cell kcellcelliiL0xc
cell iNational Institute for Basic Biology

2015 ANNUAL REPORTCONTENTS
Introduction・
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・ 1
Organization of the National Institute for Basic Biology ・
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・ 2
Goals of the National Institute for Basic Biology ・
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・ 5
Personnel Changes and Awardees in 2015 ・
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・ 7
Cell Biology
Laboratory of Cell Responses・
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・ 8
Laboratory of Neuronal Cell Biology ・
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・ 10
Laboratory of Stem Cell Biology・
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・ 12
Laboratory of Cell Sociology・
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・ 13
Developmental Biology
Division of Morphogenesis・
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・ 14
Division of Developmental Genetics・
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・ 17
Division of Molecular and Developmental Biology・
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・ 18
Division of Embryology ・
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・ 21
Division of Germ Cell Biology ・
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・ 24
Laboratory of Molecular Genetics for Reproduction ・
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・ 27
Neurobiology
Division of Molecular Neurobiology・
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・ 29
Division of Brain Biology・
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・ 32
Division of Brain Circuits・
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・ 34
Laboratory of Neurophysiology・
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・ 37
Evolutionary Biology and Biodiversity
Division of Evolutionary Biology・
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・ 38
Division of Symbiotic Systems・
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・ 41
Division of Evolutionary Developmental Biology・
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Laboratory of Morphodiversity ・
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Laboratory of Bioresources ・
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・ 47
Laboratory of Biological Diversity・
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・ 49
Environmental Biology
Division of Molecular Environmental Endocrinology・
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・ 58
Division of Environmental Photobiology ・
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・ 61
Division of Seasonal Biology (Adjunct)・
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・ 64
Theoretical Biology
Laboratory of Genome Informatics・
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・ 66
Imaging Science
Laboratory for Spatiotemporal Regulations ・
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・ 68
Okazaki Institute for Integrative Bioscience (Orion Project)
Laboratory of Nuclear Dynamics・
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・ 69
Okazaki Institute for Integrative Bioscience (Bio-Next Project)
Laboratory of Plant Development and Physiology・
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・ 70
Research Support
NIBB Core Research Facilities・
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・ 71
NIBB BioResource Center・
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・ 77
NIBB Center of the IBBP・
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・ 81
Center for Radioisotope Facilities (Okazaki Research Facilities)・
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・ 84
Research Enhancement Strategy Office・
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・ 85
Technical Division・
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Bioimaging Forum・
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NIBB Training Courses・
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NIBB Internship Program ・
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Index・
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Access to NIBB・
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・Inside back cover

The cover items are related to a study dissecting the evolution of pitcher leaf development in Sarracenia purpurea, acarnivorous plant native to North America (Fukushima et al., Nat. Commun. 2015). A graduate student working at NIBB
and his colleagues examined the process of pitcher leaf development by scanning electron microscopy, gene expression
analyses, cell division pattern analyses, and a mathematical reconstruction of pitcher morphogenesis. They showed thata tissue-specific regulation of oriented cell division is the key factor for pitcher development. A computational modelingof leaf morphogenesis also supports these results. One of the most mysterious questions in evolutionary biology is theevolution of novel complex traits. This study shows that change at the cellular level can result in quite a big change inmorphology. See page 40 of this report for details.

INTRODUCTIONI

t is my great pleasure to introduce to you all the2015 Annual Report of the National Institute for
Basic Biology (NIBB), which outlines the high level research
activities of the Institute and its effective function as a centerfor collaborative research in Japan over the last year. Given
these important missions of the Institute, I recognize mystrong responsibility to maintain the liberal atmosphere forresearch and discussion that NIBB has kept for many years,
which I believe is a basis for these remarkable activities.
To improve the economic status of the country, the Japanese
government continues to strengthen the demand that universities and inter-university research institute corporations, thelatter of which includes NIBB, should reform themselvesand draw actual profits from science. It is also necessary
for the scientific community to achieve full compliance andestablish integrity in research activities, to recover society’s
reliance on science. Under these circumstances NIBB must
stride properly. We will maintain two directions steadily.
One is that every person in NIBB should do his/her best inaccomplishing good research in basic biology. Good science,even in a basic research field, will eventually benefit humanbeings. The history of science tells us this is true. The otheris that NIBB must achieve high ethical standards for researchthat conform to the era of computer technology and data
sharing through the internet. NIBB will work hard to be truly
acknowledged as a remarkable institution by society.
Please find in this booklet a summary of the research, collaborative, educational, and international activities of NIBB
in 2015. I would like to note that we welcomed several newcolleagues in 2015, including one professor, one associate
professor, one specially appointed associate professor, two
assistant professors, and seven NIBB research fellows, whileten colleagues transferred to other institutes.
Finally I would like to congratulate Professor Emeritus
Yoshinori Ohsumi for winning several outstanding international awards, and Adjunct Professor Takashi Yoshimura
for winning the Van Meter Award in 2015 (Addendum:
Prof. Ohsumi has been elected as a 2016 Nobel Laureate in
Physiology or Medicine) . I would also like to congratulate
our young colleagues for winning awards from academic
societies as detailed on page 7.
To establish NIBB as an international leading institute in
the field of basic biology, we always welcome your suggestions, comments and queries concerning our activities, inaddition to your warm support.
Masayuki Yamamoto
Director General of NIBB
October 17, 2016

1

ORGANIZATION OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) was
founded in 1977 on a hill overlooking the old town ofOkazaki in Aichi prefecture as one of the Inter-University
Research Institutes to promote and stimulate studies of
biology. NIBB conducts first-rate research on site as well as
in cooperation with national, public and private universities
and research organizations. NIBB attempts to elucidate thegeneral and fundamental mechanisms underlying various
biological phenomena throughout a wide variety of biological fields, such as cell biology, developmental biology, neurobiology, evolutionary biology, environmental biology, andtheoretical biology.
NIBB and four other national institutes, the National
Astronomical Observatory of Japan (NAOJ), the National
Institute for Fusion Science (NIFS), the National Institutefor Physiological Sciences (NIPS), and the Institute for
Molecular Science (IMS), constitute the National Institutesof Natural Sciences (NINS), which is one of the four InterUniversity Research Institute Corporations. NIBB’s activities
are supported by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan. The Okazaki
Institute for Integrative Bioscience was established as acommon facility for the three institutes (NIBB, NIPS, andIMS) of the Okazaki campus.

■ Organization

National Institutes of Natural Sciences(NINS)President KOMORI, AkioNational AstronomicalObservatory of Japan (NAO)National Institute forFusion Science (NIFS)

National Institute for Basic Biology (NIBB)Director General YAMAMOTO, MasayukiVice-Director UENO, NaotoNational Institute for Physiological Sciences (NIPS)Advisory Committee forProgramming and Management

Research Enhancement Strategy OfficeInstitute for Molecular Science (IMS)Three Institutes in OkazakiCenter for Novel Science Initiatives
Buildings in the Myodaiji area (left) and Yamate area (right).

Policy, Decision Making, and Administration

The Director General oversees the operation of NIBB,
assisted by the Advisory Committee for Programmingand Management. The Advisory Committee, comprisedof eleven professors within NIBB and ten leading biologists outside NIBB, advises the Director General on important matters such as planning joint research programs aswell as on the scientific activities of NIBB. The Advisory
Committee makes recommendations on the appointments of
new Directors General, faculty appointments, NIBB’s annual
budget and future prospects.
The Research Enhancement Strategy Office, aimed at supporting researchers in order to improve NIBB’s abilities asa collaborative research institution, was restructured in 2013
from the former Strategic Planning Department, Office of
Public Relations, and Office of International Cooperation.
The Office is made up of five groups (p. 85) and its activities are mainly carried out by URAs (University Research
Administrators) in close collaboration with the NINS’s
Research Enhancement Promotion Headquarters.
Administration of NIBB is undertaken by the Okazaki
Administration Office under the direct auspices of the
Administration Office of NINS.

2Okazaki Research FacilitiesOkazaki Institute for Integrative Bioscience (OIIB)Department of Biodesign Research■Division of Molecular and Developmental Biology *■Division of Behavioral Neurobiology**■Division of Nuclear Dynamics■Division of Plant Development and PhysiologyDepartment of Bioorganization Research■Division of Quantitative Biology***■Division of Neuronal Cell Biology ****Divisions with researchers having concurrent positions in NIBB are shown above. Some divisions*-****) also function as NIBB’s research units†-†††)  , respectively, on the right panel.

Center for Radioisotope FacilitiesCenter for Experimental Animal
Research Center for Computational ScienceResearch Facilities run jointly by NIBB and NIPS
Electron Microscopy RoomDisposal of Waste Matter FacilityInstrument Design RoomNational Institute for Basic Biology (NIBB)  As of April 1, 2016Research Units■Division of Cellular Dynamics  Cell Biology■Division of Quantitative Biology†††■Laboratory of Cell Responses■Laboratory of Neuronal Cell Biology††††■Laboratory of Stem Cell Biology■Division of Morphogenesis  Developmental Biology■Division of Molecular and Developmental    Biology †■Division of Embryology■Division of Germ Cell Biology■Laboratory of Molecular Genetics for    Reproduction ■Division of Molecular Neurobiology  NeurobiologyEvaluation and Information GroupPublic Relations GroupInternational Cooperation GroupCollaborative Research GroupGender Equality Promotion Group■Division of Behavioral Neurobiology††■Division of Brain Circuits■Laboratory of Neurophysiology  Evolutionary Biology and Biodiversity■Division of Evolutionary Biology■Division of Symbiotic Systems■Division of Evolutionary Developmental    Biology■Laboratory of Morphodiversity■Laboratory of Bioresources■Laboratory of Biological Diversity■Division of Environmental Photobiology  Environmental Biology■Division of Seasonal Biology (Adjunct) ■Laboratory of Genome Informatics  Theoretical Biology■Laboratory for Spatiotemporal Regulations  Imaging ScienceResearch Support Facilities  NIBB Core Research FacilitiesFunctional Genomics FacilitySpectrography and Bioimaging FacilityData Integration and Analysis FacilityModel Animal Research FacilityModel Plant Research FacilityCell Biology Research Facility  NIBB BioResource Center  NIBB Center of the Interuniversity Bio-Backup Project (IBBP Center)  NIBB Center for Model Organism Development

Research Support Section  Technical DivisionOkazaki Administration Office3Research and Research Support

NIBB’s research programs are conducted in research units
and research support units. Each research unit is called either
a division, led by a professor, or a laboratory, led by anassociate professor or an assistant professor. Each research
unit forms an independent project team. The NIBB Core
Research Facilities and NIBB BioResource Center werelaunched in 2010 to support research in NIBB and also to
act as an intellectual hub to promote collaboration among
the researchers of NIBB and other institutions. The NIBB
Center of the Inter-University Bio-Backup Project (IBBP
Center) was founded in 2012 to prevent loss of invaluablebiological resources. Projects for the development of bioresource preservation technology are solicited by the IBBP
center from 2013. The NIBB Center for Model Organism
Development was founded in 2013 to promote development of new model organisms and research using them. The
Technical Division manages the activities of the technicalstaff and helps to promote the activities of each research unit
and to maintain the common resources of both the researchsupport units of NIBB and the research facilities of the
Okazaki campus. Center for Radioisotope Facilities are oneof the latter and run by the technical staff of NIBB.
The Okazaki Institute for Integrative Bioscience wasfounded to conduct interdisciplinary molecular researchon various biological phenomena and is shared by all three
institutes in Okazaki. Some of the divisions also function as
NIBB divisions.

■ Members in NIBB
Director General 1
Professors(including adjunct and specially appointed) 15
Technical
Assistants
andSecretaries

31

105

58
Graduate Students(including 7 undergraduates etc)

Associate Professors(including specially appointed) 17
Assistant Professors(including specially appointed)

65
26Technical Staff(settled forApril 2015-March 2016)
numbers are in million yen

Other Grants 88

External
Competitive
Fund

774

Grant-in-Aid
for Scientific
Research

432

Personnel

839

356

Grants forCommissioned
Research* new member from April 2016
# Chairperson## Vice-Chair

4

1,583

345

Other 43

Management Expenses
Grant for SOKENDAI■ Members of the Advisory Committee for Programming and Management

NIBB members

Management
Expenses Grant
for NIBB

Collaboration Research

58
HAKOSHIMA, Toshio ##HIGASHIYAMA, Tetsuya
KOHSHIMA, Shiro
KONDO, Shigeru
KUME, Shoen
KURUMIZAKA, Hitoshi
NISHITANI, Kazuhiko
NOSE, Akinao
OHTA, Kunihiro
TSUKITA, Sachiko
FUJIMORI, Toshihiko
HASEBE, Mitsuyasu
HIGASHIJIMA, Shin-ichi*
KAWAGUCHI, Masayoshi
MINAGAWA, Jun
NIIMI, Teruyuki*
NODA, Masaharu
TAKADA, Shinji #UENO, NaotoYOSHIDA, Shosei
YOSHIMURA, Takashi

318 in total(as of 1st April, 2016)■ Financial Configuration of NIBB

254

Non-NIBB members

Postdoctoral Fellows(11 are NIBB Research Fellows)

2,419 million yen
in total(as of April, 2016)

Professor, Nara Institute of Science and Technology
Professor, Nagoya University
Professor, Kyoto University
Professor, Osaka University
Professor, Tokyo Institute of Technology
Professor, Waseda University
Professor, Tohoku University
Professor, The University of Tokyo
Professor, The University of Tokyo
Professor, Osaka 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
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, Okazaki Institute for Integrative Bioscience
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Adjunct Professor, National Institute for Basic Biology

GOALS OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) sets fivegoals for its activities in pursuing the progress of biology.
We contribute to the world-wide community of biologists
through our efforts to accomplish these goals. This chapter
briefly explains four of these goals. The last goal, the promotion of academic research, is accomplished through our
research activities, which are introduced throughout thisbrochure.

Promotion of Collaborative Research■ Collaborative Research Support

Research activities in collaboration with NIBB’s divisions/laboratories using NIBB’s facilities are solicited from external researchers. “Individual collaborative
research projects” are the basic form of collaboration
support which provide external researchers with travel andlodging expenses to visit NIBB’s laboratories for collaborative research. For the use of NIBB’s unique and excellent
research instruments, such as the large spectrograph, theDSLM, and next generation DNA sequencers experimental projects are solicited and reviewed to provide machinetime and travel expenses. “Priority collaborative research
projects” are carried out as group research by internal andexternal researchers to develop pioneering research fields.
“Collaborative research projects for model organism/technology development” and “Collaborative research projects forbioresource preservation technology development” are fordeveloping and establishing new model organisms and new
research technology. For these projects, research expenses in
addition to travel expenses are provided. Bioimage processing and analysis collaborative research projects were started
in 2015 and are for development and application of image
analysis methods and travel expenses are supported.
year
Priority collaborative research projects
Collaborative research projects for model organisms/
technology development
Individual collaborative research projects
NIBB workshops
Collaborative experiments using the large spectrograph
Collaborative experiments using the DSLM

2012

2013

2014

3

4

2

3

89
6
14
5

89
4
15
9

87
3
12
10

88
6
10
11
14

47

41

37

46

2

1

0

1

9

10

9

174

162

190

5

2

1

Bioimage processing and analysis collaborative research projects
Collaborative experiments using
the next generation DNA sequencer
Facility Use (Training Course Facility)
Collaborative research projects for bioresource
preservation technology development
total

Collaborative Research Projects by Year

171

2015
2

International cooperation
and OutreachContribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers

large spectrograph, and provides technical support and scientific advice to researchers. These two facilities hold speciallyappointed associate professors, an expert in each field, witha mission to manage each facility as well as conducting his
own academic research. The Data Integration and Analysis
Facility supports analysis of large-scale biological data,such as genomic sequence data, gene expression data, andimaging data. For this purpose, the facility maintains highperformance computers with large-capacity storage systems.

■ Bio-Resources

The National BioResource Project (NBRP) is a national
project for the systematic accumulation, storage, and supplyof nationally recognized bio-resources (experimental animals
and plants, cells, DNA, and other genetic resources), which
are widely used as materials in life science research. To
promote this national project, NIBB has been appointed asa center for research on Medaka (Oryzia latipes) whose usefulness as a vertebrate model was first shown by Japanese
researchers. The usability of Medaka as a research material
in biology has drawn increasing attention since its fullgenome sequence recently became available. NIBB is also
a sub-center for the NBRP’s work with Japanese morning
glory. The NIBB BioResource Center has equipment, facilities, and staff to maintain Medaka and Japanese morning
glory safely, efficiently, and appropriately. The center also
maintains other model organisms, such as mice, zebraﬁsh,
Arabidopsis, Lotus japonicus, and Physcomitrella patens,
and provides technical support and advice for the appropriate
use of these organisms (p. 77).

■ NIBB Core Research Facilities

The NIBB Core Research Facilities support research in
NIBB and also act as an intellectual hub to promote collaboration between NIBB and other academic institutions. They
consist of three facilities that are developing and providing
state-of-the-art technologies through functional genomics,
bioimaging and bioinformatics (p. 71).
The Functional Genomics Facility maintains a ide array ofcore research equipment, including next generation DNA
sequencers. The facility is dedicated to fostering NIBB’s collaborative research by providing these tools as well as expertise. The current focus is supporting functional genomics
works that utilize mass spectrometers and DNA sequencers,
holding training courses as one of these undertakings (p.
90). The Spectrography and Bioimaging Facility manages
research tools, such as confocal microscopes, DSLM and theStrains of Japanese morning glory maintained in the center■ NIBB Center of the Inter-University Bio-Backup
Project (IBBP Center)

To prevent damage to important biological resources bynatural disasters, NIBB established the IBBP Center in 2012
in collaboration with seven national universities for multiplicate preservation of genetic libraries and other invaluablebioresources under cutting-edge research (p.81).

International Cooperation and Outreach■ Collaborative Programs with Overseas Institutes
5

NIBB takes a leading role in the collaborative research
programs between the European Molecular Biology
Laboratory (EMBL) and the National Institutes of Natural
Sciences (NINS) and promotes personal and technological exchange through joint meetings, exchange between
researchers and graduate students, and the introduction ofexperimental equipment.
NIBB formed an agreement with the Temasek Life Sciences
Laboratory (TLL) of Singapore and the Max Planck Institutefor Plant Breeding Research (MPIPZ) to promote joint
research projects, collaborative symposia, training courses
and student exchange programs. The 5th NIBB-MPIPZ-TLL
Joint Symposium “ Horizons in Plant Biology “ was held in
November, 2014 at NIBB.

■ NIBB Conference

The NIBB Conferences are international conferences on
hot topics in biology organized by NIBB’s professors. Since
the first conference in 1977 (the year of NIBB’s foundation), NIBB Conferences have provided researchers inbasic biology with valuable opportunities for international
exchange. The 63rd conference “ Environment to Bioresponse“ was held in November, 2015 (p.88).

■ International Practical Course

With the cooperation of researchers from Japan andabroad the NIBB international practical course is given ata laboratory specifically prepared for its use. The 8th course“Experimental Techniques Using Medaka and Xenopus The Merits of Using Both -” was held jointly with TLL and
the National University of Singapore (NUS) in September,2014 at NIBB. Graduate students and young researchers
from various areas including Taiwan, Hong Kong, India,
Indonesia, Bangladesh, Germany, the United States, andJapan, were provided with training in state-of-the-art
research techniques. International conferences and courses
are managed by the International Cooperation Group of the
Research Enhancement Strategy Office.

■ Outreach

NIBB’s outreach activities aim to present cutting edge
research results to the public via mass media through press
releases or directly through the internet, such as web pages,
Facebook, and Twitter. Our triannual open campus event washeld in 2013 at which we welcomed more than 1,300 local
citizens. NIBB also cooperates in the education of undergraduate and younger students through lectures and workshops. Outreach activities are mostly managed by the Public
Relations Group of the Research Enhancement Strategy
Office.

Development of New Fields of Biology■ Bioimaging

NIBB aims to maximize the application of modern lightmicroscopes and biophotonic probes for real time visualization of biological phenomena and to develop new imaging
techniques. As part of our collaborative work with EMBL,NIBB introduced a DSLM, which is effective for the threedimensional observation of living organisms, and has devel-6

oped an improved model using two-photon optics (p. 68).
The application of the adaptive optics to microscopy isunder way in collaboration with the National Astronomical
Observatory of Japan. The Advisory Committee on
Bioimaging, comprised of leading researchers in the bioimaging field in Japan, is organized to formulate advice on
NIBB’s imaging research. The Bioimaging Forum provides
an opportunity for researchers and company engineers tofrankly discuss practical difficulties and needs regarding
imaging. The 9th Forum “Imaging of Physical Properties”
was held in January 2015 (p. 90). A training course inbioimage analysis was also held in 2015 (p. 91).

■ Okazaki Biology Conferences

NIBB holds Okazaki Biology Conferences (OBC) that,with the endorsement of the Union of Japanese Societies
for Biological Science, aim to explore new research fields
in biology and support the formation of international communities in these fields. Dozens of top-level researchers
from Japan and abroad spend nearly one week together for
intensive discussions seeking strategies for addressing future
critical issues in biology. Past Conferences have promoted
the formation of international researcher communities.

Cultivation of Future Researchers

NIBB constitutes the Department of Basic Biology in
the School of Life Science of the SOKENDAI (Graduate
University for Advanced Studies). The department provides
a five-year course for university graduates and a three-year
doctoral course for graduate students with a master’s degree.
Graduate students enrolled in other universities and institutions can apply to be special research students eligible toconduct research under the supervision of NIBB professors.
In both cases above, graduate students can receive financial
support from NIBB based on the research assistant (RA)system from the beginning of the five-year course.
Due to the international collaboration with EMBL, graduate
students are encouraged to attend PhD student symposia held
at EMBL and provided an opportunity to give oral and poster
presentations, at least once during their master’s and doctoral
program (p. 89).
Students from Japan and abroad can also come to NIBB
through our Internship Program. Internships give students an
excellent way to build international connections while experiencing hands on research in a world class research institute(p. 92).

Special Research Student

Graduate students educated by NIBB

SOKENDAI Graduate Students

■ Personnel changes in 2015*

Newly assigned in NIBB
Name

Position

Date

Research Unit

TSUBOUCHI, Tomomi

Associate Professor

Laboratory of Stem Cell Biology

February 1

KAWADE, Kensuke

Specially Appointed
Associate Professor

Laboratory of Plant Development and Physiology(BIONEXT Project)

March 1YASUGI, Masaki

NIBB Research Fellow

Laboratory of Neurophysiology

April 1

HIRA, Seiji

NIBB Research Fellow

Division of Germ Cell Biology

April 1AIHARA, Yusuke

NIBB Research Fellow

Division of Environmental Photobiology

April 1TOYOTA, Kenji

NIBB Research Fellow

Division of Molecular Environmental Endocrinology

April 1NISHIMURA, Toshiya

NIBB Research Fellow

Laboratory of Molecular Genetics for Reproduction

April 1

OGAWA, Kota

NIBB Research Fellow

Functional Genomics Facility

April 1NIIMI, Teruyuki

Professor

Division of Evolutionary Developmental Biology

June 1

NAKAGAWA, Toshinori

Assistant Professor

Division of Germ Cell Biology

August 1

KAMIKAWA, Yasunao

NIBB Research Fellow

Laboratory of Stem Cell Biology

August 1

OHDE, Takahiro

Assistant Professor

Division of Evolutionary Developmental Biology

September 1

Newly affiliated in other universities and institutes
New Affiliation

Position

Date

YAMAMORI, Tetsuo

RIKEN, Brain Science InstituteTeam Leader

April 1

KOBAYASHI, Satoru

University of Tsukuba

Professor

April 1WATAKABE, Akiya

RIKEN, Brain Science InstituteResearcher

April 1

HAYASHI, Yoshiki

University of Tsukuba

Assistant Professor

April 1

SATO, Masanao

Keio University

Project Research Associate

April 1SADAKANE, Osamu

RIKEN, Brain Science InstituteResearcher

April 1

HARA, Kenshiro

Tohoku University

Associate Professor

April 1MIYAKAWA, Hitoshi

Utsunomiya University

Associate Professor

June 1SUZAKI, Takuya

University of Tsukuba

Associate Professor

August 16

TANAKA, Daisuke

National Institute of Agrobiological Sciences

Researcher

October 1

Name* Changes in professors, associate and assistant professors, and NIBB research fellows are shown.

■ Awardees in 2015

Name

Position

OHSUMI, Yoshinori

Professor Emeritus

YOSHIMURA, Takashi

Adjunct Professor

NAKAMURA, Yoshiaki

Postdoctoral Fellow

TOYOTA, Kenji

NIBB Research Fellow

Award
Canada Gairdner International Award, The 31st International Prize
for Biology, The 20th Keio Medical Science Prize, Bunkakorosya(Person of Cultural Merits)
2015 Van Meter Award, The Japan Society for Comparative
Endocrinology's Incentive Award
The Society for Reproduction and Development's Young
Investigator Award
Inoue Research Award for Young Scientist, The Japan Society of
Endocrine Disrupters Research's Poster Award, The Japan Society
for Comparative Endocrinology's Best Presentation Award

7

National Institute for Basic Biology

Cell Biology

LABORATORY OF CELL RESPONSES

Director GeneralSpecially Appointed Associate Professor

Postdoctoral Fellow:

OTSUBO, Yoko
SHICHINO, Yuichi
SAKAGAMI, Mari

YAMAMOTO, Masayuki

Secretary:

YAMASHITA, Akira

Cells sense the environment around them, for example theamount of nutrients and hormones, as well as the temperatureand pressure, and decide what kind of activities to undertakeusing this information. Germ cells, which produce sperm andeggs, begin halving their number of chromosomes during a
special kind of cell division called meiosis, in response to theambient conditions. In our laboratory we use the fission yeast
Schizosaccharomyces pombe, the simplest organism that
performs meiosis, to research the mechanism by which cells
switch from mitosis, the kind of cell division that divides
cells equally to create two identical cells, to meiosis, whichis essential for bringing forth genetically diverse progeny.

I. Signaling pathways that regulate the onset of
sexual differentiation

We have been trying to elucidate how fission yeast cells
switch their mode of cell cycle from mitotic to meiotic.
We focus on a highly conserved kinase, namely Target of
rapamycin (TOR) kinase, which plays key roles in the recognition of nutrition and the onset of sexual differentiationin fission yeast. TOR kinase forms two types of complexes,
namely TORC1 and TORC2. TORC1 contains Tor2 as its
catalytic subunit and is essential to suppress sexual differentiation in the presence of nitrogen. TORC2 contains Tor1
and, in contrast to TORC1, is required for onset of sexual differentiation under nitrogen starvation (Figure 1).
Temperature-sensitive tor2 mutants initiate sexual differentiation on rich medium at the restrictive temperature. To gain
insights into the TORC1 signaling pathway, we have isolated
mutants that initiate sexual differentiation ectopically undernutrient-rich conditions. We are currently characterizingthese mutants.
Nitrogen source

TORC1
Tor2

TORC2
Tor1

sexual differentiation
Figure 1. The two TOR complex pathways in S. pombe. TORC1, containing Tor2, and TORC2, containing Tor1, regulate sexual differentiation
oppositely. TORC1 suppresses sexual differentiation in the presence of
ample nitrogen.

8

II. The molecular mechanisms that establish
the meiosis-specific transcription profile

Expression of hundreds of genes are upregulated duringmeiosis. We have shown that specific control of the stabilityof meiotic transcripts, which is orchestrated by the interplay
between RNA-binding proteins and a long non-coding RNA,
contributes to the meiosis-specific gene expression in fission
yeast. Understanding precise mechanisms of this control willshed light on the regulation of timely gene expression duringmeiosis.
A YTH-family RNA-binding protein Mmi1 plays a crucialrole in the selective elimination system of meiosis-specifictranscripts during the mitotic cell cycle. Mmi1 recognizes aregion termed DSR (Determinant of Selective Removal) in
meiotic transcripts, which is enriched with repeats of hexanucleotide motifs. Meiotic transcripts bound to Mmi1 aredegraded by the RNA-degradation nuclear exosome machinery. Mmi1 also induces formation of facultative heterochromatin at a subset of its target genes.
During meiosis, a meiosis-specific nuclear body, called
Mei2 dot, blocks the Mmi1-mediated elimination system.
The Mei2 dot is composed of the RNA-binding protein Mei2
and a long non-coding RNA species termed meiRNA. Mei2
physically interacts with meiRNA and forms the dot structure
at the chromosomal sme2 locus, which encodes meiRNA.
The Mei2 dot lures Mmi1 through numerous copies of the
DSR motif on meiRNA and inhibits its function, so thatmeiotic transcripts harboring DSR are stably expressed(Figure 2).
We have shown, in collaboration with a group at the
University of Cambridge, that a conserved multifunctional
protein complex Ccr4/Not is recruited by Mmi1 to its target
transcripts and plays an essential role for heterochromatin
formation in the Mmi1-dependent pathway (Cotobal et al.,2015).

Mei2 dot

nuclearexosome

Mmi1

Mei2

meiRNADSR ts
ip

meio

nscr
tic traFigure 2. Selective elimination of meiosis-specific transcripts by theMmi1/DSR system. Mmi1 binds to DSR in meiotic transcripts andinduces their degradation by the nuclear exosome during the mitotic cell
cycle. In meiotic cells, the Mei2 dot, composed of Mei2 and meiRNA,
sequesters and inhibits Mmi1, so that DSR-harboring meiotic transcripts
escape from Mmi1-mediated selective elimination.

Num1

x

0 min

Arp1
Mug5
Jnm1

corte

During meiotic prophase in fission yeast, the nucleus
migrates back and forth between two poles of the cell. Thisoscillatory nuclear movement is called ‘horse-tail’ movement
due to its characteristic shape and motion (Figure 3). Horsetail nuclear movement enhances pairing of homologous
chromosomes and facilitates meiotic recombination. Horsetail movement is driven by cytoplasmic dynein, which forms
a huge minus-end-directed microtubule motor complex.
Cytoplasmic dynein that is anchored to the cell cortex generates a pulling force on the microtubule emanating from theleading edge of the nucleus. This dynein-mediated pullingis the major contributor to horse-tail movement. Corticalanchoring of dynein is crucial for the generation of horse-tailmovement.

Microtubule

DyneinCell

III. R egulation of nuclear oscillation driven
by cytoplasmic dynein during meiotic
prophase

Num1

Figure 4. Cortical anchoring of cytoplasmic dynein in S. pombe. Dynein
is captured by Num1 at the cell cortex. Dynactin subunits, Arp1, Mug5
and Jnm1 assemble with dynein at the cell cortex and activate dynein.
Dynactin and Num1 cooperate to establish dynein anchoring and enabledynein to generate microtubule-pulling force.

Publication List:〔Original papers〕

2 min

C., Rodríguez-López, M., Duncan, C., Hasan, A., Yamashita,• Cotobal,
A., Yamamoto, M., Bähler, J., and Mata, J. (2015). Role of Ccr4-•

Not complex in heterochromatin formation at meiotic genes andsubtelomeres in fission yeast. Epigenet. Chromatin 8, 28.
Fujita, I., Yamashita, A., and Yamamoto, M. (2015). Dynactin andNum1 cooperate to establish the cortical anchoring of cytoplasmic
dynein in S. pombe. J. Cell Sci. 128, 1555-1567.

6 min

8 min

Figure 3. Horse-tail nuclear movement during meiotic prophase in S.
pombe. Time-lapse images of nuclear membrane (Cut11, magenta) andmicrotubules (Atb2, green) in the wild-type strain. The cellular contour isshown by the dotted line.

We identified three subunits of dynactin, a protein complex
that is required for most dynein-mediated cellular activities (Fujita et al., 2015). The three subunits, namely Arp1,
Mug5 and Jnm1, transiently colocalized with dynein at thecell cortex and were essential for the cortical anchoring ofdynein. We also found that another dynein-related corticalfactor, Num1, cooperates with dynactin to establish dynein
anchoring at the cell cortex (Figure 4).

9National Institute for Basic Biology

Cell Biology

LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:
SOKENDAI Graduate Student:
Technical Assistant:

NAKAYAMA, Kei
OHASHI, Rie
KATAYAMA, Kaori
MATSUDA, Chisato

The transport of specific mRNAs and local control oftranslation in neuronal dendrites represent an important gene
expression system that provides dendritic protein synthesis
at just the right time and place. It is believed that this system
controls the location at which neurites will connect to eachother, thereby forming neural networks. Our main interest is
to understand the mechanisms and roles of mRNA transportand local translation in neuronal dendrites.
Specific mRNAs are recruited into “RNA granules” andtransported to dendrites. RNA granules are macromolecularcomplexes composed mainly of mRNAs, ribosomes andRNA-binding proteins, and mediate the transport and local
translation of their mRNA cargoes in response to synaptic
stimulation (Figure 1). We are researching RNA granulefactors regulating mRNA transport and local translation, theirtarget mRNAs, and the mechanisms of localized protein synthesis using mice in order to better understand their relation
to the formation of synapses and neural networks, memory,learning, and behavior.
Stimulation

locally translated proteinssynapsepolysome

microtubulesoma

brainstem development (Shiina et al., J. Neurosci. 30, 1281612830, 2010).
A recent study aiming to detect genetic variants in ASD bywhole-genome sequencing reported that a heterozygous denovo nonsense mutation in the Rng105/Caprin1 gene wasfound in a patient with Asperger’s syndrome, a form of ASD.
The patient’s intelligence quotient (IQ) was above average,
but adaptive behavior and sociability were delayed. The
report suggested that Rng105/Caprin1 is a candidate risk
gene for ASD, but it remained unclear as to whether there
was a causal relation between RNG105 deficiency and ASD.
To investigate the influence of RNG105 deficiency onmouse behavior, we subjected RNG105 heterozygous(Rng105+/-) mice to a comprehensive behavioral test battery.
One of the marked changes in Rng105+/- mice was sociality. Rng105 +/- mice showed reduced social interaction
with familiar mice in a home cage test and reduced social
interest/preference for novel mice over familiar mice ina three-chambered social approach test (Figure 2). The
latter change can be characterized as indistinguishable
responses of Rng105+/- mice to familiarity and novelty, as
the Rng105+/- mice also showed reduced interest/preference
for novel objects and places over familiar objects and places,
respectively. In several maze tasks, Rng105+/- mice showednormal acquisition of memory, and in some of the tasks, they
showed higher performance compared to wild-type mice.
However, they showed relative difficulty in reversal learning,

RNG105
RNA granuledendriteaxon

No stimulation

Figure 1. A model for local translation in neuronal dendrites. SpecificmRNAs are recruited into RNA granules and transported to dendrites.
Translation of the mRNAs is induced locally upon synaptic stimulation,
which modifies local postsynapses to regulate synaptic connection andnetwork formation.

I. C omprehensive behavioral analysis of
RNG105 heterozygous mice: Implications in
autism spectrum disorder (ASD)

We previously identified RNA granule protein 105(RNG105)/caprin1, an RNA-binding protein, as a component of RNA granules. RNG105 is responsible for mRNAtransport to dendrites, which is required for the encoded
proteins to be translated and function in dendrites for proper
networking of neurons. RNG105 knockout mice exhibit
reduced dendritic synapse formation and reduced dendritic
arborization, which results in poor development of neuronal
networks. The knockout neonates die soon after birth due
to respiratory failure that is associated with defects in fetal

10

Figure 2. Three-chambered social approach test. (A) Schematic diagramof the test. In the first session, one of the cages (dotted lines) contains
a stranger mouse. A test mouse is allowed to freely explore the threechambers. In the second session, a familiar mouse and a stranger mouseare put in each cage. (B and C) Heat maps showing the average traces ofwild-type mice (top panels) and Rng105+/- mice (bottom panels) in thefirst session (B) and the second session (C). Social interaction with astranger mouse is normal, but there is a lack of interest/preference for a
stranger mouse over a familiar mouse in Rng105+/- mice.

in which the target was changed from the initial position.
These results suggest that social interaction, responses tonovel situations, and flexibility to changes were reduced inRng105+/- mice.
Furthermore, RNG105-deficient neurons showed a reduction in AMPA glutamate receptor (AMPAR) cell surface
distribution in dendrites (Figure 3), which has been reported
in other ASD-like mutant mice and is thought to be relatedwith the neuropathology of ASD. The behavioral test battery,
together with the analysis of AMPAR distribution, suggestthat an RNG105 deficiency leads to ASD-like behavior. In
particular, Rng105+/- mice performed well in specific tasks,
suggesting that the phenotype of Rng105+/- mice was related
to Asperger’s syndrome-like behavior.

Figure 3. The cell surface distribution of AMPAR subunit GluR1 is
reduced in dendrites of RNG105-deficient neurons. (A) Immunostaining
for GluR1 in cultured neurons (9 DIV) from cerebral cortexes of E17.5
wild-type, Rng105+/- and Rng105-/- mice. Scale bar, 10 μm. (B and C)
The number of surface (B) and total (C) GluR1 puncta in dendrites. (D)The ratio of surface/total number of GluR1 puncta in dendrites. *p<0.05,
one-way ANOVA followed by Turkey-Kramer test.

of RNG140 in higher brain functions in adult mice, wehave started to generate RNG140 knockout mice with the
CRISPR/Cas9 system.

III. R NA granule dynamics regulated by
PRMT1

Regulation of RNA granule assembly and disassemblyis emerging as a key mechanism of translational control,
defects of which are linked with neurodegenerative diseases
such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We conducted proteomic
analyses to identify proteins associated with RNG105, andidentified PRMT1 as a factor which disassembles RNG105containing RNA granules.
PRMT1 is a major arginine methyltransferase that methylates arginine residues in the arginine and glycine-rich(RG-rich) domain. We found that the RG-rich domain of
RNG105 was bound and methylated by PRMT1. A methyltransferase activity-deficient mutant PRMT1 also bound to
RNG105, but had reduced ability to methylate RNG105 anddisassemble RNA granules. These results suggested that not
only the association with RNG105, but also the methylation
activity of PRMT1 is required to disassemble RNA granules.
Because various RNA granule components, including an
ALS and FTLD risk factor FUS, have the RG-rich domain, it
will be a future challenge to investigate whether methylationof these RNA granule factors is implicated in the regulationof RNA granule dynamics and neurodegenerative disease.

Publication List:〔Original paper〕

D., Kuroda, K., Tanaka, M., Namba, T., Iizuka, Y., Taya, S.,• Tsuboi,
Shinoda, T., Hikita, T., Muraoka, S., Iizuka, M., Nimura, A., Mizoguchi,A., Shiina, N., Sokabe, M., Okano, H., Mikoshiba, K., and Kaibuchi,
K. (2015). Disrupted-in-schizophrenia 1 regulates transport of ITPR1mRNA for synaptic plasticity. Nat. Neurosci. 18, 698-707.

II. Learning and memory deficits in RNG105
conditional knockout (cKO) mice

Although learning and memory were normal in mice witha moderate deficiency of RNG105 (Rng105+/-), learning andmemory were markedly impaired in mice with severe deficiencies of RNG105: RNG105 conditional knockout (cKO)in the brain after birth led to reduced contextual and spatial
memories.
Because memory formation is highly correlated withincreases in the size of spine heads (postsynapses) on
neurons, we measured the size of spines in the hippocampus
of RNG105 cKO mice. The size of the spine heads was significantly smaller in RNG105 cKO mice than in wild-type
mice, which may underlie the impaired memory formation in
RNG105 cKO mice.
RNG105/caprin1 has one paralog, RNG140/caprin2, which
has RNA-binding domains highly conserved with RNG105.
RNG105 and RNG140 are localized to different kinds of
RNA granules and their timing of expression is also different: RNG105 is highly expressed in embryos, but RNG140
is highly expressed in adults (Shiina and Tokunaga, J. Biol.
Chem. 285, 24260-24269, 2010). To investigate the role11

National Institute for Basic Biology

Cell Biology

LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

NIBB Research Fellow:
Technical Assistant:

KAMIKAWA, Yasunao
YASUI, Naomi

DNA is constantly damaged from both endogenous andexogenous sources. One of the most important challenges
for all living organisms is to prevent genome instability
that can lead to malfunction of a cell. Our group is interested in the strategies through which cells protect themselvesfrom alterations in the genome. To date, much information
has been gained from various model organisms and tissue
culture cells, and we are beginning to learn that the choiceof genome-maintenance strategies taken by a cell depends
on the cell type, cell cycle- and developmental stages. In the
Laboratory of Stem Cell Biology, we are currently focusing
our attention on the genome maintenance mechanisms of theembryonic stem cells, and their roles during differentiation
and reprogramming processes.

I. Self-renewal of Embryonic Stem Cells andTheir Genome-Maintenance Mechanisms

Embryonic stem (ES) cells are derived from the blastocyst
stage of embryonic development, and are capable of differentiating into all cell types that compose our body (i.e., ES cells
are “pluripotent”). Pluripotent cells exist only transiently
and are lost as development proceeds. On the other hand, ES
cells are capable of proliferating indefinitely when given an
appropriate culturing condition. Curiously, ES cells proliferate with truncated gap phases while S (DNA replication)and M (mitosis) phases are as lengthy as other cell types. ES
cells also appear to lack some of the mechanisms that ensure
genome integrity (i.e., checkpoint mechanisms), the significance of which remains a mystery.
To date, studies on cell cycle regulation in ES cells havenot been straightforward compared to that of other cell types,
as many commonly used cell-synchronization protocols are
ineffective for ES cells. We have established a protocol tosynchronize ES cells using centrifugal elutriation (Tsubouchi
et al., Cell, 2013), which allowed us to investigate specificstages of the ES cell cycle. We are currently aiming toaddress why and how ES cells maintain such unique cell
cycle regulation and how that is interlinked with pluripotency by carrying out side-by-side analyses between ES cells
and differentiated populations.

II. G enome Instability during Nuclear
Reprogramming
In order to gain a deeper understanding of the relationshipbetween the choice of genome maintenance mechanisms andpluripotency, we are investigating the behavior of factors
involved in genome maintenance mechanisms during nuclear
reprogramming towards pluripotency.
Specifically, we take advantage of the cell-to-cell fusion
approach, in which a target cell is fused to a pluripotent stem

12cell to induce pluripotency within a target nucleus (Figure 1).
The cell fusion system is a simple, versatile way to induce
reprogramming towards another lineage, not limited to pluripotency. Using this system, the first sign of reprogramming
can be detected from within a few hours to one day after
fusion, allowing us to monitor the initial events of reprogramming after induction.

Figure 1. Reprogramming induced through cellular fusion: a human
lymphoblastoid nucleus (blue, with green spots) is induced to undergo
nuclear reprogramming towards pluripotency upon fusion with mouse ES
cells (green).

Using this system, we previously found that DNA synthesis is an important event for successful reprogramming(Tsubouchi et al., Cell, 2013). Recent reports indicate that
reprogramming may cause genetic instabilities, some ofwhich are thought to arise as DNA replication errors. We
are currently investigating how such genetic instability may
arise, and how it is linked to reprogramming-specific events.

III. Future Perspective

While the fundamental mechanisms that maintain genome
integrity have been widely studied using various models, thedanger a cell might face when altering their cellular identity(through differentiation, reprogramming etc.) is unknown.
Recent studies of cancer genome sequencing repeatedly
identified mutations in the factors that govern cellular identities, leading us to hypothesize that cells may experience
genome instability when their identity is unstable. Our goal
is to uncover the nature of such genetic instability and to
gain a comprehensive understanding of the mechanisms thatmaintain genome integrity.

Publication List:〔Original paper〕

W.-K., Humphryes, N., Afshar, N., Argunhan, B., Terentyev, Y.,• Leung,
Tsubouchi, T., and Tsubouchi, H. (2015). The synaptonemal complex
is assembled by a polySUMOylation-driven feedback mechanism inyeast. J. Cell Biol. 211, 785-793.

LABORATORY OF CELL SOCIOLOGY †Assistant Professor

HAMADA, Yoshio

Technical Assistant:

GONDA, Naoko

Mammals have evolved placentae that facilitate the transport of nutrients and oxygen into the fetus. Primates androdents have an ancestral type of placenta in which maternal
blood is not contained within endothelial cell lined vessels
but rather is in direct contact with epithelial cells of the fetal
placenta (so called hemochorial placentation) that are derived
from the trophoblast cell lineage (Figure 1). How maternal
blood vascular circuits develop in the hemochorial placenta
without immunological rejection has been a longstanding
mystery in biology.

Figure 1. Fetal and maternal blood flow in the mouse placenta. Fetomaternal exchanges take place in the labyrinthine layer (green). Polyploid
trophoblasts are observed in the spongiotrophoblast layer (orange) where
Notch2 is expressed.

The Notch2 null mutant dies at mid-gestation due toimpaired maternal circuit formation. Two mechanisms for theformation of spaces for the maternal circuit have been speculated. One is by polyploid Trophoblast Giant Cells (TGCs)
that retain the ability to form cavities, analogous to endothelial cells. Notch signaling mutations reduce the population orcause dysfunction of TGCs. The other is by deletion of trophoblasts, which we have proposed. Notch mutation would
lead the trophoblasts to resist the deletion.
We carried out a survey of authentic cell death, apoptosis
and necrosis, related to the formation of the maternal blood
circuit, but failed to find meaningful signals. Therefore, we
suspected that an unknown type of cell death or deletion
might be involved. Because histological studies of developing mouse placentae show that formation of open spaces
begins at E10.5 and become bigger at later stages in the
spongiotrophoblast layer (Figure 2), it was rational to speculate that cell death or deletion occurs in the spongiotrophoblast layer. Study of the nuclear size of spongiotrophoblast
cells shows a constant and substantial population of polyploid trophoblast cells throughout development.
How is the population of polyploid trophoblast cells kept
constant? Considering that diploid spongiotrophoblast cells
differentiate into polyploidy during normal development,
the supply from diploid trophoblast cells and deletion could
maintain a constant population. Now, our working hypothesis

Figure 2. Drastic morphological change and unchanged population ofpolyploid trophoblast cells. Open spaces are barely seen at E9.5 (A) andbecome large enough to contribute to half of the spongitotrophoblast
layer at E13.5 (B). In contrast to the morphological changes, the proportion of diploid, replicating dipoloid and polyploidy is unchanged throughout E9.5-E13.5 (C).

became testable. Polyploid trophoblasts should be deleted
and Notch2 mutation should cause them to resist the deletion.
Although tetraploid embryo derived trophoblasts are able
to support ES cell derived embryos to grow to term, they
are deleted from the placenta by E11.5 if they co-exist with
diploid trophoblasts (Figure 3). Contrastingly, Notch2 nullmutant tetraploid embryo derived trophoblasts resist thedeletion and even dominate diploid trophoblasts. From theseresults we conclude that maternal blood spaces throughout
the trophoblast are created by polyploid trophoblast celldeletion.

