National Institute for Basic Biology

2020 ANNUAL REPORT

April 2020–March 2021

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 April 2020 to March 2021… …………………………… 7
Selected Publications in 2020FY from NIBB… ………………………………………………… 8Professor Emeritus… …………………………………………………………………………… 10
Cell Biology
Division of Cellular Dynamics… …………………………………………………………… 11
Division of Quantitative Biology……………………………………………………………… 14
Division of Chromatin Regulation…………………………………………………………… 17
Laboratory of Neuronal Cell Biology………………………………………………………… 20
Laboratory of Stem Cell Biology… ………………………………………………………… 23
Laboratory of Organelle Regulation………………………………………………………… 25
Developmental Biology
Division of Morphogenesis…………………………………………………………………… 27
Division of Molecular and Developmental Biology………………………………………… 30
Division of Embryology… …………………………………………………………………… 33
Division of Germ Cell Biology… …………………………………………………………… 36
Labotratory of Regeneration Biology… …………………………………………………… 39
Neurobiology
Division of Behavioral Neurobiology………………………………………………………… 41
Laboratory of Neurophysiology……………………………………………………………… 44
Evolutionary Biology and Biodiversity
Division of Evolutionary Biology… ………………………………………………………… 46
Division of Symbiotic Systems… …………………………………………………………… 49
Division of Evolutionary Developmental Biology… ……………………………………… 52
Laboratory of Evolutionary Genomics……………………………………………………… 55
Laboratory of Bioresources… ……………………………………………………………… 57
Laboratory of Morphodiversity… …………………………………………………………… 59
Laboratory of Biological Diversity…………………………………………………………… 60
Environmental Biology
Division of Environmental Photobiology… ………………………………………………… 80
Division of Plant Environmental Responses… …………………………………………… 83
Theoretical Biology
Laboratory of Genome Informatics… ……………………………………………………… 86
Imaging Science
Laboratory for Spatiotemporal Regulations………………………………………………… 88
Laboratory for Biothermology… …………………………………………………………… 89
Research Support
NIBB Core Research Facilities……………………………………………………………… 91
NIBB BioResource Center…………………………………………………………………… 96
National BioResource Project… …………………………………………………………… 99
NIBB Center for the Interuniversity Bio-Backup Project… ……………………………… 101
Center for the Development of New Model Organisms…………………………………… 103
Center for Radioisotope Facilities (Okazaki Research Facilities)… …………………… 105
Research Enhancement Strategy Office…………………………………………………… 106
Technical Division… ………………………………………………………………………… 109
Collaboration Programs with Overseas Institutions… …………………………………… 110
Collaborative Activities with Joint Usage / Research Center… ………………………… 111
NIBB Bioimaging Forum… ……………………………………………………………………… 112
NIBB Training Courses…………………………………………………………………………… 113
Advanced Bioimaging Support………………………………………………………………… 114
NIBB Internship Program………………………………………………………………………… 116
Outreach Activities… …………………………………………………………………………… 117
Access to NIBB… ………………………………………………………………… Inside back cover

The cover items are related to a paper titled “Calcium dynamics during trap closure visualized in transgenic Venus
flytrap” (Suda et al., Nature Plants 2020) from Division of Evolutionary Biology. See page 47 of this report for
details.

INTRODUCTION
t is my great pleasure to present to you all the
National Institute for Basic Biology (NIBB)’s 2020
Annual Report which outlines NIBB’s research, educational,
and international activities and its effective function as acenter for collaborative research in Japan from April 2020 toMarch 2021.

I

NIBB’s activities in 2020 were severely affected by the
COVID-19 pandemic. All planned international conferenceswere postponed and all international collaborative activities were switched to an online-based format. The first joint
meeting between NIBB and The Centre for Organismal
Studies (COS) Heidelberg was also held online in March
2021. Although researcher interactions within the country
were also limited, we could maintain research activities
within the institute at almost normal levels.
Concerning changes on the staffing front, Associate
Professor Ryuji Kodama retired in March 2021 and AssistantProfessor Ikuo Uchiyama was promoted to Associate
Professor in April 2020. Additionally, one specially appointed associate professor, four assistant professors and fourteen
specially appointed assistant professors were newly posted to
NIBB during this year.
I would like to finish this introduction by congratulating
Professor Masayoshi Kawaguchi, who was awarded the 2020
Japanese Society of Plant Physiologists Award, and Professor
Teruyuki Niimi, who was awarded the 2020 Japanese Societyof Sericulture Science Award.
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, 2021

1

ORGANIZATION OF THE NATIONAL INSTITUTE FOR BASIC BIOLOGYThe National Institute for Basic Biology (NIBB) was
founded in 1977 as one of the Inter-University ResearchInstitutes designated to promote and stimulate the study of
biology both in Japan and internationally. NIBB conducts
first-rate research on site as well as in cooperation withnational, public and private universities and research organizations. NIBB attempts to elucidate the general and fundamental mechanisms underlying various biological phenomena throughout a wide variety of biological fields, such as cellbiology, developmental biology, neurobiology, evolutionary
biology, environmental biology and theoretical biology.
NIBB and four other national institutes, the National
Astronomical Observatory of Japan (NAOJ), the National
Institute for Fusion Science (NIFS), the National Institutefor Physiological Sciences (NIPS), and the Institute for
Molecular Science (IMS), constitute the National Institutesof Natural Sciences (NINS), which is one of the four InterUniversity Research Institute Corporations. NIBB’s activities
are supported by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan.

■ 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
HASEBE, Mitsuyasu
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 andis assisted by the Advisory Committee for Programmingand Management. The Advisory Committee, comprised ofeleven professors within NIBB and ten leading biologists
from outside of it, advises the Director General on importantmatters such as planning collaborative research programs aswell as on the scientific activities of NIBB.
The Research Enhancement Strategy Office was foundedin 2013 and aims to support researchers in order to improve
NIBB’s abilities as a collaborative research institution.
Administration of NIBB is undertaken by the Okazaki
Administration Center under the direct auspices of the
Administration Office of NINS.

Research and Research Support
NIBB’s research programs are conducted in research units
called divisions and laboratories. Each research unit forms
an independent project team. The NIBB Core Research
Facilities and NIBB BioResource Center were launched in2010 to support research at NIBB and also to act as an intellectual hub to promote collaboration among the researchersof NIBB and other institutions. The NIBB Center of theInter-University Bio-Backup Project (IBBP Center) was
founded in 2012 to prevent the loss of invaluable biologicalresources. The Center for the Development of New Model
Organisms was founded in 2013 to promote the developmentof new model organisms and research that uses them. The
Technical Division manages the activities of the technical2Center 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.

International Research
Collaboration Center (IRCC)

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, 2021

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

Evolutionary Biology and Biodiversity■Division of Evolutionary Biology■Division of Symbiotic Systems■Division of Evolutionary Developmental■International Cooperation Group

Biology■Collaborative Research Group■Laboratory of Evolutionary Genomics■Young Researcher Support Group■Laboratory of Bioresources■Gender Equality Promotion Group■Laboratory of Morphodiversity■Collaborative Innovation Group■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■Functional Genomics Facility■Spectrography and Bioimaging Facility■Data Integration and Analysis Facility

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

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

Technical Division
Section of Health and Safety Management

Okazaki Administration Center3

staff and helps to promote the activities of each research
unit and research support facility to maintain the common
resources of NIBB and NINS.
The Astrobiology Center (ABC) was established in 2015as an NINS research center. Additionally, the Exploratory
Research Center on Life and Living Systems (ExCELLS)
was founded in 2018. Several NIBB researchers are alsoaffiliated with ABC and ExCELLS.

■ Members in NIBB
Director General 1
Professors(including specially-appointed) 15

Technical Assistantsand Admin Support Staff

Associate Professors(incl. sp. app.) 13

52

115

48
41

Assistant Professors(incl. sp. app.)

Postdoctoral Fellows

29

Graduate andUndergraduate Students(2 are undergraduates)

Technical Staff

314 in total(as of 1st April, 2021)■ Financial Configuration of NIBB
Indirect Costs in Grants

90

Other Grants(settled forApril 2020-March 2021)
numbers are in million yen

105
External
Competitive
Fund

821

Grant-in-Aid
for Scientific
Research

Personnel

771

420

Management
Expenses Grant
for NIBB

1,201
Grants forCommissioned Research

209

Self-Income 2

Research
Collaboration 172

256

Management Expenses
Grant for SOKENDAI

52

Donations

13

Other 22,089 million yen
in total■ Members of the Advisory Committee for Programming and Management (terms of April 2019 to March 2021)

External committee
members

Internal committee
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 BIOLOGY
In the pursuit of progress in the field of biology, The National
Institute for Basic Biology (NIBB) has set five goals regarding
its activities. We consequently contribute to the world-wide community of biologists through our efforts to achieve these goals.
International cooperation
Contribution to the community of biologists

Promotion of collaborative
research projects

Developmentof new fields

Promotion ofacademic research

Cultivation of
future researchers

Promotion of academic research
One of our goals is the promotion of academic research, andthis is accomplished through our research activities, which
we will introduce throughout this brochure.

Promotion of Collaborative Research Projects■ 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 researchers with travel and lodging expenses when visiting NIBB’s
laboratories to conduct collaborative research. “Priority
Collaborative Research Projects” are carried out as groupresearch projects by internal and external researchers todevelop pioneering research fields. The “Collaborative
research projects for new model organism development”
and “Collaborative Research Projects for Bioresource
Preservation Technology Development” projects are fordeveloping and establishing new model organisms andnew research technology. Research and travel expenses areprovided for these projects. ‘Collaborative Research Projects
for Integrative Genomics’ and ‘Collaborative Research
Projects for Integrative Bioimaging’ are projects to facilitatea 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.
year2018 2019 2020

Priority collaborative research projects

1

1

1

Collaborative research projects for newmodel organism development

―

―

2

Collaborative research projects for modelorganism and technology development

2

2

―

Individual collaborative research projects

57

60

46

Collaborative research projects for integrative genomics

67

66

56

Collaborative research projects for integrative bioimaging

23

22

19

NIBB workshops

2

3

2

Collaborative experiments using the Large
Spectrograph

9

9

9

Priority collaborative research projects

1

0

0

Collaborative research projects for bioresource preservation technology development

18

14

10

Total

180

177

145

■ National BioResource Project
The National BioResource Project (NBRP) is a national
project for the systematic accumulation, storage and supplyof nationally recognized bio-resources (i.e., experimental
animals and plants, cells, DNA and other genetic resources),
which are widely used as materials in life science research.
To promote this national project, NIBB has been selected
as a center for research on Medaka (Oryzia latipes), whose
usefulness as a vertebrate model was first demonstrated by
Japanese researchers. The usability of Medaka as a researchmaterial in biology has drawn increasing attention since its
full genome sequence became available. NIBB is also a subcenter for the NBRP’s work with Japanese morning glories
and Zebrafish (p. 99-100).

■ Interuniversity Bio-Backup Project for Basic Biology(IBBP)
To prevent damage caused by natural disasters to importantbiological resources, NIBB has managed the Interuniversity
Bio-Backup Project for Basic Biology (IBBP) from 2012
in collaboration with seven national universities for multiplicate preservation of genetic libraries and other invaluablebioresources used in cutting-edge research (p. 101-102).

■ Advanced Bioimaging Support (ABiS)
ABiS provides assistance pertaining to advanced imaging inresearch supported by Grants-in-Aid for Scientific Research.
NIBB and NIPS contribute as core institutes to the ABiS
network of domestic partner organizations that own andoperate multiple types of advanced specialized imaging
equipment. In 2018, ABiS joined the Global Bioimaging(GBI) network to represent the Japanese bioimaging community. ABiS also hosts training courses (p. 114-115).

International Cooperation■ Collaborative Programs with Overseas Institutes
NIBB plays a leading role in 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 has formed an agreement with Princeton University
and COS Heidelberg to promote joint research projects, collaborative symposia, training courses and student exchange
programs. The Kick Off (1st) Meeting for NIBB COS
Heidelberg International Collaborations was held in March
2021 (p. 110).

■ NIBB Conference
The NIBB Conferences are international conferences on
prominent topics in biology that are organized by NIBB
professors. Since the first conference in 1977 (the year
of NIBB’s founding), NIBB Conferences have provided
researchers in basic biology with valuable opportunities forinternational exchange. Unfortunately, this year’s NIBB conference was cancelled due to the COVID-19 pandemic.

5

Development of New Fields of Biology■ Establishment of new model organism
NIBB is working to establish novel model organisms inorder to advance research on biological phenomena that areinteresting but have been poorly analyzed.

Cultivation of Future Researchers■ Ph.D. program
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
doctoral course for university graduates and a three-year
doctoral course for graduate students with a master’s degree.
Additionally, graduate students enrolled in other universities
can apply to be special research students eligible to conduct
research under the supervision of NIBB professors. In any
case, graduate students can receive financial support fromNIBB based on the research assistant (RA) system from the
beginning of the five-year course.
Due to its international collaboration with EMBL, graduate
students are encouraged to attend PhD student symposia held
at EMBL at least once during their doctoral program, where
they are provided with an opportunity to give 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 class institute (p. 116).
Graduate Students Educated by NIBB
(numbers counted at the end of April each year)
60
50

7

11

13

37

2016

40

6
11

9

39

36

36

47

36

2017

2018

2019

2020

2021

4

30
20

42

10
0

2015

■ SOKENDAI Graduate Student■ Special Research Student■ 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 (i.e.
web pages, Facebook, and Twitter). Streaming live videos
concerning the development of model organisms have beenviewed numerous times. NIBB also cooperates in the education of undergraduate and young students through lectures
and workshops (p. 117-119).

6

■ Personnel changes from April 2020 to March 2021*

Newly assigned to NIBB
Name

Position

Research Unit

Date

UCHIYAMA, Ikuo

Associate Professor

Laboratory of Genome Informatics
Data Integration and Analysis Facility

April 1, 2020

SEGAMI, Shoji

Assistant Professor

Division of Evolutionary Biology

April 1, 2020

HIRA, Seiji

Specially Appointed Assistant Professor

Division of Germ Cell Biology

April 1, 2020

MORITA, Shinichi

Specially Appointed Assistant Professor

Division of Evolutionary Developmental Biology

April 1, 2020

SAKAMOTO, Joe

Specially Appointed Assistant Professor

Laboratory for Biothermology

April 1, 2020

KOSETSU, Ken

Specially Appointed Assistant Professor

Division of Evolutionary Biology

April 1, 2020

MINAMINO, Naoki

Specially Appointed Assistant Professor

Division of Cellular Dynamics

April 1, 2020

YOKE, Hiroshi

Specially Appointed Assistant Professor

Laboratory for Spatiotemporal Regulations

April 1, 2020

KOBAYASHI, Taisuke

Specially Appointed Assistant Professor

Laboratory of Neurophysiology

April 1, 2020

SHINOZUKA, Takuma

Specially Appointed Assistant Professor

Division of Molecular and Developmental Biology April 1, 2020

LIU, Meng

Specially Appointed Assistant Professor

Division of Symbiotic Systems

April 1, 2020

HAYASHI, Aki

Specially Appointed Assistant Professor

Division of Chromatin Regulation

April 1, 2020

KAWAMOTO, Nozomi

Specially Appointed Assistant Professor

Division of Plant Environmental Responses

April 1, 2020

KURASHIMA, Kiminori

Specially Appointed Assistant Professor

Laboratory of Stem Cell Biology

May 1, 2020

HUANG, Jia-Hsin

Assistant Professor

Laboratory of Evolutionary Genomics

June 1, 2020

SAITO, Nen

Specially appointed Associate Professor

Laboratory of Biological Diversity

August 1, 2020

WUDARSKI, Jakub

Specially Appointed Assistant Professor

Laboratory of Biological Diversity

August 1, 2020

YODA, Shinichi

Specially Appointed Assistant Professor

Laboratory of Evolutionary Genomics

October 1, 2020

OHASHI, Rie

Assistant Professor

Laboratory of Neuronal Cell Biology

January 1, 2021

KIM, Eunchul

Assistant Professor

Division of Environmental Photobiology

January 1, 2021

Newly affiliated with other universities and institutes
Name

New Affiliation

Date

Position

NAKAYAMA, Kei

Kobe Pharmaceutical University

Assistant Professor

September 30, 2020

TAKAHASHI, Shunichi

University of the Ryukyus

Professor

February 1, 2021

MORITA, Shinichi

Nagoya University

Specially Appointed Assistant Professor

March 1, 2021

HUANG, Jia-Hsin

AI Labs (Taiwan)

Scientist

April 1, 2021

TOKUTSU, Ryutaro

Kyoto University

Program-Specific Researcher

April 1, 2021

Mandatory Retirement
Name
KODAMA, Ryuji

Position
Associate Professor

Research Unit
Laboratory of Morphodiversity

Date
March 31, 2021

■ Awardees from January 2020 to March 2021
Name

PositionAward

KAWAGUCHI, Masayoshi Professor

Japanese Society of Plant Physiologists (JSPP) Award 2020

NIIMI, Teruyuki

Professor

Japanese Society of Sericulture Science Award 2020

OTSUBO, Yoko

Specially Appointed Assistant Professor NINS Young Researcher Award* Changes in professors, associate/ assistant professors are shown below.

7Selected Publications in 2020FY from NIBBGenomic profiling by ALaP-Seq reveals transcriptional regulation by PML bodies
through DNMT3A exclusion
Kurihara, M., Kato, K., Sanbo, C., Shigenobu, S., Ohkawa, Y., Fuchigami, T., and Miyanari, Y. (2020). Molecular Cell 78.
DOI:10.1016/j.molcel.2020.04.004

Genomes of the venus flytrap and close relatives unveil the roots of plant carnivory
Palfalvi, G., Hackl, T., Terhoeven, N., Shibata, T.F., Nishiyama,T., Ankenbrand, M., Becker, D., Förster, F., Freund, M., Iosip, A.,
Kreuzer, I., Saul, F., Kamida, C., Fukushima, K., Shigenobu, S.,
Tamada, Y., Adamec, L., Hoshi, Y., Ueda, K., Winkelmann, T., Fuchs,J., Schubert, I., Schwacke, R., Al-Rasheid, K., Schultz, J., Hasebe, M.,
Hedrich, R. (2020). Current Biology 30.
DOI:10.1016/j.cub.2020.04.051

Gravity-sensing tissues for gravitropism are required for “anti-gravitropic”
phenotypes of Lzy multiple mutants in Arabidopsis
Kawamoto, N., Kanbe, Y., Nakamura, M., Mori, A., and Morita, M.T. (2020). Plants 9.
DOI:10.3390/plants9050615

DNA damage triggers reprogramming of differentiated cells into stem
cells in Physcomitrella
Gu, N., Tamada, Y., Imai, A., Palfalvi, G., Kabeya, Y., Shigenobu, S., Ishikawa, M., Angelis, K.J.,
Chen, C., and Hasebe, M. (2020). Nature Plants 6.
DOI:10.1038/s41477-020-0745-9

Loss of symbiont infectivity following thermal stress can be afactor limiting recovery from bleaching in cnidarians
Kishimoto, M., Baird, A.H., Maruyama, S., Minagawa, J., and Takahashi, S. (2020). ISME
Journal 14.
DOI:10.1038/s41396-020-00742-8

Longin R-SNARE is retrieved from the plasma membrane by ANTH
domain-containing proteins in Arabidopsis
Fujimoto, M., Ebine, K., Nishimura, K., Tsutsumi, N., and Ueda, T. (2020). Proceedings of the National
Academy of Sciences of the United States of America 117.
DOI:10.1073/pnas.2011152117

Oral RNAi of diap1 results in rapid reduction of damage topotatoes in Henosepilachna vigintioctopunctata
Chikami, Y., Kawaguchi, H., Suzuki, T., Yoshioka, H., Sato, Y., Yaginuma, T., and Niimi, T.
(2021). Journal of Pest Science 94.
DOI:10.1007/s10340-020-01276-w

8

Implications of RNG140 (caprin2)-mediated translational
regulation in eye lens differentiation
Nakazawa, K., Shichino, Y., Iwasaki, S., and Shiina, N. (2020). Journal of Biological Chemistry 295.
DOI:10.1074/jbc.RA120.012715

Calcium dynamics during trap closure visualized in transgenic
Venus flytrap
Suda, H., Mano, H., Toyota, M., Fukushima, K., Mimura, T., Tsutsui, I., Hedrich, R., Tamada,Y., and Hasebe, M. (2020). Nature Plants 6.
DOI:10.1038/s41477-020-00773-1

MIR2111-5 locus and shoot-accumulated mature miR2111 systemicallyenhance nodulation depending on HAR1 in Lotus japonicus
Okuma, N., Soyano, T., Suzaki, T., and Kawaguchi, M. (2020). Nature Communications 11.
DOI:10.1038/s41467-020-19037-9

A step-down photophobic response in coral larvae: implications forthe light-dependent distribution of the common reef coral, Acropora
tenuisSakai, Y., Kato, K., Koyama, H., Kuba, A., Takahashi, H., Fujimori, T., Hatta, M., Negri, A.P., Baird,
A.H., and Ueno, N. (2020). Scientific Reports 10.
DOI:10.1038/s41598-020-74649-x

The liverwort oil body is formed by redirection of the secretory
pathway
Kanazawa, T., Morinaka, H., Ebine, K., Shimada, T.L., Ishida, S., Minamino,N., Yamaguchi, K., Shigenobu, S., Kohchi, T., Nakano, A., et al. (2020). Nature
Communications 11.
DOI:10.1038/s41467-020-19978-1

Intercellular and intracellular cilia orientation is coordinated by CELSR1 and
CAMSAP3 in oviduct multi-ciliated cells
Usami, F.M., Arata, M., Shi, D., Oka, S., Higuchi, Y., Tissir, F., Takeichi, M., and Fujimori, T. (2021). Journal of
Cell Science 134.
DOI:10.1242/jcs.257006

9

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

TAKEUCHI, Ikuo

SUZUKI, Yoshiaki

MOHRI, Hideo

KATSUKI, Motoya

NAGAHAMA, Yoshitaka

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

OHSUMI, Yoshinori

HORIUCHI, Takashi

OKADA, Kiyotaka

NISHIMURA, Mikio

YAMAMORI, Tetsuo

Professor Emeritus

Professor Emeritus

Professor Emeritus

IGUCHI, Taisen

YAMAMOTO, Masayuki

NODA, Masaharu〔Original Paper by Yoshitaka Nagahama〕〔Original Papers by Masaharu Noda〕

J., Takeuchi, A., Hosono, K., Fleming, T., Nagahama, Y.,• Yamashita,
and Okubo, K. (2020). Male-predominant galanin mediates androgen-T., Hiyama, T.Y., Kobayashi, K., Kobayashi, K., and Noda,• Matsuda,
M. (2020). Distinct CCK-positive SFO neurons are involved in

dependent aggressive chases in medaka. eLife 9. DOI: 10.7554/
eLife.59470

persistent or transient suppression of water intake. Nat. Commun. 11.

〔Original Paper by Mikio Nishimura〕
N., Bizan, J., Wilkens, A., Tarnawska-Glatt, K., Goto-Yamada,• Stefanik,
S., Strzalka, K., Nishimura, M., Hara-Nishimura, I., and Yamada,K. (2020). NAI2 and TSA1 drive differentiation of constitutive andinducible ER body formation in brassicaceae. Plant Cell Physiol. 61,
722–734. DOI: 10.1093/pcp/pcz236〔Original Paper by Testuo Yamamori〕
T., Komatsu, Y., and Yamamori, T. (2020). Expression patterns• Sasaki,of SLIT/ROBO mRNAs reveal a characteristic feature in the entorhinalhippocampal area of macaque monkeys. BMC Res. Notes 13, 262. DOI:
10.1186/s13104-020-05100-7

10•

DOI: 10.1038/s41467-020-19191-0
Perera, S.N., Williams, R.M., Lyne, R., Stubbs, O., Buehler, D.P.,
Sauka-Spengler, T., Noda, M., Micklem, G., Southard-Smith, E.M.,
and Baker, C.V.H. (2020). Insights into olfactory ensheathing cell
development from a laser-microdissection and transcriptome-profiling
approach. Glia 68, 2550–2584. DOI: 10.1002/glia.23870

National Institute for Basic Biology

Cell Biology
DIVISION OF CELLULAR DYNAMICS

Professor

UEDA, Takashi

Assistant Professor:

EBINE, Kazuo
KANAZAWA, TakehikoSpecially Appointed Assistant Professor:
MINAMINO, Naoki
Technical Staff:
HAYASHI, Kohji
Research Staff:
HIWATASHI, Takuma
FENG, Yihong
SOKENDAI Graduate Student: HACHINODA, Sho
Visiting Graduate Student: NORIZUKI, Takuya
Technical Assistant:
YAMAMOTO, Mayuko
OHARA, Satomi
YOSHINORI, Yumi
Admin Support Staff:
OKUBO, Masayo

the detailed molecular mechanisms and physiological
functions of membrane trafficking in higher-orderedplant 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 has 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 and specialization of membrane trafficking pathways in land plants havebeen achieved by 1) acquisition of novel machinery components, 2) relocation of 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 oilbody
The oil body is an organelle specific to liverworts, whose
origin and biogenesis mechanism remain unclear. We are
studying the oil body in M. polymorpha, as a model of newly-acquired organelles in specific lineages during evolution.
Through comprehensive analyses of SNARE members andorganelle markers in M. polymorpha, we identified a memberof the SYP1 group. Secretory cargos were targeted to the oilbody (Figure 1), which indicated that the oil body is formedby the redirection of the secretory pathway. We additionallyproposed the oil body cycle hypothesis; the periodic redirection of the secretory pathway mediates oil body development
and growth of the oil body cell. Furthermore, we revealed
that the oil body acts as a defense against potential arthropod
herbivory by using mutants of the master regulator of oilbody formation that we identified, MpERF13.
Visual overview of this lab’s work.

Membrane traffic between single membrane-boundedorganelles plays an integral role in various activities ineukaryotic cells. Recent comparative genomics has indicated that membrane trafficking pathways are diversified
among eukaryotic lineages, which are associated with thelineage-specific acquisition of new trafficking pathways
and the secondary loss of preexisting trafficking routes.
Our long-term goal is to reveal how plants have acquired
their unique membrane 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

Figure 1. The redirection of the secretory pathway in oil body cells. The
secretory cargo, sec-mRFP, is targeted to oil bodies (A) and the extracellular space (B), when expressed in hypothetical “oil body phase” and“plasma membrane phase,” respectively. Bars = 10 μm. (Kanazawa etal., 2020)11

National Institute for Basic Biology

Cell Biology

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 of oilbody morphogenesis remain to be determined. We have successfully isolated some mutants defective in oil body morphogenesis in M. polymorpha, which would lead to identification of unknown factors involved in oil body biogenesis.

Consequently, we are now investigating the molecularfunction of PICALMs with a special focus on the mechanisms of cargo recognition by PICALMs in A. thaliana.

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 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-coatedvesicle formation. ANTH proteins have been remarkably
expanded during land plant evolution, and we are investigating how this protein family has been functionally diversified in A. thaliana. We found that a pair of ANTH proteins,
PICALM1a and PICALM1b, are required for retrieving
a secretory SNARE protein, VAMP721, from the plasma
membrane. This function is required for normal vegetative
development of A. thaliana (Figure. 2). This finding also
highlighted the divergent mechanisms of VAMP7 recyclingfrom the plasma membrane between plants and animals.
We also found that another paralogous set of PICALM
proteins, PICALM5a and 5b is required for tip-localizationof ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity in A. thaliana(Figure 2). Thus, functionally differentiated ANTH proteins
underpin various physiological processes in A. thaliana.

12

Figure 2. PICALM1 and PICALM5 mediate endocytosis of distinct
plasma membrane proteins, which are required for normal vegetative
development and pollen tube integrity, respectively. (Left: Fujimoto etal., 2020, modified, right: Muro et al., 2018, modified)

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-direction of
the secretory pathway. As such, KNOLLE/SYP111 plays animportant role in membrane fusion in the formation 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.

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, especiallyspermiogenesis, using fluorescently-tagged organelle markers

in M. polymorpha. The majority of the endomembranousorganelles, such as the Golgi apparatus, were removed frommaturing spermatozoid cells, and the plasma membrane wasalso reorganized during spermiogenesis. Inspection by transmission electron microscope and live-cell imaging analyses
also indicated that the number of degradative organelles suchas the multivesicular endosome, vacuole, and autophagosome,is transiently increased during this process. To reveal themolecular mechanisms of cytoplasm removal and organelle
remodeling, we have established the analytical tools of autophagy in M. polymorpha (Norizuki et al., 2019). M. polymorpha
possesses core machineries of autophagy with lower degrees
of redundancy. The mutations in MpATG5 and MpATG7,
which are key factors for autophagosome formation, affected
the transportation of cytosolic components to the vacuole fordegradation (Figure 4).

Publication List:〔Original papers〕
Z., Tanaka, M., Kanazawa, T., Haraguchi, T., Takyu, A., Era, A.,• Duan,Ueda, T., Ito, K., and Tominaga, M. (2020). Characterization of ancestral

•
•
•
•
•
•

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.
Although a majority of organelles are removed during spermiogenesis, a specific set of organelles persists in mature
spermatozoids, which implies that there should be a mechanism for selective removal of unneeded organelles, which weare also investigating. 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 an essential role in generating fully functional flagella (Figure 5).

••

myosin XI from Marchantia polymorpha by heterologous expression in
Arabidopsis thaliana. Plant J. 104, 460–473. DOI: 10.1111/tpj.14937
Fujimoto, M., Ebine, K., Nishimura, K., Tsutsumi, N., and Ueda, T.
(2020). Longin R-SNARE is retrieved from the plasma membrane by
ANTH domain-containing proteins in Arabidopsis. Proc. Natl. Acad.
Sci. U.S.A. 117, 25150–25158. DOI: 10.1073/pnas.2011152117
Kanazawa, T., Morinaka, H., Ebine, K., Shimada, T.L., Ishida, S.,
Minamino, N., Yamaguchi, K., Shigenobu, S., Kohchi, T., Nakano A.,
Ueda, T. (2020). The liverwort oil body is formed by redirection of
the secretory pathway. Nat. Commun. 11. DOI: 10.1038/s41467-02019978-1
Lupanga, U., Roehrich, R., Askani, J., Hilmer S., Kiefer, C., Krebs, M.,
Kanazawa, T., Ueda, T., and Schumacher, K. (2020). The Arabidopis
V-ATPase is localized to the TGN/EE via a seed plant-specific motif.
eLife 9. DOI: 10.7554/eLife.60568
Otsuka, K., Mamiya, A., Konishi, M., Nozaki, M., Kinoshita, A.,
Tamaki, H., Arita, M., Saito, M., Yamamoto, K., Hachiya, T., et al.
(2021). Temperature-dependent fasciation mutants provide a link
between mitochondrial RNA processing and lateral root morphogenesis.
eLife 10. DOI: 10.7554/eLife.61611
Romani, F., Banic, E., Florent, S.N., Kanazawa, T., Goodger, J.Q.D.,
Mentink, R.A., Dierschke, T., Zachgo, S., Ueda, T., Bowman L.J.,
Tsiantis, M., et al. (2020). Oil body formation in Marchantia polymorphais controlled by MpC1HDZ and serves as a defense against arthropod
herbivores. Curr. Biol. 30, 2815+. DOI: 10.1016/j.cub.2020.05.081
Shimada, T.L., Ueda, T., and Hara-Nishimura, I. (2021). Excess
sterol accumulation affects seed morphology and physiology
in Arabidopsis thaliana. Plant Signal. & Behav. 16. DOI:
10.1080/15592324.2021.1872217
Shimizu, Y., Takagi, J., Ito, E., Ito, Y., Ebine, K., Komatsu, Y., Goto,
Y., Sato, M., Toyooka, K., Ueda, T., et al. (2021). Cargo sorting zones
in the trans-Golgi network visualized by super-resolution confocal
live imaging microscopy in plants. Nat. Commun. 12. DOI: 10.1038/
s41467-021-22267-0
Suzuki, R., Ueda, T., Wada, T., Ito, M., Ishida, T., and Sawa. S. (2021).
Identification of genes involved in Meloidogyne incognita-induced
gall formation processes in Arabidopsis thaliana. Plant Biotech. 38.
DOI:10.5511/plantbiotechnology.20.0716a〔Review article〕
T., Minamino, N., and Ueda, T. (2020). Role of autophagy in• Norizuki,
male reproductive processes in land plants. Front. Plant Sci. 11. DOI:
10.3389/fpls.2020.00756〔Book Chapter〕
E., Choi, S.W., and Ueda, T. (2020). Purification and interaction• Ito,
analysis of a plant-specific RAB5 effector by in vitro pull-down assay.
Methods Mol Biol 2177, 183-197. DOI: 10.1007/978-1-0716-07671_15

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.

13National Institute for Basic Biology

Cell Biology
DIVISION OF QUANTITATIVE BIOLOGY

Professor

AOKI, Kazuhiro

Assistant Professor:

KONDO, Yohei
GOTO, Yuhei
Technical Staff:
BINO, Takahiro
Postdoctoral Fellow:
NAKAMURA, Akinobu
SOKENDAI Graduate Student: TANII, Ryosuke
MUKAI, Masaya
YAMAMOTO, Kei
SAKAI, Keiichiro
TSURUOKA, Tatsuki
Visiting Scientist:
ITO, Reina
Technical Assistant:
EBINE, Emi
GOTO, Yoko
Admin Support Staff:
ONODA, Kaori

The intracellular signal transduction system has been extensively studied over the past few decades through approaches
utilizing both biochemistry and cell biology approaches. As
a result, many proteins and regulations have been identified,
which has resulted in an increase in the pathway’s complexity. The complicated signaling network makes it difficult tounderstand how cells process information and quantitatively
make decisions.
To address these issues, we are currently focusing on thedevelopment of two types of research tools that enable us
to (1) visualize and (2) manipulate intracellular signaling
pathways (Figure 1).

Figure 1. Information processing by intracellular signaling devices andnetworks.

I. Visualization of cell signaling

Visual overview of this lab’s work, showing fluorescence images ofmammalian cells, fission yeast, and C.elegans

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 implemented by an intracellularsignal transduction system, which is comprised of a series ofchemical reactions such as protein-protein interactions andprotein phosphorylation. Dysregulation of cell signaling by
gene mutation is widely known to result in various pathologies, such as malignant tumors.

14

With the advent of fluorescent proteins, it has become
possible to visualize kinetic reactions at a 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 fluorophore. FRET-based biosensors allow us to detect PKA, ERK,
Akt, JNK, PKC, and S6K’s kinase activity in living cells at ahigh temporal and spatial resolution (Komatsu N, Mol. Biol.
Cell, 2011). By using a FRET biosensor, we have been able
to reveal the role played by ERK activation dynamics in cellproliferation (Aoki K, Mol Cell, 2013) and collective cell
migration (Aoki K, 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 this limitation, we are currently developing single-fluorophorebased biosensors. We recently developed a red-fluorescence
dopamine (DA) reporter. DA, a neuromodulator, is involvedin many neuronal functions, and to monitor it in living
cells and animals, we have designed a red-fluorescent DA
reporter. This reporter features a circular-permutated mApple(cpmApple), a red fluorescent protein, and a cpmApple wasinserted into the third intracellular loop of a DA receptor,
DRD1 (Figure 2A). The linker sequences between DRD
and cpmApple were optimized by random mutagenesis andsubsequent live-cell screening. Finally, we picked up the best
performance reporter and named the red genetically encodedGPCR activation reporter for DA, R-GenGAR-DA. The
red fluorescence intensity decreased in response to DA andreturned to the basal level upon a DRD1 antagonist treatment (Figure 2B and 2C). In addition, by using a previously
reported green fluorescence norepinephrine (NE) reporter,
we succeeded in simultaneously visualizing DA and NE inliving neurons (Nakamoto C, Goto Y, et al., bioRxiv, 2020).

