INTRODUCTION

ur institute, the National Institute for Basic
Biology, was established in 1977, and we
proudly celebrated our 30th anniversary on June 1,
2007. We hosted a ceremony of celebration with oursister institute, the National Institute for Physiological
Sciences (NIPS), with nearly 300 friends, supporters,
former researchers and officers attending. Since its
foundation, the NIBB has developed as a center of
excellence in research, education, and inter-university
collaboration in the various fields of basic biology. In
2004, the NIBB and four national research institutes
established a new organization, the National Institutesof Natural Sciences (NINS), one of four InterUniversity Research Institute Corporations. We now
cover a wide variety of fields including cell biology,
developmental biology, neurobiology, evolutionary
biology and biodiversity, environmental biology,
theoretical biology and imaging science, and are
studying the basic action of genes and cells common to
all living organisms, as well as the mechanisms
involved in producing the diversified structures andactivities of life adapting to an ever-changing
environment. Research in these basic biological fields
is maintained at a high level while we continue tosearch for new and challenging fields of study. We arealso involved in ongoing collaboration with a numberof researchers from Japanese and foreign universities
and institutes. In addition to research, the education of
the next generation of researchers is another important
aim. As a member of the Graduate University for
Advanced Studies, we are involved in training
graduate students. We had five new doctors in Marchof 2008.
NIBB's activities during 2007, as well as our goals forthe future, are summarized in this booklet. We arealways open to questions, comments and suggestions.

O

Kiyotaka OKADA, D. Sci.
Director-general, NIBB

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, 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 wasestablished as a common facility for the three institutes(NIBB, NIPS, and IMS) of the Okazaki campus.

Research and Its Support
The NIBB conducts its research programs through thirtyone research units and three research support units. Eachresearch unit is called either a division, led by a professor, ora laboratory, led by an associate professor or an assistantprofessor. Each research unit forms an independent project
team. Some of the research units are adjunct and led byprofessors who hold joint appointments with otheruniversities. Adjunct divisions have a resident assistantprofessor. This arrangement facilitates exchange in research
activities in Japan. The Technical Division manages theactivities of the technical staff and helps to promote theresearch activities of each research unit and to maintain thecommon research resources of both the research support
units of the NIBB and some of 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 function also as
NIBB's divisions.

Members in the NIBB
(as of 1st April, 2007)
Director-General 1
Technical
AssistantandSecretary

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

Technical
Staff

Professor(incl. adjunct)

Associate Professor (incl. adj.) 15
Assistant Professor

86

31

27

92
Post doctoral fellow

51

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

2

18

Graduate Student

Financial Configuration of the NIBB
Donation 45(settled forApril 2006 - March 2007)
numbers are in million yen

Grants forCommissioned
Research

283

External
Competitive Grants-in-Aid
Fund for Scientific
Research
912

Management
expenses grant

1,248

584

Facilities
maintenancegrant 3.3
Self income 31 Expenses grantfor SOKENDAI 55

The NIBB acquires numerous competitive funds in an effort made byindividual researchers, including Grants-in-Aid for Scientific Researchand Grants for Commissioned Research, in addition to national subsidies(Management Expenses Grants and Expenses Grants for SOKENDAI).

■ Organization

President

Executive Directors

Auditors

SHIMURA, Yoshiro

Management Council

Education and Research Council

Board of Directors

National Astronomical
Observatory of Japan

National Institute for
Fusion Science

National Institute for
Basic Biology
Director-General
OKADA, Kiyotaka
Vice Director
NAGAHAMA, Yoshitaka

National Institute forPhysiological Sciences

Advisory Committee for
Programming and Management

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

Chairperson
OHSUMI, Yoshinori
IWASA, Yoh
Vice-Chair
Extra NIBB member HASEGAWA, Mariko

NIBB member

ISHINO, Fumitoshi
KATO, Kazuto
MATSUOKA, Makoto
OSUMI, Noriko
SAKANO, Hitoshi
SATOH, Noriyuki
TABATA, Satoshi
HASEBE, Mitsuyasu
HORIUCHI, Takashi
IGUCHI, Taisen
IIDA, Shigeru
NAGAHAMA, Yoshitaka
NISHIMURA, Mikio
NODA, Masaharu
TAKADA, Shinji
UENO, Naoto
YAMAMORI, Tetsuo

Professor, National Institute for Basic Biology
Professor, Kyushu University
Professor, The Graduate University for Advanced Studies (SOKENDAI)
Professor, Tokyo Medical and Dental University
Associate Professor, Kyoto University
Professor, Nagoya University
Professor, Tohoku University
Professor, The University of Tokyo
Professor, Kyoto University
Deputy Director, Kazusa DNA Research Institute
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Vice Director & Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology

3■ National Institute for Basic Biology

Research Units■ Division of Cell Mechanism

Cell Biology■ Division of Molecular Cell Biology■ Division of Cell Proliferation (adjunct)■ Laboratory of Cell Structure■ Laboratory of Cell Sociology■ Division of Reproductive Biology

Developmental Biology■ Division for Sex Differentiation■ Division for Morphogenesis■ Division of Developmental Genetics†■ Division of Molecular and Developmental Biology††■ Laboratory of Molecular Genetics for Reproduction■ Laboratory of Plant Organ Development■ Division of Molecular Neurobiology

Neurobiology■ Division of Brain Biology■ Division of Behavioral Biology (adjunct)■ Laboratory of Neurophysiology■ Laboratory of Neurochemistry

Evolutionary Biology and Biodiversity■ Division of Molecular Genetics■ Division of Genome Dynamics■ Division of Evolutionary Biology■ Division of Speciation Mechanism (adjunct)■ Laboratory of Morphodiversity■ Laboratory of Bioresource■ Division of Molecular Environmental Endocrinology†††

Environmental Biology■ Division of Plant Developmental Genetics (adjunct)■ Division of Photobiology (adjunct)■ Laboratory of Photoenvironmental Biology■ Division of Theoretical Biology

Theoretical Biology■ Laboratory of Genome Informatics■ Division of Developmental Dynamics (adjunct)

Imaging Science■ Laboratory for Spatiotemporal Regulations

Research Support Units
Research Support Facilities
Center for Transgenic Animals and Plants
Research Center for Integrative andComputational Biology

The Large Spectrograph Laboratory
Tissue and Cell Culture Laboratory
Computer Laboratory
Plant Culture Laboratory
Experimental Farm
Plant Cell Laboratory

Other Research Support Sections
Technical Division

Strategic Planning Department■ Okazaki Research Facilities

Okazaki Institute for Integrative Bioscience
Center for Radioisotope Facilities
Center for Experimental Animal
Research Center for Computational Science

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

Other divisions of the OIIB are not shown.

■ Research Facilities run jointly with National Institute for Physiological Sciences

Center for Analytical Instruments

4

Laboratory Glassware Facilities
Electron Microscope Center
Machine Shop
Low-Temperature Facilities, respectively.

GOALS OF THE NIBBNIBB has set five goals for its activities in pursuing the
progress of biology. We envision contributing to the worldwide community of biologists through our efforts toaccomplish these goals. This chapter briefly explains four ofthe goals. The last goal, the promotion of academic research,is accomplished through our research activities, which are
introduced throughout this brochure.

International
cooperation
Contribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers

Promotion of Collaborative Research■ Collaborative Research Projects

currently operating fixed (partly tunable) wavelength laser
sources, the introduction of tunable laser sources covering awide range of wavelengths from UV to IR is in preparation.

Research projects in collaboratation with the NIBB’s
divisions/laboratories and research activities to be conductedusing the NIBB’s facilities are solicited from external
researchers at other universities and institutes. In addition toconventional “individual collaborative research projects,”“collaborative experiments using the Large Spectrograph,”and “NIBB workshops,” new types of research projects are
solicited that will facilitate the strategic organization ofcollaborative research projects. “Priority collaborative
research projects” are carried out in one to three years asgroup research by internal and external researchers with the
purpose of developing pioneering research fields in biology.
Three projects have already been carried out, including“molecular mechanisms for controlling the individuals ofhigher plants.” The category of “collaborative research
projects for model organism/technology development” wasestablished in 2007 with the aim of developing andestablishing new model organisms. Two projects havealready been carried out, including “development of thetransgenic strain of Cabombaceae (primitive angiosperm).”
In the belief that the methods of conducting collaborative
research projects must be constantly modified according to
the demands of the age and the biology community, the
NIBB always encourages discussion on such projects.

The European Molecular Biology Laboratory (EMBL),
established in 1974, is a research institute funded by 18
European. It conducts comprehensive, high-level basic
research programs and leads the world in the field of
molecular biology. The NIBB takes the leading role incollaborative research programs between the 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 83 for details of the EMBL meeting
held in 2007).

Collaborative research projects by year

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

year
Priority collaborative research projects

2005

2006

2007

2

3

1

Collaborative research projects for2

model organisms/technology development
Individual collaborative research projects

41

37

34

NIBB workshops

4

1

1

19

18

12

64

59

50

Collaborative experiments using
the Large Spectrograph
totalInternational Cooperation■ Collaborative research projects with the EMBL

■ NIBB Conference

The NIBB Conference is an international conference
organized by the NIBB’s professors once or twice a yearwith the participation of a guest lecturer from abroad. Since
the first conference in 1977 (the year of the NIBB’s
foundation), the NIBB Conference has provided researchers
in basic biology with valuable opportunities for international
exchange.
■ International Practical Course■ Enhancement of the Large Spectrograph

The Large Spectrograph Laboratory, a world-leading
research facility in photobiology, has succeeded in a large
number of collaborative research projects since its
foundation in 1980. To ensure further high-level
achievements, the Laboratory has promoted theenhancement of its laboratory equipment, including
advanced control systems, the use of laser sources, andsophisticated analysis equipment. To supplement theWith the cooperation of researchers from Japan and abroad,the NIBB international practical course, a practical training
course, is given at the laboratory specifically prepared forthe course at the NIBB. The first course, titled"Developmental Genetics of Zebrafish and Medaka," washeld in 2007. Graduate students from various East Asian
nations and areas, including China, Hong Kong, Taiwan,
and India, were provided with training in state-of-the-art
techniques for small fish research.

5

■ Bio Resources

The National BioResource Project (NBRP) is a national
project for the systematic accumulation, storage, and supply
system of 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, the NIBB has
been appointed as a research center 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.
The NIBB also works as a sub-center for this national
project for research on Japanese morning glory and
Zebrafish. In addition, the NIBB provides databases
containing research data on 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 biophotonics probes have also beendeveloped. The combination of these technologies allows us
to use living samples and observe biological phenomena inreal time, which, in the past, could only be estimated basedon fragmentary information from fixed samples. The NIBB
aims to maximize the application of these techniques forvisualizing biological phenomena (bioimaging) in biologicalresearch and to develop new imaging techniques.
1) Imaging Science Laboratories The NIBB aims to be acenter for developing microscopes and biophotonics
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 in the NIBB, members of the Advisory
Committee, and company engineers to frankly discuss
practical difficulties and needs regarding imaging.
4) Introduction of DSLM (Digital Scanned laser Lightsheet Microscopy) As part of collaborative work with
the EMBL, the NIBB has introduced DSLM, which iseffective for the three-dimensional observation ofliving samples, and is the first of its kind in Japan.
5) Bioimaging Symposium This provides an opportunity
for academic exchanges with overseas cutting-edge
researchers in the imaging field, mainly from the
EMBL.

6

Optical path of the DSLM

■ Okazaki Biology Conferences

The NIBB holds Okazaki Biology Conferences (OBC) that,with the endorsement of the Union of Japanese Societies for
Biological Science, support the formation of international
communities in future biological research fields with thegoal of identifying new research issues in biology. Dozens
of top-level 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 ofinternational researcher communities. (see page 81 for
OBCs held in 2007)

Cultivation of Future Researchers
The 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
and a three-year doctoral course for graduate students
with a master’s degrees.
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 the
NIBB based on the research assistant (RA) system.

Graduate students educated by NIBB
number
80
70
60
50

19

22
4

40

16
20

18

3

5

35

36

2002

2003

6

4

12
3

18
0

30
20

43

46

44

42

37

2004

2005

2006

2007

10
0

2001

Department of Basic Biology

School of Advanced Science (educated in NIBB)

Special research student

■ Personnel changes in 2007 *Newly assigned
Date

Research Unit

Name

Position

OKADA, Kiyotaka

Director-General

NARUSE, Kiyoshi

Associate Professor

Laboratory of Bioresource

April 1WATANABE, Taka-aki

Assistant Professor

Division of Genome Dynamics

April 1OKUBO, Tadashi

Assistant Professor

Division of Molecular and Developmental Biology

August 1

April 1

Newly affiliated
Name

Date

Position

New Affiliation

KATSUKI, Motoya

National Institutes of Natural Sciences

Executive Director (Part-time)

March 31SHIGENOBU, Shuji

Japan Science and Technology Agency

PRESTO Researcher

March 31MUKAI, Masanori

Konan University

Lecturer

March 31

MOROHASHI, Ken-ichirou

Kyushu University

Professor

April 1YOSHIKUNI, Michiyasu

Kyushu University

Professor

April 1KOSHIDA, Sumito

The University of Tokyo

Associate Professor

August 1

FUKUI, Yuko

National Institute for Longevity Sciences

Laboratory Head

August 1* Changes in Director-General, professors, ascociate and assistant professors are shown. All the research associates were reaffiliated as assistant professors
on April 1.

■ Awardees in 2007

Award

Name

Position

SHINTANI, Takafumi

Assistant Professor

Japanese Association for Protein Phosphatase Research Award

WADA, Masamitsu

Professor

The Fellow of American Society of Plant Biologists Award

OHSUMI, Yoshinori

Professor

The Academic Award of the Botanical Society of Japan

HORIUCHI, Takashi

Professor

The Kihara Award of the Genetics Society of JapanYAMAGUCHI, Takahiro

Assistant Professor

The Best Poster Award of the Botanical Society of JapanOKADA, Tokindo S.

Professor Emeritus

Order of Culture

TSUKAYA, Hirokazu

Professor (adjunct)Japan Society for the Promotion of Science Prize

Note on the member list from P. 8 to P. 80: All the members who belonged to the unit during 2007 are listed irrespective of the length of the period. Those
appearing twice in the same list under different titles are those whose title was changed during 2007.

7

National Institute for Basic Biology

Cell Biology

DIVISION OF CELL MECHANISM

and protein degradation control the functionaltransformation between glyoxysomes and leaf peroxisomes.

Ⅱ. Transcriptomics, proteomics and phenomicsof plant peroxisomes
Professor

Associate Professor

NISHIMURA, Mikio

HAYASHI, Makoto

Assistant Professors
Technical Staff
Postdoctoral Fellows

Graduate Students

Technical Assistants

Secretaries

MANO, Shoji
YAMADA, Kenji
KONDO, Maki
ARAI, Yuko
KAMIGAKI, Akane
OIKAWA, Kazusato
SINGH, Tanuja
OGASAWARA, Kimi
GOTO, Shino
KANAI, Masatake
CUI, Songkui
NAKAMORI, Chihiro
YOSHINORI, Yumi
SUZUKI, Iku
FUKAZAWA, Mitsue
KATO, Kyoko
NISHINA, Momoko
SATO, Yori
UEDA, Chizuru
KUBOKI, Yuko

Since plants spread their roots in the ground, they must
survive in a given environment. To adapt to theenvironment, they positively utilize environmental changes
in the life cycle as important signals that are necessary for
their survival. Plant cells can induce, degenerate anddifferentiate their organelles to adapt to environmental
changes. The flexibility of plant organelles is the basis of thestrategy for environmental 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” was started to clarify the molecular mechanisms
underlying organelle differentiation.

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 are known to contain one of two targeting
signals (PTS1 and PTS2) within the molecules. PTS1 is aunique tripeptide sequence found in the carboxyl terminus of
the mature proteins. In contrast, PTS2 is involved in acleavable amino terminal presequence of peroxisomal
proteins that are synthesized as a precursor protein withlarger molecular mass.
We identified 256 gene candidates of PTS1- and PTS2containing proteins and another 30 genes of non-PTScontaining proteins from Arabidopsis genome. Custommade DNA microarray covering all these genes was used toinvestigate expression profiles of the peroxisomal genes invarious organs. 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 proteins existing ineither glyoxysomes or leaf peroxisomes. Some of these
proteins contain no obvious PTS1 and PTS2. Combinationof the transcriptomic and proteomic analyses is providing uswith a new insight into plant peroxisomal functions.
Bioinfomatic analysis of Arabidopsis genome predicted thepresence of 15 kinds of genes, called PEX genes, forperoxisomal biogenesis factors. We comprehensively
investigated whether or not these predicted PEX genes
function in peroxisome biogenesis by generating knockdown mutants that suppress PEX gene expression by RNAinterference (Figure 1). Phenotypes of these mutants allowed
us to identify the functional PEX genes, which can beclassified into two groups, i.e. PEX genes regulating forperoxisomal morphology and PEX genes regulating forperoxisomal protein import. These analyses revealed that
PEX5, a receptor for PTS1, is involved in both lipid
metabolism and photorespiration by regulating the import ofⅠ. 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 andmitochondria acquire the ability to oxidize glycine.
Glyoxysomes, which are peroxisomes engaged in the
degradation of reserve oil via β-oxidation and the glyoxylate
cycle, are transformed into leaf peroxisomes that function inseveral crucial steps of photorespiration. After the functionaltransition of glyoxysomes to leaf peroxisomes during thegreening of pumpkin cotyledons, the reverse transition ofleaf peroxisomes to glyoxysomes occurs during senescence.
Gene expression, alternative splicing, protein translocation

8

Figure 1. Effects of 2,4-DB and sucrose on growth of knockdown
mutants. The name of each transgenic plant at the top of the panels
indicates a silenced PEX gene or a combination of silenced PEX genes.
Homozygous T3 progenies of these transgenic plants and parental plants(control) were grown for 7 days on growth medium (GM) containing
0.25μg/z of 2,4-DB (2,4-DB) or growth medium without sucrose (sucrose) under constant illumination. Bar = 10 mm.

both PTS1- and PTS2-containing proteins. In contrast,
PEX7, a receptor for PTS2, is involved only in lipid
metabolism by regulating the import of PTS2-containing
proteins.

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 the different pattern of
GFP fluorescence from the parent plant, GFP-PTS1, in
which peroxisomes with normal sizes and numbers can bevisualized with GFP.
Of these apm mutants, apm1 mutants contain slightly larger
peroxisomes with long string-like tails. Interestingly, thedivision of mitochondria is also disturbed in apm1 mutants.
We revealed that APM1 encodes dynamin-related protein 3A(DRP3A), one of a member of the dynamin family, and that
DRP3A has a role in the division of both peroxisome andmitochondria on each membrane. Two other apm mutants,
apm2 and apm4, showed GFP fluorescence in the cytosol aswell as in peroxisomes, indicating the decrease of efficiency
of PTS1-dependent protein transport to peroxisomes.
Interestingly, both mutants are defective in PTS2-dependent
protein transport as well. APM2 and APM4 were found to
encode proteins homologous to PEX13 and PEX12,respectively. It was revealed that APM2/PEX13 andAPM4/PEX12 are localized on peroxisomal membranes, andthat APM2/PEX13 interacts with PEX7. In addition, we
found that PEX5 binds to and does not move on peroxisomal
membranes in both mutants. These results show that
APM2/PEX13 and APM4/PEX12 are components of theprotein-translocation machinery on peroxisomal membranes.
Other apm mutants are under investigation so that we canidentify the components responsible for peroxisomebiogenesis and address the regulation of its mechanism.

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 (5μm long and 0.5μmwide) that are surrounded by ribosomes. ER bodies were
widely distributed in the epidermal cells of whole seedlings.
Rosette leaves had no ER bodies, but accumulated ER
bodies after wounding or jasmonic acid treatment. Thissuggests that ER bodies function in the defense against
herbivores. ER bodies include PYK10, a β-glucosidase with
an ER retention signal, in seedlings. We have isolated acouple of Arabidopsis mutants that have a defect in ER bodyformation. Arabidopsis nai1 mutant has no ER bodies inwhole plants and does not accumulate PYK10. NAI1
encodes a transcription factor that has a basic-helix-loophelix (bHLH) domain. Transient expression of NAI1 induced
ER bodies in the nai1 mutant. These results provide direct
evidence that NAI1 plays a role in the formation of ER
bodies. We are trying to isolate additional components thatare involved in ER body formation.

Ⅴ. Vacuolar processing enzyme responsible
for programmed cell death in plants
The vacuolar processing enzyme (VPE) belongs to thecysteine protease family found in higher plants and animals.
VPE exhibits substrate specificity toward asparagine andaspartic acid residues, and is responsible for the maturationof various type of vacuolar proteins. Plant VPE homologues
are separated into three subfamilies: seed type, vegetative
type, and seed-coat type. We revealed a novel function of
VPE in various programmed cell death (PCD) in plants.
VPE is identified as the proteinase that exhibits caspase
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. Fumonisin B1 (FB1), a
fungal toxin, induced cell death in Arabidopsis. The featuresof FB1-induced cell death were completely abolished in the
VPE-null Arabidopsis mutant. Arabidopsis δVPE expresses
specifically and transiently in two cell layers of the seed coatthat causes PCD accompanying cell shrinkage. In a δvpe
mutant, shrinkage of these cell layers was delayed. These
results suggest that VPE is involved in vacuolar collapse,
which triggers PCD. Plants evolve a death strategy mediatedby a vacuolar system, which is not seen in animals.
Interestingly, a vacuolar enzyme is the key player in a plantspecific cell death system.

Ⅵ. Role of molecular chaperones on celldifferentiation
Molecular chaperones are cellular proteins that function in
the folding and assembly of certain other polypeptides into
oligomeric structures but that are not, themselves,components of the final oligomeric structure. To clarify theroles of molecular chaperones on cell differentiation, we
have purified and characterized chaperonin and Hsp70s andanalyzed their roles in the translocation of proteins into
chloroplasts.
Previously, we characterized a mitochondrial cochaperonin (Cpn10), chloroplast co-chaperonins (Cpn20 and
Cpn10) and a small heat shock protein from Arabidopsis.
Recently, we began to characterize HSP90s, using a specificinhibitor of HSP90 or transgenic plants expressing mutatedArabidopsis HSP90. HSP90 inhibitor induced heat-inducible
genes and heat acclimation in Arabidopsis seedlings (Figure2). HSP90 inhibitor induced the genes with heat shock
response element (HSE) motifs in their promoters,suggesting that heat shock transcription factor (HSF) is
involved in the response. Arabidopsis HSFs (AtHsfA1d,
AtHsfA7a and AtHsfB1) interacted with Arabidopsis
HSP90.2. Heat shock reduced cytosolic HSP90 activity invivo. Thus, it appears that in the absence of heat shock,
HSP90 actively suppresses HSF function. Upon heat shock,
HSP90 is transiently inactivated, which leads to HSF
activation. Plant survival requires the ability to acclimate toheat. This data indicates that HSP90 regulates correct gene
expression for heat acclimatization in plants. We also
observed that HSP90 is involved in hormone responses in
Arabidopsis. The evolutional and functional characterization
is now under experiment.

9National Institute for Basic Biology

Cell Biology

for Environmental Adaptation in Plants.” This databaseconsists of 3 individual parts: the organellome database, thefunctional analysis database, and external links to otherdatabases and Web pages (Figure 3). The organellome
database provides images of various plant organelles thatwere visualized with fluorescent and nonfluorescent probes
in various tissues of several plant species at different
developmental stages. The functional analysis database is acollection of protocols for plant organelle research. Thispublic database is open to all researchers. We expect that
this database will be a useful tool to help researchers gain
greater knowledge of plant organelles.

Publication List
〔Original papers〕● Kobayashi, K., Kondo, M., Nishimura, M., and Ohta, H. (2007).

Figure 2. Pretreatment with HSP90 inhibitors induces the heat shock
response and high temperature tolerance. Seedlings of Arabidopsis weretreated with 50 μM of geldanamycin (GDA) or radicicol (RAD), HSP90
inhibitors, for 6 h, and they were washed three times with fresh medium.
After plants were allowed to recover at 22 °C for 2 h, they wereincubated at 22 °C (-; left) or 45 °C (right) for 1 h. As a negative control,
plants were treated with dimethyl sulfoxide (DMSO), chemical solvent.
The panels show the 2 days after the treatment.

Galactolipid synthesis on chloroplast inner envelope is essential for
proper thylakoid biogenesis, photosynthesis and embryogenesis. Proc.
Natl. Acad. Sci, USA 104, 17216-17221.
● Morita, Y., Arai, H., Sugimoto, T., Takeuchi, T., Yamane, T., Maeda,
T., Yamamoto, Y., Nishi, K., Asano, M., Shirahama-Noda, K.,
Nishimura, M., Uzu, T., Hara-Nishimura, I., Koya, D., Kashiwagi, A.,
and Ohkubo, I. (2007). Legumain/asparaginyl endopeptidase controls
extracellular matrix remodeling through the degradation of fibronectinin mouse renal proximal tubular cells. FEBS Lett. 581, 1417-1424.
● Nito, K., Kamigaki, A., Kondo, M., Hayashi, M., and Nishimura, M.
(2007). Functional classification of Arabidopsis peroxisomebiogenesis factors proposed from analyses of knockdown mutants.
Plant Cell Physiol. 48, 763-774.
● Yamada, K., Fukazawa, M., Hayashi, M., Suzuki, I., and Nishimura,
M. (2007). Cytosolic HSP90 regulates the heat shock response that is
responsible for heat acclimation in Arabidopsis thaliana. J. Biol.
Chem. 282, 37794-37804.

〔Review article〕

Ⅶ. The Plant Organelles Database (PODB) –Databases of plant organelles visualized withfluorescent and nonfluorescent probes, andprotocols for functional analysis
The Plant Organelles Database (PODB) was built topromote a comprehensive understanding of organelle
dynamics and is maintained by the Scientific Research of
Priority Areas on “Organelle Differentiation as the Strategy

Figure 3. The graphical user interfaces of PODB
(http://podb.nibb.ac.jp/Orgenellome).

10● Kuroyanagi, M., Hatsugai, N., Nishimura, M., and Hara-Nishimura, I.

(2007). Vacuolar processing enzyme, a key molecule in both
pathogen-induced and phytotoxin-induced cell death in higher plants.
Mol. Plant Microbe Interact. 5, 208-214.

DIVISION OF MOLECULAR CELL BIOLOGY

Professor

OHSUMI, Yoshinori
Assistant Professors

Technical Staff
NIBB Research Fellow
Postdoctoral Fellows

Graduate Students
Technical Assistants

Secretaries

KAMADA, Yoshiaki
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
KABEYA, Yukiko
OBARA, Keisuke
HANADA, Takao
FUJIKI, Yuki
HARASHIMA, Toshiaki
OKU, Masahide
YAMAMOTO, Hayashi
OKAMOTO, Koji
HAMASAKI, Maho
SEKITO, Takayuki
YOSHIMOTO, Kohki
KAWAMATA, Tomoko
KAGEYAMA, Takuya
OOOKA, Kyoko
KONDO, Chika
NIIMI, KaoriISHII, Junko
OKAMOTO, Noriko
TSUKESHIBA, Kumi
ICHIKAWA, Rie
HARA, Yoko
SUZUKI, Yuko

All 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 the regulation of gene
expression. Autophagy is a bulk degradation system forcytosolic proteins and organelles in lysosomes/vacuoles thatis highly conserved in eukaryotic cells. This division aims tounderstand the physiological roles and molecularmechanisms of autophagy in yeast and higher eukaryotes.

Ⅰ. Background
Upon nutrient starvation, the autophagic process starts as abuilding up of membrane structures called autophagosomesin the cytoplasm. The autophagosome sequesters cytosol andorganelles nonselectively. It is then delivered to thevacuole/lysosome, and the cytoplasmic materials inside aredegraded by vacuolar/lysosomal hydrolases. We discovered
autophagy in a simple model organism, the budding yeast
Saccharomyces cerevisiae, and morphologically andgenetically defined the whole process.

Ⅱ. Identification and characterization of autophagyspecific proteins, Atg17, Atg29, and Atg31
In S. cerevisiae, autophagy is induced under nutrient
starvation. In growing conditions another autophagy-relatedtransport, the Cvt pathway, is observed. While most of the
Atg proteins are involved in both pathways, only a few Atg
proteins, such as Atg17 and Atg29, are uniquely required for

starvation-induced autophagy. Identification of novel
autophagy-specific Atg protein(s) and their characterization
are urgent issues for the elucidation of the induction
mechanism of autophagy.
We identified Cis1/Atg31 as a third autophagy-uniqueprotein. ATG31 null mutant cells were defective in
autophagy and lost viability under nitrogen-depleted
conditions.
Atg29 and Atg31 localize to the pre-autophagosomal
structure (PAS; a putative generation site of the
autophagosome) in an ATG17-dependent manner (Figure 1),
and Atg17-Atg29 and Atg17-Atg31 physical interactions aredetected, suggesting that these three proteins make a ternarycomplex. We further observed that this autophagy-uniqueprotein complex binds to Atg1, and this association isregulated by nutrient conditions. When the Cvt pathway is
disrupted by deletion of ATG11, encoding a Cvt-specificprotein, recruitment of the Atg17 complex to the PAS is
stimulated by starvation. In addition, in atg11Δ cells, thetrimeric complex is required for the PAS recruitment ofother Atg proteins under starvation conditions. Thesefindings indicate that the Atg17 complex plays an importantrole in the assembly of other Atg proteins to the autophagyspecific PAS to generate the autophagosome.

Figure 1. Atg31 localizes to the PAS. Yeast cells expressing Atg31-GFP
and mRFP-Ape1 were incubated with rapamycin for 3 h. Fluorescence and
DIC images are shown. Ape1 is used as a marker for the PAS. Bar: 2μm.

Ⅲ. Functions of two ubiquitin-like conjugatesin autophagosome formation
Autophagosome formation involves two ubiquitin-likeproteins, Atg8 and Atg12. We showed that Atg12 forms a
conjugate with another Atg protein, Atg5, whereas Atg8 is
conjugated to a lipid, phosphatidylethanolamine (PE). We
have reported on the functions of these conjugates using invitro reconstitution systems consisting of purified protein
components and PE-containing liposomes.
First, we showed that Atg8 mediates the tethering andhemifusion of membranes (liposomes), which are evoked bylipidation of the protein and reversibly modulated by Atg4
that catalyzes the deconjugation of Atg8-PE (Figure 2A). In
addition, mutational analyses suggested that these functionsof Atg8 observed in vitro are required for autophagosome
formation in vivo; membrane tethering and hemifusion
represent bona fide functions of Atg8. These results provide
key insights into the unique membrane dynamics ofautophagy, which cannot be explained by the mechanismsthat have been elucidated in conventional vesiculartrafficking systems.
Previous in vivo observations have suggested that two
ubiquitin-like conjugation systems have an interrelationship:11

National Institute for Basic Biology

Cell Biology

the Atg12-Atg5 conjugate is required for effective formationof Atg8-PE. We purified the Atg12-Atg5 conjugate formed
in E. coli and found that it dramatically accelerates the Atg8PE conjugation reaction in vitro, in which transfer of Atg8
from the E2 enzyme Atg3 to PE is thought to be activatedthrough the direct interaction between Atg12-Atg5 and Atg3
(Figure 2B). These results indicate that Atg12-Atg5 serves
as a novel E3 enzyme in the ubiquitin-like conjugation
reaction of Atg8. Further studies will shed light onunderstanding how these ubiquitin-like systems cooperate in
autophagosome formation in the cell.

Figure 2. Functions of two ubiquitin-like protein conjugates. (A)Liposomes clustered upon in vitro production of Atg8-PE were stainedwith phosphotungsticacid and analyzed by electron microscopy.
Arrowheads and an arrow, respectively, show junctions between
liposomes and a structure suggested to represent hemifusion ofliposomes. (B) In vitro conjugation reaction of Atg8-PE was performedin the presence or absence of the Atg12-Atg5 conjugate.

condition, which was dependent on the PI 3’-kinase
components necessary for the cell viability. Moreover, theproduct of the PI 3’-kinase, phosphatidylinositol 3’monophosphate (PI3P), was found to be enriched at the siteof vacuole invagination (Figure 3B). Subsequentmorphological studies indicated that the vacuole
invaginations are followed by a transport of lipid bodies into
vacuole, thereby showing that MIA toward lipid bodies is
induced under the stress condition.

Figure 3. Membrane dynamics of MIA. (A) Schematic drawing ofvacuolar membrane morphology undergoing MIA during glycerol
culture at 37ºC. A part of a round vacuole (1) forms an invagination (2)dependent on some components of the PI 3’-kinase complex. Within theinvagination a smaller vesicle structure emerges (3) and buds off into
vacuolar lumen (4). (B) Localization of PI3P at the site of vacuole
invagination. A tandem repeat of FYVE domain known to specificallybind to PI3P is expressed in fusion to monomeric RFP (mRFP).
Fluorescence microscopy of the mRFP-(2x)FYVE was done for the
denoted strains. Along with the mRFP signal (shown as PI3P), Nomarski
images (Nomarski) and their superimposed images (Merge) are also
shown. Bar, 2 μm.

Ⅳ. Identification of another type of autophagy(microautophagy) under a stress condition
Our efforts to elucidate the molecular mechanism of
autophagy have led to the accumulation of enormous
amounts of knowledge regarding macroautophagy(macroautophagy is here simply described as autophagy). In
contrast, little is known at present about the physiological
functions and molecular machinery of another type of
autophagic pathway, termed microautophagy (MIA). In
MIA cargo components are sequestered directly by thevacuolar (lysosomal) membrane and transported into thevacuolar lumen after an inward pinch-off of the sequestering
membrane (Figure 3A). In the yeast S. cerevisiae, MIA has
been observed under some nutrient-starved conditions, but
its biological significance remains unclear.
After phenotypic analyses of 18 ATG-gene disruptants, wefound that disruptions of genes encoding a subset ofphosphatidylinositol (PI) 3’-kinase complex led to severe
viability defects when the cells were cultured on glycerol asa solo carbon source at 37ºC and eventually exposed toextensive oxidative stress. The frequency of the cells
possessing vacuole invaginations increased under the culture

12

Ⅴ . Lap3p is a novel cargo of selective
autophagy during glycerol culture
Under growing conditions, a constitutive autophagy-likepathway, termed the Cvt (cytoplasm-to-vacuole-targeting)pathway, utilizes similar molecular machinery and vesicle
structures to those of autophagy for a biosynthetic transportof aminopeptidase I (Ape1) into the vacuole. Recently, we
discovered that in the cells grown on glycerol as a solecarbon source, the Cvt pathway was enhanced. Under thiscondition, we found that Leucyl aminopeptidase Ⅲ (Lap3),
a neutral cysteine protease, was the selective cargo proteinof the Cvt pathway (Figure 4A). Lap3 co-localized with
Ape1 (Figure 4B), and Atg19 was involved in delivering
Lap3 to the vacuole as a receptor protein. Further studies
showed that a portion of Lap3 was degraded inside vacuoles,
and thus suggested that the Cvt pathway might function as adegradative pathway and that Lap3 might be a cargo of thepathway.

Publication List
〔Original papers〕● Adachi, W., Suzuki, N.N., Fujioka, Y., Suzuki, K., Ohsumi, Y., andFigure 4. (A) A proteinase A deficient (pep4Δ) and pep4Δ atg19Δ
cells expressing GFP-Lap3 were grown to logarithmic phase in theglycerol medium and examined by differential interference contrast(DIC) and fluorescence microscopy. Lap3 was selectively delivered tovacuole by the Cvt pathway. Arrows indicate the intravacuolar structures
including GFP-Lap3 positive structures. Arrowheads indicate thepunctate structures near vacuole. (B) Cells co-expressing GFP-Lap3 and
RFP-Ape1 were examined by microscopy as described in (A). Lap3 colocalized with Ape1 near the vacuole. Bar, 4 μm.

Inagaki, F. (2007). Crystallization of Saccharomyces cerevisiaeaminopeptidase 1, the major cargo protein of the Cvt pathway. Acta
Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 63, 200-203.
● Fujiki, Y., Yoshimoto, K., and Ohsumi, Y. (2007). An Arabidopsis
homolog of Yeast ATG6/VPS30 is essential for pollen germination.
Plant Physiology 143, 1132-1139.
● Hanada, T., Noda, N.N., Satomi, Y., Ichimura, Y., Fujioka, Y., Takao,T., Inagaki, F., and Ohsumi, Y. (2007). The Atg12-Atg5 conjugate
has a novel E3-like activity for protein lipidation in autophagy. J. Biol.
Chem. 282, 37298-37302.
● Kabeya, Y., Kawamata, T., Suzuki, K., and Ohsumi, Y. (2007).
Cis1/Atg31 is required for autophagosome formation in
Saccharomyces cerevisiae. Biochem. Biophys. Res. Comm. 356, 405410.
● Matsushita, M., Suzuki, N.N., Obara, K., Fujioka, Y., Ohsumi, Y., andInagaki, F. (2007). Structure of Atg5-Atg16, a complex essential for
autophagy. J. Biol. Chem. 282, 6763-6772.
● Nakatogawa, H., Ichimura, Y., and Ohsumi Y. (2007). Atg8, aubiquitin-like protein required for autophagosome formation, mediates
membrane tethering and hemifusion. Cell 130, 165-178.
● Satoo, K., Suzuki, N.N., Fujioka, Y., Mizushima, N., Ohsumi, Y., andInagaki, F. (2007). Crystallization and preliminary crystallographic
analysis of human Atg4B-LC3 complex. Acta Crystallograph. Sect. F
Struct. Biol. Cryst. Commun. 63, 99-102.
● Suzuki, K., Kubota, Y., Sekito, T., and Ohsumi, Y. (2007). Hierarchyof Atg proteins in pre-autophagsomal structure organization. Genes
Cells 12, 209-218.
● Yamada, Y., Suzuki, N.N., Hanada, T., Ichimura, Y., Kumeta, H.,
Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2007). The crystal structureof Atg3, an autophagy-related ubiquitin carrier protein (E2) enzymethat mediates Atg8 lipidation. J. Biol. Chem. 282, 8036-8043.
● Yamaguti, M., Suzuki, N.N., Fujioka, Y., Ohsumi, Y., and Inagaki, F.
(2007). Crystallization and preliminary X-ray analysis of Atg10. Acta
Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 63, 443-445.

