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National Institute for Basic Biology

2016 ANNUAL REPORTCONTENTS
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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 2016 ・
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・ 7
Celebrating the Work of Nobel Laureate Prof. Yoshinori Ohsumi・
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・ 8
Cell Biology
Division of Cellular Dynamics ・
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・ 10
Division of Quantitative Biology・
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・ 12
Division of Chromatin Regulation・
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・ 14
Laboratory of Cell Responses・
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・ 16
Laboratory of Neuronal Cell Biology・
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・ 18
Laboratory of Stem Cell Biology・
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・ 20
Developmental Biology
Division of Morphogenesis・
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・ 21
Division of Molecular and Developmental Biology・
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・ 24
Division of Embryology・
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・ 27
Division of Germ Cell Biology・
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・ 30
Laboratory of Molecular Genetics for Reproduction・
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・ 33
Neurobiology
Division of Molecular Neurobiology・
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・ 35
Division of Behavioral Neurobiology・
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・ 38
Division of Brain Circuits・
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Laboratory of Neurophysiology・
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Evolutionary Biology and Biodiversity
Division of Evolutionary Biology・
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Division of Symbiotic Systems・
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Division of Evolutionary Developmental Biology・
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・ 49
Laboratory of Bioresources ・
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・ 51
Laboratory of Morphodiversity ・
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・ 53
Laboratory of Biological Diversity・
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・ 54
Environmental Biology
Division of Molecular Environmental Endocrinology・
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Division of Environmental Photobiology ・
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・ 66
Division of Seasonal Biology (Adjunct)・
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・ 69
Theoretical Biology
Laboratory of Genome Informatics・
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・ 71
Imaging Science
Laboratory for Spatiotemporal Regulations ・
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・ 73
Okazaki Institute for Integrative Bioscience (ORION Project)
Laboratory of Nuclear Dynamics・
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・ 74
Okazaki Institute for Integrative Bioscience (BIO-NEXT Project)
Laboratory of Plant Development and Physiology・
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・ 75
Research Support
NIBB Core Research Facilities・
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・ 76
NIBB BioResource Center・
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・ 83
NIBB Center of the IBBP・
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Bioimaging Forum・
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NIBB Training Courses・
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Access to NIBB ・
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・Inside back cover

The cover items are related to a paper titled “A blue light photoreceptor mediates the feedback regulation ofphotosynthesis” (Petroutsos, D., Tokutsu, R., et al., Nature 2016) from the laboratory of Prof. Minagawa. The paper
demonstrated the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green
alga Chlamydomonas reinhardtii. See page 66 of this report for details. See also page 79 for the instrument “Okazaki
Large Spectrograph” used in this research.

INTRODUCTIONI

t is my great pleasure to introduce to you all the2016 Annual Report of the National Institute for
Basic Biology (NIBB), which outlines the research activitiesof the Institute and its effective function as a center for collaborative research in Japan over the last year. Given theseimportant missions of the Institute, I recognize my strong
responsibility to maintain the liberal and forward-looking
atmosphere for research and discussion that NIBB has kept
for many years, which I believe is a basis for these remarkable activities.
2016 was a marvelous year for us all, as Dr. Yoshinori
Ohsumi, a Professor Emeritus of NIBB, won the Nobel Prizein Physiology or Medicine for his outstanding research on
autophagy. He ran his laboratory as a Professor in NIBB
from 1996 to 2009, and he recalls this period as the golden
age of autophagy study. We congratulate his great achievements, and are proud of the efficacy of NIBB that fostered
his science.
The Japanese government continues to ask universities andinter-university research institute corporations, the latter ofwhich includes NIBB, to strengthen performance and gain
profits from science. It is also necessary for the scientificcommunity to achieve full compliance and establish integrityin 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 in accomplishinggood research in basic biology. Good science, even in a basic
research field, will eventually benefit human beings. The
history of science tells us this is true. The other is that NIBB
must achieve high ethical standards for research that conformto the era of computer technology and data sharing throughthe 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 2016. I would like to note that we welcomed several newcolleagues in 2016, including four professors, one speciallyappointed professor, one associate professor, five assistantprofessors, one specially appointed assistant professor, andone NIBB research fellow, while nine colleagues transferred
to other institutes.
Besides Prof. Ohsumi, I would like to congratulate Professor Mitsuyasu Hasebe for winning the Japan’s
Eminent Evolutionalist Award, and Dr. Yoshiaki Nakamura
for winning an Outstanding Presentation Award from anacademic society, 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
November 28, 2017
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. 90) 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, 2017Research Units■Division of Cellular Dynamics  Cell Biology■Division of Quantitative Biology†††■Division of Chromatin Regulation■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■Division of Molecular Neurobiology  NeurobiologyEvaluation and Information GroupPublic Relations GroupInternational Cooperation GroupCollaborative Research GroupGender Equality Promotion Group■Division of Behavioral Neurobiology††■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) 16
Technical
Assistants
andSecretaries

Associate Professors(including specially appointed) 16
Assistant Professors(including specially appointed)

37

105

55
55

25

Graduate Students(including 1 undergraduates)

Technical Staff

Indirect Costs in Grants

135

Other Grants

90

External
Competitive
Fund

778

Grant-in-Aid
for Scientific
Research(settled forApril 2016-March 2017)
numbers are in million yen

Personnel

392

Grants forCommissioned Research

156

Management Expenses
Grant for SOKENDAI

54

Facility■ Members of the Advisory Committee for Programming and Management

NIBB members# Chairperson## Vice-Chair* new member from April 2017

4732

Other 3

Management
Expenses Grant
for NIBB

1,432
Research

195 Collaboration
265

42

AGATA, Kiyokazu*
HIRAOKA, Yasushi*
KOHCHI, Takayuki*
KOHSHIMA, Shiro
KUME, Shoen
NISHITANI, Kazuhiko ##NOSE, Akinao
SASAKI, Hiroyuki*
SUGIMOTO, Asako*
YAMAMOTO, Takashi
FUJIMORI, Toshihiko
HASEBE, Mitsuyasu #HIGASHIJIMA, Shin-ichi
KAWAGUCHI, Masayoshi
MINAGAWA, Jun
NIIMI, Teruyuki
NODA, Masaharu
TAKADA, Shinji
UEDA, Takashi*
UENO, NaotoYOSHIDA, Shosei

310 in total(as of 1st April, 2017)■ Financial Configuration of NIBB

Donation,

Non-NIBB members

Postdoctoral Fellows(8 are NIBB Research Fellows)237

2,306 million yen
in total(as of April, 2017)

Professor, Gakushuin University
Professor, Osaka University
Professor, Kyoto University
Professor, Kyoto University
Professor, Tokyo Institute of Technology
Professor, Tohoku University
Professor, The University of Tokyo
Professor/ Senior Vice President, Kyushu University
Professor, Tohoku University
Professor, Hiroshima University
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
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
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 withtravel and lodging expenses to visit NIBB’s laboratories
for collaborative research. “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. In 2016 two
new projects, Collaborative research projects for integrative
genomics and Collaborative research projects for integrative bioimaging, were initiated by reorganizing two former
projects to facilitate more integrated use of the NIBB Core
Research Facilities and to allow more intensive support
through the planning, experimental, data analysis, and publication stages. Travel and lodging expenses are provided forthese projects.
year
Priority collaborative research projects
Collaborative research projects for model organisms
and technology development
Individual collaborative research projects
NIBB workshops
Collaborative experiments using the Large Spectrograph
Collaborative experiments using the DSLM
Bioimage processing and analysis collaborative research projects
Collaborative experiments using the next generation
DNA sequencer
Support for NIBB training courses
Collaborative research projects for bioresource
preservation technology development
total

2013

2014

2015

4

2

3

2

89
4
15
9

87
3
12
10

88
6
10
11
14

46
6
10

41

37

46

59**

1

0

1

0

9

10

9

12

174

162

190

175

2

1

2

2016
2

38*

Collaborative Research Projects by Year*renovated as "Collaborative research projects for integrative bioimaging"**renovated as "Collaborative research projects for integrative genomics"

International cooperation
and OutreachContribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers96). The Spectrography and Bioimaging Facility manages
research tools, such as confocal microscopes, DSLM and thelarge 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 as acenter 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. 83).

■ 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. 76).
The Functional Genomics Facility maintains a wide arrayof core 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.

An example of a medaka strain in NBRP, Gaudi strain, in which individual cells in the brain and the retina are fluorescently labelled using
Brainbow system.

■ 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 invaluable5

bioresources under cutting-edge research (p. 86).

International Cooperation and Outreach■ Collaborative Programs with Overseas Institutes

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, Princeton University,and the Max Planck Institute for Plant Breeding Research(MPIPZ) to promote joint research projects, collaborative
symposia, training courses and student exchange programs.

■ NIBB Conference

The NIBB Conferences are international conferenceson 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
in basic biology with valuable opportunities for international exchange. The 64th conference “Evolution of Seasonal
Timers” was held in April, 2016 (p. 93).

■ International Practical Course

With the cooperation of researchers from Japan and abroad
the NIBB international practical course is given at a laboratory specifically prepared for its use. The 9th course “Genetics
and Imaging of Medaka and Zebrafish” was held jointly with
TLL in August, 2016 at NIBB (P. 94). Graduate students andyoung researchers from various areas including Argentina,
Korea, Singapore, Germany, India, Taiwan, UK, and Japan,
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
was held in October, 2016 at which we welcomed more than
4,700 local citizens. NIBB also cooperates in the educationof undergraduate and younger students through lectures
and workshops. Outreach activities are mostly managed by
the Public Relations Group of the Research Enhancement
Strategy Office.

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 10th Forum “ Pioneering Bioimaging in the
New Era “ was held in February, 2016 (p. 96). A training
course in bioimage analysis was also held in 2016 (p. 97).

■ 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.
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. 98).

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 developed an improved model using two-photon optics (p. 73).

6

Graduate students educated by NIBB■ Personnel changes in 2016*Newly assigned in NIBB
NamePositionResearch UnitDate

UEDA, TakashiProfessorDivision of Cellular DynamicsApril 1AOKI, KazuhiroProfessor Division of Quantitative BiologyApril 1HIGASHIJIMA, Shin-ichiProfessor Division of Behavioral NeurobiologyApril 1

SOYANO, TakashiAssociate ProfessorDivision of Symbiotic Systems

April 1EBINE, KazuoAssistant ProfessorDivision of Cellular DynamicsApril 1

KIMURA, YukikoAssistant ProfessorDivision of Behavioral NeurobiologyApril 1MORITA, ShinichiNIBB Research FellowDivision of Evolutionary Developmental BiologyApril 1ANDO, ToshiyaAssistant ProfessorDivision of Evolutionary Developmental BiologyJuly 1IBBP Center

September 1

Division of Germ Cell BiologyOctober 1

TAKETSURU, Hiroaki
NARUSE, KiyoshiSpecially Appointed Assistant Professor
Specially Appointed Professor

NAKAYAMA, Jun-ichiProfessorDivision of Chromatin RegulationOctober 1

HAMADA, KyokoAssistant ProfessorDivision of Chromatin RegulationOctober 1

KATAOKA, KensukeAssistant ProfessorDivision of Chromatin RegulationDecember 1Newly affiliated in other universities and institutesNameNew AffiliationPositionDate

NISHIMURA, ToshiyaNagoya UniversityAssistant ProfessorMarch 1OGINO, YukikoKyushu UniversityAssociate ProfessorApril 1MASAMIZU, YoshitoUniversity of TokyoAssistant ProfessorApril 1

IMAI, AkihiroHiroshima Institute of TechnologyAssistant ProfessorApril 1MATSUZAKI, Masanori University of TokyoProfessorApril 1EBINA, TeppeiUniversity of TokyoAssistant ProfessorApril 1

TANAKA, MinoruNagoya UniversityProfessorJune 1

MIYAGAWA, ShinichiWakayama Medical UniversityLecturerJune 1Tokyo University of Agriculture and TechnologyAssociate Professor (Tenure-Track)November 16

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

■ Awardees in 2016NamePositionOHSUMI, YoshinoriProfessor EmeritusHASEBE, MitsuyasuProfessor

NAKAMURA, Yoshiaki

JSPS ResearcherAward
The Nobel Prize in Physiology or Medicine, The Order of Culture (Bunka Kunsho), Dr. Paul Janssen Award for Biomedical Research, The Breakthrough Prize in Life SciencesSociety of Evolutionary Studies, Japan’s Eminent Evolutionalist Award
The Society for Reproduction and Development’s Outstanding Presentation Award

7

Celebrating the Work of Nobel Laureate Prof. Yoshinori Ohsumi

Prof. Yoshinori Ohsumi’s laboratory at NIBB:
Fermentation of yeast cell autophagy research
NIBB Professor Emeritus Yoshinori Ohsumi won the Nobel
Prize in Physiology or Medicine for 2016 for his discoveriesof the mechanisms for autophagy¶. Dr. Ohsumi presided overthe Division of Molecular Cell Biology at NIBB for 13 years,
from 1996 to 2009. It is a great honor for me, as an alumnus
of his laboratory, to introduce to you a brief summary of the
fruitful progress of Dr. Ohsumi’s work.
In April 1996, Dr. Ohsumi came to NIBB from the
University of Tokyo, with 13 yeast “apg” mutants, whichhave defects in autophagy*. At NIBB he made many
advancements with his colleagues to investigate the molecular mechanisms of autophagy: cloning of yeast ATG genes,
screening of additional ATG genes, biochemical and cellbiological characterization of Atg proteins, and extensive
autophagy studies in higher eukaryotes.
The main research achievements of Dr. Ohsumi’sLaboratory are as follows:Firstly, 18 Atg proteins were classified into 5 subgroups on
the basis of biochemical analyses:(1) Atg1 kinase and its regulators1, recognizing nutrient starvation, an inducing signal of autophagy;(2) Phosphatidylinositol 3-kinase complex2, generating phosphatidylinositol 3-phosphate (PI-3P), an important material
for autophagosome;(3) Atg12 conjugation system3;(4) Atg8 conjugation system4, both of which are ubiquitinrelated systems, revealing that two intracellular protein
degradation process, autophagy and ubiquitin-proteasome,
employ a similar molecular device;(5) Atg2-Atg18 complex and Atg95, PI-3P binding proteins
and a membrane-integrating protein, respectively.
Secondly, fluorescence microscopy studies revealed thatthese Atg proteins hierarchically assemble together at “PAS”(pre-autophagosomal structure) to generate autophagosome6.
Through these yeast studies, they established a schematic
molecular model of autophagy7. And lastly they found thatthese findings derived from yeast studies are fundamentallyconserved among another eukaryotes, especially mammals8
and plants9, to create a universal field of study of autophagy,
an important protein degradation system.

References
1. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., andOhsumi, Y. (2000) Tor-Mediated Induction of Autophagy via an Apg1
Protein Kinase Complex. J. Cell Biol. 150, 1507-1513.
2. Kihara, A., Noda, T., Ishihara, N., and Ohsumi, Y. (2001) Two
Distinct Vps34 Phosphatidylinositol 3–Kinase Complexes Functionin Autophagy and Carboxypeptidase Y Sorting in Saccharomyces
cerevisiae. J. Cell Biol. 152, 519-530.
3.† Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George,
M. D., Klionsky, D. J., Ohsumi, M., and Ohsumi, Y. (1998) A protein
conjugation system essential for autophagy. Nature 395, 395-398.
4.† Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y.,
Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M.,
Noda T., and Ohsumi, Y. (2000) A ubiquitin-like system mediates
protein lipidation. Nature 408, 488-492.
5. Noda, T., Kim, J., Huang, W.-P., Baba, M., Tokunaga, C., Ohsumi,
Y., and Klionsky, D. J. (2000) Apg9p/Cvt7p Is an Integral Membrane
Protein Required for Transport Vesicle Formation in the Cvt andAutophagy Pathways. J. Cell Biol. 148, 465-479.
6. Suzuki, K., Kirisako, T., Kamada, Y., Mizushima, N., Noda, T., andOhsumi, Y. (2001) The pre‐autophagosomal structure organized byconcerted functions of APG genes is essential for autophagosome
formation. The EMBO J. 20, 5971-5981.
7. Nakatogawa, T., Suzuki, K., Kamada, Y., and Ohsumi, Y. (2009)
Dynamics and diversity in autophagy mechanisms: lessons from yeast.
Nature Reviews Molecular Cell Biology 10, 458-467.
8. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T.,
Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000) LC3, amammalian homologue of yeast Apg8p, is localized in autophagosome
membranes after processing. The EMBO J. 19, 5720-5728.
9. Hanaoka, H., Noda, T., Shirano, Y., Kato, T., Hayashi, H., Shibata, D.,
Tabata, S., and Ohsumi, Y. (2002) Leaf Senescence and Starvationinduced chlorosis are accelerated by the disruption of an Arabidopsis
autophagy gene. Plant Physiol. 129, 1181-1193.

(Yoshiaki Kamada)¶ https://www.nobelprize.org/nobel_prizes/medicine/laureates/2016/* Autophagy-related genes including “APG” genes were uniformly renamed as “ATG” genes in 2003.
†These two papers are selected as ‘Key Publications’ by the Nobel Committee.

8Dr. Ohsumi’s 60th birthday party (2005).

Dr. Ohsumi and Dr. Ohsumi’s Lab members with Dr. Aaron
Ciechanover, a ubiquitin researcher and a Nobel Laureate (2006).

Dr. Ohsumi and Dr. Ohsumi’s Lab members posing “Ina Bauer” (2006).

Executive members of the NINS and NIBB staff gathering at the unveiling
ceremony (2017).

Dr. Ohsumi visited NIBB for the unveiling ceremony of theNobel prize monument, inspired by an image of the budding yeast(2017).

9

National Institute for Basic Biology

Cell Biology

DIVISION OF CELLULAR DYNAMICS

to distinct trafficking events, and 3) secondary loss of conserved machinery components, during evolution.

Professor

UEDA, Takashi

Assistant Professor:
Postdoctoral Fellow:

EBINE, Kazuo
EBINE, Kazuo*
SHIMADA, Takashi L.
TSUTSUI, Tomokazu
Visiting Graduate Student: TAKEMOTO, Kodai
KANAZAWA, Takehiko
MINAMINO, Naoki
NORIZUKI, Takuya
Secretary:
OKUBO, Masayo

Membrane traffic between single membrane-boundedorganelles plays integral roles in various cell activities ineukaryotic cells. Recent comparative genomics has indicatedthat membrane trafficking pathways are diversified among
eukaryotic lineages, which is associated with lineage-specificacquisition of new trafficking pathways and secondary lossof preexisting trafficking routs. Our long-term goal is unraveling how plants have acquired their unique membrane trafficking systems during evolution, which will be achievedby comparative analyses using the model plant Arabidopsis
thaliana and a model of liverworts, Marchantia polymorpha.
We also aim to elucidate detailed molecular mechanisms andphysiological functions of membrane trafficking in higherordered plant functions.

I. Diversification of membrane trafficking
pathways associated with acquisition ofnovel machinery components

Although the basic framework of membrane trafficking iswell conserved among eukaryotic lineages, recent comparative genomics has suggested that each lineage has acquired
unique membrane trafficking pathways during evolution.
RAB GTPases and SNARE proteins are evolutionarily conserved key regulators acting in tethering and/or fusion of
membrane vesicles with target membranes. It has been
proposed that lineage-specific diversification of these key
factors is tightly associated with acquisition of lineage-specific membrane trafficking pathways, whose molecular basis
remains unknown.

1-1. Characterization of RAB and SNARE proteins in
the liverwort, Marchantia polymorpha

For information on the diversification of membrane trafficking pathways during land plant evolution, we systematically
identified RAB GTPases and SNARE proteins in Marchantia
polymorpha. Comparison of organization of these protein
families with other plant lineages, followed by their functional analyses in M. polymorpha, indicated that diversification of membrane trafficking pathways in land plants
has been achieved by 1) acquisition of novel machinerycomponents, 2) relocating conserved machinery components

10

Figure 1. SYP1 genes in the genomes of green plants. Genes are indicatedas individual boxes. Numbers of SYP1 genes increased during land plant
evolution, suggesting functional diversification of SYP1 members in land
plants.

1-2. Analysis of the liverwort-specific organelle, oilbodyThrough analyses of SNARE members in M. polymorpha,
we found that a member of the SYP1 group is localized tothe membrane of an organelle specific to liverworts, the oilbody, whose origin and mechanisms of biogenesis remain
unclear. We are currently analyzing the molecular function of
the SYP1 member, as well as characterizing membrane trafficking pathways responsible for oil body biogenesis. We arealso trying to isolate mutants defective in oil body functions
by a forward-genetic approach.

Figure 2. Oil bodies in a thallus of M. polymorpha, which are visualized
by the YFP-tagged SYP1 member.

1-3. Mechanisms and dynamics of vacuolar transport

The vacuole is the largest organelle in plant cells, andoccupies over 90 % of mature plant cells. The vacuole fulfills
various functions in plant physiology and development,
which include protein degradation, protein storage, and regulation of turgor pressure. To fulfill these vacuolar functions, awide variety of vacuolar proteins and other components must
be correctly transported to the vacuole, which is mediatedby membrane trafficking. To understand molecular mecha-

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2016. The former title isindicated by an asterisk (*).

nisms of vacuolar transport in plants, we analyzed molecular functions of RAB5 and RAB7, and found that these
proteins act sequentially in the vacuolar transport pathway
in Arabidopsis cells. Furthermore, we also found that two
additional vacuolar transport pathways, the RAB5-dependent
but RAB7-independent pathway and the AP-3-dependent
pathway operate in vacuolar transport in Arabidopsis. We
are now exploring details of molecular mechanisms of thesevacuolar transport pathways, especially focusing our intereston the RAB5-dependent but RAB7-independent pathway,
because this trafficking pathway has not been described in
non-plant systems.
SNARE proteins are also key molecules of vacuolar trafficking. Defective functions in vacuolar SNAREs affect both
vacuolar transport and vacuolar morphology, which are distinctly regulated in plant cells. The sgr3-1 (shoot gravitropism3) mutant was isolated as a mutant defective in shoot gravitropism, which resulted from a point mutation in SYP22/
VAM3, one of the SNARE proteins residing on the vacuole
and acting in vacuolar transport. Intriguingly, sgr3-1 exhibits
abnormal vacuolar morphology, although vacuolar transport
is not markedly affected in this mutant. Therefore sgr3-1
should be a good tool for dissecting functions of the vacuolar
SNARE. We are exploring vacuolar dynamics regulated by
SYP22/VAM3 by analyzing the effect of the sgr3-1 mutation
in a detailed manner.

II. Significance of membrane trafficking inhigher-ordered plant functions
2-1. Analyses of functions of the plant-specific RAB
GTPase ARA6 in stress responses in Arabidopsis
thaliana

ARA6 is a plant-unique RAB GTPase, whose close
homologs are found in only green plant lineages. To elucidate why only plants harbor the ARA6 members, weanalyzed functional significance of ARA6 in biotic andabiotic stress responses. We found that ARA6 is recruited
to the extrahaustorial membranes formed by the fungal
pathogen causing powdery mildew and the oomycete causing
downy mildew, suggesting modulation of host membranetrafficking by pathogenic microbes (Inada et al., 2016). We
also found that proliferation of a pathogenic bacterium isrepressed in the ara6 mutant, which was due to an enhanced
defense response mediated by elevated sugar concentration.

Publication List:〔Original papers〕

K., Hirai, M., Sakaguchi, M., Yahata, K., Kaneko, O., and Saito• Ebine,
Nakano, Y. (2016). Plasmodium Rab5b is secreted to the cytoplasmic

•
•
•
•

face of the tubovesicular network in infected red blood cells together
with N-acylated adenylate kinase 2. Malar. J. 17, 323.
Inada, N., Betsuyaku, S., Shimada, T.L., Ebine, K., Ito, E., Kutsuna, N.,
Hasezawa, S., Takano, Y., Fukuda, H., Nakano, A., and Ueda, T. (2016).
Modulation of plant RAB GTPase-mediated membrane trafficking
pathway at the interface between plants and obligate biotrophic
pathogens. Plant Cell Physiol. 57, 1854-1864.
Mbengue, M., Bourdais, G., Gervasi, F., Beck, M., Zhou, J., Spallek,
T., Bartels, S., Boller, T., Ueda, T., Kuhn, H., and Robatzek, S. (2016).
Clathrin-dependent endocytosis is required for immunity mediated bypattern recognition receptor kinases. Proc. Natl. Acad. Sci. USA 113,11034-11039.
Sakurai, H.T., Inoue, T., Nakano, A., and Ueda, T. (2016). ENDOSOMALRAB EFFECTOR WITH PX-DOMAIN, an interacting partner of
RAB5 GTPases, regulates membrane trafficking to protein storage
vacuoles in Arabidopsis. Plant Cell 28, 1490-0503.
Yoshinari, A., Fujimoto, M., Ueda, T., Inada, N., Naito, S., andTakano, J. (2016). DRP1-dependent endocytosis is essential for polar
localization and boron-induced degradation of the borate transporter
BOR1 in Arabidopsis thaliana. Plant Cell Physiol. 57, 1985-2000.

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

K., Kobayashi, M., Sato, M., Kutsuna, N., Ueda, T., Toyooka, K.,• Akita,Nagata, N., Hasezawa, S., and Higaki, T. Accumulation of fluorescent•proteins derived from a trans-Golgi cisternal membrane marker andparamural bodies in interdigitated apoplastic space in Arabidopsis leaf
epidermis. Protoplasma 2016 Mar 9.
Cui, Y., Zhao, Q., Xie, H.T., Wong, W.S., Gao, C., Ding, Y., Tan, Y.,
Ueda, T., Zhang, Y., and Jiang, L. MON1/CCZ1-mediated Rab7
activation regulates tapetal programmed cell death and pollen
development in Arabidopsis. Plant Physiol. 2016 Oct 31.

2-2. Membrane trafficking in plant gametogenesis

Gametogenesis in plants also involves membrane trafficking-mediated processes. We are now analyzing molecularmechanisms of gametogenesis in Arabidopsis and M. polymorpha, especially focusing our interests on secretory anddegradative trafficking pathways during male gamete formation.

11

National Institute for Basic Biology

Cell Biology

Professor

AOKI, Kazuhiro

Visiting Graduate Student: KOMATSUBARA, Akira
MIURA, Haruko
UDA, Youichi
MARYU, Gembu
Technical Assistant:
ITO, Reina
Secretary:
ONODA, KaoriThe Division of Quantitative Biology has been launched in
April 2016. We are interested in the molecular mechanisms
underlying cell-fate decision in mammalian cells.
Living cells act as input-output (I/O) units, in which environment and/or internal states are recognized on the cellsurface and processed within a cell, leading to the adaptive
response to these changes. This cellular information processing is mainly controlled by intracellular signal transduction,
which is a series of chemical reactions, most commonly
protein phosphorylation. Importantly, the dysregulation of
signal transduction by gene mutation results in pathologicaldiseases such as cancer.
The intracellular signaling pathway has been extensively
studied over the last few decades, and most of its proteins
and regulations have been identified, causing the perceived
increase in the pathway’s complexity. Computer-assisted
simulation is one of the most promising approaches forthe understanding of the signal transduction pathway as asystem. Indeed, chemical and physical reactions comprising
signal transduction can be described by ordinary differential
equations, and solved numerically by computers. A numberof simulation models of signaling pathways have, in fact,
been reported to date. However, most of the kinetic parameters utilized for the simulation models were not measured
experimentally, but rather were assumed by fitting the experimental data with the simulation model or simply determined
arbitrarily. Consequently, there are substantial differences in
the kinetic parameters between these studies, thereby making
it difficult to evaluate these simulation models quantitatively.
To address these issues, we currently focus on the development of research tools enabling us to (1) visualize, (2)quantify, and (3) manipulate intracellular signaling pathways
with fluorescence imaging techniques and computational
approaches.

I. Visualization of cell signaling

With the advent of fluorescent proteins, it has been possible
to visualize kinetic reactions at the single cell level. We areusing the following two different approaches to unveil when
and where the intracellular signaling event takes place:

1-1. Development of FRET-based biosensors

Förster (or fluorescence) resonance energy transfer (FRET)
is a non-radiative process, in which excitation energy ofdonor fluorophore is transferred to a nearby acceptor fluo-

12rophore. By taking advantage of the principles of FRET, wehave developed several biosensors that allowed us to detectkinase activity of PKA, ERK, Akt, JNK, PKC, S6K, and soon in a living cell with high temporal and spatial resolution(Figure 1).

PKA

ERK

Akt

JNK

PKC

S6K

Figure 1. Visualization of PKA, ERK, Akt, JNK, PKC and S6K activities by FRET imaging. HeLa cells expressing FRET biosensors for theindicated kinases were stimulated with each ligand. Red and blue colors
indicate activation and inactivation of the kinase, respectively.

1-2. Imaging of multiple kinase activities

Although FRET biosensors enable us to monitor cell signaling events, FRET imaging is not suited for multiplexed
imaging, because these biosensors are authentically comprised of two different fluorophores in many cases. To circumvent this limitation, we are currently developing biosensors based on the principle of the kinase translocation
reporter (KTR) system; the reporter is translocated from
nucleus to cytoplasm when it is phosphorylated by its target
kinase (Figure 2A). This approach yields several kinase
reporters, e.g., ERK, Akt, p38, and JNK, and will provide
clues as to how cells emerge all-or-none and irreversible
cellular events such as cell cycle progression and apoptosis
by using analog and continuous kinase signals as an input(Figure 2B).
A

Cytoplasm
KTR

KinaseP

KTR
Phosphorylation

KTR

B

P

Dephosphorylation

KTR
Nucleus

ERK-KTR-mKO

Akt-KTR-EGFP

mCherry-Gem

0 20 40 60 80
Time (min)

0 20 40 60 80
Time (min)

Low

G1

EGF stimulation

Cells

DIVISION OF QUANTITATIVE BIOLOGY

0 20 40 60 80
Time (min)
Low

High

ERK activity

Akt activity

High

Cell cycle

S/G2/M

Figure 2. Multiplexed imaging of ERK and Akt activities and cell cycle
state. A, Schematic representation of the principle of KTR system. B,
Shown here are heat maps of ERK activity (left), Akt activity (middle),
and cell cycle (right) obtained by KTR systems.

II. Quantification of cell signaling

Classically, kinetic parameters such as protein concentration and dissociation constant have been measured by in vitro
biochemical analyses. However, some kinetic parameters
might significantly differ between in vitro and in vivo. For
instance, the Kd values measured in vivo were higher thanthe in vitro Kd values by an order of 1 or 2. Therefore, it iscritical to measure kinetic parameters in living cells. To thisend, we combine CRISPR/Cas9-mediated genome editing
with fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) to quantitatively determine the endogenous protein concentration andthe dissociation constant (Figure 3). These parameters will beapplied to the computer simulation to predict the dynamics of
singling pathways.
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
- 0.01G (∆t)

Auto correlation: ERK2-mEGFP
Auto correlation: RSK2-HaloTag:TMR
Cross correlation: ERK vs RSK2

101

102

103
104
∆t (μs)〔Original papers〕

K., Oda, K., Aoki, K., Sone, K., Ikeda, Y., Miyasaka, A.,• Inaba,
Kashiyama, T., Fukuda, T., Makii, C., Arimoto, T., Wada-Hiraike,

•
•
•

106

105

Publication List:

Figure 3. Auto- and cross-correlation functions of ERK2-mEGFP and
RSK2-HaloTag. mEGFP and HaloTag genes were knocked-in at thesite of 3’ ERK2 and RSK2 genes, respectively. These auto- and crosscorrelation functions were obtained by FCS and FCCS, providing endogenous concentration of ERK2 and RSK2 and the dissociation constant of
ERK2-RSK2 binding.

O., Kawana, K., Yano, T., Osuga, Y., and Fujii, T. (2016). Synergistic
antitumor effects of combination of PI3K/mTOR and MEK inhibition(SAR245409 and pimasertib) in mucinous ovarian carcinoma cells byfluorescence resonance energy transfer imaging. Oncotarget 7, 2957729591.
Kamezaki, A., Sato, F., Aoki, K., Asakawa, K., Kawakami, K.,
Matsuzaki, F., and Sehara-Fujiwara, A. (2016). Visualization of
Neuregulin 1 ectodomain shedding reveals its local processing in vitro
and in vivo. Sci. Rep. 6, 28873.
Maryu, G., Matsuda, M., and Aoki, K. (2016). Multiplexed fluorescenceimaging of ERK and Akt activities and cell-cycle progression. Cell
Struct. Funct. 41, 81-92.
Yamao, M., Aoki, K., Yukinawa, N., Ishii, S., Matsuda, M., and Naoki,
H. (2016). Two new FRET imaging measures: linearly proportional toand highly contrasting the fraction of active molecules. PLoS One 11,
e0164254.

III. Manipulation of cell signaling

Optogenetics is a powerful tool for the control of the intracellular signaling pathway with high spatial and temporal
precision. Our laboratory focuses on the light-induced dimerization (LID) system with plant cryptochrome 2 (CRY2) andphytochrome B (PhyB), in which blue- and red-light induce
heterodimerization to their binding partners, respectively. By
using these systems, we attempt to reconstitute the spatial
and temporal pattern of cell signaling in order to directly
validate the physiological meaning of the dynamics of cellsignaling that were observed by time-lapse fluorescenceimaging.

