INTRODUCTION
t is my great pleasure to introduce to you all the2017 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.

I

The winning of the 2016 Nobel Prize in Physiology or
Medicine by Dr. Yoshinori Ohsumi, who is a Professor
Emeritus of NIBB, was effective in reminding the society
and the government of the importance of basic research. The
government apparently compromised the trend to reduce
the fundamental budget for universities and inter-university
research institute corporations in total, and I sincerely wish
we would be finally at a turning point to the increase of
support, which would be indispensable to recovery of theleading status of Japan in science.
NIBB will stride properly to open up and foster new
research fields in biology. To this end, every person in NIBB
should do his/her best in accomplishing good research,keeping high ethics and compliance. Good science, even ina basic research field, will eventually benefit human beings.
The history of science tells us this is true. NIBB will work
hard to be truly acknowledged as a remarkable institution bysociety.
Please find in this booklet a summary of the research, collaborative, educational, and international activities of NIBB
in 2017. I would like to note that we welcomed several newcolleagues in 2017, including one associate professor, fiveassistant professors, and five NIBB research fellows, whilefour colleagues transferred to other institutes.
I would like to congratulate Associate Professor Nobuyuki
Shiina for winning the Astellas Award for the Best
Biomedical Research, Mr. Takuya Norizuki for winning an
Outstanding Poster Award from an academic society and Dr.
Takashi Matsuda for winning the 22nd Nagakura Research
Incentive Award, 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 NIBBMarch 30, 2018

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, Akio
National Astronomical
Observatory of Japan (NAO)
National Institute for
Fusion Science (NIFS)

National Institute for
Basic Biology (NIBB)
Director General
YAMAMOTO, Masayuki
Vice-Director General
UENO, Naoto
National Institute forPhysiological Sciences (NIPS)

Advisory
Committee
forProgrammingandManagement

Research
Enhancement
Strategy
OfficeInstitute for
Molecular Science (IMS)
Three Institutes in Okazaki
Center for Novel Science Initiatives■Brain Science■Imaging Science
Astrobiology Center

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 six 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 Facilities
Okazaki Institute forIntegrative 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 Physiology
Department 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 Facilities
Center for Experimental Animal
Research Center for Computational Science

Research Facilities run jointly by
NIBB and NIPS
Electron Microscopy Room
Disposal of Waste Matter Facility
Instrument Design RoomNational Institute for Basic Biology (NIBB)

As of January 1, 2018

Research 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

Neurobiology■Division of Behavioral Neurobiology††■Laboratory of Neurophysiology■Evaluation and Information Group■Public Relations Group■International Cooperation Group■Collaborative Research Group

Evolutionary Biology and Biodiversity■Division of Evolutionary Biology■Division of Symbiotic Systems■Division of Evolutionary Developmental■Young Researcher Support Group

Biology■Gender Equality Promotion Group■Laboratory of Bioresources■Laboratory of Morphodiversity■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 Science

Research Support Facilities
NIBB Core Research Facilities■Functional Genomics Facility■Spectrography and Bioimaging Facility■Data Integration and Analysis Facility

NIBB BioResource Center■Model Animal Research Facility■Model Plant Research Facility■Cell Biology Research Facility

NIBB Center of the Interuniversity Bio-Backup Project (IBBP Center)
NIBB Center for Model Organism Development

Technical Division
Section of Health and Safety Management

Okazaki 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 Centerof the Inter-University Bio-Backup Project (IBBP Center)
was founded in 2012 to prevent loss of invaluable biologicalresources. Projects for the development of bioresource preservation technology are solicited by the IBBP center from2013. The NIBB Center for Model Organism Development
was founded in 2013 to promote development of new modelorganisms and research using them. The Technical Division
manages the activities of the technical staff and helps topromote the activities of each research unit and to maintainthe common resources of both the research support units
of NIBB and the research facilities of the Okazaki campus.
Center for Radioisotope Facilities are one of the latter andrun 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, specially appointed, concurrent) 15
Associate Professors(including sp. app.) 17

Technical
Assistants
andSecretaries

36

110

Assistant Professors(including sp. app.)

60
25

Postdoctoral Fellows(12 are NIBB Research Fellows)

58

Technical Stuff
Graduate Students(3 are undergraduates)

322 in total(as of 1st January, 2018)■ Financial Configuration of NIBB
Indirect Costs in Grants

135

Other Grants(settled forApril 2016-March 2017)
numbers are in million yen

90
External
Competitive
Fund

778

Grant-in-Aid
for Scientific
Research

Personnel

732

392
Grants forCommissioned Research
Facility

54

1,432
Research

156
Management Expenses
Grant for SOKENDAIManagement
Expenses Grant
for NIBB

195 Collaboration
265

237

Other 3

Donation,42

2,306 million yen
in total■ Members of the Advisory Committee for Programming and Management# (as of April, 2017)

Non-NIBB members

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

4

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

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.

International cooperation
Contribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers

Promotion of Collaborative Research■ Collaborative Research Support■ NIBB BioResource Center

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.

The NIBB BioResource Center supports research using
model animals and plants in NIBB and other academic
institutions. The center consists of three facilities, the model
animal, the model plant, and the cell biology researchfacilities. The center has equipment, facilities, and staff to
PDLQWDLQPRGHORUJDQLVPVVXFKDVPLFHPHGDND]HEUDÀVKJapanese morning glory, Arabidopsis, Lotus japonicus, andPhyscomitrella patens, and provides technical support andadvice for the appropriate use of these organisms (p. 82).
The center also act as a hub of the National BioResource
Project (NBRP) which is a national project for the systematic
accumulation, storage, and supply of nationally recognizedbio-resources (experimental animals and plants, cells, DNA,and other genetic resources), which are widely used asmaterials in life science research. To promote this national
project, NIBB has been appointed as a center for research on
medaka (Oryzia latipes), whose usefulness as a vertebratemodel was first shown by Japanese researchers. The usability of medaka as a research material in biology has drawn
increasing attention since its full genome sequence recently
became available. NIBB is also a sub-center for the NBRP’s
work with Japanese morning glory and zebrafish (p. 85).

■ 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. 75).
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.
97). 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.

An example of a medaka strain in NBRP, Gaudi strain, in which individual cells in the brain and the retina are fluorescently labelled using
the 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 invaluablebioresources under cutting-edge research (p. 86).

■ Advanced Bioimaging Support (ABiS)
ABiS provides assistance for advanced imaging in the
research supported by Grants-in-Aid for Scientific Research.
NIBB together with NIPS contribute as core institutes to the
ABiS network of domestic partner organizations that own
and operate multiple types of advanced specialized imaging
equipment (p. 99).

5

International Cooperation and OutreachDevelopment of New Fields of Biology■ Collaborative Programs with Overseas Institutes■ Bioimaging

NIBB takes a leading role in the collaborative research
programs between the European Molecular Biology
Laboratory (EMBL) and the National Institutes of Natural
Sciences (NINS) and promotes personal and technological exchange through joint meetings, exchange between
researchers and graduate students, and the introduction ofexperimental equipment.
NIBB formed an agreement with the Temasek Life Sciences
Laboratory (TLL) of Singapore and Princeton University
to promote joint research projects, collaborative symposia,
training courses and student exchange programs. NIBBPrinceton Joint Proteomics Training Course was held in July,
2017 (p. 96).

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. 71). The
application of the adaptive optics to microscopy is under way
in collaboration with the National Astronomical Observatoryof Japan. The Advisory Committee on Bioimaging, comprisedof 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 researchersand company engineers to frankly discuss practical difficulties and needs regarding imaging. The 11th Forum “Pioneering
New Bioimaging by Fusing Optics and Biology” was held in
February, 2017 (p. 97). A training course in bioimage analysis
was also held in 2017 (p. 98).

year
Priority collaborative research projects
Collaborative research projects for model organisms andtechnology 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

2014

2015 2016

2016

1

2

2

2

3

2

2
2

87
3
12
10

88
6
10
11
14

46
6
10

51
3
9

38*

28*

37

46

59**

62**

0

1

0

0

10

9

12

12

162

190

175

169

*renovated as "Collaborative research projects for integrative bioimaging"**renovated as "Collaborative research projects for integrative genomics"■ 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 65th conference “Renaissance ofMarchantia polymorpha –the genome and beyond-” was heldin December, 2017 (p. 94).

■ 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 jointlywith TLL in August, 2016 at NIBB. Graduate students andyoung researchers from various countries and areas 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 (p. 91).

■ Outreach
NIBB’s outreach activities aim to present cutting edge
research results to the public via mass media throughpress releases or directly through the internet. 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 education of undergraduates and younger
students through lectures and workshops. Outreach activities
are mostly managed by the Public Relations Group of the
Research Enhancement Strategy Office (p. 90).

■ 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 (p. 95).
Students from Japan and abroad can experience NIBB
through our Internship Program which gives students anopportunity way to build international connections while
experiencing hands on research in a world class research
institute (p. 100).
Support for young researchers is managed by the Young
Researcher Support Group of the Research Enhancement
Strategy Office (p. 92).
Graduate Students Educated by NIBB (numbers counted at the end of April each year)
60
7
9

45
12

7

11

13

7

30

15

34

34

2011

2012

41

46

42

37

39

2016

2017

0
2013

2014

SOKENDAI Graduate Student

6

2015
Special Research Student

■ Personnel changes in 2017*

Newly assigned in NIBB
Name

Position

Research Unit

Date

NONOMURA, Keiko

Assistant Professor

Division of Embryology

January 16

KONDO, Yohei

Assistant Professor

Division of Quantitative Biology

April 1

KANAZAWA, Takehiko

Assistant Professor

Division of Cellular Dynamics

April 1

FUJITA, Hironori

Assistant Professor

Division of Symbiotic Systems

April 1

MATSUDA, Takashi

NIBB Research Fellow

Division of Molecular Neurobiology

April 1SAKAI, Yusuke

NIBB Research Fellow

Division of Morphogenesis

April 1

GOTO, Yuhei

NIBB Research Fellow

Division of Quantitative Biology

May 1

YOSHIDA, SaikoNIBB Research Fellow

Division of Evolutionary Biology

June 1

MANO, Shoji

Associate Professor

Research Enhancement Strategy OfficeNovember 1

ANSAI, Satoshi

Assistant Professor

Laboratory of Bioresources

November 1

ARATA, Masaki

NIBB Research Fellow

Division of Embryology

December 1

Newly affiliated in other universities and institutes
New Affiliation

Position

Date

TAKEHANA, Yusuke

Nagahama Institute of Bio-Science and Technology

Associate Professor

April 1

TAKEDA, Naoya

Kwansei Gakuin University

Associate Professor

April 1

OHDE, Takahiro

Kyoto University

Assistant Professor

June 1YOSHIDA, Saiko

Max Planck Institute for Plant Breeding ResearchMarie Curie Fellow

August 1

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

■ Awardees in 2017

Name

PositionAward

MATSUDA, Takashi

NIBB Research Fellow

22nd Nagakura Research Incentive AwardSHIINA, Nobuyuki

Associate Professor

Astellas Award for the Best Biomedical Research

NORIZUKI, Takuya

Graduate Student

Taiwan-Japan Plant Biology 2017 Outstanding Poster Award

7

Selected Press Releases from NIBB in 2017
■ 27 January
Discovery of a plant stem cell inducing factor having a similar structure to an animal stem cell
inducing factor
The moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming ofdifferentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced
pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and ismaintained throughout the reprogramming process and in the resultant stem cells. Genetic manipulation
experiments demonstrated a positive role of PpCSP1 in reprogramming, which is similar to the functionof mammalian Lin28 (see p. 45).
Li, C., Sako, Y., Imai, A., Nishiyama, T., Thompson, K., Kubo, M., Hiwatashi, Y., Kabeya, Y., Karlson, D., Wu, S.-H., Ishikawa, M., Murata, T., Benfey,
P.N., Sato, Y., Tamada, Y., and Hasebe, M. (2017). A Lin28 homolog reprograms differentiated cells to stem cells in the moss Physcomitrella patens. Nat.
Commun. 8, 14242.

■ 7 February
Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory
To investigate the molecular bases of carnivory, our research group sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which they succeeded in regulating the developmental
switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types
and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestionand nutrient absorption. These results imply constraints on the available routes to evolve plant carnivory(see p. 45).
Fukushima, K., Fang, X., et al. (2017). Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory. Nat. Ecol. Evol. 1,0059.

■ 10 February
A mechanism regulating self-renewal and differentiation of sperm stem cells -how stem cells areprotected from differentiation-promoting signals,Q WKH VHPLQLIHURXV WXEXOHV RI PRXVH WHVWHV D SRSXODWLRQ RI *)5ƠSRVLWLYH VSHUPDWRJRQLD KDUERUVVWHPFHOOIXQFWLRQDOLW\7KHUHVHDUFKJURXSVKRZHGWKDWDFWLYDWLRQRI:QWơFDWHQLQVLJQDOLQJSURPRWHVVSHUPGLIIHUHQWLDWLRQDQGUHGXFHVWKH*)5Ơ+ cell pool. They further discovered that SHISA6, characterL]HGDVDFHOODXWRQRPRXV:QWLQKLELWRUFRQIHUV*)5Ơ+FHOOVUHVLVWDQFHWRWKH:QWơFDWHQLQVLJQDOLQJThe difference in the effect of Wnt signaling caused by the presence or absence of SHISA6 can produce
both self-renewing stem cells or differentiating cells in the uniform tissue environment (see p. 35).
Tokue, M., Ikami, K., Mizuno, S., Takagi, C., Miyagi, A., Takada, R., Noda, C., Kitadate, Y., Hara, K., Mizuguchi, H., Sato, T., Taketo, M.M., Sugiyama,)2JDZD7.RED\DVKL68HQR17DNDKDVKL67DNHGD6DQG<RVKLGD66+,6$FRQIHUVUHVLVWDQFHWRGLIIHUHQWLDWLRQSURPRWLQJ:QWơFDWHQLQ
signaling in mouse spermatogenic stem cells. Stem Cell Reports, 8, 561-575, 2017.

■ 7 March
Fluctuation in the concentration of calcium ions contributes to neural tube shape formation
Our research group showed that during the process of neural tube formation a transient increase in
the concentration of calcium ions in cells causes morphological changes and is essential for neuraltube formation. The group observed the cell population during neural tube formation in Xenopus laevis
embryos using the fluorescent protein GECO, an intracellular Ca2+ concentration indicator. They foundthat the pattern of the fluctuation in intracellular calcium ion concentration in the cell population iscomplex. Local and transient rises in intracellular calcium ion concentrations have been found to cause
cell deformation and contribute to the formation of the neural tube (see p. 26).
Suzuki, M., Sato, M., Koyama, H., Hara, Y., Hayashi, K., Yasue, N., Imamura, H., Fujimori, T., Nagai, T., Campbell, RE., and Ueno, N. (2017). Distinct
intracellular Ca2+ dynamics regulate apical constriction and differentially contribute to neural tube closure. Development 144, 1307-1316.

8

■ 12 April
Three-dimensional computer graphic animation for studying social approach behavior in medakafish
Our research group studied social approach behavior in medaka fish using three-dimensional computer
graphic (3DCG) animations. 3DCG virtual fish lacking any combinations of four features (color, shape,
locomotion, and body motion) of real medaka fish were created and presented to live fish using acomputer display. Medaka fish presented with virtual fish with four normal features spent a long timein proximity to the display, whereas time spent near the display was decreased with other virtual fishlacking any of the four features. The results suggested that the naturalness of visual cues contributes tothe induction of social approach behavior (see p. 44).
Nakayasu, T., Yasugi, M., Shiraishi, S., Uchida, S., and Watanabe, E. (2017). Three-dimensional computer graphic animations for studying social approach
behaviour in medaka fish: Effects of systematic manipulation of morphological and motion cues. PLoS ONE 12, e0175059.

■ 17 July
Identification of PTPRZ as a drug target for cancer stem cells in glioblastoma
Glioblastoma is the most malignant brain tumor with high mortality. Cancer stem cells are thought to be
crucial for tumor initiation and its recurrence after the standard therapy with radiation and temozolomide(TMZ) treatment. An NIBB research group showed that protein tyrosine phosphatase receptor type Z(PTPRZ) is requisite for the maintenance of stem cell properties and tumorigenicity in glioblastoma cells.
The research group discovered NAZ2329, an allosteric inhibitor of PTPRZ, in collaboration with a pharmaceutical company. NAZ2329 efficiently suppressed stem cell-like properties of glioblastoma cells in
culture, and tumor growth in C6 glioblastoma xenografts. Notably, tumor growth was inhibited more effectively by co-treatment with NAZ2329 and TMZ than by the individual treatments. Thus, pharmacologicalinhibition of PTPRZ activity is a promising strategy for the treatment of malignant gliomas (see p. 38).
Fujikawa, A., Sugawara, H., Tanaka, T., Matsumoto, M., Kuboyama, K., Suzuki, R., Tanga, N., Ogata, A., Masumura, M., and Noda, M. (2017). Targeting
PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells. Sci. Rep. 7, 5609.

■ 4 September
Discovery of dynamic seasonal changes in color perception ~The small fish “medaka” shows large
differences in color perception in summer and winter~Our research group found that color perception of Medaka, a small fish inhabiting rice fields and streams,
varies greatly according to seasonal changes. Medaka in the summer conditions were strongly attracted tovirtual medaka which showed nuptial coloration, but medaka in the winter conditions were not. It further
became clear that expression of opsins, the proteins responsible for the first step in vision, and genes
related to signal transduction pathways downstream of opsins are markedly reduced in winter conditions,whereas expression of these genes rises by shifting to summer conditions. This phenomenon of seasonalchange in color perception may be a phenomenon widely preserved in various animals (see p. 67).
Shimmura, T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi, M., Watanabe, E., Shimo, T., Senga, T., Nishimura, T., Tanaka, M., Kamei, Y.,
Naruse, K., and Yoshimura, T. (2017). Dynamic plasticity in phototransduction regulates seasonal changes in color perception. Nat. Comm. 8, 412.

■ 6 September
Building a morphogen gradient by simple diffusion in a growing plant leaf
Our research group showed that a transcriptional co-activator ANGUSTIFOLIA3 (AN3) forms a signaling gradient along the leaf proximal-to-distal axis to determine cell-proliferation domain. They demonstrated that pure diffusion in a growing tissue is sufficient to explain the AN3 gradient formation. This
work provides evidence that the diffusion-based model of morphogen is viable in developmental patterning of multicellular organisms (see p. 29).

Kawade, K., Tanimoto, H., Horiguchi, G., and Tsukaya, H. (2017). Spatially different tissue-scale diffusivity shapes ANGUSTIFOlIA3 gradient in growing
leaves. Biophys. J. 113, 1109-1120.

9

■ 14 September
Identification of PTPRJ as a regulator of leptin signaling and obesity
Leptin, an adipocyte-derived hormone, strongly inhibits food intake by acting on the hypothalamus,
a part of the mid brain, and plays a crucial role in the body weight control. Circulating leptin levels
are closely correlated with the degree of adiposity. An NIBB research group demonstrated that protein
tyrosine phosphatase receptor type J (PTPRJ) negatively regulates leptin signaling by inhibiting theactivation of leptin receptor-associated JAK2, through the dephosphorylation of Y813 and Y868 in
JAK2 autophosphorylation sites. Diet-induced obesity and the leptin treatment both up-regulated PTPRJ
expression in the hypothalamus, while the overexpression of PTPRJ induced leptin resistance. Thus, theinduction of PTPRJ is a factor contributing to the development of leptin resistance, and the inhibition of
PTPRJ may be a potential strategy for improving obesity (see p. 38).
Shintani, T., Higashi, S., Suzuki, R., Takeuchi, Y., Ikaga, R., Yamazaki, T., Kobayashi, K., and Noda, M. (2017). PTPRJ inhibits leptin signaling, andinduction of PTPRJ in the hypothalamus is a cause of the development of leptin resistance. Sci. Rep. 7, 11627.

■ 20 September
Rolling dice for cell size specification in plant leaf epidermis
Because size distribution often shows a characteristically skewed pattern in a tissue, there may be somestochastic option for determining cell size. Our research group discovered that endoreduplication, which
promotes cellular enlargement in the epidermal tissue of Arabidopsis thaliana, occurs randomly as a
Poisson process throughout cellular maturation. These results link the probabilistic property of endoreduplication dynamics to cell size distribution, providing a theoretical background to explain how size
heterogeneity is established within a leaf (see p. 73).
Kawade, K., and Tsukaya, H. (2017). Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue. PLoS ONE 12, e0185050.

■ 24 October
Efficient synthesis of a photosynthetic pigment in mammalian cells for Optogenetics
The “Optogenetic” method of controlling the position of proteins in cells using light is becoming widely
employed as an important method to understand intracellular signal transduction. The Phytochrome B(PhyB)-PIF system made way to the use of red light / near infrared light, which have low toxicity andgood tissue penetrance. However in the case of animal cells the cyanobacterial photosynthetic pigment
phycocyanobilin (PCB) must be added from outside, and this step greatly hindered the use of the
PhyB-PIF system. NIBB researchers have now succeeded in synthesizing PCB in animal cells directly byintroducing four genes encoding cyanobacterial enzymes related to PCB synthesis. In addition, the groupsucceeded in increasing the amount of PCB synthesis through disruption of the gene that encodes an
enzyme called biliverdin reductase A, which is involved in the metabolism of PCB (see p. 15).
Uda, Y., Goto, Y., Oda, S., Kohchi, T., Matsuda, M., and Aoki, K. An efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control ofcell signaling. Proc. Natl. Acad. Sci. USA 2017 Oct 24. doi:10.1073/pnas.1707190114■ 7 November
Discovery of a mechanism for determining the direction of collective cell migration
A group at NIBB have found that when the activity of a molecule called ERK MAP kinase is propagatedto neighboring cells, the cells migrate in the opposite direction of ERK propagation. In cells cultured ona circular micro-patterned glass base dish, a spontaneous rotational wave of ERK activity was observed
and the cells rotated in the opposite direction. Furthermore, by artificially creating waves of ERK activity
by optogenetic techniques, the research group succeeded in making cells move collectively (see p. 15).

Aoki, K., Kondo, Y., Naoki, H., Hiratsuka, T., Itoh, R.E., and Matsuda, M. Propagating wave of ERK activation orients collective cell migration. Develop.
Cell 2017 Nov 6. doi:10.1016/j.devcel.2017.10.016

10■ 21 November
mRNA localization regulatory factor RNG105/caprin1 is essential for long-term memory formation
Researchers at NIBB have revealed that the function of RNG105 (aka Caprin1) is essential for the formation of long-term memory. Mice who have lost RNG105 in the cerebrum and hippocampus can form
short-term memories of several minutes, but the long-term memory, developed over several days to one
week, does not form in these mice. They also found that RNG105-regulated mRNA localization to dendrites is involved in the correct placement of AMPA receptors in synapses, which are known to be important for memory retention (see p. 23).
Nakayama, K., Ohashi, R., Shinoda, Y., Yamazaki, M., Abe, M., Fujikawa, A., Shigenobu, S., Futatsugi, A., Noda, M., Mikoshiba, K., Furuichi, T.,
Sakimura, K., and Shiina, N. (2017). RNG105/caprin1, an RNA granule protein for dendritic mRNA localization, is essential for long-term memory formation. eLife 6, e29677.

■ 7 December
Two types of heparan sulfate clusters may constitute a cellular platform for the distribution andsignaling of Wnt8, a morphogen molecule
Wnt proteins direct embryonic patterning, but the regulatory basis of their distribution and signal reception remain unclear. Our research group showed that endogenous Wnt8 protein is distributed in a gradedmanner in Xenopus embryo and accumulated on the cell surface in a punctate manner in association with
“N-sulfo-rich heparan sulfate (HS),” not with “N-acetyl-rich HS”. These two types of HS clusters may
constitute a cellular platform for the distribution and signaling of Wnt8 (see p. 29).

Mii, Y., Yamamoto, T., Takada, R., Mizumoto, S., Matsuyama, M., Yamada, S., Takada, S., and Taira, M. (2017). Roles of two types of heparan sulfate
clusters in Wnt distribution and signalling in Xenopus. Nat. Comm. 8, 1973.

■ 8 December
Protamine neutralizes CSPG-mediated inhibition of oligodendrocyte differentiation
Remyelination is a critical repair process in demyelinating diseases such as multiple sclerosis (MS).
Damage to the central nervous system (CNS) results in a glial reaction, leading to the formation of a glial
scar enriched with chondroitin sulfate proteoglycans (CSPGs). CSPGs also accumulate as constituentsof demyelinating plaques in MS lesions, which inhibit the migration and differentiation of oligodendrocyte precursor cells and remyelination. An NIBB research group found that a polycationic peptide,protamine (PRM) neutralized the inhibitory activity of aggrecan, a representative extracellular matrix
CSPG molecule, and that PRM functioned as an inhibitory ligand mimetic of protein tyrosine phosphatase receptor type Z (PTPRZ), a membrane-spanning CSPG predominantly expressed in oligodendrocyte
precursor cells. The intranasal administration of PRM accelerated myelination in the developing mousebrain, and its intracerebro-ventricular administration stimulated remyelination after cuprizone-induced
demyelination. These results indicate that PRM has CSPG-neutralizing activity, thereby promoting oligodendrocyte differentiation under developmental and morbid conditions (see p. 38).
Kuboyama, K., Tanga, N., Suzuki, R., Fujikawa, A., and Noda, M. (2017). Protamine neutralizes chondroitin sulfate proteoglycan-mediated inhibition ofoligodendrocyte differentiation. PLoS ONE 12, e0189164.

11

National Institute for Basic Biology

Cell Biology

DIVISION OF CELLULAR DYNAMICS

components, 2) relocating conserved machinery components
to distinct trafficking events, and 3) secondary loss of conserved machinery components, during evolution.

Professor

UEDA, Takashi

Assistant Professor:

EBINE, Kazuo
KANAZAWA, TakehikoTechnical Staff:
HAYASHI, Kohji
Postdoctoral Fellow:
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
in eukaryotic cells. Recent comparative genomics has
indicated that membrane trafficking pathways are diversified among eukaryotic lineages, which is associated withlineage-specific acquisition of new trafficking pathways
and secondary loss of preexisting trafficking routes. Our
long-term goal is unraveling how plants have acquired
their unique membrane trafficking systems during evolution, which will be achieved by comparative analyses
using the model plant Arabidopsis thaliana and a modelof liverworts, Marchantia polymorpha. We also aim toelucidate detailed molecular mechanisms and physiological functions of membrane trafficking in higher-orderedplant functions.

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

Figure 1. SYP1 and RAB11 genes in the genomes of green plants. Genes
are indicated as individual red (for SYP1) or cyan (for RAB11) boxes.
Numbers of these genes increased during land plant evolution, suggesting
secretion-related functions have been diversified 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 (Figure 2), whose origin and mechanisms of biogenesis
remain unclear. We are currently analyzing the molecularfunction of the SYP1 member, as well as characterizing
membrane trafficking pathways responsible for oil bodybiogenesis. We have succeeded in isolating several mutants
defective in the function and morphogenesis of the oil bodythrough a forward-genetic approach. Analyses of a mutant
with an increased number of oil bodies identified a transcription factor regulating oil body biogenesis, which activatestranscription of genes responsible for secondary metabolite
biosynthesis and putative transporters, as well as the SYP1
member.

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 machinery

Figure 2. Oil bodies in a thallus of M. polymorpha, which are visualized
by the YFP-tagged SYP1 member (green). Autofluorescence from chlorophyll is also shown (blue).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

12

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 mechanisms 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. A tethering complex, CORVET, is known
to act as an effector of Vps21/RAB5 to regulate endosomaltransport in yeast and animal cells. CORVET subunits arealso conserved in Arabidopsis, but molecular function of
CORVET remained unclear. We found that VPS3, one of the
CORVET subunits, acts in the RAB5-dependent and RAB7independent vacuolar transport pathway in Arabidopsis(Figure 3). Furthermore, we also found that another tethering complex sharing the core complex with CORVET,
the HOPS complex, regulates a different trafficking eventfrom CORVET. Our results further indicated that different
fusion machineries comprising distinct R-SNARE proteins
are involved in CORVET- and HOPS-mediated trafficking pathways. These findings demonstrated that the plantvacuolar transport system has been diverged from vacuolar/lysosomal transport systems in non-plant systems.

Figure 3. Subcellular localization of mRFP-SYP22 in WT-like and vps3
embryos. SYP22 is one of the cargo molecules of the RAB5-dependent
and RAB7-independent vacuolar transport pathway. mRFP-tagged
SYP22 localized to the vacuolar membrane in WT-like embryo cells(left), whereas it mislocalized to the plasma membrane in the vps3 mutant
HPEU\RFHOOV ULJKW %DUV ƫPWe also conducted detailed analyses of vacuolar SNARE
proteins. Defective functions in vacuolar SNAREs affectboth vacuolar transport and vacuolar morphology. 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 affectedin this mutant. Therefore sgr3-1 should be a good toolfor dissecting functions of the vacuolar SNARE. We are
exploring vacuolar dynamics regulated by SYP22 by analyzing the effect of the sgr3-1 mutation in a detailed manner.
Co-immunoprecipitation and MS analyses indicated that thesgr3-1 mutation specifically stabilizes a SNARE complex
comprising VAMP71, SYP22, and _SNAP. We also foundthat these molecules and HOPS components accumulate on
the vacuolar membrane in sgr3-1. These results highlighted
a unique characteristic of the sgr3-1 mutation, which will
provide valuable information to better understand mechanisms of SNARE-mediated membrane fusion.

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 only found in 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 overexpression of constitutive active ARA6
repressed the full proliferation of powdery mildew fungi(Inada et al., 2017).

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.
Cytokinesis in land plants is achieved by re-directionof the secretory pathway, and KNOLLE/SYP111 and
KEULE/SEC11 play important roles in membrane fusion
at the forming cell plate in somatic cells of Arabidopsis.
Conversely, any deleterious effects on gametogenesis havebeen reported for mutations in these genes thus far. We foundthat other SYP1 and SEC1 members are highly expressed
during male gametogenesis. The analyses of these proteinsduring male gametogenesis are currently underway.
Distinct from seed plants, basal land plants including M.
polymorpha utilize sperm as the male gamete in sexual
reproduction. We visualized the process of sperm formation, especially spermiogenesis, using fluorescently-taggedorganelle markers (Figure 4). The majority of the endomembranous organelles such as the Golgi apparatus was removed
from the maturing cells, and the plasma membrane was also
reorganized during spermiogenesis. A TEM analysis also
indicated that the number of degradative organelles suchas the multivesicular endosomes, vacuoles, and autopha-

13

National Institute for Basic Biology

Cell Biology

gosomes, was transiently increased during this process. To
reveal the molecular mechanisms of organelle degradationand cytoplasm removal, we are now analyzing the contribution of autophagy. The autophagy-defective mutations
resulted in defective morphogenesis and motility of sperm,
indicating crucial functions of autophagy during spermiogenesis in M. polymorpha.

•

Kohchi, T., Nakano, A., and Ueda, T. (2017). Dynamic reorganizationof the endomembrane system during spermatogenesis in Marchantia
polymorpha. J. Plant Res. 130, 433-441.
Ung, H., Karia, P., Ebine, K., Ueda, T., Yoshioka, K., and Moeder, W.
(2017). Triphosphate tunnel metalloenzyme function in senescencehighlights a biological diversification of this protein superfamily. Plant
Physiol. 175, 473-485.

〔Original paper (E-publication ahead of print)〕
C., Shi, Y., Kesten, C., Zhang, D., Sancho-Andrés,• Sánchez-Rodríguez,
G., Ivakov, A., Lampugnani, E.R., Sklodowski, K., Fujimoto, M.,
Nakano, A., Bacic, A., Wallace, I.S., Ueda, T., van Damme, D., Zhou,
Y., and Persson, S. The cellulose synthases are cargo of the TPLATE
adaptor complex. Mol. Plant 2017 Dec 5.

〔Review article〕
T., and Ueda, T. (2017). Exocytic trafficking pathways in• Kanazawa,plants: why and how they are redirected. New Phytol. 215, 952-957.

Figure 4. Reorganization of the plasma membrane during spermatogenesis in M. polymorpha. Spermatogenesis proceeds from A to D. A
plasma membrane-resident protein visualized with Citrine is targeted
to intracellular spherical structures, and completely removed from the
SODVPDPHPEUDQHRIPDWXUHVSHUPDWLGV%DUV ƫP $GRSWHGIURPMinamino et al., 2017)

Publication List:〔Original papers〕
K., Kobayashi, M., Sato, M., Kutsuna, N., Ueda, T., Toyooka,• Akita,K., Nagata, N., Hasezawa, S., and Higaki, T. (2017). Accumulation of

•
•
•
•
•
•
14

fluorescent proteins derived from a trans-Golgi cisternal membranemarker and paramural bodies in interdigitated apoplastic space inArabidopsis leaf epidermis. Protoplasma 254, 367-377.
Bowman, J.L., Kohchi, T., Yamato, K.T., Jenkins, J., Shu, S., Ishizaki,
K., Yamaoka, S., Nishihama, R., Nakamura, Y., Berger, F., et al.
(2017). Insights into land plant evolution garnered from the Marchantia
polymorpha genome. Cell 171, 287-304.
Cui, Y., Zhao, Q., Xie, H.T.,Wong, W.S., Gao, C., Ding, Y., Tan, Y.,
Ueda, T., Zhang, Y., and Jiang, L. (2017). MON1/CCZ1-mediatedRab7 activation regulates tapetal programmed cell death and pollen
development in Arabidopsis. Plant Physiol. 173, 206-218.
Inada, N., Ebine, K., Ito, E., Nakano, A., and Ueda, T. (2017).
Constitutive activation of plant-specific RAB5 GTPase confers
increased resistance against adapted powdery mildew fungus. Plant
Biotech. 34, 89-95.
Ito, Y., Toyooka, K., Fujimoto, M., Ueda, T., Uemura, T., and Nakano
A. (2017). The trans-Golgi network and the Golgi stacks behave
independently during regeneration after Brefeldin A treatment in
tobacco BY-2 cells. Plant Cell Physiol. 58, 811-821.
Matsui, H., Nomura, Y., Egusa, M., Hamada, T., Hyon, G.S., Kaminaka,
H., Watanabe, Y., Ueda,, T., Trujillo, M., Shirasu, K., and Nakagami, H.
(2017). The GYF domain protein PSIG1 dampens the induction of cell
death during plant-pathogen interactions. PLoS Genet. 13, e1007037.
Minamino, N., Kanazawa, T., Nishihama, R., Yamato, T.K., Ishizaki, K.,DIVISION OF QUANTITATIVE BIOLOGYA

ATP

KinaseFRET

Phosphorylation

Professor

LiganddomainAOKI, Kazuhiro

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 comprised of a series of chemical reactions, mostcommonly protein phosphorylation. Importantly, dysregulation of the signal transduction by gene mutation results inpathological diseases 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 constituting
the signal transduction can be described by a set of ordinary
differential equations, and solved numerically by computers.
A number of simulation models of signaling pathways have,in fact, been reported to date. However, most of the kinetic
parameters utilized for the simulation models were notmeasured 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 the intracellular signaling
pathways with fluorescence imaging techniques and computational approaches.

