprz-deficient
mice than wild-type mice.
We subsequently found that adult Ptprz-deficient mice areless susceptible to experimental autoimmune
encephalomyelitis (EAE) induced by active immunization
with myelin/oligodendrocyte glycoprotein (MOG) peptide
than were wild-type mice. However, the number of T-cells
and macrophages/microglia infiltrating into the spinal cord
were not decreased in Ptprz-deficient mice after MOG
immunization, suggesting that the reduced tissue damage is
not attributable to an inhibition of infiltration by
inflammatory cells.
It is known that Fyn tyrosine kinase-mediated downregulation of Rho activity through activation of
p190RhoGAP is crucial for oligodendrocyte differentiationand myelination. Here, p190RhoGAP is one of thephysiological Ptprz substrates. After EAE induction, thetyrosine phosphorylation of p190RhoGAP increased
significantly, and the EAE-induced loss of MBP wasmarkedly suppressed in the white matter of the spinal cord in
Ptprz-deficient mice (Figure 2B).
Thus, selective inhibition of Ptprz signaling could be aneffective and plausible therapeutic strategy for treating
demyelinating diseases.

Figure 2. Ptprz plays a negative role in oligodendrocyte differentiation inearly CNS development and remyelination in demyelinating CNS
diseases. A, Early myelination in Ptprz-deficient mice. Electron
micrographs of transverse sections at the corpus callosum from mice atpostnatal day 10 (left), and 3 months old (right). Scale bars, 2 μm.
Percentages of myelinated axons in total axons are shown at the lower
position of each panel. Data are the mean ± SEM *p < 0.05. B, Reduced
MBP loss in Ptprz-deficient mice after EAE induction. Anti-MBP
staining of the spinal cord sections from wild-type and Ptprz-deficient
mice 35 days after MOG immunization, or non-immunized control mice.
The lower images are enlargements of the areas enclosed by squares in
the upper images. Scale bars, 500 μm. MBP signals are expressed as therelative change compared with the non-immunized wild-type mice, andshown at the bottom. Data are the mean ± SEM. **p < 0.01.

IV. Brain systems for body-fluid homeostasis
Sodium (Na) is a major electrolyte of extracellular fluids
and the main determinant of osmolality. Na homeostasis isessential to life and Na + concentrations in plasma andcerebrospinal fluid (CSF) are continuously monitored tomaintain a physiological level of Na+ in body fluids. We havepreviously shown that Na x, which structurally resembles
voltage-gated sodium channels (Na v 1.1–1.9), is aconcentration-sensitive Na channel with a threshold of ~150mM for extracellular Na+ concentration [Na+]o in vitro.
In the brain, Nax channels are specifically expressed in37National Institute for Basic Biology

Neurobiology

a st r o c y t e s a n d e p e n d y m a l c e l l s i n t h e sens ory
circumventricular organs, such as the subfornical organ(SFO) and organum vasculosum of the lamina terminalis(OVLT), where Na x-positive glial cells are involved insensing an increase in [Na+] in body fluids. Nax-deficient
mice do not stop ingesting salt even when dehydrated, whilewild-type mice avoid salt. This behavioral defect of Naxdeficient mice is recovered by a site-directed transfer of the
Nax gene with an adenoviral vector into the SFO. Nax thus
functions as the brain’s Na+-level sensor for the homeostatic
control of [Na+] in body fluids.
Nax has a putative PSD-95/Disc-large/ZO-1 (PDZ)-binding
motif at the carboxyl-terminus. We thus hypothesized that the
Nax channel may be regulated by PDZ-scaffold proteins.
Very recently, we found that several PDZ proteins bind toNax by PDZ-array overlay assay. Among them, synapseassociated protein 97 (SAP97/DLG1) was coexpressed with
Nax in the SFO. In C6 glioblastoma cells, destruction of the
PDZ-binding motif of Na x, Na x(T1679A), resulted in adecrease in cell-surface Nax, which was attenuated with
inhibitors of endocytosis (Figure 3A). Depletion of SAP97
also led to the reduction in the surface expression of wildtype Nax (Figure 3B). Next, functional relevance of thebinding of Nax with SAP97 was confirmed by Na+-imaging
studies (Figure 3C). When the extracellular Na +concentration, [Na+]o was increased from 145 mM to 170
mM, both C6M16 cells expressing wild-type Nax andC6M(TA)8 cells expressing the Nax(T1679A) mutant showed
increases in the intracellular Na+ concentration, [Na+]i, andthe level eventually reached the same equilibrium point
between Na + influx by Na x and Na + export by Na + /K+-ATPase. However, importantly, C6M(TA)8 cells took
longer to reach this equilibrium level than control C6M16
cells. This is probably because the reduction in the number ofsurface Na x channels diminished the Na+ influx. Thus,
SAP97 appears to play an important role in sensing bodyfluid [Na+] in the SFO through regulation of the surface
expression of the sensor channel.

Figure 3. SAP97 promotes the stability of Nax channels at the plasma
membrane. A, Decrease in cell-surface expression of the Nax mutant with
Thr-1679 changed to Ala (mutation in the PDZ-binding motif) in C6 cells
and its improvement by treatment with endocytosis inhibitors,
wortmannin and dynasore. After the induction of the expression ofNax(T1679A) channels, cells were treated with 100 nM wortmannin or
200 μM dynasore for 6 h. Then the cells were fixed, permeabilized, andstained with anti-Nax. B, Reduction in the cell-surface expression of Nax
with depletion of SAP97. Non-treated C6M16 cells, or C6M16 cells
transfected with siRNA for SAP97 or scrambled siRNA were
immunostained with anti-SAP97 and anti-Nax. The fluorescence intensity
profiles along the white lines are shown in the right panel. Scale bars, 10
μm. a.u., arbitrary unit. C, Reduced sodium influx in C6 cells in theabsence of any association between Nax and SAP97. Left: Na+ imaging of
C6M16 (wild-type Nax) and C6M(TA)8 (T1679A mutant Nax) cells uponelevation of the extracellular Na+ concentration from 145 mM to 170
mM. The coordinate gives the fluorescence ratio (ΔF340/380 nm) in Na+
imaging with sodium-binding benzofuran isophthalate acetoxymethyl
ester (SBFI/AM), representing the intracellular Na+ concentration. The
physiological 145 mM Na+ solution was changed to a 170 mM solution at
0 min. Right: Summary of the time to reach 95% of the maximum
fluorescence ratio. Data represent the mean ± SE (n = 40 for each); *P <0.01, two-tailed t test.

Publication List
〔Original papers〕
K., Fujikawa, A., Masumura, M., Suzuki, R., Matsumoto,• Kuboyama,M., and Noda, M. (2012). Protein tyrosine phosphatase receptor type Z

•
•
•

38

negatively regulates oligodendrocyte differentiation and myelination.
PLoS ONE 7, e48797.
Matsumoto, M., Fujikawa, A., Suzuki, R., Shimizu, H., Kuboyama, K.,
Hiyama, T.Y., Hall, R.A., and Noda, M. (2012). SAP97 promotes thestability of Nax channels at the plasma membrane. FEBS Lett. 586,
3805-3812.
Shintani, T., Takeuchi, Y., Fujikawa, A., and Noda, M. (2012).
Directional neuronal migration is impaired in mice lacking adenomatous
polyposis coli 2. J. Neurosci. 32, 6468-6484.
Sugitani, K., Ogai, K., Hitomi, K., Nakamura-Yonehara, K., Shintani,T., Noda, M., Koriyama, Y., Tanii, H., Matsukawa, T., and Kato, S.
(2012). A distinct effect of transient and sustained upregulation ofcellular factor XIII in the goldfish retina and optic nerve on optic nerve
regeneration. Neurochem. Int. 61, 423-432.

DIVISION OF BRAIN BIOLOGY

Professor

Associate Professor

YAMAMORI, Tetsuo

WATAKABE, Akiya

Assistant Professor:

KOMINE, Yuriko
SADAKANE, Osamu
Assistant Professor (Specially appointed):
KOMATSU, Yusuke#
NIBB Research Fellow:
OHTSUKA, MasanariTechnical Staff:
OHSAWA, Sonoko
Postdoctoral Fellow:
TAKAJI, Masafumi#
OHTSUKA, Masanari*
HATA, Katsusuke#
SASAKI, Tetsuya
Visiting Scientist:
HIRAKAWA, Reiko
Graduate Student:
SHUKLA, RammohanTechnical Assistant:
NAKAMURA, Tohru
TAKEDA, Yuta#
NAKAGAMI, Yuki
MORITA, Junko
IMAI, Akiko
KOTANI, Keiko
KON, Yayoi
IMOTO, EikoHIRAYAMA, Yuka#
KAJITANI, Tomoki#
TAKAHASHI, Yoichi#

Figure 1. Cover letter for J.
Comp. Neurology, Volume520, Issue 16.

#: SRPBS (Strategic Research Program for Brain Sciences), NIPS

We continue to study genes that are expressed in specificareas of the neocortex in order to understand the principles
governing the formation of the primate brain. In addition,
this year we have reported a novel genetic manipulation
method to visualize cortical projection neurons withretrograde TET-off lentiviral vector, area-specificsubstratification of deep layer neurons in the rat cortex, andthe behavioral analysis of zfhx2-deficient mice as follows.

I. Area-specific substratification of deep layer
neurons in the rat cortex
To identify cell types for fine mapping of neuronal circuits,
we studied area-specific sublamina structures of the rat
cerebral cortex using cholecystokinin (cck) and purkinje cell
protein4 (pcp4) mRNAs as the markers for the subtypes of
excitatory neurons in layers 5 and 6. We found a segregatedexpression, especially pronounced in layer 6, where
corticothalamic and corticocortical projecting neurons reside(Figure 2). To examine the relationship between gene
expression and projection target, we injected retrograde
tracers into several thalamic subnuclei, ventral posterior(VP), posterior (PO), mediodorsal (MD), medial and lateralgeniculate nuclei (MGN and LGN); as well as into two
cortical areas (M1 and V1). This combination of tracer-in
situ hybridization (ISH) experiments revealed thatcorticocortical neurons predominantly express cck andcorticothalamic neurons predominantly express pcp4
mRNAs in all areas tested. In general, cck(+) and pcp4(+)
cells occupied the upper and lower compartment of layer 6a,
respectively. However, the sublaminar distribution and the

Figure 2. Expression patterns of cck and pcp4 mRNAs in various corticalareas. A: Low magnified views of the double-ISH of cck (red) and pcp4
(green) mRNAs in the rat brain. PL, prelimbic cortex; Cla, claustrum;
M1, primary motor cortex; S1Tr, primary somatosensory cortex, trunk
region; S1BF, primary somatosensory cortex, barrel field; S2, secondary
somatosensory cortex; V1, primary visual cortex; V2L, secondary visualcortex lateral area. B–G: The magnified cortical areas labeled in panel A.
Scale bars = 1 mm in A; 200 μm in B–G. (Watakabe et al., J Comp
Neurol. 520, 3553-3573, 2012.)

relative abundance of cck(+) and pcp4(+) cells were quitedistinctive across areas. For example, layer 6 of the prelimbic
cortex was almost devoid of cck(+) neurons, and was
occupied instead by corticothalamic pcp4(+) neurons. In thelateral areas, such as S2, there was an additional layer ofcck(+) cells positioned below the pcp4(+) compartment. The
claustrum, which has a tight relationship with the cortex,
mostly consisted of cck(+)/pcp4(−) cells. The combination ofgene markers and retrograde tracers revealed a distinct
sublaminar organization, with conspicuous cross-areavariation in the arrangement and relative density ofcorticothalamic connections. (Watakabe et al., J Comp
Neurol. 520, 3553-3573, 2012.)

II. Visualization of cortical projection neurons
with retrograde TET-off lentiviral vector.
We are interested in identifying and characterizing various
projection neurons that constitute the neocortical circuit.
For this purpose, we developed a novel lentiviral vector
that carries the tetracycline transactivator (tTA) and theNote: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

39

National Institute for Basic Biology

Neurobiology

transgene under the TET Responsive Element promoter(TRE) on a single backbone. By pseudotyping such a vector
with modified rabies G-protein, we were able to express
palmitoylated-GFP (palGFP) or turboFP635 (RFP) in thecorticothalamic, corticocortical, and corticopontine neuronsof mice. The high-level expression of the transgene achievedby the TET-Off system enabled us to observe characteristic
elaboration of neuronal processes for each cell type (Figure3). At higher magnification, we were able to observe finestructures such as boutons and spines as well. We also
injected our retrograde TET-Off vector to the marmoset
cortex, and therefore proved that this method can be used tolabel the long- long-distance cortical connectivity of themillimeter scale. In conclusion, our novel retrograde tracer
provides an attractive option to investigate the morphologies
of identified cortical projection neurons of various species(e.g., Figure 4) (Watakabe et al., PLoS One. 7, e46157,
2012).
Figure 4. Reciprocal connectivity of marmoset V1 and V2 visualized by
StTTrR/FuG-B vector.
(A) Dark field image overlaid with RFP fluorescence indicates theinjection site. The V1/V2 border (shown by the arrowhead) was clearlydelineated by the presence of the striate of Gennari (g). The arrow
indicates the injection site. (B and C) The RFP signals around theinjection site (red) are shown with NeuN counterstaining (green). The
arrow indicates the plexus of V1 terminals in layer 4. The boxed region
contains the retrogradely labeled V2 neurons with feedback projection toV1, which is magnified in panel F. The asterisk indicates the core of
injection, where NeuN expression is lost by local damage. Only the RFP
signals are shown in panel C. (D and E) RFP signals (red) and NeuN
stains (green) in the injection center. The contrast for the RFP signals isadjusted so that the cell bodies of the infected neurons can be delineated.
Note that the lamina positions of the infected cells are restricted mostly tolayers 2, 3 and 6. (F–H) Confocal images of RFP in V2. Panel F shows
the cell bodies and dendrites of V2 neurons that project back to V1. (G)The spines of the basal dendrites originating from one of these cells. (H)The plexus of axon terminals concentrated in layer 4 of V2 (shown by thearrow). Bar: 2 mm for A; 500 μm for B and C; 200 μm for D and E; 50
μm for F and H; 10 μm for G.
Figures 3 and 4 from Watakabe et al., PLoS One. 7, e46157, 2012.

III. Behavioral analysis of zfhx2-deficient mice.

Figure 3. Retrograde infection of the corticothalamic cells by StTTrG/
FuG-B vector. StTTrG/FuG-B vector was injected into the mousethalamus. (A–D) After one week (for panels A and B) or four weeks (forpanels C–G), the mouse was perfusion fixed and sliced at 40 μm
thickness. The boxed areas in panels A and C were magnified in panels B
and D, to show infection of corticothalamic neurons in layer 6 (indicatedby arrowheads). The injection site is indicated by the asterisk (*). These
images show the original green fluorescence of the infected cells. (E–G)
The immunoperoxidase detection of the GFP-expressing cells. Bar: 500
μm (A and C) , 200 μm (for others). Watakabe et al., PLoS One. 7,
e46157. The caudal block of the marmoset brain was parasagittally
sectioned to visualize the reciprocal connectivity between V1 and V2.

40

Zfhx2 (also known as zfh-5) encodes a transcription factor
containing three homeobox domains and 18 Zn-finger
motifs. We have reported that Zfhx2 mRNA is expressed
mainly in differentiating neurons in the mouse brain and its
expression level is negatively regulated by the antisensetranscripts of Zfhx2 (Figure 5). Although the expression
profile of Zfhx2 suggests that ZFHX2 might have a role in aparticular step of neuronal differentiation, the specificfunction of the gene has not been determined. We therefore
generated a Zfhx2-deficient mouse line and performed acomprehensive battery of behavioral tests to elucidate thefunction of ZFHX2. Homozygous Zfhx2-deficient mice
showed several behavioral abnormalities, namely,
hyperactivity, enhanced depression-like behaviors, and an
aberrantly altered anxiety-like phenotype (e.g., Figure 6).
These behavioral phenotypes suggest that ZFHX2 might playroles in controlling emotional aspects through the function of
monoaminergic neurons where ZFHX2 is expressed.

Moreover, considering their phenotypes, the Zfhx2-deficient
mice may provide a novel model of human psychiatricdisorders. (Komine et al., PLoS One. 7, e53114, 2012)

Figure 5. Structure and expression of mouse Zfhx2.(A) Structure of
ZFHX2 and related proteins. ZFHX2 is a protein of 2562 amino acids
containing 18 zinc fingers (green ovals) and three homeodomains (redsquares). (B) Zfhx2, Zfhx3, and Zfhx4 mRNA detected by semiquantitative RT-PCR in various RNA sources. Note that cDNAs were
amplified for 30 cycles for brains of different developmental stages,
whereas cDNAs were amplified for 35 cycles for various adult tissues.
(C–E) Expression of Zfhx2 (C), Zfhx3 (D), and Zfhx4 (E) mRNA in theparasagittal sections of an E15.5 mouse brain. These three genes wereexpressed in substantially similar patterns with the highest expression
level of Zfhx3. (F, G) Expression of Zfhx2 mRNA (F) and the ZFHX2protein (G) was compared on adjacent coronal sections of an E15.5 brain.
mRNA expressed in the thalamic region (Th) was translated, whereas
mRNA expressed in the cerebral cortex (Cx) was not translated: thissituation made the expression patterns of ZFHX2 and ZFHX3 more alikein protein level than in mRNA level. (H–J) Expression of Zfhx2 (H),Zfhx3 (I), and Zfhx4 (J) mRNA in the coronal sections of an adult brain.
Cerebral cortex (Cx), hippocampus (Hp), thalamus (Th), caudate
putamen (CP). Expression levels of all three genes were decreased
compared with those in the embryonic brain, but Zfhx2 maintained ahigher level of expression than the others. (K–P) Expression of ZFHX2protein in the adult brain. The pyramidal layer of the hippocampus (K,Py), the suprachiasmatic nucleus (L, SCN), laterodorsal thalamic nucleus(M, LD), lateral geniculate nucleus (N, LGN), substantia nigra pars
compacta (O, SNc), and magnocellular part of the red nucleus
suprachiasmatic (P, RMC). (Q–Y) Double-color in situ hybridization with
Zfhx2(red) and tyrosine hydroxylase (Th) probes (green).

Figure 6. Locomotor activity measured in open field test. Distancetraveled (A, B),stereotypic behavior (C), vertical activity (D), and timespent in the center area (E). Measurements are blocked in 5 min (A, C, D,
and E) or in 1 min (B, shown only for the first 10 min). (Komine et al.,PLoS One. 7, e53114, 2012)

Publication List
〔Original papers〕
M., Matsui, R., Kato, S., Hasegawa, T., Kasahara, H., Isa, K.,• Kinoshita,
Watakabe, A., Yamamori, T., Nishimura, Y., Alstermark, B., Watanabe,

•
•
•
•

D., Kobayashi, K., and Isa, T. (2012). Genetic dissection of the circuit
for hand dexterity in primates. Nature 487, 235-238.
Komine, Y., Takao, K., Miyakawa, T., and Yamamori, T. (2012).
Behavioral abnormalities observed in zfhx2-deficient mice. PLoS ONE
7, e53114.
Takahata, T., Shukla, R., Yamamori, T., and Kaas, J.H. (2012).
Differential expression patterns of striate cortex-enriched genes among
Old World, New World, and prosimian primates. Cereb. Cortex 22,
2313-2321.
Watakabe, A., Hirokawa, J., Ichinohe, N., Ohsawa, S., Kaneko, T.,
Rockland, K.S., and Yamamori, T. (2012). Area-specific substratificationof deep layer neurons in the rat cortex. J. Comp. Neurol. 520, 35533573.
Watakabe, A., Kato, S., Kobayashi, K., Takaji, M., Nakagami, Y.,
Sadakane, O., Ohtsuka, M., Hioki, H., Kaneko, T., Okuno, H.,
Kawashima, T., Bito, H., Kitamura, Y., and Yamamori, T. (2012).
Visualization of cortical projection neurons with retrograde TET-off
lentiviral vector. PLoS ONE 7, e46157.

41

National Institute for Basic Biology

Neurobiology

DIVISION OF BRAIN CIRCUITS

Professor

MATSUZAKI, Masanori

Assistant Professor:
NIBB Research Fellow:
Postdoctoral Fellow:
Visiting Scientist:
Graduate Student:
Technical Assistant:
Secretary:

WAKE Hiroaki
TANAKA, Yasuhiro
MASAMIZU, Yoshito
TANAKA, Yasuyo
TANAKA, Yasuhiro*
HIRA, Riichiro
OHKUBO, Fuki
HIMENO, Miki
SAITO, Junko
SUGIYAMA, Tomomi

Various firing patterns of many cortical neurons represent
information processing in the brain. The microarchitecture ofsynaptic connections control information processing in
cortical circuits. The structure and location of synapses
determine and modify the strength of this information
processing. The aim of our laboratory is to reveal how
information is formed, maintained, selected, and decoded in
the brain at the levels of single cells and of single synapses.
To do so, we mainly use two-photon microscopy that allows
us to see fluorescence signals from deep within living tissue,
developing novel photostimulation methods and animal
behavioral tasks. The aim of our recent study is to reveal
how voluntary movement is represented in cortical circuits.
One of the most important problems in neuroscience is how avariety of spatio-temporally heterogeneous neural activity inthe cortex emerges moment-by-moment at multiple stages ofa movement.

were subsequently rewarded with a drop of water from aspout near their mouth. With the reward at the cessation of
the lever pull, a magnet-controlled solenoid pole would
quickly push the lever back to the wait position. The micethen had to wait 3 s before they could receive another reward
for a lever pull. During 8-9 days of the training sessions, thetask difficulty was increased by gradually increasing thelever-pull time (100–1,000 ms) and mice were able to either
increase or maintain the number of successful trials.
Furthermore, the interval time between successful trials
decreased, indicating that mice successfully learned how topull the lever and also understood that they had to wait >3 s
to pull the lever again and obtain another reward.

II. Identification of the mouse motor forelimbareas with photostimulation mapping
We used channelrhodopsin-2 (ChR2) mice to determine theforelimb motor areas over the broad neocortical surface by
optogenetic stimulation mapping (OSM) using blue-laser
scanning of the cortical surface. Laser illumination induced
forelimb movements in two distinct areas: RFA (the rostralarea) and CFA (the caudal area) (Figures 2). These motor
forelimb areas corresponded to those reported byintracortical microstimulation in the rat and mouse cortex.

Figure 2. Dorsal view of left cortical
hemisphere. Overlaid red and blue
contours show the RFA and CFA,
respectively, as revealed by OSM.

I. Development of a novel operant task ofhead-restrained mice
To carry out two-photon calcium imaging while mice
performed a self-initiated movement, we developed a headrestrained lever-pull task (Figure 1). Mice used their rightforelimbs to pull the lever for a given amount of time andFigure 1. The lever-pull task in head-restrained mice. The head of themouse was fixed under an objective, and the right forelimb was available
to grasp and pull the movable lever. Before pulling the lever, mice had to
wait for more than 3 s. After the lever pull was maintained for Tset, a dropof water was dispensed.

42

III. Spatio-temporal representation of motor
information in the brain
We performed two-photon calcium imaging in layer 2/3 ofthe left RFA or CFA while mice executed the task (Figure 3).
Motion-corrected calcium transients reflected spiking
activity, and particularly the burst firing of individual cells.
We found two types of task-related cells in the mice: cells
whose peak activities occurred during lever pulls (pull cells)
and cells whose peak activities occurred after the end oflever pulls (post-pull cells). The activity of pull cells wasstrongly associated with lever-pull duration. In
approximately 40% of imaged fields, functional clusterings
were temporally detected during the lever pulls. Spatially,
there were ~70 μm-scale clusters that consisted of more thanfour pull cells in approximately 50% of the fields. Ensemble
and individual activities of pull cells within the cluster moreaccurately predicted lever movement trajectories thanactivities of pull cells outside the cluster. This was likely
because clustered pull cells were more often active in theindividual trials than pull cells outside the cluster. This

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

higher fidelity of activity was related to higher trial-to-trial
correlations of activities of pairs within the cluster. We
propose that strong recurrent network clusters may represent
the execution of voluntary movements.

Publication List
〔Original paper〕
R., Saiki A., Fujiwara-Tsukamoto Y., Ohkubo F., Kitamura K.,• Kimura
Matsuzaki M., Sakai Y., and Isomura Y. (2012). Reinforcing operandum:
rapid and reliable learning of skilled forelimb movements by head-fixedrodents. J. Neurophysiol. 108, 1781-1792.

Figure 3. Activities of layer 2/3 motor cortex cells during task
performance. A. A representative example of a two-photon imaged fieldin the RFA. Circles surround reconstructed neurons. Parts of fluorescent
traces of numbered, closed circles are shown in (B). Cyan, magenta,
yellow, and grey circles indicate pull cells, post-pull cells, other taskrelated cells, and non-task-related cells, respectively. B, 270-s traces of
motion-corrected calcium transients of the numbered cells shown in (A).
The trace indicated as Lever shows the lever trajectory. Shaded boxes
indicate successful trials. C, The motion-corrected traces of cells 1, 2, and3 are aligned with the start of all 30 successful lever pulls during theimaging session. Black thick lines indicate the mean traces. The lever
trajectories in individual trials are overlaid in the bottom trace.

Figure 4. Prediction accuracy of the lever movement trajectory relative toneural ensemble activity of pull cells. The linear model prediction of thelever trajectory (grey) from 15 primary-clustered cells (black) and nine
non-clustered cells (blue) in the imaged field shown in Figure 3. One of
the test segments of the trajectory is shown. The correlation coefficients
between the predicted and real trajectories for this segment were 0.55 and0.40 in the 15 primary-clustered and nine non-clustered cells,
respectively.

43

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellow:
Visiting Scientist:

NAKAYASU, Tomohiro
AONO, Sachiko

In order to interact successfully with the environment,
animals must deduce their surroundings based on sensory
information. The visual system plays a particularly criticalrole in such interactions with the environment.
“Why can we see?” This question is fundamental for athorough understanding of vision-dependent animals,
including human beings. In order to better understand thesensory systems of animals, we moved ahead from ourprevious research of the salt-sensing system to new researchon the visual system.

behavior. To generate biological motion stimuli, medaka
were videotaped and then six points were placed along the
body trunk using computer software (Figure 1). We foundthat the presentation of biological motion could prominently
induce shoaling behavior. We are now analyzing what
aspects of motion (such as movement speed and temporalorder) are critical in the induction of shoaling behavior.
Motion or behavioural characteristics can be valuable inrecognizing animal species, sex, and group members. Studies
using biological motion stimuli will enhance ourunderstanding of how non-human animals extract andprocess the information which is vital for their survival.

