NATIONAL INSTITUTE FOR BASIC BIOLOGYINTRODUCTION
The National Institute for Basic Biology (NIBB) was
founded in 1977 as one of the Inter-University ResearchInstitutes to promote and stimulate studies in the field of
biology. As a center of excellence (COE), NIBB promotes
not only basic biology but also the modern biologicalsciences by conducting first-rate research on site as wellas in cooperation with national, public and private
universities and research organizations. Research at the
NIBB covers a wide variety of biological fields, such ascell biology, developmental biology, neurobiology,
evolutionary biology, environmental biology, andtheoretical biology, and is conducted to elucidate generaland fundamental mechanisms underlying various
biological phenomena.
In 2004, NIBB, in alliance with four other national
institutes - the National Astronomical Observatory, theNational Institute for Fusion Science, the National
Institute for Physiological Sciences, and the Institute for
Molecular Science - established the National Institutes of
Natural Sciences (NINS), one of the four Inter-University
Research Institute Corporations. At this time NINS began
to promote collaboration among researchers of the five
constituting institutes and to sponsor many projects, oneof which was to hold the Imaging Science Symposium inAugust, in which cutting-edge imaging technology related
to a wide range of basic scientific fields from astronomy
to biology was discussed.
Besides being a research institute, NIBB also functions
as an institution of higher education. It constitutes the
Department of Basic Biology in the School of Life
Science of the Graduate University for Advanced Studies(SOKENDAI). The Department offers a five-year
Doctoral course (with a quota of three students) for
university graduates and a three-year senior Doctoral
course (with a quota of six students) for those students
who have completed a Master's course.
Changes in our personnel during 2005 were as follows.
Prof. Norio MURATA retired and Dr. Khoji UENO
moved to Musashino University as a professor on March
31st. Dr. Tomomichi FUJITA moved to Hokkaido
University as an assistant professor on June 30th. Dr.
Hirokazu TSUKAYA moved to the University of Tokyo
as a professor, while at the same time becoming an
adjunct professor at NIBB, and Dr. Takashi
KITSUKAWA moved to Osaka University as an
associate professor on September 30th.
Conversely, three researchers were newly appointed:
Prof. Masamitsu WADA from Tokyo Metropolitan
University and former adjunct professor at NIBB, as aprofessor, and Dr. Hitoshi NAKATOGAWA from a JSPS
Post-Doctoral fellow as a research associate, both on
April 1st. Dr. Takako TANAHASHI, a visiting scientist,
was appointed as a research associate on July 1st. Dr.
Takehiko KOBAYASHI was promoted to an associate
professor from a research associate on April 1st. In
addition, Dr. Takeshi NODA returned from his stay at UC
San Diego on December 12th.

NIBB has started a collaboration program with theEuropean Molecular Biology Laboratory (EMBL) under
the sponsorship of NINS. The agreement was signed on
July 11th by Dr. Yoshiro SHIMURA, the president of
NINS, and Dr. Iain MATTAJ, the director general of
EMBL, and is effective for at least five years. The
program aims to promote joint research activities between
the two organizations and to prompt mutual visits by
researchers and graduate students, thereby encouraging
exchange of information and intimate communication,
both of which are indispensable for the prosperity of joint
research. Activities seen this year are as follows. A
symposium on developmental and cellular biology washeld in July at EMBL, Heidelberg. Seminars were held at
NIBB by visiting researchers of EMBL, while NIBB
researchers stayed at EMBL for the preparation ofcollaboration using the microscopic facility at EMBL.
The Strategic Planning Department, a new office forthe promotion of collaborative activities, the managementof international conferences and training courses held by
NIBB, and various public relations activities such as
publications, posters, and web pages, was opened in April.
The office is chaired by the vice director of the instituteand is expected to support the director-general indeveloping a future strategy for the progress of theinstitute.
Finally, I would like to congratulate Prof. Mitsuyasu
HASEBE for winning the JSPS Prize and Japan Academy
Medal in March and Prof. Yoshinori OHSUMI for
winning the Fujihara Award in June.
We always welcome any questions or suggestions
concerning the research activities of NIBB.
Motoya KATSUKI, D. Sc.
Director-General

1

NATIONAL INSTITUTE FOR BASIC BIOLOGYORGANIZATION OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) is oneof five independent institutes making up the National
Institutes of Natural Sciences (NINS). NIBB, the Institutefor Molecular Science (IMS) and the National Institute forPhysiological Sciences (NIPS) are located on a hill
overlooking the old town of Okazaki in Aichi Prefecture.
NIBB was established in 1977 and its activities aresupported by Monbukagaku-sho (the Ministry of
Education, Culture, Sports, Science and Technology:Mext) of Japan. The Center for Integrative Bioscience(CIB) – renamed the Okazaki Institute for Integrative
Bioscience on April 1st, 2004 – was established as acommon facility for the three Okazaki Institutes in 2000
and opened in 2001.

Research
The NIBB conducts its research programs through 30
research units, 4 research support facilities including theTechnology Department, and the Research Center for
Integrative and Computational Biology. Each research
unit has its own research project and is staffed (inprinciple) by a professor, an associate professor and two
research associates. Each research unit forms an
independent project team. Three of the research units areadjunct and headed by professors who hold joint
appointments with other universities. Adjunct divisions
have a resident research associate. This arrangement
facilitates exchange in research activities in Japan. The
Technical Department manages the activities of researchtechnicians and helps to promote the research activities ofeach research unit and facility and to maintain thecommon research resources of the NIBB. The Department
also undertakes the technical education of the various

staffs.
Several members of the Okazaki Institute forIntegrative Bioscience work jointly with the NIBB.

Research Support Facilities
The Research Support Facility of the NIBB consists ofsix sub facilities: the Large Spectrograph Laboratory, theTissue 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 theTechnical Division are also research support systems of
the NIBB.
In addition, five facilities are operated jointly with
NIPS: 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 theAnimal Care Facilities are common facilities of the three
Okazaki Institutes.
CampusTogether the three Okazaki Institutes cover 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 third in June, 1983. The buildings that house
the research support facilities were also completed in
1983. A building for the Laboratory of Gene Expression
and Regulation (2,577m2) was built in 1996. A building
for the Center for Transgenic Animals and Plants(2,500m2) was completed in November, 2003.

NATIONAL INSTITUTE FOR BASIC BIOLOGYPOLICY, DECISION MAKING, AND ADMINISTRATION
Director-General to the President after hearing therecommendation from the Advisory Committee. The
Advisory Committee makes recommendations on the
Director-General, faculty appointments, the NIBB's
annual budget and future prospects.
Administration of the NIBB is undertaken by the
Administration Bureau of the Okazaki Administration
Center under the direct auspices of the Administration
Office of NINS.

The Director-General oversees the operation of the
NIBB assisted by the Advisory Committee for
Programming and Management. The Advisory Committee,
comprised of professors within the NIBB and an equal
number of leading biologists outside the NIBB, advises
the Director-General on important matters such asplanning joint research programs as well as on thescientific activities of the NIBB. The Director-General
Selection Committee makes a nomination for

MEMBERS OF THE ADVISORY COMMITTEE FOR
PROGRAMMING AND MANAGEMENT
Chairman
YAMAMORI, Tetsuo

Professor, National Institute for Basic Biology

Vice-Chairman
OKADA, Kiyotaka

Professor, Kyoto University

AIZAWA, Shinichi

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

IWASA, Yoh
KOMEDA, Yoshifumi
KONDO, Hisato
KUROSAWA,Yoshikazu
MACHIDA, Yasunori
MURAKAMI, Fujio
SAGA, Yumiko
SEHARA, Atsuko
UENO, Naoto
HASEBE, Mitsuyasu
HORIUCHI, Takashi
IIDA, Shigeru
MOROHASHI, Kenichirou
NAGAHAMA, Yoshitaka
NISHIMURA, Mikio
NODA, Masaharu
OHSUMI, Yoshinori
TAKADA, Shinji

4

GRADUATE PROGRAMSThe NIBB sponsors two graduate programs.
1. Graduate University for Advanced Studies
NIBB constitutes the Department of Basic Biology in
the School of Life Science of the Graduate University for
Advanced Studies. The University provides a five-year
Doctoral course as well as a three-year senior Doctoral
course for those students who have completed a master’scourse (or equivalent) at any university. The Department
consists of 30 Research Units (listed on 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.
At the 20th Bioscience Training Course in June 2005.

COOPERATIVE RESEARCH PROGRAM
The NIBB has a cooperative research program forresearchers throughout Japan. Two main programs areavailable. The first provides laboratory space andfacilities at NIBB while the second assists small groups

holding research meetings at NIBB. The visiting
researcher and research meeting plans are approved by the
NIBB Advisory Committee.

INTERNATIONAL COLLABORATIONThe NIBB hosts two international conferences, the
NIBB Conference and the Okazaki Biology Conference(OBC). The NIBB Conference has been held every year
since the opening of the Institute in 1977. The purpose of
the NIBB Conference is to provide a limited number ofactive researchers with the opportunity to freely discusscurrent issues in various areas of biological research. The
topic chosen for the 51st NIBB Conference was “NewAspects of Gene Amplification” and discussions were
held on the molecular mechanisms and biological
functions of gene amplification (see page 82). The OBC,
initiated in 2003, has as its long-term objective theestablishment of interdisciplinary networks in pursuit of
the solution of major biological problems.
The NIBB performs academic exchange programs with
the European Molecular Biology Laboratory (EMBL) andNational Institutes of Natural Sciences (NINS) in an effort
to promote intellectual, educational and technologicalexchange in the fields of biology and molecular biology.
Academic cooperation between NIBB and ENBL includes(1) Promotion of joint research activities; (2) Invitation
for faculty members and researchers to lectures,workshops, conferences, symposia, and other academic
activities; (3) Exchange of graduate students forconferences and training courses; and (4) Exchange of
information and academic publications.

At the 1st Okazaki Biology Conference in January 2004.

At the 1st NIBB-EMBL symposium at Heidelberg.

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

II. Transcriptomics, proteomics and phenomics ofplant peroxisomes

DIVISION OF CELL MECHANISM
Professor:
Associate Professor:
Research Associates:
Technical Staff:
NIBB Research Fellows:
Postdoctoral Fellows:

Graduate Student:
Visiting Scientist:
Technical Assistants:

Secretaries:

NISHIMURA, Mikio
HAYASHI, Makoto
MANO, Shoji
YAMADA, Kenji
KONDO, Maki
KAMADA, Tomoe
MUTSUDA, Michinori
ARAI, Yuko
KAMIGAKI, Akane
HATSUGAI, Noriyuki
OIKAWA, Kazusato
OGASAWARA, Kimi
LU, Zhongpen
NAKAMORI, Chihiro
YAGI, Mina
YOSHINORI, Yumi
SUZUKI, Iku
FUKAZAWA, Mitsue
KATO, Kyoko
NISHINA, Momoko
UEDA, Chizuru
KUBOKI, Yuko

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 (PTS1 and PTS2) within themolecules. PTS1 is a unique tripeptide sequence found in
the carboxyl terminus of the mature proteins. In contrast,
PTS2 is involved in a cleavable amino terminal
presequence of peroxisomal proteins that are synthesized
as a precursor protein with larger molecular mass.
We identified 256 gene candidates of PTS1- 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. In parallel, we madea two-dimensional protein map of glyoxysomes and leaf
peroxisomes isolated from Arabidopsis. Peptide MS
fingerprinting analyses allowed us to identify novel
proteins exists in either glyoxysomes or leaf peroxisomes.
Some of these proteins contain no obvious PTS1 and
PTS2. Combination of the transcriptomic and proteomic
analyses is providing us a new insight into plant
peroxisomal functions.

Since plants spread their roots in the ground, they must
survive in a given environment. To adapt to theenvironment, they positively utilize environmental
changes in the life cycle as important signals that arenecessary for their survival. Plant cells can induce,
degenerate and differentiate their organelles to adapt toenvironmental changes. The flexibility of plant organelles
is the basis of the strategy for environmental adaptation inplants.
The aim of this division is to clarify the molecularmechanisms underlying induction, differentiation, andinteraction of organelles, and to understand the integratedfunction of individual plants through organelle dynamics.
The Scientific Research of Priority Areas on “Organelle
Differentiation as the Strategy for Environmental
Adaptation in Plants” was started to clarify the molecularmechanisms underlying organelle differentiation.

I. Reversible transformation of plant peroxisomes
Dramatic metabolic changes that 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 -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.
Gene
expression,
alternative
splicing,
proteintranslocation and protein degradation control thefunctional transformation between glyoxysomes and leaf
peroxisomes.

Figure 1. Differential contribution of two peroxisomal protein
receptors to the maintenance of peroxisomal functions. Effectof CO2 on the growth of pex5i, PEX57 and pex7i transgenic
plants(A). Arrows indicate the top of an inflorescence apex.
Import of PTS1-containing protein is inhibited in pex5i (B)but not in pex7i (D), while import of PTS2-containing protein
is inhibited both in pex5i (C) and pex7i (E).

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 by

9

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

generating 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. These analyses revealed that PEX5, a
receptor for PTS1, is involved in both lipid metabolism
and photorespiration by regulating import of both PTS1and PTS2-containing proteins (Figure 1A, 1B, 1C). In
contrast, PEX7, a receptor for PTS2, is involved only inphotorespiration by regulating import of PTS2-containing
protein (Figure 1A, 1D, 1E).

The vacuolar processing enzyme (VPE) belongs to thecysteine protease family found in higher plants andanimals. VPE exhibits substrate specificity toward
asparagine and aspartic acid residues, the amino acid wellconserved at the processing sites of vacuolar proteins.
Plant VPE homologues are separated into threesubfamilies: seed type, vegetative type and embryogenic
type. Seed type VPE is responsible for the maturation ofseed storage proteins. On the other hand, the function ofvegetative and embryogenic type VPEs was obscure.
Recently, we revealed a novel function of VPE in various
types of programmed cell death (PCD) in plants. The

III. Identification of novel components essential for
peroxisome biogenesis
To better understand peroxisome biogenesis, we
isolated a number of Arabidopsis mutants having aberrant
peroxisome morphology (apm mutants) based on the
different pattern of GFP fluorescence from the parent
plant, GFP-PTS1, in which peroxisomes with normal size
and number can be visualized with GFP.
It was revealed that one of these mutants, apm1, whose
peroxisomes and mitochondria are long and reduced in
number, is defective in DRP3A (Dynamin-related protein
3A). This finding shows that APM1/DRP3A protein is
involved in both peroxisomal and mitochondrial division.
We revealed that other mutants, apm2 and apm4, exhibit
the GFP fluorescence in not only peroxisomes but also thecytosol, and determined that both APM2 and APM4 areresponsible for matrix protein transport on peroxisomal
membranes. Analyses of other apm mutants andidentification of APM genes will identify components
necessary for peroxisome biogenesis and address theregulation of its mechanism.

IV. ER derived organelles for protein storing anddefense strategy
Figure 2. VPE deficiency suppresses various types ofprogrammed cell death (PCD) in plants. (A) The non-silenced(WT) and VPE-silenced (silenced) Nicotiana benthamiana
plants were infected with tobacco mosaic virus on halves of
their leaves (indicated by asterisks). The photographs weretaken after 24 hours. (B) Leaves of wild-type (WT) and
VPE-null mutant (null) Arabidopsis plants were infiltrated
with FB1, a fungal toxin (indicated by asterisks). The
photographs were taken after 5 days. (C) Thickness of innerinteguments (ii) of the seed coats is reduced in wild-type(WT) seed at the early stage, whereas it is not reduced in thevpe mutant seed of Arabidopsis. PCD accompanies theshrinkage of two cell layers of the seed coat in wild typeseeds.

Plant cells develop various types of endoplasmic
reticulum (ER)-derived structures with specific functions.
ER bodies are ER-derived compartments observed in
Arabidopsis. They are rod-shaped structures (5 μm longand 0.5 μm wide) that are surrounded by ribosomes. ER
bodies were widely distributed in the epidermal cells ofwhole seedlings. Rosette leaves had no ER bodies, but
accumulated ER bodies after wounding or jasmonic acidtreatment. This suggests that ER bodies function in the
defense against herbivores. ER bodies include PYK10, a
-glucosidase with an ER retention signal, in seedlings.
Arabidopsis nai1 mutant has no ER bodies in wholeplants and does not accumulate PYK10. NAI1 encodes atranscription factor that has a basic-helix-loop-helix(bHLH) domain. Transient expression of NAI1 induced
ER bodies in the nai1 mutant. These results provide direct
evidence that NAI1 plays a role in the formation of ER
bodies. We are trying to isolate additional componentsthat are involved in ER body formation.

evidence from extensive studies indicates that caspase
activity is involved in plant PCD. VPE is identified as theproteinase that exhibits caspase activity in plants. The
plant hypersensitive response (HR), a type of defense
strategy, constitutes well-organized PCD. No HR occurs
on the tobacco mosaic virus-infected leaves of
VPE-silenced tobacco plants (Figure 2A). Fumonisin B1(FB1), a fungal toxin, induced cell death in Arabidopsis.
The features of FB1-induced cell death were completely
abolished in the Arabidopsis VPE-null mutant, which

V. Vacuolar processing enzyme responsible forprogrammed cell death in plants

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NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

lacks all VPE genes (Figure 2B). Arabidopsis VPE wasrecently identified as an embryogenic type VPE. VPE
specifically and transiently expressed in two cell layers(ii2-ii3) of the seed coat at an early stage of seeddevelopment. At this stage, PCD accompanying cell
shrinkage occurs in ii2-ii3. In a vpe mutant, shrinkage ofthese layers was delayed (Figure 2C). An ultrastructural
analysis showed that the disintegration of the vacuolar
membranes occurs before the cell death in these PCDs.
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 the key
player in a plant-specific cell death system.

VI. Role of moleculardifferentiation

chaperonesonHashimoto, K., Igarashi, H., Mano, S., Nishimura, M.,
Shimmen, T., and Yokota, E. (2005). Peroxisomal
localization of a myosin XI isoform in Arabidopsis
thaliana. Plant Cell Physiol. 46, 782-789.
Hayashi, M., Yagi, M., Nito, K., Kamada T., and Nishimura,
M. (2005). Differential contribution of two peroxisomal
protein receptors to the maintenance of peroxisomal
functions in Arabidopsis. J. Biol. Chem. 280,
14829-14835.
Kuroyanagi, M., Yamada, K., Hatsugai, N., Kondo, M.,
Nishimura, M., and Hara-Nishimura, I. (2005). Vacuolar
processing enzyme is essential for mycotoxin-induced cell
death in Arabidopsis thaliana. J. Biol. Chem. 280,
32914-32920.
Maehr, R., Hang, H.C., Mintern, J.D., Lim, Y.-M., Cuvillier,A., Nishimura, M., Yamada, K., Shirahama-Noda, K.,
Hara-Nishimura, I., and Ploegh, H.L. (2005). Asparagine
endopeptidase is not essential for class II MHC antigen
presentation but is required for processing of cathepsin L.
in mice. J. Immunol. 171, 7066-7074.
Morikami, A., Matsunaga, R., Tanaka, Y., Suzuki, S., Mano,S., and Nakamura, K. (2005). Two cis-acting regulatory
elements are involved in the sucrose-inducible expressionof the sporamin gene promoter from sweet potato intransgenic tobacco. Mol. Gen. Genomics 272, 690-699.
Nakaune, S., Yamada, K., Kondo, M., Kato, T., Tabata, S.,
Nishimura, M., and Hara-Nishimura, I. (2005). A vacuolar
processing enzyme, VPE, is involved in seed coatformation at the early stage of seed development. Plant
Cell 17, 876-887.
Tamura, I., Shimada, T., Kondo, M., Nishimura, M., andHara-Nishimura, I. (2005). Katamari1/Murus3 is a novel
Golgi membrane protein that is required forendomembrane organization in Arabidopsis. Plant Cell 17,
1764-1776.
Usami, T., Mochizuki, N., Kondo, M., Nishimura, M., andNagatani, A. (2005). Cryptochromes and phytochromes
synergetically regulate the Arabidopsis root greening
under blue light. Plant Cell Physiol. 45, 1798-1808.
Yamada, K., Fuji, K., Shimada, T., Nishimura, M., andHara-Nishimura, I. (2005). Endosomal proteases facilitate
the fusion of endosomes with vacuoles at the final step ofthe endocytotic pathway. Plant J. 41, 888-898.
Yoshida, K., Kawachi, M., Mori, M., Maeshima, M., Kondo,
M., Nishimura, M., and Kondo, T. (2005). The
involvement of tonoplast proton pumps and Na+(K+)/H +exchangers in the change of petal color during
flower-opening of morning glory, Ipomoea tricolor cv.
Heavenly Blue. Plant Cell Physiol. 46, 407-415.

cell

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 (Cpn10), chloroplast co-chaperonins(Cpn20 and Cpn10) and a small heat shock protein from
Arabidopsis. Recently, we started to characterize HSP90s,
using a specific inhibitor of HSP90 or transgenic plants
expressing mutated Arabidopsis HSP90. Preliminary datasuggests that HSP90 is involved in various cellularsignaling in Arabidopsis. The evolutional and functional
characterization is now under experiments.

VII. Organellome database – Databases of plantorganelles visualized with fluorescent protein,and protocols for functional analysis
The organellome database is a specialized databaseproject dedicated to plant organelle research. This
database is maintained by the Scientific Research of
Priority Areas on “Plant Organelles”. To support thisplant organelle research, we have been constructing a
database consisting of three individual databases: theorganellome database, the functional analysis databaseand external links about transcriptomics and proteomics.
This database will be opened to all researchers as a public
database. We expect that this database is going to be auseful analytical tool for plant organelle research.

Review articles
Publication List:

Mano, S., and Nishimura, M. (2005). Plant peroxisomes. In
Vitamins and Hormones, Gerald Litwack ed. (Califoria,
Elsevier Academic Press), pp. 111-154.
Yamada, K., Shimada, T., Nishimura, M., andHara-Nishimura, I. (2005). A VPE family supporting
various vacuolar functions in plants. Physiol. Plant. 123,369-375.

Original papers
Afitlhile, M.M., Fukushige, H., Nishimura, M., andHildebrand, D. (2005). A defect in glyoxysomal fatty acid-oxidation reduces jasmonic acid accumulation in
Arabidopsis. Plant Physiol. Biochem. 43, 603-609.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

autophagy. We also found that two hydrophobic residues
within the ubiquitin-fold region are important for
autophagy: mutations at Y149 affected conjugate
formation catalyzed by Atg10, an E2-like enzyme, while
mutations at F154 had no effect on Atg12-Atg5 conjugate
formation but its hydrophobic nature was essential for
autophagy. In the cells expressing F154 mutants, Atg8-PE
conjugation, the other ubiquitin-like conjugation in
autophagy, was severely reduced and autophagosome
formation failed. A gel filtration analysis suggested that
F154 plays a critical role in the assembly of a functional
Atg12-Atg5·Atg16 complex requisite for autophagosome
formation.
Crystal structure of Arabidopsis thaliana Atg12
homolog (AtATG12) has shown that it adopts theubiquitin-like fold (Figure 1). Residues Y57 and F62,
corresponding to Y149 and F154 in yeast, respectively,
were found to be located adjacent to each other but form
different hydrophobic patches, suggesting Atg12 utilizes
two different hydrophobic patches for autophagy: one for
conjugation reaction and the other for autophagosome
formation.

DIVISION OF MOLECULAR CELL BIOLOGY
Professor:
Research Associates:

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:
Visiting Scientists:
Technical Assistants:
Secretary:

OHSUMI, Yoshinori
KAMADA, Yoshiaki
NODA, Takeshi
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
KABEYA, Yukiko
OBARA, Keisuke
ICHIMURA, Yoshinobu
SEKITO, Takayuki
YOSHIMOTO, Kohki
HANADA, Takao
FUJIKI, Yuki
OHNEDA, Mamoru
OITA, Eiko
ONODERA, Jun
OKU, Masahide
HARASHIMA, Toshiaki
MATSUI, Makoto
KAGEYAMA, Takuya
BABA, Misuzu
KAWAMATA, Tomoko
TSUKESHIBA, Kumi
ICHIKAWA, Rie
KONDO, Chika
HARA, Yoko

This division aims to understand the physiologicalroles and molecular mechanism of autophagy in yeast andhigher eukaryotes. All cellular activity is maintained by
the balance between the synthesis and degradation ofrelated proteins. It is now well known that the degradation
process plays important roles in many physiologicalaspects. Autophagy is a bulk degradation system ofcytosolic proteins and organelles in lysosome/vacuoles.
Membrane dynamics during autophagy remain to bediscovered.

I. Background
Upon nutrient starvation, the autophagic process starts
as a building up of membrane structures(autophagosomes) in the cytoplasm. The autophagosome
sequesters cytosol and organelles nonselectively. Then it
is delivered to the vacuole/lysosome, and the cytoplasmic
materials inside are degraded by vacuolar/lysosomalhydrolases. We discovered autophagy in a simple modelorganism, Saccharomyces cerevisiae and morphologicallyand genetically defined the whole process.

Figure 1. Two hydrophobic residues important for
autophagy are located in close vicinity but form differenthydrophobic patches.

III. Atg17 plays a pivotal role in autophagosome
formation directed by Atg1 kinase activity
In the yeast S. cerevisiae, most of ATG genes are
involved in not only the process of degradative autophagy,
but also a biosynthetic process of vacuolar enzymes. In
contrast, the ATG17 gene is required specifically in
autophagy. We found that the atg17∆ mutant understarvation condition was severely impaired in
autophagosome formation. Autophagosomes with asmaller number and size were observed in the atg17∆
cells (Figure 2). We showed that Atg17 physicallyassociated with an Atg1-Atg13 complex, and that it wasenhanced under starvation conditions. The complex
formation resulted in upregulation of an Atg1 kinase

II. Hydrophobic residues in a ubiquitin-fold of
Atg12 are important for autophagy in yeast
Atg12, a post-translational modifier, is activated andconjugated to Atg5 by a ubiquitin-like conjugation system,
though it has no obvious sequence homology to ubiquitin.
The carboxyl-terminal region of Atg12 is predicted to foldinto a ubiquitin-like structure. We constructedamino-terminally truncated Atg12 mutants of the yeast S.
cerevisiae according to the predicted secondary structureand showed that the ubiquitin-fold region of Atg12 isnecessary and sufficient for both conjugation and

9NATIONAL INSTITUTE FOR BASIC BIOLOGCELL BIOLOGY

activity, suggesting that Atg17 regulates the Atg1 kinase
activity in concert with Atg13. Taken together, theseresults indicated that Atg17-Atg13 complex formation
plays an important role in normal autophagosome
formation via binding to and activating the Atg1 kinase.

V. Autophagy is essential for protein synthesis
under nitrogen starvation
Autophagy is involved in degradation of cytoplasmic
components including a significant amount of proteins,rRNA and phospholipids of organelle membranes. So far,the precise fate and the physiological importance of eachdegradation products generated by autophagy had been
unclear. Recently, we examined changes in protein
synthesis after long term nitrogen starvation and foundthat a heat shock protein (Hsp26p) and an enzyme
involved in an amino acid biosynthesis (Arg1p) were
up-regulated by nitrogen starvation in wild-type cells, butthese changes did not occur in atg mutant cells (Figure
4A). The mRNA levels in the atg mutant cells were ashigh as those in the wild-type cells under nitrogenstarvation (Figure 4B). Thus, it was suggested thatsynthesis of these proteins was inhibited at thetranslational step. We also found that bulk protein
synthesis was substantially reduced in the atg mutant cells
under nitrogen starvation compared with the wild-type
cells. The wild-type cells maintained amino acid levels tosurvive under starvation conditions. In contrast, the total
intracellular amino acid pool was reduced in the atg
mutant cells, and the levels of several amino acids fell
below critical values. These results indicate that
autophagy is required to maintain physiological aminoacid levels under nitrogen starvation conditions, andinability to synthesize proteins newly may explain mostphenotypes in autophagy-defective mutants (e.g. inabilityof spore formation and so on).

WT

atg17∆V
V

Figure 2. ATG17 is essential for normal autophagosome
formation. In a nitrogen-starvation medium, theatg17∆ mutant
generatesfewer
andsmaller
autophagosomes (arrowheads) than those in the wild-type.
Bar: 200 nm. V, vacuole.

IV. Identification and characterization of a novel
autophagy-specific gene, ATG29
At present, at least 16 autophagy-related genes (ATG)
have been identified. To search for novel genes involvedin autophagy, we performed a genome-wide screen using
a collection of ~4,500 yeast single-gene deletion mutants
and identified a novel ORF, ATG29/YPL166w. atg29∆
cells were sensitive to starvation, and induction of
autophagy was severely retarded. However, the Cvt(Cytoplasm to vacuole transport) pathway, which sharesmechanistic components with autophagy, operated
normally. Therefore, Atg29 is the second protein
specifically required for autophagy. Furthermore weobserved that an Atg29-GFP fusion protein was localized
to the PAS, at which most Atg proteins are colocalized(Figure 3). From these results, we propose that Atg29
functions in autophagosome formation at the PAS in
collaboration with other Atg proteins.

Figure 4. Autophagy is required for the expression of
nitrogen starvation-induced proteins at the translational
level. (A) Immuno-blot analysis of starvation-induced
proteins, Arg1p and Hsp26p. (B) Northern-blot analysisof starvation-induced gene, ARG1 and HSP26. ADH andrRNA were used as loading controls.

Figure 3. Localization of Atg29 at the PAS. Growing (0 h)and rapamycin treated (3 h) cells were observed byfluorescence microscopy. Atg8 and API were used as
markers for the PAS.

10

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

death. J. Biol. Chem. 280, 20722-20729.
Sugawara, K., Suzuki, N.N., Fujioka, Y., Mizushima, N.,
Ohsumi, Y., and Inagaki, F. (2005). Structural basis forthe specificity and catalysis of human Atg4B responsible
for mammalian autophagy. J. Biol. Chem., in press.
Suzuki, N.N., Yoshimoto, K., Fujioka, Y., Ohsumi, Y., andInagaki, F. (2005). The crystal structure of plant ATG12and its biological implication in autophagy. Autophagy 1,
119-126.

VI. Autophagy plays important roles in various
aspects of plant life
By establishing an autophagy-monitoring system in awhole plant we were able show that atatg mutants were
defective in autophagy. Our recent studies indicated thatthe autophagy defective mutants exhibited a reduction in
the growth rate of roots under nitrogen-starved conditions
and early senescence phenotype even in nutrient-richconditions. In addition, hypersensitive response cell
death was accelerated in the mutants during the plant
innate immune response. These results indicate that,regardless of nutrient conditions, autophagy plays
important roles in various aspects in plant life.

Review article
Bassham, D.C., Laporte, M., Marty, F., Moriyasu, Y.,
Ohsumi, Y., Olsen, L.J., and Yoshimoto, K. (2005).
Autophagy in development and stress responses of plants.
Autophagy, 2, in press.

Publication List:

Original papers
Hamasaki, M., Noda, T., Baba, M., and Ohsumi, Y. (2005).
Starvation triggers the delivery of the endoplasmic
reticulum to the vacuole via autophagy in yeast. Traffic
6, 56-65.
Hanada, T., and Ohsumi, Y. (2005). Structure-Function
relationship of Atg12, a ubiquitin-like modifier essential
for autophagy. Autophagy 1, 110-118.
Kabeya, Y., Kamada, Y., Baba, M., Takikawa, H., Sasaki, M.,
and Ohsumi, Y. (2005). Atg17 functions in cooperation
with Atg1 and Atg13 in yeast autophagy. Mol. Biol. Cell
16, 2544-2553.
Kamada, Y., Fujioka, Y., Suzuki, N.N., Inagaki, F.,
Wullschleger, S., Loewith, R., Hall, M.N., and Ohsumi, Y.
(2005). Tor2 directly phosphorylates the AGC kinase
Ypk2 to regulate actin polarization. Mol. Cell Biol. 25,
7239-7248.
Kawamata, T., Kamada, Y., Suzuki, K., Kuboshima, N.,
Akimatsu, H., Oota, S., Ohsumi, M., and Ohsumi, Y.
(2005). Characterization of a novel autophagy-specificgene, ATG29. Biochem. Biophys. Res. Comm., in press.
Komatsu, M., Waguri, S., Ueno, T., Iwata, J., Murata, S.,
Tanida, I., Ezaki, J., Mizushima, N., Ohsumi, Y.,
Uchiyama, Y., Kominami, E., Tanaka, K., and Chiba, T.
(2005). Impairment of starvation-induced and constitutive
autophagy in Atg7-deficient mice. J. Cell Biol. 169,
425-434.
Matsui, M., Yamamoto, A., Kuma, A., Ohsumi, Y., andMizushima, N. (2005). Organelle degradation during thelens and erythroid differentiation is independent of
autophagy. Biochem. Biophys. Res. Comm., in press.
Onodera, J., and Ohsumi, Y. (2005). Autophagy is required
for maintenance of amino acids levels and protein
synthesis under nitrogen starvation. J. Biol. Chem. 280,
31582-31586.
Pyo, J.O., Jang, M.H., Kwon, Y.K., Lee, H.J., Jun, J.I., Woo,
H.N., Cho, D.H., Choi, B., Lee, H., Kim, J.H., Mizushima,N., Ohsumi, Y., and Jung, Y.K. (2005). Essential roles ofAtg5 and FADD in autophagic cell death: dissection of
autophagic cell death into vacuole formation and cell

11

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYDIVISION OF Cell Structure
Associate Professor:

OGAWA, Kazuo

Dynein is a molecular motor that carries cargoes tothe direction of centriole from the cell periphery along the
microtubules in a cell. It is composed of three domains:
stem, motor, and stalk. Targeting of dynein to specificsites of cargoes may be related to the NH2-terminal
one-thirds of molecule constituting stem where aminoacid sequence diversity was found between dynein
superfamily (cytoplasmic and axonemal dynein), while
the COOH-terminal two-thirds of molecule showing
sequence conservation in distantly related species
constitutes motor. Having interrupted long heptapeptide
repeats in the sequence, the stalk takes an extended
flexible structure and binds to microtubule in an
ATP-dependent manner. Thus, dynein binds tomicrotubule at stalk domain of itself and cargoes at stemdomain of itself.
The mechanism how cytoplasmic and axonemal
dyneins target the cargoes has been gradually made clear
in terms of molecules participated in. Cytoplasmic dynein
is linked with dynactin complex containing at least 10
proteins. It is via dynactin complex that dynein targets toa receptor of cargo membranes.
In flagellar and ciliary movement, outer and innerdynein arms are projected from the A-subfiber of
peripheral doublet microtubule of axoneme corresponding
to the cargo. They bind to the B-subfiber of neighboring
doublet microtubule in an ATP-dependent manner.
Axonemal dynein is hardly detached from the A-subfiber
being the cargo, while the cargoes of cytoplasmic dynein
are thought to be detached from the motor after arrival tocell center to recruit the motor. Thus, there may bedifferent targeting mechanism for dynein superfamily to
specific sites of the cargoes.
When Triton-model sperm were exposed to hardcondition such as 0.5 M KCl or NaCl (so called chemical
dissection), outer dynein arm was detached from the
A-subfiber. Triton-sperm losing outer dynein arm swamwith a half beat frequency of control sperm. Re-binding ofouter dynein arm onto the A-subfiber was possible byremixing the extracted Triton-sperm with the extract in alow salt concentration solution. Recovered Triton-model
sperm swam with a normal beat frequency. Thus, a highsalt extract might contain some proteins necessary forcorrect positioning of outer dynein arm and scaffold
proteins to mediate binding of outer arm dynein onto the
A-subfiber. During the course of characterizing proteins
containing in the extract, we found a novel protein withmolecular mass of 58 kDa designated as ap58.
Immuno-electron microscopy using antibodies raised
against recombinant ap58 shows that gold-particles arefound at 25 nm repeat along the length of axoneme
coinciding with the repeat of outer dynein arm(longitudinal sections of Figure 1). Thus, we conclude
that ap58 is binding to in situ outer dynein arm.

Figure 1. Immuno-electron micrographs to show the
localization of ap58 within the sea urchin sperm axonemes.
a: cross section; b-d: longitudinal sections (Ogawa andInaba).

Publication List:
Original paper
Morii, H., Shimizu, T., Mizuno, N., Edamatsu, M., Ogawa,
K., Shimizu, Y., and Toyoshima, Y.Y. (2005). Removal oftightly bound ADP induces distinct structural changes of
the two tryptophan-containing regions of the ncd motor
domain. J. Biochem. 138, 95-104.

12

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYLABORATORY OF CELL SOCIOLOGY
Research Associate:

HAMADA, Yoshio

Animal organs consist of several types of cells. They
are organized in an ordered fashion wherein the
proportion of each cell type is constantly maintained. The
ordered cell arrangement and proportion are built up
during organogenesis by cell-cell interactions. Since
Notch has been postulated to play a role in cell fatedecision by mediating cell-cell interactions, we are trying
to find out cellular and molecular mechanisms working inorganogenesis by studying the 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 withmaternal deciduas. The polar trophectoderm gives rise tocells of the chorion, and ectoplacental cone. They produce
labyrinthine and spongiotrophoblast layer, respectively.
While maternal 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 embryonic day 11.5 due
to a poor maternal 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 (Figure 1). Although
inadequate formation of maternal vascular beds waspartially restored by aggregating mutant diploid embryoswith wild type tetraploid embryos (Figure 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 in
vasculogenesis. Thus, Notch2 is not cell autonomously
required for the early cell fate determination of subtypesof trophoblast cells, but plays an indispensable role incoordinated maternal and fetal vasculogenesis in thedeveloping mouse placenta.

