NATIONAL INSTITUTE FOR BASIC BIOLOGYANNUAL REPORT

2001

CONTENTS
INTRODUCTION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ... · · · · · · ·ORGANIZATION OF THE INSTITUTE · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·2

MEMBERS OF THE COUNCIL · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ....... ·......

4

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

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

DIVISION OF CELL MECHANISMS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·I0

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

DIVISION OF CELL PROLIFERATION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·15

DIVISION OF CELLULAR COMMUNICATION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·18

DIVISION OF CELL FUSION (ADJUNCT) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·20

DEPARTMENT OF DEVELOPMENTAL BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·23

DIVISION OF REPRODUCTIVE BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·24

DIVISION OF CELL DIFFERENTIATION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·27

DIVISION OF MORPHOGENESIS · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·30

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

33

DIVISION OF MOLECULAR NEUROBIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·34

DIVISION OF CELLULAR REGULATION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·38

DIVISION OF BIOLOGICAL REGULATION AND PHOTOBIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·41

DIVISION OF BEHAVIOR AND NEUROBIOLOGY··································································

43

LABORATORY OF GENE EXPRESSION AND REGULATION · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·45

DIVISION OF GENE EXPRESSION AND REGULATION I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·46

DIVISION OF GENE EXPRESSION AND REGULATION II· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·49

DIVISION OF SPECIATION MECHANISMS I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·51

DIVISION OF SPECIATION MECHANISMS II · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·53

RESEARCH SUPPORT · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·57

CENTER FOR TRANSGENIC ANIMALS AND PLANTS· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·RESEARCH CENTER FOR INTEGRATNE AND COMPUTATIONAL BIOLOGY · · · · · · · · · · · · · · · · · · · · · · · · · · · ·RESEARCH SUPPORT FACILITY · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·THE CENTER FOR ANALYTICAL INSTRUMENTS (managed by NffiB) · · · · · · · · · · · · · · · · • · · · · · · · · · · · · · · · · • • ·

58
59
60
63

TECHNOLOGY DEPARTMENT · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·64

CENTER FOR INTEGRATIVE BIOSCIENCE Gointly managed by NIB B) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·65

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION I · · • · · · · · · · · · · · · · · · · · · · · ·DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION II························

66
68

DEPARTMENT OF BIOENVIRONMENTAL RESEARCH I · · · · · · · · · · · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • • · · ·DEPARTMENT OF BIOENVIRONMENTAL RESEARCH II· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·DEPARTMENT OF BIOENVIRONMENTAL RESEARCH III · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

71
73
74

CENTER FOR RACIOISOTOPE FACILITIES (CRF) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·75

The cover photographs are FISH images which indicate amplification ofa specific DNA region, called HotA, on the genome of Escherichia coli.
For details, see p49.

IN1RODUCTION
The National Institute for Basic Biology (NIBB) is agovernment-supported research institute established in
1977. The aim of the NIBB is the promotion andstimulation of studies in the field of Biology. As a
Center of Excellence (COE), NIBB promotes not only
the basic biology but also the modern biologicalsciences by conducting first-rate research on site as wellas in cooperation with other universities and researchorganizations. Researchers at NIBB investigate cellstructure and function, reproduction and development,
neuronal and environmental biology, gene expression
and regulation, and molecular evolution of eukaryoticorganisms to elucidate the general and fundamental
mechanisms underlying various biological phenomena.
In the beginning of April, 2001, Dr. KATSUKI,
Motoya, the former Professor of the University ofTokyo and the adjunctive Professor of the NIBB, took
over the positio~ of the Director-General from theformer Director-General Dr. MOHRI, Hideo, who
retired from the NIBB due to his appointment as the
President of the Okazaki National Research Institutes.
In April, Dr. KOBAYASHI, Satoru, who had been an
Assistant Professor of Tsukuba University, became the
new Professor of the Division of Development,
Differentiation and Regulation in the Center for
Integrative Bioscience (CIB) and made a joint
appointment for the NIBB . Dr. TAKADA, Shinji, who
is a Professor of Kyoto University, was also jointappointed as a Professor of CIB and Kyoto University
in March . He will join the CIB in April, 2002, as a
Professor.
Drs. MURATA, Takashi and KINOSHITA, Noriyuki
were appointed as Associate Professors in the Divisionof Speciation Mechanisms II in April and the Divisionof Morphogenesis in September, respectively.
Drs. MUKAI, Masanori and KOSHIDA, Sumito,
were appointed as Research Associates in CIB in April.
Drs. SAKUTA, Hiraki and TSUGANE, Kazuo werealso appointed as Research Associates in NIBB in
October.
Congratulatingly, Drs. SAKAMOTO, Atsushi,
INAGAKI, Yoshishige and NISHIYAMA, Yoshitaka
were promoted to Associate Professors of Hiroshima
University , Okayama University and Ehime University,
respectively .
Associate Professor Dr. MAEDA
Nobuaki left NIBB and became a staff scientist viceDirector (equivalent to Associate Professor) of theTokyo Metropolitan Institute for Neuroscience inNovember.
Three research associates left theirpositions in NIBB to become the equivalent positions inother institutes or overseas. In addition, we replaced 2
research associates, and 2 institute research fellows
with 2 research associates and 7 institute research
fellows, and appointed 1 technician. The total numberof personnels working at NIBB including graduate
students and post doctoral 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. In March the 45'h NIBB International
Conference was sponsored by the Ministry of Education,
Culture, Sports, Science and Technology and Ministryof Environment entitled "Recent Progress in Endocrine
Disruptor Research" (Professor T. Iguchi, organizer).
The 46'h NIBB International Conference entitled"Genetics and Epigenetics: The First 100 Years"(Professors S. Ilda, M. Katsuki, S. Inagaki, et a!.,organizers) sponsored by the Ministry of Education,
Culture, Sports, Science and Technology was also heldin March. In addition, NIBB continues to sponsor
interdisciplinary symposia and study meetings oncurrent topics by inviting leading scientists from aroundthe world to the Institute. NIBB also provides a
training course in biological sciences for young
investigators. To assess our continuing improvement,
the activities and future plans of two professors who
have spent I 0 years at NIBB were subjected to peer
review by international scholars in related fields. Prof.
NISHIMURA, Mikio and Prof. HORIUCHI, Takashi
were reviewed on the I 0-year Evaluation by thecommittee organized by the former Director-General Dr.
MOHRI and the results were accepted by the Advisory
Council of the NIBB held in March. We always
welcome any suggestions concerning the research
activities of NIBB .
Finally, we performed the "Open Institute" for local
society on 13'h of October to show our activities andabout 1,600 people joined this event.

KATSUKI, Motoya, D.Sc.
Director-General

2

NATIONAL INSTITUTE

ORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology (NlBB) isan Institute in the Okazaki National Research Institutes(ONRI) that are composed of three independent
organizations, NIBB, the National Institute for Physiological Sciences (NIPS) and the Institute for Molecular
Science (IMS). They located on a hill overlooking theold town of Okazaki. NIBB was established in 1977 andits activities are supported by Monbukagaku-sho (theMinistry of Education, Culture, Sports, Science andTechnology: Mext) of Japan. The Division of Development, Differentiation and Regulation in the Center
for Integrative Bioscience that was established as acommon facility of the ONRI in 2000 began in 2001.
The Center for Integrative and Computational Biology
began in NIBB in April, 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 ofNlBB.
The Advisory Committee, comprised of professors
within the Institute and an equal number of leading
biologists outside NIBB advises the Director-General,
upon his request, on planning joint research programs
and other important matters in NIBB, as well as on thescientific 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.

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.

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.

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.

FOR BASIC BIOLOGY

3

Okazaki National Research Institutes

I

National Institute for
Basic BiologyJ

ri_Division of Cell Mechanisms
HDivision of Bioenergetics

r-JDepartment of Cell Biology~ HDivision of Cell Proliferation (adjunct)HDivision of Cell Fusion (adjunct)
YDivision of Cellular Communication (adjunct)rlDivision of Reproductive Biology
HDivision of Cell Differentiation

HDepartment of Developmental
Biology

HDivision of Morphogenesis'--

HDivision of Developmental Biology (adjunct)I

rlDivision of Molecular Neurobiologyf-HDivision of Cellular Regulation

Department of Regulation
Biology

HDivision of Biological Regulation (adjunct)yDivision of Behavior and Neurobiology(adjunct)
Division of Gene Expression and Regulation I

H Laboratory of Gene
Expression and Regulation}

HDivision of Gene Expression and Regulation II
HDivision of Speciation Mechanisms I
YDivision of Speciation Mechanisms II--iCenter for Transgenic animals and plants-{Research center for Integrative andComputational Biology_r- --iResearch support Facility

Research Support
H lnstitute for
Molecular Science
H National Institute forPhysio logical Sciences-lCenter for Analytical instrument

I

rlTechnology Department

I

H Research Facilities

I-jCenter for Integrative BioscienceI-jCenter for Radioisotope Facilities Il.. .

Admtmstratton Bureau

Center for Experimental
AnimalI ~~esearchCenter for
Computational ScienceI

4

NATIONAL INSTITUTEMEMBERS OF THE COUNCIL
ISHIGE, Naomichi
IWATSUKI, Kunio
EGUCHI, GoroOHSAKI, Hitoshi
OKADA, Masukichi
OGAWA, Tomoko
KISHIMOTO, Tadamitsu
SHIMURA, Yoshirou
SUZUKI, Akinori
TAKEICHI, Masatoshi
TAKEUCHI, lkuo

Director-General, National Museum of Ethnology
Professor, The University of Air
President, Kumamoto University
Director-General, Center for National University of FinanceVice-President, International Institute for Advanced Studies
Professor, lwate College of Nursing
President, Osaka University
Director, Biomolecular Engineering Research Institute
President, Akita Prefectural University
Professor, Kyoto University
President, Novartis Foundation (Japan) for the Promotion of
Science
Deputy Director-General, JT Biohistory Research Hall
Director-General, Research Institute for Humanity and Nature
Professor, Keio University
Director-General, National Institute of Genetics
Dean, Nagoya University
Advisor, JT Biohistory Research Hall
Director, Banyu Tsukuba Research Institute
President, Otemae University
Director-General, National Institute of Polar Research

NAKAMURA, Keiko
HIDAKA, Toshitaka
HOSHI, Motonori
HOTTA, Yoshiki
YAMASHITA, Koujun
YOSHIKAWA, Hiroshi
YOSHIDA, Mitsuaki
YONEYAMA, Toshinao
WATANABE, Okitsugu

MEMBERS OF THE ADVISORY COMMITTEE FOR
PROGRAMMING AND MANAGEMENTAIZAWA, Shinichi
OKADA, Kiyotaka
KUROSAWA, Yoshikazu
GO, Michiko
KOMEDA, Yoshifumi
KONDO, Hisato
SASAZUKI, Takehiko
HASUNUMA, Kohji
MACHIDA, Yasunori
YAMAMOTO, Masayuki
IGUCHI, Taisen
IIDA, Shigeru
UENO,NaotoOHSUMI, Yoshinori
NAGAHAMA, Yoshitaka
NISHIMURA, Mikio
NODA, Masaharu
HORIUCHI, Takashi
MURATA, Norio
MOROHASHI, Ken-ichiro
YAMAMORI, Tetsuo

Professor, Kumamoto University
Professor, Kyoto University
Professor, Fujita Health University
Professor, Nagoya University
Professor, Hokkaido University
Professor, Osaka University
Director-General, International Medical Center of Japan ResearchInstitute
Professor, Yokohama City University
Professor, Nagoya University
Professor, University of Tokyo
Professor, Okazaki National Research Institutes, Center for
Integrative Bioscience.
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National lnstitut~ for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology
Professor, Okazaki National Institute for Basic Biology

5

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

DivisionsFieldsMolecular 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:

KATSUK/, Motoya
KODAMA, Ryuji
UENO, Kohji
OHNO, Kaoru

Mechanisms determining the outline shape of theadult lepidopteran wings
KODAMA, Ryuji
Wings of the lepidopteran insects (butterflies andmoths) develop from the wing imaginal disc, which is ahollow sac made of simple epithelium. When the
pupariation is completed, the wing, which was hidden
inside the body wall of the larvae, is exposed on thesurface of the pupa, which gradually turns into the adultwing. The outline shape of the adult wing is often
different from that of the pupal wing. This differenceis brought about by the programmed cell death of themarginal area of the pupal wing, while the internal area
develops as adult wing blade. The marginal dying
area is called the degeneration region and the internalarea is called the differentiation region, hereafter.
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 regeneration region have two characteristics common with the apoptotic cell ·death in mammalian cells. These are i) the
presence of apoptotic bodies, . which are heavily
condensed cells or their fragments engulfed by othercells or macrophages, shown by transmission electron
microscopy and ii) the presence of conspicuous
accumulation of fragmented DNA evidenced by the
TUNEL histological staining (Kodama, R. et al., Raux'sArch. Dev. Bioi. 204,418-426, 1995) .
The cells in the degeneration region are actively engulfed by the macrophages in the cavity beneath thewing epithelium. Moreover, the macrophages seem tobe concentrated beneath the degeneration region by thestrong adhesion between basal surfaces of the dorsal
and ventral epithelium in the differentiation region .
By injecting the india ink or ferritin solution to the body
cavity of the pupa, we have confirmed that this adhesionis tight enough to exclude the macrophages from thedifferentiation region, because the injected probes wasfound mostly concentrated in the degeneration region
when observed several minutes later (Yoshida, A.
(Biohistory Research Hall) and Kodama, R.,
unpublished).
A collaborative work with the laboratory of Dr. K.
Watanabe (Hiroshima University) concerns mostly on
the development of trachea and tracheole pattern in theswallow tail butterflies. Trachea and trcheoles areboth important in delivering air into the wing and theirpattern coincide with that of the boundary ofdegeneration and differentiation zones at the distal endof the wing. According to the observations, the patternformation of wing epithelium is often dependent on
tracheal and tracheole patterns. Basic research on thedevelopment of tracheal pattern formation is being done
through the scanning electron microscopy and thebright field light microscopy of the fixed or fresh
specimens to describe the exact pathway and the timecourse of the formation of elaborate pattern of trachea
and tracheoles and to establish the cytological andFig. I.

The tracheole cells (on the right) depart from the primarytrachea (thick tube on the left) leaving behind tracheole, which
was stored within the cytoplasm of the tracheole cell.

developmental relationship between the formation oftracheal pattern and epithelial cell pattern, such as scale
cell pattern.
The figure depicts how the tracheoles protrude fromthe primary trachea at the pre-pupa stage (Fig. 1). The
tracheoles are arranged with even spaces within thewing lumen and may closely related with the scale cellpattern formation.
In collaboration with other developmental biologists,
a database of cellular behaviors, which are essential
steps of morphogenesis of multicellular organisms, isdeviced (S3-P7, 14th ICDB, Kyoto, 2001). An instanceof the database is basically made up of (i) the subject
cell, (ii) the input the cell receives, and (iii) the output
the cell excecutes. With the aid of methodologies in thefield of information science and computer science,
automatization of database collection ·is planned. This
database will be important in incorporating rapidly
increasing knowledge in developmental biology inhighly reusable form even by non-expert researches ofthis filed.

Protein palmitoylation and its role in neuraldevelopmental at embryogenesis
UENO, Kohji
Protein palmitoylase modifies specific cysteine residues of signalling proteins such as G proteins and G
protein-coupled receptors with palmitate via thioester
linkages. This modification is thought to be importantin the regulation of signal transduction . We have previously found that protein palmitoylase is expressed inneural cells during mouse embryogenesis.
Immunocytochemical analyses revealed that growthcone-associated protein (GAP)-43 and G0 , which is oneof G proteins, were mainly localized in growth cones
and the cell body of cultured primary neural cells. Fig.

FOR BASIC BIOLOGY

2 shows the localization of protein palmitoylase and
GAP-43 in a neural cell. Cysteine residues in the amino
terminal regions of GAP-43 and Go are palmitoylated.
Furthermore an inhibitor of protein palmitoylase reduces the axonal growth of cultured neurons. Fromthese findings, we speculated that the localization of thepalmitoylated proteins in growth cones are critical forthe development of axons.
In this study, we are attempting to elucidate themechanism that determines the localization of
palmitoylated proteins in growth cones. For this analysis, we have established a method to chemically modify
the amino terminal region of a synthetic GAP-43 peptide with palmitate or other fatty acids via a thioester
linkage. Using these acylated peptides, we plan to analyze the mechanism that regulates the localization of
palmitoylated peptide in growth cones.

7

Fig. 2

Localization of protein palmitoylase and GAP-43 in a

cultured primary neural cell. (A) and (B) A neuron was doublelabeled with anti-protein palmitoylase and anti-GAP-43 antibodies,
respectively. Protein palmitoylase (green) was widely detected in
growth cones, processes, and the cell body, whereas GAP-43 (red)was mainly localized ·in growth cones and the cell body.

8

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 Associate:
Technical Staff:
NIBB Research Fellow:
Post doctoral Fellows:

Graduate Students:

Technical Assistants:

Secretaries

NISHIMURA, Mikio
HAYASHI, Makoto
MANO, Shoji
KONDO,Maki
SHIRAHAMA, Kanae
HAYASHI, Yasuko (-March 15)
HAYASHI, Hiroshi (-March 3 I)
MITSUHASHI, Naoto (-March
31)
NITO, Kazumasa
WATANABE, Etsuko
FUK.AO, Youichiro
KAMADA, Tomoe (Aprill-)
HATSUGAI, Noriyuki (April1-)
KUROYANAG/, Miwa (-March
31)
NAKAMOR/, Chihiro
TAKE/, Rie (-April30)
YAG1, Mina (March 1-)
UEDA, Chizuru
KOMORI, Akiko

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 un-dergo atransformation of function, while other organelles may
degenerate. Because the dynamic transformation oforganellar function (differentiation of organ-elles) 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. For example, etioplasts diffe-rentiate into
chloroplasts and mitochondria acquire the ability to oxidize glycine. Glyoxysomes which are microbodies engaged in the degradation of reserve oil via ~-oxidation andthe glyoxylate cycle, are transformed into leaf
peroxisomes that function in several crucial steps ofphotorespiration. After the functional transition ofglyoxysomes to leaf peroxisomes during the greening of
pumpkin cotyledons, the reverse transition of leaf
peroxisomes to glyoxysomes occurs during senescence.
The functional transformation between glyoxysomes
and leaf peroxisomes is controlled by gene expression,
alternative splicing, protein translocation and protein degradation.
To investigate the roles of peroxisomal membrane proteins in the reversible conversion of glyoxysomes to leaf
peroxisomes, we characterized several membrance proteins of glyoxysomes. One of them (PMP38) was identified a putative ATP/ADP carrier protein. Cell fractiona-tion and immunocytochemical analysis using pumpkin
cotyledons revealed that PMP38 is localized on
peroxisomal membranes as an integral ~embrane protein.
The amount of PMP38 in pumpkin cotyledons increased
and reached the maximum protein level after 6 d in thedark but decreased thereafter. lllumination of the seedlings caused a significant decrease in the amount of theprotein. These results clearly showed that the membraneprotein, PMP38 in glyoxysomes changes dramaticallyduring transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especiallyenzymes of the fatty acid ~-oxidation cycle, that are localized in the matrix of glyoxysomes. An ascorbate peroxidase (pAPX) was also identified as one of glyoxysomal
membrane proteins. Its eDNA was isolated by immunoscreening. The deduced amino acid sequence encoded by ·the eDNA insert does not have a peroxisomal targeting
signal (PTS), suggesting that pAPX is imported by one ormore PTS-independent pathways. Subcellular fractionation of 3- and 5-d-old cotyledons of pumpkin revealed
that pAPX was localized not only in the glyoxysomal
fraction, but also in the ER fraction. A magnesium shift
experiment showed that the density of pAPX in the ER
fraction did not increase in the presence of Mg 2+, indicating that pAPX is not localized in the rough ER. Immunocytochemical analysis using a transgenic Arabidopsis
which expressed pumpkin pAPX showed that pAPX was
localized on peroxisomal membranes, and also on a unknown membranous structure in green cotyledons. The
overall results suggested that pAPX is transported toglyoxysomal membranes via this unknown membranous
structure.

II. Peroxisomes defective mutant of Arabidopsis.
It has been suggested that the functional conversion
between glyoxysomes and leaf peroxisomes is controlled
by gene expression, protein translocation, and protein
degradation. A genetic approach is an effective strategy
toward understanding the regulatory mechanism(s) ofperoxisomal function at the level of gene expression,
protein translocation, and protein degradation. We
isolated and characterized 2,4-dichloro-phenoxybutyricacid (2,4-DB)-resistant mutants. It has been demonstrated
previously that 2,4-dichlorophe-noxybutyric acid (2,4DB) is metabolized to produce a herbicide, 2,4-D, by theaction of peroxisomal fatty acid ~-oxidation in higher
plants. To isolate mutants that have defects inperoxisomal fatty acid ~-oxidation, we screened mutant
lines of Arabidopsis seedlings for growth in the presenceof toxic levels of 2,4-DB. Genetic analysis revealed thatthese mutants can be classified as carrying alleles at three
independent loci, which we designated ped1, ped2, andped3, (where ped stands for peroxisome defective). The
pedl mutant lacks the 3-ketoacyl CoA thiolase, an
enzyme involved in fatty acid ~-oxidation during
germination and subsequent seedling growth, while
AtPex14p, the PED2 gene product, is a peroxisomal
membrane protein that determines the peroxisomal
protein targeting. PED3 gene was recently identified by

FOR BASIC BIOLOGY

positional cloning. The phenotype of the ped3 mutant
indicated that the mutation in the PED3 gene inhibits theactivity of fatty acid ~-oxidation . Ped3p, the PED3 geneproduct, is a 149-kD protein that exists in peroxisomal
membranes. The amino acid sequence of Ped3p had atypical characteristic for "full-size" ATP-binding cassette(ABC) transporter consisting of two transmembrane regions and two ATP-binding regions. This protein was
divided into two parts, that had 32% identical amino acidsequences. Each domain showed a significant sequencesimilarity with peroxisomal "half' ABC transporters so
far identified in mammals and yeast. Ped3p may contribute to the transport of fatty acids and their derivatives
across the peroxisomal membrane. pedl/ped3 double
mutant showed severe defects on leaves and inflorescences, and was sterile (Fig. 1). The phenotype may tell
us unidentified function(s) of plant peroxisomes.

8

Figure I. Phenotype of pedl /ped3 double mutant.
pedl/ped3 double mutant showed vegetative and reproductive phenotypes. It had wavy leaves with irregular shapes(Fig. lA). Influorescence of the double mutant was difficult
to develop, but it occasionally had dwarf inflorescences withabnormal structure (Fig. I B). Although the inflorescences
had some flowers, it was sterile. These phenotypes were notfound in the parents, pedl and ped2.

III. ER derived organeUes for transport of proteins
to vacuoles.
Novel vesicles designated precursor-accumulating(PAC) vesicles that accumulate large amounts ofproprotein precursors of storage proteins were purified

11

and characterized from maturing pumpkin seeds. Thesevesicles had diameters of 300 to 400 nm and contained
an electron-dense core of storage proteins surroundedby an electron-translucent layer and were shown that
the PAC vesicles mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles. We found a novel membraneprotein with molecular mass of 73 kDa, MP73, on themembrane of protein storage vacuoles of pumpkinseeds. MP73 appeared during seed maturation anddisappeared rapidly after seed germination, in association with the morphological changes of the protein
storage vacuoles.
Immunocytochemistry and an
immunoblot analysis showed the PAC vesicles accumulated proMP73, but not MP73, on the membranes.
Subcellular fractionation of the pulse-labeled maturingseeds demonstrated that the proMP73 form with Nlinked oligosaccharides was synthesized on the ER andthen transported to protein storage vacuoles via PAC
vesicles. Tunicamycin-treatment of the seeds resulted
in the efficient deposition of proMP73 lacking theoligosaccharides into the PAC vesicles, but no accumulation of MP73 in vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by theaction of a vacuolar enzyme to form a 100-kD complex
on the vacuolar membranes. These results show that
PAC vesicles mediate delivery of not only storage
proteins but also membrane proteins of protein storage
vacuoles. In order to investigate the mechanism of the
PAC vesicle formation, we constructed chimeric genesthat encode fusion proteins consisting to both various
lengths of polypeptides derived from pumpkin 2S albumin
and a selectable marker enzyme, phosphinothricin
acetyltransferase and expressed in Arabidopsis. A fusion
protein expressed by one of the chimeric genes is accumulated as a proprotein-precursor form, and localized in
novel vesicles of vegetative cells, that show distinct features that well much to the PAC vesicles. Arabidopsis
mutants that defect vesicular transport of the fusion protein are now screened and characterized by using thetransgenic plants.
Plants degrade cellular materials during senescence andunder various stresses. The precursors of two stressinducible cysteine proteinases, RD21 and a vacuolar
processing enzyme (VPE), were specifically accumulated
in -0.5 f.Uil diameter x -5 Jlm long bodies in Arabidopsis
thaliana. Such bodies have previously been observed in
Arabidopsis but their function was not known. Because
these bodies contain precursors of lytic enzymes, we propose to call them ER bodies. They are surrounded withribosomes and thus are assumed to be directly derived
from the endoplasmic reticulum. ER bodies develop specifically in the epidermal cells of healthy seedlings. Thesecells are easily wounded and stressed by the external environment. When the seedlings are stressed with a concentrated salt solution, leading to death of the epidermal
cells, the ER bodies start to fuse with each other and withthe vacuoles, thereby mediating the delivery of the precursors directly to the vacuoles. This regulated, direct
pathway differs from the usual case in which proteinases

12
are transported constitutively from the endoplasmic
reticulum to the Golgi complex and then to vacuoles, withintervention of vesicle-transport machinery, such as avacuolar-sorting receptor or a syntaxin of the SNARE
family. Thus, the ER bodies appear to be a novel
proteinase-storing system that assists in cell death of thevegetative organs of higher plants.

IV. Role of molecular chaperones in organelle
differentiation.
Molecular chaperones are cellular proteins thatfunction in the folding and assembly of certain otherpolypeptides into oligomeric structures but that are not,themselves, components of the final oligomeric structure.
To clarify the roles of molecular chaperones on 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 a chloroplast co-chaperonin(Cpn20) from Arabidopsis thaliana. In 2001, we characterized a third co-chaperonin. The eDNA was 603 basepairs long, encoding a protein of 139 amino acids. From asequence analysis, the protein was predicted to have one
Cpn1 0 domain with an amino-terminal extension thatmight work as a chloroplast transit peptide. This novel
CpnlO was confirmed to be localized in chloroplasts, andwe refer to it as chloroplast CpnlO (chl-CpnlO). The
phylogenie tree that was generated with amino acid sequences of other co-chaperonins indicates that chl-CpnlO
is highly divergent from the others. In the GroEL-assisted
protein folding assay, about 30% of the substrates wererefolded with chl-CpnlO, indicating that chl-CpnlO works
as a cochaperonin. A Northern blot analysis revealed thatmRNA for chl-CpnlO is accumulated in the leaves andstems, but not in the roots. In germinating cotyledons, theaccumulation of chl-CpnlO was similar to that ofchloroplastic proteins and accelerated by light. It was
proposed that two kinds of co-chaperonins, Cpn20 and
chl-CpnlO, work independently in the chloroplast.