〔Review article〕● Suzuki, K., and Ohsumi, Y. (2007). Molecular machinery of

autophagosome formation in yeast, Saccharomyces cerevisiae. FEBS
Lett. 581, 2156-2161.

13

National Institute for Basic Biology

Cell Biology

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 microtubule,contrasting with motile cilia with a well-known 9+2 pattern.
Such cilia can been seen in cultured cells such as 3T3, 3T6,

BHK21, NRK, MDCK. They are quite common centriolar
specializations in vivo and in vitro. The incidence ofprimary cilia within a cell culture is related with the degreeof confluency. Examination of confluent cell monolayers
shows 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.
In this year, four cell lines originating from adult mouse
kidney were established to study the proteomics of the
primary cilia. They were named nibb-K1, K4, K5, and K8.
The primary cilia of cells were observed by indirect
immunofluorescence microscopy (Figure 1). In the cloned
cell, each cell has a distinct length of cilium while K5 cellhas the longest one among them (up to 10μm).

nibb-K1

nibb-K4

nibb-K5

nibb-K8

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.

14

LABORATORY OF CELL SOCIOLOGY

Assistant Professor

HAMADA, Yoshio

Animal organs consist of several types of cells. They areorganized in an ordered fashion wherein the proportion ofeach cell type is constantly maintained. The ordered cellarrangement and proportion are built up duringorganogenesis by cell-cell interactions. Since it has been
postulated that Notch plays a role in cell fate decision by
mediating cell-cell interactions, we are endeavouring 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 theoutside, forming an interface with the maternal deciduas.
The polar trophectoderm gives rise to the cells of thechorion as well as the ectoplacental cone; these produce thelabyrinthine and spongiotrophoblast layers, respectively.
While maternal red blood cells begin to perfuse into
trophoblast cell layers and reach the labyrinthine layer by
E9.5, the invasion of embryonic allantoic mesenchyme into
the labyrinthine layer and the differentiation of fetal red
blood and endothelial cells which line the fetal capillary takeplace around E9.5.
The mutation in the ankyrin repeats of mouse Notch2
results in embryonic lethality by embryonic day 11.5 due toa poor maternal vascular beds formation. The mutant
placenta showed a normal invasion of angiogenic allantoic
mesenchyme followed by premature formation of fetal bloodvessels in the mutant placentas as early as E9.0. However,the specification of trophoblast subtypes appeared not to be
drastically disturbed. Thus, in the developing mouseplacenta, Notch2 is likely not involved in cell fate decision,
but rather participates in a formation of circulatory systems
in the labyrinth layer where the expression of Notch2 wasdetected (Figure 1). Although inadequate formation ofmaternal vascular beds was partially restored by aggregating
mutant diploid embryos with wild type tetraploid embryos(Figure 2), networks of maternal vascular beds appeared still
compromised in the 4N chimeric placenta. These results
indicate that Notch2 promotes vasculogenesis. Thus, Notch2
is not required for the early cell fate determination ofsubtypes of trophoblast cells, but plays an indispensable rolein coordinated maternal and fetal vasculogenesis in thedeveloping mouse placenta.

Figure 2. Survival of Notch2 mutant embryo in a chimera with tetraploidwild type embryo. The mutant obtained in the intercrosses by theheterozygotic mice shows developmental retardation at E10.5 (A) anddie before E11.5. The mutant is able to survive in a chimera with wild
type tetraploid embryo until E13.5 (B).

Publication List
〔Original papers〕● Aoki, M., Mieda, M., Ikeda, T., Hamada, Y., Nakamura, H., andOkamoto,H. (2007). R-spondin is required for mouse placental
development. Dev. Biol. 301, 218-226.
● Hamada, Y., Hiroe, T., Suzuki, Y., Oda, M., Tsujimoto, Y., Coleman,
J.R., and Tanaka, S. (2007). Notch2 is required for formation of theplacental circulatory system, but not for cell type specification in thedeveloping mouse placenta. Differentiation 55, 268-278.
● Kokubo, H., Tomita-Miyagawa, S., Hamada, Y., and Saga, Y. (2007).
Hesr1 and Hesr2 regulate atrioventricular boundary formation in thedeveloping heart through the repression of Tbx2. Development 134,
747-755.

Figure 1. Expression of Notch2 gene in developing mouse placenta. A
cryo-section of Notch2+/LocZ placenta at E10.0 was incubated in X-gal
staining solution. Trophoblast in maternal vascular bed side expresses
the gene, but not in allantoic mesenchyme (AM) side in labyrinth layer(LB). The expression also occurs in the mesenchyme. Enucleatedmaternal red blood cells are indicated by red arrow heads. Nucleatedfetal red blood cells are by blue arrow heads.

15

National Institute for Basic Biology

Developmental Biology

DIVISION OF REPRODUCTIVE BIOLOGY

Professor

Associate Professor

NAGAHAMA, Yoshitaka

YOSHIKUNI, Michiyasu

Assistant Professors
NIBB Research Fellow
Postdoctoral Fellows

Graduate Students
Visiting Scientist
Technical Assistants

Secretary

OKUBO, Kataaki
OHNO, Kaoru
SUZUKI, Aya
CUI, Jianzhou
GUAN, Guijun
KANEKO, Hiroyo
LAU, En-Lieng
LI, Jianzhong
MATSUDA, Masaru
OHTA, Kohei
PAUL, Bindhu
SAKAI, Fumie
SHIBATA, Yasushi
USAMI, Takeshi
WANG, De-shou
ZHAO, Haobin
ZHOU, Linyan
KURITA, Kayoko
ZHOU, Linyan
SANDVIK, Guro
HARA, Ikuyo
SEKITOH, Rie
HIRAKAWA, Eri
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 using several teleost fishes focuseson (1) the identification of regulators and steroidal mediators
involved in sex determination, gonadal sex differentiationand gametogenesis, and (2) the mechanisms of synthesis andaction of these mediators.

Ⅰ. Molecular mechanisms of sex determinationand gonadal sex differentiation
We have been using medaka (Oryzias latipes) toinvestigate the molecular mechanisms of sex determination
and Nile tilapia (Oreochromis niloticus) to investigate
gonadal sex differentiation. Medaka possess a stable genetic
XX/XY sex determining system. We identified DMY as the
sex-determining gene of medaka. DMY encodes a protein of
267 amino acids including a DNA-binding motif, the DM
domain, found in other genes involved in sexual
development. A genomic DNA fragment carrying DMY was
sufficient to induce testis differentiation and subsequentmale development, producing fertile sperm (Figure 1). It is
important to note that DMY transgenic XX medaka are fully
functional and fertile males, whereas Sry transgenic mice aresterile (Koopman et al., 1991). Thus, medaka is the first16

Figure 1. DMY transgenic adult medaka (XX) with white body color (A)having testis (B)transgenic vertebrate shown to undergo complete sex
reversal. Interestingly, DMY is a homolog of DMRT1,
another DM domain gene that is involved in maledevelopment, and appears to be derived from a duplicatedcopy of autosomal DMRT1. DMY is also found in O.
curvinotus, which is most closely related to medaka, but is
not found in other Oryzias species or other fishes. Thesefindings clearly illustrate the vast diversity of sexdetermining genes in fish.
In tilapia, all genetic female (XX) or male (XY) broods areavailable. Through cDNA subtraction between XX and XY
gonads during sex differentiation and microarrayhybridization followed by gene expression analyses by RTPCR and in situ hybridization, we have concluded that intilapia, Foxl2/Cyp19a1 plays a crucial role in ovariandifferentiation and DMRT1 in testicular differentiation. The
transcripts of Foxl2 and aromatase (there are two forms ofaromatase in teleost fishes: the ovarian form (Cyp19a1) andbrain form (Cyp19a2)) were expressed only in XX gonads at
5 days after hatching (dah), with a marked elevation in
expression during the next two days. The critical role of
Foxl2 in ovarian differentiation was confirmed by male sex
reversal of XX transgenic tilapia carrying a dominantnegative mutant of Foxl2. In XY tilapia fry, DMRT1 gene isexpressed male-specifically in testicular Sertoli cells prior toand during sex differentiation. XX tilapia carrying extra
copies of tilapia DMRT1 as a transgene induced various
degrees of gonadal changes including complete sex change
to testis. It is of great interest to note that some of the sex
reversed XX tilapia produced sperm with extremely highmotility (Wang et al., unpublished).
We also investigated the expression pattern of a novel typeof P450c17 (P450c17-Ⅱ) lacking the lyase activity (seebelow) in gonads during sex differentiation. The results on
tilapia and medaka (for example, its first appearance at 1020 dah in XX tilapia and 70 dah in XY tilapia) suggest that
P450c17-Ⅱ might be involved in the initiation of meiosis
during early sex differentiation in these fishes. Further
studies on these lines are expected to reveal whether 17α,20β-DP or another C-21 steroid is involved in the initiation ofmeiosis and, consequently, sex differentiation.

Ⅱ. Molecular mechanisms of sex change
The gobiid fish, Trimma okinawae, possesses ovarian andtesticular tissues simultaneously in its gonad and is able tochange sex repeatedly in both directions depending on its
social surroundings. We examined the involvement ofgonadotropins in sex change by determining the changes in
gonadotropin (FSH and LH) receptor gene expression in
gonads during the onset of sex changes from female to maleand male to female. The expression of the GtHRs was foundto be confined to the active gonad of the corresponding
sexual phase. When the sex change was occurring fromfemale to male, the ovary initially had high levels of FSHR
and LHR, which eventually went up in the testicular tissue
once the fish had realized the fact that it was bigger than theother fish in the aquarium. The opposite of this scenario was
observed if another fish bigger than the newly sex-changed
male was introduced into the aquarium. Swapping of the
gonads started with switching of the GtHR expression thatwas discernible within 8-24 hrs of the visual cue. Further invitro culture of the transitional gonads with a supply ofexogenous gonadotropin (hCG) revealed that the to-beactive gonad acquired the ability to produce thecorresponding sex hormone within one day of the activationof GtHR. Conversely, the to-be-regressed gonad did not
respond to the exogenous gonadotropin, demonstrating theabsence of GtHR expression. A successive sex changing
fish like T. okinawae is an excellent animal model toelucidate the mysterious role of the brain in bringing out thesexuality of an individual and also for the depiction ofsexual plasticity at the organismal level (Figure 2).

Ⅳ. Endocrine regulation of oocyte maturationand ovulation
A period of oocyte growth is followed by a process called
oocyte maturation (the resumption of meiosis) which occurs
prior to ovulation and is a prerequisite for successful
fertilization. Oocyte maturation has been studied in a varietyof vertebrates and invertebrates including mammals,
amphibians, fishes, and starfishes, but the endocrine
regulation of oocyte maturation has been investigated mostextensively in fishes. 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
the hormonal agents involved (Figure 3). In both species,
three major mediators have been shown to be involved(Three step model): a gonad-stimulating substance (GSS), 1methyladenine (maturation-inducing hormone, MIH), and amaturation-promoting factor (MPF) in starfish, andgonadotropin (LH), 17α, 20β-dihydroxy-4-pregnen-3-one(17α,20β-DP) (MIH), and MPF in fish.
We recently purified GSS from the radial nerves of starfish(Asterina pectinifera), and the complete amino acidsequence was determined. GSS is a heterodimeric peptide
with a molecular weight of 4737, consisting of A and B
chains; the A chain contains 24 residues and the B chain 19
residues. Chemically synthesized GSS is as active as native
GSS in the homologous in vitro GVBD assay (M. Mita, M.
Yoshikuni et al., unpublished).

Figure 2. Gonadal sex change in T. okinawae may be triggered byvisual/brain-stimulated switching of gonadotropin (GtH) receptor gene
expression in gonads.

Ⅲ. Embryonic development of gonadotropinreleasing hormone (GnRH) neurons
Appropriate development of GnRH neurons is essential for
reproductive competence. The mechanisms underlying thisprocess are, however, poorly defined because of theunavailability of an in vivo animal model. We havegenerated transgenic medaka that express GFP under thecontrol of the GnRH gene promoters and shown that they
could provide a useful model for the study of the GnRH
neuronal development, including human disorders of GnRH
deficiency. It has recently been shown that mice deficientfor p73, a newly described homolog of the tumor suppressor
gene p53, are not interested in mating, although its
mechanism is unknown. Using the transgenic medaka, wehere examined a possible role for p73 in the development of
GnRH neurons. Antisense-mediated ablation of p73 led to
the fusion of the bilateral GnRH neuronal clusters in theterminal nerve ganglion and an inappropriate increase in thenumber of these neurons. This result identified p73 as anovel factor involving the development of GnRH neurons
and would account for the reproductive and behavioral
defects in p73-deficient mice.

Figure 3. Hormonal control of oocyte maturation in fish and starfish –Three step modelIn fish, LH acts on ovarian follicle cells to produce fish
MIH (17α,20β-DP) immediately prior to oocyte maturation.
There is a distinct shift in follicular steroidogenesis from
estradiol-17β (E2) during oocyte growth (vitellogenesis) to
17α,20β-DP during oocyte maturation. This occurs in twostages, the first being the shift in the synthesis of precursor
steroids in thecal cells, while the other is the shift in the final
steroidal mediator production in granulosa cells. We havealready shown that there is a distinct shift in thesteroidogenic enzyme genes from ovarian aromatase(Cyp19a1) to 20β-hydroxysteroid dehydrogenase (20βHSD), occurring in the granulosa cells of ovarian follicles
prior to oocyte maturation. The triggering of thesteroidogenic shift by GtHs in granulosa cells can beachieved by the differential actions of two transcription

17National Institute for Basic Biology

Developmental Biology

Publication List
factors, Ad4BP/SF-1 (aromatase) and CREB (20β-HSD).
The major remaining question was the differential
availability of precursor steroid, 17α-hydroxyprogesterone. This was essentially because, until now, a single
enzyme P450c17, possessing 17α-hydroxylase and 17, 20
lyase activities to mediate the production of estrogen and 17
α,20β-DP, has been described among the vertebrates ingeneral. Recently, we discovered a novel type of P450c17(P450c17-Ⅱ) lacking the lyase activity in several teleostspecies, and showed that P450c17-II, but not P450c17-I, is
responsible for the shift in precursor steroid fromtestosterone to 17α-hydroxyprogtesterone in both medaka
and tilapia. Thus, our studies have resolved a long-standing
question in the field of steroidogenesis with respect tooocyte maturation. As the novel type of P450c17 is found tohave only the hydroxylase activity, we investigated whether
this is the gene which is responsible for the cortisol
production by analyzing its expression pattern in the headkidney of tilapia during different developmental stages.
Interestingly, only P450c17-Ⅱ is found to be expressed in
the interregnal cells of the head kidney from very earlystages (5 dah) to adulthood (8 month old) (Figure 4). Since
one of the most important physiological functions of theinterrenal cell is to produce cortisol, our data suggests that
only P450c17-Ⅱ is responsible for the cortisol production in
the interrenal cells

Figure 4. Expression of tilapia P450c17-I (A) and – Ⅱ (B) in theinterrenal cells of the head kidney

Unlike other steroid hormones, 17α,20β-DP binds to anovel, G-protein-coupled membrane progestin receptor(non-genomic action), leading to the de novo synthesis of
cyclin B, the regulatory component of MPF, which activatesa preexisting 35-kDa cdc2 kinase via phosphorylation of its
threonine 161 by a threonine kinase, thus producing the 34
kDa active cdc2.
Ovulation is a precisely timed process by which a mature
oocyte is released from an ovarian follicle. This process is
initiated by the pituitary surge of LH and is temporally
associated with the transcriptional regulation of several
genes. The molecular mechanisms that control the complex
process of ovulation are not well understood in vertebrates.
Our recent studies (Shibata et al., unpublished) on medaka
have demonstrated that 17α,20β-DP can induce ovulation(follicle rupture) in the mature follicles in vitro. It isparticularly important to note that this action of 17α,20β-DP
is mediated through its nuclear progestin receptors (nPRs)
expressed in the granulosa cells. We were able to find thatnPR mRNA expression is induced by gonadotropin prior toovulation. Thus, 17α,20β-DP is the key hormone for the
induction of not only maturation (through its membrane
receptors), but also ovulation (through its nuclear receptors).

18〔Original papers〕● Liu, Z.H., Wu, F.R., Jiao B.W., Zhang, X.Y., Hu, C.J., Huang, B.F.,

Zhou, L.Y., Huang X.G., Wang, Z.J., Zhang, Y.G., Nagahama, Y.,
Cheng, C.H.K., and Wang, D.S. (2007). Molecular cloning of Dmrt1,
Foxl2 and Cyp19 in Southern catfish and their possible roles in sex
differentiation. J. Endocrinol. 194, 223-241.
● Matsuda, M., Shinomiya, S., Kinoshita, M., Suzuki, A., Kobayashi, T.,
Paul-Prasanth, B., Lau, E.L., Hamaguchi, S., Sakaizumi, M., andNagahama, Y. (2007). DMY gene induces male development ingenetically female (XX) medaka fish. Proc. Natl. Acad. Sci. USA 104,
3865-3870.
● Mita, M., Yamamoto, K., Yoshikuni, M., Ohno, K., and Nagahama, Y.
(2007). Preliminary study on the receptor of gonad-stimulating
substance (GSS) as a gonadotropin of starfish. Gen. Comp.
Endocrinol. 153, 299-301.
● Mittelholzer, D., Andersson, E., Consten, D., Hirai, T., Nagahama, Y.,
and Norberg, B. (2007). 20β-hydroxysteroid dehydrogenase andCYP19A1 are differentially expressed during maturation in Atlanticcod (Gadus morhua). J. Mol. Endocrinol. 39, 319-328.
● Nakamoto, M., Wang, D.S., Suzuki, A., Matsuda, M., Nagahama, Y.,
and Shibata, N. (2007). Dax1 suppresses P450arom expression in
medaka ovarian follicles. Mol. Reprod. Develop. 74, 1239-1246.
● Ohmuro-Matsuyama, Y., Okubo, K., Matsuda, M., Ijiri, S., Wang,
D.S., Guan, G.J., Suzuki, T., Matsuyama, M., Morohashi, K., andNagahama, Y. (2007). Liver receptor homologue-1 activates brain
aromatase promoter of medaka, Oryzias latipes. Mol. Reprod.
Develop. 74, 1065-1071.
● Wang, D.S., Kobayashi, T., Zhou, L.Y., Paul-Prasanth, B., Ijiri, S.,
Sakai, F., Okubo, K., Morohashi, K., and Nagahama, Y. (2007). Foxl2
up-regulates aromatase gene transcription female-specifically bybinding to the promoter as well as interacting with Ad4BP/SF-1. Mol.
Endocrinol. 21, 712-725.
● Zhou, L.Y., Wang, D.S., Kobayashi, T., Yano, A., Paul-Prasanth, B.,
Suzuki, A., Sakai, F., and Nagahama, Y. (2007). A novel type ofP450c17 lacking the lyase activity is responsible for C21-steroid
biosynthesis in the fish ovary and head kidney. Endocrinology 148,
4288-4291.
● Zhou, L.Y., Wang, D.S., Shibata, Y., Paul-Prasanth, B., Suzuki, A.,
and Nagahama, Y. (2007). Characterization, expression andtranscriptional regulation of P450c17-I and -Ⅱ in the medaka, Oryzias
latipes. Biochem. Biophys. Res. Comm. 362, 619-624.

DIVISION FOR SEX DIFFERENTIATION

Professor

MOROHASHI, Ken-ichirou
Assistant Professors
Technical Staff
NIBB Research Fellow
Postdoctoral Fellows

Graduate Students

Visiting Scientists
Technical Assistants

Secretary

FUKUI, Yuko
OGAWA, Hidesato
OKA, Sanae
ISHIMARU, Yoshiyasu
MOHAMAD, Zubair
BABA, Takashi
KOMATSU, Tomoko
SHIMA, Yuichi
MIYABAYASHI, Kanako
KATSURA, Yukako
SEKIHARA, Sanae
KOUNO, Yukie
KUSAKA, Masatomo
NISHIYAMA, Aya
ISHIKAWA, Azusa
KOIKE, Yukari
GOUNO, Hiroe
TOMIDA, Sanai
HAYASHI, Tomoko
SUGIURA, Mio

Sexual dimorphism manifests most obviously in the gonads(testis and ovary) and is thereafter observed in other parts ofthe body such as the external genitalia, muscle, and brain.
This process of sex differentiation is divided into three steps.
The first step occurs at fertilization, during which the sexesof fertilized eggs are determined genetically according to a
combination of sex chromosomes. During the second step,
mammals carrying XY and XX sex chromosomes develop
the testis and ovary, respectively. This gonad sex
differentiation usually proceeds during fetal stages, andsubsequently sex steroids synthesized in the sexuallydifferentiated gonads control the sexes of the other tissues.
Therefore, the gonad sexes are quite important for the sex
differentiation of animals.
A number of transcription factors are known to play crucial
roles in the process of gonad differentiation. Some of thesegenes, such as SRY, WT1, DAX-1, SOX9 and ARX, wereidentified as the genes responsible for human diseases that
display structural and functional defects in the gonads.
Functions of the other genes such as Ad4BP/SF-1, Emx2,
M33, and Lhx9 were elucidated by the phenotypes of genedisrupted mice. In addition, their expression profiles in thesexually differentiating gonad strongly suggested theirfunctional significance at the early stage of gonad
differentiation. However, it remains to be elucidated how thegenes are expressed by upstream regulators. Studies
considering this aspect of sex differentiation are quite
important in order to define the gene regulatory cascade andthe molecular mechanisms mediating sex differentiation of
the gonad.
Tentatively, we have hypothesized that the sexuallyindifferent gonads determine their sexes under the control of
two opposite signals: the signal for male (testicular)differentiation and the signal for female (ovarian)differentiation. It is possible to assume that the signals aretranscriptional activities driven by the transcription factors
expressed in the sexually differentiating gonads or othertypes of growth factors. This division’s research has focused
primarily on the transcriptional control of the genes
implicated in gonad sex differentiation.

Ⅰ. Function of Forkhead transcription factor
Fkhl18 during testicular vasculaturedevelopment
There is a general agreement that gonad sex determinationin mammals is a process initiated by Sry gene. Downstreamof Sry, Sox9 specifies Sertoli cell lineages, organization of
the testicular cord, and production of male hormones. In
addition, the vasculature system develops differentiallybetween the testis and ovary. Before the actions of Sry are
evoked at around embryonic day 11.0 (E11.0), the structureof the primitive vasculature in the genital ridge is similarirrespective of sex. During the early phase of gonad sex
differentiation, however, the mesonephric cells migrate
vigorously into the developing testis to form a vasculature
structure characteristic of the testis. In contrast, no suchactive cell migration is observed in the developing fetal
ovary. This difference gives rise to sexually dimorphic
vascular patterns in the gonads. Especially, in the testis, alarge artery is formed at the coelomic surface at aroundE12.5. This male-specific vascular system that develops
during fetal life is thought to be required for the export of
testosterone from the testis to the rest of the fetus to ensure
masculinization.
Vasculogenesis starts during fetal development. Precursor
cells for blood vessel endothelia, which share their origins
with hematopoietic progenitors, assemble into a primitive
vascular network of small capillaries. Subsequently, thevascular plexus progressively expands by sprouting andmatures into stable blood vessels. During this phase ofangiogenesis and arteriogenesis, nascent endothelial cells
become covered by periendothelial cells (pericytes andsmooth muscle cells) and association with these cells is
required to regulate proliferation, survival, migration,differentiation, vascular branching, blood flow and vascularpermeability.
Forkhead transcription factors are characterized by awinged helix DNA binding domain, and the members of thisfamily are classified into 20 subclasses by phylogenetic
analyses. Fkhl18 is structurally unique, and is classified in
the FoxS subfamily. We found Fkhl18 expression inperiendothelial cells of the developing mouse fetal testis. In
an attempt to clarify its function, we generated mice with
Fkhl18 gene disruption. Although KO mice developed
normally and were fertile in both sexes, we frequently
noticed unusual blood accumulation in the fetal testis.
Electron microscopic analysis demonstrated frequent gaps,
measuring 100-400 nm, between the endothelial cells ofblood vessels. To visualize the entire structure of thetesticular vasculature system, we injected carbon ink into theumbilical vein of E14.5 fetuses. Although the whole view ofbranches of the vasculature system was indistinguishable

19

National Institute for Basic Biology

Developmental Biology

between the wild-type and Fkhl18 KO testes (Figure 1a, b),
the area around the vasculature looked dark and blurred in
the Fkhl18 KO testes. The gonads were subsequently
sectioned, and we found leaking of the injected carbon ink
from the testicular vessels and coelomic vessel. The leakageof the carbon ink suggested a defect in the sealing structureof the vasculature in the Fkhl18 KO mice.
These gaps probably represented ectopic apoptosis of
testicular periendothelial cells, identified by caspase-3
expression, in KO fetuses. No apoptosis of endothelial cells
was noted. Fkhl18 suppressed the transcriptional activity ofFoxO3a and FoxO4. Considering that Fas ligand gene
expression is activated by Foxs, the elevated activity of
FoxOs in the absence of Fkhl18 probably explains themarked apoptosis of periendothelial cells in Fkhl18-KO
mice.

Figure 1. Visualization of the vasculature structure using carbon ink.
Carbon ink was injected into the testes through the umbilical vein. The
whole views of the wild-type (a) and Fkhl18 KO testes (b) are shown.

materials secreted from the seminal vesicle, a definite
functional relationship linking the seminal vesicle to malefertility has yet to be elucidated. The coagulating glandsecretes a substance that, when mixed with the secretions
from the seminal vesicle, forms a vaginal plug, and it has
been thought that the vaginal plug is required for efficient
pregnancy after insemination.
We showed age-dependent regression of the seminal
vesicles, probably together with the coagulating gland, in
AhR(-/-) male mice. The KO mice had abnormal vaginalplugs, low sperm counts in the epididymis, and low fertility.
Moreover, serum testosterone concentrations and expressionof steroidogenic 3ß hydroxysteroiddehydrogenase (3ßHsd)and steroidogenic acute regulatory protein (StAR) in
testicular Leydig cells were decreased in AhR(-/-) males.
Taken together, our results suggest that impaired
testosterone synthesis in aged mice induces regression ofseminal vesicles and the coagulating glands. Such tissue
disappearance likely resulted in abnormal vaginal plugformation, and eventually in low fertility. Together withprevious findings demonstrating AhR function in female
reproduction, AhR has essential functions in animal
reproduction in both sexes.

Publication List
〔Original papers〕● Fan, W. Q., Yanase, T., Morinaga, H., Gondo, S., Okabe, T., Nomura,

Ⅱ. Function of Dioxin Receptor (AhR) in theMale Reproductive Tract
The aryl hydrocarbon receptor (AhR) is a ligand-activatedtranscription factor belonging to the basic helix-loop-helix(bHLH)-PAS super-gene family. Since AhR can bind with
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), themolecular properties of AhR as a transcription factor havebeen extensively studied. Recently, the intrinsic functions of
AhR have been investigated with regards to animal
reproduction. Indeed, our recent study in AhR(-/-) micedemonstrated that AhR is involved in female reproduction
by regulating estradiol synthesizing Cyp19 (P450
aromatase) gene expression. Based on the essentialfunctions of estradiol in the female reproductive process
such as folliculogenesis, ovulation, and implantation, it was
concluded that AhR plays an indispensable function infemale reproduction. Its function in male reproduction,
however, remained unknown.
The accessory internal reproductive systems, derived fromthe Wolffian duct for males and from the Mullerian duct for
females, are clearly different between the two sexes. The
male internal reproductive system consists of multiple
tissues such as the epididymis, the deferens duct, the seminal
vesicle, the coagulating gland, and the ejaculatory duct.
Developmentally, all these tissues are known to be regulatedby androgen signaling. The mature seminal vesicle consistsof numerous outpouchings of alveolar glands that empty into
the ejaculatory duct. Although semen mostly contains

20

M., Komatsu, T., Morohashi, K., Hayes, T. B., Takayanagi, R., andNawata, H. (2007). Atrazine-induced aromatase expression is SF-1dependent: Implications for endocrine disruption in wildlife andreproductive cancers in humans. Environmental Health Perspectives
115, 720-727.
● Kurokawa, H., Saito, D., Katoh-Fukui, Y., Ohta, K., Baba, T.,
Morohashi, K., and Tanaka, M. (2007). Germ cells are essential forsexual dimorphism in the medaka gonad. Proc. Natl. Acad. Sci. USA
104, 16958-16963.
● Ohmuro-Matsuyama, Y., Okubo, K., Matsuda, M., Ijiri, S., Wang, D.,
Guan, G., Suzuki, T., Matsuyama, M., Morohashi, K., and Nagahama,Y. (2007). Liver receptor homologue-1 (LRH-1) activates thepromoter of brain aromatase (cyp19a2) in a teleost fish, the medaka,
Oryzias latipes. Mol. Repro. Dev. 74, 1065-1071.
● Sato, N., Kamachi, Y., Kondoh, H., Shima, Y., Morohashi, K.,
Horikawa, R., and Ogata, T. (2007). Hypogonadotropic hypogonadism
in an adult female with a heterozygous hypomorphic mutation of
SOX2. Eur. J. Endocrinol. 156, 169-173.
● Wang, D. S., Kobayashi, T., Shou, L. Y., Ohmuro-Matsuyama, Y.,
Guan, G. J., Ijiri, S., Sakai, F., Matsuda, M., Shibata, Y., Okubo, K.,
Morohashi, K., and Nagahama, Y. (2007). Foxl2 up-regulates
aromatase gene transcription in a female-specific manner by binding
to the promoter as well as interacting with Ad4BP/SF-1. Mol.
Endocrinol. 21, 712-725.

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

UENO, NaotoKINOSHITA, Noriyuki

Assistant Professors
Technical Staff
NIBB Research Fellow
Postdoctoral Fellows
Graduate Students

Visiting Scientist
Technical Assistants

Secretaries

NAKAMURA, Makoto
TAKAHASHI, Hiroki
TAKAGI, Chiyo
IIOKA, Hidekazu
CHUNG, Hyeyoung
TAO, Hirotaka
MAEDA, Masato
LEE, Rebecca
SHINDO, Asako
MORITA, Hitoshi
YOSHIKANE, Nami
YAMAMOTO, Takamasa
TERASAKA, Chie
YAMADA, Shigehiro
GODA, Tadahiro
MIYAKE, Satoko
TSUGE, Toyoko

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 maininterest is to know how pattern formation andmorphogenesis during development are regulated by thesegrowth and transcription factors. We address this problem
using several model animals, including frogs, flies andascidians, and by employing embryology, genetics,
molecular and cellular biology, and biochemistry. In
addition, we have recently introduced genomics
technologies to elucidate the precise genetic programs
controlling early development.

Ⅰ. Establishment of cell polarity duringvertebrate embryogenesis
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 thecells that acquired planar cell polarity (PCP). In theregulation of convergent extension, several growth factor
signaling pathways including Wnt/PCP pathway are
implicated.

To understand how PCP is established within the cells, wehave recently introduced a new method to visualize
microtubule (MT) dynamics based on the assumption thatcytoskeletal reorganization is one of the earliest events ofthe cell polarity formation. Using a GFP-fusion protein of
EB3 (end-binding protein) which preferentially binds to theplus-end of growing MTs, we have been able to demonstratethat MT dynamics in non-polarized and polarized cells differ
in that in polarized cells MT growth is more restricted
towards the two ends of the cells even before the cellschange their morphology to spindle shape. We also foundthat the cell polarity revealed by the MT dynamics is
influenced by extracellular environments such as thenotochord-somite tissue boundary and that physical contact
of chordamesodermal explants with heterogenous tissues has
significant impacts on the cell polarity, possibly by
establishing a new tissue boundary between the two tissues.
We are currently investigating what actually triggers the cell
polarity formation during gastrulation.

Figure 1. Tracking of EB3-GFP movements captures microtubule
growth in Xenopus explants. In an animal cap cell (a), EB3-GFP shows
radially symmetrical movements toward the rim of the cell in the rose
diagram (a’). In chordamesoderm cell (b), the movements are mostly
bidirectional toward both ends of the cell (b’).

Ⅱ. The involvement of protein ubiquitination
systems in the noncanonical Wnt signaling
pathway during gastrulation.
The noncanonical Wnt signaling pathway has been shown
to play an essential role in the regulation of gastrulation
movements. However, the molecular mechanisms of how
Wnt signals intracellularly and how it regulates the tissue
movements remain elusive. In order to clarify the Wnt signaltransduction mechanism, we searched for the proteins
essential for this signaling pathway, and identified a novel
ubiquitin ligase complex, consisting of Rab40 GTPase andCullin. This complex is localized in the Golgi apparatus andis essential for the regulation of the localization of
Dishevelled, which plays the pivotal role in the Wnt
signaling pathway. Loss-of-function of this ubiquitin ligase
resulted in the inhibition of the Wnt pathway and caused a
severe gastrulation-defective phenotype in Xenopus
embryos. We also identified another ubiquitination systemessential for gastrulation, which ubiquitinates anddestabilizes Paxillin, one of the focal adhesion components.

21

National Institute for Basic Biology

Developmental Biology

We found that the focal adhesion plays an important role inconvergent extension. Paxillin stability is regulated at the
focal adhesions, and the destabilization of Paxillin promotes
the dynamics of focal adhesions and cell movements (Figure2). Interestingly, the Wnt signaling pathway increased
Paxillin ubiquitination and destabilized focal adhesions,indicating that the Wnt pathway regulates convergent
extension movements through regulating the stability of
Paxillin. These findings implicated two previouslyunidentified ubiquitin systems in the regulation ofgastrulation movements in Xenopus embryos.

was suppressed, notochord cells failed to elongate in theembryo, even though convergence/extension took placenormally. These results indicate that many Bra-downstream
notochord genes are involved in convergence/extension of
the embryo.

Figure 2. Paxillin degradation promotes focal adhesion dynamics andcell movement. (A) EGFP-Paxillin was expressed in the cells
undergoing convergent extension. Inhibition of Paxillin degradation byantisense morpholino against XRNF185, which plays a crucial role inPaxillin degradation, decreased focal adhesion dynamics and cell
movement. (B) Statistical data of focal adhesion dynamics.

Ⅲ. Brachyury-downstream notochord genes
and convergent extension in Ciona
intestinalis embryos
Formation of the chordate body is accomplished by acomplex set of morphogenetic movements including theconvergent extension of notochord cells. In the ascidian
Ciona intestinalis, Brachyury plays a key role in theformation of the notochord, and more than thirty Bradownstream notochord genes have been identified. In thepresent study, we examined the effects of functional
suppression of nine Bra-downstream notochord genes,
which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Citrop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When thefunction of the first two genes (Ci-PTP and Ci-ACL) was
suppressed with specific morpholinos, the notochord cells
failed to converge, while functional suppression of Ciprickle resulted in a failure of intercalation, and therefore thecells in these three types of embryo remained in the middorsal region of the embryo (Fig. 3). Functional suppressionof the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and CiASAK) resulted in the partial defect of intercalation, and thenotochord did not align in a single row. In addition, when
the function of the last two genes (Ci-ERM and Ci-pellino)

22

Figure 3. Examination of intracellular localization and function ofproteins encoded by Ci-Bra-downstream notochord genes. (A)Experimental procedure. Fertilized eggs were dechorionated andelectroporated with Ci-Bra(promoter)/Bra-downstream notochord
gene:EGFP constructs. Manipulated embryos were allowed to develop at
18 ºC for 12 hrs (to the mid tailbud stage). (B) Experimental procedure.
Fertilized eggs were dechorionated and microinjected with specificmorpholino oligos against Ci-Bra downstream genes together with the
Ci-Bra(promoter)/actin:EGFP construct. Manipulated embryos wereallowed to develop at 18 ºC for 12 hrs (to the mid tailbud stage). (C-J)
Control embryos at (C) 6, (D) 7, (E) 8, (F) 9, (G) 10, (H) 11, (I) 12, and(J) 13 hrs after fertilization. Notochord cells with actin:EGFP expression
show convergence (C, D), intercalation (D-F), extension (F-H) andelongation (H-J). Scale bar, 100 μm. (K-R) Functional suppression of Cipk results in the failure of intercalation and extension of notochord cells,
which are evident with actin:EGFP expression. Embryos at (K) 6, (L) 7,(M) 8, (N) 9, (O) 10, (P) 11, (Q) 12, and (R) 13 hrs after fertilization are
shown to compare with normal embryos (C -J). Scale bar, 100 μm.

Ⅳ . Functional and genetical study of aputative translation regulator dNAT1 in
Drosophila body patterning
Translational regulation takes a major role in Drosophila
body patterning. One of the eIF4G family proteins
NAT1/p97/DAP5 has been identified and analyzed mainly
in vertebrate culture cells. NAT1 has a structural similarity
to 2/3 of eIF4G-C-terminal region. Since NAT1 deletes abinding motif to eIF4E, it has been hypothesized that thisprotein participates in CAP-independent translation or plays
an antagonistic role in translation. To elucidate in vivo
function of NAT1 we isolated Drosophila NAT1 (dNAT1)
mutant by reverse-genetical approach. We isolated fourtransposon insertion mutants as well as a 1.4 kb deletion
allele corresponding to the dNAT1 locus. One of the Pelement insertion lines dNAT1GS1 shows severe embryoniclethality with abnormal germband extension defect. The
lethality and morphological phenotype were completely
rescued by introduction of the 12 kb dNAT1 genomic DNA
fragment by germ line transformation. We also found somesimilarity in the phenotype between dNAT1 hypomorphic
mutant and mutants in ecdysone signaling cascade. Bothmutants show lethality at late 3rd instar larva to pupal stage
with various metamorphosis defects. We found that one of
the ecdysone target genes E74A is not properly translated in
the dNAT1 mutant animals. E74A is one of the rareexamples of the genes whose translation is started from nonAUG start codon. E74A is translated from CUG start
condon. Our analysis strongly suggested that dNAT1
participates in translation initiation from CUG start condon.
We also speculate that dNAT1 also regulates other target(s)translation in early embryogenesis, since dNAT1 null mutant
shows early lethality. We would like to elucidate the generalimportance of NAT1 mediated non-AUG translation
initiation in Drosophila development as well as vertebrate
development.

