National Institute for Basic Biology

2009 ANNUAL REPORTCONTENTS
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・ 1
Organization of the National Institute for Basic Biology ・
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・ 2
Goals of the National Institute for Basic Biology ・
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・ 5
Personnel Changes and Awardees in 2009 ・
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・ 7
Cell Biology
Division of Cell Mechanisms・
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・ 8
Division of Molecular Cell Biology・
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・ 11
Laboratory of Neuronal Cell Biology ・
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・ 14
Laboratory of Cell Structure ・
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・ 15
Laboratory of Cell Sociology・
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・ 16
Developmental Biology
Division of Morphogenesis・
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・ 17
Division of Developmental Genetics ・
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・ 20
Division of Molecular and Developmental Biology・
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・ 23
Division of Embryology ・
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・ 26
Division of Germ Cell Biology・
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・ 28
Division of Reproductive Biology ・
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・ 31
Laboratory of Molecular Genetics for Reproduction・
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・ 33
Laboratory of Plant Organ Development・
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・ 35
Neurobiology
Division of Molecular Neurobiology・
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・ 37
Division of Brain Biology・
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・ 40
Laboratory of Neurophysiology・
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・ 43
Laboratory of Neurochemistry・
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・ 44
Evolutionary Biology and Biodiversity
Division of Genome Dynamics・
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・ 45
Division of Molecular Genetics・
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・ 48
Division of Evolutionary Biology ・
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・ 49
Division of Symbiotic Systems ・
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・ 52
Laboratory of Morphodiversity・
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・ 55
Laboratory of Bioresources・
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・ 56
Environmental Biology
Division of Molecular Environmental Endocrinology・
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・ 58
Division of Plant Developmental Genetics・
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・ 61
Laboratory of Photoenvironmental Biology・
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・ 63
Theoretical Biology
Division of Theoretical Biology・
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・ 65
Laboratory of Genome Informatics・
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・ 67
Imaging Science
Laboratory for Spatiotemporal Regulations・
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・ 69
Laboratories Supervised by the Director-General・
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・ 70
Research Support
Research Support Facilities・
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・ 75
Center for Transgenic Animals and Plants・
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・ 77
Research Center for Integrative and Computational Biology・
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・Inside back cover
The cover photographs and illustrations are related to the study of an initial step of chromosome condensation, namely
condensin – chromatin association. The cis element and protein factors required for condensin recruitment onto
chromatin have been identified from the ribosomal RNA gene (rDNA) cluster in budding yeast, Saccharomyces
cerevisiae. Completion of chromosome segregation during mitosis requires the hypercondensation of the rDNA region
during anaphase. Any defect in condensin recruitment to the cis element will result in partial condensation andnon-disjunction of the chromosomal region containing an rDNA cluster. See Johzuka and Horiuchi, Mol. Cell 34,
26-35 (2009) and page 45 of this report for detail.

INTRODUCTIONO

ur 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 2004, NIBB, together
with four other national research institutes, establisheda new organization, the National Institutes of Natural
Sciences (NINS), one of four Inter-University
Research Institute Corporations. In order to 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.
Our works and results for 2009 are shown in thisreport.
In 2009, we welcomed six new colleagues, while six
colleagues left the institute as shown on page 7. The
results of our research groups are reported from page 8
to 74. The activities of our supporting divisions areshown on the following pages 75–84.
As international collaborative activities, we hosted
the 9th NIBB-EMBL (European Molecular Biology
Institute) joint meeting in Okazaki. We also
participated in the 1st NIBB-EMBL student symposiumin Heidelberg, and the 1st NIBB-MPIZ (Max Planck
Institute for Plant Breeding Research) joint symposiumin Cologne as shown on pages 85-87. In addition, we
held two practical training courses with lectures andtechnical training. One of the courses was held forstudents and young researchers interested in learning
about a new model plant, Physcomitrella patens. The
other newly instituted course was held for thosedesiring a fundamental knowledge of microarray
analysis, as reported on page 88.
In addition to research, the 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 happy we have theopportunity to educate graduate students. At present,
we have 39 students, as reported on page 6.

Kiyotaka OKADA, D. Sci.
Director-general, NIBBMarch 24, 2010

1

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

Research and Research Support

NIBB conducts its research programs through 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. Some of the research
units are adjunct and led by professors who hold joint
appointments with other universities. Adjunct divisions havea resident assistant professor. This arrangement facilitatesexchange in research activities in Japan. The Technical
Division manages the activities of the technical staff andhelps to promote the activities of each research unit and tomaintain the common resources of both the research support
units of the NIBB and the research facilities of the Okazaki
campus.
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 the NIBB
Director-General 1
Technical
Assitant
andSecretary

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

Technical
Staff

Professor (including adjunct, concurrent
and specially appointed) 18
Associate Professor (incl. adj.)11

89

Assistant Professor (incl. sp. app.)

30

28

82

41

Postdoctoral Fellow(as of 1st October, 2009)

Graduate Student

300 in total

Policy, Decision Making, and Administration

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

2

Financial Configuration of the NIBB
Donation 42
External
Competitive
Fund

1081

(settled forApril 2008- March 2009)
numbers are in million yen

Grants forCommissioned
Research

365

Grant-in-Aid
for Scientific
Research

3,135 million yen in total

Management
Expenses Grant

1332

674

Facilities
Maintenance
Grant

627

Self Income

36

Expenses Grant
for SOKENDAI 59

■ Organization

As of April 1, 2009

President

Executive Directors
Management Council

Auditors
Education and Research Council

Board of Directors
National Astronomical
Observatory of Japan

National Institute forPhysiological Sciences

Center for Novel Science Initiatives

National Institute for
Fusion Science

National Institute for
Basic Biology

Director-General
OKADA, Kiyotaka
Vice Director
YAMAMORI, Tetsuo

Advisory Committee for
Programming and Management

Institute for
Molecular Science
Three Institutes in Okazaki■ Members of the Advisory Committee for Programming and Management

Chairperson
Vice-Chair
Non-NIBB members

NIBB members

NODA, Masaharu
MATSUOKA, Makoto
FUKUDA, Hiroo
ISHINO, Fumitoshi
KATO, Kazuto
OSUMI, Noriko
SAKANO, Hitoshi
SATOH, Noriyuki
SIOMI, Haruhiko
TABATA, Satoshi
YANAGIDA, Toshio
FUJIMORI, Toshihiko
HASEBE, Mitsuyasu
HORIUCHI, Takashi
IGUCHI, Taisen
KOBAYASHI, Satoru
NISHIMURA, Mikio
TAKADA, Shinji
UENO, Naoto
YAMAMORI, Tetsuo
YOSHIDA, Shosei(as of April, 2009)

Professor, National Institute for Basic Biology
Professor, Nagoya University
Professor, The University of Tokyo
Professor, Tokyo Medical and Dental University
Associate Professor, Kyoto University
Professor, Tohoku University
Professor, The University of Tokyo
Faculty, Okinawa Institute of Science and Technology
Professor, Keio University
Deputy Director, Kazusa DNA Research Institute
Professor, Osaka University
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, 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

3■ National Institute for Basic Biology

As of April 1, 2009

As of April 1, 2009
Research Units
As of April 1, 2009
Research
Unitsof Cell Mechanisms■Division
Research
Units of Neuronal Cell Biology††††■Laboratory

Cell Biology
Cell Biology
Cell Biology■Division of Cell Mechanisms■Laboratory of Cell Structure■Division
of of
Cell
Mechanisms
Laboratory
Neuronal
Cell Biology††††■Laboratory of Cell Sociology■Laboratory of Neuronal
Cell Biology††††Cell Structure■Laboratory
Cell Structure
Sociology
Division forofMorphogenesis■Laboratory
Cell Sociology
Division of ofDevelopmental
Genetics †■Division for Morphogenesis■Division of Molecular and Developmental
Biology ††■Division forMorphogenesisGenetics †of Developmental■Division of Embryology■Division of Developmental
Genetics †Molecular and Developmental
Biology ††■Division of Germ Cell Biology■Division of Molecular
and Developmental Biology ††Embryology■Division of Reproductive Biology■Division of Embryology
Germ Cell Biology■Laboratory of Molecular Genetics for Reproduction■Division of Germ
Cell Biology
Reproductive
Biology■Laboratory of Plant Organ Development■Divisionof of
Reproductive
Biology for Reproduction
Laboratory
Molecular Genetics

Developmental Biology
Developmental Biology
Developmental Biology

Neurobiology
Neurobiology
Neurobiology
Evolutionary Biology and Biodiversity
Evolutionary Biology and Biodiversity
Evolutionary Biology and Biodiversity■Laboratory
Molecular
Genetics
for Reproduction
Plant Organ
Development
Division of of
Molecular
Neurobiology■Laboratory
Plant
Organ Development
Division of of
Brain
Biology■Division of Molecular Neurobiology■Laboratory of Neurophysiology■Division of Molecular
Neurobiology
Brain Biology■Laboratory of Neurochemistry■Division
of of
Brain
Biology
Laboratory
Neurophysiology

Division of of
GenomeDynamics■Laboratory
Neurophysiology
Neurochemistry

Division of of
Evolutionary
Biology■Laboratory
Neurochemistry■Division of Genome Dynamics■Division of Symbiotic Systems■Division of Genome
Dynamics
Evolutionary
Biology■Laboratory of Morphodiversity■Division of Evolutionary
Biology
Symbiotic Systems■Laboratory of Bioresource■Divisionof ofSymbiotic
Systems
Laboratory
Morphodiversity■Laboratory
Morphodiversity
Bioresource
Division of of
Molecular
Environmental Endocrinology †††■Laboratory
Bioresource
Division of of
Plant
Developmental Genetics (adjunct) †††■Division of Molecular Environmental Endocrinology■Laboratory of Photoenvironmental Biology■Division of Molecular
Environmental
Endocrinology
Plant Developmental
Genetics(adjunct) †††Environmental Biology
Environmental Biology
Environmental Biology
Theoretical Biology
Theoretical Biology
Theoretical
Biology
Imaging Science■Division
of of
Plant
Developmental Genetics
Laboratory
Photoenvironmental
Biology(adjunct)■Division of Theoretical Biology■Laboratory of Photoenvironmental Biology■Laboratory of Genome Informatics■Division of Theoretical Biology■
of ofTheoretical
Biology
Laboratory
Genome Informatics■DivisionDevelopmental
Dynamics (adjunct)■Informatics■Laboratory offorGenome
Spatiotemporal
Regulations■Division of Developmental Dynamics (adjunct)

Imaging Science
Imaging Science■Divisionof forDevelopmental
Dynamics(adjunct)Laboratory
Spatiotemporal
Regulations

Research Support
Units for Spatiotemporal Regulations■Laboratory
Research Support Units
TheUnits
Large Spectrograph Laboratory
Research Support

Research Support Facilities
Tissue and Cell Culture Laboratory
The
Large Laboratory
Spectrograph Laboratory
Research Support Facilities
Computer
Tissueand Spectrograph
Cell
Culture Laboratory
The
Laboratory
Center forSupport
Transgenic
Animals and Plants
PlantLarge
CultureLaboratory
Research
Facilities
Computer
Tissue andLaboratory
Cell Culture Laboratory
Center for Transgenic Animals and Plants
Plant Culture
Laboratory
ComputerLaboratory
Research
for Integrative
andPlants
CenterforCenter
Transgenic
Animals andPlant Culture Laboratory
Computational
Biology
Research Center for Integrative andResearch CenterBiology
for Integrative andComputational
Other Research Support Sections
Computational Biology
Technical Division
Technical Division
Technical Division

Other Research Support Sections
Other ResearchStrategic
Support
Sections
Planning
Department

Strategic Planning Department
Officeof Planning
Public Relations
and International Cooperation
Strategic
Department
Office of Public Relations and International Cooperation
Office of Public Relations and International Cooperation

Okazaki Research Facilities
OkazakiResearch
Facilities■ Okazaki
Research
Facilities
Department of Development, Differentiation and Regeneration
OkazakiOkazakiResearchInstitute Facilities
for Integrative Bioscience■Division of Developmental Genetics *Department of Development, Differentiation and Regeneration
Okazaki Institute for Integrative Bioscience**■
Division ofofMolecular
and Developmental
Biology*
Department
Development,
Differentiation
and Regeneration■
Division of Developmental
Genetics
CenterRadioisotope
FacilitiesBioscience
Okazakifor
Institutefor Integrative
Department
of
Bio-Environmental
Science*■Division of Developmental
Genetics
Molecular and Developmental
Biology *****Center for Radioisotope Facilities■Division
Divisionof
Bio-Environmental
Science■ofMolecularandDevelopmental
Biology **Department of Bio-Environmental Science
Center for Experimental
AnimalRadioisotope Facilities****■
Division ofofNeuronal
Cell Biology
Department
Bio-Environmental
Science***■
Division of Bio-Environmental
Science
Center for Experimental Animal
Department ofNeuronal
StrategicCell
Methodology**** ***■Division of Bio-Environmental
Science
Biology
Research
for Computational*-****)These divisions also function as NIBB’s divisions†-††††) , respectively.
CenterforCenter
Experimental
Animal Science■
Division ofofNeuronal
Cell
Biology ****
Department
Strategic
Methodology
Other divisions of the OIIB are not shown.
Research Center for Computational Science*-****)
Departmentof Strategic
Methodology
These divisions
also function
as NIBB’s divisions†-††††) , respectively.
ResearchCenterfor
Computational
Science*-****)These
divisionsthefunction
OIIB areshown.
divisions of
also
asnot
NIBB’sdivisions†-††††) , respectively.
Research Facilities run jointly with National Institute forOther
Physiological
Sciences
Other divisions of the OIIB are not shown.
Research Facilities run jointly with National Institute for Physiological Sciences
Laboratory
Glassware
Facilities■ Research
Facilities
run jointly
withwith
National
Institute
Sciences
Research
Facilities
run Instruments
jointly
National
Institutefor
forPhysiological
Physiological
Sciences
Centerfor
AnalyticalCenter for Analytical Instruments
Center for Analytical Instruments

4Electron
Microscope
Laboratory
GlasswareCenter
Facilities
Machine
Shop
Laboratory
GlasswareCenter
Facilities
Electron Microscope
Low-Temperature
Facilities
ElectronCenterMachine Microscope
Shop
MachineShop
Low-Temperature
Facilities
Low-Temperature Facilities

GOALS OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) has set
five goals for its activities in pursuing the progress of
biology. We contribute to the world-wide community of
biologists through our efforts to accomplish these goals. This
chapter briefly explains four of these goals. The last goal, thepromotion of academic research, is accomplished throughour research activities, which are introduced throughout thisbrochure.

Promotion of Collaborative Research■ Collaborative Research Projects

Research projects in collaboration with NIBB’s divisions/laboratories and research activities conducted using NIBB’s
facilities are solicited from external researchers at otheruniversities and institutes. In addition to conventional“individual collaborative research projects,” “collaborative
experiments using the Large Spectrograph,” and “NIBB
workshops,” new types of research projects are solicited thatfacilitate the strategic organization of collaborative research
projects. “Priority collaborative research projects” are carriedout as group research by internal and external researcherswith the purpose of developing pioneering research fields inbiology. Five projects have already been carried out and as
an outcome of the project “Molecular mechanisms forcontrolling the individuals of higher plants,” Prof. I.
Terashima (Univ. Tokyo) started a project of Grant-in-Aid for
Scientific Research on Innovative Areas in 2009. The
category “Collaborative research projects for modelorganism/technology development” was started in 2007. Six
projects have already been carried out, including those using
Cabombaceae or Pophyra as model organisms and thoserelated to methdologies in medaka genome analyses. NIBB
always encourages discussion on such projects in the belief
that our methods of conducting collaborative research
projects must be constantly modified, according to thedemands of the age and the biology community.

2006

2007 2008 2009

Priority collaborative research projects

3

1

0

1

Collaborative research projects formodel organisms/ technology development

−

2

3

2

Individual collaborative research projects

37

43

49

54

NIBB workshops

1

5

5

3

Collaborative experiments using the Large Spectrograph

18

14

11

10

Facility Use (Center for Analitical Instruments)
total

Contribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers

sources and sophisticated analysis equipment. To supplement
the currently operating fixed (partly tunable) wavelength
laser sources, we are preparing to introduce tunable laser
sources covering a wide range of wavelengths from UV to
IR.

International Cooperation■ Collaborative Programs with EMBL

The European Molecular Biology Laboratory (EMBL),
established in 1974, is a research institute funded by 18
European nations. It conducts comprehensive, high-levelbasic research programs and leads the world in the field of
molecular biology. NIBB takes a leading role in thecollaborative research programs between EMBL and the
National Institutes of Natural Sciences (NINS), which werelaunched in 2005, and promotes personal and technologicalexchange through symposia, exchange between researchersand graduate students, and the introduction of experimental
equipment (see page 85 for details of the EMBL meetings
held in 2009).

Meeting (left) and discussions (right) in EMBL, Heidelberg

Collaborative research projects by year
year

International
cooperation

0

1

0

0

59

66

68

71

■ Enhancement of the Large Spectrograph

The Large Spectrograph Laboratory, a world-leading
research facility in photobiology, has been instrumental in alarge number of collaborative research projects since its
establishment in 1980. To ensure further high-level
achievements, the Laboratory has undertaken an
enhancement of its laboratory equipment, including theaddition of advanced control systems, and the use of laser■ Collaborative Programs with MPIZNIBB formed an agreement with the Max Planck Institutefor Plant Breeding Research (MPIZ) in April 2009 to start a
new initiative aimed at stimulating academic and scholarly
exchange in the field of plant sciences. NIBB and MPIZ
work together to plan and promote joint research projects,
collaborative symposia, training courses and student
exchange programs. NIBB acts as a bridge between Japaneseand German researchers in the field of plant sciences. (seepage 87 for details of the joint meeting held in 2009).

■ NIBB Conference

The NIBB Conference is an international conference
organized by NIBB’s professors once or twice a year with theparticipation of guest lecturers from abroad. Since the first
conference in 1977 (the year of NIBB’s foundation), the
NIBB Conference has provided researchers in basic biology
with valuable opportunities for international exchange.
5

■ International Practical Course

With the cooperation of researchers from Japan and abroad
the NIBB international practical course, a practical training
program, is given at a laboratory specifically prepared for its
use at NIBB. The fourth course was held in 2009 (see page88). Graduate students and young researchers from various
areas including EU countries, Israel, India, and Singapore,
were provided with training in state-of-the-art researchtechniques.

sheet Microscope), As part of our collaborative workwith EMBL, NIBB has introduced a DSLM, which iseffective for the three-dimensional observation of living
samples, and is the first of its kind in Japan.
5) Bioimaging Symposium, This provides an opportunity
for academic exchange with cutting-edge overseas
researchers in the imaging field, mainly from EMBL.

■ Bio-Resources

The National BioResource Project (NBRP) is a national
project for the systematic accumulation, storage, and supplyof nationally recognized bio-resources (experimental animals
and plants, cells, DNA, and other genetic resources), which
are widely used as materials in life science research. To
promote this national project, NIBB has been appointed as acenter for research on “Medaka (Oryzia latipes)” whose
usefulness as a vertebrate model first developed in Japan.
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 and Zebrafish. In
addition, NIBB provides databases containing research dataon the moss Physcomitrella patens, Daphnia, Xenopus
laevis, plant cell organelles, and bacterial genomes.

Top: Medaka strain for which thefull genome sequence wasdetermined,
Middle: Transgenic Medaka with
red fluorescence,
Bottom: A strain with transparent
body.

Development of New Fields of Biology■ Bioimaging

Recently, the capability of optical microscopes has greatly
improved and biophotonic probes have been developed. The
combination of these technologies allows us to use living
samples and observe biological phenomena in real time,
which, in the past, could only be estimated based on
fragmentary information from fixed samples. NIBB aims tomaximize the application of these techniques for visualizing
biological phenomena (bioimaging) and to develop new
imaging techniques.
1) Imaging Science Laboratories, NIBB aims to be a centerfor the development of new microscopes andbiophotonic probes.
2) Advisory Committee on Bioimaging, Regular meetings
are held with several leading researchers in the
bioimaging field in Japan to formulate advice on
imaging research.
3) Bioimaging Forum, This provides an opportunity for
researchers at NIBB, members of the Advisory
Committee, and company engineers to frankly discuss
practical difficulties and needs regarding imaging.
4) Introduction of DSLMs (Digital Scanned laser Light

6

Optical path of the DSLM

■ Okazaki Biology Conferences

NIBB holds Okazaki Biology Conferences (OBC) that, withthe endorsement of the Union of Japanese Societies for
Biological Science, support the formation of international
communities in future biological research fields with the goalof identifying new research issues in biology. Dozens of toplevel researchers from Japan and abroad spend about one
week together in exhaustive discussions seeking strategies
for addressing future critical issues in biology. Past
Conferences have promoted the formation of international
researcher communities.

Cultivation of Future Researchers

NIBB sponsors two education programs for graduate
students.
1. Graduate University for Advanced Studies
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 anda three-year doctoral course for graduate students with amaster’s degree.
2. Graduate Student Training Program
Graduate students enrolled in other universities andinstitutions 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 life and receive financial support from NIBB
based on the research assistant (RA) system.

■ Personnel changes in 2009*

Newly assigned in NIBB
Date

Research Unit

Name

Position

SHIINA, Nobuyuki

Associate Professor

Laboratory of Neuronal Cell Biology

March 1

KAWAGUCHI, Masayoshi

Professor

Division of Symbiotic Systems

April 1KITADATE, Yu

Assistant Professor

Division of Germ Cell Biology

April 1MIYAGAWA, Shin-ichi

Assistant Professor

Division of Molecular
Environmental Endocrinology

June 1

SATO, Masanao

Assistant Professor

Division of Developmental Genetics

August 1

TAKEDA, Naoya

Assistant Professor

Division of Symbiotic Systems

October 1

Newly affiliated in other universitites and institutes
Name

New Affiliation

Position

Date

IIDA, Shigeru

Tokyo University of Agriculture andTechnology/ University of Shizuoka

Guest Professor

April 1OHSUMI, Yoshinori

Tokyo Institute of Technology

Professor

April 1

KATSU, Yoshinao

Hokkaido University

Associate Professor

April 1SUZUKI, Kuninori

Tokyo Institute of Technology

Assistant Professor

April 1NAKATOGAWA, Hitoshi

Tokyo Institute of Technology

Assistant Professor

April 1WATANABE, Hajime

Osaka University

Professor

June 1* Changes in professors, ascociate and assistant professors are shown.

■ Awardees in 2009

Name

PositionAward

OHSUMI, Yoshinori

Professor

The Asahi Prize

OKADA, Kiyotaka

Director-General

The Research Award of the Botanical Society of Japan

KUROKAWA, Hiromi

Graduate Student

DGD (Development, Growth & Differentiation) AwardTANAKA, Minoru et al.

Associate Professor

Awarded Publication: Kurokawa et al. DGD 48, 209-, 2006

SHINTANI, Takafumi

Assistant Professor

The JB (Journal of Biochemistry) Prize

NODA, Masaharu

Professor

Awarded Publication: Shintani and Noda JB 144, 259-, 2008

Note: On the unit member lists from P.8 to P.74 : all members who belonged to the unit during 2009 are listed irrespective of the length of the period they weremembers. Those appearing twice in the same list under different titles are those whose title was changed during 2009. The former title is indicated by anasterisk (*).

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
ARAI, Yuko
KAMIGAKI, Akane
SHIRAYA, Takeshi
OIKAWA, Kazusato*
OGASAWARA, Kimi
KANAI, Masatake
GOTO, Shino
NAKAI, Atsushi
CUI, Songkui
SHIBATA, Michitaro
NAKAMORI, Chihiro
YOSHINORI, Yumi
SUZUKI, Iku
FUKAZAWA, Mitsue
KATO, Kyoko
NISHINA, Momoko
SATO, Yori
ARAKI, Masami
TSUCHIYA, Chiaki
SAITO, Miyuki
UEDA, Chizuru
KUBOKI, Yuko

NISHIMURA, Mikio

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:

Technical Assistants:

Secretaries:

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 of Priority Areas on “Organelle
Differentiation as the Strategy for Environmental Adaptationin Plants” has just finished. The project has been
accumulating evidence of the mechanism of organelle
differentiation and has revealed the relationship between
organelle plasticities and integrated functions in plants.

I. Reversible transformation of plant 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

8cycle, 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 ofleaf 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). The overall results provide us with new insights intoplant peroxisomal functions.

Figure 1 Detection of interaction between PEX7 and PTS2-containing
proteins using BiFC. (a) Epidermal cells expressing nYFP-PEX7 andPTS2-cYFP. YFP fluorescent signal was colocalized (c) with peroxisomemarker tdTomato-PTS1 (b). (d) Epidermal cells expressing nYFP-PEX7
and PTS2(R 16G)-cYFP, which has inactive PTS2 by amino acidsubstitution from the 16th Arg to Gly. YFP signal was merged (f) withcytosolic marker, tdTomato (e).

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

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

PTS1- and PTS2-containing proteins, respectively (Figure 1).
PEX14 is a peroxisomal membrane docking protein thatc a p t u r e s t h e r e c e p t o r- c a rg o c o m p l e x . We a l s ocomprehensively investigated whether or not these predicted
PEX genes function in peroxisome biogenesis by generatingknock-down mutants that suppress PEX gene expression by
RNA-interference. 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.

and MEB2 to the ER network. These findings indicate that
NAI2 is a key factor that enables ER body formation.

Ⅲ. Identification of novel components
essential for peroxisome biogenesis

To better understand peroxisome biogenesis, we isolated anumber of Arabidopsis mutants having aberrant peroxisome
morphology (apm mutants) based on a different pattern of
GFP fluorescence from the parent plant, GFP-PTS1, in whichperoxisomes with normal sizes and numbers can bevisualized with GFP.
Of these apm mutants, APM1 gene (whose defect causes theelongation of peroxisomes and mitochondria) encodes
dynamin-related protein 3A (DRP3A), one member of the
dynamin family. In apm2 and apm4, the GFP fluorescence isobserved in the cytosol as well as in peroxisomes, showing
the defect of protein transport to peroxisomes. We
demonstrated that APM2 and APM4 encode proteinshomologous to PEX13 and PEX12, respectively, and that
APM2/PEX13 and APM4/PEX12 are components of theprotein-translocation machinery on peroxisomal membranes.
We are currently analyzing the functions of other APM
proteins. From these analyses, we will be able to identify thecomponents responsible for peroxisome biogenesis, and toaddress the mechanism at the molecular level.

IV. ER derived organelles for protein storingand defense strategy

Plant cells develop various types of endoplasmic reticulum(ER)-derived structures with specific functions. ER bodies
are ER-derived compartments observed in Arabidopsis. They
are rod-shaped structures surrounded by ribosomes. ER
bodies are widely distributed in the epidermal cells of wholeseedlings. 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.
Arabidopsis nai1 mutants have no ER bodies in the entire
plant and do not accumulate PYK10. NAI1 encodes atranscription factor that has a basic-helix-loop-helix (bHLH)
domain and regulates the expression of PYK10. Arabidopsis
nai2 mutant has no ER bodies and reduces the accumulationof PYK10. NAI2 encodes a unique protein that localizes tothe ER body. We found that the membrane protein of ER
body 1 (MEB1) and MEB2 are integral membrane proteinsof the ER body (Figure 2). NAI2 deficiency relocates MEB1

Figure 2. MEB1 and MEB2 localize to the ER body membrane.
Fluorescence images of cotyledon epidermal cells transiently expressing
tdTom-MEB1 fusion protein (left) or tdTom-MEB2 (right) in a 7-day-old
Arabidopsis transgenic plant that expresses ER-targeted GFP.

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
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 A. thaliana 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.

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.
Recently, we found that HSP90 inhibitor induced genes
with heat shock response element (HSE) motifs in their

9National Institute for Basic Biology

Cell Biology

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 absenceof heat shock, HSP90 actively suppresses HSF function.
During heat shock, HSP90 is transiently inactivated, whichleads to HSF activation. This data indicates that HSP90
regulates correct gene expression for heat acclimatization inplants. We also observed that HSP90 is involved in hormone
responses in Arabidopsis. The evolutional and functional
characterizations are now being investigated.

Ⅶ. The Plant Organelles Database 2(PODB2) – Release of version 2 for plantorganelles dynamics and methods forfunctional analysis
The Plant Organelles Database 2 (PODB2) was built topromote a comprehensive understanding of organelle
dynamics. This public database is open to all researchers.
PODB2 consists of 4 individual parts: the organelles movie
database, the organellome database, the functional analysis
database, and external links. The organelles movie database
contains videos of organelle movements and 3D structures(Figure 3). The organellome database provides images ofvarious plant organelles that were visualized with fluorescentand nonfluorescent probes in various tissues of several plant
species at different developmental stages. The functional
analysis database is a collection of protocols for plantorganelle research. The amount of included data is increasing
day by day. It is expected that PODB2 will contribute tosystems biology through the combination of the included
data with other ‘omics’ data and computational analyses. In
addition, we will release an updated version for educationaluse soon. We expect that PODB2 will be a useful tool to help
researchers gain greater knowledge of plant organelles, aswell as the general public who want to explore plant cellbiology.

Publication List

〔Original papers〕

K., Ueguchi-Tanaka, M., Kondo, M., Yano, K., Nishimura, M.,• Aya,and Matsuoka, M. (2009). Gibberellin modulates anther development in

•
•
•
•
•
•
•
•

rice via the transcriptional regulation of GAMYB. Plant Cell 21, 14531472.
Corpas, J.F., Hayashi, M., Mano, S., Nishimura, M., and Barroso, B.J.
(2009). Peroxisomes are required for in vivo nitric oxide (NO)accumulation in the cytosol following salinity stress of Arabidopsis
plants. Plant Physiol. 151, 2083-2094.
Fujimoto, M,. Arimura, S., Mano, S., Kondo, M., Saito, C., Ueda, T.,
Nakazono, M., Nakano, A., Nishimura, M., and Tsutsumi, N. (2009).
Arabidopsis dynamin-related proteins DRP3A and DRP3B arefunctionally redundant in mitochondrial fission but have distinct roles
in peroxisomal fission. Plant J. 58, 388-400.
Hatsugai, N., Iwasaki, S., Tamura, K., Kondo, M., Fuji, K., Ogasawara,
K., Nishimura, M., and Hara-Nishimura, I. (2009). A novel membranefusion-mediated plant immunity plant proteasome subunit mediates anovel defense strategy against bacterial pathogens. Genes Dev. 23,
2496-2506.
Kamigaki, A., Kondo, M., Mano, S., Hayashi, M., and Nishimura, M.
(2009). Suppression of peroxisome biogenesis factor 10 reduces
cuticular wax accumulation by disrupting the ER network in Arabidopsis
thaliana. Plant Cell Physiol. 50, 2034-2046.
Momonoi, K., Yoshida, K., Mano, S., Takahashi, H., Nakamori, C.,
Shoji, K., Nitta, A., and Nishimura, M. (2009). A vacuolar irontransporter in tulip, TgVit1, is responsible for blue coloration in petal
cells through iron accumulation. Plant J. 59, 437-447.
Nakano, R.T., Matsushima, R., Ueda, H., Tamura, K., Shimada, T., Li,
L., Hayashi, Y., Kondo, M., Nishimura, M., and Hara-Nishimura, I.
(2009). GNOM-LIKE1/ERMO1 and SEC24a/ERMO2 are required formaintenance of endoplasmic reticulum morphology in Arabidopsis
thalian. Plant Cell 21, 3672-3685.
Ogasawara, K., Yamada, K., Christeller, T.J., Kondo, M., Hatsugai, N.,
Hara-Nishimura, I., and Nishimura, M. (2009). Constitutive andinducible ER bodies of Arabidopsis thaliana accumulate distinct
b-glucosidases. Plant Cell Physiol. 50, 480-488.
Singh, T., Hayashi, M., Mano, S., Arai, Y., Goto, S., and Nishimura, M.
(2009). Molecular components required for the targeting of PEX7 toperoxisomes in Arabidopsis thaliana. Plant J. 60, 488-498.

〔Review articles〕

M., and Nishimura. M. (2009). Frontier of research on• Hayashi,organelle differentiation. Plant Cell Physiol. 50, 1995-1999.
S., Miwa, T., Nishikawa, S., Mimura, T., and Nishimura, M.
• Mano,(2009). Seeing is believing: On the use of image databases for visually
exploring plant organelle dynamics. Plant Cell Physiol. 50, 2000-2014.

K., Nagano, A.J., Ogasawara, K., Hara-Nishimura, I., and• Yamada,
Nishimura, M. (2009). The ER body, a new organelle in Arabidopsis
thaliana, requires NAI2 for its formation and accumulates specificb-glucosidases. Plant Signal. Behav. 4, 849-852.

Figure 3. The graphical user interfaces of ‘Organelles Movie Database’ in
PODB2 (http://podb.nibb.ac.jp/Orgenellome).

10

DIVISION OF MOLECULAR CELL BIOLOGY †Professor

OHSUMI, Yoshinori

Assistant Professors:
Technical Staff:
Postdoctoral Fellows:

Graduate Students:
Technical Assistants:

Secretary:

KAMADA, Yoshiaki
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
KABEYA, Yukiko
HANADA, Takao
FUJIKI, Yuki
HARASHIMA, Toshiaki
YAMAMOTO, Hayashi
OKAMOTO, Koji
OKAMOTO, Noriko
KAKUTA, Soichiro
KOBAYASHI, Takafumi
KAGEYAMA, Takuya
OH-OKA, Kyoko
KONDO, Chika
NIIMI, KaoriISHII, Junko
ICHIKAWA, Rie
SUZUKI, Yuko

Most cellular activity is maintained by the balance between
the synthesis and degradation of related proteins.
Degradation processes, therefore, play important roles inmany physiological aspects as well as in the regulation ofgene expression. Autophagy is a bulk degradation system forcytosolic proteins and organelles in lysosomes/vacuoles thatis highly conserved in eukaryotic cells. Our division aims tounderstand the physiological roles and molecularmechanisms of autophagy in yeast and higher eukaryotes.

fraction of mitochondria are exclusively sequestered andtransported to the vacuole, a lytic compartment, in post-log
phase cells under respiratory conditions (Figure 1). Using agenome-wide visual screen, we identified Atg32, a protein
essential for mitophagy in budding yeast. During respiratory
growth, Atg32 is highly expressed, likely in response tooxidative stress, and anchored on the surface of
mitochondria. We also demonstrate that Atg32 interacts with
Atg8 and Atg11, proteins critical for recognition of cargo
receptors. Notably, Atg32 contains WXXI/L/V, a conserved
motif that serves as a binding site for the Atg8 family
members. Atg32 exposes its N- and C-terminal domains tothe cytosol and mitochondrial intermembrane space,respectively. We propose that Atg32 is a transmembrane
receptor that directs autophagosome formation to
mitochondria.

Figure 1. Electron microscopy reveals electron-dense bodies containing
mitochondria accumulated in vacuoles under respiratory conditions.
Vacuolar protease-deficient cells were grown in non-fermentable medium
for 5 days. Notably, mitochondria are selectively incorporated into thevacuolar lumen in an Atg32-dependent manner.

I. BackgroundIn response to nutrient starvation, the autophagic process
starts as a building up of membrane structures called
autophagosomes in the cytoplasm. The autophagosome
sequesters a part of the cytosol and organelles nonselectively. It is then delivered to the vacuole/lysosome, andthe cytoplasmic materials inside are degraded by vacuolar/lysosomal hydrolases. We discovered autophagy in a simple
model organism, the budding yeast Saccharomyces
cerevisiae, and have morphologically and genetically defined
the whole process.

II. Mitochondria-specific autophagy requires
the transmembrane receptor Atg32

Autophagy-dependent degradation of mitochondria is afundamental process conserved from yeast to humans. In
contrast to starvation-induced, nonselective autophagy
responsible for nutrient recycling, selective autophagy, which
involves particular cues and receptors required for induction
and cargo recognition, respectively, mediates mitochondriaspecific breakdown. Although numerous studies highlightthat mitochondria-specific autophagy (mitophagy)
contributes to mitochondrial homeostasis, the molecularmechanisms underlying this selective clearance process arevery poorly understood. We establish that a substantial

III. A novel physiological role of autophagy inplants: plant autophagy puts the brakes oncell death by controlling salicylic acidsignaling

It has long been thought that autophagy in plants isimportant for nutrient recycling and plays a critical role in
the ability of plants to adapt to environmental variation suchas nutrient deprivation. Recent reverse genetic studies,
however, hint at other roles for autophagy, showing that
autophagy defects in higher plants result in early senescenceand excessive immunity-related programmed cell death(PCD), irrespective of nutrient conditions. Until now themechanisms by which cells die in the absence of autophagy
were unclear. In collaboration with RIKEN Plant Science
Center research groups, using biochemical, pharmacologicaland genetic approaches, we revealed a conserved
requirement for salicylic acid (SA) signaling for these
phenomena in autophagy-defective mutants (atg mutants).
The atg mutant phenotypes of accelerated PCD in senescenceand immunity were suppressed by inactivation of SA
signaling but not by inactivation of jasmonic acid (JA) or
ethylene (ET) signalings (Figure 2). Application of an SA
agonist restored the senescence/cell death phenotype in†: This laboratory was closed on 31 March, 2009.

