INTRODUCTION

ur institute, the National Institute for Basic
Biology (NIBB), has developed as a center of
excellence in research, education, and inter-university
collaboration in the various fields of basic biology
since its foundation in 1977. In 2004, NIBB and four
national research institutes established a neworganization, the National Institutes of Natural
Sciences (NINS), one of the four Inter-University
Research Institute Corporations. In 2008, we gladly
had two professors joining our institute, and are now
covering a wide variety of fields including cellbiology, developmental biology, neurobiology,
evolutionary biology and biodiversity, environmental
biology, theoretical biology and imaging science, andare studying the basic action of genes and cells
common to all living organisms, as well as themechanisms involved in producing the diversified
structures and activities of life adapting to an everchanging environment. Research in these basic
biological fields is maintained at a high level while wecontinue to search for new and challenging fields ofstudy. We are also involved in ongoing collaboration
with a number of researchers from Japanese andforeign universities and institutes. In addition toresearch, the education of the next generation ofresearchers is another important aim. As a member of
the Graduate University for Advanced Studies(SOKENDAI), we are involved in training graduate
students. We will have three new doctors in March of2009.
NIBB's activities during 2008, as well as our goals
for the future, are summarized in this booklet. We arealways open to questions, comments and suggestions.

O

Kiyotaka OKADA, D. Sci.
Director-General, NIBBJan 12, 2009

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
NIBB conducts its research programs through thirty-one
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 throughout Japan. The Technical Division
manages the activities of the technical staff and helps topromote the research activities of each research unit and tomaintain the common research resources of both theresearch support units of NIBB and some of the researchfacilities 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.

Personnel in NIBB
Director-General 1
Technical
Assitant
andSecretary

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

Technical
Staff

Professor (including adjunct, concurrent
and specially appointed) 18
Associate Professor (incl. adj.) 12

84

Assistant Professor (incl. sp. app.)

12

27

95
41

Postdoctoral Fellow(as of 1st August, 2008)

Graduate Student

Policy, Decision Making, and Administration
The Director-General oversees the operation of NIBB,
assisted by the Advisory Committee for Programming andManagement. The Advisory Committee, comprised ofprofessors within NIBB and an equal number of leading
biologists outside NIBB, advises the Director-General on
important matters such as planning joint research programs
as well as on the scientific activities of NIBB. The Advisory
Committee makes recommendations on the appointments of
new Director-Generals, faculty appointments, 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 long-range strategies for the institute.
Administration of NIBB is undertaken by the Okazaki
Administration Office under the direct auspices of the
Administration Office of NINS.

2305 in total

Financial Configuration of NIBB

Donation 26
External
Competitive
Fund(settled forApril 2007- March 2008)
numbers are in million yen

Grants forCommissioned
Research

318

1103

Grant-in-Aid

3,062 million yen in total

Management
Expenses Grant

1302

Research

759
Facilities
Maintenance
Grant

565
Self Income

33

Expenses Grant
for SOKENDAI 59

■ 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
YAMAMORI, Tetsuo

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
Vice-Chair
Non-NIBB member

NIBB member

OHSUMI, Yoshinori
IWASA, Yoh
HASEGAWA, Mariko
ISHINO, Fumitoshi
KATO, Kazuto
MATSUOKA, Makoto
OSUMI, Noriko
SAKANO, Hitoshi
SATOH, Noriyuki
TABATA, Satoshi
HASEBE, Mitsuyasu
HORIUCHI, Takashi
IGUCHI, Taisen
IIDA, Shigeru
KOBAYASHI, Satoru
NISHIMURA, Mikio
NODA, Masaharu
TAKADA, Shinji
UENO, Naoto
YAMAMORI, Tetsuo(as of April 1st, 2008)

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
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology

3■ National Institute for Basic Biology

Research Units■ Division of Cell Mechanisms

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

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

Neurobiology■ Division of Brain Biology■ Laboratory of Neurophysiology■ Laboratory of Neurochemistry

Evolutionary Biology and Biodiversity■ Division of Molecular Genetics■ Division of Genome Dynamics■ Division of Evolutionary Biology■ Laboratory of Morphodiversity■ Laboratory of Bioresources■ 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

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

Research Center for Integrative andComputational Biology

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 NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) has set
five goals for its activities in pursuing the progress of
biology. We envision contributing to the world-widecommunity of biologists through our efforts to accomplishthese goals. This chapter briefly explains four of the goals.
The last goal, the promotion of academic research, isaccomplished through our research activities, which are
introduced throughout this brochure.

Promotion of Collaborative Research■ Collaborative Research Projects

Research projects in collaboration with NIBB’s
divisions/laboratories and research activities to be conductedusing 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.
Six projects have already been carried out, including“molecular mechanisms for controlling the individuals ofhigher plants.” The category “collaborative research projects
for model organism/technology development” wasestablished in 2007 with the aim of developing andestablishing new model organisms. Five projects havealready been carried out, including “development of thetransgenic strain of Cabombaceae (primitive angiosperm).”
NIBB always encourages discussion on such projects in the
belief that the methods of conducting collaborative research
projects must be constantly modified, according to thedemands of the age and the biology community.

International
cooperation
Contribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers

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

International Cooperation■ Collaborative research projects with EMBL

The European Molecular Biology Laboratory (EMBL),
established in 1974, is a research institute funded by 18
European nations. It conducts comprehensive, high-levelbasic research programs and leads the world in the field of
molecular biology. NIBB takes a leading role in thecollaborative research programs between EMBL and the
National Institutes of Natural Sciences (NINS), which werelaunched in 2005, and promotes personal and technologicalexchange through symposia, exchange between researchersand graduate students, and the introduction of experimental
equipment (see page 81 for details of the NIBB-EMBL joint
meetings held in 2008).

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

Collaborative research projects by year
year

2005 2006 2007 2008

Priority collaborative research projects

2

3

1

0

Collaborative research projects formodel organisms / technology development

−

−

2

3

Individual collaborative research projects

41

37

43

49

NIBB workshops

4

1

5

5

Collaborative experiments using the Large Spectrograph

19

18

14

11

Facility Use (Center for Analitical Instruments)
total

0

0

1

0

66

59

66

68

■ NIBB Conference

The NIBB Conference is an international conference
organized by NIBB’s professors once or twice a year withthe participation of a guest lecturer from abroad. Since thefirst conference in 1977 (the year of NIBB’s foundation), the
NIBB Conference has provided researchers in basic biology
with valuable opportunities for international exchange (seepage 78 for details of the NIBB Conferences held in 2008).
■ 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
establishment 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
program, is given at a laboratory specifically prepared for its
use at NIBB. The second and third courses were held in2008 (see page 83). Graduate students and young
researchers from various areas including China, Hong Kong,
Taiwan, and Korea, were provided with training in state-ofthe-art research techniques.

5

■ Bio-Resources

The National BioResource Project (NBRP) is a national
project for the systematic accumulation, storage, and supplyof nationally recognized bio-resources (experimental
animals and plants, cells, DNA, and other genetic
resources), which are widely used as materials in life science
research. To promote this national project, NIBB has been
appointed as a center for research on "Medaka (Oryzia
latipes)" whose usefulness as a vertebrate model firstdeveloped in Japan. The usability of Medaka as a researchmaterial in biology has drawn increasing attention since its
full genome sequence recently became available. NIBB isalso a sub-center for the NBRP’s work with Japanesemorning glory and Zebrafish. In addition, NIBB provides
databases containing research data on the moss
Physcomitrella patens, Daphnia, Xenopus laevis, plant cellorganelles, and bacterial genomes.

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

Development of New Fields of Biology■ Bioimaging

Recently, the capability of optical microscopes has greatly
improved, and biophotonic probes have 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. 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, NIBB aims to be acenter for developing microscopes and biophotonic
probes.
2) Advisory Committee on Bioimaging, Regular meetings
are held with several leading researchers in the
bioimaging field in Japan to formulate advice on
imaging research.
3) Bioimaging Forum, This provides an opportunity for
researchers at NIBB, members of the Advisory
Committee, and company engineers to frankly discuss
practical difficulties and needs regarding imaging.
4) Introduction of DSLM (Digital Scanned laser Lightsheet Microscopy), As part of collaborative work with
EMBL, NIBB has introduced DSLM, which is effective
for the three-dimensional observation of living samples,and is the first of its kind in Japan.
5) Bioimaging Symposium, This provides an opportunity
for academic exchanges with cutting-edge overseas
researchers in the imaging field, mainly from EMBL.

6

Optical path of the DSLM

■ Okazaki Biology Conferences

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.

Cultivation of Future Researchers
NIBB sponsors two education programs for graduate
students.
1. Graduate University for Advanced Studies
NIBB constitutes the Department of Basic Biology in
the School of Life Science of the Graduate University
for Advanced Studies (SOKENDAI). The department
provides a five-year course for university graduates anda three-year doctoral course for graduate students witha master’s degree.
2. Graduate Student Training Program
Graduate students enrolled in other universities andinstitutions can apply to be special research students
eligible to conduct research for fixed periods of time
under the supervision of NIBB professors.
In both cases above, graduate students can live an
academic life and receive financial support from NIBB
based on the research assistant (RA) system.

Graduate students educated by NIBB
number
80
70
19

60

16

50

20

18

40

3

5

6

4

46

44

12
3

18

42

37

0

30
20

35

36

2002

2003

27

10
0

Department of Basic Biology

11
0

2004

2005

2006

School of Advanced Science (educated in NIBB)

2007

2008

Special research student

■ Personnel changes in 2008*

Newly assigned in NIBB
Name

Position

Research Unit

Date

TATEMATSU, Kiyoshi

Assistant Professor

Laboratory of Plant Organ Development

March 1

HAYASHI, Yoshiki

Assistant Professor

Division of Developmental Genetics

April 1KURATA, Tomoko

Assistant Professor(Specially appointed)

Strategic Planning Department

April 1

FUJIMORI, Toshihiko

Professor

Division of Embryology

August 1

YOSHIDA, Shosei

Professor

Division of Germ Cell Biology

August 1

TOYOOKA, Yayoi

Assistant Professor

Division of Embryology

October 1

SUZUKI, Makoto

Assistant Professor

Division of Morphogenesis

October 1

Newly affiliated in other universitites and institutes
Name

New Affiliation

Position

Date

IMAMURA, Takuya

Kyoto University

Associate Professor

February 29

OKUBO, Kataaki

University of Tokyo

Project Assistant Professor

March 31

TANAHASHI, Takako

Japan Patent Office

Jr. Patent Examination Official

March 31

NAKAMURA, Makoto

Matsuyama University

Associate Professor

March 31MOCHIZUKI, Atsushi

RIKEN Advanced Research InstituteChief Scientist

June 30

OGAWA, Hidesato

Natl. Inst. Inform. Commun. Tech.

Research Scientist

July 31* Changes in professors, ascociate and assistant professors are shown.

■ Awardees in 2008

Name

PositionAward

KITADATE, Yu

Postdoctoral Fellow

Inoue Research Award for Young Scientists

SHIMIZU, Hidetada

Postdoctoral Fellow

SOKENDAI's Nagakura Research Incentive AwardYAMAGUCHI, Takahiro

Assistant Professor

The Young Scientist Award for Plant Morphology

HASEBE, Mitsuyasu

Professor

The Research Award of the Botanical Society of Japan

FUJIKAWA, Akihiro et al.

Postdoctoral Fellow

The JB Award of the Japanese Biochemical Society

EGUCHI, Goro

Professor Emeritus

The Order of the Sacred Treasure, Gold and Silver Star

Note: On the unit member lists from P. 8 to P. 77 all members who belonged to the unit during 2008 are listed irrespective of the length of the period they weremembers. Those appearing twice in the same list under different titles are those whose title changed during 2008. The former title is indicated by an asterisk (*).

7

National Institute for Basic Biology

Cell Biology

DIVISION OF CELL MECHANISMS

Professor

Associate Professor

NISHIMURA, Mikio

HAYASHI, Makoto

Assistant 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
KANAI, Masatake
GOTO, Shino
CUI, Songkui
SHIBATA, Michitaro
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.

senescence. Gene expression, alternative splicing, proteintranslocation and protein degradation control the functionaltransformation between glyoxysomes and leaf peroxisomes.

Ⅱ. Transcriptomics, proteomics and phenomicsof plant peroxisomes
Enzymes localized in plant peroxisomes are synthesized in
the cytosol and function after their post-translationaltransport into peroxisomes. Almost all of the peroxisomal
matrix proteins contain one of two targeting signals (PTS1and PTS2) within the molecules. PTS1 is a unique tripeptide
sequence found in the carboxyl terminus of the mature
proteins. In contrast, PTS2 is involved in a cleavable amino
terminal presequence of peroxisomal proteins that aresynthesized as a precursor protein with larger molecularmass.
We identified 256 gene candidates of PTS1- and PTS2containing proteins and another 30 genes of non-PTScontaining proteins from Arabidopsis genome. Custommade DNA microarray covering all these genes was used toinvestigate expression profiles of the peroxisomal genes invarious organs. It revealed that peroxisomes in root cells
plays a pivotal role in polyamine catabolism. We also madea two-dimensional protein map of glyoxysomes and leaf
peroxisomes isolated from Arabidopsis and soybean. Peptide
MS fingerprinting analyses allowed us to identify novel
peroxisomal membrane proteins, i.e. voltage-dependentanion-selective channel and adenine nucleotide carrier 1(PNC1) (Figure 1). The overall results provide us new
insights into plant peroxisomal functions.

Ⅰ. Reversible transformation of plant peroxisomes
Dramatic metabolic changes that underlie the shift fromheterotrophic to autotrophic growth occur in the greening ofseed germination. Accompanying these metabolic changes
are the functional transformations of many constitutive
organelles. Etioplasts differentiate into chloroplasts while
mitochondria acquire the ability to oxidize glycine.
Glyoxysomes, which are peroxisomes engaged in the
degradation of reserve oil via β-oxidation and theglyoxylate cycle, are transformed into leaf peroxisomes thatfunction in several crucial steps of photorespiration. Afterthe functional transition of glyoxysomes to leaf peroxisomesduring the greening of pumpkin cotyledons, the reverse
transition of leaf peroxisomes to glyoxysomes occurs during

8

Figure 1. Proteomic identification and characterization of a novel
peroxisomal adenine nucleotide carrier 1 (PNC1). (A) 2-D map ofproteins in the digitonin-soluble fraction of purified peroxisomes. A 2-D
gel in which the first dimension was Blue Native (BN)-PAGE and thesecond was SDS-PAGE is shown. Protein spots were detected by silver
staining. (B) Phenotype of the pnc1/2 RNAi knockdown mutant.
Seedlings grown on medium without sucrose under constant illuminationfor 7 days.

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. Phenotypes of these mutants allowed us to
identify the functional PEX genes, which can be classified
into two groups: PEX genes regulating for peroxisomal
protein import and PEX genes regulating for peroxisomal
morphology. Of these, PEX5 and PEX7 form a cytosolic
receptor complex and recognize PTS1- and PTS2-containing
proteins, respectively. PEX14 is a peroxisomal membrane
docking protein that captures the receptor-cargo complex.
We continue to investigate the detailed molecular functionsof other PEX genes.

Brassicaceae. NAI2 localizes to the ER body. In the nai2
mutant, PYK10 becomes uniformly diffused throughout the
ER (Figure 2). These findings indicate that NAI2 is a key
factor that enables ER body formation and the accumulationof PYK10 in ER bodies.

Ⅲ . Identification of novel components
essential for peroxisome biogenesis
To better understand peroxisome biogenesis, we isolated anumber of Arabidopsis mutants having aberrant peroxisome
morphology (apm mutants) based on a different pattern of
GFP fluorescence than the parent plant, GFP-PTS1, in
which peroxisomes with normal sizes and numbers can bevisualized with GFP.
Of these apm mutants, APM1 gene (whose defect causes
the elongation of peroxisomes and mitochondria) encodes
dynamin-related protein 3A (DRP3A), one member of the
dynamin family. Two other apm mutants, apm2 and apm4,
showed GFP fluorescence in the cytosol as well as inperoxisomes. We demonstrated that both mutants are
defective in PTS1- and PTS2-dependent protein transport toperoxisomes, and that APM2 and APM4 encode proteinshomologous to PEX13 and PEX12, respectively. It was
revealed that APM2/PEX13 and APM4/PEX12 arecomponents of the protein-translocation machinery on
peroxisomal membranes, and that they are involved inprotein transport from the cytosol into the peroxisome. Other
APM mutants are currently under investigation in order to
identify the components responsible for peroxisomebiogenesis and to address the mechanism at the molecularlevel.

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 (5 μm long and 0.5 μm wide)surrounded by ribosomes. ER bodies are widely distributedin the epidermal cells of whole seedlings. Undamaged
rosette leaves have no ER bodies, but accumulate ER bodies
after wounding or jasmonic acid treatment. This suggeststhat ER bodies function in the defense against herbivores.
ER bodies in seedlings include PYK10, a β-glucosidasewith an ER retention signal. Arabidopsis nai1 mutant has no
ER bodies in whole plants and does not accumulate PYK10.
NAI1 encodes a transcription factor that has a basic-helixloop-helix (bHLH) domain and regulates the expression of
PYK10. Arabidopsis nai2 mutant has no ER bodies andreduces the accumulation of PYK10. NAI2 encodes amember of a unique protein family that is only found in the

Figure 2. Localization of GFP-PYK10 in nai2 mutant. GFP-PYK10
fusion gene was transiently expressed in the epidermal cells of 6-day-old
wild-type (WT) and nai2 seedlings. GFP-PYK10 is localized in ER
bodies in wild-type plants; it becomes uniformly diffused, however, in
ER in the nai2 mutant. Bars = 10 μm.

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 is responsible for the maturation of various types of
vacuolar proteins. We revealed a novel function of VPE invarious programmed cell death (PCD) in plants. VPE is
identified as the proteinase that exhibits caspase activity inplants. 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 VPEdeficient tobacco plants. These results suggest that VPE is
involved in vacuolar collapse, which triggers PCD. Plants
evolve a death strategy mediated by a vacuolar system,
which is not seen in animals. Interestingly, a vacuolar
enzyme is the key player in a plant-specific cell deathsystem.

Ⅵ. Roles of molecular chaperones on celldifferentiation
Molecular chaperones are cellular proteins that function in
the folding and assembly of certain other polypeptides into
oligomeric structures. To clarify the roles of molecularchaperones on cell differentiation, we have purified andcharacterized chaperonin and Hsp70s and analyzed theirroles in the translocation of proteins into chloroplasts.
Recently, we found that HSP90 inhibitor induced the genes
with heat shock response element (HSE) motifs in their

9National Institute for Basic Biology

Cell Biology

promoters, suggesting that heat shock transcription factor(HSF) is involved in the response. Arabidopsis HSFs
interacted with HSP90. Thus, it appears that in the absenceof heat shock, HSP90 actively suppresses HSF function.
Upon heat shock, HSP90 is transiently inactivated, whichleads to HSF activation. This data indicates that HSP90
regulates correct gene expression for heat acclimatization inplants. We also observed that HSP90 is involved in hormone
responses in Arabidopsis. The evolutional and functional
characterizations are now being investigated.

Ⅶ. The Plant Organelles Database 2 (PODB2)– Release of Version 2 for plant organelles
dynamics and methods 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
for Environmental Adaptation in Plants.” We released
Version 2 (PODB2), which incorporated time-lapse and 3D
images. PODB2 consists of 4 individual parts: the organelles
movie database, the organellome database, the functional
analysis database, and external links (Figure 3). The
organelles movie database contains the videos for organelle
movements and 3D structures. The organellome databaseprovides images of various plant organelles that werevisualized with fluorescent and nonfluorescent probes invarious 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〕● Arai, Y., Hayashi, M., and Nishimura, M. (2008). Proteomic analysis

of highly purified peroxisomes from etiolated soybean cotyledons.
Plant Cell Physiol. 49, 526-539.
● Arai, Y., Hayashi, M., and Nishimura, M. (2008). Proteomic
identification and characterization of a novel peroxisomal adenine
nucleotide transporter supplying ATP for fatty acid β-oxidation insoybean and Arabidopsis. Plant Cell 20, 3227-3240.
● Hashimoto, K., Igarashi, H., Mano, S., Takenaka, C., Shiina, T.,
Yamaguchi, M., Demura, T., Nishimura, M., Shimmen, T., andYokota, E. (2008). An isoform of Arabidopsis myosin XI interacts
with small GTPase in its C-terminal tail region. J. Exp. Bot. 59, 35233531.
● Kamada-Nobusada, T., Hayashi, M., Fukazawa, M., Sakakibara, H.,
and Nishimura, M. (2008). A peroxisomal polyamine oxidase,
AtPAO4, is involved in polyamine back-conversion pathway inArabidopsis thaliana. Plant Cell Physiol. 49, 1272-1282.
● Kunieda, T., Mitsuda, N., Ohme-Takagi, M., Takeda, S., Aida, M.,
Tasaka, M., Kondo, M., Nishimura, M., and Hara-Nishimura, I.
(2008). NAC family proteins NARS1 and NARS2 in the outer
integument regulate embryogenesis in Arabidopsis. Plant Cell 20,
2631-2642.
● Oshima, Y., Kamigaki, A., Mano, S., Hayashi, M., Nishimura, M., andEsaka, M. (2008). Plant catalase is imported into peroxisomes by
Pex5p but distinct from typical PTS1 import. Plant Cell Physiol. 49,
671-677.
● Mano, S., Miwa, T., Nishikawa, S., Mimura, T., and Nishimura, M.
(2008). The plant organelles database (PODB): A collection ofvisualized plant organelles and protocols for plant organelle research.
Nucleic Acid Res. 36, D929-D937.
● Mitsuhashi, N., Kondo, M., Nakaune, S., Ohnishi, M., Hayashi, M.,
Hara-Nishimura, I., Richardson, A., Fukaki, H., Nishimura, M., andMimura, T. (2008). Localization of myo-inositol-1-phosphate synthase
to the endosperm in developing seeds of Arabidopsis. J. Exp. Bot. 59,
3069-3076.
● Nagano, A. J., Fukao, Y., Fujiwara, M., Nishimura, M., and HaraNishimura, I. (2008). Antagonistic Jacalin-related lectins regulate thesize of ER-body-type β-glucosidase in Arabidopsis thaliana. Plant
Cell Physiol. 49, 969-980.
● Nagano A. J., Fukazawa, M., Hayashi, M., Nishimura, M., and HaraNishimura, I. (2008). AtMap1: a DNA microarray for genomic
deletion mapping in Arabidoposis thaliana. Plant J. 56, 1058-1065.
● Tani, T., Sobajima, H., Okada, K., Chujo, T., Arimura, S., Tsutumi,N., Nishimura, M., Seto, H., Nojiri, H., and Yamane, H. (2008).
Identification of the OsOPR7 gene encoding 12-oxophytodienorate
reductase involved in the biosynthesis of jasmonic acid in rice. Planta
227, 517-526.
● Yamazaki, M., Shimada, T., Takahashi, H., Tamura, K., Kondo, M.,
Nishimura, M., and Hara-Nishimura, I. (2008). Arabidopsis VPS35, a
retromer component is required for vacuolar protein sorting andinvolved in normal growth and leaf senescence. Plant Cell Physiol. 49,
142-156.
● Yamada, K., Nagano, A. J., Nishina, M., Hara-Nishimura, I., andNishimura, M. (2008). NAI2 is an endoplasmic reticulum component
that enables ER body formation in Arabidopsis thaliana. Plant Cell
20, 2529-2540.

〔Review articles〕● Arai, Y., Fukao, Y., Hayashi, M., and Nishimura, M. (2008).

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

10interfaces

of

PODB2

Peroxisome. In Plant Proteomics: Technologies, Strategies andApplications, Agrawal, G. K., and Rakwal, R. eds. (John Wiley &Sons, Inc.) pp. 377-389.
● Yamada, K., and Nishimura, M. (2008). Cytosolic heat shock protein
90 regulates heat shock transcription factor in Arabidopsis thaliana.
Plant Signaling & Behavior 3, 660-662.

DIVISION OF MOLECULAR CELL BIOLOGY

Professor

OHSUMI, Yoshinori
KAMADA, Yoshiaki
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
Technical Staff
KABEYA, Yukiko
NIBB Research Fellows OBARA, Keisuke*
HANADA, Takao
Postdoctoral Fellows
OBARA, Keisuke
HANADA, Takao*
FUJIKI, Yuki
OKU, Masahide
HARASHIMA, Toshiaki
YAMAMOTO, Hayashi
OKAMOTO, Koji
KAKUTA, Soichiro
KOBAYASHI, Takafumi
Graduate Students
KAGEYAMA, Takuya
OH-OKA, Kyoko
Technical Assistants
KONDO, Chika
NIIMI, KaoriISHII, Junko
OKAMOTO, Noriko
ICHIKAWA, Rie
Secretaries
HARA, Yoko
SUZUKI, Yuko
Assistant Professors

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

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

Ⅱ. Lap3 is a selective target of autophagy inyeast
Although autophagy is, in principle, a non-selective
degradation process, several proteins and organelles were
found to be selectively incorporated into the
autophagosome. For instance, Ald6, a soluble cytoplasmic
enzyme in yeast, is preferentially eliminated from thecytoplasm via autophagy; Little is known, however, about
the mechanisms of Ald6 targeting to autophagosomes.
Autophagy is also involved in the selective transport of
Ape1, a vacuolar hydrolase, as a biosynthetic route.
Recently, we discovered that leucine aminopeptidase III(Lap3), a soluble cytosolic cysteine protease, was spatiallyassociated with Ape1 and selectively transported to thevacuole during nitrogen starvation (Figure 1). The rate of
Lap3 transport was much higher than that of Ald6 and was
similar to that of Ape1. Moreover, Atg11 and Atg19,
essential factors for Ape1 transport, were important for Lap3transport. Most of the Lap3 was degraded within a couple ofhours in the vacuole, in contrast to Ape1; therefore, we
concluded that the machinery required for Ape1 biosynthesis
is used for the selective degradation of Lap3.

Figure 1. Microscopic analyses of Lap3. (A) Localization of GFP-Lap3 in
pep4Δ atg7Δ (A) and pep4Δ (B) cells. Cells were grown in YPD media andthen incubated in nitrogen starvation media (SD-N) for 3 h. The GFP andmRFP signals were observed simultaneously. Arrowheads indicate the colocalization of Lap3 and Ape1. Arrows point to intravacuolar structures.
Insets are high-magnification images of dots marked with asterisks. GFPLap3 and mRFP-Ape1 dots do not merge, but are localized very closely toeach other. Bars, 5 μm.

Ⅲ. Structural basis of cargo recognition by
Atg8 homologs in selective autophagy
Atg8 homologs are ubiquitin-like proteins that localize on
autophagosomal membranes and play crucial roles in theformation of the membranes. These proteins are also
involved in the selective incorporation of specific cargomolecules into autophagosomes, in which Atg8 and its
mammalian homolog, LC3, interact with Atg19 and p62,
receptor proteins for a number of vacuolar enzymes
including Ape1 and disease-related, ubiquitin-positive
protein inclusions, respectively. In collaboration with Dr.
Inagaki's group at Hokkaido University, we determined thestructures of Atg8 and LC3 in complex with Atg19 and p62
peptides, respectively, which contain amino acid residues
necessary and sufficient for complex formation.
Remarkably, Atg8 and LC3 proved to interact with Atg19
and p62 in a quite similar manner, even though the entire
sequences of Atg19 and p62 are unrelated to each other.
Hydrophobic pockets conserved among Atg8 homologs
recognized the side-chains of Trp and Leu in the WXXL
motif in Atg19 and p62 (Figure 2). Mutational analyses ofAtg8 and Atg19 showed that this interaction is indeed

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

11

National Institute for Basic Biology

Cell Biology

required for the vacuolar transport of Ape1. Thus, werevealed the fundamental interaction between receptors andAtg8 homologs by which specific cargo molecules are
efficiently captured by autophagosomes.

not observed for Atg8–PS at all. Therefore, it was suggested
that intracellular milieus, the pH of the cytosol and acidic
phospholipids in membranes, contribute to the selective
formation of Atg8–PE in the cell. We also showed thatacidic phospholipids facilitate the binding of Atg8-Atg7 andAtg8-Atg3 thioester intermediates to the membrane(Figure 3).

Ⅴ. Amino-terminal region of Atg3 is essential
for the association with PE in Atg8
lipidation

Figure 2. The structure of the interaction site of Atg8 with Atg19. The
evolutionally conserved, hydrophobic binding pocket of Atg8 (surface
model) accommodating a peptide corresponding to the Atg8-binding regionof Atg19 (stick model) is shown.

Ⅳ. The determinants of substrate specificity inlipidation of Atg8
A ubiquitin-like system consisting of the E1 enzyme Atg7
and the E2 enzyme Atg3 mediates the conjugation of Atg8
with the lipid phosphatidylethanolamine (PE). The Cterminal carboxyl group of Atg8 is activated by Atg7,
sequentially forms thioester intermediates with active site
cysteine residues of Atg7 and Atg3, and eventually forms anamide bond with the amino group of PE. Probably as thislipid-modified form (Atg8–PE), Atg8 localizes to
autophagosomal membranes and participates in theirformation. The conjugation reaction of Atg8 can bereconstituted in vitro using purified proteins and liposomes
containing PE. Whereas PE was identified as the sole in vivo
target of Atg8, in vitro studies showed that the same system
can mediate the conjugation of Atg8 with phosphatidylserine(PS) as efficiently as with PE. We found that, in contrast to
PE conjugation, the PS conjugation of Atg8 is markedly
suppressed at physiological pH (Figure 3). Furthermore,while the addition of acidic phospholipids to liposomes
significantly promotes the production of Atg8–PE, this was

We previously showed using the in vitro system that the
conjugate of the ubiquitin-like protein Atg12 with Atg5
(Atg12–Atg5) directly interacts with Atg3 and drasticallystimulates its conjugating activity (transfer of Atg8 to PE).
When PE-containing liposomes were absent from thereaction in the presence of Atg12–Atg5, Atg8 was mistransferred to the serine residues in the N-terminal region ofAtg3 from its active site cysteine, suggesting the
involvement of the region in Atg8 lipidation. To examine
the role of the N-terminal region of Atg3 in the formation of
Atg8–PE, we prepared Atg3 mutants having deletions or
mutations in this region. We found that the conjugating
activities of the Atg3 mutants lacking N-terminal 7 aminoacid residues or having the Leu-to-Asp mutation at position
6 were severely impaired both in vivo and in vitro.
Furthermore, we revealed that the N-terminal region of Atg3
is responsible for the proper interaction with PE-containing
membranes (Figure 4).

Figure 4. Floatation assay of Atg3 mutants. Purified Atg3 mutant proteins
were incubated with liposomes containing PE, and then subjected tofloatation assay. Fractions (fraction 1 represents the top fraction) wereanalyzed by western blotting with anti-Atg3 antibodies.

Ⅵ. The yeast Tor signaling pathway is involvedin G2/M transition via Polo-kinase

Figure 3. The neutral pH and the presence of acidic phospholipids result in
the preferential formation of Atg8–PE.

12

The target of rapamycin (Tor) protein plays a central rolein cell growth. Rapamycin inhibits cell growth and promotes
cell cycle arrest at G1 (G0). Little is known, however, about
whether or not Tor is involved in other stages of the cell
division cycle. Unexpectedly, we found that the rapamycinsensitive Tor complex 1 (TORC1) is involved in G2/M
transition in S. cerevisiae. Strains carrying a temperaturesensitive allele of KOG1 (kog1-105) encoding an essential
component of TORC1, as well as yeast cells treated with
rapamycin, show mitotic delay with prolonged G2 (Figure
5A). Overexpression of Cdc5, the yeast polo-like kinase,rescues the growth defect of kog1-105, and in turn, Cdc5
activity is attenuated in kog1-105 cells. The TORC1Type2A phosphatase pathway mediates nucleocytoplasmictransport of Cdc5, which is a prerequisite for its proper
localization and function (Figure 5B). The C-terminal polobox domain of Cdc5 has an inhibitory role in nuclear
translocation. Taken together, our results indicate a novel
function of Tor in the regulation of cell cycle andproliferation.

● Nakashima, A., Maruki, Y., Imamura, Y., Kondo, C., Kawamata, T.,

Kawanishi, I., Takata, H., Matsuura, A., Lee, K.S., Kikkawa, U.,
Ohsumi, Y., Yonezawa, K., and Kamada, Y. (2008). The yeast Tor
signaling pathway is involved in G2/M transition via Polo-kinase.
PLoS ONE 3, e2223.
● Noda, N.N., Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2008).
Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by thefree-interface diffusion method. J. Synchrotron Radiat. 15, 266-268.
● Noda, N.N., Kumeta, H., Nakatogawa, H., Satoo, K., Adachi, W.,
Ishii, J., Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2008). Structural
basis of target recognition by Atg8/LC3 during selective autophagy.
Genes Cells 12, 1211-1218.
● Obara, K., Noda, T., Niimi, K., and Ohsumi, Y. (2008). Transport ofphosphatidylinositol 3-phosphate into the vacuole via autophagic
membranes in S. cerevisiae. Genes Cells 13, 537-547.
● Obara, K., Sekito, T., Niimi, K., and Ohsumi, Y. (2008). The Atg18Atg2 complex is recruited to autophagic membranes via PtdIns(3)Pand exerts an essential function. J. Biol. Chem. 283, 23972-23980.
● Oh-oka, K., Nakatogawa, H., and Ohsumi, Y. (2008). PhysiologicalpH and acidic phospholipids contribute to substrate specificity inlipidation of Atg8. J. Biol. Chem. 283, 21847-21852.

