FY2017 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 four research topics: (1) retinal development, (2) photoreceptor degeneration, (3) the role of microglia in retinal development and degeneration, and (4) lens development. 

 

1. Staff

• Dr. Yuko Nishiwaki, Group leader
• Dr. Yuki Sugiyama, Staff scientist
• Dr. Yuki Takeuchi, Staff scientist (December 2017–)
• Dr. Maria Iribarne, Postdoctral scholar (–November 2017)
• Dr. Chong Yee Hang, Postdoctral scholar (HFSP) 
• Dr. Ayano Harata, Postdoctral scholar (–March 2018)
• Ms. Nishtha Ranawat, phD student
• Ms. Manana Kutsia, phD student (September 2017–)
• Ms. Mai Omar Abdulrahman Ahmad, phD student (January 2018–)
• Ms. Swathy Babu, phD student (January 2018–)
• Mr. Yutaka Kojima, Technician
• Mr. Kevin Jeff Liner, Technician
• Ms. Kimberlie Ward, Technician
• Dr. Tetsuya Harakuni, Technician
• Dr. Hiroshi Izumi, Technician (–February 2018)
• Ms. Moe Inafuku, Research Assistant
• Ms. Chitose Mizuta, Research Assistant
• Ms. Rui Inoue, Research Assistant
• Ms. Ayako Nakama, Research Assistant
• Ms. Madoka Makiya, Research Assistant (tempo)
• Ms. Maki Ishikawa, Research Assistant (tempo) 
• Ms. Satsuki Asato, Research Assistant
• 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 degeneration

3.1.1 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 photoreceptor apoptosis in zebrafish b-SNAP mutants. BNip1 is a component of the syntaxin 18 SNARE complex, which regulates retrograde transport from the Golgi apparatus to the ER. b-SNAP promotes recycle of vesicular fusion regulators, 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 zebrafish b-SNAP mutants. We found that failed disassembly of the syntaxin 18 cis-SNARE complex activates the BNip1-dependent photoreceptor apoptosis. Thus, BNip1 functions as an alarm that monitors vesicular fusion defects in photoreceptors. 

Photoreceptor apoptosis markedly occurs in 2-3 day post-fertilization (dpf) in zebrafish b-SNAP mutants. Since protein synthesis and transport to the apical photoreceptive membrane organelle, called the outer segment (OS), are most active in this period, photoreceptor apoptosis in b-SNAP mutants correlates with this OS growing period. Next, we examined a critical period of b-SNAP for photoreceptor maintenance by overexpressing b-SNAP under the control of heat shock promoter in b-SNAP mutants. Overexpression of b-SNAP in 2-5 dpf is enough to prevent photoreceptor apoptosis in zebrafish b-SNAP mutants at least until 21 dpf. These data suggest the possibility that BNip1-mediated apoptosis links to excessive activation of vesicular transport associated with rapid growth of the OS. Consistently, the knockdown of IFT88 and Kif3b, which inhibits transport to the OS, rescued photoreceptor apoptosis in b-SNAP mutants. Taken together, these data suggest that BNip1 functions as a safeguard that inhibits excessive activation of vesicular transport in photoreceptors. 

3.1.2 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 rush (gosh) is one of these zebrafish strain mutants, which show no photopic visual response, and slow progressive cone-specific photoreceptor degeneration.

We found that the gosh mutant 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 (els) mutant, a cone-specific PDE6 mutant (PDE6c). PDE6c has a central role in the metabolism of cGMP. Both zebrafish mutant strains, gosh and els, show a very similar cone-specific degeneration. We examined PDE6c functions in gosh mutants by western blot analysis. We found that the protein level of PDE6c was absent in gosh mutants. We also found that cGMP level was higher in gosh mutant than wild-type, and that gosh mutation genetically enhanced photoreceptor degeneration in els mutant. These data suggest that AIPL1 is required for PDE6 functions in zebrafish cone photoreceptors. 

3.2 The role of microglia in retinal development and degeneration

Microglia are brain-resident immune cells, which originally derive from mesoderm-derivative tissue or the hematopoietic stem cell-lineage, migrate into brain, and patrol within the brain throughout life. Microglia are thought to eliminate dead or dying neurons to prevent inflammation. To understand the role of microglia in photoreceptor degeneration, we observed cell proliferation and migration of microglia using zebrafish transgenic lines, Tg[mpeg1:EGFP] and Tg[mfap4:tdTamoto], which specifically visualize microglia. Currently, we confirmed that microglia progressively enter in the retina through the optic fissure and increase its cell number during development.  

