INTRODUCTION

The Great East Japan Earthquake hit Northern Japanon March 11, 2011 and the subsequent collapse of
nuclear power plants made us reexamine how to integratescience and technology with our culture. Though we did not
suffer any direct damage to our personnel or facilities in
Okazaki, many laboratories of basic biology lost theirbioresources and facilities in the northern regions of Japan.
We appreciated of the outpouring of help and support
provided from colleagues of other countries proposing toshare facilities and space to allow interrupted research tocontinue. Our institute also offered help and similar support
to the researchers and students who suffered damage (asdescribed on p. 5). In addition, we began preparation of anovel program of storing important bioresources as a defense
against their loss or damage by future natural disasters. We
are planning to prepare a special facility named IBBP Center(p. 5) with liquid-nitrogen tanks and freezers for protecting
the bioresources and responding to the demands ofresearchers of nationwide universities and institutes. The
program will start in 2012.
Our institute, the National Institute for Basic Biology(NIBB), has developed as a center of excellence in research,education, and inter-university collaboration in the various
fields of basic biology since its foundation in 1977. In orderto carry out our mission we have focused our efforts on five
major activities, namely Promotion of Collaborative
Research Projects, Promotion of Academic Research,
Development of New Academic Fields, Cultivation of Future
Researchers, and International Cooperation and Outreach.
Our works and results for 2011 are shown in this report.
We welcomed 11 new members to the Advisory Committee
for Programming and Management of our institute in April
2011 as shown in the list on page 3. Prof. Iguchi and Prof.
Kondo were elected to be the chairperson and the vicechairperson, respectively. Their term is 2 years.
In 2011 we welcomed several new colleagues, including 1
professor, 2 assistant professors and 10 NIBB research
fellows, while 4 colleagues left the institute as shown on
page 7. With the help of these new members, we produced avariety of high-ranked research as reported from page 8 to
73. The intensive activities of our supporting divisions areshown on the following pages 74–80.
In addition to our current research, education of the next
generation of researchers is another important aim of NIBB.
As a department of the Graduate University for Advanced
Studies, we are engaged in education of graduate students.
Our students joined the 2 nd NIBB-EMBL PhD minisymposium in November (page 81). We also continued the
NIBB Internship Program and opened our laboratories tostudents and interns from abroad (page 85).

Owing to the large earthquake and the following confusion,
many plans for our international collaborative activities wereforced to be postponed or cancelled. Nevertheless, we wereable to host the first NIBB-Princeton Symposium in Okazaki
in November (page 83), and co-sponsor the third NIBB-TLLMPIPZ joint symposium at Temasek Life Sciences
Laboratory, Singapore in November (page 82). We deeply
appreciate the efforts and support of the people involved inthese activities.
Based on our endeavors, as shown in this booklet, we hope
to develop joint activities in tight collaboration with external
researchers and supporters. We hope you enjoy reading about
the science being done at NIBB in the following pages. As
always we appreciate your suggestions and comments on our
activities.
Kiyotaka OKADA, D. Sci.
Director-General, NIBB
June 4, 2012

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 (OIIB) was established as
a common facility for the three institutes (NIBB, NIPS, andIMS) of the Okazaki campus.

■ Organization

National Institutes of
Natural Sciences(NINS)
President
SATO, Katsuhiko

National Astronomical
Observatory of Japan (NAO)
National Institute for
Fusion Science (NIFS)

National Institute for
Basic Biology (NIBB)

Advisory
Committee
forProgrammingandManagement

Director-General
OKADA, Kiyotaka
Vice Director
YAMAMORI, Tetsuo

National Institute forPhysiological Sciences (NIPS)
Institute for
Molecular Science (IMS)
Three Institutes in Okazaki
Center for Novel Science Initiatives

Buildings in the Myodaiji area (left) and Yamate area (right).

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 planninga long-range strategy for the institute. The Office of Public
Relations and the Office of International Cooperation are
central offices for public relations and the management ofconferences and other extramural activities.
Administration of NIBB is undertaken by the Okazaki
Administration Office under the direct auspices of the
Administration Office of NINS.

2Okazaki Research Facilities
Okazaki Institute forIntegrative Bioscience (OIIB)

Department of Development, Differentiation
and Regeneration■Division of Developmental Genetics *■Division of Molecular and Developmental Biology **Department of Bio-Environmental Science■Division of Bio-Environmental Science ***■Division of Neuronal Cell Biology ****
Department of Strategic Methodology*-****)These divisions also function as NIBB’s divisions†-††††) , respectively.

Other divisions of the OIIB are not shown.

Center for Radioisotope Facilities
Center for Experimental Animal
Research Center for Computational Science

Research Facilities run jointly by
NIBB and NIPS
Electron Microscopy Room
Disposal of Waste Matter Facility
Instrument Design RoomNational Institute for Basic Biology (NIBB)

As of April 1, 2012

Research Units■Division of Cell Mechanisms

Cell Biology■Division of Intercellular Signaling Biology■Laboratory of Neuronal Cell Biology††††■Laboratory of Cell Sociology■Division of Morphogenesis

Developmental Biology■Division of Developmental Genetics †■Division of Molecular and Developmental Biology ††■Division of Embryology■Division of Germ Cell Biology■Laboratory of Molecular Genetics for Reproduction■Laboratory of Plant Organ Development■Division of Molecular Neurobiology

Neurobiology■Division of Brain Biology■Division of Brain Circuits

Strategic
Planning
Department■Laboratory of Neurophysiology

Evolutionary Biology and Biodiversity■Division of Evolutionary Biology■Division of Symbiotic Systems■Laboratory of Morphodiversity■Laboratory of Bioresources■Laboratory of Biological Diversity

Environmental Biology
Theoretical Biology
Imaging Science■Division of Molecular Environmental Endocrinology †††■Division of Environmental Photobiology■Laboratory of Genome Informatics■Laboratory for Spatiotemporal Regulations

Research Support UnitsNIBB Core Research Facilities
NIBB BioResource Center

Functional Genomics Facility
Spectrography and Bioimaging Facility
Data Integration and Analysis Facility
Model Animal Research Facility
Model Plant Research Facility
Cell Biology Research Facility

NIBB Center of the Interuniversity Bio-Backup Project (IBBP Center)

This center is scheduled to open in July, 2012.

Other Research Support Sections
Technical Division

Office of Public Relations
Office of International Cooperation

Okazaki Administration Office3

Research and Research Support

NIBB’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 Interuniversity Bio-Backup Project (IBBP
Center) will be 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. The Center forRadioisotope Facilities are one of the latter and are run by
the technical 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.

■ Members in NIBB
Director-General 1
Professor 14
Associate Professor (including
specially appointed and OIIB
adjunct) 14

Technical
Assitant
andSecretary

Assistant Professor (incl. sp. app.)

34

112

69
27
Technical
Staff

Postdoctoral Fellow (15 are
NIBB Research Fellows)40
311 in total(as of 1st April, 2012)

Graduate Student■ Financial Configuration of NIBB
External
Competitive
Fund

1157

Grants forCommissioned
Research(settled forApril 2010-March 2011)
numbers are in million yen
Grant-in-Aid
for Scientific
Research

1001

54

695

Research

431

156

Management
Expenses
Grant for
SOKENDAI

Personnel

Other

25

Collaboration

Management
Expenses Grant
for NIBB

1,431

2,672 million
yen in total

310■ Members of the Advisory Committee for Programming and Management* (as of April, 2012)

Chairperson

Vice-Chair
Non-NIBB members

NIBB members* new member from April 2011** new member from April 2012

4

IGUCHI, Taisen
KONDO, Takao*
HIGASHIYAMA, Tetsuya**
MIZUSHIMA, Noboru*
MORI, Ikue*
SHIMAMOTO, Ko*
SIOMI, Haruhiko*
TAKABAYASHI, Junji*
TANAKA, Ayumi*
UEMURA, Tadashi*
YAMAMOTO, Masayuki*
FUJIMORI, Toshihiko
HASEBE, Mitsuyasu
KAWAGUCHI, Masayoshi*
KOBAYASHI, Satoru
NISHIMURA, Mikio
NODA, Masaharu
TAKADA, Shinji
UENO, Naoto
YAMAMORI, Tetsuo
YOSHIDA, Shosei

Professor, Okazaki Institute for Integrative Bioscience
Professor, Nagoya University
Professor, Nagoya University
Professor, Tokyo Medical and Dental University
Professor, Nagoya University
Professor, Nara Institute of Science and Technology
Professor, Keio University
Professor, Kyoto University
Professor, Hokkaido University
Professor, Kyoto University
Director, Kazusa DNA Research Institute
Professor, National Institute for Basic Biology
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, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology

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
contribute to the world-wide community of biologists throughour efforts to accomplish these goals. This chapter briefly
explains four of these goals. The last goal, the promotion ofacademic research, is accomplished through our research
activities, which are introduced throughout this brochure.

Promotion of Collaborative Research
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 groupresearch by internal and external researchers with the purpose ofdeveloping pioneering research fields in biology and the“collaborative research projects for model organism/technology
development” are for developing and establishing new modelorganisms 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
we began to solicit special emergency collaborative research
projects to allow researchers whose labs sufferred damage tostay at our institute to continue their work. Six projects fromTohoku University, Chiba University, and the University ofTokyo etc. were granted.
year

Facility Use (Training Course Facility)

total

2008 2009 2010 2011
0

1

4

3

3

2

6
2

49
5
11
−

54
3
10
−

68
3
8
7

88
6
9
8

−

−

11

45

−
68

−
71

1
94

0
164

Collaborative Research Projects by Year

■ NIBB Core Research Facilities

Contribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research■ Collaborative Research Support

Priority collaborative research projects
Collaborative research projects formodel organisms/ technology development
Individual collaborative research projects
NIBB workshops
Collaborative experiments using the large spectrograph
Collaborative experiments using the DSLM
Collaborative experiments using next
generation DNA sequencers

International cooperation
and Outreach

The NIBB Core Research Facilities support research in NIBB
and also act as an intellectual hub to promote collaboration
among the researchers of NIBB and other academic institutions.
They consist of three facilities that are developing and providing
state-of-the-art technologies through functional genomics,
bioimaging and bioinformatics (p. 69).
The Functional Genomics Facility maintains a wide array ofcore research equipment, including cutting edge tools such as
next generation DNA sequencers. The facility is dedicated tofostering 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 a part of this undertaking(p. 84). The Spectrography and Bioimaging Facility manages
research tools, such as confocal microscopes, the DSLM and thelarge spectrograph, and provides technical support and scientificadvice to researchers. These two facilities hold speciallyappointed associate professors, an expert in each field, with a

Developmentof new fields

Cultivation of
future researchers

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 fascility maintains highperformance computers with large-capacity storage systems.

■ NIBB Center of the Interuniversity Bio-Backup
Project (IBBP Center)

The Great East Japan Earthquake caused massive damage toimportant biological resources. To prevent such loss, NIBB will
establish the IBBP Center in 2012 in collaboration with sevennational universities for multiplicate preservation of genetic
libraries and other invaluable bioresources.

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 andtechnological exchange through joint meetings, exchange
between researchers and graduate students, and the introductionof experimental equipment. Graduate students were sent toattend the PhD students symposium held in EMBL, Heidelberg
in November, 2011 (p. 81).
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
in the field of plant sciences. NIBB and MPIPZ work together toplan and promote joint research projects, collaborative symposia,
training courses, and student exchange programs.
Collaborative programs have also been started with theTemasek Life Sciences Laboratory (TLL), of Singapore andPrinceton University. The 3 rd NIBB-TLL-MPIPZ Joint
Symposium “Cell Cycle and Development” was held in
November, 2011, at TLL (p. 82). The 1 st NIBB-Princeton
Symposium “Proteomics, Metabolomics, and Beyond” was held
in November, 2011, at NIBB (p. 83).

■ 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
conference “Gamete Stem Cells” had been planned to take placein July, 2011, but was postponed because of the earthquake. It
will be held in July, 2012, in conjunction with the 60 thconference under the title of “Germline”. The 59th conference5

“Neocortical Organization” was held March, 2012, and willappear in 2012 Annual Report.

A frame from a 3D movie ofliving Amoeba proteus firstpossible using DSLM due toits fast image taking
capability.

■ International Practical Course

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 6th course “Developmental
Genetics of Medaka IV” was held jointly with TLL in
November, 2011 (p. 84). Graduate students and young
researchers from various areas including Taiwan, Norway, India,
and Singapore, were provided with training in state-of-the-art
research techniques. International conferences and courses aremanaged by the Office of International Cooperation (see page79).

■ Bio-Resources

The National BioResource Project (NBRP) is a national project
for the systematic accumulation, storage, and supply ofnationally recognized bio-resources (experimental animals andplants, cells, DNA, and other genetic resources), which arewidely used as materials in life science research. To promote thisnational project, NIBB has been appointed as a center for
research on Medaka (Oryzia latipes) whose usefulness as avertebrate model was first shown 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 to maintain Medaka
and Japanese morning glory safely, efficiently, and appropriately.
The center also maintains other model organisms, such as mice,zebraﬁsh, Arabidopsis, Lotus japonicus, and Physcomitrella
patens, and provides technical support and advice for theappropriate use of these organisms (p. 74).

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 opend a video gallery contaning 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 aremanaged by the Office of Public Relations (p. 79).

model (p. 68). The Advisory Committee on Bioimaging,comprised of leading researchers in the bioimaging field inJapan, is organized to formulate advice on NIBB’s imaging
research. The Bioimaging Forum provides an opportunity for
researchers and company engineers to frankly discuss practicaldifficulties and needs regarding imaging.

■ Okazaki Biology Conferences

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 andsupport the formation of international communities in thesefields. Dozens of top-level researchers from Japan and abroad
spend nearly one week together for intensive discussions seeking
strategies for addressing future critical issues in biology. Past
Conferences have promoted the formation of international
researcher communities. The OBC8 “Speciation and Adaptation
II: Environment and Epigenetics” was originally planned forMarch, 2011 but was postponed due to the earthquake and washeld in March, 2012, which will appear in the 2012 Annual
Report.

Cultivation of Future Researchers

NIBB constitutes the Department of Basic Biology in the
School of Life Science of the Graduate University for Advanced
Studies (SOKENDAI). The department provides a five-year
course for university 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 are provided an opportunity to give oral and poster
presentations at least once during their master’s and doctoral
program (p. 81).
Students from Japan and abroad can also come to NIBB
through our Internship Program. Internships give students an
excellent way to build international connections while
experiencing hands on research in a world class research
institute (see page 85).

Development of New Fields of Biology■ Bioimaging

NIBB aims to maximize the application of modern lightmicroscopes and biophotonic probes for real time visualizationof 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 living samples, and has developed an improved

6

Graduate students educated by NIBB■ Personnel changes in 2011*

Newly assigned in NIBB
Name

Position

Research Unit

Date

MATSUBAYASHI, Yoshikatsu

Professor

Division of Intercellular Signaling Biology

January 1

SHINOHARA, Hidefumi

Assistant Professor

Division of Intercellular Signaling Biology

February 1

TAKIZAWA, Kenji

NIBB Research Fellow

Division of Environmental Photobiology

February 1

OHTSUKA, Masanari

NIBB Research Fellow

Division of Brain Biology

February 1

CHAKRABORTY, Tapas

NIBB Research Fellow

Division of Molecular Environmental Endocrinology

February 1

KOYAMA, Hiroshi

Assistant Professor

Division of Embryology

April 1YAMAMOTO, Yasuhiro

NIBB Research Fellow

Laboratory of Molecular Genetics for Reproduction

April 1NAKAMURA, Yoshiaki

NIBB Research Fellow

Division of Germ Cell Biology

April 1

SATO, Yasufumi

NIBB Research Fellow

Division of Embryology

April 1OSHIMA, Yusuke

NIBB Research Fellow

Laboratory for Spatiotemporal Regulations

April 1KUBOYAMA, Kazuya

NIBB Research Fellow

Division of Molecular Neurobiology

May1

NAKAYASU, Tomohiro

NIBB Research Fellow

Laboratory of Neurophysiology

July 1

OTA, Ryoma

NIBB Research Fellow

Division of Developmental Genetics

December 1

Newly affiliated in other universities and institutes
Name

New Affiliation

Position

Date

OKUBO, Tadashi

Kitasato University

Lecturer

April 1

NAGAHAMA, Yoshitaka

Ehime University

Professor (specially appointed)

April 1

MATSUNAGA, Wataru

Nara Prefectural University

Assistant Professor

July 1

KAGEYAMA, Yuji

Kobe University

Associate Professor

October 1* Changes in professors, associate and assistant professors, and NIBB research fellows are shown.

■ Awardees in 2011

Name

PositionAward

OKAMOTO, Satoru
KAWAGUCHI, Masayoshi

NIBB Research Fellow
Professor

TANAKA, Minoru

Associate Professor

JSPP Plant and Cell Physiology Award for the Paper of
Excellence (PCP Award)
The Commendation for Science and Technology by the
Minister of Education, Culture, Sports, Science and Technology

IGUCHI, Taisen

Professor

The Zoological Society of Japan Award

HIYAMA, Takeshi

Assistant Professor

Japan Neuroscience Society Young Investigator Award

Note: On the unit member lists from P. 8 all members who belonged to the unit during 2011 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 2011. The former title is indicated by an asterisk (*).

7

National Institute for Basic Biology

Cell Biology

DIVISION OF CELL MECHANISMS

Professor

Associate Professor

Assistant Professors:

MANO, Shoji
YAMADA, Kenji
KONDO, Maki
OIKAWA, Kazusato
KANAI, Masatake
WATANABE, Etsuko
TANAKA, Mina
KAMIGAKI, Akane
GOTO-YAMADA, Shino
GOTO-YAMADA, Shino*
NAKAI, Atsushi
CUI, Songkui
SHIBATA, Michitaro
NISHINA, Momoko
ARAKI, Masami
SAITO, Miyuki
NAKAYAMA, Tomomi
HIKINO, Kazumi
MATSUDA, Azusa
YOSHINORI, Yumi
YAMAGUCHI, Chinami
UEDA, Chizuru

NISHIMURA, Mikio

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:

Technical Assistants:

Secretary:

HAYASHI, Makoto

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.

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 via b-oxidation and the glyoxylate
cycle, are transformed into leaf peroxisomes that function inseveral crucial steps of photorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during thegreening of pumpkin cotyledons, the reverse transition of

8leaf peroxisomes to glyoxysomes occurs during senescence.
Gene expression, alternative splicing, protein translocationand protein 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.

Bioinfomatic 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 thereceptor-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
proposed that PEX10 is essential for the maintenance of ER
morphology and for biosynthesis of cuticular wax.

III. Identification of novel components
essential for peroxisome biogenesis

To better understand peroxisome biogenesis, we isolated anumber of Arabidopsis mutants having aberrant peroxisome
morphology (apem mutants) based on them having aNote: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

different pattern of GFP fluorescence from the parent plant,
GFP-PTS1, in which peroxisomes with normal sizes andnumbers 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, one member of the dynaminfamily. APEM2 and APEM4 (whose defects cause a decreasein the efficiency of protein transport) were revealed to
encode proteins homologous to PEX13 and PEX12,respectively, and both proteins are components of theprotein-translocation machinery on peroxisomal membranes.
In addition, we reported on characterization of other apem
mutants, apem3 and apem9. APEM3 encodes Peroxisomal
membrane protein 38, and its defect causes enlargement of
peroxisomes. APEM9 is the plant-specific PEX that has arole in tethering the PEX1-PEX6 complex on peroxisomal
membranes (Figure 1).

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
PYK10, a b-glucosidase with an ER retention signal. Whenplant cells are damaged, PYK10 forms large protein
aggregates that include other b-glucosidases (BGLUs),
GDSL lipase-like proteins (GLLs) and cytosolic jacalinrelated lectins (JALs). The aggregate formation increases
glucosidase activity, possibly producing toxic products(Figure 2). Arabidopsis nai1 mutants have no ER bodies inthe entire plant and do not accumulate PYK10. NAI1 encodes
a transcription factor that has a basic-helix-loop-helix(bHLH) domain and regulates the expression of PYK10 and
NAI2. The Arabidopsis nai2 mutant has no ER bodies andreduced accumulation of PYK10. NAI2 encodes a uniqueprotein that localizes to the ER body. We found that themembrane protein of ER body 1 (MEB1) and MEB2 areintegral membrane proteins of the ER body. NAI2 deficiency
relocates MEB1 and MEB2 to the ER network. Thesefindings indicate that NAI2 is a key factor that enables ER
body formation. We are now investigating the function of
NAI2 on ER body formation by heterologously expressing it
in onion and tobacco cells.

V. Vacuoles responsible for programmed cell
death in plants

The vacuolar processing enzyme (VPE) belongs to thecysteine protease family found in higher plants and animals.
VPE is responsible for the maturation of various types of
vacuolar proteins. We revealed a novel function of VPE invarious instances of programmed cell death (PCD) in plants.
VPE is identified as the proteinase that exhibits caspase-1

Figure 1. APEM9 is required for peroxisomal protein transport. (A)APEM9 knockdown mutants (apem9i) show growth defects. (B)Peroxisomal localization of GFP-PTS1 protein in wild type plants. (C)GFP-PTS1 protein is accumulated in the cytosol in apem9i mutants. (D)Working model of peroxisomal protein transport in plants, in which
APEM9 tethers PEX1-PEX6 complex to the peroxisomal membrane.

We are currently analyzing the functions of other APEM
proteins such as APEM10 and APEM11. From theseanalyses, we will be able to identify the components
responsible for peroxisome biogenesis, and to address themechanism at the molecular level.

Figure 2. Activation model of β-glucosidases in ER bodies of
Arabidopsis. (A) Healthy plants accumulate β-glucosidases (BGLUs) in
the ER body, GDSL lipase-like proteins (GLLs) and glucosides in thevacuole, and jacalin-related lectins (JALs) in the cytosol. (B) When cellcollapse occurs following herbivore or pathogen attack, β-glucosidases
leak from the ER body and bind to JALs, which then form a large
aggregate with increased glucosidase activity. This complex hydrolyzes
glucosides to produce toxic compounds.

9National Institute for Basic Biology

Cell Biology

activity in plants. The plant hypersensitive response (HR), a
type of defense strategy, constitutes well-organized PCD. No
HR occurs on the tobacco mosaic virus-infected leaves of
VPE-deficient tobacco plants. These results suggest that VPE
is involved in vacuolar collapse, which triggers PCD.
Using inhibitors for caspase-3 and the proteasome (alsoknown to affect animal cell death), we found that theactivities of both are required for bacterium-induced cell
death in plants. RNA interference-mediated silencing
confirmed that one of the three Arabidopsis proteasome
catalytic subunits, PBA1, is required for the fusion of thevacuolar and plasma membranes, which triggers PCD.
Plants evolve a death strategy mediated by vacuolar
systems, which are not seen in animals. Interestingly,
vacuoles are the key players in the plant-specific cell deathsystem.

Organelles World, which is based on PODB2 as an
educational tool to engage members of the non-scientificcommunity. We expect that PODB2 and Plant Organelles
World will enhance the understanding of plant organelles
among researchers and the general public who want to
explore plant biology.

VI. 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. To clarify the roles of molecularchaperones on cell differentiation, we have purified andcharacterized chaperonin and HSP70s and analyzed theirroles in the translocation of proteins into chloroplasts.
We found that HSP90 inhibitor induced genes with heatshock response element (HSE) motifs in their promoters,suggesting that heat shock transcription 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. This data indicates that HSP90 regulates correct
gene expression for heat acclimatization in plants. We also
observed that HSP90 is involved in hormone responses in
Arabidopsis. The evolutional and functional characterizations
are now being investigated.

VII. Update of The Plant Organelles Database
2 ( P O D B 2 ) a n d re l e a s e o f P l a n t
Organelles WorldThe 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 four individual units: the organelles
movie database, the organellome database, the functional
analysis database, and external links. 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 toorganelle movement and morphology in response toenvironmental stimuli, soon. It is expected that PODB2 willcontribute to systems biology through the combination of the
included data with other ‘omics’ data and computational
analyses. In addition, we released a new website, Plant

10

Figure 3. The graphical user interface of the organelles movie database in
PODB2 (http://podb.nibb.ac.jp/Orgenellome).

Publication List

〔Original papers〕

S., Mano, S., Nakamori C., and Nishimura, M. (2011).
• Goto,Arabidopsis ABERRANT PEROXISOME MORPHOLOGY 9 is a

•
•
•
•

peroxin that recruits the PEX1-PEX6 complex to peroxisomes. Plant
Cell 23, 1573-1587.
Hino, T., Tanaka, Y., Kawamukai, M., Nishimura, K., Mano, S., andNakagawa, T. (2011). Two Sec13p homologs, AtSec13A and AtSec13B,
redundantly contribute to formation of COPII transport vesicles in
Arabidopsis thaliana. Biosci. Biotechnol. Biochem. 75, 1848-1852.
Mano, S., Miwa, T., Nishikawa, S., Mimura, T., and Nishimura, M.
(2011). The Plant Organelles Database 2 (PODB2): An updated
resource containing movie data of plant organelle dynamics. Plant Cell
Physiol. 52, 244-253.
Mano, S., Nakamori, C., Fukao, Y., Araki, M., Matsuda, A., Kondo, M.,
and Nishimura, M. (2011). A defect of peroxisomal membrane protein
38 causes enlargement of peroxisomes. Plant Cell Physiol. 52, 21572172.
Masuda, S., Harada, J., Yokono, M., Yuzawa, Y., Shimojima, M.,
Murofushi, K., Tanaka, H., Masuda, H., Murakawa, M., Haraguchi, T.,
Kondo, M., Nishimura, M., Yuasa, H., Noguchi, M., Oh-oka, H.,
Tanaka, A., Tamiaki, H., and Ohta, H. (2011). A
monogalactosyldiacylglycerol synthase found in the green sulfur
bacterium Chlorobaculum tepidum reveals important roles forgalactolipids in photosynthesis. Plant Cell 23, 2644-2658.

〔Review Article〕

K., Hara-Nishimura, I., and Nishimura, M. (2011). Unique• Yamada,
defense strategy by the endoplasmic reticulum body in plants. Plant
Cell Physiol. 52, 2039-2049.

DIVISION OF INTERCELLULAR SIGNALING BIOLOGY

Professor

MATSUBAYASHI, Yoshikatsu

Assistant Professor:
Visiting Scientists:
Technical Assistants:
Secretary:

SHINOHARA, Hidefumi
MORI, Ayaka
YOSHII, Tomoaki
OGAWA-OHNISHI, MariSUMIDA, Kumiko
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.

root 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
tyrosine sulfation of secreted peptides and proteins. Thisapproach is based on the assumption that the severe short

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

11National Institute for Basic Biology

Cell Biology

III. Receptors for secreted peptide signals

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 b-1-2-linked triarabinoside involves two distinct
arabinosyltransferases. The first is responsible for theformation of a b-linkage with the hydroxyproline(hydroxyproline arabinosyltransferase), and the second forms
a b-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.

2-3 Chemical synthesis of arabinosylated peptides

Although extensive efforts have been devoted to thesynthesis of various glycopeptides, a general andstereoselective synthetic route for arabinosylated peptides
with b-1-2-linkage has yet to be developed. To address thisissue we are attempting to synthesize b-1-2-linked triarabinosylated glycopeptides in a stereoselective manner.

12

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.

3- 2 Structural basis for ligand recognition

The identification of ligand-binding sites within receptor
ectodomains provides valuable structural information
important for understanding the basis of ligand recognitionand signal transduction. We are analyzing how large
consecutive LRRs specifically recognize small peptide
ligands by focusing on CLV1/BAM family receptor kinases
as a model.

Publication List

〔Review articles〕

Y. (2011). Post-translational modifications in secreted• Matsubayashi,
peptide hormones in plants. Plant Cell Physiol. 52, 5-13.
Y. (2011). Small post-translationally modified peptide• Matsubayashi,
signals in Arabidopsis. The Arabidopsis Book 9, e0150.

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.
We are researching factors regulating mRNA transport andlocal translation, their target mRNAs, and the mechanisms of
localized protein synthesis using mice in order to better
understand its relation to the formation of synapses andneural networks, memory, learning, and behavior.

I. Regulation of transport and local translationof dendritically-targeted mRNAs

Specific mRNAs are recruited into “RNA granules” inneuronal dendrites. RNA granules are macromolecularcomplexes composed mainly of mRNAs and ribosomes, andmediate the transport and local translation of their mRNA
cargoes in response to synaptic stimulation (Figure 1).

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.

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).
To understand the regulation of transport and local
translation of dendritically-targeted mRNAs, we haveidentified RNG105 cargo mRNAs in neurons. The cargo
mRNAs, e.g., that encode Na+/K+ ATPase subunit isoform
protein (FXYD1), are recruited to RNA granules andtransported to dendrites. Furthermore, translation of themRNAs is upregulated by stimulation of neurons with brainderived neurotrophic factor (BDNF) which mimics synaptic
stimulation (Figure 2). We are currently investigating themolecular mechanism of the mRNA transport to dendrites
and BDNF-stimulated translational upregulation of the cargo
mRNAs.

Figure 2. Translation of FXYD1-GFP is activated by BDNF in mouse
cultured hippocampal neurons. Numbers indicate time (minute) after
BDNF stimulation. FXYD1-GFP is increased in membranecompartments in the cell body (an arrow) and dendrites (arrowheads)
after BDNF stimulation (right panel). Scale bar, 10 mm.

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 in the cells. They are called “stress granules” andhave common features with neuronal RNA granules, e.g.,they are macromolecular complexes containing ribosomes
and mRNAs, and repress translation of cargo mRNAs
without stimulation. We previously found that RNG105 is acomponent of not only neuronal RNA granules but also stressgranules and further that overexpression of RNG105 infibroblastic cells induced the formation of granules whichcontain mRNAs.
To identify and characterize molecular components of RNA
granules, we have performed mass spectrometric analysis of
the RNG105-induced complex. A cultured cell line A6
transfected with RNG105-green fluorescent protein(RNG105-GFP) forms prominent granules in the cytoplasm(Figure 3). The transfected cells are subjected toimmunoprecipitation with an anti-GFP antibody (Figure 3).
Proteins co-precipitated with RNG105-GFP are thenanalyzed by mass spectroscopy, which revealed that several
proteins responsible for RNA metabolism, membranetransport, posttranslational modification, etc. are contained in
the complex. We are currently investigating the localizationand function of the proteins in stress granules as well as inneuronal RNA granules.

13National Institute for Basic Biology

Cell Biology

Figure 3. Identification of components of RNG105-induced granules. (A)A6 cells expressing RNG105-GFP. RNG105-GFP induced the formationof cytoplasmic granules. Scale bar, 10 mm. (B) SDS-PAGE of
immunoprecipitants with an anti-GFP antibody from control A6 cells(left lane) and A6 cells expressing RNG105-GFP (right lane). The
positions of RNG105-GFP (an arrow) and immunoglobulin (IgG) areindicated.

