National Institute for Basic Biology

2021 ANNUAL REPORT

April 20213March 2022

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 2021 to March 2022 …………………………… 7
Selected Publications in 2021AY from NIBB ………………………………………………… 8Professor Emeritus …………………………………………………………………………… 10
Cell Biology
Division of Cellular Dynamics …………………………………………………………… 11
Division of Quantitative Biology…………………………………………………………… 14
Division of Chromatin Regulation ………………………………………………………… 18
Laboratory of Neuronal Cell Biology……………………………………………………… 21
Laboratory of Stem Cell Biology ………………………………………………………… 24
Laboratory of Organelle Regulation ……………………………………………………… 26
Developmental Biology
Division of Morphogenesis ………………………………………………………………… 28
Division of Molecular and Developmental Biology ……………………………………… 31
Division of Embryology …………………………………………………………………… 34
Division of Germ Cell Biology …………………………………………………………… 37
Labotratory of Regeneration Biology …………………………………………………… 40
Neurobiology
Division of Behavioral Neurobiology……………………………………………………… 41
Laboratory of Neurophysiology …………………………………………………………… 44
Evolutionary Biology and Biodiversity
Division of Evolutionary Biology ………………………………………………………… 47
Division of Symbiotic Systems …………………………………………………………… 50
Division of Evolutionary Developmental Biology ……………………………………… 53
Laboratory of Evolutionary Genomics …………………………………………………… 56
Laboratory of Bioresources ……………………………………………………………… 59
Laboratory of Biological Diversity ………………………………………………………… 61
Environmental Biology
Division of Environmental Photobiology ………………………………………………… 82
Division of Plant Environmental Responses …………………………………………… 85
Theoretical Biology
Laboratory of Genome Informatics ……………………………………………………… 88
Imaging Science
Laboratory for Spatiotemporal Regulations……………………………………………… 90
Laboratory for Biothermology …………………………………………………………… 91
Research Support
NIBB Core Research Facilities …………………………………………………………… 93
NIBB BioResource Center ………………………………………………………………… 98
National BioResource Project …………………………………………………………… 101
NIBB Center for the Interuniversity Bio-Backup Project ……………………………… 103
Center for the Development of New Model Organisms………………………………… 105
Center for Radioisotope Facilities (Okazaki Research Facilities) …………………… 107
Research Enhancement Strategy Office ………………………………………………… 108
Technical Division ………………………………………………………………………… 111
Collaboration Programs with Overseas Institutions …………………………………… 112
Collaborative Activities with Universities and Research Institutes in Japan ……… 113
The NIBB Genome Informatics Training Course ………………………………………… 114
The Bio-imaging Data Analysis Training Course ………………………………………… 116
Advanced Bioimaging Support ……………………………………………………………… 116
NIBB Internship Program ……………………………………………………………………… 118
Outreach Activities …………………………………………………………………………… 119
Access to NIBB ………………………………………………………………… Inside back cover

The cover items are related to and reproduced with modifications from the following papers from the Division ofGerm Cell Biology: <Transient suppression of transplanted spermatogonial stem cell differentiation restores fertility
in mice= (Nakamura et al., Cell Stem Cell 2021, https://doi.org/10.1016/j.stem.2021.03.016) and <A multistate stem
cell dynamics maintains homeostasis in mouse spermatogenesis= (Nakagawa et al., Cell Reports 2021, https://doi.
org/10.1016/j.celrep.2021.109875). See page 36 for details.

INTRODUCTION
t is my great pleasure to present all the National
Institute for Basic Biology (NIBB)9s 2021 Annual
Report which outlines NIBB9s research, educational, andinternational activities and its effective function as a centerfor collaborative research in Japan from April 2021 to March
2022.

I

The NIBB9s research activities have largely resumed tonormal in 2021, following the significant easing of restrictions caused by the COVID-19 pandemic.
Visits of researchers from overseas and travel abroad by
NIBB researchers are gradually returning to previous levels.
Online exchange activities are also continuing. The 2nd
and 3rd Meetings for NIBB-COS Heidelberg International
Collaborations were held online.
In 2021, NIBB signed a new collaboration agreementwith the Institute for Molecular and Cellular Regulation,
Gunma University. Joint research on intracellular membranetrafficking is being conducted between the two institutes.
In addition, We also initiated the seminar series <AI and
Biosystems= with the aim of active participation of AI in biological research in collaboration with Chubu University and
the National Institute for Physiological Sciences.
The well-equipped core facilities that support research
activities are one of the notable features of NIBB. It was
truly pleased that Ms. Tomoko Mori, Ms. Yumiko Makino,
Mr. Katsushi Yamaguchi, and Mr. Takahiro Bino, technical staff of the Functional Genomics Facility, wereawarded the Outstanding Support for Research Award, The
Commendation for Science and Technology by the Ministerof Education, Culture, Sports, Science, and Technology, fortheir contribution to research support. Furthermore, Professor
emeritus Yoshitaka Nagahama was awarded The Order of
the Sacred Treasure, Gold Rays with Neck Ribbon by theJapanese government. We extend our sincere congratulations
on this honorable award for his many years of outstandingachievements.
We are grateful for our status as a leading international
research institute in the field of basic biology, we are constantly striving for improvement and welcome your valuable
feedback. We would like to express our sincere gratitude foryour continued support.

Kiyokazu Agata
Director General of NIBB
December, 2022

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 Buildings in the Myodaiji area (left) and Yamate area (right).

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. NIBB9s activities
are supported by the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) of Japan.

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■ Members in NIBBTechnicla Assistantsand Admin Support
Stafffrom 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 to improve NIBB9s
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
NIBB9s 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 technicalstaff 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 a 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.

■ Financial Configuration of NIBB
Indirect Costs in Grants

Director General 1
Professors(including specially-appointed) 15

50(settled forApril 2021-March 2022)
numbers are in million yen

70

Associate Professors(incl. sp. app. RMC) 14

105

183

Other Grants

External
Competitive
Fund

Assistant Professors(incl. sp. app. RMC)

974

Grant-in-Aid
for Scientific
Research

Personnel

798

511

40
44
Graduate andUndergraduate Students(2 are undergraduates)2

Postdoctoral Fellows

28
Technical Staff

297 in total(as of 1st April, 2022)

1,333

Grants forCommissioned Research

210

Management
Expenses Grant
for NIBB

Research

129
Collaboration

Self-Income 3

404

Management Expenses
Grant for SOKENDAI

49

Donations17

Other 2

2,375 million yen
in total

■ Organization

National Institute of
Natural Sciences(NINS)

National Institute for Basic Biology (NIBB)
As of March 31, 2021

President
KOMORI, Akio

Research Units

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)
Institute for
Molecular Science (IMS)
Three Institutes in Okazaki
Center for Novel Science Initiatives■Frontier Photonic Science■Plasmabio Science
Astrobiology Center■Division of Cellular Dynamics

Cell Biology■Division of Quantitative Biology*■Division of Chromatin Regulation■Laboratory of Neuronal Cell Biology*

Advisory
Committee
forProgrammingandManagement■Laboratory of Stem Cell Biology■Laboratory of Organelle Regulation

Developmental Biology■Division of Morphogenesis■Division of Molecular and Developmental

Biology*■Division of Embryology■Division of Germ Cell Biology■Laboratory of Regeneration Biology

Research
Enhancement
Strategy
Office■ Evaluation and■Division of Behavioral Neurobiology*

Neurobiology■Laboratory of Neurophysiology

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

Biology

Information Group■ Public Relations Group■Laboratory of Evolutionary Genomics■ Liaison and■Laboratory of Bioresources

Coordinaton Group■ Collaborative
Research Group■ Young Researcher
Support Group■ Gender Equality

Promotion Group■Laboratory of Morphodiversity■Laboratory of Biological Diversity

Environmental Biology■Division of Environmental Photobiology

Theoretical Biology■Laboratory of Genome Informatics

Imaging Science■Laboratory for Spatiotemporal Regulations*■ Collaborative

Exploratory Research Center onLife and Living Systems (ExCELLS)

Innovation Group■Department of Creative Research■Division of Plant Environmental Responses■Laboratory for Biothermology

Research Support Facilities■Section for Exploration of Life inExtreme Environments
Research units of NIBB with an asterisk on theright panel also function as research groups of
ExCELLS.

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

International Research
Collaboration Center (IRCC)NIBB BioResource Center

Okazaki Research Facilities

NIBB Center of the Interuniversity Bio-Backup Project(IBBP Center)■■Model Plant Research Facility■Cell Biology Research Facility

Center for Radioisotope Facilities
Center for the Development of New Model Organisms
Center for Animal Resources and Collaborative Study
Research Center for Computational Science

Technical Division

Research Facilities run jointly by
NIBB and NIPS

Section of Health and Safety Management

Electron Microscopy Room
Waste Management Room
Instrument Design RoomOkazaki Administration Center3

■ Members of the Advisory Committee for Programming and Management (terms of April 2021 to March 2023)

External committee

Internal committee# Chairperson## Vice-Chair

4

KITANO, Jun
KYOZUKA, Junko
KUROIWA, AsatoSATAKE, Akiko
SIOMI, Mikiko C.
NIWA, Hitoshi
HANASHIMA, Carina
HIGASHIYAMA,Tetsuya
FUKUI, Manabu ##YOSHIMURA, Takashi
UEDA, Takashi
MORITA, Miyo T.
KAWAGUCHI, Masayoshi
TAKADA, Shinji
NIIMI, Teruyuki
HASEBE, Mitsuyasu
HIGASHIJIMA, Shin-ichi
FUJIMORI, Toshihiko
MINAGAWA, Jun #YOSHIDA, Shosei

Professor, National Institute of Genetics
Professor, Tohoku University
Professor, Hokkaido University
Professor, Kyushu University
Professor, Tokyo University
Professor, Kumamoto University
Associate Professor, Waseda University
Professor, Tokyo University
Professor, Hokkaido University
Professor, Nagoya University
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
Professor, National Institute for Basic Biology
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 fivegoals 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

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 NBRP9s work with Japanese morning glories
and Zebrafish (p. 101-102).

■ Inter-University Bio-Backup Project for Basic
Biology (IBBP)

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 NIBB9s facilities and in collaboration with NIBB9s 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 NIBB9s
laboratories to conduct collaborative research. The<Collaborative research projects for new model organism
development= and <Collaborative Research Projects for
Bioresource Preservation Technology Development= projects
are for developing and establishing new model organisms
and new research technology. Research and travel expenses
are provided for these projects. 8Collaborative Research
Projects for Integrative Genomics9 and 8Collaborative
Research Projects for Integrative Bioimaging9 are projects tofacilitate a more integrated use of the NIBB Core Research
Facilities and to allow more intensive support through theplanning, experimental, data analysis, and publication stages.
Travel and lodging expenses are also provided for theseprojects.
year■ National BioResource Project2019 2020 2021

Priority collaborative research projects

1

1

―

Collaborative research projects for newmodel organism development

―

2

3

Collaborative research projects for modelorganism and technology development

2

―

―

Individual collaborative research projects

60

46

42

Collaborative research projects for integrative
genomics

66

56

58

Collaborative research projects for integrative
bioimaging

22

19

24

NIBB workshops

3

2

4

Collaborative experiments using the Large
Spectrograph

9

9

11

Support for NIBB training courses

0

0

0

Collaborative research projects for bioresource
preservation technology development

14

10

10

Total

177

145

152

To prevent damage caused by natural disasters to importantbiological resources, NIBB has managed the Inter-University
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. 103-104).

■ 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. 116-117).

International Cooperation■ Collaborative Programs with Overseas Institutes
NIBB has formed an agreement with Center for Organismal
Studies (COS) Heidelberg to promote joint research projects,
collaborative symposia, and student exchange programs. The
2nd and 3rd meeting for NIBB-COS Heidelberg Internatinal
Collaborations were held in June, 2021 and March, 2022 (p.
112). In these meeting, several discussions on new collaborative research were put forward.
NIBB plays a leading role in collaborative research
programs between Princeton University and the National
Institutes of Natural Sciences (NINS) on the life science
research field. Collaborative research projects between NIBB
and Princeton University are conducted under the support of
the International Research Collaboration Center (IRCC) of
the NINS (p. 112). NIBB also promotes personal and technological exchange with the European Molecular Bioloigy
Laboratory (EMBL) based on the academic exchange agreement between the EMBL and the NINS.

■ 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 NIBB9s founding), NIBB Conferences have provided
researchers in basic biology with valuable opportunities for5international exchange. Unfortunately, this year9s NIBB conference was cancelled due to the COVID-19 pandemic.

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 master9s 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. 118).
Graduate Students Educated by NIBB
(numbers counted at the end of April each year)
60
50
40

11

13

37

6
11

9

39

36

36

2017

2018

2019

4

2

47

36

38

2020

2021

2022

30
20
10
0

2016

■ SOKENDAI Graduate Student■ Special Research Student■ Outreach
NIBB9s 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). NIBB also cooperates inthe education of undergraduate and young students throughlectures and workshops (p.119-123).

6

■ Personnel changes from April 2021 to March 2022*

Newly assigned to NIBB
Name

Position

Research Unit

Date

SUZUKI, Shinnosuke

Specially Appointed Assistant Professor

Division of Germ Cell Biology

April 1, 2021

OKUMA, Nao

Specially Appointed Assistant Professor

Division of Symbiotic Systems

April 1, 2021

YOKONO, Makio

Specially Appointed Assistant Professor

Division of Environmental Photobiology

July 1, 2021

MORITA, Shinichi

Specially Appointed Assistant Professor

Division of Evolutionary Developmental Biology

October 1, 2021

ARATA, Masaki

Specially Appointed Assistant Professor

Division of Embryology

October 1, 2021

KAWAGUCHI, Takayuki

Assistant Professor

Division of Chromatin Regulation

November 1, 2021

Change of Status in NIBBName

New Affiliation

Date

Position

NONOMURA, Keiko

Associate Professor

Division of Embryology

January 1, 2022

YOKONO, Makio

Associate Professor

Division of Environmental Photobiology

February 1, 2022

Newly affiliated with other universities and institutes
Name

New Affiliation

Date

Position

NONOMURA, KeikoTokyo Institute of Technology

Associate Professor

January 1, 2022

SHINOZUKA, Takuma

Nara Institute of Science and Technology Assistant Professor

January 1, 2022

ANDO, Toshiya

Kyoto University

Program-Specific Associate Professor

April 1, 2022

SAITO, Nen

Hiroshima University

Associate Professor

April 1, 2022

Mandatory Retirement
Name
UENO, Naoto

Position
Professor

Research Unit
Division of Morphogenesis

Date
March 31, 2022

■ Awardees from April 2021 to March 2022
Name

PositionAward

MORI, Tomoko

Chief (Technical Division)

MAKINO, YumikoVice-Unit Chief (Technical Division)

YAMAGUCHI, Katsushi

Vice-Unit Chief (Technical Division)

BINO, Takahiro

Technical Staff (Technical Division)

TOKUTSU, Ryutaro

Assistant Professor

SOYANO, Takashi

Associate Professor

FUJITA, Hironori

Assistant Professor

HASHIMOTO, Kayo

Postdoctoral Fellow

NAKAGAWA, Tomomi

Visiting Scientist

KANAZAWA, Takehiko

Assistant Professor

BSJ Young Botanist Prize, The Botanical Society of Japan Awards

KIM, Eunchul

Assistant Professor

10th NINS Award for Young Scientists

ANDO, Toshiya

Assistant Professor

Encouraging Prize, The Zoological Society of JapanNISHIUMI, Nozomi

JSPS Postdoctoral Fellow

Japan Ethological Society AwardFUKUDOME, Mitsutaka

JSPS Postdoctoral Fellow

MAEDA, Taro

Postdoctoral Fellow

Outstanding Support for Research Award,
The Commendation for Science and Technology by the Ministerof Education, Culture, Sports, Science and Technology

The Young Scientists9 Award, The Commendation for Science andTechnology by the Minister of Education, Culture, Sports,
Science and Technology

Journal of Plant Research Best Paper Award

Research Paper Award 2020 of The Microbes and Environments

KAWAGUCHI, Masayoshi Professor
NAGAHAMA, Yoshitaka

Professor Emeritus

The Order of the Sacred Treasure, Gold Rays with Neck Ribbon

HOSHINO, Atsushi

Assistant Professor

93rd Best Papers Award, The Genetics Society of Japan

AGATA, Kiyokazu

Director General

Science Lectureship Award 2021, Chiba University* Changes in professors, associate/ assistant professors are shown below.

7Selected Publications in 2021AY from NIBB
Transient suppression of transplanted spermatogonial stem celldifferentiation restores fertility in mice
Nakamura, Y., Jörg, D. J., Kon, Y., Simons, B. D., and Yoshida, S. (2021). Cell Stem Cell,
28, 1443-1456.e7.
DOI: 10.1016/j.stem.2021.03.016

Chloroplast acquisition without the gene transfer in kleptoplastic sea
slugs, Plakobranchus ocellatus
Maeda, T., Takahashi, S., Yoshida, T., Shimamura, S., Takaki, Y., Nagai, Y., Toyoda, A.,
Suzuki, Y., Arimoto, A., Ishii, H., Satoh, N., Nishiyama, T., Hasebe, M., Maruyama, T.,
Minagawa, J., Obokata, J., Shigenobu, S. (2021). eLife 10, e60176.
DOI: 10.7554/eLife.55108

Quantitative analyses reveal extracellular dynamics of Wnt ligands
in Xenopus embryos
Mii, Y., Nakazato, K., Pack, C. G., Ikeda, T., Sako, Y., Mochizuki, A., Taira, M. and Takada, S. (2021).
eLife 10, e55108.
DOI: 10.7554/eLife.60176

Structural basis of LhcbM5-mediated state transitions in green algae
Pan, X., Tokutsu, R., Li, A., Takizawa, K., Song, C., Murata, K., Yamasaki, T., Liu, Z.,
Minagawa, J., and Li, M. (2021). Nat. Plants 7, 111931131.
DOI: 10.1038/s41477-021-00960-8

Three-dimensional morphodynamics simulations of macropinocytic
cups
Saito, N., Sawai, S. (2021). iScience 24, 103087.
DOI: 10.1016/j.isci.2021.103087

A multistate stem cell dynamics maintains homeostasis in mouse
spermatogenesis
Nakagawa, T., Jörg, D. J., Watanabe, H., Mizuno, S., Han, S., Ikeda, T., Omatsu, Y.,
Nishimura, K., Fujita, M., Takahashi, S., Kondoh, G., Simons, B. D., Yoshida S. andNagasawa, T. (2021). Cell Reports 37, 109875.
DOI: 10.1016/j.celrep.2021.109875

8

Optogenetic relaxation of actomyosin contractility uncovers
mechanistic roles of cortical tension during cytokinesis
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada, S., Ueno, N.,
Sawai, S., Kondo, Y. and Aoki, K. (2021). Nat. Commun. 12, 7145.
DOI: 10.1038/s41467-021-27458-3

Near-infrared imaging in fission yeast using a genetically encodedphycocyanobilin biosynthesis system
Sakai, K., Kondo, Y., Fujioka, H., Kamiya, M., Aoki, K., and Goto, Y. (2021). J. Cell Sci. 134,
jcs259315.
DOI：10.1242/jcs.259315

Genomic and transcriptomic analyses of the subterranean termite
Reticulitermes speratus: gene duplication facilitates social evolution
Shigenobu, S., Hayashi, Y., Watanabe, D., Tokuda, G., Hojo, M.Y., Toga, K., Saiki, R.,
Yaguchi, H., Masuoka, Y., Suzuki, R., Suzuki, S., Kimura, M., Matsunami, M., Sugime,
Y., Oguchi, K., Niimi, T., Gotoh, H., Hojo, M.K., Miyazaki, S., Toyoda, A., Miura, T., andMaekawa, K. (2022). Proc. Natl. Acad. Sci. U.S.A. 119, e2110361119.
DOI: 10.1073/pnas.2110361119

Force-dependent remodeling of cytoplasmic ZO-1 condensates
contributes to cell-cell adhesion through enhancing tightjunctionsKinoshita, N., Yamamoto, T.S., Yasue, N., Takagi, C., Fujimori, T., Ueno, N.
(2022). iScience 25, 103846.
DOI: 10.1016/j.isci.2022.103846

A hemimetabolous wing development suggests the wing origin fromlateral tergum of a wingless ancestor
Ohde, T., Mito, T., and Niimi, T. (2022). Nat. Commun. 13, 979.
DOI: 10.1038/s41467-022-28624-x

Auxin methylation by IAMT1, duplicated in the legume lineage,
promotes root nodule development in Lotus japonicus
Goto, T., Soyano, T., Liu, M., Mori, T., Kawaguchi, M. (2022). Proc. Natl. Acad. Sci.
U.S.A. 119, e2116549119.
DOI: 10.1073/pnas.2116549119

Motion Illusion-like Patterns Extracted from Photo and Art Images
Using Predictive Deep Neural Networks
Kobayashi, T., Kitaoka, A., Kosaka, M., Tanaka, K., and Watanabe, E. (2022). Sci. Rep.
12, 3893.
DOI: 10.1038/s41598-022-07438-3

9

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

TAKEUCHI, Ikuo

MOHRI, Hideo

KATSUKI, Motoya

NAGAHAMA, Yoshitaka

OHSUMI, Yoshinori

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

Professor Emeritus

HORIUCHI, Takashi

OKADA, Kiyotaka

NISHIMURA, Mikio

YAMAMORI, Tetsuo

IGUCHI, Taisen

Professor Emeritus

Professor Emeritus

YAMAMOTO, Masayuki

NODA, Masaharu

Publication List of Prof. NAGAHAMA, Yoshitaka〔Original paper〕
R., Nozu, R., Mushirobira, Y., Amagai, T., Fushimi, J.," Murata,
Kobayashi, Y., Soyano, K., Nagahama, Y., and Nakamura, M. (2021).
Testicular inducing steroidogenic cells trigger sex change in groupers.
Sci. Rep. 11, 11117. DOI: 10.1038/s41598-021-90691-9

ࠝReview Article〕
Y., Chakraborty, T., Paul-Prasanth, B., Ohta, K., and" Nagahama,
Nakamura, M. (2021). Sex determination, gonadal sex differentiation,and plasticity in vertebrate species. Physiol. Rev. 101, 123731308. DOI:
10.1152/physrev.00044.201910

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
Postdoctoral Fellow:
TSUGANE, MikaNORIZUKI, Takuya
HIWATASHI, Takuma
FENG, Yihong
SOKENDAI Graduate Student: HACHINODA, Sho
Technical Assistant:
YAMAMOTO, Mayuko
YOSHINORI, Yumi
TANABE, Yoshimi
Admin Support Staff:
OKUBO, Masayo

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

Visual overview of this lab9s work.

model, Marchantia polymorpha. We also aim to elucidate
the detailed molecular mechanisms and physiological
functions of membrane trafficking in higher-orderedplant functions. In collaboration with other groups withexpertise in artificial intelligence, we are also developing aframework to deliver new findings in the basic biology field.

I. Diversification 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
families9 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 remained unclear for overa hundred years. We are studying the oil body in M. polymorpha, as a model of newly-acquired organelles in specificlineages during evolution. Through comprehensive analysesof SNARE members and organelle markers in M. polymorpha, we identified that a member of the SYP1 group, which
generally acts at the plasma membrane in the secretory
pathway, was targeted to the oil body. Together with otherlines of evidence, we concluded 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.
The morphology and distribution pattern of the oil body,e.g. its shape, color, number, and a density of oil body cells
in tissues diverge among liverwort species, which, therefore,
are regarded as important features for taxonomical classification in liverworts. However, molecular mechanisms ofoil body morphogenesis remain to be determined. We havesuccessfully isolated some mutants with abnormally shaped
oil bodies in M. polymorpha, a mutant of which harbors amutation in a subunit of the COPI coat complex (Figure 1).
Given that COPI acts around the Golgi apparatus and plays

11

National Institute for Basic Biology

Cell Biology

an essential role in secretion, our finding further supportsthat the liverwort oil body is formed by the redirection of thesecretory pathway.

Figure 1. COPI-mediated secretory activity is required for normal oil
body formation. The oil body is a complex and bumpy-surfaced structure
in wild type (A). In the Mpsec28-1 mutant, the oil body is more roundedwith a smoother-surface (B). The oil bodies were visualized using%2',3< JUHHQ %DUV ƫP

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.
Plant-specific features of the vacuolar transport werepartly achieved by acquisition of a plant-unique SNARE
protein, VAMP727. VAMP727 executes membrane fusion
between the multivesicular endosome and vacuole, assistedby the CORVET tethering complex. After execution of themembrane fusion, VAMP727 must be recycled back fromthe vacuolar membrane for sustainable vacuolar transport.
However, mechanisms of recycling from the plant vacuolar
membrane remain totally unknown. By using VAMP727 asa model cargo, we are currently exploring molecular mechanisms of recycling from the vacuolar membrane. We alreadysucceeded in isolating several candidates that could act inthis trafficking pathway.

12

II. Significance of membrane trafficking inhigher-ordered plant functions
2-1 Functions 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. The ANTH group has 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.

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)

In addition to PICALM5, PICALM2, PICALM6, andPICALM9 are also highly expressed in A. thaliana pollen and/or pollen tubes, which suggests that these PICALMs underpin
the complex endocytosis system in pollen tubes. Recently, we
found that the picalm2a/2b double mutant exhibits defective
transmission of the mutation from male plants, which suggested that PICALM2 acts in endocytic transport of importantbut still unknown cargo(s) in pollen and/or pollen tubes. Now
we are investigating molecular mechanisms of endocytosis inpollen tubes with a special focus on clathrin adaptor proteins
including PICALM members.
Our results indicate that each PICALM protein regulates
endocytosis of distinctive cargo proteins, whose molecular mechanisms remain mostly unclear in plants. We are now investigating
the molecular function of PICALMs with a special focus on themechanisms of cargo recognition by PICALMs in A. thaliana.

2-2 Membrane trafficking in plant gametogenesis
Gametogenesis in plants also involves membrane traffickingmediated processes. We are analyzing molecular mechanismsof gametogenesis in A. thaliana and M. polymorpha, and arefocusing 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 4. Impaired reorganization of mitochondria in an autophagy-defective mutant of M. polymorpha. The wild-type spermatozoid possesses two
mitochondria (green), whereas a number of mitochondria remained in the
Mpatg5-1ge mutant spermatozoid. Blue: nuclei. Bars = 5 ȝPWe also found that spermiogenesis requires the proper
function of the ESCRTs complex, which mediates multivesicular endosome formation and sorting of membraneproteins to be degraded. These results indicate that highlyorganized autophagic and non-autophagic degradation play
crucial roles to accomplish spermiogenesis in M. polymorpha.
We are also analyzing the role of RAB GTPases in flagellaformation. Through a comprehensive analysis of RAB
GTPases in M. polymorpha, we found that a RAB GTPaseplays an essential role in generating fully functional flagella(Figure 5).

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

Distinct from seed plants, basal land plants including M.
polymorpha utilize the spermatozoid with two (or more)
motile flagella as the male gamete during sexual reproduction. We visualized the spermatozoid formation process,especially spermiogenesis, using fluorescently-tagged organelle markers in M. polymorpha. The majority of the endomembranous organelles, such as the Golgi apparatus, were
removed from maturing spermatozoid cells, and the plasma
membrane was also reorganized during spermiogenesis.
Inspection by transmission electron microscope and live-cell
imaging analyses also indicated that the number of degradative organelles such as the multivesicular endosome, vacuole,and autophagosome, is transiently increased during thisprocess. To reveal the molecular mechanisms of cytoplasm
removal and organelle remodeling, we have established theanalytical tools of autophagy in M. polymorpha (Norizuki
et al., 2019). M. polymorpha possesses core machineriesof autophagy with lower degrees of redundancy. The mutations in MpATG5 and MpATG7, which are key factors forautophagosome formation, affected the transportation ofcytosolic components to the vacuole for degradation.
Autophagy-defective mutants exhibited defects regarding cytoplasm removal, spermatozoid motility, and fertility. Althougha majority of organelles are removed during spermiogenesis, a
specific set of organelles, e.g. anterior and posterior mitochondria,
persists in mature spermatozoids (Figure 4), which implies that
there should be a mechanism for selective removal of unneededorganelles. By analyzing the effect of defective autophagy during
spermiogenesis in M. polymorpha, we found that spermiogenesis
occurs through multimodal autophagic degradation.

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.

Publication List:〔Original paper〕
T.L., Ueda, T., and Hara-Nishimura, I. (2021). Excess" Shimada,sterol accumulation affects seed morphology and physiology inArabidopsis thaliana. Plant Signal. Behav. 16, 1872217. DOI:
10.1080/15592324.2021.1872217

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
JSPS Postdoctoral Fellow: SUGIYAMA, Hironori
Research Staff:
EBINE, Emi
TANII, Ryosuke
GOTO, Yoko
SOKENDAI Graduate Student: MUKAI, Masaya
YAMAMOTO, Kei
SAKAI, Keiichiro
TSURUOKA, Tatsuki
ITOH, Thoma
Visiting Scientist:
ITO, Reina
Admin Support Staff:
ONODA, Kaorigene mutation is widely known to result in various pathologies, such as malignant tumors.
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 pathway9s 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 lab9s 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

14

With the advent of fluorescent proteins, it has become
possible to visualize kinetic reactions at the single-cell level.
Förster (or fluorescence) resonance energy transfer (FRET)
is a non-radiative process in which the excitation energy of adonor fluorophore is transferred to a nearby acceptor fluorophore. FRET-based biosensors allow us to detect PKA, ERK,
Akt, JNK, PKC, and S6K9s kinase activity in living cells at ahigh temporal and spatial resolution (Komatsu N, Mol. Biol.
Cell, 2011; Aoki K, Mol Cell, 2013; 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 (Figures 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, Mol Brain, 2021).

A DRD1iRFP

A

Biliverdin

B
HO1

R-GenGAR-DA
SP
FLAG

ICL1 ICL2

GAF

PAS

Fd

Fdox

Heme

C

B HeLa cells
SCH

⊿F/F0

0.2

Fdox
Fnr

Biliverdin(BV)

cpmApple

Basal

Fd

Fnr

ICL3

R-GenGAR-DA1.2
DA

PcyA

DMSO

Phycocyanobilin(PCB)

Biliverdin

Phycocyanobilin

DIC
+
iRFP

30.6

C Primary neurons
R-GenGAR-DA1.2
Basal

DA

iRFP
SCH

0
⊿F/F0

0.3

50000

Figure 3. Phycocyanobilin (PCB) as a brighter chromophore for iRFP.
(A) Schematic representation of iRFP with its chromphore Bliverdin. (B)The metabolic pathway of PCB. (C) DIC and iRFP fluorescence images
are shown in fission yeats treated with DMSO, Biliverdin, or PCB.

30.8

Figure 2. Development of a genetically encoded red fluorescent dopamine(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.

Near-infrared fluorescent protein (iRFP) is a fluorescentprotein with near-infrared excitation and emission maxima.
Unlike the other conventional fluorescent proteins, iRFP
requires biliverdin (BV) as a chromophore (Figure 3A). We
accidentally found that phycocyanobilin (PCB) functions asa brighter chromophore for iRFP than BV and that biosynthesis of PCB allows live-cell imaging with iRFP in the fission
yeast Schizosaccharomyces pombe (Figure 3B). We initiallyfound that fission yeast cells did not produce BV and therefore did not show any iRFP fluorescence (Figure 3C). The
brightness of iRFP3PCB was higher than that of iRFP3BV
both in vitro and in fission yeast. We introduced SynPCB2.1,
a PCB biosynthesis system (Uda, Miura, ACS Chem Biol,
2020), into fission yeast, resulting in the brightest iRFP
fluorescence. These tools not only enable the easy use ofmultiplexed live-cell imaging in fission yeast with a broader
color palette, but also open the door to new opportunities
for near-infrared fluorescence imaging in a broader range ofliving organisms (Sakai, JCS, 2021).

II. Manipulation of cell signaling
Artificial manipulation of biochemical networks is wellestablished in the quantitative understanding of biologicalsystems. Multiple methods have been suggested for control-ling 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 relaxationof 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 rangeof biological processes such as cell motility and cytokinesis. Yet,it remains elusive as to how actomyosin contractility generates
force and maintains cellular morphology. To elucidate this, wehave developed a novel optogenetic tool, OptoMYPT, which
combines a catalytic subunit of type I phosphatase-binding
domain of MYPT1 with an optogenetic dimerizer so that itallows light-dependent recruitment of endogenous PP1c to theplasma membrane (Figure 4A). We confirmed that blue-lightillumination was sufficient to induce dephosphorylation of
MLC, resulting in lamellipodial membrane protrusion (Figure
4B). The OptoMYPT was further employed to understand themechanics of actomyosin-based cortical tension and contractile
ring tension during cytokinesis (Figure 4C). We found that therelaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, thus revealing that the corticaltension substantially antagonizes constriction of the cleavage
furrow. These results provide new opportunities to freely design
cell and tissue morphology through light (Yamamoto, Nat
Comm, 2021).

15

National Institute for Basic Biology

Cell Biology

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.

Publication List:
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).