Publication List
〔Original papers〕● Chung, H. A., Yamamoto, T. S., and Ueno, N. (2007). ANR5, an FGF

Target Gene Product, Regulates Gastrulation in Xenopus. Curr. Biol.
17, 932-939.
● Hotta, K., Mituhara, K., Takahashi, H., Inaba, K., Oka, K., Gojobori,T., and Ikeo, K. (2007). A web-based interactive developmental table
for ascidian Ciona intestinalis, including 3D real-image embryo
reconstructions: I. from fertilized egg to hatching larva. Dev. Dyn.
236, 1790-1805.
● Hotta, K., Yamada, S., Ueno, N., Satoh, N., and Takahashi, H. (2007).
Brachyury-downstream notochord genes and convergent extension in
Ciona intestinalis embryos. Dev. Growth. Differ. 49, 373-382.
● Iioka, H., Iemura, S., Natsume, T. and Kinoshita, N. (2007). Wnt
signalling regulates paxillin ubiquitination essential for mesodermal
cell motility. Nat. Cell Biol. 9, 813-821.
● Lee, R. H., Iioka, H., Ohashi, M., Iemura, S., Natsume, T. andKinoshita, N. (2007). XRab40 and XCullin5 form a ubiquitin ligase
complex essential for the noncanonical Wnt pathway. EMBO J. 26,
3592-3606.
● Ogata, S. Morokuma, J. Hayata, T. Kolle, G. Niehrs, C. Ueno, N.* andCho, K. W.* (*corresponding authors). (2007). TGF-beta signalingmediated morphogenesis: modulation of cell adhesion via cadherin
endocytosis. Genes Dev. 21, 1817-1831.
● Yoshikane, N., Nakamura, N., Ueda, R., Ueno, N., Yamanaka, S.,
Nakamura, M. (2007). Drosophila NAT1, a homolog of the vertebratetranslational regulator NAT1/DAP5/p97, is required for embryonic
germband extension and metamorphosis. Dev. Growth Differ. 49,
623-634.

Figure 4. Salivary gland cells of wild-type and dNAT1 mutant were
stained with antibodies against E74A (red) BR-C (green) as well as
DNA-staining dye (blue). In a dNAT1 mutant cell, nuclear signal of
E74A is completely missing while another nuclear marker BR-C isexpressed normally.

23

National Institute for Basic Biology

Developmental Biology

DIVISION OF DEVELOPMENTAL GENETICS

Professor

KOBAYASHI, Satoru
Research Associates
Technical Staff
NIBB Research Fellows
PRESTO Researchers
Postdoctoral Fellows
Graduate Students

Visiting Scientist
Technical Assistants
Secretary

MUKAI, Masanori
SHIGENOBU, Shuji
NODA, Chiyo
SATO, Kimihiro
KUMATA, Yuji
SHIGENOBU, Shuji
KAGEYAMA, Yuji
KITADATE, Yu
HASHIMOTO, Yoshiko
HAYASHI, Makoto
YATSU, Jun
MAEZAWA, Takanobu
HASHIYAMA, Kazuya
KONDO, Takefumi
SATO, KaoriIIDA, Mayu
HONDA, SatokoGerm cells are the specialized cells that can transmit thegenetic 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 they differentiate as germ cells when the organisms
are physically matured. Earlier investigators havedemonstrated that germ plasm contains maternal factors
required and sufficient for germline development. In
Drosophila, this cytoplasm is localized in the posterior poleregion of eggs, and partitioned into the germline progenitors,
or pole cells.

Ⅰ. Maternal Nanos protein is required in polecells to repress their apoptosis
In many metazoans, the germline forms early indevelopment and is maintained until the differentiation of
gametes in the adult gonads. Although genetic analyses have

Figure 1. Nanos prevents apoptosis in pole cells. Confocal images of thepole cells in control (left) and nos (right) embryos at stage 13, stainedwith TUNEL labeling (magenta) and an antibody against Vas (green).
Arrowheads show TUNEL-positive pole cells.

24

identified several mutations that eliminate pole cells, howpole cells are maintained during development is unclear.
Several components of germ plasm have been identified.
One of these components is maternal nanos (nos) RNA,
which is enriched in germ plasm during oogenesis andtranslated in situ to produce Nos protein after fertilization.
Nos is inherited by pole cells at the blastoderm stage and is
detectable in these cells throughout embryogenesis. In theabsence of maternal Nos, pole cells undergo apoptosis
during their migration to the embryonic gonads (Figure 1).
Although Nos also plays important roles in repressing
mitosis, somatic gene expression and somatic cell fate inpole cells, the primary role for Nos appears to be repressing
apoptosis in the germline, because Nos is an evolutionarily
conserved protein that is required for germline survival.
Apoptosis is mediated by caspases, a family of cysteine
proteases that cleave diverse substrates to destroy cellular
structure and integrity. Critical regulators of apoptosis
function by antagonizing the inhibitor of apoptosis protein(IAP) that directly blocks caspase action. In Drosophila,
four proapoptotic genes, reaper (rpr), head involution
defective (hid), grim and sickle (skl) encode members of afamily of related proteins that bind to and inactivate the IAP.
They are also referred to as RHG genes. Three of these RHG
genes, rpr, hid and grim, are encompassed by a genomic
region on the third chromosome, H99. Previously wereported that deletion of H99 region, Df(3L)H99, represses
apoptosis of pole cells lacking Nos, consistent with a role for
Nos in an apoptotic pathway that involves the RHG gene(s)from the H99 region.

Figure 2. A model for the regulation of apoptosis in pole cells by thematernal factors, Nos and Tao-1. (A) In normal pole cells, maternal Tao1 is inherited by pole cells and induces skl expression. Although skl
alone does not induce apoptosis, it sensitizes pole cells to induction of
apoptosis by hid. Hid mRNA is expressed in pole cells, but translation isrepressed by maternal Nos. (B) Once pole cells lack Nos, hid mRNA istranslated to produce its protein product that in turn acts together withskl to induce apoptosis.

We demonstrated that maternal Nos represses apoptosis ofpole cells by suppressing translation of hid RNA in an NREdependent manner. In the absence of Nos activity,translation of hid mRNA yields a protein product thatinduces apoptosis. In addition, we provide evidence that amaternally-provided protein kinase, Tao-1, is required toinduce apoptosis in nos pole cells by promoting skl
expression. Maternal tao-1 RNA is enriched in the germ
plasm and inherited by pole cells. Tao-1-dependent skl

expression sensitizes pole cells to induction of apoptosis byhid. We propose that pole cells express the RHG genes hid
and skl, and become competent to undergo apoptosis during
normal development. However, maternal Nos represses hidtranslation to inhibit apoptosis of pole cells for their survival(Figure 2). These findings provide the first evidence that the
germline is maintained through the regulated expression of
RHG genes.

Ⅱ. Maternal Mamo protein is required in polecells for their differentiation as thefunctional germ cells
Meiosis is a specialized cell division that produces haploid
gametes from diploid progenitors. This is accomplishedthrough two chromosomal segregation events without anintervening DNA replication. To accommodate thespecialized processes, meiotic chromosomes undergo
specific morphological changes (Figure 3). In Drosophila
oocytes, upon entry into meiosis, a synaptonemal complex(SC) is formed between homologous chromosomes tostabilize their pairing, and the meiotic recombination occurs.
Following disassembly of the SC, the meiotic chromosomescondense to form specialized prophase I chromosome
structures, “karyosomes”, and these structures aremaintained during later oogenesis. After ovulation, theFigure 3. Maternal mamo mutation affects the structure of the SC,
karyosome, and polar body. (A-F) Germarium regions (A and B), Stage6 (C and D) and stage-8 (E and F) egg chambers were stained for C(3)G(green), a marker of the SC, and DNA (propidium iodide, magenta).
(Insets) higher-magnification images of the oocyte nuclei are shown. (Aand B) In the germarium region, C(3)G protein localized to the DNA in
both the control and the mamo oocytes (arrows). (C and D) C(3)G was
not associated with the DNA, and was diffusely distributed throughout
the nuclear matrix (arrow) in a control oocyte at stage 6 (C). C(3)G
failed to dissociate from the DNA (arrow) in a mamo oocyte (D). (E andF) The karyosome was compact and spherical in a control oocyte atstage 8 (arrows) (E), while the karyosome was fragmented (arrow) in amamo oocyte at stage 8 (F). (G, H) Chromosomes were stained with ananti-PH3 antibody and TOTO-3 in control (G) and mamo eggs (H) at 0-1
hr AEL. (G) In a control egg, the rosette structure derived from polar
bodies was observed (arrow). (H) In a mamo egg, rosette structure wasindiscernible. The chromosomes were scattered, and their number was
significantly reduced. (Insets) Higher-magnification images of thechromosomes shown in (G) and (H). Dotted lines outline the eggs. Scalebars: 10 μm.

oocyte initiates meiotic division and produces both a single
female pronucleus and three polar-body nuclei which laterfuse to form a rosette structure. These dynamic changes by
the meiotic chromosomes are critical for meiosis. Genetic
screens have identified many genes involved in theregulation of the meiotic chromosomes. The mechanism bywhich the germline acquires the potential to execute meiosis,
however, remains elusive.
We demonstrated that a novel maternal factor, mamo
(maternal gene required for meiosis), is autonomously
required in pole cells to produce functional gametes. Mamo
protein which contains both a BTB/POZ (Broad Complex,
Tramtrack, Bric-a-brac/ Pox virus and Zinc finger) domain
and C2H2 zinc finger motifs is enriched in PGCs during
embryogenesis. The PGCs with reduced maternal Mamo
activity are able to undergo oogenesis, but fail to execute
meiosis properly (Figure 3). In the resulting oocytes,
meiosis-specific chromosomal configurations are impaired(Figure 3). We additionally show that the decondensation offertilized sperm nuclei is also affected in the eggs. We
propose that maternal Mamo activates downstream genes topromote specialized morphological changes within both thefemale meiotic chromosomes and the sperm nucleus, which
are critical in zygote formation.

Ⅲ. Signaling from pole cell to the gonadal
soma is required for proper formation of
the germline-stem-cell niche
The germline-stem-cell niche in Drosophila testes has
emerged as a useful model system for studying stem cells. In
the apical tip of the adult testes, the germline stem cells lie
in intimate contact with somatic hub cells, known
collectively as the niche, which causes the stem cells toretain self-renewing potential. Germline stem cells divide to
produce one daughter cell that remains associated with 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. It has been reported that the anteroposterior cellular identities within the gonads is regulated by
the homeotic genes, abdominal-A (abd-A) and Abdominal-B(Abd-B). How the formation of hub progenitors is restrictedin the anterior of embryonic gonads, however, remains
elusive. We demonstrate that a receptor tyrosine kinase,
Sevenless (Sev), provides a cue to ensure that the niche

Figure 4. Expression of sev within the embryonic gonads
The gonads in a female embryo at stage 16 (A) and in male embryos atstage 13 (B) and 16 (C). Embryos were double-stained for sev mRNA
(green) and a marker for the germline, Vasa (magenta). In all panels,
anterior is to the left, and the embryonic gonads are outlined by whitelines.

25

National Institute for Basic Biology

Developmental Biology

develops in the anterior region of the male embryonic
gonads. Sev is expressed by somatic cells within theposterior region of the gonads (Figure 4), and is activated bya ligand, Bride of sevenless (Boss), which is expressed by
the germline, to prevent ectopic niche differentiation in theposterior gonadal somatic cells. Our findings provide thefirst evidence that signal transduction from germline to soma
is essential for the proper development of a stem-cell niche.

Ⅳ. Studies on short ORF-containing transcripts
in Drosophila (Kageyama group)
Transcriptome analyses of eukaryotes, including mice andhumans, have identified poly(A)-containing transcripts that
only contains short ORFs (sORFs; less than 100 aa). ThesesORF transcripts are believed to most likely function as noncoding RNAs (ncRNAs), but their translational capacities
and biological activities have not been characterized indetail.
To elucidate the biological roles of sORF genes, we use
Drosophila as an excellent model system. By computational
filtration and expression analysis, we have identified 33
putative poly(A)-containing ncRNAs, referred as to MREs
(mRNA-like ncRNAs in embryos). In this year, we havebeen focusing on the biological roles of two MRE genes,
MRE32 and MRE29. MRE32 is specifically expressed in thecentral nervous system and detailed transcript mappingstrongly suggests that it really functions as ncRNA.
Mutational analysis revealed that the MRE32 gene isrequired for the eclosion behavior of the flies. Eclosion
timing is sexually dimorphic in Drosophila melanogaster, inwhich females eclosed one day earlier than males. In
MRE32 mutant flies, female eclosion was specificallydelayed, resulting in similar eclosion profiles in both sexes.
In addition, microarray analysis of female and male adultheads demonstrated that a lack of MRE32 severely alteredexpression of many genes in females. Thus, MRE32 is oneof the crucial regulators of the Drosophila nervous systemthat acts on developmental timing. Another MRE, MRE29,
is in fact transcribed into a polycistronic mRNA that
contains evolutionarily conserved four ORFs that encode
tiny peptides (11 and 32 aa). MRE29 is expressed in

Figure 5. Epidermal phenotype of polished rice. In contrast to wild-type
embryos (left column), pri mutant embryos exhibit complete loss ofdenticles (right, upper panel) and actin bundle formation beneathdenticles (right, lower panel).

26

epithelial tissues during embryogenesis and a lack of the
MRE29 gene eliminated apical cuticular structures,
including the epidermal denticles and tracheal taenidia(Figure 5). Considering these phenotypes, we renamed thegene as polished rice (pri). Cytological analysis demonstratethat pri is essential for the formation of specific F-actin
bundles that prefigure the formation of the denticles andtaenidium (Figure 5), indicating that pri plays essential roles
in epithelial morphogenesis by regulating F-actinorganization.
These results demonstrate that sORF genes play importantroles in Drosophila and further analysis on other MRE
members should elucidate unexplored functions of the
Drosophila genome.

Publication List
〔Original papers〕● Kitadate, Y., Shigenobu, S., Arita, K., and Kobayashi, S. (2007).

Boss/Sev signaling from germline to soma restricts germline-stemcell-niche formation in the anterior region of Drosophila male gonad.
Dev. Cell 13, 151-159.
● Kondo, T., Hashimoto, Y., Kato, K., Inagaki, S., Hayashi, S., andKageyama, Y. (2007). Small peptide regulators of actin-based cellmorphogenesis encoded by a polycistronic mRNA. Nature Cell Biol.
9, 660-665.
● Mukai, M., Hayashi, Y., Kitadate, Y., Shigenobu, S., Arita, K., andKobayashi, S. (2007). MAMO, a maternal BTB/POZ-Zn-finger
protein enriched in germline progenitors is required for the productionof functional eggs in Drosophila. Mech. Dev. 124, 570-583.
● Nakamura, Y., Kagesawa, T., Nishikawa, M., Hayashi, Y., Kobayashi,S., Niimi, T., and Matsuno, K. (2007). Soma-dependent modulations
contribute to divergence of rhomboid expression during evolution of
Drosophila eggshell morphology. Development 134, 1529-1537.
● Sato, K., Hayashi, Y., Ninomiya, Y., Shigenobu, S., Arita, K., Mukai,M., and Kobayashi, S. (2007). Maternal Nanos represses hid/skldependent apoptosis to maintain the germ line in Drosophila embryos.
Proc. Natl. Acad. Sci. USA 104, 7455-7460.

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji
Assistant Professors
Technical Staff
NIBB Research Fellows
Postdoctoral Fellows

Graduate Students

Technical Assistants

Secretary

KOSHIDA, Sumito
OKUBO, Tadashi
UTSUMI, Hideko
AKANUMA, Takashi
TAKADA, Ritsuko
CHEN, Qiuhong
YABE, Taijiro
ISHITANI, Shizuka
TAKAHASHI, Jun
TAKAHASHI, Hiroyuki
TAKASHIRO, Kayoko
SASAKI, Michiko
ISHIKAWA, Mika
SHIBATA, Chitose
TSUZUKI, 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 along some definedpattern is called “pattern formation” or “patterning.” Themost popular model to explain the patterning process is the“morphogen gradient and threshold” theory. Actually, many
genetic results indicate that secreted signal proteins such as
Wnt, BMP, and Hedgehog function as morphogens in many
aspects of patterning processes. However, in spite of theaccumulation of genetic evidence, the biochemical
characteristics, including modification and higher order
structure, of morphogens remain to be elucidated. Thus, oneof our major goals is to reveal the real image of morphogens
and the molecular mechanism underlying the formation ofmorphogen gradients, including the secretion andextracellular transport of these morphogens.
In contrast, the segmental sub-regions of the paraxial
mesoderm, or somites, appear not to be simply directed by
the morphogen gradient and threshold, but by a uniquemechanism proceeding periodically. Somites aresequentially generated in an anterior-to-posterior order by
converting oscillatory gene expression into periodical
structures. The molecular mechanism underlying thisconversion and morphological segmentation, however, has
not yet been fully understood. Thus, another goal of ourcurrent studies is to reveal the molecular mechanism of thisother and unique modes of patterning that underlies theperiodical and sequential sub-division in somite formation.

important step regulating the extracellular transport ofvarious secreted signal proteins involves post-translational
modification with lipid moieties. In the case of Wnt, Nusseand co-workers reported that murine Wnt-3a is Spalmitoylated at a conserved cysteine residue, and proposedthat palmitoylation of this cysteine residue may be required
to produce an increased local concentration of Wnt on theplasma membrane. In contrast, there is strong evidence tosuggest that lipid modification is involved in the processing
and intracellular trafficking of Wnt prior to secretion.
To resolve inconsistencies between the previous studies
and to better understand the biological significance andmolecular mechanism of lipid modification of Wnt, we
carefully examined its modification. Unexpectedly, we
found that murine Wnt-3a is modified with a monounsaturated fatty acid, palmitoleic acid, at a conserved Ser
residue. Wnt-3a defective in this modification is not secreted
from cells in culture or in Xenopus embryos, but is retainedin the endoplasmic reticulum (ER). Furthermore, Porcupine,
a protein with structural similarities to membrane-bound Oacyltransferases, 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 (Figure1). 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.

Ⅰ. Molecular mechanism for secretion 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

Figure 1. The function of palmitoleic lipid modification of Wnt protein.
Wnt proteins are modified with palmitoleic acid (C16:1) byacytransferase, Porcupine (porc), in the ER. This modification isrequired for trafficking of Wnt proteins from the ER.

27

National Institute for Basic Biology

Developmental Biology

Ⅱ. Identification and characterization ofgenes required for somite development
2-1 Paf1 complex homologs are required for Notchregulated transcription during somite segmentation
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 thepresomitic mesoderm (PSM). For instance, hairy/Enhancerof split (Espl)-related bHLH genes, including her1 and her7in zebrafish, 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.
Interestingly, many genes that exhibit cyclic expression are
regulated by Notch signaling in the PSM. Notch signaling is
also involved in other aspects of segmentation,synchronization of the oscillating phase between cells in the
PSM, and establishment of the rostral-caudal compartment
within a somite.

In previous studies, we identified numerous
ethylnitrosourea (ENU)-mutagenized zebrafish mutants withaltered somite morphogenesis. For instance, we found that
integrinα5 and fibronectin were mutated in embryos
showing defective boundary formation in their anterior
somites. Detailed analysis of these mutantas indicated that
Integrina5-directed assembly of Fibronectin appears critical
for the epithelialization and boundary maintenance ofsomites. This result indicates that our strategies are effective
for the identification of the genes involved in the somite
segmentation process.
We are also searching for other genes involved in thisprocess by both the expression screening and themutagenesis screening methods. This year, we characterized
one mutant obtained in our screens, kt641, which exhibited
reduced but still striped expression of the Notch target genes
in the PSM (Figure 2). In contrast to previously identified
Notch signaling mutants, in which the striped expression of
Notch target genes is perturbed to form salt-and-pepper
patterns, kt641 exhibited reduced but still striped expressionof the Notch target genes in the PSM. We found that thegene responsible for this phenotype is a zebrafish homologof yeast rtf1.

Figure 2. Phenotype of kt641 mutant and the structure of Rtf1. (A-F) Somite boundaries are disrupted in the posterior trunk of the kt641
mutant (bracket in D). At later stages, the kt641 mutation causes reduced pigmentation, limited tail growth, and abnormal heart (arrow) and ear(arrowhead) development (F). (G) Meiotic and physical mapping of kt641 mutation. Horizontal gray bars represent contigs deposited in LG 13.
(H) Schematic diagrams of zebrafish Rtf1 proteins encoded by wild-type and kt641 alleles.

28

Rtf1 is a member of the Paf1 complex, which has been
implicated in various processes, such as transcription
initiation and elongation, histone modification,
phosphorylation of pol II, and RNA processing and export.
Members of Paf1 complex, which is composed of at least
five components (Paf1, Rtf1, Cdc73, Leo1, and Ctr9), areconserved from yeast to human. Although these proteins
have been implicated in RNA polymerase II-mediatedtranscription, their roles in vertebrate development have notbeen elucidated. We showed that a zebrafish mutant
exhibiting a somite segmentation defect is deficient in rtf1.
In addition, embryos deficient in rtf1 or ctr9 display
abnormal development of the heart, ears, and neural crest
cells. rtf1 is required for proper RNA levels of the Notchregulated genes her1, her7, and deltaC as well as for Notchinduced her1 expression in the presomitic mesoderm.
Furthermore, the phenotype observed in rtf1-deficientmutants is enhanced by an additional deficiency in mind
bomb, which encodes an effector of Notch signaling. Thus,
zebrafish homologs of the yeast Paf1 complex appear topreferentially affect a subset of genes, including Notchregulated genes, during embryogenesis (Figure 3).

Figure 3. A model to explain Rtf1 function in the transcription of a
Notch target gene. Our results suggest that the Paf1 complex may
regulate the transcription of Notch targets in association with Spt5 andSpt6.

PSM and in several newly formed somites. Ripply1
represses specific gene expression in the PSM through aGroucho-interacting motif. In ripply1-deficient embryos,
somite boundaries do not form, the characteristic gene
expression in the PSM is not properly terminated, and theinitially established rostrocaudal polarity in the segmental
unit is not maintained, whereas paraxial mesoderm cells
become differentiated. Thus, ripply1 plays two key roles inthe transition from the PSM to somites: termination of thesegmentation program in the PSM, and maintenance of therostrocaudal polarity.
In ripply1-deficient embryos, the expression of mesp-b, a
key regulator in somite segmentation, is upregulated in acell-autonomous manner, whereas, in embryos injected withripply1mRNA, the expression of mesp-b is highly
suppressed in the anterior PSM. These results suggest that
Ripply1 regulates the proper expression of mesp-b in theanterior PSM. Taking into account that the expression ofmesp-b could be induced by T-box transcription factor(Tbx), we can speculate that Ripply1 may antagonize thefunction of Tbx in the transcription of mesp genes.
Therefore, we examined precisely the relationship between
Tbx24 and Ripply1 in the transcription of mesp-b. We
showed that Ripply1 is coprecipitated with Tbx24 andconverts it from an activator to a repressor in culture cells.
The other members of the Ripply family, Ripply2 andRipply3, can also antagonize the transcriptional activation
mediated by Tbx24. On the other hand, Ripply1 also
antagonizes the transcriptional activation of another T-box
protein, No tail (Ntl), both in vitro and in vivo. These results
indicate that the intrinsic transcriptional property of T-box
proteins is controlled by Ripply family proteins, which act
as specific adaptors that recruit the global corepressor
Groucho/TLE to T-box proteins.

Publication List
〔Original papers〕

2-2 Transcriptional repression by Groucho-associatedtranscriptional mediator Ripply1 in somite
segmentation
Prior to morphological segmentation, which is a processincluding inter-somitic boundary formation andmesenchymal-epithelial transition, a segmental pre-pattern,characterized by segmental gene expression, is establishedin 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. Concomitant with the transition
from the anterior PSM to somites, the characteristic gene
expression in the PSM is translated into the segmental
structure. However, most of the events accompanying thetransition from the anterior PSM to somites have remained
obscure.
We show that a gene identified by our in situ hybridization
screening, ripply1, encoding a nuclear protein associatedwith the transcriptional co-repressor Groucho, is required forthis transition. Zebrafish ripply1 is expressed in the anterior● Akanuma, T., Koshida, S., Kawamura, A., Kishimoto, Y., and Takada,S. (2007). Paf1 complex homologes are required for Notch-regulatedtranscription during somite segmentation. EMBO Rep. 8, 858-863.
● Ishioka, T., Katayama, R., Kikuchi, R., Nishimoto, M., Takada, S.,
Takada, R., Matsuzaka, S., Reed, J.C., Tsuruo, T., and Naito, M.
(2007). Impairment of the ubiquitin-proteasome system by cellular
FLIP. Genes Cells 12, 735-744.
● Takada, I., Mihara, M., Suzawa, M., Ohtake, F., Kobayashi, S.,
Igarashi, M., Youn, M.-Y., Takeyama, K., Nakamura, T., Mezaki, Y.,
Takezawa, S., Yogiashi, Y., Kitagawa, H., Yamada, G., Takada, S.,
Minami, Y., Shibuya, H., Matsumoto, K., and Kato, S. (2007). A
histone lysine methyltransferase activated by non-canonical Wnt
signalling suppresses PPAR-gamma transactivation. Nat. Cell Biol. 9,1273 - 1285.

29

National Institute for Basic Biology

Developmental Biology

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION

Associate Professor

TANAKA, Minoru
NIBB Research Fellow
Postdoctoral Fellow
Visiting Scientists

Technical Assistants

Secretaries

SAITO, Daisuke
KOBAYASHI, KayoAOKI, Yumiko
NAKAMURA, Shuhei
KUROKAWA, Hiromi
KINOSHITA, ChieNISHIMURA, Keiko
WATAKABE, Ikuko
UKAI, Sakie
YONEMITSU, Masako

Laboratory Scope
Our laboratory aims to reveal the molecular mechanisms of
the formation of the gonads and sex differentiation. We areusing medaka fish (Oryzias latipes) for these purposes.
Medaka has been recently established as a modelvertebrate. The entire genome sequence was determined anda variety of inbred strains with a large polymorphic genome
are available, which allows us to investigate biologicalphenomena by the means of molecular genetics.
Furthermore, an exogenous gene can be introduced into
medaka genome (transgenic medaka) and cells can betransplanted to host medaka to generate chimera medaka.
With these advantages, we have been generating transgenic
medaka enabling us to identify the different cell lineages byfluorescence and to analyze the process of gonad formationand sex differentiation in vivo. Additionally, in order to
identify the genes essential for gonadogenesis, we carriedout a mutational screening of medaka with a defect ingonads and are performing a positional cloning. With these
two unique analytical methods (visualising cells andmutants), we are attempting to unveil the fundamental
mechanisms of sex differentiation common to manyorganisms.

Figure 1. The germ cell-deficient gonad exhibits a single fundamental
structure common to both ovary and testis. A large lumen is located in
the middle and a single layer of supporting-like cells encloses the lumen,which is separated by a basement membrane from an outer stromal
region.

30

Ⅰ . Germ cells are essential for sexual
dimorphism of the gonads
The nature of somatic cells and germ cells is a topic ofbroad and long standing interest. Many studies havetherefore explored the interaction between germ cells andgonadal somatic cells. The view resulting from the previous
studies is that germ cells do not significantly affect the sex
differentiation of gonads.
We generated medaka that completely lack germ cells in
the gonad by impairment of PGC migration. The
morphology of the gonad in the germ cell-less medaka
exhibits a tube-like structure which is composed of a lumen
in the middle surrounded by a single layer of inner cells
separated by a basement membrane from an outer stromal
layer (Figure 1). This structure is common to the unit that
constitutes ovarian follicles and testicular tubules, indicatingthat germ cells are essential for organizing sex-specificstructures in the gonads.
This medaka also exhibits another interesting phenotype,male secondary sex characteristics irrespective of the genetic
sex. Novel aromatase-expressing theca cells that we
identified in ovary did not develop properly in the germ celldeficient gonads and female supporting cells that control
gametogenesis began to express male-specific genes asdevelopment of the gonad proceeded in the absence of germ
cells, suggesting trandifferentiation from female to male
supporting cells. The cells producing male sex steroid
hormone persisted in the germ cell-deficient medaka. Alllines of the data indicated that the gonadal somatic cells are
predisposed to adopting male development and the
production of male steroid hormone is responsible for malesecondary sex characteristics. Thus, we demonstrated that,
contrary to accepted thinking, germ cells are essential forsexually dimorphic gonads (Kurokawa et al., 2007, PNAS
104, 16958-16963).

Ⅱ. Successful identification of a mutated genethat causes male to female sex reversal(hotei mutant)
In collaboration with the SORST Kondoh team, we havebeen screening mutants affecting the development ofprimordial germ cells and the formation of gonads. The
screening has been performed in such a way that particularattention is paid to the presence, the number and the
distribution pattern of germ cells at a somitogenesis stage
and at ten days post hatching (10 dph). Nine mutants (19alleles) and twelve mutants (14 alleles) were identified for
PGCs and gonads, respectively.
One mutant, hotei, is of particular interest because of theexcessive number of germ cells that are arrested in the early
development of follicle growth and because male to female
sex reversal occurs irrespective of their genetic sex. As aresult of the positional cloning, we have successfully
identified a candidate gene as the gene for a typeⅡ receptorof anti-Müllerian hormone (amhrⅡ). This result reveals that
amhrⅡ, together with its ligand, anti-Müllerian hormone(amh), regulates the proliferation of germ cells. Both amh
and amhrⅡ are expressed in the gonadal somatic cells. It isinteresting to note that amhrⅡ is not detected in the absence

of germ cells, suggesting that a reciprocal cross talk between
germ cells and gonadal somatic cells is present during theterm of sex differentiation. These results also support ourcontention that the proliferation of germ cells is closely
related to sex differentiation of the gonads. (Morinaga et al.,2007, PNAS 104, 9691-9696).
Another mutant, zenzai, is a good contrast with the hotei
mutant in that germ cells are not maintained in the gonad(Figure 2). Inheritance of the phenotype indicates that theallele is recessive. We again identified one possible
candidate gene for the phenotype of zenzai mutant.
We are also characterizing other mutants in anothercategory, namely the irregular distribution of germ cells in
gonads. These mutants include, hadare, mizore, hyou andarare.

Ⅳ. Generation of transgenic medaka to identify
the cell lineages that constitute the gonads
To clarify the cell types that constitute the gonad, we are
generating transgenic fish to visualize the cell lineages. We
have established several lines of transgenic fish that allow us
to analyze how they build up the gonad during the course ofdevelopment. Crossing the transgenic fish with different
colors enabled us to successfully reconstitute novel units ofstructure that have not been reported yet in the gonad.
An attempt to monitor the process of development of eachlineage has also been made in living embryos and larva
using timelapse movies. In order to solve the difficulties invisualizing the cells located in deep positions in the embryos
and larva, confocal microscopy and SPIM have been applied
to the transgenic embryos and larva. This attempt is still inprogress in collaboration with EMBL’s Jochen Wittbrodt
Lab.

Publication List
Figure 2. Blue staining shows PGCs in the gonad. Left: zenzai mutant
that cannot maintain gerrn cells. Middle: wild type. Right: hotei mutant
that shows overproliferation of germ cells.

Ⅲ. Modes of Germ Cell Proliferation areimportant components of gonadal sex
differentiation
In order to investigate the mode of germ cell proliferation,a small number of germ cells are labeled by fluorescenceand monitored in the developing gonads of living medaka
embryos. These observations reveal that two modes ofproliferation are taking place, one type (type Ⅰ ) withintermittent division found in both male and female
developing gonads, and another type (type Ⅱ ) withsuccessive and synchronous division, leading to cystic germ
cells and subsequent meiosis and oogenesis, which is only
detected in the female (Figure 3).
Since typeⅠ-proliferation is impaired in zenzai mutants,
resulting in the depletion of germ cells, type I is responsible
for the maintenance of germ cells and typeⅡ is the mode ofgerm cell proliferation which commits to gametogenesis.
Our results also demonstrate that transition from typeⅠ to
typeⅡ is regulated in a sex-dependent manner (Saito et al.,2007, Dev. Biol. 310, 280-290).

〔Original papers〕● Hano, T., Oshima, Y., Kinoshita, M., Tanaka, M., Mishima, N.,

Ohyama, T., Yanagawa, T., Wakamatsu, Y., Ozato, K., and Honjo, T.
(2007). Quantitative bioimaging analysis of gonads in olvas-GFP/STII YI medaka (transgenic Oryzias latipes) exposed to ethinylestradiol.
Environ. Sci. Tech. 41, 1473-1479.
● Kurokawa, H., Saito, D., Nakamura, S., Katoh-Fukui, Y., Ohta, K.,
Aoki, Y., Baba, T., Morohashi, K., and Tanaka, M. (2007). Germ cells
are essential for sexual dimorphism in the medaka gonad. Proc. Natl.
Acad. Sci. USA 104, 16958-16963.
● Morinaga, C., Saito, D., Nakamura, S., Sasaki, T., Asakawa, S.,
Shimizu, N., Mitani, H., Furutani-Seiki, M., Tanaka, M(Corresponding Author), and Kondoh, H. (2007). The hotei mutationof medaka in the anti-Mullerian hormone receptor causes thedysregulation of germ cell and sexual development. Proc. Natl. Acad.
Sci. USA 104, 9691-9696.
● Saito, D., Morinaga, C., Aoki, Y., Nakamura, S., Mitani, H., FurutaniSeiki, M., Kondoh, H., and Tanaka, M. (2007). Proliferation of germ
cells during gonadal sex differentiation in medaka: insights from germ
cell depleted mutant zenzai. Dev. Biol. 310, 280-290.
● Takamatsu, N., Kurosawa, G., Takahashi, M., Inokuma, R., Tanaka,
M., Kanamori, A., and Hori, H. (2007). Duplicated Abd-B class genes
in medaka hoxAa and hoxAb clusters exhibit different expression
patterns in pectoral fin buds. Dev. Genes Evol. 217, 263-273.

Figure 3. Two types of germ cell proliferation are important componentsof sexual dimorphism of medaka developing gonads.

31

National Institute for Basic Biology

LABORATORY OF PLANT ORGAN DEVELOPMENTLABORATORY OF PLANT ORGAN DEVELOPMENT

Director-General

OKADA, Kiyotaka

Secretary

TSUCHIDA, Yuhei
HARA, Reiko
SAKAGAMI, Mari

Assistant Professor

TANAHASHI, Takako

Postdoctoral Fellow
Technical Assistant(Individual Research)

Plant organs - leaves, flowers, and roots - show
impressive, symmetrical shapes, based on an orderedarrangement of differentiated 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 oforganogenesis starts with the formation of a leaf primordium
in the peripheral zone of the shoot apical meristem at the
fixed position, following an order called phyllotaxis. Cells in
the primordium then proliferate and differentiate according
to three spatially fixed axes: the apical-basal axis, the lateralaxis (central-marginal axis), and the adaxial-abaxial(foreside-backside) axis. In the course of proliferation anddifferentiation, the plant cells are believed to exchange
information with neighboring or separated cells in order toregulate the organ architecture. We are trying to understandthe mechanism of the information exchange between plant
cells during the development of lateral organs, such asleaves, sepals, petals, stamens and carpels.

Ⅰ. Genetic approach
Recent studies of Arabidopsis mutant show a couple ofgenes are involved in the axes-dependent control of lateralorgan development. FILAMENTOUS FLOWER (FIL) andYABBY3, members of the YABBY/FIL gene family
encoding a protein with a zinc finger and an HMG-related
domains, are among them and control the specification of
the abaxial side of lateral organs. FIL gene expression wasrestricted at the abaxial side of the lateral organ primordia(Figure 1). On the contrary, PHABULOSA (PHB) geneencoding a class III homeobox-ZIP protein, and its
homologs, REVOLUTA (REV) and PHAVOLUTA (PHV)
are responsible for formation of the adaxial-side tissue. We
showed that PHB is expressed in cells of the adaxial sideand separated clearly from the abaxial side-specific FIL
gene expressing cells, by action of microRNA165/166
which targeted the PHB, REV, and PHV messenger RNAs.
A series of section analyses showed that the separation of
the adaxial side- and the abaxial side-specific genes wasobserved in the leaf primordium of stage 0, indicating thatthe microRNA action is working in the earliest stage of leaf
development.
Several lines of evidence showed that the fixing of the
adaxial-abaxial boundary in the leaf primordium is a key
step of the subsequent expression of the region-specificgenes and cell proliferation and differentiation. We thenscreened mutants with altered adaxial-abaxial boundary

32

Figure 1. Region-specific expression of the FIL gene at the abaxial sideregion of the developing cotyledons of Arabidopsis at the late heart stageembryo. Top right: light-field microscope picture. Top left: Pink signal
show auto-fluorescence of chloroplasts. Bottom right: Green signals
show the FIL promoter-drived green fluorescence protein (GFP). Bottom
left: Merged picture of chloroplast auto-fluorescence and of GFP.

position by mutagenizing seeds of transgenic Arabidopsis
carrying the abaxial-side specific FIL gene promoter::GFP.
One of the mutants, enf1-1, showed a fluctuating boundary
position, while another mutant, enf2-1, revealed a shift ofthe boundary to the adaxial side. Further analysis of themutants will unveil the molecular mechanism fordetermining the position of the boundary, an important
process of lateral organ development.