11

National Institute for Basic Biology

Cell Biology

SA-deficient atg mutants but not in atg npr1 plants,suggesting that the cell death phenotypes in the atg mutants
are dependent on the SA signal transducer NPR1. We also
showed that the SA signal can induce autophagy in plants.
These findings demonstrate a novel physiological function
for plant autophagy that operates a negative feedback loop tomodulate SA signaling.

reactions catalyzed by Atg7 and Atg3, E1 and E2 enzymes,
respectively. Atg4 also serves as a deconjugation enzyme forAtg8; it cleaves the Atg8-PE conjugate and releases Atg8
from membranes. The crystal structure of LC3 (a mammalian
Atg8 homolog) complexed with Atg4B (a mammalian Atg4
homolog) has been determined. This provided insights into

Figure 2. Accelerated PCD phenotypes in autophagy-defective plants are
SA signaling dependent but do not require intact JA and ET signaling
pathways. The early senescence phenotype of atg5 mutant was
suppressed by reducing SA biosynthesis in sid2 mutant background, or byblocking SA signaling in npr1 mutant background. Shown arephotographs of five-week-old plants grown on rockwool supplied with arich nutrient solution under long-day conditions.

IV. Characterization of the Atg17-Atg29-Atg31
c o m p l e x s p e c i f i c a l l y re q u i re d f o r
starvation-induced autophagy in
Saccharomyces cerevisiae
In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, andAtg31/Cis1 are specifically required for autophagosome
formation by acting as a scaffold complex essential for preautophagosomal structure (PAS) organization. We show thatthese proteins constitutively form an Atg17-Atg29-Atg31
ternary complex, in which phosphorylated Atg31 is included(Figure 3). Reconstitution analysis of the ternary complex in
E. coli indicates that the three proteins are included inequimolar amounts in the complex. The molecular mass of amonomeric Atg17-Atg29-Atg31 complex is calculated at 97
kDa; however, analytical ultracentrifugation shows that themolecular mass of the ternary complex is 198 kDa,
suggesting a dimeric complex. We propose that this ternarycomplex acts as a functional unit for autophagosome
formation.

Figure 3. Atg17, Atg29, and Atg31 form a stable complex in vivo. Wildtype cells (BY4741) were grown and treated with rapamycin for 1 h.
After cells were converted to spheroplasts, cell lysates were prepared byosmotic lysis. Cytosolic fractions (100,000 x g supernatant, HSS) wereseparated by size exclusion chromatography on a Superdex 200 column.
Each fraction was analyzed by immunoblotting using anti-Atg1, antiAtg17, anti-Atg29, and anti-Atg31antibodies. Positions of molecularmass standards (in kDa) are shown. Open and closed circles indicate
dephosphorylated and phosphorylated Atg31, respectively.

V. S t r u c t u r a l s t u d i e s o f A t g p ro t e i nconjugation systems

Atg proteins contain two ubiquitin-like protein conjugation
systems. In collaboration with Dr. Inagaki’s group at
Hokkaido University, understanding of these systems has
proceeded from the viewpoint of structural biology. The
ubiquitin-like protein Atg8 is synthesized as a precursor form
with an arginine at the C terminus, which is immediately
removed by the cysteine protease Atg4 to expose a glycine
residue at the new C terminus. Then, this glycine of Atg8 is
conjugated to the lipid PE (phosphatidylethanolamine) via

12

Figure 4. Model of the Atg12-Atg5-Atg16 complex.

the mechanisms of the C-terminal processing anddelipidation of Atg8 by Atg4, especially for molecular
recognition and regulation in these cleavage reactions.
We previously showed that the conjugation reaction of Atg8
is accelerated by a conjugate formed by the other ubiquitinlike protein Atg12 and its target protein Atg5. The Atg12Atg5 conjugate further forms a complex with Atg16, whichis required for the localization of this complex to the PAS.
The crystal structure of Atg16 has been solved, which
revealed that Atg16 forms a parallel coiled-coil dimer.
Together with the previously-reported structures of Atg12
and Atg5, the entire architecture of the Atg12-Atg5-Atg16
complex was modeled (Figure 4). This provided an important
structural basis to further elucidate the function of thecomplex and the mechanism of autophagosome formation.

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

Y., Noda, N.N., Nakatogawa, H., Ohsumi, Y., and Inagaki, F.
• Fujioka,
The dimeric coiled-coil structure of Saccharomyces cerevisiae Atg16
and its functional significance in autophagy. J. Biol. Chem. 2009 Nov 4.

Y., Yoshino, K., Kondo, C., Kawamata, T., Oshiro, N.,• Kamada,
Yonezawa, K., and Ohsumi, Y. Tor directly controls the Atg1 kinase
complex to regulate autophagy. Mol. Cell Biol. 2009 Dec 7.

〔Review articles〕

H., Suzuki, K., Kamada, Y., and Ohsumi, Y. (2009).
• Nakatogawa,Dynamics and diversity in autophagy mechanisms: lessons from yeast.
Nat. Rev. Mol. Cell Biol., 10, 458-467.

N.N., Ohsumi, Y., and Inagaki, F. (2009). ATG systems from the• Noda,protein structural point of view. Chem. Rev., 109, 1587-1598.
K., Kondo-Okamoto, N., and Ohsumi, Y. (2009). A landmark• Okamoto,protein essential for mitophagy. Autophagy, 5, 1203-1206.

Publication List

〔Original papers〕

T., Satomi, Y., Takao, T., and Ohsumi, Y. (2009). The amino• Hanada,terminal region of Atg3 is essential for association with

•
•
•
•
•
•
•
•
•

phosphatidylethanolamine in Atg8 lipidation. FEBS Lett., 583, 10781083.
Kabeya, Y., Noda, N.N., Fujioka, Y., Suzuki, K., Inagaki, F., andOhsumi, Y. (2009). Characterization of the Atg17-Atg29-Atg31
complex specifically required for starvation-induced autophagy in
Saccharomyces cerevisiae. Biochem. Biophys. Res. Comm., 389, 612615.
Kageyama, T., Suzuki, K., and Ohsumi, Y. (2009). Lap3 is a selective
target of autophagy in yeast, Saccharomyces cerevisiae. Biochem.
Biophys. Res. Comm., 378, 551-557.
Okamoto, K., Kondo-Okamoto, N., and Ohsumi, Y. (2009).
Mitochondria-anchored receptor Atg32 mediates degradation of
mitochondria via selective autophagy. Dev. Cell, 17, 87-97.
Satoo, K., Noda, N.N., Kumeta, H., Fujioka, Y., Mizushima, N.,
Ohsumi, Y., and Inagaki, F. (2009). The structure of Atg4B-LC3
complex reveals the mechanism of LC3-processing and delipidation
during autophagy. EMBO J., 28, 1341-1350.
Sekito, T., Kawamata, T., Ichikawa, R., Suzuki, K., and Ohsumi, Y.
(2009). Atg17 recruits Atg9 to organize the pre-autophagosomal
structure. Genes Cells, 14, 525-538.
Shin, J.H., Yoshimoto, K., Ohsumi, Y., Jeon, J.S., and An, G. (2009).
OsATG10b, an autophagosome component, is needed for cell survival
against oxidative stresses in rice. Mol. Cells., 27, 67-74.
Wada, S., Ishida, H., Izumi, M., Yoshimoto, K., Ohsumi, Y., Mae, T.,
and Makino, A. (2009). Autophagy plays a role in chloroplastdegradation during senescence in individually darkened leaves. Plant
Physiol., 149, 885-893.
Watanabe, Y., Noda, N.N., Honbou, K., Suzuki, K., Sakai, Y., Ohsumi,
Y., and Inagaki, F. (2009). Crystallization of Saccharomyces cerevisiaealpha-mannosidase, a cargo protein of the Cvt pathway. Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 65, 571-573.
Yoshimoto, K., Jikumaru, Y., Kamiya, Y., Kusano, M., Consonni, C.,
Panstruga, R., Ohsumi, Y., and Shirasu, K. (2009). Autophagy
negatively regulates cell death by controlling NPR1-dependent salicylic
acid signaling during senescence and the innate immune response in
Arabidopsis. Plant Cell, 21, 2914-2927.

13

National Institute for Basic Biology

Cell Biology

LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Our laboratory was started in March 2009. Our maininterest is to understand the mechanisms and roles of mRNAtransport and local translation in neuronal dendrites using
mice.
The transport of specific mRNAs and local control oftranslation represent an important gene expression systemthat provides localized protein synthesis in dendrites at justthe right time and place. It is believed that this system
controls the location at which neurites will connect to eachother, thereby forming neural networks. Our laboratory is
researching factors regulating mRNA transport and local
translation, their target mRNAs, and the mechanisms of
localized protein synthesis in order to better understand its
relation to the formation of synapses and neural networks,
memory, learning, and behavior.

I. Identification and characterization ofcomponents of local translational machinery

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 2. Phase contrast images of primary cultured neurons from wildtype (WT) and RNG105 knockout (KO) fetal brains. RNG105 knockout
neurons formed poor networks compared to wild-type neurons. Scale bar,
100 mm.

We have also identified an RNG105 paralog, RNG140.
RNG105 and RNG140 were localized to distinct RNA
granules and showed different expression pattern andsubcellular localization. Particularly, RNG105 was highly
expressed in the brain of embryos, whereas RNG140 was
highly expressed in the brain of adults. These results suggestthat different kinds of RNA granules function in distinct
neuronal cell types at distinct developmental stages.

II. Mechanisms regulating local translation in
dendrites

RNA granules are densely packed structures andtranslationally dormant during transport, but they becomeless compact during conversion into translating polysomes
after synaptic stimulation. We will study the mechanisms
regulating the stimulation-dependent local translation by
analyzing translational regulation of RNG105-associatedmRNAs in neuronal dendrites.

III. Roles of local translation in higher orderbrain functions

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 have identified RNG105, an RNA-binding protein, as acomponent of RNA granules. RNG105 dissociates fromRNA granules after synaptic stimulation, which isaccompanied by the induction of mRNA translation near the
granules, suggesting its involvement in local translation.
Furthermore, we have generated RNG105 knockout miceand shown that RNG105 deficiency affects the formation andmaintenance of synapses and neuronal networks (Figure 2).
We are further studying the molecular mechanism underlying
the RNG105 knockout phenotype by identifying andcharacterizing RNG105-associated mRNAs and proteins.

14

RNG105 knockout neonates were born normally but diedsoon after birth because of respiratory failure, suggesting
defects in the brainstem functions of RNG105 knockout
mice. Because of the neonatal lethality, analyses of RNG105
roles have been limited to embryonic brains and neurons. In
order to examine the roles of RNG105 in memory, learning,and behavior in adult mice, we are generating conditional
RNG105 knockout mice. We will further generate knockout
mice for RNG140 and RNG105- and RNG140-associatedmRNAs and proteins to analyze their roles in higher orderbrain functions.

LABORATORY OF CELL STRUCTURE

Associate Professor

OGAWA, Kazuo

Numerous proteins localized in the cilia are linked tohuman diseases such as PCD (primary ciliary dyskinesia)
and PCKD (polycystic kidney disease). Primary cilia aredefined as single cilia that grow out of one of the centrioles
during interphase in otherwise unciliated animal cells. Theyshow a 9+0 pattern, losing a central pair of microtubules,
contrasting with motile cilia with a well-known 9+2 pattern.
These cilia can be seen in cultured cells such as 3T3, 3T6,
BHK21, NRK, and MDCK. They are quite common
centriolar specializations in vivo and in vitro. The incidenceof primary cilia within a cell culture is related to the degreeof confluency. Examination of confluent cell monolayers
showed that the primary cilia within a single preparation of agiven cell line varied considerably in length. In most cases,
cell lines previously used were not cloned and the results
remained obscure.
Four cell lines originating from adult mouse kidneys were
established in order to study the proteomics of the primarycilia. The cell lines were named nibb-K1, K4, K5, and K8.
The primary cilia of the cells were observed by indirect
immunofluorescence microscopy (Figure 1). In the cloned
cells, each cell has a distinct length of cilium, with the K5cell having the longest one among them (up to 10 mm).
This year, we studied the relationship between primary cilia
formation and one isotype of the dynein family using thesecells.

nibb-K4

nibb-K5

nibb-K8
nibb-K1

Figure 1. Typical primary cilia of four cell lines. Cells cultured on the
cover slips were reacted with anti-acetylated tubulin antibody, followed
by FITC-labeled secondary antibody. DNA was stained with DAPI.

15

National Institute for Basic Biology

Cell Biology

LABORATORY OF CELL SOCIOLOGY

Assistant Professorrole in vasculogenesis through being involved in the processof 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.

HAMADA, Yoshio

Technical Assistants:

GONDA, Naoko
TAKAYANAGI, Noriko

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 their 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 a

16

Fig.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
TAO, Hirotaka
SHINDO, Asako
KAI, Masatake
MORITA, Hitoshi
HARA, Yusuke
MIYAGI, Asuka
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
MIYAKE, Satoko
TSUGE, Toyoko

UENO, Naoto

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

KINOSHITA, Noriyuki

Figure 1. Intracellular Ca 2+ increase at the border of mesoderm andectoderm in the conjugate culture. During the culture, several islands oftransiently elevated Ca2+ detected by a calcium indicator G-CaMP4 wereobserved in the single microscopic field (A). The Ca2+ islands appeared to
be formed by a propagation of Ca2+ wave from fewer cells with higher
Ca2+ levels (A’).

The complex morphogenesis of organisms is achieved byconsecutive cell-to-cell interactions during development.
Recent studies suggest that growth factors play crucial roles
in controlling such intercellular communications in a varietyof organisms. In addition to secretory factors that trigger
intracellular signaling, transcription factors that act in the
nucleus to regulate gene expression are thought to be
essential for the determination of cell fates. Our main interest
is to know how pattern formation and morphogenesis during
development is regulated by these growth and transcription
factors. We address this problem using several model
animals, including frogs, mice and ascidians, and by
employing embryology, genetics, molecular and cellularbiology, and biochemistry.

We also study how PCP is established within the cells using
explants of Xenopus embryonic tissues and found thatheterogenous combination culture of tissues such as
mesoderm and ectoderm triggers the cell polarity, as revealedby the live-imaging analysis of microtubule growthorientation. We have been able to demonstrate that
intracellular Ca2+ is increased prior to the oriented growth of
microtubules (Figure 1), and the transient Ca2+ increase canbe triggered not only by tissue-tissue interactions, as wereported previously, but also by physical forces
experimentally applied (A. Shindo et al., PLos ONE, 5,
e8897, 2010). We propose that mechanical forces generated
by the tissue-tissue interactions are essential for the initiationof the cell polarity and are currently investigating to identify
molecules involved in the sensing of the physical forces andtransmission of the signal to the cells.

I. Establishment of cell polarity duringvertebrate embryogenesis

II. Protein ubiquitination system involved in
the Wnt/PCP pathway

Gastrulation is one of the most important processes during
the morphogenesis of early embryos, involving dynamic cell
migration and change in embryo shape. In spite of its
importance, the mechanism underlying the event has justbegun to be studied at the molecular level. During Xenopus
gastrulation, mesodermal cells migrate to the inside of theembryo and move on the blastocoel roof. One of theimportant mechanisms for this process is the cell movement
called “convergent extension (CE)”. As convergent extension
begins, cells are polarized and aligned mediolaterally,followed by the mutual intercalation of the cells that acquired
planar cell polarity (PCP). In the regulation of vertebrate
convergent extension, the Wnt/PCP pathway is implicated.
We have recently knocked out one of two prickle-related
genes, mpk1, in mice and found that the mpk1-/- mutants are
early embryonic lethal and die between E5.5 and E6.5. The
mutants show arrested development with failure of primitive
streak and mesoderm formation and failure of distal visceralendoderm migration. At the cellular level, the mpk1-/- epiblast
tissue is disorganized, with a clear defect in cell polarization.

Although Wnt/PCP signaling has been shown to play anessential role in the regulation of gastrulation movements,
the molecular mechanisms of how Wnt signals intracellularly
and how it regulates the tissue movements remain elusive.
We have shown that Wnt/PCP signaling activates the protein
ubiquitination/degradation system, which is essential for cell
motility during Xenopus gastrulation. In order to clarify how
the ubiquitination system is involved in the Wnt/PCP
pathway, we focused on b-TrCP (transducin-repeat
containing protein), a component of E3 ubiquitin ligase
complex. b-TrCP has been shown to regulate the canonicalWnt pathway by ubiquitinating b-catenin, but it is not known
whether b-TrCP is involved in the noncanonical Wnt/PCP
pathway. Our study revealed that b-TrCP plays an essential
role in this signaling pathway. Dishevelled (Dsh) is acytoplasmic protein, which plays pivotal roles in the
canonical and noncanonical Wnt pathways. When the Wnt/
PCP pathway is activated, Dsh is translocated to the plasma
membrane. b-TrCP colocalized with Dsh both before andafter the activation of Wnt/PCP signaling (Figure 2).
Furthermore, knockdown of b-TrCP by specific antisense
morpholino oligonucleotides inhibited gastrulation

17

National Institute for Basic Biology

Developmental Biology

movements and Dsh translocation to the plasma membrane.
This translocation is essential for Dsh to activate thedownstream signaling pathway, and thus, our results strongly
suggest that b-TrCP plays a key role in this signaling
pathway. We are currently investigating the regulatory
mechanisms of b-TrCP activity and its substrates to regulate
gastrulation movements in Xenopus embryo.

Figure 2. Colocalization of b-TrCP with Dishevelled (Dsh) in ectodermal
cells of Xenopus embryo. Upper panels; Dsh showed punctate localization(green) and colocalized with b-TrCP (red). Lower panels; When Wnt/PCP
signaling was activated, b-TrCP and Dsh were translocated to the plasma
membrane.

III. Cellular morphogenesis during neural tube
formation

Closure of the neural tube, like the morphogenesis of allepithelial tissues, fundamentally requires the change andmaintenance of cell shape. Neural tube closure requires atleast two coordinated morphogenetic events, cell elongation
and apical constriction. Although cytoskeletal elements are
responsible for such changes, how actin filaments andmicrotubules are regulated in this process in vivo isunknown.
We found that neural tube closure in Xenopus criticallydepended on two proteins, MID1, the responsible protein for
Opitz G/BBB syndrome (OS) in humans, and MID2. OS ischaracterized by midline malformations, including
hypertelorism, hypospadias, cleft lip/palate, and brain
abnormalities. Depletion of the Xenopus MIDs disrupted
epithelial morphology in the neural plate, leading to neuraltube defects. In the MID-depleted neural tube, the normal
apical accumulation of the cadherin/b-catenin complex in thecells and formation of the basal lamina were perturbed(Figure 3). Furthermore, the xMID knockdown destabilized
and disorganized apicobasally-polarized microtubules in theneural plate, and these abnormalities accounted for theabnormal epithelial cell morphologies and neural tube
closure. We also found that the xMIDs and their interacting
protein Mig12 were coordinately required for microtubule
stabilization during remodeling of the neural plate. Since thedepletion of the xMIDs also affected the optic vesicle,
cement gland, and kidney, all epithelial organs, we proposethat similar mechanisms underlie the normal morphogenesis

18

of epithelial tissues and organs where MIDs are expressed,
including the tissues affected in Opitz G/BBB syndrome
patients.

Figure 3. A, Transverse sections of the neural plate containing MIDdepleted cells (magenta), stained with antibodies against C-cadherin andb-catenin. B, Functional interaction of Mig12 and MIDs. Mildknockdown of Mig12 or MIDs by antisense Morpholino (Mo) causedslight delays in neural tube closure. Double-knockdown of Mig12 andMIDs (Double-Mo) induced severe neural tube defects.

We also investigated roles of an adhesion molecule nectin,
which belongs to the immunoglobulin-like cell adhesion
molecule in the neural tube formation of Xenopus. Depletionof nectin-2, one of the nectin family members, from early
embryos resulted in incomplete neural fold formation.
Cellular analyses revealed less accumulation of F-actin at theapical surface, causing an aberrant apical constriction, a cellshape change that is required for neural tube folding.
Furthermore, we found nectin-2 functionally cooperates with
N-cadherin to synergistically enhance apical constriction,highlighting the cooperative action between spatiotemporally
upregulated nectin-2 and N-cadherin in the neural plate.

IV. Brachyury-downstream gene sets in achordate, Ciona intestinalis

Brachyury plays a pivotal role in the notochord formation in
ascidian embryos. Ciona intestinalis Noto4 (Ci-Noto4) was
isolated as a gene downstream of Ci-Bra. This gene encodes
a 307 amino-acid protein with a C-terminal phosphotyrosineinteraction domain (PTB/PID). Expression of Ci-Noto4
commences at the neural plate stage and is specific tonotochord cells. Suppression of Ci-Noto4 levels withspecific antisense morpholino oligonucleotides resulted in
the formation of two rows of notochord cells owing to a lackof midline intercalation between the bilateral populations ofprogenitor cells.
In contrast, injection of a
Ci-Bra(promoter):Ci-Noto4-EGFP construct into fertilized
eggs disrupted the localization of notochord cells in theembryonic trunk (Figure 4). Ci-Noto4 overexpression did
not affect cellular differentiation in the notochord, muscle,mesenchyme, or nervous system. Analysis of Ci-Noto4
regions that are responsible for its function suggested
significant roles for the PTB/PID and a central region, an
area with no obvious sequence similarity to other known
proteins. Genes involved in planar cell polarity, such as
Ci-prickle, did not show similar functional effects whencompared with Ci-Noto4. These results suggested that PTB/PID-containing Ci-Noto4 is essential for midline
intercalation of notochord cells in chordate embryos.

Figure 4. Effects of Ci-prickle and Ci-Noto4 overexpression. (A-A’’) A
Ci-Bra(promoter):Ci-Prickle-EGFP construct was injected into fertilized
eggs. Ci-Prickle-EGFP overexpression did not cause the notochord cells
to disperse in the trunk region as was observed in Ci-Noto4
overexpressing embryos. (A-A’’, B-B’’) Magenta marks
Ci-Bra(promoter):Ci-Prickle-EGFP expressing or Ci-Bra(promoter):CiNoto4-EGFP expressing notochord cells with anti-GFP antibody. Green
marks endogenous Ci-fibrn protein localization with anti-Ci-fibrn
antibody. Scale bars = 50μm

Publication List

〔Original papers〕

T., Takagi, C., and Ueno, N. (2009). Xenopus Rnd1 and Rnd3
• Goda,
GTP-binding proteins are expressed under the control of segmentation
clock and required for somite formation. Dev. Dyn. 238, 2867-2876.

N., Awais, M., Takeuchi, M., Ueno, N., Tashiro, M., Takagi, C.,• Hida,Singh, T., Hayashi, M., Ohmiya, Y., and Ozawa, T. (2009). High-

•
•
•
•

sensitivity real-time imaging of dual protein-protein interactions inliving subjects using multicolor luciferases. PLoS ONE 4, e5868.
Sugiura, T., Tazaki, A., Ueno, N., Watanabe, K., and Mochii, M. (2009).
Xenopus Wnt-5a induces an ectopic larval tail at injured site, suggestinga crucial role for noncanonical Wnt signal in tail regeneration. Mech.
Dev. 126, 56-67.
Tao, H., Suzuki, M., Kiyonari, H., Abe, T., Sasaoka, T., and Ueno, N.
(2009). Mouse prickle1, the homolog of a PCP gene, is essential for
epiblast apical-basal polarity. Proc. Natl. Acad. Sci. USA 106, 1442614431.
Yakushiji, N., Suzuki, M., Satoh, A., Ide, H., and Tamura, K. (2009).
Effects of activation of Hedgehog signaling on patterning, growth anddifferentiation in Xenopus froglet limb regeneration. Dev. Dyn. 238,
1887-1896.
Yamada, S., Hotta, K., Yamamoto, T.S., Ueno, N., Satoh, N., andTakahashi, H. (2009). Interaction of notochord-derived fibrinogen-likeprotein with Notch regulates the patterning of the central nervous
system of Ciona intestinalis embryos. Dev. Biol. 328, 1-12.

19

National Institute for Basic Biology

Developmental Biology

DIVISION OF DEVELOPMENTAL GENETICS

Professor

KOBAYASHI, Satoru

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

Graduate Students:
Technical Assistants:

Secretary:

HAYASHI, Yoshiki
SATO, Masanao
NODA, Chiyo
HASHIYAMA, Kazuya
SHIGENOBU, Shuji
KAGEYAMA, Yuji
KUMATA, Yuji
HASHIMOTO, Yoshiko
HAYASHI, Makoto
FUJISAWA, Chiemi
KITADATE, Yu
KONDO, Takefumi
MAEZAWA, Takanobu
KUBO Satoru
SATO, Kaori
ISHIHARA Hitomi
NIIMI Kaori
OKUDAIRA Yuichi
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 formation and function in Drosophila.

mRNAs. Recently, Gerber et al. have reported genome-wide
identification of 165 Pum-binding RNAs. Based on this data,
we started a systematic screen to identify target mRNAs for
Nos/Pum-dependent translational regulation in pole cells. We
expressed hybrid mRNAs containing GFP-coding region and
3’-UTR sequence from the Pum-binding RNAs, and thenexamined GFP expression in the pole cells with or withoutmaternal Nos activity. Among twenty hybrid mRNAs, six
were translationally repressed by Nos. In addition, we foundthat translation of two mRNAs were up-regulated by Nos.
We are now examining the roles of these mRNAs in pole cell
development.

II. Mechanism regulating sex determination ofpole cells

Germ cells must develop along distinct male or female
paths to produce eggs or sperm. It has been reported thatgermline sexual identity is regulated by a masculinizing
signal from the somatic gonadal cells to pole cells within theembryonic gonads. However, we have found that reductionof sumoylation causes apoptosis of the migrating pole cells
in a female-specific manner, suggesting that sexual identity
has already been established in the pole cells prior to gonad
formation.
Here we show that Sex lethal (Sxl) acts autonomously in the
germline to induce female development in Drosophila. Sxl istransiently expressed in the germline progenitors, or polecells, during their migration to the gonads. Its expression isdetected in a female-specific manner and is necessary forfeminization of pole cells before they form the gonads.
Furthermore, ectopic expression of Sxl in male (XY) polecells is sufficient to induce female fate in these cells, and theresulting pole cells are able to produce functional eggs within
female (XX) soma.

I. Genome-wide search for RNAs of whichtranslation is regulated by Nanos in the
germline of Drosophila embryos

Several components of germ plasm have been identified in
Drosophila. One of these components is maternal nanos(nos) RNA, which is enriched in germ plasm during
oogenesis and translated in situ to produce Nos protein after
fertilization. Nos is inherited by primordial germ cells orpole cells at the blastoderm stage and is detectable in thesecells throughout embryogenesis.
Nos acts as a translational regulator for specific RNAs in
the pole cells. Maternal Nos represses apoptosis and mitosisof pole cells by suppressing translation of cyclin-B and head
involution defective RNA, respectively. Moreover, Nos isrequired for the repression of somatic cell fate in the polecells and for the germline development within the gonads,
presumably via regulating unidentified RNAs. Thus, westarted a genome-wide identification of RNAs of whichtranslation is regulated by Nos in pole cells. Nos is known tofunction together with the Pumilio (Pum) protein, which
directly binds to distinct sequence in 3’-UTR of the target

20

Figure 1. A model for the regulation of sexual dimorphism in pole cells.

The germline sexual identity is regulated both by sex of thesurrounding soma and by a cell-autonomous cue. Ourfindings provide strong evidence that Sxl has a pivotal role in
the germline-autonomous mechanism regulating sex
determination. XX pole cells initiate female sexual identity
based on their Sxl expression, while, lacking Sxl expression
in XY pole cells, male sexual fate occurs primarily by asignal from gonadal soma (Figure 1). One remarkableexample of germline-autonomous regulation of sexual
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, and somatic control may have evolved
with the emergence of mesodermal tissues, including gonadal
soma.

decrease in the number of pole cells causes ectopic hub
differentiation, which consequently increases their chance torecruit pole cells as GSCs. When ectopic hub differentiationis repressed, the decreased number of pole cells fail tobecome GSCs. Thus we propose that SGCs sense PGC
number by the signaling from PGCs to SGCs to modulate
niche size, and this serves as a mechanism securing GSCs
(Figure 3).

III. Mechanism regulating the formation of theniche cells in male embryonic gonads

The GSC niche in Drosophila testes has emerged as a usefulmodel system for studying stem cells. In the apical tip of theadult testes, the GSCs lie in intimate contact with somatic
hub cells, known collectively as the niche cells, which causes
the stem cells to retain self-renewing potential. GSCs divide
to produce one daughter cell that remains associated with the
hub cells, while the other daughter cell detaches and initiates
spermatogenesis.
Hub cells are derived from a subset of somatic gonadal cells(SGCs) that are located in the anterior region of maleembryonic gonads. However, it remains unclear how theproper niche size and location are regulated within thedeveloping gonads. We have demonstrated that a receptor
tyrosine kinase, Sevenless (Sev), represses hub developmentin the anterior region of male embryonic gonads. Sev isexpressed by SGCs within the posterior region of the gonads,
and is activated by a ligand, Bride of sevenless (Boss), whichis expressed by pole cells, to prevent ectopic hub
differentiation in the posterior SGCs.
We further found that Notch signaling induces hub
differentiation. Notch is activated in almost all of the SGCs
within male embryonic gonads, suggesting that the posterior
SGCs, as well as the anterior SGCs, have the capacity tocontribute to hub differentiation. Since hub differentiation isrestricted in the anterior SGCs, the posterior SGCs should be
repressed to become hub cells. Although Sev acts as arepressor for hub differentiation in the posterior SGCs,
expression of a constitutive-active form of Sev is unable toinhibit hub differentiation observed in the anterior SGCs,
suggesting that Sev does not have a sufficient ability torepress hub differentiation. Thus we speculate that another
RTK signaling pathway has a key role to restrict hub
differentiation in the anterior SGCs.
We showed that epidermal growth factor receptor (Egfr) is
activated in the posterior SGCs to repress hub differentiation.
In the absence of Egfr activity, ectopic niche differentiation
is evident in the posterior SGCs. Moreover, hubdifferentiation which is normally observed in the anterior
SGCs was repressed by expressing a constitutively active
form of Egfr throughout SGCs. These observations show that
Egfr is both required and sufficient to repress hub
differentiation.
Egfr is activated in the posterior SGCs by Spitz ligand
emanating from pole cells, while a ligand for Notch, Serrate,is expressed in SGCs (Figure 2). This implies that varying
the number of pole cells alters the niche size. Indeed, aFigure 2. Hub differentiation is controlled by negative regulators, Sev
and Egfr and a positive regulator, Notch.

Figure 3. Mechanism securing the germline stem cells. Decrease in PGC
number causes niche expansion, thereby recruiting a small number of
PGCs as GSCs.

IV. The role of heparan sulfate proteoglycan 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. Drosophila GSCs are one of the mostcharacterized among animal stem cells. Recent studies showthat several signaling pathways, such as TGF-beta and JAK/STAT are essential for maintenance of the GSC niche. GSCs
are surrounded by somatic gonadal cells, named as nichecells, which form the physical area of the niche and are21

National Institute for Basic Biology

Developmental Biology

responsible for signaling molecule secretion. Despite theimportance of these molecules, it is not well understood how
these secreted molecules can precisely define the region of
the GSC niche.
Heparan Sulfate Proteoglycans (HSPGs) are a group of
glycoproteins which are expressed on the cell surface and/orin the extracellular matrix. Recent in vivo studies haveshown that HSPGs play critical roles in regulating signaling
during development by a variety of mechanisms, including
controlling extracellular ligand distribution. For example,
one of the Drosophila glypicans, dally can control
distribution of the TGF-beta ligand, Dpp, and establish a Dppmorphogen gradient during wing development.
We recently identified the Drosophila glypicans, dally anddally-like as important components of the GSC niche in both
sexes. dally and dally-like were strongly expressed in female
and male GSC niche cells, respectively. Mutant animals forthese glypicans showed significant reduction of GSC number
as a result of failure of proper signal activation. Furthermore,
ectopic expression of dally in female gonads caused anincrease in GSC number. Based on these observations, weconclude that glypicans define GSC niche region by
regulating signaling pathways involved in GSC maintenance.
We present the model that glypicans have a role in defining
the GSC niche by controlling ligand stability or distribution
in the niche region.

Figure 4. dally is an essential component of the GSC niche. (A-C)
Anterior most region of ovariole (Germarium) from control (A), dally
mutant (B) and dally-ectopically-expressed animal (C). In control female,
two to three GSCs (arrows) exist at the anterior tip of the germarium (A).
Once dally function is lost, GSC number is decreased, and consequently
germarium lacking GSCs are frequently observed (B, bracket).
Conversely, germarium in which dally is ectopically expressed possess
more than ten GSCs (C, bracket). Green signal shows germline marker,
Vasa protein, and magenta signal shows membrane-skeletal protein, Hts.
GSCs are identified as spherical shaped Vasa positive cells with dot
shaped Hts signals.

V. Transcriptional regulation in Malaria
parasites (S. Shigenobu)

Gene expression in Plasmodium parasites undergoes
significant changes in each developmental stage, but thetranscription factors (TFs) regulating these changes have notbeen identified. We identified a Plasmodium TF (AP2-O)
that activates gene expression in ookinetes, the mosquitoinvasive form, and has a DNA-binding domain structurally
related to that of a plant TF, Apetala2 (AP2). The
Plasmodium TF activates a set of genes, including all genes
reported to be required for midgut invasion, by binding to
specific six-base sequences on the proximal promoter.

22

Publication List

〔Original papers〕

K., and Kobayashi, S. (2009). Expression of genes involved• Hashiyama,
in sumoylation in the Drosophila germline. Gene Expression Patterns 9,50-53.

Y., Kobayashi, S., and Nakato, H. (2009). Drosophila• Hayashi,
glypicans regulate the germline stem cell niche. J. Cell Biol. 187, 473-

•
•

480.
Maezawa, T., Arita, K., Shigenobu, S., and Kobayashi, S. (2009).
Expression of the apoptosis inducer gene head involution defective inprimordial germ cells of the Drosophila embryo requires eiger, p53 andloki function. Develop. Growth Differ. 51, 453-461.
Yuda, M., Iwanaga, S., Shigenobu, S., Mair, G., Janse, C., Waters, A.,
Kato, T., and Kaneko I. (2009). Identification of a transcription factor in
the mosquito-invasive stage of malaria parasites. Mol. Microbiol. 71,
1402-1414.

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

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

Graduate Students:

Technical Assistants:

Secretary:

OKUBO, Tadashi
UTSUMI, Hideko
NAKAYAMA, Kei
AKANUMA, Takashi
TAKADA, Ritsuko
CHEN, Qiuhong
YABE, Taijiro
NAKAYAMA, Kei*
TAKAHASHI, Jun
ISHITANI, Shizuka
TAKAHASHI, Hiroyuki
ASANO, Takuya
WANGLAR, Chimwar
TAKASHIRO, Kayoko
TOMIDA, SanaiOHHARA, KaoriTSUZUKI, Chisa
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.” Themost popular model to explain the patterning process is the“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 patterning processes. In spite of the accumulationof genetic evidence, however, the biochemical characteristics
of morphogens, including modification and higher order
structure, remain to be elucidated. Thus, one of our major
goals is to reveal the real image of morphogens and themolecular mechanism underlying the formation ofmorphogen gradients, including the secretion andextracellular transport of these morphogens.
The segmental sub-regions of the paraxial mesoderm (orsomites), by contrast, appear not to be simply directed by themorphogen gradient and threshold, but by a uniquemechanism proceeding periodically. Somites are sequentiallygenerated in an anterior-to-posterior order by converting
oscillatory gene expression into periodical structures. The
molecular mechanism underlying this conversion andmorphological segmentation, however, is not yet fully
understood. Thus, another goal of our current studies is toreveal the molecular mechanism of this other and uniquemode of patterning that underlies the periodical andsequential sub-division in the development of somites andpharyngeal arches.

I. Secretion and modification of Wnt proteins
The Wnt family of secreted signal proteins plays a key rolein numerous aspects of embryogenesis. Most Wnt proteinstransmit their signals locally, presumably since theirsecretion and transport are under tight control. One important
step regulating the extracellular transport of various secreted
signal proteins involves post-translational modification withlipid moieties. We found that murine Wnt-3a is modified
with a mono-unsaturated fatty acid, palmitoleic acid, at aconserved Ser residue (Figure 1). Wnt-3a defective in thismodification is not secreted from cells in culture or in
Xenopus embryos, but is retained in the endoplasmic
reticulum (ER). Furthermore, Porcupine, a protein withstructural similarities to membrane-boundO-acyltransferases, is required for this Ser-dependent
modification, as well as for Wnt-3a transport from the ER for
secretion. These results strongly suggest that Wnt protein
requires a particular lipid modification for proper
intracellular transport during the secretory process. We
expect that the discovery of this unexpected lipid
modification might provide a clue regarding the higher-order
structure of secreted Wnt proteins and their gradientformation.

Figure 1. Visualization of Wnt-3a proteinssecreted from Xenopus epidermal cells. A
GFP-tagged form of Wnt-3a protein, as
well as the authentic form, can be observed
as punctate signals around the edge ofcells. We can use the GFP tagged-Wnt
proteins for visualization of extracellulartrafficking of Wnt proteins.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

23

National Institute for Basic Biology

Developmental Biology

To better understand the molecular mechanism of secretionand gradient formation of Wnt proteins, we are carefully
examining the biochemical characteristics of Wnt proteinsfrom cultured cells. We are also trying to reveal the role ofpalmitoleoylation and the molecular mechanism underlying
the extracellular transport of Wnt proteins during
embryogenesis using zebrafish and frog embryos (Figure 1).