20 min2Blue 12.5 µW/cm
light
0 µW/cm2

50

180

min
IB:

pERK1/2

37
50

ERK1/2

37

B

150

140

120

125

Light
90

60

20

30

5

0

kDa

80

60 min
Light

Light

65

A

ON

OFF

ON

OFF

Figure 4. Optogenetic manipulation of intracellular signaling. A, Bluelight-induced repetitive ERK activation with CRY2 system. B, Red andinfrared light-induced shuttling of phytochrome interacting factor (PIF)-EGFP between cytoplasm and the plasma membrane.

13National Institute for Basic Biology

Cell Biology

DIVISION OF CHROMATIN REGULATION

Professor

NAKAYAMA, Jun-ichi

Assistant Professor:
Postdoctoral Fellow:
Visiting Undergraduate:
Technical Assistant:

HAMADA, Kyoko
KATAOKA, Kensuke
KAWAGUCHI, Takayuki
HAMADA, Kyoko*
OKI, Koichi
TANAKA, Mayo

The stable inheritance of gene expression or repression stateis essential to cellular differentiation and development, andan epigenetic cellular-memory system derived from higherorder chromatin structure plays a fundamental role in thisprocess. The assembly of higher-order chromatin structure
has been linked to the covalent modifications of histone tails,
chromatin-associated proteins, and non-coding RNAs. The
exact means by which such chromatin-based epigenetic
information is established and faithfully maintained acrosscell divisions and throughout development, however, remains
incompletely understood. To try to gain a better understanding of the molecular mechanisms underlying chromatinbased epigenetic phenomena, our lab uses fission yeast,
Schizosaccharomyces pombe, and ciliate Tetrahymena asmodel organisms for studying the molecular mechanisms ofhigher-order chromatin assembly. We are also attempting todetermine the cellular functions of chromatin modifying
factors in developing a better understanding of complicatedepigenetic phenomena in higher eukaryotic cells.

I. Establishment and maintenance of higherorder chromatin structure

In eukaryotic cells, the assembly of higher-order chromatin
structure, known as heterochromatin, plays an importantrole in diverse chromosomal processes. We have previously shown that the specific methyl modification on lysine
9 of histone H3 (H3K9me) and the binding of the chromodomain (CD) proteins such as HP1 to methylated histones
are essential to the assembly of higher-order chromatin
structure. In fission yeast, the H3K9me is catalyzed by the
methyltransferase Clr4. It was previously shown that Clr4
forms a complex called CLRC, which includes Rik1, Raf1,
Raf2, and Cul4. Since Cul4 is one of the ubiquitin ligases,
and Rik1 and Raf1 show sequence similarities to mammalian DDB1 and DCAF, respectively, it is hypothesized that
CLRC has E3 ubiquitin ligase activity. However, CLRC’s
physiological targets remained unclear. We demonstrated thataffinity purified CLRC preferentially ubiquitylates histone
H3. Interestingly, H3 ubiquitylation was found to promote
H3K9 methylation by Clr4. These results suggested that
histone ubiquitylation plays a crucial role in maintaining
H3K9me. We will further characterize the crosstalk between
histone methylation and ubiquitylation by defining how
ubiquitylation promotes Clr4 and which domain(s) of Clr4 is
responsible for binding ubiquitylated histone H3.

14

II. Regulation of chromodomain proteinsin assembling higher-order chromatin
structure

The chromodomain (CD) is a conserved sequencemotif present in chromosomal proteins and functions asa binding module that targets methylated histone tails.
Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromodomain protein that binds to lysine H3K9me,
a hallmark of heterochromatin. We previously showed thatmammalian HP1α is phosphorylated at its N-terminal serine
residues and that this phosphorylation contributes to HP1α’s
specific binding to H3K9me nucleosomes and is essential
for its proper targeting to heterochromatin. We further demonstrated that Swi6, one of fission yeast HP1 homologues,
is also subjected to phosphorylation at its N-terminal conserved region, and also that this region is critical for Swi6’s
silencing function (Figure 1). We will further characterize themechanisms how Swi6’s N-terminal region contributes to its
silencing function by identifying factors that specifically or
differentially interact with the Swi6’s N-terminal region.

Figure 1. Swi6 N-terminal region is required for its silencing function.
A 10-fold serially diluted culture of indicated strain was spotted onto
nonselective medium (N/S), low adenine medium (Low Ade), or medium
lacking uracil (–Ura).

III. Characterizing nucleic acid-binding
activities associated with chromatinproteins
The CD’s best-characterized targets are methylated histone
tails. Several lines of evidence suggest that the CD, in
addition to targeting methylated histones, also binds to
DNA and RNA. Although both the nucleic acid-binding andmethylated histone-binding activities of CDs are known to
be important for CD protein functions in several biologicalprocesses, the physical and functional relationships between
these two activities are poorly understood. We previously
showed that fission yeast Chp1, a CD protein functioning
in the RNA silencing pathway, possesses unique nucleic
acid-binding activities that are essential for heterochromaticgene silencing. To obtain further evidence that nucleic acidbinding activity associated with CD plays an important rolein their chromatin targeting, we focused on mammalian CDproteins and showed that their nucleic acid-binding activity
is critical for their nucleosome binding or heterochromatin
assembly. We will further characterize the functional role of
the CD’s nucleic acid-binding activity and also try to identify
chromatin factors that specifically interact with non-coding
RNAs.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2016. The former title isindicated by an asterisk (*).

IV. Dynamic chromatin reorganization during
sexual reproduction of Tetrahymena

Tetrahymena, a unicellular protozoan, shows nuclear dimorphism. They contain the transcriptionally silent, germline
micronucleus (MIC) and the transcriptionally active, somatic
macronucleus (MAC) in a single cell (Figure 2). During
sexual reproduction, the newly synthesized MAC removes
the dispensable internal eliminated sequences (IESs) fromthe genome and adds telomeres at the ends of fragmented
chromosomes. The IES elimination requires RNA interference (RNAi)-related pathway and posttranslational histone
modifications. The methylation of histone H3 lysine 9 and/or lysine 27 catalyzed by Ezl1p acts as binding site for chromodomain proteins such as Pdd1 to form heterochromatinlike structure. Although the RNAi-related pathway to target
IES regions is extensively studied, the underlying molecularmechanisms for heterochromatin assembly remain poorly
understood. We will focus on uncharacterized heterochromatin proteins and examine their roles in dynamic changeof chromatin structure. In addition, we will try to identify
factors that are involved in chromosome fragmentation andde novo telomere addition during MAC development.

Figure 2. Developing macronuclei (MACs) during sexual reproduction of
Tetrahymena. The cells at 14 hours post-mixing were stained with antiPdd1 (green) and DAPI (magenta). Pdd1 was detected exclusively in the
new MACs.

Publication List:〔Original papers〕
K., Shinmyozu, K., Nakayama, J., Hatashita, M., Uchida, H.,• Kamata,
and Oki, M. (2016). Four domains of Ada1 form a heterochromatin••boundary through different mechanisms. Genes Cells. 21, 1125-1136.
Mitsumori, R., Shinmyozu, K., Nakayama, J., Uchida, H., and Oki,
M. (2016). Gic1 is a novel heterochromatin boundary protein in vivo.
Genes Genet. Syst. 91, 151-159.
Shimojo, H., Kawaguchi, A., Oda, T., Hashiguchi, N., Omori, S.,
Moritsugu, K., Kidera, A., Hiragami-Hamada, K., Nakayama, J., Sato,M., and Nishimura, Y. (2016). Extended string-like binding of thephosphorylated HP1α N-terminal tail to the lysine 9-methylated histone
H3 tail. Sci. Rep. 6, 22527.

15

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
the ambient conditions. In our laboratory, we use the fission
yeast Schizosaccharomyces pombe, the simplest organismthat performs meiosis, to research the mechanism by whichcells switch from mitosis, the kind of cell division that
divides cells equally to create two identical cells, to meiosis,
which is 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.

16

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 demonstrated that Mmi1 prevents untimely translation of meiotic transcripts by regulating their localization.
Multilayered suppression of meiotic genes by Mmi1 is vitalfor mitotic growth.

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.

in vivo

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. Horse-tailmovement is driven by cytoplasmic dynein, which forms ahuge minus-end-directed microtubule motor complex, anddynactin, which is a protein complex that is required for mostdynein-mediated cellular activities. Cytoplasmic dynein thatis anchored to the cell cortex generates a pulling force on the
microtubule emanating from the leading edge of the nucleus.
This dynein-mediated pulling is the major contributor tohorse-tail movement. Cortical anchoring of dynein is crucial
for the generation of horse-tail movement.
We identified novel subunits of dynactin, and showedthat dynein-related cortical factor, Num1, cooperates withdynactin to establish dynein anchoring at the cell cortex.
We are now studying how the oscillatory movement of thenucleus is accomplished by combining computational simulations with live cell imaging (Figure 4).

(µm)

(min)

(µm)

in silicoIII. Regulation of nuclear oscillation driven
by cytoplasmic dynein during meiotic
prophase(min)

Figure 4. Tracks of the leading edge of the nucleus during horse-tailnuclear movement in a living cell and in a simulation model. Coloredlines in the model indicate tracks of microtubule plus ends.

Publication List:〔Review articles〕
A., Shichino, Y., and Yamamoto, M. (2016). The long non• Yamashita,
coding RNA world in yeasts. Biochim. Biophys. Acta Gene Regul.
Mech. 1859, 147-154.

A., Sakuno, T., Watanabe, Y., and Yamamoto, M. (2016).
• Yamashita,
Analysis of Schizosaccharomyces pombe Meiosis. Fission Yeast, A
Laboratory Manual, 251-259.

0 min

2 min

A., Sakuno, T., Watanabe, Y., and Yamamoto, M. (2016).
• Yamashita,
Live imaging of chromosome segregation during meiosis in the fission••

yeast Schizosaccharomyces pombe. Fission Yeast, A Laboratory
Manual, 260-263.
Yamashita, A., Sakuno, T., Watanabe, Y., and Yamamoto, M.
(2016). Synchronous induction of meiosis in the fission yeast
Schizosaccharomyces pombe. Fission Yeast, A Laboratory Manual,
264-267.
Yamashita, A., Sakuno, T., Watanabe, Y., and Yamamoto, M. (2016). A
simple method to induce meiosis and sporulation semisynchronously
in the fission yeast Schizosaccharomyces pombe. Fission Yeast, A
Laboratory Manual, 268-270.

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.

17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, KaoriYAMASHITA, Akira
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 widely accepted that thissystem controls the location at which neurites will connect
to each other, thereby forming neural networks. Our maininterest is to understand the mechanisms and roles of mRNAtransport and local translation in neuronal dendrites.
Specific mRNAs are recruited into “RNA granules” andtransported to dendrites. RNA granules are membrane-lessmacromolecular assemblies composed mainly of mRNAs,
ribosomes and RNA-binding proteins, and mediate the transport and local translation of their mRNA cargoes in response
to synaptic stimulation (Figure 1). We are researching RNA
granule factors regulating mRNA transport and local translation, their target mRNAs, and the mechanisms of localized protein synthesis using mice in order to better understand their relation to the formation of synapses and neuralnetworks, memory, learning, and behavior.

and ASD. We then subjected Rng105+/− mice to a comprehensive behavioral test battery, and revealed the influenceof RNG105 deficiency on mouse behavior. Rng105+/− mice
exhibited a reduced sociality in a home cage and a weak
preference for novel mice. Consistently, the Rng105+/− micealso showed a weak preference for novel objects and novel
place patterns. Furthermore, although the Rng105+/− mice
exhibited normal memory acquisition in spatial referencetasks, they tended to have relative difficulty in reversal
learning in the tasks. These findings suggest that the
RNG105 heterozygous knockout leads to a reduction in sociality, response to novelty and flexibility in learning, which
are implicated in ASD-like behavior.

II. Long-term memory deficits in RNG105
conditional knockout (cKO) mice

Although learning and memory, except for reversal
learning, were normal in mice with a moderate deficiency
of RNG105 (Rng105+/−), learning and memory were remarkably impaired in mice with severe deficiency of RNG105:
RNG105 conditional knockout (cKO) postnatally in thebrain region caused reduced contextual and spatial long-term
memories.
For example, RNG105 cKO mice showed severe deficits in
the Morris water maze, a spatial long-term memory task inwhich a mouse is placed in a circular pool and learns thelocation of a hidden platform that allows the mouse to escapefrom water. Repeated trials enabled control mice to learn theplatform location and escape on the platform faster than
before the trials (Figure 2A). In contrast, the escape latencyof RNG105 cKO mice did not shorten at all over the trials(Figure 2A). Following the last trial, a probe test was conducted: the platform was removed from the pool and theswimming path of the mice was visualized by tracking themice. Control mice intensively searched around the target

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. Autism spectrum disorder (ASD)-likebehaviors in RNG105 heterozygous mice

We previously identified RNA granule protein 105(RNG105, also known as Caprin1), an RNA-binding protein,
as a component of RNA granules. RNG105 is responsible
for mRNA transport to dendrites, which is required for theencoded proteins to be translated and function in dendrites
for proper networking of neurons.
A recent study reported that a heterozygous mutation in
the Rng105/Caprin1 gene was found in an autism spectrum
disorder (ASD) patient, but it remained unclear whether
there is a causal relationship between RNG105 deficiency

18

Figure 2. Spatial memory deficits of RNG105 cKO mice in Morris water
maze. (A) Escape latency during the acquisition phase of the Morris
water maze. (B) Spatial histograms of the mice’s swimming paths during
the probe trial of the Morris water maze. T indicates the target quadrant.

place where the removed platform had existed (Figure 2B).
In contrast, RNG105 cKO mice showed a circular swimming
path along the wall of the pool and reduced the search timearound the target place (Figure 2B).

III. Reduced structural synaptic plasticity in
RNG105 cKO mice

Memory formation is generally correlated with structural
plasticity of postsynapses (spines) on dendrites, i.e., stimulation-induced long-lasting increase in the size of spines.
Therefore, we measured the stimulation-dependent changes
in spine size in RNG105 cKO cultured neurons. In response
to synaptic stimulation by glutamate uncaging, spines near
the uncaging locus increased in volume ~2.5-fold and sustained the increased state at least for 60 min in control
neurons (Figures 3A and 3B). In contrast, in RNG105 cKO
neurons, spine volume during the sustained phase was significantly reduced (Figure 3B). These results suggested thatstructural plasticity of spines was reduced by RNG105 deficiency.

mRNAs in dendrites was significantly reduced in RNG105cKO neurons, which suggested that RNG105 is responsible
for the dendritic localization of many different mRNAs.
Gene ontology enrichment analysis of the identified
mRNAs revealed that categories in which significantly
large number of mRNAs were enriched were quite different
between somatic and dendritic mRNAs. Major categories
for dendritic mRNAs were “Regulation of Arf protein signaltransduction” which included GTPase-activating proteins(GAPs) and guanine nucleotide exchange factors (GEFs)
of small G protein Arf and “Structural constituent of ribosomes” which included ribosomal subunit proteins. Arf is
known to regulate membrane trafficking between the cellsurface and endosomes through controlling endocytosis
and exocytosis, which includes glutamate receptor (AMPA
receptor) surface expression in neurons. Arf is also known
as an important regulator of actin cytoskeleton dynamics anddendritic spine formation via Rac1 activation. “Regulationof small GTPase-mediated signal transduction” was also acategory in which dendritic mRNAs were enriched, which
included regulators of small G proteins such as Ras, Rho andRac, known to be involved in synaptic and actin regulation.
Dendritic mRNAs and gene ontology categories identified
in this study provide insight into underlying mechanisms for
dendritic mRNA localization-dependent long-term synaptic
plasticity and memory, which will be addressed in the future.

Publication List:〔Original paper〕

R., Takao, K., Miyakawa, T., and Shiina, N. (2016).
• Ohashi,Comprehensive behavioral analysis of RNG105 (Caprin1) heterozygous
mice: Reduced social interaction and attenuated response to novelty.
Sci. Rep. 6, 20775.

Figure 3. Reduced structural plasticity of spines in RNG105 cKO
neurons. (A) Representative spine images before and after synaptic stimulation by glutamate uncaging. Scale bar, 1 mm. (B) Time-lapse graph ofthe effect of synaptic stimulation on spine size.

IV. Reduced localization of mRNAs in
dendrites of RNG105 cKO neurons

To investigate the mechanism of how RNG105 cKO affects
synaptic plasticity and long-term memory, we comprehensively identified mRNAs whose dendritic localization was
reduced in RNG105 cKO neurons. The soma and dendritesof neurons were isolated by microdissection of the hippocampal stratum pyramidale (SP) layer and stratum radiatum(SR) layer, respectively, and then subjected to RNA-seq
analysis using next-generation sequencing. By comparing mRNAs from the soma and dendrites, we identified
1,122 dendritically enriched mRNAs and 2,106 somatically enriched mRNAs. Enrichment of most of the dendritic

19National Institute for Basic Biology

Cell Biology

LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

NIBB Research Fellow:
Postdoctoral Fellow:
Technical Assistant:

KAMIKAWA, Yasunao
TSUBOUCHI, HideoYASUI, 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 canlead to malfunction of a cell. Our group is interested in thestrategies through which cells protect themselves from alterations in the genome. To date, much information is gained
from various model organisms and tissue culture cells, andwe are beginning to learn that the choice of 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 the embryonic 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) andM (mitosis) phases take as much time 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 now established several
protocols to synchronize ES cells (Tsubouchi et al., Cell,
2013; unpublished), which allowed us to investigate specificstages of the ES cell cycle. In 2016, we have closely investigated DNA replication and mitosis in ES cells and havefound a few features that are unique to ES cells. We are currently aiming to address how such differences are interlinked
with pluripotency by carrying out side-by-side analyses
between ES cells and differentiated populations.

II. Genome 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.

20

Specifically, we take advantage of the cell-to-cell fusion
approach, in which a target cell is fused to a pluripotent stem
cell to induce pluripotency within a target nucleus. The cellfusion 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 bedetected from within a few hours to one day after fusion,
allowing us to monitor the initial events of reprogramming
after induction.

Figure 1. Cellular fusion to study reprogramming: a human lymphoblastoid nucleus can be induced to undergo nuclear reprogramming towards
pluripotency upon fusion with mouse ES cells (green). Lamin B1 isendogenously tagged with GFP in ES cells, allowing us to distinguish ES
vs lymphoblastoid nucleus during live-imaging (unpublished).

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. To
investigate the nature of such errors and how they are linked
to reprogramming-specific events, we are in the process ofsetting up a system to isolate and track a single fused cellthrough live-imaging (Figure 1).

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:〔Review article〕

H., Argunhan, B., and Tsubouchi, T. (2016). Shaping meiotic• Tsubouchi,
chromosomes with SUMO: a feedback loop controls the assembly of
the synaptonemal complex in budding yeast. Microbial Cell 3, 126-128.

National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

UENO, Naoto

Associate Professor

KINOSHITA, Noriyuki

Assistant Professor:

TAKAHASHI, Hiroki
SUZUKI, Makoto
Technical Staff:
TAKAGI, Chiyo
NIBB Research Fellow:
YAMAGUCHI, Takeshi*
Postdoctoral Fellow:
NEGISHI, Takefumi
SUZUKI, Miho
HASHIMOTO, Yutaka
YAMAGUCHI, Takeshi
SOKENDAI Graduate Student: HAYASHI, Kentaro*
TOMINAGA, Hitoshi
Visiting Scientist:
HAYASHI, Kentaro
Technical Assistant:
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
MIYAGI, Asuka
YASUE, Naoko
Secretary:
MIYAKE, Satoko
TSUGE, Toyoko

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. Spatio-temporal regulation of traction force
during the collective chemotractic migration

During embryogenesis, the arrangement of multicellulartissue is dramatically changed to establish properly shaped
embryos. These movements of group of cells are often highlyorganized and collective. Investigating the mechanism of collective cell migration is therefore essential for understanding
embryogenesis. Xenopus leading edge mesoderm (LEM) is
one of the suitable models for studying this morphogenetic
movement, because of easy micromanipulation of tissue
excised from the embryo. During Xenopus gastrulation,
LEM moves into the blastocoel ahead of the axial mesoderm,which forms the notochord and muscles in the future. In
our previous studies, we have shown that LEM generates
the driving force of mesodermal migration, and measured
the physical value of this force with the explant. Following
this, we have started to address how each single LEM cell
generates the force for collective migration in the explant.
In order to understand this, we have decided to establish

Traction Force Microscopy (TFM) for Xenopus LEM explantmigration. Eventually, we completed the TFM system for thismulti-cellular movement with a combination of acrylamide
gel and chemoattractants. Our TFM observation revealed that
traction force was generated by the cells, which were locatedat the anterior part of explants. We are investigating howeach LEM cell generates traction force in a spatiotemporally
regulated manner.

Figure 1. A snapshot of TFM observation of LEM explant migration. LEM explant expressing membrane GFP to visualize the plasma
membrane migration toward the left side. The direction of arrow showsthat of traction force, size and color indicate the intensity of force.

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 embryogenesis, a variety of dynamic morphogenetic movements occur, which include convergent
extension of the axial mesoderm, epiboly of the ectoderm,and neural tube formation. These movements must generate
physical forces at the levels of cells and tissues. However, it
is still not fully understood how these forces influence morphogenetic processes. This project is to elucidate the mechanosensing and mechanoresponse of Xenopus embryos, and toclarify the roles of mechanotransduction in embryogenesis.
Cells sense mechanical stresses in several ways, forexample, with TRP channels, F-actin, cadherins, and/or
focal adhesions. Physical stimuli sensed by these molecules
are converted to intracellular chemical signals, which inturn induce cellular response. Phosphorylation is thought
to be one of the earliest responses to mechanical stresses.
Therefore, we attempt to profile protein phosphorylation
upon mechanical stimuli and identify the repertoire of target
proteins in Xenopus embryonic cells. In order to comprehensively analyze levels of protein phosphorylation, we took aphosphoproteomic approach. So far, we have confirmed thata mechanical stress applied to Xenopus embryos changes
phosphorylation levels of some protein kinases and cytoskeletal regulators. Particularly, some proteins that localizein the cell cortical actin network seem to be the targetsof phosphorylation. These results suggest that mechanicalstresses may regulate activities of protein kinases and/or
protein phosphatases in Xenopus embryonic cells, and thatthe cortical actin network may be involved in this mechanotransduction.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2016. The former title isindicated by an asterisk (*).

21

National Institute for Basic Biology

Developmental Biology

III. Mathematical analysis of neural tube
formation

During early development of the central nervous system,neuroepithelial cells increase their height by microtubule
rearrangement. This cell shape change is called cell elongation, which drives the tubular morphogenesis, called neuraltube formation. We previously showed that cell elongation
during Xenopus neural tube formation depends on orthologsof two microtubule-associated proteins: MID1, which is
responsible for Opitz G/BBB syndrome in humans, and its
paralog MID2. In this study, to further investigate the role ofthe cell elongation, we developed a three-dimensional multicell-based mechanical model and used it to simulate theprocess of neural tube formation. Computational simulationof our model suggests that cell elongation drove the rapid
folding of the neural plate, and reduced the lumen size of theneural tube. Then we investigated the in vivo neural tube byknocking down MID proteins, and found that cell elongation
actually reduces the lumen size of the neural tube. These data
highlight the function of MID proteins in regulation of thelumen size of the neural tube, and demonstrate the advantage
of mathematical analysis for understanding the mechanicalbasis of morphogenesis.

membrane invaginations. In the Dsh-depleted embryos, thedirection of the invagination as well as mitotic spindle orientation was randomized. In addition, epidermal cilia normally
localized in the posterior side of the cell was anteriorized.
These results support our hypothesis that the membrane
invagination controls the direction of cell division as well as
ciliary positioning via the tethering centrosome at the posterior side of the epidermal cell and highlight physiologicalimportance of this unique structure.

Figure 3. (A) The direction of membrane invagination is disturbed in
Dsh-deficient epidermal cells. The plasma membrane was labeled with
PH-GFP.
In the epidermal cells of control MO injected Ciona embryo, themembrane invaginations show the A-P polarity (Control). In the epidermal cells of Dsh MO injected embryo, the direction of invagination is
randomized (Dsh MO). Blue arrows indicate representative membrane
invaginations. Anterior: left. Bar: 10 µm.

Figure 2. Effects of cell elongation (CE) on modeled epithelial sheets andin vivo neural tube.
(A,B) Modeled epithelial sheets at the end of each simulation with cell
elongation (A) and without cell elongation (B). (C,D) F-actin stainingof control embryo (C) and MID-Mo injected embryo (D). (C’) and (D’)
indicate outlines of neural cells and lumen (gray) of (C) and (D), respectively.

IV. A novel membrane structure orchestrates
centrosome positioning and the orientationof cell division axis.

The positioning of the centrosome is critical for the polarityof cilia and the orientation of the mitotic spindle, which
are important events during morphogenesis. Previously, in
ascidian, Ciona intestinalis embryos, we found a uniquecentrosome-targeting of the membrane structure (membrane
invagination) in epidermal cells, which divide stereotypicallyalong the anterior-posterior (A–P) axis. We also reported
that membrane invaginations showed the A–P polarity anda tensile force toward the posterior end of cell. By injection of antisense morpholino oligo (MO), we revealed
that Dishevelled (Dsh), a core component of Planar Cell
Polarity (PCP) pathway was involved in the A–P polarity of

22(B) Graphical summary of our hypothesis. Anterior is left. Interphase:
nuclei (purple), cell membrane (green), centrosome (magenta), microtubule (red) and cilium (orange). The membrane invagination pulls the centrosome/cilium toward the posterior in the interphase, and then mitotic
spindle forms aligned along the A-P axis.

V. Notochord and evolution of chordates

A T-box family transcription-factor gene, Brachyury, has
two expression domains with corresponding functions during
animal embryogenesis. The primary domain, associatedwith the blastopore, is shared by most metazoans, while thesecondary domain restricted to the notochord, is specific tochordates.
Therefore, how Brachyury acquired its secondary expression domain at the mid-dorsal region of the blastopore andhow it is related to the emergence of the notochord is a
big question of evolutionary biology. To elucidate how
chordate ancestors gained the new expression domains, weused Amphioxus, the most ancestral chordates. We functionally analyzed the Amphioxus Brachyury promoter regions byreporter assay, using Ciona intestinalis embryos.
As results, we found that some fragments promoted thereporter expression in multiple regions such as blastopore,muscle and notochord, respectively, in ascidians. This result
indicates that those Amphioxus enhancers have capacities

of driving Brachyury expression in the lateral mesoderm
and notochord, in addition to the blastopore of ascidians. Furthermore, we found some other regions drive theexpression specifically in the notochord, suggesting thatAmphioxus notochord Brachyury expression is regulated by
the combination of multiple enhancers.

VI. The Ciona intestinalis cleavage clock is
independent of DNA methylation

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

VII. Cnidarian-symbiodinium Symbiosis

Corals are declining globally due to a number of stressors. Such stresses lead to a breakdown of the essential
symbiotic relationship between coral and Symbiodinium,
a process known as coral bleaching. Although the environmental stresses cause this breakdown, the molecularand cellular mechanisms of symbiosis are still unclear.
Corals are not very suitable as laboratory systems due totheir 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. Aiptasia, just as
reef-building corals, establishes a stable but temperaturesensitive symbiosis with Symbiodinium. Aiptasia can be
repeatedly bleached and repopulated with Symbiodinium,
grows rapidly, and lacks a calcareous skeleton, allowing
microscopic and cell biological analyses.

found that the symbiotic state of coral and Aiptasia is influenced by the inhibitors .

Publication List:〔Original papers〕

Y., Suzuki, M., Watanabe, T., Yasue, N., Takeo, I., Adachi, T.,• Inoue,and Ueno, N. (2016). Mechanical roles of apical constriction, cell

•
•
•
•
•
•
•

elongation, and cell migration during neural tube formation in Xenopus.
Biomech. Model Mechanobiol. 15, 1733-1746.
Nagasaka, A., Shinoda, T., Kawaue, T., Suzuki, M., Nagayama, K.,
Matsumoto, T., Ueno, N., Kawaguchi, A., and Miyata, T. (2016).
Differences in the mechanical properties of the developing cerebral
cortical proliferative zone between mice and ferrets at both the tissue
and single-cell levels. Front. Cell Dev. Biol. 4, 139.
Negishi, T., Miyazaki, N., Murata, K., Yasuo, H., and Ueno, N. (2016).
Physical association between a novel plasma-membrane structure andcentrosome orients cell division. eLife 5, e16550.
Sakamaki, K., Ishii, T.M., Sakata, T., Takemoto, K., Takagi, C.,
Takeuchi, A., Morishita, R., Takahashi, H., Nozawa, A., Shinoda,
H., Chiba, K., Sugimoto, H., Saito, A., Tamate, S., Satou, Y.,
Jung, S.K., Matsuoka, S., Koyamada, K., Sawasaki. T., Nagai,T., and Ueno, N. (2016). Dysregulation of a potassium channel,
THIK-1, targeted by caspase-8 accelerates cell shrinkage.
Biochim. Biophys. Acta 1863, 2766-2783.
Sekiguchi, T., Kuwasako, K., Ogasawara, M., Takahashi, H., Matsubara,S., Osugi, T., Muramatsu, I., Sasayama, Y., Suzuki, N., and Satake, H.
(2016). Evidence for conservation of the calcitonin superfamily andactivity-regulating mechanisms in the basal chordate Branchiostoma
floridae: insights into the molecular and functional evolution inchordates. J. Biol. Chem. 291, 2345-2356.
Session, A.M., Yamamoto, T.S., Takagi, C., Ueno, N., et al. (2016).
Genome evolution in the allotetraploid frog Xenopus laevis. Nature 538,
336–343.
Suzuki, M., Takagi, C., Miura, S., Sakane, Y., Suzuki, M., Sakuma, T.,
Sakamoto, N., Endo, T., Kamei, Y., Sato, Y., Kimura, H., Yamamoto,T., Ueno, N., and Suzuki, K.T. (2016). In vivo tracking of histone H3lysine 9 acetylation in Xenopus laevis during tail regeneration. Genes
Cells 21, 358-369.
Suzuki, M.M., Mori, T., and Satoh, N. (2016). The Ciona intestinalis
cleavage clock is independent of DNA methylation. Genomics 108,
168-176.

As the innate immune system recognizes the self and others,
it might distinguish Symbiodinium as well as pathogens
from the host. In fact, the genomic information of Acropore
digitifera, one of the reef-building corals, revealed that thecoral innate immune repertoire is far more complex thanthat of non-symbiotic cnidarians, Nematostella and hydra.
We thus speculate that the complex innate immune repertoire may reflect adaptation to the symbiotic state. We usedchemical inhibitors for the inanate immune signaling, and23

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
NIBB Research Fellow:
OKADA, Kazunori
Postdoctoral Fellow:
TAKADA, Ritsuko
FUJIMORI, Sayumi
SOKENDAI Graduate Student: SHINOZUKA, Takuma
TSUCHIYA, Yoshihiro
Visiting Scientist:
CHEN, Qiuhong
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
Secretary:
UKAI, Sakie
NOBATA, Ryuko

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 that24

underlies the periodical and sequential sub-division in thedevelopment 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 fromthe producing cell, their spread is regulated through interaction with these molecules. The heparan sulfate proteoglycan(HSPG), which is composed of a core protein with several
chains of HS glycosaminoglycans, is a major component
involved in this interaction. In collaboration with Prof. Tairaat the University of Tokyo, we found that HSPGs with different sugar chain modifications form distinct extracellular
structures, which are called heparan sulfate nanostructures(HSNSs), in the early Xenopus embryo.

II. Heterogeneity 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 (Figure1). However, it remains unclear whether Wnt proteins are
secreted only in these two forms, or also in some other forms
from the same cells, or if different forms of Wnt proteins are
secreted in a cell 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 fromeither apical or basolateral side of MDCK cells did not. In
contrast, secretion of a small amount of Wnt3a proteins via
exosomes was detected. We found that different populations

of exosomes or exosome-like vesicles were released from
MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles was detectable in culture media
collected from both apical and basolateral sides of the cells.
Basolaterally secreted Wnt3a existed with typical exosomes.
In contrast, most of apically secreted Wnt3a, as well as
Wnt11, existed with non typical exosomes, whose density
was higher than that of typical exosomes. The lipidation of
Wnt3a was required for its basolateral secretion in exosomes
but was dispensable for the apical secretion. These results
indicated that epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation
dependency.

molecules, 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, andFigure 1. Heterogeneity of Wnt transport system. Secreted Wnt proteins
are transported by several different ways, including binding with carrier
proteins, associating with exosomes, or loading on lipoprotein particles.

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-cellular

Figure 2. (A, B) Expression of ripply2 in WT embryo (A) and mesp
quadruple mutant embryo (B). In contrast to mice, the function of mesp
is not required for the ripply expression in zebrafish. (C) Scheme of theregulation of ripply expression by the segmentation clock. In the mouse,
temporal information generated by segmentation clock is converted tothe expression pattern of ripply by the functions of Notch and Mesp2.
However, this molecular machinery is not conserved between mouse andzebrafish and molecular mechanism regulating ripply expression still
remains to be elucidated.

25

National Institute for Basic Biology

Developmental Biology

the 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 zebrafish mutants
lacking all 4 zebrafish mesp genes by a genome-editing
approach. In contrast to the case for the mouse Mesp2
mutant, the positions of somite boundaries were normal in
the zebrafish mesp quadruple mutant embryos. On the other
hand, each somite was caudalized similarly to the mouse
Mesp2 mutant. These results clarify the conserved and species-specific roles of Mesp in the connection of the molecular clock to somite morphogenesis (Figure 2).