I. Psychophysical study of Medaka fish
One of our major subjects is the psychophysical andcomputational studies of medaka (Oryzias latipes). Medaka
have many advantages for behavioral work. First, genetic
examination of medaka is progressing at a rapid pace,
opening up new approaches to the understanding of genetic
control of behavior. Second, although the central nervous
system of medaka is relatively simple, its basic structure is
the same as that in mammals. Thirdly, they provide
invaluable comparative material for work on mammals.
Examination of such a relatively simple yet vertebrate
system should thus aid in the determination of the basic
mechanisms of how genes affect behavior.
This year, we have made progress in studies of the preypredator interaction using medaka and zooplankton. Visualmotion cues are one of the most important factors for
eliciting animal behavior, including predator-preyinteractions in aquatic environments. To understand theelements of motion that cause such selective predation
behavior, we used a virtual plankton system where thepredation behavior in response to computer-generated prey
was analyzed. First, we performed motion analysis ofzooplankton (Daphnia magna) to extract mathematical
functions for biologically relevant motions of prey. Next,
virtual prey models were programmed on a computer andpresented to medaka, which served as predatory fish.
Medaka exhibited predation behavior against several
characteristic virtual plankton movements, particularly
against a swimming pattern that could be characterized aspink noise motion. Analyzing prey-predator interactions via
pink noise motion will be an interesting research field in the
future.
Furthermore, we have made progress in studies of theschooling behaviors of medaka. Many fish species are known
to live in groups. Visual cues have been shown to play a
crucial role in the formation of shoals (a shoal is defined associal group of fish). Using biological motion stimuli,
depicting a moving creature by means of just a few isolated
points, we examined for the first time whether physicalmotion information is involved in the induction of shoaling
44

Figure 1. Animation sequence depicting biological motion of medaka.
Six points were automatically placed along the body trunk (Motion
tracking). Based on the tracking data, the movements of medaka wereexpressed as those of six gray dots (Stimulus presentation).

Figure 2. Sample video of Flash-lag effect. This year, we successfully
produced a new version of the 3D Flash-lag effect using Blender 3D
software. Please refer to YouTube (http://youtu.be/X8RiaNUFIaU).

II. Psychophysical study of Human vision
Another of our major subjects is the psychophysical andtheoretical studies of the visual system of human beings(Homo sapiens). One recent focus of this debate is the flashlag effect, in which a moving object is perceived to lead aflashed object when both objects are aligned in actual
physical space (Figure 2). This effect has been utilized forunderstanding human motion perception. We developed asimple conceptual model explaining the flash-lag effect(Delta model, Watanabe et al., 2010).

Publication List
〔Original paper〕
W., and, Watanabe, E. (2012). Visual motion with pink
• Matsunaga,
noise induces predation behaviour, Scientific Reports, 2, 219.

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

Associate Professor

HASEBE, Mitsuyasu

MURATA, Takashi

Assistant Professor:

HIWATASHI, Yuji
TAMADA, Yosuke
ISHIKAWA, Masaki
KABEYA, Yukiko
MANO, Hiroaki
AOYAMA, Tsuyoshi
CHENG, Chaoyang
IMAI, Akihiro
ISHIKAWA, Masaki*
ISHIKAWA, Takaaki
KUBO, Minoru
NAGASHIMA, Akitomo
NOZAWA, Masafumi
OKAMOTO, Haruko
SHIBATA, Tomoko
AOYAMA, Tsuyoshi*
FUKUSHIMA, Kenji
ICHIKAWA, Shunsuke
KAMIDA, Chiharu
LI, Chen
SUGAYA, Tomomi
ZHANG, Liechi
AOKI, Etsuko
FUKADA, Hatsumi
GOTO, Miho
GOTO, Misako
HIRAMATSU, Mika
ITO, Yukiko
KAJIKAWA, Ikumi
KAKIGI, Rieko
KOHARA, Mayuko
MASUOKA, Tomoko
MATSUZAKI, Yoko
NISHI, TayoOOI, Shoko
WAKAZUKI, Sachiko
WASHIO, Midori
OHSHIMA, Issei
NISHIYAMA, Tomoaki
VINCENTE, Jesus C.
VILLARREAL, Natalia M.
VOLK, Christian Louis
KOJIMA, Yoko

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:

Graduate Student:

Technical Assistant:

Visiting Scientist:

Secretary:

I. Evolution of Complex Adaptive Characters
The theory of natural selection and the neutral theory ofmolecular evolution are powerful concepts in evolutionary
biology. However, even with such theories, there still remain
unexplained phenomena, one of which is the evolution ofcomplexity. It is difficult to explain the mechanisms needed
to evolve complex adaptive traits at cellular and organismallevels, such as cell division machinery, regeneration, novel
organ development, host race change, and mimicry. Suchtraits comprise many components and become adaptive only
when all components are gathered together. However, basedon evolutionary theory, each component should evolve one
by one according to the accumulation of mutations. We aim
to reveal the genetic networks regulating the complex traits
and to infer the mechanisms needed to evolve complex
characters.

II. E v o l u t i o n o f R e g e n e r a t i o n :Reprogramming of Differentiated Cells to
Pluripotent Stem Cells
Different species have different morphology and also
cellular characters vary between species. Stem cells selfrenew and repeatedly produce differentiated cells during
development. Conversely, differentiated cells can beconverted into stem cells in some organisms. In plants,
regeneration of a stem cell leads to a generation of a new
individual, which is an effective strategy for propagation.
The ability of reprogramming is different from species tospecies but the reason is unknown. The moss Physcomitrella
patens has a rapid reprogramming ability (see http://www.
nibb.ac.jp/evodevo/ERATO/movie/MacMovie.mp4) and isfeasible for experiments. Cells in a dissected leaf are
reprogrammed to become chloronema apical stem cells
within 24 hours.

Figure 1. Expression changes of genes during reprogramming areclassified into nine types. Reprogramming of leaf cells started at time 0.

To understand the underlying molecular mechanisms, a
digital gene expression profiling method using mRNA 5’-end
tags (5’-DGE) was established. The 5’-DGE method
produced reproducible data with a dynamic range of fourorders that correlated well with qRT-PCR measurements.
After the excision of leaves, the expression levels of 10% of
the transcripts changed significantly within 6 h. Genes
involved in stress responses and proteolysis were induced
and those involved in metabolism, including photosynthesis,
were reduced. The later processes of reprogramming
involved photosynthesis recovery and higher macromoleculebiosynthesis, including of RNA and proteins. Comparison
with stem cell formation via callus in flowering plants
revealed that common phytohormone signaling pathways areactivated during reprogramming, although no exogenous
phytohormone is applied in the moss system, suggesting thatan intrinsic phytohormone regulatory system may be used in
the moss (Nishiyama et al. 2012).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
III. Evolution of Regeneration: Epigenetic
regulation
In differentiated cells, active modifications such as
trimethylation of histone H3 at lysine 4 (H3K4me3) areenriched at certain genes required for the cell function, andthe repressive modifications including H3K27me3 areenriched at other unnecessary genes, to ensure an expression
profile that fits the cell function. In contrast, in animal
pluripotent stem cells, most genes with H3K27me3 also have
H3K4me3, and this bivalent chromatin state is presumed tokeep genes poised for transcription and to be required for thepluripotency. Thus, changes in the genome-wide chromatin
modifications must be associated with and required for theprocess of reprogramming. We are currently attempting toanalyze the dynamics of the chromatin modifications in the
reprogramming process of P. patens with the combination ofchromatin immunoprecipitation-sequencing (ChIP-Seq)
using a next generation sequencer and live imaging ofchromatin modifications. We have successfully established a
4D (3D + time) live-imaging method of a single P. patens
nucleus, and also produced a H3K27me3 detector using
fluorescent protein fused to Drosophila melanogaster
Polycomb protein, which is known to bind to H3K27me3.
We are now analyzing the ChIP-Seq data, performing the 4D
single-nucleus live imaging for H3K27me3 during the
reprogramming, and producing live-imaging detector forother chromatin modifications including H3K4me3. This
study was mainly conducted by Yosuke Tamada.

IV. Evolution of Regeneration: Master
Regulator for Reprogramming STEMIN
Animal somatic cells can be reprogrammed to induce
pluripotent stem (iPS) cells by introducing four transcription
factors, while such factors have not been identified in plants.
On the basis of the transcriptional profile during the moss
reprogramming (Nishiyama et al., 2012), we selected genes,of which transcript levels are low in gametophores andup-regulated during reprogramming, of leaf cells after
excision, and then overexpressed those genes in intact
gametophores using the estrogen-inducible system. As aresult, we identified a gene encoding a member of plantspecific transcription factor, STEM CELL-INDUCING
FACTOR (STEMIN), that was able to induce direct
reprogramming of differentiated leaf cells into chloronema
apical stem cells without wounding signals. Using the
5’-DGE analysis, we found that many of the genes inducedby the STEMIN-expression in intact gametophores
overlapped with those upregulated during reprogramming inexcised leaves. In addition, STEMIN promoter was activatedat leaf cells that underwent reprogramming. Deletion of the
STEMIN and its two paralogous genes delayed reprograming
after leaf excision. Together, we suggest that STEMIN is asingle master regulatory transcription factor governing denovo stem cell formation. Masaki Ishikawa was this study’s
main researcher.

V. Evolution of Elaborated Cell Division
Machinery: Phragmoplast
The cells of land plants and their sister group, charophycean

46

green algae, divide by the insertion of cell plates atcytokinesis. This is in contrast to other green algae, in which
the invagination of the plasma membrane separates daughter
cells at cytokinesis. The cell plate appears in the middle ofdaughter nuclei, expands centrifugally towards the cell
periphery, and finally fuses to the parental cell wall. Cellwall materials are transported to the expanding cell platewith a phragmoplast, which is mainly composed of
microtubules. Centrifugal expansion of the phragmoplast is adriving force for that of the cell plate, although elucidating
the molecular mechanism for the expansion was a challenge.
We have found that γ-tubulin complexes on existingphragmoplast microtubules nucleate new microtubules asbranches. Although elongation of the branched microtubules
is likely a driving force of the phragmoplast expansion, themechanism by which phragmoplast microtubules redistributetowards the cell periphery is unclear. Because an inhibitor of
microtubule depolymerization inhibits phragmoplast
expansion, analyses of microtubule depolymerization might
be a key for understanding the mechanism. We developed amethod for quantifying the rate of microtubule
depolymerization in the phragmoplast, and found that therate of microtubule depolymerization gradually increases
from the outer surface to the inside of the phragmoplast.
Based on the results, we propose a hypothesis that random
branching of microtubules coupled with biased
depolymerization lead directional redistribution of
microtubules, which drives centrifugal expansion of thephragmoplast. Takashi Murata was this study’s main
researcher.

VI. Evolution of Developmental Programs
Figure 2. APB transcription
factor is indispensable for ashoot body (gametophore)formation in the moss
Physcomiterlla patens.
Deletion of four APB genes
caused no gametophores(lower panel) in comparison
to wild type (upper panel).

The basal land plant mosses have two different
developmental processes in the haploid generation, forming
hypha-like protonemata and shoot-like gametophores. We
found that four AP2-type transcription factors orthologous to
Arabidopsis thaliana AINTEGUMENTA/PLETHORA/ BABY
BOOM (APB) are indispensable for the formation of
gametophore apical cells. Quadruple disruption of all APB
genes blocked gametophore formation, even in the presenceof cytokinin, which enhances gametophore apical cell
formation in the wild type. Heat-shock induction of an APB4

transgene driven by a heat-shock promoter increased thenumber of gametophores. Expression of all APB genes was
induced by auxin but not by cytokinin. Thus, the APB genes
function synergistically with cytokinin signaling to determine
the identity of the two types of stem cells (Aoyama et al.
2012).

V. Molecular mechanisms of mimicry
An excellent example of mimicry is the flower-mimicry of
the orchid mantis Hymenopus coronatus with pink and whitecoloration and petal-like legs. HPLC and MS analyses
indicated that xanthommatin, a common red pigment of theommochrome family, almost solely contributes to the pinkcolor of late-stage nymphs. On the other hand, 1st-instar
nymph with yellowish red color contains a very labile prexanthommatin in addition to xanthommatin. These results
suggest that the orchid mantis alters its body color bychanging the composition of ommochrome pigments during
post-hatching development. This work was mainly done by
Hiroaki Mano.

VI. Molecular mechanisms of host shifting
In phytophagous insects a precise combination of
performance and preference traits for particular host plants is
crucial for host shifting because a new host plant can beincorporated into an insect’s diet if adults accept it for
oviposition and if the larvae are able to complete theirdevelopment on it. However, very little is known about thegenetic bases of the performance and preference. A QTL
analysis of a tiny moth, Acrocercops transecta revealed that
only a restricted region of a single autosome was responsible
for the larval performance, suggesting that a small number ofgenetic changes to larval performance allowed the successful
host shifting. Identification of the responsive genes is inprogress with Dr. Issei Ohshima in Kyoto Prefecture
University.

VII. M o l e c u l a r m e c h a n i s m s o f P l a n t
Movement using Mimosa pudica

Australian pitcher plant Cephalotus follicularis incollaboration with Beijing Genomics Institute. Wholegenome shotgun data corresponding to 100-fold depth were
produced by Illumina paired-end/mate-pair sequencing with180-bp to 5-kb insert sizes. A de novo assembly yielded a
total of 1.7 Gbp in 43,308 scaffolds with 15.6 kb of contig
N50 and 83.3 kb of scaffold N50. We further produced 14
Gbp of PacBio reads with 2-kb mean max subread length for
gap filling. Transcript-based gene prediction with RNA-seq
reads found 45,469 gene models. Genomic data enable us todeduce the origin and evolution of carnivory-related genes,
such as digestive enzyme genes. This study was conducted
mainly by Kenji Fukushima and Tomoko Shibata.

Publication List
〔Original papers〕
T., Hiwatashi, Y., Shigyo, M., Kofuji, R., Kubo, M., Ito, M.,• Aoyama,and Hasebe, M. (2012). AP2-type transcription factors determine stem

•
•
•
•

cell identity in the moss Physcomitrella patens. Development 139,
3120-3129.
Nishiyama, T., Miyawaki, K., Ohshima, M., Thompson, K., Nagashima,A., Hasebe, M., and Kurata, T. (2012). Digital gene expression profilingby 5’-end sequencing of cDNAs during reprogramming in the moss
Physcomitrella patens. PLoS ONE 7, e36471.
Nishimura, T., Matano, N., Morishima, T., Kakinuma, C., Hayashi, K.I.,
Komano, T., Kubo, M., Hasebe, M., Kasahara, H., Kamiya, Y., andKoshiba, T. (2012). Identification of indole-3-acetic acid transport
inhibitors including compounds affecting cellular PIN trafficking by
two chemical screening approaches using maize coleoptile systems.
Plant Cell Physiol. 53, 1671-1682.
Suetsugu, N., Sato, Y., Tsuboi, H., Kasahara, M., Imaizumi, T., Kagawa,T., Hiwatashi, Y., Hasebe, M., and Wada, M. (2012). The KAC familyof kinesin-like proteins is essential for the association of chloroplasts
with the plasma membrane in land plants. Plant Cell Physiol. 53, 18541865.
Zhao, N., Ferrer, J.L., Moon, H.S., Kapteyn, J., Zhuang, X., Hasebe, M.,
Stewart, C.N., Jr., Gang, D.R., and Chen, F. (2012). A SABATH
Methyltransferase from the moss Physcomitrella patens catalyzes
S-methylation of thiols and has a role in detoxification. Phytochemistry81, 31-41.

The molecular mechanisms and evolutionary significance ofplant movement, including seismonastic and nyctinastic
movements, are enigmatic. To introduce the sensitive plant
Mimosa pudica as a model, we established a method fortransformation. We used a cotyledonary node as a target ofAgrobacterium-mediated gene transfer because of its abilityof shoot regeneration. We obtained a large number oftransformed calluses (55-60%) and succeeded inregenerating transgenic plants with a transformation
efficiency of >5%. This study was conducted mainly by
Hiroaki Mano.

VIII. Evolution of pitcher leaves in carnivorous
plantsCarnivorous plants form specialized leaves that are capableof attracting, trapping, and digesting prey and absorbing
nutrients. The unusual plants evolved from non-carnivorous
plants but their evolutionary process is mostly unknown. To
understand the genomic changes associated with theevolution of carnivory, we sequenced 2-Gbp genome of the47National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

KAWAGUCHI, Masayoshi

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:

Graduate Student:

Technical Assistant:
Secretary:

TAKEDA, Naoya
SUZAKI, Takuya
FUKADA-TANAKA, Sachiko
OKAMOTO, Satoru
SOYANO, Takashi
YANO, Koji
FUJITA, Hironori
MIYAZAWA, Hikota
HANDA, Yoshihiro
KOBAYASHI, Yuki
TAKAHARA, Masahiro
SASAKI, Takema
YORO, EmikoTSUZUKI, Shusaku
SUZAKI, Momoyo
ICHIKAWA, Michiko
OGAWA, Yuko
SANJO, Kazuko

More than 80% of land plant families have symbiotic
relationships with arbuscular mycorrhizal (AM) fungi. The
origin of AM symbiosis is thought to have been in the early
Devonian period. On the other hand, the root nodule
symbiosis that occurs between legumes and rhizobial
bacteria, unlike AM symbiosis, involves host-specificrecognition and postembryonic development of a nitrogenfixing organ. Despite marked differences between the fungal
and bacterial symbioses, common genes required for bothinteractions have been identified using model legumes. Ourlaboratory focuses on the early stages of the interaction
between these microorganisms and Lotus japonicus in orderto reveal the molecular mechanism and the origin of these
symbiotic systems.

I. Nodulation
1-1 The site of auxin action during root nodule
development and identification of TRICOT gene in
L. japonicus
Nodulation is a form of de novo organogenesis that occurs
mainly in legumes. During early nodule development, thehost plant root is infected by rhizobia that induce
dedifferentiation of some cortical cells; these cortical cells
then proliferate to form the symbiotic root nodule
primordium. We investigated the detailed patterns of auxin
response during nodule development in L. japonicus (Figure1). Our analyses showed that cytokinin signaling positively
regulated this response. Additionally, we found that auxin
response was inhibited by a systemic negative regulatory
mechanism called autoregulation of nodulation (AON).
Analysis of the constitutive activation of LjCLE-RS genes,
which encode putative root-derived signals that function in
AON, in combination with determination of auxin response
patterns in proliferating cortical cells, indicated that
activation of LjCLE-RS genes blocked the progress of further
cortical cell division, probably through controlling auxin

48

response. Our data provide evidence for the existence of anovel fine-tuning mechanism that controls nodule
development in a cortical cell stage-dependent manner.
Recent studies on AON and hormonal controls of
nodulation have identified key mechanisms and also
indicated a possible link to other developmental processes,
such as the formation of the shoot apical meristem (SAM).
However, our understanding of this process is still limited by
the low number of nodulation-related genes that have been
identified. We found that the induced mutation tricot (tco)can suppress the activity of spontaneous nodule formation 2,
a gain-of-function mutation of the cytokinin receptor in L.
japonicus. Our analyses of tco mutant plants demonstratethat TCO positively regulates rhizobial infection and nodule
organogenesis. In addition to its role in nodulation, TCO is
involved in the maintenance of the SAM. The TCO gene wasisolated by a map-based cloning approach and found to
encode a putative glutamate carboxypeptidase with greatest
similarity to Arabidopsis ALTERED MERISTEMPROGRAM 1, which is involved in cell proliferation in the
SAM. Taken together, our analyses have not only identified anovel gene for regulation of nodule organogenesis but also
provided significant additional evidence for a common
genetic regulatory mechanism in nodulation and SAM
formation. This new data will contribute further to ourunderstanding of the evolution and genetic basis of
nodulation.

Figure 1. Auxin response patterns during nodule development in L.
japonicus. (A, B) Auxin response patterns during nodule development areindirectly shown by GFP expression (green) in DR5::GFP-NLS plants.
Fluorescence and nodule formation were observed from front (A) andside views (B) of infected roots. Red areas indicate the presence ofrhizobia. Scale bars: 100 μm.

1-2 I solation of a novel non-nodulation mutant,daphne that has a hyperinfection phenotype
The main events for making nodules are divided into
“infection” and “organogenesis.” For proper establishment of
symbiosis, it is essential that the two phenomena proceedsynchronously in different root tissues. Several nonnodulating mutants have been isolated using model legumes.
However most of those have defects in both infection andorganogenesis pathways. Therefore it has been difficult forunderstanding the molecular relationship between both
pathways independently.
We isolated a novel non-nodulating mutant, daphne fromC 6+ beam mutagenized seeds of L. japonicus. Unlikepreviously reported non-nodulation mutants, the daphne
mutant has defects only in the organogenesis pathway. The
daphne mutant has completely defective nodulation, but has
increased number of infection threads. Map-based cloning
identified the daphne mutation, reciprocal chromosomaltranslocation between chromosomal II and III which islocated on the upstream of the NODULE INCEPTION (NIN)
gene, whose expression is up-regulated dependent on
rhizobial inoculation. In daphne, the expression level of NIN
is lower than that in WT because of the mutation. Add to this,the result of the allelism test strongly suggest that daphe is a
novel nin mutant allele, being different from the nin nullmutant which cannot form any infection thread. The
phenotype of the nin null mutant indicates that NIN, putativetranscription factor, functions in both infection andorganogenesis pathways. We speculate that daphne might
have lost the specific expression of NIN that mediates the
nodule organogenesis pathway.

nodulation in a HAR1/SUNN/NARK- or KLV-dependent
manner. However, a large part of the mechanism involved atstage (iii) of AON needs to be elucidated. Previously, we
have shown that the too much love (tml) mutant in L.
japonicus (a mutant that exhibits an excessive number of
nodules) has a defect in the negative feedback regulation andthat TML functions in the roots downstream of HAR1
(Magori et al., 2009).
In this study, we found that TML acts downstream of CLERS1/RS2 and HAR1 and that TML suppresses the nodulation
signaling downstream of the cytokinin receptor LHK1/CRE1.
In addition, we identified the TML gene that encodes a Kelchrepeat-containing F-box protein with two NLSs, and weshow that TML is constitutively expressed in the root tips
independent of rhizobial infection and is expressed in theroot primordia upon rhizobial infection. With these results,
we concluded that TML functions in the CLE-RS1/RS2- andHAR1-mediated long-distance control of nodule
organogenesis. In addition, our finding that TML encodes the
F-box protein strongly suggests that AON functions via
proteasome-mediated target degradation. To our knowledge,
this is the first report of ubiquitin-related component thatfunctions during AON.

II. Arbuscular mycorrhiza symbiosis

Figure 2. Isolation of daphne mutant. (A) The 4 week old seedlings ofwild type (left) and daphne mutant (right) with rhizobial inoculation. (B,C) High-magnification images of wild type root (B) and daphne root
(C). (D, E) The infection threads with red florescence signal and root
images were merged. The number of infection threads is higher indaphne (E) than that in wild type (D). (F) The number of infection
threads is about 20-fold greater in daphne than that in wild type. Scalebars: 2cm.

1-3 TOO MUCH LOVE, a novel F-box Kelchprotein, functions in the long-distancecontrol of the root-nodule symbiosis
In the legume-rhizobia symbiosis, the nodule number istightly restricted by the host through a systemic suppression
termed autoregulation of nodulation (AON). AON provides along-distance control of nodulation via the shoot and isclassified into 3 stages: (i) an early nodulation event induces
the translocation of the root-derived signal to the shoot, (ii)the leucine-rich repeat receptor-like kinases (e.g., HAR1/
SUNN/NARK or KLV) in the shoot are activated by the rootderived signal, and the shoot-derived inhibitor (SDI) is
generated and translocated to the whole root system and (iii)
an assumed root regulator activated by the SDI inhibits thesubsequent nodule organogenesis. Earlier studies havesuggested that CLE peptides are strong candidates for the
root-derived signals, as they drastically suppresses the

Arbuscular mycorrhiza is mutualistic plant-fungalinteraction which has several similar systems to root nodule
symbiosis in host-symbiont recognition, infection process
and nutrient material exchanges. Root nodule symbiosis(RNS) is thought to evolve by sharing AM factors,
suggesting that the AM system contains a fundamental
mechanism that also regulates root nodule symbiosis.
We isolated two novel common symbiosis mutants;
cerberus and nsp1 by screening of RNS mutants in L.
japonicus. These mutant showed low symbiont infection
phenotypes in AM and RNS (Figure 3). Common symbiosis
genes isolated so far are supposed to act in the earlysignaling pathway for recognition of the symbionts andtriggering symbiosis responses. However, analysis of the AM
phenotypes revealed that CERBERUS and NSP1
quantitatively regulate AM fungal infection downstream orparallel to current models of symbiotic signaling, andextends the concept of commonality between RNS and AMS.
In order to obtain further insights about the function ofthese symbiotic factors, we performed transcriptome analysis
in nsp1 and cerberus mutant by RNAseq using next
generation sequencing technology. The analysis will reveal
effects of the mutation on the symbiotic signaling networks
during AMS and RNS. In addition, we are also analyzing
transcriptome profiles of the symbiotic partner AM fungi.
AM fungi are an obligate symbiont which requires the hostand symbiotic relationship to accomplish the life cycle,
especially for the spore formation. The transcriptome
analysis would indicate important clues about symbiotic andreproductive systems of AM fungi.

49National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Figure 3. AM fungal colonization in RNS mutant cerberus and nsp1.
Hyphae (A) and arbuscule (B) colonization in wild type, cerberus andnsp1 were examined at 4 weeks-after innoculation (wai) with AM fungi
Rhizophagus irregularis. AM fungi colonized in the root of wild type (C)and cerberus (D) were stained with ink (purple) at 3 wai. Delay or defectof hyphal elongation was observed in the cerberus root (D). Bars = 200μm.

molecules than that of the system with usual ubiquitoussynthesis. We then confirm that this complementary systemaffects pattern types in fixed and expanding two-dimensional
spaces in a similar way to the system with ubiquitoussynthesis. In addition, the two-layer system includes two
types of diffusion, lateral and transversal, and these havedistinct effects on pattern formation with lateral diffusion
mainly determining the periodicity of patterns generated andtransversal diffusion affecting pattern type. Finally, we apply
this complementary system to explain pattern formation of
the shoot apical meristem of plants (Figure 4B-E). Thesefindings provide an understanding of pattern formation
caused by the interaction between cell layers in multicellularorganisms.