Figure 3. Fate of tetraploid trophoblast cells in chimera embryos. A
tetraploid embryo was aggregated with a diploid embryo in vitro andtransplanted onto a pseudo-pregnant female’s uterus. While the 4nembryo contributes to the placenta and yolk sac, it does not contribute to
the embryo proper by E10.5, 4n embryo derived trophoblasts disappearby E11.5.

†: This laboratory was closed on 31 March, 2016.

13

National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

Assistant Professor:

TAKAHASHI, Hiroki
SUZUKI, Makoto
TAKAGI, Chiyo
YAMAGUCHI, Takeshi
NEGISHI, Takefumi
SUZUKI, Miho
NINOMIYA, Hiromasa
MIYAGI, Asuka*
HAYASHI, Kentaro
TOMINAGA, Hitoshi
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
MIYAGI, Asuka
YASUE, Naoko
WATANABE, MikaMIYAKE, Satoko
TSUGE, Toyoko

UENO, Naoto

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

Secretary:

KINOSHITA, Noriyuki

The complex morphogenesis of organisms is achieved bydynamic rearrangements of tissues during embryogenesis,
in which change in cellular morphology as well as orchestrated cell movements are involved. For cells to know how
they should change their shape and where they should move,
information called “cell polarity” is essential. How then is
the cell polarity established within cells? Is it intrinsicallyformed within the cells or triggered by extracellular cues?
Furthermore, little is known as to how coordinated andcomplex cell movements are controlled in time and space.
We attempt to understand the mechanisms underlying these
events using several model animals, including frogs, fish,
mice and ascidians, taking physical parameters such as force
in consideration, in addition to conventional molecular andcellular biology.

I. A novel signaling pathway for head
formation

The head is formed in the most anterior part of the neuraltube which is the anlage of the brain and spinal cord. How is
the head region selected in its particular region of the tube?
Patterning of the anterior-posterior axis is controlled bysecreted molecules known as growth factors whose effects
are mediated by their downstream mediators which facilitate
differentiation of the brain and eyes. It was previously shownthat the inhibition of the Wnt/β-catenin signaling pathway
by various factors is critical for the anterior specificationof head formation. We have revealed that Flop1 and Flop2(Flop1/2) G protein-coupled receptors contribute to theregulation of head formation by inhibiting Wnt/β-cateninsignaling in the amphibian Xenopus. Both the overexpression and knockdown of Flop1/2 resulted in abnormal head
formation. More interestingly, overexpression of Flop1/2 inthe absence of bone morphogenetic protein (BMP) signaling
which is inhibitory to general neural differentiation, resulted

14

in ectopic head formation in the ventral side. We have also
found that Flop1/2 inhibits Wnt/β-catenin signaling by promoting β-catenin degradation. Although the ligand for theFlops is not identified, our findings suggest that Flop1 andFlop2 are novel inhibitory components of the Wnt/β-cateninsignaling pathway and essential for proper head formation.

Figure 1. Ectopic head formation by Flop1 overexpression and BMP
inhibition.
(A) Control larvae showing normal head and tail structures.
(B) Abnormal larvae in which Flop1 is overexpressed in the absence of
BMP signals show duplicated heads with eyes at the expense of trunk
and tail.

II. M e c h a n o t r a n s d u c t i o n i n X e n o p u sembryonic cells

During early vertebrate development, many dynamic morphogenetic movements occur, which include convergent
extension of the axial mesoderm, collective migration ofmesendoderm cells, epiboly of the ectoderm, and neural tube
formation. These movements must generate physical forces
between cells and tissues. There is accumulating evidence
that forces generated by moving cells and tissues affectmorphogenetic processes. The purpose of this project is toelucidate mechanisms that sense and respond to mechanicalforce in Xenopus embryonic cells, and to clarify the roles ofmechanotransduction systems during development.
It is known that cells can sense mechanical stresses inseveral ways, for example, TRP channels, cell surface glycoproteins, flow-sensing cilia, cadherins, and focal adhesions.
Physical stimuli sensed by these molecules may be converted to intracellular signaling and induce cellular response.
Several studies using mammalian tissue culture cells haverevealed that mechanical stresses induce activation of some
protein kinases. Therefore we examined whether mechanical force might change protein phosphorylation in Xenopus
embryonic cells. For this purpose, we used the proteomic
approach to comprehensively analyze protein phosphorylation profiles. We have found that mechanical stress applied to
Xenopus embryos changes phosphorylation states of several
proteins, suggesting that Xenopus embryonic cells havea mechanism to sense mechanical forces, which involves
protein phosphorylation/dephosphorylation.

III. M athematical analysis of neural tube
closureDuring early development of the central nervous system,neuroepithelial cells decrease their apical surface area byactomyosin contractility. This cell shape change is calledapical constriction, which converts the neural plate into
a tubular structure, called the neural tube. We previously
showed that intracellular calcium ion (Ca2+) dynamicallyfluctuated throughout the Xenopus neural plate from theNote: Those members appearing in the above list twice under different titles are members whose title changed during 2015. The former title isindicated by an asterisk (*).

single-cell to whole-tissue levels with distinct patterns. Liveimaging revealed that the Ca2+ fluctuations correlated with aremodeling of F-actin and the apical constriction, suggesting that the dynamic Ca2+ signaling positively regulates theneural tube closure. In this study, to further investigate therole of the Ca2+ fluctuations, we developed a two-dimensional vertex model, in which the natures of the apical constriction and the pulsed contraction of the apical junctions wereintroduced. Computational simulation of our model showsthat the pulsed contraction accelerates the apical constriction
and decrease of the tissue size independent of its frequency. However, although dense rather than sparse pulsationinduces rapid tissue deformation, its overall effect throughout
the entire course of the simulation is not effective when the
total number of the pulses is constant. These data suggestthat the distinct patterns of Ca2+-induced local contraction
always contribute to tissue deformation.

was captured by the invagination with live-imaging analysis
and Serial block face Scanning Electron Microscopy (SBFSEM) observation. Posteriorly localized cilia are conserved
among several organisms including ascidians for establishing
the left – right (L-R) axis. Our investigations will provide a
new model of not only spindle orientation, but also ciliary
positioning.

Figure 3. Membrane invagination associated with the epidermal cilia ofacidian embryo.
(A) 3D image reconstructed from SBF-SEM data shows that tips ofinvagination approach the basal body. The basal body and daughter centriole are indicated as blue and red balls, respectively. The black-lined
square is enlarged in upper right panel. Anterior is left. (B) A frame of atime lapse video of membrane invaginations reaching cilia. PH-tdTomato(green), plasma membrane probe; Arl13b-GFP (magenta), cilia probe.
Anterior is left.

V. Notochord and evolution of chordates

Figure 2. Effects of apical constriction (AC) and pulsed contraction(Pulse) on modeled epithelial sheets.
Representative images of modeled epithelial sheets at the end of eachsimulation (top) and average tissue sizes during the time course of thesimulations (bottom) without the apical constriction and the pulsed contraction (A), with the apical constriction (B), and with the apical constriction and the pulsed contraction (C). Error bars depict s.e.m.

IV. M e m b r a n e i n v a g i n a t i o n t o w a r dcentrosome links the posterior ciliary
positioning to spindle orientation in
ascidian epidermal cells.

The role of the centrosome changes during cell cycle progression. Centrosomes form the basal bodies of cilia in interphase and the pole of the spindle in mitotic phase. Then, thepositioning of the centrosome is important for the polarity ofcilia and the orientation of the spindle.
During the last mitotic division of epidermal cells of theascidian embryo, it is already reported that most cells divide
along the anterior-posterior (A-P) axis. We found a uniquefilamentous membrane structure, which we call “invagination” elongated toward the centrosome in this division cycle.
We have proposed a model, in which this novel membrane
invagination maintains the position of the centrosome tothe posterior of the cell with tensile force and is involvedin spindle orientation along the A-P axis. Interestingly, we
also found the formation of cilia on epidermal cells during
the last division cycle. Therefore, we confirmed the positionof cilia located to the posterior of the cell and its basal body

The expression profile of Brachyury is highly conservedin metazoans from cnidarian to vertebrates. Specifically,
Brachyury is expressed in the blastopore region of early
embryos, and functions in invagination of the endomesodermal germ layer through the blastopore into the embryo. This
“primary” function of Brachyury is associated with morphogenesis.
Comparison of Brachyury expression between nonchordate invertebrates and chordates clearly shows an additional
expression domain seen only in chordates, namely, the dorsal
midline of the blastopore, an embryonic region associated with the formation of the notochord. This “secondary”
function arose during the evolution of chordates.
Therefore, it is now evident that the evolutionary origin of
the notochord is essentially the question of the molecularmechanisms underlying how Brachyury acquired its secondary expression domain at the mid-dorsal region of theblastopore. To know how chordate ancestors gained the new
expression domains, we used Amphioxus, the most ancestral chordates. We functionally analyzed the Amphioxus
Brachyury promoter regions by reporter assay, using Ciona
embryos.

VI. T he biological function of invertebrate
DNA methylation in pre-mRNA processing

DNA methylation at cytosine residues is an important
epigenetic modification found in eukaryotes ranging fromplants to humans. Invertebrates offer an interesting model forstudying evolutionary changes in the targets and the functionof DNA methylation. A marine invertebrate chordate Ciona
intestinalis has a genome-wide mosaic methylation patterncomprising methylated and unmethylated genes. It has been

15

National Institute for Basic Biology

Developmental Biology

observed that DNA methylation is targeted to the transcribedregion of ubiquitously expressed genes, and a constant targeting of the “gene body methylation” irrespective of cell
types. To reveal the function of gene body methylation ingene transcription, we analyzed newly synthesized RNA
from C. intestinalis embryos. By using 4sU labeling andsequencing methods, revealing global RNA processing
kinetics at nucleotide resolution, we obtained snapshots ofactive transcription. Significant differences were seen inco-transcriptional splicing efficiency, in connection withmethylation status of exons and introns. The splicing efficiency and DNA methylation status were also correlated tonucleosomal positions, suggesting that epigenetic states inthe bodies of transcribed genes control the pre-mRNA processing through nucleosomal positioning.

VII. Cnidarian-symbiodinium Symbiosis

Corals are declining globally due to a number of stressors. Such stresses can lead to a breakdown of the essential
symbiotic relationship between coral and Symbiodinium, aprocess known as coral bleaching. Although the environmental stresses causing this breakdown are largely known,
the molecular and cellular mechanisms of symbiosis are still
unclear. Corals are not very suitable as laboratory systems,
because they are difficult to work with due to their slow
growth, long generation times, and calcareous skeletons.
To overcome these limitations, we focused on the small sea
anemone Aiptasia as a novel experimentally tractable cnidarian model organism (Figure 4). Aiptasia, just as reef-building
corals, establishes a stable but temperature-sensitive symbiosis with Symbiodinium. Aiptasia can be repeatedly bleachedand repopulated with Symbiodinium, grows rapidly, and lacks
a calcareous skeleton, allowing microscopic and cell biological analyses. In order to further elucidate the symbiotic
mechanisms, it is necessary to establish molecular biological approaches. Therefore, we have attempted to develop amethod of gene transfection to Aiptasia. Investigating symbiosis using Aiptasia should improve our understanding of
the symbiotic mechanism.

Figure 4. Aiptasia spawning can be induced under controlled laboratory
conditions(A) Aiptasia mature female (left) and male (right). (B and C) Aiptasia
eggs and sperms, respectively.

Publication List:〔Original papers〕

M., Ueno, N., and Kinoshita, N. (2015). Phosphorylation• Kai,
dependent ubiquitination of paraxial protocadherin (PAPC) controls
gastrulation cell movements. PLoS One 10, e0115111.

A., Negishi, T., Yamamoto, T.S., and Ueno, N. (2015). G• Miyagi,
protein-coupled receptors Flop1 and Flop2 inhibit Wnt/β-cateninsignaling and are essential for head formation in Xenopus. Dev. Biol.

16407, 131-144.

T., and Yasuo, H. (2015). Distinct modes of mitotic spindle• Negishi,orientation align cells in the dorsal midline of ascidian embryos. Dev.

••

Biol. 408, 66-78.
Sakamaki, K., Iwabe, N., Iwata, H., Imai, K., Takagi, C., Chiba, K.,
Shukunami, C., Tomii, K., and Ueno, N. (2015). Conservation of
structure and function in vertebrate c-FLIP proteins despite rapid
evolutionary change. Biochem. Biophys. Rep. 3, 175-189.
Uno, Y., Nishida, C., Takagi, C., Igawa, T., Ueno, N., Sumida, M.,
and Matsuda, Y. (2015). Extraordinary diversity in the origins of sex
chromosomes in anurans inferred from comparative gene mapping.
Cytogenet. Genome Res. 145, 218-229.

〔Original paper (E-publication ahead of print)〕

T., Kuwasako, K., Ogasawara, M., Takahashi, H., Matsubara,• Sekiguchi,
S., Osugi, T., Muramatsu, I., Sasayama, Y., Suzuki, N., and Satake, H.

Evidence for conservation of the calcitonin superfamily and activityregulating mechanisms in the basal chordate Branchiostoma floridae:
insights into the molecular and functional evolution in chordates. J.
Biol. Chem. 2015 Dec 7.

DIVISION OF DEVELOPMENTAL GENETICS †Professor

KOBAYASHI, Satoru

Assistant Professor:
Technical Staff:
Postdoctoral Fellow:
SOKENDAI Graduate Student:
Technical Assistant:

Secretary:

HAYASHI, Yoshiki
SATO, Masanao
NODA, Chiyo
OTA, Ryoma
FUJISAWA, Chiemi
ASAOKA, Miho
SHINOZUKA, Yuko
SUGIMORI, Seiko
MORITA, Shunpei
SATO, Kaori
YAMAMOTO, Manami
WASHIO, Midori
KAWAMOTO, Yuri
SUGIYAMA, Arisa
HONDA, SatokoGerm cells are specialized cells that can transmit genetic
materials from one generation to the next in sexual reproduction. All of the other cells of the body are somatic 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, isinherited 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 to form eggs and sperm when the organisms are physically matured. How this developmental transition from thejuvenile stage to the adult reproductive stage is precisely
regulated in response to developmental and environmental
cues is a longstanding question in biology.

I. The mechanism regulating developmental
transition from larvae to reproductive adults

In holometabolous insects, the steroid hormone ecdysone
plays a pivotal role in metamorphosis. In Drosophila,
ecdysone is produced in the prothoracic gland (PG) and thenconverted into its active form, 20-hydroxyecdysone (20E),in the peripheral organs. The activities of 20E terminate
larval development and growth and initiate metamorphosis.
Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during
development. The stimulator of ecdysone biosynthesis is prothoracicotropic hormone (PTTH) and Insulin-like peptides(Ilps), which activate the production of ecdysone biosynthetic proteins. In addition to these neuropeptides, the larval–
prepupal transition is modulated by environmental cues suchas nutritional conditions that influence larval body size. For
example, early third-instar larvae attain the body-size checkpoint required for the transit from larva to prepupa. Although
the checkpoint is believed to ultimately modulate ecdysone
production in the PG, its downstream effectors and signaling
pathway remain elusive.
We found that monoaminergic autocrine regulation of
ecdysone biosynthesis in the PG is essential for metamorpho-

sis. PG-specific knockdown of Octb3R, resulted in arrested
metamorphosis due to lack of ecdysone. Knockdown oftyramine biosynthesis genes expressed in the PG caused
similar defects in ecdysone production and metamorphosis.
Moreover, PTTH and Ilps signaling were impaired by
Octb3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus,
monoaminergic autocrine signaling in the PG regulates
ecdysone biogenesis in a coordinated fashion upon activation
by PTTH and Ilps. We propose that monoaminergic autocrine
signaling acts downstream of a body-size checkpoint thatallows metamorphosis to occur when nutrients are sufficiently abundant.

Figure 1. Tyramine-Octb3R signaling is required for ecdysone biosynthesis.
(A and B) The PGs of control (A) and Octb3R knockdown larvae (B)were labelled for the dephosphorylated form of ERK (dpERK, green) andDNA (blue). Expression of dpERK, a downstream signaling componentof the PTTH pathway, was reduced in the PG of Octb3R knockdown.
(C and D) Our model for the regulation of ecdysone biosynthesis.
Tyramine is secreted from the PG after the attainment of body-size
checkpoint. Tyramine-Octb3R signaling activates PTTH and Ilps signaling, which leads to coordinated activation of ecdysone biosynthesis (C).
In the PG of Octb3R knockdown larvae, ecdysone biosynthesis is not
activated due to loss of PTTH and Ilps signaling activity (D).

Publication List:〔Original papers〕

M., Hira, S., Nakamura, K., Nakamura, S., Kimura, H., Sato, M.,• Mukai,and Kobayashi, S. (2015). H3K36 trimethylation-mediated epigenetic•

regulation is activated by Bam and promotes germ cell differentiation
during early oogenesis in Drosophila. Biology Open 4, 119-124.
Ohhara, Y., Shimada-Niwa, Y., Niwa, R., Kayashima, Y., Hayashi,
Y., Akagi, K., Ueda, H., Yamakawa-Kobayashi, K., and Kobayashi,
S. (2015). Autocrine regulation of ecdysone synthesis by b3octopamine receptor in the prothoracic gland is essential for Drosophila
metamorphosis. Proc. Natl. Acad. Sci. USA 112, 1452-1457.

†: This laboratory was closed on 31 March, 2015.

17

National Institute for Basic Biology

Developmental Biology

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

Assistant Professor:

YABE, Taijiro
MII, Yusuke
Technical Staff:
UTSUMI, Hideko
OKADA, Kazunori
NIBB Research Fellow:
Postdoctoral Fellow:
CHEN, Qiuhong
TAKADA, Ritsuko
FUJIMORI, Sayumi
WANGLAR, Chimwar
SOKENDAI Graduate Student: SHINOZUKA, Takuma
TSUCHIYA, Yoshihiro
Visiting Graduate Stutent: WEICHSEL, Alexander
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
Secretary:
UKAI, Sakie
The morphology of the body and tissues is established inspatio-temporarily regulated manners. A number of genes
involved in morphogenesis have been identified, but it is still
uncertain how the spatial and temporal information are established and converted to morphology during embryogenesis.
Therefore, we are challenging to understand the mechanism
by which the spatial information is established and that bywhich the temporal, or periodical, information is converted
into morphology by several different approaches.
In the development of many tissues, secreted signal molecules are important for the formation of spatial information. These molecules are secreted from producing cells andtransported to surrounding cells, resulting in the formationof concentration gradients. Given that their concentration
decreases according to the distance from the source, thegradient of the signals defines relative positions of receiving
cells in developing tissues. Many genetic studies revealed
that a limited number of secreted signal proteins, including
Wnt, BMP, and Hedgehog, function in the morphogenesis oftissues and embryos. In spite of the accumulation of genetic
evidence, however, the molecular mechanism that regulates
their spread in particular developing tissues remains to beelucidated. To this end, we started to visualize signal proteinsand monitor their movement in tissues. In addition, we are
examining structural and biochemical characteristics of thesemolecules, which appear to affect how they spread.
The segmental sub-regions of several specific tissues,
including the paraxial mesoderm (or somites) and thepharyngeal pouches, by contrast, appear not to be simply
directed by the gradient of the signal molecules, but by aunique mechanism proceeding periodically. For instance,
somites are sequentially generated in an anterior-to-posteriororder by converting the temporal periodicity created by amolecular clock into periodical structures. The molecularmechanism underlying this conversion and morphologicalsegmentation, however, is not yet fully understood. Thus,
another goal of our current studies is to reveal the molecularmechanism of this other and unique mode of patterning that
underlies the periodical and sequential sub-division in the18

development of somites and pharyngeal arches.

I. Spatial regulation of secreted Wnt proteinsin vertebrate development

The Wnt family of secreted signal proteins plays a key rolein numerous aspects of embryogenesis. In a classical view,it has been proposed that secreted signal proteins, like Wnt
proteins, spread and function over a distance of several cells
in many aspects of morphogenesis. In contrast, accumulating
evidence also implies that Wnt proteins appear to transmit
their signals locally in some particular cases. Therefore,
their secretion and transport might be differently controlleddepending on situation. Thus, for understanding the spatial
regulation of tissue morphogenesis, the molecular mechanism underlying the spreading of Wnt proteins should be
revealed.
For better understanding the spreading of Wnt proteins, westarted to visualize Wnt proteins in the extracellular space by
several different approaches. Our preliminary study indicatedthat Wnt proteins are not simply diffused during embryogenesis of the mouse. We precisely examined regulatory mechanisms and biological significance of Wnt protein distribution
in mouse embryos. These analyses revealed a novel view ofspatial regulation of Wnt signaling.
Extracellular molecules are known to regulate special distribution of Wnt proteins. Once Wnt proteins are secreted
from the producing cell, their spread is regulated throughinteraction with these molecules. The heparan sulfate proteoglycan (HSPG), which is composed of a core proteinwith several chains of HS glycosaminoglycans, is a major
component involved in this interaction. Genetic analyses
with Drosophila mutants defective in the core proteins or
GAG biosynthesis indicate the requirement of HSPG in
Wnt signaling and gradients. In collaboration with Prof.
Taira at the University of Tokyo, we found that HSPGs withdifferent sugar chain modifications form distinct extracellular structures, which are called heparan sulfate nanostructures (HSNSs), in the early Xenopus embryo. Interestingly,
Xenopus Wnt8 proteins were preferentially retained by
HSNSs with N-sulfo modification (N-sulfo HSNSs). To
understand the mechanism by which distinct distributions of
HSNSs, especially N-sulfo HSNSs are formed, we focusedon NDST1, an enzyme catalyzing N-sulfation of heparansulfate. Gain and loss of function studies of NDST1 indicated that NDST1 is necessary and sufficient for the conversion of N-acetyl HSNSs into N-sulfo HSNSs. Extracellularaccumulation of Wnt proteins and Wnt signaling were also
affected by perturbation of NDST1 expression. These results
indicated that N-sulfo modification of heparan sulfates areimportant for extracellular distribution of Wnt proteins andWnt signaling (Figure 1).

Figure 1. Schematic representation of the 2 different HSPG clusters of
Wnt signaling.

II. H eterogeneity of secreted Wnt proteinssecreted from culture cells

Although the structure of Wnt protein has already been
revealed by X-ray crystallography, its higher order structure
in extracellular space has not yet been fully understood. Oneof the proposed forms of Wnt proteins in extracellular spaceis binding to lipoprotein particles. On the other hand, recent
studies indicated that Wnt proteins are secreted on anotherlipid-based carrier, called the exosome, which is an MVB(multivesicular body)-derived membrane vesicle. However,it remains unclear whether Wnt proteins are secreted only inthese two forms, or also in some other forms from the same
cells, or if different forms of Wnt proteins are secreted in acell type specific manner.
To address this question, we systematically examined characteristics of Wnt proteins secreted from polarized MDCK(Madin-Darby Canine Kidney) cells. Although some Wnt3a
proteins from mouse L cells were secreted together withlipoprotein particles, most of the Wnt3a proteins from either
apical or basolateral side of MDCK cells did not. In contrast,
secretion of a small amount of Wnt3a proteins via exosomes
was detectable from the basolateral side of MDCK cells ina lipidation dependent manner. Interestingly, some apicallysecreted Wnt3a was secreted with an exosome marker,
CD63, but its density was higher than typical exosomes.
Signaling activity of Wnt proteins on basolaterally secreted
exosomes and apically secreted exosome-like vesicles werelower than that of Wnt3a proteins in conditioned media.
We also found that Wnt11, which is preferentially secreted
from the apical side of MDCK cells, did not associate withexosomes, but with CD63-containing exosome-like vesicles.
Our results indicate that Wnt are secreted with multiple
carriers from polarized epithelia cells and suggest that Wnts
are differently packaged depending on Wnt subtype andcellular context.

III. M o l e c u l a r m e c h a n i s m o f s o m i t edevelopment

Somites are segmental epithelial blocks located symmetrically on either side of the neural tube. Somites are periodically generated in an anterior to posterior manner from theirprecursor, the presomitic mesoderm (PSM), which is locatedposterior to newly formed somites. This periodic generation is achieved by a complex and dynamic mechanism in
the PSM. First, the molecular clock, the so-called segmentation clock, creates oscillatory expression of particulargenes, hairy and some notch-related genes, in the posterior
PSM. Because the phase of oscillation is gradually shiftedalong the posterior-to-anterior axis, a wave of the oscillation
appears to move in a posterior-to-anterior fashion. This oscillatory gene expression subsequently resulted in periodical
generation of morphologically segmented somites.
The spatial pattern of somites is defined by positioning ofintersomitic boundaries and rostro-caudal patterning within asomite. This spatial pattern is established in the anterior PSM
by converting the oscillatory gene expression into spatiallyrestricted patterns of gene expression. Several intra-cellularmolecules, as well as FGF and Wnt signaling, are involved inthis conversion. In the mouse, the prospective segmentation
boundary is periodically established in the anterior PSM at
the rostral border of the Mesp2 expression domain. Mesp2,
one of the key regulators in this conversion, is initiallyexpressed at the most anterior region of the Tbx6 protein
domain. This expression is not anteriorly extended beyond
the anterior border of the Tbx6 protein domain because
Mesp2 expression requires Tbx6 proteins. Thus, the anteriorborder formation of the Tbx6 protein domain is a more
fundamental process in the positioning of the segmentation
boundary.
Importantly, this border is not consistent with the anteriorborder of Tbx6 mRNA, rather it is regulated by a proteasome-mediated mechanism. Although the molecules directly
executing this proteolysis are still unclear, Mesp2, as wellas Ripply1 and Ripply2, have shown to be required for the
down-regulation of Tbx6 proteins by analysis of mouseembryos defective in these genes. Since expressions of
Ripply1 and 2 are eliminated in Mesp2 deficient mouseembryos, we previously proposed the following model;
Mesp2, whose expression is activated in the most anterior
part of the Tbx6 domain, causes retreat of the Tbx6 protein
domain through activation of Ripply1 and 2 expression, andthe retreated Tbx6 subsequently defines the next segmentation border and Mesp2 expression. Recently, we showed that
Ripply is a direct regulator of the Tbx6 protein level for theestablishment of intersomitic boundaries. However, it is still
to be elucidated whether Mesp is actually required for theformation of the boundaries in zebrafish.
To answer this question, we generated mesp-deficientzebrafish. Since four mesp genes have been identified in thezebrafish genome, we generated mutant fish carrying theframe shift mutation in all of the mesp genes using TALEN
mediated mutagenesis and analyzed its phenotype (Figure 2).
Unlike the mouse Mesp2 mutant, zebrafish mesps quadruple
embryo exhibited normally segmented somites and a normalboundary of the Tbx6 protein domain, although segmented
expression of epha4a was disturbed in the anterior PSM,
suggesting mesps and mesps dependent expression of epha4a
are dispensable for somite positioning and segment boundary
formation (Figure 3). Further analysis revealed zebrafish
mesp genes are required for establishment of caudal identity

19National Institute for Basic Biology

Developmental Biology

of somites similar to mouse Mesp2. mesps quadruple mutant
embryos also showed disrupted superficial horizontal myoseptum formation probably resulting in the mis-migration ofpigment cells and lateral line primordia in later development.
In contrast to mesp, double mutant for ripply1 and ripply2
revealed that these two genes are essential for segmental
boundary formation. We found that ripply1 and 2 expression is induced by a manner independent of mesp. Rather,
striped expression of ripply1 and 2 at the anterior PSM wereseverely disrupted in double mutant for her1 and 7, whichare zebrafish clock genes encoding Hairy-related transcriptional repressors, suggesting proper regulation of ripply1
and 2 expression by Hairy is essential for somite boundary
formation. Furthermore, in ripply1 and 2 double embryos,
oscillatory expression of her1 was expanded to the anterior
paraxial mesoderm, indicating that ripply1 and 2 are also
required for termination of oscillation associated with mature
somite formation.

IV. Molecular mechanism of the developmentof pharyngeal pouches

In addition to somites, metameric structures are observedin the pharyngeal region of vertebrates. Typical examples ofsuch structures are skeletal elements of jaws, gills and cranial
nerve projections. This metamerism is brought about by thesegmental development of the pharyngeal pouches, which
are generated by outpocketing of the pharyngeal endoderm.
However, the molecular mechanisms underlying the segmentation of the pharyngeal pouches and the morphogenesis of
the pharyngeal pouches still remained to be elucidated.
To understand these mechanisms, we are currently examining the function of several genes essential for the segmentation and morphogenesis of pharyngeal pouches using fishand mice as model systems.

Figure 2. Expression of four mesp genes in zebrafish embryos. The
zebrafish four mesp genes, mespaa, maspab, mespba, and mespbb, aresimilarly expressed in the anterior presomitic mesoderm during somitogenesis.

Figure 4. Segmental structure of the pharyngeal pouches in zebrafish
embryos. Endodermal gene expression of pax1 mRNA (Upper) andsox17-egfp (Lower) show the formation of segmental structures of thepharyngeal pouches.

Publication List:〔Original papers〕

Kametani, Y., Chi, N.C., Stainier, D.Y.R., and Takada, S. (2015). Notch• signaling
regulates venous arterialization during fin regeneration.

Figure 3. Phenotype of mesp quadruple mutant embryo. In contrast to themouse, zebrafish mesp genes are dispensable for the formation of segmentation boundaries

20•

Genes Cells 20, 427-438.
Okubo, T., and Takada, S. (2015). Pharyngeal arch deficiencies affecttaste bud development in the circumvallate papilla with aberrant
glossopharyngeal nerve formation. Dev. Dyn. 244, 874-887.

DIVISION OF EMBYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
SOKENDAI Graduate Student:
Technical Assistant:
Secretary:

TOYOOKA, Yayoi
KOYAMA, Hiroshi
OKA, Sanae
SHI, Dongbo
DAY, F. Timothy
NAKANOH, Shota
KAMEMIZU, Chizuru
ITO, Tomoaki
USAMI, Fumiko
HIGUCHI, Yoko
KATO, Azusa

The aim of our research is to understand the events underlying early mammalian development during the period frompre-implantation to establishment of the body axes.
Early events during embryogenesis in mammals are relatively less understood, as compared to other animals. Thisis mainly due to difficulties in approaching the developing
embryo in the oviducts and the uterus of the mother. Anothercharacteristic of mammalian embryos is their highly regulative potential. The pattern of cell division and allocation ofcells within an embryo during the early stages vary between
embryos. The timing of the earliest specification events
that control the future body axes is still under investigation.
Functional proteins or other cellular components have notbeen found that localize asymmetrically in the fertilized egg.
We want to provide basic and fundamental information about
the specification of embryonic axes, differentiation of celllineages, behaviors of cells, and the regulation of body shapein early mammalian development. We are also interested in
the relationship between embryos and their environment, theoviducts and the uterus.

I. Establishment of a live imaging system forobservation of early mouse development

To understand the mechanisms underlying embryonicdevelopment and morphogenesis, it has become common toobserve the behavior of cells and gene expression in living
embryos in a variety of animal species. Progress in GFP
technologies, genetic engineering and embryonic manipulation concomitantly with the progress in optics and microscope technologies, including development of highly sensitive photo-detectors, has allowed us to observe developing
embryos live, even in mammals. We have established a seriesof transgenic mouse lines for live imaging, which is part ofa collaborative project taking place in the Laboratory for
Animal Resources and Genetic Engineering, Riken CLST.
These mouse lines have been used to visualize nuclei, cell
shapes, the cytoskeleton and other organelles to observe cell
behaviors in living mouse embryos in many laboratories over
the world. We also established mouse lines to monitor thecell cycle.
We have also been establishing several reporter mouse lines
in the lab to study gene expression patterns during the peri-

implantation stage of mouse development. In these mice,
cDNA encoding fluorescent proteins are connected to themouse genomic DNA sequences of the enhancer/promoter
region of important genes encoding factors regulating celldifferentiation in these stages. Combining these two typesof reporter transgenic mouse lines, we have been analyzing
behaviors of cells comparing gene expression properties atthe single cell level.
For the live imaging of early mammalian embryos, acombined microscope and incubation system is an important
tool. Conventional CO2 incubators provide better conditions
compared to microscope top incubation chambers, including
stability of temperature and humidity, to support embryonic development in vitro. Incubation microscopes have also
recently become commercially available, however, these are
expensive for personal use. We have modified an incubation
microscope with wide field fluorescent illumination, whichis relatively inexpensive. We added a spinning disc confocal
system and sensitive EM-CCD camera for observation ofdeveloping mouse embryos with less photo-toxicity andhigher spatial resolution.
We have been trying to observe and reveal aspects of cell
shape, morphogenesis, cell lineage, gene expression and celldifferentiation in developing embryos and in other tissues bycombining these techniques. We reported multiple phases intranscriptional regulation of the Nanog gene, which is known
to be one of the major pluripotent regulators, during the preimplantation stages of development.

Figure 1. Multiple phases in Nanog gene expression during preimplantation mouse development. Weak expression of Nanog starts around the
4-cell stage, and is random. From the 4-cell stage to the morula, the upregulation of Nanog expression is not lineage specific. Lineage specificdifferences appear by the early blastocyst stage, and inhibition by FGF
signaling becomes evident in the late blastocyst respectively. Images are
adapted from Komatsu and Fujimori, 2015.

II. Histological observation of mouse embryos
developing in the uterus

One of the characteristics of mammalian development isthat embryos develop in the uterus after implantation. Directinteraction between embryos and the cells of the uterus isimportant. In studies on developmental biology of mammalian embryos, embryos are usually removed from theuterus, and isolated embryos are analyzed. We have been
analyzing early embryonic development of mouse comparing the changes in the uterine epithelium. To observe themorphogenesis of embryos within the uterus, serial sectionsof implanted uteruses were made, stained with hematoxylin
and eosin, and images of the embryos within the uteruses
were captured to make high resolution three-dimensional
re-constructions. Figure 2 shows an example of a section. We
are preparing samples obtained from various developmental stages. Precise positions of embryonic implantation andchanges in the uterine epithelium correlating with embryonic21National Institute for Basic Biology

Developmental Biology

development will be examined using these images. To obtain
three dimensional images of embryos within the uterus,
we have been developing a system to automatically extract
regions of the uterus where embryos localize by utilizing
image analysis after images of serial sections are captured
using a slide scanner.

parallel to the organ axis. However, in the mutant oviducts,
epithelial folds no longer run parallel to the organ axis. The
direction of the folds was randomized, and ectopic branchesof the folds were observed. This suggests that Celsr1 isimportant for the formation of multi-scale polarities in themouse oviduct ranging from the cellular to the tissue scale.
Currently, we have been trying to reveal the mechanisms ofoviduct epithelial morphogenesis by integrating the molecular functions of PCP factors, cellular shape changes, tissuemorphology, and involvement of mechanical forces. We arealso focusing on some other PCP regulators, and are trying
to understand how oviduct epithelial cells establish andmaintain their polarity.

Figure 2. A pregnant uterus 3.5 days after fertilization. Developing
embryos and the shape of uterus epithelium can be observed.

III. Polarity formation in the mouse oviductepithelium

The oviduct (Fallopian tube) is the organ connecting theperiovarian space and the uterine horn. The ova released
from the ovary are transported through the oviduct, where
the ova are fertilized by spermatozoon entering from theuterus. We are focusing on the region of the oviduct close to
the opening to the ovary.
The epithelium of the mouse oviduct consists of multi-ciliated cells and secretory cells. Ovulated oocytes are transported in oviducts by unidirectional motility of multi-cilia andthe resultant secretory fluid flow from ovary to uterus. This
cellular orientation in the oviduct is one example of planarcell polarity (PCP). PCP is seen in many animals and tissues,
where cells sense global axes of the tissue to which they
belong and orient themselves to fulfill specialized functions.
Many evolutionally conserved genes are required for theformation of PCP, and several of those encode proteins thatare localized in polarized manners within cells. We foundthat Celsr1, a PCP core member, was strongly concentrated
only at the specific domains of cell boundaries which areperpendicular to the ovary-uterus axis and that this polarized
localization appeared to precede the directional movement ofcilia.
In Celsr1-deficient mutant oviducts, cilia were generated
and those within each cell appeared to beat as in the wild
type oviduct. However, the beating direction was not coordinated along the ovary-uterus axis throughout the epithelia;
some cells were oriented orthogonally to the axis, whilesome others exhibited reverse polarity. In addition to theciliary polarity phenotype, cellular shape polarity was also
impaired in the Celsr1-deficient mice. We found a newfeature of cellular polarity in the wild type oviduct, e.g. thatthe apical surface of epithelial cells was elongated along theovary-uterus axis. In Celsr1-deficient mice, epithelial cells
showed less elongation and randomized orientation.
Furthermore, we also found that the three-dimensionalarchitecture of oviductal epithelia was dramatically disrupted. In adult wild-type mice, the epithelial cell sheet is bent
multiple times, which forms around twenty longitudinal folds

22

Figure 3. Celsr1 localization in mouse oviduct epithelium. (A-B)
Confocal microscopy images of the oviduct epithelium stained for Celsr1
and F-actin (Phalloidin) at several developmental stages. The ovary side
is to the left and the uterus side is to the right. Higher magnification ofCelsr1 images are shown in (B). P: postnatal days. Arrows indicate theaxes; L, longitudinal axis (the ovary-uterus axis); C, circumferential axis.
(C) Distribution and orientation of the cellular polarity of Celsr1 localization quantified by image processing. Scale bars: 10 micron. Images are
adapted from Shi et al., 2014.

IV. Analysis of mechanical properties of cells
and tissues during embryonic development

Mechanics is one of the essential components of biological processes, including cell shape transformation and tissuemorphogenesis etc. However, how mechanical states such as
force and material stiffness regulate these processes is poorly
understood. To approach this problem, measuring cellular
and tissue geometric information and mechanical states isnecessary. We developed image processing based techniques
to measure cellular and tissue geometric information fromfluorescent microscopic images and frameworks to theoretically estimate the mechanical states.

Figure 4. Theoretical estimation of cellular/tissue mechanical states.
Schematic illustration of estimation.

By employing image processing techniques, we haveextracted geometric information, shapes, and movements ofearly embryogenesis in C. elegans and mice. In the framework for estimating mechanical states, geometric informa-tion was combined with a mechanical simulation, which
was based on data assimilation (Figure 4). We successfully
estimated the spatio-temporal dynamics of cellular and tissue
mechanical states by systematically fitting the in vivo geometric states to the mechanical simulation. The mechanicalinformation will be useful to investigate physical mechanisms of early embryonic development and morphogenesis
during organogenesis in late stages in development.

Publication List:〔Original papers〕

S., Nishikawa-Torikai, S., Futatsugi, Y., Toyooka, Y., Yamane,• Fujii,
M., Ohtsuka, S., and Niwa, H. (2015). Nr0b1 is a negative regulator of

•
•
•
•

Zscan4c in mouse embryonic stem cells. Sci. Rep. 5, 9146.
Horikawa, S., Ishii, Y., Hamashima, T., Yamamoto, S., Mori, H.,
Fujimori, T., Shen, J., Inoue, R., Nishizono, H., Itoh, H., Majima, M.,
Abraham, D., Miyawaki, T., and Sasahara, M. (2015). PDGFRα plays acrucial role in connective tissue remodeling. Sci. Rep. 5, 17948.
Komatsu, K., and Fujimori, T. (2015). Multiple phases in regulation of
Nanog expression during pre-implantation development. Dev. GrowthDiffer. 57, 648-656.
Nakayama, S., Arima, K., Kawai, K., Mohri, K., Inui, C., Sugano, W.,
Koba, H., Tamada, K., Nakata, Y.J., Kishimoto, K., Arai-Shindo, M.,
Kojima, C., Matsumoto, T., Fujimori, T., Agata, K., and Funayama, N.
(2015). Dynamic transport and cementation of skeletal elements build
up the pole-and-beam structured skeleton of sponges. Curr. Biol. 25,
2549-2554.
Reyes de Mochel, N.S., Luong, M., Chiang, M., Javier, A.L., Luu, E.,
Fujimori, T., MacGregor, G.R., Cinquin, O., and Cho, K.W. (2015).
BMP signaling is required for cell cleavage in preimplantation-mouseembryos. Dev. Biol. 397, 45-55.

23

National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
JSPS Research Fellow:
Research Fellow:
SOKENDAI Graduate Student:
Technical Assistant:
Secretary:

KITADATE, Yu
HARA, Kenshiro
NAKAGAWA, Toshinori
MIZUGUCHI, Hiroko
HIRA, Seiji
NAKAMURA, Yoshiaki
NAKAMURA, Yoshiaki*
IKAMI, Kanako
TOKUE, Moe
NONAMI, Yuta
ISHIZAKA, Miho
HIRANO, Kodai
KON, Yayoi
MARUYAMA, Ayumi
NISHIMURA, KeikoKUBOKI, Yuko

Overview of our research

Production of numerous sperm for a long period in testis
is fundamental for continuity of life across generations.
This process of spermatogenesis is supported by the robust
function of “stem cells”, which both maintain the undifferentiated cells, while generating differentiated cells in a nicely
balanced manner. The Division of Germ Cell Biology aims
to fully understand the mouse sperm stem cell system under
the context of in vivo testicular tissue. Our particular interests have been laid on the “undifferentiated spermatogonia”,
which are responsible for the stem cell functions. Our study
has revealed several key properties of this interesting population.
First, we found that this stem cell system includes a functional hierarchy. It is comprised of an “actual” stem cellcompartment that is prone to self-renew, and a differentiation-primed, “potential” stem cell compartment. Regarding
the “actual” stem cells, we have been investigating theircellular identity, their in vivo behavior at a single-cell resolution, and the underlying mathematical principles. This leadto the discovery of neutral competition between the stem
cells. We are currently investigating the mechanisms underlying the constant size of this population, and its connection
to its tissue environment. “Potential stem cells” are also of
our enthusiastic interest: In undisturbed, steady-state spermatogenesis, they largely contribute as transient amplifying
cells and differentiate rather than self-renew. However, whentissues are insulted or testicular cells are transplanted to ahost testes, their probability of self-renewal jumps up andthey effectively replenish the lost “actual” stem cells. Sucha flexible feature of stem cell dynamics has been found paradigmatic for many other stem cell-supported tissues.
Key references for these studies that are currently public
include Nakagawa et al., Dev. Cell 2007; Yoshida et al.,Science 2007; Nakagawa et al., Science 2010; Klein et al.,Cell Stem Cell 2010; and Hara et al., Cell Stem Cell 2014.

24

I. The stem cell dynamics

Morphologically, the population of Aundiff includes singly
isolated cells (As), or syncytia consisting mainly of 2 (Apr), 4(Aal-4), 8 (Aal-8), or 16 (Aal-16) cells. The formation of syncytia
is due to incomplete cell division, a germline-specific cell
division process where cytokinesis does not complete andthe cytoplasmic connection between daughter cells persists
via intercellular bridges. The prevailing stem cell theory
proposed in 1971 states that stem cell activity is restricted
to the population of As cells, while interconnected Apr and
Aal syncytia are irreversibly committed to differentiation andno longer contribute to the stem cell pool (Huckins, 1971;
Oakberg, 1971), known as the “As model”.
Figure 1 represents our latest model for the functional
structure of the spermatogenic stem cell system, which
indeed proposes an alternative for the “A s model”. Thisis the simplest interpretation of the results of our functional analyses of GFRα1+ spermatogonia, which act as the“actual” stem cells. These include intravital live-imaging
experiments, clonal fate analysis of pulse-labeled cells, andbiophysical modeling analysis of the results.

Figure 1. A proposed stem cell dynamics. On the top of the differentiation
hierarchy, GFRα1+ spermatogonia comprise a single stem cell pool, in

which cells continually and reversibly interconvert between states of As,

Apr and Aal spermatogonia through incomplete cell division (blue arrows)

and syncytial fragmentation (red arrows), while giving rise to Ngn3+
cells. After leaving the GFRα1+ compartment, differentiation-destined

cells follow a series of transitions (GFRα1+→Ngn3+→Kit+; downward

black arrows) that accompany the extension of syncytial length (rightward black arrows). Ngn3+ and, to a lesser extent, Kit+ cells retain thecapacity to revert back into the GFRα1+ compartment in a contextdependent fashion (broken arrows). (Reprinted from Hara et al., Cell
Stem Cell 2014.)

As crystalized in this model, our results suggest that the
GFRα1+ sub-population of A undff spermatogonia, which
include both As cells and syncytia (Apr and Aal) comprises
a single stem cell pool, in which cells continually interconvert between these morphologically heterogeneous states
through stochastic incomplete division and fragmentation ofsyncytia. The incomplete division and syncytial fragmentation causes the expansion of this population, while the excess
cells over a particular “quota” would overflow to become theNote: Those members appearing in the above list twice under different titles are members whose title changed during 2015. The former title isindicated by an asterisk (*).

Ngn3+ state of Aundff, which then further differentiate into Kit+
“differentiating” spermatogonia that are largely devoid ofself-renewing potential. Currently, we are investigating themechanism that determines the quota or tissue capacity of
GFRα1+ cells, as well as the detailed nature of the heterogeneity of this population.

II. M echanisms of the different behavior of“actual” and “potential” stem cells

In 2015, we challenged one key question: Why a particular“actual” stem cell population of Aundiff (i.e., GFRα1+ cells)
remain undifferentiated, while the other, “potential” stem
cells (Ngn3+) differentiate, despite that both cells are locatedin the same tissue “open niche” environment and exposedto the same differentiation-inducing signal? (Ikami et al.,Development 2015)

2-1. Architecture of the mouse testis – the “open niche”

It was generally considered that the behavior of stem cells
is regulated by a tissue microenvironment, or the stem cellniche. In some tissues, like Drosophila gonads or mammalian small intestines, stem cells cluster in an anatomicallyspecialized (“closed” or “definitive”) niche that determines
the stem cell fates. Because niche-derived signals appear to
be spatially restricted, cells that are located within the niche
can be maintained in an undifferentiated state, and their displacement from the niche leads to differentiation. In mouse
testis, in contrast, spermatogenic stem cells appear to be distributed over the basal compartment and intermingled withdifferentiating

Figure 3. Intermingling of GFRα1+, Ngn3+, and KIT+ spermatogonia.
Representative images of triple immuno-stained whole-mount seminiferous tubules of an Ngn3-EGFP mouse. Scale bars = 50 μm. Reproduced
from Ikami et al., Development (2015)

progeny, designated as an “open” niche. Here, the details of
the mechanisms that determine whether stem cells differentiate or remain undifferentiated are unknown.
Mouse spermatogenesis occurs in seminiferous tubules andrepresents a typical example of an open niche-supported
stem cell system (Figure 2) (Russell et al., 1990; Stine andMatunis, 2013). Here, GFRα1+ (roughly corresponding to“actual” stem cells), Ngn3+ (nearly “potential” stem cells),
and Kit + (“differentiating” spermatogonia that have lost
most stem cell potential) populations of spermatogonia areintermingled with each other in the basal compartment of theseminiferous tubules (Figure 3). These cells are uniformly
exposed to retinoic acid (RA), an essential differentiationinducing signaling molecule that is synthesized in a spatiallyubiquitous but temporally periodic manner. Intriguingly,
however, all of Aundiff cell do not differentiate, rather someAundiff always remain undifferentiated. Although this process
is extremely important for the long-term maintenance of thestem cells and the integrity of spermatogenesis, the underlying mechanism has been largely unknown.

