Regulation of Retinal Ganglion Cell Regeneration - Müller glia (MG) cells function as injury responsive stem cells to enable retinal regeneration in zebrafish. Importantly, MG retain regenerative capacity in mice and potentially humans as well. Defining how MG regenerative potential is controlled could therefore lead to therapeutics for restoring visual function to patients. Many genes are known to regulate regeneration in the context of widespread retinal tissue damage. In contrast, genes controlling MG regenerative potential following limited retinal cell loss, as per degenerative disease, are unknown. Studying regeneration in the context of selective cell loss is nevertheless important as recent evidence suggests the nature of the retinal injury informs the regenerative process – i.e., MG-derived progenitor cell (MGPC) proliferation rates and fate decisions are correlated to the extent and specificity of cell loss. In a pilot screen of +100 genes for regulators of retinal ganglion cell (RGC) ablation in zebrafish, we identified 7 knockouts that inhibited and 11 that accelerated RGC regeneration kinetics. Moreover, disruption of 35 of 36 known/implicated regulators of retinal tissue regeneration either failed to impact (28 genes) or accelerated RGC regeneration kinetics (7 genes). Among the latter were proneural transcription factors, including olig2, neurog1, and ascl1a. Mechanistic analyses revealed disruption of ascl1a – a gene required for retinal tissue regeneration – accelerated RGC regeneration by increasing the propensity of MGPCs to produce RGCs; i.e., promoting RGC “fate bias”. These findings demonstrate plasticity in how MG can convert to stem cells (i.e., ascl1a-independent paths) and context specificity in how genes function to control tissue versus cellular regeneration in the retina. To rigorously test the hypothesis that the regenerative process actively adapts to the extent and specificity of cell loss, we will: 1) test ≥250 genes implicated as regulators of RGC, cone, bipolar, and/or hair cell regeneration for effects across each of these paradigms; 2) screen ~500 genes for effects on RGC replacement kinetics to define gene regulatory networks (GRNs) controlling RGC regeneration, and 3) determine if the MGPC fate bias extends to the level of RGC subtypes. Comparisons across cell regeneration paradigms – three retinal cell types, two RGC subtypes, and hair cells – will advance new knowledge of the mechanisms controlling MG regenerative potential. In particular, we will determine the degree to which paradigm-specific versus universal genetic programs govern cellular regeneration by testing how the specificity of cell loss informs the regenerative process. Our aims are designed to: 1) assign functional roles for 18 genes in RGC regeneration and define genes as paradigm/context-specific or universal per effects on RGC, cone, bipolar, and/or hair cell regeneration; 2) functionally validate entire GRNs that regulate RGC regeneration, and; 3) determine whether the propensity of MGPCs to give rise preferentially to lost cells extends to the level of RGC subtypes. We posit that defining how disease-relevant retinal cell loss parameters impact the regenerative process will support the development of disease-tailored regenerative therapeutics for restoring lost visual function to patients.