Publication List:
Fukao, Y., Y. Hayashi, S. Mano, M. Hayashi and M.
Nishimura (2001) Developmental analysis of a putative
ATP/ADP carrier protein localized on glyoxysomal
membranes during the peroxisome transition in
pumpkin. Plant Cell Physiol. 42: 835-841
Hayashi, M. and M. Nishimura (2002) Genetic
approaches to understand plant peroxisomes. in Plant
peroxisomes, edited by A. Baker and I. Graham,
Kluwer Acad. Pub. in press
Hayashi, M., K. Nito, R. Takei-Hoshi, M. Yagi, M.
Kondo, A. Suenaga, T. Yamaya and M. Nishimura(2002) Ped 3p is a peroxisomal ATP-binding cassetetransporter that might supply substrates for fatty acid ~­oxidation. Plant Cell Physiol. 43:1-11
Hayashi, Y., M. Hayashi, H. Hayashi, I. Hara-Nishimura
and M. Nishimura (2001) Direct interaction between
glyoxysomes and lipid bodies in etiolated cotyledons of

NATIONAL INSTITU1E

Arabidopsis thaliana pedl mutant . Protoplasma 218:
83-94
Hayashi, Y., K. Yamada, T. Shimada, R. Matsushima, N.
K. Nishizawa, M. Nishimura and I. Hara-Nishimura(2001) A proteinase-storing body that prepares for cell
death or stresses in the epidermal cells of Arabidopsis.
Plant Cell Physiol. 42: 894-899
Kimura, Y., S. Matsuno, S. Tsurusaki, M. Kimura, I.
Hara-Nishimura and M. Nishimura (2002) Subcellular
localization of endo-~-N-acetylglucosaminidase andhigh-mannose type free N-glycans in plant cell.
Biochim. Biophys. Acta in press
Koumoto, K., T. Shimada, M. Kondo, I. Hara-Nishimura
and M. Nishimura. (2001) Chloroplasts have a novel
Cpn10 in addition to Cpn20 as co-chaperonins in
Arabidopsis thaliana. J. Biol. Chem. 276: 29688-29694
Mano, S., C. Nakamori, M. Hayashi, A, Kato, M. Kondo
and M. Nishimura (2002) Distribution andcharacterization of peroxisomes in Arabidopsis byvisualization with GFP. Plant Cell Physiol. in press
Minamikawa, T., K. Toyooka, T. Okamoto, I. HaraNishimura and M. Nishimura (2001) Degradation ofribulose 1,5-bisphosphate carboxylase/oxygenase byvacuolar enzymes of senescing French bean leaves:
Immunocytochemical and ultrastructural observations.
Protoplasma 218: 144-153
Mitsuhashi, N., Y. Hayashi, Y: Koumoto, T. Shimada, T.
Fukasawa-Akada, M. Nishimura and I. Hara-Nishimura(2001) A novel membrane protein of protein bodies thatis transported to protein-storage vacuoles via precursoraccumulating vesicles. Plant Cell13: 2361-2372
Nito, K., K. Yamaguchi, M. Kondo, M. Hayashi and M.
Nishimura (2001) Pumpkin peroxisomal ascorbate
peroxidase is localized on peroxisomal membranes andunknown membranous structures. Plant Cell Physiol.
42: 20-27
Tanaka, H., H. Onouchi, M. Kondo, I. Hara-Nishimura,
M. Nishimura, C. Machida and Y. Machida (2001) A
subtilisin-like serine protease is required for epidermal
surface formation in Arabidopsis embryos and juvenile
plants. Development 128: 4681-4689
Watanabe, E., T. Shimada, M. Kuroyanagi, M. Nishimura
and I. Hara-Nishimura (2002) Calcium-mediatedassociation of a putative vacuolar sorting receptor
PV72 with a propeptide of 2S albumin. J. Biol. Chem.
in press
Xu, W., K. Morita, K. Yamada, M. Kondo, M. Nishimura,
H. Shioiri, M. Kojima and M. Nozue (2001)
Expression and localization of a 36-kDa vacuolar
protein (VP24) precursor in anthocyanin-producing
sweet potato cells in suspension culture. Plant
Biotechnol. 18: 203-208
Yamada, K., R. Matsushima, M. Nishimura and I. HaraNishimura (2001) A unique cysteine protease with agranulin domain that slowly matures in the vacuoles ofsenescing Arabidopsis leaves. Plant Physiol. 127:
1626-1634

FOR BASIC BIOLOGY13

DIVISION OF BIOENERGETICS
Professor:
Associate Profes sor:
Research Associates:
Technical Staff:
Postdoctoral Fellow:

Graduate Students:

Visiting Scientists:

OHSUMI, Yoshinori
YOSHIMORJ, Tamotsu
KAMADA, Yoshiaki
NODA, Takeshi
KABEYA, Yukiko
MIZUSHIMA, Noboru n
KIRISAKO, Takayoshi 21
SEKITO,Takayuki 131
YOSHIMOTO, Kohki 4!
ICHIMURA, Yoshinobu 51
SUZUKI, Kuninori 51
KOBAYASHI, Yoshinori 5•71
NARA, Atsuki 51
HANAOKA, Hideki 51
KUMA. Akiko 51
HAMASAKI, Mah o 51
SHOJI, Shisako 51
ONODERA,Jun ,5·61
OKAMOTO, Satsuki

acterized of the gene product. We drew a rough functional map of each Apg proteins this year. This is basedon the finding of novel structure called preautophagosomal structure (PAS). Many Apg proteins (Apgl ,Apg2, Apg5 , Apg8 , Apgl6) are colocalized on preautphagosomal structure. Autophagy-specific phosphatidylinositol 3-kanise complex (Apg6, Apgl4, Vps34 andVpsl5) are essential for organization of PAS. Apg8,
ubiqitin-like protein, gathers to PAS depending on lipidation reaction and the Apg5-Apg 12 conjugates. Apg5
is recruited to PAS depending on Apgl6 but independent on Apgl2-conjugation. Apg2 is localized on
PAS via Apgl. Apgl protein kinase function in transition from PAS to autophagosome. Thus, PAS is theorganizing center of autophagosome.

n PRESTO, JST, Oct 1999-.
21

fromApr2001-.
from Feb2001-.
41
from Sep2001-.
51
Graduate University for Advanced Studies.
61
from Apr2001 .
71
til Oct 2001
31

This division aims to understand the autophagy in respects to its molecular mechanism and its physiologicalrole in higher eukaryotes. Cells execute degradation
processes of their constituents together with biosynthetic processes. These two processes are well coordinated to regulate the biological activities. In other word,
we must shed light on degradation process to fully understand the cell, because the study on the degradation
has been retarded compared to the bio-synthetic process.
Autophagy is well conserved in eukaryotes and is amajor route for bulk degradation of cytoplasmic constituents and organelles in a lytic compartment,
lysosome/vacuole.

I. Background
Upon nutrient starvation, autophagic process starts asbuilding up a membrane structure, an autophagosome,in the cytoplasm. The autophagosome sequesters cytosol and organelles nonselectively. Then it is delivered to
the vacuole/lysosome, and the cytoplasmic materials
inside are degraded by vacuolar/lysosomal proteases.
We had discovered autophagy in a simple eukaryotic
model organism, Saccharomyces cerevisiae · and morphologically defined the whole process. We have isolated a set of autophagy-deficient mutants (apg ), and havecloned most of the APG genes essential for autophagy.
We are now characterizing these gene products andtheir homologues in mammals and higher plants.

2. Discovery of novel preautophagosomal structure
We have cloned the last APG genes, APG2, and char-

Fig.! Discovery of preautophagosomal structure
Most Apg proteins are gathered on preautophagosmal structure. Based on this finding, function al role of each Apg protein
can be discussed in detail.

3. Autophagosome formation depends on specificsets of coat proteinsWe revealed that autophagosme fuses with the vacuole by NSF/SNARE meditated mechanism, which iscommon through endmembrane system, such as ER and
Golgi. However, autophagosome formation is not dependent of NSF. Instead, the involvement the proteinsof early secretory pathway are revealed. Secl2, Secl6,
Sec23, Sec24 are required for autophagosome formation .
Interestingly, they are not prerequisite for Cvt pathway,
the similar pathway to autophagy. The findings may
imply that early phase of autophagosome formation isrelated to secretory pathway in some way .

4. Molecular dissection of autophagosome formation
Apgl2 and Apg5 are covalently attached in a manner
similar to the ubiquitin conjugation system. The Apg12Apg5 conjugation system is well conserved in mammalian cells. Part of the mammalian Apg12-Apg5 conjugate localized to the isolation membranes when autophagy proceeds, whereas most of it existed in the cytoplasm. Using GFP-tagged Apg5 , we revealed that the

14

NATIONAL INSTITUTE

cup-shaped isolation membrane is developed from asmall crescent-shaped compartment. Apg5 localized on
the isolation membrane throughout its elongation process. Apg5 was preferentially distributed in the outer sideof the membrane and detaches from it immediately
before or after autophagosome formation is completed.
In contrast, LC3, the mammalian homologue of Apg8,
was associated with both sides of isolation membranes
and autophagosomes as well as their precursors. To
examine the role of Apg5, we generated Apg5-deficient
ES cells. APG5 + cells are viable but bulk protein degradation was significantly reduced. Autophagosome
formation was impaired in these cells. The mutant
Apg5K 130R, which is unable to be conjugated with Apgl2,
could bind to the autophagosome precursors but could
not start elongation of the membranes. Thus, the covalent modification of Apg5 with Apg12 was not requiredfor its membrane targeting but is essential for involvement of Apg5 in elongation of the isolation membranes.
Intriguingly, Apg12-Apg5 was required for processingof LC3 and its targeting to the membranes. Therefore,
the Apg12-Apg5 conjugate plays essential roles in isolation membrane development in co-operation with LC3.
In addition, our studies provided good molecular markers, LC3 and Apg12-Apg5, for autophagic membrane at
all stages and isolation membranes, respectively, which
so far have been defined only by morphology.

5. Production of phosphatidylinositol 3-phosphate(PI3P) at the trans-Golgi network (TGN) is required for autophagy
This year, we reported that, in yeast, Vps34 PI3kinase and Apg6 form a protein complex that plays aessential role in autophagy by producing PI3P. Then,
we found in mammalian cells that the complex of the
mammalian Apg6 homologue, beclin and PI3-kinase
was distributed in the TGN rather than autophagosomes(Figure 2A). We propose that beclin functions as aregulatory-subunit of the PI3-kinase complex at the
TGN, which supplies PI3P for autophagosome formation (Figure 2B).
A

8

Fig.2
A. Beclin localizes in the TGN.
B. Model for beclin function. The beclin-PI3-kinase complex
localized at the TGN supplies PBP to the membranes in the
autophagic pathway.

Original papers :Ishihara, T., Hamasaki, M., Yokota, S., Suzuki, K.,
Kamada, Y., Kihara, A., Yoshimori, T., Noda, T.,
and Ohsumi, Y. (2001) Autophagosome requires
specific early Sec proteins for its formation and
NSF/SNARE for its fusion to the vacuole. Mol. Bioi.
Cell12, 3690-3702.
Kim, J., Kamada, Y., Stromhaug, P. E. Guan, J., Hefner-Gravinnk, A., Bevan, A., Scott, S. V., Ohsumi, Y.,
Dunn, Jr., W. A., Klionsky, D. J. (2001) Cvt9/Gsa9function in sequestering selective cytosolic cargo
destined for the vacuole. J. Cell. Bioi. 153, 381-396.
Kihara, A., Noda, T., Ishihara, N. and Ohsumi, Y.
Two distinct Vps34 Ptdlns 3-kinase complex functionin autophagy and CPY. (2001) sorting in Saccharomyces cevisiae. J. Cell. Biol. 152, 519-530.
Kihara, A., Kabeya, Y., Ohsumi, Y., and Yoshimori, T.
(2001) Beclin-phosphatidylinositol 3-kinase complex
functions at the trans-Golgi Network. EMBO Report,
2, 330-335.
Komtatsu, M., Tanida, I., Ueno, T., Ohsumi, M., Ohsurni, Y., and Kominami, E. (2001) The C-terminal
region of an Apg7p/Ctv2p is required for homodimerization and is essential for its E1-activity and E1E2 complex-formation. E. J. Biol. Chern. 276, 98469854.
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi,Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y
and Yoshimori, T. (2001) Dissection of autophagosome formation using Apg 5-deficient mouseembryonic stem cells. J. Cell. Bioi. 152, 657-667.
Shintani, T., Suzuki,K., Kamada, Y., Noda, T., Ohsumi,
Y. (2001)Apg2p functions in autophagosome formation on the perivacuolar structure. J. Bioi. Chern. 276,
30452-30460.
Suzuki, K., Kirisako, T., Kamada, Y. , Mizushima, N.,
Noda, T. and Ohsumi, Y. (2001) The preautophagosomal structure organized by concertedfunctions of APG genes is essential for autophagosome formation. EMBO. J. 20,5971-5981.

Review:
Ohsumi, Y. (2001) Molecular dissection of autophagy:
Two ubiquitin-lke systems. Nature Reviews,
Molecular Cell Bioi, 2, 211-216.

15

FOR BASIC BIOLOGYDIVISION OF CELL PROLIFERATION(ADJUNCT)
Professor:
Research Associate:
NIBB Research Fellow:
JSPS Research Fellow:
Graduate Student:

KATSUKI, Motoya
ITO, Kei
OKADA, Ryuichi
AWASAKI, Takeshi (PRESTO)TANAKA, Nobuaki(Graduate Univ. Advanced
Studies)
OTSUNA, Hideo(Nagoya University)

The aim of this adjunct division is to understand thebasic rules by which elaborate neural circuits develop
and function. With less than 105 neurones, and subject
to powerful molecular and genetic techniques, the brainof the fruit fly Drosophila melanogaster is a goodmodel system for investigating the whole of an easily
accessible nervous system that shares certain of thearchitectural and functional features of the more
complex vertebrate brains.

I. Comprehensive identification of cells in theadult brain
In spite of the hundred years of efforts using Golgi
and other 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 cloningof Drosophila, is also a powerful tool for obtaining avast array of transformant strains that label specificsubsets of brain cells. We screened such strains from astock of 4500 GAL4 lines made by the NP consortium,a joint venture of eight Japanese fly laboratories
organised by us. In the first step, all the lines werecrossed with the flies carrying the UAS-GFP transgene,which fluoresces only in GAL4-expressing cells. Their
patterns were recorded from freshly dissected, unfixed
adult brains with a high-speed confocal !nicroscope. In
June 2001, we finished making the image database of
131 ,406 photographs depicting the confocal sections of
3,939 GAL4 strains. From July 2001, we started
making a similar database of the late larval brain using
the same set of GAL4 strains.
In the second step, useful lines were selected from thedatabase, and fixed and clealised brains were subjected
to more precise serial sectioning with conventional
confocal microscopes and to three-dimensional
reconstruction with UNIX workstations. Although our
long-term aim is to get the comprehensive overview ofthe fly brain neural network, at the initial stage a few
brain regions are chosen for intensive study. The firsttarget is to identify projection interneurones thatconnect lower-level sensory processing sites andhigher-order associative regions. These fibres convey
olfactory, gustatory, auditory and visual sensory
information.
For visual pathways, we identified another 10 typesof interneurones that project from the optic lobe to thecentral brain. The total number of identified neurone
types of this kind now became 40. Their structures, aswell as their role in the controlled movement of flies,
are under investigation.
For olfactory pathways, we selected 34 strains thatlabel various subsets of antenna! lobe projection
neurones, which transmit information from the firstorder olfactory processing site (antenna! lobes) to thesecond-order sites (mushroom bodies and lateral horns).
We found that the terminals of these PNs form clear
zonal separation in both second-order sites. We
identified three zones in the mushroom bodies and ninezones in the lateral horns (Fig. 1). Projection neurones
from several glomeruli, each deriving from a single
glomerulus of the antenna! lobe, converge at selective
zones. Another type of neurones, each contributing
multiple glomeruli, terminate at zones that overlap withthe zones of uniglomerular neurones. The olfactorycode across glomeruli is thus both distributed to, andconvergent at, discrete zones of the second-order sites.
These
stereotypic projection patterns
wereestablished before adult eclosion, and surgical ablationof olfactory sense organ caused no significant
reorganisation. These suggest that distinct odortypic
pathways are established and maintained geneticallyrather than in an activity dependent manner.

II. Mapping of neurotransmitters and receptors
in the adult brain
To understand the function of the identified neuralnetwork, it is also important to get the information
about the types of neurotransmitters and receptors usedin these cells. Previously, such information is obtainedby staining brain tissue with various antibodies. Thisapproach, however, has the limitation due to theavailability of good antibodies.
Taking the advantage of the completion of
Drosophila whole genome sequence, we employed anovel approach. From the database, we first searchgenes that code receptors and enzymes associated totransmitter synthesis. Cells that express those genes arelabelled by using in-situ RNA hybridisation. We thenscreen GAL4 enhancer-trap strains that label cells in thecorresponding area, and perform double labelling tocertify the colocalisation of GAL4 expression and insitu label. Second year of this project, we concentrateon GABA, a major inhibitory transmitter, and are
mapping the GABA-generating cells and cells that
express three types of GABA receptors (Fig. 2).

m. Analysis of thecell lineage-dependent
modular structures in the brain

The central brain of Drosophila melanogaster is
produced by an average of 85 stem cells (neuroblasts)
per hemisphere. We visualised the innervation patternsof the progeny of single neuroblasts in the adult brain
using the FRT-GAL4 system. In most cases, cell bodies
form a tightly packed cluster and their neurites
fasciculate to form a single bundle to innervate a16
limited number of brain regions in a stereotypic manner.
These suggest that the progeny of a single neuroblast
often form a lineage-dependent circuit module, which
we named a "clonal unit."
To understand the mechanisms underlying this clonal
clustering and fasciculation, we focused on the role ofneural-specific homophilic cell adhesion molecules in
the cell body layer (cortex) of the developing larval
brain. DN-cadherin and Neuroglian are distributed
uniformly along the border between all the neurones.
Fasciclinll (Fasll), on the other hand, localises inseveral clusters of neurones, each of which looks likeclonally related. Double labelling of FRT-GAL4 clones
and Fasll-expressing cells revealed that Fasll clusters
indeed correspond to clones . The distribution of Fasll islimited to the cell border inside the clones. Cell surface
flanking the neighbouring clones is free of Fasii. Such
localisation might infer that Fasii would mediate cellcell adhesion within clonal cluster.
fasii mutant clones, however, showed no remarkabledefect on the formation of clonal cell clustering . Panneuronal ectopic expression of fasll caused little effect,either. The ectopically expressed Fasii showed the samecharacteristic localisation pattern: it concentrates along
the intraclonal cell borders but not along the interclonalcell borders.
Why doesn't ectopic Fasii exist at the interclonal cellborders? One possible explanation is that there might bephysical boundary that prevents direct contact ofneurones between different clones . We thus examined
the arrangement of glial cells in the larval brain. Double
labelling of glial cells and Fasll-expressing clones
showed that a type of glia send extensive processes
between neurones . In the outer area of the cell body

NATIONAL INSTITUTE
layer, which is near the brain surface and houses
neuroblasts and newly-generated cells, glial processes
wrap only the outer surface of the clonal clusters.
Processes are not observed within the cluster. Glial cells
thus physically separate the clonal border in this area.
Deeper in the cell body layer, which consists of oldcells, thin glial processes penetrate the boundary
between essentially all the neurones.

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 esearchers, is Jfly
(http ://jfly .nibb.ac.jp). Archives of research-related
images anddiscussions, experimental protocols,
movies are provided.

Publication List:
Awasaki, T. and Kimura. K. (2001) Multiple function of
poxn gene in larval PNS development and in adultappendage formation of Drosophila, Dev. Genes. Evol.
211:20-29
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 the importance of fasciclin II .
Development, in press
Verkhusha, V. V., Otsuna, H. , Awasaki, T., Oda, H.,
Tsukita, S., and Ito, K. (2001) An enhanced mutant of
red-fluorescent protein DsRed for double labelling anddevelopmental timer of neural fiber bundle formation . J
Bioi Chem 276 (32): 29621-29624.

Figure I . Zonal separation of the projection neurone terminals. The five columns show the frontal view of the antenna! lobe (green:
UAS-GFP, magenta: antenna) lobe neuropile), frontal and horizontal views of the mushroom body calyx (magenta: UAS-GFP, green:mushroom body neuropile), and frontal and horizontal views of the lateral horn (green : UAS-GFP, grey: Nomarski-contrasted
transmission). Dotted lines indicate the outer margin of the lateral horn and the border of each zone. Bar = 20 ~m . A: UniglomerularvACT neurons projecting into zone I of the mushroom body and zones MV and B of the lateral horn. B: Uniglomerular iACT neurons
innervating zone 2 and zone V3. C: Uniglomerular iACT neurons innervating zone 3 and zone DI.

17

FOR BASIC BIOLOGY

Gad1

Lcch3

Rdl

GABA·B·R2

54/54

57/60

58/59

56/57

Figure 2. GABA related gene expression in the antennal lobe local inteneurones. Green: One type of local interneurones (in total about
54-60 cells) visualised with GAL4 strain NP2426 and UAS-GFP. Magenta: in situ hybridisation against GABA synthesis enzyme Gad 1
and three types of GABA receptors (Lcch3, Rdl, GABA-B-R2). Numbers below each panel show the ratio of double-positive cells,suggesting that essentially all the interneurones of this type are GABAergic and GABA-responding.

18

NATIONAL INSTITUTEDIVISION OF CELLULAR COMMUNICATION(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
NIBB Research Fellow:
Postdoctoral Fellow:

KAMIYA, Ritsu
INABA, Kazuo (June 1-)
KATO-MINOURA, Taka/co
WAKABAYASHI, Ken-ichi
HIRAKO, Yoshiaki

The research in this laboratory is aimed at an understanding of the molecular mechanisms that regulate theassembly and function of cytoskeletal proteins. Specifically, we are currently studying the functional properties of axonemal dynein and actin in Chlamydomonas, anorganism ideally suited for genetic and molecular biological studies.

I. Function of Multiple Axonemal Dyneins
It is well established that the beating of cilia andflagella is based on sliding movements of outer-doublet
microtubules driven by motor proteins dyneins, but how
the sliding is converted into axonemal oscillatory
bending movement has not been made clear. Recently,
various lines of evidence have suggested that dynein is
crucially important also in the sliding-bending conversion mechanism. Thus our research effort is now focused on the properties of various dyneins.
Biochemical studies by us and other laboratories haveestablished that a single flagellar axoneme contains atleast eleven kinds of dynein heavy chains in inner andouter arms. An important question is how different
dynein heavy chains differ in function. To answer thisquestion, we have been isolating and characterizing
mutants that lack different kinds of axonemal dyneins.
During the last ten years, we have isolated as many as
15 genetically different mutants lacking various subsetsof dyneins. The isolation of these mutants greatly advanced our understanding of the function and organization of various dyneins within the axoneme, because
only three mutants had been known to lack dynein
heavy chains before we started mutant isolation.
The motility phenotypes of the isolated mutants haveindicated that different dynein species differ in function
in a fundamental manner. For example, the outer-arm
heavy chains are important for flagellar beating at highfrequency, whereas the inner-arm heavy chains areimportant for producing proper waveforms. Indirect
evidence also suggests that the force generation properties differ greatly among different heavy chains. Interestingly, the axoneme can beat without some of theseheavy chains, .but cannot beat if certain combinations ofheavy chains are lost. It appears that simultaneouspresence of dyneins with different properties is necessary for the axonemal beating. Thus, it should be important to understand the mechano-chemical property ofeach dynein. To this end, we are currently trying todirectly measure the force production in wild-type andmutant axonemes that lack various combinations ofdyneins.
In addition to exploring the functional properties ofindividual dyneins, we have been studying the mechanism by which various dyneins are regularly arranged
within the axoneme. In particular, we have concentrated
our effort on understanding why outer dynein arms areattached to a particular protofilament in outer doublet
microtubules, with a constant spacing of 24 nm. Such aregular arrangement of dynein should be important for
the axoneme to beat with regular waveforms. Our study
has indicated that a protein complex called the outerdynein arm docking complex (ODA-DC) is particularly
important. We found that this complex, made up of
three different protein subunits, is transported to theaxonemes independently of outer arms and attaches onto the microtubule protofilament, providing the docking
site for outer arms. Interestingly, the periodicity of 24
nm appears to originate from the periodicity in ODADC binding (Fig. 1). An attractive hypothesis is that
ODA-DC has a tendency to longitudinally associatewith each other and serves as a molecular ruler with a
24 nm spacing. Our study has also shown that the outerdoublet microtubule contains an unidentified structurethat can specify the ODA-DC binding site among different protofilaments of a single outer doublet. We are
currently trying to elucidate this structure, as well as toprove the molecular ruler hypothesis.

ll. Function of Actin and an Actin-related Protein
in Chlamydomonas
The inner dynein arms are known to contain actin as
a subunit. Hence the two independent motility systemsof eukaryotes - the actin-based and microtubule-based
motility systems - should somehow cooperate in the
inner arm dynein although the function of actin in
dynein arms is totally unknown at present. We havefound that the mutant ida5, lacking four out of the sevensubspecies of inner-arm dyneins, has a mutation in theactin-encoding gene. Intriguingly, Chlamydomonas has
been known to have only a single gene of conventional
actin, and the ·mutant ida5 was found to express noconventional actin at all. On close inspection, the cytoplasm and axonemes of this mutant were found to contain a novel actin-like protein (NAP) which displays
exceptionally low homology (64%) to conventional
actin. The mutant ida5 is deficient in the formation of
the fertilization tubule and thus has a low mating efficiency. However, it displays normal cell division andgrows as rapidly as wild type, possibly because NAP
can substitute for actin in important cellular functions.
Thus conventional actin and NAP may overlap in some,
but not all, cellular functions. It is interesting to notethat NAP is expressed in significant amount only in the
mutant lacking actin; i.e., the expression of NAP appears to depend on the presence of actin. We are currently investigating how such regulation takes place.
What is the function of NAP in wild-type cells?
Recently, we found NAP is expressed even in wild type,specifically during the re-flagellation process after
flagellar amputation. The expression of NAP mRNA

FOR BASIC BIOLOGY

precedes that of actin rnRNA. Interestingly, cell'sability to re-grow flagella is greatly impaired when ida5
is transformed with a chimeric actin gene consisting ofa 5'-UlR sequence derived · from the NAP gene and asequence that codes .for conventional actin. NAP and
actin mRNA are expressed simultaneously in this mutant, suggesting that the prior expression of NAP isimportant for efficient flagellation. Phylogenetic analysis revealed that NAP homologs exist in at least three
species in Volvocales, forming a distinct gene family of
divergent actin. Taken together, NAP may play a
specific role in the flagellation mechanism in various
species of Volvocales.

Publication List:
Hayashi, M., Hirono, M., and Kamiya, R. (2001) Recovery of flagellar dynein function in Chlamydomonas actinldynein-deficient mutant upon introduction of muscle actin by electroporation. Cell Motil.
Cytoskeleton 49, 146-153.
lnaba, K. (2001) High level of protein ubiquitination in
ascidian sperm. In The biology of ascidians.
(Sawada, Yokosawa, Lambert, eds,), pp.7479 .,Springer-Verlag
ltoh, A., Inaba, K., Fujinoki, M., and Morisawa, M.
(2001) Motility-associated and cyclic AMPdependent protein phosphorylation in the sperm of
the chum salmon, Oncorhynchus keta. Biomed. Res.

19
22, in press.
Kho, K. H., Tanimoto, S., lnaba, K., Oka, Y., and Morisawa, M. (2001) Transmembrane cell signaling forthe initiation of trout sperm motility: Roles of ionchannels and membrane hyperpolarization for
cyclic AMP synthesis. Zool. Sci. 18,919-928.
Padma, P, Hozumi, A, Ogawa, K., and Inaba, K. (2001)
Molecular cloning and characterization of a thioredoxin!NDPK related dynein intermediate chain
from the ascidian, Ciona intestinalis. Gene 275,
177-183.
Takada, S., Wilkerson, C. G., Wakabayashi, K., Kamiya,R., and Witman, G. B. (2001) The outer dynein arm
docking complex: composition and characterizationof a subunit (Oda1) necessary for outer arm assembly. Mol. Biol. Cell in press.
Wakabayashi, K., Takada, S. Witman, G. B., and Kamiya, R. (2001) Transport and arrangement of theouter-dynein-arm docking complex in the flagella ofChlamydomonas mutants that lack outer dynein arms.
Cell Motil. Cytoskeleton 48, 277-286.
Yanagisawa, H. and Kamiya, R. (2001) Association
between actin and light chains in Chlamydomonas
flagellar inner-arm dyneins. Biochem. Biophys. Res.
Commun. 288,443-447.
Yoshimura, K. and Kamiya, R. (2001) The sensitivity ofChlamydomonas photoreceptor is optimized for thefrequency of bodily rotation. Plant Cell Physiol.
42, 665-672.

Fig. I. Immunoelectron micrographs of an axoneme labeled with anti-ODA-DC antibody and secondary antibody conjugated with gold paticles.
Note that gold particles tend to align linearly along microtubules, occasionally with an apparent periodicity of -24 nm. Bar: 100 nm.

