CONTENTSINTRODUCTION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·ORGANIZATION OF THE INSTITUTE · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·2

MEMBERS OF THE COUNCIL · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·4MENBERS OF THE ADVISORY COMMITTEE FOR PROGRAMMING AND MANAGEMENTGRADUATE PROGRAMS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·5OFFICE OF DIRECTOR · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·6

DEPARTMENT OF CELL BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·9

DIVISION OF CELL MECHANISMS · · · · • · · · • · · · · · · · · · • · · · · · · · · · · · · · · • · · · · •• · · · • · • • · · • · · • · · · · • · · · · • · • · · · · • · • · •• •• · · • • · · · · · ·10

DIVISION OF BIOENERGETICS · · · · · • · · • · · · · · · · · · · · · · • · · · · · • • • • · · · · · • · · · · · • · · · • · · · · · • · · • · · • • • • • • • • · · • · · · · • · • · · · • · · · · · · · • · •13

DEPARTMENT OF DEVELOPMENTAL BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

DIVISION OF REPRODUCTIVE BIOLOGY .. · · · · · · .. · · · · · · · · .. · · · · · · .. · · · · .. · · .. · · · · · · • · · · · · .. · .... · .... · .... · .... · .. · .. · •DIVISION OF CELL DIFFERENTIATION...................................................................................

16
19

DIVISION OF MORPHOGENESIS ..........................................................................................

21

DIVISION OF DEVELOPMENTAL BIOLOGY (ADJUNCT)··································································

24

DEPARTMENT OF REGULATION BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·27

DIVISION OF MOLECULAR NEUROBIOLOGY · · • · · · · · · • · · · · · · · · · · · · · · · · • · · • • • • • • · • • · • • · • · · • · • · · • · · • · • · • · · • · · • • · • • • • • · • · · •28

DIVISION OF CELLULAR REGULATION · · · · • · · · • • · · · · · · · · · · · · · · • • • • • • • • • • · • • • • · • · · • · · · · · · · · · · · · · • · • • • · · • • • • • • • • • • · · · • · • · ·31

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

34
36

LABORATORY OF GENE EXPRESSION AND REGULATION · · .. · · .......... · .. · · · · .. · · · · · · · · · · · · · · .. ·......

39

DIVISION OF GENE EXPRESSION AND REGULATION I···································································

40

DIVISION OF GENE EXPRESSION AND REGULATION II··································································

43

DIVISION OF SPECIATION MECHANISMS I · .. · · · .... • · · · · .. · · · · · · · .. · · · · · · · · · · · .. · .. · .. · .. · .... • .. · .... · .. · · · · · · · · · .... • ·45

DIVISION OF SPECIATION MECHANISMS II • • • • · • · · · · · · · · · · • · · · · · · · • · · · · · • · · · • · • • • • • · • • · · • · · • · • • · · · · · · • · · · · · • · · • · • · · · · · · ·47

RESEARCH SUPPORT····································· ......................... ·····································

51

CENTER FOR TRANSGENIC ANIMALS AND PLANTS • • • • • • • • • • • • • • • • • • • • · • • • • • • • • • • • · • • • • • • • • • • • • · • • • • • • •RESEARCH CENTER FOR INTEGRATIVE AND COMPUTATIONAL BIOLOGY • • • • • • • • • • • • • • • • • • • • • • • • • · • · • •RESEARCH SUPPORT FACILITY •••••· ·•·••·••••··•·••·••·· ··•·••••· ··••·••••·••·•••••••· ·••·••·•·••·•••••THE CENTER FOR ANALYTICAL INSTRUMENTS (managed byNIBB) • · · ·· · · · ·· · · · · •· • · • • · · • • • • · • •• · · • •· · · ·TECHNOLOGY DEPARTMENT·············································································

52
53
58

CENTER FOR INTEGRATIVE BIOSCIENCE (jointly managed by NIBB) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·63

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·····················································

61

62

64
66
68
70

DIVISION OF BIOINFORMATICS • • • · · · • · • • · • · · • · · • • · · · · · · · • · • · · · · • · • • • • · • • · • · · • · • • • • • · · • · · · · · · · · · · · · • · • •72

CENTER FOR RACIOISOTOPE FACILITIES (CRF) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·73

The cover photograph is the central network of the auditory system
visualized in a transgenic mouse line. See also p.28

INTRODUCTION
The National Institute for Basic Biology (NIBB) is agovernment-supported research institute established in
1977. In 1988, Department of Molecular Biofunction in
School of Life Science, the Graduate University for
Advanced Studies, was established in the NIBB . The
aim of the NIBB is the promotion and stimulation ofstudies in the field of Biology. As a Center of
Excellence (COE) , NIBB promotes not only the basic
biology but also the modern biological sciences byconducting first-rate research on site as well as incooperation with other universities and researchorganizations. Researchers at NIBB investigate cellstructure and function, reproduction and development,
neuronal and environmental biology, gene expression
and regulation, molecular evolution of eukaryoticorganisms and integrative and computational biology toelucidate the general and fundamental mechanisms
underlying various biological phenomena.
In 2002, there were many doctors who wereappointed as scientific staffs in NIBB. On April 1st,
Prof. TAKADA, Shinji , who had been a Professor of
Kyoto University as well as adjunct-professor of the
NIBB , was appointed as the Professor of the Center for
Integrative Bioscience (CIB) and made a joint
appointment for the NIBB. He was also joint-appointed
as a Professor of the Graduate University of Advanced
Studies and ofKyoto University.
Prof. MIYATA, Takashi was appointed as a Professorof the Department of Integrative and Computational
Biology in the NIBB from Kyoto University on
September 1st.
Dr. MOCHIZUKI, Atsushi who was a Research
Associate of Kyushu University was appointed as an
Associate Professor of the Department of Integrativeand Computational Biology in the NIBB on September
1st.
Prof. NAKAMURA, Haruki of Osaka University
was also appointed as a Adjunct Professor of CIB on
August 1st in place of the former Prof. KANEHISA,
Minoru.
Drs. MIZUSHIMA, Noboru, YAMAMOTO, Hiroshi ,HORIGUCHI, Gorou (CIB) , YAMADA , Kenji ,HIYAMA, Takeshi and OGAWA, Hidesato wereappointed as Research Associates of the NIBB in 2002.
Drs. JOHZUKA , Masaki returned as a Research
Associate of NIBB from the University of California inplace of Dr. KODAMA, Ken-ichi, a Research AssociateofNIBB .
In congratulation, Dr. YOSHIMORI , Tamotsu , waspromoted to a Professor of the National Institute of
Genetics in March 1st, 2002. Dr. ITO , Kei, waspromoted as an Associate Professor of the University ofTokyo in April 1st, 2002 and stayed in the NIBB as aadjunct-Associate Professor of the NIBB . Dr. HIDAKA ,Masurni was promoted as an equivalent position of the
Associate Professor of the BioMolecular Engineering
Research Institute in Osaka.
Two adjunct Professors Dr. KAMIYA, Ritsu and
MABUCHI, Issei and adjunct Associate Professors
INABA, Kazuo and ABE, Hiroshi left NIBB in April
31st, 2002 just after the completion of their 5-year

appointments. Three Research Associates left theirpositions in NIBB to become the equivalent positions inother Universities and Institutes.
The total number of personnels working at NIBB
including graduate students and postdoctoral fellows
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 hosts International
Conferences. The 47'h NIBB International Conference
entitled "Protein Phosphatases in Cellular Signaling
Systems" was held in March 13-15 (Prof. M. Noda,
organizer) and the 48'h NIBB International Conference
entitled "Molecular Mechanisms of Sex Differentiation" (Prof. K. Morohashi, organizer) in October 18-20.
Those two Conferences were sponsored by the Ministryof Education, Culture, Sports, Science and Technology.
In addition, NIBB continues to sponsor interdisciplinary
symposia and study meetings on current topics byinviting leading scientists from around the world to theInstitute. NIBB also provides a training course inbiological sciences for young investigators. To assess
our continuing improvement, the activities and future
plan of the professor who have spent 10 years at NIBB
was subjected to peer review by international scholars
in related fields. Prof. NODA , Masaharu was reviewed
on the 10-year Evaluation by the committee organizedby the Director-General Dr. KATSUKI. The result was
accepted by the Advisory Council of the NIBB held inJune. We always welcome any suggestions concerning
the research activities of NIBB .
KATSUKI, Motoya, D.Sc.
Director-General

2

NATIONAL INSTITUTE

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 for Physiological 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.

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 and culture, advises the Director-General on principles andpolicies governing the activities and operations of
NIBB. The Advisory Committee, comprised of professors 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, the Institute's
annual budget and future prospects.

Administration
Administration of the Institute is undertaken by the
Administration Bureau of the Okazaki National Research Institutes under the direct auspices of the Ministry of Education, Culture, Sports, Science andTechnology.

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

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, theMachine Shop, the Laboratory Glassware Facilities andthe Low-Temperature Facilities. The Radioisotope
Facilities, the Computer Center and the Animal Care
Facilities became common facilities of ONRI.

CampusThe Okazaki National Research Institutes covers an
area of 150,000m2 with four principal buildings. The
NIBB's main research building has a floor space of
10,930m2 • Two-thirds of the space was completed in1982 and the remaining third in June, 1983. The
buildings which 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.

3

FOR BASIC BIOLOGY

Okazaki National Research Institutes

H

National Institute for
Basic Biology

I

I Department of Cell Biology

1'--

l

Department of Developmental
Biology

Department of Regulation
Biologyri_Division of Cell Mechanisms
HDivision of Bioenergetics

I
I~ HDivision of Cell Proliferation (adjunct)I

HDivision of Cell Fusion (adjunct)I

LjDivision of Cellular Communication (adjunct)I

rlDivision of Reproductive Biology

I

HDivision of Cell Differentiation

I

HDivision of Morphogenesis

I

YDivision of Developmental Biology (adjunct)I

rlDivision of Molecular Neurobiology

Ir

HDivision of Cellular Regulation

I-lDivision of Biological Regulation (adjunct)I-iDivision of Behavior and Neurobiology (adjunct) I

HExpression
Laboratory of Geneand Regulation

Division of Gene Expression and Regulation I

I-jDivision of Gene Expression and Regulation II

I-iDivision of Speciation Mechanisms I

I~Division of Speciation Mechanisms II

IrCenter for Transgenic Animals and PlantsI

1~esearch Center for Integrative andComputational Biology

I ~Research Support Facility

Research Support~Institute forl-i

Molecular Science

I

I

LjTechnology Department

I

I---iCenter for Integrative Bioscience

I~Center for Radioisotope Facilities IlCenter for Experimental
Animal~Administration Bureau I ~~esearch Center for

Computational Science

I

HCenter for Analytical Instruments

National Institute forPhysiological Sciences

I
Research Facilities

I4

NATIONAL INSTITUTEMEMBERS OF THE COUNCIL
Director-General, National Museum of Ethnology
Professor, The University of Air
President, Kumamoto University(present) Chairman, Board of Directors, Shokei Educational Institution
Director-General, Center for National University of Finance
OHSAKI, Hitoshi
Vice-President, International Institute for Advanced Studies
OKADA, Masukichi
Professor, lwate College of Nursing
OGAWA, Tomoko
President, Osaka University
KISHIMOTO, Tadamitsu
Director, Biomolecular Engineering Research Institute
SHIMURA, Yoshirou(present) Director, JSPS Liaison Office, Stockholm
President, Akita Prefectural University
SUZUKI, Akinori
Professor, Kyoto University
TAKEICHI, Masatoshi
TAKEUCHI, lkuo
President, Novartis Foundation (Japan) for the Promotion of
Science
Deputy Director-General, IT Biohistory Research Hall
NAKAMURA, Keiko
HIDAKA, Toshitaka
Director-General, Research Institute for Humanity and Nature
Professor, Keio University
HOSHI, Motonori
HOTTA, Yoshiki
Director-General, National Institute of Genetics
YAMASHITA, Koujun
Professor, Nagoya University
YOSHIKAWA, Hiroshi
Advisor, IT Biohistory Research Hall
Director, Banyu Tsukuba Research Institute
YOSHIDA, Mitsuaki
President, Otemae University
YONEYAMA, Toshinao
Director-General, National Institute of Polar Research
WATANABE, Okitsugu
ISHIGE, Naomichi
IWATSUKI, Kunio
EGUCHI, Goro

MEMBERS OF THE ADVISORY COMMITTEE FOR
PROGRAMMING AND MANAGEMENTAIZAWA, Shinichi

Professor, Kumamoto University(present) Group Director, Center for Developmental Biology, RIKEN
Professor, Kyoto University
OKADA, Kiyotaka
KUROSAWA, Yoshikazu
Professor, Fujita Health University
GO,Michiko
Professor, Nagoya University
KOMEDA, Yoshifumi
Professor, Hokkaido University(present) Professor, University ofTokyo
KONDO, Hisato
Professor, Osaka University
SASAZUKI, TakehikoDirector-General, International Medical Center of Japan ResearchInstitute
HASUNUMA, Kohji
Professor, Yokohama City University
MACHIDA, Yasunori
Professor, Nagoya University
YAMAMOTO, Masayuki
Professor, University of Tokyo
IGUCHI, Taisen
Professor, Okazaki National Research Institutes, Center for
Integrative Bioscience.
IIDA, Shigeru
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
UENO,Naoto
Professor, Okazaki National Institute for Basic Biology
OHSUMI, Yoshinori
NAGAHAMA, Yoshitaka
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
NISHIMURA, Mikio
NODA, Masaharu
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
HORIUCHI, Takashi
Professor, Okazaki National Institute for Basic Biology
MURATA, Norio
Professor, Okazaki National Institute for Basic Biology
MOROHASHI, Ken-ichiro
Professor, Okazaki National Institute for Basic Biology
YAMAMORI, Tetsuo

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 Professors :Research Associates:

KAI'SUKJ, 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 of morphogenesis.
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 areaof the pupal wing . The marginal dying area is called thedegeneration region and the internal area, which develop
into the adult wing, is called the differentiation 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
two characteristics common with the apoptotic cell deathin mammalian cells. These are i) the presence of apoptotic
bodies , which are heavily condensed cells or theirfragments engulfed by other cells or macrophages, shownby transmission electron microscopy and ii) the presenceof conspicuous accumulation of fragmented DNA
evidenced by the TUNEL histological staining. The cells
in the degeneration region are actively engulfed 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 in the
differentiation region .
A collaborative work with the laboratory of Dr. K.
Watanabe (Hiroshima University) concerns mostly on thedevelopment of trachea and tracheole pattern in theswallow tail butterflies. Trachea and trcheoles are both
important in delivering air into the wing and their patterncoincide with that of the boundary of degeneration anddifferentiation zones at the distal end of the wing .
According to the observations, the pattern formation ofwing epithelium is often dependent on tracheal andtracheole patterns. Basic research on the development oftracheal pattern formation is being done by the scanningelectron microscopy and the bright field light microscopyof the fixed or fresh specimens to describe the exact
pathway and the time course of the formation of elaborate
pattern of trachea and tracheoles and to establish thecytological and developmental relationship between theformation of tracheal pattern and epithelial cell pattern,such as scale cell pattern.
The figure depicts how the tracheoles protrude from theprimary trachea at the pre-pupa stage (Fig. 1). The
tracheoles are arranged with even spaces within the wing

Fig .I. The tracheole cells (on the right) depart fromthe primary trachea (thick tube on the left) leavi ng
behind tracheole, which was stored within the cytoplasmof the tracheole cel l.

lumen and may closely related with the scale cell patternformation.
In c:ollaboration with the Division of Molecular
Neurobiology , the localization of a sodium channel
protein was described using immunohistological staining
at the level of electron microscopy.

Publication List:
Watanabe , E. , T. Y. Hiyama, R. Kodama and M. Noda
(2002) Na,. sodium channel is expressed in nonmyelinating Schwann cells and alveolar type II cells inmice . Neu.rosc. Letters 330: 109-113

Control of the distribution of palmitoylated
proteins in neuronal growth cones
UENO, Kohji
Signalling proteins such as G proteins and G proteincoupled receptors are modified with palmitate via
thioester linkages . Protein palmitoylation is thought to beimportant 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, are mainly
concentrated in the growth cones. Addition of an inhibitorof protein palmitoylase to the medium of cultured primaryneuronal mouse cells reduces the axonal growth ofneurons . From these findings , we speculated that the
localization of the palmitoylated proteins in growth 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 this analysis,
we have established a method to chemically modify asynthetic GAP-43 peptide with palmitate or other fatty
acids via a thioester linkage. We have analyzed theinteraction of palmitoylated proteins with ERM(Ezrin/Radixin/Moesin) proteins. ERM proteins arethought to be general cross-linkers between plasma
membranes and actin filaments . ERM proteins wereFOR BASIC BIOLOGY

analyzed on the basis of evidence that radixin and moesin
are concentrated in growth cones. Furthermore, ERM
proteins contain a subdomain that possesses a similar
structure to the acyl-CoA binding protein, which has anaffinity to palmitoyl-CoA. Thus, I guessed that ERM
proteins may interact with the palmitoylated proteins.
We found that ERM proteins possessed an ability to bindto the chemically-modified palmitoylated peptides.

7

8

I

NATIONAL.INSTITUTE9

FOR BASIC BIOLOGYDEPARTMENT OF CELL BIOLOGY

Chairperson: OHSUMI, Yoshinori

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 adjunct divisions. The department conducts studies on molecular dynamics of thecell in eukaryotes such as organelle differentiation, autophagy, cell
motility, cytokinesis and neural development.

10

NATIONAL INSTITUTEDIVISION OF CELL MECHANISMS
Professor:
Associate Professor:
Research Associates:

NISHIMURA, Mikio
HAYASHI, Makoto
MANO,Shoji
YAMADA, Kenji (November I~)
Technical Staff:
KONDO, Maki
NIBB Research Fellow:
SHIRAHAMA, Kanae
JSPS Postdoctoral Fellow: NJTO, Kazumasa (April I~)
Graduate Students:
WATANABE, Etsuko (~March 3I)
FUKAO, Youichiro
KAMADA, Tomoe
HATSUGAI, Noriyuki
Technical Assistants:
NAKAMORJ, Chihiro
YAGI,Mina
YOSHINORI, Yumi (August I~)
UEDA, Chizuru
Secretaries
KOMORI, Akiko (~March 31)
IYODA, Yuri (February 15~)Higher plant cells contain several distinct organelles
that play vital roles in cellular physiology. During proliferation and differentiation of the cells, the organelles
often undergo dynamic changes. The biogenesis of neworganelles may occur, existing organelles may undergo atransformation of function, while other organelles may
degenerate. Because the dynamic transformation oforganellar function (differentiation of organelles) is responsible for flexibility of differentiation events in higher
plant cells, the elucidation of regulatory mechanisms underlying organelle transformation are currently studied inthis division.

I. Reversible transformation of plant peroxisomes
Dramatic metabolic changes which underlie the shift
from heterotrophic to autotrophic growth occur in greening of seed germination. Accompanying these metabolicchanges, many constitutive organelles are functionallytransformed. Etioplasts differentiate into chloroplasts andmitochondria acquire the ability to oxidize glycine.
Glyoxysomes which are microbodies engaged in the degradation of reserve oil via 13-oxidation and the glyoxylate
cycle, are transformed into leaf peroxisomes that function
in several crucial steps of photorespiration. After thefunctional transition of glyoxysomes to leaf peroxisomesduring 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 andprotein degradation. We now engage in proteomic andtranscriptomic analyses of the reversible peroxisomaltransition in Arabidopsis cotyledons.

II. Intracellular transport of peroxisomal proteins
Enzymes localized in plant peroxisomes are synthesized in the cytosol, and function after their posttranslational transport into peroxisomes. Almost all of
the peroxisomal matrix proteins are known to contain oneof two targeting signals (PTSl and PTS2) within themolecules. PTSI is a unique tripeptide sequence foundin carboxyl terminus of the mature proteins. The permissible combinations of amino acids for PTSl in plant
cells are [C/NS/P]-[K/R]-[1/L/M]. In contrast, PTS2 isinvolved in a cleavable amino terminal presequence ofperoxisomal proteins that are synthesized as precursor
protein with larger molecular mass. PTS2 consists of aconsensus sequence [R]-[L/Q/1]-XS-[H]-[L]. PTSI and
PTS2 are initially recognized by binding with theircytosolic receptors, AtPex5p and AtPex?p, respectively.
Since AtPex5p could also bind with AtPex7p, they mightform a receptor-cargo complex.
Catalase is a peroxisomal protein that have been know
to contain no obvious PTSIIPTS2. We recently foundthat 480QKL of the enzyme function as an alternative
peroxisomal targeting signal. Yeast two-hybrid analysis
revealed that the signal also binds with AtPex5p. The
result indicated that catalase is also imported into
peroxisomes by PTS !-dependent manner.
Arabidopsis ped2 mutant is one of the mutants thatshow resistance to 2,4-dichlorophenoxybutyric acid.
PED2 gene, responsible for this deficiency, encodes aperoxisomal membrane protein, AtPex14p. Mutation in
the PED2 gene results in pleiotropic defects on fatty aciddegradation, photorespiration, and the morphology of all
kinds of peroxisomes by disrupting both PTS 1- and PTS2dependent peroxisomal protein import pathways. Binding
assay revealed that AtPex14p directly binds with AtPex5p,
but not with AtPex?p. AtPex14p controls both PTSl- andPTS2-dependent peroxisomal protein import pathways bybinding with the receptor-cargo complex involving
AtPex5p. These data suggest that the AtPex14p has acommon role in maintaining physiological functions of all
kinds of plant peroxisomes by determining peroxisomal
protein targeting.
ill. Dynamic morphology and actin dependent

movement of plant peroxisomes
To characterize peroxisomes in vivo, we visualized
them in cells of various tissues of Arabidopsis by green
fluorescent protein (GFP) through the addition of theperoxisomal targeting signal 1 (PTSI) or PTS2. The observation using confocal laser scanning microscopy revealed that the GFP fluorescence signals were detected asspherical spots in all cells of two kinds of transgenic
plants. Interestingly, however, abnormal large peroxisomes are present as clusters at the onset of germination,and these clusters disappear in a few days. Moreover,tubular peroxisomes which looked like the dumbbell, as if
two peroxisomes were connected with the tubule, werealso observed in the hypocotyl. Peroxisomes turned to be
motile organelles whose movement might be caused bycytoplasmic flow. The movement of peroxisomes wasmore prominent in root cells than that in leaves, and divided into two categories: a relatively slow, random,
vibrational movement and a rapid movement. Treatment
with anti-actin and anti-tubulin drugs revealed that actin
filaments involve in the rapid movement of peroxisomes.
These findings indicate that peroxisomes undergo dramatic changes in size, shape, number and position within
the cell.

FOR BASIC BIOLOGY

11

bodies. We found that wound stress induced the formationof ER bodies in rosette leaves . ER bodies were also induced treatment of methyl jasmonate (MeJA), a plant
hormone involved in the defense response against
wounding. The induction of ER bodies by MeJa was suppressed by ethylene. ER bodies contained precursors of
two vacuolar proteinases, RD2I and VPE. During saltinduced cell death, ER bodies fused with each other andwith lytic vacuoles , thereby mediating the delivery of theproteinase precursors directly into the vacuoles . We isolated an Arabidopsis mutant, named nail , in which ER
bodies were hardly detected in whole plants . ER bodies
were concentrated in a 1000 x g pellet (PI) after
subcellular fractionation. PYKIO, a ~-glucosidase with an
ER-retention signal (KDEL), was specifically accumulated in the PI fraction of GFP-h plants, but not in the PI

Fig. 1 Confocal microscopic images of various tissues in
GFP-PTSl.
(A) and (B) show the merged images with blue light excitation and the difference interference contrast. (A) trichome,(B) pistil. (C) and (D) represent the tubular and the normalshape peroxisomes, respectively . Each bar indicates 50 Jtm
for (A) and (B) , and 5 Jtm for (C) and (D).

IV. ER derived organelles for transport of proteins
to vacuoles.
A novel vesicle, referred to as a precursoraccumulating (PAC) vesicle, mediates the transport ofstorage protein precursors from endoplasmic reticulum(ER) to protein storage vacuoles in maturing pumpkinseeds . PAC vesicles had diameters of 300 to 400 nm, arederived from ER and contained an electron-dense core ofstorage proteins. PV72 , a type I integral membrane protein with three repeats of epidermal growth factor (EGF)like motifs , was found on the membrane of the PAC vesicles . We expressed modified PV72 and analyzed theirability to bind to the internal propeptide of pro2S albumin(2S-I) , a storage protein precursor, by affinity chromatography and surface plasmon resonance. The recombinant
PV72 specifically bound to the 2S-I peptide with a K 0
value of 0.2 ~-tM , which was low enough for it to function
as a receptor. The EGF-like motifs modulate the Ca2+dependent conformational change of PV72 to ER bodies
are one of ER-derived compartment specific to the Brassicaceae, including Arabidopsis. ER bodies are rodshaped structures that are characteristically 5 ~-tm long and
0 .5 ~-tm wide. Electron microscopic studies revealed that
ER bodies have a fibrous pattern inside and are surrounded by ribosomes (Fig. 2). Fluorescent imaging of
ER bodies were observed in transgenic plants ofArabidopsis (GFP-h) expressing green fluorescent protein
fused with an ER retention signal (GFP-HDEL). ER bodies were widely distributed in the epidermal cells ofwhole seedlings. In contrast, rosette leaves had no ER

Fig. 2 Rod-shaped bodies that were derived from ER accumulated GFP-HDEL in the epidermal cells of Arabidopsis
cotyledons.
Fluorescent image of numerous -5-~m-long bodies in theepidermal cells of 5-day-old cotyledons of the transgenic
plants. Bar= 10 ~m. (B) Immunocytochemistry with antiGFP antibodies showing that the large spindle-shaped bodies accumulated GFP-HDEL. Bar = 0.5 ~m. (C) Electron
micrograph showing that the bodies had a characteristic fibrous pattern inside and were surrounded by ribosomes.
Asterisks show the ER bodies. M, mitochondrion; G, Golgi
complex; V, vacuole. Bar= 0.5 ~m.

12
fraction of nail plants. Compare with the accumulation of
GFP-HDEL, which was associated with both cisternal ER
and ER bodies, the accumulation of PYKlO was much
more specific to ER bodies. These findings indicate that
PYKlO is the main component of ER bodies. Taken together, these results suggest that the formation of ER
bodies is a novel and unique type of endomembrane system in the responses of certain cells to environmental
stress.

IV. Role of molecular chaperones in organelle
differentiation.
Molecular chaperones are cellular proteins that function in the folding and assembly of certain other polypeptides into oligomeric structures but that are not, themselves, components of the final oligomeric structure. To
clarify the roles of molecular chaperones on organelle
differentiation, we have purified and characterizedchaperonin and Hsp70s and analyzed their roles in thetranslocation of proteins into chloroplasts.
Previously, we characterized a mitochondrial cochaperonin (CpnlO) and chloroplast co-chaperonins(Cpn20) and (CpnlO) from Arabidopsis. In 2002, westarted to characterize molecular chaperones inperoxisomes with collaboration with Dr. A. Kato at Niigata Univ. We found that a novel small heat shock protein(sHsp) localizes in peroxisomes. Molecular and functional
characterization of the sHsp is now under experiments.

Publication List:
Fukao, Y ., Hayashi, M. and Nishimura, M. (2002) Proteomic
analysis of leaf peroxisomes in greening cotyledons ofArabidopsis thaliana. Plant Cell Physiol. 43,689-696.
Hayashi, M., Nito, K., Takei-Hoshi, R., Yagi, M., Kondo,M.,
Suenaga, A., Yamaya, T. and Nishimura, M. (2002) Ped
3p is a peroxisomal ATP-binding cassete transporter thatmight supply substrates for fatty acid ~-oxidation. Plant
Cell Physiol. 43, 1-11.
Hayashi, H., De Bellis L., Kato, A., Hayashi, Y., Nito, K.,
Hayashi, M., Hara-Nishimura, I. and Nishimura, M.
(2002) Molecular characterization of an Arabidopsis acyl
CoA synthetase localized on glyoxysomal membranes.
Plant Physiol.130, 2019-2026.
Karnigaki, A., Mano, S., Terauchi, K., Nishi, Y., TachibeKinoshita, Y., Kondo, M., Nito, K., Hayashi, M.,
Nishimura, M. and Esaka, M. (2003) Identification ofperoxisomal targeting signal of pumpkin catalase and thebinding analysis with PTSl receptor. Plant J. 33. 161-175.
Kimura, Y., Matsuno, S., Tsurusaki, S., Kimura, M., HaraNishimura, I. and Nishimura, M. (2002) Subcellular localization of endo-{3-N-acetylglucosaminidase and highmannose type free N-glycans in plant cell. Biochim.
Biophys. Acta 1570, 38-46.
Kuroyanagi, M., Nishimura, M. and Hara-Nishimura, I.
(2002) Activation of Arabidopsis vacuolar processing enzyme by self-catalytic removal of an auto-inhibitory domain of the C-terminal propeptide. Plant Cell Physiol. 43,
143-151.