The Blue light-responsive optical dimerizers are useful, but its
activation light often overlaps with the excitation light of fluorescent proteins, which in turn hampers the application of GFPor FRET biosensors together with their optogenetic tools. To
circumvent this issue, we focus on the phytochrome B (PhyB)PIF LID system. Upon red-light illumination, PhyB binds to
PIF, and the PhyB-PIF complex dissociate from each other by
far-red light exposure. One drawback of PhyB-PIF LID is thata chromophore, e.g., PCB, is required for the light sensing. To
overcome this issue, we have developed a method for PCB
biosynthesis, called SynPCB, in mammalian cells by introducing the gene products of HO1, PcyA, Fd, and Fnr into the mitochondria (Uda Y, PNAS, 2017; Uda Y, ACS Chem Biol, 2020).
To take full advantage of this system, we applied theSynPCB system to S. pombe and C. elegans, both of which
are incapable of having purified PCB delivered to them.
As we expected, the expression of HO1, PcyA, Fd, and Fnr
genes induced PCB biosynthesis in S. pombe and C. elegans.
Using this system, we developed optogenetic control of thecell 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 response to red light (Figure5) (Goto Y, and Aoki K., bioRxiv, 2020).

16〔Original papers〕
H., Ogoh, K., Kondo, Y., and Aoki, K. (2022). LIM Tracker:" Aragaki,a software package for cell tracking and analysis with advanced

"
"
"
"
"
"
"

interactivity. Sci. Rep. 12, 2702. DOI: 10.1038/s41598-022-06269-6
Ito, R.E., Oneyama, C., and Aoki, K. (2021). Oncogenic mutation oroverexpression of oncogenic KRAS or BRAF is not sufficient to confer
oncogene addiction. PLoS One 16, e0249388. DOI: 10.1371/journal.
pone.0249388
Kajiwara, K., Yamano, S., Aoki, K., Okuzaki, D., Matsumoto, K., andOkada, M. (2021). CDCP1 promotes compensatory renal growth byintegrating Src and Met signaling. Life Sci. Alliance 4, e202000832.
DOI: 10.26508/lsa.202000832
Lin, S., Hirayama, D., Maryu, G., Matsuda, K., Hino, N., Deguchi, E.,
Aoki, K., Iwamoto, R., Terai, K., and Matsuda, M. (2022). Redundantroles of EGFR ligands in the ERK activation waves during collective
cell migration. Life Sci. Alliance 5, e202101206. DOI: 10.26508/
lsa.202101206
Matsui, K., Okamoto, K., Hasegawa, T., Ohtsuka, H., Shimasaki, T.,
Ihara, K., Goto, Y., Aoki, K., and Aiba, H. (2021). Identification of ksg1
mutation showing long-lived phenotype in fission yeast. Genes to Cells
26, 9673978. DOI: 10.1111/gtc.12897
Nakamoto, C., Goto, Y., Tomizawa, Y., Fukata, Y., Fukata, M., Harpsoe,K., Gloriam, D.E., Aoki, K., and Takeuchi, T. (2021). A novel red
fluorescence dopamine biosensor selectively detects dopamine in thepresence of norepinephrine in vitro. Mol. Brain 14, 173. DOI: 10.1186/
s13041-021-00882-8
Nakayama, H., Rowland, S.D., Cheng, Z., Zumstein, K., Kang,J., Kondo, Y., and Sinha, N.R. (2021). Leaf form diversificationin an ornamental heirloom tomato results from alterations in two
different HOMEOBOX genes. Curr. Biol. 31, 4788+. DOI: 10.1016/j.
cub.2021.08.023
Sakai, K., Kondo, Y., Fujioka, H., Kamiya, M., Aoki, K., and Goto,

"
"

"

Y. (2021). Near-infrared imaging in fission yeast using a geneticallyencoded phycocyanobilin biosynthesis system. J. Cell Sci. 134,
jcs259315. DOI: 10.1242/jcs.259315
Verma, A., Jena, S.G., Isakov, D.R., Aoki, K., Toettcher, J.E., andEngelhardt, B.E. (2021). A self-exciting point process to study
multicellular spatial signaling patterns. Proc. Natl. Acad. Sci. U.S.A.
118. e2026123118. DOI: 10.1073/pnas.2026123118
Yagi-Utsumi, M., Aoki, K., Watanabe, H., Song, C., Nishimura, S.,
Satoh, T., Yanaka, S., Ganser, C., Tanaka, S., Schnapka, V., Goh, E.W.,
Furutani, Y., Murata, K., Uchihashi, T., Arakawa, K., and Kato, K.
(2021). Desiccation-induced fibrous condensation of CAHS proteinfrom an anhydrobiotic tardigrade. Sci. Rep. 11. 21328. DOI: 10.1038/
s41598-021-00724-6
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada,S., Ueno, N., Sawai, S., Kondo, Y., and Aoki, K. (2021). Optogenetic
relaxation of actomyosin contractility uncovers mechanistic roles ofcortical tension during cytokinesis. Nat. Commun. 12. 7145. DOI:
10.1038/s41467-021-27458-3〔Review Articles〕
P.E., Reed, E.H., Underhill, E.J., Aoki, K., and Toettcher, J.E.
" Farahani,(2021). Signaling, Deconstructed: Using Optogenetics to Dissect and""Direct Information Flow in Biological Systems. Annu. Rev. Biomed.
Eng. 23, 61387. DOI: 10.1146/annurev-bioeng-083120-111648
Hiiragi, T., Gladfelter, A.S., Miguel-Aliaga, I., Coller, H.A., Lau, O.S.,
McBride, H.M., Aoki, K., Linkermann, A., Santaguida, S., Wasteneys,G., Rivron, N.C., Shahbazi, M.N., Ebisuya, M., and Lancaster, M.A.
(2021). 20 years of Developmental Cell: Looking forward. Dev. Cell
56, 318533191. DOI: 10.1016/j.devcel.2021.11.017
Nakamura, A., Goto, Y., Kondo, Y., and Aoki, K. (2021). Shedding lighton developmental ERK signaling with genetically encoded biosensors.
Development 148. dev199767. DOI: 10.1242/dev.199767

17

National Institute for Basic Biology

Cell Biology
DIVISION OF CHROMATIN REGULATION

Professor

NAKAYAMA, Jun-ichi

Assistant Professor:

KATAOKA, Kensuke
KAWAGUCHI, Takayuki
Specially Appointed Assistant Professor:
HAYASHI, Aki
Technical Staff:
NISHIMOTO, Yuki
SOKENDAI Graduate Student: RAHAYU, Anisa
VALENTIROVIC, Olivera
NAKAMURA, Rinko
YOSHIDA, Takaki
OYA, Tomoyuki
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
from higher-order chromatin structure plays a fundamentalrole in this process. The assembly of higher-order chromatin
structures has been linked to the covalent modifications ofhistone tails, chromatin-associated proteins, and non-coding
RNAs. However, the exact means by which this chromatinbased epigenetic information is established and faithfully
maintained across cell divisions and throughout development remains unclear. To try to gain a better understandingof the molecular mechanisms underlying chromatin-based
epigenetic phenomena, our lab uses model systems including
mammalian culture cells, fission yeast, Schizosaccharomyces
pombe (S. pombe), and ciliate Tetrahymena for studying the18

assembly of higher-order chromatin structures, known asheterochromatin. We are also attempting to determine thecellular functions of chromatin modifying factors so that we
can develop a better understanding of complicated epigenetic
phenomena in higher eukaryotic cells.

I. Establishment and maintenance of higherorder chromatin structures
1-1 Mechanisms regulating Clr4 histone methyltransferase activity
In eukaryotic cells, the assembly of heterochromatin plays
an important role in diverse chromosomal processes andepigenetic gene regulation. Heterochromatin is characterized by the methylation of histone H3 at lysine 9 (H3K9me).
H3K9me is catalyzed by SUV39H-family histone methyltransferases, and functions as a binding site for recruiting
heterochromatin protein 1 (HP1) family proteins. In fission
yeast, H3K9me is catalyzed by histone methyltransferase Clr4. Clr4 has two functional domains, the N-terminal
chromodomain (CD), which recognizes H3K9me, and the
C-terminal SET domain responsible for Clr49s enzymatic
activity (Figure 1A). Since Clr49s uncontrolled activity leads
to inappropriate H3K9me and aberrant gene silencing, its
enzymatic activity needs to be strictly controlled. However,the detailed mechanisms regulating Clr49s activity are poorly
understood.
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 the activity of C-terminal SET domain. To examine theregulation mechanisms of Clr49s activity, we tested whether
Clr4 N-terminal region interacts with its C-terminal SET
domain. Pull-down assays using recombinant proteins revealthat the Clr4 N-terminal region physically interacts with
the SET domain and that this interaction requires both CD
and its adjacent region. To determine the regions/residues
responsible for this interaction, we performed crosslinking

Figure 1. Characterization of intramolecular interactions of Clr4. (A) Domainorganization of Clr4. The N-terminal chromodomain (CD) recognizes
H3K9me, and the C-terminal SET domain is responsible for Clr49s enzymatic activity. (B) Crosslinking mass spectrometry. After in vitro crosslinking
reaction, recombinant Clr4 proteins were separated by gel-filtration chromatography and analyzed by SDS-PAGE and silver staining. (C) Residues identified in the cross-linking mass spectrometry to be involved in the interaction
are mapped on 3D structure of Clr4 SET domain.

mass spectrometry (Figure 1B) and found that crosslinked
residues were concentrated in a region near the C-terminalend (Figure 1C). Interestingly, mutant Clr4 proteins containing amino-acid substitutions at this region exhibit higher
methyltransferase activity. These results suggest that Clr49s
enzymatic activity is negatively regulated by intramolecularinteractions.

II. Roles of HP1 family proteins in assemblinghigher-order chromatin structure
2-1 Characterizing the role Chp2 in heterochromatin
assembly in fission yeast
Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds H3K9me, and plays
a crucial role in forming higher-order chromatin structure.
HP1 family proteins share a basic structure consisting ofan N-terminal CD and a C-terminal chromoshadow domain(CSD) linked by an unstructured henge region (Figure2A). The CD functions as a binding module that target
H3K9me, whereas CSD functions as a dimerization modulethat provides a recognition surface to bind and recruit otherproteins. In fission yeast, two HP1 family proteins, Swi6
and Chp2, are involved in heterochromatic silencing. While
Swi6 is expressed abundantly and plays a dose-dependentrole, Chp2 is expressed at a lower level and displays a novel
ability to tightly bind chromatin-enriched fraction independently of H3K9me. To dissect how this Chp2-specificability contributes to its function, we performed Tandem
Affinity Purification (TAP) to identify Chp2-specific binding
partners and successfully identified novel candidates for
Chp2 binding partners. Furthermore, we examined Chp2specific DNA-binding activity by Electrophoretic Mobility
Shift Assay (EMSA) and found that both the hinge andCSD neighboring regions make distinct contribution to its
DNA binding. These results suggest that both the Chp29s
property to bind specific binding partners and its intrinsic
DNA binding ability contribute to its tight chromatin association and also to division of labor between Swi6 and Chp2.

2-2 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, theinterplay between these HP1 proteins has been elusive. Here,
we show that subset of the HP1 proteins form a complex
and play important roles to eliminate TE-related InternalEliminated Sequences (IESs) from the somatic genome
during macronuclear development in the ciliated protozoan
Tetrahymena. We tethered 7 HP1-like proteins individuallyto the artificially created locus by the LexA-LexO systemand found that only 4 of them including Pdd1 induced theelimination of the tethered site. This ectopic DNA elimination was exclusively achieved by their chromoshadow
domains (CSDs), indicating that the CSDs of the distinct typeof HP1-like proteins recruits all the proteins that are required
for DNA elimination. Immunoprecipitation of Pdd1 specifically enriched the other HP1-like proteins that were sufficientfor the ectopic DNA elimination. The chromodomains of asubset of HP1-like proteins showed strong affinity to both
H3K9me3 and H3K27me3. Overall, these results suggest thatmultiple HP1-like proteins cooperatively recognize the methylated histones and form core complex to recruit other effector
proteins for DNA elimination (Figure 3).

Figure 3. Model for how multiple HP1-like protein-containing complex
regulates DNA elimination in Tetrahymena.

2-3 The HP1-like protein Hpl8p is important for programmed DNA elimination in Tetrahymena

Figure 2. Chp2 possesses a strong DNA/RNA-binding activity. (A)Schematic diagram of Swi6 and Chp2. The chromodomain (CD) and thechromoshadow domains (CSD) are shown. (B) Representative results of
EMSAs performed with control and mutant Chp2.

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 coordinated by heterochromatin components, we focus on an
HP1-like protein named Hpl8p that localizes to the developing macronucleus during heterochromatin formation and
DNA elimination (Figure 4). The chromodomain of Hpl8p
recognized both H3K9me3 and H3K27me3 in vitro. HPL8
disrupted mutants showed defect in vegetative growth and in
the progression of sexual reproduction. FISH analyses using
a probe against one of the transposons showed that more than19

National Institute for Basic Biology

Cell Biology

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 H3K9/27 methylation 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.

Figure 5. Analysis of chromatin dynamics in spore nuclei of fission yeast.
(A) A representative image of vegetative cells (veg.) and spores in asci. (B)Heatmap representation of proteins detected in spores and vegetative cells.

Publication List:〔Original paper〕
Figure 4. 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.

III. 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 (Figure 5A). We
isolated fission yeast spores by density-gradient centrifugation and performed mass spectrometry analysis to determine
spore-specific nuclear proteins (Figure 5B). We further
examined the change in histone modifications of spore
nuclei. Chromatin immunoprecipitation analysis revealed
that the levels of H3K9me at heterochromatic regions had
not been noticeably changed, but the levels of H3K4me associated transcriptionally active regions had clearly increasedin spore nuclei. We also found that H3K4me distribution atcoding regions in spore genome clearly different from thatof control vegetative cells. These results imply that dynamicchanges in active histone marks contribute to spore formation and epigenetic inheritance.

20

R., and Nakayama, J. (2022). Multiple interfaces to" Nakamura,recognize nucleosomal targets. J. Biochem. 171, 2573259. DOI:
10.1093/jb/mvab139

LABORATORY OF NEURONAL CELL BIOLOGY

Associate Professor

SHIINA, Nobuyuki

Assistant Professor:
OHASHI, Rie
SOKENDAI Graduate Student: YAMASHITA, Akira
HORIO, Tomoyo
ISHIKURA, Yui
YOSHIDA, Sho

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 accumulation 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. Prion-like domain of ILF3 regulates gene
expression and fear memory under chronic
stressRNA granules (green) transported from the cell body (magenta) to dendrites in the hippocampus of the mouse brain.

The transport of specific mRNAs and local control of translation in neuronal dendrites are part of an important gene
expression system that provides dendritic protein synthesis
at exactly the right time and place. It is widely accepted that
this system controls the location at which neurites will stably
connect to each other, thereby forming long-term neuralnetworks and memory. Our main interest is understanding the mechanisms and roles of mRNA transport and 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 stimulation-dependent local translation of the cargo mRNAs (Figure1). We are currently using mice to research the mechanism of

LLPS is generally driven by protein intrinsically disordered regions (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
extensively studied in the context of neurodegenerative
diseases. However, little is known about the physiologicalrelevance 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 the alternative splicing isoform NFAR1. NFAR1and NFAR2 are synthesized from the Ilf3 gene, andboth possess biochemical activities that they perform incommon such as transcriptional regulation and stressdependent translational inhibition. However, they differ
in that NFAR2 alone can concentrate in the nucleoplasm
and cytoplasmic RNA granules through PrLD. To investigate the physiological relevance of PrLD, we generated
NFAR2ǻPrLD mice, in which a stop codon was introduced into the NFAR2-specific exon. This is considered
an ideal strategy for revealing the physiological role
specific to PrLD, as the splicing isoform without PrLD
is intrinsically present and maintained even after PrLD
deletion.
Deletion of the PrLD lost the nucleoplasmic localizationof NFAR1/2 and affected the genome-wide profiles of21

National Institute for Basic Biology

Cell Biology

mRNA expression and translation in the brain. In particular, PrLD deletion significantly altered chronic restraint
stress (CRS)-induced changes in mRNA expression andtranslation in the amygdala, a brain region associated withemotional events such as anxiety and fear.
Consistent with the effects on CRS-induced changes inmRNA expression and translation, PrLD deletion affected
tolerance of mice to CRS in amygdala-associated learningand memory: conditioned fear memory in wild-typemice was unaffected by CRS, but it was exacerbated in
NFAR2ǻPrLD mice due to CRS. These results indicated that
PrLD of NFAR2 is responsible for the regulation of stressresponsive gene expression and confers stress tolerance infear-associated memory formation, providing insights into
PrLD9s involvement in chronic stress adaptation.

II. Novel regulation of RNA granule dynamics
by pathogenic accumulation of FUS and
TDP-43 in RNA granules
FUS and TDP-43 are predominantly localized in the
nucleus when cells are healthy, but in neurodegenerative
diseases, they translocate to the cytoplasm and accumulate in
RNA granules. FUS and TDP-43 prone to aggregate in RNA
granules, which is thought to harden RNA granules, affectthe function of RNA granules, and impair synapse formationand function. However, little is known about the effects of
FUS and TDP-43 on the dynamics of other components of
RNA granules.
We overexpressed disease-related FUS and TDP-43 mutantsin primary cultured neurons from the mouse cerebral cortex
and analyzed their effects on the dynamics of RNA granulecomponents using fluorescence recovery after photobleach-w/ TDP-43

w/ FUS

w/o FUS or TDP-43

RNG105
SunTag (Translation)

RNG105 SunTag Merge

FUS

III. Transport and local translation of mRNAs
encoding the regulators of the small G
protein Arf in neuronal dendrites
We have previously reported that RNG105 knockout in mice
reduces mRNA transport to dendrites and impairs synaptic
long-term potentiation and long-term memory (Nakayama etal., eLife, 2017). Gene ontology enrichment analysis of mRNA
with reduced transport to dendrites in RNG105 knockout mice
revealed that a group of mRNAs encoding regulators of thesmall G protein Arf (Arf GAPs and GEFs) was remarkably
reduced in amount in the dendrites of RNG105 knockout
neurons. Of these mRNAs, Psd mRNA, which encodes an Arf
GEF, was most significantly reduced. In order to clarify the
biological significance of dendritic transport of Psd mRNA,
we are aiming to generate mice in which Psd mRNA transport
can be artificially controlled.
In this study, we identified the 39UTR of Psd mRNA as a
cis-element responsible for the transport to dendrites. WhenPsd mRNA lacking the 39UTR was expressed in mouseprimary cultured neurons, the transport of that mRNA to
dendrites was reduced. However, it was revealed that the
39UTR was required not only for dendritic transport but also
for translation. Therefore, we narrowed down the responsible
regions for the transport and translation in the 39UTR, andfound that 59 half and 39 half of the 39UTR were required
for transport and translation, respectively. Based on theseresults, we are proceeding with the generation and analysisof mice in which only the 59 half of the 39UTR of Psd mRNAis deleted to reduce only mRNA transport while maintaining
the translation level of Psd mRNA.

Publication List:
TDP-43Figure 2. Accumulation of FUS and TDP-43 reduces translation in/near
RNG105-localizing RNA granules in dendrites. Primary cultured neurons
expressing RNG105-mRFP1 with and without co-expression of FUSSirius or TDP-43-Sirius were analyzed using the SunTag system to detect
translation activity spatiotemporally. The boxed areas containing dendritic RNA granules are magnified in the right panels. The dotted lines
outline the RNA granules. Scale bar, 10 ȝm.

22

ing (FRAP) and cell permeabilization assays (Shiina, J. Biol.
Chem., 2019). FUS and TDP-43 did not affect the dynamics
of most RNA granule components such as Pum2 and Stau1
in RNA granules in dendrites, but did affect the dynamics
of RNG105 (Caprin1). In contrast to the belief model that
FUS and TDP-43 harden RNA granules, the mobility of
RNG105 was significantly increased, resulting in the releaseof RNG105 from RNA granules into the cytoplasm of dendrites. Simultaneously, the amount of mRNAs contained in
RNA granules decreased, and local translation in/near the
granules also decreased (Figure 2). Since RNG105 is responsible for mRNA recruitment to RNA granules and mRNAtransport to dendrites, increased mobility and loss of localization of RNG105 in dendritic RNA granules may affectthese functions, leading to impaired synapse formation andcognitive function.

〔Original papers〕
T., Okimura, K., Shen, J., Guh, Y.-J., Tamai, T.K., Shimada," Nakayama,A., Minou, S., Okushi, Y., Shimmura, T., Furukawa, Y., Kadofusa,N., Sato, A., Nishimura, T., Tanaka, M., Nakayama, K., Shiina, N.,
Yamamoto, N., Loudon, A.S., Nishiwaki-Ohkawa, T., Shinomiya,A., Nabeshima, T., Nakane, Y., and Yoshimura, T. (2020). Seasonalchanges in NRF2 antioxidant pathway regulates winter depression-likebehavior. Proc. Natl. Acad. Sci. U.S.A. 117, 959439603. DOI: 10.1073/
pnas.2000278117

K., Shichino, Y., Iwasaki, S., and Shiina, N. (2020).
" Nakazawa,
Implications of RNG140 (caprin2)-mediated translational regulationin eye lens differentiation. J. Biol. Chem. 295, 15029315044. 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

23

National Institute for Basic Biology

Cell Biology
LABORATORY OF STEM CELL BIOLOGY

Associate Professor

TSUBOUCHI, Tomomi

Specially Appointed Assistant Professor:
KURASHIMA, Kiminori
SOKENDAI Graduate Student: KUMAZAKI, Taisei
MATSUMOTO, Akino
Technical Assistant:
ASAI, Yuriko
NAGANUMA, Maipresence of replication obstacles. However, the direct cause
and the underlying mechanism remains to be uncovered. 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 cells.

II. Induction of Pluripotency through Nuclear
Reprogramming and Role of Cell Cycle
Regulation

Pluripotent stem cells (PSCs) are defined as stem cells
capable of producing all cell types that compose our body.
In vivo, PSCs appear only transiently during the early stageof development and are lost as development proceeds.
Molecular features that underlie pluripotency have been
extensively studied, and accumulating evidences suggest thatthe unique cell cycle regulation of PSC is tightly linked tothe maintenance of pluripotency. Curiously, they proliferatewith truncated gap phases while lengths of the S (DNA replication) and M (mitosis) phases remain similar to non-pluripotent cell types. In particular, the G1 phase of PSCs needs tobe kept short to maintain pluripotency: artificial elongationof the G1 phase makes them susceptible to differentiation.
On the other hand, active proliferation with truncated gap
phases can cause cellular stress as in some cancer cells,
leading to genome instability. Our laboratory is interested in
the role of cell cycle regulations in the maintenance of pluripotency and genome integrity of PSCs.

In mammals, PSCs are not maintained in a fully-developedorganism. However, differentiated cells can regain pluripotency upon experimental trigger, albeit at a low efficiency.
Factors that limit active reprogramming, and conditions
that potentiate reprogramming, are the subjects under active
investigation.
One of the main obstacles when investigating molecularmechanism underlying reprogramming is the time it takes tostart seeing any sign of reprogramming. In order to overcome
this problem, 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,
in a short time frame. The cell fusion system is a simple, versatile way to induce reprogramming towards another lineage,
and is not limited to pluripotency. Using this system, the firstsign of reprogramming can be detected within one day after
fusion, thus allowing us to monitor the initial events of reprogramming after induction.
Using this system, we previously found that DNA synthesis is an important event for successful reprogramming(Tsubouchi et al., Cell, 2013). Recent reports indicate that
reprogramming may cause genetic instability, some of which
are thought to arise as DNA replication errors. To investigate
the nature of such errors and how they are linked to repro-

Visual overview of this lab9s 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=). Recent reports suggest that,compared to other cell types, DNA replication machineryof ES cells proceed at lower speed, a feature generally infer

24

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).

gramming-specific events, we have set up a system to isolate
and track a single fused cell (Figure 1) through live-imaging.

III. Future Perspectives
While the fundamental mechanisms that maintain genome
integrity have been widely studied using various models, thedanger a cell might face when its identity is being altered(through differentiation, reprogramming etc.) are largely
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 gain a comprehensive understanding of how
genome integrity is maintained in ES cells and cells undergoing reprogramming.

Publication List:〔Review article〕
T., and Pereira, C.-F. (2021). Reprogramming Stars #1:" Tsubouchi,
Genome Programming Through the Cell Cycle-An Interview with Dr.
Tomomi Tsubouchi. Cell. Reprogram. 23, 1533157. DOI: 10.1089/
cell.2021.29039.tt

25

National Institute for Basic Biology

Cell Biology
LABORATORY OF ORGANELLE REGULATION

Associate Professor

MANO, Shoji

Specially Appointed Assistant Professor:
KANAI, Masatake
Visiting Scientist:
KAMIGAKI, Akane
OIKAWA, Kazusato
Technical Assistant:
HIKINO, Kazumi
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 utilized various signals generated from environmental changes as being necessary for their survival. As
such, the flexibility 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.

I. Molecular 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 `-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 demonstratethat peroxisomes contribute to pollen fertility, pollen tube
elongation, and male3female gametophyte recognition. Gene
expression, alternative splicing, protein transport, protein
degradation and degradation of peroxisomes themselvescontrol these functions.
To better understand peroxisome biogenesis and functions, we isolated a number of Arabidopsis mutants that
displayed aberrant peroxisome morphology (apem mutants)
and unusual peroxisome positioning (peup mutants) basedon them having a different pattern of GFP fluorescencecompared to their parent plant, GFP-PTS1, in which peroxisomes with normal sizes, numbers and distribution
could be visualized with GFP (Figure 1). As of writing, wehave reported the functions of APEM1, APEM2, APEM3,
APEM4, APEM9 and APEM10. Based on these results wewere 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, peup4, peup17 and peup22 mutants, which were
defective in Autophagy-related (ATG) genes.
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.

Visual overview of this lab9s work.
Figure 1. Phenotype of Arabidopsis apem11 mutants. (A) GFP fluorescence was observed in the parent plant, GFP-PTS1, and apem11 mutants.
The apem11 mutant has enlarged peroxisomes in leaf cells, but not inroot cells. Bars, 20 µm. (B) The apem11 mutant showed dwarfism undernormal atmosphere, but grew normally under high CO2 atmosphere as
did the GFP-PTS1 plant.

26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
these storage reserves in food and industrial materials.
Storage reserves vary among different types of plantseeds. Wheat, maize and rice seeds mainly accumulate
starch, whereas rapeseed, pumpkin and sesame containlarge amounts of oils. Soybeans9 major reserve areproteins. Storage oils and proteins are synthesized in theendoplasmic reticulum (ER) and accumulated in oil andprotein bodies, respectively, during seed development.
We are currently analyzing the molecular mechanisms
controlling oil and protein contents in seeds (Figure 2).
Based on the analysis of the temporal sequence of oil andprotein synthesis during seed development in Arabidopsis
thaliana, which produces seeds containing approximately
30% oil and 30% protein, we revealed that the extensionof 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
of fatty acids by degrading particular fatty acids in oil
bodies.

Lipid synthesis

IV. Construction 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, theorganellome database, the functional analysis database,and external links. The function of each database is asfollows:
・The electron micrograph database provides information
concerning the ultrastructures in plant cells
・The 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 atdifferent stages of development.
・The functional analysis database is a collection of protocols for plant organelle research
Through these databases, users can more easily comprehend plant organelle dynamics. Plant Organelles World,which is built based on PODB3, is an educational toolfor engaging members of the non-scientific community
to explore plant biology. We hope that both PODB3 andPlant Organelles World are of help to researchers as wellas 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. Development 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.

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

27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 laboratory9s works.

28

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 the

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.

plasma membrane in E4.5 embryos became much higher
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).

In contrast, migrating cells around the wound site, ones
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-19s 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. Interaction 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 in
D GLIIHUHQW ZD\ ZH FRQVWUXFWHG *)3=2Ƌ$%' ZKLFKlacks the actin-binding domain (ABD) (Figure 2A), andH[SUHVVHG LW LQ$ FHOOV *)3=2Ƌ$%' VXEVHTXHQWO\formed droplets with a smooth surface (Figure 2C). Thissuggests that cytoplasmic droplet formation is restricted by
WKH ELQGLQJ RI =2 WR )DFWLQ DQG WKDW *)3=2Ƌ$%'
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, binding to F-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.

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.

29National Institute for Basic Biology

Developmental Biology
III. Cephalochordate 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 59, 39 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 59 region for somite expression, the39 region for notochord expression, and introns for somite/
notochord expression. In contrast, sister BfBra1 became
more specialized for notochord expression using the intron
enhancers.

test whether the observed light response resulted in aggregation or dispersal under specific light fields.
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.

Figure 3. A summary of results of the present reporter assay and their
possible interpretation.
Enhancer activity of 59 upstream sequences in muscle is shown in brown
and that of the 39 downstream in notochord is in blue. The activity of
BfBra2 introns in muscle and notochord is shown in purple.

IV. A step-down photophobic response inlarvae of the common reef coral, Acropora
tenuisMany reef-building corals form a symbiotic relationship with dinoflagellate algae of the genus Symbiodinium.
Corals mostly depend on photosynthetic products fromthese symbionts as their energy source, and thus light conditions in habitats can influence post-settlement survival.
Previous studies reported that light environments play anessential role in larval habitat selection. However, due to alack of basic photobiological studies in corals, how corallarvae perceive and respond to the light in their environment
remains largely unknown. To answer these questions, weanalyzed the swimming behavior of larvae of the common
reef coral Acropora tenuis (Figure 4) under various light conditions. In addition, we developed a mathematical model to30

Publication List:〔Original papers〕
N., Yamamoto, T.S., Yasue, N., Takagi, C., Fujimori, T., and" Kinoshita,
Ueno, N. (2022). Force-dependent remodeling of cytoplasmic ZO-1"

condensates contributes to cell-cell adhesion through enhancing tightjunctions. iSCIENCE 25, 103846. DOI: 10.1016/j.isci.2022.103846
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada,S., Ueno, N., Sawai, S., Kondo, Y., and Aoki, K. (2021). Optogenetic
relaxation of actomyosin contractility uncovers mechanistic roles ofcortical tension during cytokinesis. Nat. Commun. 12, 7145. DOI:
10.1038/s41467-021-27458-3

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
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
SAKAI, Kimiko
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. Thesemolecules are secreted from the cells that produce them andtransported to surrounding cells, thus resulting in the formation of concentration gradients. Given that their concentration decreases in accordance with their distance from thesource, their specific signal gradient defines the relative positions of receiving cells in developing tissues. Many genetic
studies have revealed that a limited number of secreted signal
proteins, including Wnt, BMP, and Hedgehog, function
during tissue and embryo morphogenesis. However, in spiteof the accumulation of genetic evidence, the molecularmechanism that regulates their distribution in certain developing tissues 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.

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).

I. Regulation of spatial distribution and functionof Wnt proteins in vertebrates

Visual overview of this lab9s work.

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 Fluorescence31National Institute for Basic Biology

Developmental Biology

Correlation Spectroscopy (FCS). Several lines of evidenceshow 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).

Figure 2. Fluorescence decay after photoconversion (FDAP) at cell coundaries. A Photoconvertable fluorescent protein, mKikGR can be switch its fluorescence from green to red. Decay of red (photoconverted) fluorescence canbe fitted with a model of dissociation from cell surface scaffolds.

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 amember of sFRPs, Frzb, both of which are involved in theanteroposterior patterning of the vertebrate embryo. While
Venus-tagged Wnt8 was found on the surfaces of cells close
to Wnt-producing cells, we also detected its dispersal overa long range from the source cells. We further examined
their dynamics by FCS and fluorescence decay after photoconversion (FDAP)-based measurements in the embryonictissue. In particular , we refined FDAP-based analysis byfocusing on a limited area across the cell surface, whichenabled us to obtain dynamics comparable to those measured
by FCS (Figure 2). Combination of fluorescence correlation
spectroscopy and quantitative imaging revealed that only asmall 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 across thecell surface, showed that Wnt8 proteins that were bound tothe 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 (Figure 3). Thismodel, which is based on the dynamic exchange of the boundform of Wnt proteins, can recapitulate a graded distributionof the bound, not free, state of Wnt proteins.
In an attempt to investigate the physiological role ofWnt signaling, we collaborated with Dr. Fujimori and Dr.
Takahama at the University of Tokushima and NIH, respectively, to examine gain-of-function (GOF) and loss-of-funcWLRQ /2)  RI ơFDWHQLQ KLJKO\ VSHFLILF LQ PRXVH WK\PLF
HSLWKHOLDO FHOOV 7(&V  *2) RI ơFDWHQLQ LQ 7(&V UHVXOWVin severe thymic dysplasia and T-cell deficiency beginningfrom the embryonic period. By contrast, loss-of-function
RI ơFDWHQLQ LQ 7(&V UHGXFHV WKH QXPEHU RI FRUWLFDO 7(&Vand thymocytes modestly and only postnatally. These results
LQGLFDWH WKDW ILQHWXQLQJ RI ơFDWHQLQ H[SUHVVLRQ ZLWKLQ D
permissive range is required for TECs to generate an optimal
microenvironment to support postnatal T-cell development.