Figure 1. Expression of Notch2 gene in developing mouseplacenta. A cryo-section of Notch2+/LocZ placenta at E10.0
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.

Figure 2. Survival of Notch2 mutant embryo in a chimerawith tetraploid wild type embryo. The mutant obtained in
the intercrosses by the heterozygotic mice showsdevelopmental retardation at E10.5 (A) and die before
E11.5. The mutant is able to survive in a chimera with wild
type tetraploid embryo until E13.5 (B).

Publication List:
Original papers
Gotoh, N., Manova, K., Tanaka, S., Murohashi, M.Hadari, Y.,
Lee, A., Hamada, Y., Hiroe, T., Ito, M., Kurihara, T.,
Nakazato, H., Shibuya, M., Lax, I., Lacy, E., andSchlessinger, J. (2005). The docking protein FRS2alpha isan essential component of multiple fibroblast growth
factor responses during early mouse development. Mol.
Cell. Biol. 25, 4105-4116.
Nakamura, Y., Hamada, Y., Fujiwara, T., Enomoto, H.,
Hiroe, T., Tanaka, S., Nose, M., Nakahara, M., Yoshida,N., Takenawa, T., and Fukami, K. (2005). PhospholipaseC1 and –3 are essential in trophoblast for placental
development. Mol. Cell. Biol. 25, 10979-10988.

13

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

can be obtained by artificial fertilization of normal eggs
(XX) and sex-reversed, pseudo male sperm (XX), or
normal eggs (XX) and super male sperm (YY),
respectively. Steroidal enzymes P450scc, 3-HSD, andP450c17 are found at high levels in female gonadal
anlargen of tilapia at 7-10 days posthatching, but are only
seen weakly in males and not until 30 days posthatching.
Further, the enzyme aromatase is only detected in ovaries.
Treatment of XX fry with fadrozole (aromatase inhibitor)
or tamoxifen (estrogen receptor antagonist) caused
complete sex reversal to functional males. These results
suggest that endogenous estrogens are critical for
directing initial ovarian differentiation in tilapia. We
recently showed that Foxl2 and Ad4BP/SF-1 play
important roles in the differential expression of aromatasein XX tilapia gonads during sex differentiation. The
critical role of Foxl2 in ovarian differentiation was
confirmed by male sex reversal of XX transgenic tilapia
carrying a dominant-negative mutant of Foxl2. In XY fry,DMRT1 gene is expressed male-specifically in testicular
Sertoli cells prior to and during sex differentiation. XX
tilapia carrying extra copies of tilapia DMRT1 as atransgene induced various degrees of gonadal changes
including complete transformation to testis. These results
suggest an important role for DMRT1 in testiculardifferentiation in tilapia.

DIVISION OF REPRODUCTIVE BIOLOGY
Professor:
Associate Professor:
Research Associates:
Technical Staff:
NIBB Research Fellow:
PRESTO Researcher:
Postdoctoral Fellows:

Graduate Students:
Visiting Scientist:
Technical Assistants:

Secretary:

NAGAHAMA, Yoshitaka
YOSHIKUNI, Michiyasu
OKUBO, Kataaki
OHNO, Kaoru
KOBAYASHI, Hiroko
BASU, Dipanjan
MATSUDA, Masaru
BASU, Shakuntala
BHANDARI, Ramji Kumar
CHAUBE, Radha
GUAN, Guijun
IJIRI, Shigeho
KANEKO, Hiroyo
LAU, En-Lieng
OHTA, Kohei
PAUL, Bindhu
SAKAI, Fumie
SHIBATA, Yasushi
SUZUKI, Aya
USAMI, Takeshi
WANG, De-shou
YI, Mei-ShengZAHO, Haobin
OHMURO-MATSUYAMA, Yuki
KURITA, Kayoko
ZHOU, Linyan
TANAKA, Masaki
HARA, Ikuyo
HAYAKAWA, Rie
HIRAKAWA, Eri
SHIBATA, Emiko
TAKAKI, Chikako
SHIMADA, Yu

II. Molecular mechanisms of sex change
The gobiid fish, Trimma okinawae, possesses ovarianand testicular tissues simultaneously in its gonad and isable to change sex repeatedly in both directions
depending on its social surroundings, thus providing an
excellent animal model to investigate molecularmechanisms of sex change. Aquarium experiments werecarried out in the laboratory. Two males (M/M) or two
females (F/F) were kept in separate tanks. In the M/Maquarium, the smaller male changes its sex to female. On
the contrary, the larger female changes its sex to male in
the F/F aquarium. Behavioral changes occurred within 30
minutes of social manipulation (Figure 1). After pairing,
the larger male or female attacked the smaller fish, which
fled and often hid in a nest. After 30 min., however, thelarger fish began to court the smaller fish. These results
suggest that the brain sex of a sequential hermaphrodite isdetermined independently of gonadal effect and is
induced only by social cues.

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. Molecular mechanisms of sex determination andgonadal sex differentiation
Fish have a range of gonadal differentiation types
including gonochoristic species as well as hermaphroditic
species. We have been using two fish species toinvestigate the molecular mechanisms of sex
determination (medaka, Oryzias latipes) and gonadal sex
differentiation (Nile tilapia, Oreochromis niloticus).
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
chromosome) as a strong candidate for the
sex-determining gene of medaka. DMY encodes a proteinof 267 amino acids including the highly conserved DM
domain. Our loss- (natural mutants analysis andknock-down) and gain- (over-expression using transgenic
techniques) of-function studies indicate that DMY is the
sex-determining gene of medaka. DMY provides the firstexample of a sex-determining gene in non-mammalianvertebrates.
In tilapia, all genetic female (XX) or male (XY) broods

Figure 1. Sexual behavior
immediately after pairing

14ofTrimmaokinawae

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

4-pregnen-3-one (17,20-DP) (MIH), and MPF in fish
(Figure 3).
The primary hormone involved in starfish reproduction
has been called the gonad-stimulating hormone (GSS),
which is a peptide hormone produced in the radial nerves.
Although the hormonal activity of GSS has been known
for a long time, only recently has it been possible todetermine its primary structure. GSS has been purified
from the radial nerves of starfish (Asterina pectinifera),and the amino acid sequence was determined. GSS is aheterodimeric peptide with a molecular weight of 4737,
consisting of A and B chains; the A chain contains 24
residues and the B chain 19 residues. Chemicallysynthesized GSS is as active as native GSS in thehomologous in vitro GVBD assay.
In fish, LH acts on ovarian follicle cells to produce fish
MIH (17, 20-DP). 17, 20-DP is synthesized by a
two-step process involving two ovarian cell layers, thethecal and granulosa cells. Unlike other steroid hormones,
17, 20-DP binds to a novel, G-protein-coupledmembrane progestin receptor (non-genomic action),
leading to the de novo synthesis of cyclin B, theregulatory component of 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 isinactivated by degradation of cyclin B. We showed thatthe 26S proteasome initiates cyclin B degradation throughthe first cut of its NH2 terminus at lysine 57.
An endocrine-disrupting chemical, diethylstilbestrol(DES), a nonsteroidal estrogen, triggers oocyte maturationin goldfish (Carassius auratus) and zebrafish (Daniorerio). 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 are indistinguishable from those inducedby 17, 20-DP. Both 17, 20-DP- and DES-induced
oocyte maturation is inhibited by an antibody against themembrane progestin receptor. These results suggest that
DES may act on the membrane progestin receptor as anagonist of 17, 20-DP.

The involvement of gonadotropins in sex change was
examined by determining the changes in gonadotropin(FSH and LH) receptor gene expression in gonads during
the onset of sex changes from female to male and male tofemale. Expression appears to be related to sexual phasewith quick location switching of the two genes after social
manipulation to stimulate sex change. This differential
expression of the two gonadotropin receptor genes is anearlier event occurring in gonads after pairing and plays acritical role in the sex change.

III. Embryonic development of gonadotropinreleasing hormone (GnRH) neurons
Neurons that synthesize and release GnRH are essential
for the central regulation of reproduction. X-linked
Kallmann syndrome (X-KS), characterized by failed
gonadal function, is caused by a mutation in KAL1, which
is suggested to regulate the development of GnRH
neurons. Since rodents lack Kal1 in their genome, thepathogenesis of X-KS has been difficult to study. We
identified a KAL1 ortholog in the medaka. Antisense
knockdown of the KAL1 ortholog in the transgenic
medaka in which GnRH neurons were visualized with
GFP led us to observe the inappropriate accumulation of
GnRH neurons in the olfactory compartment and loss of
their ability to migrate into the forebrain (Figure 2). This
result was consistent with that reported in a fetus with
X-KS. Thus, our data demonstrate that X-KS can bephenocopied by antisense knockdown of kal1 and can bedirectly monitored in the transgenic medaka. Taken
together, the medaka system provides a useful in vivo
model for studying disorders of GnRH deficiency
including X-KS.



Figure 2. Knockdown of kal1 in the transgenic medaka that
express GFP in gnrh1 (panel A) and gnrh3 (panel B)
neurons resulted in the deficient migration of GnRH
neurons (arrowheads).

IV. Endocrine regulation of oocyte maturation

Fish

Starfish

LH(Pituitary)

GSS(Radial nerve)
Ovarian follicle cells

The process of oocyte maturation is the time period
between the resumption of meiosis and the second meiotic
metaphase, and thus, is a prerequisite for successful
fertilization. It has been well established that hormonesplay an important role in inducing oocyte maturation in
invertebrates as well as vertebrates. Our studies using
vertebrate (fish) and invertebrate (starfish) models haverevealed that the basic mechanisms involved in oocyte
maturation are the same in these two species, despite thediffering chemical nature of the hormonal agents involved.
In both species, three major mediators have been shown
to be involved: GSS, 1-methyladenine (maturationinducing hormone, MIH), and maturation-promoting
factor (MPF) in starfish, and LH, 17,20-dihydroxy-17  ,20 -Dihydroxy 4-pregnen -3one

Membrane receptors

1-Methyladenine
Oocyte membrane
Inhibitory G -protein

Membrane receptors

cAMP decreasec
MPF activation
MPF

Oocyte maturation
Immature

MatureOocyte maturation
30 min  2 days Figure 3. Regulation of oocyte maturation in fish andstarfish15

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

Publication List:

Insights from transgenic medaka and the relevance toX-linked Kallmann syndrome. Endocrinology, in press.
Oshima, Y., Kato, T., Wang, D.S., Murakami, T., Matsuda,Y., Nagahama, Y., and Nakamura, M. (2005). Promoter
activity and chromosomal location of the Rana rugosa
P450 aromatase (CYP19) gene. Zool. Sci., in press.
Sunobe, T., Nakamura, M., Kobayashi, Y., Kobayashi, T.,
and Nagahama, Y. (2005). Aromatase immunoreactivity
and the role of enzymes in steroid pathways for inducing
sex change in the hermaphrodite gobiid fish Trimma
Okinawae. Comp. Biochem. Physiol. Part A 141, 54-59.
Sunobe, T., Nakamura, M., Kobayashi, Y., Kobayashi, T.,
and Nagahama, Y. (2005). Gonadal structure and P450scc
and 3-HSD-like immunoreactivity in the gobiid fish
Trimma okinawae during bidirectional sex change. Ichyiol.
Res. 52, 27-32.
Tokumoto, M., Nagahama, Y., Thomas, P., and Tokumoto, T.
(2005). Cloning and identification of a membraneprogestin receptor in goldfish ovaries and evidence it is anintermediary in oocyte meiotic maturation. Gen. Comp.
Endocrinol., in press.
Wayne, N.L., Kuwahara, K., Aida, K., Nagahama, Y., andOkubo, K. (2005). Whole-cell electrophysiology of
gonadotropin releasing hormone (GnRH) neurons that
express green fluorescent protein (GFP) in the terminal
nerve of transgenic medaka (Oryzias latipes). Biol.
Reprod., in press.
Zhou, L.Y., Wang, D.S., Senthilkumaran, S., Yoshikuni, M.,
Shibata, Y., Kobayashi, T., Sudhakumari, C.C., andNagahama, Y. (2005). Cloning, expression andcharacterization of three types of 17-hydroxysteroid
dehydrogenases from the Nile tilapia, Oreochromis
niloticus. J. Mol. Endocrinol. 35, 103-116.

Original papers
Bhandari, R.K., Nakamura, M., Kobayashi, T., andNagahama, Y. (2005). Suppression of steroidogenic
enzyme expression during androgen-induced sex reversal
in Nile tilapia (Oreochromis niloticus). Gen. Comp.
Endocrinol., in press.
Bhandari, R.K., Ashraful, M.A., Higa, M., Soyano, K., andNakamura, M. (2005). Evidence that estrogen regulates
the sex change of honeycomb grouper (Epinephelus
merra), a protogynous hermaphrodite fish. J. Exp. Zool.
303A, 497-503.
Chang, X.T., Kobayashi, T., Senthilkumaran, B., KobayashiKajiura, H., Sudhakumari, C.C., and Nagahama, Y. (2005).
Two types of aromatase with different encoding genes,
tissue distribution and developmental expression in Niletilapia. Gen. Comp. Endocrinol. 141, 101-115.
Horiguchi, R., Tokumoto, M., Nagahama, Y., and Tokumoto,T. (2005). Molecular cloning and expression of cDNA
coding four spliced isoforms of casein kinase I in
goldfish oocytes. Biochim. Biophys. Acta 1727, 75-80.
Horiguchi, R., Yoshikuni, M., Tokumoto, M., Nagahama, Y.,
and Tokumoto, T. (2005). Identification of a protein
kinase which phosphorylates  subunit of the 26S
proteasome and changes in its activity during meiotic cell
cycle in goldfish oocytes. Cell. Signal. 17, 205-215.
Kobayashi-Kajiura, H., Kobayashi, T., and Nagahama, Y.
(2005). Cloning of cDNAs and the differential expressionof A-type cyclins and Dmc1 during spermatogenesis in theJapanese eel, a teleost fish. Dev. Dyn. 232, 1115-1123.
Kobayashi, Y., Sunobe, T., Kobayashi, T., Nagahama, Y.,
and Nakamura, M. (2005). Gonadal structure of theserial-sex changing fish. Dev. Growth Differ. 47, 7-13.
Kobayashi, Y., Sunobe, T., Kobayashi, T., Nakamura, M.,
Suzuki, N., and Nagahama, Y. (2005). Molecular cloning
and expression of Ad4BP/SF-1 in the serial sex changing
gobiid fish Trimma okinawae. Biochem. Biophys. Res.
Comm. 332, 1073-1080.
Nakamoto, M., Suzuki, A., Matsuda, M., Nagahama, Y., andShibata, N. (2005). Testicular type Sox9 is not involved insex determination, but might be in the development of
testicular structures in the medaka, Oryzias latipes.
Biochem. Biophys. Res. Comm. 333, 729-736.
Nakamura, I., Evans, J.C., Kusakabe, M., Nagahama, Y., andYoung, G. (2005). Changes in steroidogenic enzyme andsteroidogenic acute regulatory protein messenger RNAs in
ovarian follicles during ovarian development of rainbow
trout. Gen. Comp. Endocrinol. 144, 224-231.
Ohmuro-Matsuyama, Y., Okubo, K., Matsuda, M., Ijiri, S.,
Wang, D.S., Guan, G.J., Suzuki, T., Matsuyama, M.,
Morohashi, K., and Nagahama, Y. (2005). Liver receptor
homologue-1 activates brain aromatase promoter of
medaka, Oryzias latipes. Mol. Reprod. Develop., in press.
Okubo, K., Sakai, F., Lau, E.L. Yoshizaki, G., Takeuchi, Y.
Naruse, K., Aida, K., and Nagahama, Y. (2005). Forebrain
gonadotropin-releasing hormone neuronal development:

Review articles
Matsuda, M. (2005). Sex determination in the teleost medaka,
Oryzias latipes. Ann. Rev. Genet. 39, 293-307.
Shibata, Y. (2005). Modification of egg envelope proteinsduring fertilization in teleost fish. In New Impact on
Protein Modification in the Regulation of Reproductive
System, T. Tokumoto, ed. (Kerala, Research Signpost),
pp.115-134.

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otherwise activities of certain types of growth factors. The
study in this division has been performed mainly from the
aspect of the transcriptional control of the genes
implicated in gonadal sex differentiation.

DIVISION FOR SEX DIFFERENTIATION
Professor:
Research Associates:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:

Technical Assistants:

Secretary:

MOROHASHI, Ken-ichirou
FUKUI, Yuko
OGAWA, Hidesato
TSUCHIYA, Megumi
MOHAMAD, Zubair
BABA, Takashi
KOMATSU, Tomoko
SHIMA, Yuichi
KUSAKA, Masatomo
MATSUYAMA, Makoto
FATCHIYAH
SATO, Yuko
MIYABAYASHI, Kanako
YAMADA, Makoto
KOUTA, Ryuki
OWAKI, Akiko
ISHIKAWA, Azusa
KOIKE, Yukari
GOUNO, Hiroe
TOMIDA, Sanai
DODO, Yukiko
SUGIURA, Mio

I. Boundary of Ad4BP/SF-1 gene locus
The spatial and temporal control of gene expression isessential for establishment of cell fates in multicellularorganisms and the transcriptional control requires
appropriate interaction between enhancers and basal
promoter. In certain genes, this interaction is enhanced byadditional regulatory elements such as the locus control
region (LCR).
The LCR at the -globin locus is one of the mostintensively studied elements, and in the case of thechicken -globin LCR consists of four DNase I
hypersensitive (HS) sites, suggesting an open chromatin
structure that, unlike closed chromatin, can be easily
accessed by transcription factors. Because many
regulatory fragments including insulators show nuclease
hypersensitivity, we examined whether DNase I HS sites
are present between GCNF and Ad4BP/SF-1 gene loci.
Nuclei prepared from Y-1 cells, which are derived frommouse adrenal cortex and have robust expression of
Ad4BP/SF-1, were treated with DNase I, and three DNase
I HS sites (adHS1, 2, and 3) were found between the lastexon of GCNF and the first exon of Ad4BP/SF-1 (Figure1). CTCF, a ubiquitous nuclear protein with multiple zincfinger motifs, is regarded as an insulator-binding protein
solely known to date in vertebrates. In order to examine
whether
CTCF
contributedtothenuclease
hypersensitivity on adHS sites, we performed chromatin
immunoprecipitation (ChIP) assays using anti-CTCF
antibody. CTCF-attached DNA fragments were amplified
by PCR using primers designed against regions aroundthe adHS sites. Interestingly, CTCF was found to bindwith only a region located downstream of adHS1 but not
located in the proximity of adHS2 and 3 (CTCF in Figure1). These data suggest that the DNase I hypersensitivity of
adHS1 is most likely due to CTCF-mediated changes inchromatin architecture while the hypersensitivity at
adHS2 and 3 results from chromatin structures generated
by proteins other than CTCF.
The nuclear matrix is the fraction that is not solubilized
by treatment with high salt buffer, and is thought tocomprise the nuclear architecture that provides a platform
for nuclear events such as transcription and havesuggested that the nuclear matrix defines a transcriptional
unit by establishing a chromatin loop. In addition, acollection of DNA fragments known as a matrix
attachment region (MAR) is reported to be able to protect
a transgene from position effects. It is believed that thisphenomenon results from insulation by the MAR to block
expansion of silent heterochromatin. We therefore
examined whether a MAR exists around the adHS sites
for the insulator activity on Ad4BP/SF-1 locus.
Interestingly, a fragment corresponding to adHS2 andadHS3 exclusively bound to nuclear matrix (MAR in
Figure 1).

Sexual dimorphism manifests most obviously in the
gonads (testis and ovary), and is thereafter observed inother parts of the body such as the external genitalia,
muscle, and brain. This process of sex differentiation isdivided into three steps. The first step occurs at
fertilization, during which the sexes of fertilized eggs are
determined genetically according to a combination of sex
chromosomes. During the second step, individuals
carrying XY and XX sex chromosomes develop the testisand ovary, respectively, in the case of mammals. This
gonadal sex differentiation usually proceeds during fetal
stages, and subsequently sex steroids synthesized in thesexually differentiated gonads control the sexes of theother tissues. Therefore, the gonadal sexes are quite
important for the sex differentiation of animals.
A number of transcription factors are known to play
crucial roles in the process of gonadal differentiation.
Some of these genes such as SRY, WT1, DAX-1, SOX9
and ARX were identified as the genes responsible forhuman diseases that display structural and functional
defects in the gonads. Functions of the other genes such
as Ad4BP/SF-1, Emx2, M33, and Lhx9 were elucidated by
the phenotypes of the gene-disrupted mice. In addition,
their expression profiles in the sexually differentiating
gonad strongly suggested their functional significance atthe early stage of gonadal differentiation. However, itremains to be elucidated how the genes are expressed by
upstream regulators. Studies from the aspect above arequite important to define the gene regulatory cascade andthe molecular mechanisms mediating sex differentiationof the gonad.
Tentatively, we have hypothesized that the sexuallyindifferent gonads determine their sexes under the controlof two opposite signals, one of which is the signal for
male (testicular) differentiation and the other is the signal
for female (ovarian) differentiation. Possibly the nature of
the signals is transcriptional activities of the transcription
factors expressed in the sexually differentiating gonads, or

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

complex containing M33, Mpc2, rae28/Mph1, Bmi1, andMel18 appears to be required for stable maintenance ofthe repression state.
Previously we showed that disruption of the murine
M33 gene, a PcG member, displayed posteriortransformation of the vertebral columns and sternal ribs.
In addition, hypoplasia and sex-reversal of the gonads has
been observed. In the current study, we identified defects
in the splenic and adrenal formation of M33-knockout(KO) mice on a C57BL/6 genetic background.
Histological examination revealed that the vascularendothelium and its surrounding structures weredisorganized.
Immunohistochemical
analysesdemonstrated disturbances in vascular formation andcolonization of immature hematopoietic cells. These
splenic phenotypes observed in the M33-KO mice werequite similar to those seen in Ad4BP/SF-1 knockouts.
Moreover, we observed that the adrenal gland of M33-KO
and Ad4BP/SF-1 heterozygous KO mice are smaller thanthat of wild-type. These phenotypic similarities strongly
suggested the presence of genetic and functionalcorrelations between the two genes. In fact, western-blot,
immunohistochemistry, and RT-PCR analyses of the M33
knockouts all indicated significantly decreased expressionof Ad4BP/SF-1, indicating that M33 is an essential
upstream regulator of Ad4BP/SF-1 gene.
Upstream stimulatory factors 1 (USF-1 and USF-2),
Sox9, and Wt1(-KTS form) have all been identified as
direct-acting positive regulators of Ad4BP/SF-1 genetranscription. These factors bind recognition sites in the 5’
upstream region of the Ad4BP/SF-1 gene and thereby
activate gene transcription. However, the present genetic
analyses could not distinguish whether Ad4BP/SF-1 isregulated directly or indirectly by M33. Therefore, we
tested whether M33 directly binds to Ad4BP/SF-1 gene
locus by ChIP assay using antibody to M33. Interestingly,
the assays with Y-1 adrenocotical cells revealed thattarget sites of M33-containing PcG complex are present at
the upstream region of the first exon and the immediately
downstream region of the last exon of Ad4BP/SF-1 gene
(Figure 1). As described above, our recent study
demonstrated that the intergenic region between
Ad4BP/SF-1 and GCNF gene contains functionalarchitectures such as DNase I hypersensitive sites, a
nuclear matrix attachment region (MAR), and CTCF
binding sites. Moreover, ChIP assays showed adiscontinuous pattern of histone H3 and H4 acetylation
over the intergenic region. These observations strongly
suggested that this region forms a boundary, the so-called
insulator, between the two transcriptional units,
Ad4BP/SF-1 and GCNF. This assumption was consistent
with a previous observation that CTCF and nuclear matrix
are required for insulator activity. In this regard, it seemedinteresting to examine whether M33-containing PcG
complex binds to this intergenic region because PcG
complex is known to function in formation of inactive
chromatin arrays. Expectedly, our ChIP assays
demonstrated that M33-containing PcG complex binds tosites adjacent to and overlapped with the CTCF binding
region and MAR, respectively. Similarly, it is noteworthy

Posttranslational modifications of amino terminal tailsof histones are thought to be epigenetic marks governing
control of gene expression. Acetylation at lysine residuesof the tail is known to correlate with activation oftranscription, and chromatin associated with an active
promoter contains mostly acetylated histones. ChIP
assays with anti-AcH3 and AcH4 demonstrated thatacetylated boundary was formed around adHS1 andproximal to the transcription start site (AcH3/H4
boundary in Figure 1).
Taken together, we have demonstrated currently theexistence of an insulator defining the transcriptionalboundary between Ad4BP/SF-1 and GCNF gene loci by
DNase
I
hypersensitive
assays.
Ourfurthercharacterization indicated that these HS sites correspondto regions containing potential binding sites for CTCF andnuclear matrix. Importantly, these sites are almost
coincident with a divergence of acetylation pattern ofhistone H3 and H4 tails and constitute a boundary
between active and inactive chromatin domains. The
correlation between the binding of CTCF and nuclear
matrix attachment sites to a functional boundary fortranscription is consistent with previous reports that
CTCF and the nuclear matrix are required for insulator
activities.

Figure 1. Structure of boundary between Ad4BP/SF-1 and
GCNF gene loci. Ad4BP/SF-1 gene is localized at thedownstream of another nuclear receptor gene, GCNF, and at
the upstream of uncharacterized GPR144 gene. DNase I
hypersensitive sites, adHS1, adHS2, and adHS3, CTCF
binding site, nuclear matrix binding site (MAR), acetyl
histone H3 and H4, and M33 binding sites areschematically presented. All these sites are localized
between GCNF and Ad4BP/SF-1 gene.

II. Function of M33 as a factor bound to theboundary of Ad4BP/SF-1 locus
In mammals, multiple orthologues of PcG components
members have been identified. Polycomb (Pc) has threemouse counterparts, M33 (Mpc1), Mpc2, and Mpc3.
Likewise, other PcG members have multiple counterparts.
Therefore, mammalian PcG complexes are thought to becomprised of distinct sets of constituent group members.
In fact, mammalian PcG complexes were recently
sub-divided into two distinct types according to theirbiochemical and functional properties. The first complex,
containing Eed and Eenhancer of Zeste (Enx1 and Enx2),
appears to be required to initiate repression of target gene
expression in early development, whereas a second

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

functional properties of this region might be differentiallymodified according to developmental stages, tissues andpossibly sexes.

that M33-containing PcG complex bound to region
adjacent to the last exon of Ad4BP/SF-1 gene. Althoughfurther examination is required, it is likely thatM33-containing PcG complex is implicated in formationof intergenic boundary. This presumptive function of PcG
complex is supported by previous Drosophila studies.
Mihaly et al. (1997) showed that a cis-regulatory element,
Fab-7, consists of a boundary element and PRE, both of
which
are
implicatedinregulationofparasegmental-specific expression of bithorax genes. In
the case of gypsy insulator, Gerasimova and Corces(1998) demonstrated that a gypsy insulator binding
protein, mod(mdg4), requires Pc, a PcG component, for
the insulator function. Considering these observations, thecurrent studies strongly suggested that PcG complex
together with CTCF and nuclear matrix forms intergenic
boundary between Ad4BP/SF-1 and GCNF genes,
although functional and physical correlation of PcG
complex with CTCF and/or nuclear matrix remains to beelucidated (Figure 2).

Publication List:
Original papers
Baba, T., Mimura, J., Nakamura, N., Harada, N.,Yamamoto,M., Morohashi, K.,* and Fujii-Kuriyama, Y.* (*; The last
two authors equally contributed to this work.) (2005). Ah(dioxin) receptor as a key factor in the regulation of
female reproduction. Mol. Cell. Biol. in press.
Ishihara, S., and Morohashi, K. (2005). Identification of theboundary for histone acetylation between nuclear receptor
genes, Ad4BP/SF-1 and GCNF, aligned in tandem.
Biochem. Biophys. Res. Commun. 329, 554-562.
Kamei, Y., Aoyama, Y., Fujimoto, T., Kenmotsu, N., Kishi,C., Koushi, M., Sugano, S., Morohashi, K., Kamiyama, R.,
and Asakai, R. (2005). A steroidogenic cell line withdifferentiation potential from mouse granulosa cells,
transfected with Ad4BP and SV40 large T antigen genes. J.
Endocrinol. 185, 187-195.
Katoh-Fukui, Y., Owaki, A., Sotoyama, Y., Kusaka, M.,
Shinohara, Y., Maekawa, M., Toshimori, K., andMorohashi, M. (2005). Mouse Polycomb M33 is required
for splenic vascular and adrenal gland formation throughregulating Ad4BP/SF-1 expression. Blood 106,1612-1620.
Matsuyama, M., Mizusaki, H., Shimono, A., Mukai, T.,
Okumura, K., Abe, K., Shimada, K., and Morohashi, K.
(2005). Novel isoform of vinexin, vinexin  regulates
Sox9 gene expression through activating MAPK cascadein mouse fetal gonad. Genes Cells 10, 421-434.
Shima, Y., Mohamad, Z., Ishihara, S., Shinohara, Y., Oka, S.,
Kimura, S., Suita, S., and Morohashi, K. (2005). VMH
specific enhancer of Ad4BP/SF-1 gene. Mol. Endocrinol.
19, 2812-2823.
Toda, K., Hayashi, Y., Okada, T., Morohashi, K., andSaibara, T. (2005). Expression of the estrogen-inducible
EGFP gene in aromatase-deficient mice reveals
differential tissue-response to estrogenic compounds. Mol.
Cell. Endocrinol. 229, 119-126.
Yoshioka, H., Ishimaru, Y., Sugiyama, N., Tsunekawa, N.,
Noce, T., Kasahara, M., and Morohashi, K. (2005).
Mesonephric FGF signaling is associated with thedevelopment of sexually indifferent gonadal primordium
in chick embryos. Dev. Biol. 280, 150-161.

Figure 2. Intranuclear structure of Ad4BP/SF-1 gene locus.
Through binding of the multiple factors at the upstream anddownstream of Ad4BP/SF-1 gene, the gene locus appears
forms structurally and functionally a single transcriptionunit.

It has been well accepted that PcG complex directly
binds to target gene loci to keep silent chromatin
conformation. Nevertheless, in our current study, multiple
binding regions of the M33-containing PcG complex were
found to lie at the 5’ boundary region of thetranscriptionaly active Ad4BP/SF-1 gene. Although ourfindings seemed contradictory to the accepted notion,
recent ChIP analyses with antibodies to PcG components
have accumulated observations similar to the present
study, showing that PcG complex binds to not only
inactive but active gene loci. Accordingly, it is reasonable
to assume that PcG complex is implicated in theformation of transcriptionaly active chromatin unit. Basedon these observations together with our current results,
Ad4BP/SF-1 gene is thought to form a transcriptional unit
through forming complexes M33 containing polycomb,
CTCF, and nuclear matrix. Considering that the complex
formation is tightly coupled with the gene transcriptional
regulation, it might be noteworthy that structural and thus

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

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 that trigger intracellular signaling,transcription factors that 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 ascidian,employing embryology, genetics, molecular and cellularbiology, and biochemistry. In addition, we have recently
introduced genomics technologies to elucidate the precise
genetic programs controlling early development.

convergent extension, several growth factors are
implicated. Recent studies revealed that one of the Wnt
signaling pathways, called Wnt/JNK (c-Jun N-terminal
kinase) pathway, is shown to be important for the
regulation of convergent extension. The pathway is highly
conserved among species and initially found to be
essential for the establishment of planar cell polarity(PCP) of Drosophila wing hair and often called Wnt/PCP
pathway.
To clarify the molecular mechanism of gastrulation, we
have been attempting to identify novel regulatorycomponents controlling gastrulation cell movements by
an expression cloning based on morphology of dorsal
explant (Keller’s explant) and of embryo. One ofidentified genes was found to encode a member of thefamily of ArfGAP, GTPase activating protein (GAP) for
ADP rybosylation factors (ARFs), which we named
XGAP. Further functional analyses of XGAP revealed
that XGAP is required for gastrulation cell movements,
particularly CE and the establishment of cell polarity
which is manifested by the localization of cellular
protrusions such as lamellipodia. In addition, we foundthat XGAP is required for the proper localization of PAR
proteins that are highly conserved proteins essential forestablishing cell polarity in a wide range of organisms
from C. elegans to humans. Studies on the precise
molecular mechanism of PAR proteins’ regulation by
XGAP are currently undertaken.
Aside from the molecular mechanism, we conducted
experiments to ask how and when cell polarity isestablished in the cells participating in CE. To grasp
cellular events triggered upon the cell polarization, we
focused on microtubule (MT) formation. Based on theassumption that MT formation and direction of MT
extension are closely related to cell polarity, we observed
MT formation using GFP-fused EB1/3 proteins that are
known to bind the plus-end of MTs. The time-lapse
recording of the GFP fluorescence shows clear
bidirectional extension of MTs in cells of Keller’s explantparticipating in CE, while MTs are radially extended in
ectoderm cells. We are investigating what makes thedifference in MT dynamics and how it is related to cellmorphology and polarity formation.

I. Molecular and cellular mechanism of vertebrate
gastrulation

A

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 (CE)”. As
convergent extension begins, cells are polarized andaligned mediolaterally, followed by the mutualintercalation of the polarized cells. In the regulation of

Figure 1. Microtubule dynamics in animal cap and dorsal
mesoderm cells revealed by EB3 movement. In animal cap
cells, the direction of EB3 movements are random (A),while the EB3 moves towards both poles of cells in dorsal
mesoderm (B). Green comets: EB3-GFP, white arrows: thedirection of EB3 movements.

DIVISION OF MORPHOGENESIS
Professor:
Associate Professor:
Research Associates:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:
Graduate Students:

Visiting Scientists:
Technical Assistants:

Secretaries:

UENO, NaotoKINOSHITA, Noriyuki
NAKAMURA, Makoto
TAKAHASHI, Hiroki
TAKAGI, Chiyo
IIOKA, Hidekazu
HYODO-MIURA, Junko
TAO, Hirotaka
MIYAKOSHI, Akira
KUJIRAOKA, Masahiro
MAEDA, Masato
LEE, Rebecca
SHINDO, Asako
CHUNG, Hyeyoung
YOSHIKANE, Nami
YAMAMOTO, Takamasa
TERASAKA, Chie
YAMADA, Shigehiro
TANIYAMA, Kazumi
HAYASHI, Kazue
SHIBAMOTO, KaoriASANO, Megumi
KITABORA, Noriko
MIYAKE, Satoko
TSUGE, Toyoko

20

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

II. Roles of membrane trafficking in the regulationof gastrulation movements
Membrane trafficking has been implicated in signaltransduction, cell polarity control and cell movements. To
investigate the role of membrane trafficking in theregulation of gastrulation movements in Xenopus embryos,
we focused on Rab GTPases, key regulators for many
steps of membrane trafficking. We searched for Rab
GTPases that function in gastrulation by overexpressing
dominant-negative Rab mutants and identified four Rabs
that inhibited gastrulation movements. Loss-of-functionof one of these Rabs by the specific morpholino
oligonucleotide (Mo) also perturbed normal gastrulation,suggesting that it plays an essential role in this process.
Interestingly, the Rab Mo inhibited the non-canonicalWnt pathway which has been implicated in the regulationof convergent extension movements. In the Mo-injected
cells, the Wnt ligand and its receptor Frizzled werelocalized normally, but they were not able to activate thedownstream signal in the cells. Dishevelled, which plays
the pivotal role in the Wnt signal pathway, is known to betranslocated from the cytoplasm to the plasma membranein response to the Wnt signal activation. The Rab Mo
inhibited its translocation, suggesting that membranetrafficking might be important for signaling from
Wnt/Frizzled to Dishevelled in this pathway.
Figure 2. Distribution of Ci-Scale protein in Ciona
intestinalis embryos. Staining of tailbud stage embryos withan anti-Ci-Scale antibody shown in green and staining withphalloidin in magenta (A, B). Fusion gene constructs taggedwith EGFP to examine the role of Ci-Scale components in
its extracellular distribution. dNT mutation lacks the
N-terminal half, and 3DN was a mutation in which the threeconserved D residues in the DXDXD motif are changed to
N residues (C). Distribution of EGFP in embryos
electropolated with (D) Ci-Scale (control), (E) dNT or (F)3DN; side view of the tailbud embryos, and (D’-F’) dorsal
view of the head region of embryos. EGFP in a dNTmutated embryo was seen disused distribution while
3DNmutant was seen only in the cytoplasm of notochordcells, suggesting the NT domain and the FBG domain,
especially the DXDXD motif, is necessary for its proper
distribution. Scale bar, 50 μm.

