CONTENTSINTRODUCTION ······oo················oo········oo••oo•oo··········oo·oo········oo··oo········oo···oo······················oo·····oo············oo·

1

ORGANIZATION OF THE INSTITUTE oo•oo·············oo•oo••oo•oo•oo•••oo········oo····················oo······oo······oo····oo····4

MEMBERS OF THE ADVISORY COMMITTEE FOR PROGRAMMING AND MANAGEMENT oooooooooo.

5

GRADUATE PROGRAMS · ····················oo·oo········oo····oo•oo•oo····oo•·oo········oo····oo······ ···············oo·····oo············oo·6

CELL BIOLOGYDIVISION OF CELL MECHANISMS ooooooooooooooooo•·oo·oo····oo••oo••oo·········oo···oooo····oooo•····oo·····oooo·············oooo
DIVISION OF MOLECULAR CELL BIOLOGY oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo
LABORATORY OF CYTOSKELETON ·oo·oo····oo•oo•oo•oo•oo•oo•oo•·oo···oo···oo····oo·········oooo·······oo····oooo···········oooo
LABORATORY OF CELL SOCIOLOGY OOOOOOOOOOOOoooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

7
10
13
15

DEVELOPMENTAL BIOLOGYDIVISION OF REPRODUCTIVE BIOLOGY 0000000000000000000000000000000000000000000000 oooooooooooooooooooooooooooooooooooooooo
DIVISION FOR SEX DIFFERENTIATION oooooooo···oo········oo·oo·············oo····oo·····oo····oooo····oooo···oo·····oo···oooo
DIVISION FOR MORPHOGENESIS ···oo·oo••oo•oo················oo··oo··············oo·····oo····oo······· ······oooo···oo·····oo···
DIVISION OF DEVELOPMENTAL GENETICS oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo oooooooooo
DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY oooooooooooooooooooooooooooooooooooooooooooooooo
LABORATORY OF DEVELOPMENTAL REGULATION 000000000000000000000000000000000000000000 000000000000000000000000
LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION oooooooooooooooooooooooooooooooooooooooooo

16
19
22
25
28
31
32

NEUROBIOLOGYDIVISION OF MOLECULAR NEUROBIOLOGY oooooooooooooooooooooooo oooooooooooooooooooooooooooooooooooooooooooooooooooooo
DIVISION OF BRAIN BIOLOGY ····oo••oo•oo••oooo····oo···········oo·oo···oo····oo·•·oo············oo·············oo·············oo··
DIVISION OF BEHAVIORAL BIOLOGY oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo•
LABORATORY OF NEUROPHYSIOLOGY ···oo····oo·oo····oo··oo·oo·········oo···oo······oo····oo·····oo····oooo····oo····oo···
LABORATORY OF NEUROCHEMISTRY ·oo···oo•oo•oo•oo·····oo·oo··oo············oooo····oo····oooo •••••.•• :.oooo··········oo···

34
37
40
42
44

EVOLUTIONARY BIOLOGY AND BIODIVERSITYDIVISION OF MOLECULAR GENETICS ··oo•oo•oo•oo•oo········oo·oo·········oo····oo············oo······oo····oooo •••••.••• ,oo..
DIVISION OF GENOME DYNAMICS ·oo·oo·····oooo····oo·····oo•oo••oo········oo···oooo····oo····oooo···········oooo··········oooo
DIVISION OF EVOLUTIONARY BIOLOGY ooooooooooo·····oo·oo•·oo···oo···oo···oo·······oo····oo······oo····oooo·····oo····oo·
DIVISION OF SPECIATION MECHANISM ·····oo·oo··············oo··oo········oo·····oo·····oooo···oo·····oooo·············oo··
LABORATORY OF MORPHO DIVERSITY oo,oo.oooo .......•.•••.• oooooooo•oo····oo·····oo····oooo····oo·····oooo•••oooo···oo·····

46
49
52
55
57

ENVffiONMENTAL BIOLOGYDIVISION OF CELLULAR REGULATION oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo
DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY oooooooooooooooooooooooooooooooooooooooo•
DIVISION OF PLANT DEVELOPMENTAL GENETICS ooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo
DIVISION OF PHOTOBIOLOGY ·····oo····oo········oooo····oo·········oo··oo···oo···oo····· •..••••• oo ••••••.. ·····oo·····oo···oooo···
LABORATORY OF PHOTO ENVIRONMENTAL BIOLOGY 000000000000000000 oooooooooooooooooooooooooooooooooooooooooo•
LABORATORY OF STRESS RESPONSE oooooooooooooooooooooooooooooooooooooooooooooooooo 00000000000000000000000000000000000000000

58
61
64
66
68
70

THEORETICAL BIOLOGYDIVISION OF THEORETICAL BIOLOGY oooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo·
LABORATORY OF GENOME INFORMATICS 0000000000000000000000000000000000000000000000000000000000000000000000000000000000

7274

LABORATORY OF DIRECTOR GENERAL oo•oo····oooo············oo··oo········oo···oo······oo·····oo·······oo····oo·············

76

RESEARCH SUPPORTRESEARCH SUPPORT FACILITY ·oo·····oo··········oo·oo·oo·········oo•oooo······oo····oo·····oooooooo•oo·····oooo···oo·····oo···oo·
RESEARCH CENTER FOR INTEGRATIVE AND COMPUTATIONAL BIOLOGY 0000000000000000000000000
CENTER FOR TRANSGENIC ANIMALS AND PLANTS ooooooOOOOOoo:ooooooooooooOOOOOOOOooooOOooOOooooooooooooooooooooo
THE CENTER FOR ANALYTICAL INSTRUMENTS 000000000000000000000000000000000000000000000000000000000000000000000000
TECHNICAL DIVISION ·oo····oo····oo······oo·oo··········oo·oo········oo·oo···oo·······oo·····oo····oo·······oo······oooooooooooooooo•oo··

78
80
81
83
84

RESEARCH FACILITIESOKAZAKI INSTITUTE FOR INTEGRATIVE BIOSCIENCE Gointly managed by NIBB) oooooooooooooooo•
CENTER FOR RADIOISOTOPE FACILITIES (CRF) ooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

85
86

INDEX ························································································································································

87

The cover photograph is the panel of whole mount in situ hybridization of african clawed frog
(Xenopus laevis) neurula embryos. The blue staining represents where each gene is expressed.
These data are available in XDB3, the database of X. laevis EST ofNIBB. See also page 24.

NATIONAL INSTITUTE FOR BASIC BJOLOGYINTRODUCTIONOn April I", 2004, leighteen Inter-University ResearchInstitutes, to which the National Institute for Basic
Biology belongs, wer realigned to 4 new organizations.
Our National Inf itute for Basic Biology (NIBB)
became a member o the National Institutes of Natural
Sciences that consist of five National Institutes, National
Astronomical Obser atory, National Institute for Fusion
Science, National I~p titute for Physiological Sciences,
Institute for Molecu'Fr Science, and our-selves (NIBB).
Each Instihtte mana9ed by the Director-General, who is
assisted by the Advi fOI)' Committee in each Institute, isindependent from ea~ch other. Therefore, the NINS is a
United Research Insf 1tes of Natural Sciences. A selectionof the Director-General is performed through therecommendation frof the Advisory Committee to the
Director-General Selition Committee. Then the DirectorGeneral is appointed y the president with an approval of
the Management Co mcil and Education and Research!

Council.
The NIBB establis ed in 1977. The aim of the NIBB is
the promotion and s imulation of shtdics in the field ofBiology. As a ce~ter of excellence (COE), NIBB
promotes not only \Dasic biology but also the modern
biological sciences ~y conducting first-rate research onsite as well as in cooperation with national , public andprivateuniversitie1 andresearchorganizations.
Researchers at the NIBB investigate Cell Biology,
Developmental Bio ogy, Neurobiology, Evolutionat-y
Biology and Biodiv rsity, Environmental Biology andTheoretical BiologJ to elucidate the general andfundamental mechan sms underlying various biologicalphenomena.
In 2004 after thf establishment of NINS, NIBB
changed the researchl_unit system from 3 departments to
30 independent divisi pns and laboratories. The purpose ofthis change is to make researchers move easily to the newIfield if necessary.
On the higher edubation, NIBB became a member of
the Graduate Univer~ity for Advanced Studies when it
was established in
as a department of Molecular
Mechanics in School of Life Science. Three shtdents forfive year Doctoral c urse and six for three year seniorDoctoral course are open to applicants for entrance.
On March 31 '\ 20:04, adjunct professors Dr. MIYATA
Takashi, Dr.
YA~AGIDA
Mitsuhiro, and Dr.
NAKAMURA Haruk ~ left NIBB just after the completionof their appointmG:nts.
Research associates Dr.
ISHIHARA Satoru,J Dr. YAMAMOTO Hiroshi , Dr.
MIZUSHlMA Nobor and Dr. KOBAYASHI Tohru were
promoted to chief in fStiga tors of the Research Instihttesand on December 31", 2004, Dr. SUZUKI Iwane to
Tsukuba University a' an assistant professor.
In congrahtlation, associate professor Dr. WATANABE
Masakatsu left NIBBt n August 31" as he was promoted
to a professor of the Graduate University for Advanced
Studies.
Conversely five r searchers were newly appointed in
2004, Dr. TANAKA I inoru from Hokkaido University as
an associate professor, Dr. TSUKAMURA Hiroko from
Nagoya University as an adjunct associate professor, Dr.
YAMAUCHI Daisuke from Himeji Institute of
1

lr88,

Teclmology as an adjunct associate professor, Dr.
IMAMURA Takuya from Institut Jacques Monod (JSPS
Post-Doctoral fellow for abroad) as a research associateand Dr. SHIGENOBU Shuji from JSPS Post-Doctoral
fellow as a research associate.
In addition, a number of postdoctoral fellows, technicalassistants, secretaries, and graduate students after awarded
Ph .D. degree left, and conversely people newly joined
NIBB at the equivalent positions. In consequence, thetotal number of personnel working at NIBB has been kept
at approximately 300 for several years.
As a COE of the biological research institute, NIBB is
responsible for conducting research projects m
cooperation with various research groups.
Moreover, NIBB continues to sponsor interdisciplinary
symposia and shtdy meetings on current topics by inviting
leading scientists from around the world to the NIBB.
Based on this concept, NIBB statied to support a series of
Okazaki Biology Conference (OBC) through intimate
cooperation with a variety of biological scientificsocieties. The first OBC was held on January 25-30, 2004,
entitled "The Biology of Extinction" and the second on
September 26-30, entitled "Terra Microbiology." Bothmeetings were very successful. Thirty to fotiy scientists
from abroad and equivalent number of them from Japan
had an exciting discussion on the new field of future
biology.
NIBB also provides a training course inbiological sciences for young investigators.
Finally, I would like to celebrate the retirement of Mr.
HATTORI Hiroyuki with appreciation of his dedicatedwork as the Head Technical Staff. Toward his respectful
contribution, the NIBB awarded him a title for NlBB
Technical Staff Emeritus on March 31 ", 2004.
We always welcome any suggestions concerning the
research activities ofNIBB.
KATSUKI, Motoya, D.Sc.
Director-General

NATIONAL rNSTlTUTE FOR BASIC BIOLOGY

2

ORGANIZATION

National Institutes of Natural Sciences

I Executive Directors 11----------1

I
I

President

Auditors!

SHIMURA, Yoshiro

I

I
I

Management Council

I

I
I

Board of Directors

Education and Research Council

I

National Institute for

National Astronomical
Observatory of Japan~N/ss Basic Biology

Director-General
KATSUKI, Mataya
Vice Director
NAGAHAMA, Yoshitaka

National Institute for
Fusion Science

Institute for
Molecular Science

National Institute forPhysiological SciencesI

3

NAT IONAL INSTITUTE FOR BASIC BIOLOGY

Research Unit
Cell Biology• Division of Cell Mechanisms• Division of Molecular Cell Biology• Laboratory of Cytoskeleton• Laboratory of Cell Sociology

I Developmental Biology• Division of Reproductive Biology• Division for Sex Differentiation• Division for Morphogenesis• Division of Developmental Genetics• Division of Molecular and Developmental Biology
Laboratory of Developmental Regulation
Laboratory of Molecular Genetics for Reproduction

I Neurobiology• Division of Molecular Neurobiology• Division of Brain Biology• Division of Behavioral Biology (adjunct)Laboratory of Neurophysiology• Laboratory of Neurochemistry

Evolutionary Biology and Biodiversity• Division of Molecular Genetics• Divis ion of Genome Dynamics• Division of Evolutionary Biology• Division of Speciation Mechanism (adjunct)• Laboratory of Morphodiversity

Environmental Biology• Division of Cellular Regulation• Division of Molecular Environmental Endocrinology• Division of Plant Developmental Genetics• Division of Photobiology (adjunct)• Laboratory of Photoenvironmental Biology
Laboratory of Stress Response

I Theoretical Biology

Di vision of Theoretical Biology
Laboratory of Genome Informatics• Laboratory of Director General~-------------------------R_e__searchSupp_o_r_t________________________~
Research Support Facility
Research Center for Integrati ve andComputational Biology

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

Center for Transgenic Animals and Plants
Technical Division

Okazaki Institute for Integrative Bioscience
Center for Radioi sotope Facilities

I Center for Experimental Animal

Department of Development, Differentiation and Regeneration• Division of Developmental Genetics
Division of Molecular and Developmental Biology
Department of Bio-Environmental Science• Division of Bio-Environmental Science• Division of Plant Developmental Genetics

Research Center for Compu tational Science

I Center for Analytical Instruments• Laboratory Glassware Facilities• Electron Microscope Center• Machine Shop
• Low-Temperature Facilities

4

NATIONAL INSTITUTE FOR BASIC BIOLOGYORGANIZATION OF THE INSTITUTE
The National Institute for Basic Biology (NIBB) isan institute in the National Institutes of Natural
Sciences (NINS) that are composed of five independent
institutes. Among them, the NIBB, the Institute for
Molecular Science (IMS) and the National Institute forPhysiological Sciences (NIPS) located on a hill
overlooking the old town of Okazaki. NIBB wasestablished in 1977 and its activities are supported by
Monbukagaku-sho (the Ministry of Education, Culture,
Sports, Science and Technology: Mext) of Japan. The
Center for Integrative Bioscience (CIB) was established
as a common facility of the three Okazaki Institutes in2000 and began in 200 I. The CIB was changed its
name to Okazaki Institute for Integrative Bioscience on
April 151 , 2004, at the beginning of the neworganization.

Policy and Decision Making
The Director-General oversees the operation of the
NIBB assisted by an advisory body, the Advisory
Committee for Programming and Management. The
Advisory Committee, comprised of professors within
the NIBB and an equal number of leading biologists
outside NIBB, advises the Director-General upon his
request on planning joint research programs and otherimportant matters in NIBB as well as on the scientificactivities of the NIBB. The Director-General selection
committee makes a nomination of Director-General to
the President after hearing the recommendation fromthe Advisory Committee. The Advisory Committee
makes recommendations on the Director-General andon faculty appointments, the NIBB's annual budget andfuture prospects.

Administration
Administration of the NIBB is undertaken by the
Administration Bureau of the Okazaki Administration
Center under the direct auspices of the Administration
Office ofNINS.
Research
The NIBB conducts its research programs through 30
research units, 4 research support facilities including
Technology Department, and the Research Center for
Integrative and Computational Biology.
Each research unit has its own research project and isstaffed by a professor, an associate professor and two
research associates in principle. A research unit forms
an independent project team. Three of the research
units are adjunct and headed by professors who holdjoint appointments with other universities. Adjunctdivisions have a resident research associate. This
arrangement facilitates exchange in research activities
in Japan. The Technical Department manages theactivities of research technicians and helps to promoteresearch activities of each research unit and facility andto maintain the common research resources of the
NIBB. The Department also undertakes the technicaleducation of staffs.
Several members of the Okazaki Institute forIntegrative Bioscience jointly work with the NIBB.

Research Support Facilities
The Research Support Facility of the NIBB consistsof six sub facilities, the Large Spectrograph Laboratory,
the Tissue and Cell Culture Laboratory, the Computer
Laboratory, the Plant Culture Laboratory, the Plant Cell
Laboratory and the Experimental Farm. The ResearchCenter for Integrative and Computational Biology, theCenter for Transgenic Animals and Plants and Technical
Division are also research support system of the NIBB.
In addition, five facilities are operated jointly with
NIPS; they consist of the Electron Microscope Center,
the Center for Analytical Instruments, the Machine
Shop, the Laboratory Glassware Facilities and the
Low-Temperature
Facilities.
The
Radioisotope
Facilities, the Computer Center and the Animal Care
Facilities are common facilities of the three Okazaki
Institutes.

Campus
The three Okazaki Institutes covers an area of
164,783m2 with four principal buildings. The NIBB's
main research building has a floor space of 16,789m2•
Two-thirds of the space was completed in 1982 and theremaining one-third in June, 1983. The buildings thathave the research support facilities were also completed
in 1983. A building for the Laboratory of Gene
Expres~ion and Regulation (2,577m2) was built in 1996.
A building for Center for Transgenic Animals andPlants (2,500m2) was built in the new campus atNovember, 2003.

5

NATIONAL INSTITUTE FOR BASIC BIOLOGYMEMBERS OF THE ADVISORY COMMITTEE FOR
PROGRAMMING AND MANAGEMENT
Chairman
NAGAHAMA, Yoshitaka

Vice Director & Professor, National Institute for Basic Biology

Vice-Chairman
MACHIDA, Yasunori

Professor, Nagoya University

KOMEDA, Yoshifumi
KONDO, Hisato
KUROSAWA, Yoshikazu
MURAKAMI, Fujio
OKADA, Kiyotaka
SAGA, Yumiko
SEHARA, Atsuko
WADA, Masamitsu

Deputy Director & Group Director, RIKEN Center
for Developmental Biology
Professor, The University of Tokyo
Professor, Osaka University
Professor, Fujita Health University
Professor, Osaka University
Professor, Kyoto University
Professor, National Institute of Genetics
Professor, Kyoto University
Professor, Tokyo Metropolitan University

HASEBE, Mitsuyasu
HORIUCHI, Takashi
IIDA, Shigeru
MOROHASHI, Ken-ichirou
NISHIMURA, Mikio
NODA, Masaharu
OHSUMI, Yoshinori
TAKADA, Shinji
UENO, Naoto
YAMAMORI, Tetsuo

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

AIZAWA, Shinichi

6

NATIONAL INSTITUTE 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 five year Ph.D. course as well asa three year senior course those who have completed amaster's course or equivalent at any university are
eligible to apply.
The Department consists of the 30 Research Unitsappeared in the page 3.

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.

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGYDIVISION OF CELL MECHANISMS
Professor:
Associate Professor:
Research Associates:

NISHIMURA, Mikio
HAYASHI, Makoto
MANO, Shoji
YAMADA, Kenji
Technical Staff:
KONDO, Maki
JSPS Postdoctoral Fellows: NITO, Kazumasa
KUROYANAGI, Miwa
ARAI, Yuko
Postdoctoral Fellows:
KAMADA, Tomoe (Apr. I -)MUTSUDA, Michinori (Apr. I -)HATSUGAI, Noriyuki (Oct. I -)KAMIGAKI, Akane (Oct. I -)KAMADA, Tomoe IJ (-Mar. 3I)
Graduate Students:
HATSUGAI, Noriyuki IJ (- Sep. 30)
OGASAWARA, Kimi 2J (Apr. I-)
Technical Assistants:
NAKAMORI, Chihiro
YAGI, Mina
YOSHINORI, Yumi
SUZUKI, Iku
FUKAZAWA, Mitsue (Nov. 1 -)VEDA, Chizuru
Secretaries
IYODA, Yuri (-Mar. 3I)
IJ Graduate University for Advanced Studies
21 Tokyo University of Marine Science and Technology

Higher plant cells contain several distinct organelles
that play vital roles in cellular physiology. During
proliferation and differentiation of the cells, the organelles
often undergo dynamic changes. The biogenesis of neworganelles may occur, existing organelles may undergo atransformation of function, while other organelles may
degenerate. Because the dynamic transformation oforganellar function (differentiation of organelles) is
responsible for flexibility of differentiation events inhigher plant cells, the elucidation of regulatory
mechanisms underlying organelle transformation are
currently studied in this division.
I. Reversible transformation of plant peroxisomes
Dramatic metabolic changes which underlie the shift
from heterotrophic to autotrophic growth occur ingreening of seed germination. Accompanying thesemetabolic changes, many constitutive organelles arefunctionally transformed. Etioplasts differentiate into
chloroplasts and mitochondria acquire the ability tooxidize glycine. Glyoxysomes, which are microbodies
engaged in the degradation of reserve oil via !3-oxidationand the 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 andleaf peroxisomes is controlled by gene expression,
alternative splicing, protein translocation and protein
degradation. We now engage in proteomic andtranscriptomic analyses of the reversible peroxisomaltransition in Arabidopsis cotyledons.

7

II. Transcriptomics, proteomics and phenomics ofplant peroxisomes
Enzymes localized in plant peroxisomes aresynthesized in the cytosol, and function after theirpost-translational transport into peroxisomes. Almost allof the peroxisomal matrix proteins are known to containone of two targeting signals (PTS 1 and PTS2) within themolecules. PTS 1 is a unique tripeptide sequence found in
carboxyl terminus of the mature proteins. The permissiblecombinations of amino acids for PTS 1 in plant cells are[C/A/S/P]-[K/R]-[1/L/M]. In contrast, PTS2 is involved ina cleavable amino terminal presequence of peroxisomal
proteins that are synthesized as a precursor protein withlarger molecular mass. PTS2 consists of a consensus
sequence [R]-[L/Q/I]-Xs-[H]-[L].
We identified 256 gene candidates of PTSl- andPTS2-containing proteins and another 30 genes of
non-PTS-containing proteins from Arabidopsis genome.
Custom-made DNA microarray covering all these genes
was used to investigate expression profiles of theperoxisomal genes in various organs. Statistical analyses
revealed that the peroxisomal genes could be divided into
five groups. One group showed ubiquitous expression in
all organs examined, while the other four were classified
as showing organ-specific expression in seedlings,
cotyledons, roots and in both cotyledons and leaves.
In parallel, we made two-dimensional protein map ofglyoxysomes and leaf peroxisomes isolated from
Arabidopsis. Peptide MS fingerprinting analyses allowed
us to identifY novel proteins exists in either glyoxysomesor leaf peroxisomes. Some of these proteins contain no
obvious PTS 1 and PTS2. Of these, we characterized
GPKl as a novel protein kinase in glyoxysomes.
Bioinfomatic analysis of Arabidopsis genome predicted
the presence of 15 kinds of genes for peroxisomal
biogenesis
factors,
calledPEX
genes.
We
comprehensively investigated whether these predicted
PEX genes function in peroxisome biogenesis bygenerating knock-down mutants that suppress PEX gene
expression by RNA-interference. Phenotypes of thesemutants allowed us to identifY the functional PEX genes,
which can be classified into two groups, i.e. PEX genes
regulating for peroxisomal morphology and peroxisomal
protein import.

III. Identification of novel components essential for
peroxisome biogenesis
To better understand peroxisome biogenesis, wemutagenized seeds of transgenic Arabidopsis, GFP-PTSl,
in which peroxisomes with normal size and number canbe visualized with GFP, and isolated a number of
Arabidopsis mutants having ~berrant 12.eroxisome
morphology (apm mutants) based on the different patternof GFP fluorescence (Fig. 1).
In one of these mutants, apml, the peroxisomes arelong and reduced in number, apparently as a result of
inhibition of division. APMJ gene encodes DRP3A(Qynamin-related J2.rotein 3A). Interestingly, apml
mutation also caused inhibition of mitochondrial division.

8

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

Fig. 1 Phenotype of apm mutants.

(A) to (C) root cells. (D) to (F) leaf cells. (A) and (D) show
GFP-labelled peroxisomes in GFP-PTS I as a parent plant.
(B) apml mutants have long peroxisomes. (C) Distribution ofperoxisomes is altered in apm5 mutants. (E) In apm2 mutant,the decrease of efficiency of protein transport to peroxisomes
results in the observation of GFP fluorescence in the cytosol
and peroxisomes. (F) apm3 mutants contain larger
peroxisomes. Bar indicates 50 f.!m.
Actually APM1 /DRP3A protein is localized atperoxisomes and mitochondria. These findings showedthat APM I /DRP3A protein is involved m both
peroxisomal and mitochondrial division.
With regard to another apm mutants, apm2, apm4 andapm7, the GFP fluorescence is observed in not only
peroxisomes but also the cytosol, suggesting that theefficiency of protein transport to peroxisomes is decreasedin these mutants. apm3 and apm6 mutants have larger
peroxisomes. These phenotypes indicate that both mutants
are defective in peroxisomal division. In apm5 mutants,
the distribution of peroxisomes within cells changed tothe one position in the cell and peroxisomes hardly move,whereas peroxisomes in GFP-PTS I are dispersed andmove frequently. Analyses of these apm mutants andidentification of APM genes will identify components
necessary for peroxisome biogenesis and address theregulation of its mechanism.

ER-retention signal (KDEL), in seedlings. An electron
microscopic analysis showed that PYKlO was
accumulated in ER bodies of the wild-type plants. This
indicates that PYKlO is the main component of ER body.
We cloned NAil gene using a positional cloning strategy.
NAJJ encodes a transcnptton factor that has abasic-helix-loop-helix (bHLH) domain. The nail mutant
had a single nucleotide change at an intron acceptor siteof NAil gene. Because of this mutation , aberrant splicingof NA!l mRNA occurs in the nail mutant. Transient
expression of NAil induced ER bodies in the nail mutant.
Two-dimensional electrophoresis and RT-PCR analyses
showed that the amounts of mRNA and protein of aputative lectin in the nail mutant were decreased, as was
the case of PYKl 0. These results provide direct evidence
that this bHLH protein plays a role in the formation ofER
bodies.

V. Vacuolar processing enzyme responsible forprogrammed ceiJ death in plants.
Vacuolar processing enzyme (VPE) belongs to thecysteine protease family C 13. This family is found invarious eukaryote organisms including higher plants andanimals. VPE exhibits substrate specificity toward
asparagine and aspartic acid residues, the amino acid wellconserved at a position in the processing sites of various
vacuolar/lysosomal proteins. VPE was originallyidentified as an enzyme responsible for the processing andmaturation of seed storage proteins in plants. We haveshown that mouse VPE is responsible for the processing

A

WT

-VPE

IV. ER-derived organelles for protein storing anddefense strategy.
Plant cells develop various types of endoplasmic
reticulum (ER)-derived structures with specific functions.
ER bodies are ER-derived compartment specific to theBrassicaceae, including Arabidopsis. They are rod-shaped
structures (5 J..lm long and 0.5 J..lm wide) that is surrotmded
by ribosomes. ER bodies can be visualized in transgenic
plants of Arabidopsis expressing GFP fused with an ER
retention signal (GFP-HDEL). ER bodies were widelydistributed in the epidermal cells of whole seedlings.
Rosette leaves had no ER bodies, but accumulated ER
bodies after wounding or jasmonic acid treatment. Thissuggests that ER bodies function in the defense against
herbivores. We isolated an Arabidopsis mutant, nail ,which has no ER bodies in whole plants. The nail mutant
did not accumulate PYK 10, a ~-glucosidase with an

Fig. 2 VPE deficiency suppresses the vacuolar collapse
leading to the TMV-induced hypersensitive cell death.
(A) The non-silenced (WT) and VPE-silenced (-VPE)
Nicotiana benthamiana plants were infected with TMV on
halves of their leaves (indicated by asterisks). The
photographs were taken after 24 hours. (B) Morphologicalchanges of the TMV-infected regions of the non-silenced
leaves (WT) at 9 hours under the electron microscope. Bar
indicates I ~tm. cw, cell wall; pm, plasma membrane; vm,
vacuolar membrane; v, vacuole; ch, chloroplast; sg, starch
granule. Red triangles indicate the disintegrated regions of
vacuolar membranes.

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

of lysosomal proteases, cathepsins B, H, and L, from thesingle-chain forms into the two-chain forms, and VPE
deficient mice accumulated macromolecules in thelysosomes.
Plant VPE homologues are separated to two
subfamilies: one seed type and the other vegetative type.
Seed-type VPE is responsible for the maturation of seedstorage proteins. On the other hand, the function ofvegetative-type VPE was obscure. Recently, we found anovel function of vegetative-type VPE in programmed
cell death (PCD). The plant hypersensitive response (HR),a type of defense strategy, constitutes well-organized PCD.
The evidences from extensive studies indicate that
caspase activity is involved in plant PCD. VPE is
identified as a proteinase that exhibits caspase activity. No
visible lesions formed on the tobacco mosaic virus(TMV)-infected leaves of VPE-silenced tobacco plants(Fig. 2). VPE deficiency prevented the typicalcharacteristics of PCD, such as cell shrinkage,cytoplasmic
condensation,andnuclearDNA
fragmentation. An ultrastructural analysis showed that thedisintegration of the vacuolar membranes occurs in theleaves before visible lesions are fanned. The
disintegration of the vacuolar membranes continued,
resulting in complete vacuolar collapse in association
with plasmolysis. On the contrary, vacuoles and vacuolar
membranes remained intact in the VPE-silenced plants.
These results suggest that VPE is involved in vacuolar
collapse, which triggers PCD. Plants evolve a deathstrategy mediated by a vacuolar system, which is not seenin animals. Interestingly, a vacuolar enzyme is this key
player in a plant-specific cell death system.

VI. Role of molecular chaperones on plant celldifferentiation.
Molecular chaperones are cellular proteins thatfunction in the folding and assembly of certain otherpolypeptides into oligomeric structures, but that are not,themselves, components of the final oligomeric structure.
To clarify the roles of molecular chaperones on celldifferentiation, we have purified and characterizedchaperonin and Hsp70s and analyzed their roles in thetranslocation of proteins into chloroplasts.
Previously, we characterized a mitochondrial
co-chaperonin (Cpnl 0), chloroplast co-chaperonins(Cpn20 and CpnlO) and a small heat shock protein from
Arabidopsis. Recently, we started to characterize HSP90s.
Their evolutional and functional characterization is now
under experiments.

Publication List:
Hara-Nishimura, 1., Matsushima, R., Shimada, T., andNishimura, M. (2004) Diversity and functions of
ER-derived compartments in plants: Are thesecompartments specific to plant cells? Plant Physiol. 136,
3435-3439.
Hatsugai, N., Kuroyanagi, M., Yamada, K. Meshi, T., Tsuda,S., Kondo, M., Nishimura, M. and Hara-Nishimura, I.
(2004) A plant vacuolar protease, VPE, mediates

9virus-induced hypersensitive cell death. Science 305,
855-858.
Hayashi, M., Yagi, M., Nito, K., Kamada, T. and Nishimura,
M. (2004) Differential contribution of two peroxisomal
receptors to the maintenance of peroxisomal functions in
Arabidopsis. J. Bioi. Chern. in press
Mano, S., Nakamori, C., Kondo, M., Hayashi, M. andNishimura, M. (2004) An Arabidopsis dynamin-related
protein, DRP3A, controls both peroxisomal andmitochondrial division. Plant J. 38, 487-498.
Mano, S. and Nishimura, M. (2004) Plant peroxisomes.
Vitamins and Hormones in press
Matsushima, R., Fukao, Y., Nishimura, M. andHara-Nishimura, I. (2004) NAil gene encodes abasic-helix-loop-type transcription factor that regulates theformation of a novel ER-derived structure, the ER body.
Plant Ce/!15, 1536-1549.
Nakaune, S., Yamada, K., Kondo, M., Kato, T., Tabata, S.,
Nishimura, M., Hara-Nishimura, I. (2004) A novel
vacuolar processing enzyme is involved in seed coatformation at the early stage of seed development. Plant
Cell in press
Tamura, K., Yamada, K., Shimada, T., Hara-Nishimura, I.
(2004) Endoplasmic reticulum-resident proteins areconstitutively transported to vacuoles for degradation.
Plant J. 39, 393-402
Usami, T., Mochizuki, N., Kondo, M., Nishimura, M. andNagatani, A. (2004) Cryptochromes and phytochromes
synergetically regulate the Arabidopsis root greening
under blue light. Plant Cell Physiol. 45, 1798-1808
Watanabe, E., Shimada, T., Tamura, K., Matsushima, R.,
Koumoto, Y., Nishimura, M. and Hara-Nishimura, I.
(2004) An ER-localized form of PV72, a seed-specificvacuolar sorting receptor, interferes the transport of an
NPIR-containing proteinase in Arabidopsis leaves. Plant
Cell Physiol. 45, 9-17.
Yamada, K., Fuji, K., Shimada, T., Nishimura, M. andHara-Nishimura, I. (2004) Endosomal proteases facilitate
the fusion of endosomes with vacuoles at the final step ofthe endocytotic pathway. Plant J. in press
Yamada, K., Nishimura, M. and Hara-Nishimura, I. (2004)
The slow wound-response of VPE is regulated byendogenous salicylic acid in Arabidopsis. Planta 218,
599-605.
Yamada, K., Shimada, T., Nishimura, M. andHara-Nishimura, I. (2004) A VPE family supporting
various vacuolar functions in plants. Physiol. Plant. inpress
Yoshida, K., Kawachi, M., Mori, M., Maeshima, M., Kondo,
M., Nishimura, M. and Kondo, T. (2004) The involvementof tonoplast proton pumps and Na+(K+)/H+ exchangers in
the change of petal color during flower-opening ofmorning glory, Ipomoea tricolor cv. Heavenly Blue. Plant
Cell Physiol. in press

10

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGYDIVISION OF MOLECULAR CELL BIOLOGY
Professor:
Research Associates:

Technical Staff
NIBS Research Fellow:
JSPS Postdoctoral Fellows:

Postdoctoral Fellows:

Graduate Students:

Technical Assistants:

Secretary:

OHSUMI, Yoshinori
KAMADA, Yoshiaki
NODA, Takeshi IJ
SUZUKI, Kuninori
KABEYA , Yukiko
FUJIKI, Yuki
SEKITO, Takayuki
HANADA, Takao
NAKATOGAWA , Hitoshi
JCHIMURA , Yoshinobu
YOSHIMOTO, Kohki
BABA, Misuzu
OITA, Eiko
ONODERA , Jun
OHNEDA , Mamoru
OBARA , Keisuke
KAWAMATA , Tomoko JJ
MATSUI, Makoto 2;
KAGEYAMA, Takuya lJ
IMA MURA, Yuko
ICHJKA WA, Rie
TSUKESHJBA , Kumi
HARA , Yokotargeted to the membrane of nascent autophagosomes,
eventually sequestered into autophagosomes, and fina llytransported to the vacuolar lumen. Our previous
classification was performed under nutrient-richconditions. In order to obtain further insights for themechanism of autophagosome formation, we analyzed theintracellular localization of GFP-Atg8p in every atg
mutant under autophagy-inducing conditions. GFP-Atg8p
showed several distinct localization patterns in atg
mutants (Figure 1). Subsequent analyses revealed
epistatic relationships between Atg proteins during theprocess of autophagosome formation.
Growing

Terminal

Class~(LI.atg4)

t:..vps30

IJ Study abroad.fi·om Mar. 2003
2
) Graduate University for Advanced Studies

J) Kobe UniversityyThis division aims to understand the autophagy inrespects to its molecular mechanism and its physiologicalroles in yeast and higher eukaryotes. Cells executedegradation processes of their constituents, which are wellcoordinated to regulate the biological activities. In other
word, we must shed light on degradation process to fully
tmderstand the cell, because the study on the degradation
has been retarded compared to the biosynthetic process.
Autophagy is a major route for bulk degradation ofcytoplasmic
constituents
andorganelles
111
lysosome/vacuole, and is well conserved in eukaryotes.

I. Background
Upon nutrient starvation, autophagic process stmis asbuilding up membrane structures, autophagosomes, in thecytoplasm. The autophagosome sequesters cytosol andorganelles nonselectively. Then it is delivered to thevacuole/lysosome, and the cytoplasmic materials inside
are degraded by vacuolar/lysosomal hydrolases. We had
discovered autophagy in a simple model organism, S.
cerevisiae and morphologically and genetically defined
the whole process.

II. Interrelationships among Atg proteins during
autophagy in yeast, Saccharomyces cerevisiae
Macroautophagy is a bulk degradation process induced
nutrient starvation in eukaryotic cells. In
Saccharomyces cerevisiae, 16 ATG genes are essential for
autophagosome formation. Recently, we demonstrated
that these ATG genes can be classified into three groups
on the basis of localization of GFP-Atg8p and Atg5p-GFP
under nutrient-rich conditions. In starving cells, Atg8p isby(LI.atg9)a(LI. atg1)

Figure I. Localization of GFP-Atg8p in atg mutants before
and after 24-hour rapamycin treatment. Cell s express ing
GFP-Atg8p were grown in SD + casamino ac id med ium
and observed under the fluorescence microscope. Cells
under growing conditions (Growing; A-D) and after
24-hour rapamycin treatment (Terminal; E-H). (A and E)
t':!.atg4 cells showing the class ~ terminal phenotype. (B andF) t':!.vps30/t':!.atg6 cells showing the t':!.vps30 tenninal
phenotype. (C and G) t':!.atg9 ce lls showing a typical c lass yterminal phenotype. Arrows represent dots of GFP-Atg8p
close to the vacuole often seen in the class y mutants. (Dand H.) t':!.atgl ce ll s showing the class a. terminal phenotype.
Bar: 5 J.lll1 .

III. Starvation triggers the delivery of theendoplasmic reticulum to the vacuole via
autophagy in yeast
In the course of studying mechanistic aspect of
autophagy, we recently found that an active flow of
COPII vesicular traffic from the ER is required for
autophagosome formation. There are many studies to
fo llow the changes in expression levels of proteins under
the starvation, but it has not been studied how the protein
h·ansport pathway like early secretory pathway from theER, the production site of cell surface and secretory
proteins, is affected. Here we studied ER and Golgi
resident proteins to see if and how the starvation stress

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

modulates the early secretory pathway, and found an
autophagic degradation of ER under starvation conditionsin addition to cytoso lic protein degradation (Figure 2).
Golgi membrane protein was not engulfed by th e
autophagosome under the same conditions, indicating thatthe uptake of ER by autophagosome is the specific event.
Although the ER exists in a network structure that ismu tually connected and resides predominantly around thenucleus and beneath the plasma membrane, most of
autophagosomes engulfed ER. The extent of the ER
uptake by autophagy was nearly identical to that of thesoluble cytosolic proteins. This phenomenon was
explained by the appearance of fragmented ER membrane
structures in almost all autophagosomes. Fut1hermore, ER
dynamism is required for this process: ER uptake by
autophagosomes occurs in an actin dependent manner.
Visualizing these proteins revealed dynamic changes inthe early secretmy pathway under the starvation condition.
Significant amounts of both ER and Golgi proteins weretransported to the vacuole during starvation .

IIrecombinants and liposomes demonstrated that theefficiency of Atg8-PE conjugation was strongly affectedby lipid composition. Further, the Atg8 was linked to
the PE in liposomes, but not to the PE in the presence ofdetergent, suggesting that PE in a lipid bilayer isnecessmy for the Atg8 conjugation. These results shouldbe very useful for future works to determine w here
Atg8-PE conjugation occurs during autophagy in yeast.
Interestingly, we found that the PE-conjugated Atg8 has ahigher affinity for an antiserum raised against the
N-terminal region of the Atg8 protein. Further, Atg8
became highly sensitive to trypsin digestion after
lipidation. Hence, we concluded that PE conjugationinduces a conformational change of Atg8 .

8~r • • • • • • • • • • • •

Atg4

8
G

G¥
R

t

~

t

8 8 8
G

'A

8~

- -GD - Mrnt

AlP

G

c

(El)

(EZ)

~

G/

G

c

~PE

B

Figure 3. Atg8-PE conjugation system
DIG

FM

GFP-HDEL

MergedFigure 2. DsRed-HDEL is transp01·ted to the vacuole inan autophagy dependent manner with rapamycin
treatment. The fluorescence and the Nomarski images ofwild-type ce ll s (JK9-3da) expressing DsRed-HDEL (A)under growi ng condition and (B) und er rapamycin treatedco ndition ( I 0 hrs). The vacuolar limiting membrane waslabeled w ith FM4-64. The vacuolar lumen (noted "vac" )became tluorescent with starvation treatment.

IV. In vitro reconstitution of Atg8-PE conjugation
systemIn yeast, S. cerevisiae, Atg8 plays an important role
during autophagosome formation. We have previously
revealed that the Atg8 is covalently attached tophosphatidylethanolamine (PE) via a ubiquitin-likeconjugation system (Figure 3). The C-terminal Arg of117
newly synthesized Atg8 (Atg8R ) is removed by Atg4
protease to expose a Gly residue at the C-terminus
11 6(Atg8G 11 6). The Apg8G
is then activated by Atg7 (Elenzyme) and transferred to Atg3 (E2 enzyme).
116
Following these reactions, the Apg8G
conjugates to PE
tlu·ough an amide bond between its C-terminal Gly andthe amino group of PE. The subsequent deconjugation
reaction by Atg4 is necessary for the normal progressionof autophagy. Notably, Atg8 conjugation system, whi lesimilar to ubiqu itination mechanically, utili zes aubiquitous phospholipid, not a protein, as a target. We
further developed in vitro Atg8-PE reconstitution system.
The Atg8-PE was successfully reconstituted simply with
Atg8G 11 6 , Atg7 and Atg3 by using in vitro system. These
11 6
results confirmed that Atg8G , Atg7, and Atg3 are theminimum components for Atg8-PE conjugation reaction.
The in vitro Atg8-PE reconstitution system using

Carboxy-termi nal arginine (R) of newly synthesized Atg8 isremoved by Atg4 protease, leaving a glyc ine residue (G) at the
C terminus (Atg8G 11 6). The Atg8G11 6 is activated by El enzyme,
Atg7 , and then transfetTed to E2 enzyme, Atg3 . Finally,
Atg8G 11 6 cova lently conjugates with phosphatidylethanolamine(PE) through an amide bond between the C-tenninal glycine
and the amino group of PE. Liberation of Atg8 moiety from
Atg8-PE by the act ion of Atg4 (deconjugation) is required forthe normal progression of autophagy.

V.

Autophagy-defective
senescenceplant

exhibitearly

So far, plant autophagy has been described in reports
by morphological studies . In addition, a recent
genome-wide search revealed significant conservation
among ATG genes in yeasts and plants. It has not been
proved, however, that Arabidopsis ATG genes are
involved in plant autophagy. To evaluate this requirement,
we examined the ubiquitination-like Atg8 lipidation
system. We generated transgenic Arabidopsis expressing
GFP-ATG8 fusion proteins and established a systemmonitoring autophagy in whole plants. In wild-type plants,
GFP-ATG8s were observed as ring shapes in thecytoplasm and were delivered to vacuolar lumens under
nitrogen-starved
conditions.
By
contrast,
in
T-DNA-insertion double mutant of the ATG4s which
encodes proteases required for C-terminal cleavage of
ATG8 (atg4a4b-l) , autophagosomes were not observed
and the GFP-ATG8s were not delivered to the vacuole. In
addition, we detected autophagic bodies in the vacuoles ofwild-type roots (Figure 4 panel A) but not in those of
atg4a4b-1 (Figure 4 panel B) in the presence of
concanamycin A , a V-ATPase inhibitor. The autophagy
defective mutant exhibited early senescence of rosette

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

12

leaves even under nutri ent-rich conditions (Figure 4 panelD) and fewe r seeds under nutri ent-limiting conditions;howeve r, these plants were neverthe less able to undergo acom plete life cycle. In addition, atg mutant di splayed areduction in
the growth rate of roots undernutri ent-limiting conditions . Autophagy is required forthe m ai ntenance of cellular viability regardless of thenutrient conditions and contributes to the development ofa root system under co nditions of nutrient limitation.

form-ll formation. J. Cell Sci. 117, 2805-12 (2004)
Ichimura T, Kubota H, Goma T , Mizushima N , Ohsumi Y,
lwago M, Kakiuchi K, Shekhar HU , Shinkawa T, Taoka
M, Ito T, Isobe T. Transcriptomic and proteom ic analys isof a 14-3-3 ge ne-deficient yeast.
Biochemist1y 43,
6 149-58.
Okazaki H, Ono B, Ohsumi Y, Ohsumi M. , apg 15-l , a UGA
mutant allele in the Saccharomyces cerevisiae APG 16
gene, and its suppressio n by a cytoplas mic factor. Biosci

Biotechnol Biochem. 68, 1541-8 (2004)
Sugawara K, Suzuk i NN , Fujioka Y, Mi zushima N, Ohsumi
Y,lnagakiF.
The
crysta l
structureofm icrotub ule-associated protein li g ht chain 3, a mamma li an
ho mologue of Saccharomyces cerevisiae Atg8. Genes.
Cells 9, 6 11-8 (2004)
Ichimura Y, Imamura Y, E moto K, Umeda M , Noda T,
Ohsumi Y. In vivo and in vitro reconstitution of Atg8
conjugation essential for autophagy . J. Bioi. Chem. 279,
40584-92 (2004)
Suzuki K, Noda T, Ohsumi Y., Interre latio nships among Atg
proteins during autoph agy in Saccharomyces cerevisiae.
Yeast 21 , 1057-65 (2004)
Yoshimoto K, H anaoka H, Sato S, Kato T, Tabata S, Noda T,
Ohsumi Y., Processi ng of ATG8s, ubiquitin-li ke prote ins,
and their deconjugation by ATG4s are essentia l for plant
autophagy. Plant Cel/ 16, 2967-83 (2 004)
Kuma A, Hatano M , Matsui M, Yamamoto A, Nakaya H,
Figure 4 . atg mutant is defective in accum ul ation of
au tophagic bodies and exhibited early senescencephenotype(A) Electron micrographs of wild-type roots treated withconca namycin A.
(B) Electron mi crographs of atg4a4b-l mutant roots treatedwith concanamycin A.
Roots of one-week-old wild-type and atg4a4b-l seedlings
were treated with concanamyc in A ( 1 ~lM ) under
nitrogen-starved conditions for 6 h. The same reg ions of
the root tip were compared. Bars = 5 f.ll11(C) 7-week-old wi ld-type plant(D) 7-week-old atg4a4b-l plant

Publication List:
Mizushima, N., Methods for monitorin g autophagy. Int. J.
Biochem. Cell Bioi. 36 , 2491 -2502 (2004)

Yoshimori T, Ohsumi Y, Tokuh isa T , Mi zushima N. The
ro le of autophagy during the earl y neonata l starvati on
period. Natu re in press (2004)
Ano Y, Hattori T, Oku M, Mukaiyama H, Baba M , Ohsumi
Y, KatoN, Sakai Y ., A Sorting Nexin PpAtg24 Regul ates
Vacuo lar Membrane Dynami cs during Pexophagy via
Binding to Phosphatidylinosito l-3-Phosphate. Mol Bioi.