II. The 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 thegradient 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 periodically proceeds in an anterior-to-posterior manner from theirprecursor, the presomitic mesoderm (PSM), which is locatedat the posterior of newly formed somites. This periodic generation is achieved by a complex and dynamic mechanism
within the PSM. The molecular clock, the so-called segmentation clock, essentially creates oscillatory expression ofparticular genes, hairy and some notch-related genes, in theposterior PSM.
As the oscillatory waves of the segmentation clock movefrom posterior to anterior PSM, the temporal dynamics of thesegmentation clock are transformed into the spatial patternFigure 3. Mathematical model considering free diffusion and binding to heparan sulfate clusters. The free population (u) does not virtually contribute tospatial distribution, whereas the bound population (v) directly contributes to the graded distribution of a morphogen. This model suggests both populations
contribute to rapid formation of a stable gradient, which gives insight into a perceived dilemma between speed and stability of gradient formation.

32

of somites. One of the key processes shaping the spatial
pattern of somites is the generation of inter-somite boundaries. Evidences indicate that inter-somite boundary is definedby the anterior edge of the Tbx6 protein expression region.
It has been reported by us and other groups that this anteriorborder is newly generated in each segmentation cycle, by
periodic degradation of Tbx6 protein via physical interaction
with Ripply1 and Ripply2.
We are currently using a genetic approach in zebrafish to
elucidate the mechanisms of the interaction between thesegmental clock and the Tbx6/Ripply system. Based on
the results obtained from the genetic approach, we provide
a mathematical model showing the minimal network that
forms the metameric pattern of somites and the periodic cessation of the segmentation clock.

Publication List:〔Original papers〕
D., Wang, J., Yang, W., Li, J., Lin, X., Zha, F., Wang, X., Ma, L.," Feng,Choi, N.T., Mii, Y., Takada, S., Huen, M.S.Y., Guo, Y., Zhang, L., and

"
"
"
"
"

Gao, B. (2021). Regulation of Wnt/PCP signaling through p97/VCPKBTBD7-mediated Vangl ubiquitination and endoplasmic reticulumassociated degradation. Sci. Adv. 7, eabg2099. DOI: 10.1126/sciadv.
abg2099
Fujimori, S., Ohigashi, I., Abe, H., Matsushita, Y., Katagiri, T., Taketo,
M.M., Takahama, Y., and Takada, S. (2022). Fine-tuning of betacatenin in mouse thymic epithelial cells is required for postnatal T-celldevelopment. eLife 11, e69088. DOI: 10.7554/eLife.69088
Matsuda, S., Schaefer V, J., Mii, Y., Hori, Y., Bieli, D., Taira, M.,
Plueckthun, A., and Affolter, M. (2021). Asymmetric requirementof Dpp/BMP morphogen dispersal in the Drosophila wing disc. Nat.
Commun. 12, 6435. DOI: 10.1038/s41467-021-26726-6
Mii, Y., Nakazato, K., Pack, C.-G., Ikeda, T., Sako, Y., Mochizuki,A., Taira, M., and Takada, S. (2021). Quantitative analyses reveal
extracellular dynamics of Wnt ligands in Xenopus embryos. eLife 10,
e55108. DOI: 10.7554/eLife.55108
Okubo, T., Hara, K., Azuma, S., and Takada, S. (2021). Effect of
retinoic acid signaling on Ripply3 expression and pharyngeal archmorphogenesis in mouse embryos. Dev. Dyn. 250, 103631050. DOI:
10.1002/dvdy.301
Yamamoto, K., Miura, H., Ishida, M., Mii, Y., Kinoshita, N., Takada,S., Ueno, N., Sawai, S., Kondo, Y., and Aoki, K. (2021). Optogenetic
relaxation of actomyosin contractility uncovers mechanistic roles ofcortical tension during cytokinesis. Nat. Commun. 12, 7145. DOI:
10.1038/s41467-021-27458-3〔Review article〕
T., and Takada, S. (2021). Morphological and functional" Shinozuka,
changes of roof plate cells in spinal cord development. J. Dev. Biol. 9,
30. DOI: 10.3390/jdb9030030

33

National Institute for Basic Biology

Developmental Biology
DIVISION OF EMBRYOLOGY

Professor

FUJIMORI, Toshihiko

Associate Professor:
NONOMURA, Keiko
Assistant Professor:
KOYAMA, Hiroshi
Specially Appointed Assistant Professor:
ARATA, Masaki
Technical Staff:
OKA, Sanae
Postdoctoral Fellow:
KISHI, Kanae
SOKENDAI Graduate Student: SAKURAI, Jun
Visiting Graduate Student: MIKOSHIBA, Seiya
Visiting Scientist:
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 devel-

opment. We are also interested in the relationship between
embryos and their environment, the oviducts and the uterus.

I. Live observation of early mouse development
To understand the mechanisms underlying embryonicdevelopment and morphogenesis, it has become common toobserve the behavior of cells and gene expression in living
embryos in a variety of animal species. Progress in genetic
engineering and embryonic manipulation concomitantly withthe progress in optics and microscope technologies, including development of highly sensitive photo-detectors, hasallowed us to observe developing embryos, even mammalianones, in real time. We have established a series of transgenic
mouse lines for live imaging, which is part of a collaborative
project taking place in the Laboratory for Animal Resourcesand Genetic Engineering, Riken CLST. These mouse lines
have been used to visualize nuclei, cell shapes, the cytoskeleton and other organelles to observe cell behaviors in living
mouse embryos across many laboratories over the world. We
have also established mouse lines to monitor the cell cycle.
We have been studying the behavior of cells within embryos
by applying newly developed image analyses following theobservation of cell behaviors within embryos using thesemouse lines. We found that the proximal visceral endoderm
overlying the extra-embryonic ectoderm shows coherent cell
growth in a proximal-anterior to distal-posterior direction.
We also observed that directional cell migration is coupledwith cell elongation in the anterior region, suggesting that the
behaviors of visceral endoderm cells vary between regions
during peri-implantation stages.
We have also been establishing several reporter mouse lines
in the lab to study gene expression patterns during the periimplantation stage of mouse development. In these mice,
cDNA encoding fluorescent proteins are connected to themouse genomic DNA sequences of the enhancer/promoter
region of important genes encoding factors regulating celldifferentiation in these stages.
In addition to these reporter mouse lines, we generated transgenic (Tg) mice expressing photoactivatable-Cre(PA-Cre) proteins. Due to the limitation of high throughput
analyses in mouse embryos, we established embryonic stem
cells from the Tg mice for testing various conditions. We
then systematically investigated the conditions which enableefficient photoactivation for PA-Cre, in vitro and in silico. It
was suggested that repetitive pulsed illumination with shortpulse cycles efficiently induced DNA recombination withlow light energy. We also showed that repetitive short-pulsed
illumination induced DNA recombination in pre-implantationembryos expressing PA-Cre.

II. Histological observation of mouse embryos
developing in 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?

34

Mammalian embryos develop in the uterus after implantation. Direct interaction between embryos and the cells of theuterus is important. In studies on the developmental biology
of mammalian embryos, embryos are usually removed fromthe uterus, and those that are isolated are analyzed. We havebeen analyzing the early embryonic development of themouse by comparing the changes in the uterine epithelium.

To observe the morphogenesis of embryos within the uterus,
serial sections of implanted uteruses were made, and imagesof the embryos within it were captured to make high resolution three-dimensional re-constructions. We are preparing
samples obtained from various developmental stages. Precise
positions of embryonic implantation and changes in theuterine epithelium correlating with embryonic development
have been examined using these images. We are identifying the molecules involved in the interaction between theembryo proper and uterine cells, which may play a majorrole in embryonic development.

Figure 1. Three-dimensional image reconstruction of the pregnant uterus.
A uterus obtained 5 days after fertilization was sectioned and images 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 fromthe ovary are transported through the oviduct, where the ova
are fertilized by spermatozoon entering from the uterus. The
epithelium of the mouse oviduct consists of multi-ciliatedcells and secretory cells. Ovulated oocytes are transported in
oviducts by unidirectional motility of multi-cilia. This cellularorientation in the oviduct is one example of planar cell polarity(PCP). PCP is seen in a variety of tissues, where cells sense
global axes of the tissue to which they belong and orient themselves to fulfill specialized functions.
Many evolutionally conserved genes are required for theformation of PCP, and several of those encode proteins thatare localized in a polarized manner within cells. We found that
Celsr1, a PCP core member, was strongly concentrated only at
the specific domains of cell boundaries which are perpendicular to the ovary-uterus axis, and that this polarized localization
appeared to precede the directional movement of 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 theaxis, while some others exhibited reverse polarity. Recently,
we found that Camsap3, a microtubule 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 alignment of cilia along the ovary-uterus axis, but in distinct ways; Celsr1 orients cells, while
Camsap3 orients cilia within each cell.
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 disrupted. In adult wild-type mice, the epithelial cell sheet is bent
multiple times, which forms around twenty longitudinal folds
parallel to the organ axis. However, in the mutant oviducts,
epithelial folds no longer run parallel to the organ axis. The
direction of the folds was randomized, and ectopic branchesof the folds were observed. This suggests that Celsr1 isimportant for the formation of multi-scale polarities in themouse oviduct ranging from the cellular to the tissue scale.
Epithelial folds are observed in various organs including
the oviducts, guts, and airways etc. Longitudinally aligned
folds are also observed in the oviducts of birds and frogs. To
investigate the mechanisms of the epithelial fold pattern formation, we utilized mathematical modeling and simulations.
By considering mechanical properties of the epithelial sheets,
we reproduced the longitudinally aligned and branched folds
which are observed in wild-type and Celsr1 mutant mice,
respectively (Figure 2). In addition, our model also successfully reproduced circumferentially aligned folds and zigzag
folds observed in other organs. We are also focusing onsome other PCP regulators, and are trying to understand how
oviduct epithelial cells establish and maintain their polarity.

IV. Analysis of the mechanical properties of cells
and tissues during embryonic development
Mechanics is one of the essential components of biological processes, including cell shape transformation and tissuemorphogenesis etc. To understand how mechanical forces
contribute to various patterns of morphogenesis, measuring cellular and tissue mechanical states is necessary. We
developed statistical techniques to infer mechanical statesusing fluorescent microscopic images during morphogenesis

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

35

National Institute for Basic Biology

Developmental Biology(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.

cluster 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.

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
mechanical forces, PIEZO activation, cellular responses, andtissue morphogenesis.
Positive or
negative pressure,
shear stress

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

Publication List:〔Original papers〕
T., Tohsato, Y., Itoga, H., Shioi, G., Kiyonari, H., Oka, S.," Hirata,
Fujimori, T., and Onami, S. (2021). NeuroGT: A brain atlas of"""

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.

VI. Mechanics of cell population patterning
during development
During development, cells actively and/or passively move,resulting in various cell distribution patterns. We investigatedthe effect of passive cell movements provoked by frictional
forces from adjacent growing tissues. The passive movements generated various patterns, such as an elongated cell

36

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

"

neurogenic tagging CreER drivers for birthdate-based classification andmanipulation of mouse neurons. Cell Reports Methods 1, 100012. DOI:
10.1016/j.crmeth.2021.100012
Kinoshita, N., Yamamoto, T.S., Yasue, N., Takagi, C., Fujimori, T., andUeno, N. (2022). Article Force-dependent remodeling of cytoplasmic
ZO-1 condensates contributes to cell-cell adhesion through enhancing
tight junctions. iScience 25, 103846. DOI: 10.1016/j.isci.2022.103846
Kishi, K., Koyama, H., Oka, S., Kato, A., Sato, M., and Fujimori,T. (2021). Repetitive short-pulsed illumination efficiently activatesphotoactivatable-Cre as continuous illumination in embryonic stem
cells and pre-implantation embryos of transgenic mouse. GENESIS 59,
e23457. DOI: 10.1002/dvg.23457
Koyama, H., Suzuki, M., Yasue, N., Sasaki, H., Ueno, N., and Fujimori,T. (2022). Differential Cellular Stiffness Contributes to Tissue
Elongation on an Expanding Surface. Front. Cell Dev. Biol. 10, 864135.
DOI: 10.3389/fcell.2022.864135
Saito, H., Matsukawa-Usami, F., Fujimori, T., Kimura, T., Ide, T.,
Yamamoto, T., Shibata, T., Onoue, K., Okayama, S., Yonemura, S.,
Misaki, K., Soba, Y., Kakui, Y., Sato, M., Toya, M., and Takeichi,
M. (2021). Tracheal motile cilia in mice require CAMSAP3 for theformation of central microtubule pair and coordinated beating. Mol.
Biol. Cell 32. ar12. DOI: 10.1091/mbc.E21-06-0303

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:

KITADATE, Yu
NAKAGAWA, Toshinori
Specially Appointed Assistant Professor:
SUZUKI, Shinnosuke
Technical Staff:
MIZUGUCHI, Hiroko
JSPS Postdoctoral Fellow: IKEDA, Tatsuro
Postdoctoral Fellow:
HIRANO, Kodai
SATO, Toshiyuki
Visiting Graduate Student: BABAGUCHI, Hirotaka
Technical Assistant:
FUJITA, Miyako
NISHIMURA, Chiaki
Admin Support Staff:
KUBOKI, Yuko

As cells and syncytia together comprise a single SSC pool
Our live-imaging studies combined with mathematical andstatistical analysis evidenced that SSCs reversibly change theirmorphology between singly-isolated and syncytia. This ismediated by incomplete cell division and breakdown of syncytia(Nakagawa et al., Science 2010; Hara et al., Cell Stem Cell 2014).

Cells inclined for differentiation can revert to SSCs
It is generally thought that once stem cells have been destinedfor differentiation, they never replicate themselves again. We
found that such cells maintain the potential to self-renew up
to a particular stage of differentiation and revert to SSCs withhigh frequency, particularly during regeneration after tissue
damage (Nakagawa et al., Developmental Cell 2007).

SSCs follow variable fate behaviors
Stem cells are often thought to invariably undergo asymmetric division, which produces one stem cell and one
differentiating cell to maintain homeostasis. However, wefound that individual SSCs follow highly variable fates. Still,collectively, they achieve a perfect balance between selfrenewal and differentiation at a population level (Klein et al.,Cell Stem Cell 2010; Hara et al., Cell Stem Cell 2014).

SSCs show active motion in an <open niche=

Our research
Many animals, including mammalians, produce a large
number of sperm over a long period for reliable reproduction. On the other hand, each sperm correctly replicates andtransmits the genetic information to the next generation.
The ultimate goal of the Division of Germ Cell Biology is toreveal how seemingly contradictory, albeit essentially important, productivity and fidelity are both achieved. To this end,
we pay special attention to the sperm stem cells. We are alsointerested in other aspects of spermatogenesis.

What are the sperm stem cells?
The continual sperm production is supported by the <sperm
stem cells,= termed formally <SSCs= after spermatogonial or
spermatogenic stem cells. SSCs maintain a delicate balance
between self-renewal and differentiation to sustain life-longspermatogenesis. It has been a central question which fractionof spermatogonia, the <early stage of cells before meiosis, are
SSCs and where and how they behave in the testis.
The backbone of mammalian SSC research was establishedfrom the 1950s to the 1970s, relying on morphologicalanalyses of fixed testis specimens. This led to a proposal of
the prevailing stem cell model called the <As model.= Thismodel proposed that singly isolated spermatogonia (A-single
or As cells) act as SSCs. On the other hand, all syncytia are
all committed to differentiation.
Our lab has introduced the scale of time using updated
technologies, i.e., live-imaging and pulse-labeling studies,
which revealed many different aspects of dynamical behaviors of SSCs (Figure. 1).

In many tissues, stem cells receive self-renewing signals in
defined niche regions. We found that the mouse testis has nosuch areas, with SSCs scattered among differentiated cells
and moving around actively. Further, their distribution isbiased near blood vessels. Such microenvironment is called
an <open (or facultative) niche= in contrast to the afore-mentioned <closed (or defined) niche= (Yoshida et al., Science
2007; Hara et al., Cell Stem Cell 2014).

SSC heterogeneity underpins the asymmetric fates of SSCs
In an open niche, some SSCs remain undifferentiated whileothers differentiate in response to broadly distributed signaling molecules. We found that some SSC fractions resist
differentiation-promoting Wnt signal by expressing a Wntinhibitor, Shias 6. Another subset responds to retinoic acid(RA) by upregulating an RA receptor to differentiate (Ikami etal., Development 2015, Tokue et al., Stem Cell Reports 2017).

SSCs compete for self-renewal factors in an open niche
We also question the mechanism that maintains a constant
SSC density in the open niche of the testis. Evidence
suggests that SSC density homeostasis is achieved throughcompeting with each other for limited amounts of selfrenewal factors (e.g., FGFs). This mechanism was proposedas the <mitogen competition model= (Kitadate et al., Cell
Stem Cell 2019).

Periodic differentiation of SSCs
Strikingly, differentiation of SSCs and subsequent differentiation to mature into sperm occur synchronously every
8.6 days. This is called the seminiferous epithelial cycle.
We propose that the periodic differentiation is triggered byperiodic synthesis and degradation of RA (Sugimoto et al.,Mechanisms of Development 2012).

37

National Institute for Basic Biology

Developmental Biology

Figure 1. Different looks of mouse sperm stem cells (SSCs)
A. 3D-reconstructed seminiferous tubules, showing SSCs in green, blood vessels in red.
B. An intravital live-imaging filming of GFP+ SSC syncytia undergoing differentiation.
C. Scheme of SSC dynamics in multi-compartment, interconverting singly-isolated and syncytial states.
D. Whole-mount immunofluorescence, showing SSCs (magenta) intermingled with primed (green) and committed (blue) progenies.
E. Sectioned host testis showing repopulated by transplanted donor SSCs (green), augmented by transient RA inhibition.
F. A phase portrait showing the dynamics of homeostatic SSC density regulation in the mitogen competition model.

Clonal analysis and tuning of post-transplantation
SSC fate
When SSCs are transplanted into host animals9 seminiferous
tubules whose germ cells have been depleted, SSCs engraft
and regenerate spermatogenesis (Brinster et al., 1994). What
happens during repopulation was largely elusive. We foundthat donor SSCs follow stochastic fate with significant similarity with homeostasis. We further succeeded in tuning
the donor SSC fate to increase the repopulation efficiency,
which would promise the practical use of this technology(Nakamura et al., Cell Stem Cell 2021).

Identification of a tiny, most primitive fractionof SSCs
An important advance gained in FY2021 is that we havedetailed gene expression heterogeneity of SSCs (precisely, a
population called <undifferentiated spermatogonia=) and theirdynamical state transitioning in supporting the homeostaticand continual sperm production (Nakagawa et al., Cell Reports
2021). This is a collaborative study with Nagasawa (Osaka) andSimons (Cambridge) labs. We identified that a gene Plvap (plasmalemma vesicle associated protein) is expressed in a tiny, mostprimitive fraction of mouse SSCs (Figure 2A).

Detailing the heterogeneity of mouse SSCs
Plvap shows overlapping expression with some other genes,
including Eomes, Pdx1, Shisa6, and T (brachyury). By
dimension reduction of single-cell (sc)RNA-seq data (Laet al., 2018), Plvap+ cells cluster at the undifferentiated endof SSCs, forming a discrete subfraction of cells expressing
HVWDEOLVKHG XQGLIIHUHQWLDWHG PDUNHUV *)5Į and Id4. In
contrast, differentiation-related genes (e.g., Sox3, Ngn3, and
5$5Ȗ  LV H[SUHVVHG LQ FHOOV QHDU WKH RSSRVLWH HQG )LJXUH
2B). Based on these, Figure 2C summarizes the heterogeFigure 2. Heterogeneity of SSCs
A. Immunostaining of a mouse testis
section for Plvap protein (magenta), with
DNA counterstained in gray. Only threecells located on the periphery of seminiferous tubules express Plvap.
B. UMAP analyses of scRNA-seq data
showing the expression of indicatedgenes.
C. A summary of expression profile of aset of genes within SSCs. Genes shownin bald have been analyzed in our lab.
Adopted from Cell Reports 37 (3),109875.

38

neous expression of key SSC genes.

Contribution of multiple SSC fractions to selfrenewing pool in homeostasis
By pulse-labeling using tamoxifen-dependent CreER,
Plvap+, Sox3+, and Ngn3+ cells were found to contribute to
the self-renewing pool in homeostasis. In particular, after
being sparsely labeled with GFP expression, these cells gave
rise to cell patches comprised of GFP+ germ cells in threemonths (Figure 3).
Interestingly, while the patch-forming efficiency varied
between these fractions, the resultant individual patches
showed indistinguishable size distribution irrespective of theoriginal cell state. Further, strikingly, the same heterogeneous
SSC population was established in every such patch, again,
irrespective of the original cell state.

Figure 4. Multistate dynamics of mouse SSCs
A. A general perspective of the multistate dynamics, in which SSCs
transit between multiple states reversibly to form a single SSC pool.
B. A numerically fit dynamics of mouse SSCs in homeostasis. The flowsof cells between cell states and cell division at respective states are
VKRZQLQYDOXHVUHODWLYHWRWKHWRWDO*)5Ơ+ cells per day. Note that the*)5Ơ3 (Ngn3+ WR*)5Ơ+/Sox3+ state is not defined due to its low frequency in this model. Adopted from Cell Reports 37 (3), 109875.

Interestingly, such a multistate dynamics is found capableof saving the cell divisions in the self-renewing pool in
producing a certain number of differentiating cells. This isdue to the differential cell division frequencies between thestates; the more primitive the state is, the less frequently
the SSC divide. See Nakagawa et al., Cell Reports 2021 fordetail.
Furthermore, this quantitative model can explain the seemingly contradictory models proposed for the SSC dynamics,
and may provide inclusive insights into this long-heldproblem in the field of SSC research.
Figure 3. Pulse-labeling of multiple SSC fractions
A. Experimental design addressing the contribution of spermatogonia
expressing particular genes to homeostasis, following a pulse-labeling bytamoxifen injection.
B. Results of the pulse-labeling of the Plvap+, Sox3+, and Ngn3+ fractions. Note the differential patch forming efficiencies in contrast to thesimilarly-looking individual patches.

We reasoned that SSCs transit continually and reversibly
between multiple cell states, from the self-renewal-biased
Plvap+ state to the differentiation-primed Ngn3+ state (Figure
3B). As a result, a heterogeneous SSC pool is always established, which both maintains itself and gives rise to irreversibly committed progenitor cells of Kit+ spermatogonia as aresult of cell division and state transition.

A multistate dynamics: Proposal of an
inclusive model of mouse SSCs
Finally, we synthesized a mathematical model that captures
the multistate SSC dynamics in homeostasis. This model
assumes that the cells undergo cell division and state transition (either differentiation and reversion) in a stochastic (i.e.,
probabilistic) manner, following particular rates specific toeach state (Figure 4).

Publication List:〔Original Papers〕
T., Jörg, D.J., Watanabe, H., Mizuno, S., Han, S., Ikeda," Nakagawa,T., Omatsu, Y., Nishimura, K., Fujita, M., Takahashi, S., Kondoh, G.,""

Simons, B.D., Yoshida, S., and Nagasawa, T. (2021). A multistate stem
cell dynamics maintains homeostasis in mouse spermatogenesis. Cell
Rep. 37, 109875. DOI: 10.1016/j.celrep.2021.109875
Nakamura, Y., Jörg, D.J., Kon, Y., Simons, B.D., and Yoshida, S.
(2021). Transient suppression of transplanted spermatogonial stem celldifferentiation restores fertility in mice. Cell Stem Cell 28, 1443-1456.
e7. DOI: 10.1016/j.stem.2021.03.016
Osawa, Y., Murata, K., Usui, M., Kuba, Y., Le, H.T., Mikami, N.,
Nakagawa, T., Daitoku, Y., Kato, K., Shawki, H.H., Ikeda, Y., Kuno, A.,
Morimoto, K., Tanimoto, Y., Dinh, T.T.H., Yagami, K., Ema, M., Yoshida,S., Takahashi, S., Mizuno, S., and Sugiyama, F. (2021). EXOC1 plays an
integral role in spermatogonia pseudopod elongation and spermatocyte
stable syncytium formation in mice. eLife 10, e59759. DOI: 10.7554/
eLife.59759〔Review article〕
Y., and Yoshida, S. (2022). Regulation of spermatogenic" Kitadate,stem cell homeostasis by mitogen competition in an open nichemicroenvironment. Genes Genet. Syst. 97, 15325. DOI: 10.1266/
ggs.21-00062

39

National Institute for Basic Biology

Developmental Biology

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, NorikoComparative 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
the difference between regenerative and non-regenerative
animals to evoke abilities from non-regenerative animals.
We have already succeeded in achieving this with planarians,
which were 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 functional joint regenerationin mouse and to characterize adult pluripotent stem cells inplanarian at a single cell level.

staining with several fluorescence dyes. Recently, we found acondition to be able to isolate viable aPSC by FACS sorting
without staining with dyes. The single cell transcriptome
analyses using these isolated aPSC showed unique propertiesof planarian aPSC (Kuroki et al., unpublished).
We also found that these aPSC showed the collective migration property (branch-like patterns in Fig.1) after RNAitreatment of the MTA (Metastatic Tumor Antigen) family
genes, suggesting that planarian might have unique niche formaintain aPSC in the adult bodies (Sato et al., 2022).

Figure.1 RNAi treated animal of the MTA-A gene showed the branch-like
distribution patterns of aPSC (magenta stained with the PIWI-A antibodies).

Now, we are investigating differences of these cellular
systems between highly- and lower- regenerative animals.

Publication List:〔Original papers〕
T., Okumura, M., Bando, Y., Hagiwara, M., Hamada, Y.," Bando,Ishimaru, Y., Mito, T., Kawaguchi, E., Inoue, T., Agata, K., Noji, S., and"Trials for evoking functional joint regenerationin mouse
Mouse can9t regenerate joint structures at all. However, we
expect that functional joints might be evoked by the activation of reintegration systems since we found that the remaining tissues after amputation at the joint level possessed theability to regenerate joint tissues (Hotta et al., unpublisheddata). That is, the mouse might lose their regenerative abilities due to non-cell autonomous reasons. Therefore, we are
now investigating how to activate reintegration systems inmice after amputation at the joint level for evoking functional joint regeneration.

Isolation of the viable planarian aPSC (adultpluripotent stem cells) and characterization ofthese cells at the single cell level.
We tried to develop an isolation method for viable adultpluripotent stem cells (aPSC) from planarians using FACS.
In the previous method, isolated aPSC were unhealthy after

40

"
"
"
"

Ohuchi, H. (2022). Toll signalling promotes blastema cell proliferation
during cricket leg regeneration via insect macrophages. Development
149, dev199916. DOI: 10.1242/dev.199916
Finet, C., Kassner, V.A., Carvalho, A.B., Chung, H., Day, J.P., Day, S.,
Delaney, E.K., De Re, F.C., Dufour, H.D., Dupim, E., Izumitani, H.F.,
Gauterio, T.B., Justen, J., Katoh, T., Kopp, A., Koshikawa, S., Longdon,
B., Loreto, E.L., Nunes, Maria D.S., Raja, K.K.B, Rebeiz, M., Ritchie,
M.G., Saakyan, G., Sneddon, T., Teramoto, M., Tyukmaeva, V.,
Vanderlinde, T., Wey, E.E., Werner, T., Williams, T.M., Robe, L.J., Toda,
M.J., and Marletaz, F. (2021). DrosoPhyla: Resources for Drosophilid
Phylogeny and Systematics. GENOME Biol. Evol. 13, evab179. DOI:
10.1093/gbe/evab179
Inoue, T., and Agata, K. (2022). Quantification of planarian behaviors.
Dev. Growth & Differ. 64, 16337. DOI: 10.1111/dgd.12765
Lee, H., Hikasa, K., Umesono, Y., Hayashi, T., Agata, K., and Shibata,N. (2022). Loss of plac8 expression rapidly leads pluripotent stem cells
to enter active state during planarian regeneration. Development 149,
dev199449. DOI: 10.1242/dev.199449
Sato, Y., Umesono, Y., Kuroki, Y., Agata, K., and Hashimoto, C. (2022).
Proliferation maintains the undifferentiated status of stem cells: The
role of the planarian cell cycle regulator Cdh1. Dev. Biol. 482, 55366.
DOI: 10.1016/j.ydbio.2021.12.006
Takeuchi, T., Matsubara, H., Minamitani, F., Satoh, Y., Tozawa, S.,
Moriyama, T., Maruyama, K., Suzuki, K.T., Shigenobu, S., Inoue, T.,
Tamura, K., Agata, K., and Hayashi, T. (2022). Newt Hoxa13 has an
essential and predominant role in digit formation during development
and regeneration. Development 149, dev200282. DOI: 10.1242/
dev.200282

National Institute for Basic Biology

Neurobiology
DIVISION OF BEHAVIORAL NEUROBIOLOGY

Professor

HIGASHIJIMA, Shin-ichi

Assistant Professor:

KIMURA, Yukiko
TANIMOTO, Masashi
Technical Staff:
TAKEUCHI, Yasushi
SOKENDAI Graduate Student: SUGIOKA, Takumi
KAWANO, Kohei
SHIMIZU, Sayaka
Visiting Undergraduate: KATAYAMA, Taisei
Technical Assistant:
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. Long descending commissural V0v neurons
ensure coordinated swimming movements
along the body axis in larval zebrafish
In the early developmental stage, most animals can only
exhibit immature forms of behaviors. As development progresses, they acquire the ability to produce more mature or
refined forms of behaviors. Concurrent with such changes,
many new connections are formed in the nervous system,
which suggests that the formation of new connections islinked to the developmental maturation of behaviors. In
animals in which new neurons are generated during development (e.g., fish and amphibians), the incorporation of
new neurons into the pre-existing circuits together with theforming of new connections likely contributes to the maturation of movements.

41

National Institute for Basic Biology

Neurobiology

One example of this developmental maturation of behaviors is seen in the swimming behavior of larval zebrafish.
Older larvae (i.e., 435 dpf) exhibit more refined forms ofswimming than younger larvae (i.e., 2 dpf), During thisperiod (from 2 dpf to 435 dpf), new neurons are generated inboth the brain and spinal cord, with the latter being mainly
responsible for generating swimming outputs. One classof premotor spinal neurons that are added later to the earlyspinal neuronal circuits are MCoD neurons, a subclass of
V0v neurons (V0v neurons represent an excitatory class ofneurons derived from the p0 developmental domain of the
spinal cord). MCoD neurons are absent in the early stages
and develop in a later phase of neurogenesis in the spinal
cord. MCoD neurons are active during slow swimming. As
for their function, one study showed that they contribute to
the general excitability of spinal swimming circuits, and theirablation decreased the occurrence frequency of spontaneous
swimming. Their function in the more specific aspect of slow
swimming remains elusive, however.
One of the most characteristic features of slow swimmingin older larvae is the stability of the head in the yaw dimension. In slow swimming, the muscle contractions are mostly
confined to the trunk that is caudal to the swim bladder.
Given that the center of the mass is located near the swim
bladder in larval zebrafish (Figure 2A), it is thought thatthe head yaw displacement is produced by the recoil of theyawing moment force generated in the trunk. Considering
this, the stability of the head yaw indicates that the net
yawing moment force in the trunk that acts to the center ofthe mass is very small during slow swimming. Swimming
consists of a descending wave of muscle contraction along
the trunk. With this movement, the bending of the bodytransmits force to the surrounding water, and this region of
the body, in turn, receives reaction force. To make the net
yawing moment force minimal, the movements of the rostraland caudal parts of the trunk need to be highly coordinatedwith the diagonal dimension; when the rostral part receives
leftward force, for example, the caudal part needs to receive
rightward force. For this to occur, the swimming body formcannot be C-shaped (unilateral body bend); rather, the shapeneeds to be sinusoidal (S-shaped). MCoD neurons are a goodcandidate for implementing this coordinated movement of
the trunk in the diagonal dimension, because they are active
during slow swimming, and because they are long-distancedescending commissural excitatory neurons that make directconnections onto MNs in the caudal region of the contralateral spinal cord (Figure 2B).
In this study, we tested whether MCoD neurons play a rolein the coordinated movements of the trunk in the diagonal
dimension, thereby ensuring minimal head yaw displacement during slow swimming. Our laser ablation experiments
revealed that MCoD neurons do indeed play the expected role.
In the MCoD-ablated larvae, the normal S-shaped body form
during swimming was often lost with increased appearance of
unilateral C-shaped bends. Concurrently, the head yaw stability was greatly impaired (Figure 3). In addition to swimming,
the present study also sheds light on the evolutionarily conserved role of V0v neurons. In mice, long-distance descending
commissural V0v neurons have been implicated in interlimb

42

coordination during walking in the diagonal dimension18. We
suggest that the long-distance descending commissural V0v
neurons for the coordinated movements of the body/limbs
in the diagonal dimension are the evolutionarily conserved
pathway in spinal locomotor circuits.