III. Knockout mice for RNA granule proteins

We previously generated RNG105 knockout mice. Neurons
from the knockout mice exhibited reduced dendritic synapse
formation and reduced dendritic arborization, which resulted
in poor development of neuronal networks. The knockout
neonates died soon after birth due to respiratory failure,
which was associated with defects in fetal brainstemdevelopment. To investigate the role of RNG105 in higher
brain functions, e.g., memory and learning, in adult mice, wehave generated conditional RNG105 knockout mice using
the Cre/loxP system. Alpha-CaMKII promoter is used todrive Cre recombinase since its promoter activity is low
during embryonic stages but elevated after birth in the brain.
However, even though the Cre/loxP system is used, we have
not obtained adult RNG105 knockout mice. Therefore we areplanning to generate drug-inducible RNG105 knockout 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.

14

LABORATORY OF CELL STRUCTURE †Associate Professor

OGAWA, Kazuo

Microtubules are polymers of a- and b-tubulin heterodimer. In vitro, we can induce microtubule formation at
critical points where tubulins concentrate. In vivo, they areformed by g-TuRC at the centrosome or may be formed byunknown protein(s) in the non-centrosome region. The fastergrowing end is defined as the plus-end of the microtubules.
There are many plus-end tracking proteins of microtubules
such as EB-1, CLIP-170, and dynactin et al. The minus-ends
are considered to be the nucleation sites for microtubule
polymerization. Microtubule nucleation at the noncentrosome region remained less clear.
Several antibodies were raised for answering this unsolved
question and checked whether they stain the minus-end of
microtubules by immunofluorescence microscopy. For
simplicity, the primary cilia of three cell lines established in
our laboratory were used instead of microtubules. If
antibodies are able to react with the minus end of
microtubules, they should bind to the base of primary cilia inthe same way as g-TuRC and antigens for those antibodies
should exist there.
Finally one anti-serum successfully stained the bases of the
primary cilia of cultured cells examined so far (Figure 1). I
expect further interesting results will be forthcoming.

Figure 1. Antibodies stained the minus-end of the primary cilia. Green,candidate protein; red, acetylated tubulin (primary cilia); blue, DNA
stained with DAPI.

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

15

National Institute for Basic Biology

Cell Biology

LABORATORY OF CELL SOCIOLOGYof trophoblast cell death. We are now carrying out studies on
how Notch2 participates in the cell death process and how
the gene is activated in the trophoblast in cell culture.

Assistant Professor

HAMADA, Yoshio

Technical Assistants:

GONDA, Naoko

Animal organs are made up of several types of cells, andorganized in an orderly fashion wherein the proportion ofeach cell type is constantly maintained. This orderly cellarrangement and proportion are built up duringorganogenesis by cell-cell interactions. Since it has been
postulated that Notch plays a role in cell fate decisions by
mediating cell-cell interactions, we are endeavoring todiscover the cellular and molecular mechanisms at work
during organogenesis by studying the function of Notch.
Organogenesis of the mouse placenta occurs during earlypregnancy, embryonic days 7-9, before the establishment ofmolecular transport mechanisms in the definitive placenta
takes place. Trophoblasts not adjacent to the inner cell massdifferentiate into trophoblast giant cells and lie at the outside,
forming an interface with the maternal deciduas. The polar
trophectoderm gives rise to the cells of the chorion as well as
the ectoplacental cone; these produce the labyrinthine andspongiotrophoblast layers, respectively. While maternal red
blood cells begin to perfuse into trophoblast cell layers andreach the labyrinthine layer by E9.5, the invasion ofembryonic allantoic mesenchyme into the labyrinthine layer
and the differentiation of fetal red blood and endothelial cells
which line the fetal capillary take place around E9.5.
The Notch2 null mutation results in embryonic lethality byembryonic day 11.5 due to the formation of poor maternalvascular beds. The mutant placenta shows a normal invasionof angiogenic allantoic mesenchyme followed by prematureformation of fetal blood vessels in the mutant placentas as
early as E9.0. However, the specification of trophoblast
subtypes appears not to be drastically disturbed. Thus, in thedeveloping mouse placenta, Notch2 is likely not involved incell fate decisions, but rather participates in the formation of
circulatory systems in the labyrinth layer where theexpression of Notch2 is detected. Although inadequate
formation of maternal vascular beds was partially restored byaggregating mutant diploid embryos with wild type tetraploidembryos, networks of maternal vascular beds appeared still
compromised in the 4N chimeric placenta. These results
indicate that Notch2 promotes vasculogenesis.
How maternal vascular beds are formed in the developing
mouse placenta has yet to be explored. The simplest way toform the beds among tightly adhered labyrinthine
trophoblasts is through cell death. We studied aspatiotemporal appearance of dead cells in the developing
placenta. While vasculogenesis does not occur in thepresumptive labyrinth layer at E8.5, some dying cells weredetected. At E9.5, extensive trophoblast cell death took placearound newly forming maternal blood beds. In contrast to thewild type placenta, extensive cell death did not occur in theE9.5 mutant placenta (Fig. 1). It is likely that Notch2 plays arole in vasculogenesis through being involved in the process

16

Figure 1. Programmed cell death in the developing mouse placenta.
Dying trophoblast was visualized by staining with a fluorescent dye at
E9.5. Wild type placenta showed extensive cell death around newly
forming maternal blood beds which were surrounded by Notch2
expressing trophoblast (A), but cell death was scarce in the mutant
placenta.

National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

Assistant Professors:

TAKAHASHI, Hiroki
SUZUKI, Makoto
TAKAGI, Chiyo
HASHIMOTO, Masakazu*
HASHIMOTO, Masakazu
MORITA, Hitoshi
KAI, Masatake
MORITA, Hitoshi*
HARA, Yusuke
TAKEDA, TaroMIYAGI, Asuka
HAYASHI, Kentaro
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
SUZUKI, Atsuko
MIYAKE, Satoko
TSUGE, Toyoko

UENO, Naoto

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:
Graduate Students:

Technical Assistants:
Secretaries:

KINOSHITA, Noriyuki

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.

significant neural plate bending. We reasoned that otherforces to bend the neural plate and contribute to the complete
tube closure are generated by tissue(s) outside of the neuralplate, possibly in the non-neural ectoderm. After a series of
microscopic observations and experiments using Xenopus
laevis, we have found that non-neural deep layer cells that
underlie the non-neural ectoderm migrate actively toward thedorsal side dragging the overlying superficial layer cells andFigure 1. Tracking of cell movement of the non-neural ectoderm. Deepcells, labeled red/orange, migrate faster than superficial cells, labeled
blue/green. (observation with DSLM).

bringing them to the midline (Figure 1).
This has been confirmed by the observation that disruptionof deep cell migration by inhibiting the cell-substrateinteraction between mesoderm and deep cells, or cell-cellinteraction between deep cells and superficial cells, both ofwhich are the basis of traction forces, caused neural tube
closure defects. This study highlights the importance ofphysical force during complex organ formation (Figure 2).

I. Biological significance of force fo rmorphogenesis
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. On the other hand, we also know that such cell
shape change is necessary but not sufficient to cause
complete neural tube closure because an embryo with an
absence of these cell shape changes will still display

Figure 2. Model for the coordinated movement of non-neural ectoderm.
Deep cells (dark blue) migrate on the fibronectin ahead of superficial
cells and therefore the superficial cells are dragged toward the midline.
This also causes a stretching force for passive shape change of superficial
cells.

Another example is the axial mesoderm, which elongatesalong the anterior-posterior axis during gastrulation cell
movements by which rearrangements of the three germlayers is driven. The axial mesoderm is led by the anteriorly
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 the

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

17

National Institute for Basic Biology

Developmental Biology

following axial mesoderm. To prove the biologicalsignificance of the force generated by the LEM, we haveestimated the magnitude of the traction force by measuring
displacement of a glass needle with a known spring constantby the anterior movement of the axial mesoderm.
Furthermore, we have been examining whether the artificial
application of force, for example by tissue-stretching, cancause biological outputs such as intracellular relocalizationof signaling molecules or formation of cell polarity.

II. Regulation of cell adhesion by the ubiquitin
system during gastrulation

During gastrulation, dorsal mesoderm cells migrate toward
the midline and align along the anteroposterior axis to form
the notochord. In this process, cells show polarizedmorphological change and coordinated cell migration. To
achieve this systematic cell movement, cell-to-cellinteraction must be tightly regulated. We focus on one of thecell adhesion proteins, paraxial protocadherin (PAPC). PAPC
has been shown to be involved in gastrulation cell
movements during early embryogenesis. Using Xenopus
embryos, we found that both knock down and overexpressionof PAPC in the dorsal mesoderm impair mesoderm cell
movement. PAPC knock down reduced cell-cell interaction
and cells migrate disorderly. PAPC overexpression inhibited
cell migration toward the midline. This suggests that PAPC isessential at the early stage and then must be decreased. It has
been reported that the PAPC gene is first expressed in thedorsal marginal zone at the early gastrula stage andsubsequently down-regulated in Xenopus and zebrafish. We
found that PAPC is also regulated in the same way at theprotein level (Figure 3). PAPC is degraded via lysosome andexcluded from the plasma membrane in the axial mesoderm
at the late gastrula stage. PAPC is phosphorylated by GSK3,
providing a signal for poly-ubiquitination by b-TrCP.
Localization of PAPC to the plasma membrane at the earlygastrula stage is mediated by a novel deubiquitinating
enzyme XTI3. Taken together, our findings suggest a novel
mechanism of regulation of a cell adhesion protein by theubiquitin system, which plays a crucial role in vertebrate
embryogenesis.

Figure 3. The dynamic regulation of PAPC stability during convergent
extension. PAPC-GFP is expressed with membrane-tethered RFP in
dorsal mesoderm cells. At the early gastrula stage, PAPC localized at thecell membrane and vesicles, but was drastically reduced later at theneurula stage.

18

III. C e l l u l a r m o r p h o g e n e s i sdevelopment

during

For the morphogenesis of organs, cellular morphogenesis as
well as cellular behaviors play critical roles, as discussed
above for neural tube formation of Xenopus laevis. Unlike
Xenopus and amniote such as chicks and mammals, however,some of the teleost fish display a unique way of forming theneural tube in that rather than bending of a neuroepithelial
sheet, they adopt drastic cell rearrangement of neural cellprogenitors. During zebrafish neurulation, for example,
highly motile cells converge to the midline, elongate theirshape mediolaterally, and interdigitate with contralateral cells
to form the neural rod in a highly coordinated manner. To
date, although non-canonical, the Wnt/PCP pathway andseveral cadherins have been reported to function in thisprocess, how the cytoskeleton controls these cellularbehaviors is poorly understood.
We analyzed non-muscle myosin II (NMII) activity
focusing on its regulatory light chain (MRLC) during
zebrafish neurulation. Our analysis revealed that activated
MRLCs were enriched in the cortex of highly migratoryneural cells, suggesting that NMII functions in zebrafish
neurulation. Consistent with this, inhibition of NMII
suppressed cell elongation and interdigitation. Then we
performed live-imaging analysis with GFP-tagged MRLC
and found that its mutants that mimic the active forms wereaccumulated in the cell cortex as foci. These foci were
dynamically formed and coalesced during cell elongation andinterdigitation. We further found that these positioned
proximal to cell protrusion and partially colocalized withadherens junction and tight junction proteins. These datasuggest that NMII containing phosphorylated MRLC
controls local cell-cell adhesion as intercellular linkage,
which positively regulates highly coordinated cell shapechanges in zebrafish neurulation.

Figure 4. By injecting in vitro transcribed mRNA, we observed active
MRLC-GFP foci at the single cell level. During neural cells undergoing
convergence and cell elongation processes, these foci were dynamicallyformed in the cell cortex and coalesced, sometimes resulting filamentous
structures, which seems more stable than cell protrusive activity in the
distal region. (Left) MRLC-GFP, (middle) membrane-RFP, (right) merge
images of elongating neural cell to medial side of the neural plate.

IV. Notochord and evolution of chordates

The early embryogenesis of amphioxus, up to the lateneurula stage, provides us with useful suggestions aboutchordate origins and evolution. Its cleavage pattern, resulting
in the formation of hollow blastula, resembles those of sea
cucumbers (echinoderms) and acorn worms (hemichordates).
In contrast to the mode of non-chordate deuterostome
gastrulation, in which the archenteron invaginates into a wide
blastocoelic space and causing the embryo to become cupshaped, with a deepened archenteron. This mode ofgastrulation is seen in ascidian embryos as well. By the lategastrula stage, the embryo has become ovoid and slightly
flattened, and the neural plate is formed from the flattened
dorsal side of the embryo. As in vertebrates, neurulation
begins with enclosure of the neural plate.
During the period of neural tube formation, the notochord
develops from the adjacent chordamesodermal plate that
constitutes the roof of the archenteron (Figure 5).
Specifically, the notochord is formed by pounching off fromthe archenteron. Interestingly, Brachyury is expressed not
only in the region where the notochord pouches off, but also
in the region where the somite pouches off. Furthermore, theamphioxus notochord has properties of muscle tissue. The
morphogenetic movement of the notochord (and somites) incephalochordate embryos looks like a continuation of thearchenteron invagination. In other words, amphioxus might
have recruited the secondary Brachyury expression for thissecond invagination-like morphogenetic movement.
By investigating the cellular morphogenesis and remodelingof tissues underlying amphioxus development and by gaining
a comparative developmental biology view of the events,
specifically relevant to notochord development, we aim to
obtain new insights into the evolutionary changes that took
place in a branch of the bilaterian lineage and gave rise to thechordates more than 550 million years ago.

Publication List

〔Original papers〕

T., Ueno, K., Yonezawa, K., Mineta, K., Hotta, K., Satou, Y.,• Endo,Yamada, L., Ogasawara, M., Takahashi, H., Nakajima, A., Nakachi, M.,•••

Nomura, M., Yaguchi, J., Sasakura, Y., Yamasaki, C., Sera, M.,
Yoshizawa, A., Imanishi, T., Taniguchi, H., and Inaba, K. (2011).
CIPRO 2.5: Ciona intestinalis protein database, a unique integrated
repository of large-scale omics data, bioinformatic analyses and curated
annotation, with user rating and reviewing functionality. Nucleic Acids
Res. 39, D807-D814.
Takebayashi-Suzuki, K., Kitayama, A., Terasaka-Iioka, C., Ueno, N.,
and Suzuki, A. (2011). The forkhead transcription factor FoxB1
regulates the dorsal-ventral and anterior-posterior patterning of theectoderm during early Xenopus embryogenesis. Dev Biol. 360, 11-29.
Tao, H., Manak, J. R., Sowers, L., Mei, X., Kiyonari, H., Abe, T.,
Dahdaleh, N. S., Yang, T., Wu, S., Chen, S., Fox, M. H., Gurnett, C.,
Montine, T., Bird, T., Shaffer, L. G., Rosenfeld, J. A., McConnell, J.,
Madan-Khetarpal, S., Berry-Kravis, E., Griesbach, H., Saneto, R. P.,
Scott, M. P., Antic, D., Reed, J., Boland, R., Ehaideb, S. N., El-Shanti,
H., Mahajan, V. B., Ferguson, P. J., Axelrod, J. D., Lehesjoki, A. E.,
Fritzsch, B., Slusarski, D. C., Wemmie, J., Ueno, N., and Bassuk, A. G.
(2011). Mutations in prickle orthologs cause seizures in flies, mice, andhumans. Am. J. Hum. Genet. 88, 138-149.
Yamada, S., Ueno, N., Satoh, N., and Takahashi, H. (2011). Ciona
intestinalis Noto4 contains a phosphotyrosine interaction domain and is
involved in the midline intercalation of notochord cells. Int. J. Dev.
Biol. 55, 11-18.

Figure 5. Amphioxus embryos (Branchiostoma floridae). Side views.
Anterior at left. (A) Mid-neurula stage embryo. (B) Early larval stageembryo. The notochord runs through the dorsal side by reaching to theanterior tip of the body. Embryos were stained with Alexa 488
phalloidine (green) and CellMask (red and magenta).

19

National Institute for Basic Biology

Developmental Biology

DIVISION OF DEVELOPMENTAL GENETICS

Professor

KOBAYASHI, Satoru

Assistant Professors:

HAYASHI, Yoshiki
SATO, Masanao
Assistant Professor (Specially Appointed):
KAGEYAMA, Yuji
Technical Staff:
NODA, Chiyo
NIBB Research Fellows: HASHIYAMA, Kazuya
OTA, Ryoma
Postdoctoral Fellows:
FUJISAWA, Chiemi
INAGAKI, Sachi
Graduate Students:
KUBO, Satoru
SUGIYAMA, Arisa
SHINOZUKA, Yuko
Visiting Scientists:
FUJISAWA, Toshitaka
OHARA, Yuya
Technical Assistants:
SATO, Kaori
ISHIHARA, Hitomi
NIIMI, Kaori
YAMAMOTO, Manami
Secretary:
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. Here, we describe a genome-wide surveyof maternal transcripts that encode for transcription factors
and are enriched in the germ plasm. We isolated pole cells(primordial germ cells; PGCs) from blastodermal embryos
by fluorescence-activated cell sorting (FACS) and then used
these isolated cells in a microarray analysis. Among the 835
genes in the Gene Ontology (GO) category “transcription
regulator activity” listed in FlyBase, 68 were found to be
predominantly expressed in PGCs as compared to whole
embryos. As the early PGCs are known to betranscriptionally quiescent, the listed transcripts are predicted
to be maternal in origin. Our in situ hybridization analysis
revealed that 27 of the 68 transcripts were enriched in thegerm plasm. Among the 27 transcripts, six were found to berequired for germline-specific gene expression of vasa and/or

20

nanos by knockdown 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, which thereby
causes sterility in both female and male adults. Thus,
maternal Ovo-B has an essential role in germline
development in both sexes.

II. Mechanism regulating sex determination of
PGCs“Sex reversal” leads to infertility, because the soma andgerm cells become incompatible; male gonads are not able todeliver eggs, and vice versa. Various genetic conditions result
in sex change in the soma, but in these cases germ cells
retain their original gender, and how sex is determined ingerm cells has remained unclear. It is widely accepted in
mammals and Drosophila that male sexual development is
imposed in PGCs by the sex of the gonadal soma, and that
PGCs assume a female fate in the absence of a masculinizing
environment. How PGCs initiate female development,
however, is a long-standing question in reproductive anddevelopmental biology.
In the soma, sex determination is controlled by the Sex
lethal (Sxl) gene, which is first expressed at the blastodermal
stage. Sxl encodes an RNA binding protein involved inalternative splicing and translation. In the soma of female(XX) embryos, it functions through transformer (tra) andtransformer-2 (tra-2), which in turn regulate alternative
splicing of the doublesex (Dsx) gene to produce a femalespecific form of Dsx. In male (XY) embryos, this pathway isturned off and a male-specific form of Dsx is produced bydefault. These Dsx proteins determine the sexual identity ofsomatic tissues. Previous reports, however, suggested that Sxl
does not induce female sexual development in the germline,
as it does in the soma. Although Sxl is autonomously
required for female sexual development, constitutive
mutations in Sxl (SxlM) that cause XY animals to undergo
sexual transformation from male to female does notnecessarily interfere with male germline development.
Moreover, tra, tra-2, and dsx are not required for female
germline development. Finally, female-specific Sxl
expression has been detected later in gametogenesis, but notin early germline development.
Contrary to the previous observations, we found that Sxl
was expressed in XX, but not XY PGCs, during theirmigration to the gonads. Furthermore, we found that the
SxlM1 mutation does not result in Sxl expression in XY PGCs,

as early as in XX PGCs. To determine whether Sxl induces
female development in XY PGCs, we then induced Sxl
expression in XY PGCs using nanos-Gal4 and UAS-Sxl. We
transplanted three types of XY PGCs (Figure 1), eachcharacterized by a different duration of Sxl expression: (i)XY PGCs in which Sxl was expressed from stage 9 until
stage 16/17 using maternal nanos-Gal4 (XY-mSxl), (ii) XY
PGCs in which Sxl was expressed from stage 15/16 onward
using zygotic nanos-Gal4 (XY-zSxl), and (iii) XY PGCs inwhich Sxl was expressed from stage 9 onward using bothmaternal and zygotic nanos-Gal4 (XY-mzSxl). We found that
XY-mzSxl and XY-mSxl PGCs entered the oogenic pathway
and produced mature oocytes in XX females. These oocytes
contributed to progeny production. In contrast, XY-zSxl
PGCs did not enter the oogenic pathway. These observations
demonstrate that Sxl expression in XY PGCs during
embryogenesis induces functional egg differentiation in thefemale soma.

Figure 1. Expression of Sxl is sufficient to induce female fate in XY
PGCs.

In contrast, Sxl-specific double-stranded RNA (UAS-SxlRNAi)
under the control of maternal nanos-Gal4 was then used toreduce Sxl activity in XX PGCs during embryogenesis.
Introducing UAS-SxlRNAi resulted in tumorous and agametic
phenotypes in female adults, indicating that the XX germline
lost female characteristics.
Our findings provide powerful evidence for Sxl as a mastergene that directs a female germline fate. XX PGCs initiate
female sexual identity based on their Sxl expression, while,lacking Sxl expression in XY PGCs, male sexual fate occurs
primarily by a signal from gonadal soma (Figure 2). One
remarkable example of germline-autonomous regulation ofsexual dimorphism has been reported in a primitive animal,
cnidarian Hydra. It has long been known that sex of thegermline is not influenced by the surrounding soma, and thegermline, rather than soma, determines the phenotypic sex of
the polyp. Thus, we speculate that germline-autonomous
regulation of sex is a primitive trait conserved throughout theevolution of animals.

Figure 2. A model for the regulation of sexual dimorphism in pole cells.

III. The role of heparan sulfate proteoglycans
in the germline-stem-cell niche

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 germline-stem-cell (GSC) niche in
Drosophila gonads is a useful model system for studying thestem-cell niche, because the cellular components of thisniche have been characterized and the signaling pathways,
such as TGF-beta 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, GSC niche isdefined as the specialized region retaining a sufficientamount 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.
Heparan sulfate proteoglycans (HSPGs) are a group of
glycoproteins, which are expressed on the cell surface and/orin the extracellular matrix. Recent studies have revealed that
HSPGs play critical roles in regulating signaling pathways
during development by controlling extracellular ligand
distribution. For example, one of the Drosophila glypicans,dally controls distribution of TGF-beta ligand, or Dpp, andthereby establishing a Dpp gradient during wingdevelopment.
We recently identified Drosophila glypicans, dally anddally-like as important components of GSC niche in both
sexes. Mutations for these glypicans caused a significant
reduction in GSC number, due to the failure of proper
activation of the signaling pathway for GSC maintenance.
Conversely, ectopic expression of dally in female gonads
caused an increase in GSC number. These results strongly
suggest that these glypicans define GSC niche by regulating
distribution of niche signals.
To address this possibility, we have been trying hard tovisualize niche signals in the GSC niche. By modifying

21National Institute for Basic Biology

Developmental Biology

protocols for antibody staining and generating new
antibodies, we succeeded in visualization of niche signal
distribution. We found that Dpp distribution was significantly
expanded when dally was ectopically expressed in female
gonads, while Dpp-producing cells were unaffected (Figure3). The above results support our model that glypicans define
the GSC niche by regulating extracellular distribution ofniche signals.
We also succeeded in detecting spatial distribution of JAK/STAT ligand, Upd (Figure 3). Upd is known to act as amorphogen for proper formation of egg chambers, as well as
a niche signal in male GSC maintenance. We found that dally
was able to affect Upd distribution within the egg chambers.
This is the first evidence supporting the role of glypican inregulating extracellular distribution of Upd. Experiments
testing whether glypicans regulate Upd distribution in male
GSC niche are now ongoing.

Publication List

〔Original papers〕

K., Hayashi, Y., and Kobayashi, S. (2011). Drosophila Sex• Hashiyama,lethal gene initiates female development in germline progenitors.

•

22

Science 333, 885-888.
Mukai, M., Kato, K., Hira, S., Nakamura, K., Kita, H., and Kobayashi,
S. (2011). Innexin2 gap junctions in somatic support cells are requiredfor cyst formation and for egg chamber formation in Drosophila. Mech.
Dev. 128, 510-523.

Figure 3. Glypican regulates spatial distribution of 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 (Blackets). 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, C’) Distribution of Upd in egg
chamber. Upd (red signal) is ectopically accumulated in dally-expressing
clone (green, Bar).

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

Professor:
Assistant Professors:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:
Visiting Scientist:
Graduate Students:

Technical Assistants:
Secretary:

TAKADA, Shinji
OKUBO, Tadashi
YABE, Taijiro
UTSUMI, Hideko
CHEN, Qiuhong
KAMETANI, Yoshiko
TAKAHASHI, Jun
TAKADA, Ritsuko
ISHITANI, Shizuka
TSUNOKUNI, Hiroyuki
WANGLAR, Chimwar
SHINOZUKA, Takuma
KAMAGATA, Takanori
TAKASHIRO, Kayoko
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 (Figure 1). Wnt-3a defective in this modification is
not secreted from cells in culture or in Xenopus embryos, but
is retained in the endoplasmic reticulum (ER). Furthermore,
Porcupine (Porcn), a protein with structural similarities tomembrane-bound O-acyltransferases, is required for this Serdependent modification, as well as for Wnt-3a transport fromthe ER for secretion. These results strongly suggest that theWnt protein requires a particular lipid modification forproper 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.

Figure 1. Post-translational modification of Wnt proteins: Several
secreted signal proteins are specifically modified. These modifications
are required for proper secretion, extracellular trafficking, and binding totheir receptors. Wnt proteins are lipidated, glycosylated, and sulfated. We
have shown that one type of lipidation, palmitoleoylation, is required forproper secerion of several Wnt proteins and is dependent on an acyltransferase, Porcupine. In this figure, a schematic representation ofmouse Wnt3a, which is lipidated at Cys77 and Ser209 and glycosylated
at Asn87/298, is shown. In addition, the positions of Tyrosine residues
sulfated in Wnt5a/11 are also indicated. The conserved cysteines areshown with vertical bars.

On the other hand, recent evidence suggest that this Serdependent lipidation may not always be required for
secretion of Wnt proteins. In the case of Wingless, theDrosophila ortholog of Wnt1, this Ser residue is not required
for secretion from Drosophila S2 cells, but for the functionof this protein, suggesting that the role of the Ser-dependent
lipidation may differ for different Wnt proteins or in different
cellular contexts. To reveal the role of the Ser-dependent
lipidation in different contexts, we examined the role ofPorcn in zebrafish embryos. Porcn-deficient zebrafish
embryos only exhibited defects in convergent-extension (CE)movement, which requires non-canonical Wnt signaling.
Surprisingly, these embryos showed no sign of reduction in
canonical Wnt signaling. Consistent with this array ofphenotypes, intracellular trafficking of zebrafish Wnt5b andmouse Wnt5a, but not zebrafish and mouse Wnt3a, wasspecifically impaired in Porcn-deficient embryos. These
results indicate that Porcn is required for proper function and23National Institute for Basic Biology

Developmental Biology

trafficking of at least one Wnt protein during early
developmental stages of zebrafish, but decrease of Porcn
does not equivalently affect the trafficking and lipidation ofdifferent Wnt proteins in these embryos. Together, themechanism of trafficking and modification of Wnt proteinsappear 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 prepattern, characterized by the periodical borders between
neighboring somites and by the rostro-caudal within asomite, is established in the anterior PSM. We have alreadyshown that a gene identified by our in situ hybridization
screening, ripply1, is required for the maintenance of therostro-caudal patterning in zebrafish embryos. Ripply
proteins have been shown to associate with thetranscriptional co-repressor Groucho/TLE and the T-box
transcription factors through two distinct amino acidsequences, one termed the WRPW motif, which is a highly
conserved 4-amino acid stretch in the N-terminal half, andthe other, the Ripply homology (RH) domain, a conserved~50-amino acid stretch that interacts with the T-domain of
the T-box proteins. Therefore, Ripply is able to recruit theGroucho/TLE co-repressor to T-box proteins and control
their intrinsic transcriptional properties.
Recently, we also showed that Ripply1 and anotherstructurally related-protein, Ripply2, are required for the
positioning of intersomitic boundaries in mice. The positionsof somite boundaries are defined by the anterior limit of the
Tbx6 protein domain, which is regulated by degradation of
Tbx6 proteins. In addition, a transcription factor, Mesp2, isrequired for this degradation although it is uncertain how
Mesp2 induces this degradation. We showed that expressionof mouse Ripply1 and Ripply2 is dependent on Mesp2 andRipply1/2-deficient mouse embryos exhibit anterior
expansion of the Tbx6 protein domain, suggesting that
Ripply1/2 expression by Mesp2 is important in theestablishment of somite boundaries by regulating Tbx6
degradation. To compare the mechanism of somite
segmentation between different species, we are also
examining the mechanism of the formation of intersomitic

24boundaries in zebrafish.

III. The role of Ripply3 in the development ofpharyngeal arches.

The pharyngeal apparatus is another example of segmental
structure in the vertebrate embryo. The pharyngeal apparatus
is a transient structure formed ventrolateral to the hindbrain
in vertebrate embryos. This structure consists of bilaterally
segmented arches, and ectodermal grooves and endodermal
pouches, both of which are formed between the arches.
Components of the pharyngeal apparatus give rise to distinct
tissues in later stages of development. For instance, thepharyngeal arteries and neural crest cells in the caudal
pharyngeal arches contribute to cardiovascular development,
while the endodermal cells located in the caudal pouches
give rise to several organs, including the thymus andparathyroid gland. Thus, pharyngeal development is a key
process in the generation of these organs.
Chromosome 22q11 deletion syndrome (22q11DS), which
includes the DiGeorge syndrome (DGS), is characterized by
the abnormal development of the pharyngeal apparatus in theform of thymic hypoplasia or aplasia, hypocalcemia arisingfrom parathyroid hypoplasia, and defective cardiac outflow.
A number of mouse genetic studies and mutational analyses
in human patients have indicated that Tbx1, which encodes amember of the T-box family of transcription factors, is themost likely gene responsible for the phenotype of 22q11DS.
During murine pharyngeal development, Tbx1 is firstexpressed in the mesoderm at E7.5. Between E8.5 to 11.5,
Tbx1 also becomes located in the pharyngeal endoderm,ectoderm, and core mesoderm, but not in the neural crest
cells. Cell type-specific inactivation and analysis ofdownstream targets of Tbx1 in mice indicate that Tbx1 plays
multiple roles in endoderm, mesoderm, and ectoderm cells
during pharyngeal development. However, the molecularmechanisms underlying the cell type-specific roles of Tbx1
have not been fully elucidated.
Interestingly, another member of the Ripply family,
Ripply3, is expressed in the pharyngeal endoderm cells.
Because we found that both Ripply3 and Tbx1 were strongly
expressed in the pharyngeal endoderm and endoderm, wethen examined the role of Ripply3 by generating Ripply3deficient mice, as well as its relationship with Tbx1. Weshow that Ripply3 can modulate Tbx1 activity in in vitro
luciferase reporter assays. Furthermore, Ripply3-knock out
mouse showed hypotrophy of the caudal pharyngeal
apparatus (Figure 2). Ripply3 represses Tbx1-induced
expression of Pax9 in in vitro luciferase assays, and Ripply3deficient embryos exhibit up-regulated Pax9 expression.
Thus, our results showed that Ripply3 most likely plays itsrole in pharyngeal development by regulating Tbx1 activity.
More precise analysis revealed that the development ofpharyngeal derivatives was impaired in Ripply3 mutant mice,
including ectopic formation of the thymus and theparathyroid gland, as well as cardiovascular malformation.
For instance, the disrupted development of the caudal
pharyngeal arches seems to have resulted in two distinct
cardiovascular defects in the Ripply3 -/- embryos. One
involved an abnormality in heart development. The severeFigure 3. Cardiovascular defects in the Ripply3-deficient mice: (A-C)
Typical morphology of pharyngeal arch arteries (PAAs) in the wild-type(A), heterozygous (B), and homozygous mutant (C) mice at E10.5 asvisualized by intracardiac ink injection. The asterisk indicates theabsence of the 3rd and 4th PAAs. (D) Schematic representation of PAA
pattern.