A DRD1

R-GenGAR-DA
SP
FLAG

ICL1 ICL2

ICL3
cpmApple

B HeLa cells
Basal

R-GenGAR-DA1.2
DA

⊿ F/F

0.2

–0.6

C Primary neurons
R-GenGAR-DA1.2
Basal

DA

MYPT1 with an optogenetic dimerizer, so that it allows
light-dependent recruitment of endogenous PP1c to theplasma membrane (Figure 3A). We confirmed that blue-lightillumination was sufficient to induce dephosphorylationof MLC, resulting in lamellipodial membrane protrusion(Figure 3B). The OptoMYPT was further employed to understand the mechanics of actomyosin-based cortical tension
and contractile ring tension during cytokinesis (Figure 3C).
We found that the relaxation of cortical tension at both poles
by OptoMYPT accelerated furrow ingression rate, thus
revealing that the cortical tension substantially antagonizes
constriction of the cleavage furrow. These results provide
new opportunities to freely design cell and tissue morphology through light (Yamamoto, et al., bioRxiv, 2021).
The Blue light-responsive optical dimerizers are useful, but
its activation light often overlaps with the excitation light offluorescent proteins, which in turn hampers the applicationof GFP or FRET biosensors together with their optogenetic
tools. To circumvent this issue, we are focusing on the phytochrome B (PhyB)-PIF LID system. PhyB binds to PIF upon
red-light illumination, and the PhyB-PIF complex dissociatesfrom each other by far-red light exposure (Figure 4A). The

SCH

⊿ F/F

0.3

–0.8

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.

II. Manipulation of cell signaling
Artificial manipulation of biochemical networks is wellestablished in the quantitative understanding of biologicalsystems. Multiple methods have been suggested for controlling signal transduction, including light-induced dimerization(LID) and chemically induced dimerization (CID) systems.
Of these two, the LID system is more beneficial in termsof temporal and spatial manipulation. The photo-responsive proteins derived from fungi, cyanobacteria, plants, andmodified fluorescent proteins are used in this system.
We have reported a novel optogenetic tool to induce relaxation of actomyosin contractility with a blue light-responsive protein, such as cryptochrome 2 (CRY2) and iLID.
Actomyosin contractility, which is generated cooperatively
by nonmuscle myosin II (NMII) and actin filaments, plays
essential roles in a wide range of biological processes suchas cell motility and cytokinesis. Yet, it remains elusive as tohow actomyosin contractility generates force and maintains
cellular morphology. To elucidate this, we have developed
a novel optogenetic tool, OptoMYPT, which combines acatalytic subunit of type I phosphatase-binding domain of

Figure 3. OptoMYPT: Optogenetic relaxation of actomyosin contractility.
(A) Schematic representation of OptoMYPT. (B) Local recruitment of
OptoMYPT induced myosin regulatory light chain dephosphorylation,
leading to the induction of membrane protrusion. (C) Local relaxation ofactomyosin cortical tension at the both poles in mitotic cells accelerated
ingression of contractile ring.

15

National Institute for Basic Biology

Cell Biology

reversible control of association and dissociation by lightis a unique feature in the PhyB-PIF system because other
LID systems can only control association or dissociation by
light. One drawback is that covalent attachment of a chromophore, phycocyanobilin (PCB), is required for the lightresponsive function of PhyB-PIF. To overcome this issue,
we have developed a method for PCB biosynthesis in mammalian cells by introducing the gene products of HO1, PcyA,
Fd, and Fnr into the mitochondria (Uda Y, et al., PNAS,
2017) (Figure 4A). We recently improved the PCB synthesis system, named SynPCB2.1, which allowed establish-

ing stable cell lines that synthesize PCB and light-induced
control of protein localization at the cell population level(Figure 4B and 4C) (Uda Y, et al., ACS Chem Biol, 2020).
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. pombeand 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, and Opto-SAC
induced a spindle assembly checkpoint (SAC) in responseto red light (Figure 5) (Goto Y, and Aoki K., bioRxiv, 2020).

Publication List:〔Original papers〕
Y., Kondo, Y., Aoki, K., and Naoki, H. (2021). Hierarchical• Asakura,modeling of mechano-chemical dynamics of epithelial sheets across••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).

•••cells and tissue. Sci. Rep. 11. DOI: 10.1038/s41598-021-83396-6
Hino, N., Rossetti, L., Marin-Llaurado, A., Aoki, K., Trepat, X., Matsuda,M., and Hirashima, T. (2020). ERK-mediated mechanochemical waves
direct collective cell polarization. Dev. Cell 53, 646+. DOI: 10.1016/j.
devcel.2020.05.011
Nakamura, A., Oki, C., Kato, K., Fujinuma S., Maryu, G., Kuwata, K.,
Yoshii, T., Matsuda, M., Aoki, K., and Tsukiji, S. (2020). Engineeringorthogonal, plasma membrane-specific SLIPT systems for multiplexedchemical control of signaling pathways in living single cells. ACS
Chem. Biol. 15, 1004–1015. DOI: 10.1021/acschembio.0c00024
Uda, Y., Miura, H., Goto, Y., Yamamoto, K., Mii, Y., Kondo, Y., Takada,S., and Aoki, K. (2020). Improvement of phycocyanobilin synthesis
for genetically encoded phytochrome-based optogenetics. ACS Chem.
Biol. 15, 2896–2906. DOI: 10.1021/acschembio.0c00477
Wen, C., Miura, T., Voleti, V., Yamaguchi, K., Tsutsumi, M., Yamamoto,K., Otomo, K., Fujie, Y., Teramoto, T., Ishihara, T., et al. (2021).
3DeeCellTracker, a deep learning-based pipeline for segmenting
and tracking cells in 3D time lapse images. eLife10. DOI: 10.7554/
eLife.59187
Yano, T., Tsukita, K., Kanoh, H., Nakayama, S., Kashihara, H., Mizuno,T., Tanaka, H., Matsui, T., Goto, Y., Komatsubara, A., et al. (2021).
A microtubule-LUZP1 association around tight junction promotes
epithelial cell apical constriction. EMBO J. 40. DOI: 10.15252/
embj.2020104712〔Review Articles〕
S., Aoki, K., and Kubo, Y. (2020). Biophysical research in• Akiyama,
Okazaki, Japan. Biophys. Rev. 12, 237–243. DOI: 10.1007/s12551-•

Figure 5. Optogenetic control of cell cycle in fission yeast. (A) Lightinduced recruitment of PIF3-mNeonGreen (mNG) to the plasma
membrane (left) and nucleus (right). (B) Opto-G2/M: Optical control of
G2/M transition. (left) Opto-G2/M OFF cells showed normal morphology, while Opto-G2/M ON cells exhibited longer cell length, a typicalphenotype of G2/M arrest fission yeast. (C) Opto-SAC: Optical controlof spindle assembly checkpoint. Representative cells undergoing metaphase arrest (right) by Opto-SAC. (D) Long-term metaphase arrest by
Opto-SAC leads to a mitotic failure and cut phenotype.

16

020-00633-4
Goto Y., Kondo Y., Aoki K. (2021). Visualization and Manipulationof Intracellular Signaling. Adv. Exp. Med. Biol. 1293, 225–234. DOI:
10.1007/978-981-15-8763-4_13

DIVISION OF CHROMATIN REGULATION

Professor

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.

NAKAYAMA, Jun-ichi

Assistant Professor:
KATAOKA, Kensuke
Specially Appointed Assistant Professor:
HAYASHI, Aki
Technical Staff:
NISHIMOTO, Yuki
SOKENDAI Graduate Student: RAHAYU, Anisa
VALENTIROVIC, Olivera
NAKAMURA, Rinko
YOSHIDA, Takaki
Visiting Graduate Student: HACHISUKA, Aki
Visiting Scientist:
HAMADA, Kyoko
Technical Assistant:
YOSHIMURA, Yuriko
ASAI, Yuriko
Admin Support Staff:
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 this 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.

I. Establishment and maintenance of higherorder chromatin structures
1-1 Mechanisms regulating Clr4 histone methyltransferase activity
In eukaryotic cells, the assembly of higher-order chromatin
structures, known as heterochromatin, plays an importantrole in diverse chromosomal processes and epigenetic gene
regulation. Heterochromatin is characterized by the methylation of histone H3 at lysine 9 (H3K9me). H3K9me iscatalyzed by SUV39H-family histone methyltransferases.
In fission yeast, H3K9me is catalyzed by histone methyltransferase Clr4. Clr4 has two functional domains, theN-terminal chromodomain (CD), which recognizes H3K9me,
and the C-terminal SET domain responsible for Clr4’s enzymatic activity (Figure 1A). Since Clr4’s uncontrolled activity
leads to inappropriate H3K9me and aberrant gene silencing, its enzymatic activity needs to be strictly controlled. A
previous study showed that N-terminally deleted Clr4 mutant
exhibits a stronger activity than full-length Clr4, suggesting that the Clr4 N-terminal region negatively regulates theactivity of C-terminal SET domain. To examine the regulation mechanisms of Clr4’s activity, we tested whether Clr4
N-terminal region interacts with its C-terminal SET domain.
Pull-down assays using recombinant proteins reveal that
the Clr4 N-terminal region can potentially interact with theSET domain and that this interaction requires both CD and
its adjacent region (Figure 1B). To determine the regions/residues responsible for this interaction, we performed
cross-linking mass spectrometry analyses and found thatcrosslinked residues were concentrated in a region near the
C-terminal end of the SET domain (Figure 1C). These results
support the idea that Clr4’s enzymatic activity is regulated byinter- or intra-domain interactions.

Figure 1. Characterization of inter- and/or intra-domain interactions of
Clr4. (A) Domain organization of Clr4. The N-terminal chromodomain(CD) recognizes H3K9me, and the C-terminal SET domain is responsible
for Clr4’s enzymatic activity. (B) Pull-down assays of recombinant Clr4
to determine domains required for the interaction. Proteins were analyzed
by SDS-PAGE and CBB staining. (C) Residues identified in the crosslinking mass spectrometry to be involved in the interaction are mappedon 3D structure of Clr4 SET domain.

17

National Institute for Basic Biology

Cell Biology

1-2 C haracterization of Clr4 methyltransferase
complex (CLRC) in fission yeast
In fission yeast, Clr4 is the sole H3K9 methyltransferase
and is essential for heterochromatin assembly. Biochemicaland genetic analyses revealed that Clr4 forms a multiprotein complex called CLRC. CLRC consists of Cul4, Rik1,
Raf1, Raf2, and Rbx1, and shows structural similarity to the
CUL4-DDB1 complex involved in the repair of damaged
DNA. We previously have demonstrated that affinity-purified CLRC ubiquitylates histone H3 and that H3 lysine
14 (H3K14) is the preferred target of the complex. We
further demonstrated that CLRC-mediated H3 ubiquitylation
promotes H3K9me by Clr4, suggesting a cross-talk mechanism between histone ubiquitylation and methylation thatis involved in heterochromatin assembly (Oya et al. 2019).
Although this study uncovered one of the physiologicaltargets of CLRC, how Clr4 interacts with other components
in CLRC remains unclear. To investigate the functional relationship between Clr4 and the CLRC, we expressed FLAGtagged Clr4 in a strain expressing Rik1-myc and performed
an IP-western experiment to confirm the interaction between
Clr4 and Rik1 (CLRC). Using this assay, we expressed aseries of Clr4 deletion mutants and successfully identified adomain which is necessary for the interaction with CLRC.
Based on the above results, we performed a yeast two-hybrid
assay and identified a candidate CLRC component responsible for the binding to Clr4. These results suggest that theinteraction between Clr4 and CLRC is coupled with Clr4’s
intramolecular conformational change.

Figure 2. A representative result of Yeast two-hybrid assay using strains
expressing full-length Clr4 or Clr4-SET in combination with full-length
or truncated mutant of the candidate protein.

II. R oles of chromodomain proteins inassembling higher-order chromatin
structure
2-1 Mitotic phosphorylation of Swi6/HP1 regulates
mitotic chromosome segregation
HP1 is a conserved chromosomal protein that plays important roles in heterochromatin assembly. There is growing
evidence that HP1-family proteins undergo a variety of posttranslational modifications, including phosphorylation, acetylation, methylation, and ubiquitination. Phosphorylation,
the most extensively studied of these modifications, is widely
implicated in HP1’s dynamics and functions. We previously
showed that Swi6, one of two HP1 isoforms in S. pombe,
is constitutively phosphorylated by casein kinase II (CK2)
and this phosphorylation is essential for its function inheterochromatin assembly. Several previous studies demonstrated that HP1 is subject to additional phosphoryla-

18tion during mitosis. However, the functional importance ofHP1’s mitotic phosphorylation remains unclear. Using E.
coli co-expression system, we demonstrated that Swi6 isphosphorylated by Ark1, a solo Aurora kinase in S. pombe.
Subsequent mutation analyses revealed that serine residues
in the N-terminal region of Swi6 are efficient targets for
Ark1. We confirmed that these serine residues are phosphorylated during mitosis in vivo. Interestingly, expressionof mutant Swi6 containing amino-acid substitutions at theserine residues differentially modulates temperature-sensitive growth of the mutations for Chromosome Passenger
Complex (CPC) components. These results suggested thatArk1-mediated Swi6 phosphorylation regulates CPC’s
function during mitosis.

Figure 3. Mitotic phosphorylation of Swi6 regulates mitotic chromosome
segregation. (A) Swi6 domains, N-terminal region (N), CD, and CSD,
produced in control or control or Ark1-co-expressed E. coli cells were
purified, resolved by standard or Phos-tag PAGE, and visualized by CBB
staining. (B) Phosphorylation patterns of wild-type (wt) or mutant Swi6
proteins were analyzed by immunoblotting using anti-Swi6 antibody.
(C) Serially diluted cells of the indicated strains were spotted on nonselective media and incubated at indicated temperatures.

III. Dynamic chromatin reorganization during
sexual reproduction of Tetrahymena
3-1 Multiple HP1-like protein-containing complex
regulates DNA elimination in Tetrahymena
Heterochromatin plays important roles in transposon(TE) silencing. A major type of heterochromatin contains
chromatin, that is histone H3 methylated at lysine 9/27
(H3K9/27me), and its reader HP1 proteins, that recruit
diverse proteins onto the chromatin to silence the TEs.
Although multiple HP1 proteins are co-expressed in many
eukaryotic cells, the interplay between these HP1 proteinshas been elusive. Here, we show that subset of the HP1proteins form a complex and play important roles to eliminate TE-related Internal Eliminated Sequences (IESs) fromthe somatic genome during macronuclear development in
the ciliated protozoan Tetrahymena. We tethered 7 HP1-likeproteins individually to the artificially created locus by the
LexA-LexO system and found that only 4 of them includ-ing Pdd1 induced the elimination of the tethered site (Figure5). This ectopic DNA elimination was exclusively achievedby their chromoshadow domains (CSDs), indicating that
the CSDs of the distinct type of HP1-like proteins recruits
all the proteins that are required for DNA elimination.
Immunoprecipitation of Pdd1 specifically enriched the otherHP1-like proteins that were sufficient for the ectopic DNA
elimination. The chromodomains of a subset of HP1-likeproteins showed strong affinity to both H3K9me3 andH3K27me3. Overall, these results suggest that multiple
HP1-like proteins cooperatively recognize the methylated
histones and form core complex to recruit other effector
proteins for DNA elimination.

Figure 4. Identification of heterochromatin proteins sufficient to induce
DNA elimination. Schematics of the tethering assay (top). A representative result of the tethering assay using Pdd1p (bottom).

3-2 The HP1-like protein Hpl8p is important for programmed DNA elimination in Tetrahymena
Transposon mobility represents a threat for the genome
integrity in all living things. To counter this, the ciliatedprotozoan Tetrahymena uses heterochromatin to selectively eliminate over 10,000 transposon loci from its somatic
nucleus in the developmentally programmed process of
DNA elimination. To address how this process is coordinatedby heterochromatin components, we focus on an HP1-likeprotein named Hpl8p that localizes to the developing macronucleus during heterochromatin formation and DNA elimination (Figure 5). The chromodomain of Hpl8p recognizedboth H3K9me3 and H3K27me3 in vitro. HPL8 disrupted
mutants showed defect in vegetative growth and in the progression of sexual reproduction. FISH analyses showed that
more than 90% of HPL8 disrupted cells did not complete
its elimination, suggesting that Hpl8p plays important roles
in DNA elimination. Altogether, these results suggest that
Hpl8p binds the H3K9/27me marks over transposon lociand facilitates the heterochromatin formation by interacting with heterochromatin components required for DNA

elimination. Furthermore, we propose that Hpl8p also plays
important roles in suppression of unwanted genes during thevegetative growth and sexual reproduction process possibly
by recognizing H3K27me3 in the transcriptionally active
macronucleus.

IV. Analysis of chromatin dynamics in spore
nuclei of fission yeast
Gametogenesis is a crucial process for sexually reproducing organisms to produce haploid gametes from diploidcells. Generally, epigenetic memory is erased from zygote
nucleus, a phenomenon known as reprogramming, resulting in increased totipotency. On the other hand, it has been
demonstrated in some organisms that a part of epigenetic
memory can be inherited transgenerationally. The balance
between reprogramming and epigenetic inheritance plays a
key role in the developmental processes of offspring. It has
been also reported that some gametes, like plant sperm andmammalian spermatozoa, have highly compacted nuclei andhave altered histone modifications. However, the mechanisms underlying how epigenetic marks are regulated during
gametogenesis remain to be fully elucidated. To gain an
insight into epigenetic inheritance and chromosome structural change during gamete formation, we analyzed chromatin dynamics in spore nucleus of fission yeast. We isolated
fission yeast spores by density-gradient centrifugation andperformed mass spectrometry analysis to determine sporespecific nuclear proteins. We further examined the change inhistone modifications of spore nuclei. Chromatin immunoprecipitation analysis revealed that the levels of H3K9me atheterochromatic regions had not been noticeablely changed,
but the levels of H3K4me associated transcriptionally active
regions had clearly increased in spore nuclei. These results
imply that dynamic changes in active histone demonstrate acontribution to spore formation and epigenetic inheritance.

Figure 6. A representative image of vegetative cells (veg.) and spores in asci.

Publication List:〔Original paper〕
C., Nakagawa, R., Oyama, K., Yamamoto Y., Zhang, W., Dong, A.,• Dong,
Li, Y., Yoshimura Yuriko and Kamiya, H., Nakayama, J., Ueda, J., and Min,J. (2020). Structural basis for histone variant H3tK27me3 recognition by
PHF1 and PHF19. eLife 9, e58675. DOI: 10.7554/eLife.58675〔Book chapter〕
Figure 5. Cells expressing the Hpl8-mNeonGreen (green) and Pdd1pmCherry (red) at 8 and 13 hours post induction of mating are shown in(a) and (b), respectively. DNA was counterstained with DAPI. Arrows
and arrowheads indicate the parental and new MACs, respectively.

K., Tani, T., and Nakayama, J. (2020). Proteomics• Hiragami-Hamada,based systematic identification of nuclear proteins anchored tochromatin via RNA. Methods Mol Biol 2161, 88-99. DOI: 10.1007/9781-0716-0680-3_8

19National Institute for Basic Biology

Cell Biology
LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:

NAKAYAMA, Kei
OHASHI, Rie
SOKENDAI Graduate Student: YAMASHITA, Akira
HORIO, Tomoyo
ISHIKURA, Yui
YOSHIDA, Sho
Technical Assistant:
KATAYAMA, Kaorition-dependent local translation of the cargo mRNAs (Figure1). We are currently using mice to research the mechanism of
RNA granule assembly, RNA granule factors and their phasebehavior regulating mRNA transport and local translation,
their target mRNAs, and the roles of the locally synthesized
proteins, so we can attain a better understanding of their relationship to the formation of synapses and neural networks,
memory, learning, and behavior.
In addition to the physiological function, RNA granules
are linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar
degeneration (FTLD). The basis of these diseases is thought
to be the incorporation and aggregation of disease-causing
proteins such as FUS and TDP-43 in RNA granules. Thus
our research also aims to reveal the effects of such protein
aggregation on mRNA transport and local translation.

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. Stress tolerance of conditioned fear memory
conferred by alternatively spliced prion-likedomain of Ilf3

RNA granules (green) transported from the cell body (magenta) to dendrites in the hippocampus of the mouse brain.

The transportation 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
accepted that this system controls the location at which
neurites will stably connect to each other, thereby forming
long-term neural networks and memory. Our main interest isunderstanding the mechanisms and roles of mRNA transportand local translation in neuronal dendrites.
Specific mRNAs are recruited into “RNA granules” andtransported to dendrites. RNA granules are membranelessorganelles formed by liquid-liquid phase separation (LLPS)
of RNA-binding proteins and mRNAs, which further recruitother factors such as ribosomes. They mediate the transportof mRNAs to the vicinity of synapses and synaptic stimula-20

LLPS is generally driven by protein intrinsically disorderedregions (IDRs) that do not form three-dimensional structures.
One class of IDR is the prion-like domain (PrLD), whose
propensity to form aggregates has been well studied inregards to FUS and TDP-43 in neurodegenerative diseases.
However, little is known about the physiological relevance of
PrLDs in the brain.
We focused on an RNA-binding protein NFAR2, which
has PrLD at the C-terminus. This PrLD is spliced out in thealternative splicing isoform NFAR1. Therefore, it is possible
to investigate the role specific to PrLD by deleting PrLD of
NFAR2, since NFAR1 without PrLD is intrinsically present
and maintained even after the deletion of PrLD. NFAR1 andNFAR2 are synthesized from the Ilf3 gene and both possess
biochemical activities that they perform in common such astranscriptional regulation and stress-dependent translational
inhibition. However, they differ in that NFAR2 alone canconcentrate in the nucleoplasm and associate with cytoplasmic
RNA granules via PrLD. To investigate the physiological relevance of PrLD, we generated NFAR2ΔPrLD mice, in which
a stop codon was introduced into the NFAR2-specific exon.
Deletion of the PrLD lost the nucleoplasmic localizationof NFAR2 and affected the genome-wide profiles of mRNA

expression and translation in the brain. In particular, PrLD
deletion significantly altered chronic restraint stress (CRS)induced changes in mRNA expression and translation in the
amygdala, a brain region associated with emotional events
such as anxiety and fear.
Consistent with its effect on CRS-induced changes inmRNA expression and translation, PrLD deletion affected
tolerance of amygdala-associated learning and memory to
CRS: conditioned fear memory in wild-type mice was unaffected by CRS, but it was exacerbated in NFAR2ΔPrLD
mice due to this factor. These results suggested that PrLD of
NFAR2 conferred tolerance of fear-associated memory formation to stressful environments.

II. I ncorporation of FUS and TDP-43 into
RNA granules releases RNG105 (Caprin1)
from the granules
FUS and TDP-43 are predominantly localized in the
nucleus when the cell is healthy, but translocate to the cytoplasm and incorporate into RNA granules when stricken by
neurodegenerative diseases. This incorporation is thought
to affect the fluidity of the components of RNA granules
formed by LLPS, but little is known about their effects. We
overexpressed either disease-related FUS mutants or disease
related TDP-43 mutants in primary cultured neurons fromthe mouse cerebral cortex and analyzed their effects on thefluidity of RNA granule components using fluorescencerecovery after photobleaching (FRAP) and cell permeabilization assays (Shiina, J. Biol. Chem., 2019).
FUS and TDP-43 did not affect the fluidity of FMRP or
PUM2 in dendritic RNA granules, but did affect the fluidityof RNG105 (Caprin1) (Figure 2). The fluidity of RNG105
was significantly increased, resulting in the release of
RNG105 from RNA granules into the cytoplasm of dendrites.
Since RNG105 is required for mRNA transport to dendrites
and formation of long-term memory (Nakayama et al., eLife,2017), loss of RNG105 in dendritic RNA granules may affectthese functions. We are currently investigating the molecularmechanisms underlying the changes in fluidity specific to
RNG105 and the effects of FUS and TDP-43 incorporation into RNA granules on mRNA recruitment into RNA

granules, local translation in the vicinity of RNA granules,
and synapse formation in dendrites.

III. 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, whereas RNG140 is highly
expressed in the developing eye lens and plays a role in lensdifferentiation. Despite RNG140’s function in translational
regulation, the underlying mechanism and its role within theeye has remained unclear.
We found that RNG140 binds to the translation initiation
factor eIF3 through the application of mass spectrometryof RNG140 immunoprecipitates from cultured CHO cells.
Reporter translation assay revealed that RNG140 represses
translation through mechanisms involving the suppressionof eIF3-dependent translation initiation. Comprehensive
ribosome profiling demonstrated 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 the translation of long mRNAs. mRNAs involved in lens differentiation, such as crystallin mRNAs, are short, and were able toescape translational inhibition by RNG140 and be translated
in differentiating lenses (Figure 3). 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.

Translation

Lens

Proliferation-related mRNAs

TDP-43 PUM2

Lens differentiation-related mRNAs
RNG140

eIF3
RNG140mRNA

Inhibition

No inhibition

Figure 3. mRNA length-selective inhibition of translation by RNG140 ineye lens differentiation.

Publication List:
Maginified

MergedFigure 2. Incorporation of neurodegenerative disease-related proteins
into RNA granules. Primary cultured neurons from mouse cerebral cortex
were transfected with disease-related proteins (TDP-43-GFP in this case)
together with RNA granule components (PUM2-mRFP1 in this case).
The fluidity of these proteins in RNA granules was measured by FRAPand cell permeabilization assays.

〔Original papers〕
T., Okimura, K., Shen, J., Guh, Y.-J., Tamai, T.K., Shimada,• Nakayama,A., Minou, S., Okushi, Y., Shimmura, T., Furukawa, Y., et al. (2020).

•

Seasonal changes in NRF2 antioxidant pathway regulates winterdepression-like behavior. Proc. Natl. Acad. Sci. U.S.A. 117, 9594–
9603. DOI: 10.1073/pnas.2000278117
Nakazawa, K., Shichino, Y., Iwasaki, S., and Shiina, N. (2020).

21

National Institute for Basic Biology

Cell Biology
Implications of RNG140 (caprin2)-mediated translational regulationin eye lens differentiation. J. Biol. Chem. 295, 15029–15044. DOI:
10.1074/jbc.RA120.012715〔Review article〕
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. DOI: 10.3390/biom10020167

22

LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

Specially Appointed Assistant Professor:
KURASHIMA, Kiminori
SOKENDAI Graduate Student: KUMAZAKI, Taisei
MATSUMOTO, Akino
ASAI, Yuriko
Technical Assistant:
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.

Visual overview of this lab’s work.

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 that23

National Institute for Basic Biology

Cell Biology

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.I., Angelone, M., Yoshihashi, S., Ogawa, K., Isobe,• Kobayashi,M., Nishitani, T., Sangaroon, S., Kamio, S., Fujiwara, Y., Tsubouchi
T., Uritani, A., et al. (2020). Thermal neutron measurement bysingle crystal CVD diamond detector applied with the pulse shapediscrimination during deuterium plasma experiment in LHD. FusionEng. Des. 161. DOI: 10.1016/j.fusengdes.2020.112063

24

LABORATORY OF ORGANELLE REGULATION

Associate Professor

MANO, Shoji

Specially Appointed Assistant Professor:
KANAI, Masatake
KAMIGAKI, Akane
Visiting Scientist:
Technical Assistant:
HIKINO, Kazumi
NAGATA, Kyoko
NAKAYAMA, Tomomi
HOSHI, Rie
Admin Support Staff:
UEDA, Chizuru
ASAI, Sanae

Because plants spread their roots in the ground, they must
survive in a given environment. To adapt to their environment,
they intepret various signals generated from environmental
changes as being necessary for their survival. As such, theflexibility of plant organelles is the basis for such adaptation.
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.

Visual overview of this lab’s work, showing plant organelles.
Electronmicrograph represents Arabidopsis cotyledonary cells. Top
and lower insets show the GFP-labeled peroxisomes and oil bodies,
respectively. Peroxisomes and oil bodies are visualized with the fusion
protein of GFP and peroxisome targeting signal 1 (PTS1), and oil bodymembrane protein, oleosin, respectively. Mt: mitochondrion, N; nucleus,
Ob; oil body, P; peroxisome, V; vacuole.

I. M olecular mechanisms of peroxisome
dynamics and functions in plant cells
Peroxisomes are single-membrane-bound organelles, which
are frequently present in eukaryotic cells, and are involvedin various biological processes such as lipid metabolism andphotorespiration. These functions change dramatically duringcertain developmental stages, and when they are confronted
with environmental changes. For example, light induces thetransformation of peroxisomes from glyoxysomes, which areperoxisomes engaged in the degradation of reserve oil stored
in the oil body via β-oxidation and the glyoxylate cycle, toanother type of peroxisome, leaf peroxisomes, that function
during several crucial steps of photorespiration. In addition
to functioning in vegetative tissues such as 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, andmale–female gametophyte recognition. Gene expression, alternative splicing, protein transport, protein degradation and degradation of peroxisomes themselves control these functions.
To better understand peroxisome biogenesis and functions,
we isolated a number of Arabidopsis mutants that displayedaberrant peroxisome morphology (apem mutants) and unusual
peroxisome positioning (peup mutants) based on them having
a different pattern of GFP fluorescence compared to theirparent plant, GFP-PTS1, in which peroxisomes with normal
sizes, numbers and distribution could be visualized with GFP(Figure 1). As of writing, we have reported the functions 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 peroxisomes 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.
We are also currently investigating other apem and peupmutants. Through these analyses, we will be able to identify
the components responsible for peroxisome biogenesis, functions and maintenance, and address the mechanism underlying this phenomenon at the molecular level.
GFP-PTS1

apem1/drp3a

apem3/pmp38

apem5

apem7

apem9

Figure 1. Phenotype of Arabidopsis apem mutants. GFP fluorescence wasobserved in the parent plant, GFP-PTS1, apem1, apem3, apem5, apem7
and apem9 mutants. apem1 and apem3 have elongated and enlarged peroxisomes, respectively. In apem5 mutant, peroxisomes are aggregated. In
apem7 and apem9, GFP fluorescence is observed in the cytosol becauseof the decrease of the efficiency of protein transport to peroxisomes.

25National Institute for Basic Biology

Cell Biology
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 ofoils. Soybeans’ major reserve are proteins. Storage oils andproteins are synthesized in the endoplasmic reticulum (ER)and accumulated in oil and protein bodies, respectively,
during seed development.
We are currently analyzing the molecular mechanisms controlling oil and protein contents in seeds (Figure 2). Basedon 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 transcription factor in fatty acid biosynthesis,
expression during the mid-phase of seed development significantly enhanced seed oil content, and caused seed sizes
to enlarge.
We are also investigating the mechanisms of oil accumulation in other plant species. In the soybean (Glycine max.
L), we identified four lipases, GmSDP1s, on the oil bodymembrane. The analyses of GmSDP1s revealed that plantseeds have a mechanism required for the quality control offatty acids by degrading particular fatty acids in oil bodies.

Lipid synthesis

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 (Figure 3), and, as a public database, it is open to
all researchers. PODB3 consists of six individual sections:the electron micrograph database, the perceptive organelles
database, the organelles movie database, the organellome
database, the functional analysis database, and external links.
The function of each database is as follows:・The electron micrograph database provides information
concerning the ultrastructures in plant cells
・T he perceptive organelles database shows organelles
dynamics responding to environmental stimuli
・The organelles movie database contains time-lapse images
and 3D structural rotations
・The organellome database is a compilation of static image
data of various tissues of several plant species at different
stages of development.
・The functional analysis database is a collection of protocols for plant organelle research
Through these databases, users can more easily comprehendplant organelle dynamics. Plant Organelles World, which isbuilt based on PODB3, is an educational tool for engaging
members of the non-scientific community to explore plantbiology. We hope that both PODB3 and Plant Organelles
World are of help to researchers as well as the general public.

Lipid synthesis (µg/seed)Protein synthesis (µg/seed)Protein synthesis

Early

Mid

Late

Early

Mid

Late

Figure 2. Biosynthesis of lipids and proteins during Arabidopsis seeddevelopment. Lipid synthesis and accumulation begins before those of
seed storage proteins during seed development.

III. D evelopment of Gateway-technology
vectors for plant research
Gateway cloning is a useful and powerful technology whichallows the simultaneous generation of multiple constructs
containing a range of fusion genes. We have developed
various types of Gateway cloning-compatible vectors to
improve resources in the plant research field. Up until now,
we have provided vector sets to detect multiple proteinprotein interactions in vivo using multi-color bimolecularfluorescence complementation, and the binary vectors tofacilitate tripartite DNA assembly and promoter analysis withvarious reporters and tags in the liverwort Marchantia polymorpha. We will continue developing other useful Gateway
cloning-compatible vectors to contribute to the plant researchcommunity.

26

Figure 3. The graphical user interface of the PODB3 (http://podb.nibb.
ac.jp/Organellome).

National Institute for Basic Biology

Developmental Biology
DIVISION OF MORPHOGENESIS

place in the sea in which to live. We also investigate the evolution of the notochord using ancestral chordates.

I. ZO-1 condensates in mouse hatching embryos
Professor

Associate Professor

UENO, NaotoKINOSHITA, Noriyuki

Assistant Professor:
Technical Staff:
Technical Assistant:

TAKAHASHI, Hiroki
TAKAGI, Chiyo
YAMAMOTO, Takamasa
YASUE, Naoko
MIYAKE, Satoko
TSUGE, Toyoko

Admin Support Staff:

In addition to genes and proteins that are widely known
to govern biological phenomena and have been extensively studied over the past several decades, a growing bodyof evidence now suggests that physical environments that
include light, temperature, and internal and external forces
influence both cellular and organismal behaviors. In particular, we are currently investigating the contribution ofphysical forces using mouse embryos and light-dependent
behaviors using coral larvae to understand the impact ofphysical environments on organisms. As reported below, theinner pressure of the blastocyst cavity generates tensile force
upon trophectodermal cells in mouse embryos, and enhances
their cell junction. During this process, a physical phenomenon, liquid-liquid phase separation (LLPS) of the tightjunction protein ZO-1, plays a pivotal role. The coral planula
larvae change their swimming activities by sensing types oflight of specific wavelengths to settle upon an appropriate

A graphical overview of our laboratory’s works.

Cells sense and respond to not only molecular factors but
also mechanical forces, which in turn play various importantroles in biological events, such as tissue homeostasis, differentiation, and cell migration. During early embryogenesis,
various dynamic morphogenetic movements occur, including
the convergent extension of the axial mesoderm and epibolyof the ectoderm. These movements generate physical forcesat both the cellular and tissue levels. However, it is still not
fully understood how these forces influence morphogenetic
processes.
To investigate how embryonic cells respond to mechanicalstimuli, we artificially applied mechanical forces to Xenopus
embryos. We found that tensile force applied to ectodermal
tissue induces a mesenchymal-epithelial transition (MET)like phenotype, enhancing the junctional structure andincreasing ectodermal tissue stiffness. Among these changes,
we focused on the behavior of the tight junction protein,
ZO-1. Before the force application, ZO-1 forms cytoplasmic
granules, but the mechanical force induces its accumulation
at the tight junction. Significantly, mechanical forces generated by gastrulation movements also induce similar changes
in the developing ectodermal tissue.
To examine whether the behavior of ZO-1 protein is conserved across species, in mammals in particular, we firstobserved the localization of ZO-1 in the mouse embryo.
We focused on E3.5 and E4.5 embryos since they hatch outof the zona pellucida (ZP) and expand their shape (Figure1A). We immunostained E3.5 and E4.5 embryos with a
ZO-1 antibody and found that the cells of E3.5 embryos
showed a significantly higher number of ZO-1 puncta inthe cytoplasm relative to E4.5 embryos. As development
proceeded, the surface area of the trophectoderm (TE) cells
expanded and became thinner. The number of cytoplasmic
ZO-1 puncta was reduced, and ZO-1 signal intensity at theplasma membrane in E4.5 embryos became much higher

Figure 1. Change of ZO-1 localization in mouse hatching embryos.
A. At E3.5, the embryo is covered with the zona pellucida (ZP). During
hatching, the embryo is enlarged and emerges from the ZP. Embryos
were stained with an anti-ZO-1 antibody and Alexa Fluor 546 Phalloidin.
Scale bar, 20 µm. The inset in the E3.5 phalloidin image was acquiredwith higher laser power, demonstrating that the structure of cortical
F-actin is formed at E3.5 even though the signal intensity was weaker
than that in the E4.5 embryo. B. Schematic diagram of ZO-1 behavior
shuttling between the tight junction and cytoplasmic droplets.