Publication List

III. Pattern formation by two-layer system〔Original papers〕

Many multicellular organisms have a layered structure. Theinteraction between these layers plays an essential role inmany developmental processes, and key molecules involvedin these processes are often expressed in a layer-specificmanner. On the other hand, pattern formation of organisms
has been frequently discussed in connection with the Turingsystem. However, the Turing system has so far been studied
mainly in single-layered space. We thus investigate a twolayer Turing system with complementary synthesis, in which
two interacting molecules are exclusively synthesized indifferent layers (Figure 4A).
From a linear stability analysis, we determine the Turingcondition of the complementary system, and show that thiscondition requires stronger regulatory interactions of theJ., Moreau, C., Liu, Y., Kawaguchi, M., Hofer, J., Ellis, N., and• Chen,Chen, R. (2012). Conserved genetic determinant of motor organ identity

•
•
•
•
•

in Medicago truncatula and related legumes. Proc. Natl. Acad. Sci. USA
109, 11723-11728.
Hakoyama, T., Niimi, K., Yamamoto, T., Isobe, S., Sato, S., Nakamura,Y., Tabata, S., Kumagai, H., Umehara, Y., Brossuleit, K., Petersen, T.
R., Sandal, N., Stougaard, J., Udvardi, M. K., Tamaoki, M., Kawaguchi,
M., Kouchi, H., and Suganuma, N. (2012). The intergral membraneprotein SEN1 is required for symbiotic nitrogen fixation in Lotus
japonicus nodules. Plant Cell Physiol. 53, 225-236.
Hakoyama, T., Oi, R., Hazuma, K., Suga, E., Adachi, Y., Kobayashi, M.,
Akai, R., Sato, S., Fukai, E., Tabata, S., Shibata, S., Wu, G. J., Hase, Y,
Tanaka, A., , Kouchi, H., Umehara, Y., and Suganuma, N. (2012). The
SNARE protein SYP71 expressed in vascular tissues is involved insymbiotic nitrogen fixation in Lotus japonicus nodules. Plant Physiol.
160, 897-905.
Sandal, N., Jin, H., Rodriguez-Navarro, D.N., Temprano, F., Cvitanich,C., Brachmann, A., Sato, S., Kawaguchi, M., Tabata, S., Parniske, M.,
Ruiz-Sainz, J.E., Andersen, S.U., and Stougaard, J. (2012). A set of
Lotus japonicus Gifu x Lotus burttii recombinant inbred lines facilitate
map-based cloning and QTL mapping. DNA Research 19, 317-323.
Suzaki, T., Yano, K., Ito, M., Umehara, Y., Suganuma, N., andKawaguchi, M. (2012). Positive and negative regulation of cortical cell
division during root nodule development in Lotus japonicus isaccompanied by auxin response. Development 139, 3997-4006.
Takeda, N., Maekawa, T., and Hayashi, M. (2012). Nuclear localized
and deregulated calcium and calmodulin– dependent protein kinase
activates rhizobial and mycorrhizal responses. Plant Cell 24, 810-822.

〔Original paper (E-publication ahead of print)〕
T., Kim, C.S., Takeda, N., Szczyglowski, K., and Kawaguchi,• Suzaki,
M. TRICOT encodes an AMP1-related carboxypeptidase that regulates
root nodule development and shoot apical meristem maintenance in
Lotus japonicus. Development 2012 Dec 18.

Figure 4. Two-layer system with complementary synthesis. (A)Schematic representation of two-layer activator-inhibitor system withcomplementary synthesis of u (activator) and v (inhibitor). (B-E)
Examples of time evolution of the SAM in (B) fasciation pattern, (C)dichotomous pattern, (D) homeostasis pattern, and (E) fluctuation pattern.

50

LABORATORY OF MORPHODIVERSITY

Associate Professor

KODAMA, Ryuji

The aim of this laboratory is to observe the variety ofmorphogenetic processes that occur in the course of the
ontogenesis of multicellular organisms and to analyze themechanisms of such processes, mainly by morphologicalmethods. The accumulation of such analyses of theembryogenetic processes of related species is expected toprovide an insight into the evolution of morphogenetic
processes. This laboratory uses the wings of lepidopteraninsects as the main target of morphological studies.
The laboratory was moved from Myodaiji-area to Yamatearea in July 2012.

Figure 1. Transmission electron micrograph of the tracheole cell. Manycross-sections of the tracheoles can be observed within the cytoplasm of
the tracheole cell. As the cell migrates, the tracheole is laid behind.

I. Wing morphogenesis
The wings of the lepidopteran insects (butterflies andmoths) develop from the wing imaginal disc, which is ahollow sac made of simple epithelium. Due to its simple
construction, this tissue is a good material for studying
cellular interactions in the course of morphogenesis.
The outline shape of the adult wing is often different fromthat of the pupal wing. This difference is brought about by
the programmed cell death of the marginal area of the pupal
wing. The marginal dying area is called “the degeneration
region” and the internal area, which develops into the adultwing, is called “the differentiation region”.
Cell death in the degeneration region proceeds very rapidly
and finishes in a half-day to one-day period in Pieris rapae
and in several other examined species. It was shown that thedying cells in the degeneration region have characteristics incommon with the apoptotic cell death in mammalian cells,such as fragmented and condensed nuclei containing short
DNA fragments detected by TUNEL staining. The cells inthe degeneration region are actively en gulfed by themacrophages in the cavity beneath the wing epithelium. The
macrophages seem to be concentrated beneath thedegeneration region by the strong adhesion between the basal
surfaces of the dorsal and ventral epithelium in the
differentiation region, which occurs at the time of prominentcell death and excludes macrophages out of thedifferentiation region. This realized concentration ofmacrophages seems to accelerate the shrinkage of thedegeneration region.
A possible physiological role of the cell degeneration at thewing margin is to make space for the growth of the marginal
scales. Marginal scales are extremely elongated scales thatgrow densely on the edge of the wing. These scales are
considered to be important in stabilizing the turbulenceoccurring posterior to the wing. The movements of themarginal scales are closely monitored by sensory scales andbristles growing among them (A. Yoshida et al, unpublished).
Trachea and tracheoles are both important in delivering air
into the wing and their patterns coincide with that of theboundary of degeneration and differentiation zones at the
distal end of the wing. According to previous observations,the pattern formation of wing epithelium is often dependent on
tracheal and tracheole patterns. Basic research on thedevelopment of tracheal pattern formation is being done byscanning and transmission electron microscopy (Figure 1) to
describe the exact pathway and time course of the formationof the elaborate pattern of trachea and tracheoles and toestablish the cytological and developmental relationshipbetween the formation of the tracheal pattern and theepithelial cell pattern.

II. Other research activities
This laboratory also conducts morphological observation ofvarious animal tissues by scanning and transmission electron
microscopy and immuno-electron microscopic analyses incollaboration with other laboratories of NIBB. Training inspecimen preparation and instrument operation for suchobservations is also given.

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIORESOURCES

Associate Professor

NARUSE, Kiyoshi

Assistant Professor
NIBB Research Fellow:
Postdoctoral Fellow:

Research Fellow:
Technical Assistant:

Secretary:

TAKEHANA, Yusuke
TAKEHANA, Yusuke*
SASADO, Takao
SHIBATA, Yasushi
SHIBA, Keikun
NAKAMOTO, Masatoshi
OKUYAMA, Teruhiro
KIMURA, Tetsuaki
NOZU, Ryo
KANEKO, Hiroyo
YOSHIMURA, Yuriko
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
KOIKE, Chieko
TESHIMA, Yuuko
HARA, Ikuyo
ISHIKAWA, Hiroe
SHIBATA, Emiko
TAKAKI, Chikako
HAMATANI, Ayako
SUZUKI, Tokiko

Medaka is a small egg-laying “secondary” fresh water fish
found in brooks and rice paddies in Eastern Asia. This
species has a long history as an experimental animal,
especially in Japan. Our laboratory has conducted studies on
evolution of the sex determination system using medaka andrelatives, identification of the causal gene of mutants for
PGC migration and pigment cell development, and the
gonadal development of medaka. In addition to these
activities, our laboratory is stepping forward to lead the
National BioResource Project Medaka (NBRP Medaka).

I. Evolution of the sex chromosome and sex
determination genes in Oryzias fish
Recent studies have demonstrated that Oryzias species
have different genetic sex-determination systems (XX/XY
and ZZ/ZW) (Figure 1). Furthermore, the sex chromosomes
differ in their origin and degree of differentiation. Thesefindings suggest the repeated creation of new sex
chromosomes from autosomes during evolution of Oryzias
fishes, possibly in association with the formation of new sexdetermining genes. We are now trying to positionally clone

Figure 1. Phylogenetic relationships and sex determination mechanisms
in Oryzias fishes.

52

the novel sex-determining genes in these species.
Identification of these genes would provide a clue tounderstand the evolutionary process underlying frequent
turnover of sex determination mechanisms.

II. Genetic dissection of migration ofprimordial germ cells in medaka
Germ cells are responsible for the sustainability of life overgenerations in many multicellular animal species. To
elucidate the mechanisms underlying the development ofprimordial germ cells, we identified multiple mutations
affecting the migration and development of the primordial
germ cells in medaka in a prior large-scale mutagenesis
screening project, and have analyzed a set of them to date.
We focused on three mutants that have defects in primordial
germ cell migration, kamigamo, shimogamo, and naruto thatwere isolated in the screening project. Positional cloning andanalysis of the genes carrying the mutations are now inprogress. In addition, two mutations, kamigamo andshimogamo, cause cystic pronephric ducts simultaneouslywith abnormal positioning of the primordial germ cells.
Therefore, the analysis of these mutations will be importantin giving basal knowledge underlying the mechanisms ofhuman cystic kidney diseases.

III. The function of estrogen in the medaka
ovary
Estrogens have been generally considered to play a criticalrole in the ovarian differentiation of teleost fish by
Yamamoto’s model. In medaka, estrogen treatment has
induced functional male-to-female sex reversal. To clarify
the function of estrogen during ovarian development, we
examined the role of ovarian aromatase, which is responsible
for catalyzing the conversion of testosterone to estrogen. We
isolated two tilling mutant strains of ovarian aromatase. In
these tiling mutants, one amino acid in ovarian aromatase
ORF altered the STOP codon. Mutant females seemed todevelop normal ovaries but yolk accumulation was notobserved in the ovarian follicles and most of the ovarianfollicles undergo atresia in the adult ovary. Even more
surprisingly, spermatogenesis was observed within the
mutant ovary. These results suggest that the hypothesis that
endogenous estrogens drive ovarian differentiation needs tobe modified to the hypothesis that estrogens are essential forthe maintenance of ovarian differentiation.

IV. Positional cloning of pigment cell mutantsin medaka
All kinds of pigment cells are derived from neural crest
cells. How each type of pigment cell differentiates and what
differences are producing which of the various pigment cell
types is a very interesting question. Medaka have four typesof pigment cell (melanophore, leucophore, xanthophore andiridocyte). The leucophores are unique because only somespecies have them. To elucidate how leucophores
differentiate from neural crest cells and why they exist only
in some fishes, we have successfully identified the causal
gene of leucophore mutants (leucophore free (lf) andleucophre free 2 (lf-2)). We identified slc2a15b as the causal

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

gene of the lf mutant. The slc2a15b expression exists but
does not persist in the lf mutant. slc2a15b also exists inspecies without leucophores, but these use carotenoid as apigment. Thus, slc2a15b may have an important role in useof carotenoid as pigments. The lf-2 phenotype was rescuedby pax7a. pax7a is needed for differentiation of leucophoreand xanthophore in medaka.

IV. National BioResource Project Medaka(NBRP Medaka) (http://www.shigen.nig.ac.
jp/medaka/)
In 2007, NIBB was selected as the core facility of NBRP
Medaka. Our laboratory is taking an active part in thisproject. With the goal of facilitating and enhancing the use of
medaka as a model organism, we provide, maintain andcollect living resources such as standard strains, inbred
strains, and mutants in addition to frozen resources such as
EST/cDNA and BAC/ Fosmid clones and hatching enzymes,
as well as integrated information on medaka (Figure 2). We
have been providing the TILLING screening system to
NBRP Medaka users for promoting the reverse genetic
approach since 2011,. NBRP Medaka aims to establish a firstrate biological resource with the highest possible levels ofaccessibility and ease of use.

Publication List
〔Original papers〕
J., Zhang, X., Wang, T., Li, Z., Guan, G., and Hong, Y. (2012).
• Chen,Efficient detection, quantification and enrichment of subtle allelicalterations. DNA Research 19, 423-433.

Y., Okuyama, T., Taniguchi, Y., Kubo, T., and Takeuchi, H. (2012).
• Isoe,p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias
latipes). Biochem Biophys Res. Commun. 423, 627-631.

T., and Naruse, K. (2012). Genetic analysis of vertebral• Kimura,regionalization and number in Medaka (Oryzias latipes) inbred lines.
G3 2, 1317-1323.

Chen, W., Wang, D., Zhou, L., Sakai, F., Guan, G., and Nagahama,• Li,Y. J.,(2012).
GATA4 is involved in the gonadal development and

•
•
•
•
•
•
•
•

maturation of the teleost fish tilapia, Oreochromis niloticus. J. Reprod.
Dev. 58, 237-242.
Moriyama, Y., Kawanishi, T., Nakamura, R., Tsukahara, T., Sumiyama,
K., Suster, ML., Kawakami, K., Toyoda, A., Fujiyama, A., Yasuoka, Y.,
Nagao, Y., Sawatari, E., Shimizu, A., Wakamatsu, Y., Hibi, M., Taira,M., Okabe, M., Naruse, K., Hashimoto, H., Shimada, A., and Takeda, H.
(2012). The medaka zic1/zic4 mutant provides molecular insights into
teleost caudal fin evolution. Curr. Biol. 22, 601-607.
Myosho T., Otake, H., Masuyama H., Matuda M., Kuroki Y., Fujiyama
A., Naruse K., Hamaguchi, S., and Sakaizumi, M. (2012). Tracing the
emergence of a novel sex-determining gene in medaka, Oryzias
luzonensis. Genetics 191, 163-170.
Myosho, T., Takehana, Y., Sato, T., Hamaguchi, S., and Sakaizumi, M.
(2012). The origin of the large metacentric chromosome pair in Chinese
medaka (Oryzias sinensis). Ichthyol. Res. 59, 384-388.
Nakamoto, M., Fukasawa, M., Tanaka, S., Shimamori, K., Suzuki, A.,
Matsuda, M., Kobayashi, T., Nagahama, Y., and Shibata, N. (2012).
Expression of 3β-hydroxysteroid dehydrogenase (hsd3b), star andad4bp/sf-1 during gonadal development in medaka (Oryzias latipes).
Gen. Comp. Endocrinol. 176, 222-230.
Oshima Y., Sato H., Kajiura-Kobayashi H., Kimura T., Naruse K., andNonaka, S. (2012). Light sheet-excited spontaneous Raman imaging ofa living fish by optical sectioning in a wide field Raman microscope.
Optic Express. 20, 16195-16204.
Shibata, Y., Iwamatsu, T., Suzuki, N., Young, G., Naruse, K., Nagahama,Y., Yoshikuni, M. (2012). An oocyte-specific astacin family protease,alveolin, is released from cortical granules to trigger egg envelope
hardening during fertilization in medaka (Oryzias latipes). Develop.
Biol. 372, 239-248.
Takehana, Y., Naruse, K., Asada, Y., Matsuda, Y., Shin-I, T., Kohara, Y.,
Fujiyama, A., Hamaguchi, S., and Sakaizumi, M. (2012). Molecularcloning and characterization of the repetitive DNA sequences thatcomprise the constitutive heterochromatin of the W chromosomes of
medaka fishes. Chromosome Res. 20, 71-81.
Zhao, H., Li, M., Purwanti, Y.I., Liu, R., Chen, T., Li, Z., Hong, N.,
Guan, G., Yin, A., Xiao, L., Ge, R., Song, J., and Hong, Y. (2012). Mitf
is a transcriptional activator of medaka germ genes in culture.
Biochemie 94, 759-767.

〔Original papers (E-publication ahead of print)〕
Li, M., Guan, G., Hong, N., and Hong, Y. Multiple regulatory regions• control
the transcription of medaka germ gene vasa. Biochimie 2012
Dec 8.

H., Guan, G., Duan, J., Cheng, N., Wang, J., Matsuda, M., Paul• Zhao,
Prasanth, B., and Nagahama, Y. Ol4E-T, a eukaryotic translation
initiation factor 4E-binding protein of medaka fish (Oryzias latipes),
can interact with nanos3 and vasa in vitro. J. Exp. Zool. B (Mol. Dev.
Evol.) 2012 Sep 5.
Figure 2. NBRP Medaka website〔Review article〕
T., Kamei, Y., Takehana, Y., Sasado, T., and Naruse, K. (2012).
• Kimura,
Medaka genomics and the methods and resources for decoding genomic
functions. In Genome Mapping and Genomics in Laboratory Animals,
P. Denny and C. Kole, eds. (London Springer), pp159-182.

53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITY
KAMADA Group
Assistant Professor:

KAMADA,Yoshiaki

Nutrients are indispensable for life. Thus, perception of thenutrient environment is also essential for cells. Eukaryotic
cells employ Tor (target of rapamycin) protein kinase torecognize cellular nutrient conditions. Tor forms two distinct
protein complexes, TORC (Tor complex) 1 and TORC2.
TORC1 regulates rapamycin-sensitive branches of the TOR
pathway, such as protein synthesis, cell cycle and autophagy.
TORC1 is thought to act as a nutrient sensor, because
rapamycin, a TORC1 inhibitor, mimics a starved condition.
On the other hand, TORC2, whose function is insensitive to
rapamycin, is responsible for actin organization and cell
integrity. So far, it is not clear whether TORC2 also perceives
nutrient signals.
The aim of our research group is to reveal the molecularmechanisms of how Tor receives nutrient signals and how
the TORC1/2 pathways regulate each phenomenon. We havebeen studying Tor signaling in the budding yeast
Saccharomyces cerevisiae, and have found three novel
branches of the TOR signaling pathway (Figure 1).

I. TORC1 phosphorylates Atg13, the molecularswitch of autophagy.
Autophagy is mainly a response to nutrient starvation, andTORC1 negatively regulates autophagy. The Atg1 kinase andits regulators, i.e. Atg13, Atg17, Atg29, and Atg31
collaboratively function in the initial step of autophagy
induction, downstream of TORC1. Atg1 is a Ser/Thr protein
kinase, the activity of which is essential for autophagy and islargely enhanced following nutrient starvation or the additionof rapamycin. This regulation involves phosphorylation of
Atg13.
We found that Atg13 is directly phosphorylated by TORC1.
Phosphorylated Atg13 (during nutrient-rich conditions) loses
its affinity to Atg1, resulting in repression of autophagy. On
the other hand, under starvation conditions Atg13 isimmediately dephosphorylated and binds to Atg1 to form
Atg1 complex. Atg1 complex formation confers Atg1
activation and consequently induces autophagy. We
determined phosphorylation sites of Atg13 and generated an
unphosphorylatable Atg13 mutant (Atg13-8SA). Expressionof Atg13-8SA induces autophagy bypassing inactivation of
TORC1, such as starvation treatment or rapamycin. These
results demonstrate that Atg13 acts as a molecular switch for
autophagy induction.

II. Monitoring in vivo activity of TORC1 byphosphorylation state of Atg13.
Since Atg13 has turned out to be a substrate of TORC1, inv i v o a c t i v i t y o f TO R C 1 c a n b e m o n i t o r e d b y
Phosphorylation state of Atg13. Various conditions andmutants have been examined to determine what kind ofnutrients TORC1 recognizes and how nutrient signals aretransmitted to TORC1.

54

Figure 1. TOR signaling pathway of budding yeast. Our group has foundthree branches of the TOR pathway.

III. Localization of Polo-like kinase iscontrolled by TORC1 to regulate mitotic
entry.
It is well known that TORC1 regulates protein synthesis,
which is important for promotion of the cell cycle at G1(G0). Little is known, however, about whether or not TORC1
is involved in other stages of the cell cycle.
We generated a temperature-sensitive allele of KOG1
(kog1-105), which encodes an essential component of
TORC1. We found that this mutant, as well as yeast cells
treated with rapamycin, exhibit mitotic delay with prolonged
G2. We further demonstrated that this G2-arrest phenotype isdue to mislocalization and resultant inactivation of Cdc5, theyeast polo-kinase. These results suggest that TORC1
mediates G2/M transition via regulating polo-kinase.

IV. Ypk2 kinase acts at the downstream of
TORC2 to control actin organization.
Genetic studies have shown that TORC2 controls polarityof the actin cytoskeleton via the Rho1/Pkc1/MAPK cell
integrity cascade. However, the target (substrate) of TORC1
was not yet identified. We found that Ypk2, an AGC-type ofprotein kinase whose overexpression can rescue lethality of
TORC2 dysfunction, is directly phosphorylated by TORC2.

Publication List
〔Review article〕
D.J., Abdalla, F.C., Abeliovich, H., Abraham, R.T., Acevedo• Klionsky,
Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P.,
Aguirre-Ghiso, J.A. et al. (2012). Guidelines for the use andinterpretation of assays for monitoring autophagy. Autophagy 8, 445544.

LABORATORY OF BIOLOGICAL DIVERSITYOHNO Group
Assistant Professor:

OHNO, Kaoru

The aim of this laboratory is to research reproductive
hormones in invertebrates and to analyze the mechanisms bywhich they work. The comparisons of such molecules andmechanisms in various species are expected to provide
insights into the evolution of reproductive hormone systems.

I. Gonadotropins in the starfish, Asterina
pectinifera
Gonadotropins play important regulatory roles in
reproduction in both vertebrates and invertebrates. The
vertebrate gonadotropins, LH and FSH are structurally andfunctionally conserved across various species, whereas nosuch molecule has been identified in invertebrates. The insect
parsin hormones are assumed to be the physiologicalcounterpart of LH and FSH in mammals. Some gonadotropic
hormones, such as the egg development neurosecretory
hormone of the mosquito, the egg-laying hormone of the sea
hare, and the androgenic gland hormone of the terrestrial
isopod, have been found in invertebrate species. Morerecently, an insulin-like peptide was reported to be
responsible for the regulation of egg maturation in themosquito, Aedes aegypti, demonstrating the involvement of
insulin signaling in egg maturation among invertebrates.
The gonad-stimulating substance (GSS) of an echinoderm,the starfish, was the very first gonadotropin to be identified
in invertebrates. GSS mediates oocyte maturation in starfish
by acting on the ovary to produce the maturation-inducing
hormone (MIH), 1-methyladenine, which in turn induces thematuration of the oocytes. In this sense, GSS is functionallyidentical to vertebrate LH, especially piscine and amphibian
LHs, acting on the ovarian follicle cells to produce MIH to
induce the final maturation or meiotic resumption of theoocyte. Considering the functional similarity that GSS shareswith vertebrate LH, it is very important from an evolutionary
point of view to know the chemical and molecular structureof GSS. We cloned the gene encoding GSS referred to aminoacid sequence of purified GSS from radial nerves of the
starfish, Asterina pectinifera. Interestingly, phylogenetic
analyses revealed that it belonged to the insulin/insulin-likegrowth factor (IGF)/relaxin superfamily and, more precisely,
to the subclass of relaxin/insulin-like peptides (Figure 1).

Figure 1. Amino acid sequence of starfish GSS. (A) Comparison of theheterodimeric structure of starfish GSS with those of various
representative members of the insulin superfamily. The cysteine bridges
are shown in red. (B) Coding DNA sequence and predicted amino acidsequences of GSS. Sequences of A and B chains are shown in green andblue boxes, respectively. Characters shown in red boldface indicate basic
dipeptides that are the sites of proteolytic cleavage. Inverted triangle
shows the deduced cleavage site of the signal peptide.

II. Search for reproductive hormones ininvertebrates
In a collaborative effort with Prof. Yoshikuni’s Laboratoryof the Kyushu Univ. and Dr. Yamano and Dr. Awaji of the
National Research Institute of Aquaculture, Fisheries
Research Agency (NRIA), we are searching for reproductive
hormones in invertebrates; sea urchin, sea cucumber, oyster,and shrimp. While the collaborators are partially purifying
physiological materials which induce egg maturation fromnerve extracts and analyzing them with a tandem mass
spectrometer, we are creating and analyzing EST libraries
from nerve tissues and developing a database of the mass
analysis performed in this laboratory.

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYHOSHINO Group
Assistant Professor:
Technical Assistant:

HOSHINO, Atsushi
WATANABE, Seiko
NAKAMURA, Ryoko
TAKEUCHI, Tomoyo

While genomic structures as well as their genetic
information appear to stably transmit into daughter cells
during cell division, and also into the next generation, they
can actually vary genetically and/or epigenetically. Suchvariability has had a large impact on gene expression andevolution. To understand such genome dynamisms ineukaryotes, especially in plants, we are characterizing theflower pigmentation of morning glories.

I. Flower pigmentation patterns of themorning glories
Morning glories belong to the genus Ipomoea that is thelargest group in the family Convolvulaceae. Of these, I. nil(Japanese morning glory), I. purpurea (the common morning
glory), and I. tricolor have been domesticated well as
floricultural plants, and their various spontaneous mutations
have been isolated. The wild type morning glories produce
flowers with uniformly pigmented corolla, whereas a numberof mutants displaying particular pigmentation patterns havebeen collected (Figure 1). Because flower pigmentation
patterns are easily observed, the molecular mechanisms
underlying these phenomena provide fine model systems forinvestigating genome variability.
Margined, Rayed and Blizzard of I. nil are dominant
mutations. While these mutants show distinct flower
pigmentation patterns, the same pigmentation gene isrepressed by non-coding small RNA in the whitish parts ofthe corolla. It is suggested that distinct regulation of small
RNA cause the difference in pigmentation patterns. The
recessive mutations, duskish of I. nil and pearly-v of I.
tricolor, confer variegated flowers, and epigenetic
mechanisms are thought to regulate flower pigmentation. We
are currently characterizing detailed molecular mechanismsof these mutations.

II. de novo sequencing of Japanese morning
glory genome
Although morning glories are studied worldwide, especiallyin plant physiology and genetics, no whole nuclear genome
sequences of any Ipomoea species are available. To facilitate
the studies of our group as well as all morning glory
researchers, we are conducting de novo genome sequencingof I. nil, having a genome of about 800 Mbp. We chose theTokyo-kokei standard line for genome sequencing, andemployed not only shotgun sequencing using highthroughput DNA sequencers but also BAC end sequencing.
We are collaborating with several laboratories in Japan.

III. BioResource of morning glories
NIBB is the sub-center for National BioResource Project(NBRP) for morning glory. In this project, we are collecting,
maintaining and distributing standard lines, mutant lines forflower pigmentation, and DNA clones from EST and BAC
libraries of I. nil and its related species. I. nil has been one of
the most popular floricultural plants since the late Edo era inJapan. It has extensive history of genetic studies and also has
many advantages as a model plant; simple genome, large
number of mutant lines, and efficient self-pollination. Ourcollection includes 220 lines and 117,000 DNA clones.

Figure 2. Novel spontaneous mutants isolated in NBRP. From left toright: delayed leaf senescence, morphological alteration of flowers andleaves, flower closure failing, and bushy.

Publication List
〔Original papers〕
K.I., and Hoshino, A. (2012). A WD40-repeat protein controls• Park,proanthocyanidin and phytomelanin pigmentation in the seed coats of•

Figure 1. Flower phenotypes of the morning glories.