Cell in press (2004)
Shimazu M, Sekito T, Akiyama K, Ohsumi Y, Kakinuma Y.
A fa mil y of basic amino acid transporters of the vacuo larmembrane from Saccharomyces cerevisiae. J Bioi. Chem. ,in press (2004)
Hamasak i M, Noda T, Baba M, Ohsumi Y ., Starvation
Triggers the D eli ve1y of the Endoplasmic Reticulum to the
Vacuole via Autophagy in Yeast. Traffic

6, 56-65 (2005)

Mukaiyama H, Baba M, Osumi M, Aoyag i S, Kato N ,Ohswni Y, Sakai Y., Modification of a ubiquitin-like

Reviewprotein Paz2 conducted micropexophagy throughfo rmation of a nove l membrane stru cture. Mol. Bioi. Cell
15, 58-70 (2 004)

Ohs umi Y., Cellul ar recycling system- molec ular m echani sm
of autophagy. In Cell Growth, Cold Spring Harbor Press,
pp 412-429 (2004)

Mizushima N, Yamamoto A, Matsui M, Yoshimori T,
Ohsumi Y., In vivo ana lysis of autophagy in response tonutri ent starvation using tran sgeni c mice expressi ng afluorescent autophagosome marker. Mol. Bioi. Cell 15,
11 01-11 (2004)

Ohsumi Y. , Mizuhs ima N. , Two ubiquitin-like conjugation
systems essenti al for autophagy. Semin. Cell Dev. Bioi. IS,
231 -6 (2004)
Kamada, Y., Sekito,T., Ohsumi Y., Autophgy in yeast: a
TOR-med iated respo nse to nutri ent starvation . Curr Top
Microbial !mmunol. 279, 73-84 (2004)

Onodera J, Ohsumi Y. , A ld6p is a prefened target for
autophagy in yeast, Saccharomyces cerevisiae. J. Bioi.
Chem. 279, 1607 1-6 (2004)
Kabeya Y, Mizushima N, Yamamoto A, Os hitani-Okamoto S,
O hsum i Y, Yoshimori T., LC3, GABARAP and GATE I6
locali ze to autophagosoma l membrane depend ing on

Ohsumi , Y. and Mi zushima, N. T wo ubiquitin-li ke
conjugation systems essen ti al for auto phagy. Sem. Cell
Dev. Bioi., in press (2004)
Suzuki K. , Ohsumi Y. , A utophagy of yeast as a syste m forselective protein transpo rt to the vac uole. Jikken Jgaku 22,
150-4 . (2004)

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGYLABORATORY OF CYTOSKELETON

Associate Professor:

OGAWA, Kazuo

I. Dynein family
Dynein family is a group of microtubule-activatedaxonemal and cytoplasmic ATPases that serve to convert
chemical energy into mechanical energy. Axonemal
dynein perfonns flagellar and ciliary movements. In
contrast, cytoplasmic dynein transports organella
precursor such as vesicular membranes in a cytoplasm.
Dynein is a retrograde motor and transports the cargoes tocell center. Kinesin that is also microtubule-based motor
protein is an ortho-grade motor and transports the cargoes
to cell periphery. Dynein and kinesin are different proteinsin terms of primaty structure. However, they may becoupled partners to maintain the even distribution of
motor proteins in a cell. Figure I shows the localiza tion ofaxonemal dynein at the outer arms of sperm axonemes
and cytoplasmic dynein at the mitotic apparatus ofcleaving egg visualized by anti-axonemal dynein(Fragment A) antibodies.

13

to as ATP-binding sites in the midregion of the molecules.
The NH 2-terminal one-thirds corresponding to the stem
and may participate in the targeting of dynein to the cargo.
The probable function of the COOH-terminal one-thirds
has been unknown for a while, although our previous data
had shown that the middle one-thirds and the
COOH-terminal one-thirds make larger domain by theirintra-molecular interaction con·esponding to Fragment A
or motor. Yeast genome project have proposed for the new
family of AAA-proteins. Taking the novel idea of
AAA-protein module into consideration, there may be
two AAA-modules at the COOH-terminal one-thirds andfour AAA-modules at the midregion. As a consequence,dynein HCs may be a hexamer of an AAA-module asshown in Figure 2 (Vale 2000).
There are two, interrupted hydrophobic heptapeptide
repeats between the midregion and the COOH terminal
region, forming extended flexible structure, stalk. Stalkmay bind to microtubule in an ATP-dependent manner.

NH2

COOH

Figure 2. A hexamer model of an AAA-module for dynein
HCs.
Figure I. Dynein family. Axoneme (left) and Cleaving egg(right).

IV.ICsII. Complex structure of dynein
Dynein is a very large protein and has molecular mass
up to I to 2 mega Da. It is complex protein consisted ofheavy chains (HCs), intermediate chains (ICs), and lightchains (LCs) classified by their molecular weights.
Cytoplasmic dynein contains light-intermediate chains(LTCs) in addition to these polypeptides.

Outer arm dynein contains three intermediate chains(IC I, IC2, and IC3) that range in molecu lar mass from 70
to 120 kDa. IC2 and IC3 belong to the WD-family.
WD-containing ICs have been found in cytoplasmic
dynein . By contrast, IC I is a hybrid protein such that
the N-terminal part is homologous to the sequence ofthioredoxin and the middle part consists of three repetitive
sequences homologous to the sequence of NDP kinase.
The IC !-related proteins have been found in distantly
related species but cytoplasmic dynein may not contain it.

III. HCs
Outer arm dynein consists of two heavy chains with
ATPase activity. The motor activity is closely re lated tothese polypeptides. Dyne in is composed of three domains:
stem, motor, and stalk. The first successful molecularcloning of this huge polypeptide (520 kDa) was
performed in 1991. Since then eDNA c lones for axonemal
and cytoplasmic dyne ins from a va ri ety of organisms havebeen isolated and seq uenced. The sequences of HCs
contain, without exception , four P-loop motives referred

V.LCs
Six light chains with molecular masses of 23 .2, 20.8,
12.3, 11.5, 10.4, and 9.3 kDa are closely associating withouter arm dynein. We have a lready isolated eDNA clones
of five LCs.
Transmission ratio distortion is a dramatic exampl e ofnon-Mendelian transmission. In mice, !-haplotype males
produce dysfunctiona l +-sperm and normal /-sperm,

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

14

leading to transrmss10n in favor of t-sperm. Genetic
studies have indicated that the !-complex responder locus,
Tcr, rescues t-sperm but not +-sperm from defective
products of !-complex distorter loci, Tcds. LCl and LC3from sea urchin spem1 outer arm dynein have sequencesimilarities to Tctex2 and Tectex 1, respectively, both ofwhich are wi ld-type products of Tcds . We showed that
LC l and LC3 are ab le to make a 1:1 complex . Since Tcr
is a member of the Smok (sperm motility kinase) family(Henmann et al. , 1999) and LC I is phosphorylated at theactivation of sperm motility in a c-AMP-dependent
manner, this complex in a dynein motor molecule might
be a diTect target of Smok!Tcr kinase in a signal cascadethat regulates sperm motility. Thus, we designated it as
Smoac (sperm motility complex) .
Table I shows the polypeptide composition contained inouter arm dynein of sea urchin sperm flagella. Twopolypeptides, a-HC and LC5 have not yet been cloned.
Table I. Molecular compositions of outer arm dynein fi·om
sea urchin sperm axo nemes cloned in our laboratory.
ChainsAccession number

a -HC
P-HC
IC1
IC2
IC3
LC1
LC2
LC3
LC4
LC5
LC6

Not cloned
D01021
D63884
D38538
D28863
BAA24185
BAA24184
AB004251
BAA24152
Not cloned
AB004830

Calculated
molecular weight
51 1774.95
91622.50
79137.0268223.32
20325.81
22201.21
12535.95
12458.90

may be different targeting mechanism for dynein fami ly
to specific sites of the cargoes. Outer dynein arm is
positioned at just 24 nm interval along axis of axonemes.
The conect positioning of outer arm onto the A-subfibers
is thought to be due to the outer dynein ann-docking
complex (ODA-DC) that could link up end-to-end withsome overlap, to form a filament with a 24 nm repeat
stmcture (Kamiya, 2002).
When Triton-model sperm were exposed to hardcondition such as a high salt solution, outer arm was
detached from the A-subfibers. Extracted sperm withoutouter dynein arm swim with a beat frequency of control
sperm. Rebinding of outer dynein arm onto the
A-subfibers was possible by remjxing the extracted
Triton-sperm with the extract under a low salt condition.
Recovered Triton-model spenn swam with a normal beatfrequency (Gibbons and Gibbons, 1976). Thus, a high salt
extract may containing some proteins necessary forcorrect positioning of outer dynein arm onto the
A-subfibers. During the course of characterizing proteins
containing in it, we found a novel protein with molecularmass of 58 kDa designated as ap58. Inununo-electron
microscopy using antibodies raised against recombinant
ap58 shows that gold-particles are found at 25 nm repeat
along the length of axoneme coi nciding with the repeat ofouter dynein ann. Thus, we conclude that ap58 is binding
to in situ outer dynein arm.

10325.67

VI. Targeting of dynein to the cargo
The mechanism how dynein targets the cargoes has
been gradually made clear in terms of molecules
participated in. In cytoplasmic dynein, less characterized
IC and LIC that are closely associated with stem ofdynein are thought to target the cargoes. They are linked
with dynactin complex
consisted of at least ten
polypeptides. It is via dynactin complex that dynein
targets a receptor of cargo membranes.
In flagellar and ciliary movement, outer and irmer
dynein anns are projected from the A-subfibers ofperipheral
doub letmicrotubulesofaxonemecorrespond ing to the cargo. They bind to the B-subfibers.
They bind to the B-subfiber of neighbouring doublet
microtubu les in an ATP-dependent manner. Under thephysiological condition, in contrast to cytoplasmic dynein,
axonemal dynein is hardly detached from the A-subfibers
being the cargo. In this point, axonemal dynein are
different from cytoplasmic dynein in w hich case, thecargoes are thought to be detached from the motor after
arrival to cell center to recruit the cargoes. Thus, there

Figure 3. Localization of ap58 on outer dyne in arm
visualized by immuno-electron microscopy (Ogawa andlnaba)NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

15

LABORATORY OF CELL SOCIOLOGY

Research Associate:

HAMADA , Yoshio

Animal organs consist of several types of cells. They
are organized in an ordered fashion wherein proportion ofeach cell type is maintained constant. The ordered cellarrangement and proportion are built up duringorganogenesis by cell-cell interactions. Since Notch has
been postulated to play a role in cell fate decision by
mediating cell-cell interactions, we are h-ying to find out
cellular and molecular mechanisms working morganogenesis by studying function of Notch.
Organogenesis of the mouse placenta occurs during
early pregnancy, embryonic days 7-9, before theestablishment of molecular transport mechanisms in thedefinitive placenta takes place. Trophoblasts not adjacentto the inner cell mass differentiate into trophoblast giant
cells and lie at the outside, forming interface withmatemal deciduas. The polar trophectoderm gives rise tocells of the chorion, and ectoplacental cone. They produce
labyrinthine and spongiotrophoblast layer, respectively.
While matemal red blood cells begin to perfuse into
trophoblast cell layers and reach at labyrinthine layer by
E9.5 , invasion of embryonic allantoic mesenchyme into
labyrinthine layer and differentiation of fetal red bloodand endothelial cells which line fetal capillary takes placearound E9.5.
The mutation in the ankyrin repeats of mouse Notch2
results in embryonic lethality by emb1-yonic day 11.5 due
to a poor matemal vascular beds formation. The mutant
placenta showed an early invasion of angiogenic allantoic
mesenchyme followed by premature formation of fetal
blood vessels in the mutant placentas as early as E9.0.
However, specification of trophoblast subtypes appeared
not to be drastically disturbed. Thus, in the developing
mouse placenta, Notch2 is unlikely involved in cell fatedecision, but rather participates in a formation of
circulatory systems in the labyrinth layer where theexpression of Notch2 was detected (Fig I). Although,inadequate formation of maternal vascular beds waspartially restored by aggregating mutant diploid embryoswith wild type teteraploid embryos (Fig.2), networks ofthe mutant fetal vasculatures and maternal blood spaces
appeared still comprised in the 4N chimeric placenta.
These results indicate that Notch2 works promotive inmaternal vascular bed formation and prohibitive in fetal
vasculogenesis. Thus, Notch2 is not cell autonomously
required for the early cell fate determination of subtypesof trophoblast cells, but plays an indispensable role incoordinated matemal and fetal vasculogenesis in thedeveloping mouse placenta.

Figure I. Expression of Notch2 gene in developing mouseplacenta. A cryo-section of Notch2+!LacZ placenta at E I 0.0
was incubated in X-gal staining solution. Trophoblast inmaternal vascular bed side expresses the gene, but not inallantoic mesenchyme (AM) side in labyrinth layer (LB).
The expression also occurs in the mesenchyme. Enucleatedmaternal red blood cells are indicated by red arrow heads.
Nucleated fetal red blood cells are by blue arrow heads.

B
A

Figure. 2. Survi val of Notch2 mutant embryo in a chimerawith teteraploid wild type embryo. The mutant obtained in
the intercrosses by the heterozyotic mice showsdevelopmental retardation at E I0.5 (A) and die before
Ell.5. The mutant is able to survive in a chimera with wild
type tetraploid embryo until El3.5 (B).

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVE LOPMENTAL BIOLOGY16

DIVISION OF REPRODUCTIVE BIOLOGY

Professor:
Associate Professor:
Research Associates:
Technical Stafl
JSPS Postdoctoral Fellows:

Postdoctoral Fellows :Graduate Students:

Visiting Scientist:
Technical staffs:

Secretary:

NAGAHAMA , Yoshitaka
YOSHIKUNI, Michiyasu
OKUBO, Kataaki
OHNO, Kaoru
KOBAYASHI, Hiroko
OHTA, Kohei
YI, Mei-Sheng
BHANDARI, Ramji
CHA UBE, Radha
MATSUDA, Masaru 2J
IJJRI, Shigeho IJ
SAKAI, Fumie I}
GUAN, Guijun IJ
SHIBATA, Yasushi IJ
WANG, De-Shou IJ
LAU, En-Lieng
PAUL, Bindhu
OHMURO-MATSUYAMA , Yuki 3J
KURITA, Kayoko 3)
ZHOU, Lin-Yan 3J
TANAKA , Masaki
HAYAKAWA , Rie IJ
UNO, Eri I)
HARA, Ikuyo IJ
SUGITA, Chiyoko
TAKAKI, Chikako
SHIMADA, Yu IJ

IJ CREST, JST; 2J PREST, JST;
3)

Graduate University for Advanced Studies

Our research focuses on (1) the identification ofregulators and steroidal mediators involved in sex
determination,
gonadalsexdifferentiation
andgametogenesis, and (2) the mechanisms of synthesis andaction of these mediators.

I. Sex-determining gene
Medaka possesses a stable genetic XX/XY sex
determining system. Using positional cloning and detailed
sequence analysis of BAC clones by shotgun sequencing,
we identified DMY (DM domain gene on the Y
clu·omosome) as a strong candidate for the
sex-determining gene of medaka. DMY encodes a proteinof 267 amino acids including the highl y conserved DM
domain. The DM domain was named after a related DNA
binding motif found in two proteins, dsx and mab-3,
involved in sexual development in D rosophila and C.
elegans, respectively. Our loss- and gain-of-function
studies indicate that DMY is the sex-determining gene of
medaka. DMY provides the first example of asex-determining gene in non-mammalian vertebrates (Fig.
I). A phylogenetic tree based on the amino acid sequence
including the OM-domain shows that DMY was derived
from DMRTI immediately before speciation of 0. /atipes
and 0. curvinotus.
DMY mRNA and protein are expressed specifically inthe somatic cells surrounding primordial germ cells(PGCs) in the early gonadal primordium, before
morphological sex differences are seen. However, somatic
cells sunounding PGCs never express DMY during theearly migratory period. Expression of DMY persists in
Serioli cells, from PGC-supporting cells to Sertoli cells,

indicating that only DMY positive cells enclose PGCs
during mitotic arrest after hatching.
Medaka sex chromosome

Fig. 1 Comparison of two known sex-determinin g genes invertebrates, SRYIS1y in mammal s and DMY in medaka.

II.

Endocrinedifferentiation

regulationofgonadalsex

Nile tilapia, Oreochromis niloticus, is an excellent
example of the precise nature of steroidogenic actions
during gonadal sex differentiation. In this fish, all genetic
female (XX) or male (XY) broods can be obtained byartificial fertilization of normal eggs (XX) andsex-reversed, pseudo male sperm (XX), or normal eggs
(XX) and super male sperm (YY), respectively. In all
genetic female tilapia, steroid-producing cells in ovaries
prior to and during sex differentiation express all of thesteroidogenic enzymes required for estrogen production.
Estrogen receptors a and ~ first appear in XX gonads
prior to sex differentiation. The treatment of XX fry withfadrozole (aromatase inhibitor) or tamoxifen (estrogen
receptor antagonist) caused complete sex reversal tofunctional males. These results, together with evidence
that the XX sex reversal induced by fadrozole was
rescued completely with simultaneous estrogen treatment,suggest that endogenous estrogens are required forovarian differentiation in tilapia. In contrast to XX fry,
steroid-producing cells appear to differentiate after
gonadal differentiation in XY fry. The DMRTJ gene isexpressed male-specifically in testicular Sertoli cells
during sex differentiation. Application of DMRTJ
morpholino antisense oligonucleotides (DMRTJ knockdown) blocked androgen-induced masculinization of XX
gonads. These results suggest an important role ofDMRTJ in testicular differentiation in tilapia.
To identify the down-stream gene products of estrogen
during ovarian differentiation, we have performed
subtraction hybridization using mRNA derived fromnormal and estrogen treated XY gonads of tilapia.
Interestingly, one of the up-regulated gene products wasaromatase, indicating that endogenous aromatase activity
is up-regulated in XY fry after the induction of XY sex
reversal by estrogen. It is also of interest that one of thedown-regulated gene products was DMRTJ , providing
evidence to support that DMRTJ may function as apivotal gene for male gonadal sex differentiation.

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

III. Sex-changing fish
The goby, Trimma okinawae, can serially change sex ineither direction. Their gonads contain ovarian andtesticular portions simultaneously; however, individuals
typically produce one gamete type, change sex and thenproduce the other type. They do not produce mature
sperm and oocytes at the same time. Thus, T okinawaeprovides an ideal model for studies on the mechanisms ofsex change. Aquarium experiments were carried out in thelaboratory. Two males (M+M) or two females (F+F) were
kept in separate tanks. In the M+M aquarium, the smaller
male changed its sex to female. On the contrary, the larger
female changed its sex to male in the F+F aquarium.
Behavioral changes occurred within 30 minutes of social
manipulation. After pairing, the larger male or female
attacked the smaller fish, which fled and often hid in anest. After 30 min, however, the larger fish began to court
the smaller fish. These results suggest that the sex ofbrain/behavior changes quickly and easily, regardless of
gonadal sex. Brain sex of sequential hermaphrodite might
be determined independent of gonadal effect and were
induced only by social cues.

fV. Embryonic development
releasing hormone neuronsof gonadotropin-

Appropriate development of forebrain gonadotropinreleasing hormone (GnRH) neurons is essential forestablishing and maintaining reproducti ve competence,
yet the mechanisms underlying this process are poorly
defined . We established transgenic medaka lines that
expressed GFP under the control of the gnrhl and gnrh3
promoters, and they allowed prolonged in vivo imaging of
GnRH neuronal behavior at any stage of development In anoninvasive manner (Fig. 2). Our images revealed thatpreoptic gnrh 1 neurons and terminal nerve ganglion
onrh3 neurons originate in the olfactory region, whi le the~nedial basal telencephalic gnrhl neurons arise from therostral telencephalon. We also found that the diencephalicneuronal population transiently expresses gnrh I during
embryogenesis, that the trigeminal ganglion neurons
express gnrh3 , and that gnrh3 is maternally expressed 111
oocytes and embryos. Taken together, the transgemc
medaka provides a useful model system for studymg
GnRH neuronal development including disorders of
GnRH deficiency.

Fig. 2 Transgenic medaka e mbryos that ex press GFP under
the control of gnrh I (A) a nd gnrh3 (B) promoters.

V. Endocrine regulation of spermatogenesis
Using an organ culture system for ee l, Anguillajaponica, testes consisting of spermatogonia and inactive

17

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 Se1toli cells to stimulate theproduction of activin B. To clarify the mechanism of theinitiation and progression of spermatogenesis, we
examined the expression pattem of A-type cyclins andDmcl during gonadotropin-induced spermatogenesis ineel. Expression of cyclin A2 increased I day after the
induction of spermatogenesis and reached a plateau after
6 days when many type B spennatogonia were found. In
contrast, the expression of cyclin Al mRNA was detected
after 9 days coincident with the first appearance of
spermatocytes. Cyclin Al mRNA was localized in germ
cells of all stages from primary spermatocytes to roundspermatids, whereas cyclin A2 mRNA was spec ifically
localized in spermatogonia, secondary spermatocytes,
round spermatids, and testicular somatic cell s, includi ng
Sertoli cells. Dmc I protein was localized only in theearli er stages of primary spermatocytes; prior to this stage
cyclin A I mRNA was not detectable. These results
suggest that cyclin A2, Dmcl , and cyclin Al are
expressed in spermatogenic cells sequentially before andduring meiosis, indicating that the entry of spennatogonia
into meiotic prophase can be distinguished by Dmcl
protein and cyclin A I mRNA expression.

VI. Endocrine regulation of oocyte growth andmaturation
Two major steroidal mediators, estradiol-l 7P and 17a,
20P-dihydroxy-4-pregnen-3-one ( 17a, 20P-DP) are synthesized by ovarian follicles during oocyte growth andfin al maturation in response to FSH and LH, respecti vely.
Two cell -type models in which the thecal layer provides
precursor steroids to the granulosa layer, have been
demonstrated for estradiol-1 7P and 17a, 20P-D P
production. Ad4BP/SF-l serves as a transcriptional
regulator for ovarian cytochrome P450 arornatase(P450arom) which converts testosterone to estradiol-1 7P
in granu losa cells during vitellogenesis. LH induces adistinct shift in steroidogenesis, i.e. from estradiol-17P to
17a, 20P-DP as well as the steroidogenic enzyme genes
from P450arom to 20p-hydroxysteroid dehydrogenase(20P-HSD), in the granu losa layers of ovarian follicles
prior to oocyte maturation. The triggering of thesteroidogen ic shift by LH is manifested through two
molecul ar mechanisms, the first being the subjugation of
the expression of Ad4BP/SF-l vis a vis P450arom, andthe second, the induction of over-expression of 20P-HSD
probably via CREB.
Un li ke estradiol-1 7P (genomic action), 17a, 20P-DP
binds to a novel, G-protein-coupled membrane receptor(non-genomic action) (Fig. 3), leading to the de novo
synthesis of cyclin B, the regulatory component of
maturation-promoting factor (MPF), which activates apreexisting 35-kDa cdc2 kinase via phosphorylation of its
threonine 161 by a threonine kinase, thus producing the
34 kDa active cdc2. Upon egg activation, MPF isNATIONAL fNSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

18

inactivated by degradation of cydin B. We showed thatthe 26S proteasome initiates cyclin B degradation throughthe first cut of its NHz terminus at lysine 57.

LH

DiethylstHbestrol (DES)

Oocyte maturation

Fig. 3 Hormonal regulation of meiotic maturation in fish
oocytes. Three major mediators, LH (pituitary),
l7a ,20~ -0P (ovarian follicle) and MPF (oocyte), areinvolved. Both 17a ,20~ -DP and an endocrine-disrupting
chemical, DES, may interact with a novel membraneprogestin receptor.
An endocrine-disrupting chemical, diethylstilbestrol(DES), a nonsteroidal estrogen, triggers oocyte maturationin fish. The morphology (the time course of the change ingerminal vesicle breakdown) and an intracellularmolecular event (the de novo synthesis of cyclin B)
induced by DES were indistinguishable from thoseinduced by 17a, 20~-DP. A synergistic action of DES on
l7a., 20~ -DP-indu ced oocyte maturation was observed.
Both l7a., 20~ -DP- and DES-induced oocyte maturation
was inhibited by an antibody against the l7a., 20~-DP
receptor. These results suggest that D E S may act on thel7a., 20~-DP receptor as an agonist of 17a., 20~ -DP (Fig.

3).

Publication List:
Horiguchi, R., Tokumoto, M. , Nagahama, Y. and Tokumoto,T. (2005). Molecular cloning and expression of eDNA
coding four spliced isoforms of casein kinase Ia in
goldfish oocytes. Biochim. Biophys. Acta 1727, 75-80.
Horiguchi , R., Yoshikuni, M. , Tokumoto, M., Nagahama, Y.
and Tokumoto, T. (2004). Identification of a protein
kinase which phosphorylates a subunit of the 26S
proteasome and changes in its activity during meiotic cell
cycle in goldfish oocytes. Cell. Signal. 17, 205-215.
Kita, M., Nakamura, Y ., Okumura, Y., Ohdachi, S.D., Oba,Y., Yoshikuni, M., Kido H. and Uemura, 0. (2004).
Blarina toxin, a mammalian lethal nenom from theshort-tai led shrew Blarina brevicauda: Isolation an dcharacterization . Proc. Nat!. Acad . Sc i. USA 101 ,
7542-7547 .
Kobayashi-Kajiura, H. , Kobayashi , T. and Nagahama, Y.
(2004). The cloning of cyclin B3 and its gene expression
during hormonally induced spermatogenesis in the teleost,Anguilla japonica. Biochem. Biophys. Res. Commun. 323,

288-292.
Kobayashi-Kajiura, H., Kobayashi, T. and Nagahama, Y.
(2004). Cloning of cON As and the differential expressionof A-type cyclins and Dmcl during spennatogenesis in theJapanese eel, a teleost fish. Dev. Dyn. (in press).
Kobayashi , T., Kobayashi, H. and Nagahama, Y. (2004).
Two DM domain genes, DMY and DMRTJ, invo lved in
testicular differenti ation and development in the medaka,
01yzias talipes. Dev. Dyn . 231, 518-526.
Kobayashi, Y., Kobayashi , T., Nakamura, M., Sunobe, T. ,Money, C.E., Suzuki, N. and Nagahama, Y. (2004) .
Characterization of two types of cytochrome P450
aromatase in the serial-sex changing gobiid fish, Trimma
okinawae. Zool. Sci. 21 , 417-425 .
Mita, M., Oka, H. , Thorndyke, M.C., Shibata, Y. , Yoshikuni,
M. and Nagahama, Y. (2004). Inhibit01y effect of a
SALMFamide neuropeptide on secretion of gonadstimulating substance from radial nerves in the starfi sh
Asterina pectinifera. Zoo!. Sci . 21 , 299-303.
Nagahama, Y., Nakamura, M., Kitano, T. and Tokumoto, T.
(2004). Sexual plasticity in fish: A possible target of
endocrine disruptor action. Environ. Sci. 11, 73-82.
Senthilkumaran, B. and Nagahama, Y. (2004). A shi ft In
steroidogenesis occuning in ovarian follicles prior tooocyte maturation . Mol. Cell. Endocrinol. 215, 11-18.
Sunobe, T., Nakamura, M. , Kobayashi, Y. , Kobayashi , T.
and Nagahama, Y. (2004) . Gonadal structure and P450scc
and 3~-HSD immunoreactivity in the gobiid fish Trimma
okinawae during bi-directional sex change. Ichthyol. Res.
52 (in press).
Suzuki, A., Tanama, M., Nagahama, Y. and Shibata, N.
(2004). Expression of aromatase mRNA and effects ofaromatase inhibitor during ovarian development in the
medaka, 01yzias latipes. J. Exp. Zool. 301A, 266-273 .
Tokumoto, T., Tokumoto, M., Horiguchi, R. , Ishikawa, K.
and Nagahama, Y. (2004). Diethylstilbestrol induces fish
oocyte maturation . Proc. Nat!. Acad. Sci. USA 101,
3686-3690.
Wakata, Y., Tokumoto, M., Horiguchi , R., Ishikawa, K.,
Nagahama, Y. and Tokumoto, T. (2004). Identification ofa-type subunits of the Xenopus 20S proteasome andanalysis of their changes during the meiotic cell cycle.
BMC Biochem. 5, 18-25.
Wang, D.S., Zhou, L.Y., Kobayashi , T. and Nagahama, Y.
(2004). Molecular cloning and gene expression ofFoxl2 in
the Nile tilapia, Oreochromis niloticus. Biochem. Biophys.
Res. Comm. 320, 83 -89.

19

NATI ONA L INSTIT UTE FOR BAS IC BIOLOGYDEVE LOPMENTAL BIOLOGYDIVISION FOR SEX DIFFERENTIATION
Professor:
Research Associates:
Technical Staff:
Postdoctoral Fellows:

Graduate Students:

Technical Assistants:

Secretmy :MOROHASHI, Ken-ichirou
FUKUI, Yuko
OGAWA, !-!idesato
OKA, Sanae
TSUCHIYA , MeffL1111i
BA BA, Takashi !
MOHAMA D, Zubair 2!
KUSAKA, Masalomo 31
KOMATSU, Tomoko 31
MA TSUYAMA , Makoto 3!
FA TCHI YA H 3J
SA TO, Yuko 3!
MIYA BAYASHJ, Kanako 4!
OWAKI, Akiko
JSHIKA WA , Azusa
KOIKE, Yukari
GOUNO, Hiroe
TOMIDA, Sanai
SUGI URA , Mia 2!

'! JSPS Postdoctral Fello w
21
SORST, JST
31 Graduate University fo r Advanced Researchmechani sms that supports sex differentiation of the go nad.
Tentati vely, we hypothesize that the sexuallyindiffe rent gonads determine their sexes under the controlof two oppos ite signals, one of which is the signal fo r
testi cular differentiati on and the other is the signal fo r
ova rian differentiation (Fig. I). Probably the nature of thesignals might be a transcriptional acti vity of a certaintranscription fac tor expressed in the fetal gonads, orotherwise mi ght be activity of a certai n type of growth
factor. The study in this di vision has been performed
mainly from the aspect of the transcriptional contTO! as
the signal for the sex diffe rentiation. As indicated in F ig. I,
a number of transcription fac tors/DNA binding protei ns
are expressed in the developing gonads. Based on thesymptoms of human pati ents and the phenotypes of geneknockout mice, Ad4BPISF-l , E mx2 , and Wtl are
indispensable for the go nada l development. With respect
to the signa ls for gonadal sex differe ntiation, Sry , M33 ,Arx, Dhh , Sox 9, Pdgf, Wt I , and Fg/9 are ass umed to act as
the testicular signals while Dax-1 and Wn t4 are theovarian signals.
Indifferent Gonads(Bipotential)

4! Graduate School ofTohoku University

Although sexua l dimorphism mani fes ts most obviously
in the go nads (testi s and ovary), it can be observed in theother parts of the entire anima l body. For instance, it iswell kn own that several ti ssues such as the externa lgenitalia, muscle, and bra in exhibi t sexual dimorphisms interms of their structures and fun ctions. Thi s process of sex
differe ntiation can be di vided into three steps. The fi rst
occurs at fertili zati on, during which the sexes of fe rtili zed
eggs are determined genetically by combination of sex
chromoso mes . As the second step, the indi viduals
cany ing XY and XX sex chromosomes deve lop the testisand ovary, respecti vely, in the case of mammals. Sex
diffe rentiati on of the gonads usually proceeds during fetal
stages. Thereafter, the gonads control the sexes of thewhole body through the function of sex steroids
synthesized in the sexually diffe renti ated gonads.
Therefore, the gonadal sexes are quite important fo r sex
diffe renti ation of the animals.
It has been establi shed that a number of transcripti on
facto rs play crucial ro les in the process of go nada ldiffe rentiation. Some of these factors, such as SRY, WT I,
DAX- 1, SOX9 and ARX were identified as the productsof genes responsible for human di seases that disp lay
structural and functi onal defects in several tissues
including the gonads. T he functions of the othertranscription fac tors such as Ad4BP/SF-l , Emx2, M33 ,and Lhx9 were elucidated by the phenotypes of the genedisrupted mice. In add ition, the expression profi les interms of their gonadal distribution and sexual dimorphism
strongly suggested the functional si gnificance at the earlystage of gonada l differentiation. However, it remains to beclarifie d ho w the transcription factors above regulate theirtarget gene transcription and how the genes encoding the
tra nscri pti on factors are regu lated by upstream regulators.
Studies fro m above two directions are qui te important todefi ne the gene regulato ry cascade and the molecular·

S1gnal for TesticularDifferentiation

~

I. .,

.t.

~-v-~=;;;c====.

,___zs______,,-------~--~/'--, Ad4BP/ SF-1 r--"-.
'-.,---' Emx2 Wtl '---;/

n. ,[} •

Sry

1133

Arx

Dhh

~
Testis

,[}

~

~

•

,--~-------,

zs:=

Signal for Ovarian
Differentiationzs:=

Sox9 Pdgf
IYtl Fgf9

Dax 1 1Ynt4

q

Fig. I, Gonda! sexes are determin ed by balance of signals
fo r testicular and ovari an di ffe renti ation.

I. Function al analyses of Vinexin
In order to defi ne the function of Ad4BP/SF-1 , we havescreened a two-hybrid Iibrary prepared form mouseembryonic go nads. Yinex in was one of the clones isolatedby the screening. Two isoforms of Yinex in , Yin ex in a and~ , had previously been identified, however, the clone
iso lated in thi s study was distinct fro m those two isoforms
in the 5 ' amino ac id coding region. Thus, thi s nove lisofo rm was tentati vely designated Yinex in y. Structura lly,
Yinexin y is composed of 680 amino acids CO!Tesponding
to the 57th to 736th amino acids of Yinexin a (76 kD).
Analyses of the gene structure indicated that thi sdiffe rence is due to alternati ve usage of the first exon. In
silico screening revealed the presence of Yinexin y inother anima l species; hu man SCA M- I (SH3 -Containing
Adaptor Molecu le- !) and rat SCAM- I. In situ
hybrid ization and immunohistochemical studies showedthat Vinexin y started to be expressed in the sexauall yindiffe rent go nads (E ll.5), and thereafte r expressed
specifica lly in Se1to li cell s in the testis (Fig. 2).
For the function of Yinexin , it has been discussed that

20

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

the molecule is involved in MAPK signaling throughinteracting with ERK. Thus, we investigated whether
Vinexin y interacts with the components of MAPK
cascade. Interestingly Vincxin y was found to interact
with c-Raf as well as ERK, suggesting that Vinexin y acts
as a scaffold protein. Moreover, our data indicated that
Vinexin y activates MEK and ERK through interaction
with c-Raf and ERK. Ultimately in culture cell study, it
was revealed that Sox9 transcription was induced byVinexin y. This up-regulation of Sox9 expression
disappeared when the cells were treated with a specific
MEK inhibitor.

E11.5 MaleE12.5 MaleE11.5 Female

Vinexin ~ , it is unlikely that the function of Vinexin ~ iscompletely redundant with that of Vinexin y. Secondly,
the function s of two closely related proteins, Arg-binding
protein 2 (ArgBP2) and c-Cbl-associated protein(CAP/ponsin/SH3P 12), should be noted. Structurally,
besides the presence of three SH3 domains in common attheir C-termini, these proteins share a sorbin domain with
Vinexin y in their N-tenninal halves. Moreover,expression of ArgBP2 and CAP/pons in/SH3P 12 wasdetected in the fetal gonads (E 12.5) of both sexes by
RT-PCR and whole-mount in situ hybridization, althoughthese signals were weak. Thus, it might be possible that
their features functionally and spatially overlapped with
Vinexin y failed to display drastic gonadal defects in theVinexin y-disrupted mice. Taken together, Vinexin yseems to be implicated in regulation of Sox9 gene
expression by modulating MAPK cascade in mouse fetal
gonads.

+I-;Sox9+t-;a[iHso-/-;Sox9-/-;:J~HSD

E12.5 Female

Fig. 2, Expression of Yinexin y in the developing mouse
gonads. Vinexin y is expressed in the sexually indiffe rent
gonads (E11.5) of both sexes (Male and Female). The
ex pression continues at El2.5 gonads. In the male gonad,the expression of Yinexin y is clearly restricted in Sertoli
cell.
In order to reveal the function of Vinexin y during
gonadal development, we generated a Vinexin y-specificgene-disrupted mouse. Unexpectedly, the homozygous
mutant mice of both sexes were fertile withmorphologically normal gonads. The mice showeddecreased Sox9 expression, and the decreased levels
varied among individuals. Indeed, even in the mostaffected individuals, we failed to detect either complete
disappearance of Sox9 expression or developmental
defects in the XY gonad. Since other MAPKs, INK and
p38, were not activated ectopically in the Vinexin / - fetal
XY gonad, it is unlikely that the Vinex in y-dependentfunction of ERK was displaced by the other MAPKs.
Together, the moderate phenotype displayed by theVinexin i '- gonad suggested that MAPK activation is notcritical for testis formation . Consistent with ourobservations, it was recently reported that a MAP kinase
inhibitor did not inhibit testis cord formation in gonadalorgan culture.
Altematively, the relatively moderate phenotype
displayed by the Vinexin y-disrupted mice might be due tothe following two poss ibilities. Firstly, Vinexin a andVinexin ~ might function redundantly. Indeed, these two
isoforms share three tandem repeats of SH3 domains, andmoreover, Vinexin ~ was still evident in the gonad of
Vinexin y-dis rupted mice. However, given that Vinexin ycontains its specific N-terminal ha lf and the express ion in
the fetal testis does not appear to overlap with that ofFig. 3, Expression of Sox9 and 3~HSD in Yinexin yknockout mice. The developing gonads (E 12.5) of theknockout(-/-) and heterozygous(+/-) mice were subjected
to in situ hybridization probed with Sox9 (Sertoli cel lmarker gene) and 3 ~-h ydro xys teroid dehydrogenase(3 ~HSD) (Leydig cell marker gene). Representative
decreased expression patters are shown for Sox9 in theknockout. The expression of3~HSD in Leydig cells se<;<med
not to be affected by Yinexin y knockout.

II. Characterization of VMH-specific enhancer of
Ad4BP/SF-1 gene
It has been generally accepted that genes are divided
into two types according to their expression profiles.
Firstly, there are the so-called house-keeping genes withubiquitous expression among tissues and constitutive
expression during developmental processes. In contrast,
other genes display tissue-specific and/or developmental
stage-specific expression. Many genes have been revealed
to contain enhancer sequences, which may explain themolecular basis of regulation of these spatially andtemporally restricted-expression.
Ad4BP/SF-l is a transcription factor essential foranimal reproduction . Based on the phenotypes observedin gene-disrupted mice, Ad4BPISF-l is thought to beof
theinvolvedinestablishment
hypothalamic-pituitary-gonadal axis. However, themechanisms underlying tissue-specifi c expression of
Ad4BPISF-l are largely unknown. We have investigatedthe cis-regulatory regions of the mouse Ad4BPISF-l geneby transgenic mouse assays, and recently we identified afeta l adrenocortical-specific, pituitary-specific, andNATIONA L INSTITUTE FOR BAS IC BI OLOGYDEVELOPM ENTAL BIOLOGY
ventromed ial hypoth a lami c nucl eus (VMH)-specifi cenhancers. In th e present study, we identified a
VMH-specific enhancer in intro n 6 of the Ad4BPISF- lgene. Functiona ll y, the e nh ancer was characterized bydriving VMH-specific expression of the lacZ repo rter
gene w hen the enhancer was loca lized w ith th e basa lpromoter of Ad4BPISF-l . Moreover, th e VMH-specificexpression was a lso reproduced w ith the hsp68 basa lpromote r, indicati ng th at th e VMH-specific express ion
can be dr iven primaril y by this enhancer. Struch1rally, th esequence is conserved a mon g anima l species (mouse,
human, and chi c ken), thus strong ly indi cating th at th esequ ences
probably
functionasaconserved
VMH-specific enhancer beyond anim a l species.

E11 .5/Ad4BP

E11.5/LacZ

'

.....

'

21

phenoty pes of the gene-disrupted mi ce pro mpted us toanalyze the effect of gene disrupti o n on th e VMH
enhancer functi on . D isa ppointing ly, however, express io nof the lacZ rep orter gene dri ven by the VMH enh ancer
pers isted eve n in the Nkx2. 1 k noc ko ut backgro und,suggestin g that Nkx2. 1 is not essenti al fo r th e enh ancer
fun ction. In this co ntext, it is quite interesting to cons ider
the potenti a l redun dant functi on of anoth er fa mily
member, Nkx2 .4, of w hi c h express io n is overl apped w ith
N kx2. 1 in the developing forebrain . M ea nw hil e,cons iderin g that th e la cZ-positi ve ce ll s in the ventra l
di encepha lo n of the Nkx2.1 knockout mi ce were fewe r in
number th an m the wi ld-type from the earl ydevelo pmental stage of V MH, N kx2. 1 could be partia ll y
in vo lved in the V MH-spec ifi c enhancer functi o n. Tlu·oughth e fun cti on of th e VMH enh ancer, N kx2 . 1 mi ght regul ate
the amount of Ad4BP/ SF-l expressed, and thereby
stimul ate VMH cell p ro life ration . Indeed, it was recently
reported th at ce ll numbers in adrenocorti cal primordium
are tightl y depende nt on the amount of Ad4BP/SF-l .

Publication List:

Adult/Ad4BP

Adult/LacZ

Fig. 4, Comaprison between endogenous Ad4BP/SF-1 andtransgene-derived ex press ion of lacZ. Transgene wasconstructed with a VMH-specific enhancer DNA fragment
and IacZ gene as a reporter. Transgeni c mice at fetal age(E 11 .5) and adult were used for the study. The endogenous
expressionof
Ad4B P/SF-l
wasexamined
immunohistochemica ll y.
F urther detailed tra nsgenic an a lyses of th e VMH
enhancer revealed that th e enhancer co nta ins suppressive
and acti vating ele ments. M utation of the fo rmer e le me nt
resulted in ectopic lacZ repo rter gene expression in anarea dorsal to th e intrinsic express ion dom ain and in th eventri cul a r zone, whil e mutations in th e latter co ntaining
ATTA moti fs led to th e di sappearance of the reporter gene
express ion, suggestin g th e invo lveme nt of ho meobox
prote ins. Brain regiona lizatio n is regul ated primaril y by
the functions of growth fa ctors together with regiona ll y
expressed homeobox proteins. Moreover, it has beenshown th at combined expression of these homeobox
prote ins is essenti a l to establish proge nitor cell identi ty
and neurona l ce ll subtypes durin g fore bra in patterning.
Indeed, several homeobox prote ins are know n to be
expressed in various regions of th e developing brain .
A mong these, it is noteworthy that th e ex pression do mainof Nkx2 .l overlaps w ith that of Ad4BPISF-l until E l 8.5.
Nkx2. 1 is a memb er of the Nkx ho meo protein fa mil y, andplays important ro les in ventra l fo rebrain deve lopme nt. In
particul ar, in Nkx 2.1-null mi ce, the ve ntro medial anddorsomed ial nucle i we re underdeve loped and fused in th em id line. The express ion profi le of Nkx2.1 and th e

Hasegawa, T. , Fukami , M., Sato, N ., Sasaki, G. , Fukutan i, K.,
Moro hashi, K., Ogata, T. (2004) Testicul ar Dysgenesis
without Adrenal Insuffi ciency in a 46,XY Patient with a
Heterozygous Inacti ve Mutation of Steroidogeni c
Factor-I .J. Clin . Endocrinol. Metab. 89, 593 0-5 935 .
Komatsu, T., Mi zusaki, H., M ukai, T. , Ogawa, H., Baba, D.,
Shi rakawa, M ., Hatakeyama, S. , Nakayama, K.,
Yamamoto, H., K ikuchi , A. , Morohashi , K. (2004)
SUMO-I Modificati on of the Synergy Co ntrol Motif of
Ad4B P/SF- l Regul ates Synergisti c Tra nscription between
Ad4 BP/SF-l and Sox9. Mo l. Endocrinol. 18, 245 1-2462 .
Ogawa, H., Yu, R. T. , Haraguchi, T., Hiraoka, Y., Nakatani,Y., Morohashi K., Umesono, K. (2004) N uclear
structure-assoc iated T IF2 interacts with glucocorti coid
receptor and its targe t DNA. B iochem. Biophys. Res
Commun. 320, 2 18-225.
M itsunaga, K., Araki, K. , Mizusaki, H., Morohashi, K.,
Haruna, H., Nakaga ta, N. , Giguere, V. , Yamamura, K.,
Abe K. (2004) Loss of PGC-specific ex pression of theorphan nuc lea r receptor ERR-~ results in regul ati on ofgerm cell number in mouse emb1yos. Mech of Dev. 121,237-246.
Toda, K., Okada, Y., Zubair, M., Morohashi, K., Saibara, T.,
Okada T. (2004) Aromatase-knocko ut mouse carry ing an
estrogen-inducible enhanced green flu orescent protein
gene fac ilitates detection of estrogen actions 111
vivo.Endoc rinol. 145, 1880-1 888 .
Kato, M., Petras, D.S., Kitamura, K. , K., Morohas hi, K.,
Abuelo, D. N. , Barr, M ., Bonneau D., Brady, A. ,Ca rpenter, N.J ., Frisone, F. , Fukuda, T., Guen·ini , R., !ida,
E. , Itoh, M., Lewanda, A. F., Nanba, Y., Oka, A., Proud,
V. K., Russel, K. L. , Saugier-Veber, P., Schell ey, S. L. ,Selicorni , A., Shaner, R., Silengo, M., Stewart, F.,
Sugiyama, N., Toyama J. , Toutain, A. , Vargas, A. L. ,Yanazawa, M. , Zacka i E. H., Dobyns W. B. (2004)
Mutations of ARX are associated with striking pleiotropy
and consistent ge notype-phenotype correlati on. Human
Mutation 23, 147-1 59.