〔Review articles〕● Nakatogawa, H., Oh-oka, K., and Ohsumi, Y. (2008). Lipidation of

Atg8: How is substrate specificity determined without a canonical E3
enzyme? Autophagy 4, 911-913.
● Nakatogawa, H., and Ohsumi, Y. (2008). Starved cells eat ribosomes.
Nat. Cell Biol. 10, 505-507.
● Obara, K., and Ohsumi, Y. (2008). Dynamics and function of
PtdIns(3)P in autophagy. Autophagy 4, 952-954.
● Sekito, T., Fujiki, Y., Ohsumi, Y., and Kakinuma, Y. (2008). Novelfamilies of vacuolar amino acid transporters. IUBMB Life 60, 519525.

Figure 5. Tor regulates mitotic entry. (A) FACS analysis of wild type andtsts
kog1-105 strains. After temperature shift to 37°C, kog1-105 cells stop cell
cycle progression with 2C DNA content, suggesting mitotic delay. (B)Localization of GFP-tagged Cdc5, the yeast polo-kinase. In wild type cells,
Cdc5 localizes at the spindle pole body and the nucleus. In contrast,ts
localization of Cdc5 is disrupted in the kog1-105 mutant.

Publication List
〔Original papers〕● Fujioka, Y., Noda, N.N., Fujii, K., Yoshimoto, K., Ohsumi, Y., andInagaki, F. (2008). In vitro reconstitution of plant ATG8 and ATG12
conjugation systems essential for autophagy. J. Biol. Chem. 283,
1921-1928.
● Fujioka, Y., Noda, N.N., Matsushita, M., Ohsumi, Y., and Inagaki, F.
(2008). Crystallization of the coiled-coil domain of Atg16 essential for
autophagy. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64,
1046-1048.
● Hu, G., Hacham, M., Waterman, S.R., Panepinto, J., Shin, S., Liu, X.,
Gibbons, J., Valyi-Nagy, T., Obara, K., Jaffe, H.A., Ohsumi, Y., andWilliamson, P.R. (2008). PI3K signaling of autophagy is required for
starvation tolerance and virulence of Cryptococcus neoformans. J.
Clin. Invest. 118, 1186-1197.
● Ishida, H., Yoshimoto, K., Izumi, M., Reisen, D., Yano, Y., Makino,A., Ohsumi, Y., Hanson, M.R., and Mae, T. (2008). Mobilization of
Rubisco and stromal-localized fluorescent proteins of chloroplasts tothe vacuole by an ATG gene-dependent autophagic process. Plant
Physiol. 148, 142-155.
● Kawamata, T., Kamada, Y., Kabeya, Y., Sekito, T., and Ohsumi, Y.
(2008). Organization of the pre-autophagosomal structure responsible
for autophagosome formation. Mol. Biol. Cell 19, 2039-2050.

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 microtubules,
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, and MDCK. They are quite common

centriolar specializations in vivo and in vitro. The incidenceof primary cilia within a cell culture is related to the degreeof confluency. Examination of confluent cell monolayers
showed that the primary cilia within a single preparation of agiven cell line varied considerably in length. In most cases,
cell lines previously used were not cloned and the results
remained obscure.
This year, four cell lines originating from adult mouse
kidneys were established in order to study the proteomics ofthe primary cilia. The cell lines were named nibb-K1, K4,
K5, and K8. The primary cilia of cells were observed byindirect immunofluorescence microscopy (Figure 1). In thecloned cells, each cell has a distinct length of cilium, with
the K5 cell having the longest one among them (up to 10
μm).

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
Technical Assistant

GONDA, Naoko

Animal organs are made up of several types of cells,organized in an orderly fashion wherein the proportion ofeach cell type is constantly maintained. This orderly cellarrangement and proportion are built up duringorganogenesis by cell-cell interactions. Since it has been
postulated that Notch plays a role in cell fate 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 Notch2 null mutation results in embryonic lethality byembryonic day 11.5 due to the formation of poor maternalvascular beds. The mutant placenta showed a normal
invasion of angiogenic allantoic mesenchyme followed bypremature formation of fetal blood vessels in the mutant
placentas as early as E9.0. However, the specification oftrophoblast subtypes appeared not to be drastically
disturbed. Thus, in the developing mouse placenta, Notch2 islikely not involved in cell fate decision, but rather
participates in the formation of circulatory systems in the

Figure 2. Distribution of AO positive signals in E9.5 Notch2LacZ/LacZ
placenta (A) and Notch2+/LacZ placenta (B). Red, orange and yellow color
indicates giant cell layer, spongiotrophoblast layer and labyrinthine
trophoblast layer, respectively. Dots indicated in light blue, dark blue,and green show dying cells in three serial sections.

labyrinth layer where the expression of Notch2 was detected.
Although inadequate formation of maternal vascular beds
was partially restored by aggregating mutant diploidembryos with wild type tetraploid embryos, networks ofmaternal vascular beds appeared still compromised in the
4N chimeric placenta. These results indicate that Notch2
promotes vasculogenesis.
How maternal vascular beds are formed in the developing
mouse placenta has been unexplored. The simplest way toform the beds among tightly adhered labyrinthine
trophoblasts is through their cell death. We studied aspatiotemporal appearance of dead cells in the developing
placenta by staining with a fluorescent dye, acridine orange,
which has been employed to detect apoptotic cells in
Drosophila (Figure 1). It was found that the appearance ofdying cells correlated well with maternal vasculogenesis andwas delayed in the mutant placenta (Figure 2). Identificationof a factor which is responsible for the induction of Notch2
expression in trophoblasts around newly forming maternal
blood beds is another project in our laboratory. When bothprojects are accomplished, we should obtain some insights
into Notch2 gene function in maternal vasculogenesis in thedeveloping mouse placenta.

Publication List
〔Original paper〕● Tang, H., Brennan, J., Karl, J., Hamada, Y., Raetzman, L., and Capel,

B. (2008). Notch signaling maintains Leydig progenitor cells in themouse testis. Development 135, 3745-3753.

Figure 1. Cell death in Notch2+/LacZ mouse placenta at E8.5.
Acridine Orange (AO) positive signals were mostly observed inpresumptive labyrinthine trophobast layer.

15National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

UENO, NaotoKINOSHITA, Noriyuki

Assistant Professors

Technical Staff
Postdoctoral Fellows
Graduate Students
Technical Assistants
Secretaries

NAKAMURA, Makoto
TAKAHASHI, Hiroki
SUZUKI, Makoto
TAKAGI, Chiyo
TAO, Hirotaka
SHINDO, Asako
MORITA, Hitoshi
HARA, Yusuke
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
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 understand how pattern formation andmorphogenesis during development is regulated by thesegrowth and transcription factors. We address this problem
using several model animals, including frogs, mice andascidians, and by employing embryology, genetics,
molecular and cellular biology, and biochemistry.

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 vertebrate convergent extension, Wnt/PCP
pathway is implicated.
To understand the role of one of the core components of
the PCP pathway, we knocked out one of two prickle-related
genes mpk1 in mice. We found that the mpk1-/- mutants die inearly embryogenesis between E5.5 and E6.5. The mutants
showed arrested development with failure of primitive streak
and mesoderm formation and failure of distal visceralendoderm migration. At the cellular level, the mpk1 -/epiblast tissue is disorganized, with a clear defect in cell

16

polarization. Furthermore, we showed mpk1 geneticallyinteracts with another PCP gene Vangl2/stbm in epiblast
polarization, indicating that PCP pathway components mpk1
and Vangl2/stbm are essential for early cell polarity,
particularly the apical-basal polarity of the epiblast.

Figure 1. Fluorescent images of nuclei (stained by TOTO-3, green) in-/transverse sections of wild type (A, A’) and mpk1 mutant (B, B’)embryos. Insets are higher-magnification images of the respective
epiblast cells indicated by F-actin (red) and nuclei (green). Arrowheads
indicate the boundary between the extraembryonic and embryonicregions.

We also studied how PCP is established within the cells
using explants of Xenopus embryonic tissues and found thatheterogenous combination cultures of tissues such as
mesoderm and ectoderm trigger the cell polarity as revealedby the live-imaging analysis of microtubule growthorientation. We are currently investigating whether physicalforce generated at the interface of two cell populations is
involved in the initiation of the cell polarity.

Ⅱ. Protein ubiquitination and membranetrafficking involved in the Wnt/PC
pathway
The Wnt/PCP signaling pathway has been shown to play anessential 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 implicated protein
ubiquitination and membrane trafficking in the Wnt signaltransduction. Dishevelled is a cytoplasmic protein, whichplays a pivotal role in the Wnt pathway. When the Wnt
pathway is activated, Dishevelled is translocated to theplasma membrane. This translocation activates downstream
signaling components such as Rho GTPases, and regulates
actin cytoskeleton and cell polarity. We have identified anovel E3 ubiquitin ligase complex that binds to Dishevelled.

This E3 ligase complex is essential for the translocation of
Dishevelled. In addition, we have shown that Dishevelled
and other PCP signaling components are ubiquitinated. This
result suggests that protein ubiquitination is important for
Wnt/PCP signal transduction. We also identified a novel
SNARE family protein, xSynt2, which colocalizes with
Dishevelled and is essential for its translocation to theplasma membrane. This SNARE protein is also shown to be
essential for the gastrulation movements. SNARE family
proteins are known to regulate membrane fusion processes.
These findings show that the ubiquitin system andmembrane trafficking regulate gastrulation movements in
Xenopus embryos.

Figure 2. Membrane trafficking regulates the localization of
Dishevelled. (A) Dishevelled shows punctate localization (green), and is
colocalized with the SNARE protein xSynt1 (red). (B) Localization of
Dshevelled-GFP (green). Wnt signaling translocates Dishevelled to theplasma membrane, which is inhibited by antisense Morpholino against
xSynt2.

Ⅲ. Cellular morphogenesis during neural tube
formation
Neural tube formation is one of the most dynamic
morphogenetic processes during early embryogenesis and its
failure is known to cause malformations known as neuraltube defects. To understand cellular and molecularmechanisms regulating neural tube formation, we focusedon MIDLINE1 (MID1) and a paralogous gene MID2. MID1
is a conserved microtubule-associated protein andresponsible for Opitz G/BBB syndrome. This syndrome isassociated with midline abnormalities such as hypertelorism,
hypospadias, and heart defects, suggesting thedevelopmental functions of MID1. We found that MID1
colocalized with microtubule in embryonic cells in Xenopus,
and the combined depletion of MID1 and MID2 induced
neural tube defects. Histological and live-imaging analyses
also revealed that neuroepithelial cells in affected embryos
became rounded and failed apical constriction and cell
elongation. In addition, these defects are tightly correlatedwith the disorganization of the microtubule network along

the apicobasal axis, which should be required for cell shapechange and epithelial integrity. Theseese data suggest thatthe regulation of the microtubule network by MIDs isrequired for correct neural tube closure and that similar
mechanisms also underlie the development of other organs
affected in Opitz G/BBB syndrome patients.

Figure 3. The changes of cell shape and the microtubule network duringneural tube closure. (Aa) In control, neuroepithelial cells elongated theirshape and developed microtubule along apicobasal axis (green). (Ab) In
MIDs-depleted embryo, neuroepithelial cells became rounded and failed
microtubule reorganization. (B) Quantitative data of cell height insuperficial layer.

We also investigated the roles of an adhesion molecule,nectin, which belongs to the immunoglobulin-like celladhesion molecule in the neural tube formation of Xenopus.
Depletion of nectin-2 (one of the nectin family members)from early embryo resulted in incomplete neural foldformation. Cellular analyses revealed less accumulation of
F-actin at the apical surface, causing an aberrant apicalconstriction, a cell-shape change that is required for theneural tube folding. Furthermore, we found nectin-2functionally cooperates with N-cadherin to synergisticallyenhance apical constriction, highlighting the cooperative
action between spatiotemporally upregulated nectin-2 andN-cadherin in the neural plate.

Ⅳ. Brachyury-downstream gene sets in achordate, Ciona intestinalis
In vertebrates, Brachyury, a T-box transcription factor
gene, seems to have a dual role in the differentiation of axial
midline mesoderm cells into notochord and gastrulation cell
movements regulated by non-canonical Wnt/planar cell
polarity (Wnt/PCP) signaling. To annotate the function ofBrachyury-downstream genes in chordate embryos, based on
subtractive hybridization, dot-blot assays, EST sequences
and the expression patterns in whole-mount in situ
hybridization at embryonic stages, we developed a17

National Institute for Basic Biology

Developmental Biology

knowledge database called “CINOBI: Ciona Notochord andBrachyury-downstream gene Index” to createcomprehensive catalogues of Brachyury-downstream genesets in Ciona intestinalis. Combining genome and largescale cDNA data, we were able to characterize 450 nonredundant Brachyury-downstream genes. Twenty-four genes
were newly annotated as notochord-expressed genes.
Several genes are components of signaling pathways such as
Wnt/PCP, NfkB and TGF-beta signaling. We propose that
Brachyury is linked to these pathways, regulating theexpression of each component, and that such a regulatory
mechanism might be conserved among chordates.

Figure 4. The role of Brachyury in the transcriptional network in Ciona
intestinalis. Upstream positive regulators of Brachyury in the endoderm
include FGF and ZicL. Brachyury directly activates Ci-tropm and othernotochord-specific genes to induce differentiation of the notochord. Cellpolarity-related genes such as Ci-pk and Ci-Fz1/2/7 are involved inconvergent extension movement in gastrulation. These signaling
components are induced directly or indirectly by Brachyury.

18

Publication List
〔Original papers〕● Shindo, A., Yamamoto, T.S. and Ueno, N. Coordonation of cell

polarity during Xenopus Gastrulation. (2008). PLoS ONE 3, e1600.
● Hotta, K., Takahashi, H., Satoh, N., and Gojobori, T. (2008).

Brachyury-downstream gene sets in a chordate, Ciona intestinalis:
Intrgrating notochord specification, morphogenesis and chordate
evolution. Evo. Dev. 10, 37-51.
● Kawasaki, A., Kumasaka, M., Satoh, A., Suzuki, M., Tamura, K.,
Goto, T., Asashima, M. and Yamamoto, H. (2008). Mitf contributes tomelanosome distribution and melanophore dendricity. Pigment Cell
Melanoma Res. 21, 56-62.
● Gilchrist, M.J., Chiristensen, M.B., Harland, R., Pollet, N., Smith, J.C.,
Ueno, N. and Papalopulu, N. (2008). Evading the annotation
bottleneck: using sequence similarity to search non-sequence genedata. BMC Bioinformatics 9, 442.

DIVISION OF DEVELOPMENTAL GENETICS

Professor

KOBAYASHI, Satoru
Assistant Professor
Technical Staff
NIBB Research Fellow
PRESTO Researchers
Postdoctoral Fellows

Graduate Students

Technical Assistant
Secretary

HAYASHI, Yoshiki
NODA, Chiyo
KUMATA, Yuji
SHIGENOBU, Shuji
KAGEYAMA, Yuji
KITADATE, Yu
HASHIMOTO, Yoshiko
KONDO, Takefumi
HAYASHI, Makoto
YATSU, Jun
MAEZAWA, Takanobu
HASHIYAMA, Kazuya
SATO, KaoriHONDA, 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 germline stem cells to form eggs
and sperm when the organisms are physically matured.

Ⅰ. Maternal Nanos protein is required in polecells to repress their apoptosis
Several components of germ plasm have been identified in
Drosophila. One of these components is maternal nanos(nos) RNA, which is enriched in germ plasm during
oogenesis and translated in situ to produce Nos protein after
fertilization. Nos is inherited by primordial germ cells orpole cells at the blastoderm stage and is detectable in thesecells throughout embryogenesis. In the absence of maternalNos, pole cells undergo apoptosis during their migration to
the embryonic gonads. Although Nos also plays importantroles in repressing mitosis, somatic gene expression andsomatic cell fate in pole cells, the primary role for Nos
appears to be repressing apoptosis in the germline, because
Nos is an evolutionarily conserved protein that is requiredfor 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.

We have demonstrate that maternal Nos represses
apoptosis of pole cells by suppressing translation of hid
RNA. In the absence of Nos activity, translation of hid
mRNA yields a protein product that induces apoptosis. In
addition, a maternally-provided protein kinase, Tao-1, isrequired to induce 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 hid and skl andbecome competent to undergo apoptosis during normal
development. However, maternal Nos represses hidtranslation to inhibit apoptosis of pole cells for theirsurvival.
We recently found that hid expression requires eiger (egr), atumor necrosis factor (TNF) homolog, that is induced in polecells by decapetaplegic (dpp). In addition, p53 and loki (lok),a damage-activated kinase known to be required forphosphorylation of p53, are both required for hid expressionin pole cells. Since maternal lok mRNA is enriched in germ
plasm and then partitioned into pole cells, we speculated thatubiquitously distributed p53 may be activated in pole cells bymaternal Lok. Taken together, we propose that hid expressionis activated in a pole cell-specific manner by loki/p53 anddpp/egr during normal embryogenesis (Figure 1).

Figure 1. A model for the regulation of hid mRNA expression andapoptosis in pole cells.

Ⅱ. Expression of genes involved in sumoylationin pole cells
To identify the genes essential for germline development,
we have performed EST and microarray analyses using polecells and embryonic gonads. During the course of thisanalysis, we noticed that five genes in the SUMO (small
ubiquitin-related modifier) conjugation pathway are highly
expressed in pole cells and embryonic gonads (Hashiyama
and Kobayashi, Gene Expression Patterns, 9, 50-53, 2009).
Sumoylation regulates a wide range of cellular processes
includingtranscription,
genomic
replication,
nucleocytoplasmic signaling, and chromatin dynamics.
SUMO proteins are covalently attached to lysine residues inthe substrate protein by a series of enzymatic reactions
similar to the ubiquitination system. Briefly, SUMO protein
is processed by a SUMO-specific protease, and a SUMOactivating enzyme activates and transfers the SUMO protein
to the SUMO-conjugating enzyme. SUMO is thencovalently attached to the substrate protein by a SUMO
protein ligase. We analyzed the spatiotemporal expression

19National Institute for Basic Biology

Developmental Biology

patterns of the genes encoding the SUMO protein (smt3), the
SUMO-specific protease (Ulp1), the SUMO-activating
enzymes (Uba2 and Aos1), and the SUMO-conjugating
enzyme Ubc9 (lesswright, lwr) in Drosophila embryos andadult gonads (Figure 2).
Transcripts from all five genes are detected throughout theearly embryo by whole mount in situ hybridization, while
they are predominantly expressed in pole cells in late stageembryos. These genes are also expressed in the germline
during oogenesis and spermatogenesis. We also found that
SUMO protein is enriched in the nuclei of pole cells andgametogenic cells. Given that a large fraction of SUMO
substrates are nuclear proteins, this data suggests thatsumoylation is highly active in the germline. Our data
provide a basis for understanding how sumoylation regulates
germline development.

within the embryonic gonads. However, we found that
reduction of sumoylation causes apoptosis of the migrating
pole cells in a female-specific manner, suggesting thatsexual identity has already been established in the pole cells
prior to gonad formation. We also found that Sex lethal (Sxl)
is expressed in the migrating pole cells in a female-specificmanner, and its function is required for the feminization ofpole cells. Ectopic expression of Sxl in XY (male) pole cells
is able to direct the developmental fate of these cells to
become functional eggs. Our results show that the germlineautonomous mechanism, along with the non-autonomousmechanism, leads to sexual dimorphism in the germline(Figure 3).

Ⅳ. Mechanism regulating the formation of theniche cells in male embryonic gonads
The germline-stem-cell (GSC) niche in Drosophila testes
has emerged as a useful model system for studying stem
cells. In the apical tip of the adult testes, the GSCs lie in
intimate contact with somatic hub cells, known collectively
as the niche cells, which causes the stem cells to retain selfrenewing potential. GSCs divide to produce one daughter
cell that remains associated with the hub cells, while theother daughter cell detaches and initiates spermatogenesis.

Figure 2. Expression of sumoylation genes in embryos.
In situ hybridization of embryos with antisense probes for smt3 (sumo),
Ulp1, Uba2, Aos1 and lwr. Embryos at stage 5, stage 9/10 and stage 15
are shown. Anterior is to the left. Arrows indicate gonads.

Ⅲ. Mechanism leading to sexual dimorphismof pole cells
Germ cells must develop along distinct male or female
paths to produce eggs or sperm. It has been reported thatgermline sexual identity is regulated by a masculinizing
signal from the somatic gonadal cells (SGCs) to pole cells

Figure 3. A model for the regulation of sexual dimorphism in pole cells.

20

Figure 4. Hub and hub progenitors are marked with a molecular marker,
Fasciclin3 (Fas3). Fas3 positive cells (green) are located in the anterior
tips of both adult and embryonic gonads. Magenta shows germline cells.

Hub cells are derived from a subset of somatic gonadal
cells (SGCs) that are located in the anterior region of maleembryonic gonads (Figure 4). How the formation of hubprogenitors is restricted in the anterior of embryonic gonads,
however, remains elusive. We have demonstrated that a
receptor tyrosine kinase, Sevenless (Sev), provides a cue toensure that the hub cells develops in the anterior region of
the male embryonic gonads. Sev is expressed by SGCs
within the posterior region of the gonads, and is activated bya ligand, Bride of sevenless (Boss), which is expressed bypole cells, to prevent ectopic hub differentiation in theposterior SGCs.

Recently, we found that Egfr, like Sev, is activated by aligand from pole cells to prevent hub differentiation in theposterior SGCs. We also showed that hub formation requires
Notch, which is activated in almost all of the SGCs within
the male embryonic gonads. Thus, we propose that almost
all of the male SGCs become competent to form hub by thefunction of Notch, but niche formation is repressed in theposterior SGCs by Egfr and Sev, thereby restricting niche in
the anterior region.

GSC niche cells. We present the model that HSPGs have arole in defining the GSC niche by controling ligand stability
or distribution in[the niche region.

Publication List
〔Original paper〕● Yatsu, J., Hayashi, M., Mukai, M., Arita, K., Shigenobu, S., and

Ⅴ. The role of heparan sulfate proteoglycan in
the germline-stem-cell niche
Stem cells possess the remarkable capacity to generate bothdaughter cells that retain a stem-cell identity and others thatdifferentiate. Stem cells reside in dedicated cellular
microenvironments termed niches. These niches dictate astem-cell identity, maintain the stem cell population, andcoordinate proper homeostatic production of differentiatedcells. Recent studies have shown that several signaling
pathways, such as TGF-beta and JAK/ STAT are essential
for maintenance of the Drosophila GSCs. GSCs are
surrounded by somatic cells (niche cells) which form thephysical area of the niche and are responsible for signaling
molecule secretion. Despite the importance of thesemolecules for GSC maintenance, it is not yet wellunderstood how these secreted molecules can precisely
define the region of GSC niche.

Kobayashi, S. (2008). Identification of maternal RNAs encoding
transcription factors required for germline-specific gene expression in
Drosophila embryos. Int. J. Dev. Biol. 52, 913-923.

Figure 5. HSPG is essential for GSC maintenance in female gonads. In
HSPG mutant (dally), GSCs are missing from the anterior tip of theovary. Green and magenta show germline cells and somatic cells,
respectively.

Heparan Sulfate Proteoglycans (HSPGs) are a glycoprotein
which are expressed on the cell surface and/ or in theextracellular matrix. Recent in vivo studies have shown that
HSPGs play critical roles in regulating signaling during
development by controlling extracellular ligand distribution.
For example, one of the Drosophila glypicans, dally, cancontrol the distribution of a TGF-beta ligand, Dpp, andestablish Dpp gradient during wing development.
We showed that HSPGs are essential for GSC maintenancein both male and female gonads. In the HSPG mutant
gonads, GSCs could not maintain their number and lost thecharacteristics of undifferentiated germ cells (Figure 5). In
both male and female, HSPG genes are expressed in the21National Institute for Basic Biology

Developmental Biology

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji
Assistant Professor
Technical Staff
NIBB Research Fellow
Postdoctoral Fellows

Graduate Students

Technical Assistants

Secretary

OKUBO, Tadashi
UTSUMI, Hideko
AKANUMA, Takashi
TAKADA, Ritsuko
CHEN, Qiuhong
YABE, Taijiro
NAKAYAMA, Kei
ISHITANI, Shizuka
TAKAHASHI, Jun
TAKAHASHI, Hiroyuki
ASANO, Takuya
TAKASHIRO, Kayoko
ISHIKAWA, MikaTOMIDA, SanaiTSUZUKI, Chisa
UKAI, Sakie

various secreted signal proteins involves post-translational
modification with lipid moieties. Recently, we found thatmurine Wnt-3a is modified with a mono-unsaturated fatty
acid, palmitoleic acid, at a conserved Ser residue (Figure 1).
Wnt-3a defective in this modification is not secreted from
cells in culture or in Xenopus embryos, but is retained in theendoplasmic reticulum (ER). Furthermore, Porcupine, aprotein 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. 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.
To better understand the molecular mechanism of secretionand gradient formation of Wnt proteins, we are carefully
examining the biochemical characteristics of Wnt proteinsfrom cultured cells. We are also trying to reveal the role ofpalmotoleoylation during embryogenesis using zebrafish
embryos.

The body and tissues of the developing embryo arerepeatedly divided into sub-regions specified bycharacteristic gene expression and morphology. The processthat gives rise to these sub-regions according to a definedpattern is called “pattern formation” or “patterning.” Themost popular model to explain the patterning process is the“morphogen gradient and threshold” theory. Many genetic
results indicate that secreted signal proteins such as Wnt,
BMP, and Hedgehog function as morphogens in many
aspects of patterning processes. In spite of the accumulationof genetic evidence, however, the biochemical
characteristics of morphogens, including modification andhigher order structure, remain to be elucidated. Thus, one ofour major goals is to reveal the real image of morphogens
and the molecular mechanism underlying the formation ofmorphogen gradients, including the secretion andextracellular transport of these morphogens.
The segmental sub-regions of the paraxial mesoderm (orsomites), by contrast, appear not to be simply directed by themorphogen 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, is not
yet fully understood. Thus, another goal of our current
studies is to reveal the molecular mechanism of this otherand unique mode of patterning that underlies the periodical
and sequential sub-division in somite formation.

Ⅰ. Secretion and modification of Wnt proteins
The Wnt family of secreted signal proteins plays a key rolein numerous aspects of embryogenesis. Most Wnt proteinstransmit their signals locally, presumably since theirsecretion and transport are under tight control. One
important step regulating the extracellular transport of22

Figure 1. Lipid modifications of murine Wnt-3a protein (A, B) MASS
spectrometric profile indicating modification with palmitoleic acid
(C16:1) at Ser209, which is conserved among most Wnt proteins. (C)Schematic representation of lipid modification of murine Wnt-3a
protein. Wnt-3a is modified with palmitolate (C16:1) at Ser209 andpalmitate (C16:0) at Cys77. We found the palmitoleoylation is catalyzed
by acytransferase, Porcupine (porc), in the ER. This modification isrequired for the trafficking of Wnt proteins from the ER.

Ⅱ. Characteristics of genes required for thedevelopment of somite or pharyngeal arches– Molecular mechanism of suppression by the
Ripply family of transcriptional modulators –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 example, 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.
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.

Figure 2. The Ripply family. (A) The phylogenic tree of the Ripply
family. (B) Schematic structure of Ripply proteins.

We showed that a gene identified by our in situ
hybridization screening, ripply1, encoding a nuclear protein
associated with the transcriptional co-repressor Groucho, isrequired for this transition. Zebrafish ripply1 is expressed in
the anterior PSM and in several newly formed somites(Figure 2). Ripply1 represses specific gene expression in the
PSM through a Groucho-interacting motif. In ripply1deficient embryos, somite boundaries do not form, thecharacteristic gene expression in the PSM is not properly
terminated, and the initially established rostrocaudal polarity
in the segmental unit is not maintained, whereas paraxial
mesoderm cells become differentiated. Thus, ripply1 plays
two key roles in the transition from the PSM to somites:
termination of the segmentation program in the PSM, andmaintenance of the rostrocaudal 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 of mesp-b could be
induced by T-box transcription factor (Tbx), we can speculatethat Ripply1 may antagonize the function of Tbx in thetranscription of mesp genes. The T-box family oftranscription factors, defined by a conserved DNA binding
domain called the T-box, regulates various aspects ofembryogenesis by activating and/or repressing downstream
genes. Reduced function of T/Brachyury, the founder memberof this family, for example, causes truncated tail in the mouse.
The importance of T-box transcription factors is also shownby the fact that mutations in human T-box genes cause severecongenital disorders, such as DiGeorge, Ulnar-mammary, andHolt-Oram syndromes. Furthermore, the T-box family is
evolutionally conserved from C. elegans to insects andvertebrates, showing remarkable functional conservation
across species. In spite of the biological significance of the Tbox proteins, how they regulate transcription remains to beelucidated. Therefore, we carefully examined the relationshipbetween Tbx and Ripply family proteins.
We showed that the Groucho/TLE-associated protein,
Ripply, converts T-box proteins from activators to
repressors (Figure 3). In cultured cells, zebrafish Ripply1, anessential component in somite segmentation, and its
structural relatives, Ripply2 and Ripply3, suppress thetranscriptional activation mediated by the T-box protein
Tbx24, which is coexpressed with ripply1 during
segmentation. Ripply1 associates physically with Tbx24 andconverts it to a repressor. Ripply1 also antagonizes thetranscriptional activation of another T-box protein, No tail(Ntl), the zebrafish ortholog of Brachyury. Furthermore,injection of a high dosage of ripply1 mRNA into zebrafish
eggs causes defective development of the posterior trunk,
similar to the phenotype observed in homozygous mutantsof ntl. 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.

23

National Institute for Basic Biology

Developmental Biology

Figure 3. Transcriptional repression mediated by Ripply. Ripply
converts transcriptional property of T-box transcription factor from
activator to repressor by recruiting Groucho/TLE co-repressor complex.

To get further mechanistic insights into the interactions
between Tbx24 and Ripply1, we next prepared several
deletion or amino acid-substituted constructs of Tbx24 andRipply1 (Figure 4A). By conducting immunoprecicipitation
assays, we found that the T-domain alone, which is highly
conserved among the Tbx family proteins, is sufficient for
the association with Ripply1 but that another region of
Tbx24 is dispensable for the interactions. On the other hand,a ~50 amino acid length sequence at the carboxyl terminus,
called the Ripply homology domain, is conserved among the
Ripply family proteins. Highly conserved sequences exist in
this domain. Substitution of some of these sequences to an
alanine stretch (Ripply1-mutFPVQ) results in a significant
reduction in the transcriptional repression of the pBP-TbxX2reporter construct (Figure 4B), indicating that this conserved
sequence is required for Ripply-mediated transcriptional
repression. This reduction appears to be due to decreased
affinity of Ripply1-mutFPVQ for Tbx24 (Figure 4C).
Consistent with this result, not only Ripply1 but also its
structural relatives, Ripply2 and Ripply3, can suppress thetranscriptional activation mediated by the T-box protein
Tbx24 in cultured cells. Based on these results, many T-box
proteins are likely to be converted from activators to
repressors through specific interactions with Ripply proteins.
The identification of Ripply1 as a switching molecule forT-box genes suggests a novel transcriptional mechanism that
could participate in other aspects of development, although
the relationship between the Ripply family and T-box
factors besides Tbx24 and Ntl remains to be elucidated. The
overlapping expression of ripply and T-box genes, observedin various developing tissues and organs, supports the ideathat the two proteins cooperate in development. Further
understanding of the cooperative transcriptional regulation
by Ripply and T-box proteins could help elucidate themechanisms underlying the disproportionate activation or
repression found in human genetic disorders associated with
mutations in T-box genes.

24

Figure 4. A conserved amino acid stretch in the Ripply homology
domain required for interaction with T-box protein. (A) Schematic
representation of a Ripply1 mutant used in the analysis shown in B and
C. (B) In vitro reporter assay. (C) Immunoprecipitation to examine
physical interaction between Rippply1 mutant and Tbx24.

Publication List
〔Original papers〕● Alvarez-Medina, R., Cayuso, J., Okubo, T., Takada, S., and Marti, E.

(2008). Wnt canonical pathway restricts graded Shh/Gli patterning
activity through the regulation of Gli3 expression Development 135,237-247.
● Kawamura, A., Koshida, S., and Takada, S. (2008). Activator-torepressor conversion of T-box transcription factors by the Ripply
family of Groucho/TLE-associated mediators. Mol. Cell. Biol. 28,
3236-3244.
● Shimizu, T., Kagawa, T., Inoue, T., Nonaka, A., Takada, S.,
Aburatani, H., and Taga, T. (2008). Stabilized β-catenin functions
through TCF/LEF proteins and the Notch/RBP-Jk complex to promote
proliferation and suppress differentiation of neural precursor cells.
Mol. Cell. Biol. 28, 7427-7441.

DIVISION OF EMBYOLOGY

general behaviors of cells within embryos by utilizing live
imaging techniques in addition to morphological observationof embryos within the uterus.

Professor

Ⅱ . Molecular mechanisms regulating the celldifferentiation in preimplantation development.

