National Institute for Basic Biology2014 ANNUAL REPORTCONTENTS
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・ 1
Organization of the National Institute for Basic Biology ・
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・ 2
Goals of the National Institute for Basic Biology・
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・ 5
Personnel Changes and Awardees in 2014・
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・ 7
Cell Biology
Division of Cell Mechanisms・
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・ 8
Division of Intercellular Signaling Biology ・
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・ 11
Laboratory of Cell Responses・
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・ 13
Laboratory of Neuronal Cell Biology・
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・ 15
Laboratory of Cell Sociology ・
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・ 17
Developmental Biology
Division of Morphogenesis・
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・ 18
Division of Developmental Genetics・
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・ 21
Division of Molecular and Developmental Biology・
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・ 24
Division of Embryology・
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・ 27
Division of Germ Cell Biology・
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・ 30
Laboratory of Molecular Genetics for Reproduction・
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・ 33
Neurobiology
Division of Molecular Neurobiology・
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・ 35
Division of Brain Biology・
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・ 38
Division of Brain Circuits ・
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・ 41
Laboratory of Neurophysiology・
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・ 44
Evolutionary Biology and Biodiversity
Division of Evolutionary Biology・
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・ 45
Division of Symbiotic Systems・
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・ 48
Laboratory of Morphodiversity ・
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・ 51
Laboratory of Bioresources ・
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・ 52
Laboratory of Biological Diversity・
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・ 54
Environmental Biology
Division of Molecular Environmental Endocrinology・
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・ 62
Division of Environmental Photobiology ・
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・ 65
Division of Seasonal Biology (Adjunct)・
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・ 68
Theoretical Biology
Laboratory of Genome Informatics・
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・ 70
Imaging Science
Laboratory for Spatiotemporal Regulations ・
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・ 72
Okazaki Institute for Integrative Bioscience (Orion Project)
Laboratory of Nuclear Dynamics
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・ 73
Research Support
NIBB Core Research Facilities・
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・ 74
NIBB BioResource Center・
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・ 80
NIBB Center of the IBBP・
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・ 84
Center for Radioisotope Facilities (Okazaki Research Facilities)・
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・ 87
Research Enhancement Strategy Office・
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・ 88
Technical Division・
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Access to NIBB ・
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・ Inside back cover
The cover items are related to a study dissecting the nature and behavior of mouse spermatogenic stem cells in the testis(Hara et al., Cell Stem Cell, 2014). This study observed the in vivo fate behavior of the undifferentiated population of
spermatogonia through intravital live-imaging and pulse-labeling studies, combined with biophysical modeling. The
results showed that both singly isolated spermatogonia –which have been considered the only stem cell type– and syncytial
spermatogonia –which have been considered committed for differentiation– comprise a single stem cell pool, in whichcells stochastically interconvert between these morphologically different states. See page 30 of this report for details.

It  is  my  great  pleasure  to  introduce  to  you  all  the 201  Annual  Report  of  the  National  Institute  for Basic Biology (NIBB), which outlines the high level research activities of the Institute and its effective function as a center for collaborative research in Japan over the last year. Given these  important  missions  of  the  Institute,  I  recognize  my strong  responsibility  to  maintain  the  liberal  atmosphere  for research and discussion that NIBB has kept for many years, which I believe is a basis for these remarkable activities.
To improve the economic status of the country, the Japanese government  continues  to  strengthen  the  demand  that universities  and  inter-university  research  institute corporations,  the  latter  of  which  includes  NIBB,  should reform themselves and draw actual profits from science. It is also a serious burden to the scientific community to achieve full  compliance  in  research  activities,  excluding  any misconduct,  and  recover  the  reliance  of  the  society  to science.  These  circumstances  have  set  a  strong  headwind against  basic  sciences,  through  which  NIBB  must  stride properly.  We  will  maintain  two  directions  steadily.  One  is that  every  person  in  NIBB  should  do  his her  best  in accomplishing good research in basic biology. Good science, even in a basic research field, will eventually benefit human beings. The history of science tells us this is true. The other is that NIBB must achieve high ethical standards for research that  conform  to  the  era  of  computer  technology  and  data sharing through the internet. NIBB will work hard to be truly acknowledged as a remarkable institution in the society.
Please  find  in  this  booklet  a  summary  of  the  research, collaborative,  educational,  and  international  activities  of NIBB in 201 . I would like to note that we welcomed several new  colleagues  in  201 ,  including  one  specially  appointed professor,  one  associate  professor,  two  specially  appointed associate  professors,  one  assistant  professor,  one  specially appointed  assistant  professor,  and  five  NIBB  research fellows, while four colleagues transferred to other institutes.
Finally  I  would  like  to  congratulate  Specially Appointed Associate  Prof.  Tsuyoshi  Shimmura  for  winning  the Encouragement Award from the Japanese Society of Animal Science.  I  would  also  like  to  congratulate  young  colleagues for  winning  awards  from  academic  societies  and SOKENDAI, as detailed on page  .
To  establish  NIBB  as  an  international  leading  institute  in the  field  of  basic  biology,  we  always  welcome  your suggestions, comments and queries concerning our activities, in addition to your warm support.
Masayuki YamamotoDirector General of NIBB
July 2 , 2015

1

A

A

A

A

The  National  Institute  for  Basic  Biology  (NIBB)  was founded  in  1  on  a  hill  overlooking  the  old  town  of Okazaki  in Aichi  prefecture  as  one  of  the  Inter-University Research  Institutes  to  promote  and  stimulate  studies  of biology. NIBB conducts first-rate research on site as well as in  cooperation  with  national,  public  and  private  universities and  research  organizations.  NIBB  attempts  to  elucidate  the general  and  fundamental  mechanisms  underlying  various biological  phenomena  throughout  a  wide  variety  of biological  fields,  such  as  cell  biology,  developmental biology,  neurobiology,  evolutionary  biology,  environmental biology, and theoretical biology.
NIBB  and  four  other  national  institutes,  the  National Astronomical Observatory (NAO), the National Institute for Fusion  Science  (NIFS),  the  National  Institute  for Physiological  Sciences  (NIPS),  and  the  Institute  for Molecular  Science  (IMS),  constitute  the  National  Institutes of  Natural  Sciences  (NINS),  which  is  one  of  the  four  InterUniversity Research Institute Corporations. NIBB’s activities are supported by the Ministry of Education, Culture, Sports, Science  and  Technology  (ME T)  of  Japan.  The  Okazaki Institute  for  Integrative  Bioscience  was  established  as  a common  facility  for  the  three  institutes  (NIBB,  NIPS,  and IMS) of the Okazaki campus. BA

B■ Organization

National Institutes of Natural Sciences(NINS)President SATO, KatsuhikoNational AstronomicalObservatory of Japan (NAO)National Institute forFusion Science (NIFS)

National Institute for Basic Biology (NIBB)

Director General YAMAMOTO, Masayuki
Vice-Director
UENO, NaotoNational Institute for Physiological Sciences (NIPS)Advisory Committee forProgramming and Management

Research Enhancement Strategy OfficeInstitute for Molecular Science (IMS)Three Institutes in OkazakiCenter for Novel Science InitiativesBuildings in the Myodaiji area (left) and Yamate area (right).

Policy, Decision Making, and Administration

The  Director  General  oversees  the  operation  of  NIBB, assisted  by  the Advisory  Committee  for  Programming  and Management. The Advisory Committee, comprised of eleven professors  within  NIBB  and  ten  leading  biologists  outside NIBB,  advises  the  Director  General  on  important  matters such  as  planning  joint  research  programs  as  well  as  on  the scientific  activities  of  NIBB.  The  Advisory  Committee makes  recommendations  on  the  appointments  of  new Directors  General,  faculty  appointments,  NIBB’s  annual budget and future prospects. The  Research  Enhancement  Strategy  Office,  aimed  at supporting  researchers  in  order  to  improve  NIBB’s  abilities as  a  collaborative  research  institution,  was  restructured  in 201  from the former Strategic Planning Department, Office of Public Relations, and Office of International Cooperation. The Office is made up of five groups (p.  ) and its activities are  mainly  carried  out  by  URAs  (University  Research Administrators)  in  close  collaboration  with  the  NINS’s Research Enhancement Promotion  eadquarters. Administration  of  NIBB  is  undertaken  by  the  Okazaki Administration  Office  under  the  direct  auspices  of  the Administration Office of NINS.

2Okazaki Research FacilitiesOkazaki Institute for Integrative Bioscience (OIIB)Department of Biodesign Research■Division of Molecular and Developmental Biology *■Division of Nuclear Dynamics■Division of Plant Development and PhysiologyDepartment of Biosensing Research■Division of Bio-Environmental Science **Department of Bioorganization Research■Division of Neuronal Cell Biology ***Divisions with researchers having concurrent positions in NIBB are shown above. Some divisions*-***) also function as NIBB’s research units†-†††)  , respectively, on the right panel.

Center for Radioisotope FacilitiesCenter for Experimental Animal

Research Center for Computational ScienceResearch Facilities run jointly by NIBB and NIPS
Electron Microscopy RoomDisposal of Waste Matter FacilityInstrument Design RoomNational Institute for Basic Biology (NIBB)  As of April 1, 2015Research Units■Laboratory of Cell Responses  Cell Biology■Laboratory of Neuronal Cell Biology†††■Laboratory of Cell Sociology■Laboratory of Stem Cell Biology■Division of Morphogenesis  Developmental Biology■Division of Molecular and Developmental    Biology †■Division of Embryology■Division of Germ Cell Biology■Laboratory of Molecular Genetics for    Reproduction ■Division of Molecular Neurobiology  NeurobiologyEvaluation and Information GroupPublic Relations GroupInternational Cooperation GroupCollaborative Research GroupGender Equality Promotion Group■Division of Brain Circuits■Laboratory of Neurophysiology  Evolutionary Biology and Biodiversity■Division of Evolutionary Biology■Division of Symbiotic Systems■Laboratory of Morphodiversity■Laboratory of Bioresources■Laboratory of Biological Diversity■Division of Molecular Environmental   Environmental Biology    Endocrinology ††

■Division of Environmental Photobiology■Division of Seasonal Biology (Adjunct) ■Laboratory of Genome Informatics  Theoretical Biology■Laboratory for Spatiotemporal Regulations  Imaging ScienceResearch Support Facilities  NIBB Core Research FacilitiesFunctional Genomics FacilitySpectrography and Bioimaging FacilityData Integration and Analysis FacilityModel Animal Research FacilityModel Plant Research FacilityCell Biology Research Facility  NIBB BioResource Center  NIBB Center of the Interuniversity Bio-Backup Project (IBBP Center)  NIBB Center for Model Organism Development

Research Support Section  Technical DivisionOkazaki Administration OfficeResearch and Research Support

NIBB’s  research  programs  are  conducted  in  research  units and research support units. Each research unit is called either a  division,  led  by  a  professor,  or  a  laboratory,  led  by  an associate  professor  or  an  assistant  professor.  Each  research unit  forms  an  independent  project  team.  The  NIBB  Core Research  Facilities  and  NIBB  BioResource  Center  were launched in 2010 to support research in NIBB and also to act as  an  intellectual  hub  to  promote  collaboration  among  the researchers  of  NIBB  and  other  institutions.  The  NIBB Center  of  the  Inter-University  Bio-Backup  Project  (IBBP Center)  was  founded  in  2012  to  prevent  loss  of  invaluable biological  resources.  Projects  for  the  development  of bioresource  preservation  technology  have  been  solicited  by the  IBBP  center  from  201 .  The  NIBB  Center  for  Model Organism  Development  was  founded  in  201   to  promote development  of  new  model  organisms  and  research  using them.  The  Technical  Division  manages  the  activities  of  the technical  staff  and  helps  to  promote  the  activities  of  each research unit and to maintain the common resources of both the research support units of NIBB and the research facilities of  the  Okazaki  campus.  The  Center  for  Radioisotope Facilities is one of the latter and is run by the technical staff of NIBB.
The  Okazaki  Institute  for  Integrative  Bioscience  was founded  to  conduct  interdisciplinary  molecular  research  on various  biological  phenomena  and  is  shared  by  all  three institutes in Okazaki. Some of its divisions also function as NIBB divisions. ■ Members in NIBB
irector eneral
Pro essors
including ad unct and specially appointed

Associate Pro essors
including specially appointed
Assistant Pro essors
including specially appointed

ec nicalAssistants
andecretaries4* new member from April 2015
 Chairperson  ice-Chair5settled or
April 2arc 2 4
numbers are in million yen

ternalompetitive
Fund

24

rant in Aidor cienti ic
Researc

Personnel

4

Researc4

rants orommissioned
Researc

Facilityollaboration

t eranagement
rant orpensesN A

4■ Members of the Advisory Committee for Programming and Management

NIBB members

in totalas o st April 2

ec nical ta■ Financial Configuration of NIBB

2

AKOS IMA, Toshio IGAS IYAMA, Tetsuya
KO S IMA, Shiro*
KONDO, Shigeru*KUME, Shoen*
KURUMI AKA,  itoshiNIS ITANI, Kazuhiko*NOSE, Akinao*O TA, KunihiroTSUKITA, SachikoFUJIMORI, ToshihikoASEBE, Mitsuyasu
IGUC I, TaisenKAWAGUC I, MasayoshiMATSU AKI, MasanoriMINAGAWA, Jun*NODA, Masaharu
TAKADA, Shinji UENO, NaotoYOS IDA, ShoseiYOS IMURA, Takashi*2raduatetudents

245

Non-NIBB members

Postdoctoral Fello s
4 are N Researc Fello s

anagement
penses rant
or N

2 5

million yen
in total(as of April, 2015)Professor, Nara Institute of Science and TechnologyProfessor, Nagoya UniversityProfessor, Kyoto UniversityProfessor, Osaka UniversityProfessor, Tokyo Institute of TechnologyProfessor, Waseda UniversityProfessor, Tohoku UniversityProfessor, The University of TokyoProfessor, The University of TokyoProfessor, Osaka UniversityProfessor, National Institute for Basic BiologyProfessor, National Institute for Basic BiologyProfessor, Okazaki Institute for Integrative BioscienceProfessor, National Institute for Basic BiologyProfessor, National Institute for Basic BiologyProfessor, National Institute for Basic BiologyProfessor, National Institute for Basic BiologyProfessor, Okazaki Institute for Integrative BioscienceProfessor, National Institute for Basic BiologyProfessor, National Institute for Basic BiologyAdjunct Professor, National Institute for Basic Biology

A

A

A

BA

The  National  Institute  for  Basic  Biology  (NIBB)  sets  five goals for its activities in pursuing the progress of biology. We contribute  to  the  world-wide  community  of  biologists through  our  efforts  to  accomplish  these  goals.  This  chapter briefly  explains  four  of  these  goals.  The  last  goal,  the promotion  of  academic  research,  is  accomplished  through our research activities, which are introduced throughout this brochure. Promotion of Collaborative Research■ Collaborative Research Support Research activities in collaboration with NIBB’s divisionslaboratories  using  NIBB’s  facilities  are  solicited  from external  researchers.  Individual  collaborative  research projects   are  the  basic  form  of  collaboration  support  which provide  external  researchers  with  travel  and  lodging expenses  to  visit  NIBB’s  laboratories  for  collaborative research.  For  the  use  of  NIBB’s  unique  and  excellent research  instruments,  such  as  the  large  spectrograph,  the DSLM,  and  next  generation  DNA  sequencers  experimental projects are solicited and reviewed to provide machine time and  travel  expenses.  Priority  collaborative  research projects   are  carried  out  as  group  research  by  internal  and external  researchers  to  develop  pioneering  research  fields. Collaborative  research  projects  for  model  organismtechnology  development   and  Collaborative  research projects  for  bioresource  preservation  technology development  are for developing and establishing new model organisms  and  new  research  technology.  Projects  for  the development  of  bioresource  preservation  technology  are solicited  by  the  IBBP  center.  For  these  projects,  research expenses in addition to travel expenses are providedyear
Priority collaborative research projects
Collaborative research projects for model organisms/
technology development
Individual collaborative research projects
NIBB workshops
Collaborative experiments using the large spectrograph
Collaborative experiments using the DSLM
Collaborative experiments using
the next generation DNA sequencer
Facility Use (Training Course Facility)
Collaborative research projects for bioresource
preservation technology development
totalCollaborative Research Projects by Year■ NIBB Core Research Facilities

2011

2012

2013

2

3

4

2

88
6
9
8

89
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5

89
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15
9

87
3
12
10

45

47

41

37

0

2

1

0

9

10

174

162

6

164

5

171

2

2014
1

The NIBB Core Research Facilities were launched in 2010 to support research in NIBB and also to act as an intellectual hub  to  promote  collaboration  between  NIBB  and  other academic institutions. They consist of three facilities that are developing  and  providing  state-of-the-art  technologies through functional genomics, bioimaging and bioinformatics (p.  ). The Functional Genomics Facility maintains a wide array of core  research  equipment,  including  next  generation  DNA sequencers.  The  facility  is  dedicated  to  fostering  NIBB’s collaborative  research  by  providing  these  tools  as  well  as expertise.  The  current  focus  is  supporting  functional genomics  works  that  utilize  mass  spectrometers  and  DNA sequencers,  holding  training  courses  as  one  of  these undertakings  (p.  5).  The  Spectrography  and  Bioimaging Facility  manages  research  tools,  such  as  confocal BInternational cooperation
and OutreachContribution to the community of biologists

Promotion of collaborative
research projects

Promotion ofacademic research

Developmentof new fields

Cultivation of
future researchersmicroscopes,  DSLM  and  the  large  spectrograph,  and provides  technical  support  and  scientific  advice  to researchers.  These  two  facilities  hold  specially  appointed associate  professors,  an  expert  in  each  field,  with  a  mission to  manage  each  facility  as  well  as  conducting  his  own academic  research.  The  Data  Integration  and  Analysis Facility supports analysis of large-scale biological data, such as  genomic  sequence  data,  gene  expression  data,  and imaging  data.  For  this  purpose,  the  facility  maintains  highperformance computers with large-capacity storage systems.

■ NIBB Center of the Inter-University Bio-Backup
Project (IBBP Center)To  prevent  damage  to  important  biological  resources  by natural disasters, NIBB established the IBBP Center in 2012 in  collaboration  with  seven  national  universities  for multiplicate  preservation  of  genetic  libraries  and  other invaluable bioresources under cutting-edge research (p. ).

International Cooperation and Outreach■ Collaborative Programs with Overseas Institutes

NIBB  takes  a  leading  role  in  the  collaborative  research programs  between  the  European  Molecular  Biology Laboratory  (EMBL)  and  the  National  Institutes  of  Natural Sciences  (NINS)  and  promotes  personal  and  technological exchange  through  joint  meetings,  exchange  between researchers  and  graduate  students,  and  the  introduction  of experimental  equipment.  The  10th  NIBB-EMBL  symposium uantitative Bioimaging  was held in March, 201 . NIBB formed an agreement with the Temasek Life Sciences Laboratory (TLL) of Singapore and the Max Planck Institute for  Plant  Breeding  Research  (MPIP )  to  promote  joint research  projects,  collaborative  symposia,  training  courses and  student  exchange  programs.  The  5th  NIBB-MPIP -TLL Joint Symposium    orizons in Plant Biology   was held in November, 201  at NIBB (p. 2).

■ NIBB ConferenceThe NIBB Conferences are international conferences on hot topics in biology organized by NIBB’s professors. Since the first  conference  in  1  (the  year  of  NIBB’s  foundation), NIBB  Conferences  have  provided  researchers  in  basic biology  with  valuable  opportunities  for  international exchange. The  2nd conference  Force in Development  was held in November, 201  (p. 1).

■ International Practical CourseWith the cooperation of researchers from Japan and abroad the  NIBB  international  practical  course  is  given  at  a 5laboratory  specifically  prepared  for  its  use.  The  th  course Experimental Techniques Using Medaka and Xenopus - The Merits  of  Using  Both  -   was  held  jointly  with TLL  and  the National University of Singapore (NUS) in September, 201  at  NIBB  (p.  ).  Graduate  students  and  young  researchers from  various  areas  including  Taiwan,  ong  Kong,  India, Indonesia,  Bangladesh,  Germany,  the  United  States,  and Japan,  were  provided  with  training  in  state-of-the-art research  techniques.  International  conferences  and  courses are  managed  by  the  International  Cooperation  Group  of  the Research Enhancement Strategy Office.

■ Bio-Resources

The  National  BioResource  Project  (NBRP)  is  a  national project for the systematic accumulation, storage, and supply of nationally recognized bio-resources (experimental animals and  plants,  cells,  DNA,  and  other  genetic  resources),  which are  widely  used  as  materials  in  life  science  research.  To promote this national project, NIBB has been appointed as a center  for  research  on  Medaka  (Oryzia latipes)  whose usefulness as a vertebrate model was first shown by Japanese researchers.  The  usability  of  Medaka  as  a  research  material in  biology  has  drawn  increasing  attention  since  its  full genome sequence recently became available. NIBB is also a sub-center  for  the  NBRP’s  work  with  Japanese  morning glory.  The  NIBB  BioResource  Center  has  equipment, facilities,  and  staff  to  maintain  Medaka  and  Japanese morning  glory  safely,  efficiently,  and  appropriately.  The center  also  maintains  other  model  organisms,  such  as  mice, zebra sh,  Arabidopsis,  Lotus japonicus,  and  Physcomitrella
patens,  and  provides  technical  support  and  advice  for  the appropriate use of these organisms (p.  0). Strains of Japanese morning glory maintained in the center■ Outreach

NIBB’s  outreach  activities  aim  to  present  cutting  edge research  results  to  the  public  via  mass  media  through  press releases or directly through the internet, such as web pages, a Facebook  page,  and  a  Twitter  account.  Our  triannual  open campus event was held in 201  at which we welcomed more than  1, 00  local  citizens.  NIBB  also  cooperates  in  the education  of  undergraduate  and  younger  students  through lectures  and  workshops.  Outreach  activities  are  mostly managed  by  the  Public  Relations  Group  of  the  Research Enhancement Strategy Office.

Development of New Fields of Biology

EMBL, NIBB introduced a DSLM, which is effective for the three-dimensional  observation  of  living  organisms,  and  has developed  an  improved  model  using  two-photon  optics  (p. 2). The application of the adaptive optics to microscopy is under  way  in  collaboration  with  the  National Astronomical Observatory.  The  Advisory  Committee  on  Bioimaging, comprised  of  leading  researchers  in  the  bioimaging  field  in Japan,  is  organized  to  formulate  advice  on  NIBB’s  imaging research. The Bioimaging Forum provides an opportunity for researchers  and  company  engineers  to  frankly  discuss practical  difficulties  and  needs  regarding  imaging.  The  th Forum held in March 201  was immediately followed  by the Meeting  for  the  Future  Plan  of  Bio-Imaging  Network  and served  an  important  role  in  preparing  for  formation  of  a scientific community network in the field of imaging science (p.  ). A training course in bioimage analysis was also held in 201  (p.  5).

■ Okazaki Biology Conferences

NIBB holds Okazaki Biology Conferences (OBC) that, with the  endorsement  of  the  Union  of  Japanese  Societies  for Biological  Science,  aim  to  explore  new  research  fields  in biology  and  support  the  formation  of  international communities in these fields. Dozens of top-level researchers from  Japan  and  abroad  spend  nearly  one  week  together  for intensive discussions seeking strategies for addressing future critical  issues  in  biology.  Past  Conferences  have  promoted the  formation  of  international  researcher  communities.  The OBC10  is  being  planned  with  a  new  framework  on  the energy production in chroloplasts.

Cultivation of Future Researchers

NIBB  constitutes  the  Department  of  Basic  Biology  in  the School  of  Life  Science  of  the  SOKENDAI  (Graduate University for Advanced Studies). The department provides a five-year  course  for  university  graduates  and  a  three-year doctoral course for graduate students with a master’s degree.
Graduate  students  enrolled  in  other  universities  and institutions can apply to be special research students eligible to  conduct  research  for  fixed  periods  of  time  under  the supervision of NIBB professors.
In both cases above, graduate students can receive financial support  from  NIBB  based  on  the  research  assistant  (RA) system from the beginning of the five-year course.
Due to the international collaboration with EMBL, graduate students are encouraged to attend PhD student symposia held at EMBL and provided an opportunity to give oral and poster presentations, at least once during their master’s and doctoral program. Students  from  Japan  and  abroad  can  also  come  to  NIBB through our Internship Program. Internships give students an excellent  way  to  build  international  connections  while experiencing  hands  on  research  in  a  world  class  research institute (p.  ).

■ Bioimaging

NIBB  aims  to  maximize  the  application  of  modern  light microscopes  and  biophotonic  probes  for  real  time visualization  of  biological  phenomena  and  to  develop  new imaging  techniques. As  part  of  our  collaborative  work  with Graduate students educated by NIBB

■ Personnel changes in 2014*

Newly assigned in NIBBNameTAKAJI, Masafumi
MIYANARI, Yusuke
NISHIMURA, Mikio
YAMASHITA, Akira

Position
NIBB Research Fellow
Specially Appointed
Associate Professor
Specially Appointed
Professor
Specially Appointed
Associate Professor

Research Unit

Date

Division of Brain Biology

February 1

Laboratory of Nuclear Dynamics (Orion Project)

March 1

Research Enhancement Strategy Office

April 1

Laboratory of Cell Responses

April 1

OKADA, Kazunori

NIBB Research Fellow

Division of Molecular and Developmental Biology April 1

NOMURA, Kengo

NIBB Research Fellow

Division of Molecular Neurobiology

April 1

MASAMIZU, Yoshito

Assistant Professor

Division of Brain Circuits

June 1TATEMATSU, Kiyoshi

Specially Appointed
Assistant Professor

Research Enhancement Strategy Office

July 1

TAKAHASHI, Shunichi

Associate Professor

Division of Environmental Photobiology

October 1

EBINA, Teppei

NIBB Research Fellow

Division of Brain Circuits

November 1

SHI, Dongbo

NIBB Research Fellow

Division of Embryology

December 1

Newly affiliated in other universities and institutes
Name

New Affiliation

Position

Date

MATSUBAYASHI, Yoshikatsu Nagoya University

Professor

April 1SHINOHARA, Hidefumi

Nagoya University

Assistant Professor

April 1YAMADA, Kenji

Kyoto University

Assistant Professor

April 1

WAKE, Hiroaki

National Institute for Physiological Sciences

Associate Professor

April 1

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

■ Awardees in 2014

Name

PositionAward

SHIMMURA, Tsuyoshi

Specially Appointed
Assistant Professor

The Japanese Society of Animal Science, Encouragement Award

TOYOTA, Kenji

SOKENDAI Graduate Student

SAKAE, Yudai

SOKENDAI Graduate Student

FUKUSHIMA, Kenji

SOKENDAI Graduate Student

SOKENDAI President's Award

TOYOTA, Kenji

SOKENDAI Graduate Student

SOKENDAI President's Award

FUJIMORI, Chika

Postdoctoral Fellow

The Young Researchers' Most Valuable Oral Presentation Award,
The Japan Society for Comparative Endocrinology

The Students' Most Valuable Oral Presentation Award,
Chubu Branch Meeting, The Zoological Society of JapanThe Students' Valuable Poster Presentation Award,
Chubu Branch Meeting, The Zoological Society of Japan

National nstitute or asic iology

B

A

Professor

N

RA

i io

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

SOKENDAI Graduate Student:
Technical Assistant:

Secretary:†

MANO, Shoji
YAMADA, Kenji
KONDO, Maki
GOTO-YAMADA, Shino
KANAI, Masatake
WATANABE, Etsuko
KAMIGAKI, Akane
NITO, Kazumasa
SHIBATA, Michitaro
SAITO, Miyuki
HIKINO, Kazumi
YOSHINORI, Yumi
YAMAGUCHI, Chinami
NAKAI, Atsushi
UEDA, Chizuru

Since  plants  spread  their  roots  in  the  ground,  they  must survive in a given environment. To adapt to the environment, they  positively  utilize  environmental  changes  in  their  life cycle  as  important  signals  that  are  necessary  for  their survival. Plant cells can induce, degenerate and differentiate their  organelles  to  adapt  to  environmental  changes.  The flexibility of  plant  organelles  is  the  basis  of  the  strategy for environmental adaptation in plants.
The  aim  of  this  division  is  to  clarify  the  molecular mechanisms  underlying  the  induction,  differentiation,  and interaction  of  organelles,  and  to  understand  the  integrated function  of  individual  plants  through  organelle  dynamics. The Scientific Research on Innovative Areas  Environmental sensing of plants  Signal perception, processing and cellular responses   was  started  to  clarify  the  molecular  mechanisms underlying organelle differentiation and interaction.

I. R e v e r s i b l e t r a n s f o r m a t i o n o f p l a n t
peroxisomesDramatic  metabolic  changes  that  underlie  the  shift  from heterotrophic to autotrophic growth occur in the greening of seed  germination. Accompanying  these  metabolic  changes are  the  functional  transformations  of  many  constitutive organelles.  Etioplasts  differentiate  into  chloroplasts  while mitochondria  acquire  the  ability  to  oxidize  glycine. Glyoxysomes,  which  are  peroxisomes  engaged  in  the degradation  of  reserve  oil  stored  in  the  oil  body  via b-oxidation  and  the  glyoxylate  cycle,  are  transformed  into leaf  peroxisomes  that  function  in  several  crucial  steps  of photorespiration.  After  the  functional  transition  of glyoxysomes  to  leaf  peroxisomes  during  the  greening  of pumpkin  cotyledons,  the  reverse  transition  of  leaf peroxisomes to glyoxysomes occurs during senescence. Gene expression,  alternative  splicing,  protein  translocation  and protein  degradation  control  the  functional  transformation between glyoxysomes and leaf peroxisomes. †  This laboratory was closed on  1 March, 2015.

II. Transcriptomics, proteomics and phenomicsof plant peroxisomes

Enzymes localized in plant peroxisomes are synthesized in the  cytosol  and  function  after  their  post-translational transport  into  peroxisomes. Almost  all  of  the  peroxisomal matrix  proteins  contain  one  of  two  targeting  signals  (PTS1 and PTS2) within the molecules. PTS1 is a unique tripeptide sequence  found  in  the  carboxyl  terminus  of  the  mature proteins. In contrast, PTS2 is involved in a cleavable amino terminal  presequence  of  peroxisomal  proteins  that  are synthesized  as  a  precursor  protein  with  larger  molecular mass.  We  identified  25   gene  candidates  of  PTS1-  and  PTS2containing  proteins  and  another  0  genes  of  non-PTScontaining  proteins  from  the  Arabidopsis  genome.  Custommade  DNA  microarrays  covering  all  these  genes  were  used to investigate expression profiles of the peroxisomal genes in various organs. They revealed that peroxisomes in root cells play a pivotal role in polyamine catabolism. We also made a two-dimensional  protein  map  of  glyoxysomes  and  leaf peroxisomes isolated from Arabidopsis and soybean. Peptide MS  fingerprinting  analyses  allowed  us  to  identify  novel peroxisomal  membrane  proteins,  i.e.  voltage-dependent anion-selective  channel  and  adenine  nucleotide  carrier  1 (PNC1).  We  also  found  that  peroxisomal  membrane ATPbinding  cassette  transporter  promotes  seed  germination  by inducing  pectin  degradation  under  the  control  of  abscisic acid  signaling.  The  overall  results  provide  us  with  new insights into plant peroxisomal functions.  Bioinformatic analysis of the Arabidopsis genome predicted the  presence  of  15  kinds  of  genes,  called  PEX  genes,  for peroxisomal biogenesis factors. We demonstrated that PE 5 
and PE  form a cytosolic receptor complex and recognize PTS1- and PTS2-containing proteins, respectively. PE 1  is a  peroxisomal  membrane  docking  protein  that  captures  the receptor-cargo  complex.  We  also  comprehensively investigated  whether  or  not  these  predicted  PEX genes function  in  peroxisome  biogenesis  by  generating  knockdown  mutants  that  suppress  PEX  gene  expression  by  RNAinterference.  Phenotypes  of  these  mutants  allowed  us  to identify  the  functional  PEX  genes,  which  can  be  classified into  two  groups   PEX  genes  regulating  for  peroxisomal protein  import  and  PEX  genes  regulating  for  peroxisomal morphology.  We  continue  to  investigate  the  detailed molecular  functions  of  other  PEX  genes.  Of  these,  we recently proposed that function of PE  is maintained by a quality control mechanism involving RabE1c.

III. Identification of novel components
essential for peroxisome biogenesisTo  better  understand  peroxisome  biogenesis  and  functions, we isolated a number of Arabidopsis mutants having aberrant peroxisome  morphology  (apem  mutants)  and  peroxisome unusual  poisoning  (peup  mutants)  based  on  them  having  a different  pattern  of  GFP  fluorescence  from  the  parent  plant, GFP-PTS1,  in  which  peroxisomes  with  normal  sizes, numbers and distribution can be visualized with GFP.
Up  to  date,  we  reported  the  function  of  gene-products  of APEM1,  APEM2, APEM3,  APEM4  and  APEM9.  Recently, we  found  that  APEM10 encodes  Lon  protease  2,  which  has roles  in  chaperon  and  proteinase,  and  that  modulates peroxisome  degradation  processed  by  autophagy.  Taken together  with  the  analyses  using  peup1,  peup2  and  peup4mutants,  which  were  defective  in  Autophagy-related  2, (ATG2), ATG1 a  and ATG ,  respectively,  we  were  able  to update  the  model  for  functional  transformation  of peroxisomes (Figure 1).
We  are  currently  characterizing  other  apem  and  peup mutants. From these analyses, we will be able to identify the components  responsible  for  peroxisome  biogenesis, functions and maintenance, and to address the mechanism at the molecular level.

and herbivores. We are now investigating ER body formation and  function  using  ER  body  deficient  mutants,  and heterologously expressing NAI2 in onion and tobacco cells.

Figure 2. ER bodies in Arabidopsis thaliana. A confocal micrograph (A) and  an  electron  micrograph  (B)  of  cotyledon  and  root  epidermal  cells, respectively. Arrowheads  indicate  ribosomes  on  the  surface  of  the  ER body  membranes.  ER-localized  GFP  labels  ER  bodies  as  well  as  the typical ER network, and electron microscopy identifies ribosomes at the cytosolic  surface  of  ER  bodies,  both  of  which  indicate  the  luminal continuity  between  ER  and  ER  bodies.  Enlarged  images  of  the  squared regions are shown below. CW, cell wall   , vacuole  Mt, mitochondrion  G, Golgi body  Bars,10 m (A) and 1 m (B).

V. Roles of molecular chaperones on celldifferentiation
Figure  1.  A  model  for  the  coordinated  regulation  of  functional transformation of peroxisomes and peroxisome quality control by LON2
APEM10 and autophagy during functional transition.

IV. ER derived organelles for protein storingand defense strategy

Plant cells develop various types of endoplasmic reticulum (ER)-derived  structures  with  specific  functions.  ER  bodies are ER-derived compartments observed in Arabidopsis. They are  rod-shaped  structures  surrounded  by  ribosomes,  and  are widely  distributed  in  the  epidermal  cells  of  whole  seedlings (Figure 2). Undamaged rosette leaves have no ER bodies, but accumulate  ER  bodies  after  wounding  or  jasmonic  acid treatment.  ER  bodies  accumulate  b-glucosidase  PYK10. When  plant  cells  are  damaged,  PYK10  forms  large  protein aggregates.  The  aggregate  formation  increases  glucosidase activity.  These  findings  suggest  that  ER  bodies  function  in the  defense  against  pathogens  and  herbivores,  possibly producing toxic products. Arabidopsis nai1 mutants have no ER bodies in the entire plant and do not accumulate PYK10. NAI1  encodes  a  transcription  factor  and  regulates  the expression  of  PYK10  and  NAI2.  The  Arabidopsis  nai2 mutant  has  no  ER  bodies  and  reduced  accumulation  of PYK10. NAI2 encodes a unique protein that localizes to the ER  body.  Membrane  protein  of  ER  body  1  (MEB1)  and MEB2  are  integral  membrane  proteins  of  the  ER  body  and have iron manganese transport activity. These results suggest that  the  ER  body  has  specific  membrane  proteins  that  are involved in defense against metal stress as well as pathogens Molecular  chaperones  are  cellular  proteins  that  function  in the  folding  and  assembly  of  certain  other  polypeptides  into oligomeric  structures.  We  have  found  that  SP 0  inhibitor induces  genes  with  heat  shock  response  element  ( SE) motifs  in  their  promoters,  suggesting  that  heat  shock transcription  factor  ( SF)  is  involved  in  the  response. Arabidopsis  SFs  interacted  with  SP 0.  Thus,  it  appears that in the absence of heat shock,  SP 0 actively suppresses SF  function.  During  heat  shock,  SP 0  is  transiently inactivated,  which  leads  to  SF  activation.  These  data indicate  that  SP 0  regulates  correct  gene  expression  for heat acclimatization in plants. We also observed that  SP 0 is  involved  in  hormone  responses  in  Arabidopsis.  The evolutional  and  functional  characterizations  are  now  being investigated.

VI. Update of The Plant Organelles Database 3(PODB3) and Plant Organelles World

The  Plant  Organelles  Database    (PODB )  was  built  to promote  a  comprehensive  understanding  of  organelle dynamics.  This  public  database  is  open  to  all  researchers. PODB   consists  of  six  individual  units   the  electron micrograph database, the perceptive organelles database, the organelles  movie  database,  the  organellome  database,  the functional analysis database, and external links. The electron micrograph  database,  which  was  added  as  new  content, provides information on the ultrastructures in plant cells. The perceptive  organelles  database  shows  organelle  dynamics  in response  to  environmental  stimuli.  The  organelles  movie database  contains  time-lapse  images  and  D  structure National nstitute or asic iologyBrotations. The organellome database is a compilation of static image  data  of  various  tissues  of  several  plant  species  at different  developmental  stages.  The  functional  analysis database  is  a  collection  of  protocols  for  plant  organelle research.  Through  these  databases,  users  can  easily  grasp plant  organelle  dynamics.  Plant  Organelles  World,  which  is based  on  PODB   is  an  educational tool  to  engage  members of the non-scientific community to explore plant biology. We hope that PODB  and Plant Organelles World are of help to researchers and the general public.

〔Original paper (E-publication ahead of print)〕otomura, K., Le,  .T.,  amada, T., Kutsuna, N., Mano, S., Nishimura,  MM., and Watanabe, Y. Diffuse DCP2 accumulates in DCP1 foci under heat stress in Arabidopsis thaliana. Plant Cell Physiol. 201  Oct 22.

〔Review articles〕

oto-Yamada,  S.,  Mano,  S.,  and  Nishimura,  M.  (201 ).  The  role  of  Gperoxisomes in plant reproductive processes. In Sexual reproduction in animals and plants.   Edited by Sawada,  ., Inoue, N., and Iwano, M. Springer Japan, pp.  1 - 2 .
Goto-Yamada, S., Mano, S., Oikawa, K., Shibata, M., and Nishimura, M.  (201 ).  Interaction  between  chaperone  and  protease  functions  of LON2, and autophagy during the functional transition of peroxisomes. Plant Signal. Behav. 9, e2. Nakano,  R.T.,  Yamada,  K.,  Bednarek,  P.,  Nishimura,  M.,  and  araNishimura,  I.  (201 ).  ER  bodies  in  plants  of  the  Brassicales  order  Biogenesis and association with innate immunity. Front. Plant Sci. 5,
, doi  10.
fpls.201 .000 .
Shibata, M., Oikawa, K., Yoshimoto, K., Goto-Yamada, S., Mano, S., Yamada, K., Kondo, M.,  ayashi, M., Sakamoto, W., Ohsumi Y., and Nishimura, M. (201 ). Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality. Autophagy 10, 
.

 
 
 

Figure  .  The  graphical  user  interface  of  the  PODB   (http podb.nibb.
ac.jp Orgenellome).

Publication List

〔Original papers〕

oto-Yamada, S., Mano, S., Nakamori, C., Kondo, M., Yamawaki, R.,  GKato, A., and Nishimura, M. (201 ). Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy. Plant Cell Physiol. 55,  2.
Mano, S., Nakamura, T., Kondo, M., Miwa, T., Nishikawa, S., Mimura, T.,  Nagatani,  A.,  and  Nishimura,  M.  (201 ).  The  Plant  Organelles Database    (PODB )  update  201   integrating  electron  micrographs and new options for plant organelle research. Plant Cell Physiol. 55, e1.
Shibata,  M.,  Oikawa,  K.,  Mano,  S.,  and  Nishimura,  M.  (201 ). Measurement of the number of peroxisomes. Bio-Protoc. 4, e12 .
Yoshimoto, K., Shibata, M., Kondo, M., Oikawa, K., Sato, M., Toyooka, K. Shirasu, K. Nishimura, M., and Ohsumi, Y. (201 ).  Organ-specific quality control of plant peroxisomes is mediated by autophagy. J. Cell Sci. 127, 11 1-11 .

 
 
 

10

A

Professor

A

A A

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

A

B

†

os i atsu

SHINOHARA, Hidefumi
TABATA, Ryo
KAKITA, Mitsuru
OKAMOTO, Satoru
OGAWA-OHNISHI, MariSUMIDA, Kumiko
YASUE, Naoko
OKUBO, Masayo

Cell-to-cell  signaling  mediated  by  secreted  signals  and membrane-localized  receptors  is  one  of  the  critical mechanisms  by  which  growth  and  development  of multicellular  organisms  are  cooperatively  regulated.  Signal molecules  that  specifically  bind  receptors  are  generally referred to as ligands. Because membrane-localized receptors act  as  master  switches  of  complex  intracellular  signaling, identification of the ligand-receptor pair is one of the central issues  of  post-genome  research.  We  are  working  to  clarify the  mechanisms  by  which  plant  development  is  regulated through identification of novel ligands such as small peptides and  their  specific  receptors  using  Arabidopsis  genome information,  biochemical  analysis  and  phenotypic observation.

acid peptide originating from a C-terminal conserved domain (the  CEP  domain)  through  posttranslational  proline hydroxylation and proteolytic processing. A total of 15 CEP family genes (CEP1 through CEP15) have been found in the Arabidopsis genome.
When external N availability is lowered, CEP expression is promptly  upregulated  in  the  portion  of  the  root  system directly  experiencing  N  starvation.  CEP  acts  as  a  rootderived  ascending  N-demand  signal  to  the  shoot,  where  its perception  by  CEP  receptors  leads  to  the  production  of  a putative  shoot-derived  descending  signal  that  upregulates nitrate  transporter  genes  in  the  roots.  This  mechanism supports  N  acquisition  when  nitrate  is  unevenly  distributed within  the  soil  (Figure  1).  CEP  family  peptides  induced  on one  side  of  the  roots  by  local  N  starvation  mediate upregulation of nitrate transporter genes in the distant part of the  roots  exposed  to  N-rich  conditions  to  compensate  for  N deficiency.

A

I. Secreted peptide signals

Following complete sequencing of the Arabidopsis genome, a  number  of  genes  encoding  small  secreted  peptides  have been  identified  by  in silico  database  analysis.  Based  on  our own  analysis,  we  identified   putative  secreted  peptide genes  with  an  open  reading  frame  (ORF)  size  between  50 and 150 amino acids in the Arabidopsis genome. These  ORFs  include  many  functionally  uncharacterized  peptides. Although  estimation  of  the  total  number  of  biologically relevant  secreted  peptide  signals  is  difficult  at  present,  the presence  of  many  orphan  receptors   among  receptor-like kinases  in  Arabidopsis  suggest  that  a  substantial  number  of intercellular signals remain to be identified.
One  structurally  characteristic  group  of  peptide  signals  is post-translationally  modified  small  peptides .  These peptides  are  characterized  by  the  small  size  of  mature peptides (less than 20 amino acids) and the presence of posttranslational  modifications.  In  these  peptide  signals,  peptide chain  length  and  post-translational  modifications  are generally  very  important  for  their  receptor  binding  activity and physiological functions.

1-1 C-terminally encoded peptide (CEP)C-terminally  encoded  peptide  (CEP)  is  a  15-amino-acid peptide  involved  in  mediating  long-distance  nitrogen (N)-demand signaling. The CEP family was identified by insilico  screening  for  a  family  of  secreted  peptides  that  share short, conserved domains near the C-terminus, a feature that is  common  to  several  posttranslationally  modified  small peptide  signals  in  plants.  CEP1  is  secreted  as  a  15-amino BPR receptor

2nd signal

ompensatory andincreased nitrate upta eylem

P peptide ligand

Rootlets starved o NsecretePFigure  1.  CEP  mediates  systemic  N-demand  signaling.  (A)  Systemic N-demand  signaling  visualized  by  pNRT2.1:LUC.  N  starvation  on  one side (right) of the root system leads to an upregulation of nitrate uptake on  the  other  side  of  the  root  system  (left).  (B)  Mode  of  action  of  CEP family  peptides  in  systemic  N-demand  signaling.  N-starvation  induces CEP expression in roots. CEP acts as a root-derived ascending N-demand signal to the shoot, where its perception by CEPR leads to the production of  a  putative  shoot-derived  descending  signal  that  upregulates  nitrate transporter genes in the roots.

1-2 Other novel peptide signal candidates

The  common  feature  of  known  small  post-translationally modified peptide signals is that they are encoded by multiple paralogous genes whose primary products are approximately 0-  to  110-amino-acid  cysteine-poor  secreted  polypeptides †  This laboratory was closed on  1 March, 201 .

11National nstitute or asic iologyBthat share short conserved domains near the C-terminus. We have identified several novel polypeptide families that fulfill the above criteria by in silico screening and determined their mature  structures  by  analyzing  apoplastic  peptide  fractions by nano LC-MS MS. Functional analysis of these peptides is now going on.

II. Receptors for secreted peptide signals

The  receptors  or  putative  receptors  for  peptide  signals identified  to  date  belong  to  the  receptor  kinase  (RK)  or receptor-like protein (RLP) families. Among RKs, the largest subfamily  is  the  leucine-rich  repeat  RK  (LRR-RK)  family, which consists of 21  members in Arabidopsis. The majority of  receptors  for  small  post-translationally  modified  peptide signals  belong  to  this  family.  Especially,  an  increasing number  of  LRR    and  LRR  I  members  are  now  being confirmed as receptors for several endogenous small peptide ligands,  suggesting  that  these  subgroups  are  an  attractive target for binding analysis with novel peptide signals.
Although both genetic and biochemical methods have been used  to  identify  ligand-receptor  pairs  in  plants,  genetic redundancy  often  interferes  with  the  former  approach,  and the  low  levels  at  which  ligand  and  receptor  molecules  are often  present  in  tissues  can  make  the  latter  approach  very difficult. As described above, an increasing number of ligand candidates are being identified. If individual receptor kinases could  be  functionally  overexpressed  in  certain  cells  at sufficiently  high  levels  and  sufficiently  high  quality  for biochemical  binding  analysis,  such  a  receptor  library  would facilitate  identification  of  ligand-receptor  pairs  in  plants. To this end, we established a functional and efficient expression system  of  plant  receptor  kinases  in  tobacco  BY-2  cells  and prepared  an  expression  library  of  all  the  potential  receptor candidates.  This  approach  was  successfully  used  for identification of CEP receptors.

Publication List

〔Original paper〕

idadi,  ., Matsuoka, K., Sage-Ono, K., Fukushima, J., Pitaksaringkarn,  BW., Asahina, M., Yamaguchi, S., Sawa, S., Fukuda, ., Matsubayashi, Y., Ono, M., and Satoh, S. (201 ). CLE  expression recovers gibberellin deficiency  to  promote  shoot  growth  in Arabidopsis.  Plant  J.  78,  2 1252.
Tabata,  R.,  Sumida,  K.,  Yoshii,  T.,  Ohyama,  K.,  Shinohara,  .,  and Matsubayashi, Y. (201 ). Perception of root-derived peptides by shoot LRR-RKs  mediates  systemic  N-demand  signaling.  Science  346, .

 〔Review article〕

atsubayashi,  Y.  (201 ).  Posttranslationally  modified  small-peptide  Msignals in plants. Annu. Rev. Plant Biol. 65,  5 1 .

12

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From these observations we propose that TORC1 contributes to the suppression of sexual differentiation in the presence of rich nutrition through phosphorylation and destabilization of Mei2 (Figure 1) (Otsubo et al., 201 ).

A

Director General

A A

asayu i

Postdoctoral Fellow:
Visiting Scientist:
Secretary:

Specially Appointed Associate Professor

A A

A A ira

SHICHINO, Yuichi
OTSUBO, Yoko
SAKAGAMI, MariCells  sense  the  environment  around  them,  for  example  the amount of nutrients and hormones, as well as the temperature and pressure, and decide what kind of activities to undertake using this information. Germ cells, which produce sperm and eggs,  begin  halving  their  number  of  chromosomes  during  a special kind of cell division called meiosis, in response to the ambient conditions. In our laboratory we use the fission yeast Schizosaccharomyces pombe,  the  simplest  organism  that performs meiosis, to research the mechanism by which cells switch  from  mitosis,  the  kind  of  cell  division  that  divides cells  equally  to  create  two  identical cells,  to  meiosis, which is essential for bringing forth genetically diverse progeny. I. Signaling pathways that regulate the onset of
sexual differentiation

We  have  been  trying  to  elucidate  how  fission  yeast  cells switch  their  mode  of  cell cycle from  mitotic to  meiotic. We focus  on  a  highly  conserved  kinase,  namely  Target  of rapamycin  (TOR)  kinase,  which  plays  key  roles  in  the recognition  of  nutrition  and  the  onset  of  sexual differentiation in fission yeast. TOR kinase forms two types of complexes, TORC1 and TORC2. TORC1 contains Tor2 as its  catalytic  subunit  and  is  essential  to  suppress  sexual differentiation  in  the  presence  of  nitrogen.  owever,  the critical  effectors  of TORC1  that  function  in  the  suppression of sexual differentiation have remained largely unknown.
We  demonstrated  that  TORC1  could  phosphorylate  an RNA-binding  protein,  Mei2,  in vitro.  Mei2  is  the  master regulator that switches the cell cycle from mitotic to meiotic. Mei2 is also known to be involved in earlier steps of sexual differentiation. We found that non-phosphorylatable Mei2, in which  the  nine  phosphorylation  sites  were  changed  to alanine,  became  more  stable  than  the  wild-type  in  the presence  of  nitrogen.  We  further  showed  that  Mei2  was polyubiquitylated  in vivo  in  a  TORC1-dependent  manner. Figure  1. A  schematic  of  the  suppression  of  sexual  differentiation  by TORC1 through phosphorylation of Mei2.

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

Expression  of  hundreds  of  genes  is  upregulated  during meiosis. 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 fission yeast. Understanding precise mechanisms of this control will shed light on the regulation of timely gene expression during meiosis.
A  meiosis-specific  nuclear  body  in  fission  yeast,  called Mei2  dot,  promotes  the  progression  of  meiosis  by sequestering  and  inhibiting  Mmi1  protein,  a  crucial  factor involved  in  the  selective  elimination  of  meiosis-specific transcripts  (Figure  2).  The  Mei2  dot  is  composed  of  the RNA-binding  protein  Mei2  and  a  long  non-coding  RNA species  termed  meiRNA.  We  have  shown  previously  that Mei2  physically  interacts  with  meiRNA  and  forms  the  dot structure  at  the  chromosomal  sme2  locus,  which  encodes meiRNA, depending on the transcription of meiRNA (Figure 2).  We  have  further  shown  that  meiRNA  carries  numerous copies of the DSR motif, which is recognized specifically by Mmi1,  and  that  meiRNA  is  indeed  a  target  eliminated  by Mmi1.  Thus,  the  molecular  mechanisms  underlying  the specific localization of meiRNA to its genetic locus and the importance  of  this  localization  in  the  inactivation  of  Mmi1 provide interesting questions.
We  found  that  Mmi1  is  essential  to  anchor  meiRNA  to  its coding  locus  sme2.  Meanwhile,  overexpression  of  meiRNA promoted  accumulation  of  Mmi1  to  the  sme2  locus  and reduced active Mmi1 in the cell. These findings indicate that Mmi1 facilitates the retention of meiRNA at its genetic locus as  it  is  transcribed,  and  that  the  anchored  meiRNA  then attracts  Mmi1  and  inhibits  the  function  of  this  protein (Shichino et al., 201 ). III. Meiosis-specific cell cycle regulation

In  both  mitosis  and  meiosis,  the  accumulation  and activation of M-phase promoting factor (MPF), composed of Cdk  and  B-type  cyclin  (Cdc2  and  Cdc1   in  S. pombe, respectively) is required before nuclear division. When cells exit from M phase to enter the next phase of the cell cycle, the MPF level must be reduced. In meiosis, how is the MPF Figure 2. Co-localization of the Mei2 dot components. The upper panels display  localization  of  meiRNA,  Mei2  and  Mmi1,  and  the  lower  panels display localization of meiRNA, the sme2 locus and Mmi1. The cellular contour is shown in a dotted line.