Ⅱ. Biochemcal approach
We are taking another approach to studying theintercellular signaling system by analyzing small peptides ascandidates for intercellular signaling ligands, which arepresent in the apoplastic region of the shoot apical meristem.
Small peptides were identified by LC/MS in the apoplastic
liquid of edible cauliflower, which is a huge clump ofinflorescence and premature flowers, or of cauliflower
mutant of Arabidopsis, which makes clumped meristem
identical to the vegetable. More than 400 peptide species
were identified and their functions in lateral organ
development are now being investigated.

Publication List
〔Original papers〕● Ishida, T., Hattori, S., Sano, R., Inoue, K., Shirano, Y., Hayashi, H.,

Shibata, D., Sato, S., Kato, T., Tabata, S., Okada, K., and Wada, T.
(2007). Arabidopsis TRANSPARENT TESTA GLABRA2 is directly
regulated by R2R3 MYB transcription factors and is involved inregulation of GLABRA2 transcription in epidermal differentiation.
The Plant Cell 19, 2531-2543.
● Tominaga, R., Okada, K., and Wada, T. (2007). Functional analysis of
epidermal-specific Myb genesgenes CAPRICE and WEREWOLF in
Arabidopsis. The Plant Cell 19, 2264-2277.

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

NODA, Masaharu
Assistant Professors

Technical Staff
NIBB Research Fellow
Postdoctoral Fellows

Graduate Students

Visiting Scientist
Technical Assistants

Secretaries

SHINTANI, Takafumi
SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
IHARA, Masaru
FUJIKAWA, Akihiro
SUZUKI, Ryoko
FUKADA, Masahide
SHIMIZU, Hidetada
SHIMIZU, Hidetada
YONEHARA, Keisuke
CHOW, Pak Hong Jeremy
NAGAKURA, Ayano
ZHANG, Lanfan
AOSHIMA, Akihiro
TANI, Sachiko
SAKURABA, Jyuichi
YONEHARA, KayoMIZOGUCHI, Masae
KATAYAMA, Tomomi
HATTORI, Nobuko
TOMITA, Nao
KODAMA, Akiko
YATAGAI, Hiroko

We have been studying the molecular and cellularmechanisms underlying the development of the vertebrate
central nervous system, mainly using the visual systems ofchicks and mice. This research covers many developmental
events including the patterning of the retina, axonal
navigation, branching and targeting, synapse formation,refinement and plasticity. The scope of our interests also
encompasses the mechanisms for various functions of themature brain, including sensation, emotion, behavior,learning, and memory.

We subsequently conducted misexpression and knockdown
experiments on the embryonic chick retina by in ovo
electroporation using retroviral vectors to elucidate themolecular functions of these asymmetric molecules and thehierarchy among them. We have revealed gene cascades of
topographic molecules for the retinal patterning and for the
topographic retinotectal projection (see Annual Report2006).
Although it is known that different neuronal subtypes areorganized asymmetrically with respect to the two axes in theretina, the mechanisms of the final differentiation ofneuronal subtypes still remain unclear. The region-specificdistribution and development of these cells in the retina arealso attributable to the retinal patterning which should bedetermined by morphogens and transcription factors
expressed asymmetrically in the developing retina. Amongthe RLCS clones, we have already identified somemolecules that are expressed in a specific subtype of theretinal ganglion cells (Figure 1). Studies to explore
functional roles of these genes are currently underway.

Figure 1. SPIG1 expression in the mouse retina at P5. SPIG1-positive
retinal ganglion cells are densely distributed in the dorsotemporal retina.
In the remaining region of the retina, only a subtype of the retinal
ganglion cell appears to express SPIG1, showing a mosaic distribution.
Enlargements of the boxed regions are shown on the right. N, T, D, andV indicate nasal, temporal, dorsal, and ventral, respectively. Scale bars:
1 mm (left panel), 50μm (right panels).

Ⅰ. Mechanisms for regional specification in
the developing retina
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.
In the past ten years, we have devoted our efforts tosearching for molecules with asymmetrical distribution in
the embryonic chick retina, and to the characterization of
their roles in the topographic retinotectal projection. We
performed a large-scale screening using differential
hybridization and restriction landmark cDNA scanning(RLCS) on the embryonic day 8 (E8) chick retina, and we
successfully identified 33 asymmetric molecules along thenasotemporal (N-T) axis and 20 along the dorsoventral (DV) axis.

Ⅱ. Mechanisms for the topographic retinotectal
projection
At the onset of the retinotectal projection at E12, the axonsof retinal ganglion cells exhibit a crude topographic orderboth anteroposteriorly and mediolaterally on thetectum/superior colliculus. Next, axonal sprouting begins
predominantly in the vicinity and towards the site of thenormal terminal zone (TZ). Further axon branching andarborization lead to the formation of mature TZs, whileaberrant axon segments and branches are eliminated in an
activity-dependent manner. These processes are completed
at around E16 in chick (Figure 2) and P8 in mouse,
respectively.
General attention is now devoted to the molecularmechanisms for the axon branching and arborization andtheir selective elimination. Among the region-specific33

National Institute for Basic Biology

Neurobiology

molecules in the developing retina, we have alreadyidentified several molecules which induce abnormal
branching and arborization when their expression was
experimentally altered in vivo. We expect that our research
will shed light on these issues.

Figure 3. Tyrosine phosphorylation of ErbB4 is enhanced by PSD95
and repressed by Ptprz. (A) In vitro phosphorylation assay of ErbB4.
GST-ErbB4ICR pre-incubated with indicated amounts of GST-PSD95 or
32
GST was subjected to autophosphorylation with [γ- P]ATP, and thenanalyzed by SDS–PAGE followed by autoradiography. (B) In vitro
dephosphorylation assay of ErbB4. After indicated amounts of GST32
PtprzICR, GST-PSD95 and GST were mixed and preincubated, the Plabelled GST-ErbB4ICR was added and incubated, and then analyzed asabove. (C) Tyrosine phosphorylation of ErbB4 and its dephosphorylation
by Ptprz in HEK293T cells. The tyrosine phosphorylation of cellular
proteins and ErbB4 was analyzed by Western blotting, along with theprotein expression.
Figure 2. Development of retinotectal projection in chicks. Dorsal
axons were labeled with DiI and the feature of axon terminals on thetectum was developmentally explored.

Ⅳ. Mechanisms of Na-level sensing in thebrain for the body-fluid homeostasis
Ⅲ. Physiological roles of protein tyrosine
phosphatase receptor type Z (Ptprz)
Ptprz (also called PTPζ/RPTPβ) is a receptor-type protein
tyrosine phosphatase (RPTP) predominantly expressed in
the brain as a chondroitin sulfate proteoglycan. Among thetwenty-one RPTPs expressed in mammals, only the R5
subfamily, Ptprz and Ptprg (PTPγ), have a canonical PDZbinding motif (-S-L-V) at their carboxyl-termini. ErbB4 is amember of the ErbB-family tyrosine kinases known as aneuregulin (NRG) receptor. Ptprz and ErbB4 are reported tobind to postsynaptic density-95 (PSD95) on the second andthe first/second PDZ (PSD95/Disc large/zona occludens1)
domains, respectively, through the PDZ-binding motif of
their carboxyl termini. We found a functional interaction
between Ptprz and ErbB4.
An intracellular carboxyl-terminal region of Ptprz pulleddown PSD95 and ErbB4 from an adult rat synaptosomal
preparation. ErbB4 and Ptprz showed co-localization in cellbodies and apical dendrites of neurons in the prefrontal
cortex. In vitro experiments using the whole intracellularregion (ICR) of ErbB4 also showed that PSD95 stimulates
the autophosphorylation of ErbB4, and that the ICR of Ptprz
dephosphorylates ErbB4 independent of the presence of
PSD95 (Figure 3A, B). In HEK293T cells, phosphorylationof ErbB4 was raised by co-expression of PSD95, which wasrepressed by additional expression of Ptprz (Figure 3C).
Taken together with the finding that the tyrosine
phosphorylation level of ErbB4 was increased in Ptprzknockout mice, these results suggest that Ptprz has a role in
suppressing the autoactivation of ErbB4 by PSD95 at thepostsynaptic density in the adult brain.

34

Dehydration causes an increase in the sodium (Na)concentration and osmolarity of body fluids. For Na
homeostasis of the body, controls of Na and water intake
and excretion are of prime importance. Although it wassuggested that the circumventricular organs (CVOs) are
involved in body-fluid homeostasis, the system for sensing
Na levels within the brain, which is responsible for thecontrol of Na- and water-intake behavior, has long been an
enigma. Na x is an atypical sodium channel that is assumed to
be a descendant of the voltage-gated sodium channel family.
Our studies on the Na x-knockout mice revealed that Nax
channels are localized to the CVOs and serve as a sodiumlevel sensor of body fluids. Na x-knockout mice do not stop
ingesting salt when dehydrated, while wild-type mice avoidsalt.
As the first step toward understanding the cellularmechanism by which the information sensed by Na x
channels is reflected in the activity of the organs, we
dissected the subcellular distribution of Na x. Doubleimmunostaining and immuno-electron microscopic analyses
revealed that Na x is exclusively localized to perineuronallamellate processes extending from ependymal cells andastrocytes in the organs. In addition, glial cells isolated fromthe subfornical organ (SFO), a member of the CVOs, weresensitive to an increase in the extracellular sodium level, as
analyzed by an ion-imaging method. These results suggestthat glial cells bearing Na x channels are the first to sense aphysiological increase in the level of sodium in body fluids,
and regulate the neural activity of the CVOs by enveloping
neurons. Thus, close communication between inexcitable
glial cells and excitable neural cells is supposedly the basis

of the central control of salt homeostasis.
This year, we revealed direct interaction between Na x
channels and α subunits of Na+/K+-ATPase, which brings
about Na-dependent activation of the metabolic state of the
glial cells. The metabolic enhancement leading to extensive
lactate production was observed in the SFO of wild-type
mice, but not of the Na x-knockout mice. Furthermore,lactate, as well as Na, stimulated the activity of GABAergic
neurons in the SFO (Figure 4). These results suggest that the
information on a physiological increase of the Na level inbody fluids sensed by Na x in glial cells is transmitted toneurons by lactate as a mediator to regulate neural activitiesof the SFO. It is likely that this leads to the control of saltintake behavior (Figure 5).

Figure 5. Schematic drawing of the Na-level sensing mechanism andNa-dependent regulation of neural activity in the SFO. When animals are
dehydrated, Na concentration in plasma and cerebrospinal fluid increases
above the usual level of ~145 mM (1). When the extracellular Na
concentration exceeds ~150 mM, Na x channels open, and the intracellular
Na concentration in these glial cells is increased. This leads to activation++++of Na /K -ATPase in these cells (2). Activated Na /K -ATPase consumes++
ATP larger than the usual level to pump out Na ions (3). To fuel Na /K ATPase with ATP, the glial cells enhance the glucose uptake tostimulate the anaerobic glycolysis (4). Lactate, the end product of theanaerobic glycolysis, is released from the glial cells and supplied toneurons, including GABAergic neurons, through the processes
enveloping them (5). Lactate stimulates the activity of the GABAergic
neurons through production of ATP, which presumably regulate
hypothetic neurons involved in the control of salt-intake behavior (6).

Figure 4. Properties of GABAergic neurons in the SFO of wild-type andNa x-KO mice. There exist GABAergic neurons spontaneously firing in
the SFO. Lactate, as well as Na, stimulated the activity of GABAergic
neurons in the SFO. This activation by lactate is suppressed by αCHCA, an inhibitor of lactate transporter.

Publication List
〔Original papers〕● Fujikawa, A., Chow. J.P.H, Shimizu. H., Fukada. M., Suzuki. R., andNoda, M. (2007). Tyrosine phosphorylation of ErbB4 is enhanced by
PSD95 and repressed by protein tyrosine phosphatase receptor type Z.
J. Biochem. 142, 343-350.
● Shimizu, H., Watanabe, E., Hiyama, T.Y., Nagakura, A., Fujikawa, A.,
Okado, H., Yanagawa, Y., Obata, K., and Noda, M. (2007). Glial Na x
channels control lactate signaling to neurons for brain [Na+] sensing.
Neuron 54, 59-72.
● Yamamoto, H., Kamegaya, E., Hagino, Y., Imai, K., Fujikawa, A.,
Tamura, K., Enokita, T., Yamamoto, T., Takeshima, T., Koga, H.,
Uhl, G.R., Ikeda, K., and Sora, I. (2007). Genetic deletion of vesicularmonoamine transpoter-2 (VMAT2) reduces dopamine transporter
activity in mesencephalic neurons in primary culture. Neurochem. Int.
51, 237-244.

〔Review article〕● Noda, M. (2007). Hydromineral neuroendocrinology: Mechanism ofsensing sodium levels in the brain. Exp. Physiol. 92, 513-522.

35

National Institute for Basic Biology

Neurobiology

DIVISION OF BRAIN BIOLOGY

Professor

YAMAMORI, Tetsuo
Assistant Professors

NIBB Research Fellow
Technical Staff
Postdoctoral Fellow
Graduate Students

Technical Assistants

KOMINE, Yuriko
WATAKABE, Akiya
SADAKANE, Osamu
TAKAHATA, ToruOHSAWA, Sonoko
KOMATSU, Yusuke
TAKAJI, Masafumi
SASAKI, Tetsuya
HIROKAWA, Junya
NAKAMURA, Tohru
TOITA, Shigeko
NAKAGAMI, Yuki
ISHIKAWA, Takako
MORITA, Junko
IMAI, Akiko

In order to understand the formation and evolution of thebrain and the mechanisms underlying memory, we arefocusing primarily on two issues. Firstly, we are studying
the genes that are expressed in specific areas of the primate
neocortex. We have obtained genes that show marked
differences within primate neocortical areas. Secondly, we
study the mechanisms underlying learning behaviors by
examining gene expression.

Ⅰ. Genes expressed in specific areas andlayers of the neocortex
The neocortex is most remarkably evolved in the
anatomical areas and it has been a matter of debate to what
extent areas of the neocortex are genetically andenvironmentally determined. It is also puzzling why, during
mammalian evolution, the neocortex was markedly
expanded while the total number of genes in the mammal
was little changed. In order to answer these questions, westudied gene expression within different areas of the
neocortex. In the last several years, we reported thefollowing findings, which are schematically illustrated in
Figure 1.
1) Examining 1088 genes by microarray analysis, mostgenes showed less than two fold difference in theirexpressions among the three neocortical (frontal, motor andvisual) areas. Only one gene showed more than three folddifference and another one was between two and three folddifference within the three areas (Watakabe et al., Mol.
Brain Res., 88, 74-82, 2001). These results suggest that thegenes expressed among the different areas of the humanneocortex are very similar. The question remained, however,whether or not there are any genes that show marked
neocortical area difference.
2) In order to answer this question, we employed
differential display methods and found three genes thatindicated area-specific expressions.
i) One gene, designated occ1, is specifically expressed in36

the occipital cortex, particularly in V1 area, in the primate
brain. We also demonstrated that occ1 expression wasmarkedly increased postnataly in V1.
ii) The other gene that showed marked difference within
the neocortex is gdf7, a member of BMP/TGF-β family,
which is specifically expressed in the motor cortex of theAfrican green monkey (Watakabe et al., J. Neurochem., 76,
1455-1464, 2001).
iii) Rbp (retinol-binding protein) is preferentially expressedin association and higher areas in the neocortex (Komatsu etal., 2005). Rbp also shows characteristic features. a) Its
expression is high in sensory association and higher
association areas and limbic areas, but low in the primarysensory areas. Expression is complementary to that of occ1
and to parvalbumin immunoreactivity (PV-IR) in primarysensory areas. b) In early sensory pathways, the expression
is limited to superficial layers only (in particular, layer 2).
With progression into higher sensory areas, the expression is
expanded into layers 3 and then 5. c) In higher-orderassociation areas, Rbp is expressed throughout all layers
except layer 4. d) This characteristic distribution of Rbp ismainly formed during postnatal development. Rbp probably
regulates the concentration of retinoic acid (RA) by thedelivery of retinol, which is converted into RA in cells.
Although the role of RA in the mature brain is not yet
known, the characteristic expression of Rbp within
association areas may provide a clue to the molecular basisof the formation and function of the association areasiv) We compared the occ1 expression in subcortical areas
and found certain nuclei strongly expressed occ1.
Interestingly, in most of the nuclei that occ1 is strongly
expressed in monkeys, the authologue of occ1 is similarly
expressed in mice, which suggests that the occ1 expression
in subcortical nuclei is generally well conserved during
mammalian evolution (except for a few nuclei such as
LGN).

Figure 1. The expression of occ1 (orange color) m Rbp (blue) and gdf7
(green) are schematicaly illustrated in Brodmann’s area figure in theguenon monkey. Top and bottom views are medial and lateral surfaces,
respectively. (The figure is cited from Yamamori & Rockland, Neurosci.
Res., 55, 11-27, 2006 ).

v) occ1 is strongly expressed in the mouse LGN. We thenmonocularly deprived activity by enucleation or TTX
injection and examined occ1 expression in LGN. Contrary to
the monkey primary visual cortex, occ1 expression was notaffected by monocular deprivation. This is a clear contrast
with other well known activity dependent gene expressions
such as c-fos expression (Figure 2; Takahata et al., J. Chem
Neuroanat., in press).

vi) In the visual cortex of the mouse, it is known thatmonocular deprivation reduces the expression of immediate
early genes such as c-fos and zif268. However, theexpression of occ1/Frp in the LGN and visual cortex was
not affected by monocular deprivation experiments (Figure
3; Takahata et al., J. Chem. Neuroanat., in press).
These results suggest that activity dependency of occ1 has
been acquired during evolution of the primate brain(Takahata et al., Society for Neuroscience in North America,
2006, J. Chem Neuroanat., in press).

Ⅱ. Gene expression under a declarative and anon-declarative memory

Figure 2. The expression of c-fos (top) and occ1/Frp (bottom) in mouse
LGN are shown. In the monocular zone (MZ) of the contra LGN to thedeprived eye, c-fos mRNA expression was dramatically decreased,
whereas the occ1/Frp mRNA expression is not affected. (The original
figure is shown in Takahata et al., J. Chem Neuroanat., 2007 Sep 16;[Epub ahead of print]).

Figure 3. The expression of zif268 (top), c-fos (middle) and occ1/Frp(bottom) in mouse visual cortex are shown. In the contra visual cortex(V1MZ: V1 monocular zone) and V2L (lateral secondary visual cortex),
the expression of zif268 and c-fos to the deprived eye (MD, 1 day after
monocular deprivation), c-fos mRNA expression was dramaticallydecreased, whereas the occ1/Frp mRNA expression is not affected. (Theoriginal figure is shown in Takahata et al., J. Chem Neuroanat., 2007
Sep 16; [Epub ahead of print]).

In order to study informational processing underlying thedeclarative and non-declarative memory at molecular andcellular levels in the brain, we established two behavioral
systems.
1) We have been collaborating with professor Yoshio
Sakurai (Kyoto University) who developed an audio-visual
discrimination task (AVD-task) system. In this task, a rat
was asked to choose either an audio cue (a high tone or lowtone) or a visual cue (a light from the right or the left) toobtain a food pellet. We found that the visual and audio
tasks enhanced the specific expression of c-Fos in the visualand audio cortices, respectively. Among the early visual andauditory pathways examined, c-Fos was specifically inducedin the cortices but not in the earlier pathways, suggesting theneural modulation of the neocortex depends on the type oftask. Interestingly, the task-dependent Fos expression was
only observed in excitatory neurons in the relevant sensory
cortices.
Although this AVD task system is quite powerful for
analyzing the problem described above and useful forstudying underlying molecular and cellular mechanisms
because of the advantages of using rodents, one concern wasthat the auditory stimuli and visual stimuli were in different
positions. Thus, we could not exclude the possibility that thedifference between the auditory task and the visual task may
not completely depend on the modality (visual vs auditory)
difference.
We wanted to solve this problem by placing auditory andvisual stimuli in the same position. We use nose-poking tomeasure the reaction time in which a rat responds to stimuli;
by using this behavioral system, we were able to confirm
amodal recognition of space, which means that a rat can
respond to a different modality (visual or auditory) if thestimuli are in the same position as previously reported inother systems. We also confirmed multisensory
enhancement is indeed observed in rats. These results
suggest that this new modified AVD system can be used to
explore the molecular and cellular mechanisms underlying
multisensory processing in rats (Sakata et al., Exp. BrainRes., 159, 409-417, 2004).
We studied multisensory processing further using a c-Fos
mapping technique. We first developed a method tostandardize the cortex to quantitatively evaluate c-Fos
expression by an automatic image analyzing system. Using
this system, we found the secondary visual cortex (V2L) in
rats is specifically activated under audiovisual multisensory37

National Institute for Basic Biology

Neurobiology

stimulation. Injecting muscimol into V1, V2, V2L andsuperior colliculus (SC), we found that V2L is specificallyinvolved in the stimulation of multisensory reaction(Hirokawa et al., 2006 Society for Neuroscience in North
America).
Traditionally, multisensory integration was thought tooccurr in higher neocortical areas by merging different
modalities of primary sensory information. Our results
suggest that the multisensory integration may in fact occur at
a relatively “early sensory” area such as V2. Previous
elecrophysiological studies also show that there exist
multisensory areas in the secondary visual area and theboundary areas between two modal areas (Toldi et al.,
1986; Barth et al., 1995; Wallace et al., 2004). Thisobservation is consistent with our findings through theexperiments using the newly developed behavioral systemand c-Fos analyzing system. Therefore, we havedemonstrated evidence for a role for the lateral secondary
visual area (V2L) in multisensory informational processing
under behavior conditions.
2) The other task we developed is a wheel running system inwhich a water-deprived mouse is made to run to obtainwater because the wheel with the pegs is turning in the otherdirection (Kitsukawa et al., SFN Meeting, 2002). The task
required of the mouse can thus be regarded as representing aprocedural learning. We examined various areas of themouse brain following changes to the peg pattern. Amongthe areas examined, we found marked c-Fos expression in
the striatum, cerebral cortex. The striatum, which iscomposed of projection neurons and several distinguishedtypes of interneurons, is known to play an important role inreward-based learning. The characterization of thesesubtypes of interneurons has progressed. Their roles inbehavioral tasks, however, have remained obscure. We are
currently examining the altered behavior that appears under
the pharmacological treatments that affect the metabolism of
the interneurons in the striatum.

Ⅲ. Topological relationships between brain
and social networks
Network theory has recently revealed that many networks(gene transcription, protein-protein interaction, The Internet,
and sociological networks) share global and local properties(Albert and Barabasi, 2002; Barabasi, 2002; Barabasi andAlbert, 1999; Milo et al., 2002, 2004; Watts and Strogatz,1998). To understand the global and local design principles
of mammalian cerebral cortical networks, we applied
network-theoretical approaches to connectivity data from
macaque and cat cortical networks. Firstly, we confirmed
the “small world” property of these cortical networks.
Secondly, we then compared them, based on the significanceprofile (SP) of thirteen possible network motifs in the real
network compared to randomized networks. SPs of different
mammalian cortical networks are highly conserved androbust. Our results thus suggest that there are constraints of
neocortical development and evolution (Sakata andYamamori, Neurosci. Res., 51, 309-315, 2005).
This year, we reported on the topological similarities
between brain and social networks. The statistical relevance38

of specific tied structures differs between social “friendship”
and “disliking” networks. This suggests a relation-typespecific topology of social networks. Overrepresented
connected structures in brain networks are more similar to
those in the friendship networks than to those in othernetworks (Figure 4). We found that, unlike what could be
predicted by simply counting mutual connections, balanced
and imbalanced reciprocal connections between nodes are
significantly abundant and rare, respectively. We interpretthese results as evidence of positive selection of balanced
mutuality between nodes. These results also imply that there
exist common underlying principles between theorganization of the brain and social networks.

Figure 4. Hierarchical clustering of Triad Significance Profile (TSPs)(see thirteen possible motifs on the top panel) of complex networks.
Note that brain and friendship networks fell into the same cluster.
Abbreviations: mac. Macaque. The figures are cited from Sakata andYamamori, Neural Networks., 20, 12-21, 2007.

Publication List
〔Original papers〕● Nakamura, K., Watakabe, A., Hioki, H., Fujiyama, F., Tanaka, Y.,

Yamamori, T., and Kaneko, T. (2007). Transiently increased
colocalization of vesicular glutamate transporters 1 and 2 at single
axon terminals during postnatal development of mouse neocortex: aquantitative analysis with correlation coefficient. Eur. J. Neurosci. 26,
3054-3067.
● Sakata, S., and Yamamori, T. (2007). Topological relationships
between brain and social networks. Neural Networks 20, 12-21.
● Watakabe, A., Ichinohe, N., Ohsawa, S., Hashikawa, T., Komatsu, Y.,
Rockland, K.S., and Yamamori, T. (2007). Comparative analysis oflayer-specific genes in mammalian neocortex. Cereb. Cortex 17, 19181933.

DIVISION OF BEHAVIORAL BIOLOGY (ADJUNCT)

Professor (Adjunct)

Associate Professor (Adjunct)

MORI, Yuji

TSUKAMURA, Hiroko

Assistant Professor
NIBB Research Fellow
Visiting Scientists

IMAMURA, Takuya
TOMIKAWA, Junko
MAEDA, Maki
SATO, Hiroaki
SHIMOKAWA, Hiroko
YAMADA, Kenji

In mammals, several social behaviors are dependent onsex. These sex-dependent patterns of behavior must be
acquired through highly irreversible processes during
development. We hypothesize that the long-term effects ofsex steroids at the perinatal stage on behaviors after puberty
are somehow marked at the genome level. We are currently
investigating the epigenetic status of the discrete brain areas
responsible for the sex difference of the structure andbehaviors.

brain, ERαas expression showed tissue-, development-, andsex- dependent pattern. ERαas-Tg mice gained weight fasterthan the wild type during prepubertal stages. ERαas-Tg
females showed the significant reduction of lordorsis and thelarger volume of ventromedial hypothalamus (VMH) which
are rather reminiscent of the male-like patterns. The search
for molecular targets of ERαas revealed that the ERα
promoter was specifically demethylated, resulting in theERα upregulation. It is to be noted that, of discrete brain
areas, only the principal portion of the bed nucleus of thestria terminalis, but not the VMH, showed the sex-dependent
DNA methylation pattern in mice and rats. Thus, ERαas,acting on the ERα promoter as a noncoding RNA, is acomponent that establishes its epigenetic status for gene
expression. Spatiotemporal expression of ERαas may
mediate the functional differentiation of the female and malebrains with or without morphological alterations in rodents.

Ⅰ. DNA methylation of sex steroid receptor genes
In mammals, DNA methylation, mainly occurring on CG
dinucleotides, is a fundamental mechanism thatdifferentiates the gene expression pattern in respective cells.
DNA methylation is a restraint of the pluripotency because
once the pattern is established during development it ismaintained through cell division. On the other hand, forexample, some fish that contain much less methylation
activity are found to easily and reversibly change their sex
status according to the environmental context. In rodents,
endocrine disturbance at the fetal and/or postnatal stage
irreversibly changes behaviors such as the lordosis (infemales) and the mounting (in males) after the pubertal stage
that are normally dependent on genetic sex. In some cases,
lordosis can be observed even in males and mounting infemales. These clearly indicate that sex-dependent patternsof behavior are not directly dependent on “sex-specific”
genes but rather established through epigenetic processes.
We have found the sex- and brain area-dependent DNA
methylation pattern of rodent steroid receptor genes(estrogen receptor α, ERα; androgen receptor, AR;progesterone receptor, PR) that can be modulated by thenovel noncoding RNAs, named ERαas, ARas and PRas.
Furthermore, we have generated ERαas- and ARastransgenic (Tg) mice in which ERαas and ARas fragments
are constitutively expressed, respectively. We report hereinon the functional alteration of mouse brain by thesenoncoding RNAs.

1-1 Functional alteration of rodent brain bynoncoding RNA for ERα promoter
ERα participates in the cellular, reproductive andbehavioral mechanisms. We found novel endogenous
antisense transcripts on ERα locus, named ERαas. In the

Figure 1. Physiological alterations in ARas-Tg mice. (A) Body weights
of males. Growth graphs of the 15 weeks old ARas-Tg and the wild type.
WT denotes wildtype. (B) Body weights of females. Growth graphs of
the 15 weeks old ARas-Tg and the wild type. (C) Graphs showing thevolume of each nucleus of the PVN (paraventriclar nucleus), BNST
(principal portion of the bed nucleus of the stria terminalis), and VMH in
ARas-Tg and wild type mice. (D) lordosis behavior test in ARas-Tg andwild type female mice (LQ scores). (E) Mount behavior tests in ARas-Tg
and wild type female mice. Animals was measured with the times ofmount behaviors. (F) Testosterone levels in excrement of ARas-Tg andwild type female mice. (G) Estrogen levels in the excrement of ARas-Tg
and wild type female mice. Bars represent mean ±SE (**p < .01, .01 <*p < .05; student t-test).

39

National Institute for Basic Biology

Neurobiology

1-2 Functional alteration of rodent brain bynoncoding RNA for AR promoter
In addition to the estrogen signaling, AR-mediatedandrogen signaling participates in the cellular, reproductive
and behavioral mechanisms. We found novel endogenous
antisense transcripts on AR locus, named ARas. In the brain,
the expression showed tissue-, development-, and sexdependent pattern. ARas-Tg female mice showed thesignificant reduction of lordorsis, occurrence of mountbehavior, and larger volume of VMH which are rather
reminiscent of the male-like patterns (Figure 1). The
circulating testosterone level was lower in ARas-Tg females
than in wild type females. The search for molecular targetsof ARas revealed AR promoter to be specificallydemethylated, resulting in the AR upregulation. Of thediscrete brain areas, only VMH showed the sex-dependent
DNA methylation pattern in mice and rats. Orchidoectomy
and peripheral application of estradiol benzoate at the onsetof birth increased and decreased the methylation level atseveral CG sites in males and females, respectively,
suggesting that the sex-specific methylation of AR promoter
was established/maintained by estrogen that was producedby the aromatization of testosterone derived from the testis.
Thus, ARas, acting on the AR promoter, is a component that
establishes its epigenetic status for gene expression in a
spatiotemporal manner. It is likely that appropriate controlof AR expression by ARas mediates the estrogen-induced
sexual differentiation of the brain.

Publication List
〔Original papers〕● Iwata, E., Kikusui, T., Takeuchi, Y., and Mori, Y. (2007). Fosteringand environmental enrichment ameliorate anxious behavior inducedby early weaning in Balb/c mice. Physiol. Behav. 91, 318-324.
● Kakuma, Y., Ichimaru, T., Yonezawa, T., Momozawa, Y., Hashizume,
C., Iwata, E., Kikusui, T., Takeuchi, Y., Ohkura, S., Okamura, H., andMori, Y. (2007). Androgen induces production of male effectpheromone in female goats. J. Reprod. Dev. 53, 829-834.
● Kikusui, T., Kiyokawa, Y., and Mori, Y. (2007). Deprivation of
mother-pup interaction by early weaning alters myelin formation in
male, but not female, ICR mice. Brain. Res. 1133, 115-122.
● Kiyokawa, Y., Kikusui, T., Takeuchi, Y., and Mori, Y. (2007).
Removal of the vomeronasal organ blocks the stress-induced
hyperthermia response to alarm pheromone in male rats. Chem.
Senses 32, 57-64.
● Kiyokawa, Y., Takeuchi, Y., and Mori, Y. (2007). Two types of social
buffering differentially mitigate conditioned fear responses. Eur. J.
Neurosci. 26, 3606-3613.
● Momozawa, Y., Takeuchi, Y., Kitago, M., Masuda, K., Kakuma, Y.,
Hashizume, C., Ichimaru, T., Mogi, K., Okamura, H., Yonezawa, T.,
Kikusui, T., and Mori, Y. (2007). Gene expression profiles linked tothe hormonal induction of male-effect pheromone synthesis in goats(Capra hircus). Biol. Reprod. 77, 102-107.
● Momozawa, Y., Takeuchi, Y., Tozaki, T., Kikusui, T., Hasegawa, T.,
Raudsepp, T., Chowdhary, B. P., Kusunose, R., and Mori, Y. (2007).
SNP detection and radiation hybrid mapping in horses of ninecandidate genes for temperament. Anim. Genet. 38, 81-83.
● Momozawa, Y., Terada, M., Sato, F., Kikusui, T., Takeuchi, Y.,
Kusunose, R., and Mori, Y. (2007). Assessing equine anxiety-relatedparameters using an isolation test in combination with a questionnaire
survey. J. Vet. Med. Sci. 69, 945-950.
● Nakamura, K., Kikusui, T., Takeuchi, Y., and Mori, Y. (2007). The
critical role of familiar urine odor in diminishing territorial aggression
toward a castrated intruder in mice. Physiol. Behav. 90, 512-517.
● Shimozuru, M., Kikusui, T., Takeuchi, Y., and Mori, Y. (2007).
Discrimination of individuals by odor in male Mongolian gerbils,
Meriones unguiculatus. Zoolog. Sci. 24, 427-433.
● Shimozuru, M., Kodama, Y., Iwasa, T., Kikusui, T., Takeuchi, Y., andMori, Y. (2007). Early weaning decreases play-fighting behavior
during the postweaning developmental period of Wistar rats. Dev.
Psychobiol. 49, 343-350.
● Uematsu, A., Kikusui, T., Kihara, T., Harada, T., Kato, M., Nakano,K., Murakami, O., Koshida, N., Takeuchi, Y., and Mori, Y. (2007).
Maternal approaches to pup ultrasonic vocalizations produced by ananocrystalline silicon thermo-acoustic emitter. Brain. Res. 1163, 9199.
● Yamada, S., Uenoyama, Y., Kinoshita, M., Iwata, K., Takase, K.,
Matsui, H., Adachi, S., Inoue, K., Maeda, K. I., and Tsukamura, H.
(2007). Inhibition of metastin (kisspeptin-54)-GPR54 signaling in thearcuate nucleus-median eminence region during lactation in rats.
Endocrinology 148, 2226-2232.

〔Review article〕● Maeda, K., Adachi, S., Inoue, K., Ohkura, S., and Tsukamura, H.

(2007). Metastin/kisspeptin and control of estrous cycle in rats. Rev.
Endocr. Metab. Disord. 8, 21-29.

40

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

showed that Na x channel was exclusively localized toperineuronal lamellar processes extended from astrocytes
and tanycytes in the organs. Importantly, glial cells derived
from the organs were capable of sensing extracellularsodium-level, as analysed by the ion-imaging method.

When the correct balance between water and sodium levelsin the body fluid has been disrupted, terrestrial animals feelwater and salt appetite or satiety, and these perceptions
subsequently induce the animal behaviors referred to as
ingestion or aversion. Our research is focused onunderstanding the molecular and neural mechanisms
underlying the animal behaviors essential to homeostasis of
the body fluid.
To explain the properly regulated animal behaviors,
neurobiologists have postulated the existence of both
osmoreceptors and specific sodium receptors in the brain.
The molecular entities of these receptors, however, were notdiscovered for a long time. In 2000, by using gene-targeting
technology, we first clarified that Na x sodium channel is a
probable candidate for the specific sodium receptor in thebrain.
A line of studies using Na x-gene deficient mice showedthat 1) Na x-deficient mice ingested hypertonic sodium
chloride solution in excess in comparison with wild typemice; 2) Na x gene is expressed in the circumventricularorgans, which are the specialized central organs involved in
the sensing of sodium concentration and osmolality in the
body fluids; 3) Na x channel is an extracellular sodium-levelsensitive sodium channel (Figure 1); and 4) when Na x cDNA
was introduced into the brain of the knockout mice with an
adenoviral expression vector, only those animals thatreceived a transduction of the Na x gene into the subfornicalorgan among the circumventricular organs recovered saltavoiding behavior under dehydrated conditions. These
results suggest that the subfornical organ is the center of thecontrol of salt-intake behavior in the brain, where thesodium-level-sensitive Na x channel is involved in sensing
the physiological increase in the sodium level of body fluids.
Recent immuno-electron-microscopic studies clearly

Figure 2. Imaging analysis of the uptake of glucose in the subfornicalorgan using a fluorescent glucose derivative 2-NBDG. The subfornicalorgan was isolated from wild-type (WT; a, b, e, and f) and Nax-knockout(KO; c and d) mice, and incubated with 2-NBDG in the 145 mM (a, c,and e) or 170 mM (b, d, and f) sodium solution. In some experiments,
the extracellular solutions contained 1 mM ouabain that is a potent++
blocker of Na /K -ATPase (e and f). Scale bars: 50 mm. For details see
the following paper.

In 2007, we showed direct interaction between Na x
channels and subunits of Na +/K +-ATPase, which brings
about sodium-dependent activation of the metabolic state of
the glial cells (Figure 2). Metabolic enhancement leading toextensive lactate production was observed in the subfornicalorgan of wild-type mice, but not in the Nax-knockout mice.
Furthermore, lactate, as well as Na, stimulated the activity ofGABAergic neurons in the subfornical organ. These results
suggest that the information ofn a physiological increase of
the sodium level in body fluids sensed by Na x in glial cells istransmitted to neurons by lactate as a mediator to regulate
neural activities of the subfornical organ. These findings
suggest that the neuron-glia complex plays a key role insodium sensing in the circumventricular organs.

Publication List
〔Original paper〕
Figure 1. Na x is a sodium channel sensitive to extracellular sodium level.
When the extracellular sodium concentration increases, Na x channel
opens the gate pore and generates the sodium ion influx into the cells.
This view was hypothesized by ion-imaging studies.

● Shimizu, H., Watanabe, E., Hiyama, T.Y., Nagakura, A., Fujikawa, A.,

Okado, H., Yanagawa, Y., Obata, K., and Noda, M. (2007). Glial Na x
channels control lactate signaling to neurons for brain [Na+] sensing.
Neuron 54, 59-72.