2-2. H eterogeneous differentiation competence in
response to retinoic acid

Figure 2. Anatomy of seminiferous tubules and seminiferous epithelium.
GFRα1+ and Ngn3+ cells and KIT+ differentiating spermatogonia, residein the basal compartment (between the basement membrane and thetight junction of Sertoli cells). Modified from Ikami et al., Development(2015).

To address this issue, we tested the response of Ngn3 +and GFRα1+ spermatogonia to RA by pulse-labeling these
populations using the tamoxifen-inducible cre-loxP system.
The results indicated that Ngn3+ cells rapidly and efficiently
differentiated into KIT+ cells in response to RA. In contrast,
GFRα1+ cells did not show effective differentiation induced
by RA, but they generated Ngn3+ cells in an RA-independent
manner. Thus, GFRα1+ and Ngn3+ cells show distinctive differentiation competence in response to RA.
Next, through DNA microarray gene expression analysisof FACS-sorted GFRα1+ and Ngn3+ fractions, we found thatthe expression of Retinoic acid receptor gamma (RARγ) was
highly specific in Ngn3+ cells compared to GFRα1+ cells(Figure 4). In contrast, the levels of other transcripts related
to the RA signaling pathway were similar between thesecells, Further, we found that enforced expression of RARγ
provided the GFRα1+ cells with differentiation competence
to differentiate to KIT+ cells in response to RA.

25

National Institute for Basic Biology

Developmental Biology
Publication List:〔Original paper〕

K., Tokue, M., Sugimoto, R., Noda, C., Kobayashi, S., Hara,• Ikami,K., and Yoshida, S. (2015). Hierarchical differentiation competence in

response to retinoic acid ensures stem cell maintenance during mouse
spermatogenesis. Development 142, 1582-1592.

〔Review Article〕

S. (2015). From cyst to tubule: innovations in vertebrate• Yoshida,spermatogenesis. WIREs Developmental Biology, doi: 10.1002/
wdev.204

Figure 4. Predominant expression of RARγ in NGN3+ spermatogonia
compared with GFRα1+ cells.
Representative images of whole-mount seminiferous tubules from Ngn3EGFP mice stained for EGFP, GFRα1 and RARγ. Scale bars = 50 μm.
Reproduced from Ikami et al., Development (2015)

2-3. Differential dynamics of GFRα1+ and Ngn3+ spermatogonia

To summarize, these results inferred that, when exposed toa high concentration of RA, RARγ drives the Ngn3+ cells
to differentiation, while the absence of RARγ allows the
GFRα1+ cells to remain undifferentiated. This heterogeneous
differentiation competence, combined with the periodicallybut ubiquitously distributed RA, appears to be importantfor homeostatic spermatogenesis in the open niche environment of seminiferous tubules (Figure 5). This scenario
would be paradigmatic for other open niche-supported tissue
stem cells, since it would allow the stem cell population tomaintain the undifferentiated pool while periodically producing differentiating progeny.

Figure 5. A model of stem cell dynamics during the seminiferous epithelial cycle.
Before the elevation of RA, the entire Aundiff population is composed of a
mixture of GFRα1+ (RARγ­–) and Ngn3+ (RARγ+) cells (top). When the
RA amount increases, to which both GFRα1+ and Ngn3+ spermatogonia
appear to be equally exposed (right), only Ngn3+ cells that express RARγ
respond and differentiate into Kit + cells, while GFRα1+ cells remain
undifferentiated (bottom). Then, the GFRα1+ cells replenish Ngn3+ cells
through a RA-independent mechanism (left). Modified from Ikami et al.,Development (2015)

26

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION †Associate Professor

TANAKA, Minoru

NIBB Research Fellow:
Postdoctoral Fellow:
SOKENDAI Graduate Student:

Technical Assistant:
Secretary:

NISHIMURA, Toshiya
YAMAMOTO, Yasuhiro
FUJIMORI, Chika
NISHIMURA, Toshiya*
SAKAE, Yuta
KIKUCHI, Mariko
OTAKE, Norihito
KINOSHITA, Chie
WATAKABE, Ikuko
SHIRAISHI, Naomi

Laboratory scope
Reproduction is a universal and fundamental system fororganisms to produce new generations. To accomplish thispurpose, organisms have developed their own sexual strategies, which allow them to adapt to their environment, thereby
progressing toward maximum efficiency of reproduction.
During the embryo and larval terms, organisms develop
many cell-lineages that have special and essential roles ineach different process of reproduction. These lineages aremostly conserved among vertebrates.
Vertebrates, however, exhibit a variety of reproductive
systems. The mechanisms of sex determination and sex differentiation are some of the components of the reproductive
system which produce this variety. Actually, there are many
modes of sex determination. Sex determination genes aredifferent among vertebrates. Sex determination does not evenhave to be controlled genetically. This variety is allowed by
the different employment and different emergence of the celllineages during embryogenesis. Therefore, it is important toaddress the roles of each cell lineage for understanding thefundamental mechanism underlying a variety of reproductive
systems. Currently, our lab focuses on the core mechanisms
which are independent of sex determination genes and which
produce and maintain the sex. The core mechanism can bereferred to as cellular interaction between germ cells and surrounding somatic cells, wherein germ cells have the ability
to feminize somatic cells while the surrounding somatic cells
are predisposed to male development. These characters ofeach set of cells are totally independent of the sex determination gene on the medaka Y chromosome. We are addressing
the details of this core mechanism by analyzing each celllineage in the context of sex differentiation.
To accomplish the purpose of our study, we use medaka
fish (Oryzias latipes). We have been generating transgenic
medaka enabling us to analyze how different cell lineages
are involved in the process of gonad formation and sex differentiation in vivo. Additionally, in order to identify thegenes essential for reproduction, we carried out a mutational
screening of medaka with defective phenotypes and disrupted several candidate genes. With these two unique analyticalmethods (visualizing cells, and mutants), we are attempting
to unveil both the fundamental mechanisms and the specificmechanisms that produce a variety of reproductive systems.
Through these analyses, we have been revealing thepresence of germline stem cells in the ovary. This was thefirst proof of this system in vertebrates (Nakamura et al 2010
Science). The fluorescently labeled germline stem cells keep
producing eggs with fluorescence during the entire period of
medaka reproduction, which is a conclusive indication of thepresence of germline stem cells. The tricks employed in thisexperiment are transgenic medaka that allow heat-inducible
gene expression.

I. An issue of sex determination of germ cells

In the core mechanism of sex, germ cells are responsible forfeminization and somatic cells are for masculinization. An
important thing here is that we can determine the sex intentionally, if the core mechanism is to be modified, without any
effect from the sex determination gene. Then a big issue is
how the sex of germ cells, in other words, the fate decisionof germ cells to become sperm or eggs, is determined. Few
people have addressed this issue in vertebrates.
One way of looking at this issue is that there is no germcell-intrinsic mechanism of sex. Somatic cells might senda signal(s) to germ cells during each developmental stageof spermatogenesis/oogenesis. In this case, it would be difficult to discern the germ cell-intrinsic mechanism of sex.
Alternatively, we might assume that the sexual fate of germ
cells is determined at the initial commitment into gametogenesis. Somatic cells may only regulate the timing and/or supply an essential material(s) for germ cells to develop
into the next stage of gametogenesis. In this case, once thesomatic cells give a cue to germ cells for which way to go,
the path toward oogenesis or spermatogenesis is alreadydetermined. But nobody has shown which way of understanding is right in germ cell sex determination.
Since transplanted germ cells from testis into females
develop into eggs and the germ cells from ovaries become
sperm in testis, It is generally accepted that germline stem
cells are sexually indifferent or unfixed. No morphologicaldifference between germ cells from females or those frommales exist during early stages of gametogenesis, until theyreach the pachytene stage in meiosis. At the pachytene stage,
female germ cells are larger than male germ cells. Theseobservations have collectively suggested that germ cell-sex
determination occurs at some point between the germline
stem cell stage and that of the pachytene stage (figure 1).

Figure 1. Simplified cellular processes of gametogenesis. Given thatan intrinsic mechanism of sex determination is present in germ cells, it
should be between the stage of germline stem cells and that of pachytene
in meiosis (yellow graded area). foxl3 is found to be expressed only infemale germ cells (pink germ cells) in the yellow graded term.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2015. The former title isindicated by an asterisk (*).
†: This laboratory was closed on 31 May, 2016.

27

National Institute for Basic Biology

Developmental Biology

II. Identification of a sexually different genein early germ cells (Nishimura et al 2015
Science)

We have prepared all XX or all XY medaka larva and havepurified germ cells during early gametogenesis. Different
stages of germ cells were successfully purified using fluorescent activated cell sorter (FACS) and the transcriptomesof different stages and different sexes of germ cells wereanalyzed by next-generation sequencing.
One gene that attracted us was foxl3, a gene encoding atranscriptional factor with the forkhead domain. The geneis initially expressed in both female and male developing
gonads but male developing gonads lose its expression whilefoxl3 expression persists in female germ cells. Interestingly,
some populations of germline stem cells express this geneand, as oogenesis proceeds, expression in germ cells diminishes by the time they enter the pachytene stage. The expression pattern matched the theoretically expected expression
pattern of the sex determination gene in germ cells (figure 1).

III. foxl3 represses initiation of spermatogenesis in germ cells

To understand the function of foxl3/FOXL3, we generated
foxl3 mutant medaka using the TALEN-method.

Figure 2. Although the rate is relatively low compared with normal
sperm, sperm from mutant ovaries have the ability to fertilize eggs (leftgraph). Once the eggs are fertilized with sperm from the mutant ovary,
they develop to hatch as efficiently as normal embryos (middle graph).
Hatched larva grow into fertile medaka. The picture on the right shows
medaka larva hatched from eggs fertilized with sperm from a mutant
ovary.

Very interestingly, homozygous female medaka initiate
development of sperm in the early larval stage. Together withthe fact that foxl3 is expressed specifically in female germ
cells, this indicates that foxl3 represses initiation of spermatogenesis in germ cells in female gonads.

IV. Functional sperm develop in the ovary

We artificially inseminated sperm from female mutants into
normal eggs, and found that the fertilized eggs developed
into normal embryos. The embryos hatched and grew into
fertile adult medaka. This clearly demonstrates that sperm in
the female mutants is functional (figure 2).
Then, we analyzed the structure of the gonads in the female
mutants. The gonads display a typical ovarian structure withan ovarian cavity separated from the stromal compartment
by a germinal epithelium. In normal ovaries, the germline
stem cells with ovarian niches, Germinal Cradles, are locatedwithin the germinal epithelium and, as described, germline
stem cells express foxl3. In the female foxl3 mutants, sperm
is developed in the germinal epithelium. Probably because

28

there is no exit for the sperm to leave the germinal epithelium, the germinal epithelium with functional sperm expands
towards the stromal compartment. Therefore the mutant
ovary looks as if sperm fully fills an entire ovary (figure 3).

Figure 3. Schematic comparison of normal ovary with ovary producing
sperm in the mutant female. Sperm develop in the germinal cradles in thegerminal epithelium in the foxl3 female mutant

These results reveal several important points:
1. An intrinsic mechanism of sex determination is present ingerm cells. This mechanism can function against the direction of the sex determination gene and direct germ cells todevelop to a sex opposite from the somatic sex.
2. Repression of initiation of spermatogenesis is a main
component of the sexual switch of germ cells (the fatedecision to develop into eggs or sperm) and involves foxl3.
3. Spermatogenesis does not require the testicular environment. It can occur in the ovarian structure and environment.
This suggests that the testicular environment is required forthe coordinated regulation of spermatogenesis with otherparts of the body. This also suggests that germline stem cellswith foxl3 are ready for spermatogenesis.

Publication List:〔Original paper〕

T., Sato, T., Yamamoto, Y., Watakabe, I., Ohkawa, Y.,• Nishimura,
Suyama, M., Nakamura, S., Saito, T.L., Yoshimura, J., Morishita, S.,

Kobayashi, S., and Tanaka, M. (2015). foxl3 is a germ cell-intrinsic
factor involved in sperm-egg fate decision in medaka. Science 349,
328-331.

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

Associate Professor

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
NOMURA, Kengo
FUJIKAWA, Akihiro
SUZUKI, Ryoko
MATSUMOTO, Masahito
KUBOYAMA, Kazuya
LIN, Chia-Hao
MATSUDA, Takashi
YU, Yang
TANGA, Naomi
HIGASHI, Satoru
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
WADA, Kotoe
KONISHI, Mie
ISOSHIMA, Yoshiko
HASHIMOTO, Terumi
KODAMA, Akiko

NODA, Masaharu

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:

SOKENDAI Graduate Student:

Technical Assistant:

Secretary:

SHINTANI, Takafumi

We have been studying the molecular and cellular mechanisms underlying the development of the vertebrate central
nervous system (CNS), mainly using mice. This researchcovers many developmental events including the patterningof the retina, neuronal terminal differentiation, axonal navigation, branching and targeting, synapse formation, refinement and plasticity. The scope of our interests also encompasses the mechanisms for various functions of the mature
brain, including body-fluid regulation, behavior control,
learning, and memory.

I. Mechanisms for neural circuit formation

Adenomatous polyposis coli 2 (APC2) is preferentiallyexpressed in the nervous system from early developmental
stages through to adulthood. The knockdown of Apc2 inchick retinas reduced the stability of microtubules in retinal
axons, and yielded abnormal growth cone behaviors including a reduced response to ephrin-A2 and misprojection in thetectum without making clear target zones. In Apc2-deficient
mice, robust defects in neuronal lamination were observedin the cortex, hippocampus, cerebellum, and olfactory bulb.
These laminary abnormalities are a result of dysregulated
neuronal migration by a cell-autonomous mechanism. APC2
is distributed along actin fibers as well as microtubules inneurons. Our investigation suggests that APC2 is involved in
the signaling pathway from membrane receptors for extracellular guidance factors to the intracellular migration machinery.
Very recently, two siblings with Sotos syndrome-likefeatures caused by homozygous frame shift mutation in the
APC2 gene were found by whole exome sequencing. Sotos
syndrome (OMIM #117550) is characterized by varying

degrees of mental retardation and a combination of typicalfacial features (prominent forehead with a receding hairline,
downslanting palpebral fissures, and a pointed chin) andlarge head circumference its estimated incidence is 1/14,000
live births. Sotos syndrome has been known to be causedby haploinsufficiency in the NSD1 gene. However, thesepatients had no mutations in NSD1 or other known susceptibility genes.
We performed an etiological study on these patients. Cellbased functional assays indicated that the mutant APC2 ofthese patients was a functionally null protein. Apc2-deficient
mice exhibited significantly impaired learning and memory
abilities, together with an abnormal brain structure and head
shape, indicating a high degree of concordance between
the phenotypes observed in human patients and knockout
mice. We demonstrated for the first time that the expression of APC2 was under the control of NSD1. The impaired
migration of cortical neurons was observed when Nsd1 was
knocked down in the embryonic mouse brain, as in the Apc2deficient brain, and this defect was rescued by the forced
expression of Apc2 (Figure 1). Our study explains molecularmechanisms of the intellectual disability associated with
Sotos syndrome.

Figure 1. Effects of Nsd1 knockdown on the migration of corticalneurons during development. The control miRNA, Apc2-miRNA, or
Nsd1-miRNA construct was electroporated into the ventricular regionof the forebrain at E15.5. For the rescue experiment, Nsd1-miRNA wasco-electroporated with an Apc2-expressing plasmid. At P7 the location oftransfected cells (GFP+) was assessed in coronal sections of the cortex.
Nuclei were stained with DAPI. The layers of the cortex are indicated on
the right side. WM, white matter. Scale bars, 100 mm.

II. Physiological roles of receptor-like protein
tyrosine phosphatases

Protein tyrosine phosphorylation plays crucial roles invarious biological events such as cellular proliferation, differentiation, survival, migration, and metabolism. Cellulartyrosine phosphorylation levels are governed by the opposing
activities of protein tyrosine kinases (PTKs) and protein
tyrosine phosphatases (PTPs). The physiological functions
and regulatory mechanisms of receptor-like PTPs (RPTPs)
are not fully elucidated. We have been making efforts toreveal the functional roles of RPTPs, especially of the R3
and R5 subfamilies.

2-1 R3 RPTP subfamily

The R3 RPTP subfamily, which is comprised of PTPRB,PTPRH, PTPRJ, and PTPRO, reportedly plays pivotal rolesin the development of several tissues including the vascularand nervous systems. We performed a large scale examination of the enzyme-substrate interaction between theR3 RPTP members and representative RPTKs covering
29

National Institute for Basic Biology

Neurobiology

all RPTK subfamilies. We revealed that multiple RPTKs
are recognized as substrates by the R3 RPTPs. Among the
enzyme-substrate relationships identified, we examined theinteraction between the R3 RPTPs and insulin receptor (IR)in detail.
Co-expression of R3 RPTPs with the IR in HEK293T cells
suppressed the insulin-induced tyrosine phosphorylationof the IR. In vitro assays using synthetic phosphopeptides
revealed that R3 RPTPs preferentially dephosphorylatesparticular phosphorylation sites of the IR; Y960 in the juxtamembrane region and Y1146 in the activation loop. It has
been revealed that phosphorylation of Y1146 and Y960 are
required for the full activation and the signal transductionof the IR, respectively. Therefore, R3 RPTPs are thought toeffectively dephosphorylate these sites of IR, and thereby
suppress insulin signaling.
RT-PCR and in situ hybridization showed that only PTPRJ
was expressed together with IR in major insulin target
tissues, such as skeletal muscle, liver, and adipose tissue.
Expectedly, Ptprj-deficient mice exhibited enhanced activation of IR and Akt by insulin, and improved glucose
and insulin tolerance (Figure 2). These results indicate thatPTPRJ is a physiological enzyme attenuating insulin signaling in vivo, and selective inhibitors of PTPRJ could be
insulin-sensitizing agents.

Figure 2. Improved glucose and insulin tolerance in Ptprj-KO mice. In
glucose (A) and insulin (B) tolerance tests, glucose (2 g/kg body weight)
and insulin (1 unit/kg body weight) were administered by intraperitoneal injection to fasted mice, respectively, and blood glucose levels wereassayed. Values are shown as the mean ± SEM. The asterisks indicate
significant differences from WT mice by Student’s t-test (*p < 0.05, **p< 0.01).

2-2 R5 RPTP subfamilyProtein-tyrosine phosphatase receptor type Z (PTPRZ)
is predominantly expressed in glial and neuronal cells
during development in the central nervous system (CNS).
Oligodendrocyte precursor cells (OPCs) are the principal
source of myelinating oligodendrocytes. Deficiencies in
myelination in diseases such as multiple sclerosis (MS) leadto serious neurological disorders. Two animal disease models
have been widely accepted for studying the clinical andpathological features of MS lesions. Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated inflammatory CNS demyelination model, and the cuprizone modelof demyelination is induced by a T-cell-independent mechanism through feeding of the copper chelator cuprizone. We
previously showed that adult Ptprz-deficient mice were lesssusceptible to the induction of EAE than wild-type controls.
This year, we revealed that cuprizone-fed Ptprz-deficient
mice exhibited severe demyelination and axonal damage

30

in the corpus callosum, similar to wild-type mice, whereas
remyelination in Ptprz-deficient mice after cuprizoneinduced demyelination was significantly accelerated.
Accelerated remyelination was attributed to the higher differentiation potential of OPCs, because the number of oligodendrocyte-lineage cells recruited to the demyelinated area was
not altered. Importantly, pleiotrophin, one of the inhibitory
ligands for PTPRZ, was transiently expressed in the brain
upon demyelination, and gradually disappeared with remyelination. Pleiotrophin was detected in affected cortex neurons
and their axon fibers in the cuprizone model. Since pleiotrophin colocalized with a synaptic vesicle marker, the relevant
neurons are presumed to release pleiotrophin from theirdemyelinated axons (Figure 3). The treatment of a primaryculture of wild-type mouse brain cells with pleiotrophin itself
did not induce oligodendrocyte maturation, but enhanced
thyroid hormone-induced oligodendrocyte differentiation.
Of note, the differentiation of Ptprz-deficient cells was notfurther potentiated by pleiotrophin. Pleiotrophin released
from demyelinated fibers may stimulate the differentiation of
OPCs recruited in the demyelinated area in vivo.

Figure 3. Proposed mechanism for remyelination after demyelinating lesions. OPCs, but not mature oligodendrocytes (OLs), abundantly
express PTPRZ-A/B receptor proteins as chondroitin sulfate (CS) proteoglycans. The CS moiety of PTPRZ is important for achieving the highaffinity binding of PTN to the core protein. PTPRZ activity is requisite tomaintaining the immature state of OPCs. After cuprizone-induced oligodendrocyte death and demyelination, the expression of PTN is transiently
upregulated in damaged neurons. PTN may be released from demyelinated axons and bind to PTPRZ at the cell surface of OPCs that arerecruited to lesions, probably through PTPRZ-independent mechanisms.
The binding of PTN results in receptor dimerization or oligomerization,
thereby inhibiting its catalytic activity. PTPRZ inactivation releases theblock of differentiation in OPCs, so the remyelination of neighboringaxons is initiated.

We established immature oligodendrocytes (OL1 cells)
from p53-deficient mice. They were strongly positive forthe two receptor isoforms of PTPRZ-A and PTPRZ-B withchondroitin sulfate chains; however, their expression gradually decreased with differentiation, with only PTPRZ-B
being weakly detectable in mature oligodendrocytes. The
treatment of immature OL1 cells with pleiotrophin enhanced
the phosphorylation of p190 RhoGAP, which is a substratemolecule of PTPRZ. We found that pleiotrophin reduced
the expression of NG2 proteins in OL1 cells. Therefore, it isconceivable that the catalytic activity of PTPRZ functions tomaintain OPCs in an undifferentiated state, and the pleiotrophin-induced inactivation of PTPRZ releases this blockage(Figure 3).

III. Brain systems for body-fluid homeostasis

Sodium (Na) is a major electrolyte of extracellular fluids
and the main determinant of osmolality. Na homeostasis
is essential to life and Na+ concentrations in plasma andcerebrospinal fluid (CSF) are continuously monitored tomaintain a physiological level of Na+ in body fluids. We
have previously shown that Nax, which structurally resembles voltage-gated sodium channels (Nav1.1–1.9), is a Na+concentration ([Na+])-sensitive Na channel with a gating
threshold of ~150 mM for extracellular [Na+] ([Na+]o) in
vitro. Nax is preferentially expressed in glial cells of sensory
circumventricular organs including the subfornical organ,
and is involved in [Na+] sensing for the control of salt-intake
behavior.
Although Nax is also expressed in neurons of some brain
regions including the amygdala and cerebral cortex, thechannel properties of Na x in neurons have not yet beenadequately characterized. To investigate the properties ofNax expressed in neurons, we established an inducible cellline of Nax using the mouse neuroblastoma cell line, Neuro2a, which is endogenously devoid of the expression of Nax(N2a-Mf1 cell). Functional analyses of this cell line revealed
that the [Na+]-sensitivity of Nax in neuronal cells was similar
to that expressed in glial cells.
Furthermore, we found that Nax bound to postsynaptic
density protein 95 (PSD95) through its PSD95/Disc-large/
ZO-1 (PDZ)-binding motif at the C-terminus. Nax co-localized with PSD95 clusters along dendrites of lateral amygdala

neurons. PSD95 is known as an anchoring and scaffolding
protein for receptors and other postsynaptic proteins. Theinteraction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels by suppressing their endocytosis, because the depletion of endogenous
PSD95 resulted in a decrease in Nax at the plasma membrane(Figure 4). These results show for the first time that Nax
functions as a [Na+]-sensitive Na channel in neurons as wellas in glial cells.

Publication List:〔Original papers〕

M.*, Shintani, T.*, Fahiminiya, S.*, Fujikawa, A.,• Almuriekhi,
Kuboyama, K., Takeuchi, Y., Nawaz, Z., Nadaf, J., Kamel, H., Kitam,

•
•
•
•
•

A.K., Samiha, Z., Mahmoud, L., Ben-Omran, T., Majewski, J., andNoda, M. (2015). Loss-of-function mutation in APC2 causes Sotos
Syndrome features. Cell Repotrs 10, 1585-1598. *Co-first authors.
Fujikawa, A., Matsumoto, M., Kuboyama, K., Suzuki, R., and Noda,
M. (2015). Specific dephosphorylation at Tyr-554 of Git1 by Ptprz
promotes its association with Paxillin and Hic-5. PLoS One 10,
e0119361.
Kuboyama, K., Fujikawa, A., Suzuki, R., and Noda, M. (2015).
Inactivation of protein tyrosine phosphatase receptor type Z
by pleiotrophin promotes remyelination through activation ofdifferentiation of oligodendrocyte precursor cells. J. Neurosci. 35,
12162-12171.
Matsumoto, M., Hiyama, T.Y., Kuboyama, K., Suzuki, R., Fujikawa,A., and Noda, M. (2015). Channel properties of Nax expressed inneurons. PLoS One 10, e0126109.
Shintani, T., Higashi, S., Takeuchi, Y., Gaudio, E., Trapasso, F., Fusco,A., and Noda, M. (2015). The R3 receptor-like protein-tyrosine
phosphatase subfamily inhibits insulin signaling by dephosphorylating
the insulin receptor at specific sites. J. Biochem. 158, 235-243.
Sun, L.O., Brady, C.M., Cahill, H., Al-Khindi, T., Sakuta, H., Dhande,
O.S., Noda, M., Huberman, A.D., Nathans, J., and Kolodkin, A.L.
(2015). Functional assembly of accessory optic system circuitry criticalfor compensatory eye movements. Neuron 86, 971-984.

〔Review articles〕

Noda, M., and Hiyama, T.Y. (2015). The Na channel: What it is and• whatit does. Neuroscientist 21, 399-412.
M., and Hiyama, T.Y. (2015). Sodium sensing in the brain.
• Noda,Pflügers Arch. – Eur. J. Physiol. 467, 465-474.
x

Figure 4. Subcellular distribution of Nax in non-treated Neuro-2a (N2a)cells, or N2a-Mf1 cells transfected with control or PSD95 siRNA. Upper
panels: Immunostaining with anti-PSD95 and anti-Nax antibodies. In
order to inhibit endocytosis, cells expressing Nax were treated with 100
nM wortmannin or 200 μM dynasore for 6 h. Scale bars, 10 μm. Lowergraphs: Fluorescence intensity profiles along the white lines in the upper
panels. The profile was divided into three parts (P1 : C : P2 = 15 : 70 : 15
in length). a.u., arbitrary unit.

31

National Institute for Basic Biology

Neurobiology

DIVISION OF BRAIN BIOLOGY †Professor

Associate Professor

Assistant Professor:

KOMINE, Yuriko
SADAKANE, Osamu

YAMAMORI, Tetsuo

WATAKABE, Akiya

This year we have reported the following results: (1) Longterm two-photon calcium imaging, (2) in vivo dendritic
spines in marmoset neocortex, and (3) expression patterns ofimmediate early genes in the cerebellum.

I. Long-Term Two-Photon Calcium Imaging
of Neuronal Populations with Subcellular
Resolution in Adult Non-human Primates.

Two-photon imaging with genetically encoded calcium
indicators (GECIs) enables long-term observation ofneuronal activity in vivo. However, there are very few
studies of GECIs in primates. Here, in collaboration withProfessor Matsuzaki’s laboratory, we report a method forlong-term imaging of a GECI, GCaMP6f, expressed fromadeno-associated virus vectors in cortical neurons of theadult common marmoset (Callithrix jacchus), a small New
World primate. We used a tetracycline-inducible expression
system to robustly amplify neuronal GCaMP6f expression
and up- and downregulate it for more than 100 days. We
succeeded in monitoring spontaneous activity not only fromhundreds of neurons three-dimensionally distributed in layers
2 and 3 but also from single dendrites and axons in layer 1.
Furthermore, we detected selective activities from somata,dendrites, and axons in the somatosensory cortex responding
to specific tactile stimuli. Our results provide a way to investigate the organization and plasticity of cortical microcircuits
at subcellular resolution in non-human primates (Published
in Cell Rep. 13, 1989-1999, 2015).

II. In Vivo Two-Photon Imaging of Dendritic
Spines in Marmoset Neocortex
Two-photon microscopy in combination with a techniqueinvolving the artificial expression of fluorescent protein has
enabled the direct observation of dendritic spines in living
brains. However, the application of this method to primate
brains has been hindered by the lack of appropriate labelingtechniques for visualizing dendritic spines. Here, we developed an adeno-associated virus vector-based fluorescentprotein expression system for visualizing dendritic spines invivo in the marmoset neocortex. For the clear visualizationof each spine, the expression of reporter fluorescent proteinshould be both sparse and strong. To fulfill these requirements, we amplified fluorescent signals using the tetracycline transactivator (tTA)-tetracycline-responsive element
system and by titrating down the amount of Thy1S promoterdriven tTA for sparse expression. By this method, we wereable to visualize dendritic spines in the marmoset cortex by
two-photon microscopy in vivo and analyze the turnover of†: This laboratory was closed on 31 March, 2015.

32

Figure 1. Three-Dimensional Imaging of Neuronal Population Activity in
L2 and L3(A) Representative two-photon images of GCaMP6f at a depth of
150 μm (top), 275 μm (middle), and 400 μm (bottom) from the corticalsurface. These fields have the same horizontal location.
(B) ROI for activity analysis at each depth.
(C) ΔF/F traces of all active neurons over 400 s. Blue, green, and red
indicate the traces of neurons at a depth of 150 μm, 275 μm, and 400 μm,
respectively.
(D) Relation between pairwise correlation coefficient and horizontal
cellular distance for 1,120 pairs of active neurons located at the same
depth (from the same color traces shown in C). Red line indicates thelinear regression line. Pairs were grouped every 50 μm. The error bars
denote SEM.
(E) Relation between pairwise correlation coefficient and horizontal
cellular distance for 1,508 pairs of active neurons located at different
depths (from blue and green traces and from green and red traces; shownin C). (Cited from Sadakane et al., Published in Cell Rep. 13, 1989-1999,
2015)

spines in the prefrontal cortex. Our results demonstrated thatshort spines in the marmoset cortex tend to change more frequently than long spines. The comparison of in vivo samples
with fixed samples showed that we did not detect all existing
spines by our method. Although we found glial cell proliferation, the damage of tissues caused by window construction
was relatively small, judging from the comparison of spine
length between samples with or without window construction. Our new labeling technique for two-photon imaging tovisualize in vivo dendritic spines of the marmoset neocortex
can be applicable to examining circuit reorganization andsynaptic plasticity in primates (Published in eNeuro. 2(4)ENEURO.0019-15, 2015).

Publication List

〔Original papers〕

T., Sato, A., Kitsukawa, T., Sasaoka, T., and Yamamori, T.
• Nakamura,(2015). Expression pattern of immediate early genes in the cerebellum••
Figure 2. Dendritic spines imaged by in vivo two-photon microscopy. A,
Maximum intensity projection of the images acquired by in vivo twophoton imaging of marmoset cortex. B, Side view of three-dimensional
reconstruction of the images of the same site shown in A. The depthsof the areas shown in F and G are indicated by dashed lines. C, Image
plane near pial surface. D, Magnified image of the boxed area in C. E,
Magnified image of boxed area in D showing dendritic spines. F, Image
plane at a depth of 220 µm showing soma and basal dendrites. G, Image
plane at a depth of 330 µm. Scale bars: A, B, 100 µm; C, 50 μm; D, 5
μm; E, 2 μm; F, G, 50 µm (Published in eNeuro. 2(4) ENEURO.001915, 2015)of D1R KO, D2R KO, and wild type mice under vestibular-controlled
activity. Front. Cell Dev. Biol. 3, 38.
Sadakane, O., Masamizu, Y., Watakabe, A., Terada, S., Ohtsuka, M.,
Takaji, M., Mizukami, H., Ozawa, K., Kawasaki, H., Matsuzaki, M.,
and Yamamori, T. (2015). Long-term two-photon calcium imaging of
neuronal populations with subcellular resolution in adult non-humanprimates. Cell Rep. 13, 1989-1999.
Sadakane, O., Watakabe, A., Ohtsuka, M., Takaji, M., Sasaki, T.,
Kasai, M., Isa, T., Kato, G., Nabekura, J., Mizukami, H., Ozawa, K.,
Kawasaki, H., and Yamamori, T. (2015). In vivo two-photon imaging
of dendritic spines in marmoset neocortex. eNeuro doi: 10.1523/
ENEURO.0019-15.2015

III. E xpression pattern of immediate earlygenes in the cerebellum of D1R KO, D2R
KO, and wild type mice under vestibularcontrolled activity

We previously reported the different motor abilities of
D1R knockout (KO), D2R KO and wild-type (WT) mice.
To understand the interaction between the cerebellum andthe striatal direct and indirect pathways, we examined theexpression patterns of immediate early genes (IEG) in the
cerebellum of these three genotypes of mice. In the WT
naive mice, there was little IEG expression. However, weobserved a robust expression of c-fos mRNA in the vermisand hemisphere after running rota-rod tasks. In the vermis,
c-fos was expressed throughout the lobules except lobule 7,
and also in crus 1 of the ansiform lobule (Crus1), copula ofthe pyramis (Cop) and most significantly in the flocculus in
the hemisphere. jun-B was much less expressed but more
preferentially expressed in Purkinje cells. In addition, we
observed significant levels of c-fos and jun-B expressions
after handling mice, and after the stationary rota-rod task innaive mice. Surprisingly, we observed significant expression of c-fos and jun-B even 30 min after single weighing.
Nonetheless, certain additional c-fos and jun-B expressions
were observed in three genotypes of the mice that experienced several sessions of motor tasks 24 h after stationaryrota-rod task and on days 1 and 5 after rota-rod tasks, but
no significant differences in expressions after the runningrota-rod tasks were observed among the three genotypes. In
addition, there may be some differences 24 h after the stationary rota-rod task between the naive mice and the micethat experienced several sessions of motor tasks. (Publishedin Front Cell Dev Biol. 3:38, 2015).

33

National Institute for Basic Biology

Neurobiology

DIVISION OF BRAIN CIRCUITS †Professor

MATSUZAKI, Masanori

Assistant Professor:

HIRA, Riichiro
MASAMIZU, Yoshito
Technical Staff:
OHSAWA, Sonoko
NIBB Research Fellow:
EBINA, Teppei
Postdoctoral Fellow:
TANAKA, Yasuhiro
TANAKA, Yasuyo
KONDO, Masashi
SHINOTSUKA, Takanori
TAKEDA, Yuta
SOKENDAI Graduate Student: OHKUBO, Fuki
HASEGAWA, Ryota
SUZUKI, Ayami
Visiting Graduate Student: TERADA, Shin-Ichiro
Visiting Scientist:
HIRAKAWA, Reiko
Technical Assistant:
HIMENO, Miki
SAITO, Junko
ATSUMI, Urumi
OHARA, KaoriKOTANI, Keiko
TAKAHASHI, Yoichi
IWASE, Etsuko
IMOTO, Eiko
SUGIURA, Haruka
SUGIYAMA, Tomomi
Secretary:
Various firing patterns of many cortical neurons represent
information processing in the brain. The microarchitectureof synaptic connections control information processing in
cortical circuits. The structure and location of synapses determine and modify the strength of this information processing.
The aim of our laboratory is to reveal how information isformed, maintained, selected, and decoded in the brain at thelevels of single cells and of single synapses. To do so, wemainly use two-photon microscopy that allows us to see fluorescence signals from deep within living tissue, developing
novel photostimulation methods and animal behavioral tasks.
The goals of our recent studies are to reveal how voluntary
movement is memorized and represented in cortical circuits.
In addition, we are working to apply two-photon microscopy
to non-human primates in order to understand information
processing in the brain, which is relevant to high cognitive
functions.

I. Cortical modules for discrete & rhythmic
movements (Hira et al., J Neurosci., 2015).

Animal behavior has discrete and rhythmic components,
such as reaching and locomotion. It is unclear how these
movements with distinct dynamics are represented in the
cerebral cortex.
We examined the dynamics of complex movements offreely movable forelimbs in awake ChR2 transgenic micewith the skull coated by transparent resin (Hira et al., 2009).
The left cortical surface was illuminated by a blue laser(500-ms train of 2- or 4-ms duration pulses delivered at 50
Hz) focused through the objective lens (Fig. 1). We call thismethod pTOS. The pTOS-induced trajectory of the rightforepaw was tracked in three-dimensional space by two high†: This laboratory was closed on 31 March, 2016.

34

Figure 1. pTOS of an
awake, head-restrained,
ChR2 mouse. Right,
the dorsal view of theleft cerebral cortex. The
blue square indicates the
mapped area.

speed cameras at 100 or 200 frames/s.
pTOS induced one of two major types of right forelimb
movement. During pTOS of the anterior lateral part of the
motor cortex (ALM; Fig. 1), the right forepaw moved to anarrow space in every trial. By contrast, during pTOS of thecaudal forelimb motor area (CFA; Fig. 1), the right forepaw
moved circularly for more than one cycle and its end point
varied. We categorize the former right-forelimb movement as
a discrete movement, and the latter as a rhythmic movement.
To map the representation of the discrete and rhythmic
forelimb movements, we divided a 6 × 3 mm area of the left
dorsal cerebral cortex into 16 × 8 sites (inter-site distance of
400 μm) and performed pTOS at each site (Fig. 1). For the
first analysis, the direction and distance from the mean initialpoint to the mean end point of the forepaw were calculated
for each pTOS site. The direction and distance had clear
topography in the cortex (arrows in Fig. 2). Long arrows on
the map indicate the sites that induced forelimb movementswith a limited target space and long distance. The ALM andthe hindlimb motor area (HA), which is caudal to the CFA,
had long arrows with almost opposite directions.
For each pTOS site, the tangential velocity profile of theaveraged movement was fitted with a Gaussian function andthe coefficient of determination was defined as the ‘discreteness index’. The domains with a large discreteness index(magenta in Fig. 2) corresponded to the ALM and the HA,
which is consistent with the finding that these areas had longarrows on the map showing the direction and distance of thepTOS-induced movement (green in Fig. 2). We found thatrhythmic movement with forward rotation was represented
by the domain spanning from the medial part of the RFA
to the CFA. The domain to induce the rhythmic movement
was sandwiched by two domains to induce the discrete
movements in forward and backward directions (forward
discrete movement and backward discrete movement) (Fig.
2). When the photostimulation frequency was optimal, thetime to reach maximum tangential velocity in the discrete
movement was 136 ± 43 ms (n=4 mice) and the frequency
with the maximum power of the rhythmic movement was 8.7± 0.28 Hz (n=3 mice). Thus, low-frequency photostimulationof the ALM generated a discrete movement with an intrinsic
bell-shaped velocity profile, whereas high-frequency photo-

Figure 2. Maps of the discreteness index (magenta)
and the rhythmicity index (green) overlain on themap of the direction and distance of the forelimb
movement (arrows). The solid line indicates the FDR
border.

stimulation of the CFA generated a rhythmic movement witha relatively stable, intrinsic resonance property.
To determine whether the cortical domains to induce thediscrete and rhythmic movements were hierarchical orparallel, we applied a mixture of AMPA/kainate receptor
antagonist and NMDA receptor antagonist to the cerebral
cortex. The size of the movement induced by pTOS of the
ALM or CFA was not significantly reduced after the application of the blockers to the photostimulated areas. In addition,
unsupervised clustering of the sites with cortico-corticalsynaptic interactions obtained by the Allen Mouse Brain
Connectivity Atlas (Oh et al., 2014) showed that the borderFigure 3. Circles and gridswith the same color share cortico-cortical synaptic output
and input patterns, respectively.

separating the two different clusters in the frontal cortex
roughly corresponded to the FDR border (Fig. 3). Thus, thedomains to induce the forward discrete movement and therhythmic movement were functionally and anatomicallydistinct. We call such domains ‘modules’.
Photostimulation of either of the CFA or ALM suppressed
spontaneous activity at the other site. The distance of thediscrete movement induced by the simultaneous pTOS of the
CFA and ALM was significantly shorter than that induced
by pTOS of the ALM alone. Likewise, the power of therhythmic movement was significantly smaller than thatinduced by pTOS of the CFA alone. Thus, the two distinct
modules possess the ability to mutually weaken the neuronal
activity and the movement induction elicited by the othermodule.

II. C ortical representation of ethologically
relevant movements and lever-push/pull
movements (Hira et al., J Neurosci., 2015).

Discrete and rhythmic movements might be a part of coordinated movements that involve multiple parts of the body.
We extracted three types of ethologically relevant movements by detecting movements of other parts of the body aswell as the right forelimb. These were termed ‘forepaw-tomouth’, ‘defensive-like’, and ‘locomotion-like’ movements.
Forepaw-to-mouth and locomotion-like movements weremainly elicited by pTOS of the ALM and the CFA, respectively. Defensive-like movement was induced by pTOS of
the anterior and middle parts of the rhythmic module. The
border between forepaw-to-mouth and defensive-like movements was almost identical to the FDR border. These results
are consistent with the observation that the forepaw-to-mouth
movement had a constant goal position and that defensiveand locomotion-like movements included oscillatory components.
Although the medial part of the RFA and the CFA wereinvolved in the rhythmic module, neuronal activity in theseareas is strongly related to a voluntary lever-pull movementthat is not rhythmic (Hira et al., 2013; Masamizu et al.,2014). Therefore, these areas may not function only forrhythmic movement. To assess this possibility, we conducted
pTOS when a lever was located near the right forepaw infour mice.

Figure 4. Maps of lever-push (red) andlever-pull (cyan) movements induced
by pTOS. The solid line indicates the
FDR border.

This stimulation exclusively induced a lever-push
movement (red in Fig. 4). On the other hand, pTOS of the
CFA preferentially induced a lever-pull movement (cyanin Fig. 4), which was sometimes followed by the rhythmic
movement. The medial part of the RFA and the HA almost
exclusively induced a lever-pull movement. The border ofthe lever-push and lever-pull movements roughly corresponded to the FDR border (Fig. 4). These results indicatethat the distinct cortical modules adapted to distinct lever
movements, and that the module for the rhythmic movement
could induce a non-rhythmic movement.
Our results indicate that motor cortical neurons may not
exclusively encode unique purposes or specific complex
movements. Instead, a population of neurons in a motor
cortical area may evolve to encode various purposeful movements depending on the environmental constraints and intrinsic dynamics.

III. L ong-term two-photon calcium imagingof neuronal populations with subcellular
resolution in adult non-human primates(Sadakane et al., Cell Rep., 2015).

We developed a new method for long-term imaging of agenetically encoded calcium indicator (GECI), GCaMP6f,
expressed from adeno-associated virus (AAV) vectors incortical neurons of the adult common marmoset (Callithrix
jacchus), a small New World primate. We first cloned two
components of the TET-Off system, namely, the tetracycline-controlled transactivator (tTA) under the control of
the Thy1S promoter and GCaMP6f under the control of thetetracycline response element (TRE3) promoter, into separate
AAV vectors (Fig. 5).
Figure 5. Schematic illustration of the TET-Off
gene expression system.

In the absence of Dox, tTA constitutively activates expression of a transgene under the TRE3 promoter. Dox prevents
the binding of the tTA to the TRE3, and inhibits transgene
expression (Fig. 5).
For fluorescence imaging after AAV injection to themarmoset neocortex, marmosets were placed under the35

National Institute for Basic Biology

Neurobiology

microscope and lightly anesthetized by isoflurane inhalation. We detected strong epifluorescence signals around theinjection site through the imaging window after only 10 daysof AAV injection (Fig. 6). In many neurons, spontaneous
calcium transients were clearly detected (Fig. 6).

Consistent with the existence of stimulus-selective neurons,
we also observed stimulus-selective responses from dendrites
in L1 (Figure 8B) and axonal boutons in L1 (Figure 8C). The
responses of these dendrites and axons to each stimulus wereas robust as those of the somata.
Thus, our results demonstrate that the spatial and temporalprofile of each neuron, dendrite, and axon in the marmoset
cerebral cortex can be correlated with a variety of sensory
stimuli. Our new technique removes a major obstacle tostudying the non-human primate cortex using calcium
imaging. In future studies, the technique, combined withvarious cognitive tasks, should shed light on the organizationand plasticity of the primate cerebral cortex.

Figure 6. Left, representative two-photon image of GCaMP6f on post
injection day 10. Right, representative ΔF/F traces from five ROIs in thefield shown in left.

To test the effectiveness of Dox control in marmosets
injected with AAV-TET-Off vectors, we administered Dox indrinking water to the animals for several days, and examined
an image of the same area repeatedly over time. Twenty
eight days after the start of the 5-day Dox administration, wewere able to identify the same population of neurons that we
observed before Dox administration. Spontaneous calcium
transients were detected from the same neuron both before
and after Dox administration. We observed a set of neurons
with very similar configurations at an interval of more than
100 days after three trials of Dox administration, which aremost likely to represent identical neuronal populations (Fig.
7).
Figure 7. Representative images of
GCaMP6f fluorescent signals on
post injection days 10 and 118 fromthe same filed.

Due to a high signal-to-noise ratio of the fluorescencesignals, we were able to monitor the activity of neuronalsomata located up to 400 μm from the cortical surface. This
depth range is supposed to cover L1 and L2, as well as theupper part of layer 3 (L3). Using a piezoelectric objective
mount, we continuously recorded the activity of multiple
neurons with single-cell resolution from a relatively broad
area (625 × 625 μm) at three depths: 150 μm, 275 μm, and400 μm (from L2 to L3). In this experiment, 445 putative
neuronal somata were determined and 81 of them were spontaneously active neurons.
Next, we tested whether neuronal responses evoked bytactile stimulation could be detected in the somatosensory
cortex. For tactile stimulation, we attached vibrators to thearm and leg of marmoset B that were contralateral to thehemisphere with the imaging window, and stimulated each
body part alternately for 1 s with an interval of 7.5 s. We
found some neurons that responded to the tactile stimulation. Figure 8A shows two examples of neuronal soma in L3
whose fluorescence changes were clearly time-locked to thestimuli.