B

Dephosphorylation

PKA

ERK

Akt

JNK

PKC

S6K

Figure 1. Visualization of kinase activity in living cells. (A) Schematic
representation of intramolecular FRET biosensor of kinases. (B) PKA,
ERK, Akt, JNK, PKC and S6K activities by FRET imaging. HeLa cells
expressing FRET biosensors for the indicated kinases were stimulatedwith each ligand.

resolution (Komatsu N, Mol. Biol. Cell, 2011) (Figure 1).
By using a FRET biosensor, we revealed that intercellular propagating waves of ERK activation determined the
direction of the collective cell migration (Figure 2). Cell
migration is a fundamental process in many physiologicaland pathological contexts. Collective cell migration refers
to a movement of cell groups with physical and functional
cell-to-cell connections, and is inherently involved in theprocesses of embryonic development, wound healing, andcancer invasion. However, it remains unclear how cells ina group organize physical and chemical clues into synchronized directionality and migration to facilitate coordinatedcollective migration. We visualized ERK activity in collectively migrating Mardin-Darby canine kidney (MDCK)
cells by an ERK FRET biosensor, EKAREV, and found theA
0 hour
1 hour2 hour High
3 hour4 hour Low

ERK activity

Assistant Professor:
KONDO, Yohei
NIBB Research Fellow:
GOTO, Yuhei
Postdoctoral Fellow:
ODA, Shigekazu
Visiting Graduate Student: KOMATSUBARA, Akira
MIURA, Haruko
UDA, Youichi
MARYU, Gembu
Technical Assistant:
EBINE, Eimi
Secretary:
ONODA, Kaori

Sensordomain

5 hour
6 hourB

ERK activation wave
Epithelial cells

I. Visualization of cell signaling
With the advent of fluorescent proteins, it has been possible
to visualize kinetic reactions at the single cell level. Förster(or fluorescence) resonance energy transfer (FRET) is a
non-radiative process, in which excitation energy of donor
fluorophore is transferred to a nearby acceptor fluorophore.
Taking advantage of the principles, FRET-based biosensors
allowed us to detect kinase activity of PKA, ERK, Akt, JNK,
PKC, and S6K in a living cell with high temporal and spatial

Low
High
ERK activity

Traction force

Collective cell migration

Figure 2. ERK activation wave and collective cell migration. (A)Propagating waves of ERK activation in wound healing of MDCK cells.
(B) Schematic representation of collective cell migration driven by ERK
activation propagation.

15

National Institute for Basic Biology

Cell Biology

intercellular propagating waves of ERK activation (Figure2A). Interestingly, MDCK cells collectively migrated against
the direction of the ERK activation wave. The inhibition of
ERK activation propagation suppressed collective cell migration. The ERK activation wave spatiotemporally controlled
actomyosin contraction and cell density. Furthermore, an
artificially-generated ERK activation wave by optogenetics
reproduces induction of the collective cell migration. These
data provide new mechanistic insights into how cells sense
the direction of collective cell migration (Aoki K, Dev Cell,
2017).
Although FRET biosensors enable us to monitor cellsignaling events, FRET imaging is not suited for multiplexed imaging, because these biosensors are authenticallycomposed 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 3). 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 (Maryu
G, Cell Strut, Funct, 2016; Miura H, bioRxiv, 2017) (Figure
3).

conventional knock-in techniques. We established several S.
Pombe lines expressing cyclin and cyclin-dependent kinase
fused with GFP and RFP, respectively, and quantified these
expression levels during the process of the cell cycle by live
cell imaging. In mammalian cells, we developed an efficient knock-in system with CRISPR/Cas9-mediated genome
editing techniques (Figure 4), and combined with fluorescence correlation spectroscopy (FCS) and fluorescencecross-correlation spectroscopy (FCCS) to quantitatively
determine the protein concentration and the dissociation
constant of endogenous protein (Komatsubara A, bioRxiv,
2017)(Figure 4).

A

Cas9 + sgRNA

loxP P2A
L. arm mEGFP

loxP
R. arm

40 bp

40 bp

MMEJ-mediated KI vector
Transfection into HeLa cells
mEGFP

G418 selection, single cell cloningA

Cre expression

Cytoplasm
KTR

Kinase

P
KTR

mEGFP

Phosphorylation

Ganciclovir selection

B

P
KTR
Nucleus

ERK-KTR-mKO

Dephosphorylation

Translation
Akt-KTR-EGFP

mCherry-Gem

EGF stimulation

Cells
0 20 40 60 80
Time (min)
Low

High

ERK activity

0 20 40 60 80
Time (min)
Low

High

0 20 40 60 80
Time (min)S/G2/MG1

Akt activity

Cell cycle

Figure 3. 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, Kd, have been measured byin 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 (Sadaie
W, MCB, 2014). Therefore, it is critical to measure kinetic
parameters in living cells.
To this end, we launched a research project of quantitativecell cycle modeling in fission yeast Schizosaccharomyces
pombe and mammalian cultured cells. S. Pombe offers many
advantages for studying the cell cycle; this organism shares
most of the genes used for the cell cycle by mammaliancells and shows efficient labeling of endogenous genes with

16

ERK2 (MAPK1)
mEGFP

B

G(t)

KTR

0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
-0.01

Auto correlation(GFP)
Auto correlation(HaloTag-TMR)
Cross correlation

101

102
103
Lag time, t (μs)

104

105

Figure 4. Quantification of protein concentration and dissociation
constant of endogenous proteins. (A) Schematic illustration of geneknock-in with an MMEJ-mediated KI vector at MAPK1 locus. (B) Autoand cross-correlation functions of ERK2-mEGFP and RSK2-HaloTag.
mEGFP and HaloTag genes were knocked-in at the site of 3’ ERK2 andRSK2 genes, respectively.

III. Manipulation of cell signaling
Artificial manipulation of biochemical networks would be
useful for quantitative understanding of biological systems.
Multiple methods have been suggested for controlling signaltransduction, including light-induced dimerization (LID)
and chemically induced dimerization (CID) systems. Amongthem the LID system has strong advantages in terms of
temporal and spatial manipulations. The photo-responsivePublication List:〔Original papers〕
K., Kondo, Y., Naoki, H., Hiratsuka, T., Itoh, R.E., and Matsuda,• Aoki,M. (2017). Propagating wave of ERK activation orients collective cell•

migration. Dev. Cell 43, 305–317 e5.
Uda, Y., Goto, Y., Oda, S., Kohchi, T., Matsuda, M., and Aoki, K.
(2017). Efficient synthesis of phycocyanobilin in mammalian cells for
optogenetic control of cell signaling. Proc. Natl. Acad. Sci. USA 114,
11962–11967.

A
PCB

Apo-PhyB

PhyB (Pr)PhyB (Pfr)
Red light

Infra-red light

B HemeBiliverdin

PcyA

HO1
Fd

Fd

Fdox

Fdox
Fnr

Fnr

C

PIF binding

Phycocyanobilin(PCB)

730 nm625 nmHigh
ERK activity

-10:0

D

Low

10:00

730 nm 625 nm 730 nm 625 nm 730 nm

ERK activity(FRET/CFP)

proteins derived from fungi, cyanobacteria and plants, andmodified fluorescent proteins are used in this system.
We focus on the phytochrome B (PhyB)-PIF LID system:
Upon red-light illumination, PhyB binds to PIF, and the
PhyB-PIF complex dissociates from each other by infra-red
light exposure (Figure 5A). The reversible control of association and dissociation by light is a unique feature in the
PhyB-PIF system, because either association or dissociationis regulated by light in other LID systems. One drawback isthat covalent attachment of a chromophore, e.g. phycocyanobilin (PCB), is required for the light-responsive function of
PhyB-PIF. To overcome this issue, we developed a method
for synthesis of phycocyanobilin in mammalian cells byintroduction of the gene products of HO1, PcyA, Fd, and Fnr
into mitochondria (Uda Y, PNAS, 2017) (Figure 5B). The
amount of synthesized PCB was comparable to externallydelivered PCB. We could demonstrate light-induced ERK
activation with the PhyB-PIF LID system combined withendogenously synthesized PCB, showing the artificial oscillation of ERK activation (Figure 5C and 5D). The geneticallyencoded system of PCB synthesis will provide a potential
advantage for establishing transgenic animals that stably
synthesize PCB endogenously, thereby enabling the optogenetic manipulation of cell signaling in deeper tissues without
injecting PCB.

1.5
1.4
1.3
1.2
1.1
1.0
0.9
-20

0

20
40
Time (min)60

80

Figure 5. PhyB-PIF light-inducible dimerization (LID) system with
PhyB-PIF and its application to the manipulation of cell signaling. (A)Apo-PhyB covalently attaches its chromophore, which is phycocyanobilin (PCB) to produce holo-PhyB. There are two holo-PhyB forms, PhyB(Pr) and PhyB (Pfr), which change over in manner dependent on thechromophore status. Only PhyB (Pfr) associates with PIF. (B) The
metabolic pathway of phytochrome chromophores. (C) Light-induced
activation of ERK with PhyB-PIF LID system. Representative images of
ERK activity at the nucleus in HeLa cells. (D) The average ERK activity(normalized FRET/CFP ratio) is plotted as a function of time with thestandard deviation.

17

National Institute for Basic Biology

Cell Biology

DIVISION OF CHROMATIN REGULATION

nucleic acid–binding and H3K9me-binding activities of
Suv39h1-CD were crucial for its pericentric heterochromatin
assembly (Figure 1). These results suggest that chromatinbound RNAs contribute to creating SUV39H’s target specificity.

Professor

NAKAYAMA, Jun-ichi

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

HAMADA, Kyoko
KATAOKA, Kensuke
KAWAGUCHI, Takayuki
KAWAMURA, Manaka
OKI, Koichi
TANAKA, Mayo
YOSHIMURA, Yuriko
KONDO, Naoko

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
across cell divisions and throughout development, however,remains incompletely understood. To try to gain a better
understanding of the molecular mechanisms underlying
chromatin-based epigenetic phenomena, our lab uses fission
yeast, Schizosaccharomyces pombe, and ciliate Tetrahymena
as model organisms for studying the molecular mechanismsof higher-order chromatin assembly. We are also attempting
to determine 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 ofhistone 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.
SUV39H is the major H3K9-specific methyltransferasethat targets the pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9me3 via its CD, and enriched H3K9me3 allows
SUV39H to target specific chromosomal regions. However,the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood.
We showed that Suv39h1’s CD (Suv39h1-CD) binds nucleic
acids, and that this binding is important for its function inheterochromatin assembly. Suv39h1-CD had higher binding
affinity for single-stranded RNA than double-stranded DNA,and its ability to bind nucleic acids was independent of its
H3K9me3 recognition. Suv39h1 bound major satellite RNAsin vivo, and mutational studies demonstrated that both the18

Figure 1. Suv39h1-CD’s nucleic acid binding is required for its interaction with major satellite RNAs and chromatin. (A) Dot-blot analysis of
immunoprecipitated RNAs. RNAs associated with WT or mutant (4A)
Suv39h1 in Suv39h dn iMEFs were precipitated with the anti-FLAG
M2 antibody and subjected to dot-blot analysis using a labeled probe formajor satellite repeats (left) and 18S rRNA (right). (B) FRAP analysis of
WT or mutant GFP-Suv39h1 in suv39h dn iMEFs.

II. Regulation of chromodomain proteinsin assembling higher-order chromatin
structure0LWRWLFSKRVSKRU\ODWLRQRI+3ƠUHJXODWHVLWVFHOO
cycle-dependent chromatin binding
HP1 is an evolutionarily conserved chromosomal proteinthat plays a crucial role in heterochromatin-mediated gene
VLOHQFLQJ :H SUHYLRXVO\ VKRZHG WKDW PDPPDOLDQ +3Ơis constitutively phosphorylated at its N-terminal serine
residues by casein kinase II (CK2), and that this phosphoryODWLRQHQKDQFHV+3Ơ·VELQGLQJVSHFLILFLW\IRUQXFOHRVRPHV
containing H3K9me3. Although the presence of additional+3Ơ SKRVSKRU\ODWLRQ GXULQJ PLWRVLV ZDV UHSRUWHG PRUH
than a decade ago, its biological significance remains largely
HOXVLYH+HUHZHIRXQGWKDWPLWRVLVVSHFLILF+3ƠSKRVSKRU\ODWLRQDIIHFWHG+3Ơ·VDELOLW\WRELQGFKURPDWLQ8VLQJELRFKHPLFDO DQG PXWDWLRQDO DQDO\VHV ZH VKRZHG WKDW +3Ơ·V
mitotic phosphorylation was located in its hinge region andwas reversibly regulated by Aurora B kinase and two serine/
threonine phosphatases. In addition, chromatin fractionationand electrophoretic mobility shift assays revealed that hinge
UHJLRQSKRVSKRU\ODWHG +3Ơ ZDV SUHIHUHQWLDOO\ GLVVRFL-

ated from mitotic chromatin and exhibited a reduced DNAELQGLQJDFWLYLW\$OWKRXJK+3Ơ·VPLWRWLFEHKDYLRUZDVSUH
viously linked to H3 serine 10 phosphorylation, which blocks
WKH ELQGLQJ RI +3Ơ·V FKURPRGRPDLQ &'  WR +.PHRXUILQGLQJVVXJJHVWWKDWPLWRWLFSKRVSKRU\ODWLRQLQ+3Ơ·V
KLQJHUHJLRQDOVRFRQWULEXWHVWRFKDQJHVLQ+3Ơ·VDVVRFLDtion with mitotic chromatin.

2-2 Phosphorylation of CBX2 controls its nucleosomebinding specificity
Chromobox 2 (CBX2), a component of polycomb repressive complex 1 (PRC1), binds lysine 27–methylated histone
H3 (H3K27me3) via its chromodomain (CD) and plays acritical role in repressing developmentally regulated genes.
The phosphorylation of CBX2 has been described in several
studies, but the biological implications of this modification
remain largely elusive. We showed that CBX2’s phosphorylation plays an important role in its nucleosome binding.
CBX2 is stably phosphorylated in vivo, and domain analysis
showed that residues in CBX2’s serine-rich (SR) region
are the predominant phosphorylation sites. The serine
residues in an SR region followed by an acidic-residue(AR) cluster coincide with the consensus target of casein
kinase II (CK2), and CK2 efficiently phosphorylated theSR region in vitro. A nucleosome pull-down assay revealed
that CK2-phosphorylated CBX2 had a high specificity for
H3K27me3-modified nucleosomes (Figure 2). An electrophoretic mobility-shift assay showed that CK2-mediatedphosphorylation diminished CBX2’s AT-hook–associatedDNA-binding activity. Mutant CBX2 lacking the SR region
or its neighboring AR cluster failed to repress the transcription of p21, a gene targeted by PRC1. These results suggestthat CBX2’s phosphorylation is critical for its transcriptional
repression of target genes.

III. Proteomic approach to identify chromatinbound RNA-binding proteins
Recent advances in methodologies and technologies led tosystematic identification of more than 1,000 RNA-binding
proteins (RBPs) in mouse and human cells. Some of these
RBPs are known to locate in the nucleus. Interestingly,
previous studies suggest that RNAs together with their RBP
partners do not only contribute to transcriptional regulationof individual genes, but also play a part in three-dimensionalorganization of gene loci within the nucleus and/or in regulation of the inner-nuclear structures. However, currently
available information is rather fragmentary and molecularmechanisms by which RNA-RBP complexes mediate local
and global effects remain largely unknown. Moreover, it isunclear if RNA-RBP interactions participate in other chromatin-based biological processes. In order to grasp the whole
picture of physical or functional interactions between RNAsand RBPs in the nucleus, it is essential to identify which
RNA-RBP complexes are actually bound to chromatin/
nuclear substructures. We have developed a new proteomicsbased method to systematically identify such RBPs as potential chromatin-/nuclear matrix-associated RBPs and successfully identified previously uncharacterized RBPs (Figure 3).
We will verify their RNA-mediated chromatin interaction
and investigate their roles in chromatin organization andtranscriptional regulation.

Figure 3. Network analysis of the identified RNase-sensitive nuclear
proteins.

IV. Dynamic chromatin reorganization during
sexual reproduction of Tetrahymena

Figure 2. CK2-mediated phosphorylation of CBX2-N increases its
binding specificity for H3K27me3 nucleosomes. (A) A representative
nucleosome pull-down assay using synthesized nucleosomes containing
unmodified H3 (H3-unmod) or H3 with a K27me3 analog (H3Kc27me3).
(B) Proposed model for the role of CK2-mediated phosphorylation in thechromatin binding of CBX2.

Tetrahymena, a unicellular protozoan, shows nuclear dimorphism. It contains the transcriptionally silent, germline
micronucleus (MIC) and the transcriptionally active, somatic
macronucleus (MAC) in the single cell. During sexual reproduction, the newly developed MAC removes thousands ofinternal eliminated sequences (IESs), which are derived fromtransposable elements, from the genome. The IES elimination requires RNA interference (RNAi)-related pathway andposttranslational histone modifications. The methylation ofhistone H3 at lysine 9 and lysine 27 catalyzed by Ezl1p act
as binding sites for chromodomain proteins such as Pdd1p
to form heterochromatin-like structures. Although previous
studies identified more than 20 factors that are linked to19National Institute for Basic Biology

Cell Biology

heterochromatin formation and/or DNA elimination, theirprecise roles in chromatin reorganization remain poorly
understood. We have analyzed the cellular localization ofpotential heterochromatin factors during MAC development
and identified 3 novel proteins as heterochromatin components. We have also conducted an ectopic DNA elimination
assay and demonstrated that 10 proteins are sufficient for
inducing DNA elimination (Figure 4). We will further characterize these heterochromatin proteins and define their roles
in chromosome reorganization and IES elimination.

Figure 4. Identification of heterochromatin proteins sufficient to induce
DNA elimination. (A) Schematics of the tethering assay. (B) A representative result of the tethering assay using one of heterochromatin proteins.

Publication List:〔Original papers〕
S., Créchet, J.-B., Bouceba, T., Nakayama, J., Tanaka, M.,• Eustache,Suzuki, M., Woisard, A., Tuffery, P., Baouz, S., and Hountondji, C.

•
•
•
•

20

(2017). A functional role for the monomethylated Gln-51 and Lys-53
residues of the 49GGQTK53 motif of eL42 from human 80S ribosomes.
Open Biochem. J. 11, 8-26.
Kawaguchi, T., Machida, S., Kurumizaka, H., Tagami, H., andNakayama, J. (2017). Phosphorylation of CBX2 controls its
nucleosome-binding specificity. J. Biochem. 162, 343-355.
Mutazono, M., Morita, M., Tsukahara, C., Chinen, M., Nishioka, S.,
Yumikake, T., Dohke, K., Sakamoto, M., Ideue, T., Nakayama, J., Ishii,K., and Tani, T. (2017). The intron in centromeric noncoding RNA
facilitates RNAi-mediated formation of heterochromatin. PLoS Genet.
13, e1006606.
Shirai, A., Kawaguchi, T., Shimojo, H., Muramatsu, D., IshidaYonetani, M., Nishimura, Y., Kimura, H., Nakayama, J., and Shinkai, Y.
(2017). Impact of nucleic acid and methylated H3K9 binding activities
of Suv39h1 on its heterochromatin assembly. Elife 6, e25317.
Zafar, F., Okita, A.K., Onaka, A.T., Su, J., Katahira, Y., Nakayama, J.,
Takahashi, T.S., Nasukata, H., and Nakagawa, T. (2017). Regulation of
mitotic recombination between DNA repeats in centromeres. Nucleic
Acids Res. 45, 11222-11235.

LABORATORY OF CELL RESPONSES

Director GeneralSpecially Appointed Associate Professor

YAMAMOTO, Masayuki

YAMASHITA, Akira

Postdoctoral Fellow:

OTSUBO, Yoko
SHICHINO, Yuichi
NAKADE, Atsuko
SAKAGAMI, MariTechnical Assistant:
Secretary:

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 (Figure 1), to research the mechanism
by which cells switch from mitosis, the kind of cell divisionthat divides cells equally to create two identical cells, tomeiosis, which is essential for bringing forth geneticallydiverse progeny.

ly under nutrient-rich conditions. In most mutants identified,
TORC1 activity is downregulated and the mutated genes are
involved in tRNA expression or modification. We are currently characterizing these mutants.

Figure 2. 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.

II. The molecular mechanisms that establish
the meiosis-specific gene expression profile

Figure 1. Life cycle of the fission yeast S. pombe. S. pombe cells proliferate by mitotic growth under nutrient-rich conditions. When starvedof nutrients, especially nitrogen, S. pombe cells arrest the mitotic cell
cycle and haploid cells conjugate with cells of the opposite mating type.
Resulting diploid zygotes undergo meiosis and produce spores.

I. Signaling pathways that regulate the onset of
sexual differentiation
We have been trying to elucidate how S. pombe 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 ofnutrition and the onset of sexual differentiation in S. pombe.
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 thepresence of nitrogen. TORC2 contains Tor1 and, in contrast
to TORC1, is required for onset of sexual differentiation
under nitrogen starvation (Figure 2).
Temperature-sensitive tor2 mutants initiate sexual differentiation even on rich medium at the restrictive temperature.
To gain insights into the TORC1 signaling pathway, we haveisolated mutants that initiate sexual differentiation ectopical-

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 thefission yeast S. pombe. Understanding precise mechanismsof this control will shed light on the regulation of timely gene
expression during meiosis.
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 3).

21National Institute for Basic Biology

Cell Biology

Figure 3. 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.

We have shown that Mmi1 regulates the termination oftranscription of its target genes. Mmi1-mediated termination of an upstream non-coding RNA ensures the expressionof downstream genes, one of which encodes a mitogenactivated protein kinase kinase kinase (MAPKKK) essential
for the initiation of sexual differentiation. We have also
demonstrated that Mmi1 prevents untimely expression of
meiotic proteins by tethering their mRNAs to nuclear foci.
Multilayered suppression of meiotic genes by Mmi1 is vitalfor mitotic growth.

Publication List:〔Original paper〕
L., Shichino, Y., Dangin, M., Thierry-Mieg, N.,• Touat-Todeschini,
Gliquin, B., Hiriart, E., Sachidanandam, R., Lambert, E., Brettschneider,J., Reuter, M., Kadlec, J., Pillai R., Yamashita, A., Yamamoto., M.,
and Verdel, A. (2017). Selective termination of lncRNA transcription
promotes epigenetic silencing and cell differentiation. EMBO J. 36,
2626-2641.

〔Review article〕
Y., Yamamoto, M., and Yamashita, A. (2017). TORC1• Otsubo,
dependent phosphorylation targets in fission yeast. Biomolecules 7, 50.

22

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 thetransport and local translation of their mRNA cargoes in
response to synaptic stimulation (Figure 1). We areresearching RNA granule factors regulating mRNA transport and local translation, their target mRNAs, and themechanisms of localized protein synthesis using mice inorder to better understand their relation to the formationof synapses and neural networks, memory, learning, andbehavior.

avoidance test, in which an apparatus consisting of lightand dark chambers was used (Figure 2). Mice received footshock in the dark chamber. After that, if they remember thecontext, i.e., fear in the dark chamber, they would not enter
the dark chamber and would stay in the light chamber. At 5
min after the foot shock, RNG105 cKO mice did not enter
the dark chamber any more than control mice. However, at
1 day after the foot shock, they spent significantly longer
time in the dark chamber than control mice. Furthermore,at 1 week after the foot shock, they spent as long a time as
before the foot shock (Figure 2). These results indicated thatnot short-term memory, but long-term memory was markedly
impaired by RNG105 cKO.

Figure 2. Contextual fear conditioning test. Time spent in the darkchamber before foot shock (Pre-FS) and at 5 min, 1 day and 1 week after
the fear conditioning.

Another test was a contextual freezing test. In this test, micereceived foot shock in a single chamber and their freezing
responses were measured in the same chamber at 5 days after
the foot shock. RNG105 cKO mice showed less freezing
behavior than control mice, which indicated that RNG105cKO mice did not well remember the place they learned
to fear. These results supported the notion that RNG105 isessential for the formation of long-term memory.

II. RNG105 deficiency impairs AMPA receptor
scaling on dendrites

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. RNG105 is essential for long-term memory
formation
We previously identified RNA granule protein 105(RNG105, also known as Caprin1), an RNA-binding protein,
as a major component of RNA granules. RNG105 promotes
the assembly of RNA granules and is responsible for thetransport of its binding mRNAs in cultured cells.
To understand the role of RNG105 in learning and memory,
we subjected RNG105 conditional knockout (cKO) mice,
which lacked the Rng105 gene in the brain region, to contextual fear conditioning tests. One of the tests was a passive

To investigate the mechanism of how RNG105 cKO affects
long-term memory, we measured electrophysiologicalresponses (fEPSP) of neurons to synaptic stimulation in the
hippocampus, the essential brain region for memory formation. fEPSP in RNG105 cKO mice was reduced by half
compared to control mice both in the steady state and after
synaptic long-term potentiation (LTP). This suggested that
AMPA receptors (AMPARs), key factors for the synaptictransmission, were downregulated by RNG105 deficiency.
AMPAR downregulation by RNG105 deficiency was
further suggested by RNA-seq analysis as follows. We comprehensively identified mRNAs whose dendritic localization was reduced in hippocampal neurons from RNG105cKO mice. Subsequent gene ontology enrichment analysis
revealed that a major category of the reduced mRNAs was“Regulation of Arf protein signal transduction”, which
included GTPase-activating proteins (GAPs) and guanine
nucleotide exchange factors (GEFs) of small G protein
Arf. Arf is known to regulate membrane trafficking, which
includes the regulation of AMPAR surface expression on
dendrites. These results suggested that cell surface expres-23

National Institute for Basic Biology

Cell Biology

sion of AMPARs on dendrites was affected by RNG105
deficiency.
We then quantified AMPARs expressed on the surface ofdendrites in cultured neurons by immunostaining (Figure3). Comparison between control and RNG105-deficientneurons revealed that the number of surface AMPARs on
dendrites was significantly reduced by RNG105 deficiency.
Furthermore, the scaling mechanism for AMPARs on thesurface of dendrites was impaired in RNG105-deficientneurons: although normal neurons increase the surface
number of AMPARs upon neuronal activity deprivationin order to maintain the basal activity, RNG105-deficientneurons did not increase the surface number of AMPARs
even when their activity was blocked by tetrodotoxin (TTX)and APV (Figure 3). This impaired scaling of AMPARs is
considered to underlie the reduced synaptic responses in thebasal state, which may also attenuate synaptic responses inthe LTP state.
Taken together, our study demonstrated that an element ofRNA granules, RNG105, is essential for long-term memory
formation. Our study further revealed RNG105-dependent
dendritic localization of mRNAs as an underlying mechanism for AMPAR-dependent synaptic strength and long-term
memory formation.

Figure 3. RNG105 deficiency impairs AMPAR scaling on dendrites.
Primary cultured neurons from the cerebral cortex of control (Rng105+/+)and RNG105 knockout (Rng105-/-) littermates were immunostained
for surface-expressed AMPAR subunit GluR1. Arrowheads in the inset
indicate AMPAR puncta. The number of AMPAR puncta on dendrites
is less in Rng105-/- neurons than in Rng105+/+ neurons. Although thenumber of AMPAR puncta was increased by TTX and APV treatment in
Rng105+/+ neurons, it was not increased in Rng105-/-QHXURQV%DUƫP

Publication List:〔Original paper〕
K.*, Ohashi, R.*, Shinoda, Y., Yamazaki, M., Abe, M.,• Nakayama,
Fujikawa, A., Shigenobu, S., Futatsugi, A., Noda, M., Mikoshiba, K.,
Furuichi, T., Sakimura, K., and Shiina, N. (2017). RNG105/caprin1, an
RNA granule protein for dendritic mRNA localization, is essential for
long-term memory formation. eLife 6, e29677. (*: equal contribution)

24

LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

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

KAMIKAWA, Yasunao
TSUBOUCHI, Hideo
KUMAZAKI, Taisei
YASUI, Naomi
ASAI, Yuriko

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. Our focus is on the genome
maintenance mechanisms of the embryonic stem cells, andtheir roles during differentiation and reprogramming processes.

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

reprogramming towards pluripotency.
Specifically, we take advantage of the cell-to-cell fusion
approach, in which a target cell is fused to a pluripotent stem
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 (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).

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. So far, we have found that DNA
replication is regulated differently in ES cells, such that DNA
replication of the whole genome is more accurate in ES cells.
We are currently aiming to address how such differences areinterlinked with pluripotency by carrying out side-by-side
analyses between ES cells and differentiated populations.

〔Original paper〕

II. Genome Instability during Nuclear
Reprogramming

B., Leung, W.-K., Afshar, N., Terentyev, Y., Subramanian,• Argunhan,V., Murayama, Y., Hochwagen, A., Iwasaki, H., Tsubouchi, T.*, andIn 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

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:

Tsubouchi, H.* (2017). Fundamental cell cycle kinases collaborate toensure timely destruction of the synaptonemal complex during meiosis.
EMBO J. 36, 2488-2509. * corresponding authors

25

National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

UENO, NaotoKINOSHITA, Noriyuki

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

frequent intracellular Ca2+ transients were observed. We
have also been able to demonstrate that this spatiotemporally
controlled Ca2+ dynamics is essential for the anterior-directed
tissue migration through the regulation of the asymmetriclamellipodia formation.

Assistant Professor:

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. Cell behaviors during gastrulation
During embryogenesis, the arrangement of multicellulartissue is dramatically changed to establish properly shaped
embryos. These movements of groups of cells are often
highly organized and collective. Investigating the mechanismof 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 micromanipulationof 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, andmeasured the physical value of this force with the explant.
To address how each single LEM cell generates the force
for collective migration in the explant, we have introduced
Traction Force Microscopy (TFM) for Xenopus LEM explantmigration and found that traction force was generated by
the cells, which were located at the anterior part of explants.
Particularly in the first few rows of cells of LEM explants,

26

Figure 1. A snapshot of intracellular Ca2+ transients in a migrating LEM
explant of Xenopus laevis. Ca2+ dynamics were visualized with a FRET
probe YC-Nano2. Inhibition of these Ca2+ transients with Ca inhibitors
perturbs the tissue migration.

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 influencemorphogenetic processes. This project is to elucidate themechanisms of sensing and responding to mechanical stimuli
in Xenopus embryos.
Cells are known to sense mechanical stresses in several
ways, for example, with TRP channels, F-actin, cadherins,
and focal adhesions. Physical stimuli sensed by these molecules are converted to intracellular chemical signals, which
in turn induce cellular response. Protein phosphorylation
may be one of the earliest responses to mechanical stresses.
Therefore, we are attempting to profile protein phosphorylation upon mechanical stimuli and identify target proteinsin Xenopus embryos. In order to comprehensively analyze
levels of protein phosphorylation, we took a phosphoproteomic approach using mass spectrometry. So far, we foundthat a mechanical stress applied to Xenopus embryos changes
phosphorylation levels of many proteins related to cytoskeleton and cell adhesion. Consistently, we observed thatmechanical stimulation induced cytoskeletal remodeling.
Furthermore, some protein kinases, including ERK2, wereactivated in response to a mechanical force. As shown in
Figure 2, ERK2 was phosphorylated by the mechanical force
and its phosphorylation was rapidly attenuated after releasefrom the force. We concluded that Xenopus embryos havea system to sense and respond to mechanical stimulation,
which activates protein kinase signaling pathways and regulates cytoskeletal organization.

single-cell level more effectively accelerate apical constriction and neural tube formation than do multicellular Ca2+
fluctuations in vivo.

Figure 2. ERK2 is activated by a mechanical force in Xenopus embryoniccells.
A mechanical force was applied to Xenopus gastrula embryos by centrifugation. Embryos were stained with anti phoshorylated ERK2 antibody(green) and an actin-binding compound, phalloidin (red). Superficial cells
around the animal pole were observed. (A) control (not centrifuged). (B)centifuged at 450 x g for 15 min. (C) centrifuged as in B and then incuEDWHGIRUPLQ%DU ƫP

III. Statistical analysis of neural tube
formation based on pulsed apicalconstriction
During early development of the central nervous system,neuroepithelial cells constrict their apices by actomyosin
contractility. This cell shape change is called apical constriction, which makes the neural plate into a tubular structure,
called the neural tube. We previously showed that two typesof intracellular Ca2+-concentration changes, a single-cell anda multicellular wave-like fluctuation occurred in the Xenopus
neural plate and modulate the patterns of apical constriction
during neural tube formation. In this study, to further investigate the role of the Ca2+-induced pulsed apical constriction,
we performed statistical analysis in which we fitted a linear
mixed model with single-cell and multicellular Ca2+ transients as explanatory variables and the amount of the closing
movement of the neural tube as a response variable to data of
wild-type embryos, and obtained estimated contributions of
single-cell Ca2+ transients and multicellular Ca2+ transients.
We found that the coefficient of single-cell Ca2+ transientswas significantly larger than that of multicellular Ca2+ transients. These results suggest that the Ca2+ fluctuations at the

Figure 3. Schematic workflow and coefficients of the statistical model.
(A) Temporal profiles of closing movement, the total area of singlecell Ca2+ transients, and multicellular Ca2+ transients during neural tube
formation. (B) Generation of data set for statistical analysis by dividing
temporal profiles into time windows. (C) Fitting mixed linear model tothe data set to estimate contributions. (D) Statistics for fixed effects.