III. Notochord-derived fibrinogen-like protein
regulates a long-range patterning of nervous
system in chordate embryos
The dorsal nervous system (CNS) and the notochordunderneath CNS are two major organs characteristic tochordate body plans. Experimental embryology
demonstrated that the notochord play a critical role in thepatterning of CNS during vertebrate embryogenesis, butlittle is known about genes or molecules involved in thisinteraction. In vertebrates, a T-box gene, Brachyury (Bra),
plays a pivotal role in the formation of notochord. This is
the case of urochordate ascidian; Bra is expressed
exclusively in primodial notochord cells and its role isessential for the notochord cell differentiation. We havealready isolated nearly 40 genes that are direct or indirecttargets of Ci-Bra of Ciona intestinalis. An ascidian
homolog (Ci-scale) of the Drosophila Scabrous geneencodes a fibrinogen-like protein is specifically expressedin notochord cells, its protein product is not refined to
notochord but distributed underneath the CNS withfibril-like protrusions or vesicles. Knockdown of Ci-Scalefunction resulted in failure of convergent extention of
notochord cells and differentiation of neuronal cells andaxon guidance. A proper distribution of Ci-Scale proteinsis dependent on Notch signaling delivered by the CNS.
These results suggest that cooperative function of
Ci-Scale and Notch is essential for the patterning of CNS
and axon guidance in chordate embryos.

IV. Trithorax group components tonalli andvertebrate homolog TONAS proteins participate
roles in protein SUMO modification andepigenetic regulation
Drosophila tonalli (tna) mutant is a previously reported
mutant that is involved in epigenetic regulation. The tna
mutant phenotype mimics the homeotic loss-of-function
phenotype and this mutant shows striking genetic
interaction with the mutants of trithorax group genes. We
isolated two mutant alleles of tna as a putative
downstream target of the DPP (corresponding tovertebrate BMP) signaling. We also isolated vertebratehomologues of tna and named as tonalli related SP-RING
finger protein, TONAS-1 and TONAS-2. The role of21

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

Publication List:

Tna/TONAS proteins in the epigenetic regulation and alsoin Trithorax group function is totally unknown. The mostcharacteristic feature of Tna/TONAS proteins is theexistence of a single SP-RING finger motif in the middle.
The SP-RING motif was originally found in the PIAS
family SUMO-E3 ligase proteins. We have addressed
TONAS SUMO E3 ligase activity by in vitro experiments
as well as cultured cell over-expression study. TONAS
itself is a good substrate for SUMO-2/3 modification and
the SP-RING motif is essential for this activity. Other
SUMOylation targets of TONAS proteins are currently
unknown. We isolated TONAS binding partners from
FLAG-TONAS expressing HEK293T cell-extract byprotein affinity purification and LC-MS-MS analysis.
Some of the candidate interactors are known to be
substrates of SUMO-modification. We will confirm
whether these proteins are specific TONAS SUMOylation
targets by further analysis.

Original papers
Arima, K., Shiotsugu, J., Niu, R., Khandpur, R., Martinez,
M., Shin, Y., Koide, T., Cho, K.W., Kitayama, A., Ueno,
N., et al. (2005). Global analysis of RAR-responsive
genes in the Xenopus neurula using cDNA microarrays.
Dev. Dyn. 232, 414-431.
Baldessari, D., Shin, Y., Krebs, O., Konig, R., Koide, T.,
Vinayagam, A., Fenger, U., Mochii, M., Terasaka, C.,
Kitayama, A., et al. (2005). Global gene expression
profiling and cluster analysis in Xenopus laevis. Mech.
Dev. 122, 441-475.
Chung, H.A., Hyodo-Miura, J., Nagamune, T., and Ueno, N.
(2005). FGF signal regulates gastrulation cell movements
and morphology through its target NRH. Dev. Biol. 282,
95-110.
Kataoka, K., Tazaki, A., Kitayama, A., Ueno, N., Watanabe,K., and Mochii, M. (2005). Identification ofasymmetrically
localized
transcripts
along
the
animal-vegetal axis of the Xenopus egg. Dev. GrowthDiffer. 47, 511-521.
Kawai, N., Takahashi, H., Nishida, H., and Yokosawa, H.
(2005). Regulation of NF-kappaB/Rel by IkappaB isessential for ascidian notochord formation. Dev. Biol. 277,
80-91.
Peiffer, D.A., Von Bubnoff, A., Shin, Y., Kitayama, A.,
Mochii, M., Ueno, N., and Cho, K.W. (2005). A Xenopus
DNA microarray approach to identify novel direct BMP
target genes involved in early embryonic development.
Dev. Dyn. 232, 445-456.
Sakamaki, K., Takagi, C., Yoshino, J., Yokota, H.,
Nakamura, S., Kominami, K., Hyodo, A., Takamune, K.,
Yuge, M., and Ueno, N. (2005). Transgenic frogs
expressing the highly fluorescent protein venus under thecontrol of a strong mammalian promoter suitable formonitoring living cells. Dev. Dyn. 233, 562-569.
Shin, Y., Kitayama, A., Koide, T., Peiffer, D.A., Mochii, M.,
Liao, A., Ueno, N., and Cho, K.W. (2005). Identificationof neural genes using Xenopus DNA microarrays. Dev.
Dyn. 232, 432-444.
Takada, H., Hattori, D., Kitayama, A., Ueno, N., and Taira,M. (2005). Identification of target genes for the Xenopus
Hes-related protein XHR1, a prepattern factor specifying
the midbrain-hindbrain boundary. Dev. Biol. 283,
253-267.
Takahashi, H., Mitani, Y., and Satoh, N. (2005). Both thefunctional specificity and autoregulative activity of two
ascidian T-box genes Hr-Bra and Hr-Tbx6 are likely to be
mediated by the DNA-binding domain. Dev. GrowthDiffer. 47, 173-185.
Tazaki, A., Kitayama, A., Terasaka, C., Watanabe, K., Ueno,
N., and Mochii, M. (2005). Macroarray-based analysis of
tail regeneration in Xenopus laevis larvae. Dev. Dyn. 233,
1394-1404.

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 through the clarification of gene function
and their interactions in the system.
We constructed the comprehensive database XDB3 that
stores EST sequences, assembled sequences, full insert
sequences and WISH (whole-mount in situ hybridization)
data with the descriptions of predicted gene function. We
added a new browsing method of WISH data as 3D
animation model, a method developed under collaboration
with Drs. P. Vize and V. Gerth (University of Calgary).
We have also created several transgenic X. laevis linesthat are labeled with fluorescent proteins (EGFP, Venus,
or RFP) localized in subcellular components such as
nucleus, nuclear membrane, mitochondria, Golgi, and ER.
These frogs will be very powerful tools for the functional
analysis of genes in the post genome era.

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

22

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

Technical Assistants:
Secretary:

KOBAYASHI, Satoru
MUKAI, Masanori
SHIGENOBU, Shuji
NODA, Chiyo
ARITA, Kayo
SATO, Kimihiro
HAYASHI, Yoshiki
KITADATE, Yu
HAYASHI, Makoto
YATSU, Jun
MAEZAWA, Takanobu
HASHIYAMA, Kazuya
UEDA, Yoshiko
TSUCHIYA, Naomi
SATO, KaoriHONDA, Satoko

Figure 1. Role of mitochondrial rRNAs in polecell formation

Germ cells are the specialized cells that can transmit
the genetic materials from one generation to the next insexual reproduction. All the other cells of the body aresomatic cells. This separation of germ and somatic cells is
one of the oldest problems in developmental biology. In
many animal groups, a specialized portion of egg
cytoplasm, or germ plasm, is inherited by the cell lineage
which gives rise to germ cells. This cell lineage is called
germline. The germline progenitors eventually migrate
into the gonads, where they differentiate as germ cells
when the organisms are physically matured. Earlier
investigators have demonstrated that germ plasm contains
maternal factors required and sufficient for germline
formation. In Drosophila, this cytoplasm is histologicallymarked by the presence of polar granules, which act as arepository for the maternal factor required for germline
formation. Our molecular screens have identified several
factors stored in the polar granules.

mtlrRNA on polar granules has an essential role in polecell formation, cooperating with mtsrRNA (Figure 1).
Recently, we have found that injection of kasugamycin(KA) and chloramphenicol (CH), inhibitors forprokaryotic-type translation, disrupt pole cell formation inearly embryos. The number of mitochondrial-type
ribosomes on polar granules is significantly decreased by
KA treatment, as shown by electron microscopy (Figure2). In contrast, ribosomes in the mitochondria andmitochondrial activity are unaffected by KA and CH.
Furthermore, injection of KA and CH impairs productionof Germ cell-less (Gcl) protein, which is required for polecell formation. The above observations suggest thatmitochondrial-type translation is required for pole cell
formation, and Gcl is a probable candidate for the protein
produced by this translation system.

II. The role of maternal Nanos protein
Among the maternal components of germ plasm,
Nanos (Nos) is essential for the germline-specific eventsoccurring in pole cells. Nos mRNA is localized in thegerm plasm during oogenesis, and is translated in situ to
produce Nos protein after fertilization. Nos is onlytransiently present in the posterior half of embryos during
the preblastoderm stage, and is required there forposterior somatic patterning. Nos in the germ plasm ismore stably inherited into the pole cells at 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. Nos
represses translation of mRNAs with discrete RNA
sequences called Nos response elements (NREs). In thepathway leading to posterior somatic patterning, Nos acts
together with unlocalized Pumilio (Pum) protein 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
germline-specific events. Pum, like Nos, is required inpole cells for their migration to the gonads.

I. The role of mitochondrial ribosomal RNAs inpole cell formation
Ultrastructural studies have shown that the germ plasm
is basically composed of polar granules and mitochondria.
While the primary roles of the mitochondria are oxidative
phosphorylation and biosynthesis of many metabolites, it
has now become evident that they are also involved in theformation of the germline progenitors, or pole cells. In
Drosophila, pole cell formation requires the function of
mitochondrial ribosomal RNA 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 with polar granules ingerm plasm. Mitochondrial rRNAs remain on the polar
granules until pole cell formation and are no longer
discernible on the granules within pole cells. Reduction of
the extra-mitochondrial mtlrRNA amount results in the
failure to form pole cells and injection of mtlrRNA is able
to induce pole cells in embryos whose ability to form
these cells has been abolished by uv-irradiation. Theseobservations clearly show that the extra-mitochondrial

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

the apoptotic response in pole cells to permit their proper
migration into the gonads.
The above observations suggest that pole cells have thepotential to enter into apoptosis, which somewhat
contradicts the notion that the germline is fundamentally
immortal as it is required for the propagation of any given
species. We speculate, however, that this apoptotic
pathway may be part of a mechanism that eliminates“aberrant pole cells” that have inherited an insufficientquantity of germ plasm components, such as maternal
Nos protein.

Figure 3. Nanos prevents apoptosis in pole cells. Confocal
images of the pole cells in control (left) and nos (right)
embryos at stage 13, stained with TUNEL labeling(magenta) and an antibody against Vas (green). Arrowheads
show TUNEL-positive pole cells.
Figure 2. Injection of KA reduces the number ofmitochondrial-type ribosomes around polar granules. A,
The average number of ribosomes in an unit area (8.4 x 103nm2) is plotted against their diameter. The diameters of
ribosomes around polar granules were measured in stage 2embryos injected with DW (control) and KA(KA-treatment). The number of ribosomes smaller than 20
nm in diameter was significantly decreased by KAtreatment. B, C, Electron micrographs of sections throughpolar granules in stage 2 embryos. B, In a control embryo
injected with DW, there were two types of ribosomesaround the polar granules. C, In an embryo injected withKA, the smaller ribosome was indiscernible around polar
granules. Arrows and arrowheads point to the smaller(mitochondrial-type) and the larger (cytosolic-type)ribosomes, respectively. pg, polar granules; Scale bar = 0.2
μm.

III. The role of maternal Sva53 in meiosis
Meiosis is an obligatory step to produce haploid
gametes that can transmit the genetic materials from one
generation to the next. However, little is known about
how the germline progenitors acquire the ability toundergo meiosis. We have found that a novel maternal
factor, SVA53 is essential for meiosis. SVA53 is amember of widely conserved BTB/POZ-zinc finger
proteins, which are known to function as transcriptional
regulators by altering chromatin structures. Maternal
SVA53 is enriched in pole cells, and a reduction in its
activity prevents meiosis. We propose that SVA53 is thefirst maternal molecule that regulates a genetic pathway
leading to meiosis. Thus, our current findings provide thebasis for the understanding of a novel epigenetic
mechanism that regulates the meiotic cell cycle inDrosophila and in a variety of other animal groups.

Our unpublished observations suggest that one of thetargets of Nos and Pum is head involution defective (hid)mRNA, which also contains an NRE in its 3’UTR andencodes a protein required for the induction of apoptosis.
In the absence of Nos or Pum, migrating pole cells are
eliminated by an apoptotic mechanism which is initiatedat stage 9/10 in the developing embryo (Figure 3). We
have also found that Df(3L)H99 (H99), a small deletion
within the genomic region that includes the hid gene,suppresses apoptosis in nos pole cells. In embryos lacking
both maternal Nos and zygotic H99 activity (nos-H99
embryo), there is no apoptotic death of any pole cells. In
addition, nos-H99 pole cells have the ability to migrate
into the gonads when transplanted into normal hostembryos. Therefore, the ability of nos pole cells to
migrate into the gonads is fully restored by the
suppression of apoptosis in our transplantation
experiments. This clearly demonstrates that Nos inhibits

IV. Expression of meiotic genes in pole cells
In Drosophila, genetic screens have identified many
genes required for meiotic division. However, it remains
elusive as to when and how these meiotic genes areactivated during germline development. To obtaininsights into their regulatory mechanisms, we examinedthe expression of 38 meiotic genes in pole cells during
embryogenesis. We have found that the transcripts of 12
meiotic genes were enriched in pole cells within theembryonic gonads. Among them, bag of marbles (bam),benign gonial cell neoplasia (bgcn), deadhead (dhd),
matotopetli (topi) and twine (twe) were activated only in

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

VI. Left-right asymmetry in embryospole cells within the gonads, whereas the transcripts fromgrapes (grp), Kinesin-like protein at 3A (Klp3A),
pavarotti (pav), lesswright (lwr), mei-P26, Topoisomerase
2 (Top2) and out at first (oaf) were distributed
ubiquitously in early embryos and then became restricted
to pole cells and to a subset of somatic tissues at laterembryonic stages. These observations suggest that polecells have already acquired the potential to express
several meiotic genes. Our data will thus provide a usefulbasis for analyzing how the germline acquires a potential
to execute meiosis.

In many animal groups, left-right (LR) asymmetry
within the body is observed. The left and right sides of the
body are generally defined with reference to theanterior-posterior (AP) and dorsal-ventral (DV) axes. We
investigated whether LR asymmetry is solely dependent
on the AP and DV polarities in Drosophila embryos. We
focused on the proventriculus, a posterior part of theforegut, and the hindgut because LR asymmetry in thesebody parts is highly stable in normal embryos. In embryoswith a fully reversed AP polarity, LR asymmetry in boththe proventriculus and the hindgut was re-oriented inrelation to the reversed AP polarity (Figure 4). This
demonstrates that inversion of AP polarity does not affectLR asymmetry of these tissues, and implies that LR
asymmetry is specified in relation to the AP and DV
polarities. Our findings were not consistent with thealternative hypothesis that LR asymmetry is
pre-determined by maternal signals that localize
asymmetrically along the LR axis in the oocyte and/or
early embryo. This work was supported by the Super
Science High school (SSH) program.

V. Microarray analysis of pole cells
To explore the regulatory mechanism of germline
specification, we attempted to identify genes expressed inpole cells during embryogenesis. From the embryos
carrying EGFP-vasa transgene that express GFP only inpole cells, we isolated pole cells by using
fluorescence-activated cell sorting (FACS), and they wereused for hybridization of microarray that contains probes
for all predicted genes in Drosophila genome. Our
microarray analysis has identified approximately 300
maternal and 200 zygotic transcripts enriched in polar
plasm and pole cells. The functional analysis of thesetranscripts is now ongoing.

Publication List:
Original papers
Amikura, R., Sato, K., and Kobayashi, S. (2005). Role ofmitochondrial ribosome-dependent translation in germline
formation in Drosophila embryos. Mech. Dev. 122,
1087-1093.
Hayashi, M., Aono, H., Ishihara, J., Oshima, S., Yamamoto,H., Nakazato, Y., and Kobayashi, S. (2005). Left-rightasymmetry in the alimentary canal of the Drosophila
embryo. Develop. Growth Differ. 47, 457-460.
Mukai, M., Kitadate, Y., Arita, K., Shigenobu, S., andKobayashi, S. (2005). Expression of meiotic genes in the
germline progenitors of Drosophila embryos. Gene Expr.
Patterns, in press.
Shigenobu, S., Arita, K., Kitadate, Y., Noda, C., andKobayashi, S. (2005). Isolation of germline cells from
Drosophila embryos by flow cytometry. Develop. GrowthDiffer., in press.

Review article
Kobayashi, S., Sato, K., and Hayashi, Y. (2005). The role ofmitochondrial rRNAs and Nanos protein in germline
formation in Drosophila embryos. Zool. Sci. 22, 943-954.
Figure 4. LR asymmetries of the proventriculus and thehindgut in embryos with fully reversed AP polarities.
Dorsal views of the anterior (C, B) and the posterior region(A, D) of normal embryos (B, D) and A/P-reversed
embryos (A, C) at stage 15-16. A, B, Brackets mark theproventriculi. Note that the posterior of the proventriculi
are oriented to the right side of the embryos. C, D, The
hindguts are stained. The anterior of the hindguts areoriented to the right. Arrowheads point to the micropyles, amorphological marker for the anterior pole of the eggs.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

receptor family also consists of many members, each ofwhich shows a distinct expression pattern during
embryogenesis. However, the receptor specificity of each
Wnt ligand has been poorly understood.
To investigate systematically the combinatorial effectsof Wnts and various Fzs on canonical Wnt/-cateninsignaling, we analyzed the ability of these Wnt proteins toincrease stability of armadillo/-catenin proteins in
Drosophila S2 cells expressing vertebrate Fzs (Takada etal. 2005). Wnt-3a increases the amount of armadillo
proteins in cells expressing Fzs 4, 5, and 8, but not Fzs 3
and 6; whereas Wnt-5a does not increase it in any cell line.
In contrast, both Wnt-3a and Wnt-5a increase thephosphorylation of Dsh in combination with most of theFzs. This Dsh phosphorylation is abrogated by decreasing
the levels of casein kinase I  by double-stranded
RNA-mediatedtranslational
interference.
These
observations indicate that both Wnt proteins can interact
with the majority of Fz receptors and elicit signaling
reactions exemplified by Dsh phosphorylation but that thestabilization of -catenin/armadillo proteins in the
Wnt/-catenin signaling occurs only when specificcombinations of Wnt and Fz meet.

DIVISION OF MOLECULAR AND
DEVELOPMENTAL BIOLOGY
Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:
Technical Assistants:

Secretaries:

TAKADA, Shinji
KOSHIDA, Sumito
UTSUMI, Hideko
KURATA, Tomoko
OHBAYASHI, Norihiko
KAWAMURA, Akinori
AKANUMA, Takashi
TAKADA, Ritsuko
YAMAGUCHI, Yoshifumi
ISHITANI, Shizuka
TAKAHASHI, Jun
OHBAYASHI, Akiko
TAKASHIRO, Kayoko
SASAKI, Michiko
ODA, Ritsuko
SHIBATA, Chitose
ODA, Keiko
MATSUDA, Mitsuko

During morphogenesis of vertebrates, the embryonicbody is gradually divided into sub-regions that are
specified to give rise to functional units. Most of theprocesses of regionalization and specification are
regulated by cell signaling molecules. So far, spatial
distribution of secreted signals, called morphogens, hasbeen considered to regulate regionalization andspecification during embryogenesis. However, themolecular basis to generate a variety of function of
secreted signals, including specific interaction between
secreted signals and their receptors and specific activationof their target genes in particular cells, has not yet been
discovered. To better understand this molecular basis, weare currently examining biochemical characteristics of
Wnt proteins and the molecular mechanism to activate
specific targets.
In contrast, some regionalization processes have also
been shown to be regulated by different manners. One of
the typical examples is the segmentation of somites.
Somites are the morphologically distinct segmental units
that are transiently formed during early vertebrate
development and subsequently give rise to metameric andfundamental structures such as the vertebrae of the axial
skeleton, their associated muscles, and tendons. The
molecular mechanism underlying the periodical formationof somites is coupled to an internal oscillator, referred toas the “segmentation clock”. To gain insight intomolecular
mechanism
underlying
thisspecific
regionalization, we are also characterizing genes involved
in somite segmentation.

II. Identification and functional characteristics of
Wnt target genes during embryogenesis
To identify Wnt-target genes during mouse
development, we used the induction gene-trap approach(Yamaguchi et al. 2005). We screened 794 trapped ESlines and recovered 2 ES cell lines that contained trapped
genes responsive to WNT-3A protein. One gene wasmainly expressed in the ductal epithelium of several
developing organs, including the kidney and the salivary
glands, and the other gene was expressed in neural crest
cells and the telencephalic flexure. The spatial andtemporal expression of these two genes coincided wellwith that of several Wnt genes. Furthermore, theexpression of these two genes was significantly decreasedin embryos deficient for Wnts or in cultures of embryonictissues treated with a Wnt signal inhibitor. These results
indicate that the gene trap is an effective method forsystematic identification of Wnt-responsive genes during
embryogenesis.
We have further analyzed the developmental role of theformer gene by generating mutant embryos defective inits function from the gene-trapped ES cell line. Our
results indicated that this gene plays essential roles indifferentiation of ductal epithelial cells in the kidney andsalivary glands.

I. Specificity between Wnt ligands and their
receptors

III. Identification and characterization of genes
required for somite segmentation

In vertebrates, Wnt proteins constitute a large family ofcysteine-rich secreted glycoproteins. Secreted Wnt
proteins exert their effects on neighboring cells bybinding to 7-transmembrane Frizzled (Fz) family
receptors, as well as to LDL receptor-related family
members, LRP5 and 6, which act as co-receptors. The Fz

3-1 Hairy/Enhancer of split protein, Her13.2,
links FGF signaling to cyclic gene expression in
the periodic segmentation of somites
The somites are subdivided from the anterior end of the26

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGYof split-related gene, her13.2, links FGF signaling to the
Notch-regulated oscillation machinery in zebrafish(Kawamura et al. 2005a). Expression of her13.2 isinduced by FGF-soaked beads and decreased by an FGF
signaling inhibitor. her13.2 is required for periodic
repression of the Notch-regulated genes, her1 and her7,
and for proper somite segmentation. Furthermore,
Her13.2 augments auto-repression of her1 in association
with Her1 protein. Therefore, FGF signaling appears tomaintain the oscillation machinery by supplying a binding
partner, Her13.2, for Her1.
This result also provides a novel molecular mechanism
whereby Notch signaling, which activates the expressionof her1 and her7, cooperates with FGF signaling. This
molecular mechanism may be widely employed inregulation of developmental process where Notch
signaling cooperates with FGF signaling.

unsegmented paraxial mesoderm, called the presomitic
mesoderm (PSM), and sequentially generated in ananterior to posterior direction in a rhythmic fashion at
regular spatiotemporal intervals. The molecularmechanism underlying the periodical formation ofsomites is coupled to an internal oscillator, referred to as
the “segmentation clock”, which has been evidenced by
the cyclic expression of genes in the PSM.
Most genes that exhibit a cyclic expression pattern in
the PSM are involved in the Notch signaling pathway. For
instance, various hairy/Enhancer of split (Espl)-relatedbHLH genes, including her1 and her7 in the zebrafish, aretranscriptional targets of the Notch signaling and are
expressed in a dynamic pattern of stripes across the PSM
in a posterior to anterior direction. These cyclic genes, aswell as other components of the Notch signaling pathway,
were shown to be required for the proper somite
segmentation in mice and zebrafish. Furthermore, thecyclic expression of her1, and her7 requires their own
activity, suggesting that a negative feedback loop
involving these genes is a critical component of theoscillation machinery.
The oscillating and anteriorly propagating wave ofgene expression, which is maintained in the posterior
PSM, becomes fixed to cause segmentation in the anterior
PSM. The activity gradient of fibroblast growth factor(FGF) is proposed to regulate the differentiation of PSM
cells along the anteroposterior axis from a state permitting
the oscillating gene expression to a state driving thesegmentation program. However, the molecularmechanism by which FGF signaling permits the cyclic
gene expression in the posterior PSM has been totally
unknown.

3-2 Groucho-associated transcriptional repressor
Ripply1 is required for proper transition fromthe presomitic mesoderm to somites
Prior to morphological segmentation, which is aprocess including inter-somitic boundary formation andmesenchymal-epithelialtransition,asegmental
pre-pattern, characterized by segmental gene expression,is established in the anterior PSM. The establishment of
the segmental pre-pattern in the anterior PSM has been
revealed to require a number of processes regulated bymany transcription factors and signaling molecules.
Concomitant with the transition from the anterior PSM tosomites, the characteristic gene expression in the PSM istranslated into the segmental structure. However, most of
the events accompanying the transition from the anterior
PSM to somites have remained obscure.

Figure 1. Expression of her13.2 in zebrafish development.
Expression of zebrafish her13.2 is first observed at shieldstage. During the stages when somites are generated, its
expression is localized in the posterior PSM and the tailbud(15som. and 24hr). This expression is induced by FGF
signaling to allow the cyclic gene expression in theposterior PSM (see Kawamura et al. 2005a).

Figure 2. The structure and expression of ripply family
proteins. Three ripply genes exist commonly in zebrafish,
human and mouse genomes. All Ripply proteins possess the
WRPW motifs and conserved sequences at the carboxyl
terminus (Ripply homology domain). A typical example ofripply gene (mouse ripply2) expression is shown. Ripply1
and ripply2 are specifically expressed in the PSM and/or
somites, suggesting that they function during somitogenesis(see Kawamura et al. 2005b).

To gain insight into the molecular mechanism
underlying the segmentation process, we sought toidentify genes specifically expressed in the PSM andtailbud by performing an in situ hybridization screening
using zebrafish embryos (Figure 1). We show that a gene
identified by this screening, Hes6-related hairy/Enhancer

We show that another gene identified by our in situ
hybridization screening, ripply1, encoding a nuclear
protein associated with the transcriptional co-repressor
Groucho, is required for this transition (Figure 2,

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

indicating that both Integrin-Fibronectin and Eph-Ephrin
systems function cooperatively in maintaining somite
boundaries.

Kawamura et al. 2005b). Zebrafish ripply1 is expressed in
the anterior PSM and in several newly formed somites.
Ripply1 represses specific gene expression in the PSM
through a Groucho-interacting motif. In ripply1-deficientembryos, somite boundaries do not form, thecharacteristic gene expression in the PSM is not properly
terminated, and the initially established rostrocaudal
polarity in the segmental unit is not maintained, whereas
paraxial mesoderm cells become differentiated. Thus,
ripply1 plays two key roles in the transition from the PSM
to somites: termination of the segmentation program in
the PSM and maintenance of the rostrocaudal polarity.

The results shown in “3-1” to “3-3” indicate that our
strategies are effective for identification of genes involvedin the somite segmentation process. We are further
searching for other genes involved in this process by boththe expression screening and the mutagenesis screening
methods. This systematic screening should reveal anotherinteresting mechanism underlying somite segmentation.

Publication List:
3-3 Integrin5-dependent Fibronectin assembly isrequired for maintenance of somite boundaries.

Original papers
Kassai, Y., Munne, P., Hotta, Y., Penttila, E., Kavanagh, K.,
Ohbayashi, N., Takada, S., Thesleff, I., Jemvall, J., andItoh, N. (2005). Regulation of mammalian tooth cusp
patterning by ectodin. Science 309, 2067-2070.
Kawamura, A.*, Koshida, S.*, Hijikata, H., Sakaguchi, T.,
Kondoh, H., and Takada, S. (2005a). (* These two authors
contributed
equallytothis work.)
Zebrafish
Hairy/Enhancer of split protein links FGF signaling tocyclic gene expression in the periodic segmentation ofsomites. Genes Dev 19, 1156-1161.
Kawamura, A., Koshida, S., Hijikata, H., Ohbayashi, A.,
Kondoh, H., and Takada, S. (2005b). Groucho-associatedtranscriptional repressor Ripply1 is required for propertransition from the presomitic mesoderm to somites. Dev.
Cell 9, 735-744.
Koshida, S., Kishimoto, Y., Ustumi, H., Shimizu, T.,
Furutani-Seiki, M., Kondoh, H., and Takada, S. (2005).
Integrin5-dependent fibronectin accumulation formaintenance of somite boundaries in zebrafish embryos.
Dev. Cell 8, 587-598.
Nakagiri, S., Murakami, A., Takada, S., Akiyama, T., andYonehara, S. (2005). Viral FLIP enhances Wnt signaling
downstream of stabilized beta-catenin, leading to control
of cell growth. Mol. Cell Biol. 25, 9249-9258.
Narita, T., Sasaoka, S., Udagawa, K., Ohyama, T., Wada, N.,
Nishimatsu, S. I., Takada, S., and Nohno, T. (2005).
Wnt10a is involved in AER formation during chick limb
development. Dev. Dyn. 233, 282-287.
Riccomagno, M., Takada, S., and Epstein, D. (2005). Wnt
dependent regulation of inner ear morphogenesis isbalanced by the opposing and supposing roles of Shh.
Genes Dev. 19, 1612-1623.
Shimizu, T., Wada, T., Muroyama, Y., Takada, S., andIkenaka, K. (2005). Wnt signaling controls the timing ofoligodendrocyte development in the spinal cord. Dev. Biol.
282, 397-410.
Takada, R., Hijikata, H., Kondoh, H., and Takada, S. (2005).
Analysis of combinatorial effects of Wnts and Frizzledson -catenin/armadillo stabilization and Dishevelled
phosphorylation. Genes Cells 10, 919-928.
Yamaguchi, Y., Ogura, S., Ishida, M., Karasawa, M., andTakada, S. (2005). Gene trap screening as an effective
approach for identification of Wnt-responsive genes in the

The segmental pre-pattern established in the anterior
PSM lead to morphological segmentation. Boundary
formation and epithelialization are crucial processes in themorphological segmentation of vertebrate somites, but themolecular mechanisms underlying these processes are notyet clearly understood.

Figure 3. The role of Fibronectin assembly in somite
boundary formation. Gradual accumulation of Fibronecin
proteins (red) at somite boundaries is required formaintenance of the boundaries and for polarization ofepithelial cells. Integrin5 (green) is required for thisaccumulation (see Koshida et al. 2005).

To gain insight into the mechanism underlying somite
development, we performed an ENU mutagenesis
screening of zebrafish, in addition to in situ hybridization
screening described above. We found that integrin5 andfibronectin were mutated in embryos showing defective
boundary formation in their anterior somites (Koshida etal. 2005). Fibronectin proteins accumulate at somite
boundaries in accordance with epithelialization of thesomites. Both Fibronectin accumulation and theepithelialization are dependent on itga5, which isexpressed in the most medial part of somites. While
somite boundaries are initially formed, but not maintained,
in the anterior trunk of the mutant embryos deficient ineither gene, their maintenance is defective at all axial
levels of embryos deficient for both of these genes.
Therefore, Integrin5-directed assembly of Fibronectin
appears critical for epithelialization and boundary
maintenance of somites (Figure 3). Furthermore, with anadditional deficiency in ephrin-B2a, the segmental defectin itga5 or fn mutant embryos is expanded posteriorly,

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

mouse embryo. Dev. Dyn. 233, 484-495.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

and that PGCs exhibit three different modes of migration
to reach the gonadal area. After appearance of PGCs atearly gastrulation stage around the animal pole, timelapse
analyses show that PGCs actively move between epiblasts
and hypoblasts towards the marginal region (Figure 1).
This movement is dependent on the activity of the chemoattractant, CXCR4 (the first mode). PGCs then move
medially towards the embryonic axis with lateral somatic
cells undergoing convergent movement (the secondmode).
Afterthebilateralalignmentof
PGCs along theembryonic axis,
thePGCs
actively resume
directional
Figure 1. PGCs (asterisk)
movement
migratebetweenepiblasts(square)andhypoblasts
towards
the(triangles) during epiboly.
regionofagonadal field in the posterior lateral plate mesoderm.
SDF-1a and HMGCoA reductase are independently
involved in this posterior migration (the third mode)(Kurokawa et al. 2006).

LABORATORY OF MOLECULAR GENETICS
FOR REPRODUCTION
Associate Professor:
NIBB Research Fellow:
Visiting Scientists:

Secretary:

TANAKA, Minoru
SAITO, Daisuke
AOKI, Yumiko
NAKAMURA, Shuhei
ARITA, Kaori
KUROKAWA, Hiromi
MATSUDA, Mitsuko

Laboratory Scope
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
primordial germ cells to form the indifferent gonad.
Once indifferent gonad is formed in the gonadal area, sex
determination gene is expressed in the gonadal mesoderm
and organogenesis of the gonads (sex differentiation)
initiates in mice.
Since the sex determination gene in mice was identified
in 1990, much research has been intensively focused on
the testis development. When considering, however, thedifferentiation of the precursor cells determining sex, theinteraction of somatic cells and germ cells, thedifferentiation of primordial germ cells (PGCs) to germ
cells having the capability to undergo meiosis, and so on,
there remain many important and fascinating problems tobe solved.
Our laboratory aims to reveal the molecularmechanisms of the formation of gonads and the sex
differentiation, with an emphasis on visualizing specificcell lineages in living medaka embryos and by applying
molecular genetics to medaka embryos.

II. Origin of gonadal mesoderm
Much research has focused on the mechanisms of sex
differentiation. However, the origin and the lineages of
the gonadal precursor cells have remained to beelucidated in vertebrates.
We have successfully demonstrated the presence of the
gonadal field where the precursors of gonadal mesoderm
arise. This region is located at the most posterior end of
sdf-1a expression domain. It is known that sdf-1afunctions as a guidance cue for PGCs to migrate to the
gonadal area and the expression becomes restricted to theposterior lateral mesoderm. Our result indicates that themost posterior end of sdf-1a expression domain is theplace that coordinates PGC migration and development of
the gonadal mesoderm. We further demonstrated that

I. Analysis of movement of primordial germ cells by
timelapse movies
One of the advantages using medaka is that theembryos are transparent and the development can be seenfrom outside. This encouraged us to generate transgenic
medaka exhibiting GFP exclusively in germline andsuccesfully
establishedthetrangenic
lines(olvas-transgenic medaka). On the other hand, we havebeen screening medaka genes expressing in germline andfound that some of the 3’UTRs are critical for
maintenance and translation of their own transcripts
specifically in germline. This translational mechanism
also allows to visualize the germline by injecting
GFP-3’UTR chimeric RNA into fertilized eggs.
With the two means mentioned above, we have beenmonitoring the origin and the mode of movement of
medaka primordial germ cells (PGCs) during
embryogenesis. We revealed that presumptive PGCs are
already present by early gastrulation stage by analyzing
the expression of one of the germline-expressing genes

Figure 2. The two different precursors of gonadal
mesoderm arise from the gonadal field (orange). sdf-1a
expression domain (blue). PGCs (green).

29

NATIONAL INSTITUTE FOR BASIC BIOLOGYDEVELOPMENTAL BIOLOGY

two different populations with distinct gene expression
are spatially organized from the gonadal field along theembryonic axis and are specified before the gonadal
primordium forms (before the sex is determined)(Nakamura et al. 2006) (Figure 2).
Generally, in vertebrates, the presence andcharacteristics of gonadal precursors before sex
determination are poorly understood. Cellular interaction
among the different cell precursors, which brings aboutdevelopment of the supporting cells such as Sertoli cells
and granulosa cells, also remains to be elucidated.
We have generated medaka embryos that completely
lack germ cells in the gonadal primordium by impairmentof PGC migration and have confirmed the biased ratio of
phenotypic sex in the germ cell-less adult medaka. The
morphology of the gonad in the germ cell-less medaka
exhibits the appearance of neither testis nor ovary. We
are currently investigating the involvement of germ cells
in the development of the gonadal supporting cells.

Medaka is a small vertebrate and produces next
generations in 3 months after hatching. These
characteristics are suitable for applying molecular
genetics to this small animal.
In collaboration with ERATO Kondoh differentiation
signaling project, we have been screening mutants
affecting the development of primordial germ cells andthe formation of gonads. The screening has been
performed in such a way that particular attention is paid tothe presence, the number or the distribution pattern ofgerm cells at a somitogenesis stage and at ten days post
hatching (10 dph). Nine mutants (19 alleles) and twelve
mutants (14 alleles) were identified for PGCs and gonads,
respectively.
One mutant, totoro, is very interesting in that thephenotype has not been described before in other animals.
totoro has a swollen large abdomen filled with a gonad.
Our analyses show that the phenotype is semidominantly
inherited. Gonads are sex-reversed in genetic males andfullfilled with numerous oocytes regardless of its genetic
sex. The positional cloning is ongoing with the ERATO
project and three genes are identified as the possible
candidate genes responsible for the phenotype.

III. Generation of transgenic medaka visualizing
different cell lineages of the supporting cells in
the gonad
To clarify how many kinds of supporting cells appear
during the formation of gonadal primordium and the sex
differentiation, we are systematically identifying thedifferent types of supporting cells by in situ hybridizationand generating the transgenic medaka that visualizes eachtype of the supporting cells. Currently we are trying tomonitor different cells with GFP fluorescence driven by
the regulatory regions of several different genes. One
line has been established as a transgenic line that allows
us to observe somatic cells in gonadal primordium (Figure
3).
The transgenic medaka suggests that several

Figure 4. Blue staining shows PGCs in the gonad. Left:zenzai mutant that cannot maintain gerrn cells. Middle:
wild type. Right: totoro mutant that shows
overproliferation of germ cells.