36

Figure 8. Selective sensory responses to tactile stimulation fromneuronal somata (A), dendrites (B), and axonal boutons (C). Middle(2), the green and red lines indicate the timing of tactile stimulation
to the left arm and the left leg, respectively. Right (3), the responses
to nine stimuli to each body part were averaged. The gray band indicates the timing of the left arm and the left leg stimulation.

Publication List:〔Original papers〕
R., Terada, S., Kondo, M., and Matsuzaki, M. (2015). Distinct• Hira,functional modules for discrete and rhythmic forelimb movements in•the mouse motor cortex. J. Neurosci. 35, 13311-13322.
Sadakane, O., Masamizu, Y., Watakabe, A., Terada, S., Ohtsuka, M.,
Takaji, M., Mizukami, H., Ozawa, K., Kawasaki, H., Matsuzaki, M.,
and Yamamori, T. (2015). Long-term two-photon calcium imaging of
neuronal populations with subcellular resolution in adult non-humanprimates. Cell Rep. 13, 1989-1999.

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellow:
Postdoctoral Fellow:
Visiting Scientist:YASUGI, Masaki
NAKAYASU, Tomohiro*
NAKAYASU, Tomohiro

In order to interact successfully with the environment,
animals must deduce their surroundings based on sensory
information. The visual system plays a particularly criticalrole in such interactions with the environment.
“Why can we see?” This question is fundamental for athorough understanding of vision-dependent animals, including human beings. In order to better understand the visualsystem of animals, we are researching animal behaviors
through psychophysical and computational methods.

allow us to manipulate morphological and motion cues systematically. Therefore, we have constructed 3D computer
graphic animations of medaka based on tracking coordinate
data and photo data obtained from real medaka. These virtual
3D models will allow us to represent medaka faithfully andto undertake a more detailed analysis of the properties of thevisual stimuli that are critical for the induction of various
behaviors.
Simultaneously, we began studying “behavioral lateralization” as a characteristic which can affect the formationof shoals. Like humans, fishes have lateral differences intheir movement and perception (Bisazza & Brown, 2011).
However, there have been few studies to examine the influence of behavioral lateralization on their interaction. If eachindividual has a laterally biased response to surrounding
companions or predators, how is the united movement asshoals realized? This study is expected to bring a new viewpoint for understanding the structure and behavior of schooling.

I. Psychophysical study of Medaka fish

One of our major subjects is the psychophysical and computational study of medaka (Oryzias latipes). Recently, we
made progress in studies of prey-predator interaction using
medaka and zooplankton. Visual motion cues are one of themost important factors for eliciting animal behavior, including predator-prey interactions in aquatic environments. To
understand the elements of motion that cause such selective
predation behavior, we used a virtual plankton system where
the predation behavior in response to computer-generated
prey was analyzed. Virtual prey models were programmed
on a computer and presented to medaka, which served as
predatory fish. Medaka exhibited predation behavior against
several characteristic virtual plankton movements, particularly against a swimming pattern that could be characterized
as pink noise motion. Analyzing prey-predator interactions
via pink noise motion will be an interesting research field in
the future (Matsunaga & Watanabe, 2012).
In recent years, we have made progress in studies of theschooling behaviors of medaka. Many fish species are known
to live in groups. Visual cues have been shown to play a
crucial role in the formation of shoals. Using biologicalmotion stimuli, depicting a moving creature by means ofjust a few isolated points, we examined whether physicalmotion information is involved in the induction of shoaling
behavior. We found that the presentation of virtual biological motion can prominently induce shoaling behavior. We
have shown what aspects of this motion are critical in the
induction of shoaling behavior. Motion and behavioral characteristics can be valuable in recognizing animal species,
sex, and group members. Studies using biological motion
stimuli will enhance our understanding of how non-humananimals extract and process information which is vital for
their survival (Nakayasu & Watanabe, 2014).
This year, we have developed a novel method for behavior
analysis using 3D computer graphics. The fine control ofvarious features of living fish have been difficult to achieve
in studies of fish behavior. However, computer graphics

Figure 1. Virtual Medaka fish (Male Models) constructed of 3D polygonal models and photo textures.

II. Psychophysical study of Human vision

Another of our major subjects is the psychophysical andtheoretical studies of the visual illusions experienced byhuman beings (Homo sapiens). One recent focus of thisdebate is the flash-lag effect, in which a moving object isperceived to lead a flashed object when both objects arealigned in actual physical space. We developed a simple conceptual model explaining the flash-lag effect (Delta model,
Watanabe et al., 2010: https://www.youtube.com/eijwat).
In recent years, we have made more developed novel visual
illusions, such as the shelf-shadow illusion (http://eijwat.
blogspot.jp/).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2015. The former title isindicated by an asterisk (*).

37

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

HASEBE, Mitsuyasu

Associate Professor

MURATA, Takashi

Assistant Professor:

TAMADA, Yosuke
ISHIKAWA, Masaki
KABEYA, Yukiko
Technical Staff:
NIBB Research Fellow:
IMAI, Akihiro
Postdoctoral Fellow:
MANO, Hiroaki
SHIBATA, Tomoko
TAMAOKI, Daisuke
TORIBA, Taiyo
SOKENDAI Graduate Student: FUKUSHIMA, Kenji
KAMIDA, Chiharu
KOSHIMIZU, Shizuka
LI, Chen
MORISHITA, Mio
PALFALVI, Gergo
SUDA, Hiraku
SUGAYA, Tomomi
ZHANG, Liechi
HORIUCHI, Yuta
Visiting Graduate Student: GU, Nan
Visiting Scientist:BISOVA, KaterinaČUPEROVÁ, Zuzana
KAWASHIMA, Takeshi
LI, Liu
LI, Ping
TUROCZY, Zoltan
AOKI, Etsuko
Technical Assistant:
GOTO, Misako
HIRAMATSU, Mika
KAJIKAWA, Ikumi
MORI, Asuka
MATSUZAKI, Yoko
NISHI, TayoOOI, Shoko
KOJIMA, Yoko
Secretary:

I. Evolution of Complex Adaptive Characters

The theory of natural selection and the neutral theory ofmolecular evolution are powerful concepts in evolutionary
biology. However, even with such theories, there still remain
unexplained phenomena, one of which is the evolution ofcomplexity. It is difficult to explain the mechanisms needed
to evolve complex adaptive traits at cellular and organismallevels, such as cell division machinery, regeneration, novel
organ development, host race change, and mimicry. Suchtraits comprise many components and become adaptive only
when all components are gathered together. However, basedon evolutionary theory, each component should evolve one
by one according to the accumulation of mutations. We aim
to reveal the genetic networks regulating these complex traits
and to infer the mechanisms needed to evolve complex characters.

II. E v o l u t i o n o f R e g e n e r a t i o n :Reprogramming of Differentiated Cells to
Pluripotent Stem Cells
Different species have different morphology and also

38

cellular characters vary between species. Stem cells selfrenew and repeatedly produce differentiated cells during
development. Conversely, differentiated cells can be converted into stem cells in some organisms. In plants, regeneration of a stem cell can lead to generation of a new individual,
which is an effective strategy for propagation. The ability inreprogramming is different from species to species but thereason is unknown. The moss Physcomitrella patens has arapid reprogramming ability and is feasible for use in experiments. Cells in a dissected leaf are reprogrammed to become
chloronema apical stem cells within 24 hours. We foundthat P. patens COLD SHOCK DOMAIN PROTEIN genes(PpCSPs), homologous to mammal induced pluripotent stem
(iPS) cell factor LIN28, are positive regulators of the stemcell formation in Physcomitrella. Characterization of PpCSPs
are ongoing by Chen Li and Yosuke Tamada.
We also found that the stem cell formation required
light signaling mediated by red and blue light receptors.
In addition, the stem cell formation was facilitated in thequadruple deletion mutant of the P. patens SQUAMOSA
promoter binding protein (PpSBP) genes, some of which
are known to be repressed by the blue-light receptor
CRYPTOCHROME. In addition, the histone chaperone
genes, Histone gene repressor A (HIRA) were found to
regulate chromatin modification of PpSBPs. Characterizationof these genes are in progress mainly by Yukiko Kabeya toinvestigate the cross talk between light and chromatin modification pathways in the process of the stem cell formation.

III. E volution of Regeneration: Master
Regulator for Reprogramming STEMIN

Animal somatic cells can be reprogrammed to iPS cells
by introducing four transcription factors, while such factors
have not been identified in plants. We have previously identified a gene encoding a member of a plant-specific transcription factor, STEM CELL-INDUCING FACTOR 1(STEMIN1) that was able to induce direct reprogrammingof differentiated leaf cells into chloronema apical stem cells
without wounding signals. STEMIN1 and its two paralogous
genes (STEMIN2 and STEMIN3) were activated in leaf cells
that underwent reprogramming. In addition, deletion of
the three STEMIN genes delayed reprogramming after leaf
excision, suggesting that these genes redundantly functionin the reprogramming of cut leaves. We also found thatthree STEMIN genes function redundantly in chloronema
apical stem cell formation from chloronema cells duringchloronema development. On the other hand, in contrast to
STEMIN1, induction of STEMIN2 or STEMIN3 in gametophores and chloronemata did not induce formation of chloronema apical stem cells. These results indicate that STEMIN1
has sufficient ability to change intact leaf cells to stem cells,
but its paralogous genes do not. Masaki Ishikawa and MioMorishita were this study’s main researchers.

IV. E volution of Regeneration: Lateral
inhibition by stem cells to surrounding cells
to be stem cells

Singly isolated leaf cells are reprogramed into stem cells
in P. patens. However, only one cell of two longitudinallyisolated adjacent cells becomes a stem cell and the otherappears to be laterally inhibited by the cell to be a future
stem cell. Liechi Zhang is investigating the factors involvedin the lateral inhibition.

V. Evolution of Regeneration: Other pathways

Nan Gu is interested in DNA damage and reprogramming, and is working with the mechanisms connecting DNA
damage and reprogramming of differentiated cells to stem
cells.
We found that INHIBITOR OF GROWTH (ING) proteins
are involved in the stem cell formation of cut leaves. The
ING proteins are known to regulate an apoptosis pathway inanimals but plants do not have the corresponding pathway.
Akihiro Imai is mainly investigating the molecular functionof ING.

VI. Cell Cycle Reentry from the Late S Phasein Physcomitrella

At mitosis, all eukaryotic cells divide chromosomes to two
daughter cells using a mitotic spindle, which is composedof microtubules. Differentiated cells are in a non-dividing,quiescent state, but some differentiated cells can reenter thecell cycle in response to appropriate stimuli. Quiescent cells
are generally arrested at the G0/G1 phase, reenter the cell
cycle, and progress to the S phase to replicate their genomic
DNA. On the other hand, some types of cells are arrested
at different phases and reenter the cell cycle from there. In
the moss Physcomitrella patens, the differentiated leaf cellsof gametophores formed in the haploid generation containapproximately 2C DNA content, and DNA synthesis is necessary for reentry into the cell cycle, which is suggested to bearrested at late S phase. Masaki Ishikawa reviewed various
cell-division reactivation processes in which cells reenter
the cell cycle from the late S phase, and discussed possible
mechanisms of such unusual cell cycle reentries with special
emphasis on Physcomitrella (Ishikawa and Hasebe 2015).

VII. E volution of Elaborated Cell Division
Machinery: Spindle body

At mitosis, all eukaryotic cells divide chromosomes totwo daughter cells using a bipolar mitotic spindle, whichis composed of microtubules. The centrosomes, which act
as microtubule organizing centers, induce formation of thetwo poles in metazoan cells. In contrast, the cells of land
plants and their sister group, zygnematales green algae,
form the bipolar spindle in the absence of centrosomes.
For understanding the mechanism of acentrosomal spindle
formation, the steps of microtubule reorganization during
spindle formation should be visualized. We collaborated
with Prof. Tomomi Nemoto in Hokkaido University anddeveloped a two-photon spinning disk confocal microscope,
which enables 3-dimensional imaging of living cells withhigh temporal and spatial resolution. We found that spindle
microtubules elongate from a prospindle, that is, a microtubule cage with two poles on the nuclear envelope. Our datasuggests that the prospindle organizes the bipolar spindle,
as centrosomes do in metazoan cells. In contrast to themetazoan centrosomes, however, the prospindle disappears

before metaphase. To understand the mechanism how the
bipolar spindle is maintained in the absence of the organizer, we established a minispindle system, which involves
a bipolar microtubule complex composed of an isolated
chromosome and microtubules in tobacco cells. Analysesof microtubule behavior in the minispindle are in progress.
Daisuke Tamaoki and Takashi Murata were this study’s main
researchers.

Figure 1. Prospindle of a tobacco BY-2 cell. Left: projection image,
Right: cross section image of the same cell. Bar, 5 μm.

VIII. M o l e c u l a r m e c h a n i s m s o f P l a n t
Movement using Mimosa pudica

The sensitive plant Mimosa pudica has long attracted theinterest of researchers due to its spectacular leaf movements
in response to touch or other external stimuli. Althoughvarious aspects of the seismonastic movement have been elucidated by physiological approaches, the lack of genetic tools
has hampered the investigation of molecular mechanisms
involved in these processes. To overcome this obstacle, wedeveloped an efficient genetic transformation method for M.
pudica (Mano et al., 2014) and then established a CRISPR/
Cas9-mediated gene knock-out system. By using these newtechniques, we succeeded in generating immotile M. pudica
mutants. Further characterization of these mutants will
provide insights into the mechanism of leaf movements aswell as their adaptive meanings. Another promising approachbased on the transgenic technique is molecular imaging using
fluorescent reporter proteins. We generated transgenic plants
expressing fluorescent proteins which can visualize actin
cytoskeleton and calcium ions, respectively. We are now
attempting live-imaging of these factors, which participate
in the seismonastic movement, but their roles still remain
unclear. This study was conducted mainly by Hiroaki Mano.

IX. Molecular mechanisms of mimicry

An excellent example of mimicry is the flower-mimicryof the orchid mantis Hymenopus coronatus with pink andwhite coloration and petal-like legs. Biochemical analyses
indicated that the reduced form of xanthommatin, a common
red pigment of the ommochrome family, almost solely contributes to the pink color. We also analyzed the ultrastructureof pigment granules by transmission electron microscopy.
We found that integumentary cells of H. coronatus containa numerous number of electron-dense granules with 60-nm
diameters. This result suggests that this special intracellular
structure may contribute to the unique pink color of H. coronatus. This work was mainly done by Hiroaki Mano.

39

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

X. Oriented cell division shapes carnivorous
pitcher leaves of Sarracenia purpurea

Complex morphology is one of the major evolutionary
outcomes of phenotypic diversification. In some carnivorous plants, the planar leaves of common ancestors havebeen modified to form a pitcher shape. However, how leaf
development was altered in the evolution of pitcher leaves
remains unknown. Here we show that the pitcher leaves ofSarracenia purpurea develop through cell division patternsof adaxial tissues that are distinct from those in bifacial andpeltate leaves, subsequent to regular expression of adaxial
and abaxial marker genes. Differences in the orientation ofcell divisions in the adaxial domain cause bifacial growth in
the distal region and adaxial ridge protrusion in the middle
region. These different growth patterns in a leaf establish thepitcher morphology. A computer simulation suggests that thecell division plane is critical for the acquisition of the novel
pitcher morphology. Our results imply that tissue-specificchanges in the orientation of cell division underlie the development of a morphologically complex leaf (Fukushima et al.
2015).

involved in movable tentacle development in the sandew
Drosera spatulata. Shizuka Koshimizu is interested in
the evolution of floral homeotic genes and investigates
the function of MADS-box genes in non-flowering plants
Physcomitrella patens. The pseudanthium is a flower-likeinflorescence, the molecular mechanisms of the development of which are unknown. Tomomi Sugaya succeeded intransferring the FT gene from Arabidopsis thaliana into thepseudanthium Houttuynia cordata. Furthermore, introductionof the FT gene successfully induced flowers.

Publication List:〔Original papers〕

K., Fujita, H., Yamaguchi, T., Kawaguchi, M., Tsukaya,• Fukushima,H., and Hasebe, M. (2015). Oriented cell division shapes carnivorous

•
•
•
•

Figure 2. Schematics of leaf development in flat leaf and pitcher leaf.
Growth directions are indicated by arrows. Lines show cell division
planes.

To further investigate the evolution of pitcher leaves, weselected the Australian pitcher plant Cephalotus follicularis,
which produces both carnivorous pitcher and non-carnivorous flat leaves, enabling us to deduce carnivory-related
genes by comparative approaches in a single species. To
understand the genomic changes associated with the evolution of carnivory, we sequenced 2-Gbp genome of C.
follicularis. Whole-genome shotgun data corresponding
to 100x coverage were produced by Illumina paired-end/mate-pair sequencing with 180-bp to 5-kb insert sizes, andde novo assembly yielded 15.6 kb of contig N50 and 78 kb ofscaffold N50. 14 Gbp of PacBio reads with 2-kb mean max
subread length were produced for gap filling. Transcriptbased gene prediction with RNA-seq reads found 45,469
gene models. This study was mainly conducted by Kenji
Fukushima.

XI. Evolution of plant development

To investigate evolution of novel complex traits, the following studies are ongoing: Chiharu Kamida studies genes

40

pitcher leaves of Sarracenia purpurea. Nat. Commun. 6, 6450.
Kinoshita, A., ten Hove, C.A., Tabata, R., Yamada, M., Shimizu, N.,
Ishida, T., Yamaguchi, K., Shigenobu, S., Takebayashi, Y., Iuchi, S.,et al. (2015). A plant U-box protein, PUB4, regulates asymmetric cell
division and cell proliferation in the root meristem. Development 142,
444-453.
Otomo, K., Hibi, T., Murata, T., Watanabe, H., Kawakami, R.,
Nakayama, H., Hasebe, M., and Nemoto, T. (2015). Multi-point
scanning two-photon excitation microscopy by utilizing a high-peakpower 1042-nm laser. Anal. Sci. 31, 307-313.
Shimizu, N., Ishida, T., Yamada, M., Shigenobu, S., Tabata, R.,
Kinoshita, A., Yamaguchi, K., Hasebe, M., Mitsumasu, K., and Sawa,
S. (2015). BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2
constitute a signaling pathway and modulate CLE peptide-triggered
growth inhibition in Arabidopsis root. New Phytol. 208, 1104-1113.
Teh, O.K., Hatsugai, N., Tamura, K., Fuji, K., Tabata, R., Yamaguchi,
K., Shingenobu, S., Yamada, M., Hasebe, M., Sawa, S., et al. (2015).
BEACH-domain proteins act together in a cascade to mediate vacuolar
protein trafficking and disease resistance in Arabidopsis. Mol. Plant 8,389-398.

〔Review article〕

M., and Hasebe, M. (2015). Cell cycle reentry from the late S• Ishikawa,phase: implications from stem cell formation in the moss Physcomitrella
patens. J. Plant Res. 128, 399-419.

DIVISION OF SYMBIOTIC SYSTEMS

Professor

KAWAGUCHI, Masayoshi

Assistant Professor:

TAKEDA, Naoya
SUZAKI, Takuya
Technical Staff:
FUKADA-TANAKA, Sachiko
Visiting Scientist
NAKAGAWA, Tomomi
Postdoctoral Fellow:
FUJITA, Hironori
KOBAYASHI, Yuki
HANDA, Yoshihiro
NAGAE, Miwa
MATSUOKA, Hikaru
MAEDA, TaroKAMEOKA, Hiromu
SHIMIZU-MITAO, Yasushi
SOKENDAI Graduate Student: YORO, EmikoTSUZUKI, Shusaku
NISHIDA, Hanna
FUKUHARA, Mai
LIU, Meng		Visiting Undergraduate: MIZUTANI, Yoshiyuki
Technical Assistant:
SUZAKI, Momoyo
ICHIKAWA, Michiko
OGAWA, Yuko
YOSHINORI, Yumi		Secretary:
OKUBO, Masayo
Rhizobium–legume symbiosis is one of the most successful
mutually beneficial interactions on earth. In this symbiosis,
soil bacteria called rhizobia supply the host legumes withammonia produced through bacterial nitrogen fixation. In
return, host plants provide the rhizobia with their photosynthetic products. To accomplish this biotic interaction,
leguminous plants develop nodules on their roots. On theother hand, more than 80% of land plant families have symbiotic relationships with arbuscular mycorrhizal (AM) fungi.
Despite marked differences between the fungal and bacterial
symbioses, common genes are required for both interactions.
Using a model legume Lotus japonicus, we are trying tounveil the molecular mechanisms of both symbiotic systems.

I. AM symbiosis

Arbuscular mycorrhiza is a mutualistic plant-fungus interaction that confers great advantages to growth and survival on
the land. During the symbiosis development, AM fungi enter
into the host root and elongate the hyphae between the root
cells. The intraradical hyphae finely branch in the corticalcells and form a symbiotic structure called an arbuscule. AM
fungi supply inorganic materials, especially phosphate, to thehost plant through arbuscules and in return, they obtain photosynthetic products form the host.
1-1 Strigolactone-induced putative secreted protein 1 in
AM fungus Rhizophagus irregularis is required for symbiosis
Strigolactones (SLs) are a kind of phytohormone that wereidentified as a signal factor for AM fungi. SLs enhancement
increases hyphal branching and elongation in the AM fungi.
This process facilitates direct contact between the AM fungi

and host plants. After hyphal entrance into the host root, AM
fungi form a characteristic densely-branched hypha ‘arbuscule’ in the cortical tissue of the root, where the nutrient
exchange is undertaken. To provide novel insights into themolecular mechanisms of AM symbiosis, we screened andinvestigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants.
To compare comprehensive gene expression profiles of R.
irregularis between the pre-symbiotic and symbiotic stages,
RNA-seq analysis was performed in non-symbiotic (control),SL-treated, and symbiotic hyphae. The RNA-seq analysis
revealed that 19 genes are up-regulated by both treatment
with SL and symbiosis. Interestingly, 11 of the 19 genes
were predicted to encode small proteins with secretory signal
peptides at their N-terminal ends. Among the 11 putative
secreted protein genes, SL-induced putative secreted protein
1 (SIS1) showed the largest induction under both conditions.
To analyze the functions of SIS1 during AM symbiosis, SIS1
was then characterized by a reverse genetic approach using
host-induced gene silencing (HIGS), which leads to RNAiin the fungus via the host plant. We designed a SIS1-HIGS
construct and transformed Medicago truncatula hairy roots
with this construct. In the hairy root lines, SIS1 expression isknocked down by HIGS, resulting in significant suppressionof colonization compared with an empty vector (EV) control
root line. Futhermore, the arbuscules in EV control hairyroots were mature and finely branched (Figure 1A), whereas
the majority of arbuscules in the SIS1-HIGS hairy roots displayed defective morphology (Figure 1B). The stunted arbuscules are an indication of incomplete AM symbiosis. These
results suggest that SIS1 contributes to the establishment of
AM symbiosis in R. irregularis.
We concluded that SIS1 is a novel putative secreted proteinthat is induced by both SL treatment and AM symbiosis in R.
irregularis, and that it positively regulates AM colonization.

Figure 1. Arbuscules labeled with wheat germ agglutinin (WGA)-Alexa
Fluor 594 dye at 8 weeks after inoculation. (A) Empty vector control.
(B) SIS1-HIGS hairy roots. Bars represent 50 μm.

1-2 Gibberellin signaling interferes with AM signaling
and regulates AM-induced gene expression

We isolated the AM mutant nsp1, which has a defect in its
infection processes, through screening of root nodule symbiosis mutants of L. japonicus. The nsp1 mutant is known toshow a decrease of strigolactone biosynthesis, in addition,
our transcriptome analysis of the nsp1 mutant revealed that
expression of gibberellin biosynthesis genes are disturbed.
We have already reported that gibberellin signaling interacts with the symbiotic signaling pathway and changes theexpression of AM-induced genes. Furthermore, we havefound that some AM-induced genes were induced in the nsp1
mutant without AM fungal infection. Hormonome analysis
showed that the mutant contains a decreased amount of gib-41

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

berellins, suggesting that the AM-induced genes are induced
in low gibberellin conditions. We treated L. japonicus withgibberellin biosynthesis inhibitors, or introduced gibberellin signaling suppressor protein delta-GAI, and confirmed
significant induction of the AM-induced genes in the treatedor transformed plants. These results demonstrated that theAM-induced genes are also under the control of gibberellin signaling and the promoter of these genes is the junction
point between symbiotic and gibberellin signaling.
We expected that alternation of the gibberellin signaling conditions may suppress the nsp1 mutant phenotypes.
Actually, treatment with gibberellin biosynthesis inhibitor
or introduction of delta-GAI to the nsp1 mutant suppresses
the low infection phenotype of the nsp1 mutant. This result
indicated that abnormal gibberellin conditions caused nsp1
phenotypes and NSP1 functions in gibberellin homeostasis
and regulation for AM fungal infection in the host plant.
Gibberellin conditions in host plants are altered by various
environmental stimuli. The close interaction between bothsignaling pathways suggests that a part of regulation of AM
development in the field might be controlled through alternation of gibberellin conditions. We continue to study theregulation mechanisms by gibberellin and symbiotic signaling and, moreover, will investigate the relation between AM
symbiosis, gibberellin signaling, and various environmental
responses to understand actual regulation mechanisms of AM
in natural environments.

where they directly bind to HAR1. Application of arabinosylated CLE-RS peptides to shoots suppresses nodulation in an
HAR1-dependent manner. Furthermore, the TOO MUCH
LOVE (TML) F-box protein recently has been identified as aroot-acting AON factor that inhibits nodulation downstream
of HAR1. Although these results provide some insight into
signaling mechanisms between root and shoot, the mechanism and regulation of AON-inhibition of nodule development remains unclear. In particular, the shoot-derived inhibitor, SDI, has remained unidentified for a long time.
We focused on downstream events of the CLE-RS1/2HAR1 signaling pathway and attempted to identify SDI. We
show that the production of cytokinins in shoots is activatedby the CLE-RS1/2-HAR1 pathway, and that application ofexogenous cytokinins to shoots can inhibit nodulation ina TML-dependent manner. Our results suggest that shootderived CKs systemically regulate root nodulation in AON.

II. Long-distance control of nodulation

To establish symbiotic associations with rhizobia, a groupof nitrogen-fixing bacteria, leguminous plants form nodules
on their roots in response to rhizobial infection. The rhizobia
colonize these nodules, supplying host plants with fixed
atmospheric nitrogen while receiving photosynthates in turn.
While such symbiotic relationship generally is beneficial toboth partners, the formation of excessive numbers of nodules
inhibits the growth of the host plants. To avoid this effect,
plants perform autoregulation of nodulation (AON), which
systemically controls the number of nodules. AON is a longdistance negative-feedback system involving root-shoot communication. In L. japonicus, two leucine-rich repeat receptorlike kinases, HYPERNODULATION ABERRANT ROOTFORMATION 1 (HAR1) and KLAVIER, have been identified as key components of AON that function in shoots. The
two proteins are orthologous to Arabidopsis CLAVATA1 and
RPK2, respectively, which are involved in the maintenanceof stem cell populations in shoot apical meristems via shortrange cell-to-cell communication. As an underlying mechanism of AON, it has been postulated that signaling substances are produced in roots upon rhizobial infection which then
are transported to the shoot. The perception of these primary
signals in the shoot generates secondary signals. These
shoot-derived signals, also called shoot-derived inhibitors(SDI), are transported to the roots where they inhibit the initiation of new nodule development. In L. japonicus, the two
peptides, CLE-RS1 and CLE-RS2, are strong candidates forroot-derived mobile signaling molecules. Expression of thecorresponding genes is induced specifically in infected roots,
and CLE-RS2 glycopeptides are transported to the shoot42

Figure 2. Schematic illustration of the proposed AON model. AON is
mediated by two long-distance signal molecules, arabinosylated CLE
peptides and SDI. Cytokinins may act as SDI and play a central role in
AON.

III. E volutionary dynamics model of the Legume–
Rhizobia symbiosis

In symbiotic relationships, the participating organisms
provide mutual benefits to each other. One of the most
famous symbioses occurs between legumes and rhizobia,
in which rhizobia extract nutrients (or benefits) fromlegume plants while supplying them with nitrogen resources produced by nitrogen fixation. However, the nitrogen
fixation reaction consumes a lot of energy (or costs), andineffective rhizobia that colonize their host plants without
undertaking nitrogen fixation are ubiquitous in nature.
Here, we constructed a mathematical model to investigate
how benefit and cost influence the evolution of this symbiosis, and the conditions required for establishing the symbiotic relationship. (Figure 3A). According to our model, stablemutualism depends on the cost–benefit balance (Figure 3B).
That is, a tight symbiotic relationship emerges when the beneficial effect is much stronger than the cost (Figure 3F), but
is dissolved under the opposite condition of relatively strong
cost (Figure 3C). In the intermediate condition, where benefit
is approximately offset by cost, more complicated behaviors
emerge such as imperfect symbiotic interactions (Figure3D) and the coexistence of symbionts and cheaters (Figure3E). Our findings can explain why ineffective rhizobia arewidely distributed in nature. Our model provides a theoretical basis for understanding how the legume–rhizobia symbiosis evolves.

••characterization of LjABCG1, and ABC-binding cassette protein in
Lotus japonicus. PLoS ONE 10, e0139127.
Takeda, N., Handa, Y., Tsuzuki, S., Kojima, M., Sakakibara, H., andKawaguchi, M. (2015). Gibberellin regulates infection and colonizationof host roots by arbuscular mycorrhizal fungi. Plant Signal. Behav. 10,e1028706.
Takeda, N., Handa, Y., Tsuzuki, S., Kojima, M., Sakakibara, H., andKawaguchi, M. (2015). Gibberellins interfere with symbiosis signaling
and gene expression and alter colonization by arbuscular mycorrhizal
fungi in Lotus japonicus. Plant Physiol. 167, 545-557.

〔Review articles〕

T., and Imaizumi-Anraku, H. (2015). Rice arbuscular• Nakagawa,mycorrhiza as a tool to study the molecular mechanisms of fungal
symbiosis and a potential target to increase productivity. Rice 8, 32.

T., Nakagawa, T., Kaku, H., and Shibuya, N. (2015). Chitin• Shinya,mediated plant-fungal interactions: catching, hiding and handshaking.

•Curr. Opin. Plant Biol. 26, 64-71.
Suzaki, T., Yoro, E., and Kawaguchi, M. (2015). Leguminous plants:
inventors of root nodules to accommodate symbiotic bacteria. Int. Rev.
Cell Mol. Biol. 316, 111-158.

Figure 3. Evolutionary model of the Legume-Rhizobia symbiosis. (A)Model framework. (B-F) The evolution of the Legume-Rhizobia symbiosis depends on the cost-benefit balance. As the benefit strengthens
relative to the cost, the evolutionary outcome shifts in the order of “Nosymbiosis” (C), “Weak symbiosis” (D), “Coexistence of nitrogen-fixing
and cheating rhizobia” (E), and “Strong symbiosis” (F).

Publication List:〔Original papers〕

K., Fujita, H., Yamaguchi, T., Kawaguchi, M., Tsukaya,• Fukushima,H., and Hasebe, M. (2015). Oriented cell division shapes carnivorous

•
•
•
•

pitcher leaves of Sarracenia purpurea. Nat. Commun. 6, 6450.
Handa, Y., Nishide, H., Takeda, N., Suzuki, Y., Kawaguchi, M., andSaito, K. (2015). RNA-seq transcriptional profiling of an arbuscular
mycorrhiza provides insights into regulated and coordinated gene
expression in Lotus japonicus and Rhizophagus irregularis. Plant Cell
Physiol. 56, 1490-1511.
Horst, R.J., Fujita, H., Lee, J.S., Rychel, A.L., Garrick, J.M., Kawaguchi,
M., Peterson, K.M., and Torii, K.U. (2015). Molecular framework ofa regulatory circuit initiating two-dimensional spatial patterning ofstomatal lineage. PLoS Genet. 11, e10 05374.
Okamoto, S., Suzuki, T., Kawaguchi, M., Higashiyama, T., andMatsubayashi, Y. (2015). A comprehensive strategy for identifying
long-distance mobile peptides in xylem sap. Plant J. 84, 611-620.
Sugiyama, A., Fukuda, S., Takanashi, K., Yoshioka, M., Yoshioka, H.,
Narusaka, Y., Narusaka, M., Kojima, M., Sakakibara, H., Shitan, N.,
Sato, S., Tabata, S., Kawaguchi, M., and Yazaki, K. (2015). Molecular43

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:
OHDE, Takahiro
Visiting Graduate Student: KONISHI, Yusuke
YUZAKI, Karen
MASE, Mutsuki
YATOMI, Johichiro
Visiting Scientist:O’BRIEN, Devin Mackenzie
Technical Assistant:
KAWAGUCHI, Haruka
Secretary:
SAITO, EikoThe Division of Evolutionary Developmental Biology wasstarted in June 2015. We focus on the evolutionary novelties
acquired by insects through evolution, in order to elucidate
the molecular and evolutionary mechanisms leading to thelarge variety of traits that they display. From this wealthof exciting traits, our lab currently focuses on promoting
research into (1) the origin and diversification of insectwings, (2) wing color patterns and mimicry of ladybird
beetles, and (3) acquisition and diversification of beetle
horns.

I. Origin and diversification of insect wings

Of the various flying animals on the earth, insects have
evolved a unique flight organ. Despite over two centuries’
debate, the evolutionary origin of the insect wing is still
an enigma. We try to approach this issue using evo-devo
methods. In Drosophila melanogaster, the wing master genevestigial (vg) and its interaction partner scalloped (sd) play
pivotal roles in the formation of wing field identity. For thisreason, these genes are ideal candidates for investigating
wing origin and evolution.
One way to identify the structure from which insect wings
first evolved is to explore the function of “wing” genes inancestral wingless (apterygote) species. We chose thefirebrat, Thermobia domestica, as a model (Figure 1). T.
domestica belongs to Thysanura, phylogenetically the closest
extant relative of winged (pterygote) insects, making it ideal
for elucidating wing origin. We cloned vg and sd orthologs
from T. domestica (Td-vg and Td-sd). To examine the functions of these genes, we developed RNA interference (RNAi)
based methods for T. domestica. We are currently testing forfunctional effects of altered transcription of each of thesewing genes in the ancestrally wingless firebrats. In addition,
we are performing comparative analyses of the function ofthese same genes in “primitively winged” (hemimetabolous)
insects, to obtain additional clues relevant to understanding
the origin and evolution of insect wings.

II. W ing color patterns and mimicry of
ladybird beetles

Insect wing color patterns demonstrate a tremendous rangeof diversity and have evolved to fulfill various ecologicallyimportant functions such as intraspecific sexual signaling,mimesis, mimicry, and warning against predators. The
molecular mechanisms responsible for generating such
patterns, however, remain unknown for most species. To
investigate the developmental mechanisms of color patternformation, we chose the multicolored Asian ladybird beetle,
Harmonia axyridis, which has conspicuous and variable
wing color patterns consisting of black and red pigments(Figure 2A). Vivid wing color patterns of ladybirds function
as a warning to predators that they are distasteful, andladybird beetles are mimicked by various insect species.
Mimicry provides an exciting opportunity to study how independent lineages of insect converge on similar color patterns.
For exploring color pattern formation in a mimic, we use theleaf beetle, Argopistes coccinelliformis, which has color
patterns similar to Harmonia, and which is thought to be a
Batesian mimic of ladybird beetles (Figure 2B). To elucidate
the molecular mechanisms underlying these wing color
patterns, we have established a technique for germline transformation using a piggyBac vector and RNAi in the lady
birds. Recently, we also designed a TALEN-based method

Figure 2. The ladybird beetle, Harmonia axyridis (A) and the leaf beetle,
Argopistes coccinelliformis (B).

for genome editing in H. axyridis. Based on the knowledge
obtained from H. axyridis, we are trying to understand how
the similar wing-color patterns of model and mimic are generated – for example, do they use conserved or divergent
mechanisms?

III. Acquisition and diversification of beetle
horns

Figure 1. The firebrat, Thermobia domestica.

44

Insects show a tremendous range of diversity in “horns”,
rigid body outgrowths that function as weapons. Horns
are exciting for evo-devo studies because they have arisen
multiple times de novo, as evolutionary “novelties”.

However, the molecular mechanisms involved in sexuallydimorphic horn formation are still poorly understood. To
investigate the developmental mechanisms of horn formation, we focus on the Japanese rhinoceros beetle, Trypoxylus
dichotomus (Coleoptera), which exhibits remarkable sexual
dimorphisms in head and thoracic horns (Figure 3). The
male-specific horns of T. dichotomus are one of the bestmodels to study how an extreme, sex-specific morphology isformed. We have developed a technique for larval RNAi in
T. dichotomus, permitting us to rigorously and systematicallytest the functional roles of a large suite of candidate developmental genes, revealing for the first time the molecularmechanisms responsible for growth of male rhino beetle
horns.
To understand how sexual dimorphism in exaggerated horn
growth arises, we are currently employing two main
approaches: a candidate gene approach and a high throughput approach. To identify novel genes involved in thesexually dimorphic horn development in T. dichotomus, weare assessing mRNA of the developing horn discs using
deep-sequencing transcriptome analysis. Furthermore, tobegin to understand how, molecularly, beetle horns havediversified, we are extending our analyses to include additional beetle species with different types of exaggerated
horns, including rhinoceros beetles with diverse horn structures as well as horned beetles in other phylogenetic groups.

Figure 3. The Japanese rhinoceros beetle, Trypoxylus dichotomus. Adult
male (Left) and female (Right).

Publication List:〔Original paper (E-publication ahead of print)〕

M., Yatomi, J., Sumitani, M., Takasu, Y., Sekiné, K.,• Hatakeyama,Niimi, T., and Sezutsu, H. Knockout of a transgene by transcription
activator-like effector nucleases (TALENs) in the sawfly, Athalia
rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis(Coleoptera). Insect Mol. Biol. 2015 Oct 26.

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF MORPHODIVERSITY

Associate Professor

KODAMA, Ryuji

Visiting Scientist:

YOSHIDA, Akihiro

The aim of this laboratory is to observe the variety of morphogenetic processes that occur in the course of the ontogenesis of multicellular organisms and to analyze the mechanisms of such processes, mainly by morphological methods.
The accumulation of such analyses of the embryogenetic
processes of related species is expected to provide an insight
into the evolution of morphogenetic processes. This laboratory uses the wings of lepidopteran insects as the main targetof morphological studies.

I. Wing outline shape formed by cell death

The 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 for studying cellularinteractions in the course of morphogenesis.
The outline shape of the adult wing is often different fromthat of the pupal wing. This difference is brought about by
the programmed cell death of the marginal area of the pupal
wing. The marginal dying area is called “the degeneration
region” and the internal area which develops into the adultwing is called “the differentiation region”.
Cell death in the degeneration region proceeds very rapidly
and finishes in a half-day to one-day period in Pieris rapae
and in several other examined species. It was shown that thedying cells in the degeneration re­gion have characteristics incommon with the apop­totic cell death in mammalian cells,such as fragmented and condensed nuclei containing short
DNA fragments detected by TUNEL staining. The cells inthe degeneration region are actively en­gulfed by the macrophages in the cavity beneath the wing epithelium. The macrophages seem to be concentrated beneath the degeneration
region by the strong adhesion between the basal surfacesof the dorsal and ventral epithelium in the differentiation
region, which occurs at the time of prominent cell death andexcludes macrophages from the differentiation region. Thus
realized concentration of macrophages seems to accelerate
the shrinkage of the degeneration region.
A possible physiological role of the cell degeneration at thewing margin is to make space for the growth of the marginal
scales. Marginal scales are extremely elongated scales thatgrow densely on the edge of the wing. These scales are considered to be important in stabilizing the turbulence occurring posterior to the wing. The movements of the marginal
scales are closely monitored by sensory scales and bristles
growing among them (Yoshida and Emoto, Zool. Sci. 28,
430-437, 2011).
Trachea and tracheoles are both important in delivering
air into the wing and their patterns coincide with that of theboundary of degeneration and differentiation zones at the
distal end of the wing. According to previous observations,46

the pattern formation of wing epithelium is often dependent on
tracheal and tracheole patterns. Basic research on the development of tracheal pattern formation is being done by scanningand transmission electron microscopy to describe the exact
pathway and time course of the formation of the elaborate
pattern of trachea and tracheoles and to establish the cytological and developmental relationship between the formation of
the tracheal pattern and the epithelial cell pattern.

II. Wing morphogenesis and the growth of
marginal scales in small moths

In the small moths which have very long scales along theirwing margins, the cuticle of the pupal wings does not appear
to be large enough to house these scales. We examined thedevelopmental process of the pupal wings of three speciesof the small gelechiid moths and found that, concomitantwith the programmed cell deaths at the wing margin, there
occurs a shrinkage of the differentiation region which has
not been observed in large winged butterflies and moths. The
shrinkage concomitant with the cell deaths causes the space
between the pupal wing and the cuticle to extend, whichappears to contribute to the growth of long marginal scales.
Microscopic observation of the long marginal scales
revealed that they have a novel morphology and we are
studying the effect this morphology may have on themechanical interaction between marginal scales and further
on the air flow over the wing during flight.

Figure 1. Adult specimen of Metzneria lappella, one of the gelechiid
moths examined, with long scales along the wing margin.

III. Other research activities

This laboratory also conducts morphological observation ofvarious animal tissues by scanning and transmission electron
microscopy and immuno-electron microscopic analyses incollaboration with other laboratories of NIBB. Training inspecimen preparation and instrument operation for suchobservations is also given.

LABORATORY OF BIORESOURCES

Associate Professor

NARUSE, Kiyoshi

Assistant Professor:
Postdoctoral Fellow:
Research Fellow:
Technical Assistant:

Secretary:

TAKEHANA, Yusuke
SASADO, Takao
YOKOI, Saori
CHISADA, Shinichi
KANEKO, Hiroyo
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
KOIKE, Chieko
TESHIMA, Yuuko
HARA, Ikuyo
TAKAKI, Chikako
YOSHIMURA, Yuriko
SUZUKI, Tokiko

Medaka is a small egg-laying “secondary” fresh water
fish found in brooks and rice paddies in Eastern Asia. This
species has a long history as an experimental animal, especially in Japan. Our laboratory has conducted studies on
evolution of the sex determination system using medaka
and relatives, identification of the causal gene of mutantsfor PGC migration and pigment cell development, and the
gonadal development of medaka. In addition to these activities, our laboratory is stepping forward to lead the National
BioResource Project Medaka (NBRP Medaka).

I. Evolution of the sex chromosome and sex
determination genes in Oryzias fish

Recent studies have demonstrated that Oryzias species
have different genetic sex-determination systems (XX/XY
and ZZ/ZW) (Figure 1). Furthermore, the sex chromosomes
differ in their origin and degree of differentiation. Thesefindings suggest the repeated creation of new sex chromosomes from autosomes during evolution of Oryzias fishes,
possibly in association with the formation of new sex-determining genes. We are now trying to positionally clone thenovel sex-determining genes in these species. Identificationof these genes would provide a clue to understand the evolutionary process underlying frequent turnover of sex determination mechanisms.

Figure 1. Phylogenetic relationships and sex determination mechanisms
in Oryzias fishes.

II. G enetic dissection of migration ofprimordial germ cells in medaka

Germ cells are responsible for the sustainability of life
over generations in many multicellular animal species. To
elucidate the mechanisms underlying the development ofprimordial germ cells, we identified multiple mutations
affecting the migration and development of the primordial germ cells in medaka in a prior large-scale mutagenesis
screening project, and have analyzed a set of them to date.
We focused on three mutants that have defects in primordial
germ cell migration, kamigamo, shimogamo, and naruto thatwere isolated in the screening project. Positional cloning
and analysis of the genes carrying the mutations are now
in progress. In addition, two mutations, kamigamo and shimogamo, cause cystic pronephric ducts simultaneously withabnormal positioning of the primordial germ cells. Therefore,
the analysis of these mutations will be important in giving
basal knowledge underlying the mechanisms of human cystic
kidney diseases.

III. The study of type 2 diabetes using leptin
receptor knockout medaka

Leptin in mammals is a peptide hormone secreted byadipose tissue. It has been shown to play a key role in themaintenance of energy homeostasis through the regulationof food intake and a range of physiological functions. Micewith a deficiency of leptin or its receptor exhibit hyperphagia(an increase in food intake). The hyperphagia causes obesity
leading to type 2 diabetes-like symptoms, which is consistent with Caucasian patients. Leptin has also been isolated
from fish, including medaka, however, the amino acidsequence is poorly conserved between fish and mammals(11-30%), and fish leptins are expressed mainly in the livers.
To clarify the function of leptin on fish, we generated leptin
receptor knockout (LepRKO) medaka by the TILLING
method. The phenotypic analyses allowed us to reveal anappetite suppressive function of leptin signaling on medaka
as well as mammals, and to find new value in medaka asa novel animal model for studying type 2 diabetes. As forappetite suppressive functions; LepRKO medaka showed
high expression of the mRNA of NPY (3.5-fold) and AgRP
(6-fold), which are known to be orexigenic peptides, andan increase in food intake (1.7-fold). Next, as for glucose
metabolism; adult mutants showed signs of diabetes, suchas fasting hyperglycemia (Figure 2) and impaired insulin
secretion, which is a late-onset disorder caused by excessive feeding during post-juvenile stages. Furthermore, they
showed hyperglycemia even with the same fat level in the
blood, muscle, and liver as WT medaka (Figure 3). The
symptom is consistent with those of Asian patients, not but
Caucasian patients and mice with leptin signaling deficiencies. Now, we are investigating the gene expression associated with dysfunction of pancreatic tissues under various
feeding conditions. This will allow us to identify the factorsof diabetes that are sensitive to food intake, regardless ofobesity.

47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
NBRP Medaka aims to establish a first rate biologicalresource with the highest possible levels of accessibility andease of use.

Publication List:〔Original papers〕

H., Le, Q.D., Kinoshita, M., Takehana, Y., Sakuma, K.,• Hayakawa,Takeshima, H., Kojima S., Naruse, K., and Inoue, K. (2015). Genetic

Figure 2. Postbrandial blood glucose levels of WT and LepRKO medaka.

•
•
•
•

Figure 3. Relation between visceral fat and blood glucose levels of WT
and LepRKO medaka.

V. N ational BioResource Project Medaka(NBRP Medaka) (http://www.shigen.nig.
ac.jp/medaka/)

In 2007, NIBB was selected as the core facility of NBRP
Medaka. Our laboratory is taking an active part in thisproject. With the goal of facilitating and enhancing the useof medaka as a modelorganism, we provide,maintain and collectliving resources such as
standard strains, inbred
strains, and mutantsin addition to frozen
resources such as EST/cDNA, BAC/ Fosmid
clones, and hatching
enzymes, as well as
integrated information on medaka (Figure4). We have been providing BAC clones of
medaka related species,
a library screening
system employing a 3D
PCR strategy for evolutionary studies, and the
TILLING screening
system for promoting the
Figure 4. NBRP Medaka website.
reverse genetic approach.