II. Characteristics of genes required for thedevelopment of somite or pharyngeal arches-----The function of mouse Ripply1 and 2 in
the rostro-caudal patterning within a somite----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, which is a processincluding inter-somitic transition, a segmental pre-pattern,characterized by segmental gene expression, is established in
the anterior PSM. The establishment of the segmental prepattern in the anterior PSM has been revealed to require anumber of processes regulated by many transcription factors
and signaling molecules.
The spatial pattern of somites is characterized not only by
the periodical borders between neighboring somites, but also
by the rostral and caudal compartments within a somite.
These compartments are subsequently segregated andre-fused with adjacent compartments to form vertebra. In
addition, the rostro-caudal pattern defines the migration ofneural crest cells and motor axons. In contrast to the many
patterning processes that have already been revealed, therostro-caudal patterning of a somite is unique in that a spatial
pattern is established with temporal periodicity. However, theprecise molecular mechanism by which the rostro-caudal

pattern is established remains unclear.
We showed that a gene identified by our in situ
hybridization screening, ripply1 is required for thistransition. Ripply proteins suppress Tbx-mediatedtranscription by recruiting the Groucho/TLE co-repressor.
Rippy1 and 2 are expressed in the anterior PSM and inseveral newly formed somites in zebrafish and mouseembryos (Figure 2). In ripply1-deficient zebrafish embryos
somite boundaries do not form, the characteristic gene
expression in the PSM is not properly terminated, and theinitially established rostro-caudal patterning in the segmental
unit is not maintained, whereas paraxial mesoderm cells
become differentiated. Thus, ripply1 plays a role in themaintenance of the rostro-caudal patterning.
In addition, Notch signaling and Mesp2 activity are also
required for the rostro-caudal patterning of a somite. For
instance, mouse embryos defective in Notch signaling,caused by knocking out Dll1 or Presenilin1, show rostralized
somites, whereas those lacking Mesp2 activity exhibit
caudalization of their somites. Consistent with their roles inrostro-caudal patterning, the Notch and Mesp2 active
domains become contracted in the caudal half of S0 (theprospective somite in the most anterior PSM) and in the
rostral half of S-1 (the prospective somite posterior to S0),
respectively. Interestingly, Mesp2 is required for Ripply2
expression, indicating that Mesp2 suppresses its own
expression by activating Ripply2 expression. Thus, rostrocaudal patterning appears to be established throughmolecular interactions between these molecules.
Given that Mesp2 expression is dynamically changed in theanterior PSM in association with the traveling wave of Notch
activity, characterization of the dynamism of Mesp2
expression and Notch activity would be important forunderstanding the mechanism of rostro-caudal patterning.
Furthermore, the effect of Ripply on the dynamic movementof Mesp2 expression and Notch activity would be revealed.
Therefore, we examined the dynamic processes of thispatterning by exhaustive examination of periodical changes
in the location of the Notch active domain and the Mesp2
protein domain in wild-type and Ripply-deficient mouseembryos at several distinct phases of the segmentation cycle.
We examined the spatial movement of Notch activity with
Mesp2 protein localization in wild-type embryos and those

Figure 2. Expression patterns of mouse Ripply1 and 2
in somite development. Both Ripply1 and 2 aresegmentally expressed in the anterior presomitic
mesoderm.

24Figure 3 Segmental abnormalities observed in Ripply1 and 2 double knockout embryos. Segmental defects of somites appear in structures of vertebrae andribs. Ripply1 deficiency enhances rostralized phenotype of Ripply2 knock-out embryos in somite development. .

defective in the two Ripply genes expressed in the PSM.
Mesp2 protein appears first as a thin band in the traveling
Notch domain. In wild-type embryos, the Mesp2 band
expands anteriorly to the expression front of Tbx6, anactivator of Mesp2 transcription. Notch activity becomes
localized further anteriorly to this Mesp2 domain, but does
not pass over the anterior Mesp2 domain generated in theprevious segmentation cycle. In Ripply1/2-deficient embryos,
the Mesp2 band becomes more expanded and the Notch
domain is finally diminished. Interestingly, Ripply1/2deficient embryos exhibit anterior expansion of Tbx6 protein
domain, suggesting that Ripply1/2 regulates Mesp2
expression through Tbx6 degradation. We propose that the
rostro-caudal pattern is established by dynamic interaction of
Notch activity with two Mesp2 domains, which are definedin successive segmentation cycles by Notch, Tbx6 andRipply1/2.
In addition to these roles of Ripply1 and 2 in somite
segmentation, we have also examined the role of another
Ripply gene in the development of pharyngeal arches.

Publication List

〔Original paper〕

D., Takada, S., Agalliu, I., McMahon, A.P., and Jessell, T.M.
• Agalliu,(2009). Motor neurons with axial muscle projections specified by
Wnt4/5 signaling. Neuron 61, 708-720.

25

National Institute for Basic Biology

Developmental Biology

DIVISION OF EMBRYOLOGY

Professor

FUJIMORI, Toshihiko

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

TOYOOKA, Yayoi
OKA, Sanae
KOMATSU, Kouji
SEKI, Touhaku
HIRAO, Mayumi
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.
A complete understanding of early events during
embryogenesis in mammals, as compared to other animals,
has been relatively delayed. This is mainly due to difficulties
in the approaches to the developing embryos in the uterus of
the mother. Another characteristic of mammalian embryos istheir highly regulative potential. The pattern of cell division
and allocation of cells within an embryo during the earlystages vary between embryos. The timing of the earliest
specification events that control the future body axes is still
under investigation. Functional proteins or other cellularc o m p o n e n t s h a v e n o t b ee n fo u n d th a t l oc 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.

Ⅰ. Cell differentiation in pre-implantation
development

During the 4 days after fertilization, the mouse embryo
proceeds in its development within the zona-pellucida, an
extra-embryonic membrane surrounding the embryo proper,
and reaches the blastocyst stage. In the mouse blastocyst, twotypes of cells, namely cells of the inner cell mass (ICM) andcells of the trophectoderm (TE) can be distinguished. This is
the initial cell differentiation during mouse development.
Oct4 (Oct3/4) and Cdx2 are differentially expressed in the
ICM and TE respectively, and it is suggested that Oct4functions as a factor to maintain pluripotency of ICM cells.
The onset of the specification of these two cell types has notbeen defined. The initial stage when Oct4 and Cdx2 aredifferentially expressed is known to be early in the morula
stage. The cells located at the outside express Cdx2,
suggesting that specification has occurred in these outside
cells. It is hypothesized that the cells located inside themorula mainly contribute to the ICM and cells facing theembryonic surface will differentiate into TE cells. However,this inside-outside hypothesis is based on the analysis ofseparated blastomeres, and continuous information fromwhole embryos was very limited. We therefore analyzed
division orders of cells and expression profiles ofdifferentiation markers relating to the position of cells withinembryos to reveal the different characteristics of each
blastomere in the embryo.

26

Figure 1. Analysis of division patterns until the 8-cell stage. Blastomere
A divided earlier than blastomere B to give rise to the 3-cell stage. The
four blastomeres aa, ab, ba, bb divided to reach 5, 6, 7, and 8-cell stages,
respectively.

The dividing patterns of blastomeres until the late 8-cellstage were analyzed using bright field time-lapse recording
images. The time point of each blastomere division wasrecorded, and the division patterns were analyzed. All six
possible types of division pattern were observed. Of allcases, the type where all the daughter cells of the early
dividing cell precede in the next pattern was most common.
When the period of the 3-cell stage was examined, there wascorrelation with the appearance of different patterns and thelength of the stage, which showed a wide variety lasting up
to 220min. The most common pattern was seen in embryoswith a longer 3-cell stage period, and variant patterns
occurred more often during short 3-cell stage periods. Thissuggests that the division order might be altered when thedivisions of blastomeres occurs synchronously. We did notobserve any relationship between the division order andfuture cell fates.
We analyzed the positioning of cells within each embryousing optical section images of phalloidin and Hoechst
staining obtained by confocal microscopy. We defined anouter cell as a cell facing the embryonic surface of the

Figure 2. Localization of Oct 4 and Cdx2 protein during preimplantation
mouse development. Cell shape was visualized by phalloidin staining.

embryo and an inner cell as a cell not facing the surface ofthe embryo but surrounded by other cells. At the 8-cell stage,even after compaction, almost all the cells were outer cells.
At the 16-cell stage, inner cells were observed. The average
number of inner cells was 1.7. All the embryos contained
inner cells at the 32-cell stage. Thus, the geographicaldifferences between the inner and outer cells can be initiatedlater than the 8-cell stage. The Oct4 proteins staining signals
were observed in all embryos both in inner and outer cells
until the 32-cell stage. The Cdx2 staining signals weredifferent from that of Oct4. Cdx2 was initially observed in a
few cells of the 8-cell stage, and the numbers of Cdx2positive cells increased as development progressed. Cdx2
signals were observed in most of the outer cells at the 32-cellstage. Thus Cdx2 and Oct4 were co-expressed in most outercells at the 32-cell stage. These results suggest that thedifferentiation of the two cell types might correlate with theposition of the cells within the embryo.
We have started to prepare transgenic mice where
fluorescent protein expressions are driven by the gene
regulatory elements of genes involved in cell differentiation.
These will be used for live imaging of gene expression
during cell differentiation.

Figure 4. Observation of movements of the multiple cilia of the oviductepithelial cells. The numbers shown in bottom indicate frame numbers ofthe movie. The shape of a cilium is traced on the snap shot images taken
by high speed CCD camera. The movements of the cilium are represented
on the bottom.

carefully analyzed the movements of cilium. There was awide variety of frequency in the beating of cilia. These cilia
did not necessarily makea coordinated synchronous beating.
The effective stroke (forward movement along the ovaryuterus axis) of cilia is 2-3 folds faster than the recovery
stroke (backward movement). This differential speed of eachstroke might provide a force to generate the functional flow.
We are studying how these ciliated cells generate coordinatedand functional flow in the oviduct. To analyze the beating in
more detail, we are planning to develop a tool toautomatically analyze cilia beat frequency at multiple points
simultaneously. We are also preparing experiments touncover molecular mechanisms underlying PCP formation in
the oviducts, mainly by focusing on the involvement ofclassical PCP genes and their products.

Publication List

〔Original papers〕

T., Kurotaki, Y., Komatsu, K., and Nabeshima, Y. (2009).
• Fujimori,Morphological organization of the mouse preimplantation embryo.

Figure 3. Localization of Nanog protein at mid blastocyst stage. This
protein is one of the candidates applied for live cell imaging.

Ⅱ. Planar cell polarity in mouse oviductReprod Sci. 16, 171-177.

S., Fujimura, S., Nimura, K., Takeda, N., Toyooka, Y., Fujimura,• Yuri,Y., Aburatani, H., Ura, K., Koseki, H., Niwa, H., Nishinakamura, R.
(2009). Sall4 is essential for stabilization, but not for pluripotency, ofembryonic stem cells by repressing aberrant trophectoderm gene
expression. Stem Cells 27, 796-805.

Planar Cell Polarity (PCP) is the asymmetric organization
within the epithelial cells along the plane of the epithelium intissues. We are focusing on the epithelial cells of the mouse
oviduct. The multiple cilia on the cell surface beat back andforth along the axis from the ovary to the uterus, and thisbeating must be directionally controlled to transport eggs to
the uterus. Thus, these ciliated cells may have clear PCP
from ovary to uterus. Our aim is to reveal the molecular andcellular mechanisms of regulating PCP in the mouse oviduct.
We observed the movement of oviduct epithelial cilia by
high-speed CCD camera, and confirmed that the cilia
generally beat along the axis from ovary to uterus. To
understand how these movements of cilia can make a flow of
fluid to transport follicles from the ovary to the uterus, we27National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

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

KITADATE, Yu
MIZUGUCHI-TAKASE,
Hiroko
HARA, Kenshiro
YOSHIDA, Naoko
ICHIKAWA, Rie
INADA, Kana
SUGIMOTO, Ryo
KUBOKI, Yuko

Mammalian spermatogenesis represents a highly potent androbust stem cell system. Decades of research, including
detailed 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
mammalian spermatogenic stem cell system, mainly using
mice. In 2009, our second year at NIBB, we have tackled anumber of important issues in mouse spermatogenic stem
cells.

evaluations.
Similarly, corollaries of this model are that all the As cells
are functionally equivalent and uniformly act as the stem
cells, and that this defined population of ‘stem cells’ plays anactive role in every aspect of the stem cell functions, i.e.,maintenance of homeostasis, post-transplantation colony
formation, and post-injury tissue regeneration.
We have previously shown that no single stem cell
population acts in every aspect of stem cell function: Cells
that support the steady-state spermatogenesis are different
from those that support colony-formation and/or regeneration(Nakagawa et al., Dev. Cell, 2007). Based on this finding, we
propose the functional hierarchy in the spermatogenic stem
cell compartments as shown in Figure. 1. We propose that inaddition to the actually self-renewing population (actual stem
cells), an extended population of cells that retain the selfrenewing potential but are destined for differentiation(potential stem cells) exist, which plays an important role forthe continuity and robustness of the stem cell system. In caseof actual stem cell loss, potential stem cells might revert tothe self-renewing mode and replenish the actual stem cellpool (Figure 1).

I. Background: The mammalian spermatogenesis

Mammalian testes continually produce numerous sperm
during the reproduction period. Lines of investigations thatemerged in the 1950s and involve detailed morphologicalanalyses established the backbone of mammalianspermatogenesis research. The morphologically mostprimitive spermatogonia in the adult mouse testis are As or
Asingle spermatogonia (single, isolated spermatogonia). Theirprogeny remain interconnected due to incomplete
cytokinesis, forming syncytial cysts of 2n cells (2, 4, 8, 16etc.). It has been experimentally established that“undifferentiated spermatogonia” (or “Aundiff” hereafter),which contribute <1% of the entire testicular cells andconsist of As, Apr (Apaired; interconnected two-cell cysts), and
Aal (Aaligned; cysts of 4, 8, 16 or occasionally 32 cells) containstem cells.
Then, which subfraction of Aundiff consists of the actually
self-renewing stem cell compartment in homeostasis andhow do they behave (proliferate, self-renew or die) in thetestis? The prevailing, so-called “As model”, which was
originally proposed in 1971, proposes that As is the only cell
type that can act as stem cells, while the interconnected
population of A undiff (A pr and A al) is devoid of stem cell
capacity.

II. To w a r d t h e n a t u re o f t h e m o u s espermatogenic stem cell system that
includes reversibility

Despite that the “A s model” is comprehensive andpersuasive, this model is not based on the direct analyses of
the cells’ behavior. Thus, the As model warrants functional

28

Figure 1. Proposed model of the functional compartments in mouse
spermatogenesis (Nakagawa et al., 2007).

In 2009, by revisiting the As model, we investigated the cell
population of Aundiff. For this attempt, we introduced a gene
expression profile for describing the heterogeneity of thecells, in addition to the number of chained cells or the lengthof the cysts, on which the “As model” entirely depended on.
Several studies, including ours, have been establishing thatthe populations of cysts that compose the same number ofspermatogonia, which are classically considered to behomogeneous, are indeed heterogeneous in terms of patternsof gene expression. Therefore, a new scope has been raisedthat the entire Aundiff population is more heterogeneous thanhas previously been considered.
We have also investigated the behaviors of these A undiff
subpopulations in homeostasis (steady-state
spermatogenesis) and during regeneration after tissue insult.
We revealed that a particular subset of Aundiff changes their
behavior dynamically between homeostasis and regeneration:
In steady-state, they differentiate without self-renewal, but inregeneration, they are willing to get back into a selfrenewing stem cell state. This finding provides a novel viewfor stem cell biology and explains why the stem cells canchange the behavior between homeostasis and regeneration
so smoothly. In homeostasis, the number of stem cells should
be constant, while in regeneration, they increases to recover
the stem cell pool quickly. This feature is crucial for thecontinuity of tissue functions.

III. Investigating the temporal control ofdifferentiation and establishment of thetesticular tissue

In seminiferous tubules, spermatogenic differentiation
occurs in a periodical manner, which results in the formationof beautiful stratification of different steps of differentiating
cells within the seminiferous tubules (Figure 2A). Essentially,
this is caused by the periodical differentiation of Aundiff with aregular interval of 8.6 days (white arrows in Figure 2B).
We have investigated how this periodical event is controlled.
This question can be generalized to how the timing of Aundiff
differentiation is regulated. We paid special attention to theinvolvement of Sertoli cells and the role of retinoic acid (RA)signaling. The importance of RA has been recognized for
decades based on the finding that deficiency in vitamin A(VA), the dietary precursor of RA, causes a severe
spermatogenesis defect in which differentiation of Aundiff isaffected, and differentiating cell types are all lost.
We have revisited this VA-deficient (VAD) model andrevealed the central role of RA not only in the control of thetiming of Aundiff differentiation, but also in the regulation of thefunctional change of the supporting somatic Sertoli cells,
which are huge cells that nourish the stratified germ cells. The
local retinoid metabolism in the seminiferous tubules involves
multiple cell types of germ and somatic cells. Therefore, cooperation between different cell types that are locatedadjacently may control the differentiation of Aundiff.

IV. Investigating the nature of the stem cell
niche

Evidence suggests an intimate relationship between stem cells
and the niche microenvironment in seminiferous tubules. It isdifficult to identify the nature and function of the niche,
however, because seminiferous tubules do not exhibit
suspicious sub-structures. Moreover, actual stem cells can be
identified only functionally, and their histological detection
has not yet been achieved. Therefore, our current aim has been
to clarify the niche of Aundiff.

Figure 3. Localization of A undiff revealed by three-dimensional
reconstruction (Yoshida et al., Science, 2007)
Computationally reconstituted three-dimensional images of theseminiferous tubules based on 280 serial sections. Aundiff (green) show

biased localization to the blood vessel network (red) and the areaadjacent to the interstitium (yellow). (A, C) and (B, D), without orwith blood vessels, respectively. Roman numerals indicate the stageof the seminiferous epithelium.
Figure 2. Stratified tissue architecture in mouse seminiferous tubules.
(A) Appearance of a representative part of the seminiferous tubules. Notethe beautifully stratified germ cells at different steps ofdifferentiation.
(B) A scheme that explains the formation of the stratified architecture
shown in (A). The population of Aundiff (red) persists for a long period
and gives rise to A1 differentiating spermatogonia periodically with aregular interval of 8.6 days. As a result, a regular stratification of thedifferentiating germ cells is formed, as indicated by the rectangle.

The seminiferous tubules exhibit a convoluted tubular
structure with a diameter of ~200mm: Individual tubulesconnect to the common outlet of the mature sperm (rete testes)
with both ends and form loops. Spermatogenesis occurs
evenly throughout the inner surface of the tubules. Therefore,
in mouse testis, an overall ‘polarity’ that covers the entire
organ cannot be recognized, making a good contrast to the
Drosophila melanogaster germline stem cell system.

29

National Institute for Basic Biology

Developmental Biology

By taking advantages of live-imaging and three-dimensional
reconstruction, we previously revealed that Aundiff preferentiallylocalizes to the limited regions of seminiferous tubules thatare adjacent to blood vessels and interstitial cells that surroundthe tubules (Figure. 3). In addition, the dynamic migration of
spermatogonia from the vasculature proximity to spreadthroughout the tubules was also observed upon differentiationof Aundiff (Yoshida et al., Science, 2007). Based on thesefindings we proposed that this area may act as the niche for
Aundiff (Figure 4). We are tackling the cellular and molecular
identification of this vasculature-associated niche.

Figure 4. A proposed niche microenvironment for Aundiff.
Although the seminiferous tubule is a repeat of the same structure, theproximity to the surrounding blood vessels and interstitial cells
specializes the particular region of the basal compartment of thetubules, so that it can act as the niche for undifferentiated Aundiff
population.

Publication List

〔Original papers〕

K., Kanai-Azuma, M., Uemura, M., Shitara, H., Taya, C.,• Hara,Yonekawa, H., Kawakami, H., Tsunekawa, N., Kurohmaru, M., and•Kanai, Y. (2009). Evidence for crucial role of hindgut expansion in
directing proper migration of primordial germ cells in mouse early
embryogenesis. Dev. Biol. 330, 427-439.
Suzuki, H., Sada, A., Yoshida, S., and Saga, Y. (2009). The heterogeneityof spermatogonia is revealed by their topology and expression ofmarker proteins including the germ cell-specific proteins Nanos2 andNanos3. Dev. Biol. 336, 222-231.

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

M., Hara, K., Shitara, H., Ishii, R., Tsunekawa, N., Miura, Y.,• Uemura,
Kurohmaru, M., Taya, C., Yonekawa, H., Kanai-Azuma, M., and Kanai,

Y. (2009). Expression and function of mouse Sox17 gene in thespecification of gallbladder/bile-duct progenitors during early foregut
morphogenesis. Biochem. Biophys. Res. Commun. 2009 Nov. 12〔Review Articles〕

S. (2009). Spermatogenic stem cell system in the mouse testis.
• Yoshida,
Cold Spring Harb. Symp. Quant. Biol. 73, 25-32.
S. (2009). Casting back to stem cells. Nat. Cell Biol. 11, 118• Yoshida,
120.

30

DIVISION OF REPRODUCTIVE BIOLOGY

Professor (Specially appointed)

NAGAHAMA, Yoshitaka

Postdoctoral Fellows:

Technical Assistants:

Secretary:

GUAN, Guijun
PAUL, Bindhu
SAKAI, Fumie
SHIBATA, Yasushi
USAMI, Takeshi
ZHOU, Linyan
HORIGUCHI, Ryo
NAKAMOTO, Masatoshi
ASAO, Hisayo
ISHIKAWA, Hiroe
SAITOH, Hiromi
SHIBATA, Emiko
TAKAKI, Chikako
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/differentiation and gametogenesis invertebrates. Our research, which uses several types of teleostfish, focuses on (1) the identification of regulators andsteroidal mediators involved in sex determination, gonadal
sex differentiation, sexual plasticity, and gametogenesis(oocyte maturation and ovulation), and (2) the mechanismsof synthesis and action of these mediators.

I. Molecular mechanisms of sex determinationand gonadal sex differentiation

We identified DMY (DM-domain gene on the Y
chromosome) as the sex-determining gene of the medaka(Oryzias latipes), the first one in non-mammalianvertebrates. Recently, we have developed a simple, costeffective and gene-specific transgenic RNAi technology
for understanding the roles of the zygotic gene products
in medaka. Knockdown of DMY in XY gonads resulted
in a complete male-to-female sex-reversal in medaka(Figure 1). Importantly, we were able to continue a transgenerational knockdown effect of DMY until at least theF2 generation. Since the RNAi effect is long lasting andinheritable, this will provide a powerful tool for the
analysis of not only embryos, but also phenotypicconsequences that develop over longer periods of time.

Figure 1. Knockdown of DMY in XY gonads leads to a complete maleto-female sex reversal (C). Gonads from an XY male (A) and an XX
female (B). H&E staining was performed on gonads from 50 days after
hatching. Bars indicate 50µm.

A search for the target genes of DMY led to theidentification of gonadal soma derived factor (GSDF), a
member of the transforming growth factor-beta superfamily.
An XY-specific up-regulation was detected in the expression
levels of GSDF in the whole embryos of medaka at 6 days
post fertilization, coincident with the initiation of DMY
expression in XY gonads. Conversely, the expression of
GSDF was found to be very weak in XX gonads during
embryogenesis. Importantly, GSDF and DMY were found to
be co-localized in the same cell type in XY gonads. When
the XY embryos were treated with estradiol-17b, in order toreverse their phenotypic sex, a decline was observed in theexpression of GSDF. These results suggest that GSDF plays
an important role in testis differentiation in medaka, probably
down stream of DMY.
The molecular control of ovarian development in medaka isless understood. We examined whether R-Spondin 1 (RSPO1), a novel regulator of the Wnt/b-catenin signaling pathway,
was involved in ovarian differentiation in medaka. RSPO-1 isexpressed in XX gonads from as early as 0 day after hatching
to the adult stage, while the expression was barely detectedin XY gonads. Knockdown of RSPO-1 in XX gonads
induced female-to-male sex-reversal, while overexpressionof RSPO-1 in XY gonads induced male-to-female sexreversal. Both loss and gain of function evidence indicatesthat RSPO-1 is critical to initiate the ovary pathway inmedaka.
In the Nile tilapia (Oreochromis niloticus), we identified
that Cyp19a1/Foxl2 in XX gonads and GSDF/DMRT1 in XY
gonads during early gonadal differentiation are critical for
indifferent gonads to differentiate into either the ovary or
testis. The critical role of Foxl2 in ovarian differentiation was
confirmed by male sex reversal of XX transgenic tilapia
carrying a dominant-negative mutant of Foxl2. XX tilapia
carrying extra copies of tilapia DMRT1 as a transgene
induced various degrees of gonadal changes including
complete sex change to testis, indicating that DMRT1 plays
an important role in testicular differentiation.

II. Molecular mechanisms of sex change

The sex-changing fish Trimma okinawae can change its sex
back and forth from male to female and then back to maleserially, dependent on social status in the harem. The gonadcorresponding to the sexual status of the fish remains
functional while the other is regressed. The expression of
gonadotropin receptors (GtHR) was found to be confined tothe active gonad of the corresponding sexual phase. The
swapping of the gonads is initiated through a switching in theexpression of the GtHR, FSHR and LHR. Changing of the
gonads starts with switching of GtHR expression discernible
within 8-12 h of the visual cue. These two GtHR genes act as
mediators to convey the information about the change insocial status to the to-be-active gonad.

III. Sexual plasticity in the adult gonochoristicfishWith the exception of certain hermaphroditic species, mostvertebrates are thought to lose sexual plasticity after thedifferentiation of separate gonads/sexes with a single, distinct

31

National Institute for Basic Biology

Developmental Biology

gamete type (gonochorism). We treated females of two
species of teleost, the Nile tilapia and medaka, witharomatase inhibitors (AI) for up to five months to block theconversion of androgens to estrogens in order to investigate
whether sexual plasticity is retained in gonochoristic fish. In
both species, suppression of estradiol-17b production via AI
treatment caused a rapid degeneration of ovarian tissues,leading to the differentiation and development of testicular
tissues. The reduced expression of aromatase (P450c19a)with a rise in the expression of GSDF, indicates thedifferentiation of testicular-type somatic cells in the
AI-treated gonads. Sex-changed fish show a typical malepattern of estrogen and androgen levels, secondary sex
characteristics, producing fertile sperm in the newly formed
testes. Our results indicate that gonochoristic fish maintaintheir sexual plasticity to adulthood and that estrogens play a
critical role in maintaining the female phenotype.

M., Ito, C., Kubota, E., Nagahama, Y., and Shibata, Y. (2009).
• Mita,Expression and distribution of gonad-stimulating substance of various

IV. Endocrine regulation of oocyte maturationand ovulation•

A period of oocyte growth is followed by a process called
oocyte maturation and is a prerequisite for successful
fertilization. Our studies using vertebrate (fish) andinvertebrate (starfish) models have revealed that the basic
mechanisms involved in oocyte maturation are the same inthese two species despite the differing chemical nature of thehormonal agents involved. In both species, three major
mediators have been shown to be involved (Three stepmodel): a gonad-stimulating substance (GSS),
1-methyladenine (maturation-inducing hormone, MIH), anda maturation-promoting factor (MPF) in starfish, andgonadotropin (LH), 17a, 20b-dihydroxy-4-pregnen-3-one(17a,20b-DP) (MIH), and MPF in fish.
We recently purified GSS from the radial nerves of starfish(Asterina pectinifera) and the complete amino acid sequence
was determined. Phylogenetic analyses revealed that starfish
GSS was a relaxin-like peptide. Chemically synthesized GSS
induced not only oocyte maturation and ovulation in isolated
ovarian fragments, but also unique spawning behavior
followed by the release of gametes shortly after injection.
Thus, this study represents the first evidence of a relaxin
system in invertebrates and points towards a novel
reproductive role for this peptide in starfish. This work wasdone in collaboration with Drs. M. Mita, Tokyo Gakugei
University and M. Yoshikuni, Kyushu University.

Publication List

〔Original papers〕

W., Cao, M., Yang, Y., Nagahama, Y., and Zhao, H. (2009).
• Chen,Expression pattern of prmt5 in adult fish and embryos of medaka,

•
•

32

Oryzias latipes. Fish Physiol. Biochem. 35, 325-332.
Cui, J.Z., Shen, X.Y., Yan, Z.W., Zhao, H.B., and Nagahama, Y. (2009).
Homology-modeled ligand-binding domains of medaka estrogen
receptors and androgen receptors: a model system for the study of
reproduction. Biochem. Biophys. Res. Comm. 380, 115-121.
Kobayashi, Y., Nakamura, M., Sunobe, T., Usami, T., Kobayashi, T.,
Manabe, H., Paul-Prasanth, B., Suzuki, N., and Nagahama, Y. (2009).
Sex-change in the gobiid fish is mediated through rapid switching of
gonadotropin receptors from ovarian to testicular portion or vice-versa.

Endocrinology 150, 1503-1511.

•
•
•
•

•
•
•

organs of the starfish Asterina pectinifera. Trends in Comparative
Endocrinology and Neurobiology: Ann. N.Y. Acad. Sci. 1163, 472-474.
Mita, M., Yoshikuni, M., Ohno, K., Shibata, Y., Paul-Prasanth, B.,
Pitchayawasin, S., Isobe, M., and Nagahama, Y. (2009). A relaxin-like
peptide purified from radial nerves induces oocyte maturation andovulation in the starfish, Asterina pectinifera. Proc. Natl. Acad. Sci.
USA 106, 9507-9512.
Mittelholzer, C., Andersson, E., Taranger, G..L., Consten, D., Hirai, T.,
Senthilkumaran, B., Nagahama, Y., and Norberg, B. (2009). Molecularcharacterization and quantification of the gonadotropin receptors
FSH-R and LH-R from Atlantic cod (Gadus morhua). Gen. Comp.
Endocrinol. 160, 47-58.
Nakamoto, M., Muramatsu, S., Yoshida, S., Matsuda, M., Nagahama,Y., and Shibata, N. (2009). Gonadal sex differentiation and expressionof Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis. Genesis 47, 289299.
Ngamniyom, A., Magtoon, W., Nagahama, Y., and Sasayama, Y. (2009).
Expression levels of hormone receptors and bone morphogenic protein
in fins of medaka. Zool. Sci. 26, 74-79.
Senthilkumaran, B., Sudhakumari, C.C., Wang, D.S., Sreenivasulu, G..,
Kobayashi, T., Kobayashi, H.K., Yoshikuni, M., and Nagahama, Y.
(2008). Novel 3b-hydroxysteroid dehydrogenases from gonads of the
Nile tilapia: Phylogenic significance and expression during reproductive
cycle. Mol. Cell. Endocrinol. 299, 146-152.
Swapna, I., Sudhakumari, C.C., Sakai, F., Sreenivasulu, G., Kobayashi,T., Kagawa, H., Nagahama, Y., and Senthilkumaran, B. (2008).
Seabream GnRH immunoreactivity in brain and pituitary of XX andXY Nile tilapia, Oreochromis niloticus during early development. J.
Exp. Zool. 309A, 419-426.
Wu, F.R., Zhou, L.Y., Nagahama, Y., and Wang, D.S. (2009).
Duplication and distinct expression patterns of two thrombospondin-1
isoforms in teleost fishes. Gene Expr. Patterns 9, 436-443.
Zhang, W.L., Zhou, L.Y., Senthilkumaran, B., Huang, B.F.,
Sudhakumari, C.C., Kobayashi, T., Nagahama, Y., and Wang, D.S.
(2009). Molecular cloning of two isoforms of 11b-hydroxylase andtheir expression in the Nile tilapia, Oreochromis niloticus. Gen. Comp.
Endocrinol. 165, 34-41.

〔Review article〕

T., and Nagahama, Y. (2009). Molecular aspects of gonadal• Kobayashi,differentiation in a teleost fish, the Nile tilapia. Sex. Dev. 3, 108-117.

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION

Associate Professor

TANAKA, Minoru

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

NAKAMURA, Shuhei
KOBAYASHI, KayoAOKI, Yumiko
NISHIMURA, Toshiya
AOKI, Yumiko*
KINOSHITA, ChieNISHIMURA, Keiko
WATAKABE, Ikuko
YONEMITSU, Masako

Laboratory scope

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

I. Canalization of germ cells to feminization vs.
male predisposition of somatic cells

We have been analyzing the role of germ cells during thecourse of sex differentiation of the gonads and have revealed
an essential function of germ cells on the development ofovaries.
In wild type medaka, sex is determined genetically in an
XY manner. Without germ cells, however, both geneticallyfemale and male medaka exhibit male secondary sex
characteristics and gonadal somatic cells are masculinized interms of male-specific gene expression and production of amale steroid hormone. This can be explained by the
hypothesis that the female character of germ cells
antagonizes the autonomous masculinization of gonadal
somatic cells and that balancing between germ cells andgonadal somatic cells is essential for both sex differentiationand maintenance of sex.
In support of this claim, medaka mutants that display agerm cell-hypertrophic phenotype, hotei, exhibit female
secondary sex characteristics in approximately half ofgenetic male hotei mutants. In the germ cell-deficient hotei
mutants, none of them show any sex reversal of secondary

sex characteristics. In addition, female-specific gene
expression seen in genetic male hotei mutants is alsoabolished in the germ cell-depleted mutants. These results
indicate that feminization of the gonad in the mutant is aconsequence of hypertrophic germ cells but not of a gonadal
somatic cell-autonomous event.
The gene responsible for this phenotype is the type II
receptor gene for anti-Mullerian homone (amhrII). We foundthat both amhrII and its ligand, amh, are expressed ingonadal somatic cells (supporting cells) but not in germ cells,
indicating that Amh signaling acts autonomously of
supporting cells.

II. A m h s i g n a l i n g i s i n v o l v e d i n s e x
differentiation by modulating reciprocalinteraction between germ cells and somaticcells

The results from both germ cell-deficient medaka and germcell-hypertrophic mutants (hotei) suggest an intrinsic
mechanism of sex differentiation that is independent ofgenetic sex determination. We propose that, irrespective ofgenetic sex, germ cells have an intrinsic character thatcanalizes feminization of the gonad (canalization to female)while gonadal somatic cells are predisposed to maledevelopment (male predisposition). According to this
proposal, the possible function of the medaka testis
determination gene on the Y chromosome is enhancement ofmasculinization that conquers canalization towards ovary by
germ cells. The close interaction has to be present, thereby
reciprocal signals between germ cells and somatic cells
acting to form proper sex differentiation. Since the complete
defect of Amh signaling results in improper sex
differentiation, Amh signaling is very likely to modulate thereciprocal signals and balance the two intrinsic signals.
The balancing of the two opposing characters may be aconserved cellular interaction among vertebrates. In fact,
there have been several reports of the masculinization ofmice ovaries following the depletion of germ cells by genetic
or physical manipulations. Even in an undifferentiatedgonocholist, zebrafish, which firstly develops ovaries before
some of the population turn into males, prior to thedevelopment of testis the germ cell number decreases by
apoptosis. The suppression of the germ cell number at theearly stage of medaka gonadal sex differentiation and thedecrease in germ cells in zebrafish can be viewed as different
ways of achieving the same result (Tanaka et al., 2008 DGD:
Saito et al., Sex. Dev. 2009).

III. Estrogen-producing cells – the earliest
female-specific somatic cells develop in agerm cell-dependent manner

As mentioned above, germ cells and the surrounding
somatic cells (supporting cells) are important players in the
gonads. But there are many other functionally unknown
somatic cells in the gonads. Transgenic medaka with GFP
fluorescence are powerful tools to analyze them because they
provide fine structure and organization of the gonad at single
cell levels. We have generated transgenic medaka that enable
us to keep track of cyp19a1 (aromtatase)-expressing cells in

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

33

National Institute for Basic Biology

Developmental Biology
IV. Expression and syntenic analysis of medaka
nanos gene family

Figure 2. The cyp19a1-expressing theca cells (green) are one of theearliest female-specific cells in the female developing gonad. blue:
nuclea, red: germ cells. Picture from Nakamura et al (2009).

the gonad by the established method of using medaka BAC(bacterial artificial chromosome) (Nakamura et al., 2008
DGD). The cyp19a1-GFP transgenic medaka mimics
endogenous cyp19a1 expression by GFP fluorescence(Nakamura et al., 2009).
Once germ cells enter meiosis, they form the follicular
structure with both inner and outer somatic cells surrounding
oocytes. The aromatase (product of the gene, cyp19a1),
which catalyzes a precursor steroid, testosterone, to produce
female-specific hormone, estrogen, has generally been used
as a good marker of the inner somatic cells called granulosa
cells.
The expression of cyp19a1 in medaka has been observed as
early as 5 days post hatching (5dph) in female developing
gonads (Figure 2) but not male developing gonads. The
cyp19a1-expressing cells first appear in the most ventral sideof a stromal region adjacent to the epithelium of the female
gonad. Immediate after appearance of the cyp19a1expressing cells they were found on the outer layer offollicles but not on the inner layer. This result indicates thatthe cyp19a1-expressing cells are not the granulose cells but
theca cells. Very interestingly, another theca cell marker,
P450c17, is exclusive to cyp19a1 expression, demonstratingthat there are two different types of theca cells on thefollicles. We often observe that cyp19a1-expressing cells on
the ventral stromal region extend their cellular process
towards follicles. This observation suggests that the cyp19a1expressing cells in the ventral region are the precursors of
one of the theca cells (Figure 3).
Consistent with our result, in the absence of germ cells,
female-specific cyp19a1-expressing cells are not maintainedin the developing ovary (Nakamura et al 2009).