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY22

DIVISION FOR MORPHOGENESIS
Professor:
Associate Professor:
Research Associates:
Technical Staff:
Technical Assistants:

NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:

Secretaries:

I)

UENO, NaotoKINOSHITA, Noriyuki
NAKAMURA, Makoto
TAKAHASHI, Hiraki
TAKAGI, Chiyo
YAMAMOTO, Takamasa
TERASAKA, Chie
MOROKUMA, Yoshie
YAMADA, Shigehiro
HAYASHI, Kazue
SHIBAMOTO, KaoriASANO, Megumi
MIURA-HYODO, Junko
KITAYAMA, Atsushi
MOROKUMA, Junji
HOTTA, Kohji
IIOKA, Hidekazu I)
MIYAKOSHI, Akira IJ
CHUNG, Hyeyoung 21
KUJIRAOKA, Masahiro n
YOSHIKANE, Nami 3!
MAEDA, Masato IJ
GOTO, Masanori I!
LEE, Rebecca I)
MIYAKE, Satoko
TSUGE, Toyoko

Graduate University for Advanced Studies

2! University of Tokyo
3! Nara Institute of Science and Technology

The complex morphogenesis of organisms is achievedbyconsecutive
cell-to-cellinteractions
duringdevelopment. Recent studies suggest that growth factors
play crucial roles in controlling such intercellularcommunications in a variety of organisms. In addition tosecretory factors which trigger intracellular signaling,transcription factors which act in the nucleus to regulate
gene expression are thought to be essential for thedetermination of cell fates. Our main interest is to knowhow pattern formation and morphogenesis during
development is regulated by these growth factors andtranscription factors. We address this problem using
several model animals, including frog, fly and acidian,employing embryology, genetics, molecular and cellularbiology, and biochemistry. In addition, we have recently
introduced genomics technologies to elucidate precise
genetic program controlling early development.

I. Gastrulation
signaling

movement 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.
During Xenopus gastrulation, mesodermal cells migrate to
the inside of the embryo and move on the blastocoel roof.
One of the important mechanisms for this process is thecell movement called "convergent extension". As

convergent extension begins, cells are polarized andaligned mediolaterally, followed by the mutualintercalation of the polarized cells. Recent studies suggestthat several extracellualr signals are essential for thepolarization, one of which is fibroblast growth factor(FGF). We therefore attempted to identify FGF target
genes to understand the molecular mechanism underlying
the FGF function using our Xenopus eDNA microarray.
As a result, we identified a gene encoding a neurotrophin
receptor homolog (NRH) and demonstrated it to be an
essential component of FGF to control protrusive activityof cells undergoing gastrulation.
Another pathway regulating gastrulation is one of theWnt signaling pathways, called Wnt/JNK (c-Jun
N-terminal kinase) pathway is shown to be important for
the regulation of convergent extension. The pathway ishighly conserved among species and initially found to be
essential for the establishment of planar cell polarity(PCP) of Drosophila wing hair.
We have previously demonstrated that Xenopus prickle(Xpk), a Xenopus homologue of a Drosophila PCP gene,is an essential component for gastrulation cell movement.
Subsequently, we identified a protein that binds to XPK
by yeast two-hybrid screening. The XPK binding protein
was found to be a member of the Ste20 kinase family andnamed as Xenopus prickle-interacting kinase, XPIK.
Loss-of-function of XPIK resulted in severe defects in
gasrtulation causing spina bifida. XPIK is not only
sufficient to activate JNK in embryo, but also required forfull activation of JNK by Dishevelled. These suggest that
XPIK also plays an essential role in connecting
extracellular Wnt signal to JNK activation throughDishevelled and XPIK.
In addition, we have been attempting to identify novel
regulatory components controlling gastrulation cell
movements by an expression cloning method based onmorphology of dorsal marginal zone explant (Keller's
explant) and of embryo. After two thousand clones were
examined by overexpression in the dorsal region ofembryos, approximately 5% of clones were found to
perturb normal gastrulation cell movements. Functional
relevance of identified genes to cell movements iscurrently under investigation.

II.

Wnt signaling pathway regulates
cytoskeletal dynamics during gastrulation

actin

The noncanonical Wnt pathway has been implicated in
the regulation of Xenopus gastrulation movements. Lossof function of its signaling components, such as
Dishevelled, Rho GTPases and c-Jun N-terminal kinase,leads to a severe gastrulation defect. We investigate themolecular mechanism of how the Wnt pathway regulates
this morphogenetic process. Convergent extension of thedorsal mesoderm is one of the important mechanisms
during gastrulation. In this movement, cells are polarized,
elongated and then, intercalated. We have established atechnique to observe this process at the cellular level. Thismethod enabled us to show that loss of function of theWnt signaling components caused a defect in the

NATI ONA L INSTITUTE FOR BAS IC BIOLOG Y
DEVELOPMENTAL BIOLOGY

polari zati on of the mesodermal cell s. During convergent
extension , the formationof lamelipodiaand
fi lopodia-like protrusi ons from the mesodermal cell s areobserved. We found that the blockade of the Wn t
signaling pathway significantly reduced the protrusive
acti vity. On the contrary, overexpression of Xwntll andits receptor Frizzled pro moted the protrusive acti vity.
These results indicate that the noncanoni cal Wnt pathway
plays an essential role in the convergent extension
movements through the regul ation of actin cytoskeletal
dynamics.

Figure I The Wnt signaling pathway regulates protrusive
activity in the mesodermal ce lls. (A)The dorsal mesodermal
ce lls indi cated by the arrow were observed. Control cells(B) fo rm lamelipodi a- and fi lopodia-like protrusions,whereas this acti vity was significantly reduced by theexpress ion of the domin ant negative mutant of Xwnt ll (C)or Dishevell ed (D).

Ill. One of the DPP signal downstream components
TNA/TONAS participates roles in protein SUMO
modification and epigenetic regulation
Drosophila is one of the ideal model organisms todissect signal transducti on pathway by genetic methods.
We have can·ied out dominant suppressor screening for atransgenic mutan t fly that expresses acti vated DPP/BMP
type-! receptors in wing imaginal discs. Two mutant
alleles of the tonalli (Ina) gene were isolated in thisscreening. Ina is a previously repm1ed mutant and amember of Trithorax group genes. Trithorax groupcomponents have been shown to be involved in theepigenetic regulation including Histone modification andchromatin remodeling. We isolated ve11ebrate homologuesof Ina and named as tonalli related SP-RING fin ger
protein, TONAS-1 and TONAS-2. The most characteristicfeature of these proteins is the existence of a single
SP-RING fi nger motif in the middl e. We have been
investigating fu nction of TNA/TONAS fa mily SP-RING
fi nger proteins by genetical and biochemical approarches.
The SP-RING motif was originall y fo und in the PTAS
family SUMO-E3 ligase prote ins . We fo und that TON AS

23

can bind E2 enzyme ubc9 and has activity stimulating
SUM0-2/3 spec ific conjugation to TONAS itself.
SUM0-2/3
conjugated
TONAS
proteins
werepredominantly recovered in cytoplas mic fraction. The
result indicates thata SUM0-2/3 modification somehow
regul ates nuclear export or cytopl asmi c retention for
SUM0-2/3 modifi ed substrates. Our ongoing study alsosuggests that TNA/TONAS proteins pm1icipates a role incompl ex Histone modifications.

IV.
FunctionofBrachy ury -dow nstream
notochord-specific genes in morphogenetic
movements of the Ciona intestinalis embryo
Ascidians, urochordates, are one of the three chordategroups, and the ascidian tadpol e is thought to represent
the most simplified and primitive chordate body plan. It
contains a notochord, which is a defining characteristi c ofchordate embryo composed of only 40 cells. To
understand the morphogenesis in thi s simple system, we
have fo cused on a gene, Brachyury, which is known toplay an important role in the notochord development. ln
ascidi an, Brachyury is expressed exclusively in the
notochord and the mi sexpress ion of the Brachyury gene
(Ci-Bra) of Ciona inlestina/is is suffi cient to transform
endoderm into notochord. Thi s gene encodes asequence-specific activator that contain a T-box
DNA-binding domain , and in ve11ebrates, it is initiallyexpressed tlu·oughout the presumpti ve mesoderm andgradually restricted to the developing notochord andta ilbud . The phenotype of the Brachyury mutants in mi ceand zebrafi sh revealed that thi s gene is essential fo r
notochord differentiation. Our goa l is to elucidate thedown stream pathway of thi s impo11ant gene in ascidianin order to set the stage for understanding not only theformation and function of the notochord but how thi s
impm1ant structure has evolved.
Formation of the chordate body is accompli shed by acomplex set of morphogenetic move ments of consti tuent
cells. Convergent extension is a maj or component of ax iselongation. In the urochordate ascidian Ciona intestina/is,
Brachyury (Ci-Bra) plays a key ro le in the fonn ation of
the notochord, which undergoes typical converge nt
extension. Nearly fo rty Ci-Bra downstream notochord
genes have been identified. The present functional
analyses of 32 genes revealed that 24 (75 %) of them are
involved in convergent extension; nine genes including
Ci-prickle, Ci-PTP and Ci-lalin caused failure ofconvergent extension when their fu ncti on was suppressedwith specific morpholinos, while the function of 15 othergenes including Ci-nelrin , Ci-ERM and Ci-p e/lino wasrequ ired fo r intercalation/extension. fn
addition,knockdown of Ci-Nolo 7 resul ted in the failure ofconvergence but interca lation/extension took placenormally suggesti ng a separable phase of conve rgent
extension .

24

NATIONAL INSTITUTE FOR BASJC BIOLOGYDEVELOPME NTAL BIOLOGY

by the cell-cell interaction, we combine the microarray
technology with classical embryo manipulations including
surgical operations .

'"'"''WISHphoto

NIBH~IGINBRP X<'nOJm.<lm·l'is EST project. XOBJ is iDCiudcd~

X.vnJ i5 a web data o:at:.Lioguc ofSriiiChClone

the more th:m 250JXJO E!)l i nfom~:uion fmm the NIB B/NBRP e DNA libraries. EST St:qu~nccs.
as._-.cmbkd contig SC{jiK'I1l'C~ ami their ~impk annOfJ!Kln by B LA~I againSI nr-a.a.X.k1n·i.r UniGcm.•
cNCBIJ. Xenopus Gene lrn.icx (XGI. TIGR) and Xenopus proiC'in d:u:tb.aso:s (N IH ) an• JH=llctl . The
corrcspolldingscqucnccsofXt•lwfmJtropic'<Jiisarcnlso prcscnlt'd.

f~1 . XIJJO hO I I~
Search Contig

lnXllfll.tt>..<.'on!lt;
N'urub4-rtJ!OOO J - ~2J(,J~You can lLiiC the XDB3 for ()c:;ignlng PCR primers and MOfllholino oligo~ to clone genes or 10 knoct.:out gcncl ofyourink'rt'j;L You can also llliC lhc XDH3 for viewing Ilk" spaual cxp~si~ pattc!l':~f'?munfrniti1cd egg 10 ladpolc smge wilh !he high qu:~lhy pholo images of whole moum 10 suu hyllrkhz.:UJOn
fmm sc\·cral angle:>. The full inSC'n srqueoc-cs. of which are SC'k"Ctetl as the fu !l-kngth eDNA clones. arc
alS(litJcludcd.
Coru:um:tJI pmj«t: We are examining the global tr.mscriptomc during t'arty de\'clopmcnttl process with
4Ql: macroarrJy. :l.6l: nr-mirm:umy. 20k nr-oligo micmarray. We are :~!so ean)'in~; out !he X300U jllob~l
fuocdo rml :malrsis. It is composed of ~lobal g::~inlloss of fulll."'lion analysis b~· mc:ms of mRNA()\'Cn"ll prcs~ion. and anti~nsc ll't'hniqucs 1_,\lnrpholino oligo. RNAi. maternal dcpk·uon etc).

What's NEW?
SIDFt~. I J2002

ltbjeML!!OIIAIXD IUis nowa\'ailablc!

[iJftD]

Figure 3 Xenopus Data Base 3 (http ://xenopus.nibb.ac.jp).

Publication List
Figure 2 Failure in convergent movements caused byfunctional suppression of four genes . (A) Ci-pk, (B) Ci-FAK,(C) Ci-flbrn, (D) Ci-nmMHC. (A-D) Whole embryos with
EGFP expression, (A'-D') enlargement of notochord region,and (A" -D") their dark-field images. Scale bars, 50 ~min
A and 10 ~min A'.

V. Xenopus functional genomics (Xenomics)
Using Xenopus laevis as a model, we have been
attempting to reveal the complex regulatory network
controlling the morphological processes in early
embryogenesis that are associated with vigorous cell
pro liferation and differentiation through the clarificationof gene function and their interactions in the system.
In order to achieve the goal, we first collected morethan 170,000 ESTs from the early embryo eDNA libraries.
These ESTs were assembled into more than 30,000
contigs and singlets. Among them, we have selectedfull-length cDNAs excluding redtmdancy, and have readthe full insert sequence for about 3,000 novel genes. To
explore the spatiotemporal expression profile of eachgene, we have canied out who le mount in situ
hybridization (WISH) of embryos at different stages of
early embryogenesis. So far, we have investigated theexpression pattems of more than 2,000 genes. We havealso started to investigate the subcellular localization ofeach gene product using GFP-labeled expression vector.
We constructed the comprehensive database XDB3 that
stores EST sequences, assembled sequences, full insert
sequences and WISH data with the descriptions ofpresumed gene function. We also created microanay with
PCR-amplified cDNAs, and carried out a series of
microarray experiments on the collaboration basis. In
order to in vestigate how the gene expression is regulated

Sakamaki, K., Takagi, C., Kominami, K. , Sakata, S., Yaoita,
Y. , Kubota, H. Y. , Nozaki , M. , Yonehara, S. , and Ueno, N.
(2004) The adaptor molecule FADD from Xenopus laevis
demonstrates
evolutionary
conservationof
its
pro-apoptotic activity. Genes Cells. 9, 1249-1264.
Chung, H . A., Hyodo-Miura, J., Kitayama, A. , Terasaka, C. ,Nagamune, T., and Ueno, N. (2004) Screening of FGF
target genes in Xenopus by microanay: temporaldissection of the signalling pathway using a chemical
inhibitor. Genes Cells. 9, 749-761.
Ohkawara, B., Shirakabe, K., Hyodo-Miura, J. , Matsuo, R. ,Ueno, N. , Matsumoto, K., and Shibuya, H. (2004) Role of
the TAKI-NLK-STAT3 pathway in TGF-beta-mediatedmesoderm induction. Genes Dev. 18, 38 I -386.
Iioka, H ., Ueno, N., and Kinoshita, N. (2004) Essential roleof MARCKS in cortical actin dynamics during
gastrulation movements. J Cell Bioi. 164, 169-74.
Miyakoshi , A., Ueno, N., and Kinoshita, N. (2004) Rho
guanine nucleotide exchange factor xNETI implicated in
gastrulation movements during Xenopus development.
Differentiation. 72, 48-55.
Kawai, N. , Takahashi, H., Nishida, H. , and Yokosawa, H .
(2005) Regulation of NF-kappaB/Rel by IkappaB isessential for ascidian notochord formation . Dev Bioi. 277,
80-91.
Takatori , N. , Hotta, K. , Mochizuki, Y. , Satoh, G., Mitani, Y.,
Satoh, N. , Satou, Y. , and Takahashi , H. (2004) T-box
genes in the ascidian Ciona intestinalis: characterizationof cDNAs and spatial expression. Dev Dy n. 230, 743 -753.
Uchimura, Y., Nakamura, M. , Sugasawa, K. , Nakao, M., andSaitoh, H. (2004) Overproduction of eukaryotic SUMO-land SUM0-2-conjugated proteins in Escherichia coli .
Anal Biochem. 331 , 204-206 .

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGYDIVISION OF DEVELOPMENTAL GENETICS
Professor:
Research Associates:
Technical Staff
Postdoctoral fellows:
Graduate Students:

Technical Assistants:

Secretwy:

KOBAYASHI, Satoru
MUKAI, Masanori
SHIGENOBU, Shuji
NODA , Chiyo
SATO, Kimihiro
HAYASHI, Yoshiki
KITADATE, Ytt
HAYASHI, Makoto
YATSU, Jun
ARJTA, Kayo
UEDA, Yoshiko
TSUCHIYA , Naomi
SATO, KaoriHONDA, Satoko

The germ cells are the specialized cells, which cantransmit the genetic materials from one generation to thenext in sexual reproduction. All the other cells of the bodyare somatic cells . This separation of gem1 and somatic
cells is one of the oldest problems in developmental
biology. In many animal groups, a specialized portion ofegg cytoplasm, or germ plasm, is inherited by the celllineage which gives rise to germ cells . This cell lineage iscalled germline. The germline progenitors eventually
migrate into the gonads, where they differentiate as germ
cells when the organisms are physically matured. Earli er
investigators have demonstrated that germ plasm contains
matemal factors required and sufficient for germline
formation. In Drosophila, this cytoplasm is histologicallymarked by the presence of polar granules, which act as arepository for the maternal factor required for germline
fmmation. Our molecular screens have identified several
factors stored in the polar granu les. One of the factors is
mitochondrial large rRNA which functions to form thegermline progenitors, or pole cells. The other is nanos
mRNA, which is required for pole cell differentiation.

25

results in the failure to form pole cells and injection of
mtlrRNA is able to induce pole cells in embryos whose
ability to form these cells has been abolished by
uv-irradiation. These observations clearly show that theextra-mitochondrial mtlrRNA on polar granules has an
essential role in pole cell formation, presumably
cooperating with mtsrRNA.

Mitochondria
Mitochond1·ial
Ribosomal Proteins

Pole Cell
Formation

Fig. l Role of mitochondrial rRNAs in pole ce llformation

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 ntitochondria are oxidative
phosphorylation and biosynthesis of many metabolites, it
has now become evident that they are also involved in
germline formation .
In Drosophila, pole cell formation requires the functionof mitochondrial ribosomal .R NA in germ plasm. We havepreviously reported that mitochondrial large rRNA
(mtlrRNA) and small rRNA (mtsrRNA) are bothtransported from mitochondria to polar granules. Thistransportation occurs during early embryogenesis, when
mitochondria are tightly associated w ith polar gran ules ingerm plasm, and it depends on the function of thematernally-acting gene, tudor, that is known to berequired for pole cell formation. Mitochondrial rRNAs
remain on the polar granules until pole cell fo rmation andare no longer discemible on the granules within pole cells.
Reductio n of the extra-mitochondrial mtlrRNA amountFig. 2 Inj ection of kasugamycin causes a defect in polecell formation but not in mitochondrial activity. E mbryos
at stage 4-5 were inj ected with DW (A, C) and KA (B, D)
and photographed using a compound microscope
equipped with Nomarsk i optics. (A, B) Pole ce lls are not
di scemible in the KA-injected emb1yo, although somaticce ll s are intact. (C, D) li~ ected embryos were sta inedwith Rhodamine 123 , which was not affected by KAtrea tment. We found that CH treatment a lso inh ibi tedpole cell fom1ation , but did not affect Rhodamine 123
sta ining (data not shown). Arrowheads in (A, C) indi cate
the pol e ce ll s.

We have found that n1itochondrial rRNAs form
mitochondrial-type of ribosomes on polar granu les,cooperating with mitochondrial ribosomal proteins. Thissuggests the possibility that the protein(s) essential for

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

26

pole cell formation is produced by the mitochondrial-typeof ribosomes (Fig. 1). To address this issue, we have been
examining the effect of Chloramphenicol andKasugamycin
(KA)
on
pole
cell
formation.
Chloramphenicol and Kasugamycin are known to inhibit
mitochondrial (prokaryotic)-type of transl ation. Whenthese antibiotics were injected into the posterior poleregion of early embryos, pole cell formation was severely
affected (Fig. 2). In contrast, Chloramphenicol andKasugamycin treatment did not affect somatic cell
formation at a dose we used. These observations strongly
suggest that the mitochondrial-type of translation system
must be intact for the embryos to form pole cells.
Recently, we found that one of the target RNAstranslated on the mitochondrial-type of ribosomes wasgerm cell-less (gel) mRNA, which is stored in polar
granules and encodes a protein required for pole cell
fom1ation .

Fig. 3 nos-H99 pole cells have the ability to adopt somatic cellfate and to enter into the embryonic gonads. Pole cells fromnos (B), nos-H99 (C-F) and control embryos (A) weretransplanted into hosts, and the hosts were developed until
stage 15-1 7. The transplanted pol e cells were identified as blue
cells after heat treatment and staining with X-gal (arrowheads).
The transplanted control (A) and nos-H99 pole cells (C) wereobserved within the gonads, whil e nos pol e cells remained
outside the gonads (B). Some nos-H99 pole cells were
integrated within somatic ti ssues (D-F). (D) nos-H99 pole cellwithin midgut ep ithelium is shown. Note that nos-H9 9 polecell and the neighboring midgut ep ithe lial cells were columnar
in shape at stage 17. nos-H99 pole cells within gastric caeca(E) and trachea l epithelium (F) are a lso shown. The lower ce llpointed by arrowhead in (£) was out of focus . G-1, Drawings
trac ing the integrated nos-H 99 pole ce lls (blue) and thesurround ing somatic cells in (D-F) , respecti ve ly. Scale bar; 10~llll.

II. Role of Nanos protein in pole cell differentiation
Pole cells differ from the soma in regulation oftranscriptional activity. Pole cells are transcriptionallyquiescent unti I the onset of gastrul ation, althoughtranscription is initiated in the soma during the syncytial
blastoderm stage. Consistent with this, RNA polymerase
II (RNAP II) remains inactive in early pole cells. Thus,
the ability to express zygotic mRNA-encoding genes issuppressed only in pole cells in early embryos .
p-gal

Fig. 4 nos-H99 pole cells within midgut epithelium expressed
midgut marker genes but lost germline marker, Vas. We
examined express ion of midgut marker genes and Vas protein
in nos-H99 pole ce lls within midgut epithelium (A-E) ,haemocoe l ("lost" pole cells) (F-J) and the gonad (K-0) of hostembryos. The transplanted pol e cells were identified as thecells expressing ~-gal (marked by arrowheads). The embryos atstage 13-15 were triple-stained with probes for the midgut
marker genes (blnt-nl mexl; red) (B, G, L) and antibodies
against ~-gal (green) (A, F, A.) and Vas protein (blue) (C, H,
M). nos-H99 pol e cell integrated within midgut epithelium(mg), like the neighboring host ce lls, was cuboidal in shape atstage 14 (E). The " integrated" pole cell expressed midgut
marker genes (B) , but not Vas protein (C). ln contrast, nos-H99
pole cells, which were found within haemocoel in th e vicinityof midgut (mg) (J) , maintained Vas expression (H) , but never
expressed midgut marker genes (G). Within the gonad,nos-H99 pole cells and the host pole cells were both negati ve
for midgut markers (L). Arrows in (K) and (N) indicate
non-spec ific stai ning of tracheal lumen. Merged images (D, I,
N) and photographs taken with a compound microscope
equipped with Nomarski optics (£, J, 0) are shown. Dottedlines indicate midgut epithelia (mg) in (£) and (J) , and the
gonad (go) in (0). Scale bar; 10 ~Lm.

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 fertili zation. Nos is onlytransiently present in the posterior half of embryos during
the preblastoderm stage, and is required there forposterior somatic patterning. Nos in the genn plasm ismore stably inherited into the pole cells at the blastoderm
stage, remaining detectable in these cells throughout
embryogenesis. Pole cells that lack Nos (nos· pole cells)
are unable to follow normal germline development; they
fail to migrate properly into the embryonic gonads, andconsequentl y do not become functional germ cells. nos·pole cells fa il to establish and/or maintain transcriptionalquiescence, and ectopically express somaticallytranscribed genes, including fitshi tarazu (jiz) ,even-skipped (eve) and Sex-lethal (Sxl).
Nos represses translation ofmRNAs with di screte RNA

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

27

sequences called Nos response elements (NREs). In thepathway leading to posterior somatic patterning, Nos acts
together with unlocalized Pumilio (Pum) protein torepress translation of maternal hunchback (hb) mRNA.
This translational repression is mediated by binding of
Pum to NREs in the 3'-untranslated region (UTR) of hbmRNA. In pole cells, Nos also acts with Pum to regulate
germ line-specific events. Pum, like Nos, is required inpole cells for their migration to the gonads.
We have found that Nos, along with Pum, represses
translation of importin a2 (impa2) mRNA in early polecells. The impa2 mRNA contains an NRE-like sequencein its 3'-UTR and encodes a protein required for nuclear
import of karyophilic proteins. We found that Nos inhibits
expression of a somatically-transcribed gene, .fiz, in polecells by repressing Impa2-dependent nuclear import of atranscriptional activator for ftz, Ftz-F 1.
The above results raise the question whether the polecells lacking Nos (nos· pole cells) are able to differentiate
into somatic cells. However, it is difficult to study theirdevelopmental fate, since Nos also represses apoptosis ofpole cells, and almost all of nos· pole cells are eliminateduntil at least the end of embryogenesis. To overcome thisproblem, we used Df(3L)H99 , a deletion for tlu·ee genes
required for apoptosis. Introduction of the H99 deficiency
results in nos· pole cells being escaped from apoptosis.
We transplanted the nos--H99- pole cells into normal
embryos and observed their behavior, and found thatsome of nos--H99- pole cells were able to differentiate as
somatic cells (Fig. 3, 4). This suggests that pole cells havethe ability to differentiate as somatic cell s, but its ability is
inhibited by Nanos activity. Recently, we have found thatsomatic differentiation of nos--H99- pole cells requires
Impa-2 activity, suggesting that Nos inhibits somaticdifferentiation by repressing Impa-2 production.

III. Comprehensive analysis of genes expressed in
Drosophila gonad
Isolation of embryonic gonad
eDNA

Pole cell
brmation

Gastrulation

Migration to
mesoderm

Associa.tbn
with gondal
mesoderm

Gonadooalescence.,......;--.,............,

up

down

Fig. 6 Clustering analysis of expression profile of all of
the predicted genes in the Drosophila genome. We
divided embryonic stages into 8 groups, and isolatedpole cells from the embryos at each stage. These polecells were used for microarray analysis. As a reference,
we used RNA from whole embryos of these 8 groups.
Single line represents single transcript. Red line shows"upregulated" in pole cells. Hierarchical clustering
analysis shows that they can be di vided into two large
groups. This is due to the onset of zygotic transcription
in group-4 pole cells.
pole cells and/or in somatic cells within the gonad by acomprehensive approach. From the embryos canying
EGFP-vasa transgene that express GFP only in pole cells,
we isolated the gonads by using fluorescence-activatedcell sotiing (FACS), and costructed a gonad eDNA library.
Each eDNA clone was sequenced from both 5 ' and 3 'ends, and these Expression Sequence Tags (ESTs) werecomputationally condensed into sequence clusters, whichwere then subjected to whole-mount in situ hybridi zation(WISH) (Fig. 5). Approximately 20,227 of ESTs weregenerated, and were clustered into 2900 distinct genes.
The WISH analysis identified more than 130 genes thatwere expressed predominantly in the gonads.
We also started to identify genes expressed
predominantly in pole cells, usi ng DNA mjcroarray
technique (Fig. 6) .

mRNA or total RNA

D

D

Publication List:

DNA microarray:··:::: ::>:: ·::::··:::.:::. .. : .· .·D

Non-redundant eDNA set<::;:;

Polar Plasm

-

..·

D

Hayashi , Y., Hayashi , M. and Kobayashi, S. (2004) Nanos
suppresses somatic cell fate in Drosophila germline. Proc .
Nat!. Acad. Sci. USA. 101 , 10338-10342.

Up-regulated genes

{?

WISH

Fig. 5 Transcriptome analysis of the embryonic
gonadsTo explore the regulatory mechani sm of germline
specification, we attempted to identify genes expressed in

Hanyu-Nakamura, K. , Kobayashi , S. and Nakamura, A.
(2004) Intrinsic and extrinsic lipid phosphate phosphatasedefines cell viab ility that promotes directional migrationof Drosophila germ cells. Development 131 , 4545-4553.
Unezaki, S. , Nishizawa, M., Okuda-Ashitaka, E. , Masu, Y.,
Mukai , M., Kobayashi, S. , Sawamoto, K., Okano, H. andIto, S. (2004) Characterization of the iso[onns of MOVO
zinc fin ger protein, a mouse homologue of Drosophila
ovom as transcription factors. Gene 336, 47-58.

28

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGYDIVISION OF MOLECULAR AND
DEVELOPMENTAL BIOLOGY

Professor:
TAKADA, Shinji
KOSHIDA, Sumito
Research Associate:
Technical Staff:
UTSUMI, Hideko
NIBB Research Fellow:
KAWAMURA, Akinori
JSPS Postdoctoral Fellow: OHBAYASHI, Norihiko
KURATA, Tomoko
Postdoctoral Fellows:
AKANUMA, Takashi
TAKADA, Ritsuko
Graduate Student:
YAMAGUCHL Yoshifumi I)
Technical Assistants:
OHBAYASHI, Akiko
ODA, Ritsuko
TAKASHIRO, Kayoko
ODA, Keiko
Secretary:
I)

Graduate School of Biostudies, Kyoto University

One of the research interests of this laboratory is tounderstand molecular mechanism how a cell signaling
molecule, including members of Wnt, BMP and FGF
families, regulates different developmental events. A
number of evidence indicated that each signal is involvedin many aspects of the vertebrate development. For
instance, we have revealed that Wnt-3a, a members of
Wnt family, plays essential roles in a number of aspects ofthe mouse development, including somite development,
neural crest formation and neural development. However,cellular and molecular mechanisms how a cell signaling
molecule regulates these different events. Thus, we arefocusing on precise functional analysis of cell-to-cellsignals and identification of target genes induced by thesesignals.
Another interest is to understand molecular mechanismof development of the vertebrate trunk, especially somite.
One of interesting features of the vertebrate trunk
development is that it proceeds gradually. To understandhow metameric structures of somites are gradually
generated in an anterior to posterior order along the bothsides of embryonic body axis and how each somite arecharacterized differently along the anterio-to-posterioraxis, we are also trying genetical approach with thezebrafish.

I Functional Analysis of molecular targets of Wnt
signaling during development
Wnt signaling plays important roles in many aspects ofvertebrate development. For revealing the molecularmechanisms underlying each developmental event
triggered by Wnt signaling, identification and functional
analysis of genes activated or repressed by this signal are
important. However, only a few systematic approaches
have thus far been reported for identification andfunctional analysis of Wnt-responsive genes in vertebrate
development.
The gene-trap methodology is a powerful strategy forthe systematic identification and functional analysis ofgenes in the post genomic era, because this methodology
offers the identification of a novel gene, the analysis of its
expression pattern, and the generation of its functional
mutation in a single experimental approach as described
below. In this methodology, random insertion of agene-trap vector leads to the tagging, and frequently to thedisruption, of genes across the genome. Therefore, if ES
cells are used for the generation of insertion events,
embryonic and adult whole bodies containing tagged anddisrupted alleles can be produced. Such an insertion event
affords the following advantages for identification andfunctional analysis of a trapped gene. 1) Nucleotide
sequences of a trapped transcript and an insertion site canbe determined by 5' rapid amplification of eDNA ends
(RACE) and plasmid rescue method, respectively. The
process of identification of a trapped gene has been
substantially eased by the recent completion of the
genome database. 2) The expression pattern of a trapped
gene during development can be easily monitored by theexpression of protein tag derived from a gene-trap vector
in embryos generated from trapped ES cells. 3) An
insertion event has the potential to be mutagenic. Becauseof these strong advantages, the gene trap methodology has
been applied for several studies, including large-scale
insertional mutagenesis programs.
Although the gene trap methodology basically involves
the random integration of a gene-trap vector, this strategy
has also been expected to be available for identificationand characterization of genes regulated by particularsignals. In a modified gene-trap strategy, called induction
gene trap, genes are selected by their response to specificsecreted signal proteins, including BMP2, activin, nodal,
and FGF, added to the culture of gene-trapped cells.
Although the in vivo correlation between selected genes
and signals has remained unclear in these cases, in vitro
prescreening of trapped ES cell lines with secreted signal
proteins would be effective for enrichment of genes
regulated by specific signals even in normal development.
Here, we examined whether gene trap methodology,
which would be available for systematic identificationand functional analysis of genes, is effective for screeningof genes responsive to Wnt signaling during mouse
development. We screened out 2 individual clones (Clone
43 and Clone 5) among 794 gene-trapped ES cell lines by
their in vitro response to WNT-3A proteins.
To examine whether the in vivo expression of thesetrapped genes was actually regulated by Wnt signal, we
examined their expression precisely in mouse embryos.
Expression of Clone 43 was temporarily observed in the
inner cell mass of blastocysts. Later, Clone 43 wasspecifically expressed in ductal structures in the
mid-gestation stages. This gene was expressed during a
number of aspects of the ductal morphogenesis in kidneydevelopment. The urogenital expression of Clone 43 wasobserved first in the nephric duct and mesonephric tubules
at EI0.5 (Figure lA) and later in the Wolffian duct, theureter, the collecting duct, and the distal portion of tubulesconnected to the collecting duct in the metanephros(Figure I B, C, E). The expression was restricted to theepithelium, i.e., was not found in the mesenchyme, inNATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELO PMENTAL BIOLOGY

these organs (Fig. IE, F) . Clone 43 was also expressed in
the ductal
epithelium of the salivary glands(submandibular, sublingual, and parotid glands) atE 15 .5(Fig. 1F, G).

G

Figure I Exp ress ion of C lone 43 in the ductal deve lopmen t.
(A , B) During urogenita l deve lopm ent, lacZ repmter was
expressed first in the nephric duct and mesonep hric tubul es
at E I 0.5 (A). At E 13, lacZ reporter of c lone 43 was also
ex pressed in the Wo lffia n duct, mesonephric tubul es, theureter, and the coll ecting duct (B). (C , D) Clone 43 (C) was
co-expressed withWnt-7b (D)i n the Wo lffian d uct. (E , F)
Clone 43 is exp ressed in the epithe li al ce lls in thedeveloping kidney (E) and SMG (F). (G, H) Exp ression of
CRTR-1 mRNA (G) and Wnt-5b (H) in the SMG and S LG
at E 13.5.

29

Interestin gly, the expression of several Wnt genes was
also observed in the ductal structures in which Clone 43
was expressed . In the urogenital development, Wnt-7b ,which has been reported to be expressed in the Wolffianduct, the ureter, and the collecting duct at E 13 .5, wasco-expressed with this trapped gene (Figure I C, D). On
the other hand, in the submandibular gland (SMG), theexpression of a number of Wnt genes, including Wnt-2, 2b,3, 4, 5b, 6, JOb, 14, 16, was detected by RT-PCR analysis.
Among these Wnt genes, Wnt-5b exhibited an expression
pattern spatially and temporally correlated with that of
CRTR-1 . Wnt-5b was expressed at mid-gestation stages,
for instance E 13 , in the stalks of the SMG and thesublingual gland (SLG), where Clone 43 was expressed(Fig. l G, H). Thus, the ductal expression of Clone 43 waswell correlated with the expression of Wnt-7b and Wnt-5b
in the kidney and the salivary g land, respectively
The expression pattern of Clone 5, also suggested its
close con·elation with that of Wnt genes. Expression ofClone 5 was first observed at E 8.5 in rhombomere 5. At
E9 .5, the expression of thi s gene was still detected inrhombomere 5 although it was relatively dispersed.
Rostral to rhombomere 5, the lacZ-positive cells straggled
in the superior membrane of the rhombencephalon. At
E l 0.5-11.5 the expression was observed with strong
intensity in scattered neural crest cells over the dorsal
diencephalon and mensencephalon, as well as in relatively
weak intensity over the dorsal spinal cord. Interestingly,
these scattered signals were strong along the dorsal
midline adjacent to the roof plate where Wnt-1 and
Wnt-3a were expressed. Clone 5 gene was also expressedin mesenchyme in the telencephalic flexure. Thismesenchymal expression represents another example ofcorrelation between this gene and Wnt, since Wnt-Ja is
expressed in the neuroepithelium, adjacent to themesenchymal cells, in the telencephalic flexure . The
expression of Clone 5 gene was also observed in themeninx at El3.5 . Taken together, the close correlation
between gene expression of the 2 trapped genes and thatof several Wnt genes strongly suggests that the trapped
genes screened by their in vitro response to WNT proteins
were also responsive to Wnt signals in vivo .
Furthermore, the expression of these 2 genes was
significantly decreased in embryos deficient for several
Wnts or in cultures of embryonic tissues treated with a
Wnt signal inhibitor. For instance, the positive LacZ
signal of Clone 5 gene was absent in neural crest cells
migrating from the hindbrain and the spinal cord in the
Wnt-1/ Wnt-3a double mutant (Figure 2H). In contrast, thelacZ signal was present in the wild-type and in Wnt-1 and
Wnt-3a single mutant embryos (Figure 2E-G) . These
results strongly suggest that Wnt-1 and Wnt-3a wereredundantly required for the expression of the c lone 5
trapped gene in this region. In add ition, the X-ga l stai ningin the mesenchymal ce ll s in the telencephalic flexure wassigni ficantly reduced in the Wnt-3a null mutant, as well as
in Wnt-1/ Wnt-3a doubl e mutant, co mpared with that in
the wild-type and Wnt-1 single mutant embryos (Figure
2A-D).

30

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGYTo examine whether the trapped genes were requiredfor proper development of cells or organs where they
were expressed, we generated homozygous mutants forthe trapped genes by crossing heterozygous males andfemales. In homozygotes for the Clone 43 trapped allele,
the expression of this gene was almost completely
diminished, whereas the expression of a tmncatedtranscript fused to the ~-geo gene in the trap vector wasstrongly detected. Interestingly, 70% of homozygotes for
the Clone 43 trapped allele died before 5 weeks after birth.
Most of in the Clone 43 homozygous mice exhibited
hypoplasia of the kidney at postnatal day30, whereas theheterozygous and wild-type mice in the same littersshowed no obvious defect in their kidney. Thus, Clone 43
is likely to be required for proper development of thekidney, in which this gene is expressed. On the other hand,no obvious defect has yet been observed in homozygotes
for the clone 5 trapped allele.
Thus, the in vivo expression of the 2 trapped genes,
which were screened by their in vitro response to Wnt,
was also dependent on the Wnt activity. Furthermore,homozygotes for a trapped allele showed a morphologicalphenotype in the kidney, where the trapped gene wasexpressed overlappingly with Wnt-7b. These results
indicate that an inductive gene trap in ES cells is likely tobe effective for screening and functional analysis of genes
induced by Wnt signaling during embryogenesis.

Figure 2 Down-regulation of the expression of Clone 5 genein Wnt-3a and Wnt- J/Wnt-3a mutants. (A-D) Frontal viewsof the expression of Clone 5 in the telencephalic flexure(arrow) at El1.5 . (E-H) Dorsal views of the expression ofClone 5 gene in the hindbrain at E 11.5. Wild type (A, E),
Wnt-3a null mutant (B, F), Wnt-1 mutant (C, G), Wnt-1
and Wnt-3a double-null mutant (D, H).

II Genetical approaches for revealing molecularmechanism of trunk development in zebra fish
To identify genes involved in several aspects during
early embryogenesis of the vertebrate, we have
perfonned screening of zebrafish mutants induced by
ENU, a chemical mutagen. Until now, we havescreened 630 of F2 families and found a number ofmutants whose phenotypes are different from thosealready reported. For instance, some of these mutants
displayed defects in the somite and tailbud
development (Figure 3). Cloning of genes that are

responsible for these defects is in progress.
To complement a forward genetical approach , wehave also screened genes expressed in the tailbud andpresomitic mesoderm, in which somite progenitors
exist. Until now, we have identified 50 genes that are
expressed preferentially in these regions. To examine
developmental roles of these genes, functional analysisof these genes has been performed by injecting
morpholino anti-sense oligonucleotides.
tailbud defect (B)somite defect(D)Figure 3 Zebrafish mutant embryos which display abnormal
shape of the tailbud (kt378b) and a segmentation defect ofsomites (kt259).

Publication List:
Hasegawa H, Ashigaki S, Takamatsu M, Suzuki-Migishima
R, Ohbayashi N, ltoh N, Takada S, Tanabe Y. (2004)
Laminar patteming in the developing neocortex bytemporally coordinated fibroblast growth factor signaling.
J Neurosci. 24, 8711-8719.
Kamata T, Katsube K, Michikawa M, Yamada M, Takada S,
Mizusawa H. (2004) R-spondin, a novel gene withthrombospondin type 1 domain, was expressed in thedorsal neural tube and affected in Wnts mutants. Biochim
Biophy s A cta. 1676, 51-62
Muroyama Y, Kondoh H, Takada S. (2004) Wnt proteinspromote neuronal differentiation in neural stem cell culture.
Biochem Biophys Res Commun. 313, 915-921.
Naito M, Katayama R, Jshioka T, Suga A, Takubo K, Nanjo
M, Hashimoto C, Taira M, Takada S, Takada R, Kitagawa
M, Matsuzawa S, Reed JC, Tsuruo T. (2004) Cellular
FLIP inhibits beta-catenin ubiquitylation and enhances
Wnt signaling. Mol Cell Biol. 24, 8418-8427.
Satoh K, Kasai M, Tshidao T, Tago K, Ohwada S, Hasegawa
Y, Senda T, Takada S, Nada S, Nakamura T, Akiyama
T.(2004) Anteriorization of neural fate by inhibitor ofbeta-catenin and T cell factor (ICAT), a negative regulatorof Wnt signaling. ?roc Nat/ Acad Sci U S A . 101,
8017-8021
Usui H, Shibayama M, Ohbayashi N, Konishi M, Takada S,
Itoh N. (2004) Fgf18 is required for embryonic lungalveolar development. Biochem Biophys Res Commun. 322,
887-892.

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGYlABORATORY OF DEVELOPMENTAL REGULATION
Associate Professor:

UENO, Kohji

Protein palmitoylation during embryogenesis
Covalent attachment of fatty acids is a feature of many
proteins in eukaryotic cell. The two modifications involve
acylation with myristate and palmitate. Myristate is usually
linked through an amino-terminal glycine residue via anamide linkage which is relatively stable. Palmitate is usually
linked through cysteine residue via a thioester linkage, andthe post-translational process is referred to as protein
palmitoylation. Protein palmitoylation is a dynamic
modification. The turnover rate of bound palmitate throughthioester linkages is faster than that of the modification withmyristate through amide linkages. Most small GTP-binding,
G proteins and G protein-coupled receptor proteins are
known to be modified with palmitate via thioester linkages.
Protein palmitoylation is thought to be important in theregulation of signal transduction. However the molecularmechanisms how protein palmitoylation regulate
development is unknown.
We have previously found that protein palmitoylase isexpressed in neural cells during mouse embryogenesis. In
developing neurons growth associated protein (GAP)-43
and Go, which are palmitoylated proteins, are mainly
concentrated in growth cones. Addition of an inhibitor ofprotein palmitoylase to the medium of cultured primaryneural cells reduces the axonal growth of neurons. Fromthese findings, we speculated that palmitoylation of thesignaling proteins is critical for development of axons.
GAP-43 is a neuron-specific protein. Cys 3 and Cys 4 of
GAP-43 are modified with palmitate in developing neurons.
Transfection of GAP-43 eDNA into non-neural cells suchas COS-7 cells is reported to cause the induction offilopodia-like formation. Since Cys residues are necessary
for the induction of filopodia-like formation, protein
palmitoylation is discussed to regulate the induction
filopodia-like formation. Filopodia! processes is thought beimportant for the development of neural growth cones.
Therefore we focused to analyze the regulation mechanismsof the induction of filopodia-like formation in non-neuralcells.

Transfection of protein palmitoylase eDNA into
non-neural cells
We prepared the full-length protein palmitoy1ase eDNA
for the expression in cultured cells. The eDNA contained along open reading frame 2,504 amino acids. Protein
palmitoylase and GAP-43 cDNAs were transfected into
COS-7 and HeLa cells and the transient expression of these
proteins were induced.
We confirmed that expression of protein palmitoylase

31

reproducibly alters COS-7 morphology. Filopodia extention
is observed in about 5% of control COS cells, but in 15% ofprotein palmitoylase-expressing COS cells under ourconditions. To characterize the effect of protein
palmitoylase on non-neural cells, we examined cellularmorphology at various times after trypsinization andreplating. Before contact with a substratum allowing
adhesion, about 15% of control COS-7 cells exhibit
filopodia. Transfection with protein palmitoylase increases
this to 25%. The presence of filopodia is not significantly
altered by varying the length of time to 120 minutes during
which the cells are maintained in suspension aftertrypsinization. After contact with an adherent substratum
the percentage of cells with filopodia rapidly declines inboth the control and protein palmitoylase transfected cells.
The rate of decline is approximately the same in bothgroups. The effect of protein palmitoylase is to alter the
initial morphology after trypsinization, and not to alter the
influence of the substratum on the cell shape.
Filopodia are exhibited by few protein palmitoylase
expressing COS cells after 30 minutes of incubation with anappropriate substratum. However the shape of protein
palmitoylase expressing cells can still be distinguished fromcontrol cells by another attribute, the degree of cellspreading. Protein palmitoylase expression appears to delay
cell spreading. When control COS cells are plated onpoly-L-lysine-coated glass, cell spreading occurs over 60
minutes. The time required to achieve the same degree of
spreading is prolonged in the protein palmitoylase
expressing cells, although the degree of spreading
eventually reaches the same level. One day after plating,cell shape is indistinguishable between control and protein
palmitoylase expressing cells.

Induction of filopodia-like formation in cultured
cells by peptide transfection
We have established a method to chemically modify abiotinylated peptide composed of residues 1-15 of the
GAP-43 N-terminal with fatty acids via thioester linkages.
By using the method, we have prepared the peptides which
are modified with myristate (C14:0), palmitate (CI6:0),
stearate (CIS:O) and arachidate (C20:0) via thioester
linkages. The method also modified the peptide with
unsaturated fatty acid such as palmitoleate (C 16:1 ). The
modified peptides were separated by reverse phase HPLC
and these peptides were eluted from the column depend on
their chain length.
We are attempting to see the effect of filopodia-likeformation in COS-7 cells by peptide transfection, whichwere chemically modified with fatty acids. Since theefficiency of the induction of filopodia-like formation bytransfection with palmitoylated peptide is low, we are trying
to elevate the efficiency.

32

NATIONAL INSTITUTE FOR BASfC BiOLOGYDEVELOPMENTAL BIOLOGYLABORATORY OF MOLECULAR GENETICS
FOR REPRODUCTION
Associate Professor:
Postdoctoral Fellow:
Graduate Students:f)

TANAKA, Minoru
SAITO, Daisuke
AOKI, Yumiko IJ
NAKAMURA, Shuhei I)
ARITA, Kaori f )KUROKA WA, Hiromi IJ

Graduate School of Hokkaido University

Sexually dimorphic gonads mainly consist of two
different cell lineages, somatic gonadal cells and germ
cells. During the course of development, the gonadal
precursor cells should be specified through the process of
mesoderm patterning, which subsequently associates withprimordial germ cells to form the indifferent gonad. Oncethe indifferent gonad is observed in the gonadal area, sex
determination gene is expressed in the gonadal mesoderm
and the organogenesis of gonads (sex differentiation)
starts in mice.
Since sex detennination gene of mice was identified in
1990, research has intensively been focused on the testis
development. However, for instance, the differentiation of
the precursor cells determining sex, the interaction ofsomatic cells and germline, the differentiation of germline
to germ cells having the capability to undergo meiosis,
and so on, there remain many important and facinatingproblems to be solved.
Our laboratory aims to reveal the molecular mechanims
regarding to the formation of gonads, especiallyemphasizing on visualizing specific cell lineages inmedaka living embryos and by applying moelcular
genetics to medaka embryos.