3.3 Mechanism underlying lens development

In vertebrates, lens epithelial cells cover the anterior half of the lens fiber core. During development, lens epithelial cells proliferate, move posteriorly, and differentiate into lens fiber cells after passing through the equator. To elucidate mechanisms underlying lens epithelial cell movement, we conducted time-lapse imaging of zebrafish lens epithelium. Lens epithelial cells do not intermingle but maintain their relative positions during development. Cell division induces epithelial rearrangement, which subsequently promotes cell movement toward the equator. These data suggest that cell division is the major driving force for cell movement. In zebrafish, E-cadherin is expressed in lens epithelium, whereas N-cadherin is required for lens fiber growth. E-cadherin reduced lens epithelial cell movement, whereas N-cadherin enhanced it. Laser ablation experiments revealed that lens epithelium is governed by pulling tension, which is modulated by these cadherins. Thus, cell division and cadherin-mediated adhesion regulate lens epithelial cell movement via modulation of epithelial tension.

4. Publications

4.1 Journals

*Iribarne, M. and *Masai, I. (2017) Neurotoxicity of cGMP in the vertebrate retina: from the initial research on rd mutant mice to zebrafish genetic approaches. J. Neurogenet. 31, 88-101.

Iribarne, M. Nishiwaki, Y., Nakamura, S., Araragi, M., Oguri, E. and *Masai, I. (2017) Aipl1 is required for cone photoreceptor function and survival through the stability of Pde6c and Gc3 in zebrafish. Sci. Rep. 7:45962. 

Mochizuki, T., Luo, Y. J. Tsai, H. F. Hagiwara, A., and *Masai, I. (2017) Cell division and cadherin-mediated adhesion regulates lens epithelial cell movement in zebrafish. Development 144, 708 – 719. 

4.2 Books and other one-time publications

Nothing to report

4.3 Oral and Poster Presentations

4.3 Oral and Poster Presentations

(Oral, International conference)

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 The10^thEuropean Zebrafish conference, Budapest, Hungary, 3^rd–7^th July 2017. 

Masai, I. Endocytic regulator VSP45 suppresses lens fiber differentiation by modulating TGF-band Wnt signaling, in The 5^thInternational Conference of the Lens, Kailuna Kona, Hawaii, USA, 10^th–15^th Dec 2017. 

Sugiyama, Y. How to form eye lens with precise spheroidal shape? in The 24^thEast Asia Joint Symposium on Biomedical Research, Kyoto, Japan, 17^th–19^th Oct 2017.

(Poster, international conferences)

Sugiyama, Y. Fine tuning of ocular FGF activity regulates lens fibre cell dynamics, in The 5^thInternational Conference of the Lens, Kailuna Kona, Hawaii, USA, 10^th – 15^th Dec 2017.

Nishiwaki, Y. and Masai, I.   BNip1 induces photoreceptor apoptosis in response to excessive activation of intracellular vesicular transport. in The 10th European Zebrafish Meeting, Budapest, Hungary, 3^rd – 7^th July 2017.

Iribarne, M. Nishiwaki, Y., Nakamura, S., Araragi, M., Oguri, E. and Masai, I. Aipl1 is required for cone photoreceptor functions and survival through the stability of Pde6c and Gc3 in zebrafish. in The 10th European Zebrafish Meeting, Budapest, Hungary, 3^rd– 7^th July 2017.

Ranawat, N., Nishiwaki, Y., and Masai, I.Microglia role in neuronal cell death in pinball eye (piy) zebrafish mutant. in The 10th European zebrafish meeting, Budapest, Hungary, 3^rd – 7^th July 2017.

Nishiwaki, Y., Suenaga, M., Araragi, M., and Masai, I. ER-resident BH3-only protein, BNip1, induces apoptosis in response to excessive activation of vesicular transport in zebrafish photoreceptors. in Annual Meeting of the Association for Research in Vision and Ophthalmology 2017, Baltimore, Maryland, USA, 7^th– 11^th May 2017.