NATIONAL INSTITUTE

Mano, S., Nakamori, C., Hayashi, M., Kato, A., Kondo, M.
and Nishimura, M. (2002) Distribution and characterization of peroxisomes in Arabidopsis by visualization with
GFP: Dynamic morphology and actin dependent movement. Plant Cell Physiol. 43, 331-341.
Maser, P., Eckerman, B., Vaidyanathan, R., Fairbairn, D. J.,
Kubo, M., Yamagarni, M., Yamaguchi, K., Nishimura, M.,
Uozumi, N., Robertson, W., Sussman, M. and Schroeder, J.
I. (2002) Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruptionof the Na+transportor AtHKTJ. FEBS Lett. 531, 157-161.
Matsushima, R., Hayashi, Y., Kondo, M., Shimada, T.,
Nishimura, M. and Hara-Nishimura, I. (2002) An ERderived structure that is induced under stress conditions in
Arabidopsis. Plant Physio/.130, 1807-1814.
Matsushima, R., Kondo, M., Nishimura, M. and HaraNishimura, I. (2002) A novel ER-derived compartment,
the ER body, selectively accumulates a ~-glucosidase with
an ER retention signal in Arabidopsis. Plant J. in press
Nito, K., Hayashi, M. and Nishimura, M. (2002) Direct interaction and determination of binding domains among
peroxisomal import factors in Arabidopsis thaliana. Plant
Cell Physiol. 43, 355-366.
Ono, K., Kondo M., Osafune, T., Miyatake, K., Inui, H.,
Kitaoka, S., Nishimura, M. and Nakano, Y. (2002) Presence of Glyoxylate Cycle Enzymes in the Mitochondria ofEuglena gracilis. J. Eukaryo. Microbial. in press
Shimada, T., Watanabe, E., Tamura, K., Hayashi, Y.,
Nishimura, M., and Hara-Nishimura, I. (2002) A vacuolarsorting receptor on the membrane of the PAC vesicles that
accumulate precursors of seed storage proteins. Plant Cell
Physiol. 43, 1086-1095.
Watanabe, E., Shimada, T., Kuroyanagi, M., Nishimura, M.
and Hara-Nishimura, I. (2002) Calcium-mediated association of a putative vacuolar sorting receptor PV72 with apropeptide of 2S albumin. J. Bioi. Chern. 277, 8708-8715.

13

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS
OHSUMI, Yoshinori
YOSHIMORI, Tamotsu 11
KAMADA, Yoshiaki
NODA, Takeshi
MIZUSHIMA, Noboru 21
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellow:
YOSHIMOTO, Kohki
JSPS Postdoctral Fellow: SEKITO, Takayuki
SUZUKI, Kuninori
KIRISAKO, Takayoshi n
Postdoctoral Fellow:
ICHIMURA , Yoshinobu
HANADA, Takao
HANAOKA, Hideki 31
Graduate Students:
NARA , Atsuki 11
KUMA, Akiko 41
HAMASAKI, Maho 41
SHOJI, Shisako ' !
ONODERA, fun 41
MJWA , Masami
Technical Assistant:
TSUKESHIBA Kumi
HARA , Yohko
Secretary
Professor:
Associate Professor:
Research Associates:

11

until Mar 2002
ljrom Apr2002
31
until Jun 2002
41
Graduate University for Advanced Studies
2

I. Background
This division aims to understand the autophagy in respectsof its molecular mechanism and its physiological role within
higher eukaryotes. Biosynthetic processes and degradation
processes are well coordinated to regulate the biologicalactivities , however the study on the latter has been retarded
compared to the one on the former and we must shed light on
the degradation process to fully understand the cell . Autophagy is a well conserved degradation process in eukaryotes
and is a major route for bulk degradation of cytoplasmic
constituents in a lytic compartment, lysosome/vacuole.
Upon nutrient starvation, autophagic process starts in thecytoplasm as building up of a membrane structure, an autophagosome, which sequesters a portion of cytosol. Then it isdelivered to the vacuole/lysosome, and the cytoplasmic materials inside are degraded by the vacuolar/lysosomal proteases.
We had discovered autophagy in a simple eukaryotic modelorganism, Saccharomyces cerevisiae and morphologicallydescribed its whole process. We have isolated a set of
autophagy-deficient mutants (apg), and cloned APG genes
essential for autophagy.

plex in the cytosol. Because Apgl6 was suggested to form ahomo-oligomer, we generated an in vivo system that allowed
us to control the oligomerization state of Apg16. With thissystem, we demonstrated that formation of the approximately
350-kDa complex and autophagic activity depended on theoligomerization state of Apgl6. These results suggest that theApg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formationof the approximately 350-kDa complex is required for autophagy in yeast.

3. Cargo Delivery to the Vacuole Mediated byAutophagosomes
In the yeast Saccharomyces cerevisiae , aminopeptidase I(API) , a vacuolar hydrolase, is selectively transported to thevacuole via the autophagosome. API forms a cytosol to vacuole targeting (Cvt) complex in the cytoplasm. The complex isengulfed by the autophagosome under starvation conditions.
In this study, the Cvt complex is visualized as a dot in thecytoplasm by fluorescence microscopy with API-GFP. The
Cvt complex associates with the pre-autophagosomal structure (PAS), which plays a central role in autophagosome
formation. In a cvtl9 mutant, which is specifically defectivein API transport, but not in autophagy, the Cvt complex
forms normally but never associates with the PAS . This indicates that Cvt19p mediates association between the Cvt complex and the PAS.

Figure 1 API-GFP labels the vacuoles and dots close to thevacuole. Wild type cells expressing API-GFP under growing
conditions. (A) API-GFP, (B) vacuoles labeled with FM464, (C) merged image of API-GFP (green) and FM4-64(red), and (D) Nomarski image. Bar: 2 /.1m .

4. Studies of mammalian autophagy
2. Formation of the Apg12-Apg5-Apg16 multimericcomplex is essential for autophagy in yeast.
Autophagy requires a ubiquitin-like protein conjugation
system, in which Apg12 is covalently bound to Apg5. In theyeast Saccharomyces cerevisiae, the Apgl2-Apg5 conjugate
further interacts with a small coiled-coil protein, Apg16. The
Apgl2-Apg5 and Apgl6 are localized in the cytosol and preautophagosomal structures and play an essential role in autophagosome formation. We showed that the Apgl2-Apg5
conjugate and Apgl6 form an approximately 350-kDa com-

We cloned mouse ApglO and showed that the Apg12
conjugation system is completely conserved between yeast
and mammals . In addition, we also cloned a novel Apg5interacting protein, Apg16L. Although it was considered to
be a functional counterpart of yeast Apgl6, Apg16L has alarge C-terminal region containing a WD repeat domain thatis absent from yeast Apgl6. This unique feature of Apg16L
will provide new insights into role of Apg12-Apg5 in autophagosome formation.
Once an autophagosome is formed , it matures by fusion

14
with endosomes. Using a dominat negative mutant of mouse
SKD1, an AAA ATPase involved in the sorting and transport
from endosomes, we showed that SKD1-dependent endosomal membrane trafficking is required for further fusion withlysosome .
To monitor autophagy in vivo, we generated transgenic
mice expressing a fluorescent marker for autophagosome. It
would be a useful tool for studies on mammalian autophagy.

5. Leaf senescence is accelerated by the disruptionof an Arabi do psis autophagy gene.
The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at thecellular level, but the role that autophagy plays at the organismallevel has yet to be determined. In this study, a genomewide search revealed significant conservation between yeast
and plant autophagy genes. Twenty-five plant genes that arehomologous to 12 yeast genes essential for autophagy werediscovered . We identified an Arabidopsis mutant carrying a
T-DNA insertion within AtAPG9, which is the only orthologof yeast Apg9 in Arabidopsis (atapg9 -l). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9l mutant. Under nitrogen or carbon-starvation conditions,chlorosis was observed earlier in atapg9-l cotyledons androsette leaves compared with wild-type plants. Furthermore,
atapg9-l exhibited a reduction in seed set when nitrogenstarved. Even under nutrient growth conditions, bolting andnatural leaf senescence were accelerated in atapg9-l plants.
Senescence-associated genes SENl and YSL4 were upregulated in atapg9-l before induction of senescence, unlike
in wild type . All of these phenotypes were complemented by
the expression of wild-type AtAPG9 in atapg9-l plants.
These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.

Figure 2 The Arabidopsis during natural leaf senescence.
Wild type, atapg9-J, and atapg9- J plants transformed withwild-type AtAPG9 gene were grown under standard conditions and photographed on 35 day after germination.

NATIONAL INSTITUTE

Original papers :Hamasaki M, Noda T , Ohsumi Y. (2003) The early seretory
pathway contributes to autophagy in yeast. Cell Struct.
Funct. (in press)
Hanaoka H, Noda T, Shirano Y, Kato T , Hayashi H, Shibata
D, Tabata S, Ohsumi Y. (2002) Leaf senescence andstarvation-induced chlorosis are accelerated by the
disruption of an Arabidopsis autophagy gene. Plant
Physiol 129, 1181-1193.
Kuma A, Mizushima N , Ishihara N, Ohsumi Y . (2002) Formation of the approximately 350-kDa Apg12Apg5.Apg16 multimeric complex , mediated by Apg16
oligomerization, is essential for autophagy in yeast. J Bioi
Chem277, 18619-18625.
Mizushima N, Yoshimori T, Ohsumi Y. (2002) Mouse
ApglO as an Apg12-conjugating enzyme: analysis by the
conjugation-mediated yeast two-hybrid method. FEES
Lett. 532,450-454.
Mimura T., Reid R.J., Ohsumi Y., Smith F.A. (2002) Induction of the Na+/Pi co-transport system in the plasma
membrane of Chara corallina requires external Na+ andlow levels of Pi. Plant Cell & Environment 25:1475-1481.
Nara A, Mizushima N, Yamamoto A, Kabeya Y, Ohsumi Y,
Yoshimori T. (2002) SK.Dl AAA ATPase-dependent endosomal transport is involved in autolysosome formation.
Cell Struct Funct. 27, 29-37.
Suzuki K, Kamada Y, Ohsumi Y. (2002) Studies of cargodelivery to the vacuole mediated by autophagosomes in
Saccharomyces cerevisiae. DevCell3, 815-824.
Suzuki T, Nakagawa M, Yoshikawa A, Sasagawa N,
Yoshimori T , Ohsumi Y, Nishino I, Ishiura S , Nonaka I.
(2002) The first molecular evidence that autophagy relates
rimmed vacuole formation in chloroquine myopathy J
Biochem (Tokyo). 131 ,647-651.
Reviews:
Kamada Y. Sekito T. Ohsumi Y. (2003)Autophgy in yeast- a
Tor-mediated response to nutrient starvation- Curr. Top .
Microbiol.lmmunol. (in press)
Mizushima N, Yoshirnmi T , Ohsumi Y. (2003) Role of theApgl2 conjugation system in mammalian autophagy Int. J.
Biochem. Cell Biol. (in press)
Mizushima N, Ohsumi Y, Yoshimori T. (2002) Autophagosome formation in mammalian cells Cell Struct
Funct in press
Noda T, Suzuki K, Ohsumi Y. (2002) Yeast autophagosomes: de novo formation of a membrane structure.
Trends Cell Biol. 12,23 1-235
Ohsumi, Y. (2003) Cellular Recycling System- Molecular
Mechanism of Autophagy-.ln Cell Growth Cold Spring
Harbour Press . (in press)

15

FOR BASIC BIOLOGYDEPARTMENT OF DEVELOPMENTAL BIOLOGY

Chairperson: NAGAHAMA, Yoshitaka

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 onvarious 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.

16

NATIONAL INSTITUTEDIVISION OF REPRODUCTIVE BIOLOGY
Professor:
NAGAHAMA. Yoshitaka
YOSHIKUNI, Michiyasu
Associate Professor:
Research Associate:
KOBAYASHI, Tohru
KOBAYASHI, Hiroko
Technical Staff:
JSPS Postdoctoral Fellows:JKEUCHI, Toshitaka
SHIBATA, Yasuji
SUDHAKUMARI, Cheni Che1y
WANG, De-shou
Postdoctoral Fellows:
MATSUDA, Masaru
SAKAI, Fumie
GUAN, Gui-Jin
SENTHILKUMARAN,
Balasubramanian
LA U, En-Lieng
PAUL, Bindhu
Graduate Students:
HORIGUCHI, Ryo (Graduate
University for Advanced
Studies)
KOBAYASHI, Yasuhisa
FAN, Hai-Guang (Graduate
University for Advanced
Studies)
OHMURO, Yuki (Graduate
University for Advanced
Studies)
ZHOU, Linyan(Graduate
University for Advanced
Studies)
MATSUDA, Chika
Visiting Scientists:
MATSUOKA, Yah
YANO, Akihiro

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 of gametogenesis. Their effects are particularly profound inteleost fishes which provide several excellent models
for investigating the basic hormonal mechanisms regulating gonadal sex differentiation and gametogenesis
Our research focuses on (1) the identification of regulators and steroidal mediators involved in sex determination, gonadal sex differentiation and gametogenesis, and(2) the mechanisms of synthesis and action of these
mediators.

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. Alteration of phenotypic sex with no reproductive consequences, andrecombination over the entire sex chromosome pair,suggest that there are no major differences, other than asex-determining gene, between the X and Ychromo-

Fig. I Characteristics of madaka lacking a part of the Y
chromosome (a-c). Phenotypes of the congenic strain(XX)( a), medaka lacking a part of the Y chromosome(XY)(b) and the congenic strain (XY)(c).Histologicalcross-sections of medaka fry sampled 30 d.a.h .. XX (d)and XY(e) individuals have ovaries, whereas XY
specimen has testes with spermatogonia (f). Scale bars,
50 11m.

somes. To clone positionally the sex-determining region,
we generated a Y congenic strain to highlight the genetic differences between the X and Y chromosomesfrom inbred strains of medaka. The Y congenic strain
has a sex-determining region derived from the
HNI-strain Y chromosome on the genetic backgroundof an Hd-rR strain. Using this strain, we had previously
constructed a genetic map of the medaka sex chromosome and constructed a BAC (bacterial artificial chromosome) genomic library. We used recombinant
breakpoint analysis to restrict the sex-determining region in medaka to a 530-kb stretch of the Y chromosome. Deletion analysis of the Y chromosome of acongenic XY female further shortened the region to
250-kb (Fig.l ). Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed
during sexual differentiation. However, only one gene
PG 17 was Y specific. The full-length eDNA sequenceof PG 17 encodes a putative protein of 267 amino acids,
including the highly conserved DM domain. The DM
domain was originally described as a DNA-binding
motif shared between doublesex (dsx) in Drosophilamelanogaster and mab-3 in Caenorhabditis elegans. We
thus named it DMY (DM-domain gene on theY chromosome). To establish a role for DMY during sexual
differentiation, we screened wild medaka populations
for naturally occurring DMY mutants. Two XY femaleswith distinct mutations in DMY were found in separate
populations (Awara and Shirone). The first heritablemutant - a single insertion in exon 3 and the subsequent
truncation of DMY- resulted in all XY female offspring.
Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY
female offspring. Furthermore, during normal development, DMY is expressed only in somatic cells of XY
gonads. These findings strongly suggest that thesex-specific DMY is required for normal testicular development and is a prime candidate for the medaka
sex-determining gene.

17

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 migration.
In tilapia, first morphological sex difference is regarded as the number of germ cells between sexes.
Second sexual dimorphism in germ cells is the timing
for entry of germ cells into meiosis. Recently, we foundthat two isoforms of vasa mRNA and protein are present in tilapia. One (vas-s) lacks a part of theN-terminal
region found in the other isoform (vas). Both isoforms
are expressed in oocytes through the embryonic stage
when PGCs localize in the lateral plate mesoderm.
Thereafter vas-s expression increases and vas expression becomes undetectable. Expression of both isoforms
was observed again after morphological gonadal sex
differentiation, irrespective of genotypic sexes. We havealso generated medaka transgenic lines with green fluorescent protein (GFP) fluorescence controlled by theregulatory regions of the olvas gene in the germ cells.
The intensity of GFP fluorescence increases dramatically in PGCs located in the ventrolateral region of theposterior intestine around stage 25 (the onset of broodcirculation). Whole-mount in situ hybridization andmonitoring of ectopically located cells by GFP fluorescence suggest that 1) the increase in zygotic olvas expression occurs after PGC specification and 2) PGCs
can maintain their cell characteristics ectopically afterstages 20-25. The GFP expression persists throughout
the later stages in the mature ovary and testis.
In tilapia, mitosis of germ cells begins around 10
days post-hatching in genetic females, but can not be
confirmed until after sex differentiation in testes of genetic males. Steroid-producing cells in ovaries, but not
testes, at the undifferentiated and differentiating stages
express all of the steroidogenic enzymes required for
estradiol-17~ biosynthesis from cholesterol. Transcripts
of estrogen receptors (ER) a and~ first appear in both
female and male gonads of fry 5-10 days post-hatching.
These results, together with evidence of masculinizationof genetic females by fadrozole (an aromatase inhibitor)
or tamoxifen (an estrogen receptor antagonist), strongly
suggest that endogenous estrogens act as the natural
inducers of ovarian differentiation in tilapia. In contrast,
the ability of steroid-producing cells to synthesize steroid hormones in the testes only appears at the time oftesticular differentiation. Transcripts of androgen re-

ceptors are not present in gonads of genetic males during sex differentiation. DMRTJ is expressedmale-specifically in the gonads (Sertoli cells) during sex
differentiation, suggesting an important role of DMRTJ
in testicular differentiation.

III. Endocrine regulation of spermatogenesis
Using an organ culture system for eel testes consisting of spermatogonia and inactive somatic cells, wehave shown that the hormonal regulation of spermatogenesis in eel testes involves the gonadotropin stimulation of Leydig cells to produce 11-ketotestosterone(11-KT), a potent androgen in fish. In tum, 11-KT activates Sertoli cells to stimulate the production of activin
B. Addition of recombinant eel activin B to the culture
medium induced proliferation of spermatogonia, producing late type B spermatogonia, within 15 days in thesame manner as did 11-KT. cDNAs encoding two androgen receptors (ARa and AR~) have been cloned, forthe 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~ play different roles in spermatogenesis. Activin B binds to activin type I and II receptors on spermatogonia to stimulate de novo synthesis of G 1/S cyclins and CDKs leading to the initiation of mitosis. Interestingly, cyclin A1 transcripts are first detected inprimary spermatocytes during HCG-induced spermatogenesis in eel testes suggesting an important role forcyclin A1 in the progression to meiosis of male germ
cells.

IV. Endocrine regulation of oocyte growth andmaturation
Two follicular steroidal mediators, estradiol-17~(oocyte growth) and 17a,20~-dihydroxy-4-pregnen-3one (17a,20~-DP) (oocyte maturation) were identified
in several teleost fishes. 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. There is a distinct shift in expression of steroidogenic enzyme genes
from cytochrome P450 aromatase (P450arom) for estradiol-17~ production to 20~-hydroxysteroid dehydrogenase (20~-HSD) for 17a,20~-DP production in
granulosa cells immediately prior to oocyte maturation.
The preovulatory surge of LH-like gonadotropin is responsible for the rapid expression of 20~-HSD mRNAtranscripts in granulosa cells during oocyte maturation.
Two possible transcription factors have been identified:
Ad4BP/SF-1 (P450arom) and CREB (20~-HSD).
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 maturation-promoting factor (MPF), which activates a preex-

18

isting 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 is inactivated by degradation of cyclin B. We
showed that the 26S proteasome initiates cyclin B degradation through the first cut of its NH2 terminus at lysine 57.

Publication List:
Devlin, R.H. and Nagahama, Y. (2002). Sex determinationand sex differentiation in fish. Aquaculture 208,
191-366.
Hirai, T., Oba, Y. and Nagahama, Y. Fish gonadotropin
receptors: molecular characterization and expression
during gametogenesis. Fish. Sci. (in press).
Huang, Y.S., Yueh, W.S., Huang, J.D., Du, J.L., Sun, L.T.,
Nagahama, Y. and Chang, C.F. (2002). Cloning and expression of estrogen receptors in the protandrous black
porgy (Acanthopagrus schlegeli): Implications of sexchange mechanism. Mar. Biotechnol. 4, 236-246.
Ikeuchi, T., Todo, T., Kobayashi, T. and Nagahama, Y.
(2002). A novel progestogen receptor subtype in theJapanese eel, Anguilla japonica. FEBS Letters 510,
77-82.
Kobayashi, T., Kajiura-Kobayashi, H. and Nagahama, Y.
(2002). Two isoforms of vasa homo logs in a teleost fish:
their differential expression during germ cell differentiation. Mech. Develop.111, 167-171.
Kusakabe, M., Kobayashi, T., Todo, T., Lokman, P.M.,
Nagahama, Y. and Young, G. (2002). Molecular cloning
and expression during spermatogenesis of a eDNA encoding testicular 11j3-hydroxylase (P450 11j3) in rainbow trout (Oncorhynchus mykiss). Mol. Reprod. Dev.
(in press)
Matsuda, M. and Nagahama, Y. (2002). Positional cloningof the sex-determining region of medaka using Y congenic strain. Aquatic Genomics: Steps Toward a Great
Future. N. Shimizu, T. Aoki, I. Hirono and F. Takashima (eds.), 236-243, Springer.
Matsuda, M., Nagahama, Y., Shinomiya, A., Sato, T., Matsuda, C., Kobayashi, T., Morrey, C.E., Shibata, N.,
Asakawa, S., Shimizu, N., Hori, H., Hamaguchi, S. andSakaizumi, M. (2002). DMY is a Y -specific DM-domain
gene required for male development in the medaka fish.
Nature 417, 559-563.
Matsuda, M., Sato, T., Toyazaki, Y., Nagahama, Y.,
Hamaguchi, S. and Sakaizumi, M. (2002). Oryzias
curvinotus has DMY, a gene that is required for maledevelopment in the medaka, 0. latipes. Zoo!. Sci. (inpress).
Morrey, C.E., Nagahama, Y. and Grau, E.G. (2002). Terminal phase males stimulate ovarian function and inhibit
sex change in the protogynous wrasse Thalassoma
duperrey. Zoo!. Sci. 19, 103-109.
Ohta, K., Rhaman, M.A., Chuda, H., Yoshikuni, M., Nagahama, Y. and Matsuyama, M. Maturation-inducing
hormone and it's membrane receptor in gonads of Japanese yellowtail, Seriola quinqueradiata. Fish. Sci. (inNATIONAL INSTITUTE

press).
Rahman, M.A., Ohta, K., Yoshikuni, M., Nagahama, Y.,
Chuda, H. and Matsuyama, M. (2002). Characterizationof ovarian membrane receptor for 17a,20j3-Dihydroxy4-pregnen-3-one, a maturation-inducing hormone yellowtail, Seriola quinqueradiata. Gen. Comp. Endocrinol.
127,71-79.
Senthilkumaran, B., Sudhakumari, C.C., Chang, X.T.,
Kobayashi, T., Oba, Y., Guan, G., Yoshiura, Y., Yoshikuni, M. and Nagahama, Y. (2002). Ovarian carbonyl
20f3-hydroxysteroid
dehydrogenase
reductase-likeshows distinct surge in messenger RNA expression during natural and gonadotropin-induced meiotic maturation in Nile tilapia. Bioi. Reprod. 67, 1080-1086.
Tanaka, M., Nakajin, S., Kobayashi, D., Fukada, S., Guan,G., Todo, T., Senthilkumaran, B. and Nagahama, Y.
(2002). Teleost ovarian carbonyl reductase-like
20j3-hydroxysteroid dehydrogenase: Potential role in the
production of maturation-inducing hormone during final
oocyte maturation. Bioi. Reprod. 66, 1498-1504.
Tateno, H., Shibata, Y., Nagahama, Y., Hirai, T., Saneyoshi, M., Ogawa, T., Muramoto, K. and Kamiya, H.
(2002). Tissue specific expression of rhamnose-binding
lectins in the steelhead trout (Oncorhynchus mykiss).
Biosci. Biotechnol. Biochim. 66, 1427-1430.
Tokumoto, M., Nagahama, Y. and Tokumoto, T. (2002). A
major substrate for MPF: eDNA cloning and expressionof polypeptide chain elongation factor 1y from goldfish(Carassius auratus). DNA sequence 13, 27-31.
Tokumoto, M., Nagahama, Y. and Tokumoto, T. (2002).
Molecular cloning of eDNA encoding a polypeptide
chanin elongation factor 1a from goldfish (Carassius
auratus). DNA Sequence (in press).
Wang, D.S., Kobayashi, T., Senthilkumaran, B., Sakai, F.,
Sudhakumari, C.C., Suzuki, T., Yoshikuni, M., Matsuda,M., Morohashi, K. and Nagahama, Y. (2002). Molecularcloning of DAXJ and SHP cDNAs and their expression
patterns in the Nile tilapia, Oreochromis niloticus. Biochem. Biophys. Res. Comm. 297, 632-640.
Yokoi, H., Kobayashi, T., Tanaka, M., Nagahama, Y.,
Wakamatsu, Y., Takeda, H., Araki, K., Morohashi, K.
and Ozato, K. (2002). Sox9 in a teleost fish, medaka(Oryzias latipes): Evidence for diversified function of
Sox9 in gonad differentiation. Mol. Reprod. Develop. 63,
5-16.
Yoshiura, Y., Senthilkumaran, B., Watanabe, M., Oba, Y.,
Kobayashi, T. and Nagahama, Y. (2002). 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. (in press).

19

FOR BASIC BIOLOGYDIVISION OF CELL DIFFERENTIATION
MOROHASHI, Ken-ichirou
SHIMONO, Akihiko
ISHIHARA, Satoru
FUKUI, Yuko
OGAWA, Hidesato
OKA, Sanae
Technical Staff:
NIBB Research Fellow:
SUZUKI, Taiga
JSPS Postdoctoral Fellow: TSUCHIYA, Megumi
Postdoctoral Fellow:
MOHAMAD, Zubair n
SUGIYAMA, Noriyuki n
MIZUSAKI, Hirofumi n
SHIMA, Yuichi 2)
Graduate Student:
BABA, Takahi 3)
FUKUDA, Takayuki 4!
KUSAKA, Masatomo 5!
KOMATSU, Tomoko 5!
MATSUYAMA, Makoto 5!
FATCHIYAH 5!
MORITA, Tomoko 6!
OWAKI,Akiko 7!
Technical Assistant:
KOWA,Hiroe
SHINOHARA, Yuko 7!
ISHIKAWA, Azus 7!
KUSAKA, Hiroko 7!
SATO, Yuko
DOUZONO, Akemi 3!
Secretary:
YAMANAKA, Tae 3!
KAWASHIMA,Ryoko
HANDA, Yasushi
Visiting Scientist:
Professor:
Research Associate:

ncREST, JST Postdoctoral Fellow!Graduate School of Kyushu University
3!Graduate School ofTohoku University
4 !Graduate School of Ehime University
5!Graduate University for Advanced Studies
6!Graduate School ofTokyo University
7iCREST, JST Technical Assitant
8iCREST, JST Secretary
2

Cell and tissue differentiation proceeds systematically
based on orchestrated expressions of a battery of genes.
The expressions commence successively along with thepassage of time, and consequently a single fertilized egg
develops into a variety of tissues and organs, which consist of specialized cells in terms of their structures andfunctions. Accordingly, it is reasonable to assume thatinvestigation of the mechanisms underlying cell andtissue-specific gene expressions at a molecular level isessential for understanding molecular frameworks forgenetic cascades to support cell and tissue differentiation.
Based on the concept above, our division of Cell Differentiation has focussed on sex differentiation of the gonads
and differentiation of the steroidogneic tissues form the
aspect of functions of tissue-specific transcription factors
and growth factors.
A number of transcription factors are involved in theprocess of gonadal differentiation. Some of these factors,
such as SRY, WT-1, DAX-1, and SOX-9 have been identified as the products of genes responsible for humandiseases that display structural and functional defects intissues including the gonads. The crucial functions of theother transcription factors such as Ad4BP/SF-1, Emx-2,
M33, and Lhx-9 were identified by phenotypes of theirgene disrupted mice. In addition, the expression profiles
with respect to their distribution and sexual dimorphism
strongly suggested the functional significance at the earlystage of gonadal differentiation. However, it remains to beclarified how the transcription factors above regulate theirtarget genes transcription and how the genes encoding thetranscription factors are regulated. When considering agene regulatory cascade that supports differentiation andsex differentiation of the gonad, studies of the above two
directions are quite important. Based on this concept, weinvestigated mainly the functions of Ad4BP/SF-1 andDax-1, and the mechanism of gene regulation encoding
these factors.

I. Gene regnlatory cascade in the steroidogenic
tissue differentiation
When a differentiation process of a tissue is considered,
it is possible to assume that certain genes encoding transcription factors are involved in a gene regulatory cascadeas the critical components. As a component in the cascades required for adrenocortical and gonadal differentiation, Ad4BP/SF-l seems to be localized at the upstream ofa set of tissue-specific genes including steroidogenic Cyp
genes, while Ad4BP/SF-1 is localized at the downstreamof other transcription factors regulating Ad4BP/SF-1 genetranscription. When considering that upregulation of thecomponents occurs from upstream to downstream of thecascade along with the tissue differentiation and moreover
Ad4BP/SF-1 is an essential transcription factor in theadrenocortical and gonadal cascade, identification of thecomponents functioning with Ad4BP/SF-1 and regulating
Ad4BP/SF-1 gene transcription is quite important for
fully understanding the molecular mechanisms underlying
differentiation of the adrenal cortex and gonad. Thus, wehave investigated gene regulation of Ad4BP/SF-1 andDax-1.

Tissue-Specific Enhancers on the Ad4BPISF-1 Gene
Based on the aspect above, the regulatory region of the
Ad4BP/SF-l gene has been investigated in vivo. A mouse
YAC clone (longer than 480 kb) containing whole
Ad4BP/SF-1 gene was examined. The long fragment was
subcloned into cosmid vector carrying Lac Z gene as areporter gene, and subjected to transgenic assay to examine if they have tissues-specific enhancer element. Our
survey for the genomic DNA revealed that particular regions in the Ad4BP/SF-1 gene are responsible for thegene expression specific for the adrenal cortex,
ventromedial hypothalamic nucleus, and pituitary. Interestingly, all of them are localized at the intronic regions
and the nucleotide sequences are conserved among animal
species. The fine structures of these tissue-specific enhancers are investigating.