20

NATIONAL INSTITUTEDIVISION OF CELL FUSION (ADJUNCT)
Professor:
Associate Professor:
Research Associate:
NIBB Research Fellow:

MABUCHI, Issei
ABE, Hiroshi
FUJIMOTO, Hirotaka
YONEMURA, Izuru

Cytokinesis in animal and some primitive eukaryotic
cells is achieved by the progressive contraction of thecleavage furrow. The cleavage furrow contains a contractile apparatus, called the contractile ring, which iscomposed of a bundle of actin filaments that lies in the
furrow cortex beneath the plasma membrane. It has
been established that the contractile ring contracts as theresult of interaction between actin filaments and myosin.
However, little is known about process of its formation,mechanism that controls its formation, protein constituents, and its ultrastructure. The goal of our research is tosolve these problems and thereby clarify the molecularmechanism of cytokinesis. For this purpose, we use
three kinds of cells, namely, sea urchin egg, Xenopus
egg, and the fission yeast Schizosaccharomyces pombe.
S. pombe is an excellent system to investigate thechanges in the actin cytoskeleton during cell cycle since
F-actin patches, F-actin cables and F-actin ring are only
visible structures in the cell. The F-actin ring is considered to correspond to the contractile ring in animal cells.
It is formed during anaphase in this organism. We haveexamined process of formation of the F-actin ring by
optical sectioning and 3-D reconstitution fluorescencemicroscopy. In wild type cells, both formation of anaster-like structure composed of F-actin cables andaccumulation of F-actin cables was recognized at themedial cortex of the cell during prophase to metaphase.
The formation of the aster-like structure seemed toinitiate by branching of the longitudinal F-actin cables
at a site near the spindle pole bodies which had been
duplicated but not yet separated. A single cable extended from the aster and encircled the cell at the equator toform a primary F-actin ring during metaphase. During
anaphase, the accumulated F-actin cables were linked tothe primary F-actin ring, and then all of these structures
seemed to be packed to form the F-actin ring. Theseobservations suggest that formation of the aster-likestructure and the accumulation of the F-actin cables at
the medial region of the cell during metaphase may berequired to initiate the F-actin ring formation. We further examined F-actin structures in both cdc12 andcdc15 early cytokinesis mutants. As a result, Cdc12
seemed to be required for the primary F-actin ring formation during prophase, while Cdcl5 may be involvedin both packing the F-actin cables to form the F-actin
ring and rearrangement of the F-actin after anaphase. In
spgi, cdc7 and sid2 septum initiation mutants, the Factin ring seemed to be formed in order.
In large eggs which undergo unilateral cleavage, it
has been proposed that localized elevation of cytosolic
free calcium ions ([Ca2+]i) at the growing end of thecleavage furrow (CF) triggers CF formation by theactivation of myosin ATPase activity through the my-osin light chain kinase. However, data regarding therelevance of Ca waves in CF formation is contradictoryor incomplete. We have improved the method to visualize Ca wave in dividing Xenopus eggs: we removed
the fertilization membrane and labeled the egg surface
with rhodamine-wheat germ agglutinin (WGA) to visualize the growing ends of the early CF. We imaged
wave type Ca signal with Calcium Green-1 dextran
from the animal hemisphere of the egg. In addition to
the Ca wave, which is the orchestrated Ca release froma· global area of the cell, smaller classes of Ca signal
have been described. These are Ca puffs, which arethought to be Ca release from 10-30 of coordinately
opened Ca channels, and Ca blips, which are Ca released from single opened Ca channel. These signals
were also examined during the furrow formation. Consequently, we could not detect any Ca wave, Ca puff, or
Ca blip at the growing end of the CF, although we could
detect two Ca waves at later stages of cytokinesis.
Furthermore, we lowerd [Ca2+]i in the egg by injectionof Ca-chelators. However, it did not affect cleavages ofthe egg. Therefore, we concluded that Ca signals are not
involved in CF formation in Xenopus egg.
We also concentrate our study on function of actinregulatory proteins, including ADF/cofilin family proteins, during cytokinesis using Xenopus eggs and embryos. ADF/cofilin family proteins exist in all animals
and plants examined and have been shown to be essential. We found that ADF/cofilin family proteins are
essential for cytokinesis. Recent studies revealed that
ADF/cofilin accelerates turnover of actin filaments bothin vitro and in vivo. Most recently, we found a novel
actin-regulatory protein which induces disassembly ofactin filaments cooperatively with ADF/cofilin. eDNA
analysis revealed that this protein is a Xenopus homologue of yeast actin interacting protein 1 (AIPl).
Thus, we designated this protein as Xenopus AIPl
(XAIPl). Purified XAIPl itself binds to pure actin filaments to some extent, but it induces a rapid, drastic
disassembly of actin filaments associated with cofilin.
Microinjection of this protein into Xenopus embryos
arrested development by the resulting actin cytoskeletal
disorder. XAIPl represents the first case of a protein
cooperatively disassembling actin filaments with
ADF/cofilin family proteins. In addition, we also identified the second protein which functions with
ADF/cofilin. That was a Xenopus homologue of cyclase-associating protein (CAP), originally reported as
an actin monomer-binding protein. Our biochemical
analyses, however, suggest that XCap accelerates nucleotide exchange of actin monomer and induces actin
polymerization from actin-cofilin complex at steadystate.
Publication List:
Asano, Y., and Mabuchi, I. (2001). Calyculin-A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggs. Cell Motil. Cytoskele. 48, 245-261.

FOR BASIC BIOLOGY21

Arai, R., and Mabuchi, I. (2002). F-actin ring formation
and roles of F-actin cables in the fission yeast
Schizosaccharomyces pombe . J. Cell Sci. in press.
Bernstein, B. W., Painter, W . B., Chen, H., Minamide, L.
S., Abe, H . and Bamburg, J. R. (2000). Intracellular pH
Modulation of ADF/Cofilin Proteins. Cell Motil. Cytoskel. 47, 319-336.
Motegi, F., Arai, R., and Mabuchi, I. (2001). Identificationof two type V myosins in fission yeast, one of whichfunctions in polarized cell growth and moves rapidly inthe cell. Mol. Biol. Cell12, 1367-1380.
Nakano, K., Sato, K., Morimatsu, A., Ohnuma, M., andMabuchi, I. (2001). Interactions among a fimbrin, a capping protein, and an actin-depolymerizing factor in organization of the fission yeast actin cytoskeleton Mol.
Biol. Cell12, 3515-3526.
Nakano, K ., Muto, T., and Mabuchi, I. (2001). Characterization of GTP-activating proteins for the Rho-familysmall GTPases in the fission yeast Schizosaccharomyces
pombe. Genes to Cells 6, 1031-1042.
Noguchi, T., and Mabuchi, I. (2001). Reorganization ofFig. I

actin cytoskeleton at the growing end of the cleavagefurrow of Xenopus egg during cytokinesis. J. Cell Sci. 114,
401-412.
Noguchi, T., and Mabuchi, I. (2002). Localized calcium
signals along the cleavage furrow are not involved in the
furrow formation. Mol. Biol. Cell in press.
Takahashi, T ., Koshirnizu, U., Abe, H ., Obinata, T. andNakamura, T. (2001) . Functional Involvement of
Xenopus LIM-Kinases in Progression of Oocyte Maturation. Dev. Biol. 229, 554-567.
Toya, M. , Motegi, F. , Nakano, K., Mabuchi, I., and Yamamoto, M . (2001). Identification and functional analysis of the gene for type I myosin in fission yeast. Genes
to Cells 6, 187-199.
Yamashiro, S., Kameyama, K., Kanzawa, N., Tamiya, T .,Mabuchi, I. , and Tsuchiya, T. (2001). The gelsolin/fragrnin family protein identified in the higher
plant Mimosa pudica. J. Biochem. 130, 243-249.
Yonemura, I., and Mabuchi, I. (2001). Heterogeneity ofmRNAs coding for Caenorhabditis elegans coronin-likeproteins Gene 271, 255-259.

First cleavage of a Xenopus egg.

22

-- J

NATIONALINSTITUTE

23

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.

*Closed during 2001 and will be reinitiated on new projects.

24

NATIONAL INSTITUTEDIVISION OF REPRODUCTIVE BIOLOGY
Professor:
NAGAHAMA , Yoshitaka
Associate Professor:
YOSHIKUN!, Michiyasu
Research Associate:
KOBAYASHI, Tohru
Technical Staff:
KOBAYASHI, Hiroko
JSPS Postdoctoral Fellow: MATSUDA, Masaru
IKEUCHI, Toshitaka
CHANG, Xiao-Tian
SHIBATA, Yasuji
SUDHAKUMARI, Cheni Chery
WANG, De-Shou
Postdoctoral Fellow:
SAKAI, Fumie
GUAN, Gui-Jin
SENTHILKUMARAN,
Balasubramanian
Graduate Student:
HORIGUCHI, Ryo (Graduate
University for Advanced
Studies)
FAN, Hai-Guang (Graduate
University for Advanced
Studies)
KOBAYASHI, Yasuhisa
Visiting Scientist:
MATSUDA, Chika
MATSUOKA , Yoh
CONSTEN, Dimitori

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

I. Sex-determining gene of medaka
Although the sex-determining gene, Sry, has been
identified in mammals, no comparable genes have beenfound in non-mammalian vertebrates. We used
positional cloning to identify the sex-determining gene
ofmedaka, Oryzias latipes. Chromosome walking using
a BAC library and DNA markers derived from acongenic strain mapped the sex-determining region to a
530 kb stretch of the Y chromosome. We found acongenic XY female medaka lacking 250 kb of thisregion, further shortening the probable sex-determining
region. Shotgun sequencing of this deleted region
predicted 27 genes; however, only three were expressedin embryos during sexual differentiation. Furthermore,
only one (PG I 7) of these three genes was present
specifically on the Y chromosome (Fig. 1). We also
found a naturally-occurring XY female. Offspring of
this female inherited a Y chromosome that contained,
but did not express, PG 17 suggesting an important rolefor PG17 in testis development. Based on its homology

Fig. I Cytogenetical mapping of the sex-determining region(SD) of medaka. Left, Fluorescence in situ hybridization(FISH) of metaphase chromosomes. SL2 (sex-linked marker 2,red) localizes on the short arms of the sex chromosomes,
whereas a BAC clone containing SLJ (sex-linked marker I,
yellow) localizes on the long arms of the sex chromosomes.
Arrowheads indicate sex chromosomes. Right, FISH of one
sex chromosome with three different probes (SL2, SLJ, SD).
Signals of a BAC clone containing SD are light blue.
with the DM gene family, Y chromosome specificity,
and apparent role in testis development, PG 17 was
identified as the sex-determining gene of medaka.

II. Endocrine
differentiation

regulationofgonadalsex

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 dayof hatching, primordial germ cells (PGCs ), are locatedin the outer layer of the lateral plate mesoderm aroundthe hind gut. At 3 days post-hatching, PGCs are locatedin the gonadal anlagen after the formation of the
coelomic cavity in the lateral plate mesoderm rather
than through active migration.
The gene vasa encodes a DEAD (Asp-Giu-Aia-Asp)family of putative RNA helicase and is present in thegerm line of several animal species. Two isoforms ofvasa mRNA and protein are present in tilapia. One(vas-s) lacks a part of the N-terminal region found in
the other isoform (vas). Both isoforms are expressed inoocytes through the embryonic stage when PGCs
localize in the lateral plate mesoderm. After PGCs
localization in the gonadal anlagen, vas-s expression
increases and vas expression becomes undetectable.
Expression of both isoforms is observed again after
morphological gonadal sex differentiation, irrespectiveof genotypic sex. In ovary, compared to vas expression
vas-s expression predominates throughout.oogenesis. In
testis, vas expression is predominant compared withvas-s during spermatogenesis. These results indicatethat relative expression of two vasa isoforms is

25

FOR BASIC BIOLOGY

dependent upon germ cell differentiation and sex. We
have also generated medaka transgenic lines with green
fluorescent protein (GFP) fluorescence controlled by
the regulatory regions of the olvas gene in the germ
cells. The intensity of GFP fluorescence increases
dramatically in PGCs located in the ventrolateral regionof the posterior intestine around stage 25 (the onset ofbrood circulation). Whole-mount in situ hybridizationand monitoring 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 ectopicallyafter stages 20-25. The GFP expression persists
throughout the later stages in the mature ovary andtestis.
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 ofgenetic males. Steroid-producing cells in ovaries, but
not testes, at the undifferentiated and differentiatingstages 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-l 0 days
post-hatching. These results strongly suggest that
endogenous estrogens act as the natural inducers ofovarian differentiation in tilapia. This hypothesis isfurther supported by evidence of masculinization ofgenetic female tilapia by inhibition of estrogensynthesis using an inhibitor of cytochrome P450
aromatase. In contrast, the ability of steroid-producing
cells to synthesize steroid hormones in the testes only
appears at the time of testicular differentiation.
Transcripts of androgen receptors are not present ingonads of genetic males during sex differentiation. We
have isolated two DM (Doublesex/Mab-3 DNA-binding
motit)-domain cDNAs from tilapia testis and ovary,
named DMRTJ and DMO, respectively. DMRTJ is
expressed only in Sertoli cells and DMO is detected
only in oocytes by in situ hybridization. The correlation
between expression of DMRTJ and testiculardifferentiation of both normal XY-male and sex
reversed XX-males suggest that DMRTJ is a candidate
testis-determining gene in tilapia. In contrast, abundant
DMO expression in pre- and early vitellogenic oocytes
in XX- and sex reversed XY-females indicates a
relationship between DMO and oocyte growth.

III. Endocrine regulation of spermatogenesis
Spermatogenesis is an extended process of differentiation and maturation of germ cells resulting in haploid
spermatozoa. Using an organ culture system for eel
testes consisting of spermatogonia and inactive somatic
cells, we have shown that the hormonal regulation of
spermatogenesis in eel testes involves the gonadotropin
stimulation of Leydig cells to produce 11-ketotestosterone (11-KT), a potent androgen in fish. In turn,
11-KT activates Sertoli cells to stimulate the productionof activin B. Addition of recombinant eel activin B tothe culture medium induced proliferation of
spermatogonia, producing late type B spermatogonia,
within 15 days in the same manner as did 11-KT.
cDNAs encoding two androgen receptors (ARa andAR~) have been cloned, for the first time in any vertebrates, from eel and tilapia testes. In situ hybridization
reveals that although both AR mRNAs are present in eel
testes prior to human chorionic gonadotropin (hCG)
injection, only ARa transcripts increase duringhCG-induced spermatogenesis suggesting that ARa andAR~ 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 Al transcripts are first detected inprimary spermatocytes during hCG-induced spermatogenesiss in eel testes suggesting an important role forcyclin Al in the progression to meiosis of male germ
cells.
IY. Endocrine regulation of oocyte maturation

Meiotic maturation of fish oocytes is induced by theaction of maturation-inducing hormone (MIH). 17a,20~-dihydroxy-4-pregnen-3-one(17a,20~-DP)
hasbeen identified as the MIHs of several fish species. Theinteraction of two ovarian follilce cell layers, the thecal
and granulosa cell layers, is required for the synthesis of
17a,20~-DP. The thea! layer produces !?a-hydroxyprogesterone that is converted to 17a,20~-DP in
granulosa cells by the action of 20~-hydroxysteroid
dehydrogenase (20~-HSD). The preovulatory surge of
LH-like gonadotropin is responsible for the rapid
expression of 20~-HSD mRNA transcripts in granulosa
cells during oocyte maturation. In tilapia, Northern blot
and RT-PCR analyses reveal that the expression of
20~-HSD in ovarian follicles is not detectable inpostvitellogenic follicles. A distinct expression is evident at the day of spawning. In vitro incubation of
postvitellogenic follicles with hCG induced theexpression of20~-HSD mRNA within 1-2 hrs, followedby final oocyte maturation of oocytes. Actinomycin D
completely blocks both hCG-induced 20~-HSD
expression and final oocyte maturation, indicating the
involvement and transcriptional regulation of 20~-HSD
in final maturation.
17a,20~-DP induces oocyte maturation by acting ona pertussis toxin-sensitive G-protein-coupled membrane
receptor. The early steps of 17a,20~-DP action involvethe formation of downstream mediator of this steroid,
the maturation-promoting factor or metaphasepromoting factor (MPF) consisting of cdc2 kinase andcyclin B. 17a,20~-DP induces oocytes to synthesize
cyclin B which activates a preexisting 35-kDa cdc2
kinase via phosphorylation of its threonine 161 by athreonine kinase (M015), thus producing the 34 kDa
active cdc2. Polyadenylation of cyclin B mRNA isinvolved in 17a,20~-DP-induced initiation of cyclin B
mRNA translation. The Mos!MAPK pathway is notessential for initiating goldfish oocyte maturation

26
despite its general function as a cytostatic factor (CSF).
Upon egg activation, MPF is inactivated by degradationof 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:
Ikeuchi, T., Todo, T., Kobayashi, T. and Nagahama, Y.
(200 1) Two subtypes of androgen and progestogen
receptors in fish testes. Comp. Physiol. Biochem. Part B
129, 449-455.
Ikeuchi, T., Todo, T., Kobayashi, T. and Nagahama, Y. A
novel progestogen receptor subtype in the Japanese eel,Anguil/ajaponica. FEBS Letters (in press).
Imahara, J., Tokumoto, M., Nagahama, Y., Ishikawa, K.
and Tokumoto, T. (2001) Reconstitution of sperm nuclei
of zebrafish, Danio rerio, in Xenopus egg extracts.
Marine Biotechnology 3, 53-57.
Kitano, T., Takamune, K., Nagahama, Y. and Abe, S.
(2001) Role of P450 aromatase in gonadal sex
differentiation in Japanese flounder (Paralichthys
olivaceus). Environmental Sciences, 8, l-11.
Kobayashi, T., Kajiura-Kobayashi, H. and Nagahama, Y.
Two isoforms of vasa homologs in a teleost fish: theirdifferential expression during germ cell differentiation.
Mech. Develop. (in press).
Lee, Y.H., Du, J.L., Yueh, W.S., Lin, B. Y., Huang, J.D.,
Lee, C. Y., Lee, M. F., Lau, E. L., Lee, F. Y., Morrey, C.,
Nagahama, Y. and Chang, C. F. (200 I) Sex change in theprotandrous black porgy, Acanthopagrus sch/egeli: A
review in gonadal development, estradiol, estrogen
receptor, aromatase activity and gonadotropin. J Exp.
Zoo!. 290, 715-726.
Liu, S.J., Govoroun, M., D'Cotta, H., Ricordel, M.J.,
Lareyre, J.J., McMeel, O.M., Smith, T., Nagahama, Y.
and Guiguen, Y (200 I) Expression of cytochrome
P450J3 (1113-hydroxylase) genes during gonadal sex
differentiation and spermatogenesis of rainbow trout,
Oncorhynchus mykiss. J Steroid Biochem. Mol. Bioi.
75, 291-298.
Morrey, C.E., Nagahama, Y. and Grau, .E. G. Terminal
phase males stimulate ovarian function and inhibit sex
change in the protogynous wrasse Thalassoma duperrey.
Zoo!. Sci. (in press).
Matsuda, M. and Nagahama, Y. Positional cloning of the
sex-determining region of medaka using a Y congenic
starain. Aqua Genome (in press).
Matsuda, M., Kawato, N., Asakawa, S., Shimizu, N.,
Nagahama, Y., Hamaguchi, S., Sakaizumi, M. and Hori,
H. (2001) Construction of a BAC library derived fromthe inbred Hd-rR strains of the teleost fish, Oryzias
latipes. Gene Genetic Systems 76, 61-63.
Matsuyama, M., Sasaki, A., Nakagawa, K., Kobayashi, T.,
Nagahama, Y. and Chuda, H. (2001) Maturationinducing hormone of the tiger puffer, Takifugu rubripes(Tetraodontidae, Teleostei): biosynthesis of steroids by
the ovaries and the relative effectiveness of steroid
metabolites for germinal vesicle breakdown in vitro.
Zoo/. Sci. 18, 225-234.

NATIONAL INSTITUTE

Nakahata, S., Katsu, Y., Mita, K., Inoue, K., Nagahama, Y.
and Yamashita, M. (2001) Biochemical identification of
Xenopus Pumilio as a sequence-specific cyclin B 1
mRNA-binding protein that physiologically interacts
with a Nanos homolog, Xcat-2, and a cytoplasmic
polyadenylation element-binding protein. J Bioi. Chern.
276,20945-20953.
Nakahata, S., Mita, K., Katsu, Y., Nagahama, Y. andYamashita, M. (2001) Immunological detection andcharacterization of poly(A) polymerase, poly(A)-binding
protein and cytoplasmic polyadenylation elementbinding protein in goldfish and Xenopus oocytes. Zoo/.
Sci. 18, 337-343.
Nakamura, M., Kobayashi, T., Yoshiura, Y. and Nagahama,
Y. Masculinizing all-female tilapia by administrating
aromatase inhibitor. Mol. Reprod. Develop. (in press).
Oba, Y., Hirai, T., Yoshiura, Y., Kobayashi, T. andNagahama, Y. (2001) Fish gonadotropin and thyrotropin
receptors: The evolution of glycoprotein hormone
receptors in vertebrates. Comp. Physiol. Biochem. Part
B 129,441-448.
Ohta, K., Rhaman, M.A., Chuda, H., Yoshikuni, M.,
Nagahama, Y. and Matsuyama, M. Maturation-inducing
hormone and it's membrane receptor in gonads ofJapanese yellowtail, Seriola quenqueradiata. Fisheries
Science (in press).
Tanaka, M., Kinosita, M. and Nagahama, Y. (2001).
Establishment of medaka (Oryzias latipes) transgeniclines with the expression of GFP fluorescenceexclusively in germline cells: a useful model to monitor
germline cells in a live vertebrate. Proc. Nat/. Acad
Sci. USA 98, 2544-2549.
Tanaka, M., Nakajin, S., Kobayashi, D., Fukada, S., Guan,
G. J., Todo, T., Senthilkumaran, B. and Nagahama, Y. .
Teleost ovarian carbo~yl reductase-like 20J3-hydroxysteroid dehydrogenase: Potential role in the
production of maturation-inducing hormone in final
maturation of oocytes. Bioi. Reprod (in press).
Tokumoto, M., Nagahama, Y. and Tokumoto, T. Molecularcloning of eDNA encoding a polypeptide chain elongation factor 1a from goldfish (Carassius auratus). DNA
Sequence (in press).
Tokumoto, M., Nagahama, Y. and Tokumoto, T. A major
substrate for MPF: eDNA cloning and expression ofpolypeptide chain elongation factor ly from goldfish(Carassius auratus). DNA Sequence (in press).
Yamaguchi, A. and Nagahama, Y. Structure and protein
compositions of the nuclear matirix prepared from
germinal vesicles of goldfish (Carassius auratus)
oocytes. Mol. Reprod Develop. (in press).
Yamaguchi, A. and Nagahama, Y. Somatic lamins ingerminal vesicles of goldfish vitellogenic oocytes. Cell
Structure and Function (in press).

FOR BASIC BIOLOGY27

DIVISION OF CELL DIFFERENTIATION
Professor:
Research Associate:

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:

Technical Assistant:

Secretary:
Visiting Scientist:

MOROHASHJ, Ken-ichirou
SHIMONO, Akihiko
ISHIHARA, Satoru
FUKUI, Yuko
OKA, SanaeSUZUKI, Taiga
MOHAMAD, Zubair n
SUGIYAMA, Noriyuki n
SHIMA, Yuichi 41
BABA, Takahi 51
FUKUDA, Takayuki 61
KUSAKA, Masatomo 71
KOMATSU, Tomoko 71
MORITA, Tomoko 81
MATSUYAMA, Makoto 91
ARIMA, Tatsuya 41
MIZUSAKI, Hirofumi 21
OWAKJ, Akiko 21
KOWA, Hiroe
ISHIKAWA, Azus 21
KUSAKA, Hiroko 21
DOUZONO, Akemi 31
SUGIURA, Akemi 101

li CREST, JST Postdoctoral Fellow
21 CREST, JST Technical Assitant
31 CREST, JST Secretary
41 Graduate School of Kyushu University

51 Graduate School ofTohoku University

61 Graduate School of Ehime University
71 Graduate University for Advanced Studies
81 Graduate School of Tokyo University
91 Graduate School of Nagoya University

101 Pfizer Inc.

Cell and tissue differentiation proceeds systematically based on orchestrated expressions of sets of genes.
The expressions commence successively along with thepassage of time. As the consequence, a single fertilised egg develops into a variety of tissues and organs,which consist of specialised cells in terms of theirstructures and functions. Accordingly, it is reasonable
to assume that investigation of the mechanisms underlying the cell and tissue-specific gene expression at amolecular level is essential for understanding molecular
frameworks for genetic cascades proceeding along withcell and tissue differentiation. Based on the concept
above, our division of Cell Differentiation has focussed
on sex differentiation of the gonads and differentiationof the steroidogneic tissues form the aspect of functionsof tissue-specific transcription factors and growth factors.
Several transcription factors are involved in the process of gonadal differentiation. Some of these factors,
such as SRY, WT-1, DAX-1, and SOX-9 have been identified as the responsible genes for various human diseases that display structural and functional defects intissues including the gonads. The essential functions ofother transcription factors such as Ad4BP/SF-I, Emx-2,
M33, and Lhx-9 were identified by phenotypes of theirgene disrupted mice. In addition, the expression pro-files with respect to their distribution and sexual dimorphism strongly suggest the functional significance at theearly stage of gonadal differentiation. However, itremains to be clarified how the transcription factors
above regulate their target genes and how the genes
encoding the transcription factors are regulated. Whenconsidering a gene regulatory cascade that supports
differentiation of the gonadal tissues, studies of theabove two directions are quite important. Based on
this background, we investigated mainly the functionsof Ad4BP/SF-1 and Dax-1, and the mechanism of gene
regulation encoding these factors.

I. Gene regulatory 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 cascade as the critical components. As the component in the cascades required for the steroidogenic
adrenocortical and gonadal differentiation, Ad4BP/SF-1
is locates at the upstream of tissue-specific genes, including the steroidogenic CYP genes, and should locate
at the downstream of other transcription factors regulating Ad4BP/SF-I gene. When considering that thecascade flows from upstream to downstream along withthe tissue differentiation and moreover Ad4BP/SF-1 isan essential transcription factor in the adrenocorticaland gonadal cascade, identification of the components
functioning
with Ad4BP/SF-1
and regulating
Ad4BP/SF-1 gene transcription is essential for fully
understanding the molecular mechanisms underlying
the tissue differentiation. Thus, some of the membersin this division have investigated gene regulation of
Ad4BPISF-I and Da.x-1, both of which are quite important for the tissue differentiation.
Based on the aspect above, the regulatory region of
the Ad4BP/SF-I gene has been investigated in vivo bymaking transgenic mice. A genomic DNA fragment
longer than 480 kb containing four genes including
Ad4BPISF-I has been examined if they have tissuesspecific enhancer element. Our survey for the genomic DNA revealed that particular regions in the
Ad4BP/SF-l gene are responsible for the gene expression specific for the adrenal cortex and ventromedial
hypothalamic nucleus.
Dax-1 is another transcription factor of our interest,
which is also implicated in the steroidogenic tissue
differentiation. Our previous study revealed that thefactor acts as a suppressor against Ad4BP/SF-l. However, regulation of the suppressive effect has remained
to be clarified at the molecular level. We recently
uncovered the function of particular sequences, LXXLL
motifs, located at the amino terminal half of Dax-1.
When Dax-1 functions as the suppressor, the amino acidsequences in the repeats are essential for a proteinprotein interaction with target nuclear receptors, andthereby the transcription mediated by the nuclear receptors are largely inhibited. The LXXLL motif was

28

NATIONAL INSTITUTE

originally identified in coactivators as a motif essential
for interaction with nuclear receptors. Thus, it ishighly likely that the LXXLL motifs in Dax-1 competewith those of the coactivators for interaction with nuclear receptors such as Ad4BP/SF-l (Fig 1). Although
it remains unclear how the inhibitory activity is regulated in a variety of physiological conditions, interaction
through the LXXLL motifs should be a crucial step for
the functional regulation of the transcription facotors .

Active Form

Suppressive Form
Dax-1

Coactivator

Ad4BP/SF-1

Ad4BP/SF-1

Coactivator
Fig 1

In addition to these transcription factors, it has been
accepted that Sox-9, Wt-1 , Emx-2, and GATA-4 are
implicated . in gonad differentiation through regulatingtranscription of gene essential for gonadal structuresand functions. Although the essential functions ofthese transcription factors have been elucidated throughsymptoms of the genetic disorders and/or the phenotypes of the gene disrupted mice, characterization of
their transcriptional regulation has not yet been performed enough. Therefore, we started to investigate it
through characterizing proteins interacting with thetranscription factors . In order to isolate the interacting
factors, yeast two-hybrid screening has been performed
using a eDNA library constructed with mRNA preparedfrom mouse fetal gonads. Extensive screening resulted
in isolation of molecules including coactivators, othertype of transcription factors, proteins carrying protein
modification activities, and proteins carrying domains
capable of signal transduction. In situ hybridization
analyses with these particular molecules revealed thatsome of them are expressed in the developing gonads,
and some of them showed sex dependent expression.
For investigating their biological significance, genedisruption study has been performed.

