FY2015 Annual Report

Developmental Neurobiology Unit
Associate Professor Ichiro Masai

Abstract

The vertebrate neural retina is derived from the ventral region of the forebrain. In this region, six classes of neurons differentiate and form the neural circuit underlying visual transduction. Thus, the retina provides an excellent model for studying cell differentiation and neural circuit formation in the vertebrate brain. Furthermore, more than one hundred hereditary retinal diseases causing photoreceptor degeneration have been identified in humans. Understanding the pathological processes of photoreceptor degeneration is an important issue from a medical perspective. We are currently investigating three research topics: (1) photoreceptor differentiation, (2) photoreceptor degeneration and (3) lens development.

1. Staff

  • Dr. Yuko Nishiwaki, Group leader
  • Dr. Toshiaki Mochizuki, Staff scientist (–April 2015)
  • Dr. Sachihiro Suzuki, Staff scientist
  • Dr. Maria Iribarne, Postdoctral scholar
  • Dr. Akane Hagiwara, Postdoctral scholar
  • Ms. Nishtha Ranawat, phD student (Sept 2015–)
  • Mr. Yutaka Kojima, Technician
  • Ms. Eri Oguri, Technician (–May 2015)
  • Mr. Masato Araragi, Technician
  • Ms. Miyuki Suenaga, Technician
  • Ms. Mai Yamashita, Technician (Apr-Aug 2015)
  • Mr. Kevin Jeff Liner, Technician (Feb 2016–)
  • Ms. Lina Koronfel, Technician (Feb 2016–)
  • Ms. Yayoi Tomoyose, Research Assistant (–April 2015)
  • Ms. Kazumi Toguchi, Research Assistant
  • Ms. Moe Inafuku, Research Assistant
  • Ms. Chitose Mizuta, Research Assistant
  • Ms. Rui Inoue, Research Assistant
  • Ms. Ayako Nakama, Research Assistant (Sept 2015–)
  • Ms Ayako Gima, Research Administrator/Secretary

2. Collaborations

2.1 The Title or Name or Topic of the Collaboration

  • Theme: In vivo functional analysis of hypoxia response genes using the zebrafish retina
  • Type of collaboration: Joint research agreement
  • Researchers: Dr. Ichiro Masai (Developmental neurobiology unit, OIST), Dr. Masayuki Matsushita (Department of Medicine, Ryukyu University)

3. Activities and Findings

3.1 Mechanism underlying photoreceptor differentiation 

Perception of color requires multiple types of cone photoreceptors, each of which shows sensitivity to a distinct spectrum of light. It is prerequisite for proper functioning of color vision not only to generate appropriate numbers of individual cone types but also to arrange them spatially in a species-specific manner. Zebrafish have four cone types: red, green, blue and UV-light sensitive cones. Zebrafish four cone types form a strikingly precise lattice pattern, namely cone mosaic. In this lattice pattern, a row of double cone that are tightly associated pairs of Red- and Green-cones and row of alternating blue- and UV-cones appear in turn. Although theoretical approaches were taken to understand cone mosaic formation mechanisms previously, molecular and cellular mechanisms underlying formation of the precise lattice pattern are poorly understood. To address this question, we plan to screen genes that are expressed by a limited cone types and examine changes of the cone mosaic pattern caused by loss-of-function of each candidate gene.

In 2014, we established a quadruple transgenic line, Tg(gnat2:histone-2A-CFP; sws1:histon2A-YFP; sws2:GFP; thrb:tdTomato) in which each cone type is labeled by a distinct combination of fluorescent protein expressions. As a pilot experiments, we perturbed red cone genesis by disrupting the thyroid hormone receptor beta 2 (thrb2) gene using the CRISPR/cas9 system. This resulted in chimeric fish with retinal areas lacking red cones. In the areas without red cones, the remaining cone types still formed a regular lattice pattern and also the ratios among them were not affected. Therefore, double cone formation is dispensable for formation of the regular lattice pattern.  

3.2 Mechanism underlying photoreceptor degeneration

3.2.1 Mechanism of BNip1-mediated photoreceptor degeneration

Mitochondria-dependent apoptosis is promoted by pro-apoptotic Bcl2 family proteins, Bax, and inhibited by anti-apoptotic Bcl2 family proteins, Bcl2. BH3-only proteins promote apoptosis by modulating the balance between pro-apoptotic and anti-apoptotic Bcl2 proteins. We previously found that a BH3-only protein, BNip1, mediates thecoa-mediated photoreceptor apoptosis. BNip1 is a component of the syntaxin 18 SNARE complex, and the syntaxin 18 SNARE complex regulates retrograde transport from the Golgi apparatus to the ER. b-SNAP promotes the recycle of vesicular fusion machinery, SNARE, by disassembling the cis-SNARE complex generated by vesicular fusion. Thus, it is likely that the syntaxin 18 cis-SNARE complex is accumulated in thecoamutant. We found that failed disassembly of the syntaxin 18 cis-SNARE complex activates the BNip1-dependent photoreceptor apoptosis in the coamutant. These data suggest that the syntaxin 18 cis-SNARE complex monitors vesicular fusion competence and that BNip1 transforms vesicular fusion defects into apoptosis. From these data, we propose that BNip1 functions as an alarm that monitors abnormalities of vesicular fusion and transforms vesicular fusion defects into apoptosis in photoreceptors. 

