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National Institute for Basic Biology

2019 ANNUAL REPORT
Jan. 2019 - Mar. 2020

CONTENTS
Introduction…………………………………………………………………………………………… 1
Organization of the National Institute for Basic Biology…………………………………… 2
Goals of the National Institute for Basic Biology……………………………………………… 5
Personnel Changes and Awardees January 2019 to March 2020…………………………… 7
Selected Press Releases from NIBB……………………………………………………………… 8
Cell Biology
Division of Cellular Dynamics… …………………………………………………………… 12
Division of Quantitative Biology……………………………………………………………… 15
Division of Chromatin Regulation…………………………………………………………… 18
Laboratory of Neuronal Cell Biology………………………………………………………… 21
Laboratory of Stem Cell Biology… ………………………………………………………… 23
Laboratory of Organelle Regulation………………………………………………………… 24
Developmental Biology
Division of Morphogenesis…………………………………………………………………… 26
Division of Molecular and Developmental Biology………………………………………… 29
Division of Embryology… …………………………………………………………………… 32
Division of Germ Cell Biology… …………………………………………………………… 35
Labotratory of Regeneration Biology… …………………………………………………… 38
Neurobiology
Division of Behavioral Neurobiology………………………………………………………… 39
Laboratory of Neurophysiology……………………………………………………………… 42
Evolutionary Biology and Biodiversity
Division of Evolutionary Biology… ………………………………………………………… 44
Division of Symbiotic Systems… …………………………………………………………… 47
Division of Evolutionary Developmental Biology… ……………………………………… 50
Laboratory of Evolutionary Genomics……………………………………………………… 52
Laboratory of Bioresources… ……………………………………………………………… 54
Laboratory of Morphodiversity… …………………………………………………………… 56
Laboratory of Biological Diversity…………………………………………………………… 57
Environmental Biology
Division of Environmental Photobiology… ………………………………………………… 70
Division of Plant Environmental Responses… …………………………………………… 72
Theoretical Biology
Laboratory of Genome Informatics… ……………………………………………………… 75
Imaging Science
Laboratory for Spatiotemporal Regulations………………………………………………… 77
Laboratory for Biothermology… …………………………………………………………… 78
NINS ExCELLS (ORION Project)
Laboratory of Nuclear Dynamic……………………………………………………………… 80
NINS ExCELLS (BIO-NEXT Project)
Laboratory of Plant Development and Physiology………………………………………… 81
Research Support
NIBB Core Research Facilities……………………………………………………………… 82
NIBB BioResource Center…………………………………………………………………… 87
National BioResource Project… …………………………………………………………… 90
NIBB Center for the Interuniversity Bio-Backup Project… ……………………………… 91
Center for the Development of New Model Organisms…………………………………… 93
Center for Radioisotope Facilities (Okazaki Research Facilities)… …………………… 95
Research Enhancement Strategy Office…………………………………………………… 96
Technical Division… ………………………………………………………………………… 99
NIBB Conferences………………………………………………………………………………… 100
NIBB Princeton Symposium… ………………………………………………………………… 102
Collaboration Programs with Overseas Institutions… …………………………………… 103
Collaborative Activities with Joint Usage / Research Center… ………………………… 105
NIBB Bioimaging Forum… ……………………………………………………………………… 106
NIBB Training Courses…………………………………………………………………………… 107
Advanced Bioimaging Support………………………………………………………………… 108
NIBB Internship Program………………………………………………………………………… 110
NIBB Openhouse… ……………………………………………………………………………… 111
Outreach Activities… …………………………………………………………………………… 112
Access to NIBB… ………………………………………………………………… Inside back cover
The cover items are related to a paper titled “A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus” (Soyano et al., Science 2019) from the laboratory of Prof. Kawaguchi. The paper demonstrated that legumes co-opted a transcription factor involved in early lateral root development for producing root
nodules that house symbiotic nitrogen-fixing bacteria. See page 48 of this report for details.

INTRODUCTIONI

t is my great pleasure to present to you all the
National Institute for Basic Biology (NIBB)’s 2019
Annual Report which outlines NIBB’s research, educational,
and international activities and its effective function as acenter for collaborative research in Japan from January 2019
to March 2020.
NIBB aims to establish an international base for biologicalsciences by using not only model organisms, but also exotic
non-model organisms. Consequently, NIBB established theCenter for the Development of New Model Organisms in
2014. We have endeavored to develop a one-step pipeline toconnect genome sequencing, assembling, and annotation to
genome editing using non-model organisms such as carnivorous plants, unique insects, and regenerative newts, and thisvision was expounded upon in the April 23rd, 2019 edition of
Nature News.
NIBB also developed front bioimaging technologies toacquire high-resolution 3D images and promote quantitative biology in collaboration with Princeton University.
In keeping with this, the 66th NIBB Conference/ABiS
International Symposium “Cutting Edge Techniques of
Bioimaging” and the 2nd NIBB-Princeton Symposium“Imaging and Quantitative Biology” were held in Okazaki.
Collaborative studies by researchers from within and external
to NIBB have used our bioimaging technologies to generate
a lot of fascinating papers (see below).
Concerning changes on the staffing front, Professor
Masaharu Noda, an active researcher in the field of integrative neurosciences at NIBB since September 1991, retired
in March 2019; roughly one more prior to my appointment
as the Director-General of NIBB in April 2019. Associate
Professor Shuji Shigenobu was also promoted to the role oftaking majority responsibility for managing the Center for
the Development of New Model Organisms. Additionally,
three assistant professors, two specially appointed assistantprofessors, and four research fellows were newly posted to
NIBB within this year.
Moreover, both I and Vice-Director, Professor Naoto
Ueno, visited Heidelberg, Germany in early July and finalized an academic exchange agreement with the Centre
for Organismal Studies (COS) in the aforementioned
location, and extended the academic exchange agreementwith European Molecular Biological Laboratory (EMBL).
Following this, “The 67th NIBB Conference/The 6thInternational “Quest for Orthologs” Meeting was held in
September in Okazaki.
I would like to finish this introduction by congratulating
Professor Mitsuyasu Hasebe who was awarded the Golden
Spore Award 2019 from the International Molecular Moss
Science Society.
To establish NIBB as an internationally leading institute in
the field of basic biology, we always welcome your suggestions, comments, and queries concerning our activities, inaddition to your warm support.

Kiyokazu Agata
Director General of NIBB
December, 2020
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 designated to promote and stimulate thestudy of biology both in Japan and internationally. NIBB
conducts first-rate research on site as well as in cooperation
with national, public and private universities and researchorganizations. NIBB attempts to elucidate the general andfundamental mechanisms underlying various biologicalphenomena throughout a wide variety of biological fields,such as cell, developmental, evolutionary, environmental andtheoretical biology as well as neurobiology.
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.

■ Organization

National Institutes of
Natural Sciences(NINS)

President
KOMORI, Akio
National Astronomical
Observatory of Japan (NAOJ )National Institute for
Fusion Science (NIFS)

National Institute for
Basic Biology (NIBB)

Director General
AGATA, Kiyokazu
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
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 Programming andManagement. The Advisory Committee, comprised ofeleven professors within NIBB and ten leading biologists
from outside of it, 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 founded in 2013.
The Office is made up of seven groups (p. 96) 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.

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

2

Center for Novel Science Initiatives■Frontier Photonic Science■Plasma Bio Science
Astrobiology Center
Exploratory Research Center onLife and Living Systems (ExCELLS)■Department of Creative Research■Section for Exploration of Life inExtreme Environments

Research units of NIBB with an asterisk on theright panel also function as research groups of
ExCELLS.

Okazaki Research Facilities
Center for Radioisotope Facilities
Center for Animal Resources and Collaborative Study
Research Center for Computational Science

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

As of March 31, 2020

Research Units■Division of Cellular Dynamics

Cell Biology■Division of Quantitative Biology*■Division of Chromatin Regulation■Laboratory of Neuronal Cell Biology*■Laboratory of Stem Cell Biology■Laboratory of Organelle Regulation■Division of Morphogenesis

Developmental Biology■Division of Molecular and Developmental

Biology*■Division of Embryology■Division of Germ Cell Biology■Laboratory of Regeneration Biology■Division of Behavioral Neurobiology*■Laboratory of Neurophysiology

Neurobiology■Evaluation and Information Group■Public Relations Group■International Cooperation Group

Evolutionary Biology and Biodiversity■Division of Evolutionary Biology■Division of Symbiotic Systems■Division of Evolutionary Developmental

Biology■Collaborative Research Group■Laboratory of Evolutionary Genomics■Young Researcher Support Group■Laboratory of Bioresources■Gender Equality Promotion Group■Laboratory of Morphodiversity■Laboratory of Biological Diversity

Environmental Biology■Division of Environmental Photobiology

Theoretical Biology■Laboratory of Genome Informatics

Imaging Science■Laboratory for Spatiotemporal Regulations*■Division of Plant Environmental Responses■Laboratory for Biothermology

Research Support Facilities
NIBB Core Research Facilities
NIBB BioResource Center■Functional Genomics Facility■Spectrography and Bioimaging Facility■Data Integration and Analysis Facility■Model Animal Research Facility■Model Plant Research Facility■Cell Biology Research Facility

NIBB Center of the Interuniversity Bio-Backup Project (IBBP Center)
Center for the Development of New Model Organisms
Technical Division
Section of Health and Safety Management

Okazaki Administration Center3

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 the loss of invaluable biological resources. Projects for the development of bioresource
preservation technology are solicited by the IBBP center.
The Center for the Development of New Model Organisms
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. The
Center for Radioisotope Facilities are one of the latter andrun by the technical staff of NIBB.
The Exploratory Research Center on Life and Living
Systems (ExCELLS) was founded in 2018 to develop novel
approaches for observing biological entities, deciphering
hidden information, and creating living systems to improve
our understanding of their nature. Moreover, ExCELLS
promotes collaborative, interdisciplinary research involving
investigators exploring organisms living in extreme environments.

■ Members in NIBB
Director General 1
Professors(including specially-appointed) 15
Associate Professors(incl. sp. app.) 14

Technical
Assistants
andSecretaries

50

114

40
55

27

Graduate andUndergraduate Students(2 are undergraduates)

Assistant Professors(incl. sp. app.)

Postdoctoral Fellows

Technical Staff

316 in total(as of 1st April, 2020)■ Financial Configuration of NIBB
Indirect Costs in Grants

104

Other Grants

77

External
Competitive
Fund

863

Grant-in-Aid
for Scientific
Research

765

Management
Expenses Grant
for NIBB

1,277

Research
Collaboration 185

293

Management Expenses
Grant for SOKENDAI

50

Personnel

389

Grants forCommissioned Research(settled forApril 2019-March 2020)
numbers are in million yen

Donations

11325

Other 2

2,419 million yen
in total■ Members of the Advisory Committee for Programming and Management (terms of April 2019 to March 2021)

Non-NIBB members

NIBB members# Chairperson## Vice-Chair

4

HANASHIMA, Carina
Professor, Waseda University
HIRAOKA, Yasushi ##Professor, Osaka University
KITANO, Jun
Professor, National Institute of Genetics
KOHCHI, Takayuki
Professor, Kyoto University
KUROIWA, Asato
Professor, Hokkaido University
SATAKE, Akiko
Professor, Kyushu University
SIOMI, Mikiko
Professor, The University of Tokyo
SUGIMOTO, Asako
Professor, Tohoku University
YAMAMOTO, Takashi
Professor, Hiroshima University
YOSHIMURA, Takashi
Professor, Nagoya University
AOKI, Kazuhiro
Professor, ExCELLS and National Institute for Basic Biology
FUJIMORI, Toshihiko
Professor, National Institute for Basic Biology
HASEBE, Mitsuyasu
Professor, National Institute for Basic Biology
HIGASHIJIMA, Shin-ichi
Professor, ExCELLS and National Institute for Basic Biology
KAWAGUCHI, Masayoshi # Professor, National Institute for Basic Biology
MINAGAWA, Jun
Professor, National Institute for Basic Biology
NIIMI, Teruyuki
Professor, National Institute for Basic Biology
TAKADA, Shinji
Professor, ExCELLS and National Institute for Basic Biology
UEDA, Takashi
Professor, National Institute for Basic Biology
UENO, Naoto
Professor, National Institute for Basic Biology
YOSHIDA, Shosei
Professor, National Institute for Basic Biology

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

Promotion of Collaborative Research■ Collaborative Research Support

Research activities that are conducted using NIBB’s facilities and in collaboration with NIBB’s divisions/laboratories are solicited from external researchers. “Individual
Collaborative Research Projects” are a basic method of
supporting collaborations which provide external researcherswith travel and lodging expenses to visit NIBB’s laboratories
to conduct collaborative research. “Priority Collaborative
Research Projects” are carried out as group research projects
by internal and external researchers to develop pioneeringresearch fields. “Collaborative Research Projects for Model
Organism/Technology Development” and “Collaborative
Research Projects for Bioresource Preservation Technology
Development” are for developing and establishing newmodel organisms and new research technology. Researchexpenses in addition to travel expenses are provided for theseprojects. ‘Collaborative Research Projects for Integrative
Genomics’ and ‘Collaborative Research Projects for
Integrative Bioimaging’ are 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 also provided for these projects.
year
Priority collaborative research projects

2017 2018 20192

1

1

Collaborative research projects for modelorganisms and technology development

2

2

2

Individual collaborative research projects

51

57

60

Collaborative research projects for integrative genomics

62

67

66

Collaborative research projects for integrative bioimaging

28

23

22

NIBB workshops

3

2

3

Collaborative experiments using the Large
Spectrograph

9

9

9

Support for NIBB training courses

0

1

0

Collaborative research projects for bioresource preservation technology development

12

18

14

Total

169

180

177

■ NIBB Core Research Facilities

The NIBB Core Research Facilities support research at
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. 82).
The Functional Genomics Facility maintains a wide arrayof core research equipment, including next generation DNA
sequencers. The facility is dedicated to fostering NIBB’s

International cooperation
Contribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchers

collaborative research by providing these tools as well as
expertise. Its current focus is supporting functional genomics
projects that utilize mass spectrometers and DNA sequencers, and holding events such as training courses to achieve
this end (p. 106). The Spectrography and Bioimaging Facility
manages research tools, such as confocal microscopes,
DSLM and the large spectrograph, and provides technicalsupport and scientific advice to researchers. These two facilities task professor and specially appointed associate professor, who are experts in their respective fields, with managing
each facility as well as conducting their own academic
research. The Data Integration and Analysis Facility supports
the analysis of large-scale biological data, such as genomic
sequence data, gene expression data, and imaging data. The
facility maintains high-performance computers with largecapacity storage systems for this purpose.

■ NIBB BioResource Center

The NIBB BioResource Center supports research using
model animals and plants at 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 tomaintain model organisms, such as mice, medaka, zebraﬁsh,Japanese morning glories, Arabidopsis, Lotus japonicus, andPhyscomitrella patens, and provides technical support andadvice for the appropriate use of these organisms (p. 87).
The center also acts 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 selected as a center for research on
medaka (Oryzia latipes), whose usefulness as a vertebratemodel was first demonstrated 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 glories (p. 90).

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

5

■ 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 used in cutting-edge research (p. 91).

■ C enter for the Development of New Model
Organisms

This center was established in 2013 and employs a crossappointment researcher who developed and refined genome
editing techniques in particular in Pleurodeles waltl (Iberianribbed newt) (p. 93) and also taught these techniques in the
training course (p. 108).

■ Advanced Bioimaging Support (ABiS)

ABiS provides an assistance for advanced imaging inresearch 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. In 2018, ABiS joined the Global Bioimaging(GBI) network representing the Japanese bioimaging community. ABiS also hosts imaging competitions and training
courses (p. 108).

International Cooperation and Outreach■ Collaborative Programs with Overseas Institutes

NIBB plays 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. The NIBBPrinceton Joint Symposium “Imaging and Quantitative
Biology” was held in October, 2019 (p. 102).

■ NIBB Conference

The NIBB Conferences are international conferences on
prominent topics in biology that are 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 forinternational exchange. The 66th conference “Cutting Edge
Techniques of Bioimaging” was held jointly with ABiS in
February, 2019 (p. 100). The 67th conference “Quest for
Orthologs” was hels in July, 2019 (p. 101).

■ International Practical Course

With the cooperation of researchers from Japan and abroad,the NIBB international practical course is held in a specifically prepared laboratory. The 10th course “Genome Editing
and Imaging of Fish and Amphibians” was held jointly with
ABiS in September, 2018 at NIBB. Graduate students and6young researchers from various areas including the UK,
Columbia, Nepal, Korea, China, Taiwan, and Japan, were
provided with training in state-of-the-art research techniques. International conferences and courses are managed
by the International Cooperation Group of the Research
Enhancement Strategy Office.

■ Outreach

NIBB’s outreach activities aim to present cutting edge
research results to the public via mass media through press
releases or directly through internet-based platforms, suchas web pages, Facebook, and Twitter. Streaming live videosof the development of model organisms successfully attract
many accesses. Our triannual open campus event was heldin October, 2019 at which we welcomed nearly 3,000 local
citizens (p. 111). NIBB also cooperates in the educationof undergraduate and younger students through lectures
and workshops. Outreach activities are mostly managed by
the Public Relations Group of the Research Enhancement
Strategy Office (p. 96).

Development of New Fields of Biology■ Bioimaging

NIBB aims to maximize the application of modern lightmicroscopes and biophotonic probes for real time visualization of biological phenomena and to develop new imaging
techniques. As part of our collaborative work with EMBL,NIBB introduced a DSLM, which is effective for the threedimensional observation of living organisms, and has developed an improved model using two-photon optics (p. 77).
The Advisory Committee on Bioimaging, comprised ofleading researchers in the bioimaging field in Japan, has beenorganized to formulate advice concerning NIBB’s imaging
research. The Bioimaging Forum provides an opportunity
for researchers and company engineers to frankly discuss
practical difficulties and their needs regarding imaging. The
13th Forum “Behavioral and Recognition Research upon the
Platform of Vision and Color” was held in February, 2019 (p.
106). A training course in bioimage analysis was also held in
December, 2019 (p. 107).

■ Okazaki Biology Conferences

NIBB holds Okazaki Biology Conferences (OBC) that
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 that seek
strategies for addressing critical future 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 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 at least once during their master’s and doctoral
program, where they are provided with an opportunity togive oral and poster presentations.
Students from Japan and abroad can also come to NIBB
through our Internship Program. Internships give students
an excellent opportunity to build international connections while experiencing hands on research in a world classresearch institute (p. 110).

Support for young researchers is managed by the Young
Researcher Support Group of the Research Enhancement
Strategy Office (p. 98).

60
50

Graduate Students Educated by NIBB
(numbers counted at the end of April each year)
7

40

7

11

13

42

37

39

2015

2016

2017

6

11

9

36

36

47

2018

2019

2020

30
20
10
0

46
2014

■ SOKENDAI Graduate Student■ Special Research Student■ Personnel changes from January 2019 to March 2020*

Newly assigned to NIBB
Name

Position

Research Unit

Date

AGATA, Kiyokazu

Director General

Laratory of Regeneration Biology

April 1, 2019

SHIGENOBU, Shuji

Professor

Laboratory of Evolutionary Genomics

April 1, 2019

TANIMOTO, Masashi

Assistant Professor

Division of Behavioral Neurobiology

April 1, 2019

SHIKATA, Hiromasa

Assistant Professor

Division of Plant Environmental Responses

April 1, 2019

KOBAYASHI, Taisuke NIBB Research Fellow#

Laboratory of Neurophysiology

April 1, 2019

YOKE, Hiroshi

Laboratory for Spatiotemporal Regulations

April 1, 2019

SHINOZUKA, Takuma NIBB Research Fellow#

Division of Molecular and Developmental Biology

April 1, 2019

LIU, Meng

NIBB Research Fellow#

Division of Symbiotic Systems

October 1, 2019

GOTO, Yuhei

Assistant Professor

Division of Quantitative Biology

November 1, 2019

MANO, Hiroaki

Specially Appointed Assistant Professor

Division of Evolutionary Biology

November 1, 2019KANAI, Masatake

Specially Appointed Assistant Professor

Laboratory of Organelle Regulation

February 1, 2020

NIBB Research Fellow#

Newly affiliated with other universities and institutes
Date

PositionName

New Affiliation

NODA, Masaharu

Tokyo Institute of Technology

Specially Appointed Professor

April 1, 2019SHINTANI, Takafumi

Tokyo Institute of Technology

Specially Appointed Associate Professor

April 1, 2019

MATSUDA, Takashi

Tokyo Institute of Technology

Specially Appointed Assistant Professor

April 1, 2019

HIYAMA, Takeshi

Okayama University

Lecturer

April 1, 2019SUZUKI, Makoto

Hiroshima University

Assistant Professor

April 1, 2019

TAMADA, Yosuke

Utsunomiya University

Associate Professor

August 1, 2019

ANSAI, Satoshi

Tohoku University

Assistant Professor

November 1, 2019

MIYANARI, Yusuke

Kanazawa University

Associate Professor

April 1, 2020

MURATA, Takashi

Kanagawa Institute of Technology

Professor

April 1, 2020

* Changes in professors, associate/ assistant professors, and NIBB research fellows.
# NIBB Research Fellows were reappointed as Specially Appointed Assistant Professors on April 1, 2020.

■ Awardees from January 2019 to March 2020
Name

PositionAward

KITADATE, Yu

Assistant Professor

NINS Young Researcher Award

HASEBE, Mitsuyasu

Professor

Golden Spore Award 2019 (International Molecular Moss Science Society)7Selected Press Releases from NIBB (Jan. 2019 to Mar. 2020)
Press releases on a paper whose first and corresponding authors both have affiliation in NIBB

■ 1 January, 2019
Elucidating algal protection mechanisms against excessively strong light and its
core component, E3 ubiquitin ligase, prevailing from plants to animals
Aihara, Y., Fujimura-Kamada, K., Yamasaki, T., and Minagawa, J. (2019). Algal photoprotection is regulated by the E3 ligase
CUL4-DDB1DET1. Nat. Plants 5, 34-40. doi: 10.1038/s41477-018-0332-5■ 17 January, 2019
The characterization of a group of proteins necessary for intracellularcompartmentalization not performed by the cell membrane: Formation ofliquid and solid-like RNA granules by proteins related to long-term memory,
ALS and dementia
Shiina, N. (2019). Liquid- and solid-like RNA granules form through specific scaffold proteins and combine into biphasic
granules. J. Biol. Chem. 294, 3532-3548. doi: 10.1074/jbc.RA118.005423■ 18 January, 2019
The mechanism of left-right asymmetrical function of the brain firstly elucidated
at cellular level
Shimazaki, T., Tanimoto, M., Oda, Y., and Higashijima, S. (2019). Behavioral role of the reciprocal inhibition between a pairof Mauthner cells during fast escapes in zebrafish. J. Neurosci. 39, 1182-1194. doi: 10.1523/JNEUROSCI.1964-18.2018■ 22 January, 2019
Green fluorescence from reef-building corals attracts symbiotic algae
Aihara, Y., Maruyama, S., Baird, A.H., Iguchi, A., Takahashi, S., and Minagawa, J. (2019). Green fluorescence fromcnidarian hosts attracts symbiotic algae. Proc. Natl. Acad. Sci. USA 116, 2118-2123. doi: 10.1073/pnas.1812257116■ 7 February, 2019
The novel mechanism of intercellular signaling caused by morphological changes
in cells: Deformation of Wnt producing cells promotes the proliferation of neural
stem/progenitor cells
Shinozuka, T., Takada, R., Yoshida, S., Yonemura, S., and Takada, S. (2019). Wnt produced by stretched roof-plate cells is
required for the promotion of cell proliferation around the central canal of the spinal cord. Development 146, dev159343. doi:
10.1242/dev.159343

8

■ 7 March, 2019
Elucidating cellular responses to force: Mechanical force induces
phosphorylation-mediated signaling that underlies tissue response androbustness in Xenopus embryos
Hashimoto, Y., Kinoshita, N., Greco, T.M., Federspiel, J.D., Jean Beltran, P.M., Ueno, N., and Cristea, I.M. (2019).
Mechanical force induces phosphorylation-mediated signaling that underlies tissue response and robustness in Xenopus
embryos. Cell Syst. 8, 226-241. doi: 10.1016/j.cels.2019.01.006■ 8 April, 2019
The mechanism underlying the temporal suspension (diapose) of thedevelopment of mammalian embryos: The timing of suspension and resumptionof development differs depending on embryonic region and the individual cell
Kamemizu, C., and Fujimori, T. (2019). Distinct dormancy progression depending on embryonic regions during mouse embryonic diapause. Biol. Reprod. 100, 1204-1214. doi: 10.1093/biolre/ioz017■ 9 April, 2019
LDAIR, a lncRNA regulates seasonal changes in stress response:
Unraveling the mechanism for seasonal adaptation in animals
Nakayama, T., Shimmura, T., Shinomiya, A., Okimura, K., Takehana, Y., Furukawa, Y., Shimo, T., Senga, T.,
Nakatsukasa, M., Nishimura, T., Tanaka, M., Okubo, K., Kamei, Y., Naruse, K., and Yoshimura, T. (2019).
Seasonal regulation of the lncRNA LDAIR modulates self-protective behaviours during the breeding season. Nat.
Ecol. Evol. 3, 845-852. doi: 10.1038/s41559-019-0866-6■ 11 April, 2019
When do male and female differences appear in the development of
beetle horns?
Morita, S., Ando, T., Maeno, A., Mizutani, T., Mase, M., Shigenobu, S., and Niimi, T. (2019). Precise staging
of beetle horn formation in Trypoxylus dichotomus reveals the pleiotropic roles of doublesex depending on thespatiotemporal developmental contexts. PLoS Genet. 15, e1008063. doi: 10.1371/journal.pgen.1008063■ 26 April, 2019
Single-living-cell quantification of the concentrations
and dissociation constants of endogenous proteins bygenome editing and microscopic techniques
Komatsubara, A.T., Goto, Y., Kondo, Y., Matsuda, M., and Aoki, K. (2019). Singlecell quantification of the concentrations and dissociation constants of endogenous
proteins. J. Biol. Chem. 294, 6062-6072. doi: 10.1074/jbc.RA119.007685■ 30 April, 2019
An important function of non-nucleated sperm
Sakai, H., Oshima, H., Yuri, K., Gotoh, H., Daimon, T., Yaginuma, T., Sahara, K., and Niimi, T. (2019). Dimorphic sperm formation by Sex-lethal. Proc. Natl. Acad. Sci. USA 116, 10412-10417. doi: 10.1073/pnas.1820101116

9

■ 22 May, 2019
Identifying the neural mechanisms that inhibit slow muscle activity during
fast swimming in fish
Kimura, Y., and Higashijima, S.-I. (2019). Regulation of locomotor speed and selection of active sets of neurons by V1
neurons. Nat. Commun. 10, 2268. doi: 10.1038/s41467-019-09871-x■ 20 June, 2019
A novel mechanism for increasing the quality and quantity ofoils in soybeans
Kanai, M., Yamada, T., Hayashi, M., Mano, S., and Nishimura, M. (2019). Soybean (Glycine max
L.) triacylglycerol lipase GmSDP1 regulates the quality and quantity of seed oil. Sci. Rep. 9, 8924.
doi: 10.1038/s41598-019-45331-8■ 9 July, 2019
Finding of STEMIN (STEM CELL INDUCING FACTOR) for
feasible reprogramming in plants
Ishikawa, M., Morishita, M., Higuchi, Y., Ichikawa, S., Ishikawa, T., Nishiyama, T., Kabeya, Y.,
Hiwatashi, Y., Kurata, T., Kubo, M., Shigenobu, S., Tamada, Y., Sato, Y., and Hasebe, M. (2019).
Physcomitrella STEMIN transcription factor induces stem cell formation with epigenetic reprogramming. Nat. Plants 5, 681-690. doi: 10.1038/s41477-019-0464-2■ 10 September, 2019
The discovery of a new function of the gene (CONSTANS) which
controls the timing of flower formation in flowering plants: CONSTANS
controlls photoprotection in green algae
Tokutsu, R., Fujimura-Kamada, K., Matsuo, T., Yamasaki, T., and Minagawa, J. (2019). The CONSTANS
flowering complex controls the protective response of photosynthesis in the green alga Chlamydomonas. Nat.
Commun. 10, 4099. doi: 10.1038/s41467-019-11989-x■ 22 November, 2019
Recruitment of a lateral root developmental pathway into root nodule
formation of legumes
Soyano, T., Shimoda, Y., Kawaguchi, M., and Hayashi, M. (2019). A shared gene drives lateral root development
and root nodule symbiosis pathways in Lotus. Science 366, 1021-1023. doi: 10.1126/science.aax2153■ 26 November, 2019
The determination of the 3d structure of the supercomplex between thelight-harvesting complex II and the photosystem II: The full picture of ahuge light-harvesting machine with a molecular weight of 1.66 million
Sheng, X., Watanabe, A., Li, A., Kim, E., Song, C., Murata, K., Song, D., Minagawa, J., and Liu, Z. (2019).
Structural insight into light harvesting for photosystem II in green algae. Nat. Plants 5, 1320-1330. doi: 10.1038/
s41477-019-0543-4

10

■ 7 January, 2020
Discovery of a new factor informing the plant root of the direction of gravity:
A mechanism that makes auxin flow toward the direction of gravity
Furutani, M., Hirano, Y., Nishimura, T., Nakamura, M., Taniguchi, M., Suzuki, K., Oshida, R., Kondo, C., Sun, S., Kato,
K., Fukao, Y., Hakoshima, T., and Morita, M.T. (2020). Polar recruitment of RLD by LAZY1-like protein during gravity
signaling in root branch angle control. Nat. Commun. 11, 76. doi: 10.1038/s41467-019-13729-7■ 5 February, 2020
Discovery of a mechanism that selectively eliminates thetranscripts of meiotic genes necessary for the formation of
reproductive cells
Shichino, Y., Otsubo, Y., Yamamoto, M., and Yamashita, A. (2020). Meiotic gene silencing complex
MTREC/NURS recruits the nuclear exosome to YTH-RNA-binding protein Mmi1. PLoS Genet. 16,
e1008598. doi: 10.1371/journal.pgen.1008598■ 18 February, 2020
The oxytocin (love hormone) controls mate choice in medaka:
Oxytocin works in a contrary manner between males and females
Yokoi, S., Naruse, K., Kamei, Y., Ansai, S., Kinoshita, M., Mito, M., Iwasaki, S., Inoue, S., Okuyama, T.,
Nakagawa, S., Young, L.J., and Takeuchi, H. (2020). Sexually dimorphic role of oxytocin in medaka matechoice. Proc. Natl. Acad. Sci. USA 117, 4802-4808. doi: 10.1073/pnas.1921446117■ 4 March, 2020
The importance of the commissural inhibitory neurons that control
coordinated left-right body movement in larval zebrafish
Satou, C., Sugioka, T., Uemura, Y., Shimazaki, T., Zmarz, P., Kimura, Y., and Higashijima, S.-I. (2020). Functional
diversity of glycinergic commissural inhibitory neurons in larval zebrafish. Cell Rep. 30, 3036-3050. doi: 10.1016/j.
celrep.2020.02.015■ 18 March, 2020
Control mechanism of force-induced cell-to-cell adhesion
Kinoshita, N., Hashimoto, Y., Yasue, N., Suzuki, M., Cristea, I.M., and Ueno, N. (2020). Mechanical stress regulates epithelial
tissue integrity and stiffness through the FGFR/Erk2 signaling pathway during embryogenesis. Cell Rep. 30, 3875-3888. doi:
10.1016/j.celrep.2020.02.074

11

National Institute for Basic Biology

Cell Biology

DIVISION OF CELLULAR DYNAMICS

Professor

UEDA, Takashi

Assistant Professor:

EBINE, Kazuo
KANAZAWA, TakehikoTechnical Staff:
HAYASHI, Kohji
NIBB Research Fellow:
MINAMINO, Naoki
Postdoctoral Fellow:
MURO, Keita
SOKENDAI Graduate Student: HACHINODA, Shou
Visiting Graduate Student: NORIZUKI, Takuya
KAJIWARA, Keiji
Technical Assistant:
YAMAMOTO, Mayuko
OHARA, Satomi
Secretary:
OKUBO, Masayo
Membrane traffic between single membrane-boundedorganelles plays an integral role in various activities ineukaryotic cells. Recent comparative genomics have indicated that membrane trafficking pathways are diversified among
eukaryotic lineages, which are associated with the lineagespecific acquisition of new trafficking pathways and thesecondary loss of preexisting trafficking routes. Our longterm goal is to reveal how plants have acquired their uniquemembrane trafficking systems during evolution. This will
be achieved by comparative analyses using the model plant
Arabidopsis thaliana and a liverwort model, Marchantia
polymorpha. We also aim to elucidate the detailed molecularmechanisms and physiological functions of membrane trafficking in higher-ordered plant functions.

I. D iversification of membrane trafficking
pathways associated with the acquisition ofnovel machinery components

Although the basic framework of membrane trafficking iswell conserved among eukaryotic lineages, recent comparative genomics have suggested that each lineage has acquired
unique membrane trafficking pathways during evolution.
RAB GTPases and SNARE proteins are evolutionarily conserved key regulators active in the tethering and/or fusionof membrane vesicles with target membranes. It has been
proposed that lineage-specific diversification of these key
factors is closely associated with the acquisition of lineagespecific membrane trafficking pathways, whose molecular basis remains unknown. Comparisons of these protein
families’ organizations among plant lineages, followed byfunctional analyses of each gene product in A. thaliana andM. polymorpha, indicated that diversification of membranetrafficking pathways in land plants has been achieved by 1)
acquisition of novel machinery components, 2) relocationof conserved machinery components to distinct trafficking
events, and 3) secondary loss of conserved machinery components during evolution.

1-1 Analysis of the liverwort-specific organelle: the oil
body

Through analyses of SNARE members in M. polymorpha,a member of the SYP1 group was localized to the membrane

12of the oil body, an organelle specific to liverworts, whose
origin and biogenesis remain unclear.
Furthermore, we discovered an oil body formation master
regulator, whose molecular function is currently being investigated. We also characterized the membrane trafficking
pathway responsible for oil body biogenesis, and found thatthe trafficking pathway to the oil body should be a redirected
secretory pathway.
The morphology and distribution pattern of the oil body,e.g. its shape, color, number, and a density of oil body cells
in tissues diverge among liverwort species, which, therefore,
are regarded as important features for taxonomical classification in liverworts. However, molecular mechanisms ofoil body morphogenesis remain to be determined. We havesuccessfully isolated some mutants defective in oil bodymorphogenesis in M. polymorpha (Figure 1), which would
lead to identification of unknown factors involved in oil bodybiogenesis.

Figure 1. Oil bodies in wild-type and mutant M. polymorpha. The oilbody exhibits complex morphology in the wild-type plant, whereas
spherical in the mutant. Bars = 5 μm.

1-2 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, suchas protein degradation, protein storage, and the regulation of
turgor pressure. To perform these vacuolar functions, a wide
variety of vacuolar proteins and other components must be
properly transported to the vacuole, the entirety of whichis mediated by membrane trafficking, which is a process
distinctly regulated from non-plant systems (for example,
Takemoto et al., 2018).
Defective vacuolar SNARE functions affect both vacuolar
transport and morphology. The sgr3-1 (shoot gravitropism3)
mutant was isolated as a mutant deemed defective in shootgravitropism. This resulted from a point mutation in SYP22/
VAM3, which is one of the SNARE proteins residing on thevacuole and active in vacuolar transport. The sgr3-1 mutant
exhibits abnormal vacuolar morphology, although vacuolar
transport is not markedly affected in this mutant. We also
found that machinery components for homotypic vacuolar
membrane fusion including VAMP71, SYP22, and the tethering HOPS complex were accumulated at specific domains in
the vacuolar membrane in sgr3-1. These results suggested
that vacuolar membrane homotypic fusion is specificallyaffected by the sgr3-1 mutation.

II. S ignificance of membrane trafficking inhigher-ordered plant functions
2-1 R eorganization of the membrane trafficking
system during pathogenic fungus invasion

Membrane traffic also plays important roles in plantmicrobe interaction. Phosphoinositides, which are minor
phospholipids in endomembranes, function within
membrane traffic and signaling. Systematic observation ofphosphoinositides during the invasion of the pathogenic
fungi, Colletotrichum, revealed the strong accumulationof PtdIns(4,5)P2 in the extra-invasive hyphal membrane(EIHM) (Figure 2). During Colletotrichum infection, an
exocytic factor was recruited to the EIHM, but endocytic factors were eliminated. Furthermore, overexpression of Arabidopsis PIP5K, which catalyzes PtdIns4P into
PtdIns(4,5)P2, increased the Colletotrichum invasion, suggesting that the pathogenic fungi, Colletotrichum, could
modify PtdIns in the EIHM in the successful infection(Shimada et al., 2019).

Figure 2. Accumulation of PtdIns(4,5)P2 in the EIHM formed byColletotrichum (Ch). Bar = 5 μm. (Shimada et al., 2019)

2-2 Membrane trafficking in plant gametogenesis

Gametogenesis in plants also involves membrane trafficking-mediated processes. We are analyzing molecular mechanisms of gametogenesis in A. thaliana and M. polymorpha,
and are focusing our attention on secretory and degradative
trafficking pathways during male gamete formation in particular.
Cytokinesis in land plants is achieved by the re-directionof the secretory pathway. As such, KNOLLE/SYP111 plays
important roles in membrane fusion in the forming of cell
plates in A. thaliana somatic cells. Conversely, no deleterious effects on gametogenesis have been reported regarding
mutations in KNOLLE. We found that KNOLLE and other
SYP1 members were highly expressed during cytokinesis ingametogenesis (Figure 3). Mutant analyses of syp1 members
also supported that KNOLLE and other SYP1 regulate cytokinesis during gametogenesis in A. thaliana .

Figure 3. Expression and subcellular localization of GFP-KNOLLE
during pollen mitosis I. GFP-KNOLLE accumulates at the cell plate. Bar= 10 μm. t

Distinct from seed plants, basal land plants including M.
polymorpha utilize the spermatozoid with two (or more)
motile flagella as the male gamete during sexual reproduction. We visualized the spermatozoid formation process,especially spermiogenesis, using fluorescently-tagged organelle markers in M. polymorpha. The majority of the endomembranous organelles, such as the Golgi apparatus, were
removed from maturing spermatozoid cells, and the plasma
membrane was also reorganized during spermiogenesis.
Inspection by transmission electron microscope and live-cell
imaging analyses also indicated that the number of degradative organelles such as the multivesicular endosome, vacuole,and autophagosome, was transiently increased during thisprocess. To reveal the molecular mechanisms of cytoplasm
removal and organelle remodeling, we have established theanalytical tools of autophagy in M. polymorpha (Norizuki
et al., 2019). M. polymorpha possesses core machineriesof autophagy with lower degrees of redundancy. The mutations in MpATG5 and MpATG7, which are key factors forautophagosome formation, affected the transportation ofcytosolic components to the vacuole for degradation (Figure
4).

Figure 4. Establishment of analytical tools for the autophagy study in M.
polymorpha. YFP-tagged MpATG8a is targeted to vacuole in wild type(A), but not in the Mpatg5-1ge mutant (B). Bars = 10 μm. (Norizuki etal., 2019).

Autophagy-defective mutants exhibited defects regarding
cytoplasm removal, spermatozoid motility, and fertility. We
are also analyzing the role of RAB GTPases in flagella formation. Through a comprehensive analysis of RAB GTPases
in M. polymorpha, we found that a RAB GTPase plays anessential role in generating fully functional flagella (Figure
5).

13National Institute for Basic Biology

Cell Biology

A.M., Ozawa, Y., Furuya, T., Sakamoto, Y., Ebine, K.,• Nurani,Matsunaga, S., Ueda, T., Fukuda, H., and Kondo, Y. (2020). Deep•

Figure 5. Transverse sections of flagella in wild-type (A) and mutant (B)spermatids. The microtubule-based “9 + 2” axoneme structure is severelycompromised in the mutant. Bars = 200 nm.

2-3 F unctions of ANTH-domain proteins in plant
physiology

AP180 N-terminal homology domain-containing proteins(ANTH proteins) are thought to act as adaptors bridging
the clathrin coat and cargo proteins during clathrin-coated vesicle formation. ANTH proteins exhibit remarkable
expansion during land plant evolution, and we examined
how this protein family has been functionally diversified
in A. thaliana. We found that a pair of ANTH proteins,
PICALM5a and PICALM5b, are responsible for the tiplocalization of ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity inA. thaliana, whereas another receptor kinase PRK6 acting inpollen tube guidance is not affected (Muro et al., 2018). Now
we are looking for PICALM members regulating localizationof PRK6 and other receptors in Arabidopsis pollen tube.
We also found that another paralogous set of PICALM
proteins is required for retrieving secretory SNARE proteinsfrom the plasma membrane (under revision), which itself
is required for normal vegetative development. These lines
of evidence indicated that ANTH proteins are functionallydifferentiated, which in turn underpin various physiologicalprocesses in A. thaliana.

Publication List:〔Original papers〕

Y., Cao, W., He, Y., Zhao, Q., Wakazaki, M., Zhuang, X., Gao, J.,• Cui,
Zeng, Y., Gao, C., Ding, Y., Wong, H.Y., Wong, W.S., Lam, H.K., Wang,

•
•
•

14

P., Ueda, T., Rojas-Pierce, M., Toyooka, K., Kang, B.-H., and Jiang, L.
(2019). A whole-cell electron tomography model of vacuole biogenesis
in Arabidopsis root cells. Nat. Plants 5, 95-105. doi: 10.1038/s41477018-0328-1
D’Ippolito, R.A., Minamino, N., Rivera-Casas, C., Cheema, M.S., Bai,
D.L., Kasinsky, H.E., Shabanowitz, J., Eirin-Lopez, J.M., Ueda, T.,
Hunt, D.F., and Ausió, J. (2019). Protamines from liverwort are producedby post-translational cleavage and C-terminal di-aminopropanelation ofseveral male germ-specific H1 histones. J. Biol. Chem. 294, 1636416373. doi: 10.1074/jbc.RA119.010316
Hiwatashi, T., Goh, H., Yasui, Y., Koh, L.Q., Takami, H., Kajikawa,
M., Kirita, H., Kanazawa, T., Minamino, N., Togawa, T., Sato, M.,
Wakazaki, M., Yamaguchi, K., Shigenobu, S., Fukaki, H., Mimura,T., Toyooka, K., Sawa, S., Yamato, K.T., Ueda, T., Urano, D., Kohchi,T., and Ishizaki, K. (2019). The RopGEF KARAPPO is essential forthe initiation of vegetative reproduction in Marchantia polymorpha.
Current Biol. 29, 3525-3531. doi: 10.1016/j.cub.2019.08.071
Norizuki, T., Kanazawa, T., Minamino, N., Tsukaya, H., and Ueda,
T. (2019). Marchantia polymorpha, a new model plant for autophagy
studies. Front. Plant Sci. 10, 935. doi: 10.3389/fpls.2019.00935••

imaging analysis in VISUAL reveals the role of YABBY genes invascular stem cell fate determination. Plant Cell Physiol. 61, 255-264.
doi: 10.1093/pcp/pcaa002
Shimada, T.L., Betsuyaku, S., Inada, N., Ebine, K., Fujimoto, M.,
Uemura, T., Takano, Y., Fukuda, H., Nakano, A., and Ueda, T. (2019).
Enrichment of phosphatidylinositol 4,5-bisphosphate in the extrainvasive hyphal membrane promotes colletotrichum infection of
Arabidopsis thaliana. Plant Cell Physiol. 60, 1514-1524. doi: 10.1093/
pcp/pcz058
Shimada, T.L., Shimada, T., Okazaki, Y., Higashi, Y., Saito, K., Kuwata,
K., Oyama, K., Kato, M., Ueda, H., Nakano, A., Ueda, T., Takano,Y., and Hara-Nishimura, I. (2019). HIGH STEROL ESTER 1 is a
key factor in plant sterol homeostasis. Nat. Plants 5, 1154-1166. doi:
10.1038/s41477-019-0537-2
Uemura, T., Nakano, T.R., Takagi, J., Wang, Y., Kramere, K.,
Finkemeier, I., Nakagami, H., Tsuda, K., Ueda, T., Schulze-Lefert, P.
and Nakano, A. (2019). A Golgi-released subpopulation of the transGolgi network mediates constitutive and pathogen-inducible protein
secretion in Arabidopsis. Plant Phys. 179, 519-532. doi:10.1104/
pp.18.01228〔Review article〕

N. and Ueda, T. (2019). RAB GTPases and their effectors• Minamino,in plant endosomal transport. Curr. Opin. Plant Biol. 52, 61-68. doi:
10.1016/j.pbi.2019.07.007

National Institute for Basic Biology

Cell Biology< NINS ExCELLS >DIVISION OF QUANTITATIVE BIOLOGY

Professor

AOKI, Kazuhiro

Assistant Professor:

KONDO, Yohei
GOTO, Yuhei
Technical Staff:
BINO, Takahiro
Postdoctoral Fellow:
ITO, Reina
NAKAMURA, Akinobu
Research Staff:
MIURA, Haruko
KOMATSUBARA, Akira
SOKENDAI Graduate Student: TANII, Ryosuke
MUKAI, Masaya
YAMAMOTO, Kei
Visiting Graduate Student: UDA, Youichi
MARYU, Gembu
Visiting Scientist:
ODA, Shigekazu
Technical Assistant:
EBINE, Emi
GOTO, Yoko
Secretary:
ONODA, Kaori
Living cells act as input-output (I/O) units, in which theirenvironment and/or internal states are recognized on thecell surface and processed within the cell, thus leading toan adaptive response (Figure 1). This cellular information
processing is mainly controlled by intracellular signal transduction, which is comprised of a series of chemical reactions
such as protein-protein interaction and protein phosphorylation. Dysregulation of cell signaling by gene mutation is
widely known to result in pathological diseases, such as
malignant tumors.
The intracellular signaling pathway has been extensively
studied over the past few decades. As a result, many proteins
and regulations have been identified, which has resulted in
an increase in the pathway’s complexity. Computer-assisted
simulation is one of the most promising approaches in
the 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 can be solved numerically bycomputers. Several signaling pathway simulation models
have been reported to date. However, most of the kinetic
parameters utilized for these simulation models have notbeen measured experimentally, but have been assumed bybeing simply arbitrarily determined. Consequently, there aresubstantial differences in the kinetic parameters among thesestudies, thereby making it difficult to quantitatively evaluate
these simulation models.
To address these issues, we are currently focusing on thedevelopment of research tools that enable us to (1) visualize, (2) quantify, and (3) manipulate intracellular signaling
pathways.

Figure 1. Information processing by intracellular signaling devices andnetworks.

phore. Taking advantage of this principle, FRET-based biosensors allowed us to detect PKA, ERK, Akt, JNK, PKC, andS6K’s kinase activity in living cells with a high temporaland spatial resolution (Komatsu N, Mol. Biol. Cell, 2011).
By using a FRET biosensor, we have been able to reveal therole played by ERK activation dynamics in cell proliferation(Aoki K, Mol Cell, 2013) and collective cell migration (AokiK, Dev Cell, 2017).
Although FRET biosensors enable us to monitor cell signaling events, FRET imaging is not suitable for multiplexed
imaging, because the biosensors are composed of two different fluorophores in many cases. To circumvent thislimitation, we are currently developing single-fluorophore
based biosensors. We recently developed a red-fluorescence
dopamine (DA) reporter. DA, a neuromodulator, is involvedin many neuronal functions. To monitor DA in living cells

I. Visualization of cell signaling

With the advent of fluorescent proteins, it has become
possible to visualize kinetic reactions at the single-cell level.
Förster (or fluorescence) resonance energy transfer (FRET)
is a non-radiative process in which the excitation energy of adonor fluorophore is transferred to a nearby acceptor fluoro-

Figure 2. Development of a genetically encoded red fluorescentdopamine (DA) reporter. (A) Schematic representation of a dopamine
receptor, DRD1 (left), and Red DA reporter, R-GenGAR-DA. (B and C)
HeLa cells (B) or mouse primary hippocampal neurons (C) expressing
R-GenGAR-DA were stimulated with DA, followed by a DRD1 antagonist, SCH.

15

National Institute for Basic Biology

Cell Biology

and animals, we designed the red-fluorescent DA reporter
with circular-permutated mApple (cpmApple), a red fluorescent protein; cpmApple was inserted into the third intracellular loop of a DA receptor, DRD1 (Figure 2A). The linker
sequences between DRD and cpmApple were optimized
by random mutagenesis and subsequent live-cell screening.
Finally, we picked up the best performance reporter, andnamed the red genetically encoded GPCR activation reporter
for DA, R-GenGAR-DA. The red fluorescence intensity
decreased in response to DA, and returned to the basal levelupon a DRD1 antagonist treatment (Figure 2B and 2C). In
addition, by using previously reported green fluorescence
norepinephrine (NE) reporter, we succeeded in simultaneously visualizing DA and NE in living neurons (Nakamoto
C, Goto Y, et al., bioRxiv, 2020).

II. Quantification of cell signaling and physicalparameters

Kinetic parameters such as protein concentration and dissociation constant, Kd, have been measured by conventionalin vitro biochemical analyses. However, some kinetic parameters could 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 to 2 (Sadaie
W, et al., MCB, 2014). Therefore, it is critical to measurekinetic parameters in living cells. To this end, we combined
CRISPR/Cas9-mediated genome editing techniques withquantitative fluorescence imaging techniques.
First, we developed an efficient knock-in system, andtagged endogenous proteins with fluorescent proteins by

Figure 3. 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.

16

Cas9-mediated genome editing (Figure 3A). Next, we quantified their concentrations and Kd value by fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation
spectroscopy (FCCS) (Figure 3B). These analyses revealed
temporal changes in Kd values of the binding between ERK2
and RSK2 in response to EGF. Our approach provides arobust and efficient method for quantifying endogenous
protein concentrations and dissociation constants in living
cells (Komatsubara AT, Goto Y, et al., JBC, 2019).