Publication List

〔Original paper〕
Figure 2. Ripply3 is required for proper development of the third andfourth pharyngeal arches: (A-D) Hematoxylin-eosin-stained coronal
sections of Ripply3 +/- (A, C) and Ripply3 -/- (B, D) embryos at E9.5 (A,B)
and E10.5 (C,D). (E, F) Schematic images illustrating phenotypes of
Ripply3 +/- (E) and Ripply3 -/- (F) embryos.

T., Kawamura, A., Takahashi, J., Yagi, H., Morishima, M.,• Okubo,Matsuoka, R., and Takada, S. (2011). Ripply3, a Tbx1 repressor, isrequired for development of the pharyngeal apparatus and its derivatives
in mice. Development 138, 339-348.

reduction in neural crest cell number in the caudal
pharyngeal arches appears to have led to the abnormal
development of the outflow tract, including hypotrophy of
the aorta and incomplete formation of the ventricular septum.
The other defect involved the loss of the 3rd and 4th PAAs.
This defect appeared to result in abnormal development of
the vascular system including deletion of the aortic arch andmisshapen major blood vessels. Interestingly, the second
PAAs, which normally disappear after E10.5, persisted until
birth in the Ripply3-/- embryos (Figure 3). Persistence of the
1st and 2nd PAAs has also been reported in Endothelin-1deficient mouse embryos, where the 4th PAAs are also
poorly developed, suggesting that the formation of PAA
proximal to the heart may be a pre-requisite for the
regression of the more distal ones. We speculate thatdevelopment of the proximal PAAs resulted in a decrease inblood flow running through the existing distal PAAs, which
in turn decreased the mechanical stress caused by blood flow,
making it virtually impossible for the distal PAAs to be
maintained.

25

National Institute for Basic Biology

Developmental Biology

DIVISION OF EMBRYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professors:
Technical Staff:
NIBB Research Fellows:
Postdoctoral Fellow:
Visiting Scientist:
Technical Assistants:
Secretary:

TOYOOKA, Yayoi
KOYAMA, Hiroshi
OKA, Sanae
KOMATSU, Kouji
SATO, Yasufumi
KOBAYAKAWA, Satoru
SHI, Dongbo
HIRAO, Mayumi
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 cellular
co m p o n e n t s h a v e n o t b ee n fo u n d th a t lo c a liz e
asymmetrically in the fertilized egg. We want to provide
basic and fundamental information about the specification ofembryonic axes, behaviors of cells and the regulation ofbody shape in early mammalian development. We are alsointerested in the relationship between embryos and theirenvironment, the oviducts and the uterus.

I. Establishment of mouse lines for live imaging

Currently, it is common to observe the behavior of cells andgene expression in living embryos to understand themechanisms underlying embryonic development andmorphogenesis. Progress in GFP technologies, genetic
engineering and embryonic manipulation concomitantly withthe progress in optics and microscope technologies,
including development of highly sensitive photo-detectors,
has allowed us to observe developing embryos live, even in
mammalian species. We have previously established atransgenic mouse line ubiquitously expressing EGFP
(Enhanced Green Fluorescent Protein) fused with histone
H2B by homologous recombination in the Rosa26 locus, andembryos obtained from this mouse line have been used fortimelapse live imaging to analyze cell behavior. It would be
useful to visualize nuclei with other colors of fluorescentproteins and visualize cell shapes, cytoskeleton or otherorganelles in a similar way to observe cells and cell
behaviors in living embryos. Prof. Fujimori, as a visiting
scientist, has been participating in a project producing aseries of mouse reporter lines to express fluorescent markers,
which is taking place in the Laboratory for Animal Resources26

and Genetic Engineering, Riken CDB. This year we haveestablished 17 lines of these mice. In each mouse, cDNA
encoding fusion protein with fluorescent protein and alocalization sequence was inserted into Rosa26 locus. The
sequence for the fusion protein was following stop sequencesthat are surrounded by loxP sites on both sides. These loxPsites can be recognized by an enzyme called Cre
recombinase that catalyzes recombination between two loxPsites to remove the stop sequences. Thus, when Cre
recombinase is activated in a spatial-temporally specificmanner, the following reporter fluorescent proteins are

Figure 1. Examples of 7.5 day embryos obtained after crossing of tworeporter mouse lines. Nuclei and golgi apparatus and cell membrane andnuclei are visualized with green and red fluorescent proteins geneticallyintroduced as transgenes.

expressed in a specific way. And once this irreversible
reaction is induced in the germ line, the derived offspring
possess the transgene without the stop sequence and express
the reporter fusion protein ubiquitously. These mice will
provide useful materials for live imaging of cells in early
embryos as well as cells in the tissues of developing andadult animals. In addition to the establishment of mouselines, we have been trying to improve optical systems for live
imaging, and we could improve systems to culture in better
conditions during live imaging. The culture conditions ofperi-implantation mouse embryos have also improved. We
are planning to observe and reveal cell shape,morphogenesis, cell lineage, gene expression and celldifferentiation by combining these techniques.

Figure 2. A 4.5day rabbit embryo. The shape is very similar to othermammalian blastocysts at this stage. However, development from slightly
after this stage is different from corresponding stages of mouse embryo
development.

II. Studying early development of rabbit as a
new model 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 started
examination of morphological changes and gene expression
during early stages of development until the periimplantation stage. We will focus especially on the formationof body axes, and compare with corresponding stages in
mice.

III. Formation, maintenance of ell polarity andtissue morphogenesis 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,
wherefertilization occurs with the spermatozoon movingfrom the uterus. We are focusing on the region of the oviductclose to the opening to the ovary. In this region many multiciliated cells exist. The cilia on the surface of ovarianepithelial cells were observed with a high-speed CCD
camera and these movements were recorded. These cilia
moved in one direction along the ovary-uterus axis. This
directional movement of multi-cilia might play a major rolein the transportation of ovum from periovarian space,although muscle contractions also play roles in the region
close to the uterus. The importance of the directional ciliary
beating for the transport of ova is supported by theexperiments shown in the figure 3. An ovum was put at the
opening end of a longitudinally opened oviduct, and thisovum was moved along the fold running parallel to theovary-uterus axis of the oviduct without muscle contraction.
This suggested that the ovum was transported by the
directionally beating cilia. This directional beating of cilia
was based on the polarity of the microtubule assembly in thecilia. Skeletal microtubules in the cilia are arranged in a “9+2”array, and the central two bundles are aligned facing thesame direction in each cell. This suggests that oviductepithelial cells possess polarity along the cell surface parallelto the longitudinal axis of the oviduct; this type of cellularpolarity is called “Planner Cell Polarity (PCP)”. We havebeen studying how PCP is established during development,
and how this polarity is maintained for a long period in laterstages. Ciliary movement is one good indicator of PCP,
therefore we established a system to analyze ciliary beatswith automated image processing, and found ciliary beatfrequency was slightly different between estrus cycles.
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 known also
in mammalian species, and some of them have shown to beplaying similar roles. Disruption of functions of some ofthese 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.

Publication List

〔Original papers〕

T., Kiyonari, H., Shioi, G., Inoue, K., Nakao, K., Aizawa, S., and• Abe,
Fujimori, T. (2011). Establishment of conditional reporter mouse lines•••

at ROSA26 locus for live cell imaging. Genesis 49, 579-590.
Nakagawa, T., Izumino, K., Ishii, Y., Oya, T., Hamashima, T., Jie, S.,
Tomoda, F., Fujimori, T., Nabeshima, Y., Inoue, H., and Sasahara, M.
(2011). Roles of PDGF receptor-beta in the structure and function of
postnatal kidney glomerulus. Nephrol Dial. Transplant. 26, 458-468.
Shi, D., Komatsu, K., Uemura, T., and Fujimori, T. (2011). Analysis ofciliary beat frequency and ovum transport ability in the mouse oviduct.
Genes to Cells 16, 282-290.
Shioi, G., Kiyonari, H., Abe, T., Nakao, K., Fujimori, T., Jang, C.,
Huang, C., Akiyama, H., Behringer, R.R., and Aizawa, S. (2011). A
mouse reporter line to conditionally mark nuclei and cell membranes
for in vivo live-imaging. Genesis 49, 570-578.

Figure 3. Transport of an ovum via the movements of cilia on the oviductepithelial cells. Timelaplse images of movement of an ova on thelongitudinally opened preparation of the mouse oviduct.

27

National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professors:
NIBB Research Fellow:
Technical Staff:
Graduate Students:
Technical Assistants:

Secretary:

HARA, Kenshiro
KITADATE, Yu
NAKAMURA, Yoshiaki
MIZUGUCHI-TAKASE,
HirokoIKAMI, Kanako
TOKUE, Moe
ICHIKAWA, Rie
INADA, KanaMARUYAMA, Ayumi
SUGIMOTO, Ryo
KUBOKI, Yuko

Mammalian testes produce numerous sperm for a long
period in a constant manner, which is supported by the robust
behaviors of the stem cell population. Decades of research,
including morphological examinations, post-transplantation
repopulation, and in vitro culture, have made it one of themost intensively studied mammalian tissue stem cellsystems. However, the nature of the stem cells and theircontrol, as well as their niche, remains largely unknown.
The Division of Germ Cell Biology aims to fully
understand the mouse sperm stem cell system in vivo, i.e., in
the context of testicular tissue architectures. We haverevealed a number of characteristics of this potent stem cellsystem, including: 1) Cells believed to have irreversiblycommitted for differentiation still retain the self-renewing
potential and can contribute to stem cell pool maintenance(“potential stem cells”). 2) “Reversion” from potential stem
cells occurs at a higher frequency when testicular tissue isdamaged and regeneration is induced. 3) The undifferentiatedspermatogonia (Aundiff) population that includes both “actual”
and “potential” stem cells localized to vasculature (vascularassociated niche). 4) Stem cells turn over frequently andstochastically even under steady-state situations. (Nakagawa
et al., Dev. Cell 2007, Science 2010; Yoshida et al., Science
2007; Klein et al., Cell Stem Cell 2010)

Figure 1. Architecture of seminiferous tubules and seminiferous
epithelium. The seminiferous epithelium exhibits a stratified organizationof the differentiating germ cells (numbers in circle), which are nourishedby Sertoli cells. Bar, 20 mm.

28

In 2011, we revealed the molecular mechanisms underlying
the temporal regulation of differentiation and self-renewal ofstem cells (Aundiff) in mouse seminiferous epithelium, which
are comprised of germ cells –both stem and differentiatingcell types- and supporting somatic cells. Our study proposedthat local retinoic acid (RA) metabolism plays importantroles for the timed stem cell differentiation and coordination
with somatic cells that support the differentiation process tospermatozoa (Sugimoto et al., Mech. Dev. 2011).

I. Architecture and cyclic dynamics ofseminiferous epithelium

Spermatogenesis occurs inside the seminiferous tubule ofthe testis, where differentiating germ cells and supporting
somatic Sertoli cells comprise a composite epithelium,
termed seminiferous epithelium (Fig. 1). Sertoli cells arehuge cells that form typical epithelium. All the stages ofgerm cells (from stem cells to spermatozoa) are nourished by
Sertoli cells in a striated manner. This stratification isestablished as a result of the periodic differentiation of Aundiff
into A1 spermatogonia with an interval of 8.6 days, which isfollowed by programmed differentiating process toward
spermatozoa that takes 35 days (Fig. 2). Consequently, theresultant combination of the differentiating germ cell
observed at a particular region appears in an 8.6-day cyclicmanner, known as ‘seminiferous epithelial cycle’. In mice,
this cycle is divided into stages I to XII, while Aundiff-to-A1
differentiation occurs at stage VII to VIII.

Figure 2. Coordination of periodical events in mouse seminiferous
epithelium. (A) Germ cell differentiation, which takes 35 days from Aundiff
to spermatozoa. (B) 8.6-day cycle of gene expression in Sertoli cells. (C)Coordination of germ cell differentiation and Sertoli cell cycle.

Sertoli cells, while quiescent in their cell cycle, cyclicallychange their gene expression so that they can nourish theappropriate stages of germ cells. It is an interesting question
but remains a mystery how this periodic differentiating ofAundiff is achieved and the cycle of Sertoli cells is coordinatedwith germ cell differentiation.

II. Role of RA and regu lation o f RA
metabolism

It has been known that RA plays important roles in bothdifferentiation of A undiff and control of the seminiferous
epithelial cycle. This notion was primarily derived fromobservations made after RA signaling blockade by deficiencyof the dietary vitamin A (VA) -the only precursor of RA- andby disruption of retinoic acid receptor genes. However,results of these and other preceding studies were somewhat
puzzling and it remained a challenge to reveal how RA is
involved in the genesis of the coordinated cycling of theseminiferous epithelium.
We revisited the classical VAD model and discovered that
artificial elevation of RA signaling elicits not only
differentiation of Aundiff into A1 spermatogonia but also a resetof the cycle of Sertoli cells’ function to the appropriate stage
that supports the A1 spermatogonia in transition between
Aundiff and A1 (stage VII). Then we asked how the RA-related
metabolism is regulated in the seminiferous epithelium basedon the expression of genes encoding enzymes involved in thesynthesis of RA from VA and inactivation of RA. Based on
the data from a previous study of Manuel Mark’s group andour own analyses, it was strongly suggested that the modesof RA metabolism dynamically shift in accordance with theseminiferous epithelial cycle, with a prominent increase of
RA concentration across stage VII (Fig. 3). This is also
supported by the fact that artificially increased RA induced
Aundiff-to-A1 differentiation and reset Sertoli cells to stage VII.
Interestingly, RA metabolism involves multiple enzymes(Fig. 3A) that are expressed separately among different cell
types including meiotic and haploid germ cells and Sertoli
cells. Given that RA and its precursors are able to translocate
across adjacent cells rather freely, the different cell types
seem to cooperate in the regulation of RA metabolism. Then,the next question was raised: How is coordinated expressionof these genes among different cell types achieved? We
discovered that RA metabolism-related genes expressed in
Sertoli cells are regulated by RA signaling, forming positive
and negative feedback loops; while those expressed inmeiotic and haploid germ cells hardly respond to RA
signaling: Perhaps a differentiation step-related mechanism
controls their expression. Thus, control of RA metabolismrelated genes show a good contrast between Sertoli cells andgerm cells.

Figure 3. RA metabolism-related genes and their cyclic expression. (A)Multiple genes act together in the RA metabolic pathways, starting fromretinol (VA). (B) These genes are expressed in different cell types and indifferent stages of the seminiferous epithelial cycle.

III. Model for the seminiferous epithelial cycle

Based on these observations, we have proposed a modelthat can explain the coordinated expression of RA
metabolism-related genes that appears to cause the cyclicchange of the RA concentration (Fig. 4, 5). Importantly, thismodel suggests that meiotic and haploid germ cells regulate
the local RA metabolism to increase and decrease the RA

Figure 4. Regulation of RA metabolism-related gene expression.
Expression patterns of these genes (Fig.3) in early (A) and late (B) stages
are explained as a result of feedback regulation in Sertoli cells but not ingerm cells.

29National Institute for Basic Biology

Developmental Biology

concentration, respectively (Fig. 4). Given that RA signaling
causes the induction of Aundiff-to-A1 differentiation and resetof Sertoli cell’s cycle, it can be said that these differentiating
germ cells play a central role in the coordination of theseminiferous epithelial cycle by sending ‘go’ and ‘wait’signals to the Aundiff and Sertoli cells (Fig. 5). Therefore, thedifferentiation program of germ cells appears to determine
the timing of stem cell differentiation and makes the local
environment appropriate for germ cell differentiation.
This model nicely explains the beautiful orchestration
between germ and Sertoli cells in the seminiferous
epithelium. Generally speaking, regulation of stem celldifferentiation by their differentiating progeny that modulate
the local environment may be a common strategy of stem
cell control in other systems.

IV. Perspectives and ongoing researchmodel and future investigations will clarify the details of thetesticular environment and the stem cell response.
Besides the seminiferous epithelial cycle, we are also
investigating a number of yet-to-be elucidated problems inspermatogenesis, especially with regard to its stem cells. In
addition to temporal regulation described here, the spatial
regulation of stem cells is also a very important issue. We
previously observed that Aundiff preferentially localize to thevasculature-proximal region, and dispatch this region when
they differentiate into A1 at stage VII (Yoshida et al., Science2007), which is likely to occur in response to RA signaling.
We are also investigating the cellular and molecular nature ofthis ‘vasculature-associated niche’. We are also investigating
the behavior of Aundiff taking advantages of live imaging that
we have developed (Yoshida et al., Science 2007, Nakagawa
et al., Science 2010). We hope that these studies will throw
light to a better understanding the mouse sperm stem cellsystem.

Publication List

〔Original paper〕

T., Aiyama, Y., Ishii-Inagaki, M., Hara, K., Tsunekawa, N.,• Sato,Harikae, K., Uemura-Kamata, M., Shinomura, M., Zhu, X. B., Maeda,

S., Kuwahara-Otani, S., Kudo, A., Kawakami, H., Kanai-Azuma, M.,
Fujiwara, M., Miyamae, Y., Yoshida, S., Seki, M., Kurohmaru, M., andKanai, Y. (2011). Cyclical and patch-like GDNF distribution along thebasal surface of sertoli cells in mouse and hamster testes. PLoS ONE 6,e28367.

〔Original papers (E-publication ahead of print)〕

A., Raverdeau, M., Célébi, C., Dennefeld, C., Feret, B.,• Gely-Pernot,
Klopfenstein, M., Yoshida, S., Ghyselinck, N.B., and Mark, M.

•

Spermatogonia differentiation requires retinoic acid receptor γ.
Reproduction 2011 Nov. 1.
Sugimoto, R., Nabeshima, Y., and Yoshida, S. Retinoic acid metabolism
links the periodical differentiation of germ cells with the cycle of Sertoli
cells in mouse seminiferous epithelium. Mechanisms of Development
2011 Dec. 19.

〔Review articles〕

A., Fuller, M.T., Braun, R.E., and Yoshida, S. (2011).
• Spradling,
Germline stem cells. In Cold Spring Harbor Perspectives in Biology
Collection, “Germ Cells”, Cold Spring Harbor Laboratory Press, 1-20.

Figure 5. Coordination of germ cell differentiation and Sertoli cells’
cycle. Particular stages of meiotic cells (LP) and haploid cells (RT) sendthe ‘go’ and ‘wait’ signals to Aundff and Sertoli cells, by regulating the
local RA metabolism to increase and decrease the RA concentration,
respectively (A). These signals will occur reciprocally and periodically tomaintain the seminiferous epithelial cycle. All the figures are reproduced
from Sugimoto et al., Mech. Dev. (2011) with permission.

The suggested model for the role of RA in the regulated
stem cell differentiation warrants farther evaluation: Whilethe central role of RA is in no doubt, it is also clear that RA
solely cannot regulate the very complex and highly
orchestrated events that occur in seminiferous epithelial
cycle. In addition, stem cells should behave in response to
the cyclically changing microenvironment. We hope that our30

S. (2011). Stem cell niche system in mouse spermatogenesis.
• Yoshida,
In Stem Cell Biology and Regenerative Medicine Series, “MaleGermline Stem Cells: Developmental and Regenerative Potential”,
Humana Press, 159-175.

DIVISION OF REPRODUCTIVE BIOLOGY †Publication List

〔Original papers〕
Professor (Specially appointed)

NAGAHAMA, Yoshitaka

Postdoctoral Fellows:

Secretary:

GUAN, Guijun
SAKAI, Fumie
SHIBATA, Yasushi
NAKAMOTO, Masatoshi
SHIMADA, Yu

Fish exhibit a range of gonadal forms from gonochorism toseveral types of hermaphroditism, thus providing an
excellent animal model to study the molecular mechanismsof sex determination, gonadal sex differentiation andgametogenesis in vertebrates. Our research focuses on (1) the
identification of regulators involved in sex determination,
gonadal sex differentiation, sexual plasticity, andgametogenesis (oocyte maturation and ovulation), and (2) the
mechanisms of synthesis and action of these regulators.

Molecular mechanisms of sex determination,
gonadal sex differentiation and sex change

We previously identified DMY (DM-domain gene on the
Y chromosome) as the sex-determining gene of the
medaka (Oryzias latipes), the first in non-mammalianvertebrates. Recently, we developed a gene-specifictransgenic RNA interference (RNAi) technology for the
analysis of loss-of-function phenotypes that develop overlong periods of time, and used it to knock down the dmy
gene in genetically male (XY) fish. Knockdown of dmystrongly downregulated the expression of the only othermale-associated genes (gsdf, sox9a2 and dmrt1), andupregulated the expression of female-associated genes(foxl2 and Rspo1) in XY gonads during the early stages ofsexual differentiation. This shift in the gene expression
pattern resulted in a complete male-to-female sex-reversal
with a typical female pattern of secondary sex
characteristics, producing fertile eggs. Importantly, wewere able to continue a trans-generational knockdown
effect on dmy until at least the F3 generation. In order torescue the effect of dmy knockdown, we singularly
injected or co-injected sox9a2 (marked in cyan) and gsdf
(marked with cherry) into olvas vasa-DMY-knockdown
embryos of the F3 generation. Although singular
injections failed to complete suppression of meiosis andproliferative mitosis but co-injection re-established themale phenotype in the XY gonad leading to complete
formation of the testis, producing fertile sperm. Thisconfirms that gsdf and sox9a2 are genes downstream of
dmy which, can regulate the sexual identity of medaka
even in a DMY-independent manner. We conclude that inT., Shibata, Y., Zhou, L.Y., Katsu, Y., Iguchi, T., and• Charkraborty,
Nagahama, Y. (2011). Differential expression of three estrogen receptor

•
•
•
•
•
•
•
•

subtype mRNAs in gonads and liver from embryos to adults of the
medaka, Oryzias latipes. Mol. Cell. Endocrinol. 333, 47-54.
Charkraborty, T., Katsu, Y., Zhou, L.Y., Miyagawa, S., Nagahama, Y.,
and Iguchi, T. (2011). Estrogen receptors in medaka (Oryzias latipes)
and estrogenic environmental contaminants: An in vitro-in vivo
correlation. J. Steroid Biochem. Mol. Biol. 123, 115-121.
Fernandino, J.I., Popesku, J.T., Paul-Prasanth, B., Xiong, H., Hattori,
R.S., Oura, M., Strussmann, C.A., Somoza, G.M., Matsuda, M.,
Nagahama, Y., and Trudeau, V.L. (2011). Analysis of sexually dimorphic
expression of genes at early gonadogenesis of Pejerrey Odontesthes
bonariensis uing a heterologous microarray. Sex. Dev. 5, 89-101.
Mita, M., Yamamoto, K., and Nagahama, Y. (2011). Interaction ofrelaxin-like gonad-stimulating substance with ovarian follicle cells of
the starfish Asterina pectinifera. Zool. Sci. 28, 764-769.
Mita, M., Yamamoto, K., Nakamura, M., and Nagahama, Y. (2011).
Hormonal action of relaxin-like gonad-stimulating substance (GSS) on
starfish ovaries in growing and fully grown states. Gen. Comp.
Endocrinol. 172, 85-89
Ngamniyom, A., Magtoon, W., Nagahama, Y., and Sasayama, Y. (2011).
Expression levels of bone morphogenetic protein 2b in fins of adultJapanese medaka (Oryzias latipes) exposed to sex steroid hormones. J.
Fish. Aquat. Sci. 6, 119-129.
Okubo, K., Takeuchi, A., Chaube, R., Paul-Prasanth, B., Kanda, S.,
Oka, Y., and Nagahama, Y. (2011). Sex differences in aromatase gene
expression in the medaka brain. J. Neuroendocrinology 23, 412-423.
Paul-Prasanth, B., Shibata, Y., Horiguchi, R., and Nagahama, Y. (2011).
Exposure to diethylstilbesterol during embryonic and larval stages of
medaka fish (Oryzias latipes) leads to sex reversal in genetic males andreduced gonad weight in genetic females. Endocrinology 152, 707-717.
Raghuveer, K., Sudhakumari, C.C., Senthilkumaran, B., Kagawa, H.,
Dutta-Gupta, A., and Nagahama, Y. (2011). Gender differences intryptophan hydroxylase-2 mRNA, serotonin, and 5-hydroxytryptophan
levels in the brain of catfish, Clarias gariepinus, during sex
differentiation. Gen. Comp. Endocrinol. 171, 94-104.

〔Review Article〕

N., Nakamoto, M., Shibata, Y., and Nagahama, Y. (2011).
• Shibata,
Endocrine regulation of oogenesis in the medaka, Oryzias latipes. In

Medaka: A Model for Organogenesis, Human Disease and Evolution,
H. Takeda et al. eds. (Springer), pp. 267-283.

medaka dmy directly or indirectly upregulates the male sexdetermining pathway by activating gsdf and sox9a2
expression.

†: This laboratory was closed on 31 March, 2011.

31

National Institute for Basic Biology

Developmental Biology

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION

Associate Professor

TANAKA, Minoru

NIBB Research Fellows:
Postdoctoral Fellow:
Graduate Student:
Visiting Scientist:
Technical Assistants:
Secretary:

NAKAMURA, Shuhei*
YAMAMOTO, Yasuhiro
NAKAMURA, Shuhei
NISHIMURA, Toshiya
HAAG, Eric
KINOSHITA, Chie
WATAKABE, Ikuko
KOBAYASHI, KayoYONEMITSU, Masako

the biphasic process of sex in medaka.
We have also identified the niche structure (called germinal
cradle) that harbors germline stem cells in the medaka ovaryfor the first time in vertebrates (Nakamura et al., 2010
Science). Since the cells that constitute the cradle express the
AMH ligand and receptor, the cradles are the place not only
for regulation of proper and continuous production of eggs
but also for the biphasic process of manifestation of sex(manuscript submitted). The cradles are also characterizedwith the expression of sox9b, which is specifically expressedin male developing gonads in mammals and is essential for
mammalian testis formation (figure 2).

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.

Figure 1. Red bars indicate location of ovarian niche (germinal cradles)
expressing the sox9b gene. The germ cells are labeled with red.

I. Celluar 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 differentation 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) while mutants with an excess
number of germ cells cause complete feminization(Morinaga et al., 2007 PNAS). The sex reversal to female isthe secondary effect of the over-proliferation of germ cells
due to the impairment of an ancient type of the TGFb signal,
the anti-Müllerian hormone (AMH) system (manuscript
submitted). Importantly these sex reversals are independentof presence or absence of the sex determination gene on the
Y chromosome. This means that the balancing between germ
cells and somatic cells is essential for proper manifestation ofsex directed by the sex determination gene. We havetherefore proposed that the germ cells critically contribute to32

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

II. Sox9b is essential for germ cell maintenancebut not critically contribute to testis
determination.

As mentioned above in mammals, Sox9 is essential for
initiation of testis formation and is under transcriptional
regulation of mammalian testis determining gene, Sry, on the
Y chromosome. Consistent with the function of mammaliansox9 gene, there are several reports on the upregulation ofsox9 expression in testis of other vertebrates. These reports
may collectively suggest the conserved role of sox9 in testis
determination across the vertebrate species. However, in
medaka, sox9 is expressed not only in Sertoli cells of thetestis but also in germinal cradles with germline stem cells.
This puts into doubt the conventional story of a conservedrole of Sox9. Therefore we have isolated two sox9b mutant
medaka with different alleles.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

The phylogenetic and syntenic analyses show that sox9b is
one of the co-orphologues of mammalian Sox9 in medaka.
We have found that only sox9b is expressed in thedeveloping somatic (supporting) cells. This indicates thatsox9b is the functional homologue of mammalian Sox9 in
medaka gonads.
The detailed examination of developing gonads shows thatthe number of germ cells are reduced in the sox9b mutant.
This is consistent with a decreasing level of mitotic activity
and an increasing level of apoptosis of germ cells in the
mutant. The reduced levels parallel the mutant allele number,suggesting the dose-dependent effect of sox9b function in thephenotype. Very interestingly, even in the homozygous
mutant, the gonad is formed, indicating that identity of the
supporting cells to develop into the gonadal somatic cells is
retained with the lack of sox9b function. In addition to thedecreasing germ cell phenotype, the heterozygous mutant
with XX chromosomes (genetically female) exhibits
masculinization. This is a completely opposite phenotype ofsex reversal (male to female ) from that observed in
mammals
We have found that the extracellular matrix (ECM) that
separates interstitial regions from germ cells and supporting
cells is largely disorganized in the mutant gonads. The germ
cells often protrude blebs from the discontinuous ECM,
suggesting that cellular association is impaired in the mutant.
Chimeric analyses between the wild type and the mutant
clearly demonstrates that mutant cells, which have the ability
to develop into the gonads, are inclined to be expelled fromthe chimeric gonads (figure 3). The degree of contribution isdependent on the number of functional sox9b alleles. Fromthese results, sox9b functions to maintain the germ cells
through the regulation of ECM (Nakamura et al., 2012 PLoSONE) .

loss of germ cells might cause the sex reversal. To confirm
this hypothesis, we have tried to recover the number of germ
cells in the sox9b mutant. Since the heterozygous hotei
mutant possesses increasing mitotic activity, a compoundmutant with hotei mutant allele and sox9b mutant allele weregenerated and analyzed. The heterozygous compound mutant
had the normal number of germ cells and did not display sex
reversal. This demonstrates that the sex reversal is primarily
due to the loss of germ cells (Nakamura et al., 2012 PLoS
ONE).