41

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROCHEMISTRY

Associate Professor

SASAOKA, Toshikuni
Postdoctoral Fellow

SATO, Asako

Our major research interest is to understand thephysiological role of the dopaminergic system in animal
behavior, particularly locomotion and eating behaviors,
using genetically altered mice, both transgenic and geneknockout mice. In addition, we have developed a novel
method of conditional mutagenesis in mice in order tosubstitute the amino acid sequence of the target gene inparticular cells. We analyze the physiological roles of thecomponents of the dystrophin complex on the skeletal
muscle membrane using genetically modified mice.

Ⅰ. Role of dopaminergic transmission inlocomotion and eating behavior
The dopaminergic system is implicated in the modulationof 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 ofemotion, motivation and cognition. Dysfunction of the
dopaminergic system can result in several neurological andpsychiatric disorders, such as Parkinson’s disease andschizophrenia.

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

In mammals, five subtypes of dopamine receptor (D1R,
D2R, D3R, D4R and D5R) have been identified and divided
into two subgroups, referred to as D1-like receptors (D1R,
D5R) and D2-like receptors (D2R, D3R and D4R) on thebasis 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. D1R has an opposite property to D2R
with respect to intracellular signal transduction.
In collaboration with the Laboratory of Dr. Motoya
Katsuki, the former Director General, we have been42

investigating the involvement of dopaminergic transmission
via D1R and D2R in the regulation of locomotion and eating
behavior. We generated D1R/D2R double knockout (DKO)
mice by crossing D1R knockout (KO) with D2R KO mice,and observed that D1R/D2R DKO mice exhibited severe
impairment in locomotion, no initiation of eating, and diedby 4 weeks of age. To investigate the molecular mechanismof motor control and eating behavior, we generated
transgenic mice harboring tetracycline-regulated expressionof the D1R gene on the D1R/D2R DKO background. Severaltransgenic mouse lines successfully rescued lethal
phenotype of the D1R/D2R DKO mice and showed
doxycycline (Dox) controllable expression of transgenic
D1R gene (named as D1R/D2R DKO-D1R rescued mice).
The D1R/D2R DKO-D1R rescued mice exhibited decreases
in locomotion and food/water intake as well as a decrease in
the amount of transgene expression after Dox
administration. After the withdrawal of Dox administration,
the D1R/D2R DKO-D1R rescued mice exhibited transient
hyperactivity and recovered locomotor activity andfood/water intake. We are analyzing these results to identify
the mechanism of the relationship between the D1R
expression and altered behavior. In addition, we are also
investigating whether or not there is a critical period indevelopment for the regulation of locomotion and eating
behavior by dopaminergic transmission.

Ⅱ. Developing a novel conditional mutagenesis
method in mice
In collaboration with Prof. Yo-ichi Nabeshima of Kyoto
University, we developed a novel mouse developmental
biotechnology of introducing an amino acid substitution intoa target gene in a spatially and temporally restricted manner.
The goal of the study was to overcome the limitations of theconventional mouse molecular genetic approach in thefunctional analysis of target genes. We substituted one
critical amino acid residue (the 595th asparagines, Asp595)
of N-methyl-D-aspartate receptor (NMDAR), leading to an
aberrant activation of NMDAR. The NMDARs are widely
expressed in the nervous system, fundamental to excitatoryneurotransmission, and play a number of important roles atdifferent brain loci and time points. The NMDARs act as acoincidence detector and are not only important for neuronaldifferentiation, migration, and survival, but are also criticalfor activity dependent synapse formation. It is suggested thatthe aberrant activation of NMDAR causes excitotoxicity,
leading to neuronal death in various neurological diseases.
Our method is vastly applicable to the functional analysisof any desired gene and should contribute to studies on the
structural and functional relationships of relevant genes.

Ⅲ. Analysis of roles of the sarcoglycan complex,
dystroglycan complex and caveolin-3
Sarcoglycans (SGs) are trans-sarcolemmal glycoproteinsthat associate together to form sarcoglycan complex (SGC)and are present in the sarcolemma. SGC, together withdystrophin and the dystroglycan complex, comprises thedystrophin complex, which is considered to be themechanical link between the basement membrane and theintracellular cytoskeleton for protecting the sarcolemma
from mechanical stress during muscle contraction. Each offour SG subunits (α-,β-,γ- and δ-SG) is responsible forfour respective forms of SG-deficient muscular dystrophy,
sarcoglycanopathy (SGP). We previously generated the βSG KO and γ-SG KO mice and found that the dystrophin
complex isolated from the SG-deficient skeletal muscles wasbiochemically unstable. This indicates that SGC andsarcospan play an important role in stabilizing thedystrophin complex.

Figure 2. A schematic drawing of the dystrophin complex

Dystroglycan (DG) is a widely expressed transmembrane
glycoprotein complex which plays important roles byconnecting the intracellular cytoskeleton and theextracelluler matrix. DG is expressed as an 895 amino acidprecursor and cleaved between amino acid residues 653 and654 of the precursor to generate α- and β-DG subunits. In
collaboration with Dr. Torahiko Tanaka of Nihon University
School of Medicine, Tokyo, we performed a series ofmutation analyses to determine which amino acid residues
and which regions of DG are critical for cleavage in order toclarify the mechanisms involved in DG cleavage and subunit
association. We transfected cultured cells with wild-type andvarious mutant DGs, and confirmed the DG cleavage. We
found the following: (i) Disruption of the intramoleculardisulfide bridge between Cys669 and Cys713 in β-DG
completely abolishes cleavage; (ii) deletions in the loop
region (669-713) and in the C-terminal region of α-DG

(550-645) abolish the cleavage; (iii) disruption of thedisulfide bridge and deletions in the loop region deteriorate
the α- and β-DG association; (iv) positions P1’ (Ser654)
and P6’ (Trp659) are critical, particularly at the cleavage
site. Thus, the critical role of the Cys669-Cys713 disulfide
bridge formation is, most likely, to form a specific tertiary
structure, in which the α- and β-DG domains interact andthe cleavage site becomes susceptible to proteolytic
reactions. The Cys669 and Cys713 pair is broadly conservedin vertebrates and in some invertebrates, suggesting that thedisulfide bridge formation was established early in theevolution of DG.
Caveolin-3 is a muscle-specific membrane protein, acomponent of the dystrophin complex, and serves as a
scaffold of various molecules. Its gene mutations cause
limb-girdle muscular dystrophy (LGMD1C orcaveolinopathy) with mild clinical symptoms. In
collaboration with Dr. Yasuko Hagiwara of Musashino
University, Tokyo, we previously reported that caveolin-3
deficiency causes muscle degeneration and a decrease insarcolemmal caveolae in caveolin-3 gene-knockout (Cav3-/-)mice. To examine the pathogenic pathways and identify new
or modifying factors involved in caveolinopathy, we
examined the gene expression profiles of approximately
8,000 genes in the skeletal muscle of Cav3-/- mice using
DNA microarray technique. We found that the gene ofosteoponin (OPN), a versatile regulator of inflammation andtissue repair, was significantly down-regulated. This is incontrast to mdx mice showing a markedly up-regulated OPN
gene in their skeletal muscles. Recently, OPN has beenreported to be important in the pathogenesis of muscular
dystrophy. We examined whether up-regulated OPN gene
expression in mdx muscles is altered by the deficiency of
caveolin-3. To this end, we developed caveolin-3 anddystrophin double-deficient mice. The levels of OPN mRNAand osteopontin in the double-deficient mice clearlydecreased compared with those in mdx mice. We showedthat although the level of OPN mRNA expressed in the
double-deficient skeletal muscles was lower than that in mdx
skeletal muscles, macrophage infiltration and muscle
regeneration occurred similarly in the double-deficient andmdx skeletal muscles. There may still be other factors thatare involved in macrophage infiltration and muscle
regeneration.

Publication List
〔Original papers〕● Ohi, Y., Ishii, Y., Haji, A., Noguchi, S., Sasaoka, T., Fujimori, T.,

Nabeshima, Y., Sasahara, M., and Hattori, Y. (2007). Platelet-derived
growth factor (PDGF)-BB inhibits AMPA receptor-mediated synaptictransmission via PDGF receptor-beta in murine nucleus tractus
solitarius. Brain Research 1159, 77-85.
● Watanabe, N., Sasaoka, T., Noguchi, S., Nishino, I., and Tanaka, T.
(2007). Cys669-Cys713 disulfide bridge formation is a key todystroglycan cleavage and subunit association. Genes to Cells 12, 7588.

43

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

Graduate Student
Visiting Scientists

Technical Assistants

Secretary

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

The main interest of this division is understanding thebiology of the dynamic genome, namely, genome
organization and reorganization and its impact on gene
expression and regulation. We are also characterizing
various aspects of genetic and epigenetic gene regulations,particularly the flower pigmentation of morning glories. In
addition, we are undertaking reverse genetic approaches inorder to elucidate the nature of dynamic genome in rice, amodel plant for cereals.

Ⅰ. Spontaneous mutants in morning glories
Considerable attention has recently been paid to themorning glory genus Ipomoea because of the experimental
versatility of its floral biology including the genetics offloral variation, flavonoid biosynthesis, and transposoninduced mutations. The genus Ipomoea includes about 600
species distributed on a worldwide scale that exhibit various
flower morphologies and pigmentation patterns. Amongthem, three morning glories, Ipomoea nil (the Japanesemorning glory), Ipomoea purpurea (the common morning
glory), and Ipomoea tricolor, have been domesticated as
floricultural plants. Of these, spontaneous mutants of I. nil
and I. purpurea with various flower colors have been
isolated and cultivated since the 17th century in Japan andEurope, respectively. The wild-type I. nil and I. purpurea
display blue and dark-purple flowers, respectively, both ofwhich contain polyacylated and polyglycosylated cyanidinbased anthocyanins, and both plants exhibit red stems anddark-brown seeds. Almost all structural and regulatory

44

Figure 1. Pigmentation phenotype of the pearly-s mutant and structure of
the pearly-s mutation. (a) Wild-type cultivar Heavenly Blue. (b) A
pearly-s mutant displays stable white flowers. (c) Structure of thepearly-s mutation having the Hel-It1 transposon integrated into the DFRB gene. The boxes with numerals and the shadowed parts indicate exonsand the coding region of the DFR-B gene, respectively. The rectangle
with the filled arrowheads shows the MELS2 element. The boxes in HelIt1 indicate the predicted exons for the putative wild-type RPA-TPaseand Rep/Hel-TPase genes, respectively. The horizontal lines under theboxes indicate functional domains of these proteins, and the symbols
DB, SI, Rep, and Hel represent DNA-binding, subunit interaction, Rep,
and Hel domains, respectively. The predicted single-base substitution
and deletion are shown by vertical arrowheads with C to T and ΔT,
respectively.

genes, which encode enzymes to produce anthocyanidin 3O-sophorosides for their flower pigmentation, andtranscriptional regulators, which activate the structural genes
for anthocyanin biosynthesis, as well as their spontaneous
mutations, have been identified and characterized. The
majority of their spontaneous mutations have been shown tobe caused by insertions of various DNA transposons
belonging to the hAT, CACTA, and MuLE superfamilies.
Helitrons are newcomers of eukaryotic DNA transposons
and were originally identified by computational analysis in
the genomes of Arabidopsis, rice, and nematode. They are
distinguished from other transposons in their structuralfeatures, and their proposed transposition mechanisms are
involved in rolling circle replication. Computer-predictedautonomous Helitrons with conserved terminal sequences,
5’-TC and CTRR-3’, are presumed to encode a putativetransposase, Rep/Hel-TPase, which contains a characteristic
nuclease/ligase domain for the replication initiation protein(Rep) and a DNA helicase domain (Hel). Plant Helitrons arethought to carry an additional transposase gene, RPA-TPase,
which is related to the largest subunit of the replication
protein A (RPA70). Although Helitrons are found in diverse
genomes, neither an autonomous element nor a transposition
event has been reported. We found that a spontaneous
pearly-s mutant of Ipomoea tricolor, cultivar Pearly Gates,

exhibiting white flowers and isolated in approximately 1940,
has an 11.5-kbp novel Helitron, named Hel-It1, integrated
into the DFR-B gene for anthocyanin pigmentation (Figure1). Hel-It1 shows the predicted plant Helitron structure foran autonomous element with the conserved termini andcarries the two putative transposase genes, Rep/Hel-TPaseand RPA-TPase, which contain a nonsense and a frameshift
mutation, respectively. Hel-It1-related elements are scatteredin the Ipomoea genome, and only a fraction of the pearly-s
plants was found to carry Hel-It1 at another insertion site.
The pearly-s mutant appears to bear an autonomous element
and express the wild-type RPA-TPase transcripts, indicatingthat an Ipomoea Helitron also acts as a spontaneous
mutagen.

Ⅱ. Modification of endogenous natural genes
by homologous recombination in rice
Rice (Oryza sativa L.), with the sequenced genome of 389Mb, is an important staple food for more than half of theworld’s population and is a model plant for other cereal
species. We have developed efficient and reproducible genetargeting by homologous recombination with a large-scale
Agrobacterium-mediated transformation and a strong
positive-negative selection using the hpt and DT-A genes
and have succeeded in modifying the Waxy and Adh2 genes
to generate true gene targeting (TGT) repeatedly without theconcomitant occurrence of ectopic events, such as one-sided
invasion (OSI) and ectopic gene targeting (EGT) (Figure 2).
While Waxy is a unique gene in the rice genome, the 3 Adhgenes, Adh1, Adh2, and Adh3, reside on chromosome 11 in
the same orientation. The targeting frequencies of Waxy andAdh2 were about 1% and 2% per surviving callus,
respectively. To gain information about surviving calli thatwere not targeted homologous recombinants, a series of
PCR analyses was performed to determine the randomly
integrated transgene segments present in these calli. All thesurviving calli examined contained the selective hpt
sequence, and none of them carried the intact DT-A
sequence, indicating that none of the surviving calli was anescapee from the positive-negative selection. The analyses
prompt us to speculate the following integration processes oftransgenes associated with gene targeting in Agorbacteriummediated transformation (Figure 2).
In Agrobacterium-mediated transformation, which has
been used by gene targeting in higher plants, T-DNA
appears to integrate randomly throughout the plant genome
as a single molecule or multiple sequences ligated with eachother in various orientations. The majority of the randomly
integrated single-copy T-DNA molecules mediated bynonhomologous end-joining are known to contain the entire
T-DNA segment with a well-conserved right border, and aleft-border sequence that is either conserved or slightly
truncated, which we termed as border-associated random
integration (BARI) (Figure 2). There appears to be anothertype of random integration with relatively large deletions at
both ends of the T-DNA segment without the borderproximal regions, which we termed border-independent
random integration (BIRI) (Figure 2). BIRI appears to occur
much less frequently than BARI. Since a significant portionof single-stranded T-DNA imported into the plant nucleus
can become double-stranded in Agrobacterium-mediatedtransformation, it has been speculated that such BIRI
processes must share common recombination mechanisms
with random-integration processes by direct DNA delivery
methods using double-stranded DNA. Although thetargeting frequencies in higher plants have been thought tobe low as calculated by TGT events per BARI-mediatedtransformant, such frequencies may be too low to calculate
the proper targeting frequency because Agrobacteriummediated transformation via BARI of T-DNA is a highly
efficient process.

Figure 2. Integration events of a transgene associated with homology-dependent gene targeting with positive-negative selection. The large blackarrowheads with RB and LB indicate the right and left borders, respectively. The most efficient BARI events are shown in large parentheses
because BARI-mediated calli are killed by the action of TD-A. Zigzag lines represent breakpoints generated by nonhomologous events.

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Figure 3. Transfer of base changes from a vector to the rice genome. The rice Adh2 locus on chromosome 11 bears a Copia-like retroelement, andthe vector used contains the 6.2-kb Adh2 promoter sequence including a 0.1-kb 5’-untranslated region, hpt, 4.0-kb Adh2 region, and 2.0-kb 3’
part of a Copia-like retroelement; the DT-A genes were placed at both ends of T-DNA in order to efficiently eliminate BARI. The large blackarrowheads with RB and LB indicate the right and left borders of T-DNA, respectively. The blue boxes indicate Adh2 exons, and hpt and DT-A
are not drawn to scale. The filled and open circles indicate the base changes in the vector and the corresponding genomic sequences, respectively.
Homologous crossover sites can be deduced by determining these alternative sequences in the targeted recombinant allele.

To facilitate the molecular analysis of the recombination
processes, we began to characterize crossover sites in thetargeting of Adh2 with a vector carrying multiple basechanges (Figure 3). Preliminary results indicate that theefficient transfer of base changes (point mutations) occurs
from the vector to the rice genome.

Ⅲ. Characterization of mutable virescentallele in rice
We have identified an active nonautonomous ricetransposon nDart1 of about 0.6 kb, belonging to the hATsuperfamily, as a causative transposon of a mutablevirescent allele pyl-v (pyl-variegated) conferring pale yellow
leaves with dark green sectors in its seedlings. The
transposition of nDart1 can be controlled under natural
growth conditions; its transposition can be induced bycrossing with a line containing an active autonomous
element aDart and stabilized by segregating aDart.
Mapping data indicated that aDart resides within 170-kb
region on chromosome 6. The most likely candidate
element, Dart1-27, was cloned and reintroduced into astable virescent mutant pyl-stb (pyl-stable) exhibiting paleyellow leaves due to the a deficiency of the aDart activity.
Clear leaf variegation could be observed in a significant
portion of the pyl-stb derivatives, in which the demethylated
Dart1-27 element by growing in Escherichia coli had been
introduced. We can thus conclude that Dart1-27 is the active
autonomous aDart element in the mutable virescent pyl-v
plants. In consistent with this notion, the 5’-terminus of
Dart1-27 in the pyl-v lines is less DNA methylated than thatin the pyl-stb plants, even though the sequences of theDart1-27 elements in pyl-v and pyl-stb are found to be
identical to each other.

46

Publication List
〔Original papers〕● Choi, J.D., Hoshino, A., Park, K.I., Park, I.S., and Iida, S. (2007).

Spontaneous mutations caused by an active Helitron transposon, HelIt1, in morning glory, Ipomoea tricolor. Plant J. 49, 924-934.
● Furukawa, T., Maekawa, M., Oki, T., Suda, I., Iida, S., Shimada, H.,
Takamure, I., and Kadowaki, K. (2007). The Rc and Rd genes are
involved in proanthocyanidin synthesis in the rice pericarp. Plant J.
49, 91-102.
● Park, K.I., Ishikawa, N., Morita, Y., Choi, J.D., Hoshino, A., and Iida,
S. (2007). A bHLH regulatory gene in the common morning glory,
Ipomoea purpurea, controls anthocyanin biosynthesis in flowers,
proanthocyanidin and phytomelanin pigmentation in seeds, and seedtrichome formation. Plant J. 49, 641-659.
● Takagi, K., Ishikawa, N., Maekawa, M., Tsugane, K., and Iida, S.
(2007). Transposon display for active DNA transposons in rice. Genes
Genet. Syst. 82, 109-122.
● Terada, R., Johzuka-Hisatomi, Y., Saitoh, M., Asao, H., and Iida, S.
(2007). Gene targeting by homologous recombination as abiotechnological tool for rice functional genomics. Plant Physiol. 144,
846-856.

〔Review article〕● Iida, S., Johzuka-Hisatomi, Y., and Terada, R. (2007). Gene targetingby homologous recombination for rice functional genomics. In Rice
Functional Genomics-Challenges, Progress and Prospects, N.M.
Upadhyaya ed., (Springer), pp. 273-289.

DIVISION OF GENOME DYNAMICS

Professor

HORIUCHI, Takashi
Assistant Professors
Technical Staff
Postdoctoral Fellow
Graduate Students
Secretary

JOHZUKA, Katsuki
WATANABE, Taka-aki
MOROOKA, Naoki
KODAMA, Ken-ichi
ITAZU, Masako
OKAMOTO, Haruko
MIKAMI, Yuriko

The genomes of higher organisms contain significant
amounts of repetitive sequences which are, in general,
unstable. At present, neither the physiological function(s) ofrepeated sequence or the mechanisms producing repeatedsequences and controlling instability are fully understood.
To clarify these aspects of genomes, we are pursuing several
themes using Eschericia coli, Saccharomyces cerevisiae andChinese Hamster Ovary (CHO) cells. In 2007 we have madeadvances in understanding a new role for condensin in
maintaining a long repeated structure of rDNA in yeast anda relationship between the association of condensin withchromosomes and transcription on those chromosomes. In
addition, we also have constructed a new system of gene
amplification via DRCR (double rolling circle replication) inyeast by using the Cre-lox site-specific recombination. Fromprevious and present results, we concluded that DRCR is anactual amplification mechanism, at least in budding yeast.
There exists a possibility that DRCR might work for gene
amplification in higher eukaryotes as well.

Ⅰ . Analysis of mechanism maintaining
repeated structure of ribosomal RNA
genes
In most eukaryotic organisms, the rDNAs are clustered inlong tandem repeats on one or a few chromosomes.
Although the total number of these rDNA repeats appears to
be maintained at a level appropriate for each organism,genes with such a repeated structure are in general thought
to be unstable because of a high frequency ofrecombinational events. Thus, it might be expected thatorganisms have developed a system to regulate
recombination within rDNA repeat.
In the yeast Saccharomyces cerevisiae, 200 copies on
average are tandemly arrayed in a central position on thelongest chromosome (XII). Recombinational events within
the rDNA repeats in normal growing yeast cells appear to bemostly mediated by a FOB1-dependent system. FOB1 is thegene required for replication fork blocking activity at the
replication fork barrier (RFB) site, rDNA region-specificrecombination and expansion/contraction of rDNA repeats.
The latter two activities are likely to be triggered by adouble-strand break at the RFB site and a repairing of the
break via gene conversion. Thus, this FOB1-dependent
recombination apparently contributes to the maintenance of
the average copy number of rDNA. However, in Δfob1

cells, the repeats are still maintained without any fluctuationof copy number, thereby suggesting that another, unknown
system acts to prevent contraction of the number of repeats.
In order to understand this putative second system, wecollected a number of mutants in which the copy number ofrDNA decreased drastically under Δfob1 conditions.
Among these, we found condensin defective mutants,suggesting that, in addition to the condensation andseparation of chromosomes in M phase, condensin plays animportant role in maintaining the repeated structure of
rDNA. Each gene encoding condensin subunit is known tobe essential for growth, but here isolated condensin mutation
is not strict but leaky type. Analyzing the double mutantsand examining the specific interaction between condensin
and rDNA region revealed that (1) in the double mutants, thecopy number of rDNA in the mutant dramatically decreased,(2) condensin complex associated with the RFB region in
FOB1-dependent manner, (3) the association between
condensin and RFB was established during S phase and wasmaintained until anaphase, and (4) double mutant showedslow growth which may be caused by a defect in theseparation step of the long rDNA array in anaphase. These
results strongly suggest that FOB1-dependent condensin
association with RFB region is required for efficientsegregation of rDNA repeated region.
Recently we found that RNA polymerase I (PolI) defective
mutation suppresses dramatic reduction of the copy number
of rDNA in the condensin and fob1 double mutant. Because
PolI is an rDNA specific transcription enzyme, ΔpolI
defective mutant is non-viable. But if the defective cells
carry a plasmid, in which rDNA is inserted downstream ofGal-dependent promoter, cells can be viable in the presenceof galactose. Using the ΔpolI mutant, we examined theeffect of PolI enzyme on condensin association with rDNA.
Under fob1 conditions, PolI enzyme seems to force
condensin from transcribing to non-transcribing (IGS)
region. This characteristic change of pattern suggests that
constant PolI transcription through cell cycle prevents
condensin from associating with the transcribing region.
Thus, it is expected that, in the triple (fob1, condensin andPolI) mutant, the partial defective condensin uniformly
associated with rDNA region is responsible for successful
separation in anaphase.
In higher eucaryotes, it is well known that M phasespecific repression of gene expression (mitotic repression)
occurs, though the reason remains unknown. Our results
suggest that the mitotic repression allows condensin toassociate uniformly with whole chromosomes that ensure
their successful condensation and subsequent separation. At
any rate, genetic and molecular analyses of highly specificchromosomal regions like rDNA provide useful data which
help to understand the nature of normal (non-rDNA)
chromosomal regions.

Ⅱ. Mechanism of condensin recruitment onto
RFB site located within the tandem rDNA
repeat in budding yeast.
The primary functions of mitotic chromosome
condensation are to reduce the length of chromosomes so47National Institute for Basic Biology

Evolutionary Biology and Biodiversitythat they avoid truncation of genome during cell divisionand to ensure the proper segregation of sister chromatids.
The compaction ratio of mitotic chromosomes relative todouble stranded DNA fiber ranges from ~160-fold in
budding yeast to ~10000 – 20000-fold in mammalianchromosomes. Condensin is a multi-subunit protein complex
that plays a central role in mitotic chromosome condensationand segregation. In vertebrates, condensin has been shown
to be distributed in axial part over the whole length ofcondensed chromosomes, but only at the resolution of lightmicroscopy. The sites where condensin acts in chromatin
and the molecular mechanisms of condensin recruitment
have largely remained elusive. As described above, we
found that condensin localized at the RFB site in Fob1
dependent manner during S-phase. To date, this Fob1dependent condensin localization is the only example ofcondensin association with a specific DNA site in a specificprotein factor-dependent manner. To understandchromosome condensation in molecular resolution, we are
studying mechanisms of condensin localization at the RFB
site. Firstly, we haven't obtained any positive data indicating
direct interaction between condensin and Fob1 protein fromeither of two-hybrid or immuno-precipitation experiment,
and we therefore excluded the possibility in Figure 1 (1).
Secondly, we discovered that condensin could bind to theshort DNA fragment containing RFB sequences, even if thesequence was inserted in either orientation (fork blocking or
non-blocking orientation) at an ectopic chromosome site,

and we therefore excluded the possibility in Figure 1 (2). To
gain further information about the specific recruitment ofcondensin onto the RFB site, we isolated additional factors
by genetic approach. So far, we have identified at least three
additional factors that were necessary for condensin
recruitment to the RFB site (Figure 1 (3)). Protein-protein
interaction analysis of these three factors in addition to Fob1
and all subunits of condensin complex suggested thatcondensin was targeted at the RFB site by orderedinteractions among them.

Ⅲ. Construction of a new gene amplification
system via DRCR (double rolling circle
replication) by using the Cre-lox site
specific recombination
In addition to rDNA gene amplification in eukaryotes,
there is another type of gene amplification, which is
involved in various biological phenomena, such as cancer
development and drug-resistance. However, the mechanism
is largely unknown because of the complexity of theamplification 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 ofamplification products. Type-1 products contain 13 to ~100
copies of the amplification marker, leu2d (up to ~730 kb
increase) with novel arrangement present as randomly
oriented sequences flanked by inverted leu2d copies. Type-2

Figure 1. How to associate condensin with the specific RFB site in rDNA repeats

48

Publication List
〔Original papers〕● Cui, T., Moro-oka, N., Ohsumi, K., Kodama, K., Ohshima, T.,Ogasawara, N., Mori, H., Wanner, B., Niki, H., and Horiuchi, T.
(2007). E. coli with a linear genome. EMBO Rep. 8, 181-187.
● Ganley, A.R., and Kobayashi, T. (2007). Highly efficient concerted
evolution in the ribosomal DNA repeats: total rDNA repeat variation
revealed by whole-genome shotgun sequence data. Genome Research
17, 184-191.
● Johzuka, K., and Horiuchi, T. (2007). RNA polymerase I transcription
obstructs condensin association with 35S rRNA coding region and cancause contraction of long repeat in Saccharomyces cerevisiae. Genes
Cells 12, 759-771.

Figure 2. DRCR induced by Cre-lox system(a) Cre-lox dependent reversal of replication orientation. (a1) When thereplication fork passes through between a pair of lox sites, Cre
recombination occurs between a pair of lox sites, as shown in (a1).
Recombination changes replication orientation from un-replicated DNA(parental DNA strand) to replicated DNA (one of sister chromatids) as
shown in (a2 and a3), because DNA strand a and b are identical. (b)DRCR is induced by Cre-dependent combinational recombination of two
pairs of loxP and loxm2 as shown in (b). Recombination cannot occur
between loxP and loxm2.

products are acentric multi-copy mini-chromosomescarrying leu2d. Structures of type-1 and -2 products
resemble those of homogeneously staining region (HSR) anddouble minutes (DMs) of higher eukaryotes, respectively.
Interestingly, products analogous to these were generated at
low frequency without deliberate DNA cleavage (EMBO J
24, 190-198 (2005)).
If DRCR is an actual gene amplification mechanism inyeast, a quite different initiation reaction, which can induce
RDCR, should produce amplification products resembling to
HSR and DMs. Thus, we tried to construct a new DRCR
amplification system that is induced by another process,Cre-lox site-specific recombination. We first predicted that,
if Cre recombination occurs between the two lox sites, one
present on the replicated and the other on the unreplicatedregions, as shown in Figure 2(a1), the replication fork
should switch the template from the parental (unreplicated)to the sister-chromatid (replicated) DNA strands, as shownin Figure 2(a2) and (a3), and that the Cre recombination
system would make this process efficient, as shown in
Figure 2(a). Furthermore, a combination of the process, as
shown in Figure 2(b), can efficiently induce gene
amplification through DRCR. In actuality, this systemproduced two kinds of products; highly amplified (>100
copies) chromosome products and acentric multi-copy extrachromosomal products. The structures of these products
resemble those of HSR and DMs of higher eukaryotes,
respectively. From previous and present results, weconcluded that DRCR is an actual amplification mechanism
in budding yeast and could be naturally initiated if somestructural requirement could be satisfied.

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

Associate Professor

HASEBE, Mitsuyasu

MURATA, Takashi

Assistant Professors
Technical Staff
NIBB Research Fellow
Postdoctoral Fellows
Graduate Student
Visiting Scientist
Technical Assistants

Secretary

HIWATASHI, Yuji
TANAHASHI, Takako
SUMIKAWA, Naomi
ISHIKAWA, Takaaki
MIYAZAKI, Saori
OKANO, Yosuke
AOYAMA, Tsuyoshi
MORINAGA, Shin-ichi
HASEGAWA, Noriko
ICHIKAWA, Yukina
IKEGUCHI, Hiromi
MASUOKA, Tomoko
TSUZUKI, Yukiko
KOJIMA, Yoko

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, we hope
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 to form two different cells from a single cell
via asymmetric cell division. The first cell division of a
protoplast isolated from the protonemata of the moss
Physcomitrella patens is asymmetric regarding its shape andnature, and gives rise to an apical meristematic cell and adifferentiated non-meristematic cell. A systematic
overexpression screening for genes involved in asymmetriccell division of protoplasts in P. patens was performed. Aftereliminating genes that are not directly involved inasymmetric cell divisions, such as photosynthesis genes, weused 3000 clones as materials for the overexpression
screening. Individual cDNAs were subcloned under aconstitutive promoter and introduced into the protoplasts of
P. patens for transient expression. We observed andcategorized phenotypes of the regenerating protoplasts. We
identified 58 cDNAs whose overexpression caused thedefects in asymmetric cell divisions in two repeatedexperiments. We knocked in a cytrin gene just before thestop codon of each candidate gene and examined the cellular
localization of a fused protein under its native promoter.
Thus far, we have examined 32 of 58 candidates and nine
fused proteins were detected to be specifically localized in
an apical meristematic cell. Further characterization of thesegenes by the overexpression of the genes in protoplasts with
GFP-tubulin or GFP-talin and the loss-of-function

50

experiments using RNAi are now in progress. Functional
analyses of these genes should help us to understand themolecular mechanisms of how plants generate distinct
meristematic cell lineages to build their multicellular bodies.
This work was performed as a collaborative work with Dr.
Tomomichi Fujita (Hokkaido University).

Ⅱ. Evolution from cells to tissues based onmolecular mechanisms of cytokinesis
The cells of land plants and their sister group charophycean
green algae divide by the insertion of cell plates atcytokinesis. This is in contrast to other green algae, in which
the invagination of plasma membrane separates daughter
cells at cytokinesis. The cell plate appears in the middle ofdaughter nuclei, expands centrifugally towards a cell
periphery, and finally fuses to a parental cell wall. Cell wallmaterials are transported to the expanding cell plate with aphragmoplast, which is mainly composed of microtubules. A
centrifugal expansion of the phragmoplast is a driving force
for that of the cell plate, although the molecular mechanism
for the centrifugal expansion of the phragmoplast was achallenge. Based on live imaging of microtubules, we have
hypothesized that the formation of oblique microtubules,
which elongate to the outside of a phragmoplast, drives thecentrifugal expansion. We examined the role of γ-tubulin inphragmoplast expansion, because γ-tubulin binds onto
existing microtubules and nucleates a new microtubule as a
40 degrees branch in interphase cells. We isolated
phragmoplasts from synchronized tobacco BY-2 cells andlabeled with anti-γ-tubulin antibody.γ-tubulin was detected
at the branched points of microtubules in isolated
phragmoplasts (Figure 1). We proposed a hypothesis thatcytosolic γ-tubulin complexes are recruited onto existingphragmoplast microtubules and nucleate new microtubules
as branches, and the branched microtubules drive
phragmoplast expansion. Inhibition of γ-tubulin function
during phragmoplast expansion is in progress. T. Murata
mainly performed this study.

Figure 1. Phragmoplast development in flowering plants. γ-tubulin
detected at a microtubule end in an isolated phragmoplast by electron
microscopy (yellow arrow).

Ⅲ. Evolution of molecular mechanisms inplant development
3-1 Stem cell initiation and maintenance
Postembryonic growth of land plants occurs from themeristem, a localized region that gives rise to all adultstructures. Meristems control the continuous development ofplant organs by balancing the maintenance and proliferationof stem cells and directing their differentiation. Meristem
initiation and maintenance is a fundamental focus of plantdevelopment research. Three lines, exhibiting reporter gene(uidA) expression preferentially in the apical cells, wereisolated from previously established gene- and enhancer-traplines, and identified as encoding kinesin-like protein (API1)
and ubiquitin-like protein (PUBL1), as well as an unknown
protein. Functional analyses of these genes are currently
under investigation, mainly by Y. Hiwatashi. A distortion ofphragmoplast was observed in double disruptants of API1
and its sister gene. This suggests that these kinesin-likeproteins are indispensable for the proper formation ofphragmoplast. On the other hand, double disruption of
PUBL1 and its sister gene, PUBL2, retarded the collapse ofphragmoplast, suggesting that these ubiquitin-like genes
likely regulate the stability of phragmoplast microtubules.
Further analyses are in progress.
Formation of several types of stem cells to produce
different types of differentiated cells is properly regulated
during the development of multicellular organisms.
However, molecular mechanisms for the stem cellcharacterization have been largely unknown. We showedthat AINTEGUMENTA/PLETHORA/BABY BOOM (APB)
orthologs PpAPBs (PpAPB1, 2, 3, and 4) are involved in thestem cell characterization in the moss Physcomitrella patens.
Gametophore stem cells were induced by exogenous
cytokinin in the wild type, while the quadruple disruptants
did not form any gametophore stem cells with exogenous
cytokinin application. These results suggest that the PpAPBs
play a critical role in the characterization of a gametophore
stem cell. Meanwhile, the expression of PpAPBs is regulated
by auxin, not cytokinin. This study was mainly performed
by Tsuyoshi Aoyama and Yuji Hiwatashi.

3-3 Evolution of phytohormone regulation
Phytohormones are important regulators for plantdevelopment and we investigated their evolution, focusing
on polar auxin transport (Fujita et al. in press), cytokinin
synthesis (Sakakibara et al. submitted), and gibberellic acid(GA) signal transduction (Hirano et al. 2007).
The shoot is a repeated structure made up of stems andleaves and is the basic body plan in land plants. Vascularplants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid
generation. It is not clear whether all land plants use similarmolecular mechanisms in shoot development or how thegenetic networks for shoot development evolved. The
control of auxin distribution, especially by polar auxin
transport, is essential for shoot development in flowering
plants. We did not detect polar auxin transport in thegametophytic shoots of several mosses, but did detect it in
the sporophytes of mosses without shoot structure.
Treatment with auxin transport inhibitors resulted inabnormal embryo development, as in flowering plants, but
did not cause any morphological changes in the haploidshoots. We fused the soybean auxin-inducible promoter
GH3 with a GUS reporter gene and used it to indirectly
detect auxin distribution in the moss Physcomitrella patens.
An auxin transport inhibitor NPA did not cause any changes
in the putative distribution of auxin in the haploid shoot.
These results indicate that polar auxin transport is not
involved in haploid shoot development in mosses and thatshoots in vascular plants and mosses are most likely
regulated differently during development (Fujita et al. inpress).

3-2 Function of gametophytic MADS-box genes
Land plants are believed to have evolved from agametophyte-dominant ancestor without a multicellularsporophyte; most genes expressed in the sporophyte were
probably co-opted from those used in the gametophyte
during the evolution of land plants. To analyze the evolution
and diversification of MADS-box genes in land plants, eight
MADS-box genes predominantly expressed in Arabidopsis
thaliana pollen, male gametophyte, were analyzed. Four ofeight genes belonged to MIKC*-type MADS-box genes andquadruple disruptants of these genes were formed bymultiple crossings of four single disruptants. The quadruple
disruptants showed a defect in pollen germination both invivo and in vitro.

Figure 2. Basipetal polar auxin transport was not detected in a
gametophytic shoot (arrow) but in a sporophyte axis (arrow head) of
Dawsonia superba, which is the biggest moss in the world. Scale bar = 3
cm.