III. Manipulation of cell signaling

Artificial manipulation of biochemical networks are usefulin the quantitative understanding of biological systems.
Multiple methods have been suggested for controlling signaltransduction, including light-induced dimerization (LID) andchemically induced dimerization (CID) systems. Of thesetwo, the LID system is beneficial in terms of temporal andspatial manipulations. The photo-responsive proteins derived
from fungi, cyanobacteria, plants, and modified fluorescentproteins are used in the LID system.
Among these, we focus on the phytochrome B (PhyB)-PIF
LID system. Upon red-light illumination, PhyB binds to PIF,
and the PhyB-PIF complex dissociate from each other by
far-red light exposure (Figure 4A). The reversible control ofassociation and dissociation by light is a unique feature in
the PhyB-PIF system, because other LID systems can only
control association or dissociation by light. One drawback isthat covalent attachment of a chromophore, phycocyanobilin(PCB), is required for the light-responsive function of PhyBPIF. To overcome this issue, we have developed a method
for biosynthesis of PCB in mammalian cells by introductionof the gene products of HO1, PcyA, Fd, and Fnr into mitochondria (Uda Y, et al., PNAS, 2017) (Figure 4A). Recently,
we further improved the system for PCB synthesis, whichallowed establishing stable cell lines synthesizing PCB andlight-induced control of protein localization at the cell population level (Figure 4B and 4C).
To take full advantage of this, we applied the geneticallyencoded PCB synthesis system to S. pombe and C. elegans,
both of which are incapable of having purified PCB delivered to them. As we expected, the expression of HO1, PcyA,
Fd, and Fnr genes induced PCB biosynthesis in S. pombe

Figure 4. PhyB-PIF light-inducible dimerization (LID) system. (A) The
metabolic pathway of phytochrome chromophores, PCB. (B and C)
Light-induced translocation of PIF3-mEGFP to the plasma membrane,
where PhyB is localized (B). Membrane translocation of PIF3-mEGFP isquantified (C).

and C. elegans. Using this system, we developed optogenetic control of the cell cycle in two ways: the Opto-G2/M
checkpoint triggered G2/M cell cycle arrest in responseto red light, and Opto-SAC induced a spindle assembly
checkpoint (SAC) in response to red light and then quickly
released the SAC by far-red light (Figure 5). The geneticallyencoded system of PCB synthesis would 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.

Publication List:〔Original papers〕

A.T., Goto, Y., Kondo, Y., Matsuda, M., and Aoki, K.
• Komatsubara,(2019). Single-cell quantification of the concentrations and dissociation•

constants of endogenous proteins. J. Biol. Chem. 294, 6062-6072. doi:
10.1074/jbc.RA119.007685
Yagi, H., Yagi-Utsumi, M., Honda, R., Ohta, Y., Saito, T., Nishio, M.,
Ninagawa, S., Suzuki, K., Anzai, T., Kamiya, Y., Aoki, K., Nakanishi,
M., Satoh, T., and Kato, K. (2020). Improved secretion of glycoproteinsusing an N-glycan-restricted passport sequence tag recognized by cargo
receptor. Nat. Commun. 11, 1368. doi: 10.1038/s41467-020-15192-1〔Original paper (E-publication ahead of print)〕

A., Oki, C., Kato, K., Fujinuma, S., Maryu, G., Kuwata,• Nakamura,K., Yoshii, T., Matsuda, M., Aoki, K., and Tsukiji, S. Engineeringorthogonal, plasma membrane-specific SLIPT systems for multiplexedchemical control of signaling pathways in living single cells. ACS
Chem. Biol. 2020 Mar 20. doi: 10.1021/acschembio.0c00024

Figure 5. Optogenetic control of spindle assembly checkpoint (OptoSAC). (A) Schematic of the spindle assembly checkpoint (SAC), which
ensures the faithful chromosome segregation and anaphase onset by monitoring kinetochore-microtubule attachment. (B) Schematic of the designof Opto-SAC. Mph1ΔN, which lacks the kinetochore binding domain,is fused with PhyB (PhyB-mph1ΔN). Endogenous Nuf2 is fused with
PIF3 (nuf2-PIF3) as a kinetochore localizer. Upon red light illumination,
PhyB-Mph1ΔN is recruited to kinetochores through the binding to Nuf2PIF3, and SAC turns ON. Far-red light exposure induces dissociation of
PhyB-Mph1ΔN from kinetochores, leading to the inhibition of activatedSAC. (C) Representative cells undergoing a failure of mitosis under longterm metaphase arrest by Opto-SAC.

17

National Institute for Basic Biology

Cell Biology

DIVISION OF CHROMATIN REGULATION

Professor

NAKAYAMA, Jun-ichi

Assistant Professor:
KATAOKA, Kensuke
Postdoctoral Fellow:
HAYASHI, Aki
SOKENDAI Graduate Student: RAHAYU, Anisa
VALENTIROVIC, Olivera
Visiting Undergraduate: HACHISUKA, Aki
Visiting Scientist:
HAMADA, Kyoko
Technical Assistant:
YOSHIMURA, Yuriko
ASAI, Yuriko
Secretary
KIYOHARA, Megumi
The stable inheritance of gene expression or repression
states is essential to cellular differentiation and development,
and an epigenetic cellular-memory system derived fromhigher-order chromatin structure plays a fundamental rolein this process. The assembly of said chromatin structures
has been linked to the covalent modifications of histone
tails, chromatin-associated proteins, and non-coding RNAs.
However, the exact means by which such chromatin-based
epigenetic information is established and faithfully maintained across cell divisions and throughout development
remains unclear. To try to gain a better understanding of themolecular mechanisms underlying chromatin-based epigenetic phenomena, our lab uses mammalian culture cells, fission
yeast, Schizosaccharomyces pombe (S. pombe), in addition
to ciliate Tetrahymena as model systems for studying themolecular mechanisms of higher-order chromatin assembly.
We are also attempting to determine the cellular functions ofchromatin modifying factors so that we can develop a better
understanding of complicated epigenetic phenomena inhigher eukaryotic cells.

I. Establishment and maintenance of higherorder chromatin structures
1-1 H3K14 ubiquitylation promotes H3K9 methylation
for heterochromatin assembly

In eukaryotic cells, the assembly of higher-order chromatin
structures, known as heterochromatin, plays an importantrole in diverse chromosomal processes. Heterochromatin
assembly is intimately associated with changes in post-translational histone-tail modifications. Histone H3-lysine 9 methylation (H3K9me), a hallmark of heterochromatin structure,
is catalyzed by SUV39H-family histone methyltransferases,
and functions as a binding site for recruiting heterochromatin protein 1 (HP1) family proteins. In the fission yeast S.
pombe, Clr4, a homolog of mammalian SUV39H, plays acentral role in heterochromatin assembly. Clr4 forms a multiprotein complex called the Clr4 methyltransferase complex(CLRC) with Cul4, Rik1, Raf1, and Raf2. Cul4, Rik1, andRaf1 display a strong structural resemblance to the conserved
CUL4-DDB1-DDB2 E3 ubiquitin ligase, and it has been
demonstrated that the CLRC exhibits ubiquitin ligase activity
in vitro. However, whether the CLRC acts as an E3 ubiquitin ligase in vivo and how ubiquitylation modulates Clr4’s
activity remains unclear.

18

To identify the physiological substrate(s) ubiquitylatedby CLRC, we affinity purified the CLRC (Figure 1A) andperformed an in vitro ubiquitylation assay using histones
as candidate substrates (Figure 1B). We discovered that theaffinity-purified CLRC specifically ubiquitylates recombinant histone H3, and mass spectrometric and mutation
analyses revealed that H3-lysine 14 (H3K14) is the preferredtarget of the complex (Figure 1C).

Figure 1. Histone H3 lysine 14 is ubiquitylated in vitro by CLRC. (A)Purified TAP-tagged Rik1-containing complexes analyzed by SDS-PAGE
and silver staining. Proteins identified by LC-MS/MS are indicated on
the right. (B) In vitro ubiquitylation assays using biotinylated ubiquitin,purified CLRC, and recombinant histone H3 as the substrate. Proteins
were analyzed by Western blotting using the indicated antibodies.
Asterisks indicate ubiquitylated histone H3 species. (C) Ubiquitylation
assay using biotinylated ubiquitin and recombinant wild-type H3N-GST(WT) and arginine-substituted H3N-GST mutants as substrates. Proteins
were analyzed by Western blotting. Asterisks indicate ubiquitylated
H3N-GST proteins.

We next sought to assess the presence of K14-ubiquitylated
H3 (H3K14ub) in vivo. We performed chromatin immunoprecipitation (ChIP) assays using antibodies against
H3K9me2 and performed LC-MS/MS analyses. We confirmed that H3K14ub was detected exclusively in the
H3K9me2-associated heterochromatin.
We then examined the possible connection between
H3K14ub and H3K9me. To test whether H3K14ub modulates H3K9me, we performed in vitro histone methyltransferase (HMTase) assays using recombinant Clr4 and ubiquitylated H3N-GST as a substrate (Figure 2A). H3N-GST was
first subjected to an in vitro ubiquitylation assay using affinity-purified CLRC. After the reaction, the resultant H3N-GST
was purified and subjected to an in vitro HMTase assay.
We found that even though only 10-15% of the H3N-GST
was ubiquitylated by the CLRC under our assay condi-

tions and the rest remained unmodified, Clr4 exclusively
methylated the ubiquitylated H3N-GST (Figure 2B). These
results suggest that the CLRC-mediated H3 ubiquitylation
promotes H3K9me by Clr4 and that H3 ubiquitylation is
intimately linked to the establishment and/or maintenance of
H3K9me. These findings also demonstrate a cross-talk mechanism between histone ubiquitylation and methylation that is
involved in heterochromatin assembly (Figure 2C).

mediated phosphorylation contributed to a decrease in
HP1α’s DNA-binding activity (Figure 3C). Although HP1α’s
mitotic behavior was previously linked to H3 serine 10
phosphorylation, which blocks the binding of HP1α’s CD
to H3K9me, our findings suggest that mitotic phosphorylation in HP1α’s hinge region also contributes to changes in
HP1α’s association with mitotic chromatin.

Figure 2. H3K14 ubiquitylation promotes H3K9 methylation. (A)Experimental scheme. (B) In vitro HMTase assays. Recombinant
H3N-GST pre-ubiquitylated by the CLRC was purified and used in the
HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins
were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB)
staining (left) and autoradiography (right). (C) A cross-talk mechanism
between histone ubiquitylation and methylation involved in heterochromatin assembly.

II. 
R oles of chromodomain proteins inassembling higher-order chromatin
structure
2-1 M itotic phosphorylation of HP1α regulates its cell
cycle-dependent chromatin binding

HP1 is an evolutionarily conserved chromosomal proteinthat plays a crucial role in heterochromatin-mediated genesilencing. We previously showed that mammalian HP1αis constitutively phosphorylated at its N-terminal serine
residues by casein kinase II (CK2), and that this phosphorylation enhances HP1α’s binding specificity for nucleosomes
containing H3K9me3. Although the presence of additional
HP1α phosphorylation during mitosis was reported morethan a decade ago, its biological significance remains largely
elusive. To examine the roles played by HP1α’s mitotic
phosphorylation, we determined HP1α’s mitotic phosphorylation sites and the cellular behavior of HP1α with mitotic
phosphorylation. We found that S92 in the hinge region is
the main mitotic phosphorylation site in human HP1α andthat HP1α’s S92 phosphorylation (S92ph) was regulated by
Aurora B kinase (AURKB) and two serine/threonine phosphatases. Immunoblotting analysis using cell cycle-synchronized cells demonstrated that HP1α S92ph precedes H3S10
phosphorylation; a major hallmark of mitotic chromatin(Figure 3A). In addition, chromatin fractionation analyses
revealed that hinge region-phosphorylated HP1α was preferentially dissociated from mitotic chromatin (Figure 3B).
Furthermore, EMSA assays demonstrated that AURKB-

Figure 3. Mitotic phosphorylation of HP1α regulates its cell cycle-dependent chromatin binding. (A) The levels of HP1α_S92ph, H3K9me3_
S10ph, and H3S10ph in synchronized PRE-1 cells. (B) Chromatin fractionation assays were performed using synchronized RPE-1 cells. Wholecell lysates (WCLs) and soluble (Sup) and insoluble chromatin-enriched(Ppt) fractions were resolved by SDS-PAGE and analyzed by immunoblotting. (C) Representative results of EMSAs that were performed with
control or phosphorylated HP1α. Various concentrations of HP1α wereincubated with 193-bp 601 DNA. The protein-DNA complexes wereanalyzed by 5% native-PAGE and SYBR Gold staining.

2-2 Identification of Aurora kinase-mediated phosphorylation sites in Swi6/HP1 regulating mitotic
chromosome segregation

HP1 is a conserved chromosomal protein that plays important roles in heterochromatin assembly. We previously
showed that Swi6, one of two HP1 isoforms in S. pombe, ismultiplicatively phosphorylated by casein kinase II (CK2)
and this phosphorylation is essential for its function in heterochromatin assembly. Several previous studies demonstrated that HP1 is subjected to additional phosphorylation
during mitosis. However, the functional importance of HP1’s
mitotic phosphorylation remains unclear. We revealed that
Swi6 mitotic phosphorylation is involved in mitotic chromosomal segregation. Using E. coli co-expression system,
we demonstrated that Swi6 is phosphorylated by Ark1,
a solo Aurora kinase in S. pombe, and mutation analyses
revealed that serine residues in the N-terminal region of19

National Institute for Basic Biology

Cell Biology

Swi6 are efficient targets for Ark1. We confirmed that theseserine residues are phosphorylated during mitosis in vivo.
Interestingly, expression of mutant Swi6 containing amino-acid substitutions at the serine residues differentiallymodulates temperature-sensitive growth of the mutations for
Chromosome Passenger Complex (CPC) components. These
results suggested that Ark1-mediated Swi6 phosphorylation
regulates CPC’s function during mitosis.

III. Dynamic chromatin reorganization during
sexual reproduction of Tetrahymena

The ciliated protozoan Tetrahymena themophila has twofunctionally distinct nuclei in a single cell: the transcriptionally silent germline micronucleus (MIC) and the transcriptionally active somatic macronucleus (MAC). When thesomatic MAC differentiates from the germline MIC during
sexual reproduction, approximately 12,000 transposable
element (TE)-related sequences are eliminated from the
MAC genome. In this process, TEs are heterochromatinizedby the pathway related to RNAi/piRNA silencing and theheterochromatin acts as a mark for their elimination (Figure
4A). Although previous studies have identified more than 20
heterochromatin specific components, it remains unclear how
they orchestrate DNA elimination. Using a yeast two-hybrid
system, we analyzed the interactions between all known
heterochromatin components (Figure 4B) and found several
proteins that may act as ‘hubs’ to assemble heterochromatincomponents into functionally relevant chromatin structure
for DNA elimination. Consequently, we are currently analyzing the roles of these hub proteins both in vitro and in vivo
to understand spatiotemporal regulation of heterochromatin
formation and DNA elimination.

Publication List:〔Original papers〕

J.R., Rao, H.B.D.P., Challa, K., Higashide, M., Shinmyozu,• Bommi,K., Nakayama, J.-I., Shinohara, M., and Shinohara, A. (2019). Meiosis-

•
•
•
•
•
•

specific cohesin component, Rec8, promotes the localization of Mps3
SUN domain protein on the nuclear envelope. Genes Cells 24, 94-106.
doi: 10.1111/gtc.12653
Ding, D.-Q., Okamasa, K., Katou, Y., Oya, E., Nakayama, J.-I.,
Chikashige, Y., Shirahige, K., Haraguchi, T., and Hiraoka, Y. (2019).
Chromosome-associated RNA–protein complexes promote pairing ofhomologous chromosomes during meiosis in Schizosaccharomyces
pombe. Nat. Commun. 10, 5598. doi: 10.1038/s41467-019-13609-0
Hountondji, C., Créchet, J.-B., Tanaka, M., Suzuki, M., Nakayama,
J.-I., Aguida, B., Bulygin, K., Cognet, J., Karpova, G., and Baouz, S.
(2019). Ribosomal protein eL42 contributes to the catalytic activity of
the yeast ribosome at the elongation step of translation. Biochimie 158,
20-33. doi: 10.1016/j.biochi.2018.12.005
Nishibuchi, G., Machida, S., Nakagawa, R., Yoshimura, Y., HiragamiHamada, K., Abe, Y., Kurumizaka, H., Tagami, H., and Nakayama,
J.-I. (2019). Mitotic phosphorylation of HP1α regulates its cell cycledependent chromatin binding. J. Biochem. 165, 433-446. doi: 10.1093/
jb/mvy117
Okamoto, K., Tanaka, Y., Ogasawara, S., Obuse, C., Jun-Ichi, N., Yano,
H., and Tsuneoka, M. (2019). KDM2A-dependent reduction of rRNA
transcription on glucose starvation requires HP1 in cells, including
triple-negative breast cancer cells. Oncotarget 10, 4743-4760. doi:
10.18632/oncotarget.27092
Oya, E., Durand-Dubief, M., Cohen, A., Maksimov, V., Schurra, C.,
Nakayama, J.-I., Weisman, R., Arcangioli, B., and Ekwall, K. (2019).
Leo1 is essential for the dynamic regulation of heterochromatin andgene expression during cellular quiescence. Epigenet. Chromatin 12,
45. doi: 10.1186/s13072-019-0292-7
Oya, E., Nakagawa, R., Yoshimura, Y., Tanaka, M., Nishibuchi, G.,
Machida, S., Shirai, A., Ekwall, K., Kurumizaka, H., Tagami, H.,
and Nakayama, J.-I. (2019). H3K14 ubiquitylation promotes H3K9
methylation for heterochromatin assembly. EMBO Rep. 20, e48111.
doi: 10.15252/embr.201948111〔Review article〕

K., and Nakayama, J. (2019). Do the charges• Hiragami-Hamada,matter? - Balancing the charges of the chromodomain proteins on the
nucleosome. J. Biochem. 165, 455-458. doi: 10.1093/jb/mvz004

Figure 4. Heterochromatin formation in Tetrahymena. (A) Tetrahymena
cell (at the late stage of sexual reproduction) expressing Pdd1p-mCherry(red) and HPL4-EGFP (red) were counter stained with DAPI (blue).
The arrowheads indicate new MAC or MIC and the asterisk indicate old
MAC undergoing degradation. (B) Analyses of all possible one-to-one
interactions between heterochromatin proteins using yeast two hybrid
assay. The yeast strain expressing the Gal4 binding domain fused to aheterochromatin protein (bait) were mated to strains expressing the Gal4
activation domain linked to a heterochromatin protein (prey). Diploidcells were plated on a 48 x 48 matrix on control plates containing all of
the auxotrophic requirements (left: without threonine and leucine) and on
test plates without histidine (right).

20

National Institute for Basic Biology

Cell Biology

LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:
NAKAYAMA, Kei
SOKENDAI Graduate Student: KATAYAMA, KaoriYAMASHITA, Akira
HORIO, Tomoyo
Technical Assistant:
SHIBA, Akiho
The transport of specific mRNAs and local control of translation in neuronal dendrites are part of an important gene
expression system that provides dendritic protein synthesis
at exactly the right time and place. It is widely acceptedthat this system controls the location at which neurites willconnect to each other, thereby forming neural networks. Our
main interest is understanding the mechanisms and roles ofmRNA transport 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, which mediate thetransport and local translation of their mRNA cargoes in
response to synaptic stimulation (Figure 1). We are currently using mice to research the mechanism of RNA granuleassembly, RNA granule factors regulating mRNA transportand local translation, their target mRNAs, and the mechanisms of localized protein synthesis, so we can attain a better
understanding their relationship to the formation of synapses
and neural networks, memory, learning, and behavior.

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. Liquid and solid-phase RNA granules form
through specific proteins and combine into
biphasic granules

RNA granules are membrane-less RNA–protein condensates formed by liquid-liquid phase separation (LLPS).
They consist of not only dynamic liquid-phase shells but
also stable solid-like cores, both of which are thought tofunction in numerous processes pertaining to mRNA sorting
and translational regulation (Figure 2). Abnormalities in
RNA granule dynamics are associated with neurodegen-< NINS ExCELLS >erative diseases, such as amyotrophic lateral sclerosis (ALS)
and frontotemporal lobar degeneration (FTLD). In the caseof these diseases, solidification and aggregation of RNA
granule components are facilitated in neurons. However,
how these distinct liquid-like and solid-like substructures areformed, whether they are assembled by different scaffolds,
and whether different RNA granule scaffolds induce thesedifferent substructures remains unknown.
Cell

RNA granule(not surrounded by membrane)

Membrane
NucleusLiquidshell

RNG105
G3BP1
TDP-43

Solid
core

FUS
FMR1
Pumilio1
TIA-1
TIAR

Figure 2. Membrane-less RNA granules contain liquid-like and solidlike substructures. Liquid-phase granules induced by RNG105, G3BP1,
and TDP-43, and solid-like granules induced by FUS, FMR1, Pumilio1,
TIA-1, and TIAR, combine into biphasic RNA granules.

We expressed 8 kinds of RNA granule scaffold proteins(scaffolds) as GFP and mRFP1-tagged proteins in cultured
epithelial cells. Using these cells, we conducted fluorescencemicroscopy-based morphological and molecular-dynamics
analyses (Shiina, J. Biol. Chem., 2019). These analyses
demonstrate that the scaffolds can be largely classified into
two groups, liquid and solid types, which induce the formation of liquid-like and solid-like granules, respectively, when expressed separately in cultured cells. Liquidlike granules were induced by RNG105 (also known as
Caprin1), G3BP1, and TDP-43, whereas solid-like granules
were induced by FUS, FMR1, Pumilio1, TIA-1, and TIAR
(Figure 2). Furthermore, we found that when co-expressed,
the liquid-type and solid-type scaffolds combine and form
individual liquid and solid-like substructures in the same
granules (Figure 3). The combination of the different typesof scaffolds reduced the immobile fractions of the solid-type
scaffolds and their dose-dependent ability to inhibit translation in granules. However, it had little effect on the dynamics
of the liquid-type scaffolds or their dose-dependent ability
to increase translation in granules. These results suggest thatliquid- and solid-type scaffolds form different substructures
in RNA granules and the relative effect of each type on theirscaffold counterpart varied.
These findings provide a detailed insight into the assembly
mechanism and distinct dynamics and functions of coreand shell substructures in RNA granules. Furthermore, theyalso raise questions as to whether the liquid and solid phase
properties of RNA granules are also regulated by the scaffold
proteins in neurons, and whether such phase properties
impact mRNA transport and local translation in neuronaldendrites. We are testing these analyses using mice brains
and primary cultured neurons.

21National Institute for Basic Biology

Cell Biology

III. Behavioral analysis of mice that lack the
intrinsically disordered region (IDR) of
NFAR2, a stress responsive translation
regulatory factor

Figure 3. Liquid-type scaffolds (e.g. RNG105) and solid-type scaffolds(e.g. FUS) combine into the same granules and form individual liquidlike and solid-like substructures. The panels on the right are magnified
images of the boxed area. Top: FUS, Middle: RNG105, Bottom: merged.
The area surrounded by the dotted line contains the nucleus. Scale bar: 2μm.

II. RNG140 (Caprin2)-mediated translational
regulation in eye lens differentiation

RNG105 and RNG140 are paralogous RNA-binding
proteins that form distinct RNA granules. RNG105 is highly
expressed in neurons and regulates mRNA transport andlong-term memory formation (Nakayama et al., eLife, 2017),
whereas RNG140 is highly expressed in the developing
eye lens and plays a role in lens differentiation. Despite
RNG140’s function in translational regulation, the mechanism and its role within the eye has remained unclear.
We found that RNG140 binds to the translation initiation
factor eIF3 using mass spectrometry of RNG140 immunoprecipitates from cultured CHO cells. Reporter translation
assay revealed that RNG140 represses translation throughmechanisms involving the suppression of eIF3-dependenttranslation initiation. Comprehensive ribosome profilingdemonstrated that overexpression of RNG140 in CHO cells
reduces translation of long mRNAs, including those associated with cell proliferation. In fact, RNG140 overexpression
slowed the growth rate of CHO cells.
RNG140-mediated translational regulation also operates
in the mouse eye, where RNG140 knockout increased thetranslation of long mRNAs. mRNAs involved in lens differentiation, such as crystallin mRNAs, are short, and wereable to escape translational inhibition by RNG140 as well as
be translated in differentiating lenses. These findings provide
insight into the mechanistic basis of lens cell transition fromproliferation to differentiation via RNG140-mediated translational regulation. Moreover, the preference for long mRNAs
raised new questions about why and how RNA-binding
protein complexes distinguish mRNA lengths in the coordination of proliferation and differentiation.

22

IDRs, which do not form three-dimensional structures,
have been revealed to play key roles in LLPS. We focusedon an RNA-binding protein, NFAR2, that possesses an IDR.
NFAR1 and NFAR2 are splicing variants transcribed froma single Ilf3 gene, and inhibit translation of their binding
mRNAs in a stress-dependent (i.e. oxidative stress) manner.
However, they differ in that only NFAR2, but not NFAR1,
has an IDR and can associate with RNA granules. To investigate the physiological relevance of the IDR associated with
NFAR2, we generated NFAR2ΔIDR mice, in which a stopcodon was introduced in the exon encoding the IDR in theIlf3 gene.
Behavioral analysis demonstrated that NFAR2ΔIDR
mice specifically displayed a decrease in fear-conditioned
learning, but not in spacial learning. Furthermore, chronic
stress, which is known to induce oxidative stress in the brain,exacerbated the fear-conditioned learning of NFAR2ΔIDR
mice without affecting their spacial learning. These results
suggested that the IDR of NFAR2 is responsible for specifichigher-order brain functions such as fear-conditioned
learning under stress conditions.

Publication List:〔Original paper〕

N. (2019). Liquid- and solid-like RNA granules form through• Shiina,
specific scaffold proteins and combine into biphasic granules. J. Biol.
Chem. 294, 3532-3548. doi: 10.1074/jbc.RA118.005423〔Review articles〕

R., and Shiina, N. (2020). Cataloguing and selection of mRNAs• Ohashi,localized to dendrites in neurons and regulated by RNA-binding proteinsin RNA granules. Biomolecules 10, 167. doi: 10.3390/biom10020167

R., Shiina, N., and Wang, D.O. (2020). More dynamic, more
• Roy,
quantitative, unexpectedly intricate: Advanced understanding on

synaptic RNA localization in learning and memory. Neurobiol. Learn.
Mem. 168, 107149. doi: 10.1016/j.nlm.2019.107149

National Institute for Basic Biology

Cell Biology

LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

Postdoctoral Fellow:
KAMIKAWA, Yasunao
SOKENDAI Graduate Student: KUMAZAKI, Taisei
MATSUMOTO, Akino
Technical Assistant:
YASUI, Naomi
ASAI, Yuriko
NAGANUMA, Mai
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 cell malfunction. Our group is interested in the strategies that cells use to protect themselves from alterations in
the genome. To date, much information has been gained fromvarious model organisms and tissue culture cells, and we are
beginning to learn that the choice of genome-maintenancestrategies taken by a cell depends on the cell type, cell cycleand its developmental stage. Our focus is on the genome
maintenance mechanisms of embryonic stem cells, and theirroles during differentiation and reprogramming processes.

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

Embryonic stem (ES) cells are derived from the blastocyst
stage of embryonic development, and are capable of differentiating into all of the cell types that compose our body(i.e., ES cells are “pluripotent”). Pluripotent cells exist onlytransiently and are lost as development proceeds. However,ES cells are capable of proliferating indefinitely when given
an appropriate culturing condition. Curiously, ES cells proliferate with truncated gap phases while lengths of S (DNAreplication) and M (mitosis) phases are similar to other cell
types. ES cells also appear to lack some of the mechanismsthat ensure genome integrity (i.e., checkpoint mechanisms).
The significance of this phenomenon remains a mystery.
To date, studies on cell cycle regulation in ES cells have notbeen as 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 has allowed us to investigate
specific stages of the ES cell cycle. So far, we have foundthat DNA replication is regulated differently in ES cells,
to the extent that DNA replication of the whole genome ismore accurate in ES cells. We are currently aiming to address
how such differences are interlinked with pluripotency bycarrying out side-by-side analyses between ES cells and differentiated populations.

II. G enome Instability during Nuclear
Reprogramming
In order to gain a deeper understanding of the relationshipbetween the choice of genome maintenance mechanisms andpluripotency, we are investigating the behavior of factors

involved in genome maintenance mechanisms during nuclear
reprogramming towards pluripotency.
Specifically, we are taking advantage of the cell-to-cellfusion approach, in which a target cell is fused to a pluripotent stem cell to induce pluripotency within a target nucleus.
The cell fusion system is a simple, versatile way to induce
reprogramming towards another lineage, and is not limited topluripotency. Using this system, the first sign of reprogramming can be detected from within a few hours to one day
after fusion, thus allowing us to monitor the initial events ofreprogramming 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).

III. Future Perspectives

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

Publication List:〔Original paper〕

M., Ogawa, K., Isobe, M., Nishitani, T., Kamio, S.,• Kobayashi,
Fujiwara, Y., Tsubouchi, T., Yoshihashi, S., Uritani, A., Sakama, M.,

Osakabe, M., and LHD experiment group. Thermal neutron flux
evaluation by a single crystal CVD diamond detector in LHD deuterium
experiment. J. Instrum. 14, C09039.

23

National Institute for Basic Biology

Cell Biology

LABORATORY OF ORGANELLE REGULATION

Associate Professor

MANO, Shoji

Specially Appointed Assistant Professor:
KANAI, Masatake
Visiting Scientist:
KAMIGAKI, Akane
Technical Assistant:
HIKINO, Kazumi
NAGATA, Kyoko
NAKAYAMA, Tomomi
Secretary:
UEDA, Chizuru
ASAI, Sanae

somes via autophagy, using these apem mutants in concert
with the analyses of peup1, peup2 and peup4 mutants, whichwere defective in Autophagy-related 2 (ATG2), ATG18a and
ATG7, respectively (Figure 1).
We are currently investigating other apem and peupmutants. From these analyses, we will be able to identify thecomponents responsible for peroxisome biogenesis, functions and maintenance, and to address the mechanism at themolecular level.

Because plants spread their roots in the ground, they must
survive in a given environment. To adapt to their environment, they utilize various signals generated from environmental changes as being necessary for their survival. The
flexibility of plant organelles is the basis for environmental
adaptation in plants.
The aims of this laboratory are to clarify the molecularmechanisms underlying the induction, differentiation, andinteraction of organelles, especially peroxisomes and oil
bodies, as well as to understand the integrated functions ofindividual plants through organelle dynamics.

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

Peroxisomes are single-membrane bounded organelles,
which are ubiquitously present in eukaryotic cells, and are
involved in various biological processes such as lipid metabolism and photorespiration. These functions are dramatically changed during certain developmental stages and whenconfronted with environmental changes. For example, light
induces the transformation of peroxisomes from glyoxysomes, which are peroxisomes engaged in the degradationof reserve oil stored in the oil body via β-oxidation and theglyoxylate cycle, to another type of peroxisome, leaf peroxisomes, that function in several crucial steps of photorespiration. In addition to functioning in vegetative tissues suchas leaf and root cells, it has been revealed that peroxisomes
play essential roles in reproductive processes. Studies using
Arabidopsis mutants defective in peroxisomal functions
demonstrate that peroxisomes contribute to pollen fertility,pollen tube elongation, and male–female gametophyte recognition. Gene expression, alternative splicing, protein transport, protein degradation and degradation of peroxisomes
themselves control these peroxisomal functions.
To better understand peroxisome biogenesis and functions,
we isolated a number of Arabidopsis mutants that displayedaberrant peroxisome morphology (apem mutants) and peroxisome unusual positioning (peup mutants) based on themhaving a different pattern of GFP fluorescence compared totheir parent plant, GFP-PTS1, in which peroxisomes withnormal sizes, numbers and distribution could be visualizedwith GFP. As of writing, we have reported the function of
APEM1, APEM2, APEM3, APEM4, APEM9 and APEM10.
Based on these results we were able to update the model for
protein transport, proliferation and quality control of peroxi-

24

Figure 1. Phenotype of Arabidopsis peup1 mutant. (A) GFP fluorescencein leaf cells was observed in the parent plant, GFP-PTS1, and peup1
mutant. peup1 demonstrates aggregated peroxisomes (arrowhead). Bar
indicates 10 µm. (B) Fluorescence intensities from peroxisome-targeted
reduction-oxidation-sensitive GFP (roGFP) with two excitation wavelengths were measured, and the 405/488 nm ratio was calculated in thetransgenic plants. The 405/488 nm ratio of the peroxisome aggregates in
the peup1 roGFP-PTS1 mutant showed that the peroxisome aggregateswere more oxidative than peroxisomes in the wild type.

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 in food and industrial materials. Storage
reserves vary among different types of plant seeds. Wheat,maize and rice seeds mainly accumulate starch, whereas
rapeseed, pumpkin and sesame contain large amounts of oils.
Soybeans major reserve are proteins. Storage oils and storage
proteins are synthesized in the endoplasmic reticulum (ER)and accumulated in oil bodies and protein bodies, respectively, during the same period of seed development.
We are analyzing the molecular mechanisms controlling
oil and protein contents in seeds. Based on the analysis of
the temporal sequence of oil and protein synthesis during
seed development in Arabidopsis thaliana, which produces
seeds containing approximately 30% oil and 30% protein, we
revealed that the extension of WRINKLED1 (WRI1), a tran-

scription factor in fatty acid biosynthesis, expression during
the mid-phase of seed development significantly enhanced
seed oil content, and caused an enlargement of seed size.
We are also currently investigating the mechanisms of oil
accumulation in other plant species. In the soybean (Glycinemax. L), we identified four lipases, GmSDP1s, on the oilbody membrane. The analyses of GmSDP1s revealed thatplant seeds have the mechanism required for the quality
control of fatty acids by degrading particular fatty acids in oil
bodies (Figure 2).

PODB3 consists of six individual sections: the electron
micrograph database, the perceptive organelles database, theorganelles movie database, the organellome database, thefunctional analysis database, and external links. The functionof each database is as follows:⃝The electron micrograph database provides informationon the ultrastructures in plant cells⃝The perceptive organelles database shows organelles
dynamics responding to environmental stimuli⃝T he organelles movie database contains time-lapse
images and 3D structure rotations⃝T he organellome database is a compilation of static
image data of various tissues of several plant species atdifferent developmental stage.
⃝The functional analysis database is a collection of protocols for plant organelle research
Through these databases, users can easily grasp plant organelle dynamics. Plant Organelles World, which is built basedon PODB3, is an educational tool for engaging members of
the non-scientific community to explore plant biology. We
hope that both PODB3 and Plant Organelles World are ofhelp to researchers as well as the general public.

Publication List:〔Original papers〕

S., Oikawa, K., Bizan, J., Shigenobu, S., Yamaguchi, K.,• Goto-Yamada,Mano, S., Hayashi, M., Ueda, H., Hara-Nishimura, I., Nishimura, M.,

Figure 2. Quality and quantity control of storage oils in oil bodies by
GmSDP1 in soybean (Glycine max L.). (A) Storage oils are accumulated as triacylglycerol (TAG) in oil bodies. Four Glycine max SUGAR
DEPENDENT-1s (GmSDP1), which are types of lipase, have an activity
for releasing fatty acids from TAG. GmSDP1s degrade oleic acid (18:1),
so that the content of linoleic acid (18:2) in seeds increases (B), whereas
the content of oleic acid rises in GmSDP1-knockdown plants comparedto the wild-type plants due to reductions in the degradation of oleic acid(C, D).

III. D evelopment of Gateway-technology
vectors for plant research

Gateway cloning is a popular technology which allows thesimultaneous generation of multiple constructs containing a
range of fusion genes. We have developed various types ofGateway cloning-compatible vectors to improve resourcesin the plant research field. As of writing, we have provided
vector sets to detect multiple protein-protein interactions
in vivo using multi-color bimolecular fluorescence complementation, and the binary vectors to facilitate tripartite
DNA assembly and promoter analysis with various reporters
and tags in the liverwort Marchantia polymorpha. We willcontinue developing other useful Gateway cloning-compatible vectors to contribute to the plant research community.

•
•
•
•
•

and Yamada, K. (2019). Sucrose starvation induces microautophagy inplant root cells. Front. Plant Sci. 10, 1604. doi: 10.3389/fpls.2019.01604
Kanai, M., Yamada, T., Hayashi, M., Mano, S., and Nishimura, M.
(2019). Soybean (Glycine max L.) triacylglycerol lipase GmSDP1
regulates the quality and quantity of seed oil. Sci. Rep. 9, 8924. doi:
10.1038/s41598-019-45331-8
Kozuka, T., Sawada, Y., Imai, H., Kanai, M., Hirai, M.Y., Mano, S.,
Uemura, M., Nishimura, M., Kusaba, M., and Nagatani, A. (2020).
Regulation of sugar and storage oil metabolism by phytochrome duringde-etiolation. Plant Physiol. 182, 1114-1129. doi: 10.1104/pp.19.00535
Sultana, M.M., Dutta, A.K., Tanaka, Y., Aboulela, M., Nishimura, K.,
Sugiura, S., Niwa, T., Maeo, K., Goto-Yamada, S., Kimura, T., Ishiguro,
S., Mano, S., and Nakagawa, T. (2019). Gateway binary vectors withorganelle-targeted fluorescent proteins for highly sensitive reporter
assay in gene expression analysis of plants. J. Biotech. 297, 19-27. doi:
10.1016/j.jbiotec.2019.03.015
Watanabe, E., Mano, S., Nishimura, M., and Yamada, K. (2019).
AtUBL5 regulates growth and development through pre-mRNA
splicing in Arabidopsis thaliana. PLoS One 14, e0224795. doi: 10.1371/
journal.pone.0224795
Yamauchi, S., Mano, S., Oikawa, K., Hikino, K., Teshima, K.M.,
Kimori, Y., Nishimura, M., Shimazaki, K.I., and Takemiya, A. (2019).
Autophagy controls reactive oxygen species homeostasis in guard cells
that is essential for stomatal opening. Proc. Natl. Acad. Sci. USA 116,
19187-19192. doi: 10.1073/pnas.1910886116

IV. C onstruction of The Plant Organelles
Database 3 (PODB3) and Plant Organelles
World

The Plant Organelles Database 3 (PODB3) was built
to promote a comprehensive understanding of organelle
dynamics. This public database is open to all researchers.

25

National Institute for Basic Biology

Developmental Biology

DIVISION OF MORPHOGENESIS

Professor

Associate Professor

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

TAKAHASHI, Hiroki
TAKAGI, Chiyo
SAKAI, Yusuke
YAMAMOTO, Takamasa
YASUE, Naoko
MURAKAMI, Michiyo
MIYAKE, Satoko
TSUGE, Toyoko

UENO, Naoto

Secretary:

KINOSHITA, Noriyuki

The complex morphogenesis of organisms is achieved by
the dynamic rearrangement of tissues during embryogenesis
in which changes 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 known as “cell polarity”, which confers them
asymmetry, is essential. We have been studying how cell
polarity is established within cells and how cell polarity isinterpreted by cells to apply this information to their behaviors. We attempt to understand the mechanisms underlying
these events using several model animals, including frogs,
fish, and ascidians. In addition to genes and proteins thatare widely known to govern biological phenomena andhave been extensively studied over the past several decades,a growing body of evidence now suggests that physicalenvironments that include light, temperature, and internaland external forces influence cellular as well as organismal
behaviors. In particular, we are currently investigating thecontribution of physical forces using frog embryos and thelight-dependent behaviors using coral planula larvae tounderstand the impact of physical environments on organisms.

The mechanical force administered to embryos was appliedby centrifugation, which resulted in flattening embryos andexpanding tissues. Protein samples from embryos wereanalyzed by quantitative mass spectrometry. We detected
over 3,500 proteins in each whole proteome sample, and over
8,500 phosphorylated peptides in each sample. Enrichment
analyses based on information theory led us to identify
the enrichment in cell adhesion molecules, such as focal
adhesion and tight junction components within the upregulated phosphorylation sites during force stimulation (Figure1). In the phosphoproteome analyses, we also identified
several protein kinases contributing to these phosphorylation. Additionally, we found that centrifugal force induced amesenchymal-epithelial transition (MET)-like phenotype inthe enrichment analysis based on proteome level.
We also applied mechanical force to embryos via the use ofcompression generated by a cover glass, which is associatedwith flattening and tissue expansion. The mass spectrometry
analysis demonstrated that these compressed embryos also
showed a similar protein phosphorylation profile similar tocentrifuged embryos. This result suggests that tissue expansion induced by these forces may lead to the change inprotein phosphorylation as a response to mechanical forces.
This is the overall first study of phosphoproteome analyses
in Xenopus laevis embryos during mechanical sensing andprovides mechanistic insights into mechanical force sensing
pathways.

I. 
Mechanical stimulation induces
phosphorylation-dependent signaling in
Xenopus embryos

Cells sense and respond to not only molecular factors, butalso mechanical forces, which play various important roles inbiological events, such as tissue homeostasis, differentiationand cell migration. During early embryogenesis, a variety ofdynamic morphogenetic movements occur, which include theconvergent extension of the axial mesoderm, epiboly of theectoderm, and neural tube formation. These movements must
generate physical forces at the levels of cells and tissues.
However, it is still not fully understood how these forces
influence morphogenetic processes. The aim of this projectis to elucidate the mechanisms of sensing and responding tomechanical stimuli in Xenopus embryos.
It is assumed that protein phosphorylation, one of the immediate responses of cells to external stimuli, is dynamicallyregulated upon the detection of mechanical force. Therefore,
we used quantitative phosphoproteome analyses to investigate mechanosensing pathway in Xenopus laevis embryos.

26

Figure 1. Phosphorylation changes of tight junction and focal adhesion
components upon force stimulation in Xenopus laevis embryos.

II. Mechanical force regulates tissue stiffness
and integrity through FGFR/Erk2 signaling
pathway in Xenopus embryos

The kinase substrate enrichment analysis based on thephosphoproteome data demonstrated that the mechanicalforce applied to Xenopus embryos activates several protein
kinases. We focused on one of these protein kinases: a MAP
kinase Erk2. We first confirmed that Erk2 is activated similarly in response to both compression and centrifugal force
using antibodies against phosphorylated Erk2 (pErk2). As
shown in Figure 2, pErk2 was detected within a few minutes
after mechanical force applications.

Moreover, we demonstrated that activated Erk2 regulates
integrity and stiffness of the embryonic epithelial cells. The
centrifugal force to embryos accumulates F-actin, the tightjunctional protein ZO-1 and the cell adhesion molecule
cadherin to the apical junctional region. Furthermore, thecentrifugal force increased epithelial tissue stiffness of theembryonic surface cells, which was measured by atomic
force microscopy (AFM). Importantly, all these cellularchanges were found to be dependent on Erk2 activity.
We further examined how Erk2 was activated by this force,and found that inhibition of the FGF receptor (FGFR) diminished Erk2 activation (Figure 2). These observations demonstrated that mechanical force activates FGFR/Erk2 signaling
pathway and promotes the formation of mature epithelial
junctional structures, which is an indication of MET as predicted by the phosphoproteome analysis described above.

Figure 2. ERK2 is activated by a mechanical force (compression) in
Xenopus embryonic cells in an FGFR-dependent manner.
Membrane RFP and dominant negative FGFR (dnFGFR) were expressedand compressed for 5 minutes at the gastrula stage. Embryos were stainedwith anti phoshorylated-ERK2 antibody (green) and the ectodermal cells
were observed. Bar = 50 μm

We found that the surface elasticity of the apical side of the NP
increased during NTC and showed gradient along mediolateralaxis. The stiffening required F-actin and non-muscle myosin
II, suggesting that the increase of NT-specific actomyosin
contractility underlies this stiffening. We also measured thesurface elasticity of the basal side of the NT and surrounding
mesodermal tissues, and confirmed the highest elasticity measurable on the apical side of the NP. The mechanical properties
should change over time and space ranging from intracellular
to tissue levels, and contribute to the large-scale morphogenetic movement of the NP.

IV. C ephalochordate Brachyury enhancers
involved in evolution of the chordate
notochord and somites

The notochord and somite are the most prominent organs
in chordates, and Brachyury (Bra) plays a pivotal role intheir formation. The question how Bra gained its enhancer
activity in the notochord-specific expression is critical to ourunderstanding of chordate evolution. In cephalochordate Bra
is duplicated into two genes (Figure 3). We examined theenhancer activity of Branchiostoma floridae Bra (BfBra1 and
BfBra2) by lacZ reporter assay using a Ciona-embryo hostsystem. In the 5’, 3’ regions and in introns, the signal intensity was higher and broader in BfBra2 than BfBra1 (Figure3). In some cases lacZ expression is expanded to posterior
muscle cells in the former. The intron enhancers displayed
another difference; primary expression of BfBra1 occurs innotochord whereas both muscle and notochord manifest theexpression of BfBra2. These results along with other data
predicted an EvoDevo scenario in which BfBra2 is moreancestral and mother-like, while BfBra1 is daughter-like andduplicated by BfBra1. Originally, ancestral BfBra2 is likely
to gain enhancer machinery for gene expression in muscle
and notochord, as has been deduced from its spatial expression profile: the 5’ region for somite expression, the 3’ region
for notochord expression, and introns for somite/notochord
expression. In contrast, sister BfBra1 became more specialized for notochord expression using the intron enhancers.

III. 
M e c h a n i c a l re g u l a t i o n o f c l o s i n g
movement and morphology of Xenopus’
neural tube

Neural tube closure (NTC) is an important morphogenetic process during which an anlage of the central nervous
system is formed from a flat epithelial sheet called a neuralplate (NP). NTC involves tissue-autonomous and non-tissueautonomous cellular movements that generate physical forces
and modulate mechanical properties of the NP, but how developmental changes of those mechanical forces and properties
are integrated for NT morphogenesis is poorly understood. We
previously constructed a mathematical model explaining themechanical regulation of NTC and proposed that the surface
elasticities of the neural plate and surrounding tissues affect
NTC. In order to examine this hypothesis, we developed an
AFM system for measuring mechanical properties of tissues
and their temporal changes in developing Xenopus embryos.

Figure 3. A summary of results of the present reporter assay and their
possible interpretation.
Enhancer activity of 5’ upstream sequences in muscle is shown in brown
and that of the 3’ downstream in notochord is in blue. The activity BfBra2
introns in muscle and notochord is shown in purple.

27

National Institute for Basic Biology

Developmental Biology

V. L ight-dependent swimming behavior ofcoral larvae in Acropora tenuis

Many reef-building corals form a symbiotic relationshipwith dinoflagellate algae of the genus Symbiodinium. Corals
mostly depend on photosynthetic products from these symbionts as their energy source, and thus light conditions inhabitats can influence post-settlement survival. Previous
studies reported that light environments play an essential
role in larval habitat selection. However, due to a lack ofbasic photobiological studies in corals, how coral larvae
perceive and respond to light environment remain largely
unknown. To answer these questions, we analysed the effects
of light stimuli on larval swimming behavior. In addition, by
focusing on opsin-like photopigments, we have been examining the mechanism of photoreception in a reef-building coral,
Acropora tenuis (Figure 4).
First, we precisely observed the larval swimming activity
under fluctuating light conditions, and found that larvae
temporarily stopped swimming ~30 s after the rapid reduction of light intensity. This behavior was also observed when
we rapidly changed spectral composition of light. Further
experiments using the Okazaki Large Spectograph revealed
that the loss of short wavelengths of light (blue/green light)
induced this type of behavior. This light-dependent behavior
might lead to larval aggregation to optimal light environments in natural habitat.
As for the analysis of photoreception, we identified eightopsin-like genes on the A. tenuis genome and measured
the absorbance spectra of one of these eight opsins (s0350
opsin). We found this opsin absorbed UV light and UV
dependent activation of s0350 opsin caused the elevationof intracellular cAMP level. This indicates that s0350 opsinfunctions as a UV-sensitive photorecetor. Therefore, A. tenuis
larvae sense environmental UV light and the information
they attain may play an important role in their behavior andsettlement.

Figure 4. A wild colony of Acropora tenuis (photo taken by Masayuki
Hatta at Ochanomizu University). The inset shows a planula larvae of A.
tenuis.