1National nstitute or asic iologyBlevel regulated between the first meiotic division (meiosis I) and  the  second  meiotic  division  (meiosis  II),  during  which two  sequential  nuclear  divisions  take  place   We  are interested in the molecular mechanisms enabling the meiotic cell cycle. We have shown previously that a meiosis-specific inhibitor  of  the  anaphase-promoting  complex cyclosome (APC C),  namely  Mes1,  protects  Cdc1   from  complete destruction  at  anaphase  of  meiosis  I.  This  secures  the retention  of  a  sufficient  level  of  MPF  activity  to  conduct meiosis II.
We  isolated  mutants  defective  in  spo5,  which  encodes  a meiosis-specific RNA-binding protein, in a new screening to identify  mutants  deficient  in  the  progression  of  meiosis  II. We  found  that  spo5  mutant  cells  lost  Cdc1   prematurely prior to meiosis II (Figure  ). The defect in meiosis II in the spo5 mutants was recovered by increasing the CDK activity. Furthermore,  accumulation  of  cdc13  transcripts  during meiosis II was lower in spo5 mutant cells than in wild-type cells. These findings indicate that Spo5 is a novel factor that contributes  to  the  regulation  of  the  expression  of  Cdc1  during meiosis (Arata et al. 201 ). tsubo,  Y.,  Yamashita,  A.,  Ohno,  .,  and  Yamamoto,  M.  (201 ).  S.  Opombe TORC1 activates the ubiquitin-proteasomal degradation of the meiotic regulator Mei2 in cooperation with Pat1 kinase. J Cell Sci. 12 , 
2
-2
.
Shichino, Y., Yamashita, A., and Yamamoto, M. (201 ). Meiotic long non-coding meiRNA accumulates as a dot at its genetic locus facilitated by Mmi1 and plays as a decoy to lure Mmi1. Open Biol.  , 1 0022.
Togashi,  N.,  Yamashita,  A.,  Sato,  M.,  and  Yamamoto,  M.  (201 ). Functional significance of nuclear export and mRNA binding of meiotic regulator Spo5 in fission yeast. BMC Microbiol. 1 , 1 .

  Publication List

〔Original papers〕

S.A.,  Simanis,  ., Yamamoto,  M.,  and  Sato,  M.  A(201oi, Y., ). OKawashima, ptimization of the analogue-sensitive Cdc2 Cdk1 mutant by in vivo  selection  eliminates  physiological  limitations  to  its  use  in  cell cycle analysis. Open Biol.  , 1 00 .
Arata,  M.,  Sato,  M.,  Yamashita, A.,  and  Yamamoto,  M.  (201 ).  The RNA-binding  protein  Spo5  promotes  meiosis  II  by  regulating  cyclin Cdc1  in fission yeast. Genes Cells 1 , 225-2 .
irai,  ., Arai, K., Kariyazono, R., Yamamoto, M., and Sato, M. (201 ). The  kinetochore  protein  Kis1 Eic1 Mis1   ensures  the  integrity  of mitotic  spindles  through  maintenance  of  kinetochore  factors  Mis
CENP-I and CENP-A. PLoS One  , e111 05.
Okada,  N.,  Toda,  T.,  Yamamoto,  M.,  and  Sato,  M.  (201 ).  CDKdependent phosphorylation of Alp -Alp1  (TACC-TOG) promotes its nuclear  accumulation  and  spindle  microtubule  assembly.  Mol.  Biol. Cell 25, 1 -1 2.

 
 
 

Figure  . Cdc1  disappears prematurely in the spo5 mutants. Cdc1  and a spindle pole body marker Sfi1 were monitored in wild-type and spo5 mutant cells in the course of meiosis. In wild-type cells, Cdc1  signal diminished at anaphase of meiosis I but reappeared at the beginning of meiosis II. In spo5 mutant cells, Cdc1  signal reappeared only faintly (Cell-1) or did not reappear (Cell-2).

1

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A

B

A

Associate Professor

NA Nobuyu iAssistant Professor:
NAKAYAMA, Kei
SOKENDAI Graduate Student: OHASHI, Rie
KATAYAMA, Kaori
Technical Assistant:
MATSUDA, Chisato
The  transport  of  specific  mRNAs  and  local  control  of translation in neuronal dendrites represent an important gene expression system that provides dendritic protein synthesis at just  the  right  time  and  place.  It  is  believed  that  this  system controls  the  location  at  which  neurites  will  connect  to  each other, thereby forming neural networks. Our main interest is to understand the mechanisms and roles of mRNA transport and local translation in neuronal dendrites.
Specific  mRNAs  are  recruited  into  RNA  granules   and transported  to  dendrites.  RNA  granules  are  macromolecular complexes  composed  mainly  of  mRNAs,  ribosomes  and RNA-binding  proteins,  and  mediate  the  transport  and  local translation  of  their  mRNA  cargoes  in  response  to  synaptic stimulation  (Figure  1).  We  are  researching  RNA  granule factors regulating mRNA transport and local translation, their target  mRNAs,  and  the  mechanism  of  localized  protein synthesis using mice in order to better understand its relation to  the  formation  of  synapses  and  neural  networks,  memory, learning, and behavior. timulation

locally translated proteinssynapsepolysome

microtubule
soma

RN

5

RNA granuledendritea onbrainstem development (Shiina et al., J. Neurosci.  0, 12 1 12 0, 2010).
To investigate the role of RNG105 in the adult mouse brain, we  have  generated  RNG105  conditional  knockout  (cKO) mice  in  which  the  rng105  gene  was  disrupted  in  the  brain after  birth.  Using  RNG105  cKO  adult  mice,  we  performed comprehensive analysis of dendritic localization of mRNAs. Brain  slices  were  dissected  to  isolate  somatic  and  dendritic layers  of  hippocampal  CA1  neurons,  and  from  each  layer, RNA  was  purified  and  analyzed  by  deep  sequencing. Comparison  of  mRNAs  from  dendritic  and  somatic  layers identified  mRNAs  concentrated  in  dendrites  and  mRNAs concentrated  in  the  soma  (Figure  2).  The  difference  in  the mRNA  composition  between  dendrites  and  soma  was relatively  small  in  RNG105  cKO  mice  compared  to  control mice  (Figure  2),  which  suggested  that  the  dendritic  mRNA composition  in  RNG105  cKO  neurons  changed  toward  the somatic  type,  i.e.,  dendritic  localization  of  specific  mRNAs was reduced in RNG105 cKO mice. Gene ontology analysis revealed  that  dendritically  enriched  mRNAs  included  small G  protein  regulators  and  translation  regulators,  which  were reduced in dendrites of RNG105 cKO mice. We are going to investigate  whether  proteins  encoded  by  these  mRNAs  are involved in dendritic synapse function and neuronal network formation.
We  further  analyzed  behavior  of  RNG105  cKO  mice. Passive  avoidance  test  is  one  of  the  learning  and  memory tests, in which mice receive an electric foot shock in a room and  thereafter  they  remember  the  situation  and  do  not  enter the  room  for  more  than  several  days.  In  this  test,  RNG105 cKO mice did not enter the room at 5 minutes after the foot shock,  but  entered  the  room  after  2   hours.  We  performed another learning and memory test, Morris water maze. In this test, mice learn the location of a hidden platform that allows the mice to escape from water. After several days of training, control mice learned the platform location and escape on the platform  faster  than  before  the  training.  owever,  the training did not shorten the latency of RNG105 cKO mice to No stimulation

Figure  1. A  model  for  local  translation  in  neuronal  dendrites.  Specific mRNAs  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  and network formation.

I. Dendritic mRNA localization and learningand memory in RNG105 conditional
knockout miceWe  previously  identified  RNA  granule  protein  105 (RNG105) caprin1, an RNA-binding protein, as a component of  RNA  granules.  RNG105  is  responsible  for  mRNA transport  to  dendrites,  which  is  required  for  the  encoded proteins to be translated and function in dendrites for proper networking of neurons (Shiina et al., J. Neurosci.  0, 12 1 12 0,  2010).  RNG105  knockout  mice  exhibit  reduced dendritic  synapse  formation  and  reduced  dendritic arborization, which results in poor development of neuronal networks. The knockout neonates die soon after birth due to respiratory  failure  that  is  associated  with  defects  in  fetal Figure 2. Comparison of mRNA composition between dendrite and soma of  hippocampal  CA1  neurons.  (A)  RNAs  from  triplicate  samples  were analyzed  with  Cufflinks.  The  graph  shows  MA  plot  between  dendrite RNAs and soma RNAs from control neurons. The x-axis shows average log2 expression of dendrite RNAs and soma RNAs and the y-axis shows log2 fold change of dendrite RNAs vs. soma RNAs. Gray, all RNAs  red, mRNAs  significantly  concentrated  in  dendrites   blue,  mRNAs significantly concentrated in soma. (B) MA plot between dendrite RNAs and  soma  RNAs  from  RNG105  cKO  neurons.  The  same  mRNAs  are colored the same as in A.

15

National nstitute or asic iologyBfind the platform. These results suggested that RNG105 cKO mice had deficits in learning and memory.
RNG105 caprin1 has one paralog, RNG1 0 caprin2, which has  RNA-binding  domains  highly  conserved  with  RNG105. RNG105  and  RNG1 0  are  localized  to  different  kinds  of RNA  granules  and  their  timing  of  expression  is  also different   RNG105  is  highly  expressed  in  embryos,  but RNG1 0 is highly expressed in adults (Shiina and Tokunaga, J.  Biol.  Chem.  2 5,  2 2 0-2 2 ,  2010). We  have  obtained RNG1 0  knockout  mice  and  examined  RNG1 0  mRNA expression  by  qRT-PCR.  RNG1 0  mRNA  was  most  highly expressed in the brain and significantly reduced in the brain of  RNG1 0  knockout  mice. We  are  going  to  investigate  the role of RNG1 0 in higher brain functions in adult mice.

II. RNA granule assembly and disassembly
modulated by NFAR2 and NF45

We used proteomic analyses to identify proteins associated with RNG105. Among the identified proteins, we focused on nuclear  factor  associated  with  dsRNA  2  (NFAR2)  and  its binding  partner,  nuclear  factor  5  (NF 5).  NFAR2 co-localized  with  and  enhanced  the  assembly  of  RNG105containing  RNA  granules,  whereas  NF 5  induced  the disassembly of RNA granules (Figure  ).
NFAR2  has  a  G SY  domain  that  is  structurally  and functionally  similar  to  the  low  complexity  (LC)  sequence domain of FUS TLS, which is known to drive RNA granule assembly. We have found that the G SY domain of NFAR2 has  the  ability  to  interact  with  RNG105-containing messenger  ribonucleoprotein  (mRNP)  complexes  and enhances  the  assembly  of  RNG105-containing  RNA granules.
Another  domain  of  NFAR2,  the  D F  domain,  was  not necessary  for  the  interaction  with  the  RNG105  mRNP complexes,  but  was  involved  in  positive  and  negative r e g u l a t i o n   o f   R N A  g r a n u l e   a s s e m b l y   b y   b e i n g phosphorylated  by  PKR,  a  master  kinase  inducing  RNA granule  assembly,  and  by  association  with  NF 5, respectively (Figure  ).
Analysis  of  translation  activity  at  the  single  cell  level  by ribopuromycilation  assay  revealed  that  NFAR2  repressed translation,  but  the  NFAR2-dependent  repression  was de-repressed by NF 5.
Our results suggest a model in which NFAR2 functions as a connector  of  RNG105  mRNP  complexes  through  its multivalent  domains,  i.e.,  the  G SY  domain  and  the  D F domain, in the assembly of RNA granules that is linked with translation  repression.  The  connector  function  may  be enhanced by phosphorylation by PKR, whereas it is blocked by  NF 5  binding,  which  disassembles  RNA  granules  and de-represses  translation. We  are  going  to  elucidate  the  roles of  NFAR2  and  NF 5  in  neurons,  including  their  relation  to neurodegeneration,  because  defective  regulation  of  RNA granule  assembly  by  LC  sequence  domain-containing proteins  such  as  FUS TLS  and  TDP-   has  been  recently suggested  to  be  associated  with  neurodegenerative  diseases such  as  amyotrophic  lateral  sclerosis  (ALS)  and frontotemporal lobar degeneration (FTLD).

1

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translated proteins

NFAR2

RNA granule

P P

NFAR2

NFAR2
NF45

NFAR2
NF45

assemblytranslation repression

polysome

disassemblytranslation activation

Figure  . Effects of NFAR2 and NF 5 on the assembly of RNA granules and translation activity. (A) A  cells were co-transfected with RNG105monomeric red fluorescent protein 1 (mRFP1) and NFAR2-GFP. NFAR2 was predominantly localized to the nucleus, and also co-localized to and enlarged  RNG105-containg  RNA  granules  (arrows).  Scale  bar,  10  m. (B) A model of RNA granule assembly and translation activity modulated by  NFAR2  and  NF 5.  NFAR2  enhances  RNA  granule  assembly  and represses translation. NF 5 binds to NFAR2, disassembles RNA granules and de-represses translation. Phosphorylation of NFAR2 by PKR reduces the effect of NF 5.

Publication List

〔Original paper〕

hiina,  N.,  and  Nakayama,  Kei.  (201 ).  RNA  granule  assembly  and  Sdisassembly modulated  by  nuclear  factor  associated  with  doublestranded  RNA  2  and  nuclear  factor  5.    J.  Biol.  Chem.  289,  211
211 0.

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A

Assistant Professor

A A A

Technical Assistant:

os io

GONDA, NaokoMammalian  eggs  implant  onto  the  maternal  uterine  wall after  hatching  at  the  blastula  stage,  due  to  a  lack  of  enough nutrients  to  support  development  until  birth.  In  order  to transfer  nutrients  and  oxygen  into  the  fetus,  and  metabolic wastes into the mother, the placenta has evolved to establish close  connections  between  the  maternal  and  fetal vasculature.  There  is  amazing  diversity  from  one  animal  to another in the features, such as gross structures, microscopic arrangements and developmental processes, but the function of this organ is invariable.  Primates  and  rodents  have  the  ancestral  type  of  placenta, classified  as  heomochorial,  in  which  maternal  blood  is  not contained within endothelial cell lined vessels but rather is in direct  contact  with  epithelial  cells  of  the  fetal  placenta  that are  derived  from  the  trophoblast  cell  lineage.  In  mice, maternal  blood  coming  out  from  the  spiral  arteries  invaded by  trophoblast  cells  that  replace  the  endothelium  passes across the fetal layers of the placenta and then enters into the labyrinth  where  fetal  blood  vessels  form  and  feto-maternal exchange  occurs.  Maternal  and  fetal  vascular  circuits  in  the established  mouse  placenta  are  shown  schematically  in Figure 1.   Notch2  null  mutation  induces  lethality  around  embryonic day 11 because of impaired maternal vasculature formation. owever,  both  cellular  and  molecular  events  in  the  process of  vasculature  formation  are  little  known,  especially  as  to how  the  maternal  blood  bed  expands  among  the  tightly packed  trophoblast  mass.  Our  histological  studies  showed that  a  stream  of  maternal  blood  along  the  interface between the  labyrinthine  trophoblast  and  spongiotrophoblast  was slightly  opened  in  the  mutant  placenta.  Expression  of  the Notch2  gene  is  observed  in  spongiotrophoblast  and  Giant cells,  but  in  neither  the  labyrinthine  trophoblast  nor  in  the multi-nuclear  Syncytiotrophoblast  that  is  derived  by  cell fusion  of  the  mono-nuclear  labyrinthine  trophoblast  (Figure 2). Thus,  we  conclude  that  cellular  and  molecular  events  to form the maternal blood bed occur in the spongiotrophoblast and Giant cell layers. We  have  assumed  that  the  cellular  event  that  expands  the maternal  blood  bed  is  the  deletion  of  spongiotrophoblast cells  in  a  significant  number  from  the  developing  placenta. Thus,  we  are  trying  to  detect  trophoblast  cell  death  or detachment with various methods.

Figure  1.  Schematic  maternal  and  fetal  vascular  circuits  in  mouse placenta.  Maternal  blood  flows  in  streams  drawn  in  yellow  in  the direction  shown  by  small  red  arrows.  Fetal  blood  flowing  in  the  artery (blue  line)  reaches  the  border  of  the  spongio-  and  labyrinth  layers  and returns to the fetal vein (red line).

Figure 2. Expression of Notch2 gene in developing mouse placenta. The Notch2 gene is expressed in the ectoplacental cone at E .0, but not in the chorion. At  E .0,  the  ectoplacental  cone  and  chorion  differentiate  into  spongiotrophoblast  (Sp)  and  labyrinthine  trophoblast  (Lb),  respectively. The  expression  is detected in Sp. At E10.0, differentiation of syncytiotrophoblast (SynT) and vasculogenesis of fetal blood are obvious in the placenta. Notch2 expression occurs  in  Sp,  Giant  cell  layer  (Gc),  and  mesenchymal  cells  derived  from  allantois.  owever,  it  is  not  detected  in  SynT  and  Lb  far  away  from  maternal blood or separated by SynT.

1National nstitute or asic iology

B

Professor

N

Naoto

Associate Professor

N

A Noriyu iAssistant Professor:

TAKAHASHI, Hiroki
SUZUKI, Makoto
Technical Staff:
TAKAGI, Chiyo
NIBB Research Fellow:
YAMAGUCHI, Takeshi
Postdoctoral Fellow:
NEGISHI, Takefumi
SUZUKI, Miho
HARA, Yusuke
SOKENDAI Graduate Student: MIYAGI, Asuka
HAYASHI, Kentaro
TOMINAGA, Hitoshi
Visiting Undergraduate: TOMINAGA, Hitoshi*
Technical Assistant:
YAMAMOTO, Takamasa
MURAKAMI, Michiyo
WATANABE, Mika
YASUE, Naoko
Secretary:
MIYAKE, Satoko
TSUGE, Toyoko
The  complex  morphogenesis  of  organisms  is  achieved  by dynamic rearrangements of tissues during embryogenesis, in which change in cellular morphology as well as orchestrated cell  movements  are  involved.  For  cells  to  know  how  they should  change  their  shape  and  where  they  should  move, information  called  cell  polarity   is  essential.  ow  then  is the  cell  polarity  established  within  cells   Is  it  intrinsically formed  within  the  cells  or  triggered  by  extracellular  cues  Furthermore,  little  is  known  as  to  how  coordinated  and complex  cell  movements  are  controlled  in  time  and  space. We  attempt  to  understand  the  mechanisms  underlying  these events  using  several  model  animals,  including  frogs,  fish, mice and ascidians, taking physical parameters such as force in  consideration,  in  addition  to  conventional  molecular  and cellular biology. I. The roles of PCP core components in mouse
development

In  epithelia,  the  roles  of  planar  cell  polarity  (PCP)    have been extensively studied, whereas in non-epithelia, they have yet to be fully understood. We are exploring the roles of PCP in  the  mesenchymal  tissues  by  using  mouse  genetics. Recently,  we  generated  a  hypomorphic  allele  of  mouse Prickle1,  one  of  the  core  PCP  factors.  We  previously reported  (Tao,  .  et al.  Proc.  Natl. Acad.  Sci.,  USA,  200 ) Prickle1  null null  mice  die  around  E .0  in  gestation  due  to the  failure  of  gastrulation.  In  contrast,  the  Prickle1 hypomorphic mutant mice we generated by a partial deletion of the gene survived to P0. Interestingly, the mutant mice had shortened noses. Detailed analyses at the cell level suggested that Prickle1 governs convergent extension of nasal cartilage cells which is required for the lengthening of the nose. Wnt5, a  ligand  that  activates  the  PCP  pathway  was  found  to  be expressed forming a concentration gradient of the transcripts from  the  distal  tip  of  developing  nose.  As  ubiquitous overexpression  of  Wnt5  caused  short  noses,  the 1concentration gradient of Wnt5 may be important for proper nose  elongation.  Our  findings  further  suggests  that  PCP signaling employed multiple times at different places during development  may  be  one  of  the  universal  mechanisms  of organ  morphogenesis,  especially  for  organ  elongation.  We hope that in combination with comparative genomic analysis, these  PCP  mutant  mice  will  serve  as  good  models  that  can explain morphological variations in mammals.

Figure 1. Nose phenotype of mouse Prickle1mutant.
The  hypomorph  allele  (B)  shows  shortened  nose  cartilage  stained  with alcian blue due to insufficient convergent extension compared to that of wild type (A).

II. Regulation of cell adhesion by the ubiquitin
system during gastrulation During gastrulation, dorsal mesoderm cells migrate toward the midline and align along the antero-posterior (A-P) axis to form the notochord. In this process, cells repeatedly undergo cell  adhesion  and  detachment,  and  thus,  cell-to-cell interaction  must  be  tightly  regulated.  We  found  that  one  of the  cadherin  superfamily  cell  adhesion  molecules,  paraxial protocadherin (PAPC), plays an essential role in this process. The PAPC transcript first appears in the dorsal marginal zone at  the  early  gastrula  stage  and  is  subsequently  restricted  to the  paraxial  mesoderm  in  Xenopus  and  zebrafish.  Using Xenopus  embryos,  we  demonstrated  that  PAPC  is  also regulated  at  the  protein  level  and  is  degraded  and  excluded from the plasma membrane in the axial mesoderm by the late gastrula  stage.  This  regulation  requires  phosphorylationdependent  poly-ubiquitination.  PAPC  is  phosphorylated  by GSK   in  the  evolutionarily  conserved  cytoplasmic  domain, and this in turn is necessary for poly-ubiquitination by an E  ubiquitin ligase  -TrCP. We also show that precise control of PAPC  by  phosphorylation ubiquitination  is  essential  for normal  Xenopus  gastrulation  cell  movements.  Taken together,  our  findings  unveil  a  novel  mechanism  of regulation  of  a  cell  adhesion  protein  and  show  that  this system  plays  a  crucial  role  in  vertebrate  embryogenesis. (Kai, M. et al., PLoS One in press).

III. Cellular behavior during neural tube
closureDuring  the  formation  of  the  neural  tube,  an  anlage  of  the central  nervous  system,  neural  progenitor  cells  emerge  in  a broad  region  on  the  dorsal  side  and  gradually  change  their Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

positions toward the midline.  owever, what kind of cellular and  molecular  dynamics  occur  during  this  process  is  not clear, probably because analyses at the single cell level with high  spatial  and  temporal  resolutions  have  not  been conducted.  We  analyzed  zebrafish  neurulation  at  single  cell resolution,  and  found  that  progenitor  cells  showed  nonmuscle  myosin  II-dependent  asynchronous  and  periodic movements.  We  also  found  that  actomyosin  cytoskeleton showed  an  isotropic,  periodic  remodeling  in  the  cell  cortex, while  it  showed  directionaly  constant  movements  in peripheral cellular protrusions. Careful examination revealed that  F-actin  remodeling  was  temporarily  correlated  with  a deformation of cell morphology, suggesting that the periodic actomyosin  contractility  contributes  to  the  convergence movements. We further found that components of the planar cell  polarity  (PCP)  pathway  were  required  for  the  periodic F-actin  remodeling,  suggesting  that  the  PCP noncanonical Wnt pathway controls the periodicity of the actomyosin. Our findings  unveil  a  repertoire  of  cellular  movements  based  on periodic  actomyosin  contractility  that  contributes  to  the convergence movements of multicellular organisms.

might form this invagination. With cell cycle progression, the length of invagination became short and the centrosome was pulled to the posterior side. Thus, we hypothesize that these membrane  invaginations  are  involved  in  spindle  orientation along  the  A-P  axis.  We  would  like  to  propose  a  novel template  of  spindle  orientation  with  membrane  invagination capturing the centrosome.

 Figure  .  Novel membrane invagination structure in ascidian epidermal cell. (A)  Live  imaging  of  membrane  invagination  toward  microtubule organizing  center  (MTOC),  seemingly,  centrosome.  P -GFP  (green), plasma  membrane  probe   EB -mCherry  (magenta),  MTOC  probe  numbers  (orange),  recording  time  in  minutes.  Anterior  membrane invaginates (white arrow) toward MTOC (blue arrow) ( 0’), centrosomes are  aligned  ( 0’)  and  eventually  spindle  is  formed  ( ’)  along  the A-P axis. (B)  D images reconstructed from SBF-SEM data. Centrosomes are indicated  as  red  and  blue  balls. The  black-lined  squares  are  enlarged  in upper right panels. All bars show 10  m.

Figure 2. Live-cell imaging analyses at single cell resolution. (A) A  small  number  of  presumptive  neural  cells  expressing  fluorescent proteins  were  transplanted  to  non-labeled  host  embryos.  Then,  cellular movements in the neural plate during convergence were observed at short time  intervals.  Np,  neural  plate   Nk,  neural  keel   So,  somite   Nc, notochord.  (B)  Cortical  F-actin  was  distributed  as  a  meshwork-like structure  and  periodically  concentrated.  The  time  interval  of  the periodicity corresponds to that of the convergence movements.

IV. A novel plasma membrane structure
capturing centrosome determines theorientation of cell divisionThe orientation of the mitotic spindle has been proposed to control  cell  fate  choices,  tissue  morphogenesis  and architecture,  thus  playing  an  important  role  in  shaping embryonic forms. It is already reported that most cells divide along the A-P axis at the last division in ascidain epidermis.
With live imaging observation, we found a novel membrane structure  invaginating  along  A-P  polarity  toward  the centrosome  in  the  epidermal  last  cell  division  cycle.  Liveimaging  observation  showed  the  invagination  toward  the centrosome.  Observation  using  Serial  block  face  Scanning Electron  Microscopy  (SBF-SEM)  confirmed  this invagination reached the centrosome. The result of U  laser ablation  indicated  that  mechanical  tension,  which  was generated  between  the  centrosome  and  plasma  membrane, V. Notochord and evolution of chordates

Recently,  much  more  attention  has  been  paid  to cephalochordate amphioxus to answer an important question of  metazoan  evolution,  namely  the  origin  and  evolution  of chordates. The phylum Chordata consists of three subphyla, Cephalochrdata,  Urochordata  and  ertebrata. A  long  debate on  whether  cephalocordates  or  urochordates  are  an  early divergence  among  chordates  has  recently  reached  a consensus in which cephalochordates are believed to be more ancestral,  leaving  urochordates  and  vertebrates  as  a  sister group. While the amphioxus develop well-organized somites, their  neural  tube  lacks  a  brain-like  structure  and  the notochord  contains  myofibrils.  In  order  to  characterize  the three  organs  of  adult  amphioxus,  we  examined  their differential  gene  expression  profiles  by  RNA-seq  analysis. The  analysis  of  differential  expression  profiles  of  genes highly expressed in the notochord, somite muscle, and neural tube,  revealed  a  molecular  affinity  between  the  notochord and  the  somite  muscle,  as  previous  studies  suggested.  In addition, we could identify  5 genes that are preferentially expressed in the notochord,  11 genes in the somite muscle, and  51   in  the  neural  tube.  These  genes  play  roles  in  the formation, maintenance and function of the three organs. The repertoires  of  genes  provide  a  molecular  basis  for  further studies of amphioxus.

1National nstitute or asic iology

B

VI. Subfunctionalization of duplicated MyoD
genes in polyploid Xenopus laevis

Polyploid  organisms  offer  a  model  for  understanding  the evolutionary effects of gene duplication. The African clawed f r o g   X e n o p u s l a e v i s   h a s   e x p e r i e n c e d   r e c e n t allopolyploidization, which gave rise to the pseudotetraploid genome.  We  found  that  the  duplicated  MyoD  genes (XlMyoDM and XlMyoDZ) are under subfunctionalization as indicated  by  their  differential  expression  patterns.  The combined  expression  pattern  of  XlMyoDM  and  XlMyoDZ corresponds  to  that  of  the  unduplicated  Xenopus tropicalis XtMyoD. To test the possibility that the ancestral function of MyoD is subdivided between the duplicated copies, we asked whether  XlMyoDM  and  XlMyoDZ  regulate  different  sets  of target  genes.  An  overexpression  experiment  of  either XlMyoD  gene  followed  by  RT-qPCR  of  a  selected  MyoD target  genes  showed  that  both  XlMyoDM  and  XlMyoDZ genes  possess  comparable  activity  of  inducing  the  target genes  we  examined. To  find  the  differential  gene  regulation b y   X l M y o D M   a n d   X l M y o D Z   g e n o m e - w i d e ,   t h e overexpressed  samples  were  subjected  to  a  transcriptome analysis.  By  comparing  XlMyoDM,  XlMyoDZ  and  XtMyoD target genes, we will answer the important questions whether either  XlMyoD  gene  acquired  novel  functions  or  lost  its original functions during evolution.

VII. Cnidarian-symbiodinium Symbiosis

Corals are declining globally due to a number of stressors. Such  stresses  can  lead  to  a  breakdown  of  the  essential symbiotic  relationship  between  coral  and  Symbiodinium,  a process  known  as  coral  bleaching.  Although  the environmental  stresses  causing  this  breakdown  are  largely known, the molecular and cellular mechanisms of symbiosis are  still  unclear.  Corals  are  not  very  suitable  as  laboratory systems, because they are difficult to work with due to their slow  growth,  long  generation  times,  and  calcareous skeletons. To overcome these limitations, we focused on the small  sea  anemone  Aiptasia  as  a  novel  experimentally tractable cnidarian model organism (Figure  ). Aiptasia, just as  reef-building  corals,  establishes  a  stable  but  temperaturesensitive  symbiosis  with  Symbiodinium.  Aiptasia  can  be repeatedly  bleached  and  repopulated  with  Symbiodinium, grows  rapidly,  and  lacks  a  calcareous  skeleton,  allowing microscopic  and  cellular  biological  analyses.  In  order  to further elucidate the symbiotic mechanisms, it is necessary to Figure  .   Aiptasia  can  be  bleached  in  the  laboratory  by  raising  culture temperature. (left) Symbiotic Aiptasia cultured at 25℃. (right) Aposymbiosis induced by culturing at  0- ℃.

20establish  molecular  biological  approaches.  Therefore,  we have  attempted  to  develop  a  method  of  gene  transfection  to Aiptasia.  Investigating  symbiosis  using  Aiptasia  should improve our understanding of the symbiotic mechanism.

Publication List

〔Original paper〕

ajima,  ., Suzuki, M., Ochi,  ., Ikeda, K., Sato, S., Yamamura, K.,  YOgino, ., Ueno, N., and Kawakami, K. (201 ). Six1 is a key regulator of the developmental and evolutionary architecture of sensory neurons in craniates. BMC Biol. 12,  0.

〔Review articles〕

ashimoto,  M.,  Morita,  .,  and  Ueno,  N.  (201 ).  Molecular  and  cellular mechanisms  of  development  underlying  congenital  diseases. Congenit. Anom. (Kyoto) 54, 1- .

Yu, J.K., Kawashima, T, Takahashi,  Satoh, N., Tagawa, K., Lowe, C.J., ., Ogasawara, M., Kirschner, M.,  isata, K., Su, Y. ., and Gerhart, J. (201 ).  On  a  possible  evolutionary  link  of  the  stomochord  of hemichordates to pharyngeal organs of chordates. Genesis 52,  25.

A

Professor

A A

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

Technical Assistant:

Secretary:

†

atoru

HAYASHI, Yoshiki
SATO, Masanao
NODA, Chiyo
OTA, Ryoma
FUJISAWA, Chiemi
OHARA, Yuya
ASAOKA, Miho
SUGIYAMA, Arisa
SHINOZUKA, Yuko
SUGIMORI, Seiko
MORITA, Shunpei
SATO, Kaori
YAMAMOTO, Manami
WASHIO, Midori
KAWAMOTO, Yuri
HONDA, Satoko

Germ  cells  are  specialized  cells  that  can  transmit  genetic materials  from  one  generation  to  the  next  in  sexual reproduction. All  of  the  other  cells  of  the  body  are  somatic cells. This separation of germ and somatic cells is one of the oldest  problems  in  developmental  biology.  In  many  animal groups,  a  specialized  portion  of  egg  cytoplasm,  or  germ plasm,  is  inherited  by  the  cell  lineage  which  gives  rise  to germ cells. This cell lineage is called germline. The germline progenitors  eventually  migrate  into  the  gonads,  where  they differentiate as germline stem cells (GSC) to form eggs and sperm  when  the  organisms  are  physically  matured.  Our laboratory aims to find the molecular mechanisms regulating germline  segregation,  GSC  niche  function,  and metamorphosis in Drosophila. I. Role of maternal Ovo protein in the germlineof Drosophila embryos

It has been proposed that germline-specific gene expression is  initiated  by  the  function  of  maternal  factors  that  are enriched  in  the  germ  plasm.  owever,  such  factors  have remained  elusive.  We  have  done  a  genome-wide  survey  of maternal transcripts that are enriched in the germ plasm and encode  transcription  factors  for  germline-specific  gene expression  of  vasa  and or  nanos.  We  finally  identified   transcripts required for germline-specific gene expression by knockdown  experiments  using  RNA  interference  (RNAi). Among  the    transcripts,  we  focused  on  ovo. The  ovo  gene encodes  a  DNA-binding,  C2 2  n-finger  protein  that  is involved  in  oogenesis  and  in  epidermal  development.  The ovo  gene  produces  at  least  three  alternate  isoforms.  Ovo-A and  Ovo-B  function  as  a  negative  and  a  positive transcriptional  regulator  in  the  germline,  respectively.  OvoSvb  is  expressed  in  the  epidermal  cells  and  is  required  for their  differentiation.  We  found  that  Ovo-B  is  the  major isoform  expressed  in  primordial  germ  cells  (PGCs)  during embryogenesis.  To  understand  its  function,  we  overexpressed  the  Ovo-A  repressor  only  in  the  PGCs,  and examined  their  developmental  fate.  Our  data  shows  that  the reduction in maternal Ovo-B activity results in a decrease in the  number  of  primordial  germ  cells  during  post-embryonic stages.  Thus,  maternal  Ovo-B  has  an  essential  role  in germline development in both sexes. While  identifying  the  downstream  genes  regulated  by Ovo-B in germline, we found that Ovo is required to induce germline-enriched  genes,  and  conversely,  it  represses somatically-expressed  genes  in  PGCs.  Thus,  we  speculate that  maternal  Ovo  has  an  important  role  in  germline-fate determination. Collaboration work is now on-going to clarify the function of the ovo gene in mouse germline development. II. The role of HSPGs in germline stem cellniche of Drosophila.

Stem  cells  posses  the  remarkable  capacity  to  generate daughter cells that retain a stem-cell identity and others that differentiate.  Stem  cells  reside  in  dedicated  cellular microenvironments  termed  stem-cell  niches.  These  niches dictate  stem-cell  identity,  maintain  the  stem  cell  population, and  coordinate  proper  homeostatic  production  of differentiated cells. The GSC niche in Drosophila gonads is a useful  model  system  for  studying  the  stem-cell  niche, because  the  cellular  components  of  this  niche  have  been characterized and the signaling pathways, such as BMPs and JAK STAT  which  are  essential  for  GSC  maintenance,  are known. Ligands for these signaling pathways (niche signals) are secreted from the niche cells, and are received by GSCs to  activate  the  pathway  responsible  for  GSC  maintenance. Thus,  the  GSC  niche  is  defined  as  the  specialized  region retaining  a  sufficient  amount  of  niche  signals  for  GSC maintenance.  owever,  it  is  not  well  understood  how  the distribution of the niche signals is precisely controlled in the GSC niche.
To  address  this  question,  we  have  been  investigating  the function  of  eparan  Sulfate  Proteoglycans  ( SPGs)  in  the GSC niche.  SPGs are an evolutionally conserved family of sugar modified proteins, which are an essential component of the  extracellular  matrix.  One  of  the  important  functions  of SPGs during animal development is to regulate distribution of  growth  factors  in  extracellular  space  by  binding  to  them. Thus,  we  speculated  that  SPGs  could  retain  a  sufficient amount of niche signals for GSC maintenance. We found that Glypcan,  a  membrane-associating  type  of  SPG,  is  an essential  component  of  the  GSC  niche  both  in  female  and male  gonads.  Glypican  was  highly  expressed  in  niche  cells both  in  ovary  and  testis,  and  its  mutations  caused  a significant  reduction  in  GSC  number.  In  the  GSC  of  the mutant  ovary,  the  signaling  pathway  activated  by  Dpp  (a BMP  homologue  acting  as  a  niche  signal)  was  impaired. Conversely, ectopic expression of Glypican in female gonads caused  an  increase  in  the  number  of  GSCs  with  Dpp signaling.  These  results  strongly  suggest  that  Glypican defines  the  female  GSC  niche  by  regulating  distribution  of Dpp.
The question of whether the other  SPGs have functions in the GSC niche remains unclear. Since disrupting biosynthesis of  all  SPGs  by  knockdown  of  the  NDST  gene  caused  a more severe GSC-loss phenotype than the Glypican mutant, the  other  types  of  SPGs  could  have  functions  in  the  GSC †  This laboratory was closed on  1 March, 2015.

21National nstitute or asic iologyBniche.  We  found  that  Syndecan and  Perlecan,  two  evolutionally conserved groups of  SPGs, are essential  in  the  GSC  niche. These  SPGs  were  highly expressed  in  female  GSC  niche cells,  and  reduction  of  their function  in  niche  cells  caused  a decrease  in  GSC  number.  We further found that, in the ovaries with  reduced  Perlecan  function, ectopic GSC-like cells were also observed.  This  phenotype  has not  been  observed  in  Glypican mutants.  Thus,  we  speculated that Perlecan could regulate Dpp distribution in the GSC niche, in a  way  distinct  from  Glypican. We   h a v e   s u c c e e d e d   i n v i s u a l i z i n g   D p p   p r o t e i n distribution  in  the  female  GSC niche.  When  Glypican  was ectopically  expressed  in  female gonads,  Dpp  distribution  was e c t o p i c a l l y   o b s e r v e d   i n Syndecan  and  Perlecan  mutant ovaries.  Furthermore,  we  have also succeeded in visualizing the GSC  niche  signal  in  male gonads. This enables us to study SPG function in the male GSC niche.

Figure 1. Our model explaining the regulation of metamorphosis by Oct R
(A) Before the attainment of body-size checkpoint, tyramine is stored in PG cells, so as not to activate Oct R. Once  larvae  have  attained  body-size  checkpoint  under  nutrient-rich  conditions,  tyramine  is  secreted  from  the PG to activate Oct R signaling in an autocrine manner, leading to the larval prepupal transition via the Ilps and  PTT   signaling  pathways.  (B)  When  larvae  fail  to  attain  body-size  checkpoint    under  a  starvation condition,  tyramine  remains  unsecreted  from  the  PG   consequently,  the  Oct R,  Ilps,  and  PTT   signaling pathways fail to be activated, resulting in arrest at the larval prepupal transition.

III. The role of b3-octopamine receptor in thedevelopmental transition from larvae toreproductive adults

The  developmental  transition  is  a  well-known  biological process in which the organism alters its body morphology in order  to  proceed  from  the  juvenile  stage  to  the  adult reproductive  stage.  ow  this  processe  is  precisely  regulated in  response  to  developmental  and  environmental  cues  is  a longstanding question in biology. In  holometabolous  insects,  the  steroid  hormone  ecdysone plays  a  pivotal  role  in  metamorphosis.  In  Drosophila, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the  peripheral  organs. The  activities  of  20E  terminate  larval development  and  growth  and  initiate  metamorphosis. Ecdysone  biosynthesis  is  regulated  in  the  PG  by neuropeptides,  enabling  modulation  of  the  timing  of  20E pulses  during  development.  The  stimulator  of  ecdysone biosynthesis  is  prothoracicotropic  hormone  (PTT )  and Insulin-like peptides (Ilps), which activate the production of ecdysone  biosynthetic  proteins.  In  addition  to  these neuropeptides, the larval prepupal transition is modulated by environmental  cues  such  as  nutritional  conditions  that influence  larval  body  size.  For  example,  early  third-instar larvae attain the body-size checkpoint required for the transit from larva to prepupa. Although the checkpoint is believed to 22ultimately  modulate  ecdysone  production  in  the  PG,  its downstream effectors and signaling pathway remain elusive. We  found  that  monoaminergic  autocrine  regulation  of ecdysone  biosynthesis  in  the  PG  is  essential  for metamorphosis. PG-specific knockdown of Octb3R, resulted in  arrested  metamorphosis  due  to  lack  of  ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG  caused  similar  defects  in  ecdysone  production  and metamorphosis.  Moreover,  PTT   and  Ilps  signaling  were impaired by Octb3R knockdown in the PG, and activation of these  signaling  pathways  rescued  the  defect  in metamorphosis. Thus, monoaminergic autocrine signaling in the  PG  regulates  ecdysone  biogenesis  in  a  coordinated fashion upon activation by PTT  and Ilps. We propose that monoaminergic  autocrine  signaling  acts  downstream  of  a body-size  checkpoint  that  allows  metamorphosis  to  occur when nutrients are sufficiently abundant.

Publication List

〔Original papers〕

hanut-Delalande,  .,  ashimoto, Y.,  P lissier-Monier, A.,  Spokony,  CR., Dib, A., Kondo, T., Bohre, J., Niimi, K., Latapie, Y., Inagaki, S., Dubois,  L.,  alenti,  P.,  Polesello,  C.,  Kobayashi,  S.,  Moussian,  B., White, K., Plaza, S., Kageyama, Y., and Payre, F. (201 ). Pri peptides are  mediators  of  ecdysone  for  the  temporal  control  of  development. Nature Cell Biol. 1 , 10 5-10 .

ayashi,  M.,  Sato,  M.,  Iwasaki,  Y.,  Onozawa,  T.,  Katayama,  N.,  Nagasaka, Y.,  Sadaie,  S.,  Kobayashi,  S.,  and  Yoshizaki,  G.  (201 ) 

Enrichment  of  spermatogonial  stem  cells  using  side  population  in teleost. Biol. Reprod.  1, 1- .
Lim, R., Anand, A., Nishimiya-Fujisawa, C., Kobayashi, S., and Kai, T. (201 ). Analysis  of  ydra  PIWI  proteins  and  piRNAs  uncover  early evolutionary origins of the piRNA pathway. Dev. Biol.  , 2 -251.
Nishimura, T.,  erpin, A., Kimura, T.,  ara, I., Kawasaki, T., Nakamura, S., Yamamoto, Y., Saito, T., Yoshimura, J., Morishita, S., Tsukahara, T., Kobayashi, S., Naruse, K., Shigenobu, S., Sakai, N., Schartl, M., and Tanaka,  M.  (201 ).  Analysis  of  a  novel  gene,  Sdgc,  reveals  sex chromosome dependent  differences  of  medaka  germ  cells  prior  to gonad formation. Development 1 1, 
.

 
 

2

National nstitute or asic iology

B

A A

Professor

A A A

A

B

in i

Assistant Professor:

YABE, Taijiro
MII, Yusuke
Technical Staff:
UTSUMI, Hideko
NIBB Research Fellow:
OKADA, Kazunori
Postdoctoral Fellow:
CHEN, Qiuhong
KAMETANI, Yoshiko
TAKADA, Ritsuko
FUJIMORI, Sayumi
WANGLAR, Chimwar
SOKENDAI Graduate Student:
SHINOZUKA, Takuma
TSUCHIYA, Yoshihiro
Visiting Graduate Student: WEICHSEL, Alexander
Technical Assistant:
TAKASHIRO, Kayoko
ITO, Yukiko
ISHIJIMA, Hannosuke
Secretary:
UKAI, SakieThe  body  and  tissues  of  the  developing  embryo  are repeatedly  divided  into  sub-regions  specified  by characteristic gene expression and morphology. The process that  gives  rise  to  these  sub-regions,  according  to  a  defined pattern,  is  called  pattern  formation   or  patterning.   Our laboratories  aim  to  understand  the  molecular  mechanisms underlying pattern formation by several different approaches.
The most popular model to explain the patterning process is the  morphogen  gradient  and  threshold   theory.  Many genetic  results  indicate  that  secreted  signal  proteins  such  as Wnt, BMP, and  edgehog function as morphogens in many aspects  of  the  patterning  process.  In  spite  of  the accumulation of genetic evidence, however, the biochemical characteristics  of  morphogens,  including  modification  and higher order structure, remain to be elucidated. Thus, one of our  major  goals  is  to  reveal  the real image  of  morphogens and  the  molecular  mechanisms  underlying  the  formation  of morphogen  gradients,  including  the  secretion  and extracellular transport of these morphogens.
The  segmental  sub-regions  of  several  specific  tissues, including  the  paraxial  mesoderm  (or  somites)  and  the pharyngeal  pouches,  by  contrast,  appear  not  to  be  simply directed  by  the  morphogen  gradient  and  threshold,  but  by  a unique  mechanism  proceeding  periodically.  For  instance, somites are sequentially generated in an anterior-to-posterior order by converting oscillatory gene expression into repeated structures.  The  molecular  mechanism  underlying  this conversion and morphological segmentation, however, is not yet  fully  understood.  Thus,  another  goal  of  our  current studies  is  to  reveal  the  molecular  mechanisms  of  this otherand unique mode of patterning  that  underlies  the  periodical and  sequential  sub-division  in  the  development  of  somites and pharyngeal arches.

2

I. Spatial regulation of secreted Wnt proteinsin vertebrate development The Wnt family of secreted signal proteins plays a key role in numerous aspects of embryogenesis. In a classical view, it has  been  proposed  that  secreted  Wnt  proteins  spread  and function  over  a  distance  of  several  cells  in  many  aspects  of morphogenesis.  In  contrast,  accumulating  evidence  also implies  that  Wnt  proteins  appear  to  transmit  their  signals locally  in  some  particular  cases,  presumably  since  their secretion and transport are under tight control. To understand the  molecular  mechanisms  underlying  morphogenesis mediated by extracellular transmission of Wnt signals, it will be  required  to  reveal  manners  of  diffusion  and or accumulation of Wnt proteins in extracellular space.
For  a  better  understanding  of  the  extracellular  transport  of Wnt  proteins,  we  started  to  visualize  Wnt  proteins  in  the extracellular  space  by  a  two  different  approaches.  One  is  a live  imaging  approach  with  EGFP-tagged  Wnt  proteins  and  the  other  is  immnohistochemistry.  Our  preliminary  study indicated  that  Wnt  proteins  are  not  simply  diffused  during embryogenesis  of  the  mouse.  We  precisely  examined regulatory  mechanisms  and  the  biological  significance  of Wnt  protein  distribution  in  mouse  embryos.  These  analyses reveal a novel view of spatial regulation of Wnt signaling.

Figure 1. Accumulation of Wnt proteins between cells. Wnt proteins are acculmulated in a punctate pattern between epithelial cells in a Xenopus embryo.  (Left)  Immunostaining  of  endogenous  Wnt   proteins  (Right) The left image is merged with a bright field one.

Extracellular molecules are known to regulate special
distribution of Wnt proteins. Heparan sulfate sugar chainswith different modifications form distinct extracellular
structures, which are called heparan sulfate nanostructures(HSNSs), in the early Xenopus embryo. Interestingly,
Xenopus Wnt8 proteins were preferentially retained by
HSNSs with N-sulfo modification (N-sulfo HSNSs). To
understand the mechanism by which distinct distributions of
HSNSs, especially N-sulfo HSNSs are formed, we focusedon an enzyme catalyzing N-sulfation of heparan sulfate.
Gain and loss of function studies of this enzyme indicatedthat it is necessary and sufficient for the conversion of
N-acetyl HSNSs into N-sulfo HSNSs. Extracellularaccumulation of Wnt proteins and the convergent extension
movement, which is regulated by Wnt non-canonicalsignaling, were also affected by perturbation of expression ofthis enzyme. These results indicated that N-sulfo
modification of heparan sulfates are important for
extracellular distribution of Wnt proteins and Wnt signaling.

II. Heterogeneity of secreted Wnt proteinssecreted from cultured cells

Although  the  structure  of  Wnt  protein  has  already  been revealed, its higher order structure in extracellular space has not yet been fully understood. One of the proposed forms of Wnt  protein  in  extracellular  space  binds  to  lipoprotein particles.  On  the  other  hand,  recent  studies  indicated  that Wnt  proteins  are  secreted  on  another  lipid-based  carrier, called  the  exosome,  which  is  an  M B  (Multi esicular Body)-derived  membrane  vesicle.  owever,  it  remains unclear whether Wnt proteins are secreted in these two forms from the same cells. To  address  these  issues,  we  systematically  examined characteristics  of  Wnt  proteins  secreted  from  several different cell lines.  First,  we  fractionated conditioned media of Wnt a-expressing cells by density gradient centrifugation with Sodium Bromide. To examine whether Wnt proteins are attached  to  exosomes,  we  then  collected  exsosome-like molecules  by  ultracentrifugation  and  fractionated  the  pellet by  sucrose  density  gradient  ultracentrifugation.  Our  results indicate  that  secreted  forms  of  Wnt  proteins  differ  among different  Wnt  subtypes  and  this  diversity  is  dependent  on cellular context.

III. M o l e c u l a r m e c h a n i s m o f s o m i t edevelopment

Somites  are  segmental  epithelial  blocks  located symmetrically on either side of the neural tube. Somites are periodically  generated  in  an  anterior  to  posterior  manner from their precursor, the presomitic mesoderm (PSM), which is  located  posterior  to  newly  formed  somites.  This  periodic generation  is  achieved  by  a  complex  and  dynamic mechanism  in  the  PSM.  First,  the  molecular  clock,  the so-called  segmentation  clock,  creates  oscillatory  expression of  particular  genes,  hairy  and  some  notch-related  genes,  in the  posterior  PSM.  Because  the  phase  of  oscillation  is gradually shifted along the posterior-to-anterior axis, a wave of  the  oscillation  appears  to  move  in  a  posterior-to-anterior fashion.  This  oscillatory  gene  expression  subsequently results  in  periodical  generation  of  morphologically segmented somites.
The  spatial  pattern  of  somites  is  defined  by  positioning  of intersomitic boundaries and rostro-caudal patterning within a somite. This spatial pattern is established in the anterior PSM by  converting  the  oscillatory  gene  expression  into  spatially restricted  patterns  of  gene  expression.  Several  intra-cellular molecules, as well as FGF and Wnt signaling, are involved in this  conversion.  In  mice,  the  prospective  segmentation boundary  is  periodically  established  in  the  anterior  PSM  at the rostral border of Mesp2 expression domain. Mesp2, one of the key regulators in this conversion, is initially expressed at the most anterior region of the Tbx  protein domain. This expression  is  not  anteriorly  extended  beyond  the  anterior border  of  the  Tbx   protein  domain  because  Mesp2 expression requires Tbx  proteins. Thus, the anterior border formation of the Tbx  protein domain is a more fundamental process in the positioning of the segmentation boundary. Importantly,  this  border  is  not  consistent  with  the  anterior border  of  Tbx   mRNA,  rather  it  is  regulated  by  a proteasome-mediated  mechanism. Although  the  molecules directly executing this proteolysis are still unclear Mesp2, as well  as  Ripply1 and  2,  have  shown  to  be  required  for  the down-regulation  of  Tbx   proteins  by  analysis  of  embryos defective in these genes. Since expressions of Ripply1 and 2 are  eliminated  in  Mesp2  deficient  mouse  embryos,  we previously  proposed  the  following  model   Mesp2, whose expression is activated in the most anterior part of the Tbx  domain,  causes  retreat  of  the  Tbx   protein  domain  through activation  of  Ripply1  and  2  expression,  and  the  retreated Tbx  subsequently defines the next segmentation border and Mesp2  expression.  owever,  it  is  still  to  be  elucidated whether Mesp2 causes the retreat of the Tbx  protein border through  the  activation  of  Ripplys’  expression  or  that  Mesp2 and Ripplys regulate this retreat in parallel. To answer this question, we precisely examined function of mwsp  and  ripply  in  the  regulation  of  Tbx   proteins  in zebrafish embryos. By utilizing an antibody against zebrafish Tbx Fss, we found that the anterior border of Tbx  domain coincided with the presumptive intersomitic boundary also in the  zebrafish  and  it  shifted  dynamically  during  1  cycle  of segmentation. Consistent with the findings in mice, the tbx6
mRNA domain was located far anterior to its protein domain, indicating  the  possibility  of  posttranscriptional  regulation. When  both  ripply1/2  were  knockeddown,  the  Tbx   domain was  anteriorly  expanded.  We  further  directly  demonstrated Figure  2.  Ripply  reduces  Tbx   protein  levels   Western  blotting  with proteins  recovered  from  embryos  injected  with  Flag-tagged  zebrafish tbx6mRNA  and  Myc-tagged  zebrafish  ripply1 (A)  or  mouse  Ripply2 mRNAs  (B).  Both  zebrafish  ripply1  and  mouse  Ripply2  reduceTbx  protein levels.