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National Institute for Basic Biology

Developmental Biology

than those of E3.5 embryos at the expense of their cytoplasmic pool. Importantly, this change coincides well with theaccumulation of F-actin in the cell cortex in E4.5 embryos.
This result suggests that the shuttling of ZO-1 protein fromthe cytoplasmic puncta to cell junctions occurred as development progressed.
To confirm that the tensile force induces this ZO-1 behavior
in TE cells, we inhibited the expansion of said cells using
two methods: one was the Na+/K+ ATPase inhibitor, ouabain
treatment, and the other was piercing the embryo with aglass needle. Both methods reduced the inner pressure of thecavity and inhibited ZO-1 accumulation in the cell junction.
These results suggested that tensile force being applied to the
TE cells. Furthermore, we assumed that the ZO-1 puncta in
E3.5 embryos were formed by phase separation and treatedembryos with 1,6-hexanediol, which is known to dissolve
LLPS assembly. As expected, 5% 1,6-hexanediol treatment
reduced the number of particles within a few minutes, thus
suggesting that these puncta are liquid droplets generated byphase separation, and that mechanical force regulates ZO-1
phase separation (Figure 1B).

that were forming ZO-1 droplets, reduced cortical actin anddeveloped more stress fibers. This correlation between ZO-1
and F-actin is consistent with our previous finding that themouse E3.5 embryo with ZO-1 droplets had less F-actin thanthe E4.5 embryo with junctional ZO-1. Thus, ZO-1’s phaseseparation is regulated by cell-cell interaction and F-actin
remodeling.
We also analyzed LLPS using bacterially-produced ZO-1
in vitro. We found that ZO-1 IDR4 was sufficient to form
liquid droplets in vitro (Figure 2F), and the full-length ZO-1
can bind to F-actin (Figure 2G). This system will be usefulin analyzing detailed molecular mechanisms of ZO-1 phaseseparation.

II. I nteraction with F-actin regulates ZO-1
phase separation
To analyze the nature of ZO-1 liquid droplets in various
cell environments, we first expressed GFP-ZO1 in A6 cells.
GFP-ZO-1 is mainly localized in the cell periphery andcolocalized with F-actin bundles. We thus treated cells withlatrunculin B, which disrupted F-actin (Figure 2B). We
found that the formation and extinction of cytoplasmic ZO-1
granules depended on the destruction and development of theF-actin network. Furthermore, we confirmed that latrunculin
B-induced ZO-1 granules rapidly recovered in the FRAP
assay. These observations indicate that the efficiency andgrowth of ZO-1 condensation are negatively regulated by theinteraction with F-actin in A6 cells.
To inhibit the interaction between ZO-1 and F-actin ina different way, we constructed GFP-ZO-1ΔABD, whichlacks the actin-binding domain (ABD) (Figure 2A), andexpressed it in A6 cells. GFP-ZO-1ΔABD subsequently
formed droplets with a smooth surface (Figure 2C). Thissuggests that cytoplasmic droplet formation is restricted by
the binding of ZO-1 to F-actin and that GFP-ZO-1ΔABD
lost its capacity for the interaction. It is known that when
proteins form liquid droplets, intrinsically disordered regions(IDRs) play an important role in phase separation. In human
ZO-1, four IDRs are predicted (Figure 2A). Among these, we
found that the C-terminal IDR4, is essential for phase separation. Since IDR4 includes ABD (refer Figure 2A), binding toF-actin may affect the LLPS-inducing activity of the IDR4.
We observed ZO-1 behavior in the wound healing assay
using canine MDCK cells. Initially, most of the cells had
few condensates in a confluent cell sheet. However, after
scratching, cells started active migration toward the woundsite, forming a significant number of droplets (Figure 2D).
In these cells, F-actin distribution and intensity also changed(Figure 2E). Initially, cortical F-actin was well-developedin the non-migrating polygonal cells with junctional ZO-1.
In contrast, migrating cells around the wound site, ones

28

Figure 2. Interaction with F-actin regulates ZO-1 phase separation.
A. The domain structure and the intrinsically disordered regions (IDRs)of ZO-1. B. A6 cells were treated with latrunculin B (Lat) to disrupt
F-actin. Bar = 20 µm. C. GFP-ZO-1 lacking the actin binding domain(ABD) was expressed in A6 cells. Bar = 20 µm. D. A wound healing
assay using MDCK cells. Bar = 50 µm. E. 10 hours after the woundhealing assay, cells close to the wound site (“edge”) and in the inner
region (“inner”) were stained with phalloidin. Bar = 20 µm. F. Purified
GFP-ZO1-IDR4 formed liquid droplets in vitro. Bar = 5 µm. G. Purified
GFP-ZO-1 interacts with F-actin. Bar = 10 µm.

III. C ephalochordate Brachyury enhancers
involved in the evolution of the chordate
notochord and somites
The notochord and somite are the most central organs inchordates, and Brachyury (Bra) plays a pivotal role in theirformation. The question of how Bra gained its enhancer
activity in the notochord-specific expression is critical to ourunderstanding of chordate evolution. In cephalochordates,
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 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 occurredin the notochord, whereas both muscle and the notochordindicated BfBra2 expression. These results, along with otherdata, predicted an EvoDevo scenario in which BfBra2 ismore ancestral and mother-like, while BfBra1 is daughterlike and duplicated by BfBra1. Originally, ancestral BfBra2
was likely to gain enhancer machinery for gene expression inmuscle and notochord, as has been deduced from its spatial
expression profile: the 5’ region for somite expression, the3’ region for notochord expression, and introns for somite/
notochord expression. In contrast, sister BfBra1 became
more specialized for notochord expression using the intron
enhancers.

First, we precisely observed the larval swimming activity
under fluctuating light conditions and found that larvae temporarily stopped swimming ~30 s after rapid light intensity
reduction, thus exhibiting a step-down photophobic response.
This behavior was also observed when we rapidly changed
the spectral composition of light. Further experiments using
the Okazaki Large Spectrograph revealed that the loss of
short wavelengths of light (blue/green light) induced thistype of behavior.
The analysis of mathematical simulations of this step-down
photophobic response indicates that larvae will aggregate
in the lighter areas of two-dimensional large rectangularfields. These results suggest that the step-down photophobic
response of coral larvae may play an important role in determining where larvae settle on the reef.

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.

Publication List:〔Original paper〕
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 of
BfBra2 introns in muscle and notochord is shown in purple.

Y., Kato, K., Koyama, H., Kuba, A., Takahashi, H., Fujimori,• Sakai,T., Hatta, M., Negri, A.P., Baird, A.H., and Ueno, N. (2020). A stepdown photophobic response in coral larvae: implications for the lightdependent distribution of the common reef coral, Acropora tenuis. Sci.
Rep. 10. DOI: 10.1038/s41598-020-74649-x

IV. A step-down photophobic response in larvaeof the common reef coral, 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 rolein larval habitat selection. However, due to a lack of basic
photobiological studies in corals, how coral larvae perceiveand respond to the light in their environment remains largely
unknown. To answer these questions, we analyzed theswimming behavior of larvae of the common reef coral
Acropora tenuis (Figure 4) under various light conditions. In
addition, we developed a mathematical model to test whether
the observed light response resulted in aggregation or dispersal under specific light fields.

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National Institute for Basic Biology

Developmental Biology
DIVISION OF MOLECULAR AND DEVELOPMENTAL BIOLOGY

Professor

TAKADA, Shinji

Assistant Professor:

YABE, Taijiro
MII, Yusuke
Specially Appointed Assistant Professor:
SHINOZUKA, Takuma
Technical Staff:
UTSUMI, Hideko
Postdoctoral Fellow:
TAKADA, Ritsuko
SOKENDAI Graduate Student: HATAKEYMA, Yudai
TRAN, Thi Hong Nguyen
SUZUKI, Minako
TAKASHIRO, Kayoko
Technical Assistant:
ITO, Yukiko
Admin Support Staff:
NOBATA, Ryuko

The morphology of the body and tissues is established ina spatio-temporarily regulated manner. A number of genes
involved in the process of morphogenesis have been identified, but it is still uncertain how either spatial and temporal
information are established and converted to morphology
during embryogenesis. Therefore, our aim is to understandthe mechanism by which this spatial information is established, and how temporal and 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. These

Visual overview of this lab’s work.

30

molecules 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 is yet to be elucidated. To this end, we havevisualized signal proteins and monitored their movement intissues. Furthermore, we are also examining the biochemical characteristics and functions of these molecules, whichappear 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 research is to reveal
the molecular mechanism of this differing and unique modeof patterning that underlies the periodical and sequential subdivision in the development of somites.

I. R egulation of spatial distribution of Wnt
proteins in vertebrate embryos
By combining biochemical and structural analyses, wehave already shown that Wnt3a proteins are not secreted ina monomeric form, rather in homo-trimer and larger HMW
complexes. Secreted Wnt3a proteins were able to be dissociated via interaction with their receptor Frizzled8 and witha secreted Wnt binding protein, sFRP2, in vitro. Similarly,
this dissociation was detected in vivo by Fluorescence
Correlation Spectroscopy (FCS). Several lines of evidence

Figure 1. Model of Wnt protein diffusion: Wnt trimers are the smallest
unit of the HMW complex. Both the trimer and the HMW complex
appear to exist in the extracellular milieu. The HMW complex isprobably 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 as well as the trimer itself, can also be dissociated by interaction with soluble Wnt binding proteins (partner proteins), including sFRP.
Due to this dissociation, Wnt turns out to be more mobile and its diffusion range is expanded (diffusible action).

show that large assemblies of Wnt3a are less mobile, andWnt/sFRP2 heterodimer, which is generated through thebinding of dissociated Wnt with sFRP2, diffuse more freely.
Based on these results, we have proposed a model which
contends that the assembly and dissociation of dissociableoligomers modulate Wnt signaling range (Figure 1).
To increase our insight into the intercellular transmissionof Wnt proteins in embryonic tissue, we precisely examinedthe extracellular dynamics of Wnt, comparing with sFRP in
Xenopus embryos. Here, we focused on Wnt8 and a memberof sFRPs, Frzb, both of which are involved in the anteroposterior patterning of the vertebrate embryo.
Firstly, we visualized their localization in Xenopus embryos
by fusing them with fluorescent proteins. Monomeric Venus(mV) fused with Wnt8 and Frzb were visualized on the cellsurface when they were expressed in Xenopus embryos. By
contrast, we found that only the secreted form of mV (secmV), which was expected to have no specific binding to
the cell surface, was hardly visible along the cell boundary
under the same conditions. This difference in protein distribution can be explained by difference of affinity to docking
molecules on the cell surface because the addition of heparinbinding peptides resulted in a significant increase of thelocalization of sec-mV on the cell surface.
We subsequently examined the dispersion of these proteinsby expressing an artificial protein, called morphotrap, thatcan capture Venus-tagged proteins on the cell surface (Figure2). Because mV-Wnt8 and mV-Frzb were trapped by morphotrap, a population of mV-tagged Wnt8 and Frzb deemed
not likely to associate tightly with the cell surface, and wereable to be dispersed over a long range from the source cells.
We then examined their dynamics by FCS and fluorescencedecay after photoconversion (FDAP)-based measurementsin the embryonic tissue. In particular , we refined FDAPbased analysis by focusing on a limited area across the cellsurface, which enabled us to obtain dynamics comparable
to those measured by FCS. Combination of fluorescencecorrelation spectroscopy and quantitative imaging revealed
that only a small proportion of Wnt8 proteins diffuse freely,
whereas most of them are bound to the cell surface. FDAP
analysis, that we refined by focusing on a limited area acrossthe cell surface, showed that Wnt8 proteins that were boundto the cell surface were rapidly and exponentially decreasing, suggesting a dynamic exchange of a bound form of Wnt
proteins. Based on these results and our previous findings,
we have proposed a basic mathematical model to explain
distribution and dispersion of secreted proteins. This model,
which is based on the dynamic exchange of the bound formof Wnt proteins, can recapitulate a graded distribution of thebound, not free, state of Wnt proteins.

II. T he molecular mechanism of metameric
structures in vertebrate development
The segmental sub-regions of several specific tissues, including the paraxial mesoderm (or somites) and the pharyngeal
pouches is not likely to be simply directed by the gradient of
the signal molecules, but by a unique mechanism proceeding
periodically. For instance, somites are sequentially generated in
an anterior-to-posterior order by converting the temporal periodicity created by a molecular clock into periodical structures. The
molecular mechanism underlying this conversion and morphological segmentation, however, is not yet fully understood.
Somites are segmental epithelial blocks located symmetrically on either side of the neural tube. Somitogenesis peri-

Figure 2. Visualization of dispersed proteins by an antibody-based ligand trapping: A. Schematic representation of tethered-anti-HA Ab, which can trap
diffusing proteins with Venus tag. B. Results of trapping. The artificial ligand (sec-mV-2HA) was trapped at tethered-anti-HA Ab-expressing cells, distantfrom the source. The superficial layer of a Xenopus gastrula (st. 11.5) was imaged as a z-stack and its maximum intensity projection (MIP) was presented
for the fluorescent images. Intercellular mVenus signal (green) of sec-mV-2HA was not apparent in the vicinity of source cells, but was detected aroundthe tethered-anti-HA Ab-expressing cells (arrowheads) that are traced with memRFP (magenta).

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National Institute for Basic Biology

Developmental Biology

odically proceeds in an anterior-to-posterior manner fromtheir precursor, the presomitic mesoderm (PSM), which
is located at the posterior of newly formed somites. This
periodic generation is achieved by a complex and dynamic
mechanism within the PSM. The molecular clock, the socalled segmentation clock, essentially creates oscillatory
expression of particular genes, hairy and some notch-related
genes, in the posterior PSM. After this, the spatial pattern ofsomites is defined based on the temporal periodicity createdby the clock. We are currently investigating the transitory
mechanism concerning the temporal periodicity in the spatial
patterns of somites using zebrafish embryos.
In addition to somites, we examined the development ofanother metameric structure, the pharyngeal arches (PA),which give rise to skeletal elements of jaws and gills. This
metamerism is brought about by the segmental developmentof the pharyngeal pouches (PP), which are generated byoutpocketing of the pharyngeal endoderm. Interestingly, thetwo most anterior PAs are likely to have different characteristics from the posterior PAs. The anterior PAs express genes
involved in myogenesis and chondrogenesis whereas the posterior PAs do not undergo myogenesis and chondrogenesis inchick and mouse embryos. Furthermore, several studies havesuggested that distinct segmentation mechanisms for anterior
and posterior PAs may cooperate in establishing the entire
series of PAs. However, development of this complex PA
segmentation has been poorly understood until now. It has
been quite puzzling how the seamless array of pharyngeal
arches is generated by a combination of anterior and posterior PAs, which are formed by distinct mechanisms.
To address this issue, we examined the development ofzebrafish pharyngeal endoderm, focusing in particular on theformation of PP2, which is located at the boundary between
the anterior and posterior PAs. Precise examination by liveimaging and cell-tracing experiments performed in zebrafish showed that PP2 was formed in an unexpected manner.
Rostral and caudal aspects of PP2 were initially formed
separately, then subsequently established contact throughthe dynamic remodeling of the endoderm epithelium, uponwhich they finally became integrated (Figure 3). These
results provide an answer to the question of how a seamlessarray of PAs are generated.

32

Publication List:〔Original papers〕
K., and Takada, S. (2020). The second pharyngeal pouch• Okada,is generated by dynamic remodeling of endodermal epithelium inzebrafish. Development 147. DOI: 10.1242/dev.194738

T., Hara, K., Azuma, S., and Takada, S. (2021). Effect of• Okubo,retinoic acid signaling on Ripply3 expression and pharyngeal arch••morphogenesis in mouse embryos. Dev. Dyn. 250, 1036–1050. DOI:
10.1002/dvdy.301
Uda, Y., Miura, H., Goto, Y., Yamamoto, K., Mii, Y., Kondo, Y., Takada,S., and Aoki, K. (2020). Improvement of phycocyanobilin synthesis
for genetically encoded phytochrome-based optogenetics. ACS Chem.
Biol. 15, 2896–2906. DOI: 10.1021/acschembio.0c00477
Yasuda H, Yamamoto H, Hanamura K, Mehruba M, Kawamata T,
Morisaki H, Miyamoto M, Takada S, Shirao T, Ono Y, Mukai H. (2020)
PKN1 promotes synapse maturation by inhibiting mGluR-dependentsilencing through neuronal glutamate transporter activation. Commun.
Biol. 3, 710. doi: 10.1038/s42003-020-01435-w.

〔Review articles〕
Y. (2020). Heparan Sulfate Clusters Regulate Distribution and• Mii,
Signaling of Wnt Morphogens. Trends Glycosci. Glycotechnol. 32,
E205–E211. DOI: 10.4052/tigg.2006.7eY., and Takada, S. (2020). Heparan sulfate proteoglycan clustering• Mii,
in Wnt signaling and dispersal. Front. CELL Dev. Biol. 8. DOI:
10.3389/fcell.2020.00631

DIVISION OF EMBRYOLOGY

Professor

FUJIMORI, Toshihiko

Assistant Professor:

KOYAMA, Hiroshi
NONOMURA, Keiko
Technical Staff:
OKA, Sanae
KISHI, Kanae
Postdoctoral Fellow:
SOKENDAI Graduate Student: KATAGIRI, Saki
USAMI, Fumiko
SAKURAI, Jun
Visiting Graduate Student: KATSUTA, Hiroki
Visiting Undergraduate: MIKOSHIBA, Seiya
Visiting Scientist:
ARATA, Masaki
INOUE, Yuuki
Technical Assistant:
HIGUCHI, Yoko
KANIE, Akemi
NAKAGAWA, Mami
Admin Support Staff:
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.

A mouse fertilized egg and an embryo at 12 days after fertilization.
Embryonic body with axes of anterior-posterior, dorsal-ventral and leftright is formed from an egg with symmetrical shape. How is the information relating future body formed during early stages of development?

I. Live observation of early mouse development
To understand the mechanisms underlying embryonic development and morphogenesis, it has become common to observe
the behavior of cells and gene expression in living embryos
in a variety of animal species. Progress in genetic engineeringand embryonic manipulation concomitantly with the progressin optics and microscope technologies, including developmentof highly sensitive photo-detectors, has allowed us to observe
developing embryos, even mammalian ones, in real time. We
have established a series of transgenic mouse lines for live
imaging, which is part of a collaborative project taking place inthe Laboratory for Animal Resources and Genetic Engineering,
Riken CLST. These mouse lines have been used to visualize
nuclei, cell shapes, the cytoskeleton and other organelles toobserve cell behaviors in living mouse embryos across manylaboratories over the world. We have also established mouselines to monitor the cell cycle. We have been studying the
behavior of cells within embryos by applying newly developed
image analyses following the observation of cell behaviors
within embryos using these mouse lines. We found that theproximal visceral endoderm overlying the extra-embryonicectoderm shows coherent cell growth in a proximal-anterior to
distal-posterior direction. We also observed that directional cell
migration is coupled with 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.

33

National Institute for Basic Biology

Developmental Biology

Figure 1. Three-dimensional image reconstruction of the pregnant uterus.
A uterus obtained 5 days after fertilization was sectioned and images weredigitized by an image scanner, and reconstructed three-dimensionally toobserve the relationship between the embryo and the maternal tissue. The
right panel represents the pseudo 3D image observed from the lateral sideof 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. 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 movementof cilia.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. Recently, we found that Camsap3, a microtuble
minus-end binding protein, localized to the uterine side of
the base of cilia and its mutation disrupted cilia orientation in
individual cells without affecting Celsr1 localization. Thus,
both Celsr1 and Camsap3 are responsible for the alignmentof cilia along the ovary-uterus axis, but in distinct ways;
Celsr1 orients cells, while Camsap3 orients cilia within eachcell.
In addition to the ciliary polarity phenotype, cellular shapepolarity was also impaired in the Celsr1-deficient mice.
We found a new feature of cellular polarity in the wild typeoviduct, e.g. that the apical surface of epithelial cells waselongated along the ovary-uterus axis. In Celsr1-deficient
mice, epithelial cells showed less elongation and a randomized orientation.
Furthermore, we also found that the three-dimensionalarchitecture of oviductal epithelia was dramatically disrupt-

34

ed. 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.

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

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.

mechanical forces, PIEZO activation, cellular responses, andtissue morphogenesis.

VI. Mechanics of cell population patterning
during development
Figure 3. Theoretical inference of cellular/tissue mechanical states.
Schematic illustration of inference.

V. Mammalian tissue morphogenesis requiring
mechanosensor channel PIEZOs
Several examples have shown that mechanical stimuli
can work as key components for tissue or organ development. However, our knowledge about 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 themain 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

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.

Publication List:〔Original papers〕
T., Ishii, Y., Nguyen, L.Q., Okuno, N., Sang, Y.,• Hamashima,
Matsushima, T., Kurashige, Y., Takebayashi, H., Mori, H., Fujimori, T.,•••

Positive or
negative pressure,
shear stress

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.

•

et al. (2020). Oligodendrogenesis and myelin formation in the forebrain
require platelet-derived growth factor receptor-alpha. Neuroscience
436, 11–26. DOI: 10.1016/j.neuroscience.2020.04.001
Koyama, H., and Fujimori, T. (2020). Isotropic expansion of external
environment induces tissue elongation and collective cell alignment. J.
Theor. Biol. 496. DOI: 10.1016/j.jtbi.2020.110248
Matsumoto, Y., Yamaguchi, Y., Hamachi, M., Nonomura, K.,
Muramatsu, Y., Yoshida, H., and Miura, M. (2020). Apoptosis
is involved in maintaining the character of the midbrain and the
diencephalon roof plate after neural tube closure. Dev. Biol. 468, 101–
109. DOI: 10.1016/j.ydbio.2020.09.015
Nguyen, Q.L., Okuno, N., Hamashima, T., Dang, S.T., Fujikawa, M.,
Ishii, Y., Enomoto, A., Maki, T., Nguyen, H.N., Nguyen, V.T., et al.
(2021). Vascular PDGFR-alpha protects against BBB dysfunction after
stroke in mice. Angiogenesis 24, 35–46. DOI: 10.1007/s10456-02009742-w
Sakai, Y., Kato, K., Koyama, H., Kuba A., Takahashi, H., Fujimori,T., Hatta, M., Negri, A.P., Baird, A.H., and Ueno, N. (2020). A stepdown photophobic response in coral larvae: implications for the lightdependent distribution of the common reef coral, Acropora tenuis. Sci.
Rep. 10. DOI: 10.1038/s41598-020-74649-x
Usami, F.M., Arata, M., Shi, D., Oka S., Higuchi, Y., Tissir, F.,
Takeichi, M., and Fujimori, T. (2021). Intercellular and intracellularcilia orientation is coordinated by CELSR1 and CAMSAP3 in oviductmulti-ciliated cells. J. Cell Sci. 134. DOI: 10.1242/jcs.257006

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

35

National Institute for Basic Biology

Developmental Biology
DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:

KITADATE, Yu
NAKAGAWA, Toshinori
Specially Appointed Assistant Professor:
HIRA, Seiji
Technical Staff:
MIZUGUCHI, Hiroko
JSPS Postdoctoral Fellow: IKEDA, Tatsuro
Research Staff:
HIRANO, Kodai
SATO, Toshiyuki
SOKENDAI Graduate Student: WANG, Zhe
Visiting Undergraduate: BABAGUCHI, Hirotaka
Technical Assistant:
KON, Yayoi
MARUYAMA, Ayumi
FUJITA, Miyako
NISHIMURA, Chiaki
Admin Support Staff:
KUBOKI, Yuko

Overview of our research
The production of copious amounts of sperm over a long
period in the testis is fundamental for the continuity of life
across generations. The Division of Germ Cell Biology aims
to understand the key processes of germ cell development,that lead to the robust production of sperm and the accuratetransmission of genetic information.
In particular, our emphasis has been placed on the regulation and functional behavior of spermatogenic stem cells(SSCs) in the testes of mice. SSCs play a crucial role, notonly as the origin of long-term sperm production, but also

as the crucible of genetic and potentially epigenetic mutations, the underpinning of evolution as well as congenital
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,
Our lab has figured out a number of key properties ofmouse SSCs. One of these is that they 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 have been investigating the cellular identity of “actual” SSCs, their in vivo
behavior at a single-cell resolution, and their underlying mathematical principles, leading to the discovery ofthe dynamics of “population asymmetry” and subsequent“neutral competition” between SSCs. We have proposed thatthe aforementioned type of competition observed between
SSCs could be the result of consumption of limited supply ofself-renewal promoting extracellular ligands. The “potential
stem cells” have also fascinated us. During steady-state spermatogenesis, these cells are largely active as transient amplifying cells and differentiate rather than self-renew. However,when tissues are insulted or testicular cells are transplanted
into host testes, the probability of their self-renewal increases
greatly. Such flexible and probabilistic features of stem
cells have been found to be paradigmatic for many otherstem cell-supported 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, Tokue et al., Stem
Cell Reports 2017, and Kitadate et al., Cell Stem Cell 2019.
An important achievement we accomplished in 2020 was
the revelation of SSC behavior in the context of post-transplantation reconstruction of spermatogenesis at a clonal resolution, and the reporting of a novel strategy to enhance thetransplantation efficiency by tuning the post-transplantation
fate of SSCs (Nakamura et al., Cell Stem Cell 2021).

Figure 1. The “pulse-transplantation” experiment
In the donor mice, actual (GFRα1+) andpotential (Ngn3+) SSCs are pulse-labelled,
whose testes were dissociated into single cellsuspension for transplantation to germ celldepleted hosts. After certain times, the entire
testes of the hosts were recovered, untangled seminiferous tubules were processed
for immunostaining. All the pulse-labelled(GFP+) clones in the host tetes were scoredfor statistical analyses. Adopted from Cell
Stem Cell 5, 1443-1456 (2021).

36

P o s t - t r a n s p l a n t a t i o n re g e n e r a t i o n : aremarkable feature of stem cells promising a
wealth of applications
Tissue stem cells can restore the impaired structure andfunction of host tissues following transplantation. In blood,
hematopoietic stem cell transplantation has been established
as a radical treatment for leukemia, while trials to test theviability of stem cell transplantation in mesenchymal andepithelial tissues are ongoing. In male germline, SSC transplantation was established by Brinster and their colleagues
in 1994 using mice, which promised a wealth of applications
such as restoration of fertility to cancer patients after chemotherapy and the preservation of genetic diversity. However,
the inefficiency of this technology currently rules out its
practical application.
Indeed, our knowledge about the fate behavior of individual
SSCs and their progenies after transplantation remains poorly
understood, thus limiting the potential to develop new strategies to increase the currently low transplantation efficiencies.

Clonal fate analysis of actual and potential
stem cells following transplantation
A remarkable feature of SSCs that our group has discoveredis the presence of “potential stem cells”, which significantly
contributes to the post-transplantation regeneration in amanner similar to “actual stem cells” (Nakagawa et al.,2007). Therefore, based on an experimental setting of pulselabeling actual and potential SSCs using the specific expression of GFRα1 and Ngn3 in these cells, respectively, we firstinvestigated their detailed clonal fate following transplantation in the host testes (Figure 1). All the clones that had been
derived from labelled SSCs and resided in the host testes
were counted and scored for the number of constituent SSCs
and differentiated cells over a broad time line spanning from
2 days to 180 days after transplantation, when single donor
cell-derived clones formed robust repopulating colonies.

Actual and potential SSCs follow
indistinguishable clonal fates with comparable
repopulation efficiencies
It was clear that single SSC-derived clones follow highly
divergent fates, showing varying clone sizes (e.g., thenumber of SSCs in individual clones, or physical extension within the tissue), rather than showing a stereotypical
pattern. The clonal fate data derived from actual (GFRα1+)and potential (Ngn3+) SSCs first show that these cells form
long-term repopulating colonies at comparable, albeit notequal, efficiencies. When Ngn3+ cells were transplanted,
a significant fraction of them were quickly converted into
GFRα1+ cells within 2 days; an observation consistent withthe results of the preceding studies. Interestingly, GFRα1+cell- and Ngn3+ cell-derived clones evolve parallel to indistinguishable size distributions and kinetics over time. Thus,
actual and potential SSCs are equipotent in terms of clonal
evolution during post-transplantation repopulation, irrespective of the original state on transplantation.

Most of the initially settled SSCs are lost
through stochastic differentiation and celldeath
We subsequently looked in detail into the clonal fates ofdonor SSCs. We unexpectedly found that the number of
SSCs that initially resided in the tissue was considerablylarger than those which eventually formed colonies over thelong term (i.e., after couple of months). After being attachedto the host tissue and moved to the location in which SSCs
are normally found (on the basement membrane of seminiferous tubules), the vast majority of SSCs were lost throughdifferentiation and cell death, with only a small fraction (oneout of tens) contributing to long-term repopulation.
Furthermore, in collaboration with the Ben Simon Lab at
the University of Cambridge, we asked whether this variable
and intricate fate behavior can be explained by a minimal
mathematical model, relying on an assumption that all SSCs
are equivalent, and select their fates probabilistically at
defined rates reliant only on their current state (i.e., unrelated
to the past history). Significantly, such simple models were
found to be capable of predicting the broad range of clonalfate data with high accuracy, supporting the aforementioned
model assumption that all SSCs are equipotent. This insightis in stark contrast with the prevailing thought that only asmall fraction of GFRα1+ and/or Ngn3+ cells are definitive
SSCs that form colonies at high probabilities (presumptively,100%).

Post-transplantation fate of SSCs can be
modified through differentiation inhibition
The suggestion by the modeling analysis that all SSCs
are equivalent motivated us to test whether SSC fate canbe altered to increase repopulation efficiency. In particular,

Figure 2. Mathematical model simulation
A. The modeling scheme, in which donor SSCs are allowed to divide, differentiate or die stochastically. B-D. Examples showing the predictive capacity of themodel. The dots are the experimental measurements, while the lines indicates thesimulation. Adopted from Cell Stem Cell 5, 1443-1456 (2021).

37

National Institute for Basic Biology

Developmental Biology

given that irreversible commitment of SSCs to differentiate and lose their self-renewing potential is induced by,
and dependent on, retinoic acid (RA) signaling, we asked
if the administration of a chemical inhibitor of retinoic
acid (RA) synthesis, WIN18,446, can affect the behaviorof transplanted SSCs. We found that WIN18,446 treatment
can, in an in vivo context, not only inhibit differentiation ofdonor SSCs, but also incline them toward self-renewal andsignificantly increase the number of self-renewing pools ofsurviving SSCs. Such an effect was observed more dramatically in Ngn3+ cells that were primed for differentiation, andeffectively recruited to self-renewing pool by WIN18,446
treatment.

Tr a n s i e n t s u p p re s s i o n o f d o n o r S S C
differentiation boosts long-term repopulation
and restores host fertility
Finally, we examined if the overall long-term repopulation
efficiency could be increased, after this type of differentiation inhibition is terminated so as to allow the SSCs toproceed with spermatogenesis. Remarkably, we observed adramatic increase in long-term repopulation efficiency, (i.e.,by 5-10 times) which was capable of restoring the fertility ofa host that could not sire offspring through natural mating.
Pups obtained from the donor SSC-derived sperm generated
in the WIN18,446-treated host grew into healthy and fertile
adults without any apparent abnormalities.
These results not only support the thesis that all SSCs
are equipotent, but also offer a novel strategy to increase
the transplantation efficiency through tuning the donor
cell fate by changing the hosts’ niche environment. While
WIN18,446 treatment is effective, in theory, this efficiency
could be further increased another ten times if all transplanted SSCs successfully repopulate, Technologically, theuse of WIN18,446 and/or other reagents modifying the SSC
fate shows promise in the field of SSC transplantation acrossbroader applications, from restoring fertility to cancer patient
after therapy to the preservation of genetic diversity.

Figure 3. Restoration of host fertility using WIN18,446
A-B. Representative appearance of the host testes 90 days after transplantation of GFP+ donor testicular cells, in control (DMSO) andWIN18,446-treated hosts. Note the dramatic increase of the repopulation
efficiency. C. Quantification of the repopulation efficiency based on thenumber of repopulating colonies. D. Offspring sired by the WIN18,446treated host through natural mating with wild-type female. The green fluorescence indicates that the pups were originated from GFP+ donor testicular cells developed into functional sperm in the host testes. Adopted
from Cell Stem Cell 5, 1443-1456 (2021).

38

Publication List:〔Original Papers〕
C.A., Caldeira-Brant, A.L., Drumond-Bock A. L. and• Rezende-Melo,
Buchold, G.M., Shetty, G., Almeida, F.R.C.L., Matzuk, M.M., Hara,•••

K., Yoshida, S., Meistrich, M.L., and Chiarini-Garcia, H. (2020).
Spermatogonial asynchrony in Tex14 mutant mice lacking intercellularbridges. Reproduction 160, 205–215. DOI: 10.1530/REP-20-0118
Sznurkowska, M.K., Hannezo, E., Azzarelli R., Chatzeli, L., Ikeda, T.,
Yoshida, S., Philpott, A., and Simons, B.D. (2020). Tracing the cellularbasis of islet specification in mouse pancreas. Nat. Commun. 11. DOI:
10.1038/s41467-020-18837-3
Yeh, Y.-H., Hu, M., Nakagawa, T., Sakashita, A., Yoshida, S., Maezawa,S., and Namekawa, S.H. (2021). Isolation of murine spermatogenic
cells using a violet-excited cell-permeable DNA binding dye. JOVE-J.
Vis. Exp. DOI: 10.3791/61666
Jörg, D.J., Kitadate, Y., Yoshida, S., and Simons, B.D. (2021). Stem Cell
Populations as Self-Renewing Many-Particle Systems. Annu. Rev. Conden.
Ma. P. 12, 135-153. DOI: 10.1146/annurev-conmatphys-041720-125707〔Review Article〕
S. (2020). Mouse spermatogenesis reflects the unity and• Yoshida,
diversity of tissue stem cell niche systems. Cold Spring Harb. Perspect.
Med. 10. DOI: 10.1101/cshperspect.a036186

LABORATORY OF REGENERATION BIOLOGY

Director General

AGATA, Kiyokazu

Specially Appointed Associate Professor:
SUZUKI, Ken-ichi
Postdoctoral Fellow:
TERAMOTO, Machiko
SOKENDAI Graduate Student: SUGIURA, Nao
KUROKI, Yoshihito
BO, Kazuto
Visiting Graduate Student: ISHIDA, Miyuki
Technical Assistant:
KAJIURA-KOBAYASHI, Hiroko
Admin Support Staff:
SAKAGAMI, MariNISHIMURA, Noriko

We subsequently tried to detect enhancer RNA (eRNA)
which might be transcribed in the MFCS1 region. An interesting aspect of this was that eRNA was detected in regenerating blastema of adult newt from st. 2.0 (Pleurodeles waltl:
Figure 2A).
Conversely, expression of eRNA was suppressed in thefrog’s blastema (Xenopus laevis) after metamorphosis(Figure 2B). Thus, we subsequently planned to swap the
MFCS1 sequences between newts and frogs using CRISPR/
Cas9 technology, expecting that the eRNA will be transcribed from the newt’s MFCS1 after swapping in frogs.