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 ofmembrane 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.

V. Notochord and evolution of chordates
The notochord is the most prominent organ in chordates inwhich Brachyury (Bra) plays pivotal roles in its formation.
Since Bra is shared by non-chordate animals and expressedin the blastopore it is an intriguing EvoDevo question how
Bra acquired enhancers that promote its gene expression
in the notochord as well as in somatic muscles during theevolution of chordates. In cephalochordate lancelets Bra is
duplicated. We examined enhancer activity of Branchiostoma
floridae Bra (BfBra1 and BfBra2) using Ciona egg electroporation system. Vista analysis suggested the presence ofconserved non-coding sequences not only in 5’-upstream but
also 3’-downstream and introgenic regions. The lacZ reporter
assay showed that (1) the 5’-upstream sequences of bothgenes promoted reporter expression in muscle cells, (2) the
3’-downstream sequences also have enhancer activity andpromoted lacZ expression in notochord cells, (3) the introgenic regions of BfBra2 and BfBra1 exhibited the activity
in muscle and notochord cells, respectively, and (4) the
enhancer activity appeared as early as the gastrula stage.
These results indicate that the 5’, 3’ and introgenic regions
work together to enhance the Bra expression in muscle
and notochord cells. The acquisition of enhancer activityof BfBra is highly likely to be involved in the formation ofmuscles and notochord in chordate embryos, which in turnled to the evolution of chordates.

27

National Institute for Basic Biology

Developmental Biology
VI. Gene order and direction of transcription
contribute to the small genome size inCiona intestinalis
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. Effects of light on larval swimming
behavior in scleractinian corals
Many reef-building corals form a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium.
Corals mostly depend on photosynthetic products from these
symbionts for their energy source, and thus light conditionsin habitats can influence post-settlement survival. Several
previous studies reported that light environments play anessential role in larval habitat selection. However, due to lackof basic photobiological studies in corals, how they senselight and how light affects biological tasks remain largely
unexplored. The purpose of this study is to reveal the mechanism of phtoreception and clearly understand the effects oflight on reproduction and behavior in corals.
For this purpose, we focused on the effects of light on larvalswimming behavior of Acropora corals. We analyzed the

Figure 4. A view of stony corals around Orpheus Island, Queensland
Australia, November 201728

larval swimming activity with different light stimuli, andfound that larvae paused their swimming behavior immidiately after a change in intensity and spectral compositionof light. In addition to the behavioral assay, we surveyed
the genome of coral, Acropora digitifera and A. tenuis, fordetecting photoreceptor opsin genes using a molecular phylogenetic analysis. Five opsin gene orthologs were foundon the genome and two of the five genes were transcribedin the coral larvae. These results suggested that coral larvae
sense light with an opsin-based phototranduction cascade andchange their behavior responding to the rapid change in lightenvironments.

Publication List:〔Original papers〕
F., Ando, Y., Miyagi, A., Sugita, S., Ueno, N., and• Murakami,
Matsumoto, T. (2017). Measurement of surface topography and stiffness•••

distribution on cross-section of Xenopus laevis tailbud for estimation of
mechanical environment in embryo. Dev. Growth Differ. 59, 343-443.
Suzuki, M., Sato, M., Koyama, H., Hara, Y., Hayashi, K., Yasue, N.,
Imamura, H., Fujimori, T., Nagai, T., Cambell, R.E., and Ueno, N.
(2017). Distinct intracellular Ca2+ dynamics regulate apical constriction
and differentially contribute to neural tube closure. Development 144,
1307-1316.
Tanaka, T., Ochi, H., Takahashi, S., Ueno, N., and Taira, M. (2017).
Genes coding for cyclin-dependent kinase inhibitors are fragile in
Xenopus. Dev. Biol. 426, 291-300.
Tokue, M., Ikami, K., Mizuno, S., Takagi, C., Miyagi, A., Takada,R., Noda, C., Kitadate, Y., Hara, K., Mizuguchi, H., Sato, T., Taketo,
M.M., Sugiyama, F., Ogawa, T., Kobayashi, S., Ueno, N., Takahashi, S.,
Takeda, S., and Yoshida, S. SHISA6 confers resistance to differentiationSURPRWLQJ:QWơFDWHQLQVLJQDOLQJLQPRXVHVSHUPDWRJHQLFVWHPFHOOVStem Cell Rep. 8, 561-575.

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:
OKADA, Kazunori
TAKADA, Ritsuko
SOKENDAI Graduate Student:
SHINOZUKA, Takuma
TSUCHIYA, Yoshihiro
FUJIMORI, Sayumi
HATAKEYAMA, Yudai
TRAN, Thi Hong Nguyen
Visiting Scientist:
CHEN, Qiuhong
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
Secretary:
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 informa-tion. 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 tobe elucidated. To t his end, we started to visualize signal
proteins and monitor their movement in tissues. In addition,
we are examining structural and biochemical characteristicsof these molecules, which appear to affect how they spread.
The segmental sub-regions of several specific tissues,
including the paraxial mesoderm (or somites) and thepharyngeal pouches, by contrast, appear not to be simply
directed by the gradient of the signal molecules, but by aunique mechanism proceeding periodically. For instance,
somites are sequentially generated in an anterior-to-posteriororder by converting the temporal periodicity created by amolecular clock into periodical structures. The molecularmechanism underlying this conversion and morphologicalsegmentation, however, is not yet fully understood. Thus,
another goal of our current studies is to reveal the molecularmechanism of this other and unique mode of patterning that
underlies the periodical and sequential sub-division in 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 role

Figure 1. Colocalization of Wnt8 and N-sulfo-rich HS clusters. a-d, The colocalization of endogenous Wnt8 and a subpopulation of HS. Gastrula embryos(st. 10.5) were coimmunostained for Wnt8 and NAH46 epitopes (Nacetyl HS) or HepSS1 epitopes (Nsulfo HS) at the ventral marginal zone (a, c).
Signal intensities along white arrows were plotted (b, d), starting and ending points as indicated by yellow and cyan arrowheads, respectively, in a andc. Correlation coefficients (r) for the plots were as indicated. Of note, Wnt8 puncta inside cells were overlapped with Nsulforich HS clusters (orange
arrowheads in b) but not with Nacetyl-rich HS clusters (open arrowheads in a). Scale bars, 10 +m. e, A high correlation between distributions of Wnt8 andN-sulfo HS. Correlation coefficients of distributions between Wnt8 and N-sulfo HS were significantly higher than that between Wnt8 and N-acetyl HS(mean +/– s.d. ***p < 0.001, t-test).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

29

National Institute for Basic Biology

Developmental Biology

in 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 examine the characteristics of Wnt proteins bothin vitro and in vivo. Since Wnt proteins are easily assembled
during conventional biochemical analysis, we are trying toutilize some non-invasive methods for this characterization. Our preliminary study indicated that Wnt proteins are
secreted by forming specific complexes. In addition, analysisof the mobility of Wnt complexes in the extracellular spacesuggested that Wnt proteins are not simply diffused during
embryogenesis. 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.
We first examined the distribution of the endogenous Wnt8
protein in Xenopus embryos. Wnt8 shows a gradient in
the embryo, but at the same time, it is accumulated on thecell surface in a punctate manner. Accumulated Wnt8 is
colocalized with a specific state HS, the sulfonated state of
HS (N-sulfo HS; Figure 1). Furthermore, we found that thedefault or non/less-sulfonated state (N-acetyl HS; Figure 1a,b) and the sulfonated state of HS (N-sulfo HS; Figure 1c, d)
are separately clustered. N-sulfo HS clusters with accumulated Wnt8 recruit the receptor Frizzled (Fzd) and the transducer Dishevelled (Dvl), possibly forming a signaling complex
called the signalosome or multivesicular body, suggesting a
direct connection between the distribution of Wnt and signalling. In contrast N-acetyl HS cluster can accumulate thesecreted Wnt inhibitor Frzb. With overexpressed Frzb, Wnt8
is trapped by N-acetyl HS cluster, which is suggested tocontribute to antagonism of Wnt signaling. We further demonstrate that Gpc4, a Dlp-ortholog forms both N-sulfo andN-acetyl HS while Gpc5, a Dally-ortholog forms N-sulfo HS.
Thus, our finding of discrete clusters of HS provide a regulatory basis for the distribution and signaling of Wnt, discussed
above. We further demonstrate that internalization of N-sulfo
HS is more frequent than that of N-acetyl HS, which could
explain the differences between the short- and long-range

30

distributions of Wnt and Frzb, respectively and also rangeexpansion of Wnt8 by Frzb (Figure 2) .

Figure 2. Model of the Nsulforich and Nacetylrich HS cluster system
for the regulation of the extracellular distribution and signal receptionof the Wnt morphogen. The two types of HS clusters, N-sulfo-rich andN-acetyl-rich HS clusters differ in several aspects including core proteins,tendency of internalization and capability of signaling. Together with thespecificity of Wnt8 to the N-sulfo-rich HS clusters and that of Frzb to theN-acetyl-rich HS clusters, the two types of HS clusters could serve as asystem to regulate the extracellular distribution and signal reception of
the Wnt morphogen.

II. M o l e c u l a r m e c h a n i s m o f s o m i t edevelopment
Somites are segmental epithelial blocks located symmetrically on either side of the neural tube. Somites are periodically generated in an anterior to posterior manner from theirprecursor, the presomitic mesoderm (PSM), which is locatedposterior to newly formed somites. This periodic generation is achieved by a complex and dynamic mechanism in
the PSM. First, the molecular clock, the so-called segmentation clock, creates oscillatory expression of particulargenes, hairy and some notch-related genes, in the posterior
PSM. Because the phase of oscillation is gradually shiftedalong the posterior-to-anterior axis, a wave of the oscillation
appears to move in a posterior-to-anterior fashion. This oscillatory gene expression subsequently resulted in periodical
generation of morphologically segmented somites.
The spatial pattern of somites is defined by positioning ofintersomitic boundaries and rostro-caudal patterning within asomite. This spatial pattern is established in the anterior PSM
by converting the oscillatory gene expression into spatiallyrestricted patterns of gene expression. Several intra-cellularmolecules, as well as FGF and Wnt signaling, are involvedin this conversion. It has been generally considered that
Mesp of bHLH transcriptional regulator plays a critical rolein this conversion. In the mouse, the prospective segmentation boundary is periodically established in the anterior

PSM at the rostral border of the Mesp2 expression domain.
In contrast, recent studies by us and other groups strongly
suggested that Mesp2 does not directly define the position of
the segmentation boundary, rather other genes called Ripply1
and Ripply2 play more essential roles in this conversion in
the mouse and zebrafish. Ripply genes encode ~100 aminoacid proteins, which commonly possess 2 distinct amino acidsequences: a highly conserved WRPW stretch and a conserved ~50-amino acid stretch, called the Ripply homology(RH) domain, that interacts with the T-box proteins, including Tbx6, which is involved in the positioning of the segmentation boundary. Currently, we are examining the mechanism of this conversion by focusing on the regulation of
Ripply’s function in zebrafish.

Figure 3. Ripply3 is required for the proper morphogenesis of the posterior pharyngeal pouches in the mouse embryo. The formation of the
3rd pharyngeal arch (red arrowhead) was impaired in Ripply3 deficientembryos.

III. Molecular mechanism of pharyngeal
pouches
In addition to somites, metameric structures are observedin the pharyngeal region of vertebrates. Typical examples ofsuch structures are skeletal elements of jaws, gills and cranial
nerve projections. This metamerism is brought about by thesegmental development of the pharyngeal pouches, which
are generated by outpocketing of the pharyngeal endoderm.
However, the molecular mechanisms underlying the segmentation of the pharyngeal pouches and the morphogenesis of
the pharyngeal pouches still remained to be elucidated.
To understand the mechanism of the development of pharyngeal pouches, we sought to examine mouse embryos
defective for Ripply3, a member of the Ripply family. In
mouse embryos, Ripply3 is specifically expressed in thepharyngeal endoderm and ectoderm. We previously showedthat Ripply3-deficient embryos fail to develop the pharyngeal arches posterior to the 2nd pharyngeal pouch. However,
the molecular and cellular mechanisms by which Ripply3
regulates the morphogenesis of pharyngeal pouches have still
been unclear.
To better understand the cellular process mediated by
Ripply3, we sought to identify the primary defect observable in Ripply3-deficient mouse embryos. First, by following Ripply3 expression during the formation of the 3rdpharyngeal pouch, we found that Ripply3 expression wascorrelated with the position where epithelial bending took
place. In addition, precise histological analysis at short-term
intervals revealed that Ripply3 was required for the maintenance of a continuous monolayer of pharyngeal epithelial
sheets during their bending. Further analyses using Ripply3deficient embryos and Ripply3-expressing culture cells supported an idea that Ripply3 promotes maturation of adhesive
contacts transmitting mechanical force between the extracellular matrix and the interacting cells. Based on these results,
we propose a model in which Ripply3 plays a role for the
resistance against the mechanical stress in epithelial bending
probably by enhancing the attachment of the pharyngeal
endoderm cells to the extracellular matrix.

Publication List:〔Original papers〕
Y., Yamamoto, T., Takada, R., Mizumoto, S., Matsuyama, M.,• Mii,
Yamada, S., *Takada, S., and *Taira, M. (*Co-corresponding authors)•(2017). Roles of two types of heparan sulphate clusters in Wnt8
distribution and signalling in Xenopus. Nat. Commun. 8, 1973.
Tokue, M., Ikami, K., Mizuno, S., Takagi, C., Miyagi, A., Takada, R.,
Noda, C., Kitadate, Y., Hara, K., Mizuguchi, H., Sato, T., Taketo,
M.M., Sugiyama, F., Ogawa, T., Kobayashi, S., Ueno, N., Takahashi,S., Takada, S., and Yoshida, S. (2017). SHISA6 confers resistanceto differentiation-promoting Wnt/ơFDWHQLQ VLJQDOLQJ LQ PRXVH
spermatogenic stem cells. Stem Cell Rep. 8, 561-575.

〔Review article〕
S., Fujimori, S., Shinozuka, T., Takada, R., and Mii, Y. (2017),• Takada,
Differences in the secretion and transport of Wnt proteins. J. Biochem.
161, 1-7.

31National Institute for Basic Biology

Developmental Biology

DIVISION OF EMBYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professor:

KOYAMA, Hiroshi
NONOMURA, Keiko
Technical Staff:
OKA, SanaeNIBB Research Fellow:
ARATA, Masaki
Postdoctoral Fellow:
DAY, F. Timothy
KISHI, Kanae
NAKANOH, Shota
SOKENDAI Graduate Student: KAMEMIZU, Chizuru
ITO, Tomoaki
USAMI, Fumiko
Technical Assistant:
HIGUCHI, Yoko
KANIE, Akemi
NAKAGAWA, Mami
Secretary:
KATO, Azusa
The aim of our research is to understand the events underlying early mammalian development. One of the characteristics
of mammalian embryonic development is that embryogenesis proceeds in the oviducts and the uterus of the mother, andinteraction between an embryo and maternal tissue is essential. Another characteristic is the highly regulative potentialof embryos. The pattern of cell division and allocation ofcells within an embryo during the early stages vary between
embryos. The timing of the earliest specification events that
control the future body axes is still under investigation. We
want to provide basic and fundamental information about
the specification of embryonic axes, differentiation of celllineages, behaviors of cells, and the regulation of body shapeand tissue morphology in early mammalian development. We
are also interested in the relationship between embryos andtheir 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 genetic
engineering and embryonic manipulation concomitantlywith the progress in optics and microscope technologies,
including development of highly sensitive photo-detectors,
has allowed us to observe developing embryos live, evenin mammals. We have established a series of transgenic
mouse lines for live imaging, which is part of a collaborative
project taking place in the Laboratory for Animal Resourcesand 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 alsoestablished mouse lines to monitor the cell cycle.
We have also been establishing several reporter mouse lines
in the lab to study gene expression patterns during the periimplantation stage of mouse development. In these mice,
cDNA encoding fluorescent proteins are connected to themouse genomic DNA sequences of the enhancer/promoter
region of important genes encoding factors regulating cell32

differentiation in these stages. Combining these two typesof reporter transgenic mouse lines, we have been analyzing
behaviors of cells comparing gene expression properties atthe single cell level. We found that cells expressing Cdx2
exhibit plasticity of specification to TE and ICM lineages
through positional changes.

II. Histological observation of mouse embryos
developing in the uterus
Mammalian embryos develop in the uterus after implantation. Direct interaction between embryos and the cells of theuterus is important. In studies on developmental biology of
mammalian embryos, embryos are usually removed fromthe uterus, and isolated embryos are analyzed. We havebeen analyzing early embryonic development of mousecomparing the changes in the uterine epithelium. To observe
the morphogenesis of embryos within the uterus, serial
sections of implanted uteruses were made, and images of theembryos within the uteruses were captured to make high resolution three-dimensional re-constructions. We are preparing
samples obtained from various developmental stages. Precise

Figure 1. Three-dimensional image reconstruction of the pregnant uterus.
Uterus of 5 days after fertilization were sectioned and images were digitized by image scanner, and reconstructed three-dimensionally to observe
the relationship between embryo and the maternal tissue. Right panel
represents the pseudo 3D image observed from the lateral side of the left
image. Arrows indicate the embryo in the decidual tissue.

positions of embryonic implantation and changes in theuterine epithelium correlating with embryonic development
have been examined using these images. We are identifying
molecules involved in the interaction between embryo properand uterine cells, which may play major roles in embryonicdevelopment.

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. The epithelium of the mouse oviduct consists ofmulti-ciliated cells and secretory cells. Ovulated oocytes aretransported in oviducts by unidirectional motility of multicilia and the resultant secretory fluid flow from ovary touterus. This cellular orientation in the oviduct is one exampleof planar cell polarity (PCP). PCP is seen in a variety oftissues, 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, the beating direction
was not coordinated along the ovary-uterus axis throughout the epithelia; some cells were oriented orthogonally tothe axis, while some others exhibited reverse polarity. In
addition to the ciliary polarity phenotype, cellular shapepolarity was also impaired in the Celsr1-deficient mice.
We found a new feature of cellular polarity in the wild typeoviduct, e.g. that the apical surface of epithelial cells waselongated along the ovary-uterus axis. In Celsr1-deficient
mice, epithelial cells showed less elongation and randomized
orientation.
Furthermore, we also found that the three-dimensionalarchitecture of oviductal epithelia was dramatically disrupted. In adult wild-type mice, the epithelial cell sheet is bent
multiple times, which forms around twenty longitudinal folds
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 2). 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.

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 of morphogenesis, measuringcellular and tissue mechanical states is necessary. We developed statistic techniques to infer mechanical states using fluorescent microscopic images during morphogenesis (Figure3). By employing this method, we inferred mechanical forcesin multi-cellular systems including cultured epithelial cells,
and early embryogenesis in C. elegans and mice. Further
computational simulations based on the inferred mechanicalinformation reproduced morphological features of the multi-

Figure 3. Theoretical inference of cellular/tissue mechanical states.
Schematic illustration of inference.

cellular systems. Thus, the mechanical information will be
useful to investigate physical mechanisms of early embryonic development and morphogenesis during organogenesisin late stages of development.

V. Mammalian tissue morphogenesis requiring
mechanosensor channel Piezos
Several examples have shown that mechanical stimuli
can work as key components for tissue or organ development. However, our knowledge about involvement of
mechanotransduction in biological phenomena or theirprecise mechanisms is still limited. It is partially because
key mechanosensors are not yet identified in many cell types.
Piezos are recently identified mechanically activated cation
channels functioning in mammalian cells (Figure 4). They
are activated when mechanical forces are applied on the cell
membrane. Series of data show that Piezo2 serves as themain mechanosensor in sensory neurons for light touch sensation, proprioception and breathing. We found that Piezos
are also required for proper vascular morphogenesis. To
further elucidate how Piezo-mediated mechanotransduction
is involved in vascular development, we have been developing systems to manipulate mechanical stimuli and monitor

Positive or
negative pressure,
shear stress

Ca2+, Na+,+ +Mg2+, K+
+
+
+
+
Piezo
+ +
+ +
non-selective
cation channel

Figure 2. Epithelial fold patterns in oviduct and the reproduction of thepatterns by computational simulations.

How is Piezo-mediatedmechanotransduction
involved in tissuemorphogenesis?

Figure 4. Schematic illustration of Piezo mechanically activated nonselective cation channel and the key question of this study.

33

National Institute for Basic Biology

Developmental Biology

Piezo activity in vitro and in vivo. Analyses utilizing thesesystems and mouse lines deficient in Piezos will clarify
the relationship among mechanical forces, Piezo activation, cellular responses, and tissue morphogenesis, this may
provide a basis to understand the roles of mechanotransduction in tissue morphogenesis.

VI. Mechanics of cell population patterning
during development
During development, cells actively and/or passively move,resulting in various patterns of cell distribution. We investigated the effect of both active and passive cell movements.
First, we mathematically modeled the process of neural tube
closure in Xenopus laevis, and found that active cell contractility and its frequency are critical parameters for the tissue
shrinkage. Second, we theoretically investigated the effectof passive cell movements provoked by frictional forcesfrom surrounding tissues. The passive movements generated
various patterns, such as an elongated cell cluster, multiple
cell clusters, etc. The former situation is actually observed
during elongation of the notochord in mice. This theoreticalframework would be widely applicable to developmental
processes.

Publication List:〔Original papers〕
K., Hashimoto, M., Ajima, R., Takaoka, K., Shinohara,• Minegishi,
K., Ikawa, Y., Nishimura, H., McMahon, A.P., Willert, K., Okada, Y.,

•
•
•

34

Sasaki, H., Shi, D., Fujimori, T., Ohtsuka, T., Igarashi, Y., Yamaguchi,
T.P., Shimono, A., Shiratori, H., and Hamada, H. (2017). A Wnt5
activity asymmetry and intercellular signaling via PCP proteins polarize
node cells for left-right symmetry breaking. Dev. Cell 40, 439-452.
Nonomura, K., Woo, S.H., Chang, R.B., Gillich, A., Qiu, Z., Francisco,
A.G., Ranade, S.S., Liberles, S.D., and Patapoutian, A. (2017). Piezo2
senses airway stretch and mediates lung inflation-induced apnoea.
Nature 541, 176-181.
Shioi, G., Hoshino, H., Abe, T., Kiyonari, H., Nakao, K., Meng, W.,
Furuta, Y., Fujimori, T., and Aizawa, S. (2017). Apical constriction
in distal visceral endoderm cells initiates global, collective cell
rearrangement in embryonic visceral endoderm to form anterior visceralendoderm. Dev. Biol. 429, 20-30.
Suzuki, M., Sato, M., Koyama, H., Hara, Y., Hayashi, K., Yasue, N.,
Imamura, H., Fujimori, T., Nagai, T., Campbell, RE., and Ueno, N.
(2017). Distinct intracellular Ca2+ dynamics regulate apical constriction
and differentially contribute to neural tube closure. Development 144,
1307-1316.

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:
IKEDA, Tatsuro
TOKUE, Moe
ISHIZAKA, Miho
SOKENDAI Graduate Student: ISHIZAKA, Miho*
HIRANO, Kodai
Visiting Graduate Student: SATO, Toshiyuki
Visiting Undergraduate: YOSHIDO, Kana
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,and their connection to tissue environment. “Potential stem
cells” are also of our enthusiastic interest: In undisturbed,
steady-state spermatogenesis, they largely contribute as transient amplifying cells and differentiate rather than self-renew.
However, 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 from this division related to 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, Ikami et al., Development 2015, and Tokueet al., Stem Cell Reports 2017.

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 mitotic
division process with which cytokinesis does not complete
and the cytoplasmic connection between daughter cells
persists via intercellular bridges. The prevailing stem celltheory 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 and no 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 “As model”. Thisis the simplest interpretation of the results of our funcWLRQDO DQDO\VHV RI *)5Ơ+ 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
KLHUDUFK\ *)5Ơ+ 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+FHOOV$IWHU OHDYLQJ WKH *)5Ơ+ compartment, differentiation-destined
FHOOVIROORZDVHULHVRIWUDQVLWLRQV *)5Ơ+ĺNGN3+ĺKIT+; downward
black arrows) that accompany the extension of syncytial length (rightward black arrows). Ngn3+ and, to a lesser extent, KIT+ cells retain the
FDSDFLW\ WR UHYHUW EDFN LQWR WKH *)5Ơ FRPSDUWPHQW LQ D FRQWH[W
dependent fashion (broken arrows). (Reprinted from Hara et al., Cell
Stem Cell 2014.)

As crystalized in this model, our results suggest that the*)5Ơ + 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 overNote: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

35

National Institute for Basic Biology

Developmental Biology

a particular “quota” would overflow to become the NGN3+
state of Aundff, which then further differentiate into KIT+ “differentiating” spermatogonia that are largely devoid of selfrenewing potential.
Currently, we are investigating the mechanism that deterPLQHV WKH TXRWD RU WLVVXH FDSDFLW\ RI *)5Ơ+ cells, as wellas the detailed nature of the internal heterogeneity of the
SRSXODWLRQ RI *)5Ơ+ cells. Further, it is also becoming
DSSDUHQWWKDWWKH*)5Ơ+ population is not totally homogeneous but associated with some heterogeneity in their gene
expression and fate behavior, as described by our group (seebelow) and others. Therefore, our next challenges include
understanding the overall scheme of the stem/progenitor cell
dynamics in which short-term heterogeneity and long-term
equipotency (randomness) are both observed in a compatible
manner.

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 outside of which they differentiate (Fuller and Spradling, 2007; Morrison and Spradling,2008). However, the mouse spermatogenic stem cell systemappears to not show a defined facultative niche environmentand does not rely on asymmetric division; their fates arefound to be 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).

:QWơFDWHQLQ VLJQDOLQJ SURPRWHV GLIIHUHQWLDWLRQ
RI*)5Ơ+ to NGN3+ cells++

7R XQYHLO WKH VLJQDO WKDW SURPRWHV WKH *)5Ơ to NGN3transition. We first compared the gene expression profiles
EHWZHHQ *)5Ơ+ and NGN3+ cells by cDNA microarray.
From the microarray data, we focused on Wnt signaling. In
FXOWXUHGVSHUPDWRJRQLD YL]*6FHOO :QWơFDWHQLQVLJQDOing induced Ngn3 expression (Figure 2A). Moreover, micecarrying a gain-of-function mutation of ơFDWHQLQ showed
VSHUPDWRJHQHVLV GHIHFWV LQ ZKLFK WKH QXPEHU RI *)5Ơ+
cells was reduced (Figure 2B), whereas no significant reducWLRQ LQ UHWLQRLF DFLG UHFHSWRU JDPPD 5$5Ƣ + cells (largely
corresponding to NGN3+ cells) was observed. Consequently,
WKH 5$5Ƣ + FHOOWR*)5Ơ + cell ratio increased, consisWHQW ZLWK WKH LGHD WKDW *)5Ơ+ to NGN3+ differentiation
increased.

36)LJXUH  :QWơFDWHQLQ VLJQDO SURPRWHV WKH GLIIHUHQWLDWLRQ RI *)5Ơ+
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 P51$ OHYHO % $YHUDJH QXPEHUV RI *)5Ơ+ cells per tubule,
REVHUYHGLQPLFHZLWKLQGLFDWHGJHQRW\SHV1XPEHURI*)5Ơ+ cells was
reduced in Nanos3Cre/+;Ctnnb1 fl(ex3)/fl(ex3) miceLQZKLFKơFDWHQLQZDVDFWLvated in a germ cell specific manner. Modified from Tokue et al., Stem
Cell Reports (2017),

2-2. SHISA6, a cell-autonomous Wnt inhibitor, is
H[SUHVVHGLQDVXEVHWRI*)5Ơ+ 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 differentiationSURPRWLRQ LQ VHPLQLIHURXV WXEXOHV DQG WKDW *)5Ơ+ 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
LQKLELWRU V  PD\ FRQIHU UHVLVWDQFH WR :QWơFDWHQLQ VLJQDOLQJLQVRPHIUDFWLRQRI*)5Ơ+ cells. In our microarray data,
we found Shisa6 an interesting candidate, because Shisa6
ZDV KLJKO\ HQULFKHG LQ WKH *)5Ơ+ fraction and some ofthe 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 in about 30% of
WKH*)5Ơ+ population in vivo (Figure 3B).

2-3. SHISA6+ spermatogonia show stem cell characters
DQGFRQWULEXWHWRPDLQWHQDQFHRI*)5Ơ+ cells
We found that, although Shisa6 KO or stabilized ơFDWHQLQ
heterozygous mutants show no apparent phenotype in the
SRRO RI *)5Ơ+ cells, synthetic heterozygotes for these
DOOHOHV VKRZHG D UHGXFHG *)5Ơ+ cell pool and spermatogenesis defects. Thus, SHISA6 plays a role in the mainteQDQFH RI WKH VWHP FHOO SRRO E\ VXSSUHVVLQJ :QWơFDWHQLQ
signaling 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 continually 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 3. Function and expression of SHISA6 (A) Cell-autonomous
LQKLELWLRQ RI :QWơFDWHQLQ VLJQDOLQJ E\ 6+,6$ +(.7 FHOOV
were transfected with a Wnt reporter luciferase construct and a Shisa6
expression plasmid either simultaneously (co-transfection), or separately(transfected cells were mixed afterword), followed by stimulation of:QWơFDWHQLQ VLJQDO 7KH :QWGHSHQGHQW /XFLIHUDVH DFWLYLW\ ZDV VXSpressed only when Shisa6 was co-transfected. (B) Representative imageof dissociated testicular cells of adult mice doubly stained for Shisa6
by FISH and for GFR (B) Representative image. Note the overlapped
expression of Shisa6 and GFRsa is modified from Tokue et al., Stem Cell
Reports (2017),)LJXUH $ FRQFHSWXDO GLDJUDP RI GLIIHUHQWLDWLRQ SURPRWLRQ E\ :QWơcatenin 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:〔Original Paper〕
M., Ikami, K., Mizuno, S., Takagi, C., Miyagi, A., Takada,• Tokue,R., Noda, C., Kitadate, Y., Hara, K., Mizuguchi, H., Sato, T., Taketo,

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),

M.M., Sugiyama, F., Ogawa, T., Kobayashi, S., Ueno, N., Takahashi,S., Takada, S., and Yoshida, S. (2017). SHISA6 confers resistanceto differentiation-promoting Wnt/beta-catenin signaling in mouse
spermatogenic stem cells. Stem Cell Rep. 8, 561-575.

2-4. SHISA6 protects the stem cells from differenWLDWLRQ SURPRWLQJ :QWơFDWHQLQ VLJQDOLQJ $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).

37

National Institute for Basic Biology

Neurobiology

DIVISION OF MOLECULAR NEUROBIOLOGY

Professor

Associate Professor

NODA, Masaharu

SHINTANI, Takafumi

Assistant Professor:

SAKUTA, Hiraki
HIYAMA, Takeshi
Technical Staff:
TAKEUCHI, Yasushi
NIBB Research Fellow:
MATSUDA, Takashi
NOMURA, Kengo*
Postdoctoral Fellow:
FUJIKAWA, Akihiro
SUZUKI, Ryoko
KUBOYAMA, Kazuya
LIN, Chia-Hao
NOMURA, Kengo
YU, Yang
SOKENDAI Graduate Student: MATSUDA, Takashi*
YU, Yang*
TANGA, Naomi
HIGASHI, Satoru
Technical Assistant:
MIURA, Seiko
NAKANISHI, Norie
WADA, Kotoe
KONISHI, Mie
ISOSHIMA, Yoshiko
HASHIMOTO, Terumi
Secretary:
KODAMA, Akiko

The scope of our interests encompasses underlying mechanisms for the development of the vertebrate central nervous
system (CNS) and various functions of the mature brain,including body fluid homeostasis, blood pressure control,
food intake 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 by intellectual disability and a combination of typical facial featuresand 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 by examining
Nsd1-KO mice.

II.Physiological roles of receptor-like protein
tyrosine phosphatases
Protein tyrosine phosphorylation plays crucial roles invarious biological events such as cellular proliferation, differentiation, survival, migration, and metabolism. Cellulartyrosine phosphorylation levels are governed by the opposing
activities of protein tyrosine kinases (PTKs) and protein

38

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, play pivotal roles in the development of several tissues including the vascular and nervous
systems. Eph receptor tyrosine kinases are known to playindispensable roles in the topographic central projectionof retinal ganglion cell (RGC) axons. Using the chick retinotectal projection system, we previously showed that
PTPRO controls the sensitivity of retinal axons to ephrins
and thereby has a crucial role in the establishment of topographic projections. We also demonstrated that R3 RPTPs
dephosphorylate Eph receptors. Among R3 RPTPs, PTPRJ
and PTPRO were expressed in developing mouse RGCs.
We are now investigating the relationship between roles of
PTPRJ and PTPRO in the central projection of the retinal
axons by using gene knockout mice.
Leptin, an adipocyte-derived hormone, is a critical factor
controlling food intake: it strongly inhibits food intake
through the regulation of neuronal activities in the hypothalamic arcuate nucleus (ARC). Most obese individuals
have an increased food intake despite high circulating leptin
levels, which is referred to as leptin resistance. However,
the exact mechanisms underlying leptin resistance in obese
patients have yet to be elucidated. We recently demonstrated
that PTPRJ is a physiological enzyme attenuating insulin
signaling in vivo. In Ptprj-KO mice, the activation of insulin
receptor (IR) kinase and Akt (or protein kinase B) was
enhanced, and glucose and insulin tolerance was improved.
Furthermore, Ptprj-KO mice exhibited lower weight gain
associated with lesser food intake compared with wild-type
mice.
We have now found that PTPRJ is involved in the regulation of leptin signaling in the ARC through the dephosphorylation of JAK2, the primary tyrosine kinase in leptin signaling. In line with these results, leptin signaling is enhancedin Ptprj-KO mice. Diet-induced obesity up-regulated PTPRJ
expression in the hypothalamus, and induction of leptin resistance was strongly attenuated in Ptprj-KO mice. Upon leptin
administration, Ptprj-KO mice fed a high-fat/high-sucrose

Figure 1. Ptprj-KO mice are protected from the development of dietinduced leptin resistance. (A) Daily change in food intake by and (B)Daily change in body weights of wild-type (WT) and Ptprj-KO mice fed
high-fat/high-sucrose diet (HF/HSD for 14 weeks) upon daily administration of leptin. Leptin (500 ng) or vehicle was i.c.v. injected during
the indicated period. ANOVA was used to detect significant differencesat the same time point between the two groups (n = 8 each; *P < 0.05,**P < 0.01, ***P < 0.001).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

diet showed significant reductions in food intake and bodyweight as compared with corresponding WT mice (Figure1). Moreover, the overexpression of PTPRJ in the ARC in
non-obese mice with a viral vector induced significant leptin
resistance. Overall, our results indicated that PTPRJ plays
critical roles in the development of leptin resistance in vivo.

