Large-scale screen of novel zebrafish retinal ganglion cell ablation model reveals genetic regulation of retinal regeneration is context specific

Many genes are known to regulate Müller glia (MG)-dependent retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, per degenerative disease, are undefined. Studying regeneration in the context of selective cell loss is important as evidence suggests injury specifics inform the regenerative process. Here, transgenic zebrafish enabling inducible selective retinal ganglion cell (RGC) ablation were combined with single cell multiomics and CRISPR/Cas9-based knockout methods to screen 101 genes for effects on RGC regeneration. We identified 18 regulators of RGC regeneration- seven knockouts inhibited and eleven promoted RGC regeneration. Surprisingly, 35 of 36 known/implicated regulators of retinal tissue regeneration following widespread damage were not required for RGC regeneration, and seven of these knockouts actually enhanced RGC replacement kinetics, including sox2, olig2, and ascl1a. Mechanistic analyses revealed ascl1a knockout increased the propensity of progenitor cells to produce RGCs; i.e., biased progenitor cell fate. These data demonstrate plasticity in how MG can convert to a stem-like state and context-specificity in how genes function during regeneration. Increased understanding of how disease-relevant cell types can be selectively regenerated will, support the development of disease-tailored regenerative therapeutics. We performed single-cell RNA sequencing in larval zebrafish eyes following multiple paradigms of retinal damage including ablation of retinal ganglion cells (RGCs, 4 timepoints) and ablation of rod photoreceptors, and multiome sequencing following ablation of RGCs in fish with the ascl1a gene knocked out via CRISPR/Cas9.