Comparative Regenerative Biology
We use animals that demonstrate a high ability in regenerating body parts, such as planarians and newts, to understandthe principle of regeneration. In particular, we investigate thedifference between regenerative and non-regenerative animals
to evoke said abilities from non-regenerative animals. We havealready succeeded in achieving this with planarians, whichwere able to regenerate their heads through RNAi (Umesono
et al., 2013 Nature), and accomplishing functional joint regeneration in frogs through the activation of reintegration systems(Tsutsumi et al., 2016 Regeneration).
We are currently trying to induce limb regeneration abilities
in frogs, as they lose the capability to achieve complete limb
regeneration after metamorphosis. We are now focusing on
the Sonic hedgehog (Shh) super-enhancer MFCS1 (mammalsfishes conserved sequence 1), since it has been suggested thatthe loss of MFCS1 activity after metamorphosis might causea failure in achieving the aforementioned limb regenerationin adult frogs (Yakushiji et al., 2009). When we compared
the MFCS1 sequences between newts and frogs, newts were
found to possess several specific sequences (Figure 1), suggesting that sequence differences might affect super-enhancer
formation between newts and frogs after metamorphosis.

Figure.2 Expression of MFCS1-eRNA and Shh mRNA in the regenerating blastema of newt (P.w.) and frog (X.l.)(A) The left and right panels show expression levels of MFCS1-eRNA
and Shh mRNA, respectively. The left and right graphs in each panel
were obtained from regenerating blastema at St.2.0 and St.2.5 of adultnewts after amputation.
(B) The left and right panels shows expression levels of MFCS1-eRNA
and Shh mRNA in frogs before and after metamorphosis.

Trials in isolating viable adult pluripotent stem
cells derived from planarian using FACS
We have tried to develop an isolation method for viableadult pluripotent stem cells (aPSC) from planarians using
FACS and succeeded in conditioning the low toxic staining
method with both nuclear and cytoplasimic fluorescencedyes. 1µM Hoechst 33342 or 0.05 µg/ml Calsein AM could
be used for isolating viable aPSC of the planarian, Dugesis
japonica. We are presently investigating the effects of FGFand Wnt-morphogens to cultured aPSC to demonstrate “thedouble gradient hypothesis”, which was proposed by Thomas
Hunt Morgan (Morgan, 1901) and our group (Umesono etal., Nature, 2013).

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

39

National Institute for Basic Biology

Developmental Biology
Publication List:〔Original papers〕
M.A., Kashima, M., Nishimura, O., Hosoda, K., Motoishi,• Auwal,M., Kamimura, A., Okumura, A., Agata, K., and Umesono, Y. (2020).

•••

Identification and characterization of a fibroblast growth factor gene in
the planarian Dugesia japonica. Dev. Growth & Differ. 62, 527–539.
DOI: 10.1111/dgd.12696
Hijioka, M., Ikemoto, Y., Fukao, K., Inoue, T., Kobayakawa, T.,
Nishimura, K., Takata, K., Agata, K., and Kitamura, Y. MEK/ERK
signaling regulates reconstitution of the dopaminergic nerve circuitin the planarian Dugesia japonica. Neurochem. Res. DOI: 10.1007/
s11064-020-03226-5
Miura, S., Tsutsumi, R., Agata, K., and Endo, T. (2020). Maturating
articular cartilage can induce ectopic joint-like structures in neonatal
mice. Regen. Eng. Transl. Med. 6, 373–382. DOI: 10.1007/s40883-02000176-w
Sonpho, E., Wootthichairangsan, C., Ishida, M., Inoue, T., Agata,K., Maleehuan, A., Charngkaew, K., Chomanee, N., Moonsom,S., Wongtrakoongate, P., et al. (2020). ECM-body: A cell-free 3D
biomimetic scaffold derived from intact planarian body. Zoolog. Sci.
37, 307–313. DOI: 10.2108/zs190135〔Review article〕
M., Agata, K., and Shibata, N. (2020). What is the role of• Kashima,
PIWI family proteins in adult pluripotent stem cells? Insights from
asexually reproducing animals, planarians. Dev. Growth & Differ. 62,
407–422. DOI: 10.1111/dgd.12688

40

National Institute for Basic Biology

Neurobiology
DIVISION OF BEHAVIORAL NEUROBIOLOGY

Professor

HIGASHIJIMA, Shin-ichi

Assistant Professor:

KIMURA, Yukiko
TANIMOTO, Masashi
Technical Staff:
TAKEUCHI, Yasushi
JSPS Postdoctoral Fellow: SUZUKI, Daichi
SOKENDAI Graduate Student: SUGIOKA, Takumi
KAWANO, Kohei
SHIMIZU, Sayaka
Visiting Graduate Student: UEMURA, Yuto
Visiting Undergraduate: KATAYAMA, Taisei
Technical Assistant:
TERASAWA, Yoko
WATAKABE, Ikuko
ITO, Hiroko
TAKEUCHI, Yoshiko
SETO, Kumiko

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.

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 neuronal41

National Institute for Basic Biology

Neurobiology

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
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.

tion- and modality-selective responses to the head motion
have not been systematically studied in vivo, therefore how
the head motion signals are processed in the vestibular
system remains unclear.

Figure 5. Hair bundle polarity (arrowheads) in the utricle.

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

To visualize hair cell responses to the head motion, we built
a microscope in which an objective lens can tilt with a small
sample 360 degree by a motorized stage during Ca2+ imaging(Figure 6). A spinning-disk confocal scanner and an image
splitting optics formed green and red fluorescent images ona digital camera. This ratiometric imaging setup reduced
artifacts derived from non-uniformity of the excitation lightand optical distortion during the optics motion. With thistiltable objective microscope, we imaged neural activity inall the utricular hair cell at the single-cell level during pitch
or roll tilt/vibration in 5-day-old transgenic zebrafish larvae
expressing Ca2+ indicator, jGCaMP7f, and red fluorescentprotein, tdTomato, in the hair cells.

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

Figure 6. Tiltable objective microscope.

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

III. In vivo functional imaging analysis of thevestibular sensory organ
Maintaining head and body orientation relative to the
Earth’s vertical gravity axis is vital for survival. Vestibularorgans in the inner ear play a crucial role for this task.
Sensory hair cells in the otolith organs receive linear acceleration, e.g., head tilt, translation and vibration. Direction of
the acceleration is detected by the polarized arrangement of
hair bundles in the hair cells (Figure 5). Each otolith organ
contains hair cells with different but topographically organized hair-bundle polarity that reverses at a line of polarity
reversal (LPR). Although morphofunctional specialization of
the vestibular hair cells has been widely studied, the direc-

42

Consistent with the morphological hair-bundle polarity, haircells medial to the LPR are activated by the lateral-down roll
tilts, whereas those lateral to the LPR are activated by the
medial-down tilts (Figure 7). In response to the nose-down
pitch tilts, hair cells medial to the LPR in the rostral utricle
and those lateral to the LPR in the caudal utricle are activated, whereas the rest of the hair cells are activated by thetail-down tilts. Interestingly, hair cells in the medial utricle
exhibited larger responses to the head tilt compared to thelateral hair cells. In contrast to the responses to the head tilt,
the vibratory stimulus in the pitch or roll axis activated thehair cells only in the rostral and lateral utricle near the LPR.

Figure 7. Hair cell responses to head tilt or vibration in the utricle

Together, the tiltable objective microscope visualized, forthe first time to our knowledge, the topographically organized response selectivity for the stimulus direction andmodality in the vestibular periphery. The imaging strategy
we have established here is applicable to the central nervous
system, and thus it will provide deeper understanding of thevestibular processing in the brain.

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

Publication List:〔Original Papers〕

IV. 
F unctional diversity of glycinergic
commissural inhibitory neurons in larvalzebrafishZ., Kimura, Y., Higashijima, S., Hildebrand, D.G.C., Morgan,• Liu,
J.L., and Bagnall, M.W. (2020). Central vestibular tuning arises from

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).

••

patterned convergence of otolith afferents. Neuron 108, 748+. DOI:
10.1016/j.neuron.2020.08.019
Mizoguchi, T., Fukada, M., Iihama, M., Song X., Fukagawa, S.,
Kuwabara, S., Omaru, S., Higashijima, S., and Itoh, M. (2020).
Transient activation of the Notch-her15.1 axis plays an important role inthe maturation of V2b interneurons. Development 147. DOI: 10.1242/
dev.191312
Uemura, Y., Kato, K., Kawakami, K., Kimura Y., Oda, Y., andHigashijima, S. (2020). Neuronal circuits that control rhythmic pectoral
fin movements in zebrafish. J. Neurosci. 40, 6678–6690. DOI: 10.1523/
JNEUROSCI.1484-20.2020

43

National Institute for Basic Biology

Neurobiology
LABORATORY OF NEUROPHYSIOLOGY

Associate Professor

WATANABE, Eiji

Specially Appointed Assistant Professor :KOBAYASHI, Taisuke
JSPS Postdoctoral Fellow: NISHIUMI, Nozomi
WATABE, Mihoko
Technical Assistant:
TSURUMARU, Mayumi

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.

ing 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
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

Visual overview of this lab’s work.

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, includ-

44

Figure 1. Virtual medaka fish.

seasonal changes in color perception in medaka (Shimmura
et al., 2017), and on underwater imaging technology (Abe etal., 2019 and Yamamoto et al., 2019).

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

Figure 2. Morning Monster Illusion. Watanabe (National Institute for
Basic Biology) and Sinapayen (Sony CSL) got 10th place at the 12th
Visual Illusion and Auditory Illusion Contest, for the Morning Monster
Illusion. Itis one of the color aftereffect induced by glare effect.(demo,
https://ewatanabe.blogspot.com/2020/11/monstre-matin-illusion-10thaward-of.html).

Contest, https://doi.org/10.6084/m9.figshare.10046534),
and the Morning Monster Illusion (https://doi.org/10.6084/
m9.figshare.13125518, Figure 2).
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).
Currently, a variety of research is being conducted using
these AI systems (https://arxiv.org/abs/2106.09979, https://
arxiv.org/abs/2106.12254).

Ⅲ. Ecological study of tactics in predators andprey
We are interested in behavioral interactions between predators and prey not only concerning the psychophysical aspect
but also concerning the ecological aspect. In the process ofthe co-evolution, predators and prey have developed various
tactics to overcome each other. To elucidate the sophistication of these tactics, we have examined the mechanisms andefficacies of predatory and antipredator behaviors of several
animals such as snakes, frogs, fish, dragonflies and bats.
In 2020, we published a paper, which outlined the vulnerability of preemptive actions of both predator snakes andprey frogs, and the possibility that this vulnerability instigates a ‘waiting game’ between these animals (Nishiumi &Mori, 2020). Several on-going studies were also presented
at conferences, which were about the tactics of predatory
bats regarding echolocation. In addition, we have developed
experimental technologies for those studies, which presentinteractive virtual predators to prey animals based on animation (Figure 4) or robotics. 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 with low-price webcams and several
free computer libraries), and thus, are expected to contributewider range of animal research.

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

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.

45

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

DIVISION OF EVOLUTIONARY BIOLOGY

Professor

HASEBE, Mitsuyasu

Assistant Professor:

ISHIKAWA, Masaki
SEGAMI, Shoji
Specially Appointed Assistant Professor:
MANO, Hiroaki
KOSETSU, Ken
OHI, Shoko
Technical Staff:
Postdoctoral Fellow:
AOYAMA, Tsuyoshi
NARUKAWA, Hideki
PALFALVI, Gergo
SOKENDAI Graduate Student: SUDA, Hiraku
HORIUCHI, Yuta
DE VILLIERS, Ruan
CHEN, Peng
UEDA, Masamichi
Visiting Graduate Student: TANASE, Kuniaki
Visiting Scientist:
GU, NanTechnical Assistant:
AOKI, Etsuko
FUKADA, Hatsumi
HIRAMATSU, Mika
KAJIKAWA, Ikumi
MATSUZAKI, Yoko
MASUOKA, Tomoko
NISHI, TayoZHANG, Liechi
OGAWA, Yuko
Admin Support Staff:
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 existing, unexplained phenomena still remain, one of which is the evolution of complexity. It is difficult to explain the mechanisms
needed to evolve complex adaptive traits at the cellular andorganismal levels, such as cell division machinery, regeneration, and novel organ development. Such traits are comprisedof many components and become adaptive only when all ofthem are gathered together. However, based on evolutionary
theory, each component should evolve one by one according to the accumulation of mutations. We aim to reveal thegenetic networks regulating these complex traits and to
identify the mechanisms needed for the evolution of complex
characters.

II. S patiotemporal regulation of the cell
division axis as a grand plan of plantdevelopmental evolution
The cell division axis must 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.
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 theexpression of the latter GRAS transcription factor. This regulation seems to determine the location where the division axis ischanged from an anticlinal to a periclinal manner
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, a graduate student
in our lab, found that a transporter mutant, which belongs to
the ABC gene family, exhibited a cell shape abnormality. We
are presently investigating its underlying mechanism.
This is a collaborative project that is being undertaken by
our division and Dr. Takashi Murata (Kanagawa Institute ofTechnology) and Dr. Rumiko Kofuji (Kanazawa University),Dr. Hiroyuki Sekimoto (Japan Women’s University), andAtsushi Mochizuki (RIKEN).

III. D NA damage triggers reprogrammingof differentiated cells into stem cells withSTEMIN
Plants are better able to undergo cellular reprogrammingfrom differentiated cells to stem cells compared to animals.
The genetic regulatory networks of reprogramming inducedby phytohormones and wounding accompanying cell death
have been extensively studied. We previously investigated the molecular mechanisms underlying reprogramming46

caused by wounding in the moss Physcomitrella patens.
This year, we reported the unexpected identification of a
new genetic regulatory pathway of reprogramming inducedby moderate, repairable DNA damage in the absence ofexogenous phytohormone treatment and wounding. When
intact leafy shoots were transiently exposed to DNA damage-inducing reagents, the DNA damage was successfully
repaired and the leaf cells changed to stem cells, which
subsequently produced leafy shoots. Furthermore, we foundthat this pathway depends on the AP2/ERF transcription
factor STEMIN, whose ectopic expression changes intact
differentiated leaf cells into stem cells (Ishikawa et al. 2019
Nat. Plants 5: 681). Consequently, this response could bequite helpful to plants in nature. Since plants cannot immediately escape from severe environments that induce DNA
damage, the conversion of differentiated cells into stem cells,
which proliferate and produce new shoots, allows plants toescape from harsh environments to grow in safer places. Ourfindings shed light on an unforeseen pathway from the induction of DNA breaks to cellular reprogramming. An international joint graduate student Ms. Nan Gu from Dr. Chunli
Chen’s laboratory in the Huazhong Agricultural University
mainly worked on this project (Gu et al. 2000. Nat. Plants).

the carnivorous plant Cephalotus follicularis. We foundthat hollow formation, which was the first step in pitcherleaf development, was initiated by growth inhibition on the
adaxial side of leaf primordia. This process may be regulatedby the phytohormone cytokinin.

V. Calcium ions mediate memory in the Venus
flytrap
The leaves of the carnivorous plant Venus flytrap, Dionaea
muscipula (Dionaea) close rapidly to capture insect prey. The
closure response usually requires two successive mechanical stimuli applied to the sensory hairs on the leaf blade
within a span of approximately 30 seconds. An unknown
biological system in Dionaea is thought to memorize the firststimulus and transduce the signal from the sensory hair tothe leaf blade. A graduate student Hiraku Suda and his colleagues linked signal memory to calcium dynamics usingtransgenic Dionaea expressing a Ca2+ sensor. Stimulation of
the sensory hair caused an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) starting in the sensory hair and spreading
to the leaf blade. A second stimulus increased [Ca2+]cyt to aneven higher level, meeting a threshold that is correlated to
the leaf blade closure. Because [Ca2+]cyt gradually decreased
after the first stimulus, the [Ca2+]cyt increase induced by
the second stimulus was insufficient to meet the putative
threshold for movement after about 30 seconds. The Ca2+
wave triggered by mechanical stimulation moved an order ofmagnitude faster than that induced by wounding in petiolesof Arabidopsis thaliana and Dionaea. The capacity for rapid
movement has evolved repeatedly in flowering plants. This
study opens a path to investigate the role of Ca2+ in plantmovement mechanisms and their evolution.

Figure 1. The DNA damage reagent zeocin induces reprogramming ofleaf cells into stem cells.

IV. Evolution of Carnivory in Flowering Plants
Carnivorous plants attract, capture, digest, absorb, andnourish small animals with specialized leaves, so they cangrow in nutrient poor environments where they can gain
prominence over other plants. To better understand the evolution of botanical carnivory, we and a group of international
collaborators sequenced draft genomes of the Venus flytrap,
Dionaea muscipula, the waterwheel plant, Aldrovanda
vesiculosa, and the sundew Drosera spatulata. Mr. Gergo
Plafalvi, a graduate student and colleagues compared the
genomes and identified an early whole genome duplicationin the family as a source for carnivory-associated genes.
In particular, the recruitment of genes to the trap from theroot was a major mechanism in the evolution of carnivory,
supported by family-specific duplications. These genomes
belong to the gene poorest land plants sequenced thus far,suggesting a reduction of selective pressure on different
processes, including non-carnivorous nutrient acquisition(Plafavi et al. 2020 Curr. Biol.).
To investigate the molecular bases of carnivory, Dr. Hideki
Narukawa performed mostly comparative analysis of carnivorous pitcher leaves and non-carnivorous flat leaves in

Figure 2. Fluorescence images of a GCaMP6f Dionaea leaf blade after asensory hair was stimulated with a needle.

VI. Molecular mechanisms and evolution of
the generation and transmission of action
potential in plants
Plants lack blood flow and nerves, but have evolved uniquelong-distance intercellular signaling mechanisms. Signals
using plant hormones, peptides, proteins, and slow calcium
waves have been well elucidated; however, the molecularmechanism of long-range, rapid, intercellular signaling byaction potentials with fast calcium waves, which evolvedin parallel to similar signaling mechanisms in animals,47

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

remains largely unknown. Rapid transmission of action
potentials has been reported in specific tissues of the sensitive plant Mimosa pudica, the Venus flytrap Dionaea
muscipula, the wheel plant Aldrovanda vesiculosa, and thesundew Drosera rotundifolia. We use these four species, aswell as Arabidopsis thaliana, to study the molecular mechanisms of action potential generation and transmission. We
have obtained genome sequences for these species and established techniques for transformation. We intend to screen forgenetic factors responsible for fast intracellular and intercellular electrical signaling by action potentials, analyze theirmolecular characteristics such as effects on ion permeability
and intracellular localization, and perform genetic gain- andloss-of-function experiments. This will allow us to understand the general mechanisms of action potential transmission in plants and the evolutionary process that resulted indiversity of transmission velocity, which is adaptive in thethree plants.

Publication List:〔Original papers〕
M., Mieda, M., Sato, R., Kinoshita S., Tomoyama, T.,• Fukuda,
Ferjani, A., Maeshima, M., and Segami, S. (2020). Lack of vacuolar

•
•
•
•
•
•
•
•
•

48

H+ -pyrophosphatase and cytosolic pyrophosphatases causes fatal
developmental defects in Arabidopsis thaliana. Front. Plant Sci. 11,655. DOI: 10.3389/fpls.2020.00655
Fukushima, K., Narukawa, H., Palfalvi, G., and Hasebe, M. (2021).
A discordance of seasonally covarying cues uncovers misregulated
phenotypes in the heterophyllous pitcher plant Cephalotus follicularis.
Proc. R. Soc. B Biological Sci. 288, 20202568. DOI: 10.1098/
rspb.2020.2568
Gu, N., Tamada, Y., Imai, A., Palfalvi, G., Kabeya, Y., Shigenobu, S.,
Ishikawa, M., Angelis, K.J., Chen, C., and Hasebe, M. (2020). DNA
damage triggers reprogramming of differentiated cells into stem cells in
Physcomitrella. Nat. Plants 6, 1098-1105. DOI: 10.1038/s41477-020-0745-9
Hashida, Y., Takechi, K., Abiru, T., Yabe, N., Nagase, H., Hattori,
K., Takio, S., Sato, Y., Hasebe, M., Tsukaya, H., et al. (2020). Two
ANGUSTIFOLIA genes regulate gametophore and sporophyte
development in Physcomitrella patens. Plant J. 101, 1318–1330. DOI:
10.1111/tpj.14592
Kumar, M., Matoba, O., Quan, X., Rajput S.K. Awatsuji, Y., andTamada, Y. (2021). Single-shot common-path off-axis digits
holography: applications in bioimaging and optical metrology. Appl.
Opt. 60, A195–A204. DOI: 10.1364/AO.404208
Kumar, M., Quan, X., Awatsuji, Y., Cheng, C., Hasebe, M., Tamada, Y.,
and Matoba, O. (2020). Common-path multimodal 3D fluorescence andphase imaging system. J. Biomed. Opt. 25, 032010. DOI: 10.1117/1.
JBO.25.3.032010
Kumar, M., Quan, X., Awatsuji, Y., Tamada Y., and Matoba, O. (2020).
Single-shot common-path off-axis dual-wavelength digital holographic
microscopy. Appl. Opt. 59, 7144–7152. DOI: 10.1364/AO.395001
Palfalvi, G., Hackl, T., Terhoeven, N., Shibata, T.F., Nishiyama, T.,
Ankenbrand, M., Becker D., Foerster, F., Freund, M., Iosip, A., Kreuzer,I., et al. (2020). Genomes of the Venus flytrap and close relatives unveil
the roots of plant carnivory. Curr. Biol. 30, R700-R702. DOI: 10.1016/j.
cub.2020.04.051
Suda, H., Mano, H., Toyota, M., Fukushima, K., Mimura, T., Tsutsui,I., Hedrich, R., Tamada, Y., and Hasebe, M. (2020). Calcium dynamics
during trap closure visualized in transgenic Venus flytrap. Nat. Plants 6,
1219-1224. DOI: 10.1038/s41477-020-00773-1
Yano, H., Alam, M.Z., Rimbara, E., Shibata, T.F., Fukuyo, M., Furuta,
Y., Nishiyama, T., Shigenobu, S., Hasebe, M., Toyoda Atsushi and
Suzuki, Y., et al. (2020). Networking and specificity-changing DNA
methyltransferases in Helicobacter pylori. Front. Microbiol. 11, 1628.
DOI: 10.3389/fmicb.2020.01628〔Review article〕
H., and Hasebe, M. (2021). Rapid movements in plants. J. Plant• Mano,Res. 134, 3–17. DOI: 10.1007/s10265-020-01243-7

DIVISION OF SYMBIOTIC SYSTEMS

Professor

Associate Professor

KAWAGUCHI, Masayoshi SOYANO, Takashi

Assistant Professor:
KAWADE, Kensuke
Specially Appointed Assistant Professor:
LIU, Meng
Technical Staff:
FUKADA-TANAKA, Sachiko
Postdoctoral Fellow:
TSUGANE, Mika
YANO, Koji
HASHIMOTO, KayoJSPS Postdoctoral Fellow:FUKUDOME, Mitsutaka
Visiting Scientist:
NAKAGAWA, Tomomi
SOKENDAI Graduate Student: OKUMA, Nao
GOTO, Takashi
ODA, Akiko
Technical Assistant:

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
Systemic optimization of root nodulation throughshoot-derived miR2111.
The symbiosis with rhizobia enables legumes to thrive
under nitrogen-limiting conditions. However, sinceN2-fixation is a highly energy-consuming process, excess
nodule formation is detrimental to the host growth. Legumes,
therefore, tightly control nodule numbers using a root-shootroot, long-distance signaling mechanism: autoregulationof nodulation (AON) (Figure 1). In Lotus japonicus, theinitial step of AON is the synthesis of root-derived mobile
signals, CLV3/ESR-related (CLE) ROOT SIGNAL 1, 2,
and 3 (CLE-RS1, 2, and 3) peptides, in response to either
rhizobial infection or high nitrate concentrations in theroots. These CLE peptides are translocated into the shootthrough xylem vessels and are perceived by a shoot-acting
HYPERNODULATION ABERRANT ROOT1 (HAR1)
receptor-like kinase. Consequently, TOO MUCH LOVE
(TML) F-box/kelch repeat protein, expressing in roots,
inhibits nodulation downstream of HAR1.

AON

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.

Proposed modelThis study

HAR1

HAR1

HAR1

miR2111 genes? MIR2111-5,-2, -4, -7

miR2111
Shoot
to
Root

CLE
-RS1/2
Rhizobia

TML

TML

Nodulation Nodulation

miR2111
Shoot
to
Root

TML
Nodulation

Figure 1. The model of AON and overview of this study.AON model(Left), proposed model of miR2111 function in AON by previous study(Middle), and demonstrated in our study.

Visual overview of this lab’s work.

In this signaling system, the detailed regulatory mechanism of nodulation by the shoot in AON remains unclear. A
microRNA, miR2111, that targets TML mRNA, is the mostplausible shoot-derived factor in AON (Figure 1). Since
the promoter activity of one miR2111 gene, MIR2111-3, isdetected predominantly in leaves, the shoot-to-root translocation of miR2111 has been postulated to explain the shootmediated control of nodulation. However, whether MIR21113 is a responsible locus for AON remains unclear. Moreover,
the role of shoot-accumulating miR2111s in the systemic
regulation of nodulation is unproven thus far. Therefore, weinvestigated highly contributing miR2111 genes to AON andthe function of shoot-accumulating miR2111 to clarify theregulatory mechanism of nodulation from the shoot.

49

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Three miR2111 loci (MIR2111-1 to MIR2111-3) have beenreported in L. japonicus. To identify the loci responsible for
AON, we first searched additional potential miR2111 genes
and found four new miR2111 loci, MIR2111-4 to MIR2111-7,
on the L. japonicus genome through hairpin structure prediction by combining BLAST search and RNA-seq-based
gene prediction. Of the seven miR2111 genes, MIR2111-2,
MIR2111-4, MIR2111-5, and MIR2111-7 were expressedin leaves, and the accumulation levels of these transcripts
decreased after rhizobial inoculation in a HAR1-dependent
manner (Figure 1). MIR2111-2 and MIR2111-5 overexpression in hairy roots suppressed TML mRNA accumulationand significantly increased nodule numbers, whereas that of
MIR2111-4 did not influence nodulation.
Of the seven miR2111 loci, MIR2111-5 showed the highest
levels of its primary transcripts in leaves. Thus, we hypothesized that MIR2111-5 significantly contributes to the accumulation of mature miR2111s in leaves and roots. Using
MIR2111-5 promoter GUS assays, we found that MIR21115 was expressed predominantly in the phloem of leaves.
mir2111-5 mutants reduced mature miR2111 levels in bothleaves and roots to < 50% of those observed in the wild-type,and significantly decreased the nodule and infection thread
numbers compared to those in the wild-type. Furthermore,grafting experiments demonstrated that wild-type rootstock grafted with MIR2111-5-overexpressing scion showed
increased nodules and mature miR2111s levels (Figure 2).
The production of mature miR2111s in leaves by MIR2111-5
is therefore necessary for the systemic control of nodulation and mature miR2111 levels in roots. Taken together, weclearly showed the systemic effect of shoot-accumulating
miR2111 on nodulation and determined that MIR2111-5 is ahighly contributing locus for AON.

Figure 2. Reciprocal grafting between wild-type and MIR2111-5 overexpression plants. Nodulation at 28 days after inoculation (dai) in wild-type
and MIR2111-5ox rootstocks grafted with wild-type and MIR2111-5ox
scions. Scale bars: 1 mm.

II. Arbuscular mycorrhizal symbiosis
The roots of most terrestrial plants in the world have a
symbiotic relationship with filamentous fungi via mycorrhizae. Approximately 80% of land plants including 94% ofAngiosperms form some type of association with mycorrhizae. Arbuscular mycorrhizae, a type of endomycorrhiza in which fungal hyphae enters the plant cells andshapes highly branched structures named arbuscules, formed
symbiotic relationships with land plants more than 400
million years ago. In contrast, land plant associations with50

ectomycorrhizae, which is characterized by dense rootsurrounding hyphae and intercellular hyphae between root
cells, began about 190 million years ago. Even in modern
ecosystems, arbuscular mycorrhizae constitute the mostabundant form of mycorrhizal association with angiosperms.
Thus, arbuscular mycorrhizal (AM) symbiosis is considered
the most basic form of mycorrhizal symbiosis. AM symbiosis
is evolutionarily and agriculturally important because land
plants including bryophytes, pterophytes and many crops
colonize with AM fungi to obtain nutrients from the soil
(Figure 3).

Figure 3. AM fungi form mutualistic symbiosis with most terrestrial
plants and can generate superorganisms via their hyphae.

AM fungi are obligate biotrophic fungi that require root colonisation to complete a life cycle (Figure 4). Genome analysesof AM fungi demonstrate that they lack several important
metabolic enzymes related to the obligate biotrophy. AM fungi
have long been considered unculturable without the host.
However, co-culture of the AM fungus Rhizophagus irregularis and mycorrhiza-helper bacteria–isolates of Paenibacillus
validus–demonstrated that AM fungi can complete their life
cycle in the absence of host plants.

Figure 4. Life cycle of AM fungi. Next-generation spores are formed byroot colonization.

Recently, fatty acids have been shown to boost AM
fungal growth and sporulation under asymbiotic conditions. Palmitoleic acid and a methyl branched-chain fatty
acid isolated from P. validus asymbiotically induced infection-competent secondary spores of R. irregularis. Further,myristate initiated the asymbiotic growth of AM fungi andcan also serve as a carbon and energy source. These findings
may lead to the development of new research tools for AM
studies and novel generation system of AM fungal inoculants. However, at present, fungal biomass and spore production in the asymbiotic culture systems remain lower than

those in symbiotic co-cultures. Moreover, spores induced bypalmitoleic acid or myristate were smaller than those generated symbiotically and their performance as inoculants are
unknown.
Not only nutrients but also signalling molecules from hostplants may be crucial for AM fungal growth and reproduction. Some phytohormones show positive effects on interactions between AM fungi and hosts. Strigolactone is a major
plant-derived signal known to induce hyphal branching andelongation of AM fungi and to stimulate their mitochondrial activity in the pre-symbiotic stage. Methyl jasmonate(MeJA) was increased during AM fungal colonization inroots, consistent with the up-regulation of jasmonic acid biosynthesis genes in plant cortical cells containing arbuscules,
highly branched fungal structures for nutrient exchange.
Most research of these and other phytohormones are focusedon cell-level interactions between AM fungi and plants, andthe direct effect of phytohormones on AM fungal growth andreproduction is largely unclear.
We focused on the effect of two phytohormones, strigolactone and MeJA, on AM fungal growth and sporulation inasymbiotic culture supplemented with potassium myristate.
We used R. clarus HR1 isolated from Hazu-cho, Nishio City,
Aichi Prefecture (Figure 5) and found that hundreds of times
more secondary spores were produced in medium containing
the these two phytohormones than in media without phytohormones in R. clarus asymbiotic culture. Furthermore, we
confirmed that asymbiotically-produced spores can be subcultured and facilitates the growth of plants. Based on thesefindings, we considered the biological potential of asymbiotically-produced spores.

K., Horiguchi, G., Hirose, Y., Oikawa, A., Hirai, M.Y., Saito,• Kawade,
K., Fujita, T., and Tsukaya, H. (2020). Metabolic control of gametophore

•
•
•
•
•
•
•

shoot formation through arginine in the moss Physcomitrium patens.
Cell Rep. 32, 108127. DOI: 10.1016/j.celrep.2020.108127
Nozaki, M., Kawade, K., Horiguchi, G., and Tsukaya, H. (2020). An3Mediated Compensation is dependent on a cell-autonomous mechanism
in leaf epidermal tissue. Plant Cell Physiol. 61, 1181–1190. DOI:
10.1093/pcp/pcaa048
Okuma, N., Soyano, T., Suzaki, T., and Kawaguchi, M. (2020).
MIR2111-5 locus and shoot-accumulated mature miR2111 systemicallyenhance nodulation depending on HAR1 in Lotus japonicus. Nat.
Commun. 11, 5192. DOI: 10.1038/s41467-020-19037-9
Soyano, T., Liu, M., Kawaguchi, M., and Hayashi, M. (2021).
Leguminous nodule symbiosis involves recruitment of factors
contributing to lateral root development. Curr. Opin. Plant Biol. 59,
102000. DOI: 10.1016/j.pbi.2020.102000
Sugiura, Y., Akiyama, R., Tanaka, S., Yano, K., Kameoka, H., Marui,
S., Saito, M., Kawaguchi, M., Akiyama, K., and Saito, K. (2020).
Myristate can be used as a carbon and energy source for the asymbiotic
growth of arbuscular mycorrhizal fungi. Proc. Natl. Acad. Sci. U.S.A.
117, 25779–25788. DOI: 10.1073/pnas.2006948117
Suzuki, M., Numazaki, R., Nakagawa, T., Shibuya, N., and Kaku, H.
(2020). Cytoplasmic interaction of LysM receptors contributes to theformation of symbiotic receptor complex. Plant Biotechnol. 37, 359–
362. DOI: 10.5511/plantbiotechnology.20.0511a
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
Umnajkitikorn, K., Fukudome, M., Uchiumi, T., and Teaumroong,
N. (2021). Elevated Nitrogen Priming Induced Oxinitro-Responses
and Water Deficit Tolerance in Rice. Plants 10, 381. DOI: 10.3390/
plants10020381

Figure 5. Spore and hyphae of R. clarus HR1.

Publication List:〔Original papers〕
A., Nagae, M., Nishimura, Y., Romero Montero, D.,• Akamatsu,
Ninomiya, S., Kojima, M., Takebayashi, Y., Sakakibara, H., Kawaguchi,••M., and Takeda, N. (2021). Endogenous gibberellins affect root nodule
symbiosis via transcriptional regulation of NODULE INCEPTION in
Lotus japonicus. Plant J. 105, 1507–1520. DOI: 10.1111/tpj.15128
Aoki, T., Kawaguchi, M., Imaizumi-Anraku, H., Akao, S., Ayabe,S., and Akashi, T. (2021). Mutants of Lotus japonicus deficient inflavonoid biosynthesis. J. Plant Res. 134, 341–352. DOI: 10.1007/
s10265-021-01258-8
Greetatorn, T., Hashimoto, S., Maeda, T., Fukudome, M., Piromyou, P.,
Teamtisong, K., Tittabutr, P., Boonkerd, N., Kawaguchi, M., Uchiumi,
T., et al. (2020). Mechanisms of rice endophytic bradyrhizobial celldifferentiation and its role in nitrogen fixation. Microbes Environ. 35,
ME20049. DOI: 10.1264/jsme2.ME20049

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:

ANDO, Toshiya
NAKAMURA, TaroMIZUTANI, Takeshi
Technical Staff:
JSPS Postdoctoral Fellow: SAKAI, Hiroki
KONAGAYA, Tatsuro
TAKENAKA, Masaki
Research Staff:
KAWAGUCHI, Haruka
SOKENDAI Graduate Student: CHIKAMI, Yasuhiko
KITAZAWA, Yurina
MORITA, Shinichi
Visiting Scientist:
Technical Assistant:
MORITA, Junko
HACHISUKA, Yukari
YOKOYAMA, Michiko
TAGUCHI, Rie
Admin Support Staff:
SAITO, Eikoary origin of insect wings is still an enigma; one which weare trying to decipher by the use of evo-devo methods. In
Drosophila melanogaster, the wing master gene vestigial(vg) and its interaction partner scalloped (sd) play pivotal
roles in the formation of wing field identity. For this reason,these genes are ideal research candidates in the investigationof 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 developed RNA interference (RNAi)
based methods for T. domestica to examine the functions ofthese genes. We are currently testing the functional effects ofaltered transcription for each of these wing genes in ancestrally wingless firebrats. Furthermore, we are performing
comparative analyses of the function of these same genes in“primitively winged” (hemimetabolous) insects (Figure 1B)
to obtain additional clues relevant to us understanding theorigin and evolution of insect wings.