I. Monitering the formation of gonads throughfluorescent germline.
One of the advantages to use medaka is that embryos
are transparent and can be seen from outside. Thisencouraged us to develop transgenic medaka exhibiting
GFP exclusively in germline and succesfully establishedthe trangenic lines (a/vas-transgenic medaka). During theestablishment, we found that two genomic regions, 5'
promoter region and 3 'UTR region, are necessary forproper expression of gennline-specific gene, a/vas(medaka vasa homologue). Furthermore we identified anelement, GSE (germline-specific element), in the 3 'UTR,
which mediates both maintenance of RNA exclusively ingermline and activation of translation throughenhancement of poly(A) elongation. Both mechanisms act
independently on GSE and consequently bring about
efficient translation in germline,
Inj ection of in vitro synthesized GFP RNA with GSE at
its 3' end allows to monitor more clearly and brightly thedevelopment of germline from early embryogenesis.
Bright fluorescence enables us to identify primord ial
germ cell s easily and iso late ESTs that express 111
germ li ne . We cootinue to analyze the ESTs currently.

II. Germline Development
Primordial germ cells have been characterized as thelarge cells having electron-dense amorphas structure
called germ granules (also called germplasm or nuage).
We isolated several ESTs whose products are componentsof germ granules and revealed that 3 'UTRs of some ESTs
possess cis-element functionally similar to olvas GSE. We
synthesized RNA of EST-GFP-3 'UTR fusion genes andinjected it into fertilized eggs. The GFP-fused products
were localized in germ granules as has been detected by
antibodies.
Fig.! GSE in 3'UTR functions to translate RNA
exclusively in gem1line. The picture below right shows
fluorescent gem1line in a gonad.

By keeping track of the structure of the granules with alaser confocal microscopy, we could outline germline
development and revealed several developmental stagesof germline. Firstly, germline separates from somatic celllineages at morula stage and is suggested to be fated toprimordial germ cells as epiboly movement proceeds atearly gastrulation stage. The primordial germ cells near
the germ ring move to anterior lateral plate mesoderm andthen migrate posteriorly along the embryonic body.
Localization of the components on the genn granules
differs from that found in the germ cells of the indifferent
gonads, suggesting that an additional step is required todifferentiate into germ cells which has the capability toundergo meiosis.

III. Origin of gonadal mesoderm
Many focus have been concetrated on the mechanismsof differentiation into male gonadal mesodem1 after sex
detemination gene is activated. However, the origin andthe lineages of the gonadal precursor cells have remained
to be elucidated.
As described in section II, germ cells migrate into a
gonadal region. This suggests that cells producing
guidance substance for primordial germ cells become theprecursor of gonadal mesoderm. We constructed a fate
map showing where the precursor of gonadal mesoderm isderived using uncaging method . The result indicates thatmost posterior portions of lateral plate mesoderm
converge medially toward an embryonic body to locate
bilateral regions where migrating primordial germ cells
target. These regions correspond to posterior ends in
expression domain of guidance substance. After
primordial germ cell s meet the precursor cells of gonads
in the lateral plate mesoderm, the precursor cells andNATIONAL JNSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

primordial genn cells move dorsally around a deve loping
hindgut, and finally reach the region where the indifferent
gonad will form .
It has been suggested by the analysis of mice mutant
possessing few germ cells that somatic precursors
autonomoul y develop to gonadal mesoderm . It is,however, likely that in the process of organogenesis
gonads should interact between somatic mesoderm andgerm cells. To challenge this problem with our ski lls andbiological backgrounds, we are generating medaka
without germ cells by ablation of fluorescent germ cellswith a laser or disruption of function of guidance
substance.

IV. Analyses of mutant medaka affecting formationof gonad
Medaka is a small vertebrate and produces next
generations in 3 months after hatching. This
characteristics are suitable for app lying mo lecular
genetics to this small animal.
In collaboration with ERATO Kondoh differentiation
signaling project, we have been screening mutants
affecting primordial germ cells and the formation of
gonads. The defects were screended by expression ofolvas gene in germ cells. The screening told us thepresence, number and distribution pattern of germline at asomitogenesis stage and at ten days post hatching (I 0dph). Nine mutants ( 19 alleles) and twelve mutants ( 14alleles) were identified for primordial germ cells andgonads, respectively.
The mutants affecting formation of gonads fa ll into
four groups according to their phenotypes. 1) mutants
hav ing overproliferating germ cells, 2) mutants w ith
reduced number of germ cells, 3) mutants withfragmented gonads and 4) mutants with scattered germ
cells in the gonadal region. We are now characterizing
mutant phenotypes and the modes of inheritance.
One mutant, totoro, is very interesting in that thesimilar phenotype has not been described before in otheranima ls. totoro has a swelled large abdomen filled with agonad. Our analyses show that the phenotype issemidominantly inherited. Gonads are sex-reversed in
genetic m ales and fullfilled with numerous oocytes
regardless of its genetic sex. The positional cloning isongoning with ERATO project and the characterization of
the phenotype is also in progress.
Fig.2 sex reversed totoro mutant showing a large abdomen,
which is occupied by a gonad with numerous mu11ber of oocytes.

33

Another mutant, zenzai, is in good contrast with totoro
mutant and is unique in that germ cells are not maintainedin the gonad. The mode of inheritance of the phenotype
indicates that the allele is recessive. The characterizationand the positional cloning are also in progress.

Publication lists
Mitani, H. , Shima, A., Naruse, K. and Tanaka, M. (2004)
Medaka genome mapping for functional genomics. In Fish
Development and Genetics. pp.612-636. (eds: Gong, Z
and Korzh , V) World Scientific.
Sasado, T., Morinaga, C., Niwa, K. , Shinomiya, A. , Yasuoka,
A., Suwa, H., Hirose, Y., Yoda, H., Henrich, T. , Deguchi ,T. , Jwanami , N ., Watanabe, T., Kunimatsu, S., Osakada,M. , Okamoto, Y., Kota, Y., Yamanaka, T. , Tanaka, M. ,Kondoh H. and Furutani-Seiki , M. Mutations affecting
early distribution of primordial germ cells in Medaka(01yzias talipes) embryo. Mech. Dev. (2004) 121,
817-828.
Morinaga, C., Tomonaga, T., Sasado, T. , Suwa, H., Niwa, K. ,Yasuoka, A., Hemich, T. , Watanabe, T. , Deguchi, T. ,Yoda, H., Hirose, Y ., Iwanami, N. , Kunimatsu, S. ,Okamoto, Y. , Yamanaka, T., Shinomiya, A., Tanaka, M.,
Kondoh , H. and Furutani-Seiki. M. Mutations affecting
gonadal development in Medaka, Oryzias latipes. Mech .
Dev . (2004) 121 , 829-839.
Naruse, K. , Tanaka. M ., Mita, K., Shima, A. , Postlethwait, J.,
and Mitani, H. A Medaka Gene Map: The Trace ofAncestral Vertebrate Proto-chromosomes Revea led byComparative Gene Mapping. Genome Res. (2004) 14,
820-824.
Suzuki, A. , Tanaka, M., Shibata, N . and Nagahama, Y.
Exp ression of aromatase mRNA and effects of aromataseinhibitor during ovarian development in the medaka,
Oryzias /atip es. J. Exp. Zoo!. (2004) 301 , 266-273.

34

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

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

been devoting our efforts to searching for molecules withasymmetrical distribution in the embryonic chick retina,
and to characterization of their roles in the topographic
retinotectal projection.
We performed a large-scale screening using restriction
landmark eDNA scanning (RLCS) in the embryonic day 8(E8) chick retina several years ago. RLCS is a eDNA
display system, in which a large number of eDNA species
are displayed as two-dimensional spots with intensities
reflecting their expression levels as mRNA. We detected
about 200 spots that gave different signal intensities
between the nasal and temporal retinas or between thedorsal and ventral retinas. The asymmetric expressionof each gene was verified by Northern blotting and in situ
hybridization. By subsequent analyses using molecularcloning, DNA sequencing, and database searching, 33
asymmetric molecules along the nasotemporal (N-T) axis
and .20 along the dorsoventral (D-V) axis were finallyidentified.
This year, we published the whole of results as one
paper. These included transcription factors, secretory
factors, transmembrane proteins, and intracellular proteins
with various putative functions.
Their expression
profiles revealed by in situ hybridization are highly
diverse and individual (Fig. 1). Moreover, many ofthem
begin to be expressed in the retina from the early
developmental stages, suggesting that they are implicatedin the establishment and maintenance of regional
specificity in the developing retina. We have alreadydescribed on several molecules in published papers, but
the study to know their hierarchical order to establish theregional specification in the retina is still in progress.
The molecular repertoire revealed by this work will
provide candidates for future studies to elucidate themolecular mechanisms of topographic retinotectal map
formation.

I. Regional specification in the retina

Nasal

DIVISION OF MOLECULAR NEUROBIOLOGY
Professor:
Research Associates:

NODA, Masaharu
SHINTANI, Takafumi
SAKUTA, Hiraki
HIYAMA, Takeshi
Technical Staff:
TAKEUCHI, Yasushi
NIBB Research Fellow:
TAKAHASHI, Hiroo
JSPS Postdoctoral Fellows: SUZUKI, Ryoko
TAKAO, Motoharu
FUJIKAWA, Akihiro I)
Postdoctoral Fellows:
NAKAMURA, Takahiro I!
YAMAMOTO, Yasunori I!
FUKADA, Masahide lJ (Apr. '04 -)OHKAWARA, Takeshi (-Dec. '04)
Graduate Students:
IHARA, Masaru
ETANI, Kazuma
SHIMIZU, Hidetada
TANAKA, Rumi (-Nov. '04)
YONEHARA, Keisuke
NAKAMURA, Kayo (Apr. '04 -)Visiting Scientist:
TAMURA, Hiroshi
Secretary:
KODAMA, Akiko
Technical Assistant:
KATAYAMA, Tomomi (Dec. '04 -)JST Technical Staffs:
MIZOGUCHI, Masae
GOTOH, Megumi
YAMADA, Kaoru
AYABE, Yuko
1 CREST, JST

Topographic maps are a fundamental feature of neuralnetworks in the nervous system. Understanding themolecular mechanisms by which topographically ordered
neuronal connections are established during development
has long been a major challenge in developmental
neurobiology.
The retinotectal projection of lower
vertebrates including birds has been used as a readily
accessible model system.
In this projection, thetemporal (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.
Regional specification along the nasotemporal anddorsoventral axes precedes the topographic retinotectal
projection in the developing retina. To understand themolecular basis of topographic retinotectal projection, an
overall view of the asymmetrically expressed moleculesin the developing retinas is needed. Since 1992, we have

Temporal. . . . .•,tJ:.;dQ.'eD.····,e .., . .· ..1·N~k;. 1~~Cr 5~
T':~
T/AI!HI2

v: ' · ;._ ·,T/Aisp718#1

TJBgllll2

H.Q. ·.:.:)1

T/Stnl#1"/l h))'/ . 1(.1'

'

:_'V,

Ti85iWtl1,,~;j

TJBsiWII2'f')v_~FU.u: ~-v
TIB$p120111

TIB5p120112

T/Bsp120113

TJBsp120114

TIBG:GHU11

TIBssttHI2

NATIONAL IN STITUTE FOR BASIC BIOLOGYNEUROB IOLOGY

DorsalVentral

Double35

l'CJv"DU'"D'D'DOO-·l:YQ'C)Q'O
0 /Asp7 18 #1

OIBamHI #1

O!Bsp 1201 #1 D!Bsp1201 13

0 /Eco AI 14

0 /Mrol 13

0 /SspBI 12

V!Aflll 1 1

V/AIIH #2

VIAlw4411 1

0 /EcoRI 1 1

O/EcoAI I2

VfAiw441 15

V/Asp718 #4

oa

oa

v

V/Alw44 1#3u·( ' :)

N/Bsp 1201#1

NIHindlll 12G

TIBssHII # 1

Fig. I. In s itu hybridi zation analysis of the ex press ion ofidentified genes in E8 retina. Dig-labeled cRNA probes
were hybridized to hori zonta l sections (Nasal andTemporal) or coronal sections (Dorsal, Ventral and Double).
Hori zontal sections are oriented with nasal up and temporal
down. Coronal sections are oriented with dorsal up andventral down. The double gradient express ion of some
genes at E8 was verified in (Double). Scale bars: 500 ~Lm.

II.

Topographic retinotectal projection

In the chick embryos, the first retinal ganglion cells
become postmitotic at day E2 and their axons leave theretina at E3. The earliest axons arrive at the mostanterior part of the tectum at day E6 and advance over its
surface in the posterior direction. These ret inal axons
form the stratum opticum (SO), which covers the entire
tectum at E 12. At the onset the ch ick retinotectal
projection has a general topographic order, and axonal
sprouting begin predominantly at the vicinity and towa rds
the site of normal terminal zone . In all vertebrates
studied, however, the developing axons make trial
branches, many of which are not entirely within theretinotopic area, and elimination of ectopic branches andelaboration of appropriate branches is shaped in an
activity dependent manner. We ha ve already identified
some molecules among the region-specific molecules in
the retina that show abnormal targeti ng, branching or
synaptic arbori zation when their expression was
experimentally elevated or suppressed in vivo.

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

plays variegated roles in the brain development and brain
function.
Ptprz binds various cell adhesion molecules (Nr-CAM,
Ll , contactin , NCAM and TAG- I) and extracellularmatri x mo lecules (tenascin-C/R) . We revea led that Ptprz
binds pleiotrophin (PTN)/HB-GAM and midkine (MK),closely related heparin-binding growth factors whichshare many biological activities. The chondroitin sulfate
portion of Ptprz is essential for the high affinity binding tothese growth factors .
It is well known that the vacuo lating cytotoxin YacA
produced by Helicobacter py lori causes massive cellularvacuolation in vitro and gastric tissue damage in vivo,
leadingtogastric
ulcers,whenadministeredintragastrically. In 2002, we found that Ptprz expressedin the gastric epitheli al cells functions as a receptor for
YacA: M ice deficient in Ptprz do not show mucosal
damage by VacA, indicating that erroneous Ptprz
signaling induces gastric ulcers.
Altho ugh members of the protein tyrosine phosphatase(PTP) family are known to play critical roles in various
cellu lar processes thro ugh the regulation of protein
tyrosine phosphorylation in cooperation with protein
tyrosine kinases (PTKs), the physiological functions ofindividual PTPs are poorly understood. This is due to alack of information concerning the physiologicalsubstrates of the respective PTPs.
Therefore, thedevelopment of a standard method applicable to all PTPs
has long been awaited. We published a genetic method
to screen for PTP substrates wh ich we named the "yeast
substrate-trapping system" . This method is based on theyeast two-hybrid system with two essential modifications:
of
a
PTK
to
the
conditional
expression
tyrosine-phosphorylate the prey protein, and screening
using a substrate-trap PTP mutant as bait (Fig. 2) . Thismethod is probably applicable to all the PTPs, because itis based on PTP-substrate interaction in vivo , namely thesubstrate recogn iti on of individual PTPs. Moreover, thismethod has the advantage that continuously interacting
molecul es for a PTP are also identified, at the same time,
By using th isunder PTK-noninductive conditions.
method, we successfully identified several substratemolecules, together with many interacting molecules for
Ptprz. The identification of physiological substrates willshed light on the physiological functions of indi vidual
PTPs.
A

c

B

(a)

<MCS>
EcoR I...,,SamH I

tyrosine

Protein tyrosine phosphorylation plays crucial roles invarious biological aspects including brain development
PTP I/RPTP~/Ptprz, a nervous
and brain functions.
system-rich receptor-type PTP, is expressed as achondroitin sulfate proteoglycan in the brain from theearly developmenta l stage to the adulthood in neurons aswell as astrocytes. This suggests that this molecule'"''
Bait vector(b)

DNA-BD !LexA protein)

Prey vector

Blot: anli-pY

1· · · An CGC MC GGC GAC TGG CTG G.M..lli CCG G~GT CGA CCI...GCA..GCC MG
EcoR 1----smai BsmH I ~ Psi 1
CIA_An CCG GGC GAA ffi en ATG An TAL.G!\T m

,W o

Sir,

TAT TAT IM. ATA AGT TAT AAA •· ·llJ'\,

36

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEU ROBIOLOGY
Fig. 2. Bait and prey vectors and tyrosine phosphorylationby the PTK induction in the yeast. (A,a) Structure of thepBridgeLexA/v-src vector. The PTP substrate-trap mutant
is inserted into the multiple cloning site (MCS) in-framewith LexA. (A,b) DNA sequence of the MCS region in
the vector. Unique restriction sites are shown in bold.
The Smal and Pst1 sites are not shown in bold, because bothsites exist in the v-src sequence. (B) Structure of thepACT2 vector (CLONTECH). (C) Cellular proteins in theyeast are highly tyrosine phosphorylated when v-src is
induced in the absence of methionine (-Met), while almost
no tyrosine phosphorylation was observed in the presenceof methionine (+Met).
L40 cells containing
pBridgeLexA/v-src were cultured in the medium both withand without 1 mM methionine for 24 h, and the tyrosine
phosphorylation of cellular proteins was analyzed byWestern blotting with anti-phosphotyrosine 40 I 0 antibody.

IV.

N a+-level sensing in the brain

Dehydration causes an increase in the sodium (Na)concentration and osmolarity of body fluid in mammals.
For Na homeostasis of the body, controls ofNa and water
intake and excretion are of prime importance. However,the system for sensing the Na level within the brain that is
responsible for the control of Na- and water-intake
behavior remains to be elucidated.
We reported
previously that the Na, channel is preferentially expressedin the circumventricular organs (CVOs) in the brain andthat Na, knock-out mice ingest saline in excess underdehydrated conditions. Subsequently, we demonstrated
that Na, is aNa-level-sensitive Na channel.
We revealed this year that the subfornical organ (SFO)
is the principal site for the control of salt-intake behavior,
where the Na, channel is the Na-level sensor. Infusionof a hypertonic Na solution into the cerebral ventricle
induced extensive water intake and aversion to saline inwild-type animals but not in the knock-out mice.
Importantly, the aversion to salt was not induced by theinfusion of a hyperosmotic mannitol solution withphysiological Na concentration in either genotype of mice.
When Na, eDNA was introduced into the brain of theknock-out mice with an adenoviral expression vector,
only animals that received a transduction of the Na, gene
into the SFO among the CVOs recovered salt-avoiding
behavior under dehydrated conditions.
These results
clearly show that the SFO is the center of the control ofsalt-intake
behavior
inthebrain,
wheretheNa-level-sensitive Nax channel is invol ved in sensing thephysiological increase in the Na level of body fluids.

EGFP
(SFO)

Nax+EGFP
(SFO)

LJ
.

Nax+EGFP(OVLT).
o 2 4 6 a 10 12 hrs

Fig. 3. Abnormal salt-intake behavior of Nax knock-out
mice was rescued by introduction of the Na.. gene to SFO.
The coronal sections of the brain showing the loci infectedby the expression of EGFP (left column). Time course ofwater and saline (0.3 M NaCl) intake by the infected mice
before and after 48 hr dehydration (middle and ri ght
columns, res pecti ve ly). Behavioral data are the average ofsix mi ce that were successfully infected in a specific site in
the brain by an adenoviral vector encoding egfp (EGFP) or
by vectors encoding Nax and egfp (Na, and EGFP).

Publication List:
Nakayama, M., Kimura, M. , Wada, A. , Yahiro, K. , Ogushi ,K., Niidome, T., Fujikawa, A., Shirasaka, D., Aoyama, N .,Kurazono, H., Noda, M. , Moss, J. and Hirayama, T.
(2004) Helicobacter pylori VacA activates the
p38/activating transcription fac tor 2-mediated signal
J. Bioi. Chem. 279,
pathway in AZ-521 cells.
7024-7028.
Ohyama, K ., Ikeda, E., Kawamura, K., Maeda, N. and Noda,
M. (2004) Receptor-like protein tyrosine phosphatase
1/RPTP~ is expressed on tan gentially aligned neurons inearly mouse neocortex.
Develop. Brain Res., 148,
121-127.
Shintani , T., Kato, A., Yuasa-Kawada, J. , Sakuta, H.,
Takahashi, M ., Suzuki, R. , Ohkawara, T ., Takahashi, H.
and Noda, M. (2004) Large-scale identification andcharacterization of genes with asymmetric expression
patterns in the developing chick retina. J. Neurobiol., 59,
34-47.
Ohkawara, T., Shintani, T., Saegusa, C., Yuasa-Kawada, J. ,Takahashi , M. and Noda, M. (2004) A novel basic
helix-loop-heli x(bHLH)
transcriptional
repressor,
NeuroAB, expressed in bipolar and amacrine cells in th echick retina. Mol. Brain Res. 128, 58-74.
Hiyama, T. Y. , Watanabe, E., Okada, H. and Noda, M.
(2004) The subfornical organ is the primary locus of
sodium-level sensing by Nax sodium channels for the
J. New-asci., 24,
control of salt-intake behavior.
9276-9281.
Kiyosue, K. , Hiyama, T. Y. , Nakayama, K. , Kasai, M. andTaguchi , T. (2004) Re-expression of NR2B-containing
NMDA receptors in vitro by suppression of neuronal
activity. Int. J. Dev. New·osci., 22, 59-65 .
Muramatsu, H., Zou, P ., Suzuki, H ., Oda, Y ., Chen, G- Y .,Sakaguchi, N., Sakuma, S. , Maeda, N., Noda, M. , Takada,Y. and Muramatsu, T. (2004) a4 ~ 1 - and a 6 ~ 1 -integrins arefunctional receptors for midkine, a heparin-binding growth
factor. J. Cell Sci. , 117, 5405-5415.
Fukada, M. , Kawachi , H., Fujikawa, A. and Noda, M. (2005)
Yeast substrate-trapping system for isolating substrates ofprotein tyrosine phosphatases : Isolation of substrates forprotein tyrosine phosphatase receptor type z. Methods ,35, 54-63.
N iisato, K., Fujikawa, A., Komai , S. , Shintani, T., Watanabe,E., Sakaguchi , G., Kats uura, G., Manabe, T. and Noda, M.
(2005) Age-dependent en hancement of hippocampal LTP
and impairment of spatial learn ing through the ROCK
pathway in protein tyrosine phosphatase receptor typeZ-deficient mice. J. New·osci. , in press.

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGYDIVISION OF BRAIN BIOLOGY
Professor:
Research Associates:

Technical Staff"
Technical Assistants:
NJBB Research Fellow:
Postdoctoral Fellows:
Graduate Students:

Secretary:

YAMAMORJ, Tetsuo
KOMINE, Yuriko
WATAKABE, Akiya
KJTSUKAWA , Takashi
OOSA WA , Sonoko
MIKJ, Kazuhiko
ISHIKAWA , Takaka
Sf, Xuaohui
SAKATA, Shuzo
KOMATSU, Yusuke
TAKAHATA, Toru
SASAKI, Tetsuya
TAKAJJ, Masajiuni
NAKAMURA, Toru
HJROKAWA , Junya
HAYASHI, Hitomi

Our aim of research is to understand molecularmechanisms underlying memory, formation andevolution of the brain. For one approach to understandthese questions, we are studying the genes that are
expressed in specific areas of the primate neocortex.
We have obtained genes that showed marked
differences within primate neocotiical areas. Our
second approach is to understand informational
processing in the brain underlying learning behaviors
by examining gene expression.

37of occl tumed out to be activity dependent, because,in the monocularly deprived monkeys injected with
TTX into one of the eyes, the expression of occl ismarkedly decreased in the ocular dominance columnsof the primary visual cortex (Vl). We also
demonstrated that occl expression was markedly
increased postnataly in V 1. Interestingly, the specificexpression of excitatory cells in the primary visualcortex was only observed in primates (Takahata et al. ,in the meeting of Soceity for Neuroscience in North
America , SFN meeting, 2004).

V1

RBP

I. Genes expressed in specific areas of the

neocortex
The neocortex is most remarkably evolved in th
eprimate and plays the major role in higher brain
functions. It is divided into distinct functional andanatomical areas and has been a matter of debate what
extent areas of the neocortex are genetically andenvironmentally deter-mined. It is also puzzling why,
during the evolution of mammals, the neocortex wasmarkedly expanded while the number of the genes in
the mammal was little changed. In order to elucidate
these questions, we studied gene expression withjn
different areas of the neocortex.
1) We examined I 088 genes by macroatTay analysis
and found that most genes showed only less twofold
difference in their expressions among the threeneocotiical (frontal, motor and visual) areas. Only one
gene showed more than three fold difference andanother one was between two and three fold difference
within the three areas (Watakabe et al., Mol. BrainRes., 88, 74-82, 2001) . These results suggest that thegenes that are expressed among the different areas of
the human neocortex are very similar. However, thequestion remained whether there are any genes thatshow marked difference wi thin neo-cortical areas.
2) ln order to answer this question, we have
emp loyed differential display methods and found three
genes that indicated area specific expressions.
i) One gene, designated occl , is spec ifically
expressed in the occipita l cortex, patiicularly in V 1area, in the primate brain. Furthermore, the express ion

occ1 D

A_j
Fig. I Expression pattem of occl and Rbp in the
neocortex.
In situ hybridization pattern of occl and Rbp in the
primate neocortex. The two genes are expressed
complimentary occl is markedly expressed in the visualcortex. On the other hand Rbp is specifically expressedin association areas. ps: principal sulcus, sar: superior
arcute sulcus, cs: central sulcus, ips: inferior parietal
sulcus, lu: lunate sulcus, cal: calcarin sulcus, ios:
inferior occipital sulcus, ecal: external cal (Komatsu eta!., Cerbral Cortex, 15, 96-108, 2005).
ii) The other gene that showed marked difference
within the neocortex, is gdj7, a member of
BMP/TGF - ~ family, which is specifically expressed in
the motor cmiex of the African green monkey(Watakabe et al. , J. Neurochem. , 76, 1455-1464,
200 1).
iii) This year, we further report the third gene, Rbp(retinol-binding protein), which was preferentiallyexpressed in association and higher areas in the
neocortex (Komatsu et al., 2005). Rbp also shows
characteristic fe atures. i) Its express ion is high insensory association and higher association areas andlimbic areas, but low in the primary sensory areas.

38

Expression is complementary to that of occl and toparvalbumin immunoreactivity (PV-IR) in primarysensory areas. ii) In early sensory pathways, theexpression is limited to superficial layers only (inparticular, layer II). With progression into higher
sensory areas, the expression is expanded into layers 3
and then 5. iii) In higher-order association areas, Rbp
is expressed throughout all layers except layer 4. iv)
This characteristic distribution of Rbp is mainly
formed during postnatal development. Rbp probably
regulates the concentration of retinoic acid (RA) by
the delivery of retinol, which is converted into RA incells. Although the role of RA in the mature brain is
not yet known, the characteristic expression of Rbp
within association areas may provide a clue to themolecular basis of the formation and function of theassociation areas.
3) We have also further isolated several area
specific genes with RLCS (Restriction Landmark
eDNA Scanning). We are now characterizing thesegenes to reveal the mechanisms that form neocorticalareas.
In summary, our studies thus far revealed thefollowing points.
(1) Genes that are specifically expressed within
neocortical areas in the primate neocotex are similar
overall.
(2) We have identified several genes that are
distinctively different among neocortical areas.
(3) These genes are specific in visual, motor andassociation areas.
(4) A gene specific in the visual cortex (occl) is
activity dependent and also postnatally regulated.
(5) Rbp is expressed in association areas in acomplimentary manner to the expression of occl.
(6) These results suggest that these genes may be
useful markers to study the mechanisms underlying
neocortical formation.

II. Gene expression under a declarative and anon-declarative memory
In order to study informational processing
underlying the declarative and non-declarative
memory at molecular and cellular levels in the brain,
we employed c-Fos mapping techniques, for which weused gene expression of c-Fos. There have been an
increasing number of studies using c-Fos as markers to
examine neuronal activities ever since c-Fos induction
by electrical stimulation was found. However, since
many sensory stimuli per se cause c-Fos induction, we
should be very· careful to distinguish the c-Fos
expression that is caused by learning process from thatcaused by sensory stimuli. For this purpose, it isnecessary to use behavioral systems that are able todistinguish the difference of the two. Although a few
behavioral systems in rodents have been successfully
used for physiology, animal behavior and recently for
analyses of knockout mice, little behavioral systems infact distinguish the difference. Therefore, we prepared

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

ourselves for using two behavioral systems, which
represent declarative and non-declarative memory.
( 1) We have been collaborating with professor
Yoshio Sakurai (Kyoto University) who developed an
audio-visual discrimination task (AVD-task) system.
In this task, a rat was asked to choose either an audiocue (a high tone or low tone) or a visual cue (a lightfrom the right or the left) to obtain a food pellet . We
found that the visual and audio tasks enhanced thespecific expression of c-Fos in the visual and audio
cortices, respectively. Among the early visual andauditory pathways examined, c-Fos was specificallyinduced in the cortices but not in the earlier pathways,
suggesting the neural modulation of the neocortex
depending on the types of the tasks. Interestingly, thetask-dependent Fos expression was only observed in
excitatory neurons in the relevant sensory cortices.
Although this AVD task system is quite powerful toanalyze the problem described above and useful forstudying underlying molecular and cellularmechanisms because of advantages of using rodents,
one concern was that the auditory stimuli and visual
stimuli are in different positions. Thus we cannot
exclude the possibility that the difference between theauditory task and the visual task may not completelydepend on the modality (visual Vs auditory)
difference.
We wanted to solve this problem by placing
auditory and visual stimuli in the same position. We
also use nose-poking to measure the reaction time inwhich a rat responds to stimuli. By using thisbehavioral system, we were able to confirm amodal
recognition of space which means that a rat can
respond to a different modality (visual or auditory) if
the stimuli are in the same position and previously
reported in other systems. We also confirm
multisensory enhancement is indeed observed in rats.
These results suggest that this new modified AVD
system can be used to explore the molecular andcellularmechanisms
underlying
multisensory
processing in rats (Sakata, 2004).

Stimuli presentation(500ms)Target detection\"
Fig.2 Newly developedAVD task.
A rat faces to a panel with a visual cue and/or an
auditory cue located in the same position, being asked
to poke its nose into one of the holes to obtain reward (apellet) (The figure is cited from Sakata eta!., Exp Brain
Res, 159, 409-417, 2004).

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

Various oscillations are observed depending on
brain-states. Spike wave complexes (Sws), 7-IlHz
cortical oscillations with harmonics in awake but
immobile rats, have been widely regarded as a modelof paroxysmal activities in absence epilepsy. However,
several studies have suggested that SWs in the primarysomatosensory cortex are analogous to human murhythms. Because SWs have been frequently observed
depending on vigilance levels, SWs in rats might
represent normal brain-states related to thesleep-waking cycle. To elucidate behavioral contexts
to induce SWs and temporal relations between SWs
and neuronal ensemble activities, we recorded local
field potentials (LFP) and multi-unit activities(MUAs) in the medial prefrontal cortex andelectroencephalogram (EEG) in the bilateral regions ofrats. Long-term recordings of EEG revealed that SWs
were prominently generated in frontal and parietal
regions and that SWs frequently followed non-REM
sleeps. Occurrence probabilities of SWs significantly
increased after the rats performed cognitive tasks. Our
results suggest that SWs are one of thebrain-state-specificoscillations
ratherthanpathological activities. We also observed that MUAs
were organized into phase-locked patterns in cycles ofthese oscillations. MUAs recorded from electrodes
apart to each other were synchronized during SWs.
(Sakata eta!., SFN Meeting. 2004).
(2) The other task we developed is a wheel runningsystem in which a water-deprived mouse is asked to
run to obtain water in front because the wheel with thepegs is turning to the other direction (Kitsukawa et a!.,SFN Meeting, 2002). The pegs can be changed withvarious patterns as desired. The task required for themouse thus can be regarded to represent a procedural
learning. We examined a various areas of brains
following to the change of the peg pattern. Among theareas examined, we found marked c-Fos expression in
the striatum, cerebral cortex. The striatum, which iscomposed of projection neurons and several
distinguished types of interneurons, is known to play
an important role in a reward-based learning. The
characterization of these subtypes of interneurons has
been progressed. However, their roles in behavioral
tasks have been little known. Combining with c-Fos
mapping technique and neuronal specific markers or
Dil labeling, we revealed specific activation ofbilaterally projecting cortico-striatal neurons andparticular types of striatal neurons upon change ofsteps. These results suggesting cortico-striatal circuits
actively participate in complex motor learning(Kitsukawa eta!., SFN Meeting, 2004).

Publication list:
Sakata S, Komatasu Y, Yamamori T. (in press) Local
design principles of mammalian cortical networks.
Neurosci Res.
Komatsu Y, Watakabe A, Hashikawa T, Tochitani S,
Yamamori T. (2005) Retinol-binding Protein gene is39

Highly Expressed in Higher-order Association Areasof the Primate Neocortex. Cereb Cortex. 15, 96-108
Sakata S, Yamamori T, Sakurai Y. (2004) Behavioral
studies of auditory-visual spatialrecognition andintegration in rats. Exp Brain Res, 159, 409-417
Ichinohe N, Watakabe A, Miyashita T, Yamamori T,
Hashikawa T, Rockland KS. (2004) A voltage-gatedpotassium channel, Kv3 .I b, is expressed by a
subpopulation of large pyramidal neurons in layer 5 of
the macaque monkey cortex. Neuroscience, 129,
179-185.
Nakayama T, Mikoshiba K, Yamamori T, Akagawa K.
(2004) Activation of syntaxin I C, an alternative splicevariant of HPC-1/syntaxin lA, by phorbol
12-myristate 13-acetate (PMA) suppresses glucose
transport into astroglioma cells via the glucose
transporter-! (GLUT-1). J Bioi Chem, 279,
23728-23739.

40

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGYDIVISION OF BEHAVIORAL BIOLOGY (ADJUNCT)
Professor (Adjunct):
Associate Professor(Adjunct):
Research Associate:
NJBB Research Fellow:
Graduate Student:

MORI, Yuji
TSUKAMURA, Hiroko
IMAMURA , Tak:uya
IKEMURA, Ry ota
TAKEUCHI, Motoki IJI) Nagoya University

found that there exist the regions between - 1,000 and- 200, in which DNA methylation pattems differeddepending on brain subarea. For ERa., no differences arefound within male brain subareas examined. In contrast,
hypermethylation was specifically observed in the BNST
of female rats (Fig. 2). Similarly, we discovered thesex-dependent pattern of DNA methylation in the AR and
PR, where hyper- and hypomethylation was found in
VMH of female and male, respectively.

In mammals, several social behaviors are dependent onsex. Cooperative regulation of many autosomal genes aswell as those on the sex chromosomes constitutes theirdifferences. We are currently investigating the epigenetic
status of the critical brain subareas responsible for the sex
difference of the behaviors by analyzing genomic DNA
methylation patterns.

DNA methylation of nuclear receptors
In mammals, DNA methylation, mainly occmTing on
CG dinucleotides, is a fundamental mechanism thatdifferentiates the gene expression pattem in respective
cell. DNA methylation is a restraint of the pluripotency
because once the patterns are established during
development it is maintained through cell division. On theother hand, for example, some fishes that contain muchless methylation activity are found to easily and reversibly
change the sex status according to the environmental
context. In rodents, endocrine distmbance at the fetal
and/or postnatal stages irreversibly changes the behaviors
such as the lordosis (in females) and the mounting (inmales) after the pubet1al stages that are normallydependent on genetic sex . In some cases, lordosis can beobserved even in males and vice versa. These clearly
indicate that sex-dependent patterns of behaviors are notdirectly dependent on " sex-specific" genes but rather
establishedthrough
epigenetic
processes .
The
sex-dependent pattems of behaviors must be acquired
through highly irreversible processes dming development.
We hypothesize that the long-term effects of the sex
steroid at the perinatal stages on behaviors after puberty
are somehow marked at the genome level. In this year of
2004, we report herein the DNA methylation pattem ofsteroid receptor genes (estrogen receptor a., ERa. ;androgen receptor, AR; progesterone receptor, PR) in themale and female brain subareas.
In general , the binding affinity of the transcription
factors is reduced by hypennethylation that are frequent lyobserved on promoter region of either ERa., AR or PR intumor cells (Fig. 1). We analyzed the DNA methylation
status of their 5' -flanking regions in the rat using thebisulfite sequencing method. In either of the genes,
hypomethylated status was observed between - 200 and+ I (in relation to the transcription start site) in preoptic
area, bed nucleus of the stria terminalis (BN ST),ventromedial hypothalamic nucleus (VMH), hippocampus,
arcuate nucleus, and amygdala, suggestin g that the coreregion of the promoters are ma inta ined hypomethylated innorma l tissues in contrast to the tumor cell s. We also

Fig. I. Schematic representation of DNA methylation andchromatin structure. Upper panel shows the inacti ve statethat reduces the transcriptional activity of the associatedgene. Lower panel shows the DNA methylation-free statethat all ows DNA binding proteins, including transcription
factors , accessible to the cis-regulatory element. Lollipops
denote the methylated cytosines.

Several reports described the significance ofgonad-derived factors, such as testosterone, at theperinatal stage on brain masculinization. To determine if
the sex-dependent DNA methylation status of nuclear
receptors is established by gonadal factors, we made useof adult male rats castrated at the onset of birth . DNA
methylation pattern of AR in VMH was reversed to thatobserved in the fema les, indicating that the epigenetic
status follows the genetic sex through endocrine pathway,
at least partially. However, no significant differences were
found in the DNA methylation status of ERa. and PR in
the examined brain subregions between normal andcastrated males. Therefore, these suggest that prenatal
factors also function to set up the masculized/defemini zed
pattern of DNA methylation in the brain.
In conclusion, we found the area- and sex-specific
DNA meth ylation patterns of nuclear receptors. We are
currently ex ploring the molecul es that determine the DNA
methy lation pattern using the target cis-elements fo r
differenti al methy lation found in thi s study.

41

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

A

Ex.C

...

Ex.B

Ex.A

~~Ex.1I

ATG

8FemalePreoptic area

Hippocampus

BNST

Amygdala

VMH

Male

Male (PO cast)

-··
-·11
·-·
--1111

Arcuate nucleus.

Fig. 2. DNA methylation analysis of rat estrogen receptor aA. Genomic structure of rat estrogen receptor a. Ex.
denotes exon. Arrowheads indicate the relative positions of
the primers for the bisulfite sequence analysis. B. Bisulfite
sequencing of estrogen receptor a in adult rats. Methylation
patterns of CG dinucleotides are shown. Each row of circles
represents a single cloned allele with open circles fornon-methylated cytosines and filled circles for methylated
cytosines. PO cast, adult male castrated at postnatal day 0.

Publication List:
Kanari, K., Kikusui, T., Takeuchi, Y., Mori, Y. (2005)
Multidimensional structure of anxiety-related behavior inearly-weaned rats. Behavioral Brain Res. 156, 45-52.
Kikusui, T., Takeuchi, Y., Mori Y. (2004) Early weaning
induces anxiety and aggression in adult mice. Physiol.
Behav. 81, 37-42.
Masuda, K., Hashizume, C., Momozawa, Y., Kikusui, T.,
Takeuchi, Y., Mori, Y. (2004) Breed differences inofcatecholgenotype
andallelefrequency

0-methyltransferase gene polymorphic regions in dogs. J.
Vet. Med. Sci. 66, 183-187.
Kiyokawa, Y., Kikusui, T., Takeuchi, Y., Mori, Y. (2004)
Modulatory role of testosterone in alarm pheromone
release by male rats. Hormones Behav. 45, 122-7.
Kiyokawa, Y., Kikusui, T., Takeuchi, Y., Mori, Y. (2004)
Alarm pheromones with different functions are released
from different regions of the body surface of male rats.
Chern. Senses 29, 35-40.
Kiyokawa, Y., Kikusui, T., Takeuchi, Y., Mori, Y. (2004)
Partner's stress status influences social buffering effects in
rats. Behavioral Neurosci. 118, 798-804.
Ito, H., Nara, H., Inoue-Murayama, M., Shimada, M. K.,
Koshimura, A., Ueda, Y., Kitagawa, H., Takeuchi, Y.,
Mori, Y., Murayama, Y., Morita, M., Iwasaki, T., Ota, K.,
Tanabe, Y., Ito, S. (2004) Allele frequency distribution of
the canine dopamine receptor D4 gene exon III and I in 23
breeds. J. Vet. Med. Sci. 66, 815-820
Masuda, K., Hashizume, C., Ogata, N., Kikusui, T., Takeuchi,(2004)
Sequencing of canineY.,
Mori, Y.
5-hydroxytriptamine receptor (5-HTR) lB, 2A, 2C genes
and identification ofpolymorphisms in the 5-HTR1B gene.
J. Vet. Med. Sci. 66,965-972.
Takigami, S., Mori, Y., Tanioka, Y., Ichikawa, M. (2004)
Morphological evidence for two type of mammalianvomeronasal system. Chern. Senses 29,301-310.
Estacio, M.A., Tsukamura, H., Reyes, B. A., Uenoyama, Y.,
I' Anson, H., Maeda, K.-1. (2004) Involvement ofbrainstem catecholaminergic inputs to the hypothalamicparaventricular nucleus in estrogen receptor aexpression in this nucleus during different stressconditions in female rats. Endocrinology, 145,4917-4926.
Moriyama, R., Tsukamura, H., Kinosita, M., Okazaki, H.,
Kato, Y., Maeda, K.-1. (2004) In vitro increase in
intracellular calcium concentrations induced by low orhigh extracellular glucose levels in ependymocytes andserotonergic neurons of the rat lower brainstem.
Endocrinology 145,2507-2515.
Kinoshita, M., I' Anson, H., Tsukamura, H., Maeda, K.-1.
(2004) Fourth ventricular alloxan injection suppresses
pulsatile luteinizing hormone release in female rats. J.
Reprod. Dev. 50,279-285.
Imamura, T., Miyauchi-Senda, N., Tanaka, S, Shiota, K.
(2004) Identification of genetic and epigenetic similarities
of SPHK//Sphkl in mammals, J. Vet. Med. Sci. 66,
1387-1393.
Imamura, T., Yamamoto, S., Ohgane, J., Hattori, N., Tanaka,
S., Shiota, K. (2004) Non-coding RNA directed DNA
demethylation of Sphkl CpG island, Biochem. Biophys.
Res. Commun. 322, 593-600.
Imamura, T., Neildez, T. M., Thenevin, C., Paldi, A. (2004)
Essential role for poly (ADP-ribosyl)ation in mouse
preimplantation development, BMC Mol. Bioi. 5, 4.
Ikemura, R., Matsuwaki, T., Yamanouchi, K., Nishihara, M.
(2004) Involvement of endogenous vasopressin in highplasmaosmolality-induced
anorexia
via
VI
receptor-mediated mechanism. J. Vet. Med. Sci. 66,
951-955.

42

NATIONAL INSTITUTE FOR BAS IC BIOLOGYNEUROBIOLOGYLABORATORY OF NEUROPHYSIOLOGY
Associate Professor:
JST Technical Staff:

WATANABE, Eiji
TAKEUCHI, Kazumi

When the correct balance between water and sodium
level in the body fluid has been broken, terrestrial animals
feel water and salt appetite or satiety, and theseperceptions subsequently induce the animal behaviors
referred to as ingestion or aversion. Our research is
focus ing to understand molecular and neural mechanisms
underlying the animal behaviors essential to homeostasisof the body fluid.
To exp lain the properly regu lated animal behaviors,
neurobiologists have postulated the existence of both
osmoreceptors and specific sodium receptors in the brain.
However, the molecular entities of osmoreceptors andspecific sodium receptors have long been enigma. In 2000,
we first clarified by using gene-targeting technology that
Na, sodium cha1mel is a probable candidate for the
specific sodium receptor in the brain.
Na, sodium chmmel had long been classified as a
subfamily of voltage-gated sodium channels (NaChs) thatserve to generate action potentials in electrically excitable
cells such as neuronal and muscle cells. Comparing withthe other NaChs, however, Na, channel has unique aminoacid sequences in the regions, which are known to be
involved in ion selectivity and voltage-dependent
activation and inactivation, suggesting that it must havespecific functional properties.
To clarify the functional role of Nax channel, Na,-gene
deficient mice were generated by gene-targeting
technique and the physiological phenotypes have been
examined. Behavioral studies suggested that the Nax
channel plays an important role in the central sensing ofbody-fluid sodium level and regulation of salt intake
behavior. Na,-deficient mice ingest hypertonic sodium
chloride solution in excess in comparison with wild
type-mi ce. LacZ reporter gene knocked into Na,.-gene
locus revealed that Na, gene is expressed in thecircumventricular organs, which is the specialized central
organs involved in sensing of sodium concentration andosmosity in the body fluids .

•• •• ••• •

I

ill

!I

••
••
• •

• • e• Na+
Ext ra cellularI
Intracellular•••••

Figure 1. Na, is a sodium channel sensitive to extracellularsodium level. When the extracellular sodium concentration
is increased, Nax channel opens the gate pore and generates
the sodium ion influx into the cells. This view was
hypothesized by ion-imaging studies.

In 2002, sodium ion imaging and electrophysiologicalstudi es using cultured cells derived from the subfornicalorgans demonstrated that Na, channel is an extracellularsodium-level sensitive sodium channel (Fig. 1). Further,
we found that Na, channel is expressed in
non-myelinating Schwmm cells and alveo lar type II cells
in addition to the cells in the circumventricu lar organs .
Na, channel is thus likely to be involved in reception of
sodium-level in the body fluids at the circumventricul arorgans and sod ium absorption in the viscera l nervous
system and in the lung.
In 2003 , we fo und in collaboration with Prof.
Yamamoto 's group of Osaka University that the
periphera l nervous system bas on ly subtle effects on thehigher preference for sodium ch loride as observed in the
mutant mice. The results suggest that the mutant
phenotype is mainly due to the lack ofNa, channel in thecentral nervous system.