48

similarity of the Hainan medaka populations collected from hyperand hypo-osmotic environments in northern Vietnam. Ocean Science
Journal 50, 231-235.
Kirchmaier, S., Naruse, K., Wittbrodt, J., and Loosli, F. (2015). The
genomic and genetic toolbox of the teleost medaka (Oryzias latipes).
Genetics 199, 905-918.
Myosho, T., Takehana, Y., Hamaguchi, S., and Sakaizumi, M. (2015).
Turnover of sex chromosomes in celebensis group medaka fishes. G3Genes Genomes Genet. 5, 2685-2691.
Uemura, N., Koike, M., Ansai, S., Kinoshita, M., Ishikawa-Fujiwara, T.,
Matsui, H., Naruse, K., Sakamoto, N., Uchiyama, Y., Todo, T., Takeda,
S., Yamakado, H., and Takahashi, R. (2015). Viable neuronopathic
Gaucher disease model in medaka (Oryzias latipes) displays axonal
accumulation of alpha-synuclein. PLoS Genet. 11, e1005065.
Yokoi, S., Okuyama, T., Kamei, Y., Naruse, K., Taniguchi, Y., Ansai,S., Kinoshita, M., Young, L.J. Takemori, N., Kubo, T., and Takeuchi,
H. (2015). An essential role of the arginine vasotocin system in mateguarding behaviors in triadic relationships of medaka fish (Oryzias
latipes). PLoS Genet. 11, e1005009.

〔Original paper (E-publication ahead of print)〕N., Honda, A., Zenno, A., Omoto, R., Imanaka, S., Takehana,• Kagawa,
Y., and Naruse, K. Arginine vasotocin neuronal development and its

projection in the adult brain of the medaka. Neurosc. Lett. 2015 Dec.
29.

LABORATORY OF BIOLOGICAL DIVERSITY
KAMADA Group
Assistant Professor:

KAMADA,Yoshiaki

Nutrients are indispensable for life. Among various nutrients amino acids are the major nitrogen source; therefore,
perception of the amino acid environment is essential forcells. The cellular amino acid sensing system employs Tor(target of rapamycin) protein kinase. Tor forms two distinct
protein complexes, TORC1 (Tor complex1) and TORC2.
TORC1 is involved in amino acid sensing, regulating protein
synthesis, the cell cycle, and autophagy. On the other hand,
TORC2 is responsible for actin organization and cell integrity. So far, it is not clear whether TORC2 also perceives
nutrient signals.
The aim of our research group is to reveal the molecularmechanisms of how TORC1 receives amino acid signals
and how the TORC1/2 pathways regulate each phenomenon. We have been studying Tor signaling in the budding
yeast Saccharomyces cerevisiae, and have found three novel
branches of the TOR signaling pathways (Figure 1).

I. How do amino acids regulate TORC1?

It is confirmed that TORC1 is the master regulator in aminoacid sensing. However, little is known how TORC1 perceives
amino acid signals.
Since both amino acid sensing and TORC1 have essential
cellular functions, we assume that factor(s) transmitting
amino acid signals to TORC1 should be encoded by essential
gene(s). We identified several essential genes as candidatesof TORC1-regulators.

II. T O R C 1 p h o s p h o r y l a t e s A t g 1 3 , t h emolecular switch of autophagy

TORC1 negatively regulates autophagy, a protein degradation system induced by nutrient starvation and rapamycin (aTORC1 inhibitor).
We found the TORC1-mediated regulatory mechanism of
autophagy. Under nutrient-rich conditions, TORC1 directly
phosphorylates Atg13, a component of the Atg1 kinase
complex. Atg1 is a Ser/Thr protein kinase, the activity ofwhich is essential for autophagy and is largely enhanced in
response to TORC1 inactivation. Activation of Atg1 requires
formation of the Atg1 complex which consist of Atg1, Atg13,
Atg17, Atg29, and Atg31. Phosphorylation of Atg13 by
TORC1 plays a pivotal role in Atg1 complex formation;
phosphorylated Atg13 loses its affinity to Atg1 resulting indisassembly of the Atg1 complex and repression of autophagy. On the other hand, dephosphorylation of Atg13 triggers
formation of the Atg1 complex, activation of Atg1 kinase,and consequently induction of autophagy.
We further determined eight phosphorylation sites of Atg13
and generated an unphosphorylatable Atg13 mutant (Atg138SA). Expression of Atg13-8SA induces autophagy bypassing inactivation of TORC1, suggesting that Atg13 acts as amolecular switch for autophagy induction.

Figure 1. Tor signaling pathway of the budding yeast. Our group has
found three branches of the Tor pathway.

III. TORC1 regulates mitotic entry via pololike kinase
TORC1 regulates protein synthesis, which is important for
promotion of the cell cycle at the G1 phase.
We demonstrated that TORC1 is also involved in anotherstage of the cell cycle, mitotic entry. We generated a temperature-sensitive allele of KOG1 (kog1-105), which encodes anessential component of TORC1, and found that TORC1 plays
an important role in mitotic entry (G2/M transition). Since
Cdc5, the yeast polo-kinase is mislocalized and inactivated in
kog1-105 mutant cells, TORC1 mediates G2/M transition via
regulating polo-kinase. In addition, we discovered a physiological role of TORC1 in mitosis; autophagy negatively
controlled by TORC1 plays an important part in maintenanceof genome stability under starvation conditions.

IV. Ypk kinase acts directly downstream of
TORC2 to control actin organization
TORC2 has an essential function controlling polarity of theactin cytoskeleton.
We identified Ypk2, a member of the AGC kinase family
which acts directly downstream of TORC2 using molecular
genetics. The activated allele of YPK2 can rescue a lethality
caused by TORC2 dysfunction, suggesting that Ypk kinaseis the major downstream protein of the TORC2 pathway. We
also demonstrated that Ypk2 is directly phosphorylated by
TORC2 through a biochemical assay.

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYMANO Group
Assistant Professor:
Postdoctoral Fellow:
Visiting Scientist:
Technical Assistant:

Secretary:

MANO, Shoji
KANAI, Masatake
WATANABE, Etsuko
KAMIGAKI, Akane
HIKINO, Kazumi
YAMAGUCHI, Chinami
NAKAI, Atsushi
KATO, Kyoko
UEDA, Chizuru

Plant cells can induce, degenerate and differentiate theirorganelles to adapt to environmental changes. This flexibilityof plant organelles is the basis of the strategy for environmental adaptation in plants.
The aims of our research group are to clarify the molecularmechanisms underlying the induction, differentiation, andinteraction of organelles, and to understand the integratedfunctions of individual plants through organelle dynamics.

I. M olecular mechanisms of peroxisome
dynamics and functions in plant cells

Peroxisomes are single-membrane bounded organelles,
which are ubiquitously present in eukaryotic cells, and they
are involved in various biological processes such as lipid
metabolism and photorespiration. To understand peroxisome
dynamics and functions, we have been analyzing a numberof Arabidopsis mutants having aberrant peroxisome morphology (apem mutants) and peroxisome unusual poisoning(peup mutants). Based on analyses using these mutants a partof the mechanism of division, protein transport, and degradation of peroxisomes were revealed. In addition, we revealed
that the physical interaction between peroxisomes and chloroplasts is dependent on photosynthesis (Figure 1).
Recently, we found that peroxisome functions are required
for the reproductive process. Therefore, peroxisomes ingametes and gametophytes were visualized, and theirdynamics are currently under investigation.

II. Accumulation mechanism of seed storage
oils and proteins

Plant seeds accumulate huge amounts of storage reserves
such as oils, carbohydrates and proteins. Humans use thesestorage reserves as foods and industrial materials. Storage
reserves are different among different plant seeds. Wheat,maize and rice seeds mainly accumulate starch, whereas
rapeseed, pumpkin and sesame contain large amounts of oils.
Soybean contains proteins as a major reserve. We are analyzing the mechanisms controlling oil and protein contents inseeds, and trying to apply our knowledge and techniques forincreasing beneficial storage reserves.

III. C onstruction of The Plant Organelles
Database 3 (PODB3)

PODB3 was built to promote a comprehensive understanding of organelle dynamics. PODB3 consists of six
individual units: the electron micrograph database, the perceptive organelles database, the organelles movie database,the organellome database, the functional analysis database,and external links. Through these databases, users can obtain
information on plant organelle responses to environmental
stimuli of various tissues of several plant species, at different
developmental stages. We expect that PODB3 will enhance
the understanding of plant organelles among researchers.

Publication List:〔Original papers〕

K., Le, Q.T.N., Hamada, T., Kutsuna, N., Mano, S.,• Motomura,Nishimura, M., and Watanabe, Y. (2015). Diffuse DCP2 accumulates in•

DCP1 foci under heat stress in Arabidopsis thaliana. Plant Cell Physiol.
56, 107-115.
Oikawa, K., Matsunaga, S., Mano, S., Kondo, M., Yamada, K., Hayashi,M., Kagawa, T., Kadota, A., Sakamoto, W., Higashi, S., Watanabe, M.,
Mitsui, T., Shigemasa, A., Iino, T., Hosokawa, Y., and Nishimura, M.
(2015). Physical interaction between peroxisomes and chloroplasts
elucidated by in situ laser analysis. Nature Plants 1, 15035.

〔Original papers (E-publication ahead of print)〕

M., Mano, S., Kondo, M., Hayashi, M., and Nishimura, M.
• Kanai,
Extension of oil biosynthesis during the mid-phase of seed development••

enhances oil content in Arabidopsis seeds. Plant Biotechnol. J. 2015
Oct 26.
Kimori, Y., Hikino, K., Nishimura, M., and Mano, S. Quantifying
morphological features of actin cytoskeletal filaments in plant cells
based on mathematical morphology. J. Theor. Biol. 2015 Nov 10.
Ueda, H., Yokota, E., Kuwata, K., Kutsuna, N., Mano, S., Shimada,T., Tamura, K., Stefano, G., Fukao, Y., Brandizzi, F., Shimmen,
T., Nishimura, M., and Hara-Nishimura, I. Phosphorylation of the
C-terminus of RHD3 has a critical role in homotypic ER membranefusion in Arabidopsis. Plant Physiol. 2015 Dec 18.

〔Review article〕

S., Mano, S., Yamada, K., Oikawa, K., Hosokawa, Y.,• Goto-Yamada,Hara-Nishimura, I., and Nishimura, M. (2015). Dynamics of the light-

Figure 1. Photosynthesis-dependent chloroplast-peroxisome interaction.
Chloroplasts and peroxisomes are visualized with autofluorescence (red)and GFP (green). Morphology of peroxisomes and interaction length
between chloroplasts and peroxisomes are different in dark and light
conditions. Even in light conditions, the photosynthesis inhibitor, DCMU,
causes the dark-dependent phenotype. Bar: 10 µm.

50dependent transition of plant peroxisomes. Plant Cell Physiol. 56, 12521263.

LABORATORY OF BIOLOGICAL DIVERSITYOHNO Group
Assistant Professor:

OHNO, Kaoru

The aim of this laboratory is to research reproductive
hormones in invertebrates and to analyze the mechanisms
by which they work. The comparisons of such moleculesand mechanisms in various species are expected to provide
insights into the evolution of reproductive hormone systems.

I. G onadotropins in the starfish, Asterina
pectinifera

Gonadotropins play important regulatory roles in reproduction in both vertebrates and invertebrates. The vertebrate
gonadotropins, LH and FSH are structurally and functionallyconserved across various species, whereas no such moleculehas been identified in invertebrates. The insect parsin
hormones are assumed to be the physiological counterpartof LH and FSH in mammals. Some gonadotropic hormones,
such as the egg development neurosecretory hormone of themosquito, the egg-laying hormone of the sea hare, and theandrogenic gland hormone of the terrestrial isopod, havebeen found in invertebrate species. More recently, an insulinlike peptide was reported to be responsible for the regulationof egg maturation in the mosquito, Aedes aegypti, demonstrating the involvement of insulin signaling in egg maturation among invertebrates.
The gonad-stimulating substance (GSS) of an echinoderm,the starfish, was the very first gonadotropin to be identified
in invertebrates. GSS mediates oocyte maturation in starfish
by acting on the ovary to produce the maturation-inducing
hormone (MIH), 1-methyladenine, which in turn induces thematuration of the oocytes. In this sense, GSS is functionallyidentical to vertebrate LH, especially piscine and amphibian LHs, acting on the ovarian follicle cells to produce MIH
to induce the final maturation or meiotic resumption of theoocyte. Considering the functional similarity that GSS shareswith vertebrate LH, it is very important from an evolutionary
point of view to know the chemical and molecular structure of GSS. We cloned the gene encoding GSS referred toamino acid sequence of purified GSS from radial nerves ofthe starfish, Asterina pectinifera. Interestingly, phylogenetic
analyses revealed that it belonged to the insulin/insulin-likegrowth factor (IGF)/relaxin superfamily and, more precisely,
to the subclass of relaxin/insulin-like peptides (Figure 1).

Figure 1. Amino acid sequence of starfish GSS. (A) Comparison of theheterodimeric structure of starfish GSS with those of various representative members of the insulin superfamily. The cysteine bridges are shownin red. (B) Coding DNA sequence and predicted amino acid sequences of
GSS. Sequences of A and B chains are shown in green and blue boxes,
respectively. Characters shown in red boldface indicate basic dipeptides
that are the sites of proteolytic cleavage. Inverted triangle shows the
deduced cleavage site of the signal peptide.

II. S earch for reproductive hormones ininvertebrates

In a collaborative effort with Prof. Yoshikuni’s Laboratoryof Kyushu Univ. and Dr. Yamano and Dr. Awaji of the
National Research Institute of Aquaculture, Fisheries
Research Agency (NRIA), we are searching for reproductive
hormones in invertebrates; sea urchin, sea cucumber, oyster,and shrimp. While the collaborators are partially purifying
physiological materials which induce egg maturation fromnerve extracts and analyzing them with a tandem mass spectrometer, we are creating and analyzing EST libraries fromnerve tissues and developing a database of the mass analysis
performed in this laboratory.

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

KOMINE Group
Associate Professor:

KOMINE, Yuriko

We have been interested in the developmental and evolutional aspects of the structure of mammalian brains. In acomprehensive analysis of homeobox genes expressed in
the developing mouse neocortex, we isolated a novel gene
Zfhx2, which encodes a transcription factor containing threehomeobox domains and 18 Zn-finger motifs. Zfhx2 is highly
expressed in the developing mouse brain, particularly in differentiating neurons, and continues to be expressed throughout adulthood at a low level. Two other phylogenicallyrelated genes, Zfhx3 and Zfhx4, have been identified. The
former was reported to be expressed in a manner dependenton neural differentiation, and the latter is a candidate gene
causing congenital bilateral isolated ptosis. Although thesethree genes are expressed in substantially similar patterns in
the developing brain, common functional features have notbeen clarified. Currently we have been focusing on Zfhx2 toreveal its function and mechanisms of expression control inthe developing brain.

I. Expression of Zfhx2 is negatively regulated
by its own antisense RNA

We found that the antisense strand of Zfhx2 is also
expressed in the mouse brain in a manner complementary
to the expression of Zfhx2 mRNA (Figure 1). Although mostneurons express Zfhx2 mRNA immediately after their final
mitosis, several types of neuron (e.g., granule cells in theolfactory bulb and pyramidal and granule cells in the hippocampus) express antisense RNA prior to Zfhx2 mRNA during
the early phase of their differentiation. By generating a genetargeting mouse line in which Zfhx2 sense RNA is expressed
but not antisense RNA, we showed that this antisense RNA
has a negative regulatory role in the expression of Zfhx2
mRNA. These observations suggest that the ZFHX2 protein
might have a role in a particular step of neuronal differentiation, and in some types of neuron, this step might be delayedby the expression of antisense RNA.

Figure 1. Expression of
Zfhx2 sense RNA (mRNA)and antisense RNA in theembryonic mouse brain.
The antisense RNA wasexpressed where mRNA
was not.

52

II. Z FHX2 might play roles in controlling
emotional aspects

To elucidate the function of ZFHX2, we have also generated a Zfhx2-deficient mouse line. Although the production of the ZFHX2 protein is completely abolished in thehomozygous mutant mice, the mice appear grossly normal
and healthy. No anatomical abnormality has been observedin the mutant mouse brains so far examined. We hence subjected the Zfhx2-deficient mice to a comprehensive batteryof behavioral tests to explore the physiological functionof ZFHX2 in the nervous system. The homozygous Zfhx2
deficient mice showed several behavioral abnormalities,
namely, hyperactivity (Figure 2), enhanced depression-like behaviors, and an aberrantly altered anxiety-like phenotype. These behavioral phenotypes
suggest that ZFHX2 might play roles in controlling
emotional aspects through the function of monoaminergic neurons where ZFHX2 is expressed.

1200

Distance Traveled (cm)LABORATORY OF BIOLOGICAL DIVERSITY

1000

p=0.0003

800
600
400
200

0

30 60 90 120
Time (min)Figure 2. Locomotor activity of the Zfhx2-deficient mice. Mice weretransferred into a novel environment and the distance traveled of eachanimal was measured for 2 hours. The Zfhx2-deficient mice (●, n=19)
were significantly more active than the wild-type mice (○, n=21).

LABORATORY OF BIOLOGICAL DIVERSITYHOSHINO Group
Assistant Professor:
Technical Assistant:

HOSHINO, Atsushi
NAKAMURA, Ryoko
TAKEUCHI, Tomoyo
ITO, Kazuyo

While genomic structures as well as their genetic information appear to stably transmit into daughter cells during cell
division, and also into the next generation, they can actually
vary genetically and/or epigenetically. Such variability has
had a large impact on gene expression and evolution. To
understand these genome dynamics in eukaryotes, especiallyin plants, we are characterizing the flower pigmentation ofmorning glories.

I. F lower pigmentation patterns of themorning glories

Ipomoea nil (Japanese morning glory), I. purpurea (thecommon morning glory), and I. tricolor have been domesticated well as floricultural plants, and their various spontaneous mutations have been isolated. The wild type morning
glories produce flowers with uniformly pigmented corolla,
whereas a number of mutants displaying particular pigmentation patterns have been collected. Because flower pigmentation patterns are easily observed, the molecular mechanisms
underlying these phenomena provide fine model systems forinvestigating genome variability.
Margined, Rayed and Blizzard of I. nil are dominant mutations. While these mutants show distinct flower pigmentation patterns, the same pigmentation gene is repressed by
non-coding small RNAs in the whitish parts of the corolla.
It is suggested that distinct regulation of these small RNAs
cause the difference in pigmentation patterns. The recessive
mutations, duskish of I. nil and pearly-v of I. tricolor, confer
variegated flowers, and epigenetic mechanisms are thought
to regulate their flower pigmentation. We are currently characterizing detailed molecular mechanisms of these mutations.

II. d e novo sequencing of the Japanesemorning glory genome

Although morning glories are studied worldwide, especially in plant physiology and genetics, no whole nuclear
genome sequences of any Ipomoea species are available. To
facilitate the studies of our group as well as all morning glory
researchers, we are conducting de novo genome sequencingof I. nil. We chose a standard line for genome sequencing,and employed shotgun sequencing using a single molecule
real time sequencing system. We recently obtained a draft
genome sequence consisting of 15 pseudo-chromosomeswith reasonable size, and are going to characterize moredetails of the genome sequence.

III. BioResource of morning glories

EST and BAC libraries of I. nil and its related species. I.
nil has been one of the most popular floricultural plants
since the late Edo era in Japan. It has an extensive historyof genetic studies and also has many advantages as a model
plant; simple genome, large number of mutant lines, and efficient self-pollination. Our collection includes 235 lines and
157,000 DNA clones.

IV. Pale- and dull-colored flower formation

Anthocyanin is responsible for the colors of many flowers
and is usually glucosylated by 3GT (UDP-glucose:flavonoid
3-O-glucosyltransferase). We first demonstrated that absenceof 3GT results in pale and dull flower coloration by using
the recessive 3GT mutants of I. nil and I. purpurea (Figure1). Anthocyanin analysis revealed that 3GT is essential for
maintaining proper production quantity, acylation, and glucosylation of anthocyanin. Incomplete acylation and glucosylation of anthocyanin results in dull flower coloration in
Ipomoea. One of the I. nil mutants produces flower variegations that are thought to be epigenetically regulated (Figure1c). We are currently studying a molecular mechanism of theflower variegation.

Figure 1. Wild type (a) and 3GT deficient mutant of Japanese morning
glory (b-d). Wild type (e) and 3GT deficient mutant of common morning
glory (f).

Publication List:〔Original paper〕

Y., Ishiguro, K., Tanaka, Y., Iida, S., and Hoshino, A.
• Morita,(2015). Spontaneous mutations of the UDP-glucose: flavonoid
3-O-glucosyltransferase gene confers pale and dull colored flowers in
the Japanese and common morning glories. Planta 242, 575-587.

〔Original paper (E-publication ahead of print)〕

M., Morimoto, R., Hirose, M., Morita, Y., Hoshino, A., Iida, S.,• Azuma,
Oshima, Y., Mitsuda, N., Ohme-Takagi, M., and Shiratake, K. A petal-

specific InMYB1 promoter from Japanese morning glory: a useful tool
for molecular breeding of floricultural crops. Plant Biotechnol. J. 29
Apr 2015.

NIBB is the sub-center for the National BioResource
Project (NBRP) for morning glory. In this project, we arecollecting, maintaining and distributing standard lines,
mutant lines for flower pigmentation, and DNA clones from53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
7

LABORATORY OF BIOLOGICAL DIVERSITY1
13 1 5TSUGANE Group
Assistant Professor:

I. A Gain of Function Mutant

The nDart1-promoted gene tagging line was developed
using the endogenous nDart1/aDart1 system to generatevarious rice mutants effectively. While the dominant mutants
were occasionally isolated from the tagging line, it was
unclear what causes dominant mutations. Efficient selection
and analysis of dominant mutants to analyze the gene functions in rice is very useful. A semidominant mutant, Bushy
dwarf tiller1 (Bdt1), which has the valuable agronomic traitsof multiple tillering and dwarfism, was obtained from thetagging line. The Bdt1 mutant carried a newly inserted
nDart1 at 38-bp upstream of the transcription initiation siteof a non-protein-coding gene, miR156d. This insertion
caused an upstream shift of the miR156d transcription initiation site and, consequently, increased the functional transcripts producing mature microRNAs. These results indicate
A
BDT1/Bdt1

Bdt1/Bdt1

B

C

WT

Bdt1

Figure 1. Phenotype of Bushy dwarf tillers1 (Bdt1). (A) Three month oldplants, (B) Abnormal panicles of Bdt1/Bdt1 plants. White arrowheads
indicate overgrown bracts and leaf-like structures, respectively. (C)Morphological phenotypes of WT and Bdt1 plants. Each broken line andeach circle represents an internode and a panicle, respectively.

54

11 1

7

WT
2

TSUGANE, Kazuo

Although transposons occupying large portions of the
genome in various plants were once thought to be junk DNA,
they play an important role in genome reorganization andevolution. Active DNA transposons are important tools forgene functional analysis. The endogenous non-autonomoustransposon, nDart1-0, in rice (Oryza sativa L.) is expected togenerate various transposon-insertion mutants because
nDart1-0 elements tend to insert into genic regions undernatural growth conditions. The transpositions of nDart1-0
were promoted by an active autonomous element, aDart1-27,
on chromosome 6. By using the endogenous nDart1/aDart127 system in rice, a large-scale nDart-inserted mutant population was easily generated under normal field conditions, andthe resulting tagged lines were free of somaclonal variation.

BDT1/BDT1

Bdt1

miR156d0.1 kbnDart1-3
Figure 2. Transcription initiation sites of BDT1 gene. Major transcription initiation sites of miR156d in WT and Bdt1plants. Red and green
arrows indicate transcription initiation sites of miR156d in WT and Bdt1
plants, respectively. Numbers above the arrows represent the numbers ofclones that correspond to the transcription initiation site. The left end of
the white box indicates the reported 5’ terminal of the full-length cDNA
(AK073452) in Nipponbare (http://rapdb.dna.affrc.go.jp/). The gray andblack boxes show the corresponding positions of the pre-miR156d andmiR156d sequences.

that the total amount of miR156d is controlled not only bytranscript quantity but also by transcript quality.
Furthermore, transgenic lines introduced an miR156d
fragment that flanked the nDart1 sequence at the 5’ region,
suggesting that insertion of nDart1 in the gene promoter
region enhances gene expression as a cis-element. This study
demonstrates the ability of nDart1 to produce gain-of-function mutants as well as further insights into the function oftransposable elements in genome evolution.

Publication List:〔Original paper〕

M., Maekawa M., and Tsugane, K. (2015). A gain-of• Hayashi-Tsugane,
function Bushy dwarf tiller 1 mutation in rice microRNA gene miR156dcaused by insertion of the DNA transposon nDart1. Sci. Rep. 5, 14357.

LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Staff:

JOHZUKA, Katsuki
ISHINE, Naomi

Chromosome condensation is a basic cellular process that
ensures the faithful segregation of chromosomes in bothmitosis and meiosis. This process is required not only forshrinking the length of chromosome arms, but also for
resolving entanglements between sister-chromatids that are
created during DNA replication. Any abnormality in thisprocess leads to segregation errors or aneuploidy, resulting incell lethality. Chromosome condensation is mainly achievedby condensin, a hetero-pentameric protein complex, widelyconserved from yeast to humans. Despite its conservationand importance for chromosome dynamics, how condensin
works is not well understood. Recent studies reveal that condensin functions are not restricted to chromosome condensation and segregation during cell divisions. It is required fordiverse DNA metabolism such as genome stability, transcriptional regulation, and cell differentiation.
Our research interest is to understand the mechanism
and regulation of chromosome condensation. We havebeen studying the role of condensin in the budding yeast
Saccharomyces cerevisiae. Microscopic observation indicated the nucleolar localization of condensin. Consistent with
this, the ribosomal RNA gene (rDNA) repeat is the most condensed region in the genome during mitosis. We have foundthat condensin specifically binds to the RFB site locatedwithin the rDNA repeat. To date, the best characterized condensin binding region is the rDNA repeat on the right arm ofchromosome XII in budding yeast. We further discovered themultiple protein network required to recruit condensin to the
RFB site.

with the ectopic RFB site. To explore the role of condensin
in chromosome organization, we have constructed a strain
in which two RFB sites are inserted on an ectopic chromosome arm with an interval of 15kb distance in the cellwith complete deletion of the chromosomal rDNA repeat.
Using this strain, condensin-dependent chromatin interaction
between two RFBs was examined by chromosome conformation capture (3C) assay. We found condensin-dependent
chromatin interaction between the two RFB sites on the chromosome arm. This result indicates that condensin plays a rolein chromatin interaction between condensin binding sites,
and this interaction leads to creation of a chromatin loop
between those sites (Figure 1). It is thought that condensindependent chromatin folding is one of the basic molecular
processes of chromosome condensation. During the cell
cycle stages, the RFB - RFB interaction signal increases in
metaphase and reaches its maximum level in anaphase. In
addition to the RFB – RFB interaction, the chromatin interactions between internal regions of two RFBs increases inanaphase. Thus, the configuration of chromatin fiber changes
from a simple loop into a complicated twisted shape as thecell cycle progresses from metaphase to anaphase.

I. Dynamic relocalization of condensin duringmeiosisOur genetic screening indicated that two proteins, Csm1
and Lrs4, were required for condensin recruitment to the
RFB site. Physical interactions between Csm1/Lrs4 andsubunits of condensin are important for recruitment of condensin to the RFB site. These proteins are known as components of the monopolin complex that are required forfaithful segregation of homologous chromosomes during
meiotic division I. During meiosis I, the monopolin complex
re-localizes from the rDNA repeat to the centromere and acts
for ensuring sister-chromatid co-orientation. Re-localizationof Csm1/Lrs4 proteins suggested the re-localization of condensin from rDNA repeat to centromere. As expected, chromatin-IP experiments indicated that condensin re-localizes
to the centromere during meiosis I. Condensin might clamp
sister-chromatids together during meiosis I.

Figure 1. A Schematic model of chromosome condensation. Condensin
makes chromatin interactions between adjacent binding sites (RFB, forexample). This leads to a folding of chromatin fibers between the sites, asa basic process of chromosome condensation.

II. Condensin-dependent chromatin folding

The RFB site, which consists of a ~150bp DNA sequence,
is functioning as a cis-element for recruitment of condensin
to chromatin in the yeast genome. If the RFB site is inserted
into an ectopic chromosomal locus, condensin can associate

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITY
KATO GroupSpecially Appointed Assistant Professor:
KATO, Kagayaki
Organogenesis is accomplished by a series of deformationsof the planar cell sheet into a three-dimensional shape during
embryogenesis. This drastic structural change is an integrated
result of individual cell behaviors in response to spatio-temporally controlled mechanisms.
To better understand the programs underlying organ formation, it is required to analyze individual cells’ morphology
and dynamics quantitatively. However, due to the massive
images generated by 4D microscopy and their ambiguity, thismade it difficult to perform these analyses.
To unveil organogenesis from the point of view of distinct
cell behaviors, we are developing application software thatis capable of describing cell dynamics out of 4D time-lapse
imaging data sets by employing image processing techniques.

I. 4D cell segmentation/tracking system

Epithelial morphogenesis in the fruit fly Drosophila melanogaster embryo is considered to be an excellent model
for collective cell migrations. Drastic cell rearrangements
lead drastic structural changes to build elaborate tubularorgans such as the tracheal network. We are developing asoftware pipeline, which automatically recognizes individual
cell shapes out of 3D space and tracks them through time.
This system extracts cell boundaries and reconstitutes cell
shapes as a skeletonized chain of voxels spanning 3D space.
This abstract form of cell visualization makes it possible todescribe morphometric quantities and kinetics of cells insingle-cell resolution (Figure 1). These morphometric quantities allow us to perform comparative studies on shapes andbehaviors precisely among several experimental conditions,to gain a better understanding of the genetic programs underlying organogenesis. We are now applying this system toseveral experimental models to determine the practicality ofthe system.

II. P article tracking for tissue deformation
analysis

Besides cell boundary extraction, we also developed aderived algorithm for particle image velocimetry (PIV).
This system is designed to measure tissue deformation eventhough the imaging constraints do not allow identification ofindividual cells out of images. This implementation detects
structural characteristics, such as uneven fluorescence distributed over the specimen and tracks these patterns along
a time-series. Despite that the tissue was labeled with nontargeted cytoplasmic GFP, this tracking software successfully
outlined developmental dynamics of Xenopus neuroectoderm(Figure 2).

Figure 2. Collective cell migration of Xenopus neuroectodermal cells
visualized as optical flow along a time-series. A modified PIV method
successfully tracks uneven subcellular distribution of GFP signals overtime. Dr. M. Suzuki (Prof. Ueno’s laboratory at NIBB) performed themicroscopy.

III. A GUI application for manual image
quantification
Biologically significant image features are not always significant to computational algorithms due to their structural
instability. This kind of difficulty requires human eye inspection for feature extractions. A GUI (Graphical User Interface)application we developed can easily visualize 4D imaging
data and has made manual feature annotations easy (Figure3). This application is freely available at our website (https://is.cnsi.jp/).

Figure 3. A lightweight/native 4D stack viewer equipped with functions
for manual feature extraction.
Figure 1. Visualized apical cell surface of Drosophila embryonic epidermal cells. A time-lapse confocal microscopic data set of a fly embryo
expressing E-cadherin-GFP was subjected to our automatic cell shaperecognition system. Cell boundaries (green), center of gravity (blue) andnormal vector (magenta) are indicated for each cell.

56

LABORATORY OF BIOLOGICAL DIVERSITYKIMORI GroupSpecially Appointed Assistant Professor:
KIMORI, Yoshitaka
Image processing methods significantly contribute to visualization of biomedical targets acquired froma variety of imaging techniques, including: wide-field
optical and electron microscopy, X-ray computed tomography, magnetic resonance imaging and mammography.
Quantitative interpretation of the deluge of complicated biomedical images, however, poses many research challenges.
We have developed new computational methods based onmathematical morphology for quantitative image analysis.
One of the most important purposes of image processing
is to derive meaningful information, which is expressed as
structural properties in images. Mathematical morphology isa nonlinear image processing method based on the set theory
and is useful for the extraction of structural properties froman image. It can be used as a fundamental tool to analyze
biomedical images and provides an objective, accurate alternative to manual image analysis.

Novel image analysis method based onmathematical morphology: quantifying
morphological features of actin filaments inplant cells

Image processing is a crucial step in the quantification of
biomedical structures from images. As such, it is fundamental to a wide range of biomedical imaging fields. Image processing derives structural features, which are then numerically quantified by image analysis. We can better evaluate
complex shapes and detect subtle morphological changes inorganisms by quantifying the shape properties. Therefore,
we have developed a shape analysis method based on morphological image processing, and have applied it to image
analysis of actin cytoskeletal filaments in root-hair cellsof Arabidopsis thaliana. Actin cytoskeletal filaments havecritical roles in various cellular processes in plant cells. To
understand actin-dependent organelle motility, we analyzed
the organization of actin filaments in the cells.
We measured three shape features of the actin filaments inwild-type and mutant (root hair defective 3 (rhd3) mutant)
plants. One feature i.e. thickness (T) was extracted fromgrayscale images; the others i.e. multi-orientation index(MOI) and complexity (C) were extracted from binary
images. T of an actin filament was measured by a pattern
spectrum which provides a distribution of filament thickness.
MOI was measured by applying a series of opening operations to obtain the orientation distribution of the filament.
C was computed from the fractal dimension of the filament
network pattern. As the MOI and C quantify the complexity
properties of the filament patterns in two-dimensional space,
finally, these binary-based features were combined into asingle feature called the binarized filament pattern feature(BFPF).
The T of the wild-type and mutant were 1.88 mm and 2.33mm, respectively, and those were statistically different (p <0.001). Also, MOI of the wild-type and rhd3 mutant images
were statistically different (p < 0.01), too. Finally, we calculated the C. The mean fractal dimension of the actin filament
was significantly larger in the wild-type than in the rhd3
mutant (p < 0.001). Furthermore, the mean BFPF (obtainedby combining MOI and C) were again significantly differed
between the two groups (p < 0.001). These results are summarized in a scatter plot of BFPF (x-axis) versus T (y-axis)(Left panel of Figure 1). First, we note that actin filaments
are thicker in the rhd3 mutant than in the wild-type. Second,
both MOI and C are significantly larger in the wild-type filaments than in the rhd3 mutant filaments. Overall, thefilament patterns in two-dimensional space are more
complex in the wild-type than in the rhd3 mutant. The rhd3
mutant data in the scatter plot are divisible into two classes(Class 1 and Class 2), distinguished by linear discriminant
analysis (LDA). The right panel of Figure 1 shows three
images selected from each class.

Figure 1. Left: Scatter plot of BFPF versus T for the actin filament ofwild-type and the rhd3 mutant. Data of the rhd3 mutant is divided into
two classes. Dashed line is classification boundary obtained by LDA.
Solid line is boundary obtained by linear regression of the data in Class
1. Right: Actin filaments of the rhd3 images belonging to each class.

Publication List:〔Original paper〕

Y., Kimori, Y., Okada, H., and Ohnuki, S. (2015). Single-cell• Ohya,phenomics in budding yeast. Mol. Biol. Cell. 26, 3920-3925.

〔Original papers (E-publication ahead of print)〕

Y., Hikino K., Nishimura M. and Mano S. Quantifying• Kimori
Morphological Features of Actin Cytoskeletal Filaments in Plant Cells•

Based on Mathematical Morphology. J. Theor. Biol. 2015 Nov 10.
Yasuda, T., Kimori, Y., Nagata, K., Igarashi, K., Watanabe-Asaka,T., Oda, S., and Mitani, H. Irradiation-injured brain tissues can selfrenew in the absence of the pivotal tumor suppressor p53 in the medaka(Oryzias latipes) embryo. J. Radiat. Res. 2015 Sep 25.

57

National Institute for Basic Biology

Environmental Biology

DIVISION OF MOLECULAR ENVIRONMENTAL ENDOCRINOLOGY †Professor

IGUCHI, Taisen

Assistant Professor:

MIYAGAWA, Shinichi
OGINO, Yukiko
Technical Staff:
MIZUTANI, Takeshi
NIBB Research Fellow:
TOYOTA, Kenji
MIYAKAWA, Hitoshi*
Postdoctoral Fellow:
MIYAKAWA(OKAMOTO), Misato
MIYAKAWA, Hitoshi
SOKENDAI Graduate Student: TOYOTA, Kenji*
SUMIYA, Eri
YATSU, Ryohei
Visiting Graduate Student: TOHYAMA, Saki
Visiting Scientist:
McNABB, Nicole
Technical Assistant:
HAYASHI, Tomoko
INABA, KayoSecretary:
IMAIZUMI, Taeko

Synthetic chemicals found in the environment have thecapacity to disrupt the development and function of the endocrine system in both wildlife and humans. This has drawn
public concern since many of these chemicals may bindto estrogen receptors (ERs) and evoke estrogenic effects.
Early evidence that exposure to estrogenic chemicals during
development could pose a threat to human health came fromstudies of a synthetic hormone, diethylstilbestrol (DES),
which was used to prevent premature birth and spontaneousabortion. Laboratory experiments showed that exposure
to sex hormones during critical windows of perinatal life
caused the immune and nervous systems, bone, muscle,
and the liver of animals to be affected. Although many ofthese chemicals can bind to ERs in wildlife and humans, themolecular basis for the action of environmental estrogens
remains poorly understood. Thus, understanding the molecular mechanisms through which environmental estrogens andsex hormones act during critical developmental windows isessential.

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

I. D evelopmental origin of adult disease:
Perinatal estrogen exposure induces
persistent changes in reproductive tracts
The emerging paradigm of the “embryonic/fetal origins ofadult disease” provides a powerful new framework for con-58

Figure 2. A hypothetical model for the estrogen-independent ER activation pathway in mouse vaginae.

sidering the effects of endocrine disrupters on human andanimal health. In 1971, prenatal DES exposure was found
to result in various abnormalities of the reproductive tract
in women. This syndrome was named the DES syndrome.
Similar abnormalities have been demonstrated in experimental animals exposed perinatally to estrogens. Developmental
estrogen exposure in mice, for example, induces persistent
proliferation of vaginal epithelial cells (Figure 1). We foundthat the persistent changes in the vagina in mice exposedneonatally to estrogens result from the persistent activationof erbBs and ERa, and sustained expression of EGF-likegrowth factors (Figure 2). Currently, we are analyzing themethylation status in the mouse vagina using a microarray(MeDIP-chip). We found several differentially methylated
or demethylated DNA profiles in neonatally DES-exposedmouse vaginae and controls. We thus consider that neonatal
DES exposure affects DNA methylation profiles, resulting
in persistent abnormalities in mouse reproductive organs. We
also found that ERa is indispensable for normal vaginal epithelial cell differentiation in mice.

II. E strogen receptors of birds, reptiles,
amphibians, and fishes

Steroid and xenobiotic receptors (SXR) have been cloned
from various animal species (fish, amphibians, reptiles,
birds, and mammals) by our group and we have demonstrated species-specific differences in their responses tovarious environmental and endogenous chemicals (receptor
gene zoo). Thus, simple predictions of chemical effects
based on data from a few established model species are notsufficient to develop real world risk assessments. ER andER-like genes have been cloned from various animal species
including rockshell, Amphioxus, lamprey, catshark, whale
shark, lungfish, sturgeon, gar, polypterus, arowana, roach,stickleback, mosquitofish, mangrove Rivulus, Japanese
giant salamander, Tokyo salamander, newt, axolotl, toad,
Silurana tropicalis, American alligator, Nile crocodile,
freshwater turtle, Japanese rat snake, Okinawa habu, andvultures. Functional studies showed that the Amphioxus
ER sequence does not bind estrogen but Amphioxus steroid

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2015. The former title isindicated by an asterisk (*).
†: This laboratory was closed on 31 March, 2016.

receptor and lamprey ER exhibited ligand-dependent transactivation, proving that invertebrate and primitive vertebrates,
such as the Agnatha, have a functional ER. We found thatmedaka ER subtypes have their specific functions, andmedaka, zebrafish and stickleback ERs are more sensitive to
estrogen/estrogen-like chemical exposures than other fishes
by reporter gene assay. Thus, these approaches are efficientto evaluate the relationship between species and their sensitivities to chemicals.

III. E volutionary history and functional
characterization of androgen receptor
genes in jawed vertebrates

Vertebrates show diverse sexual characteristics which are
regulated by androgens. To elucidate the evolutionary history
and functional diversification of androgen receptor (AR)genes in vertebrates, we cloned the AR cDNAs from ashark, basal ray-finned fishes (Actinopterygii), namely bichirand sturgeon (Acipenseriformes), and teleosts including
a basal teleost, arowana (Osteoglossiformes). Molecularphylogenetic analysis revealed that a gene duplication event
gave rise to two different teleost ARs (a and b) and likely
occurred in the actinopterygian lineage leading to teleosts
after the divergence of Acipenseriformes but before thesplit of Osteoglossiformes. Functional analysis revealed
that the shark AR activates the target gene via the androgen
response element by classical androgens. The teleost ARa
showed unique intracellular localization with a significantly
higher transactivation capacity than that of teleost ARb.
These results indicate that the most ancient type of AR, asactivated by the classic androgens as ligands, emerged before
the Chondrichthyes-Osteichthyes split and the AR gene was
duplicated during a teleost-specific gene duplication event(Figure 3).

IV. Papillary process formation in medaka

Androgens play key roles in the morphological
specification of male type sex characteristics andreproductive organs, whereas little is known about thedevelopmental mechanisms. Medaka show a prominentmasculine sexual character, papillary processes in the anal
fin, which has been induced in females by exogenous
androgen exposure. We have identified androgen-dependent
expressions of Bmp7 and Lef1 are required for the bone
nodule outgrowth leading to the formation of the papillary
process in the postal region of the anal fin. We have also
developed a testing method for screening of chemicals
having androgen and anti-androgenic activity using the anal
fin in juvenile medaka.

Figure 3. Evolutionary relationships of androgen receptor sequences.

with high reproducibility and demonstrated the usefulnessof the array for the classification of toxic chemicals as well
as for the molecular understanding of chemical toxicity in
a common freshwater organism. D. magna and D. pulex
reproduce asexually (parthenogenesis) when they are in
an optimal environment for food, photoperiod and population density. Once environmental conditions become suboptimal, they alter their reproductive strategy from asexual
to sexual reproduction (Figure 4). Chemicals are able toaffect the sex determination of daphnids and we foundthat juvenile hormone (JH) agonists (insect growth regulators), for example, induce the production of male offspring.
The molecular basis of environmental sex determination islargely unknown in daphnids. To understand the molecularmechanisms of this phenomenon, we isolated sex determination-related genes. Also, we have developed a method to
inject genes into D. magna and D. pulex embryos which willallow us to study gain- and loss-of function analyses in moredetail in these species. Using these techniques, we demonstrated that DSX1 (double sex 1), one of the DM-domain
genes, is essential for male differentiation in D. magna. We
have developed an RNAi method and a TALEN method
using D. pulex. To further explore the signaling cascadeof sexual differentiation in D. magna, a gene expression
profile of JH-responsive genes is essential. We are identifying JH-responsive genes in the ovary of D. magna and D.
pulex exposed to JH agonist and methyl farnesoate (JH identified in decapods) at the critical timing of JH-induced sex
determination in D. magna and D. pulex. We have identified
a JH receptor (heterodimer of methoprene-tolerant andsteroid receptor co-activator) in daphnids and the function of
ecdysone in the molting and ovulation in D. magna.
Sex determination mechanisms can be broadly categorized

V. E nvironmental sex differentiation in
Daphnids and American alligators

Daphnia magna has been used extensively to evaluate theorganism- and population-based responses of toxicity or
reproductive toxicity tests. These tests, however, provide
no information about the mode of action of the tested compounds. Therefore, we applied an ecotoxicogenomic assessment of D. magna. We established a Daphnia EST databaseand developed an oligonucleotide-based DNA microarray

Figure 4. Life cycle of Daphnia.

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Environmental Biology

by either a genotypic or environmentally driven mechanism.
Temperature-dependent sex determination (TSD), an environmental sex determination mechanism most commonly
observed among vertebrates, has been observed especiallyamong reptiles from 1966. However, the temperature-dependent triggering mechanism of TSD and the subsequent differentiation cascade has long remained unknown. We haveisolated and cloned the thermosensitive cation channel,
TRP vanilloid subtype 4 (TRPV4) as a male-cascade trigger
for the American alligator, Alligator mississippiensis, in
response to high environmental temperature, and demonstrated its thermal activation at temperatures proximate to
TSD-related temperatures in the alligator. Furthermore, using
pharmacological exposure to manipulate TRPV4 channel
activity, we have demonstrated that TRPV4 channel activity
has a direct relationship with male differentiation gene
expression, suggesting that AmTRPV4 is involved in themale differentiation cascade, and proposed a novel mechanism for the sex determination pathway.