II. Wnt4 signal for the gonad sex differentiation
As reported previously, Ad4BP/SF-l is an indispensable component for Dax-1 gene transcription. In fact,
multiple binding sites recognized by Ad4BP/SF-l at the
upstream region of Dax-1 gene are necessary for transcriptional activation. The in vitro observation using
reporter gene assays was confirmed subsequently by anin vivo study using Ad4BPISF-1 gene disrupted mice,
which lacked Dax-1 expression in the developing geni-

tal ridge. Although these results strongly indicatedthat Ad4BPISF-1 gene is genetically located upstream
from the Dax-1 gene, their expression profiles in termsof distribution and sexual dimorphism do not necessarily agree with our findings. In this regard, it should benoticed that a recent gene disruption study indicatedimplication of Wnt4 in gonadal sex differentiation.
Normally, steroidogenic 3{3-HSD gene and MIS geneare expressed in the developing fetal gonads of males
but not females. Interestingly, however, the expression
was detected in the fetal ovary of the gene-disrupted
mice, suggesting that the Wnt4 represses 3{3-HSD and
MIS gene transcription in the fetal ovaries of the wild
type. If considering that some of Wnt signals activate
downstream gene transcription through stabilization of~-catenine, it is unlikely that the signal represses the3{3-HSD and MIS gene transcription .
To provide a rational explanation, we hypothesizedthat Wnt4 expressed in the developing gonad upregulates a suppressor molecule and thereby downregulates
3{3-HSD and MIS gene transcription. Since transcription of both genes is regulated in a positive fashion by
Ad4BP/SF- l, it was reasonable to assume that Dax-1
plays a role as the suppressor. To confirm this assumption, we examined if ~-catenin activates the Dax-1
gene transcription. As the result, Dax-1 gene transcription was activated in the presence of ~-catenin in adose-dependent manner. Interestingly the action of ~­catenin is further upregulated in the presence of
Ad4BP/SF-l, indicating that the two factors, ~-catenin
and Ad4BP/SF-l, synergistically activate the Dax-1
gene transcnpt10n. Showing a good correlation, interaction between Ad4BP/SF-l and ~-catenin was confirmed with protein-pull down and yeast tow-hybrid
analyses.
The mechanisms of Dax-1 gene regulation governing
its sexually dimorphic and spatial characteristics are
summarized in Fig. 2. As described previously, it isdifficult to explain the whole regulatory mechanism of
the Dax-1 gene transcription by Ad4BP/SF-l alone.
For instance, Ad4BP/SF-l is expressed in the maledeveloping gonads more abundantly than in the female.
Nevertheless, the amount of Dax-1 in the female developing gonad is higher than that in the male gonad. In
the case of Wnt4 expression in the developing gonads
and mesonephros, in situ examination revealed that theamount expressed in the female tissues is higher than in
the male. With respect to the distribution of Dax-1,
strong signals were detected in the gonadal regions
facing the mesonephros although such expression domain was not observed in the case of Ad4BP/SF-l. In
such inconsistent distribution, it is interesting to notethat the expression of Wnt4 in the gonads was moreabundant at the region proximal to than that distal tomesonephros . Therefore, to understand the mechanism underlying Dax-1 expression, we propose that
Ad4BP/SF-l plays basal and fundamental roles and that
the Wnt4 signal modulates the transcription mediatedby Ad4BP/SF-l . Although the regulation above islikely to function in the sexually differentiating gonads

29

FOR BASIC BIOLOGY

of both sexes, the mechanisms of other transcription
factors such as Sox-9 and Emx-2 are not fully understood. In addition, it should be noted that other growth
factors as well as other forms of Wnt molecules are
expressed in the developing gonads and mesonephros.
Further studies of the functional relationship between
growth factors and transcription factors should identify
the fine and sophisticated mechanisms underlying thesex differentiation of the gonads.

(Male Gonad ]
FGF9

.

Sox9

1A~BPr~1
~

Dax-1

j_
3~HSD and MIS(Female Gonad]

.,

>

FGF9

~

Sox9

< Wnt4> ~ Ad4BP/SF-1l< Da11j_> 313-HSD and MIS
Fig 2

Publication List:
Asoy, R., Mellgren, G., Morohashi, K., Lund, J. Activationof cAMP-dependent Protein Kinase increases the protein
level of Steroidogenic Factor-1. Endocrinol. in press.
Ikeda, Y., Takeda, Y., Shlkayama, T., Mukai, T., Hisano,S., Morohashi, K. (2001) Comparative localization of
Dax-1 and Ad4BP/SF-1 during development of the hypothalamic-pituitary-gonadal axis implies their closely
related and distinct functions. Develop. Dynam. 220,363-376.
Shibata, H., Ikeda, Y., Mukai, T., Morohashi, K., Kurihara,
1., Ando, T., Suzuki, T., Kobayashi, S., Murai, M., Saito,
1., Saruta, T. (2001) Expression profiles of COUP-TF,
DAX-1 and SF-1 in the human adrenal gland andadrenocortical tumors: Possible implications in steroidogenesis. Mol. Genet. Metab. 74, 206-216.
Suzuki, T., Mizusaki, H., Kawabe, K., Kasahara, M.,
Yoshioka, H., Morohashi, K. Concerted Regulation of
Gonadal Differentiation by Transcription Factors and
Growth Factors. In 'The Genetics and Biology of Sex
Determination' John Wiley & Sons Ltd. in press.

30

NATIONAL INSTITUTEDIVISION OF MORPHOGENESIS
Professor:
Associate Professor
Research Associates:
Technical Staff:

NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Student:

UENO, NaotoKINOSHITA, Noriyuki*
NAKAMURA, Makoto
TAKAHASHI, Hiroki
TAKAGI, Chiyo
YAMAMOTO, Takamasa
HYODO, Akiko
TERASAKA, Chie
MOROKUMA, Yoshie
NAKABAYASHI, fun
YOSHIDA , Satoru
KITAYAMA, Atsushi
MOROKUMA, Junji
KURATA, Tomoko n
OHKAWARA, Bisei n
TAKEUCHI, Masaki n
MIURA, Jyunko 21*from September I , 200I
n Graduate University for Advanced Studies
21
Graduate School of Nagoya University

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

I. Regulation of growth factor diffusion in patternformation
During early development, cells receive positional
information from neighboring cells to form tissue
patterns in initially uniform germ layers. Ligands of thetransforming growth factor (TGF-~) superfamily are

known to participate in this pattern formation. In
particular, activin has been shown to act as a long-range
dorsalizing signal to establish a concentration gradientin Xenopus. In contrast, the action of BMP-2 and BMP4, another members of the family, appear to influenceand induce ventral fates only where they are expressed.
This raises a question as to how the action of BMPs istightly restricted within and around the cells that
produce them. We have demonstrated that a basic coreof only three amino acids in the N-terminal region of
BMP-4 is required for its restriction to the non-neural
ectoderm corresponding to its expression domain . Our
results also suggest that heparan sulfate proteoglycans
bind to this basic core and thus play a role in trapping
BMP-4. We propose that restriction of BMP diffusionin vivo through the interaction with extracellularenvironment is critical for the precise definition of nonneural ectoderm during early embryogenesis.

II. Gastrulation movement
signaling

regulated

by

Wnt

Gastrulation is one of the most important processes
during morphogenesis of early embryo, involving
dynamic cell migration and change in embryo shape.
Almost all animals undergo gastrulation to form the gut.
In spite of its importance, the mechanism underlying theevent has just begun to be studied at molecular level.
The Wnt family of secretory glycoprotein is one of
the major families of developmentally importantsignaling molecules and plays important roles inembryonic induction, generation of cell polarity, andspecification of cell fate. Wnt proteins bind Frizzled(Fz) receptors, a membrane protein with seventransmembrane domains. So far, the Wnt/Fz signalingcascade has been shown to branch into at least threepathways. These are the canonical pathway, the planarcell polarity (PCP) pathway, and the pathway throughprotein kinase C (PKC). The canonical pathway is thebest-characterized signaling cascade which involves
Dishevelled (Dsh/Dvl) and ~-catenin and contributes toestablish the dorsoventral axis in Xenopus embryos. The
PCP pathway is essential for cell polarization and cell
movement during gastrulation. This pathway seems to

Figure 1. Diffusion rate of BMP is drastically modified by N-terminally localized basic amino_acid cor~. The photographs showthat wtBMP-4 (left) acts almost cell-autonomously and induces Smadl phosphorylatiOn only m the adJacent cells, whereas t..l
was deleted acquired a_ long
BMP-4 (right), a mutated BMP-4 from which N-terminally localized core of basic amino aci~srange effect affecting over many diameters of cells and induced their Smadl phosph~rylat10n. Green fluorescence md~cates
nuclear translocation of phosphorylated Smad I representing the BMP signal and cells stamed with red fluorescence are recipientanimal cap cells that received BMP signal from left-upper animal cap cells conjugated to them.

FOR BASIC BIOLOGY

involve Dsh/Dvl and c-Jun N-terminal kinase (JNK).
Although these signaling pathways have been
investigated extensively, it is stiil unknown exactly how
the Fz receptors generate signals to regulate gastrulation
cell movement. Thus, we screened for proteins whichinteract with the cytoplasmic domains of Fz using theyeast two-hybrid assay. So far, we have cloned two
Xenopus genes. One encodes a protein which contains
two PDZ domains. This gene is expressed ubiquitously
during gastrulation, and later in the central nervous
system. Our preliminary results suggest its role in theFz signaling, particularly in the PCP pathway. (a) It
binds Fz-7, an important receptor in the PCP signaling
pathway in Xenopus embryos, more strongly than otherFzs. (b) Overexpression of a predicted dominantnegative mutant, causes severe gastrulation defect, atypical phenotype seen by the inhibition of the PCP
pathway. These results suggest that this protein may bean essential component for gastrulation cell movement
acting in the PCP pathway.

m. Genetic screening for novel DPPIBMP
signaling components utilizing Drosophilamodel system
Drosophila is one of the ideal model organisms todissect signal transduction pathway by genetic methods.
We have carried out dominant suppressor screening for
two transgenic mutant flies expressed constitutively
activated PPP/BMP type-I receptors in wing imaginaldiscs. We isolated 19 suppressor mutants, Suppressor of
constitutively activated Dpp singnaling (Scad). Allelesof 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. SP-RING motif was originally found in PIASfamily proteins including Drosophila PIAS homolog
Su(var)2-l 0/Zimp. Recent studies have shown that
PIAS-family proteins function as a E3-SUMO ligase
and enhance SUMO conjugation against specific substrates. Scad67 is an evolutionary conserved moleculeand we found two Scad67 homologous genes in humanand also in other vertebrates. Homozygous Scad67 mutants show embryonic to pupal stage lethality. The most
severe zygotic mutant shows embryonic head structureand segmentation defects. Detailed Scad67 function
analysis especially in the SUMO mediated biologicalprocesses and also in DPP signalling are ongoing.
We also have been interested in the in vivo functionof the TGF-~ activated kinase-1 (TAKl) in Drosophila.
We succeed to isolate dTAKJ null mutant in collaborated with B. Lemaitre's group (CNRS, France). Interestingly, dTAKJ mutation does shows morphologicaland viability defects. However, we observed an impairment of antibacterial peptide gene expression in
dTAKJ mutants. Genetic studies of dTAKl suggested an
evolutionary conserved role for TAKl in the control ofrel/NF-KB dependent innate immune responses.

31

IV. Brachyury downstream notochorddifferentiation in the ascidian embryo
Ascidians, urochordates, are one of the three chordategroups, and the ascidian tadpole is thought to represent
the most simplified and primitive chordate body plan.
It contains a notochord, which is a defining characteristic of chordate embryo composed of only 40 cells. To
understand the morphogenesis in this simple system, we
have focused on a gene, Brachyury, which is known toplay an important role in the notochord development.
In ascidian, Brachyury is expressed exclusively in the
notochord and the misexpression of the Brachyury gene
(Ci-Bra) of Ciona intestinalis is sufficient to transform
endoderm into notochord. This gene encodes a sequence-specific activator that contains a T-box DNAbinding domain, and in vertebrates, it is initially expressed throughout the presumptive mesoderm andgradually restricted to the developing notochord andtailbud. The phenotype of the Brachyury mutants inmice and zebrafish revealed that this gene is essential
for notochord differentiation. Our goal is to elucidate
the down stream pathway of this important gene inascidian in order to set the stage for understanding not
only the formation and function of the notochord but
how this important structure has evolved. We conducted
the subtractive hybridization screens to identify potential Brachyury target genes that are induced upon CiBra overexpression. Out of 501 independent eDNA
clones that were induced cDNAs, 38 were specifically
expressed in notochord cells (Fig. 2). We characterized
20 of them by determining the complete nucleotide
sequences and in situ hybridization analyses whichshow the spatial and temporal expression patterns of thecDNAs. These potential Ci-Bra downstream genes
appear to encode a broad spectrum of divergent proteinsassociated with notochord formation and function.

V. TGF-~ family in nematodeWe have previously shown that DBL-1, a member of
TGF-~ superfamily regulates body length in C. elegans.

To understand molecular mechanism of body length
regulation by DBL-1, we examined tissue-specific requirement of a DBL-1 receptor SMA-6 by rescuingsma-6 mutation with tissue specific expression of thereceptor. We found that hypodermal expression of
SMA-6 is sufficient to rescue the sma phenotype, suggesting that hypodermis is the most critical target tissueof DBL-1 to regulate body length. Next we screened fortarget genes regulated by DBL-1 in hypodermal cells,
and identified a gene yk298h6 whose disruption by
dsRNAi resulted in long worm. yk298h6 was later
found to be identical with lon-1, a gene encoding a type
II membrane protein belonging to the PR family conserved from plant to human. LON-1 appears to suppress
hypodermal polyploidization and thus negatively regulate body length.

32

NATIONAL INSTITUTE

Publication List
Dong, W., Ogawa, S., Tsukiyama, S., Okuhara, S. Niiyama,
M., Ueno, N., Peterson, R. E. and Hiraga, T.(2002)
2,3, 7,8-Tetrachlorodibenzo-p-dioxin toxicity in thezebrafish embryo: altered regional blood flow andimpaired lower jaw development. Teraoka, H.,
Toxicological Sciences 65,192-199.
Hanazawa, M., Mochii, M. Ueno, N., Kohara, Y. and Iino,
Y. (2001) Use of eDNA subtraction and RNAinterference screens in combination reveals genes
required for germ-line development in Caenohabditis
elegans. Proc. Natl. Acad. Sci. USA 98, 8686-8691.
Ishizuya-Oka, A., Ueda, S, Amano, T., Shimizu, K.,
Suzuki, K., Ueno, N. and Yoshizato, K. (2001) Thyroidhormone-dependent and fibroblast-specific expression of
BMP-4 correlates with adult epithelial development
during amphibian intestinal remodeling. Cell Tissue Res.
303, 187-195.
ltoh, K., Udagawa, N., Katagiri, T., lemura, S., Ueno, N.,
Yasuda, H., Higashio, K., Quinn, J. M., Gillespie, M.
T., Martin, T. J., Suda, T., Takahashi, N. (2001) Bonemorphogenetic
protein2
stimulates
osteoclastdifferentiation and survival supported by receptor
activator of nuclear factor-kappab ligand. Endocrinology.
142, 3656-3662.
Kurata T., Nakabayashi, J., Yamamoto, T.S., Mochii, M.
and Ueno, N. (2001) Visualization of endogenous BMP
signaling during Xenopus development. Differentiation
67,33-40.
Mitani, Y., Takahashi, H. and Satoh, N. (2001) Regulationof the muscle-specific expression and function of an
ascidian T-box gene, As-T2. Development 128, 37173728.
Morita, K., Shimizu, M., Shibuya, H. and Ueno, N.

(2001) A Daf-1-binding protein BRA-1 is a negative
regulator of Daf-7 TGF-~ signaling. Proc. _Natl.
Acad. Sci. USA 98, 6284-6288.
Ohkawara, B., Iemura, S., ten Dijke, P. and Ueno, N.
(2002) Action range of BMP is defined by its N-terrninal
basic amino acid core. Curr. Biol, 12, 205-209.
Sakuta, H., Suzuki, R., Takahashi, H., Kato, A., Shintani,T., lemura, S.-i, Yamamoto, T. S., Ueno, N. and Noda,
M. (200 l) Ventroptin: a BMP-4 antagonist expressed ina double-gradient pattern in the retina. Science 293, 111115.
Sugawara, K. Morita, K., Ueno, N. and Shibuya, H. (2001)
BIP, a BRAM-interacting protein involved in TGF-beta
signalling, regulates body _length in Caenorhabditis
elegans. Genes Cells 6, 599-606.
Takahashi, H. and Satoh, N. (2001) Trunk lateral cellspecific genes of ascidian Halocynthia roretzi. Zoot. Sci.
18, 361-366.
Vidal, S. S., Khush, R. S., Leulier, F., Tzou, P., Nakamura,
M. and Lemaitre, B. (2001) Mutations in the Drosophila
dTAK1 gene reveal a conserved function for MAPKKKs
in the control of rel/NF-kappaB-dependent innate
immune responses. Genes. Dev. 15, 1900-12.
Yamamoto, T. S.,Takagi, C., Hyodo, A. C. and Ueno,
N.(2001) Suppression of head formation by Xmsx1through the inhibition of intracellualr nodal signaling.
Development 128, 2769-2779.
Yoshida, S., Morita, K., Mochii, M. and Ueno, N. (2001)
Hypodermal expression of Caenorhabditis elegans TGF~ type I receptor SMA-6 is essential for the growth andmaintenance of body length. Dev. Bioi. 240, 32-45.

33

FOR BASIC BIOLOGYDEPARTMENT OF REGULATION BIOLOGY

Chairperson: NODA, Masaharu

DIVISION OF MOLECULAR NEUROBIOLOGYDIVISION OF CELLULAR REGULATIONDIVISION OF BIOLOGICAL REGULATION (ADJUNCT)DIVISION OF BEHAVIOR AND NEUROBIOLOGY (ADJUNCT)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

34

NATIONAL INSTITUTEDIVISION OF MOLECULAR NEUROBIOLOGY
Professor:
Associate Professor:
Research Associates:

Technical Staff:
NmB Research Fellow:
Post£loctoral Fellows:
Graduate Students:

Visiting Scientists:

Technical Assistant:

Secretary:
JST Technical Staff:

NODA, Masaharu
MAEDA. Nobuaki (-Nov. 2001)
SHINTANI, Takafumi
YUASA, Juni chi (-Sept. 2001)
SAKUTA. Hiraki (Oct. 2001 -)TAKEUCHI.Yasushi
YAMADA, Kaoru (-July 2001)
SUZUKI, Ryoko
SAKUTA, Hiraki(- Sept. 2001/
FUJIKAWA, Akihiro 1
KATO, Akira
FUKADA, Masahide
HIYAMA. Takeshi
TAMURA, Hiroshi
OHKA WARA, Takes hi
TAKAHASHJ,Hiroo
YUASA , Junichi (Oct. 2001 -l
YAMAMOTO. Shoichi (May 2001 -l
WATAKABE.lkuko
USAMI, Suzuyo (Apr.-Dec. 2001)
AYABE. Yuko (Jun. 2001 -)YAMADA. Kaoru (Aug. 2001 -)MATSUI, Mie (Oct. 2001 -)KODAMA, Akiko
M/ZOGUCHI, Masae
GOTO, Megumi

In the first-round screening using a eDNA subtractive
hybridization technique, we identified two winged-helix
transcriptional regulators, CBF-1 and CBF-2, expressedin the nasal and temporal retina, respectively. Furthermore, our rnisexpression experiments using a retroviral vector showed that these two transcription factors
determine the regional specificity of the retinal ganglion
cells, namely, the directed axonal projections to theappropriate tecta! targets along the anteroposterior axis.
To further search for topographic molecules in the embryonic retina, we next performed a large-scale screening using a new eDNA display system called Restriction
Landmark eDNA Scanning (RLCS). With the assistance of a computer image-processing software, we
successfully identified 33 molecules along thenasotemporal axis and 20 molecules along thedorsoventral axis, with various asymmetrical expression
patterns in the developing retina. We have elucidated
the primary structures of all these eDNA clones andCREST, JST
PREST, JST3from SHIONOGI & Co. LTD.
I
2

We have been studying the molecular and cellularmechanisms underlying the development and functioning of the vertebrate central nervous system. The
scope of our interests encompasses regional specification in the retina, neuronal differentiation, cellular migration, path-finding and target recognition of axons,formation and refinement of specific synapses, and also
various functions of the matured brain.

I. Molecular
projection

mechanismoftheretinotectal

Topographic maps are a fundamental feature of neural networks 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 oflower vertebrates including birds has been used as areadily accessible model system. In this projection,
the temporal (posterior) retina is connected to the rostral(anterior) part of the contralateral optic tectum, the nasal (anterior) retina to the caudal (posterior) tectum, andlikewise the dorsal and ventral retina to the ventral anddorsal tectum, respectively. Thus, images received by
the retina are precisely projected onto the tectum in areversed manner.
Since 1992, we have been devoting our efforts tosearching for topographic molecules which show
asymmetrical distribution in the embryonic chick retina.

Fig.! Expression patterns of Ventroptin mRNA during
development(A) Coronal section in situ hybridization of the eye at stage
14 . . (B, C) Whole mount in situ hybridization of E3 (stage
16-17) chick embryos for Ventroptin (B) and BMP-4 (C).
Insets show results in coronal sections of the E3 eyes.
Ventroptin was expressed in the ventral retina (arrowheads inB) and the forebrain (arrow). BMP-4 was expressed in thedorsal retina (arrowheads in C) and the periphery of the nasal
pit (arrow) . (D) Coronal section in situ hybridization at E8 .
Ventroptin is expressed in a ventral high-dorsal low gradient(E) Horizontal section at E8. Ventroptin is expressed in anasal high-temporal low gradient. (F) Flat mount in situ
hybridization of E8 retina. (G) Schematic drawing ofVentroptin expression in the retina. Double-gradient expression is represented by the density-gradient of color. Scale
bars; 600 ~-tm (D and E). N, T, D and V indicate nasal, temporal , dorsal and ventral, respectively (D-G).

35

FOR BASIC BIOLOGY

examined their expression patterns during development.
These included many novel molecules together with the
known molecules: transcription factors (CBF-2, COUPTFII, etc.), receptor proteins (EphA3, EphB3, etc.),secretory factors, intracellular proteins, and so on.
Among them, we identified a novel retinoic acid(RA)-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 (Fig. 1). 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 (E6-8), a nasal high-temporal
low gradient expression pattern is also detected.
Ventroptin thus shows a double-gradient expression
profile along the dorsoventral and the anteroposterioraxes. Misexpression of Ventroptin altered expression
patterns of several topographic genes: BMP-4, Tbx5 andcVax along the dorsoventral axis and ephrin A2 along
the anteroposterior axis. Consistently, in these embryos , projection of the retinal ganglion cell axons to
the tectum was also changed along the both axes (Fig.
2). The topographic retinotectal projection along thedorsoventral and the anteroposterior axes thus appears
to be controlled not separately but in a highly concerted
manner by Ventroptin .

",,'~-------,Q
''
''
'

'

)j

'

:

,,,,

II

I

/

'

~I

~

<....... ___ ..../

v

D

v

D

v

Fig. 2Retinotectal projection at El8-1 9 after in ovo
electroporation(A) A typical projection pattern in the Ventroptin misexpressed
embryo. A number of the dorsal axons entered the dorsal
tectum (large arrows) and all the axons extended to theposterior end of the tectum (arrowhead).
(B) A typicalprojection pattern in the control embryo. The dorsal axons
form a tight terminal zone (*) in the middle of the ventral
tectum. (C) The arrow indicates the position of the Oil Javelin the dorsal periphery of the right retina.
(D and E)
Schematic drawings of (A) and (B), respectively. Anterior isdown and posterior is up.

Currently, with respect to the identified topographic
molecules, we are conducting over- and misexpression
experiments using viral vectors and in avo
electroporation to elucidate their molecular functions.
We expect that our studies will lead to elucidation of
the molecular mechanism underlying the retinal patterning and topographic retinotectal projection, andultimately to uncovering the basic principles for establishing complicated but extremely precise neural networks.

II. Functional roles of protein tyrosine phosphatase 1;, andy
Protein tyrosine phosphorylation plays crucial roles
in various aspects of brain development and brain functions. In 1994, we found that PTP1;/RPTP~, a nervous
system-rich RPTP, is expressed as a chondroitin sulfate
proteoglycan in the brain. The extracellular region of
PTPI,; consists of a carbonic anhydrase-like domain, afibronectin-type III-like domain and a serine-glycinerich region, which is considered to be the chondroitin
sulfate attachment region. There exist three splicevariants of this molecule: a full-length transmembrane
form (PTPI,;-A); a short transmembrane form (PTPI,;-B);
and a soluble form (PTPI,;-S), which is also known as
6B4 proteoglycan/phosphacan.
PTPI,; is expressedfrom the early developmental stage to the adulthood.
This suggests that this gene plays variegated roles in thebrain development and brain function.
We began by searching for ligand molecules of thisreceptor.
We found in 1996 that PTPI,; binds
pleiotrophin/HB-GAM and midkine, closely related
heparin-binding growth factors which share many biological activities. The chondroitin sulfate portion of
PTPI,; is essential for the high affinity binding (Kd =-0.25 nM) to these growth factors, and removal ofchondroitin sulfate chains results in a marked decreaseof binding affinity (Kd = -13 nM) . We further revealed that chondroitin sulfate interacts with Arg 78 in
Cluster I, one of the two heparin-binding sites in the Cterminal half domain of midkine. This is the first
demonstration that chondroitin sulfate plays an important regulatory role in growth factor signaling.
Next, we examined the roles of pleiotrophin/midkinePTPI;, interaction in neuronal migration using the glass
fiber assay and Boyden chamber cell migration assay.
Pleiotrophin and midkine on the substratum stimulated
migration of neurons in these assays. Experiments
using various midkine mutants with various affinities
for PTPI,; indicated that the strength of binding affinities
and the neuronal migration-inducing activities arehighly correlated. These results suggest that PTPI;, isinvolved in migration as a neuronal receptor forpleiotrophin and midkine.
In order to reveal the intracellular signaling mechanism of PTPI,;, we performed yeast two-hybrid screening using the intracellular region of PTPs as bait. We
found in 1999 that PTPI,; interacts with PSD-95/SAP90
family members, SAP102, PSD-95/SAP90 and36

NATIONAL INSTITUTESAP97/hDlg, which are concentrated in the central synapses mediating protein-protein interactions to form
large synaptic macromolecular complexes. Here, theC-terminus of PTPI;; binds to PSD-95/SAP90 proteinsthrough the second PDZ domain . This suggests that
PTPI;; is involved in the regulation of synaptic function.
However, PSD-95/SAP90 family members are not likely
to be the substrate for PTPI;; because this family
members are not tyrosine-phosphorylated.
To identify the substrate molecules of PTPI;;, we haverecently developed the yeast substrate-trapping system.
This system is based on the yeast two-hybrid systemwith two essential modifications: conditional expressionof v-src to tyrosine-phosphorylate the prey proteins andscreening using a substrate-trap mutant of PTPI;; as bait.
Using this system, we successfully isolated a number ofcandidate clones for substrate molecules or interacting
molecules (Fig. 3). Among them, we first identified
GITI/Cat-1 as a PTPI;; substrate. It is known that Cat1 regulates Pak, a serine threonine kinase which serves
as a target for the small GTP-binding proteins, Cdc42
and Rae, and is implicated in a wide range of cellular
events including the cell adhesion and cellmorphological change.
Pleiotrophin, PTPI;; andGITI/Cat-1 might regulate the neuronal migration andneurite extension by controlling the Pak signaling
pathway. We are continuing efforts to characterize theSr~..

A
PTPt tCR-D1 902A

GAL4AD~Protei n/-~
Promoter

Re porter Gene

8v-src Induction
+

+

+

+

2· 1 (PSD95]

10.1 (GIT-1)

+

pACT2

~·

..,_,

-~·

.,....,

JH

500· 11 ·1

500-8- 1

500-1- l a

Fig. 3 The yeast substrate-trapping system(A) In the absence of methionine, prey proteins could be
phosphorylated by the induced v-src , and trapped by the bait of
the whole intracelluar domain with an Aspl902Ala mutation(PTPI; ICR-D 1902A). In the presence of I mM methionine,
when v-src is not induced, only the standard two-hybrid
bindings occur. The complex formation leads to activation oftranscription of the reporter gene, LacZ . (B) ~-Galactosidase
filter-lift assay of the isolated clones. Colonies with red
asteri sks showed an increase in blue-color development upon
induction of v-src. These are candidates for the substratemolecules. The other clones, mostly containing PDZ domains,
showed no difference in the color development by v-src
induction. pACT is a negative control.

other candidate clones.
To further study the physiological function of PTPI;;in vivo, we generated PTPI;;-deficient mice in which the
PTPI;; gene was replaced with the LacZ gene in 1997.
By investigating the expression of LacZ in heterozygous mutant mice, we demonstrated that neurons as wellas astrocytes express PTPI;; in the central nervous
system. We are currently studying the phenotype of
PTPI;;-deficient mice biochemically, anatomically,physiologically and ethologically, and have alreadyfound abnormalities in behavior, learning and memory,etc.

ill. Physiological roles ofNa, sodium channel
Human Nav2.1, mouse Na.2.3 and rat NaG/SCLll
were cloned as a subfamily of voltage-gated sodium
channel (NaCh). We found that these genes are species
orthologs. This channel molecule is divergent from thepreviously cloned NaChs (Na. l.l-1.9) including theregions involved in activation, inactivation and ion
selectivity. In the end of 2000, it was renamed Na. as
a novel enigmatic member of sodium channels.
To clarify the physiological function and cells expressing Na, sodium channels in vivo, we generated
knock-out mice in which Na. channel gene was replacedwith the LacZ or neogene by gene targeting. Analysisof the targeted mice allowed us to identify Na.producing cells by examining the lacZ expression.
Besides in the lung, heart, dorsal root ganglia and
Schwann cells in the peripheral nervous system, Na.
was expressed in neurons and ependymal cells in restricted areas of the central nervous system, particularlyin the circumventricular organs that are involved inbody-fluid homeostasis (see Fig. 1 in the part of Center
for Transgenic Animals and Plants). The null mutant
mice showed markedly elevated c-fos expression inneurons in the subfornical organ and organum
vasculosum laminae terminalis compared with wildtype animals . This suggests that these neurons are in a
hyperactive state in the Na_-deficient mice. Moreover,the null mutants showed abnormal intakes of hypertonic
saline. These findings suggest that the Na. sodium
channel plays an important role in the central sensing of
the body-fluid sodium level, and in regulation of salt
intake behavior. We are currently examining differences in the electrophysiological property between thecells derived from the Na,-null mutant mouse and wildtype mouse to gain insight into the channel property of
Na_.