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 gene: twotransmembrane isoforms, PTPRZ-A and PTPRZ-B, and one
secretory isoform, PTPRZ-S (or phosphacan). All isoforms
are heavily modified with chondroitin sulfate (CS) chains,and identified as chondroitin sulfate proteoglycans (CSPGs)
in the CNS. The CS moiety on their extracellular domain of
PTPRZ is essential for achieving high-affinity binding sites
for the endogenous ligands such as pleiotrophin (PTN). We
have proposed that PTPRZ is a new molecular target for drug
development in glioblastoma and demyelinating diseases
such as multiple sclerosis (MS).
CSPGs enriched in demyelinating plaques are known to
impair remyelination by inhibiting the migration and differentiation of OPCs in MS patients. This year, we revealed
that protamine (PRM) effectively neutralizes the inhibitory
activities of CSPGs, thereby enhancing OPC differentiationand (re)myelination in mice. Cell-based assays revealed
that PRM exerted masking effects on extracellular CSPGs,
and also acted as a ligand mimetic for PTPRZ-A/B, thereby
improving oligodendrocyte differentiation even under unfavorable differentiation conditions with CSPGs (Figure 2).
Intranasal administration of PRM accelerated myelination in
the developing mouse brain, and its intracerebroventricularadministration stimulated remyelination after cuprizoneinduced demyelination.

transcription factors such as SOX2, OLIG2, and POU3F2,
along with decreased sphere-forming abilities of these cells.
We identified an allosteric PTPRZ inhibitor, NAZ2329(Figure 3A) for the first time. This compound efficiently
reduced the expression of SOX2 in C6 and U251 cells,
and abrogated the sphere-forming abilities of these cells.
Furthermore, tumor growth in the C6 xenograft mouse model
was significantly slower with the co-treatment of NAZ2329
with temozolomide, an alkylating agent, than with the individual treatments (Figure 3B). These results indicate that
PTPRZ is a promising molecular target for differentiationinducing therapy of malignant gliomas.

Figure 3. Identification of a new allosteric inhibitor, NAZ2329. A, X-ray
structure of PTPRZ1-D1 complexed with NAZ2329. Cys1933 at theactive site and Val-1911 at the bottom of the allosteric pocket are indicated in red and yellow, respectively. B, Antitumor effect of NAZ2329.
Nude mice subcutaneously implanted with C6 glioblastoma cells weretreated with DMSO, NAZ2329, temozolomide (TMZ), or the combination of NAZ2329 with TMZ until the humane endpoint (>3,000 mm3
tumor size or 40 days after the treatment). Kaplan-Meier analysis of thefour treatment groups.

III. Brain systems for body-fluid homeostasis
We have shown that Na x, which structurally resembles
voltage-gated sodium channels (Nav1.1–1.9), is the brain[Na+] sensor to detect increases in [Na+] in body fluids. Nax
is preferentially expressed in specific glial cells of sensory
circumventricular organs (sCVOs) including the subfornical organ (SFO) and organum vasculosum lamina terminalis (OVLT). We have reported that Nax signals in these brain
regions deficient in a blood-brain barrier are involved in thecontrol of water and salt intake.

3-1 Thirst control by Nax and TRPV4

Figure 2. A possible regeneration model: Protamine may neutralize
CSPG-mediated inhibition of migration of OPCs into demyelinatingplaques and differentiation of OPCs to oligodendrocytes (see text).

A recent study on primary human glioblastomas suggested aclose association between PTPRZ1 (human PTPRZ) expression and cancer stemness. However, the functional roles of
PTPRZ in glioma stem cells have remained unclear. As was
expected, sphere-forming cells from separated rat C6 andhuman U251 glioblastoma cell lines showed high expressionof PTPRZ-B, the short receptor isoform of PTPRZ. Stable
PTPRZ knockdown altered the expression levels of stem cell

We previously demonstrated that Nax signals in Nax-positive
glial cells leads to the activation of TRPV4-positive neurons
by using epoxyeicosatrienoic acids as gliotransmitters in
sCVOs to stimulate water intake. Also, we suggested that
another [Na+]-dependent signal is needed besides the NaxTRPV4 signal to explain the whole amount of water intake.
In order to identify the sensor molecule which generates theunknown [Na+]-dependent signal, we performed RNA-seq
analysis of sCVOs and identified several candidates. We arenow examining the functional roles of these candidates inwater intake.

3-2 Thirst and salt-appetite control by angiotensin II,[Na+], and cholecystokinin
Angiotensin II (Ang II) is known to drive both thirst andsalt appetite, and Ang II levels in blood are increased underboth water- and salt-depleted conditions. However, we selec-

39

National Institute for Basic Biology

Neurobiology

tively feel a thirst or salt appetite dependent on body fluid
conditions. The brain mechanisms by which we properly
take in water or salt have not been fully elucidated. Our
local deletion experiments of AT1a, an Ang II receptor gene,indicated that AT1a signals in the SFO are involved in bothwater and salt intakes, whereas those in the OVLT are only
involved in water intake. AT1a-positive neurons in the SFO
project to several nuclei, including the OVLT and ventral
part of the bed nucleus of the stria terminalis (vBNST).
By using optogenetics, we demonstrated that thirst andsalt appetite are driven by distinct groups of AT1a-positive
neurons in the SFO: Neurons projecting to the OVLT control
water intake, while those projecting to the vBNST control
salt intake. We named these two different driver neurons‘water neurons’ and ‘salt neurons’, respectively (Figure 4).
‘Water neurons’ were suppressed under Na-depleted conditions through cholecystokinin (CCK)-mediated activation of
GABA(a-aminobutyric acid)ergic neurons. As was expected,
CCK levels in the SFO were increased under Na-depleted(water-satiated) conditions. The control mechanism of CCK
secretion in the SFO remains to be elucidated. We hadreported that Nax-KO mice do not stop salt intake even underwater-depleted conditions. We herein revealed that ‘salt
neurons’ were suppressed under the water-depleted conditions through activation of another population of GABAergic
neurons by Nax signals. These studies thus provided new
insights into the central mechanisms by which body fluid
conditions control thirst and salt appetite. On the other hand,
it is also known that aldosterone enhances salt appetite
besides Ang II. Our next goal is integration mechanisms ofsalt-appetite signals from ‘salt neurons’ and ‘aldosteronesensitive neurons’ in the vBNST.
<Water-depleted condition><Na-depleted condition>

SFO

SFO

Ang II

Ang II
Na+

GABA
Water

CCK

GABA
Glia

Salt

GABA

Nax

Water

Na+

3rd ventricle

OVLT

Na+

GABA
Glia

Nax

Salt

3rd ventricle

vBNST

OVLT

vBNST

ThirstSalt appetite

Salt appetite

Water intake

Salt intake

Salt intake

Figure 4. A schematic overview of control mechanisms for thirst andsalt appetite in the SFO. Under the water-depleted condition (left), [AngII] and [Na+], but not [CCK], increase in the SFO, and “water neurons”
innervating the OVLT are selectively activated. Under the Na-depleted
condition (right), [Ang II] and [CCK], but not [Na+], increase, and “saltneurons” innervating the vBNST are selectively activated. These two
inhibitory systems by distinct GABAergic neurons are responsible for the
specific activation of “water neurons” or “salt neurons” dependent on thebody-fluid conditions. On the other hand, both water and Na intakes are
simultaneously stimulated under the water- and Na-depleted condition,
because the both neurons are active by Ang II.

40

Publication List:〔Original papers〕
A., Chow, J.P.H., Matsumoto, M., Suzuki, R., Kuboyama, K.,• Fujikawa,
Yamamoto, N., and Noda, M. (2017). Identification of novel splicing

•
•
•
•
•
•
•

variants of protein tyrosine phosphatase receptor type Z. J. Biochem.
162, 381-390.
Fujikawa, A., Sugawara, H., Tanaka, T., Matsumoto, M., Kuboyama, K.,
Suzuki, R., Tanga, N., Ogata, A., Masumura, M., and Noda, M. (2017).
Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity
in glioblastoma cells. Sci. Rep. 7, 5609.
Hiyama, T.Y., Utsunomiya, A.N., Matsumoto, M., Fujikawa, A., Lin,
C.-H., Hara, K., Kagawa, R., Okada, S., Kobayashi, M., Ishikawa, M.,
Anzo, M., Cho, H., Takayasu, S., Nigawara, T., Daimon, M., Sato, T.,
Terui, K., Ito, E., and Noda, M. (2017). Adipsic hypernatremia without
hypothalamic lesions accompanied by autoantibodies to subfornicalorgan. Brain Pathol. 27, 323-331.
Kuboyama, K., Tanga, N., Suzuki, R., Fujikawa, A., and Noda, M.
(2017). Protamine neutralizes chondroitin sulfate proteoglycanmediated inhibition of oligodendrocyte differentiation. PLoS ONE 12,e0189164.
Matsuda, T., Hiyama, T.Y., Niimura, F., Matsusaka, T., Fukamizu, A.,
Kobayashi, K., Kobayashi, K., and Noda, M. (2017). Distinct neuralmechanisms for the control of thirst and salt appetite in the subfornicalorgan. Nat. Neurosci. 20, 230-241.
Nakayama, K., Ohashi, R., Shinoda, Y., Yamazaki, M., Abe, M.,
Fujikawa, A., Shigenobu, S., Futatsugi, A., Noda, M., Mikoshiba, K.,
Furuichi, T., Sakimura, K., and Shiina, N. (2017). RNG105/caprin1, an
RNA granule protein for dendritic mRNA localization, is essential for
long-term memory formation. eLife 6, e29677.
Shintani, T., Higashi, S., Suzuki, R., Takeuchi, Y., Ikaga, R., Yamazaki,T., Kobayashi, K., and Noda, M. (2017). PTPRJ inhibits leptin
signaling, and induction of PTPRJ in the hypothalamus is a cause of thedevelopment of leptin resistance. Sci. Rep. 7, 11627.
Utsunomiya, A.N., Okada, S., Hiyama, T.Y., Noda, M., and Kobayashi,
M. (2017). Clinical characteristic features of adipsic hypernatremia
patients wit subfornical organ-targeting antibody. Clin. Pediatr.
Endocrinol. 26, 197-205.

DIVISION OF BEHAVIORAL NEUROBIOLOGY

Professor

HIGASHIJIMA, Shin-ichi

Assistant Professor:
KIMURA, Yukiko
Postdoctoral Fellow:
NISHIUMI, Nozomi
SOKENDAI Graduate Student: SUGIOKA, Takumi
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 circuitsthat 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.
In addition to zebrafish, we have also started to use medaka
as an experimental animal. Medaka has many advantages
similar to zebrafish. Because NIBB is the main center of theMedaka National Bioresource Project, we are in a perfectplace to conduct experiments using medaka. To begin with,
we explored whether knock-in fish could be efficiently
generated using the CRISPR/Cas9 system.

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

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 methodby utilizing the CRISPR-Cas9 system. The method wedeveloped is highly efficient, such that nearly one-third of
the raised 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 inhi-

41

National Institute for Basic Biology

Neurobiology

bition 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.

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 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.

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(ipsilaterally-projecting inhibitory neurons) is less clear.
En1-positive neurons constitute major components ofipsilaterally-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.

42

IV. Axially-confined in vivo single-cell labelingby primed conversion using blue andred lasers with conventional confocal
microscopes
Green-to-red photoconvertible fluorescent proteins(PCFPs), such as Kaede or Dendra2 are powerful tools tohighlight a small number of cells in a globally fluorescentcellular context. However, perfect single-cell labeling intightly packed tissues such as those that make up the nervous
system has been difficult because the traditional method
for efficient photoconversion requires the use of violet/UV
single-photon excitation (i.e., 405 nm), which is not confinedto the axial dimension. Under conditions where manycells express PCFPs in a three-dimensional manner, cells
that are located above and below the target cells undergophotoconversion when exposed to a single-photon converting
beam.
Recently, Dendra2 has been found to undergo efficientphotoconversion by a new method termed primed conversion
that employs dual wave-length illumination with blue andred/near-infrared light. By modifying a confocal laserscanning microscope (CLSM) such that two laser beams only
meet at the focal plane, confined photoconversion at the axial
dimension has been achieved. The necessity of this custom
modification to the CLSM, however, has precluded the widespread use of this method.
We investigated whether spatially-restricted primed

conversion could be achieved with CLSM without any
hardware modifications. We found that the primed
conversion of Dendra2 using a conventional CLSM withtwo visible lasers (473 nm and 635 nm) and a high NA
objective lens (NA, 1.30) resulted in a dramatic restrictionof photoconversion volume: half-width half-maximum forthe axial dimension was below 5 +m, which is comparable
to the outcome of the custom method that employed themicroscope modification. As a proof of this method’s
effectiveness, we utilized this technique in living zebrafish
embryos and succeeded in revealing the complex anatomy
of individual neurons packed between neighboring cells.
Because unmodified CLSMs are widely available, thismethod can be widely applicable for labeling cells withsingle-cell resolution.

With its simplicity, design flexibility, and high efficiency,
we propose that CRISPR/Cas9-mediated knock-in via NHEJ
will become a standard method for the generation of transgenic and mutant medaka. (Watakabe, I. et al. Zool. Lett. inpress)

Figure 7. (A) For the generation of knock-in transgenic fish, sgRNA1(for genome digestion), sgRNA2 (for plasmid digestion), donor plasmidwith a bait sequence, and Cas9 mRNA are co-injected into one-cell-stage
medaka embryos. (B) An example of transgenic medaka in which RFP
was knocked-in into the vacht gene. As with the endogenous gene, RFP isexpressed in motoneurons.

Publication List:〔Original Papers〕
A., Kawai, T., Higashijima, S., and Okamura, Y. (2017).
• Ratanayotha,
Molecular and functional characterization of the voltage-gated proton
channel in zebrafish neutrophils. Physiol. Rep. 15, e13345.

A., Kimura, Y., Mori, I., Nonaka, S., and Higashijima,• Taniguchi,
S. (2017). Axially-confined in vivo single-cell labeling by primed
conversion using blue and red lasers with conventional confocal
microscopes. Develop. Growth Differ. 59, 741-748.

Figure 6. Primed conversion of Dendra2 reveals the fine morphology
of a single motoneuron in a larval zebrafish

V. Highly efficient generation of knock-in
transgenic medaka by CRISPR/Cas9mediated genome engineering
As mentioned above, we have established an efficient(~30%) knock-in system via non-homologous end joining(NHEJ) using the CRISPR/Cas9 system in zebrafish. If thesame technique were applicable in medaka, it would greatly
expand the usefulness of this model organism. This point,
however, has not yet been addressed.
We report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ).
Donor plasmid containing a heat-shock promoter and areporter gene was co-injected with a short guide RNA(sgRNA) targeted for genome digestion, an sgRNA targeted
for donor plasmid digestion, and Cas9 mRNA. In approximately 25% of injected embryos, broad transgene expression
in the expression domain of a target gene was observed. By
raising these animals, we succeeded in establishing stable
knock-in transgenic fish with several different constructs
for five genetic loci. The efficiencies of obtaining transgenic founders among the raised animals exceeded 50% for
all five loci. Further, we show that the method is useful for
obtaining mutant alleles. In the experiments where transgene
integrations were targeted between the transcription start site
and the initiation methionine, the resultant transgenic fish
became mutant alleles.

43National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellow:
Postdoctoral Fellow:

YASUGI, Masaki
NISHIUMI, Nozomi

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.

I. Psychophysical study of medaka fish
One of our major subjects is the psychophysical and computational study of medaka (Oryzias latipes, Matsunaga and
Watanabe, 2010). Recently, we made progress in studies ofprey-predator interaction using medaka and zooplankton.
Visual motion cues are one of the most important factors for
eliciting animal behavior, including predator-prey interactions in aquatic environments. To understand the elements ofmotion that cause such selective predation behavior, we useda 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 (Nakayasu et al., 2017). The
fine control of various features of living fish have beendifficult to achieve in studies of fish behavior. However,computer graphics allow us to manipulate morphological and44

motion cues systematically. Therefore, we have constructed 3D computer graphic animations of medaka based on
tracking coordinate data and photo data obtained from real
medaka. These virtual 3D models will allow us to represent
medaka faithfully and to undertake a more detailed analysisof the properties of the visual stimuli that are critical for the
induction of various behaviors. This experimental system
was applied to studies on dynamic seasonal changes in color
perception in medaka (Shimmura et al., 2017).

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 (predictive coding, Figure 1) explaining
the flash-lag effect (Watanabe et al., 2010). In recent years,
we have made more developed novel visual illusions, suchas the shelf-shadow illusion. This year, we began studying
deep neural networks (DNNs) that represented the perceived
rotational motion for illusion images that were not moving
physically, much like human visual perception. (Figure 1).
These models of DNNs will lead us in future work on perception science.

Figure 1. A schematic diagram of a predictive coding model of the brain.
Illustration of information flow within a single layer is shown. Verticalarrows represent connections with other layers. Each layer consists of“Representation” neurons, which output a layer-specific “Prediction” ateach time step, which is subtracted with “Target” to produce an error,
which is then propagated laterally and vertically in the network. External
data or a lower-layer error signal is input to “Target”. In each layer, theinput information is not processed directly, and the prediction error signal
is processed.

Publication List:〔Original papers〕
T., Yasugi, M., Shiraishi, S., Uchida, S., and Watanabe, E.
• Nakayasu,(2017). Three-dimensional computer graphic animations for studying•

social approach behaviour in medaka fish: Effects of systematic
manipulation of morphological and motion cues. PLoS ONE 12,
e0175059.
Shimmura, T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi,
M, Watanabe, E., Shimo, T., Senga, T., Nishimura, T., Tanaka, M.,
Kamei, Y., Naruse, K., and Yoshimura, T. (2017). Dynamic plasticity inphototransduction regulates seasonal changes in color perception. Nat.
Commun. 8, 412.

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

Associate Professor

HASEBE, Mitsuyasu

MURATA, Takashi

Assistant Professor:

TAMADA, Yosuke
ISHIKAWA, Masaki
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellow:
YOSHIDA, Saiko
Postdoctoral Fellow:
MANO, Hiroaki
AOYAMA, Tsuyoshi
YOSHIDA, Saiko*
NARUKAWA, Hideki
SOKENDAI Graduate Student: KAMIDA, Chiharu*
KOSHIMIZU, Shizuka
MORISHITA, Mio
PALFALVI, Gergo
SUDA, Hiraku
SUGAYA, Tomomi
ZHANG, Liechi
HORIUCHI, Yuta
DE VILLIERS, Ruan
Visiting Graduate Student: GU, Nan
SAKAZAKI, Takuya
Visiting Scientist:
KAMIDA, Chiharu
Technical Assistant:
AOKI, Etsuko
FUKADA, Hatsumi
GOTO, Misako
HIRAMATSU, Mika
KAJIKAWA, Ikumi
MATSUZAKI, Yoko
MASUOKA, Tomoko
MORI, Asuka
NISHI, TayoOOI, Shoko
WEN, Hsin-I
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, andnovel organ development. Such traits comprise many components and become adaptive only when all components aregathered together. However, based on evolutionary theory,
each component should evolve one by one according to theaccumulation of mutations. We aim to reveal the genetic
networks regulating these complex traits and to infer themechanisms needed to evolve complex characters.

II. S p a t i o t e m p o r a l re g u l a t i o n o f c e l ldivision axis as a grand plan of plantdevelopmental 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, and Dr.
Atsushi Mochizuki in RIKEN.

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 also established aminispindle system, which involves a bipolar microtubule
complex composed of an isolated chromosome and microtubules in tobacco cells. In collaboration with Dr. Daisuke
Tamaoki (Toyama Univ.), analyses of microtubule behavior
in the minispindle are in progress. Takashi Murata is a main
researcher of this study.

IV. Evolution of Regeneration: Generic
Regulatory Networks of Reprogrammingof Differentiated Cells to Stem Cells
Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. We found that themoss Physcomitrella patens Cold-Shock Domain Protein
1 (PpCSP1) regulates reprogramming of differentiatedleaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor
Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expressionof PpCSP1 is negatively regulated by its 3’-untranslated
region (3’-UTR). Removal of the 3’-UTR stabilizes PpCSP1
transcripts, results in accumulation of PpCSP1 protein, andenhances reprogramming. A quadruple deletion mutant of
PpCSP1 and three closely related PpCSP genes exhibits
attenuated reprogramming indicating that the PpCSP genes
function redundantly in cellular reprogramming. Taken
together, these data demonstrate a positive role of PpCSP1

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

in reprogramming, which is similar to the function of mammalian Lin28 (Li et al. 2017).
We also found that histone chaperone HIRA proteinsregulate stem cell formation through histone modification on SQUAMOSA PROMOTER BINDING PROTEIN(PpSBP) genes. Characterization of HIRAs and PpSBPs arein progress mainly by Yukiko Kabeya and Yosuke Tamada.

Figure 1. Cells facing the cut of dissected gametophore leaves werereprogrammed to be stem cells in wild type (left), while both edge andinner cells were reprogrammed with PpCSP1 accumulation (right).

V. Evolution of Regeneration: Master
Regulator for Reprogramming STEMIN
Animal somatic cells can be reprogrammed to iPS cells
by introducing four transcription factors, while such factors
have not been identified in plants. We found a gene encoding
a member of a plant-specific transcription factor, STEM
CELL-INDUCING FACTOR 1 (STEMIN1) that was able
to induce direct reprogramming of differentiated leaf cells
into chloronema apical stem cells without wounding signals.
To understand the role of STEMIN1 in reprogramming,
we investigate STEMIN1-direct target genes identified byRNA-seq and ChIP-seq analyses. Masaki Ishikawa and MioMorishita are this study’s main researchers.

VI. Evolution of Regeneration: Stem cells
laterally inhibit surrounding cells
Under certain circumstances differentiated cells can be
reprogrammed to form stem cells in land plants, but only aportion of the cells reprogram successfully. A long-distanceinhibitory signal from reprogrammed cells to surrounding
cells has been reported in some ferns. We showed the existence of an anisotropic inhibitory signal to regulate stem cell
formation in the moss Physcomitrella patens. When single
cells were isolated from a gametophore leaf, over 90% ofthem were reprogrammed to stem cells with characteristic
nuclear expansion. By contrast, when two adjacent cells wereisolated, the nuclei of both cells expanded, but successful
reprogramming of both cells occurred only in approximately
one fifth of the pairs. When three aligned cells were isolated,
the reprogramming rate of both edge cells decreased witha living middle cell but did not with a dead middle cell.
Furthermore, unequal conversion into stem cells was more
prominent in cell pairs aligned parallel to the proximal-distal
leaf axis than in those perpendicular to the axis. This study
gives an insight into the role of the inhibitory signal in development and evolution as well as efficient stem cell induction
from differentiated cells (Sato et al. 2017). Liechi Zhang isinvestigating the factors involved in the lateral inhibition.

46

VII. Evolution of Regeneration: Other
pathways
Nan Gu, a 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. Evolution of Molecular Mechanisms of
Plant Movement
The sensitive plant Mimosa pudica and the carnivorous
plants sundew (Drosera spatulata) and Venus fly trap
(Dionaea muscipula) have long attracted the interest ofresearchers due to their spectacular leaf movements in
response to touch or other external stimuli. Although various
aspects of the movement have been elucidated by physiological, cytological or biochemical approaches, the lackof genetic tools hampered the investigation of molecular mechanisms involved in these processes. To overcome
this obstacle, we sequenced and analyzed their genomes.
Furthermore, we developed an efficient genetic transformation method for these plants and established a CRISPR/
Cas9-mediated gene knock-out system in M. pudica. We
isolated several candidate genes that may play roles in theseismonastic movement of M. pudica by comparing gene
expression profiles between motor organs (pulvini) andnon-motor organs and between extensor and flexor halves ofpulvini. Functional analyses of these genes with the CRISPR/
Cas9 system are in progress. The studies on M. pudica andcarnivorous plants were conducted mainly by Hiroaki Manoand Hiraku Suda, respectively.

Figure 2. Reprogramming of isolated gametophore leaf cells of the moss
Physcomitrella patens. A single cell (a, b) and two adjacent cells (c, d)were isolated by hand using a carbon knife.

IX. Evolution of Plant Development

Publication List:

To investigate evolution of novel complex traits, the following studies are ongoing with graduate students: Chiharu
Kamida studies genes involved in movable tentacle development in the sundew Drosera spatulata. 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 isa flower-like inflorescence, the molecular mechanisms of thedevelopment of which are unknown. Tomomi Sugaya succeeded in transferring the FT gene from Arabidopsis thaliana
into the pseudanthium Houttuynia cordata. Furthermore,
introduction of the FT gene successfully induced flowers.
Ruan de Villiers investigates the evolution of signaling
pathways of the phytohormone strigolactone in land plants.

〔Original papers〕

X. Evolution of Digestive Enzymes andPitcher Leaves in Carnivorous Plants
Carnivorous plants exploit animals as a nutritional sourceand have inspired long-standing questions about the origin
and evolution of carnivory-related traits. To investigate themolecular bases of carnivory, we sequenced the genome ofthe heterophyllous pitcher plant Cephalotus follicularis, inwhich we succeeded in regulating the developmental switch
between carnivorous pitcher and non-carnivorous flat leaves
by using different temperatures. Transcriptome comparisonof the two leaf types and gene repertoire analysis identified
genetic changes associated with prey attraction, capture,digestion and nutrient absorption. Analysis of digestive
fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed
repeated co-options of stress-responsive protein lineages
coupled with convergent amino acid substitutions to acquire
digestive physiology. These results imply constraints on theavailable routes to evolve plant carnivory.
To further investigate the molecular mechanisms to regulate
flat and pitcher leaves, Gergo Palfalvi analyzes transcriptomes and chromatin modifications as a collaboration workwith Dr. Kenji Fukushima in Colorado University.

K., Fang, X., Alvarez-Ponce, D., Cai, H., Carretero-Paulet,• Fukushima,
L., Chen, C., Chang, T.-H., Farr, K.M., Fujita, T., Hiwatashi, Y., Hoshi,

•
•
•
•
•

Y., Imai, T., Kasahara, M., Librado, P., Mao, L., Mori, H., Nishiyama,T., Nozawa, M., Pálfalvi, G., Pollard, S.T., Rozas, J., Sánchez-Gracia,
A., Sankoff, D., Shibata, T.F., Shigenobu, S., Sumikawa, N., Uzawa, T.,
Xie, M., Zheng, C., Pollock, D.D., Albert, V.A., Li, S., and Hasebe, M.
(2017). Genome of pitcher plant Cephalotus reveals genetic changes
associated with carnivory. Nat. Ecol. Evol. 1, 0059.
Hattori, M., Tamada, Y., Murata, T., Oya, S., Hasebe, M, Hayano,Y., and Kamei, Y. (2017). Artificial testing targets with controllable blurfor adaptive optics microscopes. Opt. Eng. 56, 080502.
Kosetsu, K., Murata, T., Yamada, M., Nishina, M., Boruc, J., Hasebe,M., Damme, DV., and Goshima, G. (2017). Cytoplasmic MTOCs
control spindle orientation for asymmetric cell division in plants. Proc.
Natl. Acad. Sci. USA 114, E8847-E8854.
Li, C., Sako, Y., Imai, A., Nishiyama, T., Thompson, K., Kubo, M.,
Hiwatashi, Y., Kabeya, Y., Karlson, D., Wu, S.-H., Ishikawa, M.,
Murata, M., Benfey, P.N., Sato, Y., Tamada, Y., and Hasebe, M. (2017).
A Lin28 homolog reprograms differentiated cells to stem cells in themoss Physcomitrella patens. Nat. Commun. 8, 14242.
Sato, Y., Sugimoto, N., Hirai, T., Imai, A., Kubo, M., Hiwatashi,Y., Nishiyama, T., and Hasebe, M. (2017). Cells reprogramming
to stem cells inhibit the reprogramming of adjacent cells in themoss Physcomitrella patens. Sci. Rep. 7, 1909.
Yilamujiang, A., Zhu, A., Ligabue-Braun, R., Bartram, S., Witte,
C.P., Hedrich, R., Hasabe, M., Schöner, C.R., Schöner, M.G., Kerth,G., Carlini, C.R., and Mithöfer, A. (2017). Coprophagous features incarnivorous Nepenthes plants: a task for ureases. Sci. Rep. 7, 11647.

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

Figure 3. Cephalotus follicularis preferentially produces flat leaves at
15ºC (left) and pitcher leaves at 25ºC.

47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

Associate Professor

KAWAGUCHI, Masayoshi SOYANO, Takashi

its tandem repeat structure. RNA-seq analysis confirmed that
all rDNA variants are actively transcribed. Observed rDNA/
rRNA polymorphism may modulate translation by using
different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic
heterogeneity of AMF rDNA may facilitate adaptation to abroad host range despite lacking a sexual life cycle.

Assistant Professor:

TAKEDA, Naoya
FUJITA, Hironori
Technical Staff:
FUKADA-TANAKA, Sachiko
Visiting Scientist
NAKAGAWA, Tomomi
Postdoctoral Fellow:
FUJITA, Hironori*
KOBAYASHI, Yuuki
NAGAE, Miwa
MAEDA, TaroKAMEOKA, Hiromu
TOKUMOTO, Yuji
TSUGANE, Mika
MURAKAMI Eiichi
Research Fellow:
OKUMA, Nao*
SOKENDAI Graduate Student: NISHIDA, Hanna
FUKUHARA, Mai
LIU, Meng
OKUMA, Nao
Technical Assistant:
SUZAKI, Momoyo
OGAWA, Yuko
YOSHINORI, Yumi
TOKAIRIN, Asami
ODA, Akiko
Secretary:
KOSUGI, Eiko

Rhizobium–legume symbiosis is one of the most successful
mutually beneficial interactions on earth. In this symbiosis,
soil bacteria called rhizobia supply the host legumes withammonia produced through bacterial nitrogen fixation. In
return, host plants provide the rhizobia with their photosynthetic products. To accomplish this biotic interaction,
leguminous plants develop nodules on their roots. On theother hand, more than 80% of land plant families have symbiotic relationships with arbuscular mycorrhizal (AM) fungi.
Despite marked differences between the fungal and bacterial
symbioses, common genes are required for both interactions.
Using a model legume Lotus japonicus, we are trying tounveil the molecular mechanisms of both symbiotic systems.

I. AM symbiosis
1-1 Non-tandemly repeated heterogeneous rDNAs ofarbuscular mycorrhizal fungi.
Arbuscular mycorrhizal fungi (AMF) are one of themost widespread symbionts of land plants. Our substantially improved reference genome assembly of a model
AMF, Rhizophagus irregularis DAOM-181602 (total contigs= 210, contig N50 = 2.3Mbp, INSDC# = BDIQ01000001-BDIQ01000210) facilitated discovery of repetitive elementswith unusual characteristics. R. irregularis has only ten
to eleven copies of the complete 45S rDNA (Figure 1A),
whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly
heterogeneous and lack a tandem repeat structure (Figure
1A, B). These findings provide evidence for the hypothesisof concerted evolution that rDNA homogeneity depends on

Figure 1. Ribosomal DNA genes of Rhizophagus irregularis DAOM181602
A. Distribution of R. irregularis rDNA units in the genome. Each
48SrDNA unit is represented as a red box. For comparison, rDNA
clusters on Saccharomyces cerevisiae chromosome XII is shown. Inset
is a magnified view of a 48rDNA unit (c311-1) with nearby proteinencoding genes (purple boxes). Genes encoded on plus strand genome are
depicted on the top side, and those encoded on minus strand are shownon the bottom side. B. Alignment of a heterogeneous region among the
48SrDNA paralogs. Partial sequences of Mafft-aligned 48SrDNAs (corresponding 2,049-2,136 bp positions on c62-1).