28

Publication List:〔Original papers〕

Y., Kinoshita, N., Greco, T.M., Federspiel, J.D., Jean• Hashimoto,
Beltran, P.M., Ueno, N., and Cristea, I.M. (2019). Mechanical force

•
•
•
•
•

induces phosphorylation-mediated signaling that underlies tissue
response and robustness in Xenopus embryos. Cell Syst. 8, 226-241.
doi: 10.1016/j.cels.2019.01.006
Ishii, Y., Maruyama, S., Takahashi, H., Aihara, Y., Yamaguchi, T.,
Yamaguchi, K., Shigenobu, S., Kawata, M., Ueno, N., and Minagawa,J. (2019). Global shifts in gene expression profiles accompanied withenvironmental changes in cnidarian-dinoflagellate endosymbiosis. G3Genes Genom. Genet. 9, 2337-2347. doi: 10.1534/g3.118.201012
Itoh, M., Dai, H., Horike, S.-I., Gonzalez, J., Kitami, Y., MeguroHorike, M., Kuki, I., Shimakawa, S., Yoshinaga, H., Ota, Y., Okazaki,T., Maegaki, Y., Nabatame, S., Okazaki, S., Kawawaki, H., Ueno, N.,
Goto, Y.-I., and Kato, Y. (2019). Biallelic KARS pathogenic variants
cause an early-onset progressive leukodystrophy. Brain 142, 560-573.
doi: 10.1093/brain/awz001
Kinoshita, N., Hashimoto, Y., Yasue, N., Suzuki, M., Cristea, I.M.,
and Ueno, N. (2020). Mechanical stress regulates epithelial tissue
integrity and stiffness through the FGFR/Erk2 signaling pathway
during embryogenesis. Cell Rep. 30, 3875-3888. doi: 10.1016/j.
celrep.2020.02.074
Sakai, Y., Hatta, M., Furukawa, S., Kawata, M., Ueno, N., andMaruyama, S. (2020). Environmental factors explain spawning day
deviation from full moon in the scleractinian coral Acropora. Biol. Lett.
16, 20190760. doi: 10.1098/rsbl.2019.0760
Virgirinia, R.P., Jahan, N., Okada, M., Takebayashi-Suzuki, K.,
Yoshida, H., Nakamura, M., Akao, H., Yoshimoto, Y., Fatchiyah, F.,
Ueno, N., and Suzuki, A. (2019). Cdc2-like kinase 2 (Clk2) promotes
early neural development in Xenopus embryos. Dev. Growth Diff. 61,
365-377. doi: 10.1111/dgd.12619

National Institute for Basic Biology

Developmental Biology

DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

Assistant Professor:

YABE, Taijiro
MII, Yusuke
Technical Staff:
UTSUMI, Hideko
NIBB Research Fellow
SHINOZUKA, Takuma
Postdoctoral Fellow:
TAKADA, Ritsuko
SOKENDAI Graduate Student: HATAKEYMA, Yudai
TRAN, Thi Hong Nguyen
SUZUKI, Minako
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
Secretary:
NOBATA, Ryuko
The morphology of the body and tissues is established ina spatio-temporarily regulated manner. A number of genes
involved in morphogenesis have been identified, but it isstill uncertain how either spatial and temporal information are established and converted to morphology during
embryogenesis. Therefore, our aim is to understand themechanism by which this spatial information is establishedand how temporal, or periodical, information is converted
into morphology through the application of several different
approaches.
Secreted signal molecules are important in forming spatial
information during the development of many tissues. Thesemolecules are secreted from the cells that produce them andtransported to surrounding cells, thus resulting in the formation of concentration gradients. Given that their concentration decreases in accordance with their distance from thesource, their specific signal gradient defines the relative positions of receiving cells in developing tissues. Many genetic
studies have revealed that a limited number of secreted signal
proteins, including Wnt, BMP, and Hedgehog, function
during tissue and embryo morphogenesis. However, in spiteof the accumulation of genetic evidence, the molecularmechanism that regulates their distribution in certain developing tissues remains to be elucidated. To this end, we havevisualized signal proteins and monitored their movement intissues. In addition to this, we are currently examining thebiochemical characteristics and functions of these molecules,
which appear to affect how they are spread.
In contrast to secreted signal proteins, the segmental subregions of several specific tissues, like somites, appear not
to be simply directed by the gradient of the signal molecules,
but by a unique mechanism that functions periodically.
Somites are sequentially generated in an anterior-to-posteriororder via the conversion of temporal periodicity, createdby a molecular clock, into periodical structures. However,
the molecular mechanism underlying this conversion andmorphological segmentation is not yet fully understood.
Therefore, another goal of our current studies is to reveal themolecular mechanism of this differing and unique mode ofpatterning that underlies the periodical and sequential subdivision in the development of somites.

< NINS ExCELLS >I. E ffect of morphologic change in signalproducing cells during signal transmission intissuesDuring morphogenesis, cells dynamically change theirshape. Marked changes in the morphology of signal producing cells during tissue morphogenesis presumably resultin the activation of signaling in neighboring cells. In early
development of the vertebrate spinal cord, the most dorsal
region, known as the roof plate, functions as an organizing
center by secreting BMP and Wnt. These signaling proteins’concentration gradients generate interneuron subtype
patterns in the dorsal spinal cord. Interestingly, as this patterning event nears completion, the spinal cord’s morphology changes dynamically. Essentially, the neural tube lumen
gradually shrinks, and a median septum forms along the dorsoventral axis. A recent study in zebrafish embryos revealed
that the roof plate stretches during this morphologic change.
This finding suggests that the processes of these stretchedroof-plate cells are involved in intercellular communicationand that the stretching of roof-plate cells induces changes incells that respond to signals from the roof plate.

Figure 1. Stretching of Wnt expressing cells during spinal cord development in the mouse. Roof plate cells act as an organizing center in early
development of spinal cord (embryonic day 10.5; E10.5). Then, these cells
are stretched along the dorso-ventral axis (E13.5) and provide Wnt ligands
to neural progenitor cells close to the tip of extended roof plate cells.

In adult mice, neural progenitor/stem cells localize aroundthe shrunken lumen of the spinal cord, and this area isreferred to as the central canal. The central canal is connected to the ventricle, where neural progenitor/stem cells
localize in the forebrain. As the proliferation of neural progenitor/stem cells in the brain are regulated by secreted
signaling molecules, including Wnt, it is plausible that spinal
cord progenitor/stem cells are regulated in a similar manner.
In the developing spinal cord, Wnt1 and Wnt3a are specifically expressed in the roof plate and perform numerous
roles, including regulating the proliferation and specification of dorsal interneurons prior to the mid-gestation stage.
However, genetic studies of Wnt1- and Wnt3a-deficientmutant mouse embryos yielded no information regarding
the roles of the respective proteins in later stages, as neither
Wnt1- nor Wnt3a-deficient single mutants exhibit a roof
plate–related phenotype, and most of the double mutants die
before embryonic day 12.5 (E12.5).
Recently, we found that the expression of Wnt1 and Wnt3a
is maintained in stretched roof-plate cells (Figure 1). Roofplate-specific cKO for Wls, which is essential for Wnt secretion, revealed that Wnt secretion from roof-plate cells was
required for their coordinated elongation. Furthermore,

29

National Institute for Basic Biology

Developmental Biology

proliferation was reduced in ependymal cells in Wls cKO
embryos during the development and regeneration of the
spinal cord (Figure 2). Thus, dynamic change in cell extension can create novel roles for signal-producing cells.

II. Regulation of spatial distribution of Wnt
proteins in vertebrate embryos

To examine the mobility of Wnt3a in an extracellular situation, we performed FCS analysis of GFP-Wnt3a in Xenopus
embryos. This examination revealed that the dynamic
behavior of Wnt3a could be divided into two distinct states.
The fast population appears to reflect quickly and freely diffusing molecules, and the slow population reflects slowly
moving molecules because of its likely interaction withextracellular matrices. Further precise analyses revealed that
HMW complexes were less mobile than relatively smallerones.
We showed that the trimer and larger HMW complexes ofWnt3a proteins could be dissociated via interaction with their
receptor Frizzled8 and with secreted Wnt binding protein,sFRP2, in vitro by utilizing AUC-FDS. Similarly, this dissociation was detected in vivo by FCS. Importantly, the resultsof FCS suggested that dissociation of the large assembly of
Wnt3a proteins by interaction with sFRP can make Wnt moremobile, probably resulting in longer diffusion distance in theembryo. These ideas are supported by a finding that statesthat the distribution range of Wnt3a was expanded by theco-expression of sFRP2 in Xenopus embryos. These results
showed that large assemblies of Wnt3a are less mobile, andsFRP2 can expand the diffusion range of Wnt proteins in
Xenopus embryos. Based on these results, we propose amodel that contends that the assembly and dissociation ofdissociable oligomers modulate Wnt signaling range (Figure
3).

Figure 2. Impaired morphology of stretched roof plate cells in Wntdefective embryos (Wls cKO). Extensions are indicated by brackets.

Figure 3. Model of Wnt protein diffusion: Wnt trimers are the smallest unit of the HMW complex. Both the trimer and the HMW complex appear toexist in the extracellular milieu although it is uncertain when the assembly to the HMW complex occurs during the process of Wnt secretion. The HMW
complex is probably less mobile when interacting with the plasma membrane, resulting in the restriction of Wnt diffusion range. Some Wnt molecules canbe dissociated by local interaction with Frizzled receptor (Fzd), resulting in a short-range signal (local action). In contrast, the HMW complex, probably aswell as the trimer itself, can also be dissociated by interaction with soluble Wnt binding protein (partner protein), including sFRP. Due to this dissociation,
Wnt turns to be more mobile and its diffusion range is expanded (diffusible action).

30

III. Molecular mechanism of somite
development

Somites are segmental epithelial blocks located symmetrically on either side of the neural tube. Somitogenesis periodically proceeds in an anterior to posterior manner from theirprecursor, the presomitic mesoderm (PSM), which is locatedat the posterior of newly formed somites. This periodic generation is achieved by a complex and dynamic mechanism
within the PSM. First, the molecular clock, the so-called
segmentation clock, creates oscillatory expression of particular genes, hairy and some notch-related genes, in theposterior PSM. Because the phase of oscillation is graduallyshifted along the posterior-to-anterior axis, a wave of oscillation appears to move in a posterior-to-anterior fashion. Thisoscillatory gene expression subsequently results in periodical
generation of morphologically segmented somites.
The spatial pattern of somites is defined by positioning ofintersomitic boundaries and rostro-caudal patterning withinthem. 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 the bHLH transcriptional regulator plays a criticalrole in this conversion. In mice, the prospective segmentation boundary is periodically established in the anterior
PSM at the rostral border of the Mesp2 expression domain.
Conversely, recent studies of this and other groups strongly
suggest that Mesp2 does not directly define the position of
the segmentation boundary, rather that other genes called
Ripply1 and Ripply2 play more essential roles in this conversion in mice and zebrafish. Ripply genes encode ~100 aminoacid proteins, that degrades Tbx6 proteins, which is involvedin the positioning of the segmentation boundary. We haveexamined the mechanism of this conversion by focusing on
the interaction between Ripply and other molecules involvedin this conversion, including Hairy and FGF pathway molecules. Currently, we are visualizing the spatio-temporal shiftsof these molecules in the PSM in wild-type and segmentation-defective zebrafish embryos.

Publication List:〔Original papers〕

H., Yokota, D., Otosaka, S., Kikuchi, M., Kinoshita, H., Fujino, Y.,• Ban,
Yabe, T., Ovara, H., Izuka, A., Akama, K., Yamasu, K., Takada, S., and••Kawamura, A. (2019). Transcriptional autoregulation of zebrafish tbx6
is required for somite segmentation. Development 146, dev177063. doi:
10.1242/dev.177063
Kikuchi, M., Nishimura, T., Saito, D., Shigenobu, S., Takada, R.,
Gutierrez-Triana, J.A., Cerdán, J.L.M., Takada, S., Wittbrodt, J.,
Suyama, M., and Tanaka, M. (2019). Novel components of germline
sex determination acting downstream of foxl3 in medaka. Dev. Biol.
445, 80-89. doi: 10.1016/j.ydbio.2018.10.019
Shinozuka, T., Takada, R., Yoshida, S., Yonemura, S., and Takada, S.
(2019). Wnt produced by stretched roof-plate cells is required for thepromotion of cell proliferation around the central canal of the spinal
cord. Development 146, dev159343. doi: 10.1242/dev.159343

31

National Institute for Basic Biology

Developmental Biology

DIVISION OF EMBRYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professor:

KOYAMA, Hiroshi
NONOMURA, Keiko
Technical Staff:
OKA, Sanae
JSPS Postdoctoral Fellow: NAKANOH, Shota
Postdoctoral Fellow:
KISHI, Kanae
SOKENDAI Graduate Student: KATAGIRI, Saki
USAMI, Fumiko
SAKURAI, Jun
Visiting Graduate Student: KATSUTA, Hiroki
MIKOSHIBA, Seiya
Visiting Scientist:
ARATA, Masaki
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,and the interaction between the embryo and maternal tissue
is essential in this process. Another characteristic is thehighly regulative potential of embryos. Cells within embryos
interact with their environment and establish their own fatesand behaviors. We want to provide basic and fundamental
information about the specification of the differentiation ofcell lineages, embryonic axes, cell behaviors, body shape
regulation and tissue morphology in early mammalian development. We are also interested in the relationship between
embryos and their environment, the oviducts and the uterus.

I. Live observation of early mouse development

To understand the mechanisms underlying embryonicdevelopment and morphogenesis, it has become common toobserve the behavior of cells and gene expression in living
embryos in a variety of animal species. Progress in genetic
engineering and embryonic manipulation concomitantly withthe progress in optics and microscope technologies, including development of highly sensitive photo-detectors, hasallowed us to observe developing embryos, even mammalianones, in real time. 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 across many laboratories over the world. We
have also established mouse lines to monitor the cell cycle.
We have been studying the behavior of cells within embryos
by applying newly developed image analyses following theobservation of cell behaviors within embryos using thesemouse lines. We found that the proximal visceral endoderm
overlying the extra-embryonic ectoderm shows coherent cell
growth in a proximal-anterior to distal-posterior direction.

32

We also observed that directional cell migration is coupledwith cell elongation in the anterior region, suggesting that the
behaviors of visceral endoderm cells vary between regions
during peri-implantation stages.
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 celldifferentiation in these stages. Combining these two types ofreporter transgenic mouse lines, we have been analyzing the
behaviors of cells and comparing gene expression properties
at the 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 the developmental biology
of mammalian embryos, embryos are usually removed fromthe uterus, and those that are isolated are analyzed. We havebeen analyzing the early embryonic development of themouse by comparing the changes in the uterine epithelium.
To observe the morphogenesis of embryos within the uterus,
serial sections of implanted uteruses were made, and imagesof the embryos within it were captured to make high resolution three-dimensional re-constructions. We are preparing
samples obtained from various developmental stages. Precise
positions of embryonic implantation and changes in theuterine epithelium correlating with embryonic development
have been examined using these images. We are identifying the molecules involved in the interaction between theembryo proper and uterine cells, which may play a majorrole in embryonic development.

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

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 a polarized manner 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 of cilia was not coordinated along the ovary-uterus
axis throughout the epithelia; some cells were oriented
orthogonally to the axis, while some others exhibited reverse
polarity. In addition to the ciliary polarity phenotype, cellular
shape polarity was also impaired in the Celsr1-deficientmice. We found a new feature of cellular polarity in thewild type oviduct, e.g. that the apical surface of epithelial
cells was elongated along the ovary-uterus axis. In Celsr1deficient mice, epithelial cells showed less elongation and arandomized 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.
Epithelial folds are observed in various organs including
the oviducts, guts, and airways etc. Longitudinally aligned
folds are also observed in the oviducts of birds and frogs. To
investigate the mechanisms of the epithelial fold pattern formation, we utilized mathematical modeling and simulations.
By considering mechanical properties of the epithelial sheets,
we reproduced the longitudinally aligned and branched folds
which are observed in wild-type and Celsr1 mutant mice,
respectively (Figure 2). In addition, our model also successfully reproduced circumferentially aligned folds and zigzag
folds observed in other organs. We are also focusing onsome other PCP regulators, and are trying to understand how
oviduct epithelial cells establish and maintain their polarity.

IV. A nalysis of the mechanical propertiesof cells and tissues during embryonicdevelopment

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, measuring cellular and tissue mechanical states is necessary. We
developed statistical techniques to infer mechanical statesusing fluorescent microscopic images during morphogenesis(Figure 3). By employing this method, we inferred mechanical forces in multi-cellular systems including cultured epithelial cells, and early embryogenesis in C. elegans and mice.
Further computational simulations based on the inferredmechanical information reproduced morphological featuresof the multi-cellular systems. Thus, the mechanical information will be useful to investigate physical mechanisms ofearly embryonic development and morphogenesis duringorganogenesis in late stages of development.

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

V. Mammalian tissue morphogenesis requiring
mechanosensor channel PIEZOs

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

Several examples have shown that mechanical stimuli
can work as key components for tissue or organ development. However, our knowledge about the involvementof 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 to the cell
membrane. Series of data show that PIEZO2 serves as the33

National Institute for Basic Biology

Developmental Biology

Positive or
negative pressure,
shear stress

Publication List:〔Original papers〕

Ca2+, Na+,
Mg2+, K+

+ +
+
+
+
+
Piezo
+ +
+ +
non-selective

cation channel

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.

main mechanosensor in sensory neurons for light touch sensation, proprioception and breathing. We recently found thatPIEZO1 in endothelial cells is required for lymphatic valve
formation (Figure 5). To further elucidate how PIEZO1mediated mechanotransduction is involved in lymphatic
valve formation, we have been developing systems to manipulate mechanical stimuli and monitor PIEZO activity in vitro
and in vivo. Analyses utilizing these systems and mouselines deficient in PIEZOs will clarify the relationship among
mechanical forces, PIEZO activation, cellular responses, andtissue morphogenesis.

T.C., Ishii, Y., Nguyen, V.D., Yamamoto, S., Hamashima, T.,• Đặng,Okuno, N., Nguyen, Q.L., Sang, Y., Ohkawa, N., Saitoh, Y., Shehata,

•
•
•
•
•
•

M., Takakura, N., Fujimori, T., Inokuchi, K., Mori, H., Andrae, J.,
Betsholtz, C., and Sasahara, M. (2019). Powerful homeostatic controlof oligodendroglial lineage by PDGFRα in adult brain. Cell Rep. 27,
1073-1089. doi: 10.1016/j.celrep.2019.03.084
Honda, H., Abe, T., and Fujimori, T. (2020). The chiral looping of
the embryonic heart is formed by the combination of three axial
asymmetries. Biophys. J. 118, 742-752. doi: 10.1016/j.bpj.2019.11.3397
Kamemizu, C., and Fujimori, T. (2019). Distinct dormancy progression
depending on embryonic regions during mouse embryonic diapause.
Biol. Reprod. 100, 1204-1214. doi: 10.1093/biolre/ioz017
Kawasoe, R., Shinoda, T., Hattori, Y., Nakagawa, M., Pham, T.Q.,
Tanaka, Y., Sagou, K., Saito, K., Katsuki, S., Kotani, T., Sano,
A., Fujimori, T., and Miyata, T. (2020). Two-photon microscopic
observation of cell-production dynamics in the developing mammalian
neocortex in utero. Dev. Growth Differ. 62, 118-128. doi: 10.1111/
dgd.12648
Kiyonari, H., Kaneko, M., Abe, T., Shioi, G., Aizawa, S., Furuta, Y., andFujimori, T. (2019). Dynamic organelle localization and cytoskeletal
reorganization during preimplantation mouse embryo development
revealed by live imaging of genetically encoded fluorescent fusion
proteins. Genesis 57, e23277. doi: 10.1002/dvg.23277
Sone, E., Noshiro, D., Ikebuchi, Y., Nakagawa, M., Khan, M., Tamura,Y., Ikeda, M., Oki, M., Murali, R., Fujimori, T., Yoda, T., Honma, M.,
Suzuki, H., Ando, T., and Aoki, K. (2019). The induction of RANKL
molecule clustering could stimulate early osteoblast differentiation.
Biochem. Biophys. Res. Commun. 509, 435-440. doi: 10.1016/j.
bbrc.2018.12.093
Yamada, K., Hamashima, T., Ishii, Y., Yamamoto, S., Okuno, N.,
Yoshida, N., Yamada, M., Huang, TT., Shioda, N., Tomihara, K.,
Fujimori, T., Mori, H., Fukunaga, K., Noguchi, M., and Sasahara, M.
(2018). Different PDGF receptor dimers drive distinct migration modesof the mouse skin fibroblast. Cell Physiol. Biochem. 51, 1461-1479.
doi: 10.1159/000495594〔Review article〕

H., Shi, D., and Fujimori, T. (2019). Biophysics in oviduct:• Koyama,
Planar cell polarity, cilia, epithelial fold and tube morphogenesis,

Figure 5. Reduced number of lymphatic valves in mice lacking PIEZO1
in endothelial cells.

VI. Mechanics of cell population patterning
during development

During development, cells actively and/or passively move,resulting in various cell distribution patterns. We investigatedthe effect of passive cell movements provoked by frictional
forces from adjacent growing tissues. The passive movements generated various patterns, such as an elongated cellcluster and multiple cell clusters, etc. Difference in cellular
stiffness was critical for these pattern formations. The former
situation is actually observed during elongation of the notochord in mice. Together with active cell movements and thedifferential adhesion hypothesis which has been recognized
as a classical concept within biology, passive cell movements
can be effective for diverse pattern formation.

34

egg dynamics. Biophys. Physicobiol. 16, 89-107. doi: 10.2142/
biophysico.16.0_89

National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:

KITADATE, Yu
NAKAGAWA, Toshinori
Technical Staff:
MIZUGUCHI, Hiroko
JSPS Researcher:
IKEDA, Tatsuro
Postdoctoral Fellow:
HIRA, Seiji
SOKENDAI Graduate Student: HIRANO, Kodai
WANG, Zhe
Visiting Graduate Student: SATO, Toshiyuki
Visiting Undergraduate: BABAGUCHI, Hirotaka
Technical Assistant:
KON, Yayoi
MARUYAMA, Ayumi
FUJITA, Miyako
Secretary:
KUBOKI, Yuko

Overview of our research

Production of numerous sperm over a long period in thetestis is fundamental for continuity of life across generations.
The Division of Germ Cell Biology aims to understand the
key processes of germ cell development that lead to robust
sperm formation.
Our emphasis has specifically been on the regulation andfunctional behavior of spermatogenic stem cells (SSCs) inthe testes of mice. SSCs play a crucial role, not only as theorigin of long-term sperm production ensuring the continuityof life, but also as the crucible of genetic and potentially epigenetic mutations, the underpinning of evolution as well ascongenital disease mechanisms. The principal and signature
strategy of our research is to directly observe the behavior
of individual germ cells in vivo in their natural (i.e. physiological) state and take advantage of intravital live-imaging,lineage analysis, and biophysical analysis
Regarding the study of SSCs, first, we found that SSCs
include a functional hierarchy, comprised of an “actual” stem
cell compartment that is able to self-renew, and a differentiation-primed, “potential” stem cell compartment. We havebeen also investigating the cellular identity of “actual” SSCs,
their in vivo behavior at a single-cell resolution, and theunderlying mathematical principles, leading to the discoveryof the dynamics of “population asymmetry” and subsequent“neutral competition” between SSCs. We are also highlyinterested in the area of “potential stem cells”. In steady-state
spermatogenesis, these largely contribute as transient amplifying cells and differentiate rather than self-renew. However,when tissues are insulted or testicular cells are transplanted
into host testes, their probability of self-renewal increases
and they effectively replenish the lost “actual” stem cells.
Such flexible and probabilistic features of stem cells havebeen found to be paradigmatic for many other stem cellsupported tissues.
Our past key references related to these studies include
Nakagawa et al., Dev. Cell 2007; Yoshida et al., Science
2007; Nakagawa et al., Science 2010; Klein et al., Cell Stem
Cell 2010; Hara et al., Cell Stem Cell 2014, Ikami et al.,Development 2015, and Tokue et al., Stem Cell Reports 2017.

Mechanism of “mitogen competition” regulates
the stem cell density homeostasis in an “open”
niche

We have developed a novel hypothesis regarding the mechanism of SSC maintenance in the mouse testis (Kitadate etal., Cell Stem Cell 2019). In contrast to better-investigatedtissues such as Drosophila testes and ovaries, as well asmammalian intestinal crypts, where physical contact madeby stem cells with an anatomically-defined niche provides aplatform to maintain homeostasis, the question of how stem
cell homeostasis is achieved in the mouse testis remains
unknown. In the mouse testes’ seminiferous tubules, theplace where spermatogenesis proceeds, SSCs are motile
and dispersed among their progeny, thus representing aclass of microenvironment designated as an “open niche”(Yoshida, Dev. Growth. Diff. 2008). Intriguingly, the densityof SSCs (defined as GFRα1+ cells) is strikingly constantwhen averaged over a certain length (e.g., >1cm), despite
local fluctuations. This suggests a presence of an unknown
mechanism that regulates the SSC homeostasis in a mannerthat stabilizes their average density.

I. FGF5 expression near the vasculature and its
mitogenic function on GFRα1+ spermatogonia

In order to find the key factors regulating the GFRα1+ celldensity, we screened the genes expressed in the tubule areathat were facing the interstitium and vasculature, where
GFRα1+ cells preferentially locate (Figure 1A). From thisscreening, we focused on Fgf5, since it is located in peritubularly located large flattened cells (termed lymphatic endo-

Figure 1. (A) Outline of the screening for genes preferentially expressedin the vasculature-associated region. (B) Representative ISH images for
Fgf5 (blue) in testis sections, counterstained with nuclear fast red.. (C)Representative image of an inter-tubular region of a GFRα1-GFP mouse
testis stained for GFP (green), FGF5 (magenta) and DNA (blue). Scalebars, 10 µm. (D) Mitogenic effect of FGF5 (red) or FGF2 (blue) on
cultured spermatogonia. Fold increase in the number of GS cells cultured
with indicated concentration of FGF5 for 8 days. Shown in average ± SEM(n = 3 independent experiments). (E) Effects of FGF5 on gene expression.
GS cells depleted of FGF2 and GDNF for 3 days were supplemented withor without FGF5 (100 ng/ml) for 24 hours, followed by cDNA microarray
analyses. * intertubular arterioles/venules All figures are reproduced frommodifications taken from Kitadate et al., Cell Stem Cell (2019).

35

National Institute for Basic Biology

Developmental Biology

thelial cells) near the interstitium (Figures 1B and C). By
using an in vitro spermatogonial culture, we found that FGF5
showed mitogenic and anti-differentiation effects (Figures
1D and E).

II. F GF5 controls GFRα1 + cell density in a
linear dosage-dependent manner

We then investigated the in vivo role of FGF5 in micecarrying a null allele (Fgf5–) or an extra copy of BACmediated transgene (BAC-Fgf5 Tg). In mutant testes, theaverage density of GFRα1+ spermatogonia showed a strikingly linear correlation with Fgf5 dosage (Figure 2A). Thesemutants notably sustained steady-state spermatogenesis withdifferent density set points of GFRα1+ cells that correlate ina manner that depends linearly on Fgf5 dosage during adulthood (Figure 2B).

the simplest form of competition consistent with the generalmechanism of FGF signal reception by target cells. We
detected speckled FGF5 signals inside GFRα1+ cells (Figure
3A). Significant portions of FGF5 cytoplasmic signals wereco-localized with SDC4 as cytoplasmic puncta on LAMP1+
lysosomes (Figures 3B). These observations indicated that
GFRα1+ cells consume extracellular FGF5, supporting theidea that GFRα1+ cells compete for extracellular FGF.

Figure 3. (A) Representative images of GFRα1-GFP+ (cyan) cells exhibiting the speckled cytoplasmic staining of FGF5 (magenta, white arrowheads). (B) Representative images of SDC4+ (magenta) cells co-stainedfor FGF5 (green) with LAMP1 (cyan). FGF5+ speckles (arrows) were
often co-localized with LAMP1+ (arrowheads) foci in SDC4+ cytoplasmic
clamp. Scale bars, 10 µm.

V. H omeostatic stem cell density regulation
follows from a model of “mitogen competition”

Figure 2. (A) Average densities ± SEM of GFRα1+ cells and representative IF images of whole-mount seminiferous tubules for GFRα1 (right)of 2.5 month-old mice with indicated genotypes. Scale bar, 50µm. (B)Average densities ± SEM of GFRα1+ cells in mice with indicated genotypes and ages. (C–E) Indexes of proliferation (EdU+ and pH3+ fractions),
differentiation [quantified as the RARγ+/KIT– (≈NGN3+) over GFRα1+cell ratio], and death (cPARP+ fraction) in GFRα1+ cells of indicatedmice at 2.5 months of age.

III. Each GFRα1+ cell receives an unchanged
level of FGF signal in Fgf mutants

Given the mitogenic and differentiation-inhibiting functions of FGF5, we first considered whether GFRα1+ cells
receive altered levels of FGF signal in mutants, which in turnchanges their fate resulting in altered densities. However, therates of proliferation, differentiation and death of GFRα1+cells, as well as their gene expression profile, surprisingly
were not different between Fgf5–/–, BAC-Fgf5Tg/+ and WT
mice (Figures 2C–E; not shown), indicating conserved fatebehavior of GFRa1+ cells between mutants.
The above findings lead to a hypothesis that FGF supply
is a limiting factor that is competed for among the GFRα1+cells. In this case, the levels of FGF signal received by each
GFRα1+ cell would be equalized among Fgf mutants harboring different densities of GFRα1+ cells.

IV. GFRα1+ spermatogonia consume FGF5

To further develop this hypothesis, we then examined
whether GFRα1 + cells consume the extracellular FGF5
when they receive its signal in vivo, as this was probably

36

To understand the mechanism of density regulation, wedeveloped a minimal theoretical model, in which GFRα1+cells are exposed to a steady supply of FGF from the lymphatic endothelial cells, which in turn elicits the concentration-dependent mitogenic and differentiation-inhibiting
activities, while also being consumed by, the GFRα1+ cells.
This model was found to be capable of capturing the counterintuitive observation that the fate behavior of GFRα1 +cells does not change in Fgf mutants (Figures 2C–E), as aconsequence of the steady-state FGF concentration being
always pinned and at which the increase (renewal) anddecrease (differentiation) of GFRα1+ cells is balanced. Given
that GFRα1+ cells effectively compete with each other forthe limited supply of FGFs, we refer to this mechanism as
the “mitogen competition”.
We then questioned whether the model could also predict
quantitatively the dynamics of stem cells, if the system isstrongly perturbed from a steady state. Analysis of the model
predicted that the recovery of GFRα1+ cell density following
a strong perturbation from its steady-state (viz. uninjured)value would show decaying oscillations. To test this prediction, we examined the kinetics during regeneration following the reduction of GFRα1+ cells by injecting a cytotoxic
reagent, busulfan. Strikingly, decaying oscillations that converged into the steady-state value over several months wereindeed observed, with a profile that quantitatively matchedwith our theory (Figure 4).

〔Review Articles〕

S. (2019). Heterogeneous, dynamic, and stochastic nature of• Yoshida,
mammalian spermatogenic stem cells. Current Topics in Developmental
Biology 135, 245-285. doi: 10.1016/bs.ctdb.2019.04.008

S. (2020). Mouse spermatogenesis reflects the unity and• Yoshida,
diversity of tissue stem cell niche systems. Cold Spring Harbor
Perspectives in Biology, a036186. doi: 10.1101/cshperspect.a036186

Figure 4. Observed kinetics of the GFRα1 + cell density following
busulfan treatment in WT (left), and examples of IF images of wholemount seminiferous tubules stained for GFRα1 (right). Model results(curves) compared to experimental measurements (dots) of the average
GFRα1+ cell density following busulfan treatment.

To summarize, our results show that the in vivo fatebehavior of GFRα1+ cells is regulated by the mitogenic andanti-differentiation effects of FGFs released in proximity
to the vasculature (Figure 5). We propose that competition
for mitogens might be a paradigmatic mechanism that can
explain the regulation of stem cell homeostasis in a wide
range of tissues.

Figure 5. A conceptual diagram of mitogen competition leading to stem
cell density homeostasis. Limited supply of FGF from a subset of lymphatic endothelial cells in proximity to vasculature plays a key role instem cell density homeostasis.

Publication List:〔Original Papers〕

Y., Jörg, D.J., Tokue, M., Maruyama, A., Ichikawa, R.,• Kitadate,
Tsuchiya, S., Segi-Nishida, E., Nakagawa, T., Uchida, A., Kimura-••

Yoshida, C., Mizuno, S., Sugiyama, F., Azami, T., Ema, M., Noda,
C., Kobayashi, S., Matsuo, I., Kanai, Y., Nagasawa, T., Sugimoto, Y.,
Takahashi, S., Simons, B.D., and Yoshida, S. (2019). Competition for
mitogens regulates spermatogenic stem cell homeostasis in an open
niche. Cell Stem Cell 24, 79-92. doi: 10.1016/j.stem.2018.11.013
Nakamura, S., Hira, S., Fujiwara, M., Miyagata, N., Tsuji, T., Kondo,
A., Kimura, H., Shinozuka, Y., Hayashi, M., Kobayashi, S., and Mukai,
M. (2019). A truncated form of a transcription factor Mamo activates
vasa in Drosophila embryos. Commun. Biol. 2, 422. doi: 10.1038/
s42003-019-0663-4
Shinozuka, T., Takada, R., Yoshida, S., Yonemura, S., and Takada, S.
(2019). Wnt produced by stretched roof-plate cells is required for thepromotion of cell proliferation around the central canal of the spinal
cord. Development 146, dev159343. doi: 10.1242/dev.159343

37

National Institute for Basic Biology

Developmental Biology

LABORATORY OF REGENERATION BIOLOGY
Director General

AGATA, Kiyokazu

SOKENDAI Graduate Student: SUGIURA, Nao
KUROKI, Yoshihito
Technical Assistant:
KAJIURA-KOBAYASHI, Hiroko
Secretary:
SAKAGAMI, Mari

Comparative Regenerative Biology

We use animals with high regenerative abilities, such asplanarians and newts, to understand the principle of regeneration. In particular, we investigate the difference between
regenerative and non-regenerative animals to evoke said
abilities from non-regenerative animals. We have alreadysucceeded in achieving this with planarians, which were able
to regenerate their heads through RNAi (Umesono et al.,2013 Nature) in addition to accomplishing functional joint
regeneration in frogs through the activation of reintegration
systems (Tsutsumi et al., 2016 Regeneration).
We are trying to induce limb-regeneration ability in frogs,
as they lose the capability to achieve complete limb regeneration after metamorphosis. Thus, we are focusing on the
Sonic hedgehog (Shh) enhancer MFCS1 (mammals-fishesconserved sequence 1), since it was suggested that the loss of
MFCS1 activity after metamorphosis might cause a failure toachieve the aforementioned limb regeneration in adult frogs(Yakushiji et al., 2009). When we compared the MFCS1
sequences between newts (C.p. and P.w.) and frogs (X.l. andX.t.), newts were found to possess several specific sequences(Figure 1). Thus, we subsequently planned to swap the
MFCS1 sequences between newts and frogs using CRISPR/
Cas9 technology.

Figure 2. Deletion patterns of the newt-MFCS1 region (ca. 1.6 kb)
obtained by microinjection of several different cocktails of gRNAs withCRISPR/Cas9.

Trial for the cultivation of planarianembryonic and adult pluripotent stem cells

We developed an isolation method of adult pluripotent stem
cells (aPSC) from planarian using FACS and tried to cultivate them under in vitro culture conditions, but we have yet
to succeed in propagating these cells. LCDM and EPSCM
media have recently been developed to cultivate mouse EPS(Expanded Potential Stem) cells, which can differentiate both
embryonic and extra-embryonic cells. Thus, it is expected thatthese media might work to cultivate embryonic pluripotent
stem cells (ePSC) derived from non-mammalian species. Thus,
we tried to cultivate planarian embryonic cells by LCDM and
EPSCM media and got healthy planarian ePSC like-aggregatesin vitro (Figure 3). We also attained similar healthy cellaggregates from planarian aPSC after isolation using FACS.
However, we unfortunately could not detect the active proliferative activity of these cells in these culture media so far.

Figure 3. planarian ePSC-aggregates formed in EPSC (left) and EPSCM(right) media.

Publication List:〔Original Papers〕

K., Sugano-Yasunaga, W., Taniguchi, A., Agata, K.,• Kishimoto,Nonaka, S., and Funayama, N. (2019). Skeleton construction upon

Figure 1. Comparison of the MFCS1 sequences between newts and frogs.

We then prepared various guide RNAs to swap thesesequences, and succeeded in the production of mosaicallytargeted Iberian ribbed newts (Pleurodeles waltl: P.w., Figure2) and frogs (Xenopus tropicalis: X.t.). Thus, we have tried
the PITch method to knock-in the newt-MFCS1 fragment
into the frog-MFCS1 site, but have yet to succeed in achieving this.

••local regression of the sponge body. Dev Growth Differ 61, 485-500.
doi:10.1111/dgd.12636
Matsunami, M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T.,
Yamaguchi, K., Uchiyama, I., Mori, K., Tashiro, K., Ito, Y., et al. (2019).
A comprehensive reference transcriptome resource for the Iberian ribbed
newt Pleurodeles waltl, an emerging model for developmental andregeneration biology. DNA Res 26, 217-229. dio:10.1093/dnares/dsz003
Mori, M., Narahashi, M., Hayashi, T., Ishida, M., Kumagai, N., Sato,Y., Bagherzadeh, R., Agata, K., and Inoue, T. (2019). Calcium ions in
the aquatic environment drive planarians to food. Zoological Lett 5, 31.
doi:10.1186/s40851-019-0147-x〔Review article〕

S., and Agata, K. (2019). Evolutionary view of pluripotency• Nakanoh,seen from early development of non-mammalian amniotes. Dev Biol
452, 95-103. doi: 10.1016/j.ydbio.2019.04.014

38

National Institute for Basic Biology

Neurobiology

DIVISION OF BEHAVIORAL NEUROBIOLOGY

Professor

HIGASHIJIMA, Shin-ichi

Assistant Professor:

KIMURA, Yukiko
TANIMOTO, Masashi
Technical Staff:
TAKEUCHI, Yasushi
Postdoctoral Fellow:
SHIMAZAKI, Takashi
JSPS Postdoctoral Fellow: SUZUKI, Daichi
SOKENDAI Graduate Student: SUGIOKA, Takumi
KAWANO, Kohei
Visiting Graduate Student: UEMURA, Yuto
Technical Assistant:
TERASAWA, Yoko
WATAKABE, Ikuko
ITO, Hiroko
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 has been very difficult to reproducibly identify cell types.
However, molecular genetic studies conducted over the past
15 years have strongly suggested that the expression of transcription factors in the developing CNS helps determine themorphological 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 instudying the functions of the corresponding neurons in theneuronal circuits.
To fully exploit the methodology described above, we
use larval zebrafish as experimental animals. The biggest
advantage of doing so is that larval zebrafish are almost
completely transparent. This allows us to utilize many opticaltechniques, including morphological/functional imaging andoptogenetics. We can also perform targeted in vivo electrophysiological recording with relative ease using this transparent model. An additional advantage of zebrafish is that their
CNS is much simpler than that of mammals. This enables us
to perform detailed functional analysis of neuronal circuits ata single cell resolution. Our hope is to reveal the operational
principles of vertebrate CNS by using this simple system.
We have been focusing on studying neuronal circuits that
control locomotion. Much of the control of locomotor movements is accomplished by neuronal circuitry located in the
spinal cord. Therefore, the focus of our studies has beenspinal neuronal circuits in larval zebrafish.
In addition to zebrafish, we have also started to use medaka
as experimental animals. Medaka have many advantages thatare similar to those of zebrafish Because NIBB is the main
hub of the Medaka National Bioresource Project, we areideally located in regards to experiments using medaka. To
begin with, we explored whether knock-in fish could be efficiently generated using the CRISPR/Cas9 technique.

< NINS ExCELLS >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 the CNS by using gene promoters/
enhancers of genes and are known to be expressed in subsetsof neurons. Most of the genes we used are transcription
factors expressed in subsets of neurons in the developing
CNS. We also used genes whose expressions are tightly
related to neurotransmitter properties of neurons (i.e., vesicular glutamate 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. By 2014, we
succeeded in establishing a reliable knock-in method by utilizing the CRISPR-Cas9 system. The method we have developed is highly efficient, so much so that nearly one-third of
the animals we raise become transgenic founders. Thus far,
we have established more than 20 knock-in transgenic fish.
Thus, this method greatly facilitates our functional analysis
on neuronal circuits.

II. N euronal circuits that control rhythmic
pectoral fin movements

Limbed vertebrates exhibit coordinated rhythmic movements of the forelimbs and hindlimbs during locomotion.
Neuronal circuits that control rhythmic limb movements in
mammals have been investigated for decades, but our knowledge of them is still limited because of the complexity of
their limbs. In this case, rhythmic movement of pectoral finsduring swimming in larval zebrafish is an attractive model(Figure 2). The pectoral fins of larval zebrafish show left andright alternated rhythmic movements, and they are actuatedonly by two types of muscles: the abductor (Ab) and the
adductor (Ad) (Figures 3). Due to the simplicity of pectoral
fins, we expect that we will be able to characterize neuronal
circuits that control rhythmic pectoral fins in more detail.
We performed electrophysiological recordings of Ab motoneurons (MNs) and Ad MNs during fictive swimming. BothAb MNs and Ad MNs show rhythmic spiking activities(Figure 4). Activities of Ab MNs and Ad MNs on the sameside alternated. Voltage clamp recordings showed that both
Ab MNs and Ad MNs received alternating excitatory andinhibitory inputs during swimming cycles. Excitation mainly
occurred in their preferential firing phase, and inhibition

39

National Institute for Basic Biology

Neurobiology

mainly occurred for the rest of the period. To obtain insights
into the source of these inputs, we are now investigating thetiming of spiking activities in possible premotor interneurons.

Figure 2. Rhythmic movements of the pectoral fin (arrow) duringswimming in larval zebrafish.
Figure 5. Ablation of spinal V1 neurons by using the Cre-loxP system.
DTA, diphtheria-toxin A.

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

Figure 6. Ventral root recordings of fictive swimming elicited byelectrical stimulations (stim) in a control and an En1-DTA fish (left).
Swimming frequency of control and En1-DTA fish during the initial
phase of swimming elicited by electrical stimulation (right).

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

III. Functional analysis of V1 neurons for axial
movements during swimming

Inhibition plays an important role in shaping motor outputs
during locomotion. In the spinal cord of larval zebrafish,
there are mainly two types of inhibitory neurons: 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 animportant role in ensuring antagonistic movements of the left
and right side of body (see Section IV). The role of the latter(ipsilaterally-projecting inhibitory neurons) is less clear.
V1 neurons are one class of neurons that are defined by theexpression of a transcription factor En1 and constitute major
components of ipsilaterally-projecting inhibitory neurons inthe spinal cord. To investigate the function of V1 neurons,
we genetically ablated spinal V1 neurons in larval zebrafish
using diphtheria toxin A (En1-DTA fish, Figure 5). From theanalysis of the En1-DTA fish, we revealed two roles of V1
neurons.
First, we found that the swimming frequency was declinedin the En1-DTA fish (Figure 6). The results show that V1

40

Figure 7. Activity of slow type motoneurons (MNs) in control andEn1-DTA fish during fictive swimming.

neurons play an important role for controlling locomotion
speed.
Second, we showed that V1 neurons play an importantrole in the selection of active sets of neurons. Slow-type
motor neurons, which project slow muscle, are known to beinactive during strong/fast movements in larval zebrafish. In
the En1-DTA fish, however, we found that slow-type motor
neurons were vigorously active during strong movements(Figure 7). We investigated the mechanism underlying thisphenotype. In the wild type, slow-type MNs were found
to receive strong inhibitory inputs. In the En1-DTA fish,these strong inhibitory inputs were found to be absent. The
results indicate that V1 neurons are the source of the strong
inhibition onto slow-type MNs and that the inhibition playa crucial role in suppressing spiking activities of slow-type
MNs during strong/fast swimming.

IV. F unctional diversity of glycinergic
commissural inhibitory neurons in larvalzebrafish

Coordinated movements of the left and right side of the
body is critical for any types of locomotion including
walking, flying and swimming. The coordination is mainly
mediated by commissural neurons located in the spinal cord.
In fish swimming, it is known that commissural inhibitory neurons in the spinal cord ensure left-right alternating
movements. The developmental origin of these commissural
inhibitory neurons, however, has been elusive. We investigated anatomy and function of two commissural inhibitory
neuron types, dI6dmrt3a and V0d, derived from the pd6 andp0 progenitor domains, respectively. We found that bothof these commissural neuron types have monosynaptic,
inhibitory connections to neuronal populations active duringswimming, supported their role in providing inhibition to thecontralateral side. V0d neurons tend to fire during faster andstronger movements, while dI6dmrt3a neurons tend to fire
more consistently during swimming. Ablation of dI6dmrt3a
neurons leads to an impairment of left-right alternating
activity through abnormal co-activation of motor neurons on
both sides of the spinal cord. Our results suggest that dI6dmrt3a and V0d commissural inhibitory neurons synergistically provide inhibition to the opposite side across different
swimming behaviors (Figure 8).

Publication List:〔Original Papers〕

R.A., Roberts, R., Sengupta, M., Kimura, Y., Higashijima,• Callahan,
S.-I., and Bagnall, M.W. (2019). Spinal V2b neurons reveal a role for

•
•
•
•

ipsilateral inhibition in speed control. eLife 8, e47837. doi: 10.7554/
eLife.47837
Frank, T., Mönig, N.R., Satou, C., Higashijima, S.-I., and Friedrich,
R.W. (2019). Associative conditioning remaps odor representations andmodifies inhibition in a higher olfactory brain area. Nat. Neurosc. 22,
1844-1856. doi: 10.1038/s41593-019-0495-z
Kimura, Y., and Higashijima, S.-I. (2019). Regulation of locomotor
speed and selection of active sets of neurons by V1 neurons. Nat.
Commun. 10, 2268. doi: 10.1038/s41467-019-09871-x
Satou, C., Sugioka, T., Uemura, Y., Shimazaki, T., Zmarz, P., Kimura,Y., and Higashijima, S.-I. (2020). Functional diversity of glycinergic
commissural inhibitory neurons in larval zebrafish. Cell Rep. 30, 30363050. doi: 10.1016/j.celrep.2020.02.015
Shimazaki, T., Tanimoto, M., Oda, Y., and Higashijima, S. (2019).
Behavioral role of the reciprocal inhibition between a pair of Mauthnercells during fast escapes in zebrafish. J. Neurosci. 39, 1182-1194. doi:
10.1523/JNEUROSCI.1964-18.2018

Figure 8. Summary of the proposed role of dI6dmrt3a and V0d neurons
during swimming. CPG, central pattern generator

41

National Institute for Basic Biology

Neurobiology

LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

NIBB Research Fellow:
Postdoctoral Fellow:
Technical Assistant:

KOBAYASHI, Taisuke
NISHIUMI, Nozomi
WATABE, Mihoko

We believe that the best way to understand mechanisms
underlying a certain system is to reconstruct the system as
it exists. By utilizing novel computer technologies, we areaccordingly trying to build the systems based on psychophysical and ecological viewpoints in particular to understand systems of animal perception and behavior. This typeof methodology based on computers will pave the way forbiology in the future.

et al., 2017). The fine control of various features of living
fish has been difficult to achieve in studies of their behavior.
However, computer graphics allow us to systematicallymanipulate morphological and motion cues. Therefore, wehave constructed 3D computer graphic animations based on
tracking coordinate data and photo data obtained from actual
medaka (Figure 1). These virtual 3D models will allow us
to represent medaka more faithfully as well as undertake amore detailed analysis of 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), and on underwater imaging technology (Abe etal., 2019 and Yamamoto et al., 2019).

I. Psychophysical study of medaka fish

One of our major subjects is the psychophysical and computational study of medaka (Oryzias latipes, Matsunaga &Watanabe, 2010). We have recently made progress in studies
of prey-predator interaction using both these organisms
and zooplankton. Visual motion cues are one of the mostimportant factors for eliciting animal behaviors, including predator-prey interactions in aquatic environments. To
understand the elements of motion that cause such selective
predation behavior, we used a virtual plankton system where
we analyzed the predation behavior in response to computergenerated plankton. To achieve this, virtual plankton models
were programmed on a computer and presented to predator
medaka. As a result of this analysis, we confirmed thatmedaka exhibited predation behavior against several characteristic movements of the virtual plankton, particularly
against a swimming pattern that could be characterized asa pink noise motion. The analysis of predator-prey interactions via pink noise motions will be a research field of greatinterest moving forward (Matsunaga & Watanabe, 2012).
In recent years, we have made progress in the study ofschooling behaviors of Medaka. Many fish species are
known to live in groups, and visual cues have been shown
to play a crucial role in the formation of shoals. By utilizing biological motion stimuli, which in this case was the
depiction of a moving creature by using just a few isolated
points, we examined whether physical motion information is involved in the induction of shoaling behavior. We
consequently found that the presentation of virtual biological motion can clearly induce shoaling behavior, and haveshown 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 further enhance our understanding of how non-humananimals extract and process information which is vital for
their survival (Nakayasu & Watanabe, 2014).
Additionally, we have developed a novel method for
behavior analysis using 3D computer graphics (Nakayasu

42

Figure 1. Virtual medaka fish.

Ⅱ. Psychophysical study of human vision

Another of our major subjects is the psychophysical andtheoretical studies of the visual illusions experienced byhuman beings. One recent focus of this debate is the flashlag effect, in which a moving object is perceived to headtowards a flashed object when both objects are aligned inan actual physical space. We developed a simple conceptual model explaining the flash-lag effect (Watanabe etal., 2010). In recent years, we have made more developed

Figure 2. Monstre Benham Illusions. Benham Top is a black and white
top that, when rotating, causes people to perceive illusory colors such as
red or blue. Illusory colors were first discovered by the German physicist
Gustav Fechner, and the Benham top was built by the English journalist
Charles Benham. We have implemented a version of the Benham top thatinstead of spinning, works like a flip book (demo, https://www.sonycsl.
co.jp/news/9164/).

novel visual illusions, such as the shelf-shadow illusion (3rdAward of the 5th Illusion Contest, https://doi.org/10.6084/
m9.figshare.6137558) and the Monstre Benham illusion(2nd Award of the 11th Visual Illusion and Auditory Illusion
Contest, https://doi.org/10.6084/m9.figshare.10046534,
Figure 2) .

with low-price webcams and several free computer libraries),
and thus, are expected to contribute wider range of animal
research.

Figure 4. A computer animation of the virtual predatory fish.

Publication List:〔Original papers〕

Figure 3. Optical flow vectors detected in the illusion. Red bars denotethe direction and magnitude of vectors, yellow dots denote the startpoints of the vectors. A and B: Illusion, C and D: Non-Illusion.

In 2018, we successfully generated deep neural networks(DNNs) that represent the perceived rotational motion for
illusion images that were not physically moving, yet similar
to what we experience in human visual perception. (Figure
3, Watanabe et al., 2018). These DNN computer models will
help to facilitate our future work on perception science. This
experimental system was applied to studies on evolutionary
illusion generator in collaborating with Dr. Lana Sinapayen(Sony CSL). (EIGen, https://github.com/LanaSina/evolutionary_illusion_generator. Sinapayen and Watanabe, 2020).