3-4 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 was not possible because of the lack ofgenome sequences in basal land plants. We established aninternational consortium for a genome project of the basal
land plant; the moss Physcomitrella patens and its entire

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

genome has been mostly sequenced as a collaborative workwith the international consortium.
We compared the features of P. patens genome to those offlowering plants, from which it is separated by more than
400 million years, and unicellular aquatic algae. This reveals
genomic changes concomitant with the evolutionary
movement to land, including a general increase in genefamily complexity, loss of genes associated with aquatic
environments, acquisition of genes for tolerating terrestrial
stresses, and the development of the auxin and abscisic acidsignaling pathways for coordinating multicellular growthand dehydration response. The P. patens genome provides aresource for phylogenetic inferences about gene function
and for experimental analysis of plant processes through thisplant's unique facility for reverse genetics (Rensing et al. inpress).
To facilitate the contig assembling and the gene annotation,
we performed (1) the EST analyses of several libraries of
cDNAs isolated from different developmental stages, (2) the
construction of full-length cDNA libraries and sequencing intheir full length, (3) the construction of BAC libraries andtheir end-sequencing, (4) 5'-end serial analysis of gene
expression (5' SAGE), and (5) a collection of 3' UTR andsmall 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 construct phylogenetic trees
efficiently with whole genome shotgun sequence data inpublic databases before their assembly. We collected
homologs of approximately 700 Arabidopsis thaliana genes
involved in development, and their phylogenetic analyses
are in progress.

3-5 Functional characterization of polycomb genes in
the moss Physcomitrella patens
Polycomb group (PcG) proteins regulate chromatin
modification and function as a cellular memory system tomaintain the repressed state of developmental genes in bothanimals and plants. PcG genes are involved in phasechanges of Arabidopsis thaliana development, such as
vegetative to reproductive and haploid to diploid transitions.
Bryophytes have dominant haploid generation, whilesporophyte generation is dominant in angiosperms. Thechange of dominant generations was one of mostconspicuous evolutionary aspects of land plants. To
elucidate the molecular mechanisms underlying theevolution in alteration of generations, we characterizedfunctions of PcG genes in P. patens. A. thaliana CLF, MSI1,
EMF2, and FIE homologs were cloned in P. patens. We
inserted a GUS reporter gene at the end of every one of the
PcG genes to investigate the expression patterns. Disruptantsfor each gene were established and their characterization isin progress.

52

Ⅳ. Molecular mechanisms of speciation
Sexual isolation is an important step for speciation,although the molecular mechanisms governing the isolationin plants are mostly unknown. A proper pollen tube
guidance is essential for reproduction in angiosperms, andsexual isolation is often related to the arrest of guidance. In
spite of the long history of studies on the pollen tube
guidance, few guidance factors have been reported becauseof the difficulty of genetic analyses. We focused on receptor
like kinases (RLKs), which function to receive extra cellular
ligands and transmit the signal into a cell. We postulated that
RLKs involved in pollen tube guidance are likely expressedmore abundantly in pollen and/or pollen tube than in othertissue. Gene expression profiles between A. thaliana pollen
and pollen tube were compared with those of other tissue
using microarray. Pollen and Pollen tube expression profiles
were similar to each other and 95 % of expressed genes wereoverlapped within the 4 fold differences. We focused on 45
RLKs predominantly expressed in pollen or germinating
pollen to characterize signaling mechanisms during
fertilization. Characterization of single and double T-DNA
insertion lines are in progress. This work was mainly doneby S. Miyazaki.

Publication List
〔Original papers〕● Hirano, K., Nakajima, M., Asano, K., Nishiyama, T., Sakakibara, H.,

Kojima, M., Katoh, E., Xiang, H., Tanahashi, T., Hasebe, M., Banks,J. A., Ashikari, M., Kitano, H., Ueguchi-Tanaka, M., and Matsuoka,
M. (2007). The GID1-mediated GA perception mechanism isconserved in the lycophyte Selaginella moellendorffii but not in the
bryophyte Physcomitrella patens. Plant Cell 19, 3058-3079.
● Odahara, M., Inouye, T., Fujita, T., Hasebe, M., and Sekine, Y.
(2007). Involvement of mitochondrial-targeted RecA in the repair ofmitochondrial DNA in the moss, Physcomitrella patens. Genes Genet.
Syst. 82, 43-51.
● Tsuji, S., Ueda, K., Nishiyama, T., Hasebe, M., Yoshikawa, S.,
Konagaya, A., Nishiuchi, T., and Yamaguchi, K. (2007). The
chloroplast genome from a lycophyte (microphyllophyte), Selaginella
uncinata, has a unique inversion, transpositions and many gene losses.
J. Plant Res. 120, 281-290.

〔Review articles〕● Murata,

T., and Hasebe, M. (2007). Microtubule-dependent
microtubule nucleation in plant cells. J. Plant Res. 120, 73-78.
● Murata, T., Tanahashi, T., Nishiyama, T., Yamaguchi, K., and Hasebe,M. (2007). How do plants organize microtubules without acentrosome? J. Integr. Pl. Biol. 49, 1455-1463.

DIVISION OF SPECIATION MECHANISM (ADJUNCT)

Professor (Adjunct)

OKADA, Norihiro
Assistant Professor (Adjunct)
NIBB Research Fellow
Postdoctoral Fellow
Technical Assistants

TAKAHASHI, Kazuhiko
SASAKI, Takeshi
MIZOIRI, Shinji
SUZUKI, Tokiko
MIURA, Seiko

During their long evolutionary history, vertebrates acquired
extensive diversity in such areas as morphology, ecology
and behavior. It is believed that many organisms inhabiting
the earth at present are derived from an ancestral species andbecame diversified in the evolutionary process withspeciation. Speciation, therefore, is an important factor ofdiversification. How, then, does speciation occur? Althoughvarious theoretical models have been proposed with respect
to speciation, its mechanism has been difficult to clarify sofar, particularly on a molecular level. The aim of our group’s
research is to propose and clarify the processes andmechanism of the speciation of vertebrates using amolecular approach. To accomplish this aim, we chose theEast African cichlid fishes as the model animals for ourstudy of speciation (Figures 1 and 2).

Ⅰ. Intention of our research focusing on
cichlids in Lake Victoria
Although cichlid fishes are broadly distributed in tropicalregions throughout the world, our group focuses on thespecies that are endemic to three great lakes – Tanganyika,
Malawi, and Victoria - and their drainages in East Africa.
One thousand or more species of cichlid fishes inhabit theselakes. It is believed that Lake Tanganyika was established
12 million years ago, Lake Malawi 2 million years ago, andLake Victoria 12,000 years ago. Thus, it is thought that thecichlids evolved in each lake after the lakes had formed. The
endemic cichlids of Lake Victoria diversified to 700 or more
species from a small number of ancestors during its shorthistory. This means that explosive adaptive radiation of thecichlid species occurred in this lake.
To date, our group has accomplished certain results inmolecular phylogenetic studies of the East African cichlids
and other vertebrate animals; for example, the elucidation of
the evolutionary history of baleen whales. From the resultsof our phylogenetic analysis of Lake Victoria cichlids, using
insertions of retroposons (SINEs: short interspersed
elements) as markers for the elucidation of theirevolutionary history, we have seen that most of theselectively-neutral polymorphic alleles (presence/absence ofretroposons at orthologous sites in the genome) are retained
both within and among the species of this lake, which is to
say that polymorphisms among Lake Victoria cichlids aretrans-specific. Such homogeneous genome within/among thespecies of Victorian cichlids provide us with the following
criterion for the elucidation of the mechanism of speciation:
if we can characterize a certain allele that is uniquely fixed
at a certain locus in natural populations of a certain species,
we can assume that this gene may possibly be related topositive selection, which may be a significant factor
promoting speciation.

Ⅱ. Field research in Lake Victoria

Figure 1. Cichlid fishes in Lake Victoria. These were caught in the fieldby our group.

The lacustrine environment of Lake Victoria is highly
diverse, thanks to area differences such as turbidity, depth,
and bottom type. Depending on such variable habitats,cichlids also show phenotypic diversity adapting torespective habitats. To obtain ecological data and natural
fish samples of Victorian cichlids adapting to various
habitats, our group and Dr. N. Okada’s laboratory (Tokyo
Inst. Tech.) have been conducting field expeditions aroundMwanza Gulf on the southern shore of Lake Victoria since2004 (Figure 3). Thousands of fish individuals have beencollected so far at various ecological habitats, and thenumber of species of these cichlids available for our study
reached approximately 150 (rough estimation). Pictures
were taken of each individual, immediately after collection,
to record their live colors (Figure 2). For the purpose ofgenetic analyses, fin clips from representative individuals
were preserved in ethanol.

Figure 2. Live color of male Pundamilia nyererei

53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Ⅲ. Analysis of candidate genes for elucidationof speciation and diversification
Cichlids’ varied body colorations are one of the examplesof their phenotypic diversity. Cichlids are known to depend
mostly on a visual system when they choose their mating
partner, and such color variations are considered to affectthe female’s choice. Therefore, it could be considered thatthe body colors of males play an important role forrecognition by the visual system of females during thecourse of reproduction. In addition, the visual system ofcichlids must have been affected by environmental
differences in their habitat such as turbidity and depth of thelake water. In a collaborative work with Dr. N. Okada’s
laboratory at the Tokyo Institute of Technology, our groupproposed that the RH1 gene, which is one of the groups of
opsin genes, evolved in parallel with the depth of theirhabitat among cichlid species in Lakes Tanganyika andMalawi. Based on actual research in the field, our groupfocused on the evolution of opsin genes for the visualsystem in several Victorian cichlid species. Lithchormis
rufus and L. rubripinnis (Figure 1A and B) inhabit shallow
water near the shoreline only in Mwanza Gulf (Figure 3,
panel B), and we found geographical clines of nuptial
coloration on the males. We are carrying out an analysis of
the opsin gene family of these species to detect genetic
variations of chromatic vision that seem to be related toadaptation for such specific male colors. Another interesting
issue concerning the evolution of the opsin gene family canbe seen for the species Haplochromis pyrrhocephalus,
which is broadly distributed in the lake (Figure 1C and D).
The light environment for this species is considerably
different among populations. To elucidate the mode ofadaptation of their vision system to such various light
conditions, we are analyzing the six types of their opsin genefamily. A more extensive analysis of the molecular
evolution of opsin genes in Victorian cichlids is in progressin our division.

Publication List
〔Original papers〕● Fujimura, K., and Okada, N. (2007). Development of the embryo,larva and early juvenile of Nile tilapia Oreochromis niloticus (Pisces:
Cichlidae). Developmental staging system. Develop. Growth Differ.
49, 301-324.
● Nikaido, M., Piskurek, O., and Okada, N. (2007). Toothed whale
monophyly reassessed by SINE insertion analysis: The absence of
lineage sorting effects suggests a small population of a common
ancestral species. Mol. Phylogenet. Evol. 43, 216-224.

54

Figure 3. Localities of collection of cichlids in Lake Victoria since 2004.
Panel A: Sampling localities in southern shore of Lake Victoria. The
area surrounded by a brown rectangle in this panel corresponds to theregion shown as a magnified map of panel B. Panel B: Sampling
localities in Mwanza gulf. Sampling was conducted by angling andtrawling and by using gill nets. Sampling was conducted in collaboration
with Tanzania Fisheries Research Institute (TAFIRI).

LABORATORY OF MORRHODIVERSITY

Associate Professor

KODAMA, Ryuji

The aim of this laboratory is to observe the variety of themorphogenetic 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.

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. Our institute’s Divisionof Sex Differentiation and the Laboratory of
Neurophysiology are both involved in these activities.

Ⅰ. Wing worphogenesis
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 to one day period in Pieris rapae and inseveral other species examined. It was shown that the dying
cells in the degeneration re-gion have characteristics incommon with the apop-totic 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 thebasal 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.
Trachea and tracheoles are both important in delivering air
into the wing and their patterns coincide with that of theboundary of degeneration and differentiation zones at the
distal end of the wing. According to previous observations,the pattern formation of wing epithelium is often dependent
on tracheal and tracheole patterns. Basic research on thedevelopment of tracheal pattern formation is being done byscanning and transmission electron microscopy (Figure 1) to
describe the exact pathway and time course of the formationof the elaborate pattern of trachea and tracheoles and toestablish the cytological and developmental relationshipbetween the formation of the tracheal pattern and theepithelial cell pattern.

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.

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIORESOURCES

Associate Professor

NARUSE, Kiyoshi
Postdoctoral Fellow
Technical Assistants

Secretary

SHEN, Xueyan
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
SUZUKI, Tokiko

Teleosts comprise about half of all vertebrate species andadapt to a variety of environments, including sea-water,
fresh water, the bottom of deep seas, and small creeks andpaddy fields. Analysis of their genome structure is importantto understand the adaptation and diversification in thisinteresting group. Medaka is a small egg-laying “secondary”
fresh water fish found in brooks and rice paddies in Eastern
Asia. This species has a long history as an experimental
animal, especially in Japan. Our laboratory has conducted acomparative genomic analysis focusing mainly on fish
chromosomes and gene evolution using medaka and otherfishes. In addition to these activities, our laboratory is
stepping ahead to lead the National BioResource Project
Medaka (NBRP Medaka).

Ⅰ . Medaka genome sequence and thevertebrate genome evolution
We have successfully completed the genome sequencing of
Hd-rR (Southern inbred strain) at the draft level. Total
nucleotide length of the generated scaffblds is 700.4Mbp.
Comparative analysis with the genome of the northern
inbred line (HNI) revealed 16.4 million single nucleotide
polymorphisms (SNPS) which allowed us to develop a highdensity SNP map that could be successfully used foraligning generated scaffblds. The genome–wide SNP rate
between Hd-rR and HNI inbred strains is 3.42 % which is, toour knowledge, the highest SNP rate seen in any vertebrate
species. We also identified 20,141 non-redundant genes with
5’-end serial analysis of gene expression (SAGE) tags
combining with Genscan. Using this medaka gene model,
we conducted four-way comparisons of the synteny oforthologous genes among the human and Tetraodon
genomes and the zebrafish gene map. Most of the medaka
and Tetraodon chromosomes have a one-by-one relationshipand about half of the medaka and zebrafish chromosomes
also correspond to a one-by-one relationship, even though
the lineage separation of these two species dates back morethan 191 million years. These results showed that the interchromosomal arrangements in the teleost lineage were lessfrequent than those of mammals whose lineage separation
dates back to 100 million years ago. We also successfullyreconstructed the pre-duplicated proto-chromosomes of theancestor of all ray-finned fish and described the scenario
leading to the present genome structure of medaka, zebrafish
and Tetraodon.

Ⅱ. 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 another sex-determining gene. In fact, it
has been demonstrated that the Y chromosome in O.
dancena differs from that in O. latipes, whereas both species
have an XX/XY sex determination system. Through aprogeny test of sex-reversed fish and a linkage analysis of
isolated sex-linked DNA markers, we showed that O.
hubbsi, which is one of the species most closely related to O.
dancena, has a ZZ/ZW system.

Figure 1. Reconstruction of the pre-duplicated proto-chromosome and scenario leading to the medaka, Tetraodon and zebrafish genome

56

Publication List
〔Original papers〕● Hojo, M., Takashima, S., Kobayashi, D., Sumeragi, A., Shimada, A.,

Figure 2. Phylogenetic relationships and sex determination mechanisms
in Oryzias fishes

Ⅲ. National BioResource Project Medaka(NBRP Medaka)
In 2007, the NIBB was selected as the core facility of
NBRP medaka. Our laboratory is taking an active part ofthis project. We provide, maintain and collect living
resources such as standard strains, inbred strains, mutants,
and frozen resources such as EST/cDNA and BAC/ Fosmid
clones, as well as the integrated information on medaka withthe goal of facilitating and enhancing the use of medaka as amodel organism. NBRP Medaka is aiming to establish a firstrate biological resource with the highest possible levels ofaccessibility and ease of use.

Tsukahara, T., Yokoi, H., Narita, T., Jindo, T., Kage, T., et al. (2007).
Right-elevated expression of charon is regulated by fluid flow in
medaka Kupffer's vesicle. Develop. Growth Differ. 49, 395-405.
● Kasahara, M., Naruse, K., Sasaki, S., Nakatani, Y., Qu, W., Ahsan, B.,
Yamada, T., Nagayasu, Y., Doi, K., Kasai, Y., et al. (2007). The
medaka draft genome and insights into vertebrate genome evolution.
Nature 447, 714-719.
● Kawaguchi, M., Yasumasu, S., Hiroi, J., Naruse, K., Suzuki, T., andIuchi, I. (2007). Analysis of the exon-intron structures of fish,
amphibian, bird and mammalian hatching enzyme genes, with specialreference to the intron loss evolution of hatching enzyme genes in
Teleostei. Gene 392, 77-88.
● Takashima, S., Shimada, A., Kobayashi, D., Yokoi, H., Narita, T.,
Jindo, T., Kage, T., Kitagawa, T., Kimura, T., Sekimizu, K., et al.
(2007). Phenotypic analysis of a novel chordin mutant in medaka.
Dev. Dyn. 236, 2298-2310.
● Takehana, Y., Demiyah, D., Naruse, K., Hamaguchi, S., andSakaizumi, M. (2007). Evolution of different Y chromosomes in two
medaka species, Oryzias dancena and O. latipes. Genetics 175, 13351340.
● Takehana, Y., Naruse, K., Hamaguchi, S., and Sakaizumi, M. (2007).
Evolution of ZZ/ZW and XX/XY sex-determination systems in theclosely related medaka species, Oryzias hubbsi and O. dancena.
Chromosoma 116, 463-470.
● Tanaka, K., Takehana, Y., Naruse, K., Hamaguchi, S., and Sakaizumi,
M. (2007). Evidence for different origins of sex chromosomes inclosely related Oryzias fishes: substitution of the master sexdetermining gene. Genetics 177, 2075-2081.
● Yokoi, H., Shimada, A., Carl, M., Takashima, S., Kobayashi, D.,
Narita, T., Jindo, T., Kimura, T., Kitagawa, T., Kage, T., et al. (2007).
Mutant analyses reveal different functions of fgfr1 in medaka andzebrafish despite conserved ligand-receptor relationships. Develop.
Biol. 304, 326-337.

Figure 3. Medaka provided from NBRP Medaka

57

National Institute for Basic Biology

Environmental Biology

DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY

Professor

Associate Professor

IGUCHI, Taisen

WATANABE, Hajime

Assistant Professor
Technical Staff
NIBB Research Fellow
Postdoctoral Fellow
Graduate Students
Visiting Scientists
Technical Assistants

Secretary

KATSU, Yoshinao
MIZUTANI, Takeshi
KATO, Yasuhiko
URUSHITANI, Hiroshi
NAKAMURA, Takeshi
KIKUCHI, Naoka
LANGE, Anke
TANIGUCHI, Ena
KOBAYASHI, Kaoru
TAKAHASHI, Eri
HINAGO, Megumi
TANEMURA, Hiroyo
IMAIZUMI, Taeko

Synthetic chemicals found in the environment have thecapacity to disrupt the development and function of theendocrine system in both wildlife and humans. This has
drawn public concern since many of these chemicals maybind to estrogen receptors (ERs) and evoke estrogenic
effects. Early evidence that exposure to estrogenic chemicals
during development could pose a threat to human healthcame from studies of a synthetic hormone, diethylstilbestrol(DES), which was used to prevent premature birth andspontaneous abortion. Laboratory experiments showed that
exposure of animals to sex hormones during critical
windows of perinatal life caused irreversible alterations to
the endocrine 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. 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 humanand animal health. In 1971, prenatal diethylstilbestrol (DES)
exposure was found to result in various abnormalities of the
reproductive tract in women and this syndrome was named
the DES syndrome. Similar abnormalities have been
demonstrated in experimental animals exposed perinatally to
estrogens: for example, developmental estrogen exposure inmice induces persistent proliferation of vaginal epithelial
cells. We found that the persistent changes in the vagina inmice exposed neonatally to estrogens result from thepersistent phosphorylation of erbB2 and estrogen receptorα(ERα), sustained expression of EGF-like growth factors
and phosphorylation of JNK1, IGF-I receptor and Akt.
Recently, we found that Wnt 4, notch1, notch 3, and othergenes also show persistent expression changes in neonatally
DES-exposed mouse vaginae. Currently, we are analyzing
the methylation status of genes showing altered expression
in the mouse vagina. The number of DES-induced genes in
the mouse vagina during the critical developmental exposure
window was smaller than after the critical period.

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 intissues of wild-type mice using a microarray approach. For
most of the identified genes, expression was induced by 17
β-estradiol (E2) in a dose-dependent manner. Subsequently,
several environmental (xeno)estrogens were tested andcharacteristic gene expression patterns were observed foreach compound tested; these patterns were distinct from thatobtained following E 2 exposure. We also found thatxenoestrogenic chemicals and dioxin have distinct effects on
the liver as well. Therefore, possible tissue-specific effects
should be considered when elucidating the distinct effects ofvarious EDCs.

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 that primitive vertebrates, such as the Agnatha, havea functional ER.

Ⅳ. Male production in Daphnids by juvenile
hormones

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

Ⅲ. Steroid hormone receptors of 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,roach, stickleback, mosquitofish, mangrove Rivulus,
Japanese giant salamander, newt, Silurana tropicalis,

Daphnia magna has been used extensively to evaluate theorganism- and population-based responses of invertebrates
to pollutants by their inclusion in acute toxicity or
reproductive toxicity tests. These tests, however, provide no
information about the mode of action of the tested
compounds. Therefore, we applied an ecotoxicogenomic
assessment of D. magna. We established a Daphnia EST
database and developed an oligonucleotide-based DNA
microarray with high reproducibility. Exposure of D. magna
to several chemicals resulted in characteristic gene
expression patterns that are chemical-specific, indicatingthat the established DNA microarray can be used for theclassification 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 suboptimal, they alter their reproductive strategy from asexual
to sexual reproduction. Chemicals are able to affect 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 theseorganisms. 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.
Recently, we have developed a method to inject silencing
genes into D. magna embryos which will allow us to study
gene function in more detail in this species.

Ⅴ. Gene zoo
We are establishing cDNA library banks of animal species
in collaboration with the University of Pretoria, South
Africa, the University of Florida, USA, San Diego Zoo,
USA, and the Asa Zoo in Hiroshima.

Ⅵ. Molecular target search

Figure. 4 Evolutionary relationships of estrogen receptor sequences

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 noanomolar affinity ligand forboth the retinoid ‘X’ receptor (RXR) and the peroxisome
proliferators activated receptor γ (PPARγ). TBT promotes
adipogenesis in the murine 3T3-L1 cell model and perturbs

59National Institute for Basic Biology

Environmental Biology

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 PPARγ ligand exposure. TBT
represents the first example of an environmental EDC that
promotes adipogenesis through RXR and PPARγ
activation. Developmental or chronic lifetime exposure toorgaotins may therefore act as a chemical stressor forobesity and related disorders.

Publication List
〔Original papers〕● Chojnowski, J.L., Franklin, J., Katsu, Y., Iguchi, T., Guillette, L.J.Jr.,

Kimball, R.T., and Braun, E.L. (2007). Patterns of vertebrate isochore
evolution revealed by comparison of expressed mammalian, avian andcrocodilian genes. J. Mol. Evolution 65, 259-266.
● Hara, A., Hirano, K., Shimizu, M., Fukada, H., Fujita, T., Itoh, F.,
Takada, H., Nakamura, M., and Iguchi, T. (2007). Carp (Cyprinus
carpio) vitellogenin: characterization of yolk proteins, development of
immunoassays and use as a biomarker of exposure to environmental
estrogens. Environ. Sci. 14, 95-108.
● Iguchi, T., Katsu, Y., Horiguchi, T., Watanabe, H., Blumberg, B., andOhta, Y. (2007). Endocrine disrupting organotin compounds arepotent inducers of imposex in gastropods and adipogenesis invertebrates. Mol. Cell. Toxicol. 3, 1-10.
● Kato, Y., Kobayashi, K., Oda, S., Tatarazako, N., Watanabe, H., andIguchi, T. (2007). Cloning and characterization of the ecdysone
receptor and ultraspiracle protein from the water flea Daphnia magna.
J. Endocrinol. 193, 183-194.
● Katsu, Y., Hinago, M., Sone, K., Guillette, L.J.Jr., and Iguchi, T.
(2007). Analysis of the ligand specificity of the estrogen and androgen
receptors of mosquitofish, Gambusia affinis affinis. Mol. Cell. Endocr.
276, 10-17.
● Katsu, Y., Lange, A., Ichikawa, R., Urushitani, H., Paull, G.C., Cahill,
L.L., Jobling, S., Tyler, C.R., and Iguchi, T. (2007). Functional
associations between two estrogen receptors, environmental estrogenand sexual disruption in the roach (Rutilus rutilus). Environ. Sci.
Technol. 41, 3360-3374.
● Kobayashi, T., Iguchi, T., and Ohta, Y. (2007). A betalipoproteinemia
induced by ORP150 over-expression in mice. Comp. Med. 57, 247254.
● Oda, S., Tatarazako, N., Dorgerloh, M., Johnson, R., Kusk, O.,
Leverett, D., Marchini, S., Nakari, T., Williams, T., and Iguchi, T.
(2007). Strain difference in sensitivity to 3,4-dichloroaniline andinsect growth regulator, fenoxycarb, in Daphnia magna. Ecotoxicol.
Environ. Safety 67, 399-405.
● Sumi, M., Kawashima, Y., Fukumaki, T., Ishibashi, H., Arizono, K.,
Iguchi, T., and Shimizu, M. (2007). Comparison of serum
vitellogenin, steroid hormone, gonad histopathology andbioaccumulation in common carp (Cyprinus carpio) between tworivers and a lake in Japan: Potential for endocrine disruption. Environ.
Sci. 14, 41-54.
● Suzuki, A., Urushitani, H., Sato, T., Watanabe, H., Ohta, Y., andIguchi, T. (2007). Gene expression change in the Müllerian duct of themouse fetus exposed to diethylstilbestrol in utero. Exp. Biol. Med.
232, 503-514.
● Suzuki, A., Urushitani, H., Watanabe, H., Sato, T., Iguchi, T.,
Kobayashi, T., and Ohta, Y. (2007). Comparison of estrogen
responsive genes in the mouse uterus, vagina and mammary gland. J.
Vet. Med. Sci. 69, 725-731.

60● Takase, M., and Iguchi, T. (2007). Molecular cloning of two isoformsof Xenopus (Silurana) tropicalis estrogen receptor mRNA and theirexpression during development. Biophys. Biochem. Acta 1769, 172181.
● Watanabe, H., Takahashi, E., Nakamura, Y., Oda, S., Tatarazako, N.,
and Iguchi, T. (2007). Development of Daphnia magna DNA
microarray for the evaluation of toxicity of environmental chemicals.
Environ. Toxicol. Chem. 26, 669-676.

〔Review articles〕● Cook, J., Iguchi, T., Linney, E., Miracle, A., Shaw, J., Viant, M., andZacharewski, T. (2007). “Omic” Approaches in the context ofenvironmental toxicology. In Cross-Species Extrapolation in
Toxicology, W.H. Benson, and R.T. Di Giulio, eds. (Taylor andFrancis, CRC Press), pp. 1-31.
● Crain, D.A., Eriksen, M., Iguchi, T., Jobling, S., Laufer, H., LeBlanc,
G.A., and Guillette, L.J.Jr. (2007). An ecological assessment ofbisphenol-A: Evidence from comparative biology. Reprod. Toxicol.
24, 225-239.
● Iguchi, T. (2007). What are the data on environmental contaminants
disrupting reproductive function? Examples of mosquitofish, roachand medaka. In Advances in Fertility Studies and Reproductive
Medicine, T.F. Kruger, Z. van der Spuy, and R.D. Kempers, eds. pp.
361-373.
● Iguchi, T., Katsu, Y., Urushitani, H., Lange, A., and Tyler, C.R.
(2007). Developmental reproductive of exposure to pharmaceuticalsteroids in the aquatic environment: Studies on mosquitofish(Gambusia affinis affinis), roach (Rutilus rutilus) and medaka (Oryzias
latipes). J. Marine Sci. Technol. 15, 29-36.
● Iguchi, T., Watanabe, H., and Katsu, Y. (2007). Toxicogenomics andecotoxicogenomics for studying endocrine disruption and basic
biology in vertebrates and invertebrates. Gen. Comp. Endocrinol. 153,
25-29.
● vom Saal, F.S., Akingbemi, B.T., Belcher, S.M., Birnbaum, L.S.,
Crain, D.A., Eriksen, M., Guillette, L.J., Hauser, R., Heindel, J.J., Ho,
S.-K., Iguchi, T., Jobling, S., Kanno, J., Keri, R.A., Knudsen, K.E.,
LeBlanc, G.A., Marcus, M., McLachlan, J.A., Myers, J.P., Nadal, A.,
Newbold, R.R., Olea, N., Prins, G.S., Richter, C.A., Rubin, B.S.,
Sonnenschein, C., Soto, A.M., Talsness, C.E., Vandernbergh, J.G.,
Vandenberg, L.N., Walser-Kuntz, D.R., Watson, C.S., Welshons,W.V., Wetherill, Y., and Zoeller, R.T. (2007). Capel Hill bisphenol A
expert panel consensus statement: Integration of mechanisms, effects
in animals and potential to impact human health at current levels of
exposure. Reprod. Toxocol. 24, 131-138.
● Takase, M., Oka, T., Tooi, O., Santoh, N., Pickford, D., and Iguchi, T.
(2007). Development of biomarkers for endocrine disrupting activity
in the emerging amphibian model, Silurana (Xenopus) tropicalis.
Environ. Sci. 14, 285-296.
● Tyler, C.R., Lange, A., Paull, G.C., Katsu, Y., and Iguchi, T. (2007).
The roach (Rutilus rutilus) as a sentinel for assessing endocrine
disruption. Environ. Sci. 14, 235-253.
● Urushitani, H., Katsu, Y., Kato, Y., Tooi, O., Santo, N., Kawashima,Y., Kisaka, Y., Ohta, Y., Lange, A., Tyler, C.R., Johnson, R.D., andIguchi, T. (2007). Medaka (Oryzias latipes) for use in evaluating
developmental effects of endocrine active chemicals with specialreference to gonadal intersex (testis-ova). Environ. Sci. 14, 211-233.

DIVISION OF PLANT DEVELOPMENTAL GENETICS (ADJUNCT)

Professor (Adjunct)

TSUKAYA, Hirokazu
Assistant Professor
Postdoctoral Fellows

Visiting Scientists

Technical Assistants

Secretary

YAMAGUCHI, Takahiro
ISHIKAWA, Naoko
USAMI, Takeshi
YANO, Satoshi
ICHIHASHI, Yasunori
NAKAYAMA, Hokuto
TAKASE, Masahide
YAMAGUCHI, Chinami
NAGURA, Masako
KADOWAKI, Tamaka
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 2006a).

Ⅰ. Mechanisms of leaf development
Focusing on the mechanisms that govern polarized growthof leaves in Arabidopsis thaliana, we have identified fourgenes 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 (reviewed in Tsukaya
2006a). In addition to the polar-dependent leaf shapecontrol, we have focused on the mechanisms of organ-wide
control of leaf size, which are reflected in the‘compensation’ phenomenon(Tsukaya 2006a).
Furthermore, we have isolated and analyzed mutants withleaf polarity defects (drl: Cho et al. 2007) and leaf epidermis
formation defects (ale2: Tanaka et al. 2007). Additionally,
the accumulation of knowledge on the basic mechanisms ofleaf shape control have enabled us to conduct Evo/Devo
studies of the mechanisms behind leaf-shape diversity.
Below is an overview of our research activities andachievements during 2007.

1-1 Polar growth of leaves in A. thaliana
AN is a member of CtBP-BARS gene family reported fromanimal genomes (reviewed in Tsukaya 2006b). Gene
products of this gene family are very curious: they act as corepressors in nuclei as a form of CtBP and also as Golgi
regulators as BARS. Is AN a functional homolog of CtBP orof BARS? Our swapping experiments and two-hybrid
analysis clearly showed that AN does not have any of themolecular functions of CtBP in Drosophila melanogaster(Stern et al. 2007). On the other hand, our detailed analysis
of intracellular localization suggested that AN have a uniquerole (or roles) in Golgi-related function. Further analyses ofAN functions are ongoing.

1-2 Evolution of establishment mechanisms of leaf
polarities in monocots
We have recently started to try to understand the genetic
basis of the development of unifacial leaves that are known
only from monocot clades. Our preliminary analyses
suggested that the unifacial character might be due to overallchanges in all polarities around leaves (i.e. adaxial-abaxial,
distal-proximal, and central-lateral polarities). Moreover,genetic controls of leaf polarities were revealed to differ, atleast in part, between eudicot and rice, a monocot species.
Understanding the differences in the genetic mechanisms forthe establishment of unifacial and normal bifacial leaves will
provide good clues as to how leaf shape is diversified. For
such purposes, comparative molecular-genetic andanatomical analyses between unifacial and bifacial leaf
development have been recently undertaken using membersof the genus Juncus.

1-3 Size control of leaves and mechanisms ofcompensation
How are cell proliferation and cell enlargement coordinatedin leaf morphogenesis? In a determinate organ - a leaf - thenumber of leaf cells is not necessarily reflected in leaf shapeor, in particular, in leaf size. Genetic analyses of leaf
development in A. thaliana show that a compensatory
system (or systems) acts in leaf morphogenesis and anincrease in cell volume might be triggered by a decrease incell number (Tsukaya 2006a). 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 specificmutants for number or size of leaf cells (Fujikura et al.
2007a, b). Moreover, we have isolated a number of new
mutants that exhibit typical compensation syndrome,
namely, decreased number of cells and increased cellvolume: fugu (Ferjani et al. 2007). Combining these mutants
as a resource, we have revealed that: (1) compensation
syndrome results from the enhancement of a particular set ofcell-expansion pathways which is required for normal leaf
expansion; (2) compensation does not depend on theenhancement of endoredupliation; (3) compensation
involves at least three different expansion pathways; and (4)compensation is not caused by the uncoupling of cell
division from cell expansion at all. Further analyses on themechanisms of compensation are in progress.

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Environmental Biology

1-4 Size control of leaves and ploidy level
Why does a high-ploidy level cause increased cell/leaf
size? The isolation of mutants with endoreduplication
defects supplied us with a good clue for understanding thelinkage mechanisms between the ploidy level and cell/organsize. bin4 mutants cannot make their leaf cells higher than
8C and show severe dwarfism. If this dwarfism - and smaller
leaves - are the direct result of lower ploidy level,
tetraploidization of this mutant should suppress thedwarfism. We found that the tetraploid bin4 showed partial
recovery in cell/organ size, and this fact showed that ploidy
level is a key factor for the determination of cell/organ size
(Breuer et al. 2007). In addition, we found that theendoreduplication mutational defects were responsible for acurious enhancement of the effects of tetraploidization interms of cell-size increase. Further studies of tetraploids ofthe other mutants are now ongoing to determine the precise
relationship between the ploidy level and cell/organ size.

Figure 1. Severe dwarfism exhibited by the bin4 mutant is partially
recovered by tetraploidization. Arrows: homozygous individuals of themutations. Bar, 1 cm.

Ⅱ. Biodiversity of leaf form
We are also interested in the biodiversity of wild plants.
Ainsliaea apiculata is a perennial weed that is widelydistributed in Japan. Some endemic varieties of this species,
which are defined by characteristic leaf shapes, have beenreported from Yakushima Island, Kyushu, Japan. We havere-examined the variations in the leaf shape of this species
from Yakushima Island, in comparison with other localities
in Japan, and found that the variations in the leaf shape were
continuous with the other forms. Thus, these variants cannot
be treated as distinct varieties. Yakushima Island wasproved, however, to be a center locality of morphologicalvariation in this species (Tsukaya et al. 2007a).
On the other hand, we have revealed that Cayratia
tenuifolia, characterized by its leaf shape and flower/fruit
characters, is a distinct species from the closely-related C.
japonica (Okada et al. 2007). Cytological and molecularphylogenetic analyses of Oxygyne shinzatoi were also
carried out (Tsukaya et al. 2007b; Yokoyama et al. 2008).

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Publication List
〔Original papers〕● Breuer, C., Stacey, N.J., Roberts, G., West, C.E., Zhao, Y., Chory, J.,

Tsukaya, H., Azumi, Y., Maxwell, A., Roberts, K., and SugimtoShirasu, K. (2007). BIN4, a novel component of the plant DNA
topoisomerase VI complex, is required for endoreduplication in
Arabidopsis. Plant Cell 19, 3655-3668.
● Cho, K.-H., Choi, H., Seki, M., Jun, S.E., Yi, Y.B., Shinozaki, K.,
Tsukaya, H., and Kim, G.-T. (2007). DRL regulates adaxial leaf
patterning and shooot apical meristem activity in Arabidopsis. J. Plant
Biol. 50, 467-474.
● Ferjani, A., Satoshi, Y., Horiguchi, G., and Tsukaya, H. (2007).
Analysis of leaf development in fugu mutants of Arabidopsis thaliana
reveals three compensation modes that modulate cell expansion in
determinate organs. Plant Physiol. 144, 988-999.
● Fujikura, U., Horiguchi, G., and Tsukaya, H. (2007a). Dissection ofenhanced cell expansion processes in leaves triggered by defect in cellproliferation, with reference to roles of endoreduplication. Plant Cell
Physiol. 48, 278-286.
● Fujikura, U., Horiguchi, G., and Tsukaya, H. (2007b). Genetic
relationship between angustifolia3 and extra-small sisters highlights
novel mechanisms controlling leaf size. Plant Signaling Behavior 2,
378-380.
● Okada, H., Tsukaya, H., and Okamoto, M. (2007). Taxonomic Notes
on Cayratia tenuifolia (Wight & Arn.) Gagnep., Vitaceae. Acta
Phytotax. Geobot. 58, 51-55.
● Stern, M.D., Aihara, H., Cho, K.-H., Kim, G.-T., Horiguchi, G.,
Roccaro, G.A., Guevara, E., Sun, J., Negeri, D., Tsukaya, H., andNibu, Y. (2007). Structurally related Arabidopsis ANGUSTIFOLIA isfunctionally distinct from the transcriptional corepressor CtBP. Dev.
Genes Evol. 217, 759-769.
● Tanaka, H., Watanabe, M., Sasabe, M., Horoe, T., Tanaka, T.,
Tsukaya, H., Ikezaki, M., Machida, C., and Machida, Y. (2007).
Novel receptor-like kinase ALE2 controls shoot development byspecifying epidermis in Arabidopsis. Development 134, 1643-1652.
● Tsukaya, H., Tsujino, R., Ikeuchi, M., Isshiki, Y., Kono, M., Takeuchi,T., and Araki, T. (2007a). Morphological variation in leaf shape in
Ainsliaea apiculata with special reference to the endemic characters of
populations on Yakushima Island, Japan. J. Plant Res. 120, 351-358.
● Tsukaya, H., Yokota, M., and Okada, H. (2007b). Chromosomal
characteristics of Oxygyne shinzatoi (Hatus.) C. Abe et Akasawa(Burmanniaceae), and its phylogenetic significance. Acta Phytotax.
Geobot. 58, 47-53.
● Yamaguchi, T., and Tsukaya, H. (2007). Evo-Devo of leaf shapecontrol with a special emphasis on unifacial leaves in monocots.
Korean J. Pl. Taxon. 37, 351-361.