II. Root nodule symbiosis
2-1 NITRATE UNRESPONSIVE SYMBIOSIS 1
mediates nitrate-induced control of root nodule
symbiosis
Symbiotic nitrogen fixation in root nodules containing
symbiotic rhizobia enables legumes to thrive under nitrogendeficient environments. However, the symbiosis is known tobe an energy consuming activity in which photosynthates areused as an energy source. Therefore, plants may cease thesymbiosis if there is a sufficient nitrogen source available intheir environment, thereby enabling plants to save the costassociated with nodulation.
Legumes control the number of nodules per root systemthrough a mechanism called autoregulation of nodulation(AON), a systemic long-range signaling between roots andshoots. In addition to their hypernodulating-phenotypes,
mutants involved in the AON retain nodule formation even

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

48

in the presence of a high nitrate concentration. Furthermore,production of CLE-RS2, a proposed root-derived signal in
AON, is induced not only by rhizobial infection but also
by nitrate application. These observations suggest that themechanism for nitrate-induced control of nodulation sharescommon elements with the AON. In contrast, some findings
suggest that fundamental knowledge of AON is insufficientto account for a regulatory mechanism, indicating that new
factors await discovery.
We identified a novel Lotus japonicus mutant, nitrate unresponsive symbiosis 1 (nrsym1) that formed mature nodules
even in nitrate-sufficient conditions. In wild type, a highconcentration of nitrate reduced infection thread number,
nodule number, nodule size and nitrogen fixation activity. In
contrast, these inhibitory effects were suppressed in nrsym1
mutants. These data indicate that NRSYM1 mediates pleiotropic nitrate-induced control of root nodule symbiosis.
In addition, analysis of the loss-of-function mutation of
CLE-RS2 and HAR1, encoding a receptor of CLE-RS2 in
AON, indicate that AON may be involved in the regulationof nitrate-induced inhibition of nodule number, but rhizobial infection, nodule growth, and nitrogen fixation activity

Figure 2. Model for the control of root nodule symbiosis in response
to nitrate. (A) Sequential progress of nodulation is shown. In response
to nitrate, NRSYM1 regulates pleiotropic phases of root nodule symbiosis, including rhizobial infection, nodule number, nodule growth,and nitrogen fixation activity. Whereas NRSYM1 activates the CLERS2>HAR1 signaling pathway leading to the negative regulation of
nodule number, NRSYM1 is likely to use different downstream targets
to achieve the regulation of other nitrate-affected processes. Red lines
and red cells respectively indicate the infection threads and rhizobia-colonized cells. (B) A model for cellular-level NRSYM1 function. NRSYM1
activated by a nitrate-dependent nuclear retention mechanism regulates
root nodule symbiosis by directly regulating nodulation-related genes
such as CLE-RS2.

are controlled through a mechanism independent of AON(Figure 2A). NRSYM1 encodes a NIN-LIKE PROTEIN transcription factor. The expression of CLE-RS2 was strongly
induced in wild type by nitrate treatment, but the induction levels were much lower in nrsym1 roots. Furthermore,
ChIP-qPCR analysis suggested that NRSYM1 can directlybind to the promoter regions of CLE-RS2 in a nitrate-dependent manner. The expression of NRSYM1 is not inducedby nitrate, suggesting that post-translational regulation of
NRSYM1 provides it with function. We then examined thesubcellular localization of NRSYM1 by immunohistochemistry. Although NRSYM1 was barely detected in nuclei innitrate-free conditions, the protein was predominantly localized in nuclei in the presence of nitrate. Our data indicatethat NRSYM1 is activated by a nitrate-dependent nuclear
retention mechanism and directly regulates the productionof CLE-RS2, thereby triggering the negative regulation of
nodule number through AON (Figure 1B).

2-2 Lotus japonicus ERN1 is essential for early processes of bacterial entry during nodulation
Bacterial entry into host tissues are essential processes toestablish the symbiotic interaction between rhizobia andhost legumes. Infection processes are initiated by adhesionof rhizobia on surfaces of root hairs and secretion of bacterial nodulation signaling molecules, Nod factors, whichcause tip deformation of root hairs. Rhizobia entrapped bycurling of the deformed root hair penetrate into inner tissue
layers through infection threads (ITs), host-membranous andtubular structures developed from infection foci. Nod factor
receptors and common symbiosis factors are necessary forsymbiotic root hair responses. However, nodulation specificfactors involved in rhizobial infection are largely unknown.
We conducted forward genetic screening to identify new
host factors involved in bacterial entry processes, andisolated two allelic mutant lines, F29 and 1699-1, that exhibit
defects in IT development, from M2 populations of EMStreated and ion beam-mutagenized L. japonicus MG-20
seeds, respectively. The causative gene encoded a member ofplant-specific APETALA2 (AP2) / ETHYLEN RESPONSE
FACTOR (ERF) family TFs, namely ERF REQUIREDFOR NODULARION1 (ERN1). Total numbers of ITs inern1 null mutants (1699-1) were less than 1% of wild type
plants. Even if ITs were developed on ern1 roots, they
were aberrantly short, and arrested in root hairs (Figure 3).
Consequently, ramified ITs that are usually observed in wild
type nodules were absent in ern1 nodules. Furthermore, significant numbers of ern1 root hairs were abnormally swollen,
and exhibited a balloon-like shape only when inoculated withrhizobia. These results suggested that ERN1 is not necessary
for Nod factor perception because root hairs were able torespond to inoculation, but is required for correct root hair
responses besides developmental processes of ITs.
ERN1 expression was upregulated within 12 hours after
inoculation. Spatial expression pattern analysis usinga ß-glucuronidase reporter gene showed that ERN1 was
expressed in infected root hairs and in developing nodules.
The expression pattern was consistent with the role of ERN1
in early processes of nodulation after Nod factor perception.

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

This expression in response to rhizobial infection required
CYCLOPS, a TF acting in the common symbiosis pathway,
suggesting that ERN1 is a factor downstream of the common
symbiosis pathway. Phylogenetic analysis showed that ERN1
is conserved in leguminous and non-leguminous plants.
ERN1 may contribute to the bacterial entry by recruiting its
downstream genes required for physiological and cellular
events widely conserved in plants.

into the theoretical and molecular basis for understanding
the phyllotaxis pattern. Our theoretical analysis strongly
predicts a diffusible molecule that is pivotal for the phyllotaxis pattern but is yet to be determined experimentally.

A
C

D

E

B

Figure 4. Examples of computer simulations in two-dimensional space.
The regular spatial pattern of auxin maxima can be generated under simplified conditions in the absence of the extracellular region (A). However,this spatial regularity is completely disrupted by introducing the extracellular region (B), but can be restored by assuming a diffusible moleculethat mutually interacts with auxin–PIN1 dynamics (C).

Publication List:〔Original papers〕
Figure 3. Root nodules formed in L. japonicus wild type (A) and ern1
(1699-1) mutant roots (B). Root hair phenotypes of wild type (C), ern1-5(F29; D), and ern1-6 (1699-1; E) mutants after inoculation with DsRedlabeled Mesorhizobium loti. Merged images of DsRed fluorescence andbright-field (C, D). Scale bars; 0.5 mm in (A, B), 50 μm in (C-E).

III. Spatial regularity control of phyllotaxis
pattern generated by the mutualinteraction between auxin and PIN1
Phyllotaxis, the arrangement of leaves on a plant stem, iswell known because of its beautiful geometric configuration, which is derived from the constant spacing between leaf
primordia. Phyllotaxis patterns are established by the mutualinteraction between a diffusible plant hormone auxin and its
efflux carrier PIN1, which cooperatively generate a regularpattern of auxin maxima, small regions with high auxin concentrations, leading to leaf primordia. However, the molecular mechanism of auxin maxima patterning is still largely
unknown. To better understand how the phyllotaxis patternis controlled, we investigated mathematical models based on
the auxin–PIN1 interaction through linear stability analysis
and numerical simulations, focusing on the spatial regularity
control of auxin maxima.
As in previous reports, we first confirmed that this spatial
regularity can be reproduced by a highly simplified andabstract model. However, this model lacks the extracellularregion and is not appropriate for considering the molecular mechanism. Thus, we investigated how auxin maxima
patterns are affected under more realistic conditions. We
found that the spatial regularity is eliminated by introducing
the extracellular region, even in the presence of direct diffusion between cells or between extracellular spaces, and thisstrongly suggests the existence of an unknown molecularmechanism. To unravel this mechanism, we assumed a diffusible molecule to verify various feedback interactions with
auxin–PIN1 dynamics. We revealed that regular patterns canbe restored by a diffusible molecule that mediates the signaling from auxin to PIN1 polarization. Furthermore, as in the
one-dimensional case, similar results are observed in thetwo-dimensional space. These results provide a great insight50

G., Chabaud, M., Miyata, K., Capozzi, M., Takeda, N.,• Carotenuto,
Kaku, H., Shibuya, N., Nakagawa, T., Barker, D., and Genre, A.

•
•
•
•

(2017). The rice LysM receptor-like kinase OsCERK1 is required forthe perception of short-chain chitin oligomers in arbuscular mycorrhizal
signaling. New Phytologist 214, 1440-1446.
Ohtsu, M., Sato, Y., Kurihara, D., Suzaki, T., Kawaguchi, M., Maruyama,D., and Higashiyama, T. (2017). Spatiotemporal deep imaging ofsyncytium induced by the soybean cyst nematode Heterodera glycines.
Protoplasma 254, 2107-2115.
Ohtsu, M., Kurihara, D., Sato, Y., Suzaki, T., Kawaguchi, M.,
Maruyama, D., and Higashiyama, T. (2017). Fluorescent labeling of thecyst nematode Heterodera glycines for deep tissue live imaging using
two-photon microscopy. Cytologia 82, 251-259.
Yano, K., Aoki, S., Liu, M., Umehara, Y., Suganuma, N., Iwasaki, W.,
Sato, S., Soyano, T., Kouchi, H., and Kawaguchi, M. (2017). Functionand evolution of a Lotus japonicus AP2/ERF family transcription factor
that is required for development of infection threads. DNA Res. 24,
193-203.
Maeda, T., Kobayashi, Y., Kameoka, H., Okuma, N., Takeda, N.,
Yamaguchi, K., Bino, T., Shigenobu, S., and Kawaguchi, M. (2017)
Evidence of non-tandemly repeated rDNAs and their intragenomic
heterogeneity in Rhizophagus irregularis. bioRxiv preprint first posted
online Oct. 19, doi: https://doi.org/10.1101/205864.

〔Original paper (E-publication ahead of print)〕
H., Shimoda, Y., Hakoyama, T., Sato, S., Kaneko, T.,• Yamaya-Ito,
Hossain, MS., Shibata, S., Kawaguchi, M., Hayashi, M., Kouchi,
H., and Umehara, Y. Loss-of-function of ASPARTIC PEPTIDASENODULE-INDUCED 1 (APN1) in Lotus japonicus restricts efficient
nitrogen-fixing symbiosis with specific Mesorhizobium loti strains.
Plant J. 2017 Dec 2.

DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:

OHDE, Takahiro
ANDO, Toshiya
Technical Staff:
MIZUTANI, Takeshi
NIBB Research Fellow:
MORITA, Shinichi
Postdoctoral Fellow:
SAKAI, Hiroki
KAWAGUCHI, Haruka
SOKENDAI Graduate Student: CHIKAMI, Yasuhiko
Visiting Graduate Student: MASE, Mutsuki
YATOMI, Johichiro
Technical Assistant:
KAWAGUCHI, Haruka*
MORITA, Junko
Secretary:
SAITO, Eiko
KIYOHARA, Megumi

The Division of Evolutionary Developmental Biology wasstarted in June 2015. We focus on the evolutionary novelties
acquired by insects through evolution, in order to elucidate
the molecular and evolutionary mechanisms leading to thelarge variety of traits that they display. From this wealthof exciting traits, our lab currently focuses on promoting
research into (1) the origin and diversification of insectwings, (2) wing color patterns and mimicry of ladybird
beetles, and (3) acquisition and diversification of beetle
horns.

I. Origin and diversification of insect wings
Of the various flying animals on the earth, insects have
evolved a unique flight organ. Despite over two centuries
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
in ancestral 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 forfunctional effects of altered transcription of each of thesewing genes in the ancestrally wingless firebrats. In addition,
we are performing comparative analyses of the function ofthese same genes in “primitively winged” (hemimetabolous)
insects, to obtain additional clues relevant to understanding
the origin and evolution of insect wings.
Interestingly, our previous work showed that vg expressing epidermal tissue forms lateral outgrowths in non-winged
segments in a 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
Insects have evolved a tremendous range of diversity ofwing color patterns which play various ecologically important roles such as intraspecific sexual signaling, mimesis,
mimicry, and warning against predators. The molecularmechanisms responsible for generating such color patterns
in most ladybird species, however, remain elusive. To investigate the developmental mechanisms of color pattern formation, we have been focusing on the multicolored Asianladybird beetle, Harmonia axyridis, which has conspicuousand variable wing color patterns consisting of black and red
pigments (Figure 2A). Vivid wing color patterns of ladybirds
function as a warning signal to convey to predators thatthey are distasteful. At the same time, various other insect
species are utilizing this ecological signal by mimicking theladybirds’ wing color patterns. Mimicry provides an exciting
opportunity to study how independent lineages of insects
have evolved convergent color patterns. To explore color
pattern formation mechanisms in mimicry, we are focusing
on the leaf beetle, Argopistes coccinelliformis, which hascolor patterns similar to Harmonia, and which is thought
to be a Batesian mimicry of ladybird beetles (Figure 2B).
To elucidate the molecular mechanisms underlying thesewing color patterns, we established a technique for germline

A

B

B
A

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).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

transformation using a piggyBac vector and RNAi in the
ladybirds.
Recently, we identified several key regulatory genes associated with the color pattern formation of ladybird beetles
using next generation sequencing technologies (RNA-seq
and de novo genome assembly), and an RNAi-based screening method that we established. Many of these genes are
expressed in specific domains of wing color patterns, suggesting that regulatory shift, such as change in enhancer
activity, at these gene loci may be crucial for evolution ofwing color patterns in ladybirds. Now, we are trying to elucidate the evolutionary origin of color patterns in ladybirds
focusing on regulatory shifts at these genetic loci. To tackle
this issue, we are establishing genome-editing technologies
using TALEN and CRISPR/Cas9, and have already established an efficient method of gene disruption. At present,
we are establishing more complicated genome editing techniques such as genomic insertion, inversion and duplication
to identify the crucial regulatory shift that may have driven
evolution of wing color patterns in ladybird beetles.
In the future, we are planning to analyze how the similar
wing color patterns of model and mimic are generated basedon the knowledge obtained from H. axyridis, – 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 best

A

B

C

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).

52

models 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 (RNA-seq). We narrowed down thegenes associated with horn formation to 40 genes, and performed RNAi-based knockdown screening to provide deep
insights into where, when, and how the head and thoracic
horns are formed during development. We successfully
identified 11 transcription factors that contribute to horn formation. These 11 genes include larval head- and appendagepatterning genes, which are involved in Onthophagus hornformation, suggesting the early redeployment of this subset
of genes during the scarab horn evolution.
To understand how, molecularly, beetle horns have diversified, we are extending our analyses to include additional
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).

Publication List:〔Original paper〕
K., Gotoh, H., Tajika, Y., Sushida, T., Aonuma, H., Niimi, T.,• Matsuda,
Akiyama, M., Inoue, Y., and Kondo, S. (2017). Complex furrows in a
2D epithelial sheet code the 3D structure of a beetle horn. Sci. Rep.,
7, 13939.

LABORATORY OF BIORESOURCES

Specially Appointed Professor

NARUSE, Kiyoshi

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

Secretary:

TAKEHANA, Yusuke
ANSAI, Satoshi
YOKOI, Saori
KAJIURA-KOBAYASHI,
HirokoKANEKO, 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 theevolution of the sex determination system using medaka andrelatives, adaptive significance of mate-guarding behavior,
The molecular genetic basis of diversified sexually dimorphic traits in Oryzias species and the identification of causal
genes for pigment cell mutants. In addition to these activities, our laboratory has stepped forward to lead the National
BioResource Project Medaka (NBRP Medaka) from 2007.

I. Evolution of the sex chromosome and sexdetermining genes in Oryzias fish
Sex chromosomes harbor a primary sex-determining
signal that 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
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 chromosomesin the marine medaka Oryzias dancena/melastgma by positional cloning. Sex reversed phenotypes in transgenic fish
and loss-of-function mutants of the Y chromosomal Sox3
allele all point to its critical role in sex determination, suggesting that the neo-Y chromosome of O. dancena arose byco-option of Sox3. Furthermore, we found the Sox3 genealso 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
sex-determining 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. Identification of the causal gene of the
medaka iridophore mutant, guanineless(gu)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.
Herein, we describe the iridophore mutant gu, which exhibitsa strong reduction in visible iridophores throughout its larval
to 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. We
sought the causal gene of gu using synteny analysis withthe zebrafish genome and found a strong candidate, purine
nucleoside phosphorylase 4a. Gene targeting and a complementation test showed that pnp4a is the 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 ofeach strain.

III. Adaptive significance of persistent mateguarding behavior in medaka

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

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.,53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity2015) 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 a 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 a closer proximity to the female by the far male and did
not block familiarization of the far male with 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 theevolutional origin of “pair-bonding”.

technologies are underway to elucidate the molecular anddevelopmental mechanisms underlying the red fin traits.

Publication List:〔Original papers〕
S., Hosokawa, H., Maegawa, S., Naruse, K., Washio, Y., Sato,• Ansai,K., and Kinoshita, M. (2017). Deficiency of serotonin in raphe neurons

•
•
•
•
•
•
•

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

IV. The molecular genetic basis of diversified
sexually dimorphic traits in Oryzias species
endemic to Sulawesi, Indonesia
Sexual dimorphism is prevalent, but often differs remarkably between closely related species. However, we know
little about which genetic changes can actually contribute to diversification of sexually dimorphic traits between
closely related species. We therefore started to investigate
the molecular genetic mechanisms underlying the diversification of sexually dimorphic body colorations in medaka(genus Oryzias) from Sulawesi, Indonesia. As a first step,
we focused on an endemic species Oryzias woworae, whose
males show red in the pectoral fins and blue in the lateralbody. Quantitative trait loci (QTL) mapping in a F2 intercrossbetween a male of O. woworae and a female of O. celebensis, which is a close relative without such sexually dimorphic
colorations, revealed that an autosomal locus controls red
pigmentation in the pectoral fins. Subsequent gene expression analysis showed that upregulation of a gene on the QTL
by the cis-regulatory mutation(s) would be responsible forthe red fins. Further analysis with targeted genome editing

54

and altered behavioral responses in tryptophan hydroxylase 2-knockout
medaka (Oryzias latipes). Zebrafish 14, 495-507.
Inoue, Y., Saga, T., Aikawa, T., Kumagai, M., Shimada, A., Kawaguchi,
Y., Naruse, K., Morishita, S., Koga, A., and Takeda, H. (2017).
Complete fusion of a transposon and herpesvirus created the Teratorn
mobile element in medaka fish. Nat. Commun. 8, 551.
Kimura, T., Takehana, Y., and Naruse, K. (2017). Pnp4a is the causal
gene of the medaka iridophore mutant guanineless. G3-Genes Genomes
Genet. 7, 1357-1363.
Murakami, Y., Ansai, S., Yonemura, A., and Kinoshita, M. (2017). An
efficient system for homology-dependent targeted gene integration in
medaka (Oryzias latipes). Zool. Letters 3, 10.
Sagai, T., Amano, T., Maeno, A., Kimura, T., Nakamoto, M., Takehana,Y., Naruse, K., Okada, N., Kiyonari, H., and Shiroishi, T. (2017).
Evolution of Shh endoderm enhancers during morphological transition
from ventral lungs to dorsal gas bladder. Nat. Commun. 8, 14300.
Sasado, T., Kondoh, H., Furutani-Seiki, M., and Naruse, K. (2017).
Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity
Factor Subunit 6) causes short 3’UTRs and disturbs gene expression indeveloping embryos, as revealed by an analysis of primordial germ cell
migration using the medaka mutant naruto. PLoS ONE 12, e0172467.
Seki, S., Kusano, K., Lee, S., Iwasaki, Y., Yagisawa, M., Ishida, M.,
Hiratsuka, T., Sasado, T., Naruse, K., and Yoshizaki, G. (2017).
Production of the medaka derived from vitrified whole testes by germ
cell transplantation. Sci. Rep. 7, 43185.
Shimmura, T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi,M., Watanabe, E., Shimo, T., Senga, T., Nishimura, T., and Tanaka,
M. (2017). Dynamic plasticity in phototransduction regulates seasonalchanges in color perception. Nat. Commun. 8, 412.

〔Original paper (E-publication ahead of print)〕
M., Shibata, Y., Ohno, K.. Usami, T., Kamei, Y., Taniguchi,• Nakamoto,Y., Todo, T., Sakamoto, T., Young, G., Swanson, P., Naruse, K., andNagahama, Y. Ovarian aromatase loss-of-function mutant medaka
undergo ovary degeneration and partial female-to-male sex reversal
after puberty. Mol. Cell Endocrinol. 2017 July 13.

〔Review article〕
T., Yokoi, S., and Takeuchi, H. (2017). Molecular basis of• Okuyama,social competence in medaka fish. Develop. Growth Differ. 59, 211–
218.

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 of
Phthorimaea operculella revealed that they have a novel
branching morphology and the branches were ubiquitously
and densely distributed within the scale array to form amesh-like architecture similar to a nonwoven fabric. The
marginal scales maintain a coherent sheet-like structureduring wingbeat (Figure 1).

Figure 1. Scanning electron microscopy of the wing margin of P. operculella. Long marginal scales show branching morphology.

Publication List:〔Original paper〕
A., Tejima, S., Sakuma, M., Sakamaki, Y., and Kodama, R.
• Yoshida,(2017). Coherent array of branched filamentary scales along the wing
margin of a small moth. Sci. Nat. 104, 27

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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

MANO, Shoji
MANO, Shoji*
KANAI, Masatake
KAMIGAKI, Akane
HIKINO, Kazumi
KATO, Kyoko
UEDA, Chizuru

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

I. 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 involved in various biological processes such as lipid
metabolism and photorespiration. To understand peroxisome
dynamics and functions, we have been analyzing a numberof Arabidopsis mutants having aberrant peroxisome morphology (apem mutants) and peroxisome unusual poisoning(peup mutants). Based on the analyses using these mutants apart of the mechanism of division, protein transport, degradation of peroxisomes, and the interactions of peroxisomeswith other organelles were revealed (Figure 1). In addition,
we found that peroxisomes are involved in the reproductive
process. Therefore, peroxisome dynamics in gametes andgametophytes are currently under investigation.

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

III. Construction of The Plant Organelles
Database 3 (PODB3)
PODB3 was built to promote a comprehensive understanding of organelle dynamics. PODB3 consists of six
individual units: the electron micrograph database, the perceptive organelles database, the organelles movie database,the organellome database, the functional analysis database,and external links. Through these databases, users can obtain
information on plant organelle responses to environmental
stimuli of various tissues of several plant species, at different
developmental stages. We expect that PODB3 will enhance
the understanding of plant organelles among researchers.

Publication List:〔Original papers〕
M., Tanaka, Y., Nishimura, K., Mano, S., Kimura, T.,• Aboulela,and Nakagawa, T. (2017). A dual-site gateway cloning system for

•
•
•
•

simultaneous cloning of two genes for plant transformation. Plasmid
92, 1-11.
Aboulela, M., Tanaka, Y., Nishimura, K., Mano, S., Nishimura, M.,
Ishiguro, S., Kimura, T., and Nakagawa, T. (2017). Development ofan R4 dual-site (R4DS) gateway cloning system enabling the efficientsimultaneous cloning of two desired sets of promoters and open reading
frames in a binary vector for plant research. PLoS ONE 12, e0177889.
Hayashi, M., Tanaka, M. Yamamoto, S., Nakagawa, T., Kanai, M.,
Anegawa, A., Ohnishi, M., Mimura, T., and Nishimura, M. (2017).
Plastidial folate prevents starch biosynthesis triggered by sugar influx
into non-photosynthetic plastids of Arabidopsis. Plant Cell Physiol. 58,
1328-1338.
Kanai, M., Mano, S., and Nishimura, M. (2017). An efficient method forthe isolation of highly purified RNA from seeds for use in quantitativetranscriptome analysis. J. Vis. Exp. 119, e55008.
Watanabe, E., Mano, S., Yamada, K., Nishimura, M., Iuchi, S.,
Kobayashi, M., Uemura, M., and Kawamura, Y. (2017). Physiologicalanalysis of Arabidopsis ecotype to investigate the freezing tolerance
after cold acclimation process. Cryobiol. Cryotechnol. 63, 161-164.

〔Review article〕

Figure 1. Physical interaction of peroxisomes and oil bodies. (A, B)
Fluorescence observation of 3-day-old cotyledons of Arabidopsis wildtype (A) and sdp1 (sucrose dependent 1) plants (B). Magenta and green
signals represent peroxisomes and oil bodies, respectively. Bar: 20
μm. (C) Model of the interaction of peroxisomes and oil bodies. PED3
(Peroxisome Defective 3) is the potential anchor protein on peroxisomal
membranes. Sucrose acts as a negative signal for the interaction between
both organelles.

E., Mano, S., Hara-Nishimura, I., Nishimura, M., and• Watanabe,
Yamada, K. (2017). HSP90 stabilizes auxin receptor TIR1 and ensuresplasticity of auxin responses. Plant Signal. Behav. 12, e1311439.

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

56

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).
Figure 1. Tor signaling pathway of the budding yeast. Ourgroup has found three branches of the Tor pathway.

desensitizes TORC1 inactivation by nitrogen starvation in
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.

II. TO R C 1 p h o s p h o r y l a t e s A t g 1 3 , t h emolecular switch of autophagy
TORC1 negatively regulates autophagy, a protein degradation system induced by nutrient starvation.
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.

III. TORC1 regulates mitotic entry via pololike kinase
TORC1 regulates protein synthesis, which is important for
promotion of the cell cycle at the G1 phase.
We demonstrated that TORC1 is also involved in anotherstage of the cell cycle, mitotic entry. 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
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

TORC2 has an essential function controlling polarity of
the actin 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 protein of the TORC2 pathway.

Publication List:〔Original paper〕
Y. (2017). Novel tRNA function in amino acid sensing of• Kamada,
yeast Tor complex1. Genes Cells 22,135-147.

57

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.

cubifrin, an IWMGY-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 and WGS
data of the NCBI database. Especially relaxin like peptide
precursor genes and insulin/IGF like peptide precursor genes
were identified from many species. We are producing theseneuropeptides by biological methods, e.g. bacterial systems
and yeast systems, for providing to collaborators for biological assays.

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).

II. Search for reproductive hormones inechinoderms
In a collaborative effort with Prof. Yoshikuni’s Laboratoryof Kyushu Univ., we are searching for reproductive
hormones in echinoderms; starfishes, brittle stars, sea
urchins, sea cucumbers, and crinoids. The collaborators havebeen able to purify physiological materials which induce
egg maturation from nerve extracts and analyze them witha protein sequencer and a tandem mass spectrometer in
the analytical center of our institute. One of them, named

58

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.

Publication List:〔Original paper (E-publication ahead of print)〕
M., Shibata, Y., Ohno, K.. Usami, T., Kamei, Y., Taniguchi,• Nakamoto,Y., Todo, T., Sakamoto, T., Young, G., Swanson, P., Naruse, K., andNagahama, Y. Ovarian aromatase loss-of-function mutant medaka
undergo ovary degeneration and partial female-to-male sex reversal
after puberty. Mol. Cell Endocrinol. 2017 July 13.

LABORATORY OF BIOLOGICAL DIVERSITYKOMINE 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. ZFHX2 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 function of
ZFHX2 in the nervous system. The homozygous Zfhx2 deficient mice showed several behavioral abnormalities, namely,
hyperactivity, enhanced depression-like behaviors, and an
aberrantly altered anxiety-like phenotype. These behavioral
phenotypes suggest that ZFHX2 might play roles in controlling emotional aspects through the function of monoaminergic neurons where ZFHX2 is expressed.

III. ZFHX2 works also in pain perception
process in human and mice
Recently, Cox’s group in University College London foundthat a point mutation in the human ZFHX2 gene segregateswith the pain insensitivity found in a family with an inherited pain insensitive phenotype. Through the collaboration ofseveral groups, including us, it has been shown that ZFHX2
works as a critical gene for pain perception in humans andmice. Further work will resolve how the mutated ZFHX2
gene contributes to the hypoalgesic phenotype and may help
development of new analgesic drugs.

Publication List:〔Original paper (E-publication ahead of print)〕
A.M., Matsuyama, A., Okorokov, A.L., Santana, S., Bras, J.T.,• Habib,
Aloisi, A.M., Emery, E.C., Bogdanov, Y., Follenfant, M., Gossage, S.J.,
Gras, M., Humphrey, J., Kolesnikov, A., Le Cann, K., Li, S., Minett,
M., Pereira, V., Ponsolles, C., Sikandar, S., Torres, J.M., Yamaoka, K.,
Zhao, J., Komine, Y., Yamamori, T., Maniatis, N., Panov, K.I., Houlden,
H., Ramirez, J.D., Bennett, D.L.H., Marsili, L., Bachiocco, V., Wood,
J.N., and Cox, J.J. A novel human pain insensitivity disorder caused bya point mutation in ZFHX2. Brain 2017 Dec 14.

Figure 1. Expression of Zfhx2
sense RNA (mRNA) andantisense RNA in the embryonic mouse brain. The antisense RNA was expressed
where mRNA was not.

59

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.

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(Figure 1). We are currently characterizing detailed molecular mechanisms of these mutations.

III. Genome sequence information of theJapanese morning glory
In 2016, we 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
are anchored to 15 pseudo-chromosomes. A genome databasefor the genome sequence was built and opened to the
public in 2017 (Figure 2). It includes a genome browserthat enables users to analyze the 3,416 scaffolds (assembly
name, Asagao_1.1), the 15 pseudo-chromosomes with the
3,095 scaffolds not anchored to the pseudo-chromosomes(Asagao_1.2), and the circular genomes of chloroplasts andmitochondria. The database provides BLAST, BLAT and
keyword search services.
The genome sequence was also archived and used byseveral online databases and tools. The National Center for
Biotechnology Information (NCBI) provides their own gene
predictions (NCBI Ipomoea nil Annotation Release 100). The
predicted genes are catalogued by the Kyoto Encyclopedia of
Genes and Genomes (KEGG), and their functional information is visualized in the PATHWAY database that contains
graphical representation of cellular processes (e.g. metabolism and signal transduction). Database Center for Life
Science (DBCLS) equipped GGGenome and CRISPRdirect
with the genome sequence. These tools facilitate ultrafast
sequence search and rational design of CRISPR/Cas based
genome editing target, respectively.

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. The end
sequences of the DNA clones can be viewed from the I. nil
genome database (Figure 2).

Figure 1. Flower phenotypes of the duskish mutant of I. nil. The phenotype is variable and displays variegated flowers (a), fully pigmented
flowers (b), and pale grayish-purple flowers (c). As the flowers havethe same genotype, epigenetic mechanisms are involved in the variablephenotypes.

II. Membrane transport of flower pigments
Anthocyanins are the most common flower pigments in
Angiosperms including I. nil. They are synthesized in thecytosols and accumulate in the central vacuole in plant cells.
Anthocyanin transport across the vacuolar membrane haslong been debated. The transcriptional regulatory network ofanthocyanin pigmentation supports involvement of an ATPbinding caste (ABC) protein in the anthocyanin transport inI. nil. We started an international collaboration with researches in the Netherlands and Switzerland to reveal the functionof the ABC protein.

60

Figure 2. The I. nil genome database.
http://viewer.shigen.info/asagao/index.php

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.). The
genome of all plants were isolated for identifying insertion
sites of nDart1.

II. Semidominant mutation in rice
The semidominant mutations produce the intermediate
phenotype in individuals heterozygous for the gene concerned. The semidominant mutations were occasionallyisolated from the MK-1, it was unclear what causes dominant
mutations. Efficient selection and analysis of dominantmutants to analyze the gene functions in rice is very useful.
Newly isolated, Bushy dwarf tiller2 (Bdt2), which has thevaluable agronomic traits of multiple tillering and dwarfism,
was obtained from the MK-1 (Figure 2). 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 effectof Bdt2a. A wild plant produces only 10 spikes or less, while
Bdt2a produced 3 times as many, Bdt2 produced 10 times theears.

I. Dominant mutation in rice
Seed size and number were controlled by various genes in
the plants. It was reported that expression changes of highcontribution genes for seed size, number and panicle shaperesulted in decrease of total yield. A strategy for boosting rice
yield based on molecular biology is to stack the finely tuned
gene expressions. The Lgg mutant bore slightly large grains(Figure 1) as a dominant inheritance. Transposon-display
identified the insertion site of the Lgg mutant.

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.

Figure 1. Phenotype of Large gain (Lgg). Harvested panicle and seeds.

61

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

62

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.

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.

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.

III. Software 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.

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.

II. Local image feature tracking for tissue
deformation analysis
Besides cell boundary extraction, we also developed a63

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYKIMORI GroupSpecially Appointed Assistant Professor:
KIMORI, Yoshitaka
Technical Assistant:
HYODO, Miwa

and shape of the target object that is to be enhanced.
This method was applied to enhancement of an abnormal
region in the chest radiograph shown in Fig. 1. The arrow
in Fig. 1(a) shows the position of the abnormality (lungnodule). Fig. 1(b) shows the image after contrast enhancement using the proposed method. From the result, the nodule
region was clearly distinguished from the surrounding
tissues.

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.

Novel contrast enhancement method basedon mathematical morphology for medical
diagnosis
Image processing is a crucial step in the analysis of medical
imaging data. As such, it is fundamental to a wide range of
biomedical imaging and clinical research fields. Image processing derives structural features, which are then numerically quantified by image analysis. Especially, in diagnostic
imaging, contrast enhancement of structural details of lesion
regions plays an important role. It improves image quality
and supports clinician diagnosis. By using the contrast
enhancement method, more accurate medical diagnosis canbe expected. However, conventional image enhancement
techniques also emphasize the noise and structure of various
normal tissues other than lesion regions.
In this study, a contrast enhancement approach based ona new type of mathematical morphology is introduced. Thismethod emphasizes only the structure of the lesion while
suppressing the emphasis of noise and normal anatomical
structure.
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 canbe derived. However, since the size of lesions vary, in manycases it is not possible to process with fixed-size SE.
This proposed method is based on aࠉmorphological subtraction method. In this new type of morphological enhancement method, h-maxima transform is applied to the original
medical image. The unwanted structures that surround thetarget are suppressed in the process of target enhancement.
Furthermore, this new method has no restrictions on the size

64

Figure 1. Contrast enhancement result for chest x-ray image: (a) Original
chest radiograph. (b) Contrast enhanced image by using the proposed
method. The image was obtained from a standard digital image databaseof chest lung nodules and non-nodules (Shiraishi et al., ARJ Am. J.
Roentgenol. 174, 2000).

Publication List:〔Original paper〕
Y., Shimizu, T., Mori, T., Yoshimura, Y., Hatanaka, N., Nambu,• Osanai,A., Kimori, Y., Koyama, S., Kobayashi, K., and Ikenaka, K. (2017).
Rabies virus-mediated oligodendrocyte labeling reveals a single
oligodendrocyte myelinates axons from distinct brain regions. Glia 65,
93-105.