Figure 3. Development of cyp19a1-expressing theca cells (green). Thesecells are different from another type of theca cell expressing P450c17(blue). The cyp19a1-expressing theca cells first arise near the epithelium(purple) and surround oocytes (pink) cells. Illustration from Nakamura etal (2009)

34

Nanos is known to be essential for germ cell development.
Availability of medaka genomic information allows us tosuccessfully identify four different nanos genes on the
medaka genome (nanos 1a, 1b, 2, and 3). Nanos3 is mostconserved among vertebrates at the levels of amino acidsequence and synteny. Nanos1a and 1b are expressed inneuronal cells in common, nanos2 in gonial cells and nanos3
specifically in germ cell-lineage (Aoki et al., 2009)

Publication List

〔Original papers〕

Y., Nakamura, S., Ishikawa, Y., and Tanaka, M. (2009).
• Aoki,Expresssion and syntenic analyses of four nanos genes in medaka. Zool.
Sci. 26, 112-118.

T., Oshima, T., Kinoshita, M., Tanaka, M., Wakamatsu, Y., Ozato,• Hano,K., Nassef, M., Shimazaki, Y., and Honjo, T. (2009). In ovo••

nanoinjection of nonylphenol affects embryonic development of atransgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI
strain. Chemosphere 77, 1594–1599.
Herpin, A., Nakamura, S., Wagner, T., Tanaka, M., and Schartl, M.
(2009). A highly conserved cis-regulatory motif directs differential
gonadal synexpression of Dmrt1 transcripts during gonad development.
Nucleic Acids Res. 35, 1510–1520.
Nakamura, S., Kurokawa, H., Asakawa, S., Shimizu, N., and Tanaka,
M. (2009). Two distinct types of theca cells in the medaka gonad: Germcell-dependent maintenance of cyp19a1-expressing cells. Dev. Dyn.
238, 2652-2657.

〔Review articles〕

M., Murata, K., Naruse, K., and Tanaka, M. (eds) (2009).
• Kinoshita,
Medaka – Biology, Management, and Experimental Protocols. 419
pages, Iowa, USA. Wiley-Blackwell. ISBN:978-0-813-818511.

D., and Tanaka, M. (2009). Comparative aspects of gonadal• Saito,differentiation in medaka: a conserved role of developing oocytes insexual canalization. Sex. Dev. 3, 99-107.

LABORATORY OF PLANT ORGAN DEVELOPMENT

Director-General

OKADA, Kiyotaka

Assistant Professor:
Postdoctoral Fellows:

Visiting Scientists:
Graduate Students:

Technical Assistants:
Secretary:

TATEMATSU, Kiyoshi
TSUCHIDA, Yuhei
IGARASHI, Hisako
URAWA, Hiroko
YABE, Kimihiko
POPRAWKA, Tomasz
WADA, Takuji
TOMINAGA, Rumi
TOYOKURA, Koichi
NAKATA, Miyuki
IWASAKI, Akira
TAMESHIGE, Toshiaki
HARA, Reiko
NAKAMORI, Chihiro
MATSUMOTO, Miwako
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 the 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.

expression domain (enf). One of them, enf1, forms leaves
with enlarged and reduced FIL-expression domains. In theextreme cases, leaves are filamentous. This phenotype
indicates that ENF1 is involved in the fixation or
maintenance of the position of the adaxial-abaxial boundary.
The structure of the SAM is often aberrant in enf1 mutants.
We revealed that the ENF1 gene encodes an enzyme
associated with primary metabolism, and that ENF1 isstrongly expressed in leaf primordia although its expression
was not found in the SAM. This indicates that ENF1 affects
the axis-dependent cell fate in leaf primordia. In contrast,
another mutant, enf2, has leaves with an enlarged FILexpression domain, and the ENF2 gene encoded a plastidlocalized unknown protein. Chloroplast development wasrepressed in a severe allele of the enf2 mutant, suggestingthat chloroplast development is required for normal
differentiation of leaf tissues.
There are methylated tandem repeat sequences in the
promoter region of another abaxial side-marker gene, YAB3.
We revealed that the methylation of the region occurred in a
Pol IV/Pol V-dependent manner. We also obtained results
suggesting the methylated sequences occur at a higher
frequency in the cells of the adaxial-side of leaves than inthose of the abaxial side.
A line of unique oblong cells is found at the marginal edgeof leaves (Fig. 1 left). We noticed that a homeobox-related
gene, PRESSED FLOWER (PRS) and its homolog, WOX1,
are required for forming the margin-specific cells. prs wox1
double mutants completely lack the margin-specific cells in
leaves, and interestingly, the abaxial side-specific epidermal
cells, which are smaller than the adaxial side-specificepidermal cells, “invade” the adaxial side surface (Fig. 1right). The results indicate that the margin-specific cells act
as a physical barrier separating the epidermal cells of the
adaxial-side surface from those of the abaxial-side surface.

I. Genetic approach

Recent studies of Arabidopsis mutants show several genes
are involved in the axes-dependent control of lateral organ
development. The adaxial-abaxial boundary in the leaf
primordium is determined by the precise expression of the
adaxial marker gene, PHABULOSA (PHB), and the abaxial
marker genes, FILAMENTOUS FLOWER (FIL) and YABBY3(YAB3). We showed that PHB is expressed in cells of the
adaxial side and separated clearly from the abaxial sidespecific FIL gene expressing cells, by the action of
microRNA165/166 which targeted the PHB, REV, and PHV
messenger RNAs. We also revealed by laser microdissectionthat specific expression of MIR165/166 genes in the abaxial
side is important for adaxial-abaxial boundary formation.
To examine the mechanisms of boundary formation, we
isolated novel mutants which show altered patterns of FIL
promoter::GFP expression, and named them enlarged fil-Figure. 1 Oblong cells located at the marginal edge of wild type
Arabidopsis leaves (painted in red in left panel). In the prs wox1double
mutant, the long margin-specific cells are absent, and the abaxial sidespecific epidermal cells seem to “invade” the adaxial side surface (shownby the arrow in right panel). A red-dashed line indicates the border
between the adaxial-side epidermal cells and those of the abaxial side.

II. Biochemical approach

We are taking another approach to study the intercellularsignaling system by analyzing small peptides as candidates
for intercellular signaling ligands, which are present in theapoplastic region of the SAM. Small peptides collected from
apoplast fractions of the heads of cauliflower (Brassica
oleracea L. var. botrytis) and Arabidopsis apetala1
cauliflower double mutants were analyzed by peptide

35National Institute for Basic Biology

Developmental Biology

sequence methods or LC-MS/MS methods. We chose about
30 peptides as the candidates, and prepared synthetic
peptides based on the obtained sequences. When we applied
the peptides to Arabidopsis seedlings, several peptides
caused morphological defects in the SAM, vascular tissue
and root development. We are currently examining themechanisms involved.

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 a few days after germination,some of the newly generated rosette leaves changed to afilamentous structure lacking the adaxial-abaxial identity.
This suggests a flow of signal(s) from the SAM to the leaf
primordia. Currently, we are ablating some of the marginspecific cells of leaf primordia to analyze the role of thesecells.

IV. One-cell gene induction approach

As a new tool for examining the intercellularcommunication system, we are developing the one-cell
induction system in planta using the InfraRed Laser Evoked
Gene Operator (IR-LEGO) system. We irradiated root
epidermal cells of a transgenic line carrying the Arabidopsis
heart-shock promoter hsp18.2::GUS fusion gene. GUS
expression was observed in irradiated cells (Fig. 2 left).
Induction of a single cell is possible by focusing theirradiation (Fig. 2 right).

Figure. 2 GUS expression induced by IR laser irradiation to 4 target cells
arranged in a diamond shape. The four irradiated cells (arrows) expressed
GUS strongly (left). However, weak staining was found in thesurrounding cells. Top right panel shows magnified image. GUS
expression was restricted to the targeted single cell (arrow in Right) using
different laser irradiation settings.

36

Publication List

〔Original papers〕

T., Itoh, M., Urawa, H., Matsumoto, T., Nakayama, S.,• Deguchi,
Kawasaki, T., Kitano, T., Oda, S., Mitani, H., Takahashi, T., Todo, T.,

•
•
•
•

Sato, J., Okada, K., Hatta, K., Yuba, S., and Kamei, Y. (2009). Infraredlaser-mediated local gene induction in medaka, zebrafish andArabidopsis thaliana. Develop. Growth Differ. 51, 769-775.
Tominaga-Wada, R., Iwata, M., Sugiyama, J., Kotake, T., Ishida, T.,
Yokoyama, R., Nishitani, K., Okada, K., and Wada, T. (2009) The
GLABRA2 homeodomain protein directly regulates CESA5 and
XTH17 gene expression in Arabidopsis roots. Plant J. 60, 564-574.
Tsugeki, R., Ditengou, F., A., Sumi, Y., Palme, K., and Okada, K.
(2009). The novel nuclear factor NO VEIN mediates auxin-dependent
specification and patterning in the embryo, shoot and root. Plant Cell
21, 3133-3151.
Yagi, N., Takeda, S., Matsumoto, N., and Okada, K. (2009). VAJ/GFA1/
CLO is involved in the directional control of floral organ growth. Plant
Cell Physiol. 50, 1-13.
Yoshida, Y., Sano, R., Wada, T., Takabayashi, J., and Okada, K. (2009).
Jasmonic acid control of GLABA3 links inducible defense and trichome
patterning in Arabidopsis. Development 136, 1039-1048.

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

NODA, Masaharu

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

Graduate Students:

Technical Assistants:

Secretaries:

SHINTANI, Takafumi
SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
IHARA, Masaru
YOSHIDA, Masahide
FUJIKAWA, Akihiro
SUZUKI, Ryoko
NISHIHARA, Eri
YOSHIDA, Masahide*
MATSUMOTO, Masahito
NAGAKURA, Ayano
ZHANG, Lanfan
TANI, Sachiko
SAKURABA, Jyuichi
YOSHINO, Jiro
MIZOGUCHI, Masae
HATTORI, Nobuko
TOMITA, Nao
ISOSHIMA, Yoshiko
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
KODAMA, Akiko
YATAGAI, Hiroko
IMAMURA, Masako

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 sensation, emotion, behavior, learning, andmemory.

I. Mechanisms for retinal patterning during
development

Topographic maps are a fundamental feature of neuralnetworks in the nervous system. The retinotectal projectionof lower vertebrates including birds has been used as areadily accessible model system of the topographic
projection. We have been studying the mechanisms forregional specification in the developing retina as the basis of
the topographic retinotectal projection.
Two winged-helix transcription factors, FoxG1 (previously
called chick brain factor1, CBF1) and FoxD1 (chick brain
factor2, CBF2), are expressed specifically in the nasal andtemporal regions of the developing chick retina, respectively.
We previously demonstrated that FoxG1 controls theexpression of topographic molecules including FoxD1, anddetermines the regional specificity of the nasal retina. FoxD1
is known to prescribe temporal specificity, however,molecular mechanisms and downstream targets have notbeen elucidated. We addressed the genetic mechanisms forestablishing temporal specificity in the developing retina
using an in ovo electroporation technique (Figure 1A).
Fibroblast growth factor (Fgf) and Wnt first play pivotal
roles in inducing the region-specific expression of FoxG1and FoxD1 in the optic vesicle. Misexpression of FoxD1
represses the expression of FoxG1, GH6, SOHo1, andephrin-A5, and induces that of EphA3 in the retina. GH6 andSOHo1 repress the expression of FoxD1. In contrast to theinhibitory effect of FoxG1 on bone morphogenic protein(BMP) signaling, FoxD1 does not alter the expression of
BMP4 or BMP2. Studies with chimeric mutants of FoxD1
showed that FoxD1 acts as a transcription repressor incontrolling its downstream targets in the retina. Taken
together with previous findings, our data suggest that FoxG1and FoxD1 are located at the top of the gene cascade forregional specification along the nasotemporal(anteroposterior) axis in the retina, and FoxD1 determines
temporal specificity (Figure 1B).

Figure 1. Expressional regulation of asymmetrically distributed
molecules along the A–P axis by FoxG1 and FoxD1. (A) The molecularmechanisms by which FoxG1 and FoxD1 control the expression of
topographic molecules. Fgf8 derived from the anterior neural ridge isrequired for both FoxG1 and FoxD1 expression in the optic vesicle, but
an excess of Fgf enhances FoxG1 expression and represses FoxD1
expression. Wnt signaling from the mesencephalon inhibits theexpression of FoxG1 and FoxD1. FoxG1 and FoxD1 counteract eachother and FoxG1 represses the transcription of negative regulators, X.
When FoxG1 is absent and FoxD1 is present, X downregulates ephrin-A5
expression and FoxD1 represses the expression of GH6 and SOHo1.
When FoxG1 is present and FoxD1 is absent, X is downregulated and theexpression of ephrin-A5, GH6, and SOHo1 is induced. GH6 and SOHo1
inhibit the expression of EphA3. The inhibitory action of FoxG1 on
BMP2 signaling is attributable to the change in the expression of
Ventroptin and BMP2 to the oblique-gradient pattern. The counteraction
between Ventroptin and BMP2 determines the expression of ephrin-A2 in
an oblique-gradient fashion. Arrows and T-bars indicate positive andnegative effects, respectively. These effects are not necessarily induced
by direct action. (B) Developmental expression of topographic moleculesalong the A–P axis. FoxG1 and FoxD1 are expressed in a countergradient manner in the optic vesicle. After the optic cup forms, theexpression domains of FoxG1 and FoxD1 are separated by reducing theirpositive regions to a narrow region of the nasal or temporal retina,
respectively. Subsequently, GH6 and SOHo1 are expressed specifically inthe nasal retina from HH stages 12–14. The expression domains of GH6
and SOHo1 fill the gap between the expression areas of FoxG1 andFoxD1. EphA3 and ephrin-A5 are homogeneously expressed in the retina

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

37

National Institute for Basic Biology

Neurobiology

at early developmental stages and become restricted to the temporal andnasal retinae, respectively, under the control of these transcription factors
at later stages. Development proceeds from left to right. Nasal (N) is left,
and temporal (T) is right in the retina.

II. Mechanisms for retinotectal projection

Special attention is now devoted to the molecularmechanisms for axon branching and arborization and theirselective elimination. Among the region-specific moleculesin the developing retina, we have already identified several
molecules which induce abnormal branching andarborization when their expression was experimentally
altered in vivo.

Figure 2. Distribution of interstitial branches of retinal axons on thetectum at E12.5. (A) The tectum was subdivided into 10 areas along theanteroposterior axis, and numbers of branches in each area were counted.
(B–D) Quantification of branch distributions of dorsotemporal (DT),dorsonasal (DN), and ventrotemporal (VT) retinal axons, respectively.
The values are shown as the mean ±SEM. In APC2 knockdown embryos,
interstitial branches from the axon shaft were more diffusely distributed
along the anteroposterior axis. A, Anterior; P, posterior.

Growth cones at the tip of growing axons are key cellular
structures that detect guidance cues and mediate axonal
growth. An increasing number of studies have suggested thatthe dynamic regulation of microtubules in the growth coneplays an essential role in growth cone steering. The dynamic
properties of microtubules are considered to be regulated byvariegated cellular factors but, in particular, throughmicrotubule-interacting proteins. We examined the functionalrole of adenomatous polyposis coli-like molecule 2 (APC2)
in the development of axonal projections by using the chickretinotectal topographic projection system. APC2 ispreferentially expressed in the nervous system from early
developmental stages through to adulthood.
Immunohistochemical analysis revealed that APC2 isdistributed along microtubules in growth cones as well asaxon shafts of retinal axons. Overexpression of APC2 incultured cells induced the stabilization of microtubules,
whereas the knockdown of APC2 in chick retinas withspecific short hairpin RNA reduced the stability of
microtubules in retinal axons. APC2 knockdown retinal

38

axons showed abnormal growth attributable to a reduced
response to ephrin-A2 in vitro. Furthermore, they showed
drastic alterations in retinotectal projections without making
clear target zones in the tectum in vivo (Figure 2). These
results suggest that APC2 plays a critical role in thedevelopment of the nervous system through the regulation of
microtubule stability.

III. Development of 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 project their axons specifically toterminal nuclei of the accessory optic system (AOS)
responsible for optokinetic reflex (OKR). We recently
generated a knock-in mouse in which SPIG1 (SPARC-related
protein containing immunoglobulin domains 1)-expressing
cells are visualized with GFP, and found that retinal ganglion
cells projecting to the medial terminal nucleus (MTN), theprincipal nucleus of the AOS, are comprised of SPIG1+ andSPIG1- ganglion cells distributed in distinct mosaic patterns
in the retina (Figure 3A, left). We examined light responsesof these two subtypes of MTN-projecting cells by targetedelectrophysiological recordings. SPIG1+ and SPIG1- ganglion
cells respond preferentially to upward motion and downward
motion, respectively, in the visual field (Figure 3B). The
direction selectivity of SPIG1 + ganglion cells develops
normally in dark-reared mice. The MTN neurons areactivated by optokinetic stimuli of only vertical motion asshown by Fos expression analysis. Combination of genetic
labeling and conventional retrograde labeling revealed thataxons of SPIG1+ and SPIG1- ganglion cells project to the
MTN via different pathways (Figure 3A). The axon terminalsof the two subtypes are organized into discrete clusters in the
MTN. These results suggest that information about upwardand downward image motion transmitted by distinct ON DS
cells is separately processed in the MTN, if not
independently. Our findings provide insights into the neuralmechanisms of OKR; how information about the direction ofimage motion is deciphered by the AOS.

V. Mechanisms of Na-level sensing in the brain
for body-fluid homeostasis

Figure 3. (A) Schematic representation of axonal connectivity between
the retina and the contralateral MTN of the AOS. Information of upwardand downward visual motion is conveyed to the MTN by distinct
neuronal pathways. This represents our findings together with those in
the previous studies on the retinal projection to the MTN in mice andrats. Upward-preferring subtype of ON DS cells (SPIG1 +; green)
predominantly projects to the MTNd via AOT-IF, whereas downwardpreferring subtype of ON DS cells (SPIG1-; red) projects to the MTNv
via AOT-SF. The fibers of the AOT-SF split from the OT and thebrachium of the SC, then they course ventrally over the surface of thecerebral peduncle (CP) and finally terminate in the MTNv. On the other
hand, the fibers of the AOT-IF leave the OT just after passing through theoptic chiasm (OC), then course caudally, and terminate in the MTNd.
MGB: medial geniculate body (B) SPIG1 marks the upward-preferring
subtype of ON DS cells. (a, b) Left, the tip of a tungsten electrode was
attached onto a GFP+ MTN-projecting (MTN-P) cell (a) or GFP- MTN-P
cell (b) to record spike discharges from the cell. Cells were identified byusing IR-DIC optics. Right, responses of a GFP+ MTN-P cell (a) and
GFP- MTN-P cell (b) to a light stimulus for 5 s. Both showed sustained
responses during the light stimulation. (c, d) Left panels, representative
spike trains and their polar plots in response to eight different directionsof the drifting square-wave gratings for 6 s. Spikes were recorded from a
GFP+ MTN-P cell (c), GFP- MTN-P cell (d), respectively. The red line
indicates the preferred direction of the cells. Right panels, preferred
direction and direction selectivity index (DSI) of individual cells are
represented by the angle and length of the red line, respectively. The
black line indicates the average of the preferred direction and DSI. The
DSI indicates the degree of asymmetry in the polar plot of responses tothe drifting gratings. The arrowhead indicates the direction of thedorsotemporal domain. Scale bars: a, b, 20 µm.

Dehydration causes an increase in the sodium (Na)concentration and osmolarity of body fluids. For Na
homeostasis of the body, control of Na and water intake andexcretion are of prime importance. Although it was suggested
that the circumventricular organs (CVOs) are involved inbody-fluid homeostasis, the system for sensing Na levels
within the brain, which is responsible for the control of Naand water-intake behavior, has long been an enigma.
Nax is an atypical sodium channel that is assumed to be adescendant of the voltage-gated sodium channel family. Ourstudies on Nax-knockout mice revealed that Nax channels are
localized to the CVOs and serve as a sodium-level sensor ofbody fluids. Nax-knockout mice do not stop ingesting salt
when dehydrated, while wild-type mice avoid salt. Veryrecently, we found that Nax-deficient mice show normal
vasopressin response to dehydration.

Publication List

〔Original papers〕

T., Ihara, M., Tani, S., Sakuraba, J., Sakuta, H., and Noda, M.
• Shintani,(2009). APC2 plays an essential role in axonal projections through theregulation of microtubule stability. J. Neurosci. 29, 11628-11640.

H., Sakuta, H., Shintani, T., and Noda, M. (2009). Functional• Takahashi,mode of FoxD1/CBF2 for the establishment of temporal retinal
specificity in the developing chick retina. Dev. Biol. 331, 300-310.

A.H., Sabirov, R.Z., Takahashi, N., Ando-Akatsuka, Y., Liu,
• Toychiev,
H., Shintani, T., Noda, M., and Okada, Y. (2009). Activation of maxi-•

anion channel by protein tyrosine dephosphorylation. Am. J. Physiol. Cell Physiol. 297, C990-C1000.
Yonehara, K., Ishikane, H., Sakuta, H., Shintani, T., NakamuraYonehara, K., Kamiji, N.L., Usui, S., and Noda, M. (2009). Identificationof retinal ganglion cells and their projections involved in central
transmission of information about upward and downward image
motion. PLoS ONE 4, e4320.

〔Review articles〕

M., Takahashi, H., and Sakuta, H. (2009). Neural patterning: Eye• Noda,fields. In Encyclopedia of Neuroscience, Squire, L.R., ed. (Elsevier),
pp. 199-204.

H., Suzuki, R., and Noda, M. (2009). Retrovirus vector• Sakuta,
mediated gene transfer into the chick optic vesicle by in ovo

electroporation. In Electroporation and Sonoporation in Developmental
Biology, Nakamura, H., ed. (Springer), pp. 105-116.

IV. Physiological roles of protein tyrosine
phosphatase receptor type Z

Protein tyrosine phosphatase receptor type Z (Ptprz, also
known as PTPz/RPTPb) is preferentially expressed in the
CNS as a major chondroitin sulfate proteoglycan. Three
splicing variants, two receptor isoforms (Ptprz-A/B) and one
secretory isoform (Ptprz-S) are known. Receptor-type Ptprz
interacts with the PSD95 family through its intracellularcarboxyl-terminal PDZ-binding motif in the postsynaptic
density. Ptprz-deficient adult mice display impairments inspatial and contextual learning. We are conducting
experiments to know molecular and cellular mechanisms forthe altered phenotypes in Ptprz-deficient mice.

39

National Institute for Basic Biology

Neurobiology

DIVISION OF BRAIN BIOLOGY

Professor

YAMAMORI, Tetsuo

Assistant Professors:

KOMINE, Yuriko
WATAKABE, Akiya
SADAKANE, Osamu
Assistant Professor (Specially Appointed):
KOMATSU, Yusuke#
NIBB Research Fellow:
HIROKAWA, Junya*
Technical Staff:
OHSAWA, Sonoko
Postdoctoral Fellows:
TAKAJI, Masafumi#
HIROKAWA, Junya
OHTSUKA, Masanari
HATA, Katsusuke
Graduate Students:
NAKAGAMI, Yuki
SHUKULA, RamohanTechnical Assistants:
SASAKI, Tetsuya
NAKAMURA, Tohru
MORITA, Junko
FURUYAMA, Makiko
IMAI, Akiko
HIRAYAMA, Yuka#
IMOTO, Eiko#
We are studying the genes that are expressed in specificareas of the neocortex in order to understand the principle forthe formation of the primate brain.

I. Genes expressed in specific areas and layersof the neocortex

The neocortex emerged in mammals and evolved mostremarkably in the primate. To understand the underlying
mechanisms, we studied gene expression patterns withindifferent areas of the neocortex. Over the last ten years

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 )40

we have reported the findings that are schematicallyillustrated in Figure 1.
In short, using differential display methods, we found threearea-specific expression genes in the primate neocortex.
Firstly, occ1 is specifically expressed in the occipital cortex
in the primate brain. Secondly, the other gene that showedmarked difference within the neocortex is gdf7, a member of
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 association andhigher 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;
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), and SPARC (an OCC1 related gene). This
year, we reported another gene (PNMA5), whose expression
is similar to RBP (an association area enriched gene) as
shown in Figure 1.

II. Paraneoplastic Antigen-Like 5 Gene
(PNMA5) is preferentially expressed in theassociation areas in a primate specificmannerAs mentioned above, this year we reported enriched
expression of the paraneoplastic antigen-like 5 gene
(PNMA5) in prefrontal and sensory association areas but notin primary sensory areas, with the lowest expression level inthe primary visual cortex. In situ hybridization in the primarysensory areas revealed PNMA5 mRNA expression isrestricted to layer II: That is, along the ventral visual
pathway, the expression gradually increased in the excitatoryneurons from the primary to higher visual areas. Thisdifferential expression pattern was very similar to that ofretinol-binding protein (RBP) mRNA, another associationarea-enriched gene which we reported previously (Figure 2).
Additional expression analysis for comparison of other genes
in the PNMA gene family, PNMA1, PNMA2, PNMA3, andMOAP1 (PNMA4), showed that they were widely expressed
across areas and layers, but without the differentiated patternof PNMA5. In mouse brains, PNMA1 was only faintly
expressed and PNMA5 was not detected. Sequence analysis
showed divergence of PNMA5 sequences among mammals.
These findings suggest that PNMA5 acquired a certain
specialized role in the association areas of the neocortex
during primate evolution (Takaji et al., Cereb Cortex. 2009,
19: 2865 - 2879).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*). Members with # belong to SRPBS (Strategic Research Program for Brain Science), NIPS.

produced by an injection of tetrodotoxin, IEGs wereexpressed in a patchy pattern that included infragranularlayers, as well as supragranular layers, where theycorresponded to the CO blobs. In addition, the expressions of
IEGs in layer 4C were especially high in narrow zones along
boundaries of ODCs, termed here, the “border strips” of the
ODCs. After longer periods of MD (> 5 hr), the border strips
were no longer apparent. When the MD was produced by abrief period of monocular eyelid suture, changes in IEG
expressions were not evident in layer 4, however, the patchy
pattern of the expression that was obvious in infragranularand supragranular layers remained. These changes of IEG
expression after MD indicate that cortical circuits involving
the CO blobs of the supragranular layers include aligned
groups of neurons in the infragranular layers, and that theborder strip neurons of layer 4C are highly active for 3 hr
after MD (Figure 3).

Figure 2. Comparison of expression patterns of PNMA5 and RBP
mRNAs in macaque brains. (A) Coronal sections at positions A, C, D, E,
F in Figure 2 are shown. (B) Higher magnification views of several areas.
The expression patterns of PNMA5 and RBP mRNAs are shown,
together with the laminar profiles of ISH signals quantified by measuring
the optical density (red, PNMA5 mRNA; green, RBP mRNA). These
profiles show the averages of the normalized values of two or threedifferent macaques. The cortical layers were identified in reference to the
Nissl staining of the nearby section. (C) Double ISH of PNMA5 and RBP
mRNAs were performed to examine the coexpression of these mRNAs.
Note that PNMA5 mRNA (left, red) is expressed in a large population of
RBP-mRNA-positive cells (middle, green) in layer II. (cited from Takaji
et al. Cereb Cortex. 19: 2865 - 2879)

III. Expression of immediate-early genes
represents anatomical compartments
within ocular dominance columns after
brief monocular deprivation. Neocorticalareas revealed by layer specific gene
expression in rats
The primary visual cortex (V1) of primates is subdivided
into compartments reflecting different neural circuits. Visual
inputs from the two eyes activate alternating bands of cortex,
thereby forming the well-studied ocular dominance columns(ODCs). In addition, the enzymatic reactivity of cytochromeoxidase (CO) reveals “blob” structures within the
supragranular layers of ODCs. This year, we presented
evidence for compartments within ODCs which have notpreviously been clearly defined (Takahata et al., 2009,
106:12151-12155). These compartments are revealed by theactivity-dependent mRNA expression of immediate-earlygenes (IEGs), zif268 and c-fos, after brief periods ofmonocular deprivation (MD). After a 1-3 hr period of MD

Figure 3. A, B: Coronal sections of ISH for IEGs, zif268 and c-fos, in V1of Monkey 0 (normal; A) and Monkey 1 (1 hr TTX; B). IEG mRNA
expression decreased in the deprived columns (open circles in B) after
MD. Note that the widths of strong IEG mRNA expression (black
brackets) are thinner than the actual widths of nondeprived columns(filled circles), and that the IEG mRNA expression remains in the centerof deprived columns (open brackets). In addition, strong IEG mRNAsignals were observed around the border of ODCs in layer 4Cβ (blackarrows). Scale bar = 500 μm (A), and 1 mm (B), C: Tangential sections of
ISH for IEGs in V1 of Monkey 1. The depth of the section from the pial
surface is indicated on each panel. Sections are arranged by increasing
depth. Scale bar = 1 mm, D: High magnification views of rectangle
windows in C (a-c) and their adjacent section of superficial layer (270μm away from the pia) stained for CO reactivity (d). Broken lines in a, cand d indicate the boundary of ODCs with reference to the patterns in b.
Black arrowheads on the photomicrographs indicate the locations of CO
blobs. Compartments of border strips (BSs), dark columns (DCs) andpale columns (PCs) are indicated in b. Insert in b represents a magnified
view of the cortex within the rectangle area, at a single cell resolution.
Scale bar = 500 μm (cited from Takahata et al., Proc Natl Acad Sci U S
A. 2009, 106:12151-12155)

From these findings, we propose a model of the functionalarchitecture of macaque V1 (Takahata et al., Proc Natl Acad
Sci U S A. 2009, 106:12151-12155; Fig 4A). The functional
unit that is seen in CO preparation in the supragranular layers
extends to layer 6, with the exception of layer 4C. In
addition, border strips reside in the vicinity of the boundaries
of ODCs in layer 4C. As for the ODCs, the normally cryptic
border strips are revealed by IEG mRNA expression after
MD with TTX injection. Border strips of the left and right41

National Institute for Basic Biology

Neurobiology

eye column adjoin each other with the boundary of ODCs
between them. Previously, Horton described CO blobs that
included layers 5/6 in macaques (Horton & Hubel, 1981),
and the existence of dim CO blobs in infragranular layers has
been suggested for squirrel monkeys and prosimian galagos(Carroll & Wong-Riley, 1984; Condo & Casagrande, 1990)).
Horton and his colleagues have also suggested the existenceof border strips along ODCs in layer 4C in terms of a gap
between anterograde tracer signals from the open eye andCO enzymatic activity after long-term MD by eye
enucleation (Horton & Hocking, 1998), and pale stripes inlayer 4C in CO staining in the strabismus monkeys (Horton,
Hocking and Adams, 1999). Although there has been no

subsequent evidence for this architecture, our data strongly
supports their model except for their suggested absence of ablob structure in layer 4A, as our data indicates that the blob
structure also includes layer 4A.

Publication List

〔Original papers〕

R.F., Inverardi, F., Regondi, M.C., Watakabe, A., Yamamori,• Moroni,T., Spreafico, R., and Frassoni, C. (2009). Expression of layer-specific•••Figure 4. A: Our schematic model of V1 architecture. Blob structure
extends from layer 2 to layer 6, with the exception of layer 4C. In
addition, there is a border strip structure in the vicinity of the boundaries
of ODCs in layer 4C. Although we did not detect strong IEG mRNAsignals in layers 4B and 4Cα, previous reports can also be referred for the
structure (12, 27). B: Our schematic view of changes of IEG expression,
showing the distribution of somas of active neurons after MD (this case,deprivation of the left eye) by TTX or ES. In MD by TTX injection, IEG
mRNA expression decreases in interblob regions in both columns,
especially in deprived columns, and increases in border strips innondeprived columns. After a period of time, IEG mRNA expression
eventually levels out within each column. This sequential pattern ismostly similar in MD by ES, except that IEG mRNA expression hardly
changes in layer 4C. L/projection from left eye, R/projection from righteye (cited from Takahata et al., Proc Natl Acad Sci U S A. 2009,
106:12151-12155).

42

markers in the adult neocortex of BCNU-Treated rat, a model of corticaldysplasia. Neuroscienc. 159, 682-691.
Takahata, T., Higo, N., Kaas, J.H., and Yamamori, T. (2009). Expressionof immediate-early genes reveals functional compartments within
ocular dominance columns after brief monocular inactivation. Proc.
Natl. Acad. Sci. USA 106, 12151-12155.
Takaji, M., Komatsu, Y., Watakabe, A., Hashikawa, T., and Yamamori,T. (2009). Paraneoplastic Antigen-Like 5 Gene (PNMA5) Is
Preferentially Expressed in the Association Areas in a Primate Specific
Manner. Cereb. Cortex. 19, 2865 - 2879
Watakabe, A. (2009). Comparative molecular neuroanatomy ofmammalian neocortex: What can gene expression tell us about areas
and layers? Dev. Growth Differ. 51, 343-354.

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellow:

MATSUNAGA, Wataru

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 to research of
the visual system, though we had previously been
researching the salt-sensing system (~March, 2008).

I. Psychobiological study of medaka fish

One of our major subjects is the psychobiological study of
medaka (Oryzias latipes). Medaka, as well as zebrafish, have
many advantages for behavioral work. First, genetic
examination of medaka is progressing at a rapid pace, likethat of the mouse, opening up new approaches to theunderstanding 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; it
is composed of the spinal cord, brainstem, cerebellum, andcerebrum. Thirdly, because they are fish, 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.

field testing over two consecutive days, locomotion on thesecond day was less than that on the first day. This
habituation-like behavior ceased with the addition or deletionof a visual cue on the second day. We also found that males
and females differed in temporal patterns of habituation tothe open field. These findings clearly show that medaka
change their behavior as they become more familiar with anovel location, as observed in ethological studies of otheranimal species. The open-field test of medaka will thus
provide findings of use in elucidating abnormal phenotypesof mutant medaka.

II. Psychophysical study of human vision

Another of our major subjects is the psychophysical studyof 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, inwhich a moving object is perceived to lead a flashed object
when both objects are aligned in actual physical space. The
discoverer of this phenomenon, Dr. Nijhawan, has proposedthat the human visual system uses motion signals to
extrapolate the position of a moving object. The differential
latencies hypothesis proposes that the flash-lag effect occurs
simply because the visual system responds with a shorter
latency to moving objects than to flash stimuli. Besides these
two major models, various modified models have been
proposed. The problem, however, has not yet been solved,
and the debate continues. How does our brain decide theposition of the moving object? We are now making anattempt to correctly interpret the flash-lag effect bydeveloping a novel motion illusion (Kabob illusion, Figure
2).

Figure 1. Temporal changes in horizontal (left) and vertical (right)
locomotion of medaka fish in an open field in 3-min bins.

This year, we examined the habituation of medaka to anovel environment by measuring long-term temporalchanges in locomotion by using open-field apparatus. The
open-field test is commonly used in behavioral studies ofrodents. However, it has not been used in most behavioral
studies of fish. We therefore examined the open-field
behavior of medaka as measured by temporal changes in two
conventional indices, locomotion and position. Considering
the characteristic motility of fish, we focused on vertical aswell as horizontal locomotion. In 7-hour as well as 24-hour
exposure of fish to the open field, the pattern of temporalchanges in vertical locomotion differed from that ofhorizontal locomotion (Figure 1). During repeated open-

Figure 2. Kabob illusion. A: Sight line of observer is fixed on a central
cross (F). A fixed time after the cross disappears, a cue (C) appears in thecenter. After an appropriate time interval (lead time of cue object), a line(L) is presented. Observer perceives illusory motion from cue side toward
the opposite side of the line (line-motion effect). B: When the linemotion effect occurs, a leading cue (gray) is perceived to move along theline (solid).

43

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROCHEMISTRY

Associate Professor

SASAOKA, Toshikuni

NIBB Research 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, both transgenic and gene knockoutmice.

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. In collaboration with Dr. Motoya Katsuki,
Executive Director, National Institute of Natural Sciences,
We generated D1R/D2R double knockout (DKO) mice andobserved that D1R/D2R DKO mice exhibited severe
impairment in locomotion, no initiation of eating, and diedby 4 weeks of age.
To investigate the role of D1R in locomotor control andeating behavior, we generated transgenic mice harboring
tetracycline-regulated expression of the D1R gene on four
different backgrounds, including wild type, D1R KO, D2R
KO, and D1R/D2R DKO. Transgenic mouse lines showed
doxycycline (Dox) controllable expression of transgenic D1R
gene.

II. Locomotor activity controlled by D1R
expression

To elucidate the effects of changing D1R expression led
by Dox administration, we applied Dox for two weeks andmonitored daily locomotor activity and food/water intake(Figure 1). In D1R/D2R DKO background, mice exhibiteddecreases in locomotion and food/water intake as well as adecrease in the amount of transgene expression after Dox
administration. After withdrawal of Dox administration, themice exhibited transient hyperactivity and then recovered
locomotor activity and food/water intake (Figure 2). We
further analyzed locomotor activities of transgenic mice inother backgrounds and found that hyperactivity after
withdrawal of Dox administration was unique to mice in
D1R/D2R KO and D1R KO background, which have no44

endogenous D1R expression.
We also examined the protein expression level of D1R in
the striatum of transgenic mice in D1R/D2R DKO
background. The striatum is considered to be a major region
responsible for control of locomotor activity and containing
abundant D1R expression. During the process of recovery of
locomotor activity after Dox withdrawal, the transgene D1R
expression gradually increased while locomotor activity
fluctuated strikingly. These results indicate that the incrementof locomotor activity is not simply in proportion to theamount of D1R expression. Instead, increase of D1R
expression from an abnormally low level is critical. To
understand mechanisms of locomotor control through D1R
signaling, we are analyzing the relationship between the D1R
expression and altered behavior. In addition, we areinvestigating whether or not there is a critical period indevelopment for the regulation of locomotion and eating
behavior by dopaminergic transmission.