Another mutant, zenzai, is a good contrast with thetotoro mutant and is unique in that germ cells are notmaintained in the gonad (Figure 4). Inheritance of thephenotype indicates that the allele is recessive. The
characterization and the positional cloning are also inprogress. We are narrowing down the genomic region
responsible for zenzai phenotype to approximately 110
Kb.
We are also characterizing other mutants in anothercategory, irregular distribution of germ cells in gonads.
These mutants include hadare, mizore, hyou and arare.

Figure 3. Somatic cells (green) surround germ cells(red) in the gonadal primordium (from in vivo
timelapse movie).

Publication List:

different types of supporting cells have already beendifferentiated before sexual differentiation (organogenesisof ovary or testis) occurs.
The attempt to monitor the process of development ofeach lineage has also been made in living embryos andlarva by timelapse movies. In order to solve thedifficulties in visualizing the cells located in the deep
positions in the embryos and larva, the confocal
microscopy and SPIM have been applied to the transgenic
embryos and larva. This attempt is still in progress incollaboration with Jochen Wittbrodt Lab. in EMBL.

Original paper
Hano, T., Oshima, Y., Oe, T., Kinoshita, M., Tanaka, M.,
Wakamatsu, Y., Ozato, K., and Honjo, H. (2005).
Quantitiative Bio-imaging analysis for evaluation ofsexual differentiation in germ cells of olvas-GFP/STII YI
medaka (Oryzias latipes) nanoinjected in ovo withethinylestradiol. Environ. Toxicol. Chem. 24, 70-77.

IV. Analysis of mutant medaka affecting formationof gonad30

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

we have been devoting our efforts to searching formolecules with asymmetrical distribution in theembryonic chick retina, and to characterization of theirroles in the topographic retinotectal projection.
We performed a large-scale screening using differential
hybridization and restriction landmark cDNA scanning(RLCS) on the embryonic day 8 (E8) chick retina.
RLCS is a cDNA display system, in which a large numberof cDNA species are displayed as two-dimensional spots
with intensities reflecting their expression levels in
mRNA. We detected ~200 spots that gave different signal
intensities between the nasal and temporal retinas or
between the dorsal and ventral retinas. The asymmetricexpression of each gene was verified by Northern blotting
and in situ hybridization. By subsequent analyses using
molecular cloning, DNA sequencing, and databasesearching, 33 asymmetric molecules along thenasotemporal (N-T) axis and 20 along the dorsoventral(D-V) axis were finally identified.
Their expression profiles revealed by in situ
hybridization are highly diverse and individual.
Moreover, some of them begin to be expressed in theretina from the early developmental stages (peaking
before E6), suggesting that they are implicated in theestablishment (and maintenance) of regional specificity inthe developing retina. We have already described somenovel molecules in published papers, but the study tounderstand the functions of the remaining molecules andthe overall hierarchical order to establish the regional
specificity in the retina is currently in progress.

DIVISION OF MOLECULAR NEUROBIOLOGY
Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:

Visiting Scientist:
Technical Assistants:

Secretary:

NODA, Masaharu
SHINTANI, Takafumi
SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
TAKAHASHI, Hiroo
FUJIKAWA, Akihiro
SUZUKI, Ryoko
FUKADA, Masahide
NAKAMURA, Takahiro
YAMAMOTO, Yasunori
IHARA, Masaru
ETANI, Kazuma
SHIMIZU, Hidetada
YONEHARA, Keisuke
NAKAMURA, Kayo
CHOW, Pak Hong Jeremy
TAMURA, Hiroshi
MIZOGUCHI, Masae
GOTOH, Megumi
YAMADA, Kaoru
AYABE, Yuko
KATAYAMA, Tomomi
HATTORI, Nobuko
KODAMA, Akiko

We have been studying the molecular and cellularmechanisms underlying the development of the vertebrate
central nervous system, mainly using the visual system of
the chick and mouse. This research covers many
developmental events including the patterning of theretina, axonal navigation, branching and targeting,
synapse formation, refinement and plasticity, and axonal
regeneration. The scope of our interests also encompasses
mechanisms for various functions of the mature brain,including sensation, emotion, behavior, learning andmemory.

II. Mechanisms for the 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 most anterior
part of the tectum at day E6 and advance over its surface
in the posterior direction. These retinal axons form thestratum opticum (SO), which covers the entire tectum at
E12.
At the onset, the chick retinotectal projection has avague topographic order, and axonal sprouting begin
predominantly at the vicinity and towards the site ofnormal terminal zone (TZ). In all vertebrates studied,however, the developing axons make trial branches, manyof which are not entirely within the retinotopic area, andelimination of ectopic branches and elaboration of
appropriate branches is shaped in an activity-dependent
manner (Figure 1).
We have already identified a battery of molecules in
the region-specific molecules in the retina (peaking afterE8) that show abnormal axonal targeting, branching or
synaptic arborization, when their expression was
experimentally enhanced or suppressed in vivo. We
expect that molecular mechanisms underlying thebranching, pruning, and synapse formation of retinal
axons shall be revealed through studies on thesemolecules.

I. Mechanisms for the regional specification in thedeveloping retina
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, the temporal(posterior) retina is connected to the rostral (anterior) partof the contralateral optic tectum, the nasal (anterior) retina
to the caudal (posterior) tectum, and likewise the dorsal
and ventral retina to the ventral and dorsal tectum,
respectively. Thus, images received by the retina are
precisely projected onto the tectum in a reversed manner.
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. In the past ten years,

31

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

hippocampus.
We then examined phenotypes of mutant mice
deficient
in
Ptprz
using
electrophysiological,
pharmacological, and behavioral approaches. Mutant
mice exhibit enhanced long-term potentiation (LTP) in
the CA1 region of hippocampal slices and impaired
spatial learning abilities in an age-dependent manner(Figure 2): Young adult (<10 weeks old) mutant miceshow normal LTP and learning abilities in the Morris
water maze task, whereas adult (>13 weeks old) mutant
mice exhibit enhanced LTP and impairment in the task.
The enhanced LTP is specifically canceled out bypharmacological inhibition of Rho-associated kinase(ROCK), a major downstream effector of Rho. Thesefindings suggest that the lack of Ptprz leads to aberrant
activation of ROCK and resultantly to enhanced LTP in
the slice and learning impairments in the animal.

Figure 1. Development of retinotectal projection. A,
Developmental processes of the topographic projection of
dorsal retinal axons.
TZ, terminal zone.
B,
Layer-specific synapse formation of retinal axons in thetectum.

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

tyrosine

Protein tyrosine phosphorylation plays crucial roles invarious biological aspects including brain development
and brain functions. PTP/RPTP/Ptprz, a nervous
system-rich receptor-type PTP, is expressed as achondroitin sulfate proteoglycan in the brain from theearly developmental stage to adulthood in neurons as wellas astrocytes. This suggests that this molecule plays
variegated roles in the brain development and brain
function.
Ptprz binds various cell adhesion molecules (Nr-CAM,
L1, contactin, NCAM and TAG-1) and extracellularmatrix molecules (tenascin-C/R). In addition to thesemolecules, we revealed that Ptprz binds pleiotrophin(PTN)/HB-GAM and midkine (MK), closely related
heparin-binding growth factors which share many
biological activities. The chondroitin sulfate portion of
Ptprz is essential for the high affinity binding to thesegrowth factors.
We recently developed a genetic method named “yeast
substrate-trapping system” to screen for substrates of
PTPs. This method carries the advantage that not only
substrates but also continuously interacting molecules arecapable of being identified at the same time. By thisscreening, we identified Rho-family GTPase related
molecules like p190 RhoGAP, Git1 (ArfGAP) and Pist as
substrates, and PDZ-domain containing molecules like
PSD-95 as continuously interacting proteins. The
expression profile and interacting molecules suggested
that Ptprz is implicated in synaptic plasticity in the

Figure 2. Age-dependent impairment of spatial learningand enhancement of hippocampal long-term potentiation(LTP) in Ptprz-deficient mice. A and C, Hidden-platform
task of Morris water maze test. B and D, The time courseof LTP in CA1 region of hippocampal slices. Tetanic
stimulation (100 Hz for 1 s) was applied at 0 min.
Ptprz-deficient mice exhibited a slower rate of spatial
learning specifically at the age of >13 weeks, but not at theage of <10 weeks, which was in accordance with theage-dependent enhancement of LTP.

IV. Mechanisms for Na+-level sensing in the brain
and body fluid homeostasis
Dehydration causes an increase in the sodium (Na)concentration and osmolarity of body fluids in mammals.
For Na homeostasis of the body, controls of Na 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 has not been fully elucidated.
We reported previously that the Nax channel ispreferentially expressed in the circumventricular organs

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NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

solution. After sodium-image recordings, cells were fixedand stained with anti-Nax (A-c), anti-GLAST (A-f) or
anti-GFAP (A-i) antibodies. All the sodium-sensitive cells
are immunopositive for Nax, GLAST and GFAP, indicatingthat these cells are glial cells. Small cells bearing shortneurites (arrows) in A-c, -f and -i appear to be neurons,
which are all insensitive to the extracellular sodiumincrease. Scale bar: 20 μm. B, Quantified intracellularsodium-ion concentrations before (open bars) and after(filled bars) the stimulation in Nax-positive (+) or -negative(-) cells, in GLAST-positive (+) or -negative (-) cells, andin GFAP-positive (+) or -negative (-) cells. Data represent
mean and SE (n=20, each).

(CVOs) in the brain, and that Nax knock-out mice do notstop ingestion of saline under dehydrated conditions.
Subsequently, we demonstrated that Nax is a
Na-level-sensitive Na channel. Last year, we showed thatthe subfornical organ (SFO), one of the CVOs, is thecenter of the control of salt-intake behavior in the brain,
where the Na-level-sensitive Nax channel is involved insensing the physiological increase in the Na level of body
fluids.
This year, we dissected the subcellular localization of
Nax. Double-immunostaining and immunoelectron
microscopic analyses revealed that Nax is exclusively
localized to perineuronal lamellate processes extended
from ependymal cells and astrocytes in the organs. In
addition, glial cells isolated from the SFO were sensitive
to an increase in the extracellular sodium level, as
analyzed by an ion-imaging method (Figure 3). Glial cells
bearing Nax channel are thus the first to sense aphysiological increase in the level of sodium in the body
fluid, and regulate the neural activity of the CVOs byenveloping neurons.

Publication List:
Original papers
Fukada, M., Kawachi, H., Fujikawa, A., and Noda, M.
(2005). Yeast substrate-trapping system for isolating
substrates of protein tyrosine phosphatases: Isolation ofsubstrates for protein tyrosine phosphatase receptor type z.
Methods 35, 54-63.
Niisato, K., Fujikawa, A., Komai, S., Shintani, T., Watanabe,E., Sakaguchi, G., Katsuura, G., Manabe, T., and Noda, M.
(2005). Age-dependent enhancement of hippocampal
long-term potentiation and impairment of spatial learning
through the Rho-associated kinase pathway in protein
tyrosine phosphatase receptor type Z-deficient mice. J.
Neurosci. 25, 1081-1088.
Noda, M., and Hiyama, T.Y. (2005). Sodium-level-sensitive
sodium channel and salt-intake behavior. Chem. Senses
30 (Supple. 1), i44-i45.
Watanabe, E., Hiyama, T.Y., Shimizu, H., Kodama, R.,
Hayashi, N., Miyata, S., Yanagawa, Y., Obata, K., andNoda, M. (2005). Sodium-level-sensitive sodium channel
Nax is expressed in glial laminate processes in the sensory
circumventricular organs. Am. J. Physiol. – Regul.
Integr. Comp. Physiol. Epub ahead of print, PMID:
16223844.

A

B

Review articles
Fukada, M., and Noda, M. (2005). Yeast substrate-trapping
system for isolating substrates of protein tyrosine
phosphatases. Methods in Mol. Biol., in press.
Noda, M. (2005). The subfornical organ, a specialized
sodium channel, and the sensing of sodium levels in thebrain. The Neuroscientist, in press.

Figure 3. A, Glial cells isolated from the SFO express Nax
channel and show sensitivity to the extracellular sodium
level. Pseudocolor images of the intracellular sodium
concentration ([Na+]i) of SFO cells in the control solution
and in the high sodium solution. A-b, -e, -h are images 20
min after stimulation with the hypertonic 170 mM [Na+]33

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGYii) The other gene that showed marked difference within
the neocortex, is gdf7, a member of BMP/TGF- family,
which is specifically expressed in the motor cortex of theAfrican green monkey (Watakabe et al. J. Neurochem. 76,
1455-1464, 2001).
iii) Rbp (retinol-binding protein) is preferentiallyexpressed in association and higher areas in the neocortex(Komatsu et al. 2005). Rbp also shows characteristicfeatures. a) Its expression is high in sensory associationand higher association areas and limbic areas, but low inthe primary sensory areas. Expression is complementary
to that of occ1 and to parvalbumin immunoreactivity(PV-IR) in primary sensory areas. b) In early sensory
pathways, the expression is limited to superficial layers
only (in particular, layer 2). With progression into higher
sensory areas, the expression is expanded into layers 3
and then 5. c) In higher-order association areas, Rbp isexpressed throughout all layers except layer 4. d) This
characteristic distribution of Rbp is mainly formed during
postnatal development. Rbp probably regulates theconcentration of retinoic acid (RA) by the delivery of
retinol, which is converted into RA in cells. Although therole of RA in the mature brain is not yet known, thecharacteristic expression of Rbp within association areas
may provide a clue to the molecular basis of the formationand function of the association areas.
In this year [2005], we also reported on the features ofocc1 expression in mammalian neocortices. Althoughocc1 is abundantly expressed in the primary visual cortex,
we found that there is another mode of expression of
occ1: sparsely expressed throughout the entire neocortex.
Therefore, there are two different modes of occ1
expression (Figure1). One mode is the expression in
excitatory cells in the primary sonsory areas, particularly
in visual cortex, which receive thalamocorticalprojections. The other mode is the expression ininhibitory GABAergic cells that are spread throughout alllayers of entire neocortex. We found that among
inhibitory neurons only Parvalbumin (PV) positive
neurons expressed occ1.

DIVISION OF BRAIN BIOLOGY
Professor:
Research Associates:
NIBB Research Fellow:
Technical Staff:
Postdoctoral Fellows:
Graduate Students:

Technical Assistants:

YAMAMORI, Tetsuo
KOMINE, Yuriko
WATAKABE, Akiya
SI, Xiaohui
OOSAWA, Sonoko
KOMATSU, Yusuke
TAKAHATA, Tohru
BOSCH, Miquel
TAKAJI, Masashi
SASAKI, Kazuya
HIROKAWA, Junya
NAKAMURA, Tohru
TOITA, Shigeko
ISHIKAWA, Takako
HAYASHI, Hitomi
MIKI, Kazuhiko
IMAI, Akiko
KUSAMA, Shunsuke

Our research is focused on understanding the formationand evolution of the brain and on the mechanisms
underlying memory.Our approach is threefold. Firstly, weare studying the genes that are expressed in specific areasof the primate neocortex. We have obtained genes thatshow marked differences within primate neocortical areas.
Secondly, we are studying the mechanisms underlying
learning behaviors by examining gene expression. Thirdly,
we are studying a new transcriptional factor in which bothsense and antisense RNA are expressed in the brain.

I. Genes expressed in specific areas of the neocortex
The neocortex is most remarkably evolved in the
primate and plays the major role in higher brain functions.
It is divided into distinct functional and anatomical areasand it has been a matter of debate what extent areas of the
neocortex are genetically and environmentally determined.
It is also puzzling why, during the evolution of mammals,
the neocortex was markedly expanded while the numberof the genes in the mammal was little changed. In order toelucidate these questions, we studied gene expression
within different areas of the neocortex. In the last several
years, we reported the following findings.
1) Examining 1088 genes by microarray analysis, mostgenes showed only less two fold difference in theirexpressions among the three neocortical (frontal, motorand visual) areas. Only one gene showed more than threefold difference and another one was between two andthree fold difference within the three areas (Watakabe etal. Mol. Brain Res. 88, 74-82, 2001). These results
suggest that the genes that are expressed among thedifferent areas of the human neocortex are very similar.
However, the question remained whether there are any
genes that show marked neocortical area difference.
2) In order to answer this question, we have employed
differential display methods and found three genes thatindicated area specific expressions.
i) One gene, designated occ1, is specifically expressed in
the occipital cortex, particularly in V1 area, in the primate
brain. We also demonstrated that occ1 expression wasmarkedly increased postnataly in V1.

Figure 1. Expression pattern of occ1. occ1 RNA ismarkedly expressed in the visual cortex and expressed inboth excitatory (A:vGlut positive) and inhibitory (B:GAD)
neurons (Takahata et al. Cerebral Cortex, 2005 Sep 8;[Epub ahead of print]).

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NATIONAL INSTITUTE FOR BASIC BIOLOGYBRAIN BIOLOGY

in rats. These results suggest that this new modified AVD
system can be used to explore the molecular and cellularmechanisms underlying multisensory processing in rats
(Sakata et al. Exp. Brain Res.,159, 409-417, 2004).

As previously reported, occ1 expression in the primaryvisual cortex is activity-dependent. In monkeys whoseeyes are monocularly deprived by TTX injection, the occ1
expression is markedly decreased in the monocularly
deprived column. We then examined which mode of occ1
expression is activity-dependent. occ1 expression in
excitatory neurons is decreased in the monocularly
deprived column but not in inhibitory interneurons. Since
primate brains have distinct features from othermammalian brains, we thought the expression profiles inprimates might be different from those of other mammals.
The most striking difference was observed in theexpression in excitatory neurons in primaly sensory areas,
particularly in the visual cortex. No clear border wasobserved in V1 and V2 in mammals (mice, rabbits,
ferrets) other than those of macaques and marmosets.
These results suggest the occ1 expression in excitatoryneurons was aquired in a specific step of evolution froman ancestor to the primate.

Figure 2. Properties of 7-12 Hz oscillations.(A) Schedule
for experiments under task conditions of EEG and EMG
recordings. (B) Illustration of one-trial schedule insequential nose-poking task. In this task, when rats were
responded to a predetermined sequence of the direction oflights (e.g., L-R-C), they were rewarded by a food pellet.
(C) Top panel: an EEG signal from a frontal region during
7-12 Hz oscillations. Middle panel: an interpolated
spectrogram of the EEG signal. These oscillations consist ofbasic frequences (7-12 Hz) with their harmonics. An arrow
indicates slow-wave activities (<4Hz) observed in the initialstage. Bottom: a stimultaneous EMG signal. (These data areshown in Sakata et al. Neuroscience 134, 1099-1111, 2005)

II. Gene expression under a declarative and anon-declarative memory
In order to study informational processing underlying
the declarative and non-declarative memory at molecularand cellular levels in the brain, we established two
behavioral systems.
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 audio cue (ahigh tone or low tone) or a visual cue (a light from theright or the left) to obtain a food pellet. We found that thevisual and audio tasks enhanced the specific expression ofc-Fos in the visual and audio cortices, respectively.
Among the early visual and auditory pathways examined,
c-Fos was specifically induced in the cortices but not in
the earlier pathways, suggesting the neural modulation of
the neocortex depending on the types of the tasks.
Interestingly, the task-dependent Fos expression was only
observed in excitatory neurons in the relevant sensory
cortices.
Although this AVD task system is quite powerful toanalyze the problem described above and useful forstudying underlying molecular and cellular mechanisms
because of the advantages of using rodents, one concern
was that the auditory stimuli and visual stimuli were indifferent positions. Thus, we could not exclude thepossibility that the difference between the auditory task
and the visual task may not completely depend on themodality (visual Vs auditory) difference.
We wanted to solve this problem by placing auditory
and visual stimuli in the same position. We also use
nose-poking to measure the reaction time in which a rat
responds to stimuli. By using this behavioral system, wewere able to confirm amodal recognition of space whichmeans that a rat can respond to a different modality(visual or auditory) if the stimuli are in the same position
as previously reported in other systems. We also
confirmed multisensory enhancement is indeed observed

Various oscillations are observed depending on
brain-states. Spike wave complexes (Sws), 7-12 Hz
cortical oscillations with harmonics in awake but
immobile rats, have been widely regarded as a model ofparoxysmal 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 the sleep-waking
cycle. To elucidate behavioral contexts to induce SWs
and temporal relations between SWs and neuronalensemble activities, we recorded local field potentials(LFP) and multi-unit activities (MUAs) in the medial
prefrontal cortex and electroencephalogram (EEG) in thebilateral regions of rats. 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-specific oscillations rather than pathologicalactivities. We also observed that MUAs were organized
into phase-locked patterns in cycles of these oscillations.
MUAs recorded from electrodes apart to each other weresynchronized during SWs (Sakata et al. 2005a).
2) The other task we developed is a wheel runningsystem in which a water-deprived mouse is made to run toobtain water because the wheel with the pegs is turning
tothe other direction (Kitsukawa et al. SFN Meeting,2002). The pegs can be changed with [?] various patterns
as desired. The task required of the mouse thus can be
regarded as representing a procedural learning. We
examined various areas of mouse brain following

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NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

Publication List:changes to the peg pattern. Among the areas examined,
we found marked c-Fos expression in the striatum,
cerebral cortex. The striatum, which is composed ofprojection neurons and several distinguished types ofinterneurons, is known to play an important role inreward-based learning. The characterization of thesesubtypes of interneurons has been progressed. However,their roles in behavioral tasks have remained obscure. We
are currently examining the altered behavior under thepharmacological treatments that affect the metabolism of
the interneurons in the striatum.

Original papers
Komatsu, Y., Watakabe, A., Hashikawa, T., Tochitani, S.,
and Yamamori, T. (2005). Retinol-binding protein geneis highly expressed in higher-order association areas of the
primate neocortex. Cereb. Cortex 15, 96-108.
Komine, Y., Nakamura, K., Katsuki, M., and Yamamori, T.
(2005). Novel transcription factor zfh-5 is negatively
regulated by its own antisense RNA in mouse brain. Mol.
Cell Neurosci. (online publication)
Sakata, S., Komatasu, Y., and Yamamori, T. (2005b). Local
design principles of mammalian cortical networks.
Neurosci. Res. 51, 309-315.
Sakata, S., Yamamori, T., and Sakurai, Y. (2005a). 7-12 Hz
cortical oscillations: Behavioral context and dynamics ofprefrontal neuronal ensembles. Neuroscience 134,
1099-1111.
Takahata, T., Komatsu, Y., Watakabe, A., Hashikawa, T.,
Tochitani,
S.,andYamamori,T.
(2005).
Activity-dependent expression of occ1 in excitatoryneurons is a characteristic feature of the primate visualcortex. Cereb. Cortex (online publication)

III. A transcriptional factor (zfh-5) both of which
sense and antisense RNA are expressed in thebrain
Antisense RNAs have recently been reported to be
expressed much more than previously thought. However,
the roles of antisense RNAs have been widely unknown,
particularly in the brain. We found that the antisense
strand of zfh-5, the gene for a novel transcription factor
that we identified, was also transcribed in the developing
mouse brain, in a manner complementary to theexpression of zfh-5 mRNA (Figure3; Komine et al. 2005).
Using gene-targeting approach, we showed that theexpression of zfh-5 mRNA was negatively regulated bythis antisense RNA. Our observations suggest that the
suppression mechanism by the zfh-5 antisense RNA
differs from those by previously known antisense RNAs.

Review article
Watakabe, A., Komatsu, W., Nawa, H., and Yamamori, T.
(2005). Gene expression profiling of primate neocortex:
molecular neuroanatomy of cortical areas. Genes Brain
Behav. in press.

Figure 3. zfh-5 expression in ASC mutant. (A) A pair of
wild-type (+/+) and homozygous (ASC/ASC: deleted
in antisence control region) littermates was used. In theASC/ASC embryo (E16.5), the antisense RNA wasalmost depleted and the amount of the sense RNA
increased in various regions of the brain compared withthe same age of +/+ embryo. Arrows: pontine nuclei. (B)In the ASC/ASC mutant mice (P8), the ectopic
expression of the sense RNA was prominent. (C) Two
sets of RNAs (each lane contains 1 μg of polyA+ RNA)
from wild-type (+/+), hetero- (+/) and homozygous(/) mice were electrophoresed and each set was
hybridized with a strand-specific probe. As the amount of
the antisense RNA decreased (+/+>+/>/), that of thesense RNA including the full-length mRNA (indicated byarrowheads) increased (D) The expression patterns ofsense and antisense RNA in RMSs (Rostal migratony
stream) (The figure is shown in Komine et al. Mol. Cell
Neurosci. 2005 Oct 27; [Epub ahead of print])

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NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

pattern differed depending on brain subarea examinedeven in male mice. These suggested the species differenceof the transcriptional regulation of the ER. Nonetheless,sex-dependent difference was specifically observed in the
BNST, suggesting that the BNST is an area important forthe sexual differentiation of the brain in rats and mice.

DIVISION OF BEHAVIORAL BIOLOGY (ADJUNCT)
Professor (Adjunct):
Associate Professor (Adjunct):
Research Associate:
NIBB Research Fellow:
Visiting Scientists:

MORI, Yuji
TSUKAMURA, Hiroko
IMAMURA, Takuya
IKEMURA, Ryota
TAKEUCHI, Motoki
SATO, Hiroaki

In mammals, several social behaviors are dependent onsex. The sex-dependent patterns of behaviors must be
acquired through highly irreversible processes during
development. We hypothesize that the long-term effects
of sex steroids at the perinatal stage on behaviors after
puberty are somehow marked at the genome level. We are
currently investigating the epigenetic status of the criticalbrain subareas responsible for the sex difference of the
behaviors.

I. DNA methylation of nuclear receptors
In mammals, DNA methylation, mainly occurring on
CG dinucleotides, is a fundamental mechanism thatdifferentiates the gene expression pattern in respective
cell. DNA methylation is a restraint of the pluripotency
because once the pattern is established during
development it is maintained through cell division. On theother hand, for example, some fish that contain much lessmethylation activity are found to easily and reversibly
change the sex status according to the environmental
context. In rodents, endocrine disturbance at the fetal
and/or postnatal stage irreversibly changes the behaviors
such as the lordosis (in females) and the mounting (inmales) after the pubertal stage 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
established through epigenetic processes. We have foundthe sex-dependent DNA methylation pattern of steroid
receptor genes (estrogen receptor , ER; androgen
receptor, AR; progesterone receptor, PR) in the male andfemale rat brain subareas. In this year of 2005, we reportherein the DNA methylation analyses of the steroid
receptor genes and identification of the naturally
occurring antisense RNAs as potent and specificepigenetic regulators in rats and mice.

1-1

Figure 1. Detection of ER-as, AR-as and PR-as. Eachupper panel shows the genomic structure of ER, AR, or
PR. Arrows denote the primers used for the PCR
amplification. Each lower panel shows the strand-specific
RT-PCR. Note that specific bands were observed whensense primers were used for RT reaction.

1-2 Identification of naturally occurring antisenseRNAs
The number of mammalian non-coding RNA genes is
rapidly expanding. We found naturally occurring
antisense RNAs for rat and mouse ER, AR, and PR,
tentatively named ER-as, AR-as, and PR-as,
respectively (Figure 1). Neither of the antisense RNAs
showed obvious open reading frames, and possible aminoacid motifs, raising the possibility that these function
without translation. Each RNA overlapped with therespective promoter region including the sex-dependent
differentially methylated region. In adult tissues,
expression patterns were tissue- and/or sex-specific. For
example, ER-as and PR-as were predominantly
expressed in the ovary. In the testis, these expressions
were under the detectable level. In contrast, AR-as was
highly expressed in the testis, but not in the ovary. It was
to be noted that expressions in the brain were also area-

Sex-dependent DNA methylation

In general, the binding affinity of the transcription
factors is reduced by hypermethylation that are frequently
observed on promoter region of either ER, AR or PR intumor cells. We analyzed the DNA methylation status of
their 5’-flanking regions in the rat using the bisulfite
sequencing method. DNA methylation patterns differeddepending on brain subarea. For ER, no differenceswere found within male rat brain subareas examined. In
contrast, hypermethylation was specifically observed in
the bed nucleus of the stria terminalis (BNST) of female
rats. The sex-dependent DNA methylation was also foundin the mouse BNST. It was to be noted that methylation

37

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

and/or sex-dependent. These structural features led us toinvestigate the possible involvement of these non-coding
RNAs on the establishment of the specific DNA
methylation pattern.
We used a rat pheochromocytoma cell line, PC12, thatdifferentiates in response to NGF, as a model system(Figure 2). Most of the CG sites located on the
sex-dependent differentially methylated region of ER
became methylated after the NGF treatment, confirmingthat the examined region was methylatable according to
the cellular status. We introduced an expression vector
driving a fragment of rat ER into PC12 cells by transienttransfection. Overexpression of a fragment of ER-as
containing the differentially methylated region
dramatically diminished genomic methylation at thecorresponding CG sites. This effect was gene-specific,
because a PR-as fragment did not reverse the DNA
methylation of ER. We are currently investigating theexpression profile of the novel antisense RNAs and theirpotential use for DNA methylation manipulation at theperinatal stage, that is thought critical period of sexual
differentiation of the brain.

cysteine-to-arginine substitution caused by a single
nucleotide polymorphism in the canine monoamineoxidase B gene. J. Vet. Med. Sci. 67, 199-201.
Imamura, T., Kerjean, A., Heams, T., Kupiec, J. J., Thenevin,C., and Paldi, A. (2005). Dynamic CpG and non-CpG
methylation of the Peg1/Mest gene in the mouse oocyte
and preimplantation embryo. J. Biol. Chem. 280,
20171-20175.
Inagawa, K., Seki, S., Bannai, M., Takeuchi, Y., Mori, Y.,
and Takahashi, M. (2005). Alleviative effects of
gamma-aminobutyric acid (GABA) on behavioral
abnormalities in aged dogs. J. Vet. Med. Sci. 67,
1063-1066.
Kanari, K., Kikusui, T., Takeuchi, Y., and Mori, Y. (2005).
Multidimensional structure of anxiety-related behavior inearly-weaned rats. Behav. Brain Res. 156, 45-52.
Kikusui, T., Isaka, Y., and Mori, Y. (2005). Early weaning
deprives mouse pups of maternal care and decreases theirmaternal behavior in adulthood. Behav. Brain Res. 162,
200-206.
Kinoshita, M., Tsukamura, H., Adachi, S., Matsui, H.,
Uenoyama, Y., Iwata, K., Yamada, S., Inoue, K., Ohtaki,T., Matsumoto, H., and Maeda, K. (2005). Involvement ofcentral metastin in the regulation of preovulatory
luteinizing hormone surge and estrous cyclicity in female
rats. Endocrinology 146, 4431-4436.
Kiyokawa, Y., Kikusui, T., Takeuchi, Y., and Mori, Y.
(2005). Alarm pheromone that aggravates stress-induced
hyperthermia is soluble in water. Chem. Senses 30,
513-519.
Kiyokawa, Y., Kikusui, T., Takeuchi, Y., and Mori, Y.
(2005). Mapping the neural circuit activated by alarm
pheromone perception by c-Fos immunohistochemistry.
Brain Res. 1043, 145-154.
Matsuyama, S., Ohkura, S., Sakurai, K., Tsukamura, H.,
Maeda, K., and Okamura, H. (2005). Activation ofmelanocortin
receptors
accelerates
thegonadotropin-releasing hormone pulse generator activity
in goats. Neurosci. Lett. 383, 289-294.
Momozawa, Y., Takeuchi, Y., Kusunose, R., Kikusui, T.,
and Mori, Y. (2005). Association between equine
temperament and polymorphisms in dopamine D4
receptor gene. Mamm. Genome 16, 538-544.
Momozawa, Y., Takeuchi, Y., Tozaki, T., Kikusui, T.,
Hasegawa, T., Raudsepp, T., Chowdhary, B. P., Kusunose,R., and Mori, Y. (2005). Sequence, detection ofpolymorphisms and radiation hybrid mapping of theequine catechol-o-methyltransferase gene. Anim. Genet.
36, 190.
Takeuchi, Y., Hashizume, C., Chon, E. M., Momozawa, Y.,
Masuda, K., Kikusui, T., and Mori, Y. (2005). Canine
tyrosine hydroxylase (TH) gene and dopamine beta
-hydroxylase (DBH) gene: their sequences, genetic
polymorphisms, and diversities among five different dog
breeds. J. Vet. Med. Sci. 67, 861-867.
Yamaguchi, H., Kikusui, T., Takeuchi, Y., Yoshimura, H.,
and Mori, Y. (2005). Social stress decreases marking
behavior independently of testosterone in Mongolian
gerbils. Horm. Behav. 47, 549-555.

Figure 2. Methylation analysis of the ER promoter in
PC12 cells. Upper panel shows the location of CG sites andthe fragment introduced into the cells. In the lower panel,each row of circles represents a single cloned allele with
open circles for non-methylated cytosines and filled circles
for methylated cytosines.

Publication List:
Original papers
Hashizume, C., Masuda, K., Momozawa, Y., Kikusui, T.,
Takeuchi, Y., and Mori, Y. (2005). Identification of an

38

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGYLABORATORY OF NEUROPHYSIOLOGY
Associate Professor:
NIBB Research Fellow:
Technical Assistant:

In 2002, sodium ion imaging and electrophysiologicalstudies using cultured cells derived from the subfornicalorgans demonstrated that Nax channel is an extracellularsodium-level sensitive sodium channel (Figure 1).
Further, we found that Nax channel is expressed in
non-myelinating Schwann cells and alveolar type II cells
in addition to the cells in the circumventricular organs.
Nax channel is thus likely to be involved in reception of
sodium-level in the body fluids at the circumventricularorgans and sodium absorption in the visceral nervous
system and in the lung.
In 2003, we found in collaboration with Prof.
Yamamoto’s group at Osaka University that the
peripheral nervous system has only subtle effects on thehigher preference for sodium chloride as observed in the
mutant mice. The results suggest that the mutant
phenotype is mainly due to the lack of Nax channel in thecentral nervous system.

WATANABE, Eiji
YAMADA, Misuzu
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
focused on understanding the molecular and neuralmechanisms underlying the animal behaviors essential tohomeostasis of the body fluid.
To explain the properly regulated animal behaviors,
neurobiologists have postulated the existence of both
osmoreceptors and specific sodium receptors in the brain.
However, the molecular entities of these receptors havenot been found for a long time. In 2000, by using thegene-targeting technology, we first clarified that Nax
sodium channel is a probable candidate for the specificsodium receptor in the brain.
Nax had long been classified as a subfamily of
voltage-gated sodium channels (NaChs) that serve togenerate action potentials in electrically excitable cells
such as neuronal and muscle cells. Compared to theother NaChs, however, Nax has unique amino acidsequences in the regions, which are known to be involvedin voltage-dependent activation and inactivation,suggesting that it must have specific functional properties.
To clarify the functional role of Nax channel, Nax-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.
Nax-deficient mice ingested hypertonic
sodium chloride solution in excess in comparison withwild type-mice.
LacZ reporter gene knocked into
Nax-gene locus revealed that Nax gene is expressed in thecircumventricular organs, which are the specialized
central organs involved in sensing of sodium
concentration and osmosity in the body fluids.

Figure 2. The Nax channel is co-localized with aglia-specific glutamate transporter GLAST in the SFO andOVLT. Coronal tissue sections of the SFO (A-C) andOVLT (D-F) were double-stained with anti-Nax (A, D) andGLAST (B, E) antibodies. Right panels (C, F) are merged
images of the left (A, D) and middle (B, E) panels.
Asterisks indicate the ventricles. A large number of round
GLAST- and Nax-negative black holes represent neuronalcell bodies. Scale bar: 10 μm.

In 2004, we developed an automatic measurement
equipment for intake volume of drinking solutions.
Using this equipment, we showed that the subfornicalorgan is the principal site for the control of salt-intake
behavior, where the Nax channel is the sodium-levelsensor. Infusion of a hypertonic sodium solution into the
cerebral ventricle induced extensive water intake andaversion to saline in wild-type animals but not in theknockout mice. Importantly, the aversion to salt was not
induced by the infusion of a hyperosmotic mannitol
solution with physiological sodium concentration in either
genotype of mice. When Nax cDNA was introduced into
the brain of the knockout mice with an adenoviral
expression vector, only animals which received atransduction of the Nax gene into the subfornical organ
among the circumventricular organs recovered
salt-avoiding behavior under dehydrated conditions.

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

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NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

These results clearly show that the subfornical organ isthe center of the control of salt-intake behavior in thebrain, where the sodium-level-sensitive Nax channel isinvolved in sensing the physiological increase in thesodium level of body fluids.
In this year of 2005, in order to understand how thecircumventricularorgantranslates
extracellularsodium-level sensed by Nax channel to the neuralactivities, we identified subcellular localization of Nax
channel in the organs.
Double immunostaining(Figure 2) and immuno-electronmicroscopic (Figure 3)
studies clearly showed that Nax channel was exclusively
localized to perineuronal lamellar processes extended
from astrocytes and tanycytes in the organs. Importantly,
glial cells derived from the organs were capable ofsensing extracellular sodium-level, as analysed byion-imaging method. In addition, we found that the
Nax-expressing glial cells enveloped multiple kinds ofneurons including GABAergic interneurons in the organs.
Finally, in the organs, neuronal population activated bywater deprivation was different from GABAergic
interneurons, as monitored by Fos immunoreactivity.
Together with previous observation that the organs of Nax
knockout mice is hyperactive under water deprivation,these results indicate that the glial Nax channel senses
increased sodium-level in the body fluid and controls theneuronal activity through glial cells.
Since we first reported aberrant behaviors found in Nax
knockout mice, a series of our studies have clarified that
Nax 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 suggests that neuron-glia
complex plays a key role on the sodium sensing in thecircumventricular organs.

