CONTENTSINTRODUCTION ........................................................................................................................................................................... .
ORGANIZATION OF THE INSTITUTE ····························································································································

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MEMBERS OF THE COUNCIL
MEMBERS OF THE ADVISORY COMMITTEE FOR PROGRAMMING AND MANAGEMENT

4GRADUATE PROGRAMS······················································································································································-

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OFFICE OF DIRECTOR ···························································································································································-

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DEPARTMENT OF CELL BIOLOGY ································································································································

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DIVISION OF CELL MECHANISMS ·······································································································································DIVISION OF BIOENERGETICS ···············································································································································DIVISION OF CELL FUSION (ADJUNCT) ·····························································································································

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DEPARTMENT OF DEVELOPMENTAL BIOLOGY ································································································

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DIVISION OF REPRODUCTIVE BIOLOGY ··························································································································DIVISION OF CELL DIFFERENTIATION ·····························································································································DIVISION OF MORPHOGENESIS ············································································································································

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DEPARTMENT OF REGULATION BIOLOGY ············································································································

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DIVISION OF MOLECULAR NEUROBIOLOGY ··················································································································DIVISION OF CELLULAR REGULATION ·····························································································································DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY (ADJUNCT) ···············································DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT) ·················································································

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LABORATORY OF GENE EXPRESSION AND REGULATION········································································DIVISION OF GENE EXPRESSION AND REGULATION I ······························································································DIVISION OF GENE EXPRESSION AND REGULATION II ·····························································································DIVISION OF SPECIATION MECHANISMS I ·····················································································································DIVISION OF SPECIATION MECHANISMS II ·····················································································································RESEARCH SUPPORT ·································································································································································CENTER FOR TRANSGENIC ANIMALS AND PLANTS ···································································································RESEARCH CENTER FOR INTEGRATIVE AND COMPUTATIONAL BIOLOGY ···················································RESEARCH SUPPORT FACILITY ············································································································································THE CENTER FOR ANALYTICAL INSTRUMENTS (managed by NIBB) ····································································TECHNOLOGY DEPARTMENT ················································································································································CENTER FOR INTEGRATIVE BIOSCIENCE (jointly managed by NffiB)···················································

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATIONDIVISION OF DEVELOPMENTAL GENETICS ··················································································································DIVISION OF MOLECULAR & DEVELOPMENTAL BIOLOGY ··················································································
DEPARTMENT OF BIO-ENVIRONMENTAL SCIENCEDIVISION OF BIO-ENVIRONMENTAL SCIENCE ···········································································································DIVISION OF PLANT DEVELOPMENTAL GENETICS ··································································································DIVISION OF BIOINFORMATICS .......................................................................................................................:............... _CENTER FOR RADIOISOTOPE FACILITIES (CRF) ······························································································

The cover photograph shows the interaction of Arabidopsis dynamin-related
protein 3A (DRP3A), which is involved in both peroxisomal and mitochondrial
division, with mitochondria. Green and red signals represent DRP3A andmitochondria, respectively. See also page 9.

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INTRODUCTION
The National Institutefor Basic Biology(NIBB) is a governmentsupported
research
institute established in
1977.
In1988 ,
Department of Molecular
Biomechanics in Schoolof Life Science, theGraduate University for
Advanced Studies, wasestablished in the NIBB .
The aim of the NIBB is
the promotion and~ ~,stimulation of studies in
the field of Biology. As a
Center of Excellence (COE) , NIBB promotes not only
the basic biology but also the modern biologicalsciences by conducting first-rate research on site as wellas in cooperation with other universities and researchorganizations. Researchers at NIBB investigate cellstructure and function , reproduction and development,
neuronal and environmental biology, gene expre.ssion
and regulation , molecular evolution of eukaryoticorganisms and integrative and computational biology toelucidate the general and fundamental mechanisms
underlying various biological phenomena.
In 2003, there were many doctors who wereappointed as scientific staffs of NIBB. On April 1st,
Profs . NAGATA, Shigekazu (Osaka University) , .
OKADA, Norihiro (Tokyo Institute of Technology) ,and YANAGIDA, Mitsuhiro (Kyoto University) wereappointed as adjunct Professors for Division of Cell
Proliferation, Division of Cellular Communication, andDivision of Cell Fusion, respectively, in the Department
of Cell Biology. Prof. MORI, Yuji of The University ofTokyo was appointed as an adjunct Professor for
Division of Behavior and Neurobiology in the
Department of Regulation Biology on September 1st.
Dr. SASAOKA, Toshikuni was appointed as an
Associate Professor of Center for Transgenic Animals
and Plants from National Center of Neurology andPsychiatry on August 1st. Drs. TAKAHASHI,
Kazuhiko, OKUBO, Kataaki, HIWATASHI, Yuji, and
SUZUKI, Kuninori were appointed as Research
Associates of the NIBB in 2003.
In congratulation, Dr. HANAMURA, Kenji (NIBB
Research Fellow) was promoted to an AssistantProfessor of Gunma University School of Medicine.
Drs. T AMADA, Atsushi and SHIMONO, Akihiko, who
had been Research Associate in NIBB , were promoted
as Research fellow of RIKEN Brain Science Instituteand RIKEN Center for Developmental Biology,
respectively.
An adjunct Professor, Dr. MURAKAMI, Fujio, andadjunct Associate Professors, ITO Kei, KIYOSUE,
Tomohiro , and NAKAFUKU , Masato, left NIBB in

March 31st, 2003 just after the completion of their 5year appointments.
In addition , a number of postdoctoral fellows,
technical assistants , secretaries, and graduate students
after awarded PhD. degree left, and conversely people
newly joined NIBB at the equivalent positions. In
consequence, the total number of personnel working at
NIBB has been kept at approximately 300 for several
years.
As a COE of the biological research institute, NIBB
is responsible for conducting research projects incooperation with various research groups. As a part ofsuch cooperative activities , NIBB has hosted
International Conferences. The 49'h NIBB International
Conference entitled "Dynamic Vacuoles in Plants" washeld in November 25-27 (Profs. Y. OHSUMI and M.
NISHIMURA, organizers). This conference wassponsored by the Ministry of Education , Culture,
Sports, Science and Technology. Moreover, NIBB
continues to sponsor interdisciplinary symposia andstudy meetings on current topics by inviting leading
scientists from around the world to the Institute. Basedon this concept, NIBB will start from the upcoming
2004 to support a series of Okazaki Biology
Conferences (OBC) through intimate cooperation with avariety of biological scientific societies. NIBB also
provides a training course in biological sciences foryoung investigators.
NIBB together with the other members of Okazaki
National Research Institutes (ONRI), the Institute for
Molecular Science and the National Institute forPhysiological Sciences, has been constructing a new
campus to prepare the facility for Center for Integrative
Bioscience (CIB). Also , NIBB built Center forTransgenic Animals and Plants as a research support
facility in the new campus . Construction of the newcampus still continues, and it is programmed that two
divisions in NIBB and a laboratory in CIB will move
there in 2004.
Finally, I would like to celebrate retirement of Dr.
MOHRI, Hideo with appreciation of his deliberate andencouraging management as the President of ONRI.
Together with his contribution as the previous DirectorGeneral of NIBB, the institute respectfully awarded atitle for Professor Emeritus MOHRI on Aprillst, 2003.
We always welcome any suggestions concerning the
research activities of NIBB.
KATSUKI, Motoya, D.Sc.
Director-General

NATIONAL INSTITUTE

2

ORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology (NIBB) isan Institute in the Okazaki National Research Institutes(ONRI) that are composed of three independent
organizations, NIBB, the Institute for Molecular
Science (IMS) and the National Institute forPhysiological Sciences (NIPS). They located on a hill
overlooking the old town of Okazaki. NIBB wasestablished in 1977 and its activities are supported by
Monbukagaku-sho (the Ministry of Education, Culture,
Sports, Science and Technology: Mext) of Japan. The
Center for Integrative Bioscience that was established
as a common facility of the ONRI in 2000 and began in
2001.

Each division has its own research project and isstaffed by a professor, an associate professor and two
research associates in principle. A division forms an
independent project team. Six of the divisions areadjunct and headed by professors who hold joint
appointments with other universities. Adjunct divisions
have a resident research associate. This arrangement
facilitates exchange in research activities in Japan. The
Technical Department manages the activities ofresearch technicians and helps to promote research
activities of each division and to maintain the common
research resources of the Institute. The Department also
undertakes the technical education of staffs.
Several members of the Center for Integrative
Bioscience jointly work with the NIB B.

Policy and Decision Making
The Director-General oversees the operation of theInstitute assisted by two advisory bodies, the Council
and the Advisory Committee for Programming andManagement. The Council, comprised of distinguishedscholars representing various fields of science andculture, advises the Director-General on principles andpolicies governing the activities and operations of
NIBB. The Advisory Committee, comprised ofprofessors within the Institute and an equal number ofleading biologists outside NIBB advises the DirectorGeneral, upon his request, on planning joint research
programs and other important matters in NIBB, as wellas on the scientific activities of the Institute. The
Council makes a nomination of Director-General andCommittee also makes recommendations on the
Director-General and on faculty appointments, theInstitute's annual budget and future prospects.

Research Support Facilities
The research support facilities of the NIBB consist of
the Large Spectrograph Laboratory, the Tissue and Cell
Culture Laboratory, the Computer Laboratory, the Plant
Culture Laboratory, the Plant Cell Culture Laboratory,
the Experimental Farm, the Laboratory of StressResistant Plants and the Center for Transgenic Animals
and Plants. In addition, five facilities are operated
jointly with NIPS; they consist of the Electron
Microscope Center, the Center for Analytical
Instruments, the Machine Shop, the Laboratory
Glassware Facilities and the Low-Temperature
Facilities. The Radioisotope Facilities, the Computer
Center and the Animal Care Facilities became common
facilities of ONRI.

CampusAdministration
Administration of the Institute is undertaken by the
Administration Bureau of the ONRI under the direct
auspices of the Ministry of Education, Culture, Sports,
Science and Technology.

Research
The Institute conducts its research programs throughthree departments and one laboratory subdivided into
17 .divisions and the Center for Integrative andComputational Biology.

The ONRI covers an area of 164,783m2 with four
principal buildings. The NIBB's main research building
has a floor space of 16,789m2• Two-thirds of the space
was completed in 1982 and the remaining third in June,
1983. The buildings that have the research support
facilities were also completed in 1983. A building for
the Laboratory of Gene Expression and Regulation(2,577m2) was built in 1996. A building for Center forTransgenic Animals and Plants (2,500m2) was dedicatedin the new campus at November, 2003.

3

FOR BASIC BIOLOGY

Okazaki National Research Institutes!National Institute for Basic Biology

I

rlDivision of Cell Mechanisms
HDivision of Bioenergetics

rl Department of Cell Biology f- HDivision of Cell Proliferation (adjunct)HDivision of Cell Fusion (adjunct)
Y Division of Cellular Communication (adjunct)rlDivision of Reproductive Biologyf---HDivision of Cell Differentiation
Department of Developmental
r-Biology---iDivision of Morphogenesis

I-lDivision of Developmental Biology (adjunct)rlDivision of Molecular Neurobiology-

Department of Regulation
Biology

H Division of Celluler Regulation
r-r-IDivision of Biological Regulation (adjunct)
Y Division of Behavior and Neurobiology (adjunct)-lDivision of Gene Expression and Regulation I-

Laboratory of Gene
Expression and Regulationr-----lDivision of Gene Expression and Regulation II---lDivision of Speciation Mechanisms I- lDivision of Speciation Mechanisms II

Research Support

I
jNational Institute for Physiological Sciences I
jlnstitute for Molecular SciencejCenter for Transgenic Animals and Plantsr-Research Center for Integrative andComputational Biology

J-WResearch Support Facility
H Center for Analytical Instruments- lTechnology Department

Research Facilities
Center for Integrative Bioscience
Center for Radioisotope Facilities
Center for Experimental Animal
Research Center for Computational Science
Administration Bureau

I

NATIONAL INSTITUTE

4

MEMBERS OF THE COUNCILANZAI, Yuichiro
IKEHATA, Setsuho
IWATSUKI, Kunio
OSAKI, Hitoshi
OGAWA, Tomoko
KAIFU, Norio
KUROKI, Toshio
GO,Mitiko(present)
SASAZUKI, Takehiko
SEKIGUCHI, Mutsuo
SUZUKI, Akinori
TAKEICHI, Masatoshi
NAKAMURA, KeikoNISHIDA, Atsuhiro
HIDAKA, Toshitaka
HOSHI, Motonori
HOTTA, Yoshiki
YOSHIKAWA, Hiroshi
YOSHIDA, Mitsuaki
WATANABE, Okitsugu

President, Keio University
President, Tokyo University of Foreign Studies
Professor, The University of the Air
Director-General, Center for National University FinanceVice-President, Iwate College of Nursing
Director-General, National Astronomical Observatory of Japan
President, Gifu University
Professor, Nagoya University
Dean & Professor, Nagahama Institute of Bio-Science andTechnology
Director-General, Research Institute, International Medical Centerof Japan
Director & Trustee, Biomolecular Engineering Research Institute
President, Akita Prefectural University
Director, RIKEN Center for Developmental Biology
Director-General, JT Biohistory Research Hall
Professor Emeritus, Institute of Space and Aeronautical Science
Director-General, Research Institute for Humanity and Nature
Professor, Keio University
Director-General, National Institute of Genetics
Advisor, JT Biohistory Research Hall
Director, Banyu Pharmaceutical Co., Ltd. Tsukuba ResearchInstitute
Director-General, National Institute of Polar Research

MEMBERS OF THE ADVISORY COMMITTEE FOR
PROGRAMMING AND MANAGEMENTAIZAW A, Shinichi
OKADA, Kiyotaka
KUROSAWA, Yoshikazu
KOMEDA, Yoshibumi
KONDO, Hisato
SAGA, Yumiko
SEHARA, Atsuko
HASUNUMA, Kohji
MACHIDA, Yasunori
MURAKAMI, Fujio
IIDA, Shigeru
UENO,NaotoOHSUMI, Yoshinori
KOBAYASHI, Satoru
NAGAHAMA, Yoshitaka
NISHIMURA, Mikio
NODA, Masaharu
HORIUCHI, Takashi
MURATA, Norio
MOROHASHI, Ken-ichirou
YAMAMORI, Tetsuo

Deputy Director & Group Director, RIKEN Center for
Developmental Biology
Professor, Kyoto University
Professor, Fujita Health University
Professor, The University of Tokyo
Professor, Osaka University
Professor, National Institute of Genetics
Professor, Kyoto University
Professor, Yokohama City University
Professor, Nagoya University
Professor, Osaka University
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Research Institutes, Center for
Integrative Bioscience
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology

5

FOR BASIC BIOLOGYGRADUATE PROGRAMSThe NIBB sponsors two graduate programs.
1. Graduate University for Advanced Studies
NIBB constitutes the Department of Molecular
Biomechanics in the School of Life Science of the
Graduate University for Advanced Studies. The
University provides a three year Ph.D. course. Those
who have completed a master's course or equivalent at
any university are eligible to apply.
The Department consists of the following Divisions
and Fields:

DIVISIONSFIELDS

Molecular Cell
Biology

Biomolecular Systems
Cell Dynamics

Developmental
Gene Expression
and Regulation

Gene Expression
Morphogenesis
Transgenic Biology

Regulation
Biology

Biological Regulation
Biological Information

2. Graduate Student Training Program
Graduate students enrolled in other Universities andInstitutions are eligible to conduct research for fixed
periods of time under the supervision of NIBB
professors.

6

NATIONAL INSTITUTE

OFFICE OF DIRECTOR
Director-General:
Associate Professor:
Research Associates:

KATSUKI, Motoya
KODAMA, Ryuji
UENO, Kohji
OHNO, Kaoru

Mechanisms working in the morphogenesis of thelepidopteran wings
KODAMA, Ryuji
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 to
study cellular interactions in the course ofmorphogenesis.
The outline shape of the adult wing is often different
from that 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 iscalled the degeneration region and the internal area,
which develop into the adult wing, is called thedifferentiation region.
The cell deaths in the degeneration region proceeds
very rapidly and completes in a half to one day period
in Pieris rapae or several other species examined. It
was shown that the dying cells in the degeneration region have characteristics common with the apop-totic
cell death in mammalian cells. The cells in thedegeneration 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
basal surfaces of the dorsal and ventral epithelium inthe differentiation region.
A collaborative work with the laboratory of Dr. K.
Watanabe (Hiroshima University) concerns mostly on
the development of trachea and tracheole pattern in theswallow tail butterflies. Trachea and tracheoles areboth important in delivering air into the wing and theirpattern coincide with that of the boundary ofdegeneration and differentiation zones at the distal endof the wing. According to the observations, the patternformation of wing epithelium is often dependent on
tracheal and tracheole patterns. Basic research on thedevelopment of tracheal pattern formation is being doneby the scanning electron microscopy and the bright fieldlight microscopy of the fixed or fresh specimens to
describe the exact pathway and the time course of theformation of elaborate pattern of trachea and tracheoles
and to establish the cytological and developmental
relationship between the formation of tracheal patternand epithelial cell pattern, such as scale cell pattern.
In collaboration with the Division of Molecular
Neurobiology, the localization of a sodium channel
protein is being investigated using immunohistologicalstaining. DAB stained Vibratome section was further
fixed and embedded in epoxy resin and then sectioned

with ultramicrotome and observed under a transmission
electron microscopy. Electron microscopic observation
was utilized in order to identify the stained cells andsurrounding cells by their ultrastructural characteristics.
Scanning electron microscopic imaging of a mutant
mouse phenotype is also being done according to arequest of another laboratory.

Control of the distribution of palmitoylated
proteins in neuronal growth cones
Kohji Ueno
Signalling proteins such as G proteins and G proteincoupled receptors are modified with palmitate via
thioester linkages. Protein palmitoylation is thought tobe important in the regulation of signal transduction.
We have. previously found that protein palmitoylase isexpressed in neural cells during mouse embryogenesis.
In developing neurons, growth associated protein(GAP)-43 and Go, which are palmitoylated proteins, aremainly concentrated in the growth cones. Addition of
an inhibitor of protein palmitoylase to the medium ofcultured primary neuronal cells reduces the axonal
growth of neurons. From these findings, we speculated
that the localization of the palmitoylated proteins ingrowth cones is critical for the development of axons.
In this study, we are attempting to elucidate themechanism that determines the localization of thepalmitoylated proteins in growth cones. For
thisanalysis, we have established a method tochemically modify a biotinylated peptide composed ofresidues 1-15 of the GAP-43 N-terminal with fatty acids
via thioester linkages. Cys 3 and Cys 4 of GAP-43 aremodified with palmitate in developing neurons. By
using the method, we have prepared the peptides which
are modified with myristate (C14:0), palmitate (Cl6:0),
palmitoleate (C16:1), stearate (C18:0) and arachidate(C20:0) via thioester linkages. The method could
modify the peptide with not only saturated but also
unsaturated fatty acids.
With the acylated peptides, we have assayed thebinding activity of ERM (Ezrin/Radixin/Moesin)proteins, which are thought to be general cross-linkers
between plasma membranes and actin filaments,
because ERM proteins are reported to be concentrated
in growth cones and ERM proteins contain a domain
which has a similar structure to a fatty acid thioesters
binding protein. Recombinant full-length ERM
proteins, which were expressed in bacteria, had thebinding activity with the palmitoylated peptides,
whereas the binding activity were less with thesynthetic peptides modified with myristate,palmitoleate, stearate and arachidate. These result
suggested that ERM proteins have a potentiality torecognize the fatty acid residue of the acylated peptides.
Recombinant erythrocyte membrane protein band 4.1,
which contains a similar structure to ERM proteins, had
low binding activity with the palmitoylated peptide.
From these results we suggested that a domain of ERM
proteins is responsible for the interaction with thepalmitoylated peptide.

FOR BASIC BIOLOGY7DEPARTMENT OF CELL BIOLOGY

Chairperson: UNO, Naoto

DIVISION OF CELL MECHANISMSDIVISION OF BIOENERGETICSDIVISION OF CELL PROLIFERATION (ADJUNCT)DIVISION OF CELL FUSION (ADJUNCT)DIVISION OF CELLULAR COMMUNICATION (ADJUNCT)The department consists of two regular divisions and three adjunctdivisions. The department conducts studies on molecular dynamics ofthe cell in eukaryotes such as organelle differentiation, autophagy, cell
motility, cytokinesis and neural development.

NATIONAL INSTITUTE8DIVISION OF CELL MECHANISMSNISHIMURA, Mikio
HAYASHI, Makoto
MANO, Shoji
YAMADA, Kenji
Technical Staff:
KONDO, Maki
NIBB Research Fellow: SHIRAHAMA, Kanae (~April30)
JSPS Postdoctoral Fellows: N!TO, Kazumasa
KUROYANAG!, Miwa (April I~)
ARAI, Yuko (April I~)
Graduate Students:
FUKAO, Youichiro (~March 31)
KAMADA, Tomoe
HATSUGAI, Noriyuki
Technical Assistants:
NAKAMORI, Chihiro
YAGI, Mina
YOSHINORI, Yumi
SUZUKI, Iku (April I~)
Secretaries:
VEDA, Chizuru
IYODA, Yuri
Professor:
Associate Professor:
Research Associates:

Higher plant cells contain several distinct organelles
that play vital roles in cellular physiology. During
proliferation and differentiation of the cells, theorganelles often undergo dynamic changes . The
biogenesis of new organelles may occur, existingorganelles may undergo a transformation of function,
while other organelles may degenerate. Because thedynamic transformation of organellar function(differentiation of organelles) is responsible forflexibility of differentiation events in higher plant cells,
the elucidation of regulatory mechanisms underlying
organelle transformation are currently studied in thisdivision.

translational transport into peroxisomes. Almost all of
the peroxisomal matrix proteins are known to containone of two targeting signals (PTS 1 and PTS2) within
the molecules. PTS 1 is a unique tripeptide sequence
found in carboxyl terminus of the mature proteins. The
permissible combinations of amino acids for PTS 1 inplant cells are [C/A/S/P]-[K/R]-[1/L/M] . In contrast,
PTS2 is involved in a cleavable amino terminal
presequence of peroxisomal proteins that aresynthesized as precursor protein with larger molecularmass . PTS2 consists of a consensus sequence [R][L/Q/I]-X5-[H]-[L].
We identified 256 gene candidates of PTSl- andPTS2-containing proteins and other 30 genes of nonPTS-containing proteins from Arabidopsis genome.
Custom-made DNA microarray covering all these genes
was used to investigate expression profiles of theperoxisomal genes in various organs. Statisticalanalyses revealed that the peroxisomal genes could be
divided into five groups in terms of their transcription.
One group showed ubiquitous expression in all organs
examined, while the other four were classified as
showing organ-specific expression in seedlings ,cotyledons, roots and in both cotyledons and leaves.
These data proposed more detailed description ofdifferentiation of plant peroxisomes (Fig. 1).

I. Reversible transformation of plant peroxisomes
Dramatic metabolic changes which underlie the shift
from heterotrophic to autotrophic growth occur ingreening of seed germination. Accompanying thesemetabolic changes, many constitutive organelles arefunctionally transformed. Etioplasts differentiate into
chloroplasts and mitochondria acquire the ability tooxidize glycine. Glyoxysomes which are microbodies
engaged in the degradation of reserve oil via f3 oxidation and the glyoxylate cycle, are transformed intoleaf peroxisomes that function in several crucial steps ofphotorespiration. After the functional transition ofglyoxysomes to leaf peroxisomes during the greening of
pumpkin cotyledons, the reverse transition of leaf
peroxisomes to glyoxysomes occurs during senescence.
The functional transformation between glyoxysomes
and leaf peroxisomes is controlled by gene expression,
alternative splicing, protein translocation and protein
deg-radation. We now engage in proteomic andtranscriptomic analyses of the reversible peroxisomaltransition in Arabidopsis cotyledons.

II. Transcriptomics and proteomics of plant
peroxisomes
Enzymes localized in pl a nt peroxisomes aresynthesized in the cytosol, and function after their post-

Novel classification of olant peroxisomes

Fig. 1 Novel nomenclature of plant peroxi some sidentified by peroxisomal gene-specific transcriptomics.

In parallel, we made two-dimensional protein map ofglyoxysomes and leaf peroxisomes isolated from
Arabidopsis. Peptide MS fingerprinting analyses
allowed us to identify novel proteins exists in either
glyoxysomes or leaf peroxisomes. Some of these
proteins contain no obvious PTS 1 and PTS2. Of these,
we characterized GPKl as a novel protein kinase inglyoxysomes.

III. Involvement of the same dynamin molecule inperoxisomal and mitochondrial division inhigher plants
To better understand peroxisome biogenesis, wemutagenized seeds of transgenic Arabidopsis , GFPPTS 1, in which peroxisomes with normal si ze andnumber can be visualized with GFP, and screened a

FOR BASIC BIOLOGY

number of Arabidopsis mutants with aberrant
12eroxisome morphology (apm mutants) based on the
different pattern of GFP. The apm mutants wereclassified into four classes. These were mutants with (1)long peroxisomes, (2) giant peroxisomes , (3) GFP
fluorescence in the cytosol as well as in peroxisomes,and (4) other distributions of GFP.

Fig. 2 Phenotype of apml mutant and subcellular
localization of APM1 /DRP3A protein.
(A) and (B) show GFP-labelled peroxisomes in GFPPTS I as a parent plant (A) and apml mutant (B). (C) and(D) represent electron microscopic observation of leaf
cells in GFP-PTS 1 (C) and apml mutants (D). Mt:
mitochondrion, Ch: chloroplast. (E) and (F) show the colocalization of DRP3A protein with peroxisomes (E) andmitochondria (F). Spherical spots in green indicate
DRP3A proteins. Red signals show peroxisomes (E) andmitochondria (F), respectively. Arrow heads represent thesites of interaction with DRP3A proteins. Each bar
indicates 50 Jtm for (A) and (B), 1 Jtm for (C) and (D),and 10 Jtm for (E) and (F).
In one of these mutants, apm1 , the peroxisomes arelong and reduced in number, apparently as a result of
inhibition of division (Fig. 2). APM1 gene encodes
DRP3A (D.ynamin related 12rotein 3A). Interestingly,
mutations in APM l/DRP3A also caused aberrant
morphology of mitochondria. The growth of
Arabidopsis, which requires the cooperation of variousorganelles including peroxisomes and mitochondria, isrepressed in apm1 , indicating that the changes ofmorphology of peroxisomes and mitochondria reduce
the efficiency of metabolism in these organelles. Thesefindings indicate that the same dynamin molecule isinvolved in peroxisomal and mitochondrial division inhigher plants.

IV. ER derived organelles for transport ofproteins to vacuoles.
Precursor-accumulating (PAC) vesicle, mediates thetransport of storage protein precursors fromendoplasmic reticulum (ER) to protein storage vacuoles

9in maturing pumpkin seeds. PAC vesicles had diametersof 300 to 400 nm, are derived from ER and contained
an electron-dense core of storage proteins. PV72, a type
I integral membrane protein was found on themembrane of the PAC vesicles. PV72 has been shownto bind to the precursor of pumpkin 2S albumin ,implying that PV72 functions as a vacuolar sorting
receptor (VSR) for storage proteins in pumpkin seeds.
Arabidopsis has seven homologue of PV72. The
homologue closely related to PV72 was designated
AtVSRl. We used a reverse-genetic approach to
explore the function of AtVSRI. Two T-DNA insertion
mutants (atvsr 1-1 and atvsr 1-2) missort storage proteinsby secreting them from cells , and abnormally
accumulate the precursors of storage proteins, together
with the mature forms of these proteins in the seeds.
These finding demonstrate a receptor-mediatedtransport of seed storage proteins to protein storage
vacuoles in higher plants.
ER bodies are another ER-derived compartment
specific to the Brassicaceae, including Arabidopsis. ER
bodies are rod-shaped structures (5 f/.111 long and 0.5 ftmwide) that is sunounded by ribosomes. ER bodies canbe visualized in transgenic plants of Arabidopsis (GFPh) expressing green fluorescent protein fused with an
ER retention signal (GFP-HDEL). ER bodies were
widely distributed in the epidermal cells of wholeseedlings. In contrast, rosette leaves had no ER bodies.
nail is an Arabidopsis mutant in which ER bodies werehardly detected in whole plants. Analysis of the nailmutant reveales that a ~-glucosidase with an ERretention signal (KDEL) , called PYKlO, is the main
component of ER bodies. The putative biological
function of PYKIO and the inducibility of ER bodies inrosette leaves by wound stress suggest that the ER bodyfunctions in the defense against herbivores.

V. Maturation of vacuolar/lysosomal proteins byvacuolar processing enzyme in animal andplant cells.
Vacuolar processing enzyme (VPE) belongs to thecysteine protease family Cl3. This family is found invarious eukaryote organisms including higher plants
and animals. VPE was originally identified as an
enzyme responsible for the processing and maturationof seed storage proteins in plants. VPE exhibit substratespecificity toward an asparagine residue, the amino acidwell conserved at the PI position in the processing sitesof various vacuolar/lysosomal proteins. Plant VPE
homologues were separated to two subfamilies: one
seed type and the other vegetative type. We haveidentified one seed type (f3VPE) and two vegetative
type (aVPE and yVPE) VPE genes from Arabidopsis.
We isolated six Arabidopsis mutants that accumulate
detectable amounts of the precursors of the storage
proteins. All mutants had a defect in the f3VPE gerle,indicating that f3VPE is involved in the processing ofstorage proteins in vivo. We further generated various
mutants lacking different VPE isoforms: aVPE, ~VPE
and/or yVPE. avpe-l!f3vpe-3/ yvpe-l triple mutant seeds

10
accumulate no properly processed mature storage
proteins. Instead, large amounts of storage protein
precursors are found in the seeds of this mutant. In
contrast to {3vpe-3 seeds, which accumulate bothprecursors and mature storage proteins, the other single(avpe-1 and yvpe-1) and double (avpe-1/yvpe-1)
mutants accumulate no precursors in their seeds at all.
Therefore, the vegetative type VPEs, aVPE and yVPE,
compensates for the deficiency in ~VPE in {3vpe mutant
seeds.
To explore the physiological function of VPE in
mammals, we generated and characterized VPEdeficient mice. VPE was abundantly expressed in
kidney and localized in the late endosomes of theproximal tuble cells. Disruption of the VPE gene led tothe enlargement of lysosomes in these cells in an agedependent manner, which suggests that the materials tobe degraded are being accumulated within thelysosomal compartments. The processing of thelysosomal proteases, cathepsins B, H, and L, from thesingle-chain forms into the two-chain forms wascompletely defected in the deficient mice. Thus, theVPE deficiency caused the accumulation ofmacromolecules in the lysosomes, highlighting apivotal role of VPE in the endosomealllysosomal
degradation system.

VI. Role of molecular chaperones on celldifferentiation.
Molecular chaperones are cellular proteins thatfunction in the folding and assembly of certain otherpolypeptides into oligomeric structures but that are not,themselves, components of the final oligomeric
structure. To clarify the roles of molecular chaperones
on cell differentiation, we have purified andcharacterized chaperonin and Hsp70s and analyzed theirroles in the translocation of proteins into chloroplasts.
Previously, we also characterized a mitochondrial cochaperonin (Cpn 10), chloroplast co-chaperonins(Cpn20 and CpnlO) and a small heat shock protein from
Arabidopsis. In 2003, we started to characterize
HSP90s. Their evolutional and functional
characterization is now under experiments.

Publication List:
Fukao, Y., Hayashi, M., Hara-Nishimura, I. andNishimura, M. (2003) Novel glyoxysomal protein
kinase, GPK 1, identified by proteomic analysis ofglyoxysomes in etiolated cotyledons of Arabidopsis
thaliana. Plant Cell Physiol. 44, 1002-1012.
Hayashi, M. and Nishimura, M. (2003) Entering a new eraof research on plant peroxisomes. Cur, Opi. Plant Sci. 6,
577-582.
K!)mada, T., Nito, K., Hayashi, H., Mano, S., Hayashi, M.
and Nishimura, M. (2003) Functional differentiation ofperoxisomes revealed by expression profiles ofperoxisomal genes in Arabidopsis thaliana. Plant Cell
Physiol. in press
Karnigaki, A., Mano, S., Terauchi, K., Nishi, Y., Tachibe-

NATIONAL INSTITUTE

Kinoshita, Y., Kondo, M., Nito, K., Hayashi, M.,
Nishimura, M. and Esaka, M. (2003) Identification ofperoxisomal targeting signal of pumpkin catalase andthe binding analysis with PTS 1 receptor. Plant J. 33,
161-175.
Kurisu, M., Morita, M., Kashiwayama, Y., Yokota, S.,
Hayashi, H., Sakai, Y., Ohkuma, S., Nishimura, M. andImanaka, T. (2003) Existence of catalase-lessperoxisomes in Sf21 insect cells. Biochern. Biophys.
Res. Cornrnun. 306, 169-176.
Matsushima, R., Hayashi, Y., Yamada, K., Shimada, T.,
Nishimura, M. and Hara-Nishimura, I. (2003) The ER
body, a novel endoplasmic reticulum-derived structure
in Arabidopsis. Plant Cell Physiol. 47, 661-666.
Matsushima, R., Kondo, M., Nishimura, M. and HaraNishimura, I. (2003) A novel ER-derived compartment,
the ER body, selectively accumulates a ~-glucosidasewith an ER retention signal in Arabidopsis. Plant J. 33,
493-502.
Okamato, T., Shimada, T., Hara-Nishimura, I., Nishimura,
M. and Minamikawa, T. (2003) C-terminal KDEL
sequence of a KDEL-tailed cysteine protease(sulfhydryl-endopeptidase) is involved in formation of
KEDL vesicle and in efficient vacuolar transport ofsulfhydryl-endopeptidase. Plant Physiol. 132, 18921900.
Ono, K., Kondo, M., Osafune, T., Miyatake, K., Inui, H.,
Kitaoka, S., Nishimura, M. and Nakano, Y. (2003)
Presence of glyoxylate cycle enzymes in the
mitochondria of Euglena gracilis. J. Eukaryo.
Microbial. 50, 92-96.
Shimada, T., Fuji, K., Kondo, M., Nishimura, M. andHara-Nishimura, I. (2003) A vacuolar sorting receptor
for seed storage proteins in Arabidopsis thaliana. Proc.
Natl. Acad. Sci. USA 100, 16095-16100.
Shimada, T., Yamada, K., Kataoka, M., Nakaune, S.,
Koumoto, Y., Kuroyanagi, M., Tabata, S., Kato, T.,
Shinozaki, K., Seki, M., Kobayashi, M., Kondo, M.,
Nishimura, M. and Hara-Nishimura, I. (2003) Vacuolar
processing enzymes are required for proper processingof seed storage proteins in Arabidopsis thaliana. J. Bioi.
Chern. 278, 32292-32299.
Shirahama-Noda, K., Yamamoto, A., Sugihara, K.,
Hashimoto, N., Asano, M., Nishimura, M. and HaraNishimura, I. (2003) Biosynthetic processing ofcathepsins and lysosomal degradation in asparaginyl
endopeptidase-deficient mice. J. Bioi. Chern. 278,
33194-33199
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani,A., Higashi, S., Watanabe, M., Nishimura, M. and HaraNishimura I. (2003) Why green fluorescent fusion
proteins have not been observed in the vacuoles ofhigher plant? Plant J. 35, 545-555.
Watanabe, E., Shimada, T., Tamura, K., Matsushima, R.,
Koumoto, Y., Nishimura, M. and Hara-Nishimura, I.
(2003) An ER-localized form of PV72, a seed-specificvacuolar sorting receptor, interferes the transport of an
NPIR-containing proteinase in Arabidopsis leaves. Plant
Cell Physiol. in press
Yamada, K., Nishimura, M. and Hara-Nishimura, I. (2003)
The slow wound-response of yVPE is regulated byendogenous salicylic acid in Arabidopsis. Planta inpress
Yamamoto, Y., Nishimura, M., Hara-Nishimura, I. andNoguchi, T. (2003) Behavior of vacuoles during pollen
development and maturation in Arabidopsis thaliana.
Plant Cell Physiol. 44, 1192-1201.