AB

0.145 M NaCI +0. 71 M Mannitol

+I+

c

+I+

E

0

I

~ ~ ~~-

0

ic:

0.2

0.15
0.1
- 0.05-/-0

0 2 4 6 8 10 12

0 2 4 6 8 10 12
hrs

Figure 2. Na_.-knockout mice are insensitive to increases ofthe sodium level in the cerebrospinal fluid. (A) Left,
Location of the cannula for intracerebroventricularinfusions. The tip of the cannula was positioned at thelateral ventricle. Right, a schematic representation of theexperimental set up for the two-bottle test. Two drinking
tubes were presented to free -moving mice infused withsodium solutions into the ventricle for 12 hrs.
(B)
Averaged time course of water and saline (0.3 M NaCl)
intake in wi ld-type (+/+) and knockout (-/-) mice during
intracerebroventricular infusions of a hypertonic (0.5 M)
NaCl solution, or hypertonic mannitol (0.145 M NaCI +0.71 M mannitol) solution. Each point shows the averagequantity per I 0-min period.
In this year of 2004, we developed an automatic
measurement equipment for intake volume of drinking
so lutions (Fig. 2A, right). Using this equipment, we
showed that the subfornical organ is the principal site forthe control of salt-intake behavior, where the Nax channel
is the sodium-level sensor. Infusion of a hypertonic
sodium solution into the cerebral ventricle induced
extensi ve water intake and aversion to saline in wild-type

NATIONAL fNSTITUTE FOR BASIC BIOLOGYNEU ROBIOLOGY

animals but not in the knockout mice (Fig 2B, left).
Impm1antly, the aversion to salt was not induced by theinfusion of a hyperosmotic mannitol solution withphysiological sodium concentration in either genotype ofmice (Fig. 2B, right). When Nax eDNA was introduced
into the brain of the knockout mice with an adenoviral
expression vector, only animals which received atransduction of the Na, gene into the subfornical organ
among
the
circumventricularorgansrecoveredsalt-avoiding behavior under dehydrated conditions .
These results clearly show that the subfomical organ isthe center of the control of salt-intake behavior in thebrain, where the sodium-level-sensitive Na, channel isinvolved in sensing the physiological increase in thesodium level of body fluids.
CutTently, in order to understand how the subfomicalorgan translates extracellular sodium-level sensed by Na,
channel to the neural activities, we identified subcellular
localization of Na, channel in the subfornical organ.
lmmuno-electronmicroscopic and double itmmmostaining
studies clearly showed that Na, channel was exclusively
localized to perineuronal lamellar processes extended
from astrocytes and tanycytes in the organs (Fig. 3).
Importantly, glial cells derived from the subfornical organ
were capable of sensing extracellular sodium-level, as
analysed by ion-imaging method. In addition, we foundthat the Na, -expressing glial cells enveloped GABAergic
interneurons in the subfornical organ. Finally, in the
subfornical organ , neuronal population activated by water
deprivation was different from GABAergic interneurons,
as monitored by Fos immunoreactivity. Together withprevious observation that the subfornical organ of Na,
knockout mice is hyperactive under water deprivation,these results indicate that the glial Na, channel senses
increased sodium-level in the body fluid and inhibit the
subfornical organ neuronal activity through control of
GABAergic
inhibitory
interneurons.
A
novelcommunication style utilized the sodium-level sensitive
sodium channel appears to occur between inexcitable glial
cells and excitable neural cell.
Since we first reported aberrant behaviors found in Na,
knockout mice, a series of our studies have clarified that
Na, channel is a sodium-level sensitive sodium channel
playing an essential role in the sodium-sensing of thecircumventricular organs and in the control of salt-intake
regulation. These works identified the molecular entity ofthe brain sodium sensor, which has long been
hypothesized as one of the important physiological issues .
In this year, we newly demonstrated that the primary
subcellular locus sensing sodium-level is perineuronalglial processes . This finding suggest that neuron-glia
complex plays a key role on the sodium sensing in thecircumventricular organs.
We are now trying to construct functional expression
systems of Na, sodium channel using vanousheterologous cell lines. The heterologous expression
system will provide us useful information on the channel
characters ofNa, channel.

43

I

·~

/

A *

8

Figm·e 3. Na, channel is localized to the perineuronalprocesses of astrocytes and tanycytes in the subfornicalorgan. (A and B) Coronal tissue sections of the subfomicalorgan are stained with anti-Na, antibody. lmmunopositive
signals are widely observed within the subfomical organ(A) and most of intensive signals are accumulated in the
shape of ring (B). An arrow indicates immunopositive
tanycyte-layer peeled off from the subfornical organ and an
asterisk indicates the choroid plexus. (C) Immuno-electron
microscopic studies using anti-Na, antibody. A core regionof the subfornical organ is shown. Neurons and theirprocesses are surrounded by immunopositive thin processesof astrocytes. In inset of C, ventricular surface of the
subfornical organ is shown. A neuronal process isenveloped with immunopositive thin processes of atanycyte. Arrows point at immunopositive signals . V,
ventricle; N, neuron; S, synapse; T, tanycyte; Ast, asb·ocyte;
Np, neural process; An asterisk in C is an artificial vo id
region occurred during fixation or staining procedures.
Scale bars, 50 ~tm for A, I0 J.lll1 for B, I J.ll11 for C and 0.4~tm for inset.

Publication List
Hiyama, T.Y., Watanabe, E., Okado, H., and Noda, M.
(2004) The subfornical organ is the primary locus of
sodium-level sensing by Na, sodium channels for thecontrol of salt-intake behavior. J. Neurosci. 24, 9276-9281
Niisato, K. , Fujikawa, A. , Komai, S., Shintani , T. , Watanabe,E., Sakaguchi , G., Katsuura, G., Manabe, T. and Noda, M.
(2004) Age-dependent enhancement of hippocampal LTP
and impairment of spatial leaming through the ROCK
pathway in protein tyrosine phosphatase receptor typeZ-deficient mice. J. New-asci. in press

44

NATIONAL rNSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGYLABORATORY OF NEUROCHEMISTRY
Associate Professor:

SASAOKA, Toshikuni

Our major research interest is to understand aphysiological role of dopaminergic system in animal
behavior, especially eating behavior using geneticallyaltered mice, both transgenic and gene knockout mice. In
addition we develop a novel method of conditionalmutagenesis in mice in order to analyze the function of
the gene of interest in detail. We analyze function ofneurotransmitter receptor complex as a whole by using
biochemical methods of analysis of the dystrophin
complex on the skeletal muscle membrane.

I. Role of dopaminergic transmission in eating
behavior
The dopaminergic system is implicated in theregulation of the several peptide hormones in the pituitary,
the modulation of locomotor activity, the modulation ofsynaptic plasticity and the neural development. The
dopaminergic system is also implicated in control ofemotion, motivation and cognition. Dysfimction of
dopaminergic system can result in several neurologicaland psychiatric disorders such as Parkinson 's disease andschizophrenia.

D2R, D3R, D4R and D5R) are identified and divided into
two subgroups referred to as D !-like (D l R, D5R) andD2-like (D2R, D3R and D4R) receptors on the basis of
their gene structure and their pharmacological andtransductional properties. D l R and D2R are mostabundantly and widely expressed in the brain and often
play a role synergistically. DlR has an opposite property
to D2R with respect to the intracellular signaltransduction.
In order to understand a role of dopaminergictransmission in behavior of interest, it is necessary todelete both Dl-like and D2-like receptors. We havegenerated DlR/D2R double knockout (DKO) mice bycrossing between D IR knockout (KO) and D2R KO mice,and observed that DlR/D2R DKO mice exhibited
impairment in eating behavior and premature death. To
investigate molecular mechanism of eating we havegenerated mutant mice in which the expression of
dopamine receptor could be controlled temporally bytetracycline system on the D IR/D2R DKO background.
We plan to establish and analyze the mutant mice.

II. Developing a novel conditional mutagenesis
method in mice.

Glutamatergic synapse

Dopaminergic synapse
Tyrosine--~ Tyrosine

t
t

DOPA

Presynaptic
terminal

NMDAR

NMDAR

INMDARs are blocked I

mutant NMDARs areaberrantly activatedFigure 2. A single amino acid substitution (595'h asparagine
to 595'h glutamine) of NR2A subunit leads to an aberrant
activation ofNMDAR.

Dopamine • •••••••••••••••••••• ••~

signal transduction~
signal transduction

Figure l. Schemati c drawing of dopamin ergi c synapse.

In mammals fi ve subtypes of dopamine receptor (DlR,

The aim of the study is to overcome the limitations of
the conventional mouse molecular genetic approach in thefunctional analysis of target genes. We substituted one
critical amino acid residue of N-methyl-D-aspartate
receptor (NMDAR), leading to NMDAR activation. The
NMDARs are widely expressed in the nervous system,
fundamental to excitatory neurotransmission, and play anumber of important roles at different brain loci and time
points. The NMDARs act as a coincidence detector andare not only important for neuronal differentiation,
migration , and survival but are also critical for activity
dependent synapse forma tion. It is suggested that theaberrant activation of NMDAR causes excitotoxicity,
leading to neuronal death in various neurological diseases.

45

NATIONAL INSTITUTE FOR BASIC BIOLOGYNE UROBIOLOGY
2That the Ca + permeability through NMDAR is blocked2
by magnesium (Mg +) in a voltage-dependent mannerindicates an essential role of NMDAR as a coincidence
detector. Functional NMDARs consist of NMDARl(NRl) subunit and at least one subunit ofNMDAR2A-2D(NR2A-NR2D). The NRl subunit is ubiquitously
expressed in the brain, while NR2 subunits have a more
specific spatial distribution. It has been shown that the
NR1/NR2A complex expressed in cultured cell is highly2
sensitive to the voltage-dependent Mg + block and that thesubstitution of asparagine (Asp595) by glutamine(Gln595) in the second transmembrane domain of the2
NR2A subunit results in a reduction of the Mg + block of
the NR1/NR2A complex (Figure 2).
Mice expressing normal
NMDAR before Cre-

Mice expressing Cre
recombinase in cellsof interest

However, the role of Asp595 of the NR2A subunit andthe effects of substitution of it with Gln595 on thefunction ofNMDAR in vivo remain to be clarified.
We develop conditional mutagenesis method in miceusing Cre-loxP recombination (Figure 3 and 4). By our
method, we accomplished conditional substitution of theamino acid in mice and our mutant mice exhibitedabenant NMDAR activation and a neurologicalphenotype, similar to that of mouse models ofneurological disorders. The development of our mutant
mice should contribute to understanding the function of
the critical amino acid residue and the molecularmechanism of neurological disorders.
Our new method is vastly applicable to functional
analysis of any desired gene and should contribute tostudies on the structural and functional relationships ofrelevant genes.

Ill. The molecular architecture and thephysiological role of the sarcoglycan complex(SGC)

Mice expressing mutant NMDAR in the specificcells after Cre-loxP recombination

Figure 3. Conditional mutagenesis in mice. First, mutant
mice expressing normal NMDAR molecule before Cre-loxP
recombination were generated. Second, transgenic miceexpressing Cre recombinase in cells of interest weregenerated. Third, these two mouse lines were crossed togenerated mice expressing mutant NMDAR molecule in thecells in which Cre-loxP recombination was executed.

Sarcoglycans (SG) are trans-sarcolemmal glycoproteins,which associate together to fmm SGC and present in thesarcolemma. SGC, together with dystrophin and thedystroglycan complex comprises the dystrophin complex,
which is considered as the mechanical link between thebasement membrane and the intracellular cytoskeleton forprotecting the sarcolemma from mechanical stress during
muscle contraction. Each of four SG subunits (a-, ~- , yand 8-SG) is responsible for four respective forms of
SG-deficient muscular dystrophy, sarcoglycanopathy(SGP). All of the SGs and sarcospan are absent in thesarcolemma in any form of SGP, suggesting that the SGC
is not assembled if a single subunit of the SGC is absent.
To understand a physiological role of the SGC, we
have generated the ~-SG KO and y-SG KO mice. The~-SG KO and y-SG KO mice developed progressive
muscular dystrophy and all SGs and sarcospan wereabsent in the sarcolemma of the ~-SG KO and y-SG KO
mice. The dystrophin complex isolated from the skeletal
muscles of ~-SG KO mice was biochemically unstable in
the absence of the SGC and sarcospan. This indicates that
SGC and sarcospan play an impm1ant role in stabilizing
the dystrophin axis connecting the basement membrane
and the cytoskeleton.
We plan to analyze a molecular architecture and aphysiological role of dopamine receptor complex as awhole by applying analytical method of the SGC.

Publication

Figure 4. Detection of cells in which recombination was
executed by Cre-IoxP system by ex pression of green
fluoresce nt protein (green) in the hippocampus. Neurons
were stained by NeuroTrace (red).

Mizuno, Y. , Guyon, J. R., Watkins, S. C. , Mizushima, K.,
Sasaoka, T. , Imamura, M. , Kunkel , L. M. , Okamoto, K.
(2004) Beta-Synemin locali zes to regions of hi gh stress inhuman skeletal myofibers. lvfuscle and Nerve 30, 337-346

46

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITYDIVISION OF MOLECULAR GENETICS
Professor:
Research Associates:

Technical Staffs:
NIBB Research Fellows:
JSPS Postdoctoral Fellows:

Postdoctoral Fellow:
Visiting Scientist:
Graduate Students:

Technical Assistants:

Secretary:

IIDA, Shigeru
TERADA, Rie
HOSHINO, Atsushi
TSUGANE, Kazuo
FUKADA-TANAKA, Sachiko
YAMAGUCHI, Katsushi
PARK, Kyeung-11
CHOI, Jeong-Doo (-Mar. '04)
EUN, Chang-Ho
COTSAFTIS, Olivier (Oct. '04 -)JIANG, Gonghao (Nov. '04 -)JOHZUKA-HJSATOMI, Yasuyo
KOUMURA, TashiroOHNJSHI, Makoto IJ
SHIMATANJ, Zenpei IJ
ONODA, Shiho If
TAKAGI, Kyoko 2;
MORITA, Yasumasa
NAGAHARA, Miki (-Mar. '04)
SAITO, Miho (-Aug. '04)
ASAO, Hisayo
IKEGAYA, Kyoko
MATSUMOTO, Miwako
SHIMAMOTO, Miki (Nov. '04 -)HASEGAWA, Yoshinobu 3J (Nov. '04 -)SANJO, Kazuko

IJ Graduate University for Advanced Studies2) Graduate School of Hokkaido University
3) OKAZAKI Silver Employment Agency

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

I. Spontaneous mutants in morning glories.
Considerable attention has recently been paid to themorning glory genus Ipomoea because of theexperimental versatility of its floral biology including thegenetics of floral variation, flavonoid biosynthesis, andtransposon-induced mutations. The genus Ipomoea
includes about 600 species distributed on a worldwide
scale that exhibit various flower morphologies andpigmentation patterns. Among them, three morning
glories, Ipomoea nil (the Japanese morning glory),
Ipomoea purpurea (the common morning glory), andIpomoea tricolor, were domesticated well as floricultural
plants, and many mutants displaying various flower
pigmentation patterns were isolated. The wild-type I. nil
displays blue flowers (Figure lA) that contain thepeonidin (3' methoxyl cyanidin) derivative named
Heavenly Blue Anthocyanin or HBA.

I. nil had been introduced into Japan from China
approximately in the 8th century as a medicinal herb, theseeds of which were used as a laxative, and the plant
became a traditional floricultural plant in Japan aroundthe 17th century. The plant has an extensive history ofgenetic studies, and a number of its spontaneous mutants
related to the color and shape of the flowers have been
isolated. Genetic studies on the color of I. nil have shownthat blue flower coloration was mainly controlled by two
genetic loci, Magenta and Purple. Recessive magenta
and purple mutants bloom magenta and purple flowers,
respectively, and double mutants carrying both magenta
and purple alleles display red flowers (Figure 1C). The
Magenta gene encodes flavonoid 3 '-hydorxylase, whichhydroxylates the 3' position of the B-ring ofanthocyanidin precursors. The Purple gene encodes avacuolar Na+IH+ anti porter called InNHX1 that increases
the vacuolar pH during flower opening, causing a shift
towards the bluer coloration. Among the various colors of
I. nil flowers, the most favorite hue for Japanesefloriculturists has been reddish-brown or purplish-grey
petals (Figure IB and D) since the early 19th century, andthe flower coloration is mainly caused by recessive dusky
mutations. We noticed that the petals in all dusky mutants
often contained intensely pigmented globules, whichappeared to affect flower hue. We found that the Dusky
gene
encodes
UDP-glucose:anthocyanidin
3-0-glucoside-2" -0-glucosyltransferase (3GGT), whichcatalyzes the the conversion of anthocyanidin
3-0-glucosides into anthocyanidin 3-0-sophorosides(Figure 1E) and that all of the dusky mutants tested carrythe 4-bp insertion mutations GGAT or CGAT at an
identical position near the 3' end of the gene, which
resulted in frameshift mutations (Figure 1F). The
expected 3GGT enzymatic activities were found in the
crude extracts of Escherichia coli, in which the 3GGT
eDNA was expressed, and the introduced 3GGT eDNA
could efficiently produce 3GGT that could convert
cyanidin 3-0-glucoside into cyanidin 3-0-sophoroside intransgenic petunia plants.
In the purple mutant deficient in the InNHXJ gene forthe vacuolar Na+IH+ anti porter, the vacuolar alkalization
occurs only partially, and reddish-purple buds become
purple open-flowers. While most of the plant NHX genes
characterized are generally expressed in leaves, stems androots and induced by NaCl treatment, the JnNHXJ gene ispredominantly expressed in the flower limbs at around 12
hour before flower-opening.
It is expressed very
scarcely in leaves, stems and roots, and no induction
occurs in response to NaCl treatment. We identified anovel vacuolarNa+/H+ antiporter gene JnNHX2, which isexpressed in leaves, stems and roots and is induced in
In addition, relatively
response to NaC1 treatment.
higher expression of InNHX2 was observed in the flower
We alsolimbs shortly before flower-opening.
discovered that both the InNHX1 and InNHX2 proteins
could catalyze both Na+ and K+ transport into vacuoles.
These results suggest that InNHX2 performs dual
functions: to confer salt tolerance on the plant and topromote partial vacuolar alkalization in the petals.

47

NATIONAL IN STITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSLTY

(E)

ex
~~

3GGT

H~~"'/ #~/V" 0
I

OH

..

OH

R
2

n---7

~t;.~~HHe( r:JH

OH
+ UDP

HO

Anthocyanidin 3-0-glucosides

Anthocyanidin 3-0-sophorosides(F)
Dusky gene
Dusky

•

0.1 kbGATCTGG.l\TTCA.n..Z\GTACATGGACTCTTTCAATCAGAAACTGCAGGATCTCCTTGGATGADLDSKYHDSFNQ

K

LQD

LL

G*

d u sky- 1 GATCTGG.~TGG.~TTCAAAGTACA"r GGAC T CTTTCAA T CAGAAAC TGCAGGATCTCCT T GGATGAATATAATATAA
DtD

G""f

KVHGLFQSETli.GSPi'JNNII•

dusky-2 GATCTGGATCGATTCAAAGTACATGGACTCTTTCAATCAGAAACTGCAGGATCTCCTTGG.A.TGAATATAATATA.Jl.
D T. D p:--p K V
H G
l.
F Q
S
E 'I'
A
G S
P ~;I
~1 N II_.

Fig.!. Flower phenotypes and the Dusky gene for the 3GGT enzyme in I. nil. A-D. Flower phenotypes (above) and microscopic
photographs of adaxial epidermal ce ll s of iJ ower peta ls (below). (A) The wild-type line with the Magenta, Pwple, and Dusky all eles.
(B) The line with the Magenta, Purple, and dus/,_)'-1 alleles. (C) The line with the magenta, pwple, and Dusky alleles. (D) The line
w ith the magenta, pwple, and dusky-/ a ll eles. Scale bars in microscopic photographs indi cate 30 [.tm. E. Reaction mediated by the
Dusky gene products, 3GGT. F. The JGGT gene and the dus/..J' mutation s. The large verti ca l arrow indicates the site of 4-bp in sertions(above) , and the 4-bp insertions underlined in the dusky mutants (below) result in fram eshift mutation s.

II. Modification of endogenous natural genes byhomologous recombination in rice.
Rice (Ot) IZG sativa L.) is an important staple food formore than half of the world 's population and a model
plant for other cereal species. We have developed alarge-scale
Agrobacterium-mediatedtransformation
procedure with a strong positive-negative se lection andsucceeded in efficient and reproducible targeting of the
Waxy gene by homologous recombination without
concomitant occunence of ectopic events, which must be
an important first step for developing a precise
modification system of the genomic sequences in rice. By

improving our transformation procedure further, we areattempting to modify the Adh I and Adh2 genes, which
belong to a small multigene family and reside adjacent torepetitive Copia- and Gypsy-like retroelements (Figure 2).
The Adhl and Adh2 genes reside on chromosome 11 in
the same orientation with the 30 kb of interval , and thecoding sequences of Adhland Adh2 are similar to eachother. The results obtained indicate that the gene targetingby homologous recombination occurred more effici ently
in Adh2 than that in Adh 1.

~?.Okb ~ ~

Ch.11

6.0kb

6.2kb

R8

D
Fig.2. Strategy for gene targeting of the Adh l and Adh2 genes in rice. The symbols hand DT-A on the T-DNA reg ions of thevectors used indicate the pos iti ve and negati ve se lection markers, respect ive ly. The hypothetica l gene flanked by th e
retroelements on chromoso me II is indicated by J-Jyp.

48

(A)

NATIONAL INSHTUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY(B)

OnoartPy/gene300 bpFig.3. A. Leaf phenotypes of the mutable pyl-v and the stablepyl-stb alleles. B. Structure of the pyl-v allele. The white andgreen boxes represent the Py l exons and cod ing region,
respectively. The upper box indi cates nDartl.

III. Characterization of a mutable virescent allele in
rice.
Leaves of seedlings in the virescent mutant of rice are
initially pale-yellow green due to partial deficient inchlorophyll and gradually become green with the growthof the mutant. We have been characterizing a spontaneous
mutable virescent allele, pale-yellow learvariegated(pyl-v), confeiTing pale yellow leaves with dark green
sectors in its seedlings (Figure 3A). The pyl-v mutant was
isolated among progeny of a hybrid between indica andjaponica rice plants. The leaf variegation is regarded as arecurrent somatic mutation from the recessive pale-yellow
allele to the dark green revertant allele. From the pyl-vline, we also obtained a stable pyl-stb (py l-stable) line thatexhibits pale-yellow leaves without variegation (Figure
3A), which appeared to cany no active autonomous
element acting on the nonautonomous DNA element
inserted into the Py l gene. The availability of the genomic
sequences of both japonica and indica subspecies
facilitates map-based cloning of the pyl-v allele. We
identified an active nonautonomous DNA transposon of
about 0.6 kb, named nDartl (nonautonomous DNA-based
active rice transposon one), in the untranslated exon I of
the Py l gene on chromosome 3 (Figure 3B), and excisionof the new DNA transposon from the pyl gene appears to
be responsible for conferring the leaf variegation. We also
showed that the transposition of nDartl could be controlled
under natural growth conditions. No somaclonal variation is
likely to occur in mutant lines induced by our newly
characterizing endogenous element, because no tissue
culture has been involved in its activation. In thjs respect,
it is impm1ant to emphasize here that tissue culture isnecessmy in all of the currently available rice reverse
genetic approaches including transposon tagging systems
employing exogenous or endogenous transposons. We are
currently attempting to develop a novel transposon
tagging system in rice.

Publication List:
Yoshida, H., Akimoto, H., Yamaguchi, M. , Shibata, M. ,Habu, Y., Iida, S. and Ozeki, Y. (2004) Alteration of
methylation profiles in distinct cell lineages of the layers
during vegetative propagation in carnation (Dianthus
caryophyllus). Euphytica 135, 247-253.
Terada, R., Asao, H. and !ida, S. (2004) A Large-scale
Agrobacterium-mediated transformation procedure wi th astrong positive-negative selection for gene targeting in rice
(01yza sativa L.). Plant Cell Reports 22, 653-659.
lida, S. , Morita, Y. , Choi, J.D., Park, K.I. and Hoshino, A.
(2004) Genetics and epigenetics in flower pigmentation
associated with transposable elements in morning glories.
Adv. Biophys. 38, 141-159.
Toki , K., Saito, N. , Morita, Y. , Hoshino, A. , Iida, S.,
Shigihara, A. and Honda, T. (2004) An acylated
pelargonidin 3-sophoroside fro m the pale-brownish red
flowers of Ipomoea nil. Heterocycles 63, 1449-1454.
!ida, S. and Terada, R. (2004) A tale of two integrations,transgene and T-DNA: gene targeting by homologous
recombination in rice. Curr. Opin. Biotechnol. 15,
132-138.
Park, K.I., Choi, J.D., Hoshino, A., Morita, Y. and !ida, S.
(2004) An intragenic tandem duplication 111 a
gene
fo r
anthocyan intranscriptional
regulatory
biosynthesis confers pale-colored flowers and seeds withfine spots in Ipomoea tricolor. Plant J. 38, 840-849.
Olmishi , M., Fukada-Tanaka, S., Hoshino, A., Takada, J. ,[nagaki, Y. and Iida, S. (2005) Characterization of a novel
Na+/H+ antiporter gene InNHX2 and comparison of
InNHX2 with InNHXI , which is responsible for blue
flower co loration by increasing the vacuolar pH in theJapanese moming glory. Plant Cell Physiol. (in press).
Morita, Y., Hoshino, A., Kikuchi, Y., Okuhara, H. , Ono, E.,
Tanaka, Y. , Fukui, Y. , Saito, N., Nitasaka, E. , Noguchi , H.
and Iida, S. (2005) Japanese morning glory duslcy mutants
displaying reddish-brown or purplish-grey flowers are
deficient in a novel glycosylation enzyme for anthocyan inbiosynthesis,
UDP-glucose:anthocyanidin
3-0-glucoside-2 " -0-glucosy ltransferase, due to 4-bp
insertions in the ge ne. Plant J. (in press).

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITYDIVISION OF GENOME DYNAMICSHORIUCHI, Takashi
KOBAYASHI, Takehiko
JOHZUKA, Katsuki
MOROOKA, Naoki
Technical Staff:
JSPS Research Fellow: AUSTEN RD Ganley
KODAMA, Ken-ichi
Postdoctoral Fellows:
OHSUMI, Katufumi
SERIZAWA, Naomi
VTSitingPostdoctora!Fellow: CUI, Tailing (Apr. 1 -)Visiting Scientist:
UJIIE, Yoshifumi
Graduate Students:
WATANABE, Takaaki

Professor:
Research Associates:

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
instability are fully understood. To clarify these aspects,
we are pursuing the following themes using E. coli and S.
cerevzswe: ( 1) the amplification mechanism of repeatedsequences or genes, especially the rRNA repeated genes,(2) the mechanism of replication fork block-dependent
recombination, a key reaction that increases or decreases
the copy number of rRNA genes, and (3) development ofin vivo artificial gene amplification systems. In 2004,
work on the following four subjects has advanced ourknowledge of the dynamics of the genome.
I. Transcription-mediated hyper-recombination in
HOTJ, a recombinational hotspot in S. cerevisiae.

Recombination hotspots are DNA sequences which
enhance recombination around that region. HOT1 is oneof the best-studied mitotic hotspots in yeast. HOTJ
consists of two elements; I and E. They are two
dis-continuous regions in an rDNA unit. The I-element
corresponds to a RNA polymerase I (Poll) transcription
promoter which is responsible for 35S ribosomal rRNA
gene (rDNA) transcription. The E-element overlaps theenhancer for Poll transcription, containing a replication
fork barrier site (FRB) where Fob! protein, required forfork blocking at RFB, binds specifically.
HOTJ
stimulates recombination when inserted at novel locationsin the genome. For example, when HOT! is integrated
into one of the repeated his4 genes, the fragment enhances
recombination between the repeats ~ 100 times. In a Poll
defective mutant the HOT1 hotspot activity is abolished,
therefore transcription of HOT1 is thought to be an
important factor for the recombination stimulation.
However, it is not clear whether the transcription itself orother pleiotropic phenotypes stimulate recombination. To
investigate the role of transcription, we made a highly
activated Pol I transcription system in HOT1 by using astrain whose rDNA repeats are totally deleted (rdn~~). In
the rdn~~ strain, HOT1 transcription was increased about
14 times compared to wild-type. Recombination activity
stimulated by HOT1 in this strain was also elevated, about
15 times, compared to wild-type. These results indicatethat the level of Pol I transcription in HOT1 determines

49

efficiency of the recombination. Moreover, Foblp, whichis essential for both the recombination stimulation activity
and transcription of HOT1, was dispensable in the rdn~~
strains. This suggests that Fob 1p is functioning as a Polltranscriptional activator in the wild-type strain.

II. SIR2 regulates recombination between different
rDNA repeats, but not recombination within
individual rRNA genes in yeast.
In most eukaryotic organisms, the rRNA genes (rDNA)
are clustered in long tandem repeats on one or a few
chromosomes. Although the total number of thesechromosomal rDNA repeats appears to be maintained at alevel appropriate for each organism, genes with such a
repeated structure are in general thought to be unstablebecause of a high frequency of recombinational events.
Thus, it might be expected that organisms have developed
systems to regulate recombination within rDNA repeats.
In the yeast S. cerevisiae, approximately 150 copies of
rDNA aremaintainedonchromosome
XII.
Recombinational events within the rDNA repeats innormal growing yeast cells appear to be mostly mediatedby a FOB1-dependent system. FOB1 is the gene required
for fork blocking activity at RFB site, recombination in
the rDNA region, and expansion/contraction of rDNA
repeats. The latter two activities are likely to be triggeredby double-strand breaks at the RFB site and repair of the
breaks via gene conversion. On the other hand, the SIR2
gene plays an important role in decreasing the frequencyof recombination in yeast rDNA. Sir2p is a protein
required for transcriptional silencing at three yeast
chromosomal regions, silent mating type loci, telomeres,and rDNA. It is generally believed that Sir2p, perhaps
through its NAD+-dependent histone deacetylase activity,
plays an essential role in forming a higher order ofrepressive chromatin structure - heterochromatin - which
prevents general access of the Po III machinery and someother macromolecules, thus causing silencing as well as
decreasing recombination in the chromosomal rDNA
repeats. Therefore, mutations in gene SIR2 increase
recombination within rDNA repeats as assayed by marker
loss or extrachromosomal rDNA circle formation.
We examined the mechanism involved in the increased
frequency of recombination in rDNA repeats that isobserved in mutants defective in SIR2 functions. We
measured the frequency of FOB1-dependent arrest of
replication forks, consequent DNA double-strand breaks,
and formation of DNA molecules with Holliday junction
structures, and found no significant difference between
sir2L1 and SIR2 strains. Formal genetic experiments
measuring mitotic recombination rates within individualrRNA genes also showed no significant difference
between these two strains. Instead, we found a significant
decrease in the association of the cohesin subunit Mcd 1p(Scclp) to the rDNA in sir2L1 relative to SIR2 strains.
From these and other experiments, we conclude that SIR2
prevents
unequalsister-chromatid recombination,

50

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUT IONARY BIOLOGY AND BIODIVERSITY

probably by forming special cohesin structures, witho ut
significant effects on recombinational events withi n
individual rRNA genes.

resistance. However, the mechanism is largely unknown
because of the complexity of the amplification process.
We developed a gene amplification system in
Saccharomyces cerevisiae that is based on double
rolling-circle replication (DRCR), uti lizing break-induced
replication (BIR), and this is depicted in the Figure below.
This system produced three types of amplification
products. Type-1 products contain 5- 7 inverted copies of
the amplificatio n marker, leu2d. Type-2 products containIII. A novel gene amplification system in yeast
based on double rolling-circle replication.
Gene amplification is involved in various biologicalphenomena such as cancer development and drug

A New Gene Amplification System

chrVI
LS20( ade3: :Ga/HO)
ARS609DSB_L~-+-J-~o-•.-cENamplification unit

+

~

c:>-a

a TEL
c> YF2c> YF4..2_~~ leu2d

BIR (~<>-e+__cc:~ BIR

URA3
HO cut site+

For~ ~ork+

Double Rolling Circle ~ ¢-Gill
amplificationc~ +..2_~-£}<>-+~by

Recombination ?

~~ ~J..
..
¢

~~

+

--~~¢~~0CJ--tli!ID

Figure: A new gene amp lification system in which ampli fication through doub le ro lling circ le rep lication (DRCR) is
triggered by break- induced replication (BIR). Top shows the structure of the right ten"ninus region of chromosome VI
where an amplification unit (-leu2d-YF4-HO- URA3-HO-YF2- leu2d-) was inserted. Induction of doub le strand breaks(DSBs: shown by the two anows) by HO endonucl ease tri ggers BIR, which initiates DRCR. An amp lification selecti ve
marker, leu2d, is used to se lect amplifi ed clones. The DRC R process is expected to tenninate by recombination between/ue2d genes on eac h bidirectiona ll y elongated arm. ARS: autonomously repli cating sequence; CEN : centromere.

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

13 to -100 copies of leu2d (up to -730 kb increase) withnovel arrangement present as randomly oriented
sequences flanked by inverted leu2d copies. Type-3
products are acentric multi-copy mini-chromosomescarrying leu2d. Structures of type-2 and -3 products
resemble those of homogeneously staining region (HSR)
and double minutes (DMs) of higher eukaryotes,
respectively. Interestingly, products analogous to thesewere generated at low frequency without deliberate DNA
cleavage. These features strongly suggest that theprocesses described here may contribute to natural gene
amplification in higher eukaryotes.

VI. Recombination enhanced by replication fork
blockage at the Ter site on E. coli plasmids.
In order to elucidate the effects of replication arrest,
which is of frequent and spontaneous occurrence,
replication blocking sites have been frequently used. In
E. coli, when both bi-directional replication forks areblocked at the two flanking replication blocking (Ter)sites, the next round of replication forks arrive at the Ter
sites, producing a giant linear DNA molecule, whose
terminus ends lead to recombination and overcome theblockage at the Ter sites through an unknown mechanism.
On the other hand, eukaryotic chromosomes, andprobably bacterial plasmids also, do not initiate such asecond round of replication, at least under normal growth
conditions. When plasmid replication is arrested at thefork blocking Ter site, it remains unknown whether
Therefore, we
recombination is enhanced or not.
investigated whether recombination is stimulated whenplasmid replication is inhibited by a 22 bp TerA site and,if the recombination is activated, what kinds ofrecombinational genes are required for the activation byusing various rec-defective mutants as hosts.
Furthermore, the recombinational genes involved in the
SOS response, which is induced by plasmid replication
blockage at the 22 bp Ter site, were also examined. The
results were ( 1) the recombination is enhanced 5 - 7 foldby the fork block, (2) this enhancement is dependent on
recA, reeF recO, recR and reel, but not recBC and recQ,(3) SOS induction is dependent on reeF absolutely, andreel and recQ partially, (4) the recombinational
enhancement is disappeared when the 22 bp TerA
sequence was replaced by the 0.6 kb original
TerA-containing E. coli chromosomal fragment, which isconsistent with the results of SOS induction. The lastresult indicates that in plasmids multiple rounds ofreplication do not occur, and furthermore fork blocking at
the Ter site induces not only recombination but also the
SOS response. This correlation suggests that there may be
a common process between the initiation steps of therecombination and the SOS induction. A mechanism bywhich recombination and the SOS response were induced
by replication blockage at the 22 bp TerA site, but not the
0.6 kb TerA containing E. coli chromosomal fragment,
was proposed.

51

Publication list:
Serizawa, N., Horiuchi, T., and Kobayashi, T. (2004)
Transcription-mediated hyper-recombination in HOTJ.
Genes Cells. 9, 305-315
Kobayashi, T., Horiuchi, T., Tongaonlar, T., Vu, L., andNomura, M. (2004) SIR2 regulates recombination between
different rDNA repeats, but not recombination within
individual rDNA genes in Yeast. Cell77, 441-453.
Kasarjian, JK., Hidaka, M., Horiuchi, T., Iida, M., and Ryu, J.
(2004) The recognition and modification sites for thebacterial type I restriction systems KpnAI, StySEAI,StySENI and StySGI. Nucleic Acids Res. 15;32(10):e82.
Komori K., Hidaka M., Horiuchi T., Fujikane R., Shinagawa
H., and Ishino Y. (2004) Cooperation of the N-terminal
Helicase and C-terminal Endonuclease Activities ofArchaeal Hef Protein in Processing Stalled Replication
Forks. J Bioi Chem. 279(51 ), 53175-53185
Watanabe, T., and Horiuchi, T. (2005) A novel gene
amplification system in yeast based on double
rolling-circle replication. EMBO J. 24, 190-198.

52

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITYDIVISION OF EVOLUTIONARY BIOLOGY
Professor:
Associate Professor:
Research Associates:
Technical Staff:
NIBB Research Fellow:
JSPS Research Fellow:
Postdoctoral Fellows:
Graduate Students:

Technical Assistants:

Secretaries:

HASEBE, Mitsuyasu
MURATA, Takashi
FUJITA, Tomomichi
HIWATASHJ, Yuji
SUMIKA WA, NaomiNISHIYAMA, Tomoaki
SATO, Yoshikatsu
AONO, Naoki
MIYAZAKI, Saari
HASHIMOTO, Kaoru
MORINAGA, Shin-ichi(Tohoku Univ.) (Apr. 1 -)HOSOKAWA, Kentaro(Tokyo Univ.) (Apr. 1 -)AOKJ, Etsuko
BITOH, Yoshimi (-Apr. 30)
NARUSE, Mayumi (-Jun. 30)
HAYAKAWA, Yuko (Aug. 1-31)
ICHIKAWA, Yuki (Jul. 16 -)KAJIKAWA, Ikumi (Aug .1-31)
KJTANI, Masakazu
MAKINO, Haruko (May. 1 -)DONO, Chikako (-Jun. 30)
SUZUKI, Yoriko
TANIKAWA, Yukiko (-Jun. 30)
WATASE, Masataka (May. 1 -)KABEYA, Kazuko (- Mar. 30)
KOJIMA, Yoko (May. 1 -)All living organisms evolved from a common ancestor
that lived more than 3.5 billion years ago, and theaccumulation of mutations in their genomes has resulted
in the present biodiversity. Traces of the evolutionary
process are found in the genomes of extant organisms. By
comparing the gene sequences and gene networks ofdifferent organisms, we can infer (1) the phylogenetic
relationships of extant organisms and (2) the genetic
changes that caused the evolution of morphology anddevelopment. The inferred phylogenetic relationships
provide important insights into problems in various fieldsof evolutionary biology. Our group focuses onbiogeography, the evolution of morphological traits, andsystematics in a wide range of taxa. Concerning theevolution of morphology and development, we hope to
explore the genetic changes that led to the evolution of theplant body plan. We have selected Arabidopsis(angiosperm),
Gnetum(gymnosperm),
Ginkgo(gymnosperm),
Ceratopteris(pteridophyte),
Physcomitrella (bryophyte), and some green algae asmodels to compare the functions of genes involved in thedevelopment of both reproductive and vegetative organs
in land plants.

I. Origin of the Plant Cell
The first green alga cell evolved via symbiosis between
an ancestral non-photosynthetic eukaryote and acyanobacterium. Cyanobacteria now exist as chloroplasts
in the host cell. The factors and mechanisms ofchloroplast movement are being investigated to reveal themolecular
mechanisms
usedto"domesticate"cyanobacteria as organelles. Analyses of (I) cytosolic

calcium icon concentration and cytoskeleton organization
during chloroplast movement in the moss Physcomitrella
patens and (2) the functional divergence ofphotoreceptors
and motor proteins involved in chloroplast movement in
the moss from angiosperms are in progress by a team
directed by Y. Sato.

II. Evolution from unicellular to multicellularorganisms
The first evolutionary step from unicellular tomulticellular organisms is to form two different cells froma single cell via asymmetric cell division. The first cell
division of a protoplast isolated from the protonemata ofthe moss Physcomitrella patens is asymmetric regarding
to its shape and nature, and gives rise to an apicalmeristematic cell and a differentiated non-meristematiccell. A systematic overexpression screening for genes
involved in asymmetric cell division of protoplasts in P.
patens is in progress by a team directed by T. Fujita. Aftereliminating genes that are not directly involved inasymmetric cell divisions, such as photosynthesis genes,
we used 3000 clones as materials for the overexpression
screening. Individual cDNAs were subcloned under aconstitutive promoter and introduced into the protoplastsof P. patens for transient expression. We observed andcategorized phenotypes of the regenerating protoplasts.
Thus far we identified 58 cDNAs, whose overexpression
caused the defects in asymmetric cell divisions in two
repeated experiments. Overexpression of the genes inprotoplasts with GFP-tubulin or GFP-talin, expression
analyses of each gene-cytrin fusion protein under its
native promoter, loss of function experiments using RNAi
are now in progress to characterize what processes thesegenes are involved in. Functional analyses of these genes
should help us to understand molecular mechanisms of
how plants generate distinct cell lineages to build theirmulticellular bodies.

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

Organization of micro tubules
Cortical arrays of microtubules are essential formorphogenesis in plants. We found that, by live imaging,microtubules in the arrays are formed as branches on
pre-existing microtubules. y-tubulin, a protein that isessential for the formation of microtubules in animal cells,
is located at the branching point and in the cytoplasm, anda loss of y-tubulin due to gene silencing causes a
malformed organ with irregularly shaped cells. In vitro
experiments using isolated plasma membrane/microtubule
complexes suggested that y-tubulin in cytoplasm attaches
onto the side of existing cortical microtubules, andinitiates a new cortical microtubule from it. The nucleus is

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODfVERSITY

known to initiate microtubules after cell division. We
hypothesize that microtubules formed around the nucleus
elongate to cell surface, and trigger initiation of cortical
microtubules via attachment of y-tubulin. Once cortical
microtubules are formed, they can tumover without
microtubules from the nucleus. Factor(s) responsible forattachment of y-tubulin onto the side of microtubules is a
key element responsible for the difference between plant
and anima l cells. Isolation of the factor(s) responsible forattachment of y-tubulin onto the side of microtubules bybiochemical and other approaches is in progress by a team
directed by T. Murata.

IV. Evolution of molecular mechanisms in thedevelopment of vegetative organs
Meristem initiation and maintenance
Postemb1yonic growth of land plants occurs from themeristem, a localized region that gives rise to all adultstructures. Meristems control the continuous developmentof plant organs by balancing the maintenance andproliferation of stem cells, and directing theirdifferentiation. Meristem initiation and maintenance is afundamental question in plant development research.
Three lines, exhibiting reporter gene (uidA) expression
preferentially in the apical cells, were isolated frompreviously established gene- and enhancer-trap lines, andidentified as encoding kinesin- and ubiquitin-like proteins,and an unknown protei11. Functional analyses of thesegenes are cunently under investigation by a team directedby Y. Hiwatashi . The disruption of the kinesin-like gene
did not show any phenotypic differences from the wild
type. This is likely caused by the functional redundancyof closely related genes, and the analyses of double
disruptions are in progress. Disruption of the geneencoding ubiquitin-like protein suggests that the gene isinvolved in cell division and elongation throughmicrotubule organization with the proteasome complex.

53

sporophytic tissues examined, which is characteristic ofclass 2 gene expression in angiosperms. Unexpectedly, allthree CRKNOX genes were not detected in gametophyte
tissues by RNA gel blot a naly~is. Arabidopsis plants
overexpressing the fern class l genes resembled plantsthat overexpress seed plant class I KNOX genes in leaf
morphology. Ectopic expression of the class 2 gene in
Arabidopsis did not result in any unusual phenotypes.
Taken together with phylogenetic analysis, our results
suggest that l) the class l and 2 KNOX genes diverged
prior to the divergence of fem and seed plant lineages; 2)
the class I KNOX genes function similarly in seed plantand fem sporophyte meristem development despite theirdifferences in structure, 3) KNOX gene expression is not
required for the development of the fern gametophyte,
and 4) the sporophyte and gametophytic meristems offerns are not regulated by the same developmental
mechanisms at the molecular level (Sano et a!. in press).
To further investigate the evolution of meristem and
KNOX
genes,
characterization
of
the
moss
Physcomitrella KNOX genes are in progress by a team
directed by T. Nishiyama.
CRKNOX1
188 rRNACRKNOX2
188 rRNACRKNOX3
188 rRNA

10

Evolution of shoot meristem
The angiosperm shoot apical meristem is a dome ofsmall , proliferating cells whose organization is highly
structured into layers and/or zones. The apical meristem
of many seed-less vascular plants contains a single apicalcell or initial that is apparent in both the gametophyte(haploid) and sporophyte (diploid) generations . Given theimportance of the apical meristem in elaborating the three
dimensional body plan of plants, the apical meristem isthought to be among the most important innovations forthe evolution of land plants . In order to gain insights into
the molecular mechanisms underlying the development
and evolution of the plant meristem, we have identified
and analyzed the expression of two class l and one class 2
KNOX (knotted-like homeobox) genes from the fern
Ceratopteris richardii. Expression of both class 1 genes
was detected in the shoot apical cell , leaf primordi a,marginal part of the leaves, and vascular bundles by insitu hybridization, a pattern that closely resembles that ofclass 1 KNOX genes in angiosperms with compound
leaves . The fern class 2 gene was expressed in all

12(Figure 1) Expression patterns of three KNOX genes in the
fem Ceratopteris richardii. The tissues from which RNAs
were extracted are shown in the schematic figures.

V. Origin and evolution of floral homeotic genes
The flower is the reproductive organ in angiosperms,
and floral homeotic genes, such as MADS-box genes and
FLO/LEAFY genes, regulate floral organ identity. To
investigate the origin of floral homeotic genes, thefuncti o n ~ of these genes in several basal angiosperms, themoss Physco mitrella, and three green algae ( Chara,
Co/eochaete, and Closterium) are being analyzed.

54

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

Land plants are believed to have evolved from agametophyte-dominant ancestor without a multicellularsporophyte; most genes expressed in the sporophyte
probably originated from those used in the gametophyte
during the evolution of land plants. To analyze theevolution and diversification of MADS-box genes in land
plants, eight MADS-box genes predominantly expressedin pollen, male gametophyte, are analyzed by a team
directed by N. Aono.

VI. Molecular mechanisms of speciation
Reproductive isolation is the first step in speciation. To
obtain insights into reproductive isolation, several
receptors specifically expressed in the pollen tube arebeing studied to screen for the receptors involved inpollen tube guidance by a team directed by S. Miyazaki.
Polyploidization is a major mode of speciation in plants,
although the changes that occur after genome duplication
are not well known. Polyploid species are usually larger
than diploids, but the mechanisms responsible for the size
difference are unknown. To investigate these mechanisms,
tetraploid Arabidopsis was established and its gene
expression patterns are being compared to those of diploidwild-type plants using microarrays.

sequences has a good statistical foundation and is not
rejected statistically by other datasets. We proposed
bryophyte monophyly as the current best hypothesis(Nishiyama et al. 2004).