Fiber-like structures radiating out from the midline andventricle were immunopositive. B, C, Immunoelectron
microscopy using anti-Nax antibody. In B, the core regionof the OVLT is shown. Neurons and their processes are
surrounded by immunopositive thin processes of astrocytes.
In C, a ventricular region in the OVLT is shown.
Ventricular side towards the upper side. Neurons arecovered by extremely thin immunopositive processes of
ependymal cells.
Arrows in B and C indicate
immunopositive signals. V, ventricle; N, neuron; S,
synapse; E, ependymal cell; Ast, astrocyte. Scale bars:
50 μm for A, and 1 μm for B and C.

Publication List:
Original papers
Niisato, K., Fujikawa, A., Komai, S., Shintani, T., Watanabe,E., Sakaguchi, G., Katsuura, G., Manabe, T., and Noda, M.
(2005). Age-dependent enhancement of hippocampal LTP
and impairment of spatial learning through the ROCK
pathway in protein tyrosine phosphatase receptor typeZ-deficient mice. J. Neurosci. 25, 1081-1088.

Currently, we are now trying to construct functional
expression systems of Nax sodium channel using various
heterologous cell lines. The heterologous expression
system will provide us useful information on the channel
characters.
Furthermore, we are studying the
involvement of Nax sodium channel in the regulation of
hormone release, using neurohypophyseal vasopressin
system. The posterior pituitary is one of simple model
systems for research of Nax sodium channel, since there
are only two kinds of cellular components, the nerve
terminals releasing neurohypophyseal hormones and glial
cells expressing Nax sodium channel. The model system
will also provide us useful information on thephysiological function of Nax channel.

Watanabe, E., Hiyama, T.Y., Shimizu, H., Kodama, R.,
Hayashi, N., Miyata, S., Yanagawa, Y., Obata, K., andNoda, M. (2005). Sodium-level-sensitive sodium channel
Nax is expressed in glial laminate processes in the sensory
circumventricular organs. Am. J. Physiol., in press.

Figure 3. The Nax channel is localized to glial processes
enveloping neurons in the OVLT. A, A coronal tissue
section of the OVLT stained with anti-Nax antibody.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROBIOLOGY

their gene structure and their pharmacological andtransductional properties. D1R and D2R are mostabundantly and widely expressed in the brain and often
play a role synergistically. D1R has an opposite property
to D2R with respect to the intracellular signaltransduction.
We have been investigating the involvement of
dopaminergic transmission via D1R and D2R in theregulation of locomotion and eating behavior incollaboration with the Laboratory of Director General.
We generated D1R/D2R double knockout (DKO) mice bycrossing D1R knockout (KO) with D2R KO mice, andobserved that D1R/D2R DKO mice exhibited impairment
in locomotion and eating behavior and died prematurely.
To investigate molecular mechanism of regulation inlocomotion and eating behavior, we generated transgenic
mice harboring tetracycline-regulated expression of the
D1R gene on the D1R/D2R DKO background. Severaltransgenic mouse lines successfully rescued lethal
phenotype of the D1R/D2R DKO mice and showed
doxycycline (Dox) controllable expression of transgenic
D1R gene (named as D1R/D2R DKO-D1R rescued mice).
The D1R/D2R DKO-D1R rescued mice exhibiteddecrease in locomotion and food/water intake as well as
decrease in amount of transgene expression after Dox
administration (Figure 2).

LABORATORY OF NEUROCHEMISTRY
Associate Professor:

SASAOKA, Toshikuni

Our major research interest is to understand thephysiological role of dopaminergic system in animal
behavior, especially locomotion and eating behavior,using genetically altered mice, both transgenic and geneknockout mice. In addition, we have developed a novel
method of conditional mutagenesis in mice in order toanalyze the function of the gene of interest in detail. We
analyze function of neurotransmitter receptor complex byusing biochemical analysis of the dystrophin complex on
the skeletal muscle membrane.

I. Role of dopaminergic transmission in locomotionand 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 development of neuron. The
dopaminergic system is also implicated in control ofemotion, motivation and cognition. Dysfunction of
dopaminergic system can result in several neurologicaland psychiatric disorders such as Parkinson’s disease andschizophrenia.

Figure 2. (A) The amount of transgene expression was
suppressed in the striatum of the D1R/D2R DKO-D1R
rescued mice by doxycycline (Dox) administration. (B)Before Dox administration the intensive expression oftransgene was seen in the striatum. Frontal sections ofmouse brains with X-gal staining were shown.
Figure 1. Schematic drawing of dopaminergic synapse

We are attempting to identify areas in which the D1R
expression was suppressed by Dox administration in orderto point out areas responsible for regulation of locomotionand eating/drinking behavior. In addition we areinvestigating whether or not there is critical period in

In mammals five subtypes of dopamine receptor (D1R,
D2R, D3R, D4R and D5R) are identified and divided into
two subgroups referred to as D1-like (D1R, D5R) andD2-like (D2R, D3R and D4R) receptors on the basis of41

NATIONAL INSTITUTE FOR BASIC BIOLOGYNEUROCHEMISTRY

acid in mice and our mutant mice exhibited aberrant
NMDAR activation and a neurological phenotype, similar
to that of mouse models of neurological disorders.
Interestingly, the phenotype of the mice was completely
suppressed by administration of NMDAR antagonists.
This clearly indicates that the NMDAR activation by thecritical amino acid substitution leads to the neurologicalphenotype.
Our method is vastly applicable to functional analysisof any desired gene and should contribute to studies on
the structural and functional relationships of relevant
genes.

development for regulation of locomotion and eating
behavior by dopaminergic transmission.

II. Developing a novel conditional mutagenesis
method in mice
In order to overcome the limitations of theconventional 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 formation. It is suggested that theaberrant activation of NMDAR causes excitotoxicity,
leading to neuronal death in various neurological diseases.
That the Ca2+ permeability through NMDAR is blockedby magnesium (Mg2+) in a voltage-dependent mannerindicates an essential role of NMDAR as a coincidence
detector. Functional NMDARs consist of NMDAR1(NR1) subunit and at least one subunit of NMDAR2A-2D(NR2A-NR2D). It has been shown that the NR1/NR2A
complex expressed in cultured cell is highly sensitive tothe voltage-dependent Mg 2+ block and that thesubstitution of asparagine (Asp595) by glutamine(Gln595) in the second transmembrane domain of the
NR2A subunit results in a reduction of the Mg2+ block of
the NR1/NR2A complex.

III. The molecular architecture and thephysiological role of the sarcoglycan complex(SGC)
Sarcoglycans
(SGs)
are
trans-sarcolemmal
glycoproteins that associate together to form SGC and arepresent in the sarcolemma. SGC, together with dystrophin
and the dystroglycan complex, comprises the dystrophin
complex, which is considered as the mechanical link
between the basement membrane and the intracellularcytoskeleton for protecting the sarcolemma frommechanical stress during muscle contraction. Each of four
SG subunits (-, -, - and -SG) is responsible for four
respective forms of SG-deficient muscular dystrophy,
sarcoglycanopathy (SGP). All of the SGs and sarcospan
are absent in the sarcolemma in any form of SGP,
suggesting that the SGC is not assembled if a single
subunit of the SGC is absent.
To analyze the function of the SGC, we generated the-SG KO and -SG KO mice. These KO mice developed
progressive muscular dystrophy and all SGs andsarcospan were absent in the sarcolemma. The dystrophin
complex isolated from the SG-deficient skeletal muscles
was biochemically unstable. This indicates that SGC andsarcospan play an important role in stabilizing thedystrophin complex connecting the basement membrane
and the cytoskeleton.
We have generated the KO mice of the other subunit ofthe dystrophin complex to analyze the physiological roleof the subunit.
Publication:
Original paper

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.

Tanaka, T., Watanabe, N., and Sasaoka, T. (2005).
Unidirectional subcloning to generate more than 109transformants from 1 microgram of vector DNA. The
Nihon University Journal of Medicine, in press.
Review article

However, the role of Asp595 of the NR2A subunit andthe effects of substitution of it with Gln595 on thefunction of NMDAR in vivo remain to be clarified.
We develop conditional mutagenesis method in miceusing Cre-loxP recombination (Figure 3). By our method,
we accomplished conditional substitution of the amino

Ozawa, E., Mizuno, Y., Hagiwara, Y., Sasaoka, T., andYoshida, M. (2005). Molecular and cell biology of thesarcoglycan complex. Muscle and Nerve. 32, 563-576.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYLABORATORY OF DIRECTOR GENERAL

can be shut off at a time point of interest. We utilized
tetracycline controllable expression system and generated
transgenic mice harboring conditional D1R expression on
the D1R/D2R DKO background (Figure 1).

LABORATORY OF DIRECTOR GENERAL
Director General:
Postdoctoral Fellow:
Technical Assistant:

KATSUKI, Motoya
ARAKAWA-KOBAYASHI, Satoko
MIYAKAWA, Atsushi

One of the largest themes of the 21st century is topromote brain research in an attempt to understand the“mind.” There are many approaches to elucidating the“mind,” including research into intelligence, memory,
cognition, emotion and volition. Research on developing an
excellent artificial system for information processing,research on the ontogeny and the development of the brain,
and research on protecting the brain from aging,neurological and psychiatric disorders are also important
themes.
Our approach to understanding the brain is to explore themolecular 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-methyl-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. Dopamine
behavior

regulates

locomotion

andeating

The 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 such as Parkinson’s disease andschizophrenia. In mammals five subtypes of dopamine
receptors (D1R-D5R) are identified and classified into two
major groups, D1-like (D1R, D5R) and D2-like (D2R, D3R,
D4R) receptors on the basis of the gene structure and thepharmacological and intracellular signaling properties. The
contribution of D1-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 lacking morethan one dopamine receptor simultaneously. We focused on
D1R and D2R, major subtypes of D1-like and D2-likereceptors, respectively, which are most widely andabundantly expressed. We found the D1R/D2R double
knockout (DKO) mice showed severe impairment inlocomotion and feeding that was not observed in D1R or
D2R KO mice. Although the D1R/D2R DKO mice wereborn normally and showed suckling behavior, the D1R/D2R
DKO mice exhibited rapid decrease in locomotion and no
initiation of eating and eventually died [of starvation?] by
the third postnatal week. These findings suggest that
dopaminergic transmission via D1R or D2R is involved inneural development of the areas that are implicated in theregulation of locomotion and eating.
To examine the involvement of [the?] dopaminergic
system in the regulation of locomotion and eating we
generated mutant mice in which dopaminergic transmission

Figure 1. D1R/D2R DKO mice were rescued by conditional
D1R expression. (A) D1R/D2R DKO mice showed
impairment in locomotion and feeding and died prematurely.
(B) To rescue D1R/D2R DKO mice, transgenic miceharboring conditional D1R expression on the D1R/D2R DKO
background were generated by tetracycline controllable
expression system.

We obtained several transgenic mouse lines rescuing
lethal phenotype of the D1R/D2R DKO mice (D1R/D2R
DKO-D1R rescued mice). The D1R/D2R DKO-D1R
rescued mice exhibited decrease in expression level oftransgene in the striatum and decrease in locomotion andfood/water intake by doxycycline (Dox) administration(Figure 2). These results indicate that areas harboring
Dox-controllable D1R expression are responsible forregulation of locomotion and eating behavior.

43

NATIONAL INSTITUTE FOR BASIC BIOLOGYLABORATORY OF DIRECTOR GENERAL

found the NR2A homozygous, NR2B heterozygous mutant(NR2A-/-,NR2B+/-) mice exhibited behavioral alteration
similar to that observed in patients with schizophrenia. We
are developing an experimental devise to assess behavioral
alteration of the NR2A-/-,NR2B+/- mice and study themolecular mechanism.

III. ras family and their roles in the brain
The ras proto-oncogene plays a critical role in cell
growth control as a central component of mitogenic signaltransduction pathways. In mammals there are H-, N-, K-ras
identified as the ras family. H-, N-, K-ras have an
overlapped spatial expression pattern as well as anoverlapped function. To investigate the distinct function of
the individual Ras protein in the brain we generated ras
DKO mice expressing a single Ras and triple KO micelacking all H-, N-, and K-Ras and analyzed developmental
aspects of these mutant mice.
Publication list:
Original papers
Hara, T., Saito, Y., Hirai, T., Nakamura, K., Nakao, K.,
Katsuki, M., and Chida, K. (2005). Deficiency of protein
kinase C alpha in mice results in impairment of epidermal
hyperplasia and enhancement of tumor formation in
two-stage skin carcinogenesis. Cancer Res. 65, 7356-7362.
Hiratsuka, S., Kataoka, Y., Nakao, K., Nakamura, K.,
Morikawa, S., Tanaka, S., Katsuki, M., Maru, Y., andShibuya, M. (2005). Vascular endothelial growth factor A(VEGF-A) is involved in guidance of VEGF
receptor-positive cells to the anterior portion of early
embryos. Mol. Cell. Biol. 25, 355-363.
Hiratsuka, S., Nakao, K., Nakamura, K., Katsuki, M., Maru, Y.,
and Shibuya, M. (2005). Membrane fixation of vascularendothelial growth factor receptor 1 ligand-binding domain
is important for vasculogenesis and angiogenesis in mice.
Mol. Cell. Biol. 25, 346-354.
Kassai, H., Aiba, A., Nakao, K., Nakamura, K., Katsuki, M.,
Xiong, W.H., Yau, K.W., Imai, H., Shichida, Y., Satomi, Y.,
Takao, T., Okano, T., and Fukada, Y. (2005). Farnesylationof retinal transducin underlies its translocation during light
adaptation. Neuron 47, 529-539.
Komine, Y., Nakamura, K., Katsuki, M., and Yamamori, T.
(2005). Novel transcription factor zfh-5 is negatively
regulated by its own antisense RNA in mouse brain. Mol.
Cell. Neurosci., in press.
Tomemori, Y., Ichiba, M., Kusumoto, A., Mizuno, E., Sato, D.,
Muroya, S., Nakamura, M., Kawaguchi, H., Yoshida, H.,
Ueno, S., Nakao, K., Nakamura, K., Aiba, A., Katsuki, M.,
and Sano, A. (2005). A gene-targeted mouse model forchorea-acanthocytosis. J. Neurochem. 92, 759-766.
Tran, A.H., Tamura, R., Uwano, T., Kobayashi, T., Katsuki,M., and Ono, T. (2005). Dopamine D1 receptors involved inlocomotor activity and accumbens neural responses toprediction of reward associated with place. Proc. Natl. Acad.
Sci. USA 102, 2117-2122.

Figure 2. Alteration of locomotion and eating of D1R/D2R
DKO-D1R rescued mice by Dox administration. Locomotive
activities of rescued (A) and control (B) mice during light anddark periods are shown as counts of movement using anactive sensor. Eating activities of rescued (C) and control (D)mice are shown as amounts of food taken during light anddark periods.

II. Analysis of the function of NMDARs
The NMDARs are widely expressed in the nervous
system, fundamental to excitatory neurotransmission, andplay a number of important roles. There are many reportson the involvement of the NMDARs in learning andmemory. According to one hypothesis schizophrenia may
involve a defect in NMDAR function. NMDARs consist of
NR1 subunit and at least one subunit of NR2A-NR2D. We
generated KO mice lacking each of NMDAR subunits andmultiple KO mice lacking two subunits simultaneously, and44

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

IIDA, Shigeru
TERADA, Rie
HOSHINO, Atsushi
TUGANE, Kazuo
Technical Staff:
FUKADA-TANAKA, Sachiko
YAMAGUCHI, Katsushi
NIBB Research Fellow:
PARK, Kyeung-Il
JSPS Postdoctoral Fellows:EUN, Chang-Ho
JIANG, Gonghao
Postdoctoral Fellows:
JOHZUKA-HISATOMI, Yasuyo
MORITOH, Masaru
Graduate Students:
SHIMATANI, Zenpei
ONODA, Shiho
Visiting Scientists:
KOUMURA, Toshiro
OHNISHI, Makoto
TAKAGI, Kyoko
OKUMURA, Yuki
YAMAUCHI, Takaki
Technical Assistants:
MORITA, Yasumasa
ONO, Akemi
ASAO, Hisayo
MATSUMOTO, Miwako
SHIMAMOTO, Miki
MATSUDA, Chisato
IKEGAYA, Kyoko
HASEGAWA, Yoshinobu
Secretary:
SANJO, Kazuko

Figure1. Flower phenotypes and the Dusky gene for the
3GGT enzyme in I. nil. A-D. Flower phenotypes(above) and microscopic photographs of adaxial
epidermal cells of flower petals (below). (A) The
wild-type line with the Magenta, Purple, and Dusky
alleles. (B) The line with the Magenta, Purple, anddusky-1 allele. (C) The line with the magenta, purple,
and Dusky alleles. (D) The line with the magenta,
purple, and dusky-1 allele. Scale bars in microscopic
photographs indicate 30 μm. E. Reaction mediated by
the Dusky gene products, 3GGT. F. The 3GGT gene andthe dusky mutations. The large vertical arrow indicatesthe site of 4-bp insertions (above), and the 4-bp
insertions underlined in the dusky mutants (below) resultin frameshift mutations.

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 the 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

number of its spontaneous mutants related to the colorand shape of the flowers have been isolated. Genetic
studies on the color of I. nil have shown that 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+/H+ antiporter called InNHX1 that increases
the vacuolar pH during flower opening, causing a shift
towards the bluer coloration. While most of the plant
NHX genes characterized are generally expressed inleaves, stems and roots and induced by NaCl treatment,
the InNHX1 gene is predominantly expressed in theflower limbs at around 12 hour before flower-opening andis very scarcely expressed in leaves, stems and roots, andno induction occurs in response to NaCl treatment. We
also found that the InNHX1 proteins could catalyze both
Na+ and K+ transport into vacuoles.

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 1A) that contain thepeonidin (3’ methoxyl cyanidin) derivative named
Heavenly Blue Anthocyanin or HBA.
I. nil was introduced into Japan from China in the 8th
century as a medicinal herb, the seeds of which were used
as a laxative, and the plant became a traditional
floricultural plant in Japan around the 17th century. The
plant has an extensive history of genetic studies, and a45

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

chromosome 11 in the same orientation with an intervalof 30 kb and flank highly repetitive Copia- andGypsy-like retroelements (Figure 2). The Adh genes playa key function in response to an anaerobic condition, andonly a single adh1 mutant has been isolated in rice. By
improving our transformation procedure further, we areattempting to modify the Adh1 and Adh2 genes, thecoding sequences of which are similar to each other. We
obtained 9 independent transformed calli having the Adh2
gene modified and subsequently isolated 8 fertiletransgenic plants without concomitant occurrence of
undesirable ectopic events. Although we were able toobtain transformed calli with modified Adh1, thefrequency of homologous recombination at Adh1
appeared to be about one magnitude lower than that at
Adh2, indicating that Adh2 contains a more active hot
spot(s) for efficient homologous recombination than
Adh1.

Among the various colors of I. nil flowers, the favorite
hue for Japanese floriculturists has been reddish-brown or
purplish-grey petals (Figure 1B and D) since the early
19th century, and the flower coloration is mainly causedby recessive dusky mutations. We noticed that the petals
in all dusky mutants often contained intensely pigmented
globules, which appeared to affect flower hue. We foundthat the Dusky gene encodes UDP-glucose:anthocyanidin
3-O-glucoside-2’’-O-glucosyltransferase (3GGT), whichcatalyzes the the conversion of anthocyanidin
3-O-glucosides into anthocyanidin 3-O-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
cDNA was expressed, and the introduced 3GGT cDNA
could efficiently produce 3GGT that could convert
cyanidin 3-O-glucoside into cyanidin 3-O-sophoroside intransgenic petunia plants.
The transcriptional regulators for anthocyanin
biosynthesis include members of proteins containing an
R2R3-MYB domain, a bHLH (basic helix-loop-helix)
domain, and conserved WD40 repeats. We also found thatthe recessive c-1 and ca alleles conferring white flowers
are frameshift mutations caused by a 2-bp deletion and7-bp insertions in the genes for the R2R3-MYB and
WD40 repeats transcriptional regulators, respectively.

III. Characterization of mutable virescent allele in
rice
Leaves of seedlings in the virescent mutant of rice are
initially pale yellow green due to partial deficient inchlorophyll and gradually become green with the growthof the mutant. We have been characterizing a spontaneous
mutable virescent allele, pale yellow leaf-variegated(pyl-v), conferring 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 (pyl-stable) line thatexhibits pale-yellow leaves without variegation (Figure
3B), which appeared to carry no active autonomous
element acting on the nonautonomous DNA element
inserted into the Pyl 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 nDart1 (nonautonomous DNA-based
active rice transposon one), in the untranslated exon 1 of
the Pyl gene on chromosome 3 (Figure 3D), and excisionof the new DNA transposon from the pyl gene appears to
be responsible for conferring the leaf variegation. We also

II. Modification of endogenous natural genes byhomologous recombination in rice
Rice (Oryza sativa L.), with the sequenced genome of
389-Mb, is an important staple food for more than half of
the world’s population and a model plant for other cereal
species.
We
havedevelopedalarge-scale
Agrobacterium-mediated transformation procedure with astrong positive-negative selection and succeeded inefficient and reproducible targeting of the Waxy gene byhomologous
recombination
without
concomitantoccurrence of ectopic events, which must be an importantfirst step for developing a precise modification system of
the genomic sequences in rice. While the Waxy gene is aunique gene in the rice genome, 3 copies of the Adh geneare present, and both Adh1 and Adh2 genes reside on

Figure 2. Strategy for gene targeting of the Adh1 and Adh2 genes in rice. The symbols h and DT-A on the
T-DNA regions of the vectors used indicate the positive and negative selection markers, respectively. The
hypothetical gene flanked by the retroelements on chromosome 11 is indicated by Hyp.

46

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

culture is necessary in all of the currently available ricereverse genetic approaches including transposon tagging
systems employing exogenous or endogenous transposons.
We are currently attempting to develop a noveltransposon tagging system in rice.

Publication List:
Original papers
Hagihara, E., Itchoda, N., Habu, Y., Iida, S., Mikami, T., andKubo, T. (2005). Molecular mapping of a fertility restorer
gene for Owen cytoplasmic male sterility in sugar beet.
Theor. Appl. Genet. 111, 250-255.
Kitazawa, D., Hatakeda, Y., Kamada, M., Fujii, N.,
Miyazawa, Y., Hoshino, A., Iida, S., Fukaki, H., Morita,
M. T., Tasaka, M., Suge, H., and Takahashi, H. (2005).
Shoot circumnutation and winding movements require
gravisensing cells. Proc. Natl. Acad. Sci. U S A, 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 dusky mutants
displaying reddish-brown or purplish-grey flowers are
deficient in a novel glycosylation enzyme for anthocyanin
biosynthesis, UDP-glucose:anthocyanidin 3-O-glucoside2’’-O-glucosyltransferase, due to 4-bp insertions in thegene. Plant J. 42, 353-363.
Ohnishi, M., Fukada-Tanaka, S., Hoshino, A., Takada, J.,
Inagaki, Y., and Iida, S. (2005). Characterization of anovel Na+/H+ antiporter gene InNHX2 and comparison of
InNHX2 with InNHX1, which is responsible for blue
flower coloration by increasing the vacuolar pH in theJapanese morning glory. Plant Cell Physiol. 46, 259-267.
Saito, N., Toki, K., Morita, Y., Hoshino, A., Iida, S.,
Shigihara, A., and Honda, T. (2005). Acylated peonidin
glycosides from duskish mutant flowers of Ipomoea nil.
Phytochemistry 66, 1852-1860.
Tsugane, K., Maekawa, M., Takagi, K., Takahara, H., Qian
Q., Eun, C.H., and Iida, S. (2005). An active DNA
transposon nDart causing leaf variegation and mutabledwarfism and its related elements in rice. Plant J., in press.

Figure 3. A-C. Leaf phenotypes of the mutable pyl-v
allele, the stable pyl-stb allele, and the stable pyl-stb
allele treated with 5-azaC. Dark-green sectors in apale-yellow leaf can be seen in pyl-v and pyl-stb treatedwith 5-azaC. D. Structure of the pyl-v allele carrying thenDart1 insertion. The white and green boxes represent
the Pyl exons and coding region, respectively. The upperbox and small horizontal arrows indicate the nDart1
element and the positions of the primers to detect thenDart1 insertion. E. A stable thl-d mutant and a mutable
thl-m plant producing a normal revertant tiller. F.
Structure of the thl-m allele carrying the nDart1
insertion. The open, black and shadowed boxes
represent the Thl exons, cording region, and a putative
esterase/lipase domain.

demonstrated that the transposition of nDart could be
induced by crossing with a line containing an active
autonomous element, aDart, and stabilized by segregatingout of aDart under natural growth conditions. Not only
pyl-stb but also the japonica cultivar Nipponbare carries
no aDart, although they contain epigenetically silenced
Dart elements that can be activated by the treatment of
5-azaC (Figure 3C). We also identified a novel mutabledwarf allele, thl-m (thambelina-mutable), which conferreda tiny and gibberellin-insensitive dwarf phenotype(around 4 cm in height, shorter than a thumb) withoccasional appearance on a normal fertile tiller (Figure3E) and was caused by an nDart1 insertion (Figure 3F).
No somaclonal variation should occur in mutant lines
induced by our newly characterizing endogenous element,
because no tissue culture is involved in its activation. In
this respect, it is important to emphasize here that tissue

Review articles
Chopra, S., Hoshino, A., Boddu, J., and Iida, S. (2005).
Flavonoid pigments as tools in molecular genetics. E.
Glotewold ed., The Science of Flavonoid, Springer
Science & Business Media, in press.
Iida, S., and Terada, R. (2005). Modification of endogenous
natural genes by gene targeting in rice and other higher
plants. Plant Mol. Biol. 59, 205-219.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

functionality of the Saccharomyces ribosomal intergenic
region and demonstrate that functional elements not
involved in gene-coding function can be identified byusing comparative genomics based on sequenceconservation.

DIVISION OF GENOME DYNAMICS
Professor:
Associate Professor:
Research Associate:
Technical Staff:
JSPS Research Fellow:
Postdoctoral Fellows:

Visiting Scientist:
Graduate Student:
Technical Assistant:
Secretary

HORIUCHI, Takashi
KOBAYASHI, Takehiko
JOHZUKA, Katsuki
MOROOKA, Naoki
CUI, Tailing
KODAMA, Ken-ichi
AUSTEN, RD Ganley
OHSUMI, Katsufumi
SERIZAWA, Naomi
WATANABE, Takaaki
UJIIE, Yoshifumi
ITAZU, Masako
IGAWA, Emi
MIKAMI, Yuriko

II. Recombination regulation by transcriptioninduced cohesin dissociation in rDNA repeats inyeast
In most eukaryotic organisms, the rDNAs are clusteredin long tandem repeats on one or a few chromosomes.
Although the total number of these chromosomal rDNA
repeats appears to be maintained at a level appropriate foreach organism, genes with such a repeated structure are in
general thought to be unstable because of a highfrequency of recombinational events. Thus, it might be
expected that organisms have developed systems toregulate 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 PolII 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
sir2 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 Mcd1p(Scc1p) to the rDNA in sir2 relative to SIR2 strains.
From these and other experiments, we conclude that SIR2
prevents unequal sister-chromatid recombination,
probably by forming special cohesin structures, without
significant effects on recombinational events within

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 is fully understood. To clarify these aspects,
we are pursuing several themes using Saccharomyces
cerevisiae and in 2005 the following four subjects haveadvanced our knowledge of the dynamics of the genome:(1) identification of non-coding functional elements
possibly involved in amplification and stability of therRNA repeated genes in yeast, (2) a finding ofrecombination regulation by transcription-induced
cohesin dissociation in rDNA, (3) a finding of a new roleof condensin in maintaining a long repeated structure of
rDNA, (4) development of in vivo artificial gene
amplification systems.

I.

Identifying
gene-independent
non-coding
functional elements in the yeast ribosomal DNA
by phylogenetic footprinting

Sequences involved in the regulation of genetic andgenomic processes are primarily located in non-coding
regions. Identifying such cis-acting sequences fromsequence data is difficult because their patterns are notreadily apparent, and, to date, identification has
concentrated on regions associated with gene-coding
functions. We used phylogenetic footprinting to look forgene-independent non-coding elements in the ribosomal
RNA gene (rDNA) repeats from several Saccharomyces
species. Similarity plots of ribosomal intergenic spaceralignments from six closely related Saccharomyces
species allowed the identification of previously
characterized functional elements, such as theorigin-of-replication (ARS) and replication-fork barrier(RFB) sites, demonstrating that this method is a powerfulpredictor of non-coding functional elements. Seventeen
previously uncharacterized elements, showing high levelof conservation, were also discovered. The conservationof these elements suggests that they are functional, andwe demonstrate the functionality of two classes of theseelements, a putative bidirectional non-coding promoter(later we named E-pro) and a series of conserved peaks
with matches to the origin-of-replication core consensus.
Our results paint a comprehensive picture of the48

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

III. Condensin loaded onto the rDNA in a
FOB1-dependent fashion to avoid contraction ofa long repeat

individual rRNA genes.
We found that bidirectional transcription actuallyoccurs from an assuming non-coding bidirectional
promoter (E-pro) within the rDNA and is absolutely
required for amplification of rDNA. E-pro transcription
stimulates the dissociation of cohesin, a DNA binding
protein complex that suppresses sister-chromatid-basedchanges in rDNA copy number. This transcription isregulated by the silencing gene, SIR2, and by copy
number. These results suggested a model of amplification
regulation where transcription of E-pro stimulates unequal
recombination by disrupting cohesin association in therDNA, thus allowing for a change in copy number (Figure1). Transcription-induced cohesin dissociation may be a
general mechanism of recombination regulation.

An average of 200 copies of the ribosomal RNA genes(rDNA) are clustered in a long tandem array in S.
cerevisiae. FOB1 is known to be required for
expansion/contraction of the repeats by stimulating
recombination, thereby contributing in the maintenance of
average copy number. In fob1 cells, the repeats are still
maintained without any fluctuation of copy number,suggesting another unknown system acts to prevent
repeats contraction. We attempted to understand theputative second unknown system by which the long rDNA
array is maintained by isolating mutants carrying unstable,
shortened rDNA tandem array in a fob1 background. We

Figure 1. Transcription-induced cohesin dissociation model of rDNA amplification. (A) In normal situation, suchas wild-type rDNA copy number, SIR2 repress E-pro activity, allowing cohesin to associates throughout the IGS(intergenic spacer; NTS1). DSBs (double stranded breaks), formed by replication forks pausing at the RFB site,
are repaired by equal sister-chromatid recombination, with no change in the rDNA copy number. (B) When SIR2
repression is removed, such as sir2 mutation or low copy number, E-pro becomes active and transcription
displaces cohesin. Unequal sister chromatids can then be used as templates for DSB repair, resulting in changes
in the rDNA copy number. Lines represent single chromatids (double stranded DNA). The IGS in which thereplication fork is paused is expanded in the bracket.

49

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

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.

found that condensin mutants showed severe contractionof rDNA tandem array in a fob1 cells, but not in FOB1+
cells, thereby suggesting that condensin acts to maintainlong repeats with FOB1 in a functionally complemented
fashion. We also found that condensin association withthe rDNA was localized to the replication fork barrier(RFB) site in a FOB1-dependent fashion. Surprisingly,
condensin association with the RFB site was establishedduring S phase and maintained until anaphase. These
results indicate that FOB1 acts in a novel role to prevent
repeats contraction by regulating condensin association,and suggest a link between replication termination, andchromosome condensation and segregation.

Publication List:
Original papers

IV. A novel gene amplification system in yeast
based on double rolling-circle replication

Ganley, A.R., Hayashi, K., Horiuchi, T., and Kobayashi, T.
(2005).
Identifying
gene-independent
noncoding
functional elements in the yeast ribosomal DNA byphylogenetic footprinting. Proc. Natl. Acad. Sci. USA 102,
11787-11792.
Kobayashi, T., and Ganley, A.R. (2005). Recombination
regulation by transcription-induced cohesin dissociation in
rDNA repeats. Science 309, 1581-1584.
Watanabe, T., and Horiuchi, T. (2005). A novel gene
amplification system in yeast based on double
rolling-circle replication. EMBO J. 24, 190-198.

Gene amplification is involved in various biologicalphenomena such as cancer development and drug
resistance. However, the mechanism is largely unknown
because of the complexity of the amplification process.
We developed a gene amplification system in S.
cerevisiae that is based on double rolling-circle
replication (DRCR), utilizing break-induced replication(BIR). This system produced three types of amplification
products. Type-1 products contain 57 inverted copies of
the amplification marker, leu2d. Type-2 products contain13 to ~100 copies of leu2d (up to ~730 kb increase) withnovel arrangement present as randomly oriented

50

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

cyanobacteria as organelles. Analyses of (1) cytosolic
calcium ion 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 between the moss and angiosperms are in
progress by a team directed by Y. Sato.

DIVISION OF EVOLUTIONARY BIOLOGY
Professor:
Associate Professor:
Research Associates:
Technical Staff:
NIBB Research Fellows:
Postdoctoral Fellows:

Graduate Students:
Visiting Scientists:
Technical Assistants:

Secretary:

HASEBE, Mitsuyasu
MURATA, Takashi
FUJITA, Tomomichi
HIWATASHI, Yuji
TANAHASHI, Takako
SUMIKAWA, NaomiNISHIYAMA, Tomoaki
SATO, Yoshikatsu
SATO, Yoshikatsu
AONO, Naoki
MIYAZAKI, Saori
TANAHASHI, Takako
HASHIMOTO, Kaoru
AOYAMA, Tsuyoshi
OBARA, MariMORINAGA, Shin-ichi
HOSOKAWA, Kentaro
AOKI, Etsuko
GOTO, Misako
HASEGAWA, Noriko
HIRAIWA, Hiroki
HIRAMATSU, MikaICHIKAWA, Yukina
ICHIKAWA, Yuki
KITANI, Masakazu
MAKINO, Haruko
MASUOKA, Tomoko
SUZUKI, Yoriko
WAKAZUKI, Sachiko
WATASE, Masahiro
KOJIMA, Yoko

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
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 the molecular mechanismsof how plants generate distinct cell lineages to build theirmulticellular bodies.

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 several land plants andsome green algae as models to compare the functions ofgenes involved in the development of both reproductive
and vegetative organs in land plants.

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.
3-1 Microtubule-dependent microtubule nucleation
Almost all microtubule arrays are organized by one ormore microtubule organizing centers, such as
centrosomes, that regulate nucleation spatially andtemporally. It has long been puzzling how, despite theabsence of conspicuous organizing centers, higher plant
cells form well-organized cortical microtubule arrays,
which are essential for cell morphogenesis. A recent
report suggests that microtubule nucleation sites for thearray are capable of associating with and dissociatingfrom the cortex. We showed that nucleation requires
extant cortical microtubules, onto which cytosolic

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"

51

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

-tubulin is recruited. Microtubule-independent nucleation
is rarely observed in living tobacco BY-2 cells andnucleation is minimal in the absence of original
microtubules in a cell-free system. In both living cells andthe cell-free system, microtubules are nucleated as
branches on the extant cortical microtubules. The branch
points contain -tubulin, which is abundant in thecytoplasm, and microtubule nucleation in the cell freesystem is prevented by inhibiting -tubulin function withspecific antibodies. When isolated plasma membrane withmicrotubules is exposed to purified neuro-tubulin, no
microtubules are nucleated, but when the membrane is
exposed to a cytosolic extract, -tubulin binds
microtubules on the membrane, and after a subsequent
incubation in neuro-tubulin, microtubules are nucleatedon the pre-existing microtubules. We propose that acytoplasmic -tubulin complex shuttles between
cytoplasm and the side of a cortical microtubule and has
nucleation activity only when bound to the microtubule(Murata et al. 2005). T. Murata mainly directed thisstudy.

and an unknown protein. Functional analyses of thesegenes are currently 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.

4-2

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.

4-3

Origin of floral homeotic MADS-box genes

The MADS-box genes of land plants are extensively
diverged to form a superfamily and are important invarious aspects of development including thespecification of floral organs as homeotic selector genes.
The closest relatives of land plants are the freshwater
green algae charophyceans. To study the origin andevolution of land plant MADS-box genes, wecharacterized these genes in three charophycean green
algae: the stonewort Chara globularis (CgMADS1), thecoleochaete Coleochaete scutata (CsMADS1), and thedesmidClosterium
peracerosum-strigosum-littorale
complex (CpMADS1). Phylogenetic analyses suggested
that MADS-box genes diverged extensively in the land
plant lineage after the separation of charophyceans fromland plants. The stonewort CgMADS1 mRNA was
specifically detected in the oogonium and antheridium
together with the egg and spermatozoid during theirdifferentiation. The expression of CpMADS1 increased
when vegetative cells began to differentiate into
gametangial cells, and decreased upon fertilization. These
expression patterns suggest that the precursors of land
plant MADS-box genes originally functioned in haploid
reproductive cell differentiation, and that the haploid
MADS-box genes were recruited into a diploid generation
during the evolution of land plants (Tanabe et al. 2005).