E., Yasugi, M., Takeuchi, H., Watanabe, E., Kamei, Y., and• Abe,
Yamamoto, H. (2019). Development of omnidirectional aerial display•••

with aerial imaging by retro-reflection (AIRR) for behavioral biology
experiments. Opt. Rev. 26, 221-229. doi: 10.1007/s10043-019-00502-w
Nishiumi, N., Mori, A. (2020). A game of patience between predator
and prey: waiting for opponent’s action determines successful captureor escape. Can. J. Zool. 98, 351-357. doi:10.1139/cjz-2019-0164.
Sinapayen, L., and Watanabe, E. (2020). Motion illusions arise fromlight source ambiguity. J. Brief Ideas doi: 10.5281/zenodo.3709097
Yamamoto, H., Abe, E., Yasugi, M., Watanabe, E., and Takeuchi, H.
(2019). Aquatic information display and its applications for behavioral
biology experiments. Proc. SPIE 10997, Three-Dimensional Imaging,
Visualization, and Display 2019, 1099707. doi: 10.1117/12.2521225

Ⅲ. Ecological study of tactics in predators andprey.

We are interested in the behavioral interactions between
predators and prey not only concerning the psychophysical aspect but also the ecological aspect. Predators and prey
have developed various tactics to overcome each other, in theprocess of the co-evolution. To elucidate the sophisticationof these tactics, we have conducted experiments to examine
the mechanisms and efficacies of predatory and antipredator behaviors of various animals such as snakes, frogs, fish,
dragonflies and bats. During this fiscal year, we published apaper, which outlined the vulnerability of preemptive actionsof both predator snakes and prey frogs, and the possibility
that this vulnerability instigates a ‘waiting game’ between
these animals (Nishiumi & Mori, 2020). Several on-goingstudies were also presented at conferences, which wereabout the tactics of predatory bats regarding echolocation. In
addition, we have developed several experimental technologies for those studies, which present interactive virtual predators to prey animals based on animation or robotics (Figure4). These technologies do not require specific uncommon
hardware or software as previous technologies have required(i.e. The present technologies work in Windows and Mac OS

43National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

HASEBE, Mitsuyasu

Associate Professor

MURATA, Takashi

Assistant Professor:

TAMADA, Yosuke
ISHIKAWA, Masaki
Specially Appointed Assistant Professor: MANO, Hiroaki
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellow:
KOSETSU, Ken
Postdoctoral Fellow:
AOYAMA, Tsuyoshi
NARUKAWA, Hideki
SOKENDAI Graduate Student: PALFALVI, Gergo
SUDA, Hiraku
ZHANG, Liechi
HORIUCHI, Yuta
DE VILLIERS, Ruan
CHEN, Peng
UEDA, Masamichi
Visiting Graduate Student: YU, Changxiu
GU, Nan
SAKAZAKI, Takuya
Visiting Undergraduate: TANASE, Kuniaki
Visiting Scientist:
YUE, Sun
Technical Assistant:
AOKI, Etsuko
FUKADA, Hatsumi
HIRAMATSU, Mika
IKEDA, Keiko
KABETANI, Keiko
KAJIKAWA, Ikumi
KAMIDA, Chiharu
MATSUZAKI, Yoko
MASUOKA, Tomoko
MORISHITA, Mio
NISHI, Tayo
OHI, Shoko
WEN, Hsin-I
SUGAYA, Tomomi
OGAWA, Yuko
Secretary:
KOJIMA, Yoko
HASEBE, Yuki

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, unexplained
phenomena still remain, one of which is the evolution ofcomplexity. It is difficult to explain the mechanisms needed
to evolve complex adaptive traits at the cellular and organismal levels, such as cell division machinery, regeneration,and novel organ development. Such traits comprise many
components and become adaptive only when all of them 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 identify themechanisms needed for the evolution of complex characters.

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

44

The cell division axis has to be properly regulated during
the development of both metazoans and land plants. Genetic
changes in the regulation of the cell division axis lead tothe development of multicellular organisms. Since they do
not have centrosomes and asteroid bodies, both of which
are involved in the axis formation of metazoans, land plants
most likely have different regulatory mechanisms. We aim toinvestigate the connecting factors between microtubules and
GRAS transcription factors that regulate periclinal cell divisions in the moss Physcomitrella patens. In addition to identifying these factors, we also intend to study the spatiotemporal regulatory mechanisms will in order to understand thebasis of body plan evolution in comparison to those found in
the flowering plant Arabidopsis thaliana and the green algae
Closterium peracerosum–strigosum-littorale.
Dr. Ken Kosetsu and his colleagues found that one GRAS
transcription factor is required for periclinal cell divisions,while another is required for anticlinal cell divisions. We
identified that the former GRAS transcription factor represses the expression of the latter GRAS transcription factor.
This regulation seems to decide the location where thedivision axis is changed from anticlinal to periclinal manner
From the observation of microtubule dynamics using the
GFP-a-tubulin as a marker in the presence or absence of the
GRAS transcription factor, Dr. Kosetsu’s team found thatthe progression of the cell cycle was regulated by the GRAS
transcription factor. This result suggests that the division axis
is determined through the cell cycle-dependent cell shape.
Regulation of local cell growth underlies the geometricshape formation of individual cells. Cell shape is an instructive factor in oriented cell division, which guides morphogenesis in land plants. Mr. Liechi Zhang found that atransporter mutant, which belongs to the ABC gene family,
exhibited a cell shape abnormality. This led to a retarded
gametophore development of Physcomitrella patens. Timelapse imaging of the fluorescent protein tagged transporter
revealed a positive correlation between the membrane localization of this transporter and local cell growth. We are presently investigating the underlying mechanism using knockout, knock-in, and inducible overexpression transgenic plants
in wild type and cytoskeleton marker lines of P. patens.
This is a collaborative project that is being undertaken by
our division and Dr. Rumiko Kofuji (Kanazawa University),Dr. Hiroyuki Sekimoto (Japan Women’s University), andAtsushi Mochizuki (RIKEN).

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

During mitosis, all eukaryotic cells divide chromosomes
into two daughter cells using a bipolar mitotic spindle, whichis composed of microtubules. The centrosomes, which act as
microtubule organizing centers, induce formation of the twopoles in metazoan cells. In contrast, the land plants cells andtheir sister group, zygnematales green algae, form a bipolar
spindle in the absence of centrosomes. In order to understandthe mechanism of acentrosomal spindle formation, the stepsof microtubule reorganization during spindle formation
should be visualized. We collaborated with Prof. Tomomi
Nemoto of Hokkaido University and developed a two-photon spinning disk confocal microscope, which enables
3-dimensional imaging of living cells with high temporal andspatial resolution. We also established a minispindle system,
which involves a bipolar microtubule complex composedof an isolated chromosome and microtubules in tobacco
cells. Analyses of microtubule behavior in the minispindle
are underway in collaboration with Dr. Daisuke Tamaoki(Toyama Univ.) with Takashi Murata being the coordinating
researcher for this study.

IV. E volution of Regeneration: Genetic
Regulatory Networks of Reprogramming ofDifferentiated Cells to Stem Cells

Both land plants and metazoa have the capacity toreprogram differentiated cells to stem cells. In the moss
Physcomitrella patens, the leaf excision induces the reprogramming of differentiated leaf cells next to the excision
to stem cells. We found that histone H3.3 chaperone HIRA
proteins are induced in the leaf cells next to the excision,and positively regulate reprogramming. The role of HIRAs
in reprogramming partly depends on the plant-specific transcription factor genes SQUAMOSA PROMOTER BINDING
PROTEINs (PpSBPs). PpSBPs are involved in the repression of the reprogramming and HIRAs are necessary for the
repression of PpSBPs through histone modifications during
the reprogramming. Characterization of HIRAs and PpSBPs
are in progress and are mainly being conducted by Yukiko
Kabeya and Yosuke Tamada.

VI. 
Evolution of Regeneration: Other
pathwaysV. 
Evolution of Regeneration: Master
Regulator for Reprogramming STEMIN

VII. E volution of Molecular Mechanisms of
Plant Movement

Epigenetic modifications, including histone modifications, stabilize cell-specific gene expression programs tomaintain cell identities in both metazoans and land plants.
Notwithstanding the existence of these stable cell states, inland plants, stem cells are formed from differentiated cells
during post-embryonic development and regeneration, indicating that land plants have an intrinsic ability to regulate
epigenetic memory to initiate a new gene regulatory network.
However, it is less well understood how epigenetic modifications are locally regulated to influence specific genes necessary for cellular changes without affecting other genes in agenome. In this study, we found that ectopic induction of theAP2/ERF transcription factor STEMIN1 in leaf cells of themoss Physcomitrella patens decreases a repressive chromatin mark, histone H3 lysine 27 trimethylation (H3K27me3),
on its direct target genes before cell division, resulting in
the conversion of leaf cells to chloronema apical stem cells.
STEMIN1 and its homologs positively regulate the formation of secondary chloronema apical stem cells from chloronema cells during development. Our results suggest that
STEMIN1 functions in an intrinsic mechanism underlying
local H3K27me3 reprogramming to initiate stem cell formation. Masaki Ishikawa is this study’s coordinating researcher.

We also found that a component of the DNA repair
machinery functions in the STEMIN1-induced stem cell
formation in leaves. This study is mainly being conductedby Ruan de Villiers.

Figure 1. STEMIN can change leaf cells into stem cells.

Nan Gu, a joint graduate student at both Huazhong
Agricultural University and NIBB whose research interestis the relationship between DNA damage and the reprogramming from differentiated cells to stem cells, has found thatDNA damage is a novel trigger to induce the reprogramming
without wounding or dead cells in P. patens.

The sensitive plant Mimosa pudica and the Venus fly
trap Dionaea muscipula have long attracted the interestof researchers due to their spectacular leaf movements in
response to touch or other external stimuli. Although various
aspects of these movements have been elucidated by physiological approaches, the lack of genetic tools available
has hampered the investigation of molecular mechanisms
involved in these processes. To overcome this obstacle,
we developed genetic transformation methods for theseplants. Functional analysis of motor organ-enriched genes
by CRISPR/Cas9-mediated knockout identified two channel
genes and a transcription factor that play various roles in
rapid leaf movements in M. pudica. We also generated atransgenic D. muscipula expressing a calcium sensor protein,which enables us to study how this plant counts the numberof mechanical stimuli for its trap closure. The studies on M.
pudica and D. muscipula were conducted mainly by Hiroaki
Mano and Hiraku Suda, respectively.

VIII. E stablishment of a new single celltranscriptome method

Next-generation sequencing technologies have made it
possible to carry out transcriptome analysis at the singlecell level. Single-cell RNA-sequencing (scRNA-seq) data
provide insights into cellular dynamics, including intercellular heterogeneity as well as inter- and intra-cellular fluctuations in gene expression that cannot be studied using populations of cells. The utilization of scRNA-seq is, however,45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

restricted to cell types that can be isolated from their original
tissues, and it can be difficult to obtain precise positional
information for these cells in situ. Here, we establishedsingle cell-digital gene expression (1cell-DGE), a methodof scRNA-seq that uses micromanipulation to extract thecontents of individual living cells in intact tissue while
recording their positional information. With 1cell-DGE, we
could detect differentially expressed genes (DEGs) during
the reprogramming of leaf cells of the moss Physcomitrella
patens, identifying 6382 DEGs between cells at 0 and 24 hafter excision. Furthermore, we identified a subpopulation of
reprogramming cells based on their pseudotimes, which were
calculated using transcriptome profiles at 24 h. 1cell- DGEwith microcapillary manipulation can be used to analyze thegene expression of individual cells without detaching themfrom their tightly associated tissues, enabling us to retain
positional information and investigate cell–cell interactions.

separating the flat leaf and pitcher leaf establishment in theprimordia. Mass sequencing of shoot apices among several
environmental conditions utilized to alter the leaf/pitcher
ratio are in progress. We are also working on refinement of
the genome especially in epigenetic studies.

Publication List:〔Original papers〕

H., Li, Q., Fang, X., Li, J., Curtis, N.E., Altenburger, A., Shibata,• Cai,T., Feng, M., Maeda, T., Schwartz, J.A., Shigenobu, S., Lundholm, N.,

•
•
•
•
•
•

Nishiyama, T., Yang, H., Hasebe, M., Li, S., Pierce, S.K., and Wang,
J. (2019). Data descriptor: A draft genome assembly of the solarpowered sea slug Elysia chlorotica. Sci. Data 6, 190022. doi: 10.1038/
sdata.2019.22
Hashida, Y., Takechi, K., Abiru, T., Yabe, N., Nagase, H., Hattori, K.,
Takio, S., Sato, Y., Hasebe, M., Tsukaya, H., and Takano, H. (2020).
Two ANGUSTIFOLIA genes regulate gametophore and sporophyte
development in Physcomitrella patens. Plant J. 101, 1318-1330. doi:
10.1111/tpj.14592
Kakishima, S., Liang, Y.-S., Ito, T., Yang, T.-Y.A., Lu, P.-L., Okuyama,Y., Hasebe, M., Murata, J., and Yoshimura, J. (2019). Evolutionary
origin of a periodical mass-flowering plant. Ecol. Evol. 9, 4373-4381.
doi: 10.1002/ece3.4881
Ishikawa, M., Morishita, M., Higuchi, Y., Ichikawa, S., Ishikawa,T., Nishiyama, T., Kabeya, Y., Hiwatashi, Y., Kurata, T., Kubo,
M., Shigenobu, S., Tamada, Y., Sato, Y., and Hasebe, M. (2019).
Physcomitrella STEMIN transcription factor induces stem cell
formation with epigenetic reprogramming. Nat. Plants 5, 681-690. doi:
10.1038/s41477-019-0464-2
Kubo, M., Nishiyama, T., Tamada, Y., Sano, R., Ishikawa, M., Murata,
T., Imai, A., Lang, D., Demura, T., Reski, R., and Hasebe, M. (2019).
Single-cell transcriptome analysis of Physcomitrella leaf cells during
reprogramming using microcapillary manipulation. Nucleic Acids Res.
47, 4539-4553. doi: 10.1093/nar/gkz181
Kumar, M., Quan, X., Awatsuji, Y., Cheng, C., Hasebe, M., Tamada, Y.,
and Matoba, O. (2020). Common-path multimodal three-dimensional
fluorescence and phase imaging system. J. Biomed. Opt. 25, 1-15. doi:
10.1117/1.JBO.25.3.032010
Sasaki, T., Tsutsumi, M., Otomo, K., Murata, T., Yagi, N., Nakamura,M., Nemoto, T., Hasebe, M., and Oda, Y. (2019). A Novel KataninTethering Machinery Accelerates Cytokinesis. Current Biol. 29, 40604070. doi: 10.1016/j.cub.2019.09.049〔Review article〕
Figure 2. Schematic representation of the workflow of single cell –digital gene expression (DGE)

IX. Evolution of Carnivory in Flowering Plants

Carnivorous plants exploit animals as a source of nutritionand have inspired long-standing questions about the origin
and evolution of carnivory-related traits. To investigate themolecular bases of carnivory, Dr. Hideki Narukawa performed mostly comparative analysis of carnivorous pitcher
leaves and non-carnivorous flat leaves in the carnivorous
plant Cephalotus follicularis. We found that hollow formation, which was the first step in pitcher leaf development,
was initiated by growth inhibition on the adaxial side of leaf
primordia. This process may be regulated by the phytohormone cytokinin.
In order to elucidate the origin of the pitcher shape, Gergo
Palfalvi, a graduate student, looked into the initiation factors

46

T., Lan, T., Farr, K.M., Ibarra-Laclette, E., Herrera-Estrella, L.,• Renner,
Schuster, S.C., Hasebe, M., Fukushima, K., and Albert, V.A. (2018).

Carnivorous plant genomes. In: Carnivorous Plants: Physiology,
Ecology, and Evolution, A.M. Ellison and L. Adamec, eds. (Oxford
University Press), pp. 135-153.

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF SYMBIOTIC SYSTEMS

Professor

Associate Professor

KAWAGUCHI, Masayoshi SOYANO, Takashi

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

KAWADE, Kensuke
FUKADA-TANAKA, Sachiko
LIU, Meng
MAEDA, TaroTSUGANE, Mika
YANO, Koji
HASHIMOTO, KayoVisiting Scientist:
NAKAGAWA, Tomomi
SOKENDAI Graduate Student: OKUMA, Nao
GOTO, Takashi
Technical Assistant:
YOSHINORI, Yumi
ODA, Akiko
Secretary:
KOSUGI, Eiko

Rhizobium–legume symbiosis is one of the most successful mutually beneficial interactions on Earth. In this typeof symbiosis, soil bacteria called rhizobia supplies the hostlegumes with ammonia produced through bacterial nitrogen
fixation. In return, host plants provide the rhizobia with theirphotosynthetic products. To accomplish this biotic interaction, leguminous plants develop nodules on their roots.
However, 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 to elucidate the molecular mechanisms of both symbiotic systems.

I. Root nodule symbiosis

I-1 Recruitment of a factor involved in lateral root
development intro root nodule organogenesis

Plants develop lateral roots to efficiently absorb water andnutrients from soil. Additionally, legumes have acquired theability to produce root nodules. We have demonstrated thata transcription factor involved in the initial stages of lateralroot development regulates nodule development in coordination with NF-Y CAATA-box binding protein complex in L.
japonicus (Figure 1A).
This factor is known as ASL18/LBD16. L. japonicus ASL18/
LBD16 is expressed in the nodule primordia as well as in
the lateral root primordia. The former originates in the root
cortex, and the latter develops from the pericycle. ASL18/
LBD16 interacts with NF-Y subunits in vitro and in planta.
The ASL18/LBD16 expression domain overlaps those of NF-Y
subunit genes in nodule primordia. ASL18/LBD16 indicatedgenetic interaction with NF-Y subunit genes in nodule primordia development (Figure 1B), whereas nf-y subunit mutations
did not affect the fewer lateral root phenotype associatedwith asl18 mutants. Co-overexpression of ASL18/LBD16 inconcert with NF-Y subunit genes partially suppressed the
nodule-defective phenotype of daphne mutants, where NIN
expression in the root cortex was repressed.

Figure 1. Recruitment of ASL18 into nodule development. (A) A model of
ASL18-mediated pathway. (B) Nodule primordia in wild-type [3 days after
inoculation (dai)] and asl18a nf-ya1 nf-yb1 triple mutant roots (10 dai).

NIN is a nodulation-specific transcription factor initiating
nodule primordia. This factor binds with the intronic ciselement in ASL18/LBD16. The binding sites and its flanking
regions are highly conserved among legume ASL18/LBD16
genes. The acquisition of this site in an ancestor of legume
lineage could have influenced the ASL18/LBD16 function in
extant legumes. The interaction with NF-Y might confer the
new function upon legume ASL18/LBD16 orthologs.

I-2 Host symbiotic transcription factor network regulates robust infection processes

Bacterial entry into root tissues is an essential process forestablishing the symbiotic relationship between rhizobia andhost legumes. Rhizobia are entrapped by curled root hairs
and penetrate into the inner tissues of roots through infection
threads (ITs), host-membranous and tubular paths, developed
from infection foci. Multiple host transcription factors are
involved in these processes.
CYCLOPS is a transcription factor commonly required forearly signaling in both root nodule and AM symbioses andis essential for IT development. This factor directly regulates two transcription factor cording genes, NIN and ERN1
(Figure 2A). Our analyses showed that ERN1 also regulated
NIN expression independently of CYCLOPS after inoculation. NIN expression was repressed more extensively incyclops ern1 than in each single mutant.
As was expected, ectopic expression of either ERN1 or NIN
suppressed the IT-defect of cyclops by the transcriptional
hierarchy (Figure 2B). However, CYCLOPS and NIN did
not recover ITs in ern1. Therefore, ERN1 has a function incommon with CYCLOPS in NIN regulation, but it also has
a different role from CYCLOPS and NIN. In keeping with
this, ern1 exhibited a unique symbiotic root hair phenotype.
A depolarized balloon-shape root hair tip was observed in
ern1, whereas cyclops and nin displayed excessive root haircurling, resulting in the rhizobia being entrapped (Figure2C). The root hair response appeared to be arrested at an
earlier stage of the deformation process in ern1 than thoseobserved in cyclops and nin.
These results highlight that transcription factors regulating symbiotic root hair responses constitute this complex
network, and that they also interact with each other. This may
reinforce the flow of the transcriptional cascade and contribute to coordinated regulation of blanching pathways.

47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Figure 2. ERN1-mediated regulation of infection processes. (A) A modelof the transcription pathway. (B) Suppression of the IT-defective cyclops3 phenotype by ectopic expression of ERN1 and NIN. (C) Root hair
deformation. Bars; 0.2 mm (B) and 50 µm (C).

II. Arbuscular mycorrhizal symbiosis

III Polygala paniculata , a new model species
for study of evolution of root nodule
symbiosis

Figure 3. Five extraradical structures of R. irregularis DAOM197198; germ
tubes (GT), runner hyphae (RH), branched absorbing structures (BAS),
immature spores (IS) and mature spores (MS), and representative genes
highly expressed in each structure detected by SMART-seq2 analysis.

Figure 4. Characteristics of a non-leguminous plant P. paniculata. (A)A two months old plant. (B) Hairy roots generated from P. paniculata
hypocotyl. (C) Deformed root hairs of P. paniculata inoculated with M.
loti broad host range strain NZP2037. Bars; 2 mm (B) and 100 µm (C).

Arbuscular mycorrhizal fungi (AMF) mainly establish symbiotic relationships with most land plants for the purpose ofnutrient exchange. Many studies have revealed the regulationof processes in AMF, such as nutrient absorption from soil,
metabolism and exchange with host plants, and the genes
involved in that process. However, the spatial regulationof the genes within the structures comprising each developmental stage is not well understood. Accordingly, we
have demonstrated the structure-specific transcriptome of
the model AMF species, Rhizophagus irregularis. We performed an ultra-low input RNA-seq analysis, SMART-seq2,
comparing five extraradical structures, germ tubes (GT),runner hyphae (RH), branched absorbing structures (BAS),
immature spores (IS) and mature spores (MS) (Figure 3).
Our results suggest the importance of RH in the absorption
and reduction of nitrate and that of the BAS in the absorption of Pi, ammonium, and carboxylate. The enrichmentof numerous GO terms involved in DNA replication andnuclear cell division in IS appears to reflect the burst ofmitosis that occurs during spore maturation. SP7, a secreted
fungal effector of R. irregularis was markedly expressed in
MS. In addition to this, we reanalyzed the recently reported
RNA-seq data comparing intraradical mycelium and arbus-

48

cule. Our analyses captured the distinct features of eachstructure and revealed the structure-specific expression
patterns of genes related to nutrient transport and metabolism. A finding of note was that the transcriptional profiles
indicated what the distinct functions performed by BAS arein nutrient absorption. These findings provide a comprehensive dataset to advance our understanding of the transcriptional dynamics of fungal nutrition in this symbiotic system.
Furthermore, we have also applied for a patent for non-symbiotic culture of AMF in cooperation with Professors Kohki
Akiyama from Osaka Prefecture University, Katsuharu Saito
from Shinshu University, and Tatsuya Ezawa from Hokkaido
University.

Plants exhibiting root nodule symbiosis are arranged only
in only four orders of angiosperms called the monophyletic nitrogen-fixing clade. This phylogenetic relationship hasraised the hypothesis which states that a predisposition facilitating acquisition of the symbiosis occurs once in a common
ancestor in this clade. However, detailed evolutionary processes related to this are largely unclear. Performing comparative analyses with non-nodulating species phylogeneticallyrelated to legumes could be a better strategy for studying theevolutionary processes of nodule symbiosis.
P. paniculata is a non-nodulating plant belonging to a
family that is different from legumes but is classified into
the same order, Fabales (Figure 4A). It has a small body size,
high fertility and a short lifecycle, whereas other families
in Fabales were found to be trees (with the exception oflegumes). These characteristics are advantageous whencultivating them in laboratories. Thus, we assessed whether
this species was suitable as a model species for comparative studies with legumes. The estimated genome size of
this species was less than that of L. japonicus. Therefore, thewhole genome sequencing of P. paniculata could be determined by comparative genomics. We further developed thehairy root transformation method (Figure 4B) and optimized
tissue culture conditions to regenerate individual specimens.
These cultivation techniques enable gene function analyses
in this species.

P. paniculata can interact with AM fungi, thus suggesting
the presence of early signaling factors in nodule symbiosis.
Deformation of root hairs was observed when they were
inoculated with Mesorhizobium loti broad host range strain,indicating that P. paniculata has the potential to respond torhizobia (Figure 4C). As such, P. paniculata is a good candidate as a model plant in the evolutionary study of root nodule
symbiosis.

Publication List:〔Original papers〕

H., Hayashi-Tsugane, M., and Kawaguchi, M. (2020). Spatial• Fujita,
regulation of resource allocation in response to nutritional availability.

•

•
•
•
•
•
•
•
•
•
•

J. Theor. Biol. 486, 110078. doi: 10.1016/j.jtbi.2019.110078
Fukudome, M., Shimada, H., Uchi, N., Osuki, K.-I., Ishizaki, H.,
Murakami, E.-I., Kawaguchi, M., and Uchiumi, T. (2020). Reactive
sulfur species interact with other signal molecules in root nodule
symbiosis in Lotus japonicus. Antioxidants 9, 145. doi: 10.3390/
antiox9020145
Kameoka, H., Maeda, T., Okuma, N., and Kawaguchi, M. (2019).
Structure-specific regulation of nutrient transport and metabolism inarbuscular mycorrhizal fungi. Plant Cell Physiol. 60, 2272-2281. doi:
10.1093/pcp/pcz122
Kameoka, H., Tsutsui, I., Saito, K., Kikuchi, Y., Handa, Y., Ezawa, T.,
Hayashi, H., Kawaguchi, M., and Akiyama, K. (2019). Stimulation ofasymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids.
Nat. Microbiol. 4, 1654-1660. doi: 10.1038/s41564-019-0485-7
Kobae, Y., Ohtomo, R., Morimoto, S., Sato, D., Nakagawa, T., Oka, N.,
and Sato, S. (2019). Isolation of native arbuscular mycorrhizal fungi
within young thalli of the liverwort Marchantia paleacea. Plants 8, 142.
doi: 10.3390/plants8060142
Liu, M., Soyano, T., Yano, K., Hayashi, M., and Kawaguchi, M. (2019).
ERN1 and CYCLOPS coordinately activate NIN signaling to promote
infection thread formation in Lotus japonicus. J. Plant Res. 132, 641653. doi: 10.1007/s10265-019-01122-w
Nishida, H., Ito, M., Miura, K., Kawaguchi, and M., Suzaki, T. (2020).
Autoregulation of nodulation pathway is dispensable for nitrate-induced
control of rhizobial infection. Plant Signal. Behav. 15, 1733814. doi:
10.1080/15592324.2020.1733814
Soyano, T., Shimoda, Y., Kawaguchi, M., and Hayashi, M. (2019). A
shared gene drives lateral root development and root nodule symbiosis
pathways in Lotus. Science 366, 1021-1023. doi: 10.1126/science.
aax2153
Suzaki, T., Takeda, N., Nishida, H., Hoshino, M., Ito, M., Misawa,F., Handa, Y., Miura, K., and Kawaguchi, M. (2019). LACK OFSYMBIONT ACCOMMODATION controls intracellular symbiont
accommodation in root nodule and arbuscular mycorrhizal symbiosis
in Lotus japonicus. PLoS Genet. 15, e1007865. doi: 10.1371/journal.
pgen.1007865
Tokumoto, Y., Hashimoto, K., Soyano, T., Aoki, S., Iwasaki, W.,
Fukuhara, M., Nakagawa, T., Saeki, K., Yokoyama, J., Fujita, H.,
and Kawaguchi, M. (2020). Assessment of Polygala paniculata(Polygalaceae) characteristics for evolutionary studies of legume–rhizobia symbiosis. J. Plant Res. 133, 109-122. doi: 10.1007/s10265019-01159-x
Yoro, E., Nishida, H., Ogawa-Ohnishi, M., Yoshida, C., Suzaki,T., Matsubayashi, Y., and Kawaguchi, M. (2019). PLENTY, ahydroxyproline O-arabinosyltransferase, negatively regulates root
nodule symbiosis in Lotus japonicus. J. Exp. Bot. 70, 507-517. doi:
10.1093/jxb/ery364
Yoro, E., Suzaki, T., and Kawaguchi, M. (2020). CLE-HAR1 systemic
signaling and NIN-mediated local signaling suppress the increased
rhizobial infection in the daphne mutant of Lotus japonicus. Mol. PlantMicrobe Interact. 33, 320-327. doi: 10.1094/MPMI-08-19-0223-R

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:

ANDO, Toshiya
NAKAMURA, Taro
Technical Staff:
MIZUTANI, Takeshi
JSPS Postdoctoral Fellow: SAKAI, Hiroki
KONAGAYA, Tatsuro
TAKENAKA, Masaki
Postdoctoral Fellow:
MORITA, Shinichi
Research Staff:
KAWAGUCHI, Haruka
SOKENDAI Graduate Student: CHIKAMI, Yasuhiko
KITAZAWA, YurinaTechnical Assistant:
MORITA, Junko
HACHISUKA, Yukari
Secretary:
SAITO, EikoThe Division of Evolutionary Developmental Biology wasstarted in June 2015. We focus on the evolutionary novelties
acquired by insects in order to elucidate the molecular andevolutionary mechanisms that lead to the large variety oftraits that they display. Among this wealth of exciting traits,
our lab is currently focused on promoting research into (1)the origin and diversification of insect wings, (2) wing color
patterns and mimicry of ladybird beetles, and (3) the acquisition and diversification of beetle horns.

I. Origin and diversification of insect wings

The flight organ of insects has uniquely evolved whencompared to that of other various flying animals on earth,.
Despite over two centuries of debate, the evolutionary originof insect wings are still an enigma; one which we are trying
to decipher it by the use of evo-devo methods. In Drosophilamelanogaster, the wing master gene vestigial (vg) and its
interaction partner scalloped (sd) play pivotal roles in theformation of wing field identity. For this reason, these genes
are ideal research candidates in the investigation of wing
origin and evolution.
One way to identify the structure from which insect wings
first evolved is to explore the function of “wing” genes inancestral wingless (apterygote) species. To achieve this end,
we chose the firebrat, Thermobia domestica, as a model(Figure 1A). T. domestica belongs to Thysanura, whichis phylogenetically the closest extant relative of winged(pterygote) insects, thus making it ideal for elucidating

wing origin. We cloned vg and sd orthologs from T. domestica (Td-vg and Td-sd), and we developed RNA interference(RNAi) based methods for T. domestica to examine the functions of these genes. We are currently testing the functional
effects of altered transcription for each of these wing genes
in ancestrally wingless firebrats. In addition, we are performing comparative analyses of the function of these same genes
in “primitively winged” (hemimetabolous) insects (Figure1B) to obtain additional clues relevant to the understandingof the origin and evolution of insect wings.
Interestingly, our previous work showed that vg expressing epidermal tissue forms lateral outgrowths in non-winged
segments in the mealworm beetle (Ohde et al., 2013). Basedon these facts, we hypothesize that ancestral lateral bodywall outgrowths evolved into functional wings. However,genetic tools available for the analysis of basally branching
wingless species are limited. To overcome these limitations, we established CRISPR/Cas9-based germline genome
editing in T. domestica. Heritable mutations were successfully introduced in white locus, an evolutionarily conserved
gene, encoding the ATP-binding cassette (ABC) membranetransporter, of T. domestica by using CRISPR/Cas9 system.
This in turn results in white-eyed firebrats. In addition to
the RNAi-mediated gene knockdown (Ohde et al., 2009),
germline genome editing using CRISPR/Cas9 in T. domestica provides a platform technology that opens new research
opportunities concerning the evolution of insects, such as
insect wing origin. We are now conducting gene knock-out/
in within various “wing” genes to identify genetic details andcell lineage analyses in T. domestica (Figure 1).

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

A tremendous range of diversity in wing color patterns has
evolved among insects, which in turn play various ecologicallyimportant roles such as intraspecific sexual signaling, mimesis,
mimicry, and as a warning signal to predators. However,
the molecular mechanisms responsible for generating suchcolor patterns in most ladybird species remain elusive. To
investigate the developmental mechanisms of color patternformation, 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). The ladybird’s vivid wing color patternfunctions as a warning signal to predators that they taste bad.
At the same time, various other insect species utilize this ecological signal by mimicking the ladybirds’ wing color patterns.

A

B

B
A

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

50

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

Mimicry provides us with 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 has color patterns similar to Harmonia,
and is thought to be a Batesian mimicry of ladybird beetles(Figure 2B). To elucidate the molecular mechanisms underlying these wing color patterns, we established a technique forgermline transformation using a piggyBac vector and RNAi in
the ladybirds.
We recently identified a key gene, pannier, which regulates
intraspecific color pattern polymorphism in H. axyridis using
next generation sequencing technologies (RNA-seq andde novo genome assembly), and an RNAi-based screening
method that we established. pannier is expressed in specificregions in the wing, which synthesizes black pigment, andsuppresses red pigmentation. The expression pattern ofpannier is diversified according to the diverse color patterntypes in H. axyridis. These findings suggest that regulatory shift, such as change in enhancer activity, at the pannier
locus may be crucial for the evolution of wing color patterns
in H. axyridis. We are currently trying to elucidate the evolutionary origin of color patterns in ladybirds with a focus on
regulatory shifts at the pannier loci. We are also establishinggenome-editing technologies using TALEN and CRISPR/
Cas9 to tackle this issue, and have already established anefficient 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 the evolution ofwing color patterns in ladybird beetles. We are also establishing cryopreservation methods for germline cells in ladybird
beetles to assist in and the high risk of losing valuablegenetic bioresources in non-model insects. We recently
established ovary transplantation and ovarian cryopreservation techniques in ladybird beetles. We hope that the genetic
tools and techniques that we have established will further
facilitate this research.
In the future, we plan to analyze how the similar wing color
patterns of model and mimic are generated based on theknowledge obtained from H. axyridis. For example, do theyuse 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 area subject of great potential for evo-devo studies because
they have arisen multiple times de novo, as evolutionary“novelties”. However, the molecular mechanisms involved
in sexually dimorphic horn formation are still poorly understood. To investigate the developmental mechanisms ofhorn formation, we are focusing on the Japanese rhinoceros
beetle, Trypoxylus dichotomus (Coleoptera), which exhibits
remarkable sexual dimorphisms in head and thoracic horns.
The male-specific horns of T. dichotomus are one of the bestmodels for studying how an extreme, sex-specific morphology is formed (Figure 3, Control).
We have recently developed a larval RNAi technique in

T. dichotomus, which allowed us to molecularly dissect therelationship between the conserved genetic pathway for sex
differentiation and sexually dimorphic horn formation during
post embryonic development. We systematically evaluatedthe function of the sex determination gene, transformer (tra)in different developmental stages, and revealed in which
tissue and developmental stage the gene regulatory network
for sex differentiation is activated to form sexual dimorphic
horn in the head and thorax. In T. dichotomus, tra regulates
sex-specific splicing of the doublesex pre-mRNA, and its
loss of function results in sex transformation in females(Figure 3). tra RNAi treatments in females at early developmental stages during metamorphosis resulted in full sexualtransformation, whereas no transformation is observed in
the treatments at later stage. Therefore, we could estimate
the onset of activation of the developmental program for thesexually dimorphic horn formation by determining the latest
RNAi treatment timing when a full sexual transformation
phenotype is observed. Based on this approach, we estimatedthat the developmental program for sexually dimorphic hornformation is activated at 29 hours after the prepupal period.

Figure 3. tra RNAi phenotypes. In the tra RNAi females, ectopic hornformation was caused by the sex transformation (masculinization).
(Adapted from Morita et al., PLOS Genet., 15: e1008063, 2019)

We are currently focusing on this developmental stage
because crucial regulatory factors for horn formation anddifferentiation are supposed to be activated at this stage in T.
dichotomus. The present study provides a good starting point
to unveil the gene regulatory network for sexually dimorphic
horn formation and to purse the evolutionary origin of such aregulatory system.

Publication List:〔Original papers〕

S., Mitsuhashi, M., Chikami, Y., Kawaguchi, H., Niimi, T., and• Hashiro,
Yasueda, H. (2019). Construction of Corynebacterium glutamicum cells••

as containers encapsulating dsRNA overexpressed for agricultural pest
control. Appl. Microbiol. Biotechnol. 103, 8485-8496. doi: 10.1007/
s00253-019-10113-9
Morita, S., Ando, T., Maeno, A., Mizutani, T., Mase, M., Shigenobu,
S., and Niimi, T. (2019). Precise staging of beetle horn formation in
Trypoxylus dichotomus reveals the pleiotropic roles of doublesex
depending on the spatiotemporal developmental contexts. PLOS Genet.
15, e1008063. doi: 10.1371/journal.pgen.1008063
Sakai, H., Oshima, H., Yuri, K., Gotoh, H., Daimon, T., Yaginuma, T.,
Sahara, K., and Niimi, T. (2019). Dimorphic sperm formation by Sexlethal. Proc. Natl. Acad. Sci. USA 116, 10412-10417. doi: 10.1073/
pnas.1820101116

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF EVOLUTIONARY GENOMICS
Professor:

SHIGENOBU, Shuji

Postdoctoral Fellow:
KOBAYASHI, Yuuki
JSPS Postdoctoral Fellow: NOZAKI, Tomonari
SOKENDAI Graduate Student: YORIMOTO, Shunta
TAN, Kathrine
Visiting Scientist:
CHUNG, Chen-yo
Technical Assistant:
SUZUKI, Miyuzu

Symbiogenomics“Nothing, it seems, exists except as part of a network ofinteractions.” (Gilbert & Epel, 2008)

Every creature on Earth exists among a network of various
biological interactions. For example, many multicellularorganisms, including humans, harbor symbiotic bacteria intheir bodies. Some of them provide their hosts with essential
nutrients deficient in the host’s diet and others digest foods
that are indigestible by the host alone. Despite 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
new interdisciplinary science known as “Symbiogenomics”,
where we aim to understand the network of biological interactions at the molecular and genetic level. To this end,
we take advantage of state-of-the-art genomics, such as
next-generation sequencing technologies and CRISPR-Cas9
genome editing.

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

Aphid species bear intracellular symbiotic bacteria in thecytoplasm of bacteriocytes, which are specialized cells forharboring said bacteria. This mutualism is so obligate thatneither can reproduce independently. The genome sequenceof the pea aphid, Acyrthosiphon pisum, in consort with thatof bacterial symbiont Buchnera aphidicola illustrates theremarkable interdependency between these two organisms(IAGC, PLOS Biol. 2010; Shigenobu et al., Nature. 2000).
The genetic capacities of the pea aphid and the symbiont
for amino acid biosynthesis are complementary. 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 aphidsin obtaining beneficial symbionts. Lineage-specific gene
duplications have occurred in genes over a broad range offunctional categories, which include signaling pathways,
miRNA machinery, chromatin modification and mitosis. The
importance of these duplications for symbiosis remains to
be determined. We found several instances of lateral genetransfer from bacteria to the pea aphid genome. Some ofthem are highly expressed in bacteriocytes.
We recently discovered a novel class of genes in the pea
aphid genome that encode small cysteine-rich proteins with52

secretion signals that are expressed exclusively in the bacteriocytes of the pea aphid, and named these bacteriocytespecific cysteine-rich proteins (BCR) (Shigenobu & Stern,
Proc. R. Soc. B 2013). The BCR mRNAs are first expressed
at a developmental time point coinciding with the incorporation of symbionts strictly in the cells that contribute to thebacteriocyte, and this bacteriocyte-specific expression is
maintained throughout the aphid’s life. Furthermore, some
BCRs showed antibiotic activity (Uchi et al., Microbes.
Environ. 2019; This paper was selected as the “Most
Valuable Paper of the Year 2019” in the journal, Microbes
and Environments). These results suggest that BCRs act
within bacteriocytes to mediate the symbiosis with bacterial
symbionts, which is reminiscent of the cysteine-rich 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., Li, Q., Fang, X., Li, J., Curtis, N.E., Altenburger, A., Shibata,• Cai,T., Feng, M., Maeda, T., Schwartz, J.A., Shigenobu, S., Lundholm,

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•

N., Nishiyama, T., Yang, H., Hasebe, M., Li, S., Pierce, S.K., and andWang, J. (2019). Data descriptor: A draft genome assembly of the solarpowered sea slug Elysia chlorotica. Sci. Data 6, 190022. doi: 10.1038/
sdata.2019.22
Hojo, M., Shigenobu, S., Maekawa, K., Miura, T., and Tokuda, G.
(2019). Duplication and soldier-specific expression of geranylgeranyl
diphosphate synthase genes in a nasute termite Nasutitermes
takasagoensis. Insect Biochem. Mol. Biol. 111, 103177. doi: 10.1016/j.
ibmb.2019.103177
Kutsukake, M., Moriyama, M., Shigenobu, S., Meng, X.-Y., Nikoh,N., Noda, C., Kobayashi, S., and Fukatsu, T. (2019). Exaggeration andcooption of innate immunity for social defense. Proc. Natl. Acad. Sci.
USA 116, 8950-8959. doi: 10.1073/pnas.1900917116
Matsunami, M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T.,
Yamaguchi, K., Uchiyama, I., Mori, K., Tashiro, K., Ito, Y., Takeuchi,T., Suzuki, K.-I.T., Agata, K., Shigenobu, S., and Hayashi, T. (2019).
A comprehensive reference transcriptome resource for the Iberianribbed newt Pleurodeles waltl, an emerging model for developmental
and regeneration biology. DNA Res. 26, 217-229. doi: 10.1093/dnares/
dsz003Morita, S., Ando, T., Maeno, A., Mizutani, T., Mase, M., Shigenobu,
S., and Niimi, T. (2019). Precise staging of beetle horn formation in
Trypoxylus dichotomus reveals the pleiotropic roles of doublesex

•
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•

depending on the spatiotemporal developmental contexts. PLoS Genet.
15, e1008063. doi: 10.1371/journal.pgen.1008063
Ohbayashi, T., Futahashi, R., Terashima, M., Barrière, Q., Lamouche,F., Takeshita, K., Meng, X.-Y., Mitani, Y., Sone, T., Shigenobu,
S., Fukatsu, T., Mergaert, P., and Kikuchi, Y. (2019). Comparative
cytology, physiology and transcriptomics of Burkholderia insecticola
in symbiosis with the bean bug Riptortus pedestris and in culture. ISME
J. 13, 1469-1483. doi: 10.1038/s41396-019-0361-8
Sekii, K., Yorimoto, S., Okamoto, H., Nagao, N., Maezawa, T., Matsui,Y., Yamaguchi, K., Furukawa, R., Shigenobu, S., and Kobayashi, K.
(2019). Transcriptomic analysis reveals differences in the regulation ofamino acid metabolism in asexual and sexual planarians. Sci. Rep. 9,6132. doi: 10.1038/s41598-019-42025-z
Sugime, Y., Oguchi, K., Gotoh, H., Hayashi, Y., Matsunami, M.,
Shigenobu, S., Koshikawa, S., and Miura, T. (2019). Termite soldier
mandibles are elongated by dachshund under hormonal and hox gene
controls. Development 146, dev171942. doi: 10.1242/dev.171942
Uchi, N., Fukudome, M., Nozaki, N., Suzuki, M., Osuki, K.-I.,
Shigenobu, S., and Uchiumi, T. (2019). Antimicrobial activities
of cysteine-rich peptides specific to bacteriocytes of the pea aphid
Acyrthosiphon pisum. Microbes Environ. 34, 155-160. doi: 10.1264/
jsme2.me18148
Yaguchi, H., Suzuki, R., Matsunami, M., Shigenobu, S., and Maekawa,
K. (2019). Transcriptomic changes during caste development throughsocial interactions in the termite Zootermopsis nevadensis. Ecol. Evol.
9, 3446-3456. doi: 10.1002/ece3.4976

53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIORESOURCES

Specially Appointed Professor

NARUSE, Kiyoshi

Assistant Professor:
Research Staff:

ANSAI, Satoshi
KANEKO, Hiroyo
HARA, Ikuyo
YANOGAWA, Azusa
Visiting Scientist:
SATO, Tadashi
Visiting Graduate Student: IMAEDA YASSUMOTO,
TamirisTechnical Assistant:
AJIOKA, Rie
KOIKE, Chieko
KOIKE, Yukari
TAKAKI, Chikako
TESHIMA, Yuuko
TORII, Naoko
YAMAZAKI, Toko
Secretary:
SUZUKI, Tokiko

The medaka is a small egg-laying fresh water fish found inbrooks and rice paddies in Eastern Asia. This species has along history as an experimental animal, especially in Japan.
Our laboratory has conducted studies on the dynamics oftranscriptional and chromatin accsssibility landscape during
medaka development, the understanding of pigment celldifferentiation using body color mutants, genome sequenceof the Javanese medaka, Oryzias javanicus and the molecular genetic basis of diversified sexually dimorphic traits inOryzias species, In addition to these activities, our laboratory was charged with the responsibility of leading the
National BioResource Project Medaka (NBRP Medaka) from2007.

much-improved gene model set including about 17,000 novel
isoforms and identified 1600 transcription factors, 1100 longnon-coding RNAs, and 150,000 potential cis-regulatory
elements as well. The work provides the first comprehensive
omics datasets of medaka embryogenesis. The data portal(http://tulab.genetics.ac.cn/medaka_omics) will serve as adaily reference tool for the entire medaka community.

II. 
I dentification of the causal gene of
the medaka body color mutant, few
melanophore(fm)The body coloration of animals is due to pigment cells
derived from neural crest cells which are multipotent anddifferentiate into diverse cell types. Medaka possess four
distinct types of pigment cells known as melanophores, xanthophores, iridophores, and leucophores. The few melanophore (fm) mutant found amongst medaka is characterized by
reduced numbers of melanophores and leucophores. We haveidentified kit-ligand as the gene whose mutation gives rise to
the fm phenotype. This identification was confirmed by generation of kit-ligand knockout medaka and the findings thatthese fish also manifest reduced numbers of melanophores
and leucophores and fail to rescue the fm mutant phenotype.
We also found that expression of sox5, pax7a, pax3a, andmitfa genes is down-regulated in both fm and kit-ligand
knockout medaka, implicating c-Kit signaling in the regulation of the expression of these genes as well as the encodedtranscription factors in pigment cell specification.

I. D aynamic transcriptional and chromatin
accesibikity landscape during medaka
development.

A high-quality genome sequence and a variety of genetic
tools are available for medaka. However, existing genome
annotation is still rudimentary, as it was mainly based oncomputational prediction and short-read RNA-seq data. To
overcome this situations, we conducted long-read RNA-seq,
short-read RNA-seq, and ATAC-seq coraboration with Dr.
Tu at the Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences. This work constructed a

Figure 1. Overview of medaka multi-omics anslysis during development

54

Figure 2. The fm mutants show a reduction in melanophore and leucophore pigmentation(A and B) and not in xanthophore and iridophore
pigmentation (C, D, E and F).

III. Genome sequence of the Javanese medaka,
Oryzias javanicus, as a model for studying
seawater adaptation

Medaka fish in the genus Oryzias are an emerging model
system for studying the molecular basis of vertebrate evolution. This genus contains approximately 35 species andexhibits great morphological, ecological and physiological differences among it’s species. Among these species,
the Java medaka, Oryzias javanicus, is the species thathas most typically adapted to seawater. We sequenced
and assembled the whole genome of O. javanicus, as amodel fish species for studying molecular mechanisms ofseawater adaptation. In teleost fish, the major osmoregulatory organs are the gills, intestine and kidney, and these playdifferent roles to maintain body fluid homeostasis. Many
genes encoding hormones, receptors, osmolytes, transporters, channels and cellular junction proteins are potentiallyinvolved in this osmotic regulation. In addition to the osmoregulation, hatching enzyme activity dramatically changes indifferent salt conditions. At the hatching stage, fish embryos
secrete a specific cocktail of enzymes in order to dissolve theenvelope. In the medaka O. latipes, digestion of the envelope
occurs after the cooperative action of two kinds of hatching
enzymes: (i) the high choriolytic enzyme (HCE) and (ii) the
low choriolytic enzyme (LCE) (Yasumasu et al., 2010).The
HCE shows higher activity in freshwater than in brackish
water (Kawaguchi et al., 2013). Thus, availability of thehigh-quality reference genome in O. javanicus would facilitate further research for investigating the molecular basis ofphysiological differences including the osmotic regulationand the hatching enzyme activity among Oryzias species.

IV. A drianichthyidae fish in Sulawesi: a
model system to explore the molecular
genetic basis of diversification in sexual
dimorphism
Sexual dimorphism is prevalent, but often differs remarkably between closely related species. However, we know
little about which genes and genetic changes can actuallycontribute to diversification of sexually dimorphic traits. The
family Adrianichthyidae are a small teleost species commonly
considered to be medaka. Although their native habitats arewidely distributed in East and South-East Asia, 20 of 37
Adrianichthyidae species are endemic to Sulawesi, Indonesia.
We have studied the molecular genetic basis of diversification in sexual dimorphism using the endemic species as amodel system, because their sexual dimorphic body colorations are significantly diversified in closely-related species.
As a research resource for the Sulawesi species, we firstly
sequenced and assembled a genome of Oryzias celebensis.
The assembly was anchored to 18 different chromosomes bylinkage mapping and was annotated using RNA-seq data fromadult and embryonic tissues. We then investigated the molecular mechanisms underlying red coloration in pectoral fins,a characteristic feature of O. woworae males. Quantitative
trait loci (QTL) mapping in a F2 intercross between a maleof O. woworae and a female of a close relative O. celebensis without any red fins revealed that an autosomal locus
controls the red pigmentation. Subsequent quantitative gene
expression analysis revealed that a gene is a strong candidate
responsible for the red fins, which is highly expressed in the
red fins of O. woworae males by the cis-regulatory mutation.
CRISPR/Cas-mediated mutagenesis in the gene caused a deficiency in the fins’pigment cells . Additionally, a behavioral
analysis showed that the mutant males had lower reproductive
success than the wild-type males. These results suggest that
ectopic expression of an autosomal gene in the pectoral finsby the cis-regulatory change will cause the red coloration in
the pectoral fins, which might spread by increasing the reproductive success in males.