Wnt4 Signal for Gonad Sex Differentiation
Dax-1 is an orphan nuclear receptor acting as a suppressor of Ad4BP/SF-1, and as an anti-Sry factor in theprocess of gonadal sex differentiation. The roles of these20
nuclear receptors in the differentiation of the gonads andthe adrenal cortex have been established through studiesof the mutant phenotype in both mice and humans. However, the mechanisms underlying transcriptional regulation of these genes remain largely unknown. We examined the relationship between Dax-1 gene transcription
and the Wnt4 pathway. Reporter gene analysis revealed
that Dax-1 gene transcription was activated by ~-catenin,
a key signal-transducing protein in the Wnt pathway ,acting in synergy with Ad4BP/SF-l . Interaction between~-catenin and Ad4BP/SF-l was observed using yeast twohybrid and in vitro pull-down assays . The region of
Ad4BP/SF-l essential for this interaction consists of anacidic amino acid cluster, which resides in the first helixof the ligand binding domain. Mutation of the amino acidcluster impaired transcriptional activation of Dax-1 aswell as interaction of Ad4BP/SF-l with ~-catenin. These
results were supported by in vivo observations using Wnt4
gene disrupted mice , where Dax-1 gene expression was
decreased significantly in sexually differentiating female
gonads . We thus conclude that Wnt4 signaling mediates
the increased expression of Dax -1 as the ovary becomessexually differentiated.

II. Characterization of factors interacting with
Ad4BP/SF-1
Arx is essntial for Leydig cell Differentiation.
We have isolated a number of clones by yeast twohybrid screening using Ad4BP/SF-1, Dax-1, Sox9 , Wt-1,
Ernx-2, and Gata-4 as baits . Arx, the a.ristaless related
homeobox gene , is one of the genes interacting with
Ad4BP/SF-1. Since the expression of Arx is specific forthe gonad and brain from the early developmental stage ,the function of Arx was investigated by characterizing
phenotype of the gene disrupted mice. In the fetal testes,
Arx is expressed in the interstitial cells such as the peritubular myoid cells , tunica albuginea, vessel endothelial
cells , and the cell lining beneath the tunica albuginea , but
not in Leydig, Sertoli, and germ cells . In the mutant testes ,Sertoli, germ cells , tunica albuginea and blood vessels
were most likely not affected, whereas the mutant testes
were usually characterized by a dysplastic interstitium.
MIS, a marker for Sertoli, was clearly detected in thetesticular cords of both wild type and mutant testes ,whereas expression of Leydig cell marker 3~-HSD wasseverely diminished in the mutant testis , indicating that
Leydig cell differentiation is blocked at a certain stage.

ARX is the responsible gene for XLAG
The structural and functional defects observed in the
mutant and the chromosomal localization of ARX on
Xp22.13 suggested ARX as a plausible candidate gene for
XLAG (X-linked lissencephaly with abnormal genitalia).
To investigate this possibility , we determined nucleotide
sequence of the ARX in eight XLAG probands , and detected eight mutations. Overall, we found one nonsense
mutation, four frame-shift mutations, two misssense mu-

NATIONAL INSTITUTE

tations , and one larger deletion . Two of the truncated
ARX proteins contain the homeodomain but lack the Cpeptide (aristaless) domain conserved in prd-like
homeoproteins. Although the function of the domain remains unknown, physiological significance is suggested
by our results. Moreover, we identified two missense
mutations that lead to amino acid substitutions , R322H
and L343Q. Since both amino acids are located in the
homeodomain and are highly conserved among the family ,it is reasonable to assume that the mutations result infunctional failure such as DNA binding.

p rd-IIQ home<~clomaln

t

P12

t t t

PU P..... P#•t

PIS

C.peptkle domaintPn

Publication List:
Asoy , R., Mellgren , G.,_Morohashi, K. and Lund, J. (2002)
Activation of cAMP-dependent Protein Kinase increases
the protein level of Steroidogenic Factor-1. Endocrinol.
143 , 295-303.
Kitamura , K., Yanazawa, M. , Sugiyama, N., Miura, H .,Iizuka-Kogo, A. , Kusaka, M., Suzuki, R ., Kato-Fukui , Y.,
Kamiirisa, K. , Omichi , K. , Kasahara, M. , Yoshioka, H.,
Ogata, T.,Fukuda, T ., Kondo , I. , Kato,M. , Dobyns , W. B ..
Yokoyama M., and Morohashi K. (2002) Mutations of
Arx/ARX cause abnormal migration and differentiation of
GABAergic interneurons and abnormal development oftestes in mice, and X-linked lissencephaly with abnormal
genitalia in humans. Nature Genet. 32, 359-369,
Meeks, J. 1., Crawford, S. E. , Russell, T. A . Morohashi, K.,
Capel , B ., Weiss , J. and Jameson , J. L. (2003) Dax1 is required for patteming of testis cords during gonadal
differentiation . Development. (in press)
Mukai, T ., Kusaka, M., Kawabe, K. , Goto, K., Nawata, H.,
Fujieda, K. and Morohashi K. (2002) Sexually dimorphic
expression of Dax-1 in the adrenal cortex . Genes Cells 7,
717-729
Suzuki , T., Kasahara , M., Yoshioka , H., Morohashi , K. andUmesono K. (2003) LXXLL motifs in Dax-1 have target
specificity for the orphan receptors Ad4BP/SF-1 andLRH-1. Mol. Cell . Bioi. 23 , 238-249
Wang, D., Kobayashi , T., Senthilkumaran, B. , Sakai , F. ,Sudhakumari, C. C. , Suzuki , T ., Yoshikuni , M ., Matsuda,
M. , Morohashi, K. and Nagahama Y. (2002) Molecularcloning of DAX-1 and SHP cDNAs and their expression
patterns in the Nile tilapia, Oreochrornis niloticus.
Biochem. Biophys. Res. Commun. 297 , 632-640,
Yokoi , H. , Kobayashi , T ., Tanaka, M ., Nagahama, Y. , Wakamatsu, Y., Takeda, H., Araki, K., Morohashi, K. andOzato K. (2002) sox9 in a teleost fish, medaka (Oryzias
latipes): evidence for diversified function of Sox9 ingonad differentiation . Mol . Reprod. Dev . 63, 5-16

21

FOR BASIC BIOLOGY

levels of Xgly4 inhibits the cell-membrane accumulationof Dishevelled (Dsh), as does the inhibition of Wntll inthe dorsal marginal zone. Finally, we provided evidence
that Xgly4 protein physically binds Wnt ligands. Thesefindings suggest that Xgly4 is serving as a coreceptor for
Wnt5A/Wntll by presenting these ligands to Frizzled
receptor. This hypothesis is further supported by thefinding that the causative gene for a zebrafish
gastrulation-defective mutant knypek encodes a glypican.

DIVISION OF MORPHOGENESIS
Professor:
Associate Professor:
Research Associates:
Technical Staff:

NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:

11

UENO , NaotoKINOSHITA , Noriyuki
NAKAMURA , Makoto
TAKAHASHI, Hiraki
TAKAGI, Chiyo
YAMAMOTO , Takamasa
HYODO, Akiko
TERASAKA , Chie
MOROKUMA, Yoshie
YAMADA , Shigehiro
MIURA, Jyunko
KITAYAMA , Atsushi
MOROKUMA , Junji
KURATA , Tomoko n
TAKEUCHI, Masaki 11
IIOKA , Hidekazun
MIYAKOSHJ , Akira 11

II. Gastrulation movement regulated by Wnt
signaling

Graduate University for Advanced Studies

The complex morphogenesis of organisms is achievedby consecutive ceil-to-cell interactions during development. Recent studies suggest that growth factors play
crucial roles in controlling such intercellular communications in a variety of organisms. In addition to secretory
factors, transcription factors which act cell-autonomously
are thought to be essential for the determination of cellfates. Our main interest is to know how pattern formationand morphogenesis during development is regulated bythese growth factors and transcription factors . We address
this problem using several model animals , including frog,
fly and acidian, employing embryology, genetics, molecular and cellular biology, and biochemistry. In addition ,we have recently introduced array technology to elucidate
precise genetic program controlling early development.

I. Regulation of growth
proteoglycans

factordiffusion

byDuring early development, cells receive positional information from neighboring cells to form tissue patterns
in initially uniform germ layers. Ligands of polypeptide
growth factors such as Wnts and TGF-~s are known toparticipate in this pattern formation as morphogens. As an
initial attempt, we searched for potential cell surface
proteoglycans which might interact with morphogens andregulate their diffusion. We identified a gene encoding
Xenopus glypican-4 (Xgly4), a member of the HSPGs
family, and analyzed its role during gastrulation. In situ
hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Xgly4
overexpression and translational inhibition by aMorpholino-oligonucleotide inhibited the gastrulation
movements of the embryo and the convergent extension(elongation) of activin-treated animal caps, but did notaffect mesoderm induction. Rescue analysis with different
mutants of Dsh an essential compomnent of Wnt pathway
and Wntll demonstrated that Xgly4 function in the noncanonical Wnt pathway, but not in the canonical Wnt/~­
catenin pathway for convergent extension movements .
Furthermore, we demonstrated that knocking down the

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 spiteof its importance, the mechanism underlying the event has
just begun to be studied at molecular level. During
Xenopus gastrulation , mesodermal cells migrate to theinside of the embryo and move along the blastocoel roof.
One of the important mechanisms for this process is convergent extension. As convergent extension begins, cells
are polarized and aligned mediolaterally , followed by theintercalation of these cells. As described above, one of theWnt signaling pathways, called Wnt/JNK (c-Jun Nterminal 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 the establishment of planar cell polarity (PCP) of
Drosophila wing hair.
We demostrated that Xenopus prickle (Xpk) , a Xenopus
homologue of a Drosophila PCP gene , is an essential
component for gastrulation cell movement. Both gain-offunction and loss-of-function of Xpk severely perturbed
gastrulation and caused spina bifida embryos without
affecting mesodermal differentiation (Figure 1). We also
demonstrated that XPK binds to Xenopus Dsh as well as
to JNK. This suggests that XPK plays a pivotal role inconnecting Dsh function to JNK activation.
uninjected,~;J~.!

~
mo-Xpk

~~4-mismo-Xpk__ . _~..

_ _Figure I. Xpk antisense morpholino oligos (mo) inhibit
convergent extension movements. Top; uninjected siblingembryo at the tadpole stage , middle panel; mo-Xpk 10pmol dorsal-injected embryo displaying spina bifida andbottom; 4mis mo-Xpk (negative control)-injected embryo
showing little effect when injected dorsally .

22

We are also studying how Wnt signal is transmitted to
Dsh from Frizzled receptors. Protein kinase C (PKC) was
implicated in the Wnt pathway, but its molecular role waspooly understood . We searched our Xenopus EST database for novel PKC genes and identified a novel geneencoding PKC6. Loss-of-function of PKC6 by adominant-negative form or Morpholino antisense
oligonucleotide has revealed that it is essential for convergent extension during gastrulation. A microscopic
analysis showed that PKC6 is requierd for the polarizationand morphological change of mesodermal cells in thisprocess. This result suggested that PKC6 might be involved in the Wnt/JNK pathway. It is known that activation of the Wnt/JNK pathway involves translocation of
Dsh from cytoplasm to the membrane and activation of
JNK. But the molecular mechanism is not clear. We tested
whether PKC6 is required for the translocation of Dsh andthe activation of JNK , and showed that Joss-of-function of
PKC6 inhibited both of these events. This result indicatesthat PKC6 is essential for Dsh function. We also showedthat it forms a complex with Dsh and is translocated to theplasma membrane by Frizzled signaling. In addition, activation of PKC6 was sufficient for Dsh translocation and
JNK activation. We concluded PKC6 plays an essential
role in convergent extension by regulating the localization
and activity ofDsh in the Wnt/JNK pathway.

III. Genetic screening for novel DPP/BMP signaling
components utilizing Drosophila model system
Drosophila is one of the ideal model organisms to dissect signal transduction pathway by genetic methods. We
have carried out dominant suppressor screening for two
transgenic mutant flies expressed constitutively activated
DPP/BMP type-I receptors in wing imaginal discs. We
isolated 19 suppressor mutants, Suppressor of constitutively activated Dpp singnaling (Scad). Alleles of punt,
Mad, shn and dCrebA were found in isolated Scad mutants. We now focus to study a novel locus Scad67 .
Scad67 encodes a protein with a SP-RING motif. SPRING motif was originally found in PIAS-family proteins
including Drosophila PIAS homolog Su(var)2-l0/Zimp .
Homozygous Scad67 mutants show embryonic to pupal
stage lethality. The most severe zygotic mutant showsabnormal embryonic head structure and segmentation
defects. Recent studies have shown that PIAS-family
proteins function as a E3-SUMO ligase and enhance
SUMO conjugation against specific substrates. Scad67 isan evolutionary conserved molecule and we found two
Scad67 homologous genes in human and also in othervertebrates. In the culture cell , transiently expressed vertebrate Scad67s are localized in the nucleus and frequently accumulate in the nuclear dots. Typical PML
body markers, such as SPlOO, CBP, SUMOl are colocalised with Scad67s. These data indicate that Scad67
facilitates PML nuclear body assemble. This PML assemble activity is clearly enhanced in the presence of SUMO!
and eliminated by inducing point mutation in the Scad67
SP-RING domain . These results strongly suggest that
Scad67s are novel family of SUMO-E3 ligases and par-NATIONAL INSTITUTE

ticipate some critical role in the SUMO-conjugation reaction in the nucleus both in vertebrate and invertebrate.

IV. Brachyury downstream notochord differentiation in the ascidian embryo
Ascidians, urochordates , are one of the three chordategroups, and the ascidian tadpole is thought to represent
the most simplified and primitive chordate body plan. It
contains a notochord , which is a defining characteristic ofchordate embryo composed of only 40 cells. To understand the morphogenesis in this simple system, we havefocused on a gene , Brachyury , which is known to play animportant role in the notochord development. In ascidian,
Brachyury is expressed exclusively in the notochord andthe misexpression of the Brachyury gene (Ci-Bra) of
Ciona intestinalis is sufficient to transform endoderm into
notochord. This gene encodes a sequence-specificactivator that contains aT-box DNA-binding domain , andin vertebrates, it is initially expressed throughout the presumptive mesoderm and gradually restricted to the developing notochord and tailbud. The phenotype of theBrachyury mutants in mice and zebrafish revealed that
this gene is essential for notochord differentiation. Our
goal is to elucidate the down stream pathway of this important gene in ascidian in order to set the stage for understanding not only the formation and function of thenotochord but how this important structure has evolved.
We conducted the subtractive hybridization screens to
identify potential Brachyury target genes that are induced
upon Ci-Bra overexpression. Out of 501 independent
eDNA clones that were induced cDNAs , 38 were specifically expressed in notochord cells. We characterized 20 ofthem by determining the complete nucleotide sequences
and in situ hybridization analyses which show the spatial
and temporal expression patterns of the cDNAs. These
potential Ci-Bra downstream genes appear to encode abroad spectrum of divergent proteins associated with notochord formation and function.

Figure 2. Subcellular localization of electroporated
notochord specific genes in ascidian embryos. The embryos
were electroporated at one-cell stage with fusion genecontaining notochord specific promoter attaced to Ci-ACLEGFP , Ci-ERM-EGFP and Ci-Trop-EGFP, and grown to
the tail bud stage of development. The EGFP fusion proteinsshown various subcellular localization in the notochordcells of the tadpole tails.

23

FOR BASIC BIOLOGY

V. Comprehensive analysis of developmentaUy
regulated genes using macro/microarray
In order to examine the global expression profile during early development of Xenopus laevis, we have collected massive EST sequences from three normalized
eDNA libraries of early gastrula, neurula and tailbud stage.
To date, more than 70,000 ESTs were produced and assembled into 12,447 contigs and 15,361 singlets. Homology analysis shows about 40% of known X. laevis proteins are included in these libraries. Moreover, a few
thousand novel X. laevis orthologue genes which sharehigh similarities (E<10' 10) with those of other animals arealso found. EST sequences and annotational information
can be accessed through the web at NIBB X. laevis EST
database XDB (http://xenopus.nibb.acjp/), and cDNAs
are available for request.
Using these resources, we generated the NIBB 40k
eDNA macroarray, and as an initial case to investigate,
we conducted a large-scale gene expression screening of
Xnr-1 regulated genes. As a result, we have isolated 74(0.16%) Xnr-1 up-regulated (ratio ;;,; 2.0), and 47 (0.10%)
down-regulated (ratio s 0.5) independent EST clones.
Whole-mount in situ hybridization was carried out assecondary screening step, showing high ratio of specificgene expression patterns. These clones encode a variety ofsignal transduction and transcription regulatory components, and also cytoskeletal components, suggesting dynamic cellular changes in response to the Nodal signals.
This also proves DNA array to be an effective screening
assay for novel genes which function in the early development of Xenopus, utilizing our ESTs.

Publication List
Hyodo-Miura, J., Urushiyama, S., Nagai, S., Nishita , M.,
Ueno, N. and Shibuya, H. (2002) Involvement of NLK
and Soxll in neural induction in Xenopus development.
Genes Cells. 7, 487-496.
Koshida, S., Shinya, M., Nikaido, M., Ueno, N., SchulteMerker, S., Kuroiwa, A. and Takeda, H. (2002) Inhibition
of BMP activity by the FGF signal promotes posterior
neural development in zebrafish. Dev Bioi. 244, 19-20.
Kurata, T. and Ueno, N. Xenopus Nbx, a novel NK-1 related
gene essential for neural crest formation Dev. Bioi. (inpress)
Morita, K., Flemming, A., Sugihara, Y., Mochii, M., Suzuki,
Y. Yoshida, S., Wood, B., Kohara, Y., Leroi, A.M. andUeno N. (2002) A C. elegans TGF-~, DBL-1, controls theexpression of LON-I that regulates polyploidization andbody length. EMBO J. 21, 1063-1073.
Nakamura, M. Baldwin, D., Hannaford, S. Palka, J. andMantell, C. (2002)DPR, a member of the immunoglobulin
superfamily required for the gustatory response to salt. J.
Neurosci. 22,3463-3472.
Ohkawara, B., Iemura, S.-I., ten Dijke, P. and Ueno, N.
(2002) Action range of BMP is defined by its N-terminal
basic amino acid core. Curr. Biol.12, 205-209.
Yuasa, T., Kataoka, H., Kinto, N., Iwamoto, M., EnomotoIwamoto, M., Iemura, S. I., Ueno, N., Shibata, Y.,
Kurosawa, H. and Yamaguchi, A. (2002) Sonic hedgehog
is involved in osteoblast differentiation by cooperatingwith BMP-2 J. Cell. Physiol. 193, 225-232.

24

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL
BIOLOGY (ADJUNCT)
Assistant Professor:
JST Research Fellow:
JST Postdoctoral Fellow:

ITO, KeiAWASAKI, Takeshi (PRESTO)OKADA, Ryuichi (BIRD)
KAMIKOUCHI,Azusa (BIRD)
JSPS Postdoctoral Fellow: KATO, Kentaro
Graduate Student:
TANAKA, Nobuaki(Graduate Univ.Advanced
Studies)
OTSUNA, Hideo(Nagoya University)

The member of this adjunct division shifted from theDivision of Cell Proliferation as of year 200 1. The aim of
this division is to understand the basic rules by which
elaborate neural circuits develop and function. With lessthan 105 neurons, and subject to powerful molecular andgenetic techniques, the brain of the fruit fly Drosophilamelanogaster is a good model system for such study.

I. Comprehensive identification of neural circuits inthe adult brain
In spite of the hundred years of efforts using Golgi andother anatomical techniques, the circuit structure ofhigher-order associative regions of the brain is still
essentially unresolved. The GAL4 enhancer-trap system,
which is widely used for mutagenesis and gene cloning ofDrosophila, is also a powerful tool for obtaining a vastarray of transformant strains that label specific subsets ofbrain cells. We screened such strains from a stock of 4500
GAL4 lines made by the NP consortium, a joint ventureof eight Japanese fly laboratories organized by us.
The database of GAL4 expression patterns in the brainof these strains is the infrastructure for further analysis.
Having completed the image database of the confocal
sections of the adult brain last year, we started making asimilar database for the larval brain and accumulated
59,723 brain images so far.
Analyses of neurons processing visual and olfactory
information continue since previous years. For the visualsystem, we identified in total 44 pathways that connect
the optic lobe and the central brain. To investigate thedifference of their roles in visual information processing,we blocked the synaptic transmission of identified
neurons with the ectopic expression of the dominantmutant form of Dynamin GTPase, which blocks therecycling of synaptic vesicles. Flies expressing thistransgene in various subsets of the 44 visual pathways
were subjected to phototaxis assay, which analyzes theflies' ability to identify the orientation of the light source.
The effect was different depending on the color of lightsource, suggesting that different pathways might convey
the visual information of different wavelength ranges.
For olfactory pathways, last year we identified thestructures of the projection neurons that transmit
information from the first-order olfactory processing site(antenna! lobes) to the second-order sites (mushroom
bodies and lateral horns). This year we screened thestrains that label neurons of these second-order sites. The
area of arborization of these neurons showed strong
spatial correlation with the distribution of the terminals of
the projection neurons. The olfactory code acrossglomeruli is thus essentially maintained in the neurons of
the second-order sites. Like projection neurons, the spatial
specification of these neurons were established before
adult eclosion and maintained after surgical ablation of
the olfactory input.
We also started the characterization of the pathways
that convey auditory information. Although the auditoryorgan of the flies had been identified as the Johnston'
Organ, which resides at the base of the antenna, theprecise projection patterns of its neurons were not known.
We screened the strains that label sensory neurons of thisorgan and are mapping the distribution of their terminals
in the brain.

II. Distribution of neurons that release and receive
inhibitory transmitters
Although inhibitory synapses play important roles for
information processing, and the GABA is the major
neurotransmitter for this function, detail of synaptic
connections mediated by GABA is essentially unknown.
By combining fluorescent in situ hybridization (FISH)
and GFP-immunostaining, we examined the precise
distributions of neurons that release and/or receive GABA
in the antenna! lobe. We found that most of the local
interneurons we identified are GABAergic and have
GABA receptors. In the case of projection neurons, wefound that majority of mACT neurons, but not iACT andoACT neurons, are GABAergic, and that mACT neurons
rarely have output sites in the antenna! lobe. Thesefindings indicate that, although a wide variety of neurons
in the antenna! lobe receive inhibitory signals, thesesignals derive essentially from only the local interneurons.

III. Cell lineage-dependent modular structures inthe brain
The brain of Drosophila melanogaster is produced byan average of 85 stem cells (neuroblasts) per hemisphere.
In previous years, we found that the progeny of eachneuroblast project to only a few regions of the adult brain
in a stereotypic manner, forming a lineage-dependentcircuit module called a clonal unit.
In order to map their distribution, we examined theclonal units in more than 800 adult brains and categorized
them. We identified 80 candidate units, among which atleast 30 have so far been confirmed to be unique. Further
characterization and categorization are under way.

IV. Cell proliferation in the adult brain
Recently, proliferation of neural stem cells in the adultbrain was reported in mammals as well as in various
insects. To see whether this is also the case in Drosophila,
we observed the cell proliferation in the adult brain
throughout their lifetime (from zero to 50 days after
eclosion). Though we found no systematic proliferation of

25

FOR BASIC BIOLOGY

neuroblasts in old brains, we identified a small number ofcells around the antenna! nerve that proliferate shortly
after eclosion. Immunohistochemistry with a glial cellmarker, REPO, and a neuron marker, ELAV, suggests thatthe proliferating cells are glia. Their progeny becomes
either glial cells or the cells of unknown type that is both
REPO negative and ELAV negative.

V. Development of identified brain structures
The neural circuits of the adult brain are formed during
the larval and pupal stages. Few is known about when andhow neurons establish their complicated morphology
during pupal metamorphosis . To address this problem , weare tracing the structural change of identified neurons inpupae. An important model circuit for this study is thebrain regions called the central complex, whose elaborate
structure in the adult brain has long been the scope ofanatomical and functional studies. By comparing theimage database of the larval and adult brain, we screened
for the strains that label putative central complex structure
already in larvae. The resulting strains are under detailed
investigation to reveal the time course of the centralcomplex composition.

Publication List:
Hayashi, S., Ito, K., Sado, Y., Taniguchi , M., Akimoto, A. ,Takeuchi , H. , Aigald , T. , Matsuzaki , F. , Nakagoshi , H.,
Tanimura , T ., Ueda, R. , Uemura , T ., Yoshihara, M., andGoto, S. (2002). GETDB, a database compiling expression
patterns and molecular locations of a collection of Ga14
enhancer traps. Genesis 34, 58-61.
Kido, A., and Ito, K. (2002). Mushroom bodies are notrequired for courtship behavior by normal and sexuallymosaic Drosophila . J Neurobiol52 , 302-11 .
Kurusu , M., Awasaki, T ., Masuda-Nakagawa, L. M.,
Kawauchi , H. , Ito, K., and Furukubo-Tokunaga, K. (2002).
Embryonic and larval development of the Drosophila
mushroom bodies: concentric layer subdivisions and therole of fasciclin II. Development 129 , 409-19.
Saito, M, Awasaki, T., and Hama, C. (2002). Genetic
analyses of essential genes in cytological region 6101-2 to
61Fl-2 of Drosophila melanogaster. Mol Genet Genomics
268: 446-54

VI. Mechanisms underlying the remodeling ofneural circuits
Little is known about the genetic mechanisms that
regulate the remodeling of neural circuits during
development and maturation of the brain. To address thisquestion, we analyze the larval mushroom body (MB),whose neurons dynamically change the pattern of axon
branches during metamorphosis . This process consists of
two phases: the degeneration or retraction of existing
axon branches, and the re-extension and re-forming of
new axon branches (Figure lA). A single cell labeling of
MB neurons revealed that, following the elimination ofsynapses on the axon branch , axon fibers break anddegenerate from proximal to distal subregions of the
branches . The synapses lose expression of Fasciclin II(Fasll) prior to their elimination (Figure lB), and theectopic expression of Fasll in the larval MB suppresses
the elimination of synapses. Ectopic expression of
dominant negative form of Rae , on the other hand,
suppresses the degeneration of axon fibers without
affecting the synapse elimination. These indicate that thedegradation of Fasll in the synapses causes theelimination of synapse, whereas the activation of Rae
causes the degeneration of axon fibers.

IV. Contribution to the science community
As a joint venture with German and US researchgroups , we maintain Flybrain, a web-based image
database of the Drosophila nervous system (http:/!flybrain.nibb.ac.jp). Another database maintained here,
mainly for Japanese-speaking fly researchers , is Jfly
(http://jfly.nibb.ac.jp). Archives of research-related
discussions, experimental protocols , images and movies
are provided.

8

GFP

~

Fasll

~

GFP + Fasll

~~

... .

Figure I. A: Remodeling of mushroom body neurons
during early pupal stages . The neurons are labeled by the
induced expression of GFP. The vertical and medial
branches are pruned during the first 24 hours of the pupal
stage (arrows in the second and third panels.) After that, thefibres re-extend only to the medial direction (arrow in the
fourth panel.) B: Close-up of the eliminating synapses at
the tip uf the vertical lobe (white rectangle in the second
panel of A.) Arrows show the regions of branches where
Fasll (magenta, labeled with anti-Fasll antibody) disappear
before the elimination of synapses of the labeled neurons(green, GFP).

26

__ _j

NA.TIQNAL tNSTI'l"UTE

i_ _

27

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairperson: NODA, Masaharu

DIVISION OF MOLECULAR NEUROBIOLOGYDIVISION OF CELLULAR REGULATIONDIVISION OF BIOLOGICAL REGULATION
AND PHOTOBIOLOGY (ADmNCT)DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADruNCT)The department is composed of two regular divisions and two adjunctdivisions.

The study of this department is focused on molecularmechanisms for the development of central nervous systems invertebrates, and also on molecular mechanisms for the response of plants
toward external environments, such as light, temperature and salinity.

28

NATIONAL INSTITUTEDIVISION OF MOLECULAR NEUROBIOLOGY
Professor:
Research Associates:

NODA, Masaharu
SHINTANI, Takafumi
SAKUTA, Hiraki
HIYAMA, Takeshi (Nov. '02 -)Technical Staff:
TAKEUCHI,Yasushi
NIBB Research Fellow:
FUKADA, Masahide
JSPS Postdoctoral Fellow: SUZUKI, Ryoko
Postdoctoral Fellows:
FUJIKAWA, Akihiron
KATO, Akiran
NAKAMURA, Takahiron
HIYAMA, Takeshi (-Oct. '02)
Graduate Students:
TAMURA, Hiroshi
OHKAWARA, Takeshi
TAKAHASHI,Hiroo
IHARA, Masaru
TORIUM/, Shigeru
YUASA, JunichPJ
Visiting Scientists:
WATAKABE, lkuko
YAMAMOTO, Shoichf! (-Apr. '02)
Secretary:
KODAMA, Akiko
Technical Assistant:
AYABE, Yuko
JSTTechnical Staff:
MIZOGUCHI, Masae
GOTO, Megumi
YAMADA, Kaoru
MATSUI,Mien CREST, JST. 2! PREST, JST!from SHIONOGI & Co. LTD.