Figure 1. Microtubules in plant cortical arrays nucleate on
existing microtubules as branches. Top panels show
microtubule nucleation visualized by GFP-alpha-tubulin in
living leaf epidermal cells of Arabidopsis thaliana. An
arrow in each panel indicates a microtubule nucleation site.
Images at 0 (left) and 8 (center) seconds, and merged image(right) showing changes of microtubules in 8 seconds areshown. Bottom panel shows proposed model of microtubule
nucleation on existing microtubules.

IV. Evolution of molecular mechanisms in the plantdevelopment
4-1

Function of gametophytic MADS-box genes

Stem cell initiation and maintenance

Postembryonic 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,

4-4 Ancestral function of the floral regulator
FLO/LFY
After fertilization, the zygote undergoes dynamicchanges in the chromosomal and cytoplasmic
organizations and begins the cell cycles that eventuallylead to formation of multicellular embryo. Specifictranscription factors that initiate this cascade of events inland plants have not been identified. We have identified
two FLO/LFY genes, PpLFY1 and PpLFY2, that regulate

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NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

the first cell division after formation of the zygote in themoss Physcomitrella patens. The deduced amino acidsequences of the two PpLFY genes are 94.8% identical toeach other and show similar expression patterns. Whilefertilization occurred in the PpLFY disruptants, thedevelopment of double disruptant zygotes was arrested at
the single-cell stage. When the double disruptants, as
female parent, were crossed with the wild type, as maleparent, normal sporophytes were formed, supporting thenotion that the PpLFY genes function after fertilization toregulate the first mitotic cell division in zygotes. The rare
sporophytes that formed on the PpLFY double disruptants
showed mostly normal organogenesis, but had
abnormalities in the pattern of cell division, supporting arole of PpLFY genes in regulating cell division. The
FLO/LFY genes in angiosperms are conserved master
regulators of floral identity without any obvious effects oncell division. In contrast, our study suggests that
FLO/LFY genes have functions throughout sporophyte
development in the basal land plant lineages (Tanahashi etal. 2005).

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.

Publication List:
Original papers
Hayashida, A., Takechi, K., Sugiyama, M., Kubo, M., Itoh,R.D., Takio, S., Fujita, T., Hiwatashi, Y., Hasebe, M., andTakano, H. (2005). Isolation of mutant lines withdecreased number of chloroplasts per cell from tagged
mutant library of moss Physcomitrella patens. Plant Biol.
54, 300-306.
Maizel, A., Bush, M.A., Tanahashi, T., Perkovic, J., Kato, M.,
Hasebe, M., and Weigel, D. (2005). The floral regulator
LEAFY evolves by substitutions in the DNA binding
domain. Science 308: 260-263.
Murata, T., Sonobe, S., Baskin, T.I., Hyodo, S., Hasezawa, S.,
Nagata, T., Horio, T., and Hasebe, M. (2005).
Microtubules are nucleated on extant microtubules via
gamma-tubulin in plant cortical arrays. Nature Cell
Biology 7, 961-968 (issue cover).
Nakamura, T., Fukuda, T., Nakano, M., Hasebe, M., Kameya,T., and Kanno, A. (2005). The modified ABC model
explains the development of the petaloid perianth of
Agapanthus praecox ssp. orientalis (Agapanthaceae)
flowers. Plant Mol. Biol. 58, 435-445.
Sano, R., Juárez, C.M., Hass, B., Sakakibara, K., Ito, M.,
Banks, J.A., 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. 7, 69-78.
Shigyo, M., Shindo, S., Hasebe, M., and Ito, M. (2005).
Phylogenetic analysis of AP2 domain-containing genes.
Gene, in press.
Tanabe*, Y., Hasebe*, M., Sekimoto, H., Nishiyama, T.,
Kitani, M., Henschel, K., Münster, T., Theißen, G.,
Nozaki, H., and Ito, M. (2005). Characterization of
MADS-box genes in charophycean green alga and its
implication for the evolution of MADS-box genes. Proc.
Natl. Acad. Sci. USA 102, 2436-2441. (*both authors
contributed equally: Selected as this week in PNAS).
Tanahashi, T., Sumikawa, N., Kato, M., and Hasebe, M.
(2005). Diversification of gene function: homologs of thefloral regulator FLO/LFY control the first zygotic cell
division in the moss Physcomitrella patens. Development
132, 1727-1736.

Figure 2. Development of the egg cell, zygote, and embryo.
Ventral parts of archegonia in the wild type (A-E) and the
PpLFY double disruptants (F-H) were observed by confocal
laser scanning microscopy. Nuclei and sperm are indicatedwith arrows and arrowheads, respectively. (A, B, F, G) An
egg cell before (A, F) or after (B, G) sperm invasion. (C, H)
An unexpanded zygote. (D) An expanded zygote at the firstcell division. (E) A two-cell embryo. An expanded zygote
and multicellular embryos were hardly observed in the
double disruptants. Bars = 25 μm.

4-5 Evolution of the floral regulator FLO/LFY inland plants
The plant-specific transcription factor FLO/LFY
controls general aspects of the life cycle in a basal plant,
the moss Physcomitrella patens. In contrast, FLO/LFY
has more specialized functions in angiosperms, where it
specifically induces floral fate during the reproductive
phase. This raises the question of a concomitant change inthe biochemical function of FLO/LFY during theevolution of land plants. We report that the DNA binding
domain of FLO/LFY, although largely conserved, hasdiverged in activity. On the contrary, other, more rapidly
evolving portions of the protein have few effects on
FLO/LFY activity (Maizel et al. 2005).

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NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

species occurred in this lake. To date, our group has
accomplished certain results in molecular phylogenetic
studies of the East African cichlids and other vertebrate
animals; for example, elucidation of the evolutionary
history of baleen whales. From the results of our
phylogenetic analysis of Lake Victoria cichlids using
insertions of retroposons (SINEs: short interspersed
elements) as markers for elucidation of their evolutionary
history, most of selectively-neutral polymorphic alleles(presence/absence of retroposons at orthologous site in
the genome) are retained both within and among speciesof this lake. Namely, polymorphisms among Lake
Victoria cichlids are trans-specific. Such homogeneous
genome within/among the species of Victorian cichlids
provide us with the following criterion for elucidation of
the mechanism of speciation. If we can characterize acertain allele that is uniquely fixed at a certain locus innatural populations of a certain species, we can assume
that this gene may possibly be related to positive selection(speciation).

DIVISION OF SPECIATION MECHANISM(ADJUNCT)
Professor (Adjunct):
Research Associate (Adjunct):
NIBB Research Fellow:
Postdoctoral Fellow:
Technical Assistant:

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

During the long evolutionary history of vertebrates,
they acquired extensive diversity in such areas asmorphology, ecology and behavior. It is believed thatmany organisms inhabiting the earth at present arederived from an ancestral species and became diversified
in the evolutionary process with speciation. Therefore,
speciation is one of the important factors ofdiversification. How then does speciation occur?
Although various theoretical models have been proposedwith respect to speciation, its mechanism has beendifficult to clarify so far, especially on a molecular level.
The aim of our group’s research is to propose theprocesses and mechanism of the speciation of vertebrates
using a molecular approach. To accomplish the aim of ourresearch, we choose the East African cichlid fishes as amodel animal for the study of speciation (Figure 1).

II. Field research in Lake Victoria
Lacustrine environment of Lake Victoria highly
diverged according to the difference of area such as
turbidity, depth and bottom. Depending on such variable
habitats, cichlids also show phenotypic diversity adapting
to respective habitats. To obtain ecological data andnatural fish samples of Victorian cichlids adapting tovarious habitat, our group and Dr. N. Okada’s laboratory(Tokyo Inst. Tech.) conducted field investigation aroundMwanza Gulf in the southern shore of Lake Victoria since2004, August (Figure 2). In 2005, this investigation was
conducted twice. The first period started in August, 2004,
and finished at the beginning of February 2005. In thisfirst research period, our group sent K. Takahashi tocollect approximately 4,000 individuals of Victoriancichlids from various ecological habitats. The second
research was conducted over the course of three months(from the beginning of June 2005 to the end of August2005). In this second research period, our group collecteda further 1,400 specimens. At present, our group has
almost 130 species of Victorian cichlids in our laboratory.

Figure 1. Cichlid fishes in Lake Victoria. These werecaught in the field by the expedition of our group.

I. Intention of our research focusing on cichlids in
Lake Victoria

III. Analysis of candidate genes for elucidation of
speciation and diversification

Although cichlid fishes are broadly distributed intropical region throughout the world, our group focuseson the species that are endemic to three great lakes andtheir drainages in East Africa. There are three lakes,
Lakes Tanganyika, Malawi and Victoria, and, in total,
1,000 or more cichlid fishes live in the lakes. It wasreported that Lake Tanganyika was established 12 million
years ago, that Lake Malawi was established 2 million
years ago, and that Lake Victoria was established 12,000
years ago. Thus, it is thought that the cichlids evolved ineach lake after the lakes formed. Endemic cichlids ofLake Victoria diversified to 700 or more species from asmall number of ancestors during its short history. This
fact means that explosive adaptive radiation of the cichlid

Varied body coloration of cichlids is one of theexamples for their phenotypic diversity. Cichlids are
known to depend mostly on visual system when theychoose their mating partner, and such color variations are
considered to affect the female’s choice. Therefore, it
could be considered that the body colors of males play animportant role for recognition by the visual system offemales during the course of reproduction. In addition, thevisual system of cichlids must have been affected byenvironmental differences in their habitat such as theturbidity and depth of lake water. In a collaborative workwith the Dr. N. Okada’s laboratory in Tokyo Institute ofTechnology, our group proposed that RH1 gene, which isone of the groups of opsin genes, evolved in parallel with54

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITY

the depth of their habitat among cichlid species in Lakes
Tanganyika and Malawi. Based on the actual research in
the field, our group focuses on the evolution of opsin
genes for visual system in several Victorian cichlid
species. Lithchormis rufus and L. rubripinnis (Figure
1A and B) inhabit shallow water near by the shoreline
only in Mwanza Gulf (Figure 2, panel B), and we foundgeographical clines of nuptial coloration on their male.
We are carrying out the analysis of the opsin gene familyof these species to detect the genetic variations ofchromatic vision as a result of adaptation for each malecolor among the populations. We also collected
Haplochromis pyrrhocephalus, which is broadly
distributed in the lake (Figure 1C and D), from several
localities and depths. The light environment for thisspecies is considerably different among the populations.
To elucidate the adaptation of vision system for various
light conditions, we are analyzing the six types of the
opsin gene family. A more extensive analysis on
molecular evolution of opsin genes in Victorian cichlids isin progress in our division.

Kijimoto, T., Watanabe, M., Fujimura, K., Nakazawa, M.,
Murakami, Y., Kuratani, S., Kohara, Y., Gojobori, T., andOkada, N. (2005). cimp1, a novel astacin family
metalloproteinase gene from East African cichlids, isdifferentially expressed between species during growth.
Mol. Biol. Evol. 22, 1649-1660.
Sasaki, T., Nikaido, M., Hamilton, H., Goto, M., Kato, H.,
Kanda, N., Pastene, L.A., Cao, Y., Fordyce, R.E.,
Hasegawa, M., and Okada, N. (2005). Mitochondrial
phylogenetics and evolution of mysticete whales. Syst.
Biol. 54, 77-90.
Sugawara, T., Terai, Y., Imai, H., Turner, G.F., Koblmuller,
S., Sturmbauer, C., Shichida, Y., and Okada, N. (2005).
Parallelism of amino acid changes at the RH1 affecting
spectral sensitivity among deep-water cichlids from Lakes
Tanganyika and Malawi. Proc. Natl. Acad. Sci. USA 102,5448-5453.

Publication List:
Original papers

Figure 2. Localities of collection of cichlids in Lake
Victoria. Panel A: Sampling localities in southern shore of
Lake Victoria. The area surrounded by a brown rectangle inthis panel corresponds to the region shown as a magnified
map of panel B. Panel B: Sampling localities in Mwanzagulf. Sampling was conducted by angling and trawling andby using gill nets. Sampling was conducted in collaboration
with Tanzania Fisheries Research Institute (TAFIRI).

55

NATIONAL INSTITUTE FOR BASIC BIOLOGYEVOLUTIONARY BIOLOGY AND BIODIVERSITYThis laboratory also conducts 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.

LABORATORY 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 on theembryogenetic processes of related species is expected togive an insight into the evolution of morphogenetic
processes. This laboratory uses the wings of lepidopteraninsects 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, whichdevelops 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 and 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
realized concentration of macrophages seems toaccelerate the shrinkage of the degeneration region.
A collaborative work with the laboratory of Dr. K.
Watanabe (Hiroshima University) is concerned mostly
with the development of trachea and tracheole pattern inswallow tail butterflies. Trachea and tracheoles are both
important in delivering air into the wing and their patterns
coincide 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 (Figure 1).

Figure 1. Transmission electron micrograph of thetracheole cell. Many cross-sections of the tracheoles can beobserved within the cytoplasm of the tracheole cell. As
the cell migrates, the tracheole is laid behind.

Publication List:
Original paper
Watanabe, E., Hiyama, T.Y., Shimizu, H., Kodama, R.,
Hayashi, N., Miyata, S., Yanagawa, Y., Obata, K., andNoda, M. (2005). Sodium-level-sensitive sodium channel
Nax is expressed in glial laminate processes in the sensory
circumventricular organs. Am. J. Physiol., in press.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

I. Perinatal estrogen exposure induces persistentchanges in reproductive tracts

DIVISION OF MOLECULARENVIRONMENTAL ENDOCRINOLOGY
Professor:
Associate Professor:
Research Associate:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Graduate Students:

Visiting Scientists:
Technical Assistants:

Secretary:

Perinatal exposure to sex hormones such as DES
induces lesions in the reproductive tracts of female mice.
In the early seventies, a close correlation between theoccurrence of vaginal clear cell adenocarcinoma in young
women and early intrauterine exposure to DES wasdemonstrated. The possible relevance of the mouse
findings to the development of this human cancer has
been emphasized. The neonatal mouse model has been
especially useful in studying the long-term effects of earlysex hormone exposure on the female reproductive tract.
Female reproductive tracts in mice exposed prenatally to
estrogen show altered expression of Hoxa genes and Wnt
genes and knockout mice lacking Hoxa-10 or Wnt7ashow uterine hypoplasia. Neonatal treatment of female
mice with estrogens induces various abnormalities of the
reproductive
tract
includingovary-independent
cervicovaginal
keratinization,adenosis,uterinehypoplasia, epithelial metaplasia, oviductal tumors,polyovular follicles and polyfollicular ovaries. The
growth response of neonatally DES-exposed reproductive
organs to estrogens is reduced, as are ER levels andepidermal growth factor (EGF) receptor levels, in addition
to other hormone receptor levels.

IGUCHI, Taisen
WATANABE, Hajime
KATSU, Yoshinao
MIZUTANI, Takeshi
KATO, Yasuhiko
URUSHITANI, Hiroshi
MIYAGAWA, Shinichi
NAGATA, Emiko
KOBAYASHI, Mika
NAKAMURA, Takeshi
KATO, Hideo
GEOGHEGAN, Fleur
LANGE, Anke
KOBAYASHI, Kaoru
TAKAHASHI, Eri
HINAGO, Megumi
GOTO, Mayu
YAMAGUCHI, Yuko
TANIGUCHI, Ena
IMAIZUMI, Taeko

Synthetic chemicals found in the environment havethe capacity to disrupt the development and function of
the endocrine system in both wildlife and humans. Thishas drawn public concern since many of these chemicals
may bind to estrogen receptors (ER) and evoke estrogenic
effects. Early evidence that exposure to estrogenic
chemicals during development could pose a threat tohuman health came from studies of a synthetic hormone,
diethylstilbestrol (DES), which was used to prevent
premature birth and spontaneous abortion. Laboratory
experiments showed that exposure of animals to sex
hormones during critical windows of perinatal life caused
irreversible alterations to the endocrine and reproductive
systems of both sexes. In the immune and nervous
systems, bone, muscle, and the liver were also affected.
Although many of these chemicals can bind to ERs inwildlife and humans, the molecular basis for the action ofenvironmental estrogens remains poorly understood. Thus,
understanding the molecular mechanisms through which
environmental estrogens and sex hormones act during
critical developmental windows is essential.

EGF-like growth factors
Cellmembrane

ErbB receptors

Raf

PI3K
Phosphorylationcascade

MEK1/2
ERK1/2

AKT

Nuclearmembrane(p90RSK)
Transcription factors

Ser122 Ser171
ER 
AF-1

GRE
Growth factors responsive genes

ERE

AF-2

Growth factors

Estrogen responsive genes

Figure2.
Ahypothetical
model
for
the
estrogen-independent ER activation pathway in mousevagina.
EGF-like
growthfactors
activatetheprotein-phosphorylation cascade via erbB receptors.
Estrogen receptor is phosphorylated on serine 122 and 171
in AF-1 domain. Furthermore, transcription factors areactivated by phosphorylation. These phosphorylations
induce the transcriptional activity of ER, and then growth
factors are expressed via estrogen-response element (ERE).
Growth factors induced by ER activate EGF-receptors.

Growth factor and ER signaling cooperate to playessential roles in cell proliferation, differentiation andtumor progression in mouse reproductive organs, yet themechanisms underlying the estrogen-dependent and-independent pathways remain unknown. EGFR anderbB2 were activated by estrogen treatment in mousevaginal epithelium. This activation was also found invaginae from neonatally DES-exposed mice, along withthe expression of EGF, TGF-, HB-EGF, amphiregulin
and neuregulin. Immunohistochemical analysis indicatedthat erbB2 was primarily expressed in vaginal epithelium.

Figure 1. Scheme of estrogen-dependent and -independent
vaginal epithelial cell proliferation in mice induced byperinatal estrogen exposure.

57

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

non-physiological estrogens, and estrogenic dioxins havedistinct effects on uterine gene expression. However,nonylphenol and dioxin activate another set of genes in
the
liver
that
were
distinctfromuterineestrogen-responsive genes. These results suggest that onlya small number of genes are directly involved in theuterotrophic effects of estrogen treatment, andnonylphenol has very similar effects to estradiol on gene
expression in uterine tissue, but not in hepatic tissue.
Therefore, potential tissue-specific effects should beconsidered in order to elucidate the distinct effects ofvarious endocrine disrupting chemicals (EDC) throughout
the body.
In order to clarify the molecular mechanisms
underlying estrogenic effects, we are studying changes ingene expression patterns induced by perinatal exposure to
estrogen and chemicals using differential display andDNA microarray techniques. We have found genes
possibly related to the ovary-independent changes bydifferential display. We also have clustered groups ofgenes that are responsive to estrogenic stimuli in uterus
by using the DNA microarray. Our goals are tounderstand the molecular background of the critical
period during development, the low dose effect of
estrogenic chemicals and the molecular metabolism ofhormones and hormone-like agents in animals andhumans.

Serine 118 and 167 located in the AF-1 domain ofER were
phosphorylated
in
thesevaginae.
Administration of antagonists for erbB2 (AG825), EGFR
(AG1478), and ER (ICI 182,780) blocked proliferation ofvaginal epithelium induced by neonatal DES exposure.
This suggests that signal transduction via EGFR anderbB2 could be related to the estrogen-induced vaginalchanges. Persistent erbB phosphorylation and sustained
expression of EGF-like growth factors would lead to ER
activation, resulting in cancerous vaginal lesions later inlife in neonatally DES-exposed mice.
We
useddifferential
displaytoidentify
estrogen-responsive genes related to the proliferation anddifferentiation of mouse vaginal epithelial cells. A novel
c-type lectin that encodes a membrane protein with ac-type lectin domain in the carboxyl-terminal region was
identified from this screening. Analysis of mRNA
expression showed that this gene is estrogen responsive inthe mouse vagina. Furthermore, it is found in epithelial,
but not stromal cells, suggesting that this novel c-type
lectin may be an important factor in the stratification
and/or cornification of the vaginal epithelium of mice. We
are continuing efforts to analyze its function during
estrogen-induced proliferation and differentiation in themouse vagina.
Estrogenic compounds such as bisphenol A (BPA)
and nonylphenol as well as dioxins and PCBs were foundin the human umbilical cord. BPA can easily cross theplacenta and enter the fetus in Japanese monkeys andmice. BPA can be found in the fetal brain, testis anduterus 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 toa low BPA dose induced acceleration of vaginal opening
in the offspring. This provides further evidence that thedeveloping mammal is sensitive to exposure to estrogenic
agents. Neonatal treatment of rats with a high dose of
BPA induced infertility in females, but no obvious effects
on males.

II. Microarray analysis of estrogen responsive genes
cDNA microarray methods have recently beendeveloped and successfully applied for genome-wide
analysis of gene expression stimulated by hormones/or
chemicals. Elucidating the expression patterns ofestrogen-responsive genes is essential to understanding
the mechanisms through which estrogenic chemicals act
on mouse reproductive organs. A large number of
estrogen-modulated genes were identified in the mouseuterus. Most of these genes were regulated in adose-dependent manner and their expression was notaltered by estrogen treatment in ER knockout mice. Thisconfirms that expression of these genes is dependent on
ER. Their activation suggests a molecular basis for themarked uterotrophic effect we observed several days
following
estrogenadministration.
Intriguingly,
characteristic gene expression patterns were observed foreach environmental estrogenic chemical and these weredistinct from those elicited by estradiol. This suggests thatspecific mechanisms of action for endocrine disruption
exist that could be different from those induced byendogenous
estrogens.
Physiologicalestrogens,

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

III. Effect of estrogen on reptiles, amphibians andfishes
Exogenous estrogen exposure during embryogenesis
induces abnormal sex differentiation and abnormal boneformation in the African clawed frog, Xenopus laevis, thecyprinodont fish, mummichog (Fundulus heteroclitus)
and mosquitofish (Gambusia affinis affinis). To analyze
the estrogen function, we isolated ER and  cDNA fromF. heteroclitus and G. affinis affinis. Exposure of roach(Rutilus rutilus – a common cyprinid fish) to effluentsfrom sewage treatment works containing complex
mixtures of EDCs has been shown to alter sexual
development and impact negatively on their reproductive
capabilities in UK rivers. To unravel the mechanisms of
disruption of sexual development in roach exposed to
EDCs, we have isolated cDNAs related to sex
determination and sex-differentiation such as ERs,

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NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

aromatase, StAR, Sox9, vasa, etc., and are examining
gene expression during gonadal differentiation with orwithout EDCs exposure. Furthermore, we have cloned
steroid hormone receptors and several oncogenes from theAmerican alligator (Alligator mississippiensis), the Nilecrocodile (Crocodilus nicotilus) and the Florida red-eared
slider turtle (Trachemys scripta) with the aim of analyzing
the ER evolution in reptilian phylogeny. We are also
isolating estrogen-responsive genes to understand themolecular physiology of estrogen action with an aim
towardunderstanding temperature-dependent sex
determination of alligators at the molecular level.
Other projects related to steroids and physiology are in
progress. The Japanese tree frog (Hyla japonica) absorbs
water through ventral skin. We found that sex steroids and
EDCs interfere with water absorption through ventral skinin frogs. Trenbolone, a potent androgenic and anabolic
steroid, induced masculinization of anal fin and sperm
production in the ovary of mosquitofish. 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.

Hiroshima.

VI. Molecular target search
Abnormalities caused by EDC have been reported but
the molecular mechanisms underlying their effects are notwell known. Although the ER is one of the most likely
candidates responsible for the endocrine disrupting
function of many chemicals, ER alone cannot explain thevariety of phenomena induced by EDC. Therefore, we arealso looking for new target molecules that may be
involved in endocrine disruption. We are also studying
the ligand-binding mechanisms of nuclear receptors tohormones and other chemicals.

Publication List:
Original papers
Mitui, N., Fujii, T., Miyahara, M., Oka, T., Kashiwagi, A.,
Kashiwagi, K., Handa, H., Urushitani, H., Santo, N., Tooi,
O., and Iguchi, T. (2005). Development of metamorphosis
assay using Silurana tropicalis for the detection of thyroid
hormone disrupting chemicals. Ecotoxicol. Environ.
Safety, in press.
Oda, S., Tatarazako, N., Watanabe, H., Morita, M., andIguchi, T. (2005). Production of male neonates in 4
cladoceran species exposed to a juvenile hormone analog,
fenoxycarb. Chemosphere 60, 74-78.
Oda, S., Tatarazako, N., Watanabe, H., Morita, M., andIguchi, T. (2005). Production of male neonates in Daphnia
magna (Cladocera, Crustacea) exposed to juvenile
hormones and their analogs. Chemosphere, 61,
1168-1174.
Sone, K., Hinago, M., Itamoto, M., Katsu, Y., Watanabe, H.,
Urushitani, H., Tooi, O., Guillette, L.J.Jr., and Iguchi, T.
(2005). Effects of an androgenic growth promoter
17-trenbolone on masculinization of mosquitofish(Gambusia affinis affinis). Gen. Comp. Endocrinol. 143,
151-160.
Todaka, E., Sakurai, K., Fukuta, H., Miyagawa, H., Uzuki,
M., Omori, M., Osada, H., Ikezuki, Y., Tsutsumi, O.,
Iguchi, T., and Mori, C. (2005). Fetal exposure tophytoestrogens – the difference in phytoestrogen status
between mother and fetus. Environ. Res. 99, 195-203.
Watanabe, H., Tatarazako, N., Oda, S., Nishide, I., Uchiyama,M., Morita, M., and Iguchi, T. (2005). Analysis ofexpressed sequence tags of the waterfrea Daphnia magna.
Genome 48, 606-609.

Figure. 4 Evolutionary relationships of estrogen receptor
sequences

IV. Male production in Daphnids by juvenile
hormones
We found that ten juvenile hormone analogs induce
production of males in Daphnia magna. Daphnids are
susceptible to the male-sex determining effects ofjuvenoids during oogenesis and the effect of juvenoids isreversible. In order to understand the molecular functional
mechanism of juvenoids in induction of male offspring,
we are analyzing juvenile hormone binding protein andestablishing a microarray system for D. magna.

Review articles
Ankley, G.T., Black, M.C., Garric, J., Hutchinson, T.H., andIguchi, T. (2005). A framework for assessing the hazard ofpharmaceutical materials to aquatic species. In Human
Pharmaceuticals-Assessing the Impacts on Aquatic
Ecosystems. (ed.) Williams, R.T. SETAC Press, Pensacola,
FL, U.S.A. pp. 183-227.
Okada, A., Sato, T., Ohta, Y., and Iguchi, T. (2005). Sex
steroid hormone receptors in the developing female
reproductive tract of laboratory rodents. J. Toxicol. Sci. 30,

V. Gene zoo
We have established cDNA libraries from various
animal species of interest including the Japanese giant
salamander (Andrias japonicus). Identifying essential
genes is indispensable for the basic study andconservation of animal species. We are establishing
cDNA library banks of animal species in collaboration
with the University of Pretoria, South Africa, theUniversity of Florida, U.S.A., and the Asa Zoo in

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75-89.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

polarized growth of leaves. Interestingly, CYP90D1, themost closely related cytochrome P450 to the
ROT3/CYP90C1 enzyme, was suggested to catalyze theother conversion steps of BR biosynthesis (Kim et al.
2005). Double mutant for the ROT3/CYP90C1 and for the
CYP90D1 exhibited extreme dwarf that is observed forthe other known mutants of genes for biosynthesis of BRs.
Since the loss-of-function 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 topic, the adaptive responses ofarabidopsis leaves against gravity and otherenvironmental factors were also analyzed and theinteraction between light signal and gravity-response inleaves was suggested (Kim et al. 2005, Tsukaya 2005).

DIVISION OF PLANT DEVELOPMENTAL
GENETICS (ADJUNCT)
Professor (Adjunct):
Research Associate:
NIBB Research Fellow:
Postdoctoral Fellows:
Graduate Students:
Technical Assistants:
Secretary:

TSUKAYA, Hirokazu
HORIGUCHI, Gorou
ISHIKAWA, Naoko
ALI, Ferjani
YAMAGUCHI, Takahiro
YANO, Satoshi
MANO, Eriko
FUJIKURA, Ushio
TAKABE, Eriko
YAMAGUCHI, Chinami
NAGURA, Masako
KOJIMA, Yoko

The leaf is the fundamental unit of the shoot system,
which is composed of the leaf and stem. The diversity ofplant forms is mostly attributable to variation of leaf andfloral organs, which are modified leaves. Moreover, leaf
shape is sensitive to environmental stimuli. The leaf istherefore the key organ for a full understanding of plantmorphogenesis. However, the genetic control ofdevelopment of these shapes has remained unclear.
Recently, studies of leaf morphogenesis reached a turning
point after our successful application of the techniques ofdevelopmental and molecular genetics using model plants,
Arabidopsis thaliana (L.) Heynh. (reviewed in Kim et al.
2005, Tsukaya 2003).

1-2

As mentioned above, we also focused on the effects ofenvironmental factors on leaf morphogenesis. In darkness,the expansion of leaf lamina is inhibited, while at thesame time petiole elongation is enhanced. Thisphenomenon is termed the shade-avoidance syndrome.
We analyzed the nature of the shade-avoidance syndrome
and found that phytochromes and cryptochromes
specifically regulate the contrasting growth patterns of theleaf blade and petiole in shade (Kozuka et al. 2005).
Differed from photomorphogenesis of hypocotyl, cell
elongation was stimulated in the petiole in dark conditions
without an increase in the ploidy level. By examining theeffects of sucrose on the growth of the leaf blade andpetiole, we revealed that the growth promotional effectsof sucrose are highly dependent on the light conditions.

I. Polarized growth of leaf blades
Focusing on the mechanisms that govern polarized
growth of leaves in Arabidopsis thaliana we haveidentified genes for polar-dependent growth of leaf
lamina: the ANGUSTIFOLIA (AN) gene regulates widthof leaves and the ROTUNDIFOLIA3 (ROT3) gene
regulates length of leaves. Both AN and ROT3 genes
control leaf cell shape. In addition to these, we added
ROT4 gene to the list of the genes responsible for thepolarity-dependent control of leaf shape. This year, wefound that the ROT3 is involved in biosynthesis ofbrassinosteroid, a steroid phytohormone (Kim et al. 2005).
In relation to the function of the ROT3, we also found thatthe different growth responses of the A. thaliana leaf
blade and the petiole during shade avoidance areregulated by photoreceptors and sugar. Moreover, we
identified the AN3 gene that regulates number of leaf cells(Horiguchi et al. 2005) and a homolog of AN from
Ipomoea nil (Cho et al. 2005).

1-1

Shade avoidance syndrome

1-3

AN3 gene regulates leaf cell numbers

How are cell proliferation and cell enlargement
coordinated in leaf morphogenesis? In a determinate
organ - a leaf- the number of leaf cells is not necessarilyreflected in leaf shape or, in particular, in leaf size.
Genetic analyses of leaf development in A. thaliana
shows that a compensatory system(s) acts in leaf
morphogenesis and an increase in cell volume might be
triggered by a decrease in cell number (Tsukaya 2003).
Thus, leaf size is, at least to some extent, uncoupled fromthe size and number of cells by the compensatory
system(s).
Recently,
wehaverevealed
that
ANGUSTIFOLIA3 (AN3) gene is involved in themaintenance/establishment of activity of cell proliferationin leaf primordia. AN3 encodes a co-activator, and isfound to control cell cycling in leaf primordia byinteracting with a transcription activator, AtGRF5(Horiguchi et al. 2005). Interestingly, the an3 shows clear“compensation”, namely, accelerated cell expansion in
relation to a decrease in the number of leaf cells (Figure
1) whereas overexpression of the AN3 caused simple
increase of leaf area in association with an increased
number of leaf cells. By using various mutants withaltered number and/or size of leaf cells, we are currently
analyzing the genetic system of the compensation.

ROT3 gene function

We found that the ROT3 gene encodes a cytochrome
P450 that catalyzes the conversion of typhasterol tocastasterone, an activation step in the biosynthesis
pathway of brassinosteroids (BRs) (Kim et al. 2005).
Differed from already known mutants of genes forbiosynthesis of BRs, the loss-of-function mutant of ROT3
has a specific defect in the length of leaves, suggesting
the importance of fine tuning the levels of BRs on the60

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

Kalimantan, Borneo, Indonesia (Tsukaya and Okada 2005,
Tsukaya et al. 2005).

Publication List:
Original papers
Cho, K.-H., Shindo, T., Kim, G.-T., Nitasaka, E., andTsukaya, H. (2005). Characterization of a member of the
AN subfamily, IAN, from Ipomoea nil. Plant Cell Physiol.
46, 250-255.
Horiguchi, G., Kim, G.-T., and Tsukaya, H. (2005). The
transcription factor AtGRF5 and the transcription
coactivator AN3 regulate cell proliferation in leaf
primordia of Arabidopsis thaliana. Plant J. 43, 68-78.
Ikeda, H., Akiyama, S., Tsukaya, H., Mohamed, M., andDarnaedi, D. (2005). A cytotaxonomic study of Impatiens(Balsaminaceae) in Java and Borneo, Malesia. J. Jap. Bot.
80, 271-277.
Kim, G.-T., Fujioka, S., Kozuka, T., Tax, F.E., Takatsuto, S.,
Yoshida, S., and Tsukaya, H. (2005). The biosynthesis ofactive forms of brassinosteroids in Arabidopsis. Plant J. 41,
710-721.
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 thepetiole during shade avoidance are regulated byphotoreceptors and sugar. Plant Cell Physiol. 46, 213-223.
Okada, H., Tsukaya, H., and Mohamed, M. (2005).
Chromosome observations in species of Cayratia(Vitaceae). II. Intraspecific polyploidy in C. trifolia. Acta
Phytotax. Geobot. 56, 203-206.
Tsukaya, H. (2005). Molecular variation of Spiranthes
sinensis (Orchidaceae) in Japan, with special reference tosystematic treatment of seasonally differentiated groups
and a dwarf form, f. gracilis, from Yakushima Island. J.
Plant Res. 118, 13-18.
Tsukaya, H., Iokawa, Y., Kondo, M., and Ohba, H. (2005).
Large-scale general collection of DNA of wild plants in
Mustang, Nepal. J. Plant Res. 118, 57-60.
Tsukaya, H., Nakajima, M., and Okada, H. (2005).
Didymoplexiella cinnabarina (Orchidaceae): a new
species from Muller Range, Central Kalimantan,
Indonesia. Acta Phytotax. Geobot. 56, 206-212.
Tsukaya, H., and Okada, H. (2005). Thismia mullerensis(Burmanniaceae), a new species from Muller Range,
Central Kalimantan. Acta Phytotax. Geobot. 56, 129-133.
Yokoyama, J., Fukuda, T., and Tsukaya, H. (2005).
Molecular identification of the mycorrhizal fungi of theepiparasitic
plant
Monotropastrum humilevar.
glaberrimum (Ericaceae). J. Plant Res. 118, 53-56.

Figure 1. Left, Columbia wild type; right, an3 mutant.
Lower panels show paradermal views of palisade tissue.
Note enlarged palisade cells in the an3 mutant that has
narrow and small leaves.

1-4

Evolution of AN gene family

How have these genes evolved the function ofleaf-shape control? The AN is a homolog of animal
CtBP/BARS gene family which has varied roles inmorphogenesis and organelle control. To understand thecommon role(s) of AN subfamily in the plant kingdom,
we analyzed 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 A.
thaliana (Cho et al. 2005).

II. Biodiversity of plant form
2-1

Biodiversity in leaf size

We are also interested in biodiversity in wild plants.
This year, Spiranthes sinensis (Orchidaceae) in Japan was
analyzed (Tsukaya 2005). S. sinensis shows a wide rangeof morphological diversity and a dwarf form has been
known on Yakushima Island. Analysis on molecularvariation of this species revealed that the dwarf form has
not yet gained any specific variations in ITS and trnL-F
loci, suggesting recent evolution of the dwarf form.

2-2

Genetic analyses of plant diversity

The so-called "Evo/Devo" study of leaf morphogenesis
is also one of our research projects. In the course of fieldresearch of natural evolution of leaf shape/size, we haverevealed some aspects of the biodiversity of plants (Ikeda
et al. 2005, Okada et al. 2005, Tsukaya and Okada 2005,
Tsukaya et al. 2005, Yokoyama et al. 2005). Thismia
mullerensis and Didymoplexiella cinnabarina are newplant species discovered in our botanical survey in central

Review articles
Kim, G.-T., Yano, S., Kozuka, T., and Tsukaya, H. (2005).
Photomorphogenesis of leaveShade-avoidance syndrome
and differentiation of sun/shade leaves. Photochem.
Photobiol. Sci. 4, 770-774.
Tsukaya, H. (2005). Leaf shape: genetic controls andenvironmental factors. Int. J. Dev. Biol. 49, 547-555.