Figure  . Schematic representation of spatial patterns of Tbx  and p-Erk domains  with  ripply1,  ripply2,  and  mesp-ab  expressions  during  a  single segmentation  cycle.  Expression  of  ripply1  and  ripply2  is  initially activated  in  the  high Tbx     low  FGF  signaling  region. These  activated Ripplys appear to suppress Tbx6 protein resulting in formation of a new
anterior border of the Tbx6 core domain and the upper band.

25National nstitute or asic iologyBthat Ripply could reduce the expression level of Tbx  protein depending on physical interaction between Ripply and Tbx  (Figure  2).  Moreover,  the  onset  of  ripply1  and  ripply2 expression  occurred  after  reduction  of  FGF  signaling  at  the anterior  PSM,  but  this  expression  initiated  much  earlier  on treatment  with  SU5 02,  a  chemical  inhibitor  of  FGF signaling.  These  results  strongly  suggest  that  Ripply  is  a direct  regulator  of  the  Tbx   protein  level  for  the establishment  of  intersomitic  boundaries  and  mediates  a reduction  in  FGF  signaling  for  the  positioning  of  the presumptive intersomitic boundary in the PSM (Figure  ).

IV. E s t a b l i s h m e n t o f g e n e k n o c k o u tmethodology based on genome editing
technologies in the zebrafish

ebrafish  are  one  of  the  most  suitable  model  animals  for genetic  analysis.  Until  now,  chemical  mutagenesis-mediated screening  provided  us  a  great  number  of  mutant  strains, which contribute to studies of morphogenesis of vertebrates.  In  addition  to  this  well-established  genetic  approach,  recent advances  in  genome  editing  technologies  have  made  it possible to create any mutation of a gene of interest. Previous  studies  revealed  mouse  Mesp2 has  essential  roles for  multiple  processes  of  somitogenesis  including determination of position of segment boundaries, generation of  somite  boundary  structure  and  establishment  of  rostrocaudal  polarity  in  each  somite. Although  four  mesp  genes have  been  identified  in  the  zebrafish  genome,  their  roles  in somitogenesis still remained unclear. To address this question we generated mutant fish carrying the frame shift mutation in all  mesp  genes  using  TALEN  mediated  mutagenesis  and analyzed  its  phenotype.  Unlike  the  mouse  Mesp2  mutant, zebrafish  mesps  quadruple  knockout  embryo  exhibited normally  segmented  somites.  We  are  currently  examining several mutants generated by TALEN mediated mutagenesis. Publication List

〔Original papers〕

imura,  T.,  Nagao,  Y.,  ashimoto,  .,  Yamamoto-Shiraishi,  Y.,  KYamamoto, S., Yabe, T., Takada, S., Kinoshita, M., Kuroiwa, A., and Naruse,  K.  (201 ).  Leucophores  are  similar  to  xanthophores  in  their specification and differentiation processes in medaka. Proc. Natl. Acd. Sci. USA 111, .
Osipovich, A.B., Long,  ., Manduchi, E., Gangula, R.,  ipkens, S.B., Schneider, J., Okubo, T., Stoeckert, C.J. Jr., Takada, S., and Magnuson, M.A.  (201 ).  Insm1  promotes  endocrine  cell  differentiation  by modulating the expression of a network of genes that includes Neurog  and Ripply . Development 141, 2 -2 .
Wanglar, C., Takahashi, J., Yabe, T., and Takada, S. (201 ). Tbx protein level critical for clock-mediated somite positioning is regulated through interaction between Tbx and Ripply. PLoS One 9, e10 2 .

 
 

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B

ProfessorFR

os i i o

Assistant Professor:

TOYOOKA, Yayoi
KOYAMA, Hiroshi
Technical Staff:
OKA, SanaeNIBB Research Fellow:
SATO, Yasufumi
SHI, Dongbo
Postdoctoral Fellow:
KOBAYAKAWA, Satoru
SOKENDAI Graduate Student: KAMEMIZU, Chizuru
ITO, Tomoaki
USAMI, Fumiko
Visiting Graduate Student: SHI, Dongbo*
Technical Assistant:
HIGUCHI, Yoko
Secretary:
KATO, Azusa

Figure  1.  Examples  of  a  morula  expressing  fucci2,    cell  cycle  markers.  The  G(1)  and  S G(2) M  phases  are  distinguished  by  mCherry(red)  and m enus (green) signals respectively.

The  aim  of  our  research  is  to  understand  the  events underlying early mammalian development during the period from pre-implantation to establishment of the body axes.
Early  events  during  embryogenesis  in  mammals  are relatively  less  understood,  as  compared  to  in  other  animals. This  is  mainly  due  to  difficulties  in  approaching  the developing  embryo  in  the  oviducts  and  the  uterus  of  the mother. Another  characteristic  of  mammalian  embryos  is their highly regulative potential. The pattern of cell division and  allocation  of  cells  within  an  embryo  during  the  early stages  vary  between  embryos.  The  timing  of  the  earliest specification events that control the future body axes is still under  investigation.  Functional  proteins  or  other  cellular co mp o n en ts  h a v e   n o t  b e e n   fo u n d   th a t  l oc a l i z e asymmetrically  in  the  fertilized  egg.  We  want  to  provide basic and fundamental information about the specification of embryonic axes, differentiation of cell lineages, behaviors of cells,  and  the  regulation  of  body  shape  in  early  mammalian development.  We  are  also  interested  in  the  relationship between embryos and their environment, the oviducts and the uterus.

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  the mouse  genomic  DNA  sequences  of  the  enhancer promoter region  of  important  genes  encoding  factors  regulating  cell differentiation in these stages. Combining these two types of reporter  transgenic  mouse  lines,  we  have  started  analysis  of behaviors  of  cells  comparing  gene  expression  properties  at the single cell level. For  the  live  imaging  of  early  mammalian  embryos,  a combined microscope and incubation system is an important tool. Conventional CO2 incubators provide better conditions compared  with  microscope  top  incubation  chambers, including  stability  of  temperature  and  humidity,  to  support embryonic  development  in vitro.  Incubation  microscopes have also recently become commercially available, however, these  are  expensive  for  personal  use.  We  have  modified  an incubation  microscope  with  wide  field  fluorescent illumination,  which  is  relatively  inexpensive.  We  added  a spinning  disc  confocal  system  and  sensitive  EM-CCD camera  for  observation  of  developing  mouse  embryos  with less photo-toxicity and higher spatial resolution.  We  are  also  trying  to  observe  and  reveal  aspects  of  cell shape, morphogenesis, cell lineage, gene expression and cell differentiation by combining these techniques. I. Establishment of a live imaging system for

II. Histological observation of mouse embryos

To  understand  the  mechanisms  underlying  embryonic development  and  morphogenesis,  it  has  become  common to observe  the  behavior  of  cells  and  gene  expression  in  living embryos  in  a  variety  of  animal  species.  Progress  in  GFP technologies,  genetic  engineering  and  embryonic manipulation  concomitantly  with  the  progress  in  optics  and microscope  technologies,  including  development  of  highly sensitive  photo-detectors,  has  allowed  us  to  observe  live developing embryos, even in mammals. We have established a series of transgenic mouse lines for live imaging, which is a  part  of  a  collaborative  project  taking  place  in  the Laboratory  for Animal  Resources  and  Genetic  Engineering, Riken CDB. These mouse lines have been used to visualize nuclei,  cell  shapes,  the  cytoskeleton  and  other  organelles  to observe  cell  behaviors  in  living  mouse  embryos  in  many laboratories over the world. We also established mouse lines to monitor the cell cycle.
We have also been establishing several reporter mouse lines One  of  the  characteristics  of  mammalian  development  is that embryos develop in the uterus after implantation. Direct interaction  between  embryos  and  the  cells  of  the  uterus  is important.  In  studies  on  developmental  biology  of mammalian embryos, embryos are usually removed from the uterus,  and  isolated  embryos  are  analyzed.  We  have  been analyzing  early  embryonic  development  of  mice  comparing the  changes  in  the  uterine  epithelium.  To  observe  the morphogenesis of  embryos  within the uterus,  serial sections of  implanted  uteruses  were  made,  stained  with  hematoxylin and  eosin,  and  images  of  the  embryos  within  the  uteruses were  captured  to  make  high  resolution  three-dimensional re-constructions. Figure 2 shows an example of a section. We are  preparing  samples  obtained  from  various  developmental stages.  Precise  positions  of  embryonic  implantation  and changes in the uterine epithelium correlating with embryonic development will be examined using these images.

observation of early mouse development

developing in the uterusNote  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

2

National nstitute or asic iologyBFigure  2. A  pregnant  uterus  .5  days  after  fertilization.  Developing embryos and the shape of uterus epithelium can be observed. III. Polarity formation in the mouse oviductepitheliumThe  oviduct  (Fallopian  tube)  is  the  organ  connecting  the periovarian  space  and  the  uterine  horn.  The  ova  released from  the  ovary  are  transported  through  the  oviduct,  where the  ova  are  fertilized  by  spermatozoon  entering  from  the uterus. We are focusing on the region of the oviduct close to the opening to the ovary. The  epithelium  of  the  mouse  oviduct  consists  of  multiciliated  cells  and  secretory  cells.  Ovulated  oocytes  are transported  in  oviducts  by  unidirectional  motility  of  multicilia  and  the  resultant  secretory  fluid  flow  from  ovary  to uterus. This cellular orientation in the oviduct is one example of  planar  cell  polarity  (PCP).  PCP  is  seen  in  many  animals and  tissues,  where  cells  sense  global  axes  of  the  tissue  to which they belong and orient themselves to fulfill specialized functions.
Many  evolutionally  conserved  genes  are  required  for  the formation  of  PCP,  and  several  of  those  encode  proteins  that are  localized  in  polarized  manners  within  cells.  We performed  immunohistochemical  analysis  of  potential  PCP regulators in postnatal oviducts. 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,  cilia  were  generated Figure  .  In  the  oviduct  of  mice  that  have  lost  the  Celsr1  protein, various polarity impairments covering multiple levels are observed.

2and  those  within  each  cell  appeared  to  beat  as  in  the  wild type  oviduct.  owever,  the  beating  direction  was  not coordinated  along  the  ovary-uterus  axis  throughout  the epithelia  some cells were oriented orthogonally to the axis, while  some  others  exhibited  reverse  polarity.  When  we closely examined orientation of cilia by electron microscopy they were not orientated in the same direction. In addition to the ciliary polarity phenotype, cellular shape polarity  was  also  impaired  in  the  Celsr1-deficient  mice. We found  a  new  feature  of  cellular  polarity  in  the  wild  type oviduct,  that  the  apical  surface  of  epithelial  cells  was elongated  along  the  ovary-uterus  axis.  In  Celsr1-deficient mice, epithelial cells showed less elongation and randomized orientation.
Furthermore,  we  also  found  that  the  three-dimensional architecture  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.  owever,  in  the  mutant oviducts, epithelial folds no longer run parallel to the organ axis. The direction of the folds was randomized, and ectopic branches of the folds were observed.
This  suggests  that  Celsr1  is  important  for  the  formation  of multi-scale polarities in the mouse oviduct ranging from the cellular  to  the  tissue  scale  (Figure  ).  Our  mosaic  analysis analyses with Celsr1-deficient cells in the wild type oviduct epithelium  suggest  that  the  geometry  of  epithelial  cells,  but not  the  morphology  of  the  epithelial  fold,  is  primarily regulated by Celsr1. Currently, we have been trying to reveal the mechanisms of oviduct  epithelial  morphogenesis  by  integrating  the molecular  functions  of  PCP  factors,  cellular  shape  changes, tissue  morphology  and  involvement  of  mechanical  forces. We are also focusing on some other PCP regulators, and are trying  to  understand  how  oviduct  epithelial  cells  establish and maintain their polarity.  IV. Analysis of mechanical properties of cells
and tissues during embryonic development Mechanics is one of the essential components of biological processes,  including  cell  shape  transformation  and  tissue morphogenesis etc.  owever, how mechanical states such as force and material stiffness regulate these processes is poorly understood.  To  approach  this  problem,  measuring  cellular and  tissue  geometric  information  and  mechanical  states  are necessary. We developed image processing based techniques to  measure  cellular  and  tissue  geometric  information  from fluorescent  microscopic  images  and  frameworks  to theoretically estimate the mechanical states.
  By  employing  image  processing  techniques,  we  have extracted  geometric  information,  shapes,  and  movements  of early  embryogenesis  in  C. elegans  and  mice.  In  the framework  for  estimating  mechanical  states,  geometric information  was  combined  with  a  mechanical  simulation, which  was  based  on  data  assimilation  (Figure  ).  We successfully  estimated  the  spatio-temporal  dynamics  of cellular and tissue mechanical states by systematically fitting the  in vivo  geometric  states  to  the  mechanical  simulation. The  mechanical  information  will  be  useful  to  investigate physical  mechanisms  of  early  embryonic  development  and morphogenesis  during  organogenesis  in  late  stages  in development.

Figure  .  Theoretical  estimation  of  cellular tissue  mechanical  states. Schematic illustration of estimation.

Publication List

〔Original papers〕

muta, Y., Koyama,  ., Shi, D., Eiraku, M., Fujimori, T., and Sasaki,  . 
 I(201).  Mechanical  control  of  notochord  morphogenesis  by  extraembryonic tissues in mouse embryos. Mech. Dev. 132, -5 .

shino, Y.,  ayashi, Y., Naruse, M., Tomita, K., Sanbo, M., Fuchigami,  IT., Fujiki, R., irose, K., Toyooka, Y., Fujimori, T., Ikenaka, K., and itoshi, S. (201 ). Bre1a, a histone  2B ubiquitin ligase, regulates the cell cycle and differentiation of neural precursor cells. J. Neurosci. 34, 0 - 0 . Nakagawa, S., Shimada, M., Yanaka, K., Mito, M., Arai, T., Takahashi, E., Fujita, Y., Fujimori, T., Standaert, L., Marine, J.C., and  irose, T. (201 ). The lncRNA Neat1 is required for corpus luteum formation and the establishment of pregnancy in a subpopulation of mice. Development 141,  1 - 2 .
Shi, D., Komatsu, K.,  irao, M., Toyooka, Y., Koyama,  ., Tissir, F., Goffinet, A.M., Uemura, T., and Fujimori, T. (201 ). Celsr1 is required for the generation of polarity at multiple levels of the mouse oviduct. Development 141,  55 - 5 .

 
 

2

National nstitute or asic iology

B

B

Professor

A

osei

Assistant Professor:

HARA, Kenshiro
KITADATE, Yu
NIBB Research Fellow:
NAKAMURA, Yoshiaki*
Technical Staff:
MIZUGUCHI, Hiroko
Postdoctoral Fellow:
NAKAMURA, Yoshiaki
IKAMI, Kanako
TOKUE, Moe
SOKENDAI Graduate Student: IKAMI, Kanako*
TOKUE, Moe*
NONAMI, Yuta
ISHIZAKA, Miho
Technical Assistant:
INADA, Kana
KON, Yayoi
MARUYAMA, Ayumi
Secretary:
KUBOKI, YukoProduction of numerous sperm for a long period in testis is fundamental  for  continuity  of  life  across  generations.  This process  of  spermatogenesis  is  supported  by  the  robust  stem cell system.
The  goal  of  the  Division  of  Germ  Cell  Biology  is  to  fully understand the mammalian sperm stem cell system under the context  of  in vivo tissue  architecture.  We  have  successfully revealed  some  characteristics  of  this  interesting  stem  cell system.  First,  differentiating  germ  cells  that  had  been believed to be irreversibly committed for differentiation still retain  their  self-renewing  potential  and  can  contribute  to stem  cell  pool  maintenance  ( potential  stem  cells ). Secondly,  reversion   from  potential  stem  cells  occurs  at  a higher  frequency  when  testicular  tissue  is  damaged  and regeneration  is  induced.  Thirdly,  the  undifferentiated spermatogonia  populations  including  both  actual   and potential   stem  cells  are  preferentially  localized  near vasculature (vasculature-associated niche). Lastly, stem cells turn over at an unexpectedly frequent and stochastic manner under  a  steady-state  situation  that  continuously  produces sperm  (Nakagawa  et al.,  Dev.  Cell  200   Yoshida  et al., Science  200   Nakagawa et al.,  Science  2010   Klein  et al., Cell Stem Cell 2010). These observations have thrown doubt on  the  prevailing  stem  cell  model,  the  A s   model , 
postulating that  stem cell function is restricted to the singly isolated spermatogonia (As cells).

I. Background: Testicular architecture andsperm stem cell theories

In  mammalian  testis,  a  huge  number  of  sperm  are continuously  produced  over  the  reproduction  period  (1-2 years  in  mice),  which  relies  on  stem  cell  activity.  For decades, the cellular identity and behavior of spermatogenic stem cells has been argued as a fundamental question in the field of spermatogenesis.
The process of spermatogenesis takes place in seminiferous tubules  of  the  testis  (Figure  1).  In  mice,  mitotic  stages  of spermatogenic cells, called  spermatogonia , are divided into undifferentiated   and  differentiating   populations. 0Undifferentiated  spermatogonia   are  found  as  singly isolated cells (As), or syncytia consisting mainly of 2 (Apr) or more  (A al)  cells.  The  formation  of  syncytia  is  due  to incomplete  cell  division,  a  germline-specific  cell  division process  where  cytokinesis  does  not  complete  and  the cytoplasmic  connection  between  daughter  cells  persists  via intercellular bridges.

Figure  1.  Spermatogenesis  in  seminiferous  tubules.  Spermatogenesis progresses  from  the  basal-  to  adluminal  compartment.  Stem  cells  are thought  to  reside  primarily  within  undifferentiated  spermatogonia (brown)  which  are  distributed  sparsely  on  the  basement  membrane. Modified from  ara et al., Cell Stem Cell (201 ). The prevailing stem cell model, called the  As model ,  was proposed  in  1 1,  suggesting  that  stem  cell  activity  is restricted  to  the  population  of  A s  spermatogonia,  while interconnected Apr and Aal syncytia are irreversibly committed to  differentiation  and  no  longer  contribute  to  the  stem  cell pool ( uckins, 1 1  Oakberg, 1 1).
On the other hand, over the last decade, studies from some groups,  including  ours,  have  shown  that  the  population  of undifferentiated spermatogonia can be divided on the basis of their  heterogeneous  gene  expression.  In  undisturbed  steadystate  spermatogenesis,  the  GFR 1   subpopulation  (mainly As, Apr, and fewer Aal) is thought to reside on the top of the hierarchy  As  well  as  maintaining  their  own  population, GFR 1   cells  also  give  rise  to  the  second,  Ngn , subpopulation of undifferentiated spermatogonia (comprised of  more Aal  and  fewer As  and Apr).  Ngn   cells,  although retaining an ability to revert into GFRα1  cells that becomes apparent  during  regeneration  after  tissue  insult,  mostly differentiate  into  differentiating  spermatogonia  and  then meiotic spermatocytes and haploid spermatids in undisturbed situations.
Seminiferous  tubules  represent  a  typical  open   stem  cell niche.  This  makes  a  stark  contrast  to  the  well-investigated closed  niche-supported tissues, such as Drosophila gonads or  mammalian  small  intestine.  The  closed   niche  is  a definitive, specified region of a tissue which typically tethers the  stem  cells,  while  cells  that  exit  this  niche  region  will differentiate. In contrast, seminiferous tubules do not harbor such  a  defined  structure,  but  the  stem  cells  (GFR 1  spermatogonia)  are  scattered  in  the  basal  compartment  with some, but significant, preference to the vasculature between the  tubules.  It  remains  an  important  unsolved  question  how Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

stem  cell  fate  is  regulated  (whether  differentiated  or remaining undifferentiated) in an open niche system. In  201 ,  we  questioned  the  cellular  identity  and  the dynamics  of  the  stem  cells,  as  well  as  the  mechanisms regulating stem cell fate in the recently proposed open niche model, as described below. would  challenge  the  As  model   that  was  proposed  over  0 years ago.

II. Cellular identity and the dynamics ofspermatogenic stem cells

The  aforementioned  studies  illustrate  that  the  dynamics  of GFR 1   spermatogonia  are  an  essential  problem  for  a  full understanding  of  the  identity  and  behavior  of  mouse  sperm stem  cells.  To  address  this,  we  performed  single-cell-level analyses  of  in vivo  dynamics  of  GFR 1   spermatogonia, taking advantage of pulse-labeling and live-imaging studies, combined  with  biophysical  modeling,  in  collaboration  with Dr. Ben Simons of the University of Cambridge, UK.. GFR 1   spermatogonia  lie  scattered  unevenly  on  the basement  membrane  of  seminiferous  tubules  (Figure  ),  but their  local  density  over  a  prolonged  tubule  length  is remarkably  constant  in  adult  mice.  By  pulse-labeling  the GFRα1   spermatogonia  with  GFP,  we  obtained  clonal  fate data of GFRα1  spermatogonia from several days to over a year,  and  showed  that,  although  the  entirety  of  GFR 1  spermatogonia maintained their own population while giving rise  to  differentiating  progeny,  the  individual  GFR 1  spermatogonia  followed  highly  variable  and  intricate  fate behaviors.  These  findings  suggest  that  the  maintenance  of GFRα1+ spermatogonia  is  achieved  by  population asymmetry ,  in  which  balanced  stem  cell  maintenance  and production  of  differentiating  progeny  are  achieved  at  a population-level. We  next  investigated  the  behavior  of  GFR 1  spermatogonia,  by  means  of  in vivo  live-imaging.  From  the numerous  -day-long observations that altogether effectively cover  one  year  of  spermatogonial  behavior,  it  is  indicated that  GFR 1   cells  continually  change  their  morphological states  between  A s,  A pr  and  A al  spermatogonia  through  a combination  of  incomplete  cell  division  (leading  to syncytial  extension)   and  syncytial  fragmentation  (through breakage  of  intercellular  bridges) ,  while  giving  rise  to differentiating  progeny  from  all  of  these  morphological states. The  live-imaging  measurements  indicate  that  the  rates  of incomplete division  and  syncytial fragmentation  may be constant  regardless  of  whether  they  are  syncytia  or As.  We were  then  motivated  to  try  to  capture  these  cells  using  a biophysical  modeling  scheme,  which  essentially  depends only  on  the  rates  of  incomplete  division   and  syncytial fragmentation .  Intriguingly,  the  model  accurately  predicted the  wide  range  of  intricate  clonal  fate  behaviors  in  steadystate over wide time scales from several days to over a year, and  the  recovery  from  tissue  insult  observed  in  laboratory experiments.
The  results  together  indicate  that  the  entirety  of  GFRα1  spermatogonia  (including  both As  and  syncytia)  comprise  a single  equipotent  stem  cell  pool  and  cells  continually interconvert  between As  and  syncytial  states,  while  giving rise  to  Ngn   progeny  (Figure  2).  This  novel  paradigm Figure  2.  A  proposed  stem  cell  dynamics.  GFR 1   spermatogonia comprise  a  single  stem  cell  pool,  in  which  cells  continually  and reversibly  interconvert  between  states  of As, Apr  and Aal  spermatogonia through  incomplete  cell  division  (blue  arrows)  and  syncytial fragmentation  (red  arrows),  while  giving  rise  to  Ngn   cells.  Thus, differentiation-destined  cells  follow  a  series  of  transitions (GFRα1 →Ngn →Kit  downward black arrows) that accompany the extension of syncytial length (rightward black arrows). Ngn  and, to a lesser  extent,  Kit   cells  retain  the  capacity  to  revert  back  into  the GFR 1   compartment  in  a  context-dependent  fashion,  such  as  after tissue  insult  or  during  regeneration  from  damage  etc.  (broken  arrows). Reproduced from  ara et al., Cell Stem Cell (201 ).

II. Undifferentiated spermatogonia show
heterogeneous differentiation competence in
response to retinoic acid.

As  described  above,  seminiferous  tubules  represent  a typical  open  niche  of  stem  cells.  In  particular,  GFRα1 , Ngn ,  and  Kit   spermatogonial  subpopulations  are intermingled  in  the  basal  compartment  (Figure  ).  In addition,  these  are  likely  to  be  ubiquitously  bathed  with retinoic  acid  (RA),  a  strong  differentiation-inducer,  that occurs  periodically  along  with  the  cycle  of  seminiferous epithelium ( .  days per a cycle). So, a fundamental question is  raised   Out  of  the  entire  population  of  undifferentiated spermatogonia  with  stem  cell  potential  on  the  basis  of  the activity  of  post-transplantation  colony  formation,  why  do Ngn  cells preferentially differentiate and why do GFR 1  cells maintain their own population (self-renewal)Figure  .  Localization  of  GFR 1 ,  Ngn ,  and  KIT   spermatogonia.
Representative  images  of  triple  immuno-stained  whole-mount seminiferous tubules of an Ngn3-EGFP mouse. Scale bars   50  m. By  pulse-labeling  the  Ngn   and  GFR 1   populations  by means of the tamoxifen-inducible cre-loxP system, we tested the response of these cells to RA. The results first indicated 1National nstitute or asic iologyBthat  Ngn   cells  rapidly  and  efficiently  differentiated  into Kit  cells in response to RA. In contrast, GFRα1+ cells did not  show  effective  differentiation  induced  by  RA,  but  they generated  the  Ngn   cells  in  an  RA-independent  manner. Thus,  GFR 1   and  Ngn   cells  show  distinctive differentiation competence in response to RA.  We  then  questioned  the  molecular  mechanism  that  caused such  differential  responses  to  RA.  To  this  end,  the transcriptomes  of  GFR 1   and  Ngn   fractions  were compared by gene expression microarray analysis. Among a number  of  genes  involved  in  RA  signal  reception, particularly, retinoic acid receptor gamma (Rarγ), expression was  preferentially  observed  in  Ngn   cells  over  GFR 1  cells. This was also true for the RAR  protein (Figure  ). On the  other  hand,  most  of  the  other  RA-related  genes  showed similar levels of expression. Figure  5.  A  scheme  for  the  fate  allocation  of  undifferentiated spermatogonia  along  the  cycle  of  seminiferous  tubules.  See  text  for details.  Publication List

〔Original paper〕

ara,  K.,  Nakagawa,  T.,  Enomoto,  .,  Suzuki,  M.,  Yamamoto,  M.,  Simons, B.D., and Yoshida, S. (201 ). Mouse spermatogenic stem cells continually  interconvert  between  equipotent  singly  isolated  and syncytial states. Cell Stem Cell 14,  5 - 2.

Figure  .  Expression  of  RAR   in  Ngn ,  but  not  in  GFR 1 , spermatogonia. Representative images of double immuno-stained wholemount  seminiferous  tubules  of  an  Ngn3-EGFP mouse.  Scale  bars    50 m. Modified from Ikami et al., Development 142, 1-11 (2015).

We then addressed the roles of RAR  by ectopic expression in  GFR 1   cells  that  normally  do  not  express  this  gene. Intriguingly,  enforced  expression  of  RAR   provided  the GFR 1  cells with differentiation competence in response to RA.  This  indicates  the  key  role  of  RAR   in  the heterogeneous differentiation competence and the subsequent fate selection of undifferentiated spermatogonia. From  these  results,  heterogeneous  differentiation competence, combined with the periodically but ubiquitously distributed RA, appears to allocate the undifferentiated cells’ fates  for  the  continuity  of  spermatogenesis  (Figure  5).  We propose  that  heterogeneous  differentiation  competence combined  with  spatially  ubiquitous  distribution  of differentiation-inducing  signals  would  be  paradigmatic  for stem cell regulation in an opened niche based model.

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

Atsumi, Y., Yorinaga, E., Ono,  ., Murayama, I., Nakane,  IY., kegami, K., Ota,  W.,  Arai,  N.,  Tega,  A.,  Iigo,  M.,  Darras,  .M.,  Tsutsui,  K., ayashi, Y., Yoshida, S., and Yoshimura, T. Low temperature-induced circulating  triiodothyronine  accelerates  seasonal  testicular  regression. Endocrinology 201  Nov 1 .

AB

A

A

Associate Professor

ANA A

inoru

NIBB Research Fellow:
Postdoctoral Fellow:

YAMAMOTO, Yasuhiro*
YAMAMOTO, Yasuhiro
FUJIMORI, Chika
SOKENDAI Graduate Student: NISHIMURA, Toshiya
SAKAE, Yuta
KIKUCHI, Mariko
OTAKE, Norihito
Technical Assistant:
KINOSHITA, Chie
WATAKABE, Ikuko
Secretary:
TAKEDA, Manami
SHIRAISHI, Naomi

Laboratory scope
Reproduction is a universal and fundamental system fororganisms to produce generations. To accomplish thispurpose efficiently, organisms develop sexual reproduction.
During the embryo and larval terms, organisms develop
many cell-lineages that have special and essential roles ineach different process of reproduction. These lineages areconserved among vertebrates.
ertebrates,  however,  exhibit  a  variety  of  reproductive systems. This variety is allowed by the different employment and  different  emergence  of  the  cell  lineages  during embryogenesis. Therefore, it is important to address the roles of  each  cell  lineage  for  understanding  the  fundamental mechanism  underlying  reproduction  and  a  variety  of reproductive  systems.  Currently,  our  lab  focuses  on  the mechanisms  of  sex  differentiation.  The  many  modes  of  sex differentiation  are  a  main  component  that  contributes  to variety, and we are addressing the role of each cell lineage in the context of sex differentiation.
We  use  medaka  fish  (Oryzias latipes)  and  have  been generating  transgenic  medaka  enabling  us  to  analyze  how different  cell  lineages  are  involved  in  the  process  of  gonad formation  and  sex  differentiation  in vivo. Additionally,  in order  to  identify  the  genes  essential  for  reproduction,  we carried out a mutational screening of medaka with defective phenotypes  and  disrupted  several  candidate  genes.  With these  two  unique  analytical  methods  (visualizing  cells,  and mutants),  we  are  attempting  to  unveil  both  the  fundamental mechanisms  and  the  specific  mechanisms  that  produce  a variety of reproductive systems. Through  these  analyses,  we  have  been  revealing  the importance  of  germ  cells  not  only  in  sex  differentiation  but also  in  reproduction.  Germ  cells  have  been  generally considered  to  be  a  permissive  cell  type  that  are  completely regulated  by  the  surrounding  somatic  cells.  But  we  have found  that  the  germ  cells  are  not  specialized  for gametogenesis  but  are  more  self-determinant  cells  in reproduction.  I. Developmental origin of primordial germcellsDevelopment of germ cells essentially rely on the presence of a cytoplasmic structure, called germplasm or alternatively nuage. The lack of germplasm results in defective or loss of the  germ  cells.  Two  separate  systems  controlling  the development of germ cells are known for vertebrates. Some vertebrate  species  possess  a  maternal  origin  of  germplasm and  the  germplasm  localized  in  a  specific  cell  type  is essential  for  germ  cell  development.  In  these  species, therefore, cells that develop into germ cells can be identified by  the  presence  of  germplasm.  On  the  other  hand  in  the second  method,  like  in  mammals,  germ  cells  are  induced  at the gastrulation stage. The germplasem-equivalent structures in these cells are also formed at a later stage. Our  previous  studies  indicated  that,  in  medaka,  germ  cells are  established  as  primordial  germ  cells  (PGCs)  during gastrulation.  owever,  it  is  not  known  how  a  small population  of  blastodermal  cells  are  specified  as  cells  to produce PGCs, or if germ cells are induced like those seen in mammals. We  have  cloned  a  cDNA  encoding  Bucky  ball,  a component  of  germplasm.  Since  we  have  found  bucky ball expressed in fertilized eggs, CFP-fused Bucky balls allow us to keep track of cells that could generate PGCs during early embryogenesis by confocal microscopy. The  RNA  encoding  cfp-fused  bucky ball  was  injected  into fertilized eggs. We found that the protein is readily translated and  was  observed  as  early  as  the  onset  of  the  first  division. The  CFP-fused  Bucky  ball  protein  is  localized  as  several particles  uniformly  in  the  blastodisc.  ery  interestingly,  the particles  seem  to  be  anchored  at  yolk  layers  until  the  late morula  stage  and  seem  independent  of  cell  division  in  the blastodisc. The inhibition of the cleavage plane by nocodazol did  not  change  any  morphology  or  distribution  of  the particles, supporting the independence of the cleavage plane.
Nanos  protein is known to be expressed in the established PGCs.  Co-injection  of  dsred-nanos ’UTR  RNA  and  cfpfused  buckey ball RNA  indicates  that  DsRed-derived fluorescence  begins  to  be  detected  in  the  cells  that  retain large  CFP-fused  Bucky  ball  particles.  This  clearly  indicates that  the  cells  retaining  the      Bucky  ball  particles  are  the presumptive  germ  cells  that  have  the  potential  to  be established as primordial germ cells.
Interestingly, at one or two stages, numerous Bucky ball
granules are present with a range of different sizes, but, asFigure 1. The localization of CFP-fused Bucky ball protein (blue) reveals presumptive primordial germ cells after fertilization. Green dots indicate histone  signals in the nuclei. The cells expressing nanos3 are established as  primordial  germ  cells  at  the  late  morula  stage,  which  are  clearly recognized  as  the  cells  possessing  large  blue  Bucky  ball  particles.  Red cells  are  nanos3-expressing  cells. The  pictures  are  time-lapse  shots  of  a live  imaging  movie.  Left     cell  stage,  Middle     cell  stage  and  Right  late morula stage.

Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

National nstitute or asic iology

Bdevelopment proceeds, small particles are getting eliminated.
As a result, at the later morula stage, approximately 20-30
cells possess granules larger than 25 m3 and become PGCs.
As the PGCs are established, the translation of nanos3 andvasa genes is activated. These translated proteins begin to be
detected on the granules of Bucky balls.

II. Sexually different characteristics ofprimordial germ cells

Since  201 ,  we  have  been  characterizing  female  and  male primordial  germ  cells  and  have  identified  genes  showing sexually  different  expression  in  the  PGCs.  Last  year  we reported  that  the  sexually  different  expression  of  one  of  the identified  genes,  sdgc,  is  due  to  the  presence  of  Y chromosomes  but  is  not  dependent  on  the  medaka  sex determination gene, DMY Dmrt1bY. This year, we continued to analyze the function of this gene and found that the gene regulates proliferation of primordial germ  cells.  Primordial  germ  cells  isolated  from  male embryos proliferate more than those from female embryos in the  culture  dish.  Loss  of  gene  function  by  injection  of morpholino  results  in  a  reduced  proliferation  rate  in  males while  injection  of  sdgc  RNA  shows  increasing  activity  of proliferation  in vitro.  These  results  suggest  that  the expression  of  sdgc  confers  primordial  germ  cells  with  the potential to regulate proliferation.  Furthermore, we have mapped sdgc on the medaka genome and  found  that  sdgc  is  located  very  close  to  the  DMYDmrt1bY locus. The DMY Dmrt1bY locus is known to be the only  region  that  is  specific  to  Y  chromosomes  and  where recombination  is  repressed.  Consistent  with  this,  we  could find  the  sex-specific  SNPs  in  the  promoter  and  the  intron regions of sdgc. Sdgc might represent the evolutional way of the  Y  chromosome  differentiating  into  a  more  specialized chromosome. Collectively, a series of analyses of sdgc indicate that cells have  the  ability  to  express  sexually  different  characteristics autonomously, which is independent of the expression of sex determination gene. Figure  2.  Sexually  different  behavior  of  primordial  germ  cells.  Upper schema shows the way of primordial germ cell isolation and culture with somatic  cells. A   Germ  cell  behavior  (incorporation  of  EdU)  is  not affected by the sex of somatic cells but is determined cell-autonomously. B   The  germ  cell  behavior  is  not  dependent  on  the  expression  of  sex determination gene, DMY Dmrt1bY. C  The germ cell behavior (activity of proliferation) is reduced by knockdown of sdgc, which is recovered by overexpression of sdgc.

 III. Sexually different development ofsteroidenic cells

Estrogen  expression  is  important  for  feminization  of  the gonad, especially forming the ovarian cavity. Aromatase is a critical  enzyme  for  producing  estrogen  from  its  precursor steroid,  testosterone,  and  is  encoded  by  the  cyp19a1  gene. Unlike  mammals,  during  development  the  first  emerging cells  that  express  cyp19a1  are  the  precursors  of  theca  cells (in mammals, granulosa cells first express cyp19a1). We have examined the development of steroidenic cells that produce  testosterone  and  found  that  ftz-f1  cells  may  be  the precursor  of  testosterone-producing  cells.  Currently,  two types  of  ftz-f1  cells  are  observed  in  the  developing  gonads and  precise  lineage  analysis  is  under  investigation  using time-lapse movies with the primary culture system.

Publication List

〔Original papers〕

T., Herpin, A., Kimura, T., Hara, I., Kawasaki, T., Nakamura, NS.,ishimura,
Yamamoto, Y., Saito, T.L., Yoshimura, J., Morishita, S., Tsukahara,T., Kobayashi, S., Naruse, K., Shigenobu, S., Sakai, N., Schartl, M., andTanaka, M.  (201 ).  Analysis of a novel gene, Sdgc, reveals sex
chromosome-dependent differences of medaka germ cells prior togonad formation. Development 141, 3363-3369.
Okuyama,  T.,  Yokoi,  S.,  Abe,  .,  Isoe,  Y.,  Suehiro,  Y.,  Imada,  ., Tanaka, M., Kawasaki, T., Yuba, S., Taniguchi, Y., Kamei, Y., Okubo, K.,  Shimada,  A.,  Naruse,  K.,  Takeda,  .,  Oka,  Y.,  Kubo,  T.,  and Takeuchi,  . (201 ).  A  neural  mechanism  underlying  mating preferences for familiar individuals in medaka fish. Science 343,  1- .

 〔Review articles〕

anaka,  M.  (201 ).  ertebrate  female  germline the  acquisition  of  Tfemaleness. WIREs Dev. Biol. doi 10.1002 wdev.1 1 ishimura, T.,  and Tanaka,  M.  (201 ).  Gonadal  development  in  fish.  NSex. Dev. 8, 252-21.
anaka, M. (201 ). Molecular and cellular bases of sexual flexibility in  Tvertebrates. In  New  Principles  in  Developmental  Processes  (eds  Kondoh,  ., and Kuroiwa, A.)’ Springer Japan, pp. 2 5-2. National nstitute or asic iologyAfunctions of the mature brain, including body-fluid
regulation, behavior control, learning, and memory.

B

I. Mechanisms for neural circuit formation

ProfessorNA

asa aru

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

SOKENDAI Graduate Student:

Technical Assistant:

Secretary:

Associate Professor

N AN

a a umiSAKUTA, Hiraki
HIYAMA, Takeshi
TAKEUCHI, Yasushi
KUBOYAMA, Kazuya*
NOMURA, Kengo
FUJIKAWA, Akihiro
SUZUKI, Ryoko
MATSUMOTO, Masahito
KUBOYAMA, Kazuya
LIN, Chia-Hao
MATSUDA, Takashi
YU, Yang
TANGA, Naomi
HIGASHI, Satoru
MIURA, Seiko
NAKANISHI, Norie
DOKYO, Yumi
WADA, Kotoe
KONISHI, Mie
ISOSHIMA, Yoshiko
HASHIMOTO, Terumi
KODAMA, Akiko

We have been studying the molecular and cellularmechanisms underlying the development of the vertebrate
central nervous system (CNS), mainly using mice. This
research covers many developmental events including thepatterning of the retina, neuronal terminal differentiation,axonal navigation, branching and targeting, synapse
formation, refinement and plasticity. The scope of ourinterests also encompasses the mechanisms for various

Topographic maps are fundamental features of neuralnetworks in the nervous system. We have long studied themolecular mechanisms for regional specification in thedeveloping retina as the basis of the topographic
retinotectal projection. We are now focusing our attention
on the molecular mechanisms underlying axonal
navigation, branching, and arborization for synapse
formation, along with elimination of mistargeted axons andbranches. Among the region-specific molecules in thedeveloping retina, we have already found several
molecules that induce abnormal branching or arborization
when their expression was experimentally manipulated invivo.
One is adenomatous polyposis coli 2 (APC2), which ispreferentially expressed in the nervous system from early
developmental stages through to adulthood. The
knockdown of Apc2 in chick retinas reduced the stabilityof microtubules in retinal axons, and yielded abnormal
behaviors of growth cones including a reduced responseto ephrin-A2 and misprojection in the tectum without
making clear target zones. In Apc2-deficient mice, robust
defects in neuronal lamination were observed in thecortex, hippocampus, cerebellum, and olfactory bulb.
These laminary abnormalities are a result of dysregulated
neuronal migration by a cell-autonomous mechanism.
APC2 is distributed along actin fibers as well as
microtubules in neurons. Our investigation suggests that
APC2 is involved in the signaling pathway frommembrane receptors for extracellular guidance factors to
the intracellular migration machinery. We are now
investigating a mutation in human APC2 gene identified inpatients with intellectual
disabilities.

F i g u r e   1 .   E f f e c t o f S p i g 1 knockdown (KD) in the developing
chick retina. A, Projection patternof dorsal RGC axons by DiI labelingin control (Con) and Spig1-KD
embryos. Higher magnification of
the boxed area is shown in the lower
part of each panel. A, anterior; P,
posterior; D, dorsal; V, ventral. Scalebars, 1 mm.  B, Effect of aneutralizing antibody for BDNF on
the elongation and branching of
RGC axons. After theelectroporation of Spig1-shRNA or
control retroviral construct at HHstage 9-10, retinal cells weredissociated from the dorsal one-thirdof the retina at E8. The branchnumber and axon length of RGC
axons were quantified. Data areshown as the mean ± SE of four
independent experiments. *p < 0.05,* * p < 0 . 0 0 1 ( A N O VA w i t h
Scheffé’s post hoc tests). Scale bars,
10 mm.

Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

5National nstitute or asic iology

Another molecule is SPARC-related protein containing
immunoglobulin domains 1 (SPIG1, also known as
Follistatin-like protein 4), which is a secretory protein
expressed in a dorsal-specific manner in the developing
chick retina. The knockdown of Spig1 in the retinal
ganglion cells (RGCs) of developing chick embryos
induced robust ectopic branching of dorsal RGC axonsand failed to form a tight terminal zone at the proper
position on the tectum (Figure 1A). The knockdown of
Spig1 in RGCs also led to enhanced axon branching invitro. However, this was canceled by the addition of aneutralizing antibody against brain-derived neurotrophic
factor (BDNF) to the culture medium (Figure 1B). SPIG1
and BDNF were colocalized in vesicle-like structures incells. SPIG1 bound with the proform of BDNF
(proBDNF) but very weakly with mature BDNF in vitro.
The expression and secretion of mature BDNF weresignificantly decreased when SPIG1 was exogenously
expressed with BDNF in HEK293T or PC12 cells. The
amount of mature BDNF proteins as well as the tyrosine
phosphorylation level of the BDNF receptor,
tropomyosin-related kinase B (TrkB), in the hippocampus
were significantly higher in Spig1-knockout mice than inwild-type mice (Figures 2A and B). Furthermore, thespine density of CA1 pyramidal neurons was consistently
increased (Figure 2C). Together, these results suggest that
SPIG1 negatively regulated BDNF maturation by binding
to proBDNF, thereby suppressing axonal branching andspine formation.

the opposing activities of protein tyrosine kinases (PTKs)
and protein tyrosine phosphatases (PTPs). The
physiological functions and regulatory mechanisms ofreceptor-like PTPs (RPTPs) are not fully elucidated. We
have been making efforts to reveal the functional roles of
the R3 and R5 subfamilies of RPTPs.
2-1 R3 RPTP subfamily
The human genome contains 58 and 20 genes for RPTKs
and RPTPs, respectively. In some studies including ours,
RPTPs have been shown to be involved in the regulationof RPTKs through dephosphorylation as substrates.
However, our understanding about the roles of individual
RPTPs in the regulation of RPTKs is still limited.
The R3 RPTP subfamily, which is comprised of Ptprb,
Ptprh, Ptprj, and Ptpro, reportedly plays pivotal roles inthe development of several tissues including the vascularand nervous systems. We performed a large scaleexamination of the enzyme-substrate interaction between
the R3 RPTP members and representative RPTKs
covering RPTK subfamilies. We revealed that multiple
RPTKs are recognized as substrates by the R3 RPTPs. We
also demonstrated that the R3 members showed
differences in substrate specificity toward individual
RPTKs. On the basis of the enzyme-substrate
relationships identified, we are now investigating thephysiological roles of the R3 RPTP subfamily by using
their knockout mice.
2-2 R5 RPTP subfamilyProtein-tyrosine phosphatase receptor type Z (Ptprz) is
predominantly expressed in glial and neuronal cells in thecentral nervous system (CNS). We are now focusing ourefforts on determining the roles of Ptprz signaling in theregulation of hippocampal synaptic plasticity, dopaminetransporter internalization, oligodendrocyte
differentiation, etc.

III. Brain systems for body-fluid homeostasis

Figure 2.  Functional interactions between SPIG1 and BDNF in
hippocampal cells. A, Comparison of BDNF expression in the
hippocampus of wild type (WT) and Spig1-knockout (KO) mice atpostnatal day 14 (P14). The amounts of BDNF are presented bydensitometric units normalized to the value for the WT. Data are shownas the mean ± SE (n = 14 for each). *p < 0.05 (Student’s t test). B,
Increased tyrosine phosphorylation of TrkB in the hippocampus of Spig1KO mice. The tyrosine phosphorylation of TrkB proteins
immunoprecipitated with an anti-Trk antibody from the hippocampus at
P14 was analyzed using 4G10 and anti-TrkB. Densitometric data arepresented as a percentage of the WT control (bottom). Data are mean ±SE (WT, n = 6; Spig1-KO, n = 8). *p < 0.05 (Student’s t test). C, Golgi
staining of the hippocampal tissues from WT and Spig1-KO mice at P10.
We analyzed the dendritic segments of CA1 pyramidal neurons. Spinedensity and dendritic length are shown as the mean ± SE (n = 6 for each).
*p < 0.01 (Student’s t test). Scale bars, 10 mm.

II. Physiological roles of receptor-like protein
tyrosine phosphatases

Protein tyrosine phosphorylation plays crucial roles invarious biological events such as cellular proliferation,differentiation, survival, migration, and metabolism.
Cellular tyrosine phosphorylation levels are governed by

Sodium (Na) is a major electrolyte of extracellular fluids
and the main determinant of osmolality. Na homeostasis
is essential to life and Na+ concentrations in plasma andcerebrospinal fluid (CSF) are continuously monitored tomaintain a physiological level of Na+ in body fluids. We
have previously shown that Na x, which structurally
resembles voltage-gated sodium channels (Nav1.1–1.9), isa concentration-sensitive Na channel.

In the brain, Nax channels are preferentially expressed ina s troc yte s a nd e pe ndyma l c e lls in the s e ns or y
circumventricular organs, such as the subfornical organ(SFO) and organum vasculosum of the lamina terminalis(OVLT), where Na x-positive glial cells are involved insensing an increase in [Na+] in body fluids. Nax-deficient
mice do not stop ingesting salt even when dehydrated, whilewild-type mice avoid salt. This behavioral defect of Naxdeficient mice is recovered by a site-directed transfer of the
Na x gene with an adenoviral vector into the SFO. The
threshold value of Nax for [Na+]o was ~150 mM in vitro. In
the SFO, however, endothelin-3 (ET-3) shifts the [Na+]odependency of Nax activation to the lower-concentration sidein a dose-dependent manner. This shift enables Nax to gateeven when [Na+]o is in the physiological range (135–145mM). Nax thus functions as the brain’s Na+-level sensor forthe homeostatic control of [Na+] in body fluids.
We can summarize the cellular mechanisms for [Na +]sensing and [Na+]-dependent regulation of neural activities in
the SFO as presented in Figure 3. The sensory CVOs,
including the SFO, are characterized by the extensive
networks of fenestrated capillaries which allow ingredientsof plasma to release to the intercellular space. Theirventricular side is partitioned by an ependymal cell layer
facing the third ventricle. Nax channels populate perineuralprocesses of astrocytes and ependymal cells in the SFO.
Even under hydrated (normal) conditions, ET-3 level
expressed in the SFO can modulate the [Na+]o dependency of
Nax and make Nax sensitive to an increase in [Na+]o in thephysiological range. When animals are dehydrated, [Na+] inplasma and CSF significantly increases above the usual level.
Under such conditions, the [Na+]o exceeds the threshold ofNax, Nax channels open, and the [Na+]i in these Nax-bearing
cells is increased. This leads to activation of Na+/K+-ATPasein these cells. Activated Na +/K +-ATPase consumes ATP
higher than the usual level to pump out Na+. To fuel Na+/
K+-ATPase with ATP, the glial cells enhance glucose uptake
to stimulate anaerobic glycolysis. Lactate, the end product of
the anaerobic glycolysis, is released from the glial cells andsupplied to neurons, including GABAergic neurons, throughthe processes enveloping them. Lactate stimulates theactivity of the GABAergic neurons through production ofATP, which presumably leads to the regulation of hypothetic
neurons involved in the control of salt-intake behavior. In
dehydrated Na x -deficient mice, the [Na + ]-dependent
stimulation of glycolysis is impaired and the activity of the
GABAergic neurons is not promoted.
Nax is also expressed in non-myelinating Schwann cells of
the adult peripheral nervous system, but thepathophysiological role remained unclear. Recently, we
found that functional recovery of the hind paw responses
from the sciatic nerve transection was delayed in Na xdeficient mice. Our studies revealed that Nax is involved in
the regeneration process of injured peripheral nerves byenhancing lactate release from non-myelinating Schwann
cells, where Na x was activated by ET-1 through ET BR
signaling. This finding may bring new strategies to promote
peripheral nerve regeneration.

Figure  . Overview of the  Na -sensing mechanism and Nax-dependent regulation of neuronal activity in the SFO. Publication List

〔Original papers〕

uzuki, R., Matsumoto, M., Fujikawa, A., Kato, A., Kuboyama, K., SShintani,T., Sakuta, H., and Noda, M. (2014). SPIG1 negatively

S., Katano, T., Hiyama, T.Y., Tu, N.H., Yoshii, S., Noda, M., Uandnezaki,
Ito, S. (2014). Involvement of Na sodium channel in peripheral
regulates BDNF maturation. J. Neurosci. 34, 3443-3453.
x

nerve regeneration via lactate signaling. Eur. J. Neurosci. 39, 720-729.

〔Review article〕

oda, M., and Hiyama, T.Y. (2014). The Na channel: What it is and Nwhatit does. The Neuroscientist DOI: 10.1177/1073858414541009.
x

National nstitute or asic iology

B A

Professor

A A

R

etsuo

B

†

Associate Professor

A A A

A iya

Assistant Professor:

KOMINE, Yuriko
SADAKANE, Osamu
NIBB Research Fellow:
OHTSUKA, Masanari*Technical Staff:
OHSAWA, Sonoko
Postdoctoral Fellow:
TAKAJI, Masafumi
OHTSUKA, Masanari
HATA, Katsusuke
NAKAGAMI, Yuki
SOKENDAI Graduate Student: SHUKLA, RammohanTechnical Assistant:
NAKAMURA, Tohru
MORITA, Junko
IMAI, Akiko
KON, Yayoi

II. Simultaneous visualization of extrinsic andintrinsic axon collaterals in Golgi-like detailfor mouse corticothalamic andcorticocortical cells: a double viral infection
methodWe  reported  a  novel  tracing  technique  to  stain  projection neurons  in  Golgi-like  detail  by  double  viral  infection.  We used retrograde lentiviral vectors and adeno-associated viral vectors  (AA )  to  drive  the  TET-ON TET-OFF  system   in neurons connecting two regions (Figure 2).