3

We have been studying the molecular and cellularmechanisms underlying the development of the vertebrate
central nervous system. It covers all the major events
including the patterning and growth of the nervous system,neuronal determination, axonal navigation and targeting,
synapse formation and plasticity, and neuronal regeneration, especially in the visual system. The scope of ourinterests also encompasses various functions of the matured brain including sensation, behavior, learning andmemory.

I. Molecular mechanism of neural development(Regional specification in the retina and topographic retinotectal projection)
Topographic maps are a fundamental feature of neuralnetworks in the nervous system. Understanding themolecular mechanisms by which topographically ordered
neuronal connections are established during development
has long been a major challenge in developmental neurobiology. The retinotectal projection of lower vertebrates
including birds has been used as a readily accessiblemodel system. In this projection, the temporal (posterior)retina is connected to the rostral (anterior) part of thecontralateral optic tectum, the nasal (anterior) retina to thecaudal (posterior) tectum, and likewise the dorsal andventral 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. We

identified 33 molecules along the nasotemporal axis and
20 molecules along the dorsoventral axis, with various
asymmetrical expression patterns in the developing retina.
We elucidated the primary structures of all these eDNA
clones and examined their expression patterns during
development. These included many novel molecules
together with the known molecules: transcription factors,
receptor proteins, secretory factors, intracellular proteins,and soon.
We first identified two winged-helix transcriptional
regulators, CBF-1 and CBF-2, expressed in the nasal andtemporal retina, respectively. Misexpression experiments
using a retroviral vector showed that these two transcription factors determine the regional specificity of theretinal ganglion cells, namely, the directed axonal projections to the appropriate tectal targets along the anteroposterior 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 a ventral
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 is also detected. Ventroptin thus shows a doublegradient expression profile along · the dorsoventral andnasotemporal axes. Misexpression of Ventroptin alteredexpression patterns of several topographic genes: BMP-4,
Tbx5 and cVax along the dorsoventral axis and ephrin-A2
along the nasotemporal axis. Consistently, in theseembryos, projection of the retinal ganglion cell axons to
the tectum was also changed along the both axes. The
topographic retinotectal projection along the dorsoventral
and anteroposterior axes thus appears to be controlled not
independently but in a highly concerted manner byVentroptin.
To understand the molecular mechanism underlying thespecificity of precise neuronal connections, detailed information on the behavior of developing axons during
their pathway selection and target recognition is essential.
For such studies, several genetic approaches to thelaveling and visualizing of neural projections in miceusing transgenic or knock-in techniques have been reported. We employed GAP-lacZ as an axon-targetedreporter protein constructed by fusing the membraneanchoring domain of the GAP-43 protein to lacZ. The
reporter gene was introduced into the genome under thecontrol of a promoter element of Brn3b transcription factor to establish transgenic mouse lines. The individual
lines thus generated afforded clear images of specificaxonal pathways of the visual (Fig. 1), vomeronasal,
pontocerebellar, and auditory systems. The reporter protein labelled the entire axonal process as well as the cellbody of developing and mature neurons on staining with
X-gal. We showed that these lines facilitate the developmental and anatomical study of these neural systems.
Moreover, this strategy must be applicable to a variety ofneural systems by using various specific promoter

29

FOR BASIC BIOLOGYA(Line 11)B

(Line 4)

Fig .l . (A) The axonal pathway of dorsal retinal ganglion
cells (RGCs) visualized in mouse line 11. The axons projectto lateral regions of the superior colliculus (SC) after
crossing at the chiasma. (B) The axonal pathway of ventral
RGCs visualized in mouse Line 4. The axons project to themedial SC.

elements.
Currently, with respect to the identified topographic
molecules, we are conducting misexpression experiments
using chick embryos and generating knock-out and transgenic mice to elucidate the molecular functions. We
expect that our studies will lead to dissection of the molecular mechanism underlying the retinal patterning andtopographic retinotectal projection, and ultimately to uncovering the basic principles for establishing complicatedbut extremely precise neural networks .

II. Physiological roles of protein
phosphatase receptor type Z (Ptprz)

tyrosine

Protein tyrosine phosphorylation plays crucial roles invarious aspects of brain development and brain functions.
In 1994, we found that Ptprz/PTPljRPTP~ , a nervous
system-rich RPTP, is expressed as a chondroitin sulfate
proteoglycan in the brain. The extracellular region of
PTPs consists of a carbonic anhydrase-like domain , afibronectin-type III-like domain and a serine-glycine-richregion , which is considered to be the chondroitin sulfate
attachment region. There exist three splice variants of thismolecule: A full-length transmembrane form (PTPs-A) ; a
short transmembrane form (PTPs-B); and a soluble form
(PTPs-S)
which
is
also
known
as
6B4
proteoglycan/phosphacan. PTPs is expressed from theearly developmental stage to the adulthood in neurons aswell as astrocytes. This suggests that this gene plays
variegated roles in the brain development and brain
function.
We found in 1996 that PTPs binds pleiotrophin/HBGAM and midkine, closely related heparin-binding
growth factors which share many biological activities.
The chondroitin sulfate portion of PTPs is essential forthe high affinity binding (Kd = ~0.25 nM) to these growth
factors, and removal of chondroitin sulfate chains results
in a marked decrease of binding affinity (Kd = ~13 nM).
We further revealed that chondroitin sulfate interacts with
Arg 78 of midkine. Pleiotrophin and midkine on thesubstratum stimulated migration of neurons in the glass
fiber assay and Boyden chamber cell-migration assay.
Experiments using various midkine mutants with various
affinities for PTPs indicated that the strength of binding
affinities and the neuronal migration-inducing activities
are highly correlated. These results suggest that PTPs isinvolved in migration as a neuronal receptor forpleiotrophin and midkine.
To identify the substrate molecules of PTPs, we haverecently developed the yeast substrate-trapping system.
This system is based on the yeast two-hybrid system withtwo essential modifications: Conditional expression of vsrc to tyrosine-phosphorylate the prey proteins andscreening using a substrate-trap mutant of PTPs as bait.
Using this system , we successfully isolated a number ofcandidate clones for substrate molecules or interacting
molecules. We found that PTPs interacts with PSD95/SAP90 family members, SAP102 , PSD-95/SAP90 and
SAP97 /hDlg , which are concentrated in the central
synapses mediating protein-protein interactions to form
large synaptic macromolecular complexes. Here, the Cterminus of PTPs binds to PSD-95/SAP90 proteinsthrough the second PDZ domain. This suggests that PTPs
is involved in the regulation of synaptic function .
Furthermore, we identified GITl/Cat-1 as a PTPs
substrate. It is known that Cat-1 regulates Pak , a serine
threonine kinase which serves as a target for the small
GTP-binding proteins, Cdc42 and Rae, and is implicatedin a wide range of cellular events including the celladhesion and cell morphological change. Pleiotrophin,
PTPs and GITl/Cat-1 might regulate the neuronalmigration and neurite extension by controlling the Pak
signaling pathway. We are continuing efforts tocharacterize the other candidate clones.
In addition , to study the physiological roles of PTPs invivo , we generated PTPs-deficient mice in which the
PTPs gene was replaced with the LacZ gene in 1997. We
are cun·ently studying the phenotype of PTPs-deficient
mice biochemically , anatomically , physiologically andethologically, and have already found abnormalities inbehavior, learning and memory, etc. Very recently , wefound that PTPs is expressed in gastric epithelial cells andfunctions as the receptor for VacA toxin secreted from

30

NATIONAL INSTITUTE

Helicobactor pylori. Surprisingly, PTPs-deficient miceare resistant to VacA and do not thereby develop gastriculcers, in contrast to the wild-type mice.

III. Physiological roles of N ax sodium channel
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 the circumventricular organs and Na,-deficient mice ingest salt in excess.
In Na,-deficient mice , c-fos expression in the subfomicalorgan (SFO) and vascular organ of the lamina terminalis(VOLT) was markedly elevated as compared with wildtype mice. Stimulation of the SFONOLT 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 suchaversion .

A

Control

170 mM+I+

extracellular sodium concentration (Fig. 2). In contrast,
these
responses
were
notobservedinN ~­immunonegative cells or neurons of Na,-deficient mutant
origin. Transfection of Na, eDNA conferred the sodium
sensitivity on Na,-deficient cells. All of the GABAimmunopositive neurons isolated from the SFO responded
to the extracellular sodium increase. Based on thesefindings, we propose that GABAergic inhibitory neurons
expressing Na, control the activity of the SFO and suppress the salt-intake behavior of animals under thirst conditions.
In addition, we examined the localization of N~
throughout the visceral organs at the cellular level. In
visceral organs including lung, heart , intestine, bladder,kidney and tongue, a subset of Schwann cells within the
peripheral nerve trunks were highly positive for N~ . An
electron microscopic study indicated that these N ~ ­positive cells were non-myelinating Schwann cells. In thelung, N~ -po si tive signals were also observed in thealveolar type II cells, which actively absorb sodium andwater to aid gas exchange through the alveolar surface . It
was thus suggested that the N ~ sodium channel is involved in controlling the local extracellular sodium level
through sodium absorption activity .

Publication List:

-/-

10

20

30 (mM)

c

3.5
3.0

-2.5
c:
!_2.0

.s 1.5~ll:: 1.0

0.5
0

-0.5
120 140 160 180 200 220
C(mM)

Fig. 2. Sodium-concentration sensitivity is lost in SFO neurons in the Na,-null mutants. (A) Pseudocolor images
showing the [Na+]; of the cells in the control and high sodium solutions. Scale bar, 50 ~-tm . (B) The [Na+]; response isdependent on [Na+] but not on extracellular [Cl"Jo or
osmotic pressure . Na, is TTX resistant. (C) Relationshipbetween the [Na+]; increase rate (R) and [Na+] RM., = 3.04
mM/min , c l/2 = 157 mM , and a = 4.67 mM .
0 ,

0 •

We showed that N~ is a sodium channel which is sensitive to the increase of extracellular sodium level : Entryof sodium ions occurred in the N ~ - immunopositive neurons from wild-type mice , in response to a rise of the

Hiyama, T.Y., Watanabe , E ., Ono , K. , Inenaga, K., Tamkun ,M.M ., Yoshida, S. and Noda , M . (2002) N~ channel
involved in CNS sodium-level sensing. Nature New·osci .,5 , 511-512.
Watanabe , E., Hiyama, T .Y., Kodama, R. and Noda, M.
(2002) Na, sodium channel is expressed non-myelinating
Schwann cells and alveolar type II cells in mice. Neurosci.
Lett. , 330, 109-11 3.
Zubair, M., Watanabe , E ., Fukada, M. and Noda, M. (2002)
Genetic labelling of specific axonal pathways in themouse central nervous system. Eur, J . Neurosci., 15, 807814.
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
Tanaka, M., Maeda, N ., Noda, M. and Marunouchi, T . (2003)
A chondroitin sulfate proteoglycan PTPS/RPTP~ regulates
the morphogenesis of Purkinje cell dendrites in thedeveloping cerebellum. J. Neurosci. (in press)
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. (in press)

31

FOR BASIC BIOLOGYDIVISION OF CELLULAR REGULATION
Professor:
Associate Professor:
Research Associates:

MURATA, Norio
MIKAMI, Koji
SUZUKI, !wane
YAMAMOTO, Hiroshi
IINUMA, Hideko
Technical Staffs:
YAMAGUCHI, Katsushi
Monka-sho Foreign Scientist:
MUSTARDY, Ldzsl6 n
NIBB Research Fellow:
KANESAKI, Yu
JSPS Research Fellow:
SHIVAJ/, S 21
JSPS Post-Doc Fellows: SULPICE, Ronan 31
JOGADHENU, S. S. Prakash 21
OHNISH/, Norikazu
Postdoctral Fellows:
TAKAHASHI, Shunichi
KANASEKI, Toku
ALLAKHVERDIEV,
Suleyman I. 41
INABA, Masami 51
Visiting Scientists:
LOS, Dmitry A. 61
SERGEYENKO, Tatiana 61
LYUKEVICH, Alexander A. 61
SHAPIGUZOV Alexey 61
SZALONTAI, Balazs n
PANICHIKIN Vladimir 71
KWON, Ohoak 81
HUBSHMANN, Thomas 91
ALLAHVERDIYEVA, Yagut 101
HALL, John J. 111
PAITHOONRANGSARID,
Kalayanee 121
DULAI, Sandor, 11
CHINTALAPAT/, Suresh
Kumar 21
OKADA,Akiko
Technical Assistants:
KOIKE, Yukari
KAIDA, Satomi
ODA,Keiko
Secretaries:
KUBOKI, Yuko
KAWAI, Masayo
ODA, Yuko
Graduate Students:
FERJANI, Ali
SUZUKI, Shingo!Jrom the Biological Research Center, Szeged, Hungary!jrom the Centre for Cellular andMolecularBiology,Hyderabad, India
3 from Universite de Rennes 1, Rennes, France
4 from the Institute of Basic Biological Problems,
Pushchino, Russia
5 from Ehime University, Matsuyama, Japan
6 from the Institute of Plant Physiology, Moscow, Russia
7 from Moscow State University, Moscow, Russia
8 from Korea Basic Science Institute, Taejeon, Korea
9Y,.om Humboldt Universitiit Berlin, Berlin, Germany
10!jrom Institute of Botany, Baku, Azerbaijan
11 !jrom University of Durham, Durham, England
12 from National Center for Genetic Engineering andBiotechnology, Bangkok, Thailand
1

plants so that they gain the ability to acclimate to and tolerate such stress conditions. In 2002, significant progress
was made in the following areas using cyanobacteria as amodel system.

I. Membrane rigidiflcation enhances the cold
inducibility of gene expression
Our previous experiments have indicated that the expression of the cold-inducible desA gene in Synechocystis
sp. PCC 6803 (hereafter Synechocystis), which encodes theM2 fatty acid desaturase, is induced by catalytic hydrogenation of fatty acids in plasma-membrane lipids. Thus,
we hypothesized that changes in membrane fluidity/rigidity
are primary signal of cold stress and are perceived by acold sensor that is localized at plasma membranes. To
evaluate this hypothesis, we examined the effect of genetic
rigidification of membrane lipids on the cold inducibilityof gene expression using DNA microarrays.
We first inactivated the desA and desD genes for M2
and ,i6 desaturases, respectively, in Synechocystis by targeted mutagenesis. The resultant desA"/desD· cells contained only mono-unsaturated lipid molecules, whereas
wild-type cells contained poly-unsaturated lipid molecules.
DNA microarray analysis demonstrated that in desA"/desD·mutant cells a large number of genes were induced by cold
than in wild-type cells. Fourier transform infrared (FTIR)
spectroscopy demonstrated that the replacement of polyunsaturated membrane lipids by mono-unsaturated ones
rigidified the membrane lipids. Thus, we conclude that therigidification of membrane lipids enhances the coldinducibility of gene expression.
By contrast, the heat inducibility of gene expression was
unaffected by mutation of the desA and desD genes, indicating that heat inducibility of gene expression is not triggered by changes in membrane rigidification. These findings suggest that the change in membrane fluidity/rigidity
is involved in the mechanism of cold perception but not ofheat perception in Synechocystis cells. [Inaba et al. (2003)
J. Biol. Chern., in press].

2

The major thrust of the research efforts of this division
is directed towards understanding the signal transduction
mechanisms operating in plants and microorganisms withrespect to perception of various kinds of stress that arise
due to changes in environmental conditions, with particularemphasis on extreme temperatures and salinity. In addition,techniques are being developed for transformation of

II. Multi-stress sensor Hik33 in Synechocystis
An increase in osmotic pressure leads to a decrease incell volume (plasmolysis) by efflux of water from the cell.
To recover from the plasmolysis, cells perceive a signal of
hyperosmotic stress to maintain physiological cellularosmolarity via the expression of osmostress-inducible
genes. Screening of Synechocystis mutant library ofhistidine kinases by DNA microarray technique identified a
histidine kinase, Hik33, as an osmosensor. Our previous
results have demonstrated that Hik33 is a cold sensor in
Synechocystis.
DNA microarrays were employed to examine whether
Hik33 perceives cold and osmostress differently. The
results indicated that Hik33 regulates the expression of alarge number of osmostress-inducible genes, although 66%
of Hik33-regulated genes encoded proteins of unknown
function. Hik33-regulated genes are involved in the
structural maintenance of cell wall and membranes and theregulation of phosphate transport, photosynthesis, signal

32

NATIONAL INSTITUTEtransduction, gene expression , and folding and turnover ofproteins (Fig. 1). Although Hik33 regulates the coldinducible expression of genes which are involved in theregulation of photosynthesis and gene expression, it isclear that Hik33 regulates distinct sets of genes underosmostress and cold conditions with the exception of asmall number of genes whose expression was induced byboth osmostress and cold (Fig. 1). Therefore, we conclude
that Hik33 senses cold and osmostress differently toregulate the expression of distinct sets of genes in a stressspecific manner [Mikami et al. (2002) Mol. Microbial. 46,
905-915].
Recently , a histidine kinase , NblS, which is a putativehomologue of Hik33 in Synechococcus elegatus PCC 7942,
was identified as a sensor of strong light and nutrient stress.
Moreover, Hik33 was originally identified as DspA, achemical sensor of drugs such as inhibitors ofphotosynthesis. These results suggest that Hik33 mightalso recognize strong light, nutrient stress and chemicals.
Confrrmation of this possibility is in progress with DNA
microarray technique .

fabG
hypA
infC
rpsl
groEL
dnaJ
htrA
ps/C
sod8
ycf21
hik31

ndkR
corR
rp/28
cyaA
groES
groEL-2 dnaK
htpG
c/p8
ndh03 psiS
pstA
ps/8
rim/
/y/8
trpS
gifA
hik34 psbW

Fig. l. Osmostress-inducible and cold-inducible genesthat are regulated by Hik33. Large and small circles
enclose genes whose expression is induced by osmostressand cold, respectively . Rectangles in these circles enclose
genes whose expression is regulated to a greater or lesser
extent by Hik33 in cells under osmostress and under cold.
Genes outside rectangles appeared to be not regulated by
Hik33 in terms of their responses to the respective
stresses. The rectangle in the overlapping region of thetwo circles encloses genes whose Hik33-regulated
expression was observed under both kinds of stress.

III. Glucosylglycerol, a compatible solute, sustains
cell division under salt stress.
When the organisms are exposed to a sudden increase in
the external concentration of salts, they accumulate compatible solutes, such as trehalose and glycinebetaine , as aresult of synthesis de novo or uptake from the environment.
Synechocystis cells accumulate glucosylglycerol (GG) andsucrose as the compatible solutes under salt stress. While
the molecular mechanism for GG biosynthesis including
regulation of the expression of the ggpS gene, which
encodes glucosylglycerol phosphate synthase, has been
intensively investigated, the role of GG in protection
against salt stress remains poorly understood. We studied

the role of GG in the tolerance to salt stress, and found thatsalt stress inhibited cell division and significantly increased
cell size in t:.ggpS mutant cells [Ferjani et al., (2003) Plant
Physiol. in press; Fig. 2]. Electron microscopy revealed
that, in t:.ggpS cells, separation of daughter cells wasincomplete and aborted division could be recognized by
the presence of a structure that resembled a division ring
(Fig . 2). Addition of GG to the culture medium protected
t:.ggpS cells against salt stress and reversed the adverse
effects of NaCl on cell division and cell size. These observations suggest that GG is important for salt tolerance andthus for the proper division of cells under salt stress conditions.

Fig. 2. Effects of 450 rnM NaCl on the ultrastructure ofwild-type (WT) and t:.ggpS cells of Synechocystis, as
examined by electron microscopy . The cells that had beengrown with 20 rnM NaCl were cultured in the presence of
20 rnM or 450 rnM NaCl for three days. (A) WT cells in
20 mM NaCI; (B) WT cells in 450 rnM NaCI; (C) t:.ggpS
cells in 20 rnM NaCl; (D) D.ggpS cells in 450 rnM NaCl.
Arrows in (D) indicate a division ring-like structure at thecell 's equator. Each scale bar represents I !J.m.

IV. Salt stress inhibits the repair of photosystem
IIbysuppressing
theactivities
oftranscriptional and translational activity
Strong light impairs the photosynthetic apparatus, inparticular that of photosystem II (PSII), via a process
known as photodamage or photoinhibition. However,photodamaged PSII can be repaired, and the repair process
involves the replacement of the damaged Dl protein (Dl) ,that is a reaction center protein of PSII , by lightdependently synthesized Dl de novo.
Under natural conditions light and salt stress are major
environmental factors that limit the efficiency of photosynthesis. However, we have found that the effects in vivoof light and salt stress on PSII are completely different in
Synechocystis [Allakhverdiev et al. Plant Physiol. (2002)
130, 1443-1453]. Strong light induced photodamage to
PSII , whereas salt stress inhibited the repair of the photodamaged PSII and did not accelerate damage to PSII
directly. The combination of both stresses inactivated PSII
more rapidly as a consequence of their synergistic effects.

33

FOR BASIC BIOLOGYRadioactive labeling of cells revealed that salt stress inhibited the synthesis of proteins de novo and, in particular, thesynthesis of the Dl. DNA microarray analysis indicatedthat the light-induced expression of various genes was
suppressed by salt stress . These results suggest that salt
stress inhibits the repair of PSTI via suppression of theactivities of the transcription and translational machinery(Fig . 3).

Figure 3. A hypothetical scheme for the inhibition of theDl protein synthesis de novo by salt stress.

List of publication:(1) Original articles
Allakhverdiev, S.I, Nishiyama, Y. , Miyairi, S., Yamamoto ,H., Inagaki, N., Kanesaki , Y. , Murata, N. (2002) Salt
stress inhibits the repair of photodamaged photosystem
IT by suppressing the transcription and translation of
psbA genes in Synechocystis. Plant Physiol., 130 , 14431453 .
Kanesaki, Y., Suzuki , I., Allakhverdiev , S.I., Mikarni, K.,
Murata , N. (2002) Salt stress and hyperosmotic stressregulate the expression of different sets of genes in
Synechocystis sp. PCC 6803. Biochem. Biophys. Res.
Commun ., 290 , 339-348.
Mikami, K ., Kanesaki, Y., Suzuki, I., Murata, N. (2002)
The histidine kinase Hik33 perceives osmotic stress andcold stress in Synechocystis sp. PCC 6803. Mol.
Microbial. , 46, 905-915.
Mohanty, A., Kathuria, H., Ferjani , A., Sakamoto, A.,
Mohanty , P., Murata, N., Tyagi, A.K. (2002)
Transgenics of an elite indica rice variety Pusa Basmati
1 harbouring the codA gene are highly tolerant to salt
stress. Theor. Appl. Genet., 106,51-57.
Simon , W.J. , Hall , J.J. , Suzuki , I. , Murata , N ., Slabas, A.R .
(2002) Proteomic study of the soluble proteins from theunicellular cyanobacterium Synechocystis sp. PCC6803
using
automated
matrix-assisted
laser
desorption/ionization-time of flight peptide mass fingerprinting. Proteomics , 2, 1735-1742.
Yamaguchi, K. , Suzuki, I., Yamamoto , H ., Lyukevich, A. ,Bodrova, I., Los, D.A., Piven, I. , Zinchenko, V .,Kanehisa, M., Murata , N. (2002) A two-component
Mn2+-sensing system negatively regulates expression of
the mntCAB operon in Synechocystis. Plant Cell , 14,2901-2913.

(2) Review articles
Chen , T.H.H., Murata , N. (2002) Enhancement oftolerance of abiotic stress by metabolic engineering ofbetaines and other compatible solutes. Curr. Opin. Plant
Bioi., 5 , 250-257.
Dmitry, D.A., Murata , N. (2002) Sensing and responses to
low temperature in cyanobacteria. In Cells and Molecular Responses to Stress (Storey, K.B. Storey , J.M. ,eds.), vol. 3, Sensing, Signaling and Cell Adaptation.
Elsevier Press, Amsterdam , pp. 139-153
Sakamoto , A., Murata , N. (2002) The role of glycine
betaine in the protection of plants from stress: clues
from transgenic plants . Plant Cell Environ., 25, 163171.
Sakamoto, T. , Murata, N. (2002) Regulation of the desaturation of fatty acids and its role in tolerance to cold andsalt stress. Curr . Opin. Microbial., 5, 208-210.

34

NATIONAL INSTITUTEDIVISION OF BIOLOGICAL REGULATION AND
PHOTOBIOLOGY (ADJUNCT)
Professor (Adjunct):
WADA, Masamitsu
Associate Professor(Adjunct):
KIYOSUE , Tomohiro
Research Associate:
KIKUCHI, Kazuhiro
NIBB Research Fellow:
KASAHARA, Masahiro
JSPS Postdoctral Fellow: CHRISTENSEN, Steen
PRESTO Fellow:
KAGAWA, Takatoshi
BRAIN Postdoctoral Fellow:
OGURA, Hironobu
MEXT Postdoctoral Fellow:
TAKAHASHI, Fumio
OIKAWA, 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, such as
cryptochrome and phototropin (phot), are the main
photoreceptors for plant photomorphogenesis. The goal of
our research is to elucidate the photoperception and thesignal transduction pathways of photomorphogenesis.

I. Chloroplast relocation movement
One of our major subjects is chloroplast photo-relocation
movement, which is thought to be one of the simplest
phenomena in this field. We use the fern Adiantum capillusveneris and the moss Physcomitrella patens as model plants
for our cell biological approach since the gametophytes arevery sensitive to light and the organization of the cells is very
simple. We also use Arabidopsis mutants to identify thegenes regulating chloroplast photo-relocation movement.

1-1

Arabidopsis

Chloroplasts accumulate at the cell surface under weak
light and escape from the cell surface to the anticlinal wallunder strong light to optimize photosynthesis. The
significance of chloroplast avoidance movement, however,has been uncertain. We analyzed the influence of strong
light using several mutants which are defective in
avoidance movement in Arabidopsis. One of the mutants
is phot2, defective in the blue-light photoreceptor
phototropin.
The other mutant is chupi which ishypothesized to lack some part of the movement
mechanism. Both mutants showed severe damage ofleaves when irradiated with strong light for more than 10
hr. The reactive oxygene-scavenging capacity in thesemutant leaves, especially the activities of scavenging
enzymes, did not differ between mutant and wild-type
plants. This suggests that the chloroplast avoidance
response is a very important physiological response inplants under high light conditions.

1- 2 Adiantum phy3
Adiantum phytochrome3 (PHY3) is a unique chimeric

protein with a phytochrome structure in the N-terminal
half and a phototropin structure in the C-terminal half.
PHY3 gene analysis of EMS-induced rap (red lightinduced aphototropic) mutants of Adiantum andcomplimentation testing
revealed that phy3 is thephotoreceptor of red light-induced phototropism andchloroplast photorelocation movement. Phy3 also
mediates these phenomena in Adiantum sporophytes aswell as gametophytes. Phylogenie analyses of PHYJ
genes in various ferns suggest that this gene is only foundin advanced ferns and is not present in primitive ferns.
Enhancement of the photosensitivity of ferns, likely an
additional function of phy3, may have contributed to fern
evolution and differentiation.
Phy3 has not yet been shown to function as a blue light
photoreceptor.

1- 3

Adiantum phot2

Adiantum mutants which do not show chloroplast
avoidance movement under strong blue light have been
isolated. Gene analysis of these mutants andcomplimentation tests with PHOT2 gene reveal that phot2
is a photoreceptor of blue light-induced avoidance
movement of chloroplasts in Adiantum, as in the case of
Arabidopsis. Particle bombardment of modified PHOT2
genes into phot2 mutants show that LOV2 domain ofphot2 functions as a chromophore binding domain but
LOVl domain does not. Gene deletions of PHOT2 from
3' region also show that an important domain of phot2
exists within the C-terminus.

II. Gene targeting and gene silencing
In order to elucidate the role of genes in Adiantum andrice whose function is unknown, we have tried toestablish new methods for gene targeting in theseorganisms.

2- 1 Miniature transposable element
Miniature inverted-repeat transposable elements (MITE)
have been found in wide range of organisms but active
MITEs have not been identified. We found a new class of
MITEs in rice and named them miniature Ping (mPing).
mPing elements are activated in cells derived from antherculture and excise efficiently from original sites to reinsert
into new loci. mPing-associated Ping element which has aputative transposase sequence was also found and shown totranspose within the rice genome.