S., Hibino, T., Minokawa, T., Naruse, K., Kamei, Y.,• Amemiya,Uemura, I., Kiyomoto, M., Hisanaga, S., and Kuraishi, R. (2019).

•
•

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•

Development of the coelomic cavities in larvae of the living isocrinid
sea lily Metacrinus rotundus. Acta Zool. 100, 414-430. doi: 10.1111/
azo.12274
Hiraki-Kajiyama, T., Yamashita, J., Yokoyama, K., Kikuchi, Y., Nakajo,
M., Miyazoe, D., Nishiike, Y., Ishikawa, K., Hosono, K., KawabataSakata, Y., Ansai, S., Kinoshita, M., Nagahama, Y., and Okubo, K.
(2019). Neuropeptide B mediates female sexual receptivity in medaka
fish, acting in a female-specific but reversible manner. eLife 8, e39495.
doi: 10.7554/eLife.39495
Ishikawa, A., Kabeya, N., Ikeya, K., Kakioka, R., Cech, J.N., Osada,N., Leal, M.C., Inoue, J., Kume, M., Toyoda, A., Tezuka, A., Nagano,
A.J., Yamasaki, Y.Y., Suzuki, Y., Kokita, T., Takahashi, H., Lucek,
K., Marques, D., Takehana, Y., Naruse, K., Mori, S., Monroig, O.,
Ladd, N., Schubert, C.J., Matthews, B., Peichel, C.L., Seehausen, O.,
Yoshizaki, G., and Kitano, J. (2019). A key metabolic gene for recurrent
freshwater colonization and radiation in fishes. Science 364, 886-889.
doi: 10.1126/science.aau5656
Nakayama, T., Shimmura, T., Shinomiya, A., Okimura, K., Takehana,Y., Furukawa, Y., Shimo, T., Senga, T., Nakatsukasa, M., Nishimura,T., Tanaka, M., Okubo, K., Kamei, Y., Naruse, K., and Yoshimura, T.
(2019). Seasonal regulation of the lncRNA LDAIR modulates selfprotective behaviours during the breeding season. Nat. Ecol. Evol. 3,
845-852. doi: 10.1038/s41559-019-0866-6
Otsuki, Y., Okuda, Y., Naruse, K., and Saya, H. (2020). Identification of
kit-ligand a as the gene responsible for the medaka pigment cell mutant
few melanophore. G3-Genes Genom. Genet. 10, 311-319. doi: 10.1534/
g3.119.400561
Stolper, J., Ambrosio, E.M., Danciu, D.-P., Buono, L., Elliott, D.A.,
Naruse, K., Martínez-Morales, J.R., Marciniak-Czochra, A., andCentanin, L. (2019). Stem cell topography splits growth and homeostatic
functions in the fish gill. eLife 8, e43747. doi: 10.7554/eLife.43747
Sugiyama, A., Schartl, M., and Naruse, K. (2019). Histopathologic
features of melanocytic tumors in Xiphophorus melanoma receptor
kinase (xmrk)-transgenic medaka (Oryzias latipes). J. Toxicol. Pathol.
32, 111-117. doi: 10.1293/tox.2018-0058
Takehana, Y., Zahm, M., Cabau, C., Klopp, C., Roques, C., Bouchez, O.,
Donnadieu, C., Barrachina, C., Journot, L., Kawaguchi, M., Yasumasu,
S., Ansai, S., Naruse, K., Inoue, K., Shinzato, C., Schartl, M., Guiguen,
Y., and Herpin, A. (2020). Genome sequence of the euryhaline javafish
medaka, Oryzias javanicus: A small aquarium fish model for studies
on adaptation to salinity. G3-Genes Genom. Genet. 10, 907-915. doi:
10.1534/g3.119.400725
Yokoi, S., Naruse, K., Kamei, Y., Ansai, S., Kinoshita, M., Mito, M.,
Iwasaki, S., Inoue, S., Okuyama, T., Nakagawa, S., Young, L.J., andTakeuchi, H. (2020). Sexually dimorphic role of oxytocin in medaka
mate choice. Proc. Natl. Acad. Sci. USA 117, 4802-4808. doi: 10.1073/
pnas.1921446117〔Review articles〕

K., Kinoshita, M., Naruse, K., Tanaka, M., and Kamei, Y. eds.
• Murata,(2020). Medaka: Biology, Managements, and Experimental Protocols,•

Volume 2. (Wiley-Blackwell, Iowa, USA). ISBN: 978-1-119-57529-0
Naruse, K., Takehana, Y., and Yamahira, K. (2020). Medaka andOryzias species as model organisms and the current status of medaka
biological resources. In Medaka: Biology, Managements, andExperimental Protocols, Volume 2, K. Murata, M. Kinoshita, K.
Naruse, M. Tanaka, and Y. Kamei, eds. pp. 31-48. ISBN: 978-1-11957529-0

Publication List:〔Original papers〕

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF MORPHODIVERSITY

Associate Professor

KODAMA, Ryuji

Visiting Scientist:

YOSHIDA, Akihiro

The aim of this laboratory is to observe the variety ofmorphogenetic processes that occur in the course of the
ontogenesis of multicellular organisms and to analyze themechanisms of such processes, mainly by morphologicalmethods. 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
subject of 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 during the course of morphogenesis.
The outline shape of an adult wing is often different fromthat of a 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 has been shownthat the dying cells in the degeneration re­gion have characteristics in common with apop­totic cell death in mammaliancells, such as fragmented and condensed nuclei containing
short DNA fragments detected by TUNEL staining. The cells
in the degeneration region are actively engulfed by the macrophages in the cavity beneath the wing epithelium. At thattime, the macrophages are excluded from the differentiation
region because the basal surfaces of the dorsal and ventral
epithelium strongly adhere to each other in the differentiation
region. The concentration of macrophages to the degeneration region seems to accelerate the removal of the dead cells
and the shrinkage of the degeneration region.
A possible physiological role of cell degeneration at thewing margin is to make space for the growth of the marginal
scales. Marginal scales are remarkably elongated scales
that grow densely on the edge of the wing. These scales
are considered to be important in stabilizing the turbulenceoccurring posterior to the wing. The movements of themarginal scales are closely monitored by sensory scales andthe bristles growing among them (Yoshida and Emoto, Zool.
Sci. 28, 430-437, 2011).

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

In small moths which have very long scales along theirwing margins, the cuticle of the pupal wing does not appear
to be large enough to house these scales. We examined thedevelopmental processes of the pupal wings of three speciesof small gelechiid moths and found that a shrinkage of thedifferentiation region occurs which has not been observedin large winged butterflies and moths. This phenomenon isconcomitant with the programmed cell deaths at the wing
margin, and causes the space between the pupal wing and the
cuticle to extend, which appears to contribute to the growthof long marginal scales.
Microscopic observation of the long marginal scales of
Phthorimaea operculella have revealed that they have anovel branching morphology and the branches were ubiquitously and densely distributed within the scale array toform a mesh-like architecture similar to a nonwoven fabric.
The marginal scales maintain a coherent sheet-like structureduring wingbeat (Figure 1).

III. Transparent wing formation by scale
removalThe wings of butterflies and moths are usually fully covered
with scales but in some species the adult wing lacks scales insome parts, thus giving the wing a transparent appearance.
We studied the emergence process of the Cephonodes hylas
moth and found that 1) the future transparent part of thepupal wing is covered with scales that are morphologicallydifferent from the scales found on the other part and 2) the
young adult moth just after emergence strongly vibrates its
wing which loosens the scales of the future transparent part
until the moth flies away. This results in the transparent wingleaving scales behind. The large size and the shape containing a small peg, which stabs the socket on the wing planeholding the scale in place, found on the scales of the futuretransparent part are key feature in enabling the formation of
the transparent wing (Yoshida et al., in preparation).

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

56

LABORATORY OF BIOLOGICAL DIVERSITY
KAMADA Group
Assistant Professor:

KAMADA,Yoshiaki

Amino acids are the most important nutrients used inprotein building; therefore, their perception is essential for
all cells’ existence. The cellular amino acid sensing system
employs Tor (target of rapamycin) protein kinase. Tor forms
two distinct protein complexes, TORC1 (Tor complex1) and
TORC2. On one hand, TORC1 is involved in amino acidsensing, regulation of protein synthesis, the cell cycle, andautophagy. On the other hand, TORC2 is responsible foractin 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 TOR
signaling pathway branches (Figure 1). Recently, we foundthat tRNA plays a pivotal role in the ability of TORC1 tosense amino acids (Figure 1).

Figure 2. A schematic model of how amino acid is sensed by TORC1.
Cytosolic free tRNA inactivates TORC1 under amino acid limited condition.

II. TORC1 phosphorylates Atg13, themolecular switch of autophagy

TORC1 negatively regulates autophagy: a protein degradation system induced by nutrient starvation.
We were consequently able to discover the TORC1mediated 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 of which is essential for
autophagy and is largely enhanced in response to TORC1
inactivation. Activation of Atg1 requires formation of theAtg1 complex. Phosphorylation of Atg13 by TORC1 playsa pivotal role in Atg1 complex formation; phosphorylated
Atg13 loses its affinity to Atg1 resulting in disassembly of
the Atg1 complex and repression of autophagy. On the other
hand, dephosphorylation of Atg13 triggers formation of theAtg1 complex, activation of Atg1 kinase, and consequently
induction of autophagy.

Publication List:〔Original Paper〕
Figure 1. Tor signaling pathway for budding yeast. Our group has foundthree branches of the Tor pathway.

I. How do amino acids regulate TORC1?

TORC1 is regulated by amino acids which in themsleves
are fundamental nutrients. 20 species of amino acids building
proteins cannot be interchanged with each other. Therefore,
each amino acid must be individually detected by TORC1.
We conducted genetic research and discovered the
involvement of (aminoacyl-) tRNA in TORC1 regulation.
Biochemical in vitro TORC1 assay also revealed that tRNA
directly inhibits TORC1 kinase activity. Reducing cellular
tRNA molecules desensitizes TORC1 inactivation by nitrogenstarvation in vivo. Based on these results, a TORC1 regulatorymodel was proposed that contends that free tRNA released
from protein synthesis under amino acid starvation inhibits
TORC1 activity. Therefore, TORC1 employs a tRNA-mediated mechanism to monitor intracellular amino acids (Figure 2).

M., Tomonaga, S., Suzuki, M., Gen, M., Takeda, E., Matsuura,• Baba,A., Kamada, Y., and Baba, N. (2019). A nuclear membrane-derived
structure associated with Atg8 is involved in the sequestration of
selective cargo, the Cvt complex, during autophagosome formation inyeast. Autophagy 15, 423-437. doi: 10.1080/15548627.2018.1525475

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.

I. G onadotropins 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 functionally conserved across various species, whereas no suchmolecule has been identified in invertebrates. The insect
parsin hormones are assumed to be the physiological counterpart of LH and FSH in mammals. Some gonadotropic
hormones (e.g. the mosquito’s egg development neurosecretory hormone, the sea hare’s egg-laying hormone, and theterrestrial isopod’s androgenic gland hormone) have beenfound in invertebrate species. More recently, an insulin-like
peptide was reported to be responsible for the regulation ofegg maturation in the mosquito, Aedes aegypti, thus 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, and acts 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 the amino acidsequence of purified GSS from radial nerves of the starfish,
Pateria pectinifera. Interestingly, phylogenetic analyses
revealed that it belonged to the insulin/insulin-like growth
factor (IGF)/relaxin superfamily and, more precisely, to thesubclass of relaxin peptides (Figure 1).

II. S earch for reproductive hormones inechinoderms

In a collaborative effort with Prof. Yoshikuni’s Laboratory
at Kyushu Univ., we are searching for reproductive
hormones in echinoderms, including starfishes, brittle stars,
sea urchins, sea cucumbers, and crinoids. The collaborating parties have been able to purify physiological materials which induce egg maturation from nerve extracts andanalyze them with a protein sequencer and a tandem mass
spectrometer in the analytical center of our institute. One of58

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 sequencesof GSS. Sequences of the A and B chains are shown in the green andblue boxes, respectively. Characters shown in red boldface indicate basic
dipeptides that are the sites of proteolytic cleavage. The inverted triangle
shows the deduced cleavage site of the signal peptide.

them, named cubifrin, which is an IWMGY-amide peptide, isin the sea cucumber Aposticopus japonicus. The others are inpreparation for publication.
We have identified many neuropeptides from our EST
analysis of nerve tissues and many from RNA-seq and WGS
data of the NCBI database. In particular, 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) to provide to collaborating researchers forbiological assays.

III. S earch for the lost mutants of female
hormones E2 in Oryzias latipes

Sequence analysis by capillary sequencer was carried out
from the tilling library of O. latipes in order to analyze thefunction of fish female reproductive hormone E2 as a partof our collaborative research. Upon the discovery of aromatase mutants, a detailed analysis was carried out using thesestrains.

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
a large impact on gene expression and evolution. To understand these genome dynamics in eukaryotes, especially inplants, we are analyzing the flower pigmentation of morning
glories including Ipomoea nil (Japanese morning glory), I.
purpurea (the common morning glory), and I. tricolor.

variety has since been lost. For this reason, it has been called
the ‘phantom morning glory’, and many efforts have been
made to reproduce it over a long period. In the yellow-flowered snapdragon, yellow pigment aurones are synthesized by
the chalcone glycosyltransferase and aurone synthase genes
from chalcone. Although I. nil produces chalcone, it lacks anability to produce large quantities of aurones. The two snapdragon genes were introduced in the I. nil mutant accumulating chalcones in its cream yellow flowers. The transgenic
plants expressing both genes exhibited yellow flowers; acharacteristic sought for many years. The flower petals of
the transgenic plants contained the snapdragon aurones and anovel acylated aurone.

I. Flower pigmentation patterns

The wild type morning glories produce flowers with uniformly pigmented corolla. However, a number of mutants
displaying particular pigmentation patterns have been collected for this study. Because flower pigmentation patterns
are easily observable, the molecular mechanisms underlying
these phenomena provide useful model systems for investigating genome variability.
The recessive mutations, duskish of I. nil and pearly-v ofI. tricolor, confer variegated flowers. They are caused bya stable insertion of a transposable element into a gene forflower pigmentation. Furthermore, epigenetic mechanisms
are thought to regulate this pigmentation (Figure 1). We are
currently analyzing the detailed molecular mechanisms ofthese mutations.

Figure 2. Flowers of the recreated ‘phantom morning glory’ (left), andthe host plant (right). The host plant often exhibits shriveled flowers withnecrotic cells, the transgenic plants produced fully opened flowers withfew necrotic cells.

III. BioResource of morning glories

NIBB is the sub-center for the National BioResource
Project (NBRP) for morning glories. In this project, we arecollecting, maintaining and distributing standard and 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, and has
a 100-year history of extensive genetic studies related to it.
Our collection includes 220 lines and 177,000 DNA clones.
The whole genome sequence, the transcriptome sequences,
as well as the end sequences of the DNA clones can beviewed via the I. nil genome database (http://viewer.shigen.
info/asagao/index.php).

Publication List:〔Original papers〕

Figure 1. The duskish mutant of I. nil shows variable flower phenotypes
and produces variegated, fully pigmented, and pale grayish-purple
flowers. It segregates offsprings that only show fully pigmented or palegrayish-purple flowers, and their phenotypes can be stably inherited byfurther generations.

II. Recreating the lost morning glory

I. nil cultivars are displayed in a wide variety of flower
colors: red, peach, purple, brown and white. However, just as
roses do not have blue flowers, morning glories do not haveyellow flowers. Yellow-flowered morning glories have beenrecorded in illustrations from the Edo period, but this color

A., Mizuno, T., Shimizu, K., Mori, S., Fukada-Tanaka, S.,• Hoshino,
Furukawa, K., Ishiguro, K., Tanaka, Y., and Iida, S. (2019). Generation•of yellow flowers of the japanese morning glory by engineering its
flavonoid biosynthetic pathway toward aurones. Plant Cell Physiol. 60,
1871-1879. doi: 10.1093/pcp/pcz101
Waki, T., Mameda, R., Nakano, T., Yamada, S., Terashita, M., Ito,K., Tenma, N., Li, Y., Fujino, N., Uno, K., Yamashita, S., Aoki,
Y., Denessiouk, K., Kawai, Y., Sugawara, S., Saito, K., YonekuraSakakibara, K., Morita, Y., Hoshino, A., Takahashi, S., and Nakayama,
T. (2020). A conserved strategy of chalcone isomerase-like protein torectify promiscuous chalcone synthase specificity. Nat. Commun. 11,870. doi: 10.1038/s41467-020-14558-959

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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 junkDNA, they play an important role in genome reorganizationand evolution. Active DNA transposons are important tools
for gene functional analysis. The endogenous non-autonomous transposon, nDart1, in rice (Oryza sativa L.) is said
to generate various transposon-insertion mutants because
nDart1 elements tend to insert into genic regions undernatural growth conditions. The transpositions of nDart1 were
promoted by an active autonomous element, aDart1-27, on
chromosome 6. By using the endogenous nDart1/aDart1-27
system in rice, a large-scale nDart-inserted mutant population
was easily generated under normal field conditions, and theresulting tagged lines were free of somaclonal variation. The
nDart1/aDart1-27 system was introduced into a rice variety,
Koshihikari, named MK-1. 3000 MK-1 plants were grown infield conditions (IPSR, Okayama Univ.). All plants’ genomes
were isolated for identifying the insertion sites of nDart1.

I. Large grain (Lgg) mutation in rice

Seed size and number were controlled by various genes
in the plants. It was reported that expression changes inhigh contribution genes for seed size, number and panicle
shape resulted in a decrease of the total yield. A strategy forboosting rice yield based on molecular biology is to stack thefinely tuned gene expressions. The Lgg mutant which wasisolated from MK-1 plants bore slightly larger grains (Figure1) as a dominant inheritance. Transposon-display identified
the insertion site of nDart1 in the Lgg mutant.

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

II. Analysis of Lgg mutants

The identified LGG gene shows similarity to RNA binding
proteins. Transgenic rice lines with knock-out (KO) andover-expressing (OE) in LGG gene showed large and small
seed sizes, respectively. The section of lemma KO andOE plant were observed, and their cell numbers counted

60(Figure2). Increased cell numbers in Lgg mutant haveinduced the Large Grain phenotype. RNA seq analysis
revealed that expression of cell-cycle and prolification
related genes was changed in transgenic plants.

Figure 2. The section and cell number of lemma in transgenic plants. Bar= 100 µm. (A) the section of lemma in rice plants. The red arrow indicates counted cells. (B) Graph of cell number of lemma

Publication List:〔Original papers〕

W.-Y., Kawamoto, T., Himi, E., Rikiishi, K., Sugimoto, M.,• Chiou,
Hayashi-Tsugane, M., Tsugane, K., and Maekawa, M. (2019). LARGE••GRAIN Encodes a putative RNA-binding protein that regulates spikelet
hull length in rice. Plant Cell Physiol. 60, 503-515. doi: 10.1093/pcp/
pcz014
Nishimura, H., Himi, E., Eun, C.-H., Takahashi, H., Qian, Q., Tsugane,
K., and Maekawa, M. (2019). Transgenerational activation of anautonomous DNA transposon, Dart1-24, by 5-azaC treatment in rice.
Theor. Appl. Genet. 132, 3347-3355. doi: 10.1007/s00122-019-03429-7
Nishimura, H., Himi, E., Rikiishi, K., Tsugane, K., and Maekawa,M. (2019). Establishment of nDart1-tagged lines of Koshihikari, anelite variety of rice in Japan. Breed. Sci. 69, 696-701. doi: 10.1270/
jsbbs.19049

LABORATORY OF BIOLOGICAL DIVERSITYSAKUTA Group
Assistant Professor:
Technical Assistant:

SAKUTA, Hiraki
ISOSHIMA, Yoshiko
KODAMA, Akiko

The homeostatic osmoregulation of body fluids (such asplasma and cerebrospinal fluid (CSF)) is vital to life. This isbecause substantial changes in cell volumes due to hypertonicity or hypotonicity cause irreversible damage to organs andlead to lethal neurological trauma. Water deprivation (loss ofwater from the body) elevates the concentration of Na+ ([Na+])and osmolality in body fluids. Animals exhibit prominent andeffective responses to water deprivation, including behavioral
responses, such as inducing water intake and avoiding sodium(Na), along with vasopressin-induced reductions in urine
volumes. The aim of our research group is to reveal the brain
systems for body-fluid homeostasis.

I. Thirst control by Nax and TRPV4[Na+] is the main factor influencing osmolality in vivo,
and is continuously monitored in the brain to be maintained
within a physiological range. We have shown that Nax,
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 expressedin specific glial cells of sensory circumventricular organs(sCVOs) including the subfornical organ (SFO) and organum
vasculosum lamina terminalis (OVLT). We have found that
Nax signals in these brain regions deficient in a blood-brain
barrier are involved in the control of salt intake.
We recently demonstrated that Nax signals are also involvedin the control of water intake behavior. Our pharmacological experiments suggested that Nax signals led to theactivation of neurons bearing TRPV4 by using epoxyeicosatrienoic acids (EETs) as gliotransmitters to stimulate
water intake. This year, we performed selective lesions ofindividual sCVOs in wild-type (WT) mice and the sitedirected rescue of Nax expression in Nax knockout (Nax-KO)
mice. These experiments revealed that the Nax channel in
the OVLT functions as a [Na+] sensor for the control ofwater intake behavior. Direct measurements of 5,6-EET and
8,9-EET in the OVLT revealed that EET levels were indeed
increased two-fold by water deprivation for two days in WT,
but not Nax-KO mice. This indicates that EETs were Naxdependently produced in the OVLT in response to increases
in [Na+] in body fluids. More importantly, the ICV injectionof 5,6-EET at the same level was effective in inducing water
intake.
The signaling mechanisms in the OVLT for water-intake
induction by increases in [Na+] in body fluids are presented
in Figure 1. When [Na+] in plasma and CSF increases, Naxchannels in glial cells in the OVLT are activated, leading
to the synthesis of EETs in Nax-positive glial cells. EETs
released from Nax-positive glial cells function as gliotransmitters to activate neurons bearing TRPV4 channels in the
OVLT, which are involved in the stimulation of water-intake
behavior.

Figure 1. Proposed mechanisms for water intake induction by increases
in [Na+] in body fluids via Nax activation in the OVLT. AA, arachidonic
acid.

II. Identification of novel sensors involved inwater intake control
Water intake by Nax-KO mice after the ICV injection of
hypertonic NaCl solution was small, but still approximately
half that by WT mice and, noteworthily, significantly higher
than that by Nax-KO and WT mice after the ICV injectionof an equimolar hypertonic sorbitol solution. These findings
suggest the existence of another unknown [Na+] sensor andosmosensor. In order to identify the novel sensors involvedin water intake control, we performed RNA-seq analysis of
OVLT and identified several candidates. We are now examining the functional roles of these candidates in water intake.

Publication List:〔Original papers〕

K., Hiyama, T.Y., Sakuta, H., Matsuda, T., Lin, C.-H.,• Nomura,
Kobayashi, K., Kobayashi, K., Kuwaki, T., Takahashi, K., Matsui, S.,•

and Noda, M. (2019). [Na+] increases in body fluids sensed by central
Nax induce sympathetically mediated blood pressure elevations via H+dependent activation of ASIC1a. Neuron 101, 60-75. doi: 10.1016/j.
neuron.2018.11.017
Winkelman, B.H.J., Howlett, M.H.C., Hölzel, M.-B., Joling, C.,
Fransen, K.H., Pangeni, G., Kamermans, S., Sakuta, H., Noda, M.,
Simonsz, H.J., McCall, M.A., De Zeeuw, C.I., and Kamermans, M.
(2019). Nystagmus in patients with congenital stationary night blindness(CSNB) originates from synchronously firing retinal ganglion cells.
PLoS Biol. 17, e3000174. doi: 10.1371/journal.pbio.3000174〔Original paper (E-publication ahead of print)〕

H., Lin, C.-H., Yamada, M., Kita, Y., Tokuoka, S.M., Shimizu,• Sakuta,T., and Noda, M. Na -positive glial cells in the organum vasculosumxlaminae terminalis produce epoxyeicosatrienoic acids to induce water
intake in response to increases in [Na+] in body fluids. Neurosci. Res.
2019 May 28. doi: 10.1016/j.neures.2019.05.006

61

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITY
KATO GroupSpecially Appointed Assistant Professor:
KATO, Kagayaki
Technical Assistant:
HYODO, Miwa
Organogenesis is accomplished by a series of deformations of the planar cell sheet into a three-dimensional shapeduring embryogenesis. This drastic structural change is theintegrated result of individual cell behaviors in response tospatio-temporally controlled mechanisms.
To better understand the programs underlying organ formation, it is necessary to quantitatively analyze individual cells’
morphology and dynamics. However, it is difficult to do sodue to the massive images generated by 4D microscopy andtheir ambiguity.
To unveil organogenesis from the point of view of distinct
cell behaviors, we are developing application software thatis capable of describing cell dynamics from 4D time-lapse
imaging data sets by employing image processing techniques.

< NINS ExCELLS >II. Image processing pipeline for 3D cell
cultureTo elucidate the relationship between mechanical forcesand epithelial deformation, we developed an image processing pipeline for segmentation of nucleus within 3D cultureof MDCK cells. This pipeline automated a segmentation/
quantification process of a large number of images acquiredby several experimental conditions for subsequent statisticalanalysis (Nishimura et al.).

III. Software for manual image quantification

Biologically significant imaging 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 have developed can easily visualize 4D
imaging data and has made manual feature annotations easier(Figure 2). This application is freely available at our website(https://bioimageanalysis.jp/).

I. 4D cell segmentation/tracking system

Epithelial morphogenesis in developing embryos is considered to be an important model for collective cell migrations.
Drastic cell rearrangements lead to drastic structural changes
in building 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. This system extracts
cell boundaries and reconstitutes cell shapes as a skeletonized chain of voxels spanning 3D space. This abstract formof cell visualization makes it possible to describe morphometric quantities and kinetics of cells in single-cell resolution (Figure 1). These morphometric quantities allow us toperform comparative studies on shapes and behaviors more
precisely among several experimental conditions, to gaina better understanding of the genetic programs underlying
organogenesis. We are now applying this system to several
experimental models to determine the practicality of thesystem (Shinoda et al.).

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.

62

Figure 2. A lightweight/native 4D stack viewer equipped with functions
for manual feature extraction.

Publication List:〔Original papers〕

I., Shitamukai, A., Kusumoto, F., Mase, S., Suetsugu, T.,• Fujita,
Omori, A., Kato, K., Abe, T., Shioi, G., Konno, D., and Matsuzaki, F.

•(2019). Endfoot regeneration restricts radial glial state and prevents
translocation into the outer subventricular zone in early mammalianbrain development. Nat. Cell Biol. 22, 26-37. doi: 10.1038/s41556019-0436-9
Furutani, M., Hirano, Y., Nishimura, T., Nakamura, M., Taniguchi,
M., Suzuki, K., Oshida, R., Kondo, C., Sun, S., Kato, K., Fukao, Y.,
Hakoshima, T., and Morita, M.T. (2020). Polar recruitment of RLD
by LAZY1-like protein during gravity signaling in root branch angle
control. Nat. Commun. 11, 76. doi: 10.1038/s41467-019-13729-7

< NINS ExCELLS >LABORATORY OF BIOLOGICAL DIVERSITY†
OHTA GroupSpecially Appointed Assistant Professor:
OHTA, Yusaku
Image analysis is an important element in understanding
life science. It makes it possible to quantify phenomena
by extracting meaningful information from a large amountof images and then appropriately expressing said information. In recent years, machine learning, including deeplearning, has changed image analysis in the field of biology.
In keeping with this, I am currently using image analysis
technology to elucidate the principles of embryo development and to provide comprehensive imaging support in life
science.

I. E lucidation of the principle of collective
migration of cells that maintain the order ofembryonic development.

During organism morphogenesis, three-dimensional remodeling of tissues by cell migration is essential. While individual cell motility depends on extracellular signals, cell-celladhesion is maintained, thereby controlling highly coordinated cell motility. To elucidate the principle of such complicated embryogenesis, it is necessary to understand the cell
dynamics of the whole embryo with single cell resolution. To
this end, I am conducting research using the following threetechnologies.
The first is a four-dimensional cell tracking analysis that
automatically tracks all cells in the early embryo. This makes
it possible to treat individual cell dynamics of the whole
embryo as digital information. The second is the automation of visualization of cell dynamics information fromlarge-scale image data. Image data displaying an entire
embryo can exceed terabytes, which in turn exceeds muchanalysis can be conducted by the visual and manual labor of
researchers. This automation makes it easier to understandthe behavior of tens of thousands of cells. The third technology is the identification of key cells that control morphogenesis by machine learning. This enables objective and quantitative analysis instead of subjective analysis that relies on the
researchers’ preconceptions .

II. Research support by image analysis

The development of imaging technology has been remarkable within life science research, and many researchers are
now able to easily acquire large and complex sets of image
data. However, image analysis is still a hurdle for researchers, and it often creates bottlenecks in research. In order tosolve this problem, I provide research support based on thefollowing three themes.
The first theme is quantitative image analysis based on a
wealth of knowledge in imaging and statistics. For many
researchers, the method of evaluating information contained in images is limited to qualitative and subjective
types. Correct analysis based on knowledge of imaging andstatistics supports quantitative and objective analysis. The
second theme is the active utilization of image analysis technology via the application of machine learning, including
deep learning. In recent years, the development of machinelearning has been remarkable, and with a little training, it ispossible to simplify analysis that is difficult to achieve withconventional image analysis technology. The third theme isrelates to the publication of explanations of image analysis
to researchers in an easy-to-understand manner on the web.
The contents of which range from the principles of image
analysis methods to the use of image analysis software andplug-ins.

Figure 2. A website with topics related to image analysis

Publication List:〔Original paper〕

H., Yagi-Utsumi, M., Honda, R., Ohta, Y., Saito, T., Nishio, M.,• Yagi,Ninagawa, S., Suzuki, K., Anzai, T., Kamiya, Y., Aoki, K., Nakanishi,

M., Satoh, T., and Kato, K. (2020). Improved secretion of glycoproteinsusing an N-glycan-restricted passport sequence tag recognized by cargo
receptor. Nat. Commun. 11, 1368. doi: 10.1038/s41467-020-15192-1

Figure 1. Example of automated image analysis

63

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITY†
SHINOMIYA GroupSpecially Appointed Assistant Professor:
SHINOMIYA, Ai
Technical Assistant:
TSURUTA, EmikoThe natural environment, which incorporates phenomena such as day length, solar radiation, temperature, andprecipitation, generates seasonal changes that affect organisms. Although animals alter their physiology and behavior
in response to seasonal changes in their environment, themechanism of seasonal adaptation remains largely unknown.
Medaka (Oryzias latipes) provide an excellent model tostudy these mechanisms because of their rapid and obvious
seasonal responses. In addition, it is also possible to apply
transgenic and genome-editing approaches when researching them, as well as reference genome sequences. In keepingwith this, we are currently using Medaka to study the molecular mechanisms of seasonal adaptation, as well as the relationship between seasonal information from the environment
and the organism’s response.

I. Underlying mechanisms that define criticalday length and temperature

Most animals living outside the tropics reproduce only
during a particular season of the year. These are calledseasonal breeders, and it is well established that day length isa crucial cue for reproduction in many of them. In addition,it has been demonstrated that temperature changes are also
important for them in the how they detect seasons. However,it remains unknown how animals measure seasonal changes
in relation to these environmental factors.
Applying forward genetic approaches, we examined criticalday length in reproduction (i. e., the duration of lighting
time required to cause gonadal development) using various
Medaka populations, that originated in various latitudes
throughout Japan to elucidate these mechanisms (Figure 1).
Geographical variation critical day length was detected, andpopulations from lower latitudes indicated a shorter criticalday length (Figure 1). To identify the genes governing
critical day length, quantitative trait loci (QTL) analysis was
conducted using F2 offspring derived from crosses between< NINS ExCELLS >populations experiencing different critical day length. We
thus identified significant QTLs using Restriction-site
Associated DNA (RAD) markers (Figure 2).

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

Whole-genome re-sequencing in various medaka populations
experiencing different critical day lengths were conducted.
We have identified potential candidate genes that define thecritical day length by analyzing this genome sequencing data.
We also performed experiments to identify critical temperature, and subsequently detected geographical variation
among Medaka populations. Significant QTLs for criticaltemperature have been detected from the genetic analysis
that was performed.

II. Analysis of the relationship between breedingrhythms and environmental information

Organisms exhibit various scales of rhythm, ranging inseconds to years. On the other hand, the natural environment provides the rhythmic changes concerning organisms.
However, the quantitative relationship between the information on environmental factors and biological rhythms ispoorly understood.
We conducted a linear regression analysis regarding annual
rhythms in gonadal development using data pertaining to
the annual changes in the gonadal size of the medaka andenvironmental information in the experimental field. The
regression model explained which environmental factors
contributed to the seasonal change in medaka gonads and towhat extent they contributed to this change.

Publication List:〔Original papers〕

Y., Shinomiya, A., Ota, W., Ikegami, K., Shimmura, T.,• Nakane,
Higashi, S., Kamei, Y., and Yoshimura, T. (2019). Action spectrum for•
Figure 1. Medaka populations used in our study (left). Differences ofcritical day length among medaka populations (right).

64

photoperiodic control of thyroid-stimulating hormone in Japanese quail(Coturnix japonica). PLoS One 14, e022106. doi: 10.1371/journal.
pone.0222106
Nakayama, T., Shimmura, T., Shinomiya, A., Okimura, K., Takehana,Y., Furukawa, Y., Shimo, T., Senga, T., Nakatsukasa, M., Nishimura,T., Tanaka, M., Okubo, K., Kamei, Y., Naruse, K., and Yoshimura, T.
(2019). Seasonal regulation of the lncRNA LDAIR modulates selfprotective behaviours during the breeding season. Nat. Ecol. Evol. 3,
845-852. doi: 10.1038/s41559-019-0866-6

< NINS CNIS Department of Plasma Sciences >LABORATORY OF BIOLOGICAL DIVERSITYYAMASHITA AND OTSUBO GroupSpecially Appointed Associate Professor

YAMASHITA, Akira

Specially Appointed Assistant Professor:
OTSUBO, Yoko
Technical Assistant:
NAKADE, Atsuko
Secretary (Senior Specialist): SAKAGAMI, Mari
Cells sense the environment around them (e.g. the amountof nutrients and hormones present, as well as the temperature and pressure), and decide what kind of activities toundertake based on this information. In response to ambientconditions, germ cells producing sperm and eggs begin
halving their number of chromosomes during a special kindof cell division called meiosis; a process for bringing forth
genetically diverse progeny. In our laboratory, we use thefission yeast Schizosaccharomyces pombe, the simplest
organism that performs meiosis (Figure 1), to research themechanisms by which cells switch from mitosis (a kind ofcell division that divides cells equally to create two identical cells) to meiosis. In addition, we have started to study
cellular responses to a novel stress: atmospheric pressureplasma.

Figure 1. The life cycle of the fission yeast S. pombe. S. pombe cells
mitotically proliferate under nutrient-rich conditions. When starved ofnutrients, especially nitrogen, S. pombe cells arrest the mitotic cell cycle
and haploid cells conjugate with cells of the opposite mating type. The
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 cell cycle mode from mitotic to meiotic. To achievethis, we focus on a highly conserved kinase, namely Targetof Rapamycin (TOR) kinase, which plays a key role in therecognition of nutrition and the onset of sexual differentiation in S. pombe. TOR kinase forms two types of complexes,
TORC1 and TORC2. TORC1 contains Tor2 as its catalytic
subunit, and is essential in suppressing sexual differentiationin the presence of nitrogen. TORC2 contains Tor1 and, in
contrast to TORC1, is required for the onset of sexual differentiation under nitrogen starved conditions.
Temperature-sensitive tor2 mutants initiate sexual differentiation even on rich mediums in restrictive temperatures. To

gain an insight into the TORC1 signaling pathway, we haveisolated mutants that initiate sexual differentiation ectopically under nutrient-rich conditions, as tor2 mutants do. We
designated these mutants as hmt, which stands for hypermating and temperature-sensitive growth. We cloned the responsible genes and found that five of the eight responsible genes
encoded tRNA-related factors. The hmt1 and hmt2 genes
encoded aminoacyl-tRNA synthetases for asparagine andproline, respectively. The hmt3 gene encodes tRNA adenosine-34 deaminase. The hmt4 is identical to rpc34, which
encodes a subunit of RNA polymerase III. The hmt5 is identical to sfc4, which encodes a subunit of the RNA polymerase
III-specific general transcription factor IIIC. In the hmt1-5
mutants, TORC1 activity is downregulated, suggesting thatthe products of these hmt genes may function upstreamof TORC1. We also found that the expression of tRNAprecursors decreases upon nitrogen starvation occurring.
Furthermore, overexpression of tRNA precursors prevents
TORC1 downregulation in response to nitrogen starvation
and represses the initiation of sexual differentiation. Basedon these observations, we have proposed that tRNA precursors act as key signaling molecules in the TORC1 pathway inresponse to nitrogen availability (Figure 2).

Figure 2. TORC1 regulation by tRNA precursors. TOR complex 1(TORC1) promotes vegetative growth and suppresses sexual differentiation under nutrient-rich conditions. tRNA precursors (pre-tRNAs)
positively regulate the TORC1 activity.

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

The expression of hundreds of genes is 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 recognizesa region termed DSR (Determinant of Selective Removal)
in meiotic transcripts, which is enriched by repeated hexanucleotide motifs. Meiotic transcripts bound to Mmi1 aredegraded by the RNA-degradation nuclear exosome machin-65

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

ery. Mmi1 also induces the formation of facultative heterochromatin at a subset of its target genes. Furthermore, Mmi1
regulates the termination of transcription of its target genes.
Mmi1-mediated termination of an upstream non-coding RNA
ensures the expression of downstream genes, one of which
encodes a mitogen-activated protein kinase kinase kinase(MAPKKK) essential for the initiation of sexual differentiation.
When S. pombe cells undergo meiosis, a meiosis-specificnuclear body, called Mei2 dot, blocks the Mmi1-mediatedelimination system. The Mei2 dot is composed of the RNAbinding protein Mei2 and a long non-coding RNA species
termed meiRNA. Mei2 physically interacts with meiRNAand forms the dot structure at the chromosomal sme2 locus,
which encodes meiRNA. The Mei2 dot lures Mmi1 throughnumerous copies of the DSR motif on meiRNA and inhibits
its function, so that meiotic transcripts harboring DSR arestably expressed.
We have shown that Mmi1 prevents untimely expression of
meiotic proteins by tethering their mRNAs to nuclear foci.
Mmi1 interacts with itself with the assistance of Erh1. Mmi1
self-interaction is crucial for nuclear foci formation, target
transcripts elimination, their nuclear retention, and protein
expression inhibition. As such, multi-layered suppression of
meiotic genes by Mmi1 is vital for mitotic growth (Figure 3).

III. Cellular responses to atmospheric pressureplasmaPlasma, which is the fourth state of matter after solid,
liquid and gas states, is ionized or electrically charged gas.
It has been known that plasma irradiation induces a wide
variety of effects on living organisms. Recently, atmosphericpressure plasma has been used in various fields including
medicine and agriculture. However, detailed mechanisms
underlying responses to plasma irradiation remains unknown.
Accordingly, we are addressing molecular mechanisms of

Figure 3. Mmi1-mediated multi-layered regulation to prevent mistimed
expression of meiotic genes. In mitotically growing cells, meiotic transcripts carrying DSR are recognized by Mmi1, and are degraded by
the RNA exosome. Mmi1 also induces heterochromatin formation.
Furthermore, Mmi1 prevents nuclear export and ectopic protein expression of its targets.

66

cellular responses to plasma by using the fission yeast S.
pombe. To this end, we have developed plasma irradiation
devices that enable control of gas temperature in collaboration with National Institute for Fusion Science.

Publication List:〔Original papers〕

S.Y., Hung, S., Esnault, C., Pathak, R., Johnson, K.R., Bankole,• Lee,O., Yamashita, A., Zhang, H., and Levin, H.L. (2020). Dense transposon••

integration reveals essential cleavage and polyadenylation factors
promote heterochromatin formation. Cell Rep. 30, 2686-2698. doi:
10.1016/j.celrep.2020.01.094
Shichino, Y., Otsubo, Y., Yamamoto, M., and Yamashita, A. (2020).
Meiotic gene silencing complex MTREC/NURS recruits the nuclear
exosome to YTH-RNA-binding protein Mmi1. PLoS Genet. 16,
e1008598. doi: 10.1371/journal.pgen.1008598
Yoshimura, S., Aramaki, M., Otsubo, Y., Yamashita, A., and Koga, K.
(2019). Controlling feeding gas temperature of plasma jet with Peltier
device for experiments with fission yeast. Japn. J. Appl. Phys. 58,
SEEG03. doi: 10.7567/1347-4065/ab1473〔Review article〕

A. (2019). meiRNA, a polyvalent player in fission yeast• Yamashita,
meiosis. non-coding RNA 5, 45. doi: 10.3390/ncrna5030045

< NINS ASTROBIOLOGY CENTER >NINS ASTROBIOLOGY CENTER
The Astrobiology Center (ABC) was established in2015 to promote interdisciplinary studies including
astronomy, earth science, and biology. Our ultimate goal
is to find a so called ‘Second Earth’ and other extraterrestrial life on this planet. Astronomers and earth scientists in the Exo-Planet Search Project and Astrobiology
Instrument Project at the main office in Tokyo are now
discovering habitable planets around the nearest stars
using the latest observation technologies. To support
these observation projects, biologists from NIBB participating in the Exo-Life Search Project are investigating
life on Earth to predict the biosignature of hypotheticallife on the aforementioned ‘Second Earth’.
This year, ABC (jointly with NAOJ) hosted the 4th ‘In
the Spirit of Lyot’ international symposium in Tokyo,
where the international research community found new
ways to work together toward direct imaging of the‘Second Earth’. The solar coronagraph, a telescopic
attachment for blocking bright light from the Sun, wasinvented by the namesake of this symposium, Bernard
Lyot, in 1931. Stellar coronagraphs enable high contrast
imaging of Earth-like planets around Sun-like stars incombination with adaptive optics and other advanced
observation technologies.

Figure 1. Lyot 2019 Poster

LABORATORY OF BIOLOGICAL DIVERSITYTAKIZAWA GroupSpecially Appointed Associate Professor

TAKIZAWA, Kenji

The Takizawa group operating out of ABC is currently
studying environmental responses of photosynthesis in orderto predict photosynthetic apparatus of ‘Alien’ plants under
extreme conditions on the ‘Second Earth’.

Remote sensing of vegetation red-edge

One of the most plausible biosignatures on habitable exoplanets is a specific reflection pattern on the land surface
named ‘red-edge’ that is caused by land vegetation. Red-edge
appears on Earth between red light which is absorbed byphotosynthetic pigments, and near infrared radiation (NIR),
which is reflected via leaf tissue structure. Vegetationindexes calculated from reflectance in red and NIR are
roughly related to photosynthetic capacity. Remote sensing
by drone-based, multiband sensors revealed light reflection
properties of plants growing in various habitats.

Floating plants in water world

The light reflection properties peculiar to land vegetation
cannot be formed by algae and plants under water. Even
though aquaplanet is rare within the universe, most of thewater-containing planets are considered ‘ocean planets’,
which are filled with a substantial amount of water. If
there are no continents, detection of vegetation red-edge isunlikely. We proposed there was a possibility remaining thata strong biosignature could be detected on ‘water planets’
due to extensive flourishing of drifting algae and floating
plants. Some species of aquatic plants have sponge-likefloating leaves and thus reflect NIR in a manner comparable
to land vegetation.

Figure 2. Normalized difference vegetation index (NDVI) survey for grassland on a riverbank. NDVI is a graphical indicator for green vegetation calculated from light reflectance in Red and NIR as (NIR-Red)/(NIR+Red). Grasscovered area showed high NDVI (red color) in contrast with low NDVI (bluecolor) for water (upper left) and soil (lower right) area. Multiband images
were obtained by drone-based sensors (pictured at upper right).

67

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Staff:

JOHZUKA, Katsuki

ISHINE Naomi

Chromosome condensation is a basic cellular process that
ensures the faithful segregation of chromosomes in bothmitosis and meiosis. This process is required not only for
decreasing chromome arm length, but also for resolving
entanglements between sister-chromatids that are createdduring DNA replication. Any abnormality in this processleads to segregation errors or aneuploidy, which results incell death. Chromosome condensation is mainly achievedby condensin, a hetero-pentameric protein complex, widelyconserved across a variety of organisms ranging from yeast
to humans. Despite its conservation and importance in chromosome dynamics, it is not fully understood how condensin
works. Recent studies have revealed that condensin functions
are not restricted to chromosome condensation and segregation during cell divisions, and is required for diverse DNA
metabolism such as genome stability, transcriptional regulation, and cell differentiation.
Our aim is to understand the mechanism and regulation of chromosome condensation. To this end, we havebeen studying the role of condensin in the budding yeast
Saccharomyces cerevisiae. Microscopic observation has 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.

< NINS ASTROBIOLOGY CENTER >into an ectopic chromosomal locus, condensin can associatewith 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 into an ectopic chromosome arm with an interval of 15kb distance in the cell with acomplete deletion of chromosomal rDNA repeat. Using thisstrain, condensin-dependent chromatin interaction between
two RFBs was examined by chromosome conformation
capture (3C) assay. We discovered the condensin-dependent
chromatin interaction between the two RFB sites on the chromosome arm. This result indicates that condensin plays a rolein chromatin interaction between condensin binding sites,
and this interaction leads to the creation of a chromatin loop
between those sites (Figure 1). It is thought that condensindependent chromatin folding is one of the basic molecular
processes of chromosome condensation. During the cell
cycle stages, the RFB - RFB interaction signal increases in
metaphase and reaches its maximum level in anaphase. In
addition to the RFB – RFB interaction, the chromatin interactions between the internal regions of the two RFBs increases in anaphase. Thus, the configuration of chromatin fiberchanges from a simple loop into a complicated twisted shapeas the cell cycle progresses from metaphase to anaphase.

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

II. Condensin-dependent chromatin folding

The RFB site, which consists of a ~150bp DNA sequence,
functions as a cis-element for the recruitment of condensin
to chromatin in the yeast genome. If the RFB site is inserted

68

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.

< NINS ASTROBIOLOGY CENTER >tal results using nitrogen nutrition, and provides a theoreticalbasis for understanding the spatial regulation of adaptive
resource allocation in response to nutritional environment.

LABORATORY OF BIOLOGICAL DIVERSITYFUJITA Group
Assistant Professor:
Technical Assistant:

FUJITA, Hironori
NIWA, Yoshimi

II. Phyllotaxis pattern formation

In nature, a variety of patterns, such as the galaxy and thesnowflake, are found on a wide range of spatiotemporalscales, and they are generated in a self-organizing manner.
Particularly in living organisms, such self-organization of
spatiotemporal patterns is both remarkable and essential.
Therefore, we aim to elucidate the mechanism of generationand control of self-organized patterns in living systems witha particular focus on plants using both mathematical andcomputational approaches.

I. Spatial regulation of resource allocation in
response to nutritional availability

It is critical for living organisms to appropriately allocate
resources among its organs, or within a specific organ,
because available resources are generally limited. For
example, in response to the nutritional environments of theirsoil, plants regulate resource allocation in their roots in orderto plastically change their root system architecture (RSA), sothey can efficiently absorb nutrients (Figure 1A). However,
it is still not understood why and how RSA is adaptively
controlled. Therefore, we modeled and investigated thespatial regulation of resource allocation by focusing on
RSA in response to nutrient availability, and provided analytical solutions to the optimal strategy in the case of simple
fitness functions (Fujita et al., J. Theor. Biol. 2020). First,
we showed that our model could explain the experimental
evidence indicating that root growth is maximized at the
optimal nutrient concentration under homogeneous conditions. Next, we extended our model to incorporate the spatial
heterogeneity of nutrient availability. This extended model
revealed that growth suppression by systemic control isrequired for adaptation to high nutrient conditions, whereas
growth promotion by local control is sufficient for adaptation to low-nutrient environments (Figure 1B). This evidence
indicates that systemic control can be evolved in the presenceof excessive amounts of nutrition, consistent with the‘N-supply’ systemic signal that is observed experimentally.
Furthermore, our model can also explain various experimen-A

Systemic signal

B

s1 , s2Phyllotaxis, the beautiful geometry of plant-leaf arrange-

ment around the stem, has long attracted attention fromresearchers of biological-pattern formation. Many mathematical models, as typified by those of Douady and Couder(alternate-specific form, DC1; more generalized form, DC2),
have been proposed for phyllotactic patterning, mostly basedon the notion that a repulsive interaction between leaf primordia spatially regulates primordium initiation. In theframework of DC models, which assume that each primordium emits a constant power that inhibits new primordium formation, and that this inhibitory effect decreases with
distance, the major (but not all) types of phyllotaxis can be
manifested as a stable pattern. Orixate phyllotaxis, which has
a tetrastichous alternate pattern with a four-cycle sequence ofthe divergence angle, is an interesting example of an unaddressed phyllotaxis type. We examined DC models regarding the ability to produce orixate phyllotaxis and found thatmodel expansion through the introduction of primordial agedependent changes of the inhibitory power is essential for theestablishment of orixate phyllotaxis (Yonekura et al., PLoS
Comput. Biol. 2019). The simulation results obtained using
the expanded version of DC2 (EDC2) fitted well the natural
distribution of phyllotactic patterns. Our findings imply thatchanging the inhibitory power is generally an important component of the phyllotactic patterning mechanism.