III. Functional divergence of sox9 explains thedifferent configuration of ovaries between
medaka and mammals.

In medaka, sox9b contributes to the maintenance of germ
cells including germline stem cells. Our results show that thetestis-determining function of mammalian sox9 appends tothe conserved role of sox9 in germ cell maintenance as
neofunctionalization. With the acquisition of maledetermining function, sox9 expression was lost in the
mammalian developing ovary. This is very well consistent
with the loss of, or low number of, germline stem cells in the
mammalian ovary. (figure 4) .

Figure 4. As sox9 acquired male determining functionality its expression
was lost in the mammalian ovary.

Figure 3. Chimeric gonad between wild type (red) and sox9b-mutant(green) cells. The mutant cells have the ability to develop into gonadal
somatic cells but contribute less to the gonad than the wild type cells.
Blue cells are germ cells.

Next we addressed the possible involvement of sox9b in thesex determination process. As mentioned above, the loss ofsox9b function results in development into male gonads. The
expression analysis demonstrates that the gonads in the XX
mutant express male-specific genes, showing that in the XX
mutants testicular development proceeds normally as in wildtype XY medaka. The sex reversal phenotype is rescued by
one copy of a functional sox9b gene, Unlike in mammals,
transgenic medaka with three copies of functional sox9b do
not show any sign of masculinization. From these data, wehave concluded that sox9b is not a critical contributor ofmale sex determination.
Then the next question is how the sex reversal to maleoccurs in the sox9b XX mutant. We have suspected that the

Publication List

〔Original paper〕

T., Oshima, Y., Kinoshita, M., Tanaka, M., Mishima, N.,• Hano,Wakamatsu, Y., Ozato, K., Shimasaki, Y., and Honjo, T. (2011).

Evaluation of the effects of ethinylestradiol on sexual differentiation in
the olvas-GFP/STII-YI medaka (transgenic Oryzias latipes) strain as
estimated by proliferative activity of germ cells. Aquatic Toxicol. 104,
177-184.

〔Review papers〕

S., Kobayashi, K., Nishimura, T., and Tanaka, M. (2011).
• Nakamura,
Ovarian germline stem cells in the teleost fish, medaka (Oryzias
K., Tanaka, M., and Takeda, H. (eds). (2011). “Medaka” a• Naruse,model for organogenesis, human disease, and evolution. total 387
latipes). Int. J. Biol. Sci. 7, 403-409.

pages. Springer-Verlag, Tokyo ISBN 978-4-431-92690-7.

33

National Institute for Basic Biology

Developmental Biology

LABORATORY OF PLANT ORGAN DEVELOPMENT

Director-General

OKADA, Kiyotaka

Assistant Professor:
Postdoctoral Fellows:

Visiting Scientists:

Technical Assistants:

Secretary:

TATEMATSU, Kiyoshi
IGARASHI, Hisako
TOYOKURA, Koichi
URAWA, Hiroko
YABE, Kimihiko
IKEUCHI, Momoko
IWASAKI, Akira
NAKATA, Miyuki
TAMESHIGE, Toshiaki
TOYOKURA, Koichi*
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,
biochemical, microsurgical and one-cell gene induction
approaches.

likely to move cell to cell. We also analyzed the function
domain of miR165 when primary transcript of MIR165A wasexpressed by the FIL-promoter. this revealed that miR165
can act in the entirety of the leaf primordia, suggesting that
there is no physical barrier interfering with miRNA
movement between the adaxial- and the abaxial-side.
To examine the mechanisms of establishment of the
adaxial-abaxial axis, we isolated novel mutants which show
altered patterns of FIL promoter::GFP expression, andnamed them enlarged fil-expression domain (enf). One ofthem, enf1, forms leaves with enlarged and reduced FILexpression domains, indicating that ENF1 is involved in the
fixation or maintenance of the position of the adaxial-abaxial
boundary (Toyokura et al., 2011). We revealed that the ENF1
gene encodes SUCCINIC SEMALDEHYDE
DEHYDROGENASE, which catalyzes the conversion of
succinic semialdehyde (SSA) to succinate, and is strongly
expressed in leaf primordia although its expression was notfound in the SAM. Exogenous application of SSA at the sideof the SAM induced the adaxial-characters on the abaxial
side of newly formed leaves (Figure 1). These results
indicate that SSA and/or its derivatives affect the axisdependent cell fate in leaf primordia. We also isolated some
suppressor mutants of enf1, which show normal leaf shapes.
We determined the genes, which have the mutation for the
suppression of enf1 phenotype, using next-generation
sequence methods, and revealed that one of them has themutation in a gene which encodes a transaminase enzyme.

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 cells locating in the abaxial side and determine theadaxial-specific expression of PHB. One of the MIR165/166
genes, MIR165A, is expressed in the abaxial epidermal cells.
We revealed by the visualizing reporter genes that MIR165A
is enough to repress the PHB expression in the cells locatedin the abaxial side. These results suggested that miR165 is34

Figure 1. Effect of exogenous application of SSA on the adaxial-abaxial
polarity in leaf primorda. We applied SSA-containing lanolin paste on theside of the Arabidopsis SAM, and checked the newly formed leaves after
1-2 weeks. Yellow arrowheads indicate the application site, and red
arrows represent the newly formed leaves. (A) The lateral views of
extreme effect (class I) of SSA taken by stereomicroscopy (left) andscanning electron microscopy (right). Newly formed leaves have theadaxial-characteristics on the abaxial-side. (B) The abaxial view of strong
effect (class II) of SSA. Newly derived leaf developed with two laminas
with upper sides facing each other. (C) The sections of the leaf petiols of
the control leaf (left) and class II leaf (right). In class II leaves, theadaxial identity was observed not only on the side facing the SAM but
also on the abaxial side.

In contrast, another mutant, enf2, has leaves with anenlarged FIL-expression domain, and the ENF2 gene

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

encoded a plastid-localized unknown protein. Chloroplastdevelopment was repressed in a severe allele of the enf2
mutant. Exogenous application of inhibitors for the gene
expression of chloroplast genome-encoding genes to
Arabidopsis seedlings mimics the defects of the FILexpression pattern by enf2 mutation. These results suggestthat the expression of chloroplast genome-encoding genes isrequired for the determination of adaxial-abaxial polarity.
A line of unique oblong cells is found at the marginal edgeof leaves. We noticed that a homeobox-related gene,
PRESSED FLOWER (PRS) and its homolog, WOX1, arerequired for forming the margin-specific cells. The analysesof prs wox1 double mutants indicate that the margin-specificcells act as a physical barrier separating the epidermal cellsof the adaxial-side surface from those of the abaxial-side
surface. Genetic analyses also showed that PRS and WOX1
function in blade outgrowth downstream of the adaxialabaxial polarity. The expression of PRS and WOX1 wasobserved in two middle mesophyll layers and the marginal
region of leaf primordia. We also revealed that the expressionof PRS and WOX1 are upregulated by the function of FIL
and YAB genes, and repressed by those of the abaxial-specificgenes, KANADI. We propose that the blade outgrowth andthe adaxial-abaxial patterning during leaf developments arecontrolled by the middle domain-specific function of PRS
and WOX1 genes.
To reveal how floral organs fix their forms throughdevelopment processes, we analyzed mutants named folded
petals (fop). In the early stage of flower development, fop
petals are similar to those of wild type, but the petals cannot
grow through the narrow space between the sepal and theanther in the flower buds. In fop mutants, petals growstraight when the sepals are removed in the early stage offlower development. We found that the petal epidermal cellsof fop petals bear epicuticular nanoridges like as those ofwild type. 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 before the epicuticularnanoridge formation.

II. Biochemical approach

We are taking a biochemical approach to study of theintercellular signaling system by analyzing small peptides ascandidates for intercellular signaling ligands, which arepresent in the apoplastic region of the SAM. Small peptides
were collected from apoplast fractions of the curds ofcauliflower (Brassica oleracea L. var. botrytis). Through abiological assay using purified fractions, we obtained afraction, which increased the number of SAMs after
exogenous application of the fraction to Arabidopsis
seedlings (Figure 2). Then we analyzed the peptide
sequences included in the fraction by LC-MS/MS methods,
and a putative lipid transfer protein was identified. We arepreparing a recombinant protein of the candidate protein andexamining effects of the recombinant protein on thedevelopment and growth of Arabidopsis seedlings.

III. Microsurgical approach

We are also carrying out microsurgical approaches usingnovel laser-ablating microscopy to investigate the cell-to-cellsignaling system working during leaf development. When weablated a small number of cells at the peripheral of the SAM
of young Arabidopsis seedlings, some of the newly generated
leaves changed to a filamentous structure lacking the
adaxial-abaxial identity, suggesting that a flow of signal(s)from the SAM to the leaf primordia has a role of fixing theabaxial-adaxial polarity. We also examined the leaf serration
mechanism by ablating some cells at the margin in serrated
leave of E. californica, which form the leaflet acropetally.
When leaflet incipient site is ablated, a new leaflet initiatedat intact tissue near leaf tip, escaping from the ablated site,
suggesting that a regular space of the leaflet initiation site is
actively kept from the leaf tip.

IV. One-cell gene induction approach

As a new tool for examining the intercellularcommunication system, we are developing a one-cell geneinduction system in planta using the InfarRed Laser Evoked
Gene Operator (IR-LEGO) system, and showed gene
expression in only a single cell of the root. When WUSSEL(WUS) gene, which functions in maintenance of the SAM,
was ectopically expressed in the root, callus or a shoot-likestructure was generated at the root tip. We observed
induction of ectopic cell division when WUS was ectopically
expressed in lateral-root-cap cells.

Publication List

〔Original papers〕

K., Watanabe, K., Oikawa, A., Kusano, M., Tameshige, T.,• Toyokura,
Tatematsu, K., Matsumoto, N., Tsugeki, R., Saito, K., and Okada, K.

Figure 2. A purified fraction increasing the number of the SAMs in
Arabidopsis seedlings. Purified fractions obtained from the apoplastic
region of the curds of cauliflower were applied to Arabidopsis CLV3
promoter:GUS transgenic lines. A purified fraction increased the numberof the SAMs (Right) compared to the control (Left). GUS expression
driven by the CLV3 promoter indicates the position of the SAM. White
dashed lines indicate the shape of the SAM. Bars indicate 200 µm.

•(2011) Succinic semialdehyde dehydrogenase is involved in the robust
patterning of Arabidopsis leaves along the adaxial-abaxial axis. Plant
Cell Physiol. 52, 1340-1353.
Ueda, M., Matsui, K., Ishiguro, S., Kato, T., Tabata, S., Kobayashi, M.,
Seki, M., Shinozaki, K., and Okada, K. (2011) Arabidopsis RPT2a
encoding the 26S proteasome subunit is required for various aspects ofroot meristem maintenance, and regulates gametogenesis redundantlywith its homolog, RPT2b. Plant Cell Physiol. 52, 1628-1640.

35

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

Associate Professor

Assistant Professors:

SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
YOSHIDA, Masahide
KUBOYAMA, Kazuya
FUJIKAWA, Akihiro
SUZUKI, Ryoko
NISHIHARA, Eri
MATSUMOTO, Masahito
TANI, Sachiko
SAKURABA, Jyuichi
MIYAZAKI, Yuriko
HATTORI, Nobuko
TOMITA, Nao
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
MIYAZAKI, Emi
WADA, Kotoe
KODAMA, Akiko

NODA, Masaharu

Technical Staff:
NIBB Research Fellows:
Postdoctoral Fellows:

Graduate Students:
Technical Assistants:

Secretary:

SHINTANI, Takafumi

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 is preferentially expressed in the nervous
system from early developmental stages through toadulthood.
APC2 is distributed along microtubules in growth cones aswell as axon shafts of retinal axons. The knockdown of Apc2
in chick retinas reduced the stability of microtubules inretinal axons and yielded abnormal behaviors including a
reduced response to ephrin-A2 and misprojection in the36

tectum without making clear target zones. Recently, we
generated Apc2-deficient mice by a gene-targeting technique.
We have found that the mutant mouse displays severe
laminary defects in some brain regions including the cerebral
cortex and cerebellum. We are now analyzing the phenotypeof the Apc2-deficient mouse in more detail to clarify thefunction of APC2 in the development of the CNS.

II. Development of direction-selective retinal
ganglion cell subtypes

Visual information is transmitted to the brain by roughly adozen distinct types of retinal ganglion cells (RGCs) definedby characteristic morphology, physiology, and central
projections. However, because few molecular markers
corresponding to individual RGC types are available, ourunderstanding of how these parallel pathways develop is stillin its infancy.
The direction of image motion is coded by directionselective (DS) ganglion cells in the retina. Particularly, theON DS ganglion cells are critical for mediating the
optokinetic reflex. We generated a knock-in mouse in which
SPIG1-expressing cells are labeled with GFP. We
successfully visualized both upward- motion-preferring anddownward-motion-preferring ON DS ganglion cells (SPIG1+and SPIG1- ganglion cells, respectively) by a combination ofgenetic labeling and conventional retrograde labeling in the
medial terminal nucleus.
A key circuit module of DS ganglion cells is a spatiallyasymmetric inhibitory input from starburst amacrine cells in
the retina. However, it was not known how and when thiscircuit asymmetry is established during development.
Therefore, we photostimulated mouse starburst cells targetedwith channelrhodopsin-2 (CR-2) while recording from single
SPIG1+ DS cells. We then followed the spatial distribution ofsynaptic strengths between starburst and DS cells during
early postnatal development before these neurons can
respond to physiological light stimuli, along withconfirmation of connectivity by monosynaptically restrictedtrans-synaptic rabies viral tracing. As a result, we found that
random or symmetric synaptic connections from starburst
amacrine cells are established as early as postnatal day 6, andthat asymmetric inhibitory synaptic inputs are subsequently
developed over a 2-day period (Figure 1).

Figure 1. CR-2-assisted circuit mapping at P6 and P8. Recordings fromSPIG1+ cells at P6 (A–C) and P8 (D–F). (A–E) Inhibitory (A, D) andexcitatory (B, E) postsynaptic currents elicited in a SPIG1+ cell by thephotostimulation of the surrounding starburst amacrine cells. Polar plots
are also shown. (C, F) Spatial asymmetry index (SAI) for inhibition andexcitation. Error bars, s.d.

Analysis of gene-expression profiles in the two types of ON
DC ganglion cells is now under way. This will shed light onmolecular mechanisms underlying the differentiation anddistinct circuit formation of the two DS ganglion cell types.

III. Physiological roles of protein tyrosine
phosphatase receptor type Z

Protein-tyrosine phosphatase receptor type Z (Ptprz, also
known as PTPz/RPTPb) is a member of the R5 receptor-likeprotein tyrosine phosphatase (RPTP) subfamily. Ptprz ispredominantly expressed in the brain 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. Although ourunderstanding of the physiological functions of Ptprz is thus
advancing, our knowledge about its biochemical properties
such as substrate specificity are still limited.
We previously identified G protein-coupled receptor kinase
interactor 1 (Git1), membrane-associated guanylate kinase,WW and PDZ domain-containing 1 (Magi1), and GTPaseactivating protein for Rho GTPase (p190RhoGAP) as
substrates for Ptprz by developing a new genetic method
named “yeast substrate-trapping system”. We had alreadyidentified a dephosphorylation site at Tyr-1105 of
p190RhoGAP; however, the structural determinants
employed for substrate recognition of Ptprz have not been
fully defined.
T h i s y e a r, w e r e v e a l e d t h a t P t p r z s e l e c t i v e l ydephosphorylates Git1 at Tyr-554, and Magi1 at Tyr-373 andTyr-858 by in vitro and cell-based assays. Of note, alignmentof the primary sequences surrounding the target
phosphotyrosine residue in these three substrates showed
considerable similarity, suggesting a consensus motif forrecognition by Ptprz (Figure 2A). We then estimated thecontribution of surrounding individual amino acid side
chains to the catalytic efficiency by using fluorescentpeptides based on the Git1 Tyr-554 sequence in vitro (Figure2B), and thereby deduced the typical substrate motif for thecatalytic domain of Ptprz (Figure 2C). Furthermore, we
found by database screening that the substrate motif ispresent in several proteins, including paxillin at Tyr-118, itsmajor phosphorylation site (Figure 2A). Expectedly, we
verified that Ptprz efficiently dephosphorylates paxillin at
this site in cells.
Although initially viewed as broad specificity“housekeeping” enzymes, PTPs are actually highly selective
enzymes. Our knowledge of substrates for Ptprz suggests that
Ptprz dephosphorylates multiple proteins associated withactin remodeling, which plays important roles in synaptic
plasticity in the adult brain and cell adhesion/migration ofepithelial cells.

Figure 2. Identification of the substrate motif for Ptprz. (A) Primary
sequences surrounding the target phosphotyrosine residues located in
Git1 (Tyr-554), p190RhoGAP (Tyr-1105), Magi1 (Tyr-373 and Tyr-858),
and paxillin (Tyr-118) aligned, together with four substrate candidates,
AFAP1L2 (Tyr-56), KIAA1486 (Tyr-277), UBR3 (Tyr-1284), and TJP2/
ZO-2 (Tyr-486). Boxed residues are conserved in at least three substratesequences. (B) Kinetic analysis for the hydrolysis of Git1-derived peptide
substrates by PtprzICR in vitro. The amino acid sequences of thesubstrate peptides (P1–10) used in this study are shown on the left of the
figure. P1, P8, and P9 peptides correspond to Git1550–556, Git1549–556,
and Git1548–556, respectively, except that the Tyr residue at 554 isreplaced with a phosphotyrosine mimic, pCAP. P1–P7 and P10 peptides
are Git1 peptide derivatives, in which the substituted amino acid residues
are shown in bold. (C) Proposed consensus substrate-site motif for the
PTP catalytic domain of Ptprz.

IV. Brain systems for body-fluid homeostasis

Mammals have a set of homeostatic mechanisms that work
together to maintain body-fluid osmolality at near 300
mOsm/kg through the intake or excretion of water and salt.
Although this homeostatic osmoregulation is vital, themechanisms for the detection of these fluctuations have notbeen fully elucidated. To date, transient receptor potential
vanilloid 1 (TRPV1), a cation channel, has been implicatedin body-fluid homeostasis in vivo based on studies with the
TRPV1-knockout mouse. However, the response of TRPV1
to hypertonic stimuli has not been demonstrated withheterologous expression systems so far, despite intense
efforts by several groups. Thus, the molecular entity of thehypertonic sensor in vivo still remains controversial.
Very recently, we found that the full-length form of TRPV1
is sensitive to an osmotic increase exclusively at around bodytemperature by using human embryonic kidney 293 cells

37National Institute for Basic Biology

Neurobiology

stably expressing rat TRPV1 (HEK293-TRPV1 cells). We
applied a hypertonic solution (350 mOsm) to HEK293TRPV1 cells at various temperatures from 24 to 40°C
(Figure 3A and B, HEK293-TRPV1). When the extracellularenvironment was changed from an isotonic (300 mOsm) to
hypertonic (350 mOsm) solution at 24°C, [Ca2+]i increased
only slightly in HEK293-TRPV1 cells, but not in native
HEK293 cells (Figure 3A and B). Surprisingly, themagnitude of the osmosensitive response markedly increased
with temperature, peaking at around 36°C, which is close tonormal mammalian body temperature (~37°C) (Figure 3A
and B). In contrast, control HEK293 cells did not respond tothe hypertonic stimulus at any temperature. Importantly, theresponse at 36°C showed a robust increase over a hypertonic
range, but a small decrease over a hypotonic range (Figure
3C). A TRPV1 antagonist, capsazepine, and a nonspecific
TRP channel inhibitor, ruthenium red, completely blocked
the increase in [Ca2+]i. These results endorse the view that the
full-length form of TRPV1 is able to function as a sensor ofhypertonic stimuli.
We also demonstrated that the osmosensitivity of TRPV1 at
36°C is further enhanced by other activating stimuli, such as
protons (pH) or capsaicin, indicating that osmosensitivity of
TRPV 1 is synergistically enhanced by these distinct
activating stimuli. (Figure 3D and E). Our findings thus
indicate that TRPV1 integrates multiple different types ofactivating stimuli, and that TRPV1 is sensitive to hypertonic
stimuli under physiologically relevant conditions.

(gray) to 350 mOsm (black). (B) Summary of the change in the
fluorescence ratio during perfusion with the hypertonic solution at
various temperatures in HEK293-TRPV1 (filled bars) and HEK293 (openbars) cells. Data are differences between fluorescence ratios before andafter the change of the solution. The maximal sensitivity of the HEK293TRPV1 cells to the hypertonic stimulation was observed at 36°C. (C)Summary of the change in the fluorescence ratio during the perfusion ofvarious solutions differing in osmolality at 36°C. Osmolality-dependentchanges in the [Ca2+]i were observed in HEK293-TRPV1 cells (filled
circles), but not in HEK293 cells (open circles). (D) Summary of thechange in the fluorescence ratio during the perfusion of solutions with therespective osmolality and pH at 36°C. The osmotic response of the [Ca2+]iin HEK293-TRPV1 cells (filled circles) was enhanced by acidification
over the entire osmolality range (compare with filled triangles). This wasevident especially in the hypertonic range. (E) Summary of the change inthe fluorescence ratio in HEK293-TRPV1 cells upon application of 1.5
nM capsaicin solutions with different osmotic strength. Response of
TRPV1 to hyperosmolality was markedly potentiated in the presence of
capsaicin (compare filled circles with filled triangles). No response wasobserved in HEK293 cells (open circles). Error bars, s.e.m.

Publication List

〔Original papers〕

A., Fukada, M., Makioka, Y., Suzuki, R., Chow, J.P.,• Fujikawa,Matsumoto, M., and Noda, M. (2011). Consensus substrate sequence

•
•
•
•

Figure 3. Osmosensitivity of TRPV 1 is synergistically enhanced by
distinct activating stimuli such as temperature and protons. (A)Temperature-dependent sensitivity to osmotic stimuli. Representative
pseudo-color images of [Ca2+]i in HEK293-TRPV1 cells at 2 min before(upper left) and 4 min after (upper right) perfusion with a hypertonic
solution (350 mOsm) at 36°C. Representative single cell traces of thefluorescence ratio at a temperature of 24, 30, 36, or 40°C (lower graph).
The line on the top indicates the timing of the change from 300 mOsm

38

for protein-tyrosine phosphatase receptor type Z. J. Biol. Chem. 286,
37137-37146.
Nayak, G., Goodyear, R.J., Legan, P.K., Noda, M., and Richardson, G.P.
(2011). Evidence for multiple, developmentally regulated isoforms of
PTPRQ on hair cells of the inner ear. Dev. Neurobiol. 71, 129-141.
Nishihara, E., Hiyama, T.Y., and Noda, M. (2011). Osmosensitivity oftransient receptor potential vanilloid 1 is synergistically enhanced by
distinct activating stimuli such as temperature and protons. PLoS ONE
6, e22246.
Sakamoto, K., Bu, G., Chen, S., Takei, Y., Hibi, K., Kodera, Y.,
McCormick, L.M., Nakao, A., Noda, M., Muramatsu, T., andKadomatsu, K. (2011). The premature ligand-receptor interaction
during biosynthesis limits the production of growth factor midkine andits receptor LDL receptor-related protein 1 (LRP1). J. Biol. Chem. 286,
8405-8413.
Yonehara, K., Balint, K., Noda, M., Nagel, G., Bamberg, E., and Roska,
B. (2011). Spatially asymmetric reorganization of inhibition establishes
a motion-sensitive circuit. Nature 469, 407-410.

DIVISION OF BRAIN BIOLOGY

Professor

YAMAMORI, Tetsuo

Associate Professor

WATAKABE, Akiya

Assistant Professors:

KOMINE, Yuriko
SADAKANE, Osamu
Assistant Professor(Specially appointed):
KOMATSU, Yusuke#
NIBB Research Fellow:
OHTSUKA, MasanariTechnical Staff:
OHSAWA, Sonoko
Postdoctoral Fellows:
TAKAJI, Masafumi#
OHTSUKA, Masanari*
HATA, Katsusuke#
GOTO, Ayako#
SASAKI, Tetsuya
Visiting Scientist:
HIRAKAWA, Reiko
Graduate Students:
NAKAGAMI, Yuki*
SHUKLA, RammohanTechnical Assistants:
NAKAMURA,Tohru
TAKEDA, Yuta#
NAKAGAMI, Yuki
MORITA, Junko
IMAI, Akiko
KOTANI, Keiko
KON, Yayoi
MIKAWA, Ryuta
IMOTO, EikoHIRAYAMA, Yuka#
TAKAFUJI, Mituse#
KAJITANI, Tomoki#
TAKAHASHI, Yoichi##: SRPBS (Strategic Research Program for Brain Sciences), NIPS

We are studying genes that are expressed in specific areasof the neocortex in order to understand the principles
governing the formation of the primate brain.

I. Genes expressed in specific areas and layersof the neocortex

The neocortex emerged in mammals and evolved mostremarkably in primates. To understand the underlying
mechanisms of the primate brain we study gene expression
patterns within different areas of the neocortex.
We have reported the findings that are schematicallyillustrated in Figure 1.
Using differential display methods, we found three areaspecific expression genes in the primate neocortex. Firstly,occ1 is specifically expressed in the occipital cortex in the
primate brain. Secondly, the other gene that showed marked
difference within the neocortex is gdf7, a member of the
BMP/TGF-b family, which is specifically expressed in the
motor cortex of the African green monkey (Watakabe et al.,J. Neurochem., 76, 1455-1464, 2001). Thirdly, RBP (retinolbinding protein) is preferentially expressed in the associationand higher areas in the neocortex (Komatsu et al., Cerebral
Cortex, 15, 96-108, 2005).
To further screen area-specific molecules systematically inthe monkey neocortex, we carried out another round ofscreening using the RLCS method (Suzuki et al. 1996;

Figure 1. The expression of visual area specific genes (orange color) andassociation area specific genes (blue) and gdf7 (green) are schematicallyillustrated. Top and bottom views are medial and lateral surfaces,
respectively. (cited from Yamamori & Rockland, Neurosci. Res., 55,
11-27, 2006 )Shintani et al. 2004). In this analysis, mRNAs were purified
from 4 distinct cortical areas, converted to cDNA by reverse
transcription and digested with a pair of restriction enzymes
for 2-dimensional analysis. Using the RLCS method weisolated genes that showed marked differences among fourareas (area 46, primary motor area, TE and V1) andcharacterized the expression patterns. Examples of suchgenes we have previously reported are testican-1, -2 (OCC1
related family genes), 5HT1B and 5HT2A (primary visualarea enriched), which are preferentially expressed in the
primary visual cortex, and SPARC (an OCC1 related gene)and PNMA5 whose expressions are similar to RBP (anassociation area enriched gene) as shown in Figure 1.
This year, I reviewed our studies over the last ten years andmore (Yamamori, 2011). The main discussions andconclusions are the functional significance of the two groupsof the genes as shown in Figure 1. The first group of genes
are those highly expressed in the primary sensory areas,
particularly in the primary visual area (V1). In collaboration
with Prof. Hiromichi Sato, Osaka University, we havepreviously shown that 5HT1B works by enhancing signal tonoise (S/N) ratio and 5HT2A works as a gain controller(Figure 2). Recently, it has been reported that, fstl1(follistatin-like 1), the mouse homologue of OCC1, is highly
expressed in mouse dorsal root ganglion (DRG). The
function in DRG has been revealed by the other group,
showing that Fstl1 is directly bound to Na, K. ATPase, which
suppresses sensory evoked presynaptic transmission byenhancing K+ influx and suppressing voltage dependent
Ca++ influx (Li et al., Neuron 69, 974-987, 2011). Thismechanism likely also works in primate V1 where OCC1
(macaque homologue of fstl1) is abundantly expressed. The

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

39

National Institute for Basic Biology

Neurobiology

major difference between macaque V1 and mouse tissues
that highly express OCC1 and fstl1, respectively, is that
expression of OCC1 in V1 is activity-dependent whereas
fstl1 expression in mouse tissues is not activity-dependent(Takahata et al., J. Chem Neuroanat., 35, 146-157, 2008).
These data of 5HT1B, 5HT2A and OCC1 expressions andfunctions strongly suggest that these genes control or
modulate inputs from the retina in activity-dependent
manners at the expression and functional levels (Figure 2).
Such expression patterns specific to primates have comeabout during the course of primate evolution (Takahata et al.,2011).

Figure 2. Roles of 5HT1B, 5HT2A and OCC1 in primate V1. Right:
Possible roles of OCC1 in primate V1. As shown in rodent DRG, OCC1
is likely bound to Na, K, ATPase (NKA) and suppresses visual stimuli
evoked synaptic transmission. The suppression is further enhanced by
activity-dependent gene expression. Left: Responses to a stimulus with alow contrast are suppressed by the activation of 5HT1B and are enhancedby that of 5HT2A. At a high stimulus contrast, the effects are reversed.
S/N ratio analysis demonstrated that the activation of 5HT1B contributes
to the increase in S/N ratio. We predict that 5HT2A works as a gain
controller to compensate for the gain or loss caused by 5HT1B receptors
at a certain stimulus contrast. The left picture is drawn by Drs. Hiromichi
Sato and Satoshi Shimegi (Osaka University) and is cited from
Yamamori, T., Progress in Neurobiology 94, 201-222, 2011.

The second group of genes is selectively expressed in theassociation areas of the primate neocortex. Among theassociation area selective-expression genes, RBP, PNMA5,SPARC, and SLT2 are expressed in layers 2, 3, 5 and 6 inassociation areas and most abundantly prefrontal cortex andhigher sensory association areas. SLIT1, on the other hand, isexpressed in layers 2, 4 and 5 and most abundantly in layer 4
in the prefrontal cortex and to a lesser extent in the sensory
association areas (Sasaki et al., Cerab. Cortex. 20, 24962510, 2010). Although the functions of these association
area-selective genes are unknown, SLIT1 works to enhance
dendritic branching in developing cortical neurons in rodents
and thus presumably has a similar function in the primate
neocortex.

II. M ultisensory Information Facilitates
Reaction Speed by Enlarging Activity
Difference between Superior Colliculus
Hemispheres in Rats

Animals can orient responses faster to multisensory

40

stimuli than to unisensory stimuli. We have been working on
studies of how animals can respond to multisensory
stimulation in collaboration with Professor Yoshio Sakurai(Kyoto University) using an experimental system that
evaluates audio-visual discrimination tasks in rats (Sakata etal., Exp Brain Res. 159, 409-417, 2004). Using this system,
we identified the brain areas of V2L that are specificallyinvolved in multisensory (visual and auditory) stimuli(Hirokawa et al., Neuroscience, 1402-1417, 2008). Thesestudies were further extended to the superior colliculus (SC).