IV. Molecular mechanism of pharyngeal
pouches

In addition to somites, metameric structures are observed in
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 examined the development of pharyngeal pouches in medaka embryos in collaboration with Prof. Wada at Tsukuba University. We foundthat the expression of pax1 in the endoderm prefigured thelocation of the next pouch before the cells bud from the epithelium. Embryos deficient for pax1 did not form the pharyngeal pouches posterior to the second arch. Precise analysis ofgene expression suggests that pax1 has a critical role in generating a primary pattern for segmentation in the pharyngeal
endoderm (Figure 3).

Figure 3. Model of molecular mechanism of the segmentation of pharyngeal pouches in the medaka embryo. Our results suggest that interaction
between pax1 and fgf3 is a key process for generation of the segmental
pattern of pharyngeal pouches.

26

Publication List:〔Original papers〕
Q., Takada, R., Noda, C., Kobayashi, S., and Takada, S. (2016).
• Chen,Different populations of Wnt-containing vesicles are individually
released from polarized epithelial cells. Sci. Rep. 6, 35562.

A., Ovara, H, Ooka, Y., Kinoshita, H., Hoshikawa, M.,• Kawamura,
Nakajo, K., Yokota, D., Fujino, Y., Higashijima, S.I., Takada, S.,

•
•
•
•

and Yamasu, K. (2016). Posterior-anterior gradient of zebrafish
hes6 expression in the presomitic mesoderm is established by the
combinatorial functions of the downstream enhancer and 3’UTR. Dev.
Biol. 409, 543-554.
Ohta, Y., Kamagata, T., Mukai, A., Takada, S., Nagai, T., and Horikawa,
K. (2016). Nontrivial effect of the color-exchange of a donor/acceptor
pair in the engineering of Förster resonance energy transfer (FRET)based indicators. ACS Chem. Biol. 11, 1816-1822.
Okada, K.*, Inohaya, K., Mise, T., Kudo, A., Takada, S.*, and Wada,
H.* (2016). Reiterative expression of pax1 directs pharyngeal pouch
segmentation in medaka. Development 143, 1800-1810. (*: Cocorresponding authors)
Takemoto, T., Abe, T., Kiyonari, H., Nakao, K., Furuta, Y., Suzuki, H.,
Takada, S., Fujimori, T., and Kondoh, H. (2016). R26-WntVis reporter
mice showing graded response to Wnt signal levels. Genes Cells 21,
661-669.
Yabe, T., Hoshijima, K., Yamamoto, T., and Takada, S. (2016). Mesp
quadruple zebrafish mutant reveals different roles of mesp genes insomite segmentation between mouse and zebrafish. Development 143,
2842-2852.

〔Review articles〕

H., Takada, S., and Takemoto, T. (2016). Axial level• Kondoh,dependent molecular and cellular mechanisms underlying the genesis•of the embryonic neural plate. Dev. Growth Differ. 58, 427-436.
Yabe, T., and Takada, S. (2016). Molecular mechanism for cyclic
generation of somites: Lessons from mice and zebrafish. Dev. GrowthDiffer. 58, 31-42.

DIVISION OF EMBYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professor:

TOYOOKA, Yayoi
KOYAMA, Hiroshi
Technical Staff:
OKA, SanaeNIBB Research Fellow:
SHI, Dongbo
Postdoctoral Fellow:
DAY, F. Timothy
NAKANOH, Shota
SOKENDAI Graduate Student: KAMEMIZU, Chizuru
ITO, Tomoaki
USAMI, Fumiko
OTAKE, Norihito
Technical Assistant:
HIGUCHI, Yoko
NAKAGAWA, Mami
Secretary:
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.
This is mainly due to difficulties in approaching developing embryos in the oviducts and the uterus of the mother.
Another characteristic of mammalian embryos is their highly
regulative potential. The pattern of cell division and allocation of cells 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 havenot been found that localize asymmetrically in the fertilized
egg. We want to provide basic and fundamental information
about the specification of embryonic axes, differentiation ofcell lineages, behaviors of cells, and the regulation of body
shape in early mammalian development. We are also interested in the relationship between embryos and their environment, the oviducts and the uterus.

I. Live observation 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 reportermouse 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 the mouse genomic DNA sequences of the enhancer/
promoter region of important genes encoding factors regulating cell differentiation in these stages. Combining these two
types of reporter transgenic mouse lines, we have been analyzing behaviors of cells comparing gene expression properties at the single cell level.
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. To monitor cell movement andthe status of the trophectoderm (TE) specification pathway
in living embryos, we established Cdx2-GFP reporter miceallowing us to visualize the expression of Caudal-type transcriptional factor (Cdx2), a key regulator of the initiationof TE differentiation. Cells localized in an outer position
initiated the expression of Cdx2. Subsequently, cells thatchanged their position from an outer to an inner position
down-regulated Cdx2 expression and contributed to thepluripotent inner cell mass (ICM). Our results indicate thatcells expressing even high levels of Cdx2 can internalize,
deactivate the TE-specification molecular pathway and be
integrated into the pluripotent cell population. This suggeststhat cells expressing Cdx2 exhibit plasticity of specification
to TE and ICM lineages through positional changes.

Figure 1. Cdx2 expressing cells in preimplantation mouse embryo
exhibit plasticity of specification. Outer positioned cells up-regulate
Cdx2 expression, but some internalized cells down-regulate its expression and contribute to both pluripotent epiblast (Epi) and extra-embryonicprimitive endoderm (PrE).

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

27

National Institute for Basic Biology

Developmental Biology

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 embryonicdevelopment 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.

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.

28

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
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.
To investigate the mechanisms of the epithelial fold patternformation, we utilized mathematical modeling and simulations. By considering mechanical properties of the epithelial
sheets we reproduced the longitudinally aligned folds andthe branched folds which are observed in wild-type and the
Celsr1 mutant mice, respectively (Figure 3). Experimental
measurements of mechanical tensions in the epithelial sheet
were consistent with the tensions predicted from the simulations. Our experimental and mathematical analyses also
successfully linked the epithelial tensions to cellular shapes.
We are also focusing on some other PCP regulators, and aretrying to understand how oviduct epithelial cells establish
and maintain their polarity.

Figure 3. Epithelial fold patterns in oviduct and the reproduction of thepatterns by computational simulations.

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. To understand how mechanical forces
contribute to various patterns, measuring cellular and tissue
mechanical states are necessary. We developed statistic techniques to infer the mechanical states by using fluorescent
microscopic images during morphogenesis (Figure 4).
By employing this method, we inferred mechanical forcesin multi-cellular systems including MDCK cultured cells,
and early embryogenesis in C. elegans and mice. Further
computational simulations based on the inferred mechanicalinformation reproduced morphological features of the multicellular systems. Thus, the mechanical information will be
useful to investigate physical mechanisms of early embry-

onic development and morphogenesis during organogenesisin late stages of development.

Figure 4. Statistical inference of cellular/tissue mechanical states.

Publication List:〔Original papers〕

H., Shi, D., Suzuki, M., Ueno, N., Uemura, T., and Fujimori,• Koyama,
T. (2016). Mechanical regulation of three-dimensional epithelial foldpattern formation in the mouse oviduct. Biophys. J. 111, 650-665.

D., Usami, F., Komatsu, K., Oka, S., Abe, T., Uemura, T., and• Shi,
Fujimori, T. (2016). Dynamics of planar cell polarity protein Vangl2 inthe mouse oviduct epithelium. Mech. Dev. 141, 78-89.

T., Abe, T., Kiyonari, H., Nakao, K., Furuta, Y., Suzuki, H.,• Takemoto,Takada, S., Fujimori, T., and Kondoh, H. (2016). R26-WntVis reporter•

mice showing graded response to Wnt signal levels. Genes Cells 21,
661-669.
Toyooka, Y., Oka, S., and Fujimori, T. (2016). Early preimplantation
cells expressing Cdx2 exhibit plasticity of specification to TE and ICM
lineages through positional changes. Dev. Biol. 411, 50-60.

〔Review article〕

D., Arata, M., Usui, T., Fujimori, T., and Uemura, T. (2016). Seven• Shi,pass transmembrane cadherin CELSRs, Fat4 and Dchs1 cadherins:

From planar cell polatiry to three-dimensional organ architecture. In
The Cadherin Superfamily, S. Suzuki, and S. Hirano, eds., pp. 251-275.

29

National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:

KITADATE, Yu
NAKAGAWA, Toshinori
Technical Staff:
MIZUGUCHI, Hiroko
NIBB Research Fellow:
HIRA, Seiji
JSPS Researcher:
NAKAMURA, Yoshiaki
Postdoctoral Fellow:
IKAMI, Kanako
TOKUE, Moe
SOKENDAI Graduate Student: NONAMI, Yuta
ISHIZAKA, Miho
HIRANO, Kodai
Visiting Graduate Student: SATO, Toshiyuki
Technical Assistant:
KON, Yayoi
MARUYAMA, Ayumi
NISHIMURA, Keiko
Secretary:
KUBOKI, 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 cell pool, while generating differentiation-destined
cells in a well balanced manner. The Division of Germ Cell
Biology aims to fully understand the mouse sperm stem cellsystem under the context of in vivo testicular tissue. Ourparticular interests have been laid on the “undifferentiatedspermatogonia”, which are responsible for the stem cell functions. Our study has revealed several key properties of thisinteresting population.
First, we found that this stem cell system includes a functional hierarchy. It is comprised of an “actual” stem cell compartment that is prone to self-renew, and a differentiationprimed, “potential” stem cell compartment. Regarding the“actual” stem cells, we have been investigating their cellular
identity, their in vivo behavior at a single-cell resolution,and the underlying mathematical principles. This lead to thediscovery of neutral competition between the stem cells. We
are currently investigating the molecular mechanism underlying their control of self-renewal and differentiation, andtheir connection to tissue environment. “Potential stem cells”
are also of our enthusiastic interest: In undisturbed, steadystate spermatogenesis, they largely contribute as transient
amplifying cells and differentiate rather than self-renew.
However, when tissues are insulted or testicular cells aretransplanted to a host testes, their probability of self-renewal
jumps up and they effectively replenish the lost “actual” stem
cells. Such a flexible, and probabilistic, 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,
and Ikami et al., Development 2015.

30

I. The identity of spermatogenic stem cells andtheir 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 proposed 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 asingle stem cell pool, in which cells continually interconvert
between these morphologically heterogeneous states throughstochastic incomplete division and fragmentation of syncytia.
The incomplete division and syncytial fragmentation causes
the expansion of this population, while the excess cells overa particular “quota” would overflow to become the 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 internal
heterogeneity of the population of GFRα1+ cells.

2. Mechanisms underlying the balanced
differentiation and self-renewal of stem cells
in an “open” stem cell niche

In general, stem cells support tissue homeostasis throughcontinual production of differentiating progeny from a poolof undifferentiated cells. This is traditionally thought tobe dependent on a couple of paradigmatic mechanisms: 1)“asymmetric cell division”, which always gives rise to one
self-renewing cell and one differentiating cell; 2) control
by “an anatomically defined niche”, inside of which stem
cells remain undifferentiated, but they differentiate outside(Fuller and Spradling, 2007; Morrison and Spradling, 2008).
However, the mouse spermatogenic stem cell system does
not show a defined niche environment and does not rely on
asymmetric division; their fates are stochastic (Hara et al.,2014; Klein et al., 2010; Klein and Simons, 2011).
In 2017, we challenged a fundamental question: How sperm
stem cells follow different fates (to differentiate or to selfrenew) in response to homogeneously distributed extracellular signals, in a facultative (or open) niche environment? The
result is published in Tokue et al., Stem Cell Reports (2017).

2-1. Wnt/β-catenin signaling promotes differentiationof GFRα1+ to NGN3+ cells

To unveil the signal that promotes the GFRα1+ to NGN3+transition, we first compared the gene expression profiles
between GFRα1+ and NGN3+ cells by cDNA microarray.
From the microarray data, we focused on Wnt signaling. In
cultured spermatogonia (viz. GS cell), Wnt/β-catenin signaling induced Ngn3 expression (Figure 2A). Moreover, micecarrying a gain-of-function mutation of β-catenin showedspermatogenesis defects, in which the number of GFRα1+cells was reduced (Figure 2B), whereas no significant reduction in retinoic acid receptor gamma (RARγ)+ cells (largely
corresponding to NGN3+ cells) was observed. Consequently,
the RARγ + cell-to-GFRα1 + cell ratio increased, consistent with the idea that GFRα1+ to NGN3+ differentiation
increased.

Figure 2. Wnt/β-catenin signal promotes the differentiation of GFRα1+
spermatogonia in vitro and in vivo. (A) Expression of Ngn3 mRNA in
GS cells in the presence or absence of GDNF or WNT3a. GS cells were
stimulated by the indicated conditions and analyzed by RT-qPCR for the
Ngn3 mRNA level. (B) Average numbers of GFRα1+ cells per tubule,observed in mice with indicated genotypes. Number of GFRα1+ cells was
reduced in Nanos3Cre/+;Ctnnb1 fl(ex3)/fl(ex3) mice, in which β-catenin was activated in a germ cell specific manner. Modified from Tokue et al., Stem
Cell Reports (2017),

the other Shisa family members have been reported as Wnt
inhibitors. Experiments using Xenopus laevis embryos andluciferase assays in HEK293T cells showed that SHISA6 isa novel Wnt inhibitor that acts autonomously (Figure 3A).
Moreover, Shisa6 expression was restricted to about 30% of
the GFRα1+ population in vivo (Figure 3B).

2-3. SHSIA6+ spermatogonia show stem cell charactersand contribute to maintenance of GFRα1+ cells

We found that, although Shisa6 KO or stabilized β-cateninheterozygous mutants show no apparent phenotype in thepool of GFRα1 + cells, synthetic heterozygotes for thesealleles showed a reduced GFRα1+ cell pool and spermatogenesis defects. Thus, SHISA6 plays a role in the maintenance of the stem cell pool by suppressing Wnt/β-cateninsignaling in a cell-autonomous manner (Figure 4A).
We then addressed the behavior of SHISA6+ cells conjectured from that of T (Brachyury)+ cells, which were found tolargely overlap with SHISA6+ cells. A pulse-label and chaseexperiment showed that T+ cells have the ability to continu-

2-2. a cell-autonomous Wnt inhibitor, is expressed in a
subset of GFRα1+ cells

We found that Wnt6 is expressed in Sertoli cells, a supporting somatic cell type, in a spatially ubiquitous manner,suggesting that WNT6 is participating in the differentiationpromotion in seminiferous tubules, and that GFRα1+ cells
are uniformly exposed to WNT6 signals. These raised a nextquestion: How do stem cells persist without exhaustion if
they are equally bathed with Wnt ligand(s)?
To address this issue, we hypothesized that a Wnt
inhibitor(s) may confer resistance to Wnt/β-catenin signaling
in some fraction of GFRα1+ cells. In our microarray data, wefound Shisa6 tobe an interesting candidate, because Shisa6
was highly enriched in the GFRα1+ fraction and some of

Figure 3. Function and expression of SHISA6 (A) Cell-autonomous
inhibition of Wnt/β-catenin signaling by SHISA6. HEK293T cells
were transfected with a Wnt reporter luciferase construct and a Shisa6
expression plasmid either simultaneously (co-transfection), or separately(transfected cells were mixed afterword), followed by stimulation of
Wnt/β-catenin signal. The Wnt-dependent Luciferase activity was suppressed only when Shisa6 was co-transfected. (B) Representative imageof dissociated testicular cells of adult mice doubly stained for Shisa6
by FISH and for GFR (B) Representative image. Note the overlapped
expression of Shisa6 and GFRsa is modified from Tokue et al., Stem Cell
Reports (2017),

31

National Institute for Basic Biology

Developmental Biology

ally produce progeny differentiating to sperm for at least 6months. Together, these results suggest that T+ (and probably
SHISA6+) cells have stem cell-related characteristics (Figure
4B).

Figure 4. SHISA6 and spermatogenic stem cells (A) SHISA6 maintains
the GFR S+ cell pool through inhibiting the Wnt/l pool through i. Average
number of GFRve+ cells per tubule section in testes with the indicatedgenotypes, based on double IF for GFR microtesticular sections. (B)(Upper) A schematic of the structure of the TnEGFP-CreERT2 allele, whichenables pulse-labeling of T+ cells using CreERT2. (Lower) A result of
pulse-labelling of T+ cells with 6 months of chase. Note that GFP (thelineage reporter) positive cells form a prominent patch in a particularsegment of seminiferous tubules. Modified from Tokue et al., Stem Cell
Reports (2017),

2-4. SHISA6 protects the stem cells from differentiation promoting Wnt/β-catenin signaling: A
proposal of a generic mechanism of stem cell regulation in facultative niches

To conclude, we propose a generic mechanism underlying the heterogeneous stem cell fates in facultative niche
environments. Different levels of cell-autonomous inhibitor(SHISA6, in this case) may confer heterogeneous resistanceto uniformly distributed extracellular signaling that promotes
differentiation (such as WNTs). Here, stem cells with higher
levels of inhibitors would remain in the undifferentiated cellpool with higher probabilities, while those with lower levels
of inhibitors are more inclined to differentiate (Figure 5).

32

Figure 5. A conceptual diagram of differentiation promotion by Wnt/βcatenin signaling to become Ngn3+ and its cell-autonomous protection
by SHISA6 in a subset of GFRd + cells. The heterogeneous expression of
SHISA6 may confer different levels of differentiation probability in an
open niche environment in which differentiation-promoting Wnt ligand
appears to distribute uniformly. Modified from Tokue et al., Stem Cell
Reports (2017),

Publication List:〔Review Article〕

S. (2016). From cyst to tubule: innovations in vertebrate• Yoshida,spermatogenesis. WIREs Developmental Biology 5, 119-131.

LABORATORY OF MOLECULAR GENETICS FOR REPRODUCTION †Associate Professor

TANAKA, Minoru

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

NISHIMURA, Toshiya
FUJIMORI, Chika
SAKAE, Yuta
KIKUCHI, Mariko
OTAKE, Norihito
KINOSHITA, Chie
WATAKABE, Ikuko
SHIRAISHI, Naomi
FUJISHO, Tamae

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. Identification of a fate switch gene in germ
cells (sex determination gene 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.
It is generally accepted that germline stem cells are sexuallyindifferent or unfixed. And the appearance of germ cells
remains the same during the process of both spermatogenesis
and oogenesis until germ cells enter meiosis. This suggeststhat the sex (the fate decision to become eggs or sperm)
might not be determined by that time.
Based on these observations, we revealed the genes which
differentially express in germ cells just before meiosis.
Among them, we successfully identified the gene that determines the sexual fate in germ cells. The gene is one of thegenes encoding forkhead transcriptional factors, foxl3. The
mutant analysis indicated that the factor functions in germ
cells autonomously and represses entering spermatogenesis
in germ cells. The mutant did not cause any defect in sex
differentiation of somatic cells – The XX mutant, genetically female medaka, develops a normal ovary but germ cells
develop into sperm in the mutant ovary during young adultperiod. Artificial insemination with sperm isolated from themutant ovary and eggs from the wildtype ovary produces
fertile eggs, demonstrating that the sperm in the mutant
ovary are functional.

II. Structural origins of ovary and testis.

Structural analysis of the mutant ovary leads to one interesting view on the origins of ovary and testis.
The view suggests that indifferent gonads have a common
unit for both germinal cradles and lobules. Germinal cradles
identify niche regions of germline stem cells in the ovary,and lobules in the testis harbor germline stem cells at theirdistal ends. Both structures develop from the common unit
composed of supporting cells that surround germ cells, whichis underlaid by the basement membrane. A mature ovary
is composed of both germinal epithelium where germinal
cradles (germline niche) are present and the stromal compartment where vitellogenic and/or mature oocytes are present.
Interestingly, the ovary in XX (genetically female) foxl3
mutant develops a huge expansion of germinal epitheliumand sperm is formed in the germinal epithelium. Although
the mutant ovary possesses the stromal compartment,†: This laboratory was closed on 31 May, 2016.

33

National Institute for Basic Biology

Developmental Biology

sperm are never observed there. In the expanded area of
the germinal epithelium, we can observe common units ofgermline stem cell-niche, supporting cells with basement
membrane. These suggest that the expanded areas within thegerminal epithelium are equivalent to testicular lobules.
All the observations mentioned above collectively suggestthat testis in the wildtype male can be viewed as the structureorganized and developed within the germinal epithelium. In
other words it may be possible to assert that mature testis
are developed from an origin (a prototype) of germinal epithelium. This is in sharp contrast with the ovary which iscomposed of germinal epithelium and stromal compartment.
Mature eggs are developed in the stromal compartment andthe germinal epithelium serves to provide an early stage of
diplotene oocytes.

Figure 1. A common unit of ovary and testis. Supporting cells (green)that surround germ cells (yellow) and basement membrane (red) can beviewed as a common unit (below illustration) which develops into theniche structure of ovary (germinal cradles) or of testis (lobules). The unit
is observed in both XX and XY gonads (upper images) where yellow
arrow heads indicate deposition of basement membrane (red) and supporting cells are stained green. purple: germ cells, white: nuclea

III. Environment during the larval period may
affect sex of medaka.

As stated in the beginning, the sex of medaka is geneticallydetermined. The sex determination gene on the Y chromosome determines the male (testicular) fate. However, ourresult suggests that the sex could be affected by the status ofnutrients during the larval stage. If medaka larva is put underfood-restricted conditions, the sex ratio of medaka is malebiased. Examination of the developing gonad indicates thatthe number of typeI germ cells (stem like germ cells) under
the restricted conditions are reduced when compared to that
under normal conditions. This result may suggest that the sex
reversal effect by food restriction may be through germ cellnumber, which we had reported in previous papers.

Publication List:〔Review articles〕

M. (2016). Germline stem cells are critical for sexual fate
• Tanaka,
decision of germ cells. BioEssays 38, 1227-1233.
Nishimura, T., Nakamura, S., and Tanaka, M. (2016). A structurally• andfunctionally common unit in testes and ovaries of medaka (Oryzias
T., and Tanaka, M. (2016). The mechanism of germline sex• Nishimura,determination in vertebrates. Biol. Reprod. 95, 1-6.
latipes), a teleost fish. Sex. Dev. 10, 159-165.

34

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

NODA, Masaharu

Associate Professor

SHINTANI, Takafumi

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
Technical Staff:
TAKEUCHI, Yasushi
NIBB Research Fellow:
NOMURA, Kengo
Postdoctoral Fellow:
FUJIKAWA, Akihiro
SUZUKI, Ryoko
KUBOYAMA, Kazuya
LIN, Chia-Hao
SOKENDAI Graduate Student:
MATSUDA, Takashi
YU, Yang
TANGA, Naomi
HIGASHI, Satoru
Technical Assistant:
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
WADA, Kotoe
KONISHI, Mie
ISOSHIMA, Yoshiko
HASHIMOTO, Terumi
Secretary:
KODAMA, Akiko
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. APC2 is distributed along actin
fibers as well as microtubules in neurons. Our investigationsuggests that APC2 is involved in the signaling pathway
from membrane receptors for extracellular guidance factors
to the intracellular migration machinery.
Sotos syndrome (OMIM #117550) is characterized byintellectual disability and a combination of typical facial
features and large head circumference. Sotos syndrome has
been known to be caused by haploinsufficiency in the NSD1
gene. Our knockdown experiments revealed that the expression of APC2 in the nervous system was under the control
of NSD1. Moreover, Apc2-knockout (KO) mice also showed
Sotos syndrome-like abnormalities. We are now investigating
the relationship between NSD1 and APC2 in more detail byexamining Nsd1-KO mice.

II. Physiological roles of receptor-like protein
tyrosine phosphatases

various 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
roles in the development of several tissues including thevascular and nervous systems. We demonstrated, for the
first time, that PTPRJ is a physiological enzyme attenuating insulin signaling in vivo. Ptprj-KO mice show lower
weight gain associated with lesser food intake comparedwith wild-type mice. Thus we are now investigating a roleof PTPRJ in the regulation of energy homeostasis.
2-2 R5 RPTP subfamily

PTPRZ is the most abundant RPTP in oligodendrocyte
precursor cells (OPCs), which are the principal source ofmyelinating oligodendrocytes. Three PTPRZ isoforms are
generated by alternative splicing from a single PTPRZ gene:two transmembrane isoforms, PTPRZ-A and PTPRZ-B, andone secretory isoform, PTPRZ-S, all of which expressedin the CNS are heavily modified with chondroitin sulfate(CS) chains. The CS moiety on their extracellular domain of
PTPRZ is essential for achieving high-affinity binding sites
for the endogenous ligands; pleiotrophin (PTN), midkine,
and interleukin-34. However, its functional significance andmolecular mechanism in regulating PTPRZ activity remains
obscure.
We have shown that PTPRZ receptors have functions tomaintain the immature status of OPCs by dephosphorylating
various substrate molecules including p190 RhoGAP. This
year, we revealed that protein expression of CS-modified
PTPRZ-A began earlier, peaking at postnatal days 5–10 (P5–
P10), and then that of PTN peaked at P10 at the developmental stage corresponding to myelination onset in the mousebrain. Ptn-KO mice consistently showed a later onset of theexpression of myelin basic protein, a major component of
the myelin sheath, than wild-type mice. Upon ligand application, PTPRZ-A/B in cultured oligodendrocyte precursor cells
exhibited punctate localization on the cell surface instead ofdiffuse distribution, causing the inactivation of PTPRZ andoligodendrocyte differentiation. Notably, the same effectwas observed with the removal of CS chains with chondroitinase ABC (chABC), but not polyclonal antibodies against
the extracellular domain of PTPRZ (Figure 1). These data
indicate that PTN-PTPRZ-A signaling controls the timingof oligodendrocyte precursor cell differentiation in vivo, in
which the negatively charged CS moiety maintains PTPRZ
in a monomeric active state, and that the positively-charged
PTN induces receptor clustering, potentially by neutralizing
electrostatic repulsion between CS chain.

Protein tyrosine phosphorylation plays crucial roles in35

National Institute for Basic Biology

Neurobiology

subfornical organ (SFO) and organum vasculosum lamina
terminalis (OVLT), and is involved in [Na+] sensing for thecontrol of salt-appetite.

CS modification

α-PTPRZ-S

chABC
PTN

3-1 Thirst control in the brain by Nax and TRPV4cell membrane
Transmembrane

Inhibition

No inhibition

Repulsive field

PTN

α-PTPRZ-SPTPRZFigure 1. Schematic representation of the role of the CS chains in regulating PTPRZ activity. The CS modification in PTPRZ was essential for
maintaining the monomeric active form, possibly by electrostatic repulsion, and, at the same time, provided ligand binding sites. The binding of
PTN appears to neutralize the negative charges of the CS chains, causing
the dimerization and inactivation of PTPase (see text).

Emerging data have indicated that PTPRZ is aberrantly
over-expressed in glioblastoma and it is a causative factor
for its malignancy. However, small molecules that selectively
inhibit the catalytic activity of PTPRZ have not been developed so far. We identified SCB4380 as the first potent inhibitor for PTPRZ by in vitro screening of a chemical library.
We determined the crystal structure of the catalytic domainof PTPRZ. Furthermore, the structural basis of the bindingof SCB4380 elucidated by a molecular docking method
was validated by site-directed mutagenesis studies (Figure2A). The intracellular delivery of SCB4380 by liposome
carriers inhibited PTPRZ activity in C6 glioblastoma cells,
and thereby suppressed their migration and proliferation invitro and tumor growth in a rat allograft model (Figure 2B).
Therefore, selective inhibition of PTPRZ represents a promising approach for glioma therapy.
ABvehicleOVLT (SFO)Nax

SCB4380

2.0

[Na+]

Na+

*

Na+-dependent signal

Water-intake
control neuron

Water intake

Ca2+
TRPV4

vehicleSCB4380

0AA

Figure 2. Development of SCB4380 and its application to a rat glioblastoma model. A, whole view of the best docking pose of SCB4380 shownby stick representation. B, rat C6 glioblastoma cells transplanted into rat
brains were allowed to grow for 7 days. One day after surgery, SCB4380/
liposome, or vehicle was icv injected daily for 5 days. Representative
sections are shown together with an enlargement of the boxed area. The
graph shows tumor volume. The horizontal bars indicate the average ofeach group. *P < 0.05, by the Student’s t-test.

III. Brain systems for body-fluid homeostasis

We have previously shown that Na x, which structurally
resembles voltage-gated sodium channels (Nav1.1–1.9), isa Na+-concentration ([Na+])-sensitive Na channel with agating threshold of ~150 mM for extracellular [Na+] ([Na+]o)in vitro. Nax is preferentially expressed in the glial cells of
sensory circumventricular organs (sCVOs) including the

36

Na+

EETs

1.0

active center

Astrocytes
EETs

Blood
Vesselvolume (mm3)SCB4380

We found that Nax is also involved in thirst control indehydrated animals (Figure 3). We investigated voluntarywater intake immediately induced after an intracerebroventricular (icv) administration of a hypertonic NaCl solution
in mice. Because the transient receptor potential vanilloid(TRPV) channels, TRPV1 and TRPV4, have been proposed
to function as osmosensors, we examined TRPV1-, TRPV4-,
Na x-, and their double-gene KO mice. The induction ofwater intake by TRPV1-KO mice was normal, whereas thatby TRPV4-KO and Nax-KO mice was significantly less thanthat by WT mice. Water intake by Nax/TRPV4-double KO
mice was similar to that by the respective single KO mice.
When TRPV4 activity was blocked with a specific antagonist(HC-067047), water intake by WT mice was significantly
reduced, whereas that by TRPV4-KO and Nax-KO mice wasnot. The same results were obtained by the administration of
miconazole, which inhibits the biosynthesis of epoxyeicosatrienoic acids (EETs), endogenous agonists for TRPV4, fromarachidonic acid (AA). Moreover, icv injection of hypertonic
NaCl with AA or 5,6-EET restored water intake by Nax-KO
mice to the WT level, but not that by TRPV4-KO mice.
These results suggest that the Na+ signal generated in Naxpositive glial cells leads to the activation of TRPV4-positive
neurons in sCVOs in order to stimulate water intake by using
EETs as gliotransmitters.

Nax

[Na+]

Ependymal cells

3rd Ventricle

Figure 3. A possible signaling pathway in sCVOs underlying the induction of water intake. When [Na+]o in plasma and cerebrospinal fluid
increases, Nax channels (red) in glial cells (astrocytes and ependymal
cells) in the SFO or OVLT are activated, leading to the synthesis of EETs.
EETs released from glial cells activate TRPV4 channels (blue) in theneighboring neurons, which conceivably control water-intake behavior.
The additional activation signal of an unknown Na+-dependent mechanism is required for the Nax/TRPV4-dependent induction of water intake.

3-2 Thirst and salt-appetite control in the brain byangiotensin II, [Na+], and cholecystokinin

Angiotensin II (Ang II) is known to drive both thirst andsalt appetite; however, the neural mechanisms underlying selective water- and/or salt-intake behaviors remain
unknown. Using optogenetics, we showed that thirst andsalt appetite are driven by distinct groups of angiotensinII receptor type 1a-positive excitatory neurons in the SFO(Figure 4). Neurons projecting to the OVLT control water
intake, while those projecting to the ventral part of the bed
nucleus of the stria terminalis (vBNST) control salt intake.
Thirst-driving neurons are suppressed under sodium-depleted
conditions through cholecystokinin (CCK)-mediated activation of GABAergic neurons. In contrast, the salt-appetitedriving neurons were suppressed under dehydrated conditions through activation of another population of GABAergic
neurons by Nax signals. These distinct mechanisms in the
SFO may underlie the selective intakes of water and/or salt
and may contribute to body fluid homeostasis.
<Water-depleted condition><Na-depleted condition>

SFO

SFO

Ang II
GABA
Water

CCK

GABA
Nax
Glia

GABA

Salt

Na+

Water

3rd ventricle

OVLT

vBNST

OVLT

•

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

GABA
Nax
Glia

Salt

3rd ventricle

Na+•

Uchiyama, S., Tomoo, T., Ogata, A., Masumura, M., and Noda, M.
(2016). Small-molecule inhibition of PTPRZ reduces tumor growth in a
rat model of Glioblastoma. Sci. Rep. 6, 20473.
Kuboyama, K., Fujikawa, A., Suzuki, R., Tanga, N., and Noda, M.
(2016). Role of chondroitin sulfate (CS) modification in the regulationof protein tyrosine phosphatase receptor type Z (PTPRZ) activity:
Pleiotrophin-PTPRZ-A signaling is involved in oligodendrocyte
differentiation. J. Biol. Chem. 291, 18117-18128.
Sakuta, H., Nishihara, E., Hiyama, T.Y., Lin, C.-H., and Noda, M.
(2016). Nax signaling evoked by an increase in [Na+] in CSF induces
water intake via EET-mediated TRPV4 activation. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 311, R299-R306.

T.Y., Utsunomiya, A.N., Matsumoto, M., Fujikawa, A., Lin,• Hiyama,
C.-H., Hara, K., Kagawa, R., Okada, S., Kobayashi, M., Ishikawa, M.,

Ang IINa+

A., Nagahira, A., Sugawara, H., Ishii, K., Imajo, S.,• Fujikawa,Matsumoto, M., Kuboyama, K., Suzuki, R., Tanga, N., Noda, M.,

vBNST

ThirstSalt appetite

Salt appetite

Water intake

Salt intake

Salt intake

Figure 4. A schematic overview of controls for thirst and salt appetitefrom SFO. Under the water-depleted condition (left), both Ang II and[Na+] increase in SFO. “Water neurons” innervating OVLT are selectively
activated under the water-depleted condition. Under the Na-depleted condition (right), both Ang II and CCK increase in SFO, and “salt neurons”
which innervate vBNST are selectively activated. Under the water- andNa-depleted condition, both water and Na intakes are stimulated, because
neither the Nax signal nor CCK signal is induced.