B
The 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.

Visual overview of this lab’s work.

I. Origin and diversification of insect wings
The flight organs of insects have uniquely evolved whencompared to that of other various flying animals on earth.
Despite more than two centuries of debate, the evolution-

52

A

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

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 this, we hypothesize that ancestral lateral body wall outgrowths evolved into functional wings. However, genetic
tools available for the analysis of basally branching winglessspecies 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) membrane transporter, ofT. domestica by using CRISPR/Cas9 system. This in turnresults in white-eyed firebrats. In addition to the RNAimediated gene knockdown (Ohde et al., 2009), germline
genome editing using CRISPR/Cas9 in T. domestica provides
a platform technology for creating new research opportunities concerning the evolution of insects, such as insect wing
origin. We are now conducting gene knock-out/in withinvarious “wing” genes to identify genetic details and celllineage 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 plays various ecologically important roles such as intraspecific sexual signaling,mimesis, mimicry, and is also used as a warning signal topredators. However, the molecular mechanisms responsible
for generating such color patterns in most ladybird species
remain elusive. To investigate the developmental mechanisms of color pattern formation, we have been focusing on
the multicolored Asian ladybird beetle, Harmonia axyridis,
which has conspicuous and variable wing color patterns
consisting of black and red pigments (Figure 2A). The ladybird’s vivid wing color pattern acts 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. 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 currently 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 RNAiin the ladybirds.

A

B

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

We recently identified a key gene, pannier, which regulates intraspecific color pattern polymorphism in H. axyridis
using next generation sequencing technologies (RNA-seq
and de novo genome assembly), and an RNAi-based screening method that we have established. pannier is expressedin specific regions in the wing, which synthesizes black
pigment, and suppresses red pigmentation. The expression
pattern of pannier is diversified according to the diverse
color pattern types in H. axyridis. These findings suggest that
regulatory shift, such as changes in enhancer activity, at thepannier 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 afocus on regulatory shifts at the pannier loci.
We are also attempting to apply genome-editing technologies such as TALEN and CRISPR/Cas9 to tackle this issue.
Thus far, we have achieved an efficient method of genedisruption using TALEN. Recently, we have begun successfully establishing the disrupting pannier gene function using
CRISPR/Cas9 system. Ribonucleoprotein (RNP) complex
of Cas9 protein and guide RNA targeting pannier codingexon was microinjected into the fertilized eggs. As a result,over 60% of G0 founders produced pannier knockout progenies (Figure 3). CRISPR/cas9 mediated gene disruption
has advantages in terms of ease of designing gRNA andalso possesses a high KO efficiency compared to TALEN
mediated gene modification in Harmonia. To this end, weare continuing to develop CRISPR/Cas9 mediated geneknock-in method. To apply this approach, we are trying toattempt 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.

Figure 3. Representative photographs of pannier mutant phenotypes in G0
wt-like (left side), G0 mosaic (middle side) and G1 KO mutant (right side).

We are also establishing cryopreservation methods for
germline cells in ladybird beetles to assist us with this.
This is due to the high risk of losing valuable genetic bioresources in non-model insects. We recently established ovarytransplantation and ovarian cryopreservation techniques inladybird beetles. We hope that the genetic tools and techniques that we have established will further facilitate thisresearch.
We plan to eventually 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 among the bestmodels for studying how an extreme, sex-specific morphology is formed (Figure 4, Control).
We have recently developed a larval RNAi technique for53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

T. dichotomus, which has allowed us to molecularly dissect
the relationship between the conserved genetic pathway forsex differentiation and sexually dimorphic horn formation
during post embryonic development. We systematicallyevaluated the function of the sex determination gene, transformer (tra) in different developmental stages, and revealedin which tissue and developmental stage the gene regulatory
network for sex differentiation is activated to form sexual
dimorphic horns in the head and thorax. In T. dichotomus,
tra regulates sex-specific splicing of the doublesex premRNA, and its loss of function results in sex transformationin females (Figure 4). tra RNAi treatments in females at
early developmental stages during metamorphosis resulted
in full sexual transformation, whereas no transformation isobserved in the treatments at later stages. Therefore, we wereable to estimate the onset of activation of the developmental program for the sexually dimorphic horn formation bydetermining the latest RNAi treatment timing when a fullsexual transformation phenotype is observed. Based on thisapproach, we estimated that the developmental program forsexually dimorphic horn formation is activated at 29 hours
after the prepupal period.

Figure 4. tra RNAi phenotypes. In the tra RNAi females, ectopic hornformation was caused by 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
in unveiling the gene regulatory network for sexually dimorphic horn formation and to pursue the evolutionary origin ofsuch a regulatory system.

Publication List:〔Original papers〕
H., Matsuda, K., Niimi, T., Kondo, S., and Gotoh, H. (2020).
• Adachi,
Genetical control of 2D pattern and depth of the primordial furrow that

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54

prefigures 3D shape of the rhinoceros beetle horn. Sci. Rep. 10. DOI:
10.1038/s41598-020-75709-y
Chikami, Y., Kawaguchi, H., Suzuki, T., Yoshioka, H., Sato, Y.,
Yaginuma, T., and Niimi, T. (2021). Oral RNAi of diap1 results in rapid
reduction of damage to potatoes in Henosepilachna vigintioctopunctata.
J. Pest Sci. 94, 505–515. DOI: 10.1007/s10340-020-01276-w
Sakai, H., Konagaya, T., Takemura, Y., Sahara, K., and Niimi, T.
(2020). Double-copulated introduction of ejaculate with dominantlarval phenotype to maintain Bombyx mori mutant with dysfunctional
apyrene sperm. J. Insect Biotechnol. Sericology 89, 2_039-2_043. DOI:
10.11416/jibs.89.2_039

LABORATORY OF EVOLUTIONARY GENOMICS

Professor:

SHIGENOBU, Shuji

Assistant Professor:
HUANGH, Jia-Hsin
Specially Appointed Assistant Professor:
YODA, Shinichi
KOBAYASHI, Yuuki
Postdoctoral Fellow:
CHUNG, Chen-yo
JSPS Postdoctoral Fellow: NOZAKI, Tomonari
MATSUDA, Naoki
SOKENDAI Graduate Student: YORIMOTO, Shunta
TAN, Kathrine
Visiting Graduate Student : TONG, Xin
Technical Assistant:
SUZUKI, Miyuzu
WEN, Hsin-I
IKEDA, Mika
Admin Support Staff:
ICHIKAWA, Mariko

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 newinterdisciplinary 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 withsecretion 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). These results suggest that BCRs act withinbacteriocytes 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.
Visual overview of this lab’s work.

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
Publication List
〔Original papers〕
M., Yamamoto, N., Shigenobu, S., Mori, H.,• Bessho-Uehara,
Kuwata, K., and Oba, Y. (2020). Kleptoprotein bioluminescence:

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Parapriacanthus fish obtain luciferase from ostracod prey. Sci. Adv. 6,
eaax4942. DOI: 10.1126/sciadv.aax4942
Fukutomi, Y., Kondo, S., Toyoda, A., Shigenobu, S., and Koshikawa,S. (2021). Transcriptome analysis reveals wingless regulates neuraldevelopment and signaling genes in the region of wing pigmentation ofa polka-dotted fruit fly. FEBS J. 288, 99–110. DOI: 10.1111/febs.15338
Tominaga, T., Yamaguchi, K., Shigenobu, S., Yamato, M., andKaminaka, H. (2020). The effects of gibberellin on the expressionof symbiosis-related genes in Paris-type arbuscular mycorrhizal
symbiosis in Eustoma grandiflorum. Plant Signal. & Behav. 15. DOI:
10.1080/15592324.2020.1784544
Mondal, S.I., Akter, A., Koga, R., Hosokawa, T., Dayi, M., Murase, K.,
Tanaka, R., Shigenobu, S., Fukatsu, T., and Kikuchi, T. (2020). Reduced
genome of the gut symbiotic bacterium “Candidatus Benitsuchiphilus
tojoi’’ provides insight into its possible roles in ecology and adaptationof the host insect. Front. Microbiol. 11. DOI: 10.3389/fmicb.2020.00840
Li, Y., Omori, A., Flores, R.L., Satterfield, S., Nguyen, C., Ota, T.,
Tsurugaya, T., Ikuta, T., Ikeo, K., Kikuchi, M., et al. (2020). Genomic
insights of body plan transitions from bilateral to pentameral symmetryin Echinoderms. Commun. Biol. 3. DOI: 10.1038/s42003-020-1091-1
Vu, T.-D., Lwasaki, Y., Shigenobu, S., Maruko Akiko and Oshima,K., Lioka, E., Huang, C.-L., Abe T., Tamaki, S., Lin, Y.-W., Chen,
C.-K., Lu Mei-Yeh and Hojo, M., et al. (2020). Behavioral and braintranscriptomic synchronization between the two opponents of a fighting
pair of the fish Betta splendens. PLoS Genet. 16. DOI: 10.1371/journal.
pgen.1008831
Cui, S., Kubota, T., Nishiyama, T., Ishida, J.K., Shigenobu, S., Shibata,T.F., Toyoda, A., Hasebe, M., Shirasu, K., and Yoshida, S. (2020).
Ethylene signaling mediates host invasion by parasitic plants. Sci. Adv.
6. DOI: 10.1126/sciadv.abc2385

LABORATORY OF BIORESOURCES

Specially Appointed Professor

NARUSE, Kiyoshi

Research Staff:

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
Admin Support Staff:
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.

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 amuch-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

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

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

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 that57

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

has 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 playdifferent 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. National BioResource Project Medaka
Our laboratory has been acting as main facility of theMedaka BioResource Project (NBRP Medaka) in the fourthphase of NBRP, which started in 2017. We are providing
wild strains, and related species, genomic resources (approximately 400,000 cDNA clones from 30 different cDNA libraries (containing approximately 23,000 different sequences) and BAC/Fosmid clones covering the entire medaka
genome), and hatchery enzymes essential for embryo manipulation and live imaging. These bioresources are available
on our website (Figure 3. https://shigen.nig.ac.jp/medaka/).
These bioresources can be searched by various methods suchas keywords, sequence homology, and expression profiles
using the database on the website. In addition, genome
browsers for the three inbred strains, phylogenetic relationships between wild strains of medaka and related species,and experiment manuals are also available. With the approvalof the second supplementary budget for FY2020, we wereable to install a cabinet-type fish tank washing machine.
This has freed up our technical support staff from the need
to wash the tanks by hand, and has allowed us to focus moreon breeding and management, which requires more humanwork. In addition, a system to remotely monitor the temperature, humidity, and illumination in the medaka breedingrooms and the water temperature in the breeding tanks was
installed. The air conditioner in the breeding room was also
upgraded. We have continuously monitored the medaka
breeding conditions using these systems.

Figure 3. NBRP Medaka website

Publication List:〔Original papers〕
S., Mochida, K., Fujimoto, S., Mokodongan, D.F., Sumarto,• Ansai,
B.K.A., Masengi, K.W.A., Hadiaty, R.K., Nagano, A.J., Toyoda, A.,

•
•

58

Naruse, K., et al. (2021). Genome editing reveals fitness effects of agene for sexual dichromatism in Sulawesian fishes. Nat. Commun. 12.
DOI: 10.1038/s41467-021-21697-0
Li, Y., Liu, Y., Yang, H., Zhang, T., Naruse, K., and Tu, Q. (2020).
Dynamic transcriptional and chromatin accessibility landscape of
medaka embryogenesis. Genome Res. 30, 924–937. DOI: 10.1101/
gr.258871.119
Takehana, Y., Zahm, M., Cabau, C., Klopp, C., Roques, C., Bouchez,O., Donnadieu C., Barrachina, C., Journot, L., Kawaguchi, M.,
Yasumasu, S., et al. (2020). Genome sequence of the euryhaline javafish
medaka, Oryzias javanicus: A small aquarium fish model for studies on
adaptation to salinity. G3: Genes Genomes Genet. 10, 907–915. DOI:
10.1534/g3.119.400725

LABORATORY OF MORPHODIVERSITY

Associate Professor

KODAMA, Ryuji

Visiting Scientist:

YOSHIDA, Akihiro

The aim of this laboratory is to observe the variety of morphogenetic processes that occur during the ontogenesis ofmulticellular organisms and to analyze the mechanisms ofsuch processes through mainly morphological methods. The
accumulation of such analyses of the embryogenetic processes of related species is expected to provide an insight into theevolution of morphogenetic processes. This laboratory uses
the wings of lepidopteran insects (butterflies and moths) as
the main subject of morphological studies.

I. Wing outline shape formed by cell death
The wings of lepidopteran insects develop from the wing
imaginal disc, which is a hollow sac made of simple epithelium. The outline shape of an adult wing often differs 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 themacrophages in the cavity beneath the wing epithelium. The
macrophages are excluded from the differentiation region bya close adherence of dorsal and ventral epithelia and concentrated in the degeneration region, thus accelerating theremoval of 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. These scales are remarkably elongated and growdensely on the edge of the wing. These scales are considered
to be important in stabilizing the turbulence occurring posterior to the wing. The movements of the marginal scales areclosely monitored by sensory scales and the bristles growing
among them.

concomitant with programmed cell death at the wing margin,
and gives rise to the space between the pupal wing andthe cuticle. This space appears to allow the growth of 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 (Yoshida, A. et al. Sci. Nat. 104, 27, 2017).

III. Transparent wing formation through scale
removalButterfly and moth wings are usually fully covered withscales. However, in some species, the adult wing lacks scales
in some 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 the wing immediately after
emergence is covered with scales that are morphologically different from the scales on the other part2) young adults detach numerous scales on the future transparent part at the initial take-off after emergence, andconsequently the transparent part appears.
We also studied the scale morphology and found that the
detaching scale is remarkably large and its proximal pedicel,
through which the scale attaches to the wing, is tapered(Figure 1). We concluded that these morphological features
facilitate the scale detachment through fluttering. (Yoshida,
A. et al., Programmed scale detachment in the wing of thepellucid hawk moth, Cephonodes hylas: Novel scale morphology, scale detachment mechanisms, and wing transparency. Zool. Sci. 38, 427-435, 2021. doi: 10.2108/zs210031).

Figure 1. Scanning electron microscopy of the wing scales in the futuretransparent part (A) and the other part left covered with scales (B) at thesame magnification. The scale in A is much larger than that in B, and thepedicel, the protrusion seen at the lower left corner of A is tapered whilepedicels in B are columnar. Scale bars: 50µm.

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 is†: This laboratory was closed on 31 March, 2021
59

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITY
KAMADA Group
Assistant Professor:

KAMADA, Yoshiaki

Tor (target of rapamycin) is a Ser/Thr protein kinase
which is well conserved in organisms spanning from yeasts
to mammals. Tor forms two distinct protein complexes,
TORC1 (Tor complex1) and TORC2. On one hand, TORC1
is involved in amino acid sensing, regulation of protein
synthesis (especially the translation step), the cell cycle, andautophagy. On the other hand, TORC2 is responsible foractin organization and cell integrity. Up until now, it’s been
deemed unlikely that TORC2 can recognize nutrient signals.
The aim of our research group is to reveal the molecularmechanisms underlying how TORC1 receives amino acidsignals and how the TORC1/2 pathways regulate phenomena
they are associated with. We have been studying Tor signaling in the budding yeast Saccharomyces cerevisiae, and havefound three novel TOR signaling pathway branches (Figure 1).

product of ARS acts as an amino acid signal rather than anamino acid itself. Biochemical in vitro TORC1 assay also
revealed that tRNA directly inhibits TORC1 kinase activity,
suggesting that uncharged tRNA, produced under aminoacid-depleted condition, functions as a starvation signal.
Reducing cellular tRNA molecules desensitizes TORC1 inactivation by nitrogen starvation in vivo.
Based on these results, a TORC1 regulatory model was
proposed which contends that free tRNA released fromprotein synthesis under amino acid starvation inhibits
TORC1 activity. Therefore, TORC1 employs a tRNAmediated mechanism to monitor intracellular amino acids(Figure 2).

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

Since tRNA directly inhibits TORC1 activity, TORC1
should have a tRNA-binding site(s). Thus, we have investigated further so as to determine the tRNA-binding site. So
far, we have obtained a good candidate for the tRNA-bindingsite in Tor protein, and we will now focus on this domain to
determine its function.

II. 
TORC1 phosphorylates Atg13, themolecular switch of autophagy

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 themselves
are fundamental nutrients. 20 species of amino acids thatbuild proteins cannot be interchanged with each other.
Therefore, each amino acid must be individually detected by
TORC1.
We have conducted genetic research and discovered the
involvement of (aminoacyl-) tRNA in TORC1 regulation.
For example, mutants of aminoacyl-tRNA synthetases(ARSs) exhibited inactivation of TORC1 even under aminoacid-rich condition, suggesting that aminoacyl-tRNA, a60

TORC1 negatively regulates autophagy: a protein degradation system induced by nutrient starvation.
We have been 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 consequent
induction of autophagy.

III. Relationships between TORC1 and eIF4Ebinding proteins
EIF4E-binding proteins (4EBPs), which binds to translation initiation factor 4E (eIF4E), are thought to negatively
regulate translation initiation, because their competition
infused binding to eIF4E prevents eIF4E-eIF4G association,
which is a primary and essential procedure in translation
initiation. In mammals, the inhibitory function of 4EBP1
is regulated by mammalian TORC1 (mTORC1). Under
nutrient-rich conditions, mTORC1 phosphorylates 4EBP1,
and the phosphorylated 4EBP1, lose affinity to eIF4E and let
eIF4E bind to eIF4G.
We examined whether yeast 4EBPs, Caf20 and Eap1, haveproperties in common with 4EBP1. Caf20, but not Eap1, is
phosphorylated in a TORC1-dependent manner, it binds toeIF4E, and it never associates to eIF4G. However, Caf20eIF4E binding was not affected by TORC1 activity, suggesting that the function of the yeast 4EBP is different from thatof its mammalian counterparts.

Publication List:〔Original paper〕
Y., Kamada, Y., and Yamashita, A. (2020). Novel links between• Otsubo,
TORC1 and traditional non-coding RNA, tRNA. Genes 11. DOI:
10.3390/genes11090956

61

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITYHOSHINO Group
Assistant Professor:
Technical Assistant:

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

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

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
impacts greatly 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.

Apart from the resequence analysis, de novo genome sequencing for the lines Violet and Africa as well as the duskish mutant
shown in Figure 1 was performed using HiFi sequencing on asingle-molecule real-time sequencer (Sequel II system). The
obtained assembly sequences were of equal or better quality
than the genome sequence previously reported, and werecompiled into a database by the NIBB Data Integration andAnalysis Facility to be made available to the public.

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, which is believed to be regulated by
epigenetic mechanisms (Figure 1). We are currently analyzing the detailed molecular mechanisms of these mutations.

II. Whole-genome analysis of various I. nil lines
NBRP Morning Glory (described below) maintains approximately 3,500 lines, including two standard lines: Tokyo
Kokei Standard and Violet. A high-quality draft whole-genome
sequence of Tokyo Kokei Standard, which is accessible fromour database, has been previously publicized. Whole genome
sequencing of 100 representative lines were performed
with the support of NBRP Genome Information Upgrading
Program. The lines were chosen to meet the needs of theresearch community, and to provide information regarding
the polymorphisms and gene mutations in the lines. This
includes the multiple mutants called ‘Henka-Asagao’ covering
the major mutations, and the wild-type plants isolated fromnatural populations outside of Japan. As a result, total 25,000
Tpn1 transposon-induced insertion polymorphisms and SNPs,
Indels, and CNVs at 25 million loci were discovered.

62

Figure 2. The I. nil lines used for the whole-genome analysis. (a) Violet:
The most widely used standard line. (b) Africa: The wild-type line
isolated in Guinea in 1956. (c) Nepal: The wild-type line isolated by Dr,
Sasuke Nakao in Nepal in 1952. (d) Danjuro, one of the most popular
cultivars. It was named after the reddish-brown color of the costume
used by Danjuro Ichikawa II in his Kabuki play ‘Shibaraku. (e) The tinymutant has small organs. White spots on the leaves are due to the variegated mutation. (f) A mutant line of ‘Henka-Asagao’ showing doubled
flowers with narrow flower petals and sepals.

III. Morning glory bio-resources
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 230 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).

LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:

TSUGANE, Kazuo

Although transposons occupying a large portion of the
genome in various plants were once thought to be junk DNA,
they play an important role in genome reorganization andevolution. Active DNA transposons are important tools forgene functional analysis. The endogenous non-autonomoustransposon, nDart1, in rice is said to generate various transposon-insertion mutants because nDart1 elements tend toinsert into genic regions under natural 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 largescale nDart-inserted mutant population was easily generated
under normal field conditions, and the resulting tagged lines
were free of somaclonal variation. The nDart1/aDart1-27
system was introduced into a rice variety, Koshihikari (Oryza
sativa subsp. japonica), and Basmati (Oryza sativa subsp.
indica). Various mutations caused by the insertion of nDart
have been screened for characteristic phenotypes.

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
for boosting rice yield based on molecular biology is tostack the finely tuned gene expressions. The Lgg mutant
which was isolated from Koshihikari-nDart tagging line bore
slightly larger grains (Figure 1) 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. To investigate the subcellular localization of LGG
protein, green fluorescent protein (GFP)-fused constructs
driven by 35S promoter, 35S:LGGNP-GFP was transformed
into rice calli. GFP fluorescence spots were observed in the
nuclei in calli (Figure 2). These results suggest that LGG islocalized to the nucleus.

NP
Figure 2. Subcellular localization of LGG. 35S:LGG -GFP isexpressed in rice calli. 40,6-Diamidino-2-phenylindole is used for nuclear
staining. Bar = 20 mm.

63National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITY
KATO GroupSpecially Appointed Assistant Professor:
KATO, Kagayaki
HYODO, Miwa
Technical Assistant:experimental conditions to gain a better understanding of
the genetic programs underlying organogenesis. We are now
applying this system to several experimental models to determine the practicality of the system (Shinoda et al.).

II. Image processing pipeline for 3D cell
cultureTo elucidate the relationship between mechanical forces
underlying the tissue deformation out of large-scale imaging
data, we have developed an image processing pipeline for
3D+T imaging datasets.
This pipeline is able to automate a segmentation/quantification process for a large number of images acquired under
several experimental conditions for subsequent statisticalanalysis in addition to building a database of acquired quantities as its final output.

Organogenesis is accomplished by a series of deformations
which causes the planar cell sheet to form itself into a threedimensional shape during embryogenesis. This drastic structural change is the integrated result of individual cell behaviors in response to spatio-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 todo so due to the massive size of the images generated by 4D
microscopy as well as their 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.

III. Software for manual image quantification
Biologically significant imaging features are not always
significant to computational algorithms due to their structural instability. This level of difficulty requires inspection conducted by human eyes to extract features from theimages gleaned. To simplify this, we have developed a GUI(Graphical User Interface) application which 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. To observe this, we are developing asoftware pipeline which will automatically recognize individual cell shapes out of 3D space and tracks them throughtime. This system extracts cell boundaries and reconstitutes
cell shapes in the form of a skeletonized chain of voxels
spanning 3D space. This abstract form of cell visualization
makes it possible to describe morphometric quantities andkinetics of cells at a single-cell resolution (Figure 1). Thesemorphometric quantities allow us to perform comparative
studies on shapes and behaviors more precisely under several

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

Publication List:〔Original papers〕
I., Kelly, D.J., Carretero, N.T., Nomori, A., Kato, K.,• Kondrychyn,
Chong, J., Nakajima, H., Okuda, S., Mochizuki, N., and Phng, L.-••
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.

64•

K. (2020). Marcksl1 modulates endothelial cell mechanoresponse
to haemodynamic forces to control blood vessel shape and size. Nat.
Commun. 11. DOI: 10.1038/s41467-020-19308-5
Kurihara, M., Kato, K., Sanbo, C., Shigenobu, S., Ohkawa, Y.,
Fuchigami, T., and Miyanari, Y. (2020). Genomic profiling by ALaP-Seq
reveals transcriptional regulation by PML Bodies through DNMT3A
exclusion. Mol. Cell 78, 493+. DOI: 10.1016/j.molcel.2020.04.004
Sakai, Y., Kato, K., Koyama, H., Kuba A., Takahashi, H., Fujimori,T., Hatta, M., Negri, A.P., Baird, A.H., and Ueno, N. (2020). A stepdown photophobic response in coral larvae: implications for the lightdependent distribution of the common reef coral, Acropora tenuis. Sci.
Rep. 10. DOI: 10.1038/s41598-020-74649-x
Uemura, Y., Kato, K., Kawakami, K., Kimura Yukiko and Oda, Y.,
and Higashijima, S. (2020). Neuronal Circuits That Control Rhythmic
Pectoral Fin Movements in Zebrafish. J. Neurosci. 40, 6678–6690.
DOI: 10.1523/JNEUROSCI.1484-20.2020

LABORATORY OF BIOLOGICAL DIVERSITYOHTA GroupSpecially Appointed Assistant Professor:
OHTA, Yusaku

Image analysis is an important element in the understanding of life science. It makes the quantification of phenomena through the extraction of meaningful information froma large amount of images, and the appropriate expression of
those information, possible. To this end, I have been developing image analysis technology aimed at analyzing the developmental process of the early zebrafish embryos at a whole
embryo scale and single cell resolution. By combining 3D
cell tracking and functional imaging, I’m currently developing an analytical technique that can simultaneously extract
multiple types of information such as cell morphology, cell
motility, and cell dynamics.

I. S imultaneous multifunctional analysis ofearly embryonic development at a whole
embryo scale and single cell resolution.
During early embryogenesis, three-dimensional remodelingof cell populations through cell migration is essential. Whileindividual cell motility depends on extracellular signals, cell-

cell adhesion are able to be maintained, thereby controllinghighly coordinated cell motility. To elucidate the principle ofsuch a complicated form of embryogenesis, it is necessary tounderstand the cell dynamics of the whole embryo at a single
cell resolution. To this end, I have been developing image
analysis technology to analyze the developmental processof the early zebrafish embryo at a whole embryo scale andsingle cell resolution.
With the development of microscopic equipment, early
embryonic imaging has evolved from two-dimensional,
fixed specimens and partial embryonic observations to threedimensional, live specimens and whole embryo scale. Thisevolution has led to an explosion in the analysis of cell
migration during early embryonic development in recent
years. However, conventional image analysis techniques
have only been able to extract information on cell migration in early development. Therefore, I have developed ananalytic method that can simultaneously extract information
concerning cell migration, cell dynamics, and cell morphology at whole embryonic scale and single cell resolution.

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 image data
sets. However, image analysis can still create hurdles forresearchers, as well as bottlenecks in research. In order tosolve this problem, I have been providing research support
based on the following three concepts.
The first concept is quantitative image analysis based ona 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 and statistics supports quantitative and objective analysis. The second
concept is the active utilization of image analysis technology via the application of machine learning, including deeplearning. In recent years, the development of machine learning
has been remarkable, and with a little training, it is possible tosimplify analysis that is difficult to achieve with conventional
image analysis technology. The third concept relates to the
publication of explanations of image analysis to researchersin an easy-to-understand manner on the web. The contents ofwhich range from the principles of image analysis methods tothe use of image analysis software and plug-ins.

Figure 2. A website that explains how to use image analysis software
Figure 1. Simultaneous multifunctional analysis of early embryonicdevelopment

65

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITYSHINOMIYA GroupSpecially Appointed Assistant Professor:
SHINOMIYA, Ai
TSURUTA, Emiko
Technical Assistant:Figure 1. Differences in critical day length (right) were detected among
medaka populations that had originated in various latitudes (left) .

The 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 keeping with this, we are currently using Medaka to
study the molecular mechanisms of seasonal adaptation, as
well as the relationship between seasonal information fromthe environment and the organism’s response.

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
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.
A medaka habitat in summer (Miyazaki, Miyazaki Prefecture) (left). A
school of wild medaka (Toyohashi, Aichi Prefecture) (right).

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.

66

Whole-genome re-sequencing in various medaka populations experiencing different critical day lengths were conducted. We have identified potential candidate genes thatdefine the critical 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 (day length, solar radiation, watertemperature) in the experimental field. The regression model
explained which environmental factors contributed to theseasonal change in medaka gonads and to what extent they
contributed to this change.

Publication List:〔Original papers〕
T., Okimura, K., Shen, J., Guh, Y.-J., Tamai, T.K., Shimada,• Nakayama,A., Minou, S., Okushi, Y., Shimmura, T., Furukawa, Y., et al. (2020).

•

Seasonal changes in NRF2 antioxidant pathway regulates winterdepression-like behavior. Proc. Natl. Acad. Sci. U.S.A. 117, 9594–
9603. DOI: 10.1073/pnas.2000278117
Yassumoto I, T., Nakatsukasa, M., Nagano, A.J., Yasugi, M., Yoshimura,T., and Shinomiya, A. (2020). Genetic analysis of body weight in wild
populations of medaka fish from different latitudes. PLoS One 15. DOI:
10.1371/journal.pone.0234803

67

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITYSAITO GroupSpecially Appointed Associate Professor

SAITO, Nen

Measurement technologies, such as live imaging and nextgeneration sequencers, have been the recipients of rapid
development. We have entered a new era in which molecular
activities and gene expression levels in living tissues canbe measured at a single-cell resolution in a high throughput
manner. Our research group aims to elucidate the theoretical logic of dynamic living systems from such data by
combining mathematical modeling and machine learning.
The research topics in our group are diverse and cover subcellular scale phenomena as well as cellular and tissue level
phenomena.

sible for the differences in cell shapes due to cell type andmutation.
We also performed three-dimensional modeling of cell
deformation in macropinocytosis, which use actin-dependentendocytosis, and one in which amoeboid cells crawl on structured substrates with an micrometer-scale ridge.

II. M athematical Modeling of multicellular
systems
In the field of active matter physics, modeling of the collective motions of birds and fish has advanced to the pointof revealing how the collective motion of flocking behaviors
self-organize according to simple rules. On the other hand,the collective motion of cells differ greatly from flocks ofbirds or fish in that the shape of individual cells can be drastically deformed. In order to understand phenomena suchas the formation, homeostasis, and breakdown of tissues bycell populations, it is very important to study soft deformableactive matter that permits deformation while keeping cellmotion extremely simple. However, this vein of research isstill in its infancy.
Based on the Fourier series expansion of cell contour,
we have developed a mathematical model of multicellular
systems. This model enables us to simulate the numberof deformable cells in the order of thousands to tens of
thousands. Using this model, we are now exploring collective phenomena that can emerge from this deformability.
Currently, we have discovered that when the deformability of
individual cells is reduced, the motion of the entire cell population stalls and they akin to a glass-like material.

III. 
M a t h e m a t i c a l M o d e l o f M i c ro b i a l
Symbiosis via Metabolite Leakage

Figure 1. Researches combining machine learning and mathematicalmodeling.

I. Simulations of amoeboid cells
We conducted mathematical modeling of the cell shape
dynamics of single cells. Migrating cells take on a varietyof complex shapes and sometimes dynamically deform toperform important biological functions. On one hand, during
the amoeboid movement of Dictyostelium discoideum, said
movement is driven by the dynamic formation and disappearance of pseudopods at the cell front. On the other hand,in the epithelial cells of fish, a network of actin fibers called
lamellipodia appear at the cell front, and migratory movements are performed while maintaining their overall shape.
Using the phase-field method, we have developed a mathematical model that can describe the two-dimensional shape
dynamics of these various forms of locomotion. We havealso applied deep learning to extract features that quantify
the shape of migrating cells, and systematically compared theresults between experiments and simulations. By combining
this type of modeling and data-driven analysis, it is possible
to estimate which physicochemical parameters are respon-

68

In microbial ecosystems, a wide variety of microbial
species sometimes can coexist even when there is only one
niche (e.g., one nutrient supply). In recent years, it has been
widely stated that nutrient symbiosis via metabolite leakage.
Said leakage is caused by cross-feeding, a phenomenon inwhich a cell leaks metabolites while simultaneously utilizing
another metabolites leaked from another cell, and is important for the formation of complex ecosystems. However, it
is unclear why cells secrete metabolites in the first place.
It makes sense if it is to dispose of unnecessary waste, butsometimes cells also secrete metabolites that are supposed tobe essential for their growth.
Based on analytical and numerical calculations, we haveillustrated that if the intracellular metabolism includes multibody (e.g., catalytic) reactions, leakage of essential metabolites can promote the leaking cell’s growth. We have also
demonstrated that mutualistic relationships among diverse
species can be established as a result of cell-level adaptationof metabolite leakage; each species cross-feeds others bysecreting essential metabolites for their own benefit, which
are usefully consumed by others, in a manner reminiscent ofgift giving. In this case, the exchange of metabolites becomesentangled, which in turn leads to the coexistence of diverse
microbes. The resultant ecosystems become resilient against
external perturbations including the removal of each coexisting species.

INTRODUCTION OF THE OPEN LABORATRY
Under the leadership of its Director General, NIBB has
been improving research environments in order to strengtheninternational collaboration, create new biological research
fields, and develop truly globalized young researchers from2019 onwards. As part of this effort, NIBB has established
the Open Laboratory: a facility whose establishment was also
supported by the National Institutes of Natural Sciences.
Researchers engaged in different research fields come to theOpen Laboratory from all around the world to conduct theirown research, to be academically stimulated, and to foster
interdisciplinary research.

The basic concepts of the Open Laboratory
1. Promoting interaction between researchers from different
research fields and countries in an open laboratory space
as well as a shared office space
2. Sharing experimental equipment, instruments, bioresources, and information among participating researchers
3. Promoting interdisciplinary collaborative research
4. Globalizing young researchers through interactions withresearchers affiliated with overseas research institutes
5. Planning symposium, seminar and other events to shareknowledge.

Unwalled Open Laboratory space

The first Open Laboratory in operation

69

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

70

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.

In a collaborative effort with Prof. Yoshikuni and AssociateProf. Kurita’s Laboratory at Kyushu Univ., we are searching for reproductive hormones in echinoderms, including
starfishes, brittle stars, sea urchins, sea cucumbers, andcrinoids. The collaborating parties have been able to purify
physiological materials which induce egg maturation fromnerve extracts and analyze them with a protein sequencer anda tandem mass spectrometer in the analytical center of ourinstitute. One of them, named cubifrin, which is an NGIWYamide peptide, is in the sea cucumber Aposticopus japonicus.
We are currently preparing for this research’s 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 in a chemical synthetic manner to provide tocollaborating researchers for biological 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 DIVERSITYSAKUTA Group
Assistant Professor:
Technical Assistant :SAKUTA, Hiraki
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.

in plasma and CSF increases, Nax channels in glial cells in
the OVLT are activated, leading to the synthesis of epoxyeicosatrienoic acids (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. Schematic drawings of sensing mechanisms of body-fluid conditions in the OVLT responsible for the induction of water intake. AA,arachidonic acid.

II. Identification of novel sensors involved inwater intake control

Visual overview: Two postulated systems for sensing body-fluid conditions.

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. The signaling mechanisms in the OVLT for water-intake induction by increases in[Na+] in body fluids are presented in Figure 1. When [Na+]Water intake by Nax-KO mice after an ICV injection of
hypertonic NaCl solution was small, but still approximately
half that of WT mice. It was noteworthy that this was significantly higher than that of Nax-KO and WT mice after an ICV
injection of an equimolar hypertonic sorbitol solution. Thesefindings suggest the existence of another unknown [Na +]sensor and osmosensor. In order to identify the novel sensors
involved in water intake control, we performed RNA-seq
analysis of OVLT and identified many candidates for said
sensors. This year, we revealed that SLC9A4 in the OVLT
functions as a [Na+] sensor for the control of water intake
behavior among these candidates, and that the signaling
pathway originating from this sensor is independent of the
Nax/TRPV4 pathway, another [Na+]-sensing pathway for thecontrol of water intake (Figure 1). Our experimental results
suggested that SLC9A4-positive neurons are activated via
ASIC1a in a H+-dependent manner. Our experimental results
also revealed that water intake induced by the increase in[Na+] in CSF was completely lost in slc9a4-knockdown
Nax-KO mice. Thus, water intake induced by [Na+] increases
in body fluids may be explained by the Nax/TRPV4 and
SLC9A4/ASIC1a pathways. In addition to these two [Na+]dependent pathways that induce water intake, another signaling pathway originating from the osmosensor may be independently involved in the induction of water intake. We arenow examining the functional roles of remaining candidates
in water intake to clarify the osmosensing system.