This  year  we  have  reported  the  following  results.  (1) Distribution  of  serotonin  receptor  subtypes  and  serotonergic terminations  in  the  marmoset  brain,  (2)  Simultaneous visualization of extrinsic and intrinsic axon collaterals in the mouse  brain,  ( )  Characterization  of  claustral  neurons,  ( ) 
Distinct motor impairments of dopamine D1 and D2 receptor knockout mice.

I. Distribution of serotonin receptor subtypes
and serotonergic terminations in the
marmoset brainWe  examined  the  mRNA  expression  patterns  of  all  the  1  members of the serotonin receptor (5 TR) family, by in situ hybridization  (IS )  and  the  distribution  of  serotonergic terminations  by  serotonin  transporter  (SERT)  protein immunohistochemical analysis. Ten of the 1  5 TRs showed significant  mRNA  expression  in  the  marmoset  brain.  Our study  showed  several  new  features  of  the  organization  of serotonergic  systems  in  the  marmoset  brain.  (1)  The thalamus  expressed  only  a  limited  number  of  receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there is layer-selective and  area-selective  mRNA  expression  of  5 TRs  (Figure  1). ( )  ighly  localized  mRNA  expressions  of  5 T1F  and 5 T A were observed. ( ) There was a conspicuous overlap of the mRNA expression of receptor subtypes known to have somatodendritic  localization  of  receptor  proteins  with  dense serotonergic  terminations  in  the  visual  cortex,  the  central lateral  (CL)  nucleus  of  the  thalamus,  the  presubiculum,  and the  medial  mammillary  nucleus  of  the  hypothalamus.  This suggests  a  high  correlation  between  serotonin  availability and  receptor  expression  at  these  locations.  (5)  The  5 TRs show  differences  in  mRNA  expression  patterns  between  the marmoset  and  mouse  cortices  whereas  the  patterns  of  both the  species  were  very  similar  in  the  hippocampus.  We discussed the possible roles of 5 TRs in the marmoset brain revealed  by  the  analysis  of  their  overall  mRNA  expression patterns (Published in Front. Neural Circuits, 1  May 201    doi  10.
fncir.201 .00052).

Figure 1. IS  expression profiles of 5 TRs in the cortex. Area  , area  , primary motor cortex (M1), primary somatosensory cortex (S1), and  5 (MT).  Layers  identified  by  Nissl  staining  (not  shown)  are  indicated  on the  left.  Note  that  all  images  of  a  given  gene  are  grouped  together  and presented  at  the  same  contrast  level.  Scale  bar   100  m.  (Cited  from Shukla et al., Front. Neural Circuits,   52, 201 ). Using  this  method,  we  successfully  labeled  the corticothalamic  (CT)  cells  of  the  mouse  somatosensory barrel  field  (S1BF)  and  motor  cortex  (M1)  in  their  entirety (Figure  ).  We  also  labeled  contra-  and  ipsilaterallyprojecting  corticocortical  (CC)  cells  of  M1  by  targeting contralateral  M1  or  ipsilateral  S1  for  retrograde  infection. The  strength  of  this  method  is  that  we  can  observe  the morphology of specific projection neuron subtypes en masse. We found that the group of CT cells extended their dendrites and  intrinsic  axons  extensively  below  but  not  within  the thalamorecipient layer in both S1BF and M1, suggesting that the  primary  target  of  this  cell  type  is  not  layer  .  We  also found that both ipsi- and contra-lateral targeting CC cells in Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).
†  This laboratory was closed on  1 March, 2015.

M1  commonly  exhibit  widespread  collateral  extensions  to contralateral M1 (layers 1 ), bilateral S1 and S2 (layers 1, 5 
and  ),  perirhinal  cortex  (layers  1,  2 ,  5,  and  ),  striatum, and  claustrum.  These  findings  not  only  strengthened  the previous  findings  of  single  cell  tracings  but  also  extended them  by  enabling  cross-area  comparison  of  CT  cells  or comparison  of  CC  cells  of  two  different  labeling  systems (Published in Watakabe et al., Front. Syst. Neurosci.   110, 201 ).

Figure  2.  Schematic  view  of  the  TET  double  infection  strategy  and  the viral  vector  constructs  used  for  the  experiments.  (A C)  Schematic  view of  our  double  infection  method,  which  utilizes  the  TET  system.  In  this scheme, AA  is injected into the cortex (represented by yellow shading) as the local infection vector, while the NeuRet vector is injected into the subcortical region (represented by blue shading) as the retrograde vector. These vectors contain either tTA or TRE-transgene as depicted in (D). In these panels, three neurons indicated as  a,   b,  and  c  are shown. In panel (A), the neurons infected with the retrograde vector ( b  and  c ) are  indicated  by  blue  nuclei.  The  arrows  represent  the  retrograde transport of the viral particles. In (B), the neurons infected with the local infection vector ( a  and  b ) are indicated by yellow nuclei. Panel (C) shows  the  consequence  of  double  infection.  Since  the  tTA  and  TREtransgene  are  both  present  in  neuron  b,   high-level  expression  of  the transgene  takes  place  and  fills  the  entire  neuron  with  the  transgene product  (represented  by  red  coloring).  No  transgene  expression  occurs when the neurons are infected by only one of the two viral vectors (e.g., neurons  a   and  c ).  (D)  Schematic  representations  of  the  viral constructs we used in this study. CFP, celulean  SynI, human synapsin I 
promoter   SYP,  synaptophysin   tTA2,  TET-Off   tetracyclin transactivator   rtT 1 ,  TET-ON   reverse  tetracyclin  transactivator  tRFP,  turboFP 5   LTR,  long  terminal  repeat   ITR,  inverted  terminal repeat   TRE,  tetracycline  responsive  element   pA,  poly(A)  site   WPRE, woodchuck hepatitis virus post-transcriptional regulatory element (Cited from Watakabe et al., Front Neural Circuits.  110, 201 ).

III. Characterization of claustral neurons bycomparative gene expression profiling anddye-injection analyses

We investigated the identity of the claustrum as a part of the cerebral  cortex,  and  in  particular  of  the  adjacent  insular cortex,  by  connectivity  features  and  patterns  of  gene expression. We mapped the cortical and claustral expression of  several  cortical  genes  in  rodent  and  macaque  monkey brains  (nurr1, latexin, cux2, and netrinG2)  to  further  assess shared  features  between  the  cortex  and  the  claustrum.  In Figure  . The identity of the claustrum as a part of the cerebral cortex, CT cells were efficiently labeled by TET (A)  Schematic  view  of  double  injection.  The  NeuRet  vector  encoding tTA  (MSC tTA)  was  injected  into  the  thalamus  (represented  by  the green  arrow)  as  a  retrograde  vector,  together  with  CTB-Alexa.  The AA   vector  carrying  TRE-tRFP  was  injected  into  S1BF.  We  observed collateral  projections  to  the  thalamus  (Th),  striatum  (St)  and  pons  (Pn). (B) The coronal section at the level of S1BF showing CT cells that were labeled  red  by  expression  of  tRFP.  The  green  signals  are  also  CT  cells retrogradely labeled by CTB-Alexa. The arrow indicates the direction of corticothalamic projections. (C)  Magnified  view  of  the  white  square  in  (B).  The  contrast  of  this confocal image is adjusted so that we can identify each labeled cell body. (D I) Coronal images of the tRFP signals that follow those in (B) were aligned  in  order  from  anterior  to  posterior.  The  arrows  indicate  the directions  of  the  collateral  projections  labeled  by  this  strategy.  (D)  The striatal  collateral  splits  at  the  level  of  (D)  *(K)  and  *(M)   higher magnification  views  in  (K,M) .  In  (E ),  the  thalamic  projection  that proceeded in the internal capsule (ic) innervate reticular thalamic nucleus (Rt),  Pm and PO, while the split collaterals proceed within the cerebral peduncle (cp). (I) Multicolor merged view for ( ), which is just around the thalamic injection site. The green signals indicate the local deposit of CTB-Alexa ,  which  mark  the  injection  center  for  the  retrograde vector. The asterisk shows the position of (J). The Paxinos atlas (Paxinos and Franklin, 200 ) was superimposed to the image in ( ) to make (I), to show  the  identification  of  the  spots  of  concentrated  tRFP  signals.  (J) A 
magnified  view  of  the  injection  site  indicated  by  asterisks  in  (I).  Note that  we  can  examine  the  fine  branches  with  boutons  even  around  the injection  site  (K)  A  magnified  view  of  the  striatal  collaterals  that branched  out  of  the  CT  bundles  of  internal  capsule  (see  D  for  low magnification view). The dotted square  denoted as *(L)  is magnified in (L) to indicate the example of collateral branching (indicated by the red arrow). (M) A magnified view of the striatal collaterals that arborized at the  final  destination  (see  D  for  low  magnification  view).  (N) A  low power  view  of  the  pontine  collaterals.  (O) A  magnified  view  of  the terminal  arborization  of  pontine  collaterals. The  boxed  region  indicated by a red arrow is magnified in the inset, which exhibits a cluster of large boutons. The images used in (C, J O) are maximal projection stacks of confocal sections. Definitions   S1BF,  somatosensory  barrel  field   Th,  thalamus   Pn,  pons  St,  striatum   ic,  internal  capsule   cp,  cerebral  peduncle   Rt,  reticular thalamic  nucleus   Pm,  ventral  posteromedial  nucleus   PO,  posterior thalamic  nuclear  group  (Cited  from  Watakabe  et  al.,  Front.  Neural Circuits 8 110, 201 ). mice,  these  genes  were  densely  expressed  in  the  claustrum, but  very  sparsely  in  the  cortex  and  not  present  in  the striatum. To  test  whether  the  cortical  vs.  claustral  cell  types National nstitute or asic iologycan  be  distinguished  by  co-expression  of  these  genes,  we performed  a  panel  of  double  IS   in  mouse  and  macaque brains.  NetrinG2  and  nurr1  genes  were  co-expressed  across the  entire  cortex  and  claustrum,  but  cux2  and  nurr1  were co-expressed  only  in  the  insular  cortex  and  claustrum. Latexin  was  expressed,  in  the  macaque,  only  in  the claustrum. The  nurr1   claustral  neurons  expressed  GluT1, a  marker  for  cortical  glutamatergic  cells  and  send  cortical projections.  Taken  together,  our  data  suggest  a  partial commonality  between  claustral  neurons  and  a  subtype  of cortical  neurons  in  the  monkey  brain.  Moreover,  in  the embryonic  (E110)  macaque  brain,  many  nurr1   neurons were scattered in the white matter between the claustrum and the  insular  cortex,  possibly  representing  their  migratory history.  In  a  second  set  of  experiments,  we  injected  Lucifer Yellow  intracellularly  in  mouse  and  rat  slices  to  investigate whether dendrites of insular and claustral neurons can cross the  border  of  the  two  brain  regions.  Dendrites  of  claustral neurons  did  not  invade  the  overlying  insular  territory.  In summary,  the  gene  expression  profile  of  the  claustrum  is similar to that of the neocortex, in both rodent and macaque brains,  but  with  modifications  in  density  of  expression  and cellular  co-localization  of  specific  genes  (Published  in Watakabe, et al., Front Syst Neurosci. , 201 ).

IV. Distinct motor impairments of dopamine
D1 and D2 receptor knockout mice
revealed by three types of motor behavior

We  examined  the  behavioral  difference  among  N10 congenic D1R and D2R KO, and wild type (WT) mice. Both D1R and D2R knock out (KO) mice of the major dopamine receptors  show  significant  motor  impairments.  owever, there  are  some  discrepant  reports,  which  may  be  due  to differences  in  genetic  background  and  experimental procedures. In addition, only a few studies directly compared the  motor  performance  of  D1R  and  D2R  KO  mice.  First,  we  examined  spontaneous  motor  activity  in  the  home cage  environment  for  5  consecutive  days.  Second,  we examined  motor  performance  using  the  rota-rod  task,  a standard  motor  task  in  rodents.  Third,  we  examined  motor ability with the Step-Wheel task in which mice were trained to  run  in  a  motor-driven  turning  wheel  adjusting  their  steps on  foothold  pegs  to  drink  water.  The  results  showed  clear differences  among  the  mice  of  three  genotypes  in  three different types of behavior. In monitoring spontaneous motor activities, D1R and D2R KO mice showed higher and lower 2   h  activities,  respectively,  than  WT  mice.  In  the  rota-rod tasks,  at  a  low  speed,  D1R  KO  mice  showed  poor performance  but  later  improved,  whereas  D2R  KO  mice showed  good  performance  early,    without  further improvement. When first subjected to a high speed task, the D2R  KO  mice  showed  poorer  rota-rod  performance  at  low speeds  than  the  D1R  KO  mice.  In  the  Step-Wheel  task, across daily sessions, D2R KO mice increased their running speed  sufficiently  to  reach  the  waterspout,  and  decreased time between touching the floor due to missing the peg, and decreased the number of times the wheel was stopped, which was  much  better  performance  than  that  of  D1R  KO  mice. These incongruent results between the two tasks for D1R and 0D2R  KO  mice  may  be  due  to  differences  in  motivation  for the rota-rod and Step-Wheel tasks, i.e. aversion- and rewarddriven,  respectively. The  Step-Wheel  system  may  become  a useful tool for assessing the motor ability of WT and mutant mice  (Published  in  Nakamura  et  al.,  Front  Integr. Neuroscience.   5 , 201 ). Publication List

〔Original papers〕

 Nand akamura, T., Sato, A., Kitsukawa, T., Momiyama, T., Yamamori, T., Sasaoka, T.  (201 ).  Distinct  motor  impairments  of  dopamine  D1 and  D2  receptor  knockout  mice  revealed  by  three  types  of  motor behavior. Front. Integr. Neurosci. 8, 5 .
Shukla, R., Watakabe, A., and Yamamori, T. (201 ). mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain. Front. Neural Circuits 8, 52.
Watakabe, A., Ohsawa, S. Ichinohe, N., Rockland, K.S., and Yamamori, T.  (201 ).  Characterization  of  claustral  neurons  by  comparative  gene expression profiling and dye-injection analyses. Front. Syst. Neurosci. 8,  . Watakabe,  A.,  Takaji,  M.,  Kato,  S.,  Kobayashi,  K.,  Mizukami,  ., Ozawa,  K.,  Ohsawa,  S.,  Matsui,  R.,  Watanabe,  D.,  and Yamamori  T. (201 ).  Simultaneous  visualization  of  extrinsic  and  intrinsic  axon collaterals  in  Golgi-like  detail  for  mouse  corticothalamic  and corticocortical  cells   a  double  viral  infection  method.  Front.  Neural Circuits 8, 110. 

 
 
 

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

A., Ohtsuka, M, Kinoshita, M. Takaji, M. Isa, K Mizukami,  W., atakabe, Ozawa,  K.  Isa,  T.,  and  Yamamori,  T.  Comparative  analyses  of adeno-associated viral vector serotypes 1, 2, 5,   and   in marmoset, mouse and macaque cerebral cortex. Neurosci. Res. 201  Sep 1 . 

B A

ProfessorAFigure  1.  Schematic  showing  the self-initiated  lever-pull  task  for  a head-restrained mouse.  Aasanori

Assistant Professor:

WAKE, Hiroaki
HIRA, Riichiro
MASAMIZU, Yoshito
NIBB Research Fellow:
TANAKA, Yasuhiro*
EBINA, Teppei
Technical Staff:
OHSAWA, Sonoko
Postdoctoral Fellow:
MASAMIZU, Yoshito*
TANAKA, Yasuyo
TANAKA, Yasuhiro
TAKEDA, Yuta
SOKENDAI Graduate Student: OHKUBO, Fuki
HASEGAWA, Ryota
SUZUKI, Ayami
Visiting Scientist:
KATO, Daisuke
KONDO, Masashi
HIRAKAWA, Reiko
Visiting Graduate Student: TERADA, Shin-Ichiro
SUGAWARA, Yuki
Technical Assistant:
HIMENO, Miki
SAITO, Junko
KOBAYASHI, Tomoko
KOTANI, Keiko
TAKAHASHI, Yoichi
IWASE, Etsuko
IMOTO, Eiko
SUGIURA, Haruka
Secretary:
SUGIYAMA, Tomomi
Various firing patterns of many cortical neurons represent
information processing in the brain. The microarchitecture ofsynaptic connections control information processing in
cortical circuits. The structure and location of synapses
determine and modify the strength of this information
processing. The aim of our laboratory is to reveal how
information is formed, maintained, selected, and decoded in
the brain at the levels of single cells and of single synapses.
To do so, we mainly use two-photon microscopy that allows
us to see fluorescence signals from deep within living tissue,
developing novel photostimulation methods and animal
behavioral tasks. The goals of our recent studies are to reveal how  voluntary  movement  is  memorized  and  represented  in cortical  circuits.  One of the most important problems inneuroscience is how a variety of spatio-temporally
heterogeneous neural activity in the cortex emerges momentby-moment at multiple stages of a movement.

I. Two distinct layer-specific dynamics ofcortical ensembles during learning of a
motor task.

The  primary  motor  cortex  (M1)  possesses  two  intermediate layers  upstream  of  the  motor-output  layer   Layer  2   (L2 ) 
and layer 5a (L5a). Although repetitive training often improves motor  performance  and  movement  coding  by  M1  neuronal ensembles,  it  is  unclear  how  neuronal  activities  in  L2   and L5a are reorganized during motor task learning. We conducted two-photon  calcium  imaging  in  the  mouse  M1  during  1  training sessions of a self-initiated lever-pull task (Figure 1). Mice were trained over 1  sessions (1 h d) to use their right forelimb to pull a lever over a distance of 5 mm for  00 ms to acquire a water reward. Training was initiated 2  weeks after  injection  of  an  adeno-associated  virus  (AA )  2 1 encoding  GCaMP   into  the  left  forelimb  M1.  Two-photon calcium imaging was performed on the left forelimb M1 of   mice  without  apparent  deterioration  of  the  neuronal responsiveness over repeated sessions in L2  or L5a. We assessed the effect of repetitive training on the neuronal coding  of  lever  movement  because  the  lever  movement provided  a  good  representation  of  forelimb  movement.  We determined  whether  the  accuracy  of  the  lever  trajectory predicted  from  neuronal  ensemble  activity  improved  with learning.  To  evaluate  the  predictive  information  carried  by the  neurons  (Iensemble),  we  calculated  the  mutual  information between  predicted  and  recorded  lever  trajectories.  In  L2 , there was no significant change in Iensemble from early sessions (sessions 1- ) to late sessions (sessions 11-1  Figure 2). By contrast,  in  L5a,  Iensemble  was  significantly  higher  in  the  late sessions than in the early sessions (Figure 2). No consistent changes  in  L2   I ensemble   were  observed  as  learning progressed, whereas increases in L5a Iensemble were associated with improved task performance.

Figure  2.  Iensemble averaged  over  early  and  late  sessions  for  each  field  in L2  (left  n ) and L5a (right n ). *P   0.05.  Next, to determine how each neuron changed its predictive information  (Isingle)  during  learning,  we  analyzed  Isingle  of neurons.  Neurons  that  increased  or  decreased  Isingle  across sessions  were  defined  as  increase-  or  decrease-neurons, respectively.  In  L2 ,  there  was  a  similar  proportion  of increase-  and  decrease-neurons  (Figure  ),  suggesting  that the  overall  change  in  L2   Isingle  was  balanced  across  all imaged  fields.  By  contrast,  in  L5a,  there  was  a  higher proportion  of  increase-neurons  than  decrease-neurons (Figure  ). Many L5a increase-neurons displayed an increase in  activity  associated  with  lever-pull  movements  during learning.  Increase-neurons  were  critical  for  the  correlation between L5a Iensemble and task performance. Each  neuron  within  a  field  in  a  given  session  was  ranked according to Isingle and the rank was then normalized between 1  (top)  and  100  (bottom).  The  Iensemble  of  the  top  20   of ranked neurons was larger than the Iensemble of the bottom 50  Note  Those members appearing in the above list twice under different titles are members whose title changed during 201 . The former title is indicated by an asterisk (*).

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National nstitute or asic iology

Figure  . Proportion of increase-, decrease-, and other-neurons across all fields in L2  (left) and L5a (right).

of  ranked  neurons  in    5   of  sessions  in  L2   and  L5a. Thus, the top 20  of ranked neurons were considered to be highly  ranked  neurons  that  strongly  contributed  to  the ensemble representation. L2  increase-neurons rose through the  ranks  from  the  early  sessions  to  occupy  1   of  the  top 20  of ranked neurons in the late sessions. Forty-six percent of the top 20  of ranked neurons in the early sessions were also  in  the  top  20   of  ranked  neurons  in  the  late  sessions. We  call  these  neurons  stable,  highly  ranked  neurons.  The mean  activity  of  the  stable,  highly  ranked  neurons  in  L2  during successful lever-pull movements did not significantly change from the early to late sessions. These results suggest that  L2   Iensemble  was  maintained  by  balanced  activity  of increase-  and  decrease-neurons,  and  by  activity  of  stable, highly  ranked  neurons.  In  contrast  to  L2 ,  the  rank  of  L5a increase-neurons  gradually  increased  during  the  middle  to the late sessions, and increase-neurons accounted for  of the  top  20   of  ranked  neurons  in  the  late  sessions.  In  L5a, only  2   of  the  top  20   of  ranked  neurons  in  the  early sessions  were  in  the  top  20   of  ranked  neurons  in  the  late sessions.  These  results  indicate  that  the  increase  in  L5a Iensemble was supported by the conversion of increase-neurons into highly ranked neurons during the middle to late sessions. We  also  found  that  1   of  the  examined  L5a  crossed corticostriatal (CCS) neurons were increase-neurons and   were  decrease-neurons.  Of  the  top  20   of  the  ranked  CCS neurons observed in the early sessions, only 1  were in the top  20   of  the  CCS  neurons  observed  in  the  late  sessions. Twenty-six  percent  of  examined  L5a  corticospinal  (CSp) neurons  were  increase-neurons  and  5   were  decreaseneurons. Of the top 20  of the ranked CSp neurons observed in the early sessions, 100  were in the top 20  of the CSp neurons  observed  in  the  late  sessions. These  results  suggest that  increase-neurons  were  more  prevalent  than  decreaseneurons for both L5a CCS and CSp neurons and that a subset of  L5a  CCS  neurons  was  likely  recruited  into  the  newly formed ensemble that represented the lever-pull movement.
The balanced changes in L2  neurons could reflect the fact that  L2   neurons  with  highly  plastic  activity  process  a variety  of  information  from  other  areas  of  the  brain  to maintain  a  constant  overall  level  of  network  activity  for homeostasis.  L2   neurons  may  play  an  important  role  in rapid acquisition of novel movements or rapid adaptation to some disturbance by combining sensory feedback and motor primitives. As  such,  L2   may  work  as  a  driver  for  motor output  throughout  learning.  L5a  Iensemble  increased  during learning, and this was maintained by a gradual increment in Isingle  ranks  of  a  subset  of  L5a  neurons.  These  neurons  were directly associated with lever-pull movement and presumably worked  as  a  potent  driver  for  motor  output  in  the  late sessions.  Changes  in  L5a  neurons  in  M1  may  represent  the 2formation  of  motor  memory  that  accompanies  memory transfer from the prefrontal area to M1 and the basal ganglia. Stable,  highly  ranked  CSp  neurons  may  be  directly associated with muscle movements or motor primitives. The distinct  dynamic  networks  in  L2   and  L5a,  the  two intermediate  layers  of  M1,  are  clearly  core  elements  that drive the L5b motor output for well-learned movements.

II. Reward-timing-dependent bidirectional
modulation of cortical microcircuits during
optical single neuron operant conditioning Animals rapidly adapt to environmental change. To reveal how  cortical  microcircuits  are  rapidly  reorganized  when  an animal  recognizes  novel  reward  contingency,  we  conducted two-photon  calcium  imaging  of  layer  2   motor  cortex neurons in mice and simultaneously reinforced the activity of a single cortical neuron with water delivery. ead-restrained  mice  were  trained  to  perform  a  selfinitiated  lever-pull  task  using  the  right  forelimb  before single-neuron  operant  conditioning  by  two-photon  calcium imaging  (2pSNOC).  After  5 1   lever-pull  task  training sessions, two-photon calcium imaging of L2  motor cortical neurons  was  performed  during  SNOC.  Each  2pSNOC session consisted of three periods  a pre-conditioning period (10  min),  a  conditioning  period  (15  min),  and  a  postconditioning  period  (10  min).  2pSNOC  was  performed during  the  conditioning  period. Active  neurons  during  the pre-conditioning  period  were  reconstructed  and  classified into  two  groups   neurons  with  high  activity  during  leverrelated  periods,  and  other  neurons  ( lever-unrelated neurons ).  One  neuron  was  targeted  in  the  conditioning period. During the conditioning period, the mouse performed a 2pSNOC task. During the 2pSNOC task, water drops were successfully  delivered  to  the  mouse  immediately  after  the F F  of  a  single  target  neuron  increased  above  a  threshold (Figure  ). During the conditioning period, for lever-unrelated target neurons, activity increased by approximately 50 , whereas, for  lever-related  target  neurons,  activity  did  not  change (Figure  5).  T he  reward  frequency  increased  by approximately  50   when  lever-unrelated  neurons  were targeted,  and  did  not  change  when  lever-related  neurons were  targeted.  The  rapid  operant  conditioning  of  single lever-unrelated  neurons  was  successful  even  though  the activity  of  non-target  neurons  slightly  changed.  When lever-related neurons were targeted, the mouse performed goal-directed lever-pull movements from the onset of the conditioning  period  without  recognition  of  SNOC.  This Figure  .  Left  A  time-averaged  image  of  a  L2   field  during  2pSNOC. Red  arrowhead,  a  target  neuron.  Scale  bar,  100  m.  Middle   Expanded image of the rectangle in left. Scale bar, 10  m. Right   F F of the target (T)  and  the  three  neighboring  (N1,  N2,  and  N )  neurons.  Cyan  bars, reward delivery.

Figure 5. Time course of the activity of lever-unrelated (left  n 11) and lever-related (right  n 1 ) target neurons during the conditioning period. The activity is Z scored for each neuron. Thick lines and shading indicate the mean   s.e.m.

may  explain  why  the  activity  gain  (the  ratio  of  the  mean activity  in  the  last  5  min  of  the  conditioning  period  to  the mean  activity  in  the  first  5  min  of  the  conditioning  period minus  1 )  for  lever-related  target  neurons  was  not significantly positive. In the following analyses, only data from  sessions  with  a  lever-unrelated  target  neuron  were used.
Non-target  neurons  whose  activity  was  temporally,  but  not spatially,  associated  with  the  target  neuron  increased  their activity. Non-target neurons should have been affected by the water  delivery  because  it  was  the  reinforcer.  For  each  nontarget neuron, the ratio of the sum of the activity in a 0. -s time bin t s after reward delivery to the sum of the remaining activity  in  the  same  bin  was  defined  as  the  reward synchronization index (t) (RSI(t)). For each time bin, activity gain  was  averaged  across  the  non-target  neurons  with  RSI values in the top 5 . The activity gain of non-target neurons with  RSI  in  the  top  5   was  significantly  larger  than  zero when  t ranged  from  0.   s  to  0.   s,  and  significantly smaller than zero when t was 2. ,  , and   s. We refer to this phenomenon  as  reward-timing-dependent  bidirectional modulation  (RTBM),  and  refer  to  neurons  that  had  RSI ( 0.  s to 0.  s) in the top 5  as SR neurons and neurons that had RSI (2 s to   s) in the top 5  as AR neurons. The activity  gain  of  SR  and  AR  neurons  was  substantially positive and negative, respectively. The percentage of leverrelated and lever-unrelated neurons was similar between SR and AR  neurons.  The  distance  of  SR  and AR  neurons  from the target neuron was similar to the distance of other neurons from  the  target  neuron.  The  pairwise  correlation  with  the target neuron was higher for SR neurons than for AR neurons and  other  neurons  during  both  the  conditioning  period  and pre-conditioning  period  without  water  delivery.  This indicates  that  SR  neurons  were  frequently  active  together with  the  target  neuron.  By  contrast, AR  neurons  did  not appear  different  from  other  non-target  neurons  in  terms  of their associations with the target neuron. Neither SR nor AR neurons  were  specifically  related  to  licking.  Likewise, neither SR nor AR neurons were specifically related to leverpull in response to the reward delivery. To  validate  whether  the  activity  timing  relative  to  the reward  delivery  is  sufficient  to  induce  the  neuronal  activity changes, we performed repetitive pairing of the activity of a 
random  set  of  neurons  and  reward  delivery  with  different time  intervals.  In  each  photostimulation  session,  0 photostimuli were delivered during a 15 min period to induce firing in channelrhodopsin-2-expressing neurons in the field of view and a reward was given 0.25 s after or 2.5 s before each  photostimulation  (potentiation  protocol  and  depression protocol, respectively). The photostimuli-induced activity of photostimuli-responsive  neurons  significantly  increased  and decreased  in  the  potentiation  and  depression  protocols, respectively (Figure  ). The activity of the other neurons did not  change  in  either  protocol.  Thus,  repetitive  pairing  of neuronal  activity  with  reward  delivery  at  different  time intervals was sufficient to recreate RTBM.
Our  results  suggest  that  the  microcircuit  has  the  capability to  strengthen  the  activity  of  neurons  that  were  previously included  in  the  same  ensemble  without  strong  spatial constrains.  RTBM  may  be  one  of  possibly  many  processes that  underlie  rapid  reorganization  of  the  L2   cortical microcircuit during fast adaptation to environmental changes, which  occurs  during  BMI BCI  learning,  motor  adaptation, and skill learning.
 Figure  .  Time  course  of t h e   m e a n  F F   o f photostimuli-responsive neurons  in  the  potentiation (green,  n
  neurons)  and depression  (orange,  n 0 neurons)  protocols.  Thick lines  and  shading  indicate the mean   s.e.m.

Publication List

〔Original papers〕 and ira, R., Ohkubo, F., Masamizu, Y., Ohkura, M., Nakai, J., Okada, T., Matsuzaki,  M.  (201 ).  Reward-timing-dependent  bidirectional modulation  of  cortical  microcircuits  during  optical  single  neuron operant conditioning. Nature Commun. 5, 5551.
Masamizu,  Y.,  Tanaka,  Y.R.,  Tanaka,  Y. .,  ira,  R.,  Ohkubo,  F., Kitamura, K., Isomura, Y., Okada, T., and Matsuzaki, M. (201 ). Two distinct layer-specific dynamics of cortical ensembles during learning of a motor task. Nature Neurosci. 17, .

 National nstitute or asic iology

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NIBB Research Fellow:

ii

NAKAYASU, Tomohirosystematically. Therefore, we have constructed  D computer graphics animations of medaka based on tracking coordinate data and photo data obtained from real medaka. These virtual D models will allow us to  represent medaka faithfully and to undertake a more detailed analysis of the properties of the visual  stimuli  that  are  critical  for  the  induction  of  various behaviors.

In  order  to  interact  successfully  with  the  environment, animals  must  deduce  their  surroundings  based  on  sensory information.  The  visual  system  plays  a  particularly  critical role in such interactions with the environment. Why  can  we  see   This  question  is  fundamental  for  a thorough  understanding  of  vision-dependent  animals, including  human  beings.  In  order  to  better  understand  the visual  system  of  animals,  we  are  researching  animal behaviors  through  psychophysical  and  computational methods.

I. Psychophysical study of Medaka fishOne  of  our  major  subjects  is  the  psychophysical  and computational  study  of  medaka  (Oryzias latipes).  Recently, we made progress in studies of the prey-predator interaction using  medaka  and  zooplankton.  isual  motion  cues  are  one of  the  most  important  factors  for  eliciting  animal  behavior, including predator-prey interactions in aquatic environments. To  understand  the  elements  of  motion  that  cause  such selective  predation  behavior,  we  used  a  virtual  plankton system  where  the  predation  behavior  in  response  to computer-generated  prey  was  analyzed.  irtual  prey  models were  programmed  on  a  computer  and  presented  to  medaka, which  served  as  predatory  fish.  Medaka  exhibited  predation behavior  against  several  characteristic  virtual  plankton movements,  particularly  against  a  swimming  pattern  that could be characterized as pink noise motion. Analyzing preypredator  interactions  via  pink  noise  motion  will  be  an interesting  research  field  in  the  future  (Matsunaga   Watanabe, 2012).
Last and this year, we have made progress in studies of the schooling behaviors of medaka. Many fish species are known to  live  in  groups.  isual  cues  have  been  shown  to  play  a crucial  role  in  the  formation  of  shoals.  Using  biological motion stimuli, depicting a moving creature by means of just a few isolated points, we examined whether physical motion information is involved in the induction of shoaling behavior. We  found  that  the  presentation  of  biological  motion  could prominently induce shoaling behavior. We have shown what aspects  of  motion  are  critical  in  the  induction  of  shoaling behavior.  Motion  or  behavioral  characteristics  can  be valuable  in  recognizing  animal  species,  sex,  and  group members.  Studies  using  biological  motion  stimuli  will enhance  our  understanding  of  how  non-human  animals extract  and  process  the  information  which  is  vital  for  their survival (Nakayasu   Watanabe, 201 ).
This  year,  we  have  developed  novel  method  for  behavior analysis by using  D computer graphics. The fine control of various features of living fish have been difficult to achieve in  studies  of  fish  behaviors.  owever,  computer  graphics allow  us  to  manipulate  morphological  and  motion  cues Figure 1.   irtual Medaka fish constructed of  D polygonal models and photo textures.

II. Psychophysical study of Human vision

Another  of  our  major  subjects  is  the  psychophysical  and theoretical  studies  of  the  visual  illusions  of  human  beings (Homo sapiens). One recent focus of this debate is the flashlag  effect,  in  which  a  moving  object  is  perceived  to  lead  a flashed  object  when  both  objects  are  aligned  in  actual physical  space.  We  developed  a  simple  conceptual  model explaining the flash-lag effect (Delta model, Watanabe et al., 2010).  This  year,  we  have  made  a  more  developed  novel visual illusion, the shelf-shadow illusion  (Figure 2).

Figure 2. Shelf-Shadow Illusion (Photograph version). Upward shadows look darker than downward shadows even when the shadows are actually the same brightness. Third place award of The 5th Illusion Contest in Japan.

Publication List

〔Original paper〕

akayasu, T., and Watanabe, E. (201 ). Biological motion stimuli are  Nattractive to medaka fish. Animal Cognition 17, 55 -5 5.

National nstitute or asic iology

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ProfessorA

itsuyasu

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Associate Professor

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a as i

Assistant Professor:

TAMADA, Yosuke
ISHIKAWA, Masaki
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellow:
IMAI, Akihiro
Postdoctoral Fellow:
MANO, Hiroaki
SHIBATA, Tomoko
TAMAOKI, Daisuke
TORIBA, Taiyo
SOKENDAI Graduate Student:
FUKUSHIMA, Kenji
KAMIDA, Chiharu
KOSHIMIZU, Shizuka
LI, Chen
MORISHITA, Mio
SUGAYA, Tomomi
ZHANG, Liechi
HORIUCHI, Yuta
Visiting Graduate Student: GU, Nan
Visiting Scientist:Čuperová, Zuzana
KAWASHIMA, Takeshi
Technical Assistant:
AOKI, Etsuko
GOTO, Misako
KAJIKAWA, Ikumi
MORI, Asuka
MATSUZAKI, Yoko
NISHI, TayoOOI, Shoko
MASUOKA, Tomoko
Secretary:
KOJIMA, Yoko

I. Evolution of Complex Adaptive Characters

The  theory  of  natural  selection  and  the  neutral  theory  of molecular  evolution  are  powerful  concepts  in  evolutionary biology.  owever, 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 at cellular and organismal levels,  such  as  cell  division  machinery,  regeneration,  novel organ  development,  host  race  change,  and  mimicry.  Such traits comprise many components and become adaptive only when all components are gathered together.  owever, based on  evolutionary  theory,  each  component  should  evolve  one by one according to the accumulation of mutations. We aim to reveal the genetic networks regulating complex traits and to  infer  the  mechanisms  needed  to  evolve  complex characters.

II. Evolution of Regeneration: Reprogrammingof Differentiated Cells to Pluripotent StemCells

Different  species  have  different  morphology  and  also cellular  characters  vary  between  species.  Stem  cells  selfrenew  and  repeatedly  produce  differentiated  cells  during development.  Conversely,  differentiated  cells  can  be converted  into  stem  cells  in  some  organisms.  In  plants, regeneration  of  a  stem  cell  leads  to  generation  of  a  new individual,  which  is  an  effective  strategy  for  propagation. The ability to reprogram is different from species to species but the reason is unknown. The moss Physcomitrella patenshas  a  rapid  reprogramming  ability  (see    http www.nibb.ac.
jp evodevo 0.TopE.html)  and  is  feasible  for  use  in experiments.  Cells  in  a  dissected  leaf  are  reprogrammed  to become  chloronema  apical  stem  cells  within  2   hours.  We reported  that  two  paralogous  P. patens WUSCHEL-related
homeobox 13-like (PpWOX13L)  genes,  homologs  of  stem cell  regulators  in  flowering  plants,  are  transiently upregulated  and  required  for  the  initiation  of  cell  growth during  stem  cell  formation.  Concordantly,  ppwox13ldeletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss  life  cycle,  most  of  the  ppwox13l mutant  zygotes  fail to expand and initiate an apical stem cell to form the embryo. Our  data  show  that  PpWOX13L genes  are  required  for  the initiation  of  cell  growth  specifically  during  stem  cell formation,  in  analogy  to  WO   stem  cell  functions  in  seed plants, but using a different cellular mechanism (Sakakibara et al. 201 ).
To  perform  fine  live  imaging  during  the  reprogramming process,  we  now  try  to  apply Adaptive  optics  (AO)  to  the observation  of  Physcomitrella  cells. AO  is  the  technique  to cancel  the  aberration  caused  by  atmospheric  turbulence  and to perform diffraction- limited observation of celestial bodies from the ground. Applying AO to microscopy, we can cancel the  aberration  caused  by  cellular  structures  and  perform high-resolution live imaging. To construct the AO system, we first  analyzed  the  optical  properties  of  Physcomitrella  cells. Live-cell  imaging  with  bright  field  and  phase  contrast microscopies  as  well  as  image  degradation  analysis  using fluorescent beads demonstrated that chloroplasts are the main source  of  the  disturbance  in  the  cell  (Tamada  et al.  201 ). According to this information, we constructed a prototype of an AO microscope that can correct the aberration caused by chloroplasts.  Images  of  chloroplasts  that  are  located  at  the opposite  side  from  the  objective  lens  in  the  leaf  cell  are degraded due to aberration from cellular structures including chloroplasts  themselves.  With  AO  microscopy,  we successfully  obtained  fine  images  of  the  chloroplast  where the  grana  structure  was  observed  more  clearly.  This  study was mainly conducted by Yosuke Tamada.

Figure 1. AO correction of a chloroplast image. Left, no correction  right, with AO. Bar, 5  m.

III. Evolution of Regeneration: Master
Regulator for Reprogramming STEMINAnimal  somatic  cells  can  be  reprogrammed  to  induce pluripotent stem (iPS) cells by introducing four transcription 5National nstitute or asic iology

BBfactors, while such factors have not been identified in plants. We have previously identified a gene encoding a member of a  plant-specific  transcription  factor,  STEM  CELLINDUCING FACTOR 1 (STEMIN1) that was able to induce direct  reprogramming  of  differentiated  leaf  cells  into chloronema  apical  stem  cells  without  wounding  signals. STEMIN1  and  its  two  paralogous  genes  (STEMIN2  and STEMIN3)  were  activated  in  leaf  cells  that  underwent reprogramming.  In  addition,  deletion  of  the  three  STEMIN genes delayed reprogramming after leaf excision, suggestingthat these genes redundantly function in the reprogrammingof cut leaves. On the other hand, differently from STEMIN1,
induction of STEMIN2 or STEMIN3 in gametophores did notchange leaf cells into chloronema apical stem cells. These
indicate that STEMIN1 has an enough ability to change intact
leaf cells to stem cells, but its paralogous  genes  do not. Masaki Ishikawa was this study’s main researcher.

IV. Evolution of Elaborated Cell Division
Machinery: Spindle bodyAt mitosis, all eukaryotic cells divide chromosomes to two daughter cells using a mitotic spindle, which is composed of microtubules. For accurate distribution of the chromosomes, the  spindle  has  two  poles.  The  centrosomes,  which  act  as microtubule organizing centers, ensure formation of the two poles  in  metazoan  cells.  In  contrast,  the  cells  of  land  plants and  their  sister  group,  charophycean  green  algae,  form  a bipolar  spindle  in  the  absence  of  centrosomes.  For understanding  the  mechanism  of  acentrosomal  spindle formation,  the  steps  of  microtubule  reorganization  during spindle  formation  should  be  visualized.  It  is  challenging, however,  to  visualize  microtubule  reorganization  during spindle formation in living plant cells, because large numbers of  microtubules  rapidly  redistribute  in  -dimensional  space. We  collaborated  with  Prof.  Tomomi  Nemoto  in  okkaido University  and  developed  a  two-photon  spinning  disk confocal  microscope,  which  enables  -dimensional  imaging of living cells with high temporal and spatial resolution. Our data  shows  that  microtubules  elongate  from  template microtubules on the nuclear envelope upon nuclear envelope breakdown. The template microtubules are distinct from the spindle  microtubules  because  they  disappear  during  spindle development. The data suggests that microtubules which had been organized before spindle formation are the organizers of the  bipolar  spindle.  Takashi  Murata  was  this  study’s  main researcher.

V. Evolution of water conducting systems

The development of a water conducting system was one of the  most  important  requirements  for  land  plants.  Recent studies  implicate  a  group  of  NAC  domain  transcription factors including  ND  and  ND  of Arabidopsis thaliana as  key  regulators  of  formation  of  xylem  vessels,  waterconducting  cells  in  vascular  plants.  owever,  molecular mechanisms  for  development  of  other  types  of  waterconducting cells are still unclear. We collaborated with Prof. Taku  Demura  in  NAIST  and  showed  that  their Physcomitrella patens  homologues,  named  Pp NS1  to Pp NS ,  play  a  crucial  role  during  the  development  of hydroids,  specialized  water-conducting  cells  in  bryophytes. PpVNS  genes  are  expressed  in  the  midrib  of  developing leaves,  in  which  hydroids  are  developed.  Overexpression  of PpVNS  genes  induced  cell  death  in  P. patens and  ectopic formation  of  vessel-like  cells  in  the  vascular  plant Arabidopsis.  From  these  observations,  we  proposed  that  the last common ancestor of bryophytes and vascular plants had developed  a  system  using  NS  homologues  to  induce  cell death  during  water-conducting  cell  formation.  Our  findings also  suggest  that  the  water-conducting  cells  in  bryophytes and  vascular  plants  are  homologous,  and  that  current  land plants  share  a  conserved  genetic  basis  with  NS  family proteins for development of water conducting systems ( u etal. 201 ).

VI. Molecular mechanisms of Plant Movement
using Mimosa pudica

The  sensitive  plant  Mimosa pudica  has  long  attracted  the interest of researchers due to its spectacular leaf movements in  response  to  touch  or  other  external  stimuli. Although various  aspects  of  the  seismonastic  movement  have  been elucidated  by  histological,  physiological,  biochemical,  and behavioral  approaches,  the  lack  of  reverse  genetic  tools  has hampered  the  investigation  of  molecular  mechanisms involved  in  these  processes.  To  overcome  this  obstacle,  we developed an efficient genetic transformation method for M.
pudica (Mano et al., 201 ). This new technique is currently applied  for  live  imaging  of  actin  cytoskeleton  and  calcium dynamics,  both  of  which  participate  in  the  seismonastic movement  through  as-yet-unidentified  mechanisms.  Our transgenic  technique  also  enabled  us  to  develop  a  CRISPR
Cas-mediated  gene  knock-out  system  in  this  species.  To investigate  adaptive  meanings  of  the  seismonastic movement,  we  now  try  to  produce  immotile  mutants  using the  CRISPR Cas  system.  This  study  was  conducted  mainly by  iroaki Mano.

Figure 2.  isualization of actin cytoskeleton in leaflets of Mimosa pudica using a Lifeact- enus proteinVII. Molecular mechanisms of mimicryAn excellent example of mimicry is the flower-mimicry of the orchid mantis Hymenopus coronatus with pink and white coloration  and  petal-like  legs.  Biochemical  analyses indicated that the reduced form of xanthommatin, a common red  pigment  of  the  ommochrome  family,  almost  solely contributes to the pink color. On the other hand, the oxidized form  of  xanthommatin  was  found  in  mantises  with  brown body  color.  To  further  elucidate  the  mechanism  underlying the  different  body  coloration,  we  are  now  analyzing  the ultrastructure  of  ommochrome  granules  in  pigment  cells  of these  mantises  by  transmission  electron  microscopy.  This work was mainly done by  iroaki Mano.

VIII. Evolution of pitcher leaves in carnivorous
plantsCarnivorous plants form specialized leaves that are capable of  attracting,  trapping,  and  digesting  prey  and  absorbing nutrients. The  unusual  plants  evolved  from  non-carnivorous plants but their evolutionary process is mostly unknown. To understand  the  genomic  changes  associated  with  the evolution  of  carnivory,  we  sequenced  2-Gbp  genome  of  the Australian  pitcher  plant  Cephalotus follicularis  in collaboration  with  Beijing  Genomics  Institute.  This  study was  conducted  mainly  by  Kenji  Fukushima  and  Tomoko Shibata.

Publication List

〔Original papers〕

Y., Namba-Fukuyo,  ., Shibata, T.F., Nishiyama, T., Shigenobu,  FS., uruta, Suzuki,  Y.,  Sugano,  S.,  asebe,  M.,  and  Kobayashi,  I.  (201 ). Methylome diversification through changes in DNA methyltransferase sequence specificity. PLoS Genet. 10, e100 2 2.
Gusev,  O.,  Suetsugu,  Y.,  Cornette,  R.,  Kawashima,  T.,  Logacheva, M.D.,  Kondrashov,  A.S.,  Penin,  A.A.,  atanaka,  R.,  Kikuta,  S., Shimura,  S.,  Kanamori, .,  Katayose,  Y.,  Matsumoto,  T., Shagimardanova,  E.,  Alexeev,  D.,  Govorun,  .,  Wisecaver,  J., Mikheyev, A., Koyanagi, R., Fujie, M., Nishiyama, T., Shigenobu, S., Shibata,  T.F.,  Golygina,  .,  asebe,  M.,  Okuda,  T.,  Satoh,  N.,  and Kikawada, T. (201 ). Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge. Nature Commun. 5, .
ieno, A., Naznin,  .A.,  yakumachi, M., Sakurai, T., Tokizawa, M., Koyama,  ., Sato, N., Nishiyama, T.,  asebe, M.,  immer, A.D., Lang, D., Reski, R., Rensing, S.A., Obokata, J., and Yamamoto, Y.Y. (201 ). ppdb   plant  promoter  database  version  .0.  Nucleic  Acids  Res. 42, D11 -11 2.
Ishida, T., Tabata, R., Yamada, M., Aida, M., Mitsumasu, K., Fujiwara, M., Yamaguchi, K., Shigenobu, S.,  iguchi, M., Tsuji,  ., Shimamoto, K.,  asebe,  M.,  Fukuda,  .,  and  Sawa,  S.  (201 ).  eterotrimeric  G 
proteins control stem cell proliferation through CLA ATA signaling in Arabidopsis. EMBO Rep. 15, 120 -1215.
Kishi-Kaboshi, M., Muto,  ., Takeda, A., Murata, T.,  asebe, M., and Watanabe, Y. (201 ). Localization of tobacco germin-like protein 1 in leaf intercellular space. Plant Physiol. Biochem. 85, 1- .
Mano,  .,  Fujii,  T.,  Sumikawa,  N.,  iwatashi,  Y.,  and  asebe,  M. (201 ).  Development  of  an  agrobacterium-mediated  stable transformation  method  for  the  sensitive  plant  Mimosa pudica.  PLoS One 9, e 11.
Sakakibara, K., Reisewitz, P., Aoyama, T., Friedrich, T., Ando, S., Sato, Y., Tamada, Y., Nishiyama, T.,  iwatashi, Y., Kurata, T., Ishikawa, M., Deguchi,  .,  Rensing,  S.A.,  Werr,  W.,  Murata,  T.,  asebe,  M.,  and Laux, T. (201 ). WO 1 -like genes are required for reprogramming of leaf  and  protoplast  cells  into  stem  cells  in  the  moss  Physcomitrella
patens. Development 141, 1 0-1 0.
Takeshita,  K.,  Shibata,  T.F.,  Nikoh,  N.,  Nishiyama,  T.,  asebe,  M., Fukatsu,  T.,  Shigenobu,  S.,  and  Kikuchi,  Y.  (201 ).  Whole-genome sequence of Burkholderia sp. strain RPE , a bacterial gut symbiont of the bean bug Riptortus pedestris. Genome Announc. 2, e0055 -1 .
Tamada, Y., Murata, T.,  attori, M., Oya, S.,  ayano, Y., Kamei, Y., and asebe, M. (201 ). Optical property analyses of plant cells for adaptive optics microscopy. International J. Optomechatronics 8, 1-11.
u, B., Ohtani, M., Yamaguchi, M., Toyooka, K., Wakazaki, M., Sato, 

 

 
 
 
 
 
 
 
 

M., Kubo, M., Nakano, Y., Sano, R.,  iwatashi, Y., Murata, T., Kurata, T.,  Yoneda,  A.,  Kato,  K.,  asebe,  M.,  and  Demura,  T.  (201 ). Contribution of NAC transcription factors to plant adaptation to land. Science 343, 1505-150 .
Yoshida,  K.,  Makino, T., Yamaguchi,  K.,  Shigenobu,  S.,  asebe,  M., Kawata, M., Kume, M., Mori, S., Peichel, C.L., Toyoda, A., Fujiyama, A.,  and  Kitano,  J.  (201 ).  Sex  chromosome  turnover  contributes  to genomic  divergence  between  incipient  stickleback  species.  PLoS Genet. 10, e100 22 .

 〔Review articles〕

ukushima,  K.,  and  asebe,  M.  (201 ). Adaxial-abaxial  polarity   the  Fdevelopmental basis of leaf shape diversity. Genesis52, 1-1 .
orio,  T.,  and  Murata,  T.  (201 )  The  role  of  dynamic  instability  in  microtubule organization. Front. Plant Sci. doi  10.
fpls.201 .00511
ofuji, R., and  asebe, M. (201 ). Eight types of stem cells in the life  Kcycle of the moss Physcomitrella patens. Curr. Opin. Plant Biol.
17, 1 21.

essa, E., Banks, J., Barker, M., Der, J., Duffy, A., Graham, S.,  asebe,  SM., Langdale, J., Li, F., Marchant, D., Pryer, K., Rothfels, C., Roux, S., Salmi, M., Sigel, E., Soltis, D., Soltis, P., Stevenson, D., and Wolf, P. (201 ). Between two fern genomes. Gigascience 3, 15.
Sparks,  E.E.,  Imai,  A.,  asebe,  M.,  and  Benfey,  P.N.  (201 ). Developmental regulation and de novo formation of stem cells in plants. In Stem Cells  From Basic Research to Therapy,  olume 1  Basic Stem Cell  Biology,  Tissue  Formation  during  Development,  and  Model Organisms. Pp.  05, CRC Press.
Tomescu, A.M., Wyatt, S.E.,  asebe, M., and Rothwell, G.W. (201 ). Early  evolution  of  the  vascular  plant  body  plan  -  the  missing mechanisms. Curr. Opin. Plant Biol. 17, 12 -1 .