Figure 3. Two-alternative spatial discrimination tasks based on auditory
and/or visual cues.
A) Timing of task events. Nose poking into the central hole initiated a
trial. After a variable foreperiod, a cue stimulus was delivered from theleft or right, randomly chosen from visual, auditory and audiovisual
stimuli. Rats responded to the stimulus by withdrawing from the central
hole and selected the direction of the cue stimulus by poking their heads
into the hole ipsilateral to the stimulus. (B) Mean reaction time for eachtype of stimulus across sessions and rats (*p<0.001 in ANOVA and post
hoc Tukey test). Error bars, standard deviation. This figure is cited from
Hirokawa et al., Plos One, 2011.

The superior colliculus (SC), which receives multiple inputs
from different sensory modalities, is thought to be involvedin the initiation of orienting responses. However, themechanism by which multisensory information facilitatesorienting responses had not yet been understood. We
demonstrate that multisensory information modulatescompetition among SC neurons to elicit faster responses. We
conducted multiunit recordings from the SC of rats
performing a two-alternative spatial discrimination taskusing auditory and/or visual stimuli. We found that a large
population of SC neurons showed direction-selective activity
before the onset of movement in response to the stimuli
irrespective of stimulation modality. Trial-by-trial correlation
analysis showed that the premovement activity of many SC
neurons increased with faster reaction speed for thecontraversive movement, whereas the premovement activity

the ipsiversive movement was depressed. Unilateralinactivation of SC using muscimol prolonged reaction timesof contraversive movements, but it shortened those ofipsiversive movements. These findings suggest that thedifference in activity between the SC hemispheres regulates
the reaction speed of orienting responses, and multisensory
information enlarges the activity difference resulting in faster
responses (Hirokawa et al., 2011).
The SC has been considered as a brain region that is
causally essential for integrating visual and auditory
information, as demonstrated by a study of excitotoxic lesionof the SC in cats. On the other hand, our study showed thatunilateral SC inactivation did not affect the facilitation ofreaction speed to multisensory stimuli. It is therefore possiblethat different neural networks are responsive for accurate
sensory detection and rapid response, respectively. In
consistent with a line of this idea, we previously showedusing the same technique as this study that the inactivation of
the secondary visual cortex (V2L) suppresses the facilitationof reaction speed (Hirokawa et al., Neuroscience, 1402-1417,
2008).

Figure 4. Direction preference preceding locomotion.
(A) Example of tetrode isolation for single unit. (Top) Average
waveforms of spikes recorded on the four tetrode channels correspond toa cluster in black in the scatter plots below. The scatter plots indicate thepeaks of waveforms from channel 3 plotted against principal component
1 (PC1) from channel 3 recorded from a tetrode. (Right) Corresponding
autocorrelation functions with a window of ±30 ms. The bin size is 0.1
ms. (B) Rasters and spike density functions (SDFs) aligned to the onsetof the target for isolated single unit (black cluster) during spatial
discrimination task. The data were derived from only correct trials in
responses to contralateral stimuli divided into visual, auditory andaudiovisual stimulus conditions. Trials in the raster plot are sorted
according to reaction time. (C) The data of the same neuron as above
were aligned to movement onset and divided into ipsiversive andcontraversive movement trials regardless of the modality of the stimulus.
(D) Direction preference index of above unit was calculated using
receiver operating characteristics (ROC) analysis for each time point(p<0.05, permutation test). (E) Direction preference curves for all cells(225 cells). Each row corresponds to one cell. Cells were sorted by thetime of center of mass of significant positive preferences. Red and blue
colors indicate preference indices with significant positive and negativevalues (p<0.05, permutation test), respectively, and gray points
correspond to insignificant values. (F) Distribution of direction
preference index in premovement period across population (225 cells).
(G) Preference calculated for correct trials plotted against preference
calculated for erroneous trials in premovement period. (H) Direction
preferences calculated for each stimulus modality in each neuron andwere averaged for each stimulus modality condition. The blue periodshows a significant modulation of the preference under the multisensory
condition (AV>unimodal, ANOVA with post hoc tukey test, p<0.05). This
figure is cited from Hirokawa et al., 2011.

Publication List

〔Original papers〕

J., Sadakane, O., Sakata, S., Bosch, M., Sakurai, Y., and• Hirokawa,
Yamamori, T. (2011). Multisensory information facilitates reaction••

speed by enlarging activity difference between superior colliculus
hemispheres in rats. PLoS ONE 6, e25283.
Kitsukawa, T., Nagata, M., Yanagihara, D., Tomioka, R., Utsumi, H.,
Kubota, Y., Yagi, T., Graybiel, A.M., and Yamamori, T. (2011). A novel
instrumented multipeg running wheel system, Step-Wheel, formonitoring and controlling complex sequential stepping in mice. J
Neurophysiol. 106, 479-487.
Rossini, L., Moroni. R.F., Tassi, L., Watakabe, A., Yamamori, T.,
Spreafico, R., and Garbelli, R. (2011). Altered layer-specific gene
expression in cortical samples from patients with temporal lobe
epilepsy. Epilepsia 52, 1928-1937.

〔Original paper (E-publication ahead of print)〕

T., Shukla, R., Yamamori, T., and Kaas, J.H. Differential• Takahata,
expression patterns of striate cortex-enriched genes among old world,
new world, and prosimian primates. Cereb. Cortex. 2011 Nov. 7.

〔Review article〕

T. (2011). Selective gene expression in regions of primate• Yamamori,neocortex: implications for cortical specialization. Prog. Neurobiol. 94,
201-222.

of another population of neurons decreased with faster
reaction speed for the ipsiversive movement. When visualand auditory stimuli were presented simultaneously, thepremovement activity of a population of neurons for thecontraversive movement was enhanced, whereas the
premovement activity of another population of neurons for41

National Institute for Basic Biology

Neurobiology

DIVISION OF BRAIN CIRCUITS

Professor

MATSUZAKI, Masanori

Postdoctoral Fellows:
Visiting Scientist:
Graduate Student:
Technical Assistants:
Secretary:

MASAMIZU, Yoshito
TANAKA, Yasuhiro
TANAKA, Yasuyo
HIRA, Riichiro
OHKUBO, Fuki*
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.

I. Development of novel techniques tophotostimulate cortical neurons in vivo

We developed a method that uses Channelrhodopsin-2(ChR2; blue-light-activated cation channel) for transcranial
optogenetic stimulation. This method is based on scanning alight beam over the brain, thereby photostimulating ChR2expressing neurons in intact mice. The laser illuminationinduced forelimb movement in two areas; rostral forelimbarea (RFA) and caudal forelimb area (CFA). The motor
forelimb areas determined by this photostimulation mapping
corresponded to those determined by intracortical
microstimulation (ICMS). We also revealed functional
synaptic connections between RFA and CFA, by recording
electrical signals in either area during the photostimulation
mapping. In addition, we developed a method that uses
Halorhodopsin (NpHR; yellow-light-activated chloride ionpump) for inactivating cortical neurons in vivo. Yellow lightillumination on the cortical surface of NpHR transgenic mice
inhibited the forelimb movement induced by ICMS in RFA.
As these photostimulation methods were effective even to
awake mice, these methods can be applied to the experiments
described below.

mice had to wait with the lever in the wait position for awhile until the next trial. During the training sessions, taskdifficulty was increased by gradually increasing the leverpull time. However, mice could increase or maintain thenumber of successful trials (>100) and the intertrial interval
time decreased. After 8-9 training sessions, mice reliably
performed this task. We are also improving this task for
future research.

III. Spatio-temporal representation of motor
information in the brain
The aim of this study is to reveal how voluntary movement
is represented in cortical circuits. One of the most important
problems in neuroscience is how a variety of spatiotemporally heterogeneous neural activity in the cortex
emerges moment-by-moment at multiple stages of amovement. To reveal how their activities are organized in thelocal circuit, we carried out two-photon calcium imaging in
the layer 2/3 RFA and CFA of mice performing the lever-pull
task after the training sessions. We have found many types ofneurons in the local circuit. We are now analyzing theactivity, distribution, and connections of the cortical neurons
involved in sequential motor phases. The activities of thecortical neurons will be modulated by using the optogenetic
stimulation methods to clarify the direction of flow of motor
information. Our results will provide insights into theprinciples of circuit operation and the cellular basis forrecovery from brain cortical damage.

Publication List

〔Original papers〕

R., Wakimoto, M., Ebisu, H., Tanno, K., Hira, R., Kasai, H.,• Ako,
Matsuzaki, M., and Kawasaki, H. (2011). Simultaneous visualization of

•
•
•
•

multiple neuronal properties with single-cell resolution in the living
rodent brain. Mol. Cell. Neurosci. 48, 246-257.
Kanemoto, Y., Matsuzaki, M., Morita, S., Hayama, T., Noguchi, J.,
Senda, N., Momotake, A., Arai, T., and Kasai, H. (2011). Spatial
distributions of GABA receptors and local inhibition of Ca2+ transients
studied with GABA uncaging in the dendrites of CA1 pyramidal
neurons. PLoS ONE 6, e22652.
Matsuzaki, M., Ellis-Davies, G.C.R., Kanemoto, Y., and Kasai, H.
(2011). Simultaneous two-photon activation of presynaptic cells andcalcium imaging in postsynaptic dendritic spines. Neural Syst. Circuits
1, 2.
Matsuzaki, M., and Kasai, H. (2011). Two-Photon Uncaging
Microscopy. Cold Spring Harbor Protocols, pdb.prot5620.
Noguchi, J., Nagaoka, A., Watanabe, S., Ellis-Davies, G.C.R., Kitamura,K., Kano, M., Matsuzaki, M., and Kasai, H. (2011). In vivo two-photon
uncaging of glutamate revealing the structure-function relationships ofdendritic spines in the neocortex of adult mice. J. Physiol. 589, 24472457.

II. 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. Mice used the right forelimb topull a lever for a given time and were rewarded with a water
drop from a spout near the mouth, while simultaneously thelever was quickly pushed back to the wait position. Then,

42

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellows:
Visiting Scientist:

MATSUNAGA, Wataru
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.

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, wehave made progress in studies of the prey-predator
interaction using medaka and zooplankton.

Figure 1. Virtual plankton system. The test aquarium was attached to a
CRT display on which virtual plankton were shown. The behaviors of the
3 fish in the test aquarium was recorded by a webcam placed above thetest aquarium.

Visual motion 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 (Figure 1) where
the predation 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 (Matsunaga and Watanabe, Scientific Reports, inpress).

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).
In order to interact successfully with the environment,
animals must know the accurate positions of objects in space,though those positions frequently change. Neural processing,
however, requires considerable time. By the time a
conclusion is reached about location, the moving object has
moved on to a new position in the actual world. Does ourvisual system compensate for this difference?
One recent focus of this debate is the flash-lag effect(Figure 2), in which a moving object is perceived to lead aflashed object when both objects are aligned in actual
physical space. Last year, we proposed a simple conceptualmodel explaining the flash-lag effect (Delta model, Watanabe
et al., 2010). This year, we have attempted to expand themodel for application to the motor control mechanisms of thebrain.

Figure 2. Flash-lag effect. A moving cube is perceived to lead in space aflashed cube when both objects are aligned in actual physical space. Thiseffect has been utilized for understanding human motion perception. This
year, we successfully produced a 3D version of the Flash-lag effect using
Blender 3D software. Please refer to YouTube (DUBM-GG0gAk).

43

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROCHEMISTRY †Professor (Concurrent)

SASAOKA, Toshikuni

Postdoctoral Fellow:

SATO, Asako

Our major research interest is to understand thephysiological role of the dopaminergic system in animal
behavior, particularly locomotion and eating behaviors, using
genetically altered mice.

I. Role of dopaminergic transmission inlocomotion and eating behavior

The dopaminergic system is implicated in the modulation of
locomotor activity, the regulation of several peptide
hormones in the pituitary, the modulation of synaptic
plasticity and the development of neurons. The dopaminergic
system is also implicated in the control of emotion,
motivation and cognition. Dysfunction of the dopaminergic
system can result in several neurological and psychiatricdisorders, such as Parkinson’s disease and schizophrenia.
In mammals, two subgroups of dopamine receptor havebeen identified, referred to as D1-like receptors (D1R, D5R)
and D2-like receptors (D2R, D3R and D4R) on the basis of
their gene structure and their pharmacological andtransductional properties. D1R and D2R are the mostabundantly and widely expressed in the brain and often playa synergistic role.
To investigate the role of D1R and D2R in locomotor
control and eating behavior, we utilized D1R knockout (KO)mice, D2R KO mice, and transgenic mice harboring
tetracycline-regulated expression of the D1R gene. Daily
motor activity and food/water intake in these mice were
continuously monitored in home cage environment for longterm (Figure 1).

short-term sessions using beam breaks or video tracking
systems. Decreased locomotor activity in D2R KO mice, andboth increase and decrease in the locomotor activity in D1R
KO mice have been reported in different studies. We
analyzed beseline activity including all the motions (walking,rearing, grooming, climbing on the lid, and so on) in thehome cages for at least 5 days. We first focused on motor
activity for a 24 hr period and found that D1R KO mice were
hyperactive and that D2R KO mice were hypoactive
compared with wild type mice (Figure 2). To elucidate if
these mice have normal circadian rhythms in activity, we
analyzed motor activity in both light and dark phases.
Results showed that all mice had normal circadian rhythms
and that the difference in motor activity was seen during thedark phase. To further analyze the extent of activities in thedark phase, percentage of time exhibiting inactive, low,
medium and high activity states were calculated.
Interestingly, both D1R KO and D2R KO mice exhibited acomparable percentage of inactive state time. D1R KO micespent more time exhibiting a high activity state, whereas
D2R KO mice spent more time exhibiting a low activity statethan wild type mice.

Figure 2. Motor activity of D1R KO and D2R KO mice in the home cagefor 24 hr.

III. Motor activity in transgenic miceharboring controllable D1R expression

Figure 1. Experimental equipment for measurement of locomotor activity
and food/water intake

II. Motor activity in D1R KO and D2R KO
mice

Our transgenic mouse lines showed doxycycline (Dox)controllable expression of transgenic D1R gene. To elucidate
the effects of altered D1R expression, we applied Dox to themice and monitored daily motor activity. We also examined
the protein expression level of D1R in the striatum oftransgenic mice. The striatum contains abundant D1R
expression and is considered to be a major region responsible
for control of motor activity. We found decrease in activity
after Dox administration in transgenic mice which had noendogenous D1R gene, suggesting that D1R is required fornormal activity. When Dox was applied for only 14 days,
transient hyperactivity was observed as D1R expression wasincreased. We are analyzing the relationship between
dopamine signaling via D1R and altered behavior.

In previous behavioral studies of D1R KO mice and D2R
KO mice, changes in walking distance were analyzed in†: Professor Sasaoka ended his term as a Concurrent Professor on 31 March, 2011.

44

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

Associate Professor

Assistant Professors:

HIWATASHI, Yuji
TAMADA, Yosuke
KABEYA, Yukiko
MANO, Hiroaki
CHENG, Chaoyang
IMAI, Akihiro
ISHIKAWA, Masaki
ISHIKAWA, Takaaki
KUBO, Minoru
NAGASHIMA, Akitomo
NOZAWA, Masafumi
OHSHIMA, Issei
OKAMOTO, Haruko
AOYAMA, Tsuyoshi
FUKUSHIMA, Kenji
ICHIKAWA, Shunsuke
LI, Chen
SUGAYA, Tomomi
ANDO, Sayuri
AOKI, Etsuko
BABA, Nayumi
FUKADA, Hatsumi
GOTO, Miho
GOTO, Misako
HIRAMATSU, MikaICHIKAWA, Michiko
ITO, Yukiko
KAJIKAWA, Ikumi
KAKIGI, Rieko
KOHARA, Mayuko
MASUOKA, Tomoko
NISHI, TayoOOI, Shoko
OHSHIMA, Masumi
WAKADSUKI, Sachiko
WASHIO, Midori
KOJIMA, Yoko

HASEBE, Mitsuyasu

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:

Technical Assistants:

Secretary:

MURATA, Takashi

I. The lycopod Selaginella moellendorffii
genomeVascular plants appeared ~410 million years ago, thendiverged into several lineages of which only two survive: theeuphyllophytes (ferns and seed plants) and the lycophytes.
We report here the genome sequence of the lycophyte
Selaginella moellendorffii (Selaginella), the first nonseed
vascular plant genome reported. By comparing gene content
in evolutionarily diverse taxa, we found that the transition
from a gametophyte- to a sporophyte-dominated life cycle
required far fewer new genes than the transition from a
nonseed vascular to a flowering plant, whereas secondary
metabolic genes expanded extensively and in parallel in thelycophyte and angiosperm lineages. Selaginella differs inposttranscriptional gene regulation, including small RNA
regulation of repetitive elements, an absence of the transacting small interfering RNA pathway, and extensive RNA
editing of organellar genes.

II. Evolution from cells to tissues based onmolecular mechanisms of cytokinesis

The cells of land plants and their sister group, charophycean
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 g-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.

III. Evolution of molecular mechanisms inplant development

Figure 1. A lycopod Selaginella moellendorffii whose draft genome waspublished.

Stem cells are formed at particular times and positions
during the development of multicellular organisms. Whereas
flowering plants form stem cells only in the sporophyte
generation, non-seed plants form stem cells in both the
sporophyte and gametophyte generations. Although themolecular mechanisms underlying stem cell formation in the
sporophyte generation have been extensively studied, only a
few transcription factors involved in the regulation of
gametophyte stem cell formation have been reported. The
45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

moss Physcomitrella patens forms a hypha-like body(protonema) and a shoot-like body (gametophore) from a
protonema apical cell and a gametophore apical cell,respectively. These apical cells have stem cell characteristics.
We found that four AP2-type transcription factors
orthologous to Arabidopsis thaliana AINTEGUMENTA/
PLETHORA/ BABY BOOM (APB) are indispensable for theformation of gametophore apical cells from protonema cells.
Quadruple disruption of all APB genes blocked gametophore
formation, even in the presence of cytokinin, which enhances
gametophore apical cell formation in the wild type. Heatshock induction of an APB4 transgene driven by a heat-shock
promoter increased the number of gametophores. Expressionof 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. The primary researchers for this study were Tsuyoshi
Aoyama and Yuji Hiwatashi.
Flowers are the most complex reproductive organs in land
plants, whose development is regulated by MADS-box
transcription factors. To understand the origin of a genetic
network of floral homeotic genes, we analyzed six MIKC
classic type MADS-box genes in P. patens. Deletion of all
six genes enhanced elongation of gametophore stem andreduced production of sporophytes. This result suggests that
the MADS-box genes function in both gametophyte andsporophyte generation. Investigation of the effects of all six
gene deletions is currently being undertaken by Yuji
Hiwatashi.
Evolution of a branched system is a conspicuous novelty inland plant evolution, although the origin and evolution of its
gene network is not known because of the lack of study inthe basal land plants. We found that a deletion mutant of apolycomb repressive complex 2 gene PpCLF forms abranched sporophyte-like organ in P. patens. Analyses ofauxin distribution and expression patterns of class 1 KNOX
genes suggest that the active site of auxin signaling islocalized to the initiation site of the branch. To elucidate how
the active site is formed in the sporophyte-like organ, spatial
expression patterns of the proteins related to auxin
biosynthesis, inactivation, and transport are underinvestigation. This work was mainly done by Yuji Hiwatashi.

IV. Molecular mechanisms of reprogrammingof gametophore leaf cells to pluripotent
stem cells in the moss Physcomitrella patens

Differentiated cells can be reprogrammed to become
undifferentiated pluripotent stem cells with abilities to bothself-renew and give rise to most cell types in the organism.
An induction of reprogramming is more easily manipulated
in plants than in animals, although the genetic and molecularbases of the difference are mostly unknown. This is likely
because the callus usually used in reprogramming studies inseed plants is a cell mixture composed of reprogrammed andunreprogrammed cells. We noticed that P. patens shouldovercome this problem by its rapid reprogramming ability
from a single cell (see http://www.nibb.ac.jp/evodevo/
ERATO/movie/MacMovie.mp4). Cells in a dissected leaf ofP. patens are reprogrammed to become chloronema apical46

cells with pluripotency within 24 hours. We can continuously
observe the reprogramming process of a specific cell under amicroscope.

Figure 2. Reprogramming of leaf cells to chloronema apical cells. (A) A
gametophore. (B) A leaf at 48 hours after excision. (C) A model showing
the dual roles of cyclin-dependent kinase A (CDKA) during
reprogramming.

One of the key factors of reprogramming is the change in
the genome-wide chromatin modification. In the
differentiated cells, a gene expression profile that fits the cellfunction is stably maintained through chromatin
modifications. The 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. In contrast, in thepluripotent stem cells, most genes are ready to be activated.
Many genes with H3K27me3 also have H3K4me3 in animal
pluripotent stem cells, and this bivalent state is presumed tokeep genes poised for transcription. Thus, in the process ofreprogramming, the epigenomic profile of differentiated cells
should be changed into a pluripotent stem cell-specificepigenomic profile. However, the mechanisms of theestablishment of such epigenomic profiles are almost
unknown. We are currently attempting to reveal thesemechanisms underlying reprogramming of P. patens leaf
cells to chloronema apical cells with the combination ofchromatin immunoprecipitation-sequencing (ChIP-Seq)
using a next generation sequencer and live imaging ofchromatin modifications. We have successfully establishedthe 4D (3D + time) live-imaging method of a single P. patens
nucleus, and also produced the H3K27me3 detector using
Drosophila melanogaster Polycomb protein, which is known
to bind to H3K27me3. We are now analyzing the ChIP-Seq
data, performing the 4D live imaging for H3K27me3 during
the reprogramming, and producing the live-imaging detector
for H3K4me3. This study is mainly conducted by Takaaki
Ishikawa and Yosuke Tamada.

V. Molecular mechanisms of mimicry

Mimicry is an intriguing phenomenon in which an organism

closely resembles another, phylogenetically distant species.
An excellent example is the flower-mimicry of the orchid
mantis Hymenopus coronatus, in which pink and whitecoloration and petal-like structures on its walking legs enable
this insect to blend perfectly into flowers. To elucidate theevolutionary mechanism of this complex mimicry at themolecular level, we first focused on the mechanism of bodycoloration in the orchid mantis. HPLC and mass
spectrometric analyses indicated that xanthommatin, acommon red pigment of the ommochrome family, almost
solely contributes to the pink body coloration of late-stage
nymphs. On the other hand, the 1st-instar nymph of theorchid mantis with yellowish red color contains threeommochrome pigments; xanthommatin, decarboxylated
xanthommatin and a labile yellow pigment which waspreviously uncharacterized. These results suggest that theorchid mantis alters its body color by changing thecomposition of ommochrome pigments during post-hatchingdevelopment. This work was mainly done by Hiroaki Mano.

VI. Molecular mechanisms of host shifting

Adaptation to a novel environment often requires evolutionof multiple traits. In phytophagous insects a precise
combination of performance and preference traits forparticular host plants is crucial for host shifting because a
new host plant can be incorporated into an insect’s diet if
adults accept it for oviposition and if the larvae are able tocomplete their development on it. However, very little is
known about the genetic bases of the performance andpreference, which are fundamental to infer the process andevolutionary consequence of host shifting. To address themolecular mechanism of host shifting, we use two host races
of a tiny moth, Acrocercops transecta, as a model system. A
QTL analysis revealed that only a restricted region of asingle autosome was responsible for the larval performance.
This indicates that host shifting from Juglans to Lyonia in A.
transecta involved changes in few genes with large effect,suggesting that a small number of genetic changes to larval
performance allowed the successful host shifting. To test
whether preference genes are physically linked withperformance genes or not, a mapping analysis of preference
genes is in progress. This study was conducted mainly by
Issei Ohshima.

shoots. We are now trying to regenerate whole plants fromthese transformed shoots in addition to further improvementof transformation efficiency. This study was conducted
mainly by Hiroaki Mano.

VIII. Evolution of pitcher leaves in carnivorous
plantsDevelopment and evolution of the unique morphology ofpitcher-shaped leaves of the carnivorous plant family
Sarraceniaceae remains problematic. Since the 1870’s, thepitcher leaves have been hypothesized to have a similar
developmental program to that of peltate leaves. However,this hypothesis could not explain the formation of the keel, a
structure specific to pitcher leaves. To understand thedevelopment and evolution of pitcher leaves, we analyzed
expression patterns of leaf developmental gene orthologs inSarracenia purpurea. Unexpectedly, the results suggested
that adaxial-abaxial patterning of pitcher leaves was different
from those of peltate leaves and have enabled us tohypothesize the evolutionary process of pitcher leaves. This
study was conducted mainly by Kenji Fukushima.

Publication List

〔Original papers〕

K., Hiwatashi, Y., Kojima, M., Sakakibara, H., Ueguchi-Tanaka,• Aya,
M., Hasebe, M., and Matsuoka, M. (2011). The Gibberellin perception•••

system evolved to regulate a pre-existing GAMYB-mediated system
during land plant evolution. Nature Communications 2, 544.
Banks, J.A., Nishiyama, T., Hasebe, M., Bowman, J.L., Gribskov, M.,
dePamphilis, C., Albert, V.A., Aono, N., Aoyama, T., Ambrose, B.A., etal. (2011). The Selaginella genome identifies genetic changes associatedwith the evolution of vascular plants. Science 332, 960-963.
Ishikawa, M., Murata, T., Sato, Y., Nishiyama, T., Hiwatashi, Y., Imai,A., Kimura, M., Sugimoto, N., Akita, A., Oguri, Y., Friedman, W.E.,
Hasebe, M., and Kubo, M. (2011). Physcomitrella cyclin-dependent
kinase A links cell cycle reactivation to other cellular changes during
reprogramming of leaf cells. Plant Cell 23, 2924-2938.
Motose, H., Hamada, T., Yoshimoto, K., Murata, T., Hasebe, M.,
Watanabe, Y., Hashimoto, T., Sakai, T., and Takahashi, T. (2011).
NIMA-related kinases 6, 4, and 5 interact with each other to regulate
microtubule organization during epidermal cell expansion in
Arabidopsis thaliana. Plant J. 67, 993-1005.

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

The molecular mechanisms and evolutionary significance ofplant movement, including seismonastic and nyctinastic
movements, are enigmatic. We are working to establish
chemically mutagenized lines that lack movement tocompare fitness to wild types. We are also attempting to set
up a method for transformation to characterize the genes
involved in movement. To achieve this goal, we use acotyledonary node explant, which can regenerate multiple
shoots in the presence of 6-benzylaminopurine (BAP), as atarget of Agrobacterium-mediated gene transfer. Although
the node explant is highly recalcitrant to Agrobacterium
infection, we successfully obtained several lines oftransformed calluses that were capable of developing new47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

KAWAGUCHI, Masayoshi

Assistant Professors:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Visiting Scientists:
Graduate Students:

Technical Assistants:
Secretary:

TAKEDA, Naoya
SUZAKI, Takuya
FUKADA-TANAKA, Sachiko
OKAMOTO, Satoru
YANO, Koji
FUJITA, Hironori
YOKOYAMA, Hiroshi
MIYAZAWA, Hikota
HANDA, Yoshihiro
YOSHIDA, Chie
MIYAZAWA, Hikota*
TAKAHARA, Masahiro
SASAKI, Takema
YORO, Emiko
NAKAJIMA, Michiko
SUZAKI, Momoyo
YOSHINORI, Yumi
SANJO, Kazuko

More than 80% of land plant families have symbiotic
relationships with arbuscular mycorrhizal (AM) fungi. AM
fungi absorb minerals, mainly phosphates, from the soil andprovide them to the plants. The origin of AM symbiosis isthought to have been in the early Devonian period. On theother hand, the root nodule symbiosis that occurs between
legumes and rhizobial bacteria, unlike AM symbiosis,
involves host-specific recognition and postembryonicdevelopment of a nitrogen-fixing organ. Root nodule
symbiosis is thought to have evolved about 60 million years
ago. Despite marked differences between the fungal andbacterial 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 Genetic analysis of molecular mechanism
regulating nodule primordia formation

In the initial developmental process of nodulation,
infection of rhizobia into the host plant root induces
dedifferentiation and division of some of the cortical cells
followed by initiation of nodule primordia. Genetic
mechanisms regulating nodule primordia formation haveremained poorly characterized due to a lack of mutants
involved in such process. Our large-scale mutant screening
approach resulted in isolation of several new non-nodulating
mutants related to nodule primordia formation in Lotus
japonicus.
In the mutant line #7-1, nodules were barely formed.
Judging from the pattern of infection thread formation,
however, the infection process of rhizobia appears to benormal, suggesting the mutation predominantly affects
nodule development. In addition, the mutation caused

48

pleiotropic defects especially in the shoot; the number ofcotyledons was increased and maintenance of the shootapical meristem (SAM) was compromised in the mutant. A
gene that is responsible for the mutation was isolated bymap-based cloning, and it turned out that the gene is aputative orthologue of the genes known to be regulators of
the SAM formation in other plants. Future detailed functional
analysis of the gene might propose the existence of a novelcommon genetic regulatory mechanism between nodule
primordia and SAM formation.
Auxin is a key phytohormone governing cell division anddifferentiation in many developmental aspects in plants. In
terms of nodulation, however, the role of auxin remains
largely unknown. As the first approach to elucidate
molecular relationship between nodule primordia formation
and auxin, we engineered transgenic plants, in which theexpression of a reporter gene (GFP-NLS) was controlled bysynthetic auxin-response promoter, DR5. Using these lines,
we monitored the spatiotemporal induction pattern of auxin
response during nodulation. It was shown that cortical cells’
division occurs concomitant with strong induction of auxin
response (Figure 1A and B). After colonization of rhizobia
into developing nodule primordia, this induction of the auxin
response halted in the infection region of nodule (Figure 1C).
Further analysis in combination with nodule symbiotic
mutants should uncover the function of auxin in nodule
primordia formation.

Figure 1. Spatial induction pattern of auxin response during nodule
primordia formation. (A) Initial cortical cell division stage. Rhizobia start
to invade into the host plant root via infection thread. (B) Beginning of
the bulge of nodule primordia stage, when cortical cells actively divide.
(C) Developing nodule primordia, into which rhizobia colonize. Greendots represent DR5::GFP-NLS expression. Red areas represent existenceof rhizobia constitutively expressing DsRED. Scale bars: 100 mm.