Publication List:〔Original papers〕

H., Hayashi-Tsugane, M., and Kawaguchi, M. (2020). Spatial• Fujita,
regulation of resource allocation in response to nutritional availability.

••

J. Theor. Biol. 486, 110078. doi: 10.1016/j.jtbi.2019.110078
Tokumoto, Y., Hashimoto, K., Soyano, T., Aoki, S., Iwasaki, W.,
Fukuhara, M., Nakagawa, T., Saeki, K., Yokoyama, J., Fujita, H., andKawaguchi, M. (2020). Assessment of plant characteristics of Polygala
paniculata (Polygalaceae) for evolutionary studies of legume-rhizobia
symbiosis. J. Plant Res. 133, 109-122. doi: 10.1007/s10265-01901159-x
Yonekura, T., Iwamoto, A., Fujita, H., and Sugiyama, M. (2019).
Mathematical model studies of the comprehensive generation of major
and minor phyllotactic patterns in plants with a predominant focus on
orixate phyllotaxis. PLoS Comput. Biol. 15, e1007044. doi: 10.1371/
journal.pcbi.1007044

···

ui

1

0.01

0.1

1

10

20

50

100

Nitrate concentration (mM)

Nitrateconc.

Optimalroot
density

un

xn

u1

u2
ui

un

x1

x2
xi

···

0.001

···

0

xi

···

0.5

x2

···

1.5

x1

··· ········ ·····

2

u2

xn

····· ·····

u1

3

2.5

···

Lateral root density(mm mm 1)

3.5

Nitrate Optimal
root
conc.
density

Figure 1. (A) Root growth in the homogeneous availability of nitrogen
nutrition. (B) Schematic representation of the spatial regulation of the
optimal resource allocation in response to nutrient availability. (left)
In the low nutrient availability, the optimal root density is promoted
by nutrient through local control (blue arrows). (right) By contrast, in
addition to local control, is suppressed through systemic control (denoted
in red) under high nutrient availability.

69

National Institute for Basic Biology

Environmental Biology

DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

MINAGAWA, Jun

Associate Professor

TAKAHASHI, Shunichi

Assistant Professor:
Technical Staff:
Postdoctoral Fellow:

TOKUTSU, Ryutaro
NODA, Chiyo
KAMADA, Konomi
SATO, Ryoichi
ISHII, Asako
SOKENDAI Graduate Student: WATANABE, Akimasa
OKAJIMA, Keisuke
KISHIMOTO, Mariko
YANAKA, Ayako
Visiting Scientist:
KIM, Eunchul
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
YOKOYAMA, Michiko
SATO, Miyu
Secretary:
TOYAMA, Mami
IIDA, Kaoru
Plants and algae have a great capacity to acclimate themselves to changing environments. We are interested in theseacclimation processes, and how they efficiently yet safely
harness sunlight for photosynthesis under fluctuating light
conditions in particular. Using a model green alga, we are
studying the molecular mechanisms underlying photoacclimation of photosynthetic machinery. We are also applying
knowledge obtained in the studies of this model green alga
to various phytoplankton, including a symbiotic dinoflagellate known to associate with coral and sea anemones,
Symbiodiniaceae, to explore how environmentally important
photosynthetic organisms thrive in their ecological niche.

I. Amphipol-associated purification method forthe highly active and stable photosystem II
Photosystem II (PSII) splits water and drives electrontransfer to plastoquinone via photochemical reactions using
light energy. It is surrounded by light-harvesting complex II(LHCII) to form the PSII-LHCII supercomplex. However,a complete characterization of its structure and function ishampered due to instability of the complex in the presenceof detergent. To overcome this problem, we developed a newprocedure for purifying the PSII-LHCII supercomplexes ofChlamydomonas reinhardtii by employing amphipol A8-35(Watanabe, A., et al. FEBS Lett. 593: 1072-1079). The
obtained supercomplexes showed little LHCII dissociationeven 4 days after purification. Oxygen-evolving activity
was retained within amphipol when the extrinsic polypeptides were kept associated by betaine. Electron microscopy
revealed that this method also improved structural uniformity
and that the major organization was C2S2M2L2.

II. 
S t r u c t u re o f l i g h t h a r v e s t i n g f o r
photosystem II in green algae
In C. reinhardtii, LHCII molecules associate with PSII toform various supercomplexes, including the C2S2M2L2 type;70

the largest PSII–LHCII supercomplex in algae and plantsthat is presently known. We reported high-resolution cryoelectron microscopy (cryo-EM) maps and structural modelsof the C2S2M2L2 and C2S2 supercomplexes from C. reinhardtii (Figure 1) (Sheng, X., Watanabe, A., et al. Nature
Plants, 5: 1320-1330). The C2S2 supercomplex contained an
LhcbM1–LhcbM2/7–LhcbM3 heterotrimer in the strongly
associated LHCII, and the LhcbM1 subunit assembled with
CP43 through two interfacial galactolipid molecules. The
loosely and moderately associated LHCII trimers interacted
closely with the minor antenna complex CP29 to form an
intricate subcomplex bound to CP47 in the C2S2M2L2 supercomplex. A notable direct pathway was established for energy
transfer from the loosely associated LHCII to the PSII reaction
centre, as well as several indirect routes. Structure-based computational analysis concerning the excitation energy transfer
within the two supercomplexes provided detailed mechanistic
insights into the light-harvesting process in green algae.

Figure 1. Top view of the C2S2M2L2 supercomplex from the stromal side. The
central elliptical symbol indicates the C2 axis running perpendicular to themembrane plane. Sheng, Watanabe et al. (2019) Nature Plants, 5: 1320-1330.

III. 
Regulation of light-inducible
photoprotection mechanism, qE, in green
algaeLight is essential for photosynthesis, but the amounts of lightthat exceed an organism’s assimilation capacity can result inoxidative stress and even cell death. Plants and microalgae
have developed a photoprotective response mechanism, qE,
that dissipates excess light energy as thermal energy. In thegreen alga C. reinhardtii, qE is regulated by light-inducible
photoprotective proteins. However, the pathway from lightperception to qE until now has remained unknown. We discovered that the transcription factors CONSTANS and Nuclear
transcription Factor Ys (NF-Ys) form a complex that governs
light-dependent photoprotective responses in C. reinhardtii(Tokutsu, R., et al. Nature Commun., 10: 4099). The qE
responses did not occur in CONSTANS or NF-Y mutants.
The signal from light perception to the CONSTANS/NF-Ys
complex was directly inhibited by the SPA1/COP1-dependent
E3 ubiquitin ligase. This negative regulation mediated by theE3 ubiquitin ligase and the CONSTANS/NF-Ys complex wascommon to photoprotective response in algal photosynthesis
and flowering in plants.

revealed a novel biological signaling mechanism between thecoral host and its potential symbionts (Aihara, Y., et al. Proc.
Natl. Acad. Sci. USA 116, 2118-2123).

Publication List:〔Original Papers〕

Y., Fujimura-Kamada, K., Yamasaki, T., and Minagawa, J.
• Aihara,(2019). Algal photoprotection is regulated by the E3 ligase CUL4-

•
•
•
•
•
Figure 2. Hypothetical signal transduction pathways and physiological
functions of CONSTANS/NF-YB/NF-YC-dependent control of the target
genes in C. reinhardtii and A. thaliana. Tokutsu et al. (2019) Nature
Commun., 10: 4099.

IV. G reen fluorescence from corals attracts
symbiotic algae Symbiodinium

Reef-building corals form an obligate symbiotic relationship with dinoflagellates from the family Symbiodiniaceae.
Most coral species recruit algal symbionts from the environment. However, it has remained unknown how they encounter each other. We focused on green fluorescence protein(GFP)-associated fluorescence, which is commonly seenin coral (Figure 3), and examined whether it attracts motile
algae. We first examined their phototaxis behavior and foundthat symbiotic algae show positive and negative phototaxis
mostly toward strong blue
and weak green light,
respectively. Attraction
shown by algae towards
green fluorescence wasobserved by using botha live coral fragment andan artificial green-fluorescence dye, but only
under blue light conditions i.e. the wavelengththat induces green florescence. We also showedthat traps painted witha green fluorescent dye
Figure 3. Emission of green fluoresattracted symbiotic algae cence from corals under the exposurein the field. Our results to blue light.

•
•
•
•
•
•

DDB1DET1. Nat. Plants 5, 34-40. doi: 10.1038/s41477-018-0332-5
Aihara, Y., Maruyama, S., Baird, A.H., Iguchi, A., Takahashi, S., andMinagawa, J. (2019). Green fluorescence from cnidarian hosts attracts
symbiotic algae. Proc. Natl. Acad. Sci. USA 116, 2118-2123. doi:
10.1073/pnas.1812257116
Burton-Smith, R.N., Watanabe, A., Tokutsu, R., Song, C., Murata,K., and Minagawa, J. (2019). Structural determination of thelarge photosystem II-light-harvesting complex II supercomplex ofChlamydomonas reinhardtii using nonionic amphipol. J. Biol. Chem.
294, 15003-15013. doi: 10.1074/jbc.RA119.009341
Ishii, Y., Maruyama, S., Takahashi, H., Aihara, Y., Yamaguchi, T.,
Yamaguchi, K., Shigenobu, S., Kawata, M., Ueno, N., and Minagawa,J. (2019). Global shifts in gene expression profiles accompanied withenvironmental changes in cnidarian-dinoflagellate endosymbiosis. G3Genes Genom. Genet. 9, 2337-2347. doi: 10.1534/g3.118.201012
Kawakami, K., Tokutsu, R., Kim, E., and Minagawa, J. (2019). Fourdistinct trimeric forms of light-harvesting complex II isolated from thegreen alga Chlamydomonas reinhardtii. Photosynth. Res. 142, 195-201.
doi: 10.1007/s11120-019-00669-y
Kim, E., Watanabe, A., Sato, R., Okajima, K., and Minagawa, J. (2019).
pH-responsive binding properties of light-harvesting complexes
in a photosystem II supercomplex investigated by thermodynamic
dissociation kinetics analysis. J. Phys. Chem. Lett. 10, 3615-3620. doi:
10.1021/acs.jpclett.9b01208
Kubota-Kawai, H., Burton-Smith, R.N., Tokutsu, R., Song, C.,
Akimoto, S., Yokono, M., Ueno, Y., Kim, E., Watanabe, A., Murata,K., and Minagawa, J. (2019). Ten antenna proteins are associatedwith the core in the supramolecular organization of the photosystem
I supercomplex in Chlamydomonas reinhardtii. J. Biol. Chem. 294,4304-4314. doi: 10.1074/jbc.RA118.006536
Sheng, X., Watanabe, A., Li, A., Kim, E., Song, C., Murata, K., Song,D., Minagawa, J., and Liu, Z. (2019). Structural insight into lightharvesting for photosystem II in green algae. Nat. Plants 5, 1320-1330.
doi: 10.1038/s41477-019-0543-4
Tokutsu, R., Fujimura-Kamada, K., Matsuo, T., Yamasaki, T., andMinagawa, J. (2019). The CONSTANS flowering complex controls theprotective response of photosynthesis in the green alga Chlamydomonas.
Nat. Commun. 10, 4099. doi: 10.1038/s41467-019-11989-x
Tokutsu, R., Fujimura-Kamada, K., Yamasaki, T., Matsuo, T., andMinagawa, J. (2019). Isolation of photoprotective signal transduction
mutants by systematic bioluminescence screening in Chlamydomonas
reinhardtii. Sci. Rep. 9, 2820. doi: 10.1038/s41598-019-39785-z
Toyoshima, M., Sakata, M., Ohnishi, K., Tokumaru, Y., Kato, Y.,
Tokutsu, R., Sakamoto, W., Minagawa, J., Matsuda, F., and Shimizu,
H. (2019). Targeted proteome analysis of microalgae under high-light
conditions by optimized protein extraction of photosynthetic organisms.
J. Biosci. Bioeng. 127, 394-402. doi: 10.1016/j.jbiosc.2018.09.001
Watanabe, A., Kim, E., Burton-Smith, R.N., Tokutsu, R., and Minagawa,J. (2019). Amphipol-assisted purification method for the highly active
and stable photosystem II supercomplex of Chlamydomonas reinhardtii.
FEBS Lett. 593, 1072-1079. doi: 10.1002/1873-3468.13394〔Original paper (E-publication ahead of print)〕

S., Perrine, Z., Friedland, N., Kumar, A., Tokutsu, R., Minagawa,• Negi,J., Berg, H., Barry, A., Govindjee, and Sayre, R. Light-regulation oflight harvesting antenna size substantially enhances photosynthetic
efficiency and biomass yield in green algae. Plant J. 2020 Mar 17. doi:
10.1111/tpj.14751

71

National Institute for Basic Biology

Environmental Biology

DIVISION OF PLANT ENVIRONMENTAL RESPONSES
Professor

MORITA, Miyo T.

Assistant Professor:

NISHIMURA, Takeshi
SHIKATA, Hiromasa
Research Staff:
NAKAMURA, Moritaka
KAWAMOTO, Nozomi
TAKASE, Wakana
Visiting Grduate Student: MORI, Shogo
Technical Assistant:
HAMADA, Mayako
MIYOSHI, Kikumi
YAMADA, Yuka
SOMA, Yoriko
MOTOMURA, Hiroe
Secretary:
KOJIMA, Yoko

Plant organs have the ability to sense various vectorial
stimuli such as light, humidity and gravity as well as reorient
their growth direction so as to be in a suitable position tosurvive and acclimate to their environment. These types of
responses are referred to as tropisms. Gravitropism is a major
determinant in the directing of plant organ growth angles.
In gravity sensing cells (statocytes), plastids accumulating starch at a high-density relocate toward the direction ofgravity. Amyloplast relocation serves as a physical signal
trigger for biochemical signal transduction, which in turnleads to the regulation of polar auxin transport necessary forchange in the direction that a given plant is growing. The
above points raise the following important questions: 1) How
is amyloplast relocation converted into a biochemical signal?
2) How do signals affect directional plant growth?
In keeping with this, we aim to understand the detailed
molecular mechanism of gravity signaling by applying
genetic and molecular biological approaches that use themodel plant Arabidopsis thaliana.

Figure 1. Role of RLDs and LZYs in gravitropic setpoint angle. A andC. Growth angle of lateral roots in lzy1 lzy2 lzy3 and rld1 rld4 mutants.
Arrows indicate the direction of gravity. B. Histochemical assay for RLD
gene promoter activities. GUS staining of the primary and lateral roots in
RLD1p, RLD2p, RLD3p or RLD4p:GUS lines are shown.

like motif repeats, a Fab1/YGL023/Vps27/EEA1 (FYVE)
domain, and a Brevis radix (BRX) domain. Promoter:GUS
analysis indicates that four RLD genes are expressed in theroot caps and vascular tissues of primary roots and lateralroots (Figure 1B). While lzy multiple mutants exhibit the
disturbed gravity setpoint angle both in primary roots andshoots, rld1 rld4 double mutants display a similar root
phenotype to that found in lzy multiple mutants (Figure
1A, C). rld1 rld4 double mutants fail to establish the asymmetric auxin distribution in roots as observed in lzy multiple
mutants. These findings suggest that both RLDs and LZYs
play an important role in gravity signaling.

I. M olecular mechanisms associated withgravity signaling

1-1 LZYs and their interactors RLDs are involved ingravitropism
LAZY1 family genes are involved in gravitropism in many
plant species. We previously found that LAZY1-LIKE (LZY)
1, LZY2, and LZY3, are required for gravity signal transduction in statocytes following amyloplast relocation, whichleads to the determination of the growth angle of roots andshoots in Arabidopsis. LZY2 and LZY3 are major contributors to root gravitropism (Figure 1A). LAZY1 family genes
encode plant specific proteins with no domain in which thefunction is inferable. To elucidate the molecular function ofthese LZY proteins, we performed yeast two-hybrid screensand immunoprecipitation coupled with mass spectrometry
to identify their interactors. We found that LZY2 and LZY3
interact with RCC1-like domain (RLD) proteins. There areeight RLD family genes in Arabidopsis genome. RLD family
genes are conserved among land plants and share a similar
domain combination containing a pleckstrin homology (PH)domain, regulator of chromosome condensation 1 (RCC1)-

72

Figure 2. Loss-of-function of RLD genes disrupt the patterns of
PIN1-GFP expression and auxin distribution. A and B. Seven-day-old
seedlings of Col wild-type (A) and rld1 rld2 rld3 rld4 (rld1; 2; 3; 4). C
and D. Embryos of Col (C) and rld1; 2; 3; 4 (D). E-H. DR5rev:GFP (E,F) and PIN1-GFP (G, H) expression in WT-like (E, G) and rld1; 2; 3; 4
(F, H) embryos dissected from ovules of plant homozygous for rld1, rld3,
and rld4 and heterozygous for rld2 at the heart stage.

1-2 LZYs recruit RLDs to the plasma membrane

rld1 rld4 mutants exhibited a significant reduction in thelevel of GFP-tagged PIN3 (PIN3-GFP), which itself is amember of auxin efflux carrier PIN family expressed in root
cap columella cells. Moreover, rld1 rld2 rld3 rld4 quadruplemutant embryos and seedlings displayed severe morphological defects (Figure 2A-D). In the quadruple mutant embryos,
a severe reduction of PIN1-GFP and abnormal auxin distribution pattern were observed (Figure 2E-H). These embryonic phenotypes closely resembled those of gnom, a lossof-function mutant of an ARF-GEF GNOM involved in the
trafficking of PIN proteins. These findings suggest that the
RLDs could regulate auxin flow in the same pathway as
GNOM to control PIN proteins not only in root gravitropism,
but also in plant development.

CCL and demonstrated the importance of the interaction for
gravitropism. These findings indicate RLDs, possible regulators of PIN trafficking, are recruited from the cytosol to theplasma membrane by LZYs through the interaction between
the BRX domain and the CCL domain.

Figure 4. The structure of the CCL-BRX complex. A. Topology diagramof RLD2 BRX domain bound to LZY3 CCL. B. Ribbon representationof the crystal structure of the BRX-CCL complex. Color codes are asin A. The RLD2 BRX domain comprises a three-stranded antiparallel
β-sheet (blue) and two α-helices (green). The LZY3 CCL peptide adopts
a β-hairpin structure (pink). Dashed lines represent inter-molecularhydrogen bonds. The colors of corresponding positions in the CCL-BRX
complex at which mutations were introduced in RLD1-GFP and LZY3mCherry are reversed.
Figure 3. LZYs recruits RLDs to the plasma membrane. A and B.
Co-expression of RLD1-GFP with mCherry (A) and LZY3-mCherry(B) in Arabidopsis protoplasts. C. Localization of RLD1-GFP (left) and
LZY2-mCherry (middle) in the primary root harboring 35Sp:RLD1-GFP
and estradiol-inducible LZY2-mCherry. Arrowheads indicate LZY2mCherry-expressing cells, where RLD1-GFP colocalized with LZY2mCherry in the plasma membrane.

While RLD-GFPs were observed in the cytoplasm in
Arabidopsis protoplast cells, they were localized on theplasma membrane only when LZY2/3-mCherry were coexpressed (Figure 3A, B). Chemical inducible expressionof LZY2-mCherry in the root cells constitutively expressing RLD1-GFP led to the localization of RLD1-GFP on theplasma membrane whereas RLD1-GFP was detected in thecytosol in cells not expressing LZY2-mCherry (Figure 3C).
We also demonstrated through yeast two-hybrid experiments and in vitro pull-down assays that the CCL domainon the C-terminus of LZY and the BRX domain on the
C-terminus of RLD are responsible for the interaction
between LZY3 and RLDs. The interaction between the
CCL domain and the BRX domain was required for theirco-localization of LZY and RLD on the plasma membranein protoplasts. To elucidate the structural basis of the interaction, we determined the crystal structures of the RLD2 BRX
domain bound to the LZY3 CCL peptide at 1.35 Å resolution. The structural analysis reveals the mode of the interaction as an intermolecular β-sheet between the CCL and the
BRX domain in addition to the structure of the BRX domain(Figure 4). Based on the structural analyses, we introduced
mutations that impaired the interaction into the BRX or the

1-3 LZY3 controls RLD1 localization in columella cellsof lateral roots

PIN3 is involved in the asymmetric auxin distribution
in roots, and has been reported to polarly localize on theplasma membrane in the columella cells. Interestingly, we
have found that LZY3-mCherry is also polarly localizedon the basal side of the plasma membrane in the columella cells of lateral roots when plants are vertically grown.
Intriguingly, 180˚ rotation of plants expressing LZY3mCherry reduces the signal of LZY3-mCherry on the basalside of the plasma membrane and increases the signal on
the apical side of plasma membrane where the latest gravity
direction is at least 30 minutes after the rotation. This observation indicates that LZY3 is polarly localized on the plasma
membrane in response to gravistimulation (Figure 5A).
RLD1-GFP exhibits the same behavior as LZY3-mCherry inthe columella cells whereas the asymmetric distribution of
PIN3-GFP is observed 300 minutes after the rotation. Taken
together, we have proposed a model in which gravity stimuli
leads to polarization of LZYs and the polarized LZYs recruit
RLDs on the plasma membrane in order to establish the polar
auxin transport (Figure 5B). Little is known about how theamyloplast sedimentation information is transduced in theform of localization of LZYs and the molecular function of
RLDs in gravity signaling. Accordingly, we are now exclusively focusing on revealing those mechanisms.

73

National Institute for Basic Biology

Environmental Biology••
Figure 5. Asymmetric localization of LZY3-mCherry toward the direction of gravity and the model on gravity signaling. A. The localizationof LZY3-mCherry in the lateral root tips of lzy1 lzy2 lzy3 seedlings
harboring LZY3p:LZY3-mCherry at the stage 2 before rotation (left)
and at 5 min (middle), 30 min (right) after 180˚ rotation. White and red
arrowheads indicate polarized LZY3-mCherry localization and amyloplasts, respectively. The yellow arrows indicate the direction of gravity
estimated from the growth orientation of lateral root tips. B. Schematic•

diagrams of LZY-RLD-mediated gravity signaling in columella cellsof lateral roots.

II. Determination mechanism of gravitropic
setpoint angle

Plant posture is controlled by various environmental cues,
such as light, temperature, and gravity. Their overall architecture is determined by the growth angles of lateral organs,such as roots and branches. The branch growth angle affectedby gravity is known as the gravitropic setpoint angle (GSA).
Many gravitropic mutants show abnormal GSA in lateralbranches; meaning they produce wider growth angle phenotypes likely due to reduced gravitropism. Lateral branchesof Arabidopsis shoot gravitropism (sgr) mutants lacking theendodermal cells, which themselves are shoot statocytes,
grow horizontally as well as those possessed by wild type
plants grown under microgravity condition.
The primary shoots of lzy1 lzy2 lzy3 triple mutants exhibited non-responsiveness to gravistimulation, so it was
expected that the lateral branches of the triple mutant wouldgrow horizontally like sgr mutants do. However, they unexpectedly grew downward and showed positive gravitropism
upon gravistimulation. Moreover, primary and lateral rootsof lzy2 lzy3 lzy4 triple mutant showed negative gravitropism.
We accordingly refer to the phenotype of reversed growth
direction observed in primary roots and lateral branches of
lzy triple mutants as “anti-gravitropic”.
It has been proposed that GSA is determined by balancing two opposing growth components: gravitropism andanti-gravitropic offset (AGO). We assumed that the balance
between gravitropism and AGO was disrupted in lzy triple
mutants, and hypothesized that AGO would be manifested as
an “anti-gravitropic” phenotype. The molecular mechanisms
underlying gravitropism have been studied extensively, butlittle is known about the nature of AGO. To investigate themechanism of GSA control, we are currently focusing onunderstanding the nature of AGO by analyzing “anti-gravitropic” phenotypes of lzy triple mutants.

Publication List:〔Original papers〕

M., Hirano, Y., Nishimura, T., Nakamura, M., Taniguchi,• Furutani,
M., Suzuki, K., Oshida, R., Kondo, C., Sun, S., Kato, K., Fukao, Y.,
Hakoshima, T., and Morita, M.T. (2020). Polar recruitment of RLD

74

by LAZY1-like protein during gravity signaling in root branch angle
control. Nat. Commun. 11, 76. doi: 10.1038/s41467-019-13729-7
Kimata, Y., Kato, T., Higaki, T., Kurihara, D., Yamada, T., Segami, S.,
Morita, M.T., Maeshima, M., Hasezawa, S., Higashiyama, T., Tasaka,M., and Ueda, M. (2019). Polar vacuolar distribution is essential for
accurate asymmetric division of Arabidopsis zygotes. Proc. Natl. Acad.
Sci. USA 116, 2338-2343. doi: 10.1073/pnas.1814160116
Mergner, J., Frejno, M., List, M., Papacek, M., Chen, X., Chaudhary, A.,
Samaras, P., Richter, S., Shikata, H., Messerer, M., Lang, D., Altmann,
S., Cyprys, P., Zolg, D.P., Mathieson, T., Bantscheff, M., Hazarika,
R.R., Schmidt, T., Dawid, C., Dunkel, A., Hofmann, T., Sprunck, S.,
Falter-Braun, P., Johannes, F., Mayer, K.F.X., Jürgens, G., Wilhelm, M.,
Baumbach, J., Grill, E., Schneitz, K., Schwechheimer, C., and Kuster,B. (2020). Mass-spectrometry-based draft of the Arabidopsis proteome.
Nature 579, 409-414. doi: 10.1038/s41586-020-2094-2
Ohbayashi, I., Huang, S., Fukaki, H., Song, X., Sun, S., Morita, M.T.,
Tasaka, M., Millar, A.H., and Furutani, M. (2019). Mitochondrial
pyruvate dehydrogenase contributes to auxin-regulated organ
development. Plant Physiol. 180, 896-909. doi: 10.1104/pp.18.01460〔Original paper (E-publication ahead of print)〕

M., Makigawa, S., Matsumoto, K., Iwata, T., Wasano, N., Kano,• Shindo,A., Morita, M.T., and Fujii, Y. Essential structural features of (2Z,4E)5-phenylpenta-2,4-dienoic acid for inhibition of root gravitropism.
Phytochemistry 2020 Feb 1. doi: 10.1016/j.phytochem.2020.112287〔Review articles〕

M., Nishimura, T., and Morita, M.T. (2019). Bridging• Nakamura,the gap between amyloplasts and directional auxin transport in plant•

gravitropism. Curr. Opin. Plant Biol. 52, 54-60. doi: 10.1016/j.
pbi.2019.07.005
Nakamura, M., Nishimura, T., and Morita, M.T. (2019). Gravity sensing
and signal conversion in plant gravitropism. J. Exp. Bot. 70, 3495-3506.
doi: 10.1093/jxb/erz158

National Institute for Basic Biology

Theoretical Biology

LABORATORY OF GENOME INFORMATICS

Assistant Professor

UCHIYAMA, Ikuo

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 this data,or finding the principles behind it, to understand complex
living systems by integrating the data with current biologicalknowledge via the use of various computational techniques.
In this laboratory we focus on developing computational
methods and tools for comparative genome analysis, which isa useful approach for finding functional or evolutionary clues
to interpret the genomic information of various species.
The current focus of our research is the comparative
analysis of microbial genomes, the number of which has
been increasing rapidly. Since different types of information
about biological functions and evolutionary processes canbe extracted by comparing genomes at different evolutionary distances, we are developing methods to conduct comparative analyses not only of distantly related but also closely
related genomes.

I. Microbial genome database for comparative
analysis

The microbial genome database for comparative analysis(MBGD, 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 combinedwith the DomRefine program. Through the application ofthese programs, MBGD not only provides comprehensive
orthologous groups among the latest genomic data, but also
allows users to create their own ortholog groups on the fly
by using a specified set of organisms. MBGD also has precalculated ortholog tables for each major taxonomic group,and provides several viewing modes to display the entiretyof each ortholog table. For some closely related taxa, MBGD
provides conserved synteny information calculated using
the CoreAligner program. MBGD additionally provides a‘MyMBGD’ mode, which allows users to add their own
genomes to MBGD.
MBGD now contains 6318 genomes, including 5861
bacteria, 254 archaea, and 203 eukaryota. These data sets areclassified based on the hierarchical ortholog classification
strategy described in the section below.

II. Hierarchical strategy for creating ortholog
tables

MBGD previously calculated all-against-all similarities
among the stored genomes and independently created 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 thisapproach enabled us to effectively calculate a set of ortholog
tables for both closely related and distantly related genome
comparisons, a considerable amount of information may belost from the standard ortholog table, given that within-species and within-genus genomic diversity is generally large.
To address this problem, we developed a stepwise protocol
to construct orthologous relationships. First, for each species
with at least two genomes, all-against-all similarities among
the genomes belonging to that species are calculated and awithin-species ortholog table is created. The species-level
pan-genome is then created by picking one representative
gene from each orthologous group. Next, for each genus
with at least two species, all-against-all similarities among
the species-level pan-genomes are calculated and a withingenus ortholog table is created. The genus-level pan-genome
is then created by picking one representative gene from eachorthologous group. Finally, all-against-all similarities among
the genus-level pan-genomes are calculated and the standard
ortholog table covering the entire taxonomic range is created.
To calculate within-species or within-genus all-against-all
similarities, we used a faster, but less sensitive, similarity
search program, UBLAST. Using this method, we can reducethe computation time required for all-against-all similarities.

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

As a core technology of our comparative genomics tools,
we have developed a rapid automated method of ortholog
grouping, named DomClust, which allows us to simultaneously compare numerous genomes. This 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 when required. As a result, the procedure cansplit genes into the domains minimally required for ortholog
grouping.
We have 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
the sum total of domain-level alignment scores. On thebasis of this idea, we have developed a refinement pipeline
to improve domain-level clustering, DomRefine, by optimizing DSP scores. 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. In particular, this data is
considered suitable for analyzing domain fusion events thathave occurred during evolution. By analyzing the domainlevel ortholog grouping data combined with taxonomic andfunctional information, we are now trying to elucidate whenand in what kind of genes domain fusion events frequently
occur.

75

National Institute for Basic Biology

Theoretical Biology

IV. 
D evelopment and application of aworkbench for comparative genomics andtranscriptomics

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 manipulate large-scale ortholog tables. The ortholog table viewer
is a spreadsheet like viewer that can display an entire
ortholog table containing more than a thousand genomes. In
RECOG, several basic operations on the ortholog table aredefined, which are classified into categories such as filtering,sorting, and coloring. By combining these basic operations,various comparative analyses can be performed. In addition,
RECOG allows the user to input arbitrary gene properties
and compare these properties among orthologs in various
genomes.
We are continuing to develop the system and apply it
to various genome comparison studies as part of various
collaborative research projects. Among these, we applied
RECOG to the comparative analyses of transcriptomic dataof Chattonella antiqua and other harmful algae causing red
tide in collaboration with Dr. Shikata (FRA). In this analysis,
we compared two newly determined and three existingRNA-seq datasets of harmful algae and 10 existing genome
sequences of various algae and a plant model organism,
Arabidopsis thaliana using RECOG, and identified common
sequence features among orthologous genes belonging toharmful algae that may be related to red tide outbreaks andtheir toxicity to fish.

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

Orthology is a key to integrating knowledge of variousorganisms through comparative analysis. In order to integrate
genomic data and various types of biological information
with this idea, we have constructed an ortholog databaseusing Semantic Web technology. To formalize the structure of the ortholog information in the Semantic Web, wedeveloped an ortholog ontology (OrthO) and described theortholog information in MBGD in the form of the Resource
Description Framework (RDF). OrthO is now superseded by
the Orthology Ontology (ORTH) that integrates OrthO andother ontologies and is adopted by other orthology resources.
On the basis of this framework, we have integrated various
kinds of microbial data using the ortholog information as ahub, as part of the MicrobeDB.jp project (http://microbedb.
jp/) under the auspices of the National Bioscience Database
Center.

VI. A novel approach for identification of
genomic islands

Genomes of bacterial species can show great variation in
their gene content, and thus systematic analysis of the entire
gene repertoire, termed the “pan-genome”, is important forunderstanding bacterial intra-species diversity. As we havealready developed a procedure (CoreAligner) to define the76

core genome as the genes conserved among the genomes of
the given species, characterizing the remaining part of the
genomes (non-core genomes) should be important for understanding the species’ diversity. To this end, we are developing a method (FindIsland) to identify a set of non-core genesthat have conserved gene order by using a modified versionof the CoreAligner algorithm (Figure 1). We applied themethod to the sets of genomes of prokaryotic species storedin MBGD and found that in many cases the resulting conserved clusters correspond to known mobile genetic elements
and/or have sequence features common to known genomic
islands.

Figure 1. Visualization of the genomic islands identified by the
FindIsland program.‑

Publication List:〔Original papers〕

M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T.,• Matsunami,
Yamaguchi, K., Uchiyama, I., Mori, K., Tashiro, K., Ito, Y., Takeuchi,•••T., Suzuki, K.-I.T., Agata, K., Shigenobu, S., and Hayashi, T. (2019).
A comprehensive reference transcriptome resource for the Iberianribbed newt Pleurodeles waltl, an emerging model for developmental
and regeneration biology. DNA Res. 26, 217-229. doi: 10.1093/dnares/
dsz003
Nagao, R., Kato, K., Suzuki, T., Ifuku, K., Uchiyama, I., Kashino,Y., Dohmae, N., Akimoto, S., Shen, J.-R., Miyazaki, N., and Akita,
F. (2019). Structural basis for energy harvesting and dissipation ina diatom PSII–FCPII supercomplex. Nat. Plants 5, 890-901. doi:
10.1038/s41477-019-0477-x
Shikata, T., Takahashi, F., Nishide, H., Shigenobu, S., Kamei, Y.,
Sakamoto, S., Yuasa, K., Nishiyama, Y., Yamasaki, Y., and Uchiyama,
I. (2019). RNA-Seq analysis reveals genes related to photoreception,
nutrient uptake, and toxicity in a noxious red-tide raphidophyte
Chattonella antiqua. Front. Microbiol. 10, 1764. doi: 10.3389/
fmicb.2019.01764
Uchiyama, I., Mihara, M., Nishide, H., Chiba, H., and Kato, M. (2019).
MBGD update 2018: Microbial genome database based on hierarchicalorthology relations covering closely related and distantly relatedcomparisons. Nucleic Acids Res. 47, D382-D389. doi: 10.1093/nar/gky1054

National Institute for Basic Biology

Imaging Science

LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

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

YOKE, Hiroshi
TANIGUCHI, Atsushi
KONDO, Akiko
ISHIBASHI, Tomoko

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

microscopes, one commercial and the other self-made,and are maintaining them for collaborations and our own
research interest (this being left-right asymmetry).
Over several years, we have developed a fast light-sheet
microscope named ezDSLM, in which the excitation lightsheet and detection objective move instead of the specimen
for z-scanning. We installed an electrically tunable lens(ETL) to achieve greater speed and the exclusion of mechanical vibrations that disturb continuous 4D imaging of floating
or loosely held specimens.
Our light-sheet microscopes are available to other researchers via NIBB’s Collaborative Research and MEXT’s
Advanced Bioimaging Support (ABiS) programs. Numerous
collaborations are in progress including observation ofmoving cell migration in zebrafish embryos, cleared mousebrains, etc.

I. Initial step for left-right asymmetry

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

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

Publication List:〔Original papers〕

T., Song, C., Murata, K., Nonaka, S., Ogawa, K., Kondo, Y.,• Kaji,Ohtsuka, S., and Palmer A.R. (2019). Evolutionary transformation••
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.

•of mouthparts from particle-feeding to piercing carnivory in Viper
copepods: Review and 3D analyses of a key innovation using advanced
imaging techniques. Front. Zool. 16, 35. doi: 10.1186/s12983-0190308-y
Sasaki, K., Shiba, K., Nakamura, A., Kawano, N., Satouh, Y.,
Yamaguchi, H., Morikawa, M., Shibata, D., Yanase, R., Jokura, K.,
Nomura, M., Miyado, M., Takada, S., Ueno, H., Nonaka, S., Baba, T.,
Ikawa, M., Kikkawa, M., Miyado, K., and Inaba, K. (2019). Calaxin
is required for cilia-driven determination of vertebrate laterality.
Commun. Biol. 2, 226. doi: 10.1038/s42003-019-0462-y
Serizawa, T., Isotani, A., Matsumura, T., Nakanishi, K., Nonaka, S.,
Shibata, S., Ikawa, M., and Okano, H. (2019). Developmental analyses
of mouse embryos and adults using a non-overlapping tracing system
for all three germ layers. Development 146, dev174938. doi: 10.1242/
dev.174938
Yoon, Y., Park, J., Taniguchi, A., Kohsaka, H., Nakae, K., Nonaka, S.,
Ishii, S., and Nose, A. (2019). System level analysis of motor-related
neural activities in larval Drosophila. J. Neurogenet. 33, 179-189. doi:
10.1080/01677063.2019.1605365

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

II. Development of light-sheet microscopy

Light-sheet microscopy has become popular during thisdecade due to benefits such as low photodamage and fast
image acquisition. We are now running two light-sheet

77

National Institute for Basic Biology

Imaging Science

LABORATORY FOR BIOTHERMOLOGYSpecially Appointed Associate Professor

KAMEI, Yasuhiro

NIBB Research Fellow:
CREST Researcher:
JSPS Postdoctral Fellow:
Technical Assistant:

SAKAMOTO, Joe
KAMIKAWA, Yuko
SUZUKI, Miyuki
KINOSHITA, Chie
TAMADA, Tomoko

Temperature is an important parameter for living organisms. Cell activity is affected by temperature since thereaction ratio and stability of molecules in cells depends on
it. Environmental temperatures which organisms function inare variable, and the biological system that regulates bodytemperature, homeostasis, differs among various organisms.
Furthermore, temperature in cells or at the molecular levelhas not been widely discussed to date. Nano-scale thermometers utilizing fluorescent proteins and dyes have beendeveloped by numerous research groups including ours, andheterogeneity of temperatures in the micro environments ofliving cells have been reported on by said groups (Okabe etal, Nat. Commun. 2014, Kiyonaka et al, Nat. Methods 2014,
and Nakano et al, PLoS One 2017). However, the meaning
and mechanisms in single cells remain unclear. Thus, ourgroup is embarking upon a new research field, biothermology, through the investigation of the nature of temperature inliving organisms ranging from nano to macro.

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

Our research group employs various and original biothermological research technologies . One of these is theinfrared (IR) laser application, which enables single-cell orsubcellular local heating by focusing IR through microscope
objective. This technology was originally developed forlaser induced gene expression in the targeting of single-cells
through the heat shock response, called IR-LEGO; InfraRed
Laser Evoked Gene Operator (Kamei et al, Nat. Methods2009) (Figure 1). This local heating technique can be used
to analyze the thermodynamics of a cell via the use of anano-scale thermometer;the second of our abovementioned
original techniques. To analyze temperature distribution at
subcellular resolutions, a nano-scale thermometer is required.
For this reason, we have focused on fluorescent proteins, anddeveloped a genetically encoded thermometer, the so calledgTEMP, with Dr. Takeharu Nagai from Osaka University(Nakano et al, PLoS One 2017). This thermometer has someunique properties such as rapid response and applicability to78

wide temperature range. By utilizing this thermometer, weare developing a high-speed thermal imaging microscope
system combined with IR irradiation optics. Through theanalysis of thermal dynamics in cells, we are trying to reveal
how temperature heterogeneity is generated within a single
cell and to consider its meaning from a biological standpoint.
Furthermore, our group is trying to improve the IR-LEGO
technique itself. We initially applied this system to manyorganisms including animals and plants, such as medaka,
nematode, flies, frogs, and Araidopsis, moss, in collaborative research projects in cooperation with other laboratories. Because the heat shock response (HSR) is a transient
response, gene expressions after IR-LEGO operation are alsotransient. Despite this, some of our collaborators wanted torealize cell fate mapping, which requires a long-term gene
expression after IR irradiation. Accordingly, we employed
Cre/LoxP recombination system. As shown in Figure 2, weestablished a cre driver line, which possesses a heat shock
promoter and loxP effector line which can permanently label
irradiated cell lineages by fluorescent proteins. By using
these transgenic lines, evidence was provided that indicated that exoskeletal tissues in trunk region came from themesoderm (Shimada et al, Nat. Commun. 2013).

Figure 2. Long-term gene expression with cre/loxP recombination system
and an example of a practical experiment in medaka ranging embryos toadults.

The HSR is a conservative stress response system found inalmost all organisms. In contrast, organisms live in various
temperatures, and HSR essentially is the upper limit of habitable temperature range for each organism. This means thatthe preset temperature of HSR may differ among organisms.
A key factor of this limitation is heat shock factor 1 (HSF1);a homologue within each organism. By substituting HSF1,
it may be possible to control the preset temperature of HSR,
and this substitution could be applied to the improvementof HSR efficiency in IR-LEGO technology. We are now
trying to lower the HSR temperature of medaka by substituting Japanese flounder HSF1. This HSF1 substitution
project provides us with various significant information andevidence related to molecular evolution and adaptation toenvironmental temperature. Therefore, this project also contributes to the study of biothermology.

We also promote other collaborative studies using microscopic techniques. One is a “clear observation project” fordeep seeing into living organisms using adaptive optics(AO), which have been thoroughly developed in the fieldof astronomy as a key technology found in large telescopes,
such as the Subaru telescope in Hawaii. Although observation using Earth based telescopes may be interfered with
due to fluctuations in the atmosphere, AO can mitigate this.
However, living materials have particular refractive indexes,
so some organelles may hinder the ideal optical path formicroscope observation, which is similar to the situation thatexists regarding the atmosphere and telescopes. AO can also
compensate for this disturbance by sensing and correcting
wave fronts by using a wave front sensor and deformablemirror. Hence, we have developed a custom-made wide-fieldmicroscope equipped with an AO system for the observationof living organisms in collaboration with Dr. Yosuke Tamada
from Utsunomiya University and Dr. Yutaka Hayano fromthe National Astronomical Observatory of Japan (NAOJ).
By utilizing this microscopic system, we have successfullyacquired high-resolution bright field and fluorescent imagesof living cells. Furthermore, AO can also be applied to
IR-LEGO technology to improve IR energy focusing of irradiation in a manner similar to improving the sight of livingorganisms.

Publication List:〔Original papers〕

T., Shimmura, T., Shinomiya, A., Okimura, K., Takehana,• Nakayama,
Y., Furukawa, Y., Shimo, T., Senga, T., Nakatsukasa, M., Nishimura,•••T., Tanaka, M., Okubo, K., Kamei, Y., Naruse, K., and Yoshimura, T.
(2019). Seasonal regulation of the lncRNA LDAIR modulates selfprotective behaviours during the breeding season. Nat. Ecol. Evol. 3,
845-852. doi: 10.1038/s41559-019-0866-6
Shikata, T., Takahashi, F., Nishide, H., Shigenobu, S., Kamei, Y.,
Sakamoto, S., Yuasa, K., Nishiyama, Y., Yamasaki, Y., and Uchiyama,
I. (2019). RNA-Seq analysis reveals genes related to photoreception,
nutrient uptake, and toxicity in a noxious red-tide raphidophyte
Chattonella antiqua. Front. Microbiol. 10, 1764. doi: 10.3389/
fmicb.2019.01764
Uchimura, T., Hara, S., Yazawa, T., Kamei, Y., and Kitano, T. (2019).
Involvement of heat shock proteins on the transcriptional regulation ofcorticotropin-releasing hormone in medaka. Front. Endocrinol. 10, 529.
doi: 10.3389/fendo.2019.00529
Yokoi, S., Naruse, K., Kamei, Y., Ansai, S., Kinoshita, M., Mito, M.,
Iwasaki, S., Inoue, S., Okuyama, T., Nakagawa, S., Young, L.J., andTakeuchi, H. (2020). Sexually dimorphic role of oxytocin in medaka
mate choice. Proc. Natl. Acad. Sci. USA 117, 4802-4808. doi: 10.1073/
pnas.1921446117〔Review articles〕

G., Hayashi, T., Yoshida, K., Yoshida, T., Kudoh, H., Sakamoto,• Abe,J., Konishi, A., Kamei, Y., Takeuchi, T., Tamura, K., and Yokoyama, H.

•(2020). Insights regarding skin regeneration in non-amniote vertebrates:
Skin regeneration without scar formation and potential step-up to ahigher level of regeneration. Semin. Cell Dev. Biol. 100, 109-121. doi:
10.1016/j.semcdb.2019.11.014
Murata, K., Kinoshita, M., Naruse, K., Tanaka, M., and Kamei, Y. eds.
(2020). Medaka: Biology, Managements, and Experimental Protocols,
Volume 2. (Wiley-Blackwell, Iowa, USA). ISBN: 978-1-119-57529-0

E., Yasugi, M., Takeuchi, H., Watanabe, E., Kamei, Y., and• Abe,
Yamamoto, H. (2019). Development of omnidirectional aerial display

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with aerial imaging by retro-reflection (AIRR) for behavioral biology
experiments. Opt. Rev. 26, 221-229. doi: 10.1007/s10043-019-00502-w
Fujikawa, Y., Ishikawa-Fujiwara, T., Kuo, T., Shinkai, N., Shogi, T.,
Kawasaki, T., Kamei, Y., Sakuraba, Y., Sato, A., Kinoshita, M., Gondo,Y., Yuba, S., Tsujimura, T., Sese, J., and Todo, T. (2020). Involvementof Rev1 in alkylating agent‐induced loss of heterozygosity in Oryzias
latipes. Genes Cells 25, 124-138. doi: 10.1111/gtc.12746
Furukawa, F., Hamasaki, S., Hara, S., Uchimura, T., Shiraishi, E.,
Osafune, N., Takagi, H., Yazawa, T., Kamei, Y., and Kitano, T. (2019).
Heat shock factor 1 protects germ cell proliferation during early ovariandifferentiation in medaka. Sci. Rep. 9, 6927. doi: 10.1038/s41598-01943472-4
Harada, Y., Matsuo, M., Kamei, Y., Goto, M., and Fukamachi, S.
(2019). Evolutionary history of the medaka long-wavelength sensitive
genes and effects of artificial regression by gene loss on behaviouralphotosensitivity. Sci. Rep. 9, 2726. doi: 10.1038/s41598-019-39978-6
Hwang, D., Wada, S., Takahashi, A., Urawa, H., Kamei, Y., andNishikawa, S. (2019). Development of a heat-inducible gene expression
system using female gametophytes of Arabidopsis thaliana. Plant Cell
Physiol. 60, 2564-2572. 10.1093/pcp/pcz148
Ikehata, H., Mori, T., Kamei, Y., Douki, T., Cadet, J., and Yamamoto,M. (2020). Wavelength- and tissue-dependent variations in the
mutagenicity of cyclobutane pyrimidine dimers in mouse skin.
Photochem. Photobiol. 96, 94-104. doi: 10.1111/php.13159
Kosugi, M., Ozawa, S., Takahashi, Y., Kamei, Y., Itoh, S., Kudoh, S.,
Kashino, Y., and Koike, H. (2020). Red-shifted chlorophyll a bands
allow uphill energy transfer to photosystem II reaction centers in anaerial green alga, Prasiola crispa, harvested in Antarctica. BBA Bioenergetics 1861, 148139. doi: 10.1016/j.bbabio.2019.148139
Nakane, Y., Shinomiya, A., Ota, W., Ikegami, K., Shimmura, T.,
Higashi, S., Kamei, Y., and Yoshimura, T. (2019). Action spectrum forphotoperiodic control of thyroid-stimulating hormone in Japanese quail(Coturnix japonica). PLoS One 14, e022106. doi: 10.1371/journal.
pone.0222106

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National Institute for Basic Biology

Exploratory Research Center on Life and Living Systems (ExCELLS), Orion Project

LABORATORY OF NUCLEAR DYNAMICS†

Specially Appointed Associate Professor

MIYANARI, Yusuke

JSPS Postdoctoral Fellow: KURIHARA, Misuzu
Research Staff:
TAGAWA, Ayako
Technical Assistant:
SANBO, Chiaki
Secretary:
HACHISUKA, Midori
A fundamental challenge in biology is the one of understanding the mechanisms underlying cell-fate decisions.
Genomic reprogramming after mammalian fertilization
reverts terminally differentiated gametes into toti- or pluripotent states when starting a new developmental program.
Cell lineage allocation in the reprogramming process isaccompanied by drastic changes in the pattern of gene
expression, epigenetic configurations, and nuclear organization. We aim to reveal the role of chromatin dynamics in celllineage-allocation by deciphering the molecular mechanisms
underlying the remodeling of nuclear organization and theireffects on developmental gene expression, using mouseembryos and embryonic stem (ES) cells as model systems.

Epigenetic reprogramming in early mouseembryos

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

Figure 1. Lineage allocation in mouse preimplantation development†: This laboratory was closed on 31 March, 2019.

80

Remodeling of nuclear architecture indevelopment

Chromatin is organized in a non-random fashion within
three-dimensional nuclear space. During developmental processes, the nuclear architecture is dramatically reconstructed,
resulting in the establishment of cell-type specific nuclear
organization. Defects in structural components of the nucleus
are responsible for developmental aberrations and several
human diseases. Remodeling of the nuclear architectureleads to spatial arrangement of genes, which could affectgenome functions including gene expression. However, regulatory mechanisms underlying nuclear reorganization duringcell-fate decisions remain largely unknown.

Chromatin structure

Spatiotemporal organization of genomic DNA within
the nucleus has been suggested as an emerging key player
to regulate gene expression. The developmental programaccompanies nuclear remodeling, resulting in construction of
nuclear architecture specific to the cell type. 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 loci frequently interact with eachother. Recent works have revealed that folding of “local”
chromatin structures such as enhancer-promoter looping
is associated with genome functions. Despite the drasticchanges of these hierarchical chromatin structures, their rolein cell-fate decisions remains largely unexplored.