56

the Japanese morning glory. J. Plant Physiol. 169, 523-528.
Tong, L., Fukuoka, H., Otaka, A., Hoshino, A., Iida, S., Nitasaka, E.,
Watanabe, N., and Kumoyama, T. (2012). Development of EST-SRR
markers of Ipomoea nil. Breed. Sci. 62, 99-104.

An active nonautonomous DNA transposon, nDart1-0,
belonging to the hAT superfamily, was identified. The
transpositions of nDart1-0 were promoted by an active
autonomous element, aDart1-27, on chromosome 6. By
using the endogenous nDart1/aDart1-27 system in rice, alarge-scale nDart-inserted mutant population could be easily
generated under normal field conditions, and the resulting
tagging lines were free of somaclonal variation. The nDart1
transposons tend to insert into the promoter, 5’ UTR region,
or exon of a gene, which suggests that the nDart1/aDart1-27
system is a powerful tool for rice functional genomics.
Furthermore, we are developing several indica lines bearing
the active nDart1/aDart1-27 system. These lines would
effectively contribute to gene functional analysis andbreeding for the indica rice varieties.

methylation status (Figure1). The aDart1-27 sequence
showed conservation between pyl-stb (pyl-v derivative line)
and Nipponbare, which both lack autonomous activity fortransposition of nDart1-3 subgroup elements. In pyl-v plants,
the promoter region of the aDart1-27 transposase gene wasmore hypomethylated than in other rice lines. Treatment withthe methylation inhibitor 5-azacytidine (5-azaC) induced
transposition of nDart1-3 subgroup elements in both pyl-stb
and Nipponbare plants; the new insertion sites were
frequently located in genic regions. 5-azaC treatment
principally induced expression of Dart1-34 transposaserather than the other 38 aDart1-related elements in both pylstb and Nipponbare treatment groups. Our observations showthat transposition of nDart1-3 subgroup elements in the
nDart1/aDart1-tagging system is correlated with the level of
DNA methylation. Our system does not cause somaclonalvariation due to an absence of transformed plants, offers thepossibility of large-scale screening in the field, and canidentify dominant mutants. We, therefore, propose that thistagging system provides a valuable addition to the tools
available for rice functional genomics.

Activation and Epigenetic Regulation of DNA
Transposon nDart1 in Rice

Publication List

LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:
Visiting Scientist:

TSUGANE, Kazuo
HAYASHI-TSUGANE, Mika

A large part of the rice genome is composed oftransposons. Since active excision/reintegration of thesemobile elements may result in harmful genetic changes,
many transposons are maintained in a genetically or
epigenetically inactivated state. However, some nonautonomous DNA transposons of the nDart1-3 subgroup,
including nDart1-0, actively transpose in specific rice lines,
such as pyl-v which carries an active
autonomous element, aDart1-27, on chromosome 6.
Although nDart1-3 subgroup elements show considerable
sequence identity, they display different excision frequencies.
The most active element, nDart1-0, had a low cytosine〔Original paper〕
C.-H., Takagi, K., Park, K.I., Maekawa, M., Iida, S., and Tsugane,• Eun
K. (2012). Activation and epigenetic regulation of DNA transposon
nDart1 in rice. Plant Cell Physiol. 53, 857-868.

〔Review article〕
H., Tsugane, K., Kanno, T., and Nishimura, T. (2012). DNA• Saze,methylation in plants: Relationship with small RNAs and histone
modifications, and functions in transposon inactivation. Plant Cell
Physiol. 53, 766-784.

Figure 1. Bisulfite sequencing of nDart1-3 subgroup elements in rice. (a) Methylation patterns of active nDart1-0 and inactive nDart1-7 elements in pylstb plants. Red, black and blue vertical lines indicate CG, CHG, and CHH, respectively. The top (upper regions) and bottom (lower regions) strands areindicated in the nDart1-0 and nDart1-7 boxes. (b) Summary of the bisulfite sequencing analysis of nDart1-3 subgroup elements in rice. Open and closed
circles define the hypomethylation or hypermethylation state of 10 sequenced clones, respectively.

57

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Assistant:

JOHZUKA, Katsuki
ISHINE, Naomi

Chromosome condensation is a basic cellular process that
ensures the faithful segregation of chromosomes in bothmitosis and meiosis. This process is required not only forshrinking the length of chromosome arms, but also for
resolving entanglements between sister-chromatids that are
created during DNA replication. Any abnormality in thisprocess leads to segregation errors or aneuploidy, resulting incell lethality. Studies in the past decade have demonstrated
that chromosome condensation is mainly achieved bycondensin, a hetero-pentameric protein complex, widelyconserved from yeast to humans. Despite its conservationand importance for chromosome dynamics, how condensin
works is not well understood. Recent studies reveal thatcondensin functions are not restricted to chromosome
condensation and segregation during cell divisions. It is
required for diverse DNA metabolism, such as genome
stability, transcriptional regulation, and cell differentiation.
Our research interest is to understand the mechanisms andregulation of chromosome condensation. We have been
studying the role of condensin in the budding yeast
Saccharomyces cerevisiae. Microscopic observation
indicated the nucleolar localization of condensin. Consistent
with this, the ribosomal RNA gene (rDNA) repeat is the mostcondensed region in the genome during mitosis. We havefound that condensin specifically binds to the RFB site
located within the rDNA repeat. By genetic screening, we
further discovered the multiple proteins interaction network
recruits condensin to the RFB site.

chromosome organization, we have constructed a strain inwhich two RFB sites are inserted on an ectopic chromosome
arm at an interval of 15kb in the cell with complete deletionof chromosomal rDNA repeat. Using this strain, condensindependent chromatin interaction between two RFBs was
examined by chromosome conformation capture (3C) assay.
We found condensin-dependent chromatin interaction
between the two RFB sites on the chromosome arm. This
chromatin interaction is already observed in interphase cells,
and its frequency increased in metaphase arrested cells.
These results indicate that condensin plays a role inchromatin interaction between condensin binding sites andthis interaction lead to creation of a chromatin loop between
those sites (Figure 2). It is thought that condensin-dependent
chromatin folding is one of basic molecular processes ofchromosome condensation. In addition, condensin-dependent
chromatin folding observed during interphase cells suggestthat other unknown reaction(s) are necessary for mitotic
chromosome condensation, in addition to simple chromatinfolding.

I. Dynamic relocalization of condensin duringmeiosisOur genetic screening indicated that two proteins, Csm1
and Lrs4, were required for condensin recruitment to the
RFB site. Physical interactions between Csm1/Lrs4 andsubunits of condensin are important for recruitment ofcondensin to the RFB site. These proteins are known ascomponents of monopolin complex that are required forfaithful segregation of homologous chromosomes during
meiotic division I. During meiosis I, monopolin complex
re-localizes from rDNA repeat to the centromere and acts for
ensuring sister-chromatid co-orientation. Re-localization of
Csm1/Lrs4 proteins suggested the re-localization ofcondensin from rDNA repeat to centromere. As expected,
chromatin-IP experiments indicated that condensin
re-localizes to the centromere during meiosis I. Condensin
might clamp sister-chromatids together during meiosis I.

II. Condensin-dependent chromatin folding
The RFB site, which consists of a ~150bp DNA sequence,
is functioning as a cis-element recruitment of condensin onto
chromatin in the yeast genome. If the RFB site is inserted
into an ectopic chromosomal locus, condensin can associatewith the ectopic RFB site. To explore the role of condensin in

58

Figure 1. A Schematic model of chromosome condensation. Condensin
makes chromatin interactions between adjacent binding sites (RFB, forexample). This lead to a folding of chromatin fiber between the sites, as abasic process of chromosome condensation.

LABORATORY OF BIOLOGICAL DIVERSITYWATANABE Group †Assistant Professor:
Technical Assistant:

WATANABE, Takaaki
YONEZAWA, Harumi

Genomes have been dynamically evolving and arecontinually changing during development, and throughdiseases and environmental stress. One type of genome
alteration, gene amplification, is involved in various
biological phenomena, such as malignant progression ofcancer, resistance to insecticides and anticancer drugs, andgene evolution. We are addressing the molecular mechanisms
underlying gene amplification from a variety of perspectives.

I. Model systems for studying mechanisms forgene amplification
Long series of studies have shown that DNA double-strand
breaks and inverted repeats play an important role in gene
amplification. However, details of the molecular mechanisms
remain to be determined. This is because previous
approaches to understanding the mechanisms were based on
the structural analysis of complex end products and very few
model systems are available that allow chromosomal
engineering and genetic analysis.
To better understand the molecular mechanisms, we havedeveloped a new approach in which we design amplification
processes and test whether the processes can produce theamplification seen in nature. Previously, we constructed asystem designed to induce a rapid amplification mode,
double rolling-circle replication, (DRCR, Figure 1A) via
chromosomal breaks induced by site-specific endonuclease(EMBO J, 2005). This system produced intra-/extrachromosomal products resembling those seen in mammaliancells; homogeneously staining regions (HSR) and double
minutes (DMs). This result strongly suggested that
amplification in mammalian cells involves DRCR.
We next examined whether recombinational processes
coupled with replication can induce gene amplification via
DRCR, using a distinct process, Cre-lox site-specificrecombination. Here, we inferred that, if Cre recombination
coupled with replication occurs, the replication fork makes
an additional copy of the replicated region (Figure 1B); andthat the processes from two pairs of lox sites could induce
DRCR (Figure 1C). In this study, we successfully detected
HSR/DM-type amplification products in yeast and Chinese
hamster ovary (CHO) cells (Figure 1D and 1E). Surprisingly,
over 10% of the Cre recombination-induced yeast cells
undergo gene amplification. In addition, scattered-type
products were also found (Figure 1F), which are frequently
seen in cancer cells. From these results, we reasoned that
DRCR and convergent replication are centrally involved in
the amplification of drug-resistance genes and oncogene.
This system can serve as a good model for amplification in
mammalian cells and contribute to a better understanding of
oncogene amplification and development of anticancer
strategies in future.

II. Intensive rearrangement in amplified region
In amplified chromosomal regions intensive chromosome
rearrangements are frequently observed, leading to anincrease in the gene copy number and to a decrease in size ofthe amplification unit. In oncogene amplification, thecomplex patterns of amplification generated by therearrangements are closely associated with poor prognosis incancer. Interestingly, we have observed the rearrangement inall our DRCR systems.
To explore the link between the rearrangements and the
DRCR process, we constructed a system that can turn on or
off the occurrence of DRCR, using yeast 2μ plasmid. Thissystem demonstrated that inversions, deletions, or
duplications could be intensively induced in a DRCRdependent manner. This result suggests that DRCR may
cause the rearrangements in amplification in nature. We
proposed a model in which DRCR markedly stimulates
recombinational events.

III. A s t r u c t u r a l p l a t f o r m f o r g e n eamplification
Based on our results, we now focus on a type of genomic
structure consisting of two sets of inverted repeats (IR),designated double IR. This structure is found in the humangenome and can be observed in the early stages of gene
amplification. The double IR constructed in yeast can induce
gene amplification as seen in our DRCR systems, suggestingthat double IR serves as a source or key intermediate of
DRCR amplification in nature. To investigate the molecularmechanisms underlying double IR-based amplification, we
have disrupted several genes involved in genome instability
in a yeast strain carrying double IR, and constructed double
IR in CHO cells.

Figure 1. DRCR process, recombinational process coupled withreplication, and amplification products in CHO cells.

†: This group was closed on 31 March, 2012.

59

National Institute for Basic Biology

Environmental Biology

DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY

Professor

IGUCHI, Taisen

Assistant Professor:
NIBB Research Fellow:
Postdoctoral Fellow:
Technical Staff:
Graduate Student:

Visiting Scientist:
Technical Assistant:
Secretary:

MIYAGAWA, Shinichi
OGINO, Yukiko
MIYAKAWA, Hitoshi
CHAKRABORTY, Tapas*
CHAKRABORTY, Tapas
HIRUTA, Chizue
MIYAKAWA, Hitoshi*
MIZUTANI, Takeshi
SATO, Masaru
TOYOTA, Kenji
SUMIYA, Eri
YATSU, Ryohei
ODA, Shigeto
HIRAKAWA, Ikumi
TOHYAMA, Saki
HAYASHI, Tomoko
IMAIZUMI, Taeko

Synthetic chemicals found in the environment have thecapacity to disrupt the development and function of theendocrine system in both wildlife and humans. This has
drawn public concern since many of these chemicals maybind to estrogen receptors (ERs) and evoke estrogenic
effects. Early evidence that exposure to estrogenic chemicals
during development could pose a threat to human healthcame from studies of a synthetic hormone, diethylstilbestrol(DES), which was used to prevent premature birth andspontaneous abortion. Laboratory experiments showed that
exposure of animals to sex hormones during critical windowsof perinatal life caused irreversible alterations to theendocrine and reproductive systems of both sexes. The
immune and nervous systems, bone, muscle, and the liver
were also affected. Although many of these chemicals canbind to ERs in wildlife and humans, the molecular basis forthe action of environmental estrogens remains poorly
understood. Thus, understanding the molecular mechanisms
through which environmental estrogens and sex hormones
act during critical developmental windows is essential.

I. Developmental origin of adult disease:
Perinatal estrogen exposure induces
persistent changes in reproductive tracts
The emerging paradigm of the “embryonic/fetal origins ofadult disease” provides a powerful new framework forconsidering the effects of endocrine disrupters on human andanimal health. In 1971, prenatal DES exposure was found toresult in various abnormalities of the reproductive tract inwomen. This syndrome was named the DES syndrome.
Similar abnormalities have been demonstrated inexperimental animals exposed perinatally to estrogens.
Developmental estrogen exposure in mice, for example,
induces persistent proliferation of vaginal epithelial cells. We
found that the persistent changes in the vagina in mice
exposed neonatally to estrogens result from the persistentactivation of erbBs and ERα, and sustained expression of
EGF-like growth factors. Currently, we are analyzing themethylation status in the mouse vagina using MeDIP(methylated DNA immunoprecipitation) coupled with amicroarray (MeDIP-chip). We found several differentiallymethylated or demethylated DNA profiles in neonatally
DES-exposed mouse vaginae and controls. We thus consider
that neonatal DES exposure affects DNA methylation
profiles, resulting in persistent abnormalities in mouse
reproductive organs.

Figure 2. A hypothetical model for the estrogen-independent ER
activation pathway in mouse vaginae.

II. Estrogen receptors of birds, reptiles,
amphibians and fishes

Figure 1. Scheme of estrogen-dependent and -independent vaginal
epithelial cell proliferation in mice induced by perinatal estrogen
exposure.

60

Steroid and xenobiotic receptors (SXR) have been cloned
from various animal species (fish, amphibians, reptiles, birds
and mammals) by our group and we have demonstrated
species-specific differences in their responses to various
environmental and endogenous chemicals (receptor genezoo). Thus, simple predictions of chemical effects based ondata from a few established model species are not sufficient
to develop real world risk assessments. ER and ER-likegenes have been cloned from various animal species
including rockshell, Amphioxus, lamprey, lungfish, sturgeon,
gar, roach, stickleback, mosquitofish, mangrove Rivulus,

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2012. The former title isindicated by an asterisk (*).

catshark, whale shark, Japanese giant salamander, Tokyo
salamander, newt, axolotl, toad, Silurana tropicalis,
American alligator, Nile crocodile, freshwater turtle,
Japanese rat snake, Okinawa habu, and vultures. Functional
studies showed that the Amphioxus ER sequence does notbind estrogen but Amphioxus steroid receptor and lamprey
ER exhibited ligand-dependent transactivation, proving that
invertebrate and primitive vertebrates, such as the Agnatha,
have a functional ER. We found that medaka ER subtypes
have their specific functions, and medaka, zebrafish andstickleback ERs are more sensitive to estrogen/estrogen-likechemical exposures than other fishes by reporter gene assay.
Thus, these approaches are efficient to evaluate therelationship between species and their sensitivities tochemicals.

III. Evolutionary history and functional
characterization of androgen receptor
genes in jawed vertebrates
Vertebrates show diverse sexual characteristics which are
regulated by androgens. To elucidate the evolutionary history
and functional diversification of androgen receptor (AR)genes in vertebrates, we cloned the AR cDNAs from a shark,basal ray-finned fishes (Actinopterygii), namely bichir andsturgeon (Acipenseriformes), and teleosts including a basal
teleost, arowana (Osteoglossiformes). Molecularphylogenetic analysis revealed that a gene duplication event
gave rise to two different teleost ARs (α and β) and likely
occurred in the actinopterygian lineage leading to teleosts
after the divergence of Acipenseriformes but before the splitof Isteoglossiformes. Functional analysis revealed that the
shark AR activates the target gene via androgen response
element by classical androgens. The teleost ARα showed
unique intracellular localization with a significantly higher
transactivation capacity than that of teleost ARβ. These
results indicate that the most ancient type of AR, as activatedby the classic androgens as ligands, emerged before theChondrichthyes-Osteichthyes split and the AR gene was
duplicated during a teleost-specific gene duplication event.

IV. Gene zoo and receptor zoo
We are establishing cDNA library banks and receptor genebanks of animal species including lancelet, lamprey,sturgeon, lungfish, gar, mangrove Rivulus, whale shark
catshark, Japanese giant salamander, newt, Rana rugosa,
Silurana tropicalis, Japanese rat snake, Okinawa habu,
Florida red berry turtle, American alligator, Nile crocodile,vulture and polar bear in collaboration with the University of
Pretoria, South Africa, University of Florida, Medical
University of South Carolina, San Diego Zoo, USA, and the
Asa Zoo in Hiroshima.

Figure 3. Evolutionary relationships of androgen receptor sequences.

database and developed an oligonucleotide-based DNA
microarray with high reproducibility and demonstrated theusefulness of the array for the classification of toxic
chemicals as well as for the molecular understanding ofchemical toxicity in a common freshwater organism. D.
magna reproduce asexually (parthenogenesis) when they are
in an optimal environment for food, photoperiod andpopulation density. Once environmental conditions become
sub-optimal, they alter their reproductive strategy from
asexual to sexual reproduction. Chemicals are able to affectthe sex determination of D. magna and we found thatjuvenile hormone (JH) agonists (insect growth regulators),
for example, induce the production of male offspring. The
molecular basis of environmental sex determination islargely unknown in D. magna. To understand the molecularmechanisms of this phenomenon we isolated sex
determination-related genes. Also, we have developed amethod to inject genes into D. magna embryos which willallow us to study gain- and loss-of function analyses in moredetail in this species. Using these techniques, wedemonstrated that DSX1 (double sex 1), one of the
DM-domain genes, is essential for male differentiation in D.
magna. To further explore the signaling cascade of sexual
differentiation in D. magna, a gene expression profile of

V. Sex differentiation mechanism in Daphnids
Daphnia magna has been used extensively to evaluate theorganism- and population-based responses of toxicity or
reproductive toxicity tests. These tests, however, provide no
information about the mode of action of the tested
compounds. Therefore, we applied an ecotoxicogenomic
assessment of D. magna. We established a Daphnia EST

Figure 4. Life cycle of Daphnia.

61National Institute for Basic Biology

Environmental Biology

JH-responsive genes is essential. Thus, DNA microarray
analysis has been performed in the gonads of D. magna
exposed to fenoxycarb (synthesized JH agonist widely used
as an insect growth regulator) and methyl farnesoate (JHidentified in decapods) at the critical timing of JH-induced
sex determination in D. magna. We are identifying
JH-responsive genes in the ovary. We are also identifying JH
receptor in daphnids.

John, J.A., Braun, E.L., Isberg, S.R., Miles, L.G., Chong, A.Y.,• St.Gongora,J., Dalzell, P., Moran, C., Bed’hom, B., Abzhanov, A.,

Publication List
〔Original papers〕
E.K., Ogino, Y., Iguchi, T., Barber, D.S., and Denslow,• Brockmeier,
N.D. (2012). Effects of 17β-trenbolone on Eastern and Western

•
•
•
•
•
•
•
•
•
•
•
•

62

mosquitofish (Gambusia holbrooki and G. affinis) and anal fin growthand gene expression patterns. Aquat. Toxicol. 128-129C, 163-170.
Goto, Y., Kajiwara, M., Yanagisawa, Y., Hirose, H., Yoshimi, T.,
Uemura, M., Nakano, H., Takahashi, S., Shida, Y., Iguchi, T., Takahashi,Y., and Miura, T. (2012). Detection of vertebrate-type steroid hormones
and their converting activities in the neogastropod Thais clavigera(Kster, 1858). J. Molluscan Studies 78, 197-204.
Haraguchi, R., Matsumaru, D., Nakagata, N., Miyagawa, S., Suzuki, K.,
Kitazawa, S., and Yamada, G. (2012). The hedgehog signal induced
modulation of bone morphogenetic protein signaling: an essential
signaling relay for urinary tract morphogenesis. PLoS ONE 7, e42245.
Hirakawa, I., Miyagawa, S., Katsu, Y., Kagami, Y., Tatarazako, N.,
Kobayashi, T., Kusano, T., Mizutani, T., Ogino, Y., Takeuchi, T., Ohta,
Y., and Iguchi, T. (2012). Gene expression profiles in the testis
associated with testis-ova in adult Japanese medaka (Oryzias latipes)
exposed to 17α-ethinylestradiol. Chemosphere 87, 668-674.
Kakuta, H., Tanaka, M., Chambon, P., Watanabe, H., Iguchi, T., andSato, T. (2012). Involvement of gonadotropins in the induction of
hypertrophy-hyperplasia in the interstitial tissues of ovaries inneonatally diethylstilbestrol-treated mice. Reprod. Toxicol. 33, 35-44.
Lange, A., Katsu, Y., Miyagawa, S., Ogino, Y., Urushitani, H.,
Kobayashi, T., Hirai, T., Shears, J.A., Nagae, M., Yamamoto, J.,
Ohnishi, Y., Oka, T., Tatarazako, N., Ohta, Y., Tyler, C.R., and Iguchi, T.
(2012). Comparative responsiveness to natural and synthetic estrogensof fish species commonly used in the laboratory and field monitoring.
Aquat. Toxicol. 109, 250-258.
Maekawa, T., Sakuma, A., Taniuchi, S., Ogo, Y., Iguchi, T., Takeuchi,
S., and Takahashi, S. (2012). Transforming growth factor-α mRNA
expression and its possible roles in mouse endometrial stromal cells.
Zool. Sci. 29, 377-383.
Myburgh, J.G., Huchzermeyer, F.W., Soley, J.T., Booyse, D.G.,
Groenewald, H.B., Bekker, L.C., Iguchi, T., and Guillette, L.J.Jr.
(2012). Technique for the collection of clean urine from the Nilecrocodile (Crocodylus niloticus). J. South African Vet. Assoc. 83, E1-6.
Nakajima, T., Iguchi, T., and Sato, T. (2012). Hedgehog signaling plays
roles in epithelial cell proliferation in the neonatal mouse uterus andvagina. Cell Tiss. Res. 348, 239-247.
Nakamura, T., Miyagawa, S., Katsu, Y., Mizutani, T., Sato, T., Takeuchi,T., Iguchi, T., and Ohta, Y. (2012). P21 and Notch signalings in thepersistently altered vagina induced by neonatal diethylstilbestrol
exposure in mice. J. Vet. Med. Sci. 74, 1589–1595.
Nakamura, T., Miyagawa, S., Katsu, Y., Watanabe, H., Mizutani, T.,
Sato, T., Morohashi, K.-I., Takeuchi, T., Iguchi, T., and Ohta, Y. (2012).
WNT family genes and their modulation in the ovary-independent andpersistent vaginal epithelial cell proliferation and keratinization inducedby neonatal diethylstilbestrol exposure in mice. Toxicology 296, 13-19.
Nakamura, T., Miyagawa, S., Katsu, Y., Sato, T., Iguchi, T., and Ohta, Y.
(2012). Sequential changes in expression of Wnt- and Notch-related
genes in the vagina and uterus of ovariectomized mice after estrogen
exposure. In Vivo 26, 899-906.
Oka, K., Kohno, S., Uruchitani, H., Guillette, L.J.Jr., Ohta, Y., Iguchi,T., and Katsu, Y. (2012). Molecular cloning and characterization of thecorticoid receptors from the American alligator. Mol. Cell. Endocrinol.
365, 153-161.

••

Burgess, S.C., Cooksey, A.M., Castoe, T.A., Crawford, N.G., Densmore,
L.D., Drew, J.C., Edwards, S.V., Faircloth, B.C., Fujita, M.K.,
Greenwold, M.J., Hoffmann, F.G., Howard, J.M., Iguchi, T., Janes,
D.E., Khan, S.Y., Kohno, S., de Koning, A.J., Lance, S.L., McCarthy,
F.M., McCormack, J.E., Merchant, M.E., Peterson, D.G., Pollock, D.D.,
Pourmand, N., Raney, B.J., Roessler, K.A., Sanford, J.R., Sawyer, R.H.,
Schmidt, C.J., Triplett, E.W., Tuberville, T.D., Venegas-Anaya, M.,
Howard, J.T., Jarvis, E.D., Guillette, L.J.Jr., Glenn, T.C., Green, R.E.,
and Ray, D.A. (2012). Sequencing three crocodilian genomes toilluminate the evolution of archosaurs and amniotes. Genome Biol. 13,
415.
Takase, M., Shinto, H., Takao, Y., and Iguchi, T. (2012). Accumulationand pharmacokinetics of estrogenic chemicals in the pre- and posthatch embryos of the frog Rana rugosa. In Vivo 26, 913-920.
Taylor, J.A., Richter, C.A., Suzuki, A., Watanabe, H., Iguchi, T., Coser,
K.R., Shioda, T., and vom Saal, F.S. (2012). Dose-related estrogeneffects on gene expression in fetal mouse prostate mesenchymal cells.
PLoS ONE 7, e48311.

〔Original paper (E-publication ahead of print)〕
I., Hirakawa, I., Miyagawa, S., Mitsui, N., Miyahara, M.,• Hirakawa,
Onishi, Y., Kagami, Y., Kusano, T., Takeuchi, T., Ohta, Y., and Iguchi, T.
Developmental disorders and altered gene expression in the tropicalclawed frog (Silurana tropicalis) exposed to 17α-ethinylestradiol. J.
Appl. Toxicol. 2012 Nov 6.

〔Review articles〕
E.G., Nishioka, R.S., Bern, A., Hirano, T., Borski, R., Clarke, C.,• Grau,Foskett, K., Guillette, L.J., Iguchi, T., Jones, L.A., Loretz, C.,•••

McCormick, S., MacLachlan, A., Mason, C.A., Mills, K.T., Nagahama,Y., Nicoll, C.S., Richman, N.H., Sheridan, M., Specker, J.L., Sullivan,
J.J., and Young, G. (2012). In memory of Professor Howard A. Bern.
Gen. Comp. Endocrinol. 176, 121-123.
Guillette, L.J.Jr., and Iguchi, T. (2012). Life in a contaminated world.
Science 337, 1614-1615.
Miyagawa, S., Yatsu, R., Sudo, T., Igarashi, K., Kanno, J., and Iguchi,
T. (2012). Irreversible effect of diethylstilbestrol on reproductive organs
and current approach for epigenetic effects of endocrine disrupting
chemicals. In: Toxicology and Epigenetics. Ed. Sahu, S.C., John Wiley& Sons, Ltd., pp. 357-364.
Tatarazako, N., and Iguchi, T. (2012). Evaluation of toxicities ofherbicides using short-term chronic tests of alga, daphnid and fish. In:
Herbiciddes – Environmental Impact Studies and Management
Approaches. Ed. Alvarez-Fernandez, R. ISBN: 978-953-307-892-2,
InTech.

DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

MINAGAWA, Jun

NIBB Research Fellow:
Postdoctoral Fellow:

Graduate Student:
Technical Assistant:
Secretary:

TAKIZAWA, Kenji
KAMADA, Konomi
OHNISHI, Norikazu
TOKUTSU, Ryutaro
AIHARA, Yusuke
KAMRANI, Yousef YariHOSHI, Rie
YONEZAWA, Harumi
KOJIMA, Yoko

Plants and algae have a large capacity to acclimate
themselves to changing environments. We are interested inthese acclimation processes, in particular, how efficiently yet
safely they harness sunlight for photosynthesis under thechanging light environment. Using a model green alga, weare studying the molecular mechanisms underlying thephotoacclimation of the photosynthetic machinery. We arealso applying the knowledge obtained in the studies of amodel green alga to various phytoplankton including diatoms
in the subarctic North Pacific, prasinophytes in thesubtropical Mediterranean Sea, and Symbiodinium in corals
in tropical oceans, to explore how these environmentally
important photosynthetic organisms thrive in their ecologicalniche.

I. Macro-organization of photosynthetic
machinery
Photosystem (PS) II is a multiprotein complex that splits
water and initiates electron transfer in photosynthesis. The
central part of PSII, the PSII core, is surrounded by lightharvesting complex II proteins (LHCIIs). In higher plants,
two or three LHCII trimers are seen on each side of the PSII
core whereas only one is seen in the corresponding positions
in a unicellular green alga Chlamydomonas reinhardtii. This

year, we re-examined the supramolecular organization of the
C. reinhardtii supercomplex by determining the effect ofdifferent solubilizing detergents. When we solubilized thethylakoid membranes with n-dodecyl-β-D-maltoside or
n-dodecyl-α-D-maltoside and subjected them to thethylakoid membranes with n-dodecyl-β-D-maltoside gelfiltration, we observed a clear difference in molecular mass.
The n-dodecyl-α-D-maltoside-solubilized PSII-LHCII
supercomplex bound twice as much LHCII than the
n-dodecyl-β-D-maltoside-solubilized supercomplex andretained higher oxygen-evolving activity. Single-particle
image analysis from electron micrographs of the n-dodecylα-D-maltoside-solubilized and negatively stained
supercomplex revealed that the PSII-LHCII supercomplex
had a novel supramolecular organization, with three LHCII
trimers attached to each side of the core.

II. Acclimation of photosynthesis
Using C. reinhardtii, we investigated the molecularmechanisms underlying the acclimation processes of thephotosynthetic complexes by means of biochemistry,
molecular genetics, absorption and fluorescence
spectroscopy, and bio-imaging.

II-1 State-transitions
The two photosystems—PSI and PSII—in the thylakoid
membranes function as charge-separation devices. Each has adistinct pigment system with distinct absorption
characteristics and a distinct action spectrum. Thus, animbalance of energy distribution between the two
photosystems tends to occur in natural environments, wherelight quality and quantity fluctuate with time. Since the two
photosystems are connected in series under normal
conditions, green plants and algae need to constantly balance
their excitation levels to ensure optimal efficiency of electron
flow. State transitions occur under such conditions toredistribute the harnessed energy to minimize its unequal
distribution.
Although state transitions have been widely accepted as ashort-term response in plants to acclimate to the fluctuating
light conditions, most of the previous investigations wereconducted in vitro, implying that the real impact on
photosynthesis remains to be characterized. We visualized
phospho-LHCII dissociation during state transitions using
fluorescence lifetime imaging microscopy (FLIM) for the
first time in vivo, where the fluorescence lifetime in live C.
reinhardtii cells was monitored under a fluorescencemicroscope during a transition from State 1 to 2. Single-cell
FLIM further indicated that the dissociated LHCII spreads
through the cell during State 2 transitions and forms several
large spotted areas. Further biochemical analyses indicatedthat dissociated phospho-LHCII formed a large aggregatedstructure, whereas unphosphorylated LHCII did not. Thus,
the free phospho-LHCII aggregates appearing during State 2transitions are in energy-dissipative form.

1I-2 Cyclic electron flow
Figure 1. A top view of the PSII-LHCII supercomplex in C. reinhardtii as
revealed by single particle analysis of electron micrographs.

In eukaryotes, photosynthesis is a process ofphotochemical energy transduction, which occurs via the63National Institute for Basic Biology

Environmental Biology

conductance of electron flow in the thylakoid membranes ofchloroplasts, resulting in the reduction of NADP+ in thestroma and the concomitant generation of a proton motive
force across the membranes. The NADPH generated by theelectron flow and the ATP synthesized by ATP synthaseutilizing the proton motive force are used to fix carbon
dioxide in the Calvin-Benson cycle. Linear electron flow(LEF) and cyclic electron flow (CEF) are known as modes ofelectron flow in photosynthesis. In the linear pathway,electrons are transferred from PSII to NADP+ by way of thecytochrome b6f complex (Cyt b6f) and PSI. In the cyclic
pathway, however, the exact pathway of electrons thatoriginate in PSI and then return to PSI has not been clear.
State transitions have long been considered as a mechanism
by which the distribution of light excitation between the two
photosystems is regulated. However, the performance of PSI
tends to overwhelm PSII under State 2 conditions in C.
reinhardtii because of its extensive ability to relocate LHCII
proteins; this implies that state transitions might represent amechanism by which the electron transfer chain in thethylakoid membranes is switched to the mechanism
exclusively employed by PSI.
We solubilized thylakoid membranes from C. reinhardtii
cells under State 2 conditions and loaded them onto a sucrose
density gradient. A “super-supercomplex” (CEF
supercomplex) with a molecular weight of approximately 1.5
million composed of the PSI-LHCI supercomplex with
LHCIIs, Cyt bf, Fd-NADPH oxidoreductase (FNR), and theintegral membrane protein PGRL1 was detected in a fraction
heavier than the PSI-LHCI supercomplex. Spectroscopic
analyses indicated that upon illumination, reducing
equivalents downstream of PSI were transferred to Cyt bf,
while the oxidized PSI was re-reduced by reducing
equivalents from Cyt bf, indicating that this supercomplex isengaged in CEF. Thus, CEF takes place in a protein
supercomplex where steps in LEF are rearranged to undergo
an alternative pathway for the flow of electrons.

studying the functional evolution of light-harvesting systems
in photosynthetic eukaryotes.
We isolated and characterized O. tauri pigment-protein
complexes to understand the diversity and the evolutional
traits of the light-harvesting systems in a primitive green
alga. Two PSI fractions were obtained by sucrose density
gradient centrifugation in addition to free LHC fraction and
PSII core fractions. The smaller PSI fraction contained thePSI core proteins, LHCI, which are conserved in all green
plants, Lhcp1, a prasinophyte-specific LHC protein, and theminor, monomeric LHCII proteins CP26 and CP29. The
larger PSI fraction contained the same antenna proteins as
the smaller, with the addition of Lhca6 and Lhcp2, and a
30% larger absorption cross-section. When O. tauri wasgrown under high-light conditions, only the smaller PSI
fraction was present. The two PSI preparations were also
found to be devoid of far-red chlorophyll fluorescence (715730 nm), a signature of PSI in oxygenic phototrophs. These
unique features of O. tauri PSI may reflect primitive lightharvesting systems in green plants and their adaptation tomarine ecosystems.
Our newest project is the study of photoacclimation of
Symbiodinium, which live in a symbiotic relationship withcorals, and other Cnidarians. We are particularly interested
in those living with corals and are trying to elucidate how
their photosynthetic machinery is acclimated to the variable
light and temperature environments in the tropical ocean.

Figure 3. Evolutionary model of LHC affinity in photosynthetic
eukaryotes as revealed by biochemical study of the LHC systems in O.
tauri.
Figure 2. Cyclic electron flow by way of the CEF supercomplex.

Publication List

64

III. Ecophysiology of marine phytoplankton〔Original paper〕

Prasinophyceae are a broad class of early-branching
eukaryotic green algae. These picophytoplankton are foundubiquitously throughout the ocean and contributeconsiderably to global carbon-fixation. Ostreococcus tauri,as the first sequenced prasinophyte, is a model species forR., Kato, N., Bui, K. H., Ishikawa, T., and Minagawa, J.
• Tokutsu,(2012). Revisiting the supramolecular organization of photosystem II in
Chlamydomonas reinhardtii. J. Biol. Chem. 287, 31574-31581.

National Institute for Basic Biology

Theoretical Biology

LABORATORY OF GENOME INFORMATICS

Assistant Professor

UCHIYAMA, Ikuo

Postdoctoral Fellow:

CHIBA, Hirokazu

The accumulation of biological data has recently been
accelerated by various high-throughput “omics” technologies
including genomics, transcriptomics, and proteomics. The
field of genome informatics is aimed at utilizing this data, or
finding the principles behind the data, for understanding
complex living systems by integrating the data with current
biological knowledge using various computational
techniques. In this laboratory we focus on developing
computational methods and tools for comparative genome
analysis, which is a useful approach for finding functional orevolutionary clues to interpreting the genomic information ofvarious species. The current focus of our research is on thecomparative analysis of microbial genomes, the number ofwhich has been increasing rapidly. Since different types of
information about biological functions and evolutionary
processes can be extracted from comparisons of genomes atdifferent evolutionary distances, we are developing methods
to conduct comparative analyses not only of distantly related
but also of closely related genomes.

I. Microbial genome database for comparative
analysis
The microbial genome database for comparative analysis(MBGD; URL http://mbgd.genome.ad.jp/) is acomprehensive platform for microbial comparative
genomics. The central function of MBGD is to create
orthologous groups among multiple genomes fromprecomputed all-against-all similarity relationships using the
DomClust algorithm (see Section II below). By means of thisalgorithm, MBGD not only provides comprehensive
orthologous groups among the latest genomic data available,
but also allows users to create their own ortholog groups on
the fly using a specified set of organisms. In addition,
MBGD also provides MyMBGD mode, which allows users
to add their own genomes to MBGD.
The database now contains more than 2000 publishedgenomes including 34 eukaryotic microbes and 4
multicellular organisms. This year, we substantially enhanced
the database functionality to explore the diversity of
microbial genomes. The enhancement includes: 1) To
efficiently explore the diversity of the microbial genomic
data, MBGD now provides summary pages for precalculated ortholog tables among various taxonomic groups;
2) For some closely related taxa, MBGD also provides theconserved synteny information (core genome alignment) precalculated using the CoreAligner program (see Section III
below); 3) An efficient incremental updating procedure was
implemented to create extended ortholog tables by adding
additional genomes to the default ortholog table generated
from the representative set of genomes (see Section II
below).

II. I m p r o v e m e n t o f t h e m e t h o d s f o r
constructing orthologous groups among
multiple genomes
As a core technology of our comparative genomics tools,
we have developed a rapid automated method of ortholog
grouping, named DomClust, which is effective enough toallow the comparison of many genomes simultaneously. The
method classifies genes based on a hierarchical clustering
algorithm using all-against-all similarity data as an input, but
it also detects domain fusion or fission events and splits
clusters into domains if required. As a result, the procedure
can split genes into the domains minimally required forortholog grouping.
Although DomClust can rapidly construct orthologous
groups, its classification quality has room for improvement
since it uses UPGMA like clustering based on pairwise
sequence alignment. We are now developing a procedure torefine the DomClust classification based on multiple
sequence alignments and phylogenetic trees. Since DomClust
constructs orthologous groups at the domain level, theprocedure refines not only sequence grouping but also
domain splitting. We developed a simple scoring system toidentify the most plausible domain boundary or to merge
multiple domains into one group by maximizing the sum of
the alignment scores of domains under this scoring system(Figure 1). We tested the scoring system using the COG
database as a reference and confirmed that changes that
increase the score generally improve the agreement with the
COG clusters. After determining domain boundaries, ourprocedure also checks whether the resulting group should be
split into subgroups by considering species overlaps between
subclusters in the phylogenetic tree (Figure 1).
We are also developing a method to update the clusteringresult incrementally, by which we can add new genomes to areference set of ortholog groups that is constructed from a
representative set of published genomes available in the
MBGD server. This approach allows us to conduct further
large-scale comparative genomics based on orthologous
groups among thousands of genomic sequences. We are also
trying to extend the algorithm for handling metagenomic
data. To infer the taxonomic position of the source organism

Figure 1. Pipeline for ortholog group refinement. The pipeline consists ofseveral modules including denoise, merge, split and divide. Merge andsplit aim at improving the domain boundary based on the sequencealignment whereas divide aims at improving the subgrouping based on
the phylogenetic tree.

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National Institute for Basic Biology

Theoretical Biologyof each metagenomic sequence, we have developed a method
to map each tree node of the hierarchical clustering tree
generated by the DomClust algorithm onto a taxonomic tree
node.

III. Identification of the core structure
conserved among moderately related
microbial genomes
Horizontal gene transfers (HGT) have played a significant
role in prokaryotic genome evolution, and the genes
constituting a prokaryotic genome appear to be divided into
two classes: a “core gene pool” that comprises intrinsic genes
encoding the proteins of basic cellular functions, and a“flexible gene pool” that comprises HGT-acquired genes
encoding proteins which function under particularconditions. Thus identification of the set of intrinsicallyconserved genes, or the genomic core, among a taxonomic
group is crucial for understanding prokaryotic diversity andevolution.
Typically “core genome” is defined as a set of genes that areconserved among all the genomes belonging to the given
species (called the “universal core”). However, this definition
can be too strict since it allows no exceptions. We consider
the core structure of related genomes as a set of sufficiently
long segments in which gene orders are conserved among
multiple genomes so that they are likely to have been
inherited mainly through vertical transfer (called “synteniccore”). To find such core structures, we developed a method
named CoreAligner, which finds the order of orthologous
groups that maximally retains the conserved gene orders.
We systematically applied our method to bacterial taxa
(family, genus and species) that contain a sufficient numberof completed genomes stored in MBGD. As a result, we cangenerally obtain more core genes as syntenic core rather thanuniversal core genes, except for some taxonomic groups thathave poor syntenic conservation. Moreover, typically the
number of syntenic core is more stable than universal corewhen the number of genomes in the given taxa increases.
The core genome data calculated for various prokaryotic
taxa is now available as part of the MBGD database (Figure
2).

IV. D e v e l o p m e n t o f a w o r k b e n c h f o rcomparative genomics
We are developing a comparative genomics workbench
named RECOG (Research Environment for COmparative
Genomics), which aims to extend the current MBGD system
by incorporating more advanced analysis functionalities
including phylogenetic pattern analysis, the ingroup/
outgroup distinction in ortholog grouping and the core
structure extraction among related genomes. The entire
RECOG system employs client-server architecture: theserver program is based on the MBGD server and contains
the database construction protocol used in MBGD so thatusers can install the server on their local machines to analyze
their own genomic data, whereas the client program is a Javaapplication that runs on a local machine by receiving datafrom any available RECOG server including the public
MBGD server.
The central function of RECOG is to display andmanipulate a large-scale ortholog table. The ortholog tableviewer is a spreadsheet like viewer that can display the entire
ortholog table, containing more than a thousand genomes.
Using the zoom in/out function, it can display the entire table
or a section of the main table with more detailed information.
In RECOG, several basic operations on the ortholog table aredefined, which are classified into categories such as filtering,sorting, and coloring, and various comparative analyses canbe done by combining these basic operations, such as“Neighborhood gene clustering” and “Phylogenetic patternclustering.” In addition, RECOG allows the user to input
arbitrary gene properties such as sequence length, nucleotide/amino acid contents and functional classes, and comparedthese properties among orthologs in various genomes.

Publication List
〔Original papers〕
H., Noguchi, H., Takaki, Y., Uchiyama, I., ,Toyoda, A., Nishi,• Takami,
S., Chee, G-J., Arai, W., Nunoura, T., Itoh, T., Hattori, M., and Takai, K.

•(2012). A deeply branching thermophilic bacterium with an ancient
acetyl-CoA pathway dominates a subsurface ecosystem. PLoS ONE 7,e30559.
Yahara, K., Kawai, M., Furuta, Y., Takahashi, N., Handa, N., Tsuru, T.,
Oshima, K., Yoshida, M., Azuma, T., Hattori, M., Uchiyama, I., andKobayashi, I. (2012). Genome-wide survey of mutual homologous
recombination in highly sexual bacterial species. Genome Biol. Evol. 4,
628-640.

〔Original paper (E-publication ahead of print)〕
I., Mihara, M., Nishide, H., and Chiba, H. MBGD update• Uchiyama,
2013: the microbial genome database for exploring the diversity of
microbial world. Nucleic Acids Res. 2012 Oct 30.

Figure 2. Core genome alignment viewer in MBGD showing the corealignment of the family Bacillaceae.

66

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

Technical Staff:
Postdoctoral Fellow:
Technical Assistant:

We took advantage of light-sheet microscopy by applying
long-term observation of intact mouse embryos at
gastrulating stages for tracking cell movements, raman
imaging of living medaka, and ultra-fast 4D data acquisitionof freely moving Amoeba proteus.

KAJIURA-KOBAYASHI, Hiroko
ICHIKAWA, Takehiko
TAKAO, Daisuke
MARUYAMA, Atsushi
SHINTANI, Atsuko
ISHIBASHI, Tomoko

Our laboratory is currently pursuing two paths of scientificinquiry of interest to developmental biology. One goal is toclarify the mechanism of left-right (L-R) determination. The
other is to develop imaging technologies for biologicalanalyses.

Figure 2. Raman signal of medaka Left: Dark-field image of a living
Quintet medaka eye taken by oblique illumination. Right: Raman imageof the same area.

I. Initial step for left-right asymmetry
In mammalian development, the initial left-right asymmetry
first appears as cilia-driven leftward fluid flow on the ventral
surface of the node, and the flow provides the initial cue forasymmetric gene expressions that actually determine
asymmetric morphogenesis. The mechanism that converts
the flow to the asymmetric gene expression, i.e. the flow
sensing mechanism, remains controversial, while several
models have been proposed, and involvement of Ca2+ has
been suggested.
We pursued this question by measuring Ca2+ dynamics inthe node and found that the node cells cause apparently
stochastic elevation of Ca 2+ , and its spatiotemporal
distribution is equal at the left and right sides but becomes
more frequent at the left after late headfold stage, when theflow-induced commitment occurs. This asymmetry was
disrupted in dynein mutant iv/iv and pkd2 -/- mutants, inaccordance to their left-right phenotypes.

Figure 3. 3D image of Amoeba Proteus reconstructed from 100 opticalsections taken in 0.5 sec.

Publication List
〔Original papers〕
H., Kajiura-Kobayashi, H., Takagi, C., Yamamoto, T.S.,• Morita,
Nonaka, S., and Ueno, N. (2012). Cell movements of the deep layer of•••

non-neural ectoderm underlie complete neural tube closure in Xenopus.
Development 139, 1417-1426.
Oshima, Y., Sato, H., Kajiura-Kobayashi, H., Kimura, T., Naruse, K.,
and Nonaka, S. (2012). Light sheet-excited spontaneous Raman
imaging of a living fish by optical sectioning in a wide field Raman
microscope. Optics Express 20, 16195-16204.
Takao, D., Taniguchi, A., Takeda, T., Sonobe, S., and Nonaka, S. (2012).
High-speed imaging of amoeboid movements using light-sheet
microscopy. PLoS ONE 7, e50846.
Yoshiba, S., Shiratori, H., Kuo, I.Y., Kawasumi, A., Shinohara, K.,
Nonaka, S., Asai, Y., Sasaki, G., Belo, J.A., Sasaki, H., et al. (2012).
Cilia at the node of mouse embryos sense fluid flow for left-rightdetermination via Pkd2. Science 338, 226-231.

Figure 1. Left: Distribution of Ca2+ elevation in a 2-somite wild-type
node. Right: Time course of Ca2+ elevation frequency at the left and theright sides.

II. Development of light-sheet microscopy
A remarkable characteristic of light-sheet microscopy ishigh efficiency of light detection against illumination power.
This feature results in several advantages in live imaging:detection of weak signals, low phototxicity and bleaching,
and fast image acquisition.

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National Institute for Basic Biology

Research Support

NIBB CORE RESEARCH FACILITIES

and various applications such as genomic re-sequencing,
RNA-seq and ChIP-seq.

Head

KOBAYASHI, Satoru

The NIBB Core Research Facilities were launched in 2010
to support basic biology research in NIBB. They consist of
three facilities that are developing and providing state-of-theart technologies to understand biological functions throughfunctional genomics, bioimaging and bioinformatics.
The NIBB Core Research Facilities also act as anintellectual hub to promote collaboration among the
researchers of NIBB and other academic institutions.

Functional Genomics Facility

Associate Professor (Specially appointed)

SHIGENOBU, Shuji

Technical Staff:
Postdoctoral Fellow:
Technical Assistant:
Secretary:

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
KITAZUME, Tatsuya
ASAO, Hisayo
FUJITA, Miyako
WAKAZUKI, Sachiko
ICHIKAWA, Mariko

The Functional Genomics Facility is a division of the NIBBCore Research Facilities and organized jointly by NIBB and
NIPS for promoting DNA and protein studies. The facility
maintains a wide array of core research equipment, fromstandard machinery like ultracentrifuges to cutting edge tools
such as next generation DNA sequencers, which amount to
60 different kinds of instrument. The facility is dedicated tofostering collaborations with researchers both of NIBB andother academic institutions worldwide by providing thesetools as well as expertise. Our current focus is supporting
functional genomics works that utilize mass spectrometersand DNA sequencers. We also act as a bridge between
experimental biology and bioinformatics.

Figure 1. Next-generation sequencer SOLiD5500xl

Proteomics
Three different types of mass spectrometer and two protein
sequencers, as listed below, are used for proteome studies in
our facility. In 2012, we analyzed approximately 248
samples with mass spectrometers and 37 samples withprotein sequencers.
- GC-Mass Spectrometer (JEOL DX-300)- MALDI-TOF-MS (Bruker Daltonics REFLEX III)- LC-Q-TOF MS (Waters Q-TOF Premier)- Protein sequencer (ABI Procise 494 HT; ABI Procise 492
cLC)

Other analytical instruments- Flow Cytometer (Coulter EPICS XL)- Bio Imaging Analyzer (Fujifilm LAS 3000 mini; GE
FLA9000)- Laser Capture Microdissection System (Arcturus XT)- DNA Sequencer (ABI PRISM 310; ABI 3130xl)- Real Time PCR (ABI 7500)- Ultra Centrifuge (Beckman XL-80XP etc.)

Representative Instruments
Genomics
The advent of next-generation sequencing (NGS)
technologies is transforming today’s biology by ultra-highthroughput DNA sequencing. Utilizing the SOLiD5500xl(Applied Biosystems), HiSeq2000 (Illumina), and MiSeq(Illumina) the Functional Genomics Facility is committed tojoint research aiming to exploring otherwise inaccessible
new fields in basic biology.
During 2012 we carried out 47 NGS projects incollaboration with NIBB laboratories as well as theresearchers of other academic institutions. These projects
cover a wide range of species (bacteria, animals, plants, andhumans) including both model and non-model organisms,

68

Figure 2. LC-Q-TOF-MS

Genome Informatics Training Course
We organize NIBB Genome Informatics Training Courses
every year. In 2012, we provided two two-day training
courses on next-generation sequence data analyses andtranscriptome analysis. These courses are designed to
introduce the basic knowledge and skills of bioinformatics
analysis to biologists who are not familiar withbioinformatics.

Figure 3. NIBB Genome Informatics Training Course● Research activity by S. Shigenobu
Associate Professor (Specially appointed)

SHIGENOBU, ShujiNIBB Research Fellow:
Technical Assistant:
Visiting Scientist:

MAEDA, TaroSUZUKI, Miyuzu
OGAWA, Kota

consort with that of bacterial symbiont Buchnera aphidicola
illustrates the remarkable interdependency between the twoorganisms. Genetic capacities of the pea aphid and thesymbiont for amino acid biosynthesis are complementary.
The genome analysis revealed that the pea aphid has
undergone characteristic gene losses and duplications. The
IMB antibacterial immune pathway is missing several criticalgenes, which might account for the evolutionary success ofaphids to obtain beneficial symbionts. Lineage-specific gene
duplications have occurred in genes in a broad range offunctional categories, which include signaling pathways,
miRNA machinery, chromatin modification and mitosis. The
importance of these duplications for symbiosis remains to be
determined. We found several instances of lateral genetransfer from bacteria to the pea aphid genome. Some ofthem are highly expressed in bacteriocytes.
Aphid research is entering the post-genome era. We
analyzed the transcriptome of aphid bacteriocytes using
RNA-seq technology featuring a next-generation DNA
sequencer. We found thousands of genes over-represented in
the symbiotic organ in comparison with the whole body.
Many genes for amino acid metabolism are found to be overrepresented as expected: the plant sap-eating insect depends
on the bacterial symbionts to supply essential amino acids. In
addition, many kinds of novel secretion proteins that arefound only in aphid species are extremely enriched in thebacteriocytes. We also found that bacteriocytes express
Distal-less (Dll), a homeodomain-containing transcription
factor throughout the life cycle. Future study should focus on
dissecting the genetic network of these components, whichshould allow us to understand the genetic basis on which
symbiosis generates evolutionary novelty.

Symbiosis Genomics“Nothing, it seems, exists except as part of a network ofinteractions.” (Gilbert & Epel, 2008)
Every creature on the earth exists among a network ofvarious biological interactions. For example, many
multicellular organisms, including humans, harbor symbiotic
bacteria in their bodies: some of them provide their hosts
with essential nutrients deficient in the host’s diet and others
digest foods indigestible by the host alone. In spite ofnumerous examples of symbioses and its intriguing
outcomes, the genetic and molecular basis underlying theseinteractions remains elusive. The goal of our group is toestablish a new interdisciplinary science “Symbiosis
Genomics”, where we aim to understand the network of
biological interactions at the molecular and genetic level. To
this end, we take advantage of state-of-the-art genomics suchas next-generation sequencing technologies.

Figure 1. Pea aphids and the bacterial symbiont, Buchnera. Adult aphids(Left). A developing viviparous embryo which symbionts are infecting(Right). Scale bar = 20um.