Figure 2. (A) One of the swim forms of a zebrafish larva at 4 to 5 dpf.
The cyan circle shows the center of mass, which is located near the swim
bladder. Muscle contractions are presumed to occur in the two locations
marked in red. (B) Projection pattern of an MCoD neuron in the spinal
cord. The axon of the MCoD neuron crosses the midline (broken line),
descends on the contralateral spinal cord, and makes mono-synaptic
excitatory connections onto caudally located MNs.

Figure 3. (A) Confocal stacked images of Tg[evx2-hs:GFP] fish before(left) and after (right) laser ablation. Images of two hemi-segments areshown. Magenta arrows show MCoD neurons that were chosen for laser
ablation. MCoD neurons can be identified by their very ventral location in
the spinal cord. Brown lines show boundaries of muscle segments. Scale
EDUƫP % 6XFFHVVLYHLPDJHVFDSWXUHGDWIUDPHVSHUVHFRQGRI
larval zebrafish swimming. Images of every three frames (3 ms interval)
are shown. Magenta bars depict the head directions in each frame. Top,
images of an intact fish. Bottom, images of an MCoD-ablated fish. Scale
EDU  ƫP &  *UDSKV RI KHDG \DZ DQJOH \ D[LV  YHUVXV WLPH [D[LV during swimming. Left, intact fish. Right, MCoD-ablated fish.

III. In vivo functional imaging analysis of thevestibular sensory organ
Maintaining head and body orientation relative to the
Earth9s 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 4). 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 direction- 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.

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

Figure 4. Hair bundle polarity (arrowheads) in the utricle.

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 5). 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.

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.

Publication List:〔Original Papers〕
U.L., Kimura, Y., Kawashima, T., Ahrens, M.B., Higashijima," Bohm,S., Engert, F., and Cohen, A.E. (2022). Voltage imaging identifies spinal""

circuits that modulate locomotor adaptation in zebrafish. Neuron 110,
1211+. DOI: 10.1016/j.neuron.2022.01.001
Kawano, K., Kato, K., Sugioka, T., Kimura, Y., Tanimoto, M., andHigashijima, S. (2022). Long descending commissural V0v neurons
ensure coordinated swimming movements along the body axis in larvalzebrafish. Sci. Rep. 12, 4348. DOI: 10.1038/s41598-022-08283-0
Suzuki, D.G., Wada, H., and Higashijima, S. (2021). Generation ofknock-in lampreys by CRISPR-Cas9-mediated genome engineering.
Sci. Rep. 11, 19836. DOI: 10.1038/s41598-021-99338-1

Figure 5. Tiltable objective microscope.

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 6). In response to the nose-down

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
Visiting Scientist:
KOMATSU, HidehikoTechnical Assistant:
WATABE, Mihoko
TSURUMARU, Mayumi
SUGINAGA, Tomomi

We believe that the best way to understand mechanisms underlying a certain system is to reconstruct the system as it exists. Byutilizing computer technologies, we are accordingly trying tobuild the systems based on psychophysical and ecological viewpoints in particular to understand systems of animal perception
and behavior. This type of methodology based on computerswill pave the way for biology 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. As a result, we confirmed that medaka
exhibited predation behavior against several characteristic
movements of the virtual plankton, particularly against aswimming pattern that could be characterized as a pink noise
motion (Matsunaga & Watanabe, 2012). As a side-story,
we analyzed the swimming behaviors of adult water flea
Daphnia magna, and found apparent sexual differences:laterally biased diffusion of males in contrast to the nondirectional diffusion of females (Toyota et al., 2022).
Many fish species are known to live in groups, and visualcues have been shown to play a crucial role in the formationof shoals. By utilizing biological motion stimuli, which inthis case was the depiction of a moving creature by using justa 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, andhave shown what aspects of this motion are critical in the
induction of shoaling behavior. Motion and behavioral characteristics can be valuable in recognizing animal species,
sex, and group members. Studies using biological motion
stimuli will further enhance our understanding of how nonhuman animals extract and process information which is vitalfor 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
seasonal changes in color perception in medaka (Shimmura
et al., 2017), and on underwater imaging technology (Abe etal., 2019 and Yamamoto et al., 2019).

I. Psychophysical study of medaka fish
One of our major subjects is the psychophysical and computational study of medaka (Oryzias latipes, Matsunaga& Watanabe, 2010). We have 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.

ɉ3V\FKRSK\VLFDOVWXG\RIKXPDQ $,YLVLRQ
Another of our major subjects is the psychophysical andtheoretical studies of the visual illusions experienced byhuman and artificial intelligence (AI). One recent focus of
this debate is the flash-lag effect, in which a moving object
is perceived to head towards a flashed object when bothobjects are aligned in an actual physical space. We developed
a simple conceptual model explaining the flash-lag effect(Watanabe et al., 2010).
In recent years, we have made more developed novel visual
illusions, such as the shelf-shadow illusion (3rd Award of theIllusion Contest), the Monstre Benham illusion (2ndAward), the Morning Monster Illusion (10th Award), and the
Genkikan Illusion (Figure 2).

static images where humans were unable to perceive any
illusory motion. Additionally, illusion-like designs withrepeating patterns were generated using areas where anomalous vectors were detected, and psychophysical experiments
were conducted, in which illusory motion perception in thegenerated designs was detected (Figure 3). The observed
inaccuracy of the networks will provide useful information
for further understanding information processing associatedwith human vision (Kobayashi et al., 2022)

Figure 3. A visual illusions extracted by AI

Ɋ(FRORJLFDOVWXG\RIWDFWLFVLQSUHGDWRUVDQG
prey.

Figure 2. Okazaki Genkikan.

Watanabe (NIBB) and Sinapayen (Sony CSL) got 2nd placeat the 13th Visual Illusion Contest, for the Okazaki Genkikan
Illusion. It is one of the Poggendorff illusions. (http://www.
psy.ritsumei.ac.jp/~akitaoka/sakkon/sakkon2021.html). It
was found in Okazaki Genkikan Facility.
In 2018, we successfully generated deep neural networks(DNNs) that represent the perceived rotational motion
for illusion images that were not physically moving, yetsimilar to what we experience in human visual perception.
(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) (Evolutionary Generation of Visual Motion
Illusions, https://arxiv.org/abs/2112.13243, Sinapayen
and Watanabe, 2021). Currently, a variety of research is
being conducted using these AI systems (https://arxiv.org/
abs/2106.12254).
In 2022, we examined the properties of the network using a
set of 1500 images, including ordinary static images of paintings and photographs and images of various types of motion
illusions (Visual Illusions Dataset, https://doi.org/10.6084/
m9.figshare.9878663). Results showed that the networks
clearly classified a group of illusory images and others andreproduced illusory motions against various types of illusions
similar to human perception. Notably, the networks occasionally detected anomalous motion vectors, even in ordinally

We are interested in behavioral interactions between predators and prey concerning the ecological aspect. In the processof the co-evolution, predators and prey have developed
various tactics to overcome each other. To elucidate thesophistication of these tactics, we have examined the mechanisms and efficacies of predatory and antipredator behaviorsof several animals such as fish, dragonflies, bats and birds.
In addition, we have developed experimental system forthose studies, which present interactive virtual predators toprey animals based on animation (Figure 4) or robotics. This
experimental system consists of three modules as a sensing, acommand generating and a virtual animal module. According
to the spatial scale, the system employs appropriate technologies in each module. For example, in a small aquarium
scale, we use high speed cameras as a sensing module andvirtual reality animation as a virtual animal module. On theother hand, in a large outdoor scale, we use real-time GPS
as a sensing module and unmanned aerial vehicle as a virtual
animal module. Therefore this system are able to cover wider
range of animals and expected to strongly support the studyof animal interactions.

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

45

National Institute for Basic Biology

Neurobiology
Publication List:〔Original paper〕
T., Kitaoka, A., Kosaka, M., Tanaka, K., and Watanabe," Kobayashi,
E. (2022). Motion illusion-like patterns extracted from photo and art
images using predictive deep neural networks. Sci. Rep. 12, 3893. DOI:
10.1038/s41598-022-07438-3

46

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
Technical Staff:
OHI, Shoko
Postdoctoral Fellow:
NARUKAWA, Hideki
PALFALVI, Gergo
SOKENDAI Graduate Student: HORIUCHI, Yuta
DE VILLIERS, Ruan
CHEN, Peng
UEDA, Masamichi
Visiting Graduate Student: TANASE, Kuniaki
Visiting Undergraduate: MATSUDA, Riku
Technical Assistant:
AOKI, Etsuko
FUKADA, Hatsumi
HIRAMATSU, Mika
KAJIKAWA, Ikumi
MATSUZAKI, Yoko
MASUOKA, Tomoko
NISHI, TayoZHANG, Liechi
OGAWA, Yuko
NAGATA, Kyoko
SAITO, Yuki
ASAOKA, Mayumi
BUSHRA, Samira
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 fundamental concepts in evolutionary biology. However, even with such theories, there still
remain unexplained phenomena, one of which is the evolution of complexity. It is difficult to explain the mechanisms
needed to evolve complex adaptive traits, because such traits
comprise plural components and become adaptive only when
all components are gathered together. However, based on
evolutionary theories, each component should evolve one by
one according to the accumulation of mutations.
To understand the molecular and cellular bases of adaptive
traits, we study the molecular and cellular mechanisms andevolution of (1) long distance signaling in plants, (2) reprogramming from differentiated cells to stem cells, and (3)development and morphology in land plants.

II. Molecular mechanisms and evolution oflong distance signaling 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 evolved inparallel to similar signaling mechanisms in animals, 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, andthe sundew Drosera rotundifolia. We use these species,
as well as Arabidopsis thaliana, to study the molecularmechanisms and evolution of action potential generation andtransmission. We have obtained genome sequences for these
species (Palfalvi et al. 2020 Curr. Biol. for Dionaea andunpublished for Mimosa pudica and Drosera rotundifolia)
and established techniques for transformation (Mano et al.
2014 PLoS ONE for Mimosa pudica; Suda et al. 2020 Nat.
Plants for Dionaea; unpublished for Drosera rotundifolia).
We intend to screen for genetic factors responsible for fast
intracellular and intercellular electrical signaling by action
potentials, analyze their molecular characteristics such as
effects on ion permeability and intracellular localization, andperform genetic gain- and loss-of-function experiments. This
will allow us to understand the general mechanisms of action
potential transmission in plants and the evolutionary processthat resulted in diversity of transmission velocity, which isadaptive in the three plants.

II-1. Molecular mechanisms of long distancesignaling of mechano-stimuli in thesensitive plant Mimosa pudica
The sensitive plant Mimosa pudica folds it leaves within acouple of seconds in response to touch. These rapid movements are mediated by specialized motor organs, pulvini,
which locate at the base of each compound leaves, pinnae,and small leaflets. When getting stimulated, motor cells in
one side of the pulvinus (extensor half) shrink while cells in
the opposite side (flexor half) do not shrink, consequently
leading to the unidirectional bending of the pulvinus. Because47National Institute for Basic Biology

Evolutionary Biology and Biodiversity

a pulvinus contains thousands of motor cells, achievementof the rapid movement at a whole pulvini requires not only
the rapid shrinkage of individual motor cells but also rapid
synchronization mechanisms among them. The molecularmechanisms underlying these rapid shrinkage and rapid cellcell communication still remain open questions.
To elucidate the molecular mechanisms for rapid leaf
movements, we have developed an efficient transformation
technique in M. pudica, and then established in planta live
imaging of Ca2+ with sensor fluorescent proteins and CRISPR/
Cas9-mediated gene knockout. To obtain candidate genes
responsible for rapid movements, we compared gene expression profiles between pulvini and non-motor organs, andbetween extensor and flexor halves of pulvini. As a result, wegot a number of extensor-enriched genes. After the systematic
knockout experiments of these candidate genes, mutants of
two channel genes, one gene encoding a membrane-associatedprotein with uncharacterized functions, and one transcription
factor showed deficiencies in touch-induced rapid movements.
Currently we are working on the detailed analysis on functionsof those genes and mutans by using transgenic techniques andelectrophysiological studies employing a heterologous expression system in Xenopus oocytes.
Rapid movements in M .pudica take only 1 to 2 seconds forthe whole process and our preliminary observations suggested
that their initial events like Ca2+ increase occurred within 100
ms or less. To achieve higher time resolution for our fluorescent
live imaging, we introduced a high-speed and high-sensitivity
camera system, and developed a method to analyze spatiotemporal propagation of Ca2+ signals robustly. We found that in
the tertiary pulvinus, a mechanical stimulus applied at the centerevokes a rapid concentric propagation of Ca2+ wave with 1.5
cm/s velocity, and it only took 30 ms to reach to the outermost
edge of excitable motor cells in the extensor half (Figure 1).
These analytical methods will facilitate future studies using
mutant lines described above. This study was mainly performed
by Dr. Hiroaki Mano with the support by technical staff.

elicited at the head by the stimulus and transmits through thestalk cells to the basal cells. The advantage of D. rotundifolia
to study transmission mechanisms of action potential is in thesimple structure of the tentacle, which is feasible for electrophysiology and bio-imaging. We succeeded to cultivate D.
rotundifolia under aseptic conditions with proper reactions
to the stimuli, and furthermore, to transform D. rotundifolia
with the green fluorescent protein gene. We improved thetransformation efficiencies with optimizing nutrient conditions in the medium.
In this fiscal year, nuclear genome of D. rotundifolia
was sequenced with long-read sequencing with Nanopore
sequencer and linked-read sequencing with TELL-seq library
and Illumina sequencer in 50 times coverage. To look forcandidate genes to function in the generation and transmission of action potential, we compared mRNA profiles
between different tissues in tentacles and between tentacles
and laminas. Loss-of-function experiments with CRISPR/
Cas9 mediated gene knockout are ongoing.
Ca2+ sensor RGECO expressed transformants were establishedto investigate the relationship between rapid calcium wave andaction potential transmission. Mechanical stimulation to thetentacle head produced a fast Ca2+ wave with similar speed tothe action potential, which was transmitted to the basal cells, but
not to laminar cells. We further investigate the molecular andcellular mechanisms with other transgenic lines.
The Venus flytrap is another carnivorous plant, whose leaf
blade bends to capture prey with two successive mechanicalstimuli applied to the sensory hairs on the surface. The mechanical stimulus is sensed at the middle cells of the hair, in which
action potential is generated to transmit to whole laminar cells.
As done in D. rotundifolia, we looked for candidate genes withcomparative transcriptomics, and gene knockout experiments
are ongoing. This study was mainly conducted by Dr. Shoji
Segami, Ms. Shoko Ooi, Mr. Peng Chen, Mr. Kuniaki Tanase,and Mr. Riku Matsuda with a the support of technical staff.
This work is collaboration work with Drs. Hiraku Suda andMasatsugu Toyota in Saitama University.

Figure 1. Fluorescence images of a red Ca2+ sensor R-GECO in a M.
pudica tertiary pulvinus after stimulated with a needle (100 fps). Right
image shows how the wave front propagates. 4 colors are repeatedly used
(R-B-Y-G) to discriminate each 10 ms frame.

II-2. Molecular mechanisms and evolution of
the generation and transmission of action
potential in the sundew Drosera rotundifolia
and the Venus flytrap Dionaea muscipula
Drosera rotundifolia is a carnivorous plant that captures
prey for nutrients by its tentacles, multicellular hairs on theleaf with sticky digestive fluid at their heads. Each tentacle
senses mechano- and chemical-stimuli at its head, and cells
in a tentacle base bend to bring the captured prey to thecenter of the trap leaf. In this process, action potential is48

Figure 2. Fluorescence images of a RGECO Drosera rotundifolia tentacle
after the tip was stimulated with a needle (red arrow in 0 sec), showingchanges of cytosolic calcium ion amounts. White arrows indicate fronts
of calcium waves.

III. Molecular mechanisms of reprogrammingfrom differentiated cells to stem cells by
STEMIN
Stem cells self-renew and give rise to differentiated cells
to form tissues or organs. Differentiated cells in land plants
can be readily reprogrammed into stem cells during postembryonic development and regeneration. Thus, land plants
have an intrinsic ability to initiate a new genetic regulatory
network, despite the existence of epigenetic memory in differentiated cells. The underlying molecular mechanism andthe changes in the network due to mutation in its components
contribute to the environmental adaptability of land plants
to survive under harsh environments and to the evolution ofcomplex adaptive traits.
To understand the molecular basis of reprogramming, wehave used the moss Physcomitrium patens, in which leaf
cells can be reprogrammed into chloronema apical stem
cells by wounding. During this process, the expressionof STEMIN1 gene, which encodes AP2/ERF transcription
factor, is activated at leaf cells facing the cut. Deletion of
STEMIN1 and its two homologs retards reprogramming,
while ectopic STEMIN1 induction changes differentiated cells into chloronema apical stem cells. We also foundthat transient DNA damage induces reprogramming ofleaf cells into stem cells, which requires the expression of
STEMIN genes. Thus, STEMIN genes play a role in both thewounding- and the DNA damage-induced cellular reprogramming (Figure 3).
We found that ectopic induction of STEMIN1 in leaf cells
decreases a repressive chromatin mark, histone H3 lysine
27 trimethylation (H3K27me3), on its direct target genes.
To elucidate the mechanism regulating the local H3K27me3
reprogramming, we screened for STEMIN1-interacting
proteins and found that some of the components have thepotential to function in the histone modification. Thesesuggest that STEMIN1 could have a role as a platform torecruit components needed for local H3K27me3 reprogramming at the specific loci of the genome. This study was
mainly performed by Dr. Masaki Ishikawa and Mr. Ruan De
Villiers with the support of the technical staff.
Genotoxic
regents

Heavy metals

IV. Evolution of Development and morphology
in land plants
Expansion of individual cells contributes to the morphogenesis of multicellular organisms, especially in land plants,
in which cell migration is restricted by the rigid cell wall.
Cell walls are mainly composed of the polysaccharides andthe outer lipid cuticle layer. Previous reports showed thatmechanisms acting on the cell wall polysaccharides, suchas cellulose and pectin, regulate cell expansion. However,
how other factors regulate cell expansion is not clear. We
found that an ATP-binding cassette (ABC) B family transporter, PpABCB14, localized on the plasma membrane of
the expanding region of gametophore initial cells and theleaf cells. Mr. Liechi Zhang and Dr. Masaki Ishikawa mainly
investigate the molecular mechanisms with support of thetechnical staff.

Publication List:〔Original papers〕
L., Inoue, N., Ishikawa, M., Gotoh, E., Teh, O.-K., Higa, T.," Bao,
Morimoto, T., Ginanjar, E.F., Harashima, H., Noda, N., Watahiki, M.,

"
"
"

"

High saltetc.

DNA damage

Wounding"

DNA repair
ATR
STEMINPpCSP

PpWOX13L

ATG

Hiwatashi, Y., Sekine, M., Hasebe, M., Wada, M., and Fujita, T. (2022).
A PSTAIRE-type cyclin-dependent kinase controls light responses inland plants. Sci. Adv. 8, eabk2116. DOI: 10.1126/sciadv.abk2116
Kamada, T., Otomo, K., Murata, T., Nakata, K., Hiruma, S., Uehara,R., Hasebe, M., and Nemoto, T. (2022). Low-invasive 5D visualizationof mitotic progression by two-photon excitation spinning-disk confocal
microscopy. Sci. Rep. 12, 809. DOI: 10.1038/s41598-021-04543-7
Kanne, J. V, Ishikawa, M., Bressendorff, S., Ansbol, J., Hasebe, M.,
Rodriguez, E., and Petersen, M. (2021). Overexpression of ATG8/LC3
enhances wound-induced somatic reprogramming in Physcomitrium
patens. Autophagy. 18, 1463-1466.DOI: 10.1080/15548627.2021.1975913
Maeda, T., Takahashi, S., Yoshida, T., Shimamura, S., Takaki, Y.,
Nagai, Y., Toyoda, A., Suzuki, Y., Arimoto, A., Ishii, H., Satoh, N.,
Nishiyama, T., Hasebe, M., Maruyama, T., Minagawa, J., Obokata, J.,
and Shigenobu, S. (2021). Chloroplast acquisition without the genetransfer in kleptoplastic sea slugs, Plakobranchus ocellatus. eLife 10,e60176. DOI: 10.7554/eLife.60176
Plant Cell Atlas Consortium, Jha, S.G., Borowsky, A.T., Cole, B.J.,
Fahlgren, N., Farmer, A., Huang, S.-S.C., Karia, P., Libault, M.,
Provart, N.J., Rice, S.L., Saura-Sanchez, M., Agarwal, P., Ahkami,
A.H., Anderton, C.R., Briggs, S.P., Brophy, J.A., Denolf, P., Di
Costanzo, L.F., Exposito-Alonso, M., Giacomello, S., Gomez-Cano,F., Kaufmann, K., Ko, D.K., Kumar, S., Malkovskiy V, A., Nakayama,N., Obata, T., Otegui, M.S., Palfalvi, G., Quezada-Rodriguez, E.H.,
Singh, R., Uhrig, R.G., Waese, J., Van Wijk, K., Wright,R.C., Ehrhardt,
D.W., Birnbaum, and K.D., Rhee, S.Y. (2021). Science Forum: Vision,
challenges and opportunities for a Plant Cell Atlas. eLife 10, e66877.
DOI: 10.7554/eLife.66877
Umeda, M., Ikeuchi, M., Ishikawa, M., Ito, T., Nishihama, R., Kyozuka,J., Torii, K.U., Satake, A., Goshima, G., and Sakakibara, H. (2021).
Plant stem cell research is uncovering the secrets of longevity andpersistent growth. Plant J. 106, 3263335. DOI: 10.1111/tpj.15184

andor

CYCD;1Autophagy
EXPB4

H3K27me3

M., and Hasebe, M. (2022). Molecular mechanisms of" Ishikawa,
reprogramming of differentiated cells into stem cells in the moss

CDKA
or
and

Cell cycle
reentry〔Review article〕

Physcomitrium patens. Curr. Opin. Plant Biol. 65, 102123. DOI:
10.1016/j.pbi.2021.102123

Tip Growth

Figure 3. A working hypothesis of a genetic regulatory network of
cellular reprogramming in Physcomitrium patens.

49National Institute for Basic Biology

Evolutionary Biology and Biodiversity
DIVISION OF SYMBIOTIC SYSTEMS

Professor

Associate Professor

KAWAGUCHI, Masayoshi SOYANO, Takashi

Assistant Professor:
KAWADE, Kensuke
Specially Appointed Assistant Professor:
OKUMA, Nao
Technical Staff:
FUKADA-TANAKA, Sachiko
Postdoctoral Fellow:
TSUGANE, Mika
YANO, Koji
HASHIMOTO, KayoJSPS Postdoctoral Fellow: FUKUDOME, Mitsutaka
Visiting Scientist:
NAKAGAWA, Tomomi
SOKENDAI Graduate Student: GOTO, Takashi
Visiting Graduate Student: NAKAGAWA, Soya
Technical Assistant:
TSUKAO, Chihaya
ODA, Akiko
Admin Support Staff:
KOSUGI, Eikorhizobia with their photosynthetic products. To accomplish this biotic interaction, leguminous plants develop
nodules on their roots. However, more than 80% of land
plant families have symbiotic relationships with arbuscular mycorrhizal (AM) fungi. Despite marked differences
between the fungal and bacterial symbioses, common
genes are required for both interactions. Using a model
legume, Lotus japonicus, we are trying to elucidate themolecular mechanisms of both symbiotic systems.

I. Root nodule symbiosis
Legumes develop de novo organs known as root nodules
to accommodate symbiotic bacteria called rhizobia.
Nodule formation involves two distinct processes: rhizobial infection in epidermis and nodule development
accompanied by cell division in cortex (Figure 1).

Figure 1. Processes for nodule formation.

Rhizobium3legume symbiosis is one of the most successful mutually beneficial interactions on Earth. In thistype of symbiosis, soil bacteria called rhizobia supplies
the host legumes with ammonia produced through bacterial nitrogen fixation. In return, host plants provide theVisual overview of this lab9s work.

50

The cortical cell division provides an indispensable
scaffold for rhizobia progression from epidermis to cortex(Figure 1; pink line), leading to successful nodule formation. Cortical cell division occurs just below the site of
rhizobial infection in epidermis. Therefore, there appears
to be a spatiotemporal coordination across epidermis
and cortex in this symbiotic organogenesis. Epidermal
expression of genes required for early symbiotic genes
is sufficient for nodule development in cortex, suggesting that some kinds of signals generated in epidermis
trigger cortical cell division (Figure 1; blue dotted arrow).
However, little is known about the mechanism that coordinates these two events.
In this study, we conducted a time-course transcriptome
analysis using a L. japonicus non-nodulation mutant<daphne=, which has uncoupled symbiotic events inepidermis and cortex, in that it promotes excessive rhizobial infection in epidermis but does not produce nodule
primordia in cortex. Among genes that showed different
expression patterns in daphne and wild type, we foundFigure 2. Auxin methylation in nodule symbiosis. IAMT1 converts IAA
into MeIAA (left). Relative MeIAA amount of wild type (WT) anddaphne at 0 (non-inoculation) or 2 d after inoculation (DAI) (right).

IAA CARBOXYL METHYLTRANSFERASE 1 (IAMT1),
which encodes the enzyme that specifically converts
auxin (indole-3-acetic acid; IAA) into its methyl ester(MeIAA). A significant MeIAA increase after rhizobial
infection was detected by using daphne roots, in which
excess rhizobia are infected (Figure 2).
In Arabidopsis, IAMT1 reportedly serves development
and differential growth of shoot. On the other hand, we
found that IAMT1 is duplicated in the legume lineage,and one of the duplicates (named IAMT1a) was expressedby rhizobial infection in root of L. japonicus. LjIAMT1aknockdown inhibited nodule formation (Figure 3).
LjIAMT1a overexpression promoted MeIAA accumulation and nodule formation. In contrast, overexpressionof LjMES17, a MeIAA esterase, decreased MeIAA levels
and nodule number. These showed that auxin methylation
is essential for nodule formation.

II. Arbuscular mycorrhizal symbiosis
Arbuscular mycorrhizal (AM) symbiosis is the oldest
beneficial mutualistic relationship between the majorityof terrestrial plants and AM fungi. AM fungal spores
germinate and elongate their hyphae toward the rootof host plants then enter the root cells and form finely
branched structures, arbuscules, for exchanging nutrientswith plants (Figure 5). AM fungi deliver mineral nutrients especially phosphorus in the soil to host plants. In
return, they obtain organic carbon sources such as sugars
which are produced through photosynthesis. AM fungi
are obligate symbionts depending on their host plants foressential nutrients, thus they are incapable of propagationby themselves.

Figure 3. LjIAMT1a-knockdown
inhibited nodule formation. Hairy
roots harboring empty vector (forcontrol; left) or LjIAMT1a-RNAi
vector (for knock-down; right).
Nodules are indicated by arrowheads. (Scale bars, 1 cm.)

Notably, application of MeIAA significantly induced
expression of a symbiotic gene, NIN, without rhizobia(Figure 4). NIN is a key regulator for cortical cell
division; therefore, auxin methylation could act on theprocess of cortical cell division. Whereas IAMT1a wasexpressed mainly in root epidermis, it would be interesting to see how MeIAA affects cortex. The result alsosuggests that auxin methylation is not simply due toalteration of auxin homeostasis, contrary to what manystudies assumed, MeIAA, as well as other auxin secondary metabolites, is also an inactive molecule of IAA.
Understanding the function of auxin methylation and
MeIAA in nodule development should open a new avenue
for auxin metabolisms in the plant biology.

Figure 4. Exogenous MeIAA significantly induced NIN expression
without rhizobia. Relative expression levels of NIN after treatment
with DMSO as mock (white bar),
IAA (gray bar), or MeIAA (bluebar) for 24 h.

Figure 5. AM fungus R. clarus HR1 growing with carrot hairy roots.
(A) Expanded extraradical hyphae and formed symbiotically generated
spores between the roots. Bar, 500 µm. (B) Fungal structures stained bygreen fluorescent (Wheat germ agglutinin Oregon green 488) in the root.
Arrowheads, arbuscules inside root cortical cells. Bar, 100 µm.

In the history of AM fungal culture research, it was
shown that an AM fungus Rhizophagus irregularis could
complete its life cycle in co-culture with mycorrhizahelper bacterium Paenibacillus validus in the absence of
host plants. Over the last several years, it was reported
that AM fungi receive not only sugars but also fatty acids.
Subsequently, from these experiments, it was demonstrated that some fatty acids such as palmitoleate andmyristate induce R. irregularis spore formation under an
asymbiotic (completely host-free) culture media.
We found that a fungus isolated from Aichi prefecture, Rhizophagus clarus HR1, whose genome has beensequenced, shows better growth on the asymbiotic culture
medium, thus is more suitable for this culture method. A
fatty acid, myristate promotes R. clarus hyphal growth,
but the addition of only myristate could not ensure induction of asymbiotically-generated spores (AS) formation.
Furthermore, the number of AS in this culture was muchsmaller than those of symbiotically-generated spores (SS)which are produced in the co-culture system with hairy roots.
Some phytohormones have been reported that they
have important roles as signals during AM symbiosis.
Especially, strigolactone is well known as a major signal
which induces hyphal elongation and branching and stimulates their mitochondrial activity in the pre-symbiotic
stage. By supplementing synthetic strigolactone, GR24
with myristic acid, the time of germination and AS formation in R. clarus was accelerated. Although not all R.
clarus spores formed AS in the medium supplemented

51

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

only with myristic acid, the addition of GR24 resulted ina quite high efficiency of AS formation to nearly 100 %.
Besides strigolactone, it was reported that there was anincrease of jasmonate levels in AM roots and the upregulation of genes involved in jasmonic acid biosynthesisin the cells containing arbuscules. Application of jasmonates in the medium with myristic acid and GR24 led R.
clarus to produce a higher number of AS with larger sizes(Figure 6).

Publication List:〔Original papers〕
T., Soyano, T., Liu, M., Mori, T., and Kawaguchi, M. (2022).
" Goto,Auxin methylation by IAMT1, duplicated in the legume lineage,

"
"
"
"

Figure 6. Produced spores on asymbiotic culture medium, supplemented
with only myristate (A), myristate and GR24 (B), myristate, GR24 andmethyl jasmonate (C). Arrowheads, parent spores. Bars, 500 µm.

Although the number of AS produced in the medium
with myristate, GR24 and jasmonate has been close
to that of SS, the size of AS is still smaller than SS.
However, AS can be subcultured with the same efficiency
as the first asymbiotic culture. Moreover, AS was capableof infecting hairy roots and plants. The growth of Welsh
onions was significantly promoted by inoculation withAS as well as with SS (Figure 7). Our findings would lead
us to more extensive studies of AM symbiosis and to theapplication of AS as an inoculum for basic research andfor sustainable agriculture.

""promotes root nodule development in Lotus japonicus. Proc. Natl.
Acad. Sci. U.S.A. 119, e2116549119. DOI: 10.1073/pnas.2116549119
Hayashi-Tsugane, M., and Kawaguchi, M. (2022). Lotus japonicus
HAR1 regulates root morphology locally and systemically under amoderate nitrate condition in the absence of rhizobia. Planta 255, 95.
DOI: 10.1007/s00425-022-03873-8
Misawa, F., Ito, M., Nosaki, S., Nishida, H., Watanabe, M., Suzuki,T., Miura, K., Kawaguchi, M., and Suzaki, T. (2022). Nitrate transport
via NRT2.1 mediates NIN-LIKE PROTEIN-dependent suppression ofroot nodulation in Lotus japonicus. Plant Cell 34, 184431862. DOI:
10.1093/plcell/koac046
Nishida, H., Nosaki, S., Suzuki, T., Ito, M., Miyakawa, T., Nomoto,M., Tada, Y., Miura, K., Tanokura, M., Kawaguchi, M., and Suzaki,
T. (2021). Different DNA-binding specificities of NLP and NIN
transcription factors underlie nitrate-induced control of root nodulation.
Plant Cell 33, 234032359. DOI: 10.1093/plcell/koab103
Tanaka, S., Hashimoto, K., Kobayashi, Y., Yano, K., Maeda, T.,
Kameoka, H., Ezawa, T., Saito, K., Akiyama, K., and Kawaguchi, M.
(2022). Asymbiotic mass production of the arbuscular mycorrhizal
fungus Rhizophagus clarus. Commun. Biol. 5, 43. DOI: 10.1038/
s42003-021-02967-5
Fukudome, M., Shimokawa, Y., Hashimoto, S., Maesako, Y., UchiFukudome, N., Niihara, K., Osuki, K.I, and Uchiumi, T. (2021).
Nitric oxide detoxification by Mesorhizobium loti affects root nodule
symbiosis with Lotus japonicus. Microbes Environ. 36, ME21038.
DOI: 10.1264/jsme2.ME21038
Kobayashi, Y., Katsuren, M., Hojo, M., Wada, S., Terashima,y., Kawaguchi, M., Tokuda, G., Kinjo, K., and Shigenobu, S. (2022).
Taxonomic revision of Termitomyces species found in Ryukyu
Archipelago, Japan, based on phylogenetic analyses with three loci.
Mycoscience 63, 33-38. DOI: 10.47371/mycosci.2021.11.001〔Review articles〕
N., and Kawaguchi, M. (2021). Systemic optimization of" Okuma,legume nodulation: A shoot-derived regulator, miR2111. Front. Plant
Sci. 12, 682486. DOI: 10.3389/fpls.2021.682486

T., Liu, M., Kawaguchi, M., and Hayashi, M. (2021).
" Soyano,
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

Figure 7. Inoculation of AM fungal spores to Welsh onions. Numbers
following AS or SS are the number of spores for inoculation. (A) An
appearance of plants inoculated with AM spores (upper). Bar, 5 cm.
Ink-stained fungal structures in the roots (bottom). Bars, 50 µm. (B) Dry
weight of Welsh onion shoots. Different letters indicate significant differences among treatments in each trial using Wilcoxon rank-sum test with
Bonferroni correction, p < 0.05.