3.2.2 Mechanism of AIPL1-mediated photoreceptor degeneration 

The photoreceptor is the neuron in charge for detecting light, and transforms this information into an electrical response. This process is known as phototransduction with the cGMP and Ca2+as the second messenger in this pathway. It has been proposed that many mutations in the components of the phototransduction produce abnormal level of cGMP and Ca2+, and finally trigger the photoreceptor death. However, the molecular mechanisms underlying the cell death remain unclear. In humans, more than a hundred genes associated with inherited photoreceptor degeneration have been mapped (RetNet,https://sph.uth.edu/Retnet).

In our lab, using different zebrafish mutant with a defect in the vision we study the degeneration process in the retina. gold rushis one of these zebrafish strain mutants, which show no photopic visual response, and slow progressive cone-specific photoreceptor degeneration. We found that the gold rushmutant gene encodes aryl hydrocarbon receptor interacting protein-like 1 (AIPL1). AIPL1 is expressed in photoreceptors and required for stability and membrane anchoring of a phototransduction molecule, cGMP-phosphodiesterase 6 (PDE6). We found that AIPL1 is an important protein not only for the phototransduction, but also for survival of cone photoreceptors in zebrafish. We previously reported that photopic vision and cone photoreceptor survival are affected in the eclipse mutant, a cone-specific PDE6 mutant (PDE6c). PDE6c has a central role in the metabolism of cGMP. Both zebrafish mutant strain, gold rush and eclipse, show a very similar cone-specific degeneration. When, we examined PDE6c functions in gold rush mutant by western blot analysis we found that the protein level of PDE6c was absent. We also found that cGMP level was higher in gold rush mutant than wild-type, and that gold rush mutation genetically enhanced photoreceptor degeneration in eclipsemutant. These data suggest that AIPL1 is required for PDE6 functions in zebrafish cone photoreceptors. Currently, we are trying to understand the role of the metabolism of the cGMP in the degeneration process using zebrafish phototransduction mutants. We expected that these findings would help to establish neuroprotection strategies for patients who are suffering photoreceptor degeneration.

 

4. Publications

4.1 Journals

Nothing to report

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

(Oral, International conference)

  1. Masai, I., Mochizuki, T., Luo, Y.-J., Tsai, H.-F. Cell division and cadherin-dependent epithelial tension regulate lens epithelial cell migration, in The 9th European Zebrafish Meeting,Oslo, Norway (2015).
  2. Nishiwaki, Y., Suenaga, M., Araragi, M., and Masai, I. Mechanism that links vesicular fusion defects and apoptosis in photoreceptors, in Academia Sinica-OIST Joint Meeting “Evolution of Complex Systems”, Taipei, Taiwan. (2015).
  3. Hagiwara, A., Mochizuki, T., Kojima, Y., Nishiwaki, Y., and Masai, I., Mechanism of ectopic lens fiber differentiation in response to early endosome trafficking defects, in Academia Sinica-OIST Joint Meeting “Evolution of Complex Systems”, Taipei, Taiwan. (2015).
  4. Masai, I. Cell division and cadherin-mediated adhesion regulate lens epithelial cell movement in zebrafish, in The 22ndEast Asia Joint Symposium on Biomedical Research, OIST, Okinawa (2015). 

(Poster, international conferences)

  1. Nishiwaki, Y., Oguri, E., Araragi, M., Suenaga, S., Nakamura, S., Kojima, Y., and Masai, I.  Molecular mechanism that converts vesicular fusion defects into apoptosis in photoreceptors, in in The 9th European Zebrafish Meeting,Oslo, Norway (2015).
  2. Iribarne, M. Nishiwaki, Y., Oguri, E., Araragi, M., Nakamura, S., Masai, I. AIPL1 mutation causes photoreceptor degeneration in zebrafish through dysfunction of PDE6c, in ARVO2015 Annual Meeting, Denver, Colo. USA (2015).

(Invited talks)

  1. Masai, I. Administration of zebrafish lines and their application to genetic research, in The 62th Annual Meeting of Japanese Association for Laboratory Animal Science, Kyoto, Japan (2015). 
  2. Masai, I., Mochizuki, T., Luo, Y.-J., Tsai, H.-F. Cell division and cadherin-mediated adhesion regulate lens epithelial cell movement in zebrafish, inThe 3rdInternational Conference of the Lens 2015, Kona, Hawaii (2015). 