Publication List

〔Original papers〕

R., Toyota, K., Miyakawa, H., Watanabe, H., Oka, T., Miyagawa,• Abe,
S., Nishide, H., Uchiyama, I., Tollefsen, E.K., Iguchi, T., and

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Tatarazako, N. (2015). Diofenolan induces male offspring production
through binding to the juvenile hormone receptor in Daphnia magna.
Aquat. Toxicol. 159, 44-51.
Abe, R. Watanabe, H., Yamamuro, M., Iguchi, T., and Tatarazako,
N. (2015). Establishment of a short-term in vivo screening methodfor detecting chemicals having juvenile hormone activity using adult
Daphnia magna. J. Appl. Toxicol. 35, 75-82.
Bain, P.A., Kumar, A., Ogino, Y., and Iguchi, T. (2015). Nortestosteronederived synthetic progestogens do not activate the progestogen receptorof Murray-Darling rainbowfish (Melanotaenia fluviatilis) but are potent
agonists of androgen receptors a and b. Aquat. Toxicol. 163, 97-101.
Bain, P.A., Ogino, Y., Miyagawa, S., Iguchi, T., and Kumar, A. (2015).
Differential ligand selectivity of androgen receptors a and b from
Murray-Darling rainbowfish (Melanotaenia fluviatilis). Gen. Comp.
Endocrinol. 212, 84-91.
Goodhead, R.M., Johnston, B., Cole, P., Baalousha, M., Hodgson,
D., Iguchi, T., Lead, J., and Tyler, C.R. (2015). Does natural organic
matter increases bioavailability of cerium dioxide nanoparticles to fish?
Environ. Chem. 12, 673-682.
Ihara, M., Kitamura, T., Kumar, V., Park, C.-B., Ihara, M.O., Lee, S.J., Yamashita, N., Miyagawa, S., Iguchi, T., Okamoto, S., Suzuki, Y.,
and Tanaka, H. (2015). Evaluation of estrogenic activity of wastewater:comparison among in vitro ERa reporter gene assay, in vivo vitellogenin
induction, and chemical analysis. Environ. Sci. Technol. 49, 6319-6326.
Kohno, S., Bernhard, M.C., Katsu, Y., Zhu, J., Byan, T.A., Doheny,
B.M., Iguchi, T., and Guillette, L.J.Jr. (2015). Estrogen receptor 1(ESR1; ERa), not ESR2 (ERb), modulates estrogen-induced sex
reversal in the American alligator, a species with temperature-dependent
sex determination. Endocrinology 156, 1887-1899.
Lange, A., Sebire, M., Rostlowski, P., Mizutani, T., Miyagawa, S.,
Iguchi, T., Hill, E.M., and Tyler, C.R. (2015). Environmental chemicals
active as human antiandrogens potentiate a fminising effect of oestrogenin fish. Aquat. Toxicol. 168, 48-59.
Miyagawa, S., and Iguchi, T. (2015). Epithelial estrogen receptor a
intrinsically mediates squamous differentiation in the mouse vagina.
Proc. Natl. Acad. Sci. USA 112, 12986-12991.
Miyagawa, S., Sato, M., Sudo, T., Yamada, G., and Iguchi, T. (2015).
Unique roles of estrogen-dependent Pten control in epithelial cellhomeostasis of mouse vagina. Oncogene 34, 1035-1043.
Miyagawa, S., Tohyama, S., Ogino, Y., Mizutani, T., Kobayashi,T., Lange, A., Tyler, C.R., Tatarazako, N., and Iguchi, T. (2015).
Characterization of Oryzias latipes glucocorticoid receptors and theirunique response to progestings. J. Appl. Toxicol. 35, 302-309.
Miyagawa, S., Yatsu, R., Kohno, S., Doheny, B.M., Ogino, Y., Ishibashi, H.,

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Katsu, Y., Ohata, Y., Guillette, L.J.Jr., and Iguchi, T. (2015). Identificationand characterization of the American alligator androgen receptor and the
intriguing role of its splice variant. Endocrinology 156, 2795-2806.
Miyakawa, H., Sato, M., Colbourne, J.K., and Iguchi, T. (2015).
Ionotropic glutamate receptors mediate inducible defense in the waterflea Daphnia pulex. PLoS One, 10, e0121324.
Miyakawa, H., Sugimoto, N., Kohyama, T.I., Iguchi, T., and Miura,
T. (2015). Repeated colonization leads to intra-specific variations
in reaction norms of predator-induced polyphenism in the water flea
Daphnia pulex. Ecol. Res. 30, 705-713.
Mohapatra, S., Chkraborty, T., Miyagawa, S., Zhou, L., Ohta, K.,
Iguchi, T., and Nagahama, Y. (2015). Steroid responsive regulation of
IFNg2 alternative splicing and its possible role in germ cell proliferation
in medaka. Mol. Cell. Endocrionol. 400, 61-70.
Nakajima, T., Tanaka, M., Chambon, P., Watanabe, H., Iguchi, T.,
and Sato, T. (2015). Neonatal ERb is important in the permanent
inhibition of epithelial cell proliferation in the female mouse uterus.
Endocrinology 156, 3317-3328.
Nakamura, A., Takanobu, H., Tamura, I., Yamamuro, M., Iguchi, T.,
and Tatarazako, N. (2015). Fish multi-generation test with preliminary
short-term reproduction assay for estrone using Japanese medaka(Oryzias latipes). J. Appl. Toxicol. 35, 11-23.
Oka, K., Hang, A., Okada, D., Iguchi, T., Baker, M.E., and Katsu,
Y. (2015). Allosteric role of the amino-terminal A/B domain on
corticosteroid transactivation of gar and human glucocorticoid
receptors. J. Steroid Biochem. Mol. Biol. 154, 112-119.
Pickford, D.B., Jones, A., Velez-Pelez, A., Orton, F., Iguchi, T., Mitsui,N., and Tooi, O. (2015). Screening breeding sites of the common toad
(Bufo bufo) in England and Wales for evidence of endocrine disrupting
activity. Ecotoxicol. Environ. Safety 117, 7-19.
Spirhanzlova, P., Leleu, M., Sébillot, A., Lemkine, G.F., Iguchi,T., Demeneix, B.A., and Tindall, A.J. (2015). Oestrogen reportertransgenic medaka for non-invasive evaluation of aromatase activity.
Comp. Biochem. Physiol. C Toxicol. Pharmacol. 179, 64-71.
Tohyama, S., Miyagawa, S., Lange, A., Ogino, Y., Mizutani, T.,
Tatarazako, N., Katsu, Y., Ihara, M., Tanaka, H., Ishibashi, H., Kobayashi,T., Tyler, C.R., and Iguchi, T. (2015). Understanding the molecular basis
for differentiation in responses of fish estrogen receptor subtypes toenvironmental estrogens. Environ. Sci. Technol. 49, 7439-7447.
Toyota, K., Miyakawa, H., Yamaguchi, K., Shigenobu, S., Ogino, Y.,
Tatarazako, N., Miyagawa, S., and Iguchi, T. (2015). NMDA receptor
activation on the upstream of methyl farnesoate signaling for short-day
induced male offspring production in water flea Daphnia pulex. BMC
Genomics 16, 186.
Toyota, K., Miyakawa, H., Hiruta, C., Furuta, K., Ogino, Y., Shinoda, T.,
Tatarazako, N., Miyagawa, S., Shaw, J.R., and Iguchi, T. (2015). Methyl
farnesoate synthesis is necessary for the environmental sex determinationin the water flea Daphnia pulex. J. Insect Physiol. 80, 22-30.
Yatsu, R., Miyagawa, S., Kohno, S., Saito, S., Lowers, R.H., Ogino,Y., Fukuta, N., Katsu, Y., Ohta, Y., Tominaga, M., Guillette, L.J.Jr., andIguchi, T. (2015). TRPV4 associates environmental temperature andsex determination in the American alligator. Sci. Rep. 5, 18581.

〔Review articles〕

Å., Becher, G., Blumberg, B., Bjerregaard, P., Bornman,• Bergman,
R., Brandt, I., Casey, S.C., Frouin, H., Giudice, L.C., Heindel, J.J.,

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Iguchi, T., Jobling, S., Kidd, K.A., Kortenkamp, A., Lind, P.M., Muir,
D., Ochieng, R., Ropstad, E., Ross, P.S., Skakkebaek, N.E., Toppari,J., Vandenberg, L.N., Woodruff, T.J., and Zoeller, R.T. (2015).
Manufacturing doubt about endocrine disrupter science - A rebuttalof industry-sponsored critical comments on the UNEP/WHO report“State of the Science of Endocrine Disrupting Chemicals 2012”. Reg.
Phamacol. Toxicol. 73, 1007-1017.
Heindel, J.J., Newbold, R.R., Iguchi, T., Tyler, C.R., and Williams, C.J.
(2015). Lou Guillette – in memorandum. Mol. Reprod. Dev. 82, Fmi-Fmv.
Helbing, C., Tyler, C.R., and Iguchi, T. (2015). In memorium: Louis J.
Guillette, Jr. Environ. Health Perspect. 123, A250.
Iguchi, T. (section editor) (2015). Handbook of Hormones: Comparative
Endocrinology for Basic and Clinical Research. (eds.) Takei, Y., Ando,
H., and Tsutsui, K., Academic Press, pp. 646.
Miyagawa, S., and Iguchi, T. (2015). Pten in mouse vagina. Oncoscience12, 749-750.

DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

MINAGAWA, Jun

Associate Professor

TAKAHASHI, Shunichi

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

TOKUTSU, Ryutaro
NODA,Chiyo
AIHARA, Yusuke
KAMADA, Konomi
KAWAI, Hisako
YAMASAKI, Hiroaki
KIM, Eunchul
OHNISHI, Norikazu
SOKENDAI Graduate Student: KAMRANI, Yousef Yari
KATO, Hiroki
KOSUGE, Kotaro
KIKUCHI, Ayaka
Visiting Graduate Student: KISHIMOTO, Mariko
Visiting Scientist:
MARUYAMA, Shin-ichiro
TAKIZAWA, Kenji
TAKAHASHI, Sara
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
KIDA, EmiHOSHI,Rie
KOJIMA, Yoko
Secretary:

the two photosystems tends to occur in natural environments,
where light quality and quantity fluctuate with time. Because
the two photosystems are functionally connected in series
under normal conditions, plants and algae must constantly
balance their excitation levels to ensure the optimal efficiency of electron flow. State transitions occur under suchconditions to balance the light-harvesting capacities of thetwo photosystems, thereby minimizing the unequal distribution of light energy. State 1 occurs when PSI is preferentially
excited and the light-harvesting capacities of PSII and PSI
are increased and decreased, respectively, to adjust the excitation imbalance; this state is indicated by a higher chlorophyll fluorescence yield at room temperature. Conversely,
State 2 occurs when PSII is preferentially excited and thelight-harvesting capacities of PSII and PSI are decreased andincreased, respectively, to readjust the excitation imbalance;
this state can be monitored as a lower chlorophyll fluorescence yield at room temperature (Figure 1).
In C. reinhardtii WT cells, the modulation of CEF normallyoccurs in parallel with state transitions. When the lightinduced reduction of Cyt bf was probed in State 1- and State

Plants and algae have a large capacity to acclimate themselves to changing environments. We are interested in theseacclimation processes, in particular, how efficiently yet
safely they harness sunlight for photosynthesis under fluctuating light conditions. Using a model green alga, we are
studying the molecular mechanisms underlying photoacclimation of the photosynthetic machinery. We are also
applying the knowledge obtained in the studies of a model
green alga to various phytoplankton, including Symbiodinium
in corals and sea anemones in tropical oceans, to explore
how environmentally important photosynthetic organisms
thrive in their ecological niche.

I. Acclimation of photosynthesis

Using a green unicellular alga C. reinhardtii, we investigate
the molecular mechanisms underlying the acclimation processes of the photosynthetic complexes such as state transitions, non-photochemical quenching, photoinhibition, andcyclic electron flow by means of biochemistry, molecular
genetics, various physiological analyses, and optical spectroscopy.

1-1 State transitions

Photosynthetic organisms respond to changes in light
quality by regulating the absorption capacity of their PSs.
These short-term acclimations use redox-controlled, reversible phosphorylation of LHCIIs to regulate the relative
absorption cross-section of the two photosystems, commonlyreferred to as state transitions. Each of the two charge-separation devices—PSI and PSII—in the thylakoid membranes
has a distinct pigment system with unique absorption characteristics. Thus, an imbalance of energy distribution between

Figure 1. Schematic representation of the regulation of electron flow
and state transitions in C. reinhardtii. Upper: when PSI is preferentially
excited, the stroma of chloroplast and the PQ pool are oxidized. Under
these conditions, LHCIIs are bound to PSII (State 1). The photosynthetic
electron flow proceeds in linear electron flow (LEF) mode, generating
NADPH as well as a proton gradient across the thylakoid membranethat is used for ATP production. Middle: when the stroma is reduced,
first, cyclic electron flow (CEF) is activated by the association of Cyt bf
and FNR with PSI to form a super-supercomplex (CEF supercomplex).
PGRL1 and possibly PGR5 are also associated with the CEF supercomplex; second, PQ pool is reduced and migration of the mobile LHCIIs(orange) from PSII to PSI occurs; Lower: the cells are in State 2, and thephotosynthetic electron flow proceeds in CEF mode.

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Environmental Biology

2-adapted cells, a differential sensitivity to the addition of
the PSII inhibitor DCMU was observed. In the presence of
DCMU, the reduction of Cyt bf was suppressed in State 1
but not in State 2. An identical sensitivity to an inhibitor of
Cyt bf, DBMIB, was observed in both State 1 and State 2,
suggesting that PSII-independent Cyt bf reduction occurs
only in State 2. However, state transitions and CEF/LEF
switching are not mechanically linked in C. reinhardtii but
the two phenomena are rather coincidental. Under anaerobic
conditions, the independent knockdown of three thylakoid
membrane proteins, PGRL1, CAS, and ANR1, resulted indecreased CEF activity, but the ability to undergo state transitions was unaffected. This was further supported by the low
CEF activity in a State 2-locked mutant of C. reinhardtii.
Although the lateral migration of mobile LHCIIs occurred in
the ptox2 mutant, which was State 2-locked due to a lack ofplastid terminal oxidase 2, the effects on P700+ re-reduction
were negligible. CEF was thus proposed to be correlatedwith a reduced state of the stroma.

1-2 Non-photochemical quenching

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosyntheticorganisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as non-photochemical
quenching (NPQ). This term comprises at least two major
processes: state transitions (qT), the change in the relative
antenna sizes of PSII and PSI as described in the previous
section, and energy-dependent quenching of excess energy(qE), the increased thermal dissipation triggered by lumen
acidification. Recently, we isolated the PSII-LHCII supercomplex from both WT C. reinhardtii and the npq4 mutant,which is qE-deficient and lacks the ancient light-harvesting
protein LHCSR. LHCSR3 was present in the PSII-LHCII
supercomplex from the high light-grown WT but not in the
supercomplex from the low light-grown WT or the npq4
mutant. The purified PSII-LHCII supercomplex containing LHCSR3 showed a normal fluorescence lifetime at aneutral pH (7.5) by single-photon counting analysis but
exhibited a significantly shorter lifetime (energy-quenching)at pH 5.5, which mimics the acidified lumen of the thylakoid
membranes in high light-exposed chloroplasts. The switching from light-harvesting mode to energy-dissipating mode
observed in the LHCSR3-containing PSII-LHCII supercomplex was inhibited by DCCD, a protein-modifying agent
specific to protonatable amino acid residues. We conclude
that the PSII-LHCII-LHCSR3 supercomplex formed in highlight-grown C. reinhardtii cells is capable of energy dissipation upon protonation of LHCSR3.

1-3 Photodamage to photosystem II

Light, the driving force of photosynthesis, damages photosynthetic machinery, primarily photosystem II (PSII), andit results in photoinhibition. A new photodamage model,
the two-step photodamage model, suggests that photodamage to PSII initially occurs at the oxygen evolving complex(OEC) because of light energy absorbed by manganese andthat the PSII reaction center is subsequently damaged fromlight energy absorbed by photosynthetic pigments due to the62limitation of electrons to the PSII reaction center. However,it was uncertain whether this model is applicable to photodamage to PSII under visible light as manganese absorbs
visible light only weakly. In our study, we demonstratedusing PSII membrane fragments isolated from spinach leaves
that visible light damages OEC prior to photodamage to the
PSII reaction center. This finding supports that the two-step
photodamage model is applicable to photodamage to PSII byvisible light.

1-4 Cyclic electron flow in plants

CEF around PS I is difficult to quantify. In our study, a
new method was introduced to measure CEF in wild-type
and pgr5 and ndh mutants of Arabidopsis. We obtained thelinear electron flux (LEFO2) through both photosystems andthe total electron flux through PS I (ETR1) in Arabidopsis
in CO2-enriched air. ΔFlux = ETR1 - LEFO2 is an upper
estimate of CEF, which consists of two components, anantimycin A-sensitive, PGR5 (proton gradient regulation 5protein)-dependent component and an insensitive component
facilitated by a chloroplastic nicotinamide adenine dinucleotide dehydrogenase-like complex (NDH). Our results
demonstrated that (1) 40% of the absorbed light was partitioned to PS I; (2) at high irradiance a substantial antimycin
A-sensitive CEF occurred in the wild type and the ndhmutant; (3) at low irradiance a sizable antimycin A-sensitive
CEF occurred in the wild type but not in the ndh mutant, suggesting an enhancing effect of NDH in low light; and (4) inthe pgr5 mutant, the wild type, and ndh mutant treated with
antimycin A, a residual ΔFlux existed at high irradiance,
attributable to charge recombination and/or pseudo-cyclic
electron flow. Therefore, in low-light-acclimated plants
exposed to high light, ΔFlux has contributions from various
paths of electron flow through PS I.

II. Ecophysiology of micro algae

Our new projects are the study of photoacclimation of dinoflagellates that can live in a symbiotic relationship with cnidarians, and the study of oil-producing Chlamydomonas. We
are particularly interested in a dinoflagellate Symbiodinium
living with corals and sea anemones (Figure 3), and the oilproducing Chlamydomonas grown under natural pond-likeenvironments. We are trying to elucidate how their photosynthetic machinery acclimates to variable light and temperature
conditions. Furthermore, our interest has been expanded tohigher plants.

2-1 Diversification of light harvesting complex genefamily via intra- and intergenic duplications in thecoral symbiotic alga Symbiodinium
The unicellular dinoflagellate alga Symbiodinium is known
to be an endosymbiont of cnidarian animals including
corals and sea anemone, and provide carbohydrates generated through photosynthesis to host animals. AlthoughSymbiodinium possesses a unique light-harvesting complex(LHC) gene family called chlorophyll a-chlorophyllc2-peridinin protein complex (acpPC), the genome-level
gene diversity and evolutionary trajectories have not been
investigated. We show phylogenetic analysis revealing that

many of the acpPC/LHCs were encoded in highly duplicated genes with the multi-subunit polyprotein structuresin the nuclear genome of Symbiodinium minutum. This
provided an extended list of the LHC gene family in a single
organism, including 82 loci encoding polyproteins composedof 164 LHC subunits recovered in the phylogenetic tree. In
S. minutum, 5 phylogenetic groups of the Lhcf-type genefamily, which is exclusively conserved in algae harboring secondary plastids of red algal origin, and 5 groups of
the Lhcr-type, of which members are known to be associated with PSI in red algae and secondary plastids of red
algal origin were identified. Notably, members classified toa phylogenetic group of the Lhcf-type (group F1) are highly
duplicated, which can explain the presence of an unusuallylarge number of LHC genes in this species. While some geneunits were homologous to other units within single loci of thepolyprotein genes, intergenic homologies between separateloci were conspicuous in other cases, implying that gene unit
‘shuffling’ by gene conversion and/or genome rearrangement
might have been a driving force for the diversification. These
results suggest that, through vigorous intra- and intergenic
gene duplication events, the genomic framework of the photosynthesis has been forged in the coral symbiont dinoflagellate algae.

synthetic oxygen production rate after high-light exposure
demonstrated that CCMP2459 is more sensitive to photoinhibition of PSII than CS-164, and tends to lose maximum photosynthetic activity faster. Measurement of photodamage to
PSII under light of different wavelength ranges demonstrated
that PSII in both Symbiodinium phylotypes was significantly
more sensitive to photodamage under shorter wavelength
regions of light spectra (<470 nm). Importantly, PSII in
CCMP2459, but not CS-164, was also sensitive to photodamage under the regions of light spectra around 470-550
and 630-710 nm, where photosynthetic antenna proteinsof Symbiodinium have light absorption peaks. This finding
indicates that the high-light-sensitive CCMP2459 has an
extra component of photodamage to PSII, resulting in higher
sensitivity to high light. Our results demonstrate that sensitivity of PSII to photodamage differs among Symbiodinium
phylotypes and this determines their sensitivity to high light.

Publication List:〔Original Papers〕

W., Seidi, A., Hill, R., Chow, W.S., Minagawa, J., Hidaka, M.,• Karim,and Takahashi, S. (2015). Novel characteristics of photodamage to

•
•
•
•
•

photosystem II in a high-light-sensitive Symbiodinium phylotype. Plant
Cell Physiol. 56, 1162-1171.
Kou, J., Takahashi, S., Fan, D-Y., Badger, M.R., and Chow, W.S. (2015).
Partially dissecting the steady-state electron fluxes in Photosystem I inwild-type and pgr5 and ndh mutants of Arabidopsis. Front. Plant Sci.
6, 758.
Maruyama, S., Shoguchi, E., Satoh, N., and Minagawa, J. (2015).
Diversification of light harvesting complex gene family via intra- andintergenic duplications in the coral symbiotic alga Symbiodinium. PLoS
One 10, e0119406.
Minagawa, J., and Tokutsu, R. (2015). Dynamic regulation ofphotosynthesis in Chlamydomonas reinhardtii. Plant J. 82, 413-428.
Xue, H., Tokutsu, R., Bergner, S., Scholz, M., Minagawa, J., andHippler, M. (2015). PSBR is required for efficient binding of LHCSR3
to photosystem II - light-harvesting supercomplexes in Chlamydomonas
reinhardtii. Plant Physiol. 167, 1566-1578.
Zavafer, A., Cheah, M.H., Hillier, W., Chow, W.S., and Takahashi, S.
(2015). Photodamage to the oxygen evolving complex of photosystem
II by visible light. Sci. Rep. 5, 16363.

Figure 2. Fluorescence image of the tiny sea anemone Aiptasia, a model
system for studies of dinoflagellate (Symbiodinium)-cnidarian symbiosis.
Each red dot is a cell of Symbiodinium.

2-2 Novel characteristics of photodamage to PSII in ahigh-light-sensitive Symbiodinium phylotype

Symbiodinium is genetically diverse, and acquiring suitable
Symbiodinium phylotypes is crucial for the host to survive
in specific environments, such as high-light conditions. The
sensitivity of Symbiodinium to high light differs among
Symbiodinium phylotypes, but the mechanism that controls
light sensitivity has not yet been fully resolved. In the present
study using high-light-tolerant and -sensitive Symbiodinium
phylotypes, we examined what determines sensitivity to highlight. In growth experiments under different light intensities,
Symbiodinium CS-164 (clade B1) and CCMP2459 (clade B2)
were identified as high-light-tolerant and -sensitive phylotypes, respectively. Measurements of the maximum quantum
yield of photosystem II (PSII) and the maximum photo-

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Environmental Biology

DIVISION OF SEASONAL BIOLOGY (ADJUNCT)

Adjunct Professor

YOSHIMURA, Takashi

Specially Appointed Assistant Professor:
SHINOMIYA, Ai
SHIMMURA, Tsuyoshi
Visiting Graduate Student: ADACHI, Daisuke
SHIMO, Takayuki
NAKATSUKASA, Mana
SENGA, Takumi
Technical Assistant:
BABA, Nayumi
AKAMA, Akiko
Secretary:
OKUBO, Masayo
Animals living outside the tropics adapt various physiology and behavior to seasonal changes in the environment.
For example, animals restrict breeding to specific seasons
to maximize survival of their offspring in temperate zones.
As animals use changes in day length and temperature asseasonal cues, these phenomena are referred to as photoperiodism and thermoperiodism, respectively. We use comparative approaches to understand these mechanisms. Japanesequail and medaka provide excellent models to study thesemechanisms because of their rapid and robust seasonal
responses. In this division, we are trying to uncover theunderlying mechanisms of seasonal adaptation.

I. Mechanism of seasonal testicular regression

Birds have evolved highly sophisticated mechanisms of
seasonal regulation, and their testicular mass can change ahundred-fold within a few weeks. Recent studies revealed
that seasonal gonadal development is regulated by central
thyroid hormone (TH) activation within the hypothalamus
depending on the photoperiodic changes. By contrast, themechanisms underlying seasonal testicular regression remain
unclear. We examined the effects of short day and low temperature on testicular regression in quail. Low temperature
stimulus accelerated short day (SD)-induced testicularregression by shutting-down the hypothalamus-pituitarygonadal (HPG) axis and inducing meiotic arrest and germ
cell apoptosis. Induction of triiodothyronine (T3) coincidedwith the climax of testicular regression. Temporal geneexpression analysis over the course of apoptosis revealed
suppression of LH response genes and activation of T 3
response genes involved in amphibian metamorphosis within
the testis. Daily i.p. administration of T3 mimicked the effectsof low temperature stimulus on germ-cell apoptosis and testicular mass. We concluded that birds utilize low temperature-induced circulating T3 not only for adaptive thermoregulation but also to trigger apoptosis in order to accelerateseasonal testicular regression.

II. O ntogeny of the saccus vasculosus, aseasonal sensor in fish

Thyroid-stimulating hormone (thyrotropin: TSH) secreted
from the pars distalis (PD) of the pituitary gland stimulates
the thyroid gland. In contrast, TSH secreted from the pars
tuberalis (PT) of the pituitary gland regulates seasonal reproduction in birds and mammals. The ontogeny of thyrotrophs
and the regulatory mechanisms of TSH are markedly different between the PD and PT. Interestingly, fish do not havean anatomically distinct PT, and the saccus vasculosus (SV)of fish is suggested to act as a seasonal sensor. Thus, it ispossible that the SV is analogous to the PT. We examined theontogeny of the pituitary gland and SV using rainbow trout.
A histological analysis demonstrated the development of thepituitary anlage followed by that of the SV. Transcription
factors Lhx3 and Pit-1, which are required for the development of PD thyrotrophs, clearly labelled the pituitary anlage.
The common glycoprotein α subunit (CGA) and TSH β
subunit (TSHB) genes were also detected in the pituitaryanlage. In contrast, none of these genes were detected in the
SV anlage. We then performed a microarray analysis andidentified Parvalbumin (Pvalb) as a marker for SV development. Because Pvalb expression was not detected in thepituitary anlage, no relationship was observed between thedevelopment of the SV and pituitary gland. In contrast toembryos, Lhx3, Pit-1, CGA, and TSHB were all expressedin the adult SV. These results suggest that the morphological differentiation of SV occurs during the embryonic stage,
but that the functional differentiation into a seasonal sensor
occurs in a later developmental stage.

III. Forward genetic analysis of seasonal timemeasurement

Figure 1. Mechanisms regulating seasonal testicular development andregression in birds.

64

It is well established that the circadian clock is somehow
involved in seasonal time measurement. However, it remains
unknown how the circadian clock measures day length.
Additionally, it is not known how animals adapt to seasonalchanges in temperature. It has been reported that medaka
populations that were caught at higher latitudes have moresophisticated responses to day length and temperature. For
example, medaka fish caught in Hokkaido have a critical

day length (i.e., duration of photoperiod required to cause
a response) of 13 h, while those caught in Okinawa havean 11.5 h critical day length. To uncover the underlying
mechanism of seasonal time measurement, we are currently
performing a forward genetic analysis in medaka populations
collected from various latitudes all over Japan.

3-1 Variation in seasonal responses with latitude in
medaka fish

To perform a forward genetic analysis, we have collected
thousands of medaka fish from all over Japan. We haveexamined the effects of changing day length to determine
the critical day lengths that will cause seasonal responses
in the gonad and we found differences in critical day length
between medaka from higher latitudes and lower latitudes(Figure 2).

3-2 Quantitative trait loci (QTL) analysis of criticalday length

To identify the genes regulating critical day length, quantitative trait loci (QTL) analysis was conducted using morethan 700 F2 medaka derived from crosses between Northernand Southern populations. As a result, we identified significant QTLs. We are now performing genome re-sequencing
using various medaka strains that show different criticalphotoperiods.

IV. Transcriptome analysis of seasonality inmedaka fish
Homeotherms such as birds and mammals do not show
clear seasonal responses to changing temperature. In
contrast, poikilothermal animals also use changing temperature as a calendar. Medaka provides an excellent model touncover this mechanism. To elucidate the signal transduction

pathway regulating seasonal reproduction in medaka fish, wehave examined transcriptome analysis using microarrays. We
identified hundreds of genes that respond to day length andtemperature changes.

Publication List:〔Original papers〕

K., Atsumi, Y., Yorinaga, E., Ono, H., Murayama, I., Nakane,• Ikegami,Y., Ota, W., Arai, N., Tega, A., Iigo, M., Darras, V.M., Tsutsui, K.,•••

Hayashi, Y., Yoshida, S., and Yoshimura, T. (2015). Low temperatureinduced circulating triiodothyronine accelerates seasonal testicularregression. Endocrinology 156, 647-659.
Maeda, R., Shimo, T., Nakane, Y., Nakao, N., and Yoshimura, T.
(2015). Ontogeny of the saccus vasculosus, a seasonal sensor in fish.
Endocrinology 156, 4238-4243.
Oshima, T., Yamanaka, I., Kumar, A., Yamaguchi, J., NishiwakiOhkawa, T., Muto, K., Kawamura, R., Hirota, T., Yagita, K., Irle,
S., Kay, S.A., Yoshimura, T., and Itami, K. (2015). C-H activation
generates period shortening molecules targeting cryptochrome in the
mammalian circadian clock. Angew. Chem. Int. Ed. 54, 7193-7197.
Shimmura, T., Ohashi, S., and Yoshimura, T. (2015). The highestranking rooster has priority to announce the break of dawn. Sci. Rep.
5, 11683.

〔Review article〕

T.J., Visser, M.E., Arnold, W., Barrett, P., Biello, S., Dawson,• Stevenson,
A., Denlinger, D.L., Dominoni, D., Ebling, F.J., Elton, S., Evans, N.,

Ferguson, H.M., Foster, R.G., Hau, M., Haydon, D.T., Hazlerigg, D.G.,
Heideman, P., Hopcraft, J.G.C., Jonsson, N.N., Kronfeld-Schor, N.,
Kumar, V., Lincoln, G.A., MacLeod, R., Martin, S.A.M., MartinezBakker, M., Nelson, R.J., Reed, T., Robinson, J.E., Rock, D., Schwartz,
W.J., Steffan-Dewenter, I., Tauber, E., Thackeray, S.J., Umstatter,C., Yoshimura, T., and Helm, B. (2015). Disrupted seasonal biology
impacts health, food security, and ecosystems. Proc. R. Soc. B. 282,
1817.

Figure 2. Different critical day length between medaka from higher latitudes and lower latitudes.

65

National Institute for Basic Biology

Theoretical Biology

LABORATORY OF GENOME INFORMATICS

Assistant Professor

UCHIYAMA, Ikuo

Postdoctoral Fellow:

CHIBA, Hirokazu

The accumulation of biological data has recently been
accelerated by various high-throughput “omics” technologies including genomics, transcriptomics, and proteomics.
The field of genome informatics is aimed at utilizing thisdata, or finding the principles behind the data, for understanding complex living systems by integrating the datawith current biological knowledge using various computational techniques. In this laboratory we focus on developing
computational methods and tools for comparative genome
analysis, which is a useful approach for finding functional orevolutionary clues to interpreting the genomic informationof various species. The current focus of our research is on
the comparative analysis of microbial genomes, the numberof which has been increasing rapidly. Since different typesof information about biological functions and evolutionary
processes can be extracted from comparisons of genomes atdifferent evolutionary distances, we are developing methods
to conduct comparative analyses not only of distantly related
but also of closely related genomes.

I. Microbial genome database for comparative
analysis

The microbial genome database for comparative analysis(MBGD; URL http://mbgd.genome.ad.jp/) is a comprehensive platform for microbial comparative genomics. The
central function of MBGD is to create orthologous groups
among multiple genomes using the DomClust program
combined with the DomRefine program (see Section II
below). By means of these programs, MBGD not only
provides comprehensive orthologous groups among thelatest genomic data, but also allows users to create their own
ortholog groups on the fly using a specified set of organisms. MBGD also has pre-calculated ortholog tables foreach major taxonomic group, and provides several views to
display the entire picture of each ortholog table. For someclosely related taxa, MBGD provides the conserved synteny
information calculated using the CoreAligner program (seeSection III below). In addition, MBGD provides MyMBGD
mode, which allows users to add their own genomes to
MBGD. Moreover, MBGD now stores recently accumulating draft genome data, and allows users to incorporate theminto a user specific ortholog database through the MyMBGD
functionality.
To cope with the explosive growth of microbial genome
data owing to next generation sequencing technology, weneed to improve the database construction procedure continuously. Since recently tens or even hundreds of genome
sequences of the same species are available for many
bacteria, we are trying to establish an efficient protocol tomaintain all-against-all similarity data by separating intraspecies comparisons and inter-species comparisons.

66

II. O rthologous gene classification among
multiple genomes at the domain level

As a core technology of our comparative genomics tools,
we developed a rapid automated method of ortholog
grouping, named DomClust, which is effective enough toallow the comparison of many genomes simultaneously. The
method classifies genes based on a hierarchical clustering
algorithm using all-against-all similarity data as an input,
but it also detects domain fusion or fission events and splits
clusters into domains if required. As a result, the procedure
can split genes into the domains minimally required forortholog grouping.
Although DomClust can rapidly construct orthologous
groups at the domain level, its classification quality has room
for improvement since it is based only on pairwise sequencealignment. We developed a procedure to refine the DomClustclassification based on multiple sequence alignments. The
method is based on a scoring scheme, the domain-specificsum-of-pairs (DSP) score, which evaluates domain-levelclassification using the sum total of domain-level alignmentscores. We developed a refinement pipeline to improvedomain-level clustering, DomRefine, by optimizing the DSP
score. DomRefine is now used to construct the standard
ortholog table covering all the representative genomes storedin MBGD.
Domain-level classification is a unique feature of ourortholog classification system in MBGD. Now, we are analyzing the database and trying to characterize domain fusion
events that occurred during prokaryotic and eukaryotic evolution.

III. I dentification of the core structure
conserved among taxonomically related
microbial genomes

Horizontal gene transfers (HGT) have played a significant
role in prokaryotic genome evolution, and the genes constituting a prokaryotic genome appear to be divided into
two classes: core and accessory. The core gene set comprises intrinsic genes encoding the proteins of basic cellularfunctions, whereas the accessory gene set comprises HGTacquired genes encoding proteins which function under particular conditions. We consider the core structure of related
genomes as a set of sufficiently long segments in which geneorders are conserved among multiple genomes so that they
are likely to have been inherited mainly through verticaltransfer, and developed a method named CoreAligner to findsuch structures. We systematically applied the method tovarious bacterial taxa to define their core gene sets.

IV. D e v e l o p m e n t o f a w o r k b e n c h f o rcomparative genomics
We have developed a comparative genomics workbench
named RECOG (Research Environment for COmparative
Genomics), which aims to extend the current MBGD system
by incorporating more advanced analysis functionalities.
The central function of RECOG is to display and manipulatea large-scale ortholog table. The ortholog table viewer is aspreadsheet like viewer that can display the entire ortholog
table, containing more than a thousand genomes. In RECOG,

several basic operations on the ortholog table are defined,
which are classified into categories such as filtering, sorting,and coloring, and various comparative analyses can bedone by combining these basic operations. In addition,
RECOG allows the user to input arbitrary gene properties
and compare these properties among orthologs in various
genomes.
We continue to develop the system and apply it to various
genome comparison studies under collaborative research
projects. In particular, we are trying to apply the RECOG
system to comparative analyses of transcriptomic data andeven metagenomic data.

V. O rtholog data representation using the
Semantic Web technology to integratevarious microbial databases

Orthology is a key to integrate knowledge about variousorganisms through comparative analysis. We have constructed an ortholog database using Semantic Web technology, aiming at the integration of numerous genomic data andvarious types of biological information. To formalize thestructure of the ortholog information in the Semantic Web,
we have constructed the Ortholog Ontology (OrthO). On thebasis of OrthO, we described the ortholog information from
MBGD in the form of Resource Description Framework(RDF) and made it available through the SPARQL endpoint.
On the basis of this framework, we are trying to integratevarious kinds of microbial data using the ortholog information as a hub, as part of the MicrobeDB.jp project developed
under the National Bioscience Database Center.
In addition, to facilitate the utilization of the RDF databases
distributed worldwide, we developed a command-line tool,
named SPANG, that simplifies querying distributed RDF
stores using the SPARQL query language.

VI. Characterization of the gene repertoire of
H. pylori pan-genome

Genomes of bacterial species can show great variation in
their gene content, and thus systematic analysis of the entire
gene repertoire, termed the “pan-genome”, is important forunderstanding bacterial intra-species diversity. We analyzed
the pan-genome identified among 30 strains of the humangastric pathogen Helicobacter pylori isolated from various
phylogeographical groups. For this purpose, we developed
a method (FindMobile) to define mobility of genes against
the reference coordinate determined by the core alignment
created by CoreAligner, and classified each non-core gene
into mobility classes. We also identified co-occurring geneclusters using phylogenetic pattern clustering combined withneighboring gene clustering implemented in the RECOG
system (Figure 1). On the basis of these analyses, we identified several gene clusters conserved among H. pylori strainsthat were characterized as mobile or non-mobile. This work
is in collaboration with Prof. Kobayashi, Univ. Tokyo.

Figure 1. The five largest co-occurring gene clusters identified among 30
strains of H. pylori using the RECOG system

Publication List:〔Original papers〕

R., Toyota, K., Miyakawa, H., Watanabe, H., Oka, T., Miyagawa,• Abe,
S., Nishide, H., Uchiyama, I., Tollefsen, K.E., Iguchi, T., and

•
•
•
•
•
•

Tatarazako, N. (2015). Diofenolan induces male offspring production
through binding to the juvenile hormone receptor in Daphnia magna.
Aquat. Toxicol. 159, 44-61.
Chiba, H., Nishide, H., and Uchiyama, I. (2015). Construction of an
ortholog database using the Semantic Web technology for integrative
analysis of genomic data. PLoS One 10, e0122802.
Fernández-Breis, J.T., Legaz-García, M.C., Chiba, H., and Uchiyama,
I. (2015). Towards the semantic standardization of orthology content.
Proc. Semant. Web Appl. Tool. Life Sci. 74-83.
Hayashi, S., Kawaguchi, A., Uchiyama, I., Kawasumi-Kita, A.,
Kobayashi, T., Nishide, H., Tsutsumi, R., Tsuru, K., Inoue, T., Ogino,
H., Agata, K., Tamura, K., and Yokoyama, H. (2015). Epigenetic
modification maintains intrinsic limb-cell identity in Xenopus limb bud
regeneration. Dev. Biol. 406, 271-282.
Kawai, M., Uchiyama, I., Takami, H., and Inagaki, F. (2015). Low
frequency of endospore-specific genes in subseafloor sedimentary
metagenomes. Environ. Microbiol. Rep. 7, 341-350.
Takami, H., Arai, W., Takemoto, K., Uchiyama, I., and Taniguchi,
T. (2015). Functional classification of uncultured “Candidatus
Caldiarchaeum subterraneum” using the Maple system. PLoS One 10,
e0132994.
Uchiyama, I., Mihara, M., Nishide, H., and Chiba, H. (2015). MBGD
update 2015: microbial genome database for flexible ortholog analysis
utilizing a diverse set of genomic data. Nucleic Acids Res. 43,
D270-D276.

67

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

NIBB Research Fellow:
Technical Assistant:

TANIGUCHI, Atsushi
ISHIBASHI, Tomoko

deterioration of contrast. A solution to these problems is
combining Light-sheet microscopy with two-photon excitation (TPE), but this results in a narrow field of view, because
generation of TPE images requires very high photon density,i.e. focusing with a high numerical aperture (NA) lens.
We utilized a new fiber laser with high peak power toovercome this problem, and enabled observation of larger
specimens using a hybrid TPE light-sheet microscope.

Our laboratory is currently pursuing two paths of scientificinquiry that are of interest to developmental biology. One
goal is to clarify the mechanism of left-right (L-R) determination. The other is to develop imaging technologies forbiological analyses.

I. Initial step for left-right asymmetry

In mammalian development, the initial left-right asymmetry
first appears as cilia-driven leftward fluid flow on the ventral
surface of the node, and the flow provides the initial cue forasymmetric gene expressions that actually determine asymmetric morphogenesis. The mechanisms that convert the flow
to asymmetric gene expression, i.e. the flow sensing mechanism, remains controversial, with several models being
proposed, and involvement of Ca2+ being suggested.
We pursued this question by measuring Ca2+ dynamics inthe node and found that the node cells cause apparently stochastic elevation of Ca2+, and its spatiotemporal distribution
is equal at the left and right sides but becomes more frequentat the left after late headfold stage, when the flow-induced
commitment occurs. This asymmetry was disrupted in dynein
mutant iv/iv and pkd2-/- mutants, in accordance to their leftright phenotypes.

Figure 2. Two-photon light-sheet microscopy. Left: Fluorescent images ofa medaka taken by a conventional (one-photon) light-sheet microscope.
Right: Images of the same area taken by our two-photon light-sheet
microscope.

Publication List:〔Original paper〕

Y., Ozeki, M., Hiraiwa, T., Tanimoto, R., Funahashi, A., Hiroi,• Nakai,N., Taniguchi, A., Nonaka, S., Boilot, V., Shrestha, R., Clark, J., Tamura,N., Draviam, V.M., and Oku, H. (2015). High-speed microscopy with
an electrically tunable lens to image the dynamics of in vivo molecularcomplexes. Rev. Sci. Instrum. 86, 013707.

Figure 1. Left: Distribution of Ca2+ elevation in a 2-somite wild-type
node. Right: Time course of Ca2+ elevation frequency at the left and theright sides.

Based on these findings, we are now examining the validityof the two major proposed models of flow sensing, mechanosensing and chemosensing, and a third novel mechanism.

II. Development of light-sheet microscopy

Light-sheet microscopy has many advantages for live
imaging including low photobleaching and phototoxicity, high penetration depth, and fast image acquisition.
This method also has peculiar disadvantages, however.
Specifically scattering of excitation light within the specimen
leading to illumination of areas besides the focal plane, and68

Okazaki Institute for Integrative Bioscience

Orion Project

LABORATORY OF NUCLEAR DYNAMICS

Specially Appointed Associate Professor

MIYANARI, Yusuke

Postdoctoral Fellow:
KURIHARA, Misuzu
SOKENDAI Graduate Student: ISHII, Satoko
Visiting Graduate Studen: KAKIZUKA, Taishi
Technical Assistant:
SANBO, Chiaki
TAGAWA, Ayako
Secretary:
HACHISUKA, Midori
A fundamental question in biology is to understand themechanisms underlying cell-fate decision. Genomic reprogramming after mammalian fertilization reverts terminallydifferentiated gametes into toti- or pluri-potent states to starta new developmental program. Cell lineage allocation in the
reprogramming process is accompanied by drastic changes
in the pattern of gene expression, epigenetic configurations,and nuclear organization. We aim to reveal the roles of chromatin dynamics in cell lineage-allocation by deciphering themolecular mechanisms underlying remodeling of nuclear
organization and their effects on developmental gene expression, using mouse embryos and embryonic stem (ES) cells as
model systems.

Epigenetic reprogramming in early mouseembryos.
Genomic reprogramming reverts fully differentiated cells
to a totipotent state to start a new developmental program. In
the early mouse embryo, terminally differentiated gametes
are reprogrammed after fertilization thereby acquiring a totipotent state. Upon the fourth cleavage, 8-cell stage embryos,
which have undergone the process of compaction, will giverise to the morula (Figure 1). The outer cells of the morula
will differentiate into the epithelial trophectoderm (TE) of
the blastocyst. The inner cells of the morula will become
the inner cell mass (ICM) of the blastocyst. The ICM subsequently leads to the formation of two lineages, epiblast (EPI)
and primitive endoderm (PE), with the former representing
pluripotency as it gives rise to the embryo itself. The reprogramming event is accompanied by epigenetic modifications and changes in chromatin structures throughout theembryo, which are essential for regulation of gene expression
involved in differentiation, and reprogramming of the EPI.

three-dimensional nuclear space. During developmental
processes, nuclear architecture is dramatically reconstructed,
resulting in establishment of cell-type specific nuclear organization. Defects in structural components of the nucleus are
responsible for developmental aberrations and several humandiseases. Remodeling of nuclear architecture leads to spatial
arrangement of genes, which could affect genome functions
including gene expression. However, regulatory mechanisms
underlying nuclear reorganization during cell-fate decision
remains largely unknown.

Chromatin structure

Spatiotemporal organization of genomic DNA within the
nucleus is suggested as an emerging key player to regulate
gene expression. The developmental program accompanies nuclear remodeling, resulting in construction of celltype specific nuclear architecture. Firstly, chromosomes are
confined in discrete nuclear spaces, “chromosome territories”(Figure 2). Within them, further levels of 3D organization,“topologically associating domains” (TADs), are observed.
TADs can be defined as linear units of chromatin containing several gene loci, and fold as discrete 3D structures inwhich gene loci frequently interact with each other. Recent
works have revealed that folding of “local” chromatin structures such as enhancer-promoter looping is associated withgenome functions. Despite the drastic changes of these hierarchical chromatin structures, their role in cell-fate decision
remains largely unexplored.

Figure 2. Hierarchical chromatin structure

Approach

We have developed a powerful imaging technology termed
TALE-mediated Genome Visualization (TGV), whichallows us to track specific genomic sequences in living cells(Miyanari Y, Nature Structural & Molecular Biology, 2013).
Importantly, this technique is versatile and can be extended
to allow many robust applications, which will be integrated
into our study to manipulate several genome functions.
Based on new technological development, we aim to understand biological roles of chromatin dynamics in cell-fatedecision.

Figure 1. Lineage allocation in mouse preimplantation development

Remodeling of nuclear architecture indevelopment

Chromatin is organized in a non-random fashion within
69

Okazaki Institute for Integrative Bioscience

Bio-Next Project

LABORATORY OF PLANT DEVELOPMENT AND PHYSIOLOGYSpecially Appointed Associate Professor

KAWADE, Kensuke

Adjunct Professor (BIO-NEXT Project, OIIB):
TSUKAYA, Hirokazu
Technical Assistant:
YAMAGUCHI, Chinami
Secretary:
HACHISUKA, Midori
There has been growing evidence that metabolic regulation has specific impacts on plant development. The picture
emerging depicts the metabolism as a dynamic system that
controls and/or supports developmental progression. Despite
these advances, the metabolic regulation behind developmental process remains largely unclear. We aim to uncoveras-yet-unknown relationships between developmental andmetabolic processes in plants and their biological meaningby elucidating molecular mechanisms for the system. To
address this, we primary use molecular genetics and metabolomics approaches using Arabidopsis thaliana as a model, in
conjunction with standard molecular biology and biochemistry techniques.

I. E xploring as-yet-unknown relationships
between development and metabolism

To explore as-yet-unknown relationships between developmental and metabolic processes, we carried out phenome
screening of Arabidopsis thaliana enzyme mutants. We
examined more than 11 traits including leaf size, primaryroot length, seed color, etc. A large number of mutants grewnormally compared to wild type, probably due to gene functional redundancy. However, we found that one mutant had ashorter primary root than wild type. Further detailed morphological analysis revealed that this could be attributed to adelay of germination and seedling establishment. Irregularpatterning of the cotyledon is also observed in the mutant(Figure 1) although the penetrance was low. Based on theseobservations, together with the expression profile, we now
hypothesize that the enzyme works during seed development
to control seed quality, and presumably embryo patterning.
To know the in vivo substrate of this enzyme and which metabolic pathway is involved in this developmental process,metabolomics analysis will be conducted.