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS

Professor:
Research Associates:

OHSUMI, Yoshinori
KAMADA, Yoshiaki
NODA, Takeshi 11
MIZUSHIMA, Noboru
SUZUKI, Kuninori
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellows: YOSHIMOTO, Kohki
FUJIK/, Yuki
JSPS Postdoctoral Fellows: SEKITO, Takayuki
HANADA, Takao
HAMASAKI, Maho
Postdoctoral Fellows:
ICHIMURA, Yoshinobu
KUMA, Akiko (~May 2003)
ICHIKAWA, Hisashi
KANAZAWA, Takumi
BABA, Misuzu
ONODERA, Jun 2)
Graduate Students:
KAWAMATA, Tomoko 3!
MATSUI, Makoto 2 )Technical Assistants:
MIWA, Mas ami
IMAMURA Yuko
TAKEUCHI, Yuka
TSUKESHIBA Kumi
HARA, Yoko
Secretary:
Study abroad from Mar. 2003
Graduate University for Advanced Studies
J) KOBE Univ.
/J

2!

This division aims to understand the autophagy inrespects to its molecular mechanism and its
physiological role in yeast and higher eukaryotes. Cells
execute degradation processes of their constituents
together with biosynthetic processes. These twoprocesses are well coordinated to regulate the biologicalactivities. In other word, we must shed light ondegradation process to fully understand the cell,because the study on the degradation has been retarded
compared to the biosynthetic process. Autophagy is amajor route for bulk degradation of cytoplasmic
constituents and organelles in lysosome/vacuole, and iswell conserved in eukaryotes.

I. Background
Upon nutrient starvation, autophagic process starts asbuilding up a membrane structure, an autophagosome,in the cytoplasm. The autophagosome sequesters
cytosol and organelles nonselectively. Then it .i'sdelivered to the vacuole/lysosome, and the cytoplasmic
materials inside are degraded by vacuolar/lysosomalhydrolases. We had discovered autophagy in a simple
model organism, S. cerevisiae and morphologically andgenetically defined the whole process.

U. In vitro reconstitution of Atg8-PE conjugation
systemIn yeast, S. cerevisiae, Atg8 plays an important role
during autophagosome formation. We have previously
reported that the Atg8 is covalently attached tophosphatidylethanolamine (PE) via a ubiquitin-likeconjugation system. The C-terminal Arg of newly

11

synthesized Atg8 (Atg8R" 7 ) is removed by Atg4
protease to expose a Gly residue at the C-terminus(Atg8° 116). The Apg8°" 6 is then activated by Atg7 (Elenzyme) and transferred to Atg3 (E2 enzyme).
Following these reactions, the Apg8° 116 conjugates to PE
through an amide bond between its C-terminal Gly andthe amino group of PE. The subsequent deconjugation
reaction by Atg4 is necessary for the normal
progression of autophagy. We developed in vitro Atg8PE reconstitution system. The Atg8-PE wassuccessfully reconstituted simply with Atg8° 11 ", Atg7
and Atg3 by using in vivo in E. coli and in vitro system.
These results suggested that Atg7 and Atg3 arenecessary and sufficient for the Atg8-PE conjugation
reaction. The in vitro Atg8-PE reconstitution systemusing recombinants and liposomes demonstrated thatthe efficiency of Atg8-PE conjugation was strongly
affected by lipid composition. Further, the Atg8 was
linked to the PE in liposomes, but not to the PE in thepresence of detergent, suggesting that the lipid bilayerof membrane is essential for the Atg8-PE conjugation.

III. Molecular dissection of Atg12
Atgl2 is activated by Atg7, transferred to AtglO andattached to Atg5 in a manner similar to theubiquitination. Although Atgl2 has scarcely sequencesimilarity with ubiquitin, its secondary structure waspredicted to have ubiquitin-like domain in the Cterminus region. We prepared N-terminally truncatedAtgl2 mutant of the yeastS. cerevisiae according to thepredicted secondary structure. Truncated form of Atgl2
having only a predicted ubiquitin-like domain
conjugated with Atg5 was still active in autophagy.
While a truncated Atg12 mutant lacking the first beta
strand in the ubiquitin-like domain didn't conjugate
with Atg5. These results showed that the ubiquitin-likedomain of Atgl2 is necessary and sufficient for
conjugation and autophagy. Furthermore, we altered
several hydrophobic amino acid residues in theubiqutin-like domain of Atg8 and found a certain aminoacid residue is critical for autophagy, even though still
active for conjugation with Atg5.

IV. Preferential degradation by autophagy
In contrast to the ubiqutin/proteasome pathway,
autophagy is thought to be non-selective protein
degradation process. We surveyed the changes ofprotein profiles after nitrogen starvation of wild-type
and !1atg7 cells using 2D-PAGE, and we found thatcytosolic acetaldehyde dehydrogenase (Ald6p; FigureI) was degraded more rapidly than other cytosolic
proteins in an autophagy dependent manner. We
observed that Ald6p was enclosed in the
autophagosome and delivered to the vacuole
preferentially. The Ald6p may be harmful to cells
during nitrogen starvation, since disruption of ALD6
improved the loss of viability of the !1atg7 mutant. Thisis the first report of the preferential degradation of a
detrimental protein via autophagy.

NATIONAL INSTITUTE

12

Nitrogen starvation 24 h
Wild-type

t:.atgl•

Ald6p

Figure I. Ald6p was degraded by autophagy. Protein
spots from 2D-PAGE using soluble lysates .of nitrogenstarved cells were shown. Ald6p, cytosolic acetaldehydedeh)'drogenase; Adhlp, alcohol dehydrogenase (cmmol).

V. Involvement of early secretory pathway
In addition to Atg proteins , we have previously
identified the involvement of secretory proteins in
autophagy. Autophagosome formation is completely
blocked when some early Sec proteins such as Sec24p, aredefective , which are involved in the formation of the
COPII coated vesicles from ER to Golgi. The autophagic
defect in sec24 deleted mutant cells was, however ,suppressed upon the recovery of ER-Golgi secretory flowby the overexpression of its homologue, Sfb2p. We foundthat the autophagic defect is not observed in sec13 andsec31 mutants, a phenomenon that can be explained by thefact that starvation stress suppresses the secretory defect ofthese mutants . These observations indicate that the active
flow in the early secretory pathway plays an important rolein autophagy. Both autophagy and its closely related
cytoplasm to vacuole-targeting (Cvt) pathway occur
through a pre-autophagosomal structure (PAS), and since
the PAS and the functional Cvt pathway exist in all sec
mutants, the early secretory pathway must be involved
specifically in autophagy, subsequent to PAS formation .
23 oc +Rap

37 oc +Rap

ubiquitin-like system, is essential for autophagosome
formation. In yeast, the Atgl2-Atg5 conjugate interacts
with a small coiled-coil protein , Atgl6, to form a ~350kDa multimeric complex . We demonstrate that the mouse
Atgl2-Atg5 conjugate forms a ~800-kDa protein complex
containing a novel WD repeat protein (Fig. 3). As the Nterminal region of this novel protein shows homology withyeast Atgl6, we have designated it mouse Atgl6-likeprotein (Atgl6L). Atgl6L, however , has a large Cterminal domain containing seven WD repeats , absent
from yeast Atgl6. Atg16L interacts with both AtgS andadditional Atgl6L monomers ; neither interaction ,however, depends on the WD-repeat domain. In
conjunction with Atgl2-Atg5 , Atgl6L associates with the
autophagic isolation membrane for the duration of
autophagosome formation. As these features are similar toyeast Atgl6 , we concluded Atgl6L is the functionalcounterpart of the yeast Atgl6 . We also found that
membrane targeting of Atgl6L requires Atg5 , but not
Atgl2. As WD repeat proteins provide a platform forprotein-protein interactions , the ~800-kDa complex is
expected to function in autophagosome formation, further
interacting with other proteins in mammalian cells.
800 kD complex~•(Apg12-Apg5 · Apg16)i•

3/

LC ~lg$ -·C -·0 ~isolation membrane

autophagosome

autolysosome

Figgure 3. Model of autophagosome formation in
mammalian cells.
The Atg12-Atg5 conjugate and Atgl6L localize to theisolation membrane throughout its elongation process.
LC3 is recruited to the membrane in the Atg5-dependent
manner. Atg12-Atg5 and Atgl6L dissociate from themembrane upon completion of autophagosome
formation , while LC3 remains on the autophagosome
membrane. Atg5 and its modification by Atg 12 arerequired for elongation of the isolation membrane.

VII. Mice with a fluorescent marker for
autophagy

Figure 2. Localization of GFP-Atg8p in sec24 mutant
cells. Wild-type (KVY55) and sec24 (MHY24) cells
haboring pRS316 GFP-Atg8p were used. GFP-Atg8p
was visualized under growing condition (a, d), starvation
condition at permissive temperature (b, e) and starvation
condition at non-permissive temperature (c, f).

VI. Mammalian Atg16-like protein: a novel WDrepeat protein
We have shown that, in yeast and mammalian cells, theAtg 12-AtgS protein conjugate, which is formed by aIn yeast, autophagy is required for cell survival during
starvation and is necessary for spore formation. In contrast,
the role of autophagy in mammals is still poorly
understood. Although the possible involvement of
autophagy in development, cell death and pathogenesis has
been repeatedly pointed out, systematic analysis has notbeen petformed, mainly due to a limitation of monitoring
methods. Our recent studies have made available several
marker proteins for autophagosomes. To understand where
and when autophagy occurs in vivo, we have generated·transgenic mice systemically expressing GFP fused toLC3 , which is a mammalian homologue of yeast
Aut7/Atg8. Cryosections of various organs were preparedand the occurrence of autophagy was examined byfluorescence microscopy (Fig. 4) . Active autophagy wasobserved in various tissues, such as the skeletal muscle,
liver, heart, exocrine glands, thymic epithelial cells, lensFOR BASIC BIOLOGY

13

epithelial cells and podocytes. Autophagy is differently
induced by nutrient starvation in most tis sues. In so meti ssues, autophagy even occurs spontaneo usly. Our results
s ugges t that the reg ulation of a utoph agy is orga ndependent and the role of autophagy is not restricted to the
starvation response. This transgenic mouse is a usefu l tool
to study mammalian autophagy .

Therefore, Atg8 is a us eful molecular marker formonitorin g autophagic process . To establish a systemmonitoring autophagy in a whole p lant, we generated
tran sge ni c Arabidopsis ex press in g GFP-AtATG8 fusion
protein. In wild-type pl ants, GFP-AtATGSs were observed
as rin g structures in the cytoplasm (Figure 5 panel A), andd e liv e red to the lumen s of vacuo le under nitrogenstarvation condition (Figure 5 pane l B). On the basis ofanalogy of yeast, we rega rded the se ring structures as
autophagosomes. In contrast, in double disruptant of
AtATG4 s which are required for C-terminal cleavage ofAt g8, GFP-AtATGS s were not locali ze d on
autophagosomes (Figure 5 panel C) and were not delivered
to the vacuole under nitrogen-starvation condition (Figure
5 pan e l D) . These re s ults indicate that AtATGS is asuitable marker for mon itoring autophagy in plant.

Publication List:
Fi g ure 4 . Muscle autophagy in res pon se to foodwithdrawal. GFP images of gastrocneniius muscles at 0 h
and 24 h starvation. Small dots indicate autophagosomes.
Bar, 10 !-lm.

VIII. Monitoring of autophagic process in a wholeplant
So far, autophagy in plant h as been de sc ribed bymorphological studies. Recent genome-wide searc h
revea led significant conservation in a utophagy ge nes
between yeast and plant. But little is known about thephysiolog ical roles and mol ecul ar mechanisms underlying
autophagy in higher pl a nts. To elucidate the plant
autophagy, we focused ubiquitination-like Atg8 lipidation
syste m. In yeast, Atg8 binds to autophagosomes and isde livered to the vacuol e through autophagic proces s .

MS

MS-N

Figure 5. Localization of GFP-AtA TG8a fusion protein
in Arabidopsis roots. The roots of !-week-old transgenic
seedlin gs were grown on MS and MS -N medium. For
sta r va tion , see dlin gs g rown on MS medium we retransferred to MS-N medium for additional 7 days. (A,B) Wild-type Arabidopsis roots stably expressing GFPAtATGSa. (C, D) atatg4ab -J di sruption mutant roots
stabl y expressing GFP-AtA TG8a. Bar: 20 Jtm.

Mi zushima, N. , Kuma , A., Kobayas hi , Y., Yamamoto, A.,
Takao, T., Natsume, T. , Ohsumi , Y., and Yoshimori , T.,
Mouse Apgl6L, a novel WD repeat protein, targets to the
autoph ag ic isolation membrane with the Apg 12-Apg5
conjugate. J. Cell Sci. , 116, 1679- 1688 (2003)
Hamasak i, M., Noda, T ., and Ohsumi Y. The early secretory
pathway contributes to autoph agoso me formation. Cell
Struc. Func., 28, 49-54 (2003)
Su gawa ra, K. , Suzuki , N ., Fujioka, Y. , Mizu shima, N .,Ohsumi , Y. , Inagaki , F. Crystalization and preliminary Xray ana lyses of LC3-I, Acta Crys. , 059, 1464- 1465 , (2003)
Mukaiyama, H. , Baba, M., Osumi , M., Aoyag i, S., Kato, N. ,Ohsumi, Y. , Sakai, Y. Modification of a Ubiquitin-likepro te in Pa z2 condu c ted mi cro pexo phagy throughformation of a membrane structure. Mol. Bioi. Cell. , inpress (2003)
Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A,
Ro se n J , E ske linen EL , Mizushima N , Oh sumi Y ,Cattoretti G, Levine B. , Promotion of tumorigenesis byheterozygous disruption of the beclin 1 autophagy gene. J.
Clin.lnvest. , 112, 1809-1820 (2003)
Klionsk y OJ, Cregg JM , Dunn WA Jr, Emr SO, Sakai Y ,Sandoval IV , Sibirny A , Subramani S , Thumm M ,Veenhuis M, Ohsumi Y. Dev. Cell. , 5, 539-45 (2003)
Mizushima, N., Yamamoto, A. , Matsui , M. , Yoshimori, T.
and Ohsumi , Y. In vivo ana lysis of autophagy in response
to nutri ent starvation using transge ni c mice expressi ng afluorescent autophagosome marker. Mol . Bioi. Cell, inpress (2004)
Ohas hi, M., Mi zushima, N., Kabeya, Y. and Yoshimori, T.
Locali za tion of mamm a lian NAD (P) H steroid
dehydrogenase-like protein on lipid droplets J. Bioi. Chem.
278, 36819-36829 (2003)
Mi zushima, N. , Yamamoto, A. , Matsui , M. , Yoshimori , T.
and Ohsumi , Y. In vivo analysis of autophagy in response
to nutrient starvation usin g tran sgenic mice expressing afluorescent autophagosome marker. Mol . Bioi. Cell inpress
Mizushima, N., Methods for monitoring autophagy. Int. J.
Biochem. Cell Bioi. In press (2004)
Mizushima, N, Yoshimori, T. , Ohsumi, Y., Role of the Apg 12
co nju ga tion sys tem in mamma lian autophagy. Int . J.
Biochem. Cell Biol. , 35, 553-561 (2003)
Ohsumi, Y. Cellular recycling system- molecular mechanismof autophagy. In Cell Growth, Cold Spring Harbor Press,
in press (2003)
Kamada, Y., Sekito,T. , Ohsumi Y. , Autophgy in yeast: a
TOR-mediated response to nutrient starvation. Curr Top
Microbiollmmunol., 279, 73-84 (2004)
Oh s umi , Y. and Mi z us him a, N . Two ubiquitin-likeconju gation sys tems essenti al for autophagy. Sem. Cell
Dev. Bioi. , in press (2004)

NATIONAL INSTITUTE14DIVISION OF CELL FUSION (ADJUNCT)

Professor (Adjunct):
Research Associate:
Graduate Student:
Technical Assistant:

OKADA, Norihiro
TAKAHASHI, Kazuhiko
SASAKI, Takeshi
MIURA, Seiko

The above members of this adjunct division havestarted a new project in this institute in April 2003.

I. Introduction
During the long history of evolution of vertebrates,
they acquired extensive diversity in characters such asmorphology, ecology and behavior. Some examples,such as conserved morphological traits of coelacanths,
show that phenotypic change during the course ofevolution can be sometimes very slow, whereas otherexamples suggest, by contrast, that their speciation anddiversification can be often accomplished very rapidly
by the process called "adaptive radiation". What factors
affect rates of phenotypic change during the evolutionof vertebrates? Although many examples of adaptive
radiation (and slow evolution) have been reported so far
for various groups of vertebrate, its mechanism has
been hardly clarified , especially from the molecularperspective.
Morphological diversification of organisms isaccomplished by differentiation of developmental
systems , which are controlled by regulation ofnumerous morphogenetic genes . This implies that, inorder to obtain insights into the mechanisms of adaptive
radiation of vertebrates , we absolutely need theviewpoint of developmental biology. However, thefields of evolutionary biology and developmental
biology have not been necessarily interactive enough so
far in spite of that such an idea has been becoming
popular in recent years. Our division aims to understandthe processes and mechanisms of diversification ofvertebrates during their evolution based on molecularapproaches of evolutionary developmental biology(evo-devo ).

(Figure 3). Evidence from the above analyses suggested
that the fauna in Lake Tanganyika as well as its
ancestor and other riverine lineages have experienced atleast two waves of a rapid radiation event more thanseveral million years ago. Interestingly, such radiations
seem now ongoing in the Victorian fauna (see the next
section for the detail) and the major groups of cichlids
in Lake Malawi, too . These results suggest that
explosion of speciation (adaptive radiation) has been acommon phenomenon during the course of evolution ofcichMs is East Africa.
Given the above results , the next primary problem tobe solved in order to clarify the background of theirevolution is how their genetic and developmental
systems have changed during the processes of adaptive
radiation .

Figure 1. African Great Lakes

II. Evolutionary history of cichlids in African
Great Lakes
Cichlid flocks of the East African Great Lakes, which
consist of Lakes Victoria , Malawi , and Tanganyika,
have attracted the interest of evolutionary biologists formore than a century (Figure 1). These species exhibit
extraordinary levels of diversity and high species
endemicity to each lake as the result of independent
explosive adaptive radiation (Figure 2).
Our group conducted phylogenetic analyses of thesefish using insertions of retroposons as markers for
elucidation of their evolutionary history. These studies
confirmed that cichlids in Lakes Victoria and Malawi
are closely related, and that species in both lakes are
related to only a portion of the lineages found in Lake
Tanganyika, the oldest Great Lake estimated at 9-12MY

Figure 2. Morphological diversity and convergent
evolution of cichlids in African Great Lakes . Leftcolumn, species in Lake Malawi ; right column, species in
Lake Tanganyika . [Figure I in Kocher, T. D. , J. A.
Conroy, K. R. McKaye and J. R. Stauffer (1993) Mol.
Bioi. Evol. 2: 158-165]

FOR BASIC BIOLOGY15

I Lake Victoria

Lake Malawi)"" '"""""'""
Figure 3. Phylogenetic relationships among cichlids inAfrica as revealed by molecular markers.

injection of the GFP (Green Fluorescent Protein) genewith a promoter known to be specific to thjs organ may
be useful. Another advantage of establishing atransgenic system is that we can newly identify genesthat are specifically expressed in jaws by using genetrap and enhancer-trap systems. However, a problem
with application of this methodology to cichlids is thattransgenic techniques have been hardly established forthese fish so far. One solution to this problem is to takeadvantage of a transposon-mediated transgenjc system,
which was recently established for zebrafish and shown
to be highly efficiently integrated into the genome
compared to other conventional systems. In order toapply this transgenic system to the above cichlid fish,
we have started control ex periments using zebrafish.

- .-.------A--

III. Analysis of Cichlids in Lake Victoria forelucidation of molecular mechanisms ofspeciation and diversification
Among the cichlid faunas in African Great Lakes, theone in Lake Victoria is considered to be the youngest
since this lake is suggested to have once been dried up
about 12,000 years ago. Such a recent origin of thisfauna is consistent with the observation by our groupthat most of selectively-neutral polymorphic alleles are
retained both within- and among species within thi slake (Figure 4). This means that, if a certain allele isuniquely fixed at a certain locus in natural populationsof a certain species , we can assume the past and/or
present existence of a selective pressure on this geneand hence can regard it as a candidate gene that was
responsible for the phenotypic differentiation of thi s
species from other related spec ie s. Therefore,
inve stiga tion of cichlids in Lake Victoria isadvantageous for the purpo se of elucidation ofmolecular mechanisms of speciation and their following
diversification during the adaptive radiation.
One of the most attractive diversification of theabove cichlids is observed in morphology of jaws.
Since they exhibit remarkable specialization to various
food resources such as algae, shrimps , fry, planktons
and fish, morphology of their jaws shows extensive
diversity among species. Another interesting point isthat all the cichlids in Lake Victoria, as well as those in
Lake Malawi, show mouth brooding of their eggs andfry . Thus, their jaw shape might be also rel ated to theirbreeding habit. Therefore, the mechanism ofdiversification of jaw shapes among species may be oneof the most important factors to be considered for thepresent purpose.
One of our strategies to identify genes that were
responsible for diversification of jaw shapes among
Victorian cichlids is to use transgenic techniques. For adetailed observation of morphogenesis of jaws,

B-,..ol!

---_- ~_. _ _ ..__ .w_, .. _----

c --- ... __ __ ...._-_..-..-.-~

Figure 4. Polymorphism of in sertion of a retroposon
seq uence at a single locu s is shared among cichlids
species in Lake Victoria. Panels A-C correspond todifferent species, and each panel includes results of PCRs
from ten different individuals. In each panel , an upperband is observed only if the sequence of the retroposon
ex ists at one or both of the alleles at the investigatedlocus whereas a lower band is observed if one or both of
the alleles did not contain a sequence of the retroposon.
Individuals showing both bands indicate heterozygote.

Original papers:
Terai Y, Takahashi K, Nishida M, Sato T, Okada N (2003)
Using SINEs to probe ancient explosive speciation:"Hidden" radiation of African cichlids? Mol. Bio. Evol.
20: 924-930.
Terai Y, Morikawa N, Kawakami K, Okada N (2003) The
complexity of alternative splicing of hagoromo mRNA
is increased in an explos ively spes;iated lineage in East
African cichlids. Proc. Nat! . Acad. Sci. USA
100:12798-12803.
Terai Y, Takezaki N, Mayer WE, Tichy H, Takahata N,
Klein J , Okada N (2003) Plylogenetic relationships
among East African haplochromine fishes as revealedby short interspersed elements (SINEs). J. Mol. Evol. inpress.
Watanabe M, Kobayashi N, Fujiyama A, Okada N (2003)
Construction of a BAC library for Haplochromi s
chi lotes, a cichlid fish from Lake Victoria. Genes Genet
Syst 78:103-105.

16

FOR BASIC BIOLOGY17

DEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairperson: MOROHASHI, Ken-ichirou

DIVISION OF REPRODUCTIVE BIOLOGYDIVISION OF CELL DIFFERENTIATIONDIVISION OF MORPHOGENESISDIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT)The department is composed of three divisions and one adjunctdivision. Department members conduct molecular analysis on various
aspects of developmental phenomena including ( 1) gonadal sex
differentiation and gametogenesis, (2) gene regulation in celldifferentiation and growth, and (3) molecular basis of body plans.

NATIONAL INSTITUTE18

DIVISION OF REPRODUCTIVE BIOLOGY

Professor:
Associate Professor:
Research Associates:

NAGAHAMA Yoshitaka
YOSHIKUNI Michiyasu
KOBAYASHI Tohru
OKUBO Kataaki
KOBAYASHI Hiroko
Technical Staff:
JSPS Postdoctoral Fellows: SHIBATA Yasushi
WANG, De-shou
YI, Mei-Sheng
BHANDARI, Ramji
MATSUDA Masaru ' 1
Postdoctoral Fellows :SAKAI Fumie 11
GUAN Guijun n
SENTHILKUMARAN
Balasubramanian n
SUDHAKUMARI, Cheni Chery n
LAU, En-Lieng
PAUL, Bindhu
Graduate Students :HORIGUCHI, Ryou 11
KOBAYASHI, Yasuhisa
FAN, Hai-Guang 31
OHMURO-MATSUYAMA, Yuki 31
KURITA, Kayoko 11
Visiting Scientists:
MATSUDA, Chika
YANO, Akihiro
ZHOU, Linyan n
JST technical staffs:
HAYAKA WA, Rie
SAGEGAMI, Reiko
UNO, Eri
Secretary:
SHIMADA, Yu n
11
CREST, JST; ' 1 PREST, JST; 11 Graduate University for
Advanced Studies, SO KENDAl

The pituitary-gonadal axis plays an important role inregulating gametogenesis in vertebrates. Gonadotropins
typically act through the biosynthesis of gonadal steroid
hormones which in turn mediate various stages ofgametogenesis. Their effects are particularly profoundin teleost fishes which provide excellent models forinvestigating the basic hormonal mechanisms regulating
gonadal sex differentiation and gametogenesis Our
research focuses on (1) the identification of regulatorsand steroidal mediators involved in sex determination,
gonadal sex differentiation and gametogenesis, and (2)the mechanisms of synthesis and action of these
mediators.

sex-determining gene , between the X and Y
chromosomes.
Medaka possesses a stable genetic XX/X Y sex
determining system. Usi ng positional clonin g anddetailed sequence analysis of BAC clones by shotgun
sequencing, we identified DMY (DM domain gene on
the Y chromosome) as a strong candidate for the sexdetermining gene of medaka. DMY encodes a protein of
267 amino acids including the highly conserved DM
domain. The DM domain was named after a related
DNA binding motif found in two proteins, dsx and mab3, involved in sexual development in Drosophila and C.
elegans, respectively . Two naturally occurring XY
female medaka showed different mutations in the DMY
gene. One of these mutants was found to carry amutation causing a frameshift and prematuretermination of the DMY protein. When mated, all XY
offspring with the mutant Y were female. The othermutant had a severe depression in DMY expression in
the embryo and 60 % of its XY offspring with the
mutant Y developed as females. More recently , weshowed that a 117-kilobase genomic DNA fragment
carrying DMY was sufficient to induce testis
differentiation and subsequent male development.
These loss- and gain-of-function studies indicate that
DMY is the sex-determining gene of medaka. DMY
provides the first example of a sex-determining gene in
non-mammalian vertebrates. 0. curvinotus also has the
DMY gene on the Y chromosome, which is homologous
to the Y chromosome of medaka, and that DMY isexpressed in XY embryos. A phylogenetic tree based on
the amino acid sequence including the DM-domainshows that DMY was derived from DMRTJ
immediately before speciation of 0. latipes and 0.
curvinotus (Fig. 1). The branch length of DMY is longer
than that of DMRTJ in the phylogenetic tree . Thismeans that DMY has more mutations than DMRTJ andsuggests that DMY accumulated these mutations after it
acquired its sex-determining function. These results
suggest that a new sex-determining gene generated by agene duplication event in the sex chromosome tends toevolve fast.

I. Sex-determining gene of medaka
Although the sex-determining gene SRY!Sry has been
identified in mammals, no comparable genes have beenfound in non-mammalian vertebrates. To identify such asex-determining gene, a positional cloning approach issuitable. The medaka, Oryzias latipes, has two majoradvantages for genetic research: a large genetic
diversity within the species and the existence of several
inbred strains. As in mammals , sex determination in
medaka is male heterogametic, although the Y
chromosome is not cytogenetically distinct. Alterationof phenotypic sex with no reproductive consequences,and recombination over the entire sex chromosome pair,suggest that there are no major differences, other than aFig. I A neighbor-joining tree based on the amino acidsequences from positions 21 to 109 of DMY proteins in
0. latipes and 0. curvinotus. The scores above eachbranch represent bootstrap values of 1000 times.

FOR BASIC BIOLOGY

II. Endocrine regulation of gonadal sex
differentiation
Nile tilapia, Oreochromis niloticus, is an excellent
example of the precise nature of steroidogenic actions
during gonadal sex differentiation . In this fish , allgenetic female (XX) or male (XY) broods can beobtained by artificial fertilization of normal eggs (XX)and sex-reversed, pseudo male sperm (XX), or normal
eggs (XX) and super male sperm (YY) , respectively .
Fertilized eggs hatch after 4 days at 26·c . On the day ofhatching, primordial germ cells (PGCs), are located in
the outer layer of the lateral plate mesoderm around thehind gut. At 3 days post-hatching, PGCs are located in
the gonadal anlagen after the formation of the coelomic
cavity in the lateral plate mesoderm rather than throughactive mi-gration.
In tilapia, ovarian differentiation is initially marked
by the appearance of an narrow space in the stromal
tissue representing the formation of the ovarian cavity.
Using all-female tilapia fries, we showed that steroidproducing cells in XX gonads prior to and during sex
differentiation express all of the steroidogenic enzymes
required for estradiol-17~ biosynthesis fromcholesterol. Transcripts of estrogen receptors (ER) a
and ~ first appear in both female and male gonads offries at 5-10 days after hatching. These results, together
with evidence of masculinization of genetic females byfadrozole (an aromatase inhibitor) or tamoxifen (anestrogen receptor antagonist) , strongly suggest that
endogenous estrogens act as the natural inducers ofovarian differentiation in tilapia. In contrast, the abilityof steroid-producing cells to synthesize steroid
hormones in the testes only appears at the time oftesticular differentiation , suggesting that unlike infemales , in males sex steroids do not play any majorroles in testicular differentiation in tilapia. This was
further confirmed by the absence of transcripts ofandrogen receptors in gonads of genetic males during
sex differentiation. We have cloned several genes whichwere reported to be involved in gonadal sex
differentiation of other vertebrates including Sox9 ,Daxl , Ad4BP/SF-1, etc. Among these genes , DMRTJ
was the only gene that was shown to be expressed malespecifically in the gonads (Sertoli cells) during sex
differentiation, suggesting an important role for DMRTJ
in testicular differentiation.

III. Endocrine regulation of spermatogenesis
Using an organ culture system for eel testes
consisting of spermatogonia and inactive somatic cells,
we have shown that the hormonal regulation of
spermatogenesis in eel testes involves the gonadotropin
stimulation of Leydig cells to produce 11ketotestosterone (11-KT), a potent androgen in fish
(Fig. 2) . In turn, 11-KT activates Sertoli cells to
stimulate the production of activin B. Addition ofrecombinant eel activin B to the culture medium
induced proliferation of spermatogonia, producing late
type B spermatogonia, within 15 days in the same19

manner as did 11-KT. cDNAs encoding two androgen
receptors (ARa and AR~) have been cloned, for the
first time in any vertebrates, from eel and tilapia testes.
In situ hybridization reveals that although both AR
mRNAs are present in eel testes prior to HCG injection,
only ARa transcripts increase during HCG-induced
spermatogenesis suggesting that ARa and AR~ playdifferent roles in spermatogenesis. Activin B binds toactivin type I and II receptors on spermatogonia tostimulate de novo synthesis of G liS cyclins and CDKs
leading to the initiation of mitosis. Interestingly, cyclin
A 1 transcripts are first detected in primary
spermatocytes during HCG-induced spermatogenesis.

Fig. 2 Hormonal regulation of gametogenesis in fish. Sex
steroid hormones produced by gonadal somatic cells
under the influence of pituitary gonadotropins stimulate
various stages of gametogenesis including
spermatogenesis, oogenesis, and final gamete maturation.

IV. Endocrine regulation of oocyte growth andmaturation
Gonadotropins (FSH and LH) require two major
steroidal mediators, estradiol-17 ~ and 17 a, 20~­
dihydroxy-4-pregnen-3-one (17a, 20~-DP) to act as
critical hormones to execute oocyte growth andmaturation, respectively (Fig. 2). Two cell-type models
in which the thecal layer provides precursor steroids to
the granulosa layer, have been demonstrated for
estradiol-17~ and 17a, 20~-DP production. Ad4BP/SF1 serves as a transcriptional regulator for ovarian
cytochrome P450 aromatase (P450arom) whichconverts testosterone to estradiol-17~ in granulosa cells
during vitellogenesis. LH induces a distinct shift insteroidogenesis, i.e. from estradiol-17~ to 17a, 20~-DP
as well as the steroidogenic enzyme genes from
P450arom to 20~-hydroxysteroid dehydrogenase (20~­HSD), in the granulosa layers of ovarian follicles prior
to oocyte maturation. The triggering of thesteroidogenic shift by LH is manifested through two
molecular mechanisms, the first being the subjugationof the expression of Ad4BP/SF-1 vis a vis P450arom,and the second, the induction of over-expression of
20~-HSD probably via CREB.

20
Unlike estradiol-17~ (genomic action), 17a., 20~-DP
binds to a novel, G-protein-coupled membrane receptor(non-genomic action), leading to the de novo synthesisof cyclin B, the regulatory component of maturationpromoting factor (MPF), which activates a preexisting
35-kDa cdc2 kinase via phosphorylation of its threonine
161 by a threonine kinase (M015), thus producing the
34 kDa active cdc2. Upon egg activation, MPF isinactivated by degradation of cyclin B. We showed thatthe 26S proteasome initiates cyclin B degradation
through the first cut of its NH2 terminus at lysine 57.
An endocrine-disrupting chemical, diethylstilbestrol(DES), a nonsteroidal estrogen, triggers oocyte
maturation in fish. The morphology (the time course of
the change in germinal vesicle breakdown) and an
intracellular molecular event (the de novo synthesis of
cyclin B) induced by DES were indistinguishable fromthose induced by 17a., 20~-DP. A synergistic action of
DES on 17a., 20~-DP-induced oocyte maturation wasobserved. Both 17a., 20~-DP- and DES-induced oocyte
maturation was inhibited by an antibody against the
17a., 20~-DP receptor. These results suggest that DES
may act on the 17a., 20~-DP receptor as an agonist of
17a., 20~-DP.