52

DIVISION OF EVOLUTIONARY DEVELOPMENTAL BIOLOGY

Professor

NIIMI, Teruyuki

Assistant Professor:

ANDO, Toshiya
NAKAMURA, Taro
Specially Appointed Assistant Professor:
MORITA, Shinichi
Technical Staff:
MIZUTANI, Takeshi
Postdoctoral Fellow:
MATSUOKA, Yuji
SAKURA, Kazuki
JSPS Postdoctoral Fellow: YANO, Koki
Research Staff:
CHIKAMI, Yasuhiko
KAWAGUCHI, Haruka
MORITA, Junko
TAGUCHI, Rie
SOKENDAI Graduate Student: KITAZAWA, Yurina
Visiting Undergraduate: ODA, Hiroki
Visiting Scientist:
TAKENAKA, Masaki
Technical Assistant:
YOKOYAMA, Michiko
SUGIYAMA, Hiroko
Admin Support Staff:
SAITO, EikoI. 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 evolutionary 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.

B
A

Figure 1. The firebrat, Thermobia domestica (A). The two-spottedcricket, Gryllus bimaculatus (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 lab9s work.

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.
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 wing53

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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. Wing 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 ladybird9s 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 ladybirds9 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.

54

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 isdue to the high risk of losing valuable genetic bioresources in
non-model insects. We recently established ovary transplantation and ovarian cryopreservation techniques in ladybird
beetles. We hope that the genetic tools and techniques that wehave established will further facilitate this research.
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 de novo multiple times 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 morphological structure is formed (Figure 4 A).

We first investigated the process of horn primordium formation to understand the developmental mechanism of hornformation. The insect body is covered by a rigid exoskeleton
mainly consisting of chitin, so changes in body size andshapes are produced by molting. Beetle horns first appear
at the pupal period (Figure 4 A). During the prepupal period(about 130 hours) which is an intermediate stage between thelast larval instar stage and pupation, the head horn primordia
are packed folded inside the larval head capsule (Figure 4B). This folded horn primordium develops into its final 3D
shape (pupal horn) during pupation. To properly make thehorn during pupation, the folding pattern (Figure 4 B) andsurface micro furrows (Figure 4 C) are precisely formed on
the horn primordium and packed within the confined spaceof the prepupal head. We recently attempt to use micro-CT toanalyze 3D pattern formation of head horn primordia during
the prepupal period. We found that horn 3D morphogenesis
is accomplished in only 72 hours from the prepupal initiation(Figure 4 B, C).

Publication List:〔Original papers〕
S., Chikami, Y., Kawaguchi, H., Krylov, A.A., Niimi, T., and" Hashiro,
Yasueda, H. (2021). Efficient production of long double-stranded RNAs"""

applicable to agricultural pest control by Corynebacterium glutamicum
equipped with coliphage T7-expression system. Appl. Microbiol.
Biotechnol. 105, 498735000. DOI: 10.1007/s00253-021-11324-9
Miyazaki, S., Fujiwara, K., Kai, K., Masuoka, Y., Gotoh, H., Niimi,
T., Hayashi, Y., Shigenobu, S., and Maekawa, K. (2021). Evolutionarytransition of doublesex regulation from sex-specific splicing to malespecific transcription in termites. Sci. Rep. 11, 15992. DOI: 10.1038/
s41598-021-95423-7
Ohde, T., Mito, T., and Niimi, T. (2022). A hemimetabolous wingdevelopment suggests the wing origin from lateral tergum of a winglessancestor. Nat. Commun. 13, 979. DOI: 10.1038/s41467-022-28624-x
Ylla, G., Nakamura, T., Itoh, T., Kajitani, R., Toyoda, A., Tomonari,S., Bando, T., Ishimaru, Y., Watanabe, T., Fuketa, M., Matsuoka, Y.,
Barnett, A.A., Noji, S., Mito, T., and Extavour, C.G. (2021). Insights
into the genomic evolution of insects from cricket genomes. Commun.
Biol. 4, 733. DOI: 10.1038/s42003-021-02197-9〔Review article〕
T., and Ando, T. (2021). Evo-devo of wing colour patterns" Niimi,in beetles. Curr. Opin. Genet. & Dev. 69, 973102. DOI: 10.1016/j.
gde.2021.02.007

Figure 4 Morphological change of horn primordia in Trypoxylus dichotomus.
(A) Developmental process of Trypoxylus dichotomus male. (B, C)
Micro-CT images of the male head horn primordium during pupal development. (Adapted from Morita and Niimi, Sanshi Konchu Biotec, 86:
1453151, 2020.)

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

55

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF EVOLUTIONARY GENOMICS

Professor:

SHIGENOBU, Shuji

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

Overview<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 host9s 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 withthat of bacterial symbiont Buchnera aphidicola illustrates
the remarkable interdependency between these two organisms (IAGC, 2010; Shigenobu et al., 2000; Shigenobu &Yorimoto, 2022). 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 criticalgenes, which might account for the evolutionary success ofaphids in obtaining beneficial symbionts. Lineage-specificgene duplications have occurred in genes over a broad rangeof functional 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,2013). The BCR mRNAs are first expressed at a developmental time point coinciding with the incorporation ofsymbionts strictly in the cells that contribute to the bacteriocyte, and this bacteriocyte-specific expression is maintained throughout the aphid9s life. Furthermore, some BCRs
showed antibiotic activity (Uchi et al., 2019). These results
suggest that BCRs act within bacteriocytes to mediate thesymbiosis with bacterial symbionts, which is reminiscent of
the cysteine-rich secreted proteins of leguminous plants that
also regulate endosymbionts. Employment of small cysteinerich peptides may be a common tactic of host eukaryotes tomanipulate 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 lab9s work.

56

Genomics of emerging model insects

Publication List

Recent remarkable technological advancements allow us
to study any organisms at molecular and genetic levels,
which has been difficult for non-model organisms. Insectscience is also benefited by the technological innovation.
New entomology is starting to study the amazing variety ofmorphology, phisiology and ethology of insects by using
these state-of-the-art technologies. Our group is developing
a platform for emerging model insects to read the genomes/transcriptomes and then manipulate their genomes.
Termites are emerging model social organisms characterized by a sophisticated caste system. We recently sequenced
the genome of the subterranean termite Reticulitermes
sepratus (Shigenobu et al., 2022). The analyses of ~1.0 Gb
genome and 15,591 genes revealed the significance of gene
duplication in social evolution. Gene duplication associated with caste-biased gene expression was prevalent in thetermite genome. The duplicate genes comprised diverse categories related to social functions, such as chemical communication, social immunity and defense, and they were often
expressed in a caste-specific organ. Gene duplication may
facilitates social evolution through regulatory diversification
leading to caste-biased expression and functional specialization.
Beyond insect genomics, we are involved in several
genome projects of emerging model organisms. We recently
published the genome information of a photosynthetic sea
slug. Some sea slugs take up chloroplasts from the algaethat they consume into their cells. These chloroplasts retain
their ability to perform photosynthetic activity within the
animal cells for several months, and thus provide them withphotosynthesis-derived nutrition. This process is called kleptoplasty. Our genome analysis of Plakobranchus ocellatus
revealed the chloroplast acquisition without gene transfers in
the photosynthetic sea slug (Maeda et al., 2021).

〔Original papers〕
K., Ikuta, T., Tame, A., Yamaguchi, K., Shigenobu, S.," Igawa-Ueda,
Hongo, Y., Takaki, Y., Fujikura, K., Maruyama, T., and Yoshida, T.

"
"
"

"
"
"
"
"

Caste development and symbiosis in the social
aphid Ceratovacuna japonica
Eusociality has evolved repeatedly among insect lineages
and represents one of the major transitions in evolution.
Molecular mechanisms underlying sociality have been extensively studied with hymenopteran lineages, such as ants,
bees, and wasps, and termites. Some aphid species show
eusociality and important groups for better understandingof the eusocial evolution, since they are one of the primitive eusocial species that belong to Hemiptera. We focus
on the social aphid Ceratovacuna japonica, because werecently established the laboratory culture system. First,
we investigated the sterility regulation in the soldier casteof the aphids (Chung and Shigenobu, 2022). We foundthat soldiers, that are completely sterile, possess a pair ofovarioles, but soldiers9 ovarioles were small and lacking gastrulating embryos. We also found that soldier reproduction
is constrained by apoptosis in maternal nutritive cells andnecrosis in oocytes and embryos. C. japonica also contains
endosymbionts. The eusocial aphid C. japonica provides us
with a unique opportunity to study how symbiosis operates ina social setting. We are currently investigating the interfaceof symbiosis and sociality.

(2021). Symbiont Transmission onto the Cell Surface of Early Oocytes
in the Deep-sea Clam Phreagena okutanii. Zoolog. Sci. 38, 1403147.
DOI: 10.2108/zs200129
Kobayashi, Y., Katsuren, M., Hojo, M., Wada, S., Terashima, Y.,
Kawaguchi, M., Tokuda, G., Kinjo, K., and Shigenobu, S. (2022).
Taxonomic revision of Termitomyces species found in Ryukyu
Archipelago, Japan, based on phylogenetic analyses with three loci.
Mycoscience 63, 33338. DOI: 10.47371/mycosci.2021.11.001
Korgaonkar, A., Han, C., Lemire, A.L., Siwanowicz, I., Bennouna, D.,
Kopec, R.E., Andolfatto, P., Shigenobu, S., and Stern, D.L. (2021). A
novel family of secreted insect proteins linked to plant gall development.
Curr. Biol. 31, 1836-1849.e12. DOI: 10.1016/j.cub.2021.01.104
Maeda, T., Takahashi, S., Yoshida, T., Shimamura, S., Takaki, Y.,
Nagai, Y., Toyoda, A., Suzuki, Y., Arimoto, A., Ishii, H., Satoh, N.,
Nishiyama, T., Hasebe, M., Maruyama, T., Minagawa, J., Obokata, J.,
and Shigenobu, S. (2021). Chloroplast acquisition without the genetransfer in kleptoplastic sea slugs, Plakobranchus ocellatus. eLife 10,e60176. DOI: 10.7554/eLife.60176
Miyazaki, S., Fujiwara, K., Kai, K., Masuoka, Y., Gotoh, H., Niimi,
T., Hayashi, Y., Shigenobu, S., and Maekawa, K. (2021). Evolutionarytransition of doublesex regulation from sex-specific splicing to malespecific transcription in termites. Sci. Rep. 11, 15992. DOI: 10.1038/
s41598-021-95423-7
Nozaki, T., Suetsugu, K., Sato, K., Sato, R., Takagi, T., Funaki, S.,
Ito, K., Kurita, K., Isagi, Y., and Kaneko, S. (2021). Development of
microsatellite markers for the geographically parthenogenetic stick
insect Phraortes elongatus (Insecta: Phasmatodea). Genes Genet. Syst.
96, 1993203. DOI: 10.1266/ggs.21-00022
Nozaki, T., and Matsuura, K. (2021). Oocyte resorption in termite
queens: Seasonal dynamics and controlling factors. J. Insect Physiol.
131, 104242. DOI: 10.1016/j.jinsphys.2021.104242
Nozaki, T., Hojo, M., Yashiro, T., and Matsuura, K. (2021). First
Record of the Soil-feeding Higher Termites (Isoptera: Termitidae) from
Okinawa Island, the Central Ryukyus, Japan. SOCIOBIOLOGY 68,
e6159. DOI: 10.13102/sociobiology.v68i2.6159
Shigenobu, S., Hayashi, Y., Watanabe, D., Tokuda, G., Hojo, M.Y.,
Toga, K., Saiki, R., Yaguchi, H., Masuoka, Y., Suzuki, R., Suzuki, S.,
Kimura, M., Matsunami, M., Sugime, Y., Oguchi, K., Niimi, T., Gotoh,
H., Hojo, M.K., Miyazaki, S., Toyoda, A., Miura, T., and Maekawa,
K. (2022). Genomic and transcriptomic analyses of the subterranean
termite Reticulitermes speratus: Gene duplication facilitates social
evolution. Proc. Natl. Acad. Sci. U.S.A. 119, e2110361119. DOI:
10.1073/pnas.2110361119

T., Matsubara, H., Minamitani, F., Satoh, Y., Tozawa, S.," Takeuchi,
Moriyama, T., Maruyama, K., Suzuki, K.T., Shigenobu, S., Inoue, T.,

"
"
"
"

Tamura, K., Agata, K., and Hayashi, T. (2022). Newt Hoxa13 has an
essential and predominant role in digit formation during development
and regeneration. Development 149, dev200282. DOI: 10.1242/
dev.200282
Tanaka, S., Hashimoto, K., Kobayashi, Y., Yano, K., Maeda, T.,
Kameoka, H., Ezawa, T., Saito, K., Akiyama, K., and Kawaguchi, M.
(2022). Asymbiotic mass production of the arbuscular mycorrhizal
fungus Rhizophagus clarus. Commun. Biol. 5, 43. DOI: 10.1038/
s42003-021-02967-5
Tominaga, T., Miura, C., Sumigawa, Y., Hirose, Y., Yamaguchi, K.,
Shigenobu, S., Mine, A., and Kaminaka, H. (2021). Conservation andDiversity in Gibberellin-Mediated Transcriptional Responses Among
Host Plants Forming Distinct Arbuscular Mycorrhizal Morphotypes.
Front. Plant Sci. 12, 795695. DOI: 10.3389/fpls.2021.795695
Tong, X., Kanzaki, N., and Akimoto, S. (2021). First record of amermithid nematode (Nematoda: Mermithidae) parasitizing winged
females of gall-forming aphids (Hemiptera: Aphididae: Eriosomatinae).
Entomol. Sci. 24, 3543360. DOI: 10.1111/ens.12486
Yashiro, T., Tea, Y.-K., van der Wal, C., Nozaki, T., Mizumoto,N., Hellemans, S., Matsuura, K., and Lo, N. (2021). Enhanced57

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
heterozygosity from male meiotic chromosome chains is superseded byhybrid female asexuality in termites. Proc. Natl. Acad. Sci. U.S.A. 118,"e2009533118. DOI: 10.1073/pnas.2009533118
Yoshida, Y., Shaikhutdinov, N., Kozlova, O., Itoh, M., Tagami, M.,
Murata, M., Nishiyori-Sueki, H., Kojima-Ishiyama, M., Noma,
S., Cherkasov, A., Gazizova, G., Nasibullina, A., Deviatiiarov, R.,
Shagimardanova, E., Ryabova, A., Yamaguchi, K., Bino, T., Shigenobu,
S., Tokumoto, S., Miyata, Y., Cornette, R., Yamada, T.G., Funahashi,A., Tomita, M., Gusev, O., and Kikawada, T. (2022). High quality
genome assembly of the anhydrobiotic midge provides insights on asingle chromosome-based emergence of extreme desiccation tolerance.
NAR Genomics Bioinforma. 4, lqac029. DOI: 10.1093/nargab/lqac029〔Review articles〕
S. & Yorimoto, S. Aphid hologenomics: current status and" Shigenobu,
future challenges. Curr Opin Insect Sci4, 100882. DOI:10.1016/j.

"
"

58

cois.2022.100882.
Shigenobu, S. (2022). Editorial overview: Insect genomics: Genomics
of emerging model insects. Curr. Opin. Insect Sci. 51, 100913. DOI:
10.1016/j.cois.2022.100913
Miura, T., Oguchi, K., Yamaguchi, H., Nakamura, M., Sato, D.,
Kobayashi, K., Kutsukake, N., Miura, K., Hayashi, Y., Hojo, M.,
Maekawa, K., Shigenobu, S., Kano, T., and Ishiguro, A. (2022).
Understanding of superorganisms: collective behavior, differentiationand social organization. Artif. Life Robot. 27, 2043212. DOI: 10.1007/
s10015-022-00754-x

LABORATORY OF BIORESOURCES

Specially Appointed Professor

NARUSE, Kiyoshi

Research Staff:

KANEKO, Hiroyo
HARA, Ikuyo
YANOGAWA, Azusa
Visiting Scientist:
SATO, Tadashi
NITTA, Masato
Visiting Graduate Student: IMAEDA YASSUMOTO,
TamirisTechnical Assistant:
AJIOKA, Rie
KOIKE, Chieko
KOIKE, Yukari
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) from 2007.

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

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

II. The Medaka Inbred Kiyosu-Karlsruhe(MIKK) panel and their genomic variationand epigenomic landscape
The Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel is thefirst near-isogenic panel of 80 inbred lines derived from awild founder population collected from Kiyosu, Toyohashi.
Inbred lines provide fixed genomes that make it possible torepeat studies, to vary both genetic information and environmental conditions in controlled studies, as well as to conduct
functional studies. MIKK will thus make it possible to investigate phenotype-to-genotype association studies of complex
genetic traits while carefully controlling interacting factors.
Its applications are numerous, and include genetic research,human health, drug development, and fundamental biology.
Using short reads from Illumina, we further characterise eachof the 80 MIKK lines according to their genetic composition,using the Southern Japanese medaka inbred strain HdrR asa reference genome. We used Oxford Nanopore long readsequencing technology to analyze 12 representative lines
from MIKK to analyze the larger and more complex structural variations. We also investigate line-specific CpG methylation and performed differential DNA methylation analysis
across these 12 lines.

III. Genome sequence of the Javanese medaka,
Oryzias javanicus, as a model for studying
seawater adaptation
Medaka fish in the genus Oryzias are an emerging model
system for studying the molecular basis of vertebrate evolution. This genus contains approximately 35 species andexhibits great morphological, ecological and physiological differences among it9s species. Among these species,
the Java medaka, Oryzias javanicus, is the species thathas most typically adapted to seawater. We sequenced
and assembled the whole genome of O. javanicus, as amodel fish species for studying molecular mechanisms ofseawater adaptation. In teleost fish, the major osmoregulatory organs are the gills, intestine and kidney, and these 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

59

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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 2. NBRP Medaka website

Publication List:〔Original papers〕
T., Brettell, I., Leger, A., Wolf, N., Kusminski, N.," Fitzgerald,
Monahan, J., Barton, C., Herder, C., Aadepu, N., Gierten, J., Becker, C.,

"

"

60

Hammouda, O.T., Hasel, E., Lischik, C., Lust, K., Sokolova, N., Suzuki,R., Tsingos, E., Tavhelidse, T., Thumberger, T., Watson, P., Welz, B.,
Khouja, N., Naruse, K., Birney, E., Wittbrodt, J., and Loosli, F. (2022).
The Medaka Inbred Kiyosu-Karlsruhe (MIKK) panel. Genome Biol.
23, 59. DOI: 10.1186/s13059-022-02623-z
Leger, A., Brettell, I., Monahan, J., Barton, C., Wolf, N., Kusminski,N., Herder, C., Aadepu, N., Becker, C., Gierten, J., Hammouda, O.T.,
Hasel, E., Lischik, C., Lust, K., Sokolova, N., Suzuki, R., Tavhelidse, T.,
Thumberger, T., Tsingos, E., Watson, P., Welz, B., Naruse, K., Loosli, F.,
Wittbrodt, J., Birney, E., and Fitzgerald, T. (2022). Genomic variations
and epigenomic landscape of the Medaka Inbred Kiyosu-Karlsruhe(MIKK) panel. Genome Biol. 23, 58. DOI: 10.1186/s13059-022-02602-4
Seleit, A., Ansai, S., Yamahira, K., Masengi, K. W., Naruse, K., &Centanin, L. (2021). Diversity of lateral line patterns and neuromast
numbers in the genus Oryzias. Journal of Experimental Biology, 224,
jeb242490. DOI: 10.1242/jeb.242490

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, it9s 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, the molecularswitch 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, a

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.

61

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
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 two yeast 4EBPs, Caf20 and Eap1,
have properties in common with 4EBP1. Caf20, but not
Eap1, is phosphorylated in a TORC1-dependent manner, itbinds to eIF4E, and it never associates to eIF4G. However,Caf20 was not directly phosphorylated by TORC1, Caf20eIF4E binding was not affected by TORC1 activity, andCaf20-eIF4E complex was found in ribosome fraction, suggesting that the function of the yeast 4EBP is different fromthat of its mammalian counterparts.

Publication List:〔Original paper〕
T., Ishii, T., Kamada, Y., Funakoshi, M., Kobayashi, H.," Sekiguchi,
and Furuno, N. (2022). Involvement of Gtr1p in the oxidative stressresponse in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res.
Commun. 598, 1073112. DOI: 10.1016/j.bbrc.2022.02.016

62

LABORATORY OF BIOLOGICAL DIVERSITYHOSHINO Group
Assistant Professor:
Technical Assistant:

HOSHINO, Atsushi
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 pale grayishpurple flowers, which can be stably inherited by further generations.

Genomic structures and their genetic information are stablytransmitted into daughter cells and future generations during
cell division; however, they can vary genetically and/or
epigenetically. Such variability impacts gene expression andevolution. To understand these genomic dynamics in eukaryotes, particularly in plants, we are analyzing the flower
pigmentation of morning glories, including Ipomoea nil(Japanese morning glory), I. purpurea (the common morning
glory) and I. tricolor.

I. Flower pigmentation patterns
The wild type morning glories produce flowers with auniformly pigmented corolla. For our study, we collectedmutants displaying pigmentation patterns. Flower pigmentation patterns are easily observable and the molecular mechanisms underlying these phenomena provide useful model
systems for investigating genomic variability.
Recessive mutations, duskish of I. nil and pearly-v of I.
tricolor, confer variegated flowers caused by a stable insertionof a transposable element into a gene for flower pigmentation,
regulated by epigenetic mechanisms (Figure 1). We intend toanalyze detailed molecular mechanisms of these mutations.

II. Whole-genome analysis of various I. nil lines
The National BioResource Project (NBRP) Morning
Glory (described below) maintains ~3,500 lines, including 2 standard lines: the Tokyo Kokei Standard and Violet.
A high-quality draft whole-genome sequence of the Tokyo
Kokei Standard, which is accessible from our database, hasbeen published. The NBRP Genome Information Upgrading
Program supported whole-genome sequencing of 100 representative lines. We chose lines based on the needs of theresearch community and to provide information regarding
their polymorphisms and gene mutations. We included themultiple mutants called <Henka-Asagao,= which covered
the major mutations, and the wildtype plants isolated fromnatural populations outside Japan. From this, we discovereda total of 25,000 Tpn1 transposon-induced insertion polymorphisms and SNPs, Indels, and CNVs at 25 million loci.
Several of those polymorphisms were tightly linked to morphological mutations, including the mutations that <HenkaAsagao= carry.

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

III. Flower opening and time control
The time of flower opening is important for the reproductive strategy of plants, and many plants are known toflower at specific times during the day. However, little is
known about the molecular mechanisms that determine
flower opening time as well as flower opening itself. As
their name suggests, morning glories typically bloom in theearly morning. There is significant physiological knowledge
about the circadian rhythm that controls flower opening time.
To elucidate these molecular mechanisms, we have begun afunctional analysis of genes for petal opening and the circadian rhythm.

IV. Morning glory bio-resources
NIBB is the subcenter for the NBRP dedicated to morning
glories. In this project, we collect, maintain, and distributestandard 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 inJapan and has a 100-year history of extensive related genetic
studies. Our collection includes 241 lines and 177,000 DNA
clones. The whole-genome sequence, the transcriptome

63

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

sequences, and the end sequences of the DNA clones can beviewed via the I. nil genome database (http://viewer.shigen.
info/asagao/index.php).

Publication List:〔Original paper〕
K., Furuta, T., Yamaji, N., Kuniyoshi, D., Ishihara, M.," Nagaki,
Kishima, Y., Murata, M., Hoshino, A., and Takatsuka, H. (2021).
Effectiveness of Create ML in microscopy image classifications: a
simple and inexpensive deep learning pipeline for non-data scientists.
Chromosome Res. 29, 3613371. DOI: 10.1007/s10577-021-09676-z

64

LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:

bore slightly larger grains (Figure 1) as a dominant inheritance. Transposon-display identified the insertion site ofnDart1 in the Lgg mutant.

TSUGANE, Kazuo

II. Analysis of Lgg mutants

Although transposons occupying a large portion of the
genome in various plants were once thought to be junkDNA, they play an important role in genome reorganization and evolution. Active DNA transposons are important tools for gene functional analysis. The endogenous
non-autonomous transposon, nDart1, in rice is said togenerate various transposon-insertion mutants because
nDart1 elements tend to insert into genic regions undernatural growth conditions. The transpositions of nDart1
were promoted by an active autonomous element, aDart127, on chromosome 6. By using the endogenous nDart1/
aDart1-27 system in rice, a large-scale nDart-inserted
mutant population was easily generated under normal
field conditions, and the resulting tagged lines were freeof 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 ofnDart have been screened for characteristic phenotypes.

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:LGGNPGFP was transformed into rice calli. GFP fluorescencespots were observed in the nuclei in calli. These results
suggest that LGG is localized to the nucleus. To verify
that LGG was responsible for the long grain phenotype, knockout (GE) and overexpression (OE) lines were
produced by transforming a CRISPR/Cas9 constructtargeting the RNA recognition motif region of LGG andan LGG construct with its WT promoter into into cv.
Nipponbare (NP), respectively (Figure 2(A)). Several
independent GE lines showed significantly increased
grain length, and three independent OE lines had shorter
grains than NP. Any significant differences of panicle
length, number of panicles per plant and number of spikelets per panicle were not observed among NP, GE andOE except for culm length. Observations of longitudinal
sections from lemmas of GE and OE lines revealed thatthe cell sizes of GE and OE lines were comparable to NP(Figure2 B-C), suggesting that LGG might regulate thelongitudinal cell number of spikelet hull and thus grain
length.

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

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

Figure 2. The spikelets morphology and longitudinal sections and cell
length of the lemma of NP, GE, and OE, respectively. (A) The spikelets.
Bar = 5 mm. (B) The Section of lemma in transgenic plants. The red andblack arrows indicate adaxial epidermis and chlorophyll-contained parenchyma cell. (C) Cell length of lemma in NP, GE and OE. n = 3. mean ± SD.

65

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITY
KATO GroupSpecially Appointed Assistant Professor:
KATO, Kagayaki
Technical Assistant:
HYODO, Miwa

Computer programs that accurately capture and measure thefeatures from images that express the phenomenon become
an inevitable tool to objectively describe and evaluate abiological phenomenon obtained as digital imaging data.
From this point of view, we are developing image analysis
programs for each experimental case and analyzing image
data that captures various biological phenomena.

I. Cell segmentation/tracking system
To better understand organ formation mechanisms, it is necessary to analyze individual cells9 morphology and dynamics
quantitatively. However, it is difficult to achieve it due tothe large image volume size generated by such as time-lapse
microscopy and its ambiguity.
To unveil organogenesis from the point of view of distinct
cell behaviors, we are developing application software
capable of describing cell dynamics from time-lapse imaging
data sets by employing image processing techniques. To
observe this, we are developing a software pipeline which
will automatically recognize individual cell shapes out
of 3D space and tracks them through time. This system
extracts cell boundaries and reconstitutes cell shapes in theform of a skeletonized chain of voxels spanning 3D space.
This abstract form of cell visualization makes it possible todescribe morphometric quantities and kinetics of cells at asingle-cell resolution (Figure 1). These morphometric quantities allow us to perform comparative studies on shapes andbehaviors more precisely under several experimental conditions to gain a better understanding of the genetic programs
underlying organogenesis. We are now applying this system

Figure 1. Visualized apical cell surface of Drosophila embryonic epidermal cells. A time-lapse confocal microscopic data set expressing
E-cadherin-GFP was processed. Cell boundaries (green), the center ofgravity (blue), and normal vector (magenta) are indicated for each cell.

66

to several experimental models to determine the practicalityof the system.

II. Image processing pipeline for morphogenesis
In complex organs, methods for identifying and tracking
the initial positions of the cell groups consisting of the final
organ morphology are essential analytical methods for elucidating the developmental program.
Here, we analyzed the details of the pathways of individual
cells in a brain primordium throughout the brain morphogenesis in chick embryos in its early stages. We identified thecell positions out of randomly fluorescently labeled brain
primordium cells from a series of time-lapse images andconstructed a database of their time evolution. Based on thisdatabase, we developed a software system for automating thevisualization and quantification of the local migration patternof brain primordium constituent cells from several experimental conditions.

III. Quantification of fish body shapeTechniques for quantitatively analyzing the movements andbody forms of model organisms are considered indispensable for phenotypic evaluation in the process of elucidating
the molecular mechanisms underlying the behavior of living
animals.
To elucidate the mechanism of neuronal development ofzebrafish, we have developed a system that extracts the
body region from each frame of a video of free migrationof fish juveniles taken at a high frame rate and describes themorphology from the head to the tail as a free curve model.
From these curve models, we developed a system that classifies and simplifies the continuous bending forms of fish
as a small number of classes. This system was applied tothe movies acquired under various experimental conditions
and successfully described and statistical evaluation of thephenotype.

Publication List:〔Original paper〕
K., Kato, K., Iida, H., Teramoto, M., Kawamura, A., Watanabe," Yoshihi,Y., Nunome, M., Nakano, M., Matsuda, Y., Sato, Y., Mizuno, H.,"

Iwasato, T., Ishii, Y., and Kondoh, H. (2022). Live imaging of avian
epiblast and anterior mesendoderm grafting reveals the complexityof cell dynamics during early brain development. Development 149,
dev199999. DOI: 10.1242/dev.199999
Kawano, K., Kato, K., Sugioka, T., Kimura, Y., Tanimoto, M., andHigashijima, S. (2022). Long descending commissural V0v neurons
ensure coordinated swimming movements along the body axis in larvalzebrafish. Sci. Rep. 12, 4348. DOI: 10.1038/s41598-022-08283-0

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

Image analysis is an important component of understanding
life sciences. It enables us to quantify phenomena by extracting meaningful information from a large number of images
and to properly represent that information. I am developing
image analysis techniques aimed at analyzing the developmental process of early zebrafish embryo at whole embryo
scale and single-cell resolution. Currently, I am developing an analysis technique that can simultaneously extract
multiple pieces of information such as cell morphology, cell
motility, and cell dynamics by combining 3D cell tracking
and functional imaging.

I. Simultaneous multifunctional analysis of early
embryonic development at a whole embryo
scale and single cell resolution.
Three-dimensional remodeling of cell populations throughcell migration is essential in early embryogenesis. Cell
migration is highly coordinated by controlling cell-celladhesion. To elucidate the principles of such complex
embryogenesis, it is necessary to understand the cellular
dynamics of the whole embryo at single cell resolution. I am

Figure 1. Simultaneous multifunctional analysis of early embryonicdevelopment

developing image analysis techniques to analyze the developmental process of early zebrafish embryogenesis at wholeembryo scale and single-cell resolution.
With the development of microscopy, imaging of early
embryonic imaging has evolved from two-dimensional,
embryonic development has evolved from two-dimensional,
fixed specimen and partial embryo observation to threedimensional, living specimen and whole embryo scale.
This evolution has led to an explosion in the analysis ofcell migration during the development of early embryos inrecent years. However, conventional image analysis techniques could only extract information on cell migration inearly development. Therefore, I have developed an analysis
method that can simultaneously extract information on cell
migration, cell dynamics, and cell morphology at whole
embryo scale and single cell resolution.

II. Research support by image analysis
The development of imaging technology in life science
research has been remarkable, allowing many researchers toeasily acquire large and complex image data sets. However,image analysis is still a hurdle for researchers and can becomea research bottleneck. To solve this problem, I provide
research support based on the following three concepts.
The first concept is quantitative image analysis based on
extensive knowledge of image processing and statistics. For
most researchers, image evaluation is limited to qualitativeand subjective analysis. Correct analysis based on knowledgeof image and statistics supports quantitative and objective
analysis. The second concept is to support image analysis
through machine learning, including deep learning. Machinelearning has made remarkable progress in recent years,
and with a little learning, it is possible to simplify analysis,
which has been difficult with conventional image analysis
techniques. The third concept relates to providing researcherswith easy-to-understand explanations of image analysis techniques on the Web. The content ranges from the principles
of image analysis methods to how to use image analysis
software and plug-ins.

Figure 2. A website that explains how to use image analysis software

67

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITYSHINOMIYA GroupSpecially Appointed Assistant Professor:
SHINOMIYA, Ai
Technical Assistant:
TSURUTA, Emiko

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 organism9s 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.