DIVISION OF PHOTOBIOLOGY (ADJUNCT)

Professor

Associate Professor (Adjunct)

WADA, Masamitsu

YAMAUCHI, Daisuke

Postdoctoral Fellows

Visiting Scientists
Technical Assistants

Secretary

KAGAWA, Takatoshi
KODAMA,Yutaka
KONG, Sam-Geun
SUETSUGU, Noriyuki
YAMASATO, Akihiro
SUETSUGU, Noriyuki
TSUBOI, Hidenori
CHUNG, Hee-Jung
HIROSE, Mana
IWATA, Ayumi
SHIMIZU, Mineko
TAKAMATSU, Kaori

Plants respond to light as an environmental factor to
optimize growth and development and to regulate otherphysiological phenomena. Phytochrome (phy) and blue light
receptors, such as cryptochrome (cry) and phototropin(phot), are the main photoreceptors for plant
photomorphogenesis. The goal of our research is to elucidate
the photoperception and signal transduction pathways ofphotomorphogenesis. One of our major subjects is
chloroplast photo-relocation movement, which is mediatedby phototropins and one of the simplest model systems tostudy photomorphogenesis. Because the phenomenon is cellautonomous, whole processes from photoperception tochloroplast movement can be accomplished in a single cellwithout any influence from surrounding neighbor cells.
Moreover, gene expression is not involved in the signaltransduction pathways, unlike in those of phy- and crymediated phenomena. Chloroplast movement is not realplant morphogenesis, but we are studying it because
chloroplast movement and photomorphobgenesis share thesame photoreceprors.

of signal transfer using Adiantum gametophytes to find aclue to the identity of any possible candidates for the signal.
The velocity in a long protonemal cell is confirmed to bedifferent between the signal moving from the base to the tip(approximately 2.3 μm min-1) and from the tip to the base(approximately 0.6 μm min-1) of the protonemata both in the
red and blue light-induced chloroplast movement. On theother hand, the velocity of signal transfer in twodimensional gametophyte cells is the same (approximately
0.7 μm min-1) irrespective of the direction of signal transfer
or the wavelength (either red or blue light) that induces
chloroplast movement. The velocities are slower than that ofcytoplasmic streaming. The velocity of the signal transfer
was very slow compared to our expectations, and we do nothave any clear idea at the moment what is the signal andwhat is the mechanism of the transfer.

1-2 Chloroplast movement under cold condition
Chloroplasts movement was induced at about 4°C in
Adiantum gametophytes. At low temperatures chloroplasts
move to the anticlinal walls under either dark or light
conditions. The position is similar to those of chloroplasts
found in the dark or under strong light. Although the
movement was even induced under darkness, it is not darkinduced movement, because the response could be inducedin the mutant gametophytes defective of dark-induced
chloroplast movement.

Ⅱ. Photoreceptor functions
2-1 Photoreceptors mediating nuclear movement in
the fern Adiantum
In gametophyte cells of the fern Adiantum capillusveneris, nuclei as well as chloroplasts change their position
according to light conditions (Kagawa and Wada 1993,
1995). Nuclei reside on anticlinal walls in darkness andmove to periclinal or anticlinal walls under weak or strong
light conditions, respectively (Figure 1).

Ⅰ. Chloroplast relocation movement
We use the fern Adiantum capillus-veneris as a model plantfor our cell biological and physiological approach tochloroplast movement since the gametophytes are very
sensitive to light and the organization of the cells is very
simple. We also use Arabidopsis mutants as well as wild
type plants to identify the genes regulating chloroplast
movement and for analyses of the genes’ functions.

1-1 Velocity of signal transfer
Phototropins (phot1and phot2) and a neochrome (which isa chimera photoreceptor of phytochrome chromophorebinding domain and phototropin) were identified as
photoreceptors for chloroplast movement (Kagawa et al.
2001, Kawai et al. 2003, Kagawa et al. 2004); however, asignal transferred from photoreceptors to chloroplasts
remained to be clarified. We therefore studied the velocity

Figure 1. The left half of an Adiantum gametophyte cultured under weak
white light was irradiated with strong white light to induce nuclear
avoidance response and then fixed with glutaraldehyde and stained with
DAPI. Nuclei irradiated with strong light moved to the anticlinal walls
from the center of cells. The response was mediated by a blue light
receptor, phototropin2 (Tsuboi et al. 2007).

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Environmental Biology

This year we tried to identify the photoreceptor(s) thatmediates nuclear movement in the gametophyte cells using
photoreceptor mutants, neo1 (neochrome1 defective), phot2(phototropin2 defective), and neo1phot2 (Figure 2) andrevealed that red and blue light-induced nuclear
accumulation movement is mediated by neo1, and possibly
phot1 and phot2, respectively, and that blue light-induced
avoidance movement is mediated by phot2 (Tsuboi et al.
2007).

2-3 Binding proteins to a putative blue light receptor
in Arabidopsis
PAS/LOV protein (PLP) is a putative blue light receptor
with a PAS domain at its N-terminal region and an LOV
domain at its C-terminal region (Crosson et al. 2003). PLP
interacting proteins were isolated by the yeast two-hybrid
system and were studied in collaboration with Dr. Kiyosue
at Kagawa University. Those were VITAMIN C
DEFECTIVE 2 (VTC2, Jander et al. 2002), and VTC2
paralog (VTC2L) and BEL1-LIKE HOMEODOMAIN 10(BLH10) (Hackbusch et al. 2004). The interaction of PLPA
with VTC2L was weakened at the intensity of >100 mmole
m-2 s-1 of blue light, while that of PLPB with VTC2L wasundetectable at that intensity (Ogura et al. 2008).

Publication List
〔Original papers〕● Kagawa, T., and Suetsugu, N. (2007). Photometrical analysis with

Figure 2. Positioning of nuclei of wild type, neo1, phot2 and neo1phot2
mutants in the dark and under weak or strong light conditions. Prothalliwere dark-adapted for 1 day (D) or irradiated with weak white light (L:-2 -1
20 - 30 mmol m-2 s-1) or strong white light (H: about 800 μmol m s )for 26 hrs. Percentages of nuclei at periclinal, anticlinal and intermediate
positions are indicated with black bars, white bars, and gray bars,
respectively.

It was also found that phot2 is necessary for dark
positioning of nucleus (Tsuboi et al. 2007). It is curious thata photoreceptor is needed for a physiological response in thedark, but it is similar to the case of Arabidopsis chloroplast
positioning wherein phot2 is indispensable in dark
positioning. Thus, both the nuclear and chloroplast
photorelocation movements may share common
photoreceptor systems.

2-2 Aureochrome as a blue light receptor in
Vaucheria
Vaucheria is a member of the group of stramenopile algae,
which includes brown algae and diatoms and shows blue
light responses, such as phototaxis, phototropism,
photomorphogenesis and chloroplast relocation (Kataoka etal. 1975). In collaboration with Professor Kataoka at
Tohoku University, we tried to find a blue light receptor(s)that might involve the blue light responses. We cloned two
genes containing sequences of one basic-region/leucinezipper (bZIP) domain and one light-oxygen-voltage (LOV)
domain that binds a flavin mononucleotide (FMN). Its bZIP
domain binds the target sequence TGACGT. We named
them AUREOCHROME1 and 2 (aureo1 and aureo2). RNAiof AUREO2 induces sex organ primordial instead ofbranches, implicating AUREO2 as a sub-switch to initiate
the development of a branch, but not a sex organ. These are
the photoreceptors for the blue light-induced branching of
Vaucheria frigida. AUREO sequences are also found in the
genome of the marine diatom but are not in green plants(Takahashi et al. 2007).

64

photosensory domains of photoreceptors in green algae. FEBS Letters581, 368-374.
● Takahashi, F., Yamagata, D., Ishikawa, M., Fukamatsu, Y., Ogura, Y.,
Kasahara, K., Kiyosue, T., Kikuyama, M., Wada, M., and Kataoka, H.
(2007). AUREOCHROME, a photoreceptor required forphotomorphogenesis in stramenopiles. Proc. Natl. Acad. Sci. USA
104, 19625 -19630.
● Tsuboi, H., Suetsugu, N., Kawai-Toyooka, H., and Wada, M. (2007).
Phototropins and neochrome1 mediate nuclear movement in the fern
Adiantum capillus-veneris. Plant Cell Physiol. 48, 892-896.

〔Review articles〕● Suetsugu, N., and Wada, M. (2007).

Phytochrome-dependent
photomovement responses mediated by phototropin family proteins incryptogam plants. Photochem. Photobiol. 83, 87-93.
● Suetsugu, N., and Wada, M. (2007). Chloroplast photorelocation
movement mediated by phototropin family proteins in green plants.
Biological Chemistry 388, 927-935.
● Wada, M. (2007). Fern as a model system to study photomorphogenesis.
J. Plant Research 120, 3-16.

LABORATORY OF PHOTOENVIRONMENTAL BIOLOGY

Professor (Adjunct)

WATANABE, Masakatsu

Photosynthetic microorganisms, such as cyanobacteria andflagellate algae, respond to light to locate themselves atappropriate photoenvironments. Our research is aimed at theelucidation of photoreceptive and signal transduction
mechanisms of the 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)
In 2002, we found a novel blue-light receptor with aneffector role from Euglena (Iseki et al., Nature 415, 10471051, 2002): Euglena gracilis, a unicellular flagellate(Figure 1), shows blue-light type photomovements. The
action spectra indicate the involvement of flavoproteins as
the photoreceptors mediating them. The paraflagellar body(PFB), a swelling near the base of the flagellum, has been
considered as a photosensing organelle for thephotomovements. To identify the photoreceptors in the PFB,
we isolated PFBs and purified the flavoproteins therein. The
purified flavoprotein (ca. 400 kDa), with noncovalently
bound FAD, seemed to be a heterotetramer of α- and βsubunits. Predicted amino acid sequences of each of thesubunits were similar to each other and contained two FADbinding domains (BLUF: sensor of blue light using FAD)
each followed by an adenylyl cyclase catalytic domain. The
flavoprotein showed an adenylyl cyclase activity, which waselevated by blue-light irradiation. Thus, the flavoprotein(PAC, photoactivated adenylyl cyclase) can directlytransduce a light signal into a change in the intracellularcyclic AMP level without any other signal transduction
proteins.

systems, X. laevis oocytes and HEK293 cells. Moreover,transgenic D. melanogaster flies demonstrated functional
PAC expression by showing blue light–induced behavioral
changes as described below.
To determine the reliance and kinetics of the light-inducedchange in behavior, the the grooming reflex15 was analysed:
when covered with a fine powder, fruit flies instantaneously
display vigorous and continuous grooming activity lasting
up to 30 min. Monitoring this behavior for a total time of 5
min with irradiation alternating between dim white light andintense blue light for 1 min each, revealed high grooming
activity in wild-type flies irrespective of stimulation by light(Figure 2). In contrast, neuronal expression of PAC (elavGal4/UAS-PAC) resulted in hyperactivity and a substantial
decline in grooming activity under blue-light stimulation.
When irradiation was switched back to dim white light, thus
turning off blue light–induced PAC activity, flies returned togrooming behavior within several seconds These results
demonstrate that transgenic expression of PAC in fruit flies
results in a functional protein that is rapidly and reversibly
activated by blue light. Moreover, the fast action observed at
the on- and offset of irradiation demonstrates the feasibilityof rapid control of cAMP levels in a freely moving animal.

Figure 1. Euglena gracilis, a unicellular flagellate alga

1-1 Fast manipulation of cellular cAMP level by lightin vivo.
In collaboration 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
functionally expressed PACs in two popular expression

Figure 2. Photostimulation of PAC alters behavior in freely moving D.
melanogaster.

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Environmental Biology

1-2 Functional transplant of photoactivated adenylyl
cyclase (PAC) into Aplysia sensory neurons.
In collaboration with Prof. T. Nagahama, (Faculty of
Pharmaceutical Science, Toho University), functionaltransplant of photoactivated adenylyl cyclase (PAC) into
Aplysia sensory neurons was performed to explore whether
PAC can produce cAMP in the neurons by light stimulation.
Serotonergic modulation of mechanoafferent sensory
neurons in Aplysia pleural ganglia has been reported toincrease intracellular cAMP level and promotes synaptictransmission to motor neurons by increasing spike width of
sensory neurons. When cAMP was directly injected into thesensory neurons, spike amplitude temporarily decreased
while spike width temporarily increased. We therefore
explored these changes as indicators of appearance of the
PAC function. PAC or the PAC expression vector (pNEXPAC) was injected into cell bodies of sensory neurons.
Spike amplitude decreased in both cases and spike width
increased in the PAC injection when the neurons were
stimulated with light (Figure 3), suggesting that thetransplanted PAC works well in Aplysia neurons. These
results indicate that we can control cAMP production in
specific neurons with light by the functional transplant of
PAC.

A

B

C
Publication List
〔Original papers〕● Nagahama, T., Suzuki, T., Yoshikawa, S., and Iseki, M. (2007).

Functional transplant of photoactivated adenylyl cyclase (PAC) into
Aplysia sensory neurons. Neurosci. Res. 59, 81-88.
● Schröder-Lang, S., Schwärzel, M., Seifert, R., Strünker, T., Kateriya,
S., Looser, J., Watanabe, M., Kaupp, U.B., Hegemann, P., and Nagel,
G. (2007). Fast manipulation of cellular cAMP level by light in vivo.
Nat. Meth. 4, 39-42.

Figure 3. Effect of blue light on spike amplitude in the Aplysia sensory
neurons into which plasmid vectors carrying PAC cDNAs were injected

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National Institute for Basic Biology

Theoretical Biology

DIVISION OF THEORETICAL BIOLOGY

Associate Professor

MOCHIZUKI, Atsushi
NIBB Research Fellow
Postdoctoral Fellows
Graduate Student
Secretary

FUJITA, Hironori
ISHIHARA, Shuji
NAKAZATO, Kenichi
IMAMURA, Hisako
UMEBAYASHI, Hiromi

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

Ⅰ. Predicting regulation of the phosphorylation
cycle of KaiC clock protein using mathematicalanalysis
Cyanobacteria are the simplest organisms exhibiting
circadian rhythms. In the bacterium, clock genes kaiA, kaiBand kaiC have been characterized as the indispensable clock
regulators. KaiC plays a central role and exhibits rhythms intranscription, translation and phosphorylation status undercontinuous illumination conditions. The other clock proteins
KaiA and KaiB modulate KaiC autophosphorylation: KaiA
enhances autophosphorylation of KaiC, and KaiB inhibits
this action of KaiA. It was recently revealed that periodic
oscillation of the phosphorylation level of KaiC persists
even under continuous dark conditions, where transcription
and translation have almost ceased. The KaiC
phosphorylation cycle was reconstituted even in vitro, thus
confirming that the interaction between Kai proteins
generates the cycle, although the specific mechanism thatdrives the clock remains unclear.
Using mathematical models, we investigated themechanism for the transcription-less KaiC phosphorylation
cycle. We developed a simple model based on possible
KaiC behavior suggested by previous experimental studies.
In the model, the KaiC-KaiA complex formation followed
by a decrease in free KaiA molecules may attenuate the
KaiC phosphorylation rate, and it acts as negative feedbackin the system. However, our mathematical analysis provedthat simple dynamics based on the experimentally suggested
model never show the KaiC phosphorylation cycle.
We then developed the generalized formulae of models anddetermined the necessary condition to generate the KaiC
phosphorylation cycle. Linear stability analysis revealed that
oscillations can occur when there is sufficient distance of
feedback between the recipient reaction and the effector.
Furthermore, we found that the negative feedback
regulations in closed systems can be classified into two
types: destabilizing inhibition and stabilizing inhibition.

Figure 1. Schematic representation of closed circuit model and thecondition for the possible oscillation by inhibition of the transition fromstate V1 to V2. Red-colored state (V1 , V4-V7) indicate that inhibition fromthe states can destabilize the system and possibly cause oscillation.
Inhibition from the blue-colored state never induces oscillation.

Table 1. Summary of the results of the general state transition modelwith conservation of molecules. The system could oscillate when theinhibiting state is more than two steps ahead of the inhibited reaction(from V1 to V2). If the inhibiting state is less than three steps ahead of
the reaction, the system is always stable. The necessary distance between
the inhibiting state and reactant state does not depend on the system size.

Figure 2. Schematic representations of "Basic model" (left) and"Multiple-phosphorelation-state model" (right). The basic model isdetermined from experimental results. It was proven that the model
never shows oscillation. The multiple-phosphorelation-state model wasdeveloped based on the mathematical analysis. The model shows clear
periodic oscillations. There are at least two different phosphorelated
states. The time-delay caused by the transition between the states isessential for generating oscillation.

Based on this result, we predicted that, in addition to theidentified states of KaiC, another unknown state must bepresent between KaiC phosphorylation and the complex
formation. By incorporating the unknown state into theprevious model, we realized the periodic pattern reminiscentof the KaiC phosphorylation cycle in computer simulation.
This result implies that the KaiC-KaiA complex formation
requires more than one step of posttranslational modification
including phosphorylation or conformational change of
KaiC. This prediction has recently been confirmed byexperimental methods.

67

National Institute for Basic Biology

Theoretical Biology

Ⅱ. Mathematical models for pattern formationof dendrites of neurons
Dendrite is a part of a neuronal cell which is specialized forreceiving and processing synaptic or sensory input. A
remarkable feature of dendrite is its morphological diversity.
The shapes of dendritic trees are characteristic of individual
neuronal types and they are highly variable from one
neuronal type to another. This diversity contributes todifferential processing of information in each type ofneuron. Therefore, patterning neuronal class-specificdendrites is a process to produce forms that realize thephysiological functions of neurons. However, acomprehensive logic of dendrite development has not beenformulated yet.
Previously proposed mathematical models to explain thepattern formation of dendrites assumed that dendrite
development is a consequence of stochastic sprouting andsubsequent growth arrest. Different forms of branching
functions were postulated and modified so that simulated
dendrograms fit the dendritic arbors of real neurons. One ofthe problems of the previous models is that thosedendrograms represent limited features of dendritic patterns
such as order of branches and degree (the number ofbranches at each order) and do not reproduce the full rangeof the morphological features of the original dendrites. Other
problems include the fact that many of the models cannot
specify experimentally confirmed mechanisms to accountfor their assumptions. To overcome these problems, wedeveloped a new class of dendrite growth model, which
represents all extension, orientation of growth and branchingof dendrites in a single scheme. In addition, this model has
explicitly incorporated an underlying biological mechanism,that is, competitive interactions between neighboring
dendrites.

diffusion (RD) model of the so-called "Activator-Inhibitor
type" (Turing, 1952; Gierer and Meinhardt, 1972). We set a
restriction in the 2D space so that the activator only diffuses
inside of the cells. These settings endow the system withfeedback loop regulations at two different levels: one
between two chemicals, and another between the dynamics
of the chemicals and the expansion of the cell compartment.
Using this formula, we study the dynamics of dendritic
branch formation. Computer simulation showed that the cellcompartment model developed dendritic branching
autonomously. The model can represent characteristicfeatures of the spatial regulation observed in actual
dendrites: tiling and regeneration. We analyzed behavior of
the model and determined the conditions for the dendrite
formation. This work has been done in collaboration with
Dr. T. Uemura and Dr. K. Sugimura of Kyoto University.

Figure 4. Regeneration after artificial severing. Patterns obtained bycomputer simulation of the model (up) and the corresponding patternobtained in an experiment (down).

Publication List
〔Original papers〕● Ishihara, S., Otsuji, M., and Mochizuki, A. (2007). Transient andFigure 3. Examples of obtained dendrite patterns by computer simulationof the model (left) and the correspondence observed in an experiment(Grueber et al., 2003; Moore et al., 2002). Dendrites from the cells
spread and cover the space. However, they never interfere with eachother.

A key point in our modeling is to couple chemical
dynamics to dendrite growth. In our model, we distinguish
two spatial compartments: inside and outside regions ofneurons. The cell compartment dynamically grows under the
regulation of a chemical reactant activator. Thus we call ourmodel a “cell compartment model”. The activator reacts
with another reactant suppressor in the way of the reaction-

68

steady state of mass-conserved reaction-diffusion systems. Phys. Rev.
E 75, 015203.
● Otsuji, M., Ishihara, S., Co, C., Kaibuchi, K., Mochizuki, A., andKuroda, K. (2007). A Mass Conserved Reaction-Diffusion System
Captures Properties of Cell Polarity. PLoS Comput. Biol. 3, 10401054.
● Sugimura, K., Shimono, K., Uemura, T., and Mochizuki, A. (2007).
Self-organizing mechanism for development of space-filling neuronaldendrites. PLoS Comput. Biol. 3, 2143-2154.

LABORATORY OF GENOME INFORMATICS

Assistant Professor

UCHIYAMA, Ikuo

The accumulation of biological data has recently been
accelerated by various high-throughput “omics”
technologies such as genomics, transcriptomics, proteomics,
and so on. The field of genome informatics is aimed at
utilizing this data, or finding some principles behind thedata, for understanding complex living systems by
integrating the data with current biological knowledge using
various computational techniques. In this laboratory wefocus on developing computational methods and tools forcomparative genome analysis, which is a useful approach forfinding functional or evolutionary clues to interpreting the
genomic information of various species. The current focus
of our research is on the comparative analysis of microbial
genomes, the number of which has been increasing rapidly.
Extracting useful information from such a growing numberof genomes is a major challenge in genomics research.
Interestingly, many of the completed genomic sequences areclosely related to each other. We are now trying to develop
methods and tools to conduct comparative analyses not onlyof distantly related genomes but also of closely related
genomes, since we can extract different types of information
about biological functions and evolutionary processes fromcomparisons of genomes at different evolutionary distances.

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 thisalgorithm, MBGD not only provides the 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 featureis especially useful when the user’s interest is focused onsome taxonomically related organisms. The constructedclassification table can be used for comparative analyses
from various points of view, such as phylogenetic patternanalysis, gene order comparison, and detailed gene structurecomparison. For researchers who are interested in ongoinggenome projects, MBGD also provides a service called “My
MBGD,” which allows users to add their own genome
sequences to MBGD for the purpose of identifying orthologs
among both the new and the existing genomes.
The database now contains more than 500 publishedgenomes and the number continues to grow. MBGD isavailable at http://mbgd.genome.ad.jp/.

Ⅱ . Hierarchical clustering algorithm forconstructing orthologous groups of multiple
genomes
As part of the core technologies of the MBGD system wehave developed a rapid automated method of ortholog
grouping, named DomClust, which is effective enough toallow the comparison of hundreds of genomes
simultaneously. The method takes as input all-against-all
similarity data and classifies genes based on the traditional
hierarchical clustering algorithm UPGMA. In the course ofclustering, the method detects domain fusion or fission
events and splits clusters into domains if required. The
subsequent procedure splits the resulting trees in such a waythat intra-species paralogous genes are divided into different
groups so as to create plausible orthologous groups. As aresult, the procedure can split genes into the domains
minimally required for ortholog grouping.
As the number of completed genomic sequences grows,
comparison among closely related as well as distantly
related genomes has become more important forunderstanding the function and evolution of various
genomes. For evolutionary analysis of a target set of relatedorganisms (ingroup), we often need another set of organisms
outside of that group (outgroup) to correctly infer
evolutionary processes. To incorporate this concept into theortholog analysis, we have enhanced the DomClustalgorithm to impose constraints on the resulting orthologous
groups such that the outgroup species should come outsideof the ingroup species (Figure 1). The resulting table has anested structure when a duplication event occurs within the
ingroup lineage.

Figure 1. Converting from a gene tree (top) to a nested ortholog table(bottom)

Ⅲ. Identification of core structures conserved
among moderately related microbial genomes
A growing body of evidence supports the theory that bothhorizontal transfer and vertical transfer have played
significant roles in prokaryotic evolution. Despite thecomplexity of evolution suggested from these observations,
it can be argued that prokaryotic phylogeny can still be

69

National Institute for Basic Biology

Theoretical Biology

inferred using a certain subset of genes (“core genes”) thathave mainly transferred vertically throughout theevolutionary process. We are trying to identify a common“core structure” of related genomes, which is defined as aset of sufficiently long consecutive genomic segments inwhich gene orders are conserved among multiple genomes
so that they are likely to have been inherited from a common
ancestor mainly through vertical transfer. For this purpose,
we have developed a graph-based algorithm for aligning
conserved regions of multiple genomes. The algorithm finds
an order of pre-identified orthologous groups so as to retain,
as much as possible, the conserved gene orders (Figure 2).
The method was applied to genome comparisons of thefamilies Bacillaceae and Enterobacteriaceae. Using
orthologous groups generated by the DomClust program, weconstructed genome alignments and identified common core
structures comprising about 1400 genes for Bacillaceae and1900 genes for Enterobacteriaceae. Despite the difference inthe overall proportions of the core genes between thesedatasets, the proportion of the core genes in each functional
category primarily exhibit a similar tendency: functional
categories related to primary metabolism, genetic
information processing, and cellular processes generally
contain a higher proportion of core genes, while categoriesof membrane transport, signal transduction and secondary
metabolism contain a lower proportion of core genes. In
addition, it also turned out that these core structures containmost of the essential genes identified in Bacillus subtilis andEscherichia coli, respectively.

The entire RECOG system employs client-server
architecture. The RECOG server program has beendeveloped based on the MBGD server and contains database
construction protocol in MBGD including all-against-all
similarity search calculation, as well as the extended versionof the DomClust program. Unlike MBGD, however, users
can install the RECOG server on their local machines toanalyze their own genomic data. Alternatively, users canconnect to the public RECOG server to analyze publicly
available data.
The RECOG client program is a Java application that runs
on a local machine by receiving data from any available
RECOG server. The main window of the RECOG clientconsists of three parts: taxonomic tree viewer (left), ortholog
table/phylogenetic pattern map viewer (center) and gene
information viewer (right) (Figure 3). In the taxonomic tree
viewer, users can specify either a set of organisms to beanalyzed (as ingroup or outgroup), or conditions to filter
phylogenetic patterns (i.e. presence or absence of each genein each genome) to be displayed. The ortholog table viewer
displays the entire ortholog table. By semantic zooming
functionality, users can see from the entire picture of thephylogenetic pattern map to the full details of the ortholog
table. In the gene information viewer, users can see detailed
information about specified orthologous groups and genes
belonging to each of the groups. Several sorting and filteringfunctions have been implemented for modifying the displayof the ortholog table viewer, including sorting by functional
category and gene name or by gene order of a specified
genome, and filtering by keywords or by phylogenetic
pattern conditions.

Figure 2. Construction of core genome alignment based on gene orderconservation
Figure 3. A snapshot of the main window of the RECOG system

Ⅳ. Research environment for comparative
genomics
We have been developing a new system named RECOG(Research Environment for COmparative Genomics), whichaims to extend the current MBGD system by incorporating
the above-described approaches including the
ingroup/outgroup distinction in ortholog grouping and thecore structure extraction among related genomes. In
addition, the RECOG system has several advanced featuresthat allow users to perform more flexible phylogenetic
pattern analyses.

70

Publication List
〔Original paper〕● Uchiyama, I. (2007). MBGD: a platform for microbial comparative

genomics based on the automated construction of orthologous groups.
Nucleic Acids Res. 35, D343-D346.

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori
Technical Staff
NIBB Research Fellow
Visiting Scientist
Secretary

KAJIURA-KOBAYASHI, Hiroko
ICHIKAWA, Takehiko
KANDA, Rieko
KAMIYA, Akemi

In spite of superficially bilateral symmetry, our bodies arehighly asymmetric along the left-right (L-R) axis, forexample in the placement of internal organs. Our main aim
is to clarify the mechanism by which mammalian embryos
generate and establish the L-R asymmetry.

The leftward flow, known as nodal flow, determines
subsequent L-R development. This idea has been confirmedby two experiments. Firstly, mutations without motile cilia
in the node result in randomized L-R asymmetry. Secondly,
and more importantly, embryos raised in a rightward
artificial flow of culture medium develop reversed L-R
asymmetry (Figure 3; Nonaka et al., Nature 418, 96-99,
2002).
While it is clear that nodal flow conveys asymmetric
information, the molecular nature of the information remains
unclear. We are now working to clarify the mechanism of
how the direction of nodal flow is converted to the subsequent
step, asymmetric gene expression.

Ⅰ. Initial step for the 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., Cell 95, 829-837,1998).
The sum of the vortical motions of the cilia, however,generates a leftward flow of the surrounding fluid rather
than a vortex. Because of the posteriorly tilted rotation axis,
the node cilia can generate leftward force without preexisting left-right asymmetry. (Figure 2; Nonaka et al.,PLoS Biology 3, e268, 2005).

Figure 3. Flow culture experiment. A, A peristaltic pump anddepulsators supply constant fluid flow in the chanber (red arrows). B,
Embryos held in the pots receive the pump-driven flow on their surface.
If the pump-driven flow reverse intrinsic nodal flow (green arrows),
expression of nodal, a master gene for the leftness, is reversed (right).

Ⅱ. New microscopy

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

Figure 2. Mechanism to generate leftward flow by a rotating cilium.
Left, fluid very close to the wall is generally reluctant to move (surface
effect). Right, this constraint negatively affects water-dragging
efficiency by the cilium. Given that the cilium rotates clockwise with aposteriorly tilted axis, fluid dragged to the right is less than that to theleft, resulting leftward force production.

Long-term live imaging techniques of early mouseembryos promise to be extremely useful for our analyses of
L-R development as well as for broader research interests.
For this purpose, we are working to introduce a digitalscanned light-sheet microscope (DSLM, Figure 4) that has
been developed by Dr. Ernst Stelzer in European Molecular
Biology Laboratory (EMBL).

Figure 4. DSLM in construction

71

National Institute for Basic Biology

Research Support

The main activities of the Department in 2007

STRATEGIC PLANNING DEPARTMENT

1) Supporting international conferences
On-line registration, web page construction, brochure
editing, hotel reservation, meal and party arrangement,
photographic recording, etc., for international conferences
held at NIBB (Table 1)

Chair

NAGAHAMA, Yoshitaka
Vice-Chairs
Professor
Associate Professor
Postdoctoral Fellow
Technical Assistants

UENO, NaotoNISHIMURA, Mikio
WADA, Masamitsu
KODAMA, Ryuji
KURATA, Tomoko
MUKOHDA, Yasuyo
OTA, Misaki
TANAKA, Megumi
EMDE, Jason R.

2) Management of education-related programs
Internship Program 2007
The First International Practical Course (Jan. 2007)

3) Press Release
News on scientific achievements are sent to newspaper andmagazine reporters via e-mail and [a? our?]password
protected web page. For some of the releases, we arranged apress conference.

4) Editing of publications
Introductory Pamphlet of NIBB (in Japanese)
Introductory Pamphlet of NIBB (in English)
Educational Booklet "Organisms sustaining research" (inJapanese)
Bulletin of NIBB 2007/08 (in Japanese, in preparation)
Annual Report of NIBB 2007 (in English, with proof
reading)

The Strategic Planning Department was founded in April
2005 as a central office for the efficient management ofcooperational activities with other organizations, thedistribution of scientific information to the public, planningand managing conferences, workshops and other extramuralactivities. The department also assists the director-general in
preparing for NIBB's evaluation procedure and in planning along-range strategy for the institute.

5) Production of posters and leaflets
Design and distribution of posters for international
conferences and advertisements for the graduate school’s
entrance examination (Figure1)6) Updating and maintenance of NIBB web page7) Publication of "NIBB News"(Intra-institutional newsletter in Japanese)

8) Maintenance of the achievement archives of NIBB
9) Assisting visitors (in collaboration with Technical
Division)

Name

Date

Title

Organizer

5th Okazaki Biology Conference

March 11-16

Speciation and Adaptation-Ecological Genomics of Model Organisms
and Beyond-

K. Shimizu
I. Dworkin

6th Okazaki Biology Conference

December 2-8

Marine Biology

N. Satoh
B.J. Swalla
D.R. Levitan

5th NIBB-EMBL Meeting

May 24-26

Cell and Developmental Biology

N. UenoS. Cohen

Table 1. International conferences managed by Strategic Planning Department in 2007

Figure 1. Examples of posters, pamphlets and abstract books produced by the Strategic Planning Department in 200772

Research Support Facilities

Head

NISHIMURA, Mikio● Large Spectrograph Laboratory

Professor (Adjunct)Technical Staff
Technical Assistant
Secretary

WATANABE, Masakatsu
HIGASHI, Sho-ichi
NAKAMURA, Takanori
ICHIKAWA, Chiaki
ISHIKAWA, Azusa● Tissue and Cell Culture Laboratory

Assistant Professor
Technical Assistant

HAMADA, Yoshio
TAKESHITA, Miyako● Computer Laboratory

Assistant Professor
Technical Staff

Technical Assistant

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

● Plant Culture, Farm, Plant Cell Laboratory

Technical Staff
Technical Assistant

NANBA, Chieko
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 ateach wavelength 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 interconnectedby 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.

Publication List on OLS Collaboration〔Original papers〕● Arimoto-Kobayashi, S., Sakata, H., Mitsu, K., and Tanoue, H. (2007).

A possible photosensitizer: Tabacco-specific nitrosamine, 4-(Nmethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induced
mutations, DNA strand breaks and oxidative and methylative damage
with UVA. Mutation Reseach 632, 111-120.
● Bolige, A., and Goto, K. (2007). Phytochrome-like responses inEuglena: A low fluence response that reorganizes the spectral
dependence of the high irradiance response in long-day photoperiodic
induction of cell division. J. Photochem. Photobiol. B. 86, 97-108.
● Bolige, A., and Goto, K. (2007). High irradiance responses involving
photoreversible multiple photoreceptors as related to photoperiodic
induction of cell devision in Euglena. J. Photochem. Photobiol. B. 86,
109-120.
● Negishi, T., Kawai, K., Arakawa, R., Higashi, S., Nakamura, T.,
Watanabe, M., Kasai, H., and Fujikawa, K. (2007). Increased levels of
8-Hydroxy-2’-Deoxyguanosine in drosophila larval DNA after
irradiation with 364-nm laser light but not with X-rays. Photochem.
Photobiol. 83, 658-663.
● Shihira-Ishikawa, I., Nakamura, T., Higashi, S-i., and Watanabe, M.
(2007). Distinct responses of chloroplasts to blue and green laser
microbeam irradiations in the centric diatom Pleurosira laevis.
Photochem. Photobiol. 83, 1101-1109.
● Xie, X., Shinomura, T., Inagaki, N., Kiyota, S., and Takano, M.
(2007). Phytochrome-mediated inhibition of coleoptile growth in rice:
age-dependency and action spectra. Photochem. Photobiol. 83, 131138.

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.
Figure 1. The Large Spectrograph

3. Computer Laboratory
The computer laboratory maintains several computers toprovide computation resources and the means of electronic73National Institute for Basic Biology

Research Support

communication within NIBB. Our main computer system,
the Biological Information Analysis System (BIAS),
consists of a shared memory parallel computer (SGI Altix
350; 8CPU, 48GB memory) with a disk array storage system(D-RAID; 1.6TBx10), a high-performance cluster system(DELL PowerEdge 1850; 2CPUx(16+1) nodes), and a datavisualization terminal (DELL Precision 370). Some personal
computers and color/monochrome printers are alsoavailable. On this system, we provide various biological
databases and data 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 ResearchInstitute (ORION). Many local services, including sequence
analysis services, file sharing services, and printer services,
are provided through this network. We also maintain apublic World Wide Web server that contains the NIBB
home page (http://www.nibb.ac.jp/).

4. Plant Culture Laboratory
This laboratory contains a large number of culture boxes
and a limited number of rooms with environmental control
for plant culture. In some of these facilities and rooms,
experiments can be carried out at the P1 physicalcontainment level under extraordinary environments such as
strong light intensity, low or high temperatures, etc.

5. Experimental FarmThis laboratory consists of two 20 m2 glasshouses with
precise temperature and humidity control, three green
houses (each 6 m2) at the P1 physical containment level, asmall farm, and two greenhouses (45 and 88 m 2) with
automatic sprinklers. The laboratory also includes a building
with storage and work space.

6. Plant Cell Laboratory
This laboratory 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.

74

RESEARCH CENTER FOR INTEGRATIVE AND COMPUTATIONAL BIOLOGYHeadTAKADA, Shinji
Associate Professor
Secretary

MOCHIZUKI, Atsushi
UMEBAYASHI, Hiromi

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 final 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 in 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
constructed from complex interactions between many genes.
To grapple with these challenges, it is essential to decipher
huge amounts of gene information, and to derive the essenceof the biological behavior of cells or organisms.
Mathematical and computational sciences have strong
capacities for dealing with these challenges. Computational
methods make it possible to process ever-increasing
amounts of data. Hypothetical experiments (including theevolution of past organisms) based on mathematical orcomputational models make it possible to consider
conditions which are impossible in real experiments. We
continue to research higher-order phenomena in biology
using mathematical or computational methods, anddeveloping new methodologies for studying such 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 of cell or gene interaction that arelikely to be responsible for the systems in real organisms.
This method gives us an integrative understanding for the
behavior of complex systems in biology, including gene
regulatory networks.