FUJIMORI, Toshihiko
Assistant Professor
NIBB Research Fellow
Visiting Scientist

TOYOOKA, Yayoi
KOMATSU, Kouji
SEKI, Touhaku

This laboratory was started in August 2008. The aim of ourresearch is to understand the events underlying earlymammalian development during the period from the preimplantation to establishment of the body axes.
An understanding of early events during embryogenesis in
mammals, as compared to other animals, has been relatively
delayed. This is mainly due to the difficulties in theapproaches to the developing embryos in the uterus of themother. The other characteristic of mammalian embryos istheir highly regulative potential. The pattern of cell division
and allocation of cells within an embryo during the earlystages vary between embryos. The timing of the earliest
specification events that control the future body axes is still
under discussion. Functional proteins or other cellularcomponents have not been found that localize
asymmetrically in the fertilized egg. We want to provide
basic and fundamental information about the specification ofembryonic axes, behaviors of cells and the regulation ofbody shape in early mammalian development. Our specificresearch interests are as follows.

Ⅰ. Characterization of the earliest event inbody axis formation
It is generally accepted that, of the three body axes, theanterior-posterior (AP) axis is actively specified earliest.
This is initiated by the movement of visceral endoderm fromthe distally located position to the future anterior side. It is
still an open question whether any differential information
relating to the future body axes already exists within theembryo before the movement of distally located visceralendoderm. We are planning to observe the behaviors of cells
and the changes of embryonic morphology to characterize
the earliest event of axis formation. We will analyze theIn early blastoycst, two types of cells - the cells of innercell mass (ICM), and the cells of trophectoderm (TE) - canbe distinguished. This is the earliest event of celldifferentiation in mouse development. By the studies ofgenes expressed in the blastocyst and in the ES cells, several
genes are known to play key roles in the regulation of celldifferentiation during preimplantation stages. In the lateblastocyst stage, expression of Oct4 is lost in TE andprimitive endoderm (PE) cells. By contrast, Cdx2 is
expressed in the TE lineage. Nanog is expressed in the ICM
of early blastocyst and will be lost in PE lineage at a laterstage. Continuous realtime analyses of the behaviors of thesegenes are unique, and will provide general basic ideas
concerning the regulation of cell differentiation.

Figure 2. Expression of key genes in the mid blastocyst

Ⅲ. Analysis of mechanisms those regulate
embryonic body shape
Mammalian early embryos change their total body size
rapidly after the implantation, whereas the total shape of theembryo is well established. We will study the mechanisms
regulating embryonic body shape by analyzing the behaviors
of individual cells within the embryo.

Publication List
〔Original papers〕● Ishii, Y., Matsumoto, Y., Watanabe, R., Elmi, M., Fujimori, T.,

Nissen, J., Cao, Y., Nabeshima, Y., Sasahara, M., and Funa, K.
(2008). Characterization of neuroprogenitor cells expressing the
PDGF beta-receptor within the subventricular zone of postnatal mice.
Mol. Cell. Neurosci. 37, 507-518.
● Katsuno, T., Umeda, K., Matsui, T., Hata, M., Tamura, A., Itoh, M.,
Takeuchi, K., Fujimori, T., Nabeshima, Y., Noda, T., Tsukita, S., andTsukita, S. (2008). Deficiency of zonula occludens-1 causes
embryonic lethal phenotype associated with defected yolk sacangiogenesis and apoptosis of embryonic cells. Mol. Biol. Cell 19,
2465-2475.
● Tokunaga, A., Oya, T., Ishii, Y., Motomura, H., Nakamura, C.,
Ishizawa, S., Fujimori, T., Nabeshima, Y., Umezawa, A., Kanamori,M., Kimura, T., and Sasahara, M. (2008). PDGF receptor beta is apotent regulator of mesenchymal stromal cell function. J. Bone Miner.
Res. 23, 1519-1528.
● Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K., and Niwa,
H. (2008). Identification and characterization of subpopulations in
undifferentiated ES cell culture. Development 135, 909-918.

Figure 1. Summary of mouse early development.

25

National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei
Postdoctoral Fellow
Visiting Scientist

YOSHIDA, Naoko
SUGIMOTO, Ryo

Mammalian spermatogenesis represents a highly potent
and robust stem cell system. Decades of research, including
detailed morphological examinations, post-transplantation
repopulation, and in vitro culture, have made it one of themost intensively studied mammalian tissue stem cellsystems. However, the nature of the stem cells, as well as
their niche, remains largely unknown in the context ofhomeostatic spermatogenesis. The Division of Germ Cell
Biology, which was launched in 2008, aims to fully
understand the mammalian spermatogenic stem cell system,
mainly using the mouse system. Being a brand-new researchdivision of NIBB, our overall research view and current fociof interests will be described. These include 1) the cellular
nature of the stem cell compartment and their behaviors in
the homeostatic spermatogenesis, and 2) the anatomicalbasis and function of the mammalian spermatogenic stem
cell niche.

Ⅰ. Identifying the mouse spermatogenic stem
cell compartments
1-1. Background: The mammalian spermatogenesis
Mammalian testes continuously produce numerous sperm
during the reproduction period. Investigations that emergedin the 1950s and involve detailed morphological analyses
established the backbone of mammalian spermatogenesis
research. The morphologically most primitive
spermatogonia in the adult mouse testis are A s or A single
spermatogonia (single, isolated spermatogonia). Theirprogeny remain interconnected due to incomplete
cytokinesis, forming syncytial chains of 2n cells (2, 4, 8, 16etc.). It has been experimentally established that“undifferentiated spermatogonia” (or “A undiff” hereafter),which contribute <1% of the entire testicular cells andconsist of As, Apr (Apaired; interconnected pairs), and Aal (Aaligned;
chains of 4, 8, 16 or occasionally 32 cells) contain stem
cells.
Which fraction of A undiff consists of the actually selfrenewing stem cell compartment in homeostasis and how dothey behave (proliferate, self-renew or die) in the testis? The
so-called “As model” is currently most widely considered to
be true. This model proposes that As is the only cell type that
can act as stem cells, while the interconnected population of
A undiff (A pr and A al) is devoid of stem cell capacity. This
comprehensive model, however, is based upon “snapshots”
of fixed specimens and involves too many theoretical limits
to be entirely conclusive regarding stem cell function
because “stem cells” by definition maintain themselves
while producing differentiating progeny for a long period.
Transplantation of a single cell suspension into the26

recipient testis enables the functional and quantitative
analyses of the “stem cells” based on their repopulating
activity in the host testis. Combined with the morphologybased As model, it is often considered to be the case that all
As spermatogonia are equivalent and act as stem cells, which
support both homeostatic spermatogenesis and posttransplantation repopulation, despite the inherent limitation
due to the lack of direct functional evidence.

1-2. Heterogeneous composition of the stem cells
In order to understand the nature of the mouse
spermatogenic stem cell system in testicular tissue, we havebeen investigating the behavior and function of A undiff.
Authentic identification of Aundiff is based on morphology
under electron or high-resolution light microscopy. Using
the genetic labeling of A undiff by means of the Ngn3
regulatory sequence (Yoshida et. al., Dev. Biol. 2004), we
have established experimental systems to investigate theirlive behaviors without disturbing normal tissue architecture.
We first asked whether the “stem cells” detected bytransplantation are identical to the “stem cells” that actually
self-renew in homeostasis, or whether these two “stem cells”
represent different groups of cells. In order to address thisquestion, a tamoxifen-dependent Cre recombinase(CreERTM) was expressed in the Ngn3+ Aundiff (Yoshida et al.,Development 2006). Together with an appropriate reportertransgene, this has enabled the irreversible pulse-labeling of
A undiff spermatogonia upon transient administration oftamoxifen, and the first quantitative detection of “actual
stem cells” (i.e., the cells that persist for a long time while
producing differentiating progeny, thus supporting tissue
homeostasis). Intriguingly, the contribution of the pulselabeled subpopulation of Aundiff to “actual stem cells” and“post-transplantation colony-forming stem cells” represents
a great difference (approximately 40 times higher in thelatter than in the former). Therefore, these two “stem cells”
represent different subpopulations of Aundiff. We concluded
that, in addition to actual stem cells, an extended population
exists that does not self-renew while retaining the potentialof self-renewal, which was defined as a population of“potential stem cells” (Nakagawa et al., Dev. Cell 2007).

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

The “potential stem cells” were shown to rapidly turn overin homeostasis, suggesting that they consist of a transitamplifying compartment.
Figure 1 shows our simplest interpretation of thehierarchical composition of the mouse spermatogenic stem
cell system (Nakagawa et al., Dev. Cell 2007). In case of
actual stem cell loss, potential stem cells might revert to theself-renewing mode and replenish actual stem cells. We are
currently investigating the cell-biological characteristics ofactual and potential stem cells.

Ⅱ. Testicular niche for Aundiff
2-1 Anatomy of the mouse testis and the researchbackgroundSpermatogenesis proceeds inside the seminiferous tubules,
a convoluted tubular structure with a diameter of ~200μm:
Individual tubules connect to the common outlet of themature sperm (rete testes) with both ends and form loops.
Spermatogenesis occurs evenly throughout the seminiferous
epithelium (the inner surface of the tubules). Therefore, inthe mouse testis, an overall ‘polarity’ that covers the entire
organ cannot be recognized, making a good contrast to the
Drosophila melanogaster germline stem cell system.
Evidence suggests an intimate relationship between stem
cells and the niche microenvironment in seminiferous
tubules. It is difficult to identify the nature and function of
the niche, however, because seminiferous tubules do notexhibit suspicious sub-structures. Moreover, actual stem
cells can be identified only functionally, and their
histological detection has not yet been achieved. Therefore,

our current aim is to clarify the niche of Aundiff.

2-2. Live imaging and the vasculature-associatedniche for Aundiff
We have developed a live imaging system in which GFPlabeled Ngn3+ Aundiff and their progeny can be continuously
filmed in undisturbed testes. It was revealed that Aundiff
preferentially localize to the area adjacent to blood vessels
and interstitial cells that surround the seminiferous tubules.
In addition, the dynamic migration of spermatogonia fromthe vasculature proximity to spread throughout the tubules
was also observed upon differentiation of Aundiff (Figure 2).
The same relocation was also supported by threedimensional reconstruction based on authentic
morphological identification of Aundiff on serial sections(Figure 3). These extend the preceding observations frommouse and rat testis sections that Aundiff shows a significant
biased localization to the interstitium, and we proposed sucharea as the niche for Aundiff (Yoshida et al., Science 2007).
We are currently investigating the cellular and molecular
identification of this vasculature-associated niche.

Figure 3. Localization of A undiff revealed by three-dimensional
reconstruction (Yoshida et al., Science 2007)
Figure 2. Localization of GFP-labeled Ngn3+ Aundiff and their relocation
upon transition into differentiating spermatogonia (Yoshida et al.,Science 2007).
(A) Live imaging of spermatogonia upon differentiation of Aundiff. Before
differentiation (0 hour; the elapsed time indicated in each panel inhours), labeled Aundiff preferentially localized to the area adjacent to theblood vessels (seen as a black line) and surrounding interstitium. Upon
differentiation, two chains of 8-cell cysts (Aal-8; indexed in yellow andorange) migrated from this position to spread all over the tubule (3660h). Subsequently, the two cysts underwent synchronous mitotic
division with 2-3 hours’ interval, resulting in the formation of two 16cell cysts of differentiating spermatogonia (73-74h).
(B-E) Examples of the vasculature-proximal localization of Aundiff. Aundiff(arrowhead) preferentially localized to areas adjacent to blood vessels,
more characteristically to their branch points. In (B-C), A undiff inneighboring seminiferous tubules show back-to-back localization overbranching vessels.

Computationally reconstituted three-dimensional images of theseminiferous tubules based on 280 serial sections. Aundiff (green) show
biased localization to the blood vessel network (red) and the areaadjacent to the interstitium (yellow). (A, C) and (B, D), without or withblood vessels, respectively. Roman numerals indicate the stage of theseminiferous epithelium.

Publication List
〔Original paper〕● Sato, Y., Watanabe, T., Saito, D., Takahashi, T., Yoshida, S.,

Kohyama, J., Ohata, E., Okano, H., and Takahashi, Y. (2008). Notch
mediates the segmental specification of angioblasts in somites andtheir directed migration toward the dorsal aorta in avian embryos.
Dev. Cell. 14, 890-901.

27

National Institute for Basic Biology

Developmental Biology

DIVISION OF REPRODUCTIVE BIOLOGY

Professor (Specially appointed)

NAGAHAMA, Yoshitaka
Professor
Assistant Professors
NIBB Research Fellow
Postdoctoral Fellows

Technical Assistants

Secretary

NAGAHAMA, Yoshitaka*
OKUBO, Kataaki
OHNO, Kaoru
SUZUKI, Aya
CUI, Jianzhou
GUAN, Guijun
LI, Jianzhong
PAUL, Bindhu
SAKAI, Fumie
SHIBATA, Yasushi
USAMI, Takeshi
ZHOU, Linyan
HARA, Ikuyo
SHIBATA, Emiko
TAKAKI, Chikako
SHIMADA, Yu

Fish exhibit a range of gonadal forms from gonochorism toseveral types of hermaphroditism, thus providing an
excellent animal model to study the molecular mechanismsof sex determination/differentiation and gametogenesis invertebrates. Our research, which uses several types ofteleost fish, focuses on (1) the identification of regulatorsand steroidal mediators involved in sex determination,
gonadal sex differentiation, sexual plasticity, andgametogenesis (oocyte maturation and ovulation), and (2)the mechanisms of synthesis and action 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. In fish with a stable genetic
XX/XY sex determining system, the sex-determining genelies on the Y chromosome and is responsible for initiating
male sex determination. We identified DMY (DM-domain
gene on the Y chromosome) as the sex-determining gene of
the medaka, the first one in non-mammalian vertebrates.
However, there is no sequence homology between the two
known vertebrate sex-determining genes, SRY/Sry(mammals) and DMY. Another important difference is that
DMY transgenic XX medaka are fully functional and fertile
males, whereas Sry transgenic mice are sterile. A search forthe target genes of DMY led to the identification of gonadal
soma derived factor (gsdf), a member of the transforming
growth factor-beta superfamily. In medaka embryos, gsdf
was predominantly expressed in the somatic cells in the XY
gonads from the day of hatching. Conversely, expression of
gsdf was found to be weaker in the XX gonads undergoingfemale sex differentiation. When the XY embryos weretreated with estrogen, in order to reverse their phenotypicsex, a decline was observed in the expression of gsdf inthose embryos. Treatment of the XX embryos with28

methytestosterone increased the expression of gsdf, provingthat the expression of this gene is linked with the phenotypicsex, not the genetic sex.
In tilapia, all genetic female (XX) and male (XY) broods
are available. Through cDNA subtraction between XX andXY gonads during sex differentiation and microarrayhybridization followed by gene expression analyses by RTPCR and in situ hybridization, we have concluded that thesex-specific expression of Cyp19a1/Foxl2 in XX gonads andDMRT1 in XY gonads during early gonadal differentiation(5 - 6 dph) is critical for indifferent gonads to differentiate
into either the ovary or testis in the Nile tilapia. The criticalrole of Foxl2 in ovarian differentiation was confirmed bymale sex reversal of XX transgenic tilapia carrying adominant-negative mutant of Foxl2. XX tilapia carrying
extra copies of tilapia DMRT1 as a transgene induced
various degrees of gonadal changes including complete sex
change to testis, indicating that DMRT1 plays an importantrole in testicular differentiation. It is of great interest to notethat some of the sex reversed XX tilapia produced spermwith extremely high motility (Wang et al., unpublished).

Ⅱ. Molecular mechanisms of sex change
The sex-changing fish Trimma okinawae can change its sex
back and forth from male to female and then back to maleserially, dependent on social status in the harem. The gonadcorresponding to the sexual status of the fish remains
functional while the other is regressed. The swapping of the
gonads is initiated through a switching in the expression of
the gonadotropin hormone receptors, FSHR and LHR. These
two genes act as mediators to convey the information about
the change in social status to the to-be-active gonad. Most
intriguingly, the sex change in these fish starts with adramatic change in their sex-specific behavior immediately
after they realize their social status. This suggests that thebrain has a primary role in sexual development and plasticity
and presents an interesting challenge for future researchers.

Ⅲ. Sexual plasticity in the adult gonochoristic fish
With the exception of certain hermaphroditic species, mostvertebrate species are thought to have lost their sexualplasticity after differentiation of separate gonads/sexes witha single, distinct gamete type. Recently, we treated adultfemale tilapia with fadrozole (AI, a non-steroidal aromataseinhibitor) for two to five months to block the conversion ofandrogens to estrogens in order to investigate whether
sexual plasticity is retained in the adult gonochoristic fish
(Nakamura et al., unpublished). Suppression of estradiol17β(E2) production via AI treatment caused a rapid
degeneration of primary oocytes, leading to testicular germ
cell differentiation in the adult ovary. Sex-changed fishshow a typical male pattern of reproductive hormone levels
and secondary sex characteristics, producing fertile sperms
in the newly formed testes. Additionally, these fish display
male-specific territorial behavior, pointing towards thechanges that might have occurred to the sex-specificneuronal circuits in the brain. Conversely, co-treatment of
E2 inhibited AI-induced sex reversal. Our results
demonstrated for the first time in any gonochoristic species

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

that tilapia retains its sexual plasticity even in the adultstage. Furthermore, this data indicates that estrogens arevital to the maintenance of female phenotype ingonochoristic species.

results demonstrate that nPR induced by gonadotropin in
granulosa cells may regulate the expression of factors
involved in follicle rupture with 17α, 20β-DP as a ligand(Shibata et al., unpublished).

Publication List
〔Original papers〕● Ijiri, S., Kaneko, H., Kobayashi, T., Wang, D.S., Sakai, F., Paul-

Figure 1. Aromatase inhibitor (AI)-induced female-to-male sex reversalof adult tilapia females. Left, Vehicle-treated gonad. Right, AI-treated
gonad.

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. Our studies using vertebrate (fish) andinvertebrate (starfish) models have revealed that the basic
mechanisms involved in oocyte maturation are the same inthese two species despite the differing chemical nature of thehormonal agents involved. In both species, three major
mediators have been shown to be involved (Three stepmodel): a gonad-stimulating substance (GSS), 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 acid sequence
was determined (M. Mita, M. Yoshikuni et al.,unpublished). Based on its cysteine motif, the purified GSS
was classified as a member of the insulin/insulin-like growth
factor (IGF)/relaxin superfamily. Phylogenetic analyses
revealed that starfish GSS was a relaxin-like peptide.
Chemically synthesized GSS induced not only oocyte
maturation and ovulation in isolated ovarian fragments, butalso unique spawning behavior followed by the release of
gametes shortly after injection. In situ hybridization showed
the transcription of GSS to occur in the periphery of radial
nerves at the side of tube-feet. Thus, the current study
represents the first evidence of a relaxin system ininvertebrates and points towards a novel reproductive role
for this peptide in starfish.
17α, 20β-DP has been shown to be involved in bothoocyte maturation and ovulation (follicle rupture).
Interestingly, these actions of 17α, 20β-DP are mediatedthrough two different progestin receptors, the membrane(mPR) and nuclear (nPR) progestin receptors expressed in
the oocyte surface and follicular granulosa cells,
respectively. nPR transiently expresses after the timing of
LH release. The expression of a protease, membrane-typematrix metalloproteinase 2 involved in follicle rupture rose
after nPR expression by gonadotropin treatment. These

Prasanth, B., Nakamura, M., and Nagahama, Y. (2008). Sexual
dimorphic expression of genes in gonads during early differentiationof a teleost fish, the Nile tilapia Oreochromis niloticus. Biol. Reprod.
78, 333-341.
● Kobayashi, T., Kajiura-Kobayashi, H., Guan, G., and Nagahama, Y.
(2008). Sexual dimorphic expression of DMRT1 and Sox9 during
gonadal differentiation and hormone-induced sex reversal in the
teleost fish Nile tilapia (Oreochromis niloticus). Dev. Dyn. 237, 297306.
● Matsuoka, Y., Kobayashi, T., Kihara, K., and Nagahama, Y. (2008).
Molecular cloning of Plk1 and Nek2 and their expression in mature
gonads of the teleost fish Nile tilapia (Oreochromis Niloticus). Mol.
Reprod. Develop. 75, 989-1001.
● Mita, M., Ito, C., Nagahama, Y., and Shibata, Y. (2008). Expression
and distribution of gonad-stimulating substance in various organs of
the starfish, Asterina pectinifera. Ann. N.Y. Acad. Sci. in press.
● Sakai, F., Kobayashi, T., Matsuda, M., and Nagahama, Y. (2008).
Stability in aromatase immunoreactivity of steroid-producing cells
during early development of XX gonads of the Nile tilapia,
Oreochromis niloticus: An organ culture study. Zool. Sci. 25, 344348.
● Swapna, I., Sudhakumari, C.C., Sakai, F., Sreenivasulu, G.,
Kobayashi, T., Kagawa, H., Nagahama, Y., and Senthilkumaran, B.
(2008). Seabream GnRH immunoreactivity in brain and pituitary of
XX and XY Nile tilapia, Oreochroomis niloticus during early
development. J. Exp. Zool. Part A, 309, 419-426.

〔Review articles〕● Okubo, K., and Nagahama, Y. (2008). Structural and functional

evolution of gonadotropin-releasing hormone in vertebrates. Acta
Physiol. 193, 3-15.
● Nagahama, Y., and Yamashita, M. (2008). Regulation of oocyte
maturation in fish. Dev. Growth Differ. 50 Suppl 1, S195-219.
● Paul-Prasanth, B., Matsuda, M., Kobayashi, T., Suzuki, A., andNagahama, Y. (2008). Functional analysis of the medaka sex
determining gene, DMY - A minireview. Cybium 32, 72-73.

29

National Institute for Basic Biology

Developmental Biology

DIVISION FOR SEX DIFFERENTIATION

Professor (Concurrent)

MOROHASHI, Ken-ichirou
Assistant Professor
Technical Staff
Postdoctoral Fellows

Graduate Students

Visiting Scientists
Technical Assistants

OGAWA, Hidesato
OKA, Sanae
BABA, Takashi
KOMATSU, Tomoko
SHIMA, Yuichi
ISHIMARU, Yoshiyasu
MIYABAYASHI, Kanako
KATSURA, Yukako
SEKIHARA, Sanae
KOUNO, Yukie
KUSAKA, Masatomo
NISHIYAMA, Aya
TOMIDA, Sanai
HAYASHI, Tomoko

A number of genes are known to play crucial roles during
gonadal differentiation. Some of them have been identified
as the genes responsible for human diseases, while thefunctions of the other genes have been elucidated by thephenotypes of gene-disrupted mice. How the genes are
regulated by upstream regulators, however, is not yet fully
understood. Studies of this aspect of genes are quite
important in order to define the molecular mechanisms
mediating sex differentiation of the gonad.

Ⅰ. Fetal Leydig enhancer
During development of the testis, fetal and adult Leydig
cells arise. The fetal mouse Leydig cells emerge at E12.5
and start to produce the male sex hormone androgen. The
number of fetal Leydig cells decreases after birth. The adult
Leydig cells start to develop at around postnatal day 10 andthereafter increase in number. The fetal and adult Leydig
cells have been revealed to be morphologically andfunctionally different, and we have discussed whether thesetwo populations originate from two distinct cells. The
origins of the two Leydig cells, however, are still unclear.
We have studied the Ad4BP/SF-1 gene locus by transgenic
mouse assays and have recently identified the fetal Leydig
cell-specific enhancer. Using the DNA fragment, a mouseline in which the lacZ reporter gene is driven wasestablished. The lacZ expression was first recognized in thetesticular interstitium at E12.5. The number of the lacZexpressing cells increased during the fetal period anddeclined after birth. In the adult male mice, only a few lacZpositive
interstitial
cells
were
detected.
Immunohistochemical analyses revealed that the lacZ
colocalized with Leydig cell-specific markers. These results
indicated that the enhancer activity is strictly confined to the
fetal Leydig cell population.

Ⅱ. Fetal to adult adrenal
As Leydig cells, adrenocortical cells synthesize steroid
hormones. Moreover, the tissue is developmentally similar30

to Leydig cells in terms of the presence of fetal and adultcells. We isolated the fetal adrenal enhancer of the
Ad4BP/SF-1 gene and, using the enhancer, a transgenic
mouse to drive Cre-recombinase was established. The
mouse was crossed with Rosa 26 mice to trace the celllineage of the fetal cells. Examination of the adult adrenal
gland of the mice demonstrated that the adult adrenocorticalcells are derived from the fetal adrenocortical cells. Further
examination with another transgenic mouse carrying Cre-ER(a fusion protein of Cre and estrogen receptor, whose
recombinase activity is induced in the presence of an
estraogen antagonist) demonstrated that the early but not thelate stage of the fetal adrenal cortex has the potential todifferentiate into adult adrenal cortex.
This division was closed and moved to the Graduate
School of Medical Sciences, Kyushu University in July,
2008. All the members of this division wish to thank themembers of NIBB for the warm and friendly collaboration
we enjoyed here. During the past 10 years at this institute,we recognized the importance of basic research and the
responsibilities of the researchers. This experience hasformed the backbone of our motivation as basic researchers.

Publication List
〔Original papers〕● Zubair, M., Parker, K. L., and Morohashi, K. (2008). Developmental

links between fetal and adult adrenal cortex revealed by lineage
tracing. Mol. Cell. Biol. 28, 7030-7040.
● Shima, Y., Zubair, M., Komatsu, T., Oka, S., Yokoyama, C.,
Tachibana, T., Hjalt, T. A, and Morohashi, K. (2008). Pitx2 directly
regulates Ad4BP/SF-1 gene transcription in the pituitary gonadotrope
via interaction with the intronic enhancer. Mol. Endocrinol. 22, 16331646.
● Sato, Y., Baba, T., Zubair, M., Miyabayashi, K., Toyama, Y.,
Maekawa, M., Owaki, A., Mizusaki, H., Sawamura, T., Toshimori, K.,
Morohashi, K., and Katoh-Fukui, Y. (2008). Importance of forkheadtranscription factor Fkhl18 for development of testicular vasculature.
Mol. Repro. Dev. 75, 1361-1371.
● Baba, T., Shima, Y., Mimura, J., Oshima, M., Fujii-Kuriyama, Y., andMorohashi, K. (2008). Disruption of aryl hydrocarbon receptor (AhR)
induces regression of the seminal vesicle in aged male mice. Sex. Dev.
2, 1-11.
● Ishimaru, Y., Komatsu, T., Kasahara, M., Katoh-Fukui, Y., Toyama,Y., Maekawa, M., Toshimori, K., Chandraratna, R. A. S., Morohashi,K., and Yoshioka, H. (2008). Mechanism of asymmetric ovariandevelopment in chick embryos. Development 135, 677-685.
● Sakai, N., Terami, H., Suzuki, S., Haga. M., Nomoto, K., Tsuchida,N., Morohashi, K., Saito, N., Asada, M., Hashimoto, M., Harada, D.,
Asahara, H., Ishikawa, T., Shimada, F., and Sakurada, K. (2008).
Identification of NR5A1 (SF-1/AD4BP) gene expression modulatorsby large-scale gain and loss of function studies. J. Endocrinol. 198,489-497.

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION

Associate Professor

TANAKA, Minoru
Associate Professor
NIBB Research Fellows
Postdoctoral Fellows
Visiting Scientists
Technical Assistants

Secretary

TANAKA, Minoru
NAKAMURA, Shuhei
SAITO, Daisuke*
KOBAYASHI, KayoSAITO, Daisuke
AOKI, Yumiko
NAKAMURA, Shuhei *KINOSHITA, ChieNISHIMURA, Keiko
WATAKABE, Ikuko
YONEMITSU, Masako

Laboratory Scope
Our laboratory aims to reveal the molecular mechanisms of
the formation of the gonads and sex differentiation. We use
medaka fish (Oryzias latipes) for these purposes and havebeen generating transgenic medaka (Figure 1) enabling us to
identify the different cell lineages by fluorescence and toanalyze the process of gonad formation and sex
differentiation in vivo. Additionally, in order to identify thegenes essential for gonadogenesis, we carried out amutational screening of medaka with a defect in gonads andare performing a positional cloning. With these two uniqueanalyticalmethods(visualising cells andmutants), we areattempting to unveil thef u n d a m e n t a lmechanisms of sex
differentiation
andplasticity common to
Figure 1. Various Transgenic medaka.
many organisms.

Ⅰ. Balancing between germ cells and gonadal
somatic cells is important for sex
differentiation of gonads.
We have generated medaka that completely lack germ cells
in the gonad by impairment of PGC migration. These
medaka reveal two important aspects of germ cell functions
on the sex differentiation of gonads. Firstly, themorphological structure of germ cell-less gonads indicatesthe importance of germ cells for the formation of sexspecific structures in the gonads, possibly organizing thecommon unit into sex-specific structures dependent on its
genetic sex. The second point is that germ cells are essential
for development of the ovary. Without germ cells, both
genetical female and male medaka exhibit male secondary
sex characteristics and gonadal somatic cells are completely
masculinized. These results suggest that the female characterof germ cells antagonizes the autonomous masculinizationof gonadal somatic cells and that balancing between germ
cells and gonadal somatic cells is essential for both sex
differentiation and maintenance of sex.

In support of this claim, medaka mutants that display agerm cell-hypertrophic phenotype, hotei, exhibit female
secondary sex characteristics. The gene responsible for thisphenotype is the type II receptor gene for anti-Müllerianhomone (amhrII). We found that both amhrII and its ligand,amh, are expressed in gonadal somatic cells but not in germ
cells.
In order to understand if male to female sex reversal in
hotei mutants occurs because of a gonadal somatic cellautonomous defect or because of a large number of germ
cells, we have generated germ cell-deficient hotei mutants.
None of the germ cell-less hotei mutants show any sex
reversal of secondary sex characteristics. In addition,
female-specific gene expression that can be seen in genetic
male hotei mutants is also abolished when the germ cells are
depleted. These results indicate that feminization of the
gonad is a consequence of hypertrophic germ cells but not ofa gonadal somatic cell-autonomous event.
The results from both germ cell-deficient medaka and germcell-hypertrophic mutants (hotei) reveal an intrinsic
mechanism of sex differentiation that is independent ofgenetic sex determination. We propose that, irrespective ofgenetic sex, germ cells have an intrinsic character thatcanalizes feminization of the gonad while gonadal somatic
cells are predisposed to male development. According to this
proposal, the function of the medaka testis determination
gene on the Y chromosome can be explained as anenhancement of masculization that conquers canalization
towards ovary by germ cells. The balancing of the two
opposing characters, canalization of germ cells towards
feminization and predisposition of gonadal somatic cells
towards masculinization, may be a conserved cellularinteraction among vertebrates. In fact, there have been
several reports of the masculization of mice ovaries
following the depletion of germ cells by genetic or physicalmanipulations. Even in an undifferentiated gonocolist,
zebrafish, which firstly develops ovaries before some of the
population turns into males, prior to the development of
testis the germ cell number decreases once by apoptosis. The
suppression of the germ cell number at the early stage of
medaka gonadal sex differentiation and the decrease in germ
cells in zebrafish can be viewed as different ways ofachieving the same result (Tanaka et al., 2008 DGD: Saito etal., Sex. Dev. in press).

Figure 2. A right lobe of sox9bp:GFP-transgenic medaka. Blue: DAPI,Red: Germ Cell and Green: GFP.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

31

National Institute for Basic Biology

Developmental Biology

Ⅱ. Gonadal morphology during early stagesof sex differentiation
Our previous results indicated that, prior to the formationof the gonadal primordium, two different precursors ofgonadal somatic cells are established in the most posterior
region of lateral plate mesoderm. These precursors express
either sox9b or ftz-f1. In mammals, sox9b is specifically
expressed in the male supporting cells surrounding germ
cells (Sertoli cells and their precursors) and is known as thegene indispensible for testis formation. In order tounderstand the types of cells arising from the precursors inmedaka gonad, we have generated transgenic medaka thatallow us to keep track of sox9b-expressing cells in the
gonad.
With the formation of the gonadal primordium, sox9bexpressing cells ensheathed primordial germ cells.
Obervation of sox9b-expressing cells revealed the change ofthe morphology of the primordium. The gonadal
primordium firstly forms as a single structure located along
the midline. The cells that do not express sox9b appear andinvade in the middle of the single gonadal primordium. As aconsequence, the gonadal primordium splits into bilaterallobes. This process occurs soon after the single gonadal
primorium forms (Figure 2).
Sox9b-expressing cells in male gonads keep surrounding
the germ cells and begin to express the male supporting cell
(Sertoli) marker, dmrt1, which is similar to those in
mammalian testis development. In female gonads, the germ
cells that undergo synchronous division enter meiosis andform follicles. Some of the population of germ cells infemale gonads enters meiosis and follicles are formed.
Unlike mammals, expression of sox9b is retained and foundin the cells surrounding germ cells. Very interestingly, thegranulosa cells of small follicles (primordial follicles) also
express sox9b, but its expression diminishes immediately
with the progress of folliculogenesis and the onset of theexpression of the granulosa cell marker, foxl2.
Collectively, the observations using sox9b-transgenic
medaka strongly suggest that the supporting cells expressing
sox9b in the gonadal primordium are present as precursors
of both Sertoli cells and granulosa cells.

Ⅲ. Germ cell development with change of thelocalization of germ-granule components
Germ granules are germ cell-specific intracellular
structures and are essential for germ cell development. An
examination of their components - olvas (vasa), nanos andtdrd1 (tudor) - reveals that they alter their localization in thecytoplasm during the early stage of sex differentiation of the
gonads. By immunohistochemical analysis, these threegermline-specific proteins were detectable on granule-likestructures in the cytoplasm of migrating primordial germ
cells (PGCs). In the germ cells of the gonadal primordia,
these granules formed a hollow area lacking these threeprotein components. During the sexual differentiation of thefemale gonads, the granules were found to be reduced in sizein the germ cells undergoing cystic division and they
showed perinuclear localization in the oocytes. The germ
cells in the male gonads, however, retained their hollow

32

granules during this early sex differentiation stage. These
results provide novel information on a distinct stage of germ
cell development and suggest different stage-specific roles
for germ granules (Figure 3).