68

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., Kadofusa,"N., Sato, A., Nishimura, T., Tanaka, M., Nakayama, K., Shiina, N.,
Yamamoto, N., Loudon, A.S., Nishiwaki-Ohkawa, T., Shinomiya,A., Nabeshima, T., Nakane, Y., and Yoshimura, T. (2020). Seasonalchanges in NRF2 antioxidant pathway regulates winter depression-likebehavior. Proc. Natl. Acad. Sci. U.S.A. 117, 959439603. 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,
e0234803. DOI: 10.1371/journal.pone.0234803

69

National Institute for Basic Biology

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

SAITO, Nen

Postdoctoral Fellow:

FUKUYAMA, Tatsuya

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.

Figure 1. Researches combining machine learning and mathematical modeling.

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 possible70

to estimate which physicochemical parameters are responsible for the differences in cell shapes due to cell type andmutation.
We also performed three-dimensional modeling of cell
deformation in micropinocytosis. Macropinocytosis refers
to the nonspecific uptake of extracellular fluid, which plays
a ubiquitous role in cell proliferation, immune surveillance,and virus invasion. Although this phenomenon is widespread, it is still unclear how the cup-shaped cell membrane
is initially formed. We demonstrated that the protrusive
force localized to the edge of the patches can give rise to aself-enclosing cup structure, without further assumptionsof local bending or contraction. Furthermore, our model
exhibits a variety of cup morphologies self-organized via acommon mutually-dependent process of reaction-diffusion
and membrane deformation.

II. Mathematical Model of Microbial Symbiosis
via Metabolite Leakage
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 cell9s 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.

III. Mathematical Model of collective cell
migration
Active matter physics, which is the study of collective
motions such as flocks of birds, has been well studied so far
in biology and physics. Considering the motion of a cell population in terms of active matter, the cells differ significantly
from existing active matter models in that the shape of individual cells can be deformed. How such deformability affects
cell collective migration is not fully understood.
This study proposes a mathematical model based on the
Fourier series expansion of cell contours to describe cell
shape. The shape is described as a superposition of Fouriermodes, and cell deformation is described as the dynamics ofeach Fourier coefficient. Unlike the cell-vertex model that
describes epithelial cells, the proposed model can describe
various shapes other than polygons. We performed a numerical analysis of a situation in which self-propelled cells with
only exclusion volume effects are densely packed using the
proposed model. We found that the cell population exhibitsa solidification-fluidization transition with only a change incell softness.

Publication List:〔Original papers〕
G., Saito, N., Fujimori, T., Hashimura, H., Nakamura, M.J.," Honda,Nakajima, A., and Sawai, S. (2021). Microtopographical guidance of"""macropinocytic signaling patches. Proc. Natl. Acad. Sci. U.S.A. 118,e2110281118. DOI: 10.1073/pnas.2110281118
Imoto, D., Saito, N., Nakajima, A., Honda, G., Ishida, M., Sugita,
T., Ishihara, S., Katagiri, K., Okimura, C., Iwadate, Y., and Sawai, S.
(2021). Comparative mapping of crawling-cell morphodynamics indeep learning-based feature space. PLoS Comput. Biol. 17, e1009237.
DOI: 10.1371/journal.pcbi.1009237
Saito, N., and Sawai, S. (2021). Three-dimensional morphodynamic
simulations of macropinocytic cups. iScience 24, 103087. DOI:
10.1016/j.isci.2021.103087
Yamagishi, J.F., Saito, N., and Kaneko, K. (2021). Adaptation of
metabolite leakiness leads to symbiotic chemical exchange and to a
resilient microbial ecosystem. PLoS Comput. Biol. 17, e1009143. DOI:
10.1371/journal.pcbi.1009143

71

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITYOHNO Group
Assistant Professor:

OHNO, Kaoru

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

I. Gonadotropins in the starfish, Patiria pectinifera
Gonadotropins play important regulatory roles in reproduction in both vertebrates and invertebrates. The vertebrate
gonadotropins, LH and FSH, are structurally and functionallyconserved across various species, whereas no such moleculehas been identified in invertebrates. Some gonadotropic
hormones have been found in invertebrate species. An insulin-like peptide was reported to be responsible for the regulation of egg 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. Search for reproductive hormones inechinoderms
In a collaborative effort with Prof. Yoshikuni and AssociateProf. Kurita9s 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 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. Search 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 part ofour collaborative research. Upon the discovery of aromatasemutants, a detailed analysis was carried out using these strains.

Publication List:〔Original papers〕
Y., Miyazoe, D., Togawa, R., Yokoyama Keiko and Nakasone," Nishiike,K., Miyata, M., Kikuchi, Y., Kamei, Y., Todo, T., Ishikawa-Fujiwara, T.,
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.

72"

Ohno, K., Usami, T., Nagahama, Y., and Okubo, K. (2021). Estrogen
receptor 2b is the major determinant of sex-typical mating behavior
and sexual preference in medaka. Curr. Biol. 31, 1699-1710.e6. DOI:
10.1016/j.cub.2021.01.089
Yuhi, T., Nishimiya, O., Ohno, K., Takita, A., Inoguchi, T., Ura, K., and Takagi,
Y. (2022). Cloning of cDNA encoding a newly recognized apolipoproteinlike protein and its expression in the northern sea urchin Mesocentrotus
nudus. Fish. Sci. 88, 2593273. DOI: 10.1007/s12562-022-01584-3

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.

body fluids are presented in Figure 1. When [Na+] 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(Nav1.131.9), 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 demonstrated that Nax signals are also involved in thecontrol of water intake behavior. The signaling mechanisms in
the OVLT for water-intake induction by increases in [Na+] in

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. As a result, 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.

73

National Institute for Basic Biology

Evolutionary Biology and Biodiversity
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; some speciesof planarian flatworms can even restore all their body 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 the availability oftransgenesis methods which I have developed during my
PhD. It enables tracking specific cells and their progenitors
during development and regeneration.

Figure 1. ERK-KTR biosensor in Macrostomum lignano red arrow points
at the cytoplasm and yellow at the nucleus.

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, becauseof the lack of transgenic methods available for these animals.
I am trying to use the ERK-KTR biosensor in Macrostomum
lignano (Fig. 1), to track ERK signaling and test the functionof genes shown to be involved in positional control during
growth and regeneration. I am also adapting the infraredlaser evoked gene operator (IR-LEGO) technology to use
with the previously established HSP20 promoter (Fig. 2).
This will enable me to track the cell fate in vivo and overexpress selected genes even on a single cell level.
The research is financed by the Mistubishi Foundation
grant.

74

Figure 2. Expression of mScarlet under the HSP20 promoter 24 hours after
induction using IR-LEGO. The lightning bolts point at the targeted sites.

Publication List:〔Original paper〕
K., Wudarski, J., Sukhikh, I., Reinoite, F., Mouton, S.," Ustyantsev,
and Berezikov, E. (2021). Proof of principle for piggyBac-mediatedtransgenesis in the flatworm Macrostomum lignano. Genetics 218,
iyab076. DOI: 10.1093/genetics/iyab076

NIBB-COS IINTERNATIONAL COLLABORATION RESEARCH PROJECTGUSE Group
Guest Professor:
Postdoctoral Fellow:

GUSE, Annika
KISHIMOTO, Mariko

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 inturn 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 Aiptasia9s 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 inmany 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 a functional symbiosis between reefbuilding corals and photosynthetic dinoflagellates of theSymbiodiniaceae family, which transfer nutrients to theircoral 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

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 areaiming to analyze the light sensitivity of the distinct Aiptasia
opsins using a heterologous cell culture-based assay. To date,
we have successfully cloned 12/18 opsins including threesymbiosis-specific ones. Using cell culture-based assays
monitoring cAMP and Calcium signaling, we were able
to detect a light-dependent response of these three opsins.
We have determined the absorbance wavelength specificityof the three symbiosis-specific opsins. Moreover, we havefound that Aiptasia anemones exhibit negative phototactic
behavior in a symbiosis-dependent manner. Together, thesefinding provide the foundation to relating distinct opsins withlight-dependent behavior including phototaxis and spawningin Aiptasia.

75

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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

Publication List:〔Original paper〕
N., Nakamura, S., Kishimoto, M., Moriyama, Y., Kawano," Urakawa,S., Higashiyama, T., and Sasaki, N. (2022). Semi-in vitro detection of
Mg2+-dependent DNase that specifically digest mitochondrial nucleoidsin the zygote of Physarum polycephalum. Sci. Rep. 12, 2995. DOI:
10.1038/s41598-022-06920-2

76

LABORATORY OF BIOLOGICAL DIVERSITYYAMASHITA AND OTSUBO GroupSpecially Appointed Associate Professor

YAMASHITA, Akira

Specially Appointed Assistant Professor:
OTSUBO, Yoko
Technical Assistant:
NAKADE, Atsuko
Admin Support Staff:
SAKAGAMI, Mari
Cells sense the environment around them (e.g. the amountof nutrients and hormones present, as well as the temperatureand pressure), and decide what kind of activities to undertake
based on this information. In response to ambient conditions, germ cells producing sperm and eggs begin halving
their number of chromosomes during a special kind of cell
division called meiosis; a process for bringing forth genetically diverse progeny. In our group, we use the fission yeast
Schizosaccharomyces pombe, the simplest organism that
performs meiosis (Figure 1), to research the mechanisms bywhich cells switch from mitosis (a kind of cell division that
divides cells equally to create two identical cells) to meiosis.
In addition, we have started to study cellular responses to anovel stress: atmospheric pressure plasma.

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 several responsible genes encoded tRNA-related factors. 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.

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

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 under restrictive temperature
conditions. To gain an insight into the TORC1 signaling
pathway, we have isolated mutants that initiate sexual differentiation ectopically under nutrient-rich conditions, as

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 hexanucleotide 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.

77

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

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.

< NINS CNIS Department of Plasma Sciences >gas temperature in collaboration with the National Institutefor Fusion Science (Figure 4).

Figure 4. Direct plasma irradiation to the fission yeast S. pombe on anagar medium.

Publication List:〔Original paper〕
H., Hatta, Y., Hayashi, K., Shimasaki, T., Otsubo, Y.," Ohtsuka,
Ito, Y., Tsutsui, Y., Hattori, N., Yamashita, A., Murakami, H., andAiba, H. (2022). Cdc13 (cyclin B) is degraded by autophagy undersulfur depletion in fission yeast. Autophagy Reports 1, 51364. DOI:
10.1080/27694127.2022.2047442

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.

III. Cellular responses to atmospheric pressureplasmaPlasma, which is the fourth state of matter after solid,
liquid and gas states, is a form of ionized or electricallycharged gas. It has been known that plasma irradiationinduces a wide variety of effects on living organisms.
Recently, atmospheric pressure plasma has been used invarious fields including medicine and agriculture. However,detailed mechanisms underlying responses to plasma irradiation remains unknown. Accordingly, we are addressing
molecular mechanisms of cellular responses to plasma
by using the fission yeast S. pombe. To this end, we havedeveloped plasma irradiation devices that enable control of78

< NINS ASTROBIOLOGY CENTER >LABORATORY OF BIOLOGICAL DIVERSITYNINS ASTROBIOLOGY CENTER
The Astrobiology Center (ABC) in NISS was established in 2015 to promote interdisciplinary studies that
include astronomy, earth science, and biology by organizational adaptation between National Astronomical
Observatory of Japan (NAOJ) and National Institutefor Basic Biology (NIBB). In the 2021 fiscal year, ABC
stayed active to encourage interdisciplinary studies
and grow the research community as the hub institute of astrobiology despite social constraints under
the COVID-19 pandemic. ABC symposium was held
online on Jan 28th, 2022, inviting speakers from diverse
research fields including an observatory mission by
James Webb Space Telescope. Life in the Universe
Workshop was also held online on Feb 17th-18th,
2022 to present the latest results from ABC-subsidized
research.

TAKIZAWA GroupSpecially Appointed Associate Professor

TAKIZAWA, Kenji

The Takizawa group operating out of ABC is currently studying
the environmental responses of photosynthesis in order to predict
photosynthetic apparatus of so-called 8Alien9 plants under
extreme conditions on the aforementioned 8Another Earth9.

Light adaptation mechanisms of photosynthesis
We investigated the flexible molecular mechanisms of lightharvesting and following photosynthetic reactions in cooperation with the division of environmental photobiology. In
photosynthesis research, we are specialized in spectroscopic
analyses which can be applied to astrobiology studies.

Characterize hypothetical plants in exo-planets

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

Three project offices in ABC are working together
to find a habitable exo-planet and a sign of life on
said planet. Exo-Planet Search Project Office andAstrobiology Instrument Project Office which arelocated in the Mitaka campus of NAOJ are preparing
direct-imaging surveys of <Another Earth= by next-generation, 30m-scale telescopes. To support future observation projects, three groups from NIBB in Okazaki, theTakizawa, Johzuka, and Fujita groups, are participatingin the Exo-Life Search Project Office to investigate
life on Earth utilizing three different approaches: 1)
assessing the biosignature of various photosyntheticorganisms to predict biosignatures of hypothetical lifeon exoplanets, 2) evaluating the effects of cosmic radiation on living cells, and 3) elucidating mathematicalprinciples in the formation of self-organizing structuresin organisms.

One of the most plausible biosignatures detected by the
exo-planet observations is a specific reflection pattern on
the land surface named 8vegetation red edge9 or VRE. VRE
arises from strong absorption of red light by photosynthetic
pigments and high reflectance of near-infrared radiation(NIR) by the developed leaf tissue structure. The wavelength
position and amplitude of VRE could be different on anotherplanet. We studied the light reflection spectrum of various
plants and predicted that substantially large VRE could beobserved even on the ocean planets if floating plants cover
the water surface (Figure 2). We also predicted the possibilityof red-sift in VRE on the planets exposed to NIR rather thanvisible light. We have revealed that several metal-containing
pigments can be functional in reaction centers under NIR
radiation conditions via quantum chemical calculations.

Figure 2. Remote sensing of light reflectance of floating leaf of waterlily.
Light reflectance in visible light (RGB) and NIR were obtained by thedrone-based multiband sensor.

Publication List:〔Original papers〕
Y., and Takizawa, K. (2021). A quantum chemical study on the" Komatsu,
effects of varying the central metal in extended photosynthetic pigments.

"

Phys. Chem. Chem. Phys. 23, 14404314414. DOI: 10.1039/d1cp00760b
Pan, X., Tokutsu, R., Li, A., Takizawa, K., Song, C., Murata, K.,
Yamasaki, T., Liu, Z., Minagawa, J., and Li, M. (2021). Structural basisof LhcbM5-mediated state transitions in green algae. Nat. Plants 7,1119+. DOI: 10.1038/s41477-021-00960-8

79National Institute for Basic Biology

Evolutionary Biology and Biodiversity
LABORATORY OF BIOLOGICAL DIVERSITY

formed under the condition of <spot inoculation=, in whichE. coli cells are inoculated in the center of soft-agar plates(Figure 2B). In order to further develop the research, wehave newly developed an assay system <uniform inoculation=, which causes a more dynamic self-organization inwhich colony spots initially emerge from the entire surfaceof agar medium, and they move and fuse with each otherrepeatedly (Figure 2C). Whereas experimental conditions
in biological experiments are usually greatly restricted, thesynthetic approach is a powerful research method that cancontrol and modify the experimental conditions in a more
flexible manner compared to the method used in standard
experiments. Therefore, we try to apply the synthetic
approach to E. coli to understand the principle of spatiotemporal self-organization of cell populations.

FUJITA Group
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).

A

Desktop darkroom

Side angle LED
ring lights

DigitalcameraE. coli
Soft-agar medium

B Spot inoculation






0 hr

24 hr

48 hr

72 hr

96 hr

 

E. coli (Soft-agar medium)

C Uniform inoculation

Hard-agar medium

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.

0 hr

I. Spatiotemporal self-organization of cell
population
One well-known example of spatiotemporal self-organization in living systems is Dictyostelium discoideum, known
as cellular slime mold. D. discoideum usually exists as aunicellular organism, but when stimulated by starvation, cells
aggregate while forming a spiral pattern and develop a sluglike multicellular body. This remarkable self-organization is
induced by chemotaxis to the signal molecule cAMP.
On the other hand, it is reported that Escherichia coli,
which is a unicellular model organism, can self-organize
spotted colony patterns (Figure 2B). This pattern formation
is induced by the positive feedback between chemotaxis toaspartate (Asp) and Asp synthesis, and can be an excellent
experimental system in the study of self-organization ofcell populations. This stationary pattern of E. coli colony is

80

2 hr

3 hr

4 hr

5 hr

Figure 2. (A) A device system for photographs and time-lapse videos ofspatiotemporal patterns of cell populations. (B) Spot inoculation; E. coli
cells are inoculated in the center of soft-agar plates. The cell population
spreads outward while forming a stable spot pattern. (C) Uniform inoculation; E. coli cells are evenly inoculated in soft-agar medium. The cell population shows a dynamic spatiotemporal self-organization in which colony
spots initially appear from the entire surface of soft-agar medium (2 hr),and they move to fuse with each other repeatedly (235 hr).

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
affects tooth root morphology potentially through the modification ofa reaction3diffusion system. Sci. Rep. 11, 5143. DOI: 10.1038/s41598021-84653-4

< NINS ASTROBIOLOGY CENTER >LABORATORY OF BIOLOGICAL DIVERSITY

changes from a simple loop into a complicated twisted shapeas the cell cycle progresses from metaphase to anaphase.

JOHZUKA Group
Assistant Professor:
Technical Staff:

JOHZUKA, Katsuki
ISHINE Naomi

I. Condensin-dependent chromatin folding
Chromosome condensation is a basic cellular process that
ensures the faithful segregation of chromosomes in bothmitosis and meiosis. Any abnormality in this process leads tosegregation errors or aneuploidy, which results in cell death.
Chromosome condensation is mainly achieved by condensin, a hetero-pentameric protein complex, widely conserved
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.
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. We have found that condensin
specifically binds to the RFB site located within the rDNA
repeat. To date, the best characterized condensin binding
region is the rDNA repeat on the right arm of chromosome
XII in budding yeast. We further discovered the multiple
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 fiber

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

II. Plasma-induced cellular responses
Plasma is an ionized gas, which consist of ions and electrons and is extremely reactive. Our question is to understandthe effect of the plasma on living organisms.
We have developed a new plasma irradiation device that cancontrol the temperature to the optimal growth temperatureof cells, and have developed a system to study the effects ofdirect plasma exposure to cells without heat stress (Figure2). Using yeast, S.cerevisiae, it was found that direct plasma
exposure treatment increases lethality as the processing timeincreases (Figure 3). Furthermore, a plasma-resistant mutant
was isolated and the causative gene responsible for plasmaresistance was identified. We are studying functional analysisof the gene to understand the molecular mechanisms of thecellular responses to plasma.

Figure 2. Schematic diagram of atomosperic pressure plasma irradiation
device (left). Direct plasma irradiation experiment (right).

Figure 3. wild-type (upper panels) and plasma-resistant mutant (lower panels)

81

National Institute for Basic Biology

Environmental Biology
DIVISION OF ENVIRONMENTAL PHOTOBIOLOGY

Professor

Associate Professor

MINAGAWA, Jun

YOKONO, Makio

Assistant Professor:
KIM, EunchulSpecially Appointed Assistant Professor:
KOSUGI, Makiko
Technical Staff:
NODA, Chiyo
Postdoctoral Fellow:
KAMADA, Konomi
ISHII, Asako
SOKENDAI Graduate Student: YANAKA, Ayako
Visiting Scientist:
DANN, Marcel
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
Admin Support Staff:
TOYAMA, Mami
SUZUKI, Yuka

I. Plant and Algal PSII3LHCII Supercomplexes:
Structure, Evolution and Energy Transfer.
Photosynthesis is the process conducted by plants and algae
to capture photons and store their energy in chemical forms.
The light-harvesting, excitation transfer, charge separation and electron transfer in photosystem II (PSII) are thecritical initial reactions of photosynthesis and thereby largely
determine its overall efficiency. Knowledge about the architectures and assemblies of plant and green algal PSII3lightharvesting complex II (LHCII) supercomplexes are rapidly
accumulating (Figure 1). We made pair-wise comparative
analyses between the supercomplexes from plants and green
algae to gain insights about the evolution of the PSII3LHCII
supercomplexes involving the peripheral small PSII subunits
that might have been acquired during the evolution (Figure

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 lab9s work

82

Figure 1. The overall architecture of PSII3LHCII supercomplexes from
C. reinhardtii and vascular plants (C2S2 from Spinacia oleracea, C2S2M2from pea). Sheng et al. (2021) Plant Cell Physiol., 62:1108-1120.

2) and about the energy transfer pathways that define theirlight-harvesting and photoprotective properties (Figure 3)(Sheng et al., Plant Cell Physiol., 62:1108-1120).

II. Structural basis of LhcbM5-mediated statetransitions in green algae.
In green algae and plants, state transitions serve as a short-termlight-acclimation process in the regulation of the light-harvesting capacity of photosystems I and II (PSI and PSII, respectively). During the process, a portion of light-harvesting complex II(LHCII) is phosphorylated, dissociated from PSII and binds
with PSI to form the supercomplex PSI3LHCI3LHCII. We
reported high-resolution structures of PSI3LHCI3LHCII
from Chlamydomonas reinhardtii, revealing the mechanism ofassembly between the PSI3LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM protein(Figure 4). Two specific LhcbM isoforms, namely LhcbM1 andLhcbM5, directly interact with the PSI core through their phosphorylated amino terminal regions. Furthermore, biochemicaland functional studies on mutant strains lacking either LhcbM1
or LhcbM5 indicate that only LhcbM5 is indispensable in supercomplex formation. The results unravel the specific interactions
and potential excitation energy transfer routes between green
algal PSI and two phosphorylated LHCIIs (Pan et al., Nat.
Plants, 7:1119-1131).

Figure 2. Superposition of green algal and plant PSII3LHCII supercomplexes and evolutionary insights from pea C2S2M2 and C. reinhardtii
C2S2M2L2 supercomplexes. Sheng et al. (2021) Plant Cell Physiol.,
62:1108-1120.

Figure 3. Förster resonance energy transfer (FRET) networks of green
algal and plant PSII-LHCII supercomplexes and major energy transfer
pathways. Sheng et al. (2021) Plant Cell Physiol., 62:1108-1120.

Figure 4. Surface representation of the CrPSI3LHCI3LHCII supercomplex from the pph1;pbcp mutant strain, with pThr residues from LhcbM1
and LhcbM5 highlighted in red. b, Detailed interaction between the
N-terminal region of LhcbM1 and PSI core subunits PsaH and PsaL. c,
Sequence alignment of the interaction-related regions of LhcbM1, PsaL
and PsaH from C. reinhardtii (Cr) with Lhcb2, PsaL and PsaH from
Zm, respectively. Conserved and variant residues involved in intersubunit interactions are indicated by blue and red asterisks, respectively. d,
Structural comparison of N-terminal five residues (shown as sticks)of CrLhcbM1 (carbon atoms coloured green) and ZmLhcb2 (carbon
atoms coloured white). The PSI core is shown in electrostatic surface
mode with red and blue representing acidic and basic regions, respectively. e, Detailed interactions between the N-terminal region of LhcbM5
and the PSI core subunit PsaH. Hydrogen bond interactions are shown as
black dashed lines. Pan et al. (2021) Nat. Plants, 7:1119-1131.

83

National Institute for Basic Biology

Environmental Biology
Publication List:〔Original Papers〕
T., Takahashi, S., Yoshida, T., Shimamura, S., Takaki, Y.," Maeda,Nagai, Y., Toyoda, A., Suzuki, Y., Arimoto, A., Ishii, H., Satoh, N.,

"
"
"
"
"

Nishiyama, T., Hasebe, M., Maruyama, T., Minagawa, J., Obokata, J.,
and Shigenobu, S. (2021). Chloroplast acquisition without the genetransfer in kleptoplastic sea slugs, Plakobranchus ocellatus. eLife 10,e60176. DOI: 10.7554/eLife.60176
Morishita, J., Tokutsu, R., Minagawa, J., Hisabori, T., and Wakabayashi,
K. (2021). Characterization of Chlamydomonas reinhardtii Mutants
That Exhibit Strong Positive Phototaxis. Plants 10, 1483. DOI: 10.3390/
plants10071483
Pan, X., Tokutsu, R., Li, A., Takizawa, K., Song, C., Murata, K.,
Yamasaki, T., Liu, Z., Minagawa, J., and Li, M. (2021). Structural basisof LhcbM5-mediated state transitions in green algae. Nat. Plants 7,1119+. DOI: 10.1038/s41477-021-00960-8
Tanno, A., Tokutsu, R., Arakaki, Y., Ueki, N., Minagawa, J., Yoshimura,K., Hisabori, T., Nozaki, H., and Wakabayashi, K.-I. (2021). The
four-celled Volvocales green alga Tetrabaena socialis exhibits weak
photobehavior and high-photoprotection ability. PLoS One 16,
e0259138. DOI: 10.1371/journal.pone.0259138
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, 189431902. DOI: 10.1093/plphys/kiab004
Zhang, X.J., Fujita, Y., Tokutsu, R., Minagawa, J., Ye, S., and Shibata,Y. (2021). High-Speed Excitation-Spectral Microscopy Uncovers In
Situ Rearrangement of Light-Harvesting Apparatus in Chlamydomonas
during State Transitions at Submicron Precision. Plant Cell Physiol. 62,8723882. DOI: 10.1093/pcp/pcab047〔Review Article〕
M., Minagawa, J., and Takahashi, Y. (2021). Photosynthesis and" Hippler,
Chloroplast Regulation-Balancing Photosynthesis and Photoprotection"

84

under Changing Environments. Plant Cell Physiol. 62, 105931062.
DOI: 10.1093/pcp/pcab139
Sheng, X., Liu, Z., Kim, E., Minagawa, J. (2021). Plant and algal PSIILHCII supercomplexes: structure, evolution and energy transfer. Plant
Cell Physiol 62: 1108-1120. DOI: 10.1093/pcp/pcab072

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
Technical Assistant:
HAMADA, Mayako
YAMADA, Yuka
SOMA, Yoriko
INOUE, Fumiko
MOTOMURA, Hiroe
Admin Support Staff:
KOJIMA, Yoko

physical 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 in gravitropic 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 LZY3-mCherry
is polarly localized on the basal side of the plasma membranein the columella cells (root statocytes) in response to inclination. This suggests the close relationship between intracellular localization of LZY and gravity sensing. To elucidate
the gravity sensing mechanism, we are analyzing regions
responsible for intracellular localization of LZY3-mCherry
and LZY4-mSacarlet by introducing mutations. A number oftransgenic lines harbouring mutated LZY-FP driven by theirown promoters in mutant background are under construction.

II. Determination mechanism of gravitropic
setpoint angle

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.

Visual overview of this lab9s work.

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 roots9
and shoots9 lateral branches maintain inclined growth angles
with respect to gravity.
It has been proposed that the GSA is determined by two
antagonistic growth components, gravitropism and antigravitropic offset (AGO). Currently, we are characterizing
Arabidopsis TAC1 (TILLER ANGLE CONTROL 1) gene asa candidate to control AGO. The TAC1 gene was originallyidentified by quantitative trait locus analysis in rice tiller
angle control (Yu et al., 2007). Later, TAC1 gene was identified in Peach tree (Prunus persica) as a causal gene of piller
phenotype (Dardick et al., 2013). In our lab, TAC1 gene
was found as a down regulated gene in agravitropic mutants
eal1 and sgr1 which lack an endodermis, a gravity sensing
tissue in shoot in Arabidopsis (Taniguchi et al., 2017). In
Arabidopsis, tac1 mutant has a compact shoot architecture
due to the upward lateral branch growth angles (Figure 1).
TAC1 encodes a protein with unknown function, but has asimilarity with LAZY1 family protein. However, while LZY
family proteins interact with RLD family proteins throughtheir conserved C-terminus in LAZY1 family protein (CCL)
domain and regulate gravitropism (Furutani et al., 2020),
TAC1 does not have a CCL domain, thereby TAC1 does notinteract with RLD family protein.
As well as characterizing TAC1 gene, currently, we are also
trying to isolate suppressor mutants of lzy1 lzy2 lzy3 by aforward genetic screening.

I. Molecular 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 the
direction of gravity. Amyloplast relocation serves as a85

National Institute for Basic Biology

Environmental Biology

Figure 1. Growth angle phenotype of shoot lateral branches. Overall
shoot architecture in wild type (A) and tac1 (B). Distribution of growth
direction of lateral branches in wild type (C) and tac1 (D). Growth angles
were plotted on histogram and distributions were evaluated in boxplot
from wild type (E) and tac1 (F).

III. Functional analysis of RLD (RCC1-likedomain) proteins
RLD proteins were isolated as interactors of LZY. RLDs
function in gravitropism and the GSA control of roots. We
have also shown that RLD1, RLD2, RLD3, and RLD4 redundantly function in plant development and morphogenesis, themorphological abnormality gradually becomes more severe
by multiplying rld mutations (Figure 3). Indeed, rld1;2;3;4
quadruple mutants displayed severe embryonic development
defects due to a reduced amount and abnormal localization of PIN proteins (auxin efflux carriers) in the plasma
membrane. It had been reported that the RLD1 fragment
containing RCC1-like domain show guanine nucleotide
exchange activity in Rab8 and Rab11. Thus, it9s likely that
RLD proteins regulate auxin transport through regulating
membrane trafficking during root gravitropism and development.
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.
To understand further function of RLDs, we made attempt86

to identify the proteins that interact with RLD using yeast
two-hybrid screening. As a result, we found several proteinsas candidates for RLD interactor in addition to LZYs.
Among them, we further analyzed the polarity protein BASL
(BREAKING OF ASYMMETRY IN THE STOMATAL
LINEAGE) which controls stomatal asymmetric cell division
as a collaboration with Dr. Dong and colleagues (Wang etal., 2021). Further analyses using yeast two-hybrid system
revealed that BASL interact with the BRX domain of RLD.
Developmental defects of rld1;2;3;4 mutant phenocopy
those of the severe allele of gnome mutants as previously
reported (Furutani et al., 2020). GNOM, a guanine nucleotide exchange factor for ARF (ADP ribosylation factor),
plays important roles in membrane trafficking. Dong andcolleagues found that RLD physically interacts with GNOM
in vitro and in vivo, and that GNOM and RLDs are requiredfor BASL polarization. Interestingly, we found GNOM in
the immunoprecipitates with LZY proteins (Furutani et al.,2020), implying that RLD-GNOM could be involved in LZY
function in the gravity signaling.
To investigate the importance of the BRX domain for
RLD function, we examined whether 5/'S5/'¨%5;
mClover3 could complement the morphological phenotype of rld2;3;4. Unexpectedly, RLD3¨BRX-mClover3
recovered the morphological abnormality of rld2;3;4(Figure 2). Interestingly, defects in gravity response
including GSA control and gravitropism were observedin 5/'S5/'¨%5;P&ORYHUUOG. Both of these
indicate that RLD3 lacking the BRX domain retains thefunction for morphogenesis but not for gravity responsein shoot. Moreover, when RLD3 was expressed under
the control of the endodermis-specific ADF9 promoter in
5/'S5/'¨%5;P&ORYHUUOG, the gravity response
was significantly restored, thus suggesting that RLD is
involved in gravity signaling in the endodermal cells inshoot gravitropism in a manner similar to LZY. These results
suggest that RLD function in several physiological events
including gravitropism, morphogenesis in BRX-dependentand -independent manner.

Figure 2. A. Phenotype of rld multiple mutants in mature plants. B. The
growth angle of lateral shoot in Wild Type (a), RLD3p:RLD3¨BRXmClover3/rld2;3;4 (b), ADF9p:RLD3/RLD3p:RLD3¨BRX-mClover3/
rld2;3;4 (c).

IV. Optogenetic approaches in plant biology
Optogenetics is a powerful, biological technique to manipulate the activity and localization of proteins in living cells,
thereby understanding their function. Many optogenetic tools
have been developed for the past three decades, however
there are few applications in plant biology because of bio-

logical and technical limitations; 1) plants use light forphotosynthesis or as their development cues and 2) most of
the tools are developed from plant light-sensitive proteins, inwhich light stimulating the tools may affect plant growth anddevelopment in an undesirable manner. Even so, optogenetic
approaches are worth introducing into plant biology because
they can control proteins of interest with a higher spatiotemporal resolution than those of conventional techniques likegene-inducible system and protein-uncaging with chemicals. We have been trying to optically control plant protein
kinases, particularly belonging to AGC kinase family, which
regulate fundamental cellular processes in plant growth anddevelopment, such as transport of plant hormone auxin andcell growth. We chose a strategy to control the kinase activity
directly by light, which can be controllable at the subcellularlevel. To carry out it, we fused light-responsive proteins/
modules to the kinases and analyzed their responsibility tolight. One of successful tools possess a blue light-sensitive
LOV2 domain from plant photoreceptor phototropin and its
interacting motif that we identified to negatively regulate
AGC kinase activity by blue light. By using this tool, we
succeeded to develop a light-sensitive kinase that directly
regulates the auxin transporter PINs, which is proven at leastby a heterogenous expression system with Xenopus oocytes.
It is expected that this tool will be a breakthrough techniquein plant biology to dissect lots of biological events mediatedby auxin (Figure 3).