VIII. Evolution of RNA editing in land plants
We sequenced transcripts from all putative genes forproteins, rRNAs, and a selection of those encoding tRNAs
in the chloroplast genome of the fern Adiantum
capillus-veneris. We detected 349 RNA editing sites whenthe eDNA sequence was compared to that of the genomic
DNA. The level of RNA editing in this fern is more thanten times that of any other vascular plant examined acrossan entire chloroplast genome. A previous study foundeven higher levels of editing in a hornwort (942 sites).
This suggests that the relatively low levels of editing inseed plants (less than 0.05%) may not be typical for land
plants, and that RNA editing may play a major role inchloroplast genome processing. Additionally, we foundthat 53 editing sites in Adiantum are homologous toediting sites in the hornwort, and some other land plants.
This implies that a major component of RNA editing sites
have been conserved for hundreds of millions of years.

Publication List:
VII. Phylogenetic analysis of land plants
Opinions on the basal relationship of land plants varyconsiderably and no phylogenetic tree with significant
statistical support has been obtained. We performed
phylogenetic analyses using 51 genes from the entire
chloroplast genome sequences of 20 representative green
plant species. The analyses, using translated amino acidsequences, indicated that extant bryophytes (mosses,
liverworts, and hornworts) form a monophyletic groupwith high statistical confidence, and that extant
bryophytes are likely sister to extant vascular plants,
although the support for monophyletic vascular plants
was not strong. Analyses at the nucleotide level could not
resolve the basal relationship with statistical confidence.
Bryophyte monophyly inferred using amino acid

Lotus comlculatus var. japonlcus
Arabldopsis thaliana- - Uverworts--Mosses
Anthoceros angustus (eDNA) - - Hornworts' - - - - Chastosphaerfdlum globosum, . - - - - - - - Nephroselmls olivacea

L------ Chiarella vulgaris

L - - - - Mesostlgma vir/de

0.1 substitutions/site(Figure 2) Phylogenetic relationship of representative

lineages in land plants inferred using 8,979 amino acidresidues for 51 genes from 20 chloroplast genomes.

Sano, R., Juarez, C. M., Hass, B., Sakakibara, K., Ito, M.,
Banks, JA, and Hasebe, M. 2005. KNOX class of
homeobox genes potentially have similar function in both
diploid unicellular and multicellular meristems, but not in
haploid meristems. Evol. Dev. in press.
Kishi, M., Murata, T., Hasebe, M., and Watanabe, Y. 2005.
An extraction method for tobacco mosaic virus movement
protein localizing in plasmodesmata. Protoplasma in press.
Hattori, M., Hasebe, M., and Sugita, M. 2004. Identificationand characterization of cDNAs encoding pentatricopeptide
repeat (PPR) proteins in the earliest land plant, the moss
Physcomitrella patens. Gene 343: 305-3 I I
Tamura, M.N., Fuse, S., Azuma, H., and Hasebe, M. 2004.
Biosystematic studies on the family Tofieldiaceae I.
Phylogeny and circumscription of the family inferred fromDNA sequences of matK and rbcL. Plant Bioi. 6: 562-567.
Rutherford, G., Tanurdzic, M., Hasebe, M., and Banks, J.A.
2004. A systemic gene silencing method suitable for highthroughput, reverse genetic analyses of gene function infern gametophytes. BMC Plant Biology 4: 6.
Wolf, P.G., Rowe, C.A., and Hasebe, M. 2004. High levelsof RNA editing in a vascular plant chloroplast genome:
analysis of transcripts from the fern Adiantum
capillus-veneris. Gene 339: 89-97.
Nishiyama, T., Wolf, P.G., Kugita, M., Sinclair, R.B., Sugita,
M., Sugiura, C., Wakasugi, T., Yamada, K., Yoshinaga, K.
Yamaguchi, K., Ueda, K., and Hasebe, M. 2004.
Chloroplast phylogeny indicates that bryophytes aremonophyletic. Mol. Bioi. Evol. 21: 1813-1819.
Aoki, S., Uehara, K., Imafuku, M., Hasebe, M., and Ito, M.
2004. Phylogeny and divergence of basal angiospenns
inferred from APETALA3- and PISTILLATA-Iike
MADS-box genes. J. Plant Res. 117: 229-244.

NATIONAL INSTIT UT E FOR BASIC BIOLOGYEVOL UTlONARY BIOLOGY AND BIODIVERSITYDIVISION OF SPECIATION MECHANISM(ADJUNCT)
Professor (Adjunct):
Research Associate:
Postdoctoral Fellow:
Technical Assistant:

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

The above members of this adjtmct division havestarted a new project in this institute in April 2003.
During the long histmy of evolution of vertebrates,
they acquired extensive diversity in characters such asmorphology, ecology and behavior. Some examples, suchas conserved morphological traits of coelacanths, showthat phenotypic change during the course of evolution
could be sometimes vety slow, whereas other examples
suggest, by contrast, that their speciation anddiversification could be often accomplished vety rapidly
by the process called adaptive radiation. What factors
affected rates of speciation and phenotypic change during
the evolution of vertebrates? Although many examples ofadapti ve radiation (and slow evolution as well) have beenreported so far for various groups of vertebrate, its
mechanism has been hardly clarified so far, especiallyfrom the molecular perspective. The final goal of theresearch of our divi sion is to understand the processes andmechanisms of speciation of vertebrates using molecularapproaches. In particular, we are dedicated to elucidate
the mechanism of speciation of cichlids in Lake Victoria(Figure 1), based on our knowledge on the phylogenetic
relationships among African cichlids as clarified by our
retroposon analysis.

Figure I. A species of cichlid fish (male Pundami!ianyererei) caught in Lake Victoria.

I. Inference of molecular phylogeny of vertebrategroups using the SINE method
Cichlid flocks of the East African Great Lakes, which
consi st of Lakes Victoria, Malawi, and Tanganyika, haveattracted interest of evolutionary biologists fo r more thana centuty. These species exhibit extraordinary levels ofdiversity and high species endemi city to each lake as aresult of explosive adaptive radiation that occurred
independently and in para llel.

55

Our group has been conducting phylogenetic analysisof these fish using insertions of retroposons (SINEs; shortinterspersed elements) as markers for elucidation of their
evo lutionaty history. Our studies confirmed that cichlids
in Lakes Victoria and Malawi are closely related to eachother, and that species in the both lakes are related to only
a portion of the major lineages found in Lake Tanganyika,
the oldest Great Lake estimated at 9-12MY. Interestingly,
evidence from the above analyses suggested that
explosive radiation is indeed in progress in the Victorian
fauna at present (see Section II for the detail)
Another group of vertebrate that we analyzed using
SINEs was the order Testudines (turtles), whichconstitutes one of the major reptile groups. Our analysis,
focusing especially on the superfamily Testudinoidea,
revealed several novel phylogenetic relationships among
members of this group. Our result indicated thatconvergence of the major morphological traits that bad
not been successfully elucidated by the pastmorphological analyses occutTed during their evolution.
This study was the first application of the SINE method toa phylogenetic analysis of reptiles .

II. Analysis of Cichlids in Lake Victoria forelucidation of molecular mechanisms of their
speciation and diversification
Among the cichlid faunas in African Great Lakes, theone in Lake Victoria is considered to be the youngest
since this lake is suggested to have once been dried up
about 12,000 years ago . Such an evolutionary very recent
origin of this fauna is consistent with the observation by
our group that most of selectively-neutral polymorphic
alleles (presence/absence of retroposons at orthologous
sites in the genome) are retained both within and among
species of this lake. This fact means that, if a certain allele
is uniquely fixed at a certain locus in natural populationsof a certain species, we can assume the past and/or
present existence of a positive selection on this gene. In
wch a case, we may be able to regard it as a candidate
gene that was responsible for the phenotypic
differentiation of this species from other related species.
Thus, investigation of cichlids in Lake Victoria isadvantageous for the purpose of elucidation of molecularmechanisms of spectatwn and their following
diversification during adaptive radiation.
The most impressive appearance of cichlids in Lake
Victoria is vivid and beautiful coloration of their body in
mal e adults (see Figure l for an example). Cichlids are
known to depend largely on their vision when they choose
individuals to mate with, and such color variations are
considered to affect mate preference by females . This fact
implies that body color of males plays an impmiant rolefor recognition of indi viduals of their own species duringmating with females. At the same time a similar logic canbe app lied to the visual system of females since the bodycolor of males are recognized by fe males through theirvisual system. Thus, if one or both of these factors
changed significantly, speciation might occur due to achange in their mate choice. Another interesting point on

56

the visual system of cichlids is that their evolution musthave been affected by environmental difference in theirhabitat such as the turbidity and depth of lake water. An
earlier study revealed that, in Lake Victoria, several
cichlid species show differently fix ed alleles, or
significant differences in allele frequencies , of a visual
pigment (LWS) gene among different populations within
the same species. Although only a limited number of
species were investigated in this research, it implies theexistence of positive selection on this gene, based on ourassumption as mentioned above.
In our division, we are interested in elucidating
evolution of genes encoding six classes (LWS, SWS-1 ,SWS-2A, SWS-2B , RH2 and rhodopsin) of visual
pigments (opsins), which are known to exist in the cichlid
genome, as factors promoting their speciation in Lake
Victoria. In order to start an extensive survey of thesegenes in Victorian cicblids, we conducted sampLing ofthese fish around Mwanza Gulf in the Southern shore ofthis lake in Tanzania (Fig. 2). In our preliminary analysisof the coding region of the rhodopsin gene that was
amplified from representative individuals by polymerase
chain reactions (PCRs), four sites were fow1d to show
variations of nucleotide so far (Fig. 3). Interestingly, all ofthese variations were considered to have arisen by
nonsynonymous mutations. This observation is notinconsistent with that the rhodopsin gene bas been understrong positive selection during the course of evolution of
the investigated species. It would be necessary toaccumulate more data on the evolution of this gene inorder to assume the environmental background that mightbe (or might have been) related to this phenomenon . A
more extensive analysis on molecular evolution of opsin
genes in Victorian cichlids is in progress in our division.

Publication List:
Shedlock, A. M., K. Takahashi, and N. Okada (2004) SINEs
of speciation: tracking lineages with retroposon . Trends
Ecol. E vol. 19: 545-553.
Sasaki, T., K. Takahashi, M. Nikaido, S. Miura, Y.
Yasukawa, and N. Okada (2004) First application of the
SINE (short interspersed repetitive element) method toInfer phylogenetic relationships in reptiles: an example
from the turtle superfamily Testudinoidea. Mol. Bioi. Evol.
21:705-715.
Sugie, A., Y. Terai , R. Ota, and N. Okada (2004) The
evolution of genes for pigmentation in African cichlid
fishes . Gene 343: 337-346.
Watanabe, M. , N. Kobayashi, T. Shin-1, Y. Tateno, Y.
Kohara, and N. Okada. (2004) Extensive analysis of ORF
sequences from two different cichlid species in Lake
Victoria provides molecular evidence for a recent
radiation event of the Victoria species flock. Gene 343:
263-269.
Terai, Y. , N. Takezaki , W. E. Mayer, H. Tichy, N . Takahata,
J. Klein, and N. Okada (2004) Plylogenetic relationships
among East African haplochromine fish es as revealed byshort interspersed elements (SINEs). J. Mol. Evo l. 58:
64-78.

NATIONAL INSTITUTE FOR BASIC BIOLOG Y
EVOLUTIONARY BIOLOGY AND BIODIVERSITY

Figure 2. Localities of collection of cichlids in Lake
Victoria. The top panel shows a map of the entire lake. The
area indicated by a red rectangle in this panel corresponds
to the region shown as a magnified map in the bottom panel.
Sampling was conducted by angling and trawling and byusing gill nets. White circles denote localities where
sampling both by using gill net and by angling was cmTied
out, and orange circles show localities where trawli.ng wasdone. Sampling was conducted in collaboration with
Tanzania Fisheries Research Institute (TAF!Rl).

Site

33

1 04

213

298

M

s

v

L

A

Codon

M
Cod onAGC G'I'C

*

~

T'I'A

GCT

1

~

~
'TCT

N

I

s

AAC

ATC

TCA

s

Figure 3. Variations observed in the rhodopsin gene that
was amplified from representative species of cichlids
collected in Lake Victoria. All the variations were ass umed
to have been caused by nonsynonymous changes that alteramino acid sequ ences. Direction of mutation was postul atedby comparison of the sequences with those from outgroupspecies in Lake Tanga nyika. AA, amino acid.

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITYLABORATORY OF MORPHODIVERSITY
Associate Professor:

KODAMA, Ryuji

The aim of this laboratory is to observe the variety ofthe morphogenetic processes in the course of the
ontogenesis of multicellular ogranisms and to analyze themechanisms of such processes mainly by morphologicalmethods. Accumulation of such analyses onembryogenetic processes of related species is expected togive an insight in the evolution of morphogenetic
processes. This laboratory uses the wings of lepidopteranspecies for the material of morphological studies.
Wings of the lepidopteran insects (butterflies andmoths) develop from the wing imaginal disc, which is ahollow sac made of simple epithelium. Due to its simple
construction, this tissue is a good material to study
cellular interactions in the course of morphogenesis.
The outline shape of the adult wing is often different
from that of the pupal wing. This difference is brought
about by the programmed cell death of the marginal areaof the pupal wing. The marginal dying area is called"the degeneration region" and the internal area, whichdevelop into the adult wing, is called "the differentiation
region".
The cell deaths in the degeneration region proceeds
very rapidly and completes in a half to one day period in
Pieris rapae or several other species examined. It was
shown that the dying cells in the degeneration region havecharacteristics common with the apoptotic cell death inmammalian cells, such as fragmented and condensed
nuclei containing short DNA fragments detected by
TUNEL staining. The cells in the degeneration region are
actively engulfed by the macrophages in the cavity
beneath the wing epithelium. The macrophages seem tobe concentrated beneath the degeneration region by thestrong adhesion between basal surfaces of the dorsal andventral epithelium in the differentiation region, which
occurs at the time of prominent cell death and excludes
macrophages out of the differentiation region. Thus
realised concentration of macrophages seems to accelerate
the shrinkage of the degeneration region.
A collaborative work with the laboratory of Dr. K.
Watanabe (Hiroshima University) concerns mostly on thedevelopment of trachea and tracheole pattern in theswallow tail butterflies. Trachea and tracheoles are both
important in delivering air into the wing and their patterncoincide with that of the boundary of degeneration anddifferentiation zones at the distal end of the wing.
According to the observations, the pattern formation ofwing epithelium is often dependent on tracheal andtracheole patterns. Basic research on the development oftracheal pattern formation is being done by the scanningelectron microscopy and the bright field light microscopyof the fixed or fresh specimens to describe the exact
pathway and the time course of the formation of elaborate
pattern of trachea and tracheoles and to establish thecytological and developmental relationship between theformation of tracheal pattern and epithelial cell pattern,such as scale cell pattern.

57

This
laboratory
also
conduct
morphologicalobservation of several animal tissues by scanning andtransmission electron microscopy and immno-electron
microscopic analyses. The training of specimen
preparation and instrument operation for suchobservations is also given. These activities include theDivision of Sex Differentiation and the Laboratory of
Neurophysiology of our institute.

58

NATIONAL INSTITUTE FOR BASfC BIOLOGYENVIRONMENTAL BIOLOGYDIVISION OF CELLULAR REGULATION
Professor:
Research Associate:
JSPS Research Fellow:
JSPS Postdoctoral Fellows:
Postdoctoral Fellows:
Visiting Scientists:

Secretmy :MURATA , NorioSUZUKi, /wan e
SHIVAJI, S n
JOGADHENU, S. S. Prakash IJ
TAKAHASHI, Shunichi
ALLAKHVERDIEV, Suleymanl. lJ
KANESAKI, Yu JJ
PANICHKJN, Vladimir 4;
PAITHOONRANGSARID,
Kaly anee 5J
LOS, Dmit1y A. 6;
SLABAS, Antoni R. 7J
PING, Xang 7;
KANESAKI, Kazuko

hspA
c/p81

hliB
hfiC
gloA
sigD
sl/1483
slr1544
ssr2016
ss/3446
s/11541dnaJ

s/10846

so dB

slr1963
htpG
slr0959
hik34 sl/1884
dnak2 slr1603
spkH
slr 1915
groES ss/2971
groEL1 slr1413
groEL2sigB
s/10528
slr1119slr0852
ssr3188

sl/0939
slr0967

htrA

rip A
rep A

ssr1853
slr0112glpD
s/11863s/11862
slf1772
slr0581s/10294slr0895
slr1501

slf0293
s/10470!Tota l: 11 1 !Total: 19 1

Induction factor;, 4.0

IJ the Centre for Cellular and Molecular Biology,

Fig. I. A hypothetical scheme for the two-component
signal transduction pathways that are activated in response
to hyperosmotic stress and the genes whose expression isregulated in the respective path ways.

Hyderabad, IndialJ the institute ofBasic Biological Problems,

Pushchino, Russia.1) Ehime University, Matsuyama, Ehime~ Moscow State University, Moscow, Russia
jJ National Center for Genetic Engineering andBiotechnology, Bangkok, Thailand
6
J Plant Physiology institute, Moscow, Russia ·lJ University ofDurham, Durham, England

The major thrust of our research efforts is directed
towards the comprehensive understanding of themolecular mechanisms that governs the responses ofplants and microorganisms to new environments. In
particular, our attention is focused on the perception andtransduction of various stress signals, such as extreme
temperatures, osmosis and salinity. Another line of ourresearch is focused on the molecular mechanisms ofphotodamage and repai r of the photosynthetic machinery
under severe stress conditions. In 2004, significant
progress was made in the following areas using the
cyanobacterium, Sy nechocystis sp. PCC 6803 (hereafter
Synechocystis).

(Figure I) . Hyperosmotic stress probably induces several
phenomena such as rigidification of membranes,
decreases in cytoplasmic volume, and changes inhydration state of intracellular proteins. It seems likelythat Hik34 and Hik2 might perceive such kinds ofstimulus and transduce the signals to Rrel to regulate theexpression of a number of genes. Genes whose expression
was regulated by RJe 1 include a number of important
genes, such as chaperons, proteases and a sigma factor.
Therefore, it is understandable that cells have multiple
pathways to activate Rrel-dependent transcriptional
regulation to synthesize proteins, which are indispensablefor the acclimation to hyperosmotic stress. These results
uncover a part of complicated mechanisms of signaltransduction via crosstalk of multiple pathways of
two-component systems.

II. The membrane rigidification regulates thecold-inducible gene expression.
I.

Crosstalk of signal
two-component systems.

transduction

in

the

Cells perceive the hyperosmotic signal and transduce it
to regulate the expression of a large number of genes.
Two-component systems that consist of a histidine kinase(Hik) and a response regulator (Rre) are widelydistributed from bacteria to higher plants as themechanism for intracellular signal transduction . The
Synechocystis genome encodes 47 Hiks and 45 Rres. To
identifY Hiks and Rres that are involved in the perception
and transduction of hyperosmotic signals, we screened
knockout libraries ofHiks and Rres by RNA slot-blot andgenome-wide DNA microarray analyses. We identified
four two-component systems, Hik33-Rre31, Hik34-Rrel ,Hikl6-Hik41-Rrel7 and Hik!O-Rre3 and an additional
potential two-component system, Hik2-Rrel. InterestiJJgly,
Rrel can perceive the hyperosmotic signals from both
Hik34 and Hik2. These results suggest that the crosstalkof hyperosmotic signals occurs in Sy nechocystis cells

Changes in the ambient temperature affect the physicalproperties of biological membranes. To obtain insights
into the role of membranes in the mechanism of cold
signal perception, we used a mutant of Synechocystis, inwhich the desA gene for the t-.12 desaturase and the desD
gene for the t-.6 desaturase were both inactive as a resultof targeted mutagenesis. Cells of the desA-;desF mutant
synthesize only a saturated C 16 fatty acid and amonounsaturated C 18 fatty acid regardless of the growthtemperature, whereas wild-type cells synthesize di-, triand tetra-unsaturated Cl8 fatty acids in addition to themonounsaturated CIS and saturated Cl6 fatty acids[Tasaka eta!. (1996) EMBO J. 15, 6416-6425]. Fouriertransform infrared spectrometry revealed that thedesA -;desF mutation rigidified the plasma membrane at
physiological temperatures. We applied DNA microarray
technique to examme effects of the membrane
rigidification on the induction of gene expression uponcold shock. The results demonstrated that the cold

59

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

inducibility of a part of cold-inducible genes, such as thecrh gene for an RNA helicase and the rbpl gene for an
RNA-binding protein, was unaffected by the rigidification.
However, the expression of the other cold-inducible genes,
such as hliA , hliB and sir 1544, was enhanced bymembrane rigidification. Moreover, the expression ofcertain heat-shock genes, namely, the hspA, htpG and
dnaK2 genes, was markedly enhanced by the membrane
rigidification. We further inactivated the genes for Hik33
and Hik34, which had been identified as sensors ofvarious signals and found that the mutation of these Hikseliminatedthemembranerigidification-dependentenhancement of expression of hli genes and heat-shock
genes, respectively. These findings suggest that Hik33 and
Hik34 perceive the change in membrane rigidification as
an initial signal of the downward shift in temperature to
regulate the cold-inducible gene expression.

HI. Genome-based systematic analysis of Ser/Thr
protein kinases and prediction of their function insignal transduction.
In addition to Hiks, cyanobacteria normally possess
Ser/Tlu· protein kinases which may function as sensors
and/or transducers of environmental signals. The
complete genome sequence of Synechocystis harbors 12
putative genes for Ser/Thr protein kinases; seven of the 12
genes encode proteins that belong to a PKN2 subfamily,
while the other five to an ABC! subfamily of Ser/Thr
protein kinases. These genes are termed in series as spkA ,spkB, spkC, spkD, spkE, spkF and spkG for kinases of
PKN2 type and spkH, spkl, spkJ, spkK and spkL forkinases of ABC I type. The role of these genes for Ser/Thr
protein kinases m Sy nechocystis remained largely
unexplored.
We systematically mutated all of these genes for
Ser/Thr kinase by insertion of antibiotic-resistance gene
cassettes and investigated the impact of these mutations
on the genome-wide expression of genes by means of
DNA micromny analysis. Mutation of seven of the genes,
namely, spkA , spkC, spkD, spkG, spkH, spkJ and spkL ,markedly changed the gene expression with increases inthe expression of some genes or with decreases in theexpression of some other genes, whereas mutation of theother five spk genes did not significantly affect the profile
of gene expression. These observations predict that atleast seven Ser/Thr protein kinases are involved
specifically in the regulation of gene expression possibly
via a signal-transduction pathway. The stress inducibilityof genes whose expression was affected by the spk
mutations predicts the signal transduction pathway towhich the individual Ser/Thr kinases belong.

characteristics of the crhR gene and its product CrhR
(RNA helicase) of Synechocystis. (!)The crhR gene was
highly induced, not only by cold, but also by
hyperosmotic and salt stress. (2) The crhR mutant cells
exhibited a phenotype of slow growth at low temperaturesindicating an important function of CrhR under cold stressconditions. (3) DNA microarray and Northem blotting
analyses indicated that the crhR mutation reduced theexpression of genes for molecular chaperonins, groES,
groELJ and groEL2. (4) CrhR regulated the level ofmRNA of groESLJ and groEL2 by stabilizing the mRNA.
(5) Further, we demonstrated that the crhR mutation
decreased the GroEL levels during cold stress. (6) The
crhR mutation dowmegulated the expression of groESLJ
and groEL2 genes under salt and hyperosmotic stressconditions.
In micro-organisms such as E. coli and Synechocystis,the activity of translation is reduced when they are
subjected to cold stress . During cold acclimation, several
cold shock proteins are synthesized and association ofthese cold shock proteins with ribosomes resumes thetranslation .
The cold-induced synthesis of GroESL
might be important for the translation machinery tomaintain the activity at a proper level under cold stressconditions. Thus the cold-induced expression of the crhR
gene is very important for Sy nechocystis cells to acclimate
cold stress.

V. Photodamage to photosystem II occurs in
two-step mechanisms.
Light is necessary for photosynthesis but it also
damages the photosynthetic machinery, in particular,
photosystem II. The current hypothesis for the mechanismof photodamage to photosystem II postulates that excess
light energy, which is absorbed by photosynthetic
pigments but not utilized in photosynthesis, produces
reactive oxygen species that damage the photochemical
reaction center of photosystem II. Using monoclu·omatic
light generated by the Okazaki Large Spectrograph, wedemonstrated that UV or blue light first inactivates theoxygen-evolving complex of photosystem II and thenblue or red light, which is absorbed by photosynthetic
pigments, inactivates the photochemical reaction center as
the second step . These results lead us to conclude that thecurrent hypothesis is not the case and that thephotodamage to photosystem II occurs in two steps
Blue orred light

UVor
strong blue light

IV. RNA helicase is required for cold acclimation in
Synechocystis.
DNA microarray analysis revealed that cold stressinduced the expression of a large number of genes. The
crhR gene for RNA helicase was one of such highly
inducible genes. We have observed the following

Functional PSII

In:.1ctivation of theoxygen evolving
complex (OEC)Inactivation of PSII
reaction center

Fig. 2. Schematic model for the two-ste p mechani sm ofphotodamage to photosystem !!.

60(Figure 2). The first step is the photodamage to theoxygen-evolving complex of photosystem II probably at
the Mn-cluster and its protein environment. The second
step is the photodamage to the photochemical reaction
center II after the oxygen-evolving complex isinactivated.

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYLight inactivates photosystem II, and this phenomenon
is referred to as the "photoinhibition". The extent ofphotoinhibition is a result of a balance between
photodamage to and repair of photosystem II. We
developed a system to monitor the photodamage andrepair separately and examined the effect of various
environmental stresses on photodamage and repair. In
contrary to the current concept that environmental stressaccelerates photodamage to photosystem II, we clearly
demonstrated that environmental stresses do not damage
photosystem II directly, but they inhibit the repair ofphotodamaged photosystem II at the level of translation of
the psbA genes which encode the D I protein, an importantcomponent of the photosystem II reaction center.
Furthermore, we found in Chlamydomonas cells that the
repair of photosystem II was inhibited when the Calvincycle was inhibited.

K. Marin, Y. Kanesaki, D. A. Los, N. Murata, I. Suzuki and
M. Hagemann (2004) Gene expression profiling reflects
physiological processes in salt acclimation m
Synechocystis sp. strain PCC 6803. Plant Physiol. 136:
1-11
E.-J. Park, Z. Jekni, A. Sakamoto, J. DeNoma, R. Yuwansiri,
N. Murata and T. H. H. Chen (2004) Genetic engineeringof glycinebetaine synthesis in tomato protects seeds,
plants, and flowers from chilling damage. Plant J. 40:
474-487
K. Paithoonrangsarid, M. Shoumskaya, Y. Kanesaki, S.
Satoh, S. Tabata, D. A. Los, V. Zinchenko, H. Hayashi, M.
Tanticharoen, I. Suzuki and N. Murata (2004) Fivehistidine kinases perceive osmotic stress and regulatedistinct sets of genes in Synechocystis. J. Bioi. Chern. 279:
53078-53086
S. I. Allakhverdiev, Y. Nishiyama, S, Takahashi, S. Miyairi,
I. Suzuki and N. Murata (2005) Systematic analyses of therelation of electron transport and ATP synthesis toregulate the photodamage and repair of photosystem II in
Synechocystis. Plant Physiol, in press
A. Shapiguzov, A. A. Lyukevich, S. I. Allakhverdiev, T. V.
Sergeyenko, I. Suzuki, N. Murata and D. A. Los (2005)
Osmotic shrinkage of cells of Synechocystis sp. PCC 6803
by water efflux via aquaporins regulates theosmostress-inducible gene expression. Microbiology 151:
in press

List of publication:(2) Review articles

VI. Environmental factors do not accelerate thephotodamage to photosystem II, but inhibit the
repair of photosystem II(1) Original articles
S. Suzuki, A Ferjani, I. Suzuki and N. Murata (2004) The
SphS-SphR two-component system is the exclusive sensor
for the induction of gene expression in response tophosphate limitation in Synechocystis. J. Bioi. Chern. 279:
13234-13240
K. Saeki, K. Matsumoto, M. Kinoshita, I. Suzuki, Y. Tasaka,
K. Kana, Y. Taguchi, K. Mikami, M. Hirabayashi, N.
Kashiwazaki, Y. Hosoi, N. Murata and A. Iritani (2004)
Functional expression of a 1'1-12 fatty acid desaturase genefrom spinach in transgenic pigs. Proc. Nat!. Acad Sci.
USA 101: 6361-6366
S. I. Allakhverdiev and N. Murata (2004) Environmental
stress inhibits the synthesis de novo of proteins involved in
the photodamage-repair cycle of photosystem II in
Synechocystis sp. PCC 6803. Biochim. Biophys. Acta
(Bioenergetics) 1657: 23-32
A. Sakamoto, R. Sulpice, T. Kaneseki, C.-X. Hou, M.
Kinoshita, S. Higashi, B. Y. Moon, H. Nonaka and N.
Murata (2004) Genetic modification of fatty acidunsatulation of chloroplastic phosphatidylglycerol alters
the sensitivity to cold stress. Plant, Cell & Environ., 27:
99-105
Y. Nishiyama, S. I. Allakhverdiev, H. Yamamoto, H.
Hayashi and N. Murata (2004) Singlet oxygen inhibits therepair of photosystem II by suppressing the elongation of
the Dl protein in Synechocystis sp. PCC 6803.
Biochemistry 43: 11321-11330

D. A. Los and N. Murata (2004) Membrane fluidity and its
roles in the perception of environmental signals. Biochim.
Biophys. Acta (Biomembrane) 1666:142-157
Y. Nishiyama, S.I. Allakhverdiev and N. Murata (2005)
Regulation by environmental conditions of the repair ofphotosystem II in cyanobacteria. Photoprotection,
Photoinhibition, Gene Regulation, and Environment (eds.,
B. Demming-Adams, W.W. Adams and A.K. Mattoo),
Kluwar Academic Publishers
Y. Nishiyama, S. I. Allakhverdiev and N. Murata (2005)
Inhibition of the repair of Photosystem II by oxidative
stress in cyanobacteria. Photosynth. Res., in press

NATIONA L INSTITUTE FOR BASIC BIOLOGYENV IRONMENTAL BIOLOGYDIVISION OF MOLECULARENVIRONMENTAL ENDOCRINOLOGY
Professor:
Associate Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Students:

Visiting Scientist:
Technical Assistants:

SecretGiy:I)

IGUCHI, Taisen
WATANABE, Hajim e
KATSU, Yoshinao
MIZUTANI, Takeshi
URUSHITANI, Hiroshi
SON£, Kiyoaki IJ (- Oct. '04)
MIYA GA WA , Shinichi
NAGATA, Emiko
KOBAYASHI, Mika
JSHJMOTO, Yoichi
KATO, Hideo 2J
SUZUKJ, Atsuko I) (- Oct. '04)
KOBAYASHI, Kaoru
TAKAHASHI, Eri
HINAGO, Megumi
JCHIKA WA , Rie IJ (- Oct. '04)
GOTO, Mayu
JMAIZUMI, Taeko

CREST. JST2) .fivm Nihon Bioresearch inc.

Synthetic chemicals found in the environment have thecapacity to disrupt endocrine system development andfunction in both wildlife and humans. This has drawn
public concern since many of these chemicals may bind to
estrogen receptors (ER) and evoke estrogenic effects.
Early evidence that estrogenic chemicals could pose athreat to human health during development came fromstudies of diethylstilbestrol (DES), which was used toprevent premature birth and spontaneous abortion.
Laboratory experiments have demonstrated that exposureof animals to sex honnones during perinatal life can cause
permanent and irreversible alterations of the endocrine
and reproductive systems as well as the immune system,
nervous system, bone, muscle, and liver in both sexes.
Although many of these chemicals may bind to ER andevoke estrogenic effects in wildlife and humans, theeffects of estrogen are not well understood even now.
Thus, understanding the effects of sex honnones at themolecular level, especially during development, is ve1y
important to resol ve these problems.
Normal Mouse

FIG. 1 Scheme of estrogen-dependent and -independent
vagin al ep itheli al ce lls in mice induced by neonatal
est:rogenization.

61

I. Estrogen-induced irreversible changes
Perinatal sex-hormone exposure has been found to
induce lesions in reproductive tracts in female mice.
The possible relevance of the mouse findings tothedevelopment of cancer in humans has beenemphasized. In the early seventies, a close correlation
between occurrence of vaginal clear cell adenocarcinoma
in young women and early intrauterine exposure to DES
was demonstrated. Many chemicals released into theenvironment have the potential to disrupt endocrine
function in wildlife and humans. Some of these chemicals
induce estrogenic activity by binding to ER.
Theneonatal mouse model has been utilized especially todemonstrate the long-term effects of early sex hormone
exposure on the female reproductive tract. Neonatal
treatment of female mice with estrogens induces various
abnormalities of the reproductive tract: ovary-independent
cervicovaginal
keratinization,adenosis,uterinehypoplasia, epithelial metaplasia, oviductal tumors,polyovular follicles (PF) and polyfollicular ovanes.
Female reproductive tracts in mice exposed prenatally to
estrogen show altered expression of Hoxa genes and Wnt
genes and the analysis of knockout mice lacking Hoxa-1 0
or Wnt7a show uterine hypoplasia. The growth responseof neonatally DES-exposed reproductive organs to
estrogen is reduced, as are ER levels and epidermal
growth factor (EGF) receptor levels, in addition to otherhormone receptor levels.
EGF-Iike growth factors · - - - - - - - - - - - ,Cell~----ErbB receptors-------------

membrane

Rat

PI3K
Phosphorylationcascade

MEK1/2
ERK112

AKT

Nuclear--------------------membrane

Ser122Ser171(p90RSK) .~.~• • • • • •Transcription factors+I
Growth factors responsive genes

AF-1

"

AF-2

Growth factors

Estrogen responsive genes

FIG.2 A hypothetical model for th e estrogen-independent
ER activation pathway in mouse vagina. EGF-like growth
factors activate the protein-phosphorylation cascade via
erbB receptors. In nuclear, estrogen receptor is
phosphorylated on serine 122 and 171 in AF-1 domain.
Furthemwre, transcnptwn factors are activated byphosphorylation. These phosphorylations induce the!Tanscriptional activity of ER, and then growth factors are
expressed via estrogen-response element (ERE). Growth
factors induced by ER activate EGF-receptors.

Growth factors and ER signaling cooperate to playessential roles in cell proliferation, differentiation andtumor progression in mouse reproductive organs . The
mechanisms of the estrogen-dependent and -independent
pathways remain unknown. EGFR and erbB2 wereactivated in the vaginal epithelium of mice by estrogen
treatment. This activation was also encountered invaginae from neonatally DES-exposed mice, along with

62

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

the expression of EGF, TGF-a, HB-EGF, amphiregulin
and neuregulin. Immunochitochemical analysis indicatedthat erbB2 was primarily expressed in vaginal epithelium.
Serine 118 and 167 located in the AF-1 domain of
ERa were phosphorylated in these vaginae. AG825,
AG1478 or ICI 182,780, that are erbB2, EGFR and ER
antagonists,
respectively,
administration
blockedproliferation of vaginal epithelium induced by neonatal
DES exposme. Signal transduction via EGFR and erbB2
could be related to the estrogen-induced vaginal changes
and persistent erbBs phosphorylation and sustained
expression of EGF-like growth factors, leading to ERa
activation that may result in cancerous lesions in vaginaefrom neonatally DES- exposed mice later in life.
To identify estrogen-responsive genes related to theproliferation and differentiation of mouse vaginal
epithelial cells, we used differential display and identified
a novel c-type lectin that encodes a membrane proteinwith a c-type lectin domain in the carboxyl-terminal
region. Characterization of mRNA expression indicatesthat estrogen regulates the gene encodeing this novel
c-type lectin in mouse vagina. Furthermore, this c-type
lectin is found in epithelial cells, but not stromal cells,suggesting that it may be an important factor in thestratification and/or cornification of the vaginal
epithelium of mice. We are continuing efforts to analyze
its function during proliferation and differentiation inmouse vagina by estrogen treatment.
Estrogenic compounds such as bisphenol A (BPA) andnonylphenol as well as dioxins and PCBs were fmmd in
the human umbilical cord. BPA can easily cross theplacenta and enter the fetus in Japanese monkey and mice.
BPA can be found in fetal brain, testis and uterus when
given to pregnant mice and monkeys.
Neonatal
exposure to a high BPA dose induced ovary-independent
vaginal changes, polyovular follciles and infertility
lacking corpora lutera. Prenatal exposure to a low BPA
dose induced acceleration of vaginal opening in theoffspring. Thus, the developing mammal is sensitive toexposme to estrogenic agents.
100000 ;, - - -- -- -- -- - - - --

--,

f

r

'\3

l •

I
~
l:j 1000 ~.
~
tt:~J

••

•

L, •••••~

l_·• ..•f. . ..

•• ·· - · :•••••[ 1000

• ·:.

t

t
: •

I

,. .

l1oo

.... -. ..

II.~ :··: - . .

10 -· - - - -... ·- · -- -- 100
1000
10

I

-- ---·-t - -- ·- ·-- - 10

10000

100000Control
FJG.3 Scatter plot of average expression levels in control
and chemical-treated uterus

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

Ill. Effect of estrogen on reptile, amphibian andfishesfQTl~§llilllO!t
0.95-1.0 0.9-0.95 <0.9-

FIG. 4 Evolutionary relationships of estrogen receptor
sequences.

During embryogenesis, exogenous estrogen exposure
induces abnormal sex differentiation and the abnormalbone formation in African clawed frog, Xenopus laevis,
the cyprinodont fish , mummichog (Fundulus heteroclitus),
mosquitofish (Gambusia a./finis). To analyze the functionof estrogen, we have isolated cDN A clones of ERa and ~from F heteroclitus, G qffinis. In UK rivers exposure ofroach (Rutilus rutilus - a common cyprinid fish) toeffluents from sewage treatment works, containing
complex mitures of endocrine disrupting chemicals(EDCs) has been shown to alter sexual development andimpact negatively on their reproductive capabilities. To
unravel the mechanisms of disruption of sexual
development in roach exposed to EDCs, we have isolated
the cDNAs related to sex determination and sex
differentiation containing estrogen receptor, aromatase,
StAR, Sox9, vasa etc. We are examining the gene
expression during gonadal differentiation with or without
EDCs exposure. Furthermore, we have isolated cDNAs ofsteroid hormone receptors from American alligators(Alligator mississippiensis), the red belly turtle(Pseudemys nelsoni) and Japanese giant salamander(Andrias japanicus). As the estrogen-responsive genes
must play important roles, we are isolating the
estrogen-responsive genes to understand the molecularphysiology of estrogen action. Japanese tree frog (HylaNATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

japonica) takes water through ventral skin. We found thatsex steroids and endocrine disruptors interfere with water
absorption through ventral skin in frogs. Further, using theamphibian and fish as model animals we aim to analyze
the effects of numerous chemicals released into theenvironment on endocrine system function in wildlife.

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

Publication List:
Adachi, T., Ono, Y., Koh, K.B., Takashima, K., Tainaka, H.,
Matsuno, Y., Nakagawa, S., Todaka, E., Sakurai, K.,
Fukata, H., Iguchi, T., Komiyama, M. and Mori, C. (2004)
Long-term alteration of gene expression withoutmorphological change in testis after neonatal exposure to
genistein in mice: toxicogenomic analysis using eDNA
microarray. Food Chern. Toxicol. 42, 445-452.
Kato, H., Iwata, T., Katsu, Y., Watanabe, H., Ohta, Y. andIguchi, T. (2004) Evaluation of estrogenic activity in dietsfor experimental animals using in vitro assay. J. Agric.
Food Chern. 52, 1410-1414.
Katsu, Y., Bermudez, D.S., Braun, E., Helbing, C.,
Miyagawa, S., Gunderson, M.P., Kohno, S., Bryan, T.A.,
Guillette, L.J. and Iguchi, T. (2004) Molecular cloning of
the estrogen and progesterone receptors of the American
alligator. Gen. Camp. Endocrinol. 136, 122-133
Kohno, S., Fujime, M., Kamishima, Y. and Iguchi, T. (2004)
Sexually dimorphic basal water absorption at the isolated
pelvic patch of Japanese tree frog, Hyla japonica. J. Exp.
Zoolog. A Camp. Exp. Bioi. 301, 428-438.
Matsuno, Y., Adachi, T., Koh, K.B., Fukata, H., Sugimura,A., Sakurai, K., Shibayama, T., Iguchi, T., Komiyama, M.
and Mori, C. (2004) Effect of neonatal exposure todiethylstilbestrol on testicular gene expression in adultmouse: comprehensive analysis with eDNA subtraction
method. Int. J. Androl. 27, 115-122.
Miyagawa, S., Katsu, Y., Watanabe, H. and Iguchi T. (2004)
Estrogen-independent activation of erbBs signaling andestrogen receptor alpha in the mouse vagina exposedneonatally to diethylstilbestrol. Oncogne 23, 340-349.
Miyagawa, S., Suzuki, A., Katsu, Y., Kobayashi, M., Goto,
M., Handa, H., Watanabe, H. and Iguchi, T. (2004)
Persistent gene expression in mouse vagina exposedneonatally to diethylstilbestrol. J. Mol. Endocrinol. 32,
663-677.
Okada, A., Ohta, Y., Brody, S.L. and Iguchi, T. (2004)
Epithelial c-jun and c-fos are temporally and spatiallyregulated by estradiol during neonatal rat oviduct63

differentiation. J. Endocrinol. 182,219-227.
Okada, A., Ohta, Y., Brody, S.L., Watanabe, H., Krust, A.,
Chambon, P. and Iguchi, T. (2004) Role of foxj1 andestrogen receptor alpha in ciliated epithelial celldifferentiation of the neonatal oviduct. J. Mol. Endocrinol.
32, 615-625.
Sato, T., Fukazawa, Y., Ohta, Y. and Iguchi, T. (2004)
Involvement of growth factors in induction of persistent
proliferation of vaginal epithelium of mice exposedneonatally to diethylstilbestrol. Reprod. Toxicol. 19,
43-51.
Seiwa, C., Nakahara, J., Komiyama, T., Katsu, Y., Iguchi, T.
and
Asou,
H.
(2004)
BisphenolA
exertsthyroid-hormone-like effects on mouse oligodendrocyte
precursor cells. Neuroendocrinology 80, 21-30.
Sone, K., Hinago, M., Kitayama, A., Morokuma, J., Ueno, N.,
Watanabe, H. and Iguchi, T. (2004) Effects of
17beta-estradiol, nonylphenol, and bisphenol-A on
developing Xenopus laevis embryos. Gen. Camp.
Endocrinol. 138, 228-236.
Uchida, D., Yamashita, M., Kitano, T. and Iguchi, T. (2004)
An aromatase inhibitor or high water temperature induce
oocyte apoptosis and depletion of P450 aromatase activity
in the gonads of genetic female zebrafish during
sex-reversal. Camp. Biochem. Physiol. A Mol. Jntegr.
Physiol. 137, 11-20.
Watanebe, H., Suzuki, A., Goto, M., Lubahn, D.B., Handa, H.
and Iguchi, T. (2004) Tissue-specific estrogenic andnon-estrogenic effects of a xenoestrogen, nonylphenol. J.
Mol. Endocrinol. 33, 243-252.
Watanabe, H., Suzuki, A., Goto, M., Ohsako, S., Tohyama,C., Handa, H. and Iguchi, T. (2004) Comparative uterine
gene expression analysis after dioxin and estradiol
administration. J. Mol. Endocrinol. 33, 763-771.
Yoshinaga, N., Shiraishi, E., Yamamoto, T., Iguchi, T., Abe,S. and Kitano, T. (2004) Sexually dimorphic expression ofa teleost homologue of Mullerian inhibiting substanceduring gonadal sex differentiation in Japanese flounder,
Paralichthys olivaceus. Biochem. Biophys. Res. Commun.
322, 508-513.

64

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

Postdoctoral Fellows:

Graduate Students:

Secretaries:

TSUKAYA , Hirokazu
HORIGUCHI, Gorou
KONDO, Makiko (-Nov. 30)
TAKABE, Erika
YAMAGUCHI, Chinami
SAKAI, Keiko
CHO, Kiuhyon
ISHIKAWA , Naoko
ALI, Ferjani
YAMAGUCHI, Takahiro
YANO, Satoshi
KOZUKA, Toshiaki
NARITA, Noriyuki
MANO, Erika
FUJIKURA, Ushio
KOJIMA, Yoko
KABEYA, Kazuko (- Mm: 30)

The leaf is the fundamental unit of the shoot system,
which is composed with leaf and stem. Diversity of plant
form is mostly attributable to variation of leaf and floralorgans, which are modified leaves. Moreover, leaf shapeis sensitive to environmental stimuli. 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 have been in a turning point, after our
successful application of the techniques of developmental
and molecular genetics to it, using model plants,
Arabidopsis thaliana (L.) Heynh . (reviewed in Tsukaya,
2003).
Focusing on mechanisms that govern polarized growthof leaves in Arabidopsis thaliana we have identified
genes for polar-dependent growth of leaf lamina: theFig. I. Intracellular localization of ROT4::GFP in onion
epidennal cells before (upper) and after (l ower) osmosis.