Publication List:
He, C.L., Du, J.L., Huang, Y.S., Lee, Y.H., Nagahama, Y.
and Chang, C.F. (2003). Differential expression ofandrogen receptor and estrogen receptor in gonad in
relation to the sex change in protandrous black porgy,
Acanthopagrus schlegeli. Bioi. Reprod. 69, 455-461.
Jiang, J.Q., Wang, D.S., Senthilkumaran, B., Kobayashi,T., Kobayashi, H.K., Yamaguchi, A., Ge, W., Young, G.
and Nagahama, Y. (2003). Isolation, characterizationand expression of 11 ~-hydroxysteroid dehydrogenase
type 2 cDNAs from the testes of Japanese eel (Anguillajaponica) and Nile tilapia (Oreochromis niloticus). J.
Mol. Endocrinol. 31, 305-315.
Kobayashi, T., Kajiura-Kobayashi, H. and Nagahama Y.
(2003). Induction of XY sex reversal by estrogen
involves altered gene expression in a teleost, tilapia.
Cytogenet. Genome Res. 101, 289-294.
Kobayashi, Y., Kobayashi, T., Nakamura, M., Sunobe T.,
Morrey, C.E., Suzuki, N. and Nagahama, Y. (2003).
Characterization of two types of cytochrmome P450
aromatase in serial-sex changing gobiid fish, Trimma
Matsuda, M. et al. okinawae. Zool. Sci. (in press).
Matsuda, M. and Nagahama, Y. (2003). Positional cloningof the sex-determining region of medaka using a Y
congenic strain. N. Shimizu, T. Aoki, I. Hirono and F.
Takashima (Eds.), Aquatic Genomics - Steps Toward a
Great Future. Springer-Verlag, Tokyo, pp. 236-243.

NATIONAL INSTITUTE

Matsuda, M., Sato, T., Toyazaki, Y., Nagahama, Y.,
Hamaguchi, S. and Sakaizumi, M. (2003). Oryzias
curvinotus has DMY, a gene that is required for maledevelopment in the medaka, 0. latipes. Zool. Sci. 20,
159-161.
Mita, M., Hirai, T., Oba, Y., Yoshikuni, M. andNagahama, Y. (2003). eDNA cloning of a novel
member of the glycoprotein hormone receptor family
from a starfish Asterina pectinifera. Mol. Reprod.
Develop. (in press).
Mita, M., Oka, H., Thorndyke, M.C., Shibata, Y.,
Yoshikuni, M. and Nagahama, Y. (2003). Inhibitory
effect of a SALMFamide neuropeptide on secretion of
gonad-stimulating substance from radial nerves in the
starfish Asterina pectinifera. Zool. Sci. (in press).
Nagahama, Y., Nakamura, M., Kitano, T. and Tokumoto,T. (2003)~ Sexual plasticity in fish: A possible target ofendorine disruptor actions. Environ. Sci. (in press).
Nakahata, S., Kotani, T., Mita, K., Kawasaki, T., Katsu,
Y., Nagahama, Y. and Yamashita, M. (2003).
Involvement of Xenopus Pumilio in the translational
regulation that is specific to cyclin Bl mRNA duringoocyte maturation. Mech. Develop. 120, 865-880.
Ohmuro-Matsuyama, Y., Matsuda, M., Kobayashi, T.,
lkeuchi, T. and Nagahama, Y. (2003). Expression of
DMY and DMRTJ in various tissues of the medaka(Oryzias latipes). Zool. Sci. 20, 1395-1398.
Ohta, K., Rhaman, M.A., Chuda, H., Yoshikuni, M.,
Nagahama, Y. and Matsuyama, M. (2003) Maturationinducing hormone and it's membrane receptor in gonads
of Japanese yellowtail, Seriola quenqueradiata.
Fisheries Science (in press).
Okubo, K., Ishii, S., Ishida, J., Mitani, H., Naruse, K.,
Kondo, M., Shima, A., Tanaka, M., Asakawa, S.,
Shimizu, N. and Aida, K. (2003). A novel third
gonadotropin-releasing hormone receptor in the medaka
Oryzias latipes: evolutionary and functional
implications. Gene. 314, 121-131.
Senthilkumaran, B., Yoshikuni, M. and Nagahama, Y.
(2003). A shift in steroidogenesis occurring in ovarianfollicles prior to oocyte maturation. Mol. Cell.
Endocrinol. (in press).
Tokumoto, T., Tokumoto, M., Horiguchi, R., Ishikawa, K.
and Nagahama, Y. (2003). Diethylstilbestrol induces
fish oocyte maturation. Proc. Natl. Acad. Sci. USA (inpress).
Yoshiura, Y., Senthilkumaran, B., Watanabe, M., Oba, Y.,
Kobayashi, T. and Nagahama, Y. (2003). Synergistic
expression of Ad4BP/SF-1 and cytochrome P-450
aromatase (Ovarian type) in the ovary of Nile tilapia,
Oreochromis niloticus, during vitellogenesis suggests
transcriptional interaction. Bioi. Reprod. 68, 1545-1553.

FOR BASIC BIOLOGYDIVISION OF CELL DIFFERENTIATIONProf essor:
Research Associates:

MOROHASHI, Ken-ichirou
FUKUI, Yuko
OGAWA, Hidesato
ISHIHARA, Satoru
Technical Staff:
OKA, Sanae
JSPS Postdoctoral Fellows: TSUCHIYA , Megumi
BABA, Takahi
Postdoctoral Fellows:
MOHAMAD, Zubair n
SUGIYAMA, Noriyuki n
MIZUSAKI, Hirofumi n
SUZUKI, Taiga n
Graduate Students :SHIMA, Yuichi 11
KUSAKA, Masatomo 31
KOMATSU, Tomoko 31
MATSUYAMA, Makoto 31
FATCHIYAH 31
SATO, Yuko 31
KURITA , Kana 31
Technical Assistants:
OWAKI, Akiko 51
KOWA, Hiroe 51
SHINOHARA, Yuko 51
ISH/ KA WA , Azusa 51
KUSAKA , Hiroko "Secretaries:
YAMANAKA , Tae 61
KAWASHIMA , Ryokon CREST, JST Postdoctoral Fellow
Graduate School of Kyushu University
31
Graduate University for Advanced Studies
41
Graduate School ofTohoku University
51
CREST, JST Technical Assistant
61
CREST, JST Secretary
11

Although sexual dimorphism manifests mostobviously in the gonads (testis and ovary), it can beobserved in the other parts of the entire animal body.
For instance, it is well known that several tissues suchas the external genitalia, muscle, and brain exhibit
sexual dimorphisms in terms of their structures andfunctions. This process of sex differentiation can bedivided into three steps. The first occurs at fertilization ,during which the sexes of fertilized eggs are determined
genetically by combination of sex chromosomes. As thesecond step, the individuals carrying XY and XX sex
chromosomes develop the testis and ovary, respectively .
Sex differentiation of the gonads usually proceeds
during fetal stages. Thereafter, the gonads control thesexes of the whole body through the function of sex
steroids synthesized in the sexually differentiatedgonads. Therefore, the gonadal sexes are quite
important for sex differentiation of the animals.
It has been established that a number of transcription
factors play crucial roles in the process of gonadal
differentiation . Some of these factors , such as SRY,
WTI , DAX-1 , SOX9 and ARX were identified as the
products of genes responsible for human diseases that
display structural and functional defects in tissues
including the gonads . The functions of the othertranscription factors such as Ad4BP/SF-l, Emx2, M33 ,and Lhx9 were defined by the phenotypes of the genedisrupted mice. In addition, the expression profiles with21
respect to their distribution and sexual dimorphism
strongly suggested the functional significance at theearly stage of gonadal differentiation . However, itremains to be clarified how the transcription factors
above regulate their target gene transcription and how
the genes encoding the transcription factors are
regulated by upstream regulators. Studies from above
two directions are quite important to define the gene
regulatory cascade and the molecular mechanisms that
supports sex differentiation of the gonad.

I. Function of AhR (dioxin receptor) in the ovary.
It has been well documented that dioxins acts as an
endocrine disruptor through estrogenic action.
However, the molecular mechanisms underlying thisaction have been largely unknown. We have clarified itby focusing on the reproductive defects displayed in the
AhR gene disrupted mouse. The gene disrupted female
mice showed significantly reduced fertility probably
due to disordered estrus cycle, low concentration ofovarian estradiol, and reduced numbers of ovulated
eggs. Since estradiol is a representative sex steroid offemale mainly produced in the ovary by successive
reactions of steroidogenic enzymes, the expression ofthese steroidogenic genes were investigated, and foundthat the expression of cytochrome P450 aromatasecatalyzing the final step of estrogen synthesis is affectedsignificantly in the gene disrupted mouse. Showing agood correlation, estrogen treatment rescued the
reduced ovulation capacity. This remarkable phenotypesuggested that AhR regulates the P450 aromatase genetranscription. Indeed, reporter gene analysis indicatedthat the mouse and human P450 aromatase genes wereactivated by AhR.

Fig. 1, During maturation process of eggs, estrogen (E2)
stimulates follicular growth and rapture. AhR together
with Ad4BP/SF-l were elucidated to regulate P450
aromatase gene expression.

NATIONAL INSTITUTE

22
Our previous study revealed that Ad4BP/SF-l, amember of nuclear receptor, is expressed specifically inthe steroidogenic tissues including the ovary, andimplicated in regulation of the steroidogenic genes .
Therefore, it was investigated whether AhR activates
the P450 aromatase gene transcription synergisticallywith Ad4BP/SF-l. Interestingly, reporter gene assays
showed synergy between the two factors. Showing agood correlation, these two factors were indicated tointeract mutually , and moreover chromatin
immunoprecipitation assays with anti-AhR and antiAd4BP/SF-l antibodies revealed that both factors bindtheir recognition sequences on the aromatase promoter
and formed a protein complex in the ovarian cells.
Thus, together with the AhR KO phenotype, it was
concluded that AhR regulates the P450 aromatase genetranscription with Ad4BP/SF-l, and thereby regulates
estrogen synthesis in the ovary (Fig. 1). This finding
clearly elucidated that dioxins through binding to AhR
exert estrogenic action by upregulating P450 aromatasegene expression in the ovary. (This study was
performed as collaboration with Prof. Fujii-Kuriyama(Tsukuba University) .)II. Characterization of factors interacting with
Ad4BP/SF-1
In order to define the function of Ad4BP/SF-l, we
have screened a two-hybrid library prepared form
mouse embryonic gonads. UBC9, PIASl and PIAS3 , allof which have been indicated as components forsumoylation reaction (conjugation of SUMO (.s.mall
ybiquitin-like ID.Qdifier)), were isolated as theinteracting proteins. Thus, we investigated whether
Ad4BP/SF-l is sumoylated in vivo and in vitro.
Immunoprecipitation study from cultured cells showedthat at least a part of Ad4BP/SF-l is sumoylated incultured cells. In vitro reconstitution study using
purified components revealed clearly that sumoylation
occurs at two lysine residues , K119 and K194 , ofActivation
E1

Conjugation
E2

Ligation
E3

SUM0-1n ____..

Ad4BP/
SF-1

Ad4BP/SF-1

Fig. 2, Sumoylation (conjugation of SUMO) consists ofthree step reactions successively catalyzed by UBA2AOSI (El), UBC9 (E2), and PIAS (E3). Reporter geneassays indicated that unsumoylated form of Ad4BP/SF-lenhances synergistic transcription. This observationsuggested the presence of a putative synergy control
factor (SCF).

Ad4BP/SF-l. Moreover, interestingly, sumoylation at
Kl94 is essential for the second sumoylation at Kll9,
suggesting that conjugation of SUMO induces
conformational alteration of Ad4BP/SF-1.
With respect to the physiological function of SUMO,
we have investigated whether sumoylated Ad4BP/SF-1
differentially activates transcription, since sumoylationconsensus sequence is overlapped with that of synergy
control region originally defined as a suppression
domain for synergistic transcriptional activation.
Ad4BP/SF-l carrying mutation at the sumoylation siteenhanced synergistic transcription with reporter gene
driven by multiple Ad4 binding sites. It was establishedthat MUllerian inhibiting substance (MIS) gene isregulated by SOX9 synergistcally with Ad4BP/SF-l.
Interestingly, SOX9 has a sumoylation consensus
sequence and in fact sumoylated as well. Therefore, we
asked whether sumoylation has effects on the synergy
between the two transcription factors. Expectedly ,unsumoylated forms activated the synergistictranscription of the MIS gene more than wild type. To
define the molecul ar mechanisms underlying thesynergy enhancement, we asked with in vitro
sumoylated Ad4BP/SF-1 whether sumoylation affects
DNA binding activity and interaction with SOX9.
However, sumoylation was found to have no effect on
both aspects, strongly suggesting the presence of aputative synergy control factor (Fig. 2). Although thephysiological significance of sumoylation has been
controversial, identification of factors interacting
preferentially with the sumoylated Ad4BP/SF-l such as
the synergy control factor will probably give us quite
valuable information. (This study has been performed
as collaboration with Profs. Kikuchi (Hiroshima Univ.),
Shirakawa (Yokohama City Univ.), and Yamanaka(Kyushu Univ.).)

III. Function of growth factors during early stagesof gonad differentiation.
The gonad as well as the reproductive tracts, kidney,
and adrenal cortex are derived from a part of theintermediate and lateral plate mesoderms. In addition,
the mesonephros essential for the early gonadal
development also originates from the intermediate
mesoderm. Although the mesonephros was elucidated
as the source of certain cell types constituting the testis,
its contribution to gonad formation has been largely
unknown. We examined in detail the expression profilesof Wnt4, Fgf8, and Fgj9 in the developing mesonephrosof chick embryos, and found that the expressions ofthese factors are spatially and temporally correlatedwith those of the marker genes for the gonad and/or
adrenal cortex such as Ad4BP!SF-1 and Dmrt-1(doublesex- and mab-3-related transcription factor). As
was shown previously with the rat fetuses , it was
confirmed with the chick embryos that these two tissues
are derived from a single cell population, the adrenogonadal primordium. Thereafter, the primordium
divided into two cell populations, one of which is the
gonadal primordium and the other of which is theFOR BASIC BIOLOGY

adrenal primordium. All of these cells are identified as
immunoreactive cells for Ad4BP/SF-l. The expressionof th e grow th factors , Wnt4 , Fgf8, a nd Fgf9, aredetectable in the nephrogi nous mesenchyme (featuremesonephros) and developing nephric tubules at thes tage of the early gonad and adre nocorticaldevelopment. Interestingly , the expressions of Wnt4
and Fgf9 are correlate with those of Ad4BP/SF-l andDmrtl . Therefore, it was assumed th at these growth
factors reg ulate the expression of th e go nadal andadrenocortical marker gene expressions.
In o rder to d efine it , Wnt4 and Fgf9 weremisexpressed with replication competent virus systemin the go n a d a nd adrenal cortex of the ea rl ydevelopmental stages. Misexpression of Wnt4 expanded
the expression of Ad4BP/SF-1 as the marker for theadrenogonadal primordium while failed to expand theexpression of Drnrt-1. Interestingly, mi sexpression of
Fgj9 expanded the expression of Dmrt-1 and probably
thereby resulted in additional gonad formation . In fact,
the expressions of marker genes for the go nad wereobserved even in the additiona l go nad (Fig . 3).
Moreover , chemical inhibitor for Fgf receptor
downregulated the Dmrt-1 expression . Both gain-offunction and loss-of-function treatments above induced
no obvious structural alteration of the adrenal cortex,
indic a tin g that F gf9 induced gonad d eve lopment
proba bly throu gh stimul a ting cell proliferation oralternatively through trans-determining cell fate into thego nad. Thi s study demonstrated that Wnt4 and Fgj9
expressed in the developing mesonephros are involvedin the early stages of the gonad and adrenocorticaldevelopment through distinct functions.

Fig. 3, Misexpression of Fgf9 induced additional gonad
formation as indicated by arrows. Ad4BP/SF-1 and Mis
are both expressed in the additional gonads.
In the case of mammals, a gene disruption study
indicated that Fgf9 is esse nti a l for testicul a rdifferentiation . However, in the chick the misexpressionof Fgf9 was unable to induce the expression of thetesticular marker gene in the female gonad . This clear
d isc repancy might be due to the difference ofexperimental condition s, one of which is genedisruption (loss-of-function) study and the other ismi sex pre ssion (gain-of-function) study , or du e todifference of animal species. (This study has been
performed as collaboration with Dr. Yoshioka in Hyogo
Univ. of Teacher Education.)
In addition to the studies described above, we haveinvestigated tissue-specific enhancers localized in the
Ad4BP/SF-1 gene. Recently , we ide ntified a f etal

23
adrenocortical-specific, pituitary-specific, and ventromedial hypothalamic nucleus-specific enhancers, andtheir fine structures have been clarified. Investigation of
the fac tors to bind the enhancers will be essential toclarify the mech a nism underlying the ti ss ue
differentiation.
It has been known that several gene disruption
s tudie s revealed their implication into gonad
differentiation and gonad sex differentiation. However,function al and genetic correlation among the genes
have remained to be investigated. As the investigationof the ge netic corre l a tion, anal yses for genetic
interaction have been performed with a certain lines ofgene di srupted mice , of which genetic backgrounds
have been fixed into C57B/6 and FVB in addition to theoriginal 129J. Some of the analyses already indicatedth e presence of the genetic interaction, and themol ec ular ba s is for the interac tion is underinvestigation.

Publication List:
Suzuki, T. , Kasahara, M., Yoshioka, H., Morohashi , K.,
Umesono K (2003) LXXLL motifs in Dax-1 have target
specificity for the orphan receptors Ad4BP/SF-l and
LRH-1. Mol. Cell. Biol. 23, 238-249.
Meeks, J. J., Crawford S. E. , TRussell, A., Morohashi, K,
Capel, B., Weiss, J. , and Jameson , J. L. (2003) Dax1
regu lates testi s cord organization during gonadal
differentiation. Development 130, 1029-1036.
Mizusaki, H., Kaw abe, K. , Mukai , T ., Ariyoshi , E .,Kasahara, M., Yoshioka, H., Swain, A., and Morohashi,
K. (2003) Dax-1 gene transcription is regulated by Wnt4
in the fem ale developing gonad. Mol. Endocrinol. 17,
507-519.
Kawajiri , K. , Ikuta, T. , Suzuki, T., Kusaka, M., Watanabe,J., Muramatsu, M. , Fujieda, K., Tac hibana, M. andMorohashi, K. (2003) NR boxes of Dax-1 participate
both in Ad4BP/SF-1 dependent nuclear import and incytoplasmic retention of Dax-1. Mol. Endocrinol. 17,
994-1004.
Kato, M., Das, S. , Petras, K. , Kitamura, K. , Morohashi , K.,
Abuelo, D. N., Barr, M ., Bonne au, D ., Brady , A .,Carpenter, N. J., Frisone, F., Fukuda, T. , Guerrini , R.,
Iida, E., Itoh, M. , Lewanda, A. F., Nanba, Y., Oka, A.,
Proud, V. K., Russel, K, L. , Saugier-Veber, P. , Schelley,
S. L., Selicomi, A., Shaner, R., Silengo, M. , Stewart, F.,
Sugiyama, N., Toyama, J., Toutain, A., Vargas, A. L,
Yanazawa, M ., Zackai, E . H . and Dobyns , W . B.
Mutations of ARX are associated with striking
pleiotropy and consistent genotype -ph enotype
correlation. Human Mutations in press
Toda, K. , Okada, Y., Zubair, M. , Morohashi, K., Saibara,T., and Okada, T . Assessment of in vivo action of
estrogen using aromatase-knockout mice which carry an
estrogen-inducible enhanced green fluorescent protein
gene. Endocrinol. in press. ·NATIONAL INSTITUTE24

DIVISION OF MORPHOGENESIS

UENO,NaotoKINOSHITA, Noriyuki
NAKAMURA, Makoto
TAKAHASHI, Hiroki
Technical Staffs:
TAKAGI, Chiyo
YAMAMOTO, Takamasa
HYODO, Akiko
TERASAKA, Chie
MOROKUMA, Yoshie
YAMADA, Shigehiro
TANABE, Fumio
NIBB Research Fellows: HYODO-MIURA, Junko
TAKEUCHI, Masaki
Postdoctoral Fellows:
KITAYAMA, Atsushi
MOROKUMA, Junji
HOTTA, Kohji
l/OKA, Hidekazu n
Graduate Students:
MIYAKOSHI, Akira 11
CHUNG, Hyeyoung 21
KUJ/RAOKA, Masahiro n
YOSHIKANE, Nami 31
Professor:
Associate Professor:
Research Associates:

Graduate University for Advanced Studies, SO KENDAl
University of Tokyo
31 Nara Institute of Science and Technology
11

21

The complex morphogenesis of organisms isachieved by consecutive cell-to-cell interactions during
development. Recent sfudies suggest that growth factors
play crucial roles in controlling such intercellularcommunications in a variety of organisms. In addition
to secretory factors, transcription factors which act cellautonomously are thought to be essential for thedetermination of cell fates. Our main interest is to knowhow pattern formation and morphogenesis during
development is regulated by these growth factors andtranscription factors. We address this problem using
several model animals, including frog, fly and ascidian,employing embryology, genetics, molecular andcellular biology, and biochemistry. In addition, we haverecently introduced genome-wide approaches to
elucidate precise genetic program controlling early
development.

I. Gastrulation movement regulated by Wnt

signaling
Gastrulation is one of the most important processes
during morphogenesis of early embryo, involving
dynamic cell migration and change in embryo shape.
Almost all animals undergo gastrulation to form the gut.
In spite of its importance, the mechanism underlying
the event has just begun to be studied at molecularlevel. During Xenopus gastrulation, mesodermal cells
migrate to the inside of the embryo and move on theblastocoel roof. One of the important mechanisms forthis process is convergent extension. As convergent
extension begins, cells are polarized and aligned
mediolaterally, followed by the intercalation of thesecells. As described above, one of the Wnt signaling
pathways, called Wnt/INK (c-Iun N-terminal kinase)pathway, is shown to be important for the regulation of

convergent extension. The pathway is highly conserved
among species and initially found to be essential for theestablishment of planar cell polarity (PCP) of
Drosophila wing hair.
We have previously demonstrated that Xenopus
prickle (Xpk), a Xenopus homologue of a Drosophila
PCP gene, is an essential component for gastrulation
cell movement. Both gain-of-function (GOF) and lossof-function (LOF) of Xpk severely perturbed
gastrulation and caused spina bi.fida embryos without
affecting mesodermal differentiation. We also
demonstrated that XPK binds to Xenopus Dsh as well as
to INK. This suggests that XPK plays a pivotal role inconnecting Dsh function to INK activation. To
understand the molecular mechanism, we identified
proteins which binds to XPK by yeast two-hybrid
screening. One of XPK binding protein was found to bea member of the Ste20 kinase family and named as
Xenopus prickle-interacting kinase, XPIK.
Developmental expression pattern of XPIK isreminiscent of that of XPIK, suggesting functional
interaction between XPK and XPIK. GOF and LOF of
XPIK resulted in perturbation of gastrulation, leading toshortened or spina bifida embryo. Furthermore, wehave found that XPIK is not only sufficient to activate
INK in embryo, but also required for full activation of
INK by Dishevelled. These suggest that XPIK also
plays an essential role in connecting extracellular Wnt
signal to INK activation through Dishevelled and
XPIK.
In addition, we have been attempting to identify
novel regulatory components controlling gastrulation
cell movements by an expression cloning method basedon morphology of dorsal marginal zone explant(Keller's explant) and of embryo. After 1,500 clones
were examined by overexpression in the dorsal regionof embryos, approximately 5% of clones were found to
perturb normal gastrulation cell movements. Functional
relevance of identified genes to cell movements iscurrently under investigation.

II. Regulation of actin cytoskeletal dynamics
during Xenopus gastrulation
Because gastrulation movements are accompanied bydynamic changes in cell polarity, morphology, andmotility, it is very likely that actin cytoskeleton iscarefully regulated. Thus, we analyzed the regulatory
mechanism of actin cytoskeletal dynamics during thisprocess. Among several factors implicated in theregulation of the actin cytoskeleton, we decided tofocus on myristoylated alanine-rich C kinase substrate(MARCKS). MARCKS is an actin-binding, membraneassociated protein previously implicated in theregulation of F-actin dynamics. It has been investigatedmainly in tissue culture cells and biochemical analyses.
MARCKS knock-out mice has been reported to showneural tube defects. Yet, its molecular mechanism has
not been elucidated. We demonstrate that MARCKS isessential for gastrulation movements and neural tube
closure in Xenopus embryos. Cell biological analyses

FOR BASIC BIOLOGY

revealed that MARCKS knock-down with Morpholino
oligo (MO) significantly reduced cortical actin
formation and caused defects in cell polarity, adhesion,
motility and protrusive activity, leading to thesedevelopmental defects. We also showed that the Wnt
pathway dramatically promoted the formation oflamelipodia- and filopodia-like protrusions and
MARCKS is required for this activity. These findings
show that the Wnt signaling pathway regulates corticalactin dynamics and MARCKS is requisite for the Wnt
function. We conclude that MARCKS is essential for
dynamic morphogenetic movements during
embryogenesis.

Figure 1 MARCKS is required for convergent extension
movements. MARCKS Mo or control Mo, Rhodamine
dextran (RhDx), and Venus mRNA were co-injected into
one of the two dorsal blastomeres. Venus mRNA alone
was injected into the other dorsal blastomere. In theabsence of MARCKS Mo , red cells and non-red cells
were polarized and intercalated. In contrast, MARCKS
Mo-injected cells were not polarized and did notparticipate in the intercalation.

III. Genetic screening for novel DPP/BMP
signaling components utilizing Drosophilamodel system
Drosophila is one of the ideal model organisms todissect signal transduction pathway by genetic methods.
We have carried out dominant suppressor screening fora transgenic mutant fly that expresses activated
DPP/BMP type-I receptors in wing imaginal discs. We
isolated 19 suppressor mutants, Suppressor of
constitutively activated Dpp singnaling (Scad). Allelesof punt, Mad, shn and dCrebA were found in isolated
Scad mutants . Most of the Scad mutants encode anuclear protein suggesting these molecules regulates
DPP signaling at nuclear level.
We now focus to study one of the mutants Scad67.
Scad67 was also isolated by a Mexican group andnamed as tonnalli (tna). We also isolated vertebratehomologs of Scad67/tna , TONAS-1 and TONAS-2 .
The most characteristic feature of these proteins is theexistence of a single SP-RING finger motif in the
middle. The SP-RING motif was originally found in the
PIAS family SUMO-E3 ligase proteins. We found thatTONAS has SUMO -E3 ligase activity and TONAS
facilitates specific SUM0-2/3 conjugation to TONAS
itself. TONAS also shows strong activity of nuclear
body formation in cultured cells. TONAS effectively
recruits transcriptional regulators including PML, CBP
and P300 to the nuclear body.
It has been shown that the Trithorax groupcomponets are essential factors in A TP-dependent

25
chromatin remodeling complex. Our results suggest arole for Scad67/TNA and TONAS in the connection of
the Trithorax/SWIISNF chromatin remodeling complex
to CBP/P300 , the relocation of these protein complexes
into the nuclear substructure, and the regulation of gene
expression.

IV. Brachyury downstream notochorddifferentiation in the ascidian embryo
Ascidians, urochordates , are one of the three chordategroups, and the ascidian tadpole is thought to represent
the most simpbfied and primitive chordate body plan.
It contains a notochord, which is a defining
characteristic of chordate embryo composed of only 40
cells. To understand the morphogenesis in this simple
system, we have focused on a gene, Brachyury, which
is known to play an important role in the notochord
development. In ascidian , Brachyury is expressed
exclusively in the notochord and the misexpression of
the Brachyury gene ( Ci-Bra) of Ciona intestinal is issufficient to transform endoderm into notochord. This
gene encodes a sequence-specific activator that containaT-box DNA-binding domain, and in vertebrates, it isinitially expressed throughout the presumptive
mesoderm and gradually restricted to the developing
notochord and tail bud. The phenotype of the Brachyury
mutants in mice and zebrafish revealed that this gene isessential for notochord differentiation. Our goal is toelucidate the down stream pathway of this importantgene in ascidian in order to set the stage forunderstanding not only the formation and function of
the notochord but how this important structure has
evolved. We conducted the subtractive hybridization
screens to identify potential Brachyury target genes thatare induced upon Ci-Bra overexpression. Out of 501
independent eDNA clones that were induced, 38 were
specifically expressed in notochord cells. We
characterized of subcellular-localizations of the 20 GFP
fusion gene products in the notochord cell. These
products observed after electroporating the embryos at
one-cell stage with GFP fusion gene containing
notochord specific promoter. They showed various
subcellular localizations in the notochord cells of thetadpole tails. In addition to investigate the actual
functions of the genes during the notochord formation ,functional analyses were performed by injecting ofantisence morphollino oligos.

V. Comprehensive analysis of developmentally
regulated genes using eDNA microarray
In order to examine the global expression profile
during early development of Xenopus laevis, we havecollected massive EST sequences from threenormalized eDNA libraries of early gastrula, neurula
and tailbud stage. To date, more than 100 ,000 ESTs(~30% of Xenopus laevis ESTs registered in the public
database) were produced. Using these eDNA clones, we
generated the NIBB 40k eDNA macroarray and 4.6k
non-redundant eDNA microarray . With the DNA

NATIONAL INSTITUTE

26
arrays, we conducted a series of large-scale screening of
the genes which expressions are regulated by sometranscription factors and signaling factors (Xnr-1,
FASTl , FGF etc.). Consequently, we have been able toisolate lots of the candidate genes that encode a variety
of signal transduction and transcription regulatorycomponents , and also cytoskeletal components,suggesting dynamic cellular changes in the early
development of Xenopus laevis. This also proves DNA
array to be an effective screening assay for novel genes
which function during early development process of
Xenopus laevis.
Adding the EST sequences , the assembled sequences
and their annotation information are available throughthe web at NIBB Xenopus laevis EST database XBD2
(Fig.2). XDB2 also provides the whole mount in situ
hybridization images as the spatial expression pattern(Fig.3).

.,

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EST p1t>jc-\'1. XDll2 ;, indu<.lc.J theth:u! 100.000
EST in funnauM fu>m lhc N lflll dlS1\ !ilmuic:>'. 10.q "'"'tuenC~:~.~"" ''J: >C<tlkl'K:~.< ~nJ lhdr •imrk ;um<lblion b)'
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I(....,.,Uin'lll l'l\l_.ilxl '.' \\'1: ~ 1:,\ \l.f\\i ll illj: Ill<' J k!h~J U:lllJCrip!HIIW: du rin~ c:"MI) ,Jncktp!n>'lll:ll JI'I'X"I:'U With .W~
niJICI\>~rr..y • .J ,I\1. ur · nli..'nOJrt:l~. ~0~ nr.()Ji~:u min'wr~y . Wr 011\' ~h., c:~nyin~ tlllt dn· Xj,()()O ~lot>;!! fun;:livn~l ~;n;~ly>~~. It ii COillfl'>.<al of fuU -kn~ lh d.lNA >fi!Utn..iny: :mtl. ~ lobal ~Jmik»< of func Jiun ltlll lp i.! hy Jn<:ln~ <>fn!((NA "wr~~prc .. ion. ~nc.l ""m.,n..., I<Xhniq~~ t ~INphnlinu oh ~u. KNt\l. nuiCm:d <lcpk tinn l"IC ).

--- ~[!l!lllllE: Latest data set:

~ · ·Figure 2 XDB2 (http://xenopus.nibb.ac.jp).

Figure 3 Whole mount in situ hybridization images.

;

I.

Publication List
Azumi, K. , Takahashi, H., Miki, Y., Fujie, M. , Usami, T.,
Ishikawa , H. , Kitayama, A., Satou, Y., Ueno , N. &Satoh , N. (2003) Construction of a eDNA microarrayderived from the ascidian Ciona intestinalis. Zoological
Science. 20, 1223-1229.
Can·eira-Barbosa, F. , Concha, M. L., Takeuchi , M., Ueno,
N. , Wilson , S . W. and Tada , M. (2003) Prickle!regulates cell movements during gastrulation andneuronal migration in zebrafish. Development 130,4037-4046.
Hotta, K. , Takahashi, H., Ueno, N. & Gojobori, T. (2003)
A genome-wide survey of the Genes for Planar Polarity
sign aling or Convergent Extention-related genes in
Ciona intestinalis and comparisons of evolutionary
conserved signaling components. Gene. 317, 165-185.
Kinoshita, N. , Iioka, H. , Miyakoshi , A. and Ueno , N.
(2003) PKC6 is essential for Dishevelled function in a
noncanonical Wnt pathway that regulates Xenopus
convergent extension movements. Genes Dev. 17, 16631676.
Kurata, T. and Ueno, N. (2003) Xenopus Nbx, a novel NK1 related gene essential for neural crest formation . Dev.
Biol. 257, 30-40 .
Ohkawara, B., Yamamoto, T. S., Tada, M. and Ueno, N.
(2003) Role of glypican 4 in the regulation of
convergent extension movements during gastrulation in
Xenopus laevis. Development 130, 2129-2138 .
Suzuki , M., Ueno, N. and Kuroiwa A. (2003) Hox proteinsfunctionally cooperate with the GC box-binding protein
system through distinct domains. J. Bioi. Chem. 278,
30148-30156.
Takeuchi, M. , Nakabayashi , J. , Sakaguchi, T. , Yamamoto,S . T. , Takahashi, H ., Takeda , H . Ueno , N . (2003)
Prickle-related gene in vertebrates is es sential for
gastrulation cell movements. Curr. Biol. , 15, 674-679.

FOR BASIC BIOLOGY27

DEPARTMENT OF REGULATION BIOLOGY

Chairperson: NODA, Masaharu

DIVISION OF MOLECULAR NEUROBIOLOGYDIVISION OF CELLULAR REGULATIONDIVISION OF BIOLOGICAL REGULATION
AND PHOTOBIOLOGY (ADJUNCT)DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)The department is composed of two regular divisions and two adjunctdivisions. The study of this department is directed towards molecularmechanisms for the development and functioning of the central nervous
systems in vertebrates, and also those for the signal transduction ofplants and microorganisms with respect to the environmental factors,
such as light, temperature and salinity.

NATIONAL INSTITUTE28
DIVISION OF MOLECULAR NEUROBIOLOGYNODA, Masaharu
SHINTANI, Takajumi
SAKUTA, Hiraki
HIYAMA , Takeshi
TAKEUCHI,Yasushi
Technical Staff:
NIBB Research Fellows: FUKADA, Masahide
TAKAHASHI, Hiroo (Oct. '03~)
JSPS Postdoctoral Fellows: SUZUKI, Ryoko
TAKAO, Motoharu (Apr. ' 03~)
Postdoctoral Fellows:
FUJIKAWA, Akihiro n
KATO, Akira' 1 (~ Sept. '03)
NAKAMURA, Takahiro ''YAMAMOTO, Yasunori n (Apr.'03-)
TAMURA, Hiroshi
Graduate Students:
OHKAWARA, Takeshi
TAKAHASHI,Hiroo (~Sept. '03)
IHARA, Masaru
TORIUMI, Shigeru (~Sept. '03)
ETANI, Kazuma (Apr. ' 03~)
SHIMIZU, Hidetada (Apr . '03~)
TANAKA , Rumi (Apr. ' 03~)
YONEHARA, Keisuke (Apr. ' 03~)
YUASA, Junichi 21 (~Mar. '03)
Visiting Scientists:
WATAKABE, lkuko (~Feb. '03)
KODAMA , Akiko
Secretary:
AYABE, Yuko (~Sept. '03)
Technical Assistant:
JST Technical Staffs:
MIZOGUCHI, Masae
GOTOH, Megumi
YAMADA, Kaoru
MATSUI, Mie (~Sept. '03)
AYABE, Yuko (Oct. '03~)

Professor:
Research Associates:

n CREST, JST 21 PREST, JST

We have been studying the molecular and cellularmechanisms underlying the development of thevertebrate central nervous system. It covers all the
major events including the patterning of the nervous
system, neuronal differentiation, axonal navigation andtargeting , synapse formation and plasticity , andneuronal regeneration, mainly in the visual system. The
scope of our interests also encompasses various
functions of the matured brain , including sensation ,behavior, learning and memory.