Figure 3. Change of localization of germ granule components olvas,
nanos and tudor.

Publication List
〔Original papers〕● Aoki, Y., Nagao, I., Saito, D., Ebe, Y., Kinjo, M., and Tanaka, M.

(2008). Temporal and spatial localization of three germline-specificproteins in medaka. Dev. Dyn. 273, 800-807.
● Nagao, I., Aoki, Y., Tanaka, M., and Kinjo, M. (2008). Analysis of themolecular dynamics of medaka nuage proteins by fluorescencecorrelation spectroscopy and fluorescence recovery after
photobleaching. FEBS J. 275, 341–349.
● Nakabayashi, T., Nagao, I., Kinjo, M., Aoki, Y., Tanaka, M., andOhta, N. (2008). Stress-induced environmental changes in a single cell
as revealed by fluorescence lifetime imaging. Photonchem. Photobiol.
Sci. 7, 671-674.
● Nakamura, S., Aoki, Y., Saito, D., Kuroki, Y., Fujiyama, A., Naruse,
K., and Tanaka, M. (2008). Sox9b/sox9a2-EGFP transgenic medaka
reveals the morphological reorganization of the gonads and a common
presursor of both the female and male supporting cells. Mol. Reprod.
Dev. 75, 472-476.
● Sasado, T., Yasuoka, A., Abe, K., Mitani, H., Furutani-Seiki, M.,
Tanaka, M., and Kondoh, H. (2008). Distinct contributions of
CXCR4b and CXCR7/RDC1 receptor systems in regulation of PGC
migration revealed by medaka mutants kazura and yanagi. Dev. Biol.
320, 328-339.

〔Review articles〕● Nakamura, S., Saito, D., and Tanaka, M. (2008). Generation oftransgenic medaka using modified bacterial artificial chromosome.
Dev. Growth Differ. 50, 415-417.
● Tanaka, M., Saito, D., Morinaga, C., and Kurokawa, H. (2008). Cross
talk between germ cells and gonadal somatic cells is critical for sex
differentiation of the gonads in the teleost fish, medaka (Oryzias
latipes). Dev. Growth Differ. 50, 273-278.

LABORATORY OF PLANT ORGAN DEVELOPMENT

Director-General

OKADA, Kiyotaka
Assistant Professor
Postdoctoral Fellows

Visiting Scientists
Graduate Students

Technical Assistant
Secretary

TATEMATSU, Kiyoshi
TSUCHIDA, Yuhei
IGARASHI, Hisako
URAWA, Hiroko
WADA, Takuji
TOMINAGA, Rumi
TOYOKURA, Koichi
IWASAKI, Akira
TAMESHIGE, Toshiaki
HARA, Reiko
SAKAGAMI, Mari

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 that a coupleof genes are involved in the axes-dependent control oflateral organ development. The adaxial-abaxial boundary in
the leaf primordium is determined by the precise expressionof the adaxial marker gene, PHABULOSA (PHB), and theabaxial marker genes, FILAMENTOUS FLOWER (FIL)and YABBY3. We showed that PHB is expressed in cells of
the adaxial side and separated clearly from the abaxial sidespecific FIL gene expressing cells by action of
microRNA165/166, which targeted the PHB, REV, andPHV messenger RNAs. Using laser microdissection
techniques we also revealed that specific expression of
miR165/166 genes in the abaxial side is important for the
adaxial-abaxial boundary formation.

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(SAM).

Ⅲ. Microsurgical approach
We are also carrying out a microsurgical approach,ablating cells localized at SAM, at the peripheral zone, andat the leaf primodium, to investigate the cell-to-cellcommunication systems working in the leaf development byusing a novel laser-ablating microscopy with UV-laser.
When we ablated cells at SAM, the plant formed afilamentous leaf which lacked the adaxial-abaxial identity(Figure 1 bottom left).

Figure 1. The disruption of cells at SAM by laser ablation system resultsin filamentous leaf formation. Top: Fluorescent images of SAM before(left) and after (right) the ablation. Middle: High magnification of thelaser ablation point before (left) and after (right) ablation. Bottom: Leaf
development of untreated control (left) and of the ablated plant (right).
Laser ablated point is within the yellow circle. Scale bar: 10μm

Publication List
〔Original papers〕● Sakai, T., Honing, van der H., Nishioka, M., Uehara, Y., Takahashi,M., Fujisawa, N., Saji, K., Seki, M., Shinozaki, K., Jones, M.,
Smirnoff, N., Okada, K., and Wasteneys, G. (2008). Armadillo repeatcontaining kinesins and a NIMA-related kinase are required for
epidermal cell morphogenesis in Arabidopsis. Plant J. 53, 157-171.
● Nagashima, A., Suzuki, G., Uehara, Y., Saji, K., Furukawa, T.,
Koshiba, T., Sekimoto, M., Fujioka, S., Kuroha, T., Kojima, M.,
Sakakibara, H., Fujisawa, N., Okada, K., and Sakai, T. (2008).
Phytochromes and cryptochromes regulate the differential growth of
Arabidopsis hypocotyls in both a PGP19-dependent and -independent
manner. Plant J. 53, 516-529.
● Tsuchida-Mayama, T., Nakano, M., Uehara, Y., Sano, M., Fujisawa,N., Okada, K., and Sakai, T. (2008). Mapping and Characterization of
the Phosphorylation Sites on the Phototropic Signal Transducer,
NPH3. Plant Sci. 174, 626-633.
● Tominaga, R., Iwata, M., Sano, R., Okada, K., and Wada, T. (2008).
Arabidopsis CAPRICE-like Myb 3 (CPL3) Controls Endoreduplication
and Flowering Development in Addition to Trichome and Root-hairFormation. Development 135, 1335-1345.
● Shimizu, K. K., Ito, T., Ishiguro, S., and Okada, K. (2008). MAA3(MAGATAMA3) helicase gene is required for female gametophyte
development and pollen tube guidance in Arabidopsis thaliana. Plant
Cell Physiol. 49, 1478-1483.

33

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

NODA, Masaharu
Assitant 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
SHIMIZU, Hidetada
NISHIHARA, Eri
YOSHIDA, Masahide
YONEHARA, Keisuke
YONEHARA, Keisuke*
CHOW, Pak Hong Jeremy
NAGAKURA, Ayano
ZHANG, Lanfan
TANI, Sachiko
SAKURABA, Jyuichi
YONEHARA, KayoMIZOGUCHI, Masae
KATAYAMA, Tomomi
HATTORI, Nobuko
TOMITA, Nao
ISOSHIMA, Yoshiko
MIURA, Seiko
KODAMA, Akiko
YATAGAI, Hiroko

We have been studying the molecular and cellularmechanisms underlying the development of the vertebrate
central nervous system (CNS), mainly using the visualsystems of chicks and mice. This research covers many
developmental events including the patterning of the retina,
axonal navigation, branching and targeting, synapse
formation, refinement and plasticity. The scope of ourinterests also encompasses the mechanisms for various
functions of the mature brain, including sensation, emotion,
behavior, learning, and memory.

for the topographic retinotectal projection (see Annual
Report 2006). We are now conducting experiments toelucidate the functional modes of CBF2, a winged-helix typetranscription factor, which specifies the temporal characterin the retina.

Ⅱ. Mechanisms for the topographic retinotectal
projection
Special attention is now devoted to the molecularmechanisms for the axon branching and arborization andtheir selective elimination. Among the region-specificmolecules in the developing retina, we have alreadyidentified several molecules which induce abnormal
branching and arborization when their expression was
experimentally altered in vivo. We have obtained promising
results indicating that our research will continue to shedlight on these issues.

Ⅲ. Development of retinal ganglion cell subtypes
Visual information is transmitted to the brain by roughly adozen distinct types of retinal ganglion cells (RGCs) definedby a characteristic morphology, physiology, and central
projections. However, because few molecular markers
corresponding to individual RGC types are available, ourunderstanding of how these parallel pathways develop is stillin its infancy. Previously, we reported a secretory protein,
SPIG1 (clone name; D/Bsp120I #1), preferentially expressedin the dorsal region in the developing chick retina (Shintani
et al., 2004). Subsequently, we generated knock-in mice tovisualize SPIG1-expressing cells with green fluorescentprotein. We found that the mouse retina is subdivided into
two distinct domains for SPIG1 expression and SPIG1
effectively marks a unique subtype of the retinal ganglion
cells during the neonatal period (Figure 1).

Ⅰ. Mechanisms for retinal patterning during
development
Topographic maps are a fundamental feature of neuralnetworks in the nervous system. The retinotectal projectionof lower vertebrates including birds has been used as areadily accessible model system of the topographic
projection. We have been studying the mechanisms forregional specification in the developing retina as the basis of
the topographic retinotectal projection.
Fifteen years ago, we performed a large-scale screening
using differential hybridization and restriction landmark
cDNA scanning (RLCS) on the embryonic day 8 (E8) chickretina, and successfully identified 33 asymmetric moleculesalong the nasotemporal (N-T) axis and 20 along thedorsoventral (D-V) axis. We have almost revealed genecascades of topographic molecules for retinal patterning and

34

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).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

SPIG1-positive RGCs in the dorsotemporal domain projectto the dorsal lateral geniculate nucleus (dLGN), superior
colliculus, and accessory optic system (AOS). In contrast,in the remaining region, here named the pan-ventronasal
domain, SPIG1-positive cells form a regular mosaic andproject exclusively to the medial terminal nucleus (MTN) of
the AOS that mediates the optokinetic nystagmus as early as
P1. Their dendrites costratify with ON cholinergic amacrine
strata in the inner plexiform layer as early as P3. Thesefindings suggest that these SPIG1-positive cells are the ON
direction selective ganglion cells (DSGCs). Moreover, theMTN-projecting cells in the pan-ventronasal domain areapparently composed of two distinct but interdependent
regular mosaics depending on the presence or absence of
SPIG1, indicating that they comprise two functionallydistinct subtypes of the ON DSGCs. The formation of theregular mosaic appears to commence at the end of theprenatal stage and be completed through the peak period of
the cell death at P6. SPIG1 will thus serve as a usefulmolecular marker for future studies on the development andfunction of ON DSGCs.

cleavage sites in the extracellular juxtamembrane region of
Ptprz by tumor necrosis factor-a converting enzyme (TACE)
and matrix metalloproteinase 9 (MMP-9). This is the firstevidence of the metalloproteinase-mediated processing of a
receptor-like protein-tyrosine phosphatase in the central
nervous system.

Ⅳ. Physiological roles of protein tyrosine
phosphatase receptor type Z
Protein tyrosine phosphatase receptor type Z (Ptprz, also
known as PTPζ/RPTPβ) is preferentially expressed in the
CNS as a major chondroitin sulfate proteoglycan. Three
splicing variants, two receptor isoforms (Ptprz-A/B) and one
secretory isoform (Ptprz-S) are known. Receptor-type Ptprz
interacts with the PSD95 family through its intracellularcarboxyl-terminal PDZ-binding motif in the postsynaptic
density. Ptprz-deficient adult mice display impairments inspatial and contextual learning.

Figure 2. Proteolytic processing of Ptprz. Generated fragments areshown with their molecular size. Ptprz is a target for activity-dependent
processing, which is supposedly involved in the synaptic remodeling
during learning and memory.

4-3 Identification of TrkA as a substrate for Ptprz
4-1 Plasmin mediated processing of Ptprz
We identified the proteolytic processing of Ptprz byplasmin in the mouse brain, which is markedly enhanced
after kainic acid (KA)-induced seizures. We mapped
plasmin cleavage sites in the extracellular region of Ptprz bycell-based assays and in vitro digestion experiments withrecombinant proteins (Figure 2). These findings indicate that
Ptprz is a physiological target for activity-dependent
proteolytic processing by the tPA/plasmin system, andsuggest that the proteolytic cleavage is involved in thefunctional processes of the synapses during learning andmemory.

4-2 Metalloproteinase- and γ-secretase-mediatedprocessing of Ptprz
We also found that the extracellular region of the receptor
isoforms of Ptprz are cleaved by metalloproteinases, and thatthe membrane-tethered fragment is subsequently cleaved bypresenilin/γ-secretase, releasing its intracellular region into
the cyto plasm (Figure 2). Notably, the intracellularfragment of Ptprz shows nuclear localization.
Administration of GM6001 (an inhibitor of
metalloproteinases) to mice demonstrated themetalloproteinase-mediated cleavage of Ptprz underphysiological conditions. Furthermore, we identified the

Tropomyosin-related kinases (Trks) are single-passtransmembrane molecules that are highly expressed in thedeveloping nervous system. Upon the ligand binding of
neurotrophins, Trk receptors are activated throughautophosphorylation of tyrosine residues; however, the PTPs
responsible for the negative regulation of Trk receptors havenot been fully elucidated. We identified Ptprz as a specific
PTP that efficiently dephosphorylates TrkA as a substrate(Figure 3). Co-expression of Ptprz with Trk receptors in
293T cells showed that Ptprz suppresses the ligandindependent tyrosine phosphorylation of TrkA, but not of
TrkB or TrkC, and that Ptprz attenuates TrkA activationinduced by nerve growth factor (NGF). Co-expression
analyses with TrkA mutants revealed that Ptprz
dephosphorylates phosphotyrosine residues in the activation
loop of the kinase domain, which are requisite for activationof the TrkA receptor. Consistent with these findings, forced
expression of Ptprz in PC12D cells markedly inhibitedneurite extension induced by a low dose of NGF. In
addition, an increment in the tyrosine phosphorylation of
TrkA was observed in the brain of Ptprz-deficient mice.
Ptprz thus appears to be one of the PTPs which regulate theactivation and signaling of TrkA receptors.

35

National Institute for Basic Biology

Neurobiology

Publication List
〔Original papers〕● Chow, J.P.H., Fujikawa, A., Shimizu, H., and Noda, M. (2008).

Figure 3. Schematic drawing of regulation of TrkA and ErbB4 by Ptprz.
ErbB4 and TrkA are activated by their specific ligands, neuregulins and
NGF, respectively. Ptprz attenuates the activation of ErbB4 (Fujikawa etal., 2007) and TrkA (Shintani et al., 2008) through dephosphorylation.

Plasmin-mediated processing of protein tyrosine phosphatase receptor
type Z in the mouse brain. Neurosci. Lett. 442, 208-212.
● Chow, J.P.H., Fujikawa, A., Shimizu, H., Suzuki, R., and Noda, M.
(2008). Metalloproteinase- and γ-secretase-mediated cleavage ofprotein tyrosine phosphatase receptor type Z. J. Biol. Chem. 283,
30879-30889.
● Shintani, T., and Noda, M. (2008). Protein tyrosine phosphatase
receptor type Z dephosphorylates TrkA receptors and attenuates NGFdependent neurite outgrowth of PC12 cells. J. Biochem. 144, 259-266.
● Tsuboi, N., Utsunomiya, T., Roberts, R.L., Ito, H., Takahashi, K.,
Noda, M., and Takahashi, T. (2008). The tyrosine phosphatase CD148
interacts with the p85 regulatory subunit of phosphoinositide 3-kinase.
Biochem. J. 413, 193-200.
● Yonehara, K., Shintani, T., Suzuki, R., Sakuta, H., Takeuchi, Y.,
Nakamura-Yonehara, K., and Noda, M. (2008). Expression of SPIG1
reveals development of a retinal ganglion cell subtype projecting to
the medial terminal nucleus in the mouse. PLoS ONE 3, e1533.

〔Review articles〕

Ⅴ. Mechanisms of Na-level sensing in thebrain for the body-fluid homeostasis
Dehydration causes an increase in the sodium (Na)concentration and osmolarity of body fluids. For Na
homeostasis of the body, control of Na and water intake andexcretion are of prime importance. Although it 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.
Nax is an atypical sodium channel that is assumed to be adescendant of the voltage-gated sodium channel family. Ourstudies on the Nax- knockout mice revealed that Nax channels
are localized to the CVOs and serve as a sodium-levelsensor of body fluids. Na x- knockout mice do not stop
ingesting salt when dehydrated, while wild-type mice avoidsalt.
Na x is exclusively localized to perineuronal lamellate
processes extending from ependymal cells and astrocytes in
the organs. Therefore, glial cells bearing Nax channels are
the first to sense a physiological increase in the level of
sodium in body fluids. We revealed that Na-dependent
activation of the metabolic state of the glial cells leads toextensive lactate production and that lactate stimulated theactivity of GABAergic neurons in the subfornical organ(SFO). Thus, information of a physiological increase of the
Na level in body fluids sensed by Na x in glial cells istransmitted to neurons by lactate as a mediator to regulate
the neural activity of the SFO to control salt-intake behavior.
Very recently, we found that Nax- deficient mice show a
reduced water intake compared with wild-type mice under
non-feeding conditions. We suppose that animals deficientin the sodium-sensing system are apt to develop
hypernatremia.

36● Noda, M., Takahashi, H., and Sakuta, H. (2008). Neural patterning:

Eye fields. In Encyclopedia of Neuroscience, Squire, L.R., ed.
(Elsevier), pp. 199-204.
● Sakuta, H., Suzuki, R., and Noda, M. (2008). Retrovirus vectormediated gene transfer into the chick optic vesicle by in ovo
electroporation. Dev. Growth Differ. 50, 453-457.

DIVISION OF BRAIN BIOLOGY

Professor

YAMAMORI, Tetsuo
Assistant Professors

NIBB Research Fellows
Technical Staff
Postdoctoral Fellows
Graduate Students

Technical Assistants

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

In order to understand the formation and evolution of thebrain, we are studying the genes that are expressed in
specific areas of the neocortex.

Ⅰ. Genes expressed in specific areas and layersof the neocortex

By differential display methods, we found three areaspecific expression genes in the primate neocortex.
Firstly, occ1 is specifically expressed in the occipital cortex
in the primate brain. Secondly, the other gene that showedmarked difference within the neocortex is gdf7, a member of
BMP/TGF-β family, which is specifically expressed in the
motor cortex of the African green monkey (Watakabe et al.,J. Neurochem., 76, 1455-1464, 2001). Thirdly, Rbp (retinolbinding protein) is preferentially expressed in associationand higher areas in the neocortex (Komatsu et al., Cerebral
Cortex, 15, 96-108, 2005).
To screen area-specific molecules systematically in themonkey neocortex, we carried out a new round of screening
using the RLCS method (Suzuki et al. 1996; Shintani et al.
2004). In this analysis, mRNAs were purified from 4 distinct
cortical areas, converted to cDNA by reverse transcription
and digested with a pair of restriction enzymes for 2dimensional analysis. Among the spots that showed area
difference, we cloned a gene that is specifically expressed in
the visual area, which turned out to be the 5-HT1B receptor
gene. (Watakabe et al., Cerebral Cortex, published online on
December 4, 2008).
The expression of 5-HT1B revealed by in situ
hybridization was strikingly high in V1 and the lateralgeniculate nucleus (LGN) (Figure 2C, E). Because themRNA expression was low in the extrastriate cortex, theabrupt change in the intensity of mRNA staining wasobserved at the border between V1 and V2 (Figure 2F).

The neocortex emerged in mammals and evolved mostremarkably in the primate. It is puzzling, however, thatduring mammalian evolution the neocortex was markedly
expanded while the total number of genes in the mammal
was little changed. In order to understand this puzzle, we
studied gene expression patterns within different areas of the
neocortex. In the last ten years, we reported the following
findings, which are schematically illustrated in Figure 1.

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 ).

Figure 2. ISH analysis of 5-HT1B receptor mRNA. (A-E) Coronal
sections of an adult monkey brain were prepared from the positions as
depicted. Bar: 1 cm. Several areas are shown: fro, frontal area; temp,
temporal area; S1, primary somatosensory area; A1, primary auditory
area. PBG, parabigeminal nucleus; Pul, pulvinar nucleus; St, striatum.
Major sulci are represented by lowercase letters: p, principal sulcus; cal,calcarin sulcus; ce, central sulcus; lf, lateral fissure; ts, superior temporalsulcus. (F) 5-HT1B receptor mRNA expression at the V1/V2 border(shown by the black arrow). Bar: 500 μm. (G) The adjacent Nissl-stained
section. (H) 5-HT1B receptor mRNA expression in V1. Bar: 200 μm.
(Watakabe et al., Cerebral Cortex, 2008)37

National Institute for Basic Biology

Neurobiology

Within V1, the expression of 5-HT1B receptor mRNA wasmostly confined to layers IVA and IVC, the major
geniculocortical input layers, and was particularly strong in
the lower part of layer IVC beta (Figure 2H). In addition, theexpression was also observed at lower intensity in layers
II/III and VI of V1, which also receive geniculocortical
inputs to a lesser extent. In the LGN, the strong mRNA
expression was observed in all 6 layers, and there was no
significant difference in staining intensity between themagnocellular and parvocellular layers. The 5-HT1B
receptor mRNA expression was by far the most conspicuousin V1, followed by that in the LGN.
Among the 13 genes of serotonin receptors, 5-HT2A
receptor mRNA exhibited area and lamina preferences
similar to those of 5-HT1B receptor mRNA, although its
expression was moderate across all areas. Both mRNA
species were highly concentrated in the geniculorecipientlayers IVA and IVC, where they were coexpressed in thesame neurons. Monocular inactivation by tetrodotoxin
injection resulted in a strong and rapid (<3 h)downregulation of these mRNAs, suggesting the retinal
activity dependency of their expression.
In collaboration with Professor Hiromichi Sato’s laboratory(Osaka University), we examined the roles of the two
serotonin receptors. The activation of 5-HT1B receptors in
V1 by specific agonist generally facilitates visual responses
but tends to suppress weak responses. Our analysis suggests
that, in geniculocortical transmission, nonsychronized
spontaneous activity (noise) from the LGN neurons would
be reduced by the suppressive effect of 5-HT1B receptors,
but the visually evoked synchronized signals would bepreserved or efficiently transferred to V1, thus, enhancing
the S/N ratio in input-output relationship. On the other hand,
neurons in the input layers of V1 which abundantly express
the 5-HT2A receptor may act as a gain controller byenhancing weak signal response and suppressing excessive
response. We therefore suggest that serotonin release in V1
exerts coordinated modulatory effects through 5-HT1B and5-HT2A receptors on the V1 neurons. It is therefore possiblethat the serotonin system has contributed to the evolution of
the elaborated function of the primate visual system.
The other gene is testican-1, which is specifically
expressed in the primate visual cortex. Since testican-1 is amember of a testican family gene which also includes occ1,
we were particularly interested in examining all the known
members of the testican family (testican-1, testican-2,
testican-3, SPARC, SC1) other than occ1 (Takahata et al.,Cerebral Cortex, published online on December 10, 2008).
Interestingly, the in situ hybridization revealed that there
are three groups of expression pattern among the occ1related (testican) family members. The expression patternsof testican-1 and testican-2 are similar to that of occ-1,
starting high level in V1, progressively decreasing along theventral visual pathway, and ending low level in the temporal
areas. Complementary to them, the neuronal expression of
SPARC mRNA is abundant in the association areas andscarce in V1. Therefore, the expression pattern of SPARC issimilar to that of Rbp. In addition, whereas occ1, testican-1,
and testican-2 mRNAs are preferentially distributed in38

thalamorecipient layers including "blobs," SPARC mRNA
expression avoids these layers. Neither SC1 nor testican-3
mRNA expression is selective to particular areas, but SC1mRNA is abundantly observed in blobs.
Lines of evidence suggest that ECM considered tomodulate neuronal development and plastic changes. Several
groups have proposed that ECM limits plasticity in therodent neocortex and ECM degeneration is required to
implement ocular dominance plasticity (Pizzorusso et al.
2002; Oray et al. 2004). Secreted glycoproteins, such as
Reelin, regulate both neuronal positioning in the developing
nervous system and synaptic plasticity in the adult (Bockand Herz 2003; Dityatev and Schachner 2006). The area
selectivity and activity dependence in expression of occ1related genes, secreted glycoproteins, raise the possibility
that OCC1-related proteins modulate synaptic plasticity inthe adult cerebral cortex, some of which mechanisms are
specific in primates.

Ⅱ . Gene expression under multisensory
enhancement
We have been collaborating with professor Yoshio Sakurai(Kyoto University) and developed an audio-visual
discrimination task (AVD-task) system, placing auditory andvisual stimuli in the same position. We use nose-poking tomeasure the reaction time in which a rat responds to stimuli.
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 (Figure 3).
Combining this system with our newly developed “Cortical
Box Methods” (Figure 3), we found the secondary visualcortex (V2L) in rats is specifically activated underaudiovisual multisensory stimulation. Injecting muscimol
into V1, V2, V2L and superior colliculus (SC), we foundthat V2L is specifically involved in the stimulation ofmultisensory reaction (Hirokawa et al., 2008).
Traditionally, multisensory integration was thought tooccur in higher neocortical areas through a merging ofdifferent modalities of primary sensory information. Our
results suggest that the multisensory integration may in factoccur 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 in
the boundary areas between two modal areas (Toldi et al.,
1986; Barth et al., 1995; Wallace et al., 2004). Thisobservation is consistent with our findings using the newly
developed behavioral system and c-Fos analyzing system.

The expression of the three layer specific genes showed
tight relationships with the functional areas. This not only
provides insight into the cortical area architecture
underlining the complex spatial expression patterns of thesegenes, but also may serve as a framework for investigating
gene expression regulation within the neocortex for future
researchers.

Publication List
〔Original papers〕● Hirokawa, J., Watakabe, A., Ohsawa, S., and Yamamori, T. (2008).

Figure 3 (a) Representative photomicrograph of cortical section stainedby antibody against c-Fos protein. (b) The local density of c-Fos-positive
cells was computed and pseudocolored. The mediodorsal end (MD),lateroventral end (LV), inner contour (IC) and outer contour (OC) weremanually chosen to extract part of the cortex. Scale bars, 1 mm (a, b) and
100 mm (insets in a, b). (c) The extracted cortex was divided into 100
bins (left), and each bin was converted into a standard rectangle (left tocenter as shown by thick arrows). The standardized rectangular bins
were reassembled into a stripe to form a standardized cortical section(right). (d) Standardized cortical sections were assembled from theposterior section to the anterior section to form a standardized corticalbox. (e) A specific layer fraction (layer 4, for example) from astandardized cortical box was extracted to construct a standardized layermap (Shown in Hirokawa et al., 2008).

Analysis of Area-Specific Expression Patterns of RORbeta, ER81 andNurr1 mRNAs in Rat Neocortex by Double In Situ Hybridization andCortical Box Method. PLoS ONE 3, e3266.
● Lyckman, A.W., Horng, S., Leamey, C.A., Tropea, D., Watakabe, A.,
Van Wart, A., McCurry, C., Yamamori, T., and Sur, M. (2008). Gene
expression patterns in visual cortex during the critical period: Synaptic
stabilization and reversal by visual deprivation. Proc. Natl. Acad. Sci.
USA 105, 9409-9414.
● Hirokawa, J., Bosch, M., Sakata, S., Sakurai, Y., and Yamamori, T.
(2008). Functional role of the secondary visual cortex in multisensory
facilitation in rats. Neuroscience 153, 1402-1417.
● Takahata, T., Hashikawa, T., Higo, N., Tochitani, S., and Yamamori,T. (2008). Difference in sensory dependence of occ1/Follistatinrelated protein expression between macaques and mice. J. Chem.
Neuroanat. 35, 146-157.

Ⅲ. Neocortical areas revealed by layer specificgene expression in rats.
To investigate area differences in rodent neocortex in termsof gene expressions, we chose three genes, RORbeta, ER81
and Nurr1, whose mRNAs are mainly expressed in layers 4,
5 and 6, respectively. To reveal their complex spatial
distribution patterns, we used double in situ hybridization
histochemistry (ISH) and cortical box method for the
analyses, as mentioned above (Hirokawa et al., 2008).
Double ISH revealed the large area differences in therelative abundance and extent of intermixing of theexpressions of these mRNAs. Based on this finding, wequantitatively analyzed the ISH patterns of the three genes
by cortical box method as described above. We made threemajor discoveries. Firstly, the three genes showed uniquearea distribution patterns that were mostly complementary to
one another. Secondly, the cortical areas defined on the basisof the expression patterns of the three genes matched wellwith the cytoarchitectonic areas defined by Nissl staining.
Finally, principal component analysis results suggested thatthe expressions of these three genes may be dictated bycommon rules that are tightly associated with the function
and topology of the cortical areas.

39

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji
NIBB Research Fellow

MATSUNAGA, Wataru

Animals respond to the physical and chemical stimuli of
their environment. Most higher vertebrates have evolved ahighly developed visual system to respond to light. “Whycan we see?” This question is a fundamental one for athorough understanding of these animals, including humanbeings. In order to more completely understand the sensory
systems of animals, we decided to move ahead to research ofvisual systems this year, though we had been researching thesalt-sensing system (~March, 2008). The research objects ofour new project were the medaka fish and human beings.
Various aspects of their behaviors rely on their visualsystems.

Ⅰ. Psychobiological studies of Medaka
One of our major subjects is the psychobiological study of
medaka (Oryzias latipes). Medaka is a world-wide teleostfish model, as is zebrafish (Danio rerio). Importantdiscoveries (including advances in the areas of heredity ofbody colour and the sex-determning gene) were brought
about through developmental studies of medaka. Medaka is
not widely used, however, in neurobiological fields, andstandardized methods of behavioral phenotyping for medaka
have been not established.

Figure 1. Representative examples of Medaka fish behaviors. A) Diving
response. When Medaka fish are transferred to a novel environment,
they immediately move to the bottom of the water tank. B) Climbing
response. Medaka move to the water surface within several minutes.

The brain structure of medaka meets the minimum
requirements for simulating the human brain. For
neurobiological or behavioral studies, small teleosts(including medaka) are sometimes more useful than rodents
thanks to their simplicity. Presently, zebrafish is more
popular as a model animal than medaka. Medaka, however,has unique behavioral characteristics. Medaka can swim
backward and hover within water flow by doing so.
Moreover, medaka show interesting optomotor responses
such as the dorsal light response under microgravity and themaintenance of their relative position to a rotating stripes
pattern around them. Medaka, therefore, is a noteworthy

40

model for the study of brain and behavior.
Our first goal is finding the medaka's behavioral
characteristics that are applicable to the behavioral
phenotyping of mutants and the study of relations between
brain and behavior. We are attempting to classify typicalnormal behaviors of medaka (Figure 1). We are also
studying the effects of acute stimulations by psychoactive
drugs such as ethanol, nicotine and caffeine on medaka.
These attempts will enable us to precisely describe thebehaviors of medaka, which will form the basis of our futurestudies.

Ⅱ. Psychobiological studies of Human
Another of our major subjects is the psychobiological studyof human beings (Homo sapiens). The visual system of ananimal can be correctly understood by comparing two or
more animals. Therefore, in our laboratory we address theissue of the human visual system in tandem with that of
medaka. Recently, it has become possible to make complex
visual stimuli easily by using computers and digital display
systems, and the importance of psychophysics is once again
beginning to be recognized.
In order to interact successfully with the environment,
animals must know the accurate positions of objects inspace, though those positions frequently change. Neuralprocessing, however, requires considerable time. By thetime a conclusion is reached about location, the moving
object has moved on to a new position in the actual world.
Does our visual system compensate for this difference?
One recent focus of this debate is the flash-lag effect, inwhich a moving object is perceived to lead a flashed object
when both objects are aligned in the actual physical space(Figure 2). The discoverer Dr. Nijhawan has proposed thatthe human visual system uses the motion signals to
extrapolate the position of a moving object (motion
extrapolation hypothesis). The differential latencies
hypothesis proposes that the flash-lag effect occurs simply
because the visual system responds with a shorter latency tomoving objects than to flash stimuli. Besides these major
two models, various modified models have been proposed.
The problem, however, has not yet been solved, and thedebate continues. How does our brain decide the position of
the moving object? In the present study, we are making anattempt to correctly interpret the flash-lag effect.

Figure 2. The flash-lag effect. A moving object is perceived to lead inspace a flashed object when both objects are aligned in the actual
physical space.

LABORATORY OF NEUROCHEMISTRY

Associate Professor

SASAOKA, Toshikuni
NIBB Research Fellow

SATO, Asako

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

been investigating the involvement of dopaminergictransmission 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 died by 4 weeks of age. To
investigate the molecular mechanism of motor control andeating behavior, we generated transgenic mice harboring
tetracycline-regulated expression of the D1R gene on the
D1R/D2R DKO background. Several transgenic mouse lines
successfully rescued lethal phenotype of the D1R/D2R DKO
mice and showed doxycycline (Dox) controllable expressionof 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 aswell 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 and food/water intake (Figure 2). We examined theexpression levels of D1R protein in the striatum, which isconsidered to be a major region responsible for control oflocomotor activity and containing abundant D1R expression.
During the process of recovery of locomotor activity afterDox withdrawal, the transgene D1R expression gradually
increased while locomotor activity fluctuated strikingly.
These results indicate that the increment of locomotor
activity is not simply in proportion to the amount of D1R
expression. We are analyzing these results to identify themechanism of the relationship between the D1R expression
and altered behavior. In addition, we are also investigating
whether or not there is a critical period in development for
the regulation of locomotion and eating behavior by
dopaminergic transmission.