  National nstitute or asic iology

B

B

B

Professor

A A

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

SOKENDAI Graduate Student:

Visiting Undergraduate:
Technical Assistant:

Secretary:

asayos i

TAKEDA, Naoya
SUZAKI, Takuya
FUKADA-TANAKA, Sachiko
SOYANO, Takashi
FUJITA, Hironori
TATEMATSU, Kiyoshi
HANDA, Yoshihiro
KOBAYASHI, Yuki
NAGAE, Miwa
MATSUOKA, Hikaru
SASAKI, Takema
YORO, EmikoTSUZUKI, Shusaku
NISHIDA, Hanna
FUKUHARA, Mai
LIU, Meng
MIZUTANI, Yoshiyuki
SUZAKI, Momoyo
ICHIKAWA, Michiko
OGAWA, Yuko
YOSHINORI, Yumi
SANJO, Kazuko
OKUBO, Masayo

Rhizobium legume symbiosis is one of the most successful mutually  beneficial  interactions  on  earth.  In  this  symbiosis, soil  bacteria  called  rhizobia  supply  the  host  legumes  with ammonia  produced  through  bacterial  nitrogen  fixation.  In return,  host  plants  provide  the  rhizobia  with  their photosynthetic  products.  To  accomplish  this  biotic interaction,  leguminous  plants  develop  nodules  on  their roots.  On  the  other  hand,  more  than  0   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  unveil  the  mechanisms  of both symbiotic systems.  I. Nodulation

1-1 Genetic mechanism involved in early and latenodule development In response to appropriate inductive conditions, plants have the capacity of forming new organs from differentiated cells. Root  nodulation  is  one  such  unique  developmental  process that  predominantly  occurs  in  leguminous  plants.  In  this process,  signaling  initiated  by  symbiotic  bacterial  infection alters  the  fate  of  differentiated  cortical  cells  and  causes formation of new organs.   The  L. japonicus vagrant infection thread 1 (vag1)  is  a novel  mutant  involved  in  nodule  development.  VAG1 encodes  a  protein  orthologous  to  Arabidopsis  ROOT AIRLESS  1,  which  functions  as  a  subunit  of  DNA topoisomerase  I. Analyses  focusing  on  nuclear  size  found the  emergence  of  a  few  cortical  cells  with  enlarged  nuclei during initiation of cortical cell division in wild type. In the vag1  mutants,  these  potentially  endoreduplicated  cells  are not observed and subsequent cortical cell division is severely compromised.  Thus,  it  is  possible  that  DNA  topoisomerase I  is  involved  in  the  endoreduplication  of  cortical  cells, which  can  trigger  the  onset  of  cortical  cell  division.  During late  nodule  development,  endoreduplication  results  in  the formation  of  enlarged  rhizobia-colonized  cells  in  mature nodules.  In  wild  type L. japonicus,  there  are  enlarged rhizobia-colonized  cells  in  the  inner  region  of  the  nodule, and  smaller  rhizobia-infected  (as  yet  uncolonized)  cells  in the  surrounding  region  (Figure  1).  In  vag1  nodules,  the number  of  these  small  rhizobia-infected  cells  is  higher whereas  the  number  of  rhizobia-colonized  cells  is  lower (Figure 1). This suggests that the vag1 mutant has a defect in differentiation from small rhizobia-infected cells to enlarged rhizobia-colonized  cells.  Overall,  our  data  indicate  that endoreduplication  mediated  by  DNA  topoisomerase  I  may be a prerequisite regulator for the control of two key nodule developmental  processes   the  first  is  related  to  the  onset  of nodule organogenesis during early nodule development, and the second is associated with the differentiation of rhizobiacolonized infected cells in late nodule development.

Figure  1. The  effect  of  the  vag1 mutation  on  nodule  structure.  Sections through nodules of wild type (A, C, D) and vag1-1 (B, E, F) at 21 days after  inoculation  with  rhizobia  that  constitutively  express  the  LacZreporter  gene.  (C,  E)  Rhizobia-colonized  infected  cells  located  at  the inner region of nodules. (D, F) In wild type, small rhizobia-infected (as yet  uncolonized)  cells  (bracket)  are  located  surrounding  the  region  of rhizobia-colonized  cells.  In  contrast,  the  inner  region  of  vag1-1  nodule comprises  a  large  number  of  these  small  rhizobia-infected  cells.  Scale bars  100  m in A, B  20  m in C-F.

1-2 A presumptive post-translational modification
enzyme, PLENTY controls nodulation and root
growth in L. japonicus

Legumes  can  survive  even  in  nitrogen-deficient environments  depending  on  root  nodules  symbiosis  with rhizobia   however,  forming  nodules  consumes  energy, requiring  nodule  number  to  be  strictly  controlled.  The previous  studies  of  hypernodulation  mutants,  har1,  klv,  and tml,  have  proposed  long-distance  control  of  nodulation (Figure 2), via systemic mobile signals, CLE-RS1 2 peptides. Recently,  we  found  that,  at  least,  the  CLE-RS2  peptide  is arabinosylated  and  this  modification  is  essential  for  nodule inhibition  (Okamoto  et  al.,  201 ).  In  Arabidopsis,  the  posttranslational  modifications  of  hormone-like  small  peptides and  their  critical  roles  in  biological  activities  have  been gradually  found.  Further,  one  of  the  modification  enzymes, hydroxyproline O-arabinosyltransferase ( PAT), is identified and  its  homolog  in  L. japonicus  is  thought  to  be  a  strong candidate for the modification enzyme of CLE-RS2. We identified the  PAT homolog in L. japonicus, PLENTY, as a responsible factor for the already isolated plenty mutant. PLENTY is localized to the Golgi, suggesting a similarity of protein  functions  between  LjPLENTY  and  At PAT. owever,  overexpression  of  CLE-RS1/2  is  still  effective  on nodule  inhibition  in  plenty,  suggesting  that  PLENTY  itself does not mainly mediate the arabinosylation of CLE-RS1 2. In  addition,  plenty har1  double  mutant  showed  an  additive nodulation.  These  suggest  a  novel  AR1-independent nodulation  controlling  pathway  mediated  by  an  unknown possible  substrate  of  PLENTY  which  is  a  putative  posttranslational modification enzyme. Figure  2.  A  model  of  the  long-distance  control  of  nodulation.  (1)Nodulation signaling pathway downstream of cytokinin receptors
activates NIN expression. The NIN transcription factor activates CLERS1/2 expression through direct binding to their promoter. (2) It is likelythat the CLE-RS1/2 peptides are posttranslationally modified withtriarabinoside, a reaction mediated by an enzyme similar to HPAT in the
Golgi apparatus. These modified CLE-RS peptides are transported to thexylem. (3) These peptides are transmitted from roots to shoots anddirectly bind to HAR1 in the phloem of leaf cells. (4) Downstream of the
CLE-RS/HAR1 signaling pathway, activated IPT3 produces cytokinin,
which is transported to roots through phloem tissue. (5) Shoot-derived
cytokinin is directly or indirectly involved in proteasome-mediateddegradation of an unidentified positive regulator of nodule organogenesis.

II. Arbuscular mycorrhiza symbiosis

Arbuscular  mycorrhiza  is  a  plant-fungus  interaction  that confers  great  advantage  to  growth  and  survival  on  the  land. AM  fungi  enter  into  the  host  root  and  elongate  the  hyphae between  the  root  cells.  The  intraradical  hyphae  form symbiotic  structures  called  arbuscule’  and  vesicle’  (Figure
3A). AM  fungi  supply  phosphate  to  the  host  plant  through the  symbiotic  structures  and  in  return,  they  obtain photosynthetic  products  form  the  host.  To  obtain  insights about  molecular  mechanisms  of AM  development,  we  are studying  symbiotic  signaling  factors  that  regulate  symbiotic gene expression and AM fungal infection.
We  performed  transcriptome  analysis  in  wild-type  and symbiotic mutants of L. japonicus. This analysis showed that plant hormone gibberellin (GA) biosynthesis and metabolism
genes were induced during AM development. The GA
biosynthesis gene expression was disturbed in the symbiotic
mutants that showed abnormal AM fungal colonization in thehost roots, indicating that de novo biosynthesis of GAs has
some function in AM development. Functional analysis of
GA in AM development revealed that GA has a negative
effect on some AM-induced gene expressions, but also has apositive effect on other expressions (Figure 3B). RAM1 andRAM2 that function in AM entry processes are suppressed by
GA signaling, on the other hand, GA signaling promotes ormaintains SbtM1 expression that is required for AM fungal
colonization in the host root. This indicated that GA
signaling interferes with the symbiotic signaling pathway,
which decreases or enhances expression levels of
AM-induced genes. In addition, treatment with GA or GA
biosynthesis inhibitor disturbed the GA signaling and caused
an inhibitory effect on AM hyphal entry into the host root or
the hyphal branching in the root cortical cell layer. Thesestudies revealed the host plant controls GA signaling level by
induction of GA biosynthesis and metabolism genes during
AM development and the GA signaling regulates AM fungal
colonization in the host root.

Figure  . AM fungal colonization and interference of GA signaling with the symbiotic gene expression. (A) AM fungal structures in the host root. (B)  GA  signaling  is  enhanced  by  GA  biosynthesis  during  AM development  and  differentially  interferes  with  symbiotic  signaling pathways. The interference affects and regulates the AM colonization in the host root.

III. Computational modeling for patternformation of stomatal differentiation in
Arabidopsis leaves

Stomata are epidermal structures that mediate gas exchange in plants, and are usually formed separately from each other (Figure  A).  In  the  initial  step  of  stomatal  development, stomatal  lineage  cells  are  self-organized  from  a homogeneous field, and thus this is a good example for twodimensional  pattern  formation  in  living  systems.  Stomatal differentiation  is  promoted  by  transcription  factors  SPC  and  SCRM,  but  in  contrast  is  suppressed  by  diffusible peptide EPF2, which interacts with its membrane receptors to stimulate  the  degradation  of  SPC SCRM.  In  addition,  this regulatory  network  involves  feedback  loops   SPC SCRM National nstitute or asic iology

BBheterodimer activates SCRM expression (positive feedback), and  also  stimulates  EPF2  expression  leading  to  suppression of  SPC SCRM  (negative  feedback).  This  regulatory framework is similar to that of the activator inhibitor system, a  well-known  model  for  pattern  formation,  and  is  predicted to be essential for stomatal patterning.
Thus, we constructed and examined a mathematical model, which  is  based  on  experimental  results.  Our  model  can explain  many  experimental  observations,  such  as  that stomatal lineage cells are formed separately in the wild type but  are  clustered  in  tmm  mutant  and  er erl1 erl2  mutants (Figure  B).  Furthermore,  our  model  also  explains  the opposite  response  that  exogenous  application  of  EPF2 peptide completely eliminates stomatal differentiation in the wild  type  but  does  not  affect  the  tmm  mutant  (Figure  5C). These  results  suggest  that  stomatal  pattern  formation  is basically  understood  by  the  activator-inhibitor  mechanism, that is, local activation and lateral inhibition.

synchronous  and  near-equivalent  uptake  of  inorganic  cations  in  an arbuscular mycorrhizal fungus. New Phytol. 204, . Kojima,T., Saito, K., Oba,  ., Yoshida, Y., Terasawa, J., Umehara, Y., Suganuma, N., Kawaguchi, M., and Ohtomo, R. (201 ). Isolation and phenotypic  characterization  of  Lotus japonicus mutants  specifically defective in arbuscular mycorrhizal formation. Plant Cell Physiol. 55, 2 - 1Nagae, M., Takeda, N., and Kawaguchi, M. (201 ). Common symbiosis genes  CERBERUS  and  NSP1  provide  additional  insight  into  the establishment  of  arbuscular  mycorrhizal  and  root  nodule symbioses. Plant Signal. Behav. 9, e2 5 .
Sasaki,  T.,  Suzaki,  T.,  Soyano,  T.,  Kojima,  M.,  Sakakibara,  .,  and Kawaguchi, M. (201 ). Shoot-derived cytokinins systemically regulate root nodulation. Nature Commun. 5, .
Soyano, T.,  irakawa,  ., Sato, S.,  ayashi, M., and Kawaguchi, M. (201 ).  NODULE  INCEPTION  creates  a  long-distance  negative feedback  loop  involved  in  homeostatic  regulation  of  nodule  organ production. Proc. Natl. Acad. Sci. USA 111, 1 0 -1 12.
Suzaki,  T.,  Ito,  M., Yoro,  E.,  Sato,  S.,  irakawa,  .  Takeda,  N.,  and Kawaguchi,  M.  (201 ).  Endoreduplication-  mediated  initiation  of symbiotic  organ  development  in  Lotus japonicus.  Development  141, 2 1-2 5.
Wakabayashi, T., Oh,  ., Kawaguchi, M.,  arada, K., Sato, S., Ikeda, ., and Setoguchi,  . (201 ). Polymorphisms of E1 and GIGANTEA in wild populations of Lotus japonicus. J. Plant Res. 127,  51- 0. Yoro,  E.,  Suzaki,  T.,  Toyokura,  K.,  Miyazawa,  .,  Fukaki,  ., and  Kawaguchi,  M.  (201 ).  A  positive  regulator  of  nodule organogenesis, NODULE INCEPTION, acts as a negative regulator of rhizobial infection in Lotus japonicus. Plant Physiol. 165, - 5 . 

 
 
 
 
 
 
 

〔Original paper (E-publication ahead of print)〕akeda,  N.,  anda, Y.,  Tsuzuki,  S.,  Kojima,  M.,  Sakakibara,  .,  and  TKawaguchi, M.  Gibberellins  interfere  with  symbiosis  signaling  and gene expression, and alter colonization by arbuscular mycorrhizal fungi in Lotus japonicus. Plant Physiol. 201  Dec 1 . 〔Review articles〕

awaguchi, M. (201 ). Genes for autoregulaton of nodulation. In The  KLotus
japonicus Genome, S. Tabata and J. Stougaard, eds. (Springer), pp.  - .
Kawaguchi,  M.,  and  Sandal,  N.  (201 ).  Wild  accessions  and  mutant resources.  In The Lotus japonicus Genome, S. Tabata and J. Stougaard, eds. (Springer), pp. 211-220.
Soyano,  T.,  and  Kawaguchi,  M.  (201 ).  Systemic  regulation  of  root nodule  formation.  Review.  In  Advances  in  Biology  and  Ecology  of Nitrogen Fixation, T. Ohyama, ed. (InTech), pp.  -10 .
Suzaki, T., and Kawaguchi, M. (2014). Root nodulation  a developmental program involving cell fate conversion triggered by symbiotic bacterial infection. Curr. Opin. Plant Biol. 21, 1 -22.   Figure  .  (A)  Regulatory  network  for  the  differentiation  of  stomatal lineage cells. Our model can generate various stomatal patterns including the  wild  type,  tmm,  and  er erl1 erl2  (B),  and  also  explains  the  opposite response to EPF2 peptide between the wild type and tmm (C).

Publication List

〔Original papers〕

S.,  irakawa,  ., Sato, S.,  anda, Y., Saito, K., Kawaguchi,  CM., hungopast, Tajima,  S.,  and  Nomura,  M.  (201 ).  Transcriptomic  profiles  of nodule  senescence  in  Lotus japonicus  and  Mesorhizobium loti symbiosis. Plant Biotech. 31,  5. Daum, G., Medzihradszky, A., Suzaki, T., and Lohmann J.U. (201 ). A mechanistic  framework  for  noncell  autonomous  stem  cell  induction in Arabidopsis. Proc. Natl. Acad. Sci. USA 111, 1 1 -1 2 . Fujita,  ., Aoki, S., and Kawaguchi, M. (201 ). Evolutionary dynamics of  nitrogen  fixation  in  the  legume-rhizobia  symbiosis.  PLoS  One  9, e0.
Kikuchi,  Y.,  ijikata,  N.,  Yokoyama,  K.,  Ohtomo,  R.,  anda,  Y., Kawaguchi,  M.,  Saito,  K.,  and  Ezawa,  T.  (201 ).  Polyphosphate accumulation  is  driven  by  transcriptome  alterations  that  lead  to  near-

 
 
 

50

 

AB

A

Associate Professor

A A Ryu i

Visiting Scientist:

YOSHIDA, Akihiro

The  aim  of  this  laboratory  is  to  observe  the  variety  of morphogenetic  processes  that  occur  in  the  course  of  the ontogenesis  of  multicellular  organisms  and  to  analyze  the mechanisms  of  such  processes,  mainly  by  morphological methods.  The  accumulation  of  such  analyses  of  the embryogenetic  processes  of  related  species  is  expected  to provide  an  insight  into  the  evolution  of  morphogenetic processes.  This  laboratory  uses  the  wings  of  lepidopteran insects as the main target of morphological studies.

I. Wing outline shape formed by cell deathThe  wings  of  the  lepidopteran  insects  (butterflies  and moths)  develop  from  the  wing  imaginal  disc,  which  is  a hollow  sac  made  of  simple  epithelium.  Due  to  its  simple construction,  this  tissue  is  a  good  material  for  studying cellular interactions in the course of morphogenesis.
The outline shape of the adult wing is often different from that  of  the  pupal  wing.  This  difference  is  brought  about  by the programmed cell death of the marginal area of the pupal wing.  The  marginal  dying  area  is  called  the  degeneration region   and  the  internal  area  which  develops  into  the  adult wing is called  the differentiation region .
Cell death in the degeneration region proceeds very rapidly and finishes in a half-day to one-day period in Pieris rapae and in several other examined species. It was shown that the dying cells in the degeneration re gion have characteristics in common  with  the  apop totic  cell  death  in  mammalian  cells, such  as  fragmented  and  condensed  nuclei  containing  short DNA  fragments  detected  by  TUNEL  staining.  The  cells  in the  degeneration  region  are  actively  en gulfed  by  the macrophages in the cavity beneath the wing epithelium. The macrophages  seem  to  be  concentrated  beneath  the degeneration region by the strong adhesion between the basal surfaces  of  the  dorsal  and  ventral  epithelium  in  the differentiation  region,  which  occurs  during  prominent  cell death  and  excludes  macrophages  from  the  differentiation region. Thus realized concentration of macrophages seems to accelerate the shrinkage of the degeneration region. A  possible  physiological  role  of  cell  degeneration  at  the wing margin is to make space for the growth of the marginal scales.  Marginal  scales  are  extremely  elongated  scales  that grow  densely  on  the  edge  of  the  wing.  These  scales  are considered  to  be  important  in  stabilizing  turbulence occurring  posterior  to  the  wing.  The  movements  of  the marginal scales are closely monitored by sensory scales and bristles growing among them (Yoshida and Emoto,  ool. Sci. 28,  0- , 2011).
Trachea and tracheoles are both important in delivering air into  the  wing  and  their  patterns  coincide  with  that  of  the boundary  of  degeneration  and  differentiation  zones  at  the distal  end  of  the  wing. According  to  previous  observations, Figure  1. Adult  specimen  of Metzneria lappella, one  of  the  gelechiid moths examined, with long scales along the wing margin.

the pattern formation of wing epithelium is often dependent on tracheal  and  tracheole  patterns.  Basic  research  on  the development  of  tracheal  pattern  formation  is  being  done  by scanning  and  transmission  electron  microscopy  to  describe the  exact  pathway  and  time  course  of  the  formation  of  the elaborate  pattern  of  trachea  and  tracheoles  and  to  establish the  cytological  and  developmental  relationship  between  the formation  of  the  tracheal  pattern  and  the  epithelial  cell pattern. II. Wing morphogenesis and the growth of
marginal scales in small moths

In the small moths which have very long scales along their wing margins, the cuticle of the pupal wings does not appear to  be  large  enough  to  house  these  scales.  We  examined  the developmental process of the pupal wings of three species of the  small  gelechiid  moths  and  found  that,  concomitant  with the programmed cell deaths at the wing margin, there occurs a shrinkage of the differentiation region which has not been observed  in  large  winged  butterflies  and  moths.  The shrinkage concomitant with the cell deaths causes the space between  the  pupal  wing  and  the  cuticle  to  extend,  which appears to contribute to the growth of long marginal scales.

III. Other research activities

This laboratory also conducts morphological observation of various animal tissues by scanning and transmission electron microscopy  and  immuno-electron  microscopic  analyses  in collaboration  with  other  laboratories  of  NIBB.  Training  in specimen  preparation  and  instrument  operation  for  such observations is also given. 51National nstitute or asic iology

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II. Genetic dissection of migration ofprimordial germ cells in medaka

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TAKEHANA, Yusuke
SASADO, Takao
NAKAMOTO, Masatoshi
OKUYAMA, Teruhiro
NOZU, Ryo
CHISADA, Shinichi
KANEKO, Hiroyo
KOIKE, Yukari
TORII, Naoko
AJIOKA, Rie
KOIKE, Chieko
TESHIMA, Yuuko
HARA, Ikuyo
ISHIKAWA, Hiroe
TAKAKI, Chikako
SUZUKI, Tokiko

Medaka is a small egg-laying  secondary  fresh water fish found  in  brooks  and  rice  paddies  in  Eastern  Asia.  This species  has  a  long  history  as  an  experimental  animal, especially in Japan. Our laboratory has conducted studies on evolution of the sex determination system using medaka and relatives,  identification  of  the  causal  gene  of  mutants  for primordial  germ  cell  (PGC)  migration  and  pigment  cell development,  and  the  gonadal  development  of  medaka.  In addition to these activities, our laboratory is stepping forward to  lead  the  National  BioResource  Project  Medaka  (NBRP Medaka).

I. Evolution of the sex chromosome and sex
determination genes in Oryzias fish  Recent  studies  have  demonstrated  that Oryzias  species have  different  genetic  sex-determination  systems  (Y and W) (Figure 1). Furthermore, the sex chromosomes differ  in  their  origin  and  degree  of  differentiation.  These findings  suggest  the  repeated  creation  of  new  sex chromosomes  from  autosomes  during  evolution  of  Oryzias fishes, possibly in association with the formation of new sexdetermining  genes.  We  are  now  trying  to  positionally  clone the  novel  sex-determining  genes  in  these  species. Identification  of  these  genes  would  provide  a  clue  to understand  the  evolutionary  process  underlying  frequent turnover of sex determination mechanisms.

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

52Germ cells are responsible for the sustainability of life over generations  in  many  multicellular  animal  species.  To elucidate  the  mechanisms  underlying  the  development  of PGCs,  we  identified  multiple  mutations  affecting  the migration  and  development  of  the  primordial  germ  cells  in medaka in a prior large-scale mutagenesis screening project, and have analyzed a set of them to date. We focused on three mutants that have defects in primordial germ cell migration, kamigamo, shimogamo, and naruto that were isolated in the screening  project.  Positional  cloning  and  analysis  of  the genes  carrying  the  mutations  are  now  in  progress.  In addition,  two  mutations,  kamigamo  and  shimogamo,  cause cystic  pronephric  ducts  simultaneously  with  abnormal positioning  of  the  primordial  germ  cells.  Therefore,  the analysis of these mutations will be important in giving basal knowledge  underlying  the  mechanisms  of  human  cystic kidney diseases.

III. The study of type 2 diabetes using leptin
receptor knockout medaka

Leptin  in  mammals  is  a  peptide  hormone  secreted  by adipose  tissue.  It  has  been  shown  to  play  a  key  role  in  the maintenance of energy homeostasis through the regulation of food  intake  and  a  range  of  physiological  functions.  Mice with a deficiency of leptin or its receptor exhibit hyperphagia (an increase in food intake). The hyperphagia causes obesity leading to type 2 diabetes-like symptoms, which is consistent with  Caucasian  patients.  Leptin  has  also  been  isolated  from fish, including medaka, however, the amino acid sequence is poorly conserved between fish and mammals (11- 0 ), and fish leptins are expressed mainly in the livers. To clarify the function  of  leptin  on  fish,  we  generated  leptin  receptor knockout  (LepRKO)  medaka  by  the TILLING  method. The phenotypic  analyses  allowed  us  to  reveal  an  appetite suppressive function of leptin signaling on medaka as well as mammals,  and  to  find  new  value  in  medaka  as  a  novel animal  model  for  studying  type  2  diabetes. As  for  appetite suppressive  functions   LepRKO  medaka  showed  high expression  of  the  mRNA  of  NPY  ( .5-fold)  and  AgRP ( -fold), which are known to be orexigenic peptides, and an increase  in  food  intake  (1. -fold).  Next,  as  for  glucose metabolism  adult mutants showed signs of diabetes, such as fasting hyperglycemia and impaired insulin secretion, which is  a  late-onset  disorder  caused  by  excessive  feeding  during post-juvenile  stages.  Furthermore,  they  showed hyperglycemia  even  with  the  same  fat  level  in  the  blood, muscle, and liver as WT medaka. The symptom is consistent with those of Asian patients, not but Caucasian patients and mice  with  leptin  signaling  deficiencies.  Now,  we  are investigating  the  gene  expression  associated  with dysfunction  of  pancreatic  tissues  under  various  feeding conditions.  This  will  allow  us  to  identify  the  factors  of diabetes  that  are  sensitive  to  food  intake,  regardless  of obesity.

screening  system  employing  a  D  PCR  strategy  for evolutionary  studies,  and      the  TILLING  screening  system for promoting the reverse genetic approach.  NBRP Medaka aims  to  establish  a  first  rate  biological  resource  with  the highest possible levels of accessibility and ease of use. Publication List

〔Original papers〕

hisada, S., Kurokawa, T., Murashita, K., Ronnestad, I., Yaniguchi, T.,  CToyoda, A.,  Sakaki,  Y.,  Takeda,  S.,  and  Yoshiura,  Y.  (201 ).  Leptin Figure 1. Postbrandial blood glucose levels of WT and LepRKO medaka.

receptor-deficient (knockout) medaka, Oryzias latipes, show chronical up-regulated  levels  of  orexigenic  neuropeptides,  elevated  food  intake and  stage  specific  effects  on  growth  and  fat  allocation.  Gen.  Comp. Endocrinol. 195,  -20.
Guan, G.,  hang,  ., Naruse, K., Nagahama, Y., and  ong, Y. (201 ). Gene  Replacement  by  inc  Finger  Nucleases  in  Medaka  Embryos. Marine Biotechnol. 16, .
Kimura,  T.,  Nagao,  Y.,  ashimoto,  .,  Yamamoto-Shiraishi,  Y., Yamamoto, S., Yabe, T., Takada, S., Kinoshita, M., Kuroiwa, A.,  and Naruse,  K.  (201 ).  Leucophores  are  similar  to  xanthophores  in  their specification and differentiation processes in medaka. Proc. Natl. Acad. Sci. USA 111, .
Maruyama,  A.,  Oshima,  Y.,  Kajiura-Kobayashi,  .,  Nonaka,  S., Imamura, T., and Naruse, K. (201 ). Wide field intravital imaging by two-photon-excitation  digital-scanned  light-sheet  microscopy (2p-DSLM)  with  a  high-pulse  energy  laser.  Biomed.  Opt.  Express  5, 11- 25.
Nagao, Y.,  Suzuki,  T.,  Shimizu, A.,  Kimura,  T.,  Seki,  R., Adachi,  T., Inoue, C., Omae, Y., Kamei, Y., and  ara, I. (201 ). Sox5 functions as a  fate  switch  in  medaka  pigment  cell  development.  PLoS  Genet.  10, e100 2 .
Nishimura, T.,  erpin, A., Kimura, T.,  ara, I., Kawasaki, T., Nakamura, S., Yamamoto, Y., Saito, T.L., Yoshimura, J., and Morishita, S. (201 ). Analysis  of  a  novel  gene,  Sdgc,  reveals  sex  chromosome-dependent differences of medaka germ cells prior to gonad formation. Development 141, .
Okuyama,  T.,  Yokoi,  S.,  Abe,  .,  Isoe,  Y.,  Suehiro,  Y.,  Imada,  ., Tanaka, M., Kawasaki, T., Yuba, S., Taniguchi, Y., Kamei, Y., Okubo, K.,  Shimada,  A.,  Naruse,  K.,  Takeda,  .,  Oka,  Y.,  Kubo,  T.,  and Takeuchi,  . (201 ).  A  neural  mechanism  underlying  mating preferences for familiar individuals in medaka fish. Science 343,  1- .
Spivakov, M., Auer, T.O., Peravali, R., Dunham, I., Dolle, D., Fujiyama, A., Toyoda, A., Aizu, T., Minakuchi, Y., and Loosli, F. (201 ). Genomic and  Phenotypic  Characterization  of  a  Wild  Medaka  Population  Towards the Establishment of an Isogenic Population Genetic Resource in Fish. G -Genes Genom. Genet. 4, - 5.
Takehana, Y., Matsuda, M., Myosho, T., Suster, M.L., Kawakami, K., Shin, T., Kohara, Y., Kuroki, Y., Toyoda, A., and Fujiyama, A. (201 ). Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena. Nature Commun. 5,  15 .
Tsuboko,  S.,  Kimura,  T.,  Shinya,  M.,  Suehiro,  Y.,  Okuyama,  T., Shimada, A., Takeda,  ., Naruse, K., Kubo, T., and Takeuchi,  . (201 ). Genetic  Control  of  Startle  Behavior  in  Medaka  Fish.  PLoS  One  9, e11252 .
hang,  .,  Guan,  G.,  Chen,  J.,  Naruse,  K.,  and  ong,  Y.  (201 ). Parameters  and  efficiency  of  direct  gene  disruption  by  inc  Finger Nucleases in Medaka Embryos. Marine Biotechnol. 16, 125-1 .

 
 
 
Figure  2.  Relation  between  visceral  fat  and  blood  glucose  levels  of WT and LepRKO medaka.

V. National BioResource Project Medaka(NBRP Medaka) (http://www.shigen.nig.ac.
jp/medaka/)Figure  . NBRP Medaka website.

In  200 ,  NIBB  was selected  as  the  core f a c i l i t y   o f   N B R P 
M e d a k a .   O u r laboratory is taking an active  part  in  this project.  With  the  goal of  facilitating  and enhancing  the  use  of medaka  as  a  model organism, we provide, maintain  and  collect living  resources  such as  standard  strains, inbred  strains,  and mutants  in  addition  to frozen  resources  such as  EST cDNA,  BAC  Fosmid  clones,  and hatching  enzymes,  as well  as  integrated i n f o r m a t i o n   o n medaka  (Figure  ). W e   h a v e   b e e n providing BAC clones of  medaka  related species,    a  library 

 
 
 
 
 
 
 

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KAMADA,YoshiakiNutrients are indispensable for life. Thus, perception of the nutrient environment is also essential for cells. To recognize cellular  nutrient  conditions,  eukaryotic  cells  employ  Tor (target  of  rapamycin)  protein  kinase. Tor  forms  two  distinct protein  complexes,  TORC  (Tor  complex)  1  and  TORC2. TORC1  regulates  rapamycin-sensitive  branches  of  the  TOR pathway, such as protein synthesis, cell cycle and autophagy. TORC1  is  thought  to  act  as  a  nutrient  sensor,  because rapamycin,  a  TORC1  inhibitor,  mimics  a  starved  condition. On the other hand, TORC2, whose function is insensitive to rapamycin,  is  responsible  for  actin  organization  and  cell integrity. So far, it is not clear whether TORC2 also perceives nutrient signals.
The  aim  of  our  research  group  is  to  reveal  the  molecular mechanisms  of  how  Tor  receives  nutrient  signals  and  how the TORC1 2 pathways regulate each phenomenon. We have been  studying  Tor  signaling  in  the  budding  yeast Saccharomyces cerevisiae,  and  have  found  three  novel branches of the TOR signaling pathway (Figure 1). 

I. TORC1 phosphorylates Atg13, the molecularswitch of autophagyAutophagy  is  mainly  a  response  to  nutrient  starvation,  and TORC1 negatively regulates autophagy. The Atg1 kinase and its  regulators,  i.e.  Atg1 ,  Atg1 ,  Atg2 ,  and  Atg 1 collaboratively  function  in  the  initial  step  of  autophagy induction, downstream of TORC1. Atg1 is a Ser Thr protein kinase, the activity of which is essential for autophagy and is largely enhanced following nutrient starvation or the addition of  rapamycin.  This  regulation  involves  phosphorylation  of Atg1 . We found that Atg1  is directly phosphorylated by TORC1. Phosphorylated Atg1  (during nutrient-rich conditions) loses its affinity to Atg1, resulting in repression of autophagy. On the  other  hand,  under  starvation  conditions  Atg1   is immediately  dephosphorylated  and  binds  to Atg1  to  form Atg1  complex.  Atg1  complex  formation  confers  Atg1 activation  and  consequently  induces  autophagy.  We determined phosphorylation sites of Atg1  and generated an unphosphorylatable Atg1   mutant  (Atg1 - SA).  Expression of Atg1 - SA  induces  autophagy  bypassing  inactivation  of TORC1,  the  same  as  through  starvation  treatment  or rapamycin.  These  results  demonstrate  that Atg1   acts  as  a molecular switch for autophagy induction.

II. Monitoring in vivo activity of TORC1 byphosphorylation state of Atg13

Since Atg1  has turned out to be a substrate of TORC1, invivo  activity  of  TORC1  can  be  monitored  by  the phosphorylation  state  of  Atg1 .  arious  conditions  and mutants  have  been  examined  to  determine  what  kind  of nutrients  TORC1  recognizes  and  how  nutrient  signals  are transmitted to TORC1.

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Figure  1.  Tor  signaling  pathway  of  the  budding  yeast.  Our  group  has found three branches of the Tor pathway. III. How and why TORC1 regulates mitotic
entry?

TORC1  regulates  protein  synthesis,  which  is  important  for promotion  of  the  cell  cycle  at  G1  (G0).  Little  is  known, however,  about  whether  or  not TORC1  is  involved  in  other stages of the cell cycle. We  generated  a  temperature-sensitive  allele  of  KOG1
(kog1-105),  which  encodes  an  essential  component  of TORC1,  and  found  that  TORC1  plays  an  important  role  in mitotic entry (G2 M transition). Since Cdc5, the yeast polokinase,  is  mislocalized  and  inactivated  in  kog1-105 mutant cells, TORC1 mediates G2 M transition via regulating polokinase.  Recently  we  discovered  a  physiological  role  of TORC1  in  mitosis   autophagy  negatively  controlled  by TORC1  plays  an  important  part  in  maintenance  of  genome stability under starvation conditions.  

IV. Ypk2 kinase acts downstream of TORC2 tocontrol actin organization

Genetic  studies  have  shown  that  TORC2  controls  polarity of  the  actin  cytoskeleton  via  the  Rho1 Pkc1 MAPK  cell integrity cascade.  owever, the target (substrate) of TORC1 was  not  yet  identified.  We  found  that Ypk2,  an  lethality  of TORC2 dysfunction, is directly phosphorylated by TORC2.

Publication List

〔Original paper〕

 Sekiguchi, T., Kamada, Y., Furuno, N., Funakoshi, M., and Kobayashi, .  (201 ).  Amino  acid  residues  required  for  Gtr1p-Gtr2p  complex formation  and  its  interactions  with  the  Ego1p-Ego p  complex  and TORC1 components in yeast. Genes Cells 19, 
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MANO, Shoji
KANAI, Masatake
WATANABE, Etsuko
KAMIGAKI, Akane
HIKINO, Kazumi
YAMAGUCHI, Chinami
NAKAI, Atsushi
KATO, Kyoko
UEDA, Chizuru

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

Peroxisomes  are  single-membrane  bounded  organelles, which  are  ubiquitously  present  in  eukaryotic  cells,  and  they are  involved  in  various  biological  processes  such  as  lipid metabolism and photorespiration. To understand peroxisome biogenesis and functions, we have been analyzing a number of  Arabidopsis  mutants  having  aberrant  peroxisome morphology  (apem  mutants)  and  peroxisome  unusual poisoning (peup mutants). To date, APEM1, 2, 3, 4, 9 and 10, and PEUP1, 2 and 4 genes were identified, and based on the characterization  using  their  gene-products  a  part  of  the mechanism of division, protein transport and degradation of peroxisomes, were revealed.
Recently,  we  found  that  peroxisome  functions  and biogenesis  are  involved  in  the  reproductive  process. Therefore,  peroxisomes  in  gametes  and  gametophytes  were visualized,  and  their  dynamics  are  currently  under investigation (Figure 1).

II. Accumulation mechanism of seed storage

proteins

Plant  seeds  accumulate  huge  amounts  of  storage  reserves such  as  oils,  carbohydrates  and  proteins.  umans  use  these storage  reserves  as  foods,  feed,  and  industrial  materials. Storage  reserves  are  different  among  different  plant  seeds. Wheat,  maize  and  rice  seeds  mainly  accumulate  starch, whereas  rapeseed,  pumpkin  and  sesame  contain  large amounts  of  oils.  Soybean  contains  proteins  as  a  major reserve.  We  are  analyzing  Arabidopsis  mutants  showing different  patterns  of  oil protein  ratios  to  elucidate  the mechanisms controlling oil and protein contents in seeds. III. Construction of The Plant Organelles
Database 3 (PODB3)PODB   was  built  to  promote  a  comprehensive understanding of organelle dynamics. PODB  consists of six individual  units   the  electron  micrograph  database,  the perceptive  organelles  database,  the  organelles  movie database,  the  organellome  database,  the  functional  analysis database,  and  external  links. Through  these  databases,  users can  obtain  information  on  plant  organelles’  responses  to environmental  stimuli  of  various  tissues  of  several  plant species,  at  different  developmental  stages.  We  expect  that PODB   will  enhance  the  understanding  of  plant  organelles among researchers.

Publication List

〔Original papers〕

oto-Yamada, S., Mano, S., Nakamori, C., Kondo, M., Yamawaki, R.,  GKato, A., and Nishimura, M. (201 ). Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy. Plant Cell Physiol. 55,  2.
Mano, S., Nakamura, T., Kondo, M., Miwa, T., Nishikawa, S., Mimura, T.,  Nagatani,  A.,  and  Nishimura,  M.  (201 ).  The  Plant  Organelles Database    (PODB )  update  201   integrating  electron  micrographs and new options for plant organelle research. Plant Cell Physiol. 55, e1.
Shibata,  M.,  Oikawa,  K.,  Mano,  S.,  and  Nishimura,  M.  (201 ). Measurement of the number of peroxisomes. Bio-Protoc. 4, e12 .

  〔Original paper (E-publication ahead of print)〕otomura,  K.,  Le,  .T.N.,  amada,  T.,  Kutsuna,  N.,  Mano,  S.,  MNishimura, M., and Watanabe, Y. Diffuse DCP2 accumulates in DCP1 foci under heat stress in Arabidopsis thaliana. Plant Cell Physiol. 201  Oct 22.

〔Review articles〕

oto-Yamada,  S.,  Mano,  S.,  and  Nishimura,  M.  (201 ).  Interaction  Gbetween chaperone  and  protease  functions  of  LON2,  and  autophagy during the functional transition of peroxisomes. Plant Signal. Behav. 9, 
e2
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Goto-Yamada, S., Mano, S., Oikawa, K., Shibata, M., and Nishimura, M. (201 ). The role of peroxisomes in plant reproductive processes. In Sexual  reproduction  in  animals  and  plants.    Edited  by  Sawada,  ., Inoue, N., and Iwano, M. Springer Japan, pp.  1 - 2 .
Shibata, M., Oikawa, K., Yoshimoto, K., Goto-Yamada, S., Mano, S., Yamada, K., Kondo, M.,  ayashi, M., Sakamoto, W., Ohsumi Y., and Nishimura, M. (201 ). Plant autophagy is responsible for peroxisomal transition and plays an important role in the maintenance of peroxisomal quality. Autophagy 10, 
.

 
 
Figure 1.  isualization of peroxisomes in pollen and pollen tube.
The fusion gene of GFP with peroxisome targeting signal 1 is expressed in the wild-type (A) and apem2 pex13 plants (B). (C) Peroxisomes move toward the tip region during pollen tube growth.

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OHNO, Kaoru

The  aim  of  this  laboratory  is  to  research  reproductive hormones in invertebrates and to analyze 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, Asterina
pectinifera

Gonadotropins  play  important  regulatory  roles  in reproduction  in  both  vertebrates  and  invertebrates.  The vertebrate  gonadotropins,  L   and  FS   are  structurally  and functionally  conserved  across  various  species,  whereas  no such molecule has been identified in invertebrates. The insect parsin  hormones  are  assumed  to  be  the  physiological counterpart of L  and FS  in mammals. Some gonadotropic hormones,  such  as  the  egg  development  neurosecretory hormone of the mosquito, the egg-laying hormone of the sea hare,  and  the  androgenic  gland  hormone  of  the  terrestrial isopod,  have  been  found  in  invertebrate  species.  More recently,  an  insulin-like  peptide  was  reported  to  be responsible  for  the  regulation  of  egg  maturation  in  the mosquito,  Aedes aegypti,  demonstrating  the  involvement  of insulin signaling in egg maturation among invertebrates.
The  gonad-stimulating  substance  (GSS)  of  an  echinoderm, the  starfish,  was  the  very  first  gonadotropin  to  be  identified in invertebrates. GSS mediates oocyte maturation in starfish by  acting  on  the  ovary  to  produce  the  maturation-inducing hormone (MI ), 1-methyladenine, which in turn induces the maturation of the oocytes. In this sense, GSS is functionally identical to vertebrate L , especially piscine and amphibian L s,  acting  on  the  ovarian  follicle  cells  to  produce  MI   to induce  the  final  maturation  or  meiotic  resumption  of  the oocyte. Considering the functional similarity that GSS shares with vertebrate L , it is very important from an evolutionary point of view to know the chemical and molecular structure of GSS. We cloned the gene encoding GSS referred to amino acid  sequence  of  purified  GSS  from  radial  nerves  of  the starfish,  Asterina pectinifera.  Interestingly,  phylogenetic analyses  revealed  that  it  belonged  to  the  insulin insulin-like growth factor (IGF) relaxin superfamily and, more precisely, to the subclass of relaxin insulin-like peptides (Figure 1).

5Figure  1. Amino  acid  sequence  of  starfish  GSS.  (A)  Comparison  of  the heterodimeric  structure  of  starfish  GSS  with  those  of  various representative members of the insulin superfamily. The cysteine bridges are  shown  in  red.  (B)  Coding  DNA  sequence  and  predicted  amino  acid sequences of GSS. Sequences of A and B chains are shown in green and blue boxes, respectively. Characters shown in red boldface indicate basic dipeptides  that  are  the  sites  of  proteolytic  cleavage.  Inverted  triangle shows the deduced cleavage site of the signal peptide.

II. Search for reproductive hormones ininvertebrates

In  a  collaborative  effort  with  Prof. Yoshikuni’s  Laboratory of  the  Kyushu  Univ.  and  Dr. Yamano  and  Dr. Awaji  of  the National  Research  Institute  of  Aquaculture,  Fisheries Research Agency (NRIA), we are searching for reproductive hormones in invertebrates  sea urchin, sea cucumber, oyster, and  shrimp.  While  the  collaborators  are  partially  purifying physiological  materials  which  induce  egg  maturation  from nerve  extracts  and  analyzing  them  with  a  tandem  mass spectrometer,  we  are  creating  and  analyzing  EST  libraries from  nerve  tissues  and  developing  a  database  of  the  mass analysis performed in this laboratory.

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While  genomic  structures  as  well  as  their  genetic information  appear  to  stably  transmit  into  daughter  cells during  cell  division,  and  also  into  the  next  generation,  they can  actually  vary  genetically  and or  epigenetically.  Such variability  has  had  a  large  impact  on  gene  expression  and evolution.  To  understand  such  genome  dynamisms  in eukaryotes,  especially  in  plants,  we  are  characterizing  the flower pigmentation of morning glories.

I. Flower pigmentation patterns of themorning glories

Ipomoea nil  (Japanese  morning  glory),  I. purpurea (the common  morning  glory),  and  I. tricolor  have  been domesticated  well  as  floricultural  plants,  and  their  various spontaneous  mutations  have  been  isolated.  The  wild  type morning  glories  produce  flowers  with  uniformly  pigmented corolla,  whereas  a  number  of  mutants  displaying  particular pigmentation  patterns  have  been  collected.  Because  flower pigmentation  patterns  are  easily  observed,  the  molecular mechanisms underlying these phenomena provide fine model systems for investigating genome variability.
Margined,  Rayed and  Blizzard of  I. nil are  dominant mutations.  While  these  mutants  show  distinct  flower pigmentation  patterns,  the  same  pigmentation  gene  is repressed  by  non-coding  small  RNA  in  the  whitish  parts  of the  corolla.  It  is  suggested  that  distinct  regulation  of  small RNA  causes  the  difference  in  pigmentation  patterns.  The recessive  mutations,  duskish of  I. nil and  pearly-v  of  I.
tricolor, confer  variegated  flowers,  and  epigenetic mechanisms are thought to regulate flower pigmentation. We are  currently  characterizing  detailed  molecular  mechanisms of these mutations. II. de novo sequencing of Japanese morning
glory genome

Although morning glories are studied worldwide, especially in  plant  physiology  and  genetics,  no  whole  nuclear  genome sequences of any Ipomoea species are available. To facilitate the  studies  of  our  group  as  well  as  all  morning  glory researchers,  we  are  conducting  de novo genome  sequencing of I. nil. We chose the Tokyo-kokei standard line for genome sequencing, and employed shotgun sequencing using a single molecule  real  time  sequencing  system.  We  could  recently obtain thousands  of assembled sequences having reasonable size,  and  are  going  to  characterize  details  of  the  genome sequence.

flower  pigmentation,  and  DNA  clones  from  EST  and  BAC libraries of I. nil and its related species. I. nil has been one of the most popular floricultural plants since the late Edo era in Japan. It has an extensive history of genetic studies and also has many advantages as a model plant  simple genome, large number  of  mutant  lines,  and  efficient  self-pollination.  Our collection includes 2 0 lines and 15 ,000 DNA clones.

IV. A novel flower color enhancing protein

Anthocyanin  is  a  type  of  flavonoid  pigment  and  is responsible  for  the  colors  of  many  flowers.  The  depth  of flower color is determined by the amount of anthocyanin. We characterized  I. nil  mutants  displaying  pale  colored  flowers (Figure  1),  and  successfully  isolated  a  novel  enhancer  of flavonoid  production  (EFP).  The  EFP  deficient  mutants  fail in  efficient  flavonoid  production  resulting  reduction  of anthocyanin  accumulation.  The  presence  and  action  of  EFP are  conserved  among  diverse  plants.  The  amino  acid sequence  of  EFP  resembles  an  enzyme  catalyzing  flavonoid biosynthesis, however, it has no enzymatic activities. We are currently  studying  a  molecular  mechanism  of  flavonoid production enhancement by EFP.

Figure  1.  Wild  type  (right)  and  EFP  deficient mutant  of  Japanese morning glory (left).

Publication List

〔Original papers〕

araco, M., Spelt, C., Bliek, M.,  erweij, W.,  oshino, A., Espen, L.,  FPrinsi, B.,  Jaarsma,  R.,  Tarhan,  E.,  de  Boer, A. .,  Di  Sansebastiano, G.-P., Koes, R., and  uattrocchio, F.M. (201 ).  yperacidification of vacuoles  by  the  combined  action  of  two  different  P-ATPases  in  the tonoplast determines flower color. Cell Rep. 6,  2- .
Morita, Y., Takagi, K., Fukuchi-Mizutani, M., Ishiguro, K., Tanaka, Y., Nitasaka, E., Nakayama, M., Saito, N., Kagami, T.,  oshino, A., and Iida, S. (201 ). A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation. Plant J. 78, 2 - 0 .
Park,  K.I.,  oshino,  A.,  Saito,  N.,  and  Tatsuzawa,  F.  (201 ). Anthocyanins in the flowers of Ipomoea tricolor Cav. (Convolvulaceae). Biochem. Syst. Ecol. 54, 15-1 .

  III. BioResource of morning glories

NIBB  is  the  sub-center  for  National  BioResource  Project (NBRP) for morning glory. In this project, we are collecting, maintaining  and  distributing  standard  lines,  mutant lines for 5National nstitute or asic iology

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mutant  (k)  exhibiting  the  neck  leaf-enveloped  panicle phenotype, like the NECK LEAF1 mutant cloned by Wang et al.  (200 ),  is  observed.  The  mutant  shown  in  (l)  displays  a phenotype similar to that of a PLASTC RON mutant. Active  DNA  transposons  are  important  tools  for  gene functional  analysis.  The  endogenous  non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate  various  transposon-insertion  mutants  because nDart1-0  elements  tend  to  insert  into  genic  regions  under natural  growth  conditions.  The  transpositions  of  nDart1-0 were promoted by an active autonomous element, aDart1-27, on chromosome  . By using the endogenous nDart1 aDart127  system  in  rice,  a  large-scale  nDart-inserted  mutant population  was  easily  generated  under  normal  field conditions,  and  the  resulting  tagged  lines  were  free  of somaclonal variation.

I. nDart1-0-Inserted Mutants  It  is  essential  to  develop  an  easy  detection  method  of  the nDart1-0-inserted gene for forward and reverse genetics. For high-copy-number  elements,  transposon  display  (TD)  based on  an  amplified  fragment-length  polymorphism  (AFLP) technique  is  a  powerful  tool  to  visualize  multiple transposons. We successfully visualized 1  copies of nDart1 of Nipponbare through an nDart1-optimized TD method. In addition,  an  nDart1-0-specific  iPCR  procedure  was  also developed  ( ayashi-Tsugane  etl  al.  201 ). Among  , lines, visible phenotypes were observed at the seedling, post- transplanting, reproductive, and mature stages. Fifty percent of  the  total  tagged  lines  showed  mutant  phenotypes. Abnormal  growth  phenotypes,  in  particular,  were  easily detectable  at  the  seedling  stage,  whereas  both  sterility  and dwarf  phenotypes  were  frequently  observed  at  the  mature stage. Some of these mutant phenotypes are shown in Figure 1. A  variegated  albino  plant  can  survive  under  normal  field conditions  (a). Although  chlorophyll  mutants  that  revert  to the  wild  phenotype  are  also  observed  at  the  seedling  stage, these  zebra  mutants  (b  and  c)  retain  a  cross-banded phenotype  even  at  the  reproductive  stage.  A  mutant  (d) abnormally elongates the leaf and stem at the seedling stage, somewhat  similar  to  the  Gibberella fujikuroi-infected  rice plant.  Fuchsia  leaf  blades  with  revertant  sectors  (e)  are observed  under  U   light.  This  is  similar  to  the  hcf  (high chlorophyll  fluorescence)  mutant  in  maize.  Under  normal growth  conditions,  this  mutant  shows  pale-green  leaves  and is  lethal  at  the  seedling  stage.  A  mutant  displaying  an extremely short-statured phenotype (f) is named Thumbelina  this  phenotype  must  be  caused  by  the  insertion  of  a  DNA transposon because of its mutable phenotype (right in f), as a normally  elongated  shoot  can  often  be  observed  in  the thumbelina  mutant  (Tsugane  et  al.,  200 ). A  semi-dominant mutant (g) shows a very short panicle with many tillers, and its  heterozygous  plant  exhibits  an  intermediate  phenotype between the wild type and the mutant. A mutant (right in h) has  an  abnormal  ligule,  resulting  in  an  erect  leaf. The  short panicle phenotype is shown in (i) (right). The mutant shown in  (j)  expresses  multiple  glumes  and  high  sterility,  and  a 

5Figure 1. Phenotypes of nDart-tagged line mutants. Scale bar   10 cm.

Publication List

〔Original paper〕

ayashi-Tsugane, M., Takahara,  ., Ahmed, N.,  imi, E., Takagi, K.,  Iida, S., Tsugane, K., and Maekawa, M. (201 ). A mutable albino allele in rice reveals that formation of thylakoid membranes requires SNOWW ITE LEAF1 gene. (201 ). Plant Cell Physiol. 55,  -15. 