National Institute for Basic Biology

Environmental Biology

DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

Associate Professor

MINAGAWA, Jun

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
BURTON-SMITH, Raymond
SATO, Ryoichi
SOKENDAI Graduate Student: KAMRANI, Yousef Yari
KATO, Hiroki
KOSUGE, Kotaro
WATANABE, Akimasa
OKAJIMA, Keisuke
KISHIMOTO, Mariko
YANAKA, Ayako
Visiting Scientist:
TAKIZAWA, Kenji
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
UENO, Yasuko
YOKOYAMA, Michiko
KIDA, Emi
Secretary:
KOJIMA, Yoko
TOYAMA, Mami

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 symbiotic
dinoflagellate, Symbiodinium, that associates with corals andsea anemones, 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 therelative 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 and the npq4 mutant, which is qE-deficient andlacks the ancient light-harvesting protein LHCSR. LHCSR3
was present in the PSII-LHCII supercomplex from the highlight-grown WT (Fig. 1) but not in the supercomplex from

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.

the low light-grown WT or the npq4 mutant. The purified
PSII-LHCII supercomplex containing LHCSR3 showeda normal fluorescence lifetime at a neutral pH (7.5) bysingle-photon counting analysis but exhibited a significantly shorter lifetime (energy-quenching) at pH 5.5, whichmimics the acidified lumen of the thylakoid membranes inhigh light-exposed chloroplasts. The switching from lightharvesting mode to energy-dissipating mode observed in
the LHCSR3-containing PSII-LHCII supercomplex was
inhibited by DCCD, a protein-modifying agent specific toprotonatable amino acid residues. We conclude that the PSIILHCII-LHCSR3 supercomplex formed in high light-grown
C. reinhardtii cells is capable of energy dissipation upon
protonation of LHCSR3. However, its molecular mechanism
remains unclear.
To investigate how LHCSR3 dissipates excitation energy,
thereby protecting the PSII supercomplex, we applied
fluorescence decay–associated spectra (FDAS) analysis
to a purified PSII–LHCII supercomplex with and without
LHCSR3 from Chlamydomonas reinhardtii. We found that,when the PSII supercomplex is associated with LHCSR3
under high-light conditions, excitation energy transferfrom light-harvesting complexes to chlorophyll binding
protein CP43 is selectively inhibited compared with thatto CP47, preventing excess excitation energy from overloading the reaction center. By analyzing femtosecond upconversion fluorescence kinetics, we further found that pHand LHCSR3-dependent quenching of the PSII—LHCII–LHCSR3 supercomplex is accompanied by a fluorescenceemission centered at 684 nm, with a decay time constant of
18.6 ps, which is equivalent to the rise time constant of thelutein radical cation generated within a chlorophyll–lutein
heterodimer. These results suggest a mechanism in which
LHCSR3 transforms the PSII supercomplex into an energydissipative state and provide critical insight into the molecular events and characteristics in LHCSR3-dependent energy
quenching (Fig. 2).

65

National Institute for Basic Biology

Environmental Biology

species 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 2. A proposed model of LHCSR3-dependent NPQ in PSII—LHCII–LHCSR3 supercomplexes of C. reinhardtii. LHCSR3 isexpressed and associated with the PSII supercomplex under high-light
conditions. The circles of orange dots represent the presumable bindingsite of LHCSR3. The binding of LHCSR3 to the PSII supercomplex
inhibits the excitation energy transfer to CP43. At acidic pH, LHCSR3dependent NPQ is activated, and it quenches excitation energy of LHCs.
NPQ values of PSII–LHCII and PSII—LHCII–LHCSR3 were calculated
from the difference between pH 7.5 and 5.5 conditions. Kim et al. (2017)
J. Biol. Chem. 17, 18951-18960.

II. Species specificity in coral-algae symbiosis
Reef-building corals harbor endosymbiotic dinoflagellates of the genus Symbiodinium and rely on the energythat the algae generate from photosynthesis for their growthand survival. Symbiodinium are genetically diverse andtheir physiological characteristics (e.g., stress sensitivity)
differ among phylotypes. Therefore, corals need to recruit
Symbiodinium phylotypes that suit the environment in orderto survive and adapt to changes (e.g., global change andwarming). Interestingly, each coral species associate onlywith specific Symbiodinium phylotypes, consequently thediversity of symbionts available differs among coral species.
However, the mechanism regulating the diversity of compatible symbionts in cnidarian organisms, including coral, wasunknown.
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 host66

Figure 3. Infection of different Symbiodinium strains into corals.
Aposymbiotic primal polyps of A. tenuis VFDOHEDUVƫP ZHUHVHSD
rately incubated with small (Mf1.05b) and large (L2469) Symbiodinium
strains. Uptake of Symbiodinium into coral polyps was monitored using astereomicroscope. Biquand et al. (2017) ISME J. 11, 1702-1712.

Publication List:〔Original Papers〕
E., Okubo, N., Aihara, Y., Rolland, V., Hayward, D., Hatta,• Biquand,M., Minagawa, J., Maruyama, T., and Takahashi, S. (2017). Acceptable•••

symbiont cell size differs among cnidarian species and may limit
symbiont diversity. ISME J. 11, 1702-1712.
Ho, S.-H., Nakanishi, A., Kato, Y., Yamasaki, H., Chang, J.-S.,
Misawa, N., Hirose, Y., Minagawa, J., Hasunuma, T., and Kondo,
A. (2017). Dynamic metabolic profiling together with transcription
analysis reveals salinity-induced starch-to-lipid biosynthesis inalga Chlamydomonas sp. JSC4. Sci. Rep. 7, 45471.
Kim, E., Akimoto, E., Tokutsu, R., Yokono, M., and Minagawa,J. (2017). Fluorescence lifetime analyses reveal how the highlight-responsive protein LHCSR3 transforms PSII light-harvesting
complexes into an energy-dissipative state. J. Biol. Chem. 292, 1895118960.
Takizawa, K., Minagawa, J., Tamura, M., Kusakabe, N., and Narita, N.
(2017). Red-edge position of habitable exoplanets around M-dwarfs.
Sci. Rep. 7, 7561.

DIVISION OF SEASONAL BIOLOGY (ADJUNCT)

latitudes required longer day lengths while those from lower
latitudes required shorter day lengths.

Adjunct Professor

YOSHIMURA, Takashi

Specially Appointed Assistant Professor:
SHINOMIYA, Ai
Visiting Graduate Student: NAKAYAMA, Tomoya
MARUYAMA, Michiyo
NAKATSUKASA, Mana
Visiting Undergraduate: MARUYAMA, Michiyo*
Visiting Scientist:
GUH, Ying-Jey
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 provide 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 a result, we found differences in the critical day length
among Medaka populations. That is, Medaka from higher

Figure 1. Result of QTL analysis for critical day length.

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 1). We have also performed whole genome
re-sequencing using various Medaka strains that show different critical photoperiods, and identified potential candidate
genes that define the critical day length.

II. Mechanism that determines seasonal
breeders and non-seasonal breeders
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
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 20 populations derived fromvarious latitudes. As a result, populations from higher latitudes showed gonadal regression, while populations fromlower latitudes did not regress their gonads (Figure 2).

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2017. The former title isindicated by an asterisk (*).

67

National Institute for Basic Biology

Environmental Biology

3-2. Seasonal changes in phototransduction pathway
The temporal pattern of gene expression in medaka eyes
associated with changes in seasons was examined by microarrays. This analysis identified summer induced genes that
include various opsin genes and genes involved in downstream phototransduction pathways (Figure 3). We next
examined the functional significance of these genes’ expression using LWS (long wavelength sensitive) opsin-null fish.
As a result, LWS opsin-null fish showed reduced negative
phototaxis to white light compared to wild type fish undersummer conditions, suggesting that summer-induced LWS
opsin is critical for the emergence of negative phototaxis. In
addition, LWS opsin-null fish showed weaker preference forvirtual fish with nuptial coloration under summer conditions,suggesting that summer-induced LWS opsin is crucial for the
emergence of mate preference observed in summer.

Figure 2. 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 also
performing a genome-wide association study to identify
responsible genes.

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.

3-1. Seasonal changes in behaviors
Medaka kept under winter conditions stayed at the bottomof the tank, whereas medaka kept under summer conditions
swam all over the tank. In general, fish avoid strong lightstimulus (i.e., negative phototaxis). Summer medaka avoided
white light stimulus, while winter medaka failed to show thisnegative phototaxis, suggesting that medaka are less sensitive to light under winter conditions, compared to summer
conditions. When color preference was examined using
three-dimensional computer graphics (3D-CG), summer
medaka exhibited a preference for virtual fish with nuptial
coloration, while winter medaka exhibited no such preference. This observation implies that the medaka’s color perception is influenced by seasons.

68

Figure 3. Genome-wide expression analysis reveals dynamic seasonalchanges in opsin gene expression within the eye. Top: Results of microarray analysis. Bottom: Expression of LWS opsin within the retina.

Publication List:〔Original paper〕
T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi,• Shimmura,
M, Watanabe, E., Shimo, T., Senga, T., Nishimura, T., Tanaka, M.,
Kamei, Y., Naruse, K., and Yoshimura, T. (2017). Dynamic plasticity inphototransduction regulates seasonal changes in color perception. Nat.
Commun. 8, 412.

〔Review articles〕
K., and Yoshimura, T. (2017). Molecular mechanism• Ikegami,
regulating seasonality. In: Biological Timekeeping: Clocks, Rhythms•

and Behaviour. 589-605.
Tamai, K.T., and Yoshimura, T. (2017). Molecular and neuroendocrine
mechanisms of avian seasonal reproduction. Adv. Exp. Med. Biol.
1001, 125-136.

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.

of the “core genes”, i.e. genes well conserved within species
or genus, can be incorporated into the standard ortholog
table, the genes that are not conserved in the representative genomes cannot be incorporated. Considering the great
diversity of microbial species/genus genomes, a considerable
amount of information may be lost in the current standard
ortholog table. To address this problem, we developed a
stepwise protocol to construct orthologous relationships. In
this approach, we first create a within-species ortholog tablefor each species and construct a species level pan-genome
by picking one representative gene from each orthologous
group (Figure. 1). Next, we create a within-genus ortholog
table for each genus using as input the species-level pangenomes generated in the previous step and construct agenus level pan-genome. Finally, we create an ortholog tablecovering the entire taxonomic range by comparison of thegenus level pan-genomes. We used a rapid similarity search
program, UBLAST, to calculate all-against-all similarities
for within-species and within-genus comparisons, which candrastically reduce the computation cost. By this approach,
we can integrate various pan-genomes into a single ortholog
table, which enables us to analyze evolutionary processes
that generate within-species or within-genus diversity of
microbes.

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 III
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

II. Hierarchical strategy for creating ortholog
tablesPreviously, MBGD provided two types of ortholog tables:
the standard ortholog table containing one representative
genome from each genus covering the entire taxonomic
range, and the taxon specific ortholog tables containing
the genomes belonging to each taxonomic group (species,
genus, family and so on). Although, by this approach, mostFigure 1. Hierarchical protocol for creating the standard ortholog table

III. 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 developed

69

National Institute for Basic Biology

Theoretical Biology

a 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.

IV. D e v e l o p m e n t o f a w o r k b e n c h f o rcomparative genomics
We have developed a comparative genomics workbench
named RECOG (Research Environment for COmparative
Genomics), which aims to extend the current MBGD system
by incorporating more advanced analysis functionalities.
The central function of RECOG is to display and manipulatea large-scale ortholog table. The ortholog table viewer is aspreadsheet like viewer that can display the entire ortholog
table, containing more than a thousand genomes. In RECOG,
several basic operations on the ortholog table are defined,
which are classified into categories such as filtering, sorting,and coloring, and various comparative analyses can bedone by combining these basic operations. In addition,
RECOG allows the user to input arbitrary gene properties
and compare these properties among orthologs in various
genomes.
We continue to develop the system and apply it to various
genome comparison studies under collaborative research
projects (including H. pylori genome comparison describedin Section VI below). In particular, in addition to microbial
genome comparison, we are trying to apply RECOG to comparative analyses of transcriptomic data and metagenomic
data.

V. 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). 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.
On the basis of this framework, we have integrated various
kinds of microbial data using the ortholog information as a70hub, as part of the MicrobeDB.jp project (http://microbedb.
jp/) under the National Bioscience Database Center.
In addition, to facilitate the utilization of the RDF databases
distributed worldwide, we developed a command-line tool,
named SPANG. 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.

VI. A novel approach for 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. In addition, by clustering the accessory OGs on
the basis of phylogenetic pattern similarity and chromosomal proximity, we identified 60 co-occurring gene clusters(CGCs). We are now trying to generalize this approach toidentify genomic islands in various bacterial species.

Publication List:〔Original papers〕
H., and Uchiyama, I. (2017). SPANG: A SPARQL client• Chiba,
supporting generation and reuse of queries for distributed RDF•••

databases. BMC Bioinformatics 18, 93.
Hayatsu, M., Tago, K., Uchiyama, I., Toyota, A., Wang, Y., Shimomura,Y., Okubo, T., Kurisu, F., Hirono, Y., Nonaka, K., Akiyama, H.,
and Takami, H. (2017). An acid-tolerant ammonia-oxidizing
ƢSURWHREDFWHULXPIURPVRLO,60(-. 11, 1130-1141.
Ikeda, T., Uchiyama, I., Iwasaki, M., Sasaki, T., Nakagawa, M., Okita,K., and Masui, S. (2017). Artificial acceleration of mammalian cell
reprogramming by bacterial proteins. Genes Cells 22, 918-928.
Takami, H., Toyoda, A., Uchiyama, I., Itoh, T., Takaki, Y., Arai, W.,
Nishi, S., Kawai, M., and Ikeda, H. (2017). Complete genome sequenceand expression profile of the commercial lytic enzyme producer
Lysobacter enzymogenes M497-1, DNA Res. 24, 169-177.

〔Review Article〕
I. (2017). Ortholog identification and comparative analysis• Uchiyama,of microbial genomes using MBGD and RECOG. In Protein function
prediction, D. Kihara ed., Humana press, pp. 147-168.

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

Postdoctoral Fellow:
Technical Assistant:

TANIGUCHI, Atsushi
ISHIBASHI, Tomoko

Our laboratory is currently pursuing two paths of scientific inquiry 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 for biological analyses.

I. Initial step for left-right asymmetry
In mammalian development, the initial left-right asymmetry first appears as cilia-driven leftward fluid flow on
the ventral surface of the node, and the flow provides theinitial cue for asymmetric gene expressions that actually
determine asymmetric morphogenesis. The mechanismsthat convert the flow to asymmetric gene expression, i.e.
the flow sensing mechanism, remains controversial, withseveral models being proposed, and involvement of Ca2+
being suggested.
We pursued this question by measuring Ca2+ dynamics
in the 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 frequent at 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 left-right phenotypes.

made, maintaining them for both collaborations and forour own research interest, left-right asymmetry.
Over several years we have developed a fast light-sheet
microscope named ezDSLM, in which the excitation
light sheet and detection objective move instead of thespecimen for z-scanning. We installed an electricallytunable lens (ETL) to achieve further speed and exclusion of mechanical vibrations 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, and marine crustaceans, etc.

Figure 2. Images of floating volvox taken by ezDSLM with
ETL. Left: Single optical section. Right: Maximum intensity
projection.

Publication List:〔Original papers〕
T., Kimura, E., Fujisawa, S., Oikawa, S., Nonaka, S.,• Hashiura,
Kurosaka, D., and Hitomi, J. (2017). Live imaging of primary ocular•vasculature formation in zebrafish. PLoS ONE 12, e0176456.
Taniguchi, A., Kimura, Y., Mori, I., Nonaka, S., and Higashijima,
S. (2017). Axially-confined in vivo single-cell labeling by primed
conversion using blue and red lasers with conventional confocal
microscopes. Dev. Growth Differ. 59, 741-748.

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 thevalidity of 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
and fast image acquisition. We are now running two lightsheet microscopes, one commercial and the other self71

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 understandthe mechanisms underlying cell-fate decision. Genomic
reprogramming after mammalian fertilization reverts terminally differentiated gametes into toti- or pluri-potent
states to start a 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 celllineage-allocation by deciphering the molecular mechanisms underlying remodeling of nuclear organization andtheir 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 differentiatedcells 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 fourthcleavage, 8-cell stage embryos, which have undergone
the process of compaction, will give rise 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 cellmass (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
the embryo, which are essential for regulation of gene
expression involved in differentiation, and reprogramming of the EPI.

Figure 1. Lineage allocation in mouse preimplantation development

72

Remodeling of nuclear architecture indevelopment
Chromatin is organized in a non-random fashion within
three-dimensional nuclear space. During developmental
processes, nuclear architecture is dramatically reconstructed, resulting in establishment of cell-type specificnuclear organization. Defects in structural componentsof the nucleus are responsible for developmental aberrations and several human diseases. 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 toregulate gene expression. The developmental programaccompanies nuclear remodeling, resulting in construction of cell-type 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 in which gene locifrequently interact with each other. Recent works haverevealed that folding of “local” chromatin structures suchas enhancer-promoter looping is associated with genome
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),
which allows us to track specific genomic sequences inliving cells (Miyanari Y, Nature Structural & Molecular
Biology, 2013). Importantly, this technique is versatileand can be extended to allow many robust applications,which will be integrated into our study to manipulate
several genome functions. Based on new technologicaldevelopment, we aim to understand biological roles ofchromatin dynamics in cell-fate decision.

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

Development and metabolism are intertwined with one
another during organogenesis. This interaction is essential
to maintain the metabolic state in a developmental context-dependent manner. There is also a growing awarenessthat metabolism plays instructive roles in developmental
processes. The emerging picture depicts metabolism as a
critical system not only for sustaining physiological conditions, but also regulating developmental patterning by
coordinating various cellular processes. However, it remains
largely unclear how this interaction is achieved in multicellular organisms. We aim to reveal as-yet-unknown relationships between developmental and metabolic processes andtheir biological meaning by elucidating molecular mechanisms of the system. To address this, we use trans-omics
approach including metabolome and transcriptome analyses
using Arabidopsis thaliana as a model, in conjunction withstandard molecular genetics and biochemistry techniques.

I. Cytochrome P450 epoxidase for embryonicpatterning
To uncover hidden relationships between development andmetabolism, we performed quantitative phenome screening using A. thaliana mutants of orphan cytochrome P450
genes. We identified the cyp77a4 mutant, which exhibits
irregular embryonic patterning as evidenced by defects, suchas a cup-shaped cotyledon morphology (Figure 1). Although
CYP77A4 is the first cytochrome P450 reported to be able tocatalyze the epoxidation of fatty acids in plants, its function
in development is completely unknown.
Through the use of auxin-related reporters, we determined that CYP77A4 is essential for polar auxin transport
via proper localization of PIN1 (an auxin efflux carrier).
Interestingly, unlike other enzyme mutants defective in auxin
dynamics in ubiquitous tissues, the cyp77a4 mutant was
associated with defects specifically in embryos. Furthermore,
our double mutant analysis clarified that CYP77A4 andCYP77A6 (the phylogenetically closest gene to CYP77A4)
are functionally independent. Based on these findings, we
proposed that the metabolic requirement for polarity establishment via auxin dynamics differ between tissues, and thatin embryos this depends on a CYP77A4-dependent metabolic pathway. These findings may augment our understandingof fatty-acid epoxidation by uncovering a novel developmental function of the epoxidase (Kawade et al., submitted).

Figure 1. Irregular arrangement of cotyledons in the cyp77a4 mutants.
(A-E) WT-like (A), abnormally arranged (B), single (C and D), and cupshaped (E) cotyledons in the cyp77a4-3 mutants. Bars = 2 mm.

II. Developmental signal intertwined withmetabolism
ANGUSTIFOLIA3 (AN3) is a transcriptional co-activator, which promotes cell proliferation in leaves. We
recently showed that AN3 forms an expression gradientalong a proximal-to-distal developmental axis to regulate
cell proliferation dynamics in time and space (Kawade
et al., 2017; Figure 2). To gain further insights into how
AN3 contributes to tissue patterning, we conductedtranscriptome and metabolome analyses using the an3
mutants. We found that AN3 regulates a transcriptional
network for oxygen homeostasis. Our metabolic profilingindeed detected characteristic features of redox disturbance in the an3 mutants. Although cell proliferation isan essential process for tissue patterning, reactive oxygen
species are generated through energy production. It would
be interesting to assume that the AN3 signal may solve
this dilemma, cell proliferation vs. oxidative stress, inplant development.

Figure 2. A merged image of the leaf
primordia (around 200-μm length)
expressing genetically engineered
mobile or immobile AN3 (cyan or red,respectively). The distribution of themobile AN3 gradually spreads along theleaf proximal-to-distal axis (from down
to top), which is broader than that of theimmobile one.

Publication List:〔Original papers〕
K., Tanimoto, H., Horiguchi, G., and Tsukaya, H. (2017).
• Kawade,
Spatially different tissue-scale diffusivity shapes ANGUSTIFOLIA3••

gradient in growing leaves. Biophys. J. 113, 1109-1120.
Kawade, K., and Tsukaya, H. (2017). Probing the stochastic propertyof endoreduplication in cell size determination of Arabidopsis thaliana
leaf epidermal tissue. PLoS ONE 12, e0185050.
Sawada, Y., Tsukaya, H., Li, Y., Sato, M., Kawade, K., and Hirai, M.Y.
(2017). A novel method for single-grain-based metabolic profiling ofArabidopsis seed. Metabolomics 13, 75.

73

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

ASTROBIOLOGY CENTER

Specially Appointed Associate Professor

TAKIZAWA, Kenji

M-dwarfs, relatively small and cool stars, are the mostabundant neighboring stars and thus could be the first candidates for searching habitable exoplanets and detecting a signof extraterrestrial life. One of the most important biosignatures on an exoplanet is a specific reflection pattern on theland surface named ‘red-edge’ that is caused by land vegetation. On the Earth, red-edge appears between visible light,
which is absorbed by photosynthetic pigments, and nearinfrared (NIR) radiation, which is reflected via leaf structure.
In previous reports, it was predicted that red-edge could be
red-shifted around M-dwarfs since phototrophs on the exoplanet should use abundant NIR radiation for photosynthesisin addition to visible light. We have proposed an alternative
prediction that red-edge could be observed as on the Eartheven on exoplanets if the initial oxygenic photosynthesis
evolved underwater.

water depth of 123 m on the AD Leo planet. If the origin oflife and its early evolution is placed underwater, Earth-type
oxygenic photosynthesis may evolve on exoplanets aroundM-dwarfs.

II. Evolution form water to land under M-starradiation
The adaptive evolution of phototrophs from water to land
may eventually also use NIR radiation, by one of two photochemical reaction centers, with the other center continuing
to use visible light. These “two-color” reaction centers canabsorb more photons, but they will encounter difficulty inadapting to drastically changing light quality and quantity atthe boundary between land and water (Figure 2). NIR photosynthesis can be more productive on land, though its evolution would be preceded by the Earth-type vegetation. Thus,
the red-edge position caused by photosynthetic organisms
on habitable M-dwarf exoplanets could initially be similar tothat on Earth. Our studies imply that red-edge is a universal
biosignature for life originated underwater.

I. Photosynthetically active radiation on the
exoplanets around M-dwarfs
We estimated the light conditions expected on habitable
planets around an M-dwarf. Assuming that an Earth-likeplanet is located in the habitable zone around AD Leo, andM4.5eV star located 16 light years away from our solar
system, the light conditions on the planet were estimatedand compared with solar irradiation on the Earth. The land
surface of the M-dwarf planet is illuminated by strong NIRradiation, while Earth’s surface is illuminated by visible
light. On the other hand, similar light conditions are expectedunderwater since only blue-green light can penetrate metersof water (Figure 1).
The visible-light intensity obtained on the land surface of ahypothetical habitable planet around AD Leo is equivalent tothat from Earth’s ocean at a depth of 15 m. The lower limitof light intensity needed to support the growth of oxygenic
marine phototrophs on Earth is about 20 nmol photon m-2 s-1,
which corresponds to a water depth of 166 m in the model
calculation. The same amount of visible light is obtained at a

Figure 2. Lighting conditions on a hypothetical habitable planet aroundan M-dwarf and the evolution of photosynthesis. Ovals and arrows
outline the flow of evolutionary paths from a two-photon reaction using
visible radiation (Vis-Vis) to a two-color reaction using visible and NIR
radiation in separate reaction centers (Vis-NIR). The area graph on theleft side shows the visible-radiation/NIR-radiation ratio on the land
surface and underwater at different depths.

Publication List:〔Original paper〕
K., Minagawa, J., Tamura, M., Kusakabe, N., and Narita, N.
• Takizawa,(2017). Red-edge position of habitable exoplanets around M-dwarfs.
Sci. Rep. 7, 7561.

Figure 1. Photon flux density spectra for Earth (a) and a hypothetical
habitable planet around AD Leo (b). Gray lines show the spectra obtainedat the top of the atmosphere. Black lines show the spectra at the land
surface passing through a 1.5 air-mass atmosphere. The underwater PFDs
at a depth of 0.1, 1, and 10 m are shown using red, green, and blue lines,
respectively.

74

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 andother academic institutions.

research aiming to explore otherwise inaccessible new fields
in basic biology.
During 2017 we carried out 56 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 organized jointly by NIBB and
NIPS for promoting DNA and protein studies. The facility
maintains a wide array of core research equipment, fromstandard machinery like ultracentrifuges to cutting edge tools
such as next generation DNA sequencers, which amount to
60 different kinds of instruments. The facility is dedicated tofostering collaborations with researchers both of NIBB andother academic institutions worldwide by providing thesetools as well as expertise. Our current focus is functional
genomics. We also act as a bridge between experimental
biology and bioinformatics through close consultation andtraining.
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 visiting scientists
can work effectively during their stay. In 2017, approximately 200 researchers visited to use our new facility and developed active collaborations, which resulted in 16 co-authoredpapers published.

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 2017, we analyzed approximately 750
samples with mass spectrometers and protein sequencers.
- LC-MS (AB SCIEX TripleTOF 5600 system)- LC-MS (Thermo Fisher SCIENTIFIC Orbtrap Elite)- MALDI-TOF-MS (Bruker Daltonics REFLEX III)- LC-Q-TOF MS (Waters Q-TOF Premier)- Protein sequencer (ABI Procise 494 HT; ABI Procise 492
cLC)

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

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

Figure 2. Triple TOF LC/MS/MS System

75

National Institute for Basic Biology

Research Support
Publication List on Cooperation:〔Original papers〕
H., Moriyama, M., Nikoh, N., Hosokawa, T., Futahashi,• Anbutsu,
R., Tanahashi, M., Meng, X.Y., Kuriwada, T., Mori, N., Oshima, K.,

•

•
•
•
•
•
•
•
•
•
•
•
•

76

Hattori, M., Fulie, M., Satoh, N., Maeda, T., Shigenobu, S., Koga, R.,
Fukatsu, T. (2017). Small genome symbiont underlies cuticle hardness
in beetles. Proc. Natl. Acad. Sci. USA 114, E8382–E8391.
Fukushima, K., Fang, X., Alvarez-Ponce, D., Cai, H., Carretero-Paulet,
L., Chen, C., Chang, T.H., Farr, K.M., Fujita, T., Hiwatashi, Y., Hoshi,
Y., Imai, T., Kasahara, M., Librado, P., Mao, L., Mori, H., Nishiyama,T., Nozawa, M., Palfalvi, G., Pollard, S.T. Rozas, J., Sanchez-Gracia,
A., Sankoff, D., Shibata, T.F., Shigenobu, S., Sumikawa, N., Uzawa,T., Xie, M., Zheng, C., Pollock, D.D., Albert, V.A., Li, S., Hasebe, M.
(2017). Genome of the pitcher plant Cephalotus reveals genetic changes
associated with carnivory. Nat. Ecol. Evol. 1, 59.
Gotoh, A., Shigenobu, S., Yamaguchi, K., Kobayashi, S., Ito, F., andTsuji, K. (2017). Transcriptome characterization of male accessory
glands in ants to identify molecules involved in their reproductive
success. Insect. Mol. Biol. 32, 382.
Gotoh, A., Shigenobu, S., Yamaguchi, K., Kobayashi, S., Ito, F., andTsuji, K. (2017). Transcriptome profiling of the spermatheca identifies
genes potentially involved in the long-term sperm storage of ant queens.
Sci. Rep. 7, 5972.
Hayashi, Y., Maekawa, K., Nalepa, C.A., Miura, T., and Shigenobu, S.
(2017). Transcriptome sequencing and estimation of DNA methylationlevel in the subsocial wood-feeding cockroach Cryptocercus punctulatus(Blattodea: Cryptocercidae). Appl. Entomol. Zool. 52, 643–651.
Hayashi, Y., Oguchi, K., Yamaguchi, K., Kitade, O., Maekawa, K.,
Miura, T., Shigenobu, S. (2017). Male-specific molecular genetic
markers in the Japanese subterranean termite Reticulitermes speratus.
Insectes Sociaux. 64, 357-364.
Ihara, K., Sato, K., Hori, H., Makino, Y., Shigenobu, S., Ando, T.,
Isogai, E., and Yoneyama, H. (2017). Expression of the alaE geneis positively regulated by the global regulator Lrp in response to
intracellular accumulation of l-alanine in Escherichia coli. J. Biosci.
Bioeng. 123, 444–450.
Kondo, S., Wakae, K., Wakisaka, N., Nakanishi, Y., Ishikawa, K.,
Komori, T., Moriyama-Kita, M., Endo, K., Murono, S., Wang,
Z., Kitamura, K., Nishiyama, T., Yamaguchi, K., Shigenobu, S.,
Muramatsu, M., Yoshizaki, T. (2017). APOBEC3A associates withhuman papillomavirus genome integration in oropharyngeal cancers.
Oncogene. 36, 1687-1697.
Kudo, A., Shigenobu, S., Kadota, K., Nozawa, M., Shibata, T.F.,
Ishikawa, Y., and Matsuo, T. (2017). Comparative analysis of the braintranscriptome in a hyper-aggressive fruit fly, Drosophila prolongata.
Insect. Biochem. Mol. Biol. 82, 11–20.
Li, B., Kamiya, T., Kalmbach, L., Yamagami, M., Yamaguchi, K.,
Shigenobu, S., Sawa, S., Danku, J.M.C., Salt, D.E., Geldner, N.,
Fujiwara, T. (2017). Role of LOTR1 in Nutrient Transport throughOrganization of Spatial Distribution of Root Endodermal Barriers. Curr.
Biol. 27, 758–765.
Matsuda, K., Mikami, T., Oki, S., Iida, H., Andrabi, M., Boss, J.M.,
Yamaguchi, K., Shigenobu, S., and Kondoh, H. (2017). ChIP-seq
analysis of genomic binding regions of five major transcription factors
highlights a central role for ZIC2 in the mouse epiblast stem cell gene
regulatory network. Development. 144, 1948-1958.
Matsui, A., Iida, K., Tanaka, M., Yamaguchi, K., Mizuhashi, K., Kim,
J.M., Takahashi, S., Kobayashi, N., Shigenobu, S., Shinozaki, K., Seki,
M. (2017). Novel Stress-Inducible Antisense RNAs of Protein-Coding
Loci Are Synthesized by RNA-Dependent RNA Polymerase. Plant.
Physiol. 175, 457–472.
Murase, K., Shigenobu, S., Fujii, S., Ueda, K., Murata, T., Sakamoto, A.,
Wada, Y., Yamaguchi, K., Osakabe, Y., Osakabe, K., Kanno, A., Ozaki,Y., Takayama, S. (2017). MYB transcription factor gene involved insex determination in Asparagus officinalis. Genes. Cells. 22, 115–123.
Nakayama, K., Ohashi, R., Shinoda, Y., Yamazaki, M., Abe, M.,
Fujikawa, A., Shigenobu, S., Futatsugi, A., Noda, M., Mikoshiba, K.,
Furuichi, T., Sakimura, K., Shiina, N. (2017). RNG105/caprin1, an
RNA granule protein for dendritic mRNA localization, is essential for

long-term memory formation. Elife. 6, e29677.

R.Z., Merzlyak, P.G., Okada, T., Islam, M.R., Uramoto, H.,• Sabirov,
Mori, T., Makino, Y., Matsuura, H., Xie, Y., Okada, Y. (2017). The•

organic anion transporter SLCO2A1 constitutes the core component of
the Maxi Cl channel. EMBO. J. 36. 3309-3324.
Suetsugu, K., Yamato, M., Miura, C., Yamaguchi, K., Takahashi,K., Ida, Y., Shigenobu, S., and Kaminaka, H. (2017). Comparison ofgreen and albino individuals of the partially mycoheterotrophic orchid
Epipactis helleborine on molecular identities of mycorrhizal fungi,
nutritional modes, and gene expression in mycorrhizal roots. Mol. Ecol.
26, 1652-1669.

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 genome sequence of the pea
aphid, Acyrthosiphon pisum, in consort with that of bacterial symbiont Buchnera aphidicola illustrates the remarkableinterdependency between the two organisms. Genetic capacities of the pea aphid and the symbiont for amino acid biosynthesis are complementary. The genome analysis revealed
that the pea aphid has undergone characteristic gene losses
and duplications. The IMB antibacterial immune pathway
is missing several critical genes, which might account for
the evolutionary success of aphids to obtain beneficial symbionts. Lineage-specific gene duplications have occurredin genes in a broad range of functional categories, which
include signaling pathways, miRNA machinery, chromatin
modification and mitosis. The importance of these duplications for symbiosis remains to be determined. We foundseveral instances of lateral gene transfer from bacteria to thepea aphid genome. Some of them are highly 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 = 20 um.

Publication List:〔Original papers〕
H., Moriyama, M., Nikoh, N., Hosokawa, T., Futahashi,• Anbutsu,
R., Tanahashi, M., Meng, X.-Y., Kuriwada, T., Mori, N., Oshima, K.,••

Shigenobu, S., et al. (2017). Small genome symbiont underlies cuticle
hardness in beetles. Proc. Natl. Acad. Sci. USA 114, E8382–E8391.
Hayashi, M., Shinozuka, Y., Shigenobu, S., Sato, M., Sugimoto, M.,
Ito, S., Abe, K., and Kobayashi, S. (2017). Conserved role of Ovo ingermline development in mouse and Drosophila. Sci. Rep. 7, 40056.
Ota, R., Morita, S., Sato, M., Shigenobu, S., Hayashi, M., andKobayashi, S. (2017). Transcripts immunoprecipitated with Sxl protein
in primordial germ cells of Drosophila embryos. Dev. Growth Differ.
59, 713–723.