Figure 1. Irregular patterning of cotyledons in the enzyme mutant. Wildtype has two symmetrically-arranged cotyledons (A). In contrast, ourenzyme mutant shows asymmetrically-arranged (B and C), fused (D-E)
and cup-shaped cotyledons (G). Asterisks indicate cotyledons. Bar = 2
mm.

70

II. C haracterizing an interaction between
development and metabolism

In addition to the phenome screening using enzyme
mutants, as another topic, we are now examining coordinatedregulation of plant growth and amino acid metabolism. We
previously found that one of key factor for plant development regulates the expression of amino acid metabolismrelated genes. Moreover, we uncovered that the mutant forthis gene shows hypersensitivity specifically to leucine.
Growth arrest is observed when this mutant is grown on
culture media containing higher concentrations of leucine.
Other amino acids such as histidine did not inhibit the
mutant’s growth, indicating that there is a specific interaction between the responsible gene and leucine metabolism.
To directly know whether the gene indeed regulates leucine
metabolism, we have established an amino acid profilingsystem with high sensitivity and accuracy using liquid chromatography-mass spectrometry (LC-MS) in collaboration
with the Functional Genomics Facility in NIBB, and are now
examining the amino acid prolife in the mutant. Although thegene is well studied as a key component for plant development, our data shed light on a novel aspect of the gene’sfunction, which could be a key to deciphering how aminoacid metabolism is interwired with vigorous plant growth.

Publication List:〔Original paper〕

H., Kawade, K., Tsukaya, H., and Kimura, S. (2015).
• Nakayama,
Detection of the cell proliferation zone in leaves by using EdU. Bioprotocol 5, 18.

〔Review articles〕

T., Kawade, K., and Tsukaya, H. (2015). Compensation:• Hisanaga,a key to clarifying the organ-level regulation of lateral organ size in•plants. J. Exp. Botany 66, 1055-1063.
Kawade, K., and Tanimoto, H. (2015). Mobility of signaling molecules:
The key to deciphering plant organogenesis. J. Plant Res. 128, 17-25.

National Institute for Basic Biology

Research Support

NIBB CORE RESEARCH FACILITIESHeadYOSHIDA, Shosei

The NIBB Core Research Facilities were launched in 2010
to support basic biology research in NIBB. They consist of
three facilities that are developing and providing state-of-theart technologies to understand biological functions throughfunctional genomics, bioimaging, and bioinformatics.
The NIBB Core Research Facilities also act as an intellectual hub to promote collaboration among the researchers of
NIBB and other academic institutions.

FUNCTIONAL GENOMICS FACILITY

Specially Appointed Associate Professor:

SHIGENOBU, Shuji

Technical Staff:

Technical Assistant:
Secretary:

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
BINO, Takahiro
ASAO, Hisayo
AKITA, Asaka
MATSUMOTO, Miwako
ICHIKAWA, Mariko

The Functional Genomics Facility is a division of the NIBBCore Research Facilities and is organized jointly by NIBB
and NIPS for promoting DNA and protein studies. The
facility maintains a wide array of core research equipment,from standard machinery like ultracentrifuges to cuttingedge tools such as next generation DNA sequencers, whichamount to 60 different kinds of instruments. The facility is
dedicated to fostering collaborations with researchers bothof NIBB and other academic institutions worldwide by providing these tools as well as expertise. Our current focus
is supporting functional genomics studies that utilize mass
spectrometers and DNA sequencers. We also act as a bridge
between experimental biology and bioinformatics.
We recently largely renovated the building of the Functional
Genomics Facility. For example, the Visitors Lab and the
Visitors Office were newly designed so that we can promotecollaboration projects. Indeed, in 2015, more than 200
researchers visited to use our new facility and developed
active collaborations.

Figure 1. Next-generation sequencer

in basic biology.
During 2015 we carried out 44 NGS projects in collaboration with NIBB laboratories as well as the researchers ofother academic institutions. These projects cover a wide
range of species (bacteria, animals, plants, and humans)
including both model and non-model organisms, and various
applications such as genomic re-sequencing, RNA-seq and
ChIP-seq.

Proteomics

Three different types of mass spectrometer and two protein
sequencers, as listed below, are used for proteome studies in ourfacility. In 2015, we analyzed approximately 620 samples withmass spectrometers and 14 samples with protein sequencers.
- LC-MS (AB SCIEX TripleTOF 5600 system)- LC-MS (Thermo Fisher SCIENTIFIC Orbtrap Elite)- MALDI-TOF-MS (Bruker Daltonics REFLEX III)- LC-Q-TOF MS (Waters Q-TOF Premier)- Protein sequencer (ABI Procise 494 HT; ABI Procise 492
cLC)

Other analytical instruments (excerpts)- Cell sorter (SONY SH800)- B ioimaging Analyzer (Fujifilm LAS 3000 mini; GE
FLA9000)- Laser Capture Microdissection System (Arcturus XT)- DNA Sequencer (ABI PRISM 310; ABI 3130xl)- Real Time PCR (ABI 7500)- Ultra Centrifuge (Beckman XL-80XP etc.)

Representative Instruments
Genomics

The advent of next-generation sequencing (NGS) technologies is transforming today’s biology by ultra-high-throughput
DNA sequencing. Utilizing HiSeq2500, HiSeq1500, andMiSeq (Illumina), and PacBio RS II (PacificBio Sciences),the Functional Genomics Facility is committed to joint
research aiming to explore otherwise inaccessible new fields

Figure 2. Triple TOF LC/MS/MS System

71

National Institute for Basic Biology

Research Support

Genome Informatics Training Course

We organize NIBB Genome Informatics Training Courses
every year. In 2014, we provided two three-day training
courses on RNA-seq data analysis. These courses aredesigned to introduce the basic knowledge and skills of bioinformatics analysis to biologists who are not familiar withbioinformatics.

••

Tatarazako, N., Miyagawa, S., and Iguchi, T. (2015). NMDA receptor
activation upstream of methyl farnesoate signaling for short dayinduced male offspring production in the water flea, Daphnia pulex.
BMC Genomics 16, 186.
Wakae, K., Aoyama, S., Wang, Z., Kitamura, K., Liu, G., Monjurul, A.M.,
Koura, M., Imayasu, M., Sakamoto, N., Nakamura, M., Shigenobu, S.,et al. (2015). Detection of hypermutated human papillomavirus type 16
genome by next-generation sequencing. Virology 485, 460–466.
Wu, T., Kamiya, T., Yumoto, H., Sotta, N., Katsushi, Y., Shigenobu,
S., Matsubayashi, Y., and Fujiwara, T. (2015). An Arabidopsis thaliana
copper-sensitive mutant suggests a role of phytosulfokine in ethylene
production. J. Exp. Bot. 66, 3657-3667.

〔Original paper (E-publication ahead of print)〕

K., Tanaka, T., Shigenobu, S., Motoi, Y., Wu, J., and Itoh, T.
• Sato,Improvement of barley genome annotations by deciphering the Haruna
Nijo genome. DNA Res. 2015 Nov 29.

● Research activity by S. Shigenobu

Figure 3. NIBB Genome Informatics Training Course

Publication List on Cooperation:

Specially Appointed Associate Professor:
SHIGENOBU, ShujiNIBB Research Fellow:
MAEDA, Taro
OGAWA, Kota
Postdoctoral Fellow:
HOJO, Masaru
OGAWA, Kota*
Technical Assistant:
SUZUKI, Miyuzu〔Original papers〕

S., Balfanz, S., Hayashi, Y., Shigenobu, S., Miura, T.,• Blankenburg,
Baumann, O., Baumann, A., and Blenau, W. (2015). Cockroach GABAB

•
•
•
•
•
•
•
•
•
72

receptor subtypes: molecular characterization, pharmacologicalproperties and tissue distribution. Neuropharmacology 88, 134–144.
Bourguignon, T., Lo, N., Cameron, S.L., Sobotník, J., Hayashi, Y.,
Shigenobu, S., Watanabe, D., Roisin, Y., Miura, T., and Evans, T.A.
(2015). The evolutionary history of termites as inferred from 66
mitochondrial genomes. Mol. Biol. Evol. 32, 406–421.
Habu, Y., Ando, T., Ito, S., Nagaki, K., Kishimoto, N., Shigenobu,
S., et al. (2015). Epigenomic modification in rice controls meiotic
recombination and segregation distortion. Mol. Breeding 35, 103.
Hojo, M.K., Ishii, K., Sakura, M., Yamaguchi, K., Shigenobu, S.,
and Ozaki, M. (2015). Antennal RNA-sequencing analysis reveals
evolutionary aspects of chemosensory proteins in the carpenter ant,
Camponotus japonicus. Sci. Rep. 5, 13541.
Kinoshita, A., ten Hove, C.A., Tabata, R., Yamada, M., Shimizu, N.,
Ishida, T., Yamaguchi, K., Shigenobu, S., Takebayashi, Y., Iuchi, S.,et al. (2015). A plant U-box protein, PUB4, regulates asymmetric cell
division and cell proliferation in the root meristem. Development 142,
444–453.
Numa, H., Yamaguchi, K., Shigenobu, S., and Habu, Y. (2015). Genebody CG and CHG methylation and suppression of centromeric CHH
methylation are mediated by DECREASE IN DNA METHYLATION1
in Rice. Mol. Plant 8, 1560-1562.
Shikanai, Y., Yamagami, M., Shigenobu, S., Yamaguchi, K., Kamiya,T., and Fujiwara, T. (2015). Arabidopsis thaliana PRL1 is involved in
low-calcium tolerance. Soil Sci. Plant Nutr. 61, 951-956.
Shimizu, N., Ishida, T., Yamada, M., Shigenobu, S., Tabata, R.,
Kinoshita, A., Yamaguchi, K., Hasebe, M., Mitsumasu, K., and Sawa,
S. (2015). BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2
constitute a signaling pathway and modulate CLE peptide-triggered
growth inhibition in Arabidopsis root. New Phytol. 208, 1104-1113.
Toyokura, K., Yamaguchi, K., Shigenobu, S., Fukaki, H., Tatematsu,
K., and Okada, K. (2015). Mutations in plastidial 5-aminolevulinic acidbiosynthesis genes suppress pleiotropic defect in shoot development of
mitochondrial GABA shunt mutant in Arabidopsis. Plant Cell Physiol.
56, 1229-1238.
Toyota, K., Miyakawa, H., Yamaguchi, K., Shigenobu, S., Ogino, Y.,

Symbiogenomics“Nothing, it seems, exists except as part of a network ofinteractions.” (Gilbert & Epel, 2008)
Every creature on the earth exists among a network ofvarious biological interactions. For example, many multicellular organisms, including humans, harbor symbiotic bacteria
in their bodies: some of them provide their hosts with essential nutrients deficient in the host’s diet and others digest
foods indigestible by the host alone. In spite of numerous
examples of symbioses and its intriguing outcomes, thegenetic and molecular basis underlying these interactions
remains elusive. The goal of our group is to establish a newinterdisciplinary science “Symbiogenomics”, where we aim
to understand the network of biological interactions at themolecular and genetic level. To this end, we take advantage
of state-of-the-art genomics such as next-generation sequencing technologies.

I. Genomic revelations of a mutualism: the pea
aphid and its obligate bacterial symbiont

Aphid species bear intracellular symbiotic bacteria in thecytoplasm of bacteriocytes, specialized cells for harboring the bacteria. The mutualism is so obligate that neither
can reproduce independently. The 464 Mb draft genome
sequence of the pea aphid, Acyrthosiphon pisum, in consort
with that of bacterial symbiont Buchnera aphidicola illustrates the remarkable interdependency between the twoorganisms. Genetic capacities of the pea aphid and thesymbiont for amino acid biosynthesis are complementary.
The genome analysis revealed that the pea aphid has undergone characteristic gene losses and duplications. The IMB

antibacterial immune pathway is missing several criticalgenes, which might account for the evolutionary success ofaphids to obtain beneficial symbionts. Lineage-specific gene
duplications have occurred in genes in a broad range of functional categories, which include signaling pathways, miRNAmachinery, chromatin modification and mitosis. The importance of these duplications for symbiosis remains to be determined. We found several instances of lateral gene transferfrom bacteria to the pea aphid genome. Some of them arehighly expressed in bacteriocytes.
We recently discovered a novel class of genes in the pea
aphid genome that encode small cysteine-rich proteins withsecretion signals that are expressed exclusively in bacteriocytes of the pea aphid, and named these bacteriocyte-specificcysteine-rich proteins (BCR). The BCR mRNAs are firstexpressed at a developmental time point coincident with theincorporation of symbionts strictly in the cells that contributeto the bacteriocyte, and this bacteriocyte-specific expression is maintained throughout the aphid’s life. Some BCRs
showed an antibiotic activity. These results suggest that
BCRs act within bacteriocytes to mediate the symbiosis withbacterial symbionts, which is reminiscent of the cysteinerich secreted proteins of leguminous plants that also regulate
endosymbionts. Employment of small cysteine-rich peptides
may be a common tactic of host eukaryotes to manipulate
bacterial symbionts.

SPECTROGRAPHY AND BIOIMAGING FACILITY

Specially Appointed Associate Professor:

KAMEI, Yasuhiro

Technical Staff:
Technical Assistant:

KONDO, Maki
TANIGUCHI-SAIDA, Misako
UCHIKAWA, Tamaki
ICHIKAWA, Chiaki
ISHIKAWA, Azusa

The Spectrography and Bioimaging Facility assists bothcollaborative and core research by managing and maintaining research tools that use “Light”. The facility also
provides technical support through management of technicalstaff assisting in the advancement of collaborative and coreresearch projects, as well as academic support to researchers
by Dr. Y. Kamei (refer to the Collaborative Research Group
Research Enhancement Strategy Office section). Among its
tools are advanced microscopes for biology and the Okazaki
Large Spectrograph for photobiology. The Okazaki Large
Spectrograph is the world’s largest wide spectrum exposure
mechanism, capable of producing a range of wavelengths
from 250 nm (ultraviolet) to 1,000 nm (infrared) along its 10
meter focal curve; allowing exposure to strong monochromatic light. The facility’s microscopes, which are cuttingedge devices such as confocal and multi-photon excitation
microscopes, are used by both internal and external researchers as vital equipment for core and collaborative research
projects.

Representative Instruments:
Okazaki Large Spectrograph (OLS)

Figure 1. Pea aphids and the bacterial symbiont, Buchnera. Adult aphids(Left). A developing viviparous embryo which symbionts are infecting(Right). Scale bar = 20um.

Publication List:〔Original papers〕

S., Balfanz, S., Hayashi, Y., Shigenobu, S., Miura, T.,• Blankenburg,
Baumann, O., Baumann, A., and Blenau, W. (2015). Cockroach GABAB••

receptor subtypes: molecular characterization, pharmacologicalproperties and tissue distribution. Neuropharmacol. 88, 134–144.
Bourguignon, T., Lo, N., Cameron, S.L., Sobotník, J., Hayashi, Y.,
Shigenobu, S., Watanabe, D., Roisin, Y., Miura, T., and Evans, T.A.
(2015). The evolutionary history of termites as inferred from 66
mitochondrial genomes. Mol. Biol. Evol. 32, 406–421.
Hojo, M.K., Ishii, K., Sakura, M., Yamaguchi, K., Shigenobu, S.,
and Ozaki, M. (2015). Antennal RNA-sequencing analysis reveals
evolutionary aspects of chemosensory proteins in the carpenter ant,
Camponotus japonicus. Sci. Rep. 5, 13541.

The spectrograph runs on a 30 kW Xenon arc lamp andprojects a wavelength spectrum from 250 nm (ultraviolet) to
1,000 nm (infrared) onto its 10 m focal curve with an intensity of monochromatic light at each wavelength more than
twice as much as that of the corresponding monochromaticcomponent of tropical sunlight at noon (Watanabe et al.,Photochem. Photobiol. 36, 491-498, 1982). The spectrographis dedicated to action spectroscopical studies of various lightcontrolled biological processes.
The NIBB Collaborative Research Program for the Use of
the OLS supports about 10 projects every year conducted by

Figure 1. An example of experiments using the Large Spectrograph.
Various color rays (monochromatic light from right side and reflected bymirrors) were irradiated simultaneously to samples in cooling chambers.

73

National Institute for Basic Biology

Research Support

both visiting scientists, including foreign researchers, as wellas those in NIBB.
Action spectroscopical studies for various regulatory anddamaging effects of light on living organisms, biologicalmolecules, and artificial organic molecules have been conducted.

Microscopes

This facility also has Bioimaging machines such as widefield microscopes (Olympus IX-81 and BX-63), confocal
microscopes (Olympus FV1000, Nikon A1R, Nikon A1Rsi
and Yokogawa CSU-X1 with EM-CCD camera), multiphoton microscopes (Olympus FV1000-MP, FV1200-MPs,
Leica TCS-SP8 MPs) and other advanced custom-madelaser microscopes with special aims (Digital Scanned Lightsheet Microscope: DSLM and Infrared Laser-Evoked Gene
Operator microscope: IR-LEGO) for users in NIBB andcollaborative guest researchers. We began Collaborative
Research Programs using these machines in 2010. In
addition, transmission electron microscope service for plantbiology has started from 2014.
The DSLM was developed by Dr. Ernst Stelzer’s group at
the European Molecular Biology Laboratory (EMBL). This
microscope can realize high-speed z-axis scanning in deeper
tissue by illuminating a specimen from the side with a lightsheet (more information is given in Dr. Nonaka’s section:
Lab. for Spatiotemporal Regulations). Dr. Shigenori Nonaka
conducted and supported 8 projects of the Collaborative
Research Program for the Use of the DSLM. The IR-LEGO
was developed by Drs. Shunsuke Yuba and Yasuhiro Kamei
at the National Institute of Advanced Industrial Science andTechnology (AIST). This microscope can induce a target
gene of interest by heating a single target cell in vivo with ahigh efficiency irradiating infrared laser (Kamei et al. Nat.
Methods, 2009). Details are described in the next section.
The IR-LEGO was also used for 10 Individual Collaborative
Research projects, including applications for animals andplants.

Workshop and Symposium

In 2015 we held courses on both medaka basic training,which focused on basic techniques for medaka research
including imaging, and the 3nd biological image processing
training course. We also have been holding a “Bioimaging
Forum” every year which discusses Bioimaging fromvarious directions such as microscopy, new photo-technology, and computer science. This year we held the 9th NIBB
Bioimaging Forum focused on physical properties on biology
including heat (temperature) and force. In addition, we held
symposiums focused on new emerging model animals andamphibians.

regeneration study. Dev. Growth Differ. 57, 601-613.

K., Fujikawa, A., Suzuki, R., and Noda, M. (2015).
• Kuboyama,
Inactivation of protein tyrosine phosphatase receptor type Z•••

by pleiotrophin promotes remyelination through activation ofdifferentiation of oligodendrocyte precursor cells. J Neurosci. 35,
12162-12171.
Nakashima, M., Suzuki, M., Saida, M., Kamei, Y., Hossain, M.B.,
and Tokumoto, T. (2015). Cell-based assay of nongenomic actionsof progestins revealed inhibitory G protein coupling to membraneprogestin receptor α (mPRα). Steroids 100, 21–26.
Oikawa, K., Matsunaga, S., Shoji Mano, S., Kondo, M., Yamada,K., Hayashi, M., Kagawa, T., Kadota, A., Sakamoto, W., Higashi, S.,
Watanabe, M., Mitsui, T., Shigemasa, A., Iino, T., Hosokawa, Y., andNishimura, M. (2015). Physical interaction between peroxisomes andchloroplasts elucidated by in situ laser analysis. Nat. Plants 1, 15035.
Takeda, N., Handa, Y., Tsuzuki, S., Kojima, M., Sakakibara, H., andKawaguchi, M. (2015). Gibberellins interfere with symbiosis signaling
and gene expression, and alter colonization by arbuscular mycorrhizal
fungi in Lotus japonicus. Plant Physiol. 167, 545-557.

〔Original paper (E-publication ahead of print)〕N., Honda, A., Zenno, A., Omoto, R., Imanaka, S., Takehana,• Kagawa,
Y., and Naruse, K. Arginine vasotocin neuronal development and its

projection in the adult brain of the medaka. Neurosci. Let. 2015 Dec.
29.

● Research activity by Y. Kamei
Specially Appointed Associate Professor:
KAMEI, Yasuhiro
NIBB Research Fellow:
HATTORI, Masayuki
Technical Assistant:
CHISADA, Eriko
Our research group promotes two cutting-edge microscope
projects; “observation” and “manipulation” using optical andbiological technologies. The aim of our “observation project”
is deep-seeing in living organisms using adaptive optics(AO) which were well-developed in the field of astronomy
as a key technology of large telescopes such as the Subaru
telescope in Hawaii. Although observation using telescopeson the earth may be disturbed by fluctuations in the atmosphere, AO technology can cancel this disturbance. On
the other hand, living materials have particular refractive
indexes, therefore, some organelles act as disturbances of
the ideal optical path for microscope observation just likethe atmosphere does for telescopes. AO technology can also
compensate for this disturbance by sensing and correcting wave fronts using a wave front sensor and deformablemirror. Hence, we developed a custom-made wide-fieldmicroscope equipped with an AO system for observation ofliving organisms in collaboration with Dr. Tamada in NIBB
and Dr. Hayano in the National Astronomical Observatoryof Japan (NAOJ) and got high-resolution bright field andPublication List on Cooperation〔Original papers (Selected)〕A., Hayashi, T., Kobayashi, T., Nagayama, C.,• Kawasumi-Kita,
Hayashi, S., Kamei, Y., Morishita, Y., Takeuchi, T., Tamura, K., andYokoyama, H. (2015). Application of local gene induction by infraredlaser-mediated microscope and temperature stimulator to amphibian74

Figure 1. Effects of adaptive optics (AO) to wide-field microscope
images (bright field and fluorescence of plant cells).

fluorescent images of living cells. Our results indicated that
improvement of optical resolution was restricted to a small
area which is called the “isoplanatic patch” (Figure 1).
Second, the aim of our “manipulation project” is to control
gene expression in vivo. Gene function analysis must be
evaluated at the cell level in vivo. To achieve spatiotemporalcontrolled gene expression we employed one of the stressresponses, the heat shock response. The heat shock promoter
is the transcription regulation region of heat shock proteins
and all organisms have this mechanism. Positioning thetarget gene downstream of the promoter, we can induce thetarget gene expression by local heating.
Infrared (IR) beams can heat water molecules, which are
the main constituent of cells, hence, we can heat a single
cell by irradiating IR to a target cell using a microscope.
We have developed a microscope, IR laser evoked gene
operator (IR-LEGO), specialized for this purpose (Figure2). The IR-LEGO microscope can irradiate an IR laser to asingle cell in vivo such as in C. elegans, Drosophila, medaka,
zebrafish, Xenopus and Arabidopsis, to induce the heatshock response at a desired timing. In 2015, additionally, we
confirmed the system was effective in the moss Marchantia
polymorpha and in the newt Pleurodeles waltl.
Optimal heating induces the heat shock response and subsequent gene expression, while an excess results in cell death.
Hence, we must precisely control laser heating. We evaluated time course and spatial heating profiles, and the resultspresented that temperature of the target area rose rapidly andkept a constant level dependant on IR laser power, additionally, the heated area was adequately as small as a typical cellsize.
With this in mind, we tried to induce gene expression invarious species. At first, we reported an IR-LEGO experiment in living C. elegans. Target gene expression in a target
cell could be induced with only 1 s-IR irradiation. Whereas
the optimal power range which can induce gene induction
without cell damage was limited. Excess laser power resulted
in cell death or cessation of cell division. We confirmed thatan optimal irradiation, e.g. 11 mW for 1 s, induced physiological gene expression in the target cell and subsequentcell division or morphogenesis underwent normal develop-

ment. Next, we tried the experiment in other animals, suchas, medaka, zebrafish and Xenopus, and the higher plant,
Arabidopsis, since all organisms have a heat shock response
system. We succeeded in local gene induction in all the
species as expected.
Studies of cell fates, cell-cell interaction, or analysis of
non-cell autonomous phenomena require a fine control
system of gene expression in experiments. IR-LEGO will
be a powerful tool for these studies in combination withmolecular biological techniques, such as the cre-loxP system.
Dr. Shimada in the University of Tokyo wanted to confirm
the cell lineage of exo-skeletal tissue such as the scales of
medaka fish. She questioned the traditional belief concerning the origin of the exo-skeleton of the body-trunk usingtransplantation studies. We then started a collaboration toestablish a local permanent labeling system in medaka and to
make clear the origin of exo-skeletal cells. The system waswell working (Figure 3), and the fate tracking results indicated that exo-skeletal tissues were mesodermal in origin, notfrom neural crest cells, as previously believed (Shimada et al,Nat. Commun, 2013).

Figure 3. Examples of Cre-loxP mediated long-term GFP marking using
IR-LEGO in living medaka individuals for cell linage tracing.

Publication List:〔Original papers〕

A., Hayashi, T., Kobayashi, T., Nagayama, C.,• Kawasumi-Kita,
Hayashi, S., Kamei, Y., Morishita, Y., Takeuchi, T., Tamura, K., and••Yokoyama, H. (2015). Application of local gene induction by infraredlaser-mediated microscope and temperature stimulator to amphibian
regeneration study. Dev. Growth Differ. 57, 601-613.
Nakashima, M., Suzuki, M., Saida, M., Kamei, Y., Hossain, B., andTokumoto, T. (2015). Cell-based assay of nongenomic actions ofprogestins revealed inhibitory G protein coupling to membraneprogestin receptor α (mPRα). Steroids 100, 21-26.
Yokoi, S., Okuyama, T., Kamei, Y., Naruse, K., Taniguchi, Y., Ansai,S., Kinoshita, M., Young, L. J., Takemori, N., Kubo, T., and Takeuchi,
H. (2015). An essential role of the arginine vasotocin system in mateguarding behaviors in triadic relationships of medaka fish (Oryzias
latipes). PLoS Genetics 11, e1005009.

〔Original paper (E-publication ahead of print)〕

R., Ishida, S., Urawa, H., Kamei, Y., and Kohchi,• Nishihama,T. Conditional gene expression/deletion systems for Marchantia

polymorpha using its own heat-shock promoter and the cre/loxPmediated site-specific recombination. Plant Cell Physiol. 2015 Jul. 6.

Figure 2. Schematic illustration of local gene induction system and an
infrared laser-evoked gene operator (IR-LEGO) microscope system in
NIBB.

75

National Institute for Basic Biology

Research Support

Data Integration and Analysis Facility
Assistant Professor:
Technical Staff:
Technical Assistant:

UCHIYAMA, Ikuo
MIWA, Tomoki
NISHIDE, Hiroyo
NAKAMURA, Takanori
OKA, Naomi

The Data Integration and Analysis Facility supports
research activities based on large-scale biological data
analysis, such as genomic sequence analysis, expression
data analysis, and imaging data analysis. For this purpose,the facility maintains high-performance computers withlarge-capacity storage systems. On the basis of this system,
the facility supports development of data analysis pipelines,
database construction and setting up websites to distributethe data worldwide as well as providing users’ basic technical support. In addition to computational analysis, the Data
Integration and Analysis Facility supports NIBB’s information infrastructure, the maintenance of the network systems
in the institute and computer/network consultation for institute members.

Representative Instruments

Our main computer system is the Biological Information
Analysis System (BIAS) (Figure 1), which consists of ahigh-performance cluster system (SGI Rackable server
C2112-4RP; 40 nodes/800 cores, 96GB memory/node), a
shared memory parallel computer (HP ProLiant DL980 G7;
80 cores, 4TB memory), a high-throughput storage system(DDN SFA7700; 480TB), and a large capacity storage
system (DELL PowerEdge R620; 720TB). All subsystems
are connected via a high-speed InfiniBand network so thatlarge amounts of data can be processed efficiently. Somepersonal computers and color/monochrome printers are alsoavailable. On this system, we provide various biological
databases and data retrieval/analysis programs, and support
large-scale data analysis and database construction for institute members and collaborative researchers. Especially, we
have supported the construction and maintenance of published databases of various model organisms including XDB(Xenopus laevis), PHYSCObase (Physcomitrella patens),
DaphniaBASE (Daphnia magna), The Plant Organelles
Database, and MBGD (microbial genomes).
The facility also provides network communication
services. Most of the PCs in each laboratory, as well as allof the above-mentioned service machines, are connectedby a local area network, which is linked to the high performance backbone network ORION connecting the threeresearch institutes in Okazaki. Many local services, including sequence analysis services, file sharing services, andprinter services, are provided through this network. We also
maintain a public World Wide Web server that hosts the
NIBB home page (http://www.nibb.ac.jp/en).

76

Figure 1. Biological Information Analysis System

Research activity by I. Uchiyama

Assistant professor I. Uchiyama is the principal investigatorof the Laboratory of Genome Informatics, which currently
focuses on microbial comparative genomics studies. For
details, please refer to the laboratory page (p. 66).

NIBB BIORESOURCE CENTERHead

FUJIMORI, Toshihiko

To understand the mechanisms of living organisms, studies
focusing on each gene in the design of life (the genome)
are required. The use of model animals and plants, such as
mice, Medaka (Oryzias latipes), zebraﬁsh, Arabidopsis,
Lotus japonicus, and Physcomitrella patens, make it possible
to produce genetically controlled organisms with markers
placed using genetic and cell engineering technology. Such
marking allows detailed studies of genes and cell functions.
The model organisms mature in a short period of time; therefore, changes in cells, organs, and individuals can be totallyand efﬁciently observed. The NIBB BioResource Center has
equipment, facilities, and staff to maintain such organisms
safely, efﬁciently, and appropriately.

Model Animal Research Facility
Associate Professor:
Technical Staff:
Technical Assistant:

WATANABE, Eiji
TANAKA, Minoru
NARUSE, Kiyoshi
HAYASHI, Kohji
NOGUCHI, Yuji
TAKAGI, Yukari
SUZUKI, Kohta
SUGINAGA, Tomomi
FUJIMOTO, Daiji
MATSUMURA, Kunihiro
ATSUMI, Miho
GODA, Misato

ment and point mutation.
The NIBB Center for Transgenic Animals and Plants wasestablished in April 1998 to support research using transgenic and gene targeting techniques at NIBB. The NIBB Center
for Transgenic Animals and Plants was integrated into the
NIBB BioResource center in April 2010, and was renamed“The Model Animal Research Facility”.
Technical and supporting staff develop and promoteresearch-supporting activities. A state-of-the-art facility fortransgenic animals opened at the end of 2003 in the Yamate
area.
The activities of the model animal research facility are as
follows:
1. The 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.
3. The development of novel techniques related to transgenic
and gene targeting technology.
4. Cryopreservation and storage of transgenic strains.

I. Research support activities (mouse)

In 2001, the NIBB mouse facility (built under specificpathogen free (SPF) conditions) opened in the Myodaiji area
and the production, breeding, analysis, cryopreservation andstorage of genetically manipulated mouse strains has been
conducted there since. The new center facility building in the
Yamate area strengthened research activities using genetically altered organisms. The building has five floors anda total floor space of 2,500 m2 in which we can generate,
breed, store and analyze transgenic, gene targeting, andmutant mice under SPF conditions. The mouse housing area
was constructed based on a barrier system. This building is
also equipped with breeding areas for transgenic small fish,
birds, and insects.
In 2015 (from January 1 to December 31), 10,209 fertilized

Figure 1. Mouse (B6C3F1)

The worldwide genome project has almost been completed
and research on basic biology has arrived at a post-genome
era in which researchers are focusing on investigating thefunctions of individual genes. To promote the functional
analysis of a gene of interest it is essential to utilize genetically altered model organisms generated using genetic engineering technology, including gene deletion, gene replace-

Figure 2. Equipment for manipulating mice eggs.

77

National Institute for Basic Biology

Research Support

eggs (in vitro fertilization; 3,585 eggs of 32 lines in which
2,552 eggs of 30 lines were frozen for long-term storage,
frozen eggs: 6,624 of 27 lines) and 4,301 mice were brought
into the facility in the Yamate area, and 61,456 mice (including pups bred in the facility) were taken out.
A number of strains of genetically altered mice from outside
the facility were brought into the mouse housing area by
microbiological cleaning using in vitro fertilization-embryotransfer techniques, and stored using cryopreservation.
A new mouse facility in the Myodaiji area opened at the
beginning of 2005. The facility provides research-supporting
activities to researchers in the Myodaiji area. In March
2009, we expanded the facility which includes areas forbreeding, behavioral tests, and transgenic studies using
various kinds of recombinant viruses. In 2015 (from January
1 to December 31), 71 mice were brought into the facility in
the Myodaiji area, and 3,362 mice (including pups bred in
the facility) were taken out.

room for insects is also available. All the rooms are qualified
to meet the criteria for transgenic animals, allowing researchers to generate and maintain these important biological tools.
In 2015 (from January 1 to December 31), 5,812 medaka
and zebrafish adults and 250 fertilized eggs (including
embryos) were brought to the facility and 121,027 medaka
and zebrafish (117,021 fertilized eggs and 4,006 adults,
including animals bred in the facility) were taken out. In thelaboratory for chick embryos there were no fertilized eggs
or chicken embryos brought in or taken out this year. These
animals were used for research activities in neurobiology anddevelopmental biology.

Figure 3. Large sized autoclave in the Myodaiji area.

Figure 4. Quarantine room for medaka and zebrafish.

II. Research support activities (small fish andbirds)

III. Research activities

The first floor of the center facility building in the Yamate
area provides space and facilities to maintain small fish andchick embryos. In the laboratory room for chick embryos, alarge incubation chamber is equipped and set at 42 degrees(suitable for chick embryogenesis). The researchers canmanipulate chick embryos under optimal conditions,removing biohazard risks. For researchers who employ fish
as an experimental model, 480 tanks (1 liter) and 450 tanks(3 liters) are available for medaka and zebrafish, respectively.
Water circulates and can be maintained to suit the conditions
desired for fish breeding in the aquarium systems. Currently,
over three mutant lines and over fifteen transgenic lines
of medaka and zebrafish are maintained in our facility. A
medaka line that allows gene induction by heat treatment,in combination with a cre/loxP system, has been developedin this facility. In addition to the rooms mentioned above, a78

In 2007 NIBB was approved as a core facility of the
National BioResource Project (NBRP) for Medaka by theJapanese Government. We have supported the activities of
NBRP Medaka by providing standard strains, mutants, transgenic lines and organizing international practical courses
for medaka. In 2014 we began providing the CRISPR/Cas9
genome editing platform as well as the TILLING library
screening system to promote the reverse genetic approach.
In 2015 we shipped 220 independent medaka strains, 155
cDNA/BAC/Fosmid clones, and 195 samples of hatching
enzyme to the scientific community worldwide.

The associate professors of this center - E. Watanabe,
T. Naruse, and M. Tanaka - are the principal investigators of the Laboratory of Neurophysiology, the Laboratoryof Bioresources, and the Laboratory of Molecular
Genetics for Reproduction, respectively. The Laboratoryof Neurophysiology is studying mechanisms of the visualsystem using a psychophysical approach. The Laboratory of
Bioresources has conducted a genetic and genomic analysis
of quantitative traits and Mendelian phenotype variations,as well as evolution of sex determination systems in medaka
related species. The Laboratory of Molecular Genetics for
Reproduction is studying the molecular mechanisms ofreproductive organ development and sex differentiation using
mutagenized or transgenic medaka. For details, please referto the pages of each laboratory (p. 37, 47, and 27).

Model Plant Research Facility● Plant Culture Laboratory
Assistant Professor:
Technical Staff:
Technical Assistant:

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
SUZUKI, KeikoThe Plant Culture Laboratory manages facilities for thecultivation of plants in general and also for the rearing ofseveral animal species that do not fit in other facilities.
The Plant Culture Laboratory equips and manages 62
culture boxes or growth chambers, 4 phytotrons, and 12
rooms with the P1P physical containment level for established and emerging model plants including the thale cress
Arabidopsis thaliana, several carnivorous plants, the rice
Oryza sativa, the moss Physcomitrella patens, green alga
Chlamydomonas reinhardtii and several other flowering
plants. Most culture space is fully used the whole year
by more than 60 researchers from both outside and insidegroups.
As well as regular culture conditions, extreme environmental conditions for light and temperature are available
for various types of experiments. Three light environmental
simulators (max 120,000 lux using xenon lamps) and threechambers (3.4 m2 each) that can control CO2 and humidity
in addition to temperature and light (max 70,000 lux) conditions are available. A tissue culture rack with dimming
LEDs and pulse-width modulation controllers are used foralgae culture under precise light control. Autotrophic andheterotrophic culture devices are also available for researchers using cyanobacteria, algae, and cultured flowering plant
cells. Aseptic experiments can be performed in an aseptic
room with clean benches. Several analytical instruments
including a flow cytometry system and a DUAL-PAM, for
DNA content and chlorophyll fluorescent measuring, respectively, are also available.
Next to the institute building of the Myodaiji area, a 386-m2
experimental farm is maintained for Japanese morning glory
and related Ipomoea species, several carnivorous plants andother flowering plants necessary to be cultivated outside.
Three greenhouses (44, 44, and 45 m2) with heating are used
for the sensitive carnivorous plants. Seven greenhouses (4,6, 6, 6, 6, 9, and 9 m2) with air-conditioning are provided forthe cultivation of rice Oryza sp., Lotus japonica and related
legume species, as well as mutant lines of the Japanesemorning glory. Two greenhouses (9 and 18 m 2) with airconditioning meet the P1P physical containment level andare available for experiments using transgenic plants. The
Plant Culture Laboratory also maintains a 46 m2 building
with storage and workspace. Part of the building is used forrearing of the orchid mantis and the japanese rhinoceros
beetle.

Figure 5. A plant culture box with LED lamps.

● Morning Glory BioResource Laboratory
Assistant Professor:

HOSHINO, Atsushi

The Japanese morning glory (Ipomoea nil) is a traditional
floricultural plant in Japan, and is studied worldwide, especially in plant physiology and genetics. NIBB collects,
develops and distributes DNA clones, mutant lines forflower pigmentation, and transgenic lines as a sub-center of
the National BioResource Project (NBRP) Morning glory,
and collaborates with the core organization center, Kyushu
University. We collected several mutant lines, and provided
31 DNA clones and 16 Ipomoea lines to both local and international biologists this year.
Research activities of the Assistant Professor A. Hoshino
are shown on the laboratory page (p. 53).

Cell Biology Research Facility
Assistant Professor:
Technical Assistant:

HAMADA, Yoshio
SUGINAGA, Tomomi

The Cell Biology Research Facility provides various equipment for tissue and cell culture. This laboratory is equippedwith safety rooms which satisfy the P2 physical containment
level, and is routinely used for DNA recombination experiments.
Research activities of Assistant Professor Y. Hamada, theprincipal investigator of the Laboratory of Cell Sociology, isshown on the laboratory page (p.13).

79

National Institute for Basic Biology

Research Support

Figure 6. Equipment for tissue and cell culture.

80

NIBB CENTER OF THE INTERUNIVERSITY
BIO - BACKUP PROJECT (IBBP CENTER)

Head

Associate Professor

KAWAGUCHI, Masayoshi NARUSE, Kiyoshi

Specially Appointed Assistant Professor:
KIMURA, Tetsuaki
TANAKA, Daisuke
Technical Assistant:
AKIMOTO-KATO, Ai
MATSUBAYASHI, Naomi
FUKUMOTO, Tatsuya
Secretary:
HAMATANI, Ayako
In order to realize a life sciences community resilient tonatural disasters and calamities, the National Institutes for
Natural Sciences (NINS) and Hokkaido University, Tohoku
University, University of Tokyo, Nagoya University, Kyoto
University, Osaka University, and Kyushu University concluded an agreement on June 1st 2012 to launch a systemto ‘back up’ the biological resources essential to the work
being done at universities and research institutions nationwide, called the ‘Interuniversity Bio-Backup Project (IBBP)’.
The IBBP Center was established as a centralized backup
and storage facility at NIBB, while IBBP member universities set up satellite hubs and work closely with the IBBP

center to put in place reciprocal systems for backing up
important biological resources that have been developed byresearchers residing in the area each university satellite hub
is responsible for.
The IBBP Center includes earthquake proof structures
capable of withstanding even very large scale quakes(equipped with emergency backup power generators),
cryopreservation facilities equipped with automatic liquid
nitrogen feeding systems, deep freezers, and refrigerated
storage (mainly for seed stocks), as well as all manner of
automated laboratory equipment, cell culture tools, and thelatest equipment necessary to back up the genetic resourcesin a collaborative manner. The specific methods of preservation are freezing of the sperm and eggs of animals, cultured
plant and animal cells, proteins and gene libraries. Plant
seeds are frozen or refrigerated.
University satellite hubs receive preservation requestsof biological resources from researchers and report to theManaging Project Committee of IBBP (constituted of facultyof NIBB and satellite institutes), where the relevance of therequest is reviewed. When the request is sustained, biological resources to be preserved will be sent to the IBBP Centerby the requesting researcher, where they will be frozen (orrefrigerated) and their particular information will be registered into a database. In the event of a disaster leading to theloss of a researcher’s own biological resources, preserved
samples will be promptly supplied back to the researcher sothey can quickly resume their work.
Through the development of this backup system biologicalresources that had only been stored at individual research
institutes can now be preserved at the state of the art facilitiesof the IBBP Center, and Japan’s research infrastructure has
been significantly strengthened.

I. Current status of back up for the biologicalresources

Figure 1. IBBP Center

Figure 2. Cryogenic storage system. Liquid nitrogen tanks are monitored
24 hours a day and are refilled automatically.

In 2015, IBBP Center stored 4,238 384-well and 69 69-wellplates consisting of 1,634,016 clones as cDNA/BAC clones,
8946 tubes for animal cells, plant and animal samples,
proteins, genes and microorganisms, 80 133mm-straw tubes
for sperm and 606 seed samples. In total 1,634,702 samples
are stored.

Figure 3. Cryo tube with 2D barcode. Each sample is printed with aunique barcode and is managed using a database.

81

National Institute for Basic Biology

Research Support

II. C ollaborative Research Project for thedevelopment of new long-term storage
technologies and cryo-biological study

As the IBBP Center can only accept biological resources
which can be cryopreserved in liquid nitrogen, researchers
cannot backup those biological resources for which cryopreservation methods are not well established, to increase
the usability of IBBP, we started a collaborative research
project for the development of new long-term storage technologies and cryo-biological study in 2013. This collaborative research includes two subjects 1) Establishment of
new storage technology for biological resources for which
long-term storage is unavailable. 2) Basic cryobiologicalresearch enabling us to improve low temperature storage forbiological resources. In 2015 we had ten applications andaccepted nine proposals. We are also working to establish
a research center for cryo-biological study thorough thisCollaborative Research Project. Accordingly, we organized
the Cryopreservation Conference 2015 on October 28-29,
2015 at the Okazaki Conference Center, Okazaki, Japan. We
had 100 participants from several fields covering physics,
chemistry, biology, and technology.

Figure 4. Group photo of Cryopreservation conference 2015

Publication List on Cooperation〔Original paper (E-publication ahead of print)〕

Figure 1. Phase diagram of vitrification of a solution.
Vitrification, a physical process, can be defined as the phase transitionof an aqueous solution from a liquid into an amorphous glassy solid, or
glass, at the glass transition temperature (Tg), while avoiding ice crystallization.

minants of survival (Figure 1).
So far cryopreservation has been considered in terms of
the liquid and solid (ice) phases of water. It is also possible
to cryopreserve tissues by the process of “vitrification”, thesolidification of liquids without crystallization (Figure 2).
This comprises a “glassy state” as the system is amorphous,lacks organized structure but possesses the mechanical andphysical properties of a solid (Taylor et al. 2004).

Figure 2. Differential Scanning Calorimetry (DSC) thermogram of
Vitrified shoot apices of a Chrysanthemum plant treated with vitrification
solution.
Glass transition temperature (Tg):-107

D., Ishizaki, K., Kohchi, T., and Yamato, K. T. Cryopreservation• Tanaka,of gemmae from the liverwort Marchantia polymorpha L. Plant Cell
Physiol.2015 Nov. 11.

Research activity by D. Tanaka
Assistant Professor:
Technical Assistant:

TANAKA, Daisuke
AKIMOTO-KATO, Ai
FUKUMOTO, Tatsuya

Cryopreservation

Cryopreservation protocols contain components which
are usually developed empirically using each biologicalresource’s specific strategy to enhance survival (Benson2008). The theory of cryopreservation encompasses several
interconnected disciplines ranging from the physiological to
cryophysical. Water status and cryopreservation, in combination with physiological factors, are the most influential deter-

82

Figure 3. Cryopreservation protocol using aluminum cryo-plate.
A: Preparation of in vitro grown gemmae of liverwort. B: Placing precultured gemmae in a cryo-plate’s wells. C: Regrowth of cryopreserved
livewort line ‘Takaragaike-1’ 30 days after rewarming. D: Control
without cooling in liquid nitrogen).

Vitrification-based protocols are known to be effective
for long-term, stable preservation of plant germplasm; thisprotocol can reduce the cost and manpower for maintaining
a large number of germplasm lines and keep many valuablegenetic lines for a long term under genetically stable conditions. However, it is still not widely employed as a reliablelong-term preservation protocol due to the lack of basic
knowledge on cellular and water behavior in tissues when
immersed in liquid nitrogen.
In the present study, electron microscopy combined withfreeze-substitution was employed to examine the ultrastructure of cells of the gemmae of the liverwort, Marchantia
polymorpha L., strain Takaragaike-1, which were cooled tothe temperature of liquid nitrogen after exposure to various
steps of the Cryo-plate protocol (Figure 3).

Publication List

〔Original paper (E-publication ahead of print)〕

D., Ishizaki, K., Kohchi, T., and Yamato, K. T. Cryopreservation• Tanaka,of gemmae from the liverwort Marchantia polymorpha L. Plant Cell

Figure 1. Model for leucophore and xanthophore development fromneural crest cell.

rise to pax7a-positive bi-potent precursor cells for xanthophores and leucophores; some of these precursor cells thenexpress sox5, and as a result of Sox5 action develop into
xanthophores (Figure 1). Our findings provide clues forrevealing diverse evolutionary mechanisms of pigment cell
formation in animals.

Physiol.2015 Nov. 11.