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

Figure 2. Locomotor activity of D1R transgenic mice that have
D1R/D2R DKO background. After withdrawal of Dox
administration, transient hyperactivity was observed.

Publication List

〔Original paper〕

H., Suzuki, M., Kiyonari, H., Abe, T., Sasaoka, T., and Ueno, N.
• Tao,(2009). Mouse prickle1, the homolog of a PCP gene, is essential for
epiblast apical-basal polarity. Proc. Natl. Acad. Sci. USA, 93, 21102115.

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION of GENOME DYNAMICS

Professor

HORIUCHI, Takashi

Assistant Professors:
Technical Staff:
Postdoctoral Fellow:
Graduate Students:
Technical Staff:
Secretary:

JOHZUKA, Katsuki
WATANABE, Taka-aki
MOROOKA, Naoki
KODAMA, Ken-ichi
ITAZU, Masako
OKAMOTO, Haruko
ISHINE, Naomi
YONEZAWA, Harumi
MATSUZAKI, Yoko
MIKAMI, Yuriko

The genomes of higher organisms contain significant
amounts of repetitive sequences which, in general, areunstable. At present, neither the physiological function(s) ofthese repeated sequences, nor the mechanism producing
repeated sequences and controlling instability are fully
understood. To clarify these aspects, we are pursuing several
themes using Saccharomyces cerevisiae, and Chinese
Hamster Ovary (CHO) cells. In 2009 we have made progressin understanding a new role of condensin in maintaining thelong repeated structure of rDNA, in which it is an essential
step that condensin is recruited to the RFB (replication fork
barrier) site in rDNA repeat units by three protein factors(Tof2p, Csm1p and Lrs4p) and Fob1 protein. In addition, we
also constructed a new system of gene amplification via
DRCR (double rolling circle replication) in yeast by using
Cre-lox site-specific recombination. Furthermore weconstructed an analogous system for the CHO chromosome
and found that it produced gene amplification products also
seen in tissue cultured cells under natural conditions.
Previously we constructed Eschericia coli cells with alinear genome and found they behave like E. coli with acircular genome (Cui et al., EMBO Rep. 8, 181-187, 2007).
We investigated how the genome behaves when the circulargenome is linearized by TelN protein.

I. Mechanism of condensin recruitment onto
the RFB site located within rDNA repeats inbudding yeast

The primary functions of chromosome condensation during
cell division are to facilitate the individualization of sister
chromatids, which remain entangled after DNA replication,and to shorten the length of chromatid arms so that they
avoid truncation during chromosome segregation to opposite
poles of the cell. The compaction ratio of mitotic
chromosomes relative to double stranded DNA ranges from~160-fold in budding yeast to ~10000-20000-fold in
mammalian chromosomes. Despite its being a fundamental
process for cell growth, we have a surprisingly poor
understanding of the higher levels of chromosome
organization. Many textbooks feature the radial loop modelof condensed mitotic chromosomes in which the chromatinfibers are bunched around a chromosome scaffold; thismodel is largely based on electron microscopy of mitotic

chromosomes under high-salt or polyanion conditions. One
clue for understanding the structure of condensed
chromosomes was the identification of components of thechromosomal scaffold, namely, Top2 and condensin.
Although, both of these are known to be required forbuilding up condensed mitotic chromosomes, theirrelationship to specific DNA elements is still poorly
understood. Condensin is a multi-subunit protein complex
that plays a central role in mitotic chromosome condensationand segregation. In vertebrates, condensin is distributed in
the axial part over the whole length of condensed
chromosomes, but this has only been shown at the resolutionof light microscopy. The sites where condensin acts on thechromatin and the molecular mechanisms of condensin
recruitment onto thereto had largely remained elusive. In
previous studies, we found that condensin localized to the
RFB site in a Fob1 (fork block protein) dependent manner
during S-phage. To date, this Fob1p-dependent condensin
localization is the only example of condensin association
with a specific DNA site in a specific protein factordependent manner. With the goal of understanding
chromosome condensation at the molecular level, we havestudied mechanisms of condensin localization at the RFB
site. This year we discovered that condensin can bind to ashort DNA fragment containing RFB sequences, even if thesequence was inserted at an ectopic chromosomal site. This
indicates that the RFB site itself acts as a site recruiting onto
chromatin. Analysis of the relationship between condensin
recruitment to the RFB site and Fob1p-dependent replication
fork blockage at the RFB site demonstrated that those two
events were completely independent phenomena. Instead, we
identified three additional protein factors, Tof2p, Csm1p, andLrs4p, necessary for both FOB1-dependent condensin
recruitment to the RFB site and for ensuring the faithfulsegregation of long rDNA repeats. We also found orderedbinding of Fob1p, Tof2p, Csm1p/Lrs4p, and condensin
complex at the RFB site, as shown in Figure 1. Finally, invivo interaction between Csm1p, Lrs4p and multiple subunitsof condensin were detected. These results suggest thatcondensin is recruited to the RFB site by the sequential
interaction of Fob1p, Tof2p, Csm1p, Lrs4p, and finallycondensin, to ensure the proper segregation of long rDNA
tandem array.

II. Condensin contributes to repressing
transcriptions by RNA polymerase II in the
rDNA cluster

As described above, we found that the replication fork
barrier (RFB) site, which is located in the intergenic spacer
region (IGS) within the ribosomal RNA gene (rDNA) cluster,
worked as a cis element for condensin recruitment tochromatin. Interestingly, recruitment of condensin to the
RFB site occurs in the DNA synthesis-phase, long before
mitotic-phase, suggesting that condensin plays some role
during interphase. We are investigating the mechanism ofcondensin recruitment to chromatin and its role inmaintaining the integrity and the higher order structure of therDNA repeated region. This year, we discovered thatcondensin contributed to silencing of RNA polymerase II

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
transcription from a reporter gene inserted close to the RFB
site. Condensin mutation leads to a decrease in the Sir2
association profile in rDNA. As well as the changes in the
Sir2 association profile, endogenous transcriptions by RNA
polymerase II within the IGS region increased in condensin
mutants. Based on these observations, we hypothesize thatcondensin has an effect on the higher-order chromatin
structures of the IGS region, resulting in the promotion ofSir2 accessibility to the region, thus contributing totranscriptional silencing.

III. Construction of a new gene amplification
system via DRCR (double rolling circle
replication) using Cre-lox site specificrecombination in budding yeast and CHO(Chinese hamster ovary) cell

In addition to rDNA gene amplification in eukaryotes, there
is another type of gene amplification, which is involved invarious biological phenomena, such as cancer development
and drug-resistance. However, the mechanism is largely
unknown because of the complexity of the amplification
process. Previously, we developed a gene amplification
system in S. cerevisiae that is based on double rolling-circle
replication (DRCR), utilizing break-induced replication(BIR). This system produced two types of amplification
products. Type-1 products contain 13 to ~100 copies of theamplification marker, lue2d (up to 730 kb increase) withnovel arrangement present as randomly oriented sequences
flanked by inverted leu2d copies. Type-2 products areacentric multi-copy mini-chromosomes, each carrying two
lue2d copies. Structures of type-1 and -2 products resemble
those of homogeneously staining regions (HSRs) and double
minutes (DMs) of higher eukaryotes, respectively.
Interestingly, products analogous to theses were generated at
low frequency without deliberate DNA cleavage.
If DRCR were an actual gene amplification mechanism inyeast, a quite different initiation reaction, which can induce
DRCR, should produce amplification products resembling
HSRs and DMs. Thus, we tried to construct a new DRCR
amplification system that is induced by another process; Crelox site-specific recombination. Cre-lox site-specificrecombination occurs between un-replicated and replicatedregions during replication, the fork replicates these alreadyreplicated regions again, and the Cre recombination system
makes this process more efficient. Furthermore, acombination of the process efficiently induces gene
amplification through DRCR. In fact this system produced
two kinds of products; the structures of these products
resemble those of HSRs and DMs of higher eukaryotes,
respectively. Furthermore, the same genetic elements
produce HSR- and DM-like products when placed incultured mammalian (CHO) cells. Thus, we concluded that
DRCR is indeed an amplification mechanism in lower andhigher eukaryotes and can be naturally initiated if somestructural requirement is satisfied.
Figure 1. Model for condensin recruitment to
the RFB site, contributing to maintenance of theintegrity of long rDNA repeats.

46

Figure 2. How do two termini of E. coli genome behave when the circular genome is linerized?

IV. E. coli with linear genome: how do two
termini of the genome behave when thecircular genome is linearized?

Previously, we constructed E. coli with a linear genome(Cui et al., EMBO Rep. 8, 181-187, 2007). Interestingly, theE. coli strain with a linear genome did not show any distinct
characteristics in comparison with circular genome E. coli.
Here, we investigate how each end of the linear genome
behaves when the circular genome is linerized with TelN
enzyme (linearization enzyme coded in N15 lysogenic phage
genome). We expected two possibilities; one was that eachend would move randomly, at least for a while, the other wasthat each end would separate to each pole position of the cell
(Figure 2).
In order to know which possibility was correct, thefollowing E. coli strain was constructed; tos sequence, atarget sequence for TelN enzyme, was inserted into the site
within the replication terminus region (the site close to dif),an opposite region of replication origin (oriC) of the circulargenome. Then, at both sides of tos, two kinds of operator(tetO and lacO) repeated sequences were inserted. Finally,
CFP-tet and YFP-lac repressor genes were inserted
downstream of arabinose promoter, and telN gene waspositioned downstream of a rhamnose promoter on the E.
coli genome.
The strain was grown in M9-succinate medium logalismicphase and then arabinose was added to the medium to induce
CFP-tet and YFP-lac repressors, which bind to the tetO andlacO sites, respectively, close to each other before
linearization. Then, by addition of rhamnose to the culture,
TelN enzyme was induced, which cut the tos site andconverted the circular genome to a linear one. During theseexperiments, we examined the behavior of two fluorescentspots under a microscope. The observations show that thetwo terminuses, after linearization, do not move randomly,
but one stays in place and the other tends to move to theother pole site of the cell. Average rate of the latter type of

movement is approximately 0.216µ/min. Because the DNA
is neither dramatically stretched nor shrunk, these results
strongly suggest participation of proteins.

Publication List

〔Original paper〕

K., and Horiuchi, T. (2009). The cis-element and factors• Johzuka,
required for condensin recruitment to chromosome. Mol. Cell 34, 2635.

47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF MOLECULAR GENETICS †Professor

IIDA, Shigeru

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

Visiting Scientist:
Technical Assistants:

Secretary:

TERADA, Rie
HOSHINO, Atsushi
TSUGANE, Kazuo
FUKADA-TANAKA, Sachiko
MORITA, Yasumasa
JOHZUKA-HISATOMI, Yasuyo
PARK, Kyeung-Il
EUN, Chang-Ho
MORITOH, Satoru
YAMAUCHI, Takaki
ONO, Akemi
TSUGANE-HAYASHI, Mika
SHIMATANI, Zenpei
SAITOH, Hiromi
ASAO, Hisayo
MATSUMOTO, Miwako
SHIMAMOTO, Miki
MATSUDA, Chisato
HASEGAWA, Yoshinobu
SANJO, Kazuko

The main interest of this division was characterizing various
aspects of genetic and epigenetic gene regulations including
the flower pigmentation of morning glories. In addition, wewere undertaking reverse genetic approaches in order toelucidate the nature of dynamic genomes in rice, a model
plant for cereals.
Because the activities of this group were terminated at theend of March, please consult the last year’s annual report formost of our activities with the exception of the following
knock-in targeting of a rice gene by homologous
recombination.

I. Knock-in targeting of endogenous naturalgenes by homologous recombination in rice

Although the analysis of the expression of a reporter gene
fused with an appropriate promoter segment in transgenic
plants, often termed promoter-reporter gene fusion analysis,
is widely used to characterize the cloned promoter
sequences, inter-individual variation of the reporter gene
expression among transgenic plants, which is attributed
mainly to the insertion sites and/or copy number of thetransgene and epigenetic gene silencing, hampers proper
spatiotemporal evaluation of the promoter activity. To
circumvent such problems, an approach known as a“promoter trap” is also employed; a transformant having a
promoterless reporter gene, which is carried by appropriately
modified T-DNA sequences or transposons, integrated into
an exon of an endogenous target gene in the proper
orientation and resulting in the reporter gene expression bytranscriptional fusion, is to be isolated for promoter analysis
among the transformants containing the randomly inserted
transgenes. Because the promoter trap is based on random
insertional mutagenesis, the isolation of an appropriate
promoter-trapped mutant relies on a fortuitous integration of
the transgene into the target gene of interest, even though the†: This laboratory was closed on 31 March, 2009.

48

promoter trap is much more likely to accurately reflect theexpression of a gene than promoter-reporter gene fusion.
Moreover, simultaneous integration of additional transgene
copies may cause a potential problem because multiple
insertions may complicate the interpretation of
spatiotemporal expression patterns. As is the case in mice,the homologous recombination-promoted knock-in-targetingstrategy is expected to be a powerful tool to monitor
promoter activity accurately in plants. Knock-in targeting
appears to offer at least two advantages over the promoter
trap; the junction sequence between a reporter gene and an
endogenous target promoter can be designed properly, andtransgenic plants carrying an identical and desired knock-in
allele can be repeatedly obtained. We have succeeded in
obtaining 15 independent and fertile transgenic knock-in rice
plants, which have only one copy of the promoterless GUS
reporter gene encoding b-glucuronidase fused with theendogenous promoter of MET1a, one of two rice MET1
genes encoding a maintenance DNA methyltransferase. As
Figure 1 shows, the spatiotemporal gene expression of GUS
was reproducibly observed in a dosage-dependent manner
among independently isolated plants.

Figure 1. Reproducible and dosage-dependent expression of GUS in
knock-in targeted plants. (Left) Embryos in T1 plants. (Right) In situ
hybridization pattern of MET1a in embryos of Nipponbare. The symbols
G/G, +/G, and +/+ indicate GUS/GUS, MET1a/GUS, and MET1a/
MET1a, respectively.

Publication List

〔Original papers〕

A., Park, K.I., and Iida, S. (2009). Identification of r mutations• Hoshino,
conferring white flowers in the Japanese morning glory, Ipomoea nil. J.
Plant Res. 122, 215-222.

K., Yasuno, N., Oikawa, T., Iida, S., Nagato, Y.,• Ikeda-Kawakatsu,
Maekawa., M., and Kyozuka, J. (2009). Expression level of ABERRANT••PANICLE ORGANIZATION determines rice inflorescence form throughcontrol of cell proliferation in the meristem. Plant Physiol. 150, 736747.
Shimatani, Z., Takagi, K., Eun, C.H., Maekawa, M., Takahara, H.,
Hoshino, A., Qian, Q., Terada, R., Johzuka-Hisatomi, Y., Iida, S., andTsugane, K. (2009). Characterization of autonomous Dart1 transposons
belonging to the hAT superfamily in rice. Mol. Gen. Genomics 281,
329-344.
Yamauchi, T., Johzuka-Hisatomi, Y., Fukada-Tanaka, S., Terada, R.,
Nakamura, I., and Iida, S. (2009). Homologous recombination-mediatedknock-in targeting of the MET1a gene for maintenance DNA
methyltransferase
reproducibly
reveals
dosage-dependent
spatiotemporal gene expression in rice. Plant J. 60, 386-396.

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

Associate Professor

Assistant Professor:
Technical Staff:
NIBB Research Fellows:

HIWATASHI, Yuji
KABEYA, Yukiko
TAMADA, Yosuke
OKANO, Yosuke
MIYAZAKI, Saori
ISHIKAWA, Takaaki
MANO, Hiroaki
OKANO, Yosuke*
OHSHIMA, Issei
AOYAMA, Tsuyoshi
FUJII, Tomomi
BABA, Nayumi
GOTO, Miho
ITO, Yukiko
KAJIKAWA, Ikumi
KIMURA, Yasuyo
OOMIZU, Yuka
TSUKAMOTO, Naho
SAKURAI-OZATO, Nami
WASHIO, Midori
YAMADA, Hiroko
BASKIN, Tobias
KOJIMA, Yoko

HASEBE, Mitsuyasu

Postdoctoral Fellows:

Graduate Students:
Technical Assistants:

Visiting Scientist:
Secretary:

MURATA, Takashi

All living organisms evolved from a common ancestor that
lived more than 3.5 billion years ago, and the accumulationof mutations in their genomes has resulted in the present
biodiversity. Traces of the evolutionary process are found in
the genomes of extant organisms. By comparing the gene
networks (and their functions) of different organisms, wehope to infer the genetic changes that caused the evolution ofcellular and developmental processes.

I. Evolution from unicellular to multicellularorganisms

The first evolutionary step from unicellular to multicellularorganisms is the formation of two different cells from asingle cell via asymmetric cell division. The first cell
division of a protoplast isolated from the protonemata of themoss Physcomitrella patens is asymmetric regarding its
shape and nature, and gives rise to an apical pluripotent stem
cell and a differentiated protonema cell. A systematic
overexpression screening for genes involved in asymmetriccell division of protoplasts in P. patens was performed for
4,000 full-length cDNA clones. We identified 58 cDNAs
whose overexpression caused defects in asymmetric cell
divisions and their functional analyses are in progress. This
work was performed as a collaboration with Dr. Tomomichi
Fujita (Hokkaido University).

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

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 centrifugal expansion of thephragmoplast was a challenge. Based on live imaging of
a-tubulin at a light microscopic level andimmunolocalization of g-tubulin at an electron microscopic
level, we proposed a hypothesis that cytosolic g-tubulin
complexes are recruited onto existing phragmoplast
microtubules and nucleate new microtubules as branches,
and that the branched microtubules drive phragmoplast
expansion. Seeing the life history of microtubules in thephragmoplast had been very difficult by live imaging of
a-tubulin, but we successfully tracked the trajectories ofgrowing microtubule ends in the phragmoplast using twophoton microscopy of a microtubule plus-end marker EB1.
Microtubules appeared in many sites in the phragmoplast andelongated obliquely towards the cell plate. We also foundthat inhibition of g-tubulin function by antibody injection
inhibited formation of new microtubules and phragmoplast
expansion. These results support our hypothesis. Takashi
Murata was this study’s main researcher.
Microtubules form arrays with parallel and antiparallel
bundles and function in various cellular processes including
cell division. The interdigitation of antiparallel microtubulesin phragmoplasts, plant-unique microtubules arrays, isformed and stabilized by microtubules-associated proteins(MAPs) including kinesins. However, mechanisms fordisassembly of the interdigitation of antiparallel
microtubules are still unclear. Here we show a type II
ubiquitin-like domain protein, PUBL1 and PUBL2 (forPhyscomitrella patens ubiquitin-like domain protein 1 and2), are novel factors regulating disassembly of the
antiparallel bundles and depolymerization of the
microtubules in the phragmoplasts. PUBL1 and PUBL2
proteins were predominantly localized to the interdigitationof the antiparallel microtubules. In the double-deletion linesof both genes, the collapse of the interdigitating microtubulesin the phragmoplasts was retarded, indicating that PUBL1
and PUBL2 are indispensable for proper loss of theinterdigitation. A kinesin, KINID1a, for generation of theinterdigitation aberrantly persisted in the phragmoplast
equator, suggesting that the crosslink between the plus endsof the antiparallel microtubules is properly lost in the doubledeletion lines. Furthermore, double-deletion lines exhibited
formation of incomplete cell plate and multinucleated cells,suggesting that PUBL1 and PUBL2, the proper disassemblyof the interdigitation, or both are required for proper lateralexpansion of the cell plate. Yuji Hiwatashi mainly performed
this study.

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

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

III. Evolution of molecular mechanisms inplant development
3-1 Stem cell initiation and maintenance

The initiation and maintenance of several types of stem
cells to produce different types of differentiated cells is
precisely regulated during the development of multicellularorganisms. Molecular mechanisms for stem cellcharacterization, however, have remained largely unknown.
We showed that AINTEGUMENTA/PLETHORA/BABY
BOOM (APB) orthologs PpAPBs (PpAPB1, 2, 3, and 4) are
involved in stem cell characterization in the moss
Physcomitrella patens. Gametophore stem cells were
induced by exogenous cytokinin in the wild type, while thequadruple disruptants did not form any gametophore stem
cells with exogenous cytokinin application. These results
suggest that the PpAPBs play a critical role in thecharacterization of a gametophore stem cell. Meanwhile, theexpression of PpAPBs is regulated by auxin, not cytokinin.
The primary researchers for this study were Tsuyoshi
Aoyama and Yuji Hiwatashi.

3-2 Nuclear genome project of the moss
Physcomitrella patens

A comparison of developmental genes among major land
plant taxa would facilitate our understanding of theirevolution, although it has not been possible because of thelack of genome sequences in basal land plants. We
established an international consortium for a genome projectof the moss Physcomitrella patens and the lycopod
Selaginella moellendorffii. After publication of the draft
genome of Physcomitrella patens, to further elaborate thecontig assembling and the gene annotation, we performed (1)EST analyses of several libraries of cDNAs isolated fromdifferent developmental stages; (2) construction of fulllength cDNA libraries and sequencing in their full length; (3)construction of BAC libraries and their end-sequencing; (4)5’-end serial analysis of gene expression (5’ SAGE); and (5)a collection of 3’ UTR and small RNA sequences as
collaborative works with groups associated with Dr. Tomoaki
Nishiyama (Kanazawa Univ.), Prof. Asao Fujiyama (National
Institute of Informatics), Prof. Sumio Sugano (Univ. Tokyo),and Prof. Yuji Kohara (National Institute of Genetics).
We developed a system to efficiently construct phylogenetic
trees with whole genome shotgun sequence data in public
databases before their assembly. We collected homologs ofapproximately 700 Arabidopsis thaliana genes involved indevelopment, and their phylogenetic analyses are in progress.

3-3 Functional characterization of polycomb
genes in the moss Physcomitrella patens

Land plants have distinct developmental programs in
haploid (gametophyte) and diploid (sporophyte) generations.
Although usually the two programs strictly alternate at
fertilization and meiosis, one program can be induced during
the other program. In a process called apogamy, cells of thegametophyte other than the egg cell initiate sporophyte
development. Here, we report for the moss Physcomitrella

50

patens that apogamy resulted from deletion of the geneorthologous to the Arabidopsis thaliana CURLY LEAF
(PpCLF), which encodes a component of polycomb
repressive complex 2 (PRC2). In the deletion lines, agametophytic vegetative cell frequently gave rise to asporophyte-like body. This body grew indeterminately froman apical cell with the character of a sporophytic pluripotent
stem cell but did not form a sporangium. Furthermore, with
continued culture, the sporophyte-like body branched.
Sporophyte branching is almost unknown among extant
bryophytes. When PpCLF was expressed in the deletion lines
once the sporophyte-like bodies had formed, pluripotent stem
cell activity was arrested and a sporangium-like organ
formed. Supported by the observed pattern of PpCLF
expression, these results demonstrate that, in thegametophyte, PpCLF represses initiation of a sporophytic
pluripotent stem cell and, in the sporophyte, represses thatstem cell activity and induces reproductive organ
development. In land plants, branching, along withindeterminate apical growth and delayed initiation of sporebearing reproductive organs were conspicuous innovations
for the evolution of a dominant sporophyte plant body. Our
study provides insights into the role of PRC2 gene regulation
for sustaining evolutionary innovation in land plants. Yosuke
Okano and Takaaki Ishikawa were this study’s primaryresearchers.

Figure 1. A PpCURLY LEAF deletion mutant formed sporophyte-likebodies with expression of a sporophyte stem cell specific MKN4 gene(left). A sporophyte-like body formed branches (center), which is similar
to extinct protracheophytes (right).

IV. Molecular mechanisms of female and maleinteractions

In sexual reproduction, proper communication andcooperation between male and female organs and tissue are
essential for male and female gametes to unite. In flowering
plants, female sporophytic tissues and gametophyte direct amale pollen tube towards an egg apparatus, which consists ofan egg cell and two synergid cells. The cell-cellcommunication between the pollen tube and the egg
apparatus, such as the reception of a signal from the egg
apparatus at the pollen tube, makes the tip of pollen tube
rupture to release the sperm cell. To detect male factors
involved in this communication, we screened mutants ofreceptor-like kinases expressed in pollen tubes andcharacterized the ANXUR1 (ANX1) and ANXUR2 (ANX2)
genes. Here we report that pollen tubes of anx1/anx2
ruptured before arriving at the egg apparatus, suggesting that
ANX1 and ANX2 are male factors controlling pollen tube

behavior by directing rupture at the proper timing.
Furthermore, ANX1 and ANX2 were the most closely relatedparalogs to a female factor, FERONIA/SIRENE, controllingpollen tube behavior expressed in synergid cells. Our findingshows that the coordinated behaviors of female and male
reproductive apparatuses are regulated by the sister genes,
whose duplication might play a role in the evolution offertilization system in flowering plants. This work was
mainly done by Saori Miyazaki.

Figure 2. Pollen tubes of wild type (left) and the anx1;anx2 mutant (right)
on culture medium. Ruptured anx1;anx2 pollen tube released sperm
nuclei together with pollen tube cytoplasm before fertilization.

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 suggested that xanthommatin, a red
pigment belonging to the ommochrome family, contributes tothe pink body coloration of the mantis. We also found thatthe orchid mantis contains large amounts of leucopterin andisoxanthopterin, both of which are known as white
compounds in other insects such as Pierid butterflies. These
results indicate that the “flower-like” coloration of the orchid
mantis is formed by pigments unrelated to those used for thecoloration of flowers.
The orchid mantis drastically changes its appearance during
post-hatching development. The first-instar nymph of themantis is colored red and black and is believed to mimicother unpalatable insects like ants. A flower-like appearance
emerges after molting into the 2nd-instar nymph. We aim tocompare the gene expression profiles between the 1st- and2nd-instar nymphs using a high-throughput DNA sequencer.
This work was mainly done by Hiroaki Mano.

VI. Molecular mechanisms of host shifting

Although plant-feeding insects as a whole utilize various
plant species, the majority of plant-feeding insect species areassociated with one or a few plant species. Such mono- andoligophagous insect species are highly specialized to theirrespective host plant species via larval physiological
adaptation (assimilability) and host preference of adultfemales. Thus, the process of host shifting to a novel plant
species involves the evolution of multiple traits. The
molecular mechanisms underlying such multi-trait evolution
are largely unknown. To address the molecular mechanism of
host shifting, we use two host races in a tiny moth,Acrocercops transecta, as a model system. Host races inplant-feeding insects are subpopulations that are specialized
to different species of host plants, so we can conduct QTL
analyses of the host-adaptation traits by crossing the two hostraces. The segregation patterns of larval assimilability andovipositing female preference in F2 and backcrossgenerations indicated that the two traits were governed by a
few major loci, but were under different genetic control. To
test whether these loci are physically linked with each otheror not, mapping analyses are in progress. This study was
conducted mainly by Issei Ohshima.

Publication List

〔Original papers〕

R., Yoshimura, T., Inoue, H., Sakakibara, K., Hiwatashi, Y.,• Kofuji,
Kurata, T., Aoyama, T., Ueda, K., and Hasebe, M. (2009). Gametangia
development in the moss Physcomitrella patens. In The Moss
Physcomitrella patens, D. Cove, F. Perroud, and C. Knight, eds.
(Wiley-Blackwell), pp. 167-181.

S., Murata, T., Sakurai-Ozato, N., Kubo, M., Demura, T.,• Miyazaki,
Fukuda, H., and Hasebe, M. (2009). ANXUR1 and 2, sister genes to••

FERONIA/SIRENE, are male factors for coordinated fertilization. Curr.
Biol. 19, 1327-1331.
Oda, Y., Hirata, A., Sano, T., Fujita, T., Hiwatashi, Y., Sato, Y., Kadota,
A., Hasebe, M., and Hasezawa, S. (2009). Microtubules regulate
dynamic organization of vacuoles in Physcomitrella patens. Plant Cell
Physiol. 50, 855-868.
Okano, Y., Aono, N., Hiwatashi, Y., Murata, T., Nishiyama, T., Ishikawa,T., Kubo, M., and Hasebe, M. (2009). A polycomb repressive complex
2 gene regulates apogamy and gives evolutionary insights into early
land plant evolution. Proc. Natl. Acad. Sci. USA 106, 16321-16326.

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

KAWAGUCHI, Masayoshi

Assistant Professor:
Technical Staff:
Postdoctoral Fellows:

Visiting Scientists:
Graduate Student:
Secretary:

TAKEDA, Naoya
FUKADA-TANAKA, Sachiko
FUJITA, Hironori
MURAKAMI, Yasuhiro
MIWA, Hiroki
OKAMOTO, Satoru
YOKOYAMA, Hiroshi
MAGORI, Shimpei
YOSHIDA, Chie
MIYAZAWA, Hikota
TAKAHARA, Masahiro
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, which was launched in April 2009, focuses on theearly stages of the interaction between these microorganisms
and Lotus japonicus in order to reveal the molecularmechanism and the origin of these symbiotic systems.

roots and transmitted to the shoot; and a shoot-derived signalthat systemically inhibits nodulation. In L. japonicus, theleucine-rich repeat receptor-like kinase, HAR1, mediates
AON and nitrate inhibition of nodulation, and ishypothesized to recognize the root-derived signal in theshoot.
Among 39 L. japonicus CLE genes, we identified LjCLERS1 and LjCLE-RS2 as strong candidates for the root-derived
signal. These genes are not expressed in shoots but strongly
up-regulated in the rhizobial-inoculated roots. A time course
analysis for gene expression revealed that the induction of
LjCLE-RS1 and -RS2 started to up-regulate 3 hours after
rhizobial inoculation and the timing for the induction of
those genes was earlier than that for the initiation of theregulatory response of nodulation that is detectable 3 days
after rhizobial inoculation in L. japonicus. By using the hairyroot transformation method, in the wild-type background, we
showed that overexpressing LjCLE-RS1 and -RS2 inhibits
nodulation and this effect was also observed in nontransformed roots, whereas overexpressing LjCLE3 did not
repress nodulation (Figure 1). By contrast, in the har1 mutant
background, overexpressing LjCLE-RS1 and -RS2 did notinhibit nodulation. Thus, overexpressing LjCLE-RS1 and-RS2 inhibits nodulation systemically and this effect depends
on HAR1 receptor-like kinase.

I. Long-distance control of nodulation

Legume plants develop root nodules to recruit nitrogenfixing bacteria called rhizobia. This symbiotic relationshipallows the host plants to grow even in nitrogen poor
environments. Since nodule development is an energeticallyexpensive process, the number of nodules must be tightly
controlled by the host plants. For this purpose, legume plants
utilize a long-distance signaling known as autoregulation of
nodulation (AON). AON signaling in legumes has been
extensively studied over decades but the underlying
molecular mechanism has remained largely unclear. We aretrying to unveil the mechanism for AON at the molecularlevel.

1-1 Exploration for root-derived long-distancesignal(s) involved in autoregulation of
nodulation

Host legumes control root nodule numbers by sensing
external and internal cues. A major external cue is soil
nitrate, whereas a feedback regulatory system in which
earlier formed nodules suppress further nodulation throughshoot-root communication is an important internal cue. The
latter, AON, is believed to consist of two long-distancesignals: a root-derived signal that is generated in inoculated

52

Figure 1. Wild type plants possessing hairy roots overexpressing LjCLE3(A to C) and overexpressing LjCLE-RS1 (D to F). Blue arrowheads
indicate normal untransformed root system. Red arrowheads indicate theinitiation site of the hairy root system. Bars = 1 cm.

Nitrogen depletion in the soil is a prerequisite for nodule
development and function, and high concentrations of
nitrogen as nitrate or ammonia abolishes nodulation.
Mutations in HAR1 exhibit nitrate-tolerant symbiotic
phenotype. Through expression analysis using 39 LjCLE
genes, we found that LjCLE-RS2 was strongly andspecifically up-regulated in the root in response to nitrate.
Exposure of seedlings to different nitrate concentrations
showed that the level of LjCLE-RS2 transcript accumulation
increased by adding 1 mM KNO3 and reached a maximum at

30 mM KNO 3, a concentration that interferes with L.
japonicus nodulation. Based on the finding, we proposed amodel in which nitrate-induced LjCLE-RS2 inhibits
nodulation via HAR1 receptor-like kinase.

1-2 TML, a root regulator associated with thelong-distance control of nodulation

too much love, tml, is a novel hypernodulating mutant
isolated by C 6+ beam mutagenesis of the seeds of L.
japonicus Miyakojima MG-20. To locate the potential site ofaction of TML, we conducted reciprocal grafting experimentswith the wild type and tml mutants. The seedlings were used
for wedge grafting surgery and the successful grafts weretransferred to vermiculite and inoculated with
Mesorhizobium loti. Grafting a tml shoot onto a wild-typeroot led to nodulation in the wild-type; in contrast, grafting awild-type shoot onto a tml root resulted in an increased
number of nodules/nodule primordia, which wasindistinguishable from that of tml self-grafts. This rootdetermined hypernodulation of tml indicates that unlike
HAR1 and KLV, TML functions in the roots rather than in theshoots.
The role of TML in the roots but not in the shoots prompted
us to ask whether a root factor TML and a shoot factor HAR1
genetically interact with each other despite the different sitesof action. For this purpose, we carried out reciprocal grafting
using tml and har1-7 mutants. We confirmed that thehypernodulation of har1-7 is regulated by the shoots,
consistent with the previous studies using different har1
alleles. This shoot-regulated har1-7 hypernodulation was not
obviously enhanced by grafting a har1-7 shoot onto a tml
root, suggesting that TML and HAR1 might constitute thesame long-distance signaling. On the other hand, grafting a
tml shoot onto a har1-7 root complemented thehypernodulation of each other, further supporting the specificroles of TML and HAR1 in the roots and the shoots,
respectively.
Based on these findings, at least two potential mechanisms
by which a root factor TML exerts its inhibitory effect on
nodulation can be speculated: TML might perceive or
mediate an unknown shoot-derived signal produced by
HAR1, or TML might generate or relay a root-derived signal.
To examine which hypothesis is more valid, we designed
inverted-Y grafting, where a sliced root is grafted into a shortslit made in a stock plant. The inverted-Y grafting
experiments suggested that the effect is likely to be local,
supporting the former hypothesis that TML might function
downstream of HAR1.

II. Arbuscular mycorrhiza symbiosis

In the arbuscular mycorrhiza (AM) symbiosis, plant roots
accommodate Glomeromycota fungi within an intracellularcompartment, the arbuscule. At this symbiotic interface,
fungal hyphae are surrounded by a plant membrane, which
creates an apoplastic compartment, the periarbuscular space(PAS) between fungal and plant cell. Despite the importanceof the PAS for symbiotic signal and metabolite exchange,
only few of its components have been identified. Part of thisreason is that the AM developmental process does not show

clear morphological changes like nodule formation. Having
no clear check point of the inoculation process makes it
difficult to characterize responses to AM impregnation in thehost plant.
In order to solve the problem, we took advantage of AM
gene marker SbtM1 and LjPT4 isolated in L. japonicus.
Subtilisin-like serine protease SbtM1 and phosphatetransporter LjPT4 were induced soon after contact of thefungal hyphae to the host epidermal cells and showed thestrongest induction in arbuscule containing cells.
Suppression of SbtM1 gene function by RNAi caused low
AM colonization in the host root. This result indicates SbtM1
has an essential role in AM development. LjPT4 was isolated
as a homolog of MtPT4. Knockdown of MtPT4 expression
was reported to cause premature death of arbuscules,
meaning that PT4 is also required for AM development.
Considering the AM specific expression patterns and theimportance of SbtM1 during AM development, we employedthese genes as an indicator of AM inoculation. Promoter
GUS fusion showed strong activation of the promoter by
infection of AM fungi (Figure 2A-C) but they were notinduced in symbiosis mutants. Fluorescent protein fusion
with SbtM1 enabled visualization of the AM inoculation
process (Figure 2D, E). Genetic screening of symbiosis
mutants which abolished AM specific signaling andregulation system is in progress. The AM specific makers
would be a powerful tool to analyze AM phenotypes in novel
mutants.

Figure 2. Transformed L. japonicus roots containing SbtM1
promoter:GUS showed staining in response to AM inoculation (A).
Stronger GUS staining was observed in arbuscule containing cells
compared with the neighboring cells (B and C). AM fungi were stainedwith WGA Alexa 488 (C). SbtM1 signal peptide:Venus fusion visualized
intraradical fungal structures in the host root (D and E).

III. Mathematical models of shoot apicalmeristem

The shoot apical meristem (SAM) of plants contains stem
cells that have the ability to renew themselves anddifferentiate all aerial tissues such as stems and leaves. theSAM consists of a central zone (CZ) and its surrounding area
named the peripheral zone (PZ) that is induced by an
unknown signal from the CZ. Maintenance of the SAM
essentially involves the interaction between WUS and CLV,
in which WUS activates itself and CLV, but CLV inhibits
WUS expression. While clv mutants show enlarged SAM
and stems with fasciation and dichotomous branching, wus
mutants generate a flattened structure of the SAM because of
reduced SAM activity but produce many ectopic shoots to

53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

generate bushy plants after prolonged incubation.
Since it is not clarified how the SAM controls its
proliferation and patterning, we constructed and analyzed amathematical model of the SAM that includes conditions of
WUS-CLV dynamics, PZ induction by CZ, and area
expansion by cell division. In numerical simulations, theSAM maintains a constant cell number under the wild typecondition because of the balance between increase of cells bycell division and departure from the SAM to the outer region(Figure 3). Whereas strong clv mutation results in an
enlarged and elongated SAM, in weak clv mutants the SAM
initially elongates and then divides into two independent
SAMs. These phenotypes correspond to fasciation anddichotomous branching of stems in clv mutants. This model
successfully generates the wild type and clv mutant
phenotypes.