37

FOR BASIC BIOLOGY

Publication List:
Kawachi, K., Fujikawa, A., Maeda, N. and Noda, M.
(2001) Identification of GIT1/Cat-1 as a substrate molecule of protein tyrosine phosphatase 1;/~ by the yeast
substrate-trapping system. Proc. Natl. Acad. Sci. USA
98, 6593-6598.
Qi, M., lkematsu, S., Maeda, N., Ichihara-Tanaka, K.,
Sakuma, S., Noda, M., Muramatsu, T. and Kadomatsu,
K. (200 1) Haptotactic migration by midkine:
Invovement of protein-tyrosine phosphatase 1;,, mitogenacti-vated protein kinase and phosphatidylinositol 3kinase. J.Biol. Chern., 276, 15868-15875.
Sakuta, H., Suzuki, R., Takahashi, H., Kato, A., Shintani,T., lemura, S., Yamamoto, T.S., Ueno, N. and Noda, M.
(2001) Ventroptin: A BMP-4 antagonist expressed in a
double-gradient pattern in the retina. Science 293, 111115.
Shintani, T., Maeda, N. and Noda, M. (2001) Receptorlike protein tyrosine phosphatase "{ (RPTPy), but not
PTPI;/RPTP~. inhibits NGF-induced neurite outgrowthin PC12D cells. Dev. Neurosci., 23, 55-69.
Sugawara, T., Tsurubuchi, Y., Agarwala, K. L., Ito, M.
Fukuma, G., Mazaki-Miyazaki, E., Nagafuji, H., Noda,
M., lmoto, K., Wada, K., Mitsudome, A., Kaneko, S.,
Montal, M., Nagata, K., Hirose, S. and Yamakawa, K.
(2001) A missense mutation of the Na+ channel ausubunit gene NaJ .2 in a patient with febrile and afebril
seizures causes channel dysfunction. Proc. Natl. Acad.
Sci. USA 98, 6384-6389.
Thornaidou, D., Coquillat, D., Meintanis, S., Noda, M.,
Rougon, G. and Matsas, R. (2001) Soluble forms of
NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein

tyrosine phosphatase 1;/~ as the putative F3 receptors on
Schwann cells. J. Neurochem. 78, 767-778.
Zubair, M., Watanabe, E., Fukada, M. and Noda, M.
(2002) Genetic labeling of specific axonal pathways inthe mouse central nervous system. Eur. J. Neurosci., inpress.

Abstracts:
Fujikawa, A., Watanabe, E., Sakaguchi, G., Katsuura, G.,
Hattori, S., Song, W.J., Noda, M. Dopmainergic dysfunction in the mice lacking the receptor tyrosine
phosphatase 1;,/RPTP~ gene. ibid.
Hiyama, T.Y., Watanabe, E., Yoshida, S., Noda, M. Na.
channel is involved in monitoring extracelular sodium
concentration. ibid.
Kawachi, H., Fujikawa, A., Maeda, N. Noda, M. Identification of GITl/Cat-1 as a substrate molecule of protein
tyrosine phosphatase 1;/~ by the yeast substrate-trapping
system. ibid.
Sakuta, H., Suzuki, R., Takahashi, H., Kato, A., Shintani,T., lemura, S., Yamamoto,T.S., Ueno, N., Noda, M.
Ventroptin: A novel BMP-4 antagonist expressed in a
double-gradient pattern in _the retina. In the abstracts of
the 3l'h Society for Neuroscience of North America.
2001.
Suzuki, R., Shintani, T., Sakuta, H., Kato, A., Ohkawara,
T., Osurni, N., Noda, M. Novel retinoid dehydrogenases expressed in the retina. ibid.
Watanabe, E., Fujikawa, A., Matsunaga, H., Yasoshima, Y.,
Sako, N., Yamamoto, T., Noda, M. Na. channel is involved in control of salt intake behavior in CNS. ibid

38

NATIONAL TNSTITUTEDIVISION OF CELLULAR REGULATION
Professor:
Associate Professor:
Research Associates:

MURATA. Norio
MIKAMI, Koji
NISHIYAMA, Yoshitaka
SUZUKI, !wane
Technical Staffs:
IINUMA , Hideko
YAMAGUCHI, Katsushi
NIBB Research Fellow: YAMAMOTO, Hiroshi
JSPS Research Fellows: SHIVAJI, S 21
MOHANTY, Prasanna 31
JSPS Post-Doc Fellow: SULPICE, Ronan 41
Graduate Students:
KANESAKI, Yu
FERJANI, Ali
SUZUKI, Shingo
Technical Assistants:
OKADA, Akiko
KOIKE, Yukari
KAIDA. Satomi
Secretaries
GOTO, MariODA. Yuko
KUBOKI, Yuko
KAWAI, Masayo
HFSP Post-Doc Fellow: INABA, MasamiMonkash o Foreign Scientist:
ALLAKHVERDIEV, Suleyman I. n
Visiting Scientists:
LOS, Dmitry A. 51
KISSELEVA , Larissa 51
SERGEYENKO, Tatiana 51
LYUKEVICH, Alexander A. 51
DEBRECZENY, Monika 61
SZALONTI, Balazs 61
MUSTARDY, Ltizsl6 61
OHAD, Itzhak 71
CHEN, Tony H. H. 81
FULDA, Martin 91
MARIN, Kay 101
CHINTALAPATI, Suresh
Kuma? 1n from the Institute of Basic Biological Problems,
Pushchino, Russia
21
from the Centre for Cellular and Molecular Biology,
Hyderabad, India
31
from the Regional Plant Resource Centre,
Bhubaneswar, India
41
from Universite de Rennes 1, Rennes, France
51
from the Institute of Plant Physiology, Moscow,
Russia61
from the Biological Research Center, Szeged,
Hungary
71
from the Hebrew University of Jerusalem, Jerusalem,
Israel81
from Oregon State University, Corvallis, OR, USA91
from Universitiit Hamburg, Hamburg, Germany
101
from Universitiit Rostock, Rostock, Germany

The major thrust of the research efforts of this division is directed towards understanding the signaltransduction mechanisms operating in plants and microorganisms with respect to perception of various
kinds of stress that arise due to changes in environmental conditions, with particular emphasis on extreme temperatures and salinity. In addition, techniques are being developed for transformation ofplants so that they gain the ability to acclimate to andtolerate such stress conditions. In 2001, significant
progress was made in the following areas using
cyanobacteria as a model system.

I. Regulation of gene expression in Synechocystis
subjected to salt and hyperosmotic stress
Salt stress and hyperosmotic stress are very important environmental factors that severely limit thegrowth and viability of plants and microorganisms.
However, these kinds of stress have been used without
proper discrimination in a number of investigations. In
this study, these effects were separately investigated on
the cytoplasmic volume and gene expression in
Synechocystis sp. PCC 6803 (hereafter Synechocystis).
Hyperosmotic stress due to 0.5 M sorbitol decreased
the cytoplasmic volume by 70% whereas salt stressdue to 0.5 M NaCI decreased it by 30% and the effectwas only transient. Furthermore, DNA microarray
analysis indicated that salt stress strongly induced theexpression of genes for some ribosomal proteins and anumber of genes for proteins of unknown function. In
contrast, hyperosmotic stress strongly induced theexpression of the genes for proteins involved in maintenance of cell wall structure (Fig. I). However, it wasalso observed that the expression of certain genes was
induced by both salt stress and hyperosmotic stress andthis category included the genes for heat-shockproteins, the genes for the enzymes that catalyze thesynthesis of a compatible solute, glucosylglycerol , andin addition a number of genes for proteins of unknown
function (Fig. lA). Our findings suggest that
Synechocystis is capable of discriminating salt stressand hyperosmotic stress as different stimuli [Kanesaki,
Suzuki, Allakhverdiev, Mikami and Murata, Biochem.
Biophys. Res. Commun . (2002) 290, 339-348] .
However, it does not preclude the possibility thatmechanisms common to the responses to each form ofstress might also exist. Both salt stress andhyperosmotic stress also repressed the expression ofcertain genes specifically, whereas certain other genes
like the genes included m the synthesis ofphycobilisome and photosystem I (PSI) subunits wererepressed by both (Fig. lB).

II. Identification of Mn 2+ sensor and signaltransduction pathway
Manganese (Mn) is an essential metal in all organisms.
It functions as a cofactor or as a prosthetic group in
A .Stress-inducible genes

B. Stress-repressible genes

Figure I. Categorization of salt and hyperosmotic stressregulated genes in Synechocystis.

39

FOR BASIC BIOLOGY

various enzymes . In particular in the photosynthetic
autotrophs which perform oxygenic photosynthesis, four
Mn atoms constitute the catalytic center of the oxygenevolving machinery in a pocket that is formed by the D I
and D2 subunits of photosystem II. Therefore, transportof Mn 2+ ions into the cell is crucial for the survival oforganisms.
It is known that the mntCAB operon that encodes an
ABC-type translocater of Mn 2+ ions is inducibly
expressed in Synechocystis, when the external supplyof Mn 2+ is limiting. However, the signal transduction
mechanism by which cyanobacterial cells recognize
the deficiency of Mn 2+ ions and induce the downstream
expression of the mntCAB operon for the translocaterof Mn 2+ ions remained to be understood. With this inview a knock-out library of histidine kinases andresponse
regulatorsofSynechocystis
wassystematically analyzed by DNA microarray technique .
The analysis revealed that mutants of a histidine kinase ,ManS, and a response regulator, ManR, exhibited
highly induced expression of the mntCAB operon under Mn2•-repleted conditions, suggesting that ManS
and ManR constitute the Mn 2+-ion sensor and response
regulator, respectively, of the signal-transducing pathway in Synechocystis . In the presence of Mn 2• , ManS
produces a signal to activate ManR and the activated
ManR represses the expression of the mntCAB operon,
whereas, under Mn 2•-depleted conditions, ManS does
not generate the signal and therefore ManR exists in its

Low concentrationofMn 2+High concentrationofMn 2+•

ManR G[J.CJ

~
Off

On

Figure 2. A hypothetical model for the regulation of the
mntCAB operon in Synechocystis.
Two membrane-spanning domains and a histidine kinasedomain in ManS (pink and green boxes) and a receiverdomain and a DNA-binding domain in ManR (red and purple
boxes) were predicted from the deduced amino-acidsequence. "H" and "D" represent histidine and aspartate
residues that might be involved in the phospho-relay.

inactive form as a consequence . of which theexpression of mntCAB operon remains active (Fig. 2).
This is the first report on the identification of thefunctional components of a two-component . signaltransduction system by the application of systematic
genomics in combination with DNA rnicroarray
analysis .

ill. Membrane rigidification enhances the coldinduced expression of heat-shock genes
Changes in the ambient temperature affect thephysical properties of membranes. To obtain insights
into the role of membranes in the mechanism of cold
signal perception, we have used a mutant ofSynechocystis, in which the desA gene for the ~12
desaturase and the desD gene for the ~6 desaturase areboth inactive as a result of targeted mutagenesis . Cellsof the desA-desD- mutant synthesizes only a saturated
C 16 fatty acid and a monounsaturated C 18 fatty acid,regardless of the growth temperature, whereas wildtype cells synthesize di-unsaturated and tri-unsaturated
C 18 fatty acids in addition to the monounsaturated
CIS fatty acid [Tasaka et al. (1996) EMBO J. 15,
6416-6425].
In the present study, Fourier transform infrared(FTIR) spectrometry revealed that the desA-desDmutation rigidified the plasma membrane of
Synechocystis at physiological temperatures. We
applied DNA rnicroarray technique to examine effectsof the membrane rigidification on the induction ofgene expression upon cold shock. The results
demonstrated that the cold inducibility of certain heatshock genes, namely, the hspA, htpG and dnaK2 genes,wasmarkedlyenhancedbythemembranerigidification. The cold inducibility of cold-shock
genes, such as the crh gene for an RNA helicase andthe rbpl gene for an RNA-binding protein, was notfurther enhanced by the rigidification. Northernblotting confirmed the results of DNA rnicroarray
analysis . Our findings suggest that the expression ofthese heat-shock genes upon cold shock might beregulated by changes in the physical properties of theplasma membrane, supporting the hypothesis thatrigidification of membrane .lipids is a primary signal
for cold sensing .

IV. Tolerance of photosynthetic machinery tolight stress depends on its ability to repair lightinduced photo-damage
Photosynthetic machinery is sensitive to various kindsof environmental stress and, in particular, thephotosystem II complex (PSII) is very sensitive to lightstress. To overcome this stress photosynthetic organisms
have the ability to rapidly repair the PSII from the lightinduced damage. We have developed a method to separately measure the damage and repair processes of PSII in
Synechocystis, and have examined the effect of various
kinds of stress on the damage and repair processes. We
have found that the rate of photodamage is proportional to40

NATIONAL INSTITUTE

light intensity and this proportionality is unaffected bystress conditions such as oxidative stress, salt stress andcold stress. In contrast, the rate of repair was fast at low
intensities of light (and reached the maximum level at
relatively low intensities of light), and this rate of repair isdepressed by stress conditions. These observations lead us
to conclude that photodamage to PSII depends solely on
the light intensity, whereas repair of PSII is dependent on
the site of regulation by various, kinds of environmental
stress which are termed "Repair-inhibitory stress".

V. Inhibition of the translational machinery byoxidative stress
Absorption of excess light energy by the photosynthetic machinery results in . the generation of reactive
oxygen species (ROS), such as. Hp 2, from thephotosystem complexes. We investigated the effects invivo of ROS to clarify the nature of the damage causedby such excess light energy to the photosynthetic
machinery in Synechocystis. ROS apparently augments
the photodamage to photosystem II (PSII) by inhibiting the repair of the damaged PSII and not by accelerating the photodamage directly. This conclusion was
confirmed by the effects of the mutation of genes for
HP 2-scavenging enzymes on the repair of PSII. Pulse
labelling experiments revealed that ROS inhibited thesynthesis of proteins de novo, in particular, that of theDl protein, a reaction center protein of PSII. Northernblotting revealed that the accumulation of transcripts
for the D 1 protein was not significantly affected by
ROS . Thus, ROS might influence the outcome ofphotodamage primarily via an effect on translation(Fig . 3).

psbA genes
Transcription~

List of publication:(1) Original articles
Allakhverdiev, S.I. , Kinoshita, M., Inaba, M., Suzuki,
I., and Murata, N. (2001) Unsaturated fatty acids inmembrane lipids protect the photosynthetic machinery against salt-induced damage in Synechococcus.
Plant Physiol. , 125, 1842-1853 .
Dilley, R.A., Nishiyama, Y. , Gombos, Z., and Murata,
N. (2001) Bioenergetics responses of Synechocystis
6803 fatty acid desaturase mutants at low temperature . J. Bioenerg. Biomemb. , 33,135-141.
Hirani, T.A., Suzuki, 1., Murata, N. , Hayashi, H., andEaton-Rye, J.J. (2001) Characterization of a twocomponent signal transduction system involved in
the induction of alkaline phosphatase underphosphate-limiting conditions in Synechocystis sp.
PCC 6803. Plant Mol. Bioi., 45, 133-144.
Inaba, M., Sakamoto, A., and Murata, N. (2001) Functional expression in Escherichia coli of low-affinity
andhigh-affinity
Na•/H+
antiportersofSynechocystis. J. Bacterial. , 183, 1376-1384.
Nishiyama, Y., Yamamoto, H., Allakhverdiev, S.I.,
Inaba, M., Yokota, A. , and Murata, N. (2001) Oxidative stress inhibits the repair of photodamage tothe photosynthetic machinery . EMBO J., 20, 55875594.
Suzuki, I., Kanesaki, Y., Mikami, K. , Kanehisa, M .,and Murata, N. , (2001) Cold-regulated genes undercontrol of the cold sensor Hik33 in Synechocystis .
Mol. Microbial. , 40, 235-244.
Turnbull, AP., Rafferty, J.B ., Sedelnikova, S.E.,
Slabas, A.R. , Schierer, T F., Kroon, J.T.M., Simon,
J.W. , Fawcett, T., Nishida, I., Murata, N., and Rice,
D.W. (2001) Analysis of the structure, substratespecificity, and mechanism of squash glycerol-3phosphate ( 1)-acyltransferase. Structure, 9, 1-20.

(2) Review articles
psbAmRNATranslation~

ROS(Oz -, HzOz , ·01-I, tQ 2)

pre-Dl protein
Processing

Dl pi"Otein

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

Sakamoto, A., and Murata, N. (2001) The use ofcholine oxidase, a glycine betaine-synthesizing enzyme, to create stress resistant transgenic plants .
Plant Physiol. (Update), 125, 180-188.
Suzuki, I., Los, D.A., and Murata, N. (2001) Perception and transduction of low-temperature signals to
induce desaturation of fatty acids. Biochem. Soc.
Trans. , 28, 628-630.

FOR BASIC BIOLOGYDIVISION OF BIOLOGICAL REGULATION AND
PHOTOBIOLOGY (ADJUNCT)
Professor:
Associate Professor:
Research Associate:
NIBB Research Fellow:
JSPS Postdoctoral Fellow:
Graduate Student:

WADA, Masamitsu
KIYOSUE, Tomohiro
KIKUCHI, Kazuhiro
KASAHARA, Masahiro
CHRISTENSEN, SteenIMAIZUMI, Takato(Tokyo Metropolitan
University)(-Mar31, 2001)
0/KAWA, Kazusato(Tokyo Metropolitan
University)
SUETSUGU, Noriyuki(Tokyo Metropolitan
University)
BRAIN Postdoctoral Fellow: TERAUCH/, Kazuki(-Mar 31.2001)
KAN, Don-lin(Apr 1- Oct.31, 2001)
PRESTO Fellow:
KAGAWA, Takatoshi
Visiting Scientist:
SHARMA, Rameshwar P.
(Hyderabad University)

Plants use light as an environmental factor which
controls their development as well as their otherphysiological phenomena. 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 clarify the signaltransduction pathways of photomorphogenesis. One of ourmajor subjects is chloroplast photo-relocation movement
which is thought to be one of the simplest phenomena inthis field. We use the fern Adiantum capillus-veneris andthe moss Physcomitrella patens as model plants for ourcell biological approach not only because the gametophytes are very sensitive to light, but also because theorganization of the cells is very simple. We also use
Arabidopsis mutants to clarify the genes regulating
chloroplast photo-relocation movement.

I. Cloning and characterization of blue-light
photoreceptors
We have described many blue-light induced
photomorphological responses in gametophytes of the fern
Adiantum capillus-veneris. As the first step in understanding the molecular mechanisms of these blue-light
responses, we are cloning and sequencing the genes of blue
light receptors, and are studying intracellular distributionsof the gene products and their function in Adiantum andPhyscomitrella

1- 1

Cryptochromes

Cryptochrome functions were studied using CRYla and
CRYlb double mutant of Physcomitrella patens. It was
revealed that blue light cryptochrome signals regulate
many steps in moss development including induction ofside branch of protonemata, and regulation of gametophyte
induction and its development. In addition, the disruptionof cryptochrome altered auxin response, including auxin-

41
inducible genes. Cryptochrome disruptants were moresensitive to external auxin than wild type in a blue lightspecific manner, suggesting that cryptochrome light signals
repress auxin signals to control plant development.

1- 2 Phototropin
Phototropin (photl) is another blue light photoreceptor
isolated recently in higher plants, and is a flavin binding
protein with light sensitive protein kinase activity. A eDNAof Adiantum PHOT2, a homologue of phototropin has beensequenced. Photocycle of FMN binding LOV domains of
the photl and phot2 expressed in E. coli were studied.

II. Chloroplast relocation movement
2- 1Arabidopsis

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 mechanism of chloroplast relocation, however, is not known. We
screened several mutants from T -DNA tagging lines aswell as EMS lines of Arabidopsis. By mutant analysis, wefound last year that photl and phot2 were photoreceptorsof chloroplast relocation movement induced by blue light.
This year, we clarified that the photl and phot2 were also
the photoreceptors redundantly working on stomatal
opening in Arabidopsis, in collaboration with the Prof.
Shimazaki's group of Kyushu University. Gene analysis ofmutants defective in chloroplast accumulation response arealso under way.

2- 2Adiantum

Adiantum phytochrome3 (PHY3) is a unique kimeric
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 light-induced
aphototropic) mutants of Adiantum which do not show
phototropic response and chloroplast photorelocation
movement under red light revealed that five rap mutant
lines tested have a mutation in the PHY3 gene. Moreover,
over expression of PHY3 genes in a rap mutant rescued the
red light-induced chloroplast movement, indicating thatphy3 is the photoreceptor of this phenomenon. PHY3 is
not yet known whether it works as a blue light photoreceptor.
Adiantum mutants which do not show chloroplast
avoidance movement under strong blue light were isolated. Gene analysis of these mutants revealed thatthey have a mutation in Adiantum phot2 gene, suggesting that Adiantum also use phot2 as a photoreceptor ofblue light-induced avoidance response as in the case of
Arabidopsis.

42

NATIONAL INSTITUTE

List of publication:
Briggs, W .R. et a! (200 1) The phototropin family of photoreceptors. Plant Cell13:993-997.
Imaizumi, T. et a! Cryptochrome light signals control
development to suppress auxin sensitivity in the moss
Physcomitrella patens. Plant Cell in press.
Kagawa, T. eta! (2001) Arabidopsis NPLI: A phototropin
homolog controlling the chloroplast high-light avoidance
response. Science 291: 2138-2141.

Kinoshita, T. et a! (2001) photl and phot2 mediate blue
light regulation of stomatal opening. Nature 414: 656660.
Sakai, T. et a! (2001) Arabidopsis nph1 and npll: Bluelight receptors that mediate both phototropism and chloroplast relocation. Proc. Nat/. Acad. Sci. USA 98:69696974.
Schultz, T. F. et a! (2001) A role for LKP2 in the circadian clock of Arabidopsis. Plant Cell13:2659-2670.

Chloroplast relocation in Arabfdopsfs tl1a!fanaweak blue light
cell 'oh'Dii~-----------~
~

phot1

phot:2&

phot2

Accumulation movement

Avoidance movement

Figure I A scheme of chloroplast photorelocation movement in Arabidopsis thaliana.

Strong blue light is absorbed by phototropin 2 (phot2)

and chloroplasts move out from the light (avoidance movement) (Kagawa et al 2001).

Under weak blue light condition, the blue light isabsorbed by photl and phot2, and chloroplasts move towards the light irradiated area (accumulation movement) (Sakai et al 2001).
from photoreceptors to chloroplasts is not yet known.

The signal

43

FOR BASIC BIOLOGYDIVISION OF BEHAVIOR AND NEUROBIOLOGY(ADJUNCT)
Professor:
Associate Professor:
Research Associate:
Postdoctoral Fellow:

Graduate Student:

MURAKAMI, Fujio
NAKAFUKU, Masato
TAMADA, Atsushi
KUMADA, Tatsuro 1
TANIGUCHI, Hiroki 4
HATANAKA, Yumiko 4
ONO, Hiroshi(from Osaka University)
KONISHI, Hiroyuki(from Osaka University)

The vertebrate nervous system contains a large number of neurons that are connected to each other by specific axonal projections. We are interested in how thecomplex but precise neuronal network, which is indispensable for functioning of the nervous system, is constructed during development. The goal of our researchin this division is to elucidate the cellular and molecularmechanisms underlying the neuronal network formationin vertebrates, with current focuses on the mechanismsof 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 cellularand molecular mechanisms of axon guidance in thevertebrate nervous system. We are particularly interested in how guidance mechanisms operating in different phases of axonal growth are assembled to generate
the complex but precise axonal wiring patterns in thebrain. To address these issues, we are first trying tounderstand axonal wiring patterns during developmentwith reference to the structural organiza-tion of thebrain. Using whole-mounted preparations of embryonic rat brains, we will reveal whole axonal trajectories
by labeling with neuronal tracers, such as Dil, or byimmunohistochemical 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 pathway have key roles in axonal guidance? 2) What
guidance cues exist in these structures? Are these
attractive cues or repulsive cues? Are these shortrange cues or long-range cues? 3) What molecules are
responsible for these cues? 4) How do multiple guidance mechanisms work in concert to generate specificwiring patterns? To answer these questions, we use
neuroanatomical techniques, in vitro culture techniques
including dissociated cell, explant and whole-mountcultures, 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
both along the radial axis and along the tangential axisof the neural tube. Another interest of our research
focuses on the cellular and molecular mechanisms underlying the radial 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 theradial migration, some neurons in the brain, such as
interneurons in the forebrain, move tangentially for along distance. We are also investigating how tangentially migrating neurons are guided for a long distancethrough specific routes to the final positions, using neurons that migrate from the rhombic lip at the dorsal rimof the hindbrain, such as cerebellar granule cells andprecerebellar 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 pieceof explant taken from a transgenic rat expressing GFP
or by introducing GFP eDNA into limited regions of thebrain by electroporation. Slices of the brain or flatmounted brain preparation are then cultured on permeable 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 molecularmechanisms of neuronal migration.

Publication List:
Imai, T., Tokunaga, A., Yoshida, T., Hashimoto, M.,
Mikoshiba, K., Weinmaster, G., Nakafuku, M., andOkano, H. (2001). The neural RNA-binding protein
Musashi1 translationally regulates mammalian numbgene expression by interacting with its mRNA. Mol.
Cell.Biol. 21, 3888-900.
Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako,
H., Nagao, M., Yoshida, S., Nabeshima, Y., Shimamura, K., and Nakafuku, M. (2001). Combinatorial
roles of olig2 and neurogenin2 in the coordinatedinduction of pan-neuronal and subtype-specificproperties ofmotoneurons. Neuron 31,757-71.
Nakashima, K., Takizawa, T., Ochiai, W., Yanagisawa,
M., Hisatsune, T., Nakafuku, M., Miyazono, K.,
Kishimoto, T., Kageyama, R., and Taga, T. (2001).
BMP2-mediated alteration in the developmental
pathway of fetal mouse brain cells from neurogenesis
to astrocytogenesis. Proc. Natl. Acad. Sci. USA 98,
5868-73.
Otsuka, T., Murakami, F., and Song, W. J. (2001). Excitatory postsynaptic potentials trigger a plateau potential in rat subthalamic neurons at hyperpolarizedstates. J. Neurophysiol. 86, 1816-25.
Sawamoto, K., Nakao, N., Kobayashi, K., Matsushita,

44

NATIONAL fNSTTTUTE

N., Takahashi, H., Kakishita, K., Yamamoto, A.,
Yoshizaki, T., Terashima, T., Murakami, F., ltakura,T., and Okano, H. (2001) . Visualization, direct isolation, and transplantation of midbrain dopaminergic
neurons. Proc. Nat/. Acad. Sci. USA 98, 6423-8.
Shirasaki, R., and Murakami, F. (2001) . Crossing thefloor plate triggers sharp turning of commissuralaxons. Develop. Bioi. 236, 99-108.
Tashiro, Y., Miyahara, M., Shirasaki, R., Okabe, M.,
Heizmann, C. W., and Murakami, F. (2001). Local
nonpermissive and oriented permissive cues guidevestibular axons to the cerebellum. Development 128,
973-81.

Figure I . 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.

45

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.

