Elucidating and bypassing molecular mechanisms that suppress Muller glia-dependent regeneration of cones in two zebrafish models of chronic retinal damage

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Project Summary: Retinal degenerative diseases are a major medical issue for society. One potentially exciting approach to restore vision is the regeneration of lost retinal neurons from an endogenous population of retinal cells, the Müller glia. We are studying this process in zebrafish, which unlike mammals, exhibits a natural Müller glia- dependent retinal regeneration response. However, there are two major gaps in our understanding of this retinal regeneration response. The first is why rapid acute damage exhibits a regeneration response and a slow chronic damage, which is what is often observed in human retinal degenerative diseases, does not induce a regeneration response in zebrafish. The second gap in our understanding is the role of the microglia, the immune cells of the central nervous system, which are the major source of inflammation resulting from damage and a known regulator of the Müller glia-dependent retinal regeneration. We will address these two gaps in three Specific Aims. Aim 1 will determine the potential of two different chronic zebrafish retinal degeneration mutants (gosh, an early onset rapid cone photoreceptor degeneration mutant and cep290, a late onset slow cone degeneration mutant) to induce Müller glia proliferation and regenerate lost cones using different stimuli. We will determine to what extent either a secondary acute damage or the introduction of molecules that stimulate Müller glia proliferation can induce cone regeneration in chronically damaged fish and how complete the regeneration process is. In Aim 2, we will conduct a comprehensive and unbiased, comparative analysis of gene expression and chromatin accessibility in Müller glia and microglia using a multiomic single-nuclear RNA- Seq and ATAC-Seq analysis in these two chronic degeneration mutants, along with two mouse chronic retinal degeneration mutants. We will determine the similarities and differences in gene expression and chromatin accessibility in the Müller glia and microglia between the acutely and chronically damaged retinas. These bioinformatic analyses will reveal transcription factors and signaling (cytokine, growth factors, ligand/ receptor pairs) molecules that are essential for regeneration following acute damage and blocking regeneration in the chronically damaged zebrafish retina. We will also determine the differences and similarities between the chronically damaged zebrafish and mouse retinas to determine how similar these regulatory components are between the zebrafish and mouse. Aim 3 will then functionally test the roles of the candidate regulators previously identified in our scRNA-Seq datasets or in Aim 2 by either modifying their expression or their activity in the chronically and acutely damaged zebrafish retina. This work will be the first molecular analysis of how retinal regeneration is regulated in the chronically damaged zebrafish retina and will be critical in the translation of Müller glial-dependent retinal regenerative therapies into human retinal degenerative diseases.