71

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
Publication List:〔Original papers〕
H., Lin, C.-H., Hiyama, T.Y., Matsuda, T., Yamaguchi, K.,• Sakuta,
Shigenobu, S., Kobayashi, K., and Noda, M. (2020). SLC9A4 in the•72

organum vasculosum of the lamina terminalis is a [Na+] sensor for thecontrol of water intake. Pflugers Arch. - Eur. J. Physiol. 472, 609–624.
DOI: 10.1007/s00424-020-02389-y
Sakuta, H., Lin, C.-H., Yamada, M., Kita Y., Tokuoka, S.M., Shimizu,T., and Noda, M. (2020). Nax-positive glial cells in the organum
vasculosum laminae terminalis produce epoxyeicosatrienoic acids to
induce water intake in response to increases in [Na+] in body fluids.
Neurosci. Res. 154, 45–51. DOI: 10.1016/j.neures.2019.05.006

LABORATORY OF BIOLOGICAL DIVERSITYWUDARSKI GroupSpecially Appointed Assistant Professor:
WUDARSKI, Jakub

Understanding the mechanisms of regeneration
using transgenic flatworms and IR-LEGO
Regeneration is the process of restoring lost or damaged
tissues and organs. Flatworms have long been considered as model organisms for studying regeneration; somespecies of planarian flatworms can even restore all theirbody parts from small pieces. In my research, I am using
the new powerful flatworm model organism, Macrostomum
lignano, to study how stem cells differentiate into various
cell types during regeneration and how body patterning
is established. The main advantage of M. lignano is theavailability of transgenesis methods which I have developed during my PhD. It enables tracking specific cells andtheir progenitors during development and regeneration.

Figure 1. Tissue specific transgenic line of Macrostomum lignano.

Positional control of regeneration in flatworms
Flatworms have remarkable regeneration capabilities. They
are able to regrow their whole body after amputation, including their reproductive organs. They can do this thanks to a
population of adult stem cells, collectively called neoblasts.
One of the fascinating aspects of flatworm regeneration is the
positional control of the process along the anterior-posterior
axis (head-tail). How cells know where specific body parts
need to be reconstructed is a question that still lacks a fullanswer. Our current state of knowledge is that Wnt pathway
and the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) signaling play major rolein this process. However, most of the research done on flatworms is based on information inferred from experiments on
gene knock-down via RNA interference (RNAi). Gene activation and overexpression studies are absent in planarians,
the more common flatworm model organisms, because of
the lack of transgenic methods available for these animals. I
am using Macrostomum lignano, to test the function of genes
shown to be involved in positional control during growth
and regeneration. I am currently adapting the infrared laser
evoked gene operator (IR-LEGO) technology to use with thepreviously established HSP20 promoter. This will enable me
to track the cell fate in vivo and overexpress selected genes
even on a single cell level.
To continue this research, I have been awarded theMistubishi Foundation grant.

Figure 2. Expression of mScarlet under the HSP20 promoter 24 hours
after induction using IR-LEGO

73

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
NIBB-COS IINTERNATIONAL COLLABORATION RESEARCH PROJECTGUSE Group
Guest Professor:
Postdoctoral Fellow:

GUSE, Annika
KISHIMOTO, Marikoturn elucidate the mechanisms of light response by opsin.
Understanding how symbiotic cnidarians perceive light tosynchronize sexual reproduction and behavior will provide
us with key insights into its evolution and ecology; a prerequisite to combat the decline of corals through climate change
which threatens reef ecosystems worldwide.

I. Light sensing changes by symbiotic status
The gene expression of Aiptasia opsins changes dependingon their symbiotic status. This suggests that symbiotic andnon-symbiotic Aiptasia have different sensitivities to light.
This could be due to the different opsin types and the light
responses in algae.
In this study, we found that symbiotic and non-symbiotic Aiptasia have different phototaxis. Irradiation withhigh levels of light caused negative-phototaxis in symbiotic Aiptasia while non-symbiotic Aiptasia did not react. The
symbiosis-specific negative phototaxis was dependent on the
wavelength specificity, and peaked in the blue region. Thus,
symbiosis with algae changes Aiptasia’s light sensitivity.

Figure 1. Wavelength specificity of negative phototaxis in symbiotic
Aiptasia.

Light dominates life for most organisms. On one hand,
sunlight acts as a key regulator for various functions including
photosynthesis and circadian clock control. Similarly, moonlight is important for synchronous gamete release in many
marine animals, such as cnidarians. Cnidarians, which include
corals and anemones, are basal, aquatic animals with immense
ecological importance. Notably, coral reefs are the most biodiverse marine ecosystems. Their productivity depends on afunctional symbiosis between reef-building corals and photosynthetic dinoflagellates of the Symbiodiniaceae family, whichtransfer nutrients to their coral host to provide a source of
fixed carbon in oligotrophic environments.
In this COS-NIBB joint project, we aim to dissect key
molecular mechanisms underlying the sensing of light insymbiotic cnidarians and how this is used in environmental
adaptation. Using the sea anemone, Aiptasia sp. (Exaiptasia
diaphana) as a model for corals, we are conducting research
to reveal the molecular mechanisms of light sensing focusing
on the evolutionary conserved photoreceptor “opsin”.
Additionally, we are aiming to establish a method of genefunction analysis using genome editing technology or genesilencing techniques in said sea anemone which will in

74

II. Functional analysis of Aiptasia opsins
Aiptasia has 18 types of opsins, and 3 of them have higher
gene expression in their symbiotic state. In this study, we
are aiming to analyze the light sensitivity of the distinct
Aiptasia opsins using a heterologous cell culture-based assay.
To date, we have successfully cloned 11/18 opsins including the 3 symbiosis-specific ones. Moreover, using the cell
culture cAMP signaling assay, we were able to detect a light
response for several (7/18) opsins. Determining of theirabsorbance wavelength specificity is key in relating distinct
opsins with light-dependent behavior including phototaxis
and spawning in Aiptasia.

Figure 2. Functional analysis of Aiptasia opsins cloned in this study.

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

YAMASHITA, Akira

Specially Appointed Assistant Professor:
OTSUBO, Yoko
NAKADE, Atsuko
Technical Assistant:Admin Support Staff:
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.

Mitotic cell cycle

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 fiveof the eight responsible gene encoded tRNA-related factors.
The hmt1 and hmt2 genes encoded aminoacyl-tRNA synthetases for asparagine and proline, 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 that the products of these hmt
genes may function upstream of TORC1. We also found thatthe expression of tRNA precursors decreases upon nitrogenstarvation occurring. Furthermore, overexpression of tRNAprecursors prevents TORC1 downregulation in response tonitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we have proposedthat tRNA precursors act as key signaling molecules in the
TORC1 pathway in response to nitrogen availability (Figure2). We are also studying how S. pombe cells respond to othertypes of starvation other than that of nitrogen starvation.
pol III
tDNA
Transcription

Nutrients

Re-supply
Nutrients

ARS
Translation

Meiosis and sporulationh+pre-tRNAaminoacyl-tRNA

Meiotic cell cycle
MatingEntry into
sexual differentiationh-Nutritional
Starvation

TORC1

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 have focused on a highly conserved kinase, namely
Target of Rapamycin (TOR) kinase, which plays a key rolein the recognition of nutrition and the onset of sexual differentiation in S. pombe. TOR kinase forms two types ofcomplexes, TORC1 and TORC2. TORC1 contains Tor2 as
its catalytic subunit, and is essential in suppressing sexual
differentiation in 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 and under restrictive
temperature conditions. To gain an insight into the TORC1
signaling pathway, we have isolated mutants that initiate

Sexual Differentiation

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. Expression of meiotic genes is strictly regulated,since untimely expression of gametogenic genes, including
meiotic genes, has a deleterious effect on somatic cells. We
have shown that specific control of the stability of 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 the fission
yeast S. pombe. Understanding precise mechanisms of thiscontrol 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 hexa-

75

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

III. Cellular responses to atmospheric pressureplasmaPlasma, which is the fourth state of matter after solid, liquidand gas states, is a form of 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 ofcellular 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 the National Institute for Fusion Science.

76

heterochromatin
formation

Mmi1

DSR

H
YT DSRH
YT DSR
YTH

D

YTHSR
D

YTH SR

YTH

R
DS

nucleotide motifs. Meiotic transcripts bound to Mmi1 aredegraded by the RNA-degradation nuclear exosome machinery. 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 theRNA-binding protein Mei2 and a long non-coding RNA
species referred to as meiRNA. Mei2 physically interacts
with meiRNA and forms the dot structure at the chromosomal sme2 locus, which encodes meiRNA. The Mei2 dotlures Mmi1 through numerous copies of the DSR motif on
meiRNA and inhibits its function, so that meiotic transcripts
harboring DSR are stably 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).
We have also demonstrated that a conserved complex called
MTREC (Mtl1-Red1 core) or NURS (nuclear RNA silencing) that consists of a zinc-finger protein, Red1, and an RNA
helicase, Mtl1, is required for the recruitment of the nuclear
exosome to Mmi1 foci.

RNA
Exosomes
Nucleusm
CytoplaTranslation
Machinery

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.

Publication List:〔Original papers〕
H., Kobayashi, M., Shimasaki, T., Sato, T., Akanuma, G.,• Ohtsuka,
Kitaura, Y., Otsubo, Y., Yamashita, A., and Aiba, H. (2021). Magnesium•

depletion extends fission yeast lifespan via general amino acid control
activation. Microbiologyopen 10. DOI: 10.1002/mbo3.1176
Yagi-Utsumi, M., Tanaka, T., Otsubo, Y., Yamashita, A., Yoshimura,S., Nishida, M., and Kato, K. (2021). Cold atmospheric plasma
modification of amyloid beta. Int. J. Mol. Sci. 22. DOI: 10.3390/
ijms22063116〔Review Articles〕
Y., Kamada, Y., and Yamashita, A. (2020). Novel links between• Otsubo,
TORC1 and traditional non-coding RNA, tRNA. Genes 11. DOI:•

10.3390/genes11090956
Yoshimura, S., Otsubo, Y., Yamashita, A., and Ishikawa, K. (2020).
Insights into normothermic treatment with direct irradiation of
atmospheric pressure plasma for biological applications. Jpn. J. Appl.
Phys. 60, 10502. DOI: 10.35848/1347-4065/abcbd2

< NINS ASTROBIOLOGY CENTER >LABORATORY OF BIOLOGICAL DIVERSITY
The Astrobiology Center (ABC) was established in2015 to promote interdisciplinary studies between fields
that include astronomy, earth science, and biology.
After 5 years of activity, ABC has come to be recognized as the hub institute of astrobiology within Japan.
Consequently, this discipline has become establishedwithin the Japanese scientific community.
Our ultimate goal is to find a planet akin to a ‘Second
Earth’ as well as extraterrestrial life on said 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 stars nearest to Earth by using thelatest observation technologies. To support these observation projects, three groups from NIBB, the Takizawa,
Johzuka, and Fujita groups, are participating in the
Exo-Life Search Project to investigate life on Earthutilizing three different approaches: 1) assessing thebiosignature of various photosynthetic organisms inorder to predict biosignatures of hypothetical life on
exoplanets, 2) evaluating the effects of cosmic radiation on living cells, and 3) elucidating mathematicalprinciples in the formation of self-organizing structuresin organisms.

TAKIZAWA GroupSpecially Appointed Associate Professor

TAKIZAWA, Kenji

The Takizawa group operating out of ABC is currently
studying the environmental responses of photosynthesis inorder to predict photosynthetic apparatus of so-called ‘Alien’
plants under extreme conditions on the aforementioned‘Second Earth’.

Vegetation red-edge of floating plants on ocean
planets
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. While red lightabsorption is a common feature of all phototrophs, NIR
reflection is more noticeable in plants at a higher elevationon land. According to the latest planet formation theory,
habitable exoplanets may have too much water to form continents. We have subsequently proposed that there is a possibility that strong biosignatures could be detected on ‘water
planets’ due to the extensive flourishing of drifting algae andfloating plants. Remote sensing of light reflection by drones
revealed that water plants with floating leaves reflect NIR ina manner comparable to land vegetation.

Figure 1. ABC mascot characters: “Exoplanet Friends” (Credit:
Hayanon Science Manga Studio)

Figure 2. Remote sensing of light reflectance of floating leaf of waterlily.
Light reflectance in visible light (BGR image on the left) and NIR (gray scale
image on the right) were obtained by the drone-based multiband sensor.

NIR driven photosynthesis around red dwarfs
Most habitable planets orbit around red dwarfs that are
predominantly exposed to NIR. In order to utilize NIR for
photosynthesis, alien plants should have NIR-absorbing,photochemically reactive pigments. We have thus evaluatedoptical and photochemical properties of various potential
photosynthetic pigments via quantum chemical calculations,and revealed that several metal-pigments can be functional inreaction centers under NIR radiation conditions.

77

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Assistant Professor:
Technical Assistant:

FUJITA, Hironori
EGAWA, Akane
TAKEKAWA, EikoIn nature, a variety of self-organized patterns, such as thegalaxy and the snowflake, are found on a wide range ofspatiotemporal scales. Particularly in living organisms, suchself-organization of spatiotemporal patterns is both remarkable and essential. Therefore, we aim to elucidate the mechanism of generation and control of self-organized patterns inliving systems with a particular focus on plants using boththeoretical and computational approaches.

Figure 1. (Left) Phyllotaxis pattern is self-organized by the feedback
regulation between auxin concentration and PIN1 polarization. (Middle)
Leaf venation pattern is self-organized by the feedback regulation
between auxin flux and PIN1 polarization. (Right) Shoot apical meristem(SAM) is self-organized by the interaction between WUS and CLV3.

Systemic signal

s1 , s2
3.5

x1

u1

x1

x2

u2

x2

ui

1

0.001

0.01

0.1

1

10

20

50

100

Nitrate concentration (mM)unxn

Nitrateconc.

Optimalroot
density

ui

un

xi

···

0

···

0.5

xi

···

1.5

u1
u2

··· ········ ·····

2

···

3

2.5

···

Escherichia coli can move towards higher concentrations of
nutrients, such as Asp and Ser, by using flagella. Chemotaxis
can induce periodic patterns in a self-organizing manner(Figure 2; Nature 349, 630–633. 1991). Whereas experimental conditions in biological experiments are usually greatly
restricted, the synthetic approach is a powerful researchmethod that can control and modify the experimental conditions in a more flexible manner compared to the method usedin standard experiments. Therefore, we apply the synthetic

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
their soil, plants regulate resource allocation in their roots
in order to plastically change their root system architecture (RSA), so they can efficiently absorb nutrients(Figure 3A). However, it is still not understood why andhow RSA is adaptively controlled. Therefore, we modeled
and investigated the spatial 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 etal., J. Theor. Biol. 486, 110078. 2020). First, we showedthat 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 is required for adaptation to high nutrient conditions, whereas growth promotion by local control is sufficient for adaptation to low-nutrient environments (Figure
3B). This evidence indicates that systemic control can be
evolved in the presence of excessive amounts of nutrition, consistent with the ‘N-supply’ systemic signal thatis observed experimentally. Furthermore, our model canalso explain various experimental results using nitrogen
nutrition, and provides a theoretical basis for understanding the spatial regulation of adaptive resource allocationin response to nutritional environment.

···

I. Spatiotemporal patterns of cell population

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

FUJITA Groupapproach to E. coli population to understand the principle of‘spatiotemporal self-organization of cell populations’.

Lateral root density(mm mm 1)

LABORATORY OF BIOLOGICAL DIVERSITY< NINS ASTROBIOLOGY CENTER >xn

Nitrate Optimal
root
conc.
density

Figure 3. (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. (right)
In the low nutrient availability, the optimal root density is promoted bynutrient through local control (blue arrows). (left) By contrast, in addition
to local control, is suppressed through systemic control (denoted in red)under high nutrient availability.

Publication List:〔Original paper〕
K., Fujita, H., Isa, M., Gotoh, S., Arasaki, A., Ishida, H.,• Kataoka,
and Kimura, R. (2021). The human EDAR 370V/A polymorphism

Figure 2. An example of self-organized colony patterns induced by chemotaxis in E. coli population.

78

affects tooth root morphology potentially through the modification ofa reaction–diffusion system. Sci. Rep. 11, 5143. DOI: 10.1038/s41598021-84653-4

< NINS ASTROBIOLOGY CENTER >LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Staff:

JOHZUKA, Katsuki
ISHINE Naomi

densin 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

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.

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
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 con-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.

79

National Institute for Basic Biology

Environmental Biology
DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

Associate Professor

MINAGAWA, Jun

TAKAHASHI, Shunichi

Assistant Professor:

TOKUTSU, Ryutaro
KIM, Eunchul
Technical Staff:
NODA, Chiyo
Postdoctoral Fellow:
SATO, Ryoichi
KAMADA, Konomi
Reseasrch Staff:
ISHII, Asako
SOKENDAI Graduate Student: WATANABE, Akimasa
OKAJIMA, Keisuke
YANAKA, Ayako
Visiting Scientist:
DANN, Marcel
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
Admin Support Staff:
TOYAMA, Mami
IIDA, Kaoru

Plants and algae have versatile abilities 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. Using a model green alga, we are studying themolecular mechanisms underlying photoacclimation of photosynthetic machinery. We are also applying knowledge
obtained in the studies of this model green alga to various
photosynthetic organisms, including phytoplankton andvascular plants, to explore how environmentally important
photosynthetic organisms thrive in their ecological niche.

Visual overview of this lab’s work.

80

I. 
Structural characterization of thephotosystems in the green alga Chlorellasorokiniana.
The photosynthetic conversion of light energy into chemicalenergy is performed by photosystems II and I (PSII and PSI)
embedded within the thylakoid membranes. In plants andgreen algae, PSII and PSI comprise the core complex andlight-harvesting complexes (LHCII and LHCI), forming
PSII–LHCII and PSI–LHCI supercomplexes, respectively. The structural information about photosystem supercomplexes of green algae has been limited to chlorophytic algae. Here, to obtain an insight into the evolution of
Chlorophyta, we determined the supramolecular organizationof the PSII–LHCII and PSI–LHCI supercomplexes from the
freshwater green alga Chlorella sorokiniana, which belongs
to Trebouxiophyceae (Fig. 1) (Watanabe and Minagawa,
Planta, 252:79). The obtained results showed that the supramolecular organizations of the photosystem supercomplexes in C. sorokiniana were essentially the same as thoseof the model green alga C. reinhardtii, which belongs to
Chlorophyceae, namely PSII–LHCII supercomplex formed
the C2S2M2L2 configuration and PSI–LHCI supercomplex
was associated with 10 LHCI subunits.

Figure 1. Single-particle EM analysis of photosystems isolated from SDG
ultracentrifugation of the α-DDM-solubilized thylakoids from C. sorokiniana cells. Watanabe and Minagawa (2020) Planta, 252:79.

II. C haracterization of a giant photosystem
I supercomplex in the symbiotic
dinoflagellate Symbiodiniaceae.
Symbiodiniaceae are symbiotic dinoflagellates that provide
photosynthetic products to corals. Because corals are
distributed across a wide range of depths in the ocean,Symbiodiniaceae species must adapt to various light environments to optimize their photosynthetic performance.
However, as few biochemical studies of Symbiodiniaceae
photosystems have been reported, the molecular mechanisms of photoadaptation in this algal family remain poorly
understood. Here, to investigate the photosynthetic machineries in Symbiodiniaceae, we purified and characterized the
PSI supercomplex from the genome- sequenced Breviolum
minutum (formerly Symbiodinium minutum) (Fig. 2) (Katoet al., Plant Physiol., 183:1725-1734). Mass spectrometry analysis revealed 25 light-harvesting complexes (LHCs),
including both LHCF and LHCR families, from the purified
PSI-LHC supercomplex. Single-particle electron microscopy

showed unique giant supercomplex structures of PSI thatwere associated with the LHCs. Moreover, the PSI-LHC
supercomplex contained a significant amount of the xanthophyll cycle pigment diadinoxanthin. Upon high light treatment, B. minutum cells showed increased nonphotochemical
quenching, which was correlated with the conversion of
diadinoxanthin to diatoxanthin, occurring preferentially in
the PSI-LHC supercomplex. The possible role of PSI-LHC inphotoprotection in Symbiodiniaceae was discussed.

Figure 3. Schematic representation of the putative arrangements of
PSII–LHCII supercomplexes in the thylakoid membrane in low-lightand high-light conditions. Kim, Watanabe et al. (2020) J. Biol. Chem.,
295:14537-14545.

IV. P hotoprotective capabilities of lightharvesting complex II trimers in a green
alga Chlamydomonas reinhardtii.

Figure 2. Single-particle EM analysis of photosystem I isolated from
SDG ultracentrifugation of the α-DDM-solubilized thylakoids from B.
minutum cells. Kato et al. (2020) Plant Physiol., 183:1725-1734.

III. Multimeric and monomeric photosystem
II supercomplexes represent structural
adaptations to low- and high-light conditions.
An intriguing molecular architecture called the “semicrystalline photosystem II (PSII) array” has been observed in
the thylakoid membranes in vascular plants. It is an array of
PSII–LHCII supercomplexes that only appears in low light,
but its functional role has not been clarified. Here, we identified PSII–LHCII supercomplexes in their monomeric andmultimeric forms in low light–acclimated spinach leaves andprepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves
were acclimated to high light, only the monomeric forms
were present, suggesting that the multimeric forms represent
a structural adaptation to low light and that disaggregationof the PSII–LHCII supercomplex represents an adaptation tohigh light. Single-particle EM revealed that the multimeric
PSII–LHCII supercomplexes are composed of two (“megacomplex”) or three (“arraycomplex”) units of PSII–LHCII
supercomplexes, which likely constitute a fraction of thesemi-crystalline PSII array. Further characterization withfluorescence analysis revealed that multimeric forms havea higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings
suggest that the configurational conversion of PSII–LHCII
supercomplexes may serve as a structural basis for acclimation of plants to environmental light (Fig. 3) (Kim and
Watanabe et al., J. Biol. Chem., 295:14537-14545).

Major light-harvesting complex (LHCII) trimers in plants
induce the thermal dissipation of absorbed excitation energy
against photooxidative damage under excess light conditions. LHCII trimers in green algae have been thought to beincapable of energy dissipation without additional quencher
proteins, although LHCIIs in plants and green algae arehomologous. In this study, we investigated the energy-dissipative capabilities of four distinct types of LHCII trimers
isolated from the model green alga Chlamydomonas reinhardtii using spectroscopic analysis. Our results revealed that
the LHCII trimers possessing LHCII type II (LHCBM5) and
LHCII type IV (LHCBM1) had efficient energy-dissipative
capabilities, whereas LHCII type I (LHCBM3/4/6/8/9) andtype III (LHCBM2/7) did not. On the basis of the amino acidsequences of LHCBM5 and LHCBM1 compared with theother LHCBMs, we propose that positively charged extra
N-terminal amino acid residues mediate the interactions
between LHCII trimers to form energy-dissipative states(Fig. 4) (Kim et al., J. Phys. Chem. Lett., 11:7755-7761).

Figure 4. Schematic representation of the extra N-terminal-dependent
photoprotective states of LHCII trimers from C. reinhardtii. Kim et al.
(2020) J. Phys. Chem. Lett., 11:7755-7761.

81

National Institute for Basic Biology

Environmental Biology
Publication List:〔Original Papers〕
D., Nisbet, R.E.R., Robledo, J.A.F., Casacuberta, E., Sudek,• Faktorova,
L., Allen, A.E., Ares Jr., M., Areste, C., Balestreri, C., Barbrook, A.C.,

•
•
•
•
•
•
•

82

et al. (2020). Genetic tool development in marine protists: emerging
model organisms for experimental cell biology. Nat. Methods 17, 481.
–494. DOI: 10.1038/s41592-020-0796-x
Kato, H., Tokutsu, R., Kubota-Kawai, H., Burton-Smith, R.N., Kim, E.,
and Minagawa, J. (2020). Characterization of a giant PSI Supercomplex
in the symbiotic dinoflagellate symbiodiniaceae. Plant Physiol. 183,
1725–1734. DOI: 10.1104/pp.20.00726
Kim, E., Kawakami, K., Sato, R., and Ishii A., and Minagawa, J. (2020).
Photoprotective capabilities of light-harvesting complex II trimers in
the green alga Chlamydomonas reinhardtii. J. Phys. Chem. Lett. 11,7755–7761. DOI: 10.1021/acs.jpclett.0c02098
Kim, E., Watanabe, A., Duffy, C.D.P., Ruban, A. V, and Minagawa, J.
(2020). Multimeric and monomeric photosystem II supercomplexes
represent structural adaptations to low- and high-light conditions. J.
Biol. Chem. 295, 14537–14545. DOI: 10.1074/jbc.RA120.014198
Kishimoto, M., Baird, A.H., Maruyama, S., Minagawa, J., andTakahashi, S. (2020). Loss of symbiont infectivity following thermal
stress can be a factor limiting recovery from bleaching in cnidarians.
ISME J. 14, 3149–3152. DOI: 10.1038/s41396-020-00742-8
Shukla, M.K., Watanabe, A., Wilson, S., Giovagnetti, V., Moustafa,
E.I., Minagawa, J., and Ruban, V, A. (2020). A novel method produces
native light-harvesting complex II aggregates from the photosynthetic
membrane revealing their role in nonphotochemical quenching. J. Biol.
Chem. 295, 17816–17826. DOI: 10.1074/jbc.RA120.016181
Tokutsu, R., Fujimura-Kamada, K., Yamasaki, T., Okajima, K.,
and Minagawa, J. (2021). UV-A/B radiation rapidly activatesphotoprotective mechanisms in Chlamydomonas reinhardtii. Plant
Physiol. 185, 1894–1902. DOI: 10.1093/plphys/kiab004
Watanabe, A., and Minagawa, J. (2020). Structural characterization of
the photosystems in the green alga Chlorella sorokiniana. Planta 252,
79. DOI: 10.1007/s00425-020-03487-y

DIVISION OF PLANT ENVIRONMENTAL RESPONSES

Professor

MORITA, Miyo T.

Assistant Professor:

NISHIMURA, Takeshi
SHIKATA, Hiromasa
Specially Appointed Assistant Professor:
KAWAMOTO, Nozomi
Research Staff:
TAKASE, Wakana
Visiting Scientist:
FURUTANI, Masahiko
Visiting Graduate Student: MORI, Shogo
Technical Assistant:
HAMADA, Mayako
MIYOSHI, Kikumi
YAMADA, Yuka
SOMA, Yoriko
INOUE, Fumiko
Admin Support Staff:
KOJIMA, Yoko

Plant organs have the ability to sense various vectorial
stimuli such as light, humidity, touch and gravity as well
as reorient their growth direction so as to be in a suitable
position to survive and acclimate to their environment. Our
aim is to understand the molecular mechanism of environmental responses as they pertain to gravity in the main.

direction of gravity. Amyloplast relocation serves as aphysical signal trigger for biochemical signal transduction,
which in turn leads to the regulation of the polar auxin transport necessary to change the direction that a given plant is
growing. We are investigating the detailed molecular mechanism of gravity sensing and signaling by using the model
plant Arabidopsis thaliana.
LAZY1 family genes have been shown to be involved ingravitropic responses in a variety of plants. LAZY1-LIKE(LZY)2, LZY3, and LZY4 are involved in root gravitropism of Arabidopsis. Previously, we have found that LZY3mCherry is polarly localized on the basal side of the plasma
membrane in the columella cells (root statocytes) in response
to inclination. At that time, we utilized fixed and clearedroot samples because LZY3-mCherry is barely detected inlive cell imaging; probably due to low expression levels andthe tissue depth of its root cap. We subsequently generated
LZY4p:LZY4-mScarlet transgenic lines which complemented
the lzy4 mutant. We then succeeded in live cell imaging of
LZY4-mScarlet in the columella cells by using verticalstage confocal microscope equipped with a high sensitivity
EMCCD camera. LZY4-mScarlet was polarly localized on
the basal side of the plasma membrane, in a similar way
to that which LZY3-mCherry was localized. In addition,
LZY4-mScarlet was found to be localized at the peripheryof amyloplasts. We are now focusing on the dynamics of
LZY4-mScarlet in response to gravistimulation and also on
the mechanism of localization to the plasma membrane andamyloplast periphery.

II. Determination mechanism of gravitropic
setpoint angle
Growth angles affected by gravity are known as the gravitropic setpoint angles (GSA). Many gravitropic mutants show
abnormal GSA in lateral branches; meaning they produce
wider growth angle phenotypes due to the likelihood of
reduced gravitropism. We are trying to understand how roots’and shoots’ lateral branches maintain inclined growth angles
with respect to gravity.

2-1 Determination of GSA in roots

Visual overview of this lab’s work.

I. M olecular mechanisms of gravity sensing
and signaling in gravitropism
Gravitropism is a major determinant in directing plant organ
growth angles. In gravity sensing cells (statocytes), plastids
accumulating starch in high-densities relocate toward theIt has been reported that primary and lateral roots of lzy2;3;4
triple mutants showed negative gravitropism. We refer to thephenotype of the reversed growth direction in the lzy triple
mutant as “anti-gravitropic”. It has been proposed that GSA
is determined by balancing two opposing growth components: gravitropism and “anti-gravitropic” offsets (AGO). We
assumed that the balance between gravitropism and AGO was
disrupted in lzy2;3;4 mutants, and hypothesized that AGO
would be manifested as an “anti-gravitropic” phenotype. To
investigate the mechanism of GSA control, we performed
genetic analyses with lzy2;3;4 mutants and pgm (Figure 1).
Since pgm is a starch-less mutant, gravity sensing ability was
greatly reduced in this type of mutant, resulting in varied
growth direction (Figure 1D and H). The pgm mutation suppressed the “anti-gravitropic” phenotype of lzy2;3;4 triple
mutants (Figure 1B, C, F, and G). This finding indicates thatgravitropic and “anti-gravitropic” growth is likely to share asimilar gravity-sensing mechanism in primary roots.

83National Institute for Basic Biology

Environmental Biology“anti-gravitropic” phenotype of lzy1;2;3 triple mutants. In
other words, the lateral branches of eal1 lzy1;2;3 quadruple
mutants grew horizontally. Overall, the endodermal tissue
appears crucial for the normal gravitropism and “anti-gravitropic” phenotypes of lateral branches in lzy1;2;3 triple
mutants, but it remains unclear whether gravitropism and“anti-gravitropic” phenotypes share a similar gravity-sensing
mechanism in shoots.

III. F unctional analysis of RLD (RCC1-likedomain) proteins

Figure 1. Starchless mutation affects “anti-gravitropic” response in roots.
Polar distribution of the growth direction of primary root in wild-type Col(A), lzy2;3;4 (B), lzy2;3;4;pgm (C), and pgm (D). Growth angles wereplotted on histograms and distributions were evaluated with boxplots fromwild-type Col (E), lzy2;3;4 (F), lzy2;3;4;pgm (G), and pgm (H). Schematic
representation of growth angle quantification (I). Each bar indicates thenumber of roots. Lugoal’s staining of primary roots of wild-type Col (J),lzy2;3;4 (K), lzy2;3;4;pgm (L), and pgm (M). Bars indicate 100 µm.

2-2 Determination of GSA in shoots
The primary shoots of lzy1;2;3 triple mutants exhibited
non-responsiveness to gravistimulation, so it was expectedthat the lateral branches of the triple mutant would grow horizontally like those of sgr mutants. However, they unexpectedly grew downward and showed positive gravitropism upon
gravistimulation (Figure 2). As amyloplast sedimentation isthought to be important for gravity sensing in shoot gravitropism as well as root gravitropism, we examined whether
starch-accumulated amyloplasts are required for the “antigravitropic” phenotype of lateral branches in the lzy1;2;3
mutants. Although no clear difference was observed in thegrowth angles of lateral branches by adding pgm mutationin lzy1;2;3 mutants, the pgm mutation delayed the “anti-gravitropic” response in lzy1;2;3 plants upon gravi-stimulation(Figure 2C, D and E).
We then investigated the relationship between the gravity
sensing tissues for shoot gravitropism and the “anti-gravitropic” phenotype of lateral branches in lzy1;2;3 plants. The
endodermis is the gravity-sensing tissue in shoots, and eal1
is classified as an agravitropic mutant due to defect in its
endodermis differentiation and because it’s lateral branches
grow horizontally. The eal1 mutation clearly suppressed

Figure 2. The gravitropic responses of lateral branches. A. Graphicalimage of the quantification of growth angles of lateral branches. Timelapse images of lateral branches from wild-type Col (B), lzy1;2;3 (C),
pgm (D), lzy1;2;3;pgm (E) plants after inversion.

84

RLD proteins were isolated as interactors of LZY. RLD1and RLD4 function in gravitropism of primary roots andthe GSA control of lateral roots. We have also shown that
RLD1, RLD2, RLD3, and RLD4 redundantly function in plantmorphogenesis, the morphological abnormality gradually
becomes more severe by multiplying rld mutations (Figure3). Indeed, rld1;2;3;4 quadruple mutants displayed severe
embryonic development defects due to a reduced amount andabnormal localization of PIN proteins (auxin efflux carriers)in the plasma membrane. Defects in root gravitropism andmorphogenesis of rld multiple mutants are associated withdefective auxin transport. It had been reported that the RLD1
fragment containing RCC1-like domain show guanine nucleotide exchange activity in Rab8 and Rab11. Thus, it’s likelythat RLD proteins regulate auxin transport through regulating membrane trafficking during root gravitropism and plantdevelopment.
In columella cells, RLD1 is polarly recruited to the plasma
membrane by interacting with LZY3 and regulates the direction of auxin transport. The interaction between RLD1 and
LZY3 occurs through the binding of the BRX domain of
RLD1 to the CCL domain of LZY3. While LZY1, LZY2,and LZY3, has been shown to play a key role in the gravity
signaling process in shoot endodermis, the role of RLDs inshoot gravitropism remains unclear because evaluating shootgravitropism in rld multiple mutants is difficult due to theirabnormal morphology and retarded growth.
To investigate the importance of the BRX domain for
RLD function, we examined whether RLD3p:RLD3∆BRXmClover3 could complement the morphological phenotypeof rld2;3;4. Unexpectedly, RLD3∆BRX-mClover3 recovered the morphological abnormality of rld2;3;4 (Figure 3).
Interestingly, a wider GSA phenotype possessing lateralbranches was observed in RLD3p:RLD3∆BRX-mClover3/
rld2;3;4. Both of these indicate that RLD3 lacking the BRX
domain retains the function for morphogenesis but not for
the GSA phenotype. Moreover, when RLD3 was expressed
under the control of the endodermis-specific ADF9 promoter
in RLD3p:RLD3∆BRX-mClover3/rld2;3;4, the GSA phenotype was significantly restored, thus suggesting that RLD is
involved in gravity signaling in the endodermal cells in shootgravitropism in a manner similar to LZYs. Furthermore,these results suggest that RLD3 without BRX domain retain
general functions in membrane trafficking required for development, and that LZYs regulates the function of RLD via
the BRX domain to conduct gravity signaling in the gravity
sensing cells.