ANGUSTIFOLIA (AN) gene regulates width of leaves andthe ROTUNDIFOLIA3 (ROT3) gene regulates length ofleaves. Both AN and ROT3 genes control leaf cell shape.
How have these genes evolved the function ofleaf-shape control? The AN is a homolog of animal
CtBPIBARS gene family which has varied roles inmorphogenesis and organelle control. To understand thecommon role(s) of AN subfamily in plant kingdom, weanalyzed a homolog of AN from Ipomoea nil, IAN, andshowed that the IAN exhibits the same function with the
AN on the control of leaf shape when introduced to
Arabidopsis thaliana (Cho et al., in press). On the other
hand, the ability of the IAN to control branching pattern oftrichomes was less efficient than the AN. Fmiher
comparative analyses of the AN homologs from various
plants are now on going.
In addition to the above factors, in this year, we added
ROT4 gene to the list of the genes responsible for thepolarity-dependent control of leaf shape (Narita et a!.,2004). ROT4 is a member of novel peptide family (RTFL:
RoT-Four-Like) which is specific to seed plants.
Overexpression of the ROT4 results in stunted leaves withnormal width, due to decrease of number of leaf cells
specifically to the longitudinal direction. All RTFL
peptide conserve RTFL domain and even truncated formsof ROT4 that have RTFL domain show the same effect onleaf shape with full-length ROT4, the RTFL family isthought to have common role in control of leaf cellproliferation to the leaf-length direction. Interestingly, theROT4 peptide appears to localize on plasma membrane
when fused with GFP marker (Fig. I) . The role of the
RTFL in the control of leaf-cell proliferation is underinvestigation.
How cell proliferation and cell enlargement arecoordinated in leaf morphogenesis? In a determinate
organ, a leaf, number of leaf cells is not necessarilyreflected on leaf shape or, in particular, leaf size. Genetic
analyses of leaf development in Arabidopsis shows that acompensatory system(s) act in leaf morphogenesis and anincrease of cell volume might be triggered by a decreasein cell number (Tsukaya, 2003). Thus, leaf size is, at least
to some extent, uncoupled from the size and number ofcells by· the compensatory system(s). Recently, we haverevealed that ANGUSTIFOLIA3 (AN3) gene is involved inmaintenance/establishment of activity of cell proliferationin leaf primordia. AN3 encodes a co-activator, and is
speculated to control cell cycling in leaf primordia(Horiguchi et a!. , submitted). Interestingly, the an3 shows
clear "compensation", namely, accelerated cell expansion
in relation to decrease of number of leaf cells. By using
various mutants with altered number and/or size of leaf
cells, we are currently analyzing genetic system of thecompensation.
On the other hand, we also focused on the effects ofenvironmental factors on leaf morphogenesis. In darkness,
expansion of leaf lamina is inhibited, while at the sametime, petiole elongation is enhanced. This phenomenon istermed the shade-avoidance syndrome. We analyzed thenature of the shade-avoidance syndrome and found thatphytochromes and cryptochromes specificall y regulate the

65

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

contrasting growth pattems of the leaf blade and petiole inshade (Kozuka et a!., in press; Fig. 2). Differed fromphotomorphogenesis of hypocotyl , cell elongation was
stimulated in the petiole in dark conditions without anincrease in the ploidy level. By examining the effects ofsucrose on the growth of the leaf blade and petiole, we
revealed that growth promotional effects of sucrose arehighly dependent on the light conditions.
Recently, we found that the ROT3 gene encodes acytochrome P450 that catalyzes the conversion oftyphasterol to castasterone, an activation step in thebiosynthesis pathway of brassinostero ids (BRs) (Kim eta!., in press). Differed from already known mutants ofgenes for biosynthesis of BRs, loss-of-function mutant of
ROT3 has specific defect in the length of leaves,suggesting importance of fine tuning of levels of BRs on
the polarized growth of leaves. Interestingly, CYP90D I ,the most closely related cytochrome P450 to the
ROT3/CYP90Cl enzyme, was suggested to catalyze theother conversion steps of BR biosynthesis (Kim et a!., inpress) . Double mutant for the ROT3/CYP90CJ and for the
CYP90D I exhibited extreme dwarf that is observed forthe other known mutants of genes for biosynthesis of BRs.
Since the Ioss-of-fi.mction mutant of ROT3 has defect in
response of petioles to dark, the ROT3 might havespecific role(s) in the shade-avoidance syndrome. In
relation to this topics, adaptive responses of arabidops isleaves against gravity and other environmental factors
were also analyzed and interaction between light signal
and gravity-response in leaves were suggested (Ts ukaya,
in press).

Fig. 2. Shade-avoidance syndrome in Arabidopsis thaliana.
Left, under white light; rigbt, under dark. Bar: 5 mm.

On the other hand, we are also interested inenvironmental adaptation of leaf size in wild plants. In theco urse of field research of natmal evolution of leaf
shape/size, we have revealed some aspects of biodiversityof plant forms (Tsukaya, 2004; in press; Tsukaya et a!. ,2004; Yokoyama et a!., in press). Interestingly, many
plants are known to have evolved small-sized leaves insome islands, such as Yakushima, Kinkazan and Miyajima
islands. Similar phenomenon is also known in plants
inhabited in precincts of shrines and temples in Japan .
Typical example of the evolution of small-sized leaves inthese environments is known in Plantago asiatica. To
understand what factors have accelerated the evolution of
the small-sized leaves, we collected P asiatica and P
major from a number of loca lities in and around Japanand established more than thirty imbred lines to ana lyze
the genetic background of the evolution. To nurse study ofthis fie ld, co-organized with Dr. Araki of Kyoto
Un iversity, we held a domestic meeting on Plantago
Research in Okazaki Conference Center, in July 3, 2004,
inviting twelve researchers from various universities . So
called "Evo/Devo" study of leaf morphogenesis is also
one of our research project in NIBB.

Publication List:
Cho, K.-H., Shindo, T., Kim, G.-T., Nitasaka, E. and Tsukaya, H.
(2005) Characterization of a member of the AN subfami ly,
JAN, fi·om ipomoea nil. Plant Cell Physiol. (in press)
Kozuka, T. , Horiguchi , G. , Kim, G.-T. , Ohgishi, M., Sakai, T.
and Tsukaya, H. (2005) The different growth responses of
the Arabidopsis thaliana leaf blade and the petiole during
shade avoidance are regulated by photoreceptors and sugar.
Plant Cell Physiol. (in press)
Narita, N. N., Moore, S., Horiguchi , G. , Kubo, M., Demura ,T. , Fukuda, H., Goodri ch J., and Tsukaya, H. (2004)
Over-expression
of
a
novel
small
peptideROTUNDIFOL!A4 decreases of cell proliferation andalters leaf shape in Arabidopsis. Plant J. 38: 699-713.
Tsukaya, H. (2004) Gene flow between Impatiens radicans
and 1. javensis (Ba lsaminaceae) in Gunung Pangrango,
central Java, Indonesia. Amer. J. Bot. 91: 2119-2123.
Tsukaya, H. (2005 ~ Leaf shape: genetic controls andenvironmental factors. Int. J. Dev. Bioi. (in press).
Tsukaya, H. (2005) Molecular variation of Spiranthes
sinensis (Orchidaceae) in Japan, with special reference tosystematic treatment of seasona ll y differentiated groups
and a dwarf f01m, f. gracilis, from Yakushima Island. J.
Plant Res. (in press).
Tsukaya, H. , lokawa, Y. , Kondo, M. and Ohba, H. (2005);
Large-scale general collection of DNA of wild plants in
Mustang, Nepal. J. Plant Res. (in press).
Tsukaya, H., Okada, H. and Mohamed, M. (2004) A novel
feature of stTUctura l variegation in leaves of tropical plant,
Schismatoglottis cafyptrata. J. Plant Res. 117: 477-480.
Yokoyam a, J. , Fukuda, T. and Tsukaya, H. (2005) Mol ecular
identification of th e mycorrhizal fungi of the epiparasitic
pl ant Mo no/ropastrum humile va r. glaberrimum(Ericaceae). J. Plant Res. (in press)

66

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYDIVISION OF PHOTOBIOLOGY (ADJUNCT)

and red light induce chloroplast movement in the moss
Physcomitrella patens, which has four phototropin genes,

WADA, Masamitsu
Professor (Adjunct):
Associate Professor (Adjunct):
YAMAUCHI, Daisuke
Research Associate:
KIKUCHI, Kazuhiro
NIBB Research Fellow:
OGURA, Yasunobu
MEXT Postdoctoral Fellow: TAKAHASHI, Fumio
TMU Postdoctoral Fellow:
CHRISTENSEN, Steen
Visiting Scientists:
OIKA WA, Kazusato
SUETSUGU, Noriyuki
UENAKA, Hidetoshi

Plants respond to light as an environmental factor to
optimize development and regulate other physiologicalphenomena. Phytochrome (phy) and blue light receptors,
such as cryptochrome (cry) and phototropin (phot), arethe main photoreceptors for plant photomorphogenesis.
The goal of our research is to elucidate thephotoperception and signal transduction pathways ofphoto-morphogenesis.

PHOTAl, PHOTA2, PHOTBl and PHOTB2. These genes
were classified into two groups (PHOTA and PHOTB) on
the basis of their deduced amino acid sequences. Then
phototropin disruptants were generated by homologous
recombination and used for analysis of chloroplast
movement. It was found that blue light-induced
chloroplast movement was mediated by phototropins.
Both photA and photB groups were able to mediate
chloroplast avoidance, although the photA groupcontributed more to the response. Red light-induced
chloroplast movement was also significantly affected in
photA2photB 1photB2 triple disruptants. Because theprimary photoreceptor for red light-induced chloroplast
movement in P patens is phytochromes, phototropins
may be downstream components of phytochromes in thesignaling pathway. The involvement of phototropins in
the phytochrome signaling pathway was reported for the
first time.

1-3 Adiantum
I. Chloroplast relocation movement

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

1-1

Arabidopsis

In Arabidopsis leaves, chloroplast movement is fluence
rate dependent. Under lower light fluence rate,
chloroplasts accumulate at the cell surface to maximize
photosynthetic potential. Under high fluence rate,
chloroplasts avoid incident light to escape photodamage.
We examined the phenomenon of chloropla~t avoidance
movement and demonstrated a proportional relationshipbetween fluence rate and the velocity of chloroplast
avoidance movement. When a small area is irradiatedwith a microbeam, the majority of chloroplasts inside thebeam begin moving towards the outside of the beam.
Some chloroplasts, however, begin to move only after arelatively long .lag time. The length of the lag period
becomes longer under higher fluence rate light andoccurred more frequently with chloroplasts locatednearest the center of the microbeam.
In addition, we
showed that the amount of light-activated phot2, thephotoreceptor for the avoidance response, likely plays arole in this phenomenon, as heterozygous mutant plants
show a reduced avoidance velocity compared to that ofhomozygous wild type plants.

1-2

Physcomitrella

Phototropin is the blue light receptor that mediates blue

The avoidance movement response in Adiantum phot2
deficient mutants can be restored by transient expressionof non-mutant AcPHOT2 eDNA, indicating thatchloroplast avoidance movement in this fern is mediatedby the Acphot2 protein. Further functional analyses of theAcphot2 protein were performed using this transient assay
for chloroplast avoidance movement. The results suggestthat the LOV2, but not the LOVl, domain of Acphot2 isessential for avoidance movement.

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

2- 1 Miniature transposable element
Transposable elements constitute a large portion of
eukaryotic genomes and contribute to their evolution anddiversification. We identified active transposable elements,
miniaturePing (mPing), Ping and Pong in rice. The mPing
element was identified as the first active MITE from anyorganism. mPing is a short 430 base pair element with 15
base pair terminal inverted repeats that lacks a transposase.
mPing elements are activated in calli derived from antherculture and excise efficiently from original sites toreinsert into new loci. Ping and Pong transposable
elements were isolated as putative autonomous elements
encodingan
IS/PIF/Harbinger
superfamilyoftransposases. Evolutionally, the number of copies ofmPing elements has increased in japonica cultivars, but
not in indica cultivars and their ancestral species, Oryza
rufipogon. Japonica cultivars are the only rice varieties inwhich the transposable Ping element can be detected.
The mPing/Ping transposon system may prove a usefulmolecular tool for gene isolation and gene knockout in
rice, the most agriculturally important crop in the world.

67

NATIONAL INSTITUTE FOR BASIC BIOLOGY
ENViRONMENTAL BIOLOGY

2- 2 DNA interference in Adiantum
Silencing of gene expression by RNA interference(RNAi) is a useful technique for determining the roles ofgenes of unknown function in a wide range of organisms.
The dramatic increase in the number of genes of unknown
function as a result of whole genome sequencing projects,
as well as EST projects, means that faster approaches areneeded for understanding gene function. We found asimple method for silencing genes using DNA fragments
homologous to the target gene rather than RNAi. We havenamed this approach DNA interference or DNAi. It has
the advantage of being faster and simpler than current
RNAi approaches, and allows simultaneous silencing ofmultiple genes.
Polarized blue light

hyg'

D

hyg'

Kagawa, T. , M. Kasahara, T. Abe, S. Yoshida and M.Wada(2004) Function analysis of Acphot2 using mutants
deficient in blue light-induced chloroplast avoidance
movement of the fern Adiantum capillus-veneris L. Plant
Cell Physiol. 45: 416-426.
Kagawa, T. and M. Wada (2004) Chloroplast avoidance
movement rate IS fluence dependent. Photochem.
Photobiol. Sci. 3:592-595.
Kasahara, M. , T. Kagawa, Y. Sato, T. Kiyosue and M. Wada
(2004) Phototropins mediate blue and red light-induced
chloroplast movements in Physcomitrel/a patens. Plant
Physiology135: 1388-1397.
Mochizuki , T. , Y. Onda, E. Fujiwara, M. Wada and Y.
Toyoshima (2004) Two independent light signals
cooperate in the activation of the plastid psbD blue
light-responsive promoter in Arabidopsis. FEBS Letters
571:26-30.
Kawai-Toyooka, H. , C. Kuramoto, K. Orui, K. Motoyama, K.
Kikuchi, T. Kanegae and M. Wada (2004) DNA
interference: a simple and efficient gene-silencing system
for high-throughput functional analysis in the fern
Adiantum. Plant Cell Physiol. 45: 1648-1657.
Ichikawa, K., M . Sugita, T. Imaizumi, M. Wada and S. Aoki
(2004) Differential expression on a daily basis of plastid
sigma factor (PpSig) genes from the moss Physcomitrella
patens: Regulatory interactions among PpSig5, thecircadian clock and blue light signaling mediated by
c1yptochromes. Plant Physiology 111 press.

Review articles
AcFtsZ1 + hygt

--

Fig.
Phenotypes by DNAi. (A) Chloroplastaccumulation and avoidance movement in prothalli induced
by weak white light (left) and strong blue light (right),
respectively. Note that the avoidance response under strong
light was inhibited by AcPHOT2 introduction. (B) Red
light-induced phototropism in protonemal cells generated
from a prothallus. Red light was irradiated from the left.
Note that phototropic response was inhibited by AcPHY3
introduction. (C) Chloroplast relocation induced bypolarized red (left) or blue light (right) in protonemal cellsof (B). Note that chloroplast accumulation response induced
by red light was inhibited by AcPHY3 introduction. (D)Chloroplasts of a prothallus. Note that chloroplast di vision
was inhibited by AcFtsz introduction. Both promoterless
AcPHOT2 (A), AcPHY3 (B,C), and AcFtsZJ(D)and hyg'
gene or hyg' gene only were introduced into thesegametophytes. Bar in A, C and D: 20 ~un , B: 200 ~Lm. See
Kawai-Tokuoka eta! (2004) in detail.

List of publication:
Original articles
Srinivas, A., RK Behera, T. Kagawa, M. Wada, and R.
Sharma (2004) High pigment! mutation negativel yregulates phototropicsignal transduction in tomato
seedlings . Plant Physiol.l34:790-800.
Lamparter, T., T. Kagawa, G. Brucker and M. Wada (2004)
Positive and negati ve tropic curvature induced by
microbeam irradiation of protonemal tip cells of the moss
Ceratodon purpureus. Plant Biology 6:165-170.

Wada, M, and N. Suetsugu (2004) Plant organelle
positioning. CwTent Opinion of Plant Biology 7:626-631.
Kasahara, M. and M. Wada (2004) Chloroplast avoidance
movement. In : Armual Plant Reviews "Piastids"
Edited by Simon Moller, Kluwer Academic Publishers.
In press .
Kanegae, T. and M. Wada (2004) Photomorphogenesis ofFerns. In, Photomorphogenesis in Plants 3rd Edition
Edited by Schafer and Nagy, Kluwer Academic Publishers,
Dordrecht. In press.
Suetsugu, N. , and M. Wada (2004) Photoreceptor Gene
Families in Lower Plants . ln, Handbook of Photosensory
Receptors. Edited by W.R. Briggs and J.L. Spudicb ,Wiley-VCH Verlag, Weinheim. In press.
Wada, M. (2005) Chloroplast movement. In: Light Sensing
in Plants, Edited by M. Wada, K. Shimazaki , and M. lino.
Springer-Verlag, Tokyo . In press.

68

NATIONAL INSTITUTE FOR BAS IC BIOLOGYENV IRONMENTAL BIOLOGYLABORATORY OF PHOTOENVIRONMENTAL BIOLOGY
Professor (Adjunct) :Associate Pro,jessor:
Postdoctoral Fellows:

Visiting Scientist:
Technical Assistants:

I}
2

J

WATANABE, Masakatsu (Sep. 1 -)WATANABE, Masakatsu (-Aug 31)
ISEKJ, Mineo IJ
YOSHIKAWA , Shinya 2J
SUZUKI, Takeshi 2;
ITO, Shinji
KO UMURA , Yoshiko 2;
ITO, Makiko

PRESTO, JST (Group Leader)
PRESTO, JST

Photoreceptive and signal transduction mechani sms ofphototaxis of unicellular algae are studied action
spectroscopically (Watanabe 2004) by measuringcomputerized-videomicrographs of the motile behavior of
the cells at the cellular and subcellular levels.
Photoreceptive and signal transduction mechanisms ofalgal gene expression were also studied by action
spectroscopy (Krabs et al. 2004) .
A novel blue-light receptor with an effector role wasfound from Euglena gracilis (Fig. I ; Iseki et a/. 2002,
Nature 415, 1047-1051): Euglena gracilis, a unicellular
flagellate, shows blue-light type photomovements. The
action spectra indicate the involve ment of flavoproteins as
the photoreceptors mediating them. The paraflagellar
body (PFB), a swelling near the base of the flagellum has
been considered as a photosensing organelle for thephotomovements . To identify the photoreceptors in the
PFB , we isolated PFBs and purified the flavoproteins
therein. The purified flavoprotein (ca. 400 kDa), with
noncova1ently bound FAD, seemed to be a heterotetramerof a- and ~-subunits . Predicted amino acid seq uences ofeach of the subunits were similar to each other andcontained two FAD-binding domains each followed by anadenylyl cyclase catalytic domain. The flavoprotein
showed an adenylyl cyclase activ ity, which was elevatedby blue-light irradiation . Thus, the fl avoprotein (PAC,
photoactivated adenylyl cyclase) can directly transduce alight signal into a change in the intracellular cyclic AMPlevel without any other signal transduction proteins.
The involvement of PAC in positive and negative
phototaxis (steering response with respect to stimulus
light direction) was also demonstrated by its knock-down
using RNA i through collaboration with Professor D .-P.
Hader's laboratory of Phillips University, Erlangen.
Germany (Fig. 2; Ntefidou et a!. 2003 Plant Physiol. 133,
1517-1521.).
Control

RNAi PACa

RNAi PAC~

RNAi PACa&~

oo

oo

oo

Fig.2 Swimming tracks of Euglena cells summarized incircular histograms under unilateral light irradiation.
Control cells swimming with high precision away from thelight source and RNAi-treated cells showing randomswimming. (From Ntefidou et a/. 2003, Plant Physiol. 133,
15I 7-1 521)

Fig.! Schematic diagram showing domain structures of
PAC subunits and working hypothesis of action
mechanism of PAC to photocontro l Eug lena flagellar
movement.

To gain an insight into the evolution of this uniqueprotein, si mil ar sequences were searched for iJ1 several
euglenoids by RT-PCR using degenerate primers. Twosimilar transcripts were detected in each of the four
phototrophic euglenoids, Euglena stellata, Co /acium
sideropus, Eutreptia viridis, Eutreptiella gymnastica, andin an osmotrophic (i. e. , obtaining nutrients by absorption)
one, Khawkinea quartana, but not in a phagotrophic
euglenoid, Petalomonas cantuscygni.
Each of themseemed to be orthologous to PACa and PAC~ ,respectively, and had the same domain structure as PAC

69

NATJONAL INSTITUTE FOR BASIC BIOLOGYENVIRONM ENTAL BIOLOGY

subunits each of which is composed of two flavin binding
domains, F 1 and F2, each follow ed by an adenylyl
cyclase catalytic domain, C I and C2, respectively. This
fact impli es that they constitute a functional
photoactivated adenylyl cyclase like PAC. Phylogenetic
analysis of the adenylyl cyclase catalytic domains
revealed that they belong to a bacterial cluster, not to atrypanosoma! one. In addition, two tJypanosome-type
adenylyl cyclases were discovered in £. gracilis. In
contrast to PAC, deduced amino acid sequences of thetrypanosome-type adenylyl cyclases indicated that they
are integral membrane proteins with a membrane
spanning region at the midpoint of them, followed by anadenylyl cyclase catalytic domain which seems
cytoplasmic. Overall, we propose that PAC might havebeen transferred to euglenoids on the occasion of
secondaty endosymbiosis (Fig. 3; Koumura et al. 2004).

=

Osmotrophoc euglenoids(secondarily derived)

I~~?

Astasia longa
Khawkinea quartana

IJ'hototrophic euglenoids) ~Acquisition ofSecondaryendosymbiont

chloroplast

I===:::>:~?

Euglena stellata

Colacium sideropus
Eutreptia viridis
Eutreptiella gymnastica(Phagotrophic euglenoid~

I(Common ancestry)

IrAe I

Euglena gracilis

ITAC I ?

Petalomonas cantuscygni

ITAC I ?

I(rrypanosomes) TAC

I

Fig.3 Working hypothesis on the origin of PAC. Presence oforthologs of PAC subunits ('PAC' in an oval) andtrypanosome-type adanylyl cyclases ('TAC' in a rectangle)
are indicated on the well accepted evolutionary history of
euglenoids. (From Koumura eta/. 2004).
Basic kinetic studies on photoactivation of PAC in vitro
and in vivo were done (Yoshikawa et al. submitted): in
vitro photoactivation by blue-light showed reciprocity
between fluence rate and duration of irradiation in the
fluence rate range of 2-50 J..tmol/m 2/s. Intermittent
irradiation caused activation of PAC in a photon fluence
dependent maru1er irrespective of cycle periods. The timecourse of the change in intracellular cAMP level agreed
well with that of the step-up photophobic response.
Overall , an increase in intracellular cAMP level evokedby photoactivation of PAC is a key event of the step-up
photophobic response.

Publication List:
Koumura, Y., Suzuki , T. , Yoshikawa, S., Watanabe, M. andlsek i, M. (2004) The origin of photoactivated adenylyl
cyclase (PAC), the Euglena blue-light receptor:
phylogenetic analysisi of orthologues of PAC subunits
from several euglenoids and ttypanosome-type adenylyl
cyclases from Euglena gracilis. Photochem. Photobio/.
Sci. , 3, 580-586.
Krabs, G., Watanabe, M. and Wieneke, C. (2004) A
monochromatic action spectrum for the photoinduction of
the UV-absorbing mycosporine-like amino acid shinorine
in the red alga Chondrus crispus. Photochem. Photobiol.
79, 515-519.
Watanabe, M. (2004) Action Spectroscopy for Photosensory
Processes. In CRC Handbook of Organic Photochemistry
and Photobiology, 2nd edition, (Horspool, W. and Lenci,
F. eds), CRC Press, Boca Raton, pp. 115. 1-115.16.

70

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYLABORATORY OF STRESS RESPONSE

Associate Professor:

MIKAMI, Koji

The aim of this laboratory is to understand molecularmechanisms of the flexibility in modification of the cell
polarity during morphogenesis in plants under thechanges in environmental conditions. As their sessile
nature, growth and development of plants are largely
influenced by environmental factors, thus plants must
percept and response to these factors to regenerate thegrowth axis. In the responses to environmental stimuli,
phosphoinositide-specific phospholipase C (PI-PLC)
plays the essential role in the generation of two second
messengers,
inositol-! ,4,5-trisphosphateandofdiacylglycerol
throughthehydrolysis
phosphatidylinositol-4,5-bisphospate (PIP 2). Plant PI-PLC
is now thought to be involved in responses to gravity andlight, both of which effect the modification of the cell
polarity by generation of a physiological asymmetry. In
moss plants, such as Physcomitrella patens andCeratodon purpureus, changes in cytosolic Ca2+
concentration in caulonemal cells are associated with thegeneration of buds, the precursors of gametophores,
which is regulated by blue light and cytokinin. In addition,
the red-light-induced phototropic responses of protonemal
tip cells are mediated by phytochrome. It has been shownthat red-light-irradiation leads to an increase of the
PI-PLC activity and the phototropic bending follows the
production of a dynamic Ca2+ gradient in the protonemal
tip cells. Moreover, Physcomitrella is now recognized as amodel system for plants with easy application ofmolecular genetic approaches such as gene-targetedmutagenesis via the homologous recombination.
Therefore, it is reasonable to employ the moss
Physcomitrella for genetical and molecular investigationsof the roles of plant PI-PLCs in the modification of cell
polarity by environmental factors.

I.

Structural
PI-PLCscharacteristics

of Physcomitre/laSearching of the plant EST databases with BLAST
algorithm against AtPLClS from Arabidiopsis thaliana
revealed four Physcomitrella eDNA clones, PPU 141114,
PPU140521, PPU070504 and PPU161218. For the first
three ESTs derived from the same mRNA, the 5'-race andthe subsequent 3 '-race reactions resulted in the isolationof a corresponding full-length eDNA of 2,424 bp
containing an ORF for a PI-PLC homologue of 633 aminoacids and calculated molecular mass of 70.8 kDa, which
was designated PpPLCI. In addition, a 2,423 bp
full-length eDNA corresponding PPU161218 was isolatedby the 5'-race reaction, which contains an ORF for a
PI-PLC homologue of 639 amino acid and calculated
molecular mass of 71.8 kDa, designated PpPLC2. The
overall structure of PpPLC I and PpPLC2 was similar to
those of known plant PI-PLCs comprising the catalytic
domain and the C2 domain. PI-PLC isoforms in mammals

have been divided into four classes, f3-, y-, 8- and s-types,
all of which contain the catalytic domain that consists of
X and Y regions and regulatory domains such aspleckstrin homology (PH), EF-hand and C2 domains. In
contrast, plant PI-PLCs analyzed so far all show 8-typeorganization but lacking the PH and typical EF-handdomains. The N domain, which is conserved in
Arabidopsis PLCs as a regulatory domain with structuralsimilarity to the second loop of the EF-hand domain of rat
PLC81, was found in the N-terminal extensions in
PpPLCl andPpPLC2, although there was an insertion in
the N domain of PpPLC2. Since the PH and EF-handdomains are responsible for membrane-localization andCa2+-binding in mammals, the mode of activation of plant
PI-PLCs is probably different from those in mammals.

II. Physcomitre/la has typical and novel types of
PI-PLCs
The in vitro actlVlty of recombinant His-tagged
PpPLCl and His-tagged PpPLC2 was examined.
His-tagged PpPLCI hydrolyzed PIP 2 with maximum
activity of 38 nmol/min/mg of protein at a physiologicalconcentration of Ca2+ around l 0 J..LM and lower activity athigher Ca2+ concentrations, consistent with otherrecombinant plant PLCs. In contrast, His-tagged PpPLC2hydrolyzed PIP2 with very low activity under the same
conditions. When phosphatidylinositol (PI) was used as a
substrate in the reactions with various concentrations ofCa2+, both His-tagged PpPLCl and His-tagged PpPLC2
showed a very low-hydrolyzing activity of 1.3 and 9.4
nmol!min/mg of protein, respectively, at 10 J..LM Ca2+.
However, at 1 mM Ca2+, His-tagged PpPLC2 hydrolyzed
PI very efficiently (22 nmol/min/mg of protein) thanHis-tagged PpPLCl (3.5 nmol/min/mg of protein).
Together with their structural characteristics, I conclude
that PpPLC I and PpPLC2 are typical and novel types ofplant PLC, respectively.
In the X and Y regions, I 1 amino acids have been
identified as essential residues for mammalian PI-PLC
activity via binding to Ca2+ and substrate. I found that allof them are well conserved in PpPLC 1, although aconserved serine residue was replaced by asparagine
residue at amino acid position 409 in PpPLC2. In fact,
PLC-like proteins with amino acid substitutions ofresidues important for PLC activity have already been
isolated from rat and human, which have no
PIP 2-dependent PLC activity. Thus, it seems that the lossof PIPrdependent activity in PpPLC2 may be due toamino acid substitution in the catalytic domain and/or its
abnormal N domain.

III. PpPLCl is involved in cytokinin and gravity
responses
To elucidate the physiological functions of PpPLC 1,
targeted knockout mutants of the PpPLCJ gene weregenerated via homologous recombination. To construct a
plasmid for gene targeting, a 35S-promotor-driven

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

neomycin phosphotransferase II (nptll) cassette providing
G418 resistance was inserted into a 3509 bp fragment of
the PpPLCJ genomic clone. After transfomation of
Physcomitrel/a protoplasts w ith the targeting construct,G418-resistance transgenic lines were obtatned andscreened by PCR to confirm the specific disruption of the
PpPLCJ gene.
Protonemal filaments grown under standard conditions
showed a c lear difference between wild type (WT) andplcl , the mutants failing to form gametophores (F ig. I).
The formation of gametophores in Physcomitre/la follows
two independent developmental steps. First, an immaturesi de-branch is induced from an interca lary caulonemal
cell, which was shown to be regulated by blue light (BL)through cryptochromes. Secondly, the side-branch initialdifferentiates into a gametophore bud. Thi s is known to bea cytokinin-dependent step in Physcomitre//a. By
checking th e response of protonemal cells to BL andcytokinin , it was concluded that the absence ofgametophores in plc l is due to a defect in cytokinin
signaling.

71

PT-PLC is based on inhibitor studies that rn ight cause
non-specifi c effects, Physcomitre//a constitutes a most
attractive experimental system for studying these points at
the molecular level, which based on the efficient targeted
gene di sruption via homologous recombinat ion to
generate pic I null-mutants. Current works are focused on
the mode of activation of PpPLCI to e lucidate how
environmental sti muli lead the modification of the cell
polarity in plants.

V\fT

plc1
1Od dark,

gravity~

5d dark,

gravity~Fig. 2. Comparison of gravitropic response of protonemalfi laments between WT and pic I knocko ut. Protonemal
filaments of WT (A, C) and pic/ (B, D) we re cu ltured onverti cal agar surfaces for ten days in the dark (A, B).
Cultures were rotated after ten days for 90° and further kept
in darkness for add itional 5 days (C, D). Scale bars
represent 500 ~Lm.

Fig. I. Comparison of visible phenotypes between WT andpic/ knockout. Leafy gametophores, which were well
produced in WT (A), were not observed in pic I (B).
In add ition, the protonemal fi laments of plcl have astrong ly reduced abi lity to response to the gravity (Fig. 2).
WT plants produce uniforml y negative-gravitropically
growtng protonemal filaments prior to unilateral red lightiiTadiation, when protonemal filaments were cultured onvertical agar plates in the dark (Fig. 2A). However, theprotonema l filaments of plcl have a strongly reduced
ability to grow negativel y grav itropically in the dark (Fig.
2B). Moreover, after rotating the cultures 90°, WT
filaments responded with a subsequent change in thegrowth direction, according to the gravitropic stimuli ,whereas the plc l continued to grow randomly (Figs. 2Cand 2D). Similar results were obtained after growth in
2
1
diffuse white li ght (1 00 Jll110 l m· s· ) for two weeks in a
vertical orientation. Therefore, PpPLC l plays animportant ro le in the detection of and/or response to
gravitropic stimuli in Physcomitrel/a.
In summary, PpPLCI has diverse functions including
the forma tion of ga metophores from side branches inprotonemal filaments and the response to gravity in apical
tip cells. To understand molecular mechanisms ofmodification of the cell polarity, it is necessary to
elucidate how plants percept cytokinin and gravity, howthese signa ls are transduced in the cells and how PpPLC 1
acts to modify the cel l polarity. Whereas most of theknowledge about the physiological function of plant

Publication list:
Original articles
Mikami , K. , Repp, A. , Graebe-Abts, E. and Hartmann, E.
(2004) Isolation of cDNAs encoding typical and novel
types of phosphoinositide-specific phospholipase C fro m
the moss Physcomitrella patens. J. Exp. Bot. 55,
1437-1439.
Repp, A., Mikami , K. , Mittmann, F. and Hartmann, E. (2004)
Phosphoinositide-specific phospholipase C is involved incytokinin and gravity responses 111 the moss
Physcomitrel/a patens. Plant J. 40, 250-259.

Review article
Mikami , K. and Hartmann, E. (2004) Lipid metabolism inmosses. In, New Frontiers in B1yology: Physiology,
Molecular Biology and Functional Genomics (A.J. Wood,
M.J. Oliver and D.J. Cove, eds.), Kluwer Academic
Publishers, Dordrecht, The Neth erlands, pp. 133 -155.

NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY72

DIVISION OF THEORETICAL BIOLOGY
Associate Professor:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Students:

Secretary:

MOCHIZUKi, Atsushi
TOHYA , Shusaku
FUJITA , Hironori
AYABE, Yoshiko
YAMAGAMI, Ay umi
IMAMURA , Hisako
UMEBAYASHI, Hiromi

I. Mathematical approach to biological phenomena
We are studying biological phenomena by using
mathematical models.
The method gives us an
integrative understanding for behavior of complex
systems in biology including gene regulatory networks.
The mathematical models are also useful to understandStudy of thepattern formation in development.
mechanisms responsible for morphological difference
between species is an important research focus of the
Theoretical studies
cLment developmental biology.
would be useful in identifying candidates of cell-cellinteraction that are likely to be responsible for the systems
in real organisms.

II. A theoretical study for gene regulatory networks
and analysis of differentiated cell-states
The highly complex behavior of organisms is based on
the interactive regulation of genes, functioning together tocreate a gene network.
The regulation of gene
expression levels is dependent on the concentrations oftranscriptional
regulators.
Typically,
multipletranscription factors can bind to the regulatory region of agene; each factor may independently affect the gene
expression levels. In this marmer, the regulatory regionof gene serves as an integration point, performing a
logical computation of these transcription factors todetermine the resulting gene expression levels.
In
addition, once translated, the encoded protein may control
the transcription of other genes .
The complexity of gene networks has been considered
to be a problem that may be solved from a theoreticalpoint of view. Since the proposal of the Boolean modelby Kauffman, multiple mathematical models have been
invented and analyzed to study complex gene networks .
There are two general classes of network models, discreteand continuous models. Continuous model s are likely
more realistic than discrete models as a representation of
biological systems. However, the dynamical behavior ofcontinuous models is analyzed much less than that ofdiscrete models .
In this paper, I have developed a model for gene
networks in a framework of continuous time and statevariabl es. I analyze steady states appearing in thi ssystem in general way. The steady sta tes specify distinct
cell states defin ed by pattems of gene activity. I
introduce three types of steady states observed in acontinuous time and space model, whose va lues andstabili ty can be eva luated from the structure of gene

regulatory interactions. The expected numbers of steadystates in a randomly generated gene network were derived
in exact forms. I determined the distribution of thesteady states in a randomly generated network.
The results were surprising; the number of steady statesin a random gene network does not depend on gene
number. In addition, the number of steady states is verysmall ; the number of RSPs, in which the activity of eachgene is either on or off, is only one. Most randomly
generated gene networks will have only a few (or no)
steady states, despite the presence of many genes in thesystem. Thus, increases in the number of genes do not
augment the number of steady states for gene activity.
The gene expression steady states may correspond todifferentiated cell type identities.
Thus, this result
implicates that the number of genes is not the driving
force increasing the diversity of differentiated cell types
or the complexity of organisms.

0.5

0.4

averagec0:s 0.3- - N=5

..0
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N=30

~ 0.2

-o

0.1
_J_...l..__

0 1

s---

__::~-------...........

5

10

- N

2

number of steady states z
Figure I. Distribution of the number of regular stationary
points (RSPs). The di stribution demonstrates a case thatthe regul ation of each gene in each domain is independent.
The two lines show the results of gene number N is 5 and30. The distribution does not hardl y depend on gene
numberCondition to bea steady state/
Number of

Number of genes N

Figure 2. Intuiti ve explanation for the independence ofsteady state number on gene number. The number ofdomain s 2N, the max imum number of RSPs, in creases
exponenti ally with gene number N. Th e probabili ty foreach domain to contain an RS P, however, decreases withgene number. The two effects are opposing and equi va lent.
Thus, the effect of N on the number of steady states
di sappears.

In rea lity, multi-cellular organisms have a wide variety
of differe ntiated cells. Why, then, do the networks inactual organisms have multi ple steady states? It seems

NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY73

likely that the networks w ithin actua l organisms are quite
biased from the average randomly generated network. A
gene network including a large number of self-regulatory
Thus,
genes has a large number of steady states.
self-regu lation within a gene network may be the main
source of cell type diversity in achm l organisms.

III. Mathematical models for pattern formation ofleaf vascular networks.
The vascular system of plant is a network of bundles
that connects within major organs. The development or
differentiation of vascular system is one of the mostimportant subjects in botany, and a lot of studies havefocused the problem. In dicot systems, veins in a leaf
are usually diverse in their size and they can be classified
distinctively by their appearance.
The largest vein
located at the center of a leaf is called mid vein (orprimary vein), which are generated first.
Veins
differentiate progressively in an early developmental
stage of leaf. A vein , which is continuous with stem
vascular bundle, extends starting from proximal edge todistal end of the leaf, and then become a midvein.
Secondary veins are generated from branches from themjdvein and are thinner than the midvein. By repeating
branching and extension , higher order veins are generated
sequentially with growth of leaf and the reticulate
network system is formed.
A lthough a lot of experimental studies have attempted
to identify the mechanism of vascular patteming in leaves,
it is still a problem to solve.
Jacobs revealed theimportance of auxin for vascular differentiation. Auxin
is one of the major plant hormones. In the higher plant,
indole acetic acid (IAA) is one of the most important
auxin chemicals. Auxin is diffusible molecule; however,
it is considered that auxin is also transported along theparticular direction, usuall y from apical to basal in a stem,from distal to proximal in a leaf. It is called auxin pol artranspm1. In a leaf, auxin is considered to be produced
in apica l margins of leaves and transported toward theMany researchers considered thatproximal regions.
such auxin flow play important role in vascularization ofplant.
Three hypotheses have been proposed so far to exp lain
the leaf venation pattern formation. One is auxin
canalization hypothesis proposed by T. Sachs that is basedon the assumption of the positive fee dback regulation
between aux in flow and flow capacity. Auxin is adiffusible plant hormone of small molecule and has
important roles in va rious developmental events including
vascul ar differentiation in plants . In this hypothesis, auxi npathways are expected to be generated from a uniform
field and are extended in a sequentia l manner. The second
is activato r-inhib itor type reaction-diffusion hypothesis
proposed by H . Meinhardt that is based on two in teractive
factors of weakly diffusible activator and strongly
diffusible inhibitor. We proposed the third hypothesis,
substrate-dep letion type reaction-diffusion model , that isbased on two interactive factors of strongly diffusible
auxin reso urce and its consumer.

Figure 3. Branching pattem generated by computer
basedon
substrate-depletion
type
simulation
reaction-diffusion model. Veins grow toward the margin
with repeating branching. In the final state, regularinterval branching pattems are formed .
On the other hand, we tried to integrate the canalization
hypothesis and the experimental know ledge. we introduce
auxin transport by PIN l efflux carrier into the auxin
cana lization hypothesis. So we investigated a model
based on the assumption of the positive feedback
regulation between auxin flow and PIN I localization.
The substrate-depletion type reaction-diffusion model
can regenerate growth and branching pattems. On theother hand, the canalization model can generate closed
circuits of leaf veins under a condition.
Both twomechanisms seem to be responsible for the leaf vein
formation.

Figure 4. Leaf ve in pattern generated by computer
simulation based on canalization model. Veins growtoward the margin with repeating branching. The final
patterns are similar to ones observed in maple leaves.

Publication List
Mochizuki , A. (2005) An Analytical Study of The Number of
Steady States in Gene Regulatory Netwo rks. J. Theor. Bioi.
(In Press).
Tohya, S. & Mochizuki, A. (2005) A new model for leaf
venation pattem. J. Th eor. Bioi. (In Press) .
Fujita, H. & Mochizuki , A. (2005) Pattern formation by the
positive feedback regul ation between flow of diffusible
signal molecule and localization of its carrier. J. Th eor.
Bioi. (ln Press).
Tohya, S., Moch izuki, A. and lwasa, Y. (2003) Random cellsorting can fonn cone mosaic patterns in fish retina andexp lain the di fferen ce between zebrafis h and medaka. J.
Theor. Bioi. 221 , 289-300.
Shoji, H. , Mochi zuki, A. , lwasa, Y. , Hi rata, M., Watanabe, T.,
Hioki, S. & Kondo, S. (2003) Origine of directionality inthe fish stripe pattern. Dev. Dyn. 226, 627-633 .
Ryohji , T. , Mochi zuki , A. & lwasa, Y. (2003) Possibil ity of
Tissue Separation Caused by Cell Adhesion . J. Theor. Bioi.
221 , 459-474.

NATIONAL INSTITUTE FOR BAS IC BIOLOGYTHEORETICAL BIOLOGY74

LABORATORY OF GENOME INFORMATICS

A
12571216
225

Research Associate:'"

UCHIYAMA, flaw

1891

ISO
134
123

Accumulation of biological data has recently been
accelerated by various high-throughput so-called "omics"
technologies such as genomics, transcriptomics,
proteomics and so on. The field of genome informatics is
aimed at utilizing these data, or finding some principles
behind the data, for understanding complex living systems
by integrating the data with current biological knowledge
using various computational techniques. In this laboratory
we focus on developing computational methods or tools
for comparative genome analysis, which is a usefulapproach for finding functional or evo lutionary clues tointerpreting genom.ic information of various species.
Especially," the current focus of our research topics is oncomparative analysis of microbial genomes, the numberof which is now beyond a hundred, as a basic model
system for understanding the variety of life through thecomparative analysis of numerous genomic sequences
simultaneously.

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109

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103
103
102

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I. Construction of microbial genome database forcomparative analysis
The number of completed microbial genome sequences
is growing rapidly, and nearly two hundreds genome
sequences in various levels of relatedness have alreadybeen available today. The role of comparative genomics
becomes much more important to utilize these large
number of sequences not only for elucidating
commonality in all of life, but also for understanding theevolutionary diversity within various groups, as well as
for understanding the evolutionary processes ormechanisms producing such diversity.
We have been developing and maintaining a databasesystem for comparative analysis of microbial genomes
named MBGD (http://mbgd.genome.ad.jp/). The central
function of MBGD is to create orthologous groups among
multiple genomes (Figure 1), which is a cmcia1 step forcomparative genome analysis. The key components of
MBGD include i) an algorithm that can classify genes
into mihologous groups using precomputed all-against-all
similarity search results, ii) a user interface that isdesigned for users to explore the resulting classification indetail, and iii) an incremental updating process for
similarities between genes and other data, which enables
the system to provide the latest data rapidly. By thisapproach, MBGD is now the world's largest database ofits kind. Moreover, by specifying a set of organisms, users
can obtain appropriate classification results that they wantusing the latest data available. This unique feature isespecially useful for users whose interests are focused onsome taxonomically related organisms. Indeed, we used
th.is feature of MBGD in our comparative genomics
studies among Bacillus-related species described below .
(see Figure 1).

Figure 1 Ortholog cluster map (A) and ortholog cluster
table (B) in MBGD . Here, the orthologous grouping was
created using genomes of 6 Bacillus-related species
including G kaustophilus. In the ortholog cluster map (A),the grouping result is sorted according to the phylogenetic
patterns, which represent presence or absence of theorthologs in each genome, and each cluster is assigned acolor according to its function category. By clicking the bar
graph of the map, the actual cluster table is shown (B),where each row represents an ortholog cluster and eachcolumn represents an organism.

We are also involved in a reannotation project forbacterial genomes that is currently in progress by theJapanese research community of pathogenic bacterial
genomics (headed by Dr. Kuhara, Kyushu University). In
this project, an orthologous grouping created by the
MBGD system is used as a prototype grouping, which
will be futiher modified and reannotated manually.

II. Revealing
thermoadaptation
trait
bycomparative genomics between thermophilic
mesophilic
Geobacillus
kaustophilus
andBacillus-related species.
How thermophilic organisms adapt to high-temperatureenvironments has long been an intriguing question in bothacademic and industrial fields. Recent studies revealed
some remarkable genomic features strongly correlatedwith thennophily such as amino acid composition, codon
usage and genomic dinucleotide composition. However
thermophiles whose genomic sequences have alreadybeen determined are somewhat phylogenetically biased;

NATIONAL fNSTJTUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY75

many of them belong to archaea or the deep-branchincr.
.
bbactena (Aquifex and Thermotoga). On the other hand,
one of the effective approaches in revea li ng thermophilic
trait is to compare genomes between closely relatedorganisms incl uding both them1ophiles and mesophiles.
This approach is also effective for understanding
thermoadaptation from the viewpoint of evolution,although the genomic sequences from an appropriate setof organisms are needed, which have not yet beenobtained.
In collaboration with Dr. Takami's g1·oup (JAMSTEC),
we determined the complete genomic sequence ofthermophilic Geobacillus kausophilus and compared itwith those of phylogenetically related 5 mesophilic bacilli ,Bacillus subtilis, Bacillus halodurans, Oceanobaci/lus
iheynsis, Bacillus cereus and Bacillus anthracis. By theprincipal component analysis of the amino ac id
composition using I 50 prokaryotic genomes including 20
thermophilic ones, we were able to find that thethermophilic G kaustophilus were distinguishable frommesophilic bacilli by the borderline distinguishingthermophiles from mesophiles on the second principal
axis, although on the whole the Bacillus-related species
were located near this borderline (Figure 2). Further
statistical analysis revealed some asymmetric amino acidsubstitutions between the them1ophilic and the mesophilicbacilli , which are possibly associated with thethermoadaptation of the organism.
In addition, upon ortho logous grouping of the 6 bacillar
sequenced genomes, we found 839 genes (24%) in th e G
kaustophilus genome being unique to that species, among
which we were able to find some candidate genes that
may contribute to thermophily presumably by enhancing
the stability of nucleic acids.
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Figure 2 Principal component analysis of the amino acidcomposition using 150 prokaryotic genomes. Thermophiles
are marked by symbols according to their optimal growthtemperatmes. An abbreviated name in upper case is
ass igned to each Bacillus-related spec ies; GK: G
kaustophilius, BH: B. halodurans, BS: B. subtilis, BA: B.
anthracis, BC: B. cereus, 01, 0. iheyensis. An abbreviatedname in lower case is assigned to each thermophile.

Ill. Identification of the common core structure ofbacillar genomes
Recently several studies showed that horizontal transfer
as well as vertical transfer has played important ro les in
the prokaryotic genome evolution. However, to obtainclearer picture of the bacterial genome evolution , we needfurther detailed investigations including extensive
comparison of multip le genomes that are closely ormoderatel y related to each other. In the collaborative
work with Dr. Takami , we also performed such extensive
comparison among Bacillus -related genomes for the
purpose of drawing general picture of the bacterial
genome evolution.
endospore-forming
gram-pos1t1ve
Aerobic
Bacillus-related species is known to be able to grow in awide range of environments. The above-mentioned 6
bacilli whose genomic sequences have been determined
include alkaliphilic B. halodurans, halotolerant 0.
iheyensis and thermophilic G kaustophilus, in addition to
we ll-known laboratory stmin B. subti/is and pathogenic B.
anthracis and B. cereus. These organisms except B.
anthracis and B. cereus are moderately diverged eachother and are belonging to distinct major clusters in the
I 6S rRNA phylogenetic tree. By simple pairwise dotplot
analyses between them, one can easi ly see some large
collinear regions along the diagonal lines of the plots sothat the overall genomic structures are primarily wellconserved between organisms. However, one can alsofind a substantial numbe r of species-specific genes thatare inserted in each of the genomes.
To investigate further, we are trying to identify
common "core structure" of bacillar genomes, which isdefined as a set of sufficiently long consecutive genomic
segments in which gene orders are conserved among
multiple genomes so that they are likely to have been
inherited from the common ancestor mainly tlu·oughvert1cal transfer. We have developed a novel algorithm forali gni ng conserved regions of mu ltiple genomes by
sorting orthologous groups so as to retain the conserved
gene orders as many as possible. From this aligrunent, wewere able to identify the conunon core structure ofbacillar genomes comprising about 1500 genes .
It appears that most of the important genes are includedin the resulting core gene set. For example, the set
contains 246 out of 271 B. subti/is essential genes thatwere primarily determined by a systematic inactivation
Fmiher characterization . of the core bcrene set
experiment.
.
IS currently in progress.