List of publication:
Original articles
Imaizumi, T., Kadota, A., Hasebe, M., Wada, M. (2002)
Cryptochrome light signals control development tosuppress auxin sensitivity in the moss Physcomitrella
patens. Plant Cel/14, 373-386.
Kasahara, M., Swartz, T. E., Olney, M. A., Onodera, A.,
Mochizuki, N., Fukuzawa, H., Asamizu, E., Tabata, S.,

35

FOR BASIC BIOLOGY

Kanegae, H., Takano, M., Christie , J. M. , Nagatani , A. ,Briggs , W . R. (2002) Photochemical properties of theflavinmononucleotide-binding
domainsofthephototropins from Arabidopsis , rice, and Chlamydomonas
reinhardtii . Plant Physiol . 129 , 762-773.
Kasahara , M ., Kagawa, T. , Oikawa, K., Suetsugu, N., Miyao ,M., Wada, M. (2002) Chloroplast avoidance movement
reduces photodamage in plants . Nature 420 , 829-832 .
Kikuchi, K., Terauchi , K. , Wada, M., Hirano , H. (2003) The
plant MITE mPing is mobilized in anther culture. Nature
421 , 167-170.
Kawai , H. , Kanegae , T. , Christensen , S., Kiyosue , T. , Sato ,Y. , Imaizumi , T., Kadota, A ., Wada, M. (2003) Responsesof ferns to red light are mediated by an unconventional
photoreceptor. Nature 421, 287-290.
Sato, Y., Wada, M., Kadota, A . (2003) Accumulation response of chloroplasts induced by mechanical stimulation

WT

in bryophyte cells. Planta. (in press)

Review articles
Kagawa, T ., Wada, M . (2002) Blue light-induced chloroplast
relocation. Plant Cell Physiol . 43, 367-371.
Kagawa, T . (2003) The phototropin family as photoreceptors
for blue light-induced chloroplast relocation . J. Plant Res .
(in press)
Suetsugu , N ., Wada, M. (2003) Cryptogam blue-light photoreceptors. Current Opinion of Plant Biology 6, 91-96 .
Sato, Y., Kadota, A. , Wada, M . (2003) Chloroplast movement: dissection of events downstream of photo- andmechano-perception. J. Plant Res. (in press)
Wada, M., Kagawa, T., Sato , Y. (2003) Chloroplast movement. Annu. Rev. Plant Bioi. (in press)

phot2-1 phot1-5 chup1-2

0 hr

10 hr

22 hr

31 hr

Figure . Phenotypes of plants exposed to continuous strong light. Wild-type, phot2-l , photl-5 and chupl-2 mutant plants (4weeks old) were irradiated with white light at 1400 ~mol m·2 s· 1 and were photographed before (0 hour) and after irradiationfor 10, 22 and 31 hours. Leaves of phot2-l and chup 1-2 plants showed some signs of bleaching after 10 hours of treatment but
were severely bleached and necrotic after 22 hours. In contrast, wild-type and photl-5 plants withstood this light stress overthe 31 hours time period of the treatment.

36

NATIONAL INSTITUTEDIVISION OF BEHAVIOR AND NEUROBIOLOGY(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Postdoctoral Fellow:

MURAKAMI, Fujio
NAKAFUKU, Masato
TAMADA, Atsushi
HANAMURA, Kenji n
TANIGUCHI, Hiraki 41
HATANAKA, Yumiko 41
KUMADA, Tatsuro 41

The vertebrate nervous system contains a large numberof neurons that are connected to each other by specificaxonal projections. We are interested in how the complex but precise neuronal network, which is indispensable
for functioning of the nervous system, is constructedduring development. The goal of our research in thisdivision is to elucidate the cellular and molecular mechanisms underlying the neuronal network formation in vertebrates, with current focuses on the mechanisms of axon
guidance and neuronal migration.

I. Mechanism of Axonal Guidance
During development of the nervous system, growing
axons are guided through specific pathways to correct
targets. Our research interest focuses on the cellular andmolecular mechanisms of axon guidance in the vertebrate
nervous system. We are particularly inter-ested in how
guidance mechanisms operating in differ-ent phases ofaxonal growth are assembled to generate the complex but
precise axonal wiring patterns in the brain. To addressthese issues, we are first trying to understand axonal wiring patterns during development with reference to thestructural organiza-tion of the brain. Using wholemounted preparations of embry-onic rat brains, we will
reveal whole axonal trajectories by labeling with neuronaltracers, such as Dil, or by immunohistochemical staining
with antibodies against molecular markers expressed in a
subset of neurons. We will next explore the axonal
guidance mechanisms generating these wiring patterns.
Following questions will be addressed. 1) What structures along the path-way have key roles in axonal guidance? 2) What guidance cues exist in these structures?
Are these attractive cues or repulsive cues? Are theseshort-range cues or long-range cues? 3) What molecules
are responsible for these cues? 4) How do multiple
guid-ance mechanisms work in concert to generate specific wiring patterns? To answer these questions, we use
neuroanatomical techniques, in vitro culture techniques
including dissociated cell, explant and whole-mount cultures, and biochemical and molecular biological techniques.

II. Mechanism of Neuronal Migration
A variety of neurons migrate from their birthplace tothe position where they finally settle. Neuronal migration in the vertebrate central nervous system occurs bothalong the radial axis and along the tangential axis of theneural tube. Another interest of our research focuses on
the cellular and molecular mechanisms un-derlying theradial and tangential neuronal migration. In many regions of the central nervous system, neurons migrate
radially from the ventricular zone where they are born
toward the pial surface. We are investigating the mechanisms of radial migration, using neurons in the neocortex
as a model system. In addition to the radial migration,some neurons in the brain, such as interneurons in theforebrain, move tangentially for a long distance. We arealso investigating how tangentially migrating neurons are
guided for a long distance through specific routes to thefinal positions, using neurons that migrate from therhombic lip at the dorsal rim of the hindbrain, such ascerebellar granule cells and precerebellar neurons.
To address these issues, we developed in vitro culture
systems that reconstruct migratory events occurring invivo. We labeled migrating neurons with green fluorescent protein (GFP) by transplantation of small piece of
explant taken from a transgenic rat expressing GFP or by
introducing GFP eDNA into limited regions of the brain
by electroporation. Slices of the brain or flat-mounted
brain preparation are then cultured on perme-able membrane filters. These culture systems enable us to analyze
migration pattern of neurons in real time (Fig. 1). Using
these in vitro culture systems, we are currently investigating the cellular and molecular mechanisms of neuronalmigration.

Publication List:
Hatanaka, Y. and Murakami, F. (2002) In vitro apalysis of
the origin, migratory behavior, and maturation of corticalpyramidal cells. J. Comp. Neural. 454, 1-14.
Saito, Y., Katsumaru, H., Wilson, C. J. and Murakami, F.
(2001) Light and electron microscopic study ofcorticorubral synapses in adult cat: evidence for extensive
synaptic remodeling during postnatal development. J.
Comp. Neural. 440,236-44.
Takemoto, M., Fukuda, T., Sonoda, R., Murakami, F.,
Tanaka, H. and Yamamoto, N. (2002) Ephrin-B3-EphA4of specificinteractions
regulatethegrowththalamocortical axon populations in vitro. Eur. J. Neurosci.
16, 1168-72.

Taniguchi, H., Tamada, A., Kennedy, T. E. and Murakami, F.
(2002) Crossing the ventral midline causes neurons tochange their response to floor plate and alar plate attractive cues during transmedian migration. Dev. Biol. 249,
321-32.
Yamamoto, N., Tamada, A. and Murakami, F. Wiring of thebrain by a range of guidance cues. Prog. Neurobiol. (inpress)

37

FOR BASIC BIOLOGY

Figure l. Migrating neocortical neurons visualized with green fluorescent protein (GFP) in vitro.
(Left) Slice culture of rat neocortex that was introduced with GFP eDNA into the ventricular zone by electroporation. A broken
line shows the outer margin of the slice. GFP-positive neurons were moving radially from the ventricular zone (bottom) toward
the pial surface (top).
(Right) High power view of labeled migrating neurons.

Leading processes of neurons extended toward the pial surface.

38

NATIONAL INSTITUTE39

FOR BASIC BIOLOGYLABORATORY OF GENE EXPRESSION AND REGULATION

Chairperson: IIDA, Shigeru

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.

40

NATIONAL INSTITUTEDIVISION OF GENE EXPRESSION AND
REGULATION I
Professor:
Research Associates:

IIDA, Shigeru
TERADA,Rie
HOSHINO , Atsushi
TSUGANE, Kazuo
Technical Staffs:
FURUKAWA , Kazuhiko
TANAKA-FUKADA, Sachiko
NIBB Research Fellow:
CHOI, Jeong-Doo
JSPS Postdoctoral Fellow : PARK, Kyeung-ll
Postdoctoral Fellows:
URAWA, Hiroko*
JOHZUKA-HISATOMI Yasuyo**
ISHIKAWA , Naoko
Visiting Scientist:
CHAREERAT Mongkolsiriwatan'!
Graduate Student:
OHNISHI, Makoto 2!
Technical Assistants:
MORITA, Yasumasa
NAGAHARA, Miki
SAITOH, Miho
Secretaries:
HOSHINO Maki***SANJO Kazuko*****until May, 2002**from February, 2002***until May, 2002****from April, 2002
1

!jrom Kasetsart University!Graduate University for Advanced Studies

2

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. Although there are many elements
affecting organization and reorganization of the genomes,
we are currently focused on mobile genetic elements ingeneral and plant transposable controlling elements inparticular. Since plant transposable elements are known to
be associated with both genetic and epigenetic gene
regulations, we are characterizing various aspects ofgenetic and epigenetic gene regulations. In addition, weare also undertaking reverse genetic approaches in order toelucidate the nature of dynamic genome in plants.
I. Spontaneous mutants in the Japanese morning

glory.
The Japanese morning glory (Ipomoea nil or Pharbitisnil), displaying blue flowers, is believed to be originatedfrom southeast Asia and has an extensive history of genetic and physiological studies. The plant had been introduced into Japan from China in about 8th century as a
medicinal herb, seeds of which were utilized as a laxative,
and has become a traditional horticultural plant in Japan
since around 17th century. A number of its spontaneous
mutants related to the color and shape of the flowers andleaves have been isolated, and about 10% of these mutants
carry mutable alleles conferring variegated phenotypes.
All of the mutants available are spontaneous mutants andmost of them were isolated more than 60 years ago. Several lines of evidence indicate that an En/Spm-relatedtransposable element Tpn1 and its relatives, which wetermed Tpnl-family elements, are common spontaneous

mutagens in the plant. Indeed, we previously identified
four mutable alleles for flower pigmentation , jlecked-1 ,jlecked-2 , speckled and purple-mutable (pr-m), which arecaused by integration of non-autonomous Tpn1-related
elements , Tpn1, Tpn9, Tpn2 and Tpn4 into the genes forantocyanin biosynthesis, DFR-B , ANS, CHI and the gene
for a vacuolar Na+;H+ exchanger lnNHX1, respectively.
An apparent stable r-1 allele conferring white flowers was
also previously found to be caused by insertion of a nonautonomous Tpn1-family element, Tpn3, into the CHS-D
gene encoding a chalcone synthase for anthocyanin biosynthesis. The transposition of these non-autonomous
elements is mediated by a Tpn1-related autonomous element, which appears to be subjected to epigenetic regulations. Thus the phenotype of the white flowers in the r-1
mutant due to very rare excision of Tpn3 from the CHS-D
gene is likely to be the result from epigenetic inactivationof the autonomous element.
We are able to identify two mutations in the UF3GT
gene encoding UDP-glucose:flavonoid 3-0-glucosyltransferase, which is the first step to form stable anthocyanin
pigments accumulating in the vacuole . One of them carries
a Tpn1-related non-autonomous element TpnlO having
integrated into its promoter region , and the other contains
an insertion of 4 bp , which generates a termination codon
in the cording region . Another mutant displaying maroon
flowers was found to carry a 4-bp insertion within thecoding region of a newly isolated glucosyltransferase gene.
The mutant is likely to be deficient in a transglucosylation
process from anthocyanidin 3-glucose to the poly-acylated
and glucosylated anthocyanin pigments accumulated in
the petal vacuole. In addition to these newly identified
mutations in the genes encoding the enzymes involved inanthocyanin biosynthesis pathway, mutants bearing whiteflowers were found to carry two different 7 bp insertions
at the same site within the coding region of a regulatory
protein containing the WD40 motif. Both insertions causing frameshifts would result in truncated regulatory protein. These 7 bp insertions are likely to be footprints generated by excision of a Tpn1-related transposable element.
The results imply that 4 bp insertions detected in the twodifferent glucosyltransferase genes are also footprints
generated by excision of Tpn1-related elements, which arespontaneous mutagens in the Japanese morning glory.

Fig. 1. Flower phenotypes of the Japanese morning glory
carrying the wild-type (A) and mutant (B) UFJGT gene in
the very similar genetic background.

41

FOR BASIC BIOLOGY

II. Spontaneous mutants in the common morning
glory.
The common morning glory (Ipomoea purpurea or
Pharbitis purpurea), originating from Central America,
was introduced to Europe probably in the late 17th century
and several cultivars with different color of flowers werealready recorded in the late 18th century . Previously , weidentified that the mutable flaked allele of the plant forflower variegation is caused by the insertion of the transposable element TiplOO into the CHS-D gene . The 3.9-kb
Tip] 00 element belongs to the AciDs family.
We are able to identify two mutant lines showing white
seeds and palely pigmented flowers were found to produce
drastically reduced amounts of the transcripts of the
JPMYC3 gene , a homologue of the ani gene for the bHLH
transcriptional factor that regulates flower and seed
pigmentation in petunia. The genomic sequences revealed
that both mutants carry two insertions of transposable
elements into the JPMYC3 gene, which comprises 8 exons .
In one mutant , two copies of TiplOO belonging to the
AciDs family are inserted into the 2nd intron and the 7th

exon . The other mutant contains two different Mu-relatedtransposable elements in the 2nd exon and the 5th intron.
Moreover, the latter mutant appears to carry an additional
mutation for flower pigmentation.

III. Spontaneous mutants in the morning glory(Ipomoea tricolor).
Like Ipomoea purpurea, the morning glory (Ipomoea
tricolor) is also originated from central America. The wild
type plant produces bright blue flowers , which is one of
the most important floricultural plants in both the UnitedStates and Europe. We have identified a mutant displaying
white flowers carries about 11.5 kb insertion into the 5th
intron of the DFR-B gene. The junction sequences of theinsertion site revealed that the 5th intron of the DFR-B
gene contains a 0.7 kb MITE-like element and that the11.5 kb sequence is integrated in the middle of the element.
Neither terminal inverted repeats of the insertion nor its
target site duplication was detected, and the transcriptionof the DFR-B gene appears to be blocked efficiently
within the inserted sequence.

Fig. 2. Strategy for the targeted disruption of the Waxy gene by homologous recombination and the Waxy phenotype of thetargeted rice plant. (A) The genomic structure of the wild-type Waxy gene region . (B) The structure between the left and rightborders (LB and RB) of the T-DNA region on the targeting vector used. (C) The structure of the targeted waxy gene byhomologous recombination. The white rectangles in the orange Waxy box represent the Waxy intron 1 sequence, and the green
bars indicate the sequences corresponding to the Waxy flanking segments carried by the targeting vector. DT-A and hpt indicate
the negative and positive selection markers employed. (D) A fertile target rice plant. (E) The Waxy phenotype in pollen . (F) The
Waxy phenotype in endosperm . The Waxy and waA-y phenotypes are indicated by the dark and light brown staining with iodine,
respectively .

42

IV. 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. It is a model
plant for other cereal species because of several characteristics: its small genome of about 430 Mb; the similarityof its sequences and constellation of the genes with othercereals; the availability of large expression sequenced tag(EST), full-length cDNAs and the entire genome sequenceof both Japonica and Indica subspecies; and its efficientuse of Agrobacterium-mediated transformation. A comparison of the genome sequences of these Japonica andIndica subspecies with that of Arabidopsis indicates that alarge proportion of rice genes hove no recognizable
homologues in Arabidopsis. Under these circumstances,the development of a method to study gene function bymodifying genomic sequences precisely becomes extremely important, and gene targeting is a powerful tool ofsuch reverse genetics. In higher plants , however, themethod is far from a common practice.
We have succeeded to develop an efficient and reproducible procedure for gene targeting in rice, based upon
the following components:(a) optimization ofAgrobacterium-mediated transformation of embryogenic
rice calli, (b) utilization of strong positive/negative selection, and (c) stringent PCR screening for targeted allele.
As a model gene to be targeted, we chose the Waxy geneencoding granule-bound starch synthase, a key enzyme inamylose synthesis, because its mutations affect the quality
and quantity of rice grain and because the associated phenotype in pollen and in endosperm can easily assessed bysimple iodine staining. All of the six independently
obtained fertile transgenic rice plants from six experiments
were heterozygotes at the Waxy locus: a wild-type Waxy
allele and a targeted recombinant waxy allele. Thesetransgenic plants segregated into a 3:1 ratio, confirming to
the Mendelian pattern of inheritance. Neither ectopic
targeting (integration of the sequence produced byhomologous recombination into a site other than the correctly targeted site in the genome) nor ectopic integration(integration by nonhomologous endjoining) of the selective marker gene could be observed. Southern blot
analysis revealed that the targeted waxy region of around35 kb comprised the anticipated structure and that the
drug-resistant marker used for selecting transformants was
integrated only within the targeted waxy gene in the
genome. Sequencing analysis of the junction regions confirmed that precise somatic homologous recombination
occurred to generate the rice plants carrying the targeted
waxy gene. We are currently examining whether the strategy we used is applicable to obtain various gene-targetedor knockout lines of rice.

NATIONAL INSTITUTE

V. Characterization of mutable virescent allele in
rice.
Leaves of seedlings in the virescent mutant of rice
(Oryza sativa L.) are initially pale yellow green due topartial deficient in chlorophyll and gradually become
green with the growth of the mutant. The mutable virescent plant (yl-v), displaying pale green leaves with darkgreen sectors, was isolated among progeny of a hybrid
between Indica and Japonica rice plants. The leaf variegation is regarded as a recurrent somatic mutation fromthe recessive pale green to the pigmented revertant allele.
During repeated crossing with the Japonica rice T -65, anapparent stable virescent mutant, yl-stb, the seedlings ofwhich bear pale green leaves, was obtained. Although theyl-stb mutant could not grow on soil, we have succeededin growing it with in vitro culture during its critical juvenile phase. Preliminary HPLC analysis indicated accumulation of a carotenoid intermediate in the yl-stb mutant.
The mutable virescent allele was mapped in the short armof the rice chromosome 3.

Fig.3 . Variegated leaves of the mutable virescent (yl-v) mutant of rice.

Publication List:
Iida, S. and Terada, R. (2002) Gene modification of an endogeneous gene in rice plants. ISB News Report December
2002 , 7-8.
Ishikawa , N., Johzuka-Hisatomi, Y., Sugita, K., Ebinuma H.
and lida , S. (2002) The transposon TiplOO from the common morning glory is an autonomous element that cantranspose in tobacco plants. Mol. Gen . Genomics 266,732-739.
Kamiunten, H., Inoue, S., Yakabe Y. and lida, S. (2002)
Characterization of ISPsy2 and ISPsy3, newly identified
insertion sequences m Pseudomonas syringae pv.
Eriobotryae. J. Gen. Plant Pathol. 68 , 75-80.
Terada , R., Urawa, H., lnagaki, Y. , Tsugane , K. and Iida, S.
(2002) Efficient gene targeting by homologous recombination in rice. Nature Biotechnol. 20, 1030-1034.

43

FOR BASIC BIOLOGYDIVISION OF GENE EXPRESSION AND
REGULATION II
Professor:
Research Associate:

Technical Staff:
JSPS Research Fellow:
Post Doctoral Fellows:
Graduate Student:
Visiting Fellows:

HORIUCHI, Takashi*HIDAKA, Masumi
KOBAYASHI, Takehiko
JOHZUKA, Katsuki
HAYASHI, Kohji**Austen RD Ganley
KODAMA, Ken-ichi
SERIZAWA, Naomi
WATANABE, Takaaki
UJIIE, Yoshifumi* to Biomolecular Engineering Research Institute, 6-2-3,
Furuedai, Suita, Osaka, 565-0874, JAPAN (Sep, 1, 2002- ).
**from Nov. 30, 2002
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, weare pursuing the following themes using E. coli and S
cereVlszae: (1) An amplification mechanism of repeatedsequences or genes, especially rRNA repeated genes, (2) amechanism of replication fork block-dependent recombination, a key reaction that increases or decreases thenumber of repeats, and (3) development of in vivo artificial gene amplification systems. Structural and functional
analyses of the E. coli genome are also being carried out.
In 2002, work on the following three subjects has advanced our knowledge of the dynamics and structure of
the genome.

I. Unequal sister-chromatid recombination induces
the instability of the rDNA in Saccharomyces
cerevisiae.
The yeast S. cerevisiae carries approximately 150
copies of the ribosomal RNA gene (rDNA) in tandem
repeats on chromosome XII. Transcription of the rDNA iscarried out specifically by RNA polymerase I (Pol I), oneof the three kinds of eukaryotic RNA polymerases. The
absence of an essential subunit of Pol I in rpa135 deletionmutants causes a gradual decrease in the rDNA repeat
number to approximately half the normal level andreintroduction of the missing RPA135 gene induces a
gradual increase in repeat number back to the normal
level. This contraction/expansion requires the FOBJ geneprotein (Foblp) which is also required for replication fork
blocking activity at the replication fork barrier (RFB) site.
The RFB site is located in a non-transcribed spacer region,
called NTS 1, in each rDNA unit. Using this artificial
induction system of amplification, we tested the effects ofseveral mutations on the pattern of copy number increase.
The gene RAD52 was essential for the increase in rDNA
repeats. mrell mutant cells showed a partial reduction in
the amplification of rDNA. In a sir2 mutant, the rate ofamplification increased by more than two-fold, although
the copy number was not stabilized. The hyper-

recombinogenic phenotype in the mutant was dependent
on FOBJ. In addition, the loss of SIR2 function reduced
the association of Mcdlp, a factor involved in sisterchromatid cohesion, with the non-transcribed spacer region, NTS1, in the rDNA repeats. A temperature sensitive
mutant of the cohesin complex, smcl-2, had an approximately 8-fold increase in the loss of a URA3 marker,
which is integrated in the rDNA repeats, at the semipermissive temperature. These results suggest that theestablishment of sister-chromatid cohesion would affectthe efficiency of the unequal sister-chromatid recombination to change the copy number of rDNA after the double
strand break at the DNA replication fork arrested at the
RFB site (see Figure).

II. Transcription-dependent recombination in yeast
rDNA.
In Escherichia coli, we previously identified a chromosome-derived EcoRI fragment, as a recombinational
hotspot, RotH, which contains the 3' end of an rRNA
operon, rrnD. Under RotH active conditions, the progressof the DNA replication fork was retarded in the latter partof the rrnD operon, the transcriptional orientation ofwhich is opposite to that of the fork. This result suggeststhat the collision between the replication fork and rRNA
gene transcription may be responsible for retardation.
Inactivation of the promoter of the rrnD operon simultaneously reduced the retardation of fork movement andRotH activity. These results indicate that RotH activation
is caused by the collision between the DNA replication
fork and the reverse-oriented rrnD transcription.
The rRNA gene (rDNA) in S. cerevisiae has a tandem
repeat structure, whose copy number is approximately
150 copies. The replication fork barrier (RFB) site is
located near the 3'-end of the 35S rDNA of each rDNA
unit. This site inhibits the progression of the DNA
replication fork corning from the opposite direction of the
35S rDNA transcription. The gene FOBJ was previously
shown to be required for the replication fork blocking
activity at the RFB site and also later for the change in the
copy number of rDNA through unequal sister chromatid
recombination. Another function of the RFB site may be
to avoid a collision between 35S rDNA transcription and
DNA replication. Although a collision has never been
observed in the wild type or even in a fobl mutant with150 copies of rDNA, it could be detected in a fobl
derivative with a reduced number of rDNA copies(approximately 20) by using two-dimensional agarose gelelectrophoresis. This result suggests that while eachrDNA of 150 copies may be weakly transcribed or a small
fraction of the rDNA may be actively transcribed, most of
the reduced copies are probably extensively transcribed.
The collision was dependent on the transcription of 35S
rDNA by Pol I. In addition, under these FOBJ defective
conditions, transcription apparently enhanced recombination to change the copy number of rDNA and also to
produce the extra-chromosomal rDNA circles (ERCs)
whose accumulation is known to be a cause of aging.
These results suggest that such a transcription-dependent

44

NATIONAL INSTITUTE

Three models showing formation of ds-DNA end------- --~)~t===occurence of replication fork arrest
~ (3)
==~"\

---!J~=~~-- -:,.~L-=~;.»1==......

direct breakat the fork

-

... J
resolution of the Holliday junction(Horiuchi et al., 1994;
Kobayashi et al., 1998)-----~)====:::t;:. . )break through Holliday structure(Seigneur et al. , 1998)

arrival of second roundof replication fork
(Bidnenko et al.. 2002)

Three models explaining the formations of a double stranded (ds) DNA end are shown. When the fork is blocked forsome reason, any of the following three mechanisms possibly work. (1) A nick or a ds-break is introduced at either
sister-chromatid . This is thought to occur when the fork is blocked at the RFB site in rDNA repeats in yeast (see text).
(2) When the fork is arrested , two newly synthesized sister-chromatid DNA strands start to pair with each other, leadin g
to regression of replication fork, resulting in a formation of the Holliday structure. A resolvase enzyme, such as RuvC in
E. coli, resolves the structure, producing a ds-DNA end . (3) When the second round of DNA replication reaches thepreviously arrested fork , two linear ds-DNA molecules with ends are produced. This event is believed to occur inprokaryotes, in which multi-round replications are allowed.
collision may function as a recombination trigger, such as
introducing a ds-break shown in Figure, for multiplicationof a single copy gene that is intensely transcribed ingeneral.

III. Complete genomic sequence of the E. coli K12
W3110 strain
Escherichia coli is one of the organisms that has been
most extensively analyzed physiologically, biochemicallyand genetically. Of all E . coli strains, E . coli K1 2 W3110
has probably been used most frequently as the wild-typestrain in these experiments . Here , we have determined thecomplete nucleotide sequence of the genome of strain
W3110 by mainly using lambda phages of Kohara 's bank.
Previously a US group determined the genomic sequenceof another K12 wild-type derivative , MG1655 . Bothstrains were derived from a common ancestor strain ,Wl485, approximately 50 years ago. After comparing thetwo sequences , the following results were obtained .
The total number of nucleotides in the W3110 genome
is 4660170 bp . There were 349 bp conflicts between thesequences of W3110 and MG1655 . Re-sequencing of theconflict sites by a PCR method using their genomic DNAs
as templates revealed that only eight sites (9 bp) were true
conflicts. Seven of them are base-change 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
rRNAgene.
Next we investigated the number and the type of
insertion sequence (IS) elements in collaboration with Dr.
Ohtsubo's laboratory (Tokyo Univ .). Thirteen different IS
elements were identified. While 12 IS elements arepresent in the W3110 genome and not in MG1655, one IS
element is in MG1655, but not in W3110.
These results indicate that the DNA sequence isalmost perfectly conserved in bacterial cells under stock
culture conditions and in Kohara ' s lambda phage bank,and during growth through repeated vegetative cycleslO .
Under stock conditions, which include transfers from thestock to fresh plates and vice versa , IS transposition
events seem to occur more frequently than base-changes .

Publication list:
Johzuka, K., and Horiuchi , T . (2002) Replication fork blockprotein , Fobl , acts as an rDNA region specificrecombinator inS. cerevisiae. Genes Cells 7:99-11 3.
Kodama, K., Kobayashi, T ., Niki, H ., Hiraga, S. , Ohshima ,T., Mori, and Horiuchi , T . (2002) Amplification of
HotDNA segments in Escherichia coli. Mol. Microbial.
45: 1575-1588.

45

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS I
YAMAMORI, Tetsuo
KOMINE, Yuriko
WATAKABE, Akiya
KITSUKAWA , Takashi
OOSAWA, Sonoko
Technical Staff:
SAWADA , Kaoru
Miki, KazuhikoTechnical Assistant:
ISHIKAWA , Takaka
NIBB Postdoctoral Fellow: Sf, Xuaohui
Postdoctral Fellow:
HATA , Katsusuke
SAKATA , Shuzo
Graduate student:
KOMATSU, Yusuke
TAKAHATA , Toru
HAYASHI, Hitomi
Secretary:

Professor:
Research Associate:

Our research is focusing to understand mechanisms underlying memory and evolution of the brain. For one approach to understand these questions , we are studying thegenes that are expressed in specific areas of the primate
neocortex . Using differential display method , we obtained
three genes that showed marked differences within areasof the primate neocortex. Our second approach is to understand informational processing in the brain underlying
learning behaviors by examining gene expression . Here,
we report our findings focusing on those of the year of
2002.

be act.J.v1ty dependent, because, in the monocularly
deprived monkeys injected with TTX into one of the eyes,
the expression of occl is markedly decreased in the oculardominance column of the primary visual cortex (Vl).
ii) The other gene that showed marked difference
within the neocortex, is gdp, a member of BMP/TGF-~
family , which is specifically expressed in the motor cortex of African green monkeys. We are currently examining the detailed expression pattern of the gene.
iii) The third gene that we designated tentatively as
134G was preferentially expressed in association andhigher sensory areas in the neocortex (Komatsu et al.,Society for Neuroscience 32"d Annual Meeting, 2002).
3) We have also further isolated several area specificgenes with RLCS (Restriction Landmark eDNA Scanning) . We are now characterizing these genes to reveal themechanisms that form neocortical areas.

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 to what
extent the area of the neocortex is genetically andenvironmentally determined . It is also puzzling why,
during the evolution of mammals, the neocortex was mostmarkedly expanded while the number of the genes inmammal phyla were little changed . To access thesequestions, we studied gene expression within different
areas of the neocortex .
1) In collaboration with Professor Hiroyuki Nawa
(Nigata university) , we used the DNA macroarray technique to examine gene expression in the areas of humanprefrontal , motor and visual cortexes. We found almost all
the genes among 1088 genes examined showed only lessthan a factor of two in the difference of 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 thegenes that are expressed among different areas of thehuman neocortex are very similar. However, the question
remained whether there are any genes that show marked
differences within areas of the neo-cortex .
2) In order to answer this question, we employed differential display methods and found at least three genesthat indicated area specific expressions .
i) One gene, designated occl , is specifically expressedin the occipital cortex, particularly in Vl , in the primate
brain. Furthermore, the expression of occl turned out toFig. 1 Expression pattern of occl in the visual cortex.
In situ hybridization pattern of occ I in the primate visualcortex. occl is markedly expressed in the layer 1Vcj3 andmoderately in the layers of II, III and IVa in area VI. The
boundary between V 1 and V2 is shown by an arrow .