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 Dr. Motoya Katsuki, Executive
Director, National Institute of Natural Sciences, we have

Figure 2. Locomotor activity of D1R/D2R DKO-D1R rescued mice. Two
weeks of Dox administration led to decreased activity followed bytransient hyperactivity.

Ⅱ. Developing a novel conditional mutagenesis
method in mice
In collaboration with Prof. Yoichi Nabeshima of Kyoto
University, we developed a novel mouse developmental
biotechnology by introducing an amino acid substitution intoa target gene in a spatially and temporally restricted manner.

41

National Institute for Basic Biology

Neurobiology

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 has beensuggested that the aberrant activation of NMDAR causes
excitotoxicity, leading to neuronal death of 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.

this end, we developed caveolin-3 and dystrophin doubledeficient mice. The levels of OPN mRNA and osteopontinin the double-deficient mice clearly decreased comparedwith those in mdx mice. We showed that although the levelof OPN mRNA expressed in the double-deficient skeletal
muscles was lower than that in mdx skeletal muscles,
macrophage infiltration and muscle regeneration occurredsimilarly in the double-deficient and mdx skeletal muscles.
There may still be other, undiscovered factors that are
involved in macrophage infiltration and muscle
regeneration.

Ⅲ. Analysis of the roles of the sarcoglycan
complex, dystroglycan complex andcaveolin-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 theintracellular 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 generated the β-SG 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.
Caveolin-3 is a muscle-specific membrane protein, acomponent of the dystrophin complex, and serves as a
scaffold for 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 reported that caveolin-3 deficiency
causes muscle degeneration and a decrease in sarcolemmal
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 of osteoponin(OPN), a versatile regulator of inflammation and tissue
repair, was significantly down-regulated. This is in contrast
to mdx mice showing a markedly up-regulated OPN gene intheir skeletal muscles. Recently, OPN has been reported 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

42

Figure 3. Immunohistochemical analysis of skeletal muscles from miceat 1 month of age. Cross sections were stained with H&E (a, e, i and m)
and antibodies against caveolin-3 (b, f, j and n), osteopontin (c, g, k, o, q
and r) and F4/80 (d, h, l and p). Note that the surrounding areas of
F4/80-positive macrophages in mdx and double-deficient mice (l and p)
are strongly stained by the anti-osteopontin antibody (k and o). In lessinfiltrated or noninfiltrated area, the sarcolemma of mdx muscle fibers ismore strongly stained than that of the double-deficient muscle fibers by
the anti-osteopontin antibody (q and r). Bar; 50 μm.

Publication List
〔Original papers〕● Hagiwara, Y., Fujita, M., Imamura, M., Noguchi, S., and Sasaoka, T.

(2006*). Caveolin-3 deficiency decreases the gene expression level ofosteopontin in mdx mouse skeletal muscle. Acta Myol. 25, 53-61.
● Kawasaki, K., Watabe, T., Sase, H., Hirashima, M., Koide, H.,
Morishita, Y., Yuki, K., Sasaoka, T., Suda, T., Katsuki, M.,
Miyazono, K., and Miyazawa, K. (2008). Ras signaling directs
endothelial specification of VEGFR2+ vascular progenitor cells. J.
Cell Biol. 181, 131-141.
* a paper published after the publication of the previous issue of theAnnual Report

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 Scientist
Technical Assistants

Secretary

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

The main interest of this division 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
The genus Ipomoea includes about 600 species, distributedon a worldwide scale, that exhibit various flower
morphologies and pigmentation patterns. Among them, threemorning glories - Ipomoea nil (the Japanese morning glory),
Ipomoea purpurea (the common morning glory), andIpomoea tricolor - have been domesticated as floricultural
plants. Of these, the Japanese morning glory has anextensive history of genetic studies, and about one-third of
more than 200 spontaneous mutants described exhibit
mainly altered flower pigmentation. The wild-type I. nil
displays blue flowers with pigmented red stems and darkbrown seeds, and its spontaneous mutants with various
flower colors have been isolated and cultivated in Japan
since the 17th century. According to classical genetic
studies, mutations conferring white flowers can be classified
into four groups: a, c, ca, and r. The recessive a and r
mutations confer white flowers with green stems and normal
colored seeds. The first such mutation was originallydesignated r, and other recessive mutations that cancomplement the r mutation were named a. One of the amutations was found to be caused by the integration of a
DNA transposon Tpn1 belonging to the CACTA
superfamily into the DFR-B gene encoding dihydroflavonol
4-reductase. The c mutants exhibit white flowers with redstems and colored seeds, whereas the ca mutants display
white flowers with green stems and ivory seeds. The c andca mutations were identified as frameshift mutations in thegenes encoding transcriptional regulators containing R2R3MYB domains and those carrying conserved WD40 repeats(WDR), designated as InMYB1 and InWDR1, respectively.
We found that the r mutants carry either 5.6-kb Tpn3 or 4.7kb Tpn6, DNA transposons of the Tpn1 family, integrated
into the CHS-D gene for chalcone synthase in theanthocyanin biosynthesis pathway (Figure 1). We designatethese two mutant alleles as r1-1 and r1-2. Because excisionof Tpn3 from CHS-D scarcely ever occurs, the r1 mutant
shows an apparent stable white flower phenotype. Thesefindings further strengthen the assumption that the Tpn1
family of elements act as major mutagens for generating
various spontaneous mutations for floriculturally important
traits in I. nil.

Figure 1. Flower phenotypes of the r1 mutants and their CHS-D
structures. (A) Flower phenotypes. (B) Mutant CHS-D genes. Boxes on
the central horizontal bar indicate exons, with their coding regions
shaded black. The transposons are shown above and under the horizontal
bar.

Ⅱ. Modification of endogenous natural genes
by homologous recombination in rice
Rice (Oryza sativa), 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. Through a gene-targeting procedure with positivenegative selection, we have reported the generation of fertiletransgenic rice plants with a positive marker inserted into the
Adh2 gene by using an Agrobacterium-mediatedtransformation vector containing the positive marker flanked
by two 6-kb homologous segments for recombination. The
vector used also contained the two constitutively expressed
DT-A genes at both ends of the T-DNA segment adjacent to
its border sequences, which acted effectively to eliminatetransformants carrying entire T-DNA segment(s) integrated
randomly and to enrich targeted transformants among theNote: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

43National Institute for Basic Biology

Evolutionary Biology and Biodiversity

surviving calli. We found that base changes within thehomologous segments in the vector could be efficiently
transferred into the corresponding genomic sequences of ricerecombinants. Interestingly, a few sequences from the hostgenome were flanked by the changed sequences derived
from the vector in most of the recombinants (Figure 2). A
likely explanation for the observation would be a result of
the mismatch correction of heteroduplex molecules formed
between the genomic and introduced T-DNA sequences
during homologous recombination. These results offer new
insights into the homologous recombination processes ofgene targeting with positive-negative selection.
Because a single-stranded T-DNA molecule inAgrobacterium-mediated transformation is imported into theplant nucleus and becomes double-stranded, both singlestranded and double-stranded T-DNA intermediates canserve in gene-targeting processes. Since the DT-A gene is acell-autonomous, nonconditional, and lethal negative

selection marker, the DT-A gene region of a single-stranded
T-DNA intermediate in the plant nucleus would neither
become double stranded nor express transiently in the
successfully targeted transformants because the transient
expression of the double-stranded DT-A gene before
integration is thought to kill host plant cells.
Several alternative models, including the occurrence of themismatch correction of heteroduplex molecules formed
between the genomic DNA and either a single-stranded or
double-stranded T-DNA intermediate, are compared in order
to explain the observation (Figure 3). Since the introductionof a double-strand break (DSB) in the plant genome greatly
stimulates the integration of transgenes flanked byhomologous sequences on T-DNA introduced into the DSB
site, we considered (A) chromosomal DSB-initiated genetargeting processes, and (B) chromosomal DSB-independent
gene-targeting processes separately. According to themodels illustrated, no heteroduplex would form in the

Figure 2. Transfer of base changes from a vector to the rice genome. The rice Adh2 locus on chromosome 11 bears a Copia-like retroelement, and thevector 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 Copialike retroelement. The large black arrowheads 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.

Figure 3. Models for gene targeting. (A) DSB-induced gene targeting processes with a single-stranded T-DNA intermediate (a) and a truncated doublestranded T-DNA intermediate (b and c). (B) DSB-independent gene targeting processes with a single-stranded T-DNA intermediate (a) and with atruncated double-stranded T-DNA intermediate (b and c).

44

simple DSB-induced synthesis-dependent strand annealing(SDSA) model involved in the annealing of the newly
synthesized DNA strands (Figure 3A c-2), whereas a longheteroduplex would likely be produced in only one of thetwo homologous regions flanking the hpt gene in the formal
double-strand break repair (DSBR) model. As the geneconversion was observed over the entire region examined,
the observation appears to be in contrast to the fact thatmajor DSB-induced conversion tracts were reported to be
bidirectional and usually shorter than 100 - 300 bp whendouble-stranded DNA molecules served in homologous
recombination with the genomic homologous sequences inmouse ES cells. We are condidering the possibility that asingle-stranded T-DNA intermediate plays an important rolein the major pathways leading to our successful GT events.

transposases. We also showed that Dart1-27 can mobilize
nDart1-0 in Arabidopsis. These findings should facilitate thedevelopment of an efficient gene-tagging system in rice andshed light on epigenetic regulatory and evolutionary aspectsof autonomous elements in the nDart/aDart system.

Ⅲ. Characterization of mutable virescent allele
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, and can be induced by crossing with aline containing an active autonomous element aDart andstabilized by segregating aDart. In the sequenced
Nipponbare genome containing no active aDart elements,
the nDart-related elements can be classified into threesubgroups of about 0.6-kb nonautonomous elements andfour subgroups of elements longer than 2 kb, which
comprise epigenetically silenced inactive iDart andgenetically defective dDart elements including 53 copies ofiDart1/dDart1 elements on the basis of their lengths andsequence characteristics. Among the 53 iDart1/dDart1
elements, 38 iDart1 elements are putative autonomous
elements silenced epigenetically because their putativetransposase genes carry no apparent nonsense or frameshift
mutations. Because the excision of a fraction of nDart1
elements can be induced by treating Nipponbare with 5azacytidine (5-azaC), Nipponbare must contain an
epigenetically silenced autonomous element or elements thatcan become aDart by 5-azaC treatment.
Mapping data indicated that aDart in the mutable pyl-v
plants resides within 170-kb region on chromosome 6, andthe 170-kb region contains a Dart1 element identical toDart1-27 in Nipponbare. We chose six representative Dart1
elements (Dart1-1, Dart1-20, Dart1-27, Dart1-28, Dart144, and Dart1-52) from Nipponbare to examine whether
they are able to excise from the GUS gene in the vectors to
be introduced into Arabidopsis by Agrobacterium-mediatedtransformation (Figure 4). Of these Dart1 elements, only
Dart1-44 is likely to be dDart1, because its putativetransposase gene contains a premature TAA stop codon.
Transposition of all of the Dart1 elements except for Dart144 can be detected and expected footprints are generated,indicating that they are potential autonomous elements
silenced epigenetically in Nipponbare and that autonomous
Dart1 elements bear a considerable sequence divergence of

Figure 4. Transposision of nDart1-0 and Dart1 elements in Arabidopsis.
(A) Structures of the T-DNA regions of the vectors used. (B) Excision
activities of the introduced nDart1-0 and Dart1 elements examined intransgenic Arabidopsis plants.

Publication List
〔Original papers〕● Johzuka-Hisatomi, Y., Terada, R., and Iida, S. (2008). Efficienttransfer of base changes from a vector to the rice genome byhomologous recombination: involvement of heteroduplex formationand mismatch correction. Nucleic Acids Res. 36, 4727-4735.
● Kitazawa, D., Miyazawa, Y., Fujii, N., Hoshino, A., Iida, S., Nitasaka,E., and Takahashi, H. (2008). The gravity-regulated growth of axillarybuds is mediated by a mechanism different from decapitation-induced
release. Plant Cell Physiol. 49, 891-900.
● Yamauchi, T., Moritoh, S., Johzuka-Hisatomi, Y., Ono, A., Terada, R.,
Nakamura, I., and Iida, S. (2008). Alternative splicing of the rice
OsMET1 genes encoding maintenance DNA methyltransferase. J.
Plant Physiol. 165, 1774-1782.
● Nishimura, H., Ahmed, N., Tsugane, K., Iida, S., and Maekawa, M.
(2008). Distribution and mapping of an active autonomous aDartelement responsible for mobilizing nonautonomous nDart1
transposons in cultivated rice varieties. Theor. Appl. Genet. 116, 395405.

〔Review article〕● Johzuka-Hisatomi, Y., Maekawa, M., Takagi, K., Eun, C.H.,

Yamauchi, T., Shimatani, Z., Ahmed, N., Urawa, H., Tsugane, K.,
Terada R., and S. Iida (2008). Homologous recombination-dependent
gene targeting and an active DNA transposon nDart-promoted genetagging for rice functional genomics. Biotechnology in Agriculture
and Forestry 62, 81-94

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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, in general, areunstable. At present, neither the physiological function(s) ofthese repeated sequences, nor the mechanism producing
them and controlling instability are fully understood. To
clarify these aspects, we are pursuing several lines ofresearch using Escherichia coli, Saccharomyces cerevisiae
and Chinese Hamster Ovary (CHO) cells. In 2008 we
discovered a new role of rDNA in yeast and reported a
relationship between condensin associating with thechromosome and gene transcription. More specifically, we
discovered a new condensin recruiting system, consisting of
the following four proteins, Tof2p, Csm1p, Lrs4p andFob1p, which recruit condensin protein complexes to the
RFB site in rDNA repeat units. In addition, we also
developed a new system of gene amplification via DRCR
(double rolling circle replication) in yeast using Cre-lox sitespecific recombination. From previous and present results,
we concluded that DRCR is an amplification mechanism
actually used at least in budding yeast. We are investigating
the possibility that DRCR might also act in gene
amplification in higher eukaryotes as well.

Ⅰ. Analysis of mechanisms maintaining
repeated structures 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 must have developed systems to regulate
recombination within rDNA repeats.
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
replication fork barrier (RFB) sites, for rDNA regionspecific recombination and for expansion/contraction of
rDNA repeats. The latter two activities are likely to be
triggered by double-strand breaks at the RFB site and repair

46of the break via gene conversion. Thus, FOB1-dependent
recombination apparently contributes to the maintenance of
average copy number of rDNA. However, in Δfob1 cells, therepeats are still maintained without any fluctuation of copy
number, suggesting that another, hitherto unknown, systemacts to prevent contraction of the number of repeats.
In order to understand this putative alternative system, wecollected a number of mutants in which the rDNA copy
number decreased drastically under Δfob1 conditions. We
have found that mutations of genes encoding condensin
caused this phenotype, suggesting that, in addition to its rolein condensation and separation of chromosomes in M phase,
condensin influences the maintenance of repeated rDNA
structures. Each gene encoding a condensin subunit is
known to be essential for growth, but the mutants isolated
here are of leaky type. Analyzing the condensin and fob1
double mutants and examining specific interactions between
condensin and rDNA regions revealed that (1) in the double
mutants, the copy number of rDNA in the mutant
dramatically decreased; (2) the condensin complex
associated with the RFB region in a FOB1-dependent
manner; (3) the association between condensin and RFB wasestablished during S phase and was maintained until
anaphase; (4) double mutants showed slow growth which
may be caused by a defect in the separation of the longrDNA array in anaphase. These results strongly suggest that
FOB1-dependent condensin association with the RFB region
is required for efficient segregation of rDNA repeat regions.

Ⅱ. Mechanism of condensin recruitment onto
RFB sites located within the tandem rDNA
repeat in budding yeast.
The primary functions of mitotic chromosome
condensation are to reduce the length of the chromosomes toprevent truncation during cell division and to ensure proper
segregation of sister chromatids. The compaction ratio of
mitotic chromosomes relative to double-stranded DNA
ranges from ~160-fold in budding yeast to ~10000 – 20000fold in mammalian chromosomes. Condensin is a multisubunit protein complex that plays a central role in mitotic
chromosome condensation and segregation. In vertebrates,
condensin is distributed in the axial part over the whole
length of condensed chromosomes, but this had only beenshown at the resolution of light microscopy. The sites wherecondensin acts on the chromatin and the molecularmechanisms of condensin recruitment thereto had largely
remained elusive. As described above, we found thatcondensin localized to the RFB site in a Fob1-dependent
manner during S-phase. To date, this Fob1p-dependent
condensin localization is the only example of condensin
association with a specific DNA site in a specific protein
factor-dependent manner. Towards understanding
chromosome condensation at the level of molecularresolution, we have studied mechanisms of condensin
localization at the RFB site. Recently, we discovered thatcondensin could bind to a short DNA fragment containing
RFB sequences, even if the sequence was inserted at anectopic chromosomal site. This indicates that the RFB site
itself acts as a site recruiting condensin onto chromatin.

Analysis of the relationship between condensin recruitment
to the RFB site and Fob1p-dependent replication fork
blockage at the RFB site demonstrated that those two eventswere completely independent phenomena. Instead, we
identified three additional protein factors, Tof2p, Csm1p,
and Lrs4p, necessary for both FOB1-dependent condensin
recruitment to the RFB site and for ensuring the faithfulsegregation of long rDNA repeats. We also found orderedbinding of Fob1p, Tof2p, Csm1p/Lrs4p, and condensin
complexes at the RFB site. Finally, in vivo interactions
between Csm1p, Lrs4P and multiple subunits of condensin
were detected. These results suggest that condensin is
recruited to the RFB site by the sequential interactions of
Fob1p, Tof2p, Csm1p, Lrs4p, and finally condensin, toensure the proper segregation of long rDNA tandem arrays(Figure 1). (Johzuka, K. and Horiuchi, T. The cis-element
and factors required for condensin recruitment tochromosome. Mol. Cell, in press)

Ⅲ. Gene amplification can be induced via
excessive silencing caused by overexpression of Sir2p and Sir3p in
Saccharomyces cerevisiae

Figure 1. Model for condensin recruitment to the RFB site, contributing
to maintenance of the integrity of long rDNA repeats.

Gene amplification is involved in various biologicalphenomena such as cancer development and drug resistance.
Furthermore, amplification can be the first step in theevolution of a new gene because the extra copies are free toacquire new functions. However, the nature of the events
causing gene amplification is poorly understood. We
focused on gene silencing, an epigenetic mechanism forcontrolling of gene expression, and examined whether genesilencing affects gene amplification. A construct with thepotential to induce gene amplification was inserted near atelomeric region in Saccharomyces cerevisiae, at whichgene silencing was enhanced by SIR2 or SIR3
overexpression. This construct contains two markers, URA3
and leu2d. The enhanced gene silencing is expected tosuppress URA3 expression and to lead to copy number
increase that confers a growth advantage on organisms
maintained in medium lacking uracil. We used theamplification marker, leu2d to detect the amplification of
URA3 with great sensitivity. The leu2d gene has only aslight transcriptional activity and complements leucine
auxotrophy only when amplified. SIR3 overexpression using
the plasmid (YRp-SIR3) gave 700-fold higher frequency of
Leu+ survivors than the control. About 50% Leu+ clones
having a construct with inverted repeats contained intrachromosomal products (>100 copies), and 16% Leu+ clones
contained extra chromosomal products (~20 copies). The
remaining 32% of Leu+ clones contained 2-3 copies of theinserted structure. On the other hand, SIR2 overexpression
could cause gene duplication, although at low frequency. In
addition, a type of chromosomal translocation was observed
through introduction of an extra copy of SIR2 (total 2 copies
per cell) into cells harboring a construct with deliberate
DNA cleavage. These results suggest that excessive levels ofgene silencing might generate selective pressure andpromote amplification of genes.

47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Ⅳ. Construction of a new gene amplification
system via DRCD (double rolling circle
replication) using Cre-lox site specificrecombination
In addition to rDNA gene amplification in eukaryotes,
there is another type of gene amplification which is involvedin 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 arrangements present as randomlyoriented sequences flanked by inverted leu2d copies. Type-2
products are acentric multi-copy mini-chromosomes, eachcarrying two leu2d copies. Structures of type-1 and -2
products resemble those of homogeneously staining regions(HSR) and double minutes (DMs) of higher eukaryotes,
respectively. Interestingly, products analogous to these weregenerated at low frequency without deliberate DNA
cleavage (EMBO J 24, 190-198 (2005)).
If DRCR were an actual gene amplification mechanism inyeast, a quite different initiation reaction, which can induce
DRCR, should produce amplification products resembling
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 recombination occurs between un-replicated andreplicated regions during replication, the fork will replicate
these already-replicated regions again, and that the Cre
recombination system would make this process more

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

48

efficient, as shown in Figure 2(a). Furthermore, acombination of the processes, as shown in Figure 2(b), could
efficiently induce gene amplification through DRCR. In fact,
this system produced two kinds of products: highly
amplified (>100 copies) chromosome products and acentric
multi-copy extra-chromosomal products. Structures of theseproducts resemble those of HSR and DMs of higher
eukaryotes, respectively. From previous and present results,
we concluded that DRCR is indeed an amplification
mechanism in budding yeast and could be naturally initiatedin the presence of the structural reguirement.

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

Associate Professor

HASEBE, Mitsuyasu

MURATA, Takashi

Assistant Professors
Technical Staff
NIBB Research Fellows
Postdoctoral Fellows

Graduate Students
Visiting Scientist:
Technical Assistants

Secretary

HIWATASHI, Yuji
TANAHASHI, Takako
SUMIKAWA, Naomi
ISHIKAWA, Takaaki *MIYAZAKI, Saori
MIYAZAKI, Saori *ISHIKAWA, Takaaki
MANO, Hiroaki
OKANO, Yosuke
OHSHIMA, Issei
AOYAMA, Tsuyoshi
FUJII, Tomomi
BASKIN, Tobias
IKEGUCHI, Hiromi
ITO, Yukiko
KAJIKAWA, Ikumi
SAKURAI-OZATO, NamiTSUZUKI, Yukiko
WASHIO, Midori
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, wehope to infer the genetic changes that caused the evolutionof cellular and developmental processes.

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

Ⅱ. Evolution from cells to tissues based onmolecular mechanisms of cytokinesis
The cells of land plants and their sister group,charophycean green algae, divide by the insertion of cell
plates at cytokinesis. 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 of daughter nuclei, expands centrifugally towards acell periphery, and finally fuses to a parental cell wall. Cellwall materials are transported to the expanding cell platewith a phragmoplast, which is mainly composed of
microtubules. A centrifugal expansion of the phragmoplast
is a driving force for that of the cell plate, althoughelucidating the molecular mechanism for the centrifugal
expansion of the phragmoplast was a challenge. Based onlive imaging of α-tubulin at a light microscopic level andimmunolocalization of γ-tubulin at an electron microscopic
level, we proposed a hypothesis that cytosolic γ-tubulin
complexes are recruited onto existing phragmoplast
microtubules and nucleate new microtubules as branches,
and that the branched microtubules drive phragmoplast
expansion. Seeing the life history of microtubules in thephragmoplast had been very difficult by live imaging of αtubulin, but we successfully tracked the trajectories ofgrowing microtubule ends in the phragmoplast using twophoton microscopy of a microtubule plus-end marker EB1.
Microtubules appeared in many sites in the phragmoplastand elongated obliquely towards the cell plate. We also
found that inhibition of γ-tubulin function by antibody
injection inhibited formation of new microtubules andphragmoplast expansion. These results support our
hypothesis. Takashi Murata was this study’s main
researcher.
In addition to the centrifugal expansion, antiparallel
bundles in phragmoplasts are mostly unexplored andpotentially offer new cellular insights. We found that the
Physcomitrella patens kinesins KINID1a and KINID1b (forkinesin for interdigitated microtubules 1a and 1b), which are
specific to land plants and orthologous to Arabidopsis
thaliana PAKRP2, are novel factors indispensable for thegeneration of interdigitated antiparallel microtubules in thephragmoplasts of the moss P. patens. KINID1a and
KINID1b are predominantly localized to the putativeinterdigitated parts of antiparallel microtubules. This
interdigitation disappeared in double-deletion mutants ofboth genes, indicating that both KINID1a and 1b are
indispensable for interdigitation of the antiparallel
microtubule array. Furthermore, cell plates formed by thesephragmoplasts did not reach the plasma membrane inapproximately 20% of the mutant cells examined. We
observed that in the double-deletion mutant lines,

Figure 1. Localization of KINID1b-Citrine and mRFP-α-tubulin fusion
proteins. The fluorescence derived from the mRFP-α-tubulin was ofhigher intensity at the phragmoplast equator where the plus ends of the
antiparallel microtubules interdigitate. Fluorescent signals of KINID1bCitrine protein overlapped with the high intensity mRFP signals. A bar =2 μm.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

chloroplasts remained between the plasma membrane andthe expanding margins of the cell plate, while chloroplasts
were absent from the margins of the cell plates in the wild
type. This suggests that the kinesins, the antiparallel
microtubule bundles with interdigitation, or both arenecessary for the proper progression of cell wall expansion.

Ⅲ. Evolution of molecular mechanisms inplant development
3-1 Stem cell initiation and maintenance
The initiation and maintenance of several types of stem
cells to produce different types of differentiated cells is
properly regulated during the development of multicellularorganisms. Molecular mechanisms for the stem cellcharacterization, however, have been largely unknown. We
showed that AINTEGUMENTA/PLETHORA/BABY BOOM
(APB) orthologs PpAPBs (PpAPB1, 2, 3, and 4) are involvedin stem 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. The primary researchers for thisstudy were Tsuyoshi Aoyama and Yuji Hiwatashi.

analysis of gene expression (5' SAGE); and (5) a collectionof 3' UTR and small RNA sequences as collaborative works
with groups associated with Dr. Tomoaki Nishiyama(Kanazawa Univ.), Prof. Asao Fujiyama (National Instituteof Informatics), Prof. Sumio Sugano (Univ. Tokyo), andProf. Yuji Kohara (National Institute of Genetics).
We developed a system to efficiently construct
phylogenetic trees with whole genome shotgun sequence
data available in public databases before their assembly. We
collected homologs of approximately 700 Arabidopsis
thaliana genes involved in development, and their
phylogenetic analyses are in progress.

3-3 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 is one of the mostconspicuous evolutionary aspects of land plants. To
elucidate the molecular mechanisms underlying theevolution in alteration of generations, the characterization of
PcG genes in P. patens is in progress. Takaaki Ishikawa and
Yosuke Okano were this study’s primary researchers.

Ⅳ. Molecular mechanisms of female and maleinteractions

Figure 2. Life cycle of the moss Physcomitrella patens. Arrows indicate
stem cells in different organs (see Sakakibara et al. 2008 in detail.)

3-2 Nuclear genome project of the moss
Physcomitrella patens
A comparison of developmental genes among major land
plant taxa would facilitate our understanding of theirevolution, although it 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; subsequently, the moss Physcomitrella patens
and its entire genome has been mostly sequenced as acollaborative work with the international consortium(Rensing et al. 2008).
To further elaborate the contig assembling and the geneannotation, 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 in their full length; (3) the construction of
BAC libraries and their end-sequencing; (4) 5'-end serial

50

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
receptors 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 arelikely expressed more abundantly in pollen and/or pollen
tube than in other tissue. Gene expression profiles between
A. thaliana pollen and pollen tube were compared withthose of other tissues using microarray. Pollen and pollen
tube expression profiles were similar to each other and 95 %of expressed genes were overlapped within the 4 folddifferences. We focused on 45 RLKs predominantly
expressed in pollen or germinating pollen to characterizesignaling mechanisms during fertilization. Characterizationof single and double T-DNA insertion lines are in progress.
This work was mainly done by Saori Miyazaki.

Ⅴ. Molecular mechanisms of mimicry
Mimicry is an intriguing phenomenon in which anorganism closely resembles another, phylogenetically distantspecies. An excellent example is the flower-mimicry of theorchid mantis, in which pink and white coloration and petallike structures on its walking legs enable this insect to blend
perfectly into flowers. We attempted to elucidate theevolutionary mechanism of this complex mimicry at themolecular level. We first focused on the orchid mantis’s
mechanism of body coloration. Mass spectrometric analysis
suggested that dihydro-xanthommatin, a red pigment
belonging to the ommochrome family, contributes to thepink body coloration of the mantis. We also isolated acDNA encoding a key enzyme for biosynthetic pathway ofommochromes. Using the sequence of this gene, we are now
trying to establish RNAi-mediated gene knock-down in theorchid mantis.
The orchid mantis drastically changes its appearance
during post-hatching development. The first-instar nymph ofthe mantis is colored red and black and is believed to mimicother unpalatable insects like ants. A flower-like appearance
emerges after the molting into the 2nd-instar nymph. We
aim to compare the gene expression profiles between the
1st- and 2nd-instar nymphs using a high-throughput DNA
sequencer. This work was mainly done by Hiroaki Mano.

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

Publication List
〔Original papers〕● Hiwatashi, Y., Obara, M., Sato, Y., Fujita, T., Murata, T., and Hasebe,M. (2008). Kinesins are indispensable for interdigitation ofphragmoplast microtubules in the moss Physcomitrella patens. Plant
Cell 20, 3094-3106.
● Morinaga, S.-I., A.J. Nagano, S. Miyazaki, M. Kubo, T. Demura, H.
Fukuda, S. Sakai, and M. Hasebe. (2008). Ecogenomics ofcleistogamous and chasmogamous flowering: genome-wide gene
expression patterns from cross-species microarray analysis in
Cardamine kokaiensis (Brassicaceae). Journal of Ecology 96, 10861097.
● Inouye, T., Odahara, M., Fujita, T., Hasebe, M., and Sekine, Y.
(2008). Expression and complementation analyses of a chloroplastlocalized homolog of bacterial RecA in the moss Physcomitrella
patens. Biosci. Biotechnol. Biochem. 72, 1340-1347.
● Sakakibara, K., Nishiyama, T., Deguchi, H., and Hasebe, M. (2008).
Class 1 KNOX genes are not involved in shoot development in themoss Physcomitrella patens but do function in sporophyte
development. Evol. Dev. 10, 555-566.
● Abe, J., Hiwatashi, Y., Ito, M., Hasebe, M., and Sekimoto, H. (2008).
Expression of exogenous genes under the control of endogenous
HSP70 and CAB promoters in the Closterium peracerosum-strigosumlittorale complex. Plant Cell Physiol. 49, 625-632.
● Fujita, T., Sakaguchi, H., Hiwatashi, Y., Wagstaff, S. J., Ito, M.,
Deguchi, H., Sato, T., and Hasebe, M. (2008). Convergent evolutionof shoots in land plants: lack of auxin polar transport in moss shoots.
Evol. Dev. 10, 176-186.
● Rensing, S. A., Lang, D., Zimmer, A., Terry, A., Salamov, A.,
Shapiro, H., Nishiyama, T., Perroud, P.-F., Lindquist, E., Kamisugi,Y., Tanahashi, T., Sakakibara, K., Fujita, T., Oishi, K., Shin-I, T.,
Kuroki, Y., Toyoda, A., Suzuki, Y, Hashimoto, S., Yamaguchi, K.,
Sugano, S., Kohara, Y., Fujiyama, A., Anterola, A., Aoki, S., Ashton,
N., Barbazuk, W. B., Barker, E., Bennetzen, J., Blankenship, R., Cho,
S. H., Dutcher, S. K., Estelle, M., Fawcett, J. A., Gundlach, H.,
Hanada, K., Hey, A., Hicks, K. A., Hughes, J., Lohr, M., Mayer, K.,
Melkozernov, A., Murata, T., Nelson, D., Pils, B., Prigge, M., Reiss,B., Renner, T., Rombauts, S., Rushton, P., Sanderfoot, A., Schween,G., Shiu, S.-H., Stueber, K., Theodoulou, F. L., Tu, H., de Peer, Y. V.,
Verrier, P. J., Waters, E., Wood, A., Yang, L., Cove, D., Cuming, A.
C., Hasebe, M., Lucas, S., Mishler, B. D., Reski, R., Grigoriev, I. V.,
Quatrano, R. S., Boore, J. L. (2008). The genome of the moss
Physcomitrella patens reveals evolutionary insights into the conquestof land by plants. Science 319, 64-69.

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF MORRHODIVERSITY

Associate Professor

KODAMA, Ryuji

The aim of this laboratory is to observe the variety ofmorphogenetic processes that occur in the course of the
ontogenesis of multicellular organisms and to analyze themechanisms of such processes, mainly by morphologicalmethods. The accumulation of such analyses of theembryogenetic processes of related species is expected toprovide an insight into the evolution of morphogenetic
processes. This laboratory uses the wings of lepidopteraninsects for our morphological studies.

Ⅰ. Wing morphogenesis
The wings of the lepidopteran insects (butterflies andmoths) develop from the wing imaginal disc, which is ahollow sac made of simple epithelium. Due to its simple
construction, this tissue is a good material for studying
cellular interactions in the course of morphogenesis.
The outline shape of the adult wing is often different fromthat of the pupal wing. This difference is brought about by
the programmed cell death of the marginal area of the pupal
wing. The marginal dying area is called "the degeneration
region" and the internal area, which develops into the adultwing, is called "the differentiation region".
Cell death in the degeneration region proceeds very rapidly
and finishes in a half-day to one-day period in Pieris rapae
and in several other examined species. It was shown that thedying cells in the degeneration region have characteristics incommon with the 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.
A possible physiological role of the cell degeneration at thewing margin is to make space for the growth of the marginal
scales. Marginal scales are extremely elongated scales thatgrow densely on the edge of the wing. These scales are
considered to be important in stabilizing the turbulenceoccurring posterior to the wing. The movements of themarginal scales are closely monitored by sensory scales andbristles growing among them (A. Yoshida et al,unpublished).
Trachea and tracheoles are both important in delivering air
into the wing and their patterns coincide with that of theboundary of degeneration and differentiation zones at the
distal end of the wing. According to previous observations,52

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.

the pattern formation of wing epithelium is often dependent
on tracheal and tracheole patterns. Basic research on thedevelopment of tracheal pattern formation is being done byscanning and transmission electron microscopy (Figure 1) to
describe the exact pathway and time course of the formationof the elaborate pattern of trachea and tracheoles and toestablish the cytological and developmental relationshipbetween the formation of the tracheal pattern and theepithelial cell pattern.