Research activity by T. Kimura
Assistant Professor:

KIMURA, Tetsuaki

Leucophores are similar to xanthophores

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neuralcrest cells (NCCs) are multipotent stem cells that give riseto numerous cell-types, including pigment cells. Medaka
has four types of NCC-derived pigment cells (xanthophores,
leucophores, melanophores and iridophores), making medaka
pigment cell development an excellent model for studying
the mechanisms controlling specification of distinct cell
types from a multipotent precursor cell. However the genetic
basis of chromatophore diversity remains poorly understood.
We reported that leucophore free-2 (lf-2) which affects
leucophore and xanthophore differentiation, encodes pax7a.
Since lf-2, a loss-of-function mutant for pax7a, causes
defects in the formation of xanthophore and leucophore
precursor cells, pax7a is critical for the development of thechromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, asthese chromatophores have purine-dependent light reflection.
Many leucophores-3 (ml-3) mutant embryos exhibit aunique phenotype characterized by excessive formation ofleucophores and an absence of xanthophores. We show thatml-3 encodes sox5, which is expressed in premigratory NCCs
and differentiating xanthophores. Cell transplantation studies
reveal a cell-autonomous role of sox5 in the xanthophore
lineage. pax7a is expressed in NCCs and required for bothxanthophore and leucophore lineages; we demonstrate that
Sox5 functions downstream of Pax7a.
We propose a model in which multipotent NCCs first give83

National Institute for Basic Biology

Research Support

CENTER FOR RADIOISOTOPE FACILITIESMyodaiji area

78

Users

29

37

MATSUDA, Yoshimi(Radiation Protection Supervisor)
SAWADA, Kaoru(Radiation Protection Supervisor)
IINUMA, Hideko
KAMIYA, Kiyomi

The technical and supporting staff of the Center forRadioisotope Facilities (CRF) maintain controlled areas
in compliance with the law. The CRF is responsible formonitoring the purchase of radioisotopes from the Japan
Radioisotope Association (JRIA) and the transfer of radioisotope wastes to JRIA.
Ms. Matsuda, Ms. Iinuma, and Ms. Kamiya maintained the
Myodaiji area. Ms. Sawada worked in the Yamate area. Dr.
Kodama worked in both areas.
The following are the CRF’s notable activities in 2015.
1. An introductory course for radioisotope handling washeld after an interval of 6 years due to the repair work of theCommon Facilities building I. The content of the course wasrenewed, allowing participants to experience handling ofseveral types of radiation and radioisotopes (Figure 1A).
2. The drain storage tanks at the Yamate area were cleaned
and checked by the maintenance workers after 4 years of use.
Both the water level indicator and the water level alarm were
exchanged. (Figure 1B)
The number of registrants and the number of users fromJanuary 2015 to December 2015 are shown in Table 1.
Users and visitors counted by the access control systemof the controlled areas numbered 2,250 during this period.
The numbers for each area are shown in Table 2. The annualchanges of registrants and the number of totals per fiscal year
are shown in Figure 2. The balance of radioisotopes received
and used at the CRF is shown in Table 3. The training
courses on radioisotope handling were given as in Table 4.

Myodaiji area
1,281
185
1,466

Users
Visitors
Total

Yamate area
665
119
784

Total
1,946
304
2,250

Table 2. Users and visitors who entered each controlled area in 2015.

350

14000

300

12000

Registrants
Days of facility use

250

10000

200

8000

150

6000

100

4000

50

2000

0

0
Fiscal year

Figure 2. Annual changes of registrants and days of facility use per fiscal
year.

Myodaiji area

Yamate area

Total

125

I Received

0

590

125

I Used

0

165

165

74,0001,110,000

1,184,000

Figure 1. The CRF’s notable activities in 2015.
A: The introductory course for radioisotope handling
B: Cleaning of the drain storage tanks at the Yamate area

84

B

590

35

S Received

35

S Used

14,706

1,343,661

1,358,367

32

P Received

216,450

0216,450

32

P Used200,540

0200,540

14

C Received

37,000037,00014

C Used18,6300

18,6303

H Received

40,7003,718,500

3,759,200

3

H Used28,075

3,715,540

3,743,615

Table 3. Balance of radioisotopes received and used (kBq) at each controlled area in 2015.

Number ofparticipants

Training course
Introductory course for beginners*

Place
MyodaijiIntroductory course for beginners*
Introductory course for experts

YamateMyodaiji3
4

Introductory course for experts
Users training course*

YamateMyodaiji6
53

Users training course

Yamate69*including English course

A

Days of facility use

Technical Staff:

KODAMA, Ryuji

Registrants

Associate Professor

Technical Assistant:62

Table 1. Numbers of registrants and users at Myodaiji area and Yamate
area in 2015.

Head

HASEBE, Mitsuyasu

Yamate area

Registrants

Table 4. Training courses for radiation workers in 2015.

1

Research Enhancement Strategy OfficeDirector

UENO, Naoto

Vice-Director (Specially
Appointed Professor (URA))NISHIMURA, Mikio

In order to fulfill two goals, to encourage cutting-edge
academic research in the field of natural sciences throughinternational joint research, and to contribute to the enhancement of research capabilities of universities etc. in Japan
using the world’s most advanced research environment forjoint utilization and joint research, NINS started in 2013a research enhancement project with the following four
approaches: 1) Support for the promotion of international
advanced research, 2) Support for the promotion of joint
utilization and joint research in Japan, 3) Dissemination of
information and enhancement of public relations in Japanand abroad, 4) Support for researchers, especially young,female or foreign researchers.
The Research Enhancement Strategy Office is aimed at
supporting researchers so that NIBB improves its ability as
a collaborative research institution, and was restructured in2013 from the former Strategic Planning Department, theOffice of Public Relations, and the Office of International
Cooperation which existed from 2005. The Office’s activities are mainly carried out by URAs (University Research
Administrators) according to the advice of the group adviser
chosen from NIBB’s professors and in close collaboration with the NINS’s Research Enhancement Promotion
Headquarters.

Evaluation and Information Group
Associate Professor:
Group Adviser:

KODAMA, Ryuji
YOSHIDA, Shosei

This group serves as a central office for assisting the
Director General in preparing for NIBB’s evaluation procedure and in planning long-range strategies for the institute.

The main activities of the group

1) Management of external evaluation processes
NIBB asks non-NIBB members of the Advisory Committee
for Programming and Management to answer a questionnaire on research achievements and collaborative research
activities of the institute. Several of the members are further
invited to participate in a meeting for the evaluation of these
activities and for proposing future directions for the institute.
This group manages these processes.
2) Editing of the Annual Report (in collaboration with the
Public Relations Group)This group edits the annual report (this print) which summarizes the annual activities of laboratories and facilities andis used in the evaluation procedures of the institute.

3) Assistance in budget requests and long-range planningof the instituteThis group also assists the Director General in preparing
long-range plans for building the most advanced researchfacilities, and in budget requests to the government to realize
and implement these plans.
4) Assistance in making the plans and reports of the institute
In addition we assist in drafting NIBB’s Medium-term
Goals and Plans (for a six-year-period), and in instituting
Annual Plans to implement them. The department also assists
in preparing Business and Performance Reports for theexternal evaluation on whether we are meeting the goals set
both annually and for the medium-term.

Public Relations GroupSpecially Appointed Assistant Professor (URA):
KURATA, Tomoko
OTA, Kyoko
Technical Assistant:
KAWAGUCHI, Colin
BAN, Misato
FUJIMORI, ToshihikoGroup Adviser:This group, in order to publicize the activities of NIBB
to the widest audience, actively facilitates communication
between NIBB and the public, school teachers, and the international community of scientific researchers.

The main activities of the group in 2015

1) Press releases
The group sends news on scientific achievements to newspaper and magazine reporters via leaflets, e-mails and web
pages. We also arrange press conferences for some releases.
2) Updating and maintenance of the NIBB web page3) Editing of publications, production of posters andleaflets
Publication of “NIBB News” (Intra-institutional newsletter, in Japanese), “NIBB English News” (Intra-institutional
newsletter, in English). Publication of leaflets introducing
the individual researchers of NIBB (in Japanese). Design anddistribution of posters of NIBB events.
4) Producing Videos
Creation of videos introducing NIBB and interviews withresearchers that are published on the web.
5) Organization of scientific outreach programs
Organizing the Summer Program for university students,
and coordinating special classes for middle school students.

85

National Institute for Basic Biology

Research Support

International Cooperation GroupSpecially Appointed Assistant Professor (URA):TATEMATSU, Kiyoshi
Technical Assistant:
TAKAHASHI, Ritsue
SANJO, Kazuko
NISHIMURA Akiko
Group Advisor:
UENO, NaotoNIBB has a mission to continually explore the leadingedge of biology and form research communities that link
Japan to the world. For this purpose, NIBB holds scientific meetings including “NIBB Conferences” and “Okazaki
Biology Conferences (OBC)”, and educational programs
such as “NIBB International Practical Courses”. Further,
NIBB is tightly interacting with the European Molecular
Biology Laboratory (EMBL, European member states) andthe Temasek Life Sciences Laboratory (TLL, Singapore)on the basis of cooperative agreements, through exchanging people and techniques and jointly holding scientificmeetings. NIBB is also conducting the “NIBB International
Collaborative Research Initiative” to promote high-levelinternational collaborations between faculty members of
NIBB and researchers around the world. NIBB invites leading-edge researchers from abroad as “Guest Professors” topromote academic exchange with NIBB members and to
start new international collaborations.
This group supports and coordinates NIBB’s activities
related to international research collaborations, through organizing the various above-mentioned international scientificmeetings and technical courses, coordination of dispatching NIBB’s researchers to international conferences, andsupport of researchers visiting from the institutes mentioned
above. This group also supports NIBB internship students
visiting from foreign countries, and the dispatching of
graduate students of SOKENDAI (the Graduate University
for Advanced Studies) to international conferences, which
are aimed at nurturing the next generation of researchers
in biology. From this year, this group, cooperating with the
Okazaki Administration Office, will begin supporting otherresearchers and students who visit NIBB.

The main activities of the group in 2015

1) Coordination of the International Conferences and theInternational Practical Course
This group coordinated the following International
Conference hosted by NIBB:The 63rd NIBB Conference “Environment to Bioresponses”
Okazaki, Japan, November 30-December 2, 2015 (p. 88)
2) Support of dispatching researchers to international conferences
This group supported sending NIBB researchers to the following events (related to the international cooperative agreements):
EMBO Workshop “Embryonic-Extraembryonic Interfaces
Emphasis on molecular control of development in amniotes”
Göttingen, Germany, May, 6-9, 2015

863) Support of visiting researchers to NIBBThis group supported visits of foreign researchers related to
the following events:
Two Guest Professors coming from Heidelberg University,
Germany (February) and Tulane University, USA (November)

4) Support of education related programs
This group supported sending and hosting students participating in the following student-related activities of NIBB:
The 17th EMBL PhD Symposium “Just by Chance? –Randomness & Variability Shaping Biology-” Heidelberg,
Germany Octorber, 22-24, 2015 (p. 89)
NIBB Internship Program 2015 (p. 92)

Collaborative Research GroupSpecially Appointed Associate Professor (URA):SHIGENOBU, Shuji
KAMEI, Yasuhiro
Technical Assistant:
ICHIKAWA, Mariko
ICHIKAWA, ChiakiGroup Adviser:
YOSHIDA, Shosei
Specially appointed associate professors of this groupbelong to the NIBB core research facilities and are responsible for managing collaborative research projects and practical courses taking advantage of their expertise in theirfield., through which this group explores further promotionof information exchange and collaboration among scientific communities, and also supports the development of new
equipment and methods.
In 2015, this group hosted a total of 123 collaboration
projects. Through these collaboration projects, 28 research
papers were published. A noteworthy achievement was
the series study of amphibian regeneration directed by Dr.
Yokoyama in Hirosaki University and his colleagues. The
NIBB collaborative research group and the NIBB Core
Facilities supported their research in bioinformatics andbioimaging technologies as individual collaborative research
projects from 2012.

A., Hayashi, T., Kobayashi, T., Nagayama, C.,• Kawasumi-Kita,
Hayashi, S., Kamei, Y., Morishita, Y., Takeuchi, T., Tamura, K. and••Yokoyama, H. (2015). Application of local gene induction by infraredlaser-mediated microscope and temperature stimulator to amphibian
regeneration study. Dev. Growth Differ. 57, 601–613.
Hayashi, S., Ochi, H., Ogino, H., Kawasumi, A., Kamei, Y., Tamura,
K. and Yokoyama, H. (2014). Transcriptional regulators in the Hippo
signaling pathway control organ growth in Xenopus tadpole tailregeneration. Dev. Biol. 396, 31-41.
Hayashi, S., Kawaguchi, A., Uchiyama, I., Kawasumi-Kita, A.,
Kobayashi, T., Nishide, H., Tsutsumi, R., Tsuru, K., Inoue, T., Ogino,
H., Agata, K., Tamura, K. and Yokoyama, H. (2015). Epigenetic
modification maintains intrinsic limb-cell identity in Xenopus limb bud
regeneration. Dev. Biol. 406, 271-82.

Gender Equality Promotion Group
Associate Professor:
Group Adviser:

TSUBOUCHI, Tomomi
TAKADA, ShinjiThis group supports the improvement of the research environment for female researchers in both research and daily
life. This includes our research support system for childbirthand childcare, etc., aiming at promoting the employment offemale researchers.
In 2015, this group contributed to the management of theresearch support system covering childbirth and childcare.
This group also organized an enlightenment lecture and published a leaflet for gender equality promotion in collaboration with other institutes of NINS.

National Institute for Basic Biology

Research Support

TECHNICAL DIVISIONHead

KAJIURA-KOBAYASHI, Hiroko

Common Facility Group

Research Support Group

Chief:● Developmental Biology
Technical Staff:
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, SanaeMIZUGUCHI, Hiroko

MIWA, Tomoki● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki
		
MORI, Tomoko
Subunit Chief:
MAKINO, Yumiko		
YAMAGUCHI, Katsushi
Technical Staff:
NISHIDE, Hiroyo
		
NAKAMURA, Takanori
		
TANIGUCHI- SAIDA, Misako
		
UCHIKAWA Tamaki		BINO, Takahiro
Technical Assistant:
ICHIKAWA, Chiaki
		
NISHIMURA, Noriko		
ICHIKAWA, Mariko		
ISHIKAWA, Azusa
		
OKA, Naomi		SHIBATA, Emiko● Neurobiology
Unit Chief:
Subunit Chief:

OHSAWA, Sonoko
TAKEUCHI, Yasushi● Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
Subunit Chief:
KABEYA, Yukiko● Environmental Biology
Unit Chief:
MIZUTANI, Takeshi
Technical Staff:
NODA, Chiyo● NIBB Bioresource Center
Subunit Chief:
HAYASHI, Kohji
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
SUZUKI, KeikoSUZUKI, Kohta● Disposal of Waste Matter Facility
Unit Chief:
MATSUDA, Yoshimi● Center for Radioisotope Facilities
Unit Chief:
MATSUDA, Yoshimi
Subunit Chief:
SAWADA, Kaoru
Technical Staff:
IINUMA, Hideko
Technical Assistant:
ITO, Takayo

The Technical Division is a support organization forresearchers and research organizations within NIBB. The
Technical Division develops and promotes the institute’s
research activities and maintains the research functions of theinstitute.
The Technical 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 research activities as describedin the reports of individual research divisions.

Reception
Secretary:
		
		
		

TSUZUKI, Shihoko
KATAOKA, Yukari
UNO, Satoko
MIYATA, Haruko

Technical staff members continually participate, throughthe Division, in self-improvement and educational activities
to increase their capabilities and expertise in technical areas.
Generally, technical staff members are attached to specificdivisions so that they may contribute their special biological and biophysical knowledge and techniques to various
research activities.
The Technical Division hosts an annual meeting for technical engineers who work in various fields of biology atuniversities and research institutes throughout Japan. At thismeeting, the participants present their own activities anddiscuss technical problems. The proceedings are publishedafter each meeting.

87

The 63rd NIBB Conference“Environment to Bioresponse”
Organizer: Taisen Iguchi (NIBB, Japan)
November 30 (Mon) - December 2 (Wed), 2015
This symposium, with researchers that have been active in
the international forefront, was intended to facilitate deep
discussions on the connection between the “environment”
and the “organism”. In addition to basic biological researchon environmental response strategies of organisms, fromwhich comes the molecular basis of evolution leading to theestablishment of a variety of species, we discussed various
issues regarding the effects of environmental chemicals tohumans and wildlife, and the mechanisms of the effects using
model species. In addition to mice and humans, a varietyof animal species such as beetles, ants, aphids, Daphnia,
medaka, zebrafish, roach, Xenopus, and alligators havebeen used. We discussed the latest findings concerning themolecular mechanisms of bioresponses such as phenotypic
plasticity, metamorphosis, defects of sexual differentiation,and reduction of reproduction, which are caused by environmental factors, hormones, and chemical substances. 15
years ago, I organized The 45th NIBB Conference in order
to discuss how to analyze the effects caused by hormonelike substances being released into the environment, on fish,
amphibians, reptiles, and mammalians, including humans.
The progress made during the 15-years of research was an
eye-opener, and our basic understanding for the elucidationof molecular evolution and the function of hormone receptors, the elucidation of sex differentiation mechanisms invarious animal species including alligators, Daphnia, andaphids, and the decoding of the genome sequence of various

animal species has been monumentally advanced. Moreover,the progress of this research has revealed the necessity forpromoting research focusing on the epigenetic changes
induced by the chemicals and the environment, the mechanism of action of “Obesogen” induced obesity, and the transgenerational and composite effects of these chemicals. Thissymposium was an opportunity to broadly discuss the impact
and bioresponses to environmental factors from a wide rangeof research fields including perspectives from basic biology
and bioinformatics.
(Taisen Iguchi)

Speakers
Bean, Tim P. (Cefas), Blumberg, Bruce (Univ. of California), Colbourne, John (Univ. of Birmingham), Guillette, Louis Jr. (Medical Univ.
of South Carolina), Helbing, Caren C. (Univ. of Victoria), Katsiadaki, Ioanna (Cefas), Kloas, Werner (IGB), Kohno, Satomi (Medical
Univ. of South Carolina), Lange, Anke (Univ. of Exeter), McLachlan, John A. (Tulane Univ.), Roberts, Mike (Defra), Shaw, Joseph R.
(Indiana Univ.), Tyler, Charles R. (Univ. of Exeter), Arizono, Koji (Pref. Univ. of Kumamoto), Horiguchi, Toshihiro (NIES), Iguchi, Taisen(NIBB), Kanno, Jun (NIHS), Katsu, Yoshinao (Hokkaido Univ.), Kobayashi, Tohru (Univ. of Shizuoka), Miura, Toru (Hokkaido Univ.),
Nagae, Masaki (Nagasaki Univ.), Niimi, Teruyuki (NIBB), Sato, Tomomi (Yokohama City Univ.), Shigenobu, Shuji (NIBB), Soyano,
Kiyoshi (Nagasaki Univ.), Tatarazako, Norihisa (NIES)

88

The 17th EMBL Ph.D Symposium “Just by Chance?
– Randomness & Variability Shaping Biology –”October 22 (Thu) – 24 (Sat), 2015
Two PhD students from SOKENDAI/NIBB were funded by
NIBB to participate in the The 17th EMBL Ph.D Symposium“Just by Chance? – Randomness & Variability Shaping
Biology –” (held on 22-24 October). Our students had thechance to give poster presentations at the symposium tointroduce their current research. They also attended EMBL’s
laboratories to exchange experimental information anddiscuss their research with PhD students, post-docs and PIs.

Comments from students (excerpts)

・Rie Ohashi
Various topics were provided in the Ph.D. Symposium, e.g.
developmental biology, evolution, structural dynamics andbioinformatics. I was very interested in the programs because
these topics covered “wet” (experimental biology) to “dry”(informatics), which reflects current biology. During thecoffee breaks, students were proactive in their communications with each other. I presented my research in a poster
session. My theme didn’t completely match the theme ofthe symposium, but many people came to my poster. I was
nervous because I had to speak only in English. However,during the poster session, I felt that we were able to understand each other through the scientific topics, much better
than through the topics during the coffee break, so it was an
enjoyable time.
Afer the symposium I visited the laboratory of Dr. Anne
Ephrussi, who aims to reveal the mechanism of local translation in Drosophila embryos. I introduced my research to thelab members in an informal seminar for about 40 min. After
that, we discussed my work and their work with a few lab
members. I was very glad that they asked many questions.
However, it was difficult for me to understand their questions
quickly, because their talking speed was very fast for me, so I
had to ask people to repeat their questions to understand. We
communicated with each other using not only language but
also drawing pictures. I reflected on these points a lot during
my lab visit, but it was still a very precious time for me. I
will make use of this experience in the future.
On the last day, I visited Dr. Kota Miura, who is an image
analysis coordinator at EMBL. He has been living inGermany for many years. I heard about the various differences between Europe and Japan, including how researchers perceive molecular biology in their research. I was very
interested in that, and it was a meaningful experience.
I felt that I have to make more of an effort to survive asa researcher, but I strongly reaffirmed my commitment
because of this EMBL visit. Finally, I would like to thank tothe staff of NIBB and EMBL for supporting my visit.

EMBL, Heidelberg
・Yousef Kamrani
The aim of the EMBL PhD student symposium was “toexplore the importance of randomness and variability inbiology”; I realized that applying bioinformatics to biology
research should be highlighted and it was a useful hint
for me. The symposium attracted mostly early-stage PhD
students and high-profile speakers spanning all continents
with diverse backgrounds. The symposium program had beenformulated with several lectures, short talks and a poster
session. At the end of each day, we had a blackboard session
and a panel discussion. At the blackboard discussions we
could choose our topic of interest and sit down with speakers
in a small group to share insights from their most recent
research. This time was a great opportunity. Moreover, I
was able to strengthen my network of connections with otherscientists. After a three day tight schedule, I had a weekend
to tour Heidelberg city. I passed through many historictreasures including Heidelberg’s Schloss (castle) and the
Neckar River’s autumn colors absolutely invigorated me.
The second part of my program was a one week training
course at the EMBL GeneCore facility headed by Dr.
Vladimir Benes. I practiced a protocol provided by Dr.
Bernd Klaus -a step by step method- using statistical bioinformatics to analyze high-throughput sequencing data such
as DNA-Seq or RNA-Seq. I was supported technically byhighly qualified staff in Dr. Benes’ group. The atmosphere of
his lab was very relaxed and friendly. I am looking forward
to having an opportunity again in the near future to visit
EMBL.

89

The 9th NIBB
Bio-Imaging Forum“Imaging of Physical Properties”
Organizer: Yasuhiro Kamei,
Shigenori Nonaka, Toshihiko Fujimori,
Naoto Ueno
January 26 (Mon) - 27 (Tue), 2015
Thirteen lectures (two from NIBB members) on temperature,kinetics, and molecular movement, as well as on how theseforces affect biological research, drew a total of 49 participants. This lead to broad multi-disciplinary discussions on
the role of physics in living systems. These physical quantities still do not draw a lot of attention in the field of biology,
but it was clear that many researchers feel we are closing inon the essence of biological activity from these processes.
Many lectures from fields rooted in engineering were given,and for researchers of biological systems this active gathering with members of remarkably different fields lead to freshexchange and new perspectives. In addition, because it was
the first symposium on biological systems in which heatand temperature were the main themes, there was hope fromthe speakers that more of these kinds of opportunities will
become available going forward.
(Yasuhiro Kamei)

The NIBB Genome
Informatics Training
CourseThe NIBB Core Research Facilities organizes a series oftraining courses on up-to-date research techniques. In 2015
we held two training courses on Genome Informatics. The
3-day programs offer lectures and hands-on tutorials to
introduce basic knowledge and skills to deal with large scale
genomic data such as DNA sequence data generated by nextgeneration sequencing (NGS). The programs are speciallydesigned for biologists who are not familiar with bioinformatics.

“Introduction to RNA-seq - from thebasics of NGS to de novo analyses”
February 25 (Wed) -27 (Fri), 2015

■O rganizer: Dr. Shuji Shigenobu (NIBB Core
Research Facilities)■L ecturers: Dr. Shuji Shigenobu, Dr. Ikuo
Uchiyama, Dr. Masanao Sato, Dr. Katsushi
Yamaguchi, Ms. Hiroyo Nishide, Dr. Taro Maeda■Participants: 22 (including 3 from NIBB)■Program:
1. UNIX for beginners
2. NGS basic data formats and NGS basic tools
3. Introduction to statistics
4. Introduction to “R”
5. RNA-seq pipeline: genome-based and transcriptome-based approaches
6. Multivariate statistics
7. Exercises“Introduction to RNA-seq - from thebasics of NGS to de novo analyses”
September 9 (Wed) -11 (Fri), 2015

■O rganizer: Dr. Shuji Shigenobu (NIBB Core
Research Facilities)■L ecturers: Dr. Shuji Shigenobu, Dr. Ikuo
Uchiyama, Dr. Masanao Sato, Dr. Katsushi
Yamaguchi, Ms. Hiroyo Nishide■Participants: 22 (including 3 from NIBB)■Program:(as above)

90

The 3rd BioImaging
Analysis Training
CourseThe NIBB
Medaka Basic
Training Course

The 3rd Bioimaging Training Course was held jointly by theCenter for Novel Science Initiatives’ Department of Imaging
Science, and NIBB. This course was designed for biologists
who are relatively new to analyzing datum obtained throughadvanced microscopy. Therefore the focus of training waslearning image processing and analytical techniques through“solving simple problems with image analysis” and “understanding appropriate methods and necessary preparation for
consulting experts in technically advanced imaging challenges”. There were applications from 83 people for thiscourse, which had a maximum capacity of 20 participants,
this clearly suggests the height of the demand for courses onthese subjects.
This course’s lectures were conducted with the aim oftraining participants to keep in mind the series of steps
leading to fundamental image processing and analysis whileobtaining the images to be used (workflows). In addition,
we loaned the participants PCs pre-installed with ImageJ, atypical software package for biological image processing andanalysis, and images to be used as teaching materials, whichwere used for practicing the basic operations and settings ofimage processing. In addition, lectures on how programmingof simple “macro-programs” which use these workflows in
ImageJ allows automation, which is essential for the large
capacity and high-dimensional throughput of microscopic
imaging which has become possible in recent years.
At the conclusion of the course each of the students gave
commentary and discussed the methods used with examplesof actual images from their own research. Every year after
the course, participants express feeling “pretty tired, andsatisfied” as part of the questionnaire, and certainly there isa true benefit in terms of their image analysis by becoming
more familiar with these techniques. In addition, we expectthat this course will increase opportunities for joint research
relating to biological image analysis.
(Kagayaki Kato)

The content of the course was decided through a questionnaire in the Medaka community’s mailing list and out of themost requested topics we chose genome editing techniques
using CRISPR/Cas9, microinjection techniques, rearing
and anesthesia methods, and advanced imaging techniques
as feasible subjects for NIBB. We originally planned toaccept 8 participants, but as we had more than 20 applicants,
we finally accepted 16. We also accepted two observers
to learn skills for managing training courses, and asked agraduate student of Kyoto University for a presentation. The
two-day course commenced with several lectures and handson training. The course was tight-scheduled and may havebeen rather brief, but we believe we informed participantsof the research potential of NIBB and of the support we canoffer through collaborative programs including techniques
needed in individual research projects. The questionnaire
after the course revealed that the Medaka rearing methods
were most appreciated.

December 7 (Mon) -9 (Wed), 2015
■O rganizer: Dr. Kagayaki Kato, Dr. Yoshitaka
Kimori, Dr. Yasuhiro Kamei, Dr. Hiroshi Koyama,
Dr. Shigenori Nonaka, Dr. Takashi Murata

August 6 (Thu) -7 (Fri), 2015
■O rganizer: Dr. Yasuhiro Kamei (NIBB Core
Research Facilities)■Lecturers: Dr. Yasuhiro Kamei, Dr. Kiyoshi Naruse,
Dr. Yusuke Takehana, Dr. Shigenori Nonaka■Presentations: Mr. Ansai Satoshi (Kyoto Univ.), Dr.
Shigenori Nonaka■Participants: 16■Program:
1. E
 gg collection and outline of injection techniques
2. Rearing and observation of embryos
3. Anesthesia of the embryo
4. Basics of genome editing techniques
5. Microinjection
6. H
 atching enzyme treatment and sample preparation for microscopy
7. Utilization of NMBR Medaka web pages
8. Microscope handling and observation

91

The NIBB Internship program
The NIBB Internship program, started in 2009, is ahands-on learning course for overseas students designed
to give high-quality experience in real world research andfocused education of biology. At the same time, this programaims to internationalize the graduate students of SOKENDAI(Graduate University for Advanced Studies), giving them theopportunity to get to know students and interns with various
cultural customs. Another goal of the program is to build
connections through providing education to the people whowill form the core of international research networks in the
future.
To participate in this program, applicants who would like toexperience research at NIBB must supply the name of the lab
they would like to visit as well as their reasons for choosing
it, and a letter of recommendation. Based on this information
applicants are chosen to spend set periods of time participating in specific research activities in the lab they applied for.
Round trip airfare and housing expenses are provided by the
NIBB Internship Program.
In FY 2015 there were 26 applicants, out of which six
interns were selected. These interns were from universities located in 4 countries (Turkey, Vientam, Thailand, andGermany) and spent periods ranging from two to twelve
weeks experiencing life as a member of a research team.
Moreover, one interns from Peru stayed at NIBB on twelve
weeks by his own travel grants.

Report from a participant
Pham Van Cuong
VNU University of Science,Vietnam
I continued my journey of science by applying to the NIBB
internship program immediately after completing my bachelor’s degree. As a newbie in this field, I expected thisprogram would be an opportunity to raise the skills of the scientist inside me: If I face a problem, how should I handle it?
How should I solve scientific questions by designing plans orexperiments?
Then I chose the laboratory of Assoc. Prof. Kamei, because
he is using medaka fish as a model, and a heat-shock-inducible gene-expression system, as my undergraduate labora-

92

tory. Also, I wanted to have a chance at IR-LEGO, the technique that uses an infra-red laser to induce expression of theheat shock promoter-driving gene in desired cells. I spentnearly 3 months working in the Kamei-lab, I was supported
enthusiastically by members of this laboratory and also the
Bioresource Laboratory. Thanks to that help, my experiments(evaluation of heat-shock response and molecular experiments) turned out well and were finished with some expected
results. I think the results I obtained were less significant tome than what I learned: techniques, experiment manipulation, and even working habits. And my questions at the
beginning were also mostly answered.
To say something about this program, in an unscientificaspect, I feel how lucky I was to be selected for this program:coming to a country I dreamed about as my first time goingabroad, being helped by the people surrounding me, andimmersing myself in Japanese culture. Thank you for givingme these precious experiences and memories.

INDEX
A ....................................................................................................
ADACHI, Daisuke
AIHARA, Yusuke
AJIOKA, Rie

AKAMA, Akiko

AKIMOTO-KATO, Ai
AKITA, Asaka
AOKI, Etsuko

ASAO, Hisayo

ASAOKA, Miho
ATSUMI, Miho

ATSUMI, Urumi

64

HASEBE, Mitsuyasu

47

HASHIMOTO, Terumi

7, 61

HASEGAWA, Ryota

64

HATTORI, Masayuki

81, 82

HAYASHI, Kentaro

71
38
17

BABA, Nayumi

64

BINO, Takahiro

71, 87

BAN, Misato

BISOVA, Katerina
CHEN, Qiuhong

18

CHISADA, Eriko

74

CHIBA, Hirokazu
CHISADA, ShinichiČUPEROVÁ, Zuzana

HIRAMATSU, MikaHIRANO, Kodai

HIYAMA, Takeshi

47

HOJO, Masaru

38

HONDA, Satoko

D ....................................................................................................
DAY, F. Timothy
DOKYO, Yumi

HORIUCHI, Yuta

21
29

E ....................................................................................................
EBINA, Teppei

34

FUJIKAWA, Akihiro

29

FUJIMORI, Sayumi

18

HOSHI, Rie

FUJIMORI, Toshihiko
FUJIMOTO, Daiji

FUJISAWA, Chiemi
FUJITA, Hironori

38

H

GU, Nan

4

13, 79HANDA, Yoshihiro

41

HAMATANI, Ayako
HARA, Ikuyo

HARA, Kenshiro

81
47

7, 24

84, 87
38
58
34
58
68

38

ISOSHIMA, Yoshiko

47
55
24
29

ITO, Kazuyo

53, 87

ITO, Yukiko

18

ITO, Tomoaki

J

41

ISHIKAWA, Masaki
ISHIZAKA, Miho

38

71, 86, 87

73, 87ISHINE, Naomi

13

HAMADA, Yoshio

61

ISHIKAWA, Azusa
ISHIKAWA, Hiroe....................................................................................................
69, 70
HACHISUKA, Midori

HAKOSHIMA, Toshio

38

24

ISHIBASHI, Tomoko

38

GOTO, Misako

17

IKAMI, Kanako

INABA, Kayo

41

77

72

58, 88IMOTO, EikoG ....................................................................................................
GONDA, Naoko

29

IGUCHI, Taisen

IMAIZUMI, Taeko

41

GODA, Misato24

73, 86, 87

IMAI, Akihiro

17

81, 82

FUKUSHIMA, Kenji

38

ICHIKAWA, Chiaki

IINUMA, Hideko

77

FUKUMOTO, Tatsuya

FUKUHARA, Mai

34

53, 79ICHIKAWA, Michiko4, 21, 77, 85, 90

41, 8734

7, 24

HOSHINO, Atsushi

ICHIKAWA, Mariko27

FUKADA-TANAKA, Sachiko50

I ....................................................................................................

F ....................................................................................................
FUJIMORI, Chika

434

HIRAKAWA, Reiko

66

4

HIMENO, Miki
HIRA, Seiji

C ....................................................................................................

29

21

HIRA, Riichiro

38

58

HIGUCHI, Yoko

HIKINO, Kazumi

85

14

7, 17

HIGASHIYAMA, Tetsuya

B ....................................................................................................

74

HAYASHI, Yoshiki

HIGASHIJIMA, Shin-ichi

34

29

77, 87HIGASHI, Satoru

77

34

HAYASHI, Kohji

HAYASHI, Tomoko

71

4, 38, 84

IWASE, Etsuko

2134....................................................................................................
JOHZUKA, Katsuki
55

K ....................................................................................................
KABEYA, Yukiko

KADOWAKI, Tamaka

38, 8761

93

INDEX

KAJIKAWA, Ikumi

38

KONISHI, Mie

29

KAKIZUKA, Taishi

69

KOSHIMIZU, Shizuka

38

KAJIURA-KOBAYASHI, Hiroko
KAMADA, Konomi

KAMADA, Yoshiaki
KAMEI, Yasuhiro

KAMEMIZU, Chizuru
KAMEOKA, Hiromu
KAMIDA, Chiharu

KAMIGAKI, Akane

61
49

73, 74, 86, 90, 91
21
41
38
50

KAMIKAWA, Yasunao

7, 12

KAMRANI, Yousef Yari61, 89KAMIYA, Kiyomi
KANAI, Masatake

KANEKO, Hiroyo

KATAOKA, Yukari

KATAYAMA, Kaori
KATO, Azusa

KATO, Hiroki

84
50
47
87
10
21
61

KATO, Kagayaki

56, 91

KAWADE, Kensuke

7, 70

KAWAGUCHI, Haruka

44

KATO, Kyoko

KAWAGUCHI, Colin

KAWAGUCHI, Masayoshi
KAWAI, Hisako

KAWAMOTO, Yuri

KAWASHIMA, Takeshi
KIDA, EmiKIKUCHI, Ayaka

KIKUCHI, Mariko
KIM, Eunchul

50
85

4, 41, 81
61
17
38
61
61
27
61

KIMORI, Yoshitaka

57, 91KINOSHITA, Chie

27

KIMURA, Tetsuaki

KINOSHITA, Noriyuki
KISHIMOTO, Mariko
KITADATE, Yu

KOBAYASHI, Satoru
KOBAYASHI, Yuki
KODAMA, Akiko

81, 83
14
61
24

7, 17
41
29

KODAMA, Ryuji

46, 84, 85

KOIKE, Chieko

47

KOJIMA, Yoko

38, 61KOMORI, Akio

2

KOHSHIMA, Shiro
KOIKE, Yukari

KOMINE, Yuriko
KON, Yayoi

KONDO, Maki

KONDO, Masashi
KONDO, Shigeru

94

87

4

47

32, 52
24

73, 8734
4KONISHI, Yusuke
KOSUGE, Kotaro
KOTANI, Keiko

KOYAMA, Hiroshi
KUBOKI, Yuko

KUBOYAMA, Kazuya
KUME, Shoen

44
61
34

21, 91
24
29
4

KURATA, Tomoko

85

KURUMIZAKA, Hitoshi

4

KURIHARA, Misuzu

69

L ....................................................................................................
LI, Chen38

LI, Ping

38

LI, Liu

LIN, Chia-Hao
LIU, Meng

38
29
41

M ....................................................................................................
MAEDA, Taro41, 72, 90

MANO, Hiroaki

38

MAKINO, Yumiko
MANO, Shoji

MARUYAMA, Ayumi

MARUYAMA, Shin-ichiro
MASAMIZU, Yoshito
MASE, Mutsuki

MATSUBAYASHI, Naomi
MATSUDA, Chisato

MATSUDA, Takashi

MATSUDA, Yoshimi

MATSUMOTO, Masahito
MATSUMOTO, Miwako

MATSUMURA, Kunihiro
MATSUOKA, Hikaru

MATSUZAKI, Masanori
MATSUZAKI, Yoko
McNABB, Nicole
MII, Yusuke

71, 87
50
24
61
34
44
81
10
29

84, 87
29
71
77
41
34
38
58
18

MINAGAWA, Jun

4, 61

MIWA, Tomoki

76, 87

MIURA, Seiko

MIYAGAWA, Shinichi
MIYAGI, Asuka

MIYAKAWA, Hitoshi

MIYAKAWA(OKAMOTO), Misato
MIYAKE, Satoko

MIYANARI, Yusuke
MIYATA, Haruko

29
58
14

7, 58
58
14
69
87

MIZUGUCHI, Hiroko

24, 87MIZUTANI, Yoshiyuki

41

MIZUTANI, Takeshi

58, 87

INDEX38

OHSUMI, Yoshinori

MORISHITA, Mio

38

OKA, Naomi

MOROOKA, Naoki

79, 87

MURATA, Takashi

38, 91

MORI, Asuka

MORI, Tomoko

MORITA, Shunpei

MURAKAMI, Michiyo

71, 87
17
14

N ....................................................................................................
NAGAE, Miwa

NAKAGAWA, Toshinori

41

7, 24

NAKAGAWA, Tomomi

41

NAKAMURA, Ryoko

53

NAKAI, Atsushi

NAKAMURA, Takanori
NAKAMURA, Yoshiaki

64

21

NAKAYAMA, Kei

10

NARUSE, Kiyoshi

47, 77, 81, 91

NISHI, TayoNISHIDA, Hanna

NISHIDE, Hiroyo

37
14

4, 7, 441438
4176, 87, 90

NISHIMURA Akiko

86

NISHIMURA, Mikio

85

NISHIMURA, Toshiya

7, 27

NISHIMURA, Keiko24

NISHIMURA, Noriko

87

NISHITANI, Kazuhiko
NODA, Chiyo

NODA, Masaharu
NOGUCHI, Yuji

NOMURA, Kengo

NONAKA, Shigenori
NONAMI, Yuta

4

17, 61, 87
4, 29

77, 8729

68, 90, 91
24

NOSE, Akinao

4O’BRIEN, Devin Mackenzie

44

O ....................................................................................................
OGAWA, Kota

7, 72

OGINO, Yukiko

58

OGAWA, Yuko

41

OHARA, Kaori

34

OHASHI, Rie

OHDE, Takahiro

OOI, Shoko

OTA, Kyoko

OTA, Ryoma

OTAKE, Norihito
OTSUBO, Yoko

18

41, 64
38
85
17
27
8

S ....................................................................................................

7, 24

NAKATSUKASA, Mana

NINOMIYA, Hiromasa

OKUBO, Masayo

21, 8776, 87

NAKANOH, Shota

NIIMI, Teruyuki

OKADA, Kazunori

476, 87

P ....................................................................................................

NAKANISHI, Norie

NEGISHI, Takefumi

OKA, Sanae

7

50

29

NAKAYASU, Tomohiro

OHTA, Kunihiro

10, 897, 44

OHKUBO, Fuki

34

OHNO, Kaoru

51

OHNISHI, Norikazu

61

OHSAWA, Sonoko

34, 87PALFALVI, Gergo38

SADAKANE, Osamu

7, 32

SAITO, EikoSAITO, Junko
SAKAE, Yuta

SAKAGAMI, Mari

44
34
27
8

SAKUTA, Hiraki

29

SANJO, Kazuko

86

SANBO, Chiaki

SASADO, Takao
SATO, Kaori

69
47
17

SATO, Masanao

7, 17, 90

SENGA, Takumi

64

SAWADA, Kaoru
SHI, Dongbo

SHIBATA, Emiko

SHIBATA, Tomoko
SHICHINO, Yuichi

SHIGENOBU, ShujiSHIINA, Nobuyuki

SHIMIZU-MITAO, Yasushi
SHIMMURA, Tsuyoshi
SHIMO, Takayuki
SHINOMIYA, Ai

SHINOTSUKA, Takanori
SHINOZUKA, Takuma
SHINOZUKA, Yuko

SHINTANI, Takafumi
SHIRAISHI, Naomi
SUDA, Hiraku

SUGAYA, Tomomi
SUGIMORI, Seiko

SUGINAGA, Tomomi
SUGIURA, Haruka

SUGIYAMA, Arisa

SUGIYAMA, Kazuya

SUGIYAMA, Tomomi
SUMIYA, Eri

SUZAKI, Momoyo

84, 872187
38
8

71, 72, 86, 90
10
41
64
64
64
34
18
17
29
27
38
38
17

77, 79
34
17
69
34
58
41

95

INDEXSUZAKI, Takuya

7, 41

TSUKAYA, Hirokazu

70

SUZUKI, Keiko79, 87TSUZUKI, Shihoko

87

SUZUKI, Makoto

14

TUROCZY, Zoltan

38

SUZUKI, Ayami
SUZUKI, Kohta
SUZUKI, Miho

SUZUKI, Miyuzu
SUZUKI, Ryoko

SUZUKI, Tokiko

34

TSUKITA, Sachiko77, 87
1472

TSUZUKI, Shusaku

UCHIKAWA Tamaki
UCHIYAMA, Ikuo
UEDA, Chizuru

47

T ....................................................................................................
69

TAKADA, Shinji

4, 18, 86

TAKAGI, Yukari

77, 87

TAKADA, Ritsuko
TAKAGI, Chiyo

TAKAHASHI, Hiroki
TAKAHASHI, Ritsue
TAKAHASHI, Sara

TAKAHASHI, Shunichi
TAKAHASHI, Yoichi
TAKAKI, Chikako

TAKASHIRO, Kayoko
TAKEDA, Naoya
TAKEDA, Yuta

TAMAOKI, Daisuke

TANAKA, Yasuyo
TANGA, Naomi

TANIGUCHI- SAIDA, Misako
TANIGUCHI, Atsushi

TATEMATSU, Kiyoshi
TERADA, Shin-Ichiro
TESHIMA, Yuuko
TOHYAMA, Saki
TOKUE, Moe

TOKUTSU, Ryutaro

TOMINAGA, Hitoshi
TORIBA, Taiyo
TORII, Naoko

TOYOOKA, Yayoi
TOYOTA, Kenji

TSUBOUCHI, Tomomi
TSUCHIYA, Yoshihiro
TSUGANE, Kazuo
TSUGE, Toyoko

14

YAMAGUCHI, Katsushi
YAMAGUCHI, Takeshi

27
50
18

1, 2, 8YAMAMOTO, Yasuhiro

27

YAMASHITA, Akira
YASUE, Naoko

86
47

YORO, Emiko14

YOSHIDA, Akihiro

38
47

Z

58
47
61
41
46

4, 24, 71, 85, 86

YOSHINORI, Yumi

41

YU, Yang

7, 12, 86

12

YOSHIDA, Shosei

YOSHIMURA, Takashi

21

7, 58

8

14

44

YONEZAWA, Harumi

61

61

YATOMI, Johichiro
YOKOI, Saori

24

14

7, 37

YATSU, Ryohei

58

17

YASUGI, Masaki
YASUI, Naomi

34

14

YAMAMOTO, Masayuki

YAMASAKI, Hiroaki

68

71, 87, 90
7, 32

YAMAMOTO, Takamasa

29

18

50, 70

YAMAMORI, Tetsuo

YAMAMOTO, Manami

73, 87

14

WATANABE, MikaYAMAGUCHI, Chinami

34

54, 791737, 77

YABE, Taijiro

27, 77

18

18

Y ....................................................................................................

38

34

29

WATANABE, Eiji

WEICHSEL, Alexander

38

TANAKA, Yasuhiro

18, 87

7, 32

WATANABE, Etsuko

61

7, 81, 82

21

WATAKABE, Akiya
WATAKABE, Ikuko

41

TANAKA, Daisuke
TANAKA, Minoru

WASHIO, Midori

1829, 87TAMADA, Yosuke

WANGLAR, Chimwar

47

TAKEUCHI, Yasushi
TAKIZAWA, Kenji

WADA, Kotoe

34

53

87

18

W ....................................................................................................

61

47, 91

UNO, Satoko

UTSUMI, Hideko

61

34

50

2 ,4, 14, 85, 86, 90

USAMI, Fumiko86

TAKEHANA, Yusuke
TAKEUCHI, Tomoyo

96

14

73, 8766, 76, 90

UENO, Naoto
UKAI, Sakie

18

14, 87

41

U ....................................................................................................

29

TAGAWA, Ayako

4

YUZAKI, Karen4, 7, 642944....................................................................................................
38
ZHANG, Liechi

Access

From Tokyo

Take the Tokaido Shinkansen and get off at Toyohashi, change to
the Meitetsu Line and get off at Higashi Okazaki Station(approximately 20 minutes from Toyohashi).

From Osaka

Take the Tokaido Shinkansen and get off at Nagoya, change to
the Meitetsu Line and get off at Higashi Okazaki Station(approximately 30 minutes from Nagoya).

From Central Japan International Airport

Get on the Meitetsu bus bound for JR Okazaki Station andget off at Higashi Okazaki Station(approximately one hour from the airport). Alternatively,
take the Meitetsu Line for Nagoya and change at Jingu-mae Station toa Toyohashi-bound train and get off at Higashi Okazaki Station(approximately 70 minutes from the airport).
City HallCity HallCity Hall S.

From Higashi Okazaki Station to Each Area
Turn left (south) at the ticket barrier and exit the station.
The Institute is a 7-minute walk up the hill
(Myodaiji-area) or 20-minute walk (Yamate-area).

By carTake the Tomei Expressway to the Okazaki Exit.
Turn left at the City Hall S. signal(approximately 10 minutes from the Exit).

National Institutes of Natural Sciences

National Institutefor Basic Biology
38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585 Japan
tel: +81 564-55-7000 fax: +81 564-53-7400
http://www.nibb.ac.jp/en/
Issued in October 2016
Edited by the Research Enhancement Strategy Office, NIBB