Figure 3. Numerical simulations under conditions of the wild type (A),weak (B) and strong (C) clv mutations.

Publication List

〔Original papers〕

T., Niimi, K., Watanabe, H., Tabata, R., Matsubara, J., Sato,• Hakoyama,S., Nakamura, Y., Tabata, S., Li, J., Matsumoto, T., Tatsumi, K.,

•
•
•
•
•
•
54

Nomura, M., Tajima, S., Ishizaka, M., Yano, K., Imaizumi-Anraku, H.,
Kawaguchi, M., Kouchi, H., and Suganuma, N. (2009). Host plant
genome overcomes a lack of bacterial gene for symbiotic nitrogen
fixation. Nature 462, 514-517.
Karas, B., Amyot, L., Johansen, C., Sato, S., Tabata, S., Kawaguchi, M.,
and Szczyglowski, K. (2009). Conservation of Lotus and Arabidopsis
basic helix- loop-helix proteins reveals new players in root hairdevelopment. Plant Physiol. 151, 1175-1185.
Kubo, M., Ueda, H., Park, P., Kawaguchi, M., and Sugimoto, Y. (2009).
Reactions of Lotus japonicus ecotypes and mutants to root parasitic
plants. J. Plant Physiol. 166, 353-362 .
Maekawa, T., Maekawa-Yoshikawa, M., Takeda, N., Imaizumi-Anraku,
H., Murooka, Y., and Hayashi, M. Gibberellin controls the nodulation
signaling pathway in Lotus japonicus. (2009). Plant J. 58, 183-194
Maekawa-Yoshikawa, M., Müller, J., Takeda, N., Maekawa, T., Sato,S., Tabata, S., Perry, J., Wang, T.L., Groth, M., Brachmann, A., andParniske, M. (2009). The temperature-sensitive brush mutant of thelegume Lotus japonicus reveals a link between root development andnodule infection by rhizobia. Plant Physiol. 149, 1785-1796.
Magori, S., Oka-Kira, E., Shibata, S., Umehara, Y., Kouchi, H., Hase,Y., Tanaka, A., Sato, S., Tabata, S., and Kawaguchi, M. (2009). TOO
MUCH LOVE, a root regulator associated with the long-distancecontrol of nodulation in Lotus japonicus. Molecular Plant-Microbe
Interactions 22, 259-268.
Okamoto, S., Ohnishi, E., Sato, S., Takahashi, H., Nakazono, M.,
Tabata, S., and Kawaguchi, M. (2009). Nod factor/nitrate-induced CLE•

genes that drive HAR1-mediated systemic regulation of nodulation.
Plant Cell Physiol. 50, 67-77.
Takeda, N., Sato S., Asamizu, E., Tabata, S., and Parniske, M. (2009).
Apoplastic plant subtilases support arbuscular mycorrhiza development
in Lotus japonicus. Plant J. 58, 766-777.

〔Review article〕

S., and Kawaguchi, M. (2009). Long-distance control of• Magori,
nodulation: molecules and models. Molecules Cells 27, 1-10.

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.

55

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
KIMURA, Tetsuaki
KANEKO, Hiroyo
YOSHIMURA, Yuriko
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
KOIKE, Chieko
HOSOMI, Azusa
TESHIMA, Yuuko
KAMATA, Akiko
SUZUKI, Tokiko

Teleosts comprise about half of all vertebrate species andhave adapted to a variety of environments, including seawater, fresh water, the bottom of deep seas, small creeks andpaddy fields. Analysis of their genome structure is importantin order to understand the adaptation and diversification inthis interesting group. Medaka is a small egg-laying“secondary” fresh water fish found in brooks and ricepaddies in Eastern Asia. This species has a long history as anexperimental animal, especially in Japan. Our laboratory has
conducted a comparative genomic analysis focusing mainly
on fish chromosomes and gene evolution using medaka andother fishes, and identification of the causal gene of mutantsfor PGC migration. In addition to these activities, ourlaboratory 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

The sex-determining gene DMY was identified on the Y
chromosome in the medaka, Oryzias latipes. However, thisgene is absent in most Oryzias fishes, suggesting that closely
related species have different sex-determining genes. We
have recently demonstrated that, in the javanicus species
group, O. dancena and O. minutillus have an XX/XY sex
determination system, while O. hubbsi and O. javanicus havea ZZ/ZW system (Figure 1). Linkage analysis and FISH
analysis showed that the sex chromosomes in these species
were not homologous, suggesting independent origins ofthese sex chromosomes. Furthermore, O. javanicus and O.

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

56

hubbsi have morphologically heteromorphic ZW sex
chromosomes, in which the W chromosome has DAPIpositive heterochromatin. These findings suggest therepeated evolution of new sex chromosomes from autosomesin Oryzias, probably through the emergence of a new sexdetermining gene.

II. Genetic dissection of migration ofprimordial germ cells in the medaka

The migratory pathways of PGCs to the gonad varydepending on the vertebrate species, yet the underlying
regulatory mechanisms guiding PGCs are believed to becommon between species. In the medaka embryo, PGC
migration follows two major steps before colonizing in
gonadal areas: (1) bilateral lineup in the trunk, and (2)posterior drift. kazura (kaz) and yanagi (yan) mutants of
medaka isolated in our mutagenesis-screening were defectivein the first and second steps, respectively. Positional cloning
identified phenotype responsible mutations in the chemokine
receptor genes cxcr4b and cxcr7, respectively. Althoughbelonging to the same chemokine receptor families,
involvement of these genes in the regulation of PGC
migration was clearly distinct. cxcr4b is expressed in the
PGCs themselves, suggesting a cell-autonomous function. In
contrast, cxcr7 is not expressed in the PGCs but in themesoderm-derived tissues connected to the route of PGC
migration, the ventral part of the somites in the area always
immediately anterior to the PGCs drifting along the bilateralroutes toward the gonad, and the pronephric ducts (Sasado etal., 2008). Further analysis of the mutants is underway toreveal the function of the two chemokine-systems in theregulation of PGC migration. kamigamo (kmg) andshimogamo (smg) are both recessive lethal mutations
showing PGC distribution defects similar to that of yan but
in different complementation groups (Sasado et al., 2004).
Positional cloning of the responsible genes of the mutations
is now in progress.

III. T h e d e v e l o p m e n t o f i n s e r t i o n a lmutagenesis system with Tol2 transposon

Mutagenesis screens by N-ethyl-N-nitrosourea have been
performed in medaka. However, the cloning of chemicallymutated genes is still laborious even after completion of
medaka genome sequencing. In order to rapidly identify acausal mutation of phenotype, we undertake insertionalmutagenesis in medaka using a transposable element, Tol2.
Tol2 is a transposable element of the hAT transposable
element family, residing in the genome of the medaka. The
Hd-rR, an inbred strain sequenced in the genome project, has
14 integration sites in its genome. These sites are stably
inherited in the Hd-rR.
We demonstrated that Tol2 can be excised in the Hd-rR
genome when transposase mRNA, synthesized in vitro, is
injected. Multiple different excisions occurred in founderfish. These mutations were highly heritable, however due tothe mosaic like nature of the germ cells progeny showedvarious new mutations. Our results show that Tol2 is usefulas a mutagen in medaka.

IV. National BioResource Project Medaka(NBRP Medaka)
4-1 Full length cDNA sequencing projectTo establish the full length cDNA resources of medaka wemade 11 full length cDNA libraries (developmental stage 22,35, 40, ovary, testes, brain, male liver, female liver, gill,kidny and spleen) and determined the sequences of both ends
of 250,000 clones in collaboration with the National Instituteof Genetics’ Kohara and Fujiyama labs. Now 499,944
sequences are availabe. After mass alignment of all
sequences, we found 21,588 independent sequences at the 3’
ends. All of the data was deposited in the DDBJ and isaccessible from the National BioResource Project Medaka
website (http://www.shigen.nig.ac.jp/medaka/).

4-2 Establishment of core facility of NBRP
medakaIn 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).
NBRP Medaka aims to establish a first rate biologicalresource with the highest possible levels of accessibility andease of use.

Publication List

〔Original papers〕

K., Klaften, M., Narita, A., Kimura, T., Imai, K., Kimura, M.,• Abe,
Rubio-Aliaga, I., Wagner, S., Jakob, T., and Hrabé de Angelis, M.

•(2009). Genome-wide search for genes that modulate inflammatory
arthritis caused by Ali18 mutation in mice. Mamm Genome. 20,152161.
Hashimoto, H., Miyamoto, R., Watanabe, N., Shiba, D., Ozato, K., et al.
(2009). Polycystic kidney disease in the medaka (Oryzias latipes) pc
mutant caused by a mutation in the Gli-similar3 (glis3) gene. PLoS ONE
4, e6299.

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

M., Takehana, Y., Sakaizumi, M., and Hamaguchi, S. A sex• Kato,
determining region on the Y chromosome controls the sex-reversal ratio

in interspecific hybrids between Oryzias curvinotus females andOryzias latipes males. Heredity. 2009 September 16.

〔Review articles〕

M., Murata, K., Naruse, K., and Tanaka, M. (2009). Medaka:• Kinoshita,
Biology, Management and Experimental Protocols. (Wiley-Blackwell,Ames, Iowa).

T. (2009). Artificial insemination using frozen medaka sperm.
• Sasado,
In: Medaka: Biology, Management and Experimental Protocols.

•

Kinoshita, M., Murata, K., Naruse, K., and Tanaka, M. eds. (WileyBlackwell, Ames, Iowa), pp. 110-116.
Sasado, T. (2009). Cryopreservation of medaka sperm. In: Medaka:
Biology, Management and Experimental Protocols. Kinoshita, M.,
Murata, K., Naruse, K., and Tanaka, M. eds. (Wiley-Blackwell, Ames,
Iowa), pp. 105-110.

Figure 2. NBRP Medaka website

57

National Institute for Basic Biology

Environmental Biology

DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY

Professor

Professor (Concurrent)

Associate Professor:
Assistant Professors:

WATANABE, Hajime*
KATSU, Yoshinao
MIYAGAWA, Shinichi
MIZUTANI, Takeshi
KOIDE, Shizuyo
KATO, Yasuhiko
URUSHITANI, Hiroshi
NAKAMURA, Takeshi
KIKUCHI, Naoka
SATO, Masaru
CHAKRABORTY, Tapas
SUGIURA, Naomi
MITSUI, Naoko
LANGE, Anke
KOBAYASHI, Kaoru
HAYASHI, Tomoko
MATSUDA, Chisato
IMAIZUMI, Taeko

IGUCHI, Taisen

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

Visiting Scientists:
Technical Assistants:
Secretary:

WATANABE, Hajime

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. In 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 diethylstilbestrol (DES)
exposure was found to result in various abnormalities of the
reproductive tract in women. This syndrome was named the
DES syndrome. Similar abnormalities have been
demonstrated in experimental animals exposed perinatally to
estrogens. Developmental estrogen exposure in mice, forexample, induces persistent proliferation of vaginal epithelial
cells. We found that the persistent changes in the vagina inmice exposed neonatally to estrogens result from thepersistent activation of erbBs and estrogen receptor a (ERa),and sustained expression of EGF-like growth factors.
Currently, we are analyzing the methylation status in themouse vagina using MeDIP (methylated DNA
immunoprecipitation) coupled with a microarray (MeDIPchip). We found several differentially methylated ordemethylated DNA profiles in neonatally DES-exposedmouse vaginae and controls. We thus consider that neonatal
DES exposure affects DNA methylation profiles, resulting inpersistent abnormalities in mouse reproductive organs.

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

II. Microarray analysis of estrogen responsive
genes

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

58

To understand the mechanisms through which estrogenic
chemicals act on mouse reproductive organs, data
documenting the temporal and spatial expression patterns ofestrogen-responsive genes is essential. A large number ofgenes affected by estrogen treatment were identified in thetissues of wild-type mice using a microarray approach. For
most of the identified genes, expression was induced by
17b-estradiol (E 2 ) in a dose-dependent manner.
Subsequently, several environmental (xeno)estrogens were
tested and characteristic gene expression patterns wereobserved for each compound tested. These patterns were

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

distinct from that obtained following E2 exposure. We also
found that xenoestrogenic chemicals and dioxin have distinct
effects on the liver as well. Therefore, possible tissuespecific effects should be considered when elucidating thedistinct effects of various EDCs.

Figure. 4 Evolutionary relationships of estrogen receptor sequences.

Figure 3. Scatter plot of average expression levels in control andchemical-treated uterus

III. Steroid hormone receptors of birds,
reptiles, amphibians and fishes

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,
gar, roach, stickleback, mosquitofish, mangrove, Rivulus,
various sharks, Japanese giant salamander, Tokyo
salamander, newt, axolotl, toad, Silurana tropicalis,
American alligator, Nile crocodile, freshwater turtle, various
snakes and vultures. Functional studies showed that therockshell ER-like sequence does not bind estrogen but
exhibits ligand-independent transactivation, whereas lamprey
ER exhibited ligand-dependent transactivation, proving thatprimitive vertebrates, such as the Agnatha, have a functional
ER. Intriguingly, we found that medaka and stickleback ERs
are more sensitive to estrogen/estrogen-like chemical
exposures than other fishes by reporter gene assay. Thus,
these approaches are efficient to evaluate the relationshipbetween species and their sensitivities to chemicals.

IV. Male production by juvenile hormones inDaphnids

Daphnia magna has been used extensively to evaluate theorganism- and population-based responses of toxicity or
reproductive toxicity tests. These tests, however, provide no
information about the mode of actionof the tested compounds. Therefore, we applied an
ecotoxicogenomic assessment of D. magna. We established aDaphnia EST database and developed an oligonucleotidebased DNA microarray with high reproducibility. Exposureof D. magna to several chemicals resulted in characteristic
gene expression patterns that are chemical-specific,
indicating that the established DNA microarray can be used
for the classification of toxic chemicals as well as for thedevelopment of a mechanistic understanding of chemical
toxicity in a common freshwater organism. D. magna
reproduce asexually (parthenogenesis) when they are in an
optimal environment for food, photoperiod and population
density. Once environmental conditions become sub-optimal,
they alter their reproductive strategy from asexual to sexual
reproduction. Chemicals are able to affect the sex
determination of D. magna and we observed that juvenile
hormone agonists (pesticides), for example, induce theproduction of male offspring. The molecular basis ofenvironmental sex determination is largely unknown in D.
magna. Therefore, we isolated sex determination-related
genes to understand the molecular mechanisms of thisphenomenon in Daphnia. DM-domain genes are well known
as sex-related genes. We identified four DM-domain genes:
DMRT11E, DMRT93B, DMRT99B and DSX. Quantitative
gene expression analysis in daphnid gonads revealed that
DMRT93B and DSX were expressed only in the testis. We
have recently developed a method to inject genes into D.
magna embryos which will allow us to study gain- and lossof function analyses in more detail in this species.

V. Gene zoo and receptor zoo

We are establishing cDNA library banks and receptor genebanks of animal species including sturgeon, gar, lamprey,lancelet, mangrove Rivulus, Japanese giant salamander, newt,
Rana rugosa, Silurana tropicalis, Florida red berry turtle,
American alligator, Nile crocodile, vulture and polar bear incollaboration with the University of Pretoria, South Africa,
the University of Florida, USA, San Diego Zoo, USA, and
the Asa Zoo in Hiroshima.

59

National Institute for Basic Biology

Environmental Biology

VI. Molecular target search

We found that the persistent and ubiquitous environmental
contaminant, tributyltin chloride (TBT), induces thedifferentiation of adipocytes in vitro and increased adipose
mass in vivo. TBT is a dual nanomolar affinity ligand forboth the retinoid ‘X’ receptor (RXR) and the peroxisome
proliferators activated receptor g (PPARg). TBT promotes
adipogenesis in the murine 3T3-L1 cell model and perturbs
key regulators of adipogenesis and lipogenic pathways invivo. Moreover, in utero exposure to TBT leads to strikingly
elevated lipid accumulation in the adipose depots, liver, andtestis of neonate mice and results in increased epididymal
adipose mass in adults. In the amphibian Xenopus laevis,
ectopic adipocytes form in and around gonadal tissues
following organotin, RXR or PPARg ligand exposure. TBT
represents the first example of an environmental EDC that
promotes adipogenesis through RXR and PPARg activation.
Developmental or chronic lifetime exposure to orgaotins may
therefore act as a chemical stressor for obesity and relateddisorders. We have recently started to use stem cells frommouse bone marrow to study the molecular mechanisms ofcell differentiation to adipocytes by environmental
chemicals.

Publication List

〔Original papers〕

K.C., Wolfe, M., Braunbeck, T., Iguchi, T., Ohta, Y. Tooi., O.,• Grim,Touart, L., Douglas, C., Wolf, D.C., and Tietge, J. (2009). Thyroid

•
•
•
•
•
•
•
•
60

histopathology assessments for the amphibian metamorphosis assay todetect thyroid-active substances. Tox. Pathol., 37, 415-424.
Hikake, T., Hayashi, S., Iguchi T., and Sato, T. (2009). The role of IGF1
on the differentiation of prolactin secreting cells in the mouse anterior
pituitary. J. Endocrinol., 203, 231-240.
Kim, H., Hayashi, S., Chambon, P., Watanabe, H., Iguchi, T., and Sato,T. (2009). Effects of diethylstilbestrol on ovarian follicle development
in neonatal mice. Reprod. Toxicol., 27, 55-62.
Kim, H., Nakajima, T., Hayashi, S., Chambon, P., Watanabe, H., Iguchi,T., and Sato, T. (2009). Effects of diethylstilbestrol on programmed
oocyte death and induction of polyovular follicles in neonatal mouseovaries. Biol. Reprod., 81, 1002-1009.
Kirigaya, A., Kim., Hayashi, S., Chambon, P., Watanabe, H., Iguchi, T.,
and Sato, T. (2009). Involvement of estrogen receptor b in the inductionof polyovular follicles in mouse ovaries exposed neonatally todiethylstilbestrol. Zool. Sci., 26, 704-712.
Lange, A., Paull, G.C., Coe, T.S., Katsu, Y., Urushitani, H., Iguchi, T.,
and Tyler, C.R. (2009). Sexual reprogramming and estrogenic
sensitization in wild fish exposed to ethinylestradiol. Environ. Sci.
Technol., 43, 1219-1225.
Miyagawa, S., Moon, A., Haraguchi, R., Inoue, C., Harada, M.,
Nakahara, C., Suzuki, K., Matsumaru, D., Kaneko, T., Matsuo, I., Yang,
L., Taketo, M.M., Iguchi, T., Evans, S.M., and Yamada, G. (2009).
Dosage dependent hedgehog signals integrated with Wnt/β-cateninsignaling regulate external genitalia formation as an appendicular
program. Development, 136, 3969-3978.
Miyagawa, S., Satoh, Y., Haraguchi, R., Suzuki, K., Iguchi, T., Taketo,
M.M., Nakagata, N., Matsumoto, T., Takeyama, K., Kato, S., andYamada, G. (2009). Genetic integrations of the androgen and Wnt/bcatenin pathways for the masculinization of external genitalia. Mol.
Endocrinol., 23, 871-880.
Oka, T., Miyahara, M., Yamamoto, J., Mitsui, N., Fujii, T., Tooi, O.,
Kashiwagi, K., Takase, M., Kashiwagi, A., and Iguchi, T. (2009).

•

Application of metamorphosis assay to a native Japanese amphibian
species, Rana rugosa, for assessing effects of thyroid system affecting
chemicals. Ecotoxicol. Environ. Safety, 72, 1400-1405.
Tyler, C.R., Filby, A.L., Bickley, L.K., Cumming, R.I., Gibson, R.,
Labadie, P., Katsu, Y., Liney, K.E., Shears, J.A., Silva-Castros, V.,
Urushitani, H., Lange, A., Winter, M.J., Iguchi, T., and Hill, E.M.
(2009). Environmental health impacts of equine estrogens derived fromhormone replacement therapy. Enviorn. Sci. Technol., 43, 3897-3904.

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

T., Urushitani, H., Ohta, Y., Iguchi, T., and Shiraishi, H.
• Horiguchi,
Establishment of a polyclonal antibody against the retinoid X receptor

•
•
•
•

of the rock shell Thais clavigera and its application to rock shell tissues
for imposex research. Ecotoxicology. 2009 Dec. 4.
Katsu, Y., Kubokawa, K., Urushitani, H., and Iguchi, T. Estrogendependent transactivation of amphioxus steroid hormone receptor via
both estrogen- and androgen-response elements. Endocrinology. 2009
Dec.4.
Kohno, S., Katsu, Y., Urushitani, H., Ohta, Y., Iguchi, T., and Guillette,
L.J.Jr. Potential contributions of heat shock proteins to temperaturedependent sex determination in the American alligator. Sex. Devel.
2009 Dec. 4.
Miyagawa, S., Katsu, Y., Ohta, Y, Sudo, T., Lubahn, D.B., and Iguchi,
T. Estrogen receptor a is indispensable for the induction of persistentvaginal change by neonatal 5a-dihydrotestosterone exposure. Biol.
Reprod. 2009 Oct. 28.
Moore, B.C. Milnes, M.R., Kohno, S., Katsu, Y., Iguchi, T., andGuillette, L.J. Influences of sex, incubation temperature, andenvironmental quality on gonadal estrogen and androgen receptor
messenger RNA expression in juvenile American alligators (Alligator
mississippiensis). Biol. Reprod. 2009 Sept. 16.

〔Review article〕

J.P., vom Saal, F.S., Akingbemi, B.T., Arizono, K., Belcher, S.,• Myers,
Colborn, T., Chahoud, I., Crain, D.A., Farabollini, F., Guillette, L.J.Jr.,

Hassold, T., Ho, S.-M., Hunt, P.A., Iguchi, T., Jobling, S., Kanno, J.,
Laufer, H., Marcus, M., McLachlan, J.A., Nadal, A., Oehlmann, J.,
Olea, N., Palanza, P., Parmigiani, S., Rubin, B.S., Schoenfelder, G.,
Sonnenschein, C., Soto, A.M., Talsness, C.E., Taylor, J.A., Vandenberg,
L.N., Vandenbergh, J.G., Vogel, S., Watson, C.S., Welshons, W.V., andZoeller, R.T. (2009). Why public health agencies cannot depend upon
good laboratory practices as a criterion for selecting data: The case ofbisphenol A. Environ. Health Perspect., 117, 309–315.

DIVISION OF PLANT DEVELOPMENTAL GENETICS (ADJUNCT)

Professor (Adjunct)

TSUKAYA, Hirokazu

Assistant Professor:
Postdoctoral Fellows:
Visiting Scientists:

Technical Assistants:

Secretary:

YAMAGUCHI, Takahiro
ISHIKAWA, Naoko
NUKAZUKA, Akira
YANO, Satoshi
GOTOH, Ayako
ICHIHASHI, Yasunori
IKEUCHI, Momoko
NAKAYAMA, Hokuto
TAKASE, Masahide
IRIE, Aki
KADOWAKI, Tamaka
NAGURA, Masako
YAMAGUCHI, Chinami
KOJIMA, Yoko

The leaf is the fundamental unit of the shoot system, whichis composed of the leaf and stem. The diversity of plant
forms is mostly attributable to variation of leaf and floralorgans, which are modified leaves. Moreover, leaf shape issensitive to environmental stimuli. The leaf is therefore thekey organ for a full understanding of plant morphogenesis.
The genetic control of the development of leaf shapes,
however, has remained unclear. Recently, studies of leaf
morphogenesis reached a turning point after our successful
application of the techniques of developmental andmolecular genetics using the model plant Arabidopsis
thaliana (L.) Heynh. (Tsukaya 2008).

I. Mechanisms of leaf development

Focusing on the mechanisms that govern the polarized
growth of leaves in Arabidopsis thaliana, we have identified
four genes for polar-dependent growth of leaf lamina: the
ANGUSTIFOLIA (AN) and AN3 genes, which regulate thewidth of leaves, and the ROTUNDIFOLIA3 (ROT3) and
ROT4 genes, which regulate the length of leaves. AN and
ROT3 genes control cell shape while AN3 and ROT4 genes
regulate cell numbers in leaves. In addition to the polardependent leaf shape control, we have focused on themechanisms of organ-wide control of leaf size, which arereflected in the ‘compensation’ phenomenon (reviewed in
Tsukaya 2008). Additionally, the accumulation of knowledge
on the basic mechanisms of leaf shape control has enabled us
to conduct Evo/Devo studies of the mechanisms behind leafshape diversity. Below is an overview of our research
activities and achievements during 2009.

1-1 Polar growth of leaves in A. thaliana

AN is a member of the CtBP-BARS gene family reported
from animal genomes; last year, we showed that homologs ofAN from Larix gmelinii, a gymnosperm, Marchantia
polymorpha and Physcomitrella patens, mosses, all fullycomplemented all known morphological phenotypes causedby an-1 mutation in Arabidopsis, suggesting that the ANfunction is conserved among land plants. Furthermore, ourdetailed analysis of intracellular localization suggested that

AN has a unique role (or roles) in Golgi-related functions.
Further analyses of AN functions are ongoing.
On the other hand, we found that constitutive overexpression of ROT4 peptide in Arabidopsis caused abnormal
protrusion of the inflorescence stem, suggesting that ROTmight be somehow involved in positional value
determination.

1-2 Evolution of establishment mechanisms ofleaf polarities in monocots

We have recently started to attempt an understanding of thegenetic basis of the development of unifacial leaves that are
known only from monocot clades. Our analyses indicatedthat the unifacial character might be due to overall changes
in all polarities around leaves (i.e. adaxial-abaxial, distalproximal, and central-lateral polarities). Moreover, thegenetic controls of leaf polarities were revealed to differ, atleast in part, between eudicots and rice, a monocot model
species. Understanding the differences in the genetic
mechanisms for the establishment of unifacial and normal
bifacial leaves will provide good clues as to how leaf-shapeis diversified.
For such purposes, comparative molecular-genetic andanatomical analyses between unifacial and bifacial leaf
development have been undertaken using members of thegenus Juncus (Yamaguchi and Tsukaya, J. Plant Res. 123,
35-41, 2010). Interestingly, molecular characterization of
unifacial leaves of Juncus revealed that they have only
abaxial identity in the leaf blades, lack leaf margins, andpossess flattened leaf lamina. Taken together, our datastrongly suggests the presence of unknown mechanisms forflat leaf organogenesis that were not previously suspectedfrom studies of model plants. We also established mutational
and transgenic approaches to analyze unifacial leaf
formation; several interesting mutants of Juncus that exhibit
abnormalities in leaf polarity have already been isolated.

1-3 Size control of leaves and mechanisms ofcompensation

We have recently noticed that leaf organogenesis depends
on the “leaf meristem” that is seen just in the border region
between leaf blade and leaf petiole. All cells required for leaf
formation seem to be supplied from this leaf meristem. We
also identified that SPT controls the size of the leaf meristem(Ichihashi et al., Plant Cell Physiol. 51, 252-261, 2010). How
are cell proliferation and cell enlargement coordinated in leaf
morphogenesis? In a determinate organ - a leaf - the numberof leaf cells is not necessarily reflected in leaf shape or, inparticular, in leaf size. Genetic analyses of leaf development
in Arabidopsis showed that a compensatory system (orsystems) acts in leaf morphogenesis in a way that an increasein cell volume might be triggered by a decrease in cellnumber (reviewed in Tsukaya 2006; 2008). Thus, leaf size is,
at least to some extent, regulated at the organ level by thecompensatory system or systems. To understand the detailsof such totally unknown regulatory mechanisms, we haveconducted a large scale screening of leaf-size and/or leafshape mutants.
As a result, we have succeeded in isolating oli mutants

61National Institute for Basic Biology

Environmental Biology

which have a specific defect in leaf-cell numbers. Using
these mutants we revealed that the compensation is triggered
only when decrease of cell numbers in leaf primordia
exceeds some threshold level; and several oli mutations are
loss-of-function mutations of ribosome biogenesis genes(Fujikura et al. 2009).
In addition, we have revealed that: (1) fugu5 phenotype iscancelled by supplying sucrose to the growth medium; (2)large-leaved gra mutations are caused by translocationdependent duplication of a segment of a chromosome(Horiguchi et al. 2009). This enlargement of leaves due to the
duplication of the chromosome segment can be explained
only partially by the increase of the gene dosage of known
gene functions in the chromosome region, suggestingpresence of some unknown genes for leaf size control; (3)“opposite-type” compensation syndrome in msc mutants was
attributed to accelerated heteroblasty (Usami et al., 2009:
Fig. 1). Detailed analyses of this phenomenon strongly
suggested that traits of heteroblasty are regulated by at least
two different pathways governed by small RNAs; (4) a newtool for studies of mechanisms of compensation, inducible,
chimeric expression systems of KRP2 or AN3, wasestablished: and (5) several candidate genes responsible forthe compensation were selected from microarray analysis of
fugu2 and an3.

for a curious enhancement of the effects of tetraploidizationin terms of cell-size increase, suggesting that some unknown
mechanisms (e.g. feedback systems) are hidden behind therelationship between the ploidy level and cell/organ size. We
also found the ratio of cell size between diploid andtetraploid varied among the mutants examined, suggestingthat an increase of cell size due to tetraploidization is notdirect or automatic. Further construction and analyses oftetraploid mutants are in progress.

II. Biodiversity of leaf form

We are also interested in the biodiversity of wild plants. We
found that P. asiatica is a cryptic allotetraploid from themolecular data, and pursued detailed molecular phylogenetic
analyses of members of the subgenus Plantago of the genus
Plantago. As a result, we found that most known species inthis section are derived from inter-species hybridization.
Surprisingly, whole species in some sections are found to be
derived from inter-section hybridization, showing thatsubgenus Plantago is derived from extensive reticulate
evolution (Ishikawa et al., 2009).

Publication List

〔Original papers〕

U., Horiguchi, G., Ponce, M.R., Micol, J.L., and Tsukaya, H.
• Fujikura,(2009). Coordination of cell proliferation and cell expansion mediated•••Figure 1. The msc3 mutant has an altered heteroblasty pattern anddevelops larger leaf blades that contain a larger number and smaller sizeof cells, due to disturbance of miR156 function. Modified from Usami etal. (2009).

In addition, in the course of studies of AN3 function, we
found that an3 mutation phenotype is drastically changed
when combined with ribosome-biogenesis mutations and/or#2047 mutation. These facts suggest that AN3 is involved invarious key aspects of organogenesis in Arabidopsis. We also
found that plant Elongator regulates auxin-related genes
during RNA polymerase II transcription elongation (Nelissen
et al., PNAS 107, 1678-1683, 2010). Further analyses on themechanisms of compensation are in progress.

1-4 Size control of leaves and ploidy level

Why does a high-ploidy level cause increased cell/leaf
size? In other words, why are tetraploid leaf cells twice aslarge in volume as diploid leaf cells? The reasons are not yet
perfectly understood (Tsukaya 2009). We found that
mutational defects in the endoreduplication were responsible

62

by ribosome-related processes in the leaves of Arabidopsis thaliana.
Plant J. 59, 499–508.
Horiguchi, G., Gonzalez, N., Beemster, G.T.S., Inzé, D., and Tsukaya,
H. (2009). Impact of segmental chromosomal duplications on leaf sizein the grandifolia-D mutants of Arabidopsis thaliana. Plant J. 60, 122133.
Ishikawa, N., Yokoyama, J., and Tsukaya, H. (2009). Molecularevidence of reticulate evolution in the subgenus Plantago(Plantaginaceae). Amer. J. Bot. 96, 1627–1635.
Usami, T., Horiguchi, G., Yano, S. and Tsukaya, H. (2009). The more
and smaller cells mutants of Arabidopsis thaliana identify novel roles
for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in thecontrol of heteroblasty. Development 136, 955-964.

〔Review article〕

H. (2009). Size regulation of plant body via control of cell
• Tsukaya,
volume by endoreduplication. Reg. Plant Growth Dev. 44, 135-141.

LABORATORY OF PHOTOENVIRONMENTAL BIOLOGY

Professor (Adjunct)

WATANABE, Masakatsu

Photosynthetic microorganisms, such as cyanobacteria andflagellate algae, respond to light in order to locate themselves
at appropriate photoenvironments. Our research is aimed at
the elucidation of the photoreceptive and signal transduction
mechanisms of light responses in microorganisms. Thisapproach has led us to the discovery, characterization, andapplication of a remarkably unique light sensor molecule as
described below.

I. Photoactivated Adenylyl Cyclase (PAC), an
algal photoreceptor protein with intrinsic
effector function to produce cAMP

In 2002, we found a novel blue-light receptor with aneffector role in Euglena (Iseki et al., Nature 415, 1047-1051,
2002): Euglena gracilis, a unicellular flagellate, which shows
blue-light type photomovements (Figure 1). The action
spectra indicate the involvement of flavoproteins as thephotoreceptors mediate them. The paraflagellar body (PFB),a swelling near the base of the flagellum, is thought to be a
photosensing organelle responsible for photomovements. To
identify the photoreceptors in the PFB, we isolated PFBs andpurified the flavoproteins therein. The purified flavoprotein(ca. 400 kDa), with noncovalently bound FAD, seemed to bea heterotetramer of α- and β-subunits. Predicted amino acidsequences for each of the subunits were similar to each otherand contained two FAD-binding domains (BLUF: sensor ofblue light using FAD) (F1 and F2) each followed by anadenylyl cyclase catalytic domain (C1 and C2). The
flavoprotein showed adenylyl cyclase activity, which waselevated by blue-light irradiation. Thus, the flavoprotein(PAC: photoactivated adenylyl cyclase) can directly

Figure 2. Domain structure of Photoactivated Adenylyl Cyclase (PAC)
and its two possible action mechanisms to mediate photoavoidance
behavior in Euglena gracilis. The green spots in the background are
fluorescence microscopical images of isolated paraflagellar bodies(PFBs), the “real eyes”.

transduce a light signal into a change in the intracellularcAMP level without any other signal transduction proteins(Figure 2).
A unique function such as this is best suited not only for the
rapid control of the flagellar motion of the Euglena cell but
also for a variety of biotechnological photocontrol of cAMPcontrolled biological functions, including neuronal functions
and developmental processes in a variety of organisms inwhich PAC can be heterologously expressed. For example, incollaboration with Max-Planck-Institut für Biophysik(Frankfurt) and other German groups, expression of PAC incells was performed, which allowed the manipulation ofcAMP with exquisite spatiotemporal control. We functionallyexpressed PACs in two popular expression systems, Xenopus
laevis oocytes and HEK293 cells. Moreover, transgenic
Drosophila melanogaster flies demonstrated functional PAC
expression by showing blue light–induced behavioral
changes (Schröder-Lang, S., Nat. Meth. 4, 39-42, 2007)

II. Flagellar motions during algal phototactic
steering
Figure 1. Euglena gracilis, a unicellular flagellate alga. It swims forward(to the left) by shaking the flagellum, the protruding whip-like structure.
The flagellar motion is controlled by ultraviolet to blue light signals
sensed by the photoreceptor molecules in the “real eye” locatedadjacently to the basal part of the flagellum, so that the cell can locate
itself in appropriate light environments for its survival. The orange spot,
so-called “eyespot” is not the “real eye” but a light shade to enable thecell to recognize the light direction.

To understand the mechanism of blue-light-induced algal
phototactic steering, we observed, using infrared high-speed
video microscopy, light-triggered transitory flagellar motionsin flagellate reproductive cells (swarmers) of a brown alga,
Scytosiphon lomentaria, under primary helical swimming
conditions before and during negative phototactic orientation
to unilateral actinic light (Figure 3).

63National Institute for Basic Biology

Environmental Biology

Figure 3. Photo-orientation of swarmer cells released from matured thalliof the brown alga, Scytosiphon lomentaria. Each swarmer has a hairylong anterior flagellum and a smooth short and autofluorescent posterior
one. Cover illustration of Photochem. Photobiol. 86 (2) drawn by Hiroko
UchidaThe posterior flagellum, which is autofluorescent andthought to be light-sensing, was passively dragged in thedark and exhibited one to several rapid lateral beats during
orientation changes for phototactic steering (Figure 4).
Notably, a brief cessation of anterior flagellar beating was
occasionally observed concomitantly with rapid beats of theposterior flagellum. This behavior caused a pause in helicalbody rotation, which may contribute to the accuracy ofphototactic steering (Figure 5). Thus, coordinated regulationof the movement of the two flagella plays a crucial role inphototactic steering.

Figure 5. A brief cessation of anterior flagellum after onset of the lightstimulus.

Publication List

〔Original paper〕

N., Suzuki, T., Watanabe, M., and Takeda, M. (2009).
• Izawa,
Characterization of arylalkylamine N-acetyltransferase (AANAT)
activities and action spectrum for suppression in the band-leggedcricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae). Comp.
Biochem. Physiol. Part B 152, 346–351.

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

S., Uchida, H., Iseki, M., Watanabe, M., and Murakami, A.
• Matsunaga,
Flagellar motions in phototactic steering in a brown algal swarmer.
Photochem. Photobiol. 2009 Dec. 14.

Figure 4. Rapid beats of posterior flagellum after onset of light stimulus
observed at 2 ms intervals.