•

Anzo, M., Cho, H., Takayasu, S., Nigawara, T., Daimon, M., Sato,T., Terui, K., Ito, E., and Noda, M. Adipsic hypernatremia without
hypothalamic lesions accompanied by autoantibodies to subfornicalorgan. Brain Pathol. 2016 Aug 2.
Matsuda, T., Hiyama, T.Y., Niimura, F., Matsusaka, T., Fukamizu,A., Kobayashi, K., Kobayashi, K., and Noda, M. Distinct neuralmechanisms for the control of thirst and salt appetite in the subfornicalorgan. Nat. Neurosci. 2016 Dec 19.

〔Review articles〕

A., and Noda, M. (2016). Role of pleiotrophin-protein• Fujikawa,tyrosine phosphatase receptor type Z signaling in myelination. Neural
Regen. Res. 11, 549-551.

T.Y., and Noda, M. (2016). Sodium sensing in the subfornical• Hiyama,
organ and body-fluid homeostasis. Neurosci. Res. 113, 1-11.

3-3 Adipsic hypernatremia accompanied by autoantibodies to the SFO

Adipsic (or essential) hypernatremia is a rare hypernatremia
caused by a deficiency in thirst regulation and vasopressin
release. In 2010, we reported a case in which autoantibodies
targeting Nax caused adipsic hypernatremia without hypothalamic structural lesions demonstrable by magnetic resonanceimaging (MRI). This year, we reported three newly identified patients with similar symptoms. Immunohistochemical
analyses using the sera of these patients revealed that antibodies specifically reactive to the mouse SFO were present
in the sera of all cases. Passive transfer to mice of the patient
immunoglobulin revealed that autoimmune destruction of
the SFO may be the cause of the adipsic hypernatremia. This
study provides a possible explanation for the pathogenesis ofadipsic hypernatremia without demonstrable hypothalamuspituitary lesions.

Publication List:〔Original papers〕

T., Kamiya, M., Obata, F., Yamaguchi, Y., Hiyama, T.Y.,• Doura,Matsuda, T., Fukamizu, A., Noda, M., Miura, M., and Urano, Y.

(2016). Detection of lacZ-positive cells in living tissue with single-cellresolution. Angew. Chem. Int. Ed. Engl. 55, 9620–9624.

37

National Institute for Basic Biology

Neurobiology

DIVISION OF BEHAVIORAL NEUROBIOLOGY

to neurotransmitter properties of neurons (i,e, vesicularglutamate transporter).

Professor

HIGASHIJIMA, Shin-ichi

Assistant Professor:
KIMURA, Yukiko
Postdoctoral Fellow:
KIMURA, Yukiko*
Visiting Graduate Student: SHIMAZAKI, Takashi
UEMURA, Yuto
Technical Assistant:
SUZUKI, Sachiko
TERASAWA, Yoko
WATAKABE, Ikuko
ITO, Hiroko
Secretary:
TAKEUCHI, Yoshiko
The vertebrate central nervous system (CNS) contains manydifferent types of neurons that form at distinct characteristic
positions, and develop specific axonal connections andfunctions. This complexity has made it difficult to perform
detailed functional analysis of neuronal circuits: in particular,
it was very difficult to reproducibly identify cell types during
investigation. For the past 15 years, however, molecular
genetic studies have strongly suggested that the expression oftranscription factors in the developing CNS helps determine
the morphological and functional properties of neurons. Thishas opened up the possibility that researchers can use thesetranscription factors as markers to identify cell types in the
CNS. Transgenic animals that express fluorescent protein in
specific subsets of neurons are particularly powerful tools to
study functions of the corresponding neurons in the neuronal
circuits.
To fully exploit the methodology described above, we
use larval zebrafish as experimental animals. The biggest
advantage of this system is that larval zebrafish are almost
completely transparent. This allows us to utilize many opticaltechniques, including morphological/functional imaging
and optogenetics. We can also perform targeted in vivo
electrophysiological recordings with relative ease in thistransparent model. An additional advantage of zebrafish isthat their CNS is much simpler than mammals. This enables
us to perform detailed functional analysis of neuronal circuits
at a single cell resolution. Our hope is to reveal operational
principles of vertebrate CNS by using this simple system.
We have been focusing on studying neuronal circuits that
control locomotion. Much of the control of locomotor movements is accomplished by neuronal circuitry located in the
spinal cord. Therefore, the focus of our studies has beenspinal neuronal circuits in larval zebrafish.

I. Generation of Transgenic zebrafish

We have been generating transgenic zebrafish that express
fluorescent proteins (GFP or RFP), Gal4, or Cre in specificclasses of neurons in CNS by using promoter/enhancerof genes that are known to be expressed in subsets ofneurons. Most of the genes we used are transcription factors
expressed in subsets of neurons in the developing CNS. We
also used those genes whose expressions are tightly related

38

Figure 1. Examples of transgenic fish expressing GFP in specific classesof neurons.

In our early studies, we used a BAC-based transgenic
technique for the generation of transgenic fish. In 2014, we
succeeded in establishing a reliable knock-in method byutilizing the CRISPR-Cas9 system. The method we developed is highly efficient, such that nearly one-third of theraised animals become transgenic founders. Thus far, wehave established more than 20 knock-in transgenic fish. The
method greatly facilitates our functional analysis on neuronal
circuits.

II. Neuronal circuits that control rhythmic
pectoral fin movements.

Limbed vertebrates exhibit coordinated rhythmic movements of forelimbs and hindlimbs during locomotion.
Neuronal circuits that control rhythmic limb movements
in mammals have been investigated for decades, but ourknowledge is still limited because of the complexity of
their limb. Rhythmic movements of pectoral fins duringswimming in larval zebrafish is an attractive model (Figure2). Pectoral fins of larval zebrafish show left and right alternated rhythmic movements, and they are actuated only by
two types of muscles, i.e., abductor (Ab) and adductor (Ad)
(Figures 3). Due to the simplicity of pectoral fins, we expectthat we well be able to characterize neuronal circuits that
control rhythmic pectoral fin more deeply.
We performed electrophysiological recordings of Ab motoneurons (MNs) and Ad MNs during fictive swimming.
Both Ab MNs and Ad MNs show rhythmic spiking activitie(Figure 4). Activities of Ab MNs and Ad MNs on the sameside essentially alternated. Voltage clamp recordings showedthat both Ab MNs and Ad MNs received alternating excitatory and inhibitory inputs in a swimming cycle. Excitations
mainly occurred in their preferential firing phase, and inhibition mainly occurred for the rest of the period. To obtaininsights into the source of these inputs, we are now investigating timings of spiking activities in possible premotor
interneurons.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2016. The former title isindicated by an asterisk (*).

Figure 2. Rhythmic movements of the pectoral fin (arrow) duringswimming in larval zebrafish.

Figure 3. Schematic of rhythmic movements of pectoral fins duringswimming. CPG, Central Pattern Generator.

Figure 4. Rhythmic firings of an abductor motoneuron during swimming.

III. Functional analysis of En1-positive
neurons for axial movements duringswimming.

Inhibitions play important roles for shaping motor outputs
during locomotion. In the spinal cord of larval zebrafish,
there are mainly two types of inhibitory neuron: commissural inhibitory neurons and ipsilaterally-projecting inhibitory neurons. The role of the former (commissural inhibitory
neurons) is easy to understand: they are likely to play important roles for ensuring antagonistic movements of the left
and the right side of body. The role of the latter (ipsilaterallyprojecting inhibitory neurons) is less clear.
En1-positive neurons constitute major components of ipsilaterally-projecting inhibitory neurons in the spinal cord.
To investigate the function of En1-positive neurons, wehave genetically-ablated En1-positive neurons by using
the Cre-loxP system (Figure 5A). In the resultant larvae,
the cycle period for the rhythmic bending of the body was
prolonged (Figure 5B), indicating that swimming speed was
slowed down. The results show that En1-positive neurons
play an important role for controlling locomotion speed.

Figure 5. Spinal En1 neurons play an important role for controllingswimming speed. A, Ablation of spinal En1 neurons by using the
Cre-loxP system. DTA, diphtheria-toxin A. B, Cycle period of the motor
bursts during regular swimming in control and En1-DTA larvae.

Publication List:〔Original Papers〕

M.-Y., Amo, R., Kinoshita, M., Cherng, B.-W., Shimazaki, H.,• Chou,Agetsuma, M., Shiraki, T., Aoki, T., Yamazaki, M., Higashijima, S., and•Okamoto, H. (2016). Social conflict resolution regulated by two dorsal
habenular subregions in zebrafish. Science 352, 87-90.
Ota, S., Taimatsu, K., Yanagi, K., Namiki, T., Ohga, R., Higashijima,S., and Kawahara, A. (2016). Functional visualization and disruption oftargeted gene using CRISPR/Cas9-mediated eGFP reporter integrationin zebrafish. Sci. Rep. 6, 34991.

39

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:
ATSUMI, Urumi
OHARA, KaoriKOTANI, Keiko
TAKAHASHI, Yoichi
IWASE, Etsuko
IMOTO, Eiko
Secretary:
SUGIYAMA, Tomomi
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 a non-human primate, the common marmoset, in orderto understand information processing in the brain, which isrelevant to high cognitive functions.

I. Measurement of thalamocortical activities
in the primary motor cortex during
voluntary movements.

The primary motor cortex (M1) integrates a variety of
information from other brain regions and transmits motor
commands to the spinal cord. Information from the basal
ganglia and cerebellum, which is thought to be critical for
motor learning and motor execution, is signaled to the M1
through the thalamus. However, little is known about thepattern of thalamocortical activities in the M1 during selfinitiated voluntary movements. Thus, we have examined
activity patterns in layer 1 (L1) and 3 (L3) thalamocorticalaxons in the M1, which presumably receive information fromthe basal ganglia and cerebellum, respectively. We conducted
two-photon calcium imaging of L1 and L3 thalamocorticalaxons in the mouse forelimb M1 during a lever pull task†: This laboratory was closed on 31 March, 2016.

40(Hira et al., 2013; Masamizu et al., 2014). We found thatthe timing of peak activity in L1 thalamocortical axons wassequentially distributed as a population during lever-pull
movement. By contrast, the peak and onset timings of theactivity in L3 thalamocortical axons were mainly seen atlever pull initiation. During learning, the population of L1
TC axonal boutons obtained sparse lever-relevant neuralcode, while the population of L3 TC axonal boutons evolved
to be robust. Optogenetic experiments showed that the thalamocortical axons have the ability to induce a forelimb
movement and modify lever pull movement. Our results
indicate that L1 and L3 thalamocortical activities convey
distinct information required for learning of voluntary movements.
The axon imaging described above is much more vulnerable to the motion of the imaging plane than imaging ofcell bodies because of the small diameters (less than 1 mm)of axons. Especially, motion in the normal direction to theimaging plane (z-motion) often causes artefactual changes
in fluorescence intensity of axons. These changes can beconfounding in interpreting fluorescence transients observedin axons of awake active animals. To solve this problem, wedeveloped a novel registration method with z-motion correction for axon imaging. In the mouse motor cortex, fourdimensional images of axons expressing fluorescent protein
were obtained with two-photon microscopy equipped witha piezoelectric objective lens positioning system. Then, theimaging data were analyzed off-line. At every time point,
two of three z-planes were blended at an appropriate ratio toobtain a composite plane similar to the target, the temporal
average image of second z-planes. The x-y displacementsof two blended images and their blending ratio were determined using a particle filter. In the particle filter, each of
three parameters decomposed to two system variables, one
obeying a second-order trend model and another a thirdorder auto-regressive model and system noise was generated
from Gaussian or Cauchy distribution. The resample weight
for each particle was calculated with the correlation between
each composite image and the target. The fluorescence intensity of axons in obtained time series of composite planes
was significantly less deviated than that in original images.
By using this method, we were able to analyze the axonal
imaging data.

II. Wide-field two-photon microscopy for
simultaneous imaging of multiple corticalareas at cellular resolution

Understanding the dynamics of cortico-cortical communications is essential because information processing in the brain
is not only performed in intra-areal circuits but also throughinter-areal interactions. To execute voluntary movements, theinteractions between M1 and the secondary motor area (M2)and between M1 and sensory-associated areas are crucial.
Recent advances in two-photon microscopy have allowed
us to image a relatively large area (up to 1 mm) at cellular
resolution. However, it is still difficult to image a continuous
large field (>2 mm) and two distant (>3 mm) brain areas atcellular resolution using a single two-photon microscope.
We developed super-wide-field two-photon microscopy witha single objective, which allows the imaging of two distant(up to 6 mm apart) cortical areas and a large continuous area
(up to 3 mm) at cellular resolution. The method depends
on placing a novel optical device under a high-NA objective with a long working distance in a standard two-photon
microscope. The device is composed of a pair of mirrors anda holder to rotate the mirrors perpendicular to the opticalaxis. By controlling the rotation timing and angle using custom-made software, the field of view can be rapidly switchedwithout moving either the objective or the sample.
By rotating the mirror pair back and forth between two
angles, we conducted sequential two-photon calcium
imaging of neuronal activities in two distant areas up to 6
mm apart and at a depth of up to 800 μm from the corticalsurface. Furthermore, by stitching the fields of view, we
succeeded in imaging a 3 mm × 1 mm continuous area.
We applied it to concurrent calcium imaging of layer 2/3
and layer 5 neurons in rostral (M2) and caudal (M1) motor
cortical areas while the mice performed a lever-pull task.
We are analyzing how the neural activities in these fields arecoordinated during motor execution. Importantly, the opticaldevice together with the controller can be easily installed ona standard two-photon microscope. Its adaptation by neuroscientists should open the door to the study of information
processing in brain networks at cellular resolution.

activities can be monitored in the neocortex of behaving marmosets with cellular and subcellular resolution over days by
two-photon calcium imaging.

Publication List:〔Original paper〕

S., Matsubara, D., Onodera, K., Matsuzaki, M., Uemura, T.,• Terada,and Usui, T. (2016). Neuronal processing of noxious thermal stimuli
mediated by dendritic Ca2+ influx in Drosophila sensory neurons. eLife
5, e12959.

III. M armoset forelimb-movement tasks for

two-photon Ca 2+ imaging of the motor
cortexRecent advances in calcium imaging have revealed cellularand subcellular mechanisms underlying a variety of brain
functions in rodents, fishes, and invertebrates. However, thecalcium imaging technique is still difficult to apply to awake
non-human primates, especially during forelimb movementtasks, which are very useful for investigating the mechanisms underlying cognitive behaviors, decision making,motor planning/execution and motor skill learning. We haveextended the technique of two-photon calcium imaging,which we established in a non-human primate, the common
marmoset, in an anesthetized state (Sadakane et al., 2015),
to record neuronal activity in the cerebral cortex of behaving
marmosets.
To do so, we developed a novel lever-manipulation device
with a chair that restrains the head and body, but not forelimbs. Three marmosets were trained with the device 10–60
min per day for 2–3 weeks. The marmosets successfully
learned a self-initiated lever-pull task, in which the animals
have to pull the lever to get a reward. The marmosets also
learned a visual cued lever-pull task, in which the reward isdelivered only when the cue is presented on the monitor andthe lever is pulled, with additional 1–2 weeks of the training.
We conducted two-photon calcium imaging in M1 of the
marmosets during the task, using novel marmoset-specifictwo-photon microscopy, which allows us to observe any
dorsal cortical area in marmosets sitting on the chair. We
detected calcium transients responding to forelimb movements from somata and dendrites.
Overall, these results indicate that forelimb movement tasks
for head-restrained marmosets are feasible and that neural

41

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellow:

YASUGI, Masaki

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.

animations of medaka based on tracking coordinate dataand photo data obtained from real medaka. These virtual 3D
models will allow us to represent medaka faithfully and toundertake a more detailed analysis of the properties of thevisual stimuli that are critical for the induction of various
behaviors. This year, we began studying artificial intelligence(AI) programmed virtual 3D models (Figure 1). The AI
models will lead us in future work on animal behaviors.
Simultaneously, we began studying “behavioral lateralization” as a characteristic which can affect the formation of
shoals. Like humans, fishes have lateral differences in their
movement and perception. However, there have been few
studies to examine the influence of behavioral lateralizationon their interaction. If each individual has a laterally biased
response to surrounding companions or predators, how is theunited movement as shoals realized? This study is expected
to bring a new viewpoint for understanding the structure andbehavior 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).
We have developed a novel method for behavior analysis
using 3D computer graphics. The fine control of various
features of living fish have been difficult to achieve instudies of fish behavior. However, computer graphics allow
us to manipulate morphological and motion cues systematically. Therefore, we have constructed 3D computer graphic

42

Figure 1. Unsupervised learning of video representations using a deeplearning method (LSTMs). The AI predicts fish position at time 1 in reference to fish position at time 0.

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. In recent years,
we have made more developed novel visual illusions, such as
the shelf-shadow illusion .

Publication List:〔Original papers〕

A., et al. (2016). Genome sequence and analysis of the• Hoshino,
Japanese morning glory Ipomoea nil. Nat. Commun. 7, 13295.
M., and Hori, M. (2016). Predominance of parallel- and cross• Yasugi,
predation in anglerfish. Marine Ecol. 37, 576-587.

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
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellow:
IMAI, Akihiro
Postdoctoral Fellow:
MANO, Hiroaki
TORIBA, Taiyo
SOKENDAI Graduate Student: KAMIDA, Chiharu
KOSHIMIZU, Shizuka
LI, Chen
MORISHITA, Mio
PALFALVI, Gergo
SUDA, Hiraku
SUGAYA, Tomomi
ZHANG, Liechi
HORIUCHI, Yuta
DE VILLIERS, Ruan
Visiting Graduate Student: GU, Nan
Visiting Undergraduate: SAKAZAKI, Takuya
Technical Assistant:
AOKI, Etsuko
GOTO, Misako
HIRAMATSU, Mika
KAJIKAWA, Ikumi
MORI, Asuka
MATSUZAKI, Yoko
NISHI, TayoOOI, Shoko
Secretary:
KOJIMA, Yoko

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. Spatiotemporal regulation of cell divisionaxis as a grand plan of plant developmental
evolution

Cell division axis has to be properly regulated during development in both metazoans and land plants. Genetic changes
in the regulation of cell division axis lead to the developmentof multicellular organisms. Since land plants do not havecentrosomes and asteroid bodies, both of which are involvedin the axis formation of metazoans, land plants should havedifferent regulatory mechanisms. We aim to investigate

the connecting factors between microtubules and GRAS
transcription factors that regulate periclinal cell divisions
in the moss Physcomitrella patens. In addition to identify
the factors, the spatiotemporal regulatory mechanisms will
be studies to understand the basis of body plan evolution
with comparison to those in the flowering plant Arabidopsis
thaliana and the green algae Closterium peracerosum–strigosum-littorale. This is a collabortion project between ourdivision and Dr. Rumiko Kofuji in Kanazawa University,
Dr. Hiroyuki Sekimoto in Japan Women’s University, andAtsushi Mochizuki in RIKEN.

Figure 1. Evolution of the regulatory mechanisms for cell division axis
appears to be the basic change leading to the subsequent divergence ofland plants.

III. Evolution 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 datasuggest 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.
Takashi Murata is a main researcher of this study.

43

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
V. E v o lu t i o n o f R eg en e r a t i o n : M a s t e r
Regulator for Reprogramming STEMIN

Figure 2. Microtubules (left) and chromosomes (right) during spindle
formation of tobacco BY-2 cells. In the right panel, Chromosomes andcentromeres are shown as magenta and green, respectively. Bar, 10 μm.

IV. Evolution of Regeneration: Reprogrammingof Differentiated Cells to Pluripotent Stem
Cells

Different species have different morphology and also
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, the reprogramming to a stem cell can lead to generation of a new
individual, which is an effective strategy for propagation.
The ability to reprogram is different from species to species
but the reason is unknown. The moss Physcomitrella patens
has a rapid reprogramming ability and is feasible for use inexperiments. Cells in a dissected leaf are reprogrammed to
become chloronema apical stem cells within 24 hours. We
found that P. patens COLD SHOCK DOMAIN PROTEIN
genes (PpCSPs) are positive regulators of the reprogrammingin Physcomitrella. Stem-cell formation was enhanced in theover-expression lines of a PpCSP gene (Figure 3). Quadruple
deletion mutants of PpCSPs exhibited attenuated reprogramming. PpCSPs share conserved domains with an induced pluripotent stem (iPS) cell factor Lin28 in mammals, indicatingthat closely related proteins function in the enhancement ofreprogramming in both land plant and metazoan lineages. This
research is mainly performed by Chen Li and Yosuke Tamada.
The stem cell formation requires light and wounding
signals. We found that the stem cell formation was facilitated in the quadruple deletion mutant of the P. patens
SQUAMOSA promoter binding protein (PpSBP) genes, someof which are known to be repressed by light signals. In
addition, we found that PpSBPs are negatively regulatedby the wounding signal. Characterization of PpSBPs are in
progress mainly by Yukiko Kabeya and Yosuke Tamada toinvestigate cross talk between light and wounding signaling
pathways in the process of stem cell formation.

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 next examined
whether the three STEMIN genes also function in stem cell
formation in regular protonemal development, formation ofchloronema side branch initial cells. We detected promoter
activities of STEMIN1 and its paralogs in chloronema cells
undergoing side branch formation (Figure 4). On the other
hand, the activities were not detected in chloronema apicalstem cells. In addition, the frequency of side branch formation in the triple deletion mutant was significantly lower thanthat in the wild type. These results suggest that STEMIN1
and its paralogs participate in the formation of chloronemaside branch initial cells, but not in the maintenance of chloronema apical stem cells. To understand the role of STEMIN1
in reprogramming, we investigate STEMIN1-direct target
genes identified by RNA-seq and ChIP-seq analyses. Masaki
Ishikawa and Mio Morishita are this study’s main researchers.

Figure 4. Promoter activity of the STEMIN1 gene in protonemata. Greenand red represent the promoter activity and plasma membrane stainedwith FM4-64, respectively. Scale bar, 100 µm.

VI. Evolution of Regeneration: Stem cells
laterally inhibit surrounding cells

Singly isolated leaf cells are reprogramed into stem cells
in P. patens. However, only one cell of two longitudinallyisolated adjacent cells becomes a stem cell and the otherappears to be laterally inhibited by the cell to be a future
stem cell. The fourth-year graduate student Liechi Zhang isinvestigating the factors involved in the lateral inhibition.

VII. Evolution of Regeneration: Other
pathways
Figure 3. Leaves of wild type (left) and PpCSP1 Bar, 200 μm.

44

Nan Gu, a fifth-year joint graduate student between Huazon
Agricultural University and NIBB 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, a former postdoc in this division and now an
Assistant Professor in Hiroshima Institute of Technology isinvestigating the molecular function of ING as a collaboration work.

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, cytological or biochemical
approaches, the lack of genetic tools hampered the investigation of molecular mechanisms involved in these processes.
To overcome this obstacle, we sequenced and analyzed
the genome in a collaborative project with Dr. Chao-Li
Huang in National Cheng Kung University. Furthermore,
we developed an efficient genetic transformation method for
M. pudica (Mano et al., 2014) and established a CRISPR/
Cas9-mediated gene knock-out system. This year we isolated
several candidate genes that may play roles in the seismonastic movement by comparing gene expression profiles
between motor organs (pulvini) and non-motor organs andbetween extensor and flexor halves of pulvini. Functional
analyses of these genes with the CRISPR/Cas9 system are in
progress.
In addition to the reverse genetic analyses, we have challenged whole-organ 3D imaging of tertiary pulvini to comprehensively understand the volume and shape changes
of individual cells during movements. To this end, we
extensively optimized fixation, staining and tissue clearing
methods and succeeded in obtaining images that covered theentire region of a tertiary pulvinus with sufficient resolution.
We have also been developing computational methods toautomatically extract and analyze the volumes and shapes of
thousands of cells in a pulvinus. This study was conducted
mainly by Hiroaki Mano.

IX. Evolution of plant development

To investigate evolution of novel complex traits, the following studies are ongoing with graduate students: The fifth-year
graduate student Chiharu Kamida studies genes involved in
movable tentacle development in the sandew Drosera spatulata. The fourth-year graduate student Shizuka Koshimizu
is interested in the evolution of floral homeotic genes andinvestigates the function of MADS-box genes in the nonflowering plant Physcomitrella patens. The pseudanthium
is a flower-like inflorescence, the molecular mechanismsof the development of which are unknown. The fifth-year
graduate student Tomomi Sugaya succeeded in transferring
the FT gene from Arabidopsis thaliana into the pseudanthium Houttuynia cordata. Furthermore, introduction of theFT gene successfully induced flowers.

Publication List:〔Original papers〕

W., Imai, A., Tabata, R., Shigenobu, S., Yamaguchi, K., Yamada,• Tong,M., Hasebe, M., Sawa, S., Motose, H., and Takahashi, T. (2016).

•••

Polyamine resistance is increased by mutations in a nitrate transporter
gene NRT1.3 (AtNPF6.4) in Arabidopsis thaliana. Front. Plant Sci. 7,
1–22.
Pereman, I., Mosquna, A., Katz, A., Wiedemann, G., Lang, D., Decker,
E.L., Tamada, Y., Ishikawa, T., Nishiyama, T., Hasebe, M., et al. (2016).
The Polycomb group protein CLF emerges as a specific tri-methylase of
H3K27 regulating gene expression and development in Physcomitrella
patens. Biochim. Biophys. Acta 1859, 860–870.
Plavskin, Y., Nagashima, A., Perroud, P.-F., Hasebe, M., Quatrano, R.S.,
Atwal, G.S., and Timmermans, M.C.P. (2016). Ancient trans-acting
siRNAs confer robustness and sensitivity onto the auxin response. Dev.
Cell 36, 276-289.
Nagata, T., Hasebe, M., Toriba, T., Taneda, H., and Crane, P.R. (2016).
Sex conversion in Ginkgo biloba (Ginkgoaceae). J. Jap. Bot. 91 Suppl.,
120-127.

〔Review article〕• Hasebe, M. (2016). Starting bell for embryos. Nat. Plants 2, 1-2.

Figure 5. Quantitative 3D image analysis of a tertiary pulvinus ofMimosa pudica at the whole organ level.
Left: Cell walls stained by a newly developed method.
Right: Automatic segmentation of the individual cells

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

Associate Professor

KAWAGUCHI, Masayoshi SOYANO, Takashi

Assistant Professor:
Technical Staff:
Visiting Scientist
Postdoctoral Fellow:

TAKEDA, Naoya
FUKADA-TANAKA, Sachiko
NAKAGAWA, Tomomi
FUJITA, Hironori
KOBAYASHI, Yuuki
NAGAE, Miwa
MAEDA, TaroKAMEOKA, Hiromu
SHIMIZU-MITAO, Yasushi
TOKUMOTO, Yuji
OKUMA, Nao
SOKENDAI Graduate Student: TSUZUKI, Shusaku
NISHIDA, Hanna
FUKUHARA, Mai
LIU, Meng
Technical Assistant:
SUZAKI, Momoyo
OGAWA, Yuko
YOSHINORI, Yumi
TOKAIRIN, Asami
ODA, Akiko
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.

FORMATION 1 (HAR1). A loss-of function mutation in the
HAR1 gene significantly increases nodule numbers. Hence,
the CLE-RS1/2–HAR1 pathway is hypothesized to play apivotal role in the negative regulation of nodulation in AON.
KLAVIER (KLV), another shoot-acting LRR-RLK, seems to
be involved in CLE-RS1/2-mediated negative regulation of
nodulation. Recently cytokinin production was reported to
be induced in the shoot by the downstream part of the CLERS1/2–HAR1 signaling pathway. In addition, shoot-applied
cytokinin is able to move to roots and inhibit nodulation.
These results suggest that shoot-derived cytokinin may be
a shoot-derived inhibitor (SDI) candidate. There might be aproteasome-mediated degradation process for an unidentified
protein in the most downstream part of AON in roots because
the negative effect of shoot-applied cytokinin is masked by
a mutation in the F-box protein TOO MUCH LOVE (TML).
Although our knowledge of AON has been furthered, identification of additional components of AON will be undoubtedly essential for a deeper understanding of the mechanism.
In order to identify new LjCLE genes, we referred to a new
database that has a reference sequence data set containing theL. japonicus genome assembly Lj2.5 and the unique de novo
assembled contigs derived from L. japonicus. A BLAST
search using the amino acid sequence of a CLE domain from
CLE-RS1 as a query enabled us to identify five new CLE
peptides. Among the CLE peptides identified, CLE-RS3 and
LjCLE40 expression was induced in inoculated roots. A hairyroot transformation study showed that constitutive expression of CLE-RS3 in the roots significantly reduced nodule
number not only in transformed but also in untransformedroots. On the other hand, the CLR-RS3-mediated suppressionof nodulation activity was masked in the har1 and tml plants.
These results suggest that CLE-RS3 is a new component of
AON in L. japonicus that may act as a potential root-derived
signal.

I. Root nodule symbiosis

1-1 A third CLE peptide systemically controls nodulation in L. japonicus.

In root nodule symbiosis, a mutual relationship between
leguminous plants and nitrogen-fixing rhizobia, the mechanism for the autoregulation of nodulation (AON) plays a
key role in preventing the production of an excess numberof nodules. AON is based on long-distance cell-to-cellcommunication between roots and shoots. Among the 39
CLAVATA3/ESR-related (CLE) peptide genes identified fromLotus japonicus, the expression of CLE-ROOT SIGNAL 1(CLE-RS1) and -RS2 is induced immediately in response torhizobial inoculation after direct activation by an RWP-RK
type transcription factor NODULE INCEPTION (NIN).
CLE-RS1 and -RS2 peptides act as putative root-derived
signals that transmit signals inhibiting further nodule development through interaction with a shoot-acting receptorlike kinase HYPERNODULATION ABERRANT ROOT

46

Figure 1. Number of nodules formed on transformed (A) and untransformed (B) roots of the wild-type plants that have transgenic hairyroots constitutively expressing GUS or CLE-RS3 (n = 14–20 plants).
The nodulation phenotype of the plants that have transgenic hairy roots
constitutively expressing GUS (C) or CLE-RS3 (D) at 21 days after
inoculation. Transgenic roots were identified by GFP fluorescence. The
Mesorhizobium loti strain that constitutively expresses DsRED was usedin these experiments. Error bars indicate SE. Scale bars 2 mm.

1-2 Thiamine biosynthesis is required for nodule
development.

Thiamine (vitamin B1) is an essential nutrient to produce
energy. Thiamine is synthesized through a multiple-step
pathway and functions in the form of a thiamine pyrophosphate. Thiamine is assembled from pyrimidine and thiazole
moieties. In L. japonicus, THI1 and THI2 (a THI1 paralog)
catalyze the biosynthesis of the thiazole moiety, and THIC
catalyzes the biosynthesis of the pyrimidine moiety.
The phenotypes of thiamine-deficient mutants of L. japonicus are summarized in Figure 2. THIC is expressed in all
tissues and is a single copy gene in L. japonicus. The thiCmutant showed chlorosis in the leaves (Figure 2A), which
is a typical and lethal phenotype observed in the thiaminedeficient plants. THIC expression was induced in nodules,
and the nodule number also showed a reduction in the thiCmutant. However, it is not clear that the nodulation defectin the thiC mutant is caused by the loss of THIC function,
because the chlorosis resulted in severe growth defects which
also caused a decrease of nodule formation. Therefore, we
also analyzed THI1 function in nodulation. THI1 is highly
expressed in roots, nodules, and seeds, whereas THI2 isexpressed mainly in shoots. The thi1 mutant did not havechlorosis in the leaves and showed no significant growthdefects, although the knockdown plants of THI2 gene displayed chlorosis and growth defects. The thi1 mutant showed
reduced nodule and seed size (Figure 2B, C), and the phenotypes were suppressed by exogenous thiamine treatment.
The analyses indicated that thiamine affects the early stage of
nodule development. These results demonstrated that THI1
is involved in both nodule development in roots and seedmaturation in shoots, excluding the effects of chlorosis andgrowth defects.

fungus.

II. Improvement of referential AM fungus
genomeArbuscular mycorrhiza (AM) is a mutualistic plant-fungus
interaction that confers great advantages to growth andsurvival on the land. However, the molecular biologicalmechanisms governing the symbiotic relationships remain
largely unknown. The fragmented genome data of AM fungi(AMF) had been one of the barriers for the molecular biological study of AM. Although the genome of a model strainof AMF, R. irregularis DAOM-181602, has been sequenced
in multiple studies, these genomic data were made up of over
28,000 short sequences (N50 = 4-16kbp). Thus, previous
data was difficult to use for comparative genomics with otherfungal species.
To facilitate the molecular biology of AMF, we improved
R. irregularis whole-genome data using PacBio-based denovo sequencing. As a result, the total size of our 210 contigs
reached 97.2 % (149.75 Mbp) of the predicted genome
size (154 Mb), and its N50 length elongated to 2.2 Mbp.
Compared to previous analyses, the genome completenessin total assembly size increased 6-39 points, the numberof assemblies decreased by135-144 fold, and N50 length
became about 140-551 times longer (Figure 3). This
improvement of the statistics validated the availability of the
PacBio sequencing to the repeat-rich genome.
From the new assemblies, we constructed 37,711 proteincoding genes. This gene model comprised 94% of the fungal
core conservative genes, suggesting high genetic completeness of our gene model set. However, our genomic data did
not contain some of the key genes for the typically present
metabolic pathway in autotrophic fungi (e.g., Thiamine synthesis). This is credible evidence of gene loss in R. irregularis genomes, and supports the previous opinion that AMF areunable to produce those essential nutrients. AMF may obtainthe nutrients from the host plant. Overall, we succeed in providing a high-quality reference genome data for the molecular understanding of AM.