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JOHZUKA, Katsuki
ISHINE, NaomiChromosome  condensation  is  a  basic  cellular  process  that ensures  the  faithful  segregation  of  chromosomes  in  both mitosis  and  meiosis.  This  process  is  required  not  only  for shrinking  the  length  of  chromosome  arms,  but  also  for resolving  entanglements  between  sister-chromatids  that  are created  during  DNA  replication. Any  abnormality  in  this process leads to segregation errors or aneuploidy, resulting in cell lethality. Chromosome condensation is mainly achieved by  condensin,  a  hetero-pentameric  protein  complex,  widely conserved  from  yeast  to  humans.  Despite  its  conservation and  importance  for  chromosome  dynamics,  how  condensin works  is  not  well  understood.  Recent  studies  reveal  that condensin  functions  are  not  restricted  to  chromosome condensation  and  segregation  during  cell  divisions.  It  is required  for  diverse  DNA  metabolism  such  as  genome stability, transcriptional regulation, and cell differentiation.
Our  research  interest  is  to  understand  the  mechanism  and regulation  of  chromosome  condensation.  We  have  been studying  the  role  of  condensin  in  the  budding  yeast Saccharomyces cerevisiae.  Microscopic  observation indicated the nucleolar localization of condensin. Consistent with this, the ribosomal RNA gene (rDNA) repeat is the most condensed  region  in  the  genome  during  mitosis.  We  have 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  II  in  budding  yeast.  We further  discovered  the  multiple  protein  network  required  to recruit condensin to the RFB site.

with the ectopic RFB site. To explore the role of condensin in chromosome  organization,  we  have  constructed  a  strain  in which two RFB sites are inserted on an ectopic chromosome arm  with  an  interval  of  15kb  distance  in  the  cell  with complete  deletion  of  the  chromosomal  rDNA  repeat.  Using this  strain,  condensin-dependent  chromatin  interaction between  two  RFBs  was  examined  by  chromosome conformation  capture  ( C)  assay.  We  found  condensindependent  chromatin  interaction  between  the  two  RFB  sites on the chromosome arm. This result indicates that condensin plays  a  role  in  chromatin  interaction  between  condensin binding  sites,  and  this  interaction  leads  to  creation  of  a chromatin  loop  between  those  sites  (Figure  1).  It  is  thought that  condensin-dependent  chromatin  folding  is  one  of  the basic  molecular  processes  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 internal regions of two RFBs increases  in  anaphase. Thus,  the  configuration  of  chromatin fiber changes from a simple loop into a complicated twisted shape  as  the  cell  cycle  progresses  from  metaphase  to anaphase. I. Dynamic relocalization of condensin duringmeiosisOur  genetic  screening  indicated  that  two  proteins,  Csm1 and  Lrs ,  were  required  for  condensin  recruitment  to  the RFB  site.  Physical  interactions  between  Csm1 Lrs   and subunits  of  condensin  are  important  for  recruitment  of condensin  to  the  RFB  site.  These  proteins  are  known  as components of the monopolin complex that are required for faithful  segregation  of  homologous  chromosomes  during meiotic division I. During meiosis I, the monopolin complex re-localizes from the rDNA repeat to the centromere and acts for  ensuring  sister-chromatid  co-orientation.  Re-localization of  Csm1 Lrs   proteins  suggested  the  re-localization  of condensin  from  rDNA  repeat  to  centromere. As  expected, chromatin-IP  experiments  indicated  that  condensin re-localizes  to  the  centromere  during  meiosis  I.  Condensin might clamp sister-chromatids together during meiosis I.

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

II. Condensin-dependent chromatin folding

The RFB site, which consists of a  150bp DNA sequence, is  functioning  as  a  cis-element  for  recruitment  of  condensin to chromatin in the yeast genome. If the RFB site is inserted into an ectopic chromosomal locus, condensin can associate 5National nstitute or asic iology

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Organogenesis is accomplished by a series of deformations of the planar cell sheet into a three-dimensional shape during embryogenesis. This drastic structural change is an integrated result  of  individual  cell  behaviors  in  response  to  spatiotemporally controlled mechanisms. To  better  understand  the  programs  underlying  organ formation,  it  is  required  to  analyze  individual  cells’ morphology  and  dynamics  quantitatively.  owever,  due  to the  massive  images  generated  by  D  microscopy  and  their ambiguity, this made it difficult to perform these analyses. To  unveil  organogenesis  from  the  point  of  view  of  distinct cell  behaviors,  we  developed  software  applications  that  are capable  of  describing  cell  dynamics  out  of  D  time-lapse imaging data sets by employing several approaches. I. Automated cell tracking system

Epithelial  morphogenesis  in  the  fruit  fly  Drosophila melanogaster embryo is considered to be an excellent model for  collective  cell  migrations.  Drastic  cell  rearrangements lead  drastic  structural  changes  to  build  elaborate  tubular organs  such  as  the  tracheal  network.  We  developed  a software pipeline, which automatically recognizes individual cell  shapes  out  of  D  space  and  tracks  them  through  time. This  system  extracts  cell  boundaries  and  reconstitutes  cell shapes as a skeletonized chain of voxels spanning  D space. This  abstract  form  of  cell  visualization  makes  it  possible  to describe  morphometric  quantities  and  kinetics  of  cells  in single-cell resolution (Figure 1). Obtained cell dynamics are subjected  to  comparative  analyses  among  wild  type  and several  mutants  to  unveil  genetic  programs  underlying organogenesis.

structural  characteristics,  such  as  uneven  fluorescence distributed over the specimen and tracks these patterns along a  time-series.  Despite  that  the  tissue  was  labeled  with  nontargeted cytoplasmic GFP, this tracking software successfully outlined developmental dynamics of Xenopus neuroectoderm (Figure 2). Figure  2.  Collective  cell  migration  of  Xenopus neuroectodermal  cells visualized  as  optical  flow  along  a  time-series. A  modified  PI   method successfully  tracks  uneven  subcellular  distribution  of  GFP  signals  over time.  Dr.  M.  Suzuki  (Prof.  Ueno’s  laboratory  at  NIBB)  performed  the microscopy.

III. A GUI application for manual image
quantification

Biologically  significant  image  features  are  not  always significant to computational algorithms due to their structural instability.  This  kind  of  difficulty  requires  human  eye inspection  for  feature  extractions. A  GUI  (Graphical  User Interface)  application  we  developed  can  easily  visualize  D imaging data and has made manual feature annotations easy (Figure  ). This application is freely available at our website (https is.cnsi.jp ).

II. Particle tracking for tissue deformation
analysisBesides  cell  boundary  extraction,  we  also  developed  a derived algorithm for particle image velocimetry (PI ). This system  is  designed  to  measure  tissue  deformation  even though the imaging constraints do not allow identification of individual  cells  out  of  images.  This  implementation  detects Figure  1.  isualized  apical  cell  surface  of  Drosophila  embryonic epidermal cells. A time-lapse confocal microscopic data set of fly embryo expressing  E-cadherin-GFP  was  subjected  to  our  automatic  cell  shape recognition system. Cell boundaries (green), center of gravity (blue) and normal vector (magenta) are indicated for each cell. 0Figure  . A lightweight native  D stack viewer equipped with functions for manual feature extraction.

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Image  processing  methods  significantly  contribute  to visualization of biomedical targets acquired from a variety of imaging  techniques,  including   wide-field  optical  and electron microscopy,  -ray computed tomography, magnetic resonance  imaging  and  mammography.  uantitative interpretation  of  the  deluge  of  complicated  biomedical images, however, poses many research challenges.  We have developed  new  computational  methods  based  on mathematical  morphology  for  quantitative  image  analysis. One of the most important purposes of image processing is to derive  meaningful  information,  which  is  expressed  as structural properties in images. Mathematical morphology is a nonlinear image processing method based on the set theory and  is  useful  for  the  extraction of  structural  properties from an  image.  It  can  be  used  as  a  fundamental  tool  to  analyze biomedical images.

chest  -ray images. In  this  study,  we  have  developed  a  novel  RMP-based contrast  enhancement  method.  The  goal  of  the  study  is  to enable enhancement of fine morphological features of a mass lesion with high suppression of surrounding tissues, such as mammary  glands.  The  method  uses  a  top-hat  contrast operator,  a  well-known  and  commonly  used  morphological operation  for  extracting  local  features  from  a  low-contrast image.  The  proposed  method  involves  three  steps   (1) selective  extraction  of  target  features  by  mathematical morphology,  (2)  enhancement  of  the  extracted  features  by two contrast modification techniques and ( ) segmentation of the  target  region  (mass  lesion)  by  using  an  automatic thresholding technique. The effectiveness of the method was quantitatively  evaluated  by  the  contrast  improvement  ratio (CIR) and its usefulness was demonstrated by applying it to various  types  of  medical  images.  The  results  prove  that  the proposed  method  enables  specific  extraction  and enhancement  of  mass  lesions,  which  is  essential  for  clinical diagnosis based on medical image analysis. Figure 1 shows a 
mammographic image enhanced by the proposed method.

I. Novel image processing method based onmathematical morphology

Image  processing  is  a  crucial  step  in  the  quantification  of biomedical  structures  from  images.  As  such,  it  is fundamental  to  a  wide  range  of  biomedical  imaging  fields. Image processing derives structural features, which are then numerically  quantified  by  image  analysis.  Contrast enhancement plays an important role in image processing  it enhances structural features that are barely detectable to the human eye and allows automatic extraction of those features. To  effectively  recognize  a  region  of  interest,  specific  target structures  must  be  enhanced  while  surrounding  objects remain unmodified. A contrast enhancement technique which uses  mathematical  morphology  enables  selective enhancement  of  target  structures.  Based  on  set  theory, mathematical  morphology  applies  shape  information  to image processing.
Mathematical  morphology  operates  by  a  series  of morphological  operations,  which  use  small  images  called structuring  elements  (typically,  a  single  structuring  element is used). The structuring element acts as a moving probe that samples  each  pixel  of  the  image.  Since  the  structuring element  moves  in  a  fixed  direction  across  the  image,  some intricate images (in particular, those whose structural details contain  a  variety  of  directional  characters)  may  not  be properly processed. Consequently, an artifact in the shape of structuring  elements  may  be  generated  at  the  object periphery.  Since  objects  in  biomedical  images  consist  of delicate  structural  features,  this  drawback  is  an  especially serious problem.
To overcome this problem, we have proposed an extension of  conventional  mathematical  morphology  called  rotational morphological processing (RMP). RMP based morphological filters  have  been  applied  to  a  wide  variety  of  biomedical images,  including  electron  micrographs,  light  micrographs and  medical  images  such  as  mammographic  images  and Figure  1.  Image  enhancement  and  target  detection  achieved  by  the proposed  method  for  a  mass  lesion  in  a  mammographic  image.  The images  were  obtained  from  the  Mammographic  Image Analysis  Society (MIAS)  database.  (a)  Original  mammographic  image  (mdb1 ).  (b) Enhancement  result  of  candidates  of  mass  lesion  obtained  by  the proposed method. (c) Detection result of the lesion region.

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MIYAGAWA, Shinichi
OGINO, Yukiko
MIYAKAWA, Hitoshi
HIRUTA, Chizue
MIYAKAWA (OKAMOTO), Misato
MIZUTANI, Takeshi
TOYOTA, Kenji
SUMIYA, Eri
YATSU, Ryohei
TOHYAMA, Saki
EVENSETH, Linn
DOHENY, Brenna
OMORI, Akiko
HAYASHI, Tomoko
INABA, KayoIMAIZUMI, TaekoSynthetic  chemicals  found  in  the  environment  have  the capacity  to  disrupt  the  development  and  function  of  the endocrine  system  in  both  wildlife  and  humans.  This  has drawn  public  concern  since  many  of  these  chemicals  may bind  to  estrogen  receptors  (ERs)  and  evoke  estrogenic effects. Early evidence that exposure to estrogenic chemicals during  development  could  pose  a  threat  to  human  health came from studies of a synthetic hormone, diethylstilbestrol (DES),  which  was  used  to  prevent  premature  birth  and spontaneous  abortion.    Laboratory  experiments  showed  that exposure of animals to sex hormones during critical windows of  perinatal  life  caused  the  immune  and  nervous  systems, bone, muscle, and the liver to be affected. Although many of these chemicals can bind to ERs in wildlife and humans, the molecular  basis  for  the  action  of  environmental  estrogens remains  poorly  understood.  Thus,  understanding  the molecular  mechanisms  through  which  environmental estrogens  and  sex  hormones  act  during  critical developmental windows is essential. Figure  1.  Scheme  of  estrogen-  dependent  and  -independent  vaginal epithelial  cell  proliferation  in  mice  induced  by  perinatal  estrogen exposure.

I. Developmental origin of adult disease:
Perinatal estrogen exposure induces
persistent changes in reproductive tracts

The  emerging  paradigm  of  the  embryonic fetal  origins  of adult  disease   provides  a  powerful  new  framework  for 2Figure  2.  A  hypothetical  model  for  the  estrogen-independent  ER activation pathway in mouse vaginae.

considering the effects of endocrine disrupters on human and animal health. In 1 1, prenatal DES exposure was found to result  in  various  abnormalities  of  the  reproductive  tract  in women.  This  syndrome  was  named  the  DES  syndrome. Similar  abnormalities  have  been  demonstrated  in experimental  animals  exposed  perinatally  to  estrogens. Developmental  estrogen  exposure  in  mice,  for  example, induces  persistent  proliferation  of  vaginal  epithelial  cells (Figure 1). We found that the persistent changes in the vagina in  mice  exposed  neonatally  to  estrogens  result  from  the persistent  activation  of  erbBs  and  ERa,  and  sustained expression of EGF-like growth factors (Figure 2). Currently, we are analyzing the methylation status in the mouse vagina using  a  microarray  (MeDIP-chip).  We  found  several differentially  methylated  or  demethylated  DNA  profiles  in neonatally  DES-exposed  mouse  vaginae  and  controls.  We thus  consider  that  neonatal  DES  exposure  affects  DNA methylation  profiles,  resulting  in  persistent  abnormalities  in mouse reproductive organs.
 II. Estrogen receptors of birds, reptiles,
amphibians, and fishes

Steroid  and  xenobiotic  receptors  (S R)  have  been  cloned from various animal species (fish, amphibians, reptiles, birds, and  mammals)  by  our  group  and  we  have  demonstrated species-specific  differences  in  their  responses  to  various environmental  and  endogenous  chemicals  (receptor  gene zoo). Thus,  simple  predictions  of  chemical  effects  based  on data from a few established model species are not sufficient to  develop  real  world  risk  assessments.  ER  and  ER-like genes  have  been  cloned  from  various  animal  species including rockshell, Amphioxus, lamprey, lungfish, sturgeon, gar,  roach,  stickleback,  mosquitofish,  mangrove,  Rivulus, catshark,  whale  shark,  Japanese  giant  salamander,  Tokyo salamander,  newt,  axolotl,  toad,  Silurana tropicalis, American  alligator,  Nile  crocodile,  freshwater  turtle, Japanese  rat  snake,  Okinawa  habu,  and  vultures.  Functional studies  showed  that  the  Amphioxus  ER  sequence  does  not bind  estrogen  but  Amphioxus  steroid  receptor  and  lamprey ER  exhibited  ligand-dependent  transactivation,  proving  that

invertebrate and primitive vertebrates, such as the Agnatha,
have a functional ER. We  found  that  medaka  ER  subtypes have  their  specific  functions,  and  medaka,  zebrafish  and stickleback ERs are more sensitive to estrogen estrogen-like chemical exposures than other fishes by reporter gene assay. Thus,  these  approaches  are  efficient  to  evaluate  the relationship  between  species  and  their  sensitivities  to chemicals.

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

ertebrates  show  diverse  sexual  characteristics  which are regulated by androgens. To elucidate the evolutionary history  and  functional  diversification  of  androgen receptor  (AR)  genes  in  vertebrates,  we  cloned  the AR cDNAs  from  a  shark,  basal  ray-finned  fishes (Actinopterygii),  namely  bichir  and  sturgeon (Acipenseriformes), and teleosts including a basal teleost, arowana  (Osteoglossiformes).  Molecular  phylogenetic analysis  revealed  that  a  gene  duplication  event  gave  rise to two different teleost ARs (a and b) and likely occurred in the actinopterygian lineage leading to teleosts after the divergence  of Acipenseriformes  but  before  the  split  of Osteoglossiformes.  Functional  analysis  revealed  that  the shark AR activates the target gene via androgen response element by classical androgens. The teleost ARa showed unique  intracellular  localization  with  a  significantly higher  transactivation  capacity  than  that  of  teleost ARb. These results indicate that the most ancient type of AR, as activated  by  the  classic  androgens  as  ligands,  emerged before  the  Chondrichthyes-Osteichthyes  split  and  the  AR gene  was  duplicated  during  a  teleost-specific  gene duplication event (Figure  ). IV. Papillary process formation in medaka

Androgens  play  key  roles  in  the  morphological specification  of  male  type  sex  characteristics  and reproductive  organs,  whereas  little  is  known  about  the developmental  mechanisms.  Medaka  show  a  prominent masculine  sexual  character,  papillary  processes  in  the  anal fin,  which  has  been  induced  in  females  by  exogenous androgen  exposure.  We  have  identified  androgen-dependent expressions  of  Bmp7  and  Lef1  are  required  for  the  bone nodule  outgrowth  leading  to  the  formation  of  the  papillary process  in  the  postal  region  of  the  anal  fin.  We  have  also developed  a  testing  method  for  screening  of  chemicals having  androgenic  and  anti-androgenic  activity  using  the anal fin in juvenile medaka.

Figure  . Evolutionary relationships of androgen receptor sequences.

microarray  with  high  reproducibility  and  demonstrated  the usefulness  of  the  array  for  the  classification  of  toxic chemicals  as  well  as  for  the  molecular  understanding  of chemical  toxicity  in  a  common  freshwater  organism.  D.
magna  and  D. pulex  reproduce  asexually  (parthenogenesis) when  they  are  in  an  optimal  environment  for  food, photoperiod  and  population  density.  Once  environmental conditions become sub-optimal, they alter their reproductive strategy  from  asexual  to  sexual  reproduction  (Figure  ). Chemicals  are  able  to  affect  the  sex  determination  of daphnids and we found that juvenile hormone (J ) agonists (insect  growth  regulators),  for  example,  induce  the production  of  male  offspring.  The  molecular  basis  of environmental  sex  determination  is  largely  unknown  in daphnids.  To  understand  the  molecular  mechanisms  of  this phenomenon,  we  isolated  sex  determination-related  genes. Also,  we  have  developed  a  method  to  inject  genes  into  D.
magna  and  D. pulex  embryos  which  will  allow  us  to  study gain-  and  loss-of  function  analyses  in  more  detail  in  these species. Using these techniques, we demonstrated that DS 1 (double sex 1), one of the DM-domain genes, is essential for male  differentiation  in  D. magna.  We  have  developed  an RNAi  method  and  a  TALEN  method  using  D. pulex.  To further explore the signaling cascade of sexual differentiation in  D. magna,  a  gene  expression  profile  of  J -responsive genes is essential. We are identifying J -responsive genes in the  ovary  of  D. magna and  D. pulex  exposed  to  J   agonist and  methyl  farnesoate  (J   identified  in  decapods)  at  the critical timing of J -induced sex determination in D. magna and D. pulex. We have identified J  receptor (heterodimer of methoprene-tolerant  and  steroid  receptor  co-activator)  in daphnids  and  the  function  of  ecdysone  in  molting  and V. Environmental sex differentiation in
Daphnids and American alligators

Daphnia magna  has  been  used  extensively  to  evaluate  the organism-  and  population-based  responses  of  toxicity  or reproductive toxicity tests. These tests, however, provide no information  about  the  mode  of  action  of  the  tested compounds.  Therefore,  we  applied  an  ecotoxicogenomic assessment  to    D. magna.  We  established  a  Daphnia  EST database  and  developed  an  oligonucleotide-based  DNA Figure  . Life cycle of Daphnia.

National nstitute or asic iologyBovulation in D. magna.
Sex determination mechanisms can broadly be categorizedby either a genotypic or environmentally driven mechanism.
Temperature-dependent sex determination (TSD), an
environmental sex determination mechanism mostcommonly observed among vertebrates, has long been
observed especially among reptiles. However, thetemperature-dependent triggering mechanism of TSD and thesubsequent differentiation cascade has long remained
unknown. We are working on the thermosensitive cation
channel, TRP vanilloid subtype 4 (TRPV4) as a malecascade trigger for the American alligator, Alligator
mississippiensis, in response to high environmental
temperature. We have successfully isolated and cloned the
TRPV4 channel, and demonstrated its thermal activation attemperatures proximate to TSD-related temperature inalligators. Furthermore, using pharmacological exposure tomanipulate TRPV4 channel activity, we have demonstrated
that TRPV4 channel activity has a direct relationship withmale differentiation gene expression, suggesting that
AmTRPV4 is involved in the male differentiation cascade,and propose a novel mechanism for the sex determination
pathway.

Publication List

〔Original papers〕

hatia,  ., Kumar, A., Ogino, Y., Gregg, A., Chapman, J., McLaughlin,  BM.J., and  Iguchi,  T.  (201 ).  Di-n-butyl  phthalate  causes  estrogenic effects  in  adult  male  Murray  rainbowfish  (Melanotaenia fluviatilis). Aquat. Toxicol. 149C, 10 -115.
Bhatia,  .,  Kumar,  A.,  Ogino,  Y.,  Du,  J.,  Gregg,  A.,  Chapman,  J., McLaughlin,  M.,  and  Iguchi,  T.  (201 ).  Effects of the commercial
antiandrogen flutamide on the biomarkers of reproduction in male
Murray rainbowfish (Melanotaenia fluviatilis).  Environ.  Toxicol. Chem. 33, 10 -110 .
Hamlin, H.J., Lowers, R.H., Kohno, S., Mitsui-Watanabe, N., Amano,
H., Hara, A., Ohta, Y., Miyagawa, S., Iguchi, T., and Guillette, L.J.Jr.
(2014). The reproductive hormone cycle of adult female American
alligator from a Barrier island population. Reproduction 147, 855-863.
Hiruta, C., Ogino, Y., Sakuma, T., Toyota, K., Miyagawa, S., Yamamoto,T., and Iguchi, T. (2014). Targeted gene disruption by use of transcription
activator-like effector nuclease (TALEN) in the water flea Daphnia
pulex. BMC Biotechnol. 14, 95.
Ichikawa, M., Murai, E., Hashiguchi, Y., Iguchi, T., and Sato, T. (2014).
Effects of diethylstilbestrol on luteinizing hormone-producing cells in
the mouse anterior pituitary. Exp. Biol. Med. 239, 311-319.
Ihara, M., Ihara, M.O., Kumar, V., Narumiya, M., Hanamoto, S.,
Nakada, N., Yamashita, N., Miyagawa, S., Iguchi, T., and Tanaka, H.
(2014). Co-occurrence of estrogenic and anti-estrogenic activities inwastewater: quantitative evaluation of balance by in vitro ERα reporter
gene assay and chemical analysis. Environ. Sci. Technol. 48, 63666373.
Miyagawa, S., Harada, M., Matsumaru, D., Tanaka, K., Inoue, C.,
Nakahara, C., Haraguchi, R., Matsushita, S., Suzuki, K., Nakagata, N.,
Ng, R.C., Akita, K., Lui, V.C., and Yamada, G. (2014). Disruption of thetemporally regulated cloaca endodermal b-catenin signaling causes
anorectal malformation. Cell Death Differ. 21, 990-997.
Miyagawa,  S.,  Lange,  A.,  irakawa,  I.,  Tohyama,  S.,  Ogino,  Y., Mizutani, T., Kagami, Y., Kusano, T., Ihara, M., Tanaka,  ., Tatarazako,N.,  Ohta,  Y.,  Katsu,  Y.,  Tyler,  C.R.,  and  Iguchi,  T.  (201 ).  Differing
species responsiveness of estrogenic contaminants in fish is conferredby the ligand binding domain of the estrogen receptor. Environ. Sci.
Technol. 48, 5254-5236.
Nakamura, A., Takanobu, H., Tamura, I., Yamamuro, M., Iguchi, T., and

 
 
 
 
 
 
 
 

Tatarazako, N. (2014). Verification of responses of Japanese medaka(Oryzias latipes) to antiandrogens, vinclozolin and flutamide, in shortterm assays. J. Appl. Toxicol. 34, 545-553.
Ogino, Y., Hirakawa, I., Inohaya, K., Sumiya, E., Miyagawa, S.,
Tatarazako, N., Denslow, N., Yamada, G., and Iguchi, T. (2014). Bmp7
and Lef1 are the downstream effectors of androgen signaling in
androgen-induced sex characteristics development in medaka.
Endocrinology 155, 449-462.
Omori, A., Miyagawa, S., Ogino, Y., Harada, M., Ishii, K., Sugimura,Y., Ogino, H., Nakagata, N., and Yamada, G. (2014). Essential roles of
epithelial bonee morphogentic protein signaling during prostatic
development. Endocrinology 155, 2534-2544.
Sébillot, A., Damdimopoulou, P., Ogino, Y., Spirhanzlova, P.,
Miyagawa, S., Du Pasquier, D., Mouatassim, N., Iguchi, T., Lemkine,G., Demeneix, B.A., and Tindall, A.J. (2014). Rapid fluorescentdetection of (anti-)androgens with spiggin-gfp medaka. Environ. Sci.
Technol. 48, 10919-10928.
Sumiya, E., Ogino Y., Miyakawa, H., Hiruta, C., Toyota, K., Miyagawa,S., and Iguchi, T. (2014). Roles of ecdysteroids for progression of
reproductive cycle in the fresh water crustacean Daphnia magna. Front.
Zool. 11, 60.
Toyota, K., Kato, Y., Miyakawa,  ., Yatsu, R., Mizutani, T., Ogino, Y., Miyagawa, S., Watanabe,  ., Nishide,  ., Uchiyama, I., Tatarazako, N., and Iguchi, T. (201 ). Molecular impact of juvenile hormone agonists on neonatal Daphnia magna. J. Appl. Toxicol. 34, 5 -5 .

 
 
 
 
 

〔Review articles〕

iruta, C., Toyota, K., Miyakawa,  ., Sumiya, E., and Iguchi, T. (201 ).  Sexual reproduction is a key element in the life history strategy of water fleas, Daphnia magna and Daphnia pulex - Casting a spotlight on male induction and its morphology -. In  El-Doma, M (ed.) Daphnia  Biology and Mathematics Perspectives, Nova Science Publishers, pp. 261-278.
Kohno, S., Parrott, B.B., Yatsu, R., Miyagawa, S., Moore, B.C., Iguchi,T., and Guillette, L.J. Jr. (2014). Gonadal differentiation in reptiles
exhibiting environmental sex determination. Sex. Devel. 8, 208-226.
Miyakawa, H., Iguchi, T., and Miura, T. (2014). Developmental processof defensive morph in Daphnia pulex. In: El-Doma, M. (ed). Daphnia:
Biology and Mathematics Perspectives. Nova Science Publishers, pp.
245-260.
Miyakawa, H., Toyota, K., Sumiya, E., and Iguchi, T. (2014).
Comparison of JH signaling in insects and crustaceans. Curr. Opinion
Insect Sci. 1, 81-87.
Zoeller, R.T., Bergman, ., Becher, G., Bjerregaard, P., Bornman, R.,
Brandt, I., Iguchi, T., Jobling, S., Kidd, K.A., Kortenkamp, A.,
Skakkebaek, N.E., Toppari, J., and Vandenberg, L.N. (2014). A pathforward in the debate over health impacts of endocrine disrupting
chemicals. Environ. Health 13, 118.

 
 
 
 

A

photochemical  quenching  by  means  of  biochemistry, molecular genetics, optical spectroscopy, small-angle neutron scattering, and bio-imaging.

B

2-1 State transitions
Professor

NA A Aun

Associate Professor

A A A

unic i

Assistant Professor:
Postdoctoral Fellow:

TOKUTSU, Ryutaro
KAMADA, Konomi
OHNISHI, Norikazu
MARUYAMA, Shin-ichiro
YAMASAKI, Hiroaki
AIHARA, Yusuke
SOKENDAI Graduate Student: KAMRANI, Yousef Yari
KATO, Hiroki
KOSUGE, Kotaro
Visiting Graduate Student: KISHIMOTO, Mariko
Technical Assistant:
YONEZAWA, Harumi
KADOWAKI, Tamaka
HOSHI, Rie
Secretary:
KOJIMA, YokoPlants  and  algae  have  a  large  capacity  to  acclimate themselves  to  changing  environments.  We  are  interested  in these acclimation processes, in particular, how efficiently yet safely  they  harness  sunlight  for  photosynthesis  under fluctuating  light  conditions.    Using  a  model  green  alga,  we are  studying  the  molecular  mechanisms  underlying photoacclimation  of  the  photosynthetic  machinery.  We  are also  applying  the  knowledge  obtained  in  the  studies  of  a model  green  alga  to  various  phytoplankton  including Symbiodinium in corals and sea anemones in tropical oceans, to explore how the environmentally important photosynthetic organisms thrive in their ecological niche.  I. Macroorganization of photosynthetic
machineryPhotosystem  (PS)  II  is  a  multiprotein  complex  that  splits water  and  initiates  electron  transfer  in  photosynthesis.  The central  part  of  PSII,  the  PSII  core,  is  surrounded  by  lightharvesting  complex  II  proteins  (L CIIs).  In  higher  plants, two or three L CII trimers are seen on each side of the PSII core  whereas  only  one  had  been  seen  in  the  corresponding positions  in  a  unicellular  green  alga  Chlamydomonas
reinhardtii.  Recently,  we  re-examined  the  supramolecular organization  of  this  PSII-L CII  supercomplex  in  C.
reinhardtii  by  solubilizing  the  thylakoid  membranes  with n-dodecyl- -D-maltoside  and  subjecting  them  to  gelfiltration.  This  newly-prepared  PSII-L CII  supercomplex bound  twice  as  much  L CII  than    previously  reported  and retained  higher  oxygen-evolving  activity.  Single-particle image analysis of the electron micrographs revealed that the PSII-L CII  supercomplex  had  a  novel  supramolecular organization, with three L CII trimers attached to each side of the core. II. Acclimation of photosynthesis

Using  C. reinhardtii,  we  investigate  the  molecular mechanisms  underlying  the  acclimation  processes  of  the photosynthetic  complexes  such  as  state  transitions  and  non-Plants respond to changes in light quality by regulating the absorption  capacity  of  their  PSs.  These  short-term a c c l i m a t i o n s   u s e   r e d o x - c o n t r o l l e d ,   r e v e r s i b l e phosphorylation of L CIIs to regulate the relative absorption cross-section of the two photosystems, commonly referred to as  state  transitions.  It  is  acknowledged  that  state  transitions induce substantial reorganizations of the PSs.  owever, their consequences  on  the  chloroplast  structure  are  more controversial.  ere,  we  investigate  how  state  transitions affect  the  chloroplast  structure  and  function  using complementary  approaches  for  the  living  cells  of Chlamydomonas reinhardtii.  Using  small-angle  neutron scattering, we found a strong periodicity of the thylakoids in State 1, with characteristic repeat distances of approximately 200  ,  which  was  almost  completely  lost  in  State  2. As revealed  by  circular  dichroism,  changes  in  the  thylakoid periodicity  were  paralleled  by  modifications  in  the  longrange  order  arrangement  of  the  photosynthetic  complexes, which  was  reduced  by  approximately  20   in  State  2 compared with State 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna  size  during  State  1  to  State  2  transition (approximately 20 ) is not commensurate to the decrease in PSII  antenna  size  (approximately  0 ).  This  implies  that  a large part of the phosphorylated L CIIs do not bind to PSI, but  instead  form  energetically  quenched  complexes,  which we  have  shown  to  be  either  associated  with  PSII supercomplexes or in a free form (Figure 1). Altogether these noninvasive in vivo approaches allowed us to present a more likely  scenario  for  state  transitions  that  explains  their molecular mechanism and physiological consequences. Figure  1.  ypothetical  model  for  chloroplast  remodeling  during  state transitions in C. reinhardtii. Side views of the membrane planes showing thylakoid  ultrastructure  and  PS  supercomplex  composition  in  State  2. A number  of  L CII  proteins  are  phosphorylated,  and  the  thylakoids  are partially  unstacked  and  undulated.  The  periodicity  of  the  thylakoid membranes  is  weak.  Most  of  the  phosphorylated  L CIIs  are  in  an energy-quenching state (red). They either remain associated with PSII so that a large part of the PSII L CII supercomplex array is preserved, or are unbound and aggregated. 5National nstitute or asic iology

B

2-2 Energy-dependent quenching of excess energy (qEquenching)Absorption  of  light  in  excess  of  the  capacity  for photosynthetic  electron  transport  is  damaging  to photosynthetic organisms. Several mechanisms exist to avoid photodamage,  which  are  collectively  referred  to  as  nonphotochemical  quenching  (NP ).  This  term  comprises  at least two major processes  state transitions (qT), the change in the relative antenna sizes of PSII and PSI as described in the  previous  section,  and  energy-dependent  quenching  of excess  energy  (qE),  the  increased  thermal  dissipation triggered  by  lumen  acidification.  Recently,  we  isolated  the PSII-L CII supercomplex from both WT C. reinhardtii and the npq4 mutant, which is qE-deficient and lacks the ancient light-harvesting protein L CSR. L CSR  was present in the PSII-L CII supercomplex from the high light-grown WT but not in the supercomplex from the low light-grown WT or the npq4  mutant.  The  purified  PSII-L CII  supercomplex containing  L CSR   showed  a  normal  fluorescence  lifetime at a neutral p  ( .5) by single-photon counting analysis but exhibited a significantly shorter lifetime (energy-quenching) at p  5.5, which mimics the acidified lumen of the thylakoid membranes in high light-exposed chloroplasts. The switching from  light-harvesting  mode  to  energy-dissipating  mode observed  in  the  L CSR -containing  PSII-L CII s u p e r c o m p l e x   w a s   i n h i b i t e d   b y   D C C D (dicyclohexylcarbodiimide),  a  protein-modifying  agent specific  to  protonatable  amino  acid  residues.  We  conclude that the PSII-L CII-L CSR  supercomplex formed in high light-grown  C. reinhardtii  cells  is  capable  of  energy dissipation upon protonation of L CSR  (Figure 2).

the C. reinhardtii genome, it was unclear how these isoforms are  differentiated  in  terms  of  transcriptional  regulation  and functionalization. We  showed  that  transcripts  of  both  of  the isoforms, LHCSR3.1 and LHCSR3.2, are accumulated under high  light  stress.  Reexamination  of  the  genomic  sequence and  gene  models  along  with  a  survey  of  sequence  motifs suggested that these two isoforms shared an almost identical but  still  distinct  promoter  sequence  and  a  completely identical  polypeptide  sequence,  with  more  divergent ’-untranscribed  regions.  Transcriptional  induction  under high  light  condition  of  both  isoforms  was  suppressed  by treatment  with  a  PSII  inhibitor,  -( , -dichlorophenyl)-1,1dimethylurea (DCMU), and a calmodulin inhibitor W . Despite  a  similar  response  to  high  light,  the  inhibitory effects  of  DCMU  and  W   to  LHCSR1  transcript accumulation  were  limited  compared  to  LHCSR3  genes. These  results  suggest  that  the  transcription  of  LHCSR paralogs  in  C. reinhardtii  are  regulated  by  light  signals  and differentially  modulated  via  photosynthetic  electron  transfer and  calmodulin-mediated  calcium  signaling  pathway(s) (Figure  ). Light
Electron flow
Calmodulin

Low CO2LHCSR1LHCSR3.1

LHCSR3.2

Figure  . A  model  of  transcriptional  regulation  of  LHCSR  genes  in  C.
reinhardtii. III. Ecophysiology of micro algaeOur  new  projects  are  the  study  of  photoacclimation  of dinoflagellates that can live in a symbiotic relationship with cnidarians,  and  the  study  of  oil-producing  Chlamydomonas. We  are  particularly  interested  in  a  dinoflagellate Symbiodinium  living  with  corals  and  sea  anemones  (Figure ),  and  the  oil-producing  Chlamydomonas  grown  under natural  pond-like  environments.  We  are  trying  to  elucidate how  their  photosynthetic  machinery  acclimates  to  variable light and temperature conditions.

Figure 2. A model for the induction of qE in C. reinhardtii. Although  it  has  been  known  that  two  very  closely  related genes  (LHCSR3.1  and  LHCSR3.2)  encoding  L CSR  protein and another paralogous gene LHCSR1 are present in Figure  . Fluorescence image of the tiny sea anemone Aiptasia, a model system for studies of dinoflagellate (Symbiodinium)-cnidarian symbiosis. Each red dot is a cell of Symbiodinium.

Publication List

〔Original papers〕

aruyama, S., Tokutsu, R., and Minagawa, J. (201 ). Transcriptional  Mregulation of  the  stress-responsive  light  harvesting  complex  genes  in Chlamydomonas reinhardtii. Plant Cell Physiol. 55, 1 0 -1 10.

agy, G.,  nnep, R.,  siros, O., Tokutsu, R., Takizawa, K., Porcar, L.,  NMoyet, L.,  Petroutsos,  D.,  Garab,  G.,  Finazzi,  G.,  and  Minagawa,  J. (201 ).  Chloroplast  remodeling  during  state  transitions  in Chlamydomonas reinhardtii as revealed by non-invasive techniques in vivo. Proc. Natl. Acad. Sci. USA 111, 50 2-50 .
Shibata, Y., Katoh, W., Chiba, T., Namie, K., Ohnishi, N., Minagawa, J., Nakanishi,  ., Noguchi, T., and Fukumura,  . (201 ). Development of a  novel  cryogenic  microscope  with  numerical  aperture  of  0.   and  its application to photosynthesis research. Biochim. Biophys. Acta 1837, 0- .
Takahashi,  ., Okamuro, A., Minagawa, J., and Takahashi, Y. (201 ). Biochemical  characterization  of  photosystem  I  associating  lightharvesting  complexes  I  and  II  isolated  from  State-2  cells  of Chlamydomonas reinhardtii. Plant Cell Physiol. 55, 1 -1
.

 

 

〔Review articles〕

inazzi, G., and Minagawa, J. (201 ).  igh light acclimation in green  Fmicroalgae. In  Non-Photochemical  uenching and Energy Dissipation in Plants, Algae, and Cyanobacteria  (B. Demmig-Adams, G. Garab G, W.  Adams  III  and  Govindjee,  Eds.),  pp. 5,  Advances  in Photosynthesis and Respiration  ol. 0, Springer, Dordrecht.
Johnson,  G.  N.,  Cardol,  P.,  Minagawa,  J.,  and  Finazzi,  G.  (201 ). Regulation of electron transport in photosynthesis. In  Plastid Biology  (Theg,  S.,  Wollman,  F.-A.,  Eds.)   pp. ,  Advances  in  Plant Biology  ol. 5, Springer, Dordrecht.

 National nstitute or asic iology

B

A

A B

Adjunct Professor

RAA

a as i

Specially Appointed Assistant Professor:
SHINOMIYA, Ai
SHIMMURA, Tsuyoshi
Visiting Graduate Student: SENGA, Takumi
ADACHI, Daisuke
SHIMO, Takayuki
Visiting Undergraduate: NAKATSUKASA, Mana
Technical Assistant:
BABA, Nayumi
AKAMA, Akiko
Secretary:
OKUBO, Masayo
Animals living outside the tropics adapt various physiology and  behavior  to  seasonal  changes  in  the  environment. As animals  use  changes  in  day  length  and  temperature  as seasonal  cues,  these  phenomena  are  referred  to  as photoperiodism  and  thermoperiodism,  respectively.  Medaka provides  an  excellent  model  to  study  these  mechanisms because  of  their  rapid  and  robust  seasonal  responses.  In addition,  genomic  sequences  and  transgenic  approaches  are available  in  this  species.  In  this  division,  we  are  trying  to uncover the underlying mechanisms of seasonal adaptation.

I. Identification of deep brain photoreceptor

It  has  been  known  for  more  than  a  century  that  nonmammalian  vertebrates  receive  light  information  directly within  the  deep  brain  to  adapt  to  seasonal  changes  in  day length.  owever, the identity of the deep brain photoreceptor remained unclear. Cerebrospinal fluid (CSF)-contacting neurons extend knoblike  dendrites  into  the  ventricular  cavity  (Figure  1).  This dendritic  structure  resembles  those  of  photoreceptor  cells  in the  developing  retina  and  the  pineal  organ.  Thus,  the  CSFcontacting neurons have been suggested to function as a deep brain photoreceptor. intrinsically photosensitive. 1-2 Involvement of OPN5-positive CSF-contacting
neuron in the seasonal reproductionWe  examined  the  effect  of  siRNA-mediated  OPN5 knockdown  on  long  day  induction  of  springtime  hormone, thyrotropin  (thyroid  stimulating  hormone   TS ).  Long  dayinduction of TS  was suppressed by the OPN5 knockdown, suggesting that OPN5 is involved in the seasonal regulation of  reproduction.  To  our  knowledge,  this  is  the  first demonstration of the intrinsic photosensitivity of deep brain CSF-contacting neurons.

II. Involvement of tissue-specific posttranslational modification in seasonal timemeasurement

TS   is  a  glycoprotein  secreted  from  the  pituitary  gland. Pars  distalis-derived  TS   stimulates  the  thyroid  gland  to produce  thyroid  hormones,  whereas  pars  tuberalis-derived TS   acts  on  the  hypothalamus  to  regulate  seasonal physiology and behavior as a springtime hormone.  owever, it  had  not  been  clear  how  these  two  TS s  avoid  functional crosstalk. We found that this regulation is mediated by tissuespecific  glycosylation. Although  pars  tuberalis-derived  TS  was released into  circulation, it did not stimulate the thyroid gland.  The  pars  distalis-derived  TS   is  known  to  have sulfated bi-antennary N-glycans, and sulfated TS  is rapidly metabolized  in  the  liver.  By  contrast,  pars  tuberalis-derived TS   had  sialylated  multi-branched  N-glycans   in  circulation,  it  formed  the  macro-TS   complex  with immunoglobulin  or  albumin,  resulting  in  the  loss  of  its bioactivity. Glycosylation is fundamental to a wide range of biological processes.  owever, this is the first demonstration of  its  involvement  in  preventing  functional  crosstalk  of signaling molecules in the body.

Figure 1. (A) Schematic drawing of the quail mediobasal hypothalamus. P O  paraventricular organ  PT  pars tuberalis of the pituitary gland    third  ventricle   OT   optic  tract.  (B)  OPN5-positive  CSF-contacting neurons  in  the  P O.  (C)  Schematic  drawing  of  representative  OPN5positive CSF-contacting neurons in the panel (B).

1-1 Intrinsic photosensitivity of OPN5-positive CSFcontacting neuron

In a previous study, we demonstrated localization of a novel photopigment, OPN5 in the CSF-contacting neurons of quail. We used whole-cell patch-clamp analysis to demonstrate that OPN5-positive  CSF-contacting  neurons  in  the  P O  are Figure 2. Medaka populations collected and used in our study.

III. Genome-wide association study of seasonal
time measurement

It  is  well  established  that  the  circadian  clock  is  somehow involved in seasonal time measurement.  owever, it remains unknown  how  the  circadian  clock  measures  day  length. Additionally, it is not known how animals adapt to seasonal changes  in  temperature.  It  has  been  reported  that  medaka populations  that  were  caught  at  higher  latitudes  have  more sophisticated  responses  to  day  length  and  temperature.  For example, medaka fish caught in  okkaido have a critical day length  (i.e.,  duration  of  light  period  required  to  cause  a response)  of  1   h,  while  those  caught  in  Okinawa  have  an 11.5  h  critical  day  length.  To  uncover  the  underlying mechanism  of  seasonal  time  measurement,  we  are  planning to  perform  a  genome-wide  association  study  in  medaka populations collected from various latitudes all over Japan.

3-1 Variation in seasonal responses with latitude in
medaka fishTo  perform  a  genome-wide  association  study,  we  have collected  thousands  of  medaka  fish  from  all  over  Japan (Figure  2).  We  have  examined  the  effects  of  changing  day length  and  temperature  to  determine  the  critical  day  lengths and  critical  temperatures  that  will  cause  seasonal  responses in the gonad and we found differences in critical day length between  medaka  from  higher  latitudes  and  lower  latitudes (Figure  ).

IV. Transcriptome analysis of seasonality inmedaka fishomeotherms  such  as  birds  and  mammals  do  not  show clear  seasonal  responses  to  changing  temperature.  In contrast,  poikilothermal  animals  also  use  changing temperature  as  a  calendar.  Medaka  provides  an  excellent model  to  uncover  this  mechanism.  To  elucidate  the  signal transduction  pathway  regulating  seasonal  reproduction  in medaka  fish,  we  have  examined  transcriptome  analysis.  We identified  hundreds  of  genes  that  respond  to  day  length  and temperature changes.

Figure  .  Different  critical  day  length  between  medaka  from  higher latitudes and lower latitudes.

Publication List

〔Original papers〕

kegami, K., Liao, X.H., Hoshino, Y., Ono, H., Ota, W., Ito, Y., INishiwaki-Ohkawa,T., Sato, C., Kitajima, K., Iigo, M., Shigeyoshi, Y.,
Yamada, M., Murata, Y., Refetoff, S., and Yoshimura, T. (2014). Tissuespecific post-translational modification allows functional targeting of
thyrotropin. Cell Reports 9, 801-809.
Nakane, Y., Shimmura, T., Abe, H., and Yoshimura, T. (2014). Intrinsic
photosensitivity of deep brain photoreceptor. Curr. Biol. 24, R596-R597.

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

K., Atsumi, Y., Yorinaga, E., Ono, H., Murayama, I., Nakane,
 IY.,kegami,Ota, W., Arai, N., Tega, A., Iigo, M., Darras, V.M., Tsutsui, K.,
Hayashi, Y., Yoshida, S., and Yoshimura, T. Low temperature-induced
circulating triiodothyronine accelerates seasonal testicular regression.
Endocrinology 2014 Nov 18.

〔Review articles〕

akane, Y., and Yoshimura, T. (2014). Universality and diversity in the Nsignaltransduction pathway that regulates seasonal reproduction in
A., Shimmura, T., Nishiwaki-Ohkawa, T., and Yoshimura, ST.hinomiya,(2014). Regulation of seasonal reproduction by hypothalamic
vertebrates. Front. Neurosc. 8, 115.

activation of thyroid hormone. Front. Endocrinol. 5, 12.

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relationships,  the  standard  ortholog  table,  which  is  first created  by  DomClust,  is  now  refined  using  the  DomRefine program (see Section II below). Assistant Professor

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The  accumulation  of  biological  data  has  recently  been accelerated by various high-throughput  omics  technologies including  genomics,  transcriptomics,  and  proteomics.  The field of genome informatics is aimed at utilizing this data, or finding  the  principles  behind  the  data,  for  understanding complex  living  systems  by  integrating  the  data  with  current biological  knowledge  using  various  computational techniques.  In  this  laboratory  we  focus  on  developing computational  methods  and  tools  for  comparative  genome analysis, which is a useful approach for finding functional or evolutionary clues to interpreting the genomic information of various  species. The  current  focus  of  our  research  is  on  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 can be extracted from comparisons of genomes at different evolutionary distances, we are developing methods to conduct comparative analyses not only of distantly related but also of closely related genomes.

I. Microbial genome database for comparative
analysisThe  microbial  genome  database  for  comparative  analysis ( M B G D   U R L  h t t p m b g d . g e n o m e . a d . j p )   i s   a comprehensive  platform  for  microbial  comparative genomics.  The  central  function  of  MBGD  is  to  create orthologous  groups  among  multiple  genomes  using  the DomClust algorithm (see Section II below). By means of this algorithm,  MBGD  not  only  provides  comprehensive orthologous  groups  among  the  latest  genomic  data,  but  also allows  users  to  create  their  own  ortholog  groups  on  the  fly using  a  specified  set  of  organisms.  MBGD  also  has  precalculated  ortholog  tables  for  each  major  taxonomic  group, and  provides  several  views  to  display  the  entire  picture  of each  ortholog  table.  For  some  closely  related  taxa,  MBGD provides the conserved synteny information calculated using the CoreAligner program (see Section III below). In addition, MBGD provides MyMBGD mode, which allows users to add their own genomes to MBGD.
Because  of  the  rapid  increase  in  microbial  genome  data owing to next generation sequencing technology, it becomes more  challenging  to  maintain  high  quality  orthology relationships  while  allowing  users  to  utilize  the  latest genomic  data  available.  Since  recently  accumulating sequences are mostly draft genome data, MBGD now stores them  and  allows  users  to  incorporate  them  into  a  user specific  ortholog  database  through  the  MyMBGD functionality.  In  MyMBGD,  draft  genome  data  as  well  as user  genome  data  are  incorporated  into  an  existing  ortholog table  created  from  complete  genome  data  in  an  incremental manner.  In  addition,  to  provide  high  quality  orthology 0Figure 1. Overview of the data construction procedure in MBGD

II. I m p r o v e m e n t o f t h e m e t h o d s f o r
constructing orthologous groups among
multiple genomes at the domain levelAs  a  core  technology  of  our  comparative  genomics  tools, we  developed  a  rapid  automated  method  of  ortholog grouping,  named  DomClust,  which  is  effective  enough  to allow the comparison of many genomes simultaneously. The method  classifies  genes  based  on  a  hierarchical  clustering algorithm using all-against-all similarity data as an input, but it  also  detects  domain  fusion  or  fission  events  and  splits clusters  into  domains  if  required. As  a  result,  the  procedure can  split  genes  into  the  domains  minimally  required  for ortholog grouping. Although  DomClust  can  rapidly  construct  orthologous groups at the domain level, its classification quality has room for  improvement  since  it  is  based  on  pairwise  sequence alignment. We developed a procedure to refine the DomClust classification  based  on  multiple  sequence  alignments.  The method  is  based  on  a  scoring  scheme,  the  domain-specific sum-of-pairs  (DSP)  score,  which  evaluates  ortholog clustering  results  at  the  domain  level  as  the  sum  total  of domain-level  alignment  scores.  We  developed  a  refinement pipeline to improve domain-level clustering, DomRefine, by optimizing  the  DSP  score.  We  applied  DomRefine  to domain-based ortholog groups created by DomClust using a dataset obtained from the MBGD database, and evaluated the results  using  COG  and  TIGRFAMs  as  the  reference  data. Thus, we observed that the agreement between the resulting classification  and  the  classifications  in  the  reference databases is improved in the refinement pipeline. Moreover, the  refined  classification  showed  better  agreement  than  the classifications  in  the  eggNOG  databases  when  TIGRFAMs was used as the reference database.

III. Identification of the core structure
conserved among moderately related
microbial genomesorizontal  gene  transfers  ( GT)  have  played  a  significant role  in  prokaryotic  genome  evolution,  and  the  genes constituting a prokaryotic genome appear to be divided into two classes  core and accessory. The core gene set comprises intrinsic  genes  encoding  the  proteins  of  basic  cellular functions,  whereas  the  accessory  gene  set  comprises  GTacquired  genes  encoding  proteins  which  function  under particular  conditions.  We  consider  the  core  structure  of related  genomes  as  a  set  of  sufficiently  long  segments  in which gene orders are conserved among multiple genomes so that  they  are  likely  to  have  been  inherited  mainly  through vertical  transfer,  and  developed  a  method  named CoreAligner  to  find  such  structures.  We  systematically applied the method to bacterial taxa to define their core gene sets,  and  are  now  trying  to  utilize  this  information  to characterize novel genomic and metagenomic data.

IV. D e v e l o p m e n t o f a w o r k b e n c h f o rcomparative genomics

We  have  developed  a  comparative  genomics  workbench named  RECOG  (Research  Environment  for  COmparative Genomics), which aims to extend the current MBGD system by incorporating more advanced analysis functionalities. The central  function  of  RECOG  is  to  display  and  manipulate  a large-scale  ortholog  table.  The  ortholog  table  viewer  is  a spreadsheet  like  viewer  that  can  display  the  entire  ortholog table, containing more than a thousand genomes. In RECOG, several  basic  operations  on  the  ortholog  table  are  defined, which are classified into categories such as filtering, sorting, and coloring, and various comparative analyses can be done by  combining  these  basic  operations.  In  addition,  RECOG allows  the  user  to  input  arbitrary  gene  properties  and compare  these  properties  among  orthologs  in  various genomes. We continue to develop the system and apply it to various  genome  comparison  studies  under  collaborative research projects.

V. Ortholog data representation using
Semantic Web technology to integratevarious microbial databases

Orthology  is  a  key  to  integrate  knowledge  about  various organisms  through  comparative  analysis.  Moreover, presence absence  of  orthologs  in  each  genome  can  be  an important  clue  to  understand  the  relationship  between  gene functions and species phenotype habitat. We  have  constructed  an  ortholog  database  using  Semantic Web  technology,  aiming  at  the  integration  of  numerous genomic data and various types of biological information. To formalize  the  structure  of  the  ortholog  information  in  the Semantic  Web,  we  have  constructed  the  Ortholog  Ontology (OrthO). While the OrthO is a compact ontology for general use,  it  is  designed  to  be  extended  to  the  description  of database-specific  concepts.  On  the  basis  of  OrthO,  we described the ortholog information from MBGD in the form of  Resource  Description  Framework  (RDF)  and  made  it available  through  the  SPAR L  endpoint,  which  accepts arbitrary queries specified by users. In this framework based on the OrthO, the biological data of different organisms can be integrated using the ortholog information as a hub.
This  is  part  of  the  MicrobeDB  project,  a  collaborative project under the National Bioscience Database Center.