(Invited talks)

Masai, I.“Molecular mechanism and cellular dynamics of lens fiber differentiation”, The 2^ndMalaysia Zebrafish Disease Model Workshop 2017, Monash University, Malaysia, 2^nd – 4^th Oct 2017.

(Oral, domestic conferences)

Iribarne, M., Nishiwaki, Y., and Masai, I. Mechanism of photoreceptor degeneration in response to dysfunction of cGMP metabolism, in ConBiol2017 (Joint meeting of 40^thAnnual Meeting of Japanese Society of Molecular Biology/90^thAnnual Meeting of Japanese Society of Biochemistry), Kobe, Japan, 6^th – 9^th Dec 2017. 

Nishiwaki, Y., and Masai, I.  BNip1 induces photoreceptor apoptosis in response to excessive activation of intracellular vesicular transport, in The 23^rdJapanese Medaka and Zebrafish Meeting, Yanamashi, Japan, 30^th–31^st Aug 2017. 

Nishiwaki, Y., Suenaga, S., Araragi, M., and Masai, I. BNip1 induces photoreceptor apoptosis in response to excessive activation of intracellular vesicular transport, in The 69^thAnnual Meeting of Japanese Society of Cell Biology, Sendai, Japan, 13^th – 15^th June 2017.

(Poster, domestic conferences)

Nishiwaki, Y. and Masai, I. ER-resident BH3-only protein, BNip1, induces apoptosis in response to excessive activation of vesicular transport in zebrafish photoreceptors, in ConBio2017 (Consortium of Biological Science 2017), Kobe, Japan, 6^th – 9^th Dec 2017.

Sugiyama, Y., Masai, I. & McAvoy, J.  Fine tuning of FGF activity in the eye regulates lens cell dynamics, in ConBio2017 (Consortium of Biological Science 2017) Kobe, Japan, 6^th – 9^th Dec 2017.

5. Intellectual Property Rights and Other Specific Achievements

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

HFSP Research Grant

PI name: Greg Stephens (Vrije Universiteit Amsterdam)

Co-PI name: Joshua Shaevitz (Princeton Univ)

Co-PI name: Ichiro Masai (OIST)

2016-2018

 

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

NA

6. Meetings and Events

6.1 Seminar Title in Full

• Date: October 10, 2017
• Venue: OIST Campus Lab1
• Speaker: Porf. David Ish-Horowicz
• Affiliation:  Medical Research Council Laboratory of Molecular Biology, University College London
• Title: “Looking back (and ahead) on my journey through in vivoRNA biology”

 

• Date: October 12, 2017
• Venue: OIST Campus Lab1
• Speaker: Porf. David Ish-Horowicz
• Affiliation:  Medical Research Council Laboratory of Molecular Biology, University College London
• Title: “Post-transcriptional regulation of stem cell maintenance during Drosophila neurogenesis”

6.2 OIST course

Title: Developmental Neurobiology Course 2017

Co-organizer:Yoko Yazaki-Sugiyama (OIST), David L. van Vactor (Harvard Medical School, OIST)

Dates: 23 July-7 August 2017

Place:OIST campus

Lecturers: Ulrike Engel (University of Heidelberg), Cecilia Moens (Fred Hutchinson Cancer Research Center), Lisa Goodrich (Harvard Medical School), Hiroshi Kawasaki (Kanazawa University), Yimin Zou (UCSD), Greg Bashaw (University of Pennsylvania), Michael Granato (University of Pennsylvania), Kang Shen (Stanford University), Tomoyuki Takahashi (OIST), Mei Zhen (University of Toronto), Jeff Wickens (OIST), Anne Hart (Brown University), Minmin Luo (National Institute of Biological Sciences), Hitoshi Okamoto (RIKEN BSI), Tadashi Isa (Kyoto University), Takao Hensch (Harvard University)

Teaching lab lectuters: Hiroshi Kawasaki (Kanazawa University), Hiroshi Kohsaka (University of Tokyo), Ichiro Masai (OIST Developmental Neurobiology Unit), David Van Vactor (Harvard Medical School/OIST Formation and Regulation of Neuronal Connectivity Unit), Yoko Yazaki-Sugiyama (OIST Neuronal Mechanism for Critical Period Unit)

 

7. Other

Nothing to report.