Ⅱ. Other research activities
This laboratory also conducts morphological observation ofvarious animal tissues by scanning and transmission electron
microscopy and immuno-electron microscopic analyses.
Training in specimen preparation and instrument operation
for such observations is also given. Our institute’s Divisionof Sex Differentiation and the Laboratory of
Neurophysiology were both involved in these activities.

LABORATORY OF BIORESOURCES

Associate Professor

NARUSE, Kiyoshi
Postdoctoral Fellows

Research Fellows
Technical Assistants

Secretary

SASADO, Takao
TAKEHANA, Yusuke
SHEN, Xueyan
KANEKO, Hiroyo
YOSHIMURA, Yuriko
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
KOIKE Chieko
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, small creeks and paddy
fields. Analysis of their genome structure is important inorder to 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, and identification of the causal gene of mutants for
PGC migration. In addition to these activities, our laboratory
is stepping forward to lead the National BioResource Project
Medaka (NBRP Medaka).

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

group, O. dancena and O. minutillus have an XX/XY sex
determination system, while O. hubbsi and O. javanicus
have a ZZ/ZW system (Figure 1). Linkage analysis and
FISH analysis showed that the sex chromosomes in these
species were not homologous, suggesting independent
origins of these sex chromosomes. Furthermore, O.
javanicus and O. hubbsi have morphologicallyheteromorphic ZW sex chromosomes, in which the W
chromosome has DAPI-positive heterochromatin. Thesefindings suggest the repeated evolution of new sex
chromosomes from autosomes in Oryzias, probably throughthe emergence of a new sex-determining gene.

Ⅲ. Involvement of two chemokine receptors
CXCR4b and CXCR7 in the regulation of
the PGC migration

Ⅰ. Construction of Polypterus Genomic DNA
Library for Analysis of the Fish Genome
Evolution
Comparative genomic analysis using medaka, zebrafish,
Tetraodon and human as an outgroup revealed that theeuteleost has experienced the teleost specific 3 rd roundgenome duplication and the common ancestor of alleuteleost should have 13 pre-duplicated proto-chromosomes.
To verify this hypothesis, it is effective to compare thegenomic structure of the reconstructed proto-chromosomes
and fish chromosomes without the 3 rd round genome
duplication. One appropriate fish for this comparison is
Polypteridae. We have started the construction of the
genomic DNA library of Polypterus senegalus using fosmid
vector and established the fosmid genomic library with
200,000 independent clones in 2008. In addition to thisgenomic resource, we are now establishing cell lines derived
from the caudal fin of Polypterus senegalus.

Ⅱ. Evolution of the sex chromosome and sex
determining genes in Oryzias fish
The sex-determining gene DMY was identified on the Y
chromosome in the medaka, Oryzias latipes. However, thisgene is absent in most Oryzias fishes, suggesting that closely
related species have different sex-determining genes. We
have recently demonstrated that, in the javanicus species

Figure 2. yanagi (yan) and kazura (kaz) mutations affect PGC migrationand lateral line positioning.

53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

The migratory pathways of PGCs to the gonad varydepending on the vertebrate species, yet the underlying
regulatory mechanisms guiding PGCs are believed to becommon between species. In teleost medaka embryo, PGC
migration follows two major steps before colonizing in
gonadal areas: (1) bilateral lineup in the trunk, and (2)posterior drift. kazura (kaz) and yanagi (yan) mutants of
medaka isolated in our mutagenesis-screening were
defective in the first and second steps, respectively (Figure2). kaz j2-15D was identified as a missense mutation inchemokine receptor gene cxcr4b expressed in PGCs.
Embryonic injection of cxcr4b mRNA with olvas 3'UTR
rescued the PGC phenotype of kaz mutant, indicating a cellautonomous function of cxcr4b in PGCs. yan j6-29C was
identified as a nonsense mutation in the cxcr7/rdc1 geneencoding another chemokine receptor. cxcr7 transgene withgenomic flanking sequences rescued the yan mutant
phenotype efficiently at the G0 generation. cxcr7 was
expressed in somites rather than PGCs. cxcr7-expressing
somitic domain expanded posteriorly with its margin
immediately anterior of posteriorly drifting PGCs, as if
PGCs were pushed toward the gonadal area. The kaz andyan mutants are also defective in the lateral line positioning,suggesting combined employment of these receptor systems
in various cell migratory processes.

Ⅳ. National BioResource Project Medaka (NBRP
Medaka) (http://www.shigen.nig.ac.jp/medaka/)
4-1 Full Length cDNA Sequencing Project
Although we published the draft genome sequence of
medaka in 2007, the annotation of medaka genome is not yet
well established. Sequencing of the full length cDNA clones
is one of the most efficient methods for genome anotation.
To do this, we made six full length cDNA libraries(developmental stage 22, 35, 40, ovary, brain and liver) anddetermined the sequences of both ends of 150, 000 clones incollaboration with the National Institute of Genetics’ Kohara
and Fujiyama labs. After mass alignment of all sequences,
we found 16, 851 independent sequences. All of the data
was deposited in the DDBJ and is accessible from the
National BioResource Project Medaka website(http://www.shigen.nig.ac.jp/medaka/).

4-2 Establishment of Core Facility of NBRP Medaka
In 2007, the NIBB was selected as the core facility of
NBRP Medaka. Our laboratory is taking an active part inthis project. With the goal of facilitating and enhancing theuse of medaka as a model organism, we provide, maintainand collect living resources such as standard strains, inbred
strains, and mutants in addition to frozen resources such as
EST/cDNA and BAC/ Fosmid clones and hatching enzymes,
as well as the integrated information on medaka (Figure 3).
NBRP Medaka is aiming to establish a first rate biologicalresource with the highest possible levels of accessibility andease of use.

54

Figure 3. NBRP Medaka website

Publication List
〔Original papers〕● Ahsan, B., Kobayashi, D., Yamada, T., Kasahara, M., Sasaki, S.,

Saito, T. L., Nagayasu, Y., Doi, K., Nakatani, Y., Qu, W., et al.
(2008). UTGB/medaka: genomic resource database for medaka
biology. Nucleic Acids Res. 36(Database issue), D747-D752.
● Miyake, A., Higashijima, S., Kobayashi, D., Narita, T., Jindo, T.,
Setiamarga, D. H., Ohisa, S., Orihara, N., Hibiya, K., Konno, S., et al.,(2008). Mutation in the abcb7 gene causes abnormal iron and fatty
acid metabolism in developing medaka fish. Dev. Growth Differ. 50,
703-716.
● Nakamura, S., Aoki, Y., Saito, D., Kuroki, Y., Fujiyama, A., Naruse,
K., and Tanaka, M. (2008). Sox9b/sox9a2-EGFP transgenic medaka
reveals the morphological reorganization of the gonads and a common
precursor of both the female and male supporting cells. Mol. Reprod.
Dev. 75, 472-476.
● Nagai, T., Takehana, Y., Hamaguchi, S., and Sakaizumi, M. (2008).
Identification of the sex-determining locus in the Thai medaka,
Oryzias minutillus. Cytogenet. Genome Res. 121, 137-142.
● Sasado, T., Yasuoka, A., Abe, K., Mitani, H., Furutani-Seiki, M.,
Tanaka, M., and Kondoh, H. (2008). Distinct contributions of
CXCR4b and CXCR7/RDC1 receptor systems in regulation of PGC
migration revealed by medaka mutants kazura and yanagi. Dev. Biol.
320, 328-339.
● Takehana, Y., Hamaguchi, S., and Sakaizumi, M. (2008). Different
origins of ZZ/ZW sex chromosomes in closely related medaka fishes,
Oryzias javanicus and O. hubbsi. Chromosome Res. 16, 801-811.

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 Fellows
Postdoctoral Fellow
Graduate Students
Visiting Scientists
Technical Assistants

Secretary

KATSU, Yoshinao
MIZUTANI, Takeshi
KATO, Yasuhiko
KOIDE, Shizuyo
URUSHITANI, Hiroshi
NAKAMURA, Takeshi
KIKUCHI, Naoka
TANIGUCHI, Ena
MITSUI, Naoko
KOBAYASHI, Kaoru
TANEMURA, Hiroyo
HAYASHI, Tomoko
IMAIZUMI, Taeko

Synthetic chemicals found in the environment have thecapacity to disrupt the development and function of theendocrine system in both wildlife and humans. This has
drawn public concern since many of these chemicals maybind to estrogen receptors (ERs) and evoke estrogenic
effects. Early evidence that exposure to estrogenic chemicals
during development could pose a threat to human healthcame from studies of a synthetic hormone, diethylstilbestrol(DES), which was used to prevent premature birth andspontaneous abortion. Laboratory experiments showed that
exposure of animals to sex hormones during critical
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. Developmental origin of adult disease:
Perinatal estrogen exposure induces
persistent changes in reproductive tracts
The emerging paradigm of the “embryonic/fetal origins ofadult disease” provides a powerful new framework forconsidering the effects of endocrine disrupters on humanand animal health. In 1971, prenatal DES exposure wasfound to result in various abnormalities of the reproductive
tract in women. This syndrome was named the DES
syndrome. Similar abnormalities have been demonstrated inexperimental animals exposed perinatally to estrogens.
Developmental estrogen exposure in mice, for example,
induces persistent proliferation of vaginal epithelial cells.
We found that the persistent changes in the vagina in mice
exposed neonatally to estrogens result from the persistentphosphorylation of erbB2 and estrogen receptorα (ERα),sustained expression of EGF-like growth factors andphosphorylation of JNK1, IGF-I receptor and Akt. Recently,
we found that Wnt 4 and other genes also show persistentexpression changes in neonatally DES-exposed mousevaginae. Currently, we are analyzing the methylation statusof 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. We have demonstrated
that neonatally exposed estrogen induced adverse effects
mainly through ERα but not ERβ using specific ligands of
ERs.

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.

To understand the mechanisms through which estrogenic
chemicals act on mouse reproductive organs, data
documenting the temporal and spatial expression patterns ofestrogen-responsive genes is essential. A large number ofgenes affected by estrogen treatment were identified in thetissues of wild-type mice using a microarray approach. For
most of the identified genes, expression was induced by 17
β-estradiol (E2) in a dose-dependent manner. Subsequently,

55

National Institute for Basic Biology

Environmental Biology

several environmental (xeno)estrogens were tested andcharacteristic gene expression patterns were observed foreach compound tested. These patterns were distinct fromthat obtained following E2 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.

Figure 4. Evolutionary relationships of estrogen receptor sequences

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

Ⅲ. Steroid hormone receptors of birds,
reptiles, amphibians and fishes
Steroid and xenobiotic receptors (SXR) have been cloned
from various animal species (fish, amphibian, reptiles, birds
and mammals) by our group and we have demonstrated
species-specific differences in their responses to various
environmental and endogenous chemicals (receptor genezoo). Thus, simple predictions of chemical effects based ondata from a few established model species are not sufficient
to develop real world risk assessments. ER and ER-likegenes have been cloned from various animal species
including rockshell, Amphioxus, lamprey, lungfish, sturgeon,
gar, roach, stickleback, mosquitofish, mangrove Rivulus,
Japanese giant salamander, newt, Silurana tropicalis,
American alligator, Nile crocodile, freshwater turtle, andvarious snakes and vultures. Functional studies showed thatthe rockshell 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 by juvenile hormones inDaphnids
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

56

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 the sex
determination of D. magna and we observed that juvenile
hormone agonists (pesticides), for example, induce theproduction of male offspring. The molecular basis ofenvironmental sex determination is largely unknown in 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. We
have recently developed a method to inject silencing genes
into D. magna embryos which will allow us to study genefunction in more detail in this species.

Ⅴ. Gene zoo and receptor zoo
We are establishing cDNA library banks and receptor genebanks of animal species including sturgeon, gar, lamprey,lancelet, mangrove Rivulus, Japanese giant salamander,
newt, Rana rugosa, Silurana tropicalis, Florida red berry
turtle, American alligator, Nile crocodile, vulture and polar
bear 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
We found that the persistent and ubiquitous environmental
contaminant, tributyltin chloride (TBT), induces thedifferentiation of adipocytes in vitro and increased adipose
mass in vivo. TBT is a dual nanomolar affinity ligand forboth 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
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 to orgaotins
may therefore act as a chemical stressor for obesity andrelated disorders. We have recently started to use stem cells
from mouse bone marrow to study the molecularmechanisms of cell differentiation to adipocytes byenvironmental chemicals.

Publication List
〔Original papers〕● Katsu, Y., Ichikawa, R,, Ikeuchi, T., Kohno, S., Guillette, L.J.Jr., andIguchi, T. (2008). Molecular cloning and characterization of estrogen,androgen and progesterone nuclear receptors from a freshwater turtle(Pseudemys nelsoni). Endocrinology 249, 161-173.
● Kato, Y., Kobayashi, K., Oda, S., Colborne, J.K., Tatarazako, N.,
Watanabe, H., and Iguchi, T. (2008). Molecular cloning and sexuallydimorphic expression of DM-domain genes in Daphnia magna.
Genomics 91, 94-101.
● Kobayashi, T., Takita, Y., Suzuki, A., Katsu, Y., Iguchi, T., and Ohta,
Y. (2008). Vacuolar degeneration of skeletal muscle in transgenic
mice overexpressing ORP150. J. Vet. Med. Sci. 70, 115-118.
● Nishida, H., Miyagawa, S., Matsumaru, D., Wada, Y., Satoh, Y.,
Ogino, Y., Iguchi, T., Fukuda, S., Taga, T., and Yamada, G. (2008).
Gene expression analyses on the embryonic expternal genitalia:
identification of regulatory genes possibly involved in masculinization
processes. Congen. Anorm. 48, 63-67.
● Milnes, M.R., Bryan, T.A., Katsu, Y., Kohno, S., Moore, B.C., Iguchi,T., and Guillette, L.J.Jr. (2008). Increased post hatching mortality andloss of sexually dimorphis gene expression in alligators (Alligator
mississippiensis) from a contaminated environment. Biol. Reprod. 78,
932-938.
● Connon, R., Hooper, H.L., Sibly, R.M., Lim, F.L., Heckmann, L.H.,
Moore, D.J., Watanabe, H., Soetaert, A., Cook, K., Maund, S.J.,
Hutchinson, T.H., Moggs, J., DeCoen, W., Iguchi, T., and Callaghan,
A. (2008). Linking molecular and population stress responses inDaphnia magna exposed to cadmium. Environ. Sci. Technol. 42,
2181-2188.
● Oka, T., Tooi, O., Mitsui, N., Miyahara, M., Ohnishi, Y., Takase, M.,
Kashiwagi, A., Shinkai, T., Santo, N., and Iguchi, T. (2008). Effect ofatrazine on metamorphosis and sexual differentiation in Xenopus
laevis. Aquat. Toxicol. 87, 215-226.
● Milnes, M.R., Garcia, A., Grossman, E., Grün, F., Shiotsugu, J., Tabb,
M.M., Kawashima, Y., Katsu, Y., Watanabe, H., Iguchi, T., andBlumberg, B. (2008). Activation of steroid and xenobiotic receptor(SXR, NR112) and its orthologs in laboratory, toxicological, andgenome model species. Environ. Health Perspect. 116, 880-885.
● Kohno, S., Bermudez, D., Katsu, Y., Iguchi, T., and Guillette, L.J.Jr.
(2008). Gene expression patterns in juvenile American alligators(Alligator mississippiensis) exposed to environmental contaminants.
Aquat. Toxicol. 88, 95-101.
● Lange, A., Katsu, Y., Ichikawa, R., Paull, G.C., Chidgey, L.L., Coe,
T.S., Iguchi, T., and Tyler, C.R. (2008). Altered sexual development

in roach (Rutilus rutilus) exposed to environmental concentrations of
the pharmaceutical 17 -ethinylestradiol and associated expression
dynamics of aromatases and estrogen receptors. Tox. Sci. 106, 113123.
● Nakamura, T., Katsu, Y., Watanabe, H., and Iguchi, T. (2008).
Estrogen receptor subtypes selectively mediate female mouse
reproductive abnormalities induced by neonatal exposure to estrogenic
chemicals. Toxicology 253, 117-124.
● Naido, V., Katsu, Y., and Iguchi, T. (2008). The influence of non-toxic
concentrations of DDT and DDE on the old world vulture estrogen
receptor a. Gen. Comp. Endocrinol. 159, 188-195.
● Watanabe, H., Kobayashi, K., Kato, Y., Oda, S., Abe, R., Tatarazako,N., and Iguchi, T. (2008). Transcriptome profiling in crustaceans as atool for ecotoxicogenomics. Cell Biol. Toxicol. 24, 641-647.
● Katsu, Y., Kohno, S., Hyodo, S., Ijiri, S., Hara, A., Guillette, L.J.Jr.,
and Iguchi, T. (2008). Molecular cloning, characterization andevolutionary analysis of estrogen receptors from phylogeneticallyancient fish. Endocrinology 149, 6300-6310.

α〔Review articles〕● Iguchi, T., Watanabe, H., Ohta, Y., and Blumberg, B. (2008).

Developmental effects: estrogen induced vaginal changes andorganotin induced adipogenesis. Int. J. Androl. 31, 263-268.
● Crain, D.A., Janssen, S.J., Edwards, T.M., Heindel, J., Ho, S.-M.,
Hunt, P., Iguchi, T., Juul, A., McLachlan, J.A., Schwartz, J.,
Skakkebaek, N., Soto, A.M., Swan, S., Walker, C., Woodruff, T.,
Woodruff, T., Giudice, L.C., and Guillette, L.J.Jr., (2008). Female
reproductive disruption: The roles of endocrine disrupting compounds
and developmental timing. Fetil. Steril. 90, 911-940.
● Iguchi, T., Watanabe, H., and Katsu, Y. (2008). Toxicogenomics andecotoxicogenomics: Studying chemical effects and basic biology invertebrates and invertebrates. In Toxicogenomics: A Powerful Tool
for Toxicology Assessment, (John Wiley & Sons), pp. 143-158.
● Iguchi, T., and Katsu, Y. (2008). Commonality and difference inmechanisms of endocrine disruption in various animal species.
BioScience 58, 1061-1067.
● Watanabe, H., Kobayashi, K., Kato, Y., Oda, S., Abe, R., Tatarazako,N., and Iguchi, T. (2008). Genomic response in Daphnia to chemical
pollutants. In Interdisciplinary Studies on Environmental Chemistry –Biological Responses to Chemical Pollutants, Y. Murakami, K.
Nakayama, S.-I. Kitamura, H. Iwata, and S. Tanabe, eds.
(TERRAPUB), pp. 133-142.

57

National Institute for Basic Biology

Environmental Biology

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
GOTOH, Ayako
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 2008).

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

1-1 Polar growth of leaves in A. thaliana
AN is a member of the CtBP-BARS gene family reported
from animal genomes; last year, however, we showed that
AN does not have any of the molecular functions of CtBP in
Drosophila melanogaster (Stern et al. 2007). If so, how
widely is the AN function conserved in plants? We haveisolated a homolog of AN from Larix gmelinii, agymnosperm, and named it LgAN. LgAN fully

58

complemented all known morphological phenotypes causedby an-1 mutation in Arabidopsis, suggesting that the ANfunction is conserved between angiosperms andgymnosperms (Li et al. 2008). Furthermore, our detailed
analysis of intracellular localization suggested that AN havea unique role (or roles) in Golgi-related functions. Further
analyses of AN functions are ongoing.
On the other hand, constitutive over-expression of deletion
series of ROT4 revealed that a 32-amino-acid core region isenough to exhibit the ROT4 function when over-expressed.

1-2 Evolution of establishment mechanisms of leaf
polarities in monocots
We have recently started to attempt an understanding of thegenetic basis of the development of unifacial leaves that are
known only from monocot clades. Our analyses indicatedthat the unifacial character might be due to overall changes
in all polarities around leaves (i.e. adaxial-abaxial, distalproximal, and central-lateral polarities). Moreover, thegenetic controls of leaf polarities were revealed to differ, atleast in part, between eudicot and rice, a monocot model
species. Understanding the differences in the genetic
mechanisms for the establishment of unifacial and normal
bifacial leaves will provide good clues as to how leaf-shapeis diversified.
For such purposes, comparative molecular-genetic andanatomical analyses between unifacial and bifacial leaf
development have been undertaken using members of thegenus Juncus. Interestingly, molecular characterization of
unifacial leaves of Juncus revealed that they have only
abaxial identity in the leaf blades, lack leaf margins, andpossess flattened leaf lamina. Taken together, our datastrongly suggests the presence of unknown mechanisms forflat leaf organogenesis that were not previously suspectedfrom studies of model plants. We also established mutational
and transgenic approaches to analyze the unifacial leaf
formation; several interesting mutants of Juncus that exhibit
abnormalities in leaf polarity were already isolated.

1-3 Size control of leaves and mechanisms ofcompensation
We have recently noticed that leaf organogenesis depends
on “leaf meristem” that is seen only in the border region
between leaf blade and leaf petiole. All cells required forleaf formation seem to be supplied from this leaf meristem.
How are cell proliferation and cell enlargement coordinatedin leaf morphogenesis? In a determinate organ - such as aleaf - the number of leaf cells is not necessarily reflected inleaf shape or, more particularly, in leaf size. Genetic
analyses of leaf development in arabidopsis showed that acompensatory system (or systems) acts in leaf
morphogenesis in a way that an increase in cell volume
might be triggered by a decrease in cell number (reviewed in
Tsukaya 2008). Thus, leaf size is, at least to some extent,
regulated at the organ level by the compensatory system orsystems. To understand the details of such totally unknown
regulatory mechanisms, we have conducted a large scale
screening of leaf-size and/or leaf-shape mutants.
As a result, we have succeeded in isolating oli mutants

which have a specific defect in the number of leaf cells, fugu
mutants that exhibit typical compensation syndrome,
namely, decreased number of cells and increased cellvolume, and msc mutants that exhibit an “opposite-type”
compensation syndrome, namely an increased number ofcells and decreased cell volumes.
This year we have revealed that: (1) fugu5 phenotype iscancelled by supplying sucrose to the growth medium; (2)several oli mutations are loss-of-function mutations ofribosome biogenesis genes; (3) “opposite-type”
compensation syndrome in msc mutants is attributed toaccelerated heteroblasty (Usami et al., 2009); detailed
analyses of this phenomenon strongly suggested that traits ofheteroblasty are regulated by several different pathways.
Furthermore, a new tool for studies of the mechanisms ofcompensation, chimeric expression system of KRP2 or AN3,
was established, and several candidate genes responsible forthe compensation were selected from microarray analysis of
fugu2 and an3.
In addition, in the course of studies of AN3 function, we
found that an3 mutation phenotype is drastically changed
when combined with ribosome-biogenesis mutations and/or#2047 mutation (Figure 1). These facts suggest that AN3 isinvolved in various key aspects of organogenesis in
Arabidopsis. Further analyses of the mechanisms ofcompensation are in progress.

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

Ⅱ. Biodiversity of leaf form
We are also interested in the biodiversity of wild plants.
This year we analyzed several achlorophyllous
mycoheterotrophs. Mophological and molecularphylogenetic analyses revealed that Monotropastrum humile
var. glaberrimum must not be conspecific to M. humile(Tsukaya et al. 2008). Molecular phylogenetic analysis of
Oxygyne shinzatoi showed that this very rare genus would
be basal taxon of tribe Thismieae (Yokoyama et al. 2008).
Moreover, we have found a new species of the genus
Oxygyne, O. yamashitae from Yakushima island (Yahara
and Tsukaya 2008). This is the third species of this genus
reported from Japan. Yakushima Island was proven once
again to be a hot spot for plant biodiversity in Japan.

Publication List
〔Original papers〕● Cho, K.-H., Tsukaya, H., and Kim, G.-T. (2008). Characterization of a

Figure 1. Ectopic root formation is seen on cotyledon region in an3#2047 double mutant. Bar, 0.5 mm.

1-4 Size control of leaves and ploidy level
Why does a high-ploidy level cause increased cell/leaf
size? In other words, why are tetraploid leaf cells twice aslarge in volume as diploid leaf cells? The reasons are not yet
perfectly understood. Curiously, plants with high-ploidy
syndrome have more than eight sets of homologous
chromosomes (8C), resulting in an increase in cell volume,
but have smaller leaves (Tsukaya 2008).
The construction of a series of tetraploids of leaf shape/size
mutants supplied us with a good clue for understanding thelinkage mechanisms between the ploidy level and cell/organsize. We found that mutational defects in theendoreduplication were responsible for a curious

dehydrogenase motif and an uORF in Arabidopsis ANGUSTIFOLIA
gene. Plant Biotech. 25, 365-368.
● Lin, X., Minamisawa, N., Takechi, K., Zhang, W., Sato, H., Takio, S.,
Tsukaya, H., and Takano, H. (2008). Isolation and characterization of
the Larix gmelini ANGUSTIFOLIA (LgAN) gene. Planta 228, 601-608.
● Nagano, A., Fukazawa, M., Hayashi, M., Ikeuchi, M., Tsukaya, H.,
Nishimura, M., and Hara-Nishimura, I. (2008). AtMap1: a DNA
Microarray for Genomic Deletion Mapping in Arabidopsis thaliana.
Plant J. 56, 1058-1065.
● Tsukaya, H., Yokoyama, J., Imaichi, R., and Ohba, H. (2008).
Taxonomic status of Monotropastrum humile, with special reference
to M. humile var. glaberrimum (Ericaceae, Monotropoideae). J. Plant
Res. 121, 271-278.
● Yahara T., and Tsukaya H. (2008). Oxygyne yamashitae, a new
species of Thismiaceae from Yaku Island, Japan. Acta Phytotax.
Geobot. 59, 97-104.
● Yokoyama, J., Koizumi, Y., Yokota, M., and Tsukaya, H. (2008).
Phylogenetic position of Oxygyne shinzatoi (Burmanniaceae) inferredfrom 18S rDNA sequences. J. Plant Res. 121, 27-32.

〔Review article〕● Tsukaya H. (2008). Controlling Size in Multicellular Organs: Focus on

the Leaf. PLoS Biol. 6, 1373-1376.

59

National Institute for Basic Biology

Environmental Biology

DIVISION OF PHOTOBIOLOGY (ADJUNCT)

Professor (Specially appointed)

Associate Professor (Adjunct)

WADA, Masamitsu

YAMAUCHI, Daisuke

Postdoctoral Fellows

Visiting Scientist
Technical Assistants

Secretary

KAGAWA, Takatoshi
KODAMA,Yutaka
KONG, Sam-Geun
SUETSUGU, Noriyuki
YAMASATO, Akihiro
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 is one of the simplest model systems forstudying photomorphogenesis. Because the phenomenon iscell autonomous, 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 photomorphogenesis share thesame photoreceptors.

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.

Figure 1. Dark-adapted A. capillus-veneris gametophyte was irradiatedwith red light (4.5 μmol m-2 s-1) to induce chloroplast movement fromanticlinal wall to periclinal wall. The change in the distribution patternof grana stucks during the movement was analyzed every 5 minutes. (a)Outlines of a chloroplast and the path of the center of the chloroplast(inset) during the movement. (b–f) Fluorescent micrographs ofchloroplasts taken every 5 minutes. (g) The distances among granastucks shown as 1, 2 and 3 were plotted (from Journal of Plant Research
Tsuboi et al. 2009).

1-1 Polarity of moving chloroplasts
Chloroplast movement can be induced by whole cell or
partial cell irradiation and is easily observed under amicroscope. Since the speed of movement is very low, thedetection of chloroplast movement during real timeobservation is not easy, so that chloroplast movement isusually recorded using time lapse movies. The figuresobtained every minute were analyzed precisely.
When A. capillus-veneris gametophytes were incubated in

60

darkness for two or three days, most chloroplasts moved tothe anticlinal wall but only a few chloroplasts could befound on the periclinal wall. Chloroplast movement was
induced by partical cell irradiation with sequential
microbeams at different areas near the chloroplasts on the
periclinal walls. Precise analyses of the chloroplast behavior
revealed that chloroplasts can change direction without
turning, although a time lag of a few minutes is required forthis. During the movement chloroplasts do not roll but slide,keeping the concave side to the plasma membrane. These
chloroplast behaviors are the same under both blue and red
light micorobeam irradiation.

1-2 Functional analysis of CHLOROPLAST
UNUSUAL POSITIONING 1 protein in chloroplast
movement
CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1)
protein has a hydrophobic domain at its N-terminus andlocalizes on a chloroplast outer envelope. CHUP1 also hasan F-actin binding domain and a proline rich region for
profilin binding, suggesting the possible role of actin
polymerization. The mutant deficient in CHUP1 protein(chup1) does not show chloroplast photo-relocation
movement, so that chloroplasts sediment at the palisade cellbottom. The function of CHUP1 was studied using thetransformants with variously truncated cDNA of CHUP1
genes in chup1 background (Oikawa et al 2008).
Interestingly, when the CHUP1 N terminus hydrophobic
region was replaced with the hydrophobic domain of achloroplast outer envelope protein 7 (OEP7)-GFP, the chup1
phenotype was complemented, although both amino acidsequences are quite different. The results indicate that theamino acid sequence of CHUP1 N terminus is not important
but that its hydrophobic character is essential for the CHUP1
function to attach the functional part of CHUP1 to thechloroplasts.
The coiled-coil region of CHUP1 anchors chloroplasts
firmly on the plasma membrane, consistent with thelocalization of coiled-coil-GFP on the plasma membrane.

Publication List
〔Original papers〕● Ogura, Y., Komatsu, A., Zikihara, K.,

Nanjo, T., Tokutomi, S., Wada,
M., and Kiyosue, T. (2008). Blue light diminishes interaction of
PAS/LOV proteins, putative blue light receptors in Arabidopsis
thaliana, with their interacting partners. J. Plant Research 121, 97 –105.
● Kodama, Y., Tsuboi, H., Kagawa, T., and Wada, M. (2008). Low
temperature-induced chloroplast relocation mediated by a blue light
receptor, phototropin 2, in fern gametophytes. J. Plant Research 121,441-448.
● Oikawa, K., Yamasato, A., Kong, S.-G., Kasahara, M., Nakai, M.,
Takahashi, F., Ogura, Y., Kagawa, T., and Wada, M. (2008).
Chloroplast outer envelope protein CHUP1 is essential for chloroplastanchorage to the plasma membrane and chloroplast movement. Plant
Physiol. 148, 829-842.

〔Review articles〕● Wada, M. (2008). Photoresponses in fern gametophytes. In: The

Biology and Evolution of Ferns and Lycophytes" Edited by Tom A.
Ranker and Christopher H. Haufler, Cambridge Univ. Press. Pp. 3-48.
● Ogura, Y., Tokutomi, S., Wada, M., and Kiyosue, T. (2008).
PAS/LOV proteins: A proposed new class of plant blue light receptor.
Plant Signaling & Behavior 3, 966-968.

Figure 2. Distribution patterns of chloroplasts in each transgenic line
under darkness (Dark), white light (WL), and strong white light (SL)conditions were compared among wild type, chup1mutant and the threetransgenic plant lines (transferred with a full length CHUP1 gene
(CHUP), with CHUP1 without N-terminus (ΔN-CHUP), and with
CHUP1 with which CHUP1 N terminus was replaced with a N terminus
hydrophobic region of the outer envelope protein 7 (OEP::ΔN-CHUP)),
CHUP1 whose N terminus was replaced with OEP7 N terminus rescued
chup1 mutant as CHUP1 transgenic plants did. The experiments wererepeated at least three times. Bar = 10 mm.

61

National Institute for Basic Biology

Environmental Biology

LABORATORY OF PHOTOENVIRONMENTAL BIOLOGY

Professor (Adjunct)

WATANABE, Masakatsu

Photosynthetic microorganisms, such as cyanobacteria andflagellate algae, respond to light in order to locate
themselves at appropriate photoenvironments. Our researchis aimed at the elucidation of the photoreceptive and signaltransduction mechanisms of light responses in
microorganisms. This approach has led us to the discovery,
characterization, and application of a remarkably uniquelight sensor molecule as described below.