46

NATIONAL INS1ITU1E

DIVISION OF GENE EXPRESSION AND
REGULATION I
Professor:
Research Associate:/IDA, Shigeru
TERADA, Rie
INAGAKI, Yoshishige*
HOSHINO, Atsushi
TSUGANE, Kazuo
Technical Staff:
FURUKAWA, Kazuhiko
TANAKA-FUKADA, Sachiko
NIBB Research Fellow:
ISHIKAWA, Naoko
Monkasho Foreign Scientist:L/, Hong-Qingl)
JSPS Postdoctoral Fellow: CHOI, Jeong-Doo
PARK, Kyeung-/l
URAWA, Hiroko
Postdoctoral Fellow:
Michael T. Clegg2)
Visiting Scientist:
Joseph N. M. Mol3)
PARK, In-Sook4)
Laurel Caitlin Coberly)
Chareerat Mongkolsiriwatan6)
Graduate Student:
YAMAGUCHI, Toshio7h*
KAGAMJ, Takash/)
OHNISHJ, Makoto 7>
KIKUCHI, YasumasaB)
SAITOH, MihdJ)
Matthew WilliamHahn4)
Technical Assistant:
MORITA, Yasumasa*until April30, 200/**until Marchl3/, 200/1)from Chinese Academy of Sciences
2'J:.om University of California
3'/:,om Vrije University
4'from Yeungnam University
51;om Duke University
6'/:om Kasetsart University
7 Graduate University for Advanced Studies
81;om University of Shizuoka
9'from Toho University

The main interest of the group is in understanding thebiology of the dynamic genome, namely, genome
organization and reorganization and its impact on gene
expression and regulation. Although there are many
elements affecting organization and reorganization of
the genomes, we are currently focused on mobile
genetic elements in general and plant transposable
controlling elements in particular. Since plant transposable elements are known to be associated with both
genetic and epigenetic gene regulations, we are characterizing various aspects of genetic and epigenetic gene
regulations. In addition, we are also undertaking reverse
genetic approaches in order to elucidate the nature of
dynamic genome in plants.

I. Spontaneous mutants in the Japanesemorning glory.
The Japanese morning glory (Ipomoea nil or Pharbitis nil), displaying blue flowers, is believed to be originated from southeast Asia and has an extensive historyof 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 alaxative, and has become a traditional horticultural plantin Japan since around 17th century._ A number of its
spontaneous mutants related to the color and shape ofthe flowers and leaves have been isolated, and about
10% of these mutants carry mutable alleles conferring
variegated phenotypes. All of the mutants available arespontaneous mutants and most of them were isolated
more than 60 years ago. Several lines of evidence
indicate that an En/Spm-related transposable element
Tpnl and its relatives, which we termed Tpnl-familyelements, are a common spontaneous mutagen in theplant. Indeed, we have succeeded to identify three ofthese mutable alleles for flower pigmentation, flecked,
speckled and purple-mutable (pr-m), which are caused
by integration of TpnJ-related elements, Tpnl, Tpn2
and Tpn4, respectively. All of them are nonautonomous elements and their transposition is mediated by a TpnJ-related autonomous element which appears to be subjected to egigenetic regulations. Due toepigenetic inactivation of the autonomous element, rare
excision of these non-autonomous elements could occur
and such lines displayed apparently stable mutant
flowers. In accordance with this notion, an apparent
stable r-1 allele conferring white flowers is also caused
by insertion of a non-autonomous Tpnl-family element,
Tpn3, into the CHS-D gene encoding a chalcone synthase for anthocyanin biosynthesis.

II. Spontaneous mutants having TpnJ-relatedtransposons inserted into the gene encoding
anthocyanidin synthase in the Japanese morning
glory.
The anthocyanidin synthase (ANS) catalyzes 2oxoglutarate-dependent oxidation of leucoanthocyanidins to yield anthocyanidins in the anthocyanin
biosynthesis pathway. The ANS gene was first isolated
from maize and snapdragon, mutants of which are
deficient in pigmentation: the A2 mutations caused byinsertion of transposons in maize control coloration in
the aleurone layer of the kernel and a large deletion
mutation including the candi gene in snapdragon confers completely acyanic flowers. Out of 23 mutants of
the Japanese morning glory displaying white flowers
examined, three were found to carry an identical insertion of a 6.6-kb TpnJ-related transposon, TpnB, at thepromoter of the ANS gene (Fig. 1). In addition, we alsocharacterized a mutable line Shibori-chidori exhibiting
white flowers with red sectors, obtained from a redflower line Beni-chidori about 3 years ago, and foundthat the mutable allele was caused by integration of a
6.4-kb transposon, Tpn9, into the ANS gene. Interestingly, Tpn9 is almost identical to the Tpnl sequence
found at the mutable flecked allele. To our knowledge,
the ANS mutations characterized here are the first ANS
insertion mutations, including the mutable allele, that
affect flower pigmentation. Since the mutable line
Shibori-chidori was isolated as a spontaneous mutant
very recently, TpnJ-related elements still act as active
spontaneous mutagens that wouid generate new interesting traits in the Japanese morning glory.

47

FOR BASIC BIOLOGY

r-3

Shibori-chidori

ATG

Beni-chidori'fAG

1.0 kb

Fig. 1. Two mutant alleles in the ANS gene of the Japanese morning glory.
The mutants r-3 and Shibori-chidori have Tpn 8 and Tpn9 inserted into the ANS gene, respectively.

ill. The transposable element TipJOO found at themutable flaked allele for flower variegation of thecommon morning glory is an autonomous element.
The mutable flaked line of the common morning
glory (Ipomoea purpurea or Pharbitis purpurea) displays white flowers with colored flakes, and the flaked
mutation is caused by the insertion of a transposable
element TipJOO into the CHS-D gene for anthocyanin
biosynthesis. The 3.9 kb TipJOO element belonging to
the AciDs family contains an open reading frame
encoding a polypeptide of 808 amino acids. The frequency and timing of the flower variegation vary in
different flaked lines and a genetic element termed
Modulator has been postulated to affect the variegation
pattern. Since the pattern of the flower variegation isdetermined by the frequency and timing of the excisionof TiplOO from the CHS-D gene, we examined whether
TipJOO is an autonomous element capable of transposition in heterologous tobacco plants. The intact TiplOO
element was able to excise from its original position in
an introduced vector and to reinsert into new sites in the
tobacco genome, whereas its internal deletion derivative
was not. Based on these results, we concluded that
TipJOO is an autonomous element.

IV. Targeted gene disruption by homologous
recombination in rice.
The modification of targeted chromosomal genes
through homologous recombination is a powerful toolof reverse genetics for characterizing gene functions . In
higher plants, however, a transgene is integrated randomly into the genome by illegitimate recombinationeven the introduced sequence contains targeted homologous region. Although generation of a single
transgenic Arabidopsis line having a targeted genedisrupted was reported, the procedure was far from acommon practice in higher flowering plants. Rice is an
important staple food for more than half of the world ' s
population and has become a model monocotyledonous
plant because of accumulating information from ricegenome projects as well as efficient transformation. To
develop a reproducible and reliable procedure fortargeted gene disruption by homologous recombinationin rice, we tried to improve the following parameters:
efficiency of A grobacterium-mediated transformation,utilization of a strong positive-negative selection, and
PCR amplification for screening and detecting a longjunction fragment produced by homologous recombination . Subsequently, we conducted detailed Southern blot
analysis to confirm the occurrence of the expected48
precise homologous recombination. We chose the single
locus gene Waxy encoding granule~bound starch synthase, a key enzyme in amylose synthesis, in rice as amodel gene to be targeted, because its mutant is ofagronomic importance and because its phenotype inendosperm and pollen can be easily monitored bysimple iodine staining. A transgenic rice plant having atargeted gene disrupted by a transgene (Fig . 2) was
isolated, which appears to be a heterozygote with only
one copy of the integrated trans gene in its geneorne.

Fig. 2. Regeneration of a targeted callus through multiple shoots, aplantlet and a fertile transgenic plant from the targeted callus.
More than 50 shoots can be usually obtained from a targeted
callus.

NATIONAL INSTITUTE

Publication List:
Fukada-Tanaka, S., Inagaki, Y., Yamaguchi T . and Iida,
S. (2001) Simplified transposon display (STD): a
new procedure for isolation of a gene tagged by atransposable element belonging to the Tpnl family in
the Japanese morning glory. Plant Biotech. 18, 143149.
Hoshino, A., Johzuka-Hisatorni Y. and Iida, S. (2001)
Gene duplication and mobile genetic elements in themorning glories. Gene 265, 1-10.
Ishikawa, N., Johzuka-Hisatorni, Y., Sugita, K. , Ebinurna H. and Iida, S. The transposon TiplOO from thecommon morning glory is an autonomous elementthat can transpose in tobacco plants. Mol. Gen.
Genet. (in press) .
Karniunten, H. , Inoue, S., Yakabe Y. and Iida, S. Characterization of ISPsy2 and ISPsy3, newly identified insertion sequences in Pseudomonas syringae pv.
Eriobotryae. Gen. Plant Pathol. (in press).
Terada, R., Ignacirnuthu, S., Bauer, P., Schultze, M .,Kondorosi, A. , Potrykus I. and Sautter, C. (2001)
Expression of early nodulin promoter gene in transgenic rice. Curr. Sci. 81, 270-276.
Toki, K., Saito, N ., Iida, S., Hoshino, A., Shigihara A.
and Honda, T. (2001) Acylated pelargonidin 3sophoroside-5-glucosides from the flowers of theJapanese morning glory cultivar 'Violet'. Heterocycles 55, 1241-1248.
Toki, K., Saito, N. , Iida, S., Hoshino, A., Shigihara A.
and Honda, T. A novel acylated pelargonidin 3sophoroside-5-glucosides from greyish-purple flowers of the Japanese morning glory. Heterocycles 55,
2261-2267 .
Yamaguchi, T., Fukada-Tanaka, S., Inagaki, Y., Saito,N., Yonekura-Sakakibara, K. , Tanaka, Y. , Kusurni T.
and Iida, S. (2001) Genes encoding the vacuolar
Na+/H+ exchanger and flower coloration. Plant
Cell Physiol. 42, 451-461.

49

FOR BASIC BIOLOGY

of an E. coli rnhA mutant. The resulting RotA DNA
transformants were analyzed using pulsed field gelelectrophoresis, fluorescence in situ hybridization, andDNA microarrays. The following results were obtained.
(1) RotA DNA is amplified by about 40-fold on average.
(2) While 90% of the cells contain 6-IO copies of RotA
DNA, the remaining IO% of cells have as many as several
hundred RotA copies (Fig. I). (3) Amplification isdetected in all other Hot DNAs, among which HotB and
HotG DNAs are amplified to the same level as RotA.
Furthermore, HotL DNA, which is activated by blocking
the clockwise oriC-starting replication fork at theartificially inserted TerL site in the fork-blocked strain
with a rnh+ background, is also amplified.

DIVISION OF GENE EXPRESSION AND
REGULATION II
Professor:
Research Associate:

HORIUCHI, Takashi
HIDAKA, Masumi
KOBAYASHI, Takehiko
KODAMA. Ken-ichi*JOHZUKA, Katsuki
HAYASHI, Kohji**URAWA, Hiroko
SERIZ4WA, Naomi
WATANABE, Takaaki
UJIIE, Yoshifumi

Technical Staff:
Post Doctoral Fellows:
Graduate Student:
Visiting Fellows:* to Department of Biological Chemistry, University ofCalifornia-Irvine, California (Feb. I, I999- Jan. 15, 2002)* to Division of Gene Expression and Regulation I (Apr. 1,
2001)
The genomes of higher organisms contain significant
amounts of repetitive sequences, which in general, areunstable. At present, neither the physiological function(s)of repeated sequences nor the mechanism controlling the
instability is fully understood. To clarify these aspects,
we are pursuing the following themes using E. coli, S.
cerevisiae and plants: (1) An amplification mechanismof repeated sequences or genes, especially rRNA repeatedgenes, (2) a mechanism of replication fork blockdependent recombination, a key reaction that increases ordecreases the number of repeats, and (3) development ofin vivo artificial gene amplification systems. Functionaland structural analyses of the E. coli genome are also
being carried out. In 2001, work on the following threesubjects has advanced our knowledge of the dynamics andstructure of the genome.

I.

Amplification

of

Hot

DNA

segments

inEscherichia coli
In yeast, about ISO copies of rDNA are located at a
specific locus on chromosome XII.
A replication forkblocking site, called a replication fork barrier (RFB), is
located in each rDNA unit. To block the replication fork
at RFB, another trans-acting factor, named Fobi protein,is required. Our recent work indicated that amplificationof rDNA repeats requires a DNA replication forkblocking event. Furthermore, Fob I protein is essential
for rDNA region specific homologous recombination andthe production of circular rDNA molecules, as well as
rDNA amplification.
Such fork blocking dependent
recombination was first identified in recombinational
hotspot (Hot) in E. coli. The eight kinds of Hot DNAs
(RotA-H) were identified using an E. coli rnhA mutant.
Among these, enhanced recombination of three kinds of
Hot DNAs (RotA, B and C) was dependent on fork
blocking events at Ter sites.
Then, we examined
whether E. coli HotA DNAs are amplified when circular
DNA (RotA plus a drug-resistance DNA segment) isinserted into the homologous region on the chromosome

II. Yeast RNA polymerase I enhancer is dispensable
for both transcription of the chromosomal rDNA
gene and cell growth, and its apparent transcription
enhancement from ectopic promoters requires Fobl
protein.
Previously we developed a system in which various
deletions can be introduced into each rDNA repeat in S.
cerevisiae. Each rDNA repeat consists of the 35S rRNA
gene, the NTS I spacer, the SS rRNA gene, and the NTS2
spacer. The FOBJ gene was previously shown to berequired for replication fork block (RFB) activity at the
RFB site in NTSl, for recombination hot spot (HOTJ)
activity, and for rDNA amplification and contraction.
We have constructed a strain in which the majority of therDNA repeats are deleted, leaving two copies of rDNA
covering the 5S-NTS2-35S region and a single intact
NTS I. Growth of the strain is supported by a helperplasmid, carrying, in addition to the SS rRNA gene, the35S rRNA coding regions fused to the GAL7 promoter.
This strain carries a fobl mutation, and an extensive
expansion of chromosomal rDNA repeats wasdemonstrated by introducing the missing FOBJ gene bytransformation. The budding yeast 35S rDNA
transcription enhancer, which is located at the end of the
35S rRNA gene within the rDNA repeats, has been shownto greatly stimulate rDNA transcription in an ectopic
reporter system.
By removing the single enhancer
region remaining in the single NTSI in the two rDNA
copy strain, followed by rDNA amplification, weconstructed a yeast strain which was deleted of all of theenhancers from the rDNA repeated genes. We foundthat this strain did not show any defect in growth or rRNA
synthesis. This result suggests that transcription activity
measured in the ectopic site is not exactly parallel to thatin the rDNA cluster.
Ectopic transcription has an effect on recombination.
HOTJ DNA enhances recombination at the nearby sitewhen it inserted into a non-rDNA region. HOTJ DNA
consist two non-contiguous DNA fragments, called E andI, which are the enhancer and initiator of 35S rDNA
transcription, respectively. Thus, it was speculated that
HOTJ recombinational enhancement was caused bytranscription from the HOTJ region. Later it was proven

50

NATIONAL INSTITUTE

that a 35S rDNA specific transcription enzyme (Poll) is
required for HOTJ recombination. On the other hand,
we had previously expected that the fork blocking event isalso involved in HOTJ recombination, because the Eelement contains an RFB site ("enhancer" describedabove is the same as E except for RFB). Actually, we
found that the FOBJ gene is also required for HOTJ activity. However, another group recently reported that
HOTJ activity is RFB orientation independent, suggestingthat the fork blocking event is not responsible for HOTJ
enhancing. We, therefore, examined the effect of thefobl mutation on HOTJ transcription.
The results
showed that the mutation abolishes transcription from
HOTJ DNA. This means that while Fob1 is a fork
blocking protein in the rDNA cluster, it acts as transcription factor as well in the ectopic HOTJ system. Analogously, Poll is essential for HOTJ recombination, but it isnever required for homologous recombination in the
rDNA cluster. The HOTJ activation mechanism cannot
explain any mechanism working in the rDNA cluster.

ill.

in Arabidopsis. Mol. Genet. Genomics 266, 546-555.
Wai, H., Johzuka, K., Vu, L., Eliason, K., Kobayashi, T.,
Horiuchi, T., and Nomura, M. (2001) Yeast RNA
polymerase I enhancer is dispensable for growth and its
apparent transcnptton enhancement for ectopic
promoters Fob 1 protein implicated in replication andrecombination of rDNA. Mol. Cell. Bioi. 21, 55415553.

E. coli genomic structure and function

We completed the whole genomic sequence of E. coli
W3110, which is a derivative of a common ancestor of
MG1655, whose entire genomic sequence was sequenced
by the US team . The two strains were separated during orright after World War II. Currently, we are comparing
their sequences to understand the micro-evolution of thesestrains.
In order to identify the minimal set of genes required
for the duplication of a single cell and to elucidate thefunction of the genes, we initiated a team project in Japan,
headed by Mori (NAIST). For the past two years, ourproject has been supported by CREST from JST and isbeing carried out by the following groups: (1) resources,(2) informatics, (3) database, and (4) functional analysis.
Until now we have had adequate results, publishing in
more than fifty papers and some of the resources
established are now available worldwide. In the Annual
Bio-training Course held in our Institute this year, ourlaboratory organized an experimental course entitled"DNA chip: focused on its data analysis" which wasstrongly supported by team members, belonging toproject group (1).

Publication list:
Johzuka, K., and Horiuchi, T . (2002) Replication forkblock protein (Fobl) acts as a rDNA region specificrecombinator inS. cerevisiae. Genes Cells (in press)
Kobayashi, T., Nomura, M., and Horiuchi, T. (2001)
Identification of DNA cis-elements essential for
expansionofribosomalDNA
repeats
in
Saccharomyces cerevisiae. Mol. Cell. Bioi. 21, 136147.
Urawa, H., Hidaka, M., Ishiguro, S., Okada, K., andHoriuchi T. (2001) Enhanced homologous recombination caused by the non-transcribed spacer of the rDNA

Figure I. Amplification of HotA DNA in individual E. coli
cells.
A. ASH image of a parental strain (KHG571) with
-HotA- structure. B. ASH image of the HotA-tet DNA
transformed KHG571 strain with -HotA-tet-HotA- structure.
These photos show that while most cells of KHG571 have one
or two red fluorescence foci, each corresponding to a single
copy of HotA DNA, about I0% of cells of the transformed
KHG571 have very large, bright signals, each corresponding toabout 350 copies (more than half a E. coli genome size) on
average.

FOR BASIC BIOLOGYDIVISION OF SPECIATION MECHANISMS I
Professor:
Research Associate:

YAMAMORI, Tetsuo
KOMINE, Yuriko
WATAKABE, Akiya
KITSUKAWA , Takashi
Technical Staff:
UTSUMI, Hideko
SAWADA, Kaoru
NIBB Postdoctoral Fellow: Vigot, Rejan
Graduate student:
TOCHITANI, Shiro(until March)
HATA, Katsusuke
KOMATSU, Yusuke
SAKATA, Shu zo(kyoto University)
Visiting scientist:
SHIRAI, Yoshinori(until June)Our research goal is to understand mechanisms
underlying evolution of the nervous system. In order
to approach this question, we are studying the genes thatare expressed in specific areas of the primate neocortex.
Using differential display method, we have obtained
genes that showed marked differences within the
primate neocortex.
Our second approach is to understand informational
processing in the brain underlying learning behaviors
with gene expression techniques. Here, we report ourfindings in the year of 200 I.

I. Genes expressed in specific areas of the
neocortex
The neocortex is most evolved in mammals, particularly in primates, and thought to play the major role inhigher functions of the brain. It is known to be divided
into distinct functional and anatomical areas and has
been a matter of debate what extent the area of the
neocortex are genetically and environmentally determined. It is also puzzling why, during the evolution of
mammals, the neocortex was most markedly expanded
while the number of the genes in the mammal was littlechanged . To access these questions, 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
less than a factor of two in the difference of their expressions. Only one gene showed more than three folddifference and another one was between two and threefold difference within the three areas. These results
suggest that the genes that are expressed among thedifferent areas of the human neocortex are very similar.
However, the question remained whether there are any
genes that show marked difference within areas of neocortex .
2) In order to answer this question, we employed differential display methods and found at least two genesthat indicated area specific expressions .
i) One gene, designated occl, is specifically51
expressed in the occipital cortex, particularly in VI area,in the primate brain. Furthermore, the expression ofoccl turned out to be activity dependent, because, in the
monocularly deprive-monkeys injected with TTX into
one of the eyes, the expression of occl is markedly
decreased in the ocular dominance columns of the primary visual cortex (VI).
ii) The other gene that showed marked difference
within the neocortex, is gdf7, a member of BMPffGF-~
family, which is specifically expressed in the motor
cortex of the African green monkey . We are currently
examining the detailed expression pattern of the gene.
3) We have also further isolated several area specificgenes with RLCS (Restriction Landmark eDNA Scanning).

II.
Gene
expression
discrimination task

underaudio-visual

We studied gene expression of c-Fos under audiovisual discrimination tasks in collaboration withprofessor Yoshio Sakurai (Kyoto University) . We foundthat the visual and audio tasks enhanced the specificexpression of c-Fos in the visual and audio cortices,
respectively. Among the early visual and auditory
pathways examined, c-Fos was specifically induced in
the cortices but not in the earlier pathways, suggesting
the neural modulation of the neocortex depending on
the types of the tasks. Interestingly, the task-dependent
Fos expression was only observed in excitatory neurons
in the relevant sensory cortices .

III CNTF is specificallydeveloping pineal glands

expressed

intheFig. I Expression pattern of occ I in the visual cortex.
In situ hybridization pattern of occl in the primate visualcortex. occ I is markedly expressed in the layer 1Vcf3 andmoderately in the layers of II, III and IVa in area VI. The
boundary between VI and V2 is shown by an arrow .

52
CNTF, a member of the IL-6 family, attracts quite
attentions of developmental neuroscientists because
it shows various effects on neurons and glial cells.
The CNTF knockout mice, however, only indicatesmoderate 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 of members of
the family and their receptors and found the specificexpression of CNTF in the embryonic pineal glands
and eyes. Since sympathetic neurons are known toinnervate pineal glands and be responsive to CNTF,
our observation suggests that CNTF plays a role
when sympathetic neurons innervate pineal glands.

Publication list:
Original Articles
Fujiwara, T., Yamamori, T. and Akagawa, K.
Suppression of transmitter release by Tat HPC1/syntaxin 1A fusion protein. (2001) Biochim.
Biophys. Acta 1539, 225-232.
Hanazawa, A., Mikamai, A., Angelika, P.S., Takenaka,0., Goto, S., Onishi, A., Koike, S., Yamamoi, T.,
Kato, k., Kondo, A., Suryobroto, B., Farajallah, A.,
Komatsu, H. (2001) Electroretinogram analysis ofrelative spectral sensitivity in genetically identified
dichromatic macaques. Proc. Natl Acad. Sci. USA.,98, 8124-8127.
Hata, K., Arakai, M. and Yamamori, T. CNTF is
specifically expressed in the developing rat pineal
gland and eyes. NeuroReport (in press)
Karachot, L., Shirai, Y., Vigot, R., Yamamori, T. andIto, M. Induction of long-term depression in

NATIONAL INSTITUTE

cerebellar Purkinje cells requires a rapidly turned
over protein. (2001) J. Neurophysiol. 86, 280-289.
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. andYamamori, T. Variations in long and middlewavelength-sensitive opsin loci m crab-eatingmonkeys. Vision Res. (in press)
Sakata, S., Kitsukawa, T., Kaneko, T., Yamamori, T.
and Sakurai, Y. Task-dependent and cell-typespecific Fos enhancement in rat sensory cortices
during audio-visual discrimination.
Eur.
J.
Neuroscience (in press)
Tochitani, S., Liang, F., Watakabe, A., Hashikawa, T.,
and Yamamori, T. (2001) occ1 is preferentiallyexpressed in the primary visual cortex in an activitydependent manner: a pattern of gene expression
related to the cytoarchitectonic area in adult macaue
neocortex. Eur. J. Neurosci. 13, 297-307.
Vigot, R., Batini, C., Kado, R.T. and Yamamori. T.
Synaptic LTD in vivo recorded on the rat cerebellar
cortex. Arch. /tal. Biol. (in press)
Watakabe A. , Sugai T., Nakaya N., Wakabayashi K.,
Takahashi H., Yamamori T. and Nawa H. (2001)
Similarity and Variation in Gene Expression Among
Human Cerebral Cortical Subregions Revealed By
DNA Macroarrays: Technical Consideration of RNA
Expression Profiling from Postmortem Samples. Mol.
Brain Res., 88, 74-82.
Watakabe, A., Fujita, H., Hayashi, M. and Yamamori T.
(2001) GDF7, a BMPffGF beta family member, isenriched in the primary motor area of monkey
neocortex. J. Neurochem., 76, 1455-1464.

FOR BASIC BIOLOGY53

DIVISION OF SPECIATION MECHANISMS II
Professor:
Associate Professor
Assistant Professor:
Technical Staff:
NIBB Research Fellow:
JSPS Research Fellow:
Graduate Students:

Technical Assistant:

Secretary:
Visiting Scientists:

HASEBE, Mitsuyasu
MURATA, Takashi
FUJITA, Tomomichi
SUMIKAWA, Naomi
MISHIMA, Misako
HIWATASH/, Yuji (Oct./-)NISHIYAMA, Tomoaki
HIWATASH/, Yuji (-Sept. 30)
KOBAYASHI-ARAKAWA,
Satoko
SAKAKIBARA, Keiko
SANO, Ryosuke (-March 31)
SAKAGUCHI, Hisako(Shinshu Univ.)TANIKAWA, Yukiko
UMEDA, Masae (-Dec. 28)
B/TOH, Yoshimi
NARUSE, Mayumi
AOKI, Etsuko (April/-)
WATANABE, Kyoko (July 11-)YANO, Kana (Nov. 19-)
KABEYA, Kazuko
JoAnn Banks n (-July 15)
George Ratherford n (-July 15)
Jean-Pierre Zryd 21 (Sept. 24-)

nfrom Purdue University, West Lafayette, IN, USA
2Yrom Universite de Lausanne, Lausanne, Switzerland

All living organisms evolved from a common ancestor more than 3 .5 billion years ago, and accumulated
mutations on their genomes caused the present biodiversity. The traces of evolutionary processes are foundin the genomes of extant organisms. By comparing the
genomes of different organisms, we can infer (1) thephylogenetic relationships of extant organisms and (2)the genetic changes having caused the evolution ofmorphology and development. The inferred phylogenetic relationships give important insights on problems in various fields of evolutionary biology, and ourgroup focuses on biogeography, 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 ofplant body plan. We selected Arabidopsis (angiosperm), Gnetum (gymnosperm), Ginkgo (gymnosperm),
Ceratopteris (pteridophyte), Physcomitrella (bryophyte),
and some green algae as models to compare the genefunctions involved in development of the reproductive
organs and shoot apical meristem of land plants.

I. Evolution of reproductive organs in land plants
A flower is the most complex reproductive organ inland plants and composed of sepals, petals, stamens,
and gynoecium. Female haploid reproductive cells arecovered with a sporangium (nucellus) and two integuments, and further enclosed in a gynoecium. Male
haploid reproductive cells (pollens) are covered with asporangium (pollen sack). On the other hand, gymnosperms and ferns have simpler reproductive organs thanangiosperms and lack sepals and petals. Female spo-

rangia (nucellus) of gymnosperms are covered with
only one integument. Sporangia of ferns have no integuments 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
MADS-box gene family. These genes are transcription factors containing the conserved MADS and K
domains. MADS-box genes of angiosperms are divided into more than 10 groups based on the gene tree.
The 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 the flowering plant lineage ? Comparisons of
MADS-box and LFY genes in vascular plants suggestthat the following sequential changes occurred in the(1) Plant-type
evolution of reproductive organs.
MADS-box genes with both MADS and K domains
were established.
(2) The number of MADS-box
genes increased, and the three ancestral MADS-box
genes that later generate A-, B-, C-functions genes werelikely originated before the divergence of ferns and seedplants. (3) Specifically expressed MADS-box genes in
reproductive organs evolved from generally expressed
ones in the seed plant lineage. (4) The ancestral geneof the AG group of MADS-box genes acquired the Cfunction before the divergence of extant gymnosperms
and angiosperms.
(5) The gene duplication thatformed the AP3 and PI groups in MADS-box genes
occurred before the diversification of extant gymnosperms and angiosperms. (6) The ancestral gene ofangiosperm A-function gene was lost in extant gymnosperm lineage. (7) LFY gene became positively regulate MADS-box genes after the divergence of ferns andseed plants, because the fern LFY gene does not directly
regulate MADS-box genes (Himi et al. 2001). The
FLOILFY gene phylogenetic tree indicates that both
duplication and loss of FLOILFY homologs occurred
during the course of vascular plant evolution. The
inductive pathway from the LFY gene to the MADSbox genes already existed in the common ancestor ofangiosperms and gymnosperms, because overexpressionof Gnetum LFY homolog in transgenic Arabidopsis
promoted a conversion of a shoot meristem to a floral
primordium and the Arabidopsis LFY null mutant, ljj;26, with a malformed flower, was complemented byoverexpression of Gnetum LFY gene. (8) Spatial andtemporal patterns of A-, B-, C-function gene expression
were established in the angiosperm lineage (Shindo et al.
2001).