Publication List:〔Original papers〕
Y., Meguriya, K., Matsuzaki, T., Sugiyama, T., Yoshikawa, H.Y.,• Abe,
Morita, M.T., and Toyota, M. (2020). Micromanipulation of amyloplasts•
Figure 3. A. Phenotype of rosette leaves of wild-type, rld2;3;4 (b) and
RLD3p:RLD3∆BRX-mClover3/rld2;3;4. B. The growth angle of shootin wild-type, rld2;3;4 (b), RLD3p:RLD3∆BRX-mClover3/rld2;3;4 (c),ADF9p:RLD3/RLD3p:RLD3∆BRX-mClover3/rld2;3;4 (d).

•IV. Re-orientation mechanism of the polarized
growing root hair cell•

Root hairs, thin tubular cells grown outward from the root
surface, increase root surface area and the uptake of nutrients
and water from soils. They are typical polarized growing
cells and grow straight and long in a cell-autonomous
manner when they are grown in open air or within a liquidmedium (Figure 4A). However soils are filled with soil
particles like grains of sand of various sizes or silt, which
may interfere with the growth of root hairs (Figure 4B). We
have developed an imaging system based on microfluidics
to observe how root hairs grow in soils, since soils are nottransparent enough to conduct observations within themthrough the use of microscopes (Figure 4C-E). Applying thissystem, paths or objects (2~250 µm) are formed in a transparent silicone-rubber to mimic the condition of soil particles(Figure 4D, E). It enables us to visualize Arabidopsis roots
including root hairs that come into contact with obstacles in
the microfluidic device. We found that root hairs grow along
the surface of the obstacles, which supports the role of root
hairs on uptake of nutrients and water from soil particles(Figure 4E). To further get insight into the mechanism of root
hair growth in soils, we have been analyzing various regulators that are important for normal root hair growth with theimaging system. We found that two protein kinases are regulators for re-orientation of root hair growth.

•with optical tweezers in Arabidopsis stems. Plant Biotechnol. 37, 405–
415. DOI: 10.5511/plantbiotechnology.20.1201a
Kawamoto, N., Kanbe, Y., Nakamura, M., Mori A., Terao Morita, M.T.
(2020). Gravity-sensing tissues for gravitropism are required for “antigravitropic’’ phenotypes of lzy multiple mutants in Arabidopsis. Plants
9. DOI: 10.3390/plants9050615
Shindo, M., Makigawa, S., Kodama, K., Sugiyama, H., Matsumoto, K.,
Iwata, T., Wasano, N., Kano, A., Morita, M.T., and Fujii, Y. (2020).
Design and chemical synthesis of root gravitropism inhibitors: Bridged
analogues of ku-76 have more potent activity. Phytochemistry 179.
DOI: 10.1016/j.phytochem.2020.112508
Shindo, M., Makigawa, S., Matsumoto, K., Iwata, T., Wasano, N.,Kano, A., Morita, M.T., and Fujii, Y. (2020). Essential structuralfeatures of (2Z,4E)-5-phenylpenta-2,4-dienoic acid for inhibitionof root gravitropism. Phytochemistry 172. DOI: 10.1016/j.
phytochem.2020.112287
Tsugawa, S., Sano, T.G., Shima, H., Morita, M.T., and Demura, T.
(2020). A mathematical model explores the contributions of bending
and stretching forces to shoot gravitropism in Arabidopsis. Quant. Plant
Biol. 1, e4. DOI: 10.1017/qpb.2020.5

Figure 4. Live-imaging of root hairs. A. An Arabidopsis root grown on theAgar medium. B. Illustration of root hairs in soils. C. A micro-fluidic device
for imaging of Arabidopsis root hairs. D. Main roots grow in a main pathwithin the device, and root hairs elongate into small chambers aligned besidethe main path. E. A root hair elongating along an obstacle set in a smallchamber. Bars indicate 200 µm or 100 µm in (A) or (D, E), respectively.

85

National Institute for Basic Biology

Theoretical Biology
LABORATORY OF GENOME INFORMATICS

Associate Professor

entry (in collaboration with Dr. Takami, Univ Tokyo) anddeveloped a novel interface to compare the presenceor absence of functional modules among specified
organisms (Figure 1).

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 thisdata, or finding the principles behind it, in order to understand complex living systems by integrating the data withcurrent biological knowledge via the use of various computational techniques. In this laboratory we focus on developing
computational methods and tools for comparative genome
analysis, which is a useful approach for finding functionalor evolutionary clues to interpret the genomic information ofvarious 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, https://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
pre-calculated ortholog tables for each major taxonomic
group, and provides several viewing modes to display theentirety of 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, and203 eukaryota. These data sets are classified based on thehierarchical ortholog classification strategy described in thesection below.
We continue to develop the database and the application
software utilizing the database to enhance its usability. As anapplication software for analyzing novel genome sequences
based on the database, we are developing a tool to predict
the functional potential of novel microbial genomes throughorthology assignment based on the MBGD ortholog table. To
evaluate the metabolic potential of the query genome fromthis assignment, we utilized the Genomaple software to calculate the module completion ratio for each KEGG Module

86

Figure 1. Comparative display of KEGG module completion among different genomes based on the MBGD-Genomaple combined analysis.

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).
To create more comprehensive ortholog classification,
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 a within-species
ortholog table is created. The species-level pan-genome isthen created by picking one representative gene from eachorthologous group. Next, for each genus with at least two
species, all-against-all similarities among the species-level
pan-genomes are calculated and a within-genus ortholog
table is created. The genus-level pan-genome is then createdby picking one representative gene from each orthologous
group. Finally, all-against-all similarities among the genuslevel pan-genomes are calculated and the standard ortholog
table covering the entire taxonomic range is created.

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 within our
ortholog 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.

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 five RNA-seq datasets of harmful algae and
10 existing genome sequences of various algae and a plantmodel organism, Arabidopsis thaliana using RECOG, andidentified common sequence features among orthologous
genes belonging to harmful algae that may be related to redtide outbreaks and their toxicity to fish. The resulting data isavailable through the database DB-HABs (https://hab.nibb.
ac.jp).

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

ture 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 thecore genome as the genes conserved among the genomesof the given species, characterizing the remaining part of
the genomes (non-core genomes) should be important forunderstanding the species’ diversity. For this purpose, wedeveloped a method (FindMobile) to define mobility ofgenes against the reference coordinate determined by thecore genome alignment. We are now developing a method(FindIsland) to identify a set of non-core genes that haveconserved gene order by using a modified version of the
CoreAligner algorithm. We applied the method to the setsof genomes of prokaryotic species stored in MBGD anddeveloped a database for analyzing their non-core genomes.
Based on the database, we found that the resulting conserved
clusters frequently correspond to known mobile genetic
elements and/or have sequence features common to known
genomic islands.

Publication List:〔Original papers〕
M., Suzuki, M., Sakane, Y., Nishide, H., Uchiyama, I., Yamamoto,• Iida,T., Suzuki, K.T., and Fujii, S. (2020). A simple and practical workflow••

for genotyping of CRISPR-Cas9-based knockout phenotypes using
multiplexed amplicon sequencing. Genes to Cells 25, 498–509. DOI:
10.1111/gtc.12775
Nagao, R., Kato, K., Ifuku, K., Suzuki, T., Kumazawa, M., Uchiyama,I., Kashino, Y., Dohmae, N., Akimoto, S., Shen, J.-R., et al. (2020).
Structural basis for assembly and function of a diatom photosystem
I-light-harvesting supercomplex. Nat. Commun. 11. DOI: 10.1038/
s41467-020-16324-3
Okubo, T., Toyoda, A., Fukuhara, K., Uchiyama, I., Harigaya, Y.,
Kuroiwa, M., Suzuki, T., Murakami, Y., Suwa, Y., and Takami, H.
(2021). The physiological potential of anammox bacteria as revealedby their core genome structure. DNA Res 28. DOI: 10.1093/dnares/
dsaa028

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 struc-87

National Institute for Basic Biology

Imaging Science
LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

Specially Appointed Assistant Professor :YOKE, Hiroshi
Postdoctoral Fellow:
TANIGUCHI, Atsushi
Visiting Scientist:
YANASE, Ryuji
Technical Assistant:
ISHIBASHI, Tomoko

the 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.
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

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.

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
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.

Visual overview of this lab’s work.

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

Publication List:〔Original papers〕
Y., Naito, Y., Tsugawa, Y., Nonaka, S., Wake, H., Nagasawa,• Hattori,T., Kawaguchi, A., and Miyata, T. (2020). Transient microglial absence•

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.

88

assists postmigratory cortical neurons in proper differentiation. Nat.
Commun. 11. DOI: 10.1038/s41467-020-15409-3
Kondow, A., Ohnuma, K., Kamei, Y., Taniguchi, A., Bise, R., Sato,Y., Yamaguchi, H., Nonaka, S. and Hashimoto, K. (2020). Lightsheet microscopy-based 3D single-cell tracking reveals a correlation
between cell cycle and the start of endoderm cell internalization inearly zebrafish development. Dev. Growth Differ. 62, 495-502. DOI:
10.1111/dgd.12695

LABORATORY FOR BIOTHERMOLOGYSpecially Appointed Associate Professor

KAMEI, Yasuhiro

Specially Appointed Assistant Professor :SAKAMOTO, Joe
Postdoctoral Fellow:
KAMIKAWA, Yuko
TOMOI, Takumi
JSPS Postdoctoral Fellow: SUZUKI, Miyuki
Technical Assistant:
KINOSHITA, Chie

Temperature is an important parameter for living organisms.
Cell activity is affected by temperature since the reaction
ratio and stability of molecules, especially proteins in cells,
depends on it. The environmental temperature which organisms function in vary, and the biological system that regulates body temperature, homeostasis, differs among variousorganisms. Furthermore, temperatures in either cells or at
the molecular level have not been widely discussed to date.
Nano-scale thermometers utilizing fluorescent proteins anddyes have been developed by numerous research groups
including our own, and heterogeneity of temperatures in the
micro environments of living cells have been reported on bysaid groups (Okabe et al, Nat Commun 2014, Kiyonaka etal, Nat. Methods 2014, and Nakano et al, PLoS One 2017).
However, the meaning and mechanisms involved in single
cells remain unclear. Therefore, our group is embarking upona new research field, biothermology, by investigating thenature of temperature in living organisms ranging from nano
to macro.
Our research group employs various and original biothermological research technologies. One of these is the infrared(IR) laser application, which enables single-cell or subcellular local heating by focusing IR through an objective lenson a microscope This technology was originally developed
for laser induced gene expression in the targeting of singlecells through the heat shock response, IR-LEGO: InfraRed
Laser Evoked Gene Operator (Kamei et al, Nat. Methods2009) (Figure 1). This local heating technique can be used

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

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 responsiveness and applicability to wide temperature range. By utilizing this thermometer, we are developing a high-speed thermal imaging microscope system combined with IR irradiation optics. Throughthe analysis of thermal dynamics in cells, we are trying toreveal how temperature heterogeneity is generated within asingle cell as well as considering its meaning from a biological standpoint.
Furthermore, our group is trying to improve the IR-LEGO
technique. We initially applied this system to many organisms including animals (i.e. medaka, nematode, flies andfrogs) and plants (i.e. 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
the Cre/LoxP recombination system. As shown in Figure 2,
we established a cre driver line, which possesses a heat shock
promoter and loxP effector line which can permanently label
irradiated cell lineages by utilizing fluorescent proteins. By
using these transgenic lines, evidence was provided that indicated that exoskeletal tissues in the trunk region came fromthe mesoderm (Shimada et al, Nat Commun. 2013).

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

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,

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National Institute for Basic Biology

Imaging Science

it may be possible to control the preset temperature of HSR,
and this substitution could be applied to the improvement of
HSR efficiency in IR-LEGO technology. We are now trying
to lower the HSR temperature of medaka by substituting
Japanese flounder HSF1. The HSF1 substitution provides us
with various types of significant information and evidence
related to molecular evolution and adaptation to environmental temperature. Thus, this is how 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〕
Y., Honma, Y., Miura, N., Shibuya, T., Kikuchi, H., Tamada,• Ashida,
Y., Kamei, Y., Matsuda, A. and Hattori, M. (2020). Imaging

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performance of microscopy adaptive-optics system using scene-based
wavefront sensing. J. Biomed. Optics-25, 123707. DOI: 10.1117/1.
jbo.25.12.123707
Kanazawa, N., Goto, M., Harada, Y., Takimoto, C., Sasaki, Y.,
Uchikawa, T., Kamei, Y. Matsuo, M. and Fukamachi, S. (2020).
Changes in a cone opsin repertoire affect color-dependent social
behavior in medaka but not behavioral photosensitivity. Front. Genet.
11. DOI: 10.3389/fgene.2020.00801
Kondow, A., Ohnuma, K., Kamei, Y., Taniguchi, A., Bise, R., Sato,Y., Yamaguchi, H., Nonaka, S. and Hashimoto, K. (2020). Lightsheet microscopy-based 3D single-cell tracking reveals a correlation
between cell cycle and the start of endoderm cell internalization inearly zebrafish development. Dev. Growth Differ. 62, 495-502. DOI:
10.1111/dgd.12695
Nishiike, Y., Miyazoe, D., Togawa, R., Yokoyama K., Nakasone, K.,
Miyata, M., Kikuchi, Y., Kamei, Y., Todo, T., Ishikawa-Fujiwara, T.,
Ohno, K., et al. (2021). Estrogen receptor 2b is the major determinantof sex-typical mating behavior and sexual preference in medaka. Curr.
Biol. 31, 1699+. DOI: 10.1016/j.cub.2021.01.089
Watanabe, Y., Okuya, K., Takada, Y., Kinoshita M., Yokoi, S., Chisada,S., Kamei, Y., Tatsukawa, H., Yamamoto, N., Abe, H., Hashimoto, H.,
et al. (2020). Gene disruption of medaka (Oryzias latipes) orthologue
for mammalian tissue-type transglutaminase (TG2) causes movement

retardation. J. Biochem. 168, 213–222. DOI: 10.1093/jb/mvaa038

C.-W., Kamei, Y., Shigenobu, S., Sheu, J.-C., and Tsai, H.-J.
• Zeng,(2021). Injury-induced Cavl-expressing cells at lesion rostral side playmajor roles in spinal cord regeneration. Open Biol. 11. DOI: 10.1098/
rsob.200304〔Review article〕
G., Hayashi, T., Yoshida, K., Yoshida, T., Kudoh, H., Sakamoto,• Abe,J., Konishi, A., Kamei, Y., Takeuchi, T., Tamura, K., et al. (2020).
Insights regarding skin regeneration in non-amniote vertebrates: Skin
regeneration without scar formation and potential step-up to a higher
level of regeneration. Semin. Cell Dev. Biol. 100, 109–121. DOI:
10.1016/j.semcdb.2019.11.014

National Institute for Basic Biology

Research Support
NIBB CORE RESEARCH FACILITIESHeadYOSHIDA, Shosei

The NIBB Core Research Facilities support basic biologicalresearch conducted at NIBB. They consist of three facilities that develop and provide state-of-the-art 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 promote collaboration among
NIBB researchers and other academic institutions.

FUNCTIONAL GENOMICS FACILITY

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), and
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.

Professor

SHIGENOBU, Shuji

Technical Staff:
Technical Assistant:Admin Support Staff:

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

Figure 1. Next-generation sequencer

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 by providing close consultation and training.
In 2020, we suffered from the effects of the COVID-19
pandemic that hampered research activities worldwide.
While we operated our facility placing the highest priority on
users’ safety, we provided remote support for the facilities’
users and online communication with collaborators to sustain
research projects. Such efforts resulted in 17 co-authoredpapers being published.

Proteomics
As is listed below, two types of mass spectrometers andtwo protein sequencers 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. LC-MS/MS system

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National Institute for Basic Biology

Research SupportG., Hackl, T., Terhoeven, N., Shibata, T.F., Nishiyama, T.,• Palfalvi,
Ankenbrand, M., Becker D., Foerster, F., Freund, M., Iosip, A., Kreuzer,

Publication List on Cooperation:•〔Original Papers〕
K.S., Nagano, A.J., Nakano, R.T., Kitazume, T., Yamaguchi,• Araki,K., Hara-Nishimura, I., Shigenobu, S., and Kudoh, H. (2020).

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of the host insect. Front. Microbiol. 11. DOI: 10.3389/fmicb.2020.00840

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)

Characterization of rhizome transcriptome and identification of arhizomatous ER body in the clonal plant Cardamine leucantha. Sci.
Rep. 10. DOI: 10.1038/s41598-020-69941-9
Cui, S., Kubota, T., Nishiyama, T., Ishida, J.K., Shigenobu, S., Shibata,T.F., Toyoda, A., Hasebe, M., Shirasu, K., and Yoshida, S. (2020).
Ethylene signaling mediates host invasion by parasitic plants. Sci. Adv.
6. DOI: 10.1126/sciadv.abc2385
Feng, Z., Nagao, H., Li, B., Sotta, N., Shikanai, Y., Yamaguchi, K.,
Shigenobu, S., Kamiya, T., and Fujiwara, T. (2020). An SMU splicing
factor complex within nuclear speckles contributes to magnesium
homeostasis in Arabidopsis. Plant Physiol. 184, 428–442. DOI:
10.1104/pp.20.00109
Shikanai, Y., Yoshida, R., Hirano, T., Enomoto, Y., Li, B., Asada,M., Yamagami, M., Yamaguchi, K., Shigenobu, S., Tabata, R., et al.
(2020). Callose synthesis suppresses cell death induced by low-calcium
conditions in leaves. Plant Physiol. 182, 2199–2212. DOI: 10.1104/
pp.19.00784
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
Tominaga, T., Yamaguchi, K., Shigenobu, S., Yamato, M., andKaminaka, H. (2020). The effects of gibberellin on the expressionof symbiosis-related genes in Paris-type arbuscular mycorrhizal
symbiosis in Eustoma grandiflorum. Plant Signal. & Behav. 15. DOI:
10.1080/15592324.2020.1784544
Vu, T.-D., Lwasaki, Y., Shigenobu, S., Maruko Akiko and Oshima,K., Lioka, E., Huang, C.-L., Abe T., Tamaki, S., Lin, Y.-W., Chen,
C.-K., Lu Mei-Yeh and Hojo, M., et al. (2020). Behavioral and braintranscriptomic synchronization between the two opponents of a fighting
pair of the fish Betta splendens. PLoS Genet. 16. DOI: 10.1371/journal.
pgen.1008831
Fukutomi, Y., Kondo, S., Toyoda, A., Shigenobu, S., and Koshikawa,S. (2021). Transcriptome analysis reveals wingless regulates neuraldevelopment and signaling genes in the region of wing pigmentation ofa polka-dotted fruit fly. FEBS J. 288, 99–110. DOI: 10.1111/febs.15338
Gu, N., Tamada, Y., Imai, A., Palfalvi, G., Kabeya, Y., Shigenobu, S.,
Ishikawa, M., Angelis, K.J., Chen, C., and Hasebe, M. (2020). DNA
damage triggers reprogramming of differentiated cells into stem cells
in Physcomitrella. Nat. Plants 6, 1098+. DOI: 10.1038/s41477-0200745-9
Li, Y., Omori, A., Flores, R.L., Satterfield, S., Nguyen, C., Ota, T.,
Tsurugaya, T., Ikuta, T., Ikeo, K., Kikuchi, M., et al. (2020). Genomic
insights of body plan transitions from bilateral to pentameral symmetryin Echinoderms. Commun. Biol. 3. DOI: 10.1038/s42003-020-1091-1
Mondal, S.I., Akter, A., Koga, R., Hosokawa, T., Dayi, M., Murase, K.,
Tanaka, R., Shigenobu, S., Fukatsu, T., and Kikuchi, T. (2020). Reduced
genome of the gut symbiotic bacterium “Candidatus Benitsuchiphilus
tojoi’’ provides insight into its possible roles in ecology and adaptation

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•

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•

I., et al. (2020). Genomes of the Venus flytrap and close relatives unveil
the roots of plant carnivory. Curr. Biol. 30, 2312+. DOI: 10.1016/j.
cub.2020.04.051
Sakuta, H., Lin, C.-H., Hiyama, T.Y., Matsuda, T., Yamaguchi, K.,
Shigenobu, S., Kobayashi, K., and Noda, M. (2020). SLC9A4 in theorganum vasculosum of the lamina terminalis is a [Na+] sensor for thecontrol of water intake. PFLUGERS Arch. J. Physiol. 472, 609–624.
DOI: 10.1007/s00424-020-02389-y
Yano, H., Alam, M.Z., Rimbara, E., Shibata, T.F., Fukuyo, M.,
Furuta, Y., Nishiyama, T., Shigenobu, S., Hasebe, M., Toyoda A.,
Suzuki, Y., et al. (2020). Networking and specificity-changing DNA
methyltransferases in Helicobacter pylori. Front. Microbiol. 11. DOI:
10.3389/fmicb.2020.01628
Kurihara, M., Kato, K., Sanbo, C., Shigenobu, S., Ohkawa, Y.,
Fuchigami, T., and Miyanari, Y. (2020). Genomic profiling by ALaP-Seq
reveals transcriptional regulation by PML Bodies through DNMT3A
exclusion. Mol. Cell 78, 493+. DOI: 10.1016/j.molcel.2020.04.004
Kanazawa, T., Morinaka, H., Ebine, K., Shimada, T.L., Ishida, S.,
Minamino, N., Yamaguchi, K., Shigenobu, S., Kohchi, T., Nakano A.,
Ueda, T. (2020). The liverwort oil body is formed by redirection of
the secretory pathway. Nat. Commun. 11. DOI: 10.1038/s41467-02019978-1
Bessho-Uehara, M., Yamamoto, N., Shigenobu, S., Mori, H., Kuwata,K., and Oba, Y. (2020). Kleptoprotein bioluminescence: Parapriacanthus
fish obtain luciferase from ostracod prey. Sci. Adv. 6, eaax4942. DOI:
10.1126/sciadv.aax4942〔Research activity by Shuji Shigenobu〕
Shuji Shigenobu is the principal investigator of the Laboratory• Professorof Evolutionary Genomics. Refer to the laboratory page for details.

SPECTROGRAPHY AND BIOIMAGING FACILITY

year conducted by both visiting scientists, including foreign
researchers, as well as members of NIBB.

Specially Appointed Associate Professor

KAMEI, Yasuhiro

Technical Staff:
Technical Assistant:

KONDO, Maki
TANIGUCHI-SAIDA, Misako
ICHIKAWA, Chiaki
ASAO, Momoko
NAKAGAWA, Mami

Figure 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 onto samples stored incooling chambers.

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.

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 have 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

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 great as that of the corresponding monochromatic component of tropical sunlight at noon (Watanabe etal., 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 both biological andartificial organic molecules have been conducted since it
has been set up. The NIBB Collaborative Research Program
for the Use of the OLS supports about 10 projects every

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

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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).Subsequently, Dr. Nonaka
has conducted and supported roughly 10 Collaborative
Research Program projects for Integrative Bioimaging. The
IR-LEGO, developed by Drs. Shunsuke Yuba and Yasuhiro
Kamei at the National Institute of Advanced Industrial
Science and Technology (AIST), can induce a target geneof interest by heating a single target cell in vivo with a highefficiency irradiating infrared laser (the details of this areprovided in the next section). IR-LEGO was also used forabout 10 Collaborative Research projects, including applications aimed at both animals and plants.

•
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•

Workshop, Symposium and Training course
In 2020, we held the 8 th biological image processing
training course in cooperation with Drs. Kagayaki Kato,
Shigenori Nonaka, Yasuhiro Kamei, Takashi Murata andHiroshi Koyama. The course was held in an online meeting
format (Figure 3) for the first time due to the COVID-19
pandemic.
We have also held annual “Bioimaging Forum” events,
which discuss bioimaging from various technical perspectives such as that of microscopy, new photo-technology, andcomputer science. This year we planned on staging the 14th
edition of this event in November with a focus on micro CT
technology. Like so many events of its type, this meeting
was also to be held in an online format due to the global
COVID-19 pandemic.

Figure 3. A scene from the 8th Bio-Imaging Analysis Training Course.
The course was held in an online meeting format to prevent COVID-19
infection. The 40 participants were able to interact with lecturers via theuse of Zoom and chat applications from their specific locations.

Publication List on Cooperation〔Original papers (Selected)〕Y., Honma, Y., Miura, N., Shibuya, T., Kikuchi, H., Tamada,• Ashida,
Y., Kamei, Y., Matsuda, A., Hattori, M. (2020) Imaging performance••94

of microscopy adaptive-optics system using scene-based wavefront
sensing. J. Biomed. Optics, 25, 123707.
Kanazawa, N., Goto, M., Harada, Y., Takimoto, C., Sasaki, Y., Uchikawa,T., Kamei, Y., Matsuo, M. and Fukamachi, S. (2020) Changes in a cone
opsin repertoire affect color-dependent social behavior in Medaka but
not behavioral photosensitivity. Front. Genetics 11, Article 801.
Kawamoto, N., Carpio, D. P., Hofmann, A., Mizuta, Y., Kurihara, D.,•

Higashiyama, T., Uchida, N., Torii, K. U., Colombo, L., Groth, G. andSimon, R. (2020) A peptide pair coordinates regular ovule initiation
patterns with seed number and fruit size. Current Biology 30, 4352–
4361.
Kondow, A., Ohuma, K., Kamei, Y., Taniguchi, A., Bise, R., Sato,Y., Yamaguchi, H., Nonaka, S., Hashimoto, K. (2020) Light-sheet
microscopy-based 3D single-cell tracking reveals a correlation between
cell cycle and the start of endoderm cell internalization in early zebrafish
development. Dev. Growth Differ. 62, 495-502.
Sakai, Y., Kato, K., Koyama, H., Kuba, A., Takahashi, H., Fujimori,T., Hatta, M., Negri, A. P., Baird, A. H. and Ueno, N. (2020) A stepdown photophobic response in coral larvae: implications for the lightdependent distribution of the common reef coral, Acropora tenuis. Sci
Rep. 10, 17680.
Sasaki, T., Tome, S. and Takei, T. (2020) Intraventricular IL-17A
administration activates microglia and alters their localization in themouse embryo cerebral cortex. Mol. Brain. 13, 93.
Tomoi, T., Kawade, K., Kitagawa, M., Sakata, Y., Tsukaya, H. andFujita, T. (2020) Quantitative imaging reveals distinct contributions of
SnRK2 and ABI3 in plasmodesmatal permeability in Physcomitrella
patens. Plant Cell Physiol. 61, 942-956.
Yokoi, S., Naruse, K., Kamei, Y., Ansai, S., Kinoshita, M., Mito,
M., Iwasaki, S., Inoue, S., Okuyama, T., Nakagawa, S., Young, L. J.,
Takeuchi, H. (2020) Sexually dimorphic role of oxytocin in medaka
mate choice. PNAS, 117, 4802-4808.

〔Research activity by Yasuhiro Kamei〕
Appointed Associate Professor Yasuhiro Kamei is the• Speciallyprincipal investigator of Laboratory for Biothermology. For details,
please refer to the laboratory page.

DATA INTEGRATION AND ANALYSIS FACILITY

Associate Professor

UCHIYAMA, Ikuo

Technical Staff:
Technical Assistant:

NISHIDE, Hiroyo
NAKAMURA, Takanori
SUGIURA, Hiroki
KOTANI, KeikoThe 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.

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(https://www.nibb.ac.jp/en).

Research activity by Ikuo Uchiyama
Associate professor Ikuo Uchiyama is the principal investigator of the Laboratory of Genome Informatics. For moredetails, please refer to the laboratory page.

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

Figure 1. Biological Information Analysis System

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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
KITAZUMI, Noriaki

The 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.

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The activities of the model animal research facility are as
follows:
1. The provision of information, materials, techniques, andanimal housing spaces to researchers.
2. T he 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.

Figure 1. Mouse breeding room in the Yamate area

Figure 2. Breeding cages for replacement in the clean corridor

From April 1, 2020 to March 31, 2021, 2,813 mice (2 transgenic lines and wild-type) were brought into the facility in
the Yamate area, and 34,445 mice (including pups bred in thefacility) were taken out.
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 (8 transgenic lines), and stored using cryopreservation(46 transgenic lines). The frozen eggs of 132 mice lines weretaken out of the facility.
Genome editing experiments were performed on two 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.

medaka and zebrafish, respectively. Additionally, water canbe 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 2020, 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.

Figure 5. Vinyl isolators in the quarantine room

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

II. Research support activities (small fish andbirds)
The first floor of the center facility building in the Yamate
area provides space and facilities to maintain small fish andchick embryos. In the laboratory room for chick embryos, alarge incubation chamber is provided and set at 37.5 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 for

Figure 4. Liquid nitrogen tank

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
Assistant Professor:
Technical Staff:
Technical Assistant:

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

The Model Plant research facility manages facilities for thecultivation of plants in general, and the rearing of several
animal species that do not qualify to be housed in otherfacilities.
This facility equips and manages around 75 culture boxes
or growth chambers, as well as 13 rooms with the P1P

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physical containment level required 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 which include several carnivorous plants. The
facilities are also used to grow the sea anemone Exaiptasia
pallida. Most culture space is used throughout the year bymore than 70 researchers from both external and internalgroups.
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 also available.
Furthermore, a tissue culture rack with dimming LEDs
and pulse-width modulation controllers are used for algae
cultures 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.
In addition, several analytical instruments, that include twoflow cytometry systems and a DUAL-PAM, for DNA content
and chlorophyll fluorescent measuring, respectively, are alsoavailable. On top of this, a liquid handling system 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 NIBB Myodaiji
area building is maintained for Japanese morning glories
and related Ipomoea species, several carnivorous plants, thecastor bean, and other flowering plants that must be cultivated outdoors. Three heated greenhouses (44, 44, and 45 m2)
are used for the sensitive carnivorous plants and a periodical
mass-flowering plant, Strobilanthes flexicaulis. Four airconditioned greenhouses (4, 6, 9, and 9 m2) are provided forthe cultivation of Japanese morning glories and several carnivorous plants. Two air-conditioned greenhouses (9 and 18m2) meet the P1P physical containment level and are available for experiments using transgenic rice plants, Japanesemorning glories, as well as carnivorous plants. The Plant
Culture Laboratory also maintains a 46 m2 building with astorage area and workspace. Part of this building is used forthe rearing of Japanese rhinoceros beetles and the common
grass yellow butterfly.

Figure 6. Webcam monitoring of Japanese rhinoceros beetle larvae.

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Between April 2020 and March 2021, 4 culture boxes were
replaced by newer ones.

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 7. Equipment for P3A experiments

NATIONAL BIORESOURCE PROJECT
The major purpose of the National BioResource Project(NBRP) is to collect, preserve, and provide bioresources(such as experimental animals and plants) that are essential
for life sciences 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 tomeet current scientific demands. NIBB serves as the coreorganization center of medaka bioresources and as a subcenter of morning glory and the zebrafish bioresources.

I. NBRP Medaka (Oryzias latipes)
Project Manager:

NARUSE, Kiyoshi

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 a genome editing platform using CRISPR/Cas9.
Using collaborative research support, researchers can visit
NIBB to generate mutants by genome editing.
With the approval of the second supplementary budget for
FY2020, we were able to install a cabinet-type fish tankwashing machine. This has freed up our technical support
staff from the need to wash the tanks by hand and has
allowed us to focus more on breeding and management,
which requires more human work. In addition, a system toremotely monitor the temperature, humidity, and illumina-tion in the medaka breeding rooms and the water temperaturein the breeding tanks was installed. The air conditioner inthe breeding room was also upgraded. We have continuously monitored the medaka breeding conditions using thesesystems. In 2020, All on-site conferences were cancelled dueto the new coronavirus pandemic, and public relations activities were shifted to online. As a new attempt under this situation, NIBB and the ICOB, Academia Sinica, Taiwan, jointly
organized the “NIBB-Academia Sinica International Webinarof Aquatic Model Organisms for Basic Biology to Human
Disease Models” Total of 100 peoples from Japan, Taiwan,
China, Indonesia, India, Singapore, Germany, Poland,
Switzerland, the Netherlands, Ukraine, the U.S., Canada,
and Argentina registered for the webinar. The active use ofwebinars, which are not limited by location, is one way topromote NBRP medaka under the new coronavirus era.

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, andis especially investigated in the fields of plant physiology
and genetics. NIBB collects, develops and distributes DNA
clones, mutant lines for flower pigmentation, and transgeniclines as a sub-center of the National BioResource Project(NBRP) Morning Glory, and collaborates with the core organization center at Kyushu University. From April 2020 toMarch 2021, we have also collected several mutant lines, andprovided 14 mutant lines and 39 DNA clones to both local
and international biologists. We also analyzed the wholegenome sequences of the 100 mutant lines using next-generation sequencers to develop databases for genetic variations.

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 obserMedaka resources provided from NBRP medaka

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vation. 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.

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

Head and Specially Appointed Professor

NARUSE, Kiyoshi

Technical Staff:
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.

⃝ 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.

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

I. C urrent status of back up available forbiological resources
In 2020, the IBBP Center stored 5,264 384-well and 112
96-well plates consisting of 2,244,864 clones as cDNA/
BAC clones, 20,163 tubes for animal cells, plant andanimal samples, proteins, genes and microorganisms, 5,114
133mm-straw tubes for sperm and 664 seed samples. In total
2.058,069 samples were stored.
Figure 1. IBBP Center.

The IBBP Center includes:⃝ earthquake proof structures capable of withstanding evenvery large-scale quakes which are equipped with emergency backup power generators,

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Research Support
Publication List on Cooperation:〔Original papers〕
H., and Akiyama, Y. (2020). Improved and reproducible cell
• Watanabe,
viability in the superflash freezing method using an automatic thawing
apparatus. Cryobiology, 96, 12-18. DOI:10.1016/j.cryobiol.2020.09.003

Y., Kohaya, N., Shibao, Y., Suyama, A., Kageyama, A.,• Kawasaki,
Fujiwara, K., Kamoshita, M., Matsumura, K., Hyon, S-H., Ito J. andKashiwazaki, N. (2020). Carboxylated ε-poly-L-lysine, a cryoprotective
agent, is an effective partner of ethylene glycol for the vitrification ofembryos at various preimplantation stages. Cryobiology, 97, 245-249.
DOI:10.1016/j.cryobiol.2020.10.004

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

II. C ollaborative Research Project for thedevelopment of new long-term storage
technologies and cryo-biological study
As the IBBP Center can only accept biological resources
which can be cryopreserved in liquid nitrogen, researchers
cannot backup 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 2020, we have conducted 10
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 2020 on November 26-27, 2020 online, becauseof the COVID-19 pandemic. We had 195 participants fromseveral fields covering physics, chemistry, biology, and technology. In the special lecture, there was a presentation on
the effect of polymeric compounds on cell cryopreservationand its mechanism of action, and other presentations related
to the development of cryopreservation technology, such as
methods for preserving insects, plants, and animal embryos.

Figure 4. Group photo of Cryopreservation conference 2020

102

CENTER FOR THE DEVELOPMENT OF NEW MODEL ORGANISMSSUZUKI GroupSpecially Appointed Associate Professor(Cross-appointment with Hiroshima University)

SUZUKI, Ken-ichi
Head

Professor

UENO, Naoto

SHIGENOBU, Shuji

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

SUZUKI, Ken-ichi

Professor:
Assistant Professor:
Postdoctoral Fellow:
Technical Assistant:

MINAGAWA, Jun
KAWAGUCHI, Masayoshi
NIIMI, Teruyuki
HOSHINO, Atsushi
KISHIMOTO, Mariko
TAKAYAMA, Ayuko
SANBO, Chiaki

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.

Technical Assistant:

TAKAYAMA, Ayuko
SANBO, Chiaki

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.

1-1 Development of genome editing techniques forvarious 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.

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.

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.

103

National Institute for Basic Biology

Research Support

1-2 Finding new model organisms and decipheringorgan 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 techniques. 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., Yasue, A., Suzuki, K.T., Fujita, H., Sato, K., Kono, H.,• Habuta,
Takayama, A., Bando, T., Miyaishi, S., Oyadomari, S., et al. (2020).