Publication List
〔Original papers〕

I. Genomic revelations of a mutualism: the pea
aphid and its obligate bacterial symbiont

M., Maekawa, K., Saitoh, S., Shigenobu, S., Miura, T., Hayashi,• Hojo,Y., Tokuda, G., and Maekawa, H. (2012). Exploration and

Aphid species bear intracellular symbiotic bacteria in thecytoplasm of bacteriocytes, specialized cells for harboring
the bacteria. The mutualism is so obligate that neither can
reproduce independently. The newly released 464 Mb draft
genome sequence of the pea aphid, Acyrthosiphon pisum, incharacterization of genes involved in the synthesis of diterpene defence
secretion in nasute termite soldiers. Insect Mol. Biol. 21, 545-557
Gallot, A., Shigenobu, S., Hashiyama, T., Jaubert-Possamai, S., andTagu, D. (2012). Sexual and asexual oogenesis require the expression ofunique and shared sets of genes in the insect Acyrthosiphon pisum.
BMC Genomics 13, 76•

69

National Institute for Basic Biology

Research Support〔Original paper (E-publication ahead of print)〕
S., and Stern, D. Aphids evolved novel secreted proteins for• Shigenobu,
symbiosis with bacterial endosymbiont. Proc. Royal Soc. B: Biol. Sci.
2012 Nov 21.

Spectrography and Bioimaging Facility

Associate Professor (Specially appointed)

KAMEI, Yasuhiro

Technical Staff:
Technical Assistant:
Secretary:

HIGASHI, Sho-ichi
TANIGUCHI-SAIDA, Misako
ICHIKAWA, Chiaki
ISHIKAWA, Azusa

The Spectrography and Bioimaging Facility assists bothcollaborative and core research by managing and maintaining
research tools that use “Light”. The facility also provides
technical support through management of technical staff
assisting in the advancement of collaborative and coreresearch projects, as well as academic support to researchers.
Among its tools are advanced microscopes for biology andthe Okazaki Large Spectrograph for photobiology. The
Okazaki Large Spectrograph is the world’s largest wide
spectrum exposure mechanism, capable of producing a rangeof wavelengths from 250 nm (ultraviolet) to 1,000 nm(infrared) along its 10 meter focal curve; allowing exposure
to strong monochromatic light. The facility’s microscopes,
which are cutting edge devices such as confocal and multiphoton excitation microscopes, are used by both internal andexternal researchers as vital equipment for core andcollaborative research projects.

Representative Instruments:
Okazaki Large Spectrograph (OLS)
The spectrograph runs on a 30 kW Xenon arc lamp andprojects a wavelength spectrum from 250 nm (ultraviolet) to
1,000 nm (infrared) onto its 10 m focal curve with anintensity of monochromatic light at each wavelength morethan twice as much as that of the corresponding
monochromatic component of tropical sunlight at noon
(Watanabe et al., Photochem. Photobiol. 36, 491-498, 1982).
The spectrograph is dedicated to action spectroscopicalstudies of various light-controlled biological processes.
The NIBB Collaborative Research Program for the Use of
the OLS supports about 10 projects every year conducted byboth visiting scientists, including foreign researchers, as wellas those in NIBB.
Action spectroscopical studies for various regulatory anddamaging effects of light on living organisms, biologicalmolecules, and artificial organic molecules have been
conducted.

Microscopes
This facility also has Bioimaging machines such as
widefield microscopes (Olympus IX-81, BX-63 and
KEYENCE BZ-8000), confocal microscopes (Olympus
FV1000, Leica TCS SP2, Nikon A1R, Nikon A1Rsi, Carl
Zeiss Duo 5 and Yokogawa CSU-X1) and other advanced
custom-made laser microscopes for special aims (Digital
Scanned Light-sheet Microscope: DSLM and Infrared LaserEvoked Gene Operator microscope: IR-LEGO) for users in
NIBB and collaborative guest researchers. We began
Collaborative Research Programs using these machines since2010.
The DSLM was developed by Dr. Ernst Stelzer’s group at
the European Molecular Biology Laboratory (EMBL). This
microscope can realize high-speed z-axis scanning in deeper
tissue by illuminating from the side of a specimen with alight sheet (more information is described in Dr. Nonaka’s
section: Lab. for Spatiotemporal Regulations). Dr. Nonaka
conducted and supported about 7 projects of the
Collaborative Research Program for the Use of the DSLM.
On the other, the IR-LEGO was developed by Drs. Shunsuke
Yuba and Yasuhiro Kamei at the National Institute ofAdvanced Industrial Science and Technology (AIST). This
microscope can induce a target gene of interest by heating asingle target cell in vivo with a high efficiency irradiating
infrared laser (Kamei et al. Nat. Methods, 2009). Details aredescribed in the next section. The IR-LEGO was also used
for about 10 Individual Collaborative Research projects,
including applications for animals and higher plant.

Workshop and Symposium
In 2012, we held workshops (training course) on IR-LEGO
for plants (Arabidopsis) and fish (medaka) in Japan andSingapore (as a joint workshop by NIBB, the National
University of Singapore, and Temasek Lifesciences
Laboratory) respectively. We also have been holding a“Bioimaging Forum” every year which discusses Bioimaging
from various directions such as microscopy, new phototechnology, and computer science. In 2012, we held the 6th
and 7th forums which focused on all imaging sciences, fromastronomy to biology, and optogenetics and adaptive optics,
respectively.
Figure 1. An example of experiments using the Large Spectrograph.
Various color rays (monochromatic light from right side and reflected bymirrors) were irradiated simultaneously to samples in cooling chambers.

70

Publication List on Cooperation〔Original papers〕
S., Eun, C-H., Ono, A., Asao, H., Okano, Y., Yamaguchi, K.,• Moritoh,
Shimatani, Z., Koizumi, A., and Terada, R. (2012). Targeted disruption

•
•
•
•

of an orthologue of DOMAINS REARRANGED METHYLASE 2,OsDRM2, impairs the growth of rice plants by abnormal DNA
methylation. Plant J. 71, 85-98.
Satoh, C., Kimura, Y., and Higashijima, S. (2012). Generation ofmultiple classes of V0 neurons in zebrafish spinal cord: Progenitor
heterogeneity and temporal control of neuronal diversity. J. Neurosci.
32, 1771-1783.
Suzaki, T., Yano, K., Ito, M, Umehara, Y., Suganuma, N., andKawaguchi, M. (2012). Positive and negative regulation of cortical cell
division during root nodule development in Lotus japonicus isaccompanied by auxin response. Development 139, 3997-4006.
Takeda, N., Maekawa, T., and Hayashi, M. (2012). Nuclear-localized
and deregulated calcium- and calmodulin-dependent protein kinase
activates Rhizobial and Mycorrhizai responses in Lotus japonicus.
Plant Cell 24, 810-822.
Watakabe, A., Kato, S., Kobayashi, K., Takaji, M., Nakagami, Y.,
Sadakane, O., Ohtsuka, M., Hioki, H., Kaneko, T., Okuno, H.,
Kawashima, T., Bito, H., Kitamura, Y., and Yamamori, T. (2012).
Visualization of Cortical Projection Neurons with Retrograde TET-Off
Lentiviral Vector. PLoS ONE 7, e46157.

〔Original paper (E-publication ahead of print)〕
T., Kim, C.S., Takeda, N., Szczyglowski, K., and Kawaguchi,• Suzaki,
M. TRICOT encodes an AMP1-related carboxypeptidase that regulates
root nodule development and shoot apical meristem maintenance in
Lotus japonicus. Development 2012 Dec. 18.

Infrared (IR) beams can heat water molecules, which are themain constituent of cells, hence, we can heat a single cell byirradiating IR to a target cell using a microscope. We havedeveloped a microscope, IR laser evoked gene operator(IR-LEGO), specialized for this purpose (Figure 1). The
IR-LEGO microscope can irradiate an IR laser to a single
cell in vivo such as C. elegans, medaka and Arabidopsis, toinduce the heat shock response at a desired timing.
Optimal heating induces the heat shock response andsubsequent gene expression, while an excess results in cell
death. Hence, we must precisely control laser heating;however, there was no way to measure temperature in a
microenvironment under microscopic observation. To
achieve this we employed green fluorescent protein (GFP) asa thermometer. Since fluorescent matter has the common
property of temperature dependent decrease of emission
intensity, we can estimate temperature shift by emission
intensity change. GFP expressing E. coli was used tomeasure temperature as a micro thermometer. Using thisprobe, we evaluated heating properties of IR-LEGO such as
speed of temperature rise and 3-dimensional distribution oftemperature during IR irradiation. In a model tissue whichcontained GFP expressing bacteria in polyacrylamide gel,temperature rose rapidly with IR irradiation and kept a
constant level dependant on IR laser power (Figure 2 left).
On the other hand, the heated area was limited to a small
volume about as large as a typical cell (Figure 2 right).

● Research activity by Y. Kamei
Associate Professor (Specially appointed)

KAMEI, Yasuhiro

Technical Assistant:

KANIE, Yuta

To investigate a gene function in each cell we have toexpress the gene in the cell in vivo, and ideally theexpression must be limited only to the single cell. Tissue orcell specific promoters were used to reveal gene functions,however promoter-driven gene expression was governed bycell fate or environment, therefore we could not control thetiming of gene expression. To achieve timing-controlled gene
expression we employed one of the stress responses, the heatshock response. The heat shock promoter is the transcription
regulation region of heat shock proteins and all organisms
have this mechanism. Positioning the target genedownstream of the promoter, we can induce the target gene
expression by heating.

Figure 1. An infrared laser-evoked gene operator (IR-LEGO) microscope
system in NIBB.

Figure 2. Heating profiles (laser power dependency of focus temperatureand 3-D temperature map) of IR irradiation.

With this in mind, we tried to induce gene expression invarious species. At first, we reported an IR-LEGO
experiment in living C. elegans. Target gene expression in atarget cell could be induced with only 1 s-IR irradiation.
Whereas the optimal power range which can induce geneinduction without cell damage was limited. Excess laser
power resulted in cell death or cessation of cell division. We
confirmed that an optimal irradiation, e.g. 11 mW for 1 s,
induced physiological gene expression in the target cell andsubsequent cell division or morphogenesis underwent normal
development. Next, we tried the experiment in animals,
medaka, zebrafish and xenopus, and the higher plant,
Arabidopsis, since all organisms have a heat shock response
system. We succeeded in local gene induction in the species
as expected.
Studies of cell fates, cell-cell interaction, or analysis of noncell autonomous phenomena require a fine control system ofgene expression in experiments. IR-LEGO will be a powerful71National Institute for Basic Biology

Research Support

the data worldwide. In addition to computational analysis,
the Data Integration and Analysis Facility supports NIBB’s
information infrastructure, the maintenance of the network
system in the institute and computer/network consultation for
institute members.

Representative Instruments
Figure 3. Cre-loxP mediated long-term GFP marking in a living medaka
individual for linage tracing.

tool for these studies in combination with molecularbiological techniques, such as the cre-loxP system. By
Applying IR-LEGO to a mutant and its rescue transgenic
strain; using hsp-cre with a rescue gene which is sandwichedby loxP sequences, we will achieve single-cell knockout
experiments in living organisms, and reveal fine interaction
between the cells. We are now testing this system using
medaka. We have already constructed a medaka TILLING
library and a screening system for reverse genetic mutant
screening, furthermore we have confirmed a system
operation of a cre-loxP system in medaka using IR-LEGO(Figure 3).

Our main computer system is the Biological Information
Analysis System (BIAS) (Figure 1), which consists of ashared memory parallel computer (DELL PowerEdge R905;
4 nodes/16 cores, 256GB memory), a high-performancecluster system (DELL PowerEdge M1000e+M610; 32
nodes/256 cores, 768GB memory) and a large-capacity
storage system (DELL Equallogic; 35TB SAS, 26TB SATA,
750GB SSD). All subsystems are connected via a high-speed
InfiniBand network so that large amounts of data can be

Publication List
〔Original papers〕
S., Ochiai, H., Kanie, Y., Kamei, Y., Gou, Y., Kitano, T.,• Ansai,
Yamamoto, T., and Kinoshita, M. (2012). Targeted disruption of•••exogenous EGFP gene in medaka using zinc-finger nucleases. Dev.
Growth Differ. 54, 546-556.
Kitano, T., Hayashi, Y., Shiraishi, E., and Kamei, Y. (2012). Estrogen
rescues masculinization of genetically female medaka by exposure tocortisol or high temperature. Mol. Reprod. Dev. 79, 719-726.
Masuyama, H., Yamada, M. Kamei, Y., Fujiwara-Ishikawa, T., Todo, T.,
Nagahama, Y., and Matsuda, M. (2012). Dmrt1 mutation causes a maleto-female sex reversal after the sex determination by Dmy in the
medaka. Chromosome Res. 20, 163-176.
Yasuda, T., Oda, S., Li, Z., Kimori, Y., Kamei, Y., Ishikawa, T., Todo, T.,
and Mitani, H. (2012). Gamma-ray irradiation promotes prematuremeiosis of spontaneously differentiating testis-ova in the testis of p53deficient medaka (Oryzias latipes). Cell Death Dis. 3, e395

〔Original paper (E-publication ahead of print)〕
K., Kamei, Y., Kinoshita, M., Czerny, T., and Tanaka, M. A
• Kobayashi,
heat-inducible cre/loxP gene induction system in medaka. Genesis 2012
Nov 3.

Data Integration and Analysis Facility
Assistant Professor:
Technical Staff:
Technical Assistant:

UCHIYAMA, Ikuo
MIWA, Tomoki
NISHIDE, Hiroyo
NAKAMURA, Takanori
YAMAMOTO, Kumi
OKA, Naomi

The Data Integration and Analysis Facility supports
research activities based on large-scale biological data
analysis, such as genomic sequence analysis, expression data
analysis, and imaging data analysis. For this purpose, thefacility maintains high-performance computers with largecapacity storage systems. On the basis of this system, thefacility supports development of data analysis pipelines,
database construction and setting up websites to distribute72

Figure 1. Biological Information Analysis Systemprocessed efficiently. Some personal computers and color/monochrome printers are also available. On this system, we
provide various biological databases and data retrieval/analysis programs, and support large-scale data analysis anddatabase construction for institute members. Especially, we
have supported the construction and maintenance of
published databases of various model organisms including
XDB (Xenopus laevis), PHYSCObase (Physcomitrella
patens), DaphniaBASE (Daphnia magna), The Plant
Organelles Database, and MBGD (microbial genomes).
The facility also provides network communication services.
Most of the PCs in each laboratory, as well as all of theabove-mentioned service machines, are connected by a local
area network, which is linked to the high performance
backbone network ORION connecting the three research
institutes in Okazaki. Many local services, including
sequence analysis services, file sharing services, and printerservices, are provided through this network. We also
maintain a public World Wide Web server that hosts the
NIBB home page (http://www.nibb.ac.jp/).

Research activity by I. Uchiyama
Assistant professor I. Uchiyama is the principal investigatorof the Laboratory of Genome Informatics, which currently
focuses on microbial comparative genomics studies. For
details, please refer to the laboratory page (p. 65).

NIBB BIORESOURCE CENTER

Head

IGUCHI, Taisen

Vice head:

FUJIMORI, Toshihiko

To understand the mechanisms of living organisms, studies
focusing on each gene in the design of life (genome) are
required. The use of model animals and plants, such as mice,
Medaka (Oryzias latipes), zebrafish, Arabidopsis, Lotus
japonicus, and Physcomitrella patens, makes it possible toproduce genetically controlled organisms with markers
placed by genetic and cell engineering technology. Such
marking allows detailed studies of genes and cell functions.
The model organisms mature in a short period of time;
therefore, changes in cells, organs, and individuals can betotally and efficiently observed. The NIBB BioResource
Center has equipment, facilities, and staff to maintain suchorganisms safely, efficiently, and appropriately.

Model Animal Research Facility
Associate Professor:
Technical Staff:
Technical Assistant:

WATANABE, Eiji
TANAKA, Minoru
NARUSE, Kiyoshi
HAYASHI, Kohji
NOGUCHI, Yuji
INADA, Junichi
INADA, Yosuke
MATSUMURA, Kunihiro
FUJIMOTO, Daiji
ICHIKAWA, Yoko
TAKAGI, Yukari
SUGINAGA, Tomomi
SUZUKI, Kohta

The worldwide genome project has almost been completed
and research on basic biology has arrived at a post-genome
era in which researchers are focusing on investigating thefunctions of individual genes. To promote the functional
analysis of a gene of interest it is essential to utilize
genetically altered model organisms generated using genetic
engineering technology, including gene deletion, genereplacement and point mutation.

Figure 1. Mouse (strain B6C3F1)

The NIBB Center for Transgenic Animals and Plants wasestablished in April 1998 to support research usingtransgenic and gene targeting techniques at NIBB. The NIBB
Center for Transgenic Animals and Plants was integrated into
the NIBB BioResource center in April 2010, and was
renamed as “The Model Animal Research Facility”.
Technical staff and supporting staff develop and promoteresearch-supporting activities. A state-of-the-art facility fortransgenic animals opened at the end of 2003 in the Yamate
area.
The activities of the model animal research facility are as
follows:
1. The provision of information, materials, techniques andanimal housing space to researchers.
2. The use of various kinds of instruments to analyze
mutant, transgenic, and gene-targeted animals.
3. The development of novel techniques related totransgenic and gene targeting technology.
4. Cryopreservation and storage of transgenic strains.

I. Research support activities (mouse)
In 2001, the NIBB mouse facility (built under specificpathogen free (SPF) conditions) opened in the Myodaiji area
and the production, breeding, analysis, cryopreservation andstorage of genetically manipulated mouse strains has been
conducted there since then. The new center facility buildingin the Yamate area strengthened research activities using
genetically altered organisms. The building has five floors
and a total floor space of 2,500 m2 in which we can generate,
breed, store and analyze transgenic, gene targeting andmutant mice under SPF conditions. The mouse housing area
was constructed based on a barrier system. This building is
also equipped with breeding areas for transgenic small fish,
birds and insects.
In 2012 (from January 1 to December 31), 2,802 fertilized
eggs (in vitro fertilization; 2,328 eggs of 8 lines in which
1,825 eggs of 8 lines were frozen for long-term storage,
frozen eggs: 474 of 11 lines) and 5,121 mice were brought
into the facility in the Yamate area, and 49,541 mice(including pups bred in the facility) and 25 fertilized eggs
were taken out.

Figure 2. Equipment for manipulating mice eggs.

73

National Institute for Basic Biology

Research Support

A number of strains of genetically altered mice from outside
the facility were brought into the mouse housing area by
microbiological cleaning using in vitro fertilization-embryotransfer techniques, and stored using cryopreservation.
A new mouse facility in the Myodaiji area opened at the
beginning of 2005. The facility provides research-supporting
activities to researchers in the Myodaiji area. In March 2009,
we expanded the facility which includes areas for breeding,behavioral tests and transgenic studies using various kinds ofrecombinant viruses. In 2012 (from January 1 to December31) 9 mice were brought into the facility in the Myodaijiarea, and 318 mice (including pups bred in the facility) weretaken out.

In 2012 (from January 1 to December 31), 4,419 medaka(1,338 eggs, 10 embryos and 3,071 adults) were brought tothe facility and 40,142 medaka (37,785 fertilized eggs and2,357 adults, including animals bred in the facility) weretaken out. In the laboratory for chick embryos there were no
fertilized eggs or chicken embryos brought in or taken out
this year. These animals were used for research activities inneurobiology and developmental biology.
In 2007 NIBB was approved as a core facility of the
National BioResource Project (NBRP) for Medaka by theJapanese Government. We have supported the activities of
NBRP Medaka by providing standard strains, mutants,
transgenic lines and organizing international practicalcourses for medaka. In 2010 we began providing the
TILLING library screening system to promote the reverse
genetic approach. In 2012 we shipped 175 independent
medaka strains, 386 cDNA/BAC/Fosmid clones, and 285
samples of hatching enzyme to the scientific community
worldwide.

Figure 3. Large sized autoclave in the Myodaiji area.
Figure 4. Medaka fish.

II. Research support activities (small fish,
birds, and insects)
The first floor of the center facility building in the Yamate
area provides space and facilities to maintain small fish,chick embryos, and insects. In the laboratory room for chickembryos, a large incubation chamber is equipped and set at
42 degrees (suitable for chick embryogenesis). The
researchers can manipulate chick embryos under optimal
conditions, removing biohazard risks. For researchers who
employ fish as an experimental model, 480 tanks (1 liter) and
450 tanks (3 liters) are available for medaka and zebrafish,
respectively. Water circulates and can be maintained to suitthe conditions desired for fish breeding in the aquarium
systems. Currently, over three mutant lines and over fifteentransgenic lines of medaka and zebrafish are maintained in
our facility. A medaka line that allows gene induction by heat
treatment, in combination with a cre/loxP system, has beendeveloped in this facility. In addition to the rooms mentioned
above, a room for insects is also available. All the rooms are
qualified to meet the criteria for transgenic animals, allowing
researchers to generate and maintain these importantbiological tools.

74

III. Research activities
The associate professors of this center - E. Watanabe, T.
Naruse and M. Tanaka - are the principal investigators of theLaboratory of Neurophysiology, the Laboratory of
Bioresources and the Laboratory of Molecular Genetics forR e p r o d u c t i o n , r e s p e c t i v e l y. T h e L a b o r a t o r y o f
Neurophysiology is studying mechanisms of the visualsystem using a psychophysical approach. The Laboratory of
Bioresources has conducted a genetic and genomic analysis
of quantitative traits and Mendelian phenotype variations aswell as evolution of sex determination systems in medaka
related species. The Laboratory of Molecular Genetics for
Reproduction is studying the molecular mechanisms ofreproductive organ development and sex differentiation using
mutagenized or transgenic medaka. For details, please referto the pages of each laboratory (p. 44, 52, and 32).

Model Plant Research Facility● Plant Culture Laboratory
Assistant Professor:
Technical Staff:
Technical Assistant:

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
SUZUKI, KeikoThe Plant Culture Laboratory manages facilities for thecultivation of plants in general and also for the rearing ofseveral animal species that do not fit in other facilities.
The Plant Culture Laboratory equips and manages 48
culture boxes or growth chambers, 6 phytotrons, and 12
rooms with the P1P physical containment level forestablished and emerging model plants including the thale
cress Arabidopsis thaliana, several carnivorous plants, therice Oryza sativa, rushes of the Juncus sp., the moss
Physcomitrella patens, and several other flowering plants.
An emerging model insect, the tiny moth Acrocercops
transecta is also reared in this laboratory. Most culture space
is fully used the whole year by more than 60 researchers
from both outside and inside groups.
As well as regular culture conditions, extreme
environmental conditions for light and temperature areavailable for various types of experiments. Three lightenvironmental simulators are available. Autotrophic andheterotrophic culture devices are also available forresearchers using cyanobacteria, algae, and cultured
flowering plant cells. Aseptic experiments can be performed
in an aseptic room with clean benches.
Next to the institute building of the Myodaiji area, a 386-m2
experimental farm is maintained for Japanese morning glory
and related Ipomoea species, several carnivorous plants andother flowering plants necessary to be cultivated outside.
Three green houses (44, 44, and 45 m2) with heating are used
for the carnivorous plants, and wild-type strains of medaka
fish Oryzias sp. Seven green houses (4, 6, 6, 6, 6, 9, and 38m2) with air-conditioning are provided for the cultivation of arice Oryza sp., Lotus japonica and related legume species, aswell as mutant lines of the Japanese morning glory. One
green house (18 m2) with air-conditioning meets the P1P
physical containment level and is available for experiments
using transgenic plants. The Plant Culture Laboratory also
maintains a 46-m2 building with storage and workspace. Partof the building is used for rearing of the orchid mantis.
In 2012, the green houses with heating were renovated andinstalled with weather sensors, and three growth chambers
were replaced. The chambers (3.4 m2 each) can control CO2
and humidity in addition to temperature and light (max
70,000 lux) conditions.

Figure 5. The growth chambers after replacement.

● Morning Glory BioResource Laboratory
Assistant Professor:

HOSHINO, Atsushi

The Japanese morning glory (Ipomoea nil) is a traditional
floricultural plant in Japan, and is studied worldwide,
especially in plant physiology and genetics. NIBB collects,
develops and distributes DNA clones, mutant lines for flower
pigmentation, and transgenic lines as a sub-center of the
National BioResource Project (NBRP) Morning glory, andcollaborates with the core organization center, Kyushu
University. We collected several mutant lines and DNA
clones, and provided 14 DNA clones and 3 BAC clone
screening systems to local biologists this year.
Research activities of the assistant professor A. Hoshino areshown on the laboratory page (p. 56).

Cell Biology Research Facility
Assistant Professor:

HAMADA, Yoshio

The Cell Biology Research Facility provides various
equipment for tissue and cell culture. This laboratory isequipped with safety rooms which satisfy the P2 physicalcontainment level, and is routinely used for DNA
recombination experiments.
Research activities of assistant professor Y. Hamada, theprincipal investigator of the Laboratory of Cell Sociology, isshown on the laboratory page (p.16).

Figure 6. Equipment for tissue and cell culture

75

National Institute for Basic Biology

Research Support

NIBB CENTER OF THE INTERUNIVERSITY
BIO - BACKUP PROJECT (IBBP CENTER)

Head

Associate Professor

NISHIMURA, Mikio

NARUSE, Kiyoshi

Postdoctoral Fellow:
Technical Assistant:

KIMURA, Tetsuaki
SAKAMOTO, Yuki

In order to realize a life sciences community resilient tonatural disasters and calamities, the National Institutes for
Natural Sciences (NINS) and Hokkaido University, Tohoku
University, University of Tokyo, Nagoya University, Kyoto
University, Osaka University, and Kyushu University
concluded an agreement on June 1st 2012 to launch a systemto ‘back up’ the biological and genetic resources essential to
the work being done at universities and research institutions
nationwide, called the ‘Interuniversity Bio-Backup Project(IBBP)’.
The IBBP Center was established as a centralized backup
and storage facility at NIBB, while IBBP member
universities set up satellite hubs and work closely with the
IBBP center to put in place reciprocal systems for backing up
important biological resources that have been developed byresearchers residing in the area each university satellite hub
is responsible for.
The IBBP Center includes earthquake proof structures
capable of withstanding even very large scale quakes(equipped with emergency backup power generators),
cryopreservation facilities equipped with automatic liquid
nitrogen feeding systems, deep freezers, and refrigerated
storage (mainly for seed stocks), as well as all manner of
automated laboratory equipment, cell culture tools, and thelatest equipment necessary to back up the genetic resourcesin a collaborative manner. The specific methods ofpreservation are freezing of the sperm and eggs of animals,
cultured plant and animal cells, and gene libraries. Plant
seeds are frozen or refrigerated. The capacity for the backup
storage facility is approximately 1.4 million samples.
University satellite hubs receive preservation requests ofbioresources from researchers and report to the Managing
Project Committee of IBBP (constituted of faculty of NIBB
and satellite institutes), where the relevance of the request isreviewed. When the request is sustained, bioresources to bepreserved will be sent to the IBBP Center by the requesting
researcher, where they will be frozen (or refrigerated) andtheir particular information will be registered into a database.
In the event of a disaster leading to the loss of a researcher’s
own bioresources, preserved samples will be promptly
supplied back to the researcher so they can quickly resume
their work.
Through the development of this backup system biologicalresources that had only been stored at individual research
institutes can now be preserved at the state of the art facilitiesof the IBBP Center, and Japan’s research infrastructure has
been significantly strengthened.