I. Regional specification in the retina andtopographic retinotectal projection
Topographic maps are a fundamental feature ofneural networks in the nervous system. Understanding
the molecular mechanisms by which topographicallyordered neuronal connections are established during
development has long been a major challenge indevelopmental neurobiology. The retinotectal
projection of lower vertebrates including birds has beenused as a readily accessible model system. In thisprojection, the temporal (posterior) retina is connectedto the rostral (anterior) part of the contralateral optic
tectum, the nasal (anterior) retina to the caudal(posterior) tectum, and likewise the dorsal and ventral
retina to the ventral and dorsal tectum, respectively.
Thus , images received by the retina are precisely
projected onto the tectum in a reversed manner.

Since 1992, we have been devoting our efforts tosearching for molecules with asymmetrical distribution
in the embryonic chick retina, and to characterization of
their roles in the topographic retinotectal projection. In
1996 , we first identified two winged-helix
transcriptional regulators, CBF-1 and CBF-2 , expressedin the nasal and temporal retina, respectively.
Misexpression experiments in the retina using a
retroviral vector showed that these two transcription
factors determine the regional specificity of the retinal
ganglion cells, namely, the directed axonal projections
to the appropriate tecta! targets along theanteroposterior axis. Secondly, we identified a novel
retinoic acid-generating enzyme, RALDH-3, which is
specifically expressed in the ventral region of the retina,
together with a dorsal-specific enzyme RALDH-1.
Furthermore, we recently identified a novel secretory
protein , Ventroptin, which has BMP-4 neutralizing
activity. Ventroptin is expressed in the retina with aventral high-dorsal low gradient at early stages. This
expression pattern is complementary to that of BMP-4.
At later stage (E6~), a nasal high-temporal low gradient
expression pattern of it is also detected. Ventroptin thus
shows a double-gradient expression profile along thedorsoventral and nasotemporal axes. Misexpression of
Ventroptin altered expression patterns of several
topographic genes and projection of the retinal
ganglion-cell axons to the tectum along the both axes.
In this year of 2003, we revealed that misexpressionof CBF-1 represses the expression of EphA3 and CBF2, and induces that of SOHo-1 , GH6, ephrin-A2 andephrin-A5. CBF-1 controls ephrin-A5 by a DNA
binding-dependent mechanism, ephrin-A2 by a DNA
binding-independent mechanism, and CBF-2 , SOHo -1 ,GH6 and EphA3 by dual mechanisms (Fig. lA). BMP-2
expression begins double-gradiently in the retina fromES instead of BMP-4 in a complementary pattern to the
Ventroptin expression. Ventroptin antagonizes BMP-2
as well as BMP-4. CBF-1 interferes in BMP-2 signaling
and thereby induces expression of ephrin-A2. Our datasuggest that CBF-1 is located at the top of the genecascade for the regional specification along thenasotemporal (NT) axis in the retina (Fig. IB) anddistinct BMP signals play pivotal roles in the
topographic projection along both axes.

A
Transcriptional Regulation
as Repressor

ephrin-A5 t
SOHo-1
t
GH6
t
CBF-2
~
EphA3
~

CBF-1
DNA-Binding Independent Mechanism(BMP signaling cascade andthe olher cascade)ephrin-A2 t
SOHo-1
t
GH6
t
CBF-2
~
EphA3
~

Fig.!. The molecular mechanisms by which CBF-1
controls the expression of topographic molecules. (A)Schematic representation of modes of actions of CBF-1.
You can see that one Eph-ephrin system is controlled as a
set by each mode of CBF-1 action.

FOR BASIC BIOLOGY

8

29

?

CBF-2
SOHo-1/ ' GHG----i EphA3

/'y
I X

CBF-1

'\,

BMP-2

ephrin-AS

I ephrin-A2

Tl
Ventroptin(B) Expressional regulation of the topographic moleculesalong the NT axis by CBF-1. EphA3 and ephrins are
directly implicated in the control of axon guidance. CBF1 and Ventroptin repre ss expression of BMP-2 byinhibiting BMP signaling as an interrupter and antagonist,
respectively , and induce ephrin-A2 expression. CBF-1
represses the transcription of negative regulators, X and Y.
SOHo-1 and GH6 inhibit the expression of EphA3. CBF-1
also represses CBF-2 expression , however , its
downstream target genes have not been identified yet.

Currently, with respect to the other identified
molecules, we are conducting misexpression
experiments using chick embryos and generatingknockout and transgenic mice to elucidate themolecular functions. We expect that our studies willlead to dissection of the molecular mechanism
underlying the retinal patterning and topographic
retinotectal projection, and ultimately to uncovering thebasic principles for establishing complicated but
extremely precise neural networks.

II. Axonal morphogenesis and behavior
During development , cells undergo dynamic
morphological changes by rearrangements of thecytoskeleton inc! uding microtu buies . However,molecular mechanisms underlying the microtubule
remodeling between orientated and disorientatedformations are almost unknown . We found that novel
subtypes of collapsin response mediator proteins(CRMP-As) in addition to the originals (CRMP-Bs),
which occur from the alternative usage of different firstcoding exons, are involved in this conversion of
microtubule patterns.
Overexpression of CRMP2A and CRMP2B in chickembryonic fibroblasts induced orientated anddisorientated patterns of microtubules , respectively.
Moreover, sequential overexpression of another subtype
overcame the effect of the former expression of thecountersubtype. Overexpression experiments incultured chick retinae showed that CRMP2B promoted
axon branching and suppressed axon elongation of
ganglion cells , while CRMP2A blocked these effects
when co-overexpressed (Fig. 2). Our findings suggestthat the opposing activities of CRMP2A and CRMP2B
contribute to the cellular morphogenesi s including
neuronal axonogenesis through remodeling of
microtubule organization.

Fig.2. Antagonistic effects of CRMP2A and -2B on axon
morphology. Retinae from chick E6 embryos were
electroporated with IGG vector to express CRMP2B (A)and CRMP2B + CRMP2A (B), and cultured for 2 days.

III. Physiological roles of protein tyrosine
phosphatase receptor type Z (Ptprz)
Protein tyrosine phosphorylation plays crucial roles
in various biological aspects including all stages ofbrain development. In 1994, we found that
PTP1;;/RPTPf3/Ptprz, a nervous system-rich receptortype PTP, is expressed as a chondroitin sulfate
proteoglycan in the brain. Ptprz is expressed from theearly developmental stage to the adulthood in neurons
as well as astrocytes. This suggests that this molecule
plays variegated roles in the brain development andbrain function.
We found in 1996 that Ptprz binds pleiotrophin(PTN)/HB-GAM and midkine (MK) , closely related
heparin-binding growth factors which share many
biological activities. The chondroitin sulfate portion of
Ptprz is essential for the high affinity binding (Kd =~0.25 nM) to these growth factors, and removal ofchondroitin sulfate chains results in a marked decreaseof binding affinity (Kd = ~ 13 nM).
To identify the substrate molecules of Ptprz , werecently developed the yeast substrate-trapping system.
This system is based on the yeast two-hybrid systemwith two essential modifications: Conditional
expression of v-src to tyrosine-phosphorylate the prey
proteins and screening using a substrate-trap mutant of
PTP as bait. Using this system, we successfully
isolated a number of candidate clones for substratemolecules (ex . GITl/Cat-1) and continuouslyinteracting molecules (ex. PSD-95/SAP90) for Ptprz.
We are now continuing efforts to characterize thesecandidate clones.
In addition, to know the physiological roles of Ptprz
in vivo, we generated Ptprz-deficient mice in which the
Ptprz gene was replaced with the LacZ gene in 1997.
We are currently studying the phenotype of Ptprzdeficient mice biochemically, anatomically ,physiologically and ethologically.
We reported this year that mice deficient in Ptprz do
not show mucosal damage by VacA, although VacA is
incorporated into the gastric epithelial cells to the same
extent as in wild-type mice (Fig. 3). The vacuolating
cytotoxin VacA produced by Helicobacter pylori causes
massive cellular vacuolation in vitro and gastric tissue
damage in vivo, leading to gastric ulcers , whenadministered intragastrically. Primary cultures ofgastric epithelial cells from Ptprz•'• and Ptprz·'· micealso showed similar incorporation of VacA, cellular

NATIONAL INSTITUTE

30
vacuolation and reduction in cellular proliferation, but
only Ptprz•'+ cells showed marked detachment from areconstituted basement membrane 24 h after treatment
with VacA. VacA bound to Ptprz, and the levels oftyrosine phosphorylation of Gitl, a Ptprz substrate,
were higher after treatment with VacA, indicating that
VacA behaves as a ligand for Ptprz. Furthermore, PTN,
an endogenous ligand of Ptprz, also induced gastritis
specifically in Ptprz•'• mice when administered orally.
Taken together, these data indicate that erroneous Ptprz
signaling induces gastric ulcers.

In contrast, these responses were not observed in Na,immunonegative cells or neurons of Na_,-deficientmutant origin . Transfection of Na, eDNA conferred thesodium sensitivity on Nax-deficient cells. All of the
GABA-immunopositive neurons isolated from the SFO
responded to the extracellular sodium increase. Basedon these findings, we proposed that GABAergic
inhibitory neurons expressing Na, control the activity of
the SFO and suppress the salt-intake behavior ofanimals under thirst conditions.

Publication List:
A

Fig.3. Pathological analyses of gastric tissues treated with
YacA. (A,a) Stereomicroscopic appearance of the insideof stomachs from Ptprz* mice 48 h after administrationof YacA (500 ~g per kg body we ight). (A ,b)Diagrammatic representation of a showing the gastriculcer (red area with arrowheads). (B) Appearance of theinside of stomachs from Ptprz·'· mice treated equally.
(C,D) Gastric sections stained with hematoxylin andeosin. Loss of epithelial cells and gastric gland structure(arrowheads) was observed in the mucosal layer in
Ptprz•'• mice (C) but not Ptpn'· mice (D). The lower
pane ls are enlargements . Sca le bars: 500 ~n1. M,
Mucosa; Sm, Submucosa; Mp, Muscularis propia.

IV. Na+-Ievel sensing in the brain
Sodium-level sensing system in the brain is essential
for the regulation of the sodium and water balance inbody fluids. Previously, we demonstrated that Na, (alsocalled Na ,.2/NaG) gene is expressed in thecircumventricular organs and Na,.-deficient mice ingest
salt in excess. In Na,-deficient mice, c-jos expression in
the subfornical organ (SFO) and organum vasculosum
laminae terminalis (OVLT) was markedly elevated ascompared with wild-type mice. Stimulation of the
SFO/OVL T of wild-type animals by infusion of a
hypertonic sodium solution into intracerebroventricule(ICY) leads to avoidance of salt intake. In contrast, Na,deficient mice did not show such aversion .
We showed that Na, is a sodium channel which issensitive to the increase of extracellular sodium level.
Entry of sodium ions occurred in response to a rise of
the extracellular sodium concentration (C 112 = 159 mM).

Asahi, M ., Tanaka, Y ., Izumi, T ., Ito , Y ., Naiki, H.,
Kersulyte, D. , Tsujikawa, K., Saito, M., Sada, K.,
Yanagai, S., Fujikawa, A., Noda, M. and Itokawa, Y.
(2003) Helicobacter pylori CagA containing IT AM-likesequences localized to lipid rafts negatively regulates
YacA-induced signaling in vivo. Helicobacter, 8, 1-14.
Fujikawa , A., Shirasaka, D ., Yamamoto , S. , Ota , H .,Yahiro, K ., Fukada , M. , Shintani, T., Wada, A. ,Aoyama, N. , Hirayama, T., Fukamachi, H. and Noda,
M. (2003) Mice deficient in protein tyrosine
phosphatase receptor type Z are resistant to gastric ulcer
induction by VacA of Helicobacter pylori. Nature
Genet., 33, 375-381.
Nakayama, M., Kimura, M. , Wada, A., Yahiro, K., Ogushi ,K., Niidome, T., Fujikawa, A. , Shirasaka, D., Aoyama,
N. , Kurazono, H. , Noda, M. and Hirayama, T. (2004)
Helicobacter pylori VacA activates the p38/activatingtranscription factor 2-mediated signal pathway in AZ521 cells. J. Biol. Chem., 279, 7024-7028.
Sakaguchi, N., Muramatsu, H ., lchihara-Tanaka, K.,
Maeda, N., Noda, M., Yamamoto, T. , Michikawa, M. ,Ikematsu, S. , Sakuma, S. and Muramatsu , T. (2003)
Receptor-type protein tyrosine phosphatase !: as acomponent of the signaling receptor complex for
midkine-dependent survival of embryonic neurons.
Neurosci. Res., 45, 219-224.
Takahashi, H., Shintani, T., Sakuta, H. and Noda, M.
(2003) CBF-1 controls the retinotectal topographicalmap along the anteroposterior axis through multiple
mechanisms. Development, 130, 5203-5215 .
Tanaka, M. , Maeda, N., Noda, M. and Marunouchi, T.
(2003) A chondroitin sulfate proteoglycan PTPtfRPTPf)
regulates the morphogenesis of Purkinje cell dendrites
in the developing cerebellum. J. Neurosci., 23, 28042814.
Watanabe, U., Shimura , T., Sako, N., Kitagawa, J .,Shingai, T. , Watanabe, E., Noda, M. and Yamamoto, T.
(2003) A comparison of voluntary salt-intake behavior
in Na,-gene deficient and wild-type mice with reference
to peripheral taste inputs. Brain Res., 967, 247-256.
Yuasa-Kawada, J., Suzuki, R., Kana, F. , Ohkawara, T. ,Murata, M. and Noda, M. (2003) Axonal morphogenesis
controlled by antagonistic roles of two CRMP subtypes
in microtubule organization. Eur . J. Nuerosci., 17,2329-2343 .
Ohyama, K., Ikeda, E. , Kawamura, K., Maeda, N. andNoda , M . (2004) Receptor-like protein tyrosine
phosphatase l:JRPTPf) is expressed on tangentiallyaligned neurons in early mouse neocortex . Develop.
Brain Res., 148, 121-127.
Shintani , T., Kato, A., Yuasa-Kawada, J ., Sakuta, H.,
Takahashi, M., Suzuki, R., Ohkawara, T., Takahashi, H.
and Noda, M. (2004) Large-scale identification andcharacterization of genes with asymmetric expression
patterns in the developing chick retina. J. Neurobiol. (inpress)

FOR BASIC BIOLOGYDIVISION OF CELLULAR REGULATION

Professor:
Associare Professor:
Research Associates:

MURATA, Norio
MJKAMJ, Koji
SUZUKI, !wane
YAMAMOTO, Hiroshi
Technical Staff:
YAMAGUCHI, Katsushi
NIBB Research Fellow: OHNISHI, Norikazu
JSPS Research Fellow: SHIVAJI, S 11
JSPS Postdoctoral Fellows: SULPICE, Ronan ''JOGADHENU, S. S. Prakash "TA KAHASHI, Shunichi
Postdoctoral Fellows:
KANESAK/, Yu
KANASEKI, Toku
ALLAKHVERDIEV, Suleyman /. 31
FERJANI, Ali
MUTSUDA , Michinori
Visiting Scientists:SZALONTAI, Balazs ' 1
ZSIROS, Otto ' 1
PANICHIKIN Vladimir 51
SHOUMSKAYA, Maria 61
PAITHOONRANGSARID,
Kalayanee 71
HUBSHMANN, Thomas s1
MODANAHALLY, Kiran 11
KOIKE, Yukari
Technical Assistants:
KAIDA , Satomi
ODA, Keiko
Secretaries:
YABUTA, Masako
KUBOKI, Yuko
SUZUKI, Shingo
Graduate Student:
11

from the Centre for Cellular and Molecular Biology,
Hyderabad, India' 1from Universite de Rennes 1, Rennes, France
31
from the Institute of Basic Biological Problems,
Pushchino, Russia
41
from the Biological Research Center, Szeged, Hungary
51 from Moscow State University, Moscow, Russia
61 from Plant Physiology In stitute, Moscow, Russia' 1from National Center for Genetic Engineering andBiotechnology, Bangkok, Thailand
81 from Humboldt Universitiit Berlin, Berlin, Germany

The major thrust of our research efforts is directed
tow a rd s the comprehensive understandin g of themolecular mechanisms that governs the responses ofpl ants and microorgani sms to new environmental. In
particular, our attention is focused on the perception
and transduction of various stress signal s, s uch as
extreme temperatures, osmosis and salinity . Anotherline of our research is focused on the repair mechani sms
from the damage to the photosynthetic machinery under
severe stress conditions. In 2003, significant progress
was made in the following areas.

I. Discovery of four kinds of the two-component
system responsible for perception andtransduction of hyperosmotic and salt signals in
Synechocystis.
Livin g organisms res pond and acc limate tohyperosmo ti c and salt stress by changin g the gene
expression . We previou s ly di scovered th at th ecyanobacterium Synechocystis sp. PCC 6803 (hereafter

31
Synechocystis) responds differently to hyperosmotic

stress and salt stress with respect to regulation of gene
expression and changes in the cytoplasmic volume.
Th ese res ults sugges ted that this organism mi ght
recognize the two kinds of stress as diffe rent signals
and perceived them by different sensors.
To inv es ti gate the components invol ved in theperception and transduction of hyperosmotic and salt
signals in thi s organism , we examined all knockout
mutants for 43 histidine kinases (Hik) and 42 response
regul ators (Rre) by a DNA microarray technique andfound, in contrast to the prediction , that the four kindsof two-component signa]] ing pathways of Hik33/Rre3 1,
Hik34/Rre l , Hikl6/Hik41 /Rrel7 and Hikl0/Rre3 are
involved in pe rceptio n and tr ans duction of both
hyperosmotic and salt signals. However, each of theI
two-component sig na ling pa th ways except
Hikl6/ Hik4l/Rrel7 was found to regulate theexpression of genes that were induced specifically byeither hyperosmotic stress or salt stress, although theyalso regul ated the expression of ge nes which werecommonly induced by these two stress signals (Fig. 1).
For example , Hik34 perceives and tran sduces both
hyperosmotic and salt signal s to the response regulator
Rre l to induce the express ion of different sets of genes.
Although the number of genes which are regulated by
the Hikl0/Rre3 pathway is small , this two-component
system induces different genes in response to diffe rent
stimuli. These findings clearly demon strate that thetwo-compon ent systems constitute complex s ignalinteg ration pathways ra ther than a simpl e " onestimulus-one output" scheme.

fabG
ss/3446

1/r/ 96.1
1/0095s/11541

1/J/16'!. groEL2 x/1()(}(}5slr/687•lr/9]6 dnaK2

IISf>A
c/pB

hhoA
lupG

slr/680

Fig. I . Schematic presentation of fo ur kinds of twocom ponent syste m involved in the p erception a ndtransduction of hyperos motic and salt sig nals . Genes
whose expression is induced by hyperosmot ic stress, bysalt stress, a nd commonly by the two kinds of stress via
each two-component syste m are included in blue, red andpurple rectangles, respective ly.

II. Discovery of four histidine kinases as sensorsof H,O, signals in Synechocystis.
Hydro ge n pe roxid e (H 20 2 ) is produc e d as aninevitable consequence of aerobic life. Microorganisms
detect in creases in the co nc entration of H,O, andregul ate the expression of certain genes to enhancetolerance against this oxidative stress. DNA microarray
analysis of genome-wide expression of genes revealed

NATIONAL INSTITUTE

32

that incubation of Synechocystis cells with 0.25 mM
H,O, dramatically changed the pattern of gene
expression. This treatment induced the expression of 77
genes and repressed the expression of 55 genes withinduction factors > 4.0. A half of the H,O,-inducible
genes were specifically induced by H,O, but by no otherstress, such as heat, cold, light, salt and hyperosmotic
stress. These findings suggest that the oxidative stress
may be distinct from the general stress. Screening formutants in the disruptant library of histidine kinases thatexhibit null response to the H,O,-inducible expressionof genes identified four histidine kinases, Hik33, Hik34,
Hik2 and Hik41, as sensors of H,O, signals. They
regulate the expression of 25, 1, 6, and 2 H,O,-inducible
genes, respectively (Fig. 2).
In Bacillus subtilis, the transcription factor PerR
perceives H,O, signals and regulates the expression of agroup of H,O,-responsible genes. We also examined the
genome-wide patterns of gene expression in the mutant
of PerR homolog in Synechocystis by DNA rnicroarray
analysis. We found that PerR regulates the expressionof five H,O,-inducible genes. These findings indicatethat histidine kinases act as major sensors of H,O,
signals in Synechocystis.

I

I

H2 0 2 stress conditions

r~ ~~t !~,T,,Trs/r/544
ssr20/6
hliA
hliB
hliC
nb/AJ
ndhD2

sl/1549
fee£
futA/
ss/1533
slr0587
ss/250/htpG

slr0967
sl/0939

ahpC
slr0589
sl/1620
sl/1550
pyrB

gifA
gijB
isiA
isiB
sill/58
ssr0692
sl/0788~ ~ UJ [2] [I] [lL]Fig.2. Schematic presentation of four histidine kinases
and a transcription factor PerR involved in the perceptionof H,O, stress and regulation of gene expression.
Numbers in each box correspond to the number of H,O,inducible genes with induction factors > 4.0 whose
expression was regulated by each histidine kinase or thetranscription factor PerR.

III.

Multi-stresssensing

by

Hik33

in

caused by heat and nutritional stresses. Nevertheless,
Hik33 regulates distinct sets of genes under different
stress conditions. Therefore, it is possible that Hik33
senses the various kinds of stress to differentiallyregulate the gene expression in a stress-specific manner.
However, it should be noted that Hik33 regulates theexpression of genes which are induced commonly by
various kinds of stress. Interestingly, Rre31 which is a
cognate response regulator of Hik33 in the gene
expression due to hyperosmotic and salt stress is not
involved in the signal transduction pathway of cold and
H,O, stresses. These results may suggest that Hik33
perceives various kinds of stimulus and sorts theirsignals to the stimulus-specific downstream pathways.
These findings suggest that the two-component signaltransduction system is more complex than a currently
accepted scheme where a single sensory histidine
kinase is tightly coupled with a cognate response
regulator.

IV. Environmental stress inhibits the repair ofphotosystem II by suppressing thetranscriptional and translational activities.
Strong light impairs the photosynthetic machinery, inparticular, photosystem II (PSII), via a process known
as photodamage or photoinhibition and the PSII
reaction center protein Dl is most sensitive target ofphotodamage. However, photodamaged PSII can be
repaired by replacement of the photo damaged Dl withlight-dependently synthesized Dl de novo.
Under natural conditions organisms are exposed to
combinations of various kinds of environmental stress,
such as light, salt, oxidative, heat, and cold stress, that
may synergistically act on the damage to PSII. We havefound that the effects in vivo of light on PSII are
completely different from the effects of the other kindsof stress. Strong light induces photodamage to PSII,
whereas the other kinds of stress inhibit the repair of thephotodamaged PSII and do not accelerate damage to
PSII directly. Results of molecular-biological analysis
reveals that these kinds of stress inhibit either or both of
the transcription and translation of several genes, inparticular, psbA genes for Dl, whose turnover isessential for the repair of PSII. These results suggestthat stress inhibits the repair of PSII via suppression of
the activities of both transcriptional and translational
machineries. We also elucidated that the requirement oflight for the repair can be explained by sustenance of
the intracellular concentration of A TP via thephotosynthetic transport of electrons.

Synechocystis.
Our previous study indicated that Hik33 is involvedin the regulation of gene expression by cold andhyperosmotic stresses. We applied the DNA microarray
technique to examine whether Hik33 also perceives
stress signals other than cold and hyperosmotic stresses.
The results clearly showed that Hik33 is involved also
in the perception of salt, H,O,, and strong-light stresses.
However, it does not regulate the gene expression

V. Transformation of plants to enhance the stress
tolerance of reproductive organs to salt andcold stress.
We showed previously that transformation with thecodA gene for choline oxidase allows plants tosynthesize glycine betaine (GB) and enhances theirability to tolerate various kinds of stress during
germination and vegetative growth. In these years, weFOR BASIC BIOLOGY

examined tolerance of transformed plants to salt stressat the reproductive stages, i.e. , the stages at whichplants are most sensitive to environmental stress. Saltshock treatment of wild-type plants for three days
resulted in the abortion of flower buds and decreased
the number of seeds per silique. Microscopic
examination of floral structures revealed that salt stressinhibited the development of anthers, pistils and petals.
In particular, the production of pollen grains and ovules
was dramatically inhibited. These effects of salt stresswere significantly reduced by transformation with thecodA gene , and our observations suggest that theenhanced tolerance of the transgenic plants was a resultof the accumulation of GB in the reproductive organs.
The cis -unsaturated molecular species ofphosphatidylglycerol (PG) in chloroplasts have been
implicated in the chilling tolerance of plants . We
established homozygous lines of transgenic tobacco(Nicotiana tabacum) that overexpressed a eDNA for
glycerol-3-phosphate acyltransferase, a key enzyme inthe determination of the extent of cis-unsaturation of the
PG , from a chilling-sensitive squash (Cucurbita
moschata) . In transgenic plants, the proportion ofsaturated plus trans-monounsaturated molecular speciesof PG increased from 24% to 65 %. However, thischange did not affect the architecture of thechloroplasts . Chilling stress also damaged
inflorescences much more severely in transgenic plants
than wild-type plants (Fig. 3). These observations
allowed us to conclude that decreases in the proportionof cis-unsaturated PG enhanced the sensitivity tochilling of reproductive organs.

Fig.3. The sensitivity of inflorescence of tobacco plants
to low temperature 5•c. Left: Wild-type plants whichcontain a high level of the cis-unsaturated molecularspecies of phosphatidylglycerol (PG). Right: Transgenic
plants which contain a low level of the cis-unsaturated
molecular species of PG . The results of this stud y
indicate that changes in the level of unsaturated PG
molecular species have a great impact on the chilling
sensitivity of plants in a broad range of the life cycle.

33

List of publication:(1) Original articles
Szalontai, B., Kota, Z. , Nonaka, H., Murata, N. (2003)
Structural consequences of genetically engineered
saturation of the fatty acids of phosphatidylglycerol in
tobacco thylakoid membranes . An FTIR Study .
Biochemistry, 42, 4292-4299.
Ferjani , A. , Mustardy, L. , Sulpice, R., Marin, K. , Suzuki,
I., Hagemann, M., Murata, N. (2003) Glucosyl-glycerol,a compatible solute, sustains cell division under salt
stress. Plant Physiol., 131 , 1628-1637.
Inaba, M., Suzuki , I. , Szalontai, B. , Kanesaki, Y. , Los,
D.A., Hayashi, H., Murata, N. (2003) Gene-engineered
rigidification of membrane lipids enhances the cold
inducibility of gene expression in Synechocystis. J. Bioi.
Chem., 278, 12191-12198.
Marin , K., Suzuki , I. , Yamaguchi, K. , Yamamoto, H. ,Ribbeck, K., Kanesaki, Y., Hagemann , M., Murata, N.
(2003) Identification of histidine kinases that act as
sensors in the perception of salt stress in Synechocystis
sp. strain PCC 6803. Proc. Nat/. Acad. Sci. USA , 100,
9061-9066.
Sulpice, R., Tsukaya, H., Nonaka , H., Chen, T .H.H .,Murata, N. (2003) Enhanced formation of flowers andsee ds in salt-stressed Arabidopsis after genetic
engineering of the accumulation of glycinebetaine.
Plant J., 36, 165-176.
Sarcina, M., Murata, N., Tobin, M.J., Mullineaux , C.W.
(2003) Lipid diffusion in the thylakoid membranes of
the cyanobacterium Synechococcus sp.: Effect of fatty
acid desaturation . FEBS Lett., 553, 295-298.
Allakhverdiev , S.I., Mohanty , P., Murata , N. (2003)
Dissection of photodamage at low temperature andrepair in darknes s suggests the existence of anintermediate form of photodamaged photosystem II.
Biochemistry, 42, 14277-14283.
Kanervo , E., Spetea, C., Nishiyama , Y. , Murata , N.,
Andersson, B ., Aro, E. (2003) Dissectin g acyanobacterial proteolytic system: efficiency ininducing degradation of the Dl protein of photosystem
II in cyanobacteria and plants. Biochim. Biophys. Acta,
1607, 131-140.

(2) Review articles
Mikami, K. , Murata, N. (2003) Membrane fluidity and theperception of environmental signals in cyanobacteria
and plants. Prog. Lipid Res. , 42, 527-543.
Mikami, K., Suzuki, I., Murata, N. (2003) Sensors of
abiotic stress in Synechocystis . Topics in Current
Genetics: Plant Response to Abiotic Stress, vol. 4(eds., Hirt, H., Shinozaki, K.), Springer, Berlin, p.l03-ll9.

NATIONAL INSTITUTE34

DIVISION OF BIOLOGICAL REGULATION AND
PHOTOBIOLOGY (ADJUNcn
Professor (Adjunct):
WADA, Masamitsu
KIKUCHI, Kazuhiro
Research Associate:
NIBB Research Fellow: OGURA, Yasunobu
MEXT Postdoctoral Fellow: TAKAHASHI, Fumio
Postdoctoral Fellow:
CHRISTENSEN, SteenOIKAWA, Kazusato
Graduate Students:(Tokyo Metropolitan University)
SUETSUGU, Noriyuki(Tokyo Metropolitan University)

Plants respond to light as an environmental factor to
optimize development and regulate other physiologicalphenomena. Phytochrome and blue light receptors, suchas cryptochrome and phototropin (phot), are the main
photoreceptors for plant photomorphogenesis. The goalof our research is to elucidate the photoperception ~nd
the signal transduction pathways of photomorphogenesis.

1· 2 Adiantum phy3
Adiantum phytochrome3 (PHY3) is a uniquechimeric protein with phytochrome structure in the Nterminal half and phototropin structure in the Cterminal half. Transient expression of PHY3 ormodified PHY3 genes in rap (red light-induced
aphototropic) mutants of Adiantum reveal that phy3 is
the photoreceptor of red light-induced chloroplast
photorelocation movement, and that the phy3 signal
might be transferred through the C-terminal,
phototropin region.
The phot region of phy3 has not yet been shown tofunction as a blue light photoreceptor, but the above
method of transient expression of modified PHY3 in rap
and phot mutants may provide experimental evidence of
this possibility soon.

II. Gene targeting and gene silencing
In order to elucidate the role of genes in Adiantum
and rice, we have tried to establish new methods forgene targeting in these organisms.

I. Chloroplast relocation movement

2· 1 Miniature transposable element

One of our major subjects is chloroplast photorelocation movement, which is thought to be one of thesimplest phenomena in this field. We use the fern
Adiantum capillus-veneris and the moss Physcomitrella
patens as model plants for our cell biological approachsince the gametophytes are very sensitive to light andthe organization of the cells is very simple. We also use
Arabidopsis mutants to identify the genes regulating
chloroplast photo-relocation movement.

Transposable elements constitute a large portion of
eukaryotic genomes and contribute to their evolution
and diversification.· Miniature inverted-repeat
transposable elements (MITE) constitute one of themain groups of transposable elements. MITEs havebeen found in wide range of organisms but active
MITEs have not been identified. We found a new classof MITEs in rice and named them miniature Ping
(mPing). mPing was identified as the first active MITE
from any organism and the first active DNA transposonfrom rice. mPing is a short 430 base pairs element with
15 base pair terminal inverted repeats that lacks atransposase. mPing elements are activated in calli
derived from anther culture and excise efficiently fromoriginal sites to reinsert into new loci. The mPingassociated Ping element which has a putativetransposase sequence was also found and shown totranspose within the rice genome.
Rice is the most agriculturally important crop in theworld. mPing!Ping transposon system is a usefulmolecular tool for gene isolation and gene knockout inrice.

1-1 Arabidopsis
Chloroplasts accumulate at the cell surface under
weak light and escape from the cell surface to theanticlinal wall under strong light to optimize
photosynthesis. We identified the photoreceptors in
Arabidopsis. Phototropin2 (phot2) mediates the
avoidance response under strong light and photl andphot2 mediate, redundantly, the accumulation response
under weak light. However, components of signaltransduction pathways still remain to be identified. A
mutant called chup1 is deficient in chloroplast
movement and consequently chloroplasts gather at thebottom of cells. CHUP 1 is a novel gene that has ahydrophobic region in the N-terminus and an actin
binding domain, a prolin-rich region, and two leucin
zippers. It was confirmed that the actin-binding domain
binds F-actin. When fusion proteins of the N-terrninal,hydrophobic region of CHUP 1 with GFP wereexpressed transiently in chup1 mutant cells,
fluorescence of the fusion proteins were found in theouter part of chloroplasts, likely on the outer membraneof chloroplasts. CHUP 1 is proposed to play animportant role on chloroplasts movement of bothaccumulation and avoidance responses.

2· 2 Expression sequence tag in Adiantum
To understand the genetic information of a fern,
Adiantum capillus-veneris, a normalized eDNA library

was constructed from prothallia grown under whitelight and analysis of expressed sequence tags (EST)
were carried out. Approximately 10,000 clones weresequenced and clustering of these obtained sequences
was performed. As a result, 7,132 non-redundantgroups were generated. These groups were subjected tosimilarity searches to identify putative function.
Approximately l ,600 EST groups were found to beFOR BASIC BIOLOGY
similar to sequences of genes registered in the public
database. About 1,100 EST groups showed similarity
to sequences of unknown function. The remaining ESTgroups showed no significant similarity and wereclassified as novel sequences.

List of publication:
Original articles
Stoelzle, S., Kagawa, T., Wada, M., Hedrich, R., Dietrich,P . (2003) Blue light activates calcium-permeablechannels in Arabidopsis mesophyll cells via thephototropin signaling pathway. Proc. Nat!. Acad. Sci.
USA 100, 1456-1461.
Sato , Y ., Wada, M., Kadota, A . (2003) Accumulation
response of chloroplasts induced by mechanicalstimulation in bryophyte cells. Planta 216, 772-777.
Iwata, T. , Nozaki, D. , Tokutomi , S., Kagawa, T., Wada,
M. , Kandori, H. (2003) Light-induced structural changes
in the LOV2 domain of Adiantum phytochrome3 studied
by low-temperature FfiR and UV-visible spectroscopy.
Biochemistry 42, 8183-8191.
Oikawa , K., Kasahara, M., Kiyosue , T ., Kagawa , T. ,Suetsugu, N. , Takahashi, F. , Kanegae, T. , Niwa, Y. ,Kadota, A., Wada, M. (2003) CHUPI is essential for
proper chloroplast positioning. Plant Cell 15 , 28052815.