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1-2

DIVISION OF PHOTOBIOLOGY (ADJUNCT)

Mougeotia

Most plant species from alga to flowering plants utilize
blue light for inducing phototropism and chloroplast
movement; many ferns, however, as well as some mosses
and green alga utilize red as well as blue light for the
regulation of these responses, resulting in efficient captureof white light under low light levels. During theirevolution, ferns have created a chimeric photoreceptor(phy3 in Adiantum) between phytochrome andphototropin enabling them to utilize red light effectively.
We have identified two genes resembling Adiantum
PHY3, NEOCHROME1 and 2 (MsNEO1 and MsNEO2),
from the green alga Mougeotia scalaris, a plant famous
for its phytochrome-dependent chloroplast movement.
Like Adiantum PHY3, both MsNEO gene products show
phytochrome-typical bilin binding and red/far-red
reversibility, the difference spectra closely matching the
known action spectra of light-induced chloroplast
movement in Mougeotia. Furthermore both rescue
red-light-induced chloroplast movement in Adiantum
phy3 mutants, indicating functional equivalence. The fern
and algal genes seem to have arisen independently inevolution, demonstrating an intriguing example ofconvergent evolution.

Professor:
WADA, Masamitsu
Associate Professor (Adjunct):
YAMAUCHI, Daisuke
Research Associate:
KIKUCHI, Kazuhiro
NIBB Research Fellow:
OGURA, Yasunobu
Postdoctoral Fellows:
TAKAHASHI, Fumio
TOYOOKA-KAWAI, Hiroko
SUETSUGU, Noriyuki
CHRISTENSEN, SteenOIKAWA, Kazusato
Visiting Scientists:
UENAKA, Hidetoshi
TSUBOI, Hidenori

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.

I. Chloroplast relocation movement
One of our major subjects is chloroplast
photo-relocation movement, which is thought to be one of
the simplest model systems to study photomorphogenesis.
We use the fern Adiantum capillus-veneris and the moss
Physcomitrella patens as model plants for our cellbiological approach since the gametophytes are very
sensitive to light and the organization of the cells is very
simple. We also use Arabidopsis mutants to identify thegenes regulating chloroplast photo-relocation movement.

A

Green alga
Mougeotia scalaris

Fern
Adiantum capillus-veneris
gametophyte

sporophyte

B
IntronMsPHY1PHYPASAtPHOT2

HKRD

1-1

Arabidopsis

LOV

MsNEO1STKD

Chloroplasts relocate in a cell according to ambient
light conditions: accumulation response occurs under
low-light condition and avoidance response underhigh-light condition. In Arabidopsis thaliana, theaccumulation response is mediated redundantly by two
blue light receptors, i.e. phototropins (phot1 and phot2)(Sakai et al. 2001) and the avoidance response ismediated only by phot2 (Kagawa et al. 2001). We
isolated a mutant, jac1 (J-domain protein required forchloroplast accumulation response 1) which lacks
accumulation response under weak light, but shows
normal avoidance response under strong light. Positional
cloning of JAC1 revealed that this gene encodes a
J-domain protein at its C-terminus similar to clathrin
uncoating factor auxilin. In dark-adapted wild type cells,
chloroplasts sediment on the cell bottom, but the jac1
mutant lacks this response. The green fluorescent protein(GFP)-JAC1 fusion protein showed the similar
localization pattern to GFP protein at the transient assay
with onion epidermal cells, suggesting that JAC1 protein
may be a soluble cytosolic protein. The results suggestthat JAC1 is one of the essential components ofphototropin-mediated chloroplast movement.

Hinge

MsNEO2Bilin
FMN

AcNEO1
100 aaFMN ?

Figure 1. A, Giant chloroplasts of Mougeotia scalaris in
their face-on (upper panel) and side-on orientation (lower
panel) and those of Adiantum capillus-veneris protonemal
cells in which the chloroplast accumulation to the upper(upper panel) or side wall (lower panel) are induced bypolarized red light vibrating parallel or perpendicular to thecell axis irradiated from horizontal direction. Scale bar 20
μm. The right panel shows A. capillus-veneris sporophyte.
B, The domain structure of fern and algal neochromes andcanonical plant phytochrome and phototropin. MsNEO1
LOV1 domain has so far failed to bind FMN in vitro, theother MsNEO LOV domains are unlikely to bind flavins.
Introns whose positions are equivalent in each gene areconnected with thin lines. PHY: phytochrome photosensory
region. HKRD: histidine kinase-related domain. STKD:
serine/threonine kinase domain. PHOT: phototropin region.

1-3

Adiantum

The avoidance movement response in Adiantum phot2
deficient mutants can be restored by transient expressionof non-mutant AcPHOT2 cDNA, indicating that62

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

are activated in calli derived from another culture andexcise efficiently from original sites to reinsert into newloci. Ping and Pong transposable elements were isolated
as putative autonomous elements encoding an
IS/PIF/Harbinger superfamily of transposases. We are
now trying to detect its transposase function.

chloroplast avoidance movement in this fern is mediatedby the Acphot2 protein as is the case in Arabidopsis. To
know whether Acphot1 functions as a photoreceptor ofblue light-induced chloroplast relocation movement, weneed either triple mutant or at least three double mutantsof Acphot1, Acphot2 and Acphy3, because phy3 mightalso be one of the blue light receptors in Adiantum. We
are screening double mutants based on Acphot2 or Acphy3
mutant lines. As some double mutants have been obtained,
the function of Acphot1 might be clarified soon.

3- 2

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.
In fern we found a simple method for gene-silencing
using DNA fragments homologous to the target gene,
called DNAi. It has the advantage of being faster andsimpler than current RNAi approaches. To make DNAi amore powerful tool to study function-unknown genes inpteridophytes, expressed sequence tags (ESTs) wereobtained from a normalized cDNA library of Adiantum
capillus-veneris constructed from prothallia grown underwhite light. Clustering of 10,552 sequences in total
resulted in 7,132 non-redundant groups. Of these, 1,608
EST groups were found to be similar to sequences ofknown function and 1,092 EST groups showedsimilarities to sequences of unknown function.

II. Photomorphogenesis
2-1

DNA interference in Adiantum

Branching in Physcomitrella

Side branch formation in the moss Physcomitrella
patens has been shown to be light dependent andcryptochrome 1a and 1b (Ppcry1a and Ppcry1b) wereassigned as the blue light receptors for this response(Imaizumi et al. 2002). Here we analyzed thisphenomenon in detail, and revealed the complex nature of
the response where multiple photoreceptors are involved.
For branch induction, blue light of a fluence rate higher
than 6μmol m-2s-1 for period longer than 3 h was required.
Number of branches is dependent on fluence rate of blue
light but further increased when red light was applied
together with the blue light, although red light alone hadmuch less effect. By partial irradiation of a cell, both
receptive sites for blue and red light were found to belocated around the nucleus.
Both red and blue light determine the position of
branches in a way affected by the vibration plane ofpolarized light. The red light effect was nullified bysimultaneous far-red light irradiation, indicating
phytochrome involvement. The blue light effect was notfound in phototropin disruptants. Thus, dichroic
phytochrome and phototropin possibly on the plasma
membrane regulate branch position. Together, at leastfour
distinct
photoreceptor
systemsnamely
cryptochromes and red light receptors around or in the
nucleus, and dichroic phytochrome and phototropin
around the cell periphery - are involved in the light
induction of side branches in the moss Physcomitrella
patens.

Publication List:
Original papers

In order to elucidate the role of genes in Adiantum andrice, we have tried to establish new methods for genetargeting in these organisms.

Suetsugu, N., Kagawa, T., and Wada, M. (2005). An
auxilin-like
J-domainprotein,
JAC1,
regulates
phototropin-mediatedchloroplast
movementinArabidopsis thaliana. Plant Physiology 139,151-162.
Suetsugu, N., Mittmann, F., Wagner, G., Hughes, J., andWada, M. (2005). A chimeric photoreceptor gene,
NEOCHROME, has arisen twice during plant evolution.
Proc. Natl. Acad. Sci. USA 102, 13705-13709.
Tucker, E.B., Lee, M., Alli, S., Sookhdeo, V., Wada, M.,
Imaizumi, T., Kasahara, M., and Hepler, P.K. (2005).
UV-A induces two calcium waves in Physcomitrella
patens. Plant Cell Physiol. 46, 1226-1236.
Uenaka, H., Wada, M., and Kadota, A. (2005). Four distinct
photoreceptors contribute to light-induced side branchformation in the moss Physcomitrella patens. Planta 222,
623-631.
Yamauchi, D., Sutoh, K., Kanegae, H., Horiguchi, T.,
Matsuoka, K., Fukuda, H., and Wada, M. (2005). Analysisof expressed sequence tags in prothallia of Adiantum
capillus-veneris. J. Plant Research 118, 223-227.

3- 1

Review articles

III. Gene targeting and gene silencing

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
(Kikuchi et al. 2003). The mPing element was identified
as the first active MITE from any organism. mPing is a
short 430 base pair element with 15 base pair terminal
inverted repeats that lacks a transposase. mPing elements

Suetsugu, N., and Wada, M. (2005). Photoreceptor genefamilies in lower plants. In Handbook of Photosensory
Receptors, W.R. Briggs, and J.L. Spudich, eds. (Weinheim,
Wiley-VCH Verlag), pp. 349-369.
Wada, M. (2005). Chloroplast movement. In Light Sensing
in Plants, M. Wada, K. Shimazaki, and M. Iino, eds.
(Tokyo, Springer-Verlag), pp. 193-199.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

irradiation on PAC activation and showed thatintermittent irradiation using pulses of light and dark ofequal length (0.1-180 s) caused activation of PAC toalmost the same extent irrespective of the cycle periods.
This means that elevation of PAC activity occurs only
during the light period and that elevated PAC activity
falls off within 0.1 s after the termination of irradiation.
Such responsiveness is fast enough for PAC to mediate
the step-up photophobic response that occurs with several
subseconds’ latency.

LABORATORY OF PHOTOENVIRONMENTAL
BIOLOGY
Professor (Adjunct):
PRESTO Researcher:
Postdoctoral Fellows:
Visiting Scientist:
Technical Assistants:

WATANABE, Masakatsu
ISEKI, Mineo
YOSHIKAWA, Shinya
SUZUKI, Takeshi
ITO, Shinji
KOUMURA-SUZUKI, Yoshiko
ITO, Makiko

Photosynthetic microorganisms, such as cyanobacteria
and flagellate algae, respond to light to locate themselvesat an appropriate photoenvironment. Our research is
aimed at the elucidation of photoreceptive and signaltransduction mechanisms of the light responses in
microorganisms. This approach has lead us to thediscovery and characterization of a remarkably uniquelight sensor molecule as described below.

I. Photoactivated Adenylyl Cyclase (PAC)
In 2002, we found a novel blue-light receptor with aneffector role from Euglena (Iseki et al., Nature 415,
1047-1051, 2002): Euglena gracilis, a unicellular
flagellate, shows blue-light type photomovements. The
action spectra indicate the involvement of flavoproteins as
the photoreceptors mediating them. The paraflagellar
body (PFB), a swelling near the base of the flagellum, hasbeen 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
noncovalently bound FAD, seemed to be a heterotetramerof - and -subunits. Predicted amino acid sequences ofeach of the subunits were similar to each other andcontained two FAD-binding domains (BLUF: sensor ofblue light using FAD) each followed by an adenylyl
cyclase catalytic domain. The flavoprotein showed an
adenylyl cyclase activity, which was elevated byblue-light irradiation. Thus, the flavoprotein (PAC,
photoactivated adenylyl cyclase) can directly transduce alight signal into a change in the intracellular cyclic AMPlevel without any other signal transduction proteins.

1-1

Figure 1. A serial recording of the step-up photophobic
response of a Euglena cell by a high-speed video camera
under infrared observation light (from top of left column tobottom of right column, 20 ms intervals).

We also reported wavelength dependency of PAC
activation between 260-650 nm at equal quanta. The
curve of wavelength dependency showed prominent peaks
at 290 nm, 390 nm and 450 nm, which agrees well bothwith the absorption spectrum of FAD and the action
spectrum for the step-up photophobic response of
Euglena. All the above properties of PAC activation
confirm that PAC actually acts as a photoreceptor for the
step-up photophobic response.

1-2 The change in intracellular cAMP levels upon
blue light irradiation

Kinetic properties of PAC photoactivation

Although PAC appeared to be a photoreceptor for the
step-up photophobic response (Figure 1), physiologicalevidence demonstrating that the photoactivation of PAC
actually causes the step-up photophobic response islacking. Recently, we reported the kinetic properties ofin vitro activation of PAC by light, comparing them withthose of the step-up photophobic response (Yoshikawa et
al. 2005). We showed that activation of PAC is dependentboth on photon fluence rate and the duration of irradiationand that reciprocity held well in the range of 2-50 mol
m-2 s-1 (total fluence of 1,200 mol m-2), suggesting that
activation of PAC is entirely dependent on total photon
fluence. We also examined the effects of intermittent

Since in vitro activity of PAC was elevated by blue
light irradiation, it is easy to think that an increase in
intracellular cAMP level evoked by photoactivation of
PAC is a major cause of the step-up photophobic response.
So far, however, there has been no experimental evidence
that blue light irradiation induces an increase in
intracellular cAMP level of Euglena. We measured
intracellular cAMP before and after onset of blue lightirradiation, which showed that the intracellular cAMPlevel remarkably increased within 1 s of the onset of
irradiation and the increased cAMP level decreased
within 10 s and gradually returned to the initial level evenif irradiation continued. The time course of intracellular64

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYhydrogen bond network to the flavin. We reported thatwhen recombinant F2 proteins were mutated at thetyrosine (Tyr472) or glutamine (Gln514) residues,
corresponding to Tyr21 and Gln63 in AppA, nophotoinduced spectral shift was observed. Thus, themechanism of photoactivation of the recombinant F2 in
PAC seems essentially the same as that of theprokaryotic BLUF proteins, though the kinetic properties
are slightly different from each other: e. g., the half-life
was 34-44 s at 25 ˚C for the recombinant PAC F2,
whereas that reported for prokaryotic BLUF domains
varies from ca. 3.5 s (Tll0078) to ca. 900 s (AppA).

cAMP coincided well with the process of the step-up
photophobic response. This strongly suggests that theincrease in intracellular cAMP evoked by photoactivationof PAC is a key event in the step-up photophobic
response.

Publication List:
Original papers
Fujita, S., Iseki, M., Yoshikawa, S., Makino, Y., Watanabe,M., Motomura, T., Kawai, H., and Murakami, A. (2005).
Identification and characterization of a flagellar
fluorescent protein of brown alga Scytosiphon lomentaria(Scytosiphonales,
Phaeophyceae):aflavoprotein
homologous to Old Yellow Enzyme. Eur. J. Phycol. 40,
159-167.
Ito, S., Murakami, A., Sato, K., Nishina, Y., Shiga, K.,
Takahashi, T., Higashi, S., Iseki, M., and Watanabe, M.
(2005). Photocycle features of heterologously expressed
and assembled eukaryotic flavin-binding BLUF domainsof photoactivated adenylyl cyclase (PAC), a blue-light
receptor in Euglena gracilis. Photochem. Photobiol. Sci. 4,
762-769.
Ohnishi, N., Allakhverdiev, S.I., Takahashi, S., Higashi, S.-I.,
Watanabe, M., Nishiyama, Y., and Murata, N. (2005).
Two-step mechanism of photodamage to photosystem II:
step 1 occurs at the oxygen-evolving complex and step 2
occurs at the photochemical reaction center. Biochemistry
44, 8494-8499.
Okajima, K., Yoshihara, S., Fukushima, Y., Geng, X.,
Katayama, M., Higashi, S.-I., Watanabe, M., Sato, S.,
Tabata, S., Shibata, Y., Itoh, S., and Ikeuchi, M. (2005).
Biochemicaland functional characterization of
BLUF-type flavin-binding proteins of two species of
cyanobacteria. J. Biochem. 137, 741-750.
Yoshikawa, S., Suzuki, T., Watanabe, M., and Iseki M.
(2005). Kinetic analysis of the activation of photoactivatedadenylyl cyclase (PAC), a blue-light receptor forphotomovements of Euglena. Photochem. Photobiol. Sci.
4, 727-731.

Figure 2. Schematic representation of PAC involvement in
the step-up photophobic response of Euglena. PAC isactivated by UV-B/C, UV-A and blue lights in a photon
fluence-dependent manner to produce cAMP, which
increases intracellular cAMP. The increase in intracellularcAMP triggers the step-up photophobic response, being
followed by a decrease in intracellular cAMP, probably
mediated by PDE, which brings about recovery from thestep-up photophobic response.

1-3

Photoinduced spectral shift of BLUF domains

The mechanism of photoactivation of PAC is a subjectof great interest. However, it is difficult to obtain enough
PAC from Euglena cells for photochemical and structural
analyses because the efficiency of PAC purification is
low; only several micrograms of PAC can be obtained
from 30 litres of Euglena culture. It is also difficult toobtain heterologously expressed PAC while keeping its
activity intact because most of the expressed protein goes
into the insoluble fraction, so-called inclusion bodies.
Recently, we succeeded in obtaining the soluble
recombinant flavin binding domain (F2) of PAC byheterologous expression in E. coli by fusing the domain toglutathion-S-transferase (GST). The recombinant F2
sample contained both FAD and FMN with trace amountsof riboflavin and showed a spectral red shift uponblue-light irradiation followed by recovery in darkness.
Such photoinduced spectral shifts were first reported andwell characterized for the BLUF domain of AppA, a
regulator of photosynthesis gene expression in
Rhodobacter sphaeroides. The spectral shift is considered
to be caused by alterations in - stacking and hydrogenbonding between FAD and the tyrosine residue (Tyr21).
Recent reports on the crystal structures of BLUF domainsof AppA and a cyanobacterial protein Tll0078 indicatethat the glutamine residue (Gln63 in AppA and Gln50 in
Tll0078) play a crucial role in rearrangement of the

Review article
Watanabe, M., and Iseki, M. (2005). Discovery andcharacterization of photoactivated adenylyl cyclase (PAC),
a novel blue-light receptor flavoprotein, from Euglena
gracilis. In Handbook of Photosensory Receptors, W.R.
Briggs, and J.L. Spudich, eds. (Weinheim, Wiley-VCH
Verlag), pp. 247-260.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

extension. There are two forms, a and b, for type III
PI4Ks; the former consists of the PH domain and catalytic
domain with a long N-terminal extension and 200 to 230
kD in size, whereas the latter is 110 kD protein composedwith LKU, NH and catalytic domains. Plants also have allforms of PI4K.
Until to date, I have obtained only one full-length
cDNA encoding a  form of type III PI4K, PpPI4K1,
whose structure is very close to those of the  form oftype III PI4Ks from other species. Interestingly, although
the repetitive sequence domain between LKU and NH
domains was reported as a unique domain in Arabidopsis
AtPI4K1, the same domain was found in PpPI4K1(Figure 2), indicating that this domain is specific in plants.
Thus, PpPI4K1 is useful to investigate the plant-specificfunction of the type III PI4K.

LABORATORY OF STRESS RESPONSE
Associate Professor:

MIKAMI, Koji

As their sessile nature, growth and development ofplants are largely influenced by environmental factors;
thus, plants must have highly sophisticated systems tosense and respond to these factors. Much effort has been
put into the analysis of stress-signal transduction in plants,
which led us to the identification of stress-responsive
genes and transcription factors responsible for the
stress-dependent transcription. However, it is still
unknown how plants sense various kinds ofenvironmental stresses separately and how environmental
stresses are converted into intracellular signals. Based on
our knowledge in mammalian cells, it is possible that, inplants, the transmission of the signals via cell surface
receptors or sensors may result in the production of
intracellular second messengers, such as inositol
1,4,5-trisphosphate (IP3) and diacylglycerol (DG), both ofwhich initiate cascades of intracellular signal transduction
in response to environmental stresses. Moreover, since
phosphatidylinositols, some of which are precursors of thesecond messengers or are second messengers themselves,are produced by sequential phosphorylation anddephosphorylation via lipid kinases and lipid
phosphatases in the PI pathway (Figure 1), these reactions
could be activated by environmental stimuli. Indeed,stress-dependent production of PI 3,4-bisphosphate[PI(3,4)P2] and IP 3 has already been reported in various
kinds of plant species. It is, therefore, important toanalyze the PI pathway to understand the molecularmechanisms of sensing of and response to extracellularsignals in plants.
Since phosphoinositide-specific phospholipase C(PI-PLC) plays an essential role in the PI pathway
through generation of IP 3 and DG by the hydrolysis ofPI(3,4)P2 (Figure 1), I have investigated the functions of
PI-PLCs, PpPLC1 and PpPLC2, isolated from the moss
Physcomitrella patens, because Physcomitrella is now
recognized as a model system for plants with easy
application of molecular genetic approaches such as
gene-targeted mutagenesis via the homologous
recombination. However, there is no information about PI
pathway-regulating enzymes other than PI-PLCs in
Physcomitrella, thus it is necessary to analyze the lipid
kinases and phosphatases to confirm the similarity of the
PI pathways between Physcomitrella and higher plants.
Accordingly,
thestructuralcharacterizationofPhyscomitrella lipid kinases and PI-PLCs were
performed.

Figure 1. Schematic representation of the PI pathway.
Light
blue
and
yellowboxes
indicate
phosphoinositides and enzymes (PI synthase, lipid
kinases or lipid phosphatases), respectively. Current
situation of the cloning of Physcomitrella full-length
cDNAs encoding enzymes involved in the PI pathway
was indicated in the large box. PM, plasma
membrane.

I. PI 4-kinase
Figure 2. Structural characteristics of plant  form of type
III PI4Ks and PIPKs. Protein domains represent in
PpPI4K1 (A) and PpPIPK1 (B), in comparison with
Arabidopsis homologues. Numbers indicate amino acidpositions. Accession nos. are as follows; AtPI4K,AJ002685; AtPI5K1, Q56YP2. RSD, repetitive sequence
domain; CD, catalytic domain.

PI 4-kinase (PI4K) phosphorylates the D4 position of
PI to yield PI 4-phosphate [PI(4)P]. Two major types of
PI4Ks, type II and type III, have been identified inanimals and yeasts. The type II PI4Ks aremembrane-bound 55 kD enzymes, which consists of theconserved catalytic domain and small N-terminal

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NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGY

II. PI-phosphate kinase
PI-phosphate kinase (PIPK) catalyzes the synthesis ofPI(5)P, PI(3,5)P2 and PI(3,4)P2 by phosphorylation of PI
and PI-phosphates. In animals, PIP5Ks are classified into
three subfamilies, type I to III, according to their substratespecificity. Type I PIPKs are PI 5-kinases that
phosphorylate PI and PI(3)P to generate PI(5)P andPI(3,5)P2, while type II enzymes produce PI(3,4)P2 from
PI(3)P by their PI 4-kinase activity. Type III enzymes
catalyze phosphorylation of the D5 position of PI as PI
5-kinase. The first plant PIPK was Arabidopsis AtPIP5K1(Mikami et al., Plant J. 15, 563-568, 1998) whosecatalytic domain is similar to both animal type I and II
enzymes, thus AtPIP5K1 was classified as type I/II.
Indeed, it has been reported that AtPIP5K1 has both the
PI 4-kinase and PI 5-kinase activities. Interestingly,
AtPIP5K1 had a long N-terminal extension containing an
unidentified repetitive sequence that was named recently
a MORN repeat; however, the function of this repeat isstill unknown. Taken together, the structure of type I/II
PIPKs is plant-specific and, thus, the activation mode ofthem may also be plant-specific. Plants have the type III
PIPKs as is in animals.
I obtained a Physcomitrella full-length cDNA encoding
PIPK, designated PpPIPK1. As shown in Figure 2,
PpPIPK1 consists of a N-terminal extension containing
the MORN repeat and the catalytic domain classified as
the type I/II enzyme. As the conservation of plant-specificstructure between Physcomitrella and Arabidopsis,
PpPIPK1 is useful to analyze the plant-specific mode ofactivation and functions of plant type I/II PIPKs.

Figure 3. Structural characteristics of five Physcomitrella
PI-PLCs. Protein domains represent in five Physcomitrella
PI-PLCs, PpPLC1 to PpPLC5, in comparison with AtPLC1
from Arabidopsis and RnPLC1 from Rattus norvegicus.
All PI-PLCs indicated have the X, Y and C2 domains,
although the PH (pleckstrin homology) domain, which
regulates membrane localization of animal PI-PLCs, is notfound in any plant PI-PLCs. Numbers indicate amino acidpositions. Accession nos. are as follows; PpPLC1,
BAD02919; PpPLC2, BAD02918; AtPLC1, BAA07547;
RnPLC1, NP_058731.

form of PI-PLC (Mikami et al., J. Exp. Bot. 55,1437-1439,
2004). In addition, I have cloned other 3 full-length
cDNAs encoding PpPLC3, PpPLC4 and PpPLC5. As
shown in Figure 3, PpPLC4 and PpPLC5 have structures
similar to that of PpPLC1, although PpPLC3 has
mutations in the Y domain and an insertion in the N
domain, which is similar to PpPLC2. Thus,
Physcomitrella seems to have three PI-PLC isozymes
whose structural organization is identical to those ofhigher plant PI-PLCs, although the inactivated forms suchas PpPLC2 and PpPLC3 are found in only Physcomitrella.
These findings indicate that Physcomitrella is useful toinvestigate the activation mode of plant PI-PLCs and thefunctional diversity among PI-PLC isoforms.

III. PI-PLC
As mentioned above, PLC catalyzes the hydrolysis ofPI(4,5)P2 to generate two second messengers in a Ca2+dependent manner. Although animal PI-PLCs wereclassified into 5 isozymes, named , , ,  and  types.
All of them contain the X and Y domains to compose
their catalytic domain and C2 domain as Ca2+-dependent
membrane-interacting
module,althoughisozymespecific-domain structures are composed by the
combination with other protein motifs in relation to theiractivation modes. It is well known that  and  isozymes
are activated by G protein-coupled receptors and receptor
tyrosine kinases, respectively, whereas the activation of 
isozymes is regulated by heteromeric G proteins and Ras.
In contrast to the variety of isozymes withisozyme-specific activation modes in animal PI-PLCs,
plant PI-PLCs reported showed the same structure that
resembles closely those of the  isozymes, which aresperm-specific enzymes composed by the two EF-handrepeats and X, Y and C2 domains. In plant PI-PLCs, there
is one EF-hand repeat called the N domain. Until to date,
it is unclear how plant PI-PLCs are activated.
It has been reported that two Physcomitrella PI-PLC,
PpPLC1 and PpPLC2, are structurally close to plant PLCs
reported, however PpPLC2 containing point mutations in
the Y domain and an insertion in the N domain is inactive

IV. Conclusion
Structures of lipid kinases and PI-PLCs involved in the
PI pathway in Physcomitrella are basically similar to
those in higher plants, suggesting the conservation of theactivation modes and, probably, functions between lowerand higher land plants. As gene-targeted disruption byhomologous recombination is available in Physcomitrella,
the functions of enzymes presented above could beanalyzed directly. In fact, since I already produced
gene-targeted mutants of PpPLC5 and PpPIPK1 inaddition to PpPLC1, analyses of these mutants may
provide new insight into the activation modes andfunctions of PIPK and PI-PLC in stress responses anddevelopment.

67

NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY

decreasingly depending on both phosphorylated andnon-phosphorylated KaiC. We examined all the possible
regulation patterns without using biological assumption
and determined the condition for oscillation by means of
the linear stability analysis. We determined that there are
only two possible patterns in the transcriptional regulation
to realize circadian oscillation (Figures 1 & 2). One of thetwo patterns does not include the direct negative feedback
regulation, but successfully exhibits the oscillatory
behavior by positive feedback of the transcriptional
regulation. Based on this result, we proposed that positive
autofeedback may be a novel mechanism for circadianoscillation.

DIVISION OF THEORETICAL BIOLOGY
Associate Professor:
NIBB Research Fellows:
Graduate Student:
Visiting Scientists:
Secretary:

MOCHIZUKI, Atsushi
TOHYA, Shusaku
FUJITA, Hironori
IMAMURA, Hisako
AYABE, Yoshiko
YAMAGAMI, Ayumi
UMEBAYASHI, Hiromi

We are studying biological phenomena using
mathematical models. This method gives us an integrative
understanding of the behavior of complex systems inbiology including gene regulatory networks.
Mathematical models are also useful in understanding
pattern formation in development. Study of themechanisms responsible for morphological differences
between species is an important research focus of current
developmental biology. Theoretical studies would be
useful in identifying candidates of cell-cell interactionthat are likely to be responsible for the systems in realorganisms.

Figure 1. Functional schemes of two models. (Left) The
Transcriptional Activation Model, where P-KaiC induces
kaiBC transcription. NP-KaiC does not affect ontranscription, or has a small effect of repressing
transcription. (Right) The Transcriptional Repression
Model, where P-KaiC represses kaiBC transcription.

I. Transcriptional autoregulation in cyanobacterial
circadian rhythms
Among the wide variety of organisms exhibiting
circadian rhythms, cyanobacteria are the simplest
organisms and may provide a model to understand thebasic mechanism of the sustained rhythmicity
physiologically. In the cyanobacterium Synechococcus
elongatus PCC 7942, clock genes kaiA, kaiB and kaiC
have been characterized as the indispensable clock
regulators. The kai genes form a gene cluster and kaiBand kaiC are co-transcribed as kaiBC mRNA. KaiC playsa central role and exhibits rhythms in transcription,translation and phosphorylation status under continuous
illumination conditions. The other clock proteins KaiA
and KaiB modulate KaiC autophosphorylation: KaiA
enhances autophosphorylation of KaiC, and KaiB inhibits
this action of KaiA.
The transcriptional regulation of kaiBC is not fully
understood yet, though experimental evidences suggest
the autoregulation by KaiC, which may work as a positiveor negative regulator depending on the phosphorylation
status. The gene of kaiB, which forms an operon with
kaiC, also seems to affect this regulation process,inhibiting KaiA-enhanced phosphorylation of KaiC.
In this study, we investigated and predicted the possible
mechanisms of the transcriptional regulation by KaiC andits phosphorylated state to realize circadian oscillation
using a mathematical model. Considering theexperimental
results,
phosphorylated
and
non-phosphorylated KaiC may play different roles in thetranscriptional regulation. We developed a dynamical
model including the concentrations of phosphorylated,
non-phosphorylated KaiC, and kaiBC mRNA. We canchoose different transcriptional regulation functions
which may switch their values increasingly or

Figure 2. The result of linear stability analysis. The
horizontal axis is , the angle of the threshold line oftranscriptional switching, and the vertical axis is thephosphorylation rate. The  specifies patterns of thetranscriptional regulation, which are shown schematicallybelow the horizontal axis. Along the horizontal axis, theregulation changes continuously; effects of NP-KaiC and
P-KaiC are positive or negative. Open circles indicate
conditions of the transcription and phosphorylation rates
where oscillation never occurs, and filled circles indicate
where oscillation can occur.

Secondly we verified if the determined conditions can
explain the cyanobacterial circadian mechanism,comparing the behaviors in computer simulation with theexperimentally observed phenotypes.
Experiments in circadian systems of Neurospora,
Drosophila, and mammals as well as theoretical studies
have long underlined the importance of the negativetranscriptional regulation for the generation of oscillations.

68

NATIONAL INSTITUTE FOR BASIC BIOLOGYENVIRONMENTAL BIOLOGYhydrogen bond network to the flavin. We reported thatwhen recombinant F2 proteins were mutated at thetyrosine (Tyr472) or glutamine (Gln514) residues,
corresponding to Tyr21 and Gln63 in AppA, nophotoinduced spectral shift was observed. Thus, themechanism of photoactivation of the recombinant F2 in
PAC seems essentially the same as that of theprokaryotic BLUF proteins, though the kinetic properties
are slightly different from each other: e. g., the half-life
was 34-44 s at 25 ˚C for the recombinant PAC F2,
whereas that reported for prokaryotic BLUF domains
varies from ca. 3.5 s (Tll0078) to ca. 900 s (AppA).

cAMP coincided well with the process of the step-up
photophobic response. This strongly suggests that theincrease in intracellular cAMP evoked by photoactivationof PAC is a key event in the step-up photophobic
response.

Publication List:
Original papers
Fujita, S., Iseki, M., Yoshikawa, S., Makino, Y., Watanabe,M., Motomura, T., Kawai, H., and Murakami, A. (2005).
Identification and characterization of a flagellar
fluorescent protein of brown alga Scytosiphon lomentaria(Scytosiphonales,
Phaeophyceae):aflavoprotein
homologous to Old Yellow Enzyme. Eur. J. Phycol. 40,
159-167.
Ito, S., Murakami, A., Sato, K., Nishina, Y., Shiga, K.,
Takahashi, T., Higashi, S., Iseki, M., and Watanabe, M.
(2005). Photocycle features of heterologously expressed
and assembled eukaryotic flavin-binding BLUF domainsof photoactivated adenylyl cyclase (PAC), a blue-light
receptor in Euglena gracilis. Photochem. Photobiol. Sci. 4,
762-769.
Ohnishi, N., Allakhverdiev, S.I., Takahashi, S., Higashi, S.-I.,
Watanabe, M., Nishiyama, Y., and Murata, N. (2005).
Two-step mechanism of photodamage to photosystem II:
step 1 occurs at the oxygen-evolving complex and step 2
occurs at the photochemical reaction center. Biochemistry
44, 8494-8499.
Okajima, K., Yoshihara, S., Fukushima, Y., Geng, X.,
Katayama, M., Higashi, S.-I., Watanabe, M., Sato, S.,
Tabata, S., Shibata, Y., Itoh, S., and Ikeuchi, M. (2005).
Biochemicaland functional characterization of
BLUF-type flavin-binding proteins of two species of
cyanobacteria. J. Biochem. 137, 741-750.
Yoshikawa, S., Suzuki, T., Watanabe, M., and Iseki M.
(2005). Kinetic analysis of the activation of photoactivatedadenylyl cyclase (PAC), a blue-light receptor forphotomovements of Euglena. Photochem. Photobiol. Sci.
4, 727-731.

Figure 2. Schematic representation of PAC involvement in
the step-up photophobic response of Euglena. PAC isactivated by UV-B/C, UV-A and blue lights in a photon
fluence-dependent manner to produce cAMP, which
increases intracellular cAMP. The increase in intracellularcAMP triggers the step-up photophobic response, being
followed by a decrease in intracellular cAMP, probably
mediated by PDE, which brings about recovery from thestep-up photophobic response.

1-3

Photoinduced spectral shift of BLUF domains

The mechanism of photoactivation of PAC is a subjectof great interest. However, it is difficult to obtain enough
PAC from Euglena cells for photochemical and structural
analyses because the efficiency of PAC purification is
low; only several micrograms of PAC can be obtained
from 30 litres of Euglena culture. It is also difficult toobtain heterologously expressed PAC while keeping its
activity intact because most of the expressed protein goes
into the insoluble fraction, so-called inclusion bodies.
Recently, we succeeded in obtaining the soluble
recombinant flavin binding domain (F2) of PAC byheterologous expression in E. coli by fusing the domain toglutathion-S-transferase (GST). The recombinant F2
sample contained both FAD and FMN with trace amountsof riboflavin and showed a spectral red shift uponblue-light irradiation followed by recovery in darkness.
Such photoinduced spectral shifts were first reported andwell characterized for the BLUF domain of AppA, a
regulator of photosynthesis gene expression in
Rhodobacter sphaeroides. The spectral shift is considered
to be caused by alterations in - stacking and hydrogenbonding between FAD and the tyrosine residue (Tyr21).
Recent reports on the crystal structures of BLUF domainsof AppA and a cyanobacterial protein Tll0078 indicatethat the glutamine residue (Gln63 in AppA and Gln50 in
Tll0078) play a crucial role in rearrangement of the

Review article
Watanabe, M., and Iseki, M. (2005). Discovery andcharacterization of photoactivated adenylyl cyclase (PAC),
a novel blue-light receptor flavoprotein, from Euglena
gracilis. In Handbook of Photosensory Receptors, W.R.
Briggs, and J.L. Spudich, eds. (Weinheim, Wiley-VCH
Verlag), pp. 247-260.

65

NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY

strongly diffusible auxin resource and its consumer(Figure 3).

The negative feedback regulation of clock genes has been
considered responsible for generating oscillation. On theother hand, the contribution of positive feedback incircadian clock is much less well understood. The
Transcriptional Activation Model we proposed in thisreport is distinct from circadian oscillator models thathave been proposed previously.

II. Mathematical models for pattern formation ofleaf vascular networks
The vascular system of a plant is a network of bundles
that connects within major organs. The development or
differentiation of vascular systems is one of the mostimportant subjects in botany, and a lot of studies havefocused on 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, locatedat the center of a leaf, is called the midvein (or primaryvein), and is generated first. Veins differentiate
progressively in an early developmental stage of the leaf.
A vein, which is continuous with the stem vascular bundle,
extends from the proximal edge to the distal end of theleaf, and then become a midvein. Secondary veins are
generated from branches of the midvein and are thinnerthan the midvein. By repeated branching and extension,
higher order veins are generated sequentially with thegrowth of the leaf and the reticulate network system isformed.
Although a lot of experimental studies have attempted
to identify the mechanism of vascular patterning in leaves,
it is still a problem to solve. It has been revealed that theauxin is important 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 a diffusible molecule; however,
it is believed that auxin is also transported from apical tobasal in a stem, from distal to proximal in a leaf. This iscalled auxin polar transport. In a leaf, auxin is thought tobe produced in the apical margins of leaves andtransported toward the proximal regions. Many
researchers believe that this kind of auxin flow plays animportant role in the vascularization of plants.
Three hypotheses have been proposed so far to explain
the leaf venation pattern formation. One is the auxin
canalization hypothesis proposed by T. Sachs (1981) thatis based on the assumption of the positive feedback
regulation between auxin flow and flow capacity. Auxin
is a diffusible plant hormone of small molecules and has
important roles in various developmental events including
vascular differentiation in plants. In this hypothesis, auxin
pathways are thought to be generated from a uniform field
and are extended in a sequential manner. The second is
the activator-inhibitor type reaction-diffusion hypothesis
proposed by H. Meinhardt (1982) that is based on two
interactive factors of weakly diffusible activator andstrongly diffusible inhibitor. We proposed the third
hypothesis, the substrate-depletion type reaction-diffusion
model, which is based on two interactive factors of

Figure 3. Branching pattern generated by computer
simulation
basedon
substrate-depletion
type
reaction-diffusion model. Veins grow toward the margin
with repeating branching. In the final state, regularinterval branching patterns are formed.