1-2 Identification of PLENTY that mediates a novel
mechanism for nodule number regulation andnon-symbiotic root development

The symbiotic relationship between legume plants andrhizobia allows the host plants to grow even in nitrogen poor
environments. However nodule development is anenergetically expensive process. So the number of nodules
must be tightly controlled by the host plants. We have been
trying to elucidate the mechanism for controlling nodule
numbers at the molecular level.
We previously isolated the novel hypernodulating mutant
plenty from C6+ beam mutagenized seeds of L. japonicus.
The number of nodules in plenty was 3-5 times more thanthat of wild type. Unlike previously reported
hypernodulating mutants (har1, klv and tml), plenty showed

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

different characteristics. Both genetic analysis and grafting
experiments using plenty and other hypernodulating mutants
indicated that PLENTY functions using different signaling forcontrolling nodule number.
PLENTY was identified by map-based cloning. The
genomic deletion was detected in the PLENTY gene.
Furthermore, both symbiotic (hypernodulating) and nonsymbiotic (short root) phenotypes of plenty werecomplemented by introducing the PLENTY gene (Figure 2).
PLENTY encodes a completely unknown protein without any
functional domain, although the protein is highly conserved
from basal plants such as algae to higher plants. PLENTY
might function as a root factor associated with an unknown
regulatory mechanism of nodulation.

Figure 2. Identification of PLENTY. (A) Map-based cloning of PLENTY.
(B-G) Complementation tests of plenty. PLENTY cDNA was introduced
into plenty mutant via A. rhizogenes-mediated hairy root transformation.
[empty] wild type indicates wild type with empty vector. [empty] and[PLENTY] plenty indicates plenty with empty vector and PLENTY(Ubiquitin pro:PLENTY cDNA), respectively. Both the symbiotic (C-F)
and non-symbiotic (B, G) phenotypes were rescued.

II. Arbuscular mycorrhiza symbiosis

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 isthought to evolve by sharing AM factors, suggesting that theAM system contains a fundamental mechanism that also
regulates root nodule symbiosis. In recent studies, AM
signaling factors that conduct host-symbiont recognition

were isolated from both host plant and AM fungi. These
results accelerated molecular analysis of the AM signalingmechanism.
We have established AM molecular marker genes SbtM1
and PT4. These genes are specifically and highly induced
during AM fungal infection. Green fluorescence protein(GFP) or beta-glucuronidase (GUS) fusions with the markers
facilitated detection and visualization of infection processesof AM fungi. Using the tools; SbtMpro:GUS derivatives, wehave identified and analyzed cis-regions which specifically
respond to AM fungal infection (Takeda et al., 2011). We
isolated candidate genes of AM trans-factor from the cisregion with the yeast one-hybrid system and we are
analyzing the genes and gene regulation system during AM
fungal infection.
We also analyzed a signaling factor calcium calmodulin
dependent protein kinase (CCaMK) which is a common
signaling factor shared with AM and root nodule symbiosis.
We found that a truncated CCaMK protein that contains only
the kinase domain of the protein shows gain of function
ability and activates a part of the AM signaling pathway
without AM fungal infection. Introduction of the gain offunction CCaMK (GOF-CCaMK) induced AM gene marker
SbtM1 expression, but not PT4 expression. Furthermore, theAM signaling induced formation of pre-penetration
apparatus (PPA). PPA is cytosolic remodeling induced by
AM fungal infection, in which ER and cytosol development,
and nuclear enlargement were observed before penetration of
fungal hypha into the host cell. In this analysis,
SbtM1pro:Venus fusion visualized formation of the PPA andenabled detailed analysis of the cytoplasmic changes (Figure3). The activation and signaling function of GOF-CCaMK
revealed a novel aspect of AM signaling systems in L.
japonicus (Takeda et al., accepted).

Figure 3. Pre-penetration apparatus-like structures induced by GOFCCaMK. (A) Hairy root co-transformed with GOF-CCaMK(CCaMK 314TD-NLS) and SbtM1 pro:Venus was observed with confocal
microscopy and laser transmission and Venus fluorescence images weremerged. (B) A stack of images obtained along the z axis of cells
corresponding to the white box region in (A) showed densely developed
cytosol. Bars = 100 µm (A) or 50 µm (B).

III. Reaction-diffusion pattern in the shootapical meristem of plants
A fundamental question in developmental biology is how
spatial patterns are self-organized from homogeneous
structures. In 1952, Turing proposed the reaction-diffusion
model in order to explain this issue. However, whether or not
this mechanism plays an essential role in developmental
events of living organisms remains elusive. Thus, we49National Institute for Basic Biology

Evolutionary Biology and Biodiversity

investigated whether reaction-diffusion dynamics can explain
the shoot apical meristem (SAM) development of plants. The
SAM resides in the top of each shoot and consists of acentral zone (CZ) and a surrounding peripheral zone (PZ)
(Figure 4B). The SAM contains stem cells and continuously
produces new organs throughout the lifespan of the plant.
The formation and maintenance of the SAM are known to be
essentially regulated by the feedback interaction between
WUSHCEL (WUS) and CLAVATA (CLV) (Figure 4A).
We developed a mathematical model of the SAM based onreaction-diffusion dynamics of the WUS-CLV interaction,
incorporating cell division and the spatial restriction of the
dynamics. Consequently, we find that SAM patterning isgoverned by only two parameters: the stem cell proliferation
mode and stem cell containment, and is classified into six
groups: the fasciation pattern, multiplication pattern,fluctuation pattern, dichotomous pattern, monopodial pattern,
and homeostasis pattern.
Next, we examined whether this theoretical prediction isconsistent with experimental observations reported so far.
Because SAM pattern formation has been intensively studied
with regard to the WUS and CLV genes in A. thaliana, theeffect of these genes on the model was investigated in detail.

As the WUS function becomes strong or the CLV function is
reduced, the SAM pattern shifts in the following order: thefluctuation pattern, the homeostasis pattern, the dichotomous
pattern, the fasciation pattern (Figure 4C). Our model
successfully explains the various SAM patterns observed inplants, for example, homeostatic control of SAM size in thewild type, enlarged or fasciated SAM in clv mutants, andinitiation of ectopic secondary meristems from an initial
flattened SAM in wus mutant (Figure 4C).
As a result, we conclude that our model captures theessence of SAM pattern formation, and furthermore thereaction-diffusion dynamics is probably indispensable for
SAM development in plants.

Publication List

〔Original papers〕

H., Toyokura, K., Okada, K., and Kawaguchi, M. (2011).
• Fujita,
Reaction-diffusion pattern in shoot apical meristem of plants. PLoS

•
•
•
•

ONE 6, e18243.
Funayama-Noguchi, S., Noguchi, K., Yoshida, C., and Kawaguchi, M.
(2011). Two CLE genes are induced by phosphate in roots of Lotus
japonicus. J. Plant Res. 124, 155-163.
Krusell, L., Sato, N., Fukuhara, I., Koch, B., Grossmann, C., Okamoto,S., Oka-Kira, E., Otsubo, Y., Aubert, G., Nakagawa, T., Sato, S., Tabata,S., Duc, G., Parniske, M., Wang, T. L., Kawaguchi, M., and Stougaard,
J. (2011). Clavata2 genes of pea and Lotus japonicus affectautoregulation of nodulation. Plant Journal 65, 861-871.
Okamoto, S., Nakagawa, T., and Kawaguchi, M. (2011). Expression
and functional analysis of a CLV3-like gene in the model legume Lotus
japonicus. Plant Cell Physiol. 52, 1211-1221.
Takeda, N., Haage, K., Sato, S., Tabata, S., and Parniske, M. (2011).
Activation of a Lotus japonicus subtilase gene during arbuscular
mycorrhiza is dependent on the common symbiosis genes and two cisactive promoter regions. Molecular Plant-Microbe Interactions 24, 662670.

〔Original paper (E-Publication ahead of print)〕

T., Niimi, K., Yamamoto, T., Isobe, S., Sato, S., Nakamura,• Hakoyama,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. The intergral membrane protein SEN1 isrequired for symbiotic nitrogen fixation in Lotus japonicus nodules.
Plant Cell Physiol. 2011 Nov. 28.

〔Review articles〕

H., and Kawaguchi, M. (2011). Strategy for shoot meristem• Fujita,
proliferation in plants. Plant Signaling Behavior 6, 1851-1854.
M. (2011). The evolution of symbiotic systems. Cellular• Kawaguchi,
and Molecular Life Sciences 68, 1283-1284.

Figure 4. (A) Schematic representation of the WUS-CLV dynamics withrespect to the activator-inhibitor system. (B) Schematic representation ofa spatially restricted SAM. The CZ is defined as cells where the activator
is highly expressed. A hypothetical molecule z is synthesized in the CZ,
and diffuses to form a gradient. The PZ is differentiated by having z
concentrations higher than a threshold Zs. (C) SAM pattern changes
predicted by the model are consistent with those of experimental results
reported in A. thaliana.

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 for our morphological studies.

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 the

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.

development 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.
Training in specimen preparation and instrument operation
for such observations is also given.

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIORESOURCES

Associate Professor

NARUSE, Kiyoshi

Postdoctoral Fellows:

NIBB Research Fellow:
Research Fellows:
Technical Assistants:

Secretary:

SASADO, Takao
TAKEHANA, Yusuke
SHIBATA, Yasushi
SHIBA, Keikun
NAKAMOTO, Masatoshi
OKUYAMA, Teruhiro
KIMURA, Tetsuaki
KANEKO, Hiroyo
YOSHIMURA, Yuriko
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
KOIKE, Chieko
TESHIMA, Yuuko
HARA, Ikuyo
ISHIKAWA, Hiroe
SHIBATA, Emiko
TAKAGI, Chikako
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
the novel sex-determining genes in these species.

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

52

Identification of these genes would provide a clue tounderstand the evolutionary process underlying frequent
turnover of the 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

Estrogen has 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 (arom), which is
responsible for catalyzing the conversion of testosterone toestrogen. We isolated two tilling mutant strains of arom. In
these tiling mutants, one amino acid in aromatase ORF
altered the STOP codon. In the tilling mutant of arom, theovaries seemed to develop normally. However, in adult fish,
yolk accumulation and formation of ovarian cavity were notobserved. In some ovaries, spermatogenesis was observed.
These results suggest that estrogen is not involved in earlyovarian differentiation but has a critical role in maintenanceof 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 cells differentiate and what
differences are producing various pigment cell types is a very
interesting question. Medaka have four types of pigment cell(melanophore, leucophore, xanthophore and iridocyte). The
leucophore is unique because only some species have it. To
elucidate how leucophore differentiate from neural crest cells
and why it exists only in some fishes, we have successfully
identified the causal gene of leucophore mutants (luecophore
free (lf) and leucophre free 2 (lf-2)). We identified slc2a15b
as the causal gene of the lf mutant. The slc2a15b expression
exists but does not persist in the lf mutant. slc2a15b also
exists in species without leucophore, but these use carotenoid
as a pigment. Thus, slc2a15b may have an important role inuse of carotenoid as pigments. The lf-2 phenotype was
rescued by pax7a. pax7a is needed for differentiation ofleucophore and xanthophore in medaka.

V. 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). In
2011, we continued providing the TILLING screening
system library to NBRP Medaka users for promoting thereverse genetic approach. NBRP Medaka aims to establish afirst rate biological resource with the highest possible levelsof accessibility and ease of use.

Publication List

〔Original papers〕

T., Shibata, Y., Zhou, L.Y., Katsu, Y., Iguchi, T., and• Chakraborty,
Nagahama, Y. (2011). Differential expression of three estrogen receptor

•
•
•
•
•
•

subtype mRNAs in gonads and liver from embryos to adults of the
medaka, Oryzias latipes. Molecular and Cellular Endocrinology 333,
47-54.
Kai, W., Kikuchi, K., Tohari, S., Chew, A.K., Tay, A., Fujiwara, A.,
Hosoya, S., Suetake, H., Naruse, K., Brenner, S., et al. (2011).
Integration of the Genetic Map and Genome Assembly of Fugu
Facilitates Insights into Distinct Features of Genome Evolution in
Teleosts and Mammals. Genome Biology and Evolution 3, 424-442.
Kato, M., Takehana, Y., Fukuda, Y., Naruse, K., Sakaizumi, M., andHamaguchi, S. (2011). An autosomal locus controls sex reversal ininterspecific XY hybrids of the medaka fishes. Heredity 107, 523-529.
Kobayashi, H., Iwamatsu, T., Shibata, Y., Ishihara, M., and Kobayashi,
Y. (2011). Effects of co-administration of estrogen and androgen on
induction of sex reversal in the medaka Oryzias latipes. Zoolog. Sci. 28,
355-359.
Koga, A., Sasaki, S., Naruse, K., Shimada, A., and Sakaizumi, M.
(2011). Occurrence of a short variant of the Tol2 transposable elementin natural populations of the medaka fish. Genetics Research 93, 13-21.
Okuyama, T., Suehiro, Y., Imada, H., Shimada, A., Naruse, K., Takeda,
H., Kubo, T., and Takeuchi, H. (2011). Induction of c-fos transcription
in the medaka brain (Oryzias latipes) in response to mating stimuli.
Biochemical and Biophysical Research Communications 404, 453-457.
Paul-Prasanth, B., Shibata, Y., Horiguchi, R., and Nagahama, Y. (2011).
Exposure to diethylstilbestrol during embryonic and larval stages of
medaka fish (Oryzias latipes) leads to sex reversal in genetic males andreduced gonad weight in genetic females. Endocrinology 152, 707-717.

〔Original paper (E-publication ahead of print)〕

Y., Naruse, K., Asada, Y., Matsuda, Y., Shin-I, T., Kohara, Y.,• Takehana,Fujiyama, A., Hamaguchi, S., and Sakaizumi, M. Molecular cloning

and characterization of the repetitive DNA sequences that comprise theconstitutive heterochromatin of the W chromosomes of medaka fishes.
Chromosome Res. 2011 Nov. 29.

〔Review articles〕

K. (2011). Genetics, Genomics, and Biological Resources in• Naruse,
the Medaka, Oryzias latipes. In: Medaka, A Model for Organogesis,•••

Human Diseases and Evolution. Naruse, K., Tanaka, M., and Takeda, H.
eds. (Springer), pp. 19-37.
Naruse, K. Tanaka, M., and Takada, H. (2011). Medaka, A Model for
Organogesis, Human Diseases and Evolution Springer Tokyo.
Shibata, N., Nakamoto, M., Shibata, Y., and Nagahama, Y. (2011)
Endocrine Regulation of Oogenesisin the Medaka, Oryzias latipes. In:
Medaka: A Model for Organogenesis, Human Disease, and Evolution.
Naruse, K., Tanaka, M., and Takeda, H. eds. (Springer), pp. 267-283.
Takehana, Y. (2011). Frequent turnover of sex chromosomes in the
medaka fishes. In: Medaka, A Model for Organogesis, Human Diseases
and Evolution. Naruse, K., Tanaka, M., and Takeda, H. eds. (Springer),
pp. 229-240.

Figure 2. NBRP Medaka website

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).

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 is 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 yphosphorylation state of Atg13. Various conditions andmutants have been examined to determine what kind ofnutrients TORC1 recognizes and how nutrient signal istransmitted to TORC1.

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.

Figure 1. TOR signaling pathway of the budding yeast. Our group havefound three branches of the TOR pathway.

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

54

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

〔Original paper〕

S., Imoto, J., Minato, T., Oouchi, R., Kamada, Y., Tomita, M.,• Yoshida,
Soga, T., and Yoshimoto, H. (2011). A novel mechanism regulates H S
and SO2 production in Saccharomyces cerevisiae. Yeast 28, 109-121.

2

LABORATORY OF BIOLOGICAL DIVERSITYOHNO Group
Assistant Professor:
Technical Assistants:

OHNO, Kaoru
ITO, Masako
NAKAMURA, Ryoko
MATSUDA, Azusa
FUJITA, Miyako

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 Assistants:

HOSHINO, Atsushi
WATANABE, Seiko
NAKAMURA, Ryoko

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 200 lines and 117,000 DNA clones.

Publication List

〔Original papers〕

Y., Sage-Ono, K., Sasaki, R., Ohtsuki, N., Hoshino, A., Iida,• Higuchi,
S., Kamada H., and Ono, M. (2011). Constitutive expression of the•••

GIGANTEA ortholog affects circadian rhythms and suppresses one-shot
induction of flowering in Pharbitis nil, a typical short-day plant. Plant
Cell Physiol. 52, 638-650.
Ohno, S., Hosokawa, M., Hoshino, A., Kitamura, Y., Morita, Y., Park,
K.I., Nakashima, A., Deguchi, A., Tatsuzawa, F., Doi, M., Iida, S., andYazawa, S. (2011). A bHLH transcription factor, DvIVS, is involved inregulation of anthocyanin synthesis in dahlia (Dahlia variabilis). J.
Exp. Bot. 62, 5105-5116.
Ohno, S., Hosokawa, M., Kojima, M., Kitamura, Y., Hoshino, A.,
Tatsuzawa, F., Doi M., and Yazawa, S. (2011). Simultaneous posttranscriptional gene silencing of two different chalcone synthase genes
resulting in pure white flowers in the octoploid dahlia. Planta 234, 945958.
Saito, N., Tatsuzawa, F., Hoshino, A., Abe, Y., Ichimura, M., Yokoi, M.,
Toki, K., Morita, Y., Iida, S., and Honda, T. (2011). The anthocyanin
pigmentation controlled by the speckled and c-1 mutations of theJapanese morning glory. J. Japan. Soc. Hort. Sci. 80, 452-460.

〔Original paper (E-publication ahead of print)〕

K.I., and Hoshino, A. A WD40-repeat protein controls• Park,proanthocyanidin and phytomelanin pigmentation in the seed coats ofwild type

Blizzard

Rayed

Marginedduskishpearly-v

Figure 1. Flower phenotypes of the morning glories.

56

the Japanese morning glory. 2011 Dec. 28.

LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:
Visiting Scientist:

TSUGANE, Kazuo
HAYASHI-TSUGANE, Mika

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.

A rice mutant displaying a heterochronicallyelongated internode carries a 100 kb deletion

We have isolated a recessive rice mutant, designated asindeterminate growth (ing), which displays creeping andapparent heterochronic phenotypes in the vegetative periodwith lanky and winding culms (Figure 1). Rough mappingand subsequent molecular characterization revealed that theing mutant carries a large deletion, which corresponds to a
103 kb region in the Nipponbare genome, containing nine
annotated genes on chromosome 3.

and the ing mutant appears to be the first characterized
mutant having the entire SLR1 sequence deleted. Our results
also suggest that the deleted region of 103 kb does notcontain an indispensable gene, whose dysfunction must
result in a lethal phenotype (Figure 2).

Figure 2. Characterization of the indeterminate growth (ing) allele. A:
Rough mapping of the ing allele. B: Structure of the ing deletion. The
large bracket indicates the deletion. C: Enlarged structures of the 5’
region of Houba, SLR1, and the region of the 3’ deletion junction. D:
Detection of PCR-amplified bands with the indicated primers. Lane 1,
Nipponbare; lane 2, ING/ING; lane 3, ING/ing; lane 4, ing/ing.

Examination of transpositional activity ofnDart1 at different stages of rice development
As a useful tool to elucidate gene functions, a ricetransposon tagging line has been developed using an active
endogenous DNA transposon, nDart1. It was highly
desirable to evaluate the transposition timing and frequencyof the nDart1 elements during rice development to facilitate
the generation of an efficient mutant isolation system.
Comparison of the detected new insertions at different stagesof rice development by transposon display analysis
demonstrated that the last heading tiller carry a higher
number of nDart1 elements than the main culm. Moreover, it
was revealed that the last heading tiller could produce
progeny that carried more new insertions of nDart1
elements, mainly as a result of the accumulation of somatic
insertions in the parental plant. This report demonstrates thatlate tillers increase the probability of producing independent
mutant lines.

Publication List

〔Original papers〕

Figure 1. Phenotypes of the wild type and ing plants. A: Three-month-old
wild-type plant (left) and ing mutant (right). The bars represent 50 cm.

Of these annotated genes, the SLR1 gene encoding a
DELLA protein is the only one that is well characterized in
its function, and its null mutation, which is caused by asingle base deletion in the middle of the intronless SLR1
gene, confers a slender phenotype that bears close
resemblance to the ing mutant phenotype. The primary causeof the ing mutant phenotype is the deletion of the SLR1 gene,M., Maekawa, M., Kobayashi H., Iida, S., and• Hayashi-Tsugane,
Tsugane, K. (2011). A rice mutant displaying a heterochronically•elongated internode carries a 100 kb deletion. J. Genet. Genomics 38,
123-128.
Hayashi-Tsugane, M., Maekawa, M., Qian, Q., Kobayashi H., Iida, S.,
and Tsugane, K. (2011). Examination of transpositional activity ofnDart1 at different stages of rice development. Genes Genet. Syst. 86,
215-219.

〔Review article〕

M., Tsugane, K., and Iida, S. (2011). Effective contribution• Maekawa,of the nDart transposon-tagging system to rice functional genomics.
Adv. Genet. Res. 4, 259-272.

57

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYYAMAGUCHI Group †Assistant Professor:
Postdoctoral Fellow:
Technical Assistant:

YAMAGUCHI, Takahiro
NUKAZAWA, Akira
YAMAGUCHI, Chinami

Angiosperm leaves generally develop as bifacial structureswith distinct adaxial and abaxial identities. However, several
monocot species, such as iris and leek, develop “unifacial
leaves”, in which leaf blades have only an abaxial identity(Figure 1). We are focusing on unifacial leaf development
and evolution to understand genetic mechanisms behind
diversity and evolution of organismal morphology.

Thus, DL is a key gene in the flattening of the unifacial leaf
blade. Interestingly, DL plays a distinct role in promoting
midrib formation during bifacial leaf development. We
suggest that morphological convergence of flattened leaf
blades in unifacial leaves has occurred via the recruitment of
DL function, which plays a similar cellular but distinct
phenotypic role in monocot bifacial leaves (Figure 2).

Figure 2. Mechanism of leaf blade flattening in unifacial leaves.

III. Leaf central–marginal and distal–proximal
polarity specification

Figure 1. Bifacial and unifacial leaf structures. Ad, Adaxial side; Ab.
Abaxial side.

I. Abaxialization of unifacial leaves

The development and evolution of unifacial leaves havelong been matters of debate. However, nothing has beenstudied at the molecular genetic level. We focused on thegenus Juncus as a model to study the evolution anddevelopment of unifacial leaves. Juncus contains species
with a wide variety of leaf forms and is amenable tomolecular genetic studies. We first characterized unifacial
leaf development by investigating gene expression patternsof adaxial and abaxial determinants. As a result, wedemonstrated that the unifacial leaf blade is abaxialized at
the gene expression level and revealed that dominant abaxial
activity leads to the unifacial leaf development.

II. Flattening of unifacial leaves

In bifacial leaves, adaxial–abaxial polarity is required forleaf blade flattening, whereas many unifacial leaves become
flattened although their leaf blades are abaxialized (Figure1).
This indicates independent mechanisms underlying flattenedleaf blade formation in bifacial and unifacial leaves.
Using two closely related Juncus species, J.
prismatocarpus, with flattened unifacial leaves, and J.
wallichianus, with radialized unifacial leaves, we revealed
that DL expression levels and patterns correlate with thedegree of laminar outgrowth. Genetic and expression studies
using interspecific hybrids of the two species revealed thatthe DL locus from J. prismatocarpus flattens the unifacial
leaf blade and expresses higher amounts of DL transcripts.
†: This group was closed on 30 September, 2011.

58

The mechanisms that regulate the central–marginal leaf
polarity remain largely unknown. We discovered that thecentral–marginal polarity differentiates in the flattened leaf
blade of J. prismatocarpus, but not in the radialized leaf
blade of J. wallichianus. This indicates that leaf blade
flattening is a key that triggers central–marginal leaf polarity
differentiation. We also found the possibility that the plant
hormone auxin is asymmetrically distributed in the flattenedleaf primordia and induces expressions of central-marginalpolarity determinants.
We also revealed that the ROTUNDIFOLIA4 (ROT4) genethat encodes a plant-specific small peptide controls cellproliferation along distal-proximal axis during leaf
development in Arabidopsis thaliana. We identified the 32
residues-long core functional region of ROT4 and showedthat ROT4 acts cell autonomously without proteolytic
processing (Ikeuchi et al., 2011).

Publication List

〔Original paper〕

M., Yamaguchi, T., Kazama, T., Ito, T., Horiguchi, G., and• Ikeuchi,
Tsukaya, H. (2011). ROTUNDIFOLIA4 regulates cell proliferation
along the body axis in Arabidopsis shoot. Plant Cell Physiol. 52, 59–69.

LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Assistants:

JOHZUKA, Katsuki
ISHINE, Naomi
MATSUZAKI, Yoko

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 createdduring DNA replication. Any abnormality in this process leadto segregation error or aneuploidy, resulting in cell lethality.
Studies in the past decade have demonstrated thatchromosome condensation is mainly achieved by condensin,
a multi-subunit protein complex widely conserved from yeast
to human.
Our major research interest is to understand the mechanism
and regulation 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 genome during mitosis. We have foundthat condensin specifically binds to the RFB site locatedwithin the rDNA repeat. We further discovered the multiple
proteins interaction network recruits condensin to the RFB
site.

I. Maintenance of the rDNA stability bycondensin

Rad52 protein was excluded from the nucleolus during themitosis in YCS4 cells, whereas it invaded into the nucleolus
in ycs4-1 mutant cells (Figure 1). This result indicates thatcondensin protects from invasion of recombination enzyme
during mitosis. It is suggested that rDNA condensation
supports the exclusion of recombination machinery, resulting
in avoidance of the accumulation of recombinationintermediates within the rDNA locus, thus contributing tomaintaining rDNA stability and faithful segregation.

II. Condensin-dependent chromatin folding

The RFB site, which consists of a ~150bp DNA sequence,
acts as the strongest cis-element for condensin recruitment
onto chromatin. If the RFB sequence is inserted into an
ectopic chromosomal locus, condensin can associate with theectopic RFB site. To explore the role of condensin inchromosome organization, we have constructed a strain inwhich two RFB sites are inserted on an ectopic chromosome
arm at an interval of 15kb distance in the cell with complete
deletion of chromosomal rDNA repeat. Using such strain,condensin-dependent chromatin interaction between two
RFBs was examined by chromosome conformation capture(3C) assay. We found condensin-dependent interaction
between two RFB sites on the chromosome arm. This result
indicates that condensin plays a role in chromatin interaction
between condensin binding sites and this interaction leads tothe creation of a chromatin loop between those sites (Figure2). It is thought that condensin-dependent chromatin folding
is one of the basic molecular processes of chromosome
condensation.

Due to its tandem repetitive structure, the rDNA locus isunstable because of frequent homologous recombination
events within the rDNA repeat. Nevertheless, most cells canmaintain rDNA stability, as indicated by their relatively
constant copy numbers of the repeats. A key recombination
protein, Rad52 is normally localized in the nucleus but is
excluded from the nucleolus. This exclusion of arecombination protein may contribute to maintaining rDNA
stability. In condensin mutants, however, aberrant
segregation of the rDNA locus is frequently observed and its
copy numbers are dramatically decreased. We found that the

Figure 2. A Schematic model of chromosome condensation. Condensin
causes 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.

Figure 1. Nucleolar localization of Rad52 protein during mitotic phase.
The wild-type (YCS4) and condensin mutant (ycs4-1) cells were arrested
at metaphase by interfering with the polymerization of microtubules(nocodazole). Nucleolar organizing protein (Nop56) was detected as red
and Rad52 protein was detected as green colors, respectively. In the ycs41 mutant, Rad52 invades in the nucleolus (yellow).

59

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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.

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

60

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 2m 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,
designated double IR. This structure is found in the humangenome and can be observed in the early stages of gene
amplification. In yeast, the double IR could induce gene
amplification. Now we try to construct the double IR in CHO
cells and to perform its detailed functional analysis in yeast.

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

Publication List

〔Original papers〕

H., Watanabe, T., and Horiuchi, T. (2011). Double rolling• Okamoto,circle replication (DRCR) is recombinogenic. Genes Cell 16, 503-513.
T., Tanabe, H., and Horiuchi, T. (2011). Gene amplification• Watanabe,
system based on double rolling-circle replication as a model foroncogene-type amplification. Nucleic Acids Res. 39, e106.

National Institute for Basic Biology

Environmental Biology

DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY

Professor

IGUCHI, Taisen

Assistant Professors:
NIBB Research Fellow:
Postdoctoral Fellow:
Technical Staff:
Graduate Students:

Visiting Scientists:

Technical Assistants:
Secretary:

MIYAGAWA, Shinichi
OGINO, Yukiko
CHAKRABORTY, Tapas
MIYAKAWA, Hitoshi
MIZUTANI, Takeshi
SATO, Masaru
TOYOTA, Kenji
SUMIYA, Eri
YATSU, Ryohei
NAKAMURA, Takeshi
ODA, Shigeto
HIRAKAWA, Ikumi
TOHYAMA, Saki
SEBILLOT, Anthony
WON, Seok
LANGE, Anke
HAYASHI, Tomoko
KOBAYASHI, Kaoru
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 ERa, 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.

Steroid and xenobiotic receptors (SXR) have been cloned
from various animal species (fish, amphibian, 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,

61

National Institute for Basic Biology

Environmental Biology

gar, roach, stickleback, mosquitofish, mangrove Rivulus,
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 and sturgeon(Acipenseriformes), and teleosts including a basal teleost,arowana (Osteoglossiformes). Molecular phylogenetic
analysis revealed that a gene duplication event gave riseto two different teleost ARs (a and b) and likely occurredin the actinopterygian lineage leading to teleosts after thedivergence of Acipenseriformes but before the split ofIsteoglossiformes. Functional analysis revealed that the
shark AR activates the target gene via androgen response
element by classical androgens. The teleost ARa showed
unique intracellular localization with a significantly
higher transactivation capacity than that of teleost ARb.
These results indicate that the most ancient type of AR, asactivated by the classic androgens as ligands, emerged
before the Chondrichthyes-Osteichthyes split and the AR
gene was duplicated during a teleost-specific gene
duplication event.

Figure 3. Evolutionary relationships of androgen receptor sequences.

information about the mode of action of the tested
compounds. Therefore, we applied an ecotoxicogenomic
assessment of D. magna. We established a Daphnia EST
database and developed an oligonucleotide-based DNA
microarray with high reproducibility and demonstrated theusefulness of thearray 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

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.

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 no62

Figure 4. Life cycle of Daphnia.

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 more

detail 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
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 currently identifying
JH-responsive genes in the ovary.