35
Srinivas , A., Behera, R. K. , Kagawa, T ., Wada, M.,
Sharma, R. (2003) High pigment! mutation negatively
regulates phototropic signal transduction in tomato
seedlings. Plant Physiol. in press
Lamparter, T., Kagawa, T. , Brucker, G., Wada, M. (2003)
Positive and negative tropic curvature induced by
microbeam irradiation of protonemal tip cells of themoss Ceratodon purpureus. Plant Biology in press.

Review articles
Sato, Y. , Kadota, A. , Wada, M. (2003) Chloroplast
movement: dissection of events downstream of photoand mechano-perception. J. Plant Res. 116, 1-5.
Wada , M. , Kagawa , T ., Sato, Y. (2003) Chloroplast
movement. Annu. Rev. Plant Bioi. 54, 455-468.
Wada, M. (2003) Blue light receptors in fern and moss.
In: ESP Comprehensive Series in Photoscience,
Photoreceptors and Light Signaling. Ed. By Batchauer,
Elsevier Science Publishers.
Kasahara, M ., Wada, M . (2004) Chloroplast avoidance
movement. In: Annual Plant Reviews "Plastids" Ed. byMoller, Kruwer Academic Publishers, in press.
Wada, M., Kanegae, T. (2004) Photomorphogenesis ofFerns . In: Photomorphogenesis in Plants 3ed Edition.
Ed. by Schafer and Nagy, Kruwer Academic Publishers,
in press.

FigureLocalization of N-CHUP-GFP in Arabidopsis palisade mesophyll cells as confocal microscopic images of the opticalsections transiently expressing GFP (A-C), pt-sGFP (D-F), AtOEP7-GFP (E-G) and N-CHUP-GFP (H-J). The green and red colors
show fluorescence from GFP and autofluorescence from chloroplasts, respectively. Images of green and red channel were merged(C, F, I and L). Scale bar: 10 Jim. OEP is a membrane protein known to distribute at the chloroplast outer membranes (Kind gift ofDr. Yasuo Niwa).

/

NATIONAL INSTITUTE36

DIVISION OF BEHAVIOR AND NEUROBIOLOGY(ADJUNCn

Professor (Adjunct):
Associate Professor(Adjunct):
Research Associate:

MORI, Yuji
TSUKAMURA, Hiroko
IMAMURA, Takuya

This adjunct division will start at April, 2004.

FOR BASIC BIOLOGY37

LABORATORY OF GENE EXPRESSION AND REGULATION

Chairperson: YAMAMOR/, Tetsuo

DIVISION OF GENE EXPRESSION AND REGULATION IDIVISION OF GENE EXPRESSION AND REGULATION II
DIVISION OF SPECIATION MECHANISMS IDIVISION OF SPECIATION MECHANISMS II

The laboratory consists of four regular divisions and conducts
researches into regulatory mechanisms of gene expression in
microorganisms, plants and animals.

NATIONAL INSTITUTE38
DIVISION OF GENE EXPRESSION AND
REGULATION I
Professor:
Research Associates:!IDA, Shigeru
TERADA,Rie
HOSHINO, Atsushi
TSUGANE, Kazuo
Technical Staffs:
FURUKAWA, Kazuhiko
TANAKA-FUKADA, Sachiko
NIBB Research Fellow: CHOI, Jeong-Doo
JSPS Postdoctoral Fellows: PARK, Kyeung-Il
EUN, Chan-Ho *Postdoctoral Fellows:
JOHZUKA-HISATOMI, Yasuyo
ISHIKAWA, Naoko **Visiting Scientist:
KOUMURA, Tashiro ***Graduate Students:
OHNISHI, Makoto n/KEUE, Natsuko 21 ****TAKAGI, Kyouko 11 *****
Technical Assistants:
MORITA, Yasumasa
NAGAHARA, Miki
SAITOH, Miho
Secretary:
SANJO, Kazuko*from October, 2003** until March, 2003***from June, 2003****from April until November, 2003*****from November, 2002
n Graduate University for Advanced Studies
21
11

from Graduate School of Tokyo University
from Graduate School of Hokkaido University

The main interest of the group is in 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 on flower pigmentation ofmorning glories. In addition, we are undertaking reverse
genetic approaches in order to elucidate the nature of
dynamic genome in plants.

I. Spontaneous mutants in morning glories.
Considerable attention has recently been paid to themorning glory genus Ipomoea because of theexperimental versatility of its floral biology including
the genetics of floral variation, flavonoid biosynthesis,
and transposon-induced mutations. The genus Ipomoea
includes about 600 species distributed on a worldwide
scale that exhibit various flower morphologies andpigmentation patterns. A large number of Ipomoea
species can be found in the Americas, particularly in
Mexico. Among the genus Ipomoea, three morning
glories, Ipomoea nil (the Japanese morning glory),
Ipomoea purpurea (the common morning glory), andIpomoea tricolor, were domesticated well as
floricultural plants, and many mutants displaying
various flower pigmentation patterns were isolated.
Among these morning .glories, I. nil and /. purpurea

belong to the same subgenera Ipomoea, whereas I.
tricolor was classified into another subgenera,
Quamoclit. Both I. nil and I. tricolor display blue
flowers (see Figure 2A) that contain the peonidin (3'methoxyl cyanidin) derivative named Heavenly Blue
Anthocyanin (HBA), and I. purpurea produces dark
purple flowers containing a cyanidin derivative thatlacks one glucose molecule and a methyl residue from
HBA. All of them produce dark-brown seeds. As
floricultural plants, spontaneous mutants of I. purpurea
and I. tricolor displaying either white or reddish flowers
were obtained while various spontaneous mutants of /.
nil exhibiting many different flower colorations weregenerated.
Although I. nil has long been believed to haveoriginated from Southeast Asia, a hypothesis that I. nil
may have arrived in Asia from tropical America wasalso recently proposed. In either case, the plant had
been introduced into Japan from China approximately
in the 8th Century as a medicinal herb. The seeds wereused as a laxative, and the plant became a traditional
floricultural plant in Japan around the 17th Century. /.
nil has an extensive history of genetic studies, and anumber of its spontaneous mutants related to the colorand shape of the flowers have been isolated. It was also
used extensively for physiological studies of thephotoperiodic induction of flowering.
The wild-type I. purpurea plant, which originatedfrom Central America, was probably introduced toEurope in the 17th Century, and cultivars with whiteand red flowers were already recorded in the late 18th
Century. Like I. purpurea, I. tricolor originated fromCentral America, and several spontaneous mutants
exhibiting various flower pigmentations were obtainedin the mid-20th Century.
As Figure 1 shows, the spontaneous mutants of I. nil,I. purpurea, and I. tricolor carrying the magenta, pink,
and fuchsia alleles, respectively, produce reddish
flowers containing pelargonidin derivatives, and all ofthem are deficient in the gene for flavonoid 3 'hydroxylase (F3'H). The magenta allele in I. nil is a
nonsense mutation caused by a single C to T basetransition generating the stop codon TGA, and thecultivar Violet carries the same mutation. Several tested
pink mutants in I. purpurea carry inserts of the 0.55-kb
DNA transposable element Tip201 belonging to the
AciDs superfamily at the identical site. No excision of
Tip201 from the F3'H gene could be detected, and bothsplicing and polyadenylation patterns of the F3'H
transcripts were affected by the Tip201 integration. The
fuchsia allele in /. tricolor is a single T insertion
generating the stop codon TAG, and the accumulationof the F3'H transcripts was drastically reduced by thenonsense-mediated RNA decay.
The I. tricolor spontaneous mutant Blue Star carrying
the mutable ivory seed-variegated allele exhibits paleblue flowers with a few fine blue spots and ivory seeds
with tiny dark-brown spots (Figure 2). The mutableallele is caused by an intragenic tandem duplication of
3.3 kb within a gene for transcriptional activator

FOR BASIC BIOLOGY

39

I. nilmagenta mutant

CtoT

I. purpurea
pink mutant

Tip201

I. tricolor
fuchsia mutantT

1 kb
Fig. I. Flower phenotypes and structures of the F3'H genes in/. nil, I. purpurea, and/. tricolor exhibiting reddish flowers. A./. nil
carrying the magenta allele. B. /. purputea carrying the pink allele. C. I. tricolor carrying the fuchsia allele.

containing a bHLH DNA-binding motif. Each of thetandem repeats is flanked by a 3-bp sequence AAT,
indicating that the 3-bp microhomology is used togenerate the tandem duplication. The transcripts in thepale-blue flower buds of the mutant contain an internal583-bp tandem duplication that results in the productionof a truncated polypeptide lacking the bHLH domain.
The mRNA accumulation of most of the structuralgenes encoding enzymes for anthocyanin biosynthesisin the flower buds of the mutant was significantly
reduced. The transcripts identical to the wild-type

mRNAs for the transcriptional activator were present
abundantly in blue spots of the variegated flowers,
whereas the transcripts containing the 583-bp tandem
duplication were predominant in the pale-bluebackground of the same flowers . The flower and seedvariegations are caused by somatic homologous
recombination between an intragenic tandem
duplication in the bHLH gene, whereas various flower
variegations in Ipomoea are known to be caused byexcision of DNA transposons inserted into pigmentation
genes.

IVS
1 2

3 45

6

7 8

1 kb
Fig.2. Flower and seed pigmentation phenotypes and structures of the bHLH transcriptional regulatortor gene for anthocyanin
pigmentation in the I. tricolor wild-type cultivar, Heavenly Blue (A and C), and its mutant, Blue Star (B, D and E).

NATIONAL INSTITUTE40

arm of the chromosome 3, and excision of a new DNA
transposon from the pyl gene appears to be responsible
for conferring the leaf variegation. It is important toemphasize here that tissue culture is necessary in all of
the currently available rice reverse genetic approaches.
No somaclonal variation is likely to occur in mutant
lines induced by our newly characterizing endogenous
element, because no tissue culture has been involved in
it s activation. Using a newly identified DNA
transposon, therefore, we are attempting to develop anovel transposon tagging system in rice.

II. Targeted gene disruption by homologous
recombination in rice.
Rice (Oryza sativa L.) is an important staple food formore than half of the world's population and a model
plant for other cereal species. We have developed alarge-scale Agrobacterium-mediated transformation
procedure with a strong positive-negative selection andsucceeded in efficient and reproducible targeting of the
Waxy gene by homologous recombination without
concomitant occurrence of ectopic events, which must
be an important first step for developing a precise
modification system of the genomic sequences in rice.
By improving our transformation procedure further, we
are attempting to modify Adh genes, which belong to asmall multigene family and reside adjacent to repetitive
retroelements.

Publication List:
Kikuchi, T., Nishimura, M. , Hoshino, A. , Morita, Y., lida,
S., Saito, N. and Honda, T . (2003) An efficientconversion of catechine into 3,4-trans-leucocyanidin.
Heterocycles 60, 1469-1475.
Hoshino, A., Morita, Y., Choi, J.D., Saito, N. , Toki, K. ,Tanaka,Y. and lida, S. (2003) Spontaneous mutations of
the flavonoid 3'-hydroxylase gene conferring reddish
flowers in the three morning glories. Plant Cell Physiol.
44, 990-100 l.
Yoshida, H., Akimoto, H. , Yamaguchi, M., Shibata, M.,
Habu, Y., !ida, S. and Ozeki, Y. (2004) Alteration of
methylation profiles in distinct cell lineages of the layers
during vegetative propagation in carnation (Dianthus
caryophyllus). Euphytica (in press).
Terada, R. , Asao, .H. and lida, S. (2004) A Large-scale
Agrobacterium-mediated transformation procedure witha strong positive-negative selection for gene targeting inrice (Oryza sativa L.). Plant Cell Reports (in press).
lida, S., Morita, Y., Choi , J.D. , Park, K.l. and Hoshino, A.
(2004) Genetics and epigenetics in flower pigmentation
associated with transposable elements in morning
glories. Advances in Biophysics (in press).

III. Characterization of mutable virescent allele in
rice.
Leaves of seedlings in the virescent mutant of rice
are initially pale yellow green due to partial deficient inchlorophyll and gradually become green with thegrowth of the mutant. We have been characterizing a
spontaneous mutable virescent allele pale yellow leafvariegated (pyl-v ), conferring pale yellow leaves withdark green sectors in its seedlings (Figure 3). The pyl-v
mutant was isolated among progeny of a hybrid
between indica and japonica rice plants . The leaf
variegation is regarded as a recurrent somatic mutationfrom the recessive pale yellow allele to the dark green
revertant allele. The availability of the genomic
sequences of both japonica and indica subspecies
facilitates map-based cloning of the pyl-v allele.
Mapping data indicate the mutation resides in the short

(B)

py/-v

D~~<=Jc::::::)

II

H30524- 1--------

CH1300·1

RM251

py/-v

(Predicted Genes)
BAC clone

MMCAPS~

RM282(SSR markers)

Fig.3. A. Leaf phenotype of the mutable pyl-v allele. B. Identification of the pyl-v allele. The pyl-v allele was mapped between the
SSR markers RM251 and RM282 on chromosome 3 and subsequently located between the SLP markers CH30524-l and
MMCAPS-1 on a single BAC clone. The horizontal pentagonals represent predicted genes.

FOR BASIC BIOLOGY41

DIVISION OF GENE EXPRESSION AND
REGULATION II
Professor:
Research Associates:
Technical Staffs:
JSPS Research Fellow:
Postdoctoral Fellows:
Visiting Scientist:
Graduate Students:

HORIUCHI, Takashi
KOBAYASHI, Takehiko
JOHZUKA, Katsuki
HAYASHI, Kohji *MOROOKA, Naoki **AUSTEN RD Ganley
KODAMA, Ken-ichi
OHSUM/, Katufumi ***UJIIE, Yoshifumi
SERIZAWA, Naomi
WATANABE, Takaaki* to Center for Trangenic Animals and Plants,
NIBB (Sep, /, 2003)**from Sep. /, 2003***from Nov. 30, 2003

The genomes of higher organisms contain significant
amounts of repetitive sequences, which in general, areunstable. At present, neither the physiological
function(s) of repeated sequences nor the mechanism
controlling the instability is fully understood. To
clarify these aspects, we are pursuing the following
themes using E. coli and S. cerevisiae: (1) the
amplification mechanism of repeated sequences orgenes, especially rRNA repeated genes, (2) the
mechanism of replication fork block-dependent
recombination, a key reaction that increases ordecreases the number of repeats, and (3) developmentof in vivo artificial gene amplification systems.
Structural and functional analyses of the E. coli genome
are also being carried out. In 2003, work on thefollowing three subjects has advanced our knowledge ofthe dynamics and structure of the genome.

I. The replication fork barrier site forms a uniquestructure with Foblp and inhibits thereplication fork.
The DNA replication fork blocking sites are found in
genomes of various organisms. In E. coli, the fork
block site, called Ter, has the fork blocking activity in apolar fashion. For the blocking activity, twocomponents are required, one is a cis-element, Ter, theother is the Ter-binding protein, called Tus protein. In
eucaryotes, the similar blocking site, called RFB(replication fork blocking barrier), has been identified
in the ribosome RNA gene (rDNA) cluster from yeast
to human. InS. cerevisiae, about 150 copies of rDNA
are clustered in a specific region on the chromosome
XII. The RFB is an approximately 100-bp DNA
sequence located near the 3' end of the rRNA genes inthe yeast Saccharomyces cerevisiae. This site inhibits

the progression of the DNA replication fork comingfrom the direction opposite to 35S rDNA transcription.
However, the RFB-binding protein has not been
identified so far. The most likely candidate for the
RFB-binding protein is Fob 1p, because it had shown tobe required for the replication fork blocking activity at
the RFB site. Furthermore, it was found later that it isessential for recombination in the ribosomal DNA(rDNA), including increase and decrease of rDNA
repeat copy number, production of extra-chromosomal
rDNA circles, and possibly homogenization of therepeats. Despite the central role that Foblp plays in both
replication fork blocking and rDNA recombination, notonly the molecular mechanism by which Foblp
mediates these activities, but also its RFB-binding
activity had not been determined. We have shown by
using chromatin immuno-precipitation, gel shift, footprinting, and atomic force microscopy assays that
Foblp directly binds to the RFB (see Figure A and B).
Foblp binds to two separated sequences in the RFB(Figure D). A predicted zinc finger motif in Foblp wasshown to be essential for the RFB binding, replication
fork blocking, and rDNA recombination activities. The
RFB seems to wrap around Fob 1p, and this wrapping
structure may be important for function in the rDNA
repeats (Figure B, C and D).

II. Transcription-mediated hyper-recombination
inHOTJ
Recombination hot-spots are DNA sequences which
enhance recombination around the region. HOTJ is oneof the well-studied recombination hot-spot in mitotic
yeast cells. Because HOT/ includes a transcription
promoter sequence of RNA polymerase I (Poll) whichis responsible for the 35S ribosomal RNA gene (rDNA)
transcription, and the HOTJ activity is abolished in a
Poll defective mutant, transcription of HOTJ wasthought to be an important factor for the recombination
stimulation. However, it is not clear whether thetranscription itself or other phenotypes shown in the
Poll mutant activates the recombination. To understandthe role of transcription, we used a highly-activated Polltranscription system for HOT/ in a strain whose rDNA
were deleted (rdnL\L\). In the rdnL\L\ cells, it is known
that the nucleolus disappears and Poll, which isnormally localized to the nucleolus, diffuses into thecytoplasm. In the rdnL\L\ strain, the HOTJ transcription
was increased by about 25 times as compared with thewild type. Recombination activity stimulated by HOTJ
was also elevated by about 15 times as compared withthat of wild type. These results indicate that frequency
of Poll transcription in HOTJ determines efficiency of
the recombination. Moreover, Foblp, which is essential
for both HOTJ recombination and transcription
activities, was dispensable in the rdnL\L\ strains, thereby
suggesting that the protein may be functioning as a Polltranscription activator in the wild type strain.

NATIONAL INSTITUTE

42

III. Gene amplification by designed double rolling
circle replication utilizing break induced
replication in Saccharomyces cerevisiae(DRCR), could amplify a gene between two directed
replication forks , and tested it on plasmid-derived minichromosomes or on a resident chromosome utilizing
break-induced replication (BIR). The latter systemproduced three kinds of amplification products; type-1
containing 5~7 copies of an amplific ation marker,
leu2d, with the expected structure, type-2 containing
several dozen copies of leu2d with a similar structure to
type-1 , but the sequences flanked by the two inverted
leu2ds are oriented randomly , and type-3 having an
acentric multi-copy mini-chromosome with leu2ds. The
type-2 and -3 products seem to correspond to HSR(homogeneously staining region) and DM (double
minute) in higher eukaryotes. Surprisingly, the twolatter types were also generated without HO cleavage,
though at low frequency. This system may provide
insights into the molecular mechanism of gene
amplification.

Gene amplification is a phenomenon widely found in
the genomes of various organisms and probably has
played an important role in the processes of gene
evolution. For example , rDNA must have amplified
during evolution and in some amphibians, further
amplification occurs during their development.
Oncogene amplification is frequently observed whencancer is in progress. Gene amplification also occurs
when cultured cells acquire drug-resistance. However,human cultured cells lack the ability to amplify drugresistance genes at a detectable level. Spontaneous
amplification also occurs in insects and plants, but themolecular mechanism remain uncertain. Thus , if anovel artificial amplification could be realized using
normal organisms , it would be helpful for better
understanding the basic mechanism of the various
amplifications. Thus, we have tried to develop a novel
designed amplification system using a normal S.
ce revi s iae strain . We assumed that a mode of
replication, named double rolling-circle replication

A

VI. Complete genomic sequence of the E. coli K12
W3110 strain
Escherichia coli is one of the organisms that has been
most extensively analyzed physiologically ,B
260 bp

RFB (l40 bp)

1

2

3

4

600 bp----~~c::Jr--------~-----

lOOObpD.._., • •RFB 1 RFB2 RFB3a------------~~~~--~ ~---------

b

a

Ill\

•

+-Foblp

c

,.,..

6

•• ,

3lnm

(nm)
Le~

Figure Observation of the Foblp-RFB complex by AFM (atomic force microscopy). (A) Structure ofRFB fragments used in AFM
analysis. (B) AFM images of the Foblp-RFB complex. (C) Frequency plot (histogram) of the length of the RFB fragment
measured from AFM images. White and hatched boxes indicate the number of free DNA molecules and Foblp-RFB complexes,
respectively. The mean fragment lengths are indicated below the graph by clotted lines. (D) Models of the Fob I p-RFB complex .

FOR BASIC BIOLOGY

biochemically and genetically. Of all E. coli strains, E.
coli K 12 W3110 has probably been used mostfrequently as the wild-type strain in these experiments.
Recently, we determined the complete nucleotide
sequence of the genome of strain W3110, mainly bysing lambda phages from Kohara's bank. Previously a
US group determined the genomic sequence fromanother K12 wild-type derivative, MG1655. Bothstrains were derived from a common ancestor strain,
W1485, probably about 50 years ago. Comparing thetwo sequences, we obtained the following results.
The total number of nucleotides in the W3110
genome is 4660170 bp. There were 349 bp conflicts
between the sequences of W3110 and MG1655. Resequencing of the conflict sites by a PCR method using
genomic DNA as templates revealed that only eightsites (9 bp) were true conflicts. Seven of them are basechange type conflicts and one is a two base frame-shift.
All of these differences reside within genes, seven in
ORFs and one in a 23S rRNA gene.
We attended the E. coli K12 annotation workshop
held at Woods Hole Marine Biology Research Institute,
Mass., USA, 2003, Nov. 13-18, which was organized
by Dr. Monica Riley. In this workshop, we discussed( 1) the definition of the starting point of the E. coligenome sequence, (2) boundaries of ORFs, that is startand end points of ORFs, and (3) the descriptions ofgene names and their function. We agreed there to makean effort to complete a new annotation version using
our revised sequence by the coming February. We cansafely say that the revised sequence and the annotation
will be among the most accurate of all the genomes
whose sequences have been determined.
Publication list:
Takeuchi, Y., Horiuchi, T., & Kobayashi, T. (2003)
Transcription-dependent recombination and the role offork collision in yeast rDNA. Genes Dev. 17, 1497-1506
Higuchi K, Katayama T, Iwai S, Hidaka M, Horiuchi T,
Maki H.(2003). Fate of DNA replication fork
encountering a single DNA lesion during oriC plasmid
DNA replication in vitro. Genes Cells 8, 437-49
Kobayashi, T. (2003) The replication fork barrier site
forms a unique structure with Foblp and inhibits thereplication fork. Mol. Cell. Bioi. 23,9178-88.

43

NATIONAL INSTITUTE44
DIVISION OF SPECIATION MECHANISMS I

Professor:
Research Associates:

YAMAMOR/, Tetsuo
KOMINE, Yuriko
WATAKABE, Akiya
KITSUKAWA, Takashi
Technical Staffs :OOSA WA, Sonoko
Miki, KazuhikoTechnical Assistant:
ISHIKAWA, Takaka
NIBB Research Fellow: Sf, Xuaohui
Postdoctoral Fellows:
HATA , Katsusuke
SAKATA, Shuzo
Graduate Students:
KOMATSU, Yusuke
TAKAHATA , Toru
Secretary:
HAYASHI, Hitomii) One gene , designated occl , is specifically
expressed in the occipital cortex , particularly in VI
area, in the primate brain. Furthermore, the expressionof occ I turned out to be activity dependent, because , inthe monocularly deprived monkeys injected with TTX
into one of the eyes, the expression of occ I is markedly
decreased in the ocular dominance co lumn s of the
primary visual cortex (VI). We also demonstrated thatocc I expression was markedly increased postnataly inV 1 (Fig l , Tochitani et al., 2003).

Our research is focusing to understand mechanisms
underlying memory, formation and evolution of thebrain . For one approach to understand these questions,
we are studying the genes that are expressed in specificareas of the primate neocortex. Using differential
display method , we obtained three genes that showedmarked differences within areas of the primate
neocortex. Our second approach is to understandinformational processing in the brain underlying
learning behaviors by examining gene expression. The
third approach is to study voles of CNTF in the nervous
system.

I. Genes expressed in specific areas of the
neocortex
The neocortex , most remarkably evolved in primates,
plays the major role in higher functions of the brain-. It
is known to be divided into distinct functional andanatomical areas and has been a matter of debate what
extent the area of the neocortex are genetically andenvironmentally deter-mined. It is also puzzling why,
during the evolution of mammals, the neocortex was
most markedly expanded while the number of the genes
in the mammal was little changed. To access thesequestions, we studied gene expression within different
areas of the neocortex.
L) In collaboration with Professor Hiroyuki Nawa
(Nigata university) , we used the DNA macroarray
technique to examine gene expression in the areas ofhuman prefrontal , motor and visual cortexes. We founda lm ost all the genes among 1088 genes examined
showed only less than a factor of two in the differenceof their expressions. Only one gene showed more than
three fold difference and another one was between two
and three fold difference within the three areas. These
results suggest that the genes that are expressed among
the different areas of the human neocortex are very
similar. However, the question remained whether there
are any genes that show marked difference within areasof neo-cortex.
2) In order to answer this question , we employed
differential display methods and found at least two
genes that indicated area specific expressions.

Fig. 1 Developmental Expression pattern of occl in thevisual cortex.
In situ hybridization pattern of occ l in the primate visualcortex. occ I is markedly expressed in the layer TV cb andmoderately in the layers of II , III and TVa in area VI (e).
This adu lt expression pattern is regulated by postnatal
development. (a, b) newborn ; (c, d) three month ; (e, f)adult monkey. (a, c, d) with occl antisense probe and (b,d, f) with thionin for cell bodies. (See. Tochitani et al.,Eur. J. Neurosci: , 13,297-307, 2001)

FOR BASIC BIOLOGYii) The other gene that showed marked difference
within the neocortex, is gdt7, a member of BMP/TGF-b
family, which is specifically expressed in the motor
cortex of the African green monkey. We are cun·ently
examining the detailed expression pattern of the gene.
iii) The third gene that we designated tentatively as
l34G which was preferentially expressed in associationand higher areas in the neocortex (Komatsu et al.,Society for Neuroscience 32"d Annual Meeting, 2002,
being submitted).
3) We have also further isolated several area specificgenes with RLCS (Restriction Landmark eDNA
Scanning) . We are now characterizing these genes toreveal the mechanisms that form neocortical areas.

45

obtain a food pellet . We found that the visual and audio
tasks enhanced the specific expression of c-Fos in thevisual and audio cortices, respectively. Among the earlyvisual and auditory pathways examined, c-Fos was
specifically induced in the cortices but not in the earlier
pathways , suggesting the neural modulation of the
neocortex depending on the types of the tasks.
Interestingly , the task-dependent Fos expression was
only observed in excitatory neurons in the relevant
sensory cortices.

A

e

/

liJ,;hl l>oo!___In summary, our studies thus far revealed thefollowing points.
(1) Genes that are specifically expressed within
neocortical areas in the primate neocotex are notdifferent overall.
(2) We have identified several genes that are
distinctively different among neocortical areas.
(3) These genes are specific in visual, motor andassociation areas.
(4) A gene specific in the visual cortex (occl) is activity
dependent and also postnatally regulated.
(5) An association area specific gene is expressed in acomplimentary manner to the expression of occl.
(6) These results suggest that these genes may be usefulmarkers to study the mechanisms underlying
neocortical formation.

II. Gene expression under a declarative and anon-declarative memory
In order to study informational processing underlying
the declarative and non-declarative memory atmolecular and cellular levels in the brain , we employedc-Fos mapping techniques, for which we used gene
expression of c-Fos. There have been an in creasing
number of studies using c-Fos as markers to examine
neuronal activities ever since c-Fos induction byelectrical stimulation was found by Morgan and Curran.
However, many sensory stimuli per se are now known
to cause c-Fos induction. So, we should be very careful
to distinguish the c-Fos expression that is caused bylearning process from that caused by sensory stimuli.
For this purpose, it is necessary to use behavioral
systems that are able to distinguish the two. Although a
few behavioral sys tems in rodents have beensuccessfully used for physiology, animal behavior andrecently for analyses of knockout mice, little behavioral
systems in fact distinguish it. Therefore, we preparedourselves for using two behavioral systems, which
represent declarative and non-declarative memory,
respective! y.
In collaboration with professor Yoshio Sakurai(Kyoto University) who developed audio-visual
discrimination task (A VD-task). In this task, a rat was
asked to choose either an audio cue (a high tone or lowtone) or a visual cue (a light from the right or the left) tolight

fl~n~ ----

/

•

-~
c

Stimulus Sees
AVD · Auditory(AV D·A) task
AVD·V!Sual(A \"D - V) tn>k

Go
Go

Go
NoGo

NoGo
Go

NoGo
NoGo

Fig.2. A YD tasks.
A) A rat face to a panel with a visual cue (a light fromright or left) or an auditory cue (a high or low tone) was
shown to push the panel with the time sequence shown in(B). The combination of possible cues by a uditory andvisual tasks to obtain a food pellet is shown in (C). (See
Sakata et al. , Eur. J. Neurosci ., 15 , 735-743 , 2002, for thedetail of the tasks)

Although this AVD task system is quite powerful toanalyze a kind of task above described and presumably
very useful for studying underlying molecular andcellular mechanisms because of advantages usingrodents, one problem is that the auditory stimuli andvisual stimuli are in different positions. Thus we cannot
exclude the possibility that the difference between theauditory task and the visual task may not completelydepend on the modality (i.e., visual Vs auditory)
difference.
We wanted to solve this problem by placing auditory
and visual stimuli in the same position. We also use
nose-poking to measure the reaction time in which a rat
responds to stimuli. By using this behavioral system, wewere able to confirm amodal recognition of space whichmeans that a rat can respond to a different modality(visual or auditory) if the stimuli is in the same positionand previously reported in other systems. We also
confirm multisensory enhancement is indeed observedin rats. These resuluts suggests that this new modified
AVD system can be used to explore the molecular andcellular mechanisms underlying multisensory
processing in rats.
The other task we developed is a wheel runningsystem in which a water-deprived mouse is asked to run
to obtain water in front because the wheel with the pegs
is turning to the other direction (Kitsukawa et a!.,

NATIONAL INSTITUTE

46
Society for Neuroscience 32nd Annual Meeting, 2002).
The pegs can be changed with various patterns as
desired. The task required for the mouse thus can be
regarded to represent a non-procedural learning. We
examined a various areas of brains following to thechange of the peg pattern. Among the areas examined,
we found marked c-Fos expression in the striatum. The
striatum, which is composed of projection neurons andseveral distinguished types of interneurons, is known toplay an important role in a reward-based learning. The
characterization of these subtypes of interneurons has
been progressed . However, their roles in behavioral
tasks have been little known. We hope our system
combined with c-Fos mapping technique reveals it. We
are cyrrently doing collaborative work with Professor
Ann Graybiel (MIT) to record with tetrode from thestriatum under the task (supported by US-Japan
exchange program). We hope our wheel running system
combined with c-Fos mapping technique andelectrophysiology reveals molecular and cellularmechanisms under the Wheel Running task.

III CNTF is specifically expressed in thedeveloping pineal gland
CNTF, a member of the IL-6 family , attracts quite
attentions of developmental neuroscientists because itshows various effects on neurons and glial cells. CNTF
knockout mice, however, only indicate moderate motor
neuron deficiency in the adult , but no apparent
phenotype in the development. In order to explore thefunction of the IL-6 family , we extensively examinedthe expression of members of the family and their
receptors and found the specific expression of CNTF in
the embryonic pineal glands and eyes . This was to ourknowledge for the first time to show a clear expression
Control+ CNTF (1 00 ng/ml)

Fig. 3. CNTF suppress Rhodopsin expression in pineal
glands in vitro. CNTF was added to the medium of rat
pineal organ culture. (Right) Control culture without
CNTF on day 10. Rhodopsin-positive photoreceptor-likecell s with man y proce sses are seen at a higher
magnification. (Left) CNTF (100 ng/ml) was added for
10 days. Photoreceptor-like cells of rhodopsin -positi vecell s are very rare. If any, they do not show longprocesses (lower, a higher magnification). These results
were published in Hata et al. , Dev. Brain Res., 143, 179187, 2003.

of CNTF in the development. Next question to be asked
is the functional role of CNTF in the pineal
development. Because of no reported phenotype of
CNTF knockout mice in development, it has beendifficult to know the developmental roles of CNTF if
any. In fact, we also found there seemed no apparent
difference in the pineal development of CNTF knockout
mice compared to that in wild type mice. This ispresumably because the CNTF gene knockout is largely
compensated by other CNTF-like factors .
We therefore studied cultured pineal organs inrodents to ask if there were any effects of CNTF on it
following to a previous study that shows that cultured
neonatal pineal organs develop photoreceptor-like cells(Araki, 1992). We found that CNTF inhibits
photoreceptor-like cells (Fig. 3, Hata et al., Dev Brain
Res. , 2003 ). Our observation raises an interesting
possibility that CNTF plays a critical role in the pineal
development to suppress a certain phenotype such as
photoreceptors . It also raises a question what roles
CNTF play in developing pineal glands if it play any
roles in the development. CNTF has been studied fornearly thirty years and still the role in vivo remains to
be studied. We hope that our study gives a clue to solve
this long standing question .