VI. Identification of mobile genes and its
application to characterizing H. pylori pangenome repertoireGene contents of the same bacterial species can have great variation and thus the whole repertoire of genes in a bacterial species,  termed  the  pan-genome,  can  be  very  large.  We analyzed the pan-genome identified among  0 strains of the human  gastric  pathogen  Helicobacter pylori isolated  from various  phylogeographical  groups.  For  this  purpose,  we developed  a  method  to  define  mobility  of  genes  against  the reference  coordinate  determined  by  the  core  alignment created  by  CoreAligner,  and  classified  each  accessory  gene into  mobility  classes.  We  also  identified  co-occurring  gene clusters using phylogenetic pattern clustering combined with neighboring  gene  clustering  implemented  in  the  RECOG system. On the basis of these analyses, we identified several gene  clusters  conserved  among  H. pylori  strains  that  were characterized  as  mobile  or  non-mobile.  This  work  is  in collaboration with Prof. Kobayashi, Univ. Tokyo.

Publication List

〔Original papers〕

hiba,  .,  and  Uchiyama,  I.  (201 ).  Improvement  of  domain-level  Cortholog clustering by optimizing domain-specific sum-of-pairs score, BMC Bioinformatics 15, 1. awai,  M.,  Futagami,  T.,  Toyoda, A.,  Takaki, Y.,  Nishi,  S.,  ori,  S.,  KArai, W., Tsubouchi, T., Morono, Y., Uchiyama, I., Ito, T., Fujiyama. A., Inagaki, F. and Takami,  . (201 ).  igh frequency of phylogenetically diverse reductive dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes. Front. Microbiol. 5,  0. Matsui,  ., Takahashi, T., Murayama, S.Y., Uchiyama, I., Yamaguchi, K., Shigenobu, S., Matsumoto, T., Kawakubo, M.,  oriuchi, K., Ota, .,  Osaki,  T.,  Kamiya,  S.,  Smet,  A.,  Flahou,  B.,  Ducatelle,  R., aesebrouck,  F.,  Takahashi,  S.,  Nakamura,  S.,  and  Nakamura,  M. (201 ). Development of New PCR Primers by Comparative Genomics for  the  Detection  of  Helicobacter suis  in  Gastric  Biopsy  Specimens. elicobacter 19, 2 0-2 1. Toyota, K., Kato, Y., Miyakawa,  ., Yatsu, R., Mizutani, T., Ogino, Y., Miyagawa, S., Watanabe,  ., Nishide,  ., Uchiyama, I., Tatarazako, N., and Iguchi, T. (201 ). Molecular impact of juvenile hormone agonists on neonatal Daphnia magna. Appl. Toxicol. 34, 5 -5 .

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

Uchiyama,  I.,  Mihara,  M.,  Nishide,  ., and  Chiba,  .  MBGD  update  2015
 microbial genome database for flexible ortholog analysis utilizing  a diverse set of genomic data. Nucleic Acids Res. 201  Nov 1 .

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Technical Assistant:igenoriTANIGUCHI, Atsushi
ISHIBASHI, Tomoko

Our laboratory is currently pursuing two paths of scientific inquiry  that  are  of  interest  to  developmental  biology.  One goal  is  to  clarify  the  mechanism  of  left-right  (L-R) determination. The other is to develop imaging technologies for biological analyses. I. Initial step for left-right asymmetry

In mammalian development, the initial left-right asymmetry first appears as cilia-driven leftward fluid flow on the ventral surface of the node, and the flow provides the initial cue for asymmetric  gene  expressions  that  actually  determine asymmetric  morphogenesis.  The  mechanisms  that  convert the flow to asymmetric gene expression, i.e. the flow sensing mechanism,  remains  controversial,  with  several  models being proposed, and involvement of Ca2  being suggested.
We  pursued  this  question  by  measuring  Ca2   dynamics  in the  node  and  found  that  the  node  cells  cause  apparently stochastic  elevation  of  Ca 2 ,  and  its  spatiotemporal distribution  is  equal  at  the  left  and  right  sides  but  becomes more  frequent  at  the  left  after  late  headfold  stage,  when  the flow-induced  commitment  occurs.  This  asymmetry  was disrupted  in  dynein  mutant  iv/iv  and  pkd2 -/-  mutants,  in accordance to their left-right phenotypes. Figure  2.  Two-photon  light-sheet  microscopy.  Left   Fluorescent  images of  a  medaka  taken  by  a  conventional  (one-photon)  light-sheet microscope.  Right   Images  of  the  same  area  taken  by  our  two-photon light-sheet microscope.

deterioration  of  contrast. A  solution  to  these  problems  is combining  Light-sheet  microscopy  with  two-photon excitation  (TPE),  but  this  results  in  a  narrow  field  of  view, because generation of TPE images requires very high photon density,  i.e.  focusing  with  a  high  numerical  aperture  (NA) lens. We  utilized  a  new  fiber  laser  with  high  peak  power  to overcome  this  problem,  and  enabled  observation  of  larger specimens using a hybrid TPE light-sheet microscope.

Publication List

〔Original papers〕

T., Nakazato, K., Keller, P.J., Kajiura-Kobayashi,  ., Stelzer,  IE.chikawa, .,  Mochizuki,  A.,  and  Nonaka,  S.  (201 ).  Live  imaging  and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and  D tracking tools. Nature Protoc. 9, 5 5-5 5.
Maruyama,  A.,  Oshima,  Y.,  Kajiura-Kobayashi,  .,  Nonaka,  S., Imamura, T., and Naruse, K. (201 ). Wide field intravital imaging by two-photon-excitation  digital-scanned  light-sheet  microscopy (2p-DSLM)  with  a  high-pulse  energy  laser,  Biomed.  Opt.  Express  5, 11- 25.
Oshima, Y., Imamura, T., Shintani, A., Kajiura-Kobayashi,  .,  ibi, T., Nagai, T., Nonaka, S., and Nemoto, T. (201 ). Ultrasensitive imaging of Ca2   dynamics  in  pancreatic  acinar  cells  of  yellow  cameleon-nano transgenic mice. Int. J. Mol. Sci. 15, 1
1-1.

 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 the right sides.

During  the  analysis  we  generated  transgenic  lines  carrying ultrasensitive  Ca2   sensor  proteins.  We  demonstrated  these lines  are  useful  for  visualizing  Ca 2   dynamics  of  adult tissues, while they did not work in the embryonic tissues.

II. Development of light-sheet microscopy

Light-sheet  microscopy  has  many  advantages  for  live imaging  including  low  photobleaching  and  phototoxicity, high  penetration  depth,  and  fast  imaging  acquisition.  This method  also  has  peculiar  disadvantages,  however. Specifically scattering of excitation light within the specimen and  illumination  of  areas  besides  the  focal  plane,  and 2 a a i nstitute or ntegrative ioscience

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SOKENDAI Graduate Student: SUGIYAMA, Kazuya
Technical Assistant:
SANBO, Chiaki
Secretary:
HACHISUKA, Midori
A  fundamental  question  in  biology  is  to  understand  the mechanisms  underlying  cell-fate  decision.  Genomic reprogramming  after  mammalian  fertilization  reverts terminally  differentiated  gametes  into  toti-  or  pluri-potent states  to  start  a  new  developmental  program.  Cell  lineage allocation  in  the  reprogramming  process  is  accompanied  by drastic changes in the pattern of gene expression, epigenetic configurations,  and  nuclear  organization.  We  aim  to  reveal the roles of chromatin dynamics in cell lineage-allocation by deciphering  the  molecular  mechanisms  underlying remodeling  of  nuclear  organization  and  their  effects  on developmental  gene  expression,  using  mouse  embryos  and embryonic stem (ES) cells as model systems.

I. Epigenetic reprogramming in early mouseembryos.

Genomic reprogramming reverts fully differentiated cells to a  totipotent  state  to  start  a  new  developmental  program.  In the  early  mouse  embryo,  terminally  differentiated  gametes are  reprogrammed  after  fertilization  thereby  acquiring  a totipotent  state.  Upon  the  fourth  cleavage,  -cell  stage embryos,  which  have undergone  the  process  of  compaction, will give rise to the morula (Figure 1). The outer cells of the morula  will  differentiate  into  the  epithelial  trophectoderm (TE)  of  the  blastocyst.  The  inner  cells  of  the  morula  will become the inner cell mass (ICM) of the blastocyst. The ICM subsequently leads to the formation of two lineages, epiblast (EPI)  and  primitive  endoderm  (PE),  with  the  former representing pluripotency as it gives rise to the embryo itself. The  reprogramming  event  is  accompanied  by  epigenetic modifications  and  changes  in  chromatin  structures throughout the embryo, which are essential for regulation of gene  expression  involved  in  differentiation,  and reprogramming of the EPI. resulting  in  establishment  of  cell-type  specific  nuclear organization. Defects in structural components of the nucleus are  responsible  for  developmental  aberrations  and  several human diseases. Remodeling of nuclear architecture leads to spatial  arrangement  of  genes,  which  could  affect  genome functions  including  gene  expression.  owever,  regulatory mechanisms  underlying  nuclear  reorganization  during  cellfate decision remains largely unknown.

III. Chromatin structureSpatiotemporal  organization  of  genomic  DNA  within  the nucleus  is  suggested  as  an  emerging  key  player  to  regulate gene  expression.  The  developmental  program  accompanies nuclear  remodeling,  resulting  in  construction  of  cell-type specific  nuclear  architecture.  Firstly,  chromosomes  are confined in discrete nuclear spaces,  chromosome territories  (Figure  2).  Within  them,  further  levels  of  D  organization, topologically  associating  domains   (TADs),  are  observed. TADs can be defined as linear units of chromatin containing several gene loci, and fold as discrete  D structures in which gene  loci  frequently  interact  with  each  other.  Recent  works have  revealed  that  folding  of  local   chromatin  structures such  as  enhancer-promoter  looping  is  associated  with genome  functions.  Despite  the  drastic  changes  of  these hierarchical  chromatin  structures,  their  role  in  cell-fate decision remains largely unexplored.

Figure 2.  ierarchical chromatin structure IV. ApproachWe have developed a powerful imaging technology termed TALE-mediated Genome  isualization (TG ), which allows us  to  track  specific  genomic  sequences  in  living  cells (Miyanari Y, Nature Structural   Molecular Biology, 201 ). Importantly,  this  technique  is  versatile  and  can  be  extended to  allow many robust applications, which will be integrated into  our  study  to  manipulate  several  genome  functions. Based  on  new  technological  development,  we  aim  to understand  biological  roles  of  chromatin  dynamics  in  cellfate decision. Publication List

〔Original paper〕

Y.  (201 ).  TAL  effector-mediated  genome  visualization  M(TGiyanari, ). Methods  . 1 -20 .

Figure 1. Lineage allocation in mouse preimplantation development

II. Remodeling of nuclear architecture indevelopment

Chromatin  is  organized  in  a  non-random  fashion  within three-dimensional  nuclear  space.  During  developmental processes, nuclear architecture is dramatically reconstructed, National nstitute or asic iology

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The NIBB Core Research Facilities were launched in 2010 to  support  basic  biology  research  in  NIBB.  They  consist  of three facilities that are developing and providing state-of-theart  technologies  to  understand  biological  functions  through functional genomics, bioimaging and bioinformatics. The  NIBB  Core  Research  Facilities  also  act  as  an intellectual  hub  to  promote  collaboration  among  the researchers of NIBB and other academic institutions.

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MORI, Tomoko
MAKINO, Yumiko
YAMAGUCHI, Katsushi
BINO, Takahiro
ASAO, Hisayo
AKITA, Asaka
MATSUMOTO, Miwako
ICHIKAWA, MarikoThe Functional Genomics Facility is a division of the NIBB Core Research Facilities and organized jointly by NIBB and NIPS  for  promoting  DNA  and  protein  studies.  The  facility maintains  a  wide  array  of  core  research  equipment,  from standard machinery like ultracentrifuges to cutting edge tools such  as  next  generation  DNA  sequencers,  which  amount  to 0 different kinds of instruments. The facility is dedicated to fostering  collaborations  with  researchers  both  of  NIBB  and other  academic  institutions  worldwide  by  providing  these tools  as  well  as  expertise.  Our  current  focus  is  supporting functional  genomics  works  that  utilize  mass  spectrometers and  DNA  sequencers.  We  also  act  as  a  bridge  between experimental biology and bioinformatics. During 201 , the Functional Genomics Facility was largely renovated.  For    example,  the  isitors  Lab  and  the  isitors Office  were  newly  designed  to  promote  collaboration projects. Figure 1. Next-generation sequencerscollaboration  with  NIBB  laboratories  as  well  as  the researchers  of  other  academic  institutions.  These  projects cover a wide range of species (bacteria, animals, plants, and humans)  including  both  model  and  non-model  organisms, and  various  applications  such  as  genomic  re-sequencing, RNA-seq and ChIP-seq. Proteomics

Three different types of mass spectrometer and two protein sequencers, as listed below, are used for proteome studies in our  facility.  In  201 ,  we  analyzed  approximately  200 samples  with  mass  spectrometers  and  50  samples  with protein sequencers. - LC-MS (AB SCIE  TripleTOF 5 00 system)- LC-MS (Thermo Fisher SCIENTIFIC Orbtrap Elite)- MALDI-TOF-MS (Bruker Daltonics REFLE  III)- LC- -TOF MS (Waters  -TOF Premier)- Protein sequencer (ABI Procise   T  ABI Procise  2 cLC)Other analytical instruments- Cell sorter (SONY S 00)
-  Bioimaging  Analyzer  (Fujifilm  LAS  000  mini   GE FLA 000)
- Laser Capture Microdissection System (Arcturus  T)
- DNA Sequencer (ABI PRISM  10  ABI  1 0xl)- Real Time PCR (ABI  500)
- Ultra Centrifuge (Beckman  L- 0 P etc.) Representative Instruments
Genomics

The  advent  of  next-generation  sequencing  (NGS) technologies  is  transforming  today’s  biology  by  ultra-highthroughput  DNA  sequencing.  Utilizing  the  SOLiD5500xl (Life  technologies),  iSeq2500,  iSeq1500,  and  MiSeq (Illumina), the Functional Genomics Facility is committed to joint  research  aiming  to  explore  otherwise  inaccessible  new fields in basic biology. During  201   we  carried  out    NGS  projects  in Figure 2. A mass spectrometry systemGenome Informatics Training CourseWe  organize  NIBB  Genome  Informatics  Training  Courses every  year.  In  201 ,  we  provided  two  three-day  training courses  on  RNA-seq  data  analysis.  These  courses  are designed  to  introduce  the  basic  knowledge  and  skills  of bioinformatics  analysis  to  biologists  who  are  not  familiar with bioinformatics.

characterization  of  an  arabidopsis  mutant  with  altered  localization  of NIP5 1, a plasma membrane boric acid channel, reveals the requirement for D-galactose in endomembrane organization. Plant Cell Physiol. 55, 0 - 1 .
Yoshida,  K.,  Makino, T., Yamaguchi,  K.,  Shigenobu,  S.,  asebe,  M., Kawata, M., Kume, M., Mori, S., Peichel, C.L., Toyoda, A., Fujiyama, A.,  and  Kitano,  J.  (201 ).  Sex  chromosome  turnover  contributes  to genomic  divergence  between  incipient  stickleback  species.  PLoS Genet. 10, e100 22 .

 ● Research activity by S. Shigenobu
Specially Appointed Associate Professor:
SHIGENOBU, ShujiNIBB Research Fellow:
MAEDA, Taro
Postdoctoral ellow:
HOJO, Masaru
OGAWA, Kota
Technical Assistant:
SUZUKI, Miyuzu

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

Figure  . NIBB Genome Informatics Training Course

Publication List of Collaborative Research〔Original papers〕

lankenburg,  S.,  Balfanz,  S.,  ayashi,  Y.,  Shigenobu,  S.,  Miura,  T.,  BBaumann, O.,  Baumann,  A.,  and  Blenau,  W.  (201 ).  Cockroach GABAB receptor subtypes  Molecular characterization, pharmacological properties and tissue distribution. Neuropharmacology. , 1 -1 .
Furuta, Y., Namba-Fukuyo,  ., Shibata, T.F., Nishiyama, T., Shigenobu, S.,  Suzuki,  Y.,  Sugano,  S.,  asebe,  M.,  and    Kobayashi,  I.  (201 ). Methylome diversification through changes in DNA methyltransferase sequence specificity. PLoS Genet. 10, e100 2 2.
Ishida, T., Tabata, R., Yamada, M., Aida, M., Mitsumasu, K., Fujiwara, M., Yamaguchi, K., Shigenobu, S.,  iguchi, M., Tsuji,  ., Shimamoto, K.,  asebe,  M.,  Fukuda,  .,  and  Sawa,  S.  (201 ).  eterotrimeric  G 
proteins control stem cell proliferation through CLA ATA signaling in Arabidopsis. EMBO Rep. 5, 1202-120 .
Kaiwa,  N.,  osokawa,  T.,  Nikoh,  N.,  Tanahashi,  M.,  Moriyama,  M., Meng,  .-Y.,  Maeda,  T., Yamaguchi,  K.,  Shigenobu,  S.,  Ito,  M.,  and Fukatsu,  T.  (201 ).  Symbiont-Supplemented  Maternal  Investment Underpinning  ost’s Ecological Adaptation. Curr. Biol. 2 , 2 5-2 0.
Kodama, Y., Suzuki,  ., Dohra,  ., Sugii, M., Kitazume, T., Yamaguchi, K.,  Shigenobu,  S.,  and  Fujishima,  M.  (201 ).  Comparison  of  gene expression  of  Paramecium  bursaria  with  and  without  Chlorella variabilis symbionts. BMC Genomics 15, 1 . Matsui,  ., Takahashi, T., Murayama, S.Y., Uchiyama, I., Yamaguchi, K., Shigenobu, S., Matsumoto, T., Kawakubo, M.,  oriuchi, K., Ota, .,  Osaki,  T.,  Kamiya,  S.,  Smet,  A.,  Flahou,  B.,  Ducatelle,  R., aesebrouck,  F.,  Takahashi,  S.,  Nakamura,  S.,  and  Nakamura,  M. (201 ).  Development  of  new  PCR  primers  by  comparative  genomics for  the  detection  of  elicobacter  suis  in  gastric  biopsy  specimens. elicobacter 1 , 2 0 2 1.
Nishimura, T.,  erpin, A., Kimura, T.,  ara, I., Kawasaki, T., Nakamura, S., Yamamoto, Y., Saito, T.L., Yoshimura, J., Morishita, S., Tsukahara, T., Kobayashi, S., Naruse, K., Shigenobu, S., Sakai, N., Schartl, M., and Tanaka,  M.  (201 ).  Analysis  of  a  novel  gene,  Sdgc,  reveals  sex chromosome-dependent  differences  of  medaka  germ  cells  prior  to gonad formation. Development 1 1, .
Uehara,  M.,  Wang,  S.,  Kamiya,  T.,  Shigenobu,  S.,  Yamaguchi,  K., Fujiwara,  T.,  Naito,  S.,  and  Takano,  J.  (201 ).  Identification  and 

 
 
 
 
 
 
 

Every  creature  on  the  earth  exists  among  a  network  of various  biological  interactions.  For  example,  many multicellular organisms, including humans, harbor symbiotic bacteria  in  their  bodies   some  of  them  provide  their  hosts with essential nutrients deficient in the host’s diet and others digest  foods  indigestible  by  the  host  alone.  In  spite  of numerous  examples  of  symbioses  and  its  intriguing outcomes,  the  genetic  and  molecular  basis  underlying  these interactions  remains  elusive.  The  goal  of  our  group  is  to establish a new interdisciplinary science  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 nextgeneration sequencing technologies.  I. Genomic revelations of a mutualism: the pea
aphid and its obligate bacterial symbiont

Aphid  species  bear  intracellular  symbiotic  bacteria  in  the cytoplasm  of  bacteriocytes,  specialized  cells  for  harboring the  bacteria.  The  mutualism  is  so  obligate  that  neither  can reproduce  independently.  The   Mb  draft  genome sequence  of  the  pea  aphid,  Acyrthosiphon pisum,  in  consort with  that  of  bacterial  symbiont  Buchnera aphidicolaillustrates  the  remarkable  interdependency  between  the  two organisms.  Genetic  capacities  of  the  pea  aphid  and  the symbiont  for  amino  acid  biosynthesis  are  complementary. The  genome  analysis  revealed  that  the  pea  aphid  has undergone  characteristic  gene  losses  and  duplications.  The IMB antibacterial immune pathway is missing several critical genes,  which  might  account  for  the  evolutionary  success  of aphids to obtain beneficial symbionts. Lineage-specific gene duplications  have  occurred  in  genes  in  a  broad  range  of functional  categories,  which  include  signaling  pathways, miRNA machinery, chromatin modification and mitosis. The importance of these duplications for symbiosis remains to be determined.  We  found  several  instances  of  lateral  gene transfer  from  bacteria  to  the  pea  aphid  genome.  Some  of 5National nstitute or asic iologythem are highly expressed in bacteriocytes. We  recently  discovered  a  novel  class  of  genes  in  the  pea aphid  genome  that  encode  small  cysteine-rich  proteins  with secretion  signals  that  are  expressed  exclusively  in bacteriocytes of the pea aphid, and named these bacteriocytespecific cysteine-rich proteins (BCR). The BCR mRNAs are first expressed at a developmental time point coincident with the  incorporation  of  symbionts  strictly  in  the  cells  that contribute  to  the  bacteriocyte  and  this  bacteriocyte-specific expression  is  maintained  throughout  the  aphid’s  life.  Some BCRs  showed  an  antibiotic  activity.  These  results  suggest that BCRs act within bacteriocytes to mediate the symbiosis with  bacterial  symbionts,  which  is  reminiscent  of  the cysteine-rich secreted proteins of leguminous plants that also regulate  endosymbionts.  Employment  of  small  cysteine-rich peptides  may  be  a  common  tactic  of  host  eukaryotes  to manipulate bacterial symbionts.

  Figure 1. Pea aphids and the bacterial symbiont, Buchnera. Adult aphids (Left). A  developing  viviparous  embryo  which  symbionts  are  infecting (Right). Scale bar   20um. Publication List

〔Original papers〕

usev,  O.,  Suetsugu,  Y.,  Cornette,  R.,  Kawashima,  T.,  Logacheva,  GM.D., Kondrashov,  A.S.,  Penin,  A.A.,  atanaka,  R.,  Kikuta,  S., Shimura,  S.,  Kanamori, .,  Katayose,  Y.,  Matsumoto,  T., Shagimardanova,  E.,  Alexeev,  D.,  Govorun,  .,  Wisecaver,  J., Mikheyev, A., Koyanagi, R., Fujie, M., Nishiyama, T., Shigenobu, S., Shibata,  T.F.,  Golygina,  .,  asebe,  M.,  Okuda,  T.,  Satoh,  N.,  and Kikawada, T. (201 ). Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge. Nature Commun. 5, .
Kaiwa,  N.,  osokawa,  T.,  Nikoh,  N.,  Tanahashi,  M.,  Moriyama,  M., Meng,  .-Y.,  Maeda,  T., Yamaguchi,  K.,  Shigenobu,  S.,  Ito,  M.,  and Fukatsu,  T.  (201 ).  Symbiont-supplemented  maternal  investment underpinning host’s ecological adaptation. Curr. Biol. 2 , 2 5-2 0.
Kodama, Y., Suzuki,  ., Dohra,  ., Sugii, M., Kitazume, T., Yamaguchi, K.,  Shigenobu,  S.,  and  Fujishima,  M.  (201 ).  Comparison  of  gene expression  of  Paramecium  bursaria  with  and  without  Chlorella variabilis symbionts. BMC Genomics 15, 1 .
Takeshita,  K.,  Shibata,  T.F.,  Nikoh,  N.,  Nishiyama,  T.,  asebe,  M., Fukatsu,  T.,  Shigenobu,  S.,  and  Kikuchi,  Y.  (201 ).  Whole-genome sequence of Burkholderia sp. strain RPE , a bacterial gut symbiont of the bean bug Riptortus pedestris. Genome Announc. 2, e0055 -1 .

 evolutionary  history  of  termites  as  inferred  from genomes. Mol. Biol. Evol. 201  Nov 10.

A

A

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  mitochondrial ASpecially Appointed Associate Professor

A

Technical Staff:
Technical Assistant:asu iroKONDO, Maki
TANIGUCHI-SAIDA, Misako
UCHIKAWA, Tamaki
ICHIKAWA, Chiaki
ISHIKAWA, Azusa

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

Representative Instruments:
Okazaki Large Spectrograph (OLS)The  spectrograph  runs  on  a  0  kW  enon  arc  lamp  and projects a wavelength spectrum from 250 nm (ultraviolet) to 1,000  nm  (infrared)  onto  its  10  m  focal  curve  with  an intensity  of  monochromatic  light  at  each  wavelength  more than  twice  as  much  as  that  of  the  corresponding monochromatic  component  of  tropical  sunlight  at  noon (Watanabe et al., Photochem. Photobiol. 36,  1, 1 2).   〔Original paper (E-publication ahead of print)〕ourguignon,  T.,  Lo,  N.,  Cameron,  S.L.,  Sobotn k,  J.,  ayashi,  Y.,  BShigenobu, S., Watanabe, D., Roisin, Y., Miura, T., and Evans, T.A. The Figure  1. An  example  of  experiments  using  the  Large  Spectrograph. arious color rays (monochromatic light from right side and reflected by mirrors) were irradiated simultaneously to samples in cooling chambers.

The  spectrograph  is  dedicated  to  action  spectroscopical studies of various light-controlled biological processes.
The  NIBB  Collaborative  Research  Program  for  the  Use  of the OLS supports about 10 projects every year conducted by both visiting scientists, including foreign researchers, as well as those in NIBB. Action  spectroscopical  studies  for  various  regulatory  and damaging  effects  of  light  on  living  organisms,  biological molecules,  and  artificial  organic  molecules  have  been conducted. Microscopes

This  facility  also  has  Bioimaging  machines  such  as widefield  microscopes  (Olympus  I - 1,  B -   and KEYENCE  B - 000),  confocal  microscopes  (Olympus F 1000,  Nikon A1R,  Nikon A1Rsi,  Carl  eiss  Duo  5  and Yokogawa  CSU- 1),  multi-photon  microscopes  (Olympus F 1000-MP,  F 1200-MPs,  Leica  SP   MPs)  and  other advanced  custom-made  laser  microscopes  with  special  aims (Digital  Scanned  Light-sheet  Microscope   DSLM  and Infrared  Laser-Evoked  Gene  Operator  microscope  IR-LEGO)  for  users  in  NIBB  and  collaborative  guest researchers.  We  began  Collaborative  Research  Programs using  these  machines  in  2010.  In  addition,  transmission electron  microscope  service  for  plant  biology  has  started from 201 .
The  DSLM  was  developed  by  Dr.  Ernst  Stelzer’s  group  at the  European  Molecular  Biology  Laboratory  (EMBL).  This microscope can realize high-speed z-axis scanning in deeper tissue by illuminating a specimen from the side with a light sheet  (more  information  is  given  in  Dr.  Nonaka’s  section  Lab. for Spatiotemporal Regulations). Dr. Nonaka conducted and  supported    projects  of  the  Collaborative  Research Program  for  the  Use  of  the  DSLM.  The  IR-LEGO  was developed by Drs. Shunsuke Yuba and Yasuhiro Kamei at the National  Institute  of  Advanced  Industrial  Science  and Technology  (AIST).  This  microscope  can  induce  a  target gene of interest by heating a single target cell in vivo with a high  efficiency  irradiating  infrared  laser  (Kamei  et al.  Nat. Methods,  200 ).  Details  are  described  in  the  next  section. The IR-LEGO was also used for 10 Individual Collaborative Research  projects,  including  applications  for  animals  and plants. Workshop and SymposiumIn  201 ,  we  held  the  th  International  training  courses  on frog and fish (refer to international practical course) and the 2nd biological image processing training course. We also have been  holding  a  Bioimaging  Forum   every  year  which discusses  Bioimaging  from  various  directions  such  as microscopy,  new  photo-technology,  and  computer  science. This  year  we  held  the  th  NIBB  Bioimaging  Forum  focused on  establishment  of  a  network  among  Imaging  Centers  in Japan.  In  addition,  we  held  a  symposium  focused  on  new microscope  methods  using  adaptive  optics,  Subaru microscope , with the National Astronomical Observatory of Japan (NAOJ).

Publication List of Collaborative Research〔Original papers (Selected)〕

., Ide, N.,  igashi, S., Kamei, Y., Toyooka, T., Ibuki, Y., Kawai,  FK., ang, Kasai, .,  Okamoto,  K., Arimoto-Kobayashi,  S.,  and  Negishi,  T. (201 ).  Somatic  cell  mutaions  caused  by  5  nm  LED-U A  doublestrand  breaks  through  oxidative  damage.  Photochem.  Photobiol.  Sci. 13, 1-1.
Goto-Yamada, S., Mano, S., Nakamori, C., Kondo, M., Yamawaki, R., Kato, A., and Nishimura, M. (201 ). Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy. Plant Cell Physiol. 55,  2.
Hayashi, S., Ochi, H., Ogino, H., Kawasumi, A., Kamei, Y., Tamura, K.,
and Yokoyama, H. (2014). Transcriptional regulators in the Hippo1
signaling pathway control organ growth in Xenopus tadpole tailregeneration. Dev. Biol. 396, 31-41.
Kimura,  T.,  Nagao,  Y.,  ashimoto,  .,  Yamamoto-Shiraishi,  Y.I., Yamamoto, S., Yabe, T., Takada, S., Kinoshita, M., Kuroiwa, A.,  and Naruse,  K.  (201 ).  Leucophores  are  similar  to  xanthophores  in  their specification  and  differentiation  processes  in  medaka.  Proc.  Natl. Accad. Sci. USA 111, .
Masamizu,  Y.,  Tanaka,  Y.R.,  Tanaka,  Y. .,  ira,  R.,  Ohkubo,  F., Kitamura, K., Isomura, Y., Okada, T., and Matsuzaki, M. (201 ). Two distinct layer-specific dynamics of cortical ensembles during learning of a motor task. Nature Neurosci. 17, .
Nagao, Y.,  Suzuki,  T.,  Shimizu, A.,  Kimura,  T.,  Seki,  R., Adachi,  T., Inoue,  C.,  Omae,  Y.,  Kamei,  Y.,  ara,  I.,  Taniguchi,  Y.,  Naruse,  K., Wakamatsu, Y., Kelsh, R.N.,  ibi, M., and  ashimoto,  . (201 ). Sox5 functions as a fate switch in medaka pigment cell development. PLoS Genetics 10, e100 2 .
Ogino,  Y.,  irakawa,  I.,  Inohaya,  K.,  Sumiya,  E.,  Miyagawa,  S., Denslow, N., Yamada, G., Tatarazako, N., and Iguchi, T. (201 ). Bmp  and  Lef1  are  the  downstream  effectors  of  androgen  signaling  in androgen-induced  sex  characteristics  development  in  medaka. Endocrinology 155, - 2. Okuyama,  T.,  Yokoi,  S.,  Abe,  .,  Isoe,  Y.,  Suehiro,  Y.,  Imada,  ., Tanaka, M., Kawasaki, T., Yuba, S., Taniguchi, Y., Kamei, Y., Okubo, K.,  Shimada,  A.,  Naruse,  K.,  Takeda,  .,  Oka,  Y.,  Kubo,  T.,  and Takeuchi,  .  (201 ).  A  neural  mechanism  underlying  mating preferences for familiar individuals in medaka fish. Science 343,  1- .  Tamada, Y., Murata, T.,  attori, M., Oya, S.,  ayano, Y., Kamei, Y., and asebe, M. (201 ). Optical property analyses of plant cells for adaptive optics microscopy. Int. Optomechatroni. 8,  - .

 
 
 
 
 
 
 
 

● Research activity by Y. Kamei
Specially Appointed Associate Professor:
KAMEI, Yasuhiro
NIBB Research Fellow:
HATTORI, Masayuki
Technical Assistant:
CHISADA, ErikoOur  research  group  promotes  two  cutting-edge  microscope projects   observation  and  manipulation  using optical and biological technologies. The aim of our  observation project  is  deep-seeing  in  living  organisms  using  adaptive  optics (AO) which were well-developed in the field of astronomy as a  key  technology  of  large  telescopes  such  as  the  Subaru telescope  in  awaii. Although  observation  using  telescopes on  the  earth  may  be  disturbed  by  fluctuations  in  the atmosphere, AO  technology  can  cancel  this  disturbance.  On the  other  hand,  living  materials  have  particular  refractive indexes, therefore, some organelles act as disturbances of the ideal  optical  path  for  microscope  observation  just  like  the atmosphere  does  for  telescopes. AO  technology  can  also compensate  for  this  disturbance  by  sensing  and  correcting wave  fronts  using  a  wave  front  sensor  and  deformable National nstitute or asic iologymirror.  ence,  we  developed  a  custom-made  wide-field microscope  equipped  with  an AO  system  for  observation  of living  organisms  in  collaboration  with  Dr. Tamada  in  NIBB and Dr.  ayano in the National Astronomical Observatory of Japan  (NAOJ)  and  got  high-resolution  bright  field  and fluorescent images of living cells. Our results indicated that improvement  of  optical  resolution  was  restricted  to  a  small area which is called the  isoplanatic patch  (Figure 1).

Figure  1.  Effects  of  adaptive  optics  (AO)  to  the  wide-field  microscope images (bright field and fluorescence of plant cells). Second, the aim of our  manipulation project  is to control gene  expression  in vivo.  Gene  function  analysis  must  be evaluated at the cell level in vivo. To achieve spatiotemporalcontrolled  gene  expression  we  employed  one  of  the  stress responses, the heat shock response. The heat shock promoter is  the  transcription  regulation  region  of  heat  shock  proteins and  all  organisms  have  this  mechanism.  Positioning  the target  gene  downstream  of  the  promoter,  we  can  induce  the target gene expression by local heating. Infrared (IR) beams can heat water molecules, which are the main constituent of cells, hence, we can heat a single cell by irradiating  IR  to  a  target  cell  using  a  microscope.  We  have developed  a  microscope,  IR  laser  evoked  gene  operator (IR-LEGO),  specialized  for  this  purpose  (Figure  2).  The IR-LEGO  microscope  can  irradiate  an  IR  laser  to  a  single cell  in vivo  such  as  C. elegans,  Drosophila,  medaka, zebrafish, Xenopus and Arabidopsis, to induce the heat shock response at a desired timing.
Optimal  heating  induces  the  heat  shock  response  and subsequent  gene  expression,  while  an  excess  results  in  cell death.  ence,  we  must  precisely  control  laser  heating.  We evaluated  time  course  and  spatial  heating  profiles,  and  the results  presented  that  temperature  of  the  target  area  rose rapidly  and  kept  a  constant  level  dependant  on  IR  laser power, additionally, the heated area was adequately as small as a typical cell size.
With  this  in  mind,  we  tried  to  induce  gene  expression  in various  species.  At  first,  we  reported  an  IR-LEGO experiment in living C. elegans. Target gene expression in a target  cell  could  be  induced  with  only  1  s-IR  irradiation. Whereas  the  optimal  power  range  which  can  induce  gene induction  without  cell  damage  was  limited.  Excess  laser power resulted in cell death or cessation of cell division. We confirmed  that  an  optimal  irradiation,  e.g.  11  mW  for  1  s, induced physiological gene expression in the target cell and subsequent cell division or morphogenesis underwent normal development. Next, we tried the experiment in other animals, such  as,  medaka,  zebrafish  and  Xenopus,  and  the  higher plant,  Arabidopsis,  since  all  organisms  have  a  heat  shock response system. We succeeded in local gene induction in all the species as expected.
Studies of cell fates, cell-cell interaction, or analysis of non-Figure  2.  Schematic  illustration  of  local  gene  induction  system  and  an infrared  laser-evoked  gene  operator  (IR-LEGO)  microscope  system  in NIBB.

cell autonomous phenomena require a fine control system of gene expression in experiments. IR-LEGO will be a powerful tool  for  these  studies  in  combination  with  molecular biological  techniques,  such  as  the  cre-loxP  system.  Dr. Shimada  in  the  University  of  Tokyo  wanted  to  confirm  the cell  lineage  of  exo-skeletal  tissue  such  as  the  scales  of medaka fish. She questioned the traditional belief concerning the  origin  of  the  exo-skeleton  of  the  body-trunk  using transplantation  studies.  We  then  started  a  collaboration  to establish a local permanent labeling system in medaka and to make  clear  the  origin  of  exo-skeletal  cells. The  system  was well  working  (Figure  ),  and  the  fate  tracking  results indicated  that  exo-skeletal  tissues  were  mesodermal  in origin,  not  from  neural  crest  cells,  as  previously  believed (Shimada  et al,  Nat.  Commun,  201 ).  This  year,  our  group and  other  collaborators  were  applying  the  same  system  to amphibians,  Xenopus  and  salamanders,  for  regeneration studies.

Figure  . Examples of Cre-loxP mediated long-term GFP marking using IR-LEGO in living medaka individuals for cell linage tracing.

Publication List

〔Original papers〕

ang,  ., Ide, N.,  igashi, S., Kamei, Y., Toyooka, T., Ibuki, Y., Kawai,  FK., Kasai, ., Okamoto, K., Arimoto- Kobayashi, S., and Negishi, T. (201 ). Somatic cell mutations caused by  5 nm LED-U A doublestrand  breaks  through  oxidative  damage.  Photochem.  Photobiol.  Sci. 13, 1-1.
ayashi, S., Ochi,  ., Ogino,  ., Kawasumi, A., Kamei, Y., Tamura, K., and  Yokoyama,  .  (201 ).  Transcriptional  regulators  in  the  ippo1 signaling  pathway  control  organ  growth  in  enopus  tadpole  tail regeneration. Dev. Biol. 396,  1- 1.

 N., Nakashima, R.,  irai, T., Kamei, Y., Ishikawa-Fujiwara,  MT., urozumi, Todo, T., and Kitano, T. (201 ). Loss of follicle-stimulating hormone receptor  function  causes  masculinization  and  suppression  of  ovarian development in genetically female medaka. Endocrinology 155,  1 1 5.
Nagao, Y.,  Suzuki,  T.,  Shimizu, A.,  Kimura,  T.,  Seki,  R., Adachi,  T., Inoue,  C.,  Omae,  Y.,  Kamei,  Y.,  ara,  I.,  Taniguchi,  Y.,  Naruse,  K., Wakamatsu, Y., Kelsh, R.N.,  ibi, M., and  ashimoto,  . (201 ). Sox5 functions as a fate switch in medaka pigment cell development. PLoS Genetics 10, e100 2 .
Okuyama,  T.,  Yokoi,  S.,  Abe,  .,  Isoe,  Y.,  Suehiro,  Y.,  Imada,  ., Tanaka, M., Kawasaki, T., Yuba, S., Taniguchi, Y., Kamei, Y., Okubo, K.,  Shimada,  A.,  Naruse,  K.,  Takeda,  .,  Oka,  Y.,  Kubo,  T.,  and Takeuchi,  .  (201 ).  A  neural  mechanism  underlying  mating preferences for familiar individuals in medaka fish. Science 343,  1- . Otozai, S., Ishikawa-Fujiwara, T., Oda, S., Kamei, Y., Ryo,  ., Sato, A., Nomura, T., Mitani,  ., Tsujimura, T., Inohara,  ., and Todo T. (201 ). p5 -Dependent suppression of genome instability in germ cells. Mutat. Res. Fundam. Mol. Mech. Mutagen. 760, 2 - 2.
Tamada, Y., Murata, T.,  attori, M., Oyac, S.,  ayano, Y.,  Kamei, Y., and  asebe,  M.  (201 ).  Optical  property  analyses  of  plant  cells  for adaptive optics microscopy. Int. J. Optomechatroni. 8,  - .

 
 
 
 

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Assistant Professor:
Technical Staff:
Technical Assistant:

UCHIYAMA, Ikuo
MIWA, Tomoki
NISHIDE, Hiroyo
NAKAMURA, Takanori
OKA, Naomi

The  Data  Integration  and  Analysis  Facility  supports research  activities  based  on  large-scale  biological  data analysis, such as genomic sequence analysis, expression data analysis,  and  imaging  data  analysis.  For  this  purpose,  the facility  maintains  high-performance  computers  with  largecapacity  storage  systems.  On  the  basis  of  this  system,  the facility  supports  development  of  data  analysis  pipelines, database  construction  and  setting  up  websites  to  distribute the  data  worldwide  as  well  as  providing  users’  basic technical  support.  In  addition  to  computational  analysis,  the Data  Integration  and  Analysis  Facility  supports  NIBB’s information  infrastructure,  the  maintenance  of  the  network systems  in  the  institute  and  computer network  consultation for institute members. Representative InstrumentsOur  main  computer  system  is  the  Biological  Information Analysis  System  (BIAS)  (Figure  1),  which  consists  of  a high-performance  cluster  system  (SGI  Rackable  server C2112- RP   0  nodes 00  cores,  GB  memory node),  a shared  memory  parallel  computer  ( P  ProLiant  DL 0  G  0  cores,  TB  memory),  a  high-throughput  storage  system (DDN  SFA 00   0TB),  and  a  large  capacity  storage system  (DELL  PowerEdge  R 20   20TB). All  subsystems are  connected  via  a  high-speed  InfiniBand  network  so  that large  amounts  of  data  can  be  processed  efficiently.  Some personal  computers  and  color monochrome  printers  are  also available.  On  this  system,  we  provide  various  biological databases  and  data  retrieval analysis  programs,  and  support large-scale  data  analysis  and  database  construction  for institute  members  and  collaborative  researchers.  Especially, Figure 1. Biological Information Analysis Systemwe  have  supported  the  construction  and  maintenance  of published  databases  of  various  model  organisms  including DB  (Xenopus laevis),  P YSCObase  (Physcomitrella
patens),  DaphniaBASE  (Daphnia magna),  The  Plant Organelles Database, and MBGD (microbial genomes).
The facility also provides network communication services. Most  of  the  PCs  in  each  laboratory,  as  well  as  all  of  the above-mentioned service machines, are connected by a local area  network,  which  is  linked  to  the  high  performance backbone  network  ORION  connecting  the  three  research institutes  in  Okazaki.  Many  local  services,  including sequence analysis services, file sharing services, and printer services,  are  provided  through  this  network.  We  also maintain  a  public  World  Wide  Web  server  that  hosts  the NIBB home page (http www.nibb.ac.jp ).

Research activity by I. Uchiyama

Assistant professor I. Uchiyama is the principal investigator of  the  Laboratory  of  Genome  Informatics,  which  currently focuses  on  microbial  comparative  genomics  studies.  For details, please refer to the laboratory page (p.  0).

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FUJIMORI, ToshihikoTo 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),  zebra sh,  Arabidopsis,  Lotus
japonicus,  and  Physcomitrella patens,  makes  it  possible  to produce  genetically  controlled  organisms  with  markers placed  by  genetic  and  cell  engineering  technology.  Such marking allows detailed studies of genes and cell functions. The  model  organisms  mature  in  a  short  period  of  time  therefore,  changes  in  cells,  organs,  and  individuals  can  be totally  and  ef ciently  observed.  The  NIBB  BioResource Center  has  equipment,  facilities,  and  staff  to  maintain  such organisms safely, ef ciently, and appropriately.

Technical Staff:
Technical Assistant:Technical  staff  and  supporting  staff  develop  and  promote research-supporting  activities. A  state-of-the-art  facility  for transgenic animals opened at the end of 200  in the Yamate area.
The  activities  of  the  model  animal  research  facility  are  as follows1.  The  provision  of  information,  materials,  techniques  and animal housing space to researchers.
2. The use of various kinds of instruments to analyze mutant, transgenic, and gene-targeted animals. . The development of novel techniques related to transgenic and gene targeting technology.
. Cryopreservation and storage of transgenic strains.

I. Research support activities (mouse)A
Associate Professor:

The  NIBB  Center  for  Transgenic Animals  and  Plants  was established  in  April  1  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 .

WATANABE, Eiji
TANAKA, Minoru
NARUSE, Kiyoshi
HAYASHI, Kohji
NOGUCHI, Yuji
TAKAGI, Yukari
SUZUKI, Kohta
SUGINAGA, Tomomi
INADA, Yosuke
FUJIMOTO, Daiji
MATSUMURA, Kunihiro
ATSUMI, MihoIn  2001,  the  NIBB  mouse  facility  (built  under  specific pathogen free (SPF) conditions) opened in the Myodaiji area and the production, breeding, analysis, cryopreservation and storage  of  genetically  manipulated  mouse  strains  has  been conducted there since then. The new center facility building in  the  Yamate  area  strengthened  research  activities  using 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  barrier  system. This  building  is also  equipped  with  breeding  areas  for  transgenic  small  fish, birds, and insects. In 201  (from January 1 to December  1), 2, 2 fertilized eggs  (in vitro  fertilization   2,  eggs  of  10  lines  in  which 2,1   eggs  of  10  lines  were  frozen  for  long-term  storage, Figure 1. Mouse (strain C5 BL J)The worldwide genome project has almost been completed and  research  on  basic  biology  has  arrived  at  a  post-genome era  in  which  researchers  are  focusing  on  investigating  the functions  of  individual  genes.  To  promote  the  functional analysis  of  a  gene  of  interest  it  is  essential  to  utilize genetically altered model organisms generated using genetic engineering  technology,  including  gene  deletion,  gene replacement and point mutation.

0Figure 2. Equipment for manipulating mouse eggs.

frozen  eggs   1 5  of    lines)  and  ,  mice  were  brought into  the  facility  in  the  Yamate  area,  and  5 ,51   mice (including pups bred in the facility) were taken out.
A number of strains of genetically altered mice from outside the  facility  were  brought  into  the  mouse  housing  area  by microbiological  cleaning  using  in vitro  fertilization-embryo transfer techniques, and stored using cryopreservation.
A  new  mouse  facility  in  the  Myodaiji  area  opened  at  the beginning  of  2005.  The  facility  supports  the  activities  of researchers  in  the  Myodaiji  area.  In  March  200 ,  we expanded  the  facility  which  includes  areas  for  breeding, behavioral tests and transgenic studies using various kinds of recombinant  viruses.  In  201   (from  January  1  to  December 1),  5   mice  were  brought  into  the  facility  in  the  Myodaiji area,  and  , 5   mice  (including  pups  bred  in  the  facility) were taken out.

researchers  to  generate  and  maintain  these  important biological tools. In  201   (from  January  1  to  December  1),  11,002  medaka adults  were  brought  to  the  facility  and  ,1 5  medaka  and zebrafish ( ,5 2 fertilized eggs and 5,5  adults, including animals bred in the facility) were taken out. In the laboratory for  chick  embryos  there  were  no  fertilized  eggs  or  chicken embryos  brought  in  or  taken  out  this  year.  These  animals were  used  for  research  activities  in  neurobiology  and developmental biology.

Figure  .  Large sized autoclave in the Myodaiji area.

Figure  .   uarantine room for medaka and zebrafish.

II. Research support activities (small fish,
birds, and insects)

III. Research activities

The first floor of the center facility building in the Yamate area  provides  space  and  facilities  to  maintain  small  fish, chick embryos, and insects. In the laboratory room for chick embryos,  a  large  incubation  chamber  is  equipped  and  set  at 2  degrees  (suitable  for  chick  embryogenesis).  The researchers  can  manipulate  chick  embryos  under  optimal conditions,  removing  biohazard  risks.  For  researchers  who employ fish as an experimental model,  0 tanks (1 liter) and 50  tanks  (   liters)  are  available  for  medaka  and  zebrafish, respectively.  Water  circulates  and  can  be  maintained  to  suit the  conditions  desired  for  fish  breeding  in  the  aquarium systems.  Currently,  over  three  mutant  lines  and  over  fifteen transgenic  lines  of  medaka  and  zebrafish  are  maintained  in our facility. A medaka line that allows gene induction by heat treatment,  in  combination  with  a  cre loxP  system,  has  been developed in this facility. In addition to the rooms mentioned above, a room for insects is also available. All the rooms are qualified to meet the criteria for transgenic animals, allowing In  200   NIBB  was  approved  as  a  core  facility  of  the National  BioResource  Project  (NBRP)  for  Medaka  by  the Japanese  Government.  We  have  supported  the  activities  of NBRP  Medaka  by  providing  standard  strains,  mutants, transgenic  lines  and  organizing  international  practical courses  for  medaka.  In  201   we  began  providing  the CRISPR Cas   genome  editing  platform  as  well  as  the TILLING  library  screening  system  to  promote  the  reverse genetic  approach.  In  201   we  shipped  20   independent medaka  strains,  2   cDNA BAC Fosmid  clones,  and  120 samples  of  hatching  enzyme  to  the  scientific  community worldwide.

The  associate  professors  of  this  center  -  E.  Watanabe,  T. Naruse and M. Tanaka - are the principal investigators of the Laboratory  of  Neurophysiology,  the  Laboratory  of Bioresources,  and  the  Laboratory  of  Molecular  Genetics  for R e p r o d u c t i o n ,   r e s p e c t i v e l y.   T h e   L a b o r a t o r y   o f Neurophysiology  is  studying  mechanisms  of  the  visual system using a psychophysical approach. The Laboratory of Bioresources  has  conducted  a  genetic  and  genomic  analysis of quantitative traits and Mendelian phenotype variations as well  as  evolution  of  sex  determination  systems  in  medaka related  species.  The  Laboratory  of  Molecular  Genetics  for Reproduction  is  studying  the  molecular  mechanisms  of reproductive organ development and sex differentiation using mutagenized  or  transgenic  medaka.  For  details,  please  refer to the pages of each laboratory (p.  , 52, and  ).

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National nstitute or asic iology● Plant Culture Laboratory
Assistant Professor:
Technical Staff:
Technical Assistant:

HOSHINO, Atsushi
TSUGANE, Kazuo
MOROOKA, Naoki
SUZUKI, KeikoThe  Plant  Culture  Laboratory  manages  facilities  for  the cultivation  of  plants  in  general  and  also  for  the  rearing  of several animal species that do not fit in other facilities.
The  Plant  Culture  Laboratory  equips  and  manages  2 culture  boxes  or  growth  chambers,    phytotrons,  and  12 rooms  with  the  P1P  physical  containment  level  for established  and  emerging  model  plants  including  the  thale cress  Arabidopsis thaliana,  several  carnivorous  plants,  the rice Oryza sativa, the moss Physcomitrella patens, green alga Chlamydomonas reinhardtii  and  several  other  flowering plants.  Most  culture  space  is  fully  used  the  whole  year  by more  than  0  researchers  from  both  outside  and  inside groups.
As  well  as  regular  culture  conditions,  extreme environmental  conditions  for  light  and  temperature  are available  for  various  types  of  experiments.  Three  light environmental  simulators  (max  120,000  lux  by  using  xenon lamp) and three chambers ( .  m2 each) that can control CO2 and  humidity  in  addition  to  temperature  and  light  (max 0,000  lux)  conditions  are  available.  Autotrophic  and heterotrophic  culture  devices  are  also  available  for researchers  using  cyanobacteria,  algae,  and  cultured flowering plant cells. Aseptic experiments can be performed in an aseptic room with clean benches.
Next to the institute building of the Myodaiji area, a -m2 experimental farm is maintained for Japanese morning glory and  related  Ipomoea species,  several  carnivorous  plants  and other  flowering  plants  necessary  to  be  cultivated  outside. Three green houses ( ,  , and  5 m2) with heating are used for the sensitive carnivorous plants, and wild-type strains of medaka fish Oryzias sp. Seven green houses ( ,  ,  ,  ,  ,  , 
and    m 2)  with  air-conditioning  are  provided  for  the cultivation  of  a  rice  Oryza sp.,  Lotus japonica  and  related legume  species,  as  well  as  mutant  lines  of  the  Japanese morning  glory.  Two  green  houses  (   and  1   m2)  with  airconditioning  meet  the  P1P  physical  containment  level  and are  available  for  experiments  using  transgenic  plants.  The Plant  Culture  Laboratory  also  maintains  a    m2  building with storage and workspace. Part of the building is used for rearing of the orchid mantis.
In  201 ,  several  plant  culture  facilities  came  into  use.  Of these,  the  multiple  precise  environment  control  plant incubator  system  is  composed  of  10  incubators.  Users  can control all the incubators synchronously. A tissue culture rack with dimming LEDs and pulse-width modulation controllers is used for algae culture under precise light control.