On the other hand, we tried to integrate the canalization
hypothesis and the experimental knowledge. We
introduce auxin transport by PIN1 efflux carrier into the
auxin canalization hypothesis. We investigated a model
based on the assumption of the positive feedback
regulation between auxin flow and PIN1 localization(Figure 4).
The substrate-depletion type reaction-diffusion model
can regenerate growth and branching patterns. On theother hand, the canalization model can generate closed
circuits of leaf veins under a condition.
Bothmechanisms seem to be responsible for the leaf vein
formation.

Figure 4. Leaf vein 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:
Original papers
Feugier, F.G., Mochizuki, A., and Iwasa, Y. (2005).
Self-organization of the vascular system in plant leaves:Inter-dependent dynamics of auxin flux and carrier
proteins. J. theor. Biol. 236, 366-375.
Mochizuki, A. (2005). An analytical study of the number ofsteady states in gene regulatory networks. J. theor. Biol.
236, 291-310.
Mochizuki, A., Yahara, K., Kobayashi, I., and Iwasa, Y.
(2005). Genetic addiction: selfish gene's strategy forsymbiosis in the genome. Genetics, in press.
Takigawa-Imamura, H., and Mochizuki, A. (2005).

69

NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGYTranscriptional autoregulation by phosphorylated andnon-phosphorylated KaiC in cyanobacterial circadianrhythms. J. theor. Biol. in press.

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NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY

We have been developing and maintaining a databasesystem for comparative analysis of microbial genomes
named MBGD (http://mbgd.genome.ad.jp/) (Figure 1).
The key components of MBGD include i) an efficientalgorithm that can classify genes into orthologous groups
using precomputed all-against-all similarity search results(see below), ii) a user interface that is designed for users
to explore the resulting classification in detail, and iii) an
incremental updating process for similarities between
genes and other data, which enables the system to provide
the latest data rapidly. By this approach, MBGD is now
the world’s largest database of its kind. Moreover, byspecifying a set of organisms, users can obtain theappropriate classification results that they require using
the latest data available.
The constructed classification table can be used forcomparative analyses from various points of view, such as
phylogenetic pattern analysis, gene order comparison anddetailed gene structure comparison (Figure 1).

LABORATORY OF GENOME INFORMATICS
Research Associate:

UCHIYAMA, Ikuo

The 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 this 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 evolutionary clues tointerpreting genomic information of various species. The
current focus of our research topics is on comparative
analysis of microbial genomes, the number of which is
now beyond a hundred, as a basic model system forunderstanding the variety of life through the comparative
analysis of numerous genomic sequences simultaneously.

II. Hierarchicalclustering
algorithm
forconstructing orthologous groups of multiple
genomesI. Construction of microbial genome database forcomparative analysis

Although ortholog identification is a crucial first step incomparative genomics, a scheme for large-scale ortholog
grouping is yet to be established. The conventional
approach to the identification of orthologs between two
genomes is the so-called bidirectional best-hit (BBH)
criterion, where two genes, a and b, in the genomes A andB, respectively, are considered to be orthologs if a is thebest hit of b in genome A and vice versa. The Clusters of
Orthologous Groups (COGs) Database, a widely usedcurated database for ortholog grouping, was constructedusing this approach, although the overall construction
process has included additional complex procedures such

The number of completed microbial genome sequences
is growing rapidly, and nearly three hundred genome
sequences of various levels of relatedness have alreadybeen made available. The role of comparative genomics
becomes much more important in order to utilize thislarge 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.

Figure 1. A flow diagram of the MBGD user interface

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NATIONAL INSTITUTE FOR BASIC BIOLOGYTHEORETICAL BIOLOGY

III. Identification of the common core structure ofphylogenetically related genomes

as the addition of species-specific paralogs, the splittingof proteins into multiple domains if required, and othercase-by-case manual modifications
As a part of the core technologies of the MBGD system,
we have developed a rapid automated method of ortholog
grouping which is effective enough to allow thecomparison of hundreds of genomes simultaneously. The
method takes as input all-against-all similarity data andclassifies genes based on the traditional hierarchicalclustering algorithm UPGMA. In the course of clustering,the method detects domain fusion or fission events, andsplits clusters into domains if required. The subsequent
procedure splits the resulting trees such that intra-species
paralogous genes are divided into different groups so as tocreate plausible orthologous groups. As a result, theprocedure can split genes into the domains minimallyrequired for ortholog grouping.
The procedure, named DomClust, was tested using the
COG database as a reference. When comparing several
clustering algorithms combined with the conventional
BBH criterion, we found that our method generally
showed better agreement with the COG classification. By
comparing the clustering results generated from datasetsof different releases, we also found that our method
showed relatively good stability in comparison to theBBH-based methods.

It is known that horizontal transfer as well as verticaltransfer has played important roles in prokaryotic
evolution. Because of this complexity, further
investigation is required in order to obtain a clearer
picture of the bacterial genome evolution. Extensive
comparison of multiple genomes that are closely ormoderately related to each other should give many clues
to understanding evolutionary processes. Such data is now
rapidly accumulating in our MBGD database.
We are trying to identify a common “core structure” ofphylogenetically related genomes, which is defined as aset of sufficiently long consecutive genomic segments inwhich gene orders are conserved among multiple
genomes so that they are likely to have been inheritedfrom the common ancestor mainly through verticaltransfer. For this purpose, we have developed agraph-based algorithm for aligning conserved regions ofmultiple genomes by ordering orthologous groups so as toretain the conserved gene orders as much as possible.
The method was applied to the comparison of
Bacillus-related species whose genome sequences havebeen determined including alkaliphilic B. halodurans,
halotolerant Oceanobacillus iheyensis and thermophilic
Geobacillus kaustophilus, in addition to well-known
laboratory strain B. subtilis and pathogenic B. anthracis
and B. cereus. These organisms – except for B. anthracis
and B. cereus – are moderately diverged each other andbelong to distinct major clusters in the 16S rRNA
phylogenetic tree. Overall genomic structures areprimarily well conserved between them, which can be
confirmed by dotplot analyses where large collinear
regions along the diagonal lines can easily be seen.
Using orthologous groups of Bacillus-related species
with Staphylococcus aureus as an outgroup generated by
the DomClust program, we constructed genomealignments by the above algorithm. From this alignment,
we were able to identify the common core structure of
Bacillus genomes comprising about 1500 genes. It
appears that most of the important genes are included in
the resulting core gene set. Indeed, the set contains mostof 271 B. subtilis essential genes that were primarily
determined by a systematic inactivation experiment.
Further investigation of the core gene set revealed
characteristic distributions of function categories in thecore and non-core gene sets.

Figure 2. Orthologous groups of RNA polymerase beta
(RpoB) and beta’ (RpoC) subunits, as an example of
DomClust classification. A) Hierarchical clustering trees
constructed by the DomClust program. Each domain isdrawn in a different color. An abbreviated species name(taken from the COG database) is shown on each leaf,
which is colored according to the kingdom: salmon,
bacteria; khaki, archaea; sky-blue, eukaryotes. B)
Schematic illustration of the gene structures of RpoB and
RpoC in selected genomes.

Publication List:
Original paper
Ishikawa, K., Watanabe, M., Kuroita, T., Uchiyama, I.,
Bujnicki, J.M., Kawakami, B., Tanokura, M., andKobayashi, I. (2005). Discovery of a novel restriction
endonuclease by genome comparison and application of awheat-germ-based cell-free translation assay: PabI
(5'-GTA/C) from the hyperthermophilic archaeon
Pyrococcus abyssi. Nucleic Acids Res. 33, e112.

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NATIONAL INSTITUTE FOR BASIC BIOLOGY

The main activities of the Department in 2005

STRATEGIC PLANNING DEPARTMENT
Chair:
Vice-Chairs:
Professor:
Associate Professor:
Technical Assistants:

1) Supporting international conferences (Table 1)
2) Management of education-related programs
20th Bioscience Training Course
Internship Program 2005
3) Editing of publications
Bulletin of NIBB 2005 (in Japanese)Annual Report of NIBB 2005 (in English)
4) Production of posters and leaflets for international
conferences andadvertisements
for entrance
examination of graduate school(Table 2, Figure1)5) Updating and maintenance of NIBB home page6) Publication of NIBB News
(Intra-institutional newsletter in Japanese)
7) Maintenance of archives8) Dealing with visitors
9) Other various performances related to NIBB activities

NAGAHAMA, Yoshitaka
UENO, Naoto
MOROHASHI, Ken-ichirou
WADA, Masamitsu
KODAMA, Ryuji
MAKIHARA, Nobuko
MUKOHDA, Yasuyo
OTA, Misaki
SUGIYAMA, Tomomi
TANAKA, Megumi
TSUGE, Toyoko

The Strategic Planning Department was founded in
April 2005 to perform and/or to support efficientmanagement for cooperation with other organizations,distribution of public information, planning conferences,
symposia and other activities and administration of the
National Institute for Basic Biology.

Table 1. International conferences managed by Strategic Planning Department

Table 2. Products of Strategic Planning Department in the occasion of events listed bellow

1Bioscience Training Course Internship Program

MBSJ Meeting

National Institute for Fusion Science, 2 Molecular Biology Society of JapanNIBB-EMBL Symposium

Figure 1. Some examples of posters and panels designed by Strategic Planning Department

72

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORT

Action spectroscopical studies for various regulatoryand damaging actions of light on living organisms,
biological molecules, and artificial organic molecules
have been conducted (Watanabe, In “CRC Handbook of
Organic Photochemistry and Photobiology, 2nd ed.”. pp.
115-1~115-16, 2004).
An advanced irradiation system composed of CW
lasers (364nm, 390-410nm, 440-460nm, 532nm, 655nm,
752nm) and uniform-fluence-rate irradiation optics
interconnected by optical fibers was constructed in 2003.
An advanced observation system for cellular andintracellular photobiological responses utilizing a
two-photon microscope (FV300-Ix71-TP with a MaiTailaser) and a microbial photomovement analyzer(WinTrack2000/Ecotox) was also introduced.

RESEARCH SUPPORT FACILITIESHead:NISHIMURA, Mikio

Large Spectrograph Laboratory
Professor (Adjunct):
WATANABE, Masakatsu
Technical Staffs:
HIGASHI, Sho-ichi
NAKAMURA, Takanori
Technical Assistant:
ICHIKAWA, Chiaki
Tissue and Cell Culture Laboratory
Research Associate:
HAMADA, Yoshio
Technical Assistant:
TAKESHITA, Miyako
Computer Laboratory
Research Associate:
Technical Staffs:
Technical Assistants:

UCHIYAMA, Ikuo
MIWA, Tomoki
NISHIDE, Hiroyo
MAKIHARA, Nobuko
YAMAMOTO, Kumi

2. Tissue and Cell Culture Laboratory
Various types of equipment for tissue and cell culture
are provided. This laboratory is equipped with safety
rooms which satisfy the P2/P3 physical containment level.
This facility is routinely used for DNA recombination
experiments.

Plant Culture, Farm, Plant Cell Laboratory
Technical Staff:
NANBA, Chieko
Technical Assistant:
SUZUKI, Keiko

1. The Large Spectrograph Laboratory
This laboratory provides, for cooperative use, thelargest spectrograph in the world, the Okazaki Large
Spectrograph (OLS), dedicated to action spectroscopicalstudies of various light-controlled biological processes.
The spectrograph runs on a 30kW Xenon arc lamp andhas a compound grating composed of 36 smaller
individual gratings. It projects a spectrum of a wavelength
range from 250nm (ultraviolet) to 1,000nm (infrared) onto
its focal curve of 10m in length. The fluence rate
(intensity) of the monochromatic light at each wavelength
is more than twice as much as that of the corresponding
monochromatic component of tropical sunlight at noon
(Watanabe et al., Photochem. Photobiol. 36, 491-498,
1982).

Figure 1.

3. Computer Laboratory
The Computer Laboratory provides computation
resources and the means for electronic communication.
Currently, the main system consists of three servers andtwo terminal workstations: biological information
analysis server (SGI Origin 2000), database server (SunEnterprise 450), file server (Sun Enterprise 220R), datavisualization terminal and molecular simulation terminal(both are SGI Octanes). Some personal computers andcolor/monochrome printers are also available. On thissystem we provide various biological databases and data
retrieval/analysis programs, and support large-scale data
analysis and database construction for the institutemembers. At the end of this year, a new computer systemwith enhanced performance will be introduced as areplacement for the current system. The new system will
be available for use at the beginning of next year.
The Computer Laboratory also provides network
communication services within the institute. Most of thecomputers in each laboratory, as well as all of the above
service machines, are connected to each other by a local
area network (LAN) which is linked to the highperformance 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/).

The Large Spectrograph

4. Plant Culture Laboratory

The NIBB Cooperative Research Program for the Useof the OLS supports about 20 projects every year
conducted by visiting scientists including foreign
sicientists as well as those in the Institute.

This laboratory contains a large number of cultureboxes and a limited number of rooms with environmental
control for plant culture. In some of these facilities androoms, experiments can be carried out at the P1 physical73

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORT

containment level under extraordinary environments suchas strong light intensity, low or high temperatures, etc.

5. Experimental FarmThis laboratory consists of two 20 m2 glass-houses with
precise temperature and humidity control, three green
houses (each 6 m2) at the P1 physical containment level, asmall farm, and two greenhouses (45 and 88 m2) with
automatic sprinklers. The laboratory also includes abuilding with storage and work space.

6. Plant Cell Laboratory
This laboratory is equipped with autotrophic andheterotrophic culture devices and equipment forexperimental cultures of plant and microbial cells.
Facilities for preparation of plant cell cultures, including
an aseptic room with clean benches, are also provided.

Publication List:
Original Papers
Hasegawa, E. (2005). Comparison of the spectral sensitivityof juvenile red sea bream investigated by the physiological
technique and by the behavioral technique. Fisheries
Science 71, 79-85.
Ohnishi, N., Allakhverdiev, I.S., Takahashi, S., Higashi, S.,
Watanabe, M., Nishiyama, Y., and Murata, N. (2005). The
two mechanism of photodamage to photosystem II: Stepone occurs at the oxygen-evolving complex and step two
occurs at the photochemical reaction center. Biochem. 44,
8494-8499.
Okajima, K., Yoshihara, S., Fukushima, Y., Geng, X.,
Katayama, M., Higashi, S., Watanabe, M., Sato, S., Tabata,S., Shibata, Y., Itho, S., and Ikeuchi, M. (2005).
Biochemicaland functional characterization of
BLUF-type flavin-binding proteins of two species of
cyanobacteria. Japanese Biochemical Society 137,
741-750.
Nagai, Y., Nakamura, D., Ueno, H., Matsumoto, N., andOhishi, F. (2005). Photodegradation mechanisms in poly
(2,6-butylenenaphthalate-co-tetramethyleneglycol)(PBN-PTMG). II: Wavelength sensitivity of thephotodegradation. Polymer Degradation and Stability 88,
256-260.

74

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORTFigure 1. A laboratory room for computational studies

RESEARCH CENTER FOR INTEGRATIVE AND

For example, integrative methods are especially
important in understanding pattern formation indevelopment. Morphological difference between species
is an important research focus of the current
developmental biology. What is the mechanism
responsible for the difference of morphogenesis between
species? Theoretical studies are useful in identifying
candidates of cell or gene interaction that are likely to be
responsible for the systems in real organisms. Thismethod gives us an integrative understanding for the
behavior of complex systems in biology, including gene
regulatory networks.

COMPUTATIONAL BIOLOGYHead:Associate Professor:
Secretary:

TAKADA, Shinji
MOCHIZUKI, Atsushi
UMEBAYASHI, Hiromi

The aims of the research center for integrative andcomputational biology are (1) investigating fundamental
principles of various biological phenomena based on themethod of integration of computational science andbiology; (2) establishing new methodologies for
integrative biology; and (3) providing the new technology
and knowledge to researchers. Our final goal is toestablish a new bioscience that includes methods
originally utilized in different fields: informatics,
mathematics, and biology.

II. Collaboration activity
Interaction between researchers is essential to make thebest use of mathematical and computational methods forexperimental biology. The theoretical methods provide
testable predictions which the experimental biologists areable to investigate before returning the results to thetheoreticians for the next round of predictions. By
repeating these predictions and tests, the integrative
methods are continually developed. The research center
for integrative and computational biology has continued
to enhance interaction between theoretical biologists andexperimental biologists.
The center provides equipment such as cluster
machines and Unix-based machines for computation andexperiments. Members can also use computational
equipment at the Research Center for Computational
Science in Okazaki. The center also provides
experimental equipment for collaborations withexperimental biologists.

I. Research activity
The research center for integrative and computational
biology was founded in 2001 in a time of rapid progressin modern biology. The success in world wide genome
projects has provided a huge amount of new information
on genes, leading to expectations of advances in thedevelopment of new medicines for intractable diseases,
the exploitation of new cultivated plants resistant tonoxious insects, and so on. In addition, it is the object ofpresent-day biology to research higher-order phenomenathat are constructed from complex interactions between
many genes. To grapple with these questions, it isessential to decipher huge amounts of gene information,and to derive the essence for the biological behavior ofcells or organisms.
Mathematical and computational sciences have strong
capacities for dealing with these questions. Computational
methods make it possible to process ever-increasing
amounts of data. Hypothetical experiments (including theevolution of past organisms) based on mathematical orcomputational models make it possible to consider
conditions which are impossible in real experiments.
We are continuing to research higher-order phenomena inbiology by using mathematical or computational methods,
and to develop new methodology for studying suchcomplex phenomena.

Figure 2. A cluster machine

In 2005, we held a symposium on March 8-9 entitled"Mathematical and Computational Approaches to
Biology." Dozens of researchers studying mathematical,
computational, or experimental biology participated. The
center is dedicated to encouraging young researchers who
can use these methods for continued research into
complex biological phenomena.

75

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORTthe Yamate area.

CENTER FOR

Technical staff and supporting staff develop andpromote research-supporting activities. In 2003 two
associate professors joined the CTAP. A new CTAP
building for transgenic animals in the Yamate area openedat the end of 2003.

TRANSGENIC ANIMALS AND PLANTS
Head:
Associate Professors:
Technical Staffs:

Supporting Staffs:

TAKADA, Shinji
WATANABE, Eiji
SASAOKA, Toshikuni
TANAKA, Minoru
HAYASHI, Kohji
ICHIKAWA, Youko
ICHIKAWA, Hiromi
TAKAGI, Yukari
KOBAYASHI-NISHIMURA,Keiko
YASUDA, Mie
YAMAGUCHI, Kyoko
NOGUCHI, Yuji
YOSHIDA, Etsuko
KAWAMURA, Motofumi

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

I. Research supporting activity (mouse)
The worldwide genome project has been almost
completed and research on basic biology has arrived at apost-genome era in which researchers focus on
investigating the function of individual genes. To promotefunctional analysis of a gene of interest it is essential toutilize genetically altered model organisms, which are
generated by the genetic engineering technology, thecreation of gene deletion, gene replacement or point
mutation.
The NIBB Center for Transgenic Animals and Plants(CTAP) was established in April 1998 to support researchusing transgenic and gene targeting techniques at NIBB.
The CTAP is managed by the head (professor, aconcurrent post) and an associate professor.

In 2001 the NIBB mouse facility (built under specificpathogen free (SPF) conditions) opened in the Myodaiji
area and the production, breeding, analysis,
cryopreservation and storage of genetically manipulatedmouse strains has been conducted there since then. The
new CTAP building in the Yamate area strengthened
research activities using genetically altered organisms.
The building has five floors and a total floor space of
2,500m2, in which we can generate, breed, store andanalyze transgenic, gene targeting and mutant mice under
SPF conditions. 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, personnel, articles of animal housing and equipmentfrom the clean area side to the semi-clean area side.

Figure 2.

Figure 1.

Autoclaves of a large scale for sterilization

In 2005, 4,122 mice were brought into the CTAP in the
Yamate area, and 20,521 mice (including pups bred in thefacility) were taken out from the CTAP from April 01,
2005 to October 31, 2005.

A new center facility for transgenic animals in

76

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORTA number of strains of genetically altered mice outside
the CTAP were brought into 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 opened in
the beginning of 2005. The facility provides
research-supporting activities to researchers in the
Myodaiji area. In 2005, 958 mice were brought into the
CTAP in the Myodaiji area, and 2,321 mice (including
pups bred in the facility) were taken out from the CTAP
from April 01, 2005 to October 31, 2005.

Figure 4.

Breeding equipments for transgenic chickens

III. Academic activity
The associate professors of this center - E. Watanabe, T.
Sasaoka, and M. Tanaka - are the principal investigatorsof the Laboratory of Neurophysiology, the Laboratory of
Neurochemistry and the Laboratory of Molecular
Genetics for Reproduction, respectively. The Laboratoryof Neurophysiology is studying the brain sensing system
for the water and sodium homeostasis in the body fluid byusing gene-targeting mice, the Laboratory of
Neurochemistry is studying a physiological role ofdopaminergic system using genetically altered mice, andthe Laboratory of Molecular Genetics for Reproduction is
studying molecular mechanism of reproductive organ
development and sex differentiation using mutagenized ortransgenic medaka. For details, please refer to theacademic activity of each laboratory.

Figure 3. Equipments to generate 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 liter) and 450
tanks (3 liters) are available for medaka and zebrafish,
respectively. Water circulates and can be maintained tosuit conditions desired for fish breeding in the aquarium
systems. Currently, three mutant lines and two transgeniclines of medaka are maintained in our facility. In
addition to the rooms mentioned above, another room isavailable for insects. All the rooms are qualified to meetthe criteria for P1A transgenic animals, allowing
researchers to generate and maintain transgenic animals.
In 2005 (as of the end of November from the beginningof January), 944 medaka (452 fertilized eggs, 440
embryos and 52 adults) were brought to the facility and46,842 medaka (45,923 fertilized eggs, 700 embryos, and219 adults, including animals bred in the facility) weretaken out from the CTAP. In a laboratory for chickembryos 44,531 fertilized chicken eggs were brought inand 452 animals (190 fertilized eggs and 262 embryos)
were taken out from the CTAP. These animals wereused for research activities in neurobiology anddevelopmental biology

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NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH SUPPORT

Representative Instruments

THE CENTER FOR ANALYTICAL INSTRUMENTS

Protein Sequencers (ABI Procise 494HT, 492cLC)
Amino Acid Analyzer (Hitachi L8500A)
Peptide Synthesizers (ABI 433A)
Plasmid Isolation Systems (Kurabo PI-100, PI-50, PI-200)
Automatic Nucleic Acid Isolation System (Kurabo NA-2000)
Genetic Anlyzer (ABI PRISM 3100, 310)
Thermal Cyclers (Perkin Elmer PJ-9600, Takara TP-300,
Biometra TGRADIENT)
Integrated Thermal Cyclers (ABI CATALYST Turbo 800)
Particle Delivery System (Bio-Rad BiolisticPDS-1000/He)
Gas Chromatograph (Shimadzu GC-14APF-SC)
Glycoprotein Analysis System (Takara Glyco-Tag)
High Performance Liquid Chromatographs (Shimadzu
LC-10AD, LC-6AD, Waters 600E, Alliance UV system)
Integrated Micropurification System (Pharmacia SMART)
Flow Cytometer (Coulter EPICS XL)
Biomolecular Interaction Analysis Systems (Pharmacia
BIACORE 2000, Affinity Sensors IAsys)
Laboratory Automation System (Beckman Coulter Biomek
2000)
ESR Spectrometer (Bruker ER-200D)
GC/Mass Spectrometer (JEOL DX-300)
MALDI/TOF-MS (Bruker Daltonics REFLEX III)
Inductively Coupled Plasma Atomic Emission
Spectrometer (Seiko SPS1200A)
Spectrofluorometers (Hitachi 850, F-4500, Simadzu
RF-5000)
Spectrophotometers (Hitachi 330, 557, U-2001,Varian Cary
5G, Perkin Elmer Lambda-Bio)
Microplate Luminometer (Berthold MicroLumat LB 96P)
Microplate Readers (Corona MTP-120, MTP-100F)
FT-IR Spectrophotometer (Horiba FT-730)
Laser Raman Spectrophotometer (JASCO R-800)
Bio Imaging Analyzers (Fujifilm BAS 2000)
Luminescent Image Analyzer (Fujifilm LAS 3000 mini)
Fluorescence Bio Imaging Analyzer (Takara FMBIO)Electrophoresis Imaging System (BIOIMAGE)
Microscopes (Carl Zeiss Axiophot, Axiovert)
Environmental Scanning Electron Microscope (PHILIPSXL30 ESEM)
Confocal Laser Scanning Microscope (Leica TCS SP2)
Color Laser 3D Profile Microscope (KEYENCE VK-8500)
High-Resolution Quick Microscope (KEYENCE VH-5000)(managed by NIBB)Head of Facility:
Technical Staffs:
Technical Assistants:
Secretary:

KOBAYASHI, Satoru
MORI, Tomoko
MAKINO, Yumiko
TAKAMI, Shigemi
MORIBE, Hatsumi
KUROYANAGI Asuka
ICHIKAWA Mariko

The Center is responsible for amino acid sequence
analysis, amino acid analysis, and chemical syntheses of
peptides, as well as supporting researchers at NIBB and
NIPS. Luminescent Image Analyzers (Fujifilm LAS 3000mini) were newly installed in 2005. The Center’s
instruments can be used by researchers outside theinstitute upon proposal.

Figure 1. Protein sequencers

Figure 2. MALDI/TOF-MS

78

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

Research Support GroupMIWA, Tomoki

Chief:

Research Support Facilities
Unit Chief:
HIGASHI, Sho-Ichi
Subunit Chief:
NANBA, Chieko
Technical Staff:
NISHIDE, Hiroyo
NAKAMURA, Takanori
Technical Assistants:
SUZUKI, Keiko
MAKIHARA, Nobuko
ICHIKAWA, Chiaki
TAKESHITA, Miyako
NISHIMURA, Noriko
YAMAMOTO, Kumi

Cell Biology
Unit Chief:
Subunit Chief:
Developmental Biology
Technical Staff:

Neurobiology
Unit Chief:
Subunit Chief:

Center for Analytical Instruments
Unit Chief:
MORI, Tomoko
Subunit Chief:
MAKINO, Yumiko
Technical Staff:
TAKAMI, Shigemi
Technical Assistants:
MORIBE, Hatsumi
DODO, Yukiko
KUROYANAGI, Asuka

KONDO, Maki
KABEYA, Yukiko

TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
NODA, Chiyo
OHSAWA, Sonoko
TAKEUCHI, Yasushi

Evolutionary Biology and Biodiversity
Unit Chief:
TANAKA, Sachiko
Subunit Chief:
YAMAGUCHI, Katsushi
Technical Staff:
MOROOKA, Naoki
SUMIKAWA, Naomi
Environmental Biology
Subunit Chief:

Transgenic Animal Facility
Subunit Chief:
HAYASHI, Kohji
Technical Assistants:
ICHIKAWA, Hiromi
ICHIKAWA, Yoko
TAKAGI, Yukari
Disposal of Waste Matter Facility
Unit Chief:
MATSUDA, Yoshimi
Radioisotope Facility
Unit Chief:
Subunit Chief:
Technical Staff:
Technical Assistant:

KOBAYASHI, Hiroko

MIZUTANI, Takeshi

Reception
Supporting Staff:

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

SAKAGAMI, MariTSUZUKI, Shihoko
KATAOKA, Yukari
NAKANE, KaoriHIRONAKA, TomieMUKOHDA, Yasuyo
UNO, Satoko
SUGIYAMA, Tomomi

Technical staff members participate, through theDivision, in mutual enlightenment and education toincrease their capabilities in technical areas. Generally,
technical staff members are attached to specificdivisions so that they may contribute their special
biological and biophysical techniques to various
research activities.
The Division hosts an annual meeting for technicalengineers who work in various fields of biology atuniversities and research institutes throughout Japan.
At this meeting, the participants present their own
activities and discuss technical problems. The
proceedings are published after each meeting.

The Technical Division is a supporting organization
for researchers and research organizations within the
NIBB. The Division develops and promotes theinstitute's research activities and maintains the researchfunctions of the institute.
The Division is organized into two groups: theCommon Facility Group, which supports and maintains
the institute's common research facilities, and the
Research Support Group, which assists the research
activities as described in the reports of individual
research divisions.

79

NATIONAL INSTITUTE FOR BASIC BIOLOGYRESEARCH FACILITIESCENTER FOR RADIOISOTOPE FACILITIESHead:Associate Professor:
Technical staff:

Supporting staff:

Table 2. The numbers of users and visitors whoentered each controlled area in 2005

NODA, Masaharu(Professor, concurrent post)
OGAWA, Kazuo(Radiation Protection Supervisor)
MATSUDA, Yoshimi(Radiation Protection Supervisor)
SAWADA, Kaoru
IINUMA, Hideko
ITO, Takayo
KATAGIRI, Izumi
KAMIYA, Kiyomi
KANEUJI, Kimie

The technical and supporting staffs of the Center forRadioisotope Facilities (CRF) maintain three controlledareas according to Japanese law. The CRF is responsible
for monitoring the purchase of radioisotopes from JRA(Japan Radioisotope Association) and the transfer ofradioisotope wastes to JRA.
 Matsuda, Iinuma, Ito, and Kaneuji maintain CFBI
(Common Facilities Building I)-branch and LGER
(Laboratory of Gene Expression and Regulation)-branchat the Myodaiji-Area, and Ogawa, Sawada, Katagiri, andKamiya work at the Yamate-Area.
The numbers of registrants and users from January2005 to November 2005 are presented in Table 1.
 Users counted by the monitoring system going in andout of the controlled areas numbered 4,485 persons during
this period. The percentages and numbers for each area
are shown in Figure 1 and Table 2. The annual changes ofregistrants and the number of totals per year are shown in
Figure 2.
 The balance of radioisotopes received at the CRF isshown in Table 3. No 125I was used at Myodaiji-Area.
The Yamate-Area has passed the periodical inspection
by the Nuclear Safety Technology Center. The sameinspection will be carried out at the Myodaiji-Area 2005
on December 16th, 2005.
The air supply system of CFBI-branch was repaired inorder to decrease the amount of dust from the air supply
duct ports (Figure 3) and increase the comfort of thoseworking at CFBI-branch’s controlled rooms.

Figure 2. Annual changes of registrants and number of
totals per fiscal year
Table 3. Balance of radioisotopes received and used(MBq) at each controlled area

Table 1. Numbers of registrants and users at
Myodaiji-Area and Yamate-Area in the 2005

MyodaijiArea(CFBI)

Yamate-Area

Figure 3. Photograph showing a controlled area
during clearing the air supply duct ports

Myodaiji-Area(LGER)

Figure 1. Percentage of users going in and out of eachcontrolled area in 2005

81

NATIONAL INSTITUTE FOR BASIC BIOLOGY

50th NIBB conferenceStructure and Dynamics of Complex Biological NetworksOrganizing Chair : Atsushi Mochizuki
February 8 (Tue) -10 (Thu), 2005

spatio-temporal patterns, ecology and evolution using
different theoretical methodologies, with a special focus
on the network structures in different fields of biology,
including gene regulation, metabolic pathway andecology. One of the notable themes was the relation
between the structural and dynamic behaviors of thesecomplex systems.
The conference featured 39 research presentations
including 8 short talks and 12 poster presentations.
Ninety one participants – including 12 reseachers fromforeign countries - enjoyed discussions and the interaction
between different fields of theoretical biology. We havereceived many mails from participants thanking us forholding this conference. We hope that this conference
leads to the start of a new theoretical bioscience in the
future.

Thanks to progress in molecular genetics technologies
the past several years have seen rapid increases in theamount of biological information available to us; the nextorder of business, therefore, is to find the essential
principles of higher-order phenomena in biology hidden
withinenormousamountsofinformation.
Non-experimental methods are now attracting attention as
new methods for integrating information or forunderstanding whole systems of biology. Many
researchers from physics, mathematics and computerscience are starting to grapple with problems in biology.
Enhanced interaction between researchers working indifferent disciplines is essential for progress in theoreticalmethods in biology. For this purpose we held aninternational conference on February 2005 in Okazaki,
Japan. For this conference we called researchers studying
various biological phenomena, network systems,

Scientific topics:
Network systems in biology
Mathematical property of network
Application and understanding for biological network
Regulations in cells, tissues and organisms
Dynamical regulation in metabolism or cellular systems
Morphogenesis in development
Ecology and evolution
Dynamics in ecological network system

Speakers
AKUTSU, Tatsuya
ALBERT, Réka
ALMAAS, Eivind
ALVAREZ-BUYLLA, Elena Roces
ARITA, Masanori
CHING, Wai Ki
CRACIUN, Gheorghe
IWASA, Yoh
KANEKO, KunihikoKING, Ross
KONDO, Shigeru
MARTINEZ, Neo
MOCHIZUKI, Atsushi
MURATOV, Cyrill
NAKAI, Kenta
PAULSSON, JohanREINITZ, JohnTOKITA, Kei
VERT, Jean-Philippe(Kyoto University, Japan)(Pennsylvania State University, USA)
(University of Notre Dame, USA)
(Universidad Nacional Autonoma de Mexico, Mexico)(University of Tokyo, Japan)(University of Hong Kong, China)(Ohio State University, USA)
(Kyushu University, Japan)(University of Tokyo, Japan)(University of Wales, UK)(Riken, Kobe / Nagoya University, Japan)(Pacific Ecoinformatics and Computational Ecology Laboratory, USA)(National Institute for Basic Biology, Japan)(New Jersey Institute of Technology, USA)
(University of Tokyo, Japan)(University of Cambridge, UK)(Stony Brook University, USA)
(Osaka University, Japan)(Ecole des Mines de Paris, France)

82

NATIONAL INSTITUTE FOR BASIC BIOLOGY

51st NIBB conferenceNew Aspects of Gene Amplification-Mechanisms and Biological Function

Organizing Chair : Takehiko Kobayashi
November 5 (Sat) - 8 (Tue), 2005
the molecular mechanisms and biological functions ofgene amplification (including maintenance mechanism of
amplified genes).
The conference featured over 50 participants (including
11 researchers from foreign countries) and included 22
oral and 11 poster presentations. Tremendous amounts of
information were exchanged during many exciting
discussions.
Now, with the enormous increases in genomic
information and the rapid progression of molecularbiology, we are in a good position to look at new aspectsof gene amplification. This timely conference, therefore,
was an excellent opportunity to develop amplification
study.

Gene amplification not only results in genome
alteration, it also plays wide-ranging roles in many
biological functions. In multicellular organisms, it isobserved in many stages of the life-cycle: development,
differentiation, senescence, and tumorgensis. In
unicellular organisms, it is one of the main strategies foradaptation to surroundings. Moreover, it is well-known
that gene amplification has played critical roles in
evolution.
Despite its involvement in these important biological
functions, gene amplification has not been a central focus
for discussion. One of the reasons is that the mechanisms
responsible have remained elusive. In this conference,researchers studying DNA recombination, DNA
replication, chromatin structure and evolution discussed
Scientific topics:
Molecular Mechanism of Gene Amplification
Adaptation and Gene Amplification
Evolution and Gene Amplification

Speakers
ARCANGIOLI, Benoit
BENSIMON, Aaron
DEBATISSE, Michelle
GANLEY, Austen
HERNANDEZ, Pablo
HISHIDA, Takashi
HORIUCHI, Takashi
IWASAKI, Hiroshi
JOHZUKA, Katsuki
KIKUCHI, Akihiko
KOBAYASHI, Takehiko
LOBACHEV, Kirill
MAKI, Hisaji
ROTH, John
SASAKI, Hiroki
SCHVARTZMAN, Jorge
SHIBATA, Takehiko
SHIMIZU, Noriaki
SHORE, David
SOGO, José
STRUNNIKOV, Alexander
TOWER, John

(Pasteur Institute, France)(Pasteur Institute, France)(Curie Institute, France)(National Institute for Basic Biology, Japan)(CSIC, Spain)(Osaka University, Japan)(National Institute for Basic Biology, Japan)(Yokohama City University, Japan)(National Institute for Basic Biology, Japan)(Nagoya University, Japan)(National Institute for Basic Biology, Japan)(Georgia Institute of Technology, USA)
(Nara Institute of Science and Technology, Japan)(University of California-Davis, USA)(National Cancer Center Research Institute, Japan)(CSIC, Spain)(RIKEN Institute, Japan)(Hiroshima University, Japan)(University of Geneva, Switzerland)(ETH, Switzerland)(NIH, USA)
(University of Southern California, USA)83