Ⅱ. Collaborative activity
Interaction between researchers is essential to make thebest use of mathematical and computational methods forexperimental biology. The theoretical methods provide
testable predictions which the experimental biologists areable to investigate before returning the results 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 1. A laboratory room for computational studies

In 2007, we conducted the arrangements for theinternational meetings on systems biology, which will beheld in April 2008 in Barcelona as a collaborative effort
with EMBL. We have also held some small meetings andenhanced the interaction between senior and juniorreseachers. The center is dedicated to encouraging young
researchers who can use these methods for continued
research into complex biological phenomena.
Figure 1. A laboratory room for computational studies

75

National Institute for Basic Biology

Research Support

CENTER FOR TRANSGENIC ANIMALS AND PLANTS

HeadTAKADA, Shinji
Associate Professors

Technical Staff

Supporting Staff

WATANABE, Eiji
SASAOKA, Toshikuni
TANAKA, Minoru
NARUSE, Kiyoshi
HAYASHI, Kohji
ICHIKAWA, Yoko
TAKAGI, Yukari
YASUDA, Mie
NOGUCHI, Yuji
KAWAMURA, Motofumi
KOBAYASHI-NISHIMURA, Keiko
OKUDA, Tadayoshi
AJIOKA, Rie
NAKASHIMA, Machiko

The worldwide genome project has almost been completed
and research on basic biology has arrived at a post-genome
era in which researchers focus on investigating the functionof individual genes. To promote the functional analysis of agene of interest it is essential to utilize genetically alteredmodel organisms, which are 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 four associate professors.
Technical staff and supporting staff develop and promoteresearch-supporting activities. A state-of-the-art facility fortransgenic animals opened at the end of 2003 in the Yamate
area.
The activities of the CTAP are as follows:
1. Provision of information, materials, techniques andanimal housing space to researchers.
2. The use of various kinds of instruments to analyze
mutant, transgenic, and gene-targeted animals and plants.
3. Development of novel techniques related to transgenic
and gene targeting technology.
4. Cryopreservation and storage of transgenic strains.

Ⅰ. Research supporting activity (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. This building is also
equipped with breeding areas for transgenic small fish, birds
and insects on the first floor. The mouse housing area of thisbuilding is constructed based on a barrier system.

Figure 2. Large scale autoclaves for sterilization

Figure 1. The new center facility for transgenic animals in the Yamate area

76

In 2007, 5,170 mice were brought into the CTAP in the
Yamate area, and 30,870 mice (including pups bred in thefacility) were taken out from the CTAP from November 1,
2006 to October 31, 2007.
A number of strains of genetically altered mice fromoutside the CTAP were brought into the mouse housing areaby microbiological cleaning using the in vitro fertilizationembryo transfer techniques, and stored using
cryopreservation.

A new mouse facility in the Myodaiji area opened at the
beginning of 2005. The facility provides research-supporting
activities to researchers in the Myodaiji area. In 2007, 85
mice were brought into the CTAP in the Myodaiji area, and1,573 mice (including pups bred in the facility) were takenout from the CTAP from November 1, 2006 to October 31,
2007.

mutants and the transgenic lines as well as the genomic
resources (such as 15,000 non-redundant cDNA/EST clones
and BAC/Fosmid clones) covering 90% of medaka genome
for the scientific community. We have provided the medakabioresources to several countries, including Germany,
United Kingdom, France, Norway, USA, Thailand, China,
Taiwan, and Japan.

Ⅱ. Research supporting activity (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 a 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 suitthe 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 aremaintained in our facility. In addition to the rooms
mentioned above, another room is available for insects. All
the rooms are qualified to meet the criteria for transgenic
animals, allowing researchers to generate and maintaintransgenic animals.

Figure 4. Medaka live resources provided from NBRP Medaka

Ⅲ. Academic activity

Figure 3. Breeding equipment for zebrafish

In 2007 (from November 1, 2006 to October 31, 2007),
4,292 medaka and zebrafish (150 eggs, 1,310 embryos and2,832 adults) were brought to the facility and 27,611 medaka
and zebrafish (26,694 fertilized eggs and 917 adults,
including animals bred in the facility) were taken out fromthe CTAP. In a laboratory for chick embryos 7,275 fertilized
chicken eggs were brought in and 7,275 fertilized eggs andchicken embryos were taken out from the CTAP. These
animals were used for research activities in neurobiology
and developmental biology.

The associate professors of this center - E. Watanabe, T.
Sasaoka, M. Tanaka and K. Naruse - are the principal
investigators of the Laboratory of Neurophysiology, theLaboratory of Neurochemistry, the Laboratory of Molecular
Genetics for Reproduction and the Laboratory of Bioresources,
respectively. The Laboratory of Neurophysiology is studying
the brain sensing system for body fluid water and sodium
homeostasis using gene-targeting mice, the Laboratory of
Neurochemistry is studying the physiological role of the
dopaminergic system using genetically altered mice, theLaboratory of Molecular Genetics for Reproduction is
studying the molecular mechanism of reproductive organ
development and sex differentiation using mutagenized ortransgenic medaka, while the Laboratory of Bioresources isstudying the genome evolution, focusing mainly on thechromosome evolution among ray-finned fish and theevolution of sex-determination systems using medakarelated species. For details, please refer to the academic
activity of each laboratory.

In 2007 NIBB was selected as a core facility of the
National BioResource Project of Medaka. We have provided
the standard strains, the inbred strains, natural and induced

77

National Institute for Basic Biology

Research Support

THE CENTER FOR ANALYTICAL INSTRUMENTS (managed by NIBB)Head of Facility

IIDA, Shigeru
Technical Staff

Technical Assistants
Secretary

MORI, Tomoko
YAMAGUCHI, Katsushi
MAKINO, Yumiko
TAKAMI, Shigemi
MORIBE, Hatsumi
KUROYANAGI, Asuka
ICHIKAWA, Mariko

Fluorescence Bio Imaging Analyzer (Takara FMBIO Ⅱ)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Environmental Scanning Electron Microscope (PHILIPSXL30 ESEM)
Confocal Laser Scanning Microscope (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)

The Center is responsible for amino acid sequence analysis,
amino acid analysis, and chemical syntheses of peptides, aswell as supporting researchers at NIBB and NIPS. A new
Liquid Chromatograph/Q-TOF Mass Spectrometer (Waters
Q-TOF Premier) was installed in 2007. The Center’s
instruments can be used by researchers from outside theinstitute upon proposal.

Representative Instruments
Protein Sequencers (ABI Procise 494HT, 492cLC)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A)
Plasmid Isolation Systems (Kurabo PI-100∑ , PI-50α, PI-50,
PI-200)Automatic Nucleic Acid Isolation System (Kurabo NA2000)
Genetic Analyzer (ABI PRISM 3130xl, 310)
Thermal Cyclers (Perkin Elmer PJ-9600, Takara TP-300,
Biometra TGRADIENT)
Particle Delivery System (Bio-Rad BiolisticPDS-1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)High Performance Liquid Chromatographs (Shimadzu LC10AD, 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 RF5000)
Spectrophotometers (Hitachi 330, 557, U-2001, Varian
Cary5G, Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB 96P)
Microplate Readers (Corona MTP-120, MTP-100F)
FT-IR Spectrophotometer (Horiba FT-730)
Bio Imaging Analyzers (Fujifilm BAS 2000)
Luminescent Image Analyzers (Fujifilm LAS 3000 mini)

78

Figure 1. Protein sequencers

Figure 2. MALDI/TOF-MS

Figure 3. LC/Q-TOF-MSTECHNICAL DIVISIONHeadFURUKAWA, KazuhikoCommon Facility Group
Chief

MIWA, Tomoki● Research Support Facilities

Unit Chief
Subunit Chief
Technical Staff
Technical Assistants

HIGASHI, Sho-Ichi
NANBA, Chieko
NISHIDE, Hiroyo
NAKAMURA, Takanori
SUZUKI, KeikoICHIKAWA, Chiaki
TAKESHITA, Miyako
NISHIMURA, Noriko
YAMAMOTO, Kumi
ISHIKAWA, Azusa

Research Support Group
Chief● Cell Biology

Unit Chief
Subunit Chief

Unit Chief
Subunit Chief
Technical Staff
Technical Assistants

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
TAKAMI, Shigemi
MORIBE, Hatsumi
KUROYANAGI, Asuka
IGHIKAWA, Mariko

KONDO, Maki
KABEYA, Yukiko● Developmental Biology

Technical Staff

TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
NODA, Chiyo● Neurobiology

Unit Chief● Center for Analytical Instruments

KAJIURA-KOBAYASHI, Hiroko

Subunit Chief

OHSAWA, Sonoko
TAKEUCHI, Yasushi● Evolutionary Biology and Biodiversity

Unit Chief
Technical Staff

FUKADA-TANAKA, Sachiko
MOROOKA, Naoki
SUMIKAWA, Naomi● Environmental Biology

Subunit Chief

MIZUTANI, Takeshi● Transgenic Animal Facility

Subunit Chief
Technical Assistants

HAYASHI, Kohji
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 supporting 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
the institute.
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.

Reception Office
Supporting Staff

TSUZUKI, Shihoko
KATAOKA, Yukari
UNO, Satoko
KONDO, Yukie
MAEDA, Yuri

Technical staff members continually participate, throughthe Division, in self-improvement and educational activities
to increase 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.

79

National Institute for Basic Biology

Okazaki Research Facilities

CENTER FOR RADIOISOTOPE FACILITIESHeadNODA, Masaharu(Professor, concurrent post)
Associate Professor

OGAWA, Kazuo(Radiation Protection Supervisor)

Technical staff

MATSUDA, Yoshimi

Figure 1. Percentage of users going in and out of each controlled area in2007(Radiation Protection Supervisor)

Supporting staff

SAWADA, Kaoru
IINUMA, Hideko
ITO, Takayo
KAMIYA, Kiyomi
KANEUJI, Kimie
Table 2. Users and visitors who entered each controlled area in 2007

The technical and supporting staff of the Center forRadioisotope Facilities (CRF) maintain three controlledareas according to Japanese law. The CRF is responsible formonitoring the purchase of radioisotopes from JRA (Japan
Radioisotope Association) and the transfer of radioisotope
wastes to JRA.
Matsuda, Iinuma, Ito, and Kaneuji maintained CFBI
(Common Facilities Building I)-branch and LGER
(Laboratory of Gene Expression and Regulation)-branch at
the Myodaiji-Area. Ogawa, Sawada and Kamiya worked at
the Yamate-Area. Kaneuji resigned from her job in March,while Kamiya moved to the Myodaiji-Area after March
2007.
The number of registrants and the number of users fromJanuary 2007 to December 2007 are presented in Table 1.
Users counted by the monitoring system going in and outof the controlled areas numbered 3,980 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. No 125I was used at Myodaiji-Area.
Due to the recent decrease in the total number of users of
the CRF and in order to prepare for a gene recombination
experiment and an animal experiment using radioisotopes,
we changed nuclides and the limit of radioactivity andprepared the rooms and applications for the genetic
recombination and animal experiments using radioisotopes
that began in August of 2007 (see Figure 3).

Table 1. Numbers of registrants and users at Myodaiji-Area and YamateArea in 2007

80

Figure 2. Annual changes of registrants and total number of days

Table 3. Balance of radioisotopes received and used (KBq) at eachcontrolled area in 2007

Figure 3. These photographs show the gene recombination experimentand the animal experiment rooms at Myodaiji-Area.
Left: The gene recombination experiment room. This room has a safetycabinet and the biohazard protect type autoclave.
Right: Signs outside of the animal experiment and the gene recombination
experiment room

The Fifth Okazaki Biology Conference
Speciation and Adaptation-Ecological Genomics of Model Organisms and BeyondOrganizing Chair : Kentaro Shimizu (University of Zurich, Switzerland)
Ian Dworkin (North Carolina State University, USA)
March 11 (Sun) – 16 (Fri), 2007
With the advent and widespread use of genomic tools, it isclear that the study of evolutionary genetics is undergoing atransformation. To promote the advancement and synthesisof the various fields of evolutionary and ecologicalgenomics, the 5th Okazaki Biological Conference was heldin March of 2007. The conference began with a social
function to welcome 70 scientists from 10 countries and was
followed by Plenary Lectures at the National Institute for
Basic Biology (NIBB). Following the plenary lectures, theparticipants moved to Yamaha Resort Tsumagoi, where thescientists had opportunities to share their work during theoral platform sessions, in addition to poster sessions by many
additional young scientists. The talks represented a broad
overview of current areas of active research in ecological andevolutionary genomics, including studies of adaptation,
speciation, domestication, genome duplication, co-evolution,
canalization and theoretical population genetics.

The real goal of such an intimate meeting was to encourageinteraction between leading researchers and to facilitate
interactions between scientists using different approaches
but asking related questions and using similar model
systems.. We believe that it is only when apparently
disparate aspects of biology are integrated that we can hopeto make real progress in our understanding of evolutionary
mechanisms. During the conference, therefore, there wasplenty of time for discussions on each of these subjects, bothformally after each session with a panel discussion as well
as more informally during social events and meals. The
feedback from the participants on all aspects of theconference, including the scientific discussions,
opportunities for developing new contacts, and the help of
the supporting staff, was very positive.

Scientific sessions:
1. Adaptive evolution
2. Genetics of speciation
3. Genome duplication and epigenetics
4. Artificial selection in domestication
5. Canalization, robustness and hidden genetic variation
6. Theory
7. Coevolution
Invited Speakers
AGUADE, Montserrat (Univ. Barcelona, Spain), ARAKI, Hitoshi (Oregon State Univ., USA), BARBASH, Daniel (Cornell Univ., USA),
BRYSTING, Anne (Univ. Oslo, Norway), CAICEDO, Ana (Univ. Massachusetts, USA), COMAI, Luca (UC Davis, USA),
COOP, Graham (Univ. Chicago, USA), DWORKIN, Ian (North Carolina State Univ., USA),
FELIX, Marie-Anne (Institut Jacques Monod, France), GROSSNIKLAUS, Ueli (Univ. Zurich, Switzerland),HERMISSON, Joachim (Univ. Munich, Germany), HIRATE, Yoshikazu (Fred Hutchinson Cancer Research Center, USA),HOEKSTRA, Hopi (Harvard Univ., USA), IGIC, Boris (Univ. Illinois, Chicago, USA), KANAOKA, Masahiro (Univ. Washington, USA),KOBAYASHI, Yasushi (MPI for Developmental Biology, Germany), KUITTINEN, Helmi (Univ. Oulu, Finland),
LAWTON-RAUH, Amy (Clemson Univ., USA), MABLE, Barbara (Univ. of Glasgow, UK), MACHADO, Carlos (Univ. Arizona, USA),OLSEN, Kenneth (Washington Univ., St. Louis, USA), PURUGGANAN, Michael (New York Univ., USA), RESCH, Alissa (NCBI, USA),RUTHERFORD, Suzannah (Fred Hutchinson Cancer Research Center, USA), SHIMIZU, Kentaro (Univ. Zurich, Switzerland),SHIMIZU-INATSUGI, Rie (Univ. Zurich, Switzerland), SHUSTER, Stephen (Northern Arizona Univ., USA),SIEGAL, Mark (New York Univ., USA), STEPHAN, Wolfgang (Univ. Munich, Germany), TANAKA, Kenta (Univ. Sheffield, UK),
TIAN, Dacheng (Nanjing Univ., China), TSIANTIS, Miltos (Univ. Oxford, UK), VERGEER, Philippine (Univ. Leeds, UK),WIDMER, Alex (ETH Zurich, Switzerland), YANG, Hsiao-Pei (Cornell Univ., USA)
FUKATSU, Takema (AIST, Japan), HARUSHIMA, Yoshiaki (NIG, Japan), HASEBE, Mitsuyasu (NIBB, Japan),
INNAN, Hideki (SOKENDAI, Japan), ISHIKAWA, Ryuji (Hirosaki Univ., Japan), IZAWA, Takeshi (NIAS, Japan),
KUDOH, Hiroshi (Kobe Univ., Japan), MOCHIZUKI, Atsushi (NIBB, Japan), OKADA, Kiyotaka (Kyoto Univ., Japan),
TAJIMA, Fumio (Univ. Tokyo, Japan), TAKAHASHI, Aya (NIG, Japan), TSUKAYA, Hirokazu (Univ. Tokyo, Japan),
TSUMURA, Yoshihiko (FFRPI, Japan), YAHARA, Tetsukazu (Kyushu Univ., Japan)

81

The Sixth Okazaki Biology Conference
Marine Biology
Organizing Chair : Noriyuki Satoh (Kyoto University, Japan)
Billie J. Swalla (University of Washington, USA)
Don R. Levitan (Florida State University, USA)
December 2 (Sun) – 8 (Sat), 2007
We conceived this conference in order to bring together
Marine Scientists from across the globe to discussapproaches to research in Marine Sciences. Most Marine
Science is carried out at Marine Biological Laboratories, so
we invited a number of Marine Laboratory Directors from
Canada, France, Italy, Norway, Sweden and Japan to get an
idea of what sort of studies are being conducted globally.
Marine Biology covers an enormous number of research
topics and we endeavored to bring together researchers in allof the main fields: reproductive biology, evolution anddevelopment, neurobiology, marine fungi/algae, marine
genomics, behavior, ecology and conservation biology. This
diverse group of speakers is united by their research on
marine systems and the fact that they are internationallyknown for the quality of their research. We expected thisgroup of brilliant leading Marine Scientists to find much incommon to share about research techniques, approaches andfuture prospects and were delighted to see the many positive

interactions that took place among the participants.
The diverse research presented at the OBC-6 Marine
Biology Conference showed the vast potential of the marine
environment in biological studies and highlighted current
ecological problems that threaten the unprecedented
biodiversity and untapped knowledge in the world’s oceans.
We heard many talks about the advances in genomics andmarine exploration applied in novel and creative ways tounderstanding the marine environment and the biologicalorganisms that live within it. The talks described research ona wide range of life forms from marine algae to invertebrates
to vertebrates.
Towards the end of conference, it was suggested that an
academic society for Marine Biologists in Japan beorganized in the very near future. This may prove to be oneof the most important achievements of this highly successful
conference.

Scientific topics:
Reproduction
Evolution and Development
Neurobiology and Physiology
Marine Algae and Fungi
Marine Genomics
Behavior
Ecology

Speakers
BERNARDI, Giorgio (Stazione Zoologica Anton Dohrn, Italy), BOYEN, Catherine (CNRS & Univ. Paris 6, France),
CHOURROUT, Daniel (Univ. Bergen, Norway), KLOAREG, Bernard (CNRS, Universit&eacute, France),
KNOWLTON, Nancy (Smithsonian Inst., USA), LEVITAN, Don R. (Florida State Univ., USA),
MATZ, Mikhail V. (Univ. Texas, Austin, USA), SARDET, Christian (CNRS UPMC, France), SPENCER,
Andrew N. (Malaspina Univ.-College, Canada), SWALLA, Billie J. (Univ. Washington, USA), THORNDYKE,
Michael C. (The Royal Swedish Academy of Sci., Sweden), VIZE, Peter D. (Univ. Calgary, Canada), WIDDER,
Edith A. (Ocean Research & Conservation Assoc., USA)AKASAKA, Koji (Univ. Tokyo, Japan), HARADA, Yoshito (Sugashima Marine Biological Laboratory, Japan),
HIDAKA, Michio (Univ. Ryukyus, Japan), HOSHI, Motonori (Open Univ., Japan), INABA, Kazuo (Univ. Tsukuba, Japan),
KIYOMOTO, Masato (Ochanomizu Univ., Japan), KUSAKABE, Takehiro G. (Univ. Hyogo, Japan),
NAKAMURA, Masaru (Univ. Ryukyus, Japan), NARUSE, Kiyoshi (NIBB, Japan), NOZAKI, Masumi (Niigata Univ., Japan),
OKAMURA, Yasushi (Okazaki Inst. Integrative Biosci., Japan), OTA, Kinya G. (RIKEN CDB, Japan),
SAGA, Naotsune (Hokkaido Univ., Japan), SAKAMOTO, Tatsuya (Okayama Univ., Japan), SATO, Katsufumi (Univ. Tokyo, Japan),
SATOH, Nori (Kyoto Univ., Japan), SAWADA, Hitoshi (Nagoya Univ., Japan), TAKEI, Yoshio (Univ. Tokyo, Japan),
TSUKAMOTO, Katsumi (Univ. Tokyo, Japan), UEDA, Hiroshi (Hokkaido Univ., Japan), UENO, Naoto (NIBB, Japan),YASUI, Kinya (Hiroshima Univ., Japan)

82

The Fifth NIBB-EMBL Meeting
Cell and Developmental Biology
Organizing Chair : Naoto Ueno (NIBB)
Stephen Cohen (EMBL Heidelberg, Germany)
May 24 (Thu) – 26 (Sat), 2007
The 5th NIBB-EMBL joint meeting, entitled “Cell andDevelopmental Biology”, was held in Okazaki on May 2426, 2007. The scientific sessions were organized inconsideration of the recent trend of understanding macrodevelopmental phenomena at the micro/single cell level.
During the two years since the 1st NIBB-EMBL joint
meeting, “Developmental Biology”, we have graduallyrealized the importance of understanding complex
developmental processes as an integration of individual cell
behaviors. During this most recent joint meeting, thecytoskeltal rearrangement underlying cellularmorphogenesis and movement, the cellular mechanism ofcell polarity, cell-to-cell interaction in organogenesis, andother related topics were discussed in a variety ofdevelopmental contexts. NIBB and EMBL share a common

interest in these topics and it is hoped that further
international collaboration between our institutions will
result from 2007’s fruitful and stimulating meeting.
Hoping to catch the big wave of genomics of many
experimental organisms now sweeping the worldwide
scientific community, we also aimed in this meeting to
explore the possibilities of genome-wide biology unveiling a“Gene Regulatory Network” (GNR) of development. GRN
is a key to understanding not only the structure of gene
regulatory pathways but also the evolutional capacity thatgenerates biodiversity. Several speakers covered this topic
and presented insightful talks, generating a great deal ofinterest in the future of “Systems Biology” among thoseattending.

Speakers
BRUNNER, Damian (EMBL Heidelberg, GERMANY), EPHRUSSI, Anne (EMBL Heidelberg, GERMANY),FURLONG, Eileen (EMBL Heidelberg, GERMANY), KNOP, Michael (EMBL Heidelberg, GERMANY),SPITZ, Francois (EMBL Heidelberg, GERMANY), WITTBRODT, Jochen (EMBL Heidelberg, GERMANY),ROSENTHAL, Nadia (EMBL Monterotondo, ITALY)AIZAWA, Shin (Riken CDB, JAPAN), HASEBE, Mitsuyasu (NIBB, JAPAN), HAYASHI, Shigeo (Riken CDB, JAPAN),
KAGEYAMA, Yuji (NIBB, JAPAN), KAIBUCHI, Kozo (Nagoya Univ., JAPAN), KINOSHITA, Noriyuki (NIBB, JAPAN),
KOBAYASHI, Satoru (NIBB, JAPAN), MURATA, Takashi (NIBB, JAPAN), NARUSE, Kiyoshi (NIBB, JAPAN),
NOJI, Sumihare (Univ. Tokushima, JAPAN), OKADA, Kiyotaka (NIBB, JAPAN), SAGA, Yumiko (NIG, JAPAN),
SATOH, Nori (Kyoto Univ., JAPAN), TABATA, Tetsuya (Univ. Tokyo, JAPAN), TAKADA, Shinji (NIBB, JAPAN),
TAKAHASHI, Yoshiko (NAIST, JAPAN), TAKEDA, Hiroyuki (Univ. Tokyo, JAPAN), TAMURA, Koji (Tohoku Univ., JAPAN),TANAKA, Minoru (NIBB, JAPAN), UEMURA, Tadashi (Kyoto Univ., JAPAN), UENO, Naoto (NIBB, JAPAN),
YAMAMORI, Tetsuo (NIBB, JAPAN)

83

INDEXA
AJIOKA, Rie56, 7658

HIRAKAWA, Eri

16

AKANUMA, Takashi

27

HIROKAWA, Junya

36

AOKI, Yumiko

30

HIROSE, Mana

63

AOSHIMA, Akihiro

33

HIWATASHI, Yuji

50

AOYAMA, Tsuyoshi

50

HIYAMA, Takeshi

33

ARAI, Yuko

8

HONDA, Satoko

ASAO, Hisayo

44

HORIUCHI, Takashi

3, 7, 47

HOSHINO, Atsushi

44

BABA, Takashi

19

CHEN, Qiuhong

27

ICHIKAWA, Chiaki

73, 79CHOW, Pak Hong Jeremy

33

ICHIKAWA, Mariko78

CHUNG, Hee-Jung

63

ICHIKAWA, Rie

11

CHUNG, Hyeyoung

21

ICHIKAWA, Takehiko

CUI, Jianzhou

16

ICHIKAWA, Yoko

CUI, Songkui

8

ICHIKAWA, Yukina

B

24

I

C

ICHIHASHI, YasunoriE

IGHIKAWA, Mariko

61

71
76, 7950
79

EMDE, Jason R.

72

IGUCHI, Taisen

3, 58

EUN, Chang-Ho

44

IHARA, Masaru

33

F

IIDA, Mayu

24
3, 44, 78

FUJIKAWA, Akihiro

33

IIDA, Shigeru

FUJIKI, Yuki

11

IINUMA, Hideko

79, 80

FUJITA, Hironori

67

IIOKA, Hidekazu

21

FUKADA, Masahide

33

IKEGUCHI, Hiromi

50

IMAI, Akiko

36

8

IMAIZUMI, Taeko

58

7, 19

IMAMURA, Hisako

67

79

IMAMURA, Takuya

39

ISHIHARA, Shuji

67

21

ISHII, Junko

11

GOTO, Shino8

ISHIKAWA, Azusa

19, 73, 79

GOUNO, Hiroe

19

ISHIKAWA, Mika

27

GUAN, Guijun

16

ISHIKAWA, Naoko

61

ISHIKAWA, Takaaki

50

ISHIKAWA, Takako

36

FUKADA-TANAKA, Sachiko
FUKAZAWA, Mitsue
FUKUI, Yuko
FURUKAWA, Kazuhiko44, 79G
GODA, Tadahiro

H
HAMADA, Yoshio

15, 73

HAMASAKI, Maho

11

ISHIMARU, Yoshiyasu

19

HANADA, Takao

11

ISHITANI, Shizuka

27

HARA, Ikuyo

16

ITAZU, Masako

HARA, Reiko

32

ITO, Takayo

HARA, Yoko

11

HARASHIMA, Toshiaki

11

47
79, 80IWATA, Ayumi

63

J

HASEBE, Mitsuyasu

3, 50

JIANG, Gonghao

44

HASEGAWA, Noriko

50

JOHZUKA, Katsuki

47

HASEGAWA, Yoshinobu

44

JOHZUKA-HISATOMI, Yasuyo

44

HASHIMOTO, Yoshiko

24

HASHIYAMA, Kazuya

24

KABEYA, Yukiko

HATTORI, Nobuko

33

KADOWAKI, Tamaka

61

76, 79KAGAWA, Takatoshi

63

HAYASHI, Makoto (Division of Cell Mechanisms)

8

KAGEYAMA, Takuya

11

HAYASHI, Makoto (Division of Developmental Genetics)

24

KAGEYAMA, Yuji

HAYASHI, Tomoko

19

KAJIURA-KOBAYASHI, Hiroko

HIGASHI, Sho-ichi

73, 79HAYASHI, Kohji

84

HINAGO, Megumi

K

KAMADA, Yoshiaki

11, 7924
71, 7911M

KAMIGAKI, Akane

8

KAMIYA, Akemi

71

MAEDA, Maki

39

KAMIYA, Kiyomi

80

MAEDA, Masato

21

KANAI, Masatake

8

MAEDA, Yumi

79

KANDA, Rieko

71

MAEZAWA, Takanobu

KANEKO, Hiroyo

16

MAKINO, Yumiko

KANEUJI, Kimie

80

MANO, Shoji

8

KATAOKA, Yukari

79

MASUOKA, Tomoko

50

KATAYAMA, Tomomi

33

MATSUDA, Chisato

44

KATO, Kyoko

8

MATSUDA, Masaru

16

KATO, Yasuhiko

58

MATSUDA, Yoshimi

79, 80KATSU, Yoshinao

58

MATSUMOTO, Miwako

44

KATSUKI, Motoya

7

MIKAMI, Yuriko

47

KATSURA, Yukako

19

MIURA, Seiko

53

KAWAMATA, Tomoko

11

MIWA, Tomoki

73, 79KAWAMURA, Motofumi

76

MIYABAYASHI, Kanako

19

KIKUCHI, Naoka

58

MIYAKE, Satoko

21

KINOSHITA, Chie

30

MIYAZAKI, Saori

50

KINOSHITA, Noriyuki

21

MIZOGUCHI, Masae

33

KITADATE, Yu

24

MIZOIRI, Shinji

KOBAYASHI, Kaoru

58

MIZUTANI, Takeshi

58, 79KOBAYASHI, Kayo30

MOCHIZUKI, Atsushi

67, 75KOBAYASHI, Satoru

24

MOHAMAD, Zubair

KOBAYASHI-NISHIMURA, Keiko76

MORI, Tomoko

KODAMA, Akiko

33

MORI, Yuji

47

MORIBE, Hatsumi

KODAMA, Ken-ichi
KODAMA, Ryuji

55, 72

KODAMA,Yutaka

24
78, 7953

1978, 7939
78, 79MORINAGA, Shin-ichi

50

63

MORITA, Hitoshi

21

KOIKE, Yukari

19, 56

MORITA, Junko

36

KOJIMA, Yoko

50, 61MORITA, Yasumasa

44
44

KOMATSU, Tomoko

19

MORITOH, Satoru

KOMATSU, Yusuke

36

MOROHASHI, Ken-ichirou

7, 19

KOMINE, Yuriko

36

MOROOKA, Naoki

47, 79KONDO, Chika

11

MUKAI, Masanori

7, 24

KONDO, Maki

8, 79

MUKOHDA, Yasuyo

72

KONDO, Takefumi

24

MURATA, Takashi

50

KONDO, Yukie

79

N

KONG, Sam-Geun

63

KOSHIDA, Sumito

7, 27

NAGAKURA, Ayano

33

KOUMURA, Toshiro

44

NAGURA, Masako

61

KOUNO, Yukie

19

NAKAGAMI, Yuki

36

KUBOKI, Yuko

8

NAKAMORI, Chihiro

8

KUMATA, Yuji

24

NAKAMURA, Makoto

21

KURATA, Tomoko

72

NAKAMURA, Shuhei

KURITA, Kayoko

16

NAKAMURA, Takanori

73, 79KUROKAWA, Hiromi

30

NAKAMURA, Takeshi

58

KUROYANAGI, Asuka

78, 79NAKAMURA, Tohru

36

NAKASHIMA, Machiko76

NAKATOGAWA, Hitoshi

11
61

KUSAKA, Masatomo

19

L

NAGAHAMA, Yoshitaka

3, 16, 72

30

LANGE, Anke

58

NAKAYAMA, Hokuto

LAU, En-Lieng16

NAKAZATO, Kenichi

LEE, Rebecca

21

NANBA, Chieko

73, 79LI, Jianzhong

16

NARUSE, Kiyoshi

7, 56, 76

67

85

INDEXNIIMI, KaoriNISHIDE, Hiroyo

11

SATO, Asako

4273, 79

SATO, Hiroaki

39

SATO, Kaori

24

SATO, Kimihiro

24

NISHIMURA, Keiko30

NISHIMURA, Mikio

3, 8, 72, 73

NISHIMURA, Noriko

79

SATO, YoriNISHINA, Momoko

8

SAWADA, Kaoru

79, 8019

SEKIHARA, Sanae

19

24, 79SEKITO, Takayuki

11

NISHIYAMA, Aya
NODA, Chiyo
NODA, Masaharu

3, 33, 80

SEKITOH, Rie

16

NOGUCHI, Yuji

76

SHEN, Xueyan

56

NONAKA, Shigenori

71

SHIBATA, Chitose

27

SHIBATA, Emiko

16

11

SHIBATA, Yasushi

16

OGASAWARA, Kimi

8

SHIGENOBU, Shuji

7, 24

OGAWA, Hidesato

19

SHIMA, Yuichi

19

14, 80SHIMADA, Yu

16O
OBARA, Keisuke

OGAWA, Kazuo

16

SHIMAMOTO, Miki

44

OHSAWA, Sonoko

36, 79SHIMATANI, Zenpei

44

OHSUMI, Yoshinori

3, 7, 11SHIMIZU, Hidetada

33

OHTA, Kohei

16

SHIMIZU, Mineko

63

OIKAWA, Kazusato

8

SHIMOKAWA, Hiroko

39

SHINDO, Asako

21

OHNO, Kaoru

OKA, Sanae

19, 79OKADA, Kiyotaka

1, 3, 7, 32

OKADA, Norihiro

53

SINGH, Tanuja

8

OKADA, Tokindo S.

7

SUETSUGU, Noriyuki

63

OKAMOTO, Haruko

47

SUGIURA, Mio

OKAMOTO, Koji

11

SUMIKAWA, Naomi

OKAMOTO, Noriko

11

SUZUKI, Aya

OKANO, Yosuke

50

SUZUKI, Iku

OKU, Masahide

11

SUZUKI, Keiko

OKUBO, Kataaki

16

SUZUKI, Kuninori

OKUBO, Tadashi

7, 27

SUZUKI, Ryoko

33

OKUDA, Tadayoshi

76

SUZUKI, Tokiko

53, 56

ONO, Akemi

44

SUZUKI, Yuko

11

OOOKA, Kyoko

11

OTA, Misaki

72

P

SHINTANI, Takafumi

7, 33

19
50, 7916
8
73, 79
11

T
TAKADA, Ritsuko

27

TAKADA, Shinji

3, 27, 75, 76
21, 79

PARK, Kyeung-Il

44

TAKAGI, Chiyo

PAUL, Bindhu

16

TAKAGI, Kyoko

44

TAKAGI, Yukari76, 79S
SADAKANE, Osamu

36

TAKAHASHI, Eri

58

SAITO, Daisuke

30

TAKAHASHI, Hiroki

21

SAITOH, Hiromi

44

TAKAHASHI, Hiroyuki

27

32, 79TAKAHASHI, Jun

27

SAKAI, Fumie

16

TAKAHASHI, Kazuhiko

53

SAKURABA, Jyuichi

33

TAKAHATA, Toru

36

SAKUTA, Hiraki

33

TAKAJI, Masafumi

36

SANDVIK, Guro

16

TAKAKI, Chikako

16

SANJO, Kazuko

44

TAKAMATSU, Kaori

SASAKI, Michiko

27

TAKAMI, Shigemi

78, 79SASAKI, Takeshi

53

TAKASE, Masahide

61

SASAKI, Tetsuya

36

TAKASHIRO, Kayoko

27

42, 76

TAKESHITA, Miyako

73, 79SAKAGAMI, Mari

SASAOKA, Toshikuni

86

8

63

TAKEUCHI, Yasushi

33, 79YAMAGUCHI, Takahiro

7, 61

TANAHASHI, Takako

32, 50

YAMAMORI, Tetsuo

3, 36

YAMAMOTO, Hayashi

11

TANAKA, Megumi

72

TANAKA, Minoru

30, 76

YAMAMOTO, Kumi

73, 79TANEMURA, Hiroyo

58

YAMAMOTO, Takamasa

21

TANI, Sachiko

33

YAMASATO, Akihiro

63

TANIGUCHI, Ena

58

YAMAUCHI, Daisuke

63

TAO, Hirotaka

21

YAMAUCHI, Takaki

44

TERADA, Rie

44

YANO, Satoshi

61

TERASAKA, Chie

21

YASUDA, Mie

76

TOITA, Shigeko

36

YATAGAI, Hiroko

33

TOMIDA, Sanai19

YATSU, Jun

24

TOMIKAWA, Junko

39

YONEHARA, Kayo33

TOMITA, Nao

33

YONEHARA, Keisuke

33

TORII, Naoko

56

YONEMITSU, Masako

30

TSUBOI, Hidenori

63

YOSHIKANE, Nami

21

TSUCHIDA, Yuhei

32

YOSHIKUNI, Michiyasu

7, 16

TSUGANE, Kazuo

44

YOSHIMOTO, Kohki

11

TSUGANE-HAYASHI, Mika

44

YOSHINORI, Yumi

8

TSUGE, Toyoko

21

TSUKAMURA, Hiroko

39

ZHANG, Lanfan

33

TSUKAYA, Hirokazu

7, 61

ZHAO, Haobin

16

TSUKESHIBA, Kumi

11

ZHOU, Linyan

16

TSUZUKI, Chisa

27

TSUZUKI, Shihoko

79

TSUZUKI, Yukiko

50

UCHIYAMA, Ikuo

69, 73ZU
8

UEDA, Chizuru
UENO, Naoto

3, 21, 72

UKAI, Sakie

27, 30UMEBAYASHI, Hiromi

67, 75

UNO, Satoko

79

URUSHITANI, Hiroshi

58

USAMI, Takeshi (Division of Reproductive Biology)

16

USAMI, Takeshi (Division of Plant Developmental Genetics)

61

UTSUMI, Hideko

27, 79

WADA, Masamitsu

7, 63, 72

W
WANG, De-shou

16

WATAKABE, Akiya

36

WATAKABE, Ikuko
WATANABE, Eiji
WATANABE, Hajime

30
41, 7658

WATANABE, Masakatsu

65, 73WATANABE, Taka-aki

7, 47

Y
YABE, Taijiro

27

YAMADA, Kenji (Division of Cell Mechanisms)

8

YAMADA, Kenji (Division of Behavioral Biology)

39

YAMADA, Shigehiro

21

YAMAGUCHI, Chinami

61

YAMAGUCHI, Katsushi

44, 78, 7987