Figure  5. A  tissue  culture  rack  with  dimming  LEDs  and  pulse-width modulation controllers. ● Morning Glory BioResource Laboratory
Assistant Professor:

HOSHINO, Atsushi

The  Japanese  morning  glory  (Ipomoea nil)  is  a  traditional floricultural  plant  in  Japan,  and  is  studied  worldwide, especially  in  plant  physiology  and  genetics.  NIBB  collects, develops and distributes DNA clones, mutant lines for flower pigmentation,  and  transgenic  lines  as  a  sub-center  of  the National  BioResource  Project  (NBRP)  Morning  glory,  and collaborates  with  the  core  organization  center,  Kyushu University.  We  collected  several  mutant  lines  and  10,000 BAC  clones,  and  provided    DNA  clones  and  2   Ipomoea
lines to both local and foreign biologists this year.
Research activities of the assistant professor A.  oshino are shown on the laboratory page (p. 5 ).

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Assistant Professor:
Technical Assistant:

HAMADA, Yoshio
SUGINAGA, Tomomi

The  Cell  Biology  Research  Facility  provides  various equipment  for  tissue  and  cell  culture.  This  laboratory  is equipped  with  safety  rooms  which  satisfy  the  P2  physical containment  level,  and  is  routinely  used  for  DNA 2recombination experiments.
Research  activities  of  assistant  professor  Y.  amada,  the principal investigator of the Laboratory of Cell Sociology, is shown on the laboratory page (p.1 ).

Figure  .  Equipment for tissue and cell culture.

National nstitute or asic iology

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Associate Professor

asayos i NAR

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Specially Appointed Assistant Professor:
KIMURA, Tetsuaki
TANAKA, Daisuke
Technical Assistant:
AKIMOTO-KATO, Ai
MATSUBAYASHI, Naomi
Secretary:
HAMATANI, Ayako
In  order  to  realize  a  life  sciences  community  resilient  to natural  disasters  and  calamities,  the  National  Institutes  for Natural  Sciences  (NINS)  and  okkaido  University,  Tohoku University,  The  University  of  Tokyo,  Nagoya  University, Kyoto University, Osaka University, and Kyushu University concluded an agreement on June 1st 2012 to launch a system to  back  up’  the  biological    resources  essential  to  the  work being  done  at  universities  and  research  institutions nationwide,  called  the  Interuniversity  Bio-Backup  Project (IBBP)’.
The  IBBP  Center  was  established  as  a  centralized  backup and  storage  facility  at  NIBB,  while  IBBP  member universities  set  up  satellite  hubs  and  work  closely  with  the IBBP center to put in place reciprocal systems for backing up important  biological  resources  that  have  been  developed  by researchers  working  in  the  area  in  which  each  university satellite hub is responsible. The  IBBP  Center  includes  earthquake  proof  structures capable  of  withstanding  even  very  large  scale  quakes (equipped  with  emergency  backup  power  generators), cryopreservation  facilities  equipped  with  automatic  liquid nitrogen  feeding  systems,  deep  freezers,  and  refrigerated storage  (mainly  for  seed  stocks),  as  well  as  all  manner  of automated  laboratory  equipment,  cell  culture  tools,  and  the latest  equipment  necessary  to  back  up  the  genetic  resources in  a  collaborative  manner.  The  specific  methods  are cryopreservation  of the sperm and eggs of animals, cultured plant  and  animal  cells,  proteins  and  gene  libraries.  Plant seeds are frozen or refrigerated. University  satellite  hubs  receive  preservation  requests  of biological  resources  from  researchers  and  report  to  the Managing Project Committee of IBBP (constituted of faculty of NIBB and satellite institutes), where the relevance of the request is reviewed. When the request is sustained, biological resources to be preserved will be sent to the IBBP Center by the  requesting  researcher,  where  they  will  be  frozen  (or refrigerated)  and  their  particular  information  will  be registered into a database. In the event of a disaster or other event  leading  to  the  loss  of  a  researcher’s  own  biological resources, preserved samples will be promptly supplied back to the researcher so they can quickly resume their work. Through the development of this backup system biological resources  that  had  only  been  stored  at  individual  research institutes can now be preserved at the state of the art facilities of  the  IBBP  Center,  and  Japan’s  research  infrastructure  has been significantly strengthened.

I. Current status of back up for biologicalresources

In  201 ,  IBBP  Center  stored  , -well  plates’ consisting of 1,5 , cDNA BACs clones, 5,0  tubes for animal cells, plant and animal samples, proteins, genes, and microorganisms. In total 1,5 ,55  samples are stored. Figure 1. IBBP Center Figure 2. Cryogenic storage system. Liquid nitrogen tanks are monitored 2  hours a day and are refilled automatically.

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

II. Collaborative Research Project for thedevelopment of new long-term storage
technologies and cryobiological studyAs  the  IBBP  Center  can  only  accept  biological  resources which  can  be  cryopreserved  in  liquid  nitrogen,  researchers cannot  backup  those  biological  resources  for  which cryopreservation  methods  are  not  well  established.  To increase  the  usability  of  IBBP,  we  started  a  collaborative research  project  for  the  development  of  new  long-term storage  technologies  and  cryobiological  study  in  201 . This collaborative research includes two subjects 1) Establishment of new storage technology for biological resources for which long-term  storage  is  unavailable.  2)  Basic  cryobiological research enabling us to improve low temperature storage for biological  resources.  In  201   we  had  ten  applications  and accepted all ten proposals. We are also working to establish a research  center  for  cryobiological  study  thorough  this Collaborative Research Project. In accordance with this aim we  organized  a  Cryopreservation  Conference  in  201   on October  2 -2 ,  201   at  the  Okazaki  Conference  Center, Okazaki, Japan. We had 10  participants from several fields from physics, chemistry, biology and technology.  Figure 5. Phase diagram of vitrification of a solution. itrification, a physical process, can be defined as the phase transition of an  aqueous  solution  from  a  liquid  into  an  amorphous  glassy  solid,  or glass,  at  the  glass  transition  temperature  (Tg),  while  avoiding  ice crystallization.

itrification-based  protocols  are  known  to  be  effective  for long-term,  stable  preservation  of  plant  germplasm   this protocol can reduce the cost and manpower for maintaining a large  number  of  germplasm  lines  and  keep  many  valuable genetic  lines  long-term  under  genetically  stable  conditions. owever,  it  is  still  not  widely  employed  as  a  reliable  longterm  preservation  protocol  due  to  the  lack  of  basic knowledge on the behavior of water in cells and tissues when immersed in liquid nitrogen. In  the  present  study,  electron  microscopy  combined  with freeze-substitution  was  employed  to  examine  the ultrastructure  of  cells  of  gemmae  of  the  liverwort, Marchantia polymorpha  L.,  strain  Takaragaike-1  that  were cooled to the temperature of liquid nitrogen after exposure to various steps of the Cryoplate protocol (Figure  ). Figure  . Group photo of Cryopreservation Conference 201Assistant Professor:
Technical Assistant:

TANAKA, Daisuke
AKIMOTO-KATO, Ai

Cryopreservation

Cryopreservation  protocols  contain  components  which  are usually  developed  empirically  using  each  biological resource’s  specific  strategy  to  enhance  survival  (Benson 200 ).  The  theory  of  cryopreservation  encompasses  several interconnected disciplines ranging from the physiological to cryophysical. Water status and cryopreservation methods, in combination  with  physiological  factors,  are  the  most influential determinants of survival. So far cryopreservation has been considered in terms of the liquid  and  solid  (ice)  phases  of  water.  It  is  also  possible  to cryopreserve  tissues  by  the  process  of  vitrification ,  the solidification  of  liquids  without  crystallization  (Figure  5). This comprises a  glassy state  as the system is amorphous, lacks  organized  structure  but  possesses  the  mechanical  and physical properties of a solid (Taylor et al. 200 ).

Figure  . Cryopreservation protocol using aluminum cryoplate. A   Preparation  of  in vitro  grown  gemmae  of  liverwort.  B   Placing precultured gemmae in a cryoplate’s wells. C  Regrowth of cryopreserved livewort  line  Takaragaike-1’  0  days  after  rewarming.  D   Control (without cooling in liquid nitrogen).

Publication List

〔Original paper〕

 T., Sakuma, T., Wada,  ., Akimoto-Kato, A., Yamamoto, T., and  Kondo, ayashi,  S.    (201 ).  TALEN-induced  gene  knock  out  in  Drosophila. Dev. Growth Differ. 56, - 1.

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Publication List

Assistant Professor:

KIMURA, Tetsuaki

Leucophores are similar to xanthophores

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells  (NCCs)  are  multipotent  stem  cells  that  give  rise  to numerous  cell-types,  including  pigment  cells.  Medaka (Oryzias Latipes)  has  four  types  of  NCC-derived  pigment cells  (xanthophores,  leucophores,  melanophores  and iridophores),  making  medaka  pigment  cell  development  an excellent  model  for  studying  the  mechanisms  controlling specification  of  distinct  cell  types  from  a  multipotent precursor  cell.  owever  the  genetic  basis  of  chromatophore diversity remains poorly understood. We  reported  that  leucophore free-2  (lf-2)  which  affects leucophore  and  xanthophore  differentiation,  encodes  pax7a. Since  lf-2,  a  loss-of-function  mutant  for  pax7a,  causes defects  in  the  formation  of  xanthophore  and  leucophore precursor  cells,  pax7a  is  critical  for  the  development  of  the chromatophores.  This  genetic  evidence  implies  that leucophores  are  similar  to  xanthophores,  although  it  was previously  thought  that  leucophores  were  related  to iridophores, as these chromatophores have purine-dependent light reflection. Many leucophores-3  (ml-3)  mutant  embryos  exhibit  a unique  phenotype  characterized  by  excessive  formation  of leucophores  and  an  absence  of  xanthophores. We  show  that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal  a  cell-autonomous  role  of  sox5  in  the  xanthophore lineage. pax7a is expressed in NCCs and is required for both xanthophore  and  leucophore  lineages   we  demonstrate  that Sox5 functions downstream of Pax a. We propose a model in which multipotent NCCs  first give rise  to  pax7a-positive  bi-potent  precursor  cells  for xanthophores and leucophores  some of these precursor cells then express sox5, and as a result of Sox5 action develop into xanthophores  (Figure  ).  Our  findings  provide  clues  for revealing  diverse  evolutionary  mechanisms  of  pigment  cell formation in animals.

Figure  .  Model  for  leucophore  and  xanthophore  development  from neural crest cells.

〔Original papers〕

imura,  T.,  Nagao,  Y.,  ashimoto,  .,  Yamamoto-Shiraishi,  Y.,  KYamamoto, S., Yabe, T., Takada, S., Kinoshita, M., Kuroiwa, A., and Naruse,  K.  (201 ).  Leucophores  are  similar  to  xanthophores  in  their specification and differentiation processes in medaka. Proc. Natl. Acad. Sci. USA 111, .
Nagao, Y.,  Suzuki,  T.,  Shimizu, A.,  Kimura,  T.,  Seki,  R., Adachi,  T., Inoue,  C.,  Omae,  Y.,  Kamei,  Y.,  ara,  I.,  Taniguchi,  Y.,  Naruse,  K., Wakamatsu, Y., Kelsh, R.N.,  ibi, M., and  ashimoto,  . (201 ). Sox5 functions as a fate switch in medaka pigment cell development. PLoS Genet. 10, e100 2 .
Nishimura, T.,  erpin, A., Kimura, T.,  ara, I., Kawasaki, T., Nakamura, S., Yamamoto, Y., Saito, T.L., Yoshimura, J., Morishita, S., Tsukahara, T., Kobayashi, S., Naruse, K., Shigenobu, S., Sakai, N., Schartl, M., and Tanaka,  M.  (201 ).  Analysis  of  a  novel  gene,  Sdgc,  reveals  sex chromosome-dependent  differences  of  medaka  germ  cells  prior  to gonad formation. Development 141, .
Tsuboko,  S.,  Kimura,  T.,  Shinya,  M.,  Suehiro,  Y.,  Okuyama,  T., Shimada, A., Takeda,  ., Naruse, K., Kubo, T., and Takeuchi,  . (201 ). Genetic  control  of  startle  behavior  in  medaka  fish.  PLoS  One  9, e11252 .

 
 
 

A

Head

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itsuyasu

Technical Staff:

Technical Assistant:A

Associate Professor

record  sheet  numbered  55 .  The  numbers  for  each  area  are shown in Table 2. The annual changes of registrants and the number  of  totals  per  fiscal  year  are  shown  in  Figure  2. The balance  of  radioisotopes  received  and  used  at  the  CRF  is shown  in  Table  .  The  training  courses  on  radioisotope handling were given as in Table  . A A Ryu i

MATSUDA, Yoshimi(Radiation Protection Supervisor)
SAWADA, Kaoru(Radiation Protection Supervisor)
IINUMA, Hideko
ITO, Takayo
KAMIYA, Kiyomi

The  technical  and  supporting  staff  of  the  Center  for Radioisotope  Facilities  (CRF)  maintain  controlled  areas  in compliance  with  the  law.  The  CRF  is  responsible  for monitoring  the  purchase  of  radioisotopes  from  the  Japan Radioisotope  Association  (JRIA)  and  the  transfer  of radioisotope wastes to JRIA. Ms.  Matsuda,  Ms.  Iinuma,  Ms.  Ito,  and  Ms.  Kamiya maintained  the  Myodaiji-Area.  Ms.  Sawada  worked  in  the Yamate-Area. Dr. Kodama worked in both areas.
The following are CRF’s notable activities in 201 . 
1.  The Common Facilities building I was repaired, therefore the  radiation  controlled  area  of  the  Myodaiji-Area  was closed from April 201  and re-opened on July 1 th 201 .
(Figure 1A, 1B, 1C)2.  Along  with  the  re-opening  of  the  Myodaiji-Area,  the following areas were closed.
2-1  The  radioisotope  handling  rooms  at  the  NIPS  building for  animal  experiments  and  gene-recombination experiments using radioisotopes.
2-2  The second radioactive waste storage room at the NIBB building.

Table 1. Numbers of registrants and users at Myodaiji-Area and YamateArea in 201 .

Table  2.  Users  and  visitors  who  entered  each  controlled  area  and radioisotope handling room in 201 .

Figure  2. Annual  changes  in  number  of  registrants  and  total  number  of days per fiscal year.

  The  number  of  registrants  and  the  number  of  users  from January 201  to December 201  are shown in Table 1. Users and visitors counted by the access control system of the controlled areas numbered  , 20 during this period. The radioisotope  handling  rooms’  users  and  visitors  counted  by 

A

B

A

B

Figure  1.  The  CRF’s  notable  activities  in  201 .  The  Myodaiji-area  was repaired.
A, A’  The radioisotope laboratory (A  before, A’  after) B, B’  The exhaust facility (B  before, B’  after)C, C’  The drainage facility (C  before, C’  after)Table  .  Balance  of  radioisotopes  received  and  used  (kBq)  at  each controlled area and radioisotope handling room in 201 .

Table  . Training courses for radiation workers in 201 .

National nstitute or asic iology

DirectorN

Naoto

Vice-Director (Specially
Appointed Professor (URA))

N

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i io

In  order  to  fulfill  two  goals,  to  encourage  cutting-edge academic  research  in  the  field  of  natural  sciences  through international  joint  research,  and  to  contribute  to  the enhancement  of  research  capabilities  of  universities  etc.  in Japan using the world’s most advanced research environment for joint utilization and joint research, NINS started in 201  a research  enhancement  project  with  the  following  four approaches   1)  Support  for  the  promotion  of  international advanced  research,  2)  Support  for  the  promotion  of  joint utilization  and  joint  research  in  Japan,  )  Dissemination  of information  and  enhancement  of  public  relations  in  Japan and  abroad,  )  Support  for  researchers,  especially  young, female or foreign researchers.
The  Research  Enhancement  Strategy  Office  is  aimed  at supporting researchers so that NIBB improves its ability as a collaborative  research  institution,  and  was  restructured  in 201   from  the  former  Strategic  Planning  Department,  the Office  of  Public  Relations,  and  the  Office  of  International Cooperation which existed from 2005. The Office’s activities are  mainly  carried  out  by  URAs  (University  Research Administrators) according to the advice of the group adviser chosen  from  NIBB’s  professors  and  in  close  collaboration with  the  NINS’s  Research  Enhancement  Promotion eadquarters.
 Associate Professor:
Group Adviser:

KODAMA, Ryuji
YOSHIDA, Shosei

This  group  serves  as  a  central  office  for  assisting  the Director  General  in  preparing  for  NIBB’s  evaluation procedure  and  in  planning  long-range  strategies  for  the institute. The main activities of the group

1) Management of external evaluation processes
NIBB asks non-NIBB members of the Advisory Committee for Programming and Management to answer a questionnaire on  research  achievements  and  collaborative  research activities of the institute. Several of the members are further invited to participate in a meeting for the evaluation of these activities and for proposing future directions for the institute. This group manages these processes.
2) Editing of the Annual Report (in collaboration with the
Public Relations Group)This  group  edits  the  annual  report  (this  print)  which summarizes the annual activities of laboratories and facilities and is used in the evaluation procedures of the institute. 3) Assistance in budget requests and long-range planningof the instituteThis  group  also  assists  the  Director  General  in  preparing long-range  plans  for  building  the  most  advanced  research facilities, and in budget requests to the government to realize and implement these plans.
4) Assistance in making the plans and reports of theinstituteIn  addition  we  assist  in  drafting  NIBB’s  Medium-term Goals  and  Plans  (for  a  six-year-period),  and  in  instituting Annual Plans to implement them. The department also assists in  preparing  Business  and  Performance  Reports  for  the external evaluation on whether we are meeting the goals set both annually and for the medium-term.

Specially Appointed Assistant Professor (URA):
KURATA, Tomoko
Technical Assistant:
OTA, Kyoko
KAWAGUCHI, Colin
BAN, Misato
Group Adviser:
FUJIMORI, Toshihiko This  group,  in  order  to  publicize  the  activities  of  NIBB  to the  widest  audience,  actively  facilitates  communication between  NIBB  and  the  public,  school  teachers,  and  the international community of scientific researchers.

The main activities of the group in 2014

1) Press releases
The  group  sends  news  on  scientific  achievements  to newspaper  and  magazine  reporters  via  leaflets,  e-mails  and web  pages.  We  also  arrange  press  conferences  for  some releases.
2) Updating and maintenance of the NIBB web page3) Editing of publications, production of posters andleafletsPublication of  NIBB News  (Intra-institutional newsletter, in  Japanese),  NIBB  English  News   (Intra-institutional newsletter, in English). Publication of leaflets introducing the individual  researchers  of  NIBB  (in  Japanese).  Design  and distribution of posters of NIBB events.
4) Producing Videos
Creation  of  videos  introducing  NIBB  and  interviews  with researchers that are published on the web.
5) Organization of scientific outreach programs
Organizing  the  Summer  Program  for  university  students, and coordinating special classes for middle school students.

Specially Appointed Assistant Professor (URA):TATEMATSU, Kiyoshi
Technical Assistant:
TAKAHASHI, Ritsue
SANJO, Kazuko
Group Adviser:
UENO, Naoto
NIBB has a mission to continually explore the leading-edge of biology and form research communities that link Japan to the world. For this purpose, NIBB holds scientific meetings including  NIBB  Conferences   and  Okazaki  Biology Conferences  (OBC) ,  and  educational  programs  such  as NIBB  International  Practical  Courses .  Further,  NIBB  is tightly  interacting  with  the  European  Molecular  Biology Laboratory  (EMBL,  European  member  states),  the  Max Planck  Institute  for  Plant  Breeding  Research  (MPIP , Germany), and the Temasek Life Sciences Laboratory (TLL, Singapore)  on  the  basis  of  cooperative  agreements,  through exchanging  people  and  techniques  and  jointly  holding scientific  meetings.  NIBB  is  also  conducting  the  NIBB International  Collaborative  Research  Initiative   to  promote high-level  international  collaborations  between  faculty members of NIBB and researchers around the world. This  group  supports  and  coordinates  NIBB’s  activities related  to  international  research  collaborations,  through organizing  the  above-mentioned  various  types  of international  scientific  meetings  and  technical  courses, coordination  of  dispatching  NIBB’s  researchers  to international conferences, and support of researchers visiting from  the  institutes  mentioned  above.  This  group  also supports NIBB’s interns visiting from foreign countries, and the  dispatching  of  graduate  students  of  SOKENDAI  (the Graduate  University  for Advanced  Studies)  to  international conferences,  which  aim  to  nurture  the  next  generation  of researchers in biology.
 The main activities of the group in 2014

1) Coordination of international conferences and theInternational Practical CourseThis  group  coordinated  the  following  international conference  and  the  International  Practical  Course  hosted  by NIBB  The  th  NIBB  International  Practical  Course  and  the  rd NIBB-TLL-DBS NUS  Joint  International  Practical  Course Experimental Techniques using Medaka and Xenopus   The Merits  of  using  both    Okazaki,  Japan,  September 22-October 1, 201  (p.  )The  2nd  NIBB  Conference  Force  in  Development  Okazaki, Japan, November 1 -1 , 201  (p.  1)2) Support for dispatching researchers to international
conferences
This  group  supported  sending  NIBB  researchers  to  the following  events  (related  to  the  international  cooperative agreements)The 5th NIBB-MPIP -TLL Joint Symposium  ORI ONS IN  PLANT  BIOLOGY   Max  Planck  Institute  for  Plant Breeding  Research,  Cologne,  Germany,  November  2 -2 , 
201  (p.  2)
EMBO  Workshop  Wnt  Signaling   Stem  Cells, Development,  Disease   Broome, Australia,  October,  - , 

201
EMBO Conference  Stem Cells in Cancer and Regenerative Medicine   EMBL,  eidelberg,  Germany,  October,  -12, 201
NIBB Investigators’ lab visits to EMBL,  eidelberg3) Support of education related programs
This  group  supported  the  following  student-related activities of NIBB
NIBB Internship Program 201  (p.  )Specially Appointed Associate Professor (URA):SHIGENOBU, Shuji
KAMEI, Yasuhiro
Technical Assistant:
ICHIKAWA, Mariko
ICHIKAWA, ChiakiGroup Adviser:
YOSHIDA, Shosei
Specially  appointed  associate  professors  of  this  group  also belong  to  NIBB  core  research  facilities  and  are  responsible for  managing  collaborative  research  projects  and  practical courses  using  their  academic  background,  knowledge,  and specialties.  Based  on  these  experiences,  this  group  explores further promotion of information exchange and collaboration among  universities  and  institutes,  both  academic  and industrial,  and  supports  the  development  and  dissemination of new experimental equipment and methods.
In  201 ,  this  group  hosted  or  managed  a  total  of   collaboration  projects. Through  these  collaboration  projects, several  research  papers  were  published.  A  noteworthy achievement was the study of a neural mechanism related to social familiarity using medaka, published in Science by Dr. Takeuchi  of  the  University  of  Tokyo.  The  NIBB Collaborative  Research  Group  supported  mutant  screening, laser  cell  ablation,  establishment  of  transgenic  lines  and discussions  through  the  Priority  Collaborative  Research Project from 2010 to 201 .
kuyama,  T.,  Yokoi,  S.,  Abe,  .,  Isoe,  Y.,  Suehiro,  Y.,  Imada,  .,  OTanaka, M., Kawasaki, T., Yuba, S., Taniguchi, Y., Kamei, Y., Okubo, K.,  Shimada,  A.,  Naruse,  K.,  Takeda,  .,  Oka,  Y.,  Kubo,  T.,  and Takeuchi,  .  (201 ).  A  neural  mechanism  underlying  mating preferences for familiar individuals in medaka fish. Science 343,  1- .

Group Adviser:

TAKADA, ShinjiThis  group  supports  the  improvement  of  the  research environment  for  female  researchers  in  both  research  and daily life. This includes our research support system for the period of childbirth and childcare, etc., aiming at promoting the employment of female researchers.
In  201 ,  this  group  contributed  to  the  management  of  the research  support  system  for  the  period  of  childbirth  and childcare. This group organized an enlightenment lecture for gender  equality  promotion  in  collaboration  with  other institutes of NINS. National nstitute or asic iology

A

Head

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iro o

MIWA, Tomoki● NIBB Core Research Facilities
Unit Chief:
KONDO, Maki
MORI, Tomoko
Subunit Chief:
MAKINO, Yumiko
YAMAGUCHI, Katsushi
Technical Staff:
NISHIDE, Hiroyo
NAKAMURA, Takanori
TANIGUCHI- SAIDA, Misako
UCHIKAWA Tamaki
BINO, Takahiro
Technical Assistant:
ICHIKAWA, Chiaki
NISHIMURA, Noriko
ICHIKAWA, Mariko
ISHIKAWA, Azusa
OKA, Naomi
SHIBATA, Emiko● NIBB Bioresource Center
Subunit Chief:
HAYASHI, Kohji
MOROOKA, Naoki
Technical Staff:
NOGUCHI, Yuji
Technical Assistant:
TAKAGI, Yukari
SUZUKI, KeikoSUZUKI, Kohta● Disposal of Waste Matter Facility
Unit Chief:
MATSUDA, Yoshimi● Center for Radioisotope Facilities
Unit Chief:
MATSUDA, Yoshimi
Subunit Chief:
SAWADA, Kaoru
Technical Staff:
IINUMA, Hideko
Technical Assistant:
ITO, Takayo
The  Technical  Division  is  a  support  organization  for researchers  and  research  organizations  within  NIBB.  The Technical  Division  develops  and  promotes  the  institute’s research activities and maintains the research functions of the institute.
The  Technical  Division  is  organized  into  two  groups   the Common  Facility  Group,  which  supports  and  maintains  the institute’s  common  research  facilities,  and  the  Research Support Group, which assists research activities as described in the reports of individual research divisions.

0● Developmental Biology
Technical Staff:
TAKAGI, Chiyo
UTSUMI, Hideko
OKA, Sanae
NODA, Chiyo
MIZUGUCHI, Hiroko● Neurobiology
Unit Chief:
Subunit Chief:

OHSAWA, Sonoko
TAKEUCHI, Yasushi● Evolutionary Biology and Biodiversity
Unit Chief:
FUKADA-TANAKA, Sachiko
Subunit Chief:
KABEYA, Yukiko● Environmental Biology
Unit Chief:
MIZUTANI, Takeshi
Technical Assistant:
INABA, KayoSecretary:

TSUZUKI, Shihoko
KATAOKA, Yukari
UNO, Satoko
MIYATA, Haruko

Technical staff members continually participate, through the Division,  in  self-improvement  and  educational  activities  to increase  their  capabilities  and  expertise  in  technical  areas. Generally,  technical  staff  members  are  attached  to  specific divisions so that they may contribute their special biological and  biophysical  knowledge  and  techniques  to  various research activities.
The  Technical  Division  hosts  an  annual  meeting  for technical engineers who work in various fields of biology at universities and research institutes throughout Japan. At this meeting,  the  participants  present  their  own  activities  and discuss  technical  problems.  The  proceedings  are  published after each meeting.

BB
rgani ers ance avidson niv Pittsburg
A os i i o Fu imori N
apan
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arl P ilipp eisenberg
Austria Austria
en i atsuno sa a niv apan
iroyu i a eda niv o yo apan
Naoto eno N
apan
Novemberoned 2 4For the past  0 years, in the field of developmental biology, the  primary  focus  has  been  on  elucidating  the  role  of  genes on  developmental  regulation,  with  biologists  pouring  the bulk of their time and effort into the investigation of genes, proteins, and their signal transduction systems, that forms the central dogma. It is indisputable that genes and proteins are absolutely  required  components  exerting  fundamental biological  actions,  however,  in  recent  years  new  light  has been shed on the importance of physical forces generated by the shape-change and movement of cells and tissues, as well as  the  responses  of  cells  and  tissues  to  such  forces  in developmental  processes  making  elucidation  of  these physical forces a rising hot-topic in the biological sciences. In  order  to  capitalize  on  this  opportunity,  the  2nd  NIBB Conference  Force  in  Development   was  planned  as  an international  convention.  The  meeting  was  held  at  the Okazaki Conference Center from November 1 th to the 1 th of 201 . With three keynote speakers, 21 lectures by invited researchers, Six sessions including seven short talks, and   poster  presentations   the  total  number  of  participants  was over  1 0  people.  The  Mecanobiology  field  has  already existed  for  a  long  time,  however,  rather  than  cultured  cells, the  effects  of  force  on  multicellular  systems,  especially dynamics  of  cells  and  tissues  which  alter  the  physical environment  of  the  embryo  and  how  this  impacts morphogenesis  was  the  main  theme  of  this  conference.  We are extremely grateful that top-class researchers from around the world were willing to gather in Okazaki during their tight schedules.  This  meeting,  by  bringing  renowned  researchers together, made it possible to deeply explore the theme of the conference,  and  we  believe  this  will  be  seen  as  an  epochmaking  meeting  for  the  future  development  of  this  field.  In addition,  with  so  many  young  researchers  from  home  and abroad  who  participated  in  the  poster  presentations  and discussions  I  feel  that  this  specific  area  is  growing exponentially.  We  are  confident  that  these  connections  will become  the  foundation  of  future  collaborative  research  and international cooperation.
The  conference  was  possible  because  of  three  research groups,  all  of  which  are  supported  by  the  Grant-in-Aid  for Scientific  Research  on  Innovative Areas  (ME T,  Japan)  From molecules and cells to organs  trans-hierarchical logic for  higher-order  pattern  and  structures   (Project  leader  iroyuki  Takeda),    Cross-talk  between  moving  cells  and microenvironment  as  a  basis  of emerging  order  in  multicellular systems   (Project  leader   Takaki Miyata,  Nagoya  University),  and C e l l   c o m m u n i t y   i n   e a r l y mammalian  development   (Project leader   Toshihiko  Fujimori),  I 
would  like  to  express  my  gratitude for  their  generous  support  for  this meeting.
(Naoto Ueno)

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avidson ance niv Pittsburg
aton u anne P
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November 24 on 2
ed 2 4The symposia jointly conducted by the National Institute for Basic  Biology  (NIBB),  the  Max  Planck  Institute  for  Plant Breeding  Research  (MPIP )  and  the  Temasek  Life  Science Laboratory  have  been  held  four  times  since  200 . Accordingly,  the  5th  NIBB-MPIP -TLL  joint  symposium titled  orizons in Plant Biology  was held from November 2 rd to November 2 th 201 , at the Max Planck Institute for Plant  Breeding  Research,  Cologne,  Germany  with researchers  gathered  from  Europe,  Singapore,  the  USA  and Japan.  In  the  joint  symposium,  1   researchers  from  Japan participated,  and  about  0  from  Germany,  Switzerland,  the USA and Singapore. The oral presentations conducted in the symposium  were  comprised  of  1   members  of  MPIP ,   from  Europe,  1  from  the  USA,    from  TLL,  1  from  NIBB and   from Japan’s other universities. At this symposium one of the major academic trends in plant science, i.e., in addition to  research  in  the  laboratory,  performing  research  that considers  ecology  and  evolution  under  the  divergent environmental  conditions  found  in  nature,  really  stood  out. The  rise  of  young  researchers  who  have  started  taking ologneermanyleading  roles  in  the  plant  science  community  was  also noticeable with many presentations and proactive discussions from  young  scientists.  For  many  of  the  participants  it  was also  a  wonderful  opportunity  to  start  new  international collaborations. Finally NIBB concluded the proceedings with a  review  of  the  conference,  as  well  as  the  ones  previously held,  and  the  announcement  of  our  future  directions  as  we step forward.
Some  Japanese  participants a l s o   h a d   m e e t i n g s   w i t h researchers  in  MPIP   about collaborative  research  projects before and after the symposium and in the afternoon of the first d a y.   I n   a d d i t i o n ,   s o m e participants  visited  other universities  in  Germany  to h a v e   d i s c u s s i o n s   a b o u t individual collaborations.
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■Period  September 22 (Mon) - October 1 (Wed), 201■P articipants   1   (   from Germany, 2 from Taiwan, one each  from  ong  Kong,  India, Indonesia,  Bangladesh,  and USA,   from Japan)■ enue   National  Institute  for Basic Biology, Japan■Lecturers  Dr. Toshinori  AYAS I (Tottori Univ.)Dr. Yasuhiro KAMEI (NIBB)Dr.  Kei  MIYAMOTO  (Wellcome  Trust Cancer  Research UK Gurdon Institute)Dr.  ajime  OGINO  (Nagahama  Institute  of  Bio-Science and Technology)Dr.  ideaki TAKEUC I (Univ. of Tokyo)Dr. Thomas T UMBERGER ( eidelberg Univ.)Dr. Christoph WINKLER (National Univ. of Singapore)Dr.  itoshi YOKOYAMA (Tohoku Univ.)Dr. Takashi YOS IMURA (Nagoya Univ. NIBB)■Course Staff
Dr.  Satoshi ANSAI,  Dr.  Shin-ichi  C ISADA,  Dr.  ideki ANADA,  Dr.  Yasuhiro  KAMEI,  Dr.  Akihiko KAS IWAGI,  Dr.  Keiko  KAS IWAGI,  Dr.  Aiko KAWASUMI,  Dr.  Masato  KINOS ITA,  Dr.  Kei MIYAMOTO,  Dr.  Kiyoshi  NARUSE,  Dr.  Shigenori NONAKA, Dr. Tetsushi SAKUMA, Dr. Takao SASADO, Dr.  Ken-ichi  T.  SU UKI,  Dr. Yusuke  TAKE ANA,  Dr. Marta  TEPEREK,  Dr.  Thomas  T UMBERGER,  Dr. itoshi YOKOYAMA ■Contents of the course  Gene  knock-out  using  TALEN  in  Xenopus,  Gene  knockout using CRISPR CAS  system in medaka, Gene knockin  using  CRISPR CAS   system  in  medaka,  in vitrofertilization  of  eggs  and  manipulation  of  embryos,  Local gene  induction  with  the  infrared  laser-evoked  gene operator  (IR-LEGO)  method,  Live  imaging  by  2-photon microscopy  and  Digital  Scaning  Light-sheet  Microscopy (DSLM),  Cryopreservation  of  sperm  and  artificial insemination for Medaka

XenopusThe  th  NIBB  International  Practical  Course    The  rd NIBB-TLL-DBS    NUS  Joint  International  Practical  Course Experimental Techniques using Medaka and Xenopus - The Merits  of  using  both  -   was  held  from  September  22  to October 1st 201 . 1  participants from Taiwan,  ong Kong, India,  Indonesia,  Bangladesh,  Germany,  the  United  States, and Japan were selected from among the 2  applicants. Unique  Features  of  this  NIBB  International  Practical Course  were  training  with  medaka  and  zebrafish,  and concurrently  training  with  Xenopus.  Through  these workshops  important  connections  were  made  between  the two communities of small fish and Xenopus researchers. The course  was  planned  with  the  intention  that  participants should  experience  research  using  a  variety  of  experimental systems.
In addition to the main project aims, there was also the subaim  of  displaying  the  rapid  development  of  genome  editing technology  (such  as  mutants  created  by  TALEN  and CRISRPR-CAS ), which can be used in Xenopus and small fish.  In  response  to  these  changes  in  technology  and  the research  environment  participants  were  allowed  to  use  both medaka and Xenopus not only in live-imaging and training in important sperm freezing technologies for long-term storage of  mutants,  but  also  in  the  TALEN    CRISRPR-CAS  genome  editing  system  to  produce  knock-out  and  knock-in mutants,  and  the  gene  induction  system  by  IR-LEGO.  The results of the questionnaire show the course was very popular to those who participated. In addition to the standard training schedule,  on  every  day  except  Sunday  and  on  the  last  day, distinguished  invited  researchers  in  the  field  conducted seminars  for  participants.  The  seminars  were  extremely interesting  because  of  both  the  cutting edge  research  using small  fish  and  amphibians,  and  the  research  using  multiple model organisms concurrently.
I want to take this opportunity to dedicate a big thank you to the staff of the Office of International Cooperation. I would also like to thank the research groups that were co-sponsors of  the  course   National  BioResource  Projects  (NBRP) Medaka,  NBRP  Xenopus,  Molecular  Mechanisms Underlying  Reconstruction  of  D  Structures  during Regeneration  (supported by ME T, Japan), and  Enhancing Application  of  Innovative  Optical  Science  and  Technology by  Making  Ultimate  Use  of  Advanced  Light  Sources  (supported  by  CREST JST,  Japan).  Finally  I  would  like  to thank  the  staff  in  the  Institute  for  Amphibian  Biology, iroshima  University,  the  Laboratory  of  BioResources, NIBB,  and  the  Spectrography  and  Bioimaging  Facility, NIBB,  whose  preparations  made  this  training  course possible.
(Kiyoshi Naruse)

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rgani ers asu iro amei
igenori Nona a Naoto eno os i i o Fu imorioyama a uyos i urata N P
os ita a imori agaya i ato
io maging Forum arc 4 ue 2 4
e eeting or t e Future Plan arc 5 edThe  th  Bio-Imaging  Forum  was  held  on  the  theme  of Directions  of  Future  Bio-Imaging,   and  was  immediately followed,    with  the  same  participants,  by  the  Meeting  for the Future Plan of Bio-Imaging Network among Universities and  Institutes  in  Japan ,  which  was  planned  by  the Department  of  Imaging  Science,  Center  for  Novel  Science Initiative, NINS. Both  the  forum  and  the  meeting  were  managed  by  the Collaborative  Research  Group,  Research  Enhancement Strategy  Office,  NIBB.  The  forum  and  the  meeting  were seamlessly  planned  by  the  same  organizers  who  undertook detailed discussions prior to the events. The aim of the forum was  to  promote  the  exchange  of  information  on  the  newest technologies  of  obtaining  and  analyzing  biological  images, and that of the meeting was to grasp the present situation of various  imaging  facilities  in  Japan  and  to  discuss  the collaborations  of  these  facilities  and  the  future  directions  of bio-imaging,  with  the  prospect  of  promoting  collaborative use of the Spectroscopy and Bioimaging Facility, NIBB.
The Forum welcomed ninety participants and included nine lectures on a wide variety of bio-image acquisition methods, ranging  from  the  newest  techniques  of  electron  and  optical microscopy  to macro bio-imaging methods such as PET and MRI,  and  also  on  the  image  analysis  methods  (Bio-Image Informatics) of acquired images.  These lectures were a part of  the  imaging  facilities’  introduction  to  the  later  meeting during  which  they  served  as  materials  of  discussion.  The meeting  started  with  self-introductions  by  the  twenty imaging facilities on their missions, present status, problems and  prospects  for  collaboration.  Dr.  Kota  Miura  of  the iros iu 2 4

Advanced Light Microscopy Facility, EMBL  eidelberg was invited  to  report  the  present  status  and  the  future  directions of  the  network  of  bio-imaging  facilities  in  Europe.  Further discussions resulted in common understanding on the present status and problems of imaging facilities and as the first step of  the  collaboration  we  agreed  to  open  a  portal  web  site  of bio-imaging  facilities  and  to  continue  to  hold  similar meetings  to  promote  formation  of  networks  in  the  imaging science  field  through  the  collaboration  of  the  Spectroscopy and  Bioimaging  Facility  and  the  Department  of  Imaging Science.
The organizers appreciate the precious information brought by  the  participants,  and  the  Spectroscopy  and  Bioimaging Facility  benefitted  much  in  enhancing  collaborative programs  and  preparing  for  improvement  of  its  equipment. The  forum  and  the  meeting  served  an  important  role  in promoting  NINS  as  a  hub  of  the  worldwide  scientific community working in the field of imaging science.
(Yasuhiro Kamei)BB

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NIBB  organizes  a  series  of  training  courses  on  up-to-date research  techniques  for  researchers  from  mainly  Japanese universities  and  institutions.  In  201   we  held  two  of  these training  courses  on  Genome  Informatics.  The  two-day programs  offer  lectures  and  hands-on  tutorials  to  introduce basic knowledge and skills to deal with genomic scale large data  such  as  those  obtained  by  next-generation  sequencing (NGS).  The  programs  are  specially  designed  for  biologists who are not familiar with bioinformatics.

Aeptember

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Fri 2

4

■O rganizer   Dr.  Shuji  Shigenobu  (NIBB  Core Research Facilities)■Lecturers  Dr. Shuji Shigenobu, Dr. Masanao Sato, Dr.  Ikuo  Uchiyama,  Dr.  Katsushi Yamaguchi,  Dr. Taro Maeda ■Participants  20 (including 2 from NIBB)■Program  1. Overview  Transcriptome data analysis2. Introduction to statistics. Introduction to  R
. Basic format and tools of NGS
5. RNA-seq 1  Basics. RNA-seq 2  Genome-based analysis. RNA-seq   de novo assembly. Multivariate statistics. Practical exercisesA

eptember

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u 2

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Fri 2

■O rganizer   Dr.  Shuji  Shigenobu  (NIBB  Core Research Facilities)■L ecturers   Dr.  Shuji  Shigenobu,  Dr.  Ikuo Uchiyama,  Dr.  Masanao  Sato,  Dr.  Katsushi Yamaguchi, Ms.  iroyo Nishide, Dr. Taro Maeda■Participants  22 (including   from NIBB) ■Program   
1. Overview
2. UNI  for beginners. NGS basic data formats. NGS basic tools 5. Introduction to statistics. Basics of RNA-seq. RSA-seq genome base. Multivariate statistics. Introduction to  R
10. RNA-seq de novo11. Practical exercisesThis  course  was  held  as  a  workshop  of  the  Department  of Imaging  Science,  the  Center  for  Novel  Science  Initiative, NINS.  The  aim  of  the  course  was  to  deliver  enough  basic knowledge  to  attendees  that  they  would  be  able  to descriminate the difficulty of the problems facing them with image  analysis   giving  the  ability  to  solve  easy  problems alone, while allowing communication with experts regarding more  challenging  analyses.  The  course  therefore  was centered  on  basic  methods  of  image  analyses,  basic techniques  of  ImageJ,  and  writing  of  simple  macros  of ImageJ. At  the  end  of  the  course,  lecturers  and  participants discussed  real  problems  some  of  the  participants  are  facing. During  the  practice  using  PCs  seven  volunteers,  some  from NIBB and others from outside, supported participants. There was also an introduction of NIBB as a collaborative research institute  by  the  vice-director  Prof.  Ueno  during  the  gettogether.  Answers  to  questionnaires  on  the  course  were mostly favorable. (Yasuhiro Kamei) ecembered

2 Fri 2

4

■Organizers   Dr.  Yoshitaka  Kimori,  Dr.  Kagayaki Kato,  Dr. Yasuhiro  Kamei,  Dr.  Shigenori  Nonaka, Dr. Takashi Murata, Dr.  iroshi Koyama■S upervisors   Dr.  Naoto  Ueno,  Dr.  Toshihiko Fujimori■Participants  21 (including 2 from NIBB)■Program
-  Basis  of  image  data  processing  and  analysis (lecture and practice)-  Usage of ImageJ software and its macros (lecture and practice)-   uantitative  analysis  of  images  (lecture  and practice)-  Selection of microscopes and important points in obtaining images (lecture)5BBThe  NIBB  Internship  program,  started in  200 ,  is  a  handson  learning  course  for  overseas  students  designed  to  give high-quality  experience  in  real  world  research  and  focused education of biology. At the same time, this program aims to internationalize  the  graduate  students  of  SOKENDAI (Graduate University for Advanced Studies), giving them the opportunity to get to know students and interns with various cultural  customs. Another  goal  of  the  program  is  to  build connections  through  providing  education  to  the  people  who will  form  the  core  of  international  research  networks  in  the future.
To participate in this program, applicants who would like to experience research at NIBB must supply the name of the lab they would like to visit as well as their reasons for choosing it, and a letter of recommendation. Based on this information applicants  are  chosen  to  spend  set  periods  of  time participating  in  specific  research  activities  in  the  lab  they applied  for.  Round  trip  airfare  and  housing  expenses  are provided by the NIBB Internship Program.
In  FY  201   there  were  12  applicants,  out  of  which  seven interns  were  selected.  These  interns  were  from  universities located in   countries (India, China,  ungary, and Germany) and  spent  periods  ranging  from  one  to  twelve  weeks experiencing life as a member of a research team. Report from a participant
Dilukshi Chinthani Perera
Shanghai Jiao Tong University, China
I  was  an  intern  student  at  Professor  Minoru  Tanaka’s  lab from  th  of August  201   to  15th  of August  201 .  My  main area of study was Gonadal sex differentiation in Medaka by observing Germ cells.
Germ  cells  are  a  type  of  biological  cell  that  involves reproduction.  In  many  animals  the  germ  cells  originate  in the primitive streak and migrate via the gut of the embryo to the  developing  gonads. There,  they  undergo  cell  division  of two  types,  mitosis  and  meiosis,  followed  by  cellular differentiation  into  mature  gametes,  either  eggs  or  sperm.
Germ  cells  play  an  essential  role  in  Sex  differentiation  in Medaka.  They  are  required  for  ovarian  formation  and  also Germ cell deficient gonads develop a testis-like structure.
In  medaka  the  sex-determining  gene  dmY/dmrt1y  has  been identified.  My  studies  were  mainly  focused  on  Genotypic Sex  Determination  (GSD),  and  two  important  genes  SDiG and figla. At the beginning of the experiment I had to dissect Medaka larva to extract gonads which was a very interesting activity.  I  enjoyed  collecting  fish  eggs  at  Myodaiji. Performing  micro  surgery  was  where  I  used  my  skills  as  a medical student who had assisted in surgery at a hospital. It was  also  an  opportunity  to  exchange  knowledge  with  the PhD  students  at  the  lab.  Therefore,  my  overall  experience was  invaluable.  I  certainly  had  a  very  useful  time  with Professor Tanaka and his team.

A ....................................................................................................
ADAC I, Daisuke

5AI ARA, Yusuke
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AKAMA, AkikoAKITA, AsakaAOKI, Etsuko5ASAOKA, Miho21ASAO,  isayo0ATSUMI, MihoB ....................................................................................................
BABA, Nayumi
BAN, Misato,  0BINO, Takahiro....................................................................................................
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C EN,  iuhong 0C IBA,  irokazu
C ISADA, Eriko52, C ISADA, Shinichiuperov ,  uzana5

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DO ENY, Brenna5DOKYO, Yumi....................................................................................................
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FUJIKAWA, AkihiroFUJIMORI, Chika FUJIMORI, Sayumi

FUJIMORI, ToshihikoFUJIMOTO, Daiji, 2 ,  0, ,  1, 2,  5
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FUJISAWA, Chiemi

21

FUKADA-TANAKA, Sachiko,  0FUJITA,  ironoriFUKU ARA, Mai

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FUKUS IMA, Kenji....................................................................................................
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GONDA, NaokoGOTO-YAMADA, Shino5GOTO, Misako5GU, Nan....................................................................................................
AC ISUKA, MidoriAKOS IMA, Toshio1 ,  2

AMADA, YoshioAMATANI, AyakoANDA, YoshihiroARA, Ikuyo52ARA, Yusuke

10ARA, KenshiroASEBE, Mitsuyasu,  5, ,  2

ASEGAWA, Ryota1AS IMOTO, Terumi

5ATA, Katsusuke

ATTORI, MasayukiAYAS I, Kentaro

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AYAS I, Kohji0,  0AYAS I, Yoshiki21

AYAS I, Tomoko 2IGAS I, Satoru5IGAS IYAMA, TetsuyaIGUC I, Yoko2IKINO, Kazumi, 55

IRA, Riichiro1IMENO, Miki

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IRAKAWA, Reiko1IRUTA, Chizue

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IYAMA, Takeshi5OJO, Masaru5ONDA, Satoko21ORIUC I, Yuta5OS I, Rie

5OS INO, Atsushi5 ,  2

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IC IKAWA, Chiaki,  ,  0IC IKAWA, Mariko, IGUC I, Taisen,  2,  0

IKAMI, Kanako0IC IKAWA, MichikoIINUMA,  ideko,  0,  0IMAI, Akihiro5IMAI, AkikoIMAI UMI, TaekoIMOTO, EikoINABA, KayoINADA, Kana

212,  00INADA, Yosuke

IS IBAS I, TomokoIS IJIMA,  annosuke
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KOTANI, KeikoKAJIKAWA, Ikumi

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KAMETANI, Yoshiko KAMIDA, Chiharu

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KAMIYA, KiyomiKAMRANI, Yousef Yari
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KATAYAMA, Kaori15KATO, Daisuke

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KATO, KyokoKAWAGUC I, ColinKAWAGUC I, MasayoshiKAWAMOTO, Yuri

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KUME, ShoenKURATA, TomokoKURUMI AKA,  itoshi....................................................................................................
LI, Chen5LIN, Chia- ao

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MAEDA, Taro5,  5

MAKINO, Yumiko,  0MANO, Shoji , 55

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MARUYAMA, Ayumi

MARUYAMA, Shin-ichiroMASAMI U, YoshitoMASUOKA, TomokoMATSUBAYAS I, NaomiMATSUBAYAS I, YoshikatsuMATSUDA, ChisatoMATSUDA, TakashiMATSUDA, Yoshimi

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NAGAE, MiwaOTA, Ryoma21OTSUBO, Yoko1MURATA, TakashiNAKAGAMI, Yuki
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NAKAMURA, Ryoko5NAKAMOTO, Masatoshi52NAKAMURA, Takanori,  0

NAKAMURA, Yoshiaki0NAKAMURA, TohruNAKANIS I, Norie5NAKAYAMA, Kei

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NIS I, TayoNIS IDA,  annaNIS IDE,  iroyo,  0,  5
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SADAKANE, Osamu

SAITO, Junko0NIS IMURA, NorikoSAKAE, YutaSAKAGAMI, Mari
SAKUTA,  iraki
SANBO, Chiaki

SANJO, KazukoSASAKI, Takema
SATO, KaoriSATO, KatsuhikoNOGUC I, Yuji

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SUGIMORI, Seiko21SUGAYA, Tomomi5SUGINAGA, Tomomi0,  2

SUGIYAMA, Arisa21SUGIURA,  aruka SUGIYAMA, Kazuya

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TANAKA, Yasuhiro1TANAKA, Minoru,  0

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TOKUTSU, Ryutaro25TOMINAGA,  itoshi1TORII, Naoko52TOYOTA, Kenji2TORIBA, TaiyoTOYOOKA, Yayoi
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WAS IO, Midori21WANGLAR, Chimwar

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WATAKABE, Akiya WATAKABE, IkukoWATANABE, Eiji,  0

WATANABE, Mika1WATANABE, Etsuko, 55WEIC SEL, Alexander

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YABE, Taijiro 2YAMADA, KenjiYAMAGUC I, Chinami

YAMAGUC I, KatsushiYAMAGUC I, Takeshi, , 55,  0,  5
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YAMAMORI, TetsuoYAMAMOTO, Manami

YAMAMOTO, MasayukiYAMAMOTO, Takamasa211, 2, 1
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YAMAMOTO, YasuhiroYAMASAKI,  iroaki
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YONE AWA,  arumi5YORO, EmikoYOS IDA, Akihiro51YOS IDA, Shosei,  0,  ,  , YOS INORI, Yumi, YOS IMURA, TakashiYU, Yang,  , 5

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ANG, Liechi5Access

From Tokyo

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

From Osaka

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

From Central Japan International Airport

Get on the Meitetsu bus bound for JR Okazaki Station andget off at Higashi Okazaki Station(approximately one hour from the airport). Alternatively,
take the Meitetsu Line for Nagoya and change at Jingu-mae Station toa Toyohashi-bound train and get off at Higashi Okazaki Station(approximately 70 minutes from the airport).
City HallCity HallCity Hall S.

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

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

National Institutes of Natural Sciences

National Institutefor Basic Biology
38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585 Japan
tel: +81 564-55-7000 fax: +81 564-53-7400
http://www.nibb.ac.jp/en/
Issued in July 2015
Edited by the Research Enhancement Strategy Office, NIBB