Ⅰ. Photoactivated Adenylyl Cyclase (PAC), an
algal photoreceptor protein with intrinsic
effector function to produce cAMP
In 2002, we found a novel blue-light receptor with an
intrinsic effector role in Euglena gracilis (Figure 1), a
unicellular flagellate alga, which shows blue-light typephotomovements (Iseki et al., Nature 415, 1047-1051,
2002). The action spectra indicate the involvement offlavoproteins as the photoreceptors mediate them. The
paraflagellar body (PFB), a swelling near the base of theflagellum, has been considered a photosensing organelle
responsible for the photomovements. To identify thephotoreceptors in the PFB, we isolated PFBs and purified
the flavoproteins therein. The purified flavoprotein (ca. 400kDa), with noncovalently bound FAD, seemed to be aheterotetramer of α- and β-subunits. Predicted amino acidsequences for each of the subunits were similar to each otherand contained two FAD-binding domains (BLUF: sensor ofblue light using FAD) (F1 and F2) each followed by anadenylyl cyclase catalytic domain (C1 and C2). The

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

62

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

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

Ⅱ. Structures and features of the photoreceptive
domains (F1 and F2) of PAC
To biophysically understand intramolecular photosignal
processing, knowledge of the three dimensional structure ofthe protein as well as spectroscopical analyses are essential.
Due to the present difficulty of preparative heterologous
expression of functional PAC, however, neither
crystallography nor spectroscopical analyses of the whole
PAC protein have been realized yet. To partially compensate
for this situation we tried homology modeling and quantum
chemical calculation of its photoreceptive domains (F1 andF2) of the α- subunit using their prokaryotic counterparts,
BLUFs, as the templates (Figure 3). It was of interest that,from the viewpoint of binding energies thus calculated, F1
appeared to bind FAD less strongly than F2 does. Thisdifference might indicate different structural and functionalroles between these very similar domains.

Figure 3. Three-dimentional structure model of the photoreceptor
domains (F1 and F2) of PACa subunit. The template for the homology
modeling is AppA, a bacterial blue-light sensor.

Publication List
〔Original papers〕● Maruyama, S., Misawa, K., Iseki, M., Watanabe, M., and Nozaki, H.

(2008). Origins of a cyanobacterial 6-phosphogluconate
dehydrogenase in plastid-lacking eukaryotes. BMC Evol. Biol. 8, 151
doi:10.1186/1471-2148-8-151.
● Ioki, M., Takahashi, S., Nakajima, N., Fujikura, K., Tamaoki, M., Saji,
H., Kubo, A., Aono, M., Kanna, M., Ogawa, D., Fukazawa, J., Oda,
Y., Yoshida, S., Watanabe, M., Hasezawa, S., and Kondo, N. (2008).
An unidentified ultraviolet-B-specific photoreceptor mediates
transcriptional activation of the cyclobutane pyrimidine dimer
photolyase gene in plants. Planta 229, 25-36.
● Mori, E., Takahashi, A., Kitagawa, K., Kakei, S., Tsujinaka, D., Unno,
M., Nishikawa, S., Ohnishi, K., Hatoko, M., Murata, N., Watanabe,M., Furusawa, Y., and Ohnishi, T. (2008). Time course and spatial
distribution of UV effects on human skin in organ culture. J. Radiat.
Res. (Tokyo) 49, 269-277.

63

National Institute for Basic Biology

Theoretical Biology

DIVISION OF THEORETICAL BIOLOGY

Professor (Concurrent)

MOCHIZUKI, Atsushi
Associate Professor
NIBB Research Fellow
Postdoctoral Fellows
Graduate Student
Secretary

MOCHIZUKI, Atsushi *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.

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

Ⅰ. Structure of regulatory networks anddiversity of gene expression patterns
The complexity of gene regulatory networks is considered
responsible for the diversity of cells. Different types of cells,
characterized by the expression patterns of genes, areproduced in early development through the dynamics ofgene activities based on the regulatory network. However,very little is known about the relationship between the
structure of regulatory networks and the dynamics of geneactivities.
In this study I introduce the new idea of "steady statecompatibility," by which the diversity of possible geneactivities can be determined from the topological structureof gene regulatory networks. The basic premise is very
simple: the activity of a gene should be a function of thecontrolling genes. Thus a gene should always show uniqueexpression activity if the activities of the controlling genes
are unique. Based on this, the maximum possible diversityof steady states is determined using only information
regarding regulatory linkages and without knowing theregulatory functions of genes.
Using the concept of "steady state compatibility," three
general properties of the relationship between the topologyof regulatory networks and the maximum number of steadystates can be derived (Figure 2). (A) Cascade structures inregulatory networks do not increase the number of possible
steady states (Figure 2a). (B) Loop structures in networks
are necessary to generate multiple steady states. The numberof separated loops increases the maximum diversity ofsteady states (Figure 2b). (C) Multiple loops that areconnected by sharing the same genes do not increase themaximum diversity of steady states (Figure 2c).
The method was applied to a gene regulatory network
responsible for early development in a sea urchin species. A
set of important genes responsible for generating diversities
of gene activities was derived based on the concept ofcompatibility of steady states.
64

Figure 2. General properties showing the relationships between the
structure of regulatory networks and the maximum diversity of steadystates.

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

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

Ⅱ. 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 may acts as negative
feedback in the system. However, our mathematical analysis
proved that simple dynamics based on the experimentallysuggested 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.

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 5. 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 a 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.

Publication List
Figure 4. 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.

〔Original paper〕● Mochizuki, A. (2008). Structure of regulatory networks and diversityof gene expression patterns. J. theor. Biol. 250, 307-321.

65

National Institute for Basic Biology

Theoretical Biology

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 developing methods
and tools to conduct comparative analyses not only of
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 III 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 around 700 published genomes
including 13 eukaryotic microbes (fungi and protozoa) andC. elegans as a reference. MBGD is available at
http://mbgd.genome.ad.jp/.

66

Ⅱ . Identification of the core structure
conserved among moderately related
microbial genomes
Horizontal gene transfers (HGT) have played a significant
role in prokaryotic genome evolution, and the genes
constituting a prokaryotic genome appear to be divided into
two classes: a "core gene pool" that comprises intrinsic
genes encoding the proteins of basic cellular functions, and a"flexible gene pool" that comprises HGT-acquired genes
encoding proteins which function under particularconditions, such as genomic islands. Therefore, theidentification of the set of intrinsically conserved genes, or
the genomic core, among a taxonomic group is crucial not
only for establishing the identity of each taxonomic group,
but also for understanding prokaryotic diversity andevolution. Here, we consider the core structure of related
genomes as a set of sufficiently long segments in whichgene orders are conserved among multiple genomes so thatthey are likely to have been inherited mainly throughvertical transfer. We developed a method for aligning
conserved regions of multiple genomes, which finds theorder of pre-identified orthologous groups (OGs) that retains
to the greatest possible extent the conserved gene orders.
The program, named CoreAligner, requires a set of wellconserved OGs. We compiled them using the MBGD server,and considered an OG "conserved" when it was present in atleast half of the genomes. Next, a neighborhood graph wasconstructed using a set of conserved neighborhood pairs,
which are defined as two conserved OGs that are locatedwithin 20 genes in at least half of the genomes. Ouralgorithm for constructing the alignments of the coregenome structures is based on finding the longest path of theconserved neighborhood graph. A similar algorithm waspreviously developed mainly for identifying much shorterbut more widely conserved gene clusters such as operons,
but unlike that method, our method considers not only genes
in the same direction but also those in the opposite direction
as neighboring genes, and thereby generally generates
longer alignments. In addition, our method uses the dynamic
programming algorithm for calculating the longest path.
The method was applied to genome comparisons of two
well-characterizedfamilies,
Bacillaceae
andEnterobacteriaceae, and identified their core structurescomprising 1438 and 2125 OGs, respectively (Figure 1),
which correspond to a third of the number of the B. subtilis
genes (4105) and half of the E. coli genes (4237),
respectively. The core sets contained most of the essential
genes (90%) and their related genes, which were primarily
included in the intersection of the two core sets comprisingaround 700 OGs. The definition of the genomic core basedon gene order conservation was demonstrated to be morerobust than the simpler approach based only on geneconservation. We also investigated the core structures interms of G+C content homogeneity and phylogenetic
congruence, and found that the core genes primarily
exhibited the expected characteristic (i.e., being indigenousand sharing the same history) more than the non-core genes.

Figure 1. Core genome alignment. (A) Part of a schematic representationof genome alignment obtained from the Bacillaceae dataset. (B)Comparative genome map showing the locations of the core (left) andnon-core (right) genes, where the latter are extensively crossed with eachother.

Ⅲ . Enhancement of the algorithm for
identifying orthologous groups among
multiple genomes
As a core technology of our comparative genomics tools,
we have developed a rapid automated method of ortholog
grouping, named DomClust, which is effective enough toallow the comparison of many genomes simultaneously. The
method classifies genes based on a hierarchical clustering
algorithm using all-against-all similarity data as an input, but
it also detects domain fusion or fission events and splits
clusters into domains if required. As a result, the procedure
can split genes into the domains minimally required forortholog grouping.
As the number of completed genomic sequences grows,
comparison among closely related as well as distantly
related genomes has become important for understanding thefunction and evolution of various genomes. For evolutionary
analysis of a target set of related organisms (ingroup), we
often need another set of organisms outside of that group(outgroup) to correctly infer evolutionary processes. To
incorporate this concept into the ortholog analysis, we haveenhanced the DomClust algorithm to impose constraints on
the resulting orthologous groups so that the outgroup species
should come outside of the ingroup species. The resulting
table has a nested structure when a duplication event occurs
within the ingroup lineage.

Ⅳ. Development of a workbench for comparative
genomics
We are developing a comparative genomics workbench
named RECOG (Research Environment for COmparative
Genomics), which aims to extend the current MBGD system
by incorporating the above-described approaches including
the ingroup/outgroup distinction in ortholog grouping andthe core structure extraction among related genomes. In
addition, the RECOG system has several advanced featuresthat allow users to perform more flexible phylogenetic
pattern analyses.
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 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). Users can choose a set ofgenomes to compare in the taxonomic tree viewer and run
the DomClust program to identify orthologous groups
among them. The result is displayed in the ortholg tableviewer, where users can see the entire picture of thephylogenetic pattern map as well as the full details of theortholog table using semantic zooming functionality. Several
sorting and filtering functions have been implemented formodifying the display of the ortholog table viewer, including
sorting by functional category or by the gene order of a
specified genome, and filtering by keywords or byphylogenetic pattern conditions. The RECOG system also
contains an interface for the CoreAligner program, by whichusers can see an alignment display and a comparative map
display of conserved core regions identified by the
CoreAligner program (Figure 2).

Figure 2. A typical usage of the RECOG system including DomClustand CoreAligner analyses.

Publication List
〔Original papers〕● Uchiyama, I. (2008). Multiple genome alignment for identifying thecore structure among moderately related microbial genomes. BMC
Genomics, 9, 515.
● Nakayama, K., Yamashita, A., Kurokawa, K., Morimoto, T., Ogawa,
M., Fukuhara, M., Urakami, H., Ohnishi, M., Uchiyama, I., Ogura, Y.,
Ooka, T., Oshima, K., Tamura, A., Hattori, M., Hayashi, T. (2008).
The Whole-genome sequencing of the obligate intracellular bacterium
Orientia tsutsugamushi revealed massive gene amplification during
reductive genome evolution. DNA Res. 15, 185-199.

67

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori
Technical Staff
NIBB Research Fellow
Visiting Scientist
Technical Assistant
Secretary

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

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

Ⅰ. Initial step for left-right asymmetry
The first L-R asymmetry in mammalian development arises
on the embryonic surface. A gastrulating mouse embryo hasa shallow hollow on its ventral surface, called ‘the node,’with hundreds of cilia moving in a clockwise rotationalmanner (Figure 1; Nonaka et al., 1998). The sum of thevortical motions of the cilia, however, generates a leftward
flow of the surrounding fluid rather than a vortex. The cilia
can generate L-R asymmetry de novo, i.e. without preexisting left-right asymmetry, by their posteriorly tilted
rotation axis (Nonaka et al., 2005).

Figure 2. Flow culture exiperiment. A) A peristaltic pump anddepulsators supply constant fluid flow in the chanber (red arrowheads).
B) Embryos held in the pots receive the pump-driven flow on theirsurface. If the pump-driven flow reverses the intrinsic nodal flow,
expression of nodal, a master gene for leftness, is reversed (Right).

Ⅱ. Imaging technologies
Long-term live imaging of large specimens, such asembryos, is very useful in developmental biology but
technically challenging, mainly because of phototoxity andthe limitations of deep imaging. The Digital Scanned Lightsheet Microscope (DSLM, Figure 3) developed by Dr. Ernst
Stelzer’s group at the European Molecular Biology
Laboratory (EMBL) is extremely suitable for this purpose,and we have introduced a set of DSLM and started tovisualize whole mouse embryo at gastrulating stages withsingle cell resolution.
Additionally, we support researchers who are interested inusing our DSLM and two-photon microscope. Several
collaborative projects are in progress.

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

The leftward flow, called nodal flow, determines
subsequent L-R development. This principle has been
confirmed by our experiments, which demonstrated thatembryos raised in a rightward artificial flow of culture
medium develop reversed L-R asymmetry (Figure 2;
Nonaka et al., 2002).
While it is clear that nodal flow conveys asymmetric
information along the L-R axis, the nature of the information
remains unclear. We are now working to clarify how thedirection of nodal flow is converted to the subsequent step,
asymmetric gene expression.

Figure 3. DSLM on a optical table.

Figure 4. Images taken with DSLM. Left: optical section of a 6-day
mouse embryo with fluorescent nuclei. Right: a juvenile zebrafish head
expressing GFP in neurons.

68National Institute for Basic Biology

Strategic Planning Department

The main activities of the Department in 2008

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

UENO, Naoto
Associate Professor

KODAMA, Ryuji

Assistant Professor(Specially appointed)

KURATA, Tomoko

2) Management of education-related programs
Internship Program 2008
The Second and Third International Practical Course (Mar.
and June, 2008)

KURATA, Tomoko*
MUKOHDA, Yasuyo
OTA, Misaki
MAEDA, Sanae
TANAKA, Megumi
OTA, Kyoko
EMDE, Jason R.

NIBB Research Fellow
Technical Assistants

3) Press Release
News on scientific achievements are sent to newspaper andmagazine reporters via leaflets, e-mails and password
protected web pages. For some of the releases, we arrangeda press conference.

4) Editing of publications
Bulletin of NIBB 2008 (in Japanese, in preparation)
Annual Report of NIBB 2008 (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 (Figure 1)

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 

February 28-29

New Frontiers for the Medaka Model-Genome, Bioresources and Biology-

M. Tanaka 



September 13-15

Frontiers of Plant Science in the 21st Century

K. Okada

November 21-22

Evolution: Genomes, Cell Types and Shapes

D. Arendt
M. Hasebe
S. Kuratani
N. Ueno       

Table 1. International conferences managed by Strategic Planning Department in 2008

54th NIBB Conference

55th NIBB Conference

8th NIBB-EMBL Meeting

Bulletin of NIBB 2008 (in Japanese)

Figure 1. Examples of posters, pamphlets and abstract books produced by the Strategic Planning Department in 2008

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

69

National Institute for Basic Biology

Research Support

Research Support Facilities

Head

TAKADA, Shinji● Large Spectrograph Laboratory

Professor (Adjunct)Technical Staff
Technical Assistant
Secretary

WATANABE, Masakatsu
HIGASHI, Sho-ichi
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 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〕● Ikehata, H., Kawai, K., Komura, J., Sakatsume, K., Wang,

L., Imai,M., Higashi, S., Nikaido, O., Yamamoto, K., Hieda, K., Watanabe,M., Kasai, H., and Ono, T. (2008). UVA1 genotoxicity is mediated notby oxidative damage but by cyclobutane pyrimidine dimers in normalmouse skin. J. Invest. Dermatol. 128, 2289-2296.
● Ioki, M., Takahashi, S., Nakajima, N., Fujikura, K., Tamaoki, M., Ioki,
M., Takahashi, S., Nakajima, N., Fujikura, K., Tamaoki, M., Saji, H.,
Kubo, A., Aono, M., Kanna, M., Ogawa, D., Fukazawa, J., Oda, Y.,
Yoshida, S., Watanabe, M., Hasezawa, S., and Kondo, N. (2008). An
unidentified ultraviolet-B-specific photoreceptor mediates
transcriptional activation of the cyclobutane pyrimidine dimer
photolyase gene in plants. Planta 229, 25-36.
● Mori, E., Takahashi, A., Kitagawa, K., Kakei, S., Tsujinaka, D., Unno,
M., Nishikawa, S., Ohnishi, K., Hatoko, M., Murata, N., Watanabe,M., Furusawa, Y., and Ohnishi, T. (2008). Time course and spacial
distribution of UV effects on human skin in organ culture. J. Radiat.
Res. 49, 269-277.
● Nagai, Y., Miyagishi, D., Akagawa, T., Ohishi, F., Ueno, H.,
Kobayashi, K., Yamashita, K., and Watanabe, J. (2008).
Photodegradation mechanisms in poly(2,6-butylenenaphthalate-cotetramethyleneglycol) (PBN-PTMG), Part III: Photodegradationinduced by the carbonyl group in n, π* excited states. Polym.
Degradat. Stabil. 93, 134-138.
● Nagao, A., Zhao, X., Takegami, T., Nakagawa, H., Matsui, S.,
Matsunaga, T., and Ishigaki, Y. (2008). Multiple shRNA expressions
in a single plasmid vector improve RNAi against the XPA gene.
Biochem. Biophys. Res. Commun. 370, 301-305.
● Simamura, E., Shimada, H., Ishigaki, Y., Hatta, T., Higashi, N., andHirai, K-I. (2008). Bioreductive activation of quinone anti-tumor
drugs by mitochondrial voltage-dependent anion channel 1. Anat. Sci.
Int. 83, 261-266.
● Suzuki, T., Takashima, T., Izawa, N., Watanabe, M., and Takeda, M.
(2008). UV radiation elevates arylalkylamine N-acetyltransferase
activity and melatonin content in the two-spotted spider mite,
Tetranychus urticae. J. Insect Physiol. 54, 1168-1174.

2. Tissue and Cell Culture Laboratory
Figure 1. The Large Spectrograph70

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

3. Computer Laboratory
The computer laboratory maintains several computers toprovide computation resources and the means of electronic
communication within NIBB. Our main computer system,
the Biological Information Analysis System (BIAS),
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; 2CPU (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
Plant culture laboratory consist of biotron, plant cell culture
facility, and experimental farm. Biotron contains a large
number of culture boxes and a limited number of rooms withenvironmental control for plant culture. In some of thesefacilities and rooms, experiments can be carried out at the P1
physical containment level under extraordinary
environments such as strong light intensity, low or hightemperatures, etc. Plant cell culture facility is equipped with
autotrophic and heterotrophic culture devices and equipment
for experimental cultures of plant and microbial cells.
Facilities for the preparation of plant cell cultures, including
an aseptic room with clean benches, are also provided.
Experimental farm consists of two 20 m2 glass-houses with
precise temperature and humidity control, four green houses
at the P1 physical containment level, a small farm, and two
greenhouses (45 and 88 m2) with automatic sprinklers. The
laboratory also includes a building with storage and work
space.
Facilities for the preparation of plant cell cultures,
including an aseptic room with clean benches, are also
provided.

71

National Institute for Basic Biology

Research Support

RESEARCH CENTER FOR INTEGRATIVE AND COMPUTATIONAL BIOLOGYHead

HASEBE, Mitsuyasu
Professor (Concurrent)
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 ultimate goal is to establish a new bioscience that
includes methods originally utilized in different fields:
informatics, mathematics, and biology.

Ⅰ. Research activity
The research center for integrative and computational
biology was founded in 2001 during a time of rapid progressin modern biology. The success of world wide genome
projects has provided a huge amount of new information on
genes, leading to expectations of advances in thedevelopment of new medicines for intractable diseases, theexploitation of new cultivated plants resistant to noxious
insects, and so on. In addition, it is the object of present-day
biology to research higher-order phenomena that are madeup of complex interactions between many genes. To grapple
with these challenges, it is necessary to decipher hugeamounts of gene information and to reveal the fundamentalsof the biological behavior of cells and organisms.
Mathematical and computational sciences have strong
capacities for dealing with these challenges. Computational
methods make it possible to process ever-increasing
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 and computational methods, as well as
developing new methodologies for studying complex
phenomena.

For example, integrative methods are especially importantfor understanding pattern formation in development.
Morphological differences between species are an importantresearch focus of current developmental biology. What is themechanism responsible for the difference of morphogenesis
between species? Theoretical studies are useful in
identifying candidates for cell or gene interaction that arelikely to be responsible for the systems in real organisms.
This method gives us an integrative understanding of 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. Theoretical methods provide testablepredictions which the experimental biologists are able toinvestigate before turning the results back over to thetheoreticians for the next round of predictions. By repeatingthese predictions and tests, the integrative methods arecontinually developed. The Research Center for Integrativeand Computational biology has continued to encourage andenhance interaction between theoretical biologists andexperimental biologists.
The center provides equipment such as cluster machines
and Unix-based machines for computation and experiments.
Members can also use computational equipment at the
Research Center for Computational Science in Okazaki. The
center also provides experimental equipment forcollaborations with experimental biologists.

Figure 2. A cluster machine

In April of 2008 we held an international meeting on
systems biology in Barcelona as a collaborative programwith EMBL. We had many participants studying biologicalsystems using different methods, including physics,
mathematics and computational science. The meetingenhanced interactions between researchers in different fields
and resulted in some collaborative works between them.
Figure 1. A laboratory room for computational studies

72

CENTER FOR TRANSGENIC ANIMALS AND PLANTS

Head

IGUCHI, Taisen
Associate Professors

Technical Staff

Supporting Staff

WATANABE, Eiji
SASAOKA, Toshikuni
TANAKA, Minoru
HAYASHI, Kohji
NOGUCHI, Yuji
ICHIKAWA, Yoko
TAKAGI, Yukari
YASUDA, Mie
KAWAMURA, Motofumi
OKUDA, Tadayoshi
INADA, Yosuke
AJIOKA, Rie
NAKASHIMA, Machiko
YAMANAKA, Megumi
WATANABE, Kaori

The worldwide genome project has almost been completed
and research on basic biology has arrived at a post-genome
era in which researchers are focusing on investigating thefunctions of individual genes. To promote the functional
analysis of a gene of interest it is essential to utilize
genetically altered model organisms generated using genetic
engineering technology, including gene deletion, genereplacement and point mutation.
The NIBB Center for Transgenic Animals and Plants(CTAP) was established in April 1998 to support researchusing transgenic and gene targeting techniques at NIBB. The
CTAP is managed by the head (professor, a concurrent post)
and three associate professors.

The activities of the CTAP are as follows:
1. The provision of information, materials, techniques andanimal housing space to researchers.
2. The use of various kinds of instruments to analyze
mutant, transgenic, and gene-targeted animals and plants.
3. The development of novel techniques related to transgenic
and gene targeting technology.
4. Cryopreservation and storage of transgenic strains.

Ⅰ. Research support activities (mouse)
In 2001 the NIBB mouse facility (built under specificpathogen free (SPF) conditions) opened in the Myodaiji area
and the production, breeding, analysis, cryopreservation andstorage of genetically manipulated mouse strains has been
conducted there since then. The new CTAP building in the
Yamate area strengthened research activities using
genetically altered organisms. The building has five floors
and a total floor space of 2,500 m2 in which we can generate,
breed, store and analyze transgenic, gene targeting andmutant mice under SPF conditions. The mouse housing area
was constructed based on a barrier system. This building is
also equipped with breeding areas for transgenic small fish,
birds and insects.

Figure 2. Liquid nitrogen storage equipment for cryopreservation

Figure 1. The new center facility for transgenic animals in the Yamate
areaTechnical staff and supporting staff develop and promoteresearch-supporting activities. A state-of-the-art facility fortransgenic animals opened at the end of 2003 in the Yamate
area.

In 2008 (from January 1 to December 31) 3,509 mice and1,126 fertilized eggs were brought into the CTAP in the
Yamate area, and 37,137 mice (including pups bred in thefacility) and 120 fertilized eggs were taken out.
A number of strains of genetically altered mice fromoutside the CTAP were brought into the mouse housing areaby microbiological cleaning using the in vitro fertilization-73

National Institute for Basic Biology

Research Support

embryo 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 2008 (fromJanuary 1 to December 31) 11 mice were brought into the
CTAP in the Myodaiji area, and 257 mice (including pups
bred in the facility) were taken out.

Figure 3. A laboratory mouse

Ⅱ. Research support activities (small fish,
birds, and insects)
The first floor of the CTAP building in the Yamate area
provides space and facilities to maintain small fish, chickembryos, and insects. In 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.
In 2008 (from January 1 to December 31), 10,442 medaka
and zebrafish (124 eggs, 3,761 embryos and 6,557 adults)
were brought to the facility and 82,929 medaka andzebrafish (75,521 fertilized eggs, 6,454 embryos and 954
adults, including animals bred in the facility) were taken out.
In a laboratory for chick embryos 7,070 fertilized chicken
eggs were brought in and 154 fertilized eggs and 10 chicken
embryos were taken out. These animals were used forresearch activities in neurobiology and developmental
biology.
In 2007 NIBB was selected as a core facility of the
National BioResource Project of Medaka. We havesupported the activities of NBRP Medaka by providing
standard strains, induced mutants and transgenic lines andtraining personnel regarding fish maintenance.

74

Figure 4. Breeding equipment for small fish

Ⅲ. Academic activities
The associate professors of this center - E. Watanabe, T.
Sasaoka and M. Tanaka - are the principal investigators of
the Laboratory of Neurophysiology, the Laboratory of
Neurochemistry and the Laboratory of Molecular Genetics
for Reproduction, respectively. The Laboratory of
Neurophysiology is studying the brain sensing system forbody fluid water and sodium homeostasis using genetargeting mice. The Laboratory of Neurochemistry is
studying the physiological role of the dopaminergic systemusing genetically altered mice. The Laboratory of Molecular
Genetics for Reproduction is studying the molecularmechanisms of reproductive organ development and sex
differentiation using mutagenized or transgenic medaka. For
details, please refer to the academic activities of eachlaboratory.

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

IIDA, Shigeru
Technical Staff

Secretary

XL30 ESEM)
Confocal Laser Scanning Microscopes (Leica TCS SP2,
OLYMPUS FV1000 )Fluorescence Microscope (KEYENCE BZ-8000)
Color Laser 3D Profile Microscope (KEYENCE VK-8500)
High-Resolution Quick Microscope (KEYENCE VH-5000)

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

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. The
Center’s instruments can be used by researchers fromoutside the institute upon proposal.

Representative Instruments
Protein Sequencers (ABI Procise 494HT, 492cLC)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizer (ABI 433A)
Plasmid Isolation Systems (Kurabo PI-100∑ , PI-50α, PI50, PI-200)
Automatic Nucleic Acid Isolation System (Kurabo NA2000)
Genetic Analyzers (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,NanoDrop ND-1000)
Microplate Luminometer (Berthold MicroLumat LB 96P)
Microplate Readers (Corona MTP-120, MTP-100F)
FT-IR Spectrophotometer (Horiba FT-730)
Bio Imaging Analyzer (Fujifilm BAS 2000)
Luminescent Image Analyzers (Fujifilm LAS 3000 mini)
Fluorescence Bio Imaging Analyzer (Takara FMBIO II)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Environmental Scanning Electron Microscope (PHILIPS

Figure 1. Protein sequencers

Figure 2. Confocal Laser Scanning Microscope

Figure 3. LC/Q-TOF-MS

75

National Institute for Basic Biology

Research Support

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

KAJIURA-KOBAYASHI, Hiroko● Cell Biology

Unit Chief
Subunit Chief

KONDO, Maki
KABEYA, Yukiko● Developmental Biology

Technical Staff

TAKAGI, Chiyo
UTSUMI, Hideko
NODA, Chiyo● Neurobiology

Unit Chief
Subunit Chief

OHSAWA, Sonoko
TAKEUCHI, Yasushi● Center for Analytical Instruments

Unit Chief
Subunit Chief
Technical Staff
Technical Assistants

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
TAKAMI, Shigemi
OKA, Sanae
ICHIKAWA, Mariko● 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 Staff
Technical Assistants

HAYASHI, Kohji
NOGUCHI,Yuji
ICHIKAWA, Yoko
TAKAGI, Yukari● Disposal of Waste Matter Facility

Unit Chief:

MATSUDA, Yoshimi

Reception
Supporting Staff● Radioisotope Facility

Unit Chief
Subunit Chief
Technical Staff
Technical Assistant

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

The Technical Division is a support organization forresearchers and research organizations within the NIBB. The
Technical Division develops and promotes the institute's
research activities and maintains the research functions of
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.

76

SAKAGAMI, MariTSUZUKI, Shihoko
KATAOKA, Yukari
UNO, Satoko
MAEDA, Yumi
MIYATA, Haruko

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.

National Institute for Basic Biology

Research Facilities

CENTER FOR RADIOISOTOPE FACILITIESHeadHASEBE, Mitsuyasu
Associate Professor

Table 2. Users and visitors who entered each controlled area in 2008

OGAWA, Kazuo(Radiation Protection Supervisor)

Technical staff

MATSUDA, Yoshimi(Radiation Protection Supervisor)

Supporting staff

SAWADA, Kaoru
IINUMA, Hideko
ITO, Takayo
KAMIYA, Kiyomi

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 JRIA (Japan
Radioisotope Association) and the transfer of radioisotope
wastes to JRIA.
Matsuda, Iinuma, Ito, and Kamiya maintained CFBI
(Common Facilities Building I)-branch and LGER
(Laboratory of Gene Expression and Regulation)-branch in
the Myodaiji-Area. Ogawa and Sawada worked in the
Yamate-Area.
The number of registrants and the number of users fromJanuary 2008 to December 2008 are presented in Table 1.
Users counted by the monitoring system going in and outof the controlled areas numbered 4,151 persons during thisperiod. The percentages and numbers for each area areshown in Figure 1 and Table 2. The annual changes ofregistrants and the number of totals per year are shown in
Figure 2.
The balance of radioisotopes received at the CRF is shownin Table 3.
Thirty years have passed since the CFBI-branch opened
and some parts of the floor and the waste water system havedeteriorated. Figure 3 shows the repair work done on Room
9 of the CFBI-branch to accommodate the law.