Publication List:〔Original paper〕
R., Uchida, N., Tameshige, T., Kawamoto, N., Hotokezaka," Fujihara,Y., Higaki, T., Simon, R., Torii, K.U., Tasaka, M., and Aida, M. (2021).

""

The boundary-expressed EPIDERMAL PATTERNING FACTOR-LIKE2
gene encoding a signaling peptide promotes cotyledon growth during
Arabidopsis thaliana embryogenesis. Plant Biotechnol. 38, 3173322.
DOI: 10.5511/plantbiotechnology.21.0508a
Kunita, I., Morita, M.T., Toda, M., and Higaki, T. (2021). A threedimensional scanning system for digital archiving and quantitativeevaluation of Arabidopsis plant architectures. Plant Cell Physiol. 62,
197531982. DOI: 10.1093/pcp/pcab068
Wang, L., Li, D., Yang, K., Guo, X., Bian, C., Nishimura, T., Le, J.,
Morita, M.T., Bergmann, D.C., and Dong, J. (2022). Connectedfunction of PRAF/RLD and GNOM in membrane trafficking controls
intrinsic cell polarity in plants. Nat. Commun. 13, 7. DOI: 10.1038/
s41467-021-27748-w〔Review article〕
M., and Morita, M.T. (2021). LAZY1-LIKE-mediated gravity" Furutani,
signaling pathway in root gravitropic set-point angle control. Plant
Physiol. 187, 108731095. DOI: 10.1093/plphys/kiab219

Figure 3. Schematic illustration of the light-sensitive AGC kinase. In
the dark, the chimeric AGC kinase is active to phosphorylate its target
proteins such as auxin transporter PINs (right). PINs are activated by thephosphorylation for efflux transport of indole-3-acetic acid (IAA). Uponblue light irradiation, the kinase activity can be reduced due to the blue
light-induced conformational change (left).

87

National Institute for Basic Biology

Theoretical Biology
LABORATORY OF GENOME INFORMATICS

Associate Professor

UCHIYAMA, Ikuo

The accumulation of biological data has recently been
accelerated by various high-throughput <omics= technologies including genomics, transcriptomics, and proteomics.
The field of genome informatics is aimed at utilizing 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 tables on the fly
by using a specified set of organisms. MBGD also has precalculated ortholog tables for each major taxonomic group,and provides several viewing modes to display the entiretyof each ortholog table. For closely related taxa, MBGD
provides conserved synteny information calculated using
the CoreAligner program. MBGD additionally provides a
8MyMBGD9 mode, which allows users to add their own
genomes to MBGD.
We continue to update the database and MBGD now
contains 15397 genomes, including 14786 bacteria, 336
archaea, and 275 eukaryota. These data sets are classified
based on the hierarchical ortholog classification strategy
described in the section below. In addition, in the latest
version of MBGD, we improved several functionalities
including a new display of the core genome alignment(Figure 1) and a novel interface for the MyMBGD mode.
As an application software for analyzing novel genome
sequences based on the database, we have developed atool to predict the functional potential of novel microbial genomes through orthology assignment based on the
MBGD ortholog table. To evaluate the metabolic potentialof the query genome from this assignment, we utilized the
Genomaple software to calculate the module completion
ratio for each KEGG Module entry (in collaboration with Dr.
Takami, Univ Tokyo). The result is displayed on a module
completion table where a user can compare the presence orabsence of functional modules among specified organisms.

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. Orthologous gene classification among
multiple genomes at the domain level

Figure 1. Comparative genome map among the family Enterobacteriaceae
on the basis of the core genome alignment constructed using a novel implementation of the CoreAligner program. The color is assigned according to
the gene order in Escherichia coli.

88

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 thesum total of domain-level alignment scores. On the basisof this idea, we have developed a refinement pipeline toimprove domain-level clustering, DomRefine, by optimizing DSP scores. DomRefine is used to construct the standard
ortholog table covering all the representative genomes storedin MBGD.
Domain-level classification is a unique feature within our
ortholog classification system. In particular, this data is
considered suitable for analyzing domain fusion events thathave occurred during evolution. By using the domain-level
ortholog grouping data combined with taxonomic and functional information, we are trying to elucidate when and inwhat kind of genes domain fusion events frequently occur
during evolution, and how the complexity of the <domainfusion network= can be associated with the phenotypic traits
in each organism.

IV. Development 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. Ortholog data representation using the
Semantic Web technology to integratevarious microbial databases
Orthology is a key to integrating knowledge of variousorganisms through comparative analysis. In order to integrate
genomic data and various types of biological information
with this idea, we have constructed an ortholog databaseusing Semantic Web technology. To formalize the structureof the ortholog information in the Semantic Web, we developed the Orthology Ontology (ORTH) and described theortholog information in MBGD in the form of the Resource
Description Framework (RDF).
On the basis of this framework, we have integrated various
kinds of microbial data using the ortholog information as 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, we are now focusing on the remaining part of the genomes (non-core genomes) that is more
directly linked to the within-species genome diversity. We
are developing a method (FindIsland) to identify a set of
non-core genes that have conserved gene order by using amodified version of the CoreAligner algorithm. We applied
the method to the sets of genomes of prokaryotic species
stored in MBGD and developed a database for analyzing
their non-core genomes. Based on the database, we foundthat the resulting conserved clusters frequently correspondto known mobile genetic elements and/or have sequence
features common to known genomic islands.

Publication List:〔Original Papers〕
M., Nishide, H., Nagao, R., Inoue-Kashino, N., Shen, J.-R.," Kumazawa,
Nakano, T., Uchiyama, I., Kashino, Y., and Ifuku, K. (2022). Molecular"phylogeny of fucoxanthin-chlorophyll a/c proteins from Chaetoceros
gracilis and Lhcq/Lhcf diversity. Physiol. Plant. 174, e13598. DOI:
10.1111/ppl.13598
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, dsaa028. DOI: 10.1093/
dnares/dsaa028〔Review Article〕
B., Ebersberger, I., McGlynn, S.E., Glover, N., Mochizuki, T.," Linard,
Patricio, M., Lecompte, O., Nevers, Y., QFO Consortium, Thomas,
P.D., Gabaldon, T., Sonnhammer, E., Dessimoz, C., and Uchiyama, I.
(2021). Ten Years of Collaborative Progress in the Quest for Orthologs.
Mol. Biol. Evol. 38, 303333045. DOI: 10.1093/molbev/msab098

89

National Institute for Basic Biology

Imaging Science
LABORATORY FOR SPATIOTEMPORAL REGULATIONSAssociate Professor

NONAKA, Shigenori

Specially Appointed Assistant Professor:
YOKE, Hiroshi
Postdoctoral Fellow:
TANIGUCHI, Atsushi
Technical Assistant:
ISHIBASHI, Tomoko

We pursued this question by measuring Ca2+ dynamics
in the node and found that the node cells apparently cause
stochastic elevation of Ca2+. The spatiotemporal distribution is equal on the left and right sides, but becomes moreprevalent on the left after the late headfold stage, when
flow-induced commitment occurs. This asymmetry was
disrupted in dynein mutant LYLY and pkd2 mutants, inaccordance to their left-right phenotypes.
Based on these findings, we are now examining thevalidity of the two major proposed models of flow
sensing, mechanosensing and chemosensing, as well as athird novel mechanism.

II. Development of light-sheet microscopy

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

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 lightsheet microscope named ezDSLM, in which the excitation light sheet and detection objective move insteadof the specimen for z-scanning. We installed an electrically tunable lens (ETL) to achieve greater speed and theexclusion of mechanical vibrations that disturb continuous 4D imaging of floating or loosely held specimens.
Our light-sheet microscopes are available to otherresearchers via NIBB9s Collaborative Researchand MEXT9s Advanced Bioimaging Support (ABiS)
programs. Numerous collaborations are in progressincluding observation of moving cell migration in zebrafish embryos, cleared mouse brains, etc.

Visual overview of this lab9s work.

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

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

Publication List:〔Original papers〕
Y., Matsuo, M., Kurihara, K., Suzuki, K., Nonaka, S., and" Hirata,
Sugawara, T. (2021). Colocalization Analysis of Lipo-Deoxyribozyme"
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.

90

Consisting of DNA and Protic Catalysts in a Vesicle-Based Protocellular
Membrane Investigated by Confocal Microscopy. Life (Basel) 11, 1364.
DOI: 10.3390/life11121364
Ohmura, T., Nishigami, Y., Taniguchi, A., Nonaka, S., Ishikawa, T., andIchikawa, M. (2021). Near-wall rheotaxis of the ciliate Tetrahymena
induced by the kinesthetic sensing of cilia. Sci. Adv. 7, eabi5878. DOI:
10.1126/sciadv.abi5878

LABORATORY FOR BIOTHERMOLOGYSpecially Appointed Associate Professor

KAMEI, Yasuhiro

Specially Appointed Assistant Professor:
SAKAMOTO, Joe
Postdoctoral Fellow:
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 bodytemperature, homeostasis, differs among various organisms.
Furthermore, temperatures in either cells or at the molecularlevel have not been widely discussed to date. Nano-scale thermometers utilizing fluorescent proteins and dyes have beendeveloped by numerous research groups including our own,
and heterogeneity of temperatures in the micro environmentsof living cells have been reported on by said groups (Okabe
et al, Nat Commun 2014, Kiyonaka et al, 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 upon a new research field,
biothermology, by investigating the nature of temperature inliving 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
to analyze the thermodynamics of a cell via the use of anano-scale thermometer; the second of our abovementioned

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

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,
it may be possible to control the preset temperature of HSR,
and this substitution could be applied to the improvement of91

National Institute for Basic Biology

Imaging Science

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〕
M., Kamei, Y., and Fukamachi, S. (2021). Behavioural red" Matsuo,
light sensitivity in fish according to the optomotor response. R. Soc.

"
"
"

92

Open Sci. 8, 210415. DOI: 10.1098/rsos.210415
Ogino, H., Kamei, Y., Hayashi, T., Sakamoto, J., Suzuki, M., and Igawa,T. (2021). Invention sharing is the mother of developmental biology
PREFACE. Dev. Growth Differ. 63, 3953396. DOI: 10.1111/dgd.12755
Tokanai, K., Kamei, Y., and Minokawa, T. (2021). An easy andrapid staining method for confocal microscopic observation andreconstruction of three-dimensional images of echinoderm larvae andjuveniles. Dev. Growth Differ. 63, 4783487. DOI: 10.1111/dgd.12758
Wu, D., Arakawa, H., Fujita, A., Hashimoto, H., Hibi, M., Naruse, K.,
Kamei, Y., Sato, C., and Kitajima, K. (2021). A point-mutation in the
C-domain of CMP-sialic acid synthetase leads to lethality of medaka
due to protein insolubility. Sci. Rep. 11, 23211. DOI: 10.1038/s41598021-01715-3

National Institute for Basic Biology

Research Support
NIBB CORE RESEARCH FACILITIES

Representative Instruments
Genomics

Head

YOSHIDA, 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

Professor

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. We have upgraded the Sequel instrument in
2021, which enabled a significant increase of the HiFi longread production.
During 2021, we carried out 53 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, single-cell
trnascriptome and ChIP-seq.

SHIGENOBU, Shuji

Technical Staff:

Technical Assistant:Admin Support Staff:

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

Figure 1. Next-generation sequencer

Proteomics

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 2021, we still suffered from the effects of the COVID-19
pandemic that hampered research activities worldwide.
While we operated our facility placing the highest priority on
users9 safety, we provided remote support for the facilities9
users and online communication with collaborators to sustain
research projects. Such efforts resulted in 11 co-authoredpapers being published.

As is listed below, two types of mass spectrometers andtwo protein sequencers are used for proteome studies in ourfacility. In 2021, we analyzed approximately 600 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

93

National Institute for Basic Biology

Research Support
Other analytical instruments (excerpts)- Cell sorter (SONY SH800)- Bioimaging analyzer (BIO-RAD ChemiDoc XRS+ ;Fujifilm LAS 3000 mini; GE FLA9000)- Laser capture microdissection system (Thermo Fisher
Scientific Arcturus XT)- Real-time PCR machine (Thermo Fisher Scientific ABI
7500, QuantStudio 3)- Ultracentrifuge (Beckman XL-80XP etc.)- Microplate reader (PerkinElmer Nivo; Hitachi
SH-9000Lab)- Single-cell analysis system (Fluidigm C1, 10x Genomics
Chromium X)

Publication List on Cooperation:〔Original papers〕"""

T., Soyano, T., Liu, M., Mori, T., and Kawaguchi, M. (2022).
" Goto,Auxin methylation by IAMT1, duplicated in the legume lineage,

"

"
"

"

"
"

"
"
"
"
94

promotes root nodule development in Lotus japonicus. Proc. Natl.
Acad. Sci. U.S.A. 119. e2116549119. DOI: 10.1073/pnas.2116549119
Hasegawa, Y., Ueno, S., Wei, F.-J., Matsumoto, A., Uchiyama, K.,
Ujino-Ihara, T., Hakamata, T., Fujino, T., Kasahara, M., Bino Takahiro
and Yamaguchi, K., Shigenobu, S., Tsumura, Y., and Moriguchi, Y.
(2021). Identification and genetic diversity analysis of a male-sterile
gene (MS1) in Japanese cedar (Cryptomeria japonica D. Don). Sci. Rep.
11, 1496. DOI: 10.1038/s41598-020-80688-1
Igawa-Ueda, K., Ikuta, T., Tame, A., Yamaguchi, K., Shigenobu, S.,
Hongo, Y., Takaki, Y., Fujikura, K., Maruyama, T., and Yoshida, T.
(2021). Symbiont Transmission onto the Cell Surface of Early Oocytes
in the Deep-sea Clam Phreagena okutanii. Zoolog. Sci. 38, 1403147.
DOI: 10.2108/zs200129
Ishida, S., Suzuki, H., Iwaki, A., Kawamura, S., Yamaoka, S., Kojima,M., Takebayashi, Y., Yamaguchi, K., Shigenobu, S., Sakakibara, H.,
Kohchi, T., and Nishihama, R. (2022). Diminished Auxin Signaling
Triggers Cellular Reprogramming by Inducing a Regeneration Factor in
the Liverwort Marchantia polymorpha. Plant Cell Physiol. 63, 3843400.
DOI: 10.1093/pcp/pcac004
Ishishita, S., Kitahara, S., Takahashi, M., Iwasaki, S., Tatsumoto, S.,
Hara, I., Kaneko, Y., Kinoshita, K., Yamaguchi, K., Harada, A., Ohmori,Y., Ohkawa, Y., Go, Y., Shigenobu, S., Matsuda, Y., and Suzuki, T.
(2022). Uterus-specific transcriptional regulation underlies eggshell
pigment production in Japanese quail. PLoS One 17, e0265008. DOI:
10.1371/journal.pone.0265008
Korgaonkar, A., Han, C., Lemire, A.L., Siwanowicz, I., Bennouna, D.,
Kopec, R.E., Andolfatto, P., Shigenobu, S., and Stern, D.L. (2021). A
novel family of secreted insect proteins linked to plant gall development.
Curr. Biol. 31, 1836-1849.e12. DOI: 10.1016/j.cub.2021.01.104
Maeda, T., Takahashi, S., Yoshida, T., Shimamura, S., Takaki, Y.,
Nagai, Y., Toyoda, A., Suzuki, Y., Arimoto, A., Ishii, H., Satoh, N.,
Nishiyama, T., Hasebe, M., Maruyama, T., Minagawa, J., Obokata, J.,
and Shigenobu, S. (2021). Chloroplast acquisition without the genetransfer in kleptoplastic sea slugs, Plakobranchus ocellatus. eLife 10,e60176. DOI: 10.7554/eLife.60176
Miyazaki, S., Fujiwara, K., Kai, K., Masuoka, Y., Gotoh, H., Niimi,
T., Hayashi, Y., Shigenobu, S., and Maekawa, K. (2021). Evolutionarytransition of doublesex regulation from sex-specific splicing to malespecific transcription in termites. Sci. Rep. 11, 15992. DOI: 10.1038/
s41598-021-95423-7
Okamoto, S., Kawasaki, A., Makino, Y., Ishida, T., and Sawa, S. (2022).
Long-distance translocation of CLAVATA3/ESR-related 2 peptide andits positive effect on roots sucrose status. Plant Physiol. 189, 23573
2367. DOI: 10.1093/plphys/kiac227
Okamoto, S., Kawasaki, A., and Makino, Y. (2022). Characterization of
Oligopeptides in Solanum lycopersicum Xylem Exudates. Life 12. 592.
DOI: 10.3390/life12040592
Sato, K., Uehara, T., Holbein, J., Sasaki-Sekimoto, Y., Gan, P., Bino,""T., Yamaguchi, K., Ichihashi, Y., Maki, N., Shigenobu, S., Ohta, H.,
Franke, R.B., Siddique, S., Grundler, F.M.W., Suzuki, T., Kadota,
Y., and Shirasu, K. (2021). Transcriptomic Analysis of Resistant andSusceptible Responses in a New Model Root-Knot Nematode Infection
System Using Solanum torvum and Meloidogyne arenaria. Front. Plant
Sci. 12, 680151. DOI: 10.3389/fpls.2021.680151
Shigenobu, S., Hayashi, Y., Watanabe, D., Tokuda, G., Hojo, M.Y.,
Toga, K., Saiki, R., Yaguchi, H., Masuoka, Y., Suzuki, R., Suzuki, S.,
Kimura, M., Matsunami, M., Sugime, Y., Oguchi, K., Niimi, T., Gotoh,
H., Hojo, M.K., Miyazaki, S., Toyoda, A., Miura, T., and Maekawa,
K. (2022). Genomic and transcriptomic analyses of the subterranean
termite Reticulitermes speratus: Gene duplication facilitates social
evolution. Proc. Natl. Acad. Sci. U.S.A. 119, e2110361119. DOI:
10.1073/pnas.2110361119
Subhankar, B., Yamaguchi, K., Shigenobu, S., and Aoki, K. (2021).
Trans-species small RNAs move long distances in a parasitic
plant complex. Plant Biotechnol. 38, 1873196. DOI: 10.5511/
plantbiotechnology.21.0121a
Takeuchi, T., Matsubara, H., Minamitani, F., Satoh, Y., Tozawa, S.,
Moriyama, T., Maruyama, K., Suzuki, K.T., Shigenobu, S., Inoue, T.,
Tamura, K., Agata, K., and Hayashi, T. (2022). Newt Hoxa13 has an
essential and predominant role in digit formation during development
and regeneration. Development 149, dev200282. DOI: 10.1242/
dev.200282
Tominaga, T., Miura, C., Sumigawa, Y., Hirose, Y., Yamaguchi, K.,
Shigenobu, S., Mine, A., and Kaminaka, H. (2021). Conservation andDiversity in Gibberellin-Mediated Transcriptional Responses Among
Host Plants Forming Distinct Arbuscular Mycorrhizal Morphotypes.
Front. Plant Sci. 12, 795695. DOI: 10.3389/fpls.2021.795695
Yoshida, Y., Shaikhutdinov, N., Kozlova, O., Itoh, M., Tagami, M.,
Murata, M., Nishiyori-Sueki, H., Kojima-Ishiyama, M., Noma,
S., Cherkasov, A., Gazizova, G., Nasibullina, A., Deviatiiarov, R.,
Shagimardanova, E., Ryabova, A., Yamaguchi, K., Bino, T., Shigenobu,
S., Tokumoto, S., Miyata, Y., Cornette, R., Yamada, T.G., Funahashi,A., Tomita, M., Gusev, O., and Kikawada, T. (2022). High quality
genome assembly of the anhydrobiotic midge provides insights on asingle chromosome-based emergence of extreme desiccation tolerance.
NAR Genomics Bioinforma. 4, lqac029. DOI: 10.1093/nargab/lqac029〔Review article〕
T., Oguchi, K., Yamaguchi, H., Nakamura, M., Sato, D.," Miura,Kobayashi, K., Kutsukake, N., Miura, K., Hayashi, Y., Hojo, M.,
Maekawa, K., Shigenobu, S., Kano, T., and Ishiguro, A. (2022).
Understanding of superorganisms: collective behavior, differentiationand social organization. Artif. Life Robot. 27, 2043212. DOI: 10.1007/
s10015-022-00754-x

SPECTROGRAPHY AND BIOIMAGING FACILITY

has been set up. The NIBB Collaborative Research Program
for the Use of the OLS supports about 10 projects every
year conducted by both visiting scientists, including foreign
researchers, as well as members of NIBB.

Specially Appointed Associate Professor

KAMEI, Yasuhiro

Technical Staff:
Visiting Undergraduate:
Technical Assistant:

KONDO, Maki
TANIGUCHI-SAIDA, Misako
YOSHIKAWA, Ryusei
ICHIKAWA, Chiaki
ASAO, Momoko
NAKAGAWA, Mami
AOYAMA, Chie

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 world9s 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 facility9s 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 (Figure2), such as 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 otheradvanced laser microscopes boasting specialized, cuttingedge technology (Light-sheet Microscope and Infrared LaserEvoked Gene Operator microscope: IR-LEGO), which canbe utilized 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 and bioimage processing/analysis techniques, and theother is the Advanced Bioimaging Support Program (ABiS)
which operates under the framework of the Grant-in-Aid for
Scientific Research on Innovative Areas.

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

Figure 2. Multi-color confocal microscope image of muscle in a transgenic medaka larva. Each muscle cell expressed randomly various color
variants of fluorescent protein, such as Cerulean, GFP, YFP and dsRed.
The transgenic line was provided by NBRP Medaka.

95

National Institute for Basic Biology

Research Support

The light-sheet microscope was developed by Dr. Ernst
Stelzer9s group at the European Molecular Biology
Laboratory (EMBL). This microscope can realize high-speed
z-axis scanning in deeper tissues by illuminating specimensfrom the side with a light sheet (more information is given in
the report submitted by Dr. Shigenori Nonaka9s 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 gene ofinterest by heating a single target cell in vivo with a high efficiency irradiating infrared laser. This microscope technology
can be applied to thermal biology through local heating (thedetails of this are provided in the next section). IR-LEGO
was also used for about 10 Collaborative Research projects,
including applications aimed at both animals and plants.

Workshop, Symposium and Training course
In 2021, we held the 9 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 due to the COVID-19 pandemic. We have also
started a new course <Optical Microscopy Principle Training
Course= cooperation with Drs. Joe Sakamoto, Yasuhiro
Kamei, Atsushi Taniguchi, Shigenori Nonaka in NIBB andMotosuke Tsutsumi and Kohei Otomo in ExCELLS. In thiscourse, participants learned microscopy principles thoroughlectures and built bright-field and fluorescent microscopes bythemselves using optical devices, such as lenses, filters, lightsources and cameras (Figure 3). We additionally hold sometraining courses and seminars related to microscopy andimage analysis, including their technologies and applications.

Figure 3. A scene from the 1st Optical Microscopy Principle Training
Course. The participants built up a basic microscope by themselves using
optical devices.

Publication List on Cooperation〔Original papers (Selected)〕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.,"96

Naruse, K., Yamahira, K., and Kitano, J. (2021). Genome editing
reveals fitness effects of a gene for sexual dichromatism in Sulawesianfishes. Nat. Commun. 12. 1350. DOI: 10.1038/s41467-021-21697-0
Beppu, K., Tsutsumi, R., Ansai, S., Ochiai, N., Terakawa, M., Mori, M.,
Kuroda, M., Horikawa, K., Tomoi, T., Sakamoto, J., Kamei, Y., Naruse,
K., and Sakaue, H. (2022). Development of a screening system for

"
"
"
"
"
"
"
"

agents that modulate taste receptor expression with the CRISPR-Cas9
system in medaka. Biochem. Biophys. Res. Commun. 601, 65372. DOI:
10.1016/j.bbrc.2022.02.082
Goto, T., Soyano, T., Liu, M., Mori, T., and Kawaguchi, M. (2022).
Auxin methylation by IAMT1, duplicated in the legume lineage,
promotes root nodule development in Lotus japonicus. Proc. Natl.
Acad. Sci. U.S.A. 119, e2116549119. DOI: 10.1073/pnas.2116549119
Kaneko, E., Sato, H., and Fukamachi, S. (2021). Validation of the threechamber strategy for studying mate choice in medaka. PLoS One 16,
e0259741. DOI: 10.1371/journal.pone.0259741
Kinoshita, N., Yamamoto, T.S., Yasue, N., Takagi, C., Fujimori,T., and Ueno, N. (2022). Article Force-dependent remodeling ofcytoplasmic ZO-1 condensates contributes to cell-cell adhesion throughenhancing tight junctions. iSCIENCE 25, 103846. DOI: 10.1016/j.
isci.2022.103846
Matsuo, M., Kamei, Y., and Fukamachi, S. (2021). Behavioural redlight sensitivity in fish according to the optomotor response. R. Soc.
Open Sci. 8, 210415. DOI: 10.1098/rsos.210415
Mohri, T., and Kyozuka, K. (2022). Starfish oocytes of A. pectinifera
reveal marked differences in sperm-induced electrical and intracellular
calcium changes during oocyte maturation and at fertilization. Mol.
Reprod. Dev. 89, 3322. DOI: 10.1002/mrd.23544
Tokanai, K., Kamei, Y., and Minokawa, T. (2021). An easy andrapid staining method for confocal microscopic observation andreconstruction of three-dimensional images of echinoderm larvae andjuveniles. Dev. Growth Differ. 63, 4783487. DOI: 10.1111/dgd.12758
Wu, D., Arakawa, H., Fujita, A., Hashimoto, H., Hibi, M., Naruse, K.,
Kamei, Y., Sato, C., and Kitajima, K. (2021). A point-mutation in the
C- domain of CMP-sialic acid synthetase leads to lethality of medaka
due to protein insolubility. Sci. Rep. 11, 23211. DOI: 10.1038/s41598021-01715-3
Zeng, C.-W., Kamei, Y., Shigenobu, S., Sheu, J.-C., and Tsai, H.-J.
(2021). Injury-induced Cavl-expressing cells at lesion rostral side playmajor roles in spinal cord regeneration. Open Biol. 11, 200304. DOI:
10.1098/rsob.200304〔Research activity by Yasuhiro Kamei〕
Specially Appointed Associate Professor Yasuhiro Kamei is the principal
investigator of Laboratory for Biothermology. For details, please refer tothe 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 NIBB9s information infrastructure, themaintenance of the institute9s 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), DB-HABs (harmful algal
blooms), and ChaetoBase (Chaetoceros gracilis).
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

97

National Institute for Basic Biology

Research Support
NIBB BIORESOURCE CENTERHead

FUJIMORI, Toshihiko

To understand the mechanisms of living organisms, studies
focusing on each gene in the design of life (the genome)
are required. The use of model animals and plants, such as
mice, Medaka (Oryzias latipes  ]HEUDÀVK Arabidopsis,
Lotus japonicus, and Physcomitrella patens, make it possible
to produce genetically controlled organisms, 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,
RUJDQV DQG LQGLYLGXDOV FDQ EH WKRURXJKO\ DQG HIÀFLHQWO\observed. On this front, the NIBB BioResource Center has
WKH HTXLSPHQW IDFLOLWLHV DQG VWDII WR VDIHO\ HIÀFLHQWO\ DQGappropriately 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.

98

The activities of the model animal research facility are as
follows:
1. The provision of information, materials, techniques, andanimal housing spaces to researchers.
2. The use of various kinds of instruments to analyze
mutant, transgenic, and gene-targeted animals.
3. The development of novel techniques related to transgenic and gene targeting technology.
4. Cryopreservation and storage of transgenic mice strains.
5. Generating genetically-engineered mice using the
CRISPR/Cas9 method.

I. Research support activities (mouse)
In 2001, the NIBB mouse facility (built under specificpathogen free (SPF) conditions) 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, 2021 to March 31, 2022, 3,388 mice (1 transgenic line and wild-type) were brought into the facility in the
Yamate area, and 30,944 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 (1 transgenic line), and stored using cryopreservation(34 transgenic lines). The frozen eggs of 43 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 2021, 0 zebrafish (0 fertilized eggs) were brought to thefacility nor were there any fertilized eggs or chicken embryos
brought in or taken from the laboratory. The animals housed
within the facility were used for research activities in neurobiology and developmental biology.

III. Research activities
The associate professor of this center, Dr. Eiji Watanabe,
is the principal investigator of the Laboratory of
Neurophysiology, which studies various mechanisms of thevisual system using a psychophysical approach. For moredetails, please refer to the laboratory9s page.

MODEL PLANT RESEARCH FACILITY
Plant Culture Laboratory

Assistant Professor:
Technical Staff:
Technical Assistant:

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

Figure 3. Large sized autoclave in the Yamate area

II. Research support activities (small fish and birds)
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. Vinyl isolators in the quarantine room

The Plant Culture Laboratory manages facilities for thegeneral cultivation of plants and the rearing of several animal
species that do not qualify for housing in other facilities.
The Plant Culture Laboratory equips and manages approximately 75 culture boxes or growth chambers and 13
rooms with the P1P physical containment level required
for established and emerging model plants, such as thale
cress (Arabidopsis thaliana), rice (Oryza sativa), moss
(Physcomitrella patens), liverwort (Marchantia polymorpha), green alga (Chlamydomonas reinhardtii), and several
other flowering plants including carnivorous plants. The
facilities are also used to grow the sea anemone Exaiptasia
pallida. The culture space is used throughout the year by >70
researchers from external and internal groups.
In addition to regular culture conditions, extreme environmental conditions in terms of light and temperature areavailable for experiments. Three chambers (3.4 m 2 each)with controllable CO2, humidity, temperature, and light (max
70,000 lux) conditions are also available. A tissue culture
rack with dimmable LEDs and pulse-width modulation con-99

National Institute for Basic Biology

Research Support

trollers is available for algae cultures that must be exposed toprecise light concentrations. Autotrophic and heterotrophic
culture devices are also available for researchers working
with cyanobacteria, algae, and cultured flowering plant cells.
Aseptic experiments can be performed in an aseptic roomwith clean benches and a safety cabinet. Various analytical
instruments, including three flow cytometry systems and a
DUAL-PAM system to measure DNA content and chlorophyll fluorescence, respectively, are also available. A liquidhandling system for fully automated and simultaneous insitu hybridization of sections of up to 60 glass slides is also
provided.
There is also a 386 m2 experimental farm next to the NIBB
Myodaiji area building for maintaining Japanese morning
glories and related Ipomoea species, several carnivorous
plants, castor beans, and other flowering plants that must be
cultivated outdoors. Three heated greenhouses (measuring
44, 44, and 45 m2) contain sensitive carnivorous plants andthe periodically mass-flowering plant Strobilanthes flexicaulis. Four air-conditioned greenhouses (4, 6, 9, and 9 m2)
are provided for the cultivation of Japanese morning glories
and additional carnivorous plants. Two of the air-conditioned
greenhouses (9 and 18 m2) meet the P1P physical containment level and are available for experiments using transgenic rice plants, Japanese morning glories, and carnivorous
plants. The Plant Culture Laboratory also maintains a 46
m2 building with a storage area and workspace. Part of thisbuilding is used for rearing Japanese rhinoceros beetles andthe common grass yellow butterfly.
Between April 2021 and March 2022, a ploidy analyzer was
introduced. It is a compact flow cytometer that is suitablefor ploidy and genome size analyses of plants, animals,
and microorganisms. The liquid handling system has been
updated to support whole-mount in situ hybridization.
There are two groups in the Laboratory of Biological
Diversity, and the assistant professors at this facility, Dr. A.
Hoshino and Dr. K. Tsugane, are the principal investigatorsof each group. For additional details, please refer to the laboratory9s webpage.

Figure 5. Newly introduced ploidy analyzer.

100

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/P3 physical containment level, and is routinely used for DNA recombination
experiments.

Figure 6. 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-

Medaka resources provided from NBRP medakation 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 the collection of individual resources, we collected 26 strains (achievement rate 173%). As a result,the entire core institute now has 730 strains (achievement
rate 125%). A total of 411 strains (achievement rate 117%)
were provided. 76 DNA clones were provided (achievement rate 38 %). As of FY2021, 395 hatching enzyme tubes(achievement rate 197%) were provided; thus the numerical targets for collection, conservation, and provisioning
were met for the entire project, except for the number ofclones provided. To enhance online contents, <The Life of
My Master Dr. Tatsuo Aida= (edited by Tetsuro Takeuchi),<Biological Experiments for Elementary, Middle, andHigh School Students= (written by Kunisuke Takeuchi),and <Medaka Experiments Anyone Can Do= (written by
Kunisuke Takeuchi) were released from NBRP Medaka in
April 2021. In October 2021, a new database, <3D atlas andsingle cell transcriptome data of medaka pituitary= was madeavailable on NBRP Medaka. On September 16-17, 2021,
we participated in the 27th Small Fish Research Meeting,as well as the Small Fish Community Meeting. The 44th
Annual Meeting of the Molecular Biology Society of Japan(December 1-3, 2021, Pacifico Yokohama) was held onsite,
and we promoted the project with Utsunomiya University
at a booth at the <National BioResource Project (NBRP) - A
lineup of bioresources.