64

National Institute for Basic Biology

Theoretical Biology

DIVISION OF THEORETICAL BIOLOGY

Professor (Concurrent)

MOCHIZUKI, Atsushi

We are studying biological phenomena using mathematicalmodels. This method gives us an integrative understanding of
the behavior of complex systems in biology including gene
regulatory networks.
Mathematical models are especially useful in understanding
pattern formation in development. The study of themechanisms responsible for morphological differences
between species is an important research focus of current
developmental biology.

I. Steepness of thermal gradient is essential toobtain a unified view of thermotaxis in C.
elegans

One of the adaptive behaviors of animals in theirenvironment is thermotaxis, by which they migrate toward apreferred temperature. This sensorimotor integration isaccomplished by choosing one of two behaviors dependingon the surrounding temperature, namely thermophilic orcryophilic movement. C. elegans exhibits thermotaxis and its
migration behavior has been analyzed experimentally at boththe population and individual levels. However, someexperimental data are inconsistent especially forthermophilic movement, which is expected to be observed inlower than favorable temperatures. There are no
experimental analyses that find thermophilic tendencies in

Figure 1. Thermophilic tendency in distribution behavior can be seen
only if we adopt the parameter sets with a thermophilic bias. Sufficiently
strong thermophilic bias is needed to observe thermophilic tendency in
distribution behavior. Red, green and purple lines show the mean of
population, the position of maximum peak and cultivated temperature
respectively.

the individual behavior of worms, despite multiple reports
supporting thermophilic movement of the population.
Although theoretical methods have been used to study
thermotaxis of C. elegans, no mathematical model provides aconsistent explanation for this discrepancy. This study wasdone by Dr. Nakazato in our group as a a collaboration workwith Drs. Mori and Kuhara in Nagoya University.
Here we develop a simple biased random walk model,
which describes population behavior, but which is based on
the results of individual assays. Our model can integrate allprevious experiments without any contradiction. We
regenerate all the population patterns reported in past studies
and give a consistent explanation for the conflicting results.
Our results suggest that thermophilic movement is observed,
even in individual movements, when the thermal gradient issufficiently slight. On the contrary, thermophilic movement
disappears when the thermal gradient is too steep. The
thermal gradient is thus essential for a comprehensive
understanding of the experimental studies of thermotaxis in
C. elegans. Our model provides insight into an integrative
understanding of the neural activity and thermotactic
behavior in C. elegans.

II. Mathematical modeling for gene expressionof vertebrate segmentation

Segmentation in the vertebrate PSM (presomitic mesoderm)is established by a series of pattern formation through the
dynamics of gene expression at different levels. Some
downstream genes suppress the activity of upstream genes.
The negative feedback seems to realize transient dynamics ofpatterning. We developed two mathematical models, where
the negative-regulator genes are different. We found that thepreviously believed regulation model cannot explain the
mutant expression patterns, but the newly proposed model
can. The mathematical model gives an integrative
understanding and a working hypothesis for a regulatory
network system including many genes. This study was done
by Dr. Saitou of our group at the RIKEN Advanced Science
Institute in collaboration with Drs. Takada and Takahashi of
NIBB.

Figure 2. Two models for the regulations of segmentation genes. Left;
Model A (Previously believed), Right; Model B (Our hypothesis).

65

National Institute for Basic Biology

Theoretical Biology

Figure 3. Dynamics of gene expressions for wild type are almost
equivalent between the two models. Left; Model A (Previously believed),
Right; Model B (Our hypothesis).

Figure 4. Dynamics of gene expressions for Ripply null-mutant arecompletely different between the two models. Left; Model A (Previously
believed), Right; Model B (Our hypothesis). Only model B can explain
the experimental results.

Figure 5. An intuitive explanation of “steady state compatibility”. (a) An
example of the regulatory links of a mono-directional loop with three
genes. (b) Another example of the regulatory links of a bi-directional
loop with three genes. (c) The shaded domains show the region whereother steady states should not appear except for the original point (0,0,0)and (1,1,1) based on the network in (a). The network (a) has two steadystates at maximum. (d) The network (b) determines the different shapesof the domains of no-steady-state except for the points (0,0,0), (0,1,1),(1,0,1) and (1,1,0). This network allows four steady states at maximum.

III. Structure of regulatory networks anddiversity of gene expression patterns

The complexity of gene regulatory networks is considered
responsible for the diversity of cells. Different types of cells,
characterized by the expression patterns of genes, areproduced in early development through the dynamics of geneactivities based on the regulatory network. However, very
little is known about the relationship between the structure of
regulatory networks and the dynamics of gene activities.
In this study I introduce the new idea of “steady statecompatibility,” by which the diversity of possible geneactivities can be determined from the topological structure ofgene regulatory networks. The basic premise is very simple:
the activity of a gene should be a function of the controllinggenes. Thus a gene should always show unique expression
activity if the activities of the controlling genes are unique.
Based on this, the maximum possible diversity of steadystates is determined using only information regarding
regulatory linkages and without knowing the regulatory
functions of genes.
Using the concept of “steady state compatibility,” three
general properties of the relationship between the topologyof regulatory networks and the maximum number of steadystates can be derived (Figure 2). (A) Cascade structures inregulatory networks do not increase the number of possible
steady states (Fig. 2a). (B) Loop structures in networks arenecessary to generate multiple steady states. The number of
separated loops increases the maximum diversity of steadystates (Fig. 2b). (C) Multiple loops that are connected bysharing the same genes do not increase the maximum
diversity of steady states (Fig. 2c).
The method was applied to a gene regulatory network
responsible for early development in a sea urchin species. A
set of important genes responsible for generating diversities
of gene activities was derived based on the concept ofcompatibility of steady states.

66

Figure 6. Analysis of an actual gene network responsible for the early
development of a sea urchin species. (a) The network is simplified fromthe one of Fig. 3 in Davidson et al. (2002). The maximum diversity
generated from this network is determined by the analysis as 64. (b) Allof the “reduced observation point” ROP genes are derived. At least one of
the ROPs should change its activities in the alternative steady states.

Publication List

〔Original paper〕

K., and Mochizuki, A. (2009). Steepness of thermal gradient• Nakazato,
is essential to obtain a unified view of thermotaxis in C. elegans. J.
theor. Biol. 260, 56-65.

LABORATORY OF GENOME INFORMATICS

Assistant Professor

UCHIYAMA, Ikuo

Postdoctoral Fellow:

KAWAI, Mikihiko

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 these 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. The latter feature isespecially useful when the user’s interest is focused on sometaxonomically related organisms.
This year, we have developed the following advanced
functionalities: 1) enhanced assignment of functionalannotation, including external database links to eachorthologous group, 2) an interface for choosing a set ofgenomes based on phenotypic properties (Figure1), 3) the
addition of more eukaryotic microbial genomes (fungi andprotists) and some higher eukaryotes as references, 4)enhancement of the MyMBGD mode, which allows users to
add their own genomes to MBGD and now accepts raw
genomic sequences without any annotation (in such cases itruns a gene-finding procedure before identifying theorthologs). Some analysis functions, such as the function tofind orthologs with similar phylogenetic patterns, have also
been improved. The database now contains around 1000
published genomes including 16 eukaryotic microbes and 4
multicellular organisms.
MBGD is available at http://mbgd.genome.ad.jp/.

Figure 1. The interface for organism selection in MBGD where aphylogenetic pattern related to “motility” is specified (top-left) and theresult of the pattern search (bottom-right).

II. Enhancement of the algorithm for
identifying orthologous groups among
multiple genomes
As a core technology of our comparative genomics tools,
we have developed a rapid automated method of ortholog
grouping, named DomClust, which is effective enough toallow the comparison of many genomes simultaneously. The
method classifies genes based on a hierarchical clustering
algorithm using all-against-all similarity data as an input, but
it also detects domain fusion or fission events and splits
clusters into domains if required. As a result, the procedure
can split genes into the domains minimally required forortholog grouping.
We are continuing to improve the algorithm. Partial
taxonomic information can be incorporated into the
DomClust algorithm by specifying ingroup/outgroup foreach input genome. The resulting table has a nested structure
when a duplication event occurs within the ingroup lineage.
We are also trying to modify the algorithm for utilizing
metagenomic analysis. In addition, to further improvescalability for comparison of thousands of genomic
sequences, we are now developing an efficient method to
update the clustering result incrementally.

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 in

67

National Institute for Basic Biology

Theoretical Biology

which 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.
The program, named CoreAligner, was successfully applied
to the genome sequences of two major bacterial families,
Bacillaceae and Enterobacteriaceae, for identifying the core
structures comprising 1438 and 2125 orthologous groups,
respectively. We are now expanding our analysis to morediverged bacterial families to examine generality of ourapproach. 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 main window of the RECOG clientconsists of a taxonomic tree viewer (left), an ortholog table/
phylogenetic pattern map viewer (center) and a gene
information viewer (right). Users can choose a set ofgenomes using the taxonomic tree viewer and run the
DomClust program to identify orthologous groups. The result
is displayed in the ortholog table viewer, where users can see
the entire picture of the phylogenetic pattern map as well as
the full details of the ortholog table using semantic zooming

functionality.
This year, we added several new functions to the RECOG
system to enhance its usability for comparative analysis. Oneof the new features is gene property analysis, in which users
can define property values for each gene and compare thesevalues among orthologous genes on the ortholog table viewer(Figure 2).

V. Comparative genomics of Helicobacter pylori

Helicobacter pylori is a major pathogen in human gastriccancer and it is known that East Asian strains of H. pylori
have a more toxic CagA protein, a major virulence factor,
than Western strains. In collaboration with Dr. Kobayashi(Univ. Tokyo) and other researchers, we have determined thecomplete genomic sequences of four H. pylori strains
isolated from Japanese patients and compared them withpublished H. pylori genomes. Although all four genomes areclearly classified into the East Asian group, they exhibit
substantial polymorphisms in genomic structure (Figure 3).
We are trying to identify genomic features specific to East
Asian strains and infer evolutionary processes andmechanisms that are related to the evolution of H. pylori.

Figure 3. Comparative genome map among H. pylori strains. Genomic
core structure is identified by CoreAligner and orthologous genes areconnected with lines. Colors are assigned according to the gene order in
one strain (26695).

Publication List

〔Original papers〕

T., Kuwahara-Arai, K., Uchiyama, I., Takeuchi, F., Ito, T., and• Baba,Hiramatsu, K. (2009). Complete genome sequence determination of a•

Macrococcus caseolyticus strain JSCS5402 reflecting the ancestralgenome of the human pathogenic staphylococci. J. Bacteriol. 191,
1180-1190.
Watanabe, S., Ito, T., Sasaki, T., Li, S., Uchiyama, I., Kishii, K.,
Kikuchi, K., Skov, R.L., and Hiramatsu, K. (2009). Genetic diversity ofstaphylocoagulase genes (coa): insight into the evolution of variable
chromosomal virulence factors in Staphylococcus aureus. PLoS One 4,
e5714.

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

I., Higuchi, T., and Kawai, M. MBGD update 2010: toward• Uchiyama,
a comprehensive resource for exploring microbial genome diversity.
Nucleic Acids Res. 2009, Nov. 11.

Figure 2. The phylogenetic pattern viewer comparing ten H. pylori strains(see section V), where each cell is colored according to G+C content at
the third codon positions using the gene property analysis functionality.

68National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

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

KAJIURA-KOBAYASHI,
HirokoTAKAO, Daisuke
ICHIKAWA, Takehiko
KANDA, Rieko
OKA, Naomi
SHINTANI, Atsuko

Stelzer’s group at the European Molecular Biology
Laboratory (EMBL) is extremely suitable for this purpose,and we have introduced a DSLM set and applied it tovisualize cell movements in intact mouse embryos at
gastrulating stages.
Additionally, we support researchers who are interested inusing our DSLM and two-photon microscope (for example,
Hashimoto et al. Nat. Cell Biol. 12, 170-186, 2010). Several
collaborative projects are in progress.

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).

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.

Figure 3. Images taken with DSLM. Left: optical section of a 6-day
mouse embryo with fluorescent nuclei. Right: a juvenile zebrafish head
expressing GFP in neurons.

Figure 1. Left: ventral view of a 7.5-day mouse embryo. Middle: thenode. Right: node cilia.

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.

Publication List

〔Review article〕

S. (2009). Modification of Mouse Nodal Flow by Applying• Nonaka,
Artificial Flow. In Methods in Cell Biology, S. King, and G. Pazour,
eds. (Academic Press), pp. 287-297.

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. The Digital Scanned Lightsheet Microscope (DSLM, Figure 2) developed by Dr. Ernst

69

National Institute for Basic Biology

Laboratories Supervised by the Director-General

KAMADA GROUP
Assistant Professor:

KAMADA,Yoshiaki

Cell growth (increase in cell mass) and proliferation(increase in cell number) are highly linked with the cell’s
perception of its nutritional environment. Tor (target of
rapamycin) protein belongs to a family ofphosphatidylinositol kinase-like protein kinases and plays acentral role in controlling cell growth. Specifically, Torsignaling couples nutrient signals to various growth-related
processes. Tor protein forms distinct Tor complexes, TORC(Tor complex) 1 and TORC2. TORC1 regulates rapamycinsensitive branches of the TOR pathway, such as translation
initiation, ribosome biogenesis, and autophagy. On the other
hand, TORC2, whose function is insensitive to rapamycin, is
responsible for actin organization and cell integrity.
The aim of this research group is to reveal the molecularmechanisms of how the Tor pathway regulates eachphenomenon, and how Tor receives nutrient signals. We havebeen studying Tor signaling in the budding yeast,
Saccharomyces cerevisiae, and have found three novel
branches of the TOR signaling pathway.

I. TORC1 is involved in the cell cycle at G2/M
transition

It is well known that inhibition of protein synthesis causes
cell growth and promotes cell cycle arrest at G1 (G0).
Rapamycin, a TORC1 inhibitor has the same effect via
inactivating TORC1. 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,
which encodes an essential component of TORC1. We foundthat this mutant, as well as yeast cells treated with
rapamycin, exhibits mitotic delay with prolonged G2. We
further demonstrated that this G2-arrest phenotype is due tomislocalization and resultant inactivation of Cdc5, the yeast
polo-kinase. These results suggest that TORC1 mediates
G2/M transition via regulating polo-kinase.

II. TORC1 regulates autophagy via Atg1
kinase complex

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 Atg13. Although
Atg13 is phosphorylated in a TORC1-dependent manner
under nutrient-replete conditions, it is immediately
dephosphorylated in response to starvation or rapamycin
treatment. Dephosphorylated Atg13 binds to Atg1 whichallows Atg17, Atg29, and Atg31 to associate with Atg1Atg13 to form Atg1 complex. Atg1 complex formation
triggers autophagy at least in two ways. First, through therecruitment of Atg proteins to the pre-autophagosomal
structure (PAS), the putative site for autophagosome

70

formation. Second, Atg1 kinase activity is enhanced by Atg1
complex formation. We assume that Atg1 phosphorylates its
substrate(s) at the PAS to trigger a downstream event in
autophagy. (Figure 1)

Figure 1. A schematic model of regulation of Atg1 complex by TORC1.
When TORC1 is inactivated following nutrient starvation, Atg13 isdephosphorylated. This allows the formation of Atg1 complex among
Atg1 and its regulators, followed by the upregulation of the Atg1 kinase
activity and assembly of other Atg proteins to the PAS to initiate
autophagosome formation.

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

Publication List

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

Y., Yoshino, K., Kondo, C., Kawamata, T., Oshiro, N.,• Kamada,
Yonezawa, K., and Ohsumi, Y. Tor directly controls the Atg1 kinase
complex to regulate autophagy. Mol. Cell Biol. 2009 Dec 7.

〔Review article〕

H., Suzuki, K., Kamada, Y., and Ohsumi, Y. (2009).
• Nakatogawa,Dynamics and diversity in autophagy mechanisms: lessons from yeast.
Nat. Rev. Mol. Cell Biol. 10, 458-467.

OHNO GROUP
Assistant Professor:
Technical Assistants:

OHNO, Kaoru
HARA, Ikuyo
FUKAZAWA, Mitsue
INAGAKI, Masako

The aim of this laboratory is to research the 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 oe 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 hormonesnare assumed to be the physiologicalcounterpart of LH and FSH in mammals. Some gonadotropic
hormones, such as egg development neurosecretory hormoneof the mosquito, egg-laying hormone of the sea hare, andandrogenic gland hormone of the terrestrial isopod, havebeen found iw invertebrate species. More recently, aninsulin-like peptide was reported to be responsible for the
regulation of egg maturation in the mosquito, 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 GSSdreferred toaminodacid sequence of purified GSS from radial nerves ofthe 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 effore 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 inducg 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 databass of the mass
analysis performed in this laboratory.

Publication List

〔Original paper〕

M., Yoshikuni, M., Ohno, K., Shibata, Y., Paul-Prasanth, B.,• Mita,Pitchayawasin, S., Isobe, M., and Nagahama, Y. (2009). A relaxin-like

peptide purified from radial nerves induces oocyte maturation andovulation in the starfish, Asterina pectinifera. Proc. Natl. Acad. Sci.
USA 106, 9507-9512.

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National Institute for Basic Biology

Laboratories Supervised by the Director-General

TERADA GROUP
Assistant Professor:
Postdoctoral Fellow:
Technical Assistants:

TERADA, Rie
SHIMATANI, Zenpei
ASAO, Hisayo
SHIMATANI, Zenpei*
MORITOH, Satoru

With the goal of developing an effective molecular breeding
method and progressing in our continuing gene function
study in rice (Oryza sativa L.), an important staple food, weare developing a new technology of gene targeting (GT)mediated by homologous recombination (HR). We havesucceeded in gene targeting of Waxy based on a strong
positive-negative (PN) selection in the cell where HR at the
Waxy locus took place. Because our GT procedure isapplicable to modify various genome regions we have beenable to target 15 individual rice genes using the publishedsequence information. We have also succeeded in making
various modifications to the target genes including geneknock-out as well as knock-in targeting of the coding
sequence of GUS to the targeted gene promoter for analysis
of its natural activity. In addition the point mutations on thevector for GT were effectively integrated into the targeted
gene locus in Alcohol dehydrogenase gene 2 (Adh2), and are-activation system of targeted-waxy by Cre-loxP
recombination (Terada et al., Plant Biotech., in press) was
achieved, similar to that used in mouse conditional GT.
These results suggest lots of possibilities for creation of ideal
mutants for GT-mediated molecular breeding and detailed
gene function studies.

I. Generation of blast fungus resistant ricebased on effective GT

OsRac1, a homolog of mammalian Rac GTPase, plays animportant role in the defense response of plants. Amino acidsubstitution of the 19 th glycine to valine (G19V) alters
OsRac1 to be constitutively active (CA) by elimination of its
GTPase activity. Increased resistance to blast fungus was
detected in random transgenic rice of CA-OsRac1 driven by
35S promoter. To examine whether the endogenous OsRac1
shows the same effect or not we generated CA-OsRac1 using
our rice GT procedure. We constructed targeting vectors to
induce the G19V substitution through a single point mutationof G to T in the first exon of OsRac1. Elimination of thepositive marker, hygromycin phospho transferase (hpt), fromtargeted OsRac1 using our Cre-loxP system created
CA-OsRac1 at the natural gene locus. Two targeting vectors,
pRac1A and pRac1B, were constructed to have homologous
sequences of OsRac1 covering slightly different regions eachother in order to insert the hpt marker flanked by the loxPsites (‘floxed’) into the first and third intron, respectively(Figure 1). After the G to T substitution by GT the hpt iseliminated by the Cre-loxP system. A single loxP remains in
the first or third intron, however, these are expected to spliceout. We have succeeded in GT of OsRac1 using both vectors(Table 1). About 14% of PN selected calli were successfullytargeted, giving us the highest efficiency of GT everobtained. Interestingly in addition to the expected true GT(TGT), one-sided invasion (OSI; HR is caused only at 5’ or72

3’ end of integrated DNA fragment) was frequently detectedduring GT using either vectors (Table 1). We selected 16
callus lines showing TGT and applied Cre mediated hpt
elimination. Finally 10 plants regenerated from 4 hpt
eliminated marker-free lines were grown all of which
revealed characteristic semi-dwarf phenotype. Precise G to T
substitution and hpt elimination in the heterozygous form
was confirmed by sequence analyses of obtained plant DNA.
Because fungus resistant phenotype is dominant, obtainedtargeted CA-OsRac1 plants are under examination forresistance to blast fungus.
This work is a collaboration with Professor Ko Shimamotoin the Laboratory of Plant Molecular Genetics, Nara Instituteof Science and Technology (NIST) and supported by the
Ministry of Education, Culture, Sports, Science andTechnology of Japan Grant-in-Aid for Scientific Research(S) (No. 19108005 to K.S.)

Figure 1. Strategy for creation of CA-OsRac1. Star indicates point
mutation of G to T.

Targeted
gene

PN
selectedCalli5’only3’only5’+3’Ratio of
TGT/PN
(％)

Waxy

638

0

0

6

0.94

OsRac1
by pRac1A

94

10

37

5

5.5

80

4

4

11

13.6

OsRac1
by pRac1B

OSI

TGT

Table I. Gene targeting of OsRac1

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2009. The former title isindicated by an asterisk (*).

HOSHINO GROUP
Assistant Professor:
Technical Assistant:

HOSHINO, Atsushi
WATANABE, Seiko

While genomic structures as well as their genetic
information appear to transmit stably 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. The morning 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.

pigmentation. While the mutations in the two groups
mentioned above are recessive, Margined and Blizzard of I.
nil are dominant mutations. Blizzard and Margined mutants
bloom pigmented corolla with irregular whitish spots andwhite edge, respectively. It was suggested that non-coding
small RNA represses the expression of a pigmentation genein the whitish parts of the corolla. We are currently
characterizing detailed molecular mechanisms of themutations in the latter two groups.

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 increased to 200 lines and 100,000 clones by theend of 2009. A web site to integrate information on theseresources is under construction (Figure 2).

Figure 2. NBRP morning glory web site.

Figure 1. Flower phenotypes of Japanese morning glories.

II. Flower pigmentation patterns

Figure 1 represents examples of such mutants showing
particular flower pigmentation patterns. Based on themolecular mechanisms conferring the particular patterns,
these mutants can be classified into three groups. The firstgroup includes the flecked and speckled mutants of I. nil thatbloom variegated flowers with pigmented spots and sectors
on whitish backgrounds. These mutations are caused by theinsertions of certain groups of DNA transposons into thegenes for flower pigmentation. Recurrent somatic mutations
due to transposon excision from the genes result inpigmented spots and sectors in white backgrounds. In thesecond group, the pearly-v mutant of I. tricolor and the
duskish mutant of I. nil also have variegated flowers, andepigenetic mechanisms are thought to regulate flower

Publication List

〔Original papers〕

A., Park, K.I., and Iida, S. (2009). Identification of r mutations• Hoshino,
conferring white flowers in the Japanese morning glory, Ipomoea nil. J.
Plant Research 122, 215-222.

Z., Takagi, K., Eun, C.H., Maekawa, M., Takahara, H.,• Shimatani,
Hoshino, A., Qian, Q., Terada, R., Johzuka-Hisatomi, Y., Iida, S., andTsugane, K. (2009). Characterization of autonomous Dart1 transposons
belonging to the hAT superfamily in rice. Mol. Gen. Genomics 281,
329-344.

73

National Institute for Basic Biology

Laboratories Supervised by the Director-General

TSUGANE GROUP
Assistant Professor:

TSUGANE, Kazuo

Although DNA transposons are one of the major
components of plant genomes, their transposition is restricted
genetically or epigenetically for genome stability. Because
insertions of transposons have contributed to the creation of
new genes and genome evolution, revealing the genome
dynamisms driven by DNA transposons is the purpose of ourresearch.
Gene tagging is an important tool for understanding genefunctions. We have constructed mutant rice lines using DNA
transposons in order to achieve functional genomics analysis
in rice, a model plant for monocots and cereals.

rice. These findings should facilitate the development of an
efficient gene-tagging system in rice and shed light on
epigenetic regulatory and evolutionary aspects ofautonomous elements in the nDart/aDart system.

I. An active DNA transposon in rice

The mutable allele virescent (or pale-yellow-leaf
variegated, pyl-v) which displays leaf variegation was causedby the integration of the 607-bp non-autonomous element
nDart1-0 belonging to the hAT superfamily into the OsClpP5
gene encoding the chloroplast protease. In the mutable pyl-v
allele, somatic excision of nDart1-0 from OsClpP5 in thepresence of an active autonomous element, aDart, results inthe pyl-v leaf variegation phenotype, a dark-green sector
consisting of somatically reverted cells, due to nDart1-0
excision, on a pale-yellow background comprising of cells
having nDart1-0 inserted into OsClpP5 in the homozygous
condition. Plants containing the pyl-v allele without an active
aDart element display pale-yellow leaves without
variegation; this has been termed as the pale-yellow leafstable (pyl-stb) phenotype. In the sequenced Nipponbare
genome containing no active aDart elements, among the 53
inactive iDart1 elements, there are 38 putative autonomous
iDart1 elements. Because their putative transposase genes
carry no apparent nonsense or frameshift mutations, they arethought to be silenced epigenetically. Therefore we felt ithighly likely that an active aDart element would be similarin structure to one of these 38 iDart1 elements. Using mapbased cloning, we found that aDart in the mutable pyl-v plant
coincides with one of the 38 iDart1 elements, iDart1-27,
residing on chromosome 6 in Nipponbare and that thetranscripts of the accumulated transposase gene in the pyl-v
leaves are predominantly from Dart1-27. Two additional
smaller transcripts were detected in pyl-v (Figure. 1). The
major transcripts detected in pyl-stb and Nipponbare were
derived from non-Dart1-27 elements, implying that theresidual expression of other Dart1 elements in both pyl-stb
and Nipponbare would be too weak to act on the nDart1-0 at
OsClpP5 and lead to their excision even though some ofthese transcripts might encode an active transposase. Whilethe longest (L) transcripts in pyl-v are the Dart1-27transcripts having intron 1 at 5’-UTR spliced, the middle (M)and the shortest (S) transcripts were mixtures that had one or
two additional introns spliced, respectively, although theexact splicing sites were often different. It is noteworthy that
all the shorter transcripts characterized in pyl-v were derived
from Dart1-27, indicating that some of the abundantly
expressed transcripts must have undergone further splicing in

74

Figure 1 Transcripts of the Dart1 transposase genes detected in rice. (A)Structure of Dart1-27 in the pyl-v genome. (B) Schematic representationof the alternatively spliced transposase transcripts observed in rice plants.
Three different sized transcripts (L, M, and S) were observed, and theirsplicing patterns are categorized.

II. Reverse genetic analysis of rice genes

Among active rice DNA transposons, nDart1-0 and its
relatives appear to be more suitable than the others fortransposon tagging in rice because (1) their transposition canbe controlled under natural growth conditions, i.e., thetransposition of nDart1-0 can be induced by crossing with aline containing an active aDartelement and stabilized bysegregating aDart, and (2)nDart1-0 and its relatives areoften found at GC-rich regionsin the genome and tend tointegrate into promoter proximal
genic regions. Because we havealso obtained dominant andsemi-dominant mutants (Figure2), nDart1-promoted mutant
lines can contribute tofunctional genomics in rice.
Figure 2. Semi-dominant bushy
dwarf tillers mutant.

Publication List

〔Original paper〕

Z., Takagi, K., Eun, C.H., Maekawa, M., Takahara, H.,• Shimatani,
Hoshino, A., Qian, Q., Terada, R., Johzuka-Hisatomi, Y., Iida, S., andTsugane, K. (2009). Characterization of autonomous Dart1 transposons
belonging to the hAT superfamily in rice. Mol. Gen. Genomics 281,
329-344.

National Institute for Basic Biology

Research Support

RESEARCH SUPPORT FACILITIESHeadTAKADA, Shinji● Large Spectrograph Laboratory
Professor (Adjunct):
WATANABE, Masakatsu
Technical Staff:
HIGASHI, Sho-ichi
Technical Assistants:
ENDOU, Seiichiro
ICHIKAWA, Chiaki
Secretary
ISHIKAWA, Azusa● Tissue and Cell Culture Laboratory
Assistant Professor:
HAMADA, Yoshio
Technical Assistant:
TAKESHITA, Miyako● Computer Laboratory
Assistant Professor:
Technical Staff:
Technical Assistant:

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

● Plant Culture Laboratory
Technical Staff:
NANBA, Chieko
Technical Assistant:
SUZUKI, Keiko

1. The Large Spectrograph Laboratory

This laboratory provides, for cooperative use, the largest
spectrograph in the world, the Okazaki Large Spectrograph(OLS), dedicated to action spectroscopical studies of various
light-controlled biological processes. The spectrograph runs
on a 30kW Xenon arc lamp and has a compound grating
composed of 36 smaller individual gratings. It projects aspectrum of a wavelength range from 250nm (ultraviolet) to
1,000nm (infrared) onto its focal curve of 10m in length. The
fluence rate (intensity) of the monochromatic light at eachwavelength is more than twice as much as that of thecorresponding monochromatic component of tropicalsunlight at noon (Watanabe et al., Photochem. Photobiol. 36,
491-498, 1982).

The NIBB Cooperative Research Program for the Use of
the OLS supports about 20 projects every year conducted byvisiting scientists, including foreign researchers, as well as
those in the Institute.
Action spectroscopical studies for various regulatory anddamaging actions of light on living organisms, biologicalmolecules, and artificial organic molecules have been
conducted (cf. Watanabe, M. In “CRC Handbook of Organic
Photochemistry and Photobiology, 2nd ed.”. pp. 115-1~11516, 2004).
An advanced irradiation system composed of CW lasers(364nm, 390-410nm, 440-460nm, 532nm, 655nm, 752nm)
and uniform-fluence-rate irradiation optics interconnected by
optical fibers was constructed in 2003. An advanced
observation system for cellular and intracellular
photobiological responses utilizing a two-photon microscope(FV300-Ix71-TP with a MaiTai laser) and a microbial
photomovement analyzer (WinTrack2000/Ecotox) was also
introduced.

2. Tissue and Cell Culture Laboratory

Various types of equipment for tissue and cell culture areprovided. This laboratory is equipped with safety rooms
which satisfy the P2/P3 physical containment level. Thisfacility is routinely used for DNA recombination
experiments.

3. Computer Laboratory

The computer laboratory maintains several computers toprovide computation resources and the means of electronic
communication within NIBB. Our main computer system,
the Biological Information Analysis System (BIAS), consistsof a shared memory parallel computer (SGI Altix 350;
8CPU, 48GB memory) with a disk array storage system(D-RAID; 1.6TB×10), a high-performance cluster system(DELL PowerEdge 1850; 2CPU×(16+1) nodes), and a datavisualization terminal (DELL Precision 370). Some personal
computers and color/monochrome printers are also available.
On this system, we provide various biological databases anddata retrieval/analysis programs, and support large-scale data
analysis and database construction for institute members.
The computer laboratory also provides network
communication services. Most of the PCs in each laboratory,
as well as all of the above-mentioned service machines, areconnected to each other by a local area network (LAN),
which is linked to the high performance multimedia
backbone network of the Okazaki National Research Institute(ORION). Many local services, including sequence analysis
services, file sharing services, and printer services, areprovided through this network. We also maintain a public
World Wide Web server that contains the NIBB home page(http://www.nibb.ac.jp/).

4. Plant Culture Laboratory
Figure 1. The Large Spectrograph

Plant culture laboratory consist of biotron, plant cell culture
facility, and experimental farm. Biotron contains a large
number of culture boxes and a limited number of rooms withenvironmental control for plant culture. In some of thesefacilities and rooms, experiments can be carried out at the P1

75

National Institute for Basic Biology

Research Support

physical containment level under extraordinary environments
such as strong light intensity, low or high temperatures, etc.
Plant cell culture facility is equipped with autotrophic andheterotrophic culture devices and equipment forexperimental cultures of plant and microbial cells. Facilities
for the preparation of plant cell cultures, including an aseptic
room with clean benches, are also provided. Experimental
farm consists of two 20 m 2 glass-houses with precise
temperature and humidity control, three green houses at theP1 physical containment level, a small farm, and two
greenhouses (45 and 88 m2) with automatic sprinklers. The
laboratory also includes a building with storage and work
space.
Facilities for the preparation of plant cell cultures, including
an aseptic room with clean benches, are also provided.

Publication List on OLS Collaboration〔Original papers〕

N., Suzuki, T., Watanabe, M., and Takeda, M. (2009).
• Izawa,
Characterization of arylalkylamine N-acetyltransferase (AANAT)

•
•

76

activities and action spectrum for suppression in the band-leggedcricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae). Comp.
Biochem. Physiol. B. 152, 346–351.
Suzuki, T., Izawa, N., Takeshima, T., Watanabe, M., and Takeda, M.
(2009). Action spectrum for the suppression of arylalkylamine
N-acetyltransferase activity in the two-spotted spider mite Tetranychus
urticae. Photochem. Pyotobiol. 85, 214-219.
Suzuki, T., Watanabe, M., and Takeda, M. (2009). UV tolerance in the
two-spotted spider mite, Tetranychus urticae. J. Insect Physiol. 55, 649654.

CENTER FOR TRANSGENIC ANIMALS AND PLANTS

Head

IGUCHI, Taisen

Associate Professors:
Technical Staff:
Postdoctoral Fellows:
Technical Assistants:

WATANABE, Eiji
SASAOKA, Toshikuni
TANAKA, Minoru
HAYASHI, Kohji
NOGUCHI, Yuji
YAMANAKA, Megumi
WATANABE, Kaori
KAWAMURA, Motofumi
INADA, Yosuke
KAJIWARA, YuyaICHIKAWA, Yoko
TAKAGI, Yukari

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.
The NIBB Center for Transgenic Animals and Plants(CTAP) was established in April 1998 to support researchusing transgenic and gene targeting techniques at NIBB. The
CTAP is managed by the head (professor, a concurrent post)
and three associate professors.

2. The use of various kinds of instruments to analyze mutant,transgenic, and gene-targeted animals and plants.
3. The development of novel techniques related to transgenic
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 CTAP building in 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 2. Liquid nitrogen storage equipment for cryopreservation

Figure 1. The new center facility for transgenic animals in the Yamate
areaTechnical 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 CTAP are as follows:
1. The provision of information, materials, techniques andanimal housing space to researchers.

In 2009 (from January 1 to December 31) 4,639 mice and1,107 fertilized eggs were brought into the CTAP in the
Yamate area, and 38,835 mice (including pups bred in thefacility) and 139 fertilized eggs were taken out.
A number of strains of genetically altered mice from outside
the CTAP 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. At March 2009,
we expanded the facility which includes areas for breeding,behavioral tests and transgenic studies using various kinds ofrecombinant viruses. In 2009 (from January 1 to December31) 44 mice were brought into the CTAP in the Myodaijiarea, and 585 mice (including pups bred in the facility) weretaken out.

77

National Institute for Basic Biology

Research Support

Japanese Government. We have supported the activities of
NBRP Medaka by providing standard strains, induced
mutants and transgenic lines and training personnel regardingfish maintenance. As the result of the NBRP project, medaka
transgenic strains have been successfully generated in the
CTAP, which allows inducing the gene of interest by heat
treatment in combination with a Cre/loxP system.

Figure 3. Breeding equipment for mice and rats in transgenic studies
using recombinant viruses

II. Research support activities (small fish,
birds, and insects)

The first floor of the CTAP building in the Yamate area
provides space and facilities to maintain small fish, chickembryos, and insects. In the laboratory room for chickembryos, a large incubation chamber is equipped and set at
42 degrees (suitable for chick embryogenesis). For
researchers who need fish, 480 tanks (1 liter) and 450 tanks(3 liters) are available for medaka and zebrafish, respectively.
Water circulates and can be maintained to suit the conditions
desired for fish breeding in the aquarium systems. Currently,
five or more mutant lines and ten or more transgenic lines of
medaka and zebrafish are maintained in our facility. In
addition to the rooms mentioned above, another room isavailable for insects. All the rooms are qualified to meet thecriteria for transgenic animals, allowing researchers togenerate and maintain these important research tools.
In 2009 (from January 1 to December 31), 4,461 medaka
and zebrafish (180 eggs, 1,230 embryos and 3,051 adults)
were brought to the facility and 91,786 medaka and zebrafish(89,710 fertilized eggs, 1,070 embryos and 1,006 adults,
including animals bred in the facility) were taken out. In thelaboratory for chick embryos 7,925 fertilized chicken eggs
were brought in and 23 fertilized eggs and 44 chicken
embryos were taken out. These animals were used forresearch activities in neurobiology and developmental
biology.
In 2007 NIBB was approved as a core facility of the
National BioResource Project (NBRP) for Medaka by the78

Figure 4. Breeding equipment for small fish

III. Academic activities

The associate professors of this center - E. Watanabe, T.
Sasaoka and M. Tanaka - are the principal investigators of
the Laboratory of Neurophysiology, the Laboratory of
Neurochemistry and the Laboratory of Molecular Genetics
for Reproduction, respectively. The Laboratory of
Neurophysiology is studying mechanisms of the visualsystem using a psychophysical approach. The Laboratory of
Neurochemistry is studying the physiological role of the
dopaminergic system using genetically altered mice. The
Laboratory of Molecular Genetics for Reproduction is
studying the molecular mechanisms of reproductive organ
development and sex differentiation using mutagenized ortransgenic medaka. For details, please refer to the academic
activities of each laboratory.

RESEARCH CENTER FOR INTEGRATIVE AND COMPUTATIONAL BIOLOGYHead

HASEBE, Mitsuyasu

Professor (Concurrent):

MOCHIZUKI, Atsushi

The aims of the research center for integrative andcomputational biology are (1) investigating the fundamental
principles of various biological phenomena based on the
integration of computational science and biology; (2)establishing new methodologies for integrative biology; and(3) providing new technology and knowledge to researchers.
Our ultimate goal is to establish a new bioscience that
includes methods originally utilized in different fields:
informatics, mathematics, and biology.