Figure 2. Hierarchical chromatin structure

Approach

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

National Institutes of Natural Sciences

Exploratory Research Center on Life and Living Systems (ExCELLS), Bio-Next Project

LABORATORY OF PLANT DEVELOPMENT AND PHYSIOLOGY†

Specially Appointed Associate Professor

KAWADE, Kensuke

Visiting Graduate Student: TOMOI, Takumi
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 contextdependent manner. There is also a growing awareness thatmetabolism plays an instructive role in developmental processes. This emerging picture depicts metabolism as a criticalsystem not only for sustaining physiological conditions,but also regulating developmental patterning by coordinating various cellular processes. However, it largely remains
unclear how this interaction is established in multicellular organisms. We aim to reveal as-yet-unknown relationships between developmental and metabolic processes, andtheir biological meaning, by elucidating molecular mechanisms in the system. To address this, we use a 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 the hidden relationships between development and metabolism, we performed quantitative phenome
screening using A. thaliana mutants of orphan cytochrome
P450 genes. As a result of this screening, we discovered thecyp77a4 mutant, which exhibits irregular embryonic patterning as evidenced by its developmental defects, such asa cup-shaped cotyledon morphology (Figure 1). Although
CYP77A4 is the first cytochrome P450 reported that is able
to catalyze the epoxidation of unsaturated fatty acids inplants, its function in development is 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 found that CYP77A4 andCYP77A6 (the phylogenetically closest gene to CYP77A4)
are functionally independent. Based on these findings, we
propose that the metabolic requirement for polarity establishment via auxin dynamics differs between tissues, and that inembryos this depends on a CYP77A4-dependent metabolic
pathway. These findings may augment our understanding of
fatty-acid epoxidation by uncovering a new developmental
function of the epoxidase (Kawade et al., 2018).

II. T he role of the developmental signal
intertwined with metabolism

ANGUSTIFOLIA3 (AN3) is a transcriptional co-activator,
which promotes cell proliferation in leaves. We recently
showed that AN3 forms an expression gradient along 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 conducted transcriptome and metabolome analyses using an3 mutants. We found that AN3 regulates a transcriptional network for oxygen homeostasis. Our
metabolic profiling detected characteristic features of redox
disturbance in the an3 mutants. Although cell proliferation
is an 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 theargument surrounding cell proliferation and oxidative stress,
in plant 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 paper (E-publication ahead of print)〕

T., Kawade, K., Kitagawa, M., Sakata, Y., Tsukaya, H., and• Tomoi,
Fujita, T. Quantitative imaging reveals distinct contributions of SnRK2and ABI3 in plasmodesmatal permeability in Physcomitrella patens.
Plant Cell Physiol. 2020 Feb. 26. doi: 10.1093/pcp/pcaa021

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.

†: This laboratory was closed on 31 March, 2019.

81

National Institute for Basic Biology

Research Support

NIBB CORE RESEARCH FACILITIESHeadYOSHIDA, Shosei

The NIBB Core Research Facilities were launched in 2010
to support basic biological research at NIBB. They consistof three facilities that develop and provide state-of-theart technologies aimed at increasing the understanding of
biological functions through the application of functional
genomics, bioimaging, and bioinformatics. The NIBB Core
Research Facilities also act as an intellectual hub to promotecollaboration among NIBB researchers and other academic
institutions.

MinION and GridION (Oxford Nanopore Technologies), theFunctional Genomics Facility is committed to joint research
aimed at exploring new yet otherwise inaccessible fields inbasic biology.
During 2019, we carried out 57 NGS projects in collaboration with researchers from academic institutions throughout
the world. These projects cover a wide range of species(bacteria, animals, plants, and fungi) including both modeland non-model organisms, and various other applications
such as genomic re-sequencing, RNA-seq and ChIP-seq.

FUNCTIONAL GENOMICS FACILITY

Professor

SHIGENOBU, Shuji

Technical Staff:
Technical Assistant:
Secretary:

MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
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 the promotion of DNA and protein studies. The
facility maintains a wide array of core research equipment,
ranging from standard machinery (e.g. ultracentrifuges)
to cutting edge tools (e.g. next generation DNA sequencers), which amount to 90 instruments in total. The facility
is dedicated to fostering collaborations with researchers
both at NIBB and other academic institutions worldwide
through the provision of these tools as well as expertise. Ourcurrent focus is functional genomics. We also act as a bridge
between experimental biology and bioinformatics throughclose consultation and training.
We recently renovated the facility building, and as partof this, the Visitors Lab and the Visitors Office were redesigned so that visiting scientists can work more effectively
during their stay. In 2019, approximately 100 researchers
came to use our facility and developed active collaborations,which consequently resulted in 21 co-authored papers published.

Representative Instruments
Genomics

The advent of next-generation sequencing (NGS) technologies is transforming modern biology thanks to ultra-highthroughput DNA sequencing. Utilizing HiSeq, NextSeq
and MiSeq (Illumina), Sequel (PacificBio Sciences), and82

Figure 1. Next-generation sequencer

Proteomics

Two types of mass spectrometer and two protein sequencers, as listed below, are used for proteome studies in ourfacility. In 2019, we analyzed approximately 1000 samples
with mass spectrometers and protein sequencers.
- LC-MS (AB SCIEX TripleTOF 5600 system)- LC-MS (Thermo Fisher SCIENTIFIC Orbtrap Elite)
Protein sequencer (ABI Procise 494 HT; ABI Procise 492
cLC)

Figure 2. Triple TOF LC/MS/MS System

Other analytical instruments (excerpts)- Cell sorter (SONY SH800)- B ioimaging analyzer (Fujifilm LAS 3000 mini; GE
FLA9000)- Laser capture microdissection system (Thermo Fisher

Scientific Arcturus XT)- Real-time PCR machine (Thermo Fisher Scientific ABI
7500)- Ultracentrifuge (Beckman XL-80XP etc.)-
M icroplate reader (PerkinElmer Nivo; Hitachi
SH-9000Lab)- Single-cell analysis system (Fluidigm C1)

Research activity by S. Shigenobu

Professor Shuji Shigenobu is the principal investigator of
the Laboratory of Evolutionary Genomics. Refer to the laboratory page for details.

Publication List on Cooperation:〔Original papers〕

M., Yamamoto, N., Shigenobu, S., Mori, H., Kuwata,• Bessho-Uehara,K., and Oba, Y. (2020). Kleptoprotein bioluminescence: Parapriacanthus

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fish obtain luciferase from ostracod prey. Sci. Adv. 6, eaax4942. doi:
10.1126/sciadv.aax4942
Cai, H., Li, Q., Fang, X., Li, J., Curtis, N.E., Altenburger, A., Shibata,T., Feng, M., Maeda, T., Schwartz, J.A., Shigenobu, S., Lundholm,N., Nishiyama, T., Yang, H., Hasebe, M., Li, S., Pierce, S.K., and andWang, J. (2019). Data descriptor: A draft genome assembly of the solarpowered sea slug Elysia chlorotica. Sci. Data 6, 190022. doi: 10.1038/
sdata.2019.22
Goto-Yamada, S., Oikawa, K., Bizan, J., Shigenobu, S., Yamaguchi, K.,
Mano, S., Hayashi, M., Ueda, H., Hara-Nishimura, I., Nishimura, M.,
and Yamada, K. (2019). Sucrose starvation induces microautophagy inplant root cells. Fronti. Plant Sci. 10, 1604. doi: 10.3389/fpls.2019.01604
Hiwatashi, T., Goh, H., Yasui, Y., Koh, L.Q., Takami, H., Kajikawa,
M., Kirita, H., Kanazawa, T., Minamino, N., Togawa, T., Sato, M.,
Wakazaki, M., Yamaguchi, K., Shigenobu, S., Fukaki, H., Mimura,T., Toyooka, K., Sawa, S., Yamato, K.T., Ueda, T., Urano, D., Kohchi,T., and Ishizaki, K. (2019). The RopGEF KARAPPO is essential forthe initiation of vegetative reproduction in Marchantia polymorpha.
Current Biol. 29, 3525-3531. doi: 10.1016/j.cub.2019.08.071
Hojo, M., Shigenobu, S., Maekawa, K., Miura, T., and Tokuda, G.
(2019). Duplication and soldier-specific expression of geranylgeranyl
diphosphate synthase genes in a nasute termite Nasutitermes
takasagoensis. Insect Biochem. Mol. Biol. 111, 103177. doi: 10.1016/j.
ibmb.2019.103177
Horikawa, A., Yoneda, T., Yaoita, E., Yamaguchi, K., Shigenobu, S.,
Kuramochi, M., Yamate, J., Inui, T., and Ishibashi, O. (2019). A novel
splicing variant of small nucleolar RNA host gene 4 is a podocyteselective non-coding RNA upregulated in response to puromycin
aminonucleoside-induced podocyte injury. J. Biochem. 165, 447-454.
doi: 10.1093/jb/mvy118
Ishii, Y., Maruyama, S., Takahashi, H., Aihara, Y., Yamaguchi, T.,
Yamaguchi, K., Shigenobu, S., Kawata, M., Ueno, N., and Minagawa,J. (2019). Global shifts in gene expression profiles accompanied withenvironmental changes in cnidarian-dinoflagellate endosymbiosis. G3Genes Genom. Genet. 9, 2337-2347. doi: 10.1534/g3.118.201012
Ishikawa, M., Morishita, M., Higuchi, Y., Ichikawa, S., Ishikawa,T., Nishiyama, T., Kabeya, Y., Hiwatashi, Y., Kurata, T., Kubo,
M., Shigenobu, S., Tamada, Y., Sato, Y., and Hasebe, M. (2019).
Physcomitrella STEMIN transcription factor induces stem cell
formation with epigenetic reprogramming. Nat. Plants 5, 681-690. doi:
10.1038/s41477-019-0464-2
Jokura, K., Shibata, D., Yamaguchi, K., Shiba, K., Makino, Y.,
Shigenobu, S., and Inaba, K. (2019). CTENO64 is required for
coordinated paddling of ciliary comb plate in Ctenophores. Current
Biol. 29, 3510-3516. doi: 10.1016/j.cub.2019.08.059
Kutsukake, M., Moriyama, M., Shigenobu, S., Meng, X.-Y., Nikoh,N., Noda, C., Kobayashi, S., and Fukatsu, T. (2019). Exaggeration andcooption of innate immunity for social defense. Proc. Natl. Acad. Sci.
USA 116, 8950-8959. doi: 10.1073/pnas.1900917116

M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T.,• Matsunami,
Yamaguchi, K., Uchiyama, I., Mori, K., Tashiro, K., Ito, Y., Takeuchi,

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T., Suzuki, K.-I.T., Agata, K., Shigenobu, S., and Hayashi, T. (2019).
A comprehensive reference transcriptome resource for the Iberianribbed newt Pleurodeles waltl, an emerging model for developmental
and regeneration biology. DNA Res. 26, 217-229. doi: 10.1093/dnares/
dsz003Morita, S., Ando, T., Maeno, A., Mizutani, T., Mase, M., Shigenobu,
S., and Niimi, T. (2019). Precise staging of beetle horn formation in
Trypoxylus dichotomus reveals the pleiotropic roles of doublesex
depending on the spatiotemporal developmental contexts. PLoS Genet.
15, e1008063. doi: 10.1371/journal.pgen.1008063
Ohbayashi, T., Futahashi, R., Terashima, M., Barrière, Q., Lamouche,F., Takeshita, K., Meng, X.-Y., Mitani, Y., Sone, T., Shigenobu,
S., Fukatsu, T., Mergaert, P., and Kikuchi, Y. (2019). Comparative
cytology, physiology and transcriptomics of Burkholderia insecticola
in symbiosis with the bean bug Riptortus pedestris and in culture. ISME
J. 13, 1469-1483. doi: 10.1038/s41396-019-0361-8
Sekii, K., Yorimoto, S., Okamoto, H., Nagao, N., Maezawa, T., Matsui,Y., Yamaguchi, K., Furukawa, R., Shigenobu, S., and Kobayashi, K.
(2019). Transcriptomic analysis reveals differences in the regulation ofamino acid metabolism in asexual and sexual planarians. Sci. Rep. 9,6132. doi: 10.1038/s41598-019-42025-z
Shikata, T., Takahashi, F., Nishide, H., Shigenobu, S., Kamei, Y.,
Sakamoto, S., Yuasa, K., Nishiyama, Y., Yamasaki, Y., and Uchiyama,
I. (2019). RNA-Seq analysis reveals genes related to photoreception,
nutrient uptake, and toxicity in a noxious red-tide raphidophyte
Chattonella antiqua. Front. Microbiol. 10, 1764. doi: 10.3389/
fmicb.2019.01764
Shimada, T.L., Yamaguchi, K., Shigenobu, S., Takahashi, H., Murase,M., Fukuyoshi, S., and Hara-Nishimura, I. (2020). Excess sterols disrupt
plant cellular activity by inducing stress-responsive gene expression. J.
Plant Res. 133, 383-392. doi: 10.1007/s10265-020-01181-4
Sugime, Y., Oguchi, K., Gotoh, H., Hayashi, Y., Matsunami, M.,
Shigenobu, S., Koshikawa, S., and Miura, T. (2019). Termite soldier
mandibles are elongated by dachshund under hormonal and hox gene
controls. Development 146, dev171942. doi: 10.1242/dev.171942
Uchi, N., Fukudome, M., Nozaki, N., Suzuki, M., Osuki, K.-I.,
Shigenobu, S., and Uchiumi, T. (2019). Antimicrobial activities
of cysteine-rich peptides specific to bacteriocytes of the pea aphid
Acyrthosiphon pisum. Microbes Environ. 34, 155-160. doi: 10.1264/
jsme2.me18148
Yaguchi, H., Suzuki, R., Matsunami, M., Shigenobu, S., and Maekawa,
K. (2019). Transcriptomic changes during caste development throughsocial interactions in the termite Zootermopsis nevadensis. Ecol. Evol.
9, 3446-3456. doi: 10.1002/ece3.4976
Yamashita, S., Kataoka, K., Yamamoto, H., Kato, T., Hara, S.,
Yamaguchi, K., Renard-Guillet, C., Katou, Y., Shirahige, K., Ochi, H.,
Ogino, H., Uchida, T., Inui, M., Takada, S., Shigenobu, S., and Asahara,
H. (2019). Comparative analysis demonstrates cell type-specificconservation of SOX9 targets between mouse and chicken. Sci. Rep. 9,12560. doi: 10.1038/s41598-019-48979-4〔Original paper (E-publication ahead of print)〕

Y., Yoshida, R., Hirano, T., Enomoto, Y., Li, B., Asada, M.,• Shikanai,
Yamagami, M., Yamaguchi, K., Shigenobu, S., Tabata, R., Sawa, S.,

Okada, H., Ohya, Y., Kamiya, T., and Fujiwara, T. Callose synthesis
suppresses cell death induced by low-calcium conditions in leaves.
Plant Physiol. 2020 Feb 5 doi: 10.1104/pp.19.00784

83

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
NAKAGAWA, Mami

The Spectrography and Bioimaging Facility assists both
The Spectrography and Bioimaging Facility assists bothcollaborative and core research by managing and maintaining research tools that use Light. The facility, under theguidance of Dr. Kamei, also provides technical support
through the management of technical staff assisting in theadvancement of collaborative and core research projects,
as well as academic support to researchers (please refer tothe Collaborative Research Group Research Enhancement
Strategy Office section for more information). Among theequipment available are advanced biological microscopes,
and the Okazaki Large Spectrograph for photobiology. The
Okazaki Large Spectrograph is the world’s largest wide
spectrum exposure mechanism, and is capable of producing a range of wavelengths from 250 nm (ultraviolet) to
1,000 nm (infrared) along its 10 meter focal curve, thus
allowing exposure to strong monochromatic light. The facility’s microscopes, which include cutting edge devices suchas confocal and multi-photon excitation microscopes, are an
indispensable part of core and collaborative projects conducted by both internal and external researchers.

Standard Instruments:
Okazaki Large Spectrograph (OLS)

The spectrograph runs on a 30 kW Xenon arc lamp andprojects a wavelength spectrum ranging from 250 nm (ultraviolet) to 1,000 nm (infrared) onto its 10 m focal curve withan intensity of monochromatic light at each wavelength morethan twice as much as that of the corresponding monochromatic component of tropical sunlight at noon (Watanabe etFigure 1. An example of an experiment using the Large Spectrograph. In
this photo, various color rays (monochromatic light from right side andreflected by mirrors) are irradiated simultaneously to samples stored incooling chambers.

84al., Photochem. Photobiol. 36, 491-498, 1982). The spectrograph is dedicated to action spectroscopical studies ofvarious light-controlled biological processes.
In addition to the other action spectroscopical studies concerning various regulatory and damaging effects of light onliving organisms, research involving biological molecules
and artificial organic molecules have been conducted since
it was set up. The NIBB Collaborative Research Program
for the Use of the OLS supports about 10 projects every
year conducted by both visiting scientists, including foreign
researchers, as well as members of NIBB.

Microscopes

This facility also provides bioimaging machinery, suchas wide-field microscopes (Olympus IX-81 and BX-63),
confocal microscopes (Leica TCS-SP8, Nikon A1R, Nikon
A1Rsi and Yokogawa CSU-X1 with EM-CCD/ CMOS
cameras), multi-photon microscopes (Olympus FV1000-MP,
FV1200-MPs, Leica TCS-SP8 MPs) and other advanced
laser microscopes boasting specialized, cutting edge technology (Light-sheet Microscope and Infrared Laser-Evoked
Gene Operator microscope: IR-LEGO), which can beutilized by researchers within NIBB, as well as collaborative guest researchers. Starting from 2016, we commenced
two new types of Collaborative Research Programs. One
is a new category within the NIBB Collaborative Research
for Integrative Bioimaging program using machinery andbioimage processing/analysis techniques, and the other isthe Advanced Bioimaging Support Program (ABiS) which
operates under the framework of the Grant-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 tissues by illuminating specimensfrom the side with a light sheet (more information is given in
the report submitted by Dr. Shigenori Nonaka’s Laboratory
for Spatiotemporal Regulations ). Dr. Nonaka conducted andsupported about 10 Collaborative Research Program projects
for Integrative Bioimaging. The IR-LEGO, developed byDrs. Shunsuke Yuba and Yasuhiro Kamei at the National
Institute of Advanced Industrial Science and Technology

Figure 2. Microscope images of green algae, Codium fragile. Sample wasstained by hoechst33342. (A) Bright and blue fluorescent marge image.
(B) Blue fluorescent and red fluorescent marge image. Images were taken
by visitor of cooperation program from Osaka City University (Seki,
Soichiro). C. fragile (KU-0654) was provided by KU-MACC, Kobe
University.

(AIST), can induce a target gene of interest by heating asingle target cell in vivo with a high efficiency irradiating
infrared laser (details are provided in the next section). The
IR-LEGO was also used for about 10 Collaborative Research
projects, including applications aimed at animals and plants.

Y., Shinomiya, A., Ota, W., Ikegami, K., Shimmura, T.,• Nakane,
Higashi, S., Kamei, Y., and Yoshimura, T. (2019). Action spectrum for•Workshop, Symposium and Training course

In 2019, we held the 7 th biological image processing
training course in cooperation with Drs. Kagayaki Kato,
Shigenori Nonaka, Takashi Murata and Hiroshi Koyama.
We also have been holding a “Bioimaging Forum” every
year, which discusses bioimaging from various technicalperspectives such as microscopy, new photo-technology, andcomputer science. This year we planned on staging the 14th
NIBB Bioimaging Forum focused on micro CT technology
in February, 2020. However, this forum was postponed until
next year due to the global COVID-19 pandemic.

Research activity by Y. Kamei

Specially Appointed Associate Professor Yasuhiro Kamei isthe principal investigator of Laboratory for Biothermology.
For details, please refer to the laboratory page.

Publication List on Cooperation

•
•
•
•

photoperiodic control of thyroid-stimulating hormone in Japanese quail(Coturnix japonica). PLoS One 14, e022106. doi: 10.1371/journal.
pone.0222106
Shikata, T., Takahashi, F., Nishide, H., Shigenobu, S., Kamei, Y.,
Sakamoto, S., Yuasa, K., Nishiyama, Y., Yamasaki, Y., and Uchiyama,
I. (2019). RNA-Seq analysis reveals genes related to photoreception,
nutrient uptake, and toxicity in a noxious red-tide raphidophyte
Chattonella antiqua. Front. Microbiol. 10, 1764. doi: 10.3389/
fmicb.2019.01764
Shinozuka, T., Takada, R., Yoshida, S. Yonemura, S., and Takada, S.
(2019). Wnt produced by stretched roof-plate cells is required for thepromotion of cell proliferation around the central canal of the spinal
cord. Development 146, dev159343. doi: 10.1242/dev.159343
Tanga, N., Kuboyama, K., Kishimoto, A., Kiyonari, H., Shiraishi, A.,
Suzuki, R., Watanabe, T., Fujikawa, A., and Noda, M. (2019). The PTNPTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway
for oligodendrocyte differentiation: Targeted inactivation of PTPRZ
activity in mice. Glia. 67, 967-984. doi: 10.1002/glia.23583
Tomoi, T., Kawade, K., Kitagawa, M., Sakata, Y., Tsukaya, H. andFujita, T. (2020). Quantitative imaging reveals distinct contributionsof SnRK2 and ABI3 in plasmodesmatal permeability in Physcomitrella
patens. Plant Cell Physiol. 61, 942-956. doi: 10.1093/pcp/pcaa021
Uchimura, T., Hara, S., Yazawa, T., Kamei, Y., and Kitano, T. (2019).
Involvement of heat shock proteins on the transcriptional regulation ofcorticotropin-releasing hormone in medaka. Front. Endocrinol. 10, 529.
doi: 10.3389/fendo.2019.00529〔Original papers〕

S., Asahi, C., Sano, K., Kimura, S., Suzuki, T., Hatano, T.
• Aoyama,
and Arimoto-Kobayashi, A. (2019). Isolation and identification of

•
•
•
•
•
•

•

photoproducts from UVA-irradiated mixture of N-nitrosoproline with
2’-deoxyadenosine. J. Photochem. Photobiol A: Chem. 377, 159-166.
doi: 10.1016/j.jphotochem.2019.03.016
Abe, E., Yasugi, M., Takeuchi, H., Watanabe, E., Kamei, Y., andYamamoto, H. (2019). Development of omnidirectional aerial display
with aerial imaging by retro-reflection (AIRR) for behavioral biology
experiments. Opt. Rev. 26, 221-229. doi: 10.1007/s10043-019-00502-w
Furukawa, F., Hamasaki, S., Hara, S., Uchimura, T., Shiraishi, E.,
Osafune, N., Takagi, H., Yazawa, T., Kamei, Y., and Kitano, T. (2019).
Heat shock factor 1 protects germ cell proliferation during early ovariandifferentiation in medaka. Sci. Rep. 9, 6927. doi: 10.1038/s41598-01943472-4
Harada, Y., Matsuo, M., Kamei, Y., Goto, M., and Fukamachi, S.
(2019). Evolutionary history of the medaka long-wavelength sensitive
genes and effects of artificial regression by gene loss on behaviouralphotosensitivity. Sci. Rep. 9, 2726. doi: 10.1038/s41598-019-39978-6
Hwang, D., Wada, S., Takahashi, A., Urawa, H., Kamei, Y., andNishikawa, S. (2019). Development of a heat-inducible gene expression
system using female gametophytes of Arabidopsis thaliana. Plant Cell
Physiol. 60, 2564-2572. 10.1093/pcp/pcz148
Ikehata, H., Mori, T., Kamei, Y., Douki, T., Cadet, J., and Yamamoto,M. (2020). Wavelength- and tissue-dependent variations in the
mutagenicity of cyclobutane pyrimidine dimers in mouse skin.
Photochem. Photobiol. 96, 94-104. doi: 10.1111/php.13159
Kitadate, Y., Jörg, D.J., Tokue, M., Maruyama, A., Ichikawa, R.,
Tsuchiya, S., Segi-Nishida, E., Nakagawa T., Uchida, A., KimuraYoshida, C., Mizuno, S., Sugiyama, F., Azami, T., Ema, M., Noda,
C., Kobayashi, S., Matsuo, I., Kanai, Y., Nagasawa, T., Sugimoto, Y.,
Takahashi S., *Simons, B.D., and Yoshida, S. (2019). Competition
for mitogens regulates spermatogenic stem cell homeostasis in an
open niche. Cell Stem Cell. Cell Stem Cell 24, 79-92. doi: 10.1016/j.
stem.2018.11.013
Kosugi, M., Ozawa, S., Takahashi, Y., Kamei, Y., Itoh, S., Kudoh, S.,
Kashino, Y., and Koike, H. (2020). Red-shifted chlorophyll a bands
allow uphill energy transfer to photosystem II reaction centers in anaerial green alga, Prasiola crispa, harvested in Antarctica. BBA Bioenergetics 1861, 148139. doi: 10.1016/j.bbabio.2019.148139

85

National Institute for Basic Biology

Research Support

DATA INTEGRATION AND ANALYSIS FACILITY
Assistant Professor:
Technical Staff:
Technical Assistant:

UCHIYAMA, Ikuo
NISHIDE, Hiroyo
NAKAMURA, Takanori
SUGIURA, Hiroki
OKA, Naomi

The Data Integration and Analysis Facility supports
research activities based on large-scale biological data
analysis, such as genomic sequence, expression data, andimaging data analysis. To achieve this, the facility maintains
high-performance computers with large-capacity storage
systems. It accordingly supports the development of data
analysis pipelines and database construction based on thesesystems, and also sets up websites to distribute data worldwide as well as providing basic technical support. In addition
to computational analysis, the Data Integration and Analysis
Facility supports NIBB’s information infrastructure, themaintenance of the institute’s network systems and provides
computer/network consultation for institute members.

Representative Instruments

Our main computer system is the Biological Information
Analysis System (BIAS) (Figure 1), which consists of ahigh-performance cluster system (HPE Apollo r2800, 20
nodes/800 cores, 192 GB memory/node), a shared memory
parallel computer (HPE ProLiant DL560, 72 cores, 3TB
memory; HP ProLiant DL980 G7, 80 cores, 4TB memory),a high-throughput storage system (DDN SFA7700X,
1.52PB+880TB), and a large capacity storage system (DELL
PowerEdge R620, 720TB). All subsystems are connected via
a high-speed InfiniBand network, so that large amounts ofdata can be efficiently processed. Some personal computersand color printers are also available for use. On this system,
we provide various biological databases and data retrieval/analysis programs, and support large-scale data analysis anddatabase construction for institute members and collaborative
researchers. We have provided support in the construction
and maintenance of published databases of various modeland non-model organisms in particular. These include XDB(Xenopus laevis), PHYSCObase (Physcomitrella patens),
iNewt (Pleurodales waltl), The Plant Organelles Database,
MBGD (microbial genomes), and DB-HABs (harmful algal
blooms).
The facility also provides network communication services.
Most of the PCs in each laboratory, as well as all of theabove-mentioned service machines, are connected by alocal area network, which is linked to the high-performance
backbone ORION network connecting the three research
institutes in Okazaki. Many local services, including
sequence analysis, file sharing, and printer services, areprovided through this network. We also maintain a public
World Wide Web server that hosts the NIBB home page(http://www.nibb.ac.jp/en).

86

Figure 1. Biological Information Analysis System

Research activity by I. Uchiyama

Assistant Professor Ikuo Uchiyama is the principal investigator of the Laboratory of Genome Informatics. For moredetails, please refer to the laboratory page.

NIBB BIORESOURCE CENTERHead

FUJIMORI, Toshihiko

To understand the mechanisms of living organisms, studies
focusing on each gene in the design of life (the genome)
are required. The use of model animals and plants, such as
mice, Medaka (Oryzias latipes), zebraﬁsh, Arabidopsis,
Lotus japonicus, and Physcomitrella patens, make it possible
to produce genetically controlled organisms, which havemarkers placed on them, using genetic and cell engineering
technologies. Such marking allows us to conduct detailed
studies of genes and cell functions. Because these modelorganisms mature in a short period of time, changes in cells,organs, and individuals can be thoroughly and efﬁciently
observed. On this front, the NIBB BioResource Center has
the equipment, facilities, and staff to safely, efﬁciently, andappropriately maintain such organisms.

MODEL ANIMAL RESEARCH FACILITY
Associate Professor:
Technical Staff:
Technical Assistant:

WATANABE, Eiji
OHSAWA, Sonoko
NOGUCHI, Yuji
TAKAGI, Yukari
SUGINAGA, Tomomi
FUJIMOTO, Daiji
TAKAHASHI, Nobuaki
MATSUMURA, Kunihiro
KITAZUMI, Noriaki

The worldwide genome project has almost been completed
aThe worldwide genome project has almost been completed
and basic biological research is now in a post-genome erain which researchers focus on investigating the functionsof individual genes. To promote the functional analysis of agene of interest, it is essential to utilize genetically alteredmodel organisms which are generated using genetic engineering technology, and harness techniques such as genedeletion, gene replacement and point mutation.
The NIBB Center for Transgenic Animals and Plants wasestablished in April 1998 to support research using transgenic and gene targeting techniques at NIBB. The NIBB Center
for Transgenic Animals and Plants was integrated into the
NIBB BioResource center in April 2010, and was renamed“The Model Animal Research Facility”; a place where technical and supporting staff develop and promote researchsupporting activities. Furthermore, a state-of-the-art facility
for transgenic animals was also opened at the end of 2003 in
the Yamate area of NIBB.
The activities of the model animal research facility are as
follows:
1. The provision of information, materials, techniques, andanimal housing spaces 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. G enerating 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) was opened in the Myodaiji
area of NIBB. Since then, the production, breeding, analysis,
cryopreservation and storage of genetically manipulatedmouse strains has been conducted there ever since. The newcenter facility building in the Yamate area has strengthened
research activities that require genetically altered organisms.
The building has five floors and a total floor space of 2,500
m2 in which we can generate, breed, store and analyze transgenic, gene targeting, and mutant mice under SPF conditions.
The mouse housing area was constructed based on a barriersystem. This building is also equipped with breeding areas
for small transgenic fish and birds.
From January 1, 2019 to March 31, 2020, 4,348 mice (13transgenic lines and wild-type) were brought into the facility
in the Yamate area, and 61,200 mice (including pups bred in
the facility) were taken out.

Figure 1. Mouse breeding room in the Yamate area

Figure 2. Large sized autoclave in the Yamate area87

National Institute for Basic Biology

Research Support

A number of strains of genetically altered mice from outside
the facility were brought into this area after microbiologicalcleaning using in vitro fertilization-embryo transfer techniques (9 transgenic lines), and stored using cryopreservation(41 transgenic lines). The frozen eggs of 32 mice lines weretaken out of the facility.
Genome editing experiments were performed on five kindsof target genes. We generated gRNAs of the target genes,
which were transferred into fertilized eggs with Cas9 protein,
and were able to introduce intended mutations into the
genome DNA.

be maintained to suit the conditions desired for fish breeding.
A medaka line that allows gene induction by heat treatment,in combination with a cre/loxP system, has been developed using this facility. All the rooms are qualified to meetthe criteria for transgenic animals, allowing researchers togenerate and maintain these important biological tools.
In 2019, 0 zebrafish (0 fertilized eggs) were brought to thefacility nor were there any fertilized eggs or chicken embryos
brought in or taken from the laboratory. The animals housed
within the facility were used for research activities in neurobiology and developmental biology.

III. Research activities

The associate professor of this center, Dr. Eiji Watanabe,
is the principal investigator of the Laboratory of
Neurophysiology, which studies various mechanisms of thevisual system using a psychophysical approach. For moredetails, please refer to the laboratory’s page.

MODEL PLANT RESEARCH FACILITY● Plant Culture Laboratory
Assistant Professor:
Technical Staff:
Technical Assistant: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 provided and set at 42 degrees(suitable for chick embryogenesis). The researchers canmanipulate these embryos under optimal conditions, thus
removing biohazard risks.
For researchers who employ fish as an experimental model,
480 tanks (1 liter) and 450 tanks (3 liter) are available formedaka and zebrafish, respectively. Additionally, water canFigure 4. Liquid nitrogen tank

88

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
YAMAGUCHI, Chinami

The Plant Culture Laboratory manages facilities for thecultivation of plants in general and for the rearing of several
animal species that do not qualify for housing in other facilities.
The Plant Culture Laboratory equips and manages around75 culture boxes or growth chambers, and 13 rooms with
the P1P physical containment level for established andemerging model plants, such as 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 up all year long by more than 70 researchers from both external and internal 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 which are exposed
to precise light concentrations. Autotrophic and heterotrophic
culture devices are also available for researchers using cyanobacteria, algae, and cultured flowering plant cells. Aseptic
experiments can be performed in an aseptic room with clean
benches and a safety cabinet. Several analytical instruments
including two flow cytometry systems and a DUAL-PAM,
for DNA content and chlorophyll fluorescent measuring,
respectively, are also available. In addition, a liquid handlingsystem for fully automated in situ hybridization of sections

of up to 60 glass slides simultaneously is also provided.
A 386-m2 experimental farm next to the institute building of
the Myodaiji area is maintained for Japanese morning glory
and related Ipomoea species, several carnivorous plants, thecastor bean, and other flowering plants that must be cultivated outdoors. Three heated greenhouses (44, 44, and 45m2) are used for the sensitive carnivorous plants and several
aquatic ferns. Four air-conditioned greenhouses (4, 6, 9, and9 m2) with air-conditioning are provided for the cultivationof rice Oryza sp., Lotus japonica and other related legumespecies, as well as Japanese morning glory mutant lines.
Two air-conditioned greenhouses (9 and 18 m2) with air-conditioning meet the P1P physical containment level and areavailable for experiments using transgenic plants. The Plant
Culture Laboratory also maintains a 46 m2 building witha storage area and workspace. Part of this building is used
for rearing of the orchid mantis and the Japanese rhinoceros
beetle and the common grass yellow butterfly.
Between January 2019 and March 2020, 10 culture boxes
were replaced and newer ones were introduced in their place.
The assistant professors of this facility, Dr. A. Hoshino and
Dr. K. Tsugane are the principal investigators of each groupin the Laboratory of Biological Diversity. For more details,
please refer to laboratory’s page.

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 6. Equipment for tissue and cell culture

Figure 5. A plant culture box with high luminance LED lamps

89National Institute for Basic Biology

Research Support

NATIONAL BIORESOURCE PROJECT
The main purpose of the National BioResource Project(NBRP) is to collect, preserve, and provide bioresources(such as experimental animals and plants) that are essential
for life science research. The project also aims to improvethese bioresources by increasing their value by enriching
their genome information and developing key preservation
technologies and other necessary procedures in order to meetcurrent scientific demands. NIBB serves as the core organization center of medaka bioresources and as a sub-center ofmorning glory and zebrafish bioresources.

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

NARUSE, Kiyoshi
KAMEI, Yasuhiro
ANSAI, Satoshi

NBRP Medaka provides three groups of resources 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 enzymes necessary for manipulation and live imaging of the medaka
embryos. Entries for these resources can be found by various
methods such as keyword searches, sequence homologies,
and by opening the expression profile on the following website (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.
As one of our main accomplishments over the last ten years,
we sequenced both ends of 260,000 clones originating from
11 kinds of full-length cDNA libraries and then sequenced
the whole length of 17,000 independent clones from2007 and 2009. We also developed strains in which CRErecombinase can be expressed in any of the cell lineages
using a heat shock promoter. We then started to provide

Medaka resources provided from NBRP medaka

90

strains (TG918, TG921, etc) established using this method.
By 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 preservation method of the testicular tissue.
We are now providing a backup service aimed at preserving
testicular tissues using this method.

II. NBRP Morning Glory (Ipomoea nil)
Project Manager: HOSHINO, Atsushi

The Japanese morning glory (Ipomoea nil) is a traditional
floricultural plant in Japan, that is studied worldwide, especially in the fields of plant physiology and genetics. NIBB
collects, develops and distributes DNA clones, mutant lines
for flower pigmentation, and transgenic lines as a sub-centerof the National BioResource Project (NBRP) Morning Glory,
and collaborates with the core organization center at Kyushu
University. From January 2019 to March 2020, we have collected several mutant lines and DNA clones, and provided 21
mutant lines and 64 DNA clones to both local and international biologists.

Left: The genome database (http://viewer.shigen.info/asagao/) contains
the whole genome sequence, the transcriptome sequences, and the end
sequences of the EST and BAC clones. The genome and mutant databases are linked by the sequence information of the mutations. Right: The
flower phenotype of a mutant line.

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 mainlycollect zebrafish strains expressing fluorescent proteins in
specific cells of the central nervous system and distributethem to researchers worldwide. The zebrafish is an importantand globally used experimental vertebrate model animal witha simple body structure. It can be genetically manipulated,
and its embryos are transparent enough for optical observations. Research using zebrafish for the studies of neuraldevelopment and neural circuit functions are growing rapidly
worldwide, and the importance of strains collected andprovided by NIBB to researchers is growing accordingly.

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

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

Head and Specially Appointed Professor

NARUSE, Kiyoshi

Research Staff:
Technical Assistant:

AKIMOTO-KATO, Ai
TANAKA, Ayako
MATSUBAYASHI, Naomi
MIZOKAMI, Yuko
TSUZUKI, Chizuru

In order to realize the vision of a life science community that can withstand natural disasters and calamities,
the National Institutes for Natural Sciences (NINS) andHokkaido University, Tohoku University, Universityof Tokyo, Nagoya University, Kyoto University, Osaka
University, and Kyushu University finalized an agreement onJune 1st 2012 to launch a system to ‘back up’ the biologicalresources essential to the work being done at universities andresearch 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 have 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 evenvery large scale quakes which are equipped with emergency backup power generators,⃝ c ryopreservation facilities equipped with automatic
liquid nitrogen feeding systems,⃝ deep freezers, and refrigerated storage (mainly for seedstocks),as well as all manner of automated laboratory equipment,
cell culture tools, and the latest equipment necessary toback up the genetic resources in a collaborative manner. The
specific preservation methods used are the freezing of animal
sperm and eggs, cultured plant and animal cells, proteins andgene libraries. Plant seeds are frozen or refrigerated.
When university satellite hubs receive preservation requests
involving biological resources from researchers, they reportto the Managing Project Committee of IBBP (which iscomprised of NIBB faculty members and other satellite institutes), where the relevance of the request is reviewed. If therequest is approved, the biological resources that are to bepreserved will be sent to the IBBP Center by the requesting
researcher, where they will be frozen (or refrigerated), andtheir particulars registered into a database. In the event of adisaster leading to the loss of a researcher’s own biologicalresources, preserved samples will be promptly returned to theresearcher so they 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 IBBP Center’s stateof the art facilities. As a result of this, Japan’s research infrastructure has been significantly strengthened.

I. Current status of back up available forbiological resources

Figure 1. IBBP Center.

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

In 2019, the IBBP Center stored 5,818 384-well and 112
96-well plates consisting of 2,244,864 clones as cDNA/
BAC clones, 16,973 tubes for animal cells, plant andFigure 3. Cryo tube with 2D barcode. Each sample is printed with aunique barcode and is managed using a database.

91

National Institute for Basic Biology

Research Support

animal samples, proteins, genes and microorganisms, 6,174
133mm-straw tubes for sperm and 728 seed samples. In total
2,110,464 samples were stored.

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 biological resources for which cryopreservation methods are not well established. In order 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 focuses on two goals: 1) The 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
for biological resources. In 2019, we have conducted 12
collaborative research projects aimed at achieving thesegoals. We also worked to establish a research center forcryo-biological study thorough this Collaborative Research
Project. Accordingly, we organized the Cryopreservation
Conference 2019 on November 18-19, 2019 at the Tsukuba
Center for Institutes, Tsukuba, Ibaraki, Japan. We had over
100 participants from several fields covering physics, chemistry, biology, and technology. We invited Prof. Tanaka at
University of Tokyo as a plenary lecturer. Prof. Tanaka gave
us a talk on the theoretical and structural view of water vitrification and crystallization processes.

Figure 4. Group photo of Cryopreservation conference 2019

Publication List on Cooperation:〔Original paper〕

T., and Nakagawa, Y. (2020). Genome editing of rodents by• Kaneko,electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stageembryos. Cryobiology 92, 231-234. doi: 10.1016/j.cryobiol.2020.01.016

92

CENTER FOR THE DEVELOPMENT OF NEW MODEL ORGANISMS

Head

UENO, Naoto

Professor

SHIGENOBU, Shuji

Specially Appointed Associate Professor(Cross-appointment with Hiroshima University)

SUZUKI, Ken-ichi

Technical Assistant:

TAKAYAMA, Ayuko

Since the beginning of life on Earth, living organisms have
evolved to adapt to various environments, and have spawned
a wide variety of species. Modern biological research has
put an emphasis on elucidating the basic principles common
to many species, and has progressed thanks to the intensive
analysis of a limited number of species known as modelorganisms, which are easy to handle in a laboratory environment. However, this development has most likely leftmany interesting biological phenomena unexamined as their
distinctive characteristics are observed only in a particulargroup of species. How we overcome this is an important
challenge for biology hereafter.
To solve these problems, we must choose a species mostsuitable to analyze the phenomenon to be researched, andthen establish it as a new model organism by developing
methods using procedures that are necessary for modern
biological analyses. These include stable raising, breedingand experimental manipulation techniques, analyses of the
genome information and gene expression, and gene manipulation techniques using gene insertion and genome editing
techniques.
To this end, The Center for the Development of New Model
Organisms was established in 2013. Through its activities,
organisms that have been out of reach of scientific researchwere designated as new model organisms. For example,
we study aphids and sea anemones to understand the symbioses, and rhinoceros beetles to get insights into the sexual
dimorphism. We are refining various techniques for studying
targeted new model organisms ranging from genome analysis
to genetic engineering to build seamless workflows that will
be shared among the research community.

Research activity by S. Shigenobu

Professor Shuji Shigenobu is the principal investigator of
the Laboratory of Evolutionary Genomics. Refer to the laboratory page for details.

Research activity by K. Suzuki

Specially Appointed Associate Professor(Cross-appointment with Hiroshima University):
SUZUKI, Ken-ichi

Figure 1. tyrosinase crispant in P. waltl. A knock-out founder of tyrosinase, a melanin synthesis enzyme, and wild newt (left and right, respectively). tyr crispant shows full albinism.

Two technical innovations have recently changed biology:
Next generation sequencing (NGS) and Genome editing.
NGS reveals whole genome sequences and gene expression
profiles from various organisms. Genome editing accelerates
the functional characterization of numerous genes involvedin the phenomenon of life. Accordingly, we are now basicallyable to choose any organism which we are interested in, andcarry out functional analyses by using these tools.

I. Development of genome editing techniquesfor various organisms

Recent advances in the CRISPR-Cas system now allow
for reverse genetics in various organisms. However, it has
been hampered by the lack of a simple and efficient method
for gene modification in most of the non-model organisms. To overcome this issue, we developed a highly-efficient workflow for gene knockout in the founder using thisCIRSPR-Cas. We call the virtually knockout founders “crispants”. Crispant assay provides us with a practical and rapid
tool for functional screening of numerous genes of interest
beyond the post-genome era (Figure 1).
Despite the practical utility of the knockout technique, there
is still room for improvement in the integration of exogenous
DNA into a target chromosomal site (i.e. knock-in), whichis still somewhat limited in various organisms. Therefore,
we are currently developing more efficient and practicalknock-in techniques than conventional ones.

II. 
F inding new model organisms anddeciphering organ regeneration

One of our missions is to discover unique organisms anddevelop them as new model organisms for basic biology.
A recent example of this is our recent establishment of thenewt Pleurodeles waltl as an experimental model animal forregenerative biology using NGS and genome editing tech-

93

National Institute for Basic Biology

Research Supportniques. P. waltl possesses several excellent characteristics
as a model animal: easy breeding, short sexual maturation
period, remarkable regenerative capacity and comparatively
high efficiency of genome editing (Figure 2). We are currently researching the molecular basis of organ regeneration
using this newt. In addition, we widely support researchers
who attempt to develop new model organisms contributing to
the up-coming biology.

Figure 2. A limb-specific enhancer (ZRS/MFCS1) of sonic hedgehog
crispant in P. waltl. Phenotypes of limb regeneration in wild and ZRS/MFCS1 crispant (left and right, respectively). Unlike in normal limb
regeneration in the wild type, severe reduction of digit formation wasseen in ZRS/MFCS1 crispant.

Publication List:〔Original papers〕

M., Suzuki, M., Haramoto, Y., Fukui, A., Inoue, T.,• Matsunami,
Yamaguchi, K., Uchiyama, I., Mori, K., Tashiro, K., Ito, Y., Takeuchi,•

94T., Suzuki, K.-I.T., Agata, K., Shigenobu, S., and Hayashi, T. (2019).
A comprehensive reference transcriptome resource for the Iberianribbed newt Pleurodeles waltl, an emerging model for developmental
and regeneration biology. DNA Res. 26, 217-229. doi: 10.1093/dnares/
dsz003
Yoshinouchi, Y., Shimizu, S., Lee, J.S., Hirano, M., Suzuki, K., Kim,
E.Y., and Iwata, H. (2019). In vitro assessment of effects of persistentorganic pollutants on the transactivation of estrogen receptor α and β(ERα and ERβ) from the Baikal seal (Pusa sibirica). Ecotox. Environ.
Safe. 181, 463-471. doi: 10.1016/j.ecoenv.2019.06.033

Nuclides Stateliquidliquid2. Changes regarding usable nuclides at Myodaiji area
Since there has been no practical use for gamma ray
nuclides at the Myodaiji area for several years, we havedecided to discontinue their use. Hence, the revised list of
usable nuclides at this facility was approved by the Nuclear
Regulation Authority in November 2019 (Table 1). We
then subsequently stopped the operation of the gamma ray
monitors and measuring instruments.
The number of registrants and the number of users at ourfacility from January 2019 to March 2020 are shown in Table
2. Users and visitors counted by the access control system inthe controlled areas numbered 1,445 during this period. The
numbers for each area are shown in Table 3. The annual changes
concerning registrants and the number of totals per fiscal year
are shown in Figure 1. The balance of radioisotopes received
and used at the CRF is shown in Table 4. The figures for training
courses on radioisotope handling are provided in Table 5.

1

4.5

500

15

500

5

4.5

liquid

all

15

all

liquidliquidDeleted

15

all

5

500

15

150

all

Deleted

2

Deleted

500

150

50

2

200

Table 1. Changes of usable nuclides at Myodaiji area
350

14000
Days of facility use

300

12000

Registants

2019

2017

2015

2013

Fiscal year

2011

0

2009

Days of facility use

2000

2007

50

2005

4000

2003

100

2001

6000

1999

150

1997

8000

1995

200

1993

10000

1991

250

1987

1. Revision of CRF’s local rules
We revised the rules as they pertain to our local facilities(referred to as “Radiation Damage Protection Rules”) in the
Myodaiji and Yamate areas to conform with amendments tonational laws concerning radioisotope protection. Major revisions that were included are as follows:
1) Preparation of the subordinate rules
We prepared the following manuals of which are deemedsubordinate to the local rules mentioned above: a) users’ guideb) management and inspection manual c) emergency responsemanual d) emergency information provision manual2) Specification of the manager and responsible party foreach action described in the rules3) Revision of the radiation safety education and training
protocols
We decided the necessary hours, standards for partial omissions, and protocols to change the necessary hours for radiation safety education and training.

day

20

all

1985

The Center for Radioisotope Facilities (CRF) is responsible
for monitoring the purchase of radioisotopes from the Japan
Radioisotope Association (JRIA) and the transfer of radioisotope waste to that organization. In keeping with this, the
CRF’s technical and support staff maintain controlled areas
in compliance with the relevant laws.
The following is an outline of the notable activities thatCRF conducted in 2019.

3 months

20

liquid1983

Technical Assistant:

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

1981

Technical Staff:

KODAMA, Ryuji

year

all
all

liquid1979

HASEBE, Mitsuyasu

Associate Professor

Registrants

Head

Maximum permissible quantity

Chemical
form

1989

CENTER FOR RADIOISOTOPE FACILITIES

0

Figure 1. Changes of registrants and days of facility use per fiscal year

Myodaiji Area

Registrants
Users

37
17

Yamate Area

30
21

Table 2. Numbers of registrants and users at the Myodaiji and Yamate
areas from 2019 January to 2020 March

Myodaiji Area

Users
Visitors
Total

641
161
802

Yamate Area

Total

460
183
643

1,101
344
1,445

Table 3. Users and visitors who entered each controlled area from 2019
January to 2020 March
125

I

Received

S

Received

125
35
35
32
32
14

I

S
P
P

C

14
3

C

H

3

H

Myodaiji Area

Used
Used

0

Yamate Area

0
0
0

0
0
0
0

Total

0
0
0
0

Received

27,75020,35048,100Received

0

0

0

Used
Used

Received
Used

22,940

0

9,250

0

29,60000052,54009,250
0Table 4. Balance of radioisotopes received and used (kBq) at each controlled area from 2019 January to 2020 Marchtraining course

place

Users training course
Users training course

Myodaiji
YamateIntroductory course for beginners
Introductory course for beginners
Introductory course for experts
Introductory course for experts

Myodaiji
YamateMyodaiji
Yamatenumbers of participant

0
0
2
1

33
19

*including English course

Table 5. Training courses for radiation workers in from 2019 January to2020 March95

National Institute for Basic Biology

Research Support

RESEARCH ENHANCEMENT STRATEGY OFFICE

Director

UENO, Naoto

Deputy Director

HASEBE, Mitsuyasu

Vice-Director

MANO, Shoji

In 2013, NINS started a research enhancement project toencourage cutting-edge academic research in the field of
natural sciences through international joint research, andto contribute to the enhancement of research capabilities ofuniversities etc. in Japan using the world’s most advanced
research environment for joint utilization and research. It
would do this by emphasizing the following: 1) Support forthe promotion of international advanced research, 2) Support
for the promotion of joint utilization and joint research inJapan, 3) Dissemination of information and enhancement ofpublic relations in Japan and abroad, 4) Support for researchers and especially young, female or foreign researchers. Inline with this strategy, the Research Enhancment Strategy
Office was founded to achieve the above mentioned four
goals at NIBB.
The Research Enhancement Strategy Office, which consistsof six groups with distinct missions, is aimed at supporting researchers so that NIBB can improve its ability as acollaborative research institution and to provide comprehensive support within the organization. It was created in2013 via the a restructuring of the former Strategic Planning
Department, the Office of Public Relations, and the Officeof International Cooperation which existed from 2005.
The Office’s activities are mainly carried out by URAs
(University Research Administrators) in accordance withrecommendations given by a group adviser chosen from theranks of NIBB’s professors and in close collaboration with
the NINS’s Research Enhancement Promotion Headquarters.