(Oral, domestic conferences)

  1. Iribarne, M. Nishiwaki, Y., Oguri, E., Araragi, M., Nakamura, S., Masai, I. AIPL1 mutation causes photoreceptor degeneration in zebrafish through dysfunction of PDE6c, in The 38thAnnual meeting of the Japan Neuroscience Society (Neuroscience 2015), Kobe, Japan (2015). 
  2. Suzuki, S., Wong, R. O., and Masai, I. Molecular mechanism underlying cone photoreceptor specification in zebrafish, in the Joint meeting of the 38thAnnual meeting of Japanese Society of Molecular Biology and the 88thAnnual meeting of Japanese Society of Biochemistry (BMB2015), Kobe, Japan (2015).
  3. Kawada, J., Kinoshita-Kawada, M., Hasegawa, J., Honmiya, N., Yanagi, S., Kanaho, Y., Masai, I., Kou, T., Rao, Y., and Wu, J. Y. Molecular basis for Slit-responsive switch of commissural axons across the midline, in the Joint meeting of the 38thAnnual meeting of Japanese Society of Molecular Biology and the 88thAnnual meeting of Japanese Society of Biochemistry (BMB2015), Kobe, Japan (2015). 

(Poster, domestic conferences)

  1. Nishiwaki, Y., Oguri, E., Araragi, M., Suenaga, S., Nakamura, S., Kojima, Y., and Masai, I.  Overexpression of ER-localized Bcl2 suppresses BNip1-dependent apoptosis, The 48th Annual meeting of Japanese Society of Developmental Biologists,Tsukuba, Japan(2015).

5. Intellectual Property Rights and Other Specific Achievements

5.1 The Category or Type of Funding, like External Funding, Awards, etc.

Funding

KAKENHI (grants from the Ministry of Education, Science and Sport/JSPS)

  1. Yuko Nishiwaki: Scientific research C (Kiban C) (2014–2016)
  2. Sachihiro Suzuki: Scientific research C (Kiban C) (2015–2017)

6. Meetings and Events

6.1 Seminars

(1) Speaker: Dr. Masaki Hiramoto

Affiliation:  Scripps Research Institute

Title: “Neurons that fire in sequence wire in sequence: “earlier” is forward, “later” is backward in visual map plasticity”.  

Date: Sept 15, 2015

(2) Speaker: Dr. Shizuya Saika

Affiliation:  Wakayama Prefectural University

Title: “EMTand myofibroblast generation in an injured lens: modulation of TGF-b/Smad signal by extracellular matrix”

Date: Aug 4, 2015

(3) Speaker: Dr. Naoki Mochizuki

Affiliation:  National Cerebral and Cardiovascular Center Research Institute

Title: “Zebrafish research uncovers preciously unidentified siganing in carviovascular development”

Date: Nov 26, 2015

6.2 OIST course

  • Title: Developmental Neurobiology Course 2015
  • Co-organizer:Yoko Yazaki-Sugiyama (OIST), David L. van Vactor (Harvard Medical School, OIST), Gordon Arbuthnott (OIST)
  • Dates:12–17 July 2015
  • Place:OIST campus
  • Speakers:David L. van Vactor (Harvard Medical School, OIST), Ichiro Masai (OIST), Brian Link (Medical College of Wisconsin), Jeffrey D. Macklis (Harvard Medical School), David Price (University of Edinburgh), Lisa Goodrich (Harvard Medical School), Tomomi Shimogori (RIKEN BSI), Gian Garriga (UC, Berkeley), Kai G. Zinn (California Institute of Technology), Mary B. Kennedy (California Institute of Technology), Christian Klämbt (University of Münster), Ian Meinertzhagen (Dalhousie University), Jay Parrish (University of Washington), Rachel Wong (University of Washington), Liqun Luo (Stanford University), Yoko Sugiyama (OIST), Dan Sanes (New York University), Steve Wilson (University College London), Ichi Maruyama (OIST), Daniel Goldman (University of Michigan), Pat Levitt (University of South California), Atsushi Miyawaki (RIKEN BSI), Hiroshi Kohsaka (University of Tokyo)

6.2 OIST workshop

  • Title: The 22ndEast Asia Joint Symposium on Biomedical Research
  • Co-organizer: Tadashi Yamamoto (OIST), Matthias Wolf (OIST), Hidetoshi Saze (OIST), Hiroki Ishikawa (OIST), Economo, E (OIST), Yoko Yazaki-Sugiyama (OIST), Sumiko Watanabe (University of Tokyo)
  • Dates:11–14 November 2015
  • Place:OIST campus
  • Participant Institutes: Institute of Biochemistry and Molecular Biology, National Taiwan University (IBMB, NTU), Institute of Molecular Biology and Genetics, Seoul National University (IMBG, SNU), Institute of Medical Science, University of Tokyo (IMSUT), Institute for Virus Research, Kyoto University (IVR, Kyoto Univ.), Okinawa Institute of Science and Technology Graduate University (OIST), Samsung Biomedical Research Institute, Sungkyunkwan University (SBRI, SKKU), Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (SIBCB, CAS), Soonchunhyang Institute of Medi-bio Science, Soonchunhyang University (SIMS, SCHU)
  • Speakers: 39 faculties and 33 young investigators from Participant Institutes

6.3 Annual meeting of the PRESTO grant group “Ninshiki-to-Keisei”

  • Co-organizer:Jun-ichi Kawada (Kyusyu University)
  • Dates:20–22 Sept 2015
  • Place:OIST Seaside House
  • Speakers: 12 PRESTO members

7. Other

Nothing to report.