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

Table 3. Balance of radioisotopes received and used (kBq) at each controlledarea in 2008

A

B

Table 1. Numbers of registrants and users at Myodaiji-Area and YamateArea in 2008

C

D

Figure 3. Repair work on room 9
A: Floor painting (waterproofing)
B: Floor construction
C: Closing floor sheet
D: Completion
Figure 1. Percentage of users going in and out of each controlled area in
2008

77

54th NIBB Conference
New Frontiers for the Medaka Model– Genome, Bioresources and Biology
Organizing Chair : Minoru Tanaka
February 28 (Thu)-29 (Fri), 2008
The medaka is an emerging model vertebrate and is
beginning to aid the exploration of new fields of biology.
The symposium was the first international symposium after
the core facility of NBRP medaka was transferred to andestablished at NIBB and the entire genomic sequence of
medaka was published. The symposium focused on not only
a variety of biological studies but also on the genome andbioresources. First rate scientists from Japan, Asia, Europe
and America gathered at the symposium and surveyed thebiology of medaka, the novel skills that support the biology,
the genomic information infrastructure and a variety of
medaka resources, and discussed the future of such studies.
Although the symposium did not set up a poster presentation
for young scientists andstudents, more than 100
researchers participated in theconference.
The presentations on thebasic sciences covered wide
areas of biology, from thefundamental issues ofdevelopment, evolution andgenome research to theanalysis of behavior. This
represents one feature of
medaka biology, together
with its application toenvironmental assessment
and toxicology. It isnoteworthy that most of thesestudies were based on orwere closely associated withgenomic infrastructures andnovel technologies that have recently become available forthe researchers.
The symposium attracted considerable attention from awide audience and was reported in the May issue of “nature
DIGEST”, vol.5 2008. Several speakers from thesymposium were interviewed by a reporter in the article.
Lastly, I’d like to briefly mention that this was the first
NIBB conference that accepted donations from several
overseas companies. An unexpectedly wide and high levelof attention to and interest in biology using medaka in thescientific community was indicated by the response.
(Minoru Tanaka)

Speakers
CHENG, Shuk Han (City Univ. Hong Kong, China SAR), CZERNY, Thomas (Univ. Applied Sciences, Austria),
HONG, Yunhan (NUS, Singapore), JOLY, Jean Stephane (INRA/CNRS, France), SCHARTL, Manfred (Univ. Wuerzburg, Germany),
TSAI, Huai-Jen (National Taiwan Univ., Taiwan), WESTERFIELD, Monte (Univ. Oregon, USA), WINKLER,
Christoph (NUS, Singapore), WINN, Richard N. (Univ. Georgia, USA), WITTBRODT, Joachim (Univ. Heidelberg / EMBL, Germany)
HAMAGUCHI, Satoshi (Niigata Univ., Japan), IWANAMI, Norimasa (Univ. Tokushima, Japan),
KAWAMURA, Shoji (Univ. Tokyo, Japan), KIKUCHI, Kiyoshi (Univ. Tokyo, Japan), KUDO, Akira (Tokyo Inst. Tech., Japan),
MORISHITA, Shinichi (Univ. Tokyo, Japan), NAGAHAMA, Yoshitaka (NIBB, Japan), NARUSE, Kiyoshi (NIBB, Japan),
NISHINA, Hiroshi (Tokyo Med. & Dental Univ., Japan), OKAMOTO, Hitoshi (RIKEN BSI, Japan), OKUBO, Kataaki (NIBB, Japan),
SASADO, Takao (Nagahama Inst. Bio-Sci. & Technology, Japan), TAKEDA, Hiroyuki (Univ. Tokyo, Japan),
TAKEUCHI, Hideaki (Univ. Tokyo, Japan), TANAKA, Minoru (NIBB, Japan), TANIGUCHI, Yoshihito (Kyoto Univ., Japan),
WAKAMATSU, Yuko (Nagoya Univ., Japan), YAMASHITA, Masakane (Hokkaido Univ., Japan), YAMAZAKI, Yukiko (NIG, Japan)

78

55th NIBB Conference
Frontiers of Plant Science in the 21st Century
Organizing Chair : Kiyotaka Okada
September 13 (Sat)-15 (Mon), 2008
For three days in mid-September 2008, from the 13th to the
15th, 147 people gathered in the Okazaki Conference Centerand discussed the present and future of Plant Science under
the title of “Frontiers of Plant Science in the 21st Century”.
The topics ranged from cellular, developmental, and genetic
biology to systems biology, evolutional biology andengineering. The program of the conference can be found on
our home page at http://www.nibb.ac.jp/conf55. We had a
special panel discussion on the afternoon of the 14th, which
was organized and held by a group of our graduate students

as part of their Graduate Student Education Program
sponsored by the Graduate University for Advanced Studies(SOKENDAI).
After the conference, we asked several attendants to give
us a frank review of the aims and execution of theconference. The comments and advice that we received will
be a great help to us in improving future NIBB conferences
and can be found below.
(Kiyotaka Okada)

Invited Speakers
BEEMSTER, Gerrit T.S. (Ghent Univ., Belgium), CAUSIER, Barry (Univ. Leeds, UK), COLLINS, Richard M. (Univ. Leeds, UK),
COPPENS, Frederik (Ghent Univ., Belgium), COUPLAND, George M. (Max Planck Inst., Germany), GIAKOUNTIS,
Antonis (Max Planck Inst., Germany), GONEHAL, Venugopala Reddy (UC, Riverside, USA), ITO,
Toshiro (Temasek Life Sciences Lab., Singapore), KEPINSKI, Stefan (Univ. Leeds, UK), KNOX, Paul (Univ. Leeds, UK),MICOL, Jose Luis (Univ. Miguel Hernandez , Spain), PEREZ-PEREZ, Jose M. (Univ. Miguel Hernandez & Inst. Bioingenieria, Spain),
SAIJO, Yusuke (Max Planck Inst., Germany), SCHULZE-LEFERT, Paul (Max Planck Inst., Germany),
SUN, Bo (Temasek Life Sciences Lab., Singapore)
ASHIKARI, Moto (Nagoya Univ., Japan), BREUER, Christian (RIKEN Plant Sci. Center, Japan),
EZURA, Hiroshi (Univ. Tsukuba, Japan), HARA-NISHIMURA, Ikuko (Kyoto Univ., Japan), HASEBE, Mitsuyasu (NIBB, Japan),
KAKUTANI, Tetsuji (NIG, Japan), KAWAGUCHI, Masayoshi (Univ. Tokyo, Japan), MACHIDA, Yasunori (Nagoya Univ., Japan),
MATSUBAYASHI, Yoshikatsu (Nagoya Univ., Japan), NISHIMURA, Mikio (NIBB, Japan), OKADA, Kiyotaka (NIBB, Japan),
SHIMAMOTO, Ko (NAIST, Japan), TAKEDA, Seiji (NAIST, Japan), TANAKA, Yoshi (Suntory Ltd, Japan), TSUKAYA,
Hirokazu (Univ. Tokyo / NIBB, Japan), YAMAGUCHI, Takahiro (NIBB, Japan)

Organizing Committee
OKADA, Kiyotaka (NIBB), TSUKAYA, Hirokazu (Univ. Tokyo / NIBB), NISHIMURA, Mikio (NIBB), HASEBE, Mitsuyasu (NIBB),YAMAGUCHI, Takahiro (NIBB)

79

55th NIBB Conference Frontiers of Plant Science in the 21st Century

Comments from the participants
Dr. Antonis Giakountis

Max Planck Institute for Plant Breeding Research, Germany.
The organizers did an excellent job, on multiple levels, of preparing the
NIBB55 conference. The invited speakers were certainly chosen wisely
and in such a way that all of the sessions were interesting. In terms oftiming the conference was also successful and there were no significant
deviations from the schedule. One suggestion would be to offer theopportunity to selected poster presenters to take 20 minutes instead of
15 minutes for their talks, especially since the discussion was also
included in that time. Perhaps the starting talk of each session could be
reduced to 30-35 minutes instead of 40 minutes so as to provide thisextra time. I appreciated and enjoyed the politeness of the hosts and theJapanese students a lot and there were many
opportunities for social interaction at the conference.
Furthermore it was a very interesting idea to have apanel discussion conducted exclusively by young scientists on topics
that the students themselves had selected. This certainly made thissession very current and up-to-date. An additional suggestion would beto perhaps split future panel discussions up over two days or to movethem to morning sessions so as to stimulate even more discussion. In
conclusion, it was a very successfully and fruitful meeting whichhighlighted research opportunities in Japan as being very attractive.

Dr. Stefan Kepinski

Centre for Plant Sciences, University of Leeds, UK.
I thought the meeting was a wonderful success. Firstly, the selection of
speakers and the quality of the presentations were excellent, making themeeting extremely satisfying scientifically and intellectually. The broad
theme of “Frontiers of plant science in the 21st century” worked very welland I imagine drew wide interest from across the diverse research
activity of NIBB. If you were to consider more focused meetings in the
future then I would suggest themes that still allow wide participation
across your institute and throughout Japan. Examples could be ‘Systems
biology and quantitative modeling’ or ‘Single-cell-level biology’ or ‘Next
generation sequencing’ because although they are focused on particularapproaches, they are useful in showing how these new approaches canboost the research capacity of a whole range of groups.
I thought that it was an excellent idea to let the younger students
organize the presentation and discussion session on the future of plantresearch and I very much enjoyed participating in the discussions. I also
enjoyed the fact that there was time to chat with other scientists both atcoffee breaks and at the poster sessions. The only thing you might follow
up on is that the younger participants felt there was enough time to‘pluck up the courage’ to approach the speakers to talk about their work.
At interesting meetings like this it can often be the case that the

speakers and senior scientists are so engaged withthe science and talking to their colleagues that it canbe difficult for the younger scientists to find the time
to talk to them.
With respect to enhancing opportunities for younger researchers tointeract with most established PIs and visiting speakers, the only
suggestion I can make is that you could consider setting up a lunch
where each PI dined with a small group of, say, 4-5 students to allow
them time to talk about life in science and research in general. As
alluded to above, it can often be daunting for early career scientists toapproach older colleagues and this might be a useful way of helping
them overcome any shyness in this regard. Having such an event earlyin the meeting would probably be most useful as it would allow them toget to know the visiting and senior scientists straight away and makethem feel more confident about continuing discussions throughout themeeting.
All in all it was a really very enjoyable and stimulating meeting. It wasvery well organized and ran very smoothly. I’d like to thank you and theorganizers again for both the invitation and your excellent hospitality
while we were with you in Okazaki.

Dr. Jose Manuel Perez-Perez

Universidad Miguel Hernandez, Spain.
The 55th NIBB Conference Arabidopsis Workshop 2008 entitled“Frontiers of Plant Science in the 21st Century” was held in Okazaki
during September 13th-15th and chaired by NIBB Director Prof. Okada
and others. The workshop consisted of 25 conferences (15 of whichwere from foreign researchers invited from outside Japan) arranged in 9
scientific sessions covering most plant topics. Fifty-two posters fromJapanese researchers were presented, with nine of them being selected
for oral discussions. The location of the posters (opposite the conferencehall) helped encourage discussion with the authors at any time and not
only during the poster sessions. About 130 participants, most of them in
the initial stages of their careers (PhD students and postdocs), attended
the meeting, which was nicely organized and properly scheduled at the
Okazaki conference centre (OCC). The meeting was the right size toallow for a fruitful flow of information and to encourage eventual
collaborations. I personally find it very useful for the young PhD students
to attend and participate in this kind of meeting because it provides themwith a good overview of the latest plant research and allows them plentyof time to discuss their favorite research topic with their colleagues. The
conference hall was very appropriate for the presentations, with very
comfortable chairs and good facilities. Lunch at the conference centre
was quite diverse and copious with enough refreshment time after eachscientific session. Furthermore, get-together dinners with our Japanese
hosts (PhD and postdocs in one case and Professors in the others) werevery enjoyable, not only for the food but also for the relaxed and friendly

80

environment. In this 55th NIBB edition, the students
organized a session about the future of plantresearch that began after short introductions by theinvited young researchers. Although the aim of this panel discussion
was to enhance participation from the young students in the audience, it
was mostly the senior PIs who participated. In my opinion, the topic list
chosen by the students was too broad. Maybe focusing only on a few
hot topics (the future of transgenic plants, climate change, etc.) andbuilding a real debate table around them would have helped to improveaudience participation. Although most of the talks I attended were notrelated to my main research topic (leaf development in Arabidopsis), allof the presentations were clear and concise with a definite time forenough questions too. Maybe it would help the students if they wereable to ask their questions in Japanese first, with an English translation
afterwards. I really liked that most of the speakers presented
unpublished or ongoing work from their laboratories, which gives a good
impression about the kind of science that’s being performed nowadays
in Japan. The travel and accommodation for the invited speakers washandled very efficiently, and I did not have to worry at all about flighttickets, etc., since everything was arranged in advance from Japan. This
meeting has been my first contact with Japanese culture and as far as I
can say the most interesting one. I am looking forward to the opportunity
to come back again in 2010!

NIBB-EMBL JOINT MEETINGS
The 6th NIBB-EMBL Meeting: “Evolution of Epigenetic Regulations”
March 17 (Mon)-19 (Wed), 2008

EMBL, Heidelberg

One of the most important questions in biology is how
gene activity is temporally and spatially controlled. It has
been several decades since we learned that not only DNA
sequences but also other aspects of genetic regulation such
as DNA/protein modifications influence chromatin
remodeling and gene expression and thus the developmentof a variety of organisms. Epigenetic regulation involving
methylation and acetylation of DNA or histone protein
appears to be evolutionary conserved among species.
However, it also seems that each organism evolves its own
mechanism of epigenetic regulation of gene activity as
shown by the fact that bacteria such as E. coli, yeast, thenematode, and the fruit fly lack DNA methylation. As
organizers, Jürg Müller (EMBL), Kunio Shiota (Univ.
Tokyo), and Shigeru Iida (NIBB) arranged this meeting inorder to exchange the latest information on transcriptional
regulation and remodeling of chromatin structure, regulationof cell differentiation, genomic imprinting, and so on. The
sessions enabled discussion about both epigenetic regulation
at different levels as well as the evolutional conservation of

the epigenetic mechanism. As a result, species-specificmechanisms were highlighted through a series ofpresentations with a variety of organisms from yeast, plants,
arthropods, to mammals. In addition, this meetingemphasized that genome-wide approaches to understanding
the epigenetic state of the whole genome is important. We
thank Drs. David Lane and Shigeru Iida for their stimulating
plenary lectures.

Speakers
AKHTAR, Asifa (EMBL) , ALLSHIRE, Robin (Welcome Trust Univ. Edinburgh), LADURNER, Andreas (EMBL),
MOSHER, Becky (The Sainsbury Laboratory), MUELLER, Juerg (EMBL), MUELLER, Christoph (EMBL),PASZKOWSKI, Jerzy (Univ. Geneva), REINBERG, Danny (HHMI at NYU School of Medicine), STANCHEVA, Irina (Univ. Edinburgh),
HAMADA, Kyoko (RIKEN CDB), HIROSE, Susumu (National Institute of Genetics), IIDA, Shigeru (NIBB), IMAMURA,
Takuya (Kyoto Univ.) , ISHINO, Fumitoshi (Tokyo Med. & Dent. Univ.), KAKUTANI, Tetsuji (National Institute of Genetics),
MATSUI, Yasuhisa (Tohoku Univ.), NAKAYAMA, Jun-ichi (RIKEN CDB), OHGANE, Jun (Univ. Tokyo),
SASAKI, Hiroyuki (National Institute of Genetics), SHINKAI, Yoichi (Kyoto Univ.), SHIOTA, Kunio (Univ. Tokyo),
TANAKA, Satoshi (Univ. Tokyo)

The 7th NIBB-EMBL Meeting: “Systems Biology and Functional Genomics”
April 18 (Fri)-19 (Sat), 2008

Center for Genomic Regulation, Barcelona

Center for Genomic Regulation (CRG) is located at theBarcelona Biomedical Research Park (PRBB), one of thelargest research clusters in southern Europe. This meeting
was held at the new CRG/PRBB building overlooking thebeautiful Catalonian coast. The main purpose of the meeting
was to bring Japanese and European scientists in the field
together to grasp the current status of systems biology andfunctional genomics, and to discuss the future direction of
the field. We also aimed to stimulate international
collaborations between scientists. The meeting included
excellent presentations on gene and protein networks,
biological systems controlling higher-order phenomena suchas complex morphogenesis of organs of animals and plants,
and gene regulatory networking of circadian rhythm. As aresult of the meeting we realized some key issues that need
to be addressed are the setting-up of experimental systems
by which data with minimum noise can be obtained,81

comprehensive collection of data sets with different
conditions or genetic backgrounds, a well-designed
presentation of data that allows the extraction of biologicalsignificance, and the necessity of mathematical modeling topredict the principles behind biological phenomena. There isan ever increasing amount of biological data being
accumulated by the achievements of large-scale biology

such as genomic, proteomics, and phenomics, and therefore
we certainly need sophisticated “Systems Biology” to makethe best use of them.
Organizers of this meeting were Luis Serrano(EMBL/CRG), Eileen Furlong (EMBL), and Atsushi
Mochizuki (NIBB).

Speakers
FURLONG, Eileen (EMBL), HUFNAGEL, Lars (EMBL), ISALAN, Mark (CRG), LEHNER, Ben (CRG), LEMAIRE, Patrick (IBDM),NEDELEC, Francois (EMBL), OLIVERI, Paola (UCL), PAPONOV, Ivan (Univ. Freiburg), RUSSELL, Rob (EMBL),SHARPE, James (CRG), STEINMETZ, Lars (EMBL), WITTBRODT, Jochen (EMBL)HORIKAWA, Kazuki (Hokkaido Univ.), ISHIHARA, Shuji (Univ. Tokyo), ITO, Takashi (Univ. Tokyo), KURODA, Shinya (Univ. Tokyo),
MIURA, Takashi (Kyoto Univ.), MOCHIZUKI, Atsushi (NIBB), SATOU, Yutaka (Kyoto Univ.), SHIGENOBU, Shuji (NIBB),
UEDA, Hiroki (RIKEN, CDB)

The 8th NIBB-EMBL Meeting: "Evolution: Genomes, Cell Types and Shapes"
November 21 (Fri)-23 (Sun), 2008

Okazaki Conference Center, Okazaki

This meeting was planned by three organizers, Detlev
Arendt (EMBL), Mitsuyasu Hasebe (NIBB) and Shigeru
Kurtani (RIKEN, CDB), to address current problems inevolutionary biology at different hierarchical levels ofanimal and plant life. The levels discussed included
genomes, gene regulatory networks at the micro-level andorganelles, cell types, organs, and morphology of species at
the macro-level. Among the many meetings in evolutionary
biology meetings, in which typically only a few limited
topics of similar levels are discussed, this meeting was
unique in that it overviewed the evolution of organismal lifeand brought the interconnectivity of the logic underlying
different layers of evolutional events into focus. The life andevolutionary strategies of a variety of organisms, fromsingle-celled organisms such as bacteria and yeast to higher
plants and vertebrate including mammals, were presented
and discussed not only with mechanistic insights but also
from the aspect of their impact on the history of organismal
life.

Four graduate students from EMBL were invited tocontribute to the meeting and presented their work. They
also had chances to meet with graduate students of NIBB
and exchange thoughts on their scientific life at each
institution.

Speakers
AKAM, Michael (Univ. Cambridge), ARENDT, Detlev (EMBL Heidelberg), BORK, Peer (EMBL Heid.),
BOWMAN, John (Monash Univ.), CHOURROUT, Daniel (Univ. Bergen), DESPLAN, Claude (New York Univ.),
FRIEDMAN, William (Univ. Colorado), FURLONG, Eileen (EMBL Heid.), HASELOFF, Jim (Univ. Cambridge),
HASTINGS, Nicola (EMBL Heid.), KNOP, Michael (EMBL Heid.), LIU, Ya-Hsin (EMBL Heid.), LOWE, Christopher (Univ. Chicago),
SPITZ, Francois (EMBL Heid.), STEINMETZ, Lars (EMBL Heid.), TECHNAU, Ulrich (Univ. Vienna), TOMER, Raju (EMBL Heid.),
TRACHANA, Kalliopi (EMBL Heid.)
AGATA, Kiyokazu (Kyoto Univ.), HASEBE, Mitsuyasu (NIBB), ISHINO, Fumitoshi (Tokyo Med. & Dental Univ.),
KURATANI, Shigeru (RIKEN CDB), NOJI, Sumihare (Univ. Tokushima), OKABE, Masataka (The Jikei Univ. School of Med.),OKADA, Norihiro (Tokyo Inst. Tech.), SATOH, Nori (Kyoto Univ.), TAMURA, Koji (Tohoku Univ.), TANAKA, Mikiko (Tokyo Inst. Tech.),
TSUKAYA, Hirokazu (Univ. Tokyo), UENO, Naoto (NIBB), YAMAMORI, Tetsuo (NIBB)

82

THE NIBB INTERNATIONAL PRACTICAL COURSE
The first NIBB International Practical Course was held in2007 with the aim of providing young scientists around theworld with opportunities to learn cutting-edge experimental
skills and to communicate with experts in the field. The
course replaced our Bioscience Training Course, which had
been held for twenty years and which had, for the most part,
only accepted participants from universities and institutes in

The Second International Practical
Course: "Developmental Genetics
of Zebrafish and Medaka II"■Period: March 3 (Sat)- 12 (Wed), 2008
■Participants: 12 (three from Hong Kong, two eachfrom China and Taiwan, one each from Australia,
Germany, Korea, Norway, and Japan)■Lecturers:
Dr. Shinichi Higashijima (National Institute forPhysiological Sciences)Dr. Kiyoshi Naruse (NIBB)
Dr. Kohei Hatta (University of Hyogo)
Dr. Shoji Fukamachi (University of Konstanz)■Research Results Presentations:Dr. Jochen Wittbrodt (University of Heidelberg)
Dr. Kataaki Okubo (NIBB)
Dr. Masahiko Hibi (RIKEN CDB)
Dr. Mikiko Tanaka (Tokyo Institute of Technology)
Dr. Koichi Kawakami (National Institute for
Genetics)
Contents of the course: basic technologies, including
gene knockdown by injecting anitisense morpholino
oligo, as well as a number of advanced techniquessuch as BAC homologous recombination mediatedtransgenesis, genomic/bioinformatic techniques, andcell tracking using photoconvertible fluorescentproteins

Japan. The Second and Third International Practical Courses
were held in 2008 as summarized below. The courses were
given in a laboratory provided by NIBB and equipped withall the necessary instruments and tools and a good supply ofexperimental materials. We hope that the techniques andknowledge provided in these courses will prove useful in the
future work of all of the participants.

The Third International Practical
Course: "2008 NIBB Laboratory
Course and Workshops on
Physcomitrella patens"■Period: June 30 (Mon)- July 4 (Fri), 2008
■ Participants: 11 (six from Japan, two from
America., and one each from Korea, Spain, andSweden)■Lecturers:
Dr. Andrew C. Cuming (University of Leeds)
Dr. Yasuko Kamisugi (University of Leeds)
Dr. Tetsuya Kurata (JST)Dr. Yoshikatsu Sato (JST)Dr. Yuji Hiwatashi (NIBB)
Dr. Minoru Kubo (JST)Dr. Takashi Murata (NIBB)
Dr. Mitsuyasu Hasebe (NIBB)
Contents of the course: basic techniques including
cultivation, observation of developmental processes atthe cellular level, gene targeting, bioimaging, andbioinformatics used in the biology of the moss
Physcomitrella patens

83

INDEXAHAYASHI, Tomoko
HAYASHI, Yoshiki

7, 19

AKANUMA, Takashi

22

HIGASHI, Sho-ichi

70, 76

AOKI, Yumiko

31

HIROKAWA, Junya

37

AOYAMA, Tsuyoshi

49

HIROSE, Mana

60

ARAI, Yuko

8

HIWATASHI, Yuji

49

ASANO,Takuya

22

HIYAMA, Takeshi

34

ASAO, Hisayo

43

HONDA, Satoko

B

19

HORIUCHI, Takashi

3, 46

HOSHINO, Atsushi

43

BABA, Takashi

30

BASKIN, Tobias

49

CHEN, Qiuhong

22

ICHIKAWA, Chiaki

70, 76CHOW, Pak Hong Jeremy

34

ICHIKAWA, Mariko

75, 76CHUNG, Hee-Jung

60

ICHIKAWA, Rie

11

CUI, Jianzhou

28

ICHIKAWA, Takehiko

68

CUI, Songkui

8

ICHIKAWA, Yoko

I

C

ICHIHASHI, Yasunori

E

IGARASHI, Hisako
EGUCHI, Goro

7

IGUCHI, Taisen

EMDE, Jason R.

69

IHARA, Masaru

EUN, Chang-Ho

43

IIDA, Shigeru

F

IINUMA, Hideko

58

73, 7633
3, 55, 73
34
3, 43, 75
76, 77

IKEGUCHI, Hiromi

49

IMAI, Akiko

37

11

IMAIZUMI, Taeko

55

7, 25

IMAMURA, Hisako

64

64

IMAMURA, Takuya

7

43, 76INADA, Yosuke

73

FUKAZAWA, Mitsue

8

ISHIHARA, Shuji64

FURUKAWA, Kazuhiko

76

ISHII, Junko

FURUYAMA, Makiko

37

ISHIKAWA, Azusa

70, 76ISHIKAWA, Mika

22

GONDA, Naoko

15

ISHIKAWA, Naoko

58

GOTO, Shino8

ISHIKAWA, Takaaki

49

GOTOH, Ayako

58

ISHIKAWA, Takako

37

GUAN, Guijun

28

ISHIMARU, Yoshiyasu

30

ISHINO, Fumitoshi

7

FUJII, Tomomi
FUJIKAWA, Akihiro

49
7, 34

FUJIKI, Yuki
FUJIMORI, Toshihiko
FUJITA, Hironori
FUKADA-TANAKA, Sachiko

G

H

11

HAMADA, Yoshio

15, 70ISHITANI, Shizuka

22

HANADA, Takao

11

ISOSHIMA, Yoshiko34

HARA, Ikuyo

28

ITAZU, Masako

46

HARA, Reiko

33

ITO, Takayo

76, 77

HARA, Yoko

11

ITO, Yukiko

49

HARA, Yusuke

16

IWASA, Yoh

3

11

IWASAKI, Akira

33

HASEBE, Mitsuyasu

3, 7, 49, 72, 77

IWATA, Ayumi

60

HASEGAWA, Mariko

3

HASEGAWA, Yoshinobu

43

JOHZUKA, Katsuki

46

HASHIMOTO, Yoshiko

19

JOHZUKA-HISATOMI, Yasuyo

43

HASHIYAMA, Kazuya

19

HATTORI, Nobuko

34

HARASHIMA, Toshiaki

JK
KABEYA, Yukiko

11, 76

KADOWAKI, Tamaka

58

8

KAGAWA, Takatoshi

60

HAYASHI, Makoto (Division of Developmental Genetics) 19

KAGEYAMA, Takuya

11

HAYASHI, Kohji

73, 76

HAYASHI, Makoto (Division of Cell Mechanisms)

84

30, 5553, 73

AJIOKA, Rie

KAGEYAMA, Yuji

19

MAEZAWA, Takanobu

KAJIKAWA, Ikumi

49

MAKINO, Yumiko

KAJIURA-KOBAYASHI, Hiroko

68, 76

MANO, Hiroaki

1975, 7649

KAKUTA, Soichiro

11

MANO, Shoji

8

KAMADA, Yoshiaki

11

MATSUDA, Chisato

43

KAMIGAKI, Akane

8

MATSUDA, Yoshimi

76, 77KAMIYA, Akemi

68

MATSUMOTO, Miwako

43

KAMIYA, Kiyomi

77

MATSUNAGA, Wataru

40

KANAI, Masatake

8

MATSUOKA, Makoto

3

KANDA, Rieko

68

MIKAMI, Yuriko

46

KANEKO, Hiroyo

53

MITSUI, Naoko

55

KATAOKA, Yukari

76

MIURA, Seiko

34

KATAYAMA, Tomomi

34

MIWA, Tomoki

70, 76

KATO, Kazuto

3

MIYABAYASHI, Kanako

30

KATO, Kyoko

8

MIYAKE, Satoko

16

KATO, Yasuhiko

55

MIYATA, Haruko

76

KATSU, Yoshinao

55

MIYAZAKI, Saori

49

KATSURA, Yukako

30

MIZOGUCHI, Masae

34

KAWAMURA, Motofumi

73

MIZUTANI, Takeshi

55, 76KIKUCHI, Naoka

55

MOCHIZUKI, Atsushi

7, 64, 72

KINOSHITA, Chie

31

MORI, Tomoko

KINOSHITA, Noriyuki

16

MORITA, Hitoshi

16

7, 19

75, 76

MORITA, Junko

37

KOBAYASHI, Kaoru

55

MORITA, Yasumasa

43

KOBAYASHI, Kayo

31

MORITOH, Satoru

43

KOBAYASHI, Satoru

3, 19

KOBAYASHI, Takafumi

11

MOROOKA, Naoki

46, 76

KODAMA, Akiko

34

MUKOHDA, Yasuyo

69

46

MURAKAMI, Michiyo

16

MURATA, Takashi

49

KITADATE, Yu

KODAMA, Ken-ichi

MOROHASHI, Ken-ichirou

30

KODAMA, Ryuji

52, 69KODAMA,Yutaka

60

KOIDE, Shizuyo

55

NAGAHAMA, Yoshitaka

28

KOIKE Chieko

53

NAGAKURA, Ayano

34

KOIKE, Yukari

53

NAGURA, Masako

58

KOJIMA, Yoko

49, 58NAKAGAMI, Yuki

37

N

KOMATSU, Kouji

25

NAKAMORI, Chihiro

8

KOMATSU, Tomoko

30

NAKAMURA, Makoto

7, 16

KOMATSU, Yusuke

37

NAKAMURA, Shuhei

KOMINE, Yuriko

37

NAKAMURA, Takanori

70, 76KONDO, Chika

11

NAKAMURA, Takeshi

55

KONDO, Maki

31

8, 76

NAKAMURA, Tohru

37

KONDO, Takefumi

19

NAKASHIMA, Machiko73

KONG, Sam-Geun

60

NAKATOGAWA, Hitoshi

11

KOUNO, Yukie

30

NAKAYAMA, Hokuto

58

KUBOKI, Yuko

8

NAKAYAMA, Kei

22

KUMATA, Yuji

19

NAKAZATO, Kenichi

64

KURATA, Tomoko

7, 69

NANBA, Chieko

KUSAKA, Masatomo

30

NARUSE, Kiyoshi

L

NIIMI, Kaori70, 76
53
11

NISHIDE, Hiroyo

70, 76NISHIHARA, Eri

34

LI, Jianzhong

28

MAEDA, Sanae69

NISHIMURA, Keiko

31

MAEDA, Yumi

76

NISHIMURA, Mikio

3, 8

M

85

INDEXNISHIMURA, Noriko76

SATOH, Noriyuki

3

NISHINA, Momoko

8

SAWADA, Kaoru

76, 77NISHIYAMA, Aya

30

SEKI, Touhaku

25

NODA, Chiyo

19, 76

SEKIHARA, Sanae

30

NODA, Masaharu

3, 34

SHEN, Xueyan

53

NOGUCHI, Yuji

73, 76SHIBATA, Emiko28

NONAKA, Shigenori

68

O

8

SHIBATA, Yasushi

28

OBARA, Keisuke

11

SHIGENOBU, Shuji

19

OGASAWARA, Kimi

8

SHIMA, Yuichi

30

OGAWA, Hidesato

7, 30

SHIMADA, Yu

28

OGAWA, Kazuo

14, 77SHIMAMOTO, Miki

43

28

SHIMATANI, Zenpei

43

OHNO, Kaoru
OH-OKA, Kyoko
OHSAWA, Sonoko
OHSHIMA, Issei

11

SHIMIZU, Hidetada

7, 34

37, 76SHIMIZU, Mineko

60

49

SHIMURA, Yoshiro

3

OHSUMI, Yoshinori

3, 11

SHINDO, Asako

16

OIKAWA, Kazusato

8

SHINTANI, Takafumi

34

OKA, Naomi

68

SINGH, Tanuja

8

OKA, Sanae

30, 75, 76

SUETSUGU, Noriyuki

60

OKADA, Kiyotaka

1, 3, 33

SUGIMOTO, Ryo

26
49, 76

OKAMOTO, Haruko

46

SUMIKAWA, Naomi

OKAMOTO, Koji

11

SUZUKI, Aya

OKAMOTO, Noriko

11

SUZUKI, Iku

OKANO, Yosuke

49

SUZUKI, Keiko

OKU, Masahide

11

SUZUKI, Kuninori

11

OKUBO, Kataaki

7, 28

SUZUKI, Makoto

7, 16

OKUBO, Tadashi

22

SUZUKI, Ryoko

34

OKUDA, Tadayoshi

73

SUZUKI, Tokiko

53

ONO, Akemi

43

SUZUKI, Yuko

11

OSUMI, Noriko

3

OTA, Kyoko

69

TABATA, Satoshi

3

OTA, Misaki

69

TAKADA, Ritsuko

22

P

28
8
70, 76T

TAKADA, Shinji

3, 22, 70
16, 76

PARK, Kyeung-Il

43

TAKAGI, Chiyo

PAUL, Bindhu

28

TAKAGI, Kyoko

43

TAKAGI, Yukari73, 76SSADAKANE, Osamu

37

TAKAHASHI, Hiroki

16

SAITO, Daisuke

31

TAKAHASHI, Hiroyuki

22

SAITOH, Hiromi

43

TAKAHASHI, Jun

2233, 76

TAKAHATA, Toru

37

SAKAI, Fumie

28

TAKAJI, Masafumi

37

SAKANO, Hitoshi

3

TAKAKI, Chikako

28

SAKURABA, Jyuichi

34

TAKAMATSU, Kaori

SAKURAI-OZATO, Nami

49

TAKAMI, Shigemi

75, 76SAKUTA, Hiraki

34

TAKASE, Masahide

58

SANJO, Kazuko

43

TAKASHIRO, Kayoko

22

SASADO, Takao

53

TAKEHANA, Yusuke

53

SASAKI, Tetsuya

37

TAKESHITA, Miyako

70, 7641, 73

TAKEUCHI, Yasushi

34, 76SAKAGAMI, Mari

SASAOKA, Toshikuni

86

SHIBATA, Michitaro

60

SATO, Asako

41

TAMESHIGE, Toshiaki

33

SATO, Kaori

19

TANAHASHI, Takako

7, 49

SATO, Yori

8

TANAKA, Megumi

69

YAMAMOTO, Hayashi

31, 73

TANAKA, Minoru

11

TANEMURA, Hiroyo

55

YAMAMOTO, Kumi

TANI, Sachiko34

YAMAMOTO, Takamasa

16

TANIGUCHI, Ena

55

YAMANAKA, Megumi

73

TAO, Hirotaka

16

YAMASATO, Akihiro

60

7, 33

YAMAUCHI, Daisuke

60

TERADA, Rie

43

YAMAUCHI, Takaki

43

TOITA, Shigeko

37

YANO, Satoshi

58

TOMIDA, Sanai

22, 30YASUDA, Mie

73

TATEMATSU, Kiyoshi

70, 76TOMINAGA, Rumi

33

YATAGAI, Hiroko

34

TOMITA, Nao

34

YATSU, Jun

19

TORII, Naoko

53

YONEHARA, Kayo34

TOYOKURA, Koichi

33

YONEHARA, Keisuke

34

TOYOOKA, Yayoi

7, 25

YONEMITSU, Masako

31

TSUBOI, Hidenori

60

YOSHIDA, Masahide

34

TSUCHIDA, Yuhei

33

YOSHIDA, Naoko

26

TSUGANE, Kazuo

43

YOSHIDA, Shosei

7, 26

TSUGANE-HAYASHI, Mika

43

YOSHIMURA, Yuriko

53

TSUGE, Toyoko

16

YOSHINORI, Yumi

8

TSUKAYA, Hirokazu

58

TSUZUKI, Chisa

22

ZHANG, Lanfan

34

TSUZUKI, Shihoko

76

ZHOU, Linyan

28

TSUZUKI, Yukiko

49

UCHIYAMA, Ikuo

66, 70Z

U
8UEDA, Chizuru
UENO, Naoto

3, 16, 69

UKAI, Sakie

22

UMEBAYASHI, Hiromi

64, 72

UNO, Satoko

76

URAWA, Hiroko

33

URUSHITANI, Hiroshi

55

USAMI, Takeshi (Division of Reproductive Biology)

28

USAMI, Takeshi (Division of Plant Developmental Genetics)

58

UTSUMI, Hideko

22, 76W
WADA, Masamitsu

60

WADA, Takuji

33

WASHIO, Midori

49

WATAKABE, Akiya

37

WATAKABE, Ikuko
WATANABE, Eiji
WATANABE, Hajime
WATANABE, KaoriWATANABE, Masakatsu

31
40, 7355
73
62, 70WATANABE, Taka-aki

46

YABE, Taijiro

22

Y
YAMADA, Kenji

8

YAMAGUCHI, Chinami

58

YAMAGUCHI, Katsushi

75, 76YAMAGUCHI, Takahiro

7, 58

YAMAMORI, Tetsuo

3, 37

87