II. Homology of reproductive organs in seedplantsThe morphological variation among reproductive organs of extant seed plants makes assessment of organ
homology difficult. Comparisons of expression patterns of homeotic genes that control organ development
will yield new information about the homology of organs to assess inferences deduced from previous mor-

54

NATIONAL INSTITUTE

phological studies. To provide insights into the evolution of reproductive organs in seed plants, a Gnetum
LFY homolog was cloned and its expression patterswere compared to the conifer LFY homolog. The
comparison suggests that the Gnetum collar and ovule
are homologous with the conifer bract and ovuleovuliferous scale complex , respectively. This inference is concordant with our previous comparisons ofexpression patters of orthologous MADS-box genes
between Gnetum and conifers (Shindo eta!. 2001).

[A]caphytic to sporophytes in seed plant lineage. Molecularmechanisms of development in a diploid generation
have been well studied in some model angiosperms, but
we have scarce information on those in a gametophyte
generation. For example, mosses have leaf- and stemlike organs in their haploid generation, but it is completely unknown whether similar genes involved inangiosperm leaf and stem development are used in thegametophytic generation of mosses or not. To understand the evolution of body plans in diploid and haploid
generation at the molecular level, we focus on the comparison of molecular mechanisms governing shoot development between Arabidopsis and the moss Physcomitrella patens. P. patens is known by its high rateof homologous recombination and suitable for analyze
gene functions using the gene targeting, and should be agood model lower land plants .

111-11. Characterization of homeobox genes in themoss Physcomitrella patens
0•

FLO/LFY•

FLO/LFY & MADS-box

coFigure I . Expression patterns of gymnosperm FLOILFY homologs and MADS-box genes are shown schematically in a
scenario of the evolution of ovule-bearing s.tructures in conifers and Gnetum from a Cordaitales-like prototype. A, Fertileshoot subtended by a bract in the Cordaitales-like ancestor. B,
Ovuliferous structure of a conifer. C, Ovuliferous structure of
Gnetum . The ovuliferous scale of conifers should correspondto several sterile scales of the Cordaites-like ancestor based on
fossil records. Outer and middle envelopes likely correspondto two sterile scales of the Cordaites-like ancestor, because
these envelopes develop from a pair of opposite primordia. b,
bract; ca, cone axis; co, collar; in, integument; me, middle
envelope; n, nucellus; o, ovule; oe, outer envelope; os, ovuliferous scale; ss, sterile scale.

111-1. 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 became interpolated into the life cycle. In the earlystages of land plant evolution, sporophytes are epiphytic
to gametophytes, a~ observed in extant bryophytes.
During the course of evolution , both generations started
to grow independently at the stage of pteridophytes.
Finally gametophytes became much reduced and epi-

Homeobox genes encode transcnptwn factors involved in many aspects of developmental processes
including shoot development in angiosperms.
The
homeodomain-leucine zipper (HD-Zip) genes, which
are characterized by the presence of both ahomeodomain and a leucine zipper motif, form a clade
within the homeobox superfamily and previously reported only from vascular plants. We isolated 10 HDZip genes from P. patens (Pphbl-10 genes). Based ona phylogenetic analysis of the 10 Pphb genes and previously reported vascular plant HD-Zip genes, all thePphb genes except Pphb3 belong to three of the four
HD-Zip subfamilies (HD-Zip I, II, and III), indicatingthat these subfamilies originated before the divergenceof the vascular plant and moss lineages. Pphb3 issister to HD-Zip II subfamily, and has some distinctive
characteristics, including the difference of a 1 and d 1 sitesof its leucine zipper motif, which are well conserved ineach HD-Zip subfamily. Comparison of the genetic
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 (Sakakibara et a!.
2001).
The moss homologs of SHOOTMERISTEMLESS andZWILLE genes, which are involved in Arabidopsis
shoot development, have been cloned and their characterization is in progress.

111-111. Establishment of enhancer and gene traplines in the moss Physcomitrella patens
We also established enhancer and gene trap lines
and tagged mutant libraries of P. patens to clone genes
involved in the leafy shoot development in haploid generation (Hiwatashi et a!. 2001). Elements for gene-trap
and enhancer-trap systems were constructed using theuidA reporter gene with either a splice acceptor or aminimal promoter, respectively. Through a high rateof transformation conferred by a method utilizing homologous recombination, 235 gene-trap and 1073

55

FOR BASIC BIOLOGY

enhancer-trap lines were obtained from 5637 and 3726
transgenic lines, respectively. Expression patterns ofthese trap lines in the moss gametophyte varied . The
candidate gene trapped in a gene-trap line YH209,
which shows rhizoid-specific expression, was obtainedby 5' and 3' RACE. This gene was named PpGLU,
and forms a clade with plant acidic alpha-glucosidase
genes. Thus, these gene-trap and enhancer-trap systems
should prove useful to identify tissue- and cell-specificgenes in Physcomitrella.

Publication List:
Fujita, T., Ikeda, M., Kusano, S., Yamazaki, S., Ito, S.,
Obayashi, M., Yanagi, K. (2001) Amino acid substitution analyses of the DNA contact region, twoamphipathic a-helices and a recognition-helix-likehelix outside the dimeric b-barrel of Epstein-Barr virus nuclear antigen I. Intervirology. 44: 271-282.
Himi, S., Sano, R., Nishiyama, T. , Tanahashi, T., Kato,
M., Ueda, K. and Hasebe, M. (2001) Evolution of
MADS-box gene induced by FLOILFY genes. J. Mol.
Evol. 53: 387-393.
Hiwatashi, Y., Nishiyama, T., Fujita, T. and Hasebe, M.
(200 I) Establishment of gene-trap and enhancer-trapsystems in the moss Physcomitrella patens. Plant J.
28: 1-14.
Kurumatani, M., Yagi, K., Murata, T ., Tezuka, M.,
Mander, L. N., Nishiyama, M. and Yamane, H.
(200 1) Isolation and identification of antheridiogens
in the ferns, Lygodium microphyllum and Lygodium
reticulatum . Biosci. Biotechnol. Biochem. 65: 23112314.
Sakakibara, K., Nishiyama, T., Kato, M. and Hasebe, M.
(2001) Isolation of Homeodomain-Leucine Zipper
Genes from the Moss Physcomitrella patens and the
Evolution of Homeodomain-Leucine Zipper Genes inLand Plants. Mol. Bioi. Evol. 18: 491-502 .
Shindo, S., Sakakibara, K. , Sano, R., Ueda, K. andM.
(200 1)
Characterizatinof aHasebe,FLORICAULAILEAFY homologue of Gnetum parvifolium, and its implications for the evolution of reproductive organs in seed plants. Int. J. Plant Sci.
162: 1199-1209.
Yoshihara, S., Geng, X. X. , Okamoto, S., Yura, K. ,Murata, T., Go, M., Ohmori, M . and Ikeuchi, M .
(2001) Mutational analysis of genes involved in pilus
structure, motility and transformation competency inthe unicellular motile cyanobacterium Synechocystis
sp. PCC 6803. Plant Cell Physiol. 42: 63-73 .

Figure 2. Histochemical GUS activity in representative trap lines of the moss Physcomitre/la patens. (a) Chloronema cells of gene-trap line
YH261. (b) A bud of enhancer-trap line ET77. Leaf-primordial cell s (arrows) are predominantly stained. (c) A bud of gene-trap line YH727 . An
apical cell (arrow) and its surrounding cells are stained. (d) Apical portion of a gametophore in gene-trap line YH8 . Axillary hairs thatdifferentiate at the adaxial base of the leaf are stained. (e) A gametophore of enhancer-trap line ET63. The apical portion of the gametophore is
predominantly stained. (f) A gametophore of gene-trap line YH560.

Leaves are stained. (g) A gametophore of enhancer-trap line ET21 . A stem

is stained . LinesYH261 , YH727, YH8, and YH560 were generated using HI-GT, lines ET63 and ET21 were generated using HI-ET, and line
ET77 was generated using NHI-ET. Bars in (a), (c), and (d)= 100 !Lm. in (b) = 20 !Lm. and in (e) , (f), and (g)= 300 !Lm.

56

NATIONAL INSTITUTE

57

FOR BASIC BIOLOGYRESEARCH SUPPORTCENTER FOR 'IRANSGENIC ANIMALS AND PLANTSRESEARCH CENTER FOR INTEGRATIVE
AND COMPUTATIONAL BIOLOGYRESEARCH SUPPORT FACILITYTHE CENTER FOR ANALYTICAL INSTRUMENTSTECHNOLOGY DEPARTMENT

58

NATIONAL INSTITUTE

II. Academic activity

CENTER FOR
TRANSGENIC ANIMALS AND PLANTS
Head:
Associate Professor:
Supporting Staff:

NODA, Masaharu
WATANABE, Eiji
YASUDA, Mie
TOZAKI, Ayako
SHIBATA, Mariko (Jun, 2001-)
EGUSA, Chizu (Sep, 2001-)
NARUSE, Aki (Jan, 2002-)
YAGI, Eri (Feb, 2002-)

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 centerbuilding.
The expected activities of the Center are as follows :We are studying the functional role of Na,. sodium
channel in collaboration with Division of Molecular
Neurobiology. Na, has belonged to a subfamily of
voltage-gated sodium channels (NaChs) that serve togenerate action potentials in electrically excitable cells
such as neuronal and muscle cells. Comparing withthe other NaChs, Na,. has unique amino acid sequencesin the regions, which are known to be involved in ion
selectivity and voltage-dependent activation and inactivation, suggesting that it must have specific functional
properties. To clarify the functional role Na,. in vivo,
the Na,-deficient mice were generated by gene targetingand the physiological phenotypes have been examined.
It was suggested that the Na, channel plays an importantrole in the central sensing of body-fluid sodium level
and regulation of salt intake behavior. Details of thisstudy are described in the part of Division of Molecular
Neurobiology .

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 .

Publication List
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,
in press.

Cerebral Cortex

Fig. I Na, sodium channel is a candidate molecule for the sodium-level sensor in the brain.
The schematic representation indicates a sagittal plane of the mouse brain at midline level. The boxed regions are the circum ventricular organs,which are tissues situated outside of the blood-brain barrier and surrounded the ventricle of brain, and a site convenient for monitoring the levelsof various substances in both plasma and CSF. Na, sodium channel is expressed in specialized neurons and ependymal cells of the circumventricular organs as shown in red. We also found abnormal salt ingestion in the Na,-deficient mice .

FOR BASIC BIOLOGYRESEARCH CENTER FOR INTEGRATIVE ANDCOMPUTATIONAL BIOLOGY(Staff of the Center are in the process of selection.)

This center aims to integrate biology and bioinformatics through elucidation of various biologicalphenomena at the molecular level employing the following: (l) Computational analysis using accumulating data
related to biological information; (2) analysis using
mathematically model and simulations; (3) comprehensive functional analysis of genes, genomes and proteinsby new technology and application of novel concepts.

59

60

NATIONAL INSTITUTERESEARCH SUPPORT FACILITY
Head of Facility:
Associate Professor:
Research Associates:

Technical Staff:

MURATA, Norio
WATANABE, Masakatsu
HAMADA, Yoshio(Tissue and Cell Culture)
UCHIYAMA, lkuo
(Computer)
HIGASHI, Sho-ichi(Large Spectrograph)
MIWA, Tomoki (Computer)
NANBA, Chieko(Plant Culture, Farm, Plant Cell)NISHIDE, Hiroyo(Computer)
HATTORI, Nobuko(Large Spectrograph. -April
3, 2001)
ITO, Makiko(Large Spectrograph)
KAMIYA, Yasuko (Tissue andCell Culture.- February 28
2001)
KATA GIRl, Izumi (Tissue andCell Culture. March I, 2001-)
HARADA, Miyuki (Computer.
July 16, 2001 -)MAKIHARA, Nobuko (Computer.
April], 2001 -)SUZUKI, Keiko(Plant Culture, Farm, Plant Cell)

I. Facilities
1. The Large Spectrograph Laboratory

This laboratory provides, for cooperative use, theOkazaki Large Spectrograph (OLS), which is the largest
spectrograph in the world, dedicated to action spectroscopical studies of various light-controlled biologicalprocesses. The spectrograph runs on a 30kW Xenon arc
lamp and has a compound grating composed of 36 smaller
individual gratings. It projects a spectrum of a wavelength·range from 250nm (ultraviolet) to l,OOOnm (infrared) onto
its focal curve of 1Om 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-lOOOmW output), NO
modulators (up to 40MHz) to chop the laser beam, a beam
expander, and a tracking microbeam irradiator (up to 200
J.tm s·' in tracking speed, down to 2 J.tm in beam diameter)with an infrared phase-contrast observation system.

2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell culture areprovided. This laboratory is equipped with safely rooms
which satisfy the P2/P3 physical containment level. 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 PI physical containment level under extraordinary environments such as
strong light intensity, low or high temperatures.
5. Experimental FarmThis laboratory consists of two 20 m 2 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 In-

61

FOR BASIC BIOLOGY

stitute 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 andinternational collaborations devorted to the topic of stressresistant transgenic plants.

IT. 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 arestudied action spectroscopically (Watanabe 1995, In CRC
Handbook of Organic Photochemistry and Photobiology)
by measuring computerized-videomiceographs of the
motile behavior of the cells at the cellular and subcellularlevels. Photo-receptive and signal transduction mechanisms of algal gene expression were also studied by action
spectroscopy.
(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 thetetraploid chimeras beyond E12.5 because of insurfficientdevelopment of umbilical cord, indicating another role of
Notch2 signaling in the mouse development. Chimericanalysis with diploid wild type, however, revealed
contribution of mutant cells to these affected tissues by
El3.5. Thus, Notch2 are not cell autonomously required
for the early cell fate determination of labyrinthine
trophoblast cells and allantoic mesodermal cells, but plays
an indispensable role in the further formation offunctional 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 databasesystem for comparative analysis of many of microbial
genomes ever sequenced and are developing new computational techniques for large-scale genome sequencecomparison. Especially, we are developing a method fororthologous grouping among multiple genomes, which isa crucial step for comparative genomics. In addition to

splitting fusion genes into orthologous domains, we arealso enhancing the grouping algorithm to incorporate theinformation of gene arrangement on the genome.
In parallel, we have developed a tool to incorporate
various sequence features such as G+C contents, codon
usage bias and locations of repetitive elements into the
genome comparison. By this approach, we made detailed
comparison of closely related microbial genomes toinvestigate the genomic polymorphisms or evolutionarychanges in collaboration with Dr. I. Kobayashi's group(Univ. Tokyo), including comparative genome analysis of
two strains of Staphylococcus aureus.

2. Cooperative Research Program for the Okazaki
Large Spectrograph
The NIBB Cooperative Research Program for the Useof the OLS supports about 30 projects every year
conducted by visiting scientists including foreign scientists as well as those in the Institute.
Action spectroscopical studies for various regulatoryand damaging actions of light on living organisms,
biological molecules, and organic molecules have been
conducted (Watanabe, 1995, In "CRC Handbook of
Organic Photochemistry and Photobiology". pp, 1276 1288).

Publication List:
I. Faculty

Kondou, Y., Nakazawa, M., Higashi, S., Watanabe, M. andManabe, K. (2001) Equal-quantum action spectra indicate
fluence-rate-selective action of multiple photoreceptors forphotomovement of the thermophilic cyanobacterium
Synechococcus elongatus. Photochem. Photobiol. 73, 9095.
Kumano, K., Chiba, S., Shimizu, K., Yamagata, T., Hosoya,N., Saito, T., Takahashi, T., Hamada, Y., Hirai, H. (2001)
Notch1 inhibits differentiation of hematopoietic cells bysustaining GAT A-2 expression. Blood 98: 3238-9.
Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H.,
Kobayashi, I., Cui, L, Oguchi, A., Aoki, K., Nagai, Y.,
Lian, J., Ito, T., Kanamori, M., Matsumaru, H., Maruyama,A., Murakami, H., Hosoyama, A., Mizutani-Ui, Y.,
Takahashi, N.K., Sawano, T., Inoue, R., Kaito, C,
Sekimizu, K., Hirakawa, H., Kuhara, S, Goto, S., Yabuzaki,J., Kanehisa, M., Yamashita, A., Oshima, K., Furuya, K.,
Yoshino, C., Shiba, T., Hattori, M., Ogasawara, N.,
Hayashi, H., Hiramatsu, K. (2001) Whole genome
sequencing of meticillin-resistant Staphylococcus aureus,
The Lancet 357, 1225-1240.
Lenci, F. and Watanabe, M. (200 1) Photomovements andlight sensing systems. In "Photobiology in the 21"
Century" (eds. T. Coohil, D.-P. Hader, D. Valenceno)
Valdemaer Press
Nakamura, S., Mikamori, M., Hiramatsu, M., Eura, S., and
Watanabe, M. (200 1) Specutacular fluorescence emission
in sea urchin larvae. Zoo/. Sci. 18, 807-810.
Nakamura, S., Ogihara, H., Jinbo, K., Tateishi, M.,
Takahashi, T., Yoshimura, K., Kubota, M., Watanabe, M.,

62
and Nakamura, S. (2001) Chalamydomonas reinhardtii
Dangeard (Chlamydomonadales, Chlorophyceae) mutant
with multiple eyespots. Phycol. Res. 49, 115-122.
Negishi, T., Nagaoka, C., Hayatsu, H., Suzuki, K., Hara, T.,
Kubota, M., Watanabe, M., and Hieda, K. (2001) Somaticcell mutation induced by UVA and monochromatic UV
radiation in repair-proficient and -deficient Drosophilamelanogaster. Photochem. Photobiol. 73, 493-498.
Watanabe, M. and Erata, M. (2001) Yellow-light sensing
phototaxis in cryptomonad algae. In "Photomovement"(eds. D.-P. Hader and M. Lebert) pp. 343-373. Elsevier

II. Cooperative Research Program for the Okazaki
Large Spectrograph
Hasegawa, E., Sawada, K., Akamatsu, T., Fujita, K. andTarnai, M. (2001) Fish behaviour control by the opticalfiber optomotor reaction device. Bull. Nat. Res. Inst. Fish.
Eng. 22, 27-33.
lnada, C., Kudo, T., Yoshizumi, K., Takatori, K., Hedge, A.
(2001) Effect of light irradiation wavelength on theinactivation of Penicillium. B~kin Bobai. 12, 757-762.

NATIONAL INSTITUTE

Kakiuchi, Y., Takahashi,T., Murakami, A. and Ueda, T.
(2001) Light irradiation induces fragmentation of theplasmodium, a novel photomorphogenesis in the true slime
mold Physarum polycephalum: action spectra andevidence for involvement of the phytochrome. Photochem
Photobiol. 73, 324-329.
Kondou, Y., Nakazawa, M., Higashi, S., Watanabe, M. andManabe, K. (2001) Equal-quantum action spectra indicate
fluence-rate-selective action of multiple photoreceptors forphotomovement of the thermophilic cyanobacterium
Synechococcus elongatus. Photochem. Photobiol. 73, 9095.
Negishi, T., Nagaoka, C., Hayatsu, H., Suzuki, K.,Hara, T.,
Kubota, M., Watanabe, M. and Hieda, K. (2001) Somaticcell mutation induced by UVA and monochromatic UV
radiation in repair-proficient and deficient Drosophilamelanogaster. Photochem. Photobiol. 73, 493-498.
Yoshizumi, K., Kudo, T.,Imaizurni, A., Fujinarni, N.(2001)
Action spectrum of light fastness of cartharnin dyed on silk
fabric under the irradiation of UV and visible light. SEN/GAKKAISHI 57 No.9

63

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

Techni cal Assistant:

MOROHASHI, Ken-ichirou
OHSAWA, Sonoko
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
to support researchers in NIBB and NIPS . Newly installed instrument in 2001 is MALDI/TOF-MS . Instruments of the Center can be used by researchers outside
the Institute upon proposal.

Figure I. MALDI!TOF-MS.

Figure 2. Biomek 2000 Laboratory Automation System.

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-1 00 2:, PI-50)
Automatic Nucleic Acid Isolation System (Kurabo NA2000)
DNA Sequencers (ABI 377, 373S, 310)
DNA/RNA Synthesizers (ABI 394, 392)
Thermal Cyclers (Perkin Elmer PJ-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)
MALDIITOF-MS (Bruker Daltonics REFLEX III)
Inductively Coupled Plasma Atomic Emission
Spectrometer (Seiko SPS 1200A)
Spectrofluorometers (Hitachi 850, Simadzu RF-5000)
Spectrophotometers (Hitachi 330, 557, Varian Cary 5G,
Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB
96P)Time-resolved Fluorescence Microplate Reader(Pharmacia DELFIA Research)
Microplate Readers (Corona MTP-120, MTP-1 OOF)
Spectropolarimeter (JASCO J-40S)
FT-IR Spectrophotometer (Horiba FT-730)
Laser Raman Spectrophotometer (JASCO R-800)
Bio Imaging Analyzers (Fujifilm BAS2000)
Fluorescence Bio Imaging Analyzer (Takara FMBIO)Electrophoresis Imaging Systems (BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)

64

NATIONAL INSTITUTETECHNOLOGY DEPARTMENT

Head: HAITOR/, Hiroyuki

Common Facility Group

Research Support Group

Chief: FURUKAWA, Kazuhiko

Chief: KOBAYASHI, Hiroko

Reseach Support Facilities
HIGASHI, Shoichi (Unit Chief)
NANBA, Chieko (Subunit Chief
NISHIDE, Hiroyo
ITOH, Makiko (TeGhnical Assistant)
SUZUKI, Keiko (Technical Assistant)
KATA GIRl, Izumi (Technical Assistant)
MAKIHARA, Nobuko (Technical Assistant)
HARADA, Miyuki (Technical Assistant)
TOSAKI, Ayako (Technical Assistant)
SHIBATA, Mariko (Technical Assistant)

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

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

Developmental Biology Group
TAKAGI, Chiyo
OKA, Sanae
Regulation Biology GroupIINUMA, Hideko
YAMAGUCHI, Katsushi
TAKEUCHI, Yasushi
Gene Expression and Regulation GroupMIWA, Tomoki (Unit Chief)
TANAKA, Sachiko (Subunit Chief)
SAWADA, Kaoru (Subunit Chief)
HAYASHI, Koji
UTSUMI, Hideko
SUMIKAWA, Naomi
Integrated Bioscience GroupMIZUTANI, Takeshi
NODA, Chiyo
KONDO, Makiko (Technical Assistant)

Glassware Washing Facility(MORI, Tomoko)(FURUKAWA, Kazuhiko)

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

in mutual enligtement and education increase theircapability in technical area. Each technical staff is proceeded to the fixed division usually and they support thevarious research with their special biological and biophysical techniques.
The Department hosts an annual meeting for technical engineers who work in various fields of biology atuniversities and research institutes throughout Japan.
At this meeting, the participants present their own activities and discuss technical problems.The Proceedings
are published soon after the meeting.

65

FOR BASIC BIOLOGYCENTER FOR INTEGRATIVE BIOSCIENCE(jointly managed by NIBB)Interim Head: NAGAYAMA, Kuniaki

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION IDEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION II
DEPARTMENT OF BIO-ENVIRONMENTAL RESEARCH I
DEPARTMENT OF BIO-ENVIRONMENTAL RESEARCH II
DEPARTMENT OF BIO-ENVIRONMENTAL RESEARCH III

The center is jointly managed by NIBB and two other institutes in Okazaki,
IMS (Institute for Molecular Science) and NIPS (National Institute forPhysiological Sciences).
center:

The following projects will be the main focus of the1) Development, Differentiation and Regeneration. 2) Strategic

Methodology.

3) Bio-environmental Science.

66

NATIONAL INSTITUTE

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION I
Professor:
KOBAYASHI, Satoru
Research Associate:
MUKAI, Masanori
Technical Staffs:
NODA, Chiyo
NIBB Research Fellow:
SATO, Kimihiro
Post doctoral fellows :AMIKURA, Reiko
JSPS Postdoctral Fellow: SHIGENOBU, Shuji
Graduate Students:
HAYASHI, Yoshiki
UNNAI, Kohei 11
KITADATE, Yu n
Secretary:
SUZUKI, Masako
Technical Assistant:
VEDA , Yoshiko
TSUCHIYA, Naomin Graduate School of Biological Sciences,
University ofTsukuba

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

Fig. I. Presence of m1tochondnal rRNAs and nbosomal
proteins in the polar granule polysomes at stage 2. (A) An
electron micrograph showing well developed polysomes(arrow) on the surface of polar granules. (B and C) Electron
micrographs of sections hybridized with probes for mtlrRNA(B) and mtsrRNA (C) (arrowheads). (Scale bar=0.25 Jlrn.)(D-G) Distribution of SI2-EGFP (D), L7/LI2-EGFP (E),
L7/LI2-HA (F). Signals were detected at the periphery ofpolar granules (arrows). mt, mitochondria; pg, polar
granules. Scale bar= 0.2 Jlm.

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 arealso involved in germline formation.
In Drosophila, pole cell formation requires the function of mitochondrial ribosomal RNA in germ plasm.
We have previously reported that mitochondrial largerRNA (mtlrRNA) and small rRNA (mtsrRNA) are bothtransported from mitochondria to polar granules . Thistransportation occurs during early embryogenesis, when
mitochondria are tightly associated with polar granules
in germ plasm, and it depends on the function of thematernally-acting gene, tudor, that is known to be required for pole cell formation . Mitochondrial rRNAs
remain on the polar granules until pole cell formation
and are no longer discernible on the granules withinpole cells. Reduction of the extra-mitochondrial
mtlrRNA amount results in the failure to form pole cells
and injection of mtlrRNA is able to induce pole cells inembryos whose ability to form these cells has beenabolished by uv-irradiation. These observations clearlyshow that the extra-mitochondrial mtlrRNA on polar
granules has an essential role in pole cell formation,
presumably cooperating with mtsrRNA.
Since both mtlrRNA and mtsrRNA are major components of ribosomes within mitochondria, we speculated
that these rRNAs function to form ribosomes on thepolar granules. We reported that mtlrRNA and2. Distribution of mitochondrial ribosomal proteinsin oocytes and early embryos. (A-H) Developmental
changes in the distribution of S 12-EGFP (A-D) andL7/LI2-EGFP (E-H) detected by EGFP fluorescence.
(Aand E) Mature oocytes, (B and F) stage- I embryos, (Cand G) stage-2 embryos, and (Dand H) stage-3 embryos.
(1-L) Immunohistochemical detection of the Gal4induced SI2-EGFP (I) and L7/LI2-EGFP (J) by an antiGFP antibody and L7/LI2-HA by an anti-HA antibody(K). (L) A wild-type embryo stained with anti-S 12
antibody. Scale bar= 50 Jlrn .

67

FOR BASIC BIOLOGY

mtsrRNA are both localized in the polysomes formedon the surface of polar granules during a short period(embryonic stage 2) prior to pole cell formation (Fig. 1).
Furthermore, mitochondrial ribosomal proteins (S 12and L7/L12) are enriched in germ plasm (Fig. 2). They
are present in the polysomes on the polar granules aswell as in mitochondria. In the polysomes around polar
granules are there smaller ribosomes, of which size isalmost identical to that of mitochondrial ribosomes but
is smaller than that of cytosolic ones. We conclude thatmitochondrial rRNAs form mitochondrial-type of ribosomes on pol.ar granules, cooperating with mitochondrial ribosomal proteins.

II. Role ofdifferentiation

Nanos

protein

in

pole

cell

Pole cells differ from the soma in regulation ofmitosis and transcriptional activity. Pole cells ceasemitosis at gastrulation and remain quiescent in the G2phase of the cell cycle throughout their migration to the
gonads, while somatic cells continue to proliferate
during the rest of embryogenesis. Furthermore, polecells are transcriptionally quiescent until the onset ofgastrulation, although transcription is initiated in thesoma during the syncytial blastoderm stage. Consistent
with this, RNA polymerase II (RNAP II), but not RNA
polymerase I, remains inactive in early pole cells. Thus,
the ability to express zygotic mRNA-encoding genes issuppressed only in pole cells in early 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 onlytransiently present in the posterior half of embryos
during the preblastoderm stage, and is required there forposterior somatic patterning. Nos in the germ plasm ismore stably inherited into the pole cells at theblastoderm stage, remaining detectable in these cells
throughout embryogenesis. Pole cells that lack Nos (nospole cells) are unable to follow normal germline
development; they fail to migrate properly into theembryonic gonads, and consequently do not become
functional germ cells. In nos pole cells, mitotic arrest at
G2 phase is impaired, and they undergo prematuremitosis. Furthermore, nos pole cells fail to establish
and/or maintain transcriptional quiescence, andectopically express somatically-transcribed genes,
including fushi tarazu (ftz), even-skipped (eve) and Sexlethal (Sxl).
Nos represses translation of mRNAs with discrete
RNA sequences called Nos response elements (NREs).
In the pathway leading to posterior somatic patterning,
Nos acts together with unlocalized Pumilio (Pum)protein to repress translation of maternal hunchback(hb) mRNA. This translational repression is mediatedby binding of Pum to NREs in the 3 '-untranslated
region (UTR) of hb mRNA. In pole cells, Nos also actswith Pum to regulate germline~specific events. Pum,
like Nos, is required in pole cells for their migration to
the gonads and their mitotic quiescence. We havereported that a regulatory target for Nos-dependenttranslational repression in pole cells is maternal cyclin B
mRNA which contains an NRE-like sequence within its
3'-UTR. Nos, cooperating with Pum, inhibits mitosis ofpole cells by repressing translation of maternal cyclin B
mRNA. In contrast, pole cell migration and gene expression in pole cells are independent of thetranslational repression of cyclin B, suggesting the existence of another target mRNA for Nos-dependenttranslational repression in pole cells.
We found that Nos, along with Pum, represses
translation of importin a2 (impa2) mRNA in early polecells. The impa2 mRNA contains an NRE-likesequence in its 3'-UTR and encodes a Drosophila
importin a. homologue that plays a role in nuclear
import of karyophilic proteins. We found that Nos
inhibits expression of a somatically-transcribed gene,ftz,
in pole cells by repressing Impa.2-dependent nuclear
import of a transcriptional activator for ftz, Ftz-Fl.
Furthermore, the expression of another somatic gene,eve, and RNA Polymerase II activity are also repressedby Nos in pole cells through its effects on Impa.2dependent nuclear import. Finally, we found that the
repression of Impa.2 production in pole cells is needed
for proper migration of pole cells and the expression ofa germline-specific marker, vasa (vas).