III. Pattern density control in self-organizedpattern formation

Figure 2. Thiamine-deficient phenotypes in thiC and thi1 mutants of L.
japonicus. The thiC mutant showed chlorosis in leaves (A). The nodule
size decreased in the thi1 mutant (B). The thi1 mutant also showedabnormal phenotypes in seed formation (C).

On the other hand, we could not observe obvious AM colonization phenotypes in the thi1 mutant or thiamine-treatedplants. However, it has been reported that the AM fungus
Rhizophagus irregularis lacks thiamine biosynthesis genes.
Thiamine is essential for living organisms, therefore, AM
fungus should require a thiamine supply from the host plant.
We think that further analysis is required to reveal thiaminefunction and effect on AM in both the host plant and AM

Figure 3. Assembly statistics of R. irregularis and other fungus genomic
data. The correspondence between the symbols and the assembly setsare presented in the boxed legend. The statistics (total assembly size andN50 length) from R. irregularis were presented with larger symbols. The
other 153 fungal genomes from the RefSeq database are presented withblue dots. The horizontal and vertical red lines represent averages of total
assembly numbers and N50 length of the 153 fungal genomes.

47National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Many self-organized patterns have been explained by theconcept of the Turing mechanism, in which interactions
between diffusible molecules initiate spatial instability totranslate into stable patterns. Whether or not spatial instability is induced can be determined by conventional linear stability analysis. In contrast, resulting spatial patterns producedby such instability depend on nonlinear effects of the model
dynamics and are difficult to be predicted without numericalsimulations.
In two-dimensional space, patterns generated by the Turingsystem are divided into three types: spot patterns, stripe
patterns, and reverse spot patterns (Figure 4, upper panels).
It is reported that these pattern types are associated with therelative position of the equilibrium between lower and upper
constraints in the activator–inhibitor system, one of the bestknown Turing systems. That is, spot, stripe, and reverse spot
patterns are formed when the equilibrium is closer to thelower limit, around the middle of the two limits, and closer toupper limit, respectively.
We here report that pattern density, proportion of area withhigh concentrations of the activator molecule, is strongly
correlated with Req, the relative position of the equilibrium
between the upper and lower constraints, in linear dynamics
of the activator–inhibitor system (Figure 4). Furthermore,
we demonstrate that this finding can be successfully applied
to the well-known phenomenon of animal skin color patternformation and to the patterning of stomatal lineage. This
relationship between equilibrium position and pattern density
is also observed in various nonlinear dynamics. Accordingly,
this finding could be widely applicable to self-organizedpatterns and would be a powerful and reliable tool for elucidating the underlying mechanism of self-organized patternformations in biological systems.

Publication List:〔Original papers〕

H., Dun, E.A., Lopez-Obando, M., Brewer P.B., de Saint• Kameoka,
Germain, A., Rameau, C., Beveridge, C.A., and Kyozuka, J. (2016).

•
•
•
•
•
•
•
•
•
•
•

Phloem transport of the receptor DWARF14 protein is required for fullfunction of strigolactones. Plant Physiol. 172, 1844–1852.
Kikuchi, Y., Hijikata, N., Ohtomo, R., Handa, Y., Kawaguchi, M.,
Saito, K., Masuta, C., and Ezawa, T. (2016). Aquaporin-mediatedlong-distance polyphosphate translocation directed towards the host inarbuscular mycorrhizal symbiosis: application of virus induced genesilencing. New Phytol. 211, 1202-1208.
Malolepszy, A., Mun, T., Sandal, N., Gupta, V., Dubin, M., Urbański, D.,
Shah, N., Bachmann, A., Fukai, E., Hirakawa, H., Tabata, S., Nadzieja,M., Markmann, K., Su, J., Umehara, Y., Soyano, T., Miyahara, A.,
Satoh, S., Hayashi, M., Stougaard, J., and Andersen, S. (2016). The
LORE1 insertion mutant resource. Plant J. 88, 306-317.
Miyata, K., Hayafune, M., Kobae, Y., Kaku, H., Nishizawa, Y., Masuda,Y., Shibuya, N., and Nakagawa, T. (2016). Evaluation of the role of
the LysM receptor-like kinase, OsNFR5/OsRLK2 for AM symbiosis in
rice. Plant Cell Physiol. 57, 2283-2290.
Miyata, K., and Nakagawa, T. (2016). The Lotus intrinsic ethylene
receptor regulates both symbiotic and non-symbiotic responses. Plant
Biotech. 33, 27-32.
Nagae, M., Parniske, M., Kawaguchi, M., and Takeda, N. (2016). The
thiamine biosynthesis gene THI1 promotes nodule growth and seedmaturation. Plant Physiol. 172, 2033-2043.
Nagae, M., Parniske, M., Kawaguchi, M., and Takeda, N. (2016).
The relationship between thiamine and two symbioses: root nodule
symbiosis and arbuscular mycorrhiza. Plant Signal. Behav. 11,
e1265723.
Nishida, H., Handa, Y., Tanaka, S., Suzaki, T., and Kawaguchi, M.
(2016). Expression of the CLE-RS3 gene suppresses root nodulation in
Lotus japonicus. J. Plant Res. 129, 909-919.
Sakurai, K., Tomiyama, K., Kawakami, Y., Ochiai, N., Yabe, S.,
Nakagawa, T., and Asakawa, Y. (2016). Volatile components emitted
from the liverwort Marchantia paleacea subsp. diptera. Nat. Prod.
Commun. 11, 263-264.
Tokumoto, Y., and Nakagawa, M. (2016). Climate-induced abortionand predation: reproductive success of the pioneer shrub Dillenia
suffruticosa in Malaysian Borneo. J. Trop. Ecol. 32, 50-62.
Tokumoto, Y., Kajiura, H., Takeno, S., Harada, Y., Suzuki, N., Hosaka,T., Gyokusen, K., and Nakazawa, Y. (2016). Induction of tetraploid
hardy rubber tree, Eucommia ulmoides, and phenotypic differencesfrom diploid. Plant Biotechnol. 33, 51-57.
Tsuzuki, S., Handa, Y., Takeda, N., and Kawaguchi, M. (2016).
Strigolactone-induced putative secreted protein 1 is required fortheestablishment of symbiosis by the arbuscular mycorrhizal fungus
Rhizophagusirregulars. Mol. Plant Microbe Interact. 29, 277-286.

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

K., Aoki, S., Liu, M., Umehara, Y., Suganuma, N., Iwasaki, W.,• Yano,Sato, S., Soyano, T., Kouchi, H., and Kawaguchi, M. Function andevolution of a Lotus japonicus AP2/ERF family transcription factor thatis required for development of infection threads. DNA Res. 2016 Dec
27.

Figure 4. Pattern density control by activator-inhibitor dynamics withupper and lower constraints in two-dimensional space. Pattern density(proportion of area with high activator concentrations) increases as
equilibrium becomes apart from lower limit and close to upper limit (i.e.
Req increases). Insets show activator distributions that correspond to spot
pattern (Req = 0.2), tripe pattern (Req = 0.5), and reverse spot pattern (Req
= 0.8).

48

DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:

OHDE, Takahiro
ANDO, Toshiya
Technical Staff:
MIZUTANI, Takeshi
NIBB Research Fellow:
MORITA, Shinichi
Visiting Graduate Student: MASE, Mutsuki
YATOMI, Johichiro
YUZAKI, Karen
KONISHI, Yusuke
Technical Assistant:
KAWAGUCHI, Haruka
MORITA, Junko
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.

functional 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.
Interestingly, our previous work showed that vg expressing epidermal tissue forms lateral outgrowths in non-winged
segments in the mealworm beetle (Ohde et al., 2013). Fromthese facts, we hypothesize that ancestral lateral body wall
outgrowths evolved into functional wings. To test thishypothesis, we are now comparing the role of vg and other“wing genes” between wings and lateral outgrowths in abasal winged insect, Gryllus bimaculatus, and non-winged
insect, Thermobia domestica (Figure 1).

II. Wing color patterns and mimicry of
ladybird beetles

Of the various flying animals on the earth, insects have
evolved a unique flight organ. Despite over two centuries
of 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 1A). 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 for

Insect wing color patterns demonstrate a tremendous rangeof diversity and have evolved to fulfill various ecologically important 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 signal 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 insects converge on similar color
patterns. For exploring color pattern formation in a mimic,
we use the leaf 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 wingcolor patterns, we have established a technique for germline
transformation using a piggyBac vector and RNAi in the
ladybirds.
Recently, genome-editing technologies using TALEN and
CRISPR/Cas9 provide rapid and potent genetic analysis
systems including not only gene knockout but also geneknock-in. To establish TALEN-mediated genome editingin Harmonia, we used a transgenic ladybird strain that has
single copies of exogenous EGFP and N-acetyl transferase(NAT, anti-melanin synthesis enzyme) genes. Messenger

Figure 1. The firebrat, Thermobia domestica (A). the two-spotted cricket,
Gryllus bimaculatus (B).

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

I. Origin and diversification of insect wings

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

RNAs of TALENs targeting only the EGFP transgene weremicroinjected into the fertilized eggs. As a result, 20.8% of
G0 founders produced EGFP-negative and NAT-positive
knockout progenies. This efficiency was higher than that ofgermline transformation by piggyBac vectors (about 5%). We
are continuing to develop both CRISPR/Cas9 and piggyBac
methods in our lab and will use both approaches to perturb
expression of candidate genes putatively involved in wingcolor pattern formation.
Based on the knowledge obtained from H. axyridis, we aretrying to understand how the similar wing-color patterns ofmodel and mimic are generated – for example, do they use
conserved or divergent mechanisms?

III. Acquisition and diversification of beetle
horns
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 3A). 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. In addition, we are employing a high throughput
approach. To identify novel genes involved in the sexuallydimorphic horn development in T. dichotomus, mRNA of thedeveloping horn discs has been assessed by deep-sequencing
transcriptome analysis.
To understand how, molecularly, beetle horns have diversified, we are extending our analyses to include additional

Figure 3. The Japanese rhinoceros beetle, Trypoxylus dichotomus (A),the Atlas beetle, Chalcosoma atlas (B) and the broad-horned flour beetle,
Gnatocerus cornutus (C). Adult male (Left) and female (Right).

50

beetle species with different types of exaggerated horns,
including rhinoceros beetles with diverse horn structures aswell as horned beetles in other phylogenetic groups (Figure
3).
Previously, we have shown that the sex-determination genedoublesex (dsx) plays crucial roles regulating horn development in T. dichotomus (Scarabaeoidea, Scarabaeidae)(Ito et al., 2013). The function of dsx on weapon traits in
Scarabaeoidea is conserved among beetles tested so far.
Although sex-specific weapon traits have evolved independently in various Coleopteran groups, developmental
mechanisms of sex-specific expression have not been studied
outside of the Scarabaeoidea. Therefore, we focused on thesexually dimorphic broad-horned flour beetle Gnatocerus
cornutus (Tenebrioidea, Tenebiriodae). In this beetle, only
males have well-developed mandibles and genae, and a pairof short horns on the head (Figure 3C). We investigated thefunction of dsx in this beetle using larval RNAi. Our results
show that sex-specific G. cornutus dsx isoforms have anantagonistic function for all sexually dimorphic head structures. Combining these results with previous studies in othersexually-dimorphic weaponed beetles in the Scarabaeoidea,
indicated that sex-specific regulation of weapon development
via differential Dsx isoforms is a common mechanism sharedby sexually-dimorphic beetles, even though their specificweapon traits have evolved independently.

Publication List:〔Original papers〕

H., Ishiguro, M., Nishikawa, H., Morita, S., Okada, K.,• Gotoh,Miyatake, T., Yaginuma T., and Niimi, T. (2016). Molecular cloning and•••functional characterization of the sex-determination gene doublesex
in the sexually dimorphic broad-horned beetle Gnatocerus cornutus(Coleoptera, Tenebrionidae). Sci. Rep. 6, 29337.
Gotoh, H., Zinna, R.A., Warren, I., DeNieu, M., Niimi, T., Dworkin,
I., Emlen, D.J., Miura, T., and Lavine, L.C. (2016). Identificationand functional analyses of sex determination genes in the sexuallydimorphic stag beetle Cyclommatus metallifer. BMC Genom. 17, 250.
Ozawa, T., Mizuhara, T., Arata, M., Shimada, M., Niimi, T., Okada, K.,
Okada, Y., and Ohta, K. (2016). Histone deacetylases control modulespecific phenotypic plasticity in beetle weapons. Proc. Natl. Acad. Sci.
USA 113, 15042-15047.
Zinna, R., Gotoh, H., Brent, C.S., Dolezal, A., Kraus, A., Niimi, T.,
Emlen, D., and Lavine, L.C. (2016). Endocrine control of exaggerated
trait growth in rhinoceros beetles. Integr. Comp. Biol. 56, 247-259.

LABORATORY OF BIORESOURCES

Specially Appointed Professor

NARUSE, Kiyoshi

Assistant Professor:
Postdoctoral Fellow:
Research Fellow:
Visiting Scientist:
Technical Assistant:

Secretary:

TAKEHANA, Yusuke
CHISADA, Shinichi
SASADO, Takao
YOKOI, Saori
KANEKO, Hiroyo
KIMURA, Tetsuaki
AJIOKA, Rie
HARA, Ikuyo
KOIKE, Chieko
KOIKE, Yukari
TAKAKI, Chikako
TESHIMA, Yuuko
TORII, Naoko
YAMAZAKI, TokoYANOGAWA, Azusa
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 mutants for
PGC migration and pigment cell development, developmentof human disease models, and the genetic and neural basisof sexual behavior in 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 sexdetermining genes in Oryzias fish
Sex chromosomes harbor a primary sex-determining signalthat triggers sexual development of the organism. In
mammals, Sry is the dominant male-determining genelocated on the Y chromosome, and has evolved from theneural gene Sox3 on the X chromosome probably through aregulatory mutation. However, independent evolution of sex
chromosomes is widespread in non-mammalian vertebrates,suggesting that sex determination mechanisms are regulatedby different genes and have evolved rapidly. Medaka fishes

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

in the genus Oryzias have different sex chromosomes withdifferent systems (XY and ZW), providing ideal conditions
for investigating the mechanisms that lead to the rapid
turnover of sex chromosomes (Figure 1). So far, different
sex-determining genes, Dmy and GsdfY, have been isolated
from the Oryzias species, demonstrating that turnover of sex
chromosomes is associated with the substitution of mastersex-determining genes. Recently, we identified Sox3 as anovel sex-determining gene on the XY sex chromosomes in
the marine medaka Oryzias dancena/melastgma by positional cloning. Sex reversed phenotypes in transgenic fish andloss-of-function mutants of the Y chromosomal Sox3 allele
all point to its critical role in sex determination, suggestingthat the neo-Y chromosome of O. dancena arose by cooption of Sox3. Furthermore, we found the Sox3 gene also on
the XY sex chromosomes in distantly related Oryzias
species, O. marmoratus and O. profundicola. Fine mapping
and association analysis identified the Y chromosome-specific 430-bp insertion at the Sox3 locus, which appeared to be
involved in its male determination function. The Sox3dependent sex determination system in Oryzias species ispolyphyletic, and the Y-specific insertion has not been foundin O. dancena, suggesting that Sox3 has evolved as the sexdetermining gene independently in different lineages ofOryzias. These results suggest that Sox3 might have acquired
the novel male-determining function repeatedly and independently during vertebrate evolution.

II. 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, such as
fasting hyperglycemia 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, andliver as WT medaka. The symptom is consistent with thoseof Asian patients, not but Caucasian patients and mice withleptin signaling deficiencies. Now, we are investigating the51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

gene expression associated with dysfunction of pancreatic
tissues under various feeding conditions. This will allow us
to identify the factors of diabetes that are sensitive to foodintake, regardless of obesity.

III. Adaptive significance of persistent mateguarding behavior in medaka

Males of various animals exhibit mate-guarding behavior
to prevent rival males from mating with the female. Most ofthem, however, exhibit this behavior during only the mating
period, because persistent mate-guarding is thought to havea high energy cost, which would reduce male survival rate.
Previously, we reported that medaka males exhibit mateguarding irrespective of the mating period (Yokoi et al.,2015) and it remains unknown whether there is some benefitof the persistent mate-guarding, such as enhancement ofmale reproductive success, in medaka fish. In addition,
medaka females tend to choose visually-familiarized males
as their mating partner and the adaptive significance of thisfemale mating preference is totally unknown. Recently, we
found that mate-guarding led to familiarization with thefemale while at the same time blocking the female’s visual
familiarization with other males in medaka fish. We put
three fish (female, male, male) separately in a transparent
three-chamber tank, which allowed the male in the center(near male) to maintain closer proximity to the female thanthe other male (far male). Placement of the wild-type malein the center blocked visual familiarization of the far maleby the female via mate-guarding. In contrast, placement ofan arginine-vasotocin receptor mutant male, which exhibits
mate-guarding deficits, in the center, allowing for maintaining close proximity to the female, did not block familiarization of the far male by the female. This finding suggested
that persistent mate-guarding allows males to gain familiarity
with the female over their rivals, which may enhance female
preference for the dominant male (Figure 2). We hope that
this study helps us understand the evolutional origin of “pairbonding”.

collect living resources such as standard strains, inbred
strains, and mutants in addition to frozen resources such as
EST/cDNA, BAC/ Fosmid clones, and hatching enzymes,
as well as integrated information on medaka. 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 and
CRISPR-Cas9 genome editing platform for promoting thereverse genetic approach. NBRP Medaka aims to establish afirst rate biological resource with the highest possible levelsof accessibility and ease of use.

Publication List:〔Original papers〕

E., Terao, O., and Naruse, K. (2016). Empirical evidence for• Edeline,
competition-driven semelparity in wild medaka. Popul. Ecol. 33, 246-

•
•
•
•
•
•
•

254.
Isoe, Y., Konagaya, Y., Yokoi, S., Kubo, T., and Takeuchi, H. (2016).
Ontogeny and sexual differences in swimming proximity to conspecifics
in response to visual cues in medaka fish. Zool. Sci. 33, 246-254.
Kagawa, N., Honda, A., Zenno, A., Omoto, R., Imanaka, S., Takehana,Y., and Naruse, K. (2016). Arginine vasotocin neuronal development
and its projection in the adult brain of the medaka. Neurosc. Lett. 613,
47–53.
Komine, R., Nishimaki, T., Kimura, T., Oota, H., Naruse, K., Homma,N., and Fukamachi, S. (2016). Transgenic medaka that overexpress
growth hormone have a skin color that does not indicate the activation
or inhibition of somatolactin-α signal. Gene 584, 38-46.
Takehana, Y., Sakai, M., Narita, T., Sato, T., Naruse, K., and Sakaizumi,
M. (2016). Origin of the boundary populations in medaka (Oryzias
latipes species complex). Zool. Sci. 33, 125-131.
Takehana, Y., Matsuda, Y., Ikuta, J., Kryukov, A.P., and Sakaizumi,
M. (2016). Genetic population structure of the Japanese grass lizard,
Takydromus tachydromoides (Reptilia: Squamata), inferred frommitochondrial cytochrome b variations. Curr. Herpetol. 35, 22-32.
Yokoi, S., Ansai, S., Kinoshita, M., Naruse, K., Kamei, Y., Young,
L.J., Okuyama, T., and Takeuchi, H. (2016). Mate-guarding behavior
enhances male reproductive success via familiarization with mating
partners in medaka fish. Front. Zool. 13, 21.
Zhang, X., Guan, G., Li, M., Zhu, F., Liu, Q., Naruse, K., Herpin, A.,
Nagahama, Y., Li, J., and Hong, Y. (2016). Autosomal gsdf acts as amale sex initiator in the fish medaka. Sci. Rep. 6, 19738.

〔Review article〕

K., Chisada, S., Sasado, T., and Takahana, Y. (2016). Medaka as• Naruse,model animal and current atatus of medaka biological resources. Res.
& Knowl. 2, 31-34.

Figure 2. Persistent mate-guarding blocks familiarization between thefemale and the rival male, which may enhance female preference for thedominant male.

IV. National 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 model organism, we provide, maintain and

52

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,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 branching morphology
and the branches were ubiquitously and densely distributed within the scale array to form a mesh-like architecture
similar to a nonwoven fabric. The marginal scales maintaina coherent sheet-like structure during wingbeat. (Yoshida, A.,
Tejima, S., Sakuma, M., Sakamaki, Y., and Kodama, R. Sci.
Nat. in press)

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.

53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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).

vivo. Based on these results, a TORC1 regulatory modelis proposed that free tRNA released from protein synthesis under amino acid starvation inhibits TORC1 activity.
Therefore, TORC1 employs a tRNA-mediated mechanism tomonitor intracellular amino acids (Kamada, in press 2017).

II. TORC1 phosphorylates Atg13, themolecular switch of autophagy

TORC1 negatively regulates autophagy, a protein degradation system induced by nutrient starvation.
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. 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.

III. TORC1 regulates mitotic entry via pololike kinase

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

I. How do amino acids regulate TORC1?

TORC1 is regulated by amino acids. Amino acids arefundamental nutrients, and 20 species of amino acids
building proteins are not interchangeable with each other.
Therefore, TORC1 should sense each amino acid individually. Mammalian mTORC1 is believed to be controlled by Rag
GTPases and their regulators. However, Rag-factors are dispensable for amino acid sensing by TORC1 in the budding
yeast, suggesting an alternative mechanism of TORC1 regulation.
Genetic investigation was done to discover the involvementof (aminoacyl-)tRNA in TORC1 regulation. Biochemical in
vitro TORC1 assay also revealed that tRNA directly inhibits
TORC1 kinase activity. Reducing cellular tRNA molecules
desensitizes TORC1 inactivation by nitrogen starvation in

54

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. Cdc5, the yeast polokinase mediates this regulation, and the nuclear localizationof Cdc5 at G2/M transition is controlled by TORC1. In
addition, we discovered a physiological role of TORC1 inmitosis; autophagy negatively controlled by TORC1 plays
an important part in maintenance of genome stability understarvation conditions.

IV. Ypk kinase acts directly downstream of
TORC2 to control actin organization

TORC2 has an essential function controlling polarity of theactin cytoskeleton.
We found that Ypk2, a member of the AGC kinase family is
directly phosphorylated by TORC2. The activated allele of
YPK2 can rescue a lethality caused by TORC2 dysfunction,suggesting that Ypk kinase is the major downstream proteinof the TORC2 pathway.

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

MANO, Shoji
KANAI, Masatake
WATANABE, Etsuko
KAMIGAKI, Akane
HIKINO, Kazumi
KATO, Kyoko
NAKAYAMA, Tomomi
UEDA, Chizuru

Plant cells can induce, degenerate and differentiate their organelles
to adapt to environmental changes. This flexibility of plant organelles
is the basis of the strategy for environmental adaptation in plants.
The aims of our research group are to clarify the molecular mechanisms underlying the induction, differentiation, and interaction oforganelles, and to understand the integrated functions of individual
plants through organelle dynamics.

I. Molecular 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 involvedin various biological processes such as lipid metabolism and photorespiration. To understand peroxisome dynamics and functions,
we have been analyzing a number of Arabidopsis mutants having
aberrant peroxisome morphology (apem mutants) and peroxisome
unusual poisoning (peup mutants). Based on the analyses using
these mutants a part of the mechanism of division, protein transport,
degradation of peroxisomes, and the interactions of peroxisomeswith other organelles were revealed. In addition, we found thatperoxisomes are involved in the reproductive process. Therefore,
peroxisome dynamics in gametes and gametophytes are currently
under investigation.

II. Accumulation mechanism of seed storage oils
and proteins

Plant seeds accumulate huge amounts of storage reserves such asoils, carbohydrates and proteins. Humans use these storage 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 containlarge amounts of oils. Soybean contains proteins as a major reserve.
We are analyzing the mechanisms controlling oil and protein
contents in seeds, and trying to apply our knowledge and techniques for increasing beneficial storage reserves (Figure 1).

III. Construction of The Plant Organelles Database
3 (PODB3)

PODB3 was built to promote a comprehensive understanding oforganelle dynamics. PODB3 consists of six individual units: theelectron 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.

Figure 1. Biosynthesis of lipids and proteins during Arabidopsis seed development and characterization of transgenic seeds overexpressing WRI1 during the
middle and late phases. (A, B) Lipid biosynthesis and accumulation begins
before that of seed storage proteins (SSP) during seed development. (C) A
master transcription factor regulating seed oil biosynthesis, WRINKLED1(WRI1), is expressed under the control of the FUSCA3 (FUS3) promoter,
which specifically expresses during the middle phase, in the wild type (middle
panel) and SSP knockout mutants (right panel). Bar: 0.5 mm.

Publication List:〔Original papers〕

S., Hayashi, Y., Otomo, M., Mano, S., Oikawa, K., Hayashi,• Cui,
M., and Nishimura, M. (2016). Sucrose production mediated by lipid

•
•
•
•
•
•
•

metabolism suppresses physical interaction of peroxisomes and oil
bodies during germination of Arabidopsis thaliana. J. Biol. Chem. 291,
19734-19745.
Hosokawa, Y., Iino, T., Oikawa, K., Mano, S., Yamada, K., andNishimura, M. (2016). Quantification of the adhesion strength between
peroxisomes and chloroplasts by femtosecond laser technology. BioProtoc. 6, e1834.
Kamigaki, A.*, Nito, K.*, Hikino, K., Goto-Yamada, S., Nishimura, M.,
Nakagawa, T., and Mano, S. (2016). Gateway vectors for simultaneous
detection of multiple protein−protein interactions in plant cells using
bimolecular fluorescence complementation. PLoS One 11, e0160717.
(*Co-first authors)
Kanai, M., Mano, S., Kondo, M., Hayashi, M., and Nishimura,
M. (2016). Extension of oil biosynthesis during the mid-phase of
seed development enhances oil content in Arabidopsis seeds. Plant
Biotechnol. J. 14, 1241-1250.
Kimori, Y., Hikino, K., Nishimura, M., and Mano, S. (2016).
Quantifying morphological features of actin cytoskeletal filaments inplant cells based on mathematical morphology. J. Theor. Biol. 389, 123131.
Oikawa, K., Mano, S., Yamada, K., Hosokawa, Y., and Nishimura,
M. (2016). Measuring the interactions between peroxisomes andchloroplasts by in situ laser analysis. Bio-Protoc. 6, e1790.
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. (2016). Phosphorylation of the
C-terminus of RHD3 has a critical role in homotypic ER membranefusion in Arabidopsis. Plant Physiol. 170, 867-880.
Watanabe, E., Mano, S., Nomoto, M., Tada, Y., Hara-Nishimura,I., Nishimura, M., and Yamada, K. (2016). HSP90 stabilizes auxinresponsive phenotypes by masking a mutation in the auxin receptor
TIR1. Plant Cell Physiol. 57, 2245-2254.

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

M., Mano, S., and Nishimura, M. An efficient method for the• Kanai,isolation of highly purified RNA from seeds for use in quantitativetranscriptome analysis. J. Vis. Exp. 2016 Aug 24.

〔Review articles〕

M., Mano, S., Hayashi, M., and Nishimura, M. (2016).
• Kanai,
Designing novel breeding strategies for producing high-oil crops based•on the molecular understanding of triacylglycerol metabolism. In New
Challenges in Seed Biology -Basic and translational research driving
seed technology-, Araújo, S. and Balestrazzi, A., eds. (Croatia, InTech)
pp. 137-153.
Oikawa, K., Mano, S., Hosokawa, Y., and Nishimura, M. (2016).
Analysis of physical interaction between peroxisomes and chloroplast
induced by dynamic morphological changes of peroxisomes using
femtosecond laser impulsive force. Plant Morphol. 28, 29-34.

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYOHNO Group
Assistant Professor:

OHNO, Kaoru

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

I. Gonadotropins in the starfish, Patiria
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, Pateria 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 peptides (Figure 1).

56

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. Search 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; starfishes, brittle stars, sea
urchins, sea cucumbers, crinoids, oyster, and shrimp. The
collaborators have been able to purify physiological materials which induce egg maturation from nerve extracts andanalyze them with a protein sequencer and a tandem mass
spectrometer in the analytical center of our institute. One ofthem, named cubifrin, an NGIWY-amide peptide, in the sea
cucumber Aposticopus japonicus, the others are in preparation for publications.
We have identified many neuropeptides from our EST
analysis of nerve tissues and many from RNA-seq data of
the NCBI database. Especially relaxin like peptide precursor genes and insulin/IGF like peptide precursor genes wereidentified from many species. We are testing production ofthese neuropeptides by biological methods, e.g. bacterial
systems and yeast systems, for providing to collaborators forbiological assays.

KOMINE Group
Assistant 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.

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-likebehaviors, and an aberrantly altered anxiety-like phenotype.
These behavioral phenotypes suggest that ZFHX2 might playroles 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).

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

57

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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 including Ipomoea nil (Japanese morning
glory), I. purpurea (the common morning glory), and I.
tricolor.

the InWDR1 gene carrying an insertion of a Stowaway-like
transposon, InSto1. InSto1 is the first example of an active
transposon inducing spontaneous mutations other than the
Tpn1 family transposons in I. nil.

IV. BioResource of morning glories

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 from
EST and BAC libraries of I. nil and its related species. I. nil
is one of the most popular floricultural plants in Japan, andhas a 100 year history of extensive genetic studies. Our collections include 240 lines and 160,000 DNA clones.
ab

I. Flower pigmentation patterns

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 for investigating genome variability.
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 mechanismsof these mutations.

II. Genome sequence of the Japanese morningglory

To facilitate the studies of our group as well as all morning
glory researchers, we conducted de novo genome sequencingof I. nil. We chose a standard line, Tokyo Kokei Standard,for genome sequencing, and employed shotgun sequencing
using a single molecule real time sequencing system. We
successfully reported an I. nil draft genome sequence witha scaffold N50 of 2.88 Mb, covering 98% of the 750 Mb
genome. Scaffolds covering 91% of the genome sequence areanchored to 15 pseudo-chromosomes.
From the draft genome, we could identified around 340
Tpn1 family transposons, known as the major mutagen of
I. nil, and found a putative autonomous Tpn1 transposon,named TpnA1. We also identified the CONTRACTED genelocated on the genetic map published in 1956. Comparative
genomic analysis suggested that a whole genome duplicationin Convolvulaceae, distinct from the recent whole genome
duplication in Solanaceae, has occurred after the divergenceof the two sister families.

III. A novel active transposon in the Japanesemorning glory

InWDR1 is the multifunction transcriptional regulator of
I. nil. Characterization of a medicinal herb line showing
white flowers and whitish seeds revealed that the line has

58

Figure 1. Tokyo Kokei Standard used for genome sequencing (a), and amutant carrying contracted and star mutations (b). The CONTRACTED
and STAR genes encode enzymes catalyzing brassinosteroid hormone
biosynthesis.

Publication List:〔Original paper〕

M., Morimoto, R., Hirose, M., Morita, Y., Hoshino, A., Iida, S.,• Azuma,
Oshima, Y., Mitsuda, N., Ohme-Takagi, M., and Shiratake, K. (2016). A••

petal-specific InMYB1 promoter from Japanese morning glory: a useful
tool for molecular breeding of floricultural crops. Plant Biotechnol. J.
14, 354-363.
Hoshino, A., Jayakumar, V., Nitasaka, E., Toyoda, A., Noguchi, H.,
Itoh, T., Shin-I, T., Minakuchi, Y., Koda, Y., Nagano, A., Yasugi, M.,
Honjo, M., Kudoh, H., Seki, M., Kamiya, A., Shiraki, T., Carninci, P.,
Asamizu, E., Nishide, H., Tanaka, S., Park, K.I., Morita, Y., Yokoyama,
K., Uchiyama, I., Tanaka, Y., Tabata, S., Shinozaki, K., Hayashizaki,
Y., Kohara, Y., Suzuki, Y., Sugano, S., Fujiyama, A., Iida, S., andSakakibara, Y. (2016). Genome sequence and analysis of the Japanesemorning glory Ipomoea nil. Nat. Commun. 7, 13295.
Hoshino, A., Yoneda, Y., and Kuboyama, T. (2016). A Stowaway
transposon disrupts the InWDR1 gene controlling flower and seedcoloration in a medicinal cultivar of the Japanese morning glory. Genes
Genet. Syst. 91, 37-40.

LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:

TSUGANE, Kazuo

Although transposons occupying a large portion 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
to generate 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,and the resulting tagged lines were free of somaclonal variation. The nDart1/aDart1-27 system was introduced into arice variety, Koshihikari, named MK-1. 3000 MK-1 plants
were grown in field conditions (IPSR, Okayama Univ.)(Figure 1). The genome of all plants were isolated for identifying insertion sites of nDart1.

Figure 2. Phenotype of Bushy dwarf tillers2 (Bdt2). (A) Two-month-oldplants in the field (B) Segregants of Bdt2 mutants. Wild type (left), Bdt2a(middle), Bdt2(right). (C) Abnormal bract of Bdt2.

Publication List:〔Original paper〕
Figure 1. Mk-1 plants were grown at normal field condition. Various
mutants were observed.