〔Review article〕
P., Kikuchi, Y., Shigenobu, S., and Nowack, E.C.M. (2017).
• Mergaert,
Metabolic integration of bacterial endosymbionts through antimicrobial
peptides. Trends Microbiol. 25, 703–712.

77

National Institute for Basic Biology

Research Support

SPECTROGRAPHY AND BIOIMAGING FACILITY

Specially Appointed Associate Professor:

KAMEI, Yasuhiro

Technical Staff:
Technical Assistant:

KONDO, Maki
TANIGUCHI-SAIDA, Misako
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 light-

controlled biological processes.
The NIBB Collaborative Research Program for the Use of
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 laser microscopeswith special aims (Light-sheet Microscope and Infrared
Laser-Evoked Gene Operator microscope: IR-LEGO) for
users in NIBB and collaborative guest researchers. We began
two new types of Collaborative Research Program from2016. One is a new category of the NIBB Collaborative
Research for Integrative Bioimaging using machines andbioimage processing/analysis techniques, and the other isthe Advanced Bioimaging Support Program (ABiS) of theGrant-in-aid for Scientific Research on Innovative Areas.
The light-sheet microscope was developed by Dr. Ernst
Stelzer’s group at the European Molecular Biology
Laboratory (EMBL). This microscope can realize high-speed
z-axis scanning in deeper tissue by illuminating a specimen
from the side with a 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 2017 we held the 5th biological image processing training
course with the Department of Imaging Science, CNSI. 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 11th 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 Biothermology:
heat and temperature in biology.

Publication List on Cooperation
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.

78〔Original papers (Selected)〕• Homma, N., Harada, Y., Uchikawa, T., Kamei, Y., and Fukamachi, S.

•
•
•
•
•
•
•
•

(2017). Protanopia (red color-blindness) in medaka: a simple system forproducing color-blind fish and testing their spectral sensitivity. BMC
Genetics 18, 10.
Ishikawa, T., Kashima, M., Nagano, A.J., Ishikawa-Fujiwara, T., Kamei,Y., Todo T., and Mori, K. (2017). Unfolded protein response transducer
IRE1-mediated signaling independent of XBP1 mRNA splicing is not
required for growth and development of medaka fish. eLife 6, e26845.
Li, C., Sako, Y., Imai, A., Nishiyama, T., Thompson, K., Kubo, M.,
Hiwatashi, Y., Kabeya, Y., Karlson, D., Wu, S. H., Ishikawa, M.,
Murata, T., Benfey, P.N., Sato, Y., Tamada, Y., and Hasebe, M. (2017).
A Lin28 homologue reprograms differentiated cells to stem cells in themoss Physcomitrella patens. Nat. Commun. 8, 14242.
Nakano, M., Arai, Y., Kotera, I., Okabe, K., Kamei, Y., and Nagai, T.
(2017). Genetically encoded ratiometric fluorescent thermometer withwide range and rapid response. PLoS ONE 12, e0172344.
Nakayasu, T., Yasugi, M., Shiraishi, S., Uchida, S., and Watanabe, E.
(2017). Three-dimensional computer graphic animations for studying
social approach behaviour in medaka fish: Effects of systematic
manipulation of morphological and motion cues. PLoS ONE 12,
e0175059.
Osanai, Y., Shimizu, T., Mori, T., Yoshimura, Y., Hatanaka, N., Nambu,
A., Kimori, Y., Koyama, S., Kobayashi, K., and Ikenaka, K. (2017).
Rabies virus-mediated oligodendrocyte labeling reveals a single
oligodendrocyte myelinates axons from distinct brain regions. Glia 65,
93-105.
Shimmura, T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi,M., Watanabe, E., Shimo, T., Senga, T., Nishimura, T., Tanaka, M.,
Kamei, Y., Naruse, K., and Yoshimura, T. (2017). Dynamic plasticity inphototransduction regulates seasonal changes in color perception. Nat.
Commun. 8, 412.
Suzuki, M., Sato, M., Koyama, H., Hara, Y., Hayashi, K., Yasue, N.,
Imamura, H., Fujimori, T., Nagai, T., Campbell, R.E., and Ueno, N.
(2017). Distinct intracellular Ca2+ dynamics regulate apical constriction
and differentially contribute to neural tube closure. Development. 144,
1307-1316.
Taniguchi, A., Kimura, Y., Mori, I., Nonaka, S., and Higashijima, S.I. (2017). Axially-confined in vivo single-cell labeling by primed
conversion using blue and red lasers with conventional confocal
microscopes. Dev. Growth Differ. 59, 741-748.

〔Original paper (E-publication ahead of print)〕
M., Shibata, Y., Ohno, K.. Usami, T., Kamei, Y., Taniguchi,• Nakamoto,Y., Todo, T., Sakamoto, T., Young, G., Swanson, P., Naruse, K., andNagahama, Y. Ovarian aromatase loss-of-function mutant medaka
undergo ovary degeneration and partial female-to-male sex reversal
after puberty. Mol. Cell Endocrinol. 2017 July 13.

● Research activity by Y. Kamei
Specially Appointed Associate Professor:
KAMEI, Yasuhiro
NIBB Research Fellow:
SAKAMOTO, Joe
Visiting Scientist:
HATTORI, Masayuki
Technical Assistant:
NAKAGAWA, Mami
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

Figure 1. Schematic illustration of heat shock response of cells (left) andan infrared laser-evoked gene operator (IR-LEGO) microscope system.

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”.
Second, the aim of our “manipulation project” is to control
gene expression in vivo. Gene function analysis must be
evaluated at the cell level in vivo. To achieve spatiotemporalcontrolled gene expression we employed one of the stressresponses, the heat shock response. The heat shock promoter
is the transcription regulation region of heat shock proteins
and all organisms have this mechanism. Positioning thetarget gene downstream of the promoter, we can induce thetarget gene expression by local heating.
Infrared (IR) beams can heat water molecules, which are
the main constituent of cells, hence, we can heat a single
cell by irradiating IR to a target cell using a microscope.
We have developed a microscope, IR laser evoked gene
operator (IR-LEGO), specialized for this purpose (Figure1). The IR-LEGO microscope can irradiate an IR laser to asingle cell in vivo such as in C. elegans, Drosophila, medaka,
zebrafish, Xenopus and Arabidopsis, to induce the heatshock response at a desired timing. In 2015, additionally, we
confirmed the system was effective in the moss Marchantia
polymorpha and in the newt Pleurodeles waltl.
Optimal heating induces the heat shock response and subsequent gene expression, while an excess results in cell death.
Hence, we must precisely control laser heating. We evaluated time course and spatial heating profiles, and the resultspresented that temperature of the target area rose rapidly andkept a constant level dependant on IR laser power, additionally, the heated area was adequately as small as a typical cellsize.
With this in mind, we tried to induce gene expression invarious species. At first, we reported an IR-LEGO experiment in living C. elegans. Target gene expression in a target
cell could be induced with only 1 s-IR irradiation. Whereas
the optimal power range which can induce gene induction
without cell damage was limited. Excess laser power resulted
in cell death or cessation of cell division. We confirmed thatan optimal irradiation, e.g. 11 mW for 1 s, induced physiological gene expression in the target cell and subsequent79

National Institute for Basic Biology

Research Support

cell 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
expression (Figure 2).
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 biothermology, a new field thinking about temperature or heat in biological systems, because spatiotemporal micrometer order local heating is difficult without thissystem. Now we are trying to estimate thermal properties ofcells and biomaterials in vivo using a newly developed thermo-probe (Nakano et al. PLoS One 2017) and the IR-LEGO
microscope system.

Figure 2. Long-term gene expression system with cre/loxP recombination system and its example of practical experiment in medaka embryo
to adult.

Publication List:〔Original papers〕
M., Tamada Y., Murata T., Oya S., Hasebe M., Hayano Y., and• Hattori
Kamei Y. (2017). Artificial testing targets with controllable blur for

•
•
•

80

adaptive optics microscopes. Optical Engineering 56, 080502.
Homma, N., Harada, Y., Uchikawa, T., Kamei, Y., and Fukamachi, S.
(2017). Protanopia (red color-blindness) in medaka: a simple system forproducing color-blind fish and testing their spectral sensitivity. BMC
Genetics 18, 10.
Ishikawa, T., Kashima, M., Nagano, A.J., Ishikawa-Fujiwara, T., Kamei,
Y,, Todo T., and Mori, K. (2017). Unfolded protein response transducer
IRE1-mediated signaling independent of XBP1 mRNA splicing is not
required for growth and development of medaka fish. eLife 6, e26845.
Ishikawa, T., Toyama, T., Nakamura, Y., Tamada, K., Shimizu, H.,
Ninagawa, S., Okada, T., Kamei, Y., Ishikawa-Fujiwara, T., Todo, T.,
Aoyama, E., Takigawa, M., Harada, A., and Mori, K. (2017). UPRtransducer BBF2H7 allows export of type II collagen in a cargo- anddevelopmental stage-specific manner. J. Cell Biol. 216, 1761-1774.

M., Arai, Y., Kotera, I., Okabe, K., Kamei, Y., and Nagai, T.
• Nakano,(2017). Genetically encoded ratiometric fluorescent thermometer with•wide range and rapid response. PLoS ONE 12, e0172344.
Shimmura, T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi,M., Watanabe, E., Shimo, T., Senga, T., Nishimura, T., Tanaka, M.,
Kamei, Y., Naruse, K., and Yoshimura, T. (2017). Dynamic plasticity inphototransduction regulates seasonal changes in color perception. Nat.
Commun. 8, 412.

〔Original paper (E-publication ahead of print)〕
M., Shibata, Y., Ohno, K.. Usami, T., Kamei, Y., Taniguchi,• Nakamoto,Y., Todo, T., Sakamoto, T., Young, G., Swanson, P., Naruse, K., andNagahama, Y. Ovarian aromatase loss-of-function mutant medaka
undergo ovary degeneration and partial female-to-male sex reversal
after puberty. Mol. Cell Endocrinol. 2017 July 13.

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.

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. 69).

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).

81National Institute for Basic Biology

Research Support

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  ]HEUDÀVK 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 totally
DQGHIÀFLHQWO\REVHUYHG7KH1,%%%LR5HVRXUFH&HQWHUKDV
equipment, facilities, and staff to maintain such organisms
VDIHO\HIÀFLHQWO\DQGDSSURSULDWHO\Model Animal Research Facility
Associate Professor:
Technical Staff:
Technical Assistant:

WATANABE, Eiji
OHSAWA, Sonoko
HAYASHI, Kohji
NOGUCHI, Yuji
TAKAGI, Yukari
SUGINAGA, Tomomi
FUJIMOTO, Daiji
TAKAHASHI, Nobuaki
MATSUMURA, Kunihiro
NOMOTO, Yoshihiro

established 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 mice strains.
5. Generating genetically-engineered mice using the
CRISPR/Cas9 method.

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 breeding room in the Yamate area

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.
The NIBB Center for Transgenic Animals and Plants was

82

Figure 2. Large sized autoclave in the Yamate area.

In 2017 (from January 1 to December 21), 3,940 mice (7transgenic lines and wild-type) were brought into the facility
in the Yamate area, and 40,428 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 (12 transgenic lines), and stored using
cryopreservation (28 transgenic lines). Frozen eggs of 33
mice lines were taken out of the facility.
Genome editing experiments were performed on threekinds of target genes. We generated gRNAs of the target
genes, which were transferred into fertilized eggs with Cas9
protein. We were able to introduce intended mutations into
the genome DNA.
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 2017 (from January
1 to December 21), 0 mice were brought into the facility in
the Myodaiji area, and 1,246 mice (including pups bred in
the facility) were taken out.

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

Figure 4. Liquid nitrogen tank.

III. Research activities
The associate professor of this center - E. Watanabe - is theprincipal investigators of the Laboratory of Neurophysiology.
The Laboratory of Neurophysiology is studying mechanismsof the visual system using a psychophysical approach. For
details, please refer to the page of the laboratory (p. 44).

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

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
KOTANI, Keiko

Figure 3. Equipment for gene transfer.

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 2017 (from January 1 to December 21), 75 zebrafish (75The 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 73
culture boxes or growth chambers, and 13 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 70 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 are

83National Institute for Basic Biology

Research Support

also 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.
The building renovation work was completed in May
2017, and the temporally closed facilities including the threechambers that can produce extreme environmental conditions
were renewed and opened.

Cell Biology Research Facility
Associate Professor:

WATANABE, Eiji

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.

84

NATIONAL BIORESOURCE PROJECT
The major purpose of the National BioResource Project(NBRP) is to collect, preserve, and provide bioresources(such as experimental animals and plants) that are essential
experimental materials for life sciences research. The project
also aims to improve the bioresources by increasing theirvalue through enriching genome information and developing
fundamental technologies for preservation and other necessary procedures, in order to meet to the current scientificdemands. NIBB serves as the core organization center of the
medaka bioresource and as sub-centers of the morning glory
and the zebrafish bioresources.

I. NBRP Medaka (Oryzias latipes)
Project Manager:
Sub Managers:

NARUSE, Kiyoshi
KAMEI, Yasuhiro
ANSAI, Satoshi

NBRP medaka provides three groups of resource worldwide, including 1) live medaka resources comprising morethan 600 strains (strains for general use, wild populations,related species, inbred strains, mutants, and transgenics), 2)
genome resources (ca. 400 thousand cDNA clones originatedfrom 33 cDNA libraries, and BAC/Fosmid clones covering
the whole medaka genome), and 3) hatching enzyme necessary for manipulation and live imaging of the medaka
embryo. Entries for these resources can be searched byvarious methods such as keywords, sequence homology, andexpression profile on the web site (https://shigen.nig.ac.jp/
medaka/).
We provide mutant gene screening using the HRM methodin the TILLING library, and also provide a genome editing
platform using CRISPR/Cas9. Using collaborative researchsupport, researchers can visit NIBB to generate mutants bygenome editing. We developed and provided a new genome
browser (http://viewer.shigen.info/medaka/index.php) using
a recently published genome assembly by PacBioRSII.
Summary of previous accomplishments are as follows.
In 2007-2009, we sequenced both ends of 260,000 clones
originated from 11 kinds of full-length cDNA libraries andsequenced the whole length of 17,000 independent clones.
We also developed strains in which CRE-recombinase can be
expressed in any cell lineages using a heat shock promoter,and started to provide strains (TG918, TG921, etc) established using this method. In 2010, we re-sequenced the
genomes of five inbred strains by coverage corresponding
to genome 100X (http://medaka.lab.nig.ac.jp/service/menu).
In 2012, we developed a vitrification freezing preserva-

NBRP Medaka fish facility to collect, maintain and supply the five
medaka.

tion method of the testicular tissue. We are now providing
a backup preservation service of testicular tissues using thismethod.

II. NBRP Morning Glory (Ipomoea nil)
Project Manager: 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 for flower
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
122 mutant lines and 4 DNA clones to both local and international biologists in 2017. A database for the whole genome
sequence of I. nil was updated.

Various colors of morning glory flowers. The color depends not only on
the molecular structure of the pigment, but also on the pH of the vacuole
where the pigment is accumulated.

III. NBRP Zebrafish (Danio rerio)
Project Manager: HIGASHIJIMA, Shin-ichi
NIBB is a sub-center of the National BioResource Project(NBRP) Zebrafish, and collaborates with the core organization center, RIKEN Brain Science Institute. We at NIBB
mainly collect zebrafish strains expressing fluorescentproteins in specific cells of the central nervous system anddistribute them to researchers worldwide. Zebrafish is an
important and globally used experimental vertebrate model
animal with a simple body structure, it allows genetic manipulations, and its embryos are transparent enough for opticalobservations. Researches using zebrafish for the studies ofneural development and neural circuit functions are growing
rapidly worldwide, and the importance of strains collected
and provided by NIBB to researchers is growing accordingly.

An example of transgenic fish generated by the CRISPR/Cas9-mediatedknock-in method.

85

National Institute for Basic Biology

Research Support

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

Specially Appointed Professor

NARUSE, Kiyoshi

Specially Appointed Assistant Professor: TAKETSURU, Hiroaki
Specially Appointed Senior Specialist: AKIMOTO-KATO, Ai
Technical Assistant:
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
In 2017, IBBP Center stored 4,478 384-well and 69 96-wellplates consisting of 1,726,176 clones as cDNA/BAC clones,
12,225 tubes for animal cells, plant and animal samples,
proteins, genes and microorganisms, 1769 133mm-straw
tubes for sperm and 654 seed samples. In total 1,728,599
samples are stored.

Figure 1. IBBP Center.

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

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

86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 2017, we also worked to establish
a research center for cryo-biological studies thorough thisCollaborative Research Project. Accordingly, we organized
the Cryopreservation Conference 2017 on November 1-2,
2017 at the Tsukuba Center for Institutes, Tsukuba, Ibaraki,
Japan. We had 132 participants from several fields covering
physics, chemistry, biology, and technology.

Pronuclear, 2-cell and morula stage embryos were vitrified
using a solution containing 10% propylene glycol, 30%
ethylene glycol, 20% Percoll and 0.3 M sucrose in PB1.

Figure 5. Vitrification and transfer of rat embryos.

Each embryo was warmed using a solution containing 0.3
M sucrose in PB1. Morphologically normal embryos weretransferred into the oviducts of the pseudopregnant females(Figure 5). Pronuclear embryos were vitrified after culturingfor 0, 4 and 7 hours. After warming, the embryos that developed to the 2-cell stage were transferred into the oviducts ofthe pseudopregnant females.
The development to offspring of vitrified-warmed pronuclear, 2-cell and morula stage embryos were 16%, 33% and52%, respectively (Figure 6). When the pronuclear embryos
were vitrified after culturing for 0, 4 and 7 hours, the developmental rates to offspring were 12%, 25% and 24%, respectively. These results indicated that tolerance to vitrification
was increased with embryo development. Vitrified embryos
can be used for producing genetically engineered rats andknockout/in rats.

Figure 4. Group photo of Cryopreservation Conference 2017.

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

Figure 6. Development to offspring of vitrified embryos.

The level of ovulation in various rat strains
and development of embryos after vitrification
Development of rat vitrified embryos in eachdevelopmental stage
Vitrification of embryos is a simple method and is used invarious animals. Although vitrification of 2-cell embryos ispossible, only a few reports were published about vitrification of embryos in various developmental stages. Vitrified
pronuclear and morula stage embryos are used for producing
genetically engineered rats and knockout/in rats. This study
examined the development of rat embryos in pronuclear,
2-cell and morula stages after vitrification.
Wistar female rats aged more than 8 weeks were injectedwith 150 IU/kg PMSG and 75 IU/kg hCG. Females werethen mated with mature male rats of the same strain.
Pronuclear and 2-cell stage embryos were collected from
oviducts. Morula embryos were collected from the uterus.

In rats, it is now possible to produce genetically engineered
strains, not only as transgenic animals but also using geneknockout techniques. Reproductive technologies have beenused as indispensable tools to produce and maintain thesenovel valuable strains. Here we studied the sensitivity to
PMSG/hCG for oocyte collection, and the development ofembryos after cryopreservation in some strain rats (Figure 7).

Figure 7. Rat strains.

87National Institute for Basic Biology

Research Support

The response to pregnant mare serum gonadotropin(PMSG) and human chorionic gonadotropin (hCG) was
examined by injection of 150IU/kg PMSG + 75IU/kg hCGor 300IU/kg PMSG + 300IU/kg hCG. The numbers of 2-cellembryos collected from F344/Stm female rats were high(31.0). On the other hand, the number of 2-cell embryos
collected from BN/SsNSlc female rats were low (2.4) inthese strains. Of cryopreserved 2-cell embryos collectedfrom these strains, more than 65% of embryos survived
after warming. After transfer, offspring were obtained fromcryopreserved 2-cell embryos in all strains. However, the offspring rate was different for strains, F344/Stm and Jcl: Wistar
were more than 50%, and ACI/Nkyo was 20% (Figure 8).
In summary, there are differences in sensitivity to superovulation and the development to offspring rate from cryopreserved embryos in these strains.

Figure 8. Development to offspring of vitrified embryos in some strains.

88room in Myodaiji area for saving energy at Summer 2017. As
the result, radioisotopes received apparently increased. But
the new radioisotope received and radioisotope experimental
handling user was only 1 at the Myodaiji-area from April
2017. The training courses on radioisotope handling were
given as in Table 4.

Associate Professor

HASEBE, Mitsuyasu

KODAMA, Ryuji

Technical Staff:

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

Technical Assistant:

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 2017.
1. The CRF usage charge system was started.
At April 2017, the users of the CRF must pay the facility
usage charge which equals the disposal cost of radioactive
wastes generated in the user’s experiment or in the decontamination processes of the contamination apparently causedby the user. The CRF prepared each laboratory’s radioactive
waste container. (Figure 1A)
2. The “User’s guide” in English was renewed.
Main points changed were the explanation and the documents about the CRF usage charge.
3. The CRF website was renewed.
The main point changed was the usage of new web design
software, which allows users to access the contents moreconveniently http://www.nibb.ac.jp/ricenter/ (Figure 1B)
The number of registrants and the number of users fromJanuary 2017 to December 2017 are shown in Table 1.
Users and visitors counted by the access control systemof the controlled areas numbered 1,302 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. Yamate
area storage radioisotopes were transferred to the storage

Myodaiji Area
Registrants
Users

Yamate Area
47
21

43
24

Table 1. Numbers of registrants and users at Myodaiji area and Yamate
area in 2017.

Myodaiji Area

Yamate Area

Total

Users

527

488

Visitors

125

162

1,015
287

Total

652

650

1,302

Table 2. Users and visitors who entered each controlled area in 2017.

14000

350
Resistrants

300

12000

Days of facility use
250

10000

200

8000

150

6000

100

4000

50

2000

Days of facility use

HeadRegistrants

CENTER FOR RADIOISOTOPE FACILITIES

00
Fiscal year

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

Myodaiji Area
125

I Received

125

I UsedYamate Area

Total

111

221

332

0

3,329

3,329

35

S Received

35

S Used32

P Received

9,250

9,250

18,50032

P Used

9,250

9,250

18,50014

C Received

3,913

3,913

7,827

14

C Used

2,079

0

2,079

3

H Received

676,104

676,104

1,352,209

3

H Used1,480

933,917935,397

73,186

073,186025,43825,438

Table 3. Balance of radioisotopes received and used (kBq) at each controlled area in 2017.

Table 4. Training courses for radiation workers in 2017.

$%

Figure 1. The CRF’s notable activities in 2017.
A: the radioactive waste containers for each laboratory.
B: renewal of website of the CRF

89

National Institute for Basic Biology

Research Support

RESEARCH ENHANCEMENT STRATEGY OFFICE

Director

Vice-Director:

UENO, Naoto

MANO, Shoji (Nov. 1~)

Vice-Director:

NISHIMURA, Mikio ~Sep. 30)

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.
In 2017, Young Researcher Support Group was newly
added as the 6th group to the Research Enhancement
Strategy Office to provide comprehensive support for young
researchers in NIBB.

Evaluation and Information Group
Associate Professor:
Group Adviser:

KODAMA, Ryuji
KAWAGUCHI, Masayoshi

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.

902) Editing of the Annual Report (in collaboration with the
Public Relations Group)This group edits the annual report (this print) which summarizes the annual activities of laboratories and facilities andis used in the evaluation procedures of the institute.
3) Assistance in budget requests and long-range planningof the instituteThis group also assists the Director General in preparing
long-range plans for building the most advanced researchfacilities, and in budget requests to the government to realize
and implement these plans.
4) Assistance in making the plans and reports of the institute
In addition we assist in drafting NIBB’s Medium-term
Goals and Plans (for a six-year-period), and in instituting
Annual Plans to implement them. The department also assists
in preparing Business and Performance Reports for external
evaluation on whether we are meeting the goals set bothannually 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 2017
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

・One Dean for Research coming from Princeton
University (October)Specially Appointed Assistant Professor (URA):TATEMATSU, Kiyoshi
Technical Assistant:
TAKAHASHI, Ritsue
KAWAGUCHI, ColinNISHIMURA, Akiko
Group Advisor:
UENO, Naoto

3) Support of education-related programs
This group supported graduate students participating in thefollowing student exchange programs of NIBB/EMBL and
NIBB internship:

NIBB 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), theTemasek 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, supports other researchers and students who
visit NIBB.

The main activities of the group in 20171) Coordination of international conferences and theInternational Practical Course
This group coordinated the following international conference hosted by NIBB:
・The 65th NIBB Conference “Renaissance of Marchantia
polymorpha – the genome and beyond” Okazaki, Japan,
December 16 – 18, 2017 (p. 94)
2) Support of visiting researchers to NIBBThis group supported visits of foreign researchers related to
the following events:
・Two researchers coming from Princeton University
as lecturers for The NIBB-Princeton Joint Proteomics
Training Course (July 19-21, 2017; p. 96)

・The 19th EMBL International Ph.D. Symposium“Bridging the Gaps – Interdisciplinary Approaches inLife Sciences –” Heidelberg, Germany, October 19-21,
2017(p. 95)
・NIBB Internship Program 2017 (p. 100)

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 2017, this group hosted a total of 99 collaboration
projects. The success of these collaborations are provedby 30 research papers published in this year. One of theexamples is a 5-year collaboration of the group of Drs.
Kondoh (Osaka Univ. and Kyoto Sangyo Univ.) and
Shigenobu (NIBB): they established a technique of ChIP-seq
using in vivo biotinylated transcription factors (TFs), whichenabled them to reveal the gene regulatory network inmouse epiblast stem cells where ZIC2 TF plays a central
role (Matsuda et al., 2017). Another noteworthy achievement is a series of publications about development of aplatform to study the psychology of vision using medaka
fish. Dr. Fukamachi (Japan Woman’s University) conducted the Priority Collaborative Research Project “Study ofmolecular mechanisms for vision and color perceptiondependent sexual behavior” with many collaborators including Dr. Kamei and other NIBB members. The collaborationteam created a red opsin KO medaka and analyzed the
red-color perception using the Okazaki Large Spectrograph(Homma et al., 2017). Dr. Watanabe (NIBB) used a 3D
virtual imaging system and found that movement is crucial
for medaka to discriminate each other (Nakayasu et al.,2017). Dr. Yoshimura (NIBB and Nagoya Univ.) revealed
seasonal changes in color perception of medaka by using
the resources and the platform developed as described above(Shimmura et al., 2017).

91

National Institute for Basic Biology

Research SupportN., Harada, Y., Uchikawa, T., Kamei, Y., and Fukamachi, S.
• Homma,(2017). Protanopia (red color-blindness) in medaka: a simple system for•••producing color-blind fish and testing their spectral sensitivity. BMC
Genetics 18, 10.
Matsuda, K., Mikami, T., Oki, S., Iida, H., Andrabi, M., Boss, J.M.,
Yamaguchi, K., Shigenobu, S., and Kondoh, H. (2017). ChIP-seq
analysis of genomic binding regions of five major transcription factors
in mouse epiblast stem cells that highlights a central role for ZIC2.
Development 144, 1948-1958.
Nakayasu, T., Yasugi, M., Shiraishi, S., Uchida, S., and Watanabe, E.
(2017). Three-dimensional computer graphic animations for studying
social approach behaviour in medaka fish: Effects of systematic
manipulation of morphological and motion cues. PLoS ONE 12,
e0175059.
Shimmura, T., Nakayama, T., Shinomiya, A., Fukamachi, S., Yasugi,M., Watanabe, E., Shimo, T., Senga, T., Nishimura, T., Tanaka, M.,
Kamei, Y., Naruse, K., and Yoshimura, T. (2017). Dynamic plasticity inphototransduction regulates seasonal changes in color perception. Nat.
Commun. 8, 412.

Young Researcher Support Group
Assistant Professor:
Group Adviser:

KOMINE, Yuriko
FUJIMORI, Toshihiko

Cultivation of future researchers is also an importantmission of NIBB, as an international leading institute in thefield of basic biology. Constituting the Department of Basic
Biology in the School of Life Science of SOKENDAI (theGraduate University for Advanced Studies), NIBB provides
PhD courses for graduate students. The young researcher
support group was organized in 2017 aiming to support
young researchers in NIBB, including graduate students(SOKENDAI students and special research students fromother universities), to help make their study and researchexperiences in NIBB effective and fruitful for their future
careers.

The main activities of this group1) Coordination and management of courses provided by
the Department of Basic Biology (lectures, research presentation, etc.), in collaboration with all faculty members
in NIBB.
2) Support in organizing NIBB’s programs related to the
PhD courses, such as the Open Campus Day for prospective students.
3) Cooperation in the interdepartmental programs offeredby the SOKENDAI headquarters, including the Freshman
Course.
4) Gathering and providing information useful for bothstudents and faculty members.

Gender Equality Promotion Group
Associate Professor:
Group Adviser:

TSUBOUCHI, Tomomi
TAKADA, Shinji

NIBB is committed to promoting gender equality in the scientific community. To help scientists maintain their work-lifebalance and progress their carriers during life events, such as
having children, this group manages the research assistant92

system for those in need, and promotes networking amongfemale researchers.
In 2017, this group established a mailing list for female
researchers working in NIBB, held several meetings to facilitate networking and provided opportunities to discuss theirconcerns on career development. This group has also beenworking towards setting up a room where NIBB members
can bring their children to work.

TECHNICAL DIVISIONHeadMIWA, Tomoki

Common Facility Group

Research Support Group

Chief:

Chief:

MORI, Tomoko● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki
Subunit Chief:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
NISHIDE, Hiroyo
Technical Staff:
NAKAMURA, Takanori
TANIGUCHI-SAIDA, Misako
BINO, Takahiro
Technical Assistant:
ICHIKAWA, Chiaki
ICHIKAWA, Mariko
OKA, Naomi
SHIBATA, Emiko● NIBB Bioresource Center
Unit Chief:
OHSAWA, Sonoko
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
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

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.

● Cell Biology
Subunit Chief:

MIZUTANI, Takeshi

HAYASHI, Kohji● Developmental Biology
Subunit Chief:
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
Technical Staff:
MIZUGUCHI, Hiroko●Neurobiology
Subunit Chief:

TAKEUCHI, Yasushi●Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
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.

93

The 65th NIBB Conference“Renaissance of Marchantia polymoropha – the genome and beyond”
Organizers: John L. Bowman (Monash University, Australia),Takayuki Kohchi (Kyoto
University, Japan),Takashi Ueda (National Institute for Basic Biology, Japan)
December 16 (Sat) – 18 (Mon), 2017
The 65th NIBB Conference “Renaissance of Marchantia
polymorpha ~ the genome and beyond” was held at the
Okazaki Conference Center from 16th December to 18th
December 2017. Professor Takayuki Kohchi (Kyoto
University, Japan), Professor John L. Bowman (Monash
University, Australia) and I organized the conference. The
liverwort “Zenigoke” (Marchantia polymorpha) was alreadydescribed in the literature of pharmaceutical sciences inancient Greece, and in the past 200 years Marchantia has
been used as a model plant in modern biology. In the latter
half of the 20th century its position as a major model plant
was temporarily taken over by other plant species such as
Arabidopsis thaliana, but in recent years Marchantia has
been reevaluated because of its merits in the life cycle andbody plan as well as its manageability as a research material.
Combined with the establishment of various tools and techniques for molecular genetic studies, this plant is now getting
hot attention from plant scientists. Furthermore, the paperon the draft genome of M. polymorpha was published in the
October 5th issue of Cell, 2017. Under these circumstances,
the NIBB Conference was planned to discuss deeply fundamental biological functions and physiologies of plants aswell as to promote future collaborative research.
The conference was held with 30 invited speakers from10 countries and 115 general participants from 9 countries. Three plenary lectures, 27 invited talks, 16 short talks
selected from posters, 6 topics in a technology workshop,and 58 posters were presented. Throughout the entire conference program, including scientific sessions and the banquet
on the last day, all participants enjoyed deep discussions
from various perspectives, ranging from individual biologicalphenomena, to the future roles to be played by Marchantia,
and the future of plant science. After the meeting, we
received a lot of messages from both foreign and domestic
participants, many of which said “the conference was very
meaningful and stimulating for future research”. It is a great
pleasure for the organizers to have such a fruitful international conference as the NIBB conference.
The organizers appreciate all the participants for their
exciting talks and lively discussion. We are also thankful
to the Grant-in-Aid for Scientific Research on Innovative
Areas (MEXT) “Multidimensional Exploration of Logics
of Plant Development”, the Interuniversity Bio-Backup
Project for Basic Biology (IBBP), the Support Programfor Interaction-based Initiative Team Studies (SPIRITS)
by Kyoto University, and the Daiko Foundation for financial support. We are also most grateful to the Office ofInternational Cooperation, NIBB for the intensive support.
I would like to conclude this message with words fromJohn Bowman, “after the Renaissance at NIBB/OCC… theEnlightenment!”.
Takashi Ueda (On behalf of the Organizers)

Speakers
Arteaga-Vazquez, Mario A. (Universidad Veracruzana), Berger, Frederic (Gregor Mendel Institute), Bowman, John L. (Monash Univ.),
Chen, Feng (Univ. Tennessee), Dolan, Liam (Univ. Oxford), Grossniklaus, Ueli (Univ. Zurich), Harrison, Jill (Univ. Bristol), Lin, ShihShun (National Taiwan Univ.), Nakagami, Hirofumi (MPIPZ), Solano, Roberto (CNB-CSIC), Weijers, Dolf (Wageningen Univ.
Research),Aki, Shiori S. (NAIST), Araki, Takashi (Kyoto Univ.), Demura, Taku (NAIST), Hasebe, Mitsuyasu (NIBB), Ishizaki, Kimitsune(Kobe Univ.), Kodama, Yutaka (Utsunomiya Univ.), Kohchi, Takayuki (Kyoto Univ.), Kuchitsu, Kazuyuki (Tokyo Univ. Science),
Nakamura, Yasukazu (NIG), Naramoto, Satoshi (Tohoku Univ.), Nishihama, Ryuichi (Kyoto Univ.), Nishitani, Kazuhiko (Tohoku Univ.),
Ohta, Hiroyuki (Tokyo Tech.), Sakayama, Hidetoshi (Kobe Univ.), Sakakibara, Keiko (Rikkyo Univ.), Sekimoto, Hiroyuki (Japan:RPHQ·V8QLY 6KLPDPXUD0DVDNL +LURVKLPD8QLY 8HGD7DNDVKL 1,%% <DPDWR.DWVX\XNL7 .LQGDL8QLY

94

The 19th EMBL International Ph.D. Symposium “Bridging theGaps – Interdisciplinary Approaches in Life Sciences –”October 19 (Thu) – 21 (Sat), 2017
Five PhD students from SOKENDAI/NIBB were funded
by NIBB to participate in the 19th EMBL International
Ph.D. Symposium “Bridging the Gaps – Interdisciplinary
Approaches in Life Sciences –” (held on 19-21 October).
Our students had the chance to give poster presentations
at the symposium to introduce their current research. They
also attended EMBL’s laboratories to exchange experimental
information and discuss their research with PhD students,
post-docs and PIs.