Publication List:
Amikura, R., K. Hanyu, M. Kashikawa and S.
Kobayashi (2001) Tudor protein is essential for the
localization of mitochondrial ribosomal RNAs inpolar granules in germ plasm of Drosophila embryos.
Mech. Dev. 107,97-104.
Amikura, R., M. Kashikawa, A. Nakamura and S.
Kobayashi (2001) Presence of mitochondrial-type
ribosomes outside mitochondria in germ plasm ofDrosophila embryos. Proc. Natl. Acad. Sci. USA. 98,
9133-9138.
Kashikawa, M., R. Amikura and S. Kobayashi (2001)
Mitochondrial small ribosomal RNA is a componentof germinal granules in Xenopus embryos. Mech.
Dev., 101:71-77.
Nakamura, A., R. Amikura, K. Hanyu and S. Kobayashi(2001) Me31B silences translation of oocytelocalizing RNAs through the formation ofcytoplasmic RNP complex during Drosophila
oogenesis. Development.128, 3233-3242.
Sano, H., A. Nakamura and S. Kobayashi (2001)
Identification of a transcriptional regulatory region
for germline-specific expression of vasa gene in
Drosophila melanogaster. Mech. Dev. (in press)
Sano, H., M. Mukai and S. Kobayashi (2001) Maternal
Nanos and Pumilio regulate zygotic vasa expression
autonomously in the germline progenitors ofDrosophila embryos. Develop. Growth & Differ. 43,
545-552.

68

NATIONAL INSTITUTE

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATION II
Professor:
Research Associate:

TAKADA, Shinji
KOSHIDA , Sumito

The research interest of this laboratory is to understand molecular mechanism of the vertebrate development. Particularly, roles of cell-to-cell signals, including
members of Wnt and fibroblast growth factor (FGF)
families, are characterized. Evidence indicated that eachsignal is involved in many aspects of the vertebrate
development. For instance, we have revealed that Wnt3a, a members of Wnt family , plays essential roles in a
number of aspects of the mouse development, including
somite development, neural crest formation and neuraldevelopment. However, cellular and molecular mechanisms how a cell signaling molecule regulates thesedifferent events. Thus, we are focusing on precise
functional analysis of cell-to-cell signals and identification of target genes induced by these signals. In addition,
to reveal molecular networks in which these signals are
involved, we are also trying genetical approach with thezebrafish .

I Roles of Wnt signals in somite development
The Wnt family of genes that encode cysteine richsecreted 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, Wnt-3a, a member of the Wnt
family genes, is expressed in the primitive streak ectoderm during gastrulation and in the tailbud in later
development of the mouse (Fig . 1). For dissecion of thecomplex developmental events regulated by Wnt-3a
signaling in these regions, it is important to identify

8.5dpc

9.5dpcgenes regulated by this signal. It has already been
demonstrated that T (Brachy ury) is a direct target of
Wnt-3a in the anterior primitive ectoderm, which isfated to give rise to the paraxial mesoderm, suggestingthat Wnt-3a modulates a balance between mesodermal
and neural cell fates via T.
To gain more insight into the role of Wnt-3a during
gastrulation, we searched for potential target genes of
Wnt-3a. We found that Wnt signaling stimulated the invitro expression of cdx-1 , and that Wnt-3a was required
for full activation of cdx-1 expression in the primitive
ectoderm and tailbud in vivo. Moreover, Wnt-3a mutants displayed a defect in their anteroposterior patterning similar to that in the cdx-1 mutant mouse, in
addition to another homeotic transformation that was
not observed in cdx-1 mutants (FIG . 2). These results
suggest that cdx-1 is one of the mediator genes of Wnt3a signaling in the anteroposterior patterning of thevertebrae and that Wnt-3a is also involved in a Cdx-1
independent process in anteroposterior patterning

II A roles of Wnt signaling in neural development
In the dorsal half of the spinal cord, three subclasses
of interneurons called D 1, D2 and D3 from the dorsal
side. These interneuron subclasses are thought to be
derived from distinct neural progenitor domains. Recent
studies indicate that the roof plate is a source of inductive signals that control the generation of D1 and D2classes of dorsal interneurons.
Interestingly, Wnt-1 and Wnt-3a are expressed inlargely overlapping regions within the central nervous
system, predominantly in the roof plate from diencephalon to spinal cord. We show that mouse embryos
lacking both Wntl and Wnt3a are indeed defective indetermination of the dorsal interneurons. Generation of
D 1 and D2 classes of dorsal interneurons and theirprogenitors was markedly disrupted; this loss of thedorsal interneurons was compensated by a dorsal
expansion D3 interneuron populations. Most importantly, expression of TGF (3 family members was not
significantly affected in these mutant embryos. Moreover, we demonstrate that WNT3A can induce D 1 andD2 class interneurons in the isolated intermediate regionof the neural tube. Together, these observations clearly
indicate that Wnt signaling has a critical role in thegeneration of dorsal interneurons specific D 1 and D2
cell types .

III Screening of target gen,es of Wnt signaling by
an gene trap approach

AG. l Expression of Wnt-3a at 8.5 and 9.5 days post coitus(dpc) in the mouse embryo. Wnt-3a is expressed in theprimitive streak ectoderm (8.5 dpc) and the dorsal neural tub
and the tail bud (9.5 dpc).

To gain more insight into roles of Wnt signaling
during embryogenesis, we searched for potential target
genes of this signaling by an induction gene trap
screening in mouse ES cells. In at least three ES cellclones among 794 clones screened, expression of betageo reporter genes was dramatically changed in response to the conditioned medium of Wnt-3a expressing

69

FOR BASIC BIOLOGY

suture closure is delayed. Furthermore, terminal differentiation of the osteoblast is specifically delayed in developing calvaria and long bones. Proliferation of calvarial
osteogenic mesenchymal cells is transiently decreased. On
the other hand, the number of proliferating and differentiated chondrocytes is increased in the limbs. F gfl8 is thus
essential for distinct aspects of cell proliferation anddifferentiation in bone formation . Taken together, Fgf18
plays essential roles in the osteogenesis and the chondrogenesis of the mammal and regulates differently cellproliferation and differentiation in these two processes ..

cells. The expression analysis of the reporter genes inembryos generated from these ES cell clones revealed
that the spatiotemporal expression patterns of thesereporter 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.

IV 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 onnull mutant mice have not yet fully shown the role of thisfamily in the skeletal development.
As an attempt to identify a candidate Fgf gene essential
for bone formation, we have examined the expression ofmouse Fgfl8 gene using in situ hybridisation and demonstrated expression during calvarial and long bone development. Furthermore, to investigate the role of Fgfl8 in vivo,
we generated a null allele of the Fgfl8 locus by homologous recombination in ES cells. In Fgfl8-deficient mouseembryos generated by gene targeting, progress of the

V Screening of mutations affecting mesoderm
development in zebrafish
To understand the molecular mechanism of mesoderm development in the tail bud , we have been
screening mutations affecting mesoderm developmentin zebrafish.

Wnt-3a vt/vt

WT

a

b

T10

-&

T9

-&

c

FIG. 2 Skeletal analysis of Wnt-3a mutants. a, In the cervical region, C2 to Cl transformation was observed in Wnt-Janeo/neo and
Wnt-3avtlvt mutants. b, Side view of the midthoracic region. Anterior is to the left. The transitional vertebra, which was normallyobserved in TIO, was observed in T9 in the Wnt-3avtlvt mutant. c, From the posterior thoracic to the anterior caudal region. In Wnt3a vtlvt, the TI3 vertebra, which normally has ribs on both sides, did not have rib on the right side (partially transformed to Ll) and the
6th lumbar vertebra fused together at the transverse processes to form sacral bone (L6 to S I transformation). Black arrow indicates arudimentary rib. White arrow indicates that the morphology of S4 was altered, its transverse processes being fused to those of S3(partially transformed to S3).

70

Publication List:
Hino, S.-1., Kishida, S., Michiue, T., Fukui, A., Sakamoto,I., Takada, S., Asashima, M. & Kikuchi, A. (2001) Inhibition of the Wnt signaling pathway by ldax, a novel
Dvl-binding protein. Mol. Cell. Bioi., 21, 330-342.
lkeya, M. & Takada, S. (2001) Wnt-3a is required forsomite specification along anteroposterior axis of themouse embryo and regulates cdx-1 expression. Mech.
Dev. 103, 27-33.
Jin, E.J., Erickson, C.A., Takada, S,. & Burrus, L.W.
(2001) Wnt and bmp signaling govern lineage segregation of melanocytes in the avian embryo. Dev. Bioi. 233,
22-37.
Kawamura, Y., Kikuchi, A., Takada, R., Takada, S., Sudoh,S., Shibamoto, S., Yanagisawa K., & Komano, H.
(2001) Inhibitory effect of presenilin 1 mutation in theWnt signaling pathway---presenilin 1 mutations enhance the phosphorylation of 13-catenin--- Eur. J. Biochem.
268 3036-3041.
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. (inpress).
Mao, J., Wang, J., Liu, B., Pan, W., Farr, G.H., Flynn, C.,
Yuan, H., Takada, S., Kimelman, D., Li, L., & Wu, D.

NATIONAL INSTITUTE(2001) Low-density lipoprotein receptor-related protein-S binds to axin and regulates the canonical Wnt
signaling pathway. Mol. Cell1, 801-809.
Muroyama, Y., Fujihara, M., lkeya, M,. Kondoh,. H., &Takada, S.(2002) Wnt signaling plays an essential rolein neuronal specification of the dorsal spinal cord.
Genes Dev. 16, 548-55.
Nomi M., Oishi 1., KaniS., Suzuki H., Natsuda T.,Yoda A.,
Kitamura M., ltoh K., Takeuchi S Tkeda K., Akira S.,
lkeya M., Takada S., & Mnami Y. (2001) Loss ofmRor1 enhances the heart and skeletal abnormalities inmRor2-deficient mice: kinase. Mol Cell. Bioi. 21, 83298335.
Ohbayashi, N., Shibayama, M., Kurotaki, Y., Imanishi, M.,
Fujimori, T., Itoh, N., & Takada, S. (2002) Fgf18 isrequired for normal cell proliferation and differentiation
during osteogenesis and chondrogeneses. Genes Dev.
(in press)
Shinya, M., Koshida, S., Sawada, A., Kuroiwa, A., &Takeda, H. (2001) Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos. Development 128,4153-4164.
Yamane, T., Kunisada, T., Tsukamoto, H., Yamazaki, H.,
Niwa, H., Takada, S. & Hayashi, S.-1. (2001) Wnt signaling regulates hematopoiesis through stromal cells. J.
1mmunol. 167, 765-772.

71

FOR BASIC BIOLOGYDEPARTMENT OF BIOENVIRONMENTALRESEARCH I
Professor:
IGUCHI, Taisen
WATANABE, Hajime
Associate Professor:
KATSU, Yoshinao
Research Associate:
MIZUTANI, Takeshi
Technical Staff:
Institute Research Fellow: SONE, Kiyoaki
KHONO, Satomi n
Graduate Students:
MIYAGA WA , Shinichi nTechnical Assistants:
KAJIYAMA , Chieko
SUZUKI, Atsuko
KOBAYASHI, Kaoru
TAKASU, Eri
Secretary:
IMAIZUMI, Taeko'! Graduate School of Yokohama City University

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 and evoke estrogenic effects . Early
evidence that estrogenic chemicals could pose a threat tohuman health during development came from studies ofdiethylstilbestrol (DES), which was · used to prevent
premature birth and spontaneous abortion . Laboratory
experiments have demonstrated that exposure of animals
to sex hormones during perinatal life can cause permanentand irreversible alterations of the endocrine and reproductive systems as well as the immune system, nervous
system, bone, muscle, and liver in both sexes. Althoughmany of these chemicals may bind to estrogen receptors
and evoke estrogenic effects in wildlife and humans, theeffects of estrogen are not well understood even now.
Thus, understanding the effects of sex hormones at themolecular level, especially during development, is very
important to resolve these problems.

I. Estrogen-induced irreversible changes
Perinatal sex-hormone exposure has been found to induce lesions in reproductive tracts in female mice . The
possible relevance of the mouse findings to the develop-

ment of cancer in humans has been emphasized. In theearly seventies, a close correlation between occurrence 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 the estrogen receptor (ER) . The
neonatal mouse model has been utilized especially todemonstrate the long-term effects of early sex hormone
exposure on the female reproductive tract.
Neonataltreatment of female mice with estrogens induces various
abnormalities of the reproductive tract: ovary-independent
cervicovaginal keratinization, adenosis, uterine hypoplasia,
epithelial metaplasia, oviductal tumors, polyovular follicles (PF) and polyfollicular ovaries . Female reproductive
tracts in mice exposed prenatally to estrogen show alteredexpression of Hoxa genes and Wnt genes and the analysis
of knockout 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 EGF receptor levels, in addition to otherhormone receptor levels.
Estrogenic compounds such as bisphenol A (BP A) andnonylphenol as well as dioxins and PCBs were found in
the human umbilical cord. BP A 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 BP A dose induced ovary-independent vaginalchanges, PF and infertility lacking corpora lutera. Prenatal exposure to a low BP A dose induced acceleration ofvaginal 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 microarraytechiniques. We have found genes possibly related to theovary-independent changes by differential display . We
also have clustered groups of genes that are responsive toNormal Mouse

FIG. 2 Fluorescence image of an array
FIG. I72

NATIONAL INSTITUTE

100000 : ~~-: 100000

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

Publication List:·-.... .... -..
10 ----A....r••--'---'' -·-~
10
100
1000
10000

- -- 10
100000Control
FIG.3 Scatter plot of average expression levels in control
and chemical-treated uterus
estrogenic stimuli in uterus by using the DNA microarraysystem . We need to understand the molecular background of the critical period during development, the lowdose 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 laevis
and the cyprinodont fish, mummichog (Fundulus heteroclitus). To analyze the function of estrogen, we haveisolated eDNA clones of estrogen receptor a and~ from F.
heteroclitus and American alligators. The estrogenresponsive genes must play important roles. We tried toisolate the estrogen-responsive genes to understand themolecular physiology of estrogen action. Vitellogenin has
been well characterized in avian, amphibian and fish as aprecursor of egg yolk. As the vitellogenin gene is responsive to estrogen, we can examine the effect of endocrine
disruptors in the environment using a vitellogenin-specificand sensitive enzyme-linked immunosorbent assay(ELISA) . Japanese tree frog (Hyla japonica) takes water
through ventral skin. We found that sex steroids andendocrine disruptors interfere with water absorption
through ventral skin in frogs. Further, using the amphibianand fish 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

Buchanan, D.L., S. Ohsako, C. Tohyama, P.S . Cooke andT. Iguchi : 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. (in press)
Honma, S., A. Suzuki, D.L. Buchanan,Y. Katsu, H. Watanabe
and T. Iguchi: Low dose effect of in utero exposure tobisphenol A and diethylstilbestrol onfemale mouse reproduction. Reprod. Toxicol. (in press)
Iguchi, T ., H. Watanabe and Y. Katsu (2001) Developmental effects of estrogenic agents on mice, fish andfrogs : a mini review. Horm. Behav. 40: 248-251.
Miyagawa, S., D.L. Buchanan, T. Sato, Y. Ohta, Y. Nishina
and T. Iguchi: Characterization of diethylstilbestrolinduced hypospadias in female mice. Anat. Rec. (in press)
Okada, A., T . Sato: Y. Ohta, D. L. Buchanan and T . Iguchi(2001) Effect of diethylstilbestrol on cell proliferationand expression of epidermal growth factor in the developing female rat reproductive tract. J. Endocrinol.,
170:539-554.
Okada, A., Y. Ohta, D.L. Buchanan, T . Sato, S. Inoue, H.
Hori, M. Muramatsu and T. Iguchi: Changes in ontogenetic expression of estrogen receptor a and not of estrogen receptor ~ in the female rat reproductive tract. J.
Mol. Endocrinol. (in press)
Suzuki, A., A. Sugihara, K. Uchida, T . Sato, Y. Ohta, Y.
Katsu, H. Watanabe and T. Iguchi: Developmental effects of perinatal exposure to bisphenoi-A and diethylstilbestrol on reproductive organs in female mice.
Reprod. Toxicol. (in press)
Yamamura, Y., M. Tamano, T. Iguchi and Y. Ohta (2001)
Methallothionein expression and tumor growth in thetransplantable pregnancy-independent mouse mammary
tumor. J. Vet. Med. Sci., 63: 687-689.
Yamamura, Y., Y. Ohta, T. Iguchi and A. Matsuzawa (2001)
Metallothionein expression and apoptosis in pregnancydependent and -independent mouse mammary tumors.
Anticancer Res., 21 : 1145-1150.
Watanabe, H., A. Suzuki, T. Mizutani, H. Handa and T .
Iguchi : Large-scale gene expression analysis forevaluation of endocrine disruptors. Proceedings of T'
International Forum for Toxicogenomics. (in press)
Watanabe, H., D.L.Buchanan, H. Handa and T. Iguchi(2001) Global analysis of gene expression induced byenvironmental endocrine disruptors. Perspective in
Comparative Endocrinology, Goos, H.J.Th., et al. (eds.),
Monduzzi Editore, pp.147-151.

73

FOR BASIC BIOLOGYDEPARTMENT OF BIOENVIRONMENTALRESEARCH II
Associate Professor:
Assistant Professor:
Technical Staff:
Post Doctoral Fellow:
Graduate Student:
Secretary:

TSUKAYA, Hiroka zu, Ph.D.
KIM, Gyung-Tae, Ph.D.
KONDO, Makiko
CHO, Kiuhyon, Ph.D.
KOZUKA, Toshiaki
KABEYA, Kazuko

relative length of leaf to width, is also the most diverse
factor of leaf shape, and is affected by environmental
factors in some plants (Kuwahara et al. , 200 1). Are AN
and ROT3 genes are involved in regulation of adaptivechange of leaf 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:
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 (Dengler and Tsukaya, 200 1) .
Based on it, a new perspective on understanding of
mechanisms for leaf morphogenesis is proposed (Tsukaya,
in press).
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 AN gene
regulates width of leaves and the ROT3 gene regulates
length of leaves . The AN gene controls the width of leaf
blades and the ROT3 gene controls length. Cloning of the
AN gene revealed that the gene is a member of gene family found from animal kingdom (Kim et al., submitted).
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).
Apart from polar elongation, we identified the following genes involved in leaf expansion process . The ASJ
and AS2 genes are needed for proportional growth of theleaf. Molecular and anatomical analysis of the as2 mutant
is now underway, in collaboration with a research team ofProf. Machida, Nagoya University (Endang et al., 2001).
We also started analysis of blp mutation which strongly
enhances the as2 phenotype, in collaboration with areaseach team of Prof. Narn, POSTECH, Korea.
On the other hand, we are trying to identify molecularmechanisms which distinguish developmental pathway ofleaves from that of shoots. For such purposes, we introduced tropical plants having queer developmental program for leaf morphogenesis, namely, Chisocheton,
Guarea and Monophyllaea, as materials for molecularstudies.
In addition, we are interested in environmental adaptation of leaves, from view point of biodiversity (e.g., Tsukaya and Tsuge, 2001; Tsukaya, in press) . Leaf index,

Dengler, N. and Tsukaya, H. (200 1). Leaf morphogenesis in
dicotyledons: current issues. Int. J. Plant Sci. 162: 459464.
Kuwahara, A., Tsukaya, H., and Nagata, T. (2001) Identifca
tion of factors that cause heterophylly in Ludwigia arcuata
Walt. (Onagraceae) Plant Biology 3: 98-105.
Nakaya, M., Tsukaya, H. , Murakami, N., and Kato, M.
(2002) Brassinosteroids control the proliferation of the leaf
cells in Arabidopsis thaliana. Plant Cell Physiol. 43: 239244

Semiarti, E., Ueno, Y. , Tsukaya, H. , Iwakawa H. , Machida C.
and Machida, Y. (2001). The ASYMMETRIC LEAVES2
gene of Arabidopsis thaliana regulates formation of symmetric lamina, establishment of venation and repressionof meristem-related homeobox genes in leaves . Development 128: 1771-1783.
Tsukaya, H. and Tsuge, T. (2001) Morphological adaptation of inflorescences in plants that bloom at low temperatures in early spring : the convergent evolution of"downy plants". Plant Biology 3: 536-543.
Tsukaya, H. (2002). 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 (in press)
Tsukaya, H. (2002) Optical and anatomical characteristics ofbracts from the Chinese "Glasshouse" plant, Rheum
alexandrae Batalin (Polygonaceae), in Yunnan, China. J.
Plant Res. 115: 59-64

74

NATIONAL INSTITUTE

DEPARTMENT OF BIOENVIRONMENTALRESEARCH Ill
Professor (Adjunct):

KANEHISA, Minoru(Kyoto University)

Though intermediary metabolism common to most organisms has been deeply investigated so far, variety of
species specific pathways in secondary metabolism,
which may work only in specific environmental conditions, are still unclear. The aim of this laboratory is todevelop a database system for environmental biology,
which integrates knowledge about organic compounds,chemical reactions between these compounds in vivo,
enzymes (genes) involved in these reactions, and species
whose genomes contain these genes. Through this data-base combining with data from transcriptome or proteome
analyses in various environmental conditions, we intend
to elucidate the principle of interactions between organisms and environmental chemical compounds to predict ordesign novel interactions.

Publication list:
Kanehisa, M. (2001) Prediction of higher order functional
networks from genomic data. Pharmacogenomics 2, 373385.
Nakaya, A., Goto, S., and Kanehisa, M. (2001) Extraction ofcorrelated gene clusters by multiple graph comparison.
Genome Informatics 12,44-53.

75

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

Associate Professor:
Technical Staffs:

Supporting Staff:

OGAWA. Kazuo
MATSUDA, Yoshimi(Radiation Protection
Supervisor)
KATO, Yosuke(Radiation Protection
Supervisor)
MOROOKA, Naoki(Radiation Protection
Supervisor)
ITO, Takayo!IDA, Yumi

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
one of our business.
The CRF consists of four controlled areas: Center,
NIBB-sub, LGER (Laboratory of Gene Expression andRegulation)-sub, and NIPS (National Institute forPhysiological Science)-sub. Users going in and out thecontrolled areas counted by the monitoring system are
7,559 in 200 I. The items in each controlled area ispresented in Figure I.

NIPS-subCenterNIBB-sub
LGER-sub
NIPS-subtotal

Actual counts
2,359
956
3,320
924
7,559

Figure I . Percentage of users going in and out the controlledareas in 200 I.

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 and cytoplasmic dyneins. Figure 2 shows the localization of two
isoforms of dynein in the outer arms of sperm axonemes(Ogawa et a!., J 977) and the mitotic apparatus ofcleaving egg (Mohri et a!., 1976) visualized by antiaxonemal dynein (Fragement 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 lightchains defined by the molecular mass. Our prensent
project is the molecular cloning of polypeptides contained in outer arm dynein of sea urchin sperm flagellato understand the mechanism how dynein interacts withrnicrotubules, resulting in producing the force.
Outer arm dynein consists of two heavy chainswith ATPase activity. The motor activity is closely
related to this polypeptide. The first successful
molecular cloning of this huge polypeptide (520 kDa)
was performed in our laboratory in 1991. Since thencDN A clones for axonemal and cytoplasmic dyneins
have been isolated in a variety of organisms. The sequences of heavy chains, without exception, containfour P-loop motives referred to as ATP-binding sites in
the midregion of the molecules. Figure 3A and B draw
the structure of heavy chain deduced from the aminoacid sequence (Ogawa, 1992). Taking the recent works
by Koonce et a!. (1998) and Vallee et a!. ( 1998) into
consideration, this model might be seen as depicted in
Figure 3C. In particular, Vallee et a!. (1998) have described the importance of a hairpin structure formed
between M and C domains which binds to microtubules
and presented a novel mechanism for dynein force production different from that of myosin and kinesin.
Outer arm dynein contains three intermediate chains(ICI , IC2, and IC3) that range in molecular mass from70 to 120 kDa. IC2 and IC3 were cloned by Ogawa et a!.
(1995) and contain the WD repeats in the carboxyterminal halves of the molecules. By contrast, IC1 is nota member of the WD family. IC1 has a unique sequencesuch that the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three
repetitive sequences homologous to the sequence of
NDP kinase, and the C-terminal part contains a highproportion of negatively charged glutamic acid residues(Ogawa et a!., 1996). Thus, IC I is a novel dynein intermediate chain distinct from IC2 and IC3 and may be amultifunctional protein.
Six light chains with molecular masses of 23.2, 20.8,
12.3, 11.5, I 0.4, and 9.3 kDa are associating with outerarm dynein. We have already isolated eDNA clone offive LCs. LCI (23 .2 kDa) and LC3 (12.3 kDa) arehighly homologous to mouse Tctex2 and Tctexl , respectively. These mouse proteins are encoded by the tcomplex region that is involved in transmission ratio
distortion (TRD), male sterility and the development ofgerm cells. Our finding raises the possibility that axonemal dynein proteins are involved in this phenomenon. TRD may be caused by the dysfunction of multiple
axonemal dynein proteins.

76

NATIONAL INSTITUTE

Sperm
Axoneme

Mitotic
Egg

Figure 2. Localization of two dynein isotypes on outer arm of sperm axonemes and mitotic apparatus ofcleaving egg.

A

N

M

c

B

c
Figure 3. Structure of dynein heavy chain. A; Analysis of aminoacid sequence of heavy chain reveals that it consists of three major
domains referred to as N, M, and C from theN-terminus. B; M andC domains make larger domain (motor domain) by intramolecularassociation. C, According to Koonce et al. ( 1998) showthat recombinant motor domain would be spherical. Vallee et al. (1998)
propose that a2 region corresponds to the B-link which is the stalkprojected from the globular head structure of dynein, by demonstrating that the recombinant a2 actually binds to microtubules.

Route 248

CD :g~5t;lttil~*T-Jt.­® I£:£!'#:PJT~pJT

Meitetsu Okazaki Hotel

National Institute for Physiological Sciences

cv ltti]~±.PX® ~:£!f.8®

® ·5Ff-H'#:PJT~fiJT

Okazaki Castle

Administration Bureau

lttil~=_l-1'7/f:';J;T-Jt.­

Okazaki New Grand Hotel@lttil~1'7/i<;J; T-Jt.­

Okazaki Grand Hotel@

Institute for Molecular Science® =~o·;; ::/·lttil~:::J/7rv/.:::Zt/:}1Mishima Lodge ·Okazaki Conference Center® =~tj\IB1¥r.:8

Mishima Elementary School

Post Office@ lttil~rEit~® ~f'rOkazaki High School

Bank

(J)

.:::Z-J'\--;:>-7·:;1-- ":l=-"@) ~~*~f1$®

Super Market "Uny"ffi:f£):pJT
Okazaki City Office@) ~;iij$® 7';:fiJT1$t±

Jr. High School affiliated to Aichi Univ. of Education
Ryukai Jr. High School@ :::J/t::'=I/.:::Z.:::Zi'-Y

Convenience Store
Rokusho Shrine® ~f,j)@) lttil~OO.lL~I'i>HiJT~1f!IH~ UlH&)
Signboard of Okazaki National Research Institutes
Pharmacy@ U-1-'F-r:::J ·;;:;'@ £~1£4~J'¥':PJT~pJT
Yamate Lodge
National Institute of Basic Biology@) *ft.g,;\-{;;f-tj--{I/.:::Zi:z/:)1Center for Integrative Bioscience

National Institute for Basic Biology
Okazaki 444-8585, Japan