Publication list:
Tochitani, S., Hashikawa, T. and Yamamori, T. (2003)
Expression of occ1 mRNA in the visual cortex during
postnatal development in macaques. Neurosci. Lett. 337,
114-116
Su ga, K., Yamamori , T. and Akagawa , K. (2003)
Identification of Carboxyl- Terminal MembraneAnchoring Region of HPC-1 /Syntaxin 1 A by
Substituted-Cysteine-Accessibility Method andMonoclonal Antibody . J. Biolchem. 133, 325-334
Nakayana, T. , Mikoshiba, K. , Yamamori, T. and Akazawa,K. (2003) Expression of syntaxin 1C, an alternive splicevariant of HPC-1/syntaxin 1A, is activaed by phorbolester stimulaton in astrglioma. FEES Lett. 536, 209-214
Tochitani, S., Hashikawa, T. and Yamamori, T. (2003)
Occl mRNA expression reveals a characteristic featurein the hippocampal CA2 field of adult mazaques.
Neuroscience Lett. 346, 105-108
Hata, K. , Araki , M. , and Yamamori , T. (2003) Ciliary
neurotrophic factor inhibits differentiation ofphotoreceptor-like cells in rat pineal glands in vitro.
Dev. Brain Res. 143, 179-187

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS II

Professor:
Associate Professor:
Research Associates:

HAS£8£, Mitsuyasu
MURATA , Takashi
FUJITA , Tomomichi
HIWATASHI, Yuji (Oct. 1~)
Technical Staff:
SUMIKAWA, Naomi
N/88 Research Fellows: H!WATASH/, Yuji (~Sept. 30)
NISHIYAMA, Tomoaki (Sept. 16~)
JSPS Research Fellow: SATO, Yoshikatsu
Postdoctoral Fellows:
NISHIYAMA, Tomoaki (~Sept . 15)
AONO, Naoki (April 1 ~)MIYAZAKI, Saari (May 19~)
K08AYASHI-ARAKA WA, Satoko
Graduate Students:(~Sept. 30)
SAKAK/8ARA, Keiko(~March 31)
NAKAMURA, Tohru(Niigata Univ.) (~March 31)
KITANI, Masakazu (April I~)
Technical Assistants:
TAN1KA WA, Yukiko
8/TOH, Yoshimi
NARUSE, Mayumi
AOKJ, Etsuko
OONO, Chikako (Feb. I ~)SUZUKI, Yoriko (Feb. 1 ~)Paul G. Wolf " (Sept. 22~Dec. 22)
Visiting Scientists:
Carol A. Rowe '1 (Sept. 22~Dec. 22)
KA8EYA , Kazuko
Secretary:11

j i·01n Utah State University, USA

All living organisms evolved from a common
ancestor that lived more than 3.5 bi!Jion years ago, andthe accumulation of mutations in their genomes has
resulted in the present biodiversity. Traces of theevolutionary process are found in the genomes of extant
organisms. By comparing the gene seq uences and gene
networks of different organisms, we can infer (1) thephylogenetic relationships of extant organisms and (2)the genetic changes that caused th e evolution ofmorphology and development. The inferred
phylogenetic relationships provide important insights
into problem~ in various field~ of evolutionary biology.
Our group focuses on biogeography , the evolution ofmorphological traits , and systematics in a wide range oftaxa. Concerning the evolution of morphology anddevelopment, we hope to explore the genetic changes
that led to the evolution of the plant body plan. We havese lec ted Arabidopsis (angiosperm), Gnetum(gy mnosperm) , Ginkgo (gy mnosperm), Ceratopteris(pteridophyte), Physcomitrell a (bryophyte), and somegree n algae as models to compare the functions ofgenes involved in the development of both reproductive
and vegetative organs in land plants.

47
of cytosolic calcium icon concentration andcytoskeleton organization during chloroplast movementin the moss Physcomitrella patens is in progress by ateam directed by Y. Sato.

II. Evolution from unicellular to multicellularorganisms
The first evolutionary step from unicellular tomulticellular organisms is to form two different cells
from a single cell via asymmetric cell division. The firstcell division of a protoplast isolated from theprotonemata of the moss Physcomitrella patens isasymmetric regarding to its shape and nature, and gives
rise to an apical meristematic cell and a differentiatednon-meristematic cell. A systematic overexpression
sc reening for genes involved in asymmetric cell
division of protoplasts in P. patens is in progress by ateam directed by T . Fujita. We constructed three fulllength eDNA libraries from non-treated, auxin-treated,and cytokinin-treated protonemal cells of P . patens,
then determined the seq ue nces of more than 40,000
cDNAs from the both ends (Nishiyama, Fujita et al.
2003). We used these clones as materials for theoverexpression screening. Individual cDNAs werese lected based on their sequence, subcloned under aconstitutive promoter and introduced into theprotoplasts of P. patens for transient expression. We
observed and categorized phenotypes of theregenerating protoplasts. Thus far we identified many
cDNAs, whose overexpression resulted in symmetriccell division rather than asymmetric cell division,
isotropic outgrowth with no polarity or curved cellss howin g incorrect direction of growth. The sepreliminary results indicate that some of these genes
likely function for polarity formation and/or asymmetriccell division of the protoplasts. Functional analyses ofthese genes should help u s to understand molecularmechanisms of how plants ge nerate distinct celllineages to build their multicellular bodies.

I. Origin of the Plant Cell
The first green alga cell evo lv ed via sy mbiosis
between an ancestral non-photosy nthetic eukaryote anda cyanobacterium. Cyanobacteria now exist as
chloroplasts in the ho st ce ll. The factors andm ec hani s ms of chloroplast movement are being
investigated to reveal the molecular mechanisms used
to "domesticate" cyanobacteria as organelles. Analyses(Figure 1) Asymmetric cell division of a Physcomitrella
protoplast. Regular asymmetric cell division (I , 2) anddefective cell division with overexpression (3 , 4). Cellwa ll of the cell in (I) is visuali zed with calcoflor in (2).

NATIONAL INSTITUTE

48

III. Evolution from cells to tissues
The most prominent difference between plant andanimal cells is that plant cells have a cell wall and do
not move during development. Therefore, the plane ofcell division and the direction of cell elongation , which
are regulated by cortical microtubules, determine themorphology of differentiated tissues and organs.

Organization of y-tubulin
y-tubulin is a protein that is essential for theformation of microtubules in animal cells. We foundthat y-tubulin is located at the end of cortical
microtubules, and a loss of y-tubulin due to genesilencing causes a malformed organ with irregularly
shaped cells. In vitro experiments using isolated plasma
membrane/ microtubule complexes suggested that ytubulin attaches onto the side of existing cortical
microtubules , and initiates a new cortical microtubulefrom it. The nucleus is known to initiate microtubules
after cell division. We hypothesize that microtubules
formed around the nucleus elongate to cell surface, andtrigger initiation of cortical microtubules via attachmentof y-tubulin. Once cortical microtubules are formed ,they can turnover without microtubules from thenucleus . Factor(s) responsible for attachment of ytubulin onto the side of microtubules is a key element
responsible for the difference between plant and animalcells. I s olation of the factor(s) responsible forattachment of y-tubulin onto the side of microtubules bybiochemical and other approaches is inprogress by ateam directed by T. Murata.

Dynamics of actin filaments and microtubules in themoss P. patens

Physcomitrella patens, the developmental process ofmeristem is well defined at the cellular level, and genetargeting based on homologous recombination is
feasible . Thus , meristem development in P. patens isused as a model system for studies of meristem
development in land plants. We establishedapproximately 20,000 gene- or enhancer-trap lines in P.
patens to clone genes involved in meristem
development. Seven lines, exhibiting reporter gene(uidA) expression preferentially in the apical cells, wereisolated. Corresponding genes in three of these traplines were identified as encoding kinesin- and ubiquitinlike proteins, and an unknown protein. Disruption of thegene encoding ubiquitin-like protein suggests that thegene be involved in cell division and elongation throughmicrotubule organization. The functions of other genes
in the meristem are currently under investigation by ateam directed by Y. Hiwatashi .
The morphology of the shoot meristem in land plants
varies. To investigate whether the molecularmechanisms of shoot development in angiosperms are
con served in other land plants, the functions of the
KNOX homeobox, and the ZWILLE, NAC, and PIN
genes , which are indispensable for shoot meristem
development in angiosperms, are being studied in thefern Ceratopteris and the moss Physcomitrella.

Rhizoid differentiation
Rhizoids are multicellular filaments with similarfunctions to root hairs, and are observed in a wide rangeof plants. A team directed by K. Sakakibara, who was a
graduate student in our lab, examined mechanisms
underlying rhizoid development in the moss ,Physcomitrella patens (Sakakinara eta!. 2003).

Cells of the moss P. patens gametophytes are an
excellent model to study dynamics of cytoskeleton
because of the easiness for observation and thefeasibility of gene-targeting. Transformants with
reporter constructs by fusing a GFP with P. patens atubulin or an actin binding domain of mouse talin were
established by Y. Sato and collaborators to visualize
microtubules and actin filaments. These transformants
will be useful to investigate the dynamics of
microtubules and actin filaments in the processes of cell
division, cell elongation, and chloroplast movement.

IV. Evolution of molecular mechanisms in thedevelopment of vegetative organs
Meristem initiation and maintenance
Postembryonic growth of land plants occurs from themeristem, a localized region that gives rise to all adultstructures. Meristems control the continuous
development of plant organs by balancing themaintenance and proliferation of stem cells , anddirecting their differentiation . Meristem initiation andmaintenance is a fundamental question in plantdevelopment research. However , the molecularmechanisms involved in meristem initiation andmaintenance have not been studied in detail because
most loss-of-function mutants are lethal. In the moss

Figure. 2 (I) a gametophyte of P. patens. (2) an auxintreated gametophyte with plenty of rhizoids.

V. Origin and evolution of floral homeotic genes
The flower is the reproductive organ in angiosperms ,and floral homeotic genes , such as MADS-box genes
and FLO/LEAFY genes, regulate floral organ identity .
To investigate the origin of floral homeotic genes, thefunctions of MADS -box genes and the FLO/LEAFY
genes in gymnosperms (Gnetum, Ginkgo, and cycads), a
fern (Ceratopteris) , a moss (Physcomitrella), and threegreen algae (Chara, Coleochaete , and Closterium) are49

FOR BASIC BIOLOGY

being analyzed.
Land plants are believed to have evolved from agametophyte (haploid)-dominant ancestor without amulticellular sporophyte (diploid plant body); mostgenes expressed in the sporophyte probably originatedfrom those used in the gametophyte during theevolution of land plants. To analyze the evolution anddiversification of MADS-box genes in land plants,
gametophytic MADS-box genes were screened using
macroarray analyses for 105 MADS-box genes found in
the Arabidopsis genome (Kofuji et al. 2003). Eight
MADS-box genes were predominantly expressed inpollen, male gametophyte, and analyses of theirfunction are in progress by a team directed by N. Aono.

VI. Evolution of life cycles
The mosses and flowering plants diverged more than
400 million years ago. The mosses have haploiddominant life cycles, while the flowering plants are
diploid-dominant. The common ancestors of land plants
are inferred to have been haploid-dominant, suggestingthat genes used in the diploid body of flowering plants
were recruited from the genes used in the haploid bodyof their ancestors during the evolution of land plants.
To assess the evolutionary hypothesis that genes usedin diploid body of flowering plants were recruited fromthe genes used in haploid body of the ancestors, a team
directed by T. Fujita constructed an expressed sequencetag (EST) library of Physcomitrella, and T. Nishiyama
mainly worked on the comparison of the mosstranscriptome to the genome of Arabidopsis. We
constructed full-length enriched eDNA libraries fromauxin-treated, cytokinin-treated, and untreatedgametophytes of Physcomitrella, and sequenced bothends of more than 40,000 clones. These data, together
with the mRNA sequences in the public databases, wereassembled into 15,883 putative transcripts. Sequencecomparisons of Arabidopsis and Physcomitrella showedthat the haploid transcriptome of Physcomitrellaappears to be quite similar to the Arabidopsis genome,
supporting the evolutionary hypothesis. Our study also
revealed that a number of genes are moss specific andwere lost in flowering plant lineage. Our full-length
eDNA library will be a good resource for functional
genomic studies in plants. Our EST data together withsome information on the moss is open to public at
PHYSCObase (http://moss.nibb.ac.jp ).

VII. Molecular mechanisms of speciation
Reproductive isolation is the first step in speciation.
To obtain insight into reproductive isolation, several
receptors specifically expressed in the pollen tube arebeing studied to screen for the receptors that are

involved in pollen tube guidance by a team directed by
S. Miyazaki.
Polyploidization is a major mode of speciation inplants, although the changes that occur after genome
duplication are not well known. Polyploid species areusually larger than diploids, but the mechanisms
responsible for the size difference are unknown. To
investigate these mechanisms, tetraploid Arabidopsis
was established and its gene expression patterns arebeing compared to those of diploid wild-type plants
using microarrays.

Vill. Phylogenetic analysis of land plants
We determined the complete nucleotide sequence ofthe chloroplast genome of the leptosporangiate fern,
Adiantum capillus-veneris L. (Pteridaceae) as acollaboration work with P. Wolf and C. Rowe from
Utah State University (Wolf et al. 2003). Phylogenetic
analysis of basal land plants using the sequence is inprogress by T. Nishiyama. Many unusual start codons
and internal stop codons were found, suggesting that
extensive number of RNA editing exists in the genome.

Publication List:
Kofuji, R., Sumikawa, N., Yamasaki, M., Kondo, K.,
Ueda, K., Ito, M. and Hasebe, M. 2003. Evolution anddivergence of MADS-box gene family based on genome
wide expression analyses. Mol. Bioi. Evol. 20: 19631977.
Sakakibara, K., Nishiyama, T., Surnikawa, N., Kofuji, R.,
Murata, T. and Hasebe, M. 2003. Involvement of auxin
and a homeodomain-leucine zipper I gene in rhizoid
development of the moss Physcomitrella patens.
Development 130: 4835-4846.
Nishiyama, T., Fujita, T., Shin-1, T., Seki, M., Nishide, H.,
Uchiyama, 1., Kamiya, A., Carninci, P., Hayashizaki,Y., Shinozaki, K., Kohara, Y., and Hasebe, M. 2003.
Comparative genomics of Physcomitrella patens
gemetophytic transcriptorne and Arabidopsis thaliana:
Implication for land plant evolution. Proc. Natl. Acad.
Sci. USA 100: 8007-8012.
Wolf, P.G., Rowe, C.A., Sinclair, R.B., and Hasebe, M.
2003. Complete nucleotide sequence of the chloroplast
genome from a leptosporangiate fern, Adiantum
capillus-veneris L. DNA Res. 10: 59-65.
Tanabe, Y., Uchida, M. Hasebe, M., and Ito, M. 2003.
Characterization of the Selaginella remotifolia MADSbox gene. J. Plant Res. 116: 71-75.
Itoh, Y., Hasebe, M., Davies, E., Takeda, J., and Ozeki, Y.
2003. Survival of Tdc transposable elements of the
En!Spm superfamily in the carrot genome. Mol. Gen.
Genornics 269: 49-59.
Rivadavia, F., Kondo, K., Kato, M., and Hasebe, M. 2003.
Phylogeny of the sundews, Drosera (Droseraceae)
based on chloroplast rbcL and nuclear 18S ribosomal
DNA sequences. Amer. J. Bot. 90: 123-130.

50

NATIONAL INSTITUTE

FOR BASIC BIOLOGY

51

RESEARCH SUPPORTCENTER FOR TRANSGENIC ANIMALS AND PLANTSRESEARCH CENTER FOR INTEGRATIVE
AND COMPUTATIONAL BIOLOGYRESEARCH SUPPORT FACILITYTHE CENTER FOR ANALYTICALINSTRUMENTS (managed by NIBB)TECHNOLOGY DEPARTMENTNATIONAL INSTITUTE52
CENTER FOR
TRANSGENIC ANIMALS AND PLANTS
Head:
Associate Professors:

Technical Staffs:
Supporting Staffs:

NAGAHAMA , Yoshitaka
WATANABE, Eiji
SASAOKA, Toshikuni (Aug. 03- )TANAKA , Minoru (Feb . 04-)
l/NUMA, Hideko (-Oct. 03)
HAYASHI, Kohji (Spt. 03-)
YASUDA, Mie
NOGUCHI, Yuji
SHIMIZU, Naoki
TAKEUCHI, Kazwni
YAMAGUCHI, Kyoko

I. Research supporting activity
NIBB Center for Transgenic Animals and Plants wasestablished in April 1998 to support researches usingtransgenic and gene targeting techniques in NIBB . The
center building for transgenic anima ls has been
completed in the end of 2003 (Figure 1). The building
has a floor space of 2,300m', in which we can breed,develop, store and analyze transgenic , gene targetingand mutant mice under condition of "specific pathogen
free" . It is also equipped with breeding areas fortransgenic aquatic animals, birds and insects.

Figure I. A new center facility for tran sgenic animal s
built at the area E approximately l km away from thearea A .

The expected activities of the Center are as follows:
1. Provision of information, materials, experimental
techniques and breeding space of trausgenic animals
and plants to researchers .
2. Equipment of various kinds of instruments to analyze
mutant, trangenic, and gene-targeted animals andplants.
3. Development of novel techniques related totransgenic and gene targeting technology.

11-1. Academic activity (Watanabe Lab,
Physiological role of Nax sodium channel)
We are studying the functional role of Nax (alsocalled Nav2 or NaG) sodium channel in collaboration
with Division of Molecular Neurobiology. Nax sodium
channel was long classified as a subfamily of voltagegated sodium channels (NaChs) that serve to generateaction potentials in electrically excitable cells such asneuronal and muscle cells . Comparing with the other
NaChs, however, Nax has unique amino acid sequencesin the regions, which are known to be involved in ion
selectivity and voltage-dependent activation andinactivation, suggesting that it must have specificfunctional properties.
To clarify the functional role of Nax , Nax-deficient
mice were generated by gene-targeting technique andthe physiological phenotypes have been examined.
Behavioral studies suggested that the Nax channel plays
an important role in the central sensing of body-fluid
sodium level and regulation of salt intake behavior.
Nax-deficient mice ingest hypertonic sodium ch loride
sol ution in excess in comparison with wild type-mice.
LacZ reporter gene knocked into Nax-gene locus
revealed that Nax gene is expressed in thecircumventricular organ s (CVOs), which is the
specia li zed central organs involved in sensing of
sodium concentration and osmosity in body flu id s.
Sodium ion imaging and electrophysiological studies
using cultured CVO neurons also suggested that Nax is
an extracellular sodium-level sensitive sodium channel.
Recently, we fou nd that Nax is expressed in nonmyelinating Schwann cells and alveolar type II cell s inaddition to the neurons and ependymal cells in the
CVOs. Nax is thus like ly to be involved in reception of
sod ium-level in body fluids at the CVOs and sodium
absorption in the visceral nervous system and in thelung (Figure 2).
More recently , we found in coll aboration with Prof.
Yamamoto's group of Osaka University that the
peripheral nervous system has only subtle effects on thehigher preference for sodium chloride as ovserved in
the mutant mice. The results suggest that the mutant
phenotype is mainly due to the lack of Nax channels in
the central nervous system .
We are now trying to construct functional expression
systems of Nax sodium chan ne l usi ng various
heterologous cell lines. The heterologous expression
system will provide us useful information on thechannel charactors of Nax.

FOR BASIC BIOLOGY53

11-11. Academic activity (Sasaoka Lab)
(1) Studying the function of dopaminergic transmission

The dopamine receptors are divided into two
subgroups, referred to as Dl-like (Dl , D5) and D2-like(D2, D3 and D4) receptors on the basis of their genestructure and their pharmacological and transductional
properties. The dopaminergic system is implicated in
the regulation of the several peptide hormones in thepituitary, the modulation of motor activity, themodulation of synaptic plasticity , and the neuraldevelopment. The dopaminergic system is also
implicated in motivated behaviors, several neurologicaland psychiatric disorders, such as Parkinson's disease
and schizophrenia. Since Dl-like and D2-like receptors
often work synergistically, it is necessary to delete both
Dl-like and D2-like receptors in order to understand therole of dopaminergic transmission. We have generated
multiple dopamine receptor-deficient mice by
combination of single mutant mice, and are studying
phenotype of the multiple dopamine receptor mutant
m.ice.
(2) Developing a novel conditional mutagenesis
technique in mice
The aim of the study is to overcome the limitations of
the conventional mouse molecular genetic approach in
the functional analysis of target genes. We substituted
one critical amino acid residue of N-methyl-D-aspartate
receptor (NMDAR) , leading to NMDAR activation. By
our technique, we accomplished conditional substitutionof the amino acid in mice and our mutant mice
exhibited NMDAR activation and a neurologicalphenotype, similar to that of mouse models forneurological disorders. The development of our mutant
mice should contribute to understanding the function of
the critical amino acid residue and the mechanism ofneurological disorders. Our new technique is vastly
applicable to functional analysis of any desired geneand should contribute to studies on the structural andfunctional relationships of relevant genes.

To understand a physiological role of the SGC , wegenerated the P-SG-deficient (BSG-/-) and y-SGdeficient (GSG-/-) mice. The dystrophin complex
isolated from the skeletal muscles of BSG-1- mice wasunstable in the absence of the SGC and sarcospan. This
indicates that SGC and sarcospan play an important rolein stabilizing the dystrophin axis connecting thebasement membrane and the cytoskeleton. The BSG-/and GSG -1- mice developed progressive muscular
dystrophy with notable muscle hypertrophy. We foundthat the number of muscle fibers increased with age andmost of the fibers were regenerating fibers in their
hypertrophic muscle. Therefore, muscle hypertrophy isa consequence of excess regenerating process but notdue to fibrous and fat tissue replacement, which has
been assumed to be present in so - called
pseudohypertrophy in human diseased muscle.

Publication List
Watanabe , U. , Shimura, T. , Sako, N. , Kitagawa , J. ,Shingai, T., Watanabe, E., Noda, M., and Yamamoto, T.
(2003) A comparison of voluntary salt-intake behavior
in Nax-gene deficient and wild-type mice with reference
to peripheral taste inputs. Brain Res. 964, 274-256
Sasaoka, T ., Imamura, M ., Araishi, K. , Noguchi, S .,Mizuno, Y. , Takagoshi, N., 'Hama, H. , WakabayashiTakai , E. , Yoshimoto-Matsuda, Y., Nonaka, L, Kaneko,
K., Yoshida, M., and Ozawa, E. (2003) Pathologicalanalysis of muscle hypertrophy and degeneration in
muscular dystrophy in gamma-sarcoglycan-deficientmice. Neuromuscular Disorders 13, 193-206

• Na+
••
•••
• e

•.• • .• Na+

cxt ra -~..:c llu lar

Nax

~

••••
•• • •••

Nax

inlro:H;cllul ar

a

_...

X

Salt ln<akeBehav ior

inhibit••••C VO lll!uron.-.
lntra-alwolar(3) Studying of the function of the sarcoglycan complex(SGC)\\,Iterch.lll tti.!l

I N.tKpump

Sarcoglycans (SG) are trans-sarcolemmal
glycoproteins , which associate together to form SGC
and present in the sarcolemma. SGC, together withdystrophin and the dystroglycan complex comprises thedystrophin complex, which has been considered as themechanical link between the basement membrane andthe intracellular cytoskeleton for protecting thesarcolemma from mechanical stress during muscle
contraction. Each of four SG subunits (a-, p-, y- andSG) is responsible for four respective forms of SOdeficient muscular dystrophy, sarcoglycanopathy(SGP) . All of the SGs and sarcospan are absent in thesarcolemma in any form of SGP, suggesting that the
SGC is not assembled, if a single subunit of the SGC is
absent.

o-

(
t

water

:\T- Il ce ll•
Non-111ycliu:ui ng schw:mn ce ll.-.

C

Al veolar l)'JX.' II cdb

Figure 2. A hypothetical scheme for the physiologicalrole of Nax sodium channel. (a) Nax channel is activatedby increase of extracellular sodium concentration. The
activated Nax may promote the excitability ofGABAergic neurons in SFO. The activated GABAergic
neurons in SFO will inhibit the salt-intake-promoting
neuron s. (b) Repetitive action potential s cause
extracellular increase of potassium concentration. Nax
may contribute to the potassium uptake by Schwann
cells. (c) The osmotic gradient between intra-alveolar
space and capiHary is a major driving force for theremoval of water through water channel expressed in
ATI cells. Nax may be one of the molecules generating
the osmotic gradientNATIONAL INSTITUTE

54

RESEARCH CENTER FOR INTEGRATIVE ANDCOMPUTATIONAL BIOLOGYHead:Professo (Adjunct):

YAMAMORJ, Tetsuo
MIYATA, Takashi(Kyoto University)

The neutral theory of molecular evolution, the basic
theory for explaining evolutionary changes at themolecular level, claims that most evolutionary changes
at the molecular level are caused by random drift ofneutral mutants. This is sharply contrasted to theevolution at the organismal level, where it is generally
thought that natural selection is the major driving force
by which evolutionary changes occur. How can we
explain the evolutionary changes at the two levels in a
unified way. This is the major problem that remains
unsolved for molecular evolutionists. As a first step forunderstanding the fmal problem, we are investigating
relationships of evolutionary diversifications between
molecular and organismal levels, based on molecularphylogenetic approach.

I. Divergence pattern of animal gene families andrelationship with the Cambrian explosion
In multicellular animals, a variety of gene families
involved in cell-cell communication and developmental
control have evolved through gene duplication and gene
shuffling, basic mechanisms for generating diverse
genes with novel functions. Each of these animal genefamilies is thought to have originated either from a few
ancestral genes which are shared with plants and fungior from an ancestral gene created uniquely in animallineage. The major groups of bilateral animals arethought to have diverged explo-sively at or prior to theVendian - Cambrian boundary. No direct molecularevidence has been provided to date as to whether the
Cambrian explosion was triggered by a dramaticalincrease in the number of genes involved in cell-cellcommu-nication and developmental control either
immediately prior to or in concert with the Cambrian
explosion.
A molecular phylogeny-based analysis of several
animal-specific gene families has revealed that the genediversification by gene duplication occurred during two
active periods interrupted by a long intervening
quiescent period. Intriguingly, the Cambrian explosion
is situated in the silent period, indicating that there is no
direct link between the first burst of gene diversificationand the Cambrian explosion itself. The above result alsosuggests the importance of gene recruitment as apossible molecular mechanism for morphologicaldiversity.
To understand a possible origin of animal-specificgene families, we have carried out cloning andsequencing of genes related to the animal-specificprotein tyrosine kinase (PTK) genes fromchoanoflagellates, a group of unicellular plotists known
to be the closest relatives of multicellular animals.
Many PTK related genes including both the receptor
and non-receptor type genes have been identified, someof which are likely to be orthologous to animal PTKs. A
molecular phylogenetic tree of the PTK family
members including those from animals andchoanoflagellates showed an unexpected pattern of the
PTK gene family: Most gene duplications that gave rise
to different PTK subfamilies recognized in all animalgroups occurred at very ancient times before thedivergence of animals and choanoflagellates.

II. The origin of land vertebrates andevolutionary relationships among thecoelacanth, lungfishes, and tetrapods based on
nuclear DNA-coded genes
The phylogenetic relationships among tetrapods andtwo living groups of lobe-finned fishes, the coelacanthand the lungfishes are still unsolved and debated,
despite many studies based on morphological andmolecular data. Recent analyses based on complete
mitochondrial sequences resulted in confusing
phylogenetic trees, which are obviously inconsistent
with generally accepted trees. To resolve this difficult
phylogenetic question at statistically solid bases, wehave cloned and sequenced ten nuclear DNA-coded
genes from fourteen major groups of vertebrates,
including the coelacanth and the three lungfish species.
These sequences, together with sequences available
from databases, have been subjected to phylogenetic
analyses based on the maximum likelihood (ML)method, using a cyclostome and a lancelet as an
outgroup. The obtained ML tree supports the close
association of the coelacanth and the lungfish,suggesting that the coelacanth and the lungfish equallyclosely related as sister groups of tetrapods.

Publication List
Hashimoto-Gotoh, T., Tsujimura, A., Watanabe, Y., lwabe,N., Miyata, T. and Tabira, T. (2003) Differential
involvements of presenilins-1 and -2 in the genesis of
Alzheimer's desease. Gene in press.
Terakita, A., Koyanagi, M., Tsukamoto, H., Yamashita, T.,
Miyata, T. and Shichida, Y. (2003) Counterion
displacement in the molecular evolution of rhodopsin
family. Nature Structure & Molecular Biology in press.
Kikugawa, K., Katoh, K., Kuraku, S., Sakurai, H., Ishida,0., Iwabe, N., and Miyata, T. (2003) Basal jawedvertebrate phylogeny inferred from multiple nuclear
DNA-coded genes.BMC evolutionary biology in press.

FOR BASIC BIOLOGY

Associate Professor:
NIBB Research Fellow :Postdoctoral Fellow:
Graduate Students:

55
MOCHIZUKI, Atsushi
TOHYA, Shusaku
FUJITA, Hironori
AYABE, Yoshiko
YAMAGAMI, Ayumi

I. Mathematical method for biological phenomena
We are studying biological phenomena of higher
orders by using mathematical models . Mathematicalmodels give us integrative understanding for complex
behavior of biological systems including a lot offactors.
Study of the mechanisms responsible formorphological difference between species is an
important research focus of the current developmental
biology. One way to answer this question is to model
the pattern formation process and to show how thespecies-specific pattern can be formed by the samemodel with different parameter values. Such atheoretical study would be useful in identifying
candidates of cell-cell interaction that are likely to be
responsible, to which future experimental study can befocused.

II. Pattern formation of the cone mosaic in thezebrafish retina
In teleost fish, there are several subtypes of cone
cells, which are sensitive to different wave-lengths oflight. In retinas of some species of teleost fish, regulararrangements of cone cells are observed, where eachsubtype of cone cells appears periodically in the twodimensional retinal sheet. These patterns are called"cone mosaics." The biological mechanism of thepattern formation is still under examination. Some
species show quite different mosaic patterns between
peripheral region and more central region in the retina,
which suggests mobility of cone cells in retinal space.
Different patterns are observed in different species.
For example, in the zebrafish retina, there are four
subtypes of cones, maximally sensitive to blue, red,green and ultra-violet. A green-sensitive cone cell and a
red-sensitive cone cell are in tight contact and form a
double cone. The cells of the other two subtypes arecalled single cones. The blue-, ultra violet-, red-, andgreen-sensitive cones are also called long single, short
single, long double, and short double cones,
respectively. In an adult zebrafish retina, rows of single
cones and those of double cones appear alternatively.
This pattern is called a "row mosaic".

Figure l. Cone mosaic of fish retina, (Left) in zebrafish,and (Right) in medaka. In fish retina, there are four kindsof differentiated cells. A pair of red-sensitive and greensensitive cones is in close contact, constituting a double
cone. Here four kinds of cones are indicated by capitals(R, G, B, U). In the regular mosaic pattern of zebrafish(Left), the rows of double cones and those of single cones(blue- and UV-sensitive cones) are in parallel andalternately. Two small cones of a red-light sensitive anda green-light sensitive cones form a "double cone".
. In contrast, in the medaka retina, four types of cones
are arranged in a different manner as illustrated in Fig.
1. In the medaka pattern, each blue-sensitive cone issurrounded by four double cones, and the red-sensitive
part of a double cone is close to the green-sensitive partof another. This is called a "square mosaic".
We have studied the mechanism of the patternformation by using mathematical models. In this study,
we examined a process of cell-cell interaction to
generate the regular mosaic pattern -- namely cell
rearrangement. In the model , cells have already been
determined as to their final subtypes, but they change
their locations in the pattern formation process and thecell movement is affected by their neighbours.

.

.

I.
.

.

.

::z.Lt
::I:
.
.
.

.

""T · ~-f · .. -, · ~1·.

-~ ~ ~ - ~

.

.

.

.

.

l. ::l:tl. ::l:.
.

Figure 2. The examples of obtained patterns by cell
rearrangement model. The obtained patterns werecompletely the same as the actual zebrafish retina, when
the used adhesion was appropriate.
We show that the same model can produce both row
and square mosaic pattern. If the cell-cell interaction isrestricted to nearest neighbors only, the square mosaic
pattern cannot be generated. We studied the model
considering the adhesion working between nearest
neighbors and next nearest neighbors, with different
weighting between them. Two "shape factors"
specifying how to combine adhesion in different
geometric are very important in determining whether
row mosaic (zebrafish pattern) or square mosaic(medaka pattern) is to be formed .

~2
2

NATIONAL INSTITUTE

56

Figure 3. The region in which zebrafish pattern isformed. Dots indicate the parameters that generated thezebrafish pattern obtained by computer simulations.
Planes specified the borders separating the parameterregion by comparing the zebrafish pattern with four otherpatterns. A region determined by theoretical analysis wasconsistent with the numerical results.

••zebrafish mosaic pattem<BI

We also studied the direction of stripes in moregeneral situations in which the diffusive direction may
differ between the two substances. We derived aformula for the direction of stripes, based on a heuristic
argument of unstable modes of deviation from theuniform steady state. We confirm the accuracy of theformula by computer simulations. When the diffusive
direction is different between two substances, thedirections of stripes in the spatial pattern changesmoothly with the magnitude of anisotropy of two
substances. When the diffusive direction of the two
substances is the same, the stripes are formed either
parallel to or perpendicular to the common diffusive
direction, depending on the relative magnitude of theanisotropy. The transition between these two phases
occurs sharply.

.- : ..--

8

.e:;:

e

e e
Figure 4. Alternative irregular patterns obtained theparameter regions around the area shown in (A).

We study the conditions for generating mosaic
pattern. The condition for generating zebrafish mosaic
are shown in Fig. 3, 4. They are the necessary
condition and that should be satisfied even in actualorganisms if they make the arrangement based on celladhesion and cell movement.

...

.......~"""-.---

Figure 5. The stripe formation in the skin of Genicanthus.
These two species are closely related.

o.

{j 1 : Anlsolropv o l v

0.

-0.9

-o.5

-o.
·0.

III. Directionality of Stripes Formed by
Anisotropic Reaction-Diffusion Models.
The pattern formation of animal coating has beenstudied mathematically by a pair of partial differential
equations, named a reaction-diffusion (RD) model. By
the model, starting from an initial distribution very
close to uniformity, a spatial heterogeneity emerges anda stable periodic pattern is formed spontaneously. This
simple mechanism suggests that the reaction of a small
number of chemicals and their diffusion might create
stable non-uniform patterns . When we analyze themodel in a two dimensional plane, striped patterns inaddition to spotted patterns often emerge. This wasconsidered as the basic mechanism explaining the stripe
patterns observed among animal coating.
We focused on the directionality of the stripes. Mostof the stripes observed in the fish skins are either
parallel or perpendicular to their anterior-posterior (AP)axis. The direction of stripes is considered ofimportance in the behavioral and ecological viewpoints.
However, very little is known about the mechanismsthat makes the strong directionality either in the actual
fish skin or in the theoretical models. The standard RD
model doesn't determine the direction of stripes. To
explain the. directionality of stripes on fish skin inclosely related species, we have studied the effect ofanisotropic diffusion of the two substances on the
direction of stripes , in the cases in which bothsubstances have the high diffusivity in the same
direction .

Figure 6. Summary of the direction of stripe patterns
obtained by the anisotropic diffusion model. Horizontal
and vertical axes indicate anisotropy of activator and thatof inhibitor, respectively. The left-upper corner indicatesthe distortion of diffusion range. Each point indicates the
direction of the observed stripe: horizontal; vertical; or
not-determined. The direction is determined only by thedifference between anisotropies . A small difference in
diffusion anisotropy can alter the final pattern to one with
opposite directionality. Note the transition of the stripes
by changing the anisotropies from 0 to A (or I).

Publication List
Tohya, S., Mochizuki, A. and Iwasa, Y. (2003) Random
cell sorting can form cone mosaic patterns in fish retina
and explain the difference between zebrafish andmedaka. J. Theor. Bioi. 221, 289-300.
Shoji, H. , Mochizuki, A., lwasa, Y., Hirata, M., Watanabe,
T ., Hioki , S . & Kondo, S. (2003) Origine of
directionality in the fish stripe pattern. Dev. Dyn. 226,627-633.
Ryohji, T., Mochizuki, A. & lwasa, Y. (2003) Possibilityof Tissue Separation Caused by Cell Adhesion. J.
Theor. Biol. 221, 459-474.