I. Semidominant mutation in rice

The semidominant mutations produce the intermediate phenotype in individuals heterozygous for the geneconcerned. The semidominant mutations were occasionally isolated from the MK-1, it was unclear what causes
dominant mutations. Efficient selection and analysis of
dominant mutants to analyze the gene functions in rice isvery useful. Newly isolated, Bushy dwarf tiller2 (Bdt2),
which has the valuable agronomic traits of multiple tillering and dwarfism, was obtained from the MK-1 (Figure2). Genetic analysis revealed the Bdt2 mutation was controlled by two genetic elements. One Bdt2 element, Bdt2a
showed weak dwarf phenotype, another element, Bdt2b
strengthened the effect of Bdt2a. A wild plant produces
only 10 spikes or less, while Bdt2a produced 3 times as
many, Bdt2 produced 10 times the ears.

E., Himi, E., Takahashi, H., Zhu, S., Doi, K., Tsugane,• Gichuhi,K., and Maekawa, M. (2016). Identification of QTLs for yield-related

traits in RILs derived from the cross between pLIA-1 carrying Oryza
longistaminata chromosome segments and Norin 18 in rice. Breed. Sci.
66, 720-733.

59

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Assistant:

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 cell withcomplete deletion of chromosomal rDNA repeat. Using thisstrain, condensin-dependent chromatin interaction between
two RFBs was examined by chromosome conformation
capture (3C) assay. We found the condensin-dependent chromatin interaction between the two RFB sites on the chromosome arm. This result indicates that condensin plays a role inchromatin interaction between condensin binding sites, andthis interaction leads to creation of a chromatin loop between
those sites (Figure 1). It is thought that condensin-dependent
chromatin folding is one of the basic molecular processes ofchromosome condensation. During the cell cycle stages, the
RFB - RFB interaction signal increases in metaphase andreaches its maximum level in anaphase. In addition to the
RFB – RFB interaction, the chromatin interactions between
internal regions of two RFBs increases in anaphase. Thus,
the configuration of chromatin fiber changes from a simple
loop into a complicated twisted shape as the cell cycle progresses from metaphase to anaphase.

Chromatin folding
RFB

Condensin

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 monopolin complex that are required for faithfulsegregation of homologous chromosomes during meiotic
division I. During meiosis I, monopolin complex re-localizes
from rDNA repeat to the centromere and acts for ensuringsister-chromatid co-orientation. Re-localization of Csm1/
Lrs4 proteins suggested the re-localization of condensin
from rDNA repeat to centromere. As expected, chromatinIP experiments indicated that condensin re-localizes to thecentromere during meiosis I. Condensin might clamp sisterchromatids together during meiosis I.

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

60

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.

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 developing embryo is
considered to be an essential model for collective cell
migrations. Drastic cell rearrangements lead drastic structural changes to build elaborate organs such as the tubular
network of Drosophila trachea. We are developing a software
pipeline, which automatically recognizes individual cell
shapes out of 3D space and tracks them through time. Thissystem 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.

derived 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.nins.jp/).

Figure 3. A lightweight/native 4D stack viewer equipped with functions
for manual feature extraction.

Publication List:〔Original Paper〕
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.

K., Dong, B., Wada, H., Tanaka-Matakatsu, M., Yagi, Y., and• Kato,Hayashi, S. (2016). Microtubule-dependent balanced cell contraction

and luminal-matrix modification accelerate epithelial tube fusion. Nat.
Commun. 7, 11141.

II. Particle tracking for tissue deformation
analysis
Besides cell boundary extraction, we also developed a61

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYKIMORI GroupSpecially Appointed Assistant Professor:
KIMORI, Yoshitaka
Image processing methods significantly contribute tovisualization of biomedical targets acquired from a varietyof imaging techniques, including: wide-field optical andelectron 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 on mathematicalmorphology for quantitative image analysis. One of the mostimportant purposes of image processing is to derive meaningful information, which is expressed as image structuralproperties. Mathematical morphology is a nonlinear image
processing method based on set theory and is useful for the
extraction of the structural properties from an image. It canbe used as a fundamental tool to analyze biomedical images.

the target object that is to be segmented.
This method was applied to segmentation of an abnormal
region in the chest x-ray image shown in Fig. 1. The arrow
in Fig. 1(a) shows the position of the abnormality (lungnodule). Fig. 1(b) shows the image after segmentation using
the proposed method. Candidates of lung nodule regions
were extracted. Fig. 1(c) is the resultant image of nodule
detection. The nodule region was clearly distinguished fromthe surrounding tissues.

Novel image segmentation method based onmathematical morphology

Image processing is a crucial step in the quantification of
biomedical imaging data. As such, it is fundamental to awide range of biomedical imaging fields. Image processing derives structural features, which are then numericallyquantified by image analysis. Image segmentation plays animportant role in image processing. Segmentation is the partitioning of an image into sub-regions and the extraction of atarget that is to be analyzed.
In this study, an image segmentation approach based ona new type of mathematical morphology is introduced.
Mathematical morphology is a methodology for extracting
shape and size information from an image. It involves configuration of a set of nonlinear operators that act on imagesby using structuring elements (SE). The SE, which indicatesthe shape characteristics in an image, is generally a small andsimple binary image. The two basic morphological operators
are dilation and erosion, from which many operations can bederived.
This segmentation method is based on a double morphological subtraction method. Generally, morphological subtraction methods may include top-hat
transform, rolling-ball transform, and h-maximatransform. These methods extract target objects thatare brighter than the surrounding areas. However,these methods may also extract unwanted structures
which are located in the neighborhood of the target
object. Thus, an identification process between thetarget regions and other object regions is required.
Commonly, this task is difficult due to intensity andsize similarities among these extracted regions.

In this new type of morphological segmentation method,h-maxima transform is applied twice to the original image.
The unwanted structures that surround the target are suppressed in the process of target segmentation. Furthermore,this new method has no restrictions on the size and shape of62

Figure 1. Segmentation results for chest x-ray image: (a) test image.
(b) Segmentation using the proposed method. The image was obtained
from a standard digital image database of chest lung nodules and nonnodules (Shiraishi et al., ARJ Am. J. Roentgenol. 174, 2000). (a) Original
chest x-ray image. (b) Segmentation result of candidates of lung nodule
obtained by the proposed method. (c) Detection of lung nodule.

Publication List:〔Original paper〕

K., Hagiwara, S., Kimori, Y., and Kaneko, Y. (2016).
• Murata,
Ultrastructure of compacted DNA in cyanobacteria by high-voltage
cryo-electron tomography. Sci. Rep. 6, 34934.

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

Y., Shimizu, T., Mori, T., Yoshimura, Y., Hatanaka, N.,• Osanai,
Nambu, A., Kimori, Y., Koyama, S., Kobayashi, K., and Ikenaka,

K. Rabies virus-mediated oligodendrocyte labeling reveals a single
oligodendrocyte myelinates axons from distinct brain regions. Glia
2016 Oct 19.

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
Postdoctoral Fellow:
MIYAKAWA(OKAMOTO), Misato
SOKENDAI Graduate Student: SUMIYA, Eri
YATSU, Ryohei
Visiting Graduate Student: TOHYAMA, Saki
Technical Assistant:
HAYASHI, Tomoko
INABA, KayoSecretary:
IMAIZUMI, Taeko

Synthetic chemicals found in the environment have thecapacity to disrupt the development and function of theendocrine system in both wildlife and humans. This has
drawn public concern since many of these chemicals maybind to estrogen receptors (ERs) and evoke estrogenic
effects. Early evidence that exposure to estrogenic chemicals during development could pose a threat to humanhealth came from studies of a synthetic hormone, diethylstilbestrol (DES), which was used to prevent prematurebirth and spontaneous abortion. 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 beaffected. Although many of these chemicals can bind to
ERs in wildlife and humans, the molecular basis for theaction of environmental estrogens remains poorly understood. Thus, understanding the molecular mechanisms
through which environmental estrogens and sex hormones
act during critical developmental windows is essential.

I. Developmental 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 considering 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. We found that thepersistent changes in the vagina in mice exposed neonatallyto estrogens result from the persistent activation of erbBs
and ERα, and sustained expression of EGF-like growth
factors. Currently, we are analyzing the methylation status
in the mouse vagina using a microarray (MeDIP-chip). We
found several differentially methylated or demethylated
DNA profiles in neonatally DES-exposed mouse vaginae
and controls. We thus consider that neonatal DES exposure
affects DNA methylation profiles, resulting in persistentabnormalities in mouse reproductive organs. We also foundthat ERα is indispensable for normal vaginal epithelial celldifferentiation in mice, and retinoic acid signaling determines
the fate of uterine stroma in the mouse Müllerian duct.

II. Estrogen 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
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. Squamae ER is the most sensitivein animal species. Thus, these approaches are efficient toevaluate the relationship between species and their sensitivities to chemicals.

III. Evolutionary history and functional
characterization of androgen receptor
genes in jawed vertebrates

Vertebrates show diverse sexual characteristics
which are regulated by androgens. To elucidate theevolutionary history and functional diversification ofandrogen receptor (AR) genes in vertebrates, wecloned the AR cDNAs from a shark, basal ray-finned
fishes (Actinopterygii), namely bichir and sturgeon(Acipenseriformes), and teleosts including a basal teleost,arowana (Osteoglossiformes). Molecular phylogenetic
analysis revealed that a gene duplication event gave riseto two different teleost ARs (α and β) and likely occurredin the actinopterygian lineage leading to teleosts after
the divergence of Acipenseriformes but before the splitof Osteoglossiformes. Functional analysis revealed thatthe shark AR activates the target gene via the androgen
response element by classical androgens. The teleost ARαshowed unique intracellular localization with a significantly higher transactivation capacity than that of teleost
ARβ. Therefore, the most ancient type of AR emerged
before the Chondrichthyes-Osteichthyes split and the AR
gene was duplicated during a teleost-specific gene duplication event.

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

63

National Institute for Basic Biology

Environmental Biology

VI. Environmental sex differentiation in
Daphnids and American alligators

Daphnia magna has been used extensively to evaluate theorganism- and population-based responses to 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. D. magna and D. pulex reproduce asexually (parthenogenesis) when they are in an optimal environment for food, photoperiod and population density. Onceenvironmental conditions become sub-optimal, they alter
their reproductive strategy from asexual to sexual reproduction. Chemicals are able to affect the sex determination ofdaphnids and we found that juvenile hormone (JH) agonists(insect growth regulators), for example, induce the production of male offspring. The molecular basis of environmental sex determination is largely unknown in daphnids. To
understand the molecular mechanisms of this phenomenon,
we isolated sex determination-related genes. Also, we havedeveloped a method to inject genes into D. magna and D.
pulex embryos which will allow us to study gain- and loss-offunction analyses in more detail in these species. Using thesetechniques, we demonstrated that DSX1 (double sex 1), oneof the DM-domain genes, is essential for male differentiationin D. magna. We have developed an RNAi method and a
TALEN method using D. pulex. To further explore the signaling cascade of sexual differentiation in D. magna, a gene
expression profile of JH-responsive genes is essential. We
identified JH-responsive genes in the ovary of D. magna andD. pulex exposed to JH agonist and methyl farnesoate (JHidentified 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 functionof ecdysone in molting and ovulation in D. magna. We
established a reliable induction system of female and maleoffspring in D. pulex WTN6 strain in response to photoperiod differences, and reported developmental staging of
female and male D. pulex and D. magna during embryogenesis. Metabolomics revealed involvement of pantothenate formale production responding to the short-day stimulus, andalso protein kinase C also has an essential role in inductionof males in D. pulex
Sex determination mechanisms can be broadly categorizedby 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

64

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〕

T., Zhou, L.Y., Chaudhari, A., Iguchi, T., and Nagahama,• Chakraborty,
Y. (2016). Dmy initiates masculinity by altering Gsdf/Sox9a2/Rspo1

•
•
•
•
•
•
•
•
•
•
•
•
•
•

expression in medaka (Oryzias latipes). Sci. Rep. 6, 19480.
Haselman, J.T., Kosian, P.A., Korte, J.J., Olmstead, A.W., Iguchi, T.,
Johnson, R.D., and Degitz, S.J. (2016). Developemnt of the larval
amphibian growth and development assay: Effects of chronic 4-tertoctylphenol or 17β-trenbolone exposure in Xenopus laevis fromembryo to juvenile. J. Appl. Toxicol. 36, 1639-1650.
Haselman, J.T., Sakurai, M., Watanabe, N., Goto, Y., Onishi, Y., Ito,Y., Onoda, Y., Kosian, P.A., Korte, J.J., Johnson, R.D., Iguchi, T., andDegitz, S.J. (2016). Development of the larval amphibian growth anddevelopment assay: Effects of benzophenone-2 exposure in Xenopus
laevis from embryo to juvenile. J. Appl. Toxicol. 36, 1651-1661.
Katsu, Y., Cziko, P.A., Chandsawangbhuwana, C., Thornton, J.W., Sato,R., Oka, K., Takei, Y., Baker, M.E., and Iguchi, T. (2016). A second
estrogen receptor from Japanese lamprey (Lethenteron japonicum) does
not have activities for estrogen binding and transcription. Gen. Comp.
Endocrinol. 236, 105-114.
Nakajima, T., Iguchi, T., and Sato, T. (2016). Retinoic acid signallingdetermines the fate of uterine stroma in the mouse Müllerian duct. Proc.
Natl. Acad. Sci. USA 113, 14354-14359.
Ogino, Y., Kuraku, S., Ishibashi, H., Sumiya, E., Miyagawa, S.,
Matsubara, H., Yamada, G., Baker, M.E. and Iguchi, T. (2016).
Neofunctionalization of androgen receptor by gain-of-function
mutations in teleost fish lineage. Mol. Biol. Evol. 33, 228-244.
Oka, K., Kohno, S., Ohta, Y., Guillette, L.J. Jr., Iguchi, T., and Katsu, Y.
(2016). Molecular cloning and characterization of the aryl hydrocarbon
receptors and aryl hydrocarbon receptor nuclear translocators in theAmerican alligator. Gen. Comp. Endocrinol. 238, 13-22.
Song, Y., Rundberget, J.T., Xie, L., Gomes, T.C., Høgåsen, T., Iguchi,T., and Tollefsen, K.E. (2016). Whole-organism transcriptomic analysis
provides mechanistic insight into the acute toxicity of emamectinbenzoate in Daphnia magna. Environ. Sci. Technol. 50, 11994–12003.
Sumiya, E., Ogino, Y., Toyota, K., Miyakawa, H., Miyagawa, S., andIguchi, T. (2016). Neverland regulates embryonic moltings through theregulation of ecdysteroid synthesis in the water flea Daphnia magna,
and may thus act as a target for chemical disruption of molting. J. Appl.
Toxicol. 36, 1476-1485.
Terauchi, K.J., Shigeta, Y., Iguchi, T., and Sato, T. (2016). Role of
Notch signaling in granulosa cell proliferation and polyovular follicle
induction during folliculogenesis in mouse ovary. Cell Tissue Res. 365,
197-206.
Tohyama, S., Miyagawa, S., Lange, A., Ogino, Y., Mizutani, T., Ihara,
M., Tanaka, H., Tatarazako, N., Kobayashi, T., Tyler, C.R., and Iguchi,
T. (2016). Evolution of estrogen receptors in ray-finned fish and theircomparative responses to estrogenic substances. J. Steroid Biochem.
Mol. Biol. 158, 189-197.
Toyota, K., Gavin, A., Miyagawa, S., Viant, M.R., and Iguchi, T. (2016).
Metabolomics analysis reveals an involvement of pantothenate in maleoffspring production in response to the short-day stimulus in the water
flea, Daphnia pulex. Sci. Rep. 6, 25125.
Toyota, K., Hiruta, C., Ogino, Y., Miyagawa, S., Okamura, Y., Onishi,Y., Tatarazako, N., and Iguchi, T. (2016). Comparative developmental
staging of the female and male water fleas Daphnia pulex and Daphnia
magna during embryogenesis. Zool. Sci. 33, 31-37.
Toyota, K., McNabb, N.A., Spyropoulos, D.D., Iguchi, T., and Kohno,
S. (2016). Toxic effects of chemical dispersant Corexit 9500 on waterflea Daphnia magna. J. Appl. Toxicol. 37, 201-206.
Yatsu, R., Katsu, Y., Kohno, S., Mizutani, T., Ogino, Y., Guillette,
L.J. Jr., Miyagawa, S., and Iguchi, T. (2016). Characterization ofevolutionary trend in squamate estrogen receptor sensitivity. Gen.

Comp. Endocrinol. 238, 88-95.

R., Miyagawa, S., Kohno, S., Parrott, B.B., Yamaguchi, K.,• Yatsu,
Ogino, Y., Miyakawa, H., Lowers, R.H., Shigenobu, S., Guillette,

L.J. Jr., and Iguchi, T. (2016). RNA-seq analysis of the gonadaltranscriptome during Alligator mississippiensis temperature-dependent
sex determination and differentiation. BMC Genomics 17, 77.

〔Review article〕

T., Bergman, Å., Becher, G., Bjerregaard, P., Bornman,• Zoller,R., Brandt, I., Iguchi, T., Jobling, S., Kidd, K., Kortenkamp, A.,

Slallaebaek, N., Toppari, J., and Vandenberg, L. (2016). The pathforward on endocrine disruptors requires focus on the basics. Toxicol.
Sci. 149, 272.

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National Institute for Basic Biology

Environmental Biology

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
YAMASAKI, Tomohito
KAWAI, Hisako
YAMASAKI, Hiroaki
KIM, Eunchul
SOKENDAI Graduate Student:
KAMRANI, Yousef Yari
KATO, Hiroki
KOSUGE, Kotaro
WATANABE, Akimasa
OKAJIMA, Keisuke
Visiting Scientist:
TAKIZAWA, Kenji
TAKAHASHI, Sara
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
KIDA, Emi
Secretary:
KOJIMA, Yoko
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. 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, 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 andthe 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 WTor the npq4 mutant. The purified PSII-LHCII supercomplex
containing LHCSR3 showed a normal fluorescence lifetimeat a neutral 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

66

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. However, the environmental cue that triggers the expression of LHCSR3 protein
has been elusive.

Figure 1. Purification of the PSII-LHCII-LHCSR3 supercomplex fromWT C. reinhardtii. (A) Thylakoids from WT cells grown under highlight conditions were subjected to sucrose density gradient centrifugation (SDG). (B) Polypeptides in the SDG fractions shown in (A) wereanalyzed by SDS/PAGE. (C) Polypeptides in the SDG fractions were
subjected to immunoblotting with an antibody against LHCSR3.

In plants and algae, light serves both as the energy sourcefor photosynthesis and as a biological signal that triggers
cellular responses via specific sensory photoreceptors. Red
light is perceived by bilin-containing phytochromes and blue
light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory
light, oxygen, or voltage (LOV) domains. Photoperceptionspans several orders of light intensity, ranging from far
below the threshold for photosynthesis to values beyond thecapacity of photosynthetic CO2 assimilation. We revealed
that PHOT controls qE by inducing the expression of
LHCSR3 in high light intensities. This control requires
blue-light perception by LOV domains on PHOT, LHCSR3
induction through PHOT kinase, and light dissipation inphotosystem II via LHCSR3. Mutants deficient in the PHOT
gene display severely reduced fitness under excessive light
conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital rolein microalgal acclimation to environments of variable lightintensities. Here we demonstrated the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii.

III. Species specificity in coral-algae symbiosis

Figure 2. PHOT controls induction of LHCSR3 and qE and is crucial
for survival of C. reinhardtii in high light. NPQ in WT (A) and phot (B)cells after exposure for 4 h to different wavelengths of high light). (C)Erlenmeyer flasks containing WT, acry, phot and npq4 cells after 16 h of
exposure to light of 20, 200 and 750 μmol photons m-2 s-1. (D) Schematic
representation of the relationship between photoreception, photosynthesis and photoprotection in C. reinhardtii. cp, chloroplast; cNMP, cyclic
nucleotide mono phosphate (cAMP or cGMP); ET, electron transport;eye, eyespot; nuc, nucleus; PSII, photosystem II.

II. Photoprotection mechanism in symbiotic
algae

Reef-building corals harbor endosymbiotic dinoflagellatesof the genus Symbiodinium and rely on the energy that thealgae generate from photosynthesis for their growth andsurvival. Symbiodinium within corals is a major producer
in coral reef ecosystems, which represent one of the most
biologically rich environments on earth. When the photosynthesis in Symbiodinium is damaged, corals bleach andthe sustainability of the reefs is endangered. Photosynthesis
in Symbiodinium is sensitive to small increases in seawatertemperature, resulting in photoinhibition of photosynthesis.
The thermal sensitivity of Symbiodinium to photoinhibitiondiffers among Symbiodinium strains but its mechanism has
not been well understood.
Previous studies have demonstrated that increase in
seawater temperature enhances cyclic electron flow (CEF),
which sustains photoprotective thermal energy dissipation(qE), in Symbiodinium. However, the result was still controversial. Furthermore, it was uncertain whether this ability
differs among Symbiodinium strains. We therefore examinedthe effect of increased temperature on CEF using different
Symbiodinium strains. The light-dependent reduction of the
primary electron donor PSI, i.e., P700+, was enhanced in all
Symbiodinium strains by increasing temperatures, indicating
CEF was induced by heat, which is accompanied by qE activation. However the critical temperatures for inducing CEF
were different among Symbiodinium strains. The clade A
strains with greater susceptibility to photoinhibition, OTcH-1
and Y106, exhibited higher CEF activities under moderateheat stress than a more phototolerant clade B strain Mf1.05b,
suggesting that the observed CEF induction was not a preventive protection mechanism, but was a stress response in
Symbiodinium.

Symbiodinium are genetically diverse and their physiological characteristics (e.g., stress sensitivity) differ among phylotypes. Therefore, corals need to recruit Symbiodinium
phylotypes that suit the environment in order to survive
and adapt to changes (e.g., global change and warming).
Interestingly, each coral species associate only with specificSymbiodinium phylotypes, consequently the diversity ofsymbionts available differs among coral species. However,
the mechanism regulating the diversity of compatible symbionts in cnidarian organisms, including coral, was unknown.
We studied how corals select symbionts and what determines symbiont diversity in each coral species. In our study,
we focus on the difference of cell size among Symbiodinium
strains. Using a model Aiptasia-Symbiodinium system, wefirst found that, of Symbiodinium strains tested, only largesized strains failed to infect the Aiptasia host. This sizedependency was supported by experiments using fluorescent
microspheres of different sizes. We then tested the uptakeof different sized Symbiodinium strains into aposymbiotic
polyps from two different coral species. Acropora tenuis
showed the same preference as Aiptasia, with no infection by the large-sized Symbiodinium strains. However,for Cyphastrea serailia all Symbiodinium strains tested,
including the large-sized strains, were able to infect thehost. Our results demonstrated that the infectivity of each
Symbiodinium strains in a host is primarily determined bytheir cell size and that the diversity of symbionts in each hostspecies is determined by their maximum acceptable symbiont
cell size. We proposed that corals with a higher maximum
threshold for symbiont cell size may have the opportunity
to associate with more diverse Symbiodinium phylotypes.
Such coral species may be better able to adapt to changing
environmental conditions, and more specifically might bemore suited to avoiding bleaching under increasing ocean
temperatures.

Figure 3. Infection of different Symbiodinium strains into corals.
Aposymbiotic primal polyps of A. tenuis (scale bars, 500 μm) were separately incubated with small (Mf1.05b) and large (L2469) Symbiodinium
strains. Uptake of Symbiodinium into coral polyps was monitored using astereomicroscope.

Publication List:〔Original Papers〕

L., Yamano, T., Takane, S., Niikawa, Y., Toyokawa, C., Ozawa,• Wang,S., Tokutsu, R., Takahashi, Y., Minagawa, J., Kanesaki, Y., Yoshikawa,H., and Fukuzawa, H. (2016). Chloroplast-mediated regulation of CO2concentrating mechanism by Ca2+-binding protein CAS in the green

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Environmental Biology

•
•
•
•
•

68

alga Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 113,
12586-12591.
Petroutsos, D.*, Tokutsu, R.*, Maruyama, S., Flori, S., Greiner,A., Magneschi, L., Cusant, L., Kottke, T., Mittag, M., Hegemann,
P., Finazzi, G., and Minagawa, J. (2016). A blue light photoreceptor
mediates the feedback regulation of photosynthesis. Nature 537, 563566. (*: Co-first authors)
Ueki, N., Ide, T., Mochiji, S., Kobayashi, Y., Tokutsu, R., Ohnishi, N.,
Yamaguchi, K., Shigenobu, S., Tanaka, K., Minagawa, J., Hisabori,T., Hirono, and M., Wakabayashi, K. (2016). Eyespot-dependent
determination of the phototactic sign in Chlamydomonas reinhardtii.
Proc. Natl. Acad. Sci. USA 113, 5299-5304.
Aihara, Y., Takahashi, S., and Minagawa, J. (2016). Heat inductionof cyclic electron flow around photosystem I in the symbiotic
dinoflagellate Symbiodinium. Plant Physiol. 171, 522-529.
Watanabe, C.K., Yamori, W., Takahashi, S., Terashima, I., andNoguchi, K. (2016). Mitochondrial alternative pathway-associatedphotoprotection of photosystem II is related to the photorespiratory
pathway. Plant Cell Physiol. 57, 1426-1431.
Yamamoto, H., Takahashi, S., Murray, R.B., and Shikanai, T. (2016).
Artificial remodelling of alternative electron flow by flavodiiron
proteins in Arabidopsis. Nature Plants 2, 16012.

DIVISION OF SEASONAL BIOLOGY (ADJUNCT)

Adjunct Professor

YOSHIMURA, Takashi

Specially Appointed Assistant Professor:
SHINOMIYA, Ai
SHIMMURA, Tsuyoshi
Visiting Graduate Student: NAKAYAMA, Tomoya
NAKATSUKASA, Mana
SHIMO, Takayuki
ADACHI, Daisuke
Visiting Undergraduate: MARUYAMA, Michiyo
Technical Assistant:
AKAMA, Akiko
KINOSHITA, Chie
BABA, Nayumi
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. Medaka
fish provides an excellent model to study these mechanisms
because of their rapid and robust seasonal responses. In thisdivision, we are trying to uncover the underlying mechanisms of seasonal adaptation.

I. Underlying mechanism that defines thecritical photoperiod

It is well established that the circadian clock (i.e., aninternal biological clock with a period of approximately 24hrs) is somehow involved in seasonal time measurement.

However, it remains unknown how the circadian clock
measures day length. It has been reported that Medaka populations that were caught at higher latitudes have more sophisticated responses to day length (Sawara and Egami, 1977).
For example, Medaka fish caught in Hokkaido have a longer
critical day length (i.e., duration of photoperiod required tocause a response) than those caught in Okinawa. To uncover
the underlying mechanism of seasonal time measurement,
we are currently performing a forward genetic analysis inMedaka populations collected from various latitudes all overJapan.

1-1 Variation in critical photoperiod with latitude inMedaka fish

To perform a forward genetic analysis, we have obtained
11 populations including wild populations, closed colonies,
and inbred strains from all over Japan. We have examinedthe effects of changing day length to determine the criticalday lengths that will cause seasonal responses in the gonad.
In winter, fish were subjected to 10, 11, 12, 13, and 14 h day
lengths with warm temperatures. Then gonadal development was examined to determine the critical day length. As aresult, we found differences in the critical day length among
Medaka populations. That is, Medaka from higher latitudes
required longer day length while those from lower latitudes
required shorter day length (Figure 1).

1-2 Quantitative trait loci (QTL) analysis of criticalday length

To identify the genes regulating critical day length, quantitative trait loci (QTL) analysis was conducted using
F 2 medaka derived from crosses between Northern andSouthern populations. As a result, we identified significant QTLs using Restriction-site Associated DNA (RAD)markers (Figure 2). We are now performing whole genome
re-sequencing using various Medaka strains that show different critical photoperiods.

Figure 2. Result of QTL analysis for critical day length.

II. Mechanism that determines seasonal
breeders and non-seasonal breeders

Figure 1. Different critical day length between Medaka from higher andlower latitudes.

Animals that reproduce year-round (e.g., human beingsand laboratory mice) are so-called non-seasonal breeders. In
contrast most animals living outside of tropical zones reproduce only during a particular period of the year. Therefore,
they are called seasonal breeders. However, the underlying

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National Institute for Basic Biology

Evolutionary Biology

mechanism that determines seasonal breeders and nonseasonal breeders remains unknown. To uncover this mechanism, we performed a forward genetic approach.

2-1 Geographic variations in the responses to short
day stimulus

When we transferred Medaka fish from summer conditions
to winter conditions, we noticed that Medaka from lower
latitudes do not regress their gonads even under short day
conditions. Accordingly, we next examined the responses
to short day conditions using 19 populations derived fromvarious latitudes. As a result, populations from higher latitudes showed gonadal regression, while populations fromlower latitudes did not regress their gonads (Figure 3).

III. Transcriptome analysis of seasonality inMedaka fish

In addition to the forward genetic approach, we have performed genome-wide transcriptome analysis of brain, eye,and liver of Medaka fish to understand the underlying mechanism of seasonal adaptation. Results of the eyes revealed
dynamic seasonal changes in expression of genes encoding
photopigments and the downstream phototransduction
pathway. Functional analysis suggested that seasonally regulated plasticity in the phototransduction pathway is critical
for the emergence of seasonally regulated behavior.

Publication List:〔Review articles〕

K., and Yoshimura, T. (2016). Comparative analysis reveals• Ikegami,
the underlying mechanism of vertebrate seasonal reproduction. Gen.
Comp. Endocrinol. 227, 64-68.

T., and Yoshimura, T. (2016). Molecular basis for• Nishiwaki-Ohkawa,regulating seasonal reproduction in vertebrates. J. Endocrinol. 229,
R117-R127.

Figure 3. Medaka from lower latitudes (Solid black symbols) do notregress their gonads even under short day conditions.

2-2 QTL analysis of genes determining seasonal
breeders and non-seasonal breeders

To identify genes that determine seasonal breeders andnon-seasonal breeders, we performed QTL analysis using F2generations and identified a significant QTL that determines
seasonal breeders and non-seasonal breeders. We are currently trying to identify responsible genes (Figure 4).

Figure 4. Result of QTL analysis that determines seasonal breeder andnon-seasonal breeder.

70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. 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 them into a user specificortholog database through the MyMBGD functionality.
To cope with the rapid growth of microbial genome data,
we are trying to establish an efficient protocol to maintainthe ortholog tables. We are now developing a progressive
strategy to create ortholog data: it first creates an intraspecies ortholog table and generates a species pan-genome;
next it creates an intra-genera ortholog table and generates
a genus pan-genome; and finally it conducts an inter-genera
comparison to create an ortholog table covering the entire
taxonomic range. This strategy can integrate the multiple

ortholog tables currently created: the standard ortholog tablecreated from representative species and the taxon-specificortholog tables for major taxa. The strategy can also reducethe computation time to calculate all-against-all similarities
because it calculates accurate similarities only for intergenera comparisons and uses a much more rapid (but lessaccurate) program to calculate intra-species and intra-genus
similarities.

II. Orthologous 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.
We also developed a procedure to refine the DomClustclassification based on multiple sequence alignments insteadof pairwise sequence alignments. The method is based ona scoring scheme, the domain-specific sum-of-pairs (DSP)
score, which evaluates domain-level classification using thesum total of domain-level alignment scores. We developeda refinement pipeline to improve domain-level clustering,
DomRefine, by optimizing the DSP score. DomRefine is
now used to construct the standard ortholog table covering
all the representative genomes stored in MBGD.
Domain-level classification is a unique feature of ourortholog classification system. In fact, it is different fromconventional domain databases like Pfam in that it is basedon orthology instead of homology. Particularly, this datais considered to be suitable for analyzing domain fusion
events that occurred during evolution. Now, by analyzing
the domain-level ortholog grouping data combined withtaxonomic and functional information, we are trying to elucidate when and in what kind of genes domain fusion events
frequently occurred.

III. Development of a workbench forcomparative 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.

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National Institute for Basic Biology

Theoretical Biology

We continue to develop the system and apply it to various
genome comparison studies under collaborative research
projects (including H. pylori genome comparison describedin Section V below). In particular, in addition to microbial
genome comparison, we are trying to apply RECOG to comparative analyses of transcriptomic data and metagenomicdata.

IV. Ortholog 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
and various types of biological information. To formalize
the structure of the ortholog information in the Semantic
Web, we developed an ortholog ontology (OrthO) anddescribed the ortholog information in MBGD in the form of
the Resource Description Framework (RDF). On the basis of
this framework, we have integrated various kinds of microbial data using the ortholog information as a hub, as part of
the MicrobeDB.jp project under the National Bioscience
Database Center.
This year, to further standardize the ontology, we developed the Orthology Ontology (ORTH) in collaboration with
Dr. Fernandez-Breis (Univ.Murcia) by integrating OrthO
and OGO (another ortholog ontology developed by Dr.
Fernandez-Breis) and reusing other existing ontologies.
In addition, to facilitate the utilization of the RDF databases
distributed worldwide, we developed a command-line tool,
named SPANG. SPANG simplifies querying distributed RDF
stores using the SPARQL query language, and provides aframework for reusing and sharing queries across the Web,
thereby reducing the burden of writing complex queries in
SPARQL.

V. H . p y l o r i p a n - g e n o m e a n a l y s i s f o r
identification of genomic islands
Genomes of bacterial species can show great variationin their gene content, and thus systematic analysis of theentire gene repertoire, termed the “pan-genome”, is important for understanding bacterial intra-species diversity. We
analyzed the pan-genome identified among 30 strains of thehuman gastric pathogen Helicobacter pylori isolated fromvarious phylogeographical groups. 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 (Figure 1). In addition, by clustering the accessory OGs on the basis of phylogenetic pattern similarity andchromosomal proximity, we identified 60 co-occurring geneclusters (CGCs). Besides known genomic islands including
cag pathogenicity island, bacteriophages, and integrating
conjugative elements, we identified some novel ones, including TerY-phosphorylation triad and that containing a reversetranscriptase homolog.