76

Figure 1. Cryogenic storage system. Liquid nitrogen tanks are monitored
for 24 hours and are refilled automatically.

Figure 2. Cryo tube with 2D barcode. Each sample is printed with uniquebarcode and is managed using database.

CENTER FOR RADIOISOTOPE FACILITIES

Table 2. Users and visitors who entered each controlled area in 2012

Head

HASEBE, Mitsuyasu

Associate Professor

Associate Professor

OGAWA, Kazuo

KODAMA, Ryuji(Radiation Protection Supervisor)

Technical Staff:

Technical Assistant:

MATSUDA, Yoshimi(Radiation Protection Supervisor)
SAWADA, Kaoru(Radiation Protection Supervisor)
IINUMA, Hideko
ITO, Takayo
KAMIYA, Kiyomi

The technical and supporting staff of the Center forRadioisotope Facilities (CRF) maintain controlled areas incompliance with the law. The CRF is responsible formonitoring the purchase of radioisotopes from JRIA (Japan
Radioisotope Association) and the transfer of radioisotope
wastes to JRIA.
Ms. Matsuda, Ms. Iinuma, Ms. Ito, and Ms. Kamiya
maintained the Myodaiji-Area. Dr. Ogawa and Ms. Sawada
worked in the Yamate-Area. Dr. Ogawa, the associate
professor of the radioisotope facilities, retired in March 2012and Dr. Kodama arrived at the post in April, working in bothareas.
The number of registrants and the number of users fromJanuary 2012 to December 2012 are shown in Table 1.
Users counted by the access control system of thecontrolled areas numbered 2,491 during this period. The
numbers for each area are shown in Table 2. The annualchanges of registrants and the number of totals per fiscal year
are shown in Figure 1. The balance of radioisotopes received
and used at the CRF is shown in Table 3. The training
courses on radioisotope handling were given as in Table 4.
The followings are CRF’s notable activities in 2012.
1.A lecture was given on the relationship of Radon
concentration in the air and earthquakes by an inviteds p e a k e r ( D r. Yu m i Ya s u o k a , L e c t u r e r, K o b e
Pharmaceutical University) as the 2012 special lecture
for radiation workers.
2. At the Myodaiji-area, the exhaust fan of line 2 expired
and was exchanged. (See Figure 2, A, B.)
3. At the Yamate-area, the access control system of thecontrolled areas was updated. The new system uses IC
cards meeting the FeliCa standard and will be compatible
in the future with the access control system of all Yamatearea buildings.

Table 1. Numbers of registrants and users at Myodaiji-Area and YamateArea in 2012

Figure 1. Annual changes of registrants and number of totals per fiscal
year

Table 3. Balance of radioisotopes received and used (kBq) at eachcontrolled area in 2012

Table 4. Training courses for radiation workers in 2012

A

B

C

D

Figure 2. The CRF’s notable activities in 2012
A, B: The exhaust fan was exchanged. A shows the old fan and B shows
the new fan.
C, D: The access control system of the controlled areas was updated. Cshows the card reader of the radioisotope storage room entrance. Dshows a card reader for the FeliCa standard card.

77

National Institute for Basic Biology

Research Support

STRATEGIC PLANNING DEPARTMENTChair

NISHIMURA, Mikio

Associate Professor:

KODAMA, Ryuji

The Strategic Planning Department was founded in April
2005 as a central office for assisting the Director General in
preparing for NIBB’s evaluation procedure and in planning along-range strategy for the institute. Formerly the department
also managed activities relating to public relations andinternational cooperation, however those roles of thedepartment were separated in 2009 and are now managed by
Office of Public Relations and Office of International
Cooperation, respectively.

The main activities of the Department
1) Management of external evaluation processes
NIBB asks non-NIBB members of the Advisory Committee
for Programming and Management to answer a questionnaire
on research achievements and collaborative research
activities of the institute. Several of the members are further
invited to participate in a meeting for the evaluation of these
activities and for proposing future directions for the institute.
The department manages these processes.

782) Editing of the Annual Report (in collaboration with the
Office of Public Relations)
The department edits the annual report (this issue) whichsummarizes the annual activities of laboratories and facilities
and is used in the evaluation procedures of the institute.
3) Assistance in fund application and long-range planningof the institute
The department assists the Director-General and Vice
Director in preparing long-range plans for building the mostadvanced research facilities, and in application for funds
from the government to realize these plans.
4) Assistance in making the Plans and Reports of theinstitute
The department assists in making NIBB’s Medium-term
Goals and Plans (for a six-year-period), and in instituting
Annual Plans to realize them. The department also assists in
preparing required Business and Performance Reports toanswer whether we are meeting the goals set both annuallyand for the medium-term.

OFFICE OF PUBLIC RELATIONS

Chair

FUJIMORI, Toshihiko

Assistant Professor (Specially appointed):
KURATA, Tomoko
Technical Assistant:
OTA, Kyoko
KAWAGUCHI, Colin
BAN, Misato
OKUBO, Masayo
The Office of Public Relations, in order to communicate theactivities of NIBB to the widest audience, performs bothstandard public relations duties as well as communication
with scientific publications and organizations worldwide.

The main activities of the office in 2012
1) Press releases
The office sends news on scientific achievements tonewspaper and magazine reporters via leaflets, e-mails andweb pages. We also arrange press conferences for somereleases.

OFFICE OF INTERNATIONAL COOPERATION

2) Updating and maintenance of the NIBB web page3) Editing of publications, production of posters andleaflets
Publication of the pamphlet “An introduction to the
National Institute for Basic Biology”. Publication of leaflets
introducing the individual researchers of NIBB (in Japanese).
Publication of “NIBB News” (Intra-institutional newsletter,in Japanese), “NIBB English News” (Intra-institutional
newsletter, in English). Design and distribution of posters forinternational conferences and advertisements for the graduate
school’s entrance examination.
4) Producing Videos
Creation of videos introducing NIBB and interviews withresearchers that are published publicly on the web.
5) Organization of scientific outreach programs
Planning the Summer Program for university students, andcoordinating special classes for middle school students.

The main activities of the office in 2012
1) Supporting coordination of international conferences
The Office of International Cooperation coordinated thefollowing international conferences hosted by NIBB:ChairYOSHIDA, Shosei

Assistant Professor:
Technical Assistant:

KAMADA, Yoshiaki
NAKANE, KaoriSANJO, Kazuko
TAKAHASHI, Ritsue

The Office of International Cooperation coordinatesinternational collaboration in the field of biology and forms
cooperative agreements with research institutes from aroundthe world, exchanges researchers, and holds various
academic meetings.
NIBB manages international cooperation with the European
Molecular Biology Laboratories EMBL (since 2005),
Germany’s Max Planck Institute for Plant Breeding Research(MPIPZ) (since 2009), Singapore’s Temasek Life Sciences
Laboratory (TLL) (since 2010), and Princeton University of
the USA (since 2010).

The 59th NIBB Conference “Neocortical Organization” Mar.
10-13, 2012
8th Okazaki Biology Conference “Speciation and Adaptation
II - Environment and Epigenetics -” Mar. 18-23, 2012
The 58th/60th NIBB Conference “Germline -Specification,
Sex, and Stem Cells-” Jul. 17-21, 2012
9th Okazaki Biology Conference “Marine Biology II” Oct.
14-19, 2012
The 4th NIBB-MPIPZ-TLL Symposium “Arabidopsis andEmerging Model Systems” Nov. 19-21, 20122) Supporting dispatching of researchers and students tointernational conferences
The Office of International Cooperation coordinatedsending NIBB researchers, and those from the Japanesescientific community outside NIBB, to the following course:
The NUS/TLL/NIBB joint practical workshop on“Genetics, Genomics and Imaging in Medaka & Zebrafish”
Jul. 22-31, 2012 (held in Singapore).
3) Management of education related programs
The Office of International Cooperation co-organized thefollowing international training course:
The NUS/TLL/NIBB joint practical workshop on“Genetics, Genomics and Imaging in Medaka & Zebrafish”
Jul. 22-31, 2012 (held in Singapore).

79

National Institute for Basic Biology

Research Support

TECHNICAL DIVISIONHeadFURUKAWA, KazuhikoCommon Facility Group

Research Support Group

Chief:

Chief:

MIWA, Tomoki● NIBB Core Research Facilities
Unit Chief:
HIGASHI, Sho-ichi
MORI, Tomoko
Subunit Chief:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
Technical Staff:
NISHIDE, Hiroyo
NAKAMURA, Takanori
TANIGUCHI-SAIDA, Misako
Technical Assistant:
ICHIKAWA, Chiaki
NISHIMURA, Noriko
YAMAMOTO, Kumi
OKA, Naomi
Secretary:
ICHIKAWA, Mariko
ISHIKAWA, Azusa● NIBB Bioresource Center
Subunit Chief:
HAYASHI, Kohji
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
ICHIKAWA, Yoko
TAKAGI, Yukari
SUZUKI, KeikoSUZUKI, Kohta● Disposal of Waste Matter Facility
Unit Chief:
MATSUDA, Yoshimi● Center for Radioisotope Facilities
Unit Chief:
MATSUDA, Yoshimi
Subunit Chief:
SAWADA, Kaoru
Technical Staff:
IINUMA, Hideko
Technical Assistant:
ITO, Takayo

The Technical Division is a support organization forresearchers and research organizations within NIBB. The
Technical Division develops and promotes the institute’s
research activities and maintains the research functions of theinstitute.
The Technical Division is organized into two groups: theCommon Facility Group, which supports and maintains theinstitute’s common research facilities, and the Research
Support Group, which assists research activities as describedin the reports of individual research divisions.

80● Cell Biology
Unit Chief:

KAJIURA-KOBAYASHI, Hiroko

KONDO, Maki

● Developmental Biology
Technical Staff:
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
NODA, Chiyo
MIZUGUCHI, Hiroko● Neurobiology
Unit Chief:
Subunit Chief:

OHSAWA, Sonoko
TAKEUCHI, Yasushi● Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
Subunit Chief:
KABEYA, Yukiko● Environmental Biology
Unit Chief:
MIZUTANI, Takeshi

Reception
Secretary:

TSUZUKI, Shihoko
KATAOKA, Yukari
UNO, Satoko
MIYATA, Haruko

Technical staff members continually participate, through theDivision, in self-improvement and educational activities toincrease their capabilities and expertise in technical areas.
Generally, technical staff members are attached to specificdivisions so that they may contribute their special biologicaland biophysical knowledge and techniques to various
research activities.
The Technical Division hosts an annual meeting fortechnical engineers who work in various fields of biology atuniversities and research institutes throughout Japan. At thismeeting, the participants present their own activities anddiscuss technical problems. The proceedings are publishedafter each meeting.

The Eighth Okazaki Biology Conference“Speciation and Adaptation II - Environment andEpigenetics”
Organizers: Aya Takahashi (National Inst. Genetics), Michael Purugganan (New York Univ.),
Fumitoshi Ishino (Tokyo Medical and Dental Univ.), Mitsuyasu Hasebe (NIBB)March 18 (Sun)-23 (Fri), 2012
Building on 2007’s 5th OBC’s topic of “Speciation and
Adaptation - Ecological Genetics of Model Organisms andBeyond,” the 8th OBC was held with the theme of“Speciation and Adaptation - Environment and Epigenetics.”
At OBC 8, the foundations were laid for frontier expanding,mold-breaking research investigating how environmental
responses relate to evolution, and the relationship epigenetic
factors may have on them. Though the event was originallyplanned for March, 2011, the unfortunate events of the 2011
Tōhoku earthquake and Tsunami forced its postponement
until March of the following year. This dark cloud did have asmall silver lining for the participating researchers however,as they were granted an additional year within which togather and analyze data, resulting in more and finer results

than may have been presented otherwise.
The topics of discussion included: “major and minor QTL’srole in relation to adaptation and wild mutation,” “thenecessity of analyzing wild populations,” “research using
fruit flies on the genes controlling speciation andpolymorphism in wild populations,” “multi-generational
polymorphism maintenance mechanisms and their adaptive
significance,” and “the connection between macro-evolution
and epigenetic change,” taking place through 7 sessions over
5 days. Through combining the fields of evolutionary,developmental, and ecological science in a single event,researchers joining OBC 8 were able to transcend theboundaries of their own research areas to gain a newunderstanding of unfamiliar fields, make new ties withcolleagues, and create
fertile ground for theplanning and execution of
new research ventures. We
at NIBB could not be more
pleased.
(Mitsuyasu Hasebe)

Speakers
Chong, Suyinn (Queenlsand Inst. of Medical Research), Colot, Vincent (IBENS), Comai, Luca (UC Davis), Feder, Martin (Univ. of
Chicago), Gibson, Greg (Georgia Inst. of Tech.), Graves, Jennifer (La Trobe Inst. of Molecular Sciences), Gresham, David (New York
Univ.), Guerrero-Bosagna, Carlos (WSU), Hanzawa, Yoshie (UIUC), Leakey, Andrew (UIUC), Muotri, Alysson (UCSD), Newfeld, Stuart(ASU), Olsen, Kenneth (WUSTL), Presgraves, Daven (Univ. of Rochester), Purugganan, Michael (New York Univ.), Renfree, Marilyn(The Univ. of Melbourne), Schott, Daniel (Harvard Univ.), Shimizu, Kentaro (Univ. of Zurich), Stephan, Wolfgang (LMU Munich),
Zhang, Cuicui (HZAU)
Akashi, Hiroshi (NIG), Araki, Kiwako (Kyoto Univ.), Innan, Hideki (SOKENDAI), Ishii, Shunsuke (RIKEN Tsukuba Inst.), Ishino,
Fumitoshi (Tokyo Medical and Dental Univ.), Ito, Hidetaka (Hokkaido Univ.), Kakutani, Tetsuji (NIG), Kanaoka, Masahiro (Nagoya
Univ.), Kawamura, Shoji (The Univ. of Tokyo), Kinoshita, Tetsu (NAIST), Kitano, Jun (NIG), Kohda, Takashi (Tokyo Medical and Dental
Univ.), Kudoh, Hiroshi (Kyoto Univ.), Kuwabara, Tomoko (AIST), Matsui, Takeshi (iCeMS, Kyoto Univ.), Okada, Norihiro (Tokyo Inst.
Tech.), Osada, Naoki (NIG), Sawamura, Kyoichi (Univ. of Tsukuba), Takahashi, Aya (NIG), Takahashi, Kazuo (Okayama Univ.),
Tamada, Yosuke (NIBB), Tanaka, Kenta (Univ. of Tsukuba)

81

The Ninth Okazaki Biology Conference“Marine Biology II”
Organizers: Noriyuki Satoh (Okinawa Inst. Sci. Tech.), Thomas C.G. Bosch (Univ. Kiel), Jun Minagawa (NIBB)
October 14 (Sun)-19 (Fri), 2012The Okazaki Biology Conferences (OBC) are unique retreat
style international conferences that focus on discovering newthemes in the field of basic biology and forming newcommunities of researchers.
The 9th OBC, “Marine Biology II”, was a continuation of
OBC 6 (Marine Biology), focusing on cnidarians, especiallycorals, and their symbionts, with the first half of theconference taking part in Okazaki from October 14th to the
16th, and the second half in Okinawa from the 17th throughthe 19 th . 44 researchers (24 fromoverseas) invited from the forefront of 7
fields showing remarkable advancement(Ecophysiology, Genomics, Circadian
Rhythms, Photosynthesis, Symbiotics,
Evo&Devo), came together to discuss,
give presentations, and exchange
information on the current challenges
and future development of their fields.
The conference was organized by Dr.
Jun Minagawa of NIBB, Dr. Noriyuki
Satoh of the Okinawa Institute of
Science and Technology, and Dr.
Thomas C. G. Bosch of the Universityof Kiel. With few opportunities for
cutting edge researchers working withcnidarians and their symbionts to gather

and share their work, the 9th OBC was an ideal chance forthe participants to rethink the striking new developments andnew possibilities opening in their respective fields. With aconclusion that Eco-Devo should be advocated as animportant direction in the post genomic era, this conference
was a perfect example of the OBCs’ goal of developing new
fields of biology research.
(Jun Minagawa)Speakers
Allemand, Denis (CSM), Ball, Eldon (ANU), Bosch, Thomas (CAU), Foret, Sylvain (ANU), Fraune, Sebastian (CAU), Gates, Ruth
(Univ. of Hawaii, Manoa), Holstein, Thomas (Univ. of Heidelberg), Houliston, Evelyn (UPMC/CNRS), Khalturin, Konstantin (CAU),
Lallier, François (UPMC), Larkum, Anthony (Univ. of Sydney), Levy, Oren (BIU), Manuel, François (UPMC), Miller, David (JCU),
Pringle, John (Stanford Univ.), Ralph, Peter (UTS), Rosenberg, Eugene (TAU), Smith, Joel (MBL), Takahashi, Shunichi (ANU), Tarrant,
Ann (WHOI), Technau, Ulrich (Univ. of Vienna), Vize, Peter (Univ. of Calgary), Weis, Virginia (OSU)
Fujisawa, Toshitaka (SOKENDAI), Hamada, Shun (FWU), Hatta, Masayuki (Ochanomizu Univ.), Hidaka, Michio (Univ. of the
Ryukyus), Kawaguchi, Masayoshi (NIBB), Kondo, Takao (Nagoya Univ.), Kurihara, Haruko (Univ. of the Ryukyus), Maruyama, Tadashi(JAMSTEC), Minagawa, Jun (NIBB), Sakai, Kazuhiko (Univ. of the Ryukyus), Satoh, Nori (OIST), Shinzato, Chuya (OIST), Shoguchi,
Eiichi (OIST)82

The 59th NIBB Conference“Neocortical Organization”
Organizers: Tetsuo Yamamori (NIBB), Yukiko Gotoh (The Univ. of Tokyo), Takuya
Shimazaki (Keio Univ.), Ikue Mori (Nagoya Univ.)
March 10 (Sat)-13 (Tue), 2012
From March 10th to the 13th 2012 the 59th NIBB
Conference, in partnership with the Grant-in-Aid for
Scientific Research on Inovative Areas “Neural Diversity and
Neocortical Organization” was held at the Okazaki
Conference Center, with 137 members (31 speakers)
gathered together from all around the world.
This conference was held with a special focus on the
cerebral neocortex, with the objective of shedding light on
the current state of research and to spur on more activity inthe research field. This was probably the first international
symposium on Neocortical Organization to be held over a
period of 4 days in Japan, with top researchers from aroundthe world gathering to discuss the newest research in thefield.
The poster session, focusing primarily on younger
researchers, was also quite fruitful, with 48 researchers
making presentations. Their discussions extended on into theevening dinner parties, some of which went on to become thebasis of actual research collaborations.
Though much of the current advances in this field of
research pertain to the processes by which diverse controls inneural stem cells give rise to the different types of cells ineach layer of the neocortex,
as research moves forward
we expect that elucidation ofthese control mechanisms
will contribute to ourunderstanding of thalamo –cortical projections andcortico– cortical connectionsin the brain, and theformation of different areas
within the neocortex.
( Tetsuo Yamamori)Speakers
Guillemot, Francois (NIMR), Hensch, Takao (Harvard Univ.), Kriegstein, Arnold (UCSF), Nieto, Marta (CSIC), Polleux, Franck (TSRI),
Shi, Song-Hai (MSKCC), Yuste, Rafael (Columbia Univ.)
Aizawa, Shinichi (RIKEN CDB), Ikegaya, Yuji (The Univ. of Tokyo), Okano, Hideyuki (Keio Univ.), Osumi, Noriko (Tohoku Univ.),
Kaibuchi, Kozo (Nagoya Univ.), Kageyama, Ryoichiro (Kyoto Univ.), Kano, Masanobu (The Univ. of Tokyo), Kawaguchi, Yasuo (NIPS),
Gotoh, Yukiko (The Univ. of Tokyo), Sakano, Hitoshi (The Univ. of Tokyo), Shimazaki, Takuya (Keio Univ.), Tagawa, Yoshiaki (Kyoto
Univ.), Nabekura, Junichi (NIPS), Noda, Makoto (Kyoto Univ.), Hanashima, Carina (RIKEN CDB), Masu, Masayuki (Univ. of Tsukuba),
Matsuzaki, Masanori (NIBB), Matsuzaki, Fumio (RIKEN CDB), Mishina, Masayoshi (The Univ. of Tokyo), Murakami, Fujio (Osaka
Univ.), Mori, Ikue (Nagoya Univ.), Yamamori, Tetsuo (NIBB)

83

The 58th/60th NIBB Conference“Germline -Specification, Sex, and Stem Cells-”
Organizers: Robert E. Braun (The Jackson Laboratory), Mark Van Doren (The Jackson Laboratory),
Satoru Kobayashi (NIBB), Shosei Yoshida (NIBB)
July 17 (Tue) - 21 (Sat), 2012

Originally the 58th NIBB conference on “Gamete StemCells” was to be held in July, 2011, however, due to the
March 11th earthquake and tsunami the meeting was
postponed. Fortunately, the 60th NIBB conference on“Germline Development” was planned for this year and wewere able to hold this unprecedented “58th and 60th ‘joint’
NIBB conference”.
At the symposium we focused on three subjects central togermline research, the formation of the germline, the sexual
characteristics of the germline, and gamete stem cells, andthoroughly discussed the universal and distinctive aspects ofa wide range of species, including mammals (primates,mice), birds, fish, ascidians, hydra, nematodes, planarians,
and fruit flies. Over 130 participants from around the world
took part, with four scientists who are pushing the bounds ofpresent research giving special lectures based on data fromthe cutting edge of this field, which lead to impassioned
debate from the beginning to the end of the conference.
Following the special lectures, lectures on the mechanisms
regulating specification and sexual development of thegermline continued. The topics included identification of thegenes that induce feminization or masculinization ofprimordial germ cells, somatic control of germline sex, andthe regulatory mechanisms of gene expression in germline
cells. Research using birds, planarians, ascidians, and hydra
was also presented, allowing for participants to share theinvaluable viewpoint of evolutional conservation anddivergence of the regulatory mechanism underlying germline
development, and to feel the true breadth of the germline
research field.
On the following days, researchers investigating the stem
cells of gametogenesis in a variety of animals, including
mice, primates, fish and fruit flies, were able to cometogether and discuss their research in a single venue, making
this a historic opportunity. Many debates were presented
based on similarities in the gamete stem cell systems in mice,
fruit flies, and other animals that display substantial
differences in gonadal morphology, as well as computer

simulations and quantitative statistical analyses of stem cell
dynamics. A variety of revolutionary germ cell manipulating
techniques motivated by medical science, animal husbandry,
and fisheries science was another important theme of theconference, with much lively debate on the subject to be had.
Though the schedule for the whole event was rather tight,
we believe the conference provided ample ground forparticipants to meet between talk and poster sessions, and atmeals and other get-togethers, and to strengthen and broaden
their personal bonds. It is our hope that these newconnections might become the seeds for new breakthroughresearch in the field.
Finally, we would like to give our thanks to all theparticipants for this year’s conference for attending, theOffice of International Cooperation and the members of
Kobayashi and Yoshida Laboratories of NIBB for managing
the conference, Grant-in-Aid
for Scientific Research on
Innovative Areas, “Regulatory
Mechanisms of Gamete StemCells” from the Ministry of
Education, Culture, Sports,
Science, and Technology forco-sponsoring the event, and to
the Inoue Foundation for
Science and the Daiko
Foundation for their support.
(Shosei Yoshida)

Speakers
Braun, Robert (The Jackson Laboratory), de Rooij, Dirk (Univ. of Amsterdam), Han, Jae Yong (SNU), Koopman, Peter (Univ. of
Queensland), Lehmann, Ruth (NYU), Matunis, Erika (Johns Hopkins Univ.), Newmark, Phillip (UIUC), Orwig, Kyle (Univ. of
Pittsburgh), Page, David (Whitehead Institute), Seydoux, Geraldine (Johns Hopkins Univ.), Simons, Benjamin (Univ. of Cambridge),
Spradling, Allan (CIW), Van Doren, Mark (Johns Hopkins Univ.), Yamashita, Yukiko (Univ. of Michigan Ann Arbor)
Kobayashi, Kazuya (Hirosaki Univ.), Kobayashi, Satoru (NIBB), Kumano, Gaku (Osaka Univ.), Matsui, Yasuhisa (Tohoku Univ.),
Nakamura, Akira (RIKEN CDB), Niki, Yuzo (Ibaraki Univ.), Ogawa, Takehiko (YCU), Saga, Yumiko (NIG), Saitou, Mitinori (Kyoto Univ.),
Shinohara, Takashi (Kyoto Univ.), Tanaka, Minoru (NIBB), Yoshida, Shosei (NIBB), Yoshizaki, Goro (TUMSAT),

84

The 4th NIBB-MPIPZ-TLL Symposium“Arabidopsis and Emerging Model Systems”
Organizers: Masayoshi Kawaguchi(NIBB), Yoshikatsu Matsubayashi (NIBB),
Kiyoshi Tatematsu (NIBB), Mitsuyasu Hasebe (NIBB), Mikio Nishimura (NIBB)
November 19 (Mon) - 21 (Wed), 2012
The National Institute for Basic Biology (NIBB), with thegoal of advancing research and providing a venue foracademic exchange, held the 4th NIBB-MPIPZ-TLL joint
symposium from November 19th ~ November 21st 2012, at
the Okazaki Conference Center in Okazaki City, Aichi
Prefecture, Japan. This series of symposia began in Cologne
in 2009, as a joint venture between NIBB and The Max
Planck Institute for Plant Breeding Research (MPIPZ), withwhich NIBB has formed an academic exchange agreement,
the Temasek Life Science Laboratory, which also enteredinto an academic exchange agreement with NIBB, laterjoined. The symposia are now being held in rotation between
all three institutes. The theme for this year’s conference was“Arabidopsis and Emerging Model Systems”. Arabidopsis iswell known as an excellent model organism, playing acentral role in the elucidation of the molecular mechanisms
involved in development, physiology, environmental
response, and other biological processes. For this year’s
symposium, we invited many researchers on the forefront of
the field to present their research results and engage in lively
discourse regarding their work. Notably, Okazaki is known
as one of the founding centers of the Arabidopsis community
in Japan. In 1990 and 1991, Dr. Kiyotaka Okada, (at thetime, an assistant professor at the Dr. Yoshiro Shimura’s Lab
of NIBB) planned and held the first and second workshops
promoting Arabidopsis as a model organism in NIBB, after
which its use within Japan rapidly expanded.
The world of plants, however, is quite diverse just like theworld of animals or microbes. Many biological phenomena
remain which cannot be understood or elucidated throughArabidopsis, and even whether the insights into plant biology
provided by Arabidopsis are universal remains unknown. In
addition, with recent years bringing revolutions in next
generation sequencer technology and genetic analysis
techniques, new model lines continue to spring forth at a
rapid pace. Thus, at this symposium, we also asked for theparticipation of researchers engaged in dynamic researchutilizing new model plants such as Marchantia Polymorpha,
seaweed, algae, or other experimental lines in order to further
mutual understanding of research new and old.
This year marks the final year of General Director Okada’s
tenure at NIBB, and a quarter century of his work in the fieldof plant biology. The symposium was a lively one, thanks not
only to the researchers engaged in pushing forward the stateof plant research while expanding the Arabidopsis
community, but also thanks to the researchers connected to
NIBB, and internationally active women researchers, whowere kind enough to take part in this symposium. There werea total of 186 participants, with 67 poster presentations of
new research, primarily
featuring the work of younger
researchers, on exhibit aswell. With question andanswer sessions running on
i n t o l e n g t h y, a n i m a t e ddiscussions, the 4th NIBBMPIPZ-TLL joint symposium
was a wonderful experience
for all.
(Masayoshi Kawaguchi)