FOR BASIC BIOLOGY

57

RESEARCH SUPPORT FACILITY
Head of Facility:

Associate Professor:
Research Associates:
Technical Staffs:

MURATA, Norio (~Mar. 2003),
NODA,Masaharu (Apr.~Nov. 2003),
NISHIMURA, Mikio (Dec. 2003~)
WATANABE, Masakatsu
HAMADA, Yoshio(Tissue and Cell Culture)
UCHIYAMA, lkuo (Computer)
HIGASHI, Sho-ichi(Large Spectrograph)
NAKAMURA,Takanori(Large Spectrograph;Oct. 2003~)
MIWA, Tomoki (Computer)
NANBA, Chieko(Plant Culture, Farm, Plant Cell)NISHIDE, Hiroyo (Computer)
ICHIKAWA. Chiaki(Large Spectrograph)
TAKESHITA, Miyako(Tissue and Cell Culture)
MAKIHARA,Nobuko (Computer)
SUZUKI, Keiko(Plant Culture, Farm, Plant Cell)

I. Facilities

1. The Large Spectrograph Laboratory
This laboratory provides, for cooperative use, theOkazaki Large Spectrograph (OLS), which is the largest
spectrograph in the world, dedicated to action spectroscopical studies of various light-controlled biologicalprocesses. The spectrograph runs on a 30kW Xenon arc
lamp and has a compound grating composed of 36
smaller individual gratings. It projects a spectrum of awavelength range from 250nm (ultraviolet) to 1,000nm(infrared) onto its focal curve of lOrn in length. The
fluence rate (intensity) of the monochromatic light ateach wavelength is more than twice as much as that of
the corresponding monochromatic component of
tropical sunlight at noon (Watanabe et al. 1982,
Photochem. Photobiol., 36, 491-498).
An advanced irradiation system composed of CW
lasers (364nm, 390-410nm, 440-460nm, 532nm,
655nm, 752nm) and uniform-fluence-rate irradiation
optics interconnected by optical fibers was constructed
in 2003. An advanced observation system for cellularand intracellular photobiological responses utilizing a
two-photon microscope (FV300-Ix71-TP with a MaiTailaser) and a microbial photomovement analyzer(WinTrack2000/Ecotox) etc. was also introduced.
2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell culture areprovided. This laboratory is equipped with safely rooms
which satisfy the P2/P3 physical containment level.
This facility is routinely used for DNA recombination
experiments.
3. Computer Laboratory
Computer laboratory maintains several computers toprovide computation resources and means of electronic
communication in this Institute. Currently, the main

system consists of three servers and two terminal workstations: biological information analysis server (SGI
Ori-gin 2000), database server (Sun Enterprise 450),
file server (Sun Enterprise 3000), data visualization
terminal and molecular simulation terminal (both are
SGI Octanes). Some personal computers andcolor/monochrome printers are also equipped. On thissystem, we provide various biological databases anddata retrieval/analysis programs, and support large-scaledata analysis and database con-struction for the Institutemembers.
Computer laboratory also provides network
communi-cation services in the Institute. Most of PCs
in each laboratory as well as all of the above service
machines are connected each other with local area
network (LAN), which is linked to the highperformance multimedia back-bone network of Okazaki
National Research Institute (ORION). Many local
services including sequence analysis service, filesharing service and printer service are provided throughthis LAN. We also maintain a public World Wide Web
server that contains the NIBB home pages(http://www.nibb.ac.jp).
4. Plant Culture Laboratory
There are a large number of culture boxes, and a limited number of rooms with environmental control forplant culture. In some of these facilities and rooms, experiments can be carried out at the Pl physical containment level under extraordinary environments such as
strong light intensity, low or high temperatures.
5. Experimental FarmThis laboratory consists of two 20 m2 glass-houses
with precise temperature and humidity control, threegreen houses (each 6m2) at the PI physical containment
level, a small farm, two greenhouses (45 and 88 m 2)
with automatic sprinklers. The laboratory also includes
a building with storage and work space.
6. Plant Cell Laboratory
Autotrophic and heterotrophic culture devices andequipment for experimental cultures of plant and microbial cells in this laboratory. A facility for preparation ofplant cell cultures including an aseptic room with clean
benches, is also provided.
7. Laboratory of Stress-Resistant Plants
This laboratory was. found to study transgenic plants
with respect to tolerance toward various environmental
stresses. It is located in the Agricultural Experimental
Station of Nagoya University (30 km from National Institute for Basic Biology). The laboratory provides avari-ety of growth chambers that precisely control thecondi-tions of plant growth and dacilities for molecularbiologi-cal and physiological enaluations of transgenic
plants.
The laboratory is also a base of domestic andinternational collaborations devoted to the topic ofstress resistant transgenic plants.

58

II. Research Activities
1. Faculty
The faculty of the Research Support Facility
conducts its own research as well as scientific andadministrative public services.
(1) Photobiology: Photoreceptive and signal transduction mechanisms of phototaxis of unicellular algae
are studied action spectroscopically (Watanabe 2004, In"CRC Handbook of Organic Photochemistry andPhotobiology, 2nd ed.") by measuring computerizedvideomiceographs of the motile behavior of the cells at
the cellular and subcellular levels. Photoreceptive 'andsignal transduction mechanisms of algal gene
expression were also studied by action spectroscopy.
A novel blue-light receptor with an effector role wasfound from Euglena gracilis (Fig. ; Iseki et al. 2002,
Nature 415, 1047-1051): Euglena gracilis, a unicellular
flagellate , shows blue-light type photomovements. The
action spectra indicate the involvement of flavoproteinsas the photoreceptors mediating them . The
paraflagellar body (PFB), a swelling near the base of
the flagellum has been considered as a photosensing
organelle for the photomovements. To identify thephotoreceptors in the PFB , we isolated PFBs andpurified the fla voproteins therein. The purified
flavoprotein (ca. 400 kDa), with noncovalently bound
FAD, seemed to be a heterotetramer of a- and bsubunits. Predicted amino acid sequences of each of thesubunits were similar to each other and contained two
FAD-binding domains each followed by an adenylyl
cyclase catalytic domain. The flavoprotein showed an
adenylyl cyclase activity, which was elevated by bluelight irradiation. Thus, the flavoprotein (PAC,

NATIONAL INSTITUTE

photoactivated adenylyl cyclase) can directly transduce
a light signal into a change in the intracellular cyclic
AMP level without any other signal transduction
proteins.
The involvement of PAC in positive and negative
phototaxis (steering response with respect to stimulus
light direction) was also demonstrated by its knockdown using RNAi (Ntefidou et al. 2003). Orthologuesof PAC a and ~ were detected in several relatives ofEuglena and their phylogenetic analysis indicated thatthey were trtansfered to euglenoids on the occasion of
secondary endosymbiosis (Koumura et al. submitted).
(2) Developmental Biology: Replacement of theankyrin repeats of mouse Notch2 gene with E. coli bgactosidase gene induces early embryonic lethality
around El0 .5. The lethality was suggested due todefects in extraembryonic tissues , because the mutant
embryo grew and differentiated further in vitro .
Histological examination and in situ hybridization
analysis with trophoblast subtype-specific probes
revealed that the development of giant andspongiotrophoblast cell layers are normal in the mutant
placenta, while vasculogenesis in the labyrinth layer
apperaed compromised at E9.5. Since the lethality wascircumvented by production of chimeric mice withtetraploidy wild type embryos, we concluded that theembryonic lethality is due to defect in growth and/or
differentiation of labyrinthine trophoblast cells. The
mutant embryo, however, could not be rescued in thetetraploid chimeras beyond El2.5 because of
insurfficient development of umbilical cord, indicating
another role of Notch2 signaling in the mouse
development. Chimeric analysis with diploid wild type,however, revealed contribution of mutant cells to these
affected tissues by El3 .5. Thus , Notch2 are not cellautonomously required for the early cell fate
determination of labyrinthine trophoblast cells andallantoic mesodermal cells, but plays an indispensablerole in the further formation of functional labyrinth
layer and umbilical cord.
(3) Computational Biology: Comparative genomics isa useful approach to find clues to understanding
complex and diverse biological systems. Our research
aim is to develop new methods as well as practicalanalysis systems to compare a large number of genomic
sequences, and to apply them to real data analyses.
Our main developing system is a workbench formicrobial genome comparison named MBGD, which
now contains more than a hundred of microbial genome
sequences . We have continued to enhance theefficiency of the database to treat such large number ofgenomes. As a key component of MBGD, we have also
continued to develop an automated method fororthologous grouping among multiple genomes. In
addition to splitting fusion genes into orthologous
domains, we are also trying to enhance the algorithm toincorporate some other information such as genearrangement and species phylogeny.

FOR BASIC BIOLOGYIn addition to these developments, we are also trying
to apply the computational methods to real data
analyses, especially to the comparative genomics withsome closely related organisms. In collabolation with
Dr. Takami's group (JAMSTEC), we are comparing
genomic sequences of some Bacillus-related organismsthat live in several different environmental conditions.

2. Cooperative Research Program for the Okazaki
Large Spectrograph
The NIBB Cooperative Research Program for the
Use of the OLS supports about 20 projects every year
conducted by visiting scientists including foreign scientists as well as those in the Institute.
Action spectroscopical studies for various regulatoryand damaging actions of light on living organisms,
biological molecules, and artificial organic molecules
have been conducted (Watanabe, 2004, In "CRC
Handbook of Organic Photochemistry andPhotobiology, 2nded.". pp. 115-1-115-16).

Publication List:
I. Faculty
Mikami, K. and Murata, N. (2003) Membrane fluidity andthe perception of environmental signals in cyanobacteria
and plants. Progress Lipid Res. 42, 527-543.
Mikami, K., Suzuki, I. and Murata, N. (2003) Sensors of
abiotic stress in Synechocystis. In "Topics in Current
Genetics, vol. 4, Plant Stress Response" (H. Hirt and K.
Shinozaki eds.), pp 103-119.
Nishiyama T, Fujita T, Shin-I T, Seki M, Nishide H,
Uchiyama I, Kamiya A, Carninci P, Hayashizaki Y,
Shinozaki K, Kohara Y, Hasebe M. (2003) Comparative
genomics of Physcomitrella patens gametophytictranscriptome and Arabidopsis thaliana: implication forland plant evolution. Proc. Nat/. Acad. Sci. USA. 100,
8007-8012.
Ntefidou, M., Iseki, M., Watanabe, M., Lebert, M., Raeder,
D.-P. (2003) Photoactivated adenylyl cyclase (PAC)
controls phototaxis in the flagellate Euglena gracilis.
Plant Physiol. 133, 1517-21.

59
Suzuki, T., Yamasaki, K., Fujita, S., Oda, K., Iseki, M.,
Yoshida, K., Watanabe, M., Daiyasu, H., Toh, H.,
Asamizu, E., Tabata, s., Miura, K., Fukuzawa, H.,
Nakamura, S., Takahashi, T. (2003) Archaeal-type
rhodopsins in Chlamydomonas: model structure andintracellular localization. Biochem. Biophys. Res.
Commun. 301,711-717.
Tamura, K., Shimada, T., Ono, Y., Nagatani, A., Higashi,
S-I, Watanabe, M., Nishimura, M., Hara-Nishimura, I.
(2003) Why green fluorescencent fusion proteins havenot been observed in the vacuoles of higher plants. Plant
J. 35, 545-555.
Uchiyama, I. (2003) MBGD: microbial genome databasefor comparative analysis. Nucleic Acids Res. 31, 58-62.
Watanabe, M. (2003) Action spectroscopy forphotosensory processes. In "CRC Handbook of Organic
Photochemistry and Photobiology, 2nd ed." (W.
Horspool and F. Lenci eds.) pp. 115-1-115-16, CRC
Press, Boca Raton
II. Cooperative Research Program for the Okazaki

Large Spectrograph
Andrady, A.L., Halis, S. H., and Torikai, A. (2003) Effectof climate change and UV-B on materials. Photochem.
Photobiol. Sci. 2, 68-72.
Andrady, A.L., Hamid, S. H., and Torikai, A., (2003)
Effect of climate change and UV-B on materials. In"
Environmental Effect of Ozone Depletion andInteractions with Climate Change:2002 Assessment" .
Arimoto-Kobayashi, S., Ando, Y., Horai, Y., Okamoto, K.,
Hayatsu, H and Michael H. L. G. (2002) Mutation,
DNA strand cleavage and nitric oxide formation causedby N-nitroproline with UV A & UVB. J. Photosci. 9, 4950.
Sasaki, M., Takeshita, S., Oyanagi, T., Miyake, Y. and
Sakata, T. (2002). Increasing trend of biologically active
solar ultraviolet-B irradiance in mid-latitude Japan in
the 1990s. Optical Engineering 41,3062-3069.
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani,A., Higashi, S-i., Watanabe, M., Nishimura, M. andHara-Nishimura, I. (2003) Why green fluorescent fusion
proteins have not been observed in the vacuoles ofhigher plants. Plant J. 35, 545-555.
·

NATIONAL INSTITUTE

60

THE CENTER FOR ANALYTICAL INSTRUMENTS(managed by NIBB)

Representative instruments are listed below.
Protein Sequencers (ABI Procise 494HT,492cLC)
Amino Acid Analyzer (Hitachi L8500A)

Head of Facility:
Technical Staff's:

Technical Assistant:

KOBAYASHI, Satoru
MORJ, Tomoko
MAKINO, Yumiko
TAKAMI, Shigemi
MORIBE, Hatsumi

Peptide Synthesizers (ABI 433A, 432A)
Plasmid Isolation Systems (Kurabo PI-1002: , PI-50, PI200)
Automatic Nucleic Acid Isolation System (K urabo NA2000)
DNA Sequencers (ABI 377, 373S, 310, 3100)

The Center serves for amino acid sequence analysis,
and chemical syntheses of peptides and nucleotids tosupport researchers in NIBB and NIPS . Newly installed
instruments in 2003 are Protein Sequencer (ABI Procise
492cLC) , Color Laser 3D Profile Microscope(KEYENCE VK-8500) and so on. Instruments of theCenter can be used by researchers outside the Instituteupon proposal.

Thermal Cyclers (Perkin Elmer PJ-9600 , Takara TP-300,
Biometra TGRADIENT)
Integrated Thermal Cyclers (ABI CATALYST Turbo 800)
Particle Delivery System (B io-Rad BiolisticPDS-1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Analysis System (Takara Glyco-Tag)
High Performance Liquid Chromatographs (Shimadzu LCIOAD, LC-6AD, Waters 600E, Alliance UV system)
Integrated Micropurification System (Pharmacia SMART)
Flow Cytometer (Cou lter EPICS XL)
Biomolecular Interaction Analysis Systems (Pharmacia
BIACORE 2000, Affinity Sensors IAsys)
Laboratory Automation System (Beckman Coulter Biomek
2000)
NMR Spectrometer (Bruker AMX-360wb)
EPR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)

Figure I. Protein Sequencers.

MALDI/TOF-MS (Bruker Daltonics REFLEX III)
Inductively Coupled Plasma
Spectrometer (Seiko SPS1200A)

Atomic Em is sion

Spectrofluorometers (Hitachi 850, F-4500, Simadzu RF5000)
Spectrophotometers (Hitachi 330, 557, U-2001,Varian
Cary 5G,Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB 96P)
Microplate Readers (Corona MTP-120, MTP-lOOF)
Spectropolarimeter (JASCO J-40S)
FT-IR Spectrophotometer (Horiba FT-730)
Laser Raman Spectrophotometer (JASCO R-800)
Bio Imaging Analyzers (Fujifilm BAS 1500, 2000, 5000)
Fluorescence Bio Imaging Analyzer (Takara FMBIO)
Figure 2. MALDI/TOF-MS

Electrophoresis Imaging System (BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Environmental Scanning Electron Microscope (PHILIPSXL30 ESEM)
Confocal Laser Scanning Microscope (Leica TCS SP2)
Color Laser 3D Profile Microscope (KEYENCE VK-8500)
High-Resolution Quick Microscope (KEYENCE VH5000)

61

FOR BASIC BIOLOGYTECHNOLOGY DEPARTMENT

Head: HATTORI, Hiroyuki

Common Facility Group

Research Support Group

Chief: FURUKAWA, Kazuhiko

Chief: KOBAYASHI, Hiroko

Reseach Support Facilities
HIGASHI, Sho-Ichi (Unit Chief) <Large Spectrograph>
NANBA, Chieko (Subunit Chief) <Plant Culture>
NISHIDE, Hiroyo <Computer>
NAKAMURA, Takanori <Large Spectrograph>SUZUKI, Keiko (Technical Assistant)
MAKIHARA, Nobuko (Technical Assistant)
ICHIKAWA, Chiaki (Technical Assistant)
HARADA, Miyuki (Technical Assistant)

Cell Biology Group
KONDO, Maki (Unit Chief)
KABEYA, Yukiko (Subunit Chief)

Center for Analytical Instruments
MORI, Tomoko (Unit Chief)
MAKINO, Yumiko
TAKAMI, Shigemi
MORIBE, Hatsumi (Technical Assistant)
Transgenic Animal Facility
HAYASHI, Koji (Subunit Chief)
YAMAMOTO, Mamiko (Technical Assistant)
Disposal of Waste Matter Facility
MORI, Tomoko (Unit Chief)
FURUKAWA, KazuhikoRadioisotope Facility
MATSUDA, Yoshimi (Unit Chief)
SAWADA, Kaoru (Subunit Chief)l/NUMA, Hideko
ITOH, Takayo (Technical Assistant)

The Technology Department is a supportingorganization for researchers and research organization
within the NIBB. The Department develops andpromotes the institute's research activities and at thesame time, maintains the research functions of theinstitute.
The department is organized into two groups: one,
the Common Facility Group, which supports andmaintains the institute's common research facilities andthe other, the Research Support Group, which assists
the research activities as described in individual reports.
Technical staffs participate, through the department,

Developmental Biology Group
TAKAGI, Chiyo
OKA, Sanae
Regulation Biology GroupYAMAGUCHI, Katsushi
TAKEUCHI, Yasushi
Gene Expression and Regulation Group I
TANAKA, Sachiko (Unit Chief)
MOROOKA, Naoki
Gene Expression and Regulation Group IIOHSAWA, Sonoko (Unit Chief)
SUMIKAWA, Naomi
Integrated Bioscience GroupMIWA, Tomoki (Unit Chief)
MIZUTANI, Takeshi
UTSUMI, Hideko
NODA, Chiyo
KONDO, Makiko (Technical Assistant)in mutual enlightenment and education increase theircapability in technical area. Each technical staff isproceeded to the fixed division usually and they support
the various research with their special biological andbiophysical techniques.
The Department hosts an annual meeting fortechnical engineers who work in various fields of
biology at universities and research institutes
throughout Japan. At this meeting, the participantspresent their own activities and discuss technicalproblems. The Proceedings are published soon after the
meeting.

62

NATIONAL INS':rt:I't:1TE

63

FOR BASIC BIOLOGYCENTER FOR INTEGRATIVE BIOSCIENCE(jointly managed by NIBB)
Chairperson: KITAGAWA Teizo

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATIONDIVISION OF DEVELOPMENTAL GENETICSDIVISION OF MOLECULAR & DEVELOPMENTAL BIOLOGYDEPARTMENT OF BIO-ENVIRONMENTAL SCIENCEDIVISION OF BIO-ENVIRONMENTAL SCIENCEDIVISION OF PLANT DEVELOPMENTAL GENETICSDIVISION OF BIOINFORMATICSThe center is jointly managed by NIBB and two other institutes in
Okazaki, IMS (Institute for Molecular Science) and NIPS (National
Institute for Physiological Sciences). The following projects will be themain focus of the center: 1) Development, Differentiation andRegeneration. 2) Strategic Methodology. 3) Bio-Environmental Science.

64

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL GENETICS

Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
JSPS Postdoctral Fellow:
Postdoctoral Fello w:
Graduate Students:

Technical Assistants:

Secretary:

KOBAYASHI, Satoru
MUKAI, Masanori
NODA, Chiyo
SATO, Kimihiro
SHIGENOBU, ShujiAMIKURA, Reiko
HAYASHI, Yoshiki
KITADATE, Yu
HAYASHI, Makoto n
ARITA KayoUEDA , Yoshiko
TSUCHIYA, Naomi
SATO, KaoriSUZUKI, Masako'' Graduate School of Biological Sciences,
University ofTsukuba

The sperm and egg, or the germ cells are thespecialized cells , which can transmit the genetic
materials from one generation to the next in sexual
reproduction. All the other cells of the body are somatic
cells. This separation of germ and somatic cells is oneof the oldest problems in developmental biology. In
many animal groups , a specialized portion of egg
cytoplasm , or germ plasm , is inherited by the celllineage which gives rise to germ cells. This cell lineage
is called germline. The germline progenitors eventually
migrate into the gonads, where they differentiate as
germ cells when the organisms are physically matured .
Earlier investigators have demonstrated that germ plasm
contains maternal factors required and sufficient for
germline formation. In the fruit fly, Drosophila , thiscytoplasm is histologically marked by the presence ofpolar granules , which act as a repository for thematernal factor required for germline formation. Our
molecular screens have identified several factors stored
in the polar granule s . One of the factor s is
mitochondrial large rRNA which functions to form thegermline progenitors, or pole cells. The other is nanos
mRNA, which is required for pole cell differentiation.

granules in germ plasm, and it depends on the functionof the maternally-acting gene, tudor, that is known to berequired for pole cell formation. Mitochondrial rRNAs
remain on the polar granules until pole cell formation
and are no longer discernible on the granules withinpole cells . Reduction of the extra-mitochondrial
mtlrRNA amount results in the failure to form pole cells
and injection of mtlrRNA is able to induce pole cells inembryos whose ability to form these cells has beenabolished by uv-irradiation. These observations clearlyshow that the extra-mitochondrial mtlrRNA on polar
granules has an essential role in pole cell formation ,presumably cooperating with mtsrRNA.
Recently, we found that mitochondrial rRNAs form
mitochondrial-type of ribosomes on polar granules ,cooperating with mitochondrial ribosomal proteins.
This s uggests the possibility that the protein (s)essential for pole cell formation is produced by the
mitochondrial-type of ribosomes. To address this issue,
we examined the effect of Chloramphenicol andKasugamycin on pole cell formation. Chloramphenicol
and Kasugamycin are known to inhibit mitochondrial(prokaryotic)-type of translation. When these antibiotics
were injected into the posterior pole region of early
embryos, pole cell formation was severely affected. In
contrast, Chloramphenicol and Kasugamycin treatment
did not affect somatic cell formation at a dose we used.
These observations strongly suggest that the
mitochondrial-type of translation system must be intact
for the embryos to form pole cells. The project toidentify a targer mRNA for the mitochondrial-type oftranslation machinery is now on going.

Mitochondria
Mitochondriul
R i bosonm l l»r·otl'in!>

Po larGr.:mult•Polt•Cell
Fo1·nmlion

Fig . I Role of mitochondrial rRNAs in pole cell
formation

I. Role of Mitochondrial Ribosomal RNAs in Pole

Cell Formation
Ultrastructural studies have shown that the germ
plasm is basically composed of polar granules andmitochondria. While the primary roles of themitochondria are oxidative phosphorylation andbiosynthesis of many metabolites, it has now become
evident that they are also involved in germline
formation.
In Drosophila , pole cell formation requires thefunction of mitochondrial ribosomal RNA in germ
plasm. We have previously reported that mitochondrial
large rRNA (mtlrRNA) and small rRNA (mtsrRNA) areboth transported from mitochondria to polar granules .
This transportation occurs during early embryogenesis,
when mitochondria are tightly associated with polar

II. Role of Nanos protein in pole celldifferentiation
Pole cells differ from the soma in regulation ofmitosis and transcriptional activity . Pole cells ceasemitosis at gastrulation and remain quiescent in the G2phase of the cell cycle throughout their migration to the
gonads , while somatic cells continue to proliferate
during the rest of embryogenesis . Furthermore , polecells are transcriptionally quiescent until the onset ofgastrulation , although transcription is initiated in thesoma during the syncytial blastoderm stage. Consistent
with this, RNA polymerase II (RNAP II), but not RNA
polymerase I, remains inactive in early pole cells. Thus,
the ability to express zygotic mRNA-encoding genes is

65

FOR BASIC BIOLOGY

suppressed only in pole cells in early embryos.
Among the maternal components of germ plasm,
Nanos (Nos) is essential for the germline-specific
events occurring in pole cells. nos mRNA is localized in
the gerrri plasm during oogenesis, and is translated insitu to produce Nos protein after fertilization. Nos isonly transiently present in the posterior half of embryos
during the preblastoderm stage, and is required there forposterior ·somatic patterning. Nos in the germ plasm ismore sta bly inherited into the pole cells at theblastoderm stage, remaining detectable in these cells
throughout embryogenesis. Pole ce ll s that lack Nos(nos· pole cells) are unable to follow normal germline
development; they fail to migrate properly into theembryonic gonads, and consequently do not become
functional germ cells. In nos· pole cells, mitotic arrest at
G2 ph ase is impaired , and they undergo prematuremitosis. Furthermore, nos· pole cells fail to establish
and / or m ai nt ain tr anscription a l quiescence, andectopically ex press somatically-tran scribed genes,
including.fushi tarazu (jtz), even-skipped (eve) and Sexlethal (Sxl).
Nos represses translation of mRNAs with discrete
RNA sequences called Nos response elements (NREs).
In the pathway leading to posterior somatic patterning,
Nos ac ts together with unlocalized Pumilia (Pum)protein to repress translation of maternal hunchback(hb) mRNA. This translational repression is mediatedby binding of Pum to NREs in th e 3'-untranslated
region (UTR) of hb mRNA. In pole cells, Nos also actswith Pum to regulate germline-specific events. Pum,
like Nos , is required in pole cells for their migration to
the gonads.

We found that No s, along with Pum, repres ses
translation of importin a2 (impa2) mRNA in early polecells. The impa2 mRNA contains an NRE-likeseq uence in its 3'-UTR and encodes a Drosophila
importin a homologue that plays a role in nuclear
import of karyophilic proteins. We found that Nos
inhibits expression of a somatically-transcribed gene,jtz, in pole cells by repressing Impa2-dependent nuclear
import of a transcriptional activator for ftz , Ftz-Fl.
Furthermore, the expression of another somatic gene,eve, and RNA Polymerase II activity are also repressedby Nos in pole cells through its effects on Impa2dependent nuclear import.
The above results raise the question whether the polecells lacking Nos (nos · pole cells) are able todifferentiate into somatic cells. However, it is difficult
to study their developmental fate, since Nos also
represses apoptosis of pole cells, and almost all of nos·pole cells are eliminated until at least the end ofembryogenesis. To overcome this problem, we used Df(3 L) H99 , a deletion for three ge ne s required for
apoptosis. Introduction of the H99 deficiency results in
nos - pole cells being escaped from apoptosis. We
transplanted the nos· H99 · pole cells into normal
embryos and observed their behavior, and found thatsome of nos· H99· pole cells were able to differentiate as
somatic cells. This suggests that pole cells have theability to differentiate as somatic cells, but its ability is
inhibited by Nanos activity. Recently , we have foundthat somatic differenti ation of nos· H99· pole cells
requires Impa-2 activity, suggesting that Nos inhibits
so mati c differentiation by repre ssi ng Impa-2
production.

Isolation of embryonic gonad

D
eDNAmRNA or total RNA

DD
DNA microarray.. . ..
.· .

. . ·::, .">:· :::·· ': : :·

D
<;:;

. .
..

D

Non-redundant eDNA set-

Up-regulated genes

{1

WISH

Fig. 3 Transcriptome analysis of the embryonic gonads

III. Comprehensive analysis of genes expressed in
Drosophila gonad

Apoplosis\,.].)l
Somatic diffcrcntialion

Fig. 2 Somatic differentiation of pole cells lacki ng Nos

Pole cells migrating into the gonads are specified tobe the primordial germ cells (PGCs). It has been
believed that zygotic genes expressed in pole cells
within the gonads are required for their fatespecification. To explore the regulatory mechanism ofgermline specification, we attempted to identify genes

66

NATIONAL INSTITUTE

expressed in pole cells and/or in somatic cells within
the gonad by a comprehensive approach. From theembryos carrying EGFP-vasa transgene that express
GFP only in pole cells, we isolated the gonads by using
fluorescence-activated cell sorting (FACS), andcostructed a gonad eDNA library. Each eDNA clone
was sequenced from both 5' and 3' ends, and these
Expression Sequence Tags (ESTs) werecomputationally condensed into sequence clusters,
which were then subjected to whole-mount in situ
hybridization (WISH). Approximately 20,000 of ESTs
were generated, and were clustered into 2900 distinct
genes . The WISH analysis identified more than 130
genes that were expressed predominantly in the gonads.
In addition, we found gonad-specific splicing form insome transcripts. These transcriptome data will allow us
to illustrate genetic networks governing the gerrnline
specification.
We also started to identify genes expressed
predominantly in the embryonic gonads, using DNA
microarray technique.

1o'

u

rn

8,1&

10'

101
101
control

Fig. 4 Scatter plot of expressin levels in control (whole
embryos) and the embryonic gonads.

IV. sva53, a Maternal Gene Required for Meiosis
It has been believed that maternal factors localized ingerm plasm may ultimately trigger germline-specific
events, such as meiosis. We have isolated an X-linked
maternal mutation, sva53 that affects meiosis. Pole cells
that were formed in the embryos derived from sva53
homozygous germline clone (sva53 pole cells) wereable to develop into the oocytes, but they failed to
execute meiosis. We also found that the germlinespecific expression of vasa gene was severely affectedin sva53 pole cells. These results suggest that thematernal factor encoded by sva53 gene may activate
gene expression, which is essential for meiosis. In order
to identify sva53 gene, we mapped sva53 mutation to
200 kb-genomic region of 11 C by using duplications
and deficiencies. Within this region, we found a geneencoding a Zn-finger transcription factor, of whichmRNA is maternally supplied into embryos.

Publication List:
Tsuda, M., Sasaoka, Y., Kiso, M., Abe, K., Haraguchi, S.,
Kobayashi, S. and Saga, Y. (2003) Conserved role ofnanos proteins in germ cell development. Science 301,

1239-1241.

FOR BASIC BIOLOGYDIVISION OF MOLECULAR &DEVELOPMENTAL BIOLOGY
Professor:
TAKADA , Shinji
Research Associates:
KOSHIDA, Sumito
Technical Staff:
UTSUMI, Hideko
NIBB Research Fellows: OHBAYASHI, Norihiko
KAWAMURA, Akinori
MUROYAMA , Yuko
Postdoctoral Fellows:
KURATA, Tomoko
AKANUMA, Takashi
TAKADA, Ritsuko
Graduate Students:
YAMAGUCHI, Yoshifumi"
Technical Assistant:
ODA, Ritsuko

67
at the cellular level has been poorly understand. In vitro
stem cell culture is a powerful tool for examining theeffect of an external signal on neural stem cells. In
vitro clonal analysis has shown that, in a serum-freedefined medium supplemented with FGF-2, single cells
derived from the embryonic or adult brain proliferate
and form floating spherical colonies , calledneurospheres. Single cells derived from neurospheres
self-renew to generate a new neurosphere ordifferentiate into neurons or glia depending on theculture condition, indicating that they havecharacteristics of stem cells.

A" Graduate School of Biostudies, Kyoto University

One of the research interests of this laboratory is tounderstand molecular mechanism how a cell signaling
molecule, including members of Wnt, BMP and FGF
families, regulates different developmental events. A
number of evidence indicated that each signal isinvolved in many aspects of the vertebrate
development. For instance, we have revealed that Wnt3a, a members of Wnt family , plays essential roles in a
number of aspects of the mouse development, including
somite development, neural crest formation and neuraldevelopment. However, cellular and molecularmechanisms how a cell signaling molecule regulates
these different events. Thus, we are focusing on precise
functional analysis of cell-to-cell signals andidentification of target genes induced by these signals.
Another interest is to understand molecularmechanism of development of the vertebrate trunk,especially somite. One of interesting features of thevertebrate trunk development is that it proceeds
gradually. To understand how metameric structures ofsomites are gradually generated in an anterior toposterior order along the both sides of embryonic body
axis and how each somite are characterized differently
along the anterio-to-posterior axis, we are also trying
genetical approach with the zebrafish.
I. Roles of Wnt signals during neural development

The Wnt family of genes that encode cysteine richsecreted proteins consists of at least 17 members in thevertabrate. It has already been shown that some of them
are expressed and play important roles during neuraldevelopment. For instance, we showed that Wnt-1 and
Wnt-3a, which are expressed in the most dorsal region
within the developing central nervous system, direct
specification of the dorsal interneurons. Analysis ofmouse embryos lacking both Wntl and Wnt3a andculture of explants from the neural plate indicated thatthese Wnt signals promote generation of the most dorsal
subclass of the interneuron, called Dl and D2, at theexpense of that of more ventral subclass, called D3.
Wnt signaling is also implicated in the control of cell
growth and differentiation during CNS developmentfrom studies of mouse and chick models, but its action

c

B
A

c

B

Wnt

Nestm

~tAP2

DAPI

Control

Fig.!. Inhibition of neurosphere formation and promotionof neural cell differentiation in the presence of Wnt3a
CM.
A: Cells dissociated from the E I I .5 mou se forebrain
were cultured for 2 days in low attachment multiwellplates under various conditions: with Wnt3a conditioned
medium (Wnt-3a CM) (A), with immunoabsorbed Wnt3a CM (B), with control conditioned medium (controlCM) (C), and without conditioned medium (D). Manylarge spheres were generated in the culture with control
CM (D). In contrast, treatment with Wnt-3a CM
significantly increased the number of differentiated cells
and almost wiped out neurospheres (A). This effect wasdependent on Wnt-3a concentration (data not shown).
These effects of Wnt-3a CM were almost eliminated byimmunoabsorption, indicating that these effects are dueto the Wnt-3a protein (B). Total 10 fields were counted
under phase-contrast microscope using I OO x
magnification.
B: Dissociated cells from the primary neuro spheres

obtained from the Ell.5 telencephalon were cultured fortwo days on poly 0-Iysine coated coverslips in thepresence of Wnt3a CM and FGF2. Then, these cells were
continuously cultured for an additional four days with
Wnt3a CM (A-C) or control CM (D-F) , but without
FGF2. After 4 days in culture, cells were immunostainedfor Nestin or MAP2. Immunofluorescence microscopy
for Nestin (red) (A, D), MAP2 (green) (B , E) and DAPI
(blue) (C, F).

NATIONAL INSTITUTE

68
Thus, to understand function of Wnt signaling on theneural stem cells, we examined the in vitro function of
Wnt signaling in embryonic neural stem cells,
dissociated from neurospheres derived from Ell.5
mouse telencephalon. Conditioned media containing
active Wnt-3a proteins were added to the neural stem
cells and its effect on regeneration of neurospheres anddifferentiation into neuronal and glial cells was
examined. Wnt-3a proteins inhibited regeneration ofneurospheres, but promoted differentiation into MAP2positive neuronal cells (FIG. 1). Wnt-3a proteins also
increased the number of GFAP- positive astrocytes but
suppress the number of oligodendroglia! lineage cells
expressing PDGFR or 04. These results indicate that
Wnt-3a signaling can inhibit the maintenance of neuralstem cells , but rather promote the differentiation ofneural stem cells into several cell lineages.