II. Gene expression under a declarative and a nondeclarative memory
In order to study informational processing underlying
the declarative and the non-declarative memory atmolecular and cellular levels in the brain , we employed cFos mapping techniques. There have been an increasing
number of studies using c-Fos as markers to examine
neuronal activities since c-Fos induction by electricalstimulation was found by Morgan and Curran. However,many sensory stimuli per se are now known to cause cFos induction. So , we should be very careful todistinguish the c-Fos expression that is caused by learning
process from that caused by sensory stimuli. For thispurpose, it is necessary to use behavioral systems that areable to distinguish the two . Although a few behavioral
systems in rodents have been successfully used forphysiology , animal behavior and recently for analyses ofknockout mice , little behavioral systems in fact
distinguish them. Therefore, we prepared ourselves forusing two behavioral systems, each of which represent a
declarative and a non-declarative memory.
In one system, we collaborated with professor Yoshio

46

NATIONAL INSTITUTE

Sakurai (Kyoto University) who developed audio-visual
discrimination task (AVD-task) . In this task, a rat was
asked to choose either an audio cue (a high or low tone)
or a visual cue (a light from the right or the left) to obtaina food pellet (Fig. 2) . 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 c-Fos expression was
only observed in excitatory neurons in the relevant
sensory cortices.
A

•.. .

l1 g hl Dnu.!_--.

,.

hghl

•et_

c
Stimulus Sets
AVD-Auditory(A VD-A) task
AVD.Visual(f\ Y D· V) task

Go
Go

Go
NoGo

NoGo
Go

NoGo
NoGo

Fig.2. AVD tasks.
A) A rat faces to a panel with a visual cue (a light from rightor left) or an auditory cue (a high or low tone) was shown topush the panel with the time sequence shown in (B) . The
combination of possible cues by auditory and visual tasks toobtain a food pellet is shown in (C) . (See Sakata eta!. , Eur.
1. Neurosci. , 15,735-743 , 2002, for the detail of the tasks)
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 withpegs is turning to the other direction (Kitsukawa et a!.,Society for Neuroscience 32"d 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 procedural learning . We
examined 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
to play an important role in a reward-based learning.
The characterization of these subtypes of interneurons
has been progressed. However, the roles in behavioral
tasks have been little known. We hope our system
combined with c-Fos mapping technique solve thesequestions.

III CNTF is specifically expressed in the developing
pineal gland
CNTF, a member of the IL-6 family, attracts quite attentions of developmental neuroscientists because itshows various effects on neurons and glias. CNTF

knockout mice, however, only indicate moderate motor
neuron deficiency in the adult, but no apparent phenotype
in the development. In order to explore the function of the
IL-6 family, we extensively examined the expression ofmembers of the family and their receptors and found thespecific expression of CNTF in the embryonic pineal
glands and eyes. This was to our knowledge for the firsttime to show a clear expression 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 their development,
it has been difficult to know the developmental roles of
CNTF if any. In fact, we confirmed that there seemed noapparent difference in the pineal development of CNTF
knockout mice compared to that in wild type mice. This ispresumably because the CNTF gene knockout wascompensated by other CNTF-like factors .
We therefore studied cultured pineal organs in rodents
to ask if there were any effects of CNTF on themfollowing 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 (Rata et al., Society for Neuroscience 32"d Annual Meeting, 2002). Our observation raises
an interesting possibility that CNTF plays a critical role in
the pineal development to suppress a certain phenotype
such as photoreceptors. We are currently working toprove it.

Publication list:
Rata, K., Arakai , M. and Yamamori, T. (2002) CNTF is
specifically expressed in the developing rat pineal glandand eyes. NeuroReport 13, 735-739
Nakayana, T., Mikoshiba, K., Yamamori, T. and Akazawa, K.
Expression of syntaxin lC , an alternive splice variant of
HPC-1/syntaxin lA, is activaed by phorbol-ester
stimulaton in astrglioma. FEBS Lett. (in press)
Onishi, A. , Koike, S., lda-Hosonuma , M., Imai, H., Shichida,Y., Takenaka, 0. , Hanazawa, A., Komatsu, H. , Mikamai,A., Goto., S., Suryobroto , B., Farajallah , A., Varavudhi, P. ,Ekavhibata, C., Kitahara, K. and Yamamori, T. (2002)
Variations in ling-term and middle-wavelength-sensitive
opsin loci in crab-eating monkeys. Vision Res., 42, 281292
Sakata, S., Kitsukawa, T., Kaneko, T ., Yamamori , T. and
Sakurai, Y. (2002) Task-dependent and cell-type-specificFos enhancement in rat sensory cortices during audiovisual discrimination. Eur. J. Neuroscience.15, 735-743
Suga, K., Yamamori , T. and Akagawa, K. Identification of
Carboxyl-Terminal Membrane-Anchoring Region of
HPC-1 /Syntaxin IA by Substituted-Cysteine-Accessibility
Method and Monoclonal Antibody. J. Biolchem (in press)
Tochitani, S., Hashikawa, T. and Yamamori, T. Expressionof occl mRNA in the visual cortex during postnatal development in macaques . Neurosci. Lett. (in press)
Vigot, R. , Batini, C. , Kado, R.T . and Yamamori. T. (2002)
Synaptic LTD in vivo recorded on the rat cerebellar cortex.
Arch . !tal. Bioi. , 140 , 1-12.

47

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS II

HASEBE, Mitsuyasu
MURAl'A , Takashi
FUJITA, Tomomichi
SUMIKAWA, Naomi
MISHIMA , Misako (-March 31)
HIWATASHI, Yuji
NISHIYAMA, Tomoaki
JSPS Research Fellow:
SATOH, Yoshikatsu
KOBAYASHI-ARAKAWA ,Graduate Students :Satoko
SAKAKIBARA, Keiko
SAKAGUCHI, Hisako(Shinshu Univ.)(-March 31)
NAKAMURA , Tohru(Niigata Univ.)(April I-)TANIKAWA, Yukiko
Technical Assistant:
B/TOH, Yoshimi
NARUSE, Mayumi
AOKI, Etsuko
WATANABE, Kyoko (-Feb. 4)
YANO, Kana (-Nov. 8)
KABETANI, Keiko (Feb.l-)
OGURA, Youko (Nov. I-)
Secretary:
KABEYA, Kazuko
Visiting Scientists:
Jean-Pierre Zryd !)(-Jan . 18)
1l_{rom Universite de Lausanne , Lausanne , Switzerland

Professor:
Associate Professor:
Assistant Professor:
Technical Staff:
NIBB Research Fellow:

analyses of DNA sequences of the chloroplast gene rbcL
for 59 species of Drosera covering all sections except one.
These analyses revealed that 5 sections in 11 including 3
monotypic sections are polyphyletic. Combined data of
the rbcL and 18S rDNA sequences were used to infer
phylogenetic relationships among Drosera, Dionaea, andAldrovanda . This analysis revealed that all Drosera species form a clade sister to a clade including Dionaea andAldrovanda, suggesting that snap traps of Aldrovanda andDionaea are homologous despite their morphologicaldifferences. MacClade reconstructions indicated thatmultiple episodes of aneuploidy occurred in a clade including mainly Australian species, although chromosome
numbers in other clades are not so variable. D. regia
native to South Africa and most species native to Australia were basally clustered, suggesting that Drosera originated in Africa or Australia. The rbcL tree indicates that
Australian species expanded their distribution to SouthAmerica, and then to Africa . Expansion of distribution to
the North Hemisphere from the South Hemispere occurred in a few different lineages.
D. montana tomentosa.------·D. platypoda.____ __All living organisms evolved from a common ancestor
more than 3.5 billion years ago, and accumulated mutations on their genomes , which caused the present biodiversity . The traces of evolutionary processes are found in
the genomes of extant organisms. By comparing the genomes of different organisms , we can infer (1) the phylogenetic relationships of extant organisms and (2) thegenetic changes having caused the evolution of morphology and development. The inferred phylogenetic relationships give important insights on problems in various
fields of evolutionary biology, and our group focuses onbiogeography , evolution of morphological traits , and systematics in wide range of taxa. On the evolution of morphology and development, we aim to explore genetic
changes led the evolution of plant body plan . We selectedArabidopsis (angiosperm) , Gnetum (gymnosperm),
Ginkgo (gymnosperm), Ceratopteris (pteridophyte), Physcomitrella (bryophyte) , and some green algae as models
to compare the gene functions involved in development ofboth reproductive and vegetative organs in land plants.
I. Molecular phylogeny of the sundews Drosera
Carnivorous plants have long attracted the attention of
botanists, because of their highly specialized morphology
and curious trapping mechanisms. However, their evolutionary processes are still unknown . The sundew genus
Drosera consists of carnivorous plants with active flypaper traps and contains nearly 150 species mainly distributed in Australia, Africa , and South America, with somespecies in the Northern Hemisphere. In addition to theconfusion of intrageneric classification of Drosera , intergeneric relationships among Drosera and two other genera in the Droseraceae with snap traps, Dionaea and Aldrovanda are problematic . We conducted phylogenetic

D. pygmaea.____ _ _ D. glandullgera

L - - - - Nepenthes

Figure.l. The most parsimonious tree resulting from parsimony analysis of the combined sequences of rbcL and ISS
rONA. Branch lengths correspond to the number of nucleotide substitutions (ACC1RAN optimization). Numbers above branches represent bootstrap values more than
50% of 10,000 bootstrap replicates , and numbers below
branches are decay indecies (Bremer, 1988) .

II. Evolution of reproductive organs in land plants
A flower is the most complex reproductive organ inland plants and composed of sepals, petals, stamens, andgynoecium . Female haploid reproductive cells are covered with a sporangium (nucellus) and two integuments ,and further enclosed in a gynoecium. Male haploid reproductive cells (pollens) are covered with a sporangium(pollen sack). On the other hand, gymnosperms and ferns
have simpler reproductive organs than angiosperms andlack sepals and petals. Female sporangia (nucellus) ofgymnosperms are covered with only one integument.
Sporangia of ferns have no in-teguments and are naked on
the abaxial side of a leaf.
The development of floral organs is mainly regulatedby A-, B-, C-function genes , which are members of the
MIKC-type MADS-box genes . These genes are transcription factors containing four domains, MADS, I, K, and C
domains. MADS-box genes of angiosperms are divided
into more than 10 groups based on the gene tree. The

48

FLO/LEAFY gene is the positive regulator of the MADSbox genes in flower primordia.
What kind of changes of the MADS-box genes caused
the evolution of the complex reproductive organs in theflowering plant lineage? Comparisons of MADS-box and
FLO/LFY genes in vascular plants suggest that the following sequential changes occurred in the evolution of
reproductive organs. (1) Origin of MIKC-type MADSbox genes. (2) The number of MADS-box genes increased ,and the three ancestral MADS-box genes that later generate A-, B-, C-functions genes were likely originated before the divergence of ferns and seed plants. (3) Specification of MADS-box gene expression in reproductive
organs occurred in seed plant lineage. (4) The ancestralgene of the AG group of MADS-box genes acquired the
C-function before the divergence of extant gymnosperms
and angiosperms. (5) The gene duplication that formed
the AP3 and PI groups in MADS-box genes occurred before the diversification of extant gymnosperms and angiosperms. (6) The ancestral gene of angiosperm A-function
gene was lost in extant gymnosperm lineage. (7) LFY
gene became positively regulate MADS-box genes in thecommon ancestor of angiosperms and gymnosperms after
the divergence of ferns and seed plants. (8) Spatial andtemporal patterns of A-, B-, C-function gene expression
were established in the angiosperm lineage .

NATIONAL INSTITUTE

elude the A-, B- , and C-function genes . In contrast,
LAMBJ from the clubmoss was identified as the only
other MIKC*-type gene published so far. Our fmdings
strongly suggest that the last common ancestor of mosses
and vascular plants contained at least one MIKCc- andone MIKC*-type gene.

III-I. Evolution of vegetative organs
The ancestor of land plants was primarily haploid.
The only diploid cell was the zygote, which immediately
underwent meiosis. It is believed that early during land
plant evolution , zygotic meiosis was delayed and a multicellular diploid sporophytic generation be-came interpolated into the life cycle. In the early stages of land plant
evolution , sporophytes are epiphytic to gametophytes, as
observed in extant bryophytes. During the course ofevolution, both generations started to grow independently
at the stage of pteridophytes. Finally gametophytes became much reduced and epiphytic to sporophytes in seedplant lineage. Molecular mechanisms of development in a
diploid generation have been well studied in some model
angiosperms , but we have scarce information on those ina gametophyte generation . For example, mosses haveleaf- and stem-like organs in their haploid generation, but
it is completely unknown whether similar genes involvedin angiosperm leaf and stem development are used in thegametophytic generation of mosses or not. To understandthe evolution of body plans in diploid and haploid generation at the molecular level , we focus on the comparisonof molecular mechanisms governing shoot development
between Arabidopsis and the moss Physcomitrella patens.
Physcomitrella is known by its high rate of homologous
recombination and suitable for analyze gene functions
using the gene targeting, and should be a good model
lower land plants.

111-11. Characterization of homeobox genes in themoss Physcomitrella patens

Figure 2. Expression of Physcomitrella patens MADS-box
gene. GUS activity of PpMADSI-uidA protein was detected in transgenic Physcomitrella patens. Localization of
PpMADSI-GUS protein in an archegonium (a-d), and anantheridium (e-h) is shown.
MIKC-type MADS-box genes have been reported only
from vascular plants, but not from non-vascular plants. To
investigate the origin of MIKC-type MADS-box genes,
eight MADS-box genes (PPM1-PPM5 and PPMADSJ-3)
were characterized in the moss Physcomitrella patens(Henschel et al. 2002). Phylogenetic reconstructions andcomparison of exon-intron structures revealed that thesemoss genes represent two different classes of homologous,
yet distinct MIKC-type MADS-box genes. We named
them MIKCc-type and MIKC*-type genes. MIKCc-typegenes are abundantly present in all land plants, and in-

Homeobox genes encode transcription factors involvedin many aspects of developmental processes including
shoot development in angiosperms. The homeodomainleucine zipper (HD-Zip) genes , which are characterized
by the presence of both a homeodomain and a leucine
zipper motif, form a clade within the homeobox
superfamily and previously re-ported only from vascularplants . We isolated 10 HD-Zip genes from P. patens(Pphbl-10 genes). Based on a phylogenetic analysis of
the 10 Pphb genes and previ-ously reported vascular plant
HD-Zip genes, all the Pphb genes except Pphb3 belong tothree of the four HD-Zip subfamilies (HD-Zip I, II, andill) , indicating that these subfamilies originated before thedivergence of the vascular plant and moss lineages .
Pphb3 is sister to HD-Zip II subfamily , and has somedistinctive characteristics , including the difference of al
and dl sites of its leucine zipper motif, which are wellconserved in each HD-Zip subfamily. Comparison of thegenetic divergence of representative HD-Zip I and II
genes showed that the evolutionary rate of HD-Zip I
genes was faster than HD-Zip II genes .

FOR BASIC BIOLOGY

The moss homologs of SHOOTMERISTEMLESS andZWILLE genes, which are involved in Arabidopsis shootdevelopment, have been cloned and their characterization
is in progress.

ill-ill. Establishment of enhancer and gene traplines in the moss Physcomitrella patens
We also established enhancer and gene trap lines andtagged mutant libraries of P. patens to clone genes involved in the leafy shoot development in haploid generation. Elements for gene-trap and enhancer-trap systems
were constructed using the uidA reporter gene with eithera splice acceptor or a minimal promoter, respectively.
Through a high rate of transformation conferred by amethod utilizing homologous recombination, 235 genetrap and 1073 enhancer-trap lines were obtained from5637 and 3726 transgenic lines, respectively. Expression patterns of these trap lines in the moss gametophyte
varied. The candidate gene trapped in a gene-trap line
YH209, which shows rhizoid-specific expression, wasobtained by 5' and 3' RACE. This gene was named
PpGLU, and forms a clade with plant acidic alphaglucosidase genes. Thus, these gene-trap and enhancertrap systems should prove useful to identify tissue- andcell-specific genes in Physcornitrella.

Publication List:
Henschel, K., Kofuji, R., Hasebe, M., Saedler, H., Munster,T. and Theissen, G. (2002) Two ancient classes of MIKCtype MADS-box genes are present in the moss Physcomitrella patens. Mol. Bioi. Evol. 19, 801-814.

49
Imaizumi, T., Kadota, A., Hasebe, M. and Wada, M. (2002)
Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens.
Plant Celll4, 373-386.
Ishikawa, H., Ueno, Y., Semiarti, E., Onouchi, H., Kojima, S.,
Tsukaya, H., Hasebe, M., Soma, T., Ikezaki, M., Machida,
C. and Machida, Y. (2002) The ASYMMETRIC LEAVES2
gene of Arabidopsis thaliana, required for formation of asymmetric flat leaf lamina, encodes a member of a novel
family of proteins characterized by cysteine repeats and aleucine zipper. Plant Cell Phys. 43,467-478.
Kitakura, S., Fujita, T., Ueno, Y., Terakura, S., Wabiko, H.
and Machida, Y. (2002) The protein encoded by oncogene
6b from Agrobacterium tumefaciens interacts with a putative transcription factor in tobacco cells. Plant Cell 14,
451-463.
Maggio, A., Miyazaki, S., Veronese, P., Fujita, T., lbeas, J.I.,
Damsz, B., Narasimhan, M.L., Hasegawa, P.M., Joly, R.J.
and Bressan R.A. (2002) Does proline accumulation play
an active role in stress-induced growth reduction? Plant J.
31,699-712.
Murata, T., Karahara, I., Kozuka, T., Giddings Jr., T.H.,
Staehelin, L.A. and Mineyuki, Y. (2002) Improved
methodd for visualizing coated pits, microfilaments, andmicrotubules in cryofixed and freeze-substituted plant
cells. J. Electron microscopy 51, 133-136.
Rivadavia, F., Kondo, K., Kato, M. and Hasebe, M. (2003)
Phylogeny of the sundews, Drosera (Droseraceae) basedon chloroplast rbcL and nuclear 18S ribosomal DNA sequences. Amer. J. Bot. 90, 123-130.

50

NATIONAL INSTITUTE

51

FOR BASIC BIOLOGYRESEARCH SUPPORTCENTER FOR 1RANSGENIC ANIMALS AND PLANTSRESEARCH CENTER FOR INTEGRATIVE
AND COMPUTATIONAL BIOLOGYRESEARCH SUPPORT FACILITYTHE CENTER FOR ANALYTICAL
INS1RUMENTS (managed by NIBB)TECHNOLOGY DEPARTMENT

52

NATIONAL INSTITUTECENTER FOR
TRANSGENIC ANIMALS AND PLANTS
Head:
Associate Professor:
Technical Staff:
Supporting Staff:

NODA, Masaharu
WATANABE, Eiji
IINUMA , Hideko
YASUDA,Mie
UNOU, Satsuki
SHIMIZU, Naoki
NOGUCHI, Yuuji

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. We are
now planning on the construction of the center building.
The expected activities of the Center are as follows:
1. Provision of information, materials and techniques toresearchers.
2. Equipment of various instruments to analyze mutant
animals and plants.
3. Development of novel techniques related to transgenic
and gene targeting technology .

II. Academic activity
We are studying the functional role of Nax sodium
channel in collaboration with Division of Molecular Neurobiology. Nax was long classified as a subfamily of
voltage-gated sodium channels (NaChs) that serve togenerate action potentials in electrically excitable cells
such as neuronal and muscle cells. Comparing with theother NaChs, however, Nax has unique amino acidsequences in 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 and the physiologicalphenotypes have been examined . Behavioral studies
suggested that the Nax channel plays an important role inthe central sensing of body-fluid sodium level andregulation of salt intake behavior. Ion imaging andelectrophysiological studies in vitro also suggested that
Nax is an extracellular sodium-level sensitive sodium
channel. More recently, we found that Nax is expressed in
non-myelinating Schwann cells and alveolar type II cells
in addition to the neurons and ependymal cells in thecircumventricular organs (CVOs). Nax is thus likely to be
involved in reception of sodium-level in body fluids at the
CVOs and sodium absorption in the visceral nervous
system and in the lung.

Publication List
Hiyama, T.Y., Watanabe, E., Ono, K., Inenaga, K ., Tamkun ,M.M. , Yoshida, S. and Noda, M. (2002) Nax channel involved in CNS sodium-level sensing. Nat. NeUI·osci. 5
511-512
Watanabe , E., Hiyama, T.Y. , Kodama, R . and Noda, M.
(2002) Nax sodium channel is expressed in nonmyelinating Schwarm cells and alveolar type II cells inmice. New ·osci. Lett. 330, 109-113
Zubair, M., Watanabe, E., Fukada, M. and Noda, M. (2002)
Genetic labeling of specific axonal pathways in the mouse
central nervous system. Eur . J. Neurosci . 15, 807-814

Fig. I Nax sodium channel is expressed in non-myelinating Schwann cells.
Immuno-electron micrographs of a region underneath fungiform taste buds stained with anti-Nax antibody . Arrowheads indicate
immuno-positive non-myelinating Schwann cells . Arrows indicate the myelin sheath, and asterisks indicate myelinated axons.
Scale bars: I f.tm in (A) and 400 nm in (B) , respectively. We also found the Nax-expression in the alveolar type II cells . Nax
expressed in these cells is likely to be involved in sodium absorption in the peripheral nervous system and in the lung.

53

FOR BASIC BIOLOGYRESEARCH CENTER FOR INTEGRATIVE ANDCOMPUTATIONAL BIOLOGYHead:Professor:

HORIUCHI, Takashi
MIYATA, Takashi(kyoto University)

The neutral theory of molecular evolution, the basic
theory for explaining evolutionary changes at the molecular level, claims that most evolutionary changes at themolecular level are caused by random drift of neutral
mutants. This is sharply contrasted to the evolution at theorganismallevel, where it is generally thought that natural
selection is the major driving force by which evolutionarychanges occur. How can we explain the evolutionarychanges at the two levels in a unified way. This is the
major problem that remains unsolved for molecular
evolutionists. As a first step for understanding the final
problem, we are investigating relationships of evolutionary diversifications between molecular and organismallevels, based on molecular phylogenetic 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
shuffiing, basic mechanisms for generating diverse genes
with novel functions. Each of these animal gene families
has originated either from a few ancestral genes which areshared with plants and fungi or from an ancestral genecreated uniquely in animal lineage. The major groups ofbilateral animals are thought to have diverged explosively
at or prior to the Vendian- Cambrian boundary. No direct
molecular evidence has been provided to date as towhether the Cambrian explosion was triggered by adramatical increase in the number of genes involved incell-cell communication 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 the genediversification by gene duplication occurred during two
active periods interrupted by a long intervening quiescentperiod. Intriguingly, the Cambrian explosion is situated in
the silent period, indicating that there is no direct link
between the first burst of gene diversification and theCambrian explosion itself. The above result also suggests
the importance of gene recruitment as a possible molecular mechanism for morphological diversity.

II. Episodic divergence of eukaryote-specific genes
and evolution of Giardia! membrane boundedorganella
To understand a question of whether divergence ofeukaryote-specific gene families recognized commonly inall eukaryotes by gene duplication and domain shuffiing
proceeded intermittently during evolution, as found inanimal-specific gene families, a further molecular phylogeny-based analysis has been conducted for kinesin family
as a typical example for eukaryote-specific gene families.
We have cloned and sequenced cDNAs encoding kinesins
and kinesin-related proteins (KRPs) Giardia Iamblia, themost primitive protist that represents the earliest branching among extant eukaryotes and have obtained 13
kinesin-related cDNAs, some of which are likely
homologs of vertebrate kinesins involved in vesicle transfer to ER, Golgi, and plasma membrane. A phylogenetic
tree of the kinesin family revealed that most gene duplications that gave rise to different kinesin subfamilies with
distinct functions have been completed before the earliest
divergence of extant eukaryotes. This suggests that thecomplex endomembrane system has arisen very early ineukaryotic evolution, and diminutive ER and Golgi
apparatus recognized in the Girdial cells, together withthe absence of mitochondria, might be characters acquired
secondarily during evolution of parasitism. To understandthe divergence pattern of the kinesin family in the lineage
leading to vertebrates, seven more Unci 04-related
cDNAs have been cloned from sponge, amphioxus, hagfish, and lamprey. The divergence pattern of animal
Unc104/KIF1 subfamily is characterized by two active
periods in gene duplication interrupted by silent periodsof considerable long, but not proceeded gradually: Thus,in the eukaryotic lineage leading to vertebrates, the divergence of kinesin family might occurred episodically, as
demonstrated by at least three periods of extensive gene
duplications, instead of proceeding gradually. Extensive
subfamily-generating duplications in ancient times before
the earliest branching among extant eukaryotes may have
implications to the evolution of Giardial membranebounded organella.

JL---..L...lEukaryotes

JL-....-flllll---_..

Gnathostomes
Eumetazoans

Figure 1. Episodic divergence of kinesin family ineukaryotic lineage leading to vertebrates. Circles I, II and
III, periods in which extensive gene duplications occurred.
In the period I, basic kinesin genes corresponding to
subfamilies with different structures and functions were created by gene duplication and domain shuffling. In the later
periods II and III, the multiplicity of members in the same
subfamily increased by further gene duplications. Box, theCambrian explosion.

54

NATIONAL INSTITUTE

Publication List
lnokuchi R., Kuma, K., Miyata, T. and Okada, M. (2002)
Nitrogen-assimilating enzymes in land plants and algae:
Phylogenetic and physiological perspectives. Physiologia
Plantarum 116, 1-11.
Iwabe, N. and Miyata, T. (2002) Kinesin-Related Genes from
Diplomonad, Sponge, Amphioxus and Cyclostomes:
Divergence Pattern of Kinesin Family and Evolution ofGiardia! Membrane Bounded Organella. Mol. Bioi. Evol.
19, 1524-1533.

Katoh, K., Misawa, K., Kuma, K. and Miyata, T. (2002)
MAFFT: a novel method for rapid multiple sequencealignment based on fast Fourier transformation. Nucleic
Acids Res 30, 3059-3066.
Katoh, K. and Miyata,T. (2002) Cyclostome hemoglobins
are possibly paralogous to gnathostome hemoglobins ..!.
Mol. Evol. 55,246-249.

55

FOR BASIC BIOLOGY

Associate Professor:
MOCHIZUKI, Atsushi
JSPS Postdoctral Fellow: TOHYA , Shusaku
Graduate Student:
TAKANO, Ryohjic) .: Anlsotropyol v
1-0.9

I. 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 themodel , starting from an initial distribution very close touniformity, a spatial heterogeneity emerges and a stable
periodic pattern is formed spontaneously . This simple
mechanism suggests that the reaction of a small numberof chemicals and their diffusion might create stable nonuniform patterns. When we analyze the model in a twodimensional plane, striped patterns in addition to spotted
patterns often emerge. This was considered as the basic
mechanism explaining the stripe patterns observed among
animal coating.
We focused on the directionality of the stripes. Most of
the stripes observed in the fish skins are either parallel orperpendicular to their anterior-posterior (AP) axis. The
direction of stripes is considered of importance in the
behavioral and ecological viewpoints. However, very
little is known about the mechanisms that makes thestrong directionality either in the actual fish skin or in thetheoretical models . The standard RD model doesn't determine the direction of stripes. To explain the directionality of stripes on fish skin in closely related species, wehave studied the effect of anisotropic diffusion of the two
substances on the direction of stripes, in the cases inwhich both substances have the high diffusivity in thesame direction.
We also studied the direction of stripes in more generalsituations in which the diffusive direction may differ between the two substances. We derived a formula for the
direction of stripes , based on a heuristic argument of unstable modes of deviation from the uniform steady state.
We confirm the accuracy of the formula by computer
simulations. When the diffusive direction is different between two substances, the directions of stripes in the spatial pattern change smoothly 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.

-

-""~
...._

4-

·~

'

---

Figure 1. The stripe formation in the skin of Genicanthus .
These two species are closely related.

-<1.5:-'·::·~~· -1·-...... -Wflfl~;•::mn·. . . . .::~
. .::::. ~: ~~···~GGGOIOOG~...
···· - ···· ~.........
· - ····· ~~:::~··"'::
"0

(I"..
0

0

......

.. "0

0...

<>

......

"0

0.

Figure 2. Summary of the direction of stripe patterns obtained by the anisotropic diffusion model. Horizontal andvertical axes indicate anisotropy of activator and that of inhibitor, respectively. The left-upper corner indicates the
distortion of diffusion range. Each point indicates the direction of the observed stripe: horizontal; vertical ; or notdetermined. The direction is determined only by the difference 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).

II. Comparative Study of Circadian Clock Models
Many species living in an environment fluctuating witha strong daily rhythm have evolved an internal clock. According to molecular biological studies of circadian clock,many genes and proteins are involved in the biochemical
dynamics generating their stable rhythm. Although theidentity of genes and proteins may differ between species ,the basic mechanism is the same -- there is one or moregenes whose products may enter to the nucleus and thensuppress the transcription of their own gene(s). This
negative feedback with a long time delay can generate thecircadian rhythm.
In this study , we attempt to identify those aspects ofbiochemical systems for the circadian rhythm which promote sustainable oscillation. We discussed models ofdifferent complexity, and examined the effects of cellcompartmentalization, protein modification steps inchemical reactions, having multiple clock genes in a cell ,and cooperativity in the inhibition of transcription and in
the protein transport. To derive general biological conclusions , we focused on mathematical analyses of relatively
simple models, rather than computer simulations of complex realistic models .
First, using a Lyapunov function , we proved under very
general conditions that two-variable models always converge to a stable equilibrium , implying that additional
structures are needed for the model to generate a sustainable rhythm. Second, we compared several models ofdifferent complexity using the Routh-Hurwitz criteria of
stability. We showed that a sustainable oscillation is more
likely to occur; if the cell is compartmentalized so that theproteins need to be transported from the cytosol to the
nucleus ; if the proteins have to be modified before entering the nucleus; if the kinetics of transcription inhibitionor the transport to the nucleus have a nonlinear depend-

56

NATIONAL INSTITUTE

ence on the substrate concentration (cooperativity); or if
the products of two clock genes form a heterodimer thatsuppresses both of their own genes.
Specifically we analyzed the local stability mathematically for the cases: (1) there is the reaction reverse toprotein modification, (2) there are two clock genes, perand tim, instead of one -- which have been analyzed only
by computer simulations; and also the cases (3) the protein transport to the nucleus has cooperativity, which has
not been examined before.