Publication List:
Takami , H. , Takaki, Y. , Chee, G-J. , Nishi , S. , Shimamura, S.,
Suzuki, H., Matsui , S., Uchiyama, I. (2004)
Thermoadaptation trait revealed by the genome sequenceof thermophilic Geobacillus kaustophilus. Nucleic Acids
Res., 32, 6292-6303.

76

NATIONAL INSTJTUTE FOR BASIC BIOLOGYLABO RATORY OF DIRECTOR GENERALLABORATORY OF DIRECTOR GENERAL
Director General:
Postdoctoral Fellovv:
Graduate Student:
Supporting Staffs:

KATSUKJ, Mataya
ARAKAWA-KOBAYASHI, Satoko
MO/UTA, Tomoko (Ap1: 'OJ- Mm: '03)
ETOH, Tomoo (Ap1: 'O J- Mm: '03)
MJYAKAWA, Atsushi
KATSUKJ, Kuniko
MJYAKAWA, Yuko

To examine involvement of dopaminergic system inneural development we generated mutant mice in which
dopaminergic transmission can be shut off at a time point ofinterest. We utilized tetracycline controllable expression
system and generated transgenic mice harboring conditional
DIR expression on the DJR!D2R DKO background(Figures I and 2).

One of the largest themes of the 21 " century is topromote the brain research, for example, researches onunderstanding a "mind." There are many approaches to
elucidate the "mind," such as intelligence, memory,
cognition, emotion and volition. Researches on developing
an excellent artificial system for information-processing,researches on the ontogeny and the development of thebrain, and researches on the protection of the brain fromaging, neurological and psychiatric disorders are also
important themes.
Our approach to understanding of the brain is to explore
the molecular mechanism of higher brain function by
employing genetic modification of living organisms. Major
research interests of the laboratory are to elucidate aphysiological role of dopamine receptors in animal
behavior, an implication of N-methyi-D-aspartate receptors(NMDARs) in psychiatric disorders and the roles of the ras
family in the brain by producing genetically altered mice,
both gene targeted and transgenic mice.
I. Doparninergic system and feeding behavior
Dopaminergic system is considered to be involved inlocomotor control, emotional behavior, reward, motivationand thought process. Hypoactivity or hyperactivity of
dopaminergic system can result in neurological andpsychiatric disorders like Parkinson 's disease andschizophrenia. In mammals five subtypes of dopamine
receptors (DlR-D5R) are identified and classified into two
major groups, Dl-like (DlR, DSR) and D2-like (D2R, D3R,
D4R) receptors on the basis of the gene structure and thepharmacological and intracellular signaling properties. The
contribution of D 1-like and D2-like receptors to behaviors
is determined pharmacologically.
We generated knockout (KO) mice lacking each of five
dopamine receptors and multiple KO mice lacbng morethan one dopamine receptor simultaneously. We focus on
DlR and D2R, major subtypes of Dl-like and D2-likereceptors, respectively, which are most widely andabundantly expressed. D 1R KO and D 2R KO rnice werefertile and exhibited characteristic locomotion. We foundthe Dl RID2R double knockout (DKO) mice showed severe
impairment in feeding that was not observed in DJ R or
D2R KO mice. Although the DJR/D2R DKO mice werebom normally and showed suck ling behavior, the DJRID2R
DKO mice exhibited rapid decrease in locomotion and no
initiation of feeding aro und weaning period, and evenhmlly
died with emaciation by the third postnatal week. Thesefindings suggest that dopaminergic system is involved inneura l development in the areas wh ich are implicated in theregulation of locomotion and feeding .

Figure l. DJRID2R DKO mice were rescued by conditional
DIR expression. (A) DIRID2R DKO mi ce showed impaired
feeding and prematu re death. (B) To rescue DJRID2R DKO
mice transgenic mi ce harboring cond itional D I R express ionon the D I RID2R DKO background were generated bytetracycline controllable expression system.

We screened potential h·ansgenic lines with respect totetracycline controllable expressiOn and successfully
established the transgenic mice harboring conditional D 1R
expression (Figure 3). We are producing the conditiona l
Dl R expressing mice on the Dl RID2R DKO backgroundand plan to examine the mice by molecular biological ,morphological and behavioral analyses.

NATIONAL INST ITUTE FOR BAS IC BIOLOGYLABORATORY OF DIRECTOR GENERA L

77

play a number of important roles. There are many reportson the in volvement of th e NMDARs in learn ing andmemory. According to one hypothes is schi zophrenia may
involve a defect in NMDAR function . NMDAR s consist of
NRI subuni t and at least one subunit ofNR2A-NR2D. The
NRI is ubiquitous ly expressed in th e brain, while NR2
subunits have a more specific spati a l distribution. We
generated KO mice lacking each of NMDAR subunits andmul tipl e KO mice lacking two subunits s imultaneousl y, a nd
found the NR2A homozygous, NR2B heterozygous mutant(NR2A ·'-, NR2B+1-) mice exhibited behav ioral alteration
similar to that observed in patients with schi zophrenia. We
plan to develop a new experimenta l devise to assessbehavioral alteration of the NR2A·'-,NR2B+t- mi ce and study
the mol ec ular mechani sm.
Figure 2. Tetracycline controllable gene expression system.
(A) Transgenic mice we re generated by introducing of the
D IR promoter-driven tetracycli ne trasnactivator (tTA) and thetetracyc line operator-dri ven D I R gene. In the absence of
doxycyc line the DIR gene was expressed by the action oftTA
and the tetracycline operator. (B) By administration of
doxycycline the binding of doxycycline to tTA led to the
suppression of the expression of D I R.

III. ras family and their roles in the brain
The ras proto-oncogene plays a criti ca l role in cell
growth control as a central component of mitogenic s igna ltransduction pathways. In ma mmals the re are H-, N -, K-ras
identified as the ras fa mily. H-, N -, K-ras have an
overl apped spatial expression pattem as well as anoverlapped function . We generated and analyzed KO micelacking each of H-, N-, an d K-ras. We found H-Ras was
implicated in the regulation of long-term po te nti atio n in the
hippocamp al
CA I
reg ionthrough
NMDAR
phosphorylation. To investigate the distinct function of theindi vidua l Ras prote in in the brain we generated ras DKO
mice expressing a si ng le Ras and triple KO mice lacking a ll
H-, N-, and K-Ras and analyzed developmental aspects ofthese mutant mice.

Publication list:

Figure 3. Mice carrying cond itional D I R express ion weregenerated using teh·acycline system. Frontal secti ons ofmouse brains with X-gal staining were shown. (A) The
expression of tra nsgene was seen in the striatum by theexpression of a marker gene, /acZ (b lue color). (B) By
doxycycline adm inistration the expression of transgene wassuppressed in the stri atum .

11. Analysis of the function of NMDA Rs
The NMDARs are wide ly expressed in the nervo us
system, fundamental to exci tatory neurotransmission, andKonishi S, Naora H , Kimura M, Sato M, Nagasaki M,
Yokoya ma M, Otani H, Moritake K, Kats uki M. (2004)
Expression of SV40 T anti gen gene in the oligodendrog lia
induced primitive neuroectodermal tumor-like tumors in themouse brai n. Congenit Anom (Kyoto). 44, 2 I 5-224.
Sato M, Tabata T, Has himoto K, Nakamura K, Nakao K,
Katsuki M, Kitano J, Moriyoshi K, Kano M, Nakanishi S.
(2004) Altered agoni st sensiti vity and desensitization ofneuronal mGluRI responses in knock-in mice by a singleamino ac id substi tuti on at the PKC phosp horyl ation site.
Eur J New·osci. 20, 947-955.
Kuriwaki J, Nishij o H, Kondoh T, Uwano T, Torii K, Katsuki
M, Ono T. (2004) Comparison of brain activity between
dopamine 02 receptor-knockout and wild mice in response
to dopamine agonist and antagonist assessed by fMRI.
Neurosigna/s. 13, 227-240.
Tran AH , Tamura R, Uwano T, Kobayash i T, Katsuki M,
Matsumoto G, Ono T. (2002) Altered accumbens neural
response to prediction of reward associated with pl ace indopamine D2 receptor knockout mice. Proc Nat/ A cad Sci U
SA . 99, 8986-8991.

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORT78

RESEARCH SUPPORT FACILITY
Head:
Associate Professor:
Research Associates:

Technical Staffi:

NISHIMURA, Mikio
WATANABE, Masakatsu
HAMADA, Yoshio(Tissue and Cell Culture)
UCHIYAMA, Ikuo (Computer)
HIGASHI, Sho-ichi(Large Spectrograph)
NAKAMURA, Takanori(Large Spectrograph)
MIWA, Tomoki (Computer)
NANBA, Chieko(Plant Culture, Farm, Plant Cell)NISHIDE, Hiroyo (Computer)
ICHIKAWA, Chiaki(Large Spectrograph)
TAKESHITA, Miyako(Trssue and Cell Culture)
MAKIHARA, Nobuko (Computer)
SUZUKI, Keiko(Plant Culture, Farm, Plant Cell)

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
spectro-scopical studies of various light-controlled
biological processes. The spectrograph runs on a 30kW
Xenon arc lamp and has a compound grating composed of
36 smaller individual gratings. It projects a spectrum of awavelength 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 eachwavelength is more than twice as much as that of thecorresponding monochromatic component of tropicalsunlight at noon (Watanabe et a/. 1982, Photochem.
Photobiol., 36, 491-498).
An advanced irradiation system composed of CW
lasers (364nm, 390-41 Onm, 440-460nm, 532nm, 655nm,
752nm) and uniform-fluence-rate irradiation optics
interconnected by optical fibers was constructed in 2003.
An advanced observation system for cellular andintracellular photobiological responses utilizing a
two-photon microscope (FV300-Ix71-TP with a MaiTailaser) and a microbial photomovement analyzer(WinTrack2000/Ecotox) etc. was also introduced.

2. Tissue and Cell Culture Laboratory
Various equipments for tissue and cell culture areprovided. This laboratory is equipped with safely rooms
which satisfy the P2/P3 physical containment level. 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 220R), 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. At the end of thisyear, a new computer system with enhanced performance
was introduced as a replacement for the current system.
The service of the new system will start at the beginningof the next year.
Computer laboratory
also provides
network
communication services in the institute. Most of PCs ineach laboratory as well as all of the above service
machines are connected 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 are provided 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 alimited number of rooms with environmental control forplant culture. In some of these facilities and rooms,
experiments can be carried out at the PI physicalcontainment level under extraordinary environments suchas strong light intensity, low or high temperatures.

5. Experimental FarmThis laboratory consists of two 20m2 glass-houses with
precise temperature and humidity control, three green
houses (each 6 m2) at the P 1 physical containment level, asmall farm, two greenhouses (45 and 88 m2) with
automatic sprinklers. The laboratory also includes abuilding with storage and work space.

6. Plant Cell Laboratory
Autotrophic and heterotrophic culture devices andequipment for experimental cultures of plant andmicrobial cells in this laboratory. A facility for preparationof plant cell cultures including an aseptic room with clean
benches, is also provided.

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORT

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

Publication List:
Cooperative Research Program for the Okazaki
Large SpectrographAoki, M., Furusawa, Y., Higashi, S-1. And Watanabe, M.
(2003) Action spectra of apoptosis induction andreproductive cell death in L5178Y cells in the UV-B
region. Photochem. Photobiol. Sci. 2004, 3, 268-272.
Kami, C., Mukougawa, K., Muramoto, T., Yokota, A.,
Shinomura, T., Lagarias, J. C. And Kohchi, T. (2004)
Complementation of phytochrome chromophore-deficientArabidopsis by expression of phycocyanobilin: ferredoxin
oxidoreductase. Proc. Nat/. Acad. Sci. 101, 1099-1104.
Kraebs, G., Watanabe, M. And Wieneke, C. (2004) A
monochromatic action spectrum for the photoinduction of
the uv-absorbing mycosporine-like amino acid shinorine
in the red alga Chondrus crispus. Photochem. Photobiol.
Sci. 79, 515-519.
Ohmori, M. And Okamoto, S. (2004) Photoresponsive cAMPsignal transduction m cyanobacteria. Photochem.
Photobiol. Sci. 3, 503-511.
Terauchi, K. And Ohmori, M. (2004) Blue light stimulates
cyanobacterial motility via a cAMP signal transduction
system. Molecular Microbial. 52, 303-309.
Watanabe, M. (2004) Action spectroscopy for photosensory
processes. In CRC Handbook of Organic Photochemistry
and Photobiology, 2nd ed.(Horspool, W. and Lenci, F.
eds.), CRC Press, Boca Raton, pp. 115.1-115.16.
Yoshihara, S. And Ikeuchi, M. (2004) Phototactic matility inthe unicellular cyanobacterium Synechocystis sp. PCC
6803. Photochem. Photobiol. Sci. 3, 512-518.

79

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORT80
RESEARCH CENTER FOR INTEGRATIVE ANDCOMPUTATIONAL BIOLOGYHead:Associate Professor:
Supporting Staff'

TAKADA , Shinji
MOCHIZUKI, Atsushi
UMEBAYASHJ, Hiromi

The aim of the research center for integrative andcomputational biology is (I) investigating fundamental
principles of various biological phenomena based on themethod of integration of computational science andbiology; (2) establishing new methodology for the
integrative biology; and (3) providing the new technology
and knowledge for the researchers. Our final goal is toestablish new bioscience including methods originally ofdifferent fields ; informatics, mathematics, and biology.

I. Research activity
The research center for integrative and computational
biology was founded in 2001 under the situation ofrapid
progress in modem biology. The success in world wide
genome projects has provided a huge amount of
information of genes. Following this, it is expected todevelop new medicine for intractable diseases or to
exploit new cultivated plants resistant to noxious insects,
and so on. In addition, it is the subject of present
biology to solve higher-order phenomena that are
constructed from complex interaction between many
genes. To answer these requests, it is essential todecipher the enigma of huge amount of gene information,and to derive the essence for the biological behavior ofcells or organisms.
Mathematical and computational sciences have strong
property for this condition.
Computational methods
make it possible to process much larger data than humanability.
Hypotheticalexperiments
basedonmathematical or computational models make it possible toconsider conditions which are impossible in realexperiments including the evolution of past organisms.
We are continuing to research higher-order phenomena inbiology by using mathematical or computational methods,
and to develop new methodology for studying suchcomplex phenomena.

For examp le, the integrative methods are espec ially
important to understand pattem fonnation in development.
Morphological difference between species is an importantresearch focus of the current developmental biology.
What is the mechanism responsible for the difference ofmorphogenesis between species? Theoretical studies
would be useful in identifying candidates of cell or geneinteraction that are likely to be responsible for the systems
in real organisms. The method gives us an integrative
understanding for behavior of complex systems in biology
including gene regulatory networks.

II. Collaboration activity
Interaction between researchers is essential to make thebest use of mathematical and computational methods forexperimental biology. The theoretical methods should
provide testable predictions, and the experimental
biologists should test the predictions and return the results
to theoretician for the next predictions. By repeatingthese predictions and tests, the integrative methods should
be developed. The research center for integrative andcomputational biology has continued to enhanceinteraction
betweentheoreticalbiologists
andexperimental biologists.
The center provides equipments for computation andexperiments.
We have some cluster machines andenough number of Unix based machines for our member.
The member can also use computational equipments in
Research Center for Computational Science in Okazaki.
The center also provides experimental equips for thecollaborations with experimental biologists.

Figure 2.

Figure 1.

A laboratory room for computational studies.

one of equipm ent, a cluster machine.

We have held research conferences repeatedly. [n2004, we held two days symposium in March 8-9, entitl ed
as "Mathematica l and Computational Approaches to
Bio logy" , where dozens of researchers studying
mathematical, computational , or experimental biology
The history of mathematical orparticipated .
computational methods for b iology is still short. The
center has conti nued to bring up and to encourage yo ung
researchers who can use these methods for biologicalphenomena.

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORTCENTER FOR
TRANSGENIC ANIMALS AND PLANTS
Head:
Associate Professors:

Technical Staffs:
Supporting Staffs:

TAKADA, Shinji
WATANABE, Eiji
SASAOKA, Toshikuni
TANAKA , Minoru
HAYASHI, Kohji
ICHIKAWA , Hiromi (Jun. '04 -)YASUDA, Mie
NOGUCHI, Yuji
SHIMIZU, Naoki (-Jul. '04)
YAMAGUCHI, Kyoko
YOSHIDA , Etsuko
KAWAMURA, Motofioni (Jun. '04-)

The worldwide genome project has been almost
completed, and research on basic biology comes to post
genome era in which researches focus on investigating ofa function of an individual gene. To promote functional
analysis of a gene of interest it is essential to utilize
genetically altered model organisms, which are generated
by the genetic engineering technology, the creation ofgene deletion, gene replacement or point mutation .
NIBB Center for Transgenic Animals and Plants(CTAP) was established in April 1998 to support
researches using transgenic and gene targeting techniques
in NIBB. The CTAP began to be managed by the head(professor, a concmTent post) and an associ ate professor.

81

Technical staffs and supporting staffs develop andpromote research supporting activities. In 2003 two
associate professors joined in the CTAP. A new CTAP
building for transgenic animals in the Yamate area has
opened in the end of 2003 (Figure I) .
The expected activities of the CTAP are as follows:
I. Provision of information, materials, techniques andanimal housing space to researchers.
2. Equipment of various kinds of Lnstruments to analyze
mutant, transgenic, and gene-targeted animals andplants.
3. Development of novel techniques related to transgenic
and gene targeting teclmology.
4. Cryopreservation and storage of transgenic strains.

l. Research supporting activity (mouse)
In 200 I the NIBB mouse facility under the specificpathogen free (SPF) condition opened in the Myodaiji
area
and
the
production,
breeding,
analysis,
cryopreservation and storage of genetically manipulatedmouse strains has been conducted. The new CTAP
building in the Yamate area strengthened research
activities using genetically altered organisms (Figure I).
The building has five floors and a total floor space of
22,500m , in which we can generate, breed, store andanalyze transgenic, gene targeting and mutant mice under
SPF condition. This building is also equipped withbreeding areas for transgenic small fish , birds and insects
on the first floor. The mouse housing area of this building
is constructed based on a barrier system in which theclean area and the semi-clean area are clearly separated
and designed for an efficient and one-directional flow of
mice, persormel , articles of animal housing andequipments from the clean area side to the semi-clean
area side.

Figure 2.

Figure 1. A new center fac ili ty for transge nic an imals in
the Yamate area.

Autoc laves of a large scal e for sterili zation.

Since research activities in the CTAP building in the
Yamate area stat1cd in 2004, 3,138 mice brought into the
CTAP, and 7,894 mice (including pups bred in thefac ility) were ta ken out from the CTAP from April 01,
2004 to December 14, 2004.

82

NATIONAL INSTITUTE FOR BASIC BIOLOGYRES EARCH SUPPORTA number of strains of genetically altered mice outside
the CTAP were brought in the mouse housing area by
microbiologicalcleaning
using
the
in
vitro
fertilization-embryo transfer techniques, and stored bycryopreservation.
A new mouse facility in the Myodaiji area is under theconstruction and will be open in the beginning of 2005.
The facility provides research suppotiing activities toresearchers in the Myodaiji area.

ln 2004 (as of the end of November) , 1,190 medaka(552 embryos and 638 adults) were brought to the facilityand 19,776 medaka (15 ,757 fertili zed eggs, 1,662
embryos, and 2,357 adults, including animals bred in thefacility) were taken out from the CTAP. In a laboratory
for chick embryos 20,349 fertilized chicken eggs werebrought in and 213 animals (123 fertili zed eggs, 85embryos and 5 broods) were taken out from the CTAP.
These animals were used for the research activities inneurobiology and developmental biology.

Academic activity

Figure 3. Breeding equipments for mutant mice undercondition of specific pathogen-free.

II. Research supporting activity (small fish, birds,
and insects)
The first floor of the CTAP building in the Yamate area
provides space and facilities to maintain small fish , chickembryos and insects. In a laboratory room for chickembryos, a large incubation chamber is equipped and set
at 42 degrees suitable for chick embryogenesis. For
researchers who need fish , 480 tanks (1 litter) and 450
tanks (3 litters) are available for medaka and zebrafish,
respectively. Water circulates and can be maintained tosuit conditions desired for fish breeding in the aquarium
systems. In addition to the rooms mentioned above,
another room is available for insects. All the rooms are
qualified to meet the criteria for PIA transgenic animals,
allowing researchers to generate and maintain transgenic
animals.

Figure 4.

Breeding equipments for transgenic small fi sh.

The associate professors of this center, E. Watanabe, T.
Sasaoka, M. Tanaka, are the principal investigators ofLaboratoryof Neurophysiology,
Laboratoryof
Neurochemistry and Laboratory of Molecular Genetics
for
Reproduction,
respectively.
Laboratoryof
Neurophysiology is studying the brain sensing system forthe water and sodium homeostasis in the body fluid byusing gene-targeting mice, Laboratory of Neurochemistry
is sh1dying a physiological role of dopaminergi c systemusing genetically altered mice, and Laboratory of
Molecular Genetics for Reproduction is sh1dying
molecular mechanism of reproductive organ development
and sex differentiation using mutagenized or transgenic
medaka . For details, please refer to academic activity ofeach laboratory.

NATIONAL INST IT UTE FOR BASIC BIOLOGYRESEARCH SUPPORTTHE CENTER FOR ANALYTICAL INSTRUMENTS(managed by NIBB)Head of Facility :Technical Staffs:

Technical Assistants:

Secretmy:

KOBAYASHI, Saton1
MORI, Tomoko
MAKINO, Yumiko
TAKAMJ, Sh igem i
MORIBE, Hatsumi
DODO, Yukiko
KA J, Tsutomu
HATTORI, Nobuko

The Center serves for a mino acid seque nce ana lysis,
a mino ac id analys is, and chemica l sy nth eses of peptides
and to support researchers in NIBB and NIPS . Newly
installed instruments in 2004 is Genetic Anlyzer(ABI
PRISM 3 100). Instruments of the Center can be used byresearchers outside the Institute upon proposal.

83

Represe ntative instruments are listed below.
Protei n Seq uencers (ABI Procise 494HT,492cLC)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A)
Plasmid Isolation Systems (Kurabo Pl- IOOI, Pl-50, Pl-200)
Automati c Nucl eic Acid Isolation System (Kurabo NA-2000)
Genetic Anlyzer(AB l PRISM 3 100,3 10)
Therm al Cyclers (Perkin Elmer PJ-9600, Takara TP-300,
Biometra TGRAD!ENT)
Integrated Them1al Cyclers (ABJ CATALYST Turbo 800)
Parti cle Deli very System (Bio-Rad Bioli sticPDS-1 000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Ana lys is System (Takara G lyco-Tag)
High Perf01mance L iquid Chromatograp hs (S himadzu
LC-IOAD, LC-6AD, Waters 600E, Alli ance UV system)
Integrated Micropurification System (Phannac ia SMART)
Flow Cytometer (Co ulter EPICS XL)
Biomolecular Interaction Analysis Systems (P hannacia
BlACORE 2000, Affi nity Sensors IAsys)
Laboratory Autom ation System (Beckma n Co ulter Biomek
2000)
NMR Spectrometer (B ru ker AMX-3 60wb)
EPR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)
MALDI/TOF-MS (B ru ke r Daltonics REFLEX Ill)

Figure I. Protein Sequencers.

Ind uctive ly Co upl ed Plasma Atomi c Emission Spectrometer(Seiko SPS 1200A)
Spectrofluorometers (Hitachi 850, F-4500, Simadzu
RF-5000)
Spectrophotometers (H itachi 33 0, 557, U-200l ,Varian Cary
5G,Perki n Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB 96P)
Microplate Readers (Corona MTP-1 20, MTP-lOOF)
Spectropolarimeter (JASCO J-40S)
FT-IR Spectrophotometer (Horiba FT-730)
Laser Raman Spectrophotometer (JASCO R-800)
Bio Imag ing Anal yzers (F ujifilm BAS 1500, 2000, 5000)
Fluorescence Bio imag ing A nalyzer (Takara FMBIO)Electrophoresis Imaging System (B IOIMAGE)

Figure 2. MALDJ/TOF-MS

Microscopes (Carl Zeiss Axiophot, Ax iovert)
Environ menta l Scanning E lech·on Microscope (PHILIPSXL30 ESEM)
Confocal Laser Scanning Microscope (Leica TCS SP2)
Color Laser 3D Profile Microscope (KEYENCE VK-8500)
Hi gh-Reso luti on Qu ick Microscope (KEYENCE VH-5000)

84

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORTTECHNICAL DIVISION
Head: FURUKAWA, KazuhikoCommon Facility Group

Research Support Group
Chief KOBAYASHI, Hiroko

Research Support Facilities
HIGASHI, Sho-ichi (Unit Chief) <Large Spectrograph>
NANBA, Chieko (Subunit Chief) <Plant Culture>
NISHIDE, Hiroyo <Computer>
NAKAMURA, Takanori <Large Spectrograph>SUZUKI, Keiko (Technical Assistant)
MAKIHARA, Nobuko (Technical Assistant)
ICHIKAWA, Chiaki (Technical Assistant)
TAKESHITA, Miyako (Technical Assistant)
NISHIMURA, Noriko (Technical Assistant)Center for Analytical Instruments
MORI, Tomoko (Unit Chief)
MAKINO, Yumiko (Subunit Chief)
TAKAMI, Shigemi
MORIBE, Hatsumi (Technical Assistant)
DODO, Yukiko (Technical Assistant)
Transgenic Animal Facility
HAYASHI, Kohji (Subunit Chief)
KAWAMURA, Mamiko (Technical Assistant)
ICHIKAWA, Hiromi (Technical Assistant)
Disposal of Waste Matter Facility(MATSUDA, Yoshimi (Unit Chief))
Radioisotope Facility
MATSUDA, Yoshimi (Unit Chief)
SAWADA, Kaoru (Subunit Chief)
IINUMA, Hideko
ITO, Takayo (Technical Assistant)

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

Department of Cell Biology
KONDO, Maki (Unit Chief)
KABEYA, Yukiko (Subunit Chief)
Department of Developmental Biology
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
NODA, Chiyo
Department ofNeurobiology
OHSAWA, Sonoko (Unit Chief)
TAKEUCHI, Yasushi (Subunit Chief)
Department of Evolutionary Biology and Biodiversity
TANAKA, Sachiko (Unit Chief)
YAMAGUCHI, Katsushi (Subunit Chief)
MOROOKA, Naoki
SUMIKAWA, Naomi
Department ofEnvironmental Biology
MIZUTANI, Takeshi (Subunit Chief)
KONDO, Makiko (Technical Assistant)
Department of Theoretical Biology
MIWA, Tomoki (Unit Chief)

Reception
KATAOKA, Yukari
NAKANE, KaoriTSUZUKI, Shihoko
HIRONAKA, TomieMUKOHDA, Yasuyo
UNO, Satoko

Technical staffs participate, through the department,
in mutual enlightenment and education increase theircapability in technical area. Each technical staff isproceeded to the fixed division usually and they support
the various research with their special biological andbiophysical techniques.
The Department hosts an annual meeting fortechnical engineers who work in various fields of
biology at universities and research institutes throughout
Japan. At this meeting, the participants present theirown activities and discuss technical problems. The
Proceedings are published soon after the meeting.

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH FACILITIES85

OKAZAKI INSTITUTE FOR INTEGRATIVE BIOSCIENCE
Oointly managed by NIBB)

DEPARTMENT OF DEVELOPMENT, DIFFERENTIATION AND REGENERATIONDIVISION OF DEVELOPMENTAL GENETICS

See page. 25
DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

See page. 28

DEPARTMENT OF BIO-ENVIRONMENTAL SCIENCEDIVISION OF BIO-ENVIRONMENTAL SCIENCE

See page. 61
DIVISION OF PLANT DEVELOPMENTAL GENETICS

Seepage. 64

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH FACILITI ES

86

CENTER FOR RADIOISOTOPE FAGILITIES (CRF)

Myodaiji-Area

Yamate-Area

total

CFBI- branch LGER- branch

Head:
Associate Professor:
Technical staffs:

Supporting Staffs:

TAKADA, Shinji(Projess01; concurrent post)
OGAWA, Kazuo(Radiation Protection Supervisal)
MATSUDA, Yoshimi(Radiation Protection Supervisot)
SAWADA, Kaoru
l!NUMA, Hideko
ITO, Takayo
KATAGIRI, Izumi
KAMIYA , Kiyomi (Dec. 16-)
KANEUJI, Kimie (Ap1c 16 -)

4516

users

1382

1675

1459

visitors

225

86

2 19

530

total

1 607

1761

1678

5046

Table 2. The numbers of users and visitors who entered into
an each controlled area in 2004

~I""' I
12000

J

I

""l

I(

8000 ~, ~200Technical and suppmiing staffs of the CRF maintain
three controlled areas being adaptable for the law. The
purchase of radioisotopes from JRA (Japan Radioisotope
Association) and the transfer of radioisotope wastes to
JRA is under our surveillance.
Matsuda, Iinuma, Ito, and kaneuji are maintai ning
CFBI(Common Facilities Building I)-branch and LGER
(Laboratory of Gene Expression and Regulation)-branchin the Myodaiji-Area, and Ogawa, Sawada, Katagiri , andKamiya working in the Yamate-Area.
Number of registrants and users in 2004 April to
November are shown in table 1.
Users going in and out the controlled areas counted by
the monitoring system are 5,046 persons in 2004 Apri l to
November. The items in each area is shown in F igure I
and table 2. Annual changes of registrants and number of
totals per year is shown in figure 2. A nd amounts ofradioisotopes used in the CRF is shown in Table 3.
At 2004 December ,the animal radiation wastes drying
system was renewed. (figure 3) This system can dry
animal bodies etc. with heat, and can perform thepretreatment handed over to JRA.

~ ~ 150
I~
i~

6000

::::~I

100'-----------~-~-~-------;~;~;~~~;=;_:__:_:__:_:__:_:_~_~_:_:__'~1'----

Fisic:;lll year.

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

Yamate-Area

Myodaiji-Area

Total

CFBI- branch

LGER-branch

mi Received

1110 . 000

0 . 000

1110.000

Used

1110.0000 . 000

740 . 000

1850 .000

1217000.000

259000.000

523180.000

1999180. ODD"~ I"S Received

2220 . 000

Used969500.000

65380 . 000

734161.826

1769041.826

n p Received

558750.000

1 14 7000 . 000

1017000. 000

2722750.000

1Hg' P Used

394217 . 000

912020 . 000

78834 0. 000

2094577.000"C Received

114 70 . 000

111 00 . 000

0 . 000

22570 . 000

Used

1917.060

5651 . 000

0 . 000

7568.060

H Received

74740. 000

0. 000925000.000

999740 . 000'H Used48148.840

0. 00074 7400 . 000

795548 . 840~< c1

Table 3

Balance of radioisotopes received and used

(MBq)

Myodaiji-Area Yamate - Area
registrants

199

113

users

118

50

Table 1. Number of registrants and users in the 2004.

MyodaijiArea(CFBI)

MyodaijiArea(LGER)

Figure l. Percentage of users go ing in and out thecontrolled areas in 2004.

i

Figure 3. the animal radiation wastes drying system(Wakaida Engneering Co. type WINDY2000)

NATIONAL INSTITUTE FOR BASIC BIOLOGY

87

H

INDEX

A
AIZAW A, Shinichi
AKANUMA, Takashi
ALI, Ferjani
ALLAKHVERDIEV, Suleyman I.
AOKI, Etsuko
AOKI, Yumiko
AONO, Naoki
ARAI, Yuko
ARAKAWA-KOBAYASHI, Satoko
ARITA, Kaori
ARITA, KayoASANO, Megumi
ASAO, Hisayo
AUSTEN RD Ganley
AYABE, Yoshiko
AYABE, Yuko

5
28
64
58
52
32
52
7
76
32
25
22
46
49
72

34

B
BABA, Misuzu
BABA, Takashi
BHANDARI, Ramji
BITOH, Yoshimi

10
19
16
52

c
CHAUBE, Radha
CHO, Kiuhyon
CHOI, Jeong-Doo
CHRISTENSEN, SteenCHUNG, Hyeyoung
COTSAFTIS, Olivier
CUI, Tailing

16
64
46
66
22
46
49

D
DODO, Yukiko

83,84

E
ETANI, Kazuma
ETOH, Tomoo
EUN, Chang-Ho

34
76
46

F
FATCHIYAH
FUnKAWA, Akihiro
FUJIKI, Yuki
FUJIKURA, Ushio
FUTITA, Hironori
FUJITA, Tomomichi
FUKADA, Masahide
FUKADA-TANAKA, Sachiko
FUKAZAWA, Mitsue
FUKUI, Yuko
FURUKAWA, Kazuhiko

19
34
10
64
72

52
34
46
7
19
84

G
GOTO, Masanori
GOTO,Mayu
GOTOH, Megumi
GOUNO, Hiroe
GUAN, Guijun

22
61
34
19
16

HAMADA, Yoshio
HANADA, Takao
HARA, Ikuyo
HARA, Yoko
HASEBE, Mitsuyasu
HASEGAWA, Yoshinobu
HASHIMOTO, Kaoru
HATSUGAI, Noriyuki
HATTORI, Nobuko
HAYAKAWA, Rie
HAYAKAWA, Yuko
HAYASHI, Hitomi
HAYASHI, Kazue
HAYASHI, Kohji
HAYASHI, Makoto (Div. Cell Mechanisms)
HAYASHI, Makoto (Div. Developmental Genetics)
HAYASHI, Yoshiki
HIGASHI, Sho-ichi
HINAGO, Megumi
HIROKAWA, Junya
HIRONAKA, TomieHIWATASHI, Yuji
HIYAMA, Takeshi
HONDA, Satoko
HORIGUCHI, Gorou
HORIUCHI, Takashi
HOSHINO, Atsushi
HOSOKAWA, Kentaro
HOTTA, Kohji

15, 78
10
16
10
5,52
46
52
7
83
16
52
37
22
81,84
7
25
25
78,84
61
37
84
52
34
25
64
5,49
46
52
22

I
ICHIKAWA, Chiaki
ICHIKAWA, Hiromi
ICHIKAWA, Rie
ICHIKAWA, Yuki
ICHIMURA, Yoshinobu
IGUCHI, Taisen
IHARA, Masaru
IIDA, Shigeru
IINUMA, Hideko
IIOKA, Hidekazu
IJIRI, Shigeho
IKEGAYA, Kyoko
IKEMURA, Ryota
IMAIZUMI, Taeko
IMAMURA, Hisako
IMAMURA, Takuya
IMAMURA, Yuko
ISEKI, Mineo
ISHIKAWA, Azusa
ISHIKAWA, Naoko
ISHIKAWA, Takako
ISHIMOTO, Yoichi
ITO, Makiko
ITO, Shinji
ITO, Takayo
IYODA, Yuri

78,84
81, 84
10,61
52
10

61
34
5,46
84,86
22
16
46
40
61
72

40
10

68
19
64
37
61
68
68
84,86
7

J
JIANG, Gonghao
JOGADHENU, S. S. Prakash
JOHZUKA, Katsuki
JOHZUKA-HISATOMI, Yasuyo

46
58
49
46

K
KABEYA, Kazuko

52,64

NATIONAL INSTITUTE FOR BASIC BIOLOGY88

KABEYA, Yukiko
KAGEYAMA, Takuya
KAI, Tsutomu
KAJIKAWA, Ikumi
KAMADA, Tomoe
KAMADA, Yoshiaki
KAMIGAKI, Akane
KAMIYA, Kiyomi
KANESAKI , Kazuko
KANESAKI, Yu
KANEUJI, Kimie
KATAGIRI, Izumi
KATAOKA, Yukari
KATAYAMA, Tomomi
KATO,Hideo
KATSU, Yoshinao
KATSUKI, Kuniko
KATSUKI, Motoya
KAWAMATA, Tomoko
KAWAMURA, Akinori
KAWAMURA, Mamiko
KAWAMURA, Motofumi
KIKUCHI, Kazuhiro
KINOSHITA, Noriyuki
KITADATE, Yu
KITANI,Masakazu
KITAYAMA, Atsushi
KITSUKAWA, Takashi
KOBAYASHI, Hiroko
KOBAYASHI, Kaoru
KOBAYASHI, Mika
KOBAYASHI, Satoru
KOBAYASHI, Takehiko
KODAMA, Akiko
KODAMA, Ken-ichi
KODAMA, Ryuji
KOIKE, Yukari
KOJIMA, Yoko
KOMATSU, Tomoko
KOMATSU, Yusuke
KOMEDA, Yoshifumi
KOMINE, Yuriko
KONDO, Hisato
KONDO,Maki
KONDO, Makiko
KOSHIDA, Sumito
KOUMURA, Toshiro
KOUMURA, Yoshiko
KOZUKA, Toshiaki
KUJIRAOKA, Masahiro
KURATA, Tomoko
KURITA, Kayoko
KUROKAWA, Hiromi
KUROSAWA, Yoshikazu
KUROYANAGI, Miwa
KUSAKA, Masatomo

10,84
10
83
52
7
10
7
86
58
58
86
86
84
34
61
61
76
1, 76
10

28
84
81
66
22
25
52
22
37
16,84
61
61
25,83
49
34
49
57
19
52,64
19
37
5
37
5
7,84
64,84
28
46
68
64
22
28
16
32
5
7
19

L
LAU, En-Lieng
LEE, Rebecca
LOS, Dmitry A.

16
22
58

M
MACHIDA, Yasunori
MAEDA, Masato
MAKIHARA, Nobuko
MAKINO, Haruko
MAKINO, Yumiko

5
22
78,84
52
83,84

MANO, Eriko
MANO, Shoji
MATSUDA, Masaru
MATSUDA, Yoshimi
MATSUI, Makoto
MATSUMOTO, Miwako
MATSUYAMA, Makoto
MIKAMI, Koji
MIKI, Kazuhiko
MIURA, Seiko
MIURA-HYODO, Junko
MIWA, Tomoki
MIYABAYASHI, Kanako
MIYAGAWA, Shinichi
MIYAKAWA, Atsushi
MIYAKAWA, Yuko
MIYAKE, Satoko
MIYAKOSHI, Akira
MIYAZAKI, Saori
MIZOGUCHI, Masae
MIZUTANI, Takeshi
MOCHIZUKI, Atsushi
MOHAMAD, Zubair
MORI, Tomoko
MORI, Yuji
MORIBE, Hatsumi
MORINAGA, Shin-ichi
MORITA, Tomoko
MORITA, Yasumasa
MOROHASHI, Ken-ichirou
MOROKUMA, Junji
MOROKUMA, Yoshie
MOROOKA, Naoki
MUKAI, Masanori
MUKOHDA, Yasuyo
MURAKAMI, Fujio
MURATA, Norio
MURATA, Takashi
MUTSUDA, Michinori

64
7
16
84,86
10
46
19
70
37
55
22
78,84
19
61
76
76
22
22
52
34
61,84
72,80
19
83,84
40
83,84
52
76
46
5, 19
22
22
49,84
25
84
5
58
52
7

N
NAGAHAMA, Yoshitaka
NAGAHARA, Miki
NAGATA, Emiko
NAKAMORI, Chihiro
NAKAMURA, Kayo
NAKAMURA, Makoto
NAKAMURA, Shuhei
NAKAMURA, Takahiro
NAKAMURA, Takanori
NAKAMURA, Torn
NAKANE, Kaori
NAKATOGAWA, Hitoshi
NANBA, Chieko
NARITA, Noriyuki
NARUSE, Mayumi
NISHIDE, Hiroyo
NISHIMURA, Mikio
NISHIMURA, Noriko
NISHIYAMA, Tomoaki
NITO, Kazumasa
NODA,Chiyo
NODA, Masaharu
NODA, Takeshi
NOGUCHI, Yuji

5, 16
46
61
7
34
22
32
34
78,84
37
84
10
78, 8464
52
78,84
5, 7, 78
84
52
7
25,84
5,34
10
81

0
OBARA, Keisuke

10

89

NATIONAL INSTITUTE FOR BASIC BIOLOGYODA,Keiko
ODA, Ritsuko
OGASAWARA, Kimi
OGAWA, Hidesato
OGAWA, Kazuo
OGURA, Yasunobu
OHBAYASHI, Akiko
OHBAYASHI, Norihiko
OHKAWARA, Takeshi
OHMURO-MATSUYAMA, Yuki
OHNEDA, Mamoru
OHNISHI, Makoto
OHNO,Kaoru
OHSAWA, Sonoko
OHSUMI, Katufumi
OHSUMI, Yoshinori
OHTA,Kohei
OIKAWA, Kazusato
OITA, Eiko
OKA, Sanae
OKADA, Kiyotaka
OKADA, Norihiro
OKUBO, Kataaki
ONODA, Shiho
ONODERA, Jun
OONO, Chikako
OOSAWA, Sonoko
OWAKI, Akiko

28
28
7
19
13,86
66
28
28
34
16
10
46
16
84
49
5, 10
16
66
10
19,84
5
55
16
46
10
52
37
19

p
PAITHOONRANGSARID, Kalyanee
PANICHKIN, Vladimir
PARK, Kyeung-II
PAUL, Bindhu
PING, Xang

58
58
46
16
58

s
SAGA, Yumiko
SAITO, Daisuke
SAITOH, Miho
SAKAI, Fumie
SAKAI, Keiko
SAKATA, Shuzo
SAKUTA, Hiraki
SANJO, Kazuko
SASAKI, Takeshi
SASAKI, Tetsuya
SASAOKA, Toshikuni
SATO, Kaori
SATO, Kimihiro
SATO, Yoshikatsu
SATO, Yuko
SAWADA, Kaoru
SEHARA, Atsuko
SEKITO, Takayuki
SERIZAWA, Naomi
SHIBAMOTO, KaoriSHIBATA, Yasushi
SHIGENOBU, Shuji
SHIMADA,Yu
SHIMAMOTO, Miki
SHIMATANI, Zenpei
SHIMIZU, Hidetada
SHIMIZU, Naoki
SHINTANI, Takafumi
SHIVAJI, S
SI, Xuaohui
SLABAS, Antoni R.

5
32
46
16
64
37
34
46
55
37
44,81
25
25
52
19
84,86
5
10
49
22
16
25
16
46
46
34
81
34
58
37
58

SONE, Kiyoaki
SUETSUGU, Noriyuki
SUGITA, Chiyoko
SUGIURA, Mio
SUMIKAWA, Naomi
SUZUKI, Atsuko
SUZUKI, Iku
SUZUKI, Iwane
SUZUKI, KeikoSUZUKI, KuninoriSUZUKI, Ryoko
SUZUKI, Takeshi
SUZUKI, Yoriko

61
66
16
19
52,84
61
7
58
78,84
10
34
68
52

T
TAKABE, Erika
TAKADA, Ritsuko
TAKADA, Shinji
TAKAGI, Chiyo
TAKAGI, Kyoko
TAKAHASHI, Eri
TAKAHASHI, Fumio
TAKAHASHI, Hiraki
TAKAHASHI, Hiroo
TAKAHASHI, Kazuhiko
TAKAHASHI, Shunichi
TAKAHATA, Toru
TAKAJI, Masafumi
TAKAKI, Chikako
TAKAMI, Shigemi
TAKAO, Motoharu
TAKASHIRO, Kayoko
TAKESHITA, Miyako
TAKEUCHI, Kazumi
TAKEUCHI, Motoki
TAKEUCHI, Yasushi
TAMURA, Hiroshi
TANAKA, Masaki
TANAKA, Minoru
TANAKA, Rumi
TANAKA, SachikoTANIKAWA, Yukiko
TERADA, Rie
TERASAKA, Chie
TOHYA, Shusaku
TOMIDA, Sanai
TSUCHIYA, Megumi
TSUCHIYA, Naomi
TSUGANE, Kazuo
TSUGE, Toyoko
TSUKAMURA, Hiroko
TSUKAYA, Hirokazu
TSUKESHIBA, Kumi
TSUZUKI, Shihoko

64
28
5, 28, 80, 81, 86
22,84
46
61
66
22
34
55
58
37
37
16
83, 84
34
28
78,84
42
40
34,84
34
16
32,81
34
84
52
46
22
72
19
19
25
46
22
40
64
10
84

u
UCHIYAMA, Ikuo
UEDA, Chizuru
UEDA, Yoshiko
UENAKA, Hidetoshi
UENO, Kohji
UENO, Naoto
UJIIE, Yoshifumi
UMEBAYASHI, Hiromi
UNO, Eri
UNO, Satoko
URUSHITANI, Hiroshi
UTSUMI, Hideko

74, 78
7
25
66
31
5,22
49
72,80
16
84
61
28,84

90

NATIONAL INSTITUTE FOR BASIC BIOLOGYw
WADA, Masamitsu
WANG, De-Shou
WATAKABE, Akiya
WATANABE, Eiji
WATANABE, Hajime
WATANABE, Masakatsu
WATANABE, Takaaki
WATASE, Masataka

5,66
16
37
42,81
61
68,78
49
52

y
YAGI,Mina
YAMADA, Kaoru
YAMADA, Kenji
YAMADA, Shigehiro
YAMAGAMI, Ayumi
YAMAGUCHI, Chinami
YAMAGUCHI, Katsushi
YAMAGUCHI, Kyoko
YAMAGUCHI, Takahiro
YAMAGUCHI, Yoshifumi
YAMAMORI, Tetsuo
YAMAMOTO, Takamasa
YAMAMOTO, Yasunori
YAMAUCHI, Daisuke
YANO, Satoshi
YASUDA, Mie
YATSU,JunYl, Mei-Sheng
YONEHARA, Keisuke
YOSHIDA, Etsuko
YOSHIKANE, Nami
YOSHIKAWA, Shinya
YOSHIKUNI, Michiyasu
YOSHIMOTO, Kohki
YOSHINORI, Yumi

7
34
7
22
72

64
46,84
81
64
28
5,37
22
34
66
64
81
25
16
34
81
22
68
16
10

7

z
ZHOU, Lin-Yan

16

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