Publication List

〔Original papers〕

D.S., Skotko, J.P., Ohta, Y., Boggs, A.S.P., Iguchi, T., and• Bermudez,
Guillette, L.J.Jr. (2011). Sex steroid and thyroid hormone receptor

•
•
•
•
•
•
•
•
•
•
•
•

expressions in the thyroid of the American alligator (Alligator
mississippiensis) during different life stages. J. Morphol. 272, 698-703.
Chakraborty, T., Katsu, Y., Zhou, L.Y., Miyagawa, S., Nagahama, Y.,
and Iguchi, T. (2011). Estrogen receptors in medaka (Oryzias latipes)
and estrogenic environmental contaminants: an in vitro-in vivo
correlation. J. Steroid Biochem. Mol. Biol. 123, 115-121.
Chakraborty, T., Shibata, Y., Zhou, L.Y., Katsu, Y., Iguchi, T., andNagahama, Y. (2011). Differential expression of three estrogen receptor
subtype mRNAs in gonads and liver from embryos to adults of the
medaka, Oryzias latipes. Mol. Cell. Endocrinol. 333, 47-54.
Kato, Y., Kobayashi, K., Watanabe, H., and Iguchi, T. (2011).
Environmental sex determination in the branchiopod crustacean
Daphnia magna: Deep conservation of a Doublesex gene in the sexdetermining pathway. PLoS Genetics 7, e1001345.
Kato, Y., Shiga, Y., Kobayashi, K., Tokishita, S., Yamagata, H., Iguchi,T., and Watanabe, H. (2011). Development of an RNA interference
method in the cladoceran crustacean Daphnia magna. Devel. Genes
Evol. 220, 337-345.
Lange, A., Paull, G.C., Hamilton, P.B., Iguchi, T., and Tyler, C.R.
(2011). Implications of persistent exposure to treated wastewater
effluent for breeding in wild roach (Rutilus rutilus) populations.
Environ. Sci. Technol. 45, 1673-1679.
Matsumura, D., Haraguchi, R., Miyagawa, S., Motoyama, J., Nakagata,N., Meijlink, F., and Yamada, G. (2011). Genetic analysis of hedgehog
signaling in ventral body wall development and the onset of omphalocele
formation. PLoS ONE 20, e16260.
Miyagawa, S., Matsumaru, D., Murashima, A., Omori, A., Satoh, Y.,
Haraguchi, R., Motoyama, J., Iguchi, T., Nakagata, N., Hui, C.C., andYamada, G. (2011). The role of sonic hedgehog-Gli2 pathway in the
masculinization of external genitalia. Endocrinology 152, 2894-2903.
Moore, B.C., Milnes, M.R., Kohno, S., Katsu, Y., Iguchi, T., Woodruff,
T.K., and Guillette, L.J.Jr. (2011). Altered gonadal expression of TGF-β
superfamily signaling factors in environmental contaminant-exposed
juvenile alligators. J. Steroid Biochem. Mol. Biol. 127, 58-63.
Murashima, A., Miyagawa, S., Ogino, Y., Nishida-Fukuda, H., Araki,K., Matsumoto, T., Kaneko, T., Yoshinaga, K., Yamamura, K.I., Kurita,T., Kato, S., Moon, A.M., and Yamada, G. (2011). Essential roles of
androgen signaling in Wolffian duct stabilization and epididymal celldifferentiation. Endocrinology 152, 1640-1651.
Nakajima, T., Hayashi, S., Iguchi, T., and Sato, T. (2011). The role offibroblast growth factors on the differentiation of vaginal epithelium of
neonatal mice. Differentiation 82, 28-37.
Nakajima, T., Iguchi, T., and Sato, T. (2011). Involvement of activin
signaling in abnormalities of the mouse vagina exposed neonatally todiethylstilbestrol. Cell Tiss. Res. 344, 527-538.
Southam, A.D., Lange, A., Hines, A., Hill, E.M., Katsu, Y., Iguchi, T.,
Tyler, C.R., and Viant, M.R. (2011). Metabolomics reveals target and••off-target toxicities of a model organophosphate pesticide to roach(Rutilus rutilus): Implications for biomonitoring. Environ. Sci. Technol.
45, 3759-3767.
Urushitani, H., Katsu, Y., Miyagawa, S., Kohno, S., Ohta, Y., Guillette,
L.J.Jr., and Iguchi, T. (2011). Molecular cloning of anti-Müllerian
hormone from the American alligator, Alligator mississippiensis. Mol.
Cell. Endocrinol. 333, 190-199.
Urushitani, H., Katsu, Y., Ohta, Y., Shiraishi, H., Iguchi, T., andHoriguchi, T. (2011). Cloning and characterization of retinoid X
receptor (RXR) isoforms in the rock shell, Thais clavigera. Aquat.
Toxicol. 103, 101-111.

〔Review articles〕

B., Iguchi, T., and Odermatt, A. (2011). Editorial: Endocrine• Blumberg,
disrupting chemicals. J. Steroid Biochem. Mol. Biol. 127, 1-3.
M.C., Goldstone, J.V., Denslow, N.D., Iguchi, T., Kille, P.,• Celander,
Meyerhoff, R.D., Smith, B.A., Hutchinson, T.H., and Wheeler, J.R.

•
•
•
•

(2011). Species extrapolation for the 21st century. Environ. Toxicol.
Chem. 30, 52-63.
Hayes, T.B., Beasley, V.R., de Solla, S., Iguchi, T., Ingraham, H.,
Kestemont, P., Kniewald, J., Kniewald, Z., Langlois, V.S., Luque, E.H.,
McCoy, K.A., Muñoz-de-Toro, M., Oka, T., Oliveira, C.A., Orton, F.,
Ruby, S., Suzawa, M., Tavera-Mendoza, L.E., Trudeau, V.L., VictorCosta, A.B., and Willingham, E. (2011). Demasculinization andfeminization of male gonads by atrazine: consistent effects acrossvertebrate classes. J. Steroid Biochem. Mol. Biol. 127, 64-73.
Miyagawa, S., Sato, M., and Iguchi, T. (2011). Molecular mechanisms
of induction of persistent changes by estrogenic chemicals on female
reproductive tracts and external genitalia. J. Steroid Biochem. Mol.
Biol. 127, 51-57.
Ogino, Y., Miyagawa, S., Katoh, H., Prins, G.S., Iguchi, T., and Yamada,G. (2011). Essential functions of androgen signaling emerged throughthe developmental analysis of vertebrate sex characteristics. Evol.
Devel. 13, 315–325.
Watanabe, H., Kato, Y., and Iguchi, T. (2011). Application of
ecotoxicogenomics for understanding mode of action of chemicals andspecies extrapolation. In: Handbook of Systems Toxicology. Eds.
Casciano, D.A. and Sahu, S.C., John Wiley & Sons, Ltd. pp.67-72.

63

National Institute for Basic Biology

Environmental Biology

DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

MINAGAWA, Jun

NIBB Research Fellow:
Postdoctoral Fellows:
Visiting Scientist:
Technical Assistant:
Secretary:

TAKIZAWA, Kenji
TAKIZAWA, Kenji*
OHNISHI, Norikazu
TOKUTSU, Ryutaro
CHEN, Yang
HOSHI, Rie
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.

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 afluorescence microscope during a transition from State 1 to
2. Initially, the average lifetime of fluorescence emitted
between 680-700 nm was 170 psec, which was largely due tothe PSII-bound LHCII, but it shifted to 250 psec when thecells were in transition to State 2 after 5 min. 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.
The molecular mechanism for q E quenching has been aheated issue during the last two decades, and it still remains
controversial. Because the unexpectedly short fluorescence
lifetime of the phospho-LHCII aggregates during the statetransition described above was not caused by high lightillumination, they are not exhibiting qE quenching, but rather
exhibiting qT (state transition) quenching. However, it is now
tempting to speculate that LHCII aggregates are a commonsite of energy dissipation, i.e., that both qE and qT quenching
are causally related by the energy-dissipative LHCII
aggregates.

I. Acclimation of photosynthesis

Using a unicellular green alga Chlamydomonas reinhardtii,
we investigate the molecular mechanisms underlying theacclimation processes of the photosynthetic complexes bymeans of biochemistry, molecular genetics, absorption andfluorescence spectroscopy, and bio-imaging.

1-1 State-transitions

The two photosystems—photosystem I (PSI) and II(PSII)—in the thylakoid membranes function as chargeseparation devices. Each has a distinct pigment system with
distinct absorption characteristics (PSI has a broad
absorption peak in the far-red region as well as peaks in theblue and red regions, whereas PSII has absorption peaks inthe blue and red, but not in the far-red region) and a distinct
action spectrum. Thus, an imbalance of energy distribution
between the two photosystems tends to occur in naturalenvironments, where light quality and quantity fluctuate withtime. Since the two photosystems are connected in series
under normal conditions, green plants and algae need toconstantly balance their excitation levels to ensure optimal
efficiency of electron flow. State transitions occur under suchconditions to redistribute 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. This year, we
visualized phospho-LHCII dissociation during state

64

Figure 1. Visualization of the progress of a state 1-to-2 transition bymeans of FLIM. Blue and red dots correspond to 170 and 250 psec
lifetime components, respectively.

1-2 Cyclic electron flow

In eukaryotes, photosynthesis is a process ofphotochemical energy transduction, which occurs via theconductance 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

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

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.

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 was grown underhigh-light conditions, only the smaller PSI fraction waspresent. The two PSI preparations were also found to be
devoid of far-red chlorophyll fluorescence (715-730 nm), a
signature of PSI in oxygenic phototrophs. These uniquefeatures of O. tauri PSI may reflect primitive light-harvesting
systems in green plants and their adaptation to marine
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 inthose living with corals and are trying to elucidate how theirphotosynthetic machinery is acclimated to the variable lightand 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.

II. Ecophysiology of marine phytoplankton

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 forstudying 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 contains the PSI
core proteins, LHCI, which are conserved in all green plants,
Lhcp1, a prasinophyte-specific LHC protein, and the minor
monomeric LHCII proteins CP26 and CP29. The larger PSI

Publication List

〔Review article〕

J. (2011). State transitions — The molecular remodeling of• Minagawa,
photosynthetic supercomplexes that controls energy flow in thechloroplast. Biochim. Biophys. Acta 1807, 897-905.

65

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) is a comprehensive platform for microbial
comparative genomics. The central function of MBGD is tocreate 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. These features areespecially useful when the user’s interest is focused oncomparison of genomes that are taxonomically related tosome specific organisms.
The database now contains well over 1000 publishedgenomes including 27 eukaryotic microbes and 4
multicellular organisms. To cope with further increase of
genomic data in the future, we have begun to develop an
efficient system for updating ortholog data, which includes arefinement procedure for constructing reference ortholog
groups and an efficient procedure for incremental update oforthologous relationships (see Section II below).
MBGD is available at http://mbgd.genome.ad.jp/.

II. Development of methods for constructing
orthologous groups among multiple
genomes

As a core technology of our comparative genomics tools,

66

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.
We are continuing to improve the algorithm. To improve thescalability of the algorithm for comparison of thousands ofgenomic sequences, we are developing an efficient method to
update the clustering result incrementally. By this method,
one can add new genomes to a reference set of ortholog
groups that is constructed from a representative set of
published genomes available in the MBGD server. We arealso trying to extend the algorithm for handling metagenomic
data. To infer the taxonomic position of the source organismof 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. The identification of the set of intrinsicallyconserved genes, or the genomic core, among a taxonomic
group is crucial not only for establishing the identity of eachtaxonomic group, but also for understanding prokaryotic
diversity and evolution. We consider the core structure of
related genomes as a set of sufficiently long segments inwhich gene orders are conserved among multiple genomes sothat they are likely to have been inherited mainly throughvertical transfer. We developed a method for aligning
conserved regions of multiple genomes, which finds theorder of pre-identified orthologous groups that retains to thegreatest possible extent the conserved gene orders.
We are now expanding our analysis to more diverged
bacterial families to examine generality of our approach. We
are also developing an enhanced algorithm that can
incorporate phylogenetic relationships among input genomes.

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 (Figure 1). The
ortholog table viewer is a spreadsheet like viewer that candisplay the entire ortholog table, containing more than athousand genomes. Using the zoom in/out function, it candisplay the entire table or a section of the main table withmore detailed information. In RECOG, several basic
operations on the ortholog table are defined, which areclassified into categories such as filtering, sorting, andcoloring, and various comparative analyses can be done bycombining these basic operations, such as “Neighborhood
gene clustering” and “Phylogenetic pattern clustering” In
addition, RECOG allows the user to input arbitrary geneproperties such as sequence length, nucleotide/amino acidcontents and functional classes, and compared theseproperties among orthologs in various genomes.

detailed examination of the boundary of these and otherchromosomal inversions identified among H. pylori
genomes, we were able to find a novel mechanism of
genome evolution named DNA duplication associated withinversion.

Figure 2. Identification of the genes characteristic to the East Asianstrains of H. pylori based on phylogenetic tree topologies. Several
characteristic genes were identified in addition to well-known virulence
factors, cagA and vacA.

Publication List

〔Original papers〕

Y., Kawai, M., Uchiyama, I., and Kobayashi, I. (2011). Domain• Furuta,
movement within a gene: a novel evolutionary mechanism for protein•
Figure 1. The RECOG system displaying ortholog groups of outermembrane proteins identified among 20 H. pylori strains.

V. Comparative genomics of Helicobacter pylori

Helicobacter pylori is a major pathogen in human gastriccancer and it is known that the East Asian strains of H. pylori
have a stronger subtype of a major virulence factor, CagA
protein, than Western strains. In collaboration with Dr.
Kobayashi (Univ. Tokyo) and other researchers, we havedetermined the complete genomic sequences of four H.
pylori strains isolated from Japanese patients and comparedthem with other published H. pylori genomes. Using the
RECOG system and other tools (Figure 1), we tried to
identify characteristic genomic features of the East Asianstrains from various points of view and infer evolutionary
processes and mechanisms that are related to the evolution of
H. pylori (Figure 2). As a result, we were able to identify
several genes that characterize the East Asian strains.
In addition, we found that some outer membrane proteinsthat are specifically duplicated in the East Asian strains arelocated at the boundary of the chromosomal inversion
identified between the East Asian and other strains. After••diversification. PLoS ONE, 6, e18819.
Furuta, Y., Kawai, M., Yahara, K., Takahashi, N., Handa, N., Tsuru, T.,
Oshima, K., Yoshida, M., Azuma, T., Hattori, M., Uchiyama, I., andKobayashi, I. (2011). Birth and death of genes linked to chromosomal
inversion, Proc. Natl. Acad. Sci. USA 108, 1501-1506.
Kawai, M., Furuta, Y., Yahara, K., Tsuru, T., Oshima, K., Handa, N.,
Takahashi, N., Yoshida, M., Azuma, T., Hattori, M., Uchiyama, I., andKobayashi, I. (2011). Evolution in an oncogenic bacterial species withextreme genome plasticity: Helicobacter pylori East Asian genomes.
BMC Microbiology 11, 104.
Yamamoto, K., Tanaka, H., Nishitani, Y., Nishiumi, S., Miki, I.,
Takenaka, M., Nobutani, K., Mimura, T., Ben Suleiman, Y., Mizuno, S.,
Kawai, M., Uchiyama, I., Yoshida, M., and Azuma, T. (2011).
Helicobacter suis KB1 derived from pig gastric lymphoid follicles
induces the formation of gastric lymphoid follicles in mice through theactivation of B cells and CD4 positive cells. Mocrobes Infect. 13, 697708.

〔Review article〕

I. (2011). Functional inference in microbial genomics based• Uchiyama,
on large-scale comparative analysis. In Omics Approaches to Protein
Function Prediction, D. Kihara ed., (Springer), pp.55-92.

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National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

Technical Staff:
NIBB Research Fellows:
Postdoctoral Fellows:
Visiting Scientist:
Technical Assistants:

KAJIURA-KOBAYASHI, Hiroko
OSHIMA, Yusuke*
TAKAO, Daisuke*
ICHIKAWA, Takehiko
TAKAO, Daisuke
OSHIMA, Yusuke
SHINTANI, Atsuko
ISHIBASHI, Tomoko
OKA, Naomi

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 developmental
biology.

I. Initial step for left-right asymmetry

The first L-R asymmetry in mammalian development arises
on the embryonic surface. A gastrulating mouse embryo hasa shallow hollow on its ventral surface, called ‘the node,’with hundreds of cilia moving in a clockwise rotationalmanner (Figure 1; Nonaka et al., 1998). The sum of thevortical motions of the cilia, however, generates a leftward
flow of the surrounding fluid rather than a vortex. The cilia
can generate L-R asymmetry de novo, i.e. without preexisting left-right asymmetry, by their posteriorly tilted
rotation axis (Nonaka et al., 2005).
The leftward flow, called nodal flow, determines subsequent
L-R development. This principle has been confirmed by ourexperiments, which demonstrated that embryos raised withan artificial rightward flow develop reversed L-R asymmetry(Nonaka et al., 2002).
While it is clear that nodal flow conveys asymmetric
information along the L-R axis, the nature of the information
remains unclear. We are now working to clarify how thedirection of nodal flow is converted to the subsequent step,
asymmetric gene expression.

Figure 2. Principle of light-sheet microscopy. Left: light path ofconventional fluorescent microscopes using single objective lens for both
illumination (Ex) and detection (Em). Right: light-sheet microscopes
including DSLM, where illumination light is limited to the focal plane ofthe detection objective.

including Digital Scanned Light-sheet Microscope (DSLM,
Figure 2) is extremely suitable for this purpose, and we haveapplied it to analyze cell movements in intact mouseembryos at gastrulating stages (Figure 3). We have been also
developing another light-sheet microscope for wider
application of living samples: our embed-free and fast image
acquisition system enables 4D data acquisition of freely
moving Amoeba proteus.

Figure 3. 6.5-day mouse embryos visualized by DSLM. Left: an opticaltransverse section of a fixed embryo. Right: cross section of a living one
expressing GFP in the neurons.

In addition to our own research projects, we support
researchers who are interested in using our DSLM and twophoton microscopes. Several collaborations including live
imaging of developing brains, hair bulbs, salivary glands andrenal tubules are in progress.

Publication List

〔Original paper〕

N., Alfaro-Cervello, C., Shimizu, K., Asakawa, K., Urasaki,• Kishimoto.,
A., Nonaka, S., Kawakami, K., Garcia-Verdugo, JM., and Sawamoto,Figure 1. Left: ventral view of a 7.5-day mouse embryo. Middle: thenode. Right: node cilia.

K. (2011). Migration of neuronal precursors from the telencephalicventricular zone into the olfactory bulb in adult zebrafish. Journal ofComparative Neurology 519, 3549-3565.

II. Imaging technologies

Long-term live imaging of large specimens, such asembryos, is very useful in developmental biology but
technically challenging, mainly because of phototoxicity andthe limitations of deep imaging. Light-sheet microscopy

68

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2011. The former title isindicated by an asterisk (*).

National Institute for Basic Biology

Research Support

NIBB CORE RESEARCH FACILITIESHeadKOBAYASHI, Satoru

RNA-seq and ChIP-seq. A successful example is the mutant
screening of Arabidopsis. We successfully identified
causative mutations in EMS mutant screening by deep
sequencing quickly at low cost, which is much more
effective than conventional mapping-based cloning methods.

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 Assistants:
Secretary:

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
KITAZUME, Tatsuya
ASAO, Hisayo
FUJITA, Miyako
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 2011, we analyzed approximately 350 samples
with mass spectrometers and 40 samples with protein
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) and HiSeq2000 (Illumina), which was
installed this year, the Functional Genomics Facility iscommitted to joint research aiming to exploring otherwise
inaccessible new fields in basic biology.
During 2011 we carried out 45 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,
and various applications such as genomic re-sequencing,

Figure 2. LC-Q-TOF-MS

69

National Institute for Basic Biology

Research Support

Genome Informatics Training Course

We organize NIBB Genome Informatics Training Courses
every year. In 2011, we provided a two-day training courseon next-generation sequence data analyses. This course isdesigned to introduce the basic knowledge and skills of nextgeneration sequence data analysis to biologists who are notfamiliar with bioinformatics (p. 84).

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

SHIGENOBU, Shuji

Technical Assistants:

HASHIYAMA, Tomomi
SUZUKI, Miyuzu

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.

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

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, inconsort with that of bacterial symbiont Buchnera aphidicola

70

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 paper〕

D., Duncan, R., Shigenobu, S., and Wilson, A. (2011). Genome• Price,
expansion and differential expression of amino acid transporters at theaphid/Buchnera symbiotic interface. Mol. Biol. Evol. 28, 3113–3126.

〔Review article〕

S., and Wilson, A.C.C. (2011). Genomic revelations of a
• Shigenobu,
mutualism: the pea aphid and its obligate bacterial symbiont. Cell. Mol.
Life Sci. 68, 1297-1309.

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 confocal microscopes and the Okazaki
Large Spectrograph. The Okazaki Large Spectrograph is theworld’s largest wide spectrum exposure mechanism, capableof producing a range of 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 suchas confocal and two-photon excitation microscopes, are usedby both internal and external researchers as vital equipment
for core and collaborative 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 and Nikon A1R, Carl Zeiss Duo 5)
and other custom-made laser microscopes (Digital ScannedLight-sheet Microscope: DSLM and Infrared Laser-Evoked
Gene Operator microscope: IR-LEGO) for users in NIBB
and collaborative guest researchers. We began Collaborative
Research Programs using these machines since 2010.
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 7 projects of the Collaborative
Research Program for the Use of the DSLM.
The IR-LEGO was developed by Drs. Shunsuke Yuba andYasuhiro Kamei at the National Institute of Advanced
Industrial Science and Technology (AIST). This microscope
can induce a target gene of interest by heating a single target
cell in vivo with a high efficiency irradiating infrared laser(Kamei et al. Nat. Methods, 2009). Details are described in
the next section. The IR-LEGO was also used for 8
Individual Collaborative Research projects, including
applications for higher plants and small fish.

Publication List on Cooperation〔Original papers〕

G., Muto, T., and Uozumi, Y. (2011). Molecular-architecture• Hamasaka,
based administration of catalysis in water: self-assembly of anT., Kawamura, A., Takahashi, J., Yagi, H., Morishima, M.,• Okubo,Matsuoka, R., and Takada, S. (2011). Ripply3, a Tbx1 repressor, isamphiphilic palladium pincer complex. Angew. Chem. 50, 4876-4878.

•••
Figure 1. An example of experiments using the Large Spectrograph.
Various color rays (monochromatic light) were irradiated simultaneously
to samples in cooling chambers.

•

required for development of the pharyngeal apparatus and its derivatives
in mice. Development 138, 339-348.
Moritoh, S., Sato, K., Okada, Y., and Koizumi, A. (2011). Endogenous
arginine vasopressin–positive retinal cells in arginine vasopressin–
eGFP transgenic rats identified by immunohistochemistry and reverse
transcriptase– polymerase chain reaction. Mol.Vis. 17, 3254-3261.
Inada, H., Watanabe, M., Uchida, T., Ishibashi, H., Wake, H., Nemoto,T., Yanagawa, Y., Fukuda, A., and Nabekura, J. (2011). GABA regulate
the multidirectional tangential migration of GABAergic interneurons inliving neonatal mice. PLoS ONE 6, e277048.
Osada, H., Yoshitake, Y., Ikeda, T., Ishigaki, Y., Takata, T., Tomosugi,N., Sasaki, H., and Yonekura, H. (2011). Ultraviolet B-induced
expression of amphiregulin and growth differentiation factor 15 inhuman lens epithelial cells. Mol. Vis. 17, 159-169.
Shikata, T., Iseki, M., Matsunaga, S., Higashi, S., Kamei, Y., and
Watanabe, M. (2011). Blue and red light-induced germination of resting
spores in the red-tide diatom Leptocylindrus danicus. Photochem.
Photobiol. 87, 590-597.

71

National Institute for Basic Biology

Research Support

A.L., Hamid, H., and Torikai, A. (2011). Effects of solar UV• Andrady,
and climate change on materials. Photochem. Photobiol. Sci. 10, 292-•

300.
Karahara, I., Takaya, E., Fujibayashi, S., Inoue, H., Weller, J. L., Reid,
J.B., and Sugai, M. (2011). Development of the Casparian strip isdelayed by blue light in pea stems. Planta 234, 1019-1030.

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

KAMEI, Yasuhiro

Technical Assistant:measure temperature as a micro thermometer. Using thisprobe, we evaluated heating properties of IR-LEGO such as
time course of temperature rise and 3-dimensional
distribution of temperature during IR irradiation. In a model
tissue which contained GFP expressing bacteria inpolyacrylamide 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 waslimited to a small volume about as large as a typical cell
(Figure 2 right).

KANIE, Yuta

To investigate a gene function in each cell we have toexpress the gene in the cell in vivo, ideally the expression
must be limited only to the single cell. Tissue or cell specificpromoters were used to reveal gene functions, however
promoter-driven gene expression was governed by cell fateor environment, therefore we could not control the timing ofgene 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.
In infrared (IR) beams can heat water molecules, which are
the main constituent of cells, hence, we can heat a single cellby irradiating 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.

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 fishes,
medaka and zebrafish, 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.

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

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 to72

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

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 powerfultool for these studies in combination with molecularbiological techniques, such as the cre-loxP system (Figure 3).
By Applying IR-LEGO to a mutant and its rescue transgenic
strain; using hsp-cre with a rescue gene which is sandwichedby 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 started establishment of acre-loxP system in medaka.

Publication List

〔Original paper〕

T., Iseki, M., Matsunaga, S., Higashi, S., Kamei, Y., and• Shikata,
Watanabe, M. (2011). Blue and red light-induced germination of resting

spores in the red-tide diatom Leptocylindrus danicus. Photochem.
Photobiol. 87, 590-597.

〔Original paper (E-publication ahead of print)〕

H., Yamada, M. Kamei, Y., Fujiwara-Ishikawa, T., Todo, T.,• Masuyama,
Nagahama, Y., and Matsuda, M. Dmrt1 mutation causes a male-to-database 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/).

female sex reversal after the sex determination by Dmy in the medaka.
Chromosome Res. 2011 Dec. 21.

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

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

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 distributethe 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.

Figure 1. Biological Information Analysis System

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. 66).

Representative Instruments

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 beprocessed 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 and73National Institute for Basic Biology

Research Support

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 Professors:
Technical Staff:
Technical Assistants:

WATANABE, Eiji
TANAKA, Minoru
NARUSE, Kiyoshi
HAYASHI, Kohji
NOGUCHI, Yuji
INADA, Junichi
INADA, Yosuke
KAJIWARA, Yuya
MATSUMURA, Kunihiro
ICHIKAWA, Yoko
TAKAGI, Yukari
SUGINAGA, Tomomi

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.

Figure 1. The Model Animal Research Facility in the Yamate area.

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.

74

Figure 2. Equipment for manipulating mice eggs.

In 2011 (from January 1 to December 11) 3,807 mice and717 fertilized eggs were brought into the facility in the
Yamate area, and 48,862 mice (including pups bred in thefacility) and 18,139 fertilized eggs were taken out.

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 2011 (from January 1 to December
11) 48 mice were brought into the facility in the Myodaijiarea, and 1,828 mice (including pups bred in the facility)
were taken out.

biological tools.
In 2011 (from January 1 to the date as of December 11),4,490 medaka and zebrafish (340 eggs, 650 embryos and3,500 adults) were brought to the facility and 51,954 medaka
and zebrafish (50.591 fertilized eggs, 473 embryos and 890
adults, including animals bred in the facility) were taken out.
In the laboratory for chick embryos 180 fertilized chicken
eggs were brought in and there were no fertilized eggs orchicken embryos taken out this year. These animals wereused for research activities in neurobiology anddevelopmental 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 2011 we shipped 219 independent
medaka strains, 478 cDNA/BAC/Fosmid clones, and 120
samples of hatching enzyme to the scientific community
worldwide.

Figure 3. Large sized autoclave in the Myodaiji area.

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 important

Figure 4. Gapdh-loxP[DsRed]-GFP transgenic medaka.

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 (p. 43) is studying mechanisms of thevisual system using a psychophysical approach. The
Laboratory of Bioresources (p. 52) has conducted a genetic
and genomic analysis of quantitative traits and Mendelian
phenotype variations as well as evolution of sex
determination systems in medaka related species. The
Laboratory of Molecular Genetics for Reproduction (p. 32) isstudying the molecular mechanisms of reproductive organ
development and sex differentiation using mutagenized ortransgenic medaka. For details, please refer to the pages ofeach laboratory.

75

National Institute for Basic Biology

Research Support

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

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
KAJIURA-KOBAYASHI, Hiroko
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, 6 phytotrons, and 12 rooms with the P1P
physical containment level for established and emerging
model plants including a thale cress Arabidopsis thaliana,
several carnivorous plants, a rice Oryza sativa, rushes Juncus
sp., a moss Physcomitrella patens, and several otherflowering plants. An emerging model insect, a tiny mothAcrocercops transecta is also reared in this laboratory. Most
culture space is fully used the whole year by more than 70
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 sensitive plant Mimoza pudica, carnivorous plants,
and wild-type strains of medaka fish Oryzias sp. Seven green
houses (4, 6, 6, 6, 6, 9, and 38 m2) with air-conditioning areprovided for the cultivation of a rice Oryza sp., Lotus
japonica and related legume species, as well as mutant linesof 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-m 2 building
with storage and workspace. Part of the building is used forrearing of the orchid mantis.
In the spring of 2011, a bench-top bioreactor and three
growth chambers (7 m2 each) were introduced by the Japan
Advanced Plant Science Network. This equipment is widely
opened for outside research groups. The chambers have web
cameras on each plant rack, and users can monitor theirsamples via the Internet. One of the cambers can control CO2
level in addition to temperature and light conditions.

Figure 5. The growth chambers newly introduced.

● 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, constructed a
BAC clone screening system, and provided 27 DNA clones
and 11 mutant lines to both local and foreign biologists thisyear.
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 the assistant professor Y. Hamada, theprincipal investigator of the Laboratory of Cell Sociology, isshown on the laboratory page (p. 16).

Figure 6. A user operating a cell storage tank.

76

CENTER FOR RADIOISOTOPE FACILITIESHeadHASEBE, Mitsuyasu

Technical Staff:

Technical Assistants:

Associate Professor

OGAWA, Kazuo(Radiation Protection Supervisor)

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
according to Japanese law. The CRF is responsible formonitoring the purchase of radioisotopes from JRIA (Japan
Radioisotope Association) and the transfer of radioisotope
wastes to JRIA.
Matsuda, Iinuma, Ito, and Kamiya maintained the MyodaijiArea. Ogawa and Sawada worked in the Yamate-Area.
The number of registrants and the number of users fromJanuary 2011 to December 2011 are presented in Table 1.
Users counted by the monitoring system going in and out of
the controlled areas numbered 3,760 persons during thisperiod. The percentages and numbers for each area areshown in Table 2. The annual changes of registrants and thenumber of totals per year are shown in Figure 1.
The balance of radioisotopes received at the CRF is shownin Table 3.
The followings are CRF’s special activities in 2011.
1. A special lecture was performed covering NBC (Nuclear,
Biological, Chemical) hazards and the Great East Japan
Earthquake disaster rescue operations by invited speakers(Okazaki fire station officers) as the 2011 training lesson
for radiation workers. (See Figure 2, photo A, B.)
2. At the Myodaiji-area, the exhaust fan had deteriorated
and has subsequently been exchanged. The repair work
was done in accordance with Japanese law. (See Figure 2,
photo C, D.)