Comments from Students (excerpts)
・Liu Meng
I got some nice advice from my host lab and had an impressive experience in the following symposium.
My host PI, Dr. Rainer Pepperkok and his team members
introduced their research to me, and discussed my current
studies as well, during the one-day-stay in their lab. My
research is generally genetic analysis while Dr. Pepperkok’s
team is good at cell biology, specifically on ER vesicle
traffic. They helped me in making a clear picture of what
I can possibly do for the next step and also gave me somedetailed advice like what kind of marker might be suitable
for me; they literally guided me to build a bridge between my
genetic studies and further cellular level analysis. The discussion on their seminar impressed me so much. They are very
open to giving and accepting advice from others. At somepoints the discussion turned heated and they were neverafraid of speaking their minds. This was my first time joining
a western style discussion and it was really like a brainstorm
for me.
For the next three days we attended the PhD symposiumwith other EMBL students. Besides all those fantastic eyeopening lectures and talks, I want to talk about our exciting
experience at the party on the last day of conference. In Asia
we never had this type of party. Seeing all other western
students singing and dancing, we debated for a while ofwhether we would join them or not. Eventually we jumped
into the crowd when we realized just standing there wasworse than dancing in front of a bunch of people and we
fit in right away. We kept drinking and dancing with other
EMBL students, enjoying the rare opportunity for relaxing.
Though it was a small stepfor me, it was an interesting
starter to attempt to integrate
into western culture.
The four-day trip was short
but its effect on me was significant. Besides the usefuladvice and tips I got fromthe lab tour and conference,
I love my research more thanever before. I’m more com-

EMBL, Heidelberg
fortable to learn new things in my studies and am not caring
so much about loneliness anymore. I hope this trip is a goodstarter for my two years deferment study here.
・Haruko Miura
The symposium wasentirely organized by 2 ndyear EMBL PhD students,
who did a really great job
in compiling a fantastic meeting. About 100
PhD students from all over
the world and 15 invited
speakers participated in
the symposium. Many
students came from Finlandand Australia, since EMBL has partnerships and sites inthose countries as well. Surprisingly, there were only a few
German students, which might reflect the very international
character of EMBL. This year’s theme was “Bridging theGaps - Interdisciplinary Approaches in Life Sciences”, sospeakers from interdisciplinary fields gave lectures, e.g. synthetic biology, systems biology, tissue engineering, microfluidic technology, and state-of-art fluorescence imaging.
Furthermore, there were interesting ‘Science and Society’talks and a plenary discussion on topics like talking to journalists, women in science, and career paths. I had the chance
to present my research in a poster presentation and short talkto a broad audience and got positive feedback from many
fellow students. Inspired by the lectures, I want to apply 3Dtissue culture, microfluidics, and new fluorescent tools in myfuture research. The meeting was not only a good opportunity to network with peers from various countries, but also toconnect with the other NIBB students.
On the last day, I visited the Schultz group, who work on
the development of chemical tools for live cell imaging andmanipulating cellular signaling. Since our fields of study
were related, we had lively scientific discussions and theygave me valuable advice for my research. All in all, I had theimpression that EMBL is a great place for postdoc studies,due to the cutting edge research, excellent core-facilities withsuperb technical support, and high internationality. Finally,
I want to thank all the involved EMBL and NIBB members
for making these valuable experiences possible which willdefinitely help me in my future research and career.

95

NIBB-Princeton Joint Proteomics Training Course“– Protein Identification,
Quantification and Characterization –”■Period: July 19 (Wed) – 21 (Fri), 2017
■Participants: 17 (including 4 from NIBB)■Organizer:
Dr. Naoto Ueno (NIBB), Dr. Shuji Shigenobu (NIBB)
Dr. Ileana Cristea (Princeton Univ.)■Lecturers:
Dr. Ileana Cristea (Princeton Univ.)
Dr. Todd Greco (Princeton Univ.)■Program:
Lecture 1: Introduction to Mass Spectrometry andProteomics
Lecture 2: Protein quantification
Lecture 3: Protein interactions
Lecture 4: Protein posttranslational modifications
Tutorial 1: Manual interpretation on MS/MS Spectra
Tutorial 2: Quantification: introduction to software
Tutorial 3: Hands-on data analysis
Hands-on 1: SDS-PAGE staining and imaging, Gel bandexcision and dehydration, In-gel digestion
Hands-on 2: Peptide extraction, LC/MS/MS analysis,
Demonstration of Mass SpectrometerAs part of international collaboration and cooperation with
Princeton University, we held a proteomics training course“NIBB - Princeton Joint Proteomics Training Course 2017
- Protein identification, quantification and characterization-” from July 19 to 21, 2017. Over the three-day course, we
conducted lectures and hands-on training to master principles and measurement techniques on protein identificationand quantification by mass spectrometry. From Princeton
University, Professor Ileana Cristea, who is a head instructorof the summer Proteomics Course at the Cold Spring Harbor
Laboratory and a prominent virus researcher using proteomic
approaches, was invited as one of the organizers. Dr. Todd
Greco, who is a researcher at Cristea’s Laboratory, participated in the course as a lecturer. Participants were selectedbased on their applications. From 26 domestic and overseas
applicants, 17 people, including four NIBB members, participated in the course. There was a wide range of participants
including graduate students, postdoctoral researchers, and96

faculty members and staff of various universities and institutes. Three of them were graduate students and postdoctoral
researchers belonging to universities in Japan from foreigncountries.
Professor Cristea was mainly responsible for lectures onproteomics from theoretical background to applied research.
The hands-on training was supported by the NIBB faculty
and staff, and the participants themselves prepared andanalyzed protein samples extracted from Xenopus embryos.
The lectures and hands-on training for bioinformatics ofproteomics analysis was handled by Dr. Greco, and theparticipants conducted data analysis exercises based on the
results obtained from the hands-on training using a computer
deployed for each group. Due to being divided among fourgroups, participants deeply interacted with their partners, andlectures and hands-on training were held with active discussion.
Through the preparation and conduction of the course, I
believe that the connection between Princeton University and
NIBB researchers has deepened. It was also a good opportunity for us to learn the cutting edge analytical techniques
used by Princeton University. We gained a lot of hints that I
believe will be useful for Collaborative Research Projects for
Integrative Genomics utilizing mass spectrometers.
Shuji Shigenobu (On behalf of the Organizer)

The 11th NIBB
Bioimaging Forum“Pioneering New Bioimaging by
Fusing Optics and Biology”
Organizers: Yasuhiro Kamei, Hideki
Takami, Yutaka Hayano, Mitsuo Takeda,
Hirotsugu Yamamoto, Masayuki Hattori,
Takashi Murata, Shigenori Nonaka,
Yosuke Tamada
February 14 (Tue) -15 (Wed), 2017
We held the 11th NIBB Bioimaging Forum on February 14thand 15th 2017. The subtitle for this meeting was “Pioneering
New Bioimaging by Fusing Optics and Biology”, and aimed
at the development of new fields in bioimaging by inviting
experts in various disciplines including optical engineeringand astronomy to give lectures. Fifty four participants (8were from NIBB, 46 from outside in which 24 were fromprivate companies) from a wide variety of specialties, suchas optics, engineering, mathematics, astronomy, biology,
medicine, and agricultural science. The first session of theforum, therefore, included descriptions of characteristics ofeach field by specialists in biology, optical engineering, andastronomical optics to enhance understanding each other.
The succeeding 4 sessions included 15 lectures (30 minutes
each with 5 minutes discussion) and 10 posters. Dr. Mitsuo
Takeda, a famous theoretical optics specialist, summarizedat the end of the forum how we came to understand the “philosophy and condition” of each field, which is necessary tofuse different fields. Forums aiming to establish new methodsof bioimaging by fusing different fields are still rare and webelieve our forum is functioning well as a start point.
(Yasuhiro Kamei)

The NIBB Genome Informatics
Training ourse
The 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 with bioinformatics. In 2017, we held two sets of training courses on
RNA-seq analysis, a UNIX learning course and a course on
BLAST analysis. We provided the RNA-seq analysis course
divided into two 2-day programs: one was a preparatory
course to learn basics of UNIX and R and the other was apractical course to learn pipelines of RNA-seq analysis using
next-generation sequence data. The UNIX learning course
was a 2-day program for beginners who are starting bioinformatics. The BLAST analysis course was designed foradvanced users aiming to enable participants to get familiar
with a large scale sequence database search in the local environment using BLAST software in a robust and systematicmanner. These GITC courses offered lectures and hands-on
tutorials.

Introduction to RNA-seq – from thebasics of NGS to de novo analyses■Organizers: Dr. Shuji Shigenobu and Dr. Ikuo
Uchiyama (NIBB Core Research Facilities)■Lecturers: Dr. Shuji Shigenobu,
Dr. Ikuo Uchiyama, Dr. Masanao Sato (Hokkaido
Univ.), Mr. Tomoki Miwa, Dr. Katsushi
Yamaguchi, Ms. Hiroyo Nishide, Mr. Takanori
Nakamura (NIBB Core Research Facilities)

February 23(Thu)-24(Fri), 2017
(Preparatory Course) Basics of UNIX, R, and NGS■Participants: 23 (including 4 from NIBB)■Program:
1. UNIX for beginners
2. Introduction to “R”
3. NGS basic data formats and NGS basic tools
4. Text processing
5. Shell scripting
6. Exercises

March 9(Thu)-10(Fri), 2017
(Practical Course) RNA-seq analysis pipeline■Participants: 21 (including 3 from NIBB)■Program:
1. Introduction to statistics
2. RNA-seq for beginners: genome-based and transcriptome-based approaches
3. Multivariate statistics
4. Exercises

97

The 5th BioImage Analysis
Training Course
Introduction to UNIX for analysesof large scale biological data sets
June 22(Thu)-23(Fri), 2017
■Organizer: Dr. Ikuo Uchiyama (NIBB Core
Research Facilities)■Lecturers: Dr. Ikuo Uchiyama, Mr. Tomoki Miwa
(Head of Technical Division), Ms. Hiroyo Nishide,
Mr. Takanori Nakamura (NIBB Core Research
Facilities)■Participants: 22 (including 6 from NIBB)■Program:
1. UNIX for beginners
2. Introduction to NGS data analysis
3. Text processing
4. Shell scripts
5. Usage of job management system
6. Exercises

Mastering BLAST, the essenceof sequence analyses
September 14(Thu)-15(Fri), 2017
■Organizers & Lecturers: Dr. Shuji Shigenobu, Dr.
Ikuo Uchiyama (NIBB Core Research Facilities)■Participants: 21 (including 5 from NIBB)■Program:
1. BLAST for beginners
2. Local BLAST search with command-line
3. BLAST inside – theoretical background of
sequence search
4. Large scale BLAST search
5. Annotation of genes and ortholog analysis
6. Beyond BLAST

98

Organizers: Dr. Kagayaki Kato,
Dr. Yoshitaka Kimori, Dr. Yasuhiro Kamei,
Dr. Hiroshi Koyama, Dr. Shigenori Nonaka,
Dr. Takashi Murata.
Supervisors: Prof. Naoto Ueno, Prof.
Toshihiko Fujimori.
November 20 (Mon) -22 (Wed), 2017
The 5th BioImage Analysis Training Course washeld jointly by the Center for Novel Science Initiatives’
Department of Imaging Science, JSPS KAKENHI Platforms
for Advanced Bioimaging Support (ABiS), and NIBB. This
course was designed for biologists who are relatively new
to analyzing datum obtained through advanced microscopy. Therefore the focus of training was learning image
processing and analytical techniques through “solving simple
problems with image analysis” and “understanding appropriate methods and necessary preparation for consulting experts
in technically advanced imaging challenges”. 47 people
applied for the course, which had a maximum capacity of 18
participants, this clearly suggests the height of the demandfor courses on these 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)

Advanced Bioimaging Support(ABiS)In the field of life science, the demand for bioimaging
has increased in recent years. However, due to advances inimaging technologies, i.e., the diversification and specialization of imaging equipment and increasingly complex operations, increased costs of equipment and running costs, andthe growing need for image data analysis, individual research
institutes and universities have encountered increasing difficulties related to the introduction, maintenance, and operation of imaging equipment.
ABiS was launched as one of the “Platforms for Advanced
Technologies and Research Resources” in fiscal year (FY)2016 under the new framework of the Grant-in-Aid for
Scientific Research on Innovative Areas (Leader: Prof.
Masanobu KANO, NIPS/The University of Tokyo). This
program aims to contribute the further development of
academic research in Japan through providing cutting-edge
equipment and methodologies to individual KAKENHI(Grants-in-Aid for Scientific Research) research by the
Ministry of Education, Culture, Sports, Science andTechnology (MEXT) under the Grant-in-Aid for Scientific
Research on Innovative Areas (FY2016-FY2021).
ABiS provides cutting-edge instruments for light microscopy, electron microscopy, magnetic resonance imaging,and other methods through the network in cooperation withdomestic partner organizations that own and operate multiple
types of advanced specialized imaging equipment, in orderto provide comprehensive support for advanced imaging in
the field of life science. NIBB together with NIPS contributeas core institutes to the ABiS network. Of the various ABiS
support activities, NIBB is in charge of the following:■Light microscopy• 4D microscopy by Prof. Toshihiko FUJIMOIRI• IR-LEGO microcopy by Assoc. Prof. Yasuhiro KAMEI• DSLM by Assoc. Prof. Shigenori NONAKA■Imaging analysis• Development of image processing/analysis algorithms
for biological data by Prof. Naoto UENO and Assist.
Prof. Kagayaki KATO
■Training• Training for image analysis by Assist. Profs. Yoshitaka
KIMORI and Hiroshi KOYAMA(Platform of Supporting Cohort Study and Biospecimen
Analysis, Platform of Advanced Animal Model Support, andPlatform for Advanced Genome Science) to provide multidisciplinary and international support.

The 1st ABiS Symposium “Towards the Futureof Advanced Bioimaging for Life Sciences”
February 19 (Sun) ~ 20 (Mon), 2017
Sponsored by: Advanced Bioimaging Support■Co-sponsored by:
National Institutes of Natural Sciences
National Institute for Physiological Sciences
National Institute for Basic Biology
Center for Novel Science Initiatives
Grant-in-Aid for Scientific Research on Innovative
Areas-‘Resonance Bio’
Grant-in-Aid for Scientific Research on Innovative
Areas-‘The Birth of New Plant Species’
Grant-in-Aid for Scientific Research on Innovative
Areas ― Platforms for Advanced Technologies
and Research Resources “Committee on Promoting
Collaboration in Life Sciences”■Participants: 151■Oral presentation: 18■Poster presentation: 54
The 1st ABiS symposium was held in Okazaki toexchange information on the state-of-the-art technologies for imaging. This symposium was open
to researchers who are interested in imaging techniques in addition to researchers involved in the ABiS
network. In this symposium, we introduced ABiS
activities and the research results of the support. In
addition, two research groups of ‘Grant-in-Aid for
Scientific Research on Innovative Areas’ in which
bio-imaging is a core methodology, and two foreign
programs, Global Bioimaging promoted by EuroBioimaging and Center for Bioimaging Sciencesin National University of Singapore, introduced
their activities related to the promotion of imaging
sciences.

To organize and coordinate ABiS activities, two secretariat offices were established in NIBB (Assoc. Prof. Shoji
MANO) and NIPS, respectively, under the control of thegeneral support group (The persons in charge in NIBB;
Director-general Prof. Masayuki YAMAMOTO, Prof. Naoto
UENO, Prof. Shinji TAKADA, Assoc. Prof. Shoji MANO).
General support includes the budget planning and management of ABiS activities. In particular, we promote ABiS
activities via the website and other media so that KAKENHI
researchers make full use of the ABiS platform to accelerate
their research projects. In addition, we organize technology
training sessions, workshops, and symposia to disseminate
advanced imaging technologies and share information about
them. We also coordinate with the other three platforms

99

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 2017 there were 57 applicants, out of which seveninterns were selected. These interns were from universities
located in six countries (Colombia, Germany, Indonesia,
Malaysia, Republic of Serbia, and Japan) and spent periods
ranging from two to twelve weeks experiencing life as amember of a research team. Moreover, two interns from
China stayed at NIBB for twelve weeks and one intern from
France stayed one week by their own travel grants.

Report from a participant
2OLYHUD9DOHQWLURYLþ
University of Belgrade, Serbia
0\QDPHLV2OLYHUD9DOHQWLURYLþDQG,DPDQXQGHUJUDGXDWH
student at Faculty of Biology at the University of Belgrade,
Serbia. Thanks to prof. Dr Nakayama, who suggested the
NIBB internship program to me, I was able to experience
working at his laboratory for one month. It was a great
adventure and opportunity for me, since I had a chance towork in the field of epigenetics and to travel to a breathtaking far away country, that was something I only dreamt
about before.
The research I performed during my internship at NIBB wason the ciliate Tetrahymena thermophila. This was my firsttime working in a laboratory as well as with Tetrahymena. I
found its life cycle and the extreme processes happening on
its genome during its sexual reproduction process called conjugation extremely fascinating. Those peculiarities make it areally favorable and attractive model organism.
My project was to characterize the proteins involved in theformation of heterochromatin and DNA elimination during
genome rearrangement within the macronucleus. In one
experiment I characterized several cell lines that express
proteins tagged with green fluorescent protein to analyze
localizations of heterochromatin protein candidates. It wasa great experience seeing gene gun transformation for the
first time. I also learned a lot about culturing, storing and100

transferring Tetrahymena and how to perform a selection of
the transformed cells and phenotype assortments using different concentrations of selective substances in the medium.
In a parallel experiment, I aimed to reveal the sufficiency
of few proteins for DNA elimination by a tethering assay. I
faced some difficulties during this experiment, but I foundit a good experience for my future work, that I may face andneed to solve on a daily basis. Every day I was presented
with interesting challenges and new chances to find a way tountangle some problem. In the end, I managed to go throughall the intended steps and successfully finish the experimentswith some of the samples. I wish I could have had a longer
period of time to do more experiments with different conditions, and finish all of the initially planned experiments.
Accommodation at Myodaiji Lodge was extremely comfortable which made my stay very pleasant. I didn’t lackanything and everything was professionally organized,
thanks to the International Corporation Office.
All of the lab members of the Division of Chromatin
Regulation were always willing to help me with my work, aswell as with my stay. They gave me a lot of precious advices
for which I am immensely grateful. It is a great honor forme to have worked with such polite, kind and hard-working
people. I would especially like to thank prof. Dr Nakayama
who gave me this opportunity to join his lab and offered mean unforgettable experience of Japanese culture, its delicious
cuisine and its people. I am eternally thankful to Dr Kataoka
from whom I’ve learned many valuable things, who patiently
and carefully watched and guided me through every step andwho showed me how wonderful and incredibly fun working
in the laboratory can be. I really appreciate friendly and welcoming atmosphere during my stay.
This was a life-changing adventure for me. In addition to
the exciting bench work in the actual lab, I’ve gained many
unforgettable memories, visited amazing places, felt one
unique culture, met remarkable people and made lasting
friendships. The whole experience inspired me to pursue mycareer in science even more than before.

101

INDEX
A ....................................................................................................
4

HAYASHI, Kentaro

AIHARA, Yusuke

65

HAYASHI, Kohji

AJIOKA, Rie

53

HAYASHI, Tomoko

AKAMA, Akiko

67

HIGASHI, Satoru

AKIMOTO-KATO, Ai

86

HIGASHIJIMA, Shin-ichi

AKITA, Asaka

75

HIGUCHI, Yoko

32

ANDO, Toshiya

51

HIKINO, Kazumi

56

ANSAI, Satoshi

7, 53, 85

HIRA, Seiji

35

26
12, 82, 93
89
38
4, 41, 85

AOKI, Etsuko

45

HIRAMATSU, Mika

45

AOKI, Kazuhiro

15

HIRANO, Kodai

35

AOYAMA, Tsuyoshi

45

HIRAOKA, Yasushi

4

ARATA, Masaki

7, 32

HIYAMA, Takeshi

38

ASAI, Yuriko

25

HORIUCHI, Yuta

45

ASAO, Hisayo

75

HOSHINO, Atsushi

60, 83, 85

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

HSIAO, Yi-Min

76

67

HYODO, Miwa

64

BABA, Nayumi
BAN, Misato
BINO, Takahiro
BURTON-SMITH, Raymond90

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

75, 93ICHIKAWA, Chiaki

78, 91, 93

65

ICHIKAWA, Mariko

75, 91, 93

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

IINUMA, Hideko

89, 93

CHEN, Qiuhong

29

IKEDA, Tatsuro

35

CHIBA, Hirokazu

69

ISHIBASHI, Tomoko

71

CHIKAMI, Yasuhiko

51

ISHII, Satoko

72

D ....................................................................................................

ISHIKAWA, Azusa

78

DAY, F. Timothy

32

ISHIKAWA, Masaki

45

DE VILLIERS, Ruan

45

ISHINE Naomi

62

ISHIZAKA, Miho35

E ....................................................................................................
F

EBINE, Eimi

15

ISOSHIMA, Yoshiko

38

EBINE, Kazuo

12

ITO, Hiroko

41....................................................................................................
38
FUJIKAWA, Akihiro

ITO, Kazuyo

60

ITO, Tomoaki

32

ITO, Yukiko

29

FUJIMORI, Sayumi
FUJIMORI, Toshihiko
FUJIMOTO, DaijiG294, 32, 82, 90, 92, 98, 99
82

FUJITA, Hironori

7, 48

FUKADA-TANAKA, Sachiko48, 93

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

45, 93

FUKADA, Hatsumi

45

KADOWAKI, Tamaka

65

FUKUHARA, Mai

48

KAJIKAWA, Ikumi

45

KAJIURA-KOBAYASHI, Hiroko

53

KAKIZUKA, Taishi

72
65....................................................................................................
GOTO, Misako
45

GOTO, Yuhei

7, 15

KAMADA, Konomi

GU, Nan45

KAMADA, Yoshiaki

GUH, Ying-Jey

67

KAMEI, Yasuhiro

H ....................................................................................................
HACHISUKA, Midori
HAMADA, Kyoko
HARA, Ikuyo

57
78, 79, 85, 91, 97, 98, 99

KAMEMIZU, Chizuru

32

KAMEOKA, Hiromu

48

18

KAMIDA, Chiharu

45

53

72, 73

KAMIGAKI, Akane

56

4, 45, 89, 94

KAMIKAWA, Yasunao

25

HASHIMOTO, Terumi

38

KAMRANI, Yousef Yari65

HASHIMOTO, Yutaka

26

KANAI, Masatake

HATAKEYAMA, Yudai

29

KANAZAWA, Takehiko

HASEBE, Mitsuyasu

HATTORI, Masayuki

102J....................................................................................................
JOHZUKA, Katsuki
6279, 97KANEKO, Hiroyo

56
7, 12
53

INDEXKANIE, Akemi

32

KATAOKA, Kensuke

18

KATAOKA, Yukari

93

KATAYAMA, Kaori

23

KATO, Azusa

32

KATO, Hiroki

65

KATO, Kagayaki

63, 98, 99

KURIHARA, Misuzu

LM....................................................................................................
MAEDA, Taro48

LIU, Meng

MAKINO, Yumiko

KATO, Kyoko

56

MANO, Hiroaki

KAWADE, Kensuke

73

MANO, Shoji

KAWAGUCHI, Colin90, 91

KAWAGUCHI, Haruka

5172....................................................................................................
LIN, Chia-Hao
38

48, 9575, 9345
7, 56, 90, 99

MARUYAMA, Ayumi

35

MARUYAMA, Michiyo

67

KAWAGUCHI, Masayoshi

4, 48, 90

MARYU, Gembu

15

KAWAGUCHI, Takayuki

18

MASE, Mutsuki

51

KAWAI, Hisako

65

MASUOKA, Tomoko

45

KAWAMURA, Manaka

18

MATSUBAYASHI, Naomi

86

KIDA, Emi

65

MATSUDA, Chisato

23

KIM, Eunchul

65

MATSUDA, Takashi

7, 38

KIMORI, Yoshitaka

64, 98, 99

MATSUDA, Yoshimi

89, 93

KIMURA, Tetsuaki

53

MATSUMOTO, Miwako

75

KIMURA, Yukiko

41

MATSUMURA, Kunihiro

82

KINOSHITA, Chie

67

MATSUZAKI, Yoko

45

KINOSHITA, Noriyuki

26

MII, Yusuke

29

KISHI, Kanae

32

MINAGAWA, Jun

KISHIMOTO, Mariko

65

MINAMINO, Naoki

KITADATE, Yu

35

MIURA, Haruko

KIYOHARA, Megumi

51

MIURA, Seiko38

KOBAYASHI, Yuuki

48

MIWA, Tomoki

81, 93, 97

KODAMA, Akiko

38

MIYAKE, Satoko

26

KODAMA, Ryuji

55, 89, 90

4, 65
12
15, 95MIYANARI, Yusuke

72

KOHCHI, Takayuki

4

MIYATA, Haruko

93

KOHSHIMA, Shiro

4

MIZOKAMI, Yuko

86

KOIKE, Chieko

53

MIZUGUCHI, Hiroko

35, 93

KOIKE, Yukari

53

MIZUTANI, Takeshi

51, 93

KOJIMA, Yoko

45, 65

KOMATSUBARA, Akira

15

KOMINE, Yuriko

59, 92KOMORI, Akio
KON, Yayoi

MORI, Asuka
MORI, Tomoko

4575, 93

MORISHITA, Mio

45

2

MORITA, Junko

51

35

MORITA, Shinichi

51

KONDO, Maki78, 93

MOROOKA, Naoki

83, 93

KONDO, Naoko

18

MURAKAMI Eiichi

48

KONDO, Yohei

7, 15

MURAKAMI, Michiyo

KONISHI, Mie

38

KOSHIMIZU, Shizuka

45

KOSUGE, Kotaro

65

NAGAE, Miwa

KOSUGI, Eiko48

NAKADE, Atsuko

KOTANI, Keiko83, 93

KOYAMA, Hiroshi

MURATA, Takashi

26
45, 97, 98

N ....................................................................................................
NAKAGAWA, Mami

48
2132, 7932, 98, 99

NAKAGAWA, Tomomi

48

KUBOKI, Yuko

35

NAKAGAWA, Toshinori

35

KUBOYAMA, Kazuya

38

NAKAMURA, Ryoko

60

KUMAZAKI, Taisei

25

NAKAMURA, Takanori

81, 93, 97

KUME, Shoen

4

NAKAMURA, Yoshiaki

35

KURATA, Tomoko

90

NAKANISHI, Norie

38

103

INDEXNAKANOH, Shota

32

NAKATSUKASA, Mana

67

SAITO, Eiko51

NAKAYAMA, Jun-ichi

18

SAKAGAMI, Mari

21

NAKAYAMA, Kei

23

SAKAI, Hiroki

51

NAKAYAMA, Tomoya

67

SAKAI, Yusuke

7, 26

NARUKAWA, Hideki

45

SAKAMOTO, Joe

79

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

NARUSE, Kiyoshi

53, 85, 86

SAKAZAKI, Takuya

45

NEGISHI, Takefumi

26

SAKUTA, Hiraki

38

4, 51

SANBO, Chiaki

72

NISHI, Tayo

45

SASAKI, Hiroyuki

4

NISHIDA, Hanna

48

SATO, Ryoichi

65

NISHIDE, Hiroyo

81, 93, 97

SATO, Toshiyuki

35

NISHIMURA, Akiko

91

SAWADA, Kaoru

89, 93NISHIMURA, Keiko

35

SHIBATA, Emiko93

NISHIMURA, Mikio

90

SHICHINO, Yuichi

21

NISHITANI, Kazuhiko

4

SHIGENOBU, Shuji

75, 76, 91, 96, 97

41, 44SHIINA, Nobuyuki

7, 23

SHIMAZAKI, Takashi

41

65, 93

SHINOMIYA, Ai

67

NODA, Masaharu

4, 38

SHINOZUKA, Takuma

29

NOGUCHI, Yuji

82, 93SHINTANI, Takafumi

38

82

SOYANO, Takashi

48

38

NIIMI, Teruyuki

NISHIUMI, Nozomi
NOBATA, Ryuko
NODA, Chiyo

NOMOTO, Yoshihiro

29

SUDA, Hiraku

45

NONAKA, Shigenori

71, 97, 98, 99

SUGAYA, Tomomi

45

NONOMURA, Keiko7, 32

SUGIMOTO, Asako

4

NORIZUKI, Takuya

7, 12SUGINAGA, Tomomi

NOMURA, Kengo

82, 93

NOSE, Akinao

4

SUGIOKA, Takumi

41

NOZAKI, Mamoru

73

SUZAKI, Momoyo

48

O ....................................................................................................

SUZUKI, Makoto

26

ODA, Akiko

48

SUZUKI, Miho

26

ODA, Shigekazu

15

SUZUKI, Miyuzu

76

OGAWA, Kota

76

SUZUKI, Ryoko

38

OGAWA, Yuko

48

SUZUKI, Sachiko

41

OHASHI, Rie

23

SUZUKI, Tokiko

53

OHDE, Takahiro

7, 51

OHNO, Kaoru

58

TAGAWA, Ayako

72

OHSAWA, Sonoko

82, 93TAKADA, Ritsuko

29

OKA, Naomi

81, 93TAKADA, Shinji

4, 29, 92, 99

OKA, Sanae32, 93

TAKAGI, Chiyo

26, 93

OKADA, Kazunori

29

TAKAGI, Yukari

82, 93

OKAJIMA, Keisuke

65

TAKAHASHI, Hiroki

26

OKI, Koichi

18

TAKAHASHI, Nobuaki

82

TAKAHASHI, Ritsue

91

OKUBO, Masayo

12, 67

T ....................................................................................................

OKUMA, Nao

48

TAKAHASHI, Shunichi

65

ONODA, Kaori

15

TAKAKI, Chikako

53

OOI, Shoko

45

TAKASHIRO, Kayoko

29

OTA, Kyoko

90

TAKEDA, Naoya

7, 48

OTSUBO, Yoko

21

TAKEHANA, Yusuke

7, 53

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

TAKEMOTO, Kodai

1245

TAKETSURU, Hiroaki

R ....................................................................................................

TAKEUCHI, Tomoyo

60

26

TAKEUCHI, Yasushi

38, 93

PALFALVI, Gergo
RAJESHWAR, Raj

104

86, 87

INDEXTAKEUCHI, Yoshiko

41

YAMAMOTO, Takashi

4

TAKIZAWA, Kenji

65, 74

YAMASAKI, Hiroaki

65

TAMADA, Yosuke

45, 97YAMASAKI, Tomohito

65

TANAKA, Mayo

18

YAMASHITA, Akira

21

TANGA, Naomi

38

YAMASHITA, Akira

2378, 93

YAMAZAKI, Toko53

TANIGUCHI, Atsushi

71

YANAKA, Ayako

65

TATEMATSU, Kiyoshi

91

YANOGAWA, Azusa

53

TERASAWA, Yoko

41

YASUE, Naoko

26

TESHIMA, Yuuko

53

YASUGI, Masaki

44

TOKAIRIN, Asami

48

YASUI, Naomi

25

TOKUE, Moe

35

YATOMI, Johichiro

51

TOKUMOTO, Yuji

48

YOKOI, Saori

53

TOKUTSU, Ryutaro

65

YOKOYAMA, Michiko65

TOMINAGA, Hitoshi

26

YONEZAWA, Harumi

65

TOMOI, Takumi

73

YOSHIDA, Akihiro

55

TORII, Naoko

53

YOSHIDA, Saiko7, 45

TOYAMA, Mami

65

YOSHIDA, Shosei

4, 35, 75, 91

TRAN, Thi Hong Nguyen

29

YOSHIDO, Kana

35

TANIGUCHI-SAIDA, Misako

TSUBOUCHI, Hideo

U

25

YOSHIMURA, Takashi

67

TSUBOUCHI, Tomomi

25, 92YOSHIMURA, Yuriko

18

TSUCHIYA, Yoshihiro

29

YOSHINORI, Yumi

48

YU, Yang

38

TSUGANE, Kazuo

61, 83TSUGANE, Mika

48

TSUGE, Toyoko

26

TSUKAYA, Hirokazu

73

TSUTSUI, Tomokazu

12

TSUZUKI, Chizuru

86

TSUZUKI, Shihoko

93

Z ....................................................................................................
ZHANG, Liechi

45....................................................................................................
UCHIYAMA, Ikuo
69, 81, 97

UDA, Youichi

15

UEDA, Chizuru

56

UEDA, Takashi

4, 12, 94

UEMURA, Yuto

41

UENO, Naoto

2, 4, 26, 90, 91, 96, 98, 99

UENO, Yasuko

65

UNO, Satoko

93

USAMI, Fumiko

32

UTSUMI, Hideko

29, 93

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

38

WATAKABE, Ikuko

41

WATANABE, Akimasa
WATANABE, Eiji
WEN, Hsin-I

65
44, 82, 84
45

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

29

YAMAGUCHI, Chinami

73

YAMAGUCHI, Katsushi

75, 93, 97

YAMAMOTO, Masayuki

2, 21, 99

YAMAMOTO, Takamasa

26105