II. Functional Analysis of molecular targets of
Wnt signaling during development
Wnt-3a is also expressed in the primitive streak
ectoderm during gastrulation and in the tailbud in later

Oh

12h

development of the mouse. For dissecion of thecomplex developmental events regulated by Wnt-3a
signaling in these regions , it is important to identify
genes regulated by this signal. It has already been
demonstrated that T (Brachyury) is a direct target of
Wnt-3a in the anterior primitive ectoderm, which isfated to give rise to the paraxial mesoderm, suggestingthat Wnt-3a modurates a balance between mesodermal
and neural cell fates via T.
To gain more insight into roles of Wnt signaling
during embryogenesis, we searched for potential target
genes of this s ignaling by an induction gene trap
screening in mouse ES cells. In at least three ES cellclones among 794 clones screened, expression of betageo reporter genes was dramatically changed in
response to the conditioned medium of Wnt-3a
expressing cells. The expression analysis of the reporter
genes in embryos generated from these ES cell clones
revealed that the spatiotemporal expression patterns ofthese reporter genes were well correlated to those of
several Wnt genes. These results suggested that an
induction gene trap approach is effective for screeningof target genes of Wnt signaling during embryogenesis.

24h

..

48h

Control

WNT-3A
Fig. 2. An example of the expression of trapped genes which are repressed by Wnt signal in ES cells. In vivo expression and in vivo
function of this gene have been examined by generating mice carrying this trapped allele.

69

FOR BASIC BIOLOGY

Fig. 3. Zebrafish mutant embryos which display abnormal shape of the tail bud (B) and a segmentation defect of somites (C).

ill. Genetical approaches for revealing molecularmechanism of trunk development in zebrafish
To identify genes involved in several aspects during
early embryogenesis of the vertebrate , we have
performed screening of zebrafish mutants induced by
ENU, a chemical mutagen. Until now , we havescreened 630 of F2 families and found a number ofmutants whose phenotypes are different from thosealready reported. For instance, some of these mutants
displayed defects in the somite and tailbud
development. Cloning of genes that are responsible forthese defects is in progress.
To complement a forward genetical approach, wehave also screened genes expressed in the tailbud andpresomitic mesoderm, in which somite progenitors
exist. Until now, we have identified 50 genes that are
expressed preferentially in these regions. To examine
developmental roles of these genes, functional analysisof these genes has been performed by injecting
morpholino anti-sense oligonucleotides.

Publication List:
Fujino T, Asaba H, Kang MJ, Ikeda Y, Sone H, Takada S,
Kim DH, Ioka RX, Ono M, Tomoyori H, Okubo M,
Murase T, Kamataki A, Yamamoto J, Magoori K,
Takahashi S, Miyamoto Y, Oishi H, Nose M, Okazaki
M , Usui S, Imaizumi K, Yanagisawa M , Sakai J,
Yamamoto TT . (2003) Low-density lipoprotein
receptor-related protein 5 (LRP5) is essential for normalcholesterol metabolism and glucose-induced insulin
secretion. Proc Natl Acad Sci US A. 100, 229-234
Muroyama Y. Kondoh H. Takada S. Wnt proteins promote
neuronal differentiation in neural stem cell culture.
Biochem. Biophys. Res. Commun. In press
Nakagawa S, Takada S, Takada R, Takeichi M. (2003)
Identification of the laminar-inducing factor: Wnt-signal
from the anterior rim induces correct laminar formationof the neural retina in vitro. Dev Bioi. 260, 414-425.
Oishi I, Suzuki H, Onishi N, Takada R, Kani S, Ohkawara
B, Koshida I, Suzuki K, Yamada G, Schwabe GC,
Mundlos S, Shibuya H, Takada S, Minami Y. (2003)
The receptor tyrosine kinase Ror2 is involved in noncanonical Wnt5a/JNK signalling pathway. Genes Cells.
8, 645-654

70

NATIONAL INSTITUTEDIVISION OF BIO-ENVIRONMENTAL SCIENCE

Professor:
Associate Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Students:

Visiting Scientist:
Technical Assistants:

Secretary:

IGUCHI, Taisen
WATANABE, Hajime
KATSU, Yoshinao
MIZUTANI, Takeshi
URUSHJTANI Hiroshi
SONE, Kiyoaki
MIYAGA WA, Shinichi
NAGATA, Emiko
KOBAYASHI, Mika
KATO, Hideo n
SUZUKI, Atsuko
KOBAYASHI, Kaoru
TAKAHASHI, Eri
ITAMOTO, Misaki
HINAGO, Megumi
ICHIKAWA , Rie
GOTO, Mayu
IMAIZUMI, Taeko

nfrom Nihon Bioresearch Inc.

Synthetic chemicals found in the environment havethe capacity to disrupt endocrine system development
and function in both wildlife and humans. This has
drawn public concern since many of these chemicals
may bind to estrogen receptors (ER) and evoke
estrogenic effects. Early evidence that estrogenic
chemicals could pose a threat to human health during
development came from studies of diethylstilbestrol ·(DES), which was used to prevent premature birth andspontaneous abortion . Laboratory experiments havedemonstrated that exposure of animals to sex hormones
during perinatal life can cause permanent andirreversible alterations of the endocrine andreproductive systems as well as the immune system,
nervous system, bone, muscle, and liver in both sexes.
Although many of these chemicals,may bind to ER andevoke estrogenic effects in wildlife and humans, theeffects of estrogen are not well understood even now.
Thus , understanding the effects of sex hormones at themolecular level, especially during development, is very
important to resolve these problems.

I. Estrogen-induced irreversible changes

Perinatal sex-hormone exposure has been found to
induce lesions in reproductive tracts in female mice.
The possible relevance of the mouse findings tothedevelopment of cancer in humans has beenemphasized. In the early seventies, a close correlation
between occurrence of vaginal clear celladenocarcinoma in young women and early intrauterine
exposure to DES was demonstrated. Many chemicals
released into the environment have the potential todisrupt endocrine function in wildlife and humans .
Some of these chemicals induce estrogenic activity bybinding to ER. The neonatal mouse model has beenutilized especially to demonstrate the long-term effectsof early sex hormone exposure on the female
reproductive tract. Neonatal treatment of female micewith estrogens induces various abnormalities of the
reproductive tract: ovary-independent cervicovaginal
keratinization, adenosis, uterine hypoplasia, epithelial
metaplasia, oviductal tumors, polyovular follicles (PF)and polyfollicular ovaries. Female reproductive tracts
in mice exposed prenatally to estrogen show alteredexpression of Hoxa genes and Wnt genes and theanalysis of knockout mice lacking Hoxa-10 or Wnt7ashow uterine hypoplasia. The growth response ofneonatally DES-exposed reproductive organs to
estrogen is reduced, as are ER levels and epidermal
growth factor (EGF) receptor levels, in addition to otherhormone receptor levels.

Normal Mouse
FIG .2 A hypothetical model for the estrogen-independent
ER activation pathway in mouse vagi na. EGF-likegrowth factors activate the protein-phosp horylationcascade via erbB receptors . In nuclear, estrogen receptor
is phosphorylated on serine 122 and 171 in AF-1 domain.
Furthermore , transcription factors are activated byphosphorylation . These phosphorylations induce thetranscriptional activity of ER, and then growth factors are
expressed via estrogen-response element (ERE). Growth
factors induced by ER activate EGF-receptors.

FIG. 1 Scheme of estrogen-dependent and -independent
vaginal epithelial cells in mice induced by neonatal
estrogenization.

Growth factors and ER signaling cooperate to playessential roles in cell proliferation, differentiation andtumor progression in mouse reproductive organs. The
mechanisms of the estrogen-dependent and independent pathways remain unknown. EGFR anderbB2 were activated in the vaginal epithelium of miceby estrogen treatment. This activation was also

71

FOR BASIC BIOLOGY

encountered in vaginae from neonatally DES-exposed
mice, along with the expression of EGF, TGF-a, HBEGF,
amphiregulin
andneuregulin .
Immunochitochemical analysis indicated that erbB2
was primarily expressed in vaginal epithelium. Serine
118 and 167 located in the AF-1 domllin of ERa were
phosphorylated in these vaginae. AG825, AG 1478 or
ICI 182,780, that are erbB2, EGFR and ER antagonists,
respectively, administration blocked proliferation ofvaginal epithelium induced by neonatal DES exposure.
Signal tran sd uction via EGFR and erbB2 could be
related to the estrogen-induced vaginal changes andpersistent erbBs phosphoryl atio n a nd sustained
expression of EGF-like growth factors, leading to ERa
activation that may result in cancerous lesions invaginae from neonatally DES- exposed mice later inlife.
To identify estrogen-responsive genes related to theproliferation and differentiation of mouse vaginal
epithelial cells, we used differential display andidentified a novel c-type lectin that encodes amembrane protein with a c-type lectin domain in the
carboxyl-terminal region. Characterization of mRNA
expression indicates that estrogen regulates the geneencodeing this novel c-type lectin in mouse vagina.
Furthermore, this c-type lectin is found in epithelial
cells, but not stromal cells, suggesting that it may be an
important factor in the stratification and/or cornificationof the vaginal epithelium of mice. We are continuing
efforts to analyze its function during proliferation anddifferentiation in mouse vagina by estrogen treatment.
Estrogenic compounds such as bisphenol A (BPA)
and nonylphenol as well as dioxins and PCB s were
found in the human umbilical cord. BPA can easily
cross the placenta and enter the fetus in Japanesemonkey and mice . BPA can be found in fetal brain ,testis and uterus when given to pregnant mice andmonkey s. Neonatal exposure to a high BPA dose
induced ovary-independent vaginal changes, polyovularfollciles and infertility lacking corpora lutera. Prenatal
exposure to a low BPA dose induced acceleration ofvaginal opening in the offspring. Thus, the developing
mammal is sensitive to exposure to estrogenic agents.
100000 : --- - - ---

- , 100000

-

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10000

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; 10000... . .:.
~
...
··: ~
,..· ....
~1000 ~
/~·
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FIG.3 Scatter plot of average expression levels in control
and chemical-treated uterus

II. MicroArray analysis
In order to clarify the molecular mechanisms of theseeffects, we are studying changes in gene expression
patterns induced by perinatal exposure to chemicals or
estrogen using differential display and DNA microarraytechiniques. We have found genes possibly related tothe ovary-independent changes by differential display.
We also have clustered groups of genes that are
responsive to estrogenic stimuli in uterus by using the
DNA microarray system. We need to understand themolecular background of the critical period during
development , the low dose effect of estrogenic
chemicals and the molecular metabolism of hormones
and hormone-like agents in animals including humans.

III. Effect of estrogen on reptile, amphibian andfishesDuring embryogenesis, exogenous estrogen exposure
induces abnormal sex differentiation and the abnormalbone formation in African clawed frog, Xenopus laevis,
the cyprinodont fish, mummichog (Fundulus
heteroclitus), mosquitofi sh (Gambusia affinis). To
analyze the function of estrogen, we have isolated
eDNA clones of ERa and ~ from F. heteroclitus, G.
affinis. In UK rivers exposure of roach (Rutilus rutilusa common cyprinid fish) to effluents from sewage
treatment works, containing complex mitures of
endocrine disrupting chemicals (EDCs) has been shown
to alter sexual development and impact negatively on
th eir reproductive capabilities. To unravel themechanisms of disruption of sex ual development inroach exposed to EDCs, we have isolated the cDNAs
related to sex determination and sexdifferentiation
containing estrogen receptor, aromatase, StAR, Sox9,
vasa etc. We are examining the gene expression during
gonadal differentiation with or without EDCs exposure.
Furthermore, we have isolated cDNAs of steroid
hormone receptors from American alligators (Alligator
mississippiensis). As the estrogen-responsive genes
must play important roles, we are isolating the estrogenresponsive genes to understand the molecularphysiology of estrogen action. Japanese tree frog (Hylajaponica) takes water through ventral skin. We foundthat sex steroids and endocrine disrupters interfere withwater absorption through ventral skin in frogs . Further,using the amphibian and fish as model animals we aim
to analyze the effects of numerous chemicals released
into the environment on endocrine system function in
wildlife.

NATIONAL INSTITUTE

72
1JJB ~5
Alligator!

AlB

I

87

Human!

60

I

74
31
31

C hicken

Xenopus

Zebrafish l
Roach

I

345

LBO

I c 245I D 345I
E
11oo 1 89 I"''"' 98
165 251351

179

1100 1 73I9:)539 587IF I

539 5119

1761

5<15

595

is<J I~r42~r88
l1ooi 65 jJ
218
319
513 569
195 I 42 I " 68 "' 1281
15'1 220319
512 559
69
195 I 42 I
1251
1

152

FIG. 4 Comparison of alligator and roach ERa proteins
with estrogen receptors of several species. The functional
A/B to F domains are schematically represented with thenumbers of amino acid residues indicated.

IV. Molecular Target Search
Abnormalities caused by endocrine disrupting
chemicals are reported but the molecular mechanismsof the effects are not well studied. Although estrogen
receptor is one of the strongest candidates possibly
responsible for the endocrine disrupting function ofmany chemicals, it alone cannot explain the variety ofphenomena induced by endocrine disrupting chemicals.
Thus, we are also looking for new target molecules that
may be responsible for endocrine disruption. In
parallel, we also are studying the ligand-binding
mechanisms of nuclear receptors to hormones and otherchemicals.

FIG. 5 Evolutionary relationships of estrogen receptor
sequences.

Publication List:
Adachi, T., Matsuno, Y., Sugimura, A., Takano, K., Koh,K.B., Sakurai, K. , Shibayama, T. , Iguchi, T. , Mori, C.
and Komiyama, M. (2003) ADAM? (a disintegrin andmetalloprotease 7) mRNA is suppressed in mou se
epididymis by neonatal exposure to Diethylstilbestrol.
Mol Reprod Dev. 64, 414-421.
Inui, M., Adachi, T. , Takenaka, S. , lnui, H., Nakazawa, M.,
Ueda, M., Watanabe , H. , Mori, C., Iguchi , T . and
Miyatake, K . (2003) Effect of UV screens andpreservatives on vitellogenin and choriogenin
production in male medaka (Oryz ias latipes).
Toxicology 194, 43-50.
Kato, H., Ota, T., Furuhashi, T, Ohta, Y. and Iguchi T .
(2003) Changes in reproductive organs of female rats
treated with bisphenol A during the neonatal period.
Reprod. Toxicol. 17, 283-288.

Katsu, Y., Lubahn, D.B . and Iguchi T. (2003) Expressionof a novel c-type lectin in the mouse vagina .
Endocrinology 144, 2597-2605 .
Kohno, S., Kamishima, Y. and Iguchi, T. (2003) Molecularcloning of an anuran V (2) type [Arg (8) ] vasotocin
receptor and mesotocin receptor: functional
characterization and tissue expression in the Japanesetree frog (Hyla japonica). Gen. Camp. Endocrinol. 132,
485-498.
Miyahara, M., Ishibashi , H., Inudo, M. , Nishijima, H. ,Iguchi, T ., Guillette , L.J. and Arizona , K. (2003)
Estrogenic activity of a diet to estrogen receptors -alphaand -beta in an experimental animal. J. Health Sci. 49,
481 -491.
Nakahata, S. , Kotani, T., Mita, K., Kawasaki, T ., Katsu,
Y ., Nagahama , Y . and Yam as hita, M . (2003)
Involvement of Xenopus Pumilia in the translational
regulation that is specific to cyclin B 1 mRNA duringoocyte maturation. Mech. Develop. 120, 865-880.
Okada, A. , Ohta, Y., Inoue, S., Hirai, H., Muramatsu, M.
and Iguchi , T . (2003) Expression of estrogen,progesterone and androgen receptors in the oviduct ofdeveloping, cycling and pre-implantation rats . J. Mol.
Endocrinol. 30, 301-315.
Sato, T., Fukazawa, Y., Kojima, H., Ohta, Y. and Iguchi,
T . (2003) Mu ltiple mechanisms are involved in
apoptotic cell death in the mouse uterus and vagina after
ovariectomy. Reprod. Toxicol. 17, 289-297.
Tatarazako, N., Oda, S., Watanabe, H., Morita, M. andIguchi , T. (2003) Juvenile hormone agonists affect theoccurrence of male Daphnia. Chemosphere 53, 827-33.
Tominaga, N., Ura, K., Kawakami, M., Kawaguchi , T.,
Kohra, S., Mitsui, Y. , Iguchi, T. and Arizona, K. (2003)
Caenorhabditis elegans responses to specific steroid
hormones. J. Health Sci. 49, 28-33.
Tominaga, N ., Kohra, S. , Iguchi , T . and Ari zo na , K.
(2003) A multi-generation sublethal assay of phenols
using the nematode Caenorhabditis elegans. J. Heath
Sci. 49, 459-463.
Urushitani, H., Nakai, M. , Inanaga, H., Shimohigashi, Y. ,Shimizu, A., Katsu, Y. and Iguchi , T. (2003) Cloning
and characterization of estrogen receptor alpha inmummichog , Fundulus hetetoclitus . Mol. Cell.
Endocrinol. 203, 41-50.
Watanabe H. , Suzuki , A. , Kobayashi , M., Lubahn D.B. ,Handa , H. and Iguchi , T. (2003) Similarities anddifferences in uterine gene expression patterns causedby treatment with physiological and non-physiologicalestrogens. J. Mol. Endocrinol. 31, 487-497.
Watanabe H., Suzuki, A., Kobayashi , M., Takahashi , E.,ltamoto, M. , Lubahn D.B. , Handa, H. and Iguchi, T .
(2003) Analysis of temporal changes in the expressionof estrogen-regulated genes in the uterus . J. Mol.
Endocrinol. 30, 347-358.
Adachi, T., Koh, K.B., Tainaka, H., Matsuno, Y. , Ono, Y.,
Sakurai, K. , Fukata, H., Iguchi, T., Kamiyama, M. andMori , C. (2004) Toxicogenomic difference between
diethylstilbestro l and !?beta-estradiol in mouse
testicular gene expression by neonatal exposure. Mol.
Reprod. Dev. 67, 19-25.
Katsu , Y. , Bermudez, D.S. , Braun , E ., Helbing , C.,
Miyagawa, S., Gunderson, M .P., Kohno, S., Bryan ,T.A., Guillette, L.J . and Iguchi, T. Molecular cloning of
the estrogen and progesterone receptors of the American
alligator. Gen. Comp. Endocrinol. (in press).
Miyagawa, S. , Katsu, Y., Watanabe, H. and Iguchi T.
Estrogen-independent activation of erbBs signaling andestrogen receptor alpha in the mouse vagina exposedneonatally to diethylstilbestrol. Oncogene (in press)

73

FOR BASIC BIOLOGYDIVISION OF PLANT DEVELOPMENTAL
GENETICS
Associate Professor:
Research Associate:
Technical Staffs:

Postdoctoral Fellows:
Graduate Students:

Secretary:

TSUKAYA, Hirokazu, Ph.D.
HORIGUCHI, Gorou, Ph.D.
KONDO, Makiko
TAKABE, Erika
YAMAGUCHI,Chinami
SAKAI, Keiko
CHO, Kiuhyon, Ph.D.
ISHIKAWA , Naoko, Ph.D.
KOZUKA, Toshiaki
NARJTA, Noriyuki
MANO, Erika
KABEYA, Kazuko

Diversity of plant form is mostly attributable tovariation of leaf and floral organs, which are modified
leaves. The leaf is the fundamental unit of the shootsystem, which is composed with leaf and stem. So theleaf is the key organ for a full understanding of plantmorphogenesis. However, the genetic control ofdevelopment of these shapes had remained unclear.
Recently, studies of leaf morphogenesis has been in aturning point, after our successful application of thetechniques of developmental and molecular genetics toit, using model plants, Arabidopsis thaliana (L.) Heynh.
(reviewed in Tsukaya 2003).
In a determinate organ, a leaf, number of leaf cells is
not necessarily reflected on leaf shape or, in particular,
leaf size. Genetic analyses of leaf development inArabidopsis shows that a compensatory system(s) act inleaf morphogenesis and an increase of cell volume
might be triggered by a decrease in cell number. Thus,
leaf size is, at least to some extent, uncoupled from thesize and number of cells by the compensatory
system(s). Based on these facts, a new perspective on
understanding of mechanisms for leaf morphogenesis,
Neo-Cell theory, is proposed (Tsukaya, 2003). Neo-Celltheory is the Cell theory which stipulated positional
cooperation of the behavior of cells. In short, Neo-Celltheory hypothesizes as follows: cells are the unit ofmorphogenesis; however, each cell is also controlled byfactors that govern the morphogenesis of the organ ofwhich the cells are a part.
Focusing on mechanisms that govern polarized
growth of leaves in a model plant, Arabidopsis thaliana ,we found that the two genes act independently to eachother on the processes of polar growth of leaves: the
ANGUSTJFOLIA (AN) gene regulates width of leaves
and the ROTUNDJFOLIA3 (ROT3) gene regulates
length of leaves. The AN gene controls the width of leaf
blades and the ROT3 gene controls length. Cloning of
the AN gene (Kim et al. , 2002) revealed that the gene isa member of CtBP (C-terminal binding protein) genefamily which had been found from animal kingdom. In
the animal kingdom, CtBPs self-associate and act as aco-repressor of transcription. We found that also the
AN protein can self-associate in yeast two-hybrid
system (Kim et al. 2002). Furthermore, microarray
analysis suggested that the AN gene might regulate theexpression of certain genes, for example , the gene
involved in formation of cell walls , MER/5. We also
found that the abnormal arrangement of cortical MTs inan-1 mutant leaf cells appeared to account entirely forthe abnormal shape of the cells. It suggested that the ANgene might regulate the polarity of cell growth bycontrolling the arrangement of cortical MTs (below).

Arrangement of cortical MTs in leaf epidermis of wi ld
type (left) and the an-1 mutant (right).

The ROT3 gene encodes a cytochrome P450.
Transgenic experiments proved that the ROT3 gene
regulates leaf-length without affect on leaf-width via
biosynthesis of steroids. In relation to it, we recently
revealed that a steroid hormone , brassinosteroid,
controls both proliferation and expansion of leaf cells(Nakaya et al. , 2002). To understand the role of the
ROT3/CYP90Cl enzyme in biosynthesis ofbrassinosteroid , biochemical analysis of the rot3
mutants has been carried out. As a result, we found that
CYP90Cl and its closely related homolog, CYP90Dl,
are involved in the critical steps of biosynthesis ofbrassinolide (Kim et al., submitted).
On the other hand, by studying role of light conditionin leaf development, we found that the length of leaf
petioles was related exclusively to genetic control of thelength of individual cells in all leaf morphologicalmutants examined. In contrast, both the size and thenumber of cells were affected by the mutations
examined in leaf blades (Tsukaya et al. , 2002).
Leaf expansion is controlled not only by polar cell
expansion but also by polar cell proliferation. We haverevealed that ANGUSTJFOLIA3 (AN3) gene is involvedin maintenance/establishment of activity of cel l
proliferation in leaf primordia. AN3 encodes a coactivator, and is speculated to control cell cycling inleaf primordia (Horiguchi et al., in prep.). Interestingly,
the an3 shows clear "compensation ", namely ,accelerated cell expansion in relation to decrease of
number of leaf cells. Using various mutants with alterednumber and/or size of leaf cells, we are currently
analyzing genetic system of the compensation.
We also identified a novel gene that regulates
number of leaf cells: ROT4. ROT4 is a member ofnovel peptide family which is specific to seed plants.
Overexpression of the ROT4 results in stunted leaves
with normal width (Narita et al., submitted), suggesting
importance of peptides in control of leaf shape.
Apart from the aspects of leaf expansion, we also
analyzed genes for identification of leaf primordia. The
ASJ, AS2 and BOP genes are needed for determinate
growth of leaf. We isolated and analyzed a novel
mutant, bop (blade-on-petiole) mutant, which strongly

74
enhances the as2 phenotype , in coll aboration with areaseach teams of Prof. Nam, POSTECH , Korea andProf. Machida, Nagoya Univ. (Ha et al. 2003). Threeclass I knox genes, namely, KNATJ , KNAT2 and
KNAT6, were misexpressed in the leaves of the bopl-1
mutant as in those of the as2 mutant. The bopl single
mutant results in ectopic, lobed blades along the adaxial
side of petioles of the cotyledon and foliage leaves. The
bop mutation strongly enhances the morphologicalabnormality of leaves when combined with the as] or
as2 mutation , resulting in repeated compound leaves.
We, thus, suggest that BOP1 promotes or maintains adevelopmentally determinate state of leaf cells throughregulation of class I knox] genes (Ha et al. 2003).
On the other hand, we are trying to identify molecularmechanisms which distinguish developmental pathwayof leaves from that of shoots by studying tropical plants
with 'fuzzy ' morphology. For s uch purposes , weintroduced tropical plants having queer developmental
program for leaf morphogenesis, namely, Chisocheton,
Guarea and Mon.ophyllaea, as materials for the studies.
The indeterminate compound leaves of members of thegenus Chisocheton in Southeast As ia and of the gen us
Guarea in the New World and Africa, in the fami ly
Meliaceae, are unique and can develop indeterminately
as a result of the activity of the leaf apical meristem,
which can function very similarly to a shoot apicalmer istem. We performed a molecular phylogenetic
study of these genera with a research team of Dr.
Yokoyama, Tohoku Univ ., and the result suggested thatindeterminate program in the leaves of members ofthese two genera might have evo lved only once inMeliaceae (Fukuda et al. 2003). Cloning of apicalmeristem-specific genes from these species are now
underway. Similarly, we are trying to clone key gene(s)which may support the indeterminate leaf growth inMonophyllaea spp. (Gesneriaceae).
On the other hand, leaf shape variation is sometimes
linked to variation of ploidy level. In Cayratiajaponica,
Vitaceae, trifoliate leaves are rarely observed in specificstrains . Detailed analysis of morphological features
including fertility of pollen grains and chromosome
numbers in collaboration with Prof. okada, Osaka City
Univ., revealed that there are two types of Cayratia
japonica in Japan: ferti le diploid type that sometimes
develops trifoliates and steri le triploid type thatdevelops only quiquefoliates (Okada eta!., 2003). In thecourse of this study, we have found several curious
features in diploid type of Cayratia japonica, e.g., lowfertility. To understand why, analyses of closely related
species are underway (Okada and Tsukaya, 2003).
In addi tion, we are interested in env ironmenta ladaptation of leaves. Adaptive responses of leaves
against light, gravity and other environmental factors
are also analyzed in a model plant, Arabidopsis thaliana
and anatomical a n a lyses of various types ofmorphological adaptation of leaves against certain kindsof environments were also performed. To obtain clues
for understanding of natural variation in plant form,
molecular systematic analyses of wild plants are also

NATIONAL INSTITUTE

carri ed o ut (Yokoyama et a l. , 2003; Tsukaya et al.,2003). So called "Evo/Devo" study of leaf
morphogenesis is also one of our research project.

Publication List:
Fukuda, T. , Yokoyama, J. and Tsukaya, H. (2003) The
evo luti onary origin of indeterminate leaves in
Meliaceae: phylogenetic relationships among species in
the genera Chisocheton and Guw·ea, as inferred fromsequences of ch loroplast DNA. Int. J. Plant Sci. 164:
13-24.
Ha, C.-H., Kim, G.-T., Kim, B.-C. , Jun, 1.-H., Soh, M.-S .,Ueno, Y., Machida, Y., Tsukaya, H. arid Nam, H.-G .
(2003) The BLADE-ON-PETIOLE gene controls leaf
pattern formation through regu lation of meri stematic
activi ty. Development 130: 161-172.
Machida , C. , Iwakawa , H. , Ueno , Y., Semiarti, E.,
Tsukaya, H., Hasebe, H. , Kojima, S. , and Machida, Y.
(2003) Formation of a symmetric flat leaf lamin a in
Arabidopsis. In: Sekimura, T. , Noji, S., Ueno, N. andmaini, P.K. eds., Morphogenesis and Pattern Formationin Biological Systems: Experiments and Models .
Springer-Verlag Tokyo, Tokyo.
Okada, H. , Tsukaya, H. and Okamoto, M. (2003) Intraspecific polyploidy and possiblity of occurrence of some
genetic types for pollen development in Cayrat ia
japonica, Vitaceae. Acta Phytobotanica et Geobotanica
54: 69-75.
Okada, H. and Tsukaya, H. (2003) Chromosome
observations of Cayratia species, Vitaceae . Acta
Phytobot. Geobot. 54: 177-179.
Sulpice, R., Tsukaya, H., Nonaka, H. , Mustardy, L., Chen,
T ., and Murata , N. (2003) E nhanced form ation of
flowers in sa lt-stressed Arabidopsis after genetic
engineering of the acc umulation of glycine betaine.
Plant]. 36: 165-176.
Tsukaya, H. (2003) A simple method for collecting DNA
samples in the field. Newslet. Him. Bot. 32: 15-17.
Tsukaya, H. (2003) Organ shape and size: a lesson fromstudies of leaf morphogenesis. Curr. Opin. Plant Bioi. 6:
57-62.
Tsukaya, H., Fukuda, T., and Yokoyama, J. (2003)
Hybridization and introgression between Callicarpa
japonica and C. mollis (Verbenaceae) in central Japan,
as inferred from nuclear and chloropl ast DNA
sequences. Mol. Ecol. 12: 3003-30 11.
Yokoyama, J ., Fukuda, T. and Tsukaya, H. (2003)
Morphological and molecular variation of Mitchella
undulata Siebold et Zucc., with special reference tosystematic treatment of the dwarf form from YakushimaIsland. J. Plant Res. 116: 309-315.

Leaf shape control by various genes in Arabidopsis thaliana.

75

FOR BASIC BIOLOGYDIVISION OF BIOINFORMATICS

Professor (Adjunct):

NAKAMURA, Haruki(Osaka University)

The aim of this laboratory is structural bioinformatics
covering molecular modeling and design of proteins
and other biological macromolecules: Development of a
new database, eF-site, for protein surface geometry withthe physicochemical properties, and identification ofprotein functions using the database, and Developmentof new algorithms and softwares for large scale
simulation calculations by parallel computers to
examine free energy landscapes of biomolecularsystems. A new hybrid method of Quantum Mechanics
and Molecular Mechanics has also been developed foranalysis of the electronic state in biologicalmacromolecular systems.

Publication list:
Kinoshita, K., Nakamura, H. (2003) Protein informatics
towards function identification. Curr. Opin. Struct. Bioi.
13, 396-400.
Kinoshita, K., Nakamura, H. (2003) Identification ofprotein biochemical functions by similarity search using
the molecular surface database, eF-site. Protein Science,
12, 1589-1595.
Kim, J. G., Fukunishi, Y., Kidera, A., Nakamura, H.
(2003) Determination of multicanonical weight based onstochastic model of sampling dynamics. Phys. Rev. E
68,21110.
Fukunishi, Y., Mikami, Y., Nakamura, H. (2003) The
filling potential method: a method for estimating thefree energy surface for protein-ligand docking. J. Phys.
Chern. B, 107, 13201-13210.
Fukuda, I., Nakamura, H. (2003) Deterministic generationof the Boltzmann-Gibbs distribution and the free energy
calculation form the Tsallis distribution. Chern. Phys.
Lett. 382, 367-373.

76
CENTER FOR RADIOISOTOPE FACILITIES (CRF)

Head: Takada, Shinji(Professor, concurrent post)
Associate Professor:
Technical Assistant:

Supporting Staff

OGAWA, Kazuo(Radiation Protection Supervisor)
MATSUDA, Yoshimi(Radiation Protection Supervisor)
SAWADA, Kaoru(from Apri/12)
1/NUMA, Hideko(from October 16)
ITO, Takayo
IJDA, Yumi
KATAGJRI, Izumi

I. Research supporting activity
Technical and supporting staffs of the CRF maintainfive controlled areas being adaptable for the law . The
purchase of radioisotopes from JRA (Japan
Radioisotope Association) and the transfer ofradioisotope wastes to JRA is under our surveillance.
In this year, we discontinued two controlled areas;
NIBB (National Institute for Basic Biology)-branch and
NIPS (National Institute for Physiological Science)branch. The name of Center located in Common
Building I of the area A has been changed to Common
Building I (COBI)-branch . Matsuda, Iinuma, Ito , andIida are maintaining COBI-branch and LGER
(Laboratory of Gene Expression and Regulation)branch in the area A, and Ogawa, Sawada, and Katagiri
working in the area E.
Users going in and out the controlled areas counted
by the monitoring system are 6,965 persons in 2003 .
The items in each area is shown in Figure 1.

AreaA(NIPS)

AreaA(NI BB)

AreaA(LGER)

Figure 1. Percentage of users going in and out thecontrolled areas in 2003.

II. Academic activity
Dyneins are a group of microtubule-activatedATPases that serve to convert chemical energy intomechanical energy and divided into axonemal andcytoplasmic dyneins. Figure 2 shows the localization of
two isoforms of dynein in the outer arms of sperm
axonemes and the mitotic apparatus of cleaving egg
visualized by anti-axonemal dynein (Fragment A)
antibodies (Fig. 2).

e.

Figure 2. Dynein fami ly. Axoneme (left) and Cleaving
egg (right).
Dyneins are very large and range in molecular mass
up to 1 to 2 mega Da. They are complex proteins
containing heavy, intermediate, and light chains definedby the molecular mass. The project is the molecularcloning of polypeptides contained in outer arm dyneinof sea urchin sperm flagella to understand themechanism how dynein interacts with microtubules ,resulting in producing the force.
Outer arm dynein consists of two heavy chains with
ATPase activity . The motor activity is closely related tothis polypeptide. The first successful molecular cloningof this huge polypeptide (520 kDa) was performed in1991. Since then eDNA clones for axonema l andcytoplasmic dyneins have been isolated in a variety oforganisms. The sequences of heavy chains , without
exception, contain four P-loop motives referred to as
ATP-binding sites in the midregion of the molecules .
Outer arm dynein contains three intermediate chains(IC1 , IC2, and IC3) that range in molecular mass from70 to 120 kDa. IC2 and IC3 belong to the WD-family .
By contrast, IC1 is a hybrid protein such that the Nterminal part is homologous to the sequence ofthioredoxin and the middle part consists of three
repetitive sequences homologous to the sequence of
NDP kinase . The IC1-related proteins are found in
distantly related species.
Six light chains with molecular masses of 23.2, 20.8 ,
12.3, 11.5, 10.4, and 9.3 kDa are associating with outerarm dynein. We have already isolated eDNA clones offive LCs. LC1 (23.2 kDa) and LC3 (12.3 kDa) arehighly homologous to mouse Tctex2 and Tctex 1,
respectively , which are encoded by the t complex
region that is involved in transmission ratio distortion(TRD) , male sterility and the development of germ
cells. In this year, we found that LCl and LC3 are able
to making sperm motility activating complex (Smoac).
Our finding raises the possibility that axonemal dynein
proteins are involved in this phenomenon. TRD may becaused by the dysfunction of multiple axonemal dynein
proteins.

Publication
K. Ogawa and K. Inaba. Biochem. Biophys. Res.
Commun . 310 (2003) 1155-1159. Smoac : Spermmotility activ ating complex formed by t-comp lex
distortors

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