72

Figure 1. Definition of the mobility classes in FindMobile. Boxes in dark
blue and pale blue represent fully conserved and partially conserved coregenes, respectively. Boxes in the other colors are non-core genes that are
here classified into four classes: A) stable, B) mobile, C) intermediate, D)
unique.

Publication List:〔Original papers〕

J.T. Chiba, H., Legaz-García, M.C., and Uchiyama,• Fernández-Breis,
I. (2016). The orthology ontology: development and applications. J.
Biomed. Semant. 7, 34.

A., Jayakumar, V., Nitasaka, E., Toyoda, A., Noguchi, H.,• Hoshino,
Itoh, T., Shin-I, T., Minakuchi, Y., Koda, Y., Nagano, A.J., Yasugi, M.,

•
•
•
•

Honjo, M.N., Kudoh, H., Seki, M., Kamiya, A., Shiraki, T., Carninci, P.,
Asamizu, E., Nishide, H., Tanaka, S., Park, K., Morita, Y., Yokoyama,
K., Uchiyama, I., Tanaka, Y., Tabata, S., Shinozaki, K., Hayashizaki,
Y., Kohara, Y., Suzuki, Y., Sugano, S., Fujiyama, A., Iida, S., andSakakibara, Y. (2016). Genome sequence and analysis of the Japanesemorning glory Ipomoea nil. Nat. Commun. 7, 13295.
Matsui, H., Takahashi, T., Murayama, S., Uchiyama, I., Yamaguchi,
K., Shigenobu, S., Suzuki, M., Rimbara, E., Shibayama, K., Overby,A., and Nakamura, M. (2016). Draft genome sequence of Helicobactersuis strain SNTW101, isolated from a Japanese patient with nodular
gastritis. Genome Announc. 5, e00934-16.
Nakai, R., Fujisawa, T., Nakamura, Y., Nishide, H., Uchiyama, I.,
Baba, T., Toyoda, A., Fujiyama, A., Naganuma, T., and Niki, H. (2016).
Complete genome sequence of Aurantimicrobium minutum type strain
KNCT, a planktonic ultramicrobacterium isolated from river water.
Genome Announc. 4, e00616.
Uchiyama, I., Albritton, J., Fukuyo, M., Kojima, K., Yahara, K., andKobayashi, I. (2016). A novel approach to Helicobacter pylori pangenome analysis for identification of genomic islands. PLoS One 11,
e0159419.
Yahara, K., Furuta, Y., Morimoto, S., Kikutake, C., Komukai, S.,
Matelska, D., Dunin-Horkawicz, S., Bujnicki, J.M., Uchiyama, I., andKobayashi, I. (2016). Genome-wide survey of codons under diversifying
selection in a highly recombining bacterial species, Helicobacter pylori.
DNA Res. 23, 135-143.

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

NIBB Research Fellow:
Postdoctoral Fellow:
Technical Assistant:

TANIGUCHI, Atsushi*
TANIGUCHI, Atsushi
ISHIBASHI, Tomoko

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.

Over several years we have developed a fast light-sheet
microscope named ezDSLM, in which the excitation lightsheet and detection objective move instead of the specimen
for z-scanning. We installed an electrically tunable lens(ETL) to achieve further speed and exclusion of mechanicalvibrations that disturb continuous 4D imaging of floating or
loosely held specimens.
Our light-sheet microscopes are open to other researchers
via NIBB’s Collaborative Research and MEXT’s Advanced
Bioimaging Support (ABiS) programs. Numerous collaborations are in progress including observation of moving
Amoeba proteus, neuronal activity in Drosophila larvae, cell
migration in zebrafish embryos, cleared mouse brains, andmarine crustaceans, etc.

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. Images of floating volvox taken by ezDSLM with ETL. Left:
Single optical section. Right: Maximum intensity projection.

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 become popular over thisdecade for its advantages including low photodamage andfast image acquisition. We are now running two light-sheet
microscopes, one commercial and the other self-made, maintaining them for both collaborations and for our own researchinterest, left-right asymmetry.
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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 Student: 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.

Publication List:〔Review article〕

Y. (2016). TAL effector-mediated Genome Visualization• Miyanari,(TGV). Methods 69. 198-204.

Figure 1. Lineage allocation in mouse preimplantation development

Remodeling of nuclear architecture indevelopment

Chromatin is organized in a non-random fashion within

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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
Postdoctoral Fellow:
NOZAKI, Mamoru
Visiting Graduate Student: TOMOI, Takumi
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, it remains largely unclear how metabolism isregulated behind developmental process. 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 trans-omics approach including
metabolome and transcriptome analyses using Arabidopsis
thaliana as a model, in conjunction with standard molecular
genetics and biochemistry techniques.

I. Functional screening of orphan metabolic
enzymes from phenome data

To explore as-yet-unknown relationships between developmental and metabolic processes, we carried out functional
screening of Arabidopsis thaliana mutants of orphan genes
encoding metabolic enzymes. We examined 12 non-biased
traits including leaf size, primary root length, seed color, etc.
A large number of mutants grew normally compared to wild
type (WT), probably due to gene functional redundancy.
However, we found that cotyledon size is smaller and morevariable in one mutant line. A more severe morphologicaldefect in this mutant is irregular arrangement of cotyledons.
Although bilateral arrangement of cotyledons was observedin WT, this mutant forms non-bilateral, single or cup-shaped
cotyledon (Figure 1). This defect in cotyledon morphology/
arrangement could be detected from early embryo development. Confocal microscopy using various auxin-related
markers revealed that cellular polarity is often disordered bythis mutation, resulted in abnormal distribution of auxin in

the mutant embryos. To identify the in vivo metabolic targetof this enzyme, metabolome analysis using gas-chromatography mass-spectrometry (GC-MS) will be performed.

II. Characterization of developmental signal
intertwined with metabolism control

We recently uncovered that one of the plant developmental
signals forms an expression gradient along the leaf proximalto-distal axis to determine the cell-proliferation domain(Kawade et al., submitted). By metabolome and transcriptome analyses, we have uncovered that this developmental
signal regulates not only cell proliferation but also leucine
metabolism and/or tricarboxylic acid (TCA) cycle (Figure2). When we cultured an Arabidopsis thaliana mutant of
this developmental signal on culture media with higher
leucine, this mutant showed hypersensitivity to this aminoacid, resulted in growth arrest. Interestingly, we found thatredox condition in this mutant was perturbed at the transcriptome level. We are now re-confirming the change in metabolic profile using liquid chromatography-mass spectrometry (LC-MS) analysis in collaboration with the Functional
Genomics Facility in NIBB, and also examining the mutant’s
redox status to clarify an interaction of leucine metabolism
and/or TCA cycle with redox homeostasis. This study would
provide new insights into how a developmental signal coordinately regulates primary metabolism and redox condition,and then how this metabolic homeostasis has functional
importance on developmental processes including cell proliferation.

Figure 2. Metabolic change in the mutant of the developmental signaling
detected by GC-MS metabolomics technique. Colors indicate change in
metabolite content in dry seed, normally cultured seedling and seedlingtreated with leucine (left to right). Sugars, amino acids and organic acidsin the TCA cycle are mainly summarized here.

Figure 1. Irregular arrangement of cotyledons in the enzyme mutant. Wildtype has two bilateral cotyledons (A). In contrast, our enzyme mutant
shows abnormally-arranged (B), fused (C), single (D) and cup-shaped
cotyledons (E). Bars = 2 mm.

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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.

in basic biology.
During 2016 we carried out 52 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.

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 2016, more than 200
researchers visited to use our new facility and developed
active collaborations.

Figure 1. Next-generation sequencer

Proteomics

Three different types of mass spectrometer and two protein
sequencers, as listed below, are used for proteome studies
in our facility. In 2016, we analyzed approximately 700
samples with mass spectrometers and protein sequencers.
- LC-MS (AB SCIEX TripleTOF 5600 system)- LC-MS (Thermo Fisher SCIENTIFIC Orbitrap 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, BD FACS Aria II)- Bioimaging Analyzer (Fujifilm LAS 3000 mini; GE
FLA9000)- Laser Capture Microdissection System (Arcturus XT)- DNA Sequencer (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

76

Figure 2. Triple TOF LC/MS/MS System

Genome Informatics Training Course

We organize NIBB Genome Informatics Training Courses
every year. These courses are designed to introduce basic
knowledge and skills of bioinformatics to biologists who are
not familiar with bioinformatics. In 2016, we provided twocourses on RNA-seq data analysis and on BLAST sequence
analysis, where a total of 107 researchers and students joinedfrom all over Japan.

R., Miyagawa, S., Kohno, S., Parrott, B.B., Yamaguchi, K.,• Yatsu,
Ogino, Y., Miyakawa, H., Lowers, R.H., Shigenobu, S., Guillette,
L.J. Jr., and Iguchi, T. (2016). RNA-seq analysis of the gonadaltranscriptome during Alligator mississippiensis temperature-dependent
sex determination and differentiation. BMC Genomics 17, 77〔Original paper (E-publication ahead of print)〕

S., Wakae, K., Wakisaka, N., Nakanishi, Y., Ishikawa, K.,• Kondo,Komori, T., Moriyama-Kita, M., Endo, K., Murono, S., Wang,•

Z., Kitamura, K., Nishiyama, T., Yamaguchi, K., Shigenobu,
S., Muramatsu, M., and Yoshizaki, T. APOBEC3A associates withhuman papillomavirus genome integration in oropharyngeal cancers.
Oncogene 2016 Oct 3.
Murase, K., Shigenobu, S., Fujii, S., Ueda, K., Murata, T., Sakamoto,A., Wada, Y., Yamaguchi, K., Osakabe, Y., Osakabe, K., et al. MYB
transcription factor gene involved in sex determination in Asparagus
officinalis. Genes Cells 2016 Nov 21.

● Research activity by S. Shigenobu

Figure 3. NIBB Genome Informatics Training Course

Publication List on Cooperation:〔Original papers〕

•
•
•
•
•
•
•
•
•

Akashi, H.D., Cádiz Díaz, A., Shigenobu, S., Makino, T., and Kawata,
M. (2016). Differentially expressed genes associated with adaptation todifferent thermal environments in three sympatric cuban anolis lizards.
Molec. Ecol. 25, 2273–2285.
Higo, A., Niwa, M., Yamato, K.T., Yamada, L., Sawada, H., Sakamoto,T., Kurata, T., Shirakawa, M., Endo, M., Shigenobu, S., et al. (2016).
Transcriptional framework of male gametogenesis in the liverwort
Marchantia polymorpha L. Plant Cell Physiol. 57, 325–338.
Ide, T., Mochiji, S., Ueki, N., Yamaguchi, K., Shigenobu, S., Hirono,M., and Wakabayashi, K. (2016). Identification of the agg1 mutation
responsible for negative phototaxis in a “wild-type” strain ofChlamydomonas reinhardtii. Biochem. Biophys. Rep. 7, 379-385.
Koga, H., Fujitani, H., Morino, Y., Miyamoto, N., Tsuchimoto, J.,
Shibata, T.F., Nozawa, M., Shigenobu, S., Ogura, A., Tachibana, K.,
et al. (2016). Experimental approach reveals the role of alx1 in theevolution of the echinoderm larval skeleton. PLoS ONE 11, e0149067.
Matsui, H., Takahashi, T., Murayama, S.A., Uchiyama, I., Yamaguchi,
K., Shigenobu, S., Suzuki, M., Rimbara, E., Shibayama, K., Øverby,A., and Nakamura, M. (2016). Draft genome sequence of Helicobactersuis strain SNTW101, isolated from a Japanese patient with nodular
gastritis. Genome Announc. 4, e00934-16.
Oda, K., Kamiya, T., Shikanai, Y., Shigenobu, S., Yamaguchi, K.,
and Fujiwara, T. (2016). The arabidopsis Mg transporter, MRS2-4, isessential for Mg homeostasis under both low and high Mg conditions.
Plant Cell Physiol. 57, 754-763.
Sato, K., Tanaka, T., Shigenobu, S., Motoi, Y., Wu, J., and Itoh, T.
(2016). Improvement of barley genome annotations by deciphering theHaruna Nijo genome. DNA Res. 23, 21–28.
Tong, W., Imai, A., Tabata, R., Shigenobu, S., Yamaguchi, K., Yamada,M., Hasebe, M., Sawa, S., Motose, H., and Takahashi, T. (2016).
Polyamine resistance is increased by mutations in a nitrate transporter
gene NRT1.3 (AtNPF6.4) in Arabidopsis thaliana. Front. Plant Sci. 7,834.
Ueki, N., Ide, T., Mochiji, S., Kobayashi, Y., Tokutsu, R., Ohnishi, N.,
Yamaguchi, K., Shigenobu, S., Tanaka, K., Minagawa, J., Hisabori,T., Hirono, M., and Wakabayashi, K. (2016). Eyespot-dependent
determination of the phototactic sign in Chlamydomonas reinhardtii.
Proc. Natl. Acad. Sci. USA 113, 5299-5304.

Specially Appointed Associate Professor:
SHIGENOBU, ShujiNIBB Research Fellow:
OGAWA, Kota
Visiting Graduate Student: HSIAO, Yi-Min
Technical Assistant:
SUZUKI, Miyuzu

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

77

National Institute for Basic Biology

Research Support

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.

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 paper〕

Y., Ikuta, T., Takaki, Y., Shimamura, S., Shigenobu, S.,• Hongo,Maruyama, T., and Yoshida, T. (2016). Expression of genes involved in

the uptake of inorganic carbon in the gill of a deep-sea vesicomyid clamharboring intracellular thioautotrophic bacteria. Gene 585, 228-240.

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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)

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

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.

the OLS supports about 10 projects every year conducted byboth 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 two new types ofCollaborative Research Program. One is a new category of
the NIBB Collaborative Research for Integrative Bioimaging
using machines and bioimage processing/analysis techniques, and the other is the Advanced Bioimaging Support
Program (ABiS) of the Grant-in-aid for Scientific Researchon Innovative Areas.
The DSLM was developed by Dr. Ernst Stelzer’s groupat the European Molecular Biology Laboratory (EMBL).
This microscope can realize high-speed z-axis scanningin deeper tissue by illuminating a specimen from theside with a light sheet (more information is given in Dr.
Nonaka’s section: Lab. for Spatiotemporal Regulations).
Dr. Shigenori Nonaka conducted and supported about 10
projects as Collaborative Research Programs for Integrative
Bioimaging. The IR-LEGO was developed by Drs. Shunsuke
Yuba and Yasuhiro Kamei at the National Institute ofAdvanced Industrial Science and Technology (AIST). This
microscope can induce a target gene of interest by heating
a single target cell in vivo with a high efficiency irradiating
infrared laser. Details are described in the next section. The
IR-LEGO was also used for about 10 Collaborative Research
projects, including applications for animals and plants.

Workshop and Symposium

In 2016 we held courses during both the 9 th NIBBInternational Practical Course which focused on basic techniques for medaka and zebrafish research including imaging,and the 4th biological image processing training course. We
also have been holding a “Bioimaging Forum” every year
which discusses Bioimaging from various directions such as
microscopy, new photo-technology, and computer science.
This year we held the 10th NIBB Bioimaging Forum focusedon adaptive optics for microscopy. In addition, we held three
symposiums focused on new emerging model animals, next
generation research using amphibians, and heat and temperature in biology.

Publication List on Cooperation〔Original papers (Selected)〕

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National Institute for Basic Biology

Research Support

K., Inohaya, K., Mise, T., Kudo, A., Takada, S., and Wada,• Okada,H. (2016). Reiterative expression of pax1 directs pharyngeal pouch
segmentation in medaka. Development 143, 1800-1810.

D., Tokutsu, R., Maruyama, S., Flori, S., Greiner, A.,• Petroutsos,
Magneschi, L., Cusant, L., Kottke, T., Mittag, M., Hegemann, P.,

•
•
•
•
•

Finazzi, G., and Minagawa, J. (2016). A blue-light photoreceptor
mediates the feedback regulation of photosynthesis. Nature 537, 563566.
Shikata, T., Matsunaga, S., Kuwahara, Y., Iwahori, S., and Nishiyama,
Y. (2016). Light spectrum regulates cell accumulation during daytime
in the raphidophyte Chattonella antiqua causing noxious red tides. J.
Photochem. Photobiol. B: Biology 160, 128-133.
Suthaparan, A., Solhaug, K. A., Stensvand, A., and Gislerod, H.R.
(2016). Determination of UV action spectra affecting the infection
process of Oidium neolycopersici, the cause of tomato powdery mildew.
J. Photochem. Photobiol. B: Biology 156, 41-49.
Utagawa, U., Higashi, S., Kamei, Y., and Fukamachi, S. (2016).
Characterization of assortative mating in medaka: Mate discrimination
cues and factors that bias sexual preference. Horm. Behav. 84, 9-17.
Yokoyama, R., Yamamoto, H., Kondo, M., Takeda, S., Ifuku, K., Fukao,Y., Kamei, Y., Nishimura, M., and Shikanai, T. (2016). Grana-localized
proteins, RIQ1 and RIQ2, affect the organization of light-harvesting
complex II and grana. Plant Cell 28, 2261–2275.
Zeng, C.W., Kamei, Y., Wang, C.T., and Tsai, H.J. (2016). Subtypesof hypoxia-responsive cells differentiate into neurons in spinal cord ofzebrafish embryos after hypoxic stress. Biol. Cell 108, 1–21.

● Research activity by Y. Kamei

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

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.

Specially Appointed Associate Professor:
KAMEI, Yasuhiro
NIBB Research Fellow:
HATTORI, Masayuki
Postdoctoral Fellow:
ANDO, Yoriko
Technical Assistant:
CHISADA, Eriko
ATSUMI, Urumi
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 andfluorescent 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

80

Figure 2. Schematic illustration of heat shock response of cells (left) andan infrared laser-evoked gene operator (IR-LEGO) microscope system.

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 development. 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. Moreover, this system can be combinedto the cre/loxP recombination technique for long-term gene

Figure 3. Long-term gene expression system with cre/loxP recombination system and its example of practical experiment in medaka embryo
to adult.

expression (Figure 3).
As mentioned above, excess irradiation resulted in cell
damage, so we utilized the system to ablate target cells withstrong pulsed irradiation. In collaboration with National
Taiwan University, we used the system for neuronal regeneration study in zebrafish and revealed that a kind of neuronalprecursor cell played an important role within the neuron
regeneration step the in zebrafish spinal cord (Zeng et al.
Biol. Cell 2016). In addition, the IR-LEGO system can beutilized for thermo-biology because spatiotemporal micrometer order local heating is difficult without this system. Now
we are trying to estimate thermal properties of cells and biomaterials in vivo using the IR-LEGO system.

Publication List:〔Original papers〕

R., Ishida, S., Urawa, H., Kamei, Y., and Kohchi, T.
• Nishihama,(2016). Conditional gene expression/deletion systems for Marchantia

•
•
•
•
•

polymorpha using its own heat-shock promoter and the cre/loxPmediated site-specific recombination. Plant Cell Physiol. 57, 271-280.
Suzuki, M., Takagi, C., Miura, S., Sakane, Y., Suzuki, M., Sakuma, T.,
Sakamoto, N., Endo, T., Kamei, Y., Sato, Y., Kimura, H., Yamamoto,T., Ueno, N., and Suzuki, K.T. (2016). In vivo tracking of histone H3lysine 9 acetylation in Xenopus laevis during tail regeneration. Genes
Cells 21, 358-369.
Utagawa, U., Higashi, S., Kamei, Y., and Fukamachi, S. (2016).
Characterization of assortative mating in medaka: Mate discrimination
cues and factors that bias sexual preference. Horm. Behav. 84, 9-17.
Yokoi, S., Ansai, S., Kinoshita, M., Naruse, K., Kamei, Y., Young,
L.J., and Takeuchi, H. (2016). Mate-guarding behavior enhances male
reproductive success via familiarization with mating partners in medaka
fish. Front. Zool. 13, 21.
Yokoyama, R., Yamamoto, H., Kondo, M., Takeda, S., Ifuku, K., Fukao,Y., Kamei, Y., Nishimura, M. and Shikanai, T. (2016). Grana-localized
proteins, RIQ1 and RIQ2, optimize the dynamics of light-harvesting
complex II and grana stacking in Arabidopsis. Plant Cell 28, 2261-2275.
Zeng, C.W., Kamei, Y., Wang, C.T., and Tsai, H.J. (2016). Subtypesof hypoxia-responsive cells differentiate into neurons in spinal cord ofzebrafish embryos after hypoxic stress. Biol. Cell, 108, 357-377.

81

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 printers are also available. On
this system, we provide various biological databases anddata retrieval/analysis programs, and support large-scale data
analysis and database construction for institute members andcollaborative researchers. Especially, we have supported
the construction and maintenance of published databases ofvarious 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).

82

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. 71).

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
Specially Appointed Professor:
NARUSE, Kiyoshi
Associate Professor:
WATANABE, Eiji
TANAKA, Minoru
Technical Staff:
OHSAWA, Sonoko
HAYASHI, Kohji
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
SUZUKI, Kohta
SUGINAGA, Tomomi
FUJIMOTO, Daiji
MATSUMURA, Kunihiro
GODA, Misato
NOMOTO, Yoshihiro

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
and birds.

Figure 1. Mouse (MCH(ICR))

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 replacement and point mutation.

Figure 2. Equipment for manipulating mice eggs.

In 2016 (from January 1 to December 31), 2,907 fertilized
eggs (in vitro fertilization; 2,546 eggs of 14 lines in which
1,747 eggs of 9 lines were frozen for long-term storage,
frozen eggs: 361 of 7 lines) and 4,175 mice were brought

83

National Institute for Basic Biology

Research Support

into the facility in the Yamate area, and 59,019 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 2016 (from January
1 to December 31), 25 mice were brought into the facility in
the Myodaiji area, and 1,961 mice (including pups bred in
the facility) were taken out.

facility) were taken out. In the laboratory for chick embryos
there were no fertilized eggs or chicken embryos brought inor taken out this year. These animals were used for research
activities in neurobiology and developmental biology.
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.

Figure 4. Quarantine room for medaka and zebrafish.

III. Research activities
Figure 3. Large sized autoclave in the Myodaiji area.

II. Research support activities (small fish andbirds)

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 developed inthis facility. All the rooms are qualified to meet the criteria
for transgenic animals, allowing researchers to generate andmaintain these important biological tools.
In 2016 (from January 1 to December 31), 600 medaka (300
adults and 300 fertilized eggs) were brought to the facility
and 23,691 medaka and zebrafish (2,796 adults, 90 embryos
and 20,805 fertilized eggs, including animals bred in the84

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. 42, 51, and 33).

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

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
SUZUKI, Keiko
KOTANI, 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, and 12 rooms with theP1P physical containment level for established and emerging
model plants, for example the thale cress Arabidopsis
thaliana, the rice Oryza sativa, the moss Physcomitrella
patens, the liverwort Marchantia polymorpha, the green
alga Chlamydomonas reinhardtii and several other flowering plants including several carnivorous plants. Most culture
space is fully used the whole year by more than 80 researchers from both outside and inside groups.
As well as regular culture conditions, extreme environmental conditions for light and temperature are available forvarious types of experiments. Three chambers (3.4 m2 each)that can control CO2 and humidity in addition to temperatureand light (max 70,000 lux) conditions are available. A tissue
culture rack with dimming LEDs and pulse-width modulation controllers are used for algae culture under precise lightcontrol. Autotrophic and heterotrophic culture devices arealso available for researchers using cyanobacteria, algae,
and cultured flowering plant cells. Aseptic experiments canbe performed in an aseptic room with clean benches and asafety cabinet. Several analytical instruments including aflow cytometry system and a DUAL-PAM, for DNA content
and chlorophyll fluorescent measuring, respectively, are alsoavailable.
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. Four greenhouses (4, 6,
9, and 9 m2) with air-conditioning are provided for the cultivation of rice Oryza sp., Lotus japonica and related legumespecies, as well as mutant lines of the Japanese morning
glory. Two greenhouses (9 and 18 m2) with air-conditioning
meet the P1P physical containment level and are available
for experiments using transgenic plants. The Plant Culture
Laboratory also maintains a 46 m2 building with storage andworkspace. Part of the building is used for rearing of theorchid mantis and the Japanese rhinoceros beetle.
Due to the building renovation work, 4 phytotrons and threelight environmental simulators were removed, and some of
the facilities are temporally closed. The closed facilities will
be renewed and opened in May 2017.

● 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
47 DNA clones to both local and international biologists thisyear. A database for the whole genome sequence of I. nil wasbuilt and opened to the public.
Research activities of the Assistant Professor A. Hoshino
are shown on the laboratory page (p. 58).

Cell Biology Research Facility
Associate Professor:
Assistant Professor:
Technical Assistant:

WATANABE, Eiji
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.

Figure 5. Equipment for tissue and cell culture.

85

National Institute for Basic Biology

Research Support

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

Head

Specially Appointed Professor

KAWAGUCHI, Masayoshi NARUSE, Kiyoshi

Specially Appointed Assistant Professor:
KIMURA, Tetsuaki
TAKETSURU, Hiroaki
Technical Assistant:
AKIMOTO-KATO, Ai
MATSUBAYASHI, Naomi
MIZOKAMI, Yuko
TSUZUKI, Chizuru
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.

86

In 2016, IBBP Center stored 4,478 384-well and 69 96-wellplates consisting of 1,726,176 clones as cDNA/BAC clones,
11,792 tubes for animal cells, plant and animal samples, proteins,
genes and microorganisms, 541 133mm-straw tubes for spermand 654 seed samples. In total 1,727,371 samples are stored.

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

II. Collaborative 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 technologies for biological resources for which
long-term storage is unavailable. 2) Basic cryobiologicalresearch enabling us to improve low temperature storage forbiological resources. In 2016, we also worked to establish
a research center for cryo-biological study thorough thisCollaborative Research Project. Accordingly, we organized
the Cryopreservation Conference 2016 on November 10-11,
2016 at the Okazaki Conference Center, Okazaki, Japan. We
had 128 participants from several fields covering physics,
chemistry, biology, and technology.

Figure 1. Number of MII oocytes or OGCs collected from ovaries offemales. aSignificantly different at P < 0.05.

Figure 2. In vitro maturation of immature rat oocytes.

Research activity by H. Taketsuru
Specially Appointed Assistant Professor:
TAKETSURU, Hiroaki

In vitro maturation of immature rat oocytes

Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative
method to produce numerous matured oocytes. Furthermore,establishment of an in vitro maturation system with simple
culture conditions is cost effective and leads to easy handlingof oocytes. This study examined developmental ability of
rat germinal vesicle (GV) oocytes maturing in vitro undersimple culture conditions. Significantly different numbers ofovulated oocytes reached the second metaphase of meiosis(MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/
SsNSlc (2.2) rats in whom superovulation was induced by
pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (Figure 1). However, similar numbers of
GV oocytes were obtained from ovaries of PMSG-injected

Wistar (27.7), F344 (34.7), and BN (24.7) rats (Figure 1).
These GV oocytes were cultured in vitro in HTF, αMEM,
and a 1:1 HTF+αMEM or TYH+αMEM mixture (Figure 2).
High proportions of Wistar and F344 oocytes that matured
to MII in αMEM were parthenogenetically activated bystrontium chloride treatment (78% and 74%, respectively).
Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injectionand embryo transfer to foster mothers (Figure 3). Although
BN oocytes cultured in αMEM could be parthenogenetically activated and developed into offspring, the success rate
was lower than that for Wistar and F344 oocytes. This study
demonstrated that numerous GV oocytes were produced in
rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed
to maintain valuable rat strains experiencing reproductive
difficulties.

Figure 3. Offspring were obtained from IVM oocytes.

87National Institute for Basic Biology

Research Support

Publication List:〔Original paper〕

H., and Kaneko., T. (2016). In vitro maturation of• Taketsuru,
immature rat oocytes under simple culture conditions and subsequentdevelopmental ability. J. Reprod. Dev. 62, 521-526.

Research activity by T. Kimura
Specially Appointed Assistant Professor:
KIMURA, Tetsuaki

Positional cloning of iridophores mutant

See-through medaka lines are suitable for the observation of internal organs throughout life. They were bred bycrossings of multiple color mutants. However, some of thecausal genes for these mutants have not been identified. The
medaka has four pigment cell types: black melanophores,
yellow xanthophores, white leucophores, and silvery iridophores. To date, causal genes of melanophore, xanthophore,and leucophore mutants have been elucidated, but the causal
gene for the iridophore mutant remains unknown. Iridophoremutant guanineless (gu) exhibits a strong reduction in visible
iridophores throughout its larval and adult stages. The gu
locus was previously mapped on chromosome 5, but was
located near the telomeric region, making it difficult to integrate into the chromosome. To circumvent this, I perfomed
synteny analysis using the zebrafish genome and found astrong candidate gene, pnp4a. Gene targeting and a complementation test showed that the candidate gene is a causal
gene of gu. This result will allow the establishment of inbred
medaka strains or other useful strains with see-through phenotypes, without disrupting the majority of the genetic background of each strain.

Figure 1. gu and wild-type.

88

CENTER FOR RADIOISOTOPE FACILITIESHeadAssociate Professor

Technical Staff:

MATSUDA, Yoshimi(Radiation Protection Supervisor)
SAWADA, Kaoru(Radiation Protection Supervisor)
IINUMA, Hideko
HAYASHI, Tomoko

HASEBE, Mitsuyasu

Technical Assistant:

KODAMA, Ryuji

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. Hayashi 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 2016.
1. The facility’s emergency manual was updated. Main pointsof modification are the following.
1) When the fire alarm goes off.
2) When the warning declaration about an earthquake
disaster is issued.
The emergency manual file is placed in the contamination
check room.
2. The cameras of the video recording system at the Yamate
area were superannuated, and were exchanged in February
2016. (Figure 1A)
3. The pumps of the radioisotope waste water treatment
facility at the Myodaiji area could not start draining when
the water level was low. By adding an electric valve andexchanging flap valves in February 2016, the pumps operate
normally. (Figure 1B)
4. As the number of users decreases, the ventilating installations, the air conditioners, and the low temperature rooms
are deactivated when there are no users. The ice makers weredeactivated after June 2016.
The number of registrants and the number of users fromJanuary 2016 to December 2016 are shown in Table 1.

Figure 1. The CRF’s notable activities in 2016.
A: exchanging of the video camera at the Yamate area
B: adding the electric valve at the Myodaiji area

Users and visitors counted by the access control systemof the controlled areas numbered 1,531 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.

Table 1. Numbers of registrants and users at Myodaiji area and Yamate
area in 2016.

Table 2. Users and visitors who entered each controlled area in 2016.

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

Table 3. Balance of radioisotopes received and used (kBq) at each controlled area in 2016.

Table 4. Training courses for radiation workers in 2016.

89

National Institute for Basic Biology

Research Support

RESEARCH ENHANCEMENT STRATEGY OFFICE

Director

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.
903) 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
Technical Assistant:
OTA, Kyoko
KAWAGUCHI, Colin
BAN, Misato
Group Adviser:
FUJIMORI, ToshihikoThis 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 2016

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). Publication of leaflets introducing the individual researchers of NIBB (in Japanese). Design and distributionof 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.

INTERNATIONAL COOPERATION GROUP
Specially 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),
the Temasek Life Sciences Laboratory (TLL, Singapore)and Princeton University, USA on the basis of cooperative
agreements, through exchanging people and techniques andjointly holding scientific meetings. NIBB is also conducting
the “NIBB International Collaborative Research Initiative”
to promote high-level international collaborations between
faculty members of NIBB and researchers around the world.
NIBB invites leading-edge researchers from abroad as“Guest Professors” to promote academic exchange withNIBB 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. This group, cooperating with the Okazaki
Administration Office and International Affairs Division,
City of Okazaki, also supports researchers and students who
visit NIBB.

The main activities of the group in 2016

1) Coordination of the International Conferences and theInternational Practical Course
This group coordinated the following International
Conference hosted by NIBB:The 64th NIBB Conference “Evolution of Seasonal Timers”
Okazaki, Japan, April 22 - 24, 2016 (p. 93)
The 9th NIBB International Practical Course and the 4th
NIBB-TLL Joint International Practical Course “Genetics
and Imaging of Medaka and Zebrafish” Okazai, Japan,
Augusut 18 – 31, 2016 (p. 94)
2) Support of visiting researchers to NIBBThis group supported visits of foreign researchers related to
the following events:
One Guest Professor coming from Academia Sinica,

Taiwan (March) with the financial support of NINS and
two researchers coming from EMBL, Germany (July) and
Princeton University, USA (September)3) Support of education related programs
This group supported hosting students participating in thefollowing student-related activities of NIBB:NIBB Internship Program 2016 (p. 98)

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 2016, this group hosted a total of 114 collaboration
projects. Through these collaborations, 28 research papers
were published. A noteworthy achievement was the awardwinning publication by Prof. Wakabayashi (Tokyo Instituteof Technology) and his colleagues on the phototactic
response of the green alga Chalmydomonas reinharditii.
Through the close collaboration, the NIBB team analyzed the
genome of an alga mutant by using next-generation sequencing technology resulting in the identification of the gene
responsible for the phenotype.
T., Mochiji, S., Ueki, N., Yamaguchi, K., Shigenobu, S., Hirono,• Ide,
M., and Wakabayashi, K. (2016). Identification of the agg1 mutation•

responsible for negative phototaxis in a “wild-type” strain ofChlamydomonas reinhardtii. Biochem. Biophys. Rep. 7, 379-385.
Ueki, N., Ide, T., Mochiji, S., Kobayashi, Y., Tokutsu, R., Ohnishi, N.,
Yamaguchi, K., Shigenobu, S., Tanaka, K., Minagawa, J., Hisabori,T., Hirono, M., and Wakabayashi, K. (2016). Eyespot-dependent
determination of the phototactic sign in Chlamydomonas reinhardtii.
Proc. Natl. Acad. Sci. USA 113, 5299-5304.