I. Research activity

The research center for integrative and computational
biology was founded in 2001 during a time of rapid progressin modern biology. The success of world wide genome
projects has provided a huge amount of new information on
genes, leading to expectations of advances in thedevelopment of new medicines for intractable diseases, theexploitation of new cultivated plants resistant to noxious
insects, and so on. In addition, it is the object of present-day
biology to research higher-order phenomena that are made up
of complex interactions between many genes. To grapple
with these challenges, it is necessary to decipher hugeamounts of gene information and to reveal the fundamentalsof the biological behavior of cells and organisms.
Mathematical and computational sciences have strong
capacities for dealing with these challenges. Computational
methods make it possible to process ever-increasing amountsof data. Hypothetical experiments (including the evolution of
past organisms) based on mathematical or computationalmodels make it possible to consider conditions which are
impossible in real experiments. We continue to researchhigher-order phenomena in biology using mathematical andcomputational methods, as well as developing new
methodologies for studying complex phenomena.

For example, integrative methods are especially importantfor understanding pattern formation in development.
Morphological differences between species are an importantresearch focus of current developmental biology. What is themechanism responsible for the difference of morphogenesis
between species? Theoretical studies are useful in identifying
candidates for cell or gene interaction that are likely to be
responsible for the systems in real organisms. This method
gives us an integrative understanding of the behavior ofcomplex systems in biology, including gene regulatory
networks.

II. Collaborative activity

Interaction between researchers is essential to make the bestuse of mathematical and computational methods forexperimental biology. Theoretical methods provide testablepredictions which the experimental biologists are able toinvestigate before turning the results back over to thetheoreticians for the next round of predictions. By repeatingthese predictions and tests, the integrative methods arecontinually developed. The Research Center for Integrativeand Computational Biology has continued to encourage andenhance interaction between theoretical biologists andexperimental biologists.
The center provides equipment such as cluster machines
and Unix-based machines for computation and experiments.
Members can also use computational equipment at the
Research Center for Computational Science in Okazaki. The
center also provides experimental equipment forcollaborations with experimental biologists.

Figure 2. A cluster machine

Figure 1. The laboratory room for computational studies

Over the last five years we have organized four meetings,
including two international meetings on systems biology andmathematical biology that had many participants studying
biological systems using different methods, including
physics, mathematics and computational science. The
meetings enhanced interactions between researchers in manydifferent fields and resulted in several collaborative research
projects.

79

National Institute for Basic Biology

Research Support

CENTER FOR ANALYTICAL INSTRUMENTS (managed by NIBB)HeadTAKADA, Shinji

Technical Staff:

Secretary:

Environmental Scanning Electron Microscope (PHILIPSXL30 ESEM)
Confocal Laser Scanning Microscopes (Leica TCS SP2,
OLYMPUS FV1000)
Fluorescence Microscope (KEYENCE BZ-8000)
Color Laser 3D Profile Microscope (KEYENCE VK-8500)
High-Resolution Quick Microscope (KEYENCE VH-5000)

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
TAKAMI, Shigemi
OKA, SanaeTANIGUCHI, Misako
ICHIKAWA, Mariko

The Center is responsible for amino acid sequence analysis,
amino acid analysis, chemical syntheses of peptides, andnext generation sequencing with SOLiD system (ABI) as
well as supporting researchers at NIBB and NIPS. The
Center’s instruments can be used by researchers from outside
the institute upon proposal.

Representative Instruments

Protein Sequencers (ABI Procise 494HT, 492cLC)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizer (ABI 433A)
Plasmid Isolation Systems (Kurabo PI-50α, PI-50, PI-200)
Automatic Nucleic Acid Isolation System (Kurabo NA-2000)
Isoelectric Focusing System (Amarsham Biotech IPGphor)
Genetic Analyzers (ABI PRISM 3130xl, 310)
Real Time PCR System (ABI 7500)
Thermal Cyclers (ABI 9700, Perkin Elmer PJ-9600,
Biometra TGRADIENT)
Particle Delivery System (Bio-Rad BiolisticPDS-1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)High Performance Liquid Chromatographs (Shimadzu
LC-10AD, LC-6AD, Waters 600E, Alliance UV system)
Integrated Micropurification System (Pharmacia SMART)
Flow Cytometer (Coulter EPICS XL)
Biomolecular Interaction Analysis Systems (Pharmacia
BIACORE 2000, Affinity Sensors IAsys)
Laboratory Automation System (Beckman Coulter Biomek
2000)
ESR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)
MALDI-TOF-MS (Bruker Daltonics REFLEX III)
LC/Q-TOF MS (Waters Q-TOF Premier)
Inductively Coupled Plasma Atomic Emission
Spectrometer (Seiko SPS1200A)
Spectrofluorometers (Hitachi 850, F-4500, Simadzu
RF-5000)
Spectrophotometers (Hitachi 330, 557, U-2001, Varian
Cary5G, Perkin Elmer Lambda-Bio, NanoDrop ND-1000)
Microplate Luminometer (Berthold MicroLumat LB 96P)
Microplate Readers (Corona MTP-120, MTP-100F)
FT-IR Spectrophotometer (Horiba FT-730)
Bio Imaging Analyzer (Fujifilm BAS 2000)
Luminescent Image Analyzers (Fujifilm LAS 3000 mini)
Fluorescence Bio Imaging Analyzer (Takara FMBIO II)

80

Figure 1. Protein sequencers

Figure 2. Confocal Laser Scanning Microscope

Figure 3. LC/Q-TOF-MSCENTER FOR RADIOISOTOPE FACILITIES(OKAZAKI RESEARCH FACILITIES)HeadHASEBE, Mitsuyasu

Associate Professor:
Technical Staff:

Technical Assistants:

Table 2. Users and visitors who entered each controlled area in 2009

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

The technical and supporting staff of the Center forRadioisotope Facilities (CRF) maintain three 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 CFBI
(Common Facilities Building I)-branch and LGER
(Laboratory of Gene Expression and Regulation)-branch in
the Myodaiji-Area. Ogawa and Sawada worked in the
Yamate-Area.
The number of registrants and the number of users fromJanuary 2009 to December 2009 are presented in Table 1.
Users counted by the monitoring system going in and out of
the controlled areas numbered 3,764 persons during thisperiod. The percentages and numbers for each area areshown in Figure 1 and Table 2. The annual changes ofregistrants and the number of totals per year are shown in
Figure 2.
The balance of radioisotopes received at the CRF is shownin Table 3.
Thirty years have passed since the CFBI-branch opened andsome parts of the air supply and exhaust system havedeteriorated. Figure 3 shows the repair work done on the
CFBI-branch to accommodate the law.

Figure 2. Annual changes of registrants and number of totals per fiscal
year

Table 3. Balance of radioisotopes received and used (kBq) at eachcontrolled area in 2009

Table 1. Numbers of registrants and users at Myodaiji-Area and YamateArea in 2009

Figure 1. Percentage of users going in and out of each controlled area in2009

Figure 3. Repair work on air supply & exhaust system at CFBI-branch
A: The room re-heaters were replaced.
B: The air supply system’s eliminators were replaced.
C: The exhaust fan was repaired.

81

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 international cooperationand public relations, however those aspects of the department
became the “Office of Public Relations and International
Cooperation” in April 2009.

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.

822) Editing of the Annual Report (in collaboration with the
Office of Public Relations and International Cooperation)
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.

The main activities of the office in 2009

OFFICE OF PUBLIC RELATIONS AND INTERNATIONAL COOPERATION

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.
2) Supporting international conferences
On-line registration, web page construction, brochure
editing, hotel reservation, banquet and party arrangement,
photography and video recording, etc., for international
conferences held at NIBB (Table 1)
3) Management of education related programs
The Fourth International Practical Course (June, 2009)
Bioinformatics training course (August and September,
2009)4) Updating and maintenance of the NIBB web page5) Editing of publications, production of posters andleaflets
Design and distribution of posters for international
conferences and advertisements for the graduate school’s
entrance examination. Publication of the pamphlet “An
introduction to the National Institute for Basic Biology”.
Publication of leaflets introducing the individual researchersof NIBB (in Japanese). Publication of “NIBB News” (Intrainstitutional newsletter, in Japanese).
6) Organization of scientific outreach programs
Management of collaborative events with Japan’s National
Museum of Emerging Science and Innovation. Assisting
exhibitions held at the Nagoya City Scientific Museum.
7) Aiding visitors (in collaboration with the Technical
Division)

Chair

UENO, Naoto

Assistant Professor(Specially appointed) :Technical Assistants:

KURATA, Tomoko
OTA, Misaki
TANAKA, Megumi
MIYATA, Hiroe
ADACHI, Shoko
OTA, Kyoko
EMDE, Jason R.
KAWAGUCHI, Colin

The Office of Public Relations and International
Cooperation, in order to communicate the activities of NIBB
to the widest audience, performs both standard public
relations duties as well as communication with scientificpublications and organizations worldwide. The Office of
Public Relations and International Cooperation also manages
the planning and administration of the international
symposiums and international training courses held at NIBB.

Name

Date

Title

Organizer

9th NIBB-EMBL joint symposium

April 20-22

Functional Imaging from Atoms toOrganisms

1st NIBB-MPIZ joint symposium

August 25-27

Japanese-German Symposium on Evolution
and Development

J. Ellenberg,
K. Nagayama,
N. UenoM. Koornneef
and K. Okada

Table 1. International conferences managed by the Office of Public Relations and International Cooperation in 2009

Ninth NIBB-EMBL Symposium

Open Campus 2009

Graduate School Showcase 2009

Passionate about Science

Figure 1. Examples of posters, pamphlets and abstract books produced by the Office of Public Relations and International Cooperation in
2009

83

National Institute for Basic Biology

Research Support

TECHNICAL DIVISIONHeadFURUKAWA, KazuhikoCommon Facility Group

Research Support Group

Chief:

Chief:

MIWA, Tomoki● Research Support Facilities
Unit Chief:
HIGASHI, Sho-ichi
Subunit Chief:
NANBA, Chieko
Technical Staff:
NISHIDE, Hiroyo
NAKAMURA, Takanori
Technical Assistants:
SUZUKI, KeikoICHIKAWA, Chiaki
TAKESHITA, Miyako
NISHIMURA, Noriko
YAMAMOTO, Kumi
ENDOU, Seiichiro
Secretary:
ISHIKAWA, Azusa● Center for Analytical Instruments
Unit Chief:
MORI, Tomoko
Subunit Chief:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
Technical Staff:
TAKAMI, Shigemi
TANIGUCHI, Misako
Secretary:
ICHIKAWA, Mariko● Transgenic Animal Facility
Subunit Chief:
HAYASHI, Kohji
Technical Staff:
NOGUCHI, Yuji
Technical Assistants:
ICHIKAWA, Yoko
TAKAGI, Yukari● Disposal of Waste Matter Facility
Unit Chief:
MATSUDA, Yoshimi● Radioisotope Facility
Unit Chief:
Subunit Chief:
Technical Staff:
Technical Assistant:

MATSUDA, Yoshimi
SAWADA, Kaoru
IINUMA, Hideko
ITO, Takayo

The Technical Division is a support organization forresearchers and research organizations within the 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 the research activities as
described in the reports of individual research divisions.

84● Cell Biology
Unit Chief:

KAJIURA-KOBAYASHI,
HirokoKONDO, 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
Technical Staff:
MOROOKA, Naoki● 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.

NIBB-EMBL Meetings
The 9th NIBB-EMBL Joint Meeting: “Functional Imaging from Atoms to Organisms”
April 20 (Mon)-22 (Wed), 2009

Okazaki Conference Center, Okazaki

This joint meeting was the second bioimaging-related
meeting of the NIBB-EMBL joint meeting series following
the first symposium “Frontiers of bioimaging” held in 2006,
and aimed to overview the recent advance of bioimaging
from the molecular to the organismal level. In contrast to theprevious meeting in which light microscopy technologies
focusing on cell functions and animal and plant development
were discussed, in this meeting many cutting edge
technologies of single molecule imaging at high resolutions
with electron as well as light microscopy combined withquantitative analyses to capture molecular and cellular
dynamics were presented. The talks illustrated the rapid
advance of bioimaging during the last three years andoutlined the future direction of bioimaging using new
methodologies of electron and light microscopy, imagingbased large scale screening, and image processing forquantitative analysis of a diverse range of biologicalphenomena. Several short oral presentations were selectedfrom posters and four young scholars including graduate
students of EMBL participated in the meeting as a part of thestudent exchange program of the NIBB-EMBL collaboration,
both of which further stimulated the meeting and contributed
to the success of the symposium.

Speakers
Cusack, Stephen (EMBL Grenoble), Ellenberg, Jan (EMBL Heidelberg), Frangakis, Achilleas (Univ. Frankfurt), Keller, Philipp (EMBL
Heidelberg), Mueller-Reichert, Thomas (Max Planck Inst.), Patterson, George (NIH), Sattler, Michael (Helmholtz Zentrum Munchen &TU Munchen), Schultz, Carsten (EMBL Heidelberg), Sedat, John (UCSF), Stelzer, Ernst (EMBL Heidelberg), Weiss, Matthias (DKFZ)
Fujimori, Toshihiko (NIBB), Goshima, Gohta (Nagoya Univ.), Hamaguchi, Hiro-o (Univ. Tokyo), Haraguchi, Tokuko (KARC), Kikkawa,
Masahide (Kyoto Univ.), Maeshima, Kazuhiro (NIG), Miyawaki, Atsushi (RIKEN / JST), Nabekura, Junichi (NIPS), Nagai, Takeharu(Hokkaido Univ.), Nagayama, Kuniaki (OIB), Nakano, Akihiko (Univ. Tokyo), Nonaka, Shigenori (NIBB), Onami, Shuichi (RIKEN ASI),
Sadato, Norihiro (NIPS), Ueno, Naoto (NIBB)

Invited Students from EMBL
Blattmann, Peter; Cobos Correa, Amanda; Heiligenstein, Xavier; Wuensche, Annelie

Comments from the Students

Xavier Heiligenstein (EMBL)

I found the content of the conference really interesting andof a great level. The comments, questions and discussion tome really facilitated going deeper in to each presentation. I
got a great and deep overview of what light microscopy canoffer us today in complement to electron microscopy andalso enjoyed the external activities a lot, such as visiting the
NIBB institute, its spectrophotometer and its brand newLight Sheet Microscope. Having the opportunity to give ashort talk was also nice and lead to good feed-back during
the poster session. Complementing this nice meeting, we hadsome great interaction with the Post-doc and Students andalso greatly enjoyed the side parts of this symposium (a very
nice party on Tuesday evening). And what to say about theexcursion that lead us to see wonderful landscapes, cultural,and scientific Japanese pride. Again I would like to thankyou again for such a nice welcome.

Peter Blattmann (EMBL)

I enjoyed the 9th NIBB-EMBL Symposium on Functional Imaging
a lot. For me, it was an invaluable experience to come to Japan andattend this conference. I was amazed by the high standards of all thetalks and discussions throughout the conference and profited a lot
from them. The program was very well chosen and I think I received
a good and interesting overview of the cutting edge research goingon in this field. The hospitality with which we were received wasoutstanding and I felt very welcome at all times. However, for me
personally, the greatest experience was to meet the Japanese
students, post-docs and the speakers and thus to learn about the
Science and technology conducted in Japan. I am very grateful tohave received the opportunity to present both my poster and also ashort talk at this conference. The following excursion was also very
enjoyable and for the first time I experienced Japanese culture andmountains, which I liked very much. I am very glad, that this fruitfulcollaboration between NIBB and EMBL is continuing and lookforward to further opportunities to interact with NIBB members.
Thank you again so much for the hospitality and all your efforts!

85

NIBB-EMBL Meetings
The 1st NIBB-EMBL PhD Mini-Symposium and 11th International EMBL PhD Students Symposium
October 28 (Wed)-31 (Sat), 2009

EMBL, Heidelberg

The first NIBB-EMBL PhD Mini-Symposium was held at
EMBL, Heidelberg on 28 October. Seven PhD students fromNIBB and three from Nagoya University got together with
EMBL pre-docs to exchange and discuss their recent results.
This symposium and subsequent “lab tour” also connected
our students with EMBL PIs who are in the field of theirrespective interests. On 29-31 October the 11th International
EMBL PhD Students Symposium “Puzzles in Biology;
Putting the Pieces Together” was held at EMBL. The
program contained three sessions; the first aimed to shedlight on the latest technical innovations in the field of
biology, the second session focused on complex puzzles
which had been resolved by integrating data from different
scientific fields, and the final session covered several issues
which illustrated beneficial relationships between basic
biology and applied studies to understand the mechanisms
causing human diseases. Our PhD students presented theirresearch work during the poster and the short-talk session. It
was a great opportunity for our PhD students to meet
students from a variety of European countries and hear about
their interesting work. Moreover, they were very much
impressed by the scientific atmosphere and cutting-edge
researches at EMBL. We hope to continue this collaborationin the future.

Participating students from Japan

Hara, Yusuke; Morita, Hitoshi; Okamoto, Haruko; Goto,
Shino; Tameshige, Toshiaki; Sugimoto, Ryo; Takahashi,
Hiroyuki (NIBB); Yamamoto, Haruki; Mollah, Md.Bazlur
Rahman; Tanimoto, Masashi (Nagoya Univ.)

Comments from students (excerpts)

・Takahashi, Hiroyuki
This was my first experience of an English oralpresentation in a foreign country and its preparation was a
tough job for me. This opportunity of an oral presentation
was, however, a precious one and gave me experience indiscussing my research with EMBL people. A student of
EMBL was assigned as a guide for each student from Japan,
which made it easier to communicate with EMBL students
and to have a glimpse of the students’ life at EMBL and theirview of research. I found students at EMBL are much moreaggressive and positive in their research than I had expected,
which I think we should follow.
・Tameshige, Toshiaki
At the 11 th International EMBL PhD Student
Symposium, there were a lot of interesting presentations byresearchers from various fields, but the most surprising thing
was that the whole symposium was organized by the EMBL

86

students. From the invited speakers, I can easily imagine how
widely the EMBL students’ knowledge and interest range.
And I thought it was a fruitful experience for them toorganize such a big symposium while still learning as
students.
・Haruko, Okamoto
The best thing I obtained at this Ph.D symposium was that I
had an opportunity to communicate with students of similarages from various countries. I spoke with students fromcountries unfamiliar to me, such as Israel and Nigeria. I
experienced the everyday life of students in Europe, such as
dance parties and talks over drinks. I found, contrary to my
expectation, we have much in common.

The 1st NIBB-MPIZ Joint Symposium
Japanese-German Symposium on Evolution
and Development
Max Planck Institute for Plant Breeding Research, Cologne, Germany
August 25(Tue)-27(Thu), 2009

NIBB formalized an Agreement with Max-Planck Institutefor Plant Breeding Research (MPIZ, at Cologne, Germany)in April, 2009. This agreement aims to promote intellectual,educational and technological exchange between researchersand students of Japan and Germany by joining NIBB and
MIPZ as national hubs of scientific cooperation in the fieldof plant science. As the first joint activity, a JapaneseGerman Symposium on Evolution and Development washeld at MPIZ in Cologne from August 25 to 26.
NIBB began gathering applicants who wished to attend thesymposium and are planning possible collaborative researchwith researchers of MPIZ through e-mail networks, andselected seven researchers from the applicants. At the
symposium the Japanese delegation, consisting of the sevenapplicants and five PIs of NIBB plant science laboratories,
participated in discussions with researchers and students of
MPIZ, Cologne University, and other universities andinstitutes of Germany. After the symposium, on the morningof August 27, individual talks were held between Japaneseand German researchers with the aim of starting possible
collaborative research. In the afternoon of August 27,
Japanese researchers visited the Demonstration Garden and
Wissenschaftsscheune (Science Barn) of MIPZ, which was
founded for the purpose of giving the public an opportunity
to experience several aspects of plant science including
Mendel’s law, domestication of crops, and GMO plants.
As a continuing activity, NIBB supports the traveling costsof three Japanese researchers who visit MIPZ forcollaborative research projects which arose from attending
the Symposium.

87

The NIBBInternational
Practical 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
course replaced our Bioscience Training Course, which had
been held for twenty years and which had, for the most part,
accepted participants from universities and institutes inJapan. The Fourth International Practical Courses was held in2009 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. We hope that the techniques andknowledge provided in these courses will prove useful in the
future work of all of the participants.

NIBB started a series of training courses on up-todate research techniques for researchers from mainly
Japanese universities and institutions. In 2009 we helda bioinformatics training course on the statisticalanalyses of data obtained by microarray analyses.

The Fourth International Practical
Course: “ 2009 NIBB Laboratory
Course and Workshops on
Physcomitrella patens”

Microarray analysis is one of the most frequently
used comprehensive methods for researching gene
expression. But it is not easy to properly translate
results from microarray data for ordinary biologists
who are not familiar with bioinformatics. The aim of
this course was to give lectures on the fundamentalsof microarray analysis using the statisticalenvironment “R”, and practical methods for gene
ontology analysis using free software.

■Period: June 29 (Mon)- July 3 (Fri), 2009
■Participants: 17 (five from Japan, two from the UK,
Germany, and Israel, and one each from Belgium,
Czech, France, Sweden, Singapore, and India)■Lectures:
Dr. Andrew C. Cuming (Univ. Leeds)
Dr. Yasuko Kamisugi (Univ. Leeds)
Dr. Tomoaki Nishiyama (Kanazawa Univ.)
Dr. Tetsuya Kurata (JST)Dr. Yoshikatsu Sato (JST)Dr. Yuji Hiwatashi (NIBB)
Dr. Minoru Kubo (JST)Dr. Takashi Murata (NIBB)
Dr. Mitsuyasu Hasebe (NIBB)
Contents of the course: basic techniques including
cultivation, observation of developmental processes atthe cellular level, gene targeting, bioimaging, andbioinformatics used in the biology of the moss
Physcomitrella patens

88

The NIBB Training Course“How to translate results frommicroarray analysis”■Period:August 19 (Wed) -21 (Fri), 2009
September 8 (Tue) -10 (Thu), 2009
■L ecturers: Drs. Koji Kadota and Yuji Nakai(University of Tokyo)■Participants:
August course: 16 (including 5 from NIBB)
September course: 17 (including 4 from NIBB)

1.
2.
3.
4.

Fundamentals of microarray analysis
How to use the statistical environment “R” formicroarray analysis
Fundamentals of gene ontology analysis
Practical methods for gene ontology analysis

INDEX
A ....................................................................................................
ADACHI, Shoko

83

HASHIYAMA, Kazuya

20

AKANUMA, Takashi

23

HATTORI, Nobuko

37

AJIOKA, Rie

AOKI, Yumiko

AOYAMA, Tsuyoshi
ARAI, Yuko

ARAKI, Masami

ASANO, Takuya

56

HATA, Katsusuke

33

HAYASHI, Kohji

HAYASHI, Makoto (Division of Cell Mechanisms)

49
8

31, 48, 72

BABA, Nayumi

49

B ....................................................................................................
BASKIN, Tobias

CHAKRABORTY, Tapas

58

CUI, Songkui

8CHEN, Qiuhong

D

ENDOU, SeiichiroF

EUN, Chang-Ho

83

75, 8448....................................................................................................
49
FUJII, Tomomi

FUJIKAWA, Akihiro

37

FUJIMORI, Toshihiko

26

FUJISAWA, Chiemi

20

FUJIKI, Yuki

FUJIMORI, Toshihiko
FUJITA, Hironori

FUKADA-TANAKA, Sachiko
FUKAZAWA, Mitsue
FUKUDA, Hiroo

FURUKAWA, Kazuhiko
FURUYAMA, Makiko

11
3

52

48, 52, 84
8, 71
3

84
40

G ....................................................................................................
GONDA, Naoko

16

GOTO, Shino

8

GOTO, Miho

GOTOH, Ayako

H

GUAN, Guijun

49
61
31

....................................................................................................
16, 75
HAMADA, Yoshio

HANADA, Takao

11

HARA, Ikuyo

71

HARA, Reiko

35

HARA, Kenshiro
HARA, Yusuke

HARASHIMA, Toshiaki
HASEBE, Mitsuyasu

HASEGAWA, Yoshinobu
HASHIMOTO, Yoshiko

28
17
11

3, 49, 79, 81
48
20

75, 84HIRAYAMA, Yuka#

40

HIYAMA, Takeshi
HONDA, Satoko

HORIGUCHI, Ryo

HORIUCHI, Takashi....................................................................................................
37
DOKYO, Yumi

EMDE, Jason R.

20

HOSHINO, Atsushi

I

58

HIGASHI, Sho-ichi

HIWATASHI, Yuji

23

E ....................................................................................................

HAYASHI, Yoshiki

HIROKAWA, Junya

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

8

20

HIRAO, Mayumi

49

77, 84

HAYASHI, Makoto (Division of Developmental Genetics)
HAYASHI, Tomoko

8

23

ASAO, Hisayo

40

HOSOMI, Azusa

26
40
49
37
20
31

3, 45

48, 7356....................................................................................................
ICHIHASHI, Yasunori61

ICHIKAWA, Chiaki

75, 84ICHIKAWA, Rie

11, 28ICHIKAWA, Mariko
ICHIKAWA, Takehiko80, 8469

ICHIKAWA, Yoko

77, 84

IGUCHI, Taisen

3, 58, 77

IGARASHI, Hisako
IHARA, Masaru
IIDA, Shigeru

IINUMA, Hideko

IKEUCHI, Momoko
IMAI, Akiko

IMAIZUMI, Taeko

IMAMURA, Masako
IMOTO, Eiko#INADA, Kana

INADA, Yosuke

INAGAKI, Masako
IRIE, Aki

ISHIHARA Hitomi
ISHII, Junko

35
37

7, 48

81, 84
61
40
58
37
40
28
77
71
61
20
11

ISHIKAWA, Azusa

75, 84

ISHIKAWA, Naoko

61

ISHIKAWA, Hiroe

ISHIKAWA, Takaaki
ISHINE, Naomi

ISHINO, Fumitoshi

ISHITANI, Shizuka

ISOSHIMA, Yoshiko
ITAZU, Masako
ITO, Takayo

31
49
45
3

23
37
45

81, 8489

INDEX

ITO, Yukiko

IWASAKI, Akira

49

KONDO, Chika

35

KONDO, Maki

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

JOHZUKA-HISATOMI, Yasuyo

KONDO, Takefumi

45

KUBO Satoru

48

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

KADOWAKI, Tamaka

KAGEYAMA, Takuya
KAGEYAMA, Yuji
KAI, Masatake

KAJIKAWA, Ikumi

KAJIURA-KOBAYASHI, Hiroko
KAJIWARA, Yuya

KAKUTA, Soichiro69, 8477
1156

KAMIYA, Kiyomi

81

KANDA, Rieko

69

KATAOKA, Yukari

84

KATO, Kazuto
KATO, Kyoko

KATO, Yasuhiko

8

56
26
3
8

58

KATSU, Yoshinao

7, 58

KAWAGUCHI, Masayoshi

7, 52

KAWAMURA, Motofumi

77

KAWAGUCHI, Colin
KAWAI, Mikihiko
KIKUCHI, Naoka

KIMURA, Tetsuaki
KIMURA, Yasuyo

KINOSHITA, Chie

KINOSHITA, Noriyuki
KITADATE, Yu

KOBAYASHI, Kaoru
KOBAYASHI, Kayo

83
67
58
56
49
33
17

7, 20, 2858
33

KOBAYASHI, Satoru

3, 20

KODAMA, Akiko

37

KODAMA, Ryuji

55, 82

KOIKE, Chieko

56

KOBAYASHI, Takafumi
KODAMA, Ken-ichi
KOIDE, Shizuyo
KOIKE, Yukari

KOJIMA, Yoko

KOMATSU, Kouji

KOMATSU, Yusuke#
KOMINE, Yuriko

7

M

49

8

KATO Azusa

83

KUROKAWA, Hiromi

17

KAMIGAKI, Akane

KANEKO, Hiroyo

KURATA, Tomoko

20

L ....................................................................................................

20

11, 70KANAI, Masatake

20

8, 28

11

KAMADA,Yoshiaki
KAMATA, Akiko

90

61

20

KUBOKI, Yuko
KUMATA, Yuji

11, 49, 84

11

8, 84

11

45
58
56

49, 6126
40
40

LANGE, Anke58....................................................................................................
MAEZAWA, Takanobu
20

MAGORI, Shimpei
MAKINO, Yumiko
MANO, Hiroaki
MANO, Shoji

MATSUDA, Chisato

MATSUDA, Yoshimi

MATSUMOTO, Masahito
MATSUMOTO, Miwako
MATSUNAGA, Wataru
MATSUOKA, Makoto

52

80, 8449
848, 5881, 8437

35, 4843
3

MATSUZAKI, Yoko

45

MITSUI, Naoko

58

MIKAMI, Yuriko
MIURA, Seiko
MIWA, Hiroki

45
37
52

MIWA, Tomoki

75, 84MIYAGI, Asuka

17

MIYAGAWA, Shinichi
MIYAKE, Satoko

MIYATA, Haruko
MIYATA, Hiroe

MIYAZAKI, Saori

MIYAZAWA, Hikota

MIZOGUCHI, Masae

7, 58
17
84
83
49
52
37

MIZUGUCHI-TAKASE, Hiroko

28, 84MOCHIZUKI, Atsushi

65, 79MIZUTANI, Takeshi
MORI, Tomoko

MORITA, Hitoshi
MORITA, Junko

MORITA, Yasumasa
MORITOH, Satoru

MOROOKA, Naoki

MURAKAMI, Michiyo

MURAKAMI, Yasuhiro
MURATA, Takashi

58, 8480, 8417
40
48

48, 7245, 8417
52
49

N ....................................................................................................
NAGAHAMA, Yoshitaka

31

NAGURA, Masako

61

NAGAKURA, Ayano

37

NAKAGAMI, Yuki

40

NAKAMORI, Chihiro

8, 35

NAKAMURA, Shuhei

33

NAKAI, Atsushi

8

NAKAMOTO, Masatoshi

31

NAKAMURA, Takanori

75, 84NAKAMURA, Takeshi

58

NAKANISHI, Norie

37

NAKAMURA, Tohru
NAKATA, Miyuki

NAKATOGAWA, Hitoshi
NAKAYAMA, Hokuto
NAKAYAMA, Kei
NANBA, Chieko

NARUSE, Kiyoshi
NIIMI KaoriNISHIDE, Hiroyo
NISHIHARA, Eri

NISHIMURA, KeikoNISHIMURA, Mikio

40
35

7, 11
61
23

75, 8456

11, 2075, 8437
33

3, 8, 82NISHIMURA, Noriko

84

NISHINA, Momoko

8

NISHIMURA, Toshiya
NODA, Chiyo

NODA, Masaharu
NOGUCHI, YujiNONAKA, Shigenori

33
20, 843, 7, 37
77, 8469

NUKAZUKA, Akira

61

OGASAWARA, Kimi

8

O ....................................................................................................
OGAWA, Kazuo

15, 81OHHARA, Kaori

23

OH-OKA, Kyoko

11

OHNO, Kaoru

71

OHSAWA, Sonoko

40, 84

OHSUMI, Yoshinori

7, 11

OIKAWA, Kazusato

8

OHSHIMA, Issei

49

OHTSUKA, Masanari

40

OKA, Naomi

69

OKA, Sanae

26, 80, 84

OKADA, Kiyotaka

1, 3, 7, 35

OKAMOTO, Koji

11

OKAMOTO, Satoru

52

OKUBO, Tadashi

23

ONO, Akemi

48

OSUMI, Noriko

3

OKAMOTO, Haruko

45

OKAMOTO, Noriko

11

OKANO, Yosuke

49

OKUDAIRA Yuichi

20

OOMIZU, Yuka

49

OTA, Kyoko

OTA, Misaki

83
83

P ....................................................................................................
PARK, Kyeung-Il

48

POPRAWKA, Tomasz

35

PAUL, Bindhu

31

S ....................................................................................................
SADAKANE, Osamu
SAITO, Miyuki

SAITOH, Hiromi

SAKAGAMI, Mari
SAKAI, Fumie

SAKANO, Hitoshi

SAKURABA, Jyuichi

SAKURAI-OZATO, Nami
SAKUTA, Hiraki
SANJO, Kazuko

SASADO, Takao

SASAKI, Tetsuya

SASAOKA, Toshikuni
SATO, Asako
SATO, Kaori

SATO, Masanao
SATO, Masaru
SATO, Yori

SATOH, Noriyuki

40
831, 48
35
31
3

37
49
37

48, 5256
40

44, 77
44
20

7, 20
58
8
3

SAWADA, Kaoru

81, 84SHIBATA, Emiko31

SEKI, Touhaku

SHIBATA, Michitaro

26
8

SHIBATA, Yasushi

31

SHIINA, Nobuyuki

7, 14

SHIGENOBU, Shuji
SHIMADA, Yu

SHIMAMOTO, Miki

SHIMATANI, Zenpei
SHIMURA, Yoshiro
SHINDO, Asako

SHINTANI, Atsuko

20
31
48

48, 72
3

17
69

SHINTANI, Takafumi

7, 37

SHUKULA, Ramohan

40

SUGIMOTO, Ryo

28

SUZUKI, Iku

8

SHIRAYA, Takeshi
SIOMI, Haruhiko

SUGIURA, Naomi

8
3

58

SUZUKI, Keiko75, 84SUZUKI, Makoto

17

SUZUKI, KuninoriSUZUKI, Ryoko

SUZUKI, Tokiko
SUZUKI, Yuko

7, 11

37
56
11

91

INDEX

T ....................................................................................................
TABATA, Satoshi

TAKADA, Ritsuko
TAKADA, Shinji
TAKAGI, Chiyo

TAKAGI, Yukari

TAKAHARA, Masahiro
TAKAHASHI, Hiroki

TAKAHASHI, Hiroyuki
TAKAHASHI, Jun

TAKAJI, Masafumi#
TAKAKI, Chikako

317, 8477, 8423

TAKASHIRO, Kayoko

TAKAYANAGI, Noriko

16

TAKEDA, Naoya

7, 52

TAKESHITA, Miyako

75, 84

TAKEHANA, Yusuke
TAKEUCHI, Yasushi
TAMADA, Yosuke

TAMESHIGE, Toshiaki
TANAKA, Megumi

56

37, 8449
35
83

TANAKA, Minoru

7, 33, 77

TANIGUCHI, Misako

80, 84

TATEMATSU, Kiyoshi

35

TANI, Sachiko
TAO, Hirotaka
TERADA, Rie

TESHIMA, Yuuko
TOMIDA, SanaiTOMINAGA, Rumi
TOMITA, Nao
TORII, Naoko

TOYOKURA, Koichi
TOYOOKA, Yayoi

TSUCHIDA, Yuhei

TSUCHIYA, Chiaki
TSUGANE, Kazuo

TSUGANE-HAYASHI, MikaTSUGE, Toyoko

TSUKAMOTO, Naho
TSUKAYA, Hirokazu
TSUZUKI, Chisa

TSUZUKI, Shihoko

37
17

48, 72
56
23
35
37
56
35
26
35
8

48, 74
48
17
49
61
23
84

U ....................................................................................................
UCHIYAMA, Ikuo
UEDA, Chizuru

8UENO, Naoto

3, 17, 83

UNO, Satoko

84

UKAI, Sakie

9267, 7523

23

WATAKABE, Akiya

40

WATANABE, KaoriWATANABE, Masakatsu

69

WATANABE, SeikoY23, 84WANGLAR, Chimwar

WATANABE, Hajime

31

23

UTSUMI, Hideko

58....................................................................................................
WADA, Takuji
35

WATANABE, Eiji

40

61

31

WATAKABE, Ikuko

23

TAKASE, Masahide

USAMI, Takeshi

WASHIO, Midori

1780, 84

TAKAO, Daisuke

W

52

TAKAMI, Shigemi

35

URUSHITANI, Hiroshi

23

3, 23, 75, 80

URAWA, Hiroko

WATANABE, Taka-aki

49
33

43, 777, 58
77

63, 757345....................................................................................................
YABE, Kimihiko
35

YABE, Taijiro

23

YAMADA, Kenji

8

YAMADA, Hiroko
YAMAGUCHI, Chinami

49
61

YAMAGUCHI, Katsushi

80, 84

YAMAMORI, Tetsuo

3, 40

YAMAMOTO, Kumi

75, 84

YAMAGUCHI, Takahiro
YAMAMOTO, Hayashi
YAMAMOTO, Takamasa
YAMANAKA, Megumi
YAMAUCHI, Takaki
YANAGIDA, Toshio

61
11

17
77
48
3

YANO, Satoshi

61

YOKOYAMA, Hiroshi

52

YATAGAI, Hiroko

YONEMITSU, Masako
YONEZAWA, Harumi
YOSHIDA, ChieYOSHIDA, Masahide
YOSHIDA, Naoko

37
33
45
52
37
28

YOSHIDA, Shosei

3, 28

YOSHINO, Jiro

37

YOSHIMURA, Yuriko
YOSHINORI, Yumi

56
8

Z ....................................................................................................
ZHANG, Lanfan
ZHOU, Linyan

37
31

Access

From Tokyo

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

From Osaka

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

From Central Japan International Airport

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

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

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

National Institutes of Natural Sciences

National Institutefor Basic Biology
38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585 Japan
tel: +81 564-55-7000 fax: +81 564-53-7400
http://www.nibb.ac.jp/en/
Issued in April 2010
Edited by the Strategic Planning Department, NIBB