EVALUATION AND INFORMATION GROUP
Associate Professor:
Group Adviser:

KODAMA, Ryuji
KAWAGUCHI, Masayoshi

This group is tasked with assisting the Director Generalin the preparation of NIBB’s evaluation procedure and inplanning long-range strategies for the institute.

The main activities of the group

1) Management of external evaluation processes
Every year, NIBB asks non-NIBB members of the Advisory
Committee for Programming and Management to submit
a questionnaire concerning research achievements, collaborative research, as well as other activities conducted at theinstitute. Furthermore, several of these members, and a few
of other leading scientists in basic biology, are then invited toparticipate in an evaluation session pertaining to these activities and to propose future directions for the institute. The
record of this meeting is published annually.

962) Editing of the Annual Report (in collaboration with the
Public Relations Group)This group edits the annual report (this publication) whichsummarizes the annual activities of all of the institute’s laboratories and facilities. The annual report is also used to publicize the institute and also for evaluation purposes.
3) Assistance in making the plans and reports of the instituteThis group assists in the preparation of NIBB’s Mediumterm Goals and Plans which in themselves span a period of
six years. We are also tasked with formulating Annual Plans
in order to set yearly benchmarks in regards to the progressof the above mentioned Goals and Plans. The department
also assists in preparing Business and Performance Reports
for external evaluation on whether we are meeting the goals
set both annually and over the medium-term.

PUBLIC RELATIONS GROUP
Specially Appointed Assistant Professor (URA):
KURATA, Tomoko
Technical Assistant:
BAN, Misato
HOSHINO, Maki
UCHIMURA, Ai
OTA, Kyoko
Group Adviser:
FUJIMORI, ToshihikoThis group actively facilitates communication between
NIBB and the public, school teachers, and the international
community of scientific researchers in order to publicize theactivities of NIBB to the widest audience possible.

The group’s main activities in 20191) Press releases
The group issues news items concerning 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) E diting of publications, production of posters andleaflets
Publication of “NIBB News” (Intra-institutional newsletter,in Japanese). Publication of brochures introducing NIBB.
Design and distribution of posters of NIBB events.
4) Production of Videos
Creation of videos introducing NIBB and interviews withresearchers that are published on the web.
5) Organization of scientific outreach programs
Organization of the Summer Program for university
students, and coordination of special classes for middle school
students. Organization of the 27th NINS symposium for the
public. Coordination of NIBB open house 2019 (p. 111).

INTERNATIONAL COOPERATION GROUP
Specially Appointed Assistant Professor (URA):TATEMATSU, Kiyoshi
Technical Assistant:
TAKAHASHI, Ritsue
COWAN, GlenGroup Advisor:
UENO, Naoto
Two of NIBB’s missions is to continually explore the leading-edge of biology and to form research communities thatlink Japan to the world. To achieve this, it conducts scientificmeetings such as the “NIBB Conference”, as well as educational programs such as the “NIBB International Practical
Course”.
Furthermore, NIBB is coordinating closely with theEuropean Molecular Biology Laboratory (EMBL, European
member states), the Temasek Life Sciences Laboratory (TLL,
Singapore) and Princeton University (USA), and has also
signed a new cooperative agreement with the Center for
Organismal Studies (COS) Heidelberg (Germany) in 2019.
Working on the basis of cooperative agreements signed
between NIBB and the above listed organizations, NIBB
coordinates the exchange of personnel and techniques andthe joint staging of scientific meetings. NIBB also conducts
the “NIBB International Collaborative Research Initiative”
to promote high-level international collaborations between
faculty members and researchers from around the world.
It also invites researchers, who are at forefront in research
fields, from abroad to promote academic exchange withNIBB members in addition to starting new international collaborations.
This group supports and coordinates NIBB’s activities
related to international research collaborations by organizing the various above-mentioned international scientificmeetings and technical courses, coordinating the dispatchof researchers to international conferences, and supporting researchers visiting NIBB from the institutes mentioned above. This group also supports NIBB internshipstudents visiting from foreign countries, and the dispatchof SOKENDAI graduate students to international conferences 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 engaged in by this groupfrom Jan. 2019 to March 2020

1) C oordination of international conferences and theInternational Practical Course
This group coordinated and supported the following international events organized by NIBB:・T he 66th NIBB Conference/ABiS International
Symposium “Cutting Edge Techniques of Bioimaging”
Okazaki, Japan, February 17-18, 2019 (p. 100)・T he 67th NIBB Conference “Quest for Orthologs”
Okazaki, Japan, July 31 – August 2, 2019 (p. 101)

・T he 2nd NIBB-Princeton Symposium “Imaging andQuantitative Biology” Okazaki, Japan, October 28–30,
2019 (p. 102)2) Support of visiting researchers to NIBBThis group supported visits of foreign researchers related to
the following events (p. 103):
・O ne professor and one manager of Corporate
Engagement Team coming from Princeton University(January and April, 2019)
・Managing Director and two junior professors comingfrom COS Heidelberg (May and September, 2019)
・Director coming from EMBL-EBI (August, 2019)3) Support of education-related programs
This group supported graduate students participating in thefollowing NIBB student exchange and internship programs:
・NIBB Internship Program 2019 (p. 110)

COLLABORATIVE RESEARCH GROUP
Professor:
SHIGENOBU, Shuji
Specially Appointed Associate Professor (URA):
KAMEI, Yasuhiro
Technical Assistant:
ICHIKAWA, Mariko
ICHIKAWA, ChiakiGroup Adviser:
YOSHIDA, Shosei
Specially appointed associate professors within this groupbelong to the NIBB core research facilities, and are responsible for managing collaborative research projects and practical courses that take advantage of their expertise in their
particular field. It is through this that we are able to further
promote the exchange of information and greater collaboration among scientific communities, as well as support thedevelopment of new equipment and methods.
In 2019, this group hosted a total of 115 collaborative
projects. The success of these collaborations is best illustrated by the 36 research papers that were jointly publishedduring this year thanks to a number of these projects. Oneof the more remarkable achievements produced by thisgroup was a transcriptome analysis of a harmful red tide
alga Chattonella antiqua. This analysis was conducted by
Dr. Tomoyuki Shikata (FEIS, FRA) and his colleagues incollaboration with the members of the NIBB core researchfacilities, Ikuo Uchiyama, Shuji Shigenobu and Yasuhiro
Kamei. In this project, the abovementioned researchers conducted RNA-seq analysis of C. antiqua and identified several
genes with functions possibly related to the generation anddynamics of red tide, such as photosynthesis, photoreception,
nutrient uptake and superoxide production. The identified
genes were further characterized by comparative analysis
with orthologs in other harmful and non-harmful algae. The
obtained data and analysis results are open to the public

97

National Institute for Basic Biology

Research Support

through the website HAB-DB (http://hab.nibb.ac.jp/) in orderto provide a basis for the understanding of the mechanism
behind the development of red tide.
T., Takahashi, F., Nishide, H., Shigenobu, S., Kamei, Y.,• Shikata,
Sakamoto, S., Yuasa, K., Nishiyama, Y., Yamasaki, Y., and Uchiyama,

I. (2019). RNA-Seq analysis reveals genes related to photoreception,
nutrient uptake, and toxicity in a noxious red-tide raphidophyte
Chattonella antiqua. Front. Microbiol. 10, 1764. doi: 10.3389/
fmicb.2019.01764

YOUNG RESEARCHER SUPPORT GROUP
Assistant Professor:
Group Adviser:

KOMINE, Yuriko
FUJIMORI, Toshihiko

As a leading international institute in the field of basic
biology, one of NIBB’s more important missions is thecultivation of future researchers. As such, NIBB provides
PhD courses for graduate students while acting as the
Department of Basic Biology in the School of Life Sciences
at SOKENDAI (The Graduate University for Advanced
Studies). To complement this, The Young Researcher
Support Group is aimed at supporting young researchers
at NIBB (including graduate students such as SOKENDAIand special research students from other universities) to help
make their study and research experiences at NIBB beneficial and valuable for their career.

The main activities of the group1) Coordination and management of courses provided by
the Department of Basic Biology (lectures, researchpresentations, etc.), in collaboration with all faculty
members in NIBB.
2) Support in organizing NIBB’s programs related to PhD
courses, such as the Open Campus Day for prospective
students.
3) C ooperation with the interdepartmental programs
offered by the SOKENDAI headquarters, including theFreshman Course and the Life Science Retreat.
4) Gathering and providing information that is useful toboth students 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 advance their careers while enduring the stressesof life events, such as having children, this group manages
the research assistant system for those in need, and promotes
networking among female researchers.
In 2018, a multi-purpose room within the NIBB building
was refurbished to suit the needs of NIBB members who
may occasionally want to bring their children to work. The

98

room has been particularly useful for those wishing to holdmeetings with small children by their side or to wait for one’s
partner/spouse while s/he is in the lab in cases where alternative child care service cannot be arranged.
In 2019, the room had 18 registered users and also accommodated visitors attending meetings held in Okazaki. To
create a safer environment, a surveillance camera and an air
purifier were also installed.

TECHNICAL DIVISIONHeadMIWA, Tomoki

Common Facility Group

Research Support Group● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki
Subunit Chief:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
NISHIDE, Hiroyo
Technical Staff:
NAKAMURA, Takanori
TANIGUCHI-SAIDA, Misako
NISHIMOTO, Yuki
SUGIURA, Hiroki
echnical Assistant:
ICHIKAWA, Chiaki
ICHIKAWA, Mariko
OKA, Naomi
KOTANI, Keiko
SHIBATA, Emiko● Cell Biology
Subunit Chief:
Technical Staff:● NIBB Bioresource Center
Unit Chief:
OHSAWA, Sonoko
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
SUGINAGA, Tomomi
YAMAGUCHI, Chinami● Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
KABEYA, Yukiko

Chief:

MORI, Tomoko● 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.

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

TAKEUCHI, Yasushi● Environmental Biology
Technical Staff:
NODA, Chiyo

Reception
Secretary:

TSUZUKI, Shihoko
KATAOKA, Yukari
UNO, Satoko
KOTANI, Keiko

Technical staff members continually participate in selfimprovement and educational activities through the Division
to increase their capabilities and expertise in technical areas.
Technical staff members are attached to specific common
research facilities and research divisions so that they may
contribute their special biological and related knowledge andtechniques to various research activities.
The Technical Division hosts an annual meeting for technical engineers who work in various biological fields atuniversities and research institutes throughout Japan. At thismeeting, the participants present activities they have recently
participated in and discuss technical problems. The proceedings are published after each meeting.

99

The 66th NIBB Conference / ABiS International Symposium"Cutting Edge Techniques of Bioimaging"
Organizers: Naoto Ueno (NIBB), Toshihiko Fujimori (NIBB), Junichi Nabekura (NIPS),
Masanobu Kano (NIPS / The University of Tokyo)
February 17 (Sun)–18 (Mon), 2019
Imaging technology has been widely applied to areas
ranging from molecules, cells, tissues and all the way up to
individual organisms. It has consequently become an essential analytical tool in the field of life science research. The
development of new probes, microscopes and quantitative
analytical methods, as well as the ability of being able toextract biological information from images, has progressed
at a remarkable pace. As a result, we can now observe both
multiple objects and the spatial temporal dynamics of thesubjects simultaneously at both high speed and resolution.
NIBB has taken on a major role within the field of bioimaging in Japan as evidenced by its involvement in the advancement of various activities. These roles have involved thedevelopment of new observation technology and bioimage
analysis methods and their circulation through bioimaging
training courses, conducting the NIBB collaborative research
projects for integrative bioimaging, and taking on the roleof core institute in coordinating the Advanced Bioimaging
Support (ABiS) activities in conjunction with NIPS. Against
this backdrop, the 66th NIBB Conference, entitled "Cutting
Edge Techniques of Bioimaging", was held in cooperation
with ABiS at the Okazaki Conference Center from February
17th to 18th.

This conference aimed to deepen discussion concerning
cutting edge bioimaging as well as sharing information
regarding this area. In particular, there was a focus on superhigh resolution and live cell imaging. Events included:●L ectures conducted by both domestic and international
researchers engaged in cutting edge projects in the abovementioned fields● Imaging related poster presentations●S hort talks about various posters chosen from those on
display
Additionally, much passionate discussion was also
exchanged prior to the lecture’s Q and A and poster sessions.
A total of 102 participants were able to obtain information on results and analytical techniques concerning the useof advanced bioimaging equipment. They were also able
to think more deeply about the usage of advanced imaging
techniques in their research. Furthermore, it was also a good
opportunity to rethink cooperation in the field of bioimaging
with other institutes and researchers.
Due to problems such as budgetary constraints, it is
becoming progressively more difficult to introduce advanced
equipment and operate it under suitable conditions. Network
support activities such as ABiS, which was founded three
years ago and has provided advanced bioimaging technologies, are necessary to support bioimaging research in Japan.
In closing, we would like to express our gratitude to themembers of NIBB and NIPS for their efforts in hosting thisconference.
Shoji Mano (NIBB /ABiS Administration Office)Speakers
Chew, Teng-Leong (HHMI Janelia, USA), McDole, Kate (HHMI Janelia, USA)
Higashiyama, Tetsuya (Nagoya University, Japan), Okada, Yasushi (The University of Tokyo / RIKEN, Japan), Tamada, Yosuke (NIBB,
Japan), Wake, Hiroaki (Kobe University, Japan), Watanabe, Naoki (Kyoto University, Japan)

100The 67th NIBB Conference / The 6th Quest for Orthologs Meeting"Quest for Orthologs"
Organizers: Ikuo Uchiyama (NIBB), Christophe Dessimoz (UNIL), Toni Gabaldón
Estevan (CRG), Erik Sonnhammer (SU), Paul D. Thomas (USC), Wataru Iwasaki(UTokyo), Shigehiro Kuraku (RIKEN BDR), Shuji Shigenobu (NIBB)
July 31 (Wed)–August 2 (Fri), 2019
The 67th NIBB conference “Quest for Orthologs” was heldwith a focus on orthology analysis as a fundamental technology in the field of comparative genomics; something that is
becoming more important as genomic technology is applied
to a broader range of organisms. This meeting originatedfrom the international consortium of the same name “Quest
for Orthologs,” which was established in 2009 by developersof orthology inference methods. Since then, the consortium
has held a meeting biennially to discuss common subjects
in the development of orthology inference methods such as
benchmark tests for evaluating methods, a reference sequence
set commonly used as input, and a common data format torepresent orthology relationships, in addition to presenting thelatest results from this field. This meeting, which marked thetenth anniversary of the abovementioned events being held,
was hosted in Japan. This was the first time that it had been
held outside of Europe and United States, and about half of theparticipants were from local institutions. As a part of the NIBB
conference series, application of orthology inference methods
to gene function predictions and/or evolutionary studies withapplication to biological studies using various organisms werealso featured during this meeting.
The conference consisted of four sessions: the developmentof orthology inference methods and databases, application tofunction prediction, their application to evolutionary analysis,
and new challenges in orthology inference issues. There were
22 invited speakers including 4 keynote lectures, as well as
8 oral presentations and 14 poster presentations by young
researchers. During the round table discussion session held at
the end of the second day, lively discussions were conducted
between developers and users of orthology inference methods.
While this meeting was focused on the specific field of
orthology inference methods, it covered a broad range of
relevant studies from the basics to specific applications, andprovided a good opportunity to review the current and future
status of this field as a basis of various genomic studies. A
large portion of the event’s budget was used to financially
support the travel expenses of students and young researchers, who themselves were active contributors to the discussions held during the conference.
We would like to thank all the presenters and participants
for helping to stage this conference, the Japanese Society of
Bioinformatics and Daiko Foundation for financial support,
and the International Cooperation Group of the Research
Enhancement Office for organizing the conference.
Ikuo Uchiyama (Laboratory of Genome Informatics)

Speakers
Altenhoff, Adrian (ETH Zurich / SIB Swiss Institute of Bioinformatics, Switzerland), Capella-Gutierrez, Salvador (Barcelona
Supercomputing Center, Spain), Dessimoz, Christophe (University of Lausanne, Switzerland), Durand, Dannie (Carnegie Mellon
University, USA), Ebersberger, Ingo (Goethe University Frankfurt, Germany), Hansen, Madison (American Museum of Natural History,USA), Jones, Tamsin (EMBL-EBI, UK), Kelly, Steven (University of Oxford, UK), Kriventseva, Evgenia (University of Geneva,
Switzerland), Lecompte, Odile (ICUBE/University of Strasbourg, France), Linard, Benjamin (LIRMM, France), Mendes, de Farias
Tarcisio (University of Lausanne, Switzerland), Moi, David (UNIL, Switzerland), Nevers, Yannis (ICube laboratory UMR7357, France),
Ouangraoua, Aida (University of Sherbrooke, Canada), Patricio, Mateus (EMBL-EBI, UK), Pearson, William (University of Virginia,
USA), Sonnhammer, Erik (Stockholm University, Sweden), Szklarczyk, Damian (SIB Swiss Bioinformatics Institute / University of
Zurich, Switzerland), Thomas, Paul D. (University of Southern California, USA), Toni, Gabaldón (Centre for Genomic Regulation(CRG), Spain), Warwick, Vesztrocy Alex (University College London, UK), Chiba, Hirokazu (Research Organization of Information and
Systems, Japan), Hara, Yuichiro (Nagoya University, Japan), Horiike, Tokumasa (Shizuoka University, Japan), Iwasaki, Wataru (The
University of Tokyo, Japan), Kanehisa, Minoru (Kyoto University, Japan), Kuraku, Shigehiro (RIKEN BDR, Japan), Shigenobu, Shuji(National Institute for Basic Biology, Japan), Uchiyama, Ikuo (National Institute for Basic Biology, Japan)

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The 2nd NIBB–Princeton Symposium"Imaging and Quantitative Biology"
Organizers: Michael S. Levine (Princeton Univ.), Danelle Devenport (Princeton Univ.),
Kazuhiro Aoki (NIBB), Naoto Ueno (NIBB)
October 28 (Mon)–30 (Wed), 2019
The second NIBB-Princeton Joint Symposium, entitled'Imaging and Quantitative Biology', was held on October
28-30th, 2019 at the Okazaki Conference Center. The
National Institutes for Natural Sciences (NINS) and
Princeton University have been conducting academic
exchange and collaborative research in the life sciences
based on an academic cooperation agreement that had beensigned by these two parties in 2010.
This NIBB symposium was jointly organized not onlywith Princeton University，but also with the Division of
Quantitative and Imaging Biology (QIB) at the International
Research Collaboration Center (IRCC), NINS, the National
Institute for Physiological Science (NIPS), the Institute for
Molecular Science (IMS), and the Exploratory ResearchCenter on Life and Living Systems (ExCELLS), and was
supported by the Advanced Bioimaging Platform (ABiS).
Although the scientific sessions covered a rather broad
spectrum of biological phenomena such as molecular
dynamics, cell biology, embryogenesis, symbiosis between
organisms and viral infection, and information processing
within the brain, the key concept ‘Imaging and Quantitative
Biology’ was underlying all the presentations. The latest
methodologies such as state of the art imaging technologies,
optogenetics, single cell sequencing, mass spectrometry, liquid-liquid phase separation and machine learning presented

in the symposium were indeed a showcase of the recent technical advances in life sciences.
Eighty-five participants, who were researchers from thevarious institutes and research centers of NINS, as well as
those from other universities enjoyed lively discussions
during poster presentation sessions over the three days ofthis symposium. In particular, four professors from Princeton
University gave insightful comments to the presenters andled the discussions.
In closing, we would like to note that the success of thesymposium is something that proves the significant progressof the collaboration between NINS and Princeton University
up until now. We would also like to express our gratitude
to all participants for taking part in a variety of active discussions, and the IRCC-QIB, the Office of International
Cooperation and the individuals from the organizer’s laboratories for coordinating and cooperating with this symposium.
Kazuhiro Aoki, Naoto Ueno (On behalf of the organizers)

Speakers
Cristea, Ileana (Princeton Univ., USA), Levine, Michael S. (Princeton Univ., USA), Toettcher, Jared E. (Princeton Univ., USA), Yang,
Haw (Princeton Univ., USA)
Aoki, Kazuhiro (NIBB/ExCELLS/IRCC-QIB, Japan), Chikazoe, Junichi (NIPS, Japan), Fujimori, Toshihiko (NIBB, Japan),
Fukaya, Takashi (Univ. Tokyo, Japan), Go, Yasuhiro (NIPS/ExCELLS, Japan), Iino, Ryota (IMS/IRCC-QIB, Japan), Kinoshita, Noriyuki(NIBB, Japan), Kitadate, Yu (NIBB, Japan), Morita, Miyo T. (NIBB, Japan), Nemoto, Tomomi (ExCELLS/ NIPS, Japan), Ohsawa,
Shizue (Nagoya Univ., Japan), Shigenobu, Shuji (NIBB, Japan), Shiina, Nobuyuki (NIBB/ExCELLS, Japan), Soyano, Takashi (NIBB,
Japan), Takada, Shinji (NIBB/ExCELLS, Japan), Takahashi, Shunichi (NIBB, Japan), Uchihashi, Takayuki (ExCELLS/Nagoya Univ.,
Japan),

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Collaboration Programs with
Overseas Institutions
NIBB-Princeton University Collaboration

Professor Michael Levine’s visit to NIBB
Professor Michael Levine, the Director of the Lewis-Sigler
Institute for Integrative Genomics and professor from the
Department of Molecular Biology at Princeton University,
visited NIBB from January 15th to 16th 2019 to deliver aseminar and to meet with NIBB researchers. Prof. Levine’s
visit was supported by NINS’s International Research
Collaboration Center (IRCC) program to promote international
academic exchange between NINS and Princeton University.
Prof. Levine gave a special seminar entitled “Single Cell
Resolution of Animal Development” to NIBB researchers.
In addition, Prof. Levine had discussions with several NIBB
researchers and toured NIBB facilities to observe next-generation sequencers and a light-sheet microscope that has been
newly developed at NIBB.

Mr. Dean Edelman’s visit to NIBB
Mr. Dean Edelman, the manager of the Office of Corporate
Engagement and Foundation Relations at Princeton
University, visited NIBB on April 1st 2019. Mr. Edelman’svisit was supported by the international academic exchange
program between NINS and Princeton University.
Mr. Edelman visited three laboratories affiliated with the
NINS’s Division of Quantitative and Imaging Biology,
International Research Collaboration Center (IRCC-QIB)
which are the laboratories administered by Prof. Ryota
Iino (IMS), Prof. Kazuhiro Aoki (Division of Quantitative
Biology, NIBB) and Prof. Naoto Ueno (Division ofMorphogenesis, NIBB). After brief introductions of theirrespective fields of research, Mr. Edelman discussed futurecollaborative/cooperative projects between NINS and
Princeton University in the field of life science field.

NIBB-EMBL Collaboration

EMBL and the National Institutes of Natural Sciences
renew their research exchange agreement.
Since 2005, NIBB has taken a leading role in the academic
exchange and collaborative research programs forged
between EMBL and the National Institutes of Natural
Sciences (NINS), under the auspices of the academic
exchange agreement. It has also promoted personal and technological exchanges through symposia, exchanges between
researchers and graduate students, as well as the introductionof experimental equipment.
NIBB Director General, Dr. Kiyokazu Agata, and ViceDirector General, Dr. Naoto Ueno, visited EMBL to meet the
Director General of EMBL, Dr. Edith Heard from July 1st
through 2nd, 2019. The purpose of their visit was to bring a
letter renewing the academic exchange agreement between
the two institutes signed by Akio Komori, the President of the
National Institutes of Natural Sciences. Upon this agreement
being signed by Dr. Heard, they exchanged views on international cooperation, collaborative research and the exchange ofindividuals between the two institutes.

Dr. Ewan Birney’s visit to NIBB
Dr. Ewan Birney, the Director of EMBL-EBI and the seniorscientist in charge of research at that institute, visited NIBB
on August 21st 2019 to deliver a seminar as well as to meetwith NIBB researchers. Dr. Birney gave a special seminar
titled “Outbred Genetics in Medaka Fish and Humans Bringing Models and Medicine Together” to NIBB researchers, and had discussions with the NIBB Director General, Dr.
Kiyokazu Agata, to promote an international collaboration
between NIBB and EMBL-EBI.

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NIBB-COS Collaboration

Prof. Jan Lohmann’s visit to NIBB
Starting from 2019FY, NIBB will be entering into an international research collaboration program with the Centre for
Organismal Studies (COS) Heidelberg aimed at understanding the adaptation strategies of organisms to environments.
Prior to its commencement, the Director of COS, Prof. Jan
Lohmann visited NIBB from May 21st through 22nd, 2019
to discuss upcoming international collaborative research
projects and international cooperation between both institutes
with NIBB Director General, Dr. Kiyokazu Agata, and ViceDirector General, Dr. Naoto Ueno.
Prof. Lohmann introduced COS and presented his research
entitled ‘Signal Integration in Plant Stem Cells’ to NIBB
researchers and graduate students. In addition, he toured
the NIBB facilities while exchanging opinions with fellow
researchers. This in turn contributed to a closer relationshipbetween our institutions.

Dr. Alexis Meizel’s visit to NIBB
Dr. Alexis Meizel from COS Heidelberg visited NIBB
on May 30th, 2019 during his research posting at Nagoya
University. He had a discussion on collaborative research
projects using the IR-LEGO system with Associated Prof.
Yasuhiro Kamei and Dr. Joe Sakamoto (NIBB Research
Fellow) at the Spectrography and Bioimaging Facility.

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NIBB signs academic exchange agreement with COS
Heidelberg, Germany
NIBB has signed an academic exchange agreement with
COS Heidelberg, Germany. NIBB and COS Heidelberg haveconducted collaborative research in several research fields
using small fish, cnidarians, plants and microscopies. In
addition to this, researchers and graduate students from both
institutes have previously visited the other for the purposesof this collaboration.
NIBB Director General, Dr. Kiyokazu Agata, and ViceDirector General, Dr. Naoto Ueno, visited COS Heidelberg
from July 1st through 2nd, 2019 to sign an agreement facilitating academic exchange between the institutes. They
also used this opportunity to present their research projects
during seminars for COS researchers and students, as wellas to exchange ideas concerning future collaborative/cooperative programs with COS PIs including the COS Managing
Director, Prof. Dr. Jan Lohmann.

Dr. Annika Guse’s visit to NIBBNIBB will undertake an international research collaborative
program with COS Heidelberg aimed at understanding the adaptation strategies of organisms to environments. In this program,
researchers who belong to NIBB and COS Heidelberg will starta collaborative research program focusing on the mechanisms
and evolution of light sensing in cnidarians. Dr. Annika Guse,
who is currently analyzing the mechanisms and evolution oflight sensing in cnidarians, visited NIBB from September 8th
through 9th, 2019 regarding this.
On this occasion, she gave a presentation entitled Molecular
Mechanisms of Coral-Algal Endosymbiosis, and was able tostrengthen her relationship with multiple NIBB researchers
via academic discussion.

Collaborative Activities with
Joint Usage / Research Center
Domestic Institutions
NIBB Signs Agreement of Cooperation
with Institute of Low Temperature Science,
Hokkaido University (ILTS)NIBB has signed an agreement with the Institute of Low
Temperature Science, Hokkaido University (ILTS) aimed
at promoting collaboration and cooperation between these
two institutions. A signing ceremony was held at NIBB on
December 9, 2019.

NIBB Priority Collaborative Research Projects● M olecular and physiological mechanisms for understanding mammalian hibernation and their comparative
analysis among species
Prof. Yoshifumi Yamaguchi (ILTS)
Prof. Shuji ShigenobuToshihiko Fujimori (NIBB)
Prof. Shuji Shigenobu (NIBB)NIBB researchers and ILTS researchers have been working
together on several collaborative projects, NIBB and ILTS
have been engaged in cooperative and collaborative research
activities within their respective standard operations forsome time, and both institute NIBB and ILTS aim to further
develop research in the field of low-temperature science andbasic biology with the signing of this agreement.

Dr. Kiyokazu Agata, the Director General of NIBB, and Dr. Manabu
Fukui, the Director of ILTS, signing the agreement.

Following the signing ceremony, the Director of ILTS, Prof.
Manabu Fukui, provided an outline of ILTS. Additionally,
Prof. Fukui and Prof. Yoshifumi Yamaguchi of ILTS gave
seminars concerning their research project. The signingceremony and seminars were attended by a large number of
NIBB members, and provided an opportunity for friendly
exchanges to occur which will hopefully promote further
cooperation and collaboration.

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The 13th NIBB Bioimaging Forum“Behavioral and Recognition
Research upon the Platform of
Vision and Color-Recognition
Studies and its Interdisciplinary
Merging with Bioimaging”
Organizing Committee: Yasuhiro Kamei,
Shoji Fukamachi, and Hideaki Takeuchi
February 12 (Tue)–13 (Wed), 2019
The 13th NIBB Bioimaging Forum was held in conjunction with the achievement briefing meeting conductedby the priority collaborative research project led by Prof.
Fukamachi entitled "Toward Construction of a Research
Platform of Vision and Color-Recognition Studies -Uponthe Model of Medaka Whose Behaviors are Dominated by
Vision- ". This collaborative research project was aimed at
establishing methodologies for analyzing issues ranging fromindividual behaviors to molecular mechanisms of vision,
which also use imaging technologies such as virtual reality
and aerial imaging. As bioimaging aims to improve observation technologies, this joint meeting was viewed as a good
opportunity for exchanging study results and other information.
In particular, rhodopsin (and other opsins) is a key substance combining the two participant groups. This is due to it
being directly related to vision. It also can be used as a toolin optogenetics, itself a cutting-edge bioimaging methods.
Additionally, Medaka have been used as a model organism
for more than a hundred years in Japan, and have become
popular as pets due to the many color variations they produce
through cross-breeding.
We made this symposium open to public for the purposes ofdisseminating of knowledge on the research uses of Medaka
and their history as a model organism, something which has
likely benefitted historically. It was unfortunate that two of
the invited speakers withdrew due to cancelling caused byan influenza epidemic, but their absence was covered by two
young participants in a similar research field. This shows alarge pool of researchers active in this field.
I would like to thank all the speakers and the project
members for their contribution.
(Yasuhiro Kamei)

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The NIBB Genome Informatics
Training Course

The NIBB Core Research Facilities regularly organizes aseries of training 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 2019, we held three sets of training
courses on RNA-seq analysis. Each set of the RNA-seq
analysis course was made up of two 2-day programs: one
was a preparatory course concerning the basics of UNIX andR, and the other was a practical course to learn the pipelinesof RNA-seq analysis using next-generation sequencing data.
These GITC courses offered lectures and hands-on tutorials.

Introduction to RNA-seq: From the
Basics 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.),
Dr. Katsushi Yamaguchi, Ms. Hiroyo Nishide,
Mr. Takanori Nakamura, Mr. Takahiro Bino, Mr.
Hiroki Sugiura (NIBB Core Research Facilities)

February 21 (Thu)–22 (Fri), 2019
(Preparatory Course) Basics of UNIX, R, and NGS■ 30 participants (including 2 from NIBB)■ Program:
1. UNIX for Beginners
2. Editor and Scripts
3. Introduction to “R”
4. NGS Basic Data Formats and NGS Basic Tools
5. Text Processing
6. Exercises

March 14 (Thu)–15 (Fri), 2019
(Practical Course) RNA-seq Analysis Pipeline■ 28 participants (including 1 from NIBB)■ Program:
1. Introduction to RNA-seq
2. NGS Basic Data Format and Basic Tools
3. Visualization of NGS Data
4. Introduction to Statistics
5. RNA-seq Pipelines: Genome-Based andTranscriptome-Based Approaches
6. Multivariate Statistics
7. Functional Annotation and Gene Ontology
8. Exercises

May 16 (Thu)–17 (Fri), 2019
(Preparatory Course) Basics of UNIX, R, and NGS■ 28 participants (including 3 from NIBB)The 7th Bio-imaging Data
Analysis Training Course(Practical Course) RNA-seq Analysis Pipeline■ 30 participants (including 4 from NIBB)

Organizers: Dr. Kagayaki Kato, Dr. Hiroshi
Koyama, Dr. Takashi Murata, Dr. Yasuhiro
Kamei, and Dr. Shigenori Nonaka
Supervisors: Prof. Naoto Ueno, Prof.
Toshihiko Fujimori, and Prof. Shinji
TakadaMay 16 (Thu)–17 (Fri), 2019

December 10 (Tue)–12 (Thu), 2019

May 30 (Thu)–31 (Fri), 2019

(Preparatory Course) Basics of UNIX, R, and NGS■ 26 participants (including 6 from NIBB)* Practical Course was postponed due to the COVID-19.

The 7th Bio-imaging Data Analysis Training Course washeld jointly by the Exploratory Research Center on Life andLiving Systems (ExCELLS), 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 the training related to learning about
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”. Forty
three people applied for the course, which had an announced
capacity of 16 participants. Considering the high demand forcourses on these subjects, we accepted 18 participants.
This course's lectures were conducted with the aim of
guiding participants towards keeping in mind the series ofsteps essential to fundamental image processing and analysis
while obtaining images to be used (workflows). In addition,
we loaned PCs pre-installed with ImageJ, a typical opensource software package for biological image processing
and analysis, to the participants in addition to images whichwere used for practice. Also, lectures were given on how
programming of simple “macro language”, which uses theseworkflows in ImageJ allows automation; itself being a necessity for the large capacity and high-dimensional throughputof 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, butsatisfied”. It certainly is beneficial in terms of their image
analysis as they became more familiar with these techniques.
In addition, we expect that this course will increase opportunities for joint research relating to biological image analysis.
(Kagayaki Kato)

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The Center for the Development of
New Model Organisms Training Course"Technical Workshop on Microinjection
Techniques Using New Model Organisms"
Instructors: Ken-ichi Suzuki, Shuji
Shigenobu, Teruyuki Niimi, Kiyoshi Naruse
Lecurers: Ken-ichi Suzuki, Tatsuma Mohri,
Masato Ohtsuka
Organizers: Ken-ichi Suzuki, Shuji
Shigenobu, Naoto Ueno
The development of new model organisms has enabled theoutstanding recent progress of genome sequencing and editing
technologies. In keeping with this, the stable rearing and reproduction of animals, the preparation of genetic information,and especially the establishment of experimental protocols forfunctional analyses is necessary for the establishment of newmodel organisms. Microinjection is a fundamental techniquefound among these protocols, which itself is used in many functional operations, e.g. genome editing and RNAi. However, thedevelopment of microinjection techniques, presents an obstacle
to developing new organisms, due to these type of techniques
requiring intensive optimization for each new organism. With
this in mind, The Center for the Development of New Model
Organisms of NIBB organized a technical workshop during
2019 concerning microinjection techniques in order to promoteand share said techniques among the scientific community.
We welcomed fourteen participants from various backgrounds,
including academic researchers, students, and researchers fromprivate companies. Participants were divided into three groups,
each of which worked for two days with either amphibians(Iberian ribbed newt), insects (aphids and silverfish), or fish(medaka). Participants learned and practiced the basics of injection techniques as they were optimized for each organism.
They ultimately joined together for a debriefing session where
their individual experiences were shared. We also had a lecture
session regarding functional analyses techniques by the application of microinjection. Four lectures, that were mostly run byinvited extramural researchers, outlined the history of microinjection and genome editing. Researchers from a privatecompany developing and manufacturing microinjection instruments also joined in with these sessions, thereby facilitating an
informative exchange between users and developers.
This course successfully provided a valuable opportunity forparticipants to share microinjection techniques. One of the participants said those present could “acquire skills not provided inpublished textbooks or papers. As such, I felt the course was very
informative". Another attendee stated that they "could exchange
information with lecturers and other participants, and felt the conversations I was able to have were valuable". Although microinjection is a classic technique, it no doubt grows even more important as it is required in the application of cutting-edge technologies
such as genome editing. We at the Center for the Development of
New Model Organisms, NIBB, continue to focus on the improvement and promotion of this both old yet new technique, while
keeping an eye on the collective needs of researchers.
(Shuji Shigenobu)

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Advanced Bioimaging Support(ABiS)The demand for bioimaging has increased in recent years
in the field of life science. However, due to advances inimaging technologies, such as the diversification and specialization of imaging equipment, increasingly complex operations, increased equipment and running costs, as well as thegrowing need for image data analysis, individual research
institutes and universities are increasingly encountering difficulties related to the introduction, maintenance, and operation of imaging equipment.
ABiS was launched as one of the designated "Platforms for
Advanced Technologies and Research Resources" during
fiscal year (FY) 2016 under the new framework of the Grantin-Aid for Scientific Research on Innovative Areas (Leader:Prof. Masanobu KANO, NIPS/The University of Tokyo).
This program aims to contribute to the further development of academic research in Japan through the provisionof cutting-edge equipment and methodologies to individual
KAKENHI (Grants-in-Aid for Scientific Research) research
projects by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) under the Grant-in-Aid for
Scientific Research on Innovative Areas (FY2016-FY2021).
Cooperating with domestic partner organizations that own
and operate multiple types of advanced specialized imaging
equipment, ABiS provides cutting-edge instruments forlight microscopy, electron microscopy, magnetic resonanceimaging, and other methods through its network. It aims toprovide comprehensive support for advanced imaging in thefield of life science. NIBB, together with NIPS, contributes
as a core institute in the ABiS network. Among the various
support activities that ABiS performs, NIBB is tasked withthe following:■ Light microscopy• 4 D microscopy administered by Prof. Toshihiko
FUJIMOIRI• IR-LEGO microcopy administered by Assoc. Prof.
Yasuhiro KAMEI• D SLM administered by Assoc. Prof. Shigenori
NONAKA■ Imaging analysis• Development of image processing/analysis algorithms
for biological data administered by Prof. Naoto UENO,
Assist. Prof. Kagayaki KATO and Assist. Prof. Yusaku
OHTA.
■ Training• Training for image analysis administered by Assist. Prof.
Hiroshi KOYAMA and Prof. Naoto UENO
To organize and coordinate ABiS activities, two secretariat offices were established at NIBB (Assoc. Prof. Shoji
MANO) and NIPS, respectively, under the control of thegeneral support group (Individuals in charge at NIBB;
Director-general Prof. Kiyokazu AGATA, Prof. Naoto
UENO, Prof. Shinji TAKADA, Assoc. Prof. Shoji MANO).
General support provided includes budget planning and management of ABiS activities. In particular, we promote ABiS

activities via its associated website and other media, so that
KAKENHI researchers make full use of the ABiS platformto 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 otherthree platforms (Platform of Supporting Cohort Study andBiospecimen Analysis, Platform of Advanced Animal Model
Support, and Platform for Advanced Genome Science) to
provide both multidisciplinary and international support.
Euro-Bioimaging (EuBI) is the largest and most well-established imaging network in Europe, and has been expanding
globally to form the Global Bioimaging (GBI) network
which boasts participants from areas such as India, Australia,
Singapore, the Republic of South Africa, Canada, Mexico,USA, and some Latin American countries. In 2018, ABiS
joined the GBI project representing the Japanese bioimaging
community. It is hoped that through GBI, ABiS will be able
to raise the quality of present support to that of the currently
accepted international benchmarks, and that further observations, data analyses and research methods will be able to bebetter shared globally. Furthermore, it is also hoped that thiscollaboration will lead to set up a better environment for bioimaging research in Japan. This will be achieved not only byproviding international training courses for young researchers and the staff of imaging facilities, but also throughdiscussions about the implementation of career paths; acommon problem in this field.
GBI also aims to establish an image data sharing infrastructure system, promote academic/industrial cooperationand formulate a global strategy for bioimaging research. It
has been decided that the GBI event “The Fifth Exchange ofExperience (EoE)” hosted by ABiS, will be held in Okazaki,
Japan during the autumn of 2020.

Image Competition Held During
The 57th Annual Meeting of The
Biophysical Society of Japan (BSJ)

Figure 1. Award giving ceremony. Prof. UENO (Center) awarding
prizes to winners of the image competition. Osaka University’s
Prof. Takeharu NAGAI (Left) chaired the meeting.

Training Course for AI-based
Bio-image Analysis
August 29 (Thu)–30 (Fri) 2019
In addition to the support given by cutting-edge
imaging research technologies, ABiS holds various
training courses for image acquisition, processing andanalysis to further disseminate imaging techniques
every year. ABiS held 10 training courses on lightmicroscopy, immunoelectron microscopy, MRI andimaging analysis in 2019. Among them, AI-based bioimage analysis was held at Kumamoto University,
which was organized by Assoc. Prof. Takumi HIGAKI(Kumamoto Univ.), Assist. Prof. Kagayaki KATO(NIBB), Assist. Prof. Yusaku OHTA (NIBB) and Prof.
Naoto UENO (NIBB). Prof. Seiichi UCHIDA (Kyushu
Univ.) and Prof. Akira FUNAHASHI (Keio Univ.),
who are recognized for their expertise in software andalgorithm creation in the field of imaging analysis, andAI-based systems biology, respectively, were invitedlecturers (Figure 2).

September 24 (Tue)–26 (Thu) 2019
ABiS held the ‘ABiS Image Competition’ as a
special joint event in collaboration with BSJ during
the 57th annual meeting of BSJ in Miyazaki (Chair:
Dr. Takeharu NAGAI) to encourage researchers, especially students, graduate students, and young researchers, to present their work to the research community. Winners were selected from the categories of‘Humor’, ‘Intellectual’ and ‘Beauty’ on the basis of
votes collated from attending participants, and were
publicly commended by ABiS and the 57th Annual
meeting of BSJ (Figure 1). All photographs and videos
are available to the public on the annual meeting’s
website (https://www2.aeplan.co.jp/bsj2019/abisicontest/57_abisimagecontest_list).

Figure 2. Training course session at Kumamoto University. Fifteen
participants were selected via examination of their respective applications, and received lectures on AI-based machine learning forbio-image analysis.

109

The NIBB Internship Program
The NIBB Internship program, which started in 2009, isa hands-on learning course for overseas students designed
to give high-quality experience in real world research and afocused education in biology. At the same time, this programaims to internationalize graduate students from The Graduate
University for Advanced Studies SOKENDAI, giving themthe opportunity to get to know students and interns with differing cultural customs. Another goal of the program is tobuild connections through providing education to the people
who will form the core of international research networks inthe future.
To participate in this program, applicants who would liketo experience research at NIBB must supply the name of
the lab they would like to visit as well as their reasons forchoosing it, and a letter of recommendation. Based on thisinformation, applicants are chosen to spend set periods oftime participating in specific research activities in the lab
they applied for. Round trip airfare and housing expenses areprovided by the NIBB Internship Program.
In FY 2019 there were 29 applicants, out of which 11
interns were selected. These interns were from universities located in six countries (Vietnam, Thailand, Germany,
Hungary, USA, and Japan) and spent periods ranging fromtwo to twelve weeks experiencing life as a member of a
research team. Moreover, two interns from the UK and
Germany, who were funded by travel grants they had appliedfor, were also posted to NIBB.

Report from a participant
Neen Phan-udom
Mahidol University, Thailand
My name is Neen Phan-udom. I am a third-year undergraduate student from Mahidol University Thailand, majoringin Bioresources and Environmental Biology (International
Program). I had the opportunity to do a 2-week internshipat Professor Ueda’s lab- the Division of Cellular Dynamicswhich works on plant membrane trafficking proteins in
Arabidopsis thaliana and Marchantia polymorpha.
I was under the supervision of Asst. Prof. Ebine during myinternship. My work mainly consisted of performing transient assays for Arabidopsis thaliana leaves and visualizing
the localization of the targeted proteins via confocal microscopy. I learned to find and take pictures of the cells expressing GFP/RFP-tagged proteins to later measure their activities
and possible interactions. I also got to attend a 2-day summer
practical course on oil bodies in liverwort which included
mini fieldwork to collect liverwort samples and visualize
their oil bodies under the microscope.
In addition to gaining more knowledge and laboratory
skills, this internship was one of the most wonderful experiences I’ve ever had. I received a very warm welcome fromall the lab members as well as other staff at NIBB. I was
pleasantly surprised when the lab organized a welcoming

110

party for me and my lab partner on our first day (which
has gotten me really into soumen). Everyone was so niceand friendly which made me feel very welcomed and lookforward to my tasks every day. I was on the verge of crying
on my last day here when I had to say goodbye. I also had
many wonderful moments learning about Japanese culture
both inside and outside of the lab, not to mention having theprivilege to live in Okazaki city and do some traveling on theweekends. Japan really is a beautiful country.
I will never forget my two weeks here. If anything, I wish I
could have stayed longer and learned more about membranetrafficking in plants. The internship has shown me the beautyof basic research of exploring the unknowns and left me
wanting for more. Really, I can’t thank everyone enough forthis amazing experience. I am forever grateful for this internship opportunity.

The NIBB Openhouse 2019
October 5 (Sat), 2019
The triennial NIBB Open House was held on 5 October
2019, and was attended by 2,980 people.
This event saw us opening our laboratories and facilities in
the Myodaiji area to the public in order to introduce cuttingedge biological research.

Exhibition of the Nobel Prize Monument andNobel Medal (official replica) of dedicated toProfessor Emeritus Yoshinori Ohsumi

Science Workshop and Research Poster
Exhibit by Okazaki High School SSH Club

Stamp Rrally
Talks LiveThe event combined talks and demonstrations run by NIBB
researchers. Four researchers, including the Director General
Kiyokazu Agata, gave talks about why living things are so
fascinating, which were followed by a demonstration and
Q&A session with the audience.

Unique badges were given to visitors who had collected adesignated number of stamps at various locations within theOpen House event.

Booth Exhibits

There were 31 laboratories or departments in two separate
venues:, the NIBB main building and the conference hall.

111

Outreach Activities
NIBB widely disseminates our research and results to thegeneral public, and we conduct activities to convey the
excitement of studying biology to students at elementary,junior high, and high schools to nurture the next generationof scientists.

July 18 (Thu), 2019

Special class at Iwazu Junior High School■ Assist. Prof. Yusuke Mii

Visits to schools
July 2 (Tue), 2019

Special class at Kouzan Junior High School■ Assist. Prof. Keiko Nonomura

October 24 (Thu), 2019

Special class at Ryunan Junior High School■ Prof. Teruyuki Niimi

July 10 (Wed), 2019

Special class at Nukata Junior High School■ S. A. Assist. Prof. Tomoko Kurata

November 14 (Thu), 2019

Special class at Mikawa Junior High School■ S. A. Assoc. Prof. Kenji Takizawa

July 11 (Thu), 2019

Special class at Yahagi Junior High School■ Assist. Prof. Ryutaro Tokutsu

December 3 (Tue), 2019

Special class at Aoi Junior High School■ Prof. Teruyuki Niimi

112

January 14 (Tue), 2020

Special class at Okazaki High School■ Director General Kiyokazu Agata

December 20 (Fri), 2019

Special class at Aichi Sangyo University Mikawa HighSchool■ Assoc. Prof. Shigenori Nonaka

June 19 (Wed), 2019

Feedback session regarding research presentations by highschool students■ S. A. Assist. Prof. Kiyoshi Tatematsu

December 27 (Fri), 2019

Feedback session regarding research presentations by highschool students■ S. A. Assist. Prof. Kiyoshi Tatematsu

Seminars for school teachers
October 29 (Tue), 2019
■ Assist. Prof. Atsushi Hoshino

Work experience for junior high school
students at NIBB
■ Honan Junior High School students
Ryujin Junior High School students

Programs broadcast live online
August 23 (Fri)–31 (Sat), 2019

"Observing the Iberian ribbed newt developmental process
over a period of 200 hours"
This was a 200 hour live broadcast displaying the developmental process of the Iberian ribbed newt. Researcher
commentaries were provided via the live internet broadcast
platform, NicoNico■ S. A. Assoc. Prof. Ken-ichi T. Suzuki
Dr. Miyuki Suzuki
Director General Kiyokazu Agata
Assist. Prof. Yuki Moriyama (Chuo Univ)
Prof. Toshinori Hayashi (Hiroshima Univ.)
S. A. Assist. Prof. Tomoko Kurata■ Number of accesses: 452174

113

October 20 (Sun), 2019

Inter-University Research Institute Corporation
Symposium 2019
■ Prof. Shuji Shigenobu

Lectures for the public
June 2 (Sun), 2019

Lecture at the Natural History Museum/Sea of Life
■ Prof. Teruyuki Niimi

Workshop at Natural History Museum/Sea of Life
■ Dr. Tatsuro Konagaya
S. A. Assist. Prof. Tomoko Kurata

April 16 (Tue), 2019

Open lecture to commemorate the appointment of the
Director General of NIBB
■ Director General Kiyokazu Agata

114

July 7 (Sun), 2019

National Institute of Natural Sciences
The 8th Young Researcher Award Commemorative Lecture■ Assist. Prof. Yu KitadateFebruary 8 (Fri), 2020

Presentation by OKASHIN (Okazaki Credit Union) scholarship students■ Dr. Mariko Kishimoto■ Special lecture at OKASHIN
Director General Kiyokazu Agata

March 11 (Wed), 2020

Online lecture■ Assist. Prof. Toshiya Ando115

Access

From Tokyo

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

From Osaka

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

From Central Japan International Airport

Take the Meitetsu Line for Nagoya and change at Jingu-mae Station toa Toyohashi-bound train and get off at Higashi Okazaki Station(approximately 70 minutes from the airport).

City HallCity HallCity Hall S.

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

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

National Institutes of Natural Sciences

National Institutefor Basic Biology
38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585 Japan
tel: +81 564-55-7000 fax: +81 564-53-7400
http://www.nibb.ac.jp/en/
Issued in December 2020
Edited by the Research Enhancement Strategy Office, NIBB