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 2016, this group contributed to the management of theresearch support system covering childbirth and childcare
and to establishing a network between female researchers.
91

National Institute for Basic Biology

Research Support

TECHNICAL DIVISIONHead

KAJIURA-KOBAYASHI, Hiroko

Common Facility Group

Research Support Group

Chief:

Chief:

MIWA, Tomoki● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki
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● NIBB Bioresource Center
Unit Chief:
OHSAWA, Sonoko
Subunit Chief:
HAYASHI, Kohji
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
SUZUKI, KeikoSUZUKI, Kohta
SUGINAGA, Tomomi
KOTANI, Keiko● 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.

92

MORI, Tomoko● Developmental Biology
Technical Staff:
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, SanaeMIZUGUCHI, Hiroko● Neurobiology
Subunit Chief:

TAKEUCHI, Yasushi● Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
Unit Chief:
MIZUTANI, Takeshi
Subunit Chief:
KABEYA, Yukiko● Environmental Biology
Technical Staff:
NODA, Chiyo

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.

The 64th NIBB Conference“Evolution of Seasonal Timers”
Organizers: Andrew Loudon (Univ. Manchester, UK), David Burt (Roslin Inst., UK), David
Hazlerigg (UiT –Arctic Univ. Norway, Norway), Takashi Yoshimura (NIBB, Japan)
April 22 (Fri) - 24 (Sun), 2016
Until now how animals recognize and adapt to seasonalchanges in the environment has remained a mystery thathas long fascinated human beings. In addition, the impact
of sudden climate change caused by global warming etc. on
the ecosystem is also a concern, and societal interest in thismystery is great.
With recent research understanding of the mechanisms bywhich plants and animals perceive seasons has progressed,and both the universality and diversity of the seasonal sensing
mechanisms of living organisms has come to light. With thisbackground, the 64th NIBB Conference entitled “Evolution ofseasonal timers” was held with the aim of promoting in-depth
understanding of the design principles and evolution of theseasonal sensing mechanisms of living things.
Although the keyword of the conference was “season”,
topics ranged from “mathematical analysis of the flowering of wild plants in the field”, “mechanisms of migratory behavior and spawning activity of eels”, “hibernationof squirrels”, and “chromatin remodeling and chemical
genetics”. Leading researchers in their respective fields
came together from across the world. The approximately
100 participants made for an ideal size for the meeting, andfacilitated deep discussions in face-to-face settings for every
participant throughout the three day period. Members who
would normally never have a chance to meet face to face at ausual academy gathered together for an opportunity to deeply
consider seasonal sensation from an interdisciplinary point ofview, and we were able to obtain a lot of inspiration toward
future research. We received kind acknowledgements such as“the most stimulating symposium I ever had” and “an unforgettable symposium”
I’d like to express my gratitude to the members of the
Office of International Cooperation and the Division of
Seasonal Biology for the efforts they made to the conference,and I very much appreciate the support from the National
Institute for Basic Biology, the Daiko foundation, and the
Human Frontier Science Program.
Takashi Yoshimura (On behalf of the organizers)

Speakers
Barnes, Brian (UAF), Burt, David (The Roslin Institute/Univ. of Edinburgh), Foulkes, Nicholas (KIT), Hazlerigg, David (UiT The Arctic
Univ. of Norway), Loudon, Andrew (The Univ. of Mancheste), McClung, C. Robertson (Dartmouth College), Menaker, Michael (Univ. ofVirginia), Simonneaux, Valerie (INCI), Tessmar-Raible, Kristin (Univ. of Vienna)
Izawa, Takeshi (The Univ. of Tokyo), Kitano, Jun (NIG), Kudoh, Hiroshi (Kyoto Univ.), Myung, Jihwan (RIKEN), Nakamichi, Norihito(Nagoya Univ.), Niwa, Ryusuke (Univ. of Tsukuba), Numata, Hideharu (Kyoto Univ.), Satake, Akiko (Kyushu Univ.), Takumi, Toru
(RIKEN), Tsukamoto, Katsumi (Nihon Univ.), Yoshimura, Takashi (NIBB)

93

The 9th NIBB International Practical Course,
The 4th NIBB-TLL Joint International Practical Course“Genetics and Imaging of Medaka and Zebrafish”■P eriod: August 18 (Thu) 31(Wed), 2016
■Participants: 17 (2 from
Argentina, 2 Korea, 2Singapore, 1 Germany, 1India, 1 Taiwan, 1 UK, and7 Japan)■Venue: National Institute for
Basic Biology, Japan■Lecturers:
Dr. Tomonori DEGUCHI(AIST)
Dr. Rie GOTO (Ehime
Univ.)
Dr. Masayuki HATTORI (NIBB)
Dr. Shinya KOMOTO (EMBL)
Dr. Akiko KONDOW (Fujita Health Univ.)
Dr. Laszlo ORBAN (TLL)
Dr. Sachihiro SUZUKI (OIST)Dr. Saori YOKOI (NIBB)
Dr. Goro YOSHIZAKI (TUMSAT)
Dr. Zoltán VARGA (Zebrafish International Resource
Center/ Univ. of Oregon)■Course Staff:
Dr. Yoriko ANDO (NIBB), Dr. Satoshi ANSAI (NIG),
Dr. Shoji FUKAMACHI (Japan Women’s Univ.), Dr.
Akiko KONDOW (Fujita Health Univ.), Dr. Shin-ichi
HIGASHIJIMA (NIBB), Dr. Yasuhiro KAMEI (NIBB),
Dr. Shinya KOMOTO (EMBL), Dr. Kiyoshi NARUSE(NIBB), Dr. Shigenori NONAKA (NIBB), Dr. Sachihiro
SUZUKI (OIST), Dr. Yusuke TAKEHANA (NIBB), Dr.
Saori YOKOI (NIBB)■Contents of the course:
Gene knock-in using the CRISPR/Cas9 system in zebrafish, Gene knock-out using the CRISPR/Cas9 system inmedaka, Highly efficient transgenesis using the PhiC31
system in medaka, Optomotor response in medaka
mutant, Local gene induction with infrared laser-evoked
gene operator (IR-LEGO) in medaka, in vivo imaging,
Cryopreservation of sperm and artificial insemination in
medaka94

The 9th NIBB International Practical Course - 4th
NIBB-TLL Joint International Practical Course was heldfrom August 18 to 30, 2016. We received 28 applications,and 17 (8 males, 9 females) passed the screening process.
The participants comprised a group of 7 from Japan and 10
from overseas of 10 regions in 7 countries. Dr. Laszlo Orban,
one of the organizers of the program, attended as a special
lecturer as well, and we invited other researchers to give
special lectures during the course such as Dr. Zoltan Varga,
who is the director of the Zebrafish International Resource
Center, the University of Oregon, Dr. Rie Goto from
Ehime University, and Dr. Goro YOSHIZAKI from Tokyo
University of Marine Science and Technology. Moreover
we invited 7 lecturers from both Japan and abroad to giveseminars. The course was covered the topics “Highly efficient transgenesis using the PhiC31 system”, “Gene knock-in
using the CRISPR/Cas9 system in zebrafish as well as
medaka”, “Cryopreservation of sperm and artificial insemination in medaka”, “Imaging using light-sheet microscopes”,
and “Gene expression induction method by IR-LEGO”.
We managed the course without supporting the travel costs
for students, however in spite of having supported travel
costs in the past we had just under twice as many applicantsfor this year’s course. It was the first time we hosted participants from South America, of which we had two. We werealso pleased that we received students from both EMBL and
TLL, two of NIBB’s research cooperation institutes.
We plan to continue to conduct NIBB International
Practical Courses which feature small fish research with a
combination of basic laboratory techniques and the latest
technology in the future.
(Kiyoshi Naruse on behalf of the organizing committee)NIBB-EMBL Collaboration
Dr. Anne Ephrussi’svisit to NIBB

NIBB-Princeton University
Collaboration
Prof. Ileana M. Cristea’s visit to NIBB

Dr. Anne Ephrussi, the head of the Developmental Biology
Unit of EMBL, visited on July 12 nd, 2016. She gave aseminar titled “An atypical RNA-binding Tropomyosin
recruits kinesin-1 to oskar mRNA for transport in the
Drosophila oocyte” and visited the Laboratory of Neuronal
Cell Biology to have a discussion with the members of Prof.
Shiina’s team about intracellular RNA transport.
In addition, Dr. Ephrussi had a meeting with Dr. Masayuki
Yamamoto, Director General of the National Institute for
Basic Biology, and Prof. Naoto Ueno on the future of collaborative activities between EMBL and NIBB.

Prof. Ileana M. Cristea from the Department of Molecular
Biology, Princeton University, visited on September 23rd,
2016. Prof. Cristea has been conducting an international
joint research project, financially supported by NINS, with
the Division of Morphogenesis at NIBB. She gave a seminar
titled “Virology meets Proteomics: Understanding humanorganelle remodeling in space and time during viral infection” exclusively for NIBB’s researchers, and had a meetingwith the members of the Division of Morphogenesis, Prof.
Ueno’s group, to discuss the future of the joint research
project.

95

The 10th NIBB
Bioimaging Forum“Pioneering Bioimaging in the
New Era”
Organizer: Yasuhiro Kamei, Hideki
Takami, Yutaka Hayano, Mitsuo Takeda,
Hirotsugu Yamamoto, Masayuki Hattori,
Takashi Murata, Shigenori Nonaka,
Yosuke Tamada
We held the 10th NIBB Bioimaging Forum on February 16th
and 17th 2016. The subtitle for this meeting was “Pioneering
Bioimaging in the New Era”, and aimed at the developmentof new fields in bio-imaging by asking experts in various
disciplines (optics, engineering, mathematics, astronomy,
biology) to give lectures on new and anticipated imaging
methods on the horizon. The forum was divided into 5
sessions; on the first day “The Present State and Problemsof Imaging in the Fields of Biology and Astronomy” wasaddressed, followed by “Optical Theory Opening Up the
Future of Imaging”, which covered the latest optical theory
on holography and coherence, and the lecture “Creationof New Imaging Technology 1” on topics such as tomography was given. On the second day, we held the lecture“State-of-the-Art Image-Sensors and Devices” on cuttingedge camera technology and “Creation of new Imaging
Technology 2” on the sparse modeling theory. There were 52
people in the audience, and more than 10 industry attendees.
At the end of the forum a comprehensive discussion was heldwith all members, including the audience, and a consensus
was reached on the necessity and difficulties of fusing theimaging fields. In addition, nearly 30 participants remainedin the venue after the forum, and enthusiastic discussions andconsultations on joint research took place. Although meetings
aiming at establishing new bioimaging methods throughfusion of different fields are uncommon in Japan the active
interaction of participants at this forum lead to its success.
(Yasuhiro Kamei)

The NIBB Genome
Informatics Training
CourseThe NIBB Core Research Facilities organizes a series oftraining courses on up-to-date research techniques. The
NIBB Genome Informatics Training Course (GITC) is specially designed for biologists who are not familiar withbioinformatics. In 2016, we held two sets of training courses
on RNA-seq analysis and one course on BLAST analysis.
While we previously provided a 3-day program on RNA-seq
analysis, we rearranged the course this year dividing it into
two 2-day programs: one was a preparatory course to learn
basics of UNIX and R and the other was a practical course
to learn pipelines of RNA-seq analysis using next-generation
sequence data. The new BLAST analysis course aimed toenable participants to get familiar with a large scale sequence
database search in the local environment using BLAST
software for systematic sequence data analyses. These GITC
courses offered lectures and hands-on tutorials.

Introduction to RNA-seq – from thebasics of NGS to de novo analyses
February 25(Thu)-26(Fri), 2016

(Preparatory Course) Basics of UNIX and R■O rganizer: Dr. Ikuo Uchiyama (NIBB Core
Research Facilities)■L ecturers: Dr. Ikuo Uchiyama, Dr. Shuji
Shigenobu, Mr. Tomoki Miwa, Ms. Hiroyo
Nishide, Mr. Takanori Nakamura (NIBB Core
Research Facilities)■Participants: 22■Program:
1. UNIX for beginners
2. Introduction to “R”
3. Text processing
4. Shell scripting
5. Exercises

March 10(Thu)-11(Fri), 2016

(Practical Course) RNA-seq analysis pipeline■O rganizer: Dr. Shuji Shigenobu (NIBB Core
Research Facilities)■L ecturers: Dr. Shuji Shigenobu, Dr. Ikuo
Uchiyama, Dr. Katsushi Yamaguchi, Ms. Hiroyo
Nishide (NIBB Core Research Facilities), Dr.
Masanao Sato (Keio Univ.)■Participants: 21 (including 1 from NIBB)■Program:
1. NGS basic data formats and NGS basic tools
2. Introduction to statistics
3. RNA-seq pipeline: genome-based and transcriptome-based approaches
4. Multivariate statistics
5. Exercises

96Introduction to RNA-seq – from thebasics of NGS to de novo analyses
August 25(Thu)-26(Fri), 2016

(Preparatory Course) Basics of UNIX, R, and NGS■O rganizer: Dr. Shuji Shigenobu, Dr. Ikuo
Uchiyama (NIBB Core Research Facilities)■L ecturers: Dr. Shuji Shigenobu, Dr. Ikuo
Uchiyama, Mr. Tomoki Miwa, Dr. Katsushi
Yamaguchi, Ms. Hiroyo Nishide, Mr. Takanori
Nakamura (NIBB Core Research Facilities), Dr.
Masanao Sato (Hokkaido Univ.)■Participants: 22 (including 3 from NIBB)■Program:
1. UNIX for beginners
2. Introduction to “R”
3. NGS basic data formats and NGS basic tools
4. Text processing in UNIX
5. Introduction to bias4 system
6. Exercises

September 8(Thu)-9(Fri), 2016

(Practical Course) RNA-seq analysis pipeline■Participants: 22 (including 3 from NIBB)■Program:
1. Review NGS basic data formats and NGS basic
tools
2. Introduction to statistics
3. RNA-seq pipeline: genome-based and transcriptome-based approaches
4. Multivariate statistics
5. Exercises

Mastering BLAST, the essenceof sequence analyses
December 1(Thu), 2016

■Organizers & Lecturers: Dr. Shuji Shigenobu, Dr.
Ikuo Uchiyama (NIBB Core Research Facilities)■Participants: 20 (including 4 from NIBB)■Program:
1. BLAST for beginners
2. Local BLAST search with command-line
3. BLAST inside
4. Large scale BLAST search
5. Annotation of genes and ortholog analysis
6. Beyond BLAST

The 4th BioImaging
Analysis Training
CourseOrganizers: Dr. Kagayaki Kato,
Dr. Yoshitaka Kimori, Dr. Yasuhiro Kamei,
Dr. Hiroshi Koyama, Dr. Shigenori Nonaka,
Dr. Takashi Murata
December 5 (Mon) -7 (Wed), 2016
The 4th Bioimaging Training Course was held jointly by
the Center for Novel Science Initiatives’ Department of
Imaging Science, JSPS KAKENHI Platforms for Advanced
Bioimaging Support (ABiS) and NIBB. This course wasdesigned for biologists who are relatively new to analyzing
datum obtained through advanced microscopy. Therefore
the focus of training was learning image processing andanalytical techniques through “solving simple problems withimage analysis” and “understanding appropriate methods andnecessary preparation for consulting experts in technicallyadvanced imaging challenges”. 58 people applied for thecourse, which had a maximum capacity of 21 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
essential to fundamental image processing and analysis
while obtaining images to be used (workflows). In addition,
we loaned the participants PCs pre-installed with ImageJ, atypical open-source software package for biological image
processing and analysis, and images which were used forpracticing the basic operations and settings of image processing. Also, lectures were given on how programming of
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 common 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)

97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 2016 there were 23 applicants, out of which fiveinterns were selected. These interns were from universities
located in 5 countries (India, Germany, Turkey, USA, andVietnam) and spent periods ranging from four to twelve
weeks experiencing life as a member of a research team.

Report from a participant
Tran Thi Hong Nguyen
VNU University of Science, Vietnam
I am Tran T.H. Nguyen, from Hanoi, Vietnam. I have justcompleted my Bachelor’s in Microbiology from Vietnam
National University, University of Science. I was so glad
to be an NIBB internship student under the guidance ofProfessor Takada from 10th Oct to 28th Dec, 2016. During
my internship in the molecular and developmental laboratory, I focused on the expression of EGFP fluorescently
tagged Wnt3a protein during the embryogenesis of Xenopus
embryos.

98

In my research, I planned to visualize Wnt proteins by
the addition of fluorescent tags because it is known to bequite difficult to generate anti-Wnt antibodies available for
immunohistochemistry. Prior to visualization of taggedWnt proteins in embryos, in the beginning, I focused on
the optimization of linker length to minimize the effect oftags on the activities of Wnt proteins. According to somerecent research, it was shown that activities of Wnt proteinsare frequently damaged by the addition of fluorescent tags.
Therefore, I tried to optimize the design of tagged proteinsby changing the length of the liker peptide connecting Wnt
to EGFP tag. Specifically, I generated 5 constructs in which
the lengths varied from 9 to 55 amino acids. These constructs
were expressed in culture cells and in Xenopus embryos andexamined to what extent Wnt3a activity and EGFP fluorescence were retained.
During my three months of stay in Okazaki, I had lots of
unforgettable memories with labmates and other friends
here. I was able to learn some interesting research topics in
my internship under the valuable guidance and inspirationof Prof. Takada, my mentors – Ritsuko san, Mii san, and thewarm hearts of Nobata-san, Utsumi-san, Takashiro-san andother labmates who encouraged me to successfully complete
my internship here. During my first visit in Japan, my flight
was delayed by 2 hours and I arrived in Nagoya at midnight.
It was so touching that Prof. Takada and his wife came toreceive me from the airport so late at night.
The NIBB internship program is really meaningful forinternational students who want an experience with one of
the highest reputations in education as well as the Japaneseculture. Thanks all, for everything I experienced here!

INDEX
A ....................................................................................................
ADACHI, Daisuke

69

HATTORI, Masayuki

AIHARA, Yusuke

66

HAYASHI, Kohji

AKAMA, Akiko

69

AGATA, Kiyokazu
AJIOKA, Rie

AKIMOTO-KATO, Ai
AKITA, Asaka

ANDO, Toshiya

4

51

HIGASHI, Satoru

HIGASHIJIMA, Shin-ichi

76

HIGUCHI, Yoko

7, 49

80, 94

AOKI, Kazuhiro

7, 12

ATSUMI, Urumi

40, 80

BABA, Nayumi

69

BINO, Takahiro

76, 92

CHEN, Qiuhong

24

CHISADA, Eriko80

ASAO, Hisayo

HAYASHI, Tomoko

86

ANDO, Yoriko
AOKI, Etsuko

HAYASHI, Kentaro

HIKINO, Kazumi
HIRA, Riichiro

43

HIRA, Seiji

HIRAKAWA, Reiko76

HIRAMATSU, MikaHIRANO, Kodai

B ....................................................................................................
BAN, Misato

90

C ....................................................................................................
CHIBA, Hirokazu
CHISADA, Shinichi

HIRAOKA, Yasushi

71

DOKYO, Yumi

35

DE VILLIERS, Ruan

IGUCHI, Taisen

FUKUHARA, Mai

43

ISHIZAKA, Miho

33

ISOSHIMA, Yoshiko

46

ITO, Hiroko

46, 92

ITO, Kazuyo

46

GU, Nan43

H ....................................................................................................
HACHISUKA, Midori
HAMADA, Kyoko

HAMADA, Yoshio
HARA, Ikuyo

HASEBE, Mitsuyasu
HASEGAWA, Ryota

HASHIMOTO, Terumi
HASHIMOTO, Yutaka

74, 757, 14
85
51

4, 7, 43, 89
40
35
21

63

40

ISHINE Naomi

83

92

IMOTO, EikoISHIKAWA, Azusa

4, 27, 83, 90

43

77

7, 43

ISHII, Satoko

33

83

43

IMAI, Akihiro

INABA, Kayo

ITO, Reina

G ....................................................................................................
GOTO, Misako

489, 92IKAMI, Kanako

7, 10

GODA, Misato30

IINUMA, Hideko

ISHIKAWA, Masaki

FUKADA-TANAKA, Sachiko43

76, 91, 92

24

FUJITA, Hironori

40

ICHIKAWA, MarikoF

FUJISHO, Tamae

30

79, 91ICHIKAWA, Chiaki

ISHIBASHI, Tomoko

FUJIMOTO, Daiji

40

ICHIKAWA, Chiaki....................................................................................................
35
FUJIKAWA, Akihiro

FUJIMORI, Toshihiko55

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

E

FUJIMORI, Sayumi

27

58, 85IMAIZUMI, Taeko

FUJIMORI, Chika

35

4, 7, 38, 94

HOSHINO, Atsushi....................................................................................................
7, 40
EBINA, Teppei

EBINE, Kazuo

89, 6335

HSIAO, Yi-Min

D ....................................................................................................
27

21

83, 92

HIYAMA, Takeshi
HORIUCHI, Yuta

51

DAY, F. Timothy

80, 94, 96

ITO, Tomoaki
ITO, Yukiko

IWASE, Etsuko

30
63
63
73
74

79, 92
43
60
30
35
38
58
12
27
24
40

J ....................................................................................................
K

JOHZUKA, Katsuki

60....................................................................................................
KABEYA, Yukiko
43, 92

KADOWAKI, Tamaka

66

KAJIURA-KOBAYASHI, Hiroko

92

KAJIKAWA, Ikumi

KAKIZUKA, Taishi

KAMADA, Konomi

KAMADA, Yoshiaki

43
74
66

8, 54

99

INDEXKAMEI, Yasuhiro

KAMEMIZU, Chizuru
KAMEOKA, Hiromu
KAMIDA, Chiharu

KAMIGAKI, Akane

KAMIKAWA, Yasunao
KAMIYA, Kiyomi

KAMRANI, Yousef Yari
KANAI, Masatake

KANAZAWA, Takehiko
KANEKO, Hiroyo

KOTANI, Keiko40, 85, 92

46

KUBOKI, Yuko

30

27

89
66
55
10
51

18

KATO, Kagayaki
KATO, Kyoko

KAWADE, Kensuke

KAWAGUCHI, Colin

KAWAGUCHI, Haruka

KAWAGUCHI, Masayoshi
KAWAGUCHI, Takayuki
KAWAI, Hisako
KIDA, EmiKIKUCHI, Mariko
KIM, Eunchul

92
27
66

61, 9755
75
90
49

4, 46, 86
14
66
66
33
66

KIMORI, Yoshitaka

62, 97

KIMURA, Tetsuaki

86, 88KINOSHITA, Chie33, 69KIMURA, Tetsuaki
KIMURA, Yukiko

KINOSHITA, Noriyuki
KITADATE, Yu

KOBAYASHI, Yuuki
KODAMA, Akiko
KODAMA, Ryuji

KOHCHI, Takayuki
KOHSHIMA, Shiro

51

7, 38
21
30
46
35

53, 89, 90
4
4

KOIKE, Chieko

51

KOJIMA, Yoko

43, 66

KOIKE, Yukari

KOMATSUBARA, Akira
KOMINE, Yuriko
KOMORI, Akio
KON, Yayoi

51
12
57
2

30

KONDO, Maki79, 92KONISHI, Mie

35

KONDO, Masashi
KONISHI, Yusuke

KOSHIMIZU, Shizuka
KOSUGE, Kotaro

KUME, Shoen

20

KATAYAMA, Kaori
KATO, Hiroki

KUBOYAMA, Kazuya

55

7, 14

KATO, Azusa

KOYAMA, Hiroshi

43

KATAOKA, Kensuke
KATAOKA, Yukari

100

79, 80, 91, 94, 96, 97

40
49
43
66

KURATA, Tomoko

L

KURIHARA, Misuzu

27, 9735
49074....................................................................................................
LI, Chen43

LIN, Chia-Hao
LIU, Meng

35
46

M ....................................................................................................
MAEDA, Taro

MAKINO, Yumiko
MANO, Hiroaki
MANO, Shoji

MARUYAMA, Ayumi

MARUYAMA, Michiyo
MARYU, Gembu

MASAMIZU, Yoshito
MASE, Mutsuki

MATSUBAYASHI, Naomi
MATSUDA, Chisato

MATSUDA, Takashi

MATSUDA, Yoshimi

MATSUMOTO, Miwako

MATSUMURA, Kunihiro
MATSUZAKI, Masanori
MATSUZAKI, Yoko
MII, Yusuke

46

76, 92
43
55
30
69
12

7, 40
49
86
18
35

89, 92
76
83

7, 40
43
24

MINAGAWA, Jun

4, 66

MIURA, Haruko

12

MINAMINO, Naoki
MIURA, Seiko

10
35

MIWA, Tomoki

82, 92, 96

MIYAGI, Asuka

21

MIYAGAWA, Shinichi
MIYAKAWA(OKAMOTO), Misato
MIYAKE, Satoko

MIYANARI, Yusuke
MIYATA, Haruko

MIZOKAMI, Yuko

MIZUGUCHI, Hiroko
MIZUTANI, Takeshi
MORI, Asuka

MORI, Tomoko

MORISHITA, Mio
MORITA, Junko

MORITA, Shinichi

7, 63
63
21
74
92
86

30, 9249, 63, 92
43

76, 92
43
49

7, 49

MOROOKA, Naoki

85, 92

MURATA, Takashi

43, 96, 97

MURAKAMI, Michiyo

21

INDEX

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

46

NAKAGAWA, Tomomi

46

NAKAMURA, Ryoko

58

NAKAGAWA, Mami

27

NAKAGAWA, Toshinori

30

NAKAMURA, Takanori

82, 92, 96

NAKAMURA, Yoshiaki

OKADA, Kazunori

24

OKI, Koichi

14

OKAJIMA, Keisuke
OKUBO, Masayo
OKUMA, Nao

ONODA, KaoriOOI, Shoko

7, 30

OTA, Kyoko

66
10, 46, 69
46
12
43
90

NAKANISHI, Norie

35

NAKATSUKASA, Mana

69

P

NAKAYAMA, Kei

18

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

NAKAYAMA, Tomoya

69

NAKANOH, Shota

27

NAKAYAMA, Jun-ichi

7, 14

NAKAYAMA, Tomomi

55

NARUSE, Kiyoshi

NEGISHI, Takefumi
NIIMI, Teruyuki
NISHI, TayoNISHIDA, Hanna

NISHIDE, Hiroyo

7, 51, 83, 86, 94
21

4, 49
43
46

82, 92, 96

NISHIMURA, Akiko

91

NISHIMURA, Mikio

90

NISHIMURA, Toshiya

7, 33

NISHIMURA, Keiko30

NISHIMURA, Noriko

92

NISHITANI, Kazuhiko
NOBATA, Ryuko

4

24

NODA, Chiyo

66, 92NOGUCHI, Yuji

83, 92

NODA, Masaharu

4, 35

NOMOTO, Yoshihiro

83

NONAKA, Shigenori

73, 94, 96, 97

NORIZUKI, Takuya

10

NOZAKI, Mamoru

75

ODA, Akiko

46

OGAWA, Yuko

46

NOMURA, Kengo

35

NONAMI, Yuta

30

NOSE, Akinao

4

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

77

OGINO, Yukiko

7, 63

OHASHI, Rie

18

OHKUBO, Fuki

40

OHARA, Kaori40

OHDE, Takahiro

49

OHNO, Kaoru

56

OHSAWA, Sonoko

40, 83, 92

OKA, Naomi

82, 92, 96

OHSUMI, Yoshinori
OKA, Sanae

727, 92OTAKE, Norihito
OTSUBO, Yoko

27, 3316....................................................................................................
PALFALVI, Gergo43

SAITO, Eiko49

SAKAGAMI, Mari

16

SAKAE, Yuta

SAKAZAKI, Takuya
SAKUTA, Hiraki
SANBO, Chiaki

SANJO, Kazuko

SASADO, Takao

SASAKI, Hiroyuki
SATO, Toshiyuki

33
43
35
74
91
51
4

30

SAWADA, Kaoru

89, 92SHIBATA, Emiko92

SHI, Dongbo

SHICHINO, Yuichi

SHIGENOBU, ShujiSHIINA, Nobuyuki

SHIMADA, Takashi L.
SHIMAZAKI, Takashi

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

SHINOTSUKA, Takanori
SHINOZUKA, Takuma
SHINTANI, Takafumi
SHIRAISHI, Naomi

27
16

76, 77, 91, 96
18
10
38
46

7, 69
69
69
40
24
35
33

SOYANO, Takashi

7, 46

SUGAYA, Tomomi

43

SUDA, Hiraku

SUGIMOTO, Asako

SUGINAGA, Tomomi

SUGIYAMA, Tomomi
SUMIYA, Eri

SUZAKI, Momoyo
SUZUKI, Ayami

43483, 85, 92
40
63
46
40

SUZUKI, Keiko85, 92SUZUKI, Makoto

21

SUZUKI, Kohta
SUZUKI, Miho

83, 9221

101

INDEXSUZUKI, Miyuzu
SUZUKI, Ryoko

SUZUKI, SachikoTSUZUKI, Tokiko

TSUGANE, Kazuo

35

TSUGE, Toyoko

38

TSUKAYA, Hirokazu

51

TSUTSUI, Tomokazu
TSUZUKI, Chizuru....................................................................................................
TAGAWA, Ayako
74

TAKADA, Ritsuko

24

TAKADA, Shinji

4, 24, 91

TAKAGI, Yukari

83, 92TAKAGI, Chiyo

TAKAHASHI, Hiroki
TAKAHASHI, Ritsue
TAKAHASHI, Sara

TAKAHASHI, Shunichi
TAKAHASHI, Yoichi
TAKAKI, Chikako

TAKASHIRO, Kayoko
TAKEDA, Naoya
TAKEDA, Yuta

21, 92
21
91
66
66
40
51
24
46
40

TAKEHANA, Yusuke

51, 94

TAKETSURU, Hiroaki

7, 86, 87

TAKEUCHI, Yasushi

35, 92TAKEMOTO, Kodai

TAKEUCHI, Tomoyo
TAKEUCHI, Yoshiko
TAKIZAWA, Kenji
TAMADA, Yosuke
TANAKA, Mayo

TANAKA, Minoru

TANAKA, Yasuhiro
TANAKA, Yasuyo
TANGA, Naomi

TANIGUCHI-SAIDA, Misako
TANIGUCHI, Atsushi

TATEMATSU, Kiyoshi
TERADA, Shin-Ichiro
TERASAWA, Yoko
TESHIMA, Yuuko
TOHYAMA, Saki

TOKAIRIN, Asami
TOKUE, Moe

TOKUMOTO, Yuji

TOKUTSU, Ryutaro

TOMINAGA, Hitoshi
TOMOI, Takumi
TORIBA, Taiyo
TORII, Naoko

TOYOOKA, Yayoi
TOYOTA, Kenji

TSUBOUCHI, Hideo

TSUBOUCHI, Tomomi
TSUCHIYA, Yoshihiro

102

77

10
58
38
66

43, 9614

7, 33, 834040
3579, 92
73
91
40
38
51
63
46
30
46
66
21
75
43
51
27
63
20

20, 9124

TSUZUKI, Shihoko

U

TSUZUKI, Shusaku

59, 85
21
75
10
86
92
46

....................................................................................................
79, 92UCHIKAWA, Tamaki

UCHIYAMA, Ikuo
UDA, Youichi

UEDA, Chizuru

71, 82, 96
12
55

UEDA, Takashi

4, 7, 10

UENO, Naoto

2, 4, 21, 90, 91

UNO, Satoko

92

UEMURA, Yuto
UKAI, Sakie

USAMI, Fumiko

UTSUMI, Hideko

38
24
27

24, 92

W ....................................................................................................
WADA, Kotoe

WATAKABE, Ikuko

WATANABE, Akimasa
WATANABE, Eiji

WATANABE, Etsuko

35

33, 3866

42, 83, 85
55

Y ....................................................................................................
YABE, Taijiro

YAMAGUCHI, Chinami

24
75

YAMAGUCHI, Katsushi

76, 92, 96

YAMAMOTO, Masayuki

1, 2, 16

YAMAGUCHI, Takeshi

YAMAMOTO, Takamasa
YAMAMOTO, Takashi

21
21
4

YAMASAKI, Hiroaki

66

YAMASHITA, Akira

16

YAMASAKI, Tomohito
YAMASHITA, Akira
YAMAZAKI, TokoYANOGAWA, Azusa
YASUE, Naoko

YASUGI, Masaki
YASUI, Naomi

YATOMI, Johichiro
YATSU, Ryohei
YOKOI, Saori

YONEZAWA, Harumi
YOSHIDA, Akihiro

66
18
51
51
21
42
20
49
63

51, 94
66
53

YOSHIDA, Shosei

4, 30, 76, 90, 91

YOSHINORI, Yumi

46

YOSHIMURA, Takashi
YU, Yang

YUZAKI, Karen69, 9335
49

INDEXZ ....................................................................................................
ZHANG, Liechi

43

103

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 December 2017
Edited by the Research Enhancement Strategy Office, NIBB