I also compared the condition of cell-cell interaction
for generating the mosaic pattern with the different model,
the fate transition model. In the fate transition model, celldifferentiation regulated by interaction of neighbouring
cells is assumed to be responsible for the mosaic pattern.
The condition for obtaining the focal pattern is looser inthe cell rearrangement model than in the fate transition
model.

ill. 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 of light. In
retinas of some species of teleost fish, regular arrangements of cone cells are observed, where each subtype ofcone cells appears periodically in the two-dimensional
retinal sheet. Different patterns are observed in different
species. These patterns are called "cone mosaics." Thebiological mechanism of the pattern 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.
We have studied the mechanism of the pattern formation by using mathematical models. In this study, I examined a process of cell-cell interaction to generate theregular mosaic pattern -- namely cell rearrangement. In
the model, cells have already been determined as to theirfmal subtypes, but they change their locations in the pattern formation process and the cell movement is affectedby their neighbours. I study the conditions for generatingmosaic pattern.
First, an assumption regarding "double cone" is shown
to be necessary, whereby the two component cells of adouble cone behave as if they are a single cone, i.e. agreen cone and a red cone are never detached during thepattern formation process. Second, the correct pattern isderived by computer simulation only when the chosen
magnitude of cell adhesion is appropriate. I determine theconditions of adhesive strength for generating the observed pattern. These conditions lead to the testable predictions.

Figure 3. The example of obtained pattern by cell rearrangement model. The obtained patterns were completely
the same as the actual zebrafish retina, when the used adhesion was appropriate.

Figure 4. The successful values of adhesion parameters incell rearrangement model (a) and fate transition model (b).
The successful region in cell rearrangement model distribute linearly. On the other hand, horizontal and verticalvalues have to be balanced in fate transition model. The line
segments indicate the boundaries between the region ofzebrafish pattern and that of non-zebrafish patterns, which
are determined by analytical method. The lines explain the
distribution derived computer simulation very well.

IV. Formation and Maintenance of Distinctive Cell
Patterns by Co-expression of Membrane-botind
Ligands and Their Receptors
We showed that graded or checkerboard-like cell patterns, and segmental domains along a body axis, can begenerated by cell behaviors involving differences in intercellular repulsion. A membrane-bound signal transduction
system mediating contact-dependent cell interactions includes membrane-bound ligands (ephrins) and their receptors with tyrosine-kinase activity (Eph proteins). Thesemolecules mediate both repulsive and attractive interactions under bilateral threshold control: Cells expressing
the receptors adhere to a surface bearing a critical densityof ligand reciprocal to the density of receptor, but are
repelled by a surface with other densities of ligand. The
present paper extends this model: General membranebound ligands (not always ephrins) and their receptors are
assumed to be co-expressed in a single cell under bilateralthreshold control. Computer simulations of cell patternformation showed that when co-expression of the ligand
and receptor is reciprocal, the cells self-organize into apattern of segmental domains or a graded cell arrangement along the body axis. The latter process interprets
positional information in terms of protein molecules.
When co-expression of the two species of molecules is
not always reciprocal, the cells generate various patterns
including checkerboard and kagome (star) patterns. The
case of separate expression of ligands and receptors in
different cells is also examined. The mechanism of differences in cell repulsion is compared to the differential celladhesion hypothesis, which has been used to explain cellsorting.

FOR BASIC BIOLOGY

Publication List
Honda, H. and Mochizuki, A. (2002) Formation and maintenance of distinctive cell patterns by co-expression of
membrane-bound ligands and their receptors. Dev. Dyn.
223, 180-192.
Kurosawa, G., Mochizuki, A. and Iwasa, Y. (2002) Processes
promoting oscillations -- comparative study of circadianclock models. J. Theor. Biol. 216, 193-208.
Mochizuki, A. (2002) Pattern formation of cone mosaic inzebrafish retina: A Cell rearrangement model. J. Theor.
Bioi. 215, 345-361.

57
Shoji, H., Mochizuki, A., Iwasa, Y. and Kondo, S. (2002)
Directionality of Stripes Formed by Anisotropic ReactionDiffusion Models. J. Theor. Bioi. 214,549-561.
Tohya, S., Mochizuki, A. and Iwasa, Y. Random cell sortingcan form cone mosaic patterns in fish retina and explain
the difference between zebrafish and medaka. J. Theor.
Biol. (In Press)
Ryohji, T., Mochizuki, A. & Iwasa, Y. Possibility of Tissue
Separation Caused by Cell Adhesion. J. Theor. Biol. (InPress)

58

NATIONAL INSTITUTERESEARCH SUPPORT FACILITY
Head of Facility:
Associate Professor:

Research Associates:

Technical Staff:

MURATA, Norio
WATANABE, Masakatsu
MIKAMI, Koji
(Stress-Resistant Plants)
HAMADA, Yoshio(Tissue and Cell Culture)
UCHIYAMA, Ikuo
(Computer)
HIGASHI, Sho-ichi(Large Spectrograph)
MIWA, Tomoki (Computer)
NANBA, Chieko(Plant Culture, Farm, Plant Cell)NISHIDE, Hiroyo(Computer)
ITO,Makiko(Large Spectrograph.-June 2002)
ICHIKAWA. Chiaki(Large Spectrograph. November
2002-)YAGI, Eri (Tissue andCell Culture. June 2002 -)HARADA, Miyuki (Computer)
MAKIHARA, Nobuko(Computer)
SUZUKI, Keiko(Plant Culture, Farm, Plant Cell)

I. Facilities
1. 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 a wavelength
range from 250nm (ultraviolet) to 1 ,OOOnm (infrared) onto
its focal curve of lOrn in length. The fluence rate (intensity) of the monochromatic light at each wavelength ismore than twice as much as that of the corresponding
monochromatic component of tropical sunlight at noon
(Watanabe et al., 1982, Photochem. Photobiol., 36, 491498).
A tunable two-wavelength CW laser irradiation system
is also available as a complementary light source to OLS
to be used in irradiation experiments which specificallyrequire ultra-high fluence rates as well as ultra-high spectral-, time-and spatial resolutions. It is composed of ahigh-power Ar-ion laser (Coherent, Innova 20) (336.6528.7 nm, 20W output), two CW dye lasers (Coherent,
CR599-01) (420-930nm, 250-1000mW output), A/0
modulators (up to 40MHz) to chop the laser beam, a beam
expander, and a tracking microbeam irradiator (up to 200
pm s· 1 in tracking speed, down to 2 pm in beam diameter)with an infrared phase-contrast observation system.
2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell culture are provided. This laboratory is equipped with safely rooms
which satisfy the P2/P3 physical containment level. Thisfacility 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 Origin 2000), database server (Sun Enterprise 450), fileserver (Sun Enterprise 3000), data visualization terminal
and molecular simulation terminal (both are SGI Octanes).
Some personal computers and color/monochrome printers
are also equipped. On this system, we provide various
biological databases and data retrieval/analysis programs,
and support large-scale data analysis and database construction for the Institute members.
Computer laboratory also provides network communication services in the Institute. Most of PCs in eachlaboratory as well as all of the above service machines areconnected each other with local area network (LAN),
which is linked to the high performance multimedia backbone network of Okazaki National Research Institute(ORION). Many local services including sequence analysis service, file sharing service and printer service areprovided through this 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 P1 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, three green
houses (each 6m2) at the PI physical containment level, asmall farm, two greenhouses (45 and 88 m2) 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 a variety of growth chambers that precisely control the conditions of plant growth and dacilities for molecular biological and physiological enaluations of transgenic plants.
The laboratory is also a base of domestic and international collaborations devorted to the topic of stress resistant transgenic plants.

FOR BASIC BIOLOGY

II. Research Activities
1. Faculty
The faculty of the Research Support Facility conducts
its own research as well as scientific and administrative
public services.
(1) Photobiology: Photoreceptive and signal transduction mechanisms of phototaxis of unicellular algae
are studied action spectroscopically (Watanabe 1995,
In "CRC Handbook of Organic Photochemistry andPhotobiology")bymeasuring
computerizedvideomicrographs 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 glacilis (Fig . 1; Iseki et al. 2002 ,Nature 415 , 1047-1051): Euglena gracilis, a unicellular flagellate , shows blue-light type photomovements.
The action spectra indicate the involvement offlavoproteins as the photoreceptors mediating them.
The paraflagellar body (PFB) , a swelling near the baseof the flagellum has been considered as a photosensing
organelle for the photomovements. To identify thephotoreceptors in the PFB , we isolated PFBs andpurified the flavoproteins therein. The purified
flavoprotein (ca. 400 kDa), with noncovalently bound
FAD seemed to be a heterotetramer of a- and bsubu~its . Predicted amino acid sequences of each of
the subunits were similar to each other and contained
two FAD-binding domains each followed by anadenylyl cyclase catalytic domain. The flavoprotein
showed an adenylyl cyclase activity, which was

Fig .l Photoactivated adenylyl cyclase (PAC): a novel
blue-light receptor mediating photoavoidance inEuglena gracilis.

59
elevated by blue-light irradiation. Thus, theflavoprotein (PAC, photoactivated adenylyl cyclase)can directly transduce a light signal into a change in
the intracellular cyclic AMP level without any othersignal transduction proteins.
(2) Developmental Biology: Replacement of theankyrin repeats of mouse Notch2 gene with E.coli bgactosidase gene induces early embryonic lethality aroundE10.5. The lethality was suggested due to defects inextraembryonic 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 ofgiant and spongiotrophoblast cell layers are normal in the
mutant placenta, while vasculogenesis in the labyrinth
layer apperaed compromised at E9 .5. Since the lethality
was circumvented 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 the tetraploidchimeras 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 ofmutant cells to these affected tissues by El3 .5. Thus ,Notch2 are not cell autonomously required for the earlycell fate determination of labyrinthine trophoblast cells
and allantoic mesodermal cells, but plays an indispensablerole in the further formation of functional labyrinth layer
and umbilical cord.
(3) Computational Biology: Comparative genomics is a
useful approach to find clues to understanding complex
and diverse biological systems from rapidly growing
genome database . We have constructed a workbenchsystem for comparative analysis of microbial genomes
named MBGD . Since the number of completed microbial
genome sequences has now become more than a hundred ,we have enhanced the efficiency of the database greatly totreat such large number of genomes. One of the uniquefeatures of MBGD is to allow users to create their own
orthologous classification table with specified set oforganisms. By this approach, MBGD accommodates
comparative analysis for both closely related and distantly
related genomes.
We also continue to develop an automated method fororthologous grouping among multiple genomes , which isa key component of MBGD . ln addition to splitting fusion
genes into orthologous domains, we are also trying toenhance the algorithm to incorporate the information ofgene arrangement on each genome.
2. Cooperative Research Program for the Okazaki
Large Spectrograph.
The NIBB Cooperative Research Program for the Useof the OLS supports about 20 projects every year conducted by visiting scientists including foreign scien-tists
as well as those in the Institute.
Action spectroscopical studies for various regulatoryand damaging actions of light on living organisms, bio-

60
logical molecules, and organic molecules have been conducted (Watanabe, 1995, In "CRC Handbook of Organic
Photochemistry and Photobiology". pp, 1276- 1288).

Publication List:
I. Faculty

lseki, M., Matsunaga, S., Murakami, A., Ohno, K., Shiga, K.,
Yoshida, K., Sugai, M., Takahashi, T., Hori, T. and Watanabe, M. (2002). A blue-light-activated adenylyl cyclase
mediates photoavoidance in Euglena gracilis. Nature 415,
1047-1051.
Kanesaki, Y., Suzuki, 1., Allakhverdiev, Sl., Mikami, K. and
Murata, N. (2002) Salt strees and hyperosmotic stressregulate the expression of different sets of genes in
Synechocystis sp. PCC 6803. Biochem. Biophys. Res.
Commun. 290, 339-48.
Mikami, K., Kanesaki, Y., Suzuki, I. and Murata, N. (2002)
The histidine kinase Hik33 perceives osmotic stress andcold stress in Synechocystis sp.PCC 6803. Mol. Microbial.
46,905-15.
Ohmori, M., Terauchi, K., Okamoto, S. and Watanabe, M.
(2002). Regulation of cAMP-mediated photosignaling bya phytochrome in the cyanobacterium Anabaena
cylindrica. Photochem. Photobiol. 75,675-679.

NATIONAL INSTITUTE

Takami, H., Takaki, Y., Uchiyama, I. (2002) Genome sequence of Oceanobacillus iheyensis isolated from theIheya Ridge and its unexpected adaptive capabilities toextreme environments. Nucleic Acids Res. 30, 3927-3935.
Uchiyama, I. (2003) MBGD: microbial genome database forcomparative analysis. Nucleic Acids Res. 31, 58-62.
Il. Cooperative Research Program for the Okazaki Large
Spectrographlseki, M., Matsunaga, S., Murakami, A., Ohno, K., Shiga, K.,
Yoshida, K., Sugai, M., Takahashi, T., Hori, T. and Watanabe, M. (2002). A blue-light-activated adenylyl cyclase
mediates photoavoidance in Euglena gracilis. Nature 415,
1047-1051.
Ohmori, M., Terauchi, K., Okamoto, S. and Watanabe,M.(2002). Regulation of cAMP-mediated photosignaling
by a phytochrome in the cyanobacterium Anabaena
cylindrica. Photochem. Photobiol. 75, 675-679.
Toyoshima, M., Takinami, S., Hieda, K., Furusawa, Y. andNegishi, T.(2002).The involvement of cell cycle checkinduced inpoint-mutations in the mutagenesis
Drosophila by a longer wavelength light band of solar UV.
Photochem. Photobiol. Sci., 1, 178- 183.

61

FOR BASIC BIOLOGYTHE CENTER FOR ANALYTICAL INSTRUMENTS(managed by NIBB)Head of Facility:
Technical Staffs:

Technical Assistant:

MOROHASHI, Ken-ichirou
MORI, Tomoko
MAKINO, Yumiko
TAKAMI, Shigemi
NAKAMURA , Takanori
MORIBE, Hatsumi

The Center serves for amino acid sequence analysis,
and chemical syntheses of peptides and nucleotids tosupport researchers in NIBB and NIPS. Newly installed
instrument m 2002 is Confocal Laser Scanning
Microscope and Environmental Scanning Electron
Microscope . Instruments of the Center can be used byresearchers outside the Institute upon proposal.

Figure 1. MALDifTOF-MS .

Figure 2 . Environmental Scanning Electron Microscope.

Representative instruments are listed below.
Protein Sequencers (ABI Procise 494 , 473A)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A, 432A)
Plasmid Isolation Systems (Kurabo PI-100 L:, PI-50)
Automatic Nucleic Acid Isolation System (Kurabo NA2000)
DNA Sequencers (ABI PRISM 310 , ABI PRISM 3100,
ABI 377)
DNA/RNA Synthesizers (ABI 394 , 392)
Thermal Cyclers (Perkin ElmerPJ-9600 , Takara TP300)
Integrated Thermal Cyclers (ABI CATALYST Turbo
800)
Particle Delivery System (Bio-Rad BiolisticPDS1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Analysis System (Takara Glyco-Tag)
High Performance Liquid Chromatographs(Shimadzu LC-lOAD , LC-6AD, Waters 600E)
Integrated Micropurification System (Pharmacia
SMART)
Flow Cytometer (Coulter EPICS XL)
Biomolecular Interaction Analysis Systems(Pharmacia BIACORE 2000 , Affinity Sensors IAsys)
Laboratory Automation System(Beckman Coulter Biomek 2000)
NMR Spectrometer (Bruker AMX-360wb)
EPR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)
MALDI/TOF-MS (Bruker Daltonics REFLEX ill)
Inductively Coupled Plasma Atomic Emission
Spectrometer (Seiko SPS 1200A)
Spectrofluorometers (Hitachi 850, Simadzu RF-5000)
Spectrophotometers (Hitachi 330 , 557 , Varian Cary SG ,Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB
96P)Time-resolved Fluorescence Microplate Reader(Pharmacia DELFIA Research)
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 5000, BAS 2000)
Fluorescence Bio Imaging Analyzer (Takara FMBIO)Electrophoresis Imaging Systems (BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Environmental Scanning Electron Microscope(PHILIPS XL30 ESEM)
Confocal Laser Scanning Microscope (Leica TCS SP2)

62

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT
Head: HATI'ORI, Hiroyuki

Common Facility Group

Research Support Group

Chief: FURUKAWA, Kazuhiko

Chief: KOBAYASHI, Hiroko

Reseach Support Facilities
HIGASHI, Sho-Ichi (Unit Chief)
NANBA, Chieko (Subunit Chief)
NISHIDE, Hiroyo
IINUMA, Hideko
SUZUKI, Keiko (Technical Assistant)
MAKIHARA, Nobuko (Technical Assistant)
YAGI, Eri (Technical Assistant)
ICHIKAWA, Chiaki (Technical Assistant)
YAMAMOTO, Mamiko (Technical Assistant)
HARADA, Miyuki (Technical Assistant)

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

Radioisotope Facility
MATSUDA, Yoshimi (Unit Chief)
KATO, Yousuke (Subunit Chief)
ITOH, Takayo (Technical Assistant)Center for Analytical Instruments
MORI, Tomoko (Unit Chief)
MAKINO, Yumiko
TAKAMI, Shigemi
NAKAMURA, Takanori
MORIBE, Hatsumi (Technical Assistant)

Developmental Biology Group
TAKAGI, Chiyo
OKA,Sanae
Regulation Biology GroupYAMAGUCHI, Katsushi
TAKEUCHI, Yasushi
Gene Expression and Regulation Group I
MIWA, Tomoki (Unit Chief)
TANAKA, Sachiko (Subunit Chief)
HAYASHI, Koji
MOROOKA, Naoki
Gene Expression and Regulation Group IIOHSAWA, Sonoko (Unit Chief)
SAWADA, Kaoru (Subunit Chief)
SUMIKA WA, Naomi
MIKI, Kazuhiko

Glassware Washing Facility(KATO, Yousuke)(FURUKAWA, Kazuhiko)Integrated Bioscience GroupMIZUTANI, Takeshi
UTSUMI, Hideko
NODA, Chiyo
KONDO, Makiko (Technical Assistant)

The Technology Department is a supporting organization for researchers and research organization within the
NIBB. The Department develops and promotes the institute's research activities and at the same time, maintains
the research functions of the institute.
The department is organized into two groups: one, theCommon Facility Group, which supports and maintains
the institute's common research facilities and the other,
the Research Support Group, which assists the research
activities as described in individual reports.
Technical staffs participate, through the department, inmutual enlightenment and education increase their capability in technical area. Each technical staff is proceeded
to the fixed division usually and they support the various
research with their special biological and biophysicaltechniques.
The Department hosts an annual meeting for technicalengineers who work in various fields of biology at universities and research institutes throughout Japan. At thismeeting, the participants present their own activities anddiscuss technical problems. The Proceedings are published soon after the meeting.

63

FOR BASIC BIOLOGYCENTER FOR INTEGRATIVE BIOSCIENCE(jointly managed by NIBB)

Head: NAGAYAMA, Kuniaki

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 forPhysiological Sciences). The following projects will be the main focus of thecenter:1) Development, Differentiation and Regeneration. 2) Strategic

Methodology. 3) Bio-Environmental Science.

64

NATIONAL INSTITUTEDIVISION OF DEVELOPMENTAL GENETICS
Professor:
Research Associate:
Technical Staffs:
NIBB Research Fellow:
JSPS Postdoctral Fellow:
Postdoctoral fellow:
Graduate Students:

Technical Assistant:

Secretary:
1!

KOBAYASHI, Satoru
MUKAI, Masanori
NODA,Chiyo
SATO, Kimihiro
SHIGENOBU, ShujiAMIKURA, Reiko
HAYASHI, Yoshiki
UNNAI, Kohei 1J
KITADATE, Yu 1i
HAYASHI, Makoto 1!
UEDA, Yoshiko
TSUCHIYA, Naomi
ARITAKayo
SUl:UKl,Masako

Graduate School of Biological Sciences,
University ofTsukuba

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

I. Role of Mitochondrial Ribosomal RNAs in Pole
Cell Formation
Ultrastructural studies have shown that the germ plasm
is basically composed of polar granules and mitochondria.
While the primary roles of the mitochondria are oxidative
phosphorylation and biosynthesis of many metabolites, it
has now become evident that they are also involved in
germline formation.
In Drosophila, pole cell formation requires the functionof mitochondrial ribosomal RNA in germ plasm. We havepreviously reported that mitochondrial large rRNA
(mtlrRNA) and small rRNA (mtsrRNA) are both transported from mitochondria to polar granules. This transportation occurs during early embryogenesis, when mitochondria are tightly associated with polar granules ingerm plasm, and it depends on the function of the maternally-acting gene, tudor, that is known to be required forpole cell formation. Mitochondrial rRNAs remain on thepolar granules until pole cell formation and are no longer
discernible on the granules within pole 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 in embryos whose ability to form
these cells has been abolished by uv-irradiation. Theseobservations clearly show that the extra-mitochondrial
mtlrRNA on polar granules has an essential role in polecell 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 suggests the possibility that the protein(s) essential for polecell formation is produced by the mitochondrial-type ofribosomes. To address this issue, we examined the effectof Chloramphenicol and Kasugamycin on pole cell formation. Chloramphenicol and Kasugamycin are known to
inhibit mitochondrial (prokaryotic)-type of translation.
When these antibiotics were injected into the posteriorpole 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 suggestthat the mitochondrial-type of translation system must be
intact for the embryos to form pole cells.
ll. Role of Nanos protein in pole cell differentiation
Pole cells differ from the soma in regulation of mitosis
and transcriptional activity. Pole cells cease mitosis at
gastrulation and remain quiescent in the G2 phase of thecell cycle throughout their migration to the gonads, whilesomatic cells continue to proliferate during the rest ofembryogenesis. Furthermore, pole cells are transcriptionally quiescent until the onset of gastrulation, althoughtranscription is initiated in the soma during the syncytial
blastoderm stage. Consistent with this, RNA polymerase
II (RNAP II), but not RNA polymerase I, remains inactivein early pole cells. Thus, the ability to express zygotic
mRNA-encoding genes is suppressed only in pole cells inearly embryos.
Among the maternal components of germ plasm,
Nanos (Nos) is essential for the germline-specific eventsoccurring in pole cells. nos mRNA is localized in thegerm plasm during oogenesis, and is translated in situ to
produce Nos protein after fertilization. Nos is only transiently present in the posterior half of embryos during thepreblastoderm stage, and is required there for posterior
somatic patterning. Nos in the germ plasm is more stably
inherited into the pole cells at the blastoderm stage, remaining detectable in these cells throughout embryogenesis. Pole cells that lack Nos (nos- pole cells) are unable tofollow normal germline development; they fail to migrate
properly into the embryonic gonads, and consequently do
not become functional germ cells. In nos- pole cells, mitotic arrest at G2 phase is impaired, and they undergo
premature mitosis. Furthermore, nos- pole cells fail toestablish and/or maintain transcriptional quiescence, andectopically express somatically-transcribed genes, including fushi tarazu (jtz), even-skipped (eve) and Sex-

65

FOR BASIC BIOLOGY

lethal (Sxl).
Nos represses translation of mRNAs with discrete RNA
sequences called Nos response elements (NREs). In thepathway leading to posterior somatic patterning, Nos acts
together with unlocalized Pumilio (Pum) protein to repress translation of maternal hunchback (hb) mRNA. Thistranslational repression is mediated by binding of Pum to
NREs in the 3'-untranslated region (U1R) of hb mRNA.
In pole cells, Nos also acts with Pum to regulate germlinespecific events. Pum, like Nos, is required in pole cells fortheir migration to the gonads.
We found that Nos, along with Pum, represses translation of importin a2 (impa2) mRNA in early pole cells.
The impa2 mRNA contains an NRE-like sequence in its
3'-UTR and encodes a Drosophila importin a homologuethat plays a role in nuclear import of karyophilic proteins.
We found that Nos inhibits expression of a somaticallytranscribed gene, ftz, in pole cells by repressing Impa2dependent 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
repressed by Nos in pole cells through its effects on
Impa2-dependent nuclear import.
The above results raise the question whether the polecells lacking Nos (nos· pole cells) are able to differentiate
into somatic cells. However, it is difficult to study theirdevelopmental fate, since Nos also represses apoptosis ofpole cells, and almost all of nos· pole cells are eliminateduntil at least the end of embryogenesis. To overcome thisproblem, we used Df(3L)H99, a deletion for three genes
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 that some ofnos· H99. pole cells were able to differentiate as somatic
cells. This suggests that pole cells have the ability to differentiate as somatic cells, but its ability is inhibited by
Nanos activity.

m. Comprehensive analysis of genes expressed in
Drosophila gonad

Pole cells migrating into the gonads are specified to be
the primordial germ cells (PGCs). It has been believedthat zygotic genes expressed in pole cells within the gonads are required for their fate specification. To explore
the regulatory mechanism of germline specification, we
attempted to identify genes expressed in pole cells and/or
in somatic cells within the gonad by a comprehensive
approach. From the embryos carrying EGFP-vasa transgene that express GFP only in pole cells, we isolated the
gonads by using fluorescence-activated cell sorting(FACS), and costructed a gonad eDNA library. EacheDNA clone was sequenced from both 5' and 3' ends, andthese Expression Sequence Tags (ESTs) were computationally condensed into sequence clusters, which werethen subjected to whole-mount in situ hybridization(WISH). As of July 2002, approximately 10,000 of ESTs
were generated. The WISH analysis identified more than
30 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 germline
specification.

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) were able todevelop into the oocytes, but they failed to execute meiosis. We also found that the germline-specific expressionof vasa gene was severely affected in sva53 pole cells.
These results suggest that the maternal 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 llC
by using duplications and deficiencies. Within this region,
we found a gene encoding a Zn-finger transcription factor,of which mRNA is maternally supplied into embryos.

Publication List:
Inoue, S., Shimoda, M., Nishinokubi, 1., Siomi, M., Okamura,M., Nakamura, A., Kobayashi, S., Ishida, N. and Siomi, H.
(2002) A Role for the Drosophila Fragile X-Related Genein Circadian Output. Current Biology 12, 1331-1335.
Sarro, H., Nakamura, A. and Kobayashi, S (2002) Identification of a transcriptional regulatory region for germlinespecific expression of vasa gene in Drosophila melanogaster. Mech. Dev.112, 129-139.

66

NATIONAL INSTITUTEDIVISION OF MOLECULAR &DEVELOPMENTAL BIOLOGY
Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:

Technical Assistant:
11

TAKADA, Shinji
KOSHIDA , Sumito
UTSUMI, Hideko
OHBAYASHI, Norihiko
KAWAMURA , Akinori
MUROYAMA, Yuko
YAMAGUCHI, Yoshifumi 11
ISHII)A , Mio n
OKITAAkiyoshi 11
OGURA Satoshi 11
OHBAYASHI, Akiko
ODA , Ritsuko

ance 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 ofthis signaling by an induction gene trap screening inmouse ES cells. In at least three ES cell clones among 794
clones screened, expression of beta-geo 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 fromthese ES cell clones revealed that the spatiotemporal expression patterns of these reporter genes were well correlated to those of several Wnt genes. These results suggested that an induction gene trap approach is effective
for screening of target genes of Wnt signaling during
embryogenesis.

Graduate School of Biostudies, Kyoto University

The research interest of this laboratory is to understandmolecular mechanism of the vertebrate development. Particularly, roles of cell-to-cell signals, including membersof Wnt and fibroblast growth factor (FGF) families , arecharacterized. Evidence indicated that each signal is involved in many aspects of the vertebrate development.
For instance, we have revealed that Wnt-3a, a members of
Wnt family, plays essential roles in a number of aspectsof the mouse development, including somite development,
neural crest formation and neural development. However,cellular and molecular mecha-nisms how a cell signaling
molecule regulates these different events. Thus , we arefocusing on precise functional analysis of cell-to-cell signals and identifica-tion of target genes induced by thesesignals. In addition , to reveal molecular networks inwhich these signals are involved, we are also trying
genetical approach with the zebrafish.