Speakers
Albani, Maria (MPIPZ), Benfey, Philip (Duke Univ.), Berger, Frederic (TLL), Bowman, John (Monash Univ.), Coupland, George(MPIPZ), Grossniklaus, Ueli (Univ. of Zurich), Ito, Toshiro (TLL), Juergens, Gerd (Univ. of Tuebingen), Koornneef, Maarten (MPIPZ),
Laux, Thomas (Univ. of Freiburg), Nishii, Ichiro (TLL), Palme, Klaus (Univ. of Freiburg), Sarojam, Rajani (TLL), Schulze-Lefert, Paul(MPIPZ), Shimizu, Kentaro (Univ. of Zurich), Somerville, Chris (Univ. of California Berkeley), Theres, Klaus (MPIPZ), Torii, Keiko (Univ.
of Washington), Tsuda, Kenichi (MPIPZ)
Fukuda, Hiroo (The Univ. of Tokyo), Hara-Nishimura, Ikuko (Kyoto Univ.), Hasebe, Mitsuyasu (NIBB), Kawaguchi, Masayoshi (NIBB),
Komeda, Yoshibumi (The Univ. of Tokyo), Kondo, Takao (Nagoya Univ.), Kyozuka, Junko (The Univ. of Tokyo), Machida, Yasunori(Nagoya Univ.), Matsubayashi, Yoshikatsu (NIBB), Minagawa, Jun (NIBB), Nagatani, Akira (Kyoto Univ.), Nakamura, Kenzo (Chubu
Univ.), Nishimura, Mikio (NIBB), Okada, Kiyotaka (NIBB), Sawa, Shinichiro (Kumamoto Univ.), Shimamoto, Ko (NAIST), Sugimoto,
Keiko (RIKEN), Tasaka, Masao (NAIST), Tatematsu, Kiyoshi (NIBB), Tsukaya, Hirokazu (The Univ. of Tokyo)

85

The NUS/TLL/NIBB Joint Practical Workshop“Genetics, Genomics and Imaging inMedaka and Zebrafish”■Period: July 22(Sun) - July 31(Tue), 2012
■Participants: 16 (3 from Germany, 2 from China, 2from India, 2 from Italy, one each from Australia,
Austria, Canada, France, Japan, Norway, and USA)
■Venue: Natl. Univ. Singapore (NUS) and TLL,
Singapore■Lecturers:
Dr. Kiyoshi Naruse (NIBB)
Dr. Atsuko Shimada (Univ. Tokyo)
Dr. Suresh Jesuthasan (Duke-NUS NRP, Singapore)Dr. Zhiyuan Gong (NUS)
Dr. Thorsten Wohland (NUS)
Dr. Dipanjan Bhattacharya (NUS)
Dr. Paul Matsudaira (NUS)
Dr. Tom Carney (Inst. Mol. Cell Biol., Singapore)
Dr. Vladimir Korzh (Inst. Mol. Cell Biol.,
Singapore)
Dr. Atsuko Sehara-Fujisawa (Kyoto University)
Dr. Yasuhiro Kamei (NIBB)
Dr. Hiroyuki Takeda (Univ. Tokyo)
Dr. Tetsuaki Kimura (NIBB)
Dr. Sinnakarupan Mathavan (Genome Inst.
Singapore)
Dr. Sudipto Roy (Inst. Mol. Cell Biol., Singapore)■Course Staff:
Dr. Mireia Perez Camps, Dr. Takuya Kaneko, Dr.
Toru Kawanishi, Dr. Karuna Sampath, Dr. Anand
Singh, Dr. Sahar Tavakoli, Dr. Yan Tong, Dr. Yin
Wang, Dr. Christoph Winkler, Dr. Chang Woei
■Contents of the course:
Somite transplantation, Cell transplantation,
Cryopreservation of Medaka sperm: In-vitro
fertilization, TALENs: Targeted gene modificationin zebrafish,
Establishment of
BAC transgenic
medaka, IR-LEGO,
SPIM

86

The NIBB Genome
Informatics Training
CourseNIBB organizes a series of training courses on up-to-date
research techniques for researchers from mainly Japanese
universities and institutions. In 2012 we held two training
courses on Genome Informatics. The two-day programs offer
lectures and hands-on tutorials to introduce basic knowledge
and skills to deal with genomic scale large data such as nextgeneration sequences (NGS). The programs are speciallydesigned for biologists who are not familiar withbioinformatics.

“Introduction to Transcriptome
Data Analysis”
March 22 (Thu) -23 (Fri), 2012
■Organizer: Dr. Shuji Shigenobu(NIBB Core
Research Facilities)■Lecturers: Dr. Ikuo Uchiyama, Dr. Masanao Sato,
Dr. Katsushi, Yamaguchi, Dr. Shuji Shigenobu■Participants: 19 (including 6 from NIBB)■Program:
1. Overview: Transcriptome data analysis
2. Introduction to statistics
3. Introduction to “R”
4. RNA-seq analysis pipeline
5. Basic format of mapping data and basic tools
6. Expression data analysis I: Normalization,differential expression analysis
7. Expression data analysis II: Multivariate analysis
8. Excercise“Introduction to Next-generation
DNA Sequence Data Analysis”
September 6 (Thu) -7 (Fri), 2012
■Organizer: Dr. Shuji Shigenobu(NIBB Core
Research Facilities)■Lecturers: Dr. Ikuo Uchiyama, Mr. Tomoki Miwa,
Dr. Katsushi, Yamaguchi, Dr. Shuji Shigenobu■Participants: 16 (including 1 from NIBB)■Program:
1. Overview: NGS data analysis
2. UNIX for beginners
3. NGS basic data formats
4. NGS basic tools I: Mapping
5. NGS basic tools II: Visualization tools
6. NGS basic tools III: Samtools
7. Methods for text data processing
8. Excercise

The NIBBInternship Program
The NIBB Internship program is a hands on learning
experience started in 2009 as a way to promote NIBB withoverseas students and to build connections through providing
education to the people who will form the core of future
research networks. At the same time, this program aims tointernationalize the graduate students of the Graduate
University for Advanced Studies (SOKENDAI), giving themthe opportunity to get to know students and interns withvarious cultural customs.
To participate in this program, applicants who would like toexperience research at NIBB must supply the name of the lab
they would like to visit as well as their reasons for choosing
it and a letter of recommendation. Based on this information
applicants are chosen to spend set periods of time
participating in specific research activities in the lab they
applied for. Round trip airfare and housing expenses areprovided by the NIBB Internship Program.
In FY 2012 there were 26 applicants, of which seveninterns were selected. These interns were from universities
located in 3 countries (India, Germany and China) and spent
periods ranging from 1 week to two months experiencing life
as a member of a research team.

Report from a participant
Ji Zhongzhong
Capital Normal University, China
From Sep. 1st to 30th, I joined Prof. Hasebe’s
laboratory as an intern and focused on roles of asingle transcription factor, STEMIN, in the
reprogramming of differentiated cells to stem cells. To
address how STEMIN induces reprogramming, I
investigated spatiotemporal expression patterns of
STEMIN in excised leaf cells using the GUS knock-in
line and changes in expression of genes involved in
reprogramming after the STEMIN induction withadvanced techniques.
I successfully completed the work described above
and obtained some experimental data. On the last dayof my internship program, I gave a short talk about
these results and discussed them with Prof. Hasebeand his lab members. It helped me to understand plantstem cell formation.
During my stay at NIBB, everything went well;everyone here was friendly, enthusiastic andaccommodating, I felt very lucky for joining Prof.
Hasebe’s lab. NIBB offered many seminars forstudents and staff, it is a good platform and helpful
for enhancing
communicationand opening themind to researchhere.

The 7th Bio-Imaging Forum“Innovation of the Microscope”
Organizers: Yasuhiro Kamei, Shigenori Nonaka,
Masayuki Hattori (NAO), Yosuke Tamada, Takeshi Hiyama
Supervisors: Naoto Ueno, Toshihiko Fujimori
November 26 (Mon)-27 (Tue), 2012
The 7 th Bio-Imaging Forum was held on the theme of“Innovation of the Microscope,” as a collaboration with the
NINS Program for Cross-disciplinary Study by Young
Researchers. This NINS Program includes two projects from
NIBB: “The establishment of observation techniques forplant cells, utilizing adaptive optics developed for
astronomical observation,” (lead by Dr. Tamada) focusing on
the application of adaptive optics to biological microscopic
imaging, and the “Development of 3D optogenetics utilizing
new femtosecond ultrashort pulse lasers” project (lead by Dr.
Hiyama), focusing on the development and application of
new methodologies within the rapidly expanding
optogenetics field. For this year’s Bio-Imaging Forum, we
provided a shared venue for presenting the results of theseresearch projects, and also invited 16 researchers to givelectures on the effects of direct, visual observation in
microscopy that adaptive optics provides, the new paradigm
shift from “observation” to “manipulation” and the related
new potentialities for microscopy made possible by
optogenetics, as well as expected future research anddevelopments that may come from these two fields of study.
The forum had 60 participants, with 39 coming from outsideof NIBB, including 10 participants from optics-relatedindustries (Olympus, Nikon, Hamamatsu Photonics, SigmaKoki), and 3 participants from the field of astronomy(National Astronomical Observatory of Japan (NAO), Hawaii
Observatory, Subaru Telescope). As a cross-disciplinary
gathering of researchers, the forum was host to lively
academic exchange, with topics ranging from basic questions
about unfamiliar fields, through to rigorous, in depth
discussions focusing on specialty research. This exchange
continued on into the evening’s get-together dinner, withindustry affiliated participants providing technical advice andtips, and scientists giving ideas and requests for possible
future industrial research. At the end of the forum Professor
Ueno also introduced the Center for Novel Science
Initiative’s (CNSI) Department of Imaging Science stressing
the importance of furthering imaging research in the future.
Through this year’s forum, the necessity of further uniting
the field of imaging, and its research, was made clear. It is
our hope that the Bio-Imaging Forum will continue to play anactive role in bringing the field of imaging research closer
together, as well as promoting new front-line imaging
research.
(Yasuhiro Kamei)

87

INDEX
A ....................................................................................................
AIHARA, Yusuke

63

HIGASHI, Sho-ichi

AJIOKA, Rie

52

HIGASHIYAMA, Tetsuya

4

AOKI, Etsuko

45

HIGUCHI, Yoko

26

AONO, Sachiko44

HIKINO, Kazumi

8

AOYAMA, Tsuyoshi

45

HIMENO, Miki

42

ASAO, Hisayo

68

HIRA, Riichiro

42

B ....................................................................................................

HIRAKAWA, Ikumi

60

79

HIRAKAWA, Reiko39

C ....................................................................................................

HIRAMATSU, Mika

45

CHAKRABORTY, Tapas

60

HIRAYAMA, Yuka

39

CHEN, Qiuhong

23

HIRUTA, Chizue

60

CHENG, Chaoyang

45

HIWATASHI, Yuji

7, 45

CHIBA, Hirokazu

65

HIYAMA, Takeshi

36, 87

CUI, Songkui

8

HONDA, Satoko

20

HOSHI, Rie

63

BAN, Misato

D ....................................................................................................
DOKYO, Yumi

36

F ....................................................................................................
FUJIKAWA, Akihiro

36

FUJIMORI, ToshihikoI56, 75....................................................................................................
ICHIKAWA, Chiaki
70, 804, 26, 73, 79, 87

ICHIKAWA, Mariko

68, 80

FUJIMOTO, Daiji

73

ICHIKAWA, Michiko48

FUJISAWA, Chiemi

20

ICHIKAWA, Shunsuke

45

FUJITA, Hironori

48

ICHIKAWA, Takehiko

FUJITA, Miyako

68

ICHIKAWA, Yoko

73, 8048, 80IGARASHI, Hisako

34

FUKADA-TANAKA, Sachiko67

FUKADA, Hatsumi

45

IGUCHI, Taisen

4, 60, 73

FUKUSHIMA, Kenji

7, 45

IINUMA, Hideko

77, 80FURUKAWA, Kazuhiko

80

IKAMI, Kanako

29

G ....................................................................................................

IKEUCHI, Momoko

34

GONDA, Naoko

16

IMAI, Akihiro

45

GOTO-YAMADA, Shino

7, 8

IMAI, Akiko

39

GOTO, Ayako

20

IMAIZUMI, Taeko

60

GOTO, Miho

45

IMOTO, Eiko39

GOTO, Misako

45

INADA, Junichi

73

INADA, Kana

29

INADA, Yosuke

73

ISHIBASHI, Tomoko

67

H ....................................................................................................
HAKOSHIMA, Toshio

4

HAMADA, Yoshio

16, 75

HAMATANI, Ayako

52

ISHIKAWA, Azusa

HANDA, Yoshihiro

48

ISHIKAWA, Hiroe

52

HARA, Ikuyo

52

ISHIKAWA, Masaki

7, 45

HARA, Kenshiro

29

ISHIKAWA, Takaaki

45

HARA, Reiko34

ISHINE, Naomi

HARA, Yusuke

17

ITO, Takayo

HASEBE, Mitsuyasu

4, 45, 77, 81, 85

70, 8058
77, 80

ITO, Yukiko (Div. Mol. Dev. Biol.)

23

HASHIMOTO, Masakazu

17

ITO, Yukiko (Div. Evol. Biol.)

45

HATA, Katsusuke

39

IWASAKI, Akira

34

HATTORI, Nobuko

36

HAYASHI-TSUGANE, Mika

57

HAYASHI, Kentaro

17

J ....................................................................................................
JOHZUKA, Katsuki

58

K ....................................................................................................

73, 80KABEYA, Yukiko

45, 80HAYASHI, Makoto

8

KAJIKAWA, Ikumi

45

HAYASHI, Tomoko

60

KAJITANI, Tomoki

HAYASHI, Yoshiki

20

KAJIURA-KOBAYASHI, Hiroko

HAYASHI, Kohji

88

HOSHINO, Atsushi

70, 8039
67, 80INDEXKAKIGI, Rieko

45

MARUYAMA, Atsushi

67

KAKITA, Mitsuru

11

MARUYAMA, Ayumi

29

KAMADA, Konomi

63

MASAMIZU, Yoshito

42

KAMADA, Yoshiaki

54, 79MASUOKA, Tomoko

KAMAGATA, Takanori
KAMEI, Yasuhiro

23

MATSUBAYASHI, Yoshikatsu

45
11, 8570, 71, 86, 87

MATSUDA, Chisato

KAMETANI, Yoshiko

23

MATSUDA, Takashi

36

KAMIDA, Chiharu

45

MATSUDA, Yoshimi

77, 80KAMIGAKI, Akane

8

MATSUMOTO, Masahito

36

KAMIYA, Kiyomi

77

MATSUMOTO, Miwako

34

KAMRANI, Yousef Yari

63

MATSUMURA, Kunihiro

73

KANAI, Masatake

8

MATSUZAKI, Masanori

42, 83

KANEKO, Hiroyo

52

MATSUZAKI, Yoko

KANIE, Yuta

71

MII, Yusuke

KATAOKA, Yukari

80

MINAGAWA, Jun

KATO, Azusa

26

MIURA, Seiko

36

KAWAGUCHI, Colin

79

MIWA, Tomoki

72, 80, 86

KAWAGUCHI, Masayoshi

4, 48, 82, 85

MIYAGAWA, Shinichi

45
7, 23
63, 82, 85

7, 60

KIMURA, Tetsuaki

52, 76, 86

KINOSHITA, Chie32

MIYAKAWA, Hitoshi

KINOSHITA, Noriyuki

17

MIYAKE, Satoko

17

KITADATE, Yu

29

MIYATA, Haruko

80

KITAZUME, Tatsuya

68

MIYAZAKI, Yuriko

36

KOBAYAKAWA, Satoru

26

MIYAZAWA, Hikota

48
29, 80

KOBAYASHI, Satoru

MIYAGI, Asuka

14

4, 20, 68, 84

MIZUGUCHI, Hiroko

KOBAYASHI, Yuki

48

MIZUSHIMA, Noboru

KODAMA, Akiko

36

MIZUTANI, Takeshi

KODAMA, Ryuji

51, 77, 78

MORI, Ayaka

KOHARA, Mayuko

45

MORI, Ikue

KOIKE, Chieko

52

MORI, Tomoko

KOIKE, Yukari

52

MORITA, Junko

KOJIMA, Yoko

45, 63

KOMATSU, Yusuke

39

KOMINE, Yuriko

39

KON, Yayoi

39

KONDO, Maki

8, 80

MOROOKA, Naoki

N

17
7, 60

4
60, 8011
4
68, 8039
75, 80

MURAKAMI, Michiyo

17

MURATA, Takashi

45....................................................................................................
NAGASHIMA, Akitomo
45

KONDO, Takao

4

NAKAGAMI, Yuki

KONISHI, Mie

36

NAKAI, Atsushi

8

KOTANI, Keiko39

NAKAMORI, Chihiro

34

KOYAMA, Hiroshi

26

NAKAMOTO, Masatoshi

52

KUBO, Minoru

45

NAKAMURA, Ryoko

KUBOKI, Yuko

29

NAKAMURA, Takanori

7, 36

NAKAMURA, Tohru

39

KURATA, Tomoko

79

NAKAMURA, Yoshiaki

29

KURUMIZAKA, Hitoshi

4

NAKANE, Kaori79

NAKANISHI, Norie

36

45

NAKATA, Miyuki

34

M ....................................................................................................

NAKAYAMA, Kei

14
8

KUBOYAMA, Kazuya

L ....................................................................................................
LI, Chen
MAEDA, Taro

7, 69

NAKAYAMA, Tomomi

MAKINO, Yumiko68, 80NAKAYASU, Tomohiro

MANO, Hiroaki

45

NARUSE, Kiyoshi

MANO, Shoji

8

NISHI, Tayo

39

56
72, 8044
52, 73, 76, 86
45

89

INDEX72, 80

SASADO, Takao

52

23

SASAKI, Takema

48

NISHIMURA, Mikio

1,2, 4, 8, 76, 78, 85

SASAKI, Tetsuya

39

NISHIMURA, Noriko

80

SATO, Kaori20

NISHIMURA, Toshiya

32

SATO, Katsuhiko

2

NISHIYAMA, Tomoaki

45

SATO, Masanao

20, 86

8

SATO, Masaru

60

NISHIDE, Hiroyo
NISHIGUCHI, Kimiko

NITO, Kazumasa
NODA, Chiyo

20, 80

SATO, Yasufumi

26

NODA, Masaharu

4, 36

SAWADA, Kaoru

77, 80NOGUCHI, Yuji

73, 80

SHI, Dongbo

26

NONAKA, Shigenori

67, 87SHIBA, Keikun

52

NONAMI, Yuta

29

SHIBATA, Emiko52

NOZAWA, Masafumi

45

SHIBATA, Michitaro

7, 8

NOZU, Ryo52

SHIBATA, Tomoko

45

O ....................................................................................................

SHIBATA, Yasushi

52

ODA, Shigeto

60

SHIGENOBU, Shuji

68, 69, 86

OGAWA-OHNISHI, Mari11

SHIINA, Nobuyuki

14

OGAWA, Kazuo

77

SHINOHARA, Hidefumi

11

OGAWA, Kota

69

SHINOZUKA, Takuma

23

OGAWA, Yuko

48

SHINOZUKA, Yuko

20

OGINO, Yukiko

60

SHINTANI, Atsuko

67

OHARA, Yuya

20

SHINTANI, Takafumi

36

OHHARA, Kaori23

SHUKLA, Rammohan

OHKUBO, Fuki

42

SOYANO, Takashi

7, 48

OHNISHI, Norikazu

63

SUGAYA, Tomomi

45

OHNO, Kaoru

55

SUGIMOTO, Nagisa

34

SUGIMOTO, Ryo

29

OHSAWA, SonokoS9039, 8039

OHSHIMA, Issei

45

SUGINAGA, Tomomi

73

OHSUMI, Yoshinori

7

SUGIYAMA, Arisa

20

OHTA, Kunihiro

4

SUGIYAMA, Tomomi

42

OHTSUKA, Masanari

39

SUMIYA, Eri

60

OIKAWA, Kazusato

8

SUZAKI, Momoyo

48
48

OKA, Naomi

72, 80SUZAKI, Takuya

OKA, Sanae

26, 80SUZUKI, Atsuko

17

OKADA, Kiyotaka

34, 85SUZUKI, Keiko75, 80OKAMOTO, Haruko

45

SUZUKI, Kohta

73, 80OKAMOTO, Satoru

11, 48SUZUKI, Makoto

17

OKUBO, Masayo

11, 79SUZUKI, Miho

17

OKUYAMA, Teruhiro

52

SUZUKI, Miyuzu

69

OOI, Shoko

45

SUZUKI, Ryoko

36

OTA, Kyoko

79

SUZUKI, Tokiko

52

OTA, Ryoma

20....................................................................................................
39
SADAKANE, Osamu

T ....................................................................................................
TAKABAYASHI, Junji

4

TAKADA, Ritsuko

23

SAITO, Junko

42

TAKADA, Shinji

4, 23

SAITO, Miyuki

8

TAKAGI, Chiyo

17, 80

SAKAE, Yuta

32

TAKAGI, Yukari

73, 80SAKAGAMI, Mari

34

TAKAHARA, Masahiro

48

SAKAMOTO, Yuki

76

TAKAHASHI, Hiroki

17

SAKURABA, Jyuichi

36

TAKAHASHI, Ritsue

79

SAKUTA, Hiraki

36

TAKAHASHI, Yoichi

39

SANJO, Kazuko

48, 79TAKAJI, Masafumi

39

INDEXTAKAKI, Chikako

52

WATAKABE, Akiya

TAKAO, Daisuke

67

WATAKABE, Ikuko

TAKASHIRO, Kayoko

23

WATANABE, Eiji

TAKEDA, Naoya

48

WATANABE, Estuko

8

TAKEDA, Yuta

39

WATANABE, Seiko

56

7, 52

WATANABE, Takaaki

7, 59

TAKEHANA, Yusuke
TAKEUCHI, Tomoyo

56

TAKEUCHI, Yasushi

36, 80

TAKIZAWA, Kenji

63

TAMADA, Yosuke

45, 81, 87

32
44, 73

Y ....................................................................................................
YABE, Kimihiko

34

YABE, Taijiro

23

YAMADA, Kenji

8

TAMESHIGE, Toshiaki

34

YAMAGUCHI, Chinami

8

TANAKA, Ayumi

4

YAMAGUCHI, Katsushi

68, 80, 86

8

YAMAMORI, Tetsuo

4, 39, 83

TANAKA, Minoru

32, 73, 84

YAMAMOTO, Kumi

72, 80TANAKA, Yasuhiro

7, 42

YAMAMOTO, Manami

TANAKA, Mina

20

42

YAMAMOTO, Masayuki

2

TANIGUCHI-SAIDA, Misako

70, 80

YAMAMOTO, Takamasa

17

TATEMATSU, Kiyoshi

34, 85

YAMAMOTO, Yasuhiro

32
48

TANAKA, Yasuyo

TESHIMA, Yuuko

52

YANO, Koji

TOHYAMA, Saki

60

YASUE, Naoko

11

TOKUE, Moe

29

YATSU, Ryohei

60

TOKUTSU, Ryutaro

63

YONEMITSU, Masako

32

TOMIDA, Sanai

23

YONEZAWA, Harumi

59, 63TORII, Naoko

52

YORO, EmikoTOYOKURA, Koichi

34

YOSHIDA, Shosei

TOYOOKA, Yayoi

26

YOSHII, Tomoaki

11

TOYOTA, Kenji

60

YOSHIMURA, Yuriko

52

TSUGANE, Kazuo

U

39

57, 75

TSUGE, Toyoko

17

TSUKITA, Sachiko4

TSUNOKUNI, Hiroyuki

23

TSUZUKI, Shihoko

80

TSUZUKI, Shusaku

48

TSUZUKI, Yumiko

34

48
4, 29, 79, 84

YOSHINORI, Yumi

8

YU, Yang

36

Z ....................................................................................................
ZHANG, Liechi

45....................................................................................................
UCHIYAMA, Ikuo
65, 72, 86

UEDA, Chizuru

8UENO, Naoto

4, 17, 87

UKAI, Sakie

23

UNO, Satoko

80

URAWA, Hiroko

34

UTSUMI, Hideko

23, 80

V ....................................................................................................
VILLARREAL, Natalia M.

45

VINCENTE, Jesus C.

45

VOLK, Christian Louis45

W ....................................................................................................
WADA, Kotoe

36

WAKAZUKI, Sachiko45, 68

WAKE, Hiroaki

7, 42

WANGLAR, Chimwar
WASHIO, Midori

23
20, 4591

Access

From Tokyo
Take the Tokaido Shinkansen and get off at Toyohashi, change to
the Meitetsu Line and get off at Higashi Okazaki Station(approximately 20 minutes from Toyohashi).

From Osaka
Take the Tokaido Shinkansen and get off at Nagoya, change to
the Meitetsu Line and get off at Higashi Okazaki Station(approximately 30 minutes from Nagoya).

From Central Japan International Airport
Get on the Meitetsu bus bound for JR Okazaki Station andget off at Higashi Okazaki Station(approximately one hour from the airport). Alternatively,
take the Meitetsu Line for Nagoya and change at Jingu-mae Station toa Toyohashi-bound train and get off at Higashi Okazaki Station(approximately 70 minutes from the airport).

From Higashi Okazaki Station to Each Area
Turn left (south) at the ticket barrier and exit the station.
The Institute is a 7-minute walk up the hill
(Myodaiji-area) or 20-minute walk (Yamate-area).

By carTake the Tomei Expressway to the Okazaki Exit.
Turn left at the City Office S.E. signal(approximately 10 minutes from the Exit).