The mammalian retina does not repair itself following retinal cell loss. This fact led to the assumption that the mammalian retina is incapable of self-repair. However, recent studies suggest the potential for the retina to regenerate is intact, even in humans;human M?ller glia cell cultures are capable of giving rise to retinal neurons, and M?ller glia cells can function as injury-induced retinal stem cells in mammalian models when stimulated with exogenous factors, albeit functional repair remains elusive. Together, these studies suggest that: 1) the regenerative potential of M?ller glia cells is conserved in humans and;2) an understanding of how retinal stem cells are regulated?in particular, M?ller glia responses to cell loss?could aid development of regenerative therapies for diseases causing vision loss and blindness. M?ller glia recently emerged as the stem cells responsible for robust retinal regeneration in zebrafish, providing an excellent model system for investigating how the regenerative potential of M?ller glia cells is regulated. To date, zebrafish studies have implicate only a few molecular regulators of retinal regeneration. To expand mechanistic understanding of retinal repair, we propose to use unbiased genetic and chemical screening approaches to: 1) identify regeneration deficient zebrafish mutants that develop a normal retina but fail to regenerate rod photoreceptors following cell-specific ablation (Aim 1), and 2) discover compounds that promote retinal regeneration?increase the pace of rod cell replacement kinetics or promote rod cell regeneration in mutants (Aim 2). We have established a transgenic line in which selective ablation of rod photoreceptor cells can be induced. Using this line for an ongoing pilot screen, we have succeeded in identifying three regeneration deficient mutants and numerous potential mutants that display incomplete rod cell replacement, demonstrating proof of principle of the genetic screening strategy. Chemical screens will use an in vivo high-throughput screening (HTS) system we developed for measuring changes in fluorescent reporter levels in individual fish. This system allows us to discover compounds that effect rod cell regeneration by quantifying the kinetics of cell loss and replacement in thousands of fish per day. Defining cellular and molecular mechanisms that underlie how mutants disrupt and compounds modulate the regenerative process will serve to further our understanding of retinal stem cell biology. Additionally, this project will generate/validate new and useful resources for the research community: 1) novel mutant zebrafish lines for defining how regeneration is controlled?ranging from cell-specific repair to mechanisms regulating whole tissue regeneration, and;2) an in vivo HTS platform for drug discovery that is applicable to a broad range of research programs.

Public Health Relevance

Loss of cells in the retina of the eye (e.g., photoreceptors) leads to loss of vision and eventually blindness. Our lab focuses on discovering mechanisms which stimulate the ability of the retina to replace lost cells as a means of promoting the development of human therapeutics. Using large-scale genetic and chemical screens in zebrafish, we will: 1) identify regeneration defective mutant fish to define genes required for photoreceptor replacement, and;2) discover compounds which promote photoreceptor regeneration information and resources that could lead to reparative therapies for the human eye.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CB-Z (55))
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Neuhold, Lisa
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Johns Hopkins University
United States
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White, David T; Sengupta, Sumitra; Saxena, Meera T et al. (2017) Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina. Proc Natl Acad Sci U S A 114:E3719-E3728
White, David T; Eroglu, Arife Unal; Wang, Guohua et al. (2016) ARQiv-HTS, a versatile whole-organism screening platform enabling in vivo drug discovery at high-throughput rates. Nat Protoc 11:2432-2453
Sengupta, Sumitra; Zhang, Liyun; Mumm, Jeff S (2015) Chemical genetics and regeneration. Future Med Chem 7:2263-83