I Roles of Wnt signals during embryogenesis
The Wnt family of genes that encode cysteine rich secreted proteins consists of at least 15 members in thevertabrate. It has already been shown that some of them
are expressed and play important roles during gasturulation. For instance, we showed that Wnt-1 and Wnt-3a,which are expressed in the most dorsal region within thedeveloping central nervous system, direct specification of
the dorsal interneurons. Analysis of mouse embryos
lacking both Wntl and Wnt3a and culture of explantsfrom the neural plate indicated that these Wnt signals
promote generation of the most dorsal subclass of theinterneuron, called D1 and D2, at the expense of that ofmore ventral subclass, called D3 (FIG. 1).
Wnt-3a is also expressed in the primitive streak ectoderm during gastrulation and in the tailbud in later development of the mouse. For dissecion of the complex developmental events regulated by Wnt-3a signaling inthese regions, it is important to identify genes regulatedby this signal. It has already been demonstrated that T(Brachyury) is a direct target of Wnt-3a in the anteriorprimitive ectoderm , which is fated to give rise to the paraxial mesoderm, suggesting that Wnt-3a modurates a bal-A

wild typeB
Wnt1-/-;Wnt3a-/-

FIG .I . Summary of the phenotype of dorsal interneuron development in Wnt-r'·/ Wnt-3a·'· embryos at EIO.S. Dl , D2 ,and D3 interneurons are indicated by the expression ofLH2,
Isll , and Pax2, respectively. The progenitors ofinterneurons are also subdivided by the expression of
Math! , Ngnl, and Mash!, respectively. Absence of Wnt-1
and Wnt-3a leads to diminished development of Dl and D2
neurons and to a compensatory increase of D3 neurons.

II Roles of Fgf signals in bone development
The Fgf family of cell signaling molecules is composedof at least 22 members in the mouse . Although Fgf signaling has been implicated in the bone development,
study on null mutant mice have not yet fully shown therole of this family in the skeletal development.
As an attempt to identify a candidate Fgf gene essential
for bone formation, we have examined the expression ofmouse Fgf18 gene using in situ hybridisation and demonstrated expression during calvarial and long bone development. Furthermore, to investigate the role of Fgfl8 invivo, we generated a null allele of the Fgf18 locus byhomologous recombination in ES cells. In Fgf18-deficientmouse embryos generated by gene targeting, progress ofthe suture closure is delayed (FIG . 2). Furthermore, terminal differentiation of the osteoblast is specifically delayed in developing calvaria and long bones. Proliferationof calvarial osteogenic mesenchymal cells is transiently
decreased. On the other hand , the number of proliferating
and differentiated chondrocytes is increased in the limbs.
Fgfl8 is thus essential for distinct aspects of cell

67

FOR BASIC BIOLOGY

proliferation and differentiation in bone formation. Thus ,Fgf18 plays essential roles in the osteogenesis and thechondrogenesis of the mammal and regulates differently
cell proliferation and differentiation in these twoprocesses.
To reveal the molecular mechanism that regulates theosteogenesis and the chondrogenesis, we are currently
analyzing function of several genes, which act on theseprocesses with Fgf-18 .

A

Fgf18

Fgfr2

III Screening of mutations affecting mesoderm
development in zebrafish
To identify genes involved m several aspects during
early embryogenesis of the vertebrate, we have performed
screening of zebrafish mutants induced by ENU, a chemical mutagen. Until now, we have screened 630 of F2
families and found a number of mutants whose phenotypes are different from those already reported . For instance, some of these mutants displayed defects in thesomite development. Cloning of genes that are responsible for these defects is in progress.

Publication List:
l[)

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0

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t osteogenic front
FIG.2 Fgf18 is required for normal bone development. (A)Expression of Fgf18 and its receptor, Fgfr2 , in the calvarialbone development. The expression of Fgf18 is first detectedin osteogenic mesenchymal cells surrounding the brain at
El2.5. At this stage, Fgfr2 is expressed in ventral
mesenchyme, where ossification is initiated. In later stages,
Fgf18 is also expressed in differentiating osteoblasts while
Fgfr2 is expressed intensely in cells at the osteogenic front.
(B) The phenotype of Fgns·'· embryos in the calvarial bonedevelopment. In the absense of Fgfl8 , calvarial ossification
is delayed.

limbo , T, Kawasaki, Y, Koyama, R, Sato , R, Takada, S,
Haraguchi , K, & Akiyama, T. (2002) Identification of alink between the tumour suppressor APC and the kinesin
superfamily. Nat. Cell Bioi . 4 323-327
Koshida, S., Shinya, M., Nikaido, M. , Ueno, N., SchulteMerker, S., Kuroiwa, A., & Takeda, H . (2002) Inhibition
of BMP activity by the FGF signal promotes posterior
neural development in zebrafish Dev. Bioi. 244,9-20.
Muroyama, Y., Fujihara, M. , Ikeya, M, Kondoh,. H., &Takada, S. (2002) Wnt signaling plays an essential role inneuronal specification of the dorsal spinal cord. Genes
Dev . 16, 548-55.
Ohbayashi , N., Shibayama, M. , Kurotaki, Y., Imanishi , M.,
Fujimori, T., Itoh, N., & Takada, S. (2002) Fgf18 is required for normal cell proliferation and differentiation
during osteogenesis and chondrogeneses. Genes Dev . 16,
870-879.
Ueda, Y, Hijikata, M , Takagi, S, Takada, R, Takada , S,
Chiba, T, & Shimotohno , K. (2002) Wnt/beta-cateninsignaling suppresses apoptosis in low serum medium andinduces morphologic change in rodent fibroblasts. Int . J.
Cancer. 99,681-688
Yamanaka, H, Moriguchi , T, Masuyama, N, Kusakabe , M,
Hanafusa, H, Takada, R, Takada, S, & Nishida E. (2002)
JNK functions in the non-canonical Wnt pathway toregulate convergent extension movements in vertebrates.
EMBO Rep. 3, 69-75

68

NATIONAL INSTITUTEDIVISION OF BIO-ENVIRONMENTAL SCIENCE
Professor:
IGUCHI, Taisen
Associate Professor:
WATANABE, Hajime
Research Associate:
KATSU, Yoshinao
Technical Staff:
MIZUTANI, Takeshi
Institute Research Fellow: SONE, Kiyoaki
Graduate Students:
KHONO , Satomi 11
MIYAGAWA , Shinichi
NAGATA, Emiko
Visiting Scientist:
KATO, Hideo 2!
Technical Assistants:
KAJIYAMA, Chieko
SUZUKI, Atsuko
KOBAYASHI, Kaoru
KOBAYASHI, Mika
TAKAHASHI, Eri
ITAMOTO, Misaki
HINAGO, Megumi
Secretary:
IMAIZUM/, Taekon Graduate School of Yokohama City University
21from Nihon Bioresearch Inc.

Synthetic chemicals found in the environment have thecapacity to disrupt endocrine system development andfunction 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 athreat to human health during development came fromstudies of diethylstilbestrol (DES), which was used toprevent premature birth and spontaneous abortion.
Laboratory experiments have demonstrated that exposureof animals to sex hormones during perinatal life can cause
permanent and ineversible alterations of the endocrine
and reproductive 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, the effects 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.
Normal Mouse

FIG . 1

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 to the development of cancer in humans has been emphasized. In theearly seventies, a close conelation between occunence ofvaginal clear cell adenocarcinoma in young women andearly intrauterine exposure to DES was demonstrated .
Many chemicals released into the environment have thepotential to disrupt endocrine function in wildlife andhumans . Some of these chemicals induce estrogenic
activity by binding to ER . The neonatal mouse model has
been utilized especially to demonstrate the long-term
effects of 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 inmice exposed prenatally to estrogen show altered expression of Hoxa genes and Wnt genes and the analysis ofknockout mice lacking Hoxa-10 or Wnt7a show uterine
hypoplasia. The growth response of neonatally DESexposed reproductive organs to estrogen is reduced, as are
ER levels and epidermal growth factor (EGF) receptor
levels , in addition to other hormone receptor levels.
Estrogenic compounds such as bisphenol A (BPA) andnonylphenol as well as dioxins and PCBs were found in
the human umbilical cord. BPA can easily cross theplacenta and enter the fetus in Japanese monkey and mice.
BPA can be found in fetal brain, testis and uterus when
given to pregnant mice and monkeys. Neonatal exposure
to a high BPA dose induced ovary-independent vaginalchanges , PF and infertility lacking corpora lutera.
Prenatal exposure to a low BP A dose induced accelerationof vaginal opening in the offspring. Thus, the developing
mammal is sensitive to exposure to estrogenic agents.
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 microarray

FIG. 2

Fluorescence image of an array

69

FOR BASIC BIOLOGY- - - - - -- --100000 ~
,

It

10000

"";::::;

for new target molecules that may be responsible for
endocrine disruption. In parallel, we also are studying theligand-binding mechanisms of nuclear receptors tohormones and other chemicals using Surface Plasmon
Resonance technology.

--: 10000.··• •.....J.

j•

<)..)

..

~

t •

~

•• :

~1000 [

; 1000

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100

)
10

-

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100

1000

: 100

-··---J1o
10000

100000Control
FIG .3 Scatter plot of average expression levels incontrol and chemical-treated utems
techiniques. We have found genes possibly related to theovary-independent changes by differential display. We
also have clustered groups of genes that are responsive to
estrogenic stimuli in uterus by using the DNA microarraysystem. We need to understand the molecular backgroundof the critical period during development, the low dose
effect of estrogenic chemicals and the molecular
metabolism of hormones and hormone-like agents inanimals including humans.

II. Effect of estrogen on amphibian and fishes
During embryogenesis , exogenous estrogen exposure
induces abnormal sex differentiation and the abnormalbone formation in African clawed frog, Xenopus laevi, thecyprinodont fish, mummichog (Fundulus heteroclitus)
and mosquitofish (Gambusia affinis). To analyze thefunction of estrogen, we have isolated eDNA clones of
ERa and ~ from F . heteroclitus, G. affinis and American
alligators . The estrogen-responsive genes must play important roles . We try to isolate the estrogen-responsive
genes to understand the molecular physiology of estrogenaction . Japanese tree frog (Hyla japonica) takes water
through ventral skin. We found that sex steroids and endocrine disruptors interfere with water absorption throughventral skin in frogs. Further, using the amphibian andfish as model animals we aim to analyze the effects ofnumerous chemicals released into the environment on
endocrine system function in wildlife .

III. Molecular Target Search
Abnormalities caused by endocrine disrupting chemicals are reported but the molecular mechanisms of theeffects are not well studied. Although estrogen receptor is
one of the strongest candidates possibly responsible forthe endocrine disrupting function of many chemicals , italone cannot explain the variety of phenomena induced by
endocrine disrupting chemicals. Thus , we are also looking

Publication List:
Buchanan , D.L. , Ohsako, S., Tohyama, C., Cooke, P.S . andIguchi , T . (2002) 2,3 ,7 ,8-Tetrachlorodibenzo-p-dioxin(TCDD) inhibition of estrogen-induced mouse uterine
epithelial mitogenesis involves changes in cylin and transforming growth factor-beta expression. Toxicol . Sci. 66,
62-68 .
Honma, S., Suzuki, A., Buchanan , D.L. , Katsu, Y ., Watanabe,
H. and Iguchi, T. (2002) Low dose effect of in utero exposure to bisphenol A and diethylstilbestrol on female mouse
reproduction . Reprod. Toxicol . 16, 117-122.
Katsu, Y., Takasu , E. and Iguchi , T . (2002) Estrogenindependent expression of neuropsin , a serine protease in
the vagina of mice exposed neonatally to diethylstilbestrol.
Mol . Cell. Endocr.l95, 99-107.
Miyagawa, S., Buchanan , D.L. , Sato , T. , Ohta, Y., Nishina,
Y. and Iguchi , T . (2002) Characterization of diethylstilbestrol-induced hypospadias in female mice . Anat. Rec. 266,
43-50.
Okada, A., Ohta, Y., Buchanan, D.L., Sato , T . and Iguchi , T.
(2002). Effect of estrogens on ontogenetic expression ofprogesterone receptor in the female rat reproductive tract.
Mol. Cell. Endocr. 195 , 55-64.
Shimamura, M., Kodaira, K., Hino , K., lshimoto , Y ., Tamura,
H. and Iguchi , T. (2002) Comparison of antiandrogenic
activities of vinclozolin and D,L-camphorquinone inandrogen receptor gene transcription assay in vitro andmouse in utero exposure assay in vivo. Toxicology 174,
97-107.
Suzuki , A., Sugihara, A., Uchida , K., Sato , T ., Ohta, Y.,
Katsu , Y., Watanabe , H. and Iguchi, T. (2002) Developmental effects of perinatal exposure to bisphenol-A anddiethylstilbestrol on reproductive organs in female mice .
Reprod. Toxicol . 16, 107-116.
Uchida , K., Suzuki, A., Kobayashi , Y ., Buchanan , D.L. , Sato ,T. , Watanabe , H. , Katsu , Y., Suzuki , J., Asaoka, K., Mori,C. , Arizona, K. and Iguchi, T. (2002). Bisphenol-A administration during pregnancy results in female exposurein mice and monkeys. J . Health Sci. 48, 579-582.
Uchida, D. , Yamashita , M., Kitano , T . and Iguchi, T . (2002)
Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile
zebrafish. J. Exp. Bioi. 205 , 711-718 .
Urushitani , H., Shimizu, A. , Katsu , Y. and Iguchi , T . (2002)
Early estrogen exposure induces abnormal development ofFundulus heteroclitus. J. Exp. Zoot . 293 , 693-702.
Watanabe , H. , Suzuki , A. , Mizutani , T ., Khono S. , Lubahn ,D.B. , Handa, H. and Iguchi, T. (2002) Genome-wide
analysis of changes in early gene expression induced byoestrogen . Genes to Cells 7, 497-507.

70

NATIONAL INSTITUTEDIVISION OF PLANT DEVELOPMENTAL
GENETICS

Associate Professor:
Research Associate:
Technical Staff:
Post Doctoral Fellow:
Graduate Student:
Secretary:

TSUKAYA, Hirokazu, PhD.
HORIGUCHI, Gorou, PhD.
KONDO, Makiko
TAKABE, Eriko
CHO, Kiuhyon, PhD.
KOZUKA, Toshiaki
NARITA, Noriyuki
KABEYA, Kazuko

Diversity of plant form is mostly attributable to variation of leaf and floral organs, which are modified leaves.
The leaf is the fundamental unit of the shoot system,
which is composed with leaf and stem. So the leaf is the
key organ for a full understanding of plant morphogenesis.
However, the genetic control of development of theseshapes had remained unclear. Recently, studies of leaf
morphogenesis has been in a turning point, after our successful application of the techniques of developmental
and molecular genetics to it, using model plants, Arabidopsis thaliana (L.) Heynh (reviewed in Tsukaya 2002a,b).

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 in Arabidopsis shows that a compensatory system(s) act in leaf
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 the size and number of cells by the compensatory system(s). Based onthese facts, a new perspective on understanding of
mechanisms for leaf morphogenesis, Neo-Cell theory, is
proposed (Tsukaya, 2002a; Tsukaya, 2003). Neo-Celltheory is the Cell theory which stipulated positional cooperation of the behavior of cells. In short, Neo-Cell theory
hypothesizes as follows: cells are the unit of morphogenesis; however, each cell is also controlled by factors thatgovern the morphogenesis of the organ of which the cells
are apart.
Focusing on mechanisms that govern polarized growthof leaves in a model plant, Arabidopsis thaliana, wefound that the two genes act independently to each otheron the processes of polar growth of leaves: the ANGUSTIFOLIA (AN) gene regulates width of leaves and the
ROTUNDIFOLIA3 (ROT3) gene regulates length ofleaves. The AN gene controls the width of leaf blades andthe ROT3 gene controls length. Cloning of the AN gene
(Kim et al., 2002) revealed that the gene is a member of
CtBP (C-terminal binding protein) gene family which had
been found from animal kingdom. In the animal kingdom,
CtBPs self-associate and act as a co-repressors of transcription. We found that also the AN protein can selfassociate in yeast two-hybrid system (Kim et al. 2002).
Furthermore, microarray analysis suggested that the ANgene might regulate the expression of certain genes, forexample, the gene involved in formation of cell walls,
MER/5. We also found that the abnormal arrangement ofcortical MTs in an-I mutant leaf cells appeared to accountentirely for the abnormal shape of the cells. It suggested
that the AN gene might regulate the polarity of cell growthby controlling the arrangement of cortical MTs.
The ROT3 gene was cloned by us in 1998. Transgenic
experiments proved that the ROT3 gene regulates leaflength without affect on leaf-width via biosynthesis ofsteroids (Kim et al., 1999). In relation to it, we recently
revealed that a steroid hormone, brassinosteroid, controls
both proliferation and expansion of leaf cells (Nalaya etal., 2002). In addition, 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 morphological mutants examined. In contrast, both the size and the numberof cells were affected by the mutations examined in leaf
blades. Studies of the roles of light signal perception inleaf development are now underway.
Apart from the aspects of leaf expansion, we also analyzed genes for identification of leaf primordia. The AS!,AS2 and BOP genes are needed for determinate growth ofleaf. Molecular and anatomical analysis of the AS2 genehas been carried out in collaboration with a research teamof Prof. Machida, Nagoya University (Endang et al.,
2001; Iwakawa et al. 2002). AS2 gene is a member of anovel gene family, including 42 putative genes in Arabidopsis genome, encoded proteins with cysteine repeats
and a leucine zipper (Iwakawa et al. 2002). The geneproduct also represses the expression of class 1 knox homeobox genes in leaves (Endang et al. 2001; Iwakawa et
al. 2002), as known for ASI. We also isolated and analyzed a novel mutant, bop (blade-on-petiole) mutant,which strongly enhances the as2 phenotype, in collaboration with a reaseach team of Prof. Nam, POSTECH, Korea (Ha et al. 2003). Three class I knox genes, namely,
KNJITJ, KNJIT2 and KNJIT6, were misexpressed in theleaves 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. We, thus, suggest that BOPJ
promotes or maintains a developmentally determinate
state of leaf cells through regulation of class I knox]
genes (Ha et al. 2003).
On the other hand, we are trying to identify molecularmechanisms which distinguish developmental pathway ofleaves from that of shoots by studying tropical plants with'fuzzy' morphology. For such purposes, we introduced
tropical plants having queer developmental program forleaf morphogenesis, namely, Chisocheton, Guarea andMonophyllaea, as materials for the studies. The indeterminate compound leaves of members of the genus Chisocheton in Southeast Asia and of the genus Guarea in the
New World and Mrica, in the family Meliaceae, areunique and can develop indeterminately as a result of theactivity of the leaf apical meristem, which can function
very similarly to a shoot apical meristem. We performed amolecular phylogenetic study of these genera, and theresult suggested that indeterminate program in the leavesof members of these two genera might have evolved only
once in Meliaceae (Fukuda et al. 2003).
In addition, we are interested in environmental adapta-

71

FOR BASIC BIOLOGY

tion of leaves , from view point of biodiversity. For example , thermograph analysis of function of downy bracts ofa so-called snowball plant, Saussurea medusa that habits
in alpine zone of Yunnan , China, revealed that the downybracts of S . medusa have two functions: thermal insulation to protect the inside of flowers and the accumulation of heat on the upper surfaces of the inflorescence(Tsukaya et a! . 2002a). Anatomical analyses of various
types of morphological adaptation of leaves against certain kinds of environments were also performed (Tsukaya,
2002d, e, f, g) . In particular, leaf index, relative length ofleaf to width , is also the most diverse factor of leaf shape,and is affected by environmental factors in some plants(Tsukaya, 2002c; Tsukaya et a!. b) . Are AN and ROT3
genes are involved in regulation of adaptive change ofleaf index in natural condition? Are these genes the responsible for evolution of rheophytes? So called"Evo/Devo" study of leaf morphogenesis is also one of
our research project in NIBB .

Publication List:
Fukuda, T. , Yokoyama, J. and Tsukaya, H . (2003) The evolutionary origin of indeterminate leaves in Meliaceae:
phylogenetic relationships among species in the genera
Chisocheton and Guarea, as inferred from sequences ofchloroplast DNA . Int. J. Plant Sci. 164, 13-24.
Ha, C.-H ., Kim, G.-T. , Kim, B.-C., Jun , J.-H ., Soh , M.-S. ,Ueno, Y., Machida, Y., Tsukaya, H. and Nam, H.-G .
(2003) The BLADE-ON-PETIOLE gene controls leaf pattern formation through regulation of meristematic activity.
Development 130,161-172.
Iwakawa , H., Ueno , Y ., Semiarti, E., Onouchi, H., Kojima, S. ,Tsukaya, H., Hasebe,M ., Soma, T ., Ikezaki, M. ,Machida,C., and Machida, Y. (2002) The ASYMMETRICLEA VES2 gene of Arabidopsis thaliana, required for formation of a symmetric flat leaf lamina , encodes a memberof a novel family of proteins characterized by cysteine repeats and a leucine zipper. Plant Cell Physiol. 43, 467478 .
Kim, G.-T ., Shoda,K., Tsuge, T., Cho , K.-H ., Uchimiya, H.,
Yokoyama, R ., Nishitani, K. and Tsukaya, H. (2002) The
ANGUSTIFOLIA gene of Arabidopsis , a plant CtBP gene,
regulates leaf-cell expansion, the arrangement of cortical
microtubules in leaf cells and expression of a gene involved in cell-wall formation . EMBO J. 21, 1267-1279.
Kim , G.-T. and Tsukaya , H. (2002) Regulation of the Biosynthesis of Plant Hormones by Cytochrome P450s . J.
Plant Res. 115,169-177.
Nakaya, M., Tsukaya, H. , Murakami , N. , and Kato , M.
(2002) Brassinosteroids control the proliferation of theleaf cells in Arabidopsis thaliana. Plant Cell Physiol. 43,
239-244.
Stoynova-Bakalova, E., Petrov, P ., and Tsukaya, H. (2002)
Cell division and cell enlargement in isolated Cucurbita
cotyledons grown in darkness and in light. J. Plant Res.
115, 375-380.
Tsukaya, H. (2002a) Interpretation of mutants in leaf morphology: genetic evidence for a compensatory system inleaf morphogenesis that provides a new link between Celland Organismal theory . Int. Rev. Cytol. 217,1-39.
Tsukaya, H . (2002b) Leaf Development, The Arabidopsis
Book, eds. C.R. Somerville and E .M. Meyerowitz, American Society of Plant Biologists, Rockville, MD ,doi/10.1199/tab.0072 ,http://www .aspb.org/downloads/arabidopsis/tsukaya.pdf
Tsukaya, H . (2002c) The Leaf Index: Heteroblasty , Natural
Variation, and the Genetic Control of Polar Processes ofLeaf Expansion. Plant Cell Physiol. 43,372-378 .
Tsukaya, H. (2002d) Morphological adaptation of bracts in
Sino-Himalayan plants that bloom at low temperatures Downy plants and glasshouse plants - Newslet. Him . Bot.
No. 30,1-7 .
Tsukaya, H. (2002e) Molecular mechanisms related to theevolution of leaf shape , with a special emphasis on leaf
index . In Himalayan Botany in the Twentieth and Twentyfirst Centuries. Edited by Noshiro, S. and Rajbhandari ,K.R. pp. 152-154, The Society of Himalayan Botany
Tokyo , Tokyo.
Tsukaya, H . (2002f) Optical and anatomical characteristics ofbracts from the Chinese "Glasshouse" plant, Rheum
alexandrae Batalin (Polygonaceae) , in Yunnan, China. J.
Plant Res. 115,59-64.
Tsukaya, H. (2002g) Leaf anatomy of a rheophyte,
Dendranthema yoshinaganthum (Asteraceae), and of hybrids between D . yoshinaganthum and a closely related
non-rheophyte species , D. indicum. J. Plant Res.115, 329333 .
Tsukaya, H. (2003) Organ shape and size: a Jesson fromstudies of leaf morphogenesis . Curr. Opin. Plant Bioi. 61,
57-62.
Tsukaya,H ., Fujikawa , K. and Wu , S.-G. (2002a) Thermal
Insulation and Accumulation of Heat in the Downy Inflorescences of Saussurea medusa (Asteraceae) at High Elevation in Yunnan, China. J. Plant Res. 115,263-268.
Tsukaya, H., Kozuka, T. and Kim , G.-T. (2002b) Genetic
control of petiole length in Arabidopsis thaliana . Plant
Cell Physiol. 43, 1221-1228.

Leaf shape control by various genes in Arabidopsis

thaliana.

72

NATIONAL INSTITUTEDIVISION OF BIOINFORMATICS

Professor (Adjunct):

NAKAMURA, Haruki(Osaka University)

The aim of this laboratory is structural bioinformatics
covering molecular modeling and design of proteins andother biological macromolecules: Development of a new
database, eF-site, for protein surface geometry with thephysicochemical properties, and identification of proteinfunctions using the database, and Development of new
algorithms and so:ftwares for large scale simulation
calculations by parallel computers to examine free energy
landscapes ofbiomolecular systems.

Publication list:
Fukuda, I. and Nakamura, H. (2002) Tsallis Dynamics Using
the Nose-Hoover Approach. Physical Review E., 65,
26105.
Kamiya, N., Higo, J., Nakamura, H. (2002) Conformational
Transition States of Beta-hairpin Peptide between the Ordered and Disordered Conformations in Explicit Water.
Protein Science, 11, 2297-2307.
Ono, S., Kuroda, M., Higo, J., Nakajima, N., and Nakamura,
H. (2002) Calibration of Force Field Dependency in Free
Energy Landscapes of Peptide Conformations by Quantum Chemical Calculations. Journal of Computational
Chemistry, 23, 470-476.
Tsuchiya, Y., Kinoshita, K., and Nakamura ,H. (2002)
Analysis of Complementarity of Protein-DNA Interactions
Using the Electrostatic Potential and the Molecular Surface Geometry. Genome Informatics, 13, 310-311.

73

FOR BASIC BIOLOGYCENTER FOR RADIOISOTOPE FACILITIES (CRF)
Head:YAMAMORI, Tetsuo(Professor, concurrent post)
Associate Professor:

Technical Staffs:

Supporting Staff:

OGAWA , Kazuo(Radiation Protection
Supervisor)
MATSUDA, Yoshimi(Radiation Protection
Supervisor)
KATO, Yosuke(Radiation Protection
Supervisor)
MOROOKA , Naoki(Radiation Protection
Supervisor)
ITO, Takayo/IDA , Yumi
KATA GIRl, Izumi

I. Research supporting activity
Technical and supporting staffs of the CRF are serving
the purchase of radioisotopes from JRA (Japan
Radioisotope Association) and the transfer of radioisotope
wastes to JRA. The physical maintenance of thecontrolled areas where radioisotopes are used is also oneof our business.
In this summer (July 1, 2002) , a new radioisotope
facility opened at the area E about 1km apart from thearea A where four controlled areas have been built: Center,
NIBB-sub, LGER (Laboratory of Gene Expression andRegulation)-sub, and NIPS (National Institute forPhysiological Science)-sub. Each facility is maintained
under the two radiation protection supervisors . Matsuda
and Kato are named to the supervisors in the area A, andOgawa and Morooka in the area E. Ito and Iida support
them at the area A, and Katagiri at the area E.
Users going in and out the controlled areas counted by
the monitoring system are 6 ,700 persons in 2002. The
items in each controlled area is presented in Figure 1.

(NIBS-sub)
Actual counts
Area A
Center
NIBS-sub
LGER-sub
NIPS-subArea E
total

2,359
956
3,320
924
700
6,700

Figure 1. Percentage of users going in and out thecontrolled areas in 2002.

II. Academic activity
Academic activity by teaching staff is focused on theanalysis of the structure and function of a dynein motor
protein. 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 (Ogawa et a!., 1977) and the mitotic apparatus
of cleaving egg (Mohri et a!., 1976) visualized by antiaxonemal dynein (Fragment A) antibodies.
The native dyneins are very large and range inmolecular mass up to 1 to 2 mega Da. They are complex
proteins containing heavy, intermediate, and light chains
defined by the molecular mass. Our present project is themolecular cloning of polypeptides contained in outer arm
dynein of 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 cloning of
this huge polypeptide (520 kDa) was performed in ourlaboratory in 1991. Since then eDNA clones for axonemal
and cytoplasmic dyneins have been isolated in a variety oforganisms. The sequences of heavy chains , without
exception , contain four P-loop motives referred to as ATPbinding sites in the midregion of the molecules. Figure 3A
and B draw the structure of heavy chain deduced from theamino acid sequence (Ogawa, 1992) . Outer arm dynein
contains three intermediate chains (IC1, IC2, and IC3)that range in molecular mass from 70 to 120 kDa. IC2 andIC3 were cloned by Ogawa et a!. (1995) and contain the
WD repeats in the carboxy-terminal halves of themolecules. By contrast, IC1 is not a member of the WD
family . ICl has a unique sequence such that the Nterrninal part is homologous to the sequence ofthioredoxin, the middle part consists of three repetitive
sequences homologous to the sequence of NDP kinase ,and the C-terminal part contains a high proportion ofnegatively charged glutamic acid residues (Ogawa et a!. ,1996) . Thus, IC1 is a novel dynein intermediate chain
distinct from IC2 and IC3 and may be a multifunctional
protein.
Six light chains with molecular masses of 23.2, 20.8,
12.3, ll.5 , 10.4, and 9.3 kDa are associating with outerarm dynein . We have already isolated eDNA clone of fiveLCs. LCl (23.2 kDa) and LC3 (12 .3 kDa) are highly
homologous to mouse Tctex2 and Tctex 1, respectively,
which are encoded by the t complex region that is
involved in transmission ratio distortion (TRD), malesterility and the development of germ cells. Our finding
raises the possibility that axonemal dynein proteins are
involved in this phenomenon. TRD may be caused by thedysfunction of multiple axonemal dynein proteins.

74

NATIONAL INSTITUTE

Mitotic
Egg

Sperm
AxonemeFigure 2. Localization of two dynein isotypes on outer arm of sperm axonemes andmitotic apparatus of cleaving egg.

A----~~Pl P2 P3

N

M

P4

a2

-==-

c

B

c
Figure 3 . Structure of dynein heavy chain. A; Analysis ofamino acid sequence of heavy chain reveals that it consistsof three major domains referred to as N, M, and C from the
N-terminus. B and C; M and C domains make larger
domain (motor domain) by intramolecular association .

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