•
•

104

Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related lossof the accessory lobe and decrease in the number of alveolar type 2
epithelial cells in mouse lung. PLoS One 15. DOI: 10.1371/journal.
pone.0240333
Iida, M., Suzuki, M., Sakane, Y., Nishide, H., Uchiyama, I., Yamamoto,T., Suzuki, K.T., and Fujii, S. (2020). A simple and practical workflow
for genotyping of CRISPR-Cas9-based knockout phenotypes using
multiplexed amplicon sequencing. Genes to Cells 25, 498–509. DOI:
10.1111/gtc.12775
Sanoh, S., Hanada, H., Kashiwagi, K., Mori Tsukasa and Goto-Inoue,N., Suzuki, K.T., Mori, J., Nakamura, N., Yamamoto, T., Kitamura, S.,
Kotake, Y., et al. (2020). Amiodarone bioconcentration and suppressionof metamorphosis in Xenopus. Aquat. Toxicol. 228. DOI: 10.1016/j.
aquatox.2020.105623

CENTER FOR RADIOISOTOPE FACILITIESHeadAssociate Professor

HASEBE, Mitsuyasu

KODAMA, Ryuji

Technical Staff:

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

Technical Assistant:

A

B

C

D

Figure 2. Absence of radiation contamination at the Yamate branch
Inspection of the floor (A), drainage pipes (B), filter units (C), and exhaust
ducts (D).

Table 1. Training courses offered for radiation workers during fiscal year 2020.

The Center for Radioisotope Facilities (CRF) provides awell-established and comfortable environment for naturalscience researchers across Japan as part of the InterUniversity Research Institute Corporation. Center staff
maintain controlled areas in compliance with relevant laws
(Table 1) and monitor the purchase and transfer of radioisotopes. This year, the staff also worked to close down the CRF
Yamate branch (Figure 1, Tables 2-4).
With the emergence of non-radioisotope alternative technologies, the number of CRF users has gradually declined
over the past 20 years (Figure 1). To improve efficiency,
the director general of the NIBB proposed concentrating
the CRF to the Myodaiji branch and closing the Yamate
branch. This possibility was discussed by the CRF Steering
Committee; it was decided during the Directors’ Meetingof the three Okazaki Institutes (NIBB, NIPS, and IMS) in
September 2020 that the Yamate branch would be closed andthe CRF would be consolidated to the Myodaiji branch. The
CRF center staff confirmed that there was no contamination
at the Yamate branch and transferred radioisotopes to the
Myodaiji branch for further use or to the Japan Radioisotope
Association for proper disposal. In March 2021, the Yamate
branch was successfully decommissioned, and the CRF wascentralized to the Myodaiji branch (Figure 2).

Training course

Branch# of participants

Introductory course for beginners
Introductory course for experts
User training course
User training course

Myodaiji
Myodaiji
Myodaiji
Yamate5128
9Table 2. Number of registrants and users of the CRF during fiscal year 2020.

14000

I Received

8000

150

6000

100

4000

50

2000

0

0

0

0

S Received

0

0

0

S Used000

P Received

37,00012,95049,950P Used22,274

12,95035,224

C Received

3,913

0

3,913

15

0

15

612,3500612,350

0

0

0

35
35
32

H

Received
Used

2019

2017

2015

2013

2011

2009

2007

2005

2003

2001

1999

1997

1995

1993

1991

1989

1987

1985

1983

1981

0
1979

0

Total

I Used125

3

Yamate branch0

C UsedTotal number of entrances# of registrants

200

265
44

0

H

10000

Yamate branch

631
119

Myodaiji branch
125

3
250

Myodaiji branch

Table 4. Radioisotopes received and used (in kBq) at each branch during
fiscal year 2020.

14

12000

Total number of entrances

96# of users# of visitors

14# of registrants
300

Yamate-branch

33
14

Table 3. Total number of uses by individual users and visitors during
fiscal year 2020.

32
350

Myodaiji-branch# of registrants# of users

Fiscal year

Figure 1. Annual number of registrants and total number of entrances to
the CRF105

National Institute for Basic Biology

Research Support
RESEARCH ENHANCEMENT STRATEGY OFFICE

Director

Vice-Director

Vice-Director

HASEBE, Mitsuyasu

YOSHIDA, Shosei

MANO, Shoji

6) S ummarized 2019 NIBB activities for the annual
external reviewer meeting on 29 January 2020, managed
the meeting, and published the report (https://www.nibb.
ac.jp/pressroom/pdf/NIBBgaibu_19.pdf).
7) Prepared the proposal on mid-term objectives and plansfrom 2022 to 2027.
8) Published the Annual Report 2019.

The Research Enhancement Strategy Office (RESO)
was established in 2013 to manage the NIBB action plans
proposed by the Director and the Advisory Committee
for Programming and Management as an Inter-University
Research Institute: (1) promoting internationally top-notch
research and (2) providing opportunities for cutting-edge
researchers throughout Japan to gather and engage in activities aimed at exploring future academic fields and creating
new principles. RESO is composed of six groups with thefollowing missions: (1) planning and evaluation, (2) joint utilization of NIBB facilities and joint research between NIBB
members and researchers throughout the world, (3) domestic
and international cooperation, (4) promotion of the diversity and education of young researchers, (5) dissemination of
information and enhancement of public relations, and (6) collaboration between academia and industry. RESO is mainly
managed by University Research Administrators who hold
PhDs and have experience in both research and management.
RESO cooperates with the mother institute NINS’s Research
Enhancement Promotion Headquarters. The activities of thisoffice are partly supported by the Program for Promoting theEnhancement of Research Universities of the Ministry of
Education, Culture, Sports, Science and Technology.

PUBLIC RELATIONS GROUP
Professor:
FUJIMORI, Toshihiko
Specially Appointed Assistant Professor (URA):
KURATA, Tomoko
Technical Assistants:
BAN, Misato
HOSHINO, Maki
UCHIMURA, Ai

This group actively facilitates communication between
NIBB and the public, school teachers, and scientific researchcommunities and describes the activities of NIBB to a wider
audience.

The group’s main activities in 2020 were as follows:
1) Press releases
The group issued press releases concerning NIBB’s scientific achievements to newspaper and magazine reporters
via leaflets, e-mails and web pages. We also arranged press
conferences.
2) Updated and maintained the NIBB web page

PLANNING AND EVALUATION GROUP
Professor:
Associate Professor:
Assistant Professor:

HASEBE, Mitsuyasu
KAWAGUCHI, Masayoshi
MANO, Shoji
KODAMA, Ryuji
JOZUKA, Katsuki
FUJITA, Hironori

This group compiles reports describing NIBB activities forevaluation and budget proposals.

The group’s main activities in 2020 were as follows:
1) Prepared reports on 2019 NIBB activities for MEXT.
2) Prepared reports for 3rd medium-term objectives for theevaluation of education and research.
3) Prepared application forms for the MEXT FY2020 third
supplementary budget and FY2021 budget demands.
4) Prepared reports for the NINS auditors.
5) Conducted follow-up investigation of the activity of the
Research University Strengthening Promotion project.

1063) Edited publications, produced posters and leaflets
We published “NIBB News” (an intra-institutional newsletter in Japanese) and brochures introducing NIBB. We also
designed and distributed posters about NIBB events.
4) Managed social media
This group operated Twitter and Facebook accounts tocommunicate the activities of NIBB to the public. We also
created videos introducing NIBB’s research that are posted
on YouTube.
5) Public relations activities to recruit graduate students
This group organized four graduate school information
sessions for prospective students during the year.
6) Organized scientific outreach programs
We organized online outreach events for the public andcoordinated science classes for middle and high school
students.

LIAISON AND COOPERATION GROUP
Professor:

UENO, Naoto
HASEBE, Mitsuyasu
NARUSE, Kiyoshi
MORITA, Miyo T.
Specially Appointed Assistant Professor (URA):TATEMATSU, Kiyoshi
Technical Staff:
AKIMOTO-KATO, Ai
Research Staff:
TANAKA, Ayako
Technical Assistant:
TAKAHASHI, Ritsue
COWAN, GlenThis group supports and coordinates NIBB’s activities
related to research collaborations and cooperation with universities and research institutes in Japan and abroad.

The main activities performed by this group in2020 were as follows:
1) Coordinated the following conferences/symposia:
・C ryopreservation Conference 2020. Zoom online,
November 26–27, 2020 (p. 102)・NIBB-Academia Sinica International Webinar of Aquatic
Model Organisms for Basic Biology to Human Disease
Models. Zoom online, March 5, 2021 (p. 110-111)
・The Kick Off (1st) meeting for NIBB-COS Heidelberg
International Collaborations Lecture series #1 “Plant RootDevelopment”. Zoom online, March 30, 2021 (p. 110)2) Supported the acceptance of researchers to NIBB fromabroad related to the following events:
・O ne young researcher from the Netherlands to beappointed as a specially appointed assistant professor(November 2020)

The main activities of this group in 2020 wereas follows:
1) Hosted 142 collaborative projects.
2) Operated and managed NOUS (NINS Operation Use
System), an online platform to manage collaborative
projects.
3) Developed remote and automated systems to support collaborative research during the COVID-19 pandemic, as
well as online discussion meetings for the collaborative
research projects.
4) Organized the Genome Informatics Training Course(GITC) and the BioImage Analysis Training Course(BIATC), both of which were held online.
5) Made public announcements of the Cooperative andCollaborative Research System in NIBB to the basic
biology research communities at research meetings andconferences.

YOUNG RESEARCHER SUPPORT GROUP
Professor:
Assistant Professor:

NIIMI, Teruyuki
KOMINE, Yuriko

NIBB provides PhD courses for graduate students while
acting as Department of Basic Biology, School of Life
Science, The Graduate University for Advanced Studies,
SOKENDAI (SOKENDAI is the commonly used namefor the university). The Young Researcher Support Group
provides support for graduate students (SOKENDAI students
and special research students from other universities).

・One young researcher from Germany to conduct collaborative research with the Division of Environmental
Photobiology (January 2021)

The group’s main activities in 2020 were as follows:3) Supported the following education-related programs:
・NIBB Internship Program 2020 (p. 116)2) Organized NIBB’s programs related to PhD courses,
such as the orientation program for new students andOpen Campus Day for prospective students.

COLLABORATIVE RESEARCH GROUP
Professor:

TAKADA, Shinji
SHIGENOBU, Shuji
Specially Appointed Associate Professor (URA):
KAMEI, Yasuhiro
Associate Professor:
UCHIYAMA, Ikuo
Technical Assistant:
ICHIKAWA, Mariko
ICHIKAWA, Chiaki1) Coordinated and managed lectures and other activities,
including periodical research presentations by students.

3) Cooperated the organization of the Freshman Course
and the Life Science Retreat as interdepartmental
programs in SOKENDAI.
4) G athered and provided useful information for bothstudents and faculty members.

GENDER EQUALITY PROMOTION GROUP
This group acts as a hub to promote collaborative projects
among multiple researchers through the exchange of information and the development of new equipment and methods.

Professor:
Associate Professor:

HIGASHIJIMA, Shin-ichi
TSUBOUCHI, Tomomi

This group manages gender equality promotion activities.

107

National Institute for Basic Biology

Research Support
The group’s main activities in 2020 were as follows:1) Managed a multi-purpose room for use by researcherswith children.
2) M aintained two group mailing lists for (1) female
researchers and (2) researchers with young children inorder to share useful information.

COLLABORATIVE INNOVATION GROUP
Professor:
MINAGAWA, Jun
Associate Professor:
KODAMA, Ryuji
Specially Appointed Assistant Professor:
KANAI, Masatake

This group is responsible for activities that bridge the gap
between NIBB researchers and the business community.

The group’s main activities in 2020 were as follows:
1) Supported the application for JST’s A-step, a research
grant for practical use.
2) Supported participation in Innovation Japan, a trade fairfor university seeds.
3) Held a seminar on the support system for industry-university collaboration.
4) Negotiated a license agreement with a company.
5) Provided support for joint research agreements between
NIBB researchers and companies.

108

TECHNICAL DIVISIONHeadMIWA, Tomoki

Common Facility Group

Research Support Group

Chief:

Chief:

MORI, Tomoko● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki		Vice-Unit Chief:
Technical Staff:
Technical Assistant:

MAKINO, Yumiko
YAMAGUCHI, Katsushi
NISHIDE, Hiroyo
NAKAMURA, Takanori
TANIGUCHI-SAIDA, Misako
SUGIURA, Hiroki
ICHIKAWA, Chiaki
ICHIKAWA, Mariko
KOTANI, Keiko
SHIBATA, Emiko● NIBB Bioresource Center
Unit Chief:
OHSAWA, Sonoko
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
SUGINAGA, Tomomi
YAMAGUCHI, Chinami● IBBPTechnical Staff:● Cell Biology
Vice-Unit Chief:
Technical Staff:

MIZUTANI, Takeshi

HAYASHI, Kohji
BINO, Takahiro
NISHIMOTO, Yuki

● Developmental Biology
Vice-Unit Chief:
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
Technical Staff:
MIZUGUCHI, Hiroko● Neurobiology
Vice-Unit Chief:

TAKEUCHI, Yasushi● Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
OHI, Shoko● Environmental Biology
Technical Staff:
NODA, Chiyo

AKIMOTO-KATO, Ai● Waste Management Room
Unit Chief:
MATSUDA, Yoshimi● Center for Radioisotope Facilities
Unit Chief:
MATSUDA, Yoshimi
Vice-Unit 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.

Reception
Admin Support Staff:

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.

109Collaboration Programs with
Overseas Institutions
N I B B - C e n t re f o r O r g a n i s m a l S t u d i e s
Heidelberg (COS Heidelberg), Germany
NIBB and COS Heidelberg are conducting collaborative
activities based on the academic exchange agreement signed
in 2019.
NIBB-COS Heidelberg international collaboration research
project focusing on the mechanisms and evolution of lightsensing in cnidarians
NIBB and COS Heidelberg started an international collaborative research project focusing on the mechanism and evolution of light sensing in cnidarians. This project was financially supported by the NINS. In this collaborative research
project, a new emerging model organism, sea anemone(Aiptasia sp.), was used. Professor Annika Guse, who has
been conducting research analyzing the mechanisms and evolution of light sensing in sea anemones at COS Heidelberg,
joined the “Open Laboratory” established in NIBB. In
October 2020, a post-doc researcher, Dr. Mariko Kishimoto
who received PhD degree in NIBB, joined this research
project and has been conducting research using molecularbiological and molecular genetic approaches (p. 74).
The Kick Off (1st) meeting for NIBB–COS Heidelberg
International Collaborations
Lecture series #1 “Plant Root Development”
The “Kick Off (1st) meeting for NIBB–COS Heidelberg
International Collaborations” was held on March 30, 2021.
We had planned to hold an NIBB–COS Joint Workshop
in Okazaki in 2020, but this was postponed due to the
COVID-19 pandemic and was changed to an online exchange
program between the two institutes. The program consistedof two events: a lecture series at which researchers from both
institutes presented their research projects, and a break-out
discussion session using an online communication tool for
all members in attendance. The theme of the lecture series
was “Plant Root Development”. Dr. Takashi Soyano fromthe Division of Symbiotic Systems and Dr. Alexis Maizel
from COS Heidelberg gave presentations on rhizogenesis andlateral root formation, respectively. 58 members from NIBB
and 32 members from COS (both totals include post-docs
and students) participated in this event, and lively discussions
took place.

At the break-out discussion session, we used the Remo
online meeting platform, and the PIs from both sides were110

grouped into seven research themes (New Model Organisms,
Stem Cells / Regeneration, Development, Cell Biology/ Signaling, Neurobiology, Bioimaging, Modeling, andQuantitative Biology) to facilitate discussions. To ourdelight, this event generated new research ideas and possibilities for collaborative research.
As the overall feedback was very positive, we will continue
this format of meetings in 2021, namely the lecture series
combined with the break-out discussion session. The themeof the lecture series will change every time to attract a
broader range of participants for the lectures as well as forthe break-out discussion session.

NIBB-Princeton University, USA
Collaborative activities between NIBB and Princeton
University are conducted under the support of theInternational Research Collaboration Center (IRCC) of the
National Institutes of Natural Sciences (NINS), based on
the academic exchange agreement between the NINS and
Princeton University. In AY2020, Dr. Ellen Reed, an IRCC's
specially appointed research employee and postdoctoral
research fellow of Princeton University, promoted a collaborative research project with Professor Kazuhiro Aoki of
NIBB and Professor Jared Toettcher of Princeton University
on "Dissolving biomolecular condensates using opticalor chemical recruitment of soluble proteins" at Princeton
University. Although it was difficult to travel between NIBB
and Princeton University due to the pandemic of COVID-19,
collaborative research activities were conducted using online
communication tools.

NIBB–Institute of Cellular and Organismic
Biology (ICOB), Academia Sinica, Taiwan
NIBB–ICOB, Academia Sinica, Taiwan, International
Webinar of Aquatic Model Organisms for Basic Biology to
Human Disease Models
The “NIBB–Academia Sinica International Webinar of
Aquatic Model Organisms for Basic Biology to Human
Disease Models” was held online on March 5, 2021. A
training course jointly organized by NIBB and Academia
Sinica was initially planned in 2020, but was postponed toAutumn 2022 due to the pandemic. In response to this, thecourse’s organizers held the above-mentioned webinar as aninternational cooperative activity when international travel
was limited. A total of 11 lecturers were invited from China,
Singapore, Taiwan, and Japan to attend the online webinar.
The lectures covered a wide variety of topics and aquaticorganisms, from corals and sea urchins to amphibians andbony fishes, in the fields of developmental biology, evolutionary biology, genomics, neurophysiology, and immunology.
A total of 100 people from Japan, Taiwan, China,
Indonesia, India, Singapore, Germany, Poland, Switzerland,
the Netherlands, Ukraine, the United States, Canada, andArgentina registered to this webinar. An advantage of theonline format was that the number of registered participants
had expanded worldwide. The webinar itself saw roughly
50–60 people consistently staying online to participate in all
sessions.
In the post-event questionnaire, 97% of respondents
answered that the webinar content was interesting, andmany gave high marks to the lecture content and management. During this webinar, we attempted to foster realtime dialogue via Slack in the form of a typed chat format
enabling week-long question-and-answer sessions to encourage our participants to discuss the issues. 86% of participants
felt that Slack was easy to use, while others preferred direct
discussions with all lecturers and participants. In addition,
there were useful comments regarding the timing of breaks,
which will be helpful in organizing future webinars. We
would like to organize scientific events in this format, notonly because travel bans remain in effect, but also because
this format attracts participants from more diverse countries.

Webinar information:■ Organizers
Kiyoshi Naruse (NIBB / NBRP-medaka / IBBP)Ai Shinomiya (ExCELLS / NIBB)
Yasuhiro Kamei (NIBB)
Chen-Hui Chen (ICOB, Academia Sinica, Taiwan)
Sheng-Ping Hwang (ICOB, Academia Sinica, Taiwan)
Date: March 5, 2021

Collaborative Activities with
Joint Usage/Research Center
NIBB–Institute of Low Temperature Science(ILTS), Hokkaido University
NIBB and ILTS are conducting collaborative research basedon the agreement of cooperation signed in 2019.
Accomplishment of the NIBB Priority Collaborative
Research Project■ Project Title:
M olecular and physiological mechanisms for understanding mammalian hibernation and their comparative
analysis among species■ Co-Principal Investigators:
Prof. Yoshifumi Yamaguchi (ILTS)
Prof. Toshihiko Fujimori (NIBB)
Prof. Shuji Shigenobu (NIBB)NIBB–Institute of Molecular Embryology andGenetics (IMEG), Kumamoto University
Agreement of Cooperation with IMEG, Kumamoto
University
NIBB and IMEG at Kumamoto University have signed anagreement of cooperation, which is deemed effective fromMay 26, 2020. To prevent the further spread of COVID-19
infection, the signing ceremony was held in a remotecapacity via an online connection between Kumamoto andOkazaki.

Dr. Kiyokazu Agata, the Director General of NIBB, and Dr. Hitoshi
Niwa, the Director of IMEG, signing the agreement.

This agreement will promote research and education
efforts at these two top research institutes, leading the way
in advanced research of the highest international standards
through mutual collaboration in the fields of developmental
medicine and basic biology. The results of the collaborative
activities will be used for research on both sides. In addition,
the agreement aims to strengthen the support infrastructure
for joint usage and joint research nationwide and to revitalizeinternational academic exchange.
Young researchers and graduate students in NIBB and
IMEG have organized regular joint mini-meetings focusedon developmental biology.

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NIBB–Institute of Advanced Medical Sciences(IAMS), Tokushima University
Agreement of Cooperation with IAMS, Tokushima
University
NIBB and IAMS at Tokushima University have signed
an agreement of cooperation, which is deemed effectivefrom November 26, 2020. To prevent the further spreadof COVID-19 infection, the signing ceremony was held
remotely via an online connection between Tokushima andOkazaki.

Dr. Kiyokazu Agata, the Director General of NIBB, and Dr. Toyomasa
Katagiri, the Director of IAMS, signing the agreement.

The aims of this agreement are promotion of mutual collaborative activities in the fields of research related to medical
science and basic biology and leading the way in advanced
research of the highest international standards. This will
promote research and education efforts at these two top
research institutes to strengthen the support infrastructure forjoint usage and joint research nationwide and to revitalizeinternational academic exchange.
NIBB researchers and IAMS researchers collaborate on
several projects using bioimaging and genome editing
methods.

The 14th NIBB Bioimaging Forum“A New Aspect of Bioimaging
by Non-optical Modality”
Organizing Committee: Shinya Komoto,
Kagayaki Kato, Joe Sakamoto
Supervisors: Yasuhiro Kamei, Kiyoshi
Tatematsu
November 6 (Fri), 2020
The 14th NIBB Bioimaging Forum supported by Advanced
Bioimaging Support (ABiS) and entitled “A New Aspectof Bioimaging by Non-optical Modality” was held on
November 6, 2020.
NIBB Bioimaging Forums have previously focused on
optical observation techniques such as super-resolution
microscopy using adaptive optics, and the imaging of biophysical properties. Therefore, during the 14th version of thisevent, we focused on Computed Tomography (CT) especiallythrough the utilization of X-ray, synchrotron radiation andmagnetic resonance, as a non-optical observation method
instead. CT is a powerful tool for the visualization of thethree-dimensional structure of samples without destroying
them. Nevertheless, it is difficult for newcomers to engage
in research due to the embryonic nature of the existing CT
user community, and the difficulty of accessing devices.
Therefore, we held this forum to provide opportunities forcommunication with both existing and potential CT users, aswell as to share information.
When people hear the term CT, many imagine machines
in hospitals that medically diagnose humans. However, CT
is not used only in the medical field, but also in various
non-medical fields, such as industry, biology and so on.
Therefore, we invited lecturers from both academia andcompany. Lecture topics were diverse and included areas
such as sample preparation, observation methods, recent
trends in CT device, and applications to academic research.
Although the forum was held on-site in 2019, it was postponed because COVID-19 outbreak. The organizing committee subsequently decided to merge the 2019 and 2020
versions of the event and hold the forum online using theZoom online meeting platform. To organize and manage
the form both quickly and effectively, the three organizers
gathered at NIBB to live stream the forum. Communicationdifficulties were experienced by some participants, among
several other things that needed to be improved. In contrast,
lecturers who could not attend on the day of the forum took
advantage of the format and gave their presentation by prerecording it and streaming it on the day of the event.
In closing, I would like to express my appreciate to all participants and organizers, and hope that the forum was helpful
for the former’s research,(Joe Sakamoto)112

The NIBB Genome Informatics
Training Course
The NIBB Core Research Facilities regularly organizes
a series of training courses on the most recently developed research techniques. The NIBB Genome Informatics
Training Course (GITC) is specially designed for biologists
who are unfamiliar with bioinformatics. In 2020, we held
two sets of training courses on RNA-seq analysis. Eachversion of the RNA-seq analysis course was basically madeup of two 2-day programs: one being a preparatory course(Introduction to NGS Analysis) concerning the basics ofUNIX and R, and the other a practical course (Introduction
to RNA-seq) for learning about the pipelines to RNA-seq
analysis using next-generation sequencing data. These GITC
courses offered lectures and hands-on tutorials. This year, we
initially held a practical course on RNA-seq analysis, that
had been postponed due to the COVID-19 pandemic, only.
However, we subsequently held a full version of the training
course. All courses were held online to prevent the spread of
COVID-19 infection.

Introduction to RNA-seq: From the
Basics of NGS to de novo Analyses

Introduction to NGS Analysis:
Basics of UNIX, R, and NGS
November 26 (Thu)–27 (Fri), 2020
■ 32 participants and 9 auditors (including 3 from NIBB)■ Program:
1. UNIX for Beginners
2. Introduction to “R”
3. Introduction to Statistics
4. NGS Basic Data Formats and NGS Basic Tools
5. Editor and Scripts
6. Text Processing
7. Exercises

Introduction to RNA-seq: RNAseq Analysis Pipeline
March 10 (Wed)–11 (Thu), 2021
(This program was the same as the above listed practical course)■ 31 participants and 10 auditors (including 2 from NIBB)■ O rganizers: 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. Hiroki Sugiura (NIBB Core
Research Facilities)

June 4 (Thu)–5 (Fri), 2020
(Practical Course) RNA-seq Analysis Pipeline■ 33 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. RNA-seq Pipelines: Genome-Based andTranscriptome-Based Approaches
5. Multivariate Statistics
6. Functional Annotation and Gene Ontology
7. Exercises

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The Bio-imaging Data Analysis
Training Course 2020
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
TakadaDecember 9 (Wed)–11 (Fri), 2020
The 8th Bio-imaging Data Analysis Training Course was
jointly held 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 was related to learning
about image processing and analytical techniques throughsolving simple problems with image analysis, and understanding appropriate methods and necessary preparation
when consulting experts in technically advanced problems
concerning imaging. 114 people applied for the course,which had an announced capacity of 16 participants. Given
the high demand for courses on these subjects, we accepted
18 participants.
This course's lectures were conducted with the aim of
guiding participants towards an awareness of the seriesof steps essential for fundamental image processing andanalysis while also obtaining images for eventual use (workflows). In addition to this, participants independently worked
on practical image analysis exercises using ImageJ; a typicalopen-source software package for biological image processing and analysis. Lectures were also given on how the
programming of simple “macro language”, which uses theaforementioned workflows in ImageJ, allows for automation; a necessity for the large capacity and high-dimensional
throughput of microscopic imaging which has more becomecommon over recent years.
Upon the course’s conclusion, each student offered commentary about and discussed the methods taught by providing examples of images gleaned from their own research.
Unfortunately, although this year's course was held online
due to issues associated with COVID-19, we utilized technological innovations within the lectures and exercises, suchas a chat service to support individual participants, so as toallow them to feel as if the course was being held as an inperson event.
Every year following the course’s completion, participants
often report feeling pretty tired, but satisfied. In a similarvein, we believe that this course is beneficial to its participants thanks to the associated increase in their familiarity
with image analysis techniques Thus, we expect that thiscourse will increase opportunities for joint research relating
to biological image analysis moving forward.
(Kagayaki Kato)

114

Advanced Bioimaging Support(ABiS)The demand for bioimaging has increased in recent years in
the field of life science. However, due to advances in imaging
technologies, such as the diversification and specialization ofimaging equipment, increasingly complex operations, increased
equipment and running costs, as well as the growing need forimage data analysis, individual research institutes and universities are increasingly encountering difficulties related to theintroduction, 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 for lightmicroscopy, electron microscopy, magnetic resonance imaging,and other methods through its network. It aims to provide
comprehensive support for advanced imaging in the field 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 with the following:■ Light microscopy• 4D microscopy administered by Prof. Toshihiko FUJIMOIRI• IR-LEGO microcopy administered by Assoc. Prof.
Yasuhiro KAMEI• DSLM administered by Assoc. Prof. Shigenori NONAKA■ Imaging analysis• Development of image processing/analysis algorithms forbiological data administered by Prof. Naoto UENO, Assist.
Prof. Kagayaki KATO and Assist. Prof. Yusaku OHTA.
■ Training• Training for image analysis administered by Prof. Naoto
UENO and Assist. Prof. Hiroshi KOYAMA.
To organize and coordinate ABiS activities, two secretariat offices were established at NIBB (Assoc. Prof.
Shoji MANO) and NIPS, respectively, under the controlof the general support group (Individuals in charge at
NIBB; Director-general Prof. Kiyokazu AGATA, Prof.
Naoto UENO, Prof. Shinji TAKADA, and 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 makefull use of the ABiS platform to accelerate their research
projects. In addition, we organize technology training
sessions, workshops, and symposia to disseminate advanced
imaging technologies and share information about them.

We also coordinate with the other three platforms (Platformof Supporting Cohort Study and Biospecimen 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 acceptedinternational benchmarks, and that further observations, data
analyses and research methods will be able to be better sharedglobally. Furthermore, it is also hoped that this collaboration
will lead to set up a better environment for bioimaging researchin Japan. This will be achieved not only by providing international training courses for young researchers and the staff ofimaging facilities, but also through discussions about the implementation of career paths; a common problem in this field.
GBI also aims to establish an image data sharing infrastructure system, promote academic/industrial cooperation andformulate a global strategy for bioimaging research. The
GBI event “The Fifth Exchange of Experience (EoE V)”
to be hosted by ABIS, was initially planned to be held in
Okazaki during autumn of 2020, but was held online insteadto prevent the spread of COVID-19.

ABiS Symposium “The Present andFuture of Advanced Bioimaging-Research Strategy in Japan-”February 24 (Wed), 2021
The ABiS symposium “The Present and Future ofAdvanced Bioimaging -Research Strategy in Japan-”
was held as an online event, and saw four researchers, who have received support from ABiS in the past,
introduce their research and achievements which themselves were aided by the aforementioned organization. Another four researchers, who currently study
cutting-edge life science via the application of new
imaging technologies, also gave additional lectures.
On top of this, a panel discussion entitled “Promotionof Bioimaging Research and Support” was carried out
to discuss the future of bioimaging in Japan (Figure 1).

Figure 1. Lectures and chairpersons of ABiS Symposium "The Present
and Future of Advanced Bioimaging -Research Strategy in Japan-.

115

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 FY2020, due to the global spread of COVID-19 and subsequent attempts at preventing further infection, this program
mainly accepted international students attending universities in Japan. Consequently, two international students, one
from the Philippines and the other from the United Kingdom,
were selected. Because of the situation in Japan, one ofthem came to NIBB in March 2021, and the other will comeduring FY 2021. Furthermore, we received applications fromtwo other students who belonged to overseas universities andwere staying in Japan due to circumstances unrelated to theInternship Program; one of whom was subsequently selected
and stayed at NIBB for two months.

Report from a participant
Sea Hee Yook
Imperial College London
I immersed myself in the NIBB internship program as
an undergraduate student who had just completed her firstyear of a Medical Biosciences degree at Imperial College
London. Although I had limited experience in conducting
experiments in a professional laboratory, I was motivated tointern at Professor Aoki`s laboratory specializing in quantitative biology upon reading one of his publications. Amongthe other host labs, Professor Aoki lab`s unique approachin presenting scientific results through microscopy visualization and precisely quantified data appealed to me as anopportunity to obtain skills in both practical research andscientific communication. Throughout the course of the next
month and a half, I was able to get a deep insight into using
advanced techniques and equipment to visualize and model
various intracellular mechanisms.
My research primarily focused on examining the proteins
involved in controlling the cell cycle of Schizosaccharmyces
pombe, otherwise known as fission yeast. The progression ofbiochemical events from G1 to M phase in its cell cycle is
known to be regulated through a single CDK-cyclin complex
composed of Cdc2 and Cdc13, alongside the activationof additional protein substrates at distinct timepoints. To

116

examine such properties, I conducted confocal fluorescenceimaging and quantitative analysis of the images.
Whilst being engaged in using various imaging tools, I
was also introduced to basic laboratory techniques suchas western blotting, transforming plasmids into cell lines,
designing PCR primers, and conducting swift but precise
DNA work. Additionally, I learned about the importance ofperforming detailed experimental planning, careful observation, interactive discussions with other researchers, reliabledata analysis, and effective troubleshooting to develop as awell-established researcher capable of presenting influential
scientific discoveries.
Undoubtedly, the internship program provided me with anopportunity to further explore Japanese culture by encountering people with different values, interests, and lifestyles.
I want to thank the NIBB for providing me with such aninvaluable opportunity, and the people at Professor Aoki’slaboratory for their unconditional support throughout theinternship. I am confidently returning home with newly
acquired skills and attributes that have equipped me tocontinue pursuing my passion for research.

Outreach Activities
November 12 (Thu), 2020
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.

Special class at Kawai Junior High School■ S. A. Prof. Kiyoshi Naruse

Visits to Junior High Schools
NIBB, in collaboration with the Okazaki City Board of
Education, visited junior high schools during 2020 to give
special classes.

November 2 (Mon), 2020
Special class at Ryukai Junior High School■ Director General Kiyokazu Agata

November 17 (Tue), 2020

November 5 (Thu), 2020

December 1 (Tue), 2020

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

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

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

December 4 (Fri), 2020
November 11 (Wed), 2020
Special class at Ryunan Junior High School■ Prof. Teruyuki Niimi

Special class at Mutsumi Junior High School■ Assist. Prof. Kazuo Tsugane117

Visit to High Schools

Seminar for school teachers

February 5 (Fri), 2021

February 9 (Tue), 2021

Special class at Okazaki High School■ Prof. Takashi Ueda

■ Prof. Shinji Takada

August 4 (Tue), 2020
Special class at Okazaki Kita High School■ Assist. Prof. Hiroki Takahashi

Feedback session regarding research presentations by high school students
August 28 (Fri), December 25 (Fri),2020■ S. A. Assist. Prof. Kiyoshi Tatematsu

December 7 (Mon), 2020
Special class at Okazaki Kita High School■ Prof. Jun-ichi Nakayama

Programs broadcast live online
June13 (Sat)-20 (Sat), 2020
“Watching Both the Laying of Medaka Eggs and Their
Hatching”
This was a 200 hour live broadcast displaying the process
entailed in the birth of medaka ranging from egg laying
to hatching. Commentaries were provided by researchers
studying medaka via the live internet broadcast platform,
NicoNico

March 16 (Tue), 2021
Special class at Okazaki Kita High School■ Dr. Gergo Palfalvi

118■ S. A. Prof. Kiyoshi Naruse■ Dr. Tadashi Sato
■ S. A. Assist. Prof. Tomoko Kurata

August 9 (Sun)–16 (Sun), 21 (Fri), 2020

March 13 (Sat), 2021

"Watching the Planarian Regeneration Process over the
Course of 200 Hours"
This was a 200 hour live broadcast displaying the processof planarian regeneration. Commentaries were provided byresearchers studying planarians via the live internet broadcast
platform, NicoNico

The 31st National Institutes of Natural Sciences
Symposium■ Prof. Kazuhiro Aoki

■ Director General Kiyokazu Agata■ S. A. Assist. Prof. Tomoko Kurata

June 14 (Sun), 2020
National Institute of Natural Sciences
The 9th Young Researcher Award Commemorative Lecture■ S. A. Assist. Prof. Yoko Otsubo

Lectures for the Public
February 23 (Tue), 2021
Online lecture at Natural History Museum/Sea of Life■ Dr. Tatsuro Konagaya■ Mr. Yasuhiko Chikami

February 24 (Wed), 2021
Presentation by OKASHIN (Okazaki Credit Union) scholarship students■ Mr. Akimasa Watanabe

February 28 (Sun), 2021
Science Day in Toki (Online Lecture)■ Prof. Teruyuki Niimi■ Mr. Kohei Kawano

119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
https://www.nibb.ac.jp/en/
Issued in December 2021
Edited by the Research Enhancement Strategy Office, NIBB