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 2011

Table 4. Training course for radiation workers in 2011

A

B

C

D

Table 1. Numbers of registrants and users at Myodaiji-Area and YamateArea in 2011

Table 2. Users and visitors who entered each controlled area in 2011

Figure 2. The CRF’s special acticities in 2011
A, B: Okazaki fire station officers lecture about NBC hazards. Photo A
shows a lecture by the head of the Okazaki fire station. Photo B shows aparticipant wearing NBC hazard protective wear.
C, D: The exhaust fan was exchanged. Photo C shows the new fan (left)
and the old fan (right). Photo D shows CRF’s staff checking radiation
contamination inside of the fan.

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):
Technical Assistants:

KURATA, Tomoko
OTA, Kyoko
KAWAGUCHI, Colin

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 2011

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 COOPERATIONChair

YOSHIDA, Shosei

Assistant Professor:
Technical Assistants:

KAMADA, Yoshiaki
NAKANE, Kaori
TAKAHASHI, Ritsue
SANJO, Kazuko

The Office of International Cooperation coordinatesinternational collaboration in the field of biology, 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).

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 2011

1) Supporting coordination of international conferences
The Office of International Cooperation coordinated thefollowing international conferences hosted by NIBB: The 1st
NIBB - Princeton Symposium “Proteomics, Metabolomics,
and Beyond” (November 2011).
Other conferences scheduled for 2011 were postponed dueto the earthquake on March 11th.
2) Supporting dispatching researchers and students tointernational conferences
The Office of International Cooperation coordinatedsending NIBB researchers and graduated students to thefollowing symposia: 3 rd NIBB-TLL-MPIPZ Joint
Symposium “Cell cycle and Development” (November 2011)and EMBL PhD symposium “Rhythm of Life” (November2011).
3) Management of education related programs
The Office of International Cooperation also coordinated
The 6th NIBB International Practical Course“Developmental Genetics of Medaka IV” (November, 2011)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 Chiefs:
HIGASHI, Sho-ichi
MORI, Tomoko
Subunit Chiefs:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
Technical Staff:
NISHIDE, Hiroyo
NAKAMURA, Takanori
TANIGUCHI-SAIDA, Misako
Technical Assistants:
ICHIKAWA, Chiaki
NISHIMURA, Noriko
YAMAMOTO, Kumi
Secretaries:
ICHIKAWA, Mariko
ISHIKAWA, Azusa● NIBB Bioresource Center
Subunit Chiefs:
HAYASHI, Kohji
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistants:
ICHIKAWA, Yoko
TAKAGI, Yukari
SUZUKI, Keiko● 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-TAKASE, 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
Secretaries:

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 2nd NIBB-EMBL PhD Mini-Symposium and13th International EMBL PhD Students Symposium“The Rhythm of Life: Cycles in Biology”
November 16 (Wed) - 19 (Sat), 2011
Seven PhD students from NIBB, three from Nagoya
University, and one from Kyoto University (total 11 students)
were funded by NIBB to participate in the 2nd NIBB-EMBL
PhD Mini-Symposium 2011 (held on 16 November) and the13th International EMBL PhD Symposium “The Rhythm ofLife”(held on 17-19 November). Our students had the chance
to give oral presentations at the mini-symposium and poster
presentations at the PhD symposium to introduce theircurrent research. They also attended Lab tours to visit some
EMBL laboratories and facilities to exchange experimental
information and discuss their research with PhD students,
post-docs and PIs. (Y. Kamada)Participating students from Japan

Nakata, Miyuki; Ohara, Yuya; Shi, Dongbo; Shibata,
Michitaro; Tokue, Moe; Wanglar, Chimwar; Yoro, Emiko(NIBB); Ikegami, Keisuke; Imai, Saki; Ishida, Kai (Nagoya
Univ.); Muranaka, Tomoaki (Kyoto Univ.)

Comments from students (excerpts)

・Nakata, Miyuki
Among the many great experiences I had During the EMBL
PhD Symposium were chances to meet and interact withother students like myself, to tour the EMBL facilities, and to
give a talk in professor Marcus Heisler’s lab. Because he has
been such a pioneer in my field of plant development, I senta personal email to Professor Heisler to ask for the chance totalk with him during the symposium. He replied andsuggested I give a presentation to the members of his lab.
Because I have never been very comfortable giving
presentations in English I was worried whether I would beunderstood. However, I was relieved as my presentation
seemed to be received with great interest. In addition,
because my ideas were approved of it has given me more
confidence in my research.

EMBL, Heidelberg
EMBL students were wonderful and co-operative. We, the
NIBB students got to present our works both in oral andposter presentations. We got lots of feedback from the EMBL
students and some Professors during the 13th EMBL PhD
Symposium. Many researchers around the globe working on
Biology had come to advise and expose their wonderfulworks. Their works encourage us even more to love science.
Not to forget the good food over there, Heidelberg is a small,beautiful city, very much similar to Okazaki. Such a
wonderful and peaceful city, no wonder many researchers
come up with good inspirations and works. I would
encourage many of our aspiring NIBB students not to missthis opportunity of knowledge sharing/exchange amongst
different Universities and to make many new friends.
・Yoro, EmikoThis visit to EMBL was my first trip to Europe and my first
English oral presentation. First I must thank the people who
took the time to help me in my preparations. Of course I
received a lot of advice from the members of my lab, but I
also had help from the other students who visited EMBL as
we repeatedly practiced together, and from the international
students at NIBB who helped make my presentation easier tounderstand. It was a lot of work but it was also an excellent
experience. Because of the help of all these people I think I
gave my best presentation possible. I also feel that mymotivation to give good English presentations, as well as to
do good research has grown remarkably due to thisexperience. Going forward I hope to use this experience asan encouragement to work toward my future.

・Tokue, Moe
In addition to the students from EMBL many students fromaround the world participated in this symposium. Seeing thatpeople from Germany and places like Kazakhstan which isso much colder than the already very cold Germany, andItaly where fashion is so much more a part of their culture
than in Germany, all work hard at their research through trial
and error in the same way gave me a new feeling of
connection to them all. Seeing that people from various
countries with different cultures and different languages can
all discuss their research together influenced me greatly.
・Wangler, Chimwar
I highly appreciate NIBB for providing us an opportunity toattend the mini PhD symposium at EMBL. It was an eye
opening experience. The travelling and lodging were great.

81

3rd NIBB-TLL-MPIPZ Joint Symposium 2011
Cell Cycle and Development
Organizers: Frederic Berger (TLL), Karuna Sampath (TLL), Toshie Kai (TLL), Toshiro Ito (TLL)
November 21 (Mon) - 22 (Tue), 2011
Temasek Life Sciences Laboratory, Singapore
The 3rd NIBB-TLL (Temasek Life Sciences Laboratory)-MPIPZ (Max Planck Institute for Plant Breeding Research)
Joint Symposium 2011 was held at TLL’s facilities in
Singapore. Despite it being winter, Singapore’s close
position to the equator keeps it quite warm, with theconference seeing 30℃ temperatures both days, and humidityof over 80%.
Through lectures on the cell cycle, development, and related
fields of basic biology by researchers from Japan, Singapore,and Germany, this joint symposium set out to promote aninternational exchange of scientific ideas unhampered byboundaries of nation or field. The Japanese lecturers for the
symposium consisted of 6 lecturers from NIBB, as well as 3other researchers from throughout the Japanese communityof basic biology scientists. Including the talks by the three
keynote speakers, Prof. Mitsuyasu Hasebe (NIBB), Prof.
Yoshiaki Ito (TLL), and Prof. George Coupland (MPIPZ),
the topics covered included the cell cycle, cell division,
fertilization, development, and differentiation, among others,with research drawn from a large pool of model species suchas mice, Arabidopsis thaliana, Physcomitrella patens,
zebrafish, Xenopus laevis, yeast, and Volvox. Each
presentation by the researchers was both interesting, and fullof personal flavor, with the question and answer sessions
afterwards blossoming into fruitful cross-disciplinary
discussions lasting past the initially allotted time.
The poster presentations were also quite lively, consistingof ten presentations, primarily by younger researchers, whospoke passionately about their subjects until the end of thesymposium. With additional activities, such as tours of the
TLL facilities and botanical gardens, as well as several
lunches and dinners spent getting to know each other whilesampling the delicious local cuisine, this symposiumprovided a wonderful opportunity for the strengthening ofresearch ties on both a personal and organizational level, andwas ultimately a very fruitful endeavor. (Y. Matsubayashi)

Speakers
Balasubramanian, Mohan (TLL), Berger, Fred (TLL), Coupland, George (MPIPZ), Nishii, Ichiro (TLL), Ingham, Phil (IMCB), Ito,
Yoshiaki (NUS), Ito, Toshiro (TLL), Kai, Toshie (TLL), Kaldis, Phil (IMCB), Rorth, Pernille (IMCB), Sampath, Karuna (TLL), Simon,
Ruediger (Heinrich-Heine Univ.), Turck, Franziska (MPIPZ), Yu, Cai (TLL)
Goshima, Gohta (Nagoya Univ.), Goto, Koji (RIBS Okayama), Hasebe, Mitsuyasu (NIBB), Ikui, Amy (Brooklyn College), Kamada,
Yoshiaki (NIBB), Kawaguchi, Masayoshi (NIBB), Matsubayashi, Yoshikatsu (NIBB), Ueno, Naoto (NIBB), Yoshida, Shosei (NIBB)

82

The 1st NIBB - Princeton SymposiumProteomics, Metabolomics, and Beyond
Organizers: James Broach, Joshua Rabinowitz, Shosei Yoshida, Shuji Shigenobu, Satoru Kobayashi
November 1 (Tue) - 2 (Wed), 2011
The 1st NIBB-Princeton Symposium, “Proteomics,
Metabolomics, and Beyond” was held on November 1st and2nd, 2011 at the Okazaki Conference Center. This was theinaugural symposium of the academic exchange program
between the National Institutes of Natural Sciences andPrinceton University, which aims to increase scientificinteraction between these institutions, with NIBB and
Princeton’s Department of Molecular Biology playing central
roles. This symposium was originally scheduled for July
2011, but was forced to be postponed due to the hardships
following the earthquake in March. This symposium
gathered researchers performing unique biologicalinvestigations taking advantages of “-omics” approaches
such as proteomics and metabolomics. Cutting edge researchsuch as the interplay between extracellular nutrient andintracellular metabolism in yeast, the structural basis of
metabolic enzyme localization and origin of the cytoskeleton
in bacteria, comprehensive analyses of spatial distribution oflipid and protein molecules (i.e., imaging massspectrometry), identification of plant hormones with uniqueprotein modifications, and real-time imaging of ATP in living

cells, have made us realize that biology is changing
dramatically at this very moment. NIBB’s ongoing research
was also introduced by sort talks and poster presentations,which stimulated intensive discussions. The participants
came to an understanding of each other’s ideas and interests,
which we hope will ignite future communications andscientific interactions. After the symposium, participantsfrom Princeton University and invitees from outside NIBB
deepened their communications by touring around the labs
and core facilities of NIBB On behalf of the organizers, wethank all the participants, the staff of the Office ofInternational Cooperation, and the organizers’ lab members,
who have worked hard to conduct this symposium
successfully. (Shosei Yoshida, Satoru Kobayashi, and Shuji
Shigenobu)

Speakers
Broach, James (Princeton Univ.), Cristea, Ileana (Princeton Univ.), Gitai, Zemer (Princeton Univ.), Imamura, Hiromi (Kyoto Univ.)
Cui, Songkui (NIBB), Kitadate, Yu (NIBB), Matsubayashi, Yoshikatsu (NIBB), Sato, Masanao (NIBB), Shigenobu, Shuji (NIBB),
Taguchi, Ryo (Chubu Univ.), Tanaka, Minoru (NIBB), Yao, Ikuko (Kansai Medical Univ.)

83

The NIBBInternational
Practical Course

The NIBB
Genome Informatics
Training Course

The first NIBB International Practical Course was held in2007 with the aim of providing young scientists around theworld with opportunities to learn cutting-edge experimental
skills and to communicate with experts in the field. The 6th
course was held as a joint course with TLL, Singapore, in2011 as summarized below. The course was held in alaboratory provided by NIBB and equipped with thenecessary instruments and tools and a good supply ofexperimental materials.

NIBB organizes a series of training courses on up-to-date
research techniques for researchers from mainly Japanese
universities and institutions. In 2011 we held training courses
on genome informatics. The two-day programs offer lecturesand hands-on tutorials to introduce basic knowledge andskills to deal with genomic scale large data such as nextgeneration sequences (NGS). The programs are speciallydesigned for biologists who are not familiar withbioinformatics.

The 6th NIBB International Practical Course
and The 1st NIBB - TLL Joint International
Practical Course“Developmental Genetics of Medaka IV”■Period: November 14(Mon) - November 21(Mon), 2011
■Participants: 15(4 from Japan, 2 from Taiwan, oneeach from Hong Kong, South Korea, China,
Singapore, Norway, Canada, USA, Italy, India)■Lecturers:
Dr. Christoph Winkler (National University of Singapore,
Singapore)
Dr. Jun KITANO (National Institute of Genetics, Japan)
Dr. Minoru TANAKA (NIBB, Japan)
Dr. Rie KUSAKABE (Kobe University, Japan)
Dr. Minori SHINYA (National Institute of Genetics, Japan)
Dr. Tomonori DEGUCHI (National Institute of Advanced
Industrial Science and Technology, Japan)
Dr. Yoshihito TANIGUCHI (Keio University, Japan)■Course Staff from NIBB:
Kiyoshi Naruse, Takao Sasado, Yusuke Takehana,
Yasuhiro Kamei, Masatoshi Nakamoto, Tetsuaki
Kimura, Yukiko Ogino■Contents of the course:
Tr a n s g e n i c M e d a k a ( B A C h o m o l o g o u srecombination and transposon mediated genetransfer)
Cryopreservation of sperm and artificial insemination
Local gene induction with infrared laser-evoked gene
operator (IR-LEGO) method Forward genetics
approaches with ENU mutagenesis (Demonstration)Reverse genetics approaches with TILLING(Targeting Induced Local Lesion IN Genome) method

84■Period: 1st course March 10 (Thu) -11 (Fri), 2011
2nd course September 8 (Thu) -9 (Fri), 2011
■Organizer: Dr. Shuji Shigenobu (NIBB)■Lecturers: Dr. Ikuo Uchiyama, Dr. Katsushi
Yamaguchi, Dr. Shuji Shigenobu (NIBB
Core Research Facilities)■Participants: 1st course: 23 (including 3 from NIBB)
2nd course: 15 (including 4 from NIBB)■Topic: “Introduction to Next-generation Sequence
Data Analysis”
1. Overview: NGS data analysis
2. UNIX for beginners
3. Programming for biologists
4. NGS data formats
5. NGS tools
6. Exercise

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 2011 there were 17 applicants, of which nine interns
were selected. These interns were from universities located in
7 countries (Taiwan, Australia, India, the United States,
Singapore, Egypt, and Iran) and spent periods ranging from 1
week to 1 month experiencing life as a member of a researchteam. Since then one of these interns has enrolled as a student
of SOKENDAI.

Report from a participant
Yousef Yari Kamrani
Justus Liebig University, Giessen, Germany

My project in the lab of Professor Minagawa was
about designing a new vector for gene transformationof a microalgae Chlamydomonas reinhardtii. The
genes of interest for Professor Minagawa were those
involved in photosynthetic machinery. The newly
designed vector contained all the necessary elements
including an antibiotic/resistance gene as a selectable
marker and an appropriate inducible promoter. The
gene encoding Cyan Fluorescent Protein (CFP) was
selected as a reporter gene and was cloned between
the Hsp70-RBCS promoter/terminator.
I liked the sense of community and the friendly
atmosphere in NIBB: not only students but also thestaff and professors were outstandingly supportive.
Furthermore, the great lodge program and wellorganized student organization allowed me a verysmooth transition to life in Okazaki during myinternship.
I am inspired by the people I worked with and I
hope to have another opportunity to come back NIBB
in the future.

85

INDEX
A ....................................................................................................
AJIOKA, Rie

52

HAYASHI, Kohji

AOKI, Etsuko

45

HAYASHI, Tomoko

61

HIGASHI, Sho-ichi

71, 80

ANDO, Sayuri
AONO, SachikoAOYAMA, Tsuyoshi
ARAKI, Masami

B

ASAO, Hisayo

45

HAYASHI, Makoto

43

HAYASHI, Yoshiki

45
8

HIGUCHI, Yoko

69

HIKINO, Kazumi....................................................................................................
45
BABA, Nayumi

CHAKRABORTY, Tapas
CHEN, Qiuhong
CHEN, Yang

CHENG, Chaoyang
CHIBA, Hirokazu

7, 61

HIRAYAMA, Yuka

66

CUI, Songkui

8, 83

DOKYO, Yumi

36

HIWATASHI, Yuji

HIYAMA, Takeshi

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

3, 26, 74, 79

FUJISAWA, Toshitaka

20

FUJISAWA, Chiemi
FUJITA, Hironori
FUJITA, Miyako

FUKADA-TANAKA, Sachiko
FUKADA, Hatsumi

FUKUSHIMA, Kenji

G

36

FUJIMORI, Toshihiko

FURUKAWA, Kazuhiko

HONDA, Satoko
HOSHI, Rie

20

ICHIKAWA, Yoko

IGARASHI, Hisako

45
80

HAMADA, Yoshio

HANDA, Yoshihiro
HARA, Ikuyo

HARA, Kenshiro
HARA, Reiko

HARA, Yusuke

HASEBE, Mitsuyasu

HASHIMOTO, Masakazu
HASHIYAMA, Kazuya

HASHIYAMA, Tomomi
HATA, Katsusuke

HATTORI, Nobuko

HAYASHI-TSUGANE, Mika
HAYASHI, Kentaro

IMAIZUMI, Taeko
IMOTO, EikoINADA, Junichi

86

INADA, Kana

32

INADA, Yosuke

16, 76INAGAKI, Sachi

48

ISHIHARA Hitomi

52

ISHIKAWA Hiroe

28
34
17

ITO, Takayo

39

ITO, Yukiko

J74, 8034

77, 80
34
45
39
61
39
74
28
74
20
20
52

45

ITO, Masako

70

45

ISHIKAWA, Takaaki
ISHITANI, Shizuka

20

28

71, 80ISHINE, Naomi

17

45

ISHIKAWA, Azusa

ISHIKAWA, Masaki

3, 45, 77, 82

69, 8028

IMAI, Akiko

31

17

64

IKAMI, Kanako
IMAI, Akihiro

45

57

20

3, 7, 61, 74

IKEUCHI, Momoko

8

36

45

7, 36

IGUCHI, Taisen

IINUMA, Hideko

H ....................................................................................................
HAAG, Eric

39

71, 80ICHIKAWA, Shunsuke

45

45

GUAN, Guijun

26

ICHIKAWA, Chiaki

ICHIKAWA, Rie

48, 80GOTO, Misako

GOTO, Miho

4556, 76ICHIKAWA, Michiko55, 6939

39

HOSHINO, Atsushi

ICHIKAWA, Mariko48

GOTO, Ayako

42

I ....................................................................................................

....................................................................................................
16
GONDA, Naoko

GOTO-YAMADA, Shino

8

61

HIRAO, Mayumi

45

26

HIRAKAWA, Ikumi

HIRAMATSU, Mika

64

20

42

HIRAKAWA, Reiko

23

8

HIMENO, Miki
HIRA, Riichiro

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

74, 80IWASAKI, Akira

45
59
23
55

77, 804534....................................................................................................
JOHZUKA, Katsuki
59

INDEX

K ....................................................................................................
KABEYA, Yukiko

KAGEYAMA, Yuji
KAI, Masatake

KAJIKAWA, Ikumi

KAJITANI, Tomoki

KAJIURA-KOBAYASHI, Hiroko
KAJIWARA, Yuya
KAKIGI, Rieko

KAMADA, Yoshiaki

KAMAGATA, Takanori
KAMEI, Yasuhiro

KAMETANI, Yoshiko
KAMIGAKI, Akane

7, 20

39

KATO, Azusa

KAWAGUCHI, Colin

KAWAGUCHI, Masayoshi
KIMURA, Tetsuaki
KINOSHITA, Chie

KINOSHITA, Noriyuki
KITADATE, Yu

KITAZUME, Tatsuya

KOBAYAKAWA, Satoru
KOBAYASHI, Kaoru
KOBAYASHI, KayoKOBAYASHI, Satoru
KODAMA, Akiko

KOMATSU, Yusuke
KOMINE, Yuriko
KON, Yayoi

KONDO, Maki

KONDO, Takao

KOTANI, Keiko

KOYAMA, Hiroshi
KUBO, Minoru
KUBO, Satoru

KUBOKI, Yuko

KUBOYAMA, Kazuya
KURATA, Tomoko

MATSUMURA, Kunihiro

832

MIYAZAKI, Emi

26

MIYAZAKI, Yuriko

61

MIYAZAWA, Hikota

32

3, 20, 69

MORITA, Hitoshi
MORITA, Junko

26
39
39

8, 80
3

39

7, 26
45
20
28

7, 36
79

....................................................................................................
LANGE, Anke
61

N

61
61
17
80
36
36
48
3

11

3

69, 80
17
39

MOROOKA, Naoki

76, 80

MURATA, Takashi

45

MURAKAMI, Michiyo

39

36

61, 80

MORI, Tomoko

45, 6464

MIZUTANI, Takeshi
MORI, Ikue

52

39

28, 80

MORI, Ayaka

45

42

MIZUGUCHI-TAKASE, Hiroko
MIZUSHIMA, Noboru

36

74

17

MIYATA, Haruko

69

34

MIYAGI, Asuka

MIYAKE, Satoko

28, 8336

73, 80MIYAKAWA, Hitoshi

17

13

77, 80

MIWA, Tomoki

MIYAGAWA, Shinichi

52, 84

8, 55

59

MIURA, Seiko3, 7, 48, 82

45

7, 11, 82, 83

MATSUZAKI, Yoko
MINAGAWA, Jun

79

42

7, 43

MIKAWA, Ryuta

26

8

MATSUNAGA, Wataru

MATSUZAKI, Masanori

72

52

KOMATSU, Kouji

MATSUMOTO, Miwako

8

KOIKE, Chieko
KOJIMA, Yoko

MATSUMOTO, Masahito

23

51, 78

KOIKE, Yukari

MATSUDA, Yoshimi

71, 72, 84

KODAMA, Ryuji

KOHARA, Mayuko

MATSUDA, Chisato

23

80

28

MATSUDA, Azusa

54, 79, 81, 82

KATAOKA, Yukari

MARUYAMA, Ayumi

MATSUBAYASHI, Yoshikatsu

45

52

45

MASUOKA, Tomoko

74

45

MANO, Hiroaki

MASAMIZU, Yoshito

76, 80KANEKO, Hiroyo

LI, Chen....................................................................................................
MAKINO, Yumiko69, 80

MANO, Shoji

45

77

KANIE, Yuta

M

17

KAMIYA, Kiyomi
KANAI, MasatakeL45, 8017....................................................................................................
NAGAHAMA, Yoshitaka
7, 31

NAGASHIMA, Akitomo

45

NAKAI, Atsushi

8

NAKAGAMI, Yuki

NAKAJIMA, Michiko
NAKAMORI, Chihiro

NAKAMOTO, Masatoshi
NAKAMURA, Ryoko

NAKAMURA, Shuhei

NAKAMURA, Takanori

39
48
34

31, 52, 84
55, 5632

73, 8087

INDEX

NAKAMURA, Takeshi

61

SAITO, Miyuki

NAKAMURA, Yoshiaki

7, 28

SAKAI, Fumie

NAKAMURA, Tohru

39

NAKANE, Kaori

79

NAKATA, Miyuki

34, 81NAKANISHI, Norie
NAKAYAMA, Kei

NAKAYAMA, Tomomi

NAKAYASU, Tomohiro
NARUSE, Kiyoshi
NIIMI, KaoriNISHI, Tayo

36

7, 43

3, 8, 78

NISHIMURA, Toshiya

32

NISHIMURA, Noriko

80

NISHINA, Momoko

8

NODA, Chiyo

20, 80NOGUCHI, Yuji

74, 80

NODA, Masaharu
NOZAWA, Masafumi

ODA, Shigeto

61

OGAWA-OHNISHI, Mari

15, 77

OHARA, Yuya

20, 81OHKUBO, Fuki

42

OHNO, Kaoru

55

OGINO, Yukiko

61, 84OHHARA, Kaori23

OHNISHI, Norikazu

64

OHSAWA, Sonoko

39, 80OHSHIMA, Masumi

45

OHSHIMA, Issei

OHTSUKA, Masanari

7, 39

OKA, Sanae

26, 80OIKAWA, Kazusato
OKADA, Kiyotaka

1, 3, 34

OKAMOTO, Satoru

7, 48

OKUBO, Tadashi

7, 23

OKAMOTO, Haruko
OKUBO, Masayo

OKUYAMA, Teruhiro
OOI, Shoko

45

OTA, Kyoko

79

OSHIMA, Yusuke
OTA, Ryoma

SADAKANE, Osamu
SAITO, Junko

88

SHIINA, Nobuyuki
SHIMADA, Yu

SHIMAMOTO, Ko

SHINOHARA, Hidefumi
SHINOZUKA, Takuma
SHINOZUKA, Yuko

SHINTANI, Takafumi

SHUKLA, Rammohan
SIOMI, Haruhiko

39
42

52
52

8, 81

31, 5269, 70, 83, 84
13
31
3

7, 11
23
20
36
39
3

SUGIMOTO, Ryo

28

SUGIYAMA, Arisa

SUGIYAMA, Tomomi
SUMIDA, Kumiko

34
74
20
42
11

SUMIYA, Eri

61

SUZAKI, Takuya

48

SUZAKI, Momoyo
SUZUKI, Atsuko

48
17

SUZUKI, Keiko76, 80SUZUKI, Miyuzu

70

SUZUKI, Makoto
SUZUKI, RyokoT26, 8145

SUGINAGA, Tomomi

7, 20

61

SUGAYA, Tomomi

SUGIMOTO, Nagisa

7

S ....................................................................................................

3

SHIGENOBU, Shuji

52

61

SEHARA, Atsuko

SHIBATA, Yasushi

11

3

77, 80SHIBATA, Michitaro

45

20

SAWADA, Kaoru

SHIBATA, Emiko

8

44

7, 26

SHIBA, Keikun

45

44

SATO, Yasufumi

SHI, Dongbo

11

OGAWA, Kazuo

39

20, 83

SEBILLOT, Anthony

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

52, 84

SATO, Masanao
SATO, Masaru

45
58

48

SATO, Katsuhiko

3, 36

NUKAZAWA, Akira

SASAKI, Takema

SATO, Kaori

36

36

48, 79

SATO, Asako

45

31

SANJO, Kazuko

SASAOKA, Toshikuni

20

NISHIMURA, Mikio

NISHIHARA, Eri

36

SASAKI, Tetsuya

52, 74, 84

NISHIDE, Hiroyo

SAKURABA, Jyuichi

SASADO, Takao

8

73, 8034

SAKUTA, Hiraki

13

8

SAKAGAMI, MariSUZUKI, Tokiko

17
36
52

....................................................................................................
TAKABAYASHI, Junji3

INDEXTAKADA, Ritsuko
TAKADA, Shinji

TAKAFUJI, Mituse
TAKAGI, Chikako
TAKAGI, Chiyo

TAKAGI, Yukari

TAKAHARA, Masahiro
TAKAHASHI, Hiroki
TAKAHASHI, Jun

TAKAHASHI, Ritsue

TAKAHASHI, Yoichi
TAKAJI, Masafumi

TAKASHIRO, Kayoko
TAKEDA, Naoya
TAKEDA, Taro
TAKEDA, Yuta

39
52

17, 8074, 80
48
17
23
79
39
39
23
48
17
39

TAKEHANA, Yusuke

52, 84TAKIZAWA, Kenji

7, 64

TAKEUCHI, Yasushi
TAMADA, Yosuke

TAMESHIGE, Toshiaki
TANAKA, Ayumi
TANAKA, Mina36, 80453438TANAKA, Minoru

7, 32, 74, 83, 84

TANAKA, Yasuyo

42

TANAKA, Yasuhiro
TANI, Sachiko

TANIGUCHI-SAIDA, Misako
TATEMATSU, Kiyoshi
TESHIMA, Yuuko
TOHYAMA, Saki
TOKUE, Moe

TOKUTSU, Ryutaro
TOMIDA, SanaiTOMITA, Nao
TORII, Naoko

TOYOKURA, Koichi
TOYOOKA, Yayoi
TOYOTA, Kenji

TSUGANE, Kazuo
TSUGE, Toyoko

TSUNOKUNI, Hiroyuki
TSUZUKI, Shihoko

U

23

3, 23

TSUZUKI, Yumiko

42
36

71, 8034
52
61

28, 81
64
23
36
52
34
26
61

W

UTSUMI, Hideko

23, 80....................................................................................................
WADA, Kotoe
36

WAKADSUKI, Sachiko
WANGLAR, Chimwar
WASHIO, Midori

WATAKABE, Akiya
WATAKABE, Ikuko
WATANABE, Eiji

WATANABE, Etsuko

45

23, 814539
32

43, 74
8

WATANABE, Seiko

56

WON, Seok

61

WATANABE, Takaaki

60

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

YAMADA, Kenji

YAMAGUCHI, Chinami

YAMAGUCHI, Katsushi

YAMAGUCHI, Takahiro
YAMAMORI, Tetsuo
YAMAMOTO, Kumi

YAMAMOTO, Manami

YAMAMOTO, Masayuki

YAMAMOTO, Takamasa
YAMAMOTO, Yasuhiro
YANO, Koji

YATSU, Ryohei

YOKOYAMA, Hiroshi

YONEMITSU, Masako
YONEZAWA, Harumi
YORO, EmikoYOSHIDA, ChieYOSHIDA, Masahide
YOSHIDA, Shosei
YOSHII, Tomoaki

YOSHIMURA, Yuriko
YOSHINORI, Yumi

34
23
8

8, 58

69, 80, 84
58

3, 39

73, 80
20
3

17

7, 32
48
61
48
32
60

48, 8148
36

3, 28, 79, 82

11

52

8, 48

57, 7617
23
80
34....................................................................................................
UCHIYAMA, Ikuo
66, 73, 84

UEDA, Chizuru

UEMURA, Tadashi

83UENO, Naoto

3, 17, 82

UNO, Satoko

80

UKAI, Sakie

URAWA, Hiroko

23
34

89