II. NBRP Morning Glory (Ipomoea nil)
Project Manager: HOSHINO, Atsushi
Japanese morning glory (Ipomoea nil) is a traditional floricultural plant in Japan. Studied worldwide, it is commonly
investigated in the fields of plant physiology and genetics.
NIBB collects, develops, and distributes DNA clones, mutant
lines for flower pigmentation, and transgenic lines as a subcenter of the National BioResource Project (NBRP) Morning
Glory in collaboration with the core organization center at
Kyushu University. From April 2021 to March 2022, we collected 10 mutant lines and 6 transgenic lines. Additionally,
we provided 10 mutant lines, 2 transgenic lines, and 13 DNA
clones to both local and international biologists. Finally, we
analyzed whole-genome sequences of 100 mutant lines using
next-generation sequencers to develop genetic variation databases.

Left: The genome database (http://viewer.shigen.info/asagao/) containing
the whole-genome sequence, transcriptome sequences, and end sequencesof the EST and BAC clones. Mutation sequence information links the
genome and mutant databases. Right: A mutant flower phenotype101

National Institute for Basic Biology

Research Support
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 observation. 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.

102

NIBB CENTER OF 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
HAMATANI, Ayako

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 8back up9 the biologicalresources essential to the work being done at universities andresearch institutions nationwide, called the 8Interuniversity
Bio-Backup Project (IBBP)9.
The IBBP Center was established as a centralized backup
and storage facility at NIBB, while IBBP member universities have set up satellite hubs and work closely with the
IBBP center to put in place reciprocal systems for backing up
important biological resources that have been developed byresearchers residing in the area each university satellite hub
is responsible for.

The IBBP Center includes:
earthquake proof structures capable of withstanding evenvery large-scale quakes which are equipped with emergency backup power generators,
cryopreservation 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 researcher9s 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 Center9s stateof the art facilities. As a result of this, Japan9s research infrastructure has been significantly strengthened.

I. Current status of back up available forbiological resources

Figure 1. IBBP Center.

In 2021, the IBBP Center stored 5,264 384-well and 112
96-well plates consisting of as cDNA/BAC clones, 17,669
tubes for animal cells, plant and animal samples, proteins,
genes and microorganisms, 8,498 133mm-straw tubes forsperm and 671 seed samples. In total 2,049,024 samples
were stored.

103

National Institute for Basic Biology

Research Support

during freezing and drying of aqueous solutions of lyophilized pharmaceuticals, the analysis of amino acid and genecomposition, elucidation of biochemical and physicochemical properties, structure-function relationship analysis, anddevelopment of mass production technology for several
antifreeze proteins from Japanese plants and animals and thefundamentals of successful cryopreservation as well as recent
preservation methods and facilities.

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

II. Collaborative Research Project for thedevelopment of new long-term storage
technologies and cryo-biological study
As the IBBP Center can only accept biological resources
which can be cryopreserved in liquid nitrogen, researchers
cannot backup 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 2021, 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 2021 on November 11-12, 2021 online, becauseof the COVID-19 pandemic. We had 227 participants fromseveral fields covering physics, chemistry, biology, and technology. As the special lectures, We had 4 special lectures,the current status of the preservation of cultured cells andgerm cells of endangered species at the National Institutefor Environmental Studies, the relationship between protein
conformational changes and additive properties that occur

Figure 4. Group photo of Cryopreservation conference 2021

104

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

SUZUKI, Ken-ichi

Postdoctoral Fellow:
Technical Assistant:

KISHIMOTO, Mariko
SHIBATA, Yuki
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.

Postdoctoral Fellow:
Technical Assistant:

SHIBATA, Yuki
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.

105

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〕
T., Matsubara, H., Minamitani, F., Satoh, Y., Tozawa, S.," Takeuchi,
Moriyama, T., Maruyama, K., Suzuki, K.T., Shigenobu, S., Inoue, T.,
Tamura, K., Agata, K., and Hayashi, T. (2022). Newt Hoxa13 has an
essential and predominant role in digit formation during development
and regeneration. Development 149, dev200282. DOI: 10.1242/
dev.200282

106

CENTER FOR RADIOISOTOPE FACILITIESHeadHASEBE, Mitsuyasu

Technical Staff:

Technical Assistant:

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

3. Cleaning and checking the radioactive
wastewater storage tanks
All wastewater from the CRF is stored in the radioactive
wastewater storage tanks. Wastewater is discharged after
confirming that the concentration of RIs in the water isbelow the legal concentration limit. In February 2022, we
commissioned contractors to clean and inspect the inside of
the tanks. By this checking, the wastewater storage tanks
were diagnosed to continue to be usable.
The figures for training courses of RI handling are shown in
Table 1. The annual changes of the CRF registrants and thetotal number of entrances to the CRF per fiscal year are shownin Figure 1. The numbers of the CRF registrants and users
from April 2021 to March 2022 are shown in Table 2. During
this period the total number of the users and visitors counted
by the access control system was 802 (Table 3). The balance of
RIs received and used at the CRF is shown in Table 4.
14000

350
# of registrants

1. Revision of the CRF9s rules
The CRF9s rules were revised twice in 2021. In April 2021,
to respond to the elimination of the seal and signature and thereorganization of the IMS technical organization, the CRF9s
two rules, the Center for Radioisotope Facilities Myoudaiji
District Experimental Facilities Radiation Damage
Protection Rules and the Center for Radioisotope Facilities
Myoudaiji District Experimental Facilities Radiology Worker
Registration Procedure Guide, were revised accordingly. In
July 2021, to meet the revision of the Japanese relevant laws,
the CRF9s rule (Center for Radioisotope Facilities Myoudaiji
District Experimental Facilities Radiation Damage Protection
Rules) was revised. The main changes include:
1) the measurement of exposure doses in the lens of theeyes (from <150mSv per year= to <50mSv per year and
100mSv per 5 years=)
2) the method of recording radiation dose measurements andcalculations

2. Renewal of the RI handling management
systemThe relevant laws require the handling (acceptance, use,
dispose and dispensing) of RIs to be recorded. The CRF has
used a PC software (the RI handling management system,
Aloka ISR1000), but the software is no longer compatible
with the current situation. With the approval of the CRF
Steering Committee, we renewed the software in March 2022
using budget allocated from NIBB and NIPS.

2021

2017

2019

2015

2011

2013

2007

2009

2003

1989

2005

0
1999

0
2001

2000

1995

4000

50

1997

100

1991

6000

1993

150

1987

8000

1983

200

1985

10000

1981

250

Total number of entrances

12000

Total number of entrances"

1979

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
and monitor the purchase and transfer of radioisotopes (RIs).
The following is an outline of the major activities that CRF
conducted in 2021.

# of registrants

300

Figure 1. Annual number of the CRF registrants and the total number ofentrances to the CRF
Table 1. Training courses offered for radiation workers during fiscal year 2021.

Training course# of participants

Introductory course for beginners
Introductory course for experts

05

Users training course
Radioisotope handling practice32
1

Table 2. Number of the CRF registrants and users during fiscal year 2021.

Type# of registrants

The number of persons
37# of users

16

Table 3. Total number of uses by individual users and visitors during
fiscal year 2021.

Type# of users# of visitors

The number of persons
688
114

Table 4. RIs received and used (in kBq) during fiscal year 2021.

NuclideSituation

Radioactivity

P

Received

59,200P

Used

46,450C

Received

18,500C

Used

23,865H

Received

0

Used

37

32
32
14
14
3
3

H

107

National Institute for Basic Biology

Research Support
RESEARCH ENHANCEMENT STRATEGY OFFICE

Director

Vice-Director

Vice-Director

HASEBE, Mitsuyasu

YOSHIDA, Shosei

MANO, Shoji

5) Conducted follow-up investigation of the activity of the
Research University Strengthening Promotion project.
6) Summarized 2020 and 2021 NIBB activities for theannual external reviewer meeting on 8 March 2022,
managed the meeting, and prepared the report.
7) Prepared the proposal on mid-term objectives and plansfrom 2022 to 2027.

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 NINS9s 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.

PLANNING AND EVALUATION GROUP
Professor:

HASEBE, Mitsuyasu
KAWAGUCHI, Masayoshi
Specially Appointed Assistant Professor (URA):
KURATA, Tomoko
TATEMATSU, Kiyoshi
Assistant Professor:
JOHZUKA, Katsuki
FUJITA, Hironori

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

The group9s main activities in 2021 were as
follows:
1) Prepared reports on 2020 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 FY2022
budget demands.
4) Prepared reports for the NINS auditors.

1088) Published the Annual Report 2020.

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 group9s main activities in 2021 were as follows:
1) Press releases
The group issued press releases concerning NIBB9s 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 page3) Edited publications, produced posters and leaflets
The group 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
The group operated Twitter and Facebook accounts tocommunicate the activities of NIBB to the public. We also
created videos introducing NIBB9s research that are posted
on YouTube.
5）Public relations activities to recruit graduate students
The group organized four graduate school information
sessions for prospective students during the year.
6) Organized scientific outreach programs
The group organized online outreach events for the publicand coordinated science classes for middle and high school

LIAISON AND COOPERATION GROUP
Professor:

udtse.snt

UENO, Naoto
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 NIBB9s activities
related to research collaborations and cooperation with universities and research institutes in Japan and abroad.

The main activities performed by this groupfrom April 2021 to March 2022 were as follows:
1) Coordinated the following conferences/symposia:
・The 2nd meeting for NIBB-Center for Organismal Studies(COS) Heidelberg International Collaborations Lecture
series #2 <Stem Cell=. Zoom online, June 25, 2021 (p. 112)
・NIBB-Institute of Advanced Medical Sciences(IAMS), Tokushima University, joint seminar <Toward
Strengthening Collaboration and Cooperation=. Zoom
online, October 22, 2021 (p. 113)
・Cryopreservation Conference 2021. Zoom online,
November 11312, 2021 (p. 104)
・The 3rd Chubu University-NIPS-NIBB joint seminar <AI
to Life System=. Okazaki Conference Center together
with Zoom online (Hybrid), Okazaki, Japan. March 28,2022 (p. 114)
・The 3rd meeting for NIBB-COS Heidelberg International
Collaborations Lecture series #3 <Cell Signaling / Cell
Biology=. Zoom online, March 31, 2021 (p. 112)2) Supported the acceptance of researchers to NIBB fromabroad related to the following events:
・Family of a specially appointed assistant professor from
Poland (September 2021)
3) Supported the following education-related programs:
・NIBB Internship Program 2021 (p. 118)COLLABORATIVE RESEARCH GROUP
Professor:

TAKADA, Shinji
SHIGENOBU, Shuji
Specially Appointed Associate Professor (URA):
KAMEI, Yasuhiro
Associate Professor:
UCHIYAMA, Ikuo
Specially Appointed Assistant Professor:
KURATA,Tomoko
TATEMATSU,Kiyoshi
Technical Assistant:
ICHIKAWA, Mariko
ICHIKAWA, ChiakiThis 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.
The main activities of this group in 2021 were as follows:
1) Hosted collaborative projects.
2) Operated and managed NOUS (NINS Operation Use
System), an online platform to manage collaborative
projects.
3) Organized the Genome Informatics Training Course(GITC) and the BioImage Analysis Training Course(BIATC).
4) 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).

The group9s main activities in 2021 were as follows:
1) Coordinated and managed lectures and other activities,
including periodical research presentations by students.
2) Organized NIBB9s programs related to PhD courses,
such as the orientation program for new students andOpen Campus Day online for prospective students.
3) Cooperated to organize interdepartmental programs in
SOKENDAI such as the Life Science Retreat.
4) Gathered and provided useful information for bothstudents and faculty members.

109

National Institute for Basic Biology

Research Support

COLLABORATIVE INNOVATION GROUP
Professor:
MINAGAWA, Jun
Specially Appointed Associate Professor:
KANAI, Masatake
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 group9s main activities in 2021 were as follows:
1) Supported the application for JST9s 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.

110

TECHNICAL DIVISIONHeadMIWA, Tomoki

Common Facility Group

Research Support Group

Chief:

Chief:

MORI, Tomoko● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki
Vice-Unit Chief:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
NISHIDE, Hiroyo
Technical Staff:
NAKAMURA, Takanori
TANIGUCHI-SAIDA, Misako
SUGIURA, Hiroki
MORI, Shogo
Technical Assistant:
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
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, SachikoTechnical Staff:
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 institute9s
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 institute9s 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.

111Collaboration Programs with
Overseas Institutions
NIBB-Center for Organismal Studies (COS),
Heidelberg, Germany
NIBB-COS international collaboration research projectfocusing on the mechanisms and evolution of light sensing
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. More detail areprovided on the page of the collaborative research project (p.
75).

We also organized the social meetup events in the meetings
and used the video chat tool, Spatial Chat, as an online
meeting platform to facilitate it. Principal investigators,
researchers, and students from both institutes were thus able
to meet and have lively exchanges. By manipulating thespecially assigned icon given to each of them, participants
were able to freely join any discussions they chose to partakein with no limitations on the number of people involved.
Consequently, the event was highly praised.

Group discussion after the lectures in the 3rd meeting.

The meeting for NIBB-COS Heidelberg International
Collaborations
Lecture series #2 <Stem Cell=
Lecture series #3 <Cell Signaling / Cell Biology=
The meeting for NIBB-COS Heidelberg International
Collaborations was organized in conjunction with COS
Heidelberg in Germany. During the Lecture Series held inthis program, two researchers from both institutes gave apresentation on the specific theme. In the 2nd meeting heldon June 25, 2021, the lecture series theme was <Stem Cell=.
Dr. Tomomi Tsubouchi from the Laboratory of Stem Cell
Biology and Dr. Sergio P. Acebrón from COS Heidelberg
talked about their research projects. Thirty-nine members
from NIBB and 33 from COS Heidelberg (both totals
included post-docs and students) participated in this event.
The theme of the 3rd meeting held on March 31, 2022, was<Cell Signaling / Cell Biology=. Dr. Kazuhiro Aoki from theDivision of Quantitative Biology and Dr. Gislene Pereira
from COS Heidelberg gave their presentations to 36 NIBB
members and 26 COS members. During both events, lively
discussions took place.

Lecture by Tomomi Tsubouchi (NIBB) in the 2nd meeting.

112

In these meetings, several discussions on new collaborative research were put forward, and as a result, ties between
NIBB and COS seemed to have deepened. Some participants suggested that the exchange of young researchers andstudents from both institutes would be considered and thata poster session would be held at the meeting. In light ofthese comments, we are considering holding an online poster
session for the next meeting.

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), basedon the academic exchange agreement between the NINS
and Princeton University. In FY2020, Dr. Ellen Reed, an
IRCC's specially appointed research staff 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
optical or chemical recruitment of soluble proteins" at
Princeton University. In FY2021, due to the COVID-19 situation, it was difficult to travel between NIBB and Princeton
University. However, collaborative research activities wereconducted using online communication tools and we agreed
that Dr. Reed would visit NIBB and carry out the collaboration in Prof. Aoki's laboratory in the next year.

Collaborative Activities with Universities
and Research Institutes in Japan
NIBB-Institute of Low Temperature Science(ILTS), Hokkaido University
Accomplishment of the NIBB Priority Collaborative
Research Project■ Project Title:
Molecular 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
International Online Seminar by IMEG, Kumamoto University
IMEG at Kumamoto University has been holding a series ofmonthly seminars called <IMEG Seminar Series - The road
WRJOREDOVFLHQFHʮDVSDUWRILWVDFWLYLWLHVDWWKH,QWHUQDWLRQDOAdvanced Research Center. As part of the collaborationagreement between IMEG and NIBB, NIBB members has
participated in this seminar series.
■ Seminar series:
2021 May: Jacqueline Tabler (Max Planck Institute of
Molecular Cell Biology and Genetics, Germany)
2021 June: Vikas Trivedi (EMBL Barcelona)
2021 July: Patrick PL Tam (University of Sydney, Australia)
2021 Aug: Eran Meshorer (The Hebrew University ofJerusalem, Israel)2021 Sep: Jacob H. Hanna (Weizmann Institute of
Science, Israel)2021 Oct: Kathy Niakan (The Francis Crick Institute andthe University of Cambridge, UK)
2021 Nov: Xin Chen (Johns Hopkins University, Howard
Hughes Medical Institute, USA)2021 Dec: Peter K. Todd (University of Michigan, USA)2022 Jan: Peter S. Zammit (King9s College London, UK)
2022 Feb: John B. Wallingford (University of Texas atAustin, USA)2022 Mar: Qi-Long Ying (University of South California,
USA)

NIBB-Institute of Advanced Medical Sciences(IAMS), Tokushima University
NIBB- IAMS, Tokushima University, joint seminar<Toward Strengthening Collaboration and Cooperation=
Following the collaboration agreement between NIBB
and IAMS, a joint seminar was held on October 22, 2021,
to strengthen cooperation and budding new collaborative
research between the two institutions. The seminar introduced
the facilities and equipment of both institutes, as well as efforts
for their joint usage/research programs. A total of 76 people
attended the seminar, 49 from NIBB and 27 from IAMS.
After the seminar, an opinion exchange meeting was heldwith the participation of directors and faculty from bothsides. They actively discussed future cooperation and col-laboration in joint usage/research programs, the exchangeof technologies and methods in which both sides havestrengths, and training and exchanging of staff involvedin the implementation and operation of the joint usage/
research programs. This would be the first step toward deepening cooperation between NIBB and IAMS and leading to
the development of collaborative research and joint usage/
research programs between the two institutes in the future.
■ Seminar information:
Presentation from NIBB
Kiyokazu Agata (Introduction of NIBB)
Shuji Shigenobu (Introduction of NGS and mass spectrometry analyses)
Yasuhiro Kamei (Introduction of bioimaging analysis)
Eiji Watanabe (Introduction of model animal researchfacility)■ Presentation from IAMS
Hidetaka Kosako (Introduction of mass spectrometry analysis)
Tomohide Saio (Introduction of nuclear magnetic resonance (NMR) analysis)
Yosuke Matsushita (Introduction of NGS analysis)
Tatsuya Takemoto (Introduction of genome editing analysis)

NIBB-Institute for Molecular and Cellular
Regulation, Gunma University
Agreement of Cooperation with IMCR, Gunma University
NIBB and IMCR at Gunma University have signed an agreement of cooperation which is deemed effective from April 7,
2021. To prevent the further spread of COVID-19 infection,
the signing ceremony was held in a remote capacity via an
online connection between Maebashi and Okazaki.

Dr. Kiyokazu Agata, the Director-General of NIBB, and Dr. Ken Sato, theDirector of IMCR, singing the agreement.

This agreement aims to promote mutual collaborative activities in the capacity of research and education centers thatlead the way in world class, cutting edge research in the fieldof endocrinology, metabolism research and basic biology, as
well as to utilize the results of these activities for research ona mutual basis. It also strengthens the support infrastructure
for collaborative use and research nationwide, and to stimulate international academic exchange.
As part of the collaboration agreement, Director-general
Kiyokazu Agata and Prof. Takashi Ueda present theirresearch activities in the 7th IMCR Symposium that was heldat September 9-10.

113

NIBB-Chubu University
The 3rd Chubu University-NIPS-NIBB joint seminar <AI
to Life System=NIBB and NIPS have been promoting exchanges of personnel and information with Chubu University through joint
seminars on the theme of "AI to Life System" since the fallof 2021, aiming for emergence through interdisciplinary
research and the discovery and cultivating of young researchers. The first joint seminar was hosted by Chubu University
in November 2021, and the 2nd seminar was organized by
NIPS in January 2022.
On March 28, 2022, NIBB organized the 3rd joint seminar
held as a hybrid meeting with Zoom online system at the
Okazaki Conference Center. In addition to the lectures ofongoing research utilizing AI analysis methods at each institute,a small group discussion was held to promote new collaborative research between the three institutes. The group discussions
were divided into two categories, "technology" and "science".
In each category, four topics were set up, and researchers andstudents discussed each topic separately. This event helped todeepen mutual understanding between information science andlife science, and promoted the budding of collaboration researchleading to the "elucidation of life systems using AI.

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 2021, 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, allcourses were held online to prevent the spread of COVID-19
infection. By virtue of the online system, we expanded thecourse audiences by accepting some of them as <auditors=
who could receive only limited support during the hands-on
practice.

Introduction to RNA-seq: From the
Basics of NGS to de novo Analyses■ Organizers: Dr. Shuji Shigenobu and Dr. Ikuo
Uchiyama (NIBB Core Research Facilities)■ Lecturers: Dr. Shuji Shigenobu, Dr. Ikuo Uchiyama,
Dr. Masanao Sato (Hokkaido Univ.), Dr. Katsushi
Yamaguchi, Ms. Hiroyo Nishide, Mr. Takanori
Nakamura, Mr. Hiroki Sugiura (NIBB Core
Research Facilities)

August 25 (Thu)326 (Fri), 2021
Opening remarks by Dr, Kiyokazu Agata, the Director-General of NIBB.

Group discussion on the topics of gene and protein.

■ Seminar information
LecturesTakashi Ueda (NIBB)
Junichi Chikazoe (NIPS)
Seine Shintani (Chubu University)■ Group discussion
1) Technology: Computer vision, Robotics, fMRI, andElectron Microscopy.
2) Science: Gene and protein, Cell, Brain and sensory,
and Brain and behavior.

114(Practical Course) Introduction to NGS Analysis:
Basics of UNIX, R, and NGS■ 31 participants and 16 auditors (including 2 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

September 15 (Thu)316 (Fri), 2021
(Practical Course) RNA-seq Analysis Pipeline■ 28 participants and 16 auditors (including 3 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

Introduction to NGS Analysis:
Basics of UNIX, R, and NGS
February 9 (Wed)310 (Thu), 2022
(This program was the same as the above listed practical course)■ 27 participants and 16 auditors (including 5 from NIBB)

Introduction to RNA-seq: RNAseq Analysis Pipeline
March 2 (Wed)33 (Thu), 2022
(This program was the same as the above listed practical course)■ 27 participants and 12 auditors (including 5 from NIBB)

115

The Bio-imaging Data Analysis
Training Course 2021
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 Takada

November 24 (Wed)326 (Fri), 2021
The 9th 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. 134 people applied for the course,which had an announced capacity of 16 participants. Given
the high demand for courses on these subjects, we accepted
33 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 course9s 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 course9s 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)

116

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

KAKENHI researchers make full use of the ABiS platformto accelerate their research projects. In addition, we organize
technology training sessions, workshops, and symposia
to disseminate advanced imaging technologies and share
information about them. We also coordinate with the otherthree platforms (Platform of Supporting Cohort Study andBiospecimen Analysis, Platform of Advanced Animal Model
Support, and Platform for Advanced Genome Science) to
provide both multidisciplinary and international support.
Euro-Bioimaging (EuBI) is the largest and most well-established imaging network in Europe, and has been expanding
globally to form the Global Bioimaging (GBI) network
which boasts participants from areas such as India, Australia,
Singapore, the Republic of South Africa, Canada, Mexico,USA, and some Latin American countries. In 2018, ABiS
joined the GBI project representing the Japanese bioimaging
community. It is hoped that through GBI, ABiS will be able
to raise the quality of present support to that of the currently
accepted international benchmarks, and that further observations, data analyses and research methods will be able to bebetter shared globally. Furthermore, it is also hoped that thiscollaboration will lead to set up a better environment for bioimaging research in Japan. This will be achieved not only byproviding international training courses for young researchers and the staff of imaging facilities, but also throughdiscussions about the implementation of career paths; acommon problem in this field.
Since the ABiS project that spanned 6 years was finishedat the end of this fiscal year, we self-evaluated the past
ABiS activities and forecast the future demands, andapplied for the Grant-in-Aid for Transformative Research
Areas, Platforms for Advanced Technologies and Research
Resources (FY2022) as the next ABiS project. In February2022, our past activities were highly evaluated by the committee and plans for the next project have been approved forthe second phase of the ABiS project (FY2022-FY2027). As
in the past, the ABiS will continue to support light microscopy, electron microscopy, magnetic resonance imaging, andimage analysis as the core institute together with the Institutefor Physiological Sciences.

ABiS Symposium "Image Data
Analysis to Open Up A New Era
for Life Science"
March 1 (Tue) 2022
The ABiS symposium "Imaging Data Analysis toOpen Up A New Era for Life Science" was held thisyear as an online event. The following 8 researchers,
experts in image data analysis and database construction, gave lectures.
" Naomi KAMASAWA (The Max Planck Florida
Institute for Neuroscience Imaging Center)" Nobuhiko OHNO (Jichi Medical University/
National Institute for Physiological Sciences)" Tatsumi HIRATA (National Institute of Genetics)" Kazuo IABA (Tsukuba University)" Naohiro OKADA (The University of Tokyo)" Saori TANAKA (Brain Information Communication
Research Laboratory Group)" Shu-ichi ONAMI (RIKEN Center for Biosystems
Dynamics Research)" Yo s h i t a k a K I M O R I ( F u k u i U n i v e r s i t y o f
Technology)
There were 168 participants, and a lively question
and answer session was held for each presentation. A
participant survey and other information about this
symposium is available on the website. (https://www.
nibb.ac.jp/abis/ev20220301/)

117

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 FY2021, considering the situation of COVID-19, we
called the applications from students attending universities
abroad with the explicit understanding that the internshipprogram might cancel if the COVID-19 situation worsened.
Consequently, one student who belongs to a Vietnamese university was selected. However, due to border control by theJapanese Government, the student was unable to visit Japan.
In contrast, four international students attending universities
in Japan were accepted, and three of them came to NIBB
during FY2021.

Report from a participant
Piyusha Mongia
I am Piyusha Mongia, a graduate student from Osaka
University, Japan. I am grateful that I could take part inthis internship program and be a part of Prof. Jun-Ichi
Nakayama9s laboratory. The lab works on epigenetics- how
it regulates gene expression, the establishment of epigenetic
markers and so on.
The bigger question was to understand how epigenetic
information is inherited and what are the factors involved.
Epigenetics refers to heritable changes- not coded by DNA.
Eukaryotic chromosomes can be divided into 2 broad categories: euchromatin and heterochromatin. Euchromatin refers
to the regions on chromosomes which are transcriptionallyactive. Heterochromatin, on the hand, is generally transcriptionally silent, present at repetitive sequences or transposons. Methylation of lysine 9 of histone H3 (H3K9me) isa hallmark of heterochromatin. I created and assessed the
involvement of four candidate gene deletions in heterochromatin inheritance in fission yeast spores. Fission yeast
is a simple eukaryote having three chromosomes, making
its study relatively easier. I first replaced the 4 genes with
nourseothricin by PCR and then visualized swi6-egfp localization by fluorescence microscopy. One of the candidate
genes seems to be important for the inheritance of heterochromatin but more studies need to be done to conclude so.
In a parallel project, I tried to express three of Tetrahymena118

proteins using a bacterial expression system. These proteins
are involved in heterochromatin assembly. Producing recombinant proteins allows us to purify and biochemically analyze
individual proteins activities and interactions in-vitro. In
the end, I managed to express all, but purify one of theproteins due to time constraints. I learned many biochemical techniques, like Gel Filtration Chromatography and Ion
Exchange Chromatography. I wish I could have had a longer
time period to do more fun experiments.
Every day I was faced with challenges, and learnt something new. All the members were very patient with me andtaught me something or the other. I9m extremely grateful to
Dr. Hayashi and Dr. Kataoka, who guided me throughout the
project. Prof. Nakayama was very supportive and encouraging. The research atmosphere in the lab is very exciting and it
was truly an enriching experience for me.

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

November 10 (Wed), 2021
Special class at Iwazu Junior High School■ Prof. Teruyuki Niimi<Full of Wonders in Diverse Insects=

Visits to Elementary and Junior High Schools
NIBB, in collaboration with the Okazaki City Board of
Education, visited elementary and junior high schools during2021 to give special classes.
October 13 (Wed), 2021
Special class at Ryukai Junior High School■ Prof. Toshihiko Fujimori<The Beginning of Forming Animals - Thinking from the Cell=

November 15 (Mon), 2021
Special class to Tokiwa Junior High School (online)■ S. A. Assist. Prof. Tomoko Kurata<Virtual Tour in NIBB=■ Assist. Prof. Takehiko Kanazawa<The Micro World of Plants=

November 9 (Tue), 2021
Special class to All junior high schools in Okazaki (online)■ Director General Kiyokazu Agata<Planarian No Matter How Many Cuts=

February 14 (Mon), 2022
Special class to Atago elementary school (online)■ Assoc. Prof. Makio Yokono<How Do Plants Use Light to Build Bodies?=

119

Work experience for junior high school
students at NIBB
Kita Junior High School

March 4(Fri), 2022
Special class at Okazaki Kita High School■ Dr. Yakub Wudarski<Science and English=

June 22 (Tue), 2021
Special class at Meiwa High School■ Prof. Teruyuki Niimi<Exploring the Diversity of Insect Patterns and Forms=

Visit to High Schools
February 17 (Thu), 2022
Special class at Okazaki High School■ Prof. Miyo Morita T.
<Plants that Rise Up After Falling Down: Mechanism ofGravity Sensing=

July 2 (Fri), 2021
Special class at Okazaki Kita High School■ Assoc. Prof. Nobuyuki Shiina<Mechanism of Long-Term Memory Formation=

120

July 27 (Tue), 2021
Special class to Tondabayashi High School (online)■ S. A. Assist. Prof. Tomoko Kurata

October 27 (Wed), 2021
Special class to Kanagawa Hakuyo High School (online)■ S. A. Prof. Kiyoshi Naruse<Where Did Medaka Came From?=

November 2 (Tue), 2021
Special class at Aichi Sangyo University Mikawa High School■ Prof. Teruyuki Niimi<Exploring the Diversity of Insect Patterns and Forms=

August 20(Fri),2021
December 24(Fri),2021
Feedback session regarding research presentations by highschool students (online)■ S. A. Assist. Prof. Kiyoshi Tatematsu

Programs broadcast live online
April 24 (Sat)3 May 1 (Sat), 2021
May 21 (Fri), 2021 (Sequel)Live broadcast at niconico live
<Watching the complete metamorphosis of ladybirds.=■ Director General Kiyokazu Agata■ Prof. Teruyuki Niimi■ Prof. Shuji Shigenobu■ Assis. Prof. Toshiya Ando
■ Graduate Student Yasuhiko Chikami■ S. A. Assist. Prof. Tomoko Kurata

121

January 15 (Sat)3 January 16 (Sun), 2022
January 28 (Fri), 2022 (Sequel)Live broadcast at niconico live
■ Director General Kiyokazu Agata■ Graduate Student Miyuki Ishida■ S. A. Assist. Prof. Tomoko Kurata<Is it possible to hijack and regenerate? 4Transplanting
planarian stem cells.=

March 27 (Sun), 2022
Public lecture at the event held by Ohsumi Frontier Science
Foundation■ S. A. Prof. Kiyoshi Naruse<Biodiversity and Utilization of Wild Medaka and Related
Species 4Perspectives from Biogenetic Resources=

July 20 (Tue), 2021
National Institute of Natural Sciences The 9th Young
Researcher Award Commemorative Lecture (online)■ Assist. Prof. Kim Eunchul<Even Plants Feel Dazzling at Times=

Lectures for the public
September 25(Sat), 2021
Public lecture at the Kihara Institute of Biology■ Assist. Prof. Atsushi Hoshino<How to Create the One and Only Morning Glory in the World=

122

August 21 (Sat), 2021
The 32nd National Institutes of Natural Sciences
Symposium (online)■ S. A. Assist. Prof. Yoko Otsubo<Close Encounters of Yeast with Plasma=February 25 (Fri), 2022
Online lecture by NIBB and NIPS<The discovery of the PIEZO channel that solved themystery of skin sensation and the world of mechanosensing
research opened up through them=■ Assoc. Prof. Keiko Nonomura

October 24 (Sun), 2021
Inter-university Research Institute Symposium 2021 (online)■ Prof. Shin-ichi Higashijima<Exploring the Mechanisms for Sensing Balance andPostural Control=

Exhibition support

February 24 (Wed)3March 31 (Thu), 2022
Presentation by OKASHIN (Okazaki Credit Union) scholarship students (online)■ Graduate Student Masamichi Ueda

July 9 (Fri)3September 5 (Sun), 2021
Toyohashi Museum of Natural History
The 35th Special exhibition <The Wonderful World of Insects=■ Prof. Teruyuki Niimi■ S. A. Assist. Prof. Tomoko Kurata

July 10 (Sat)3September 20 (Mon), 2021
National Museum of Nature and Science, Tokyo
January 14 (Fri)3April 3 (Sun), 2022
Osaka Museum of Natural History
Special exhibition <PLANTS Mainstays of the Planet=■ Prof. Mitsuyasu Hasebe (Supervisory support)■ Prof. Takashi Ueda
■ Assist. Prof. Atsushi Hoshino123

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).

From Higashi Okazaki Station to Each Area
Take the South Exit of the station and walk approximately
7 minutes to Myodaiji-area, 20 minutes to 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).

AgrwF_jj

AgrwF_jj
AgrwF_jjQ,

Myodaiji-area
38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585 Japan

Yamate-area
5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787 Japan

National Institutes of Natural Sciences

National Institutefor Basic Biology
38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585 Japan
phone: +81 564-55-7000 fax: +81 564-53-7400
https://www.nibb.ac.jp/en/
Issued in December 2022
Edited by the Research Enhancement Strategy Office, NIBB