Mechanistic assessment of retinal ganglion cell genesis - Project Summary/Abstract Retinal ganglion cells (RGCs) connect the eyes to the brain. They are essential for vertebrate vision as the sole output neuron of the retina and are the pathogenic targets in glaucoma. A key goal of vision scientists is to fully understand the factors required for RGC development, so these cells can be generated in vitro for cell transplantation or replaced through dedifferentiation of existing cells. RGC genesis begins with transient expression of the proneural basic helix-loop-helix protein ATOH7 in a subpopulation of early retinal progenitor cells, which give rise to the 7 major cell types of the retina. Despite its broad lineage, only RGCs absolutely require ATOH7 for their genesis as its loss of function causes optic nerve aplasia and severe secondary retinovascular malformations. Interestingly, overexpression of ATOH7 can only generate ectopic RGCs from certain lineages. Why do only some ATOH7+ cells become RGCs? Downstream of ATOH7 in the RGC lineage are POU4F2 (BRN3B) and ISL1, two transcription factors that are also required for RGC development. Ectopic expression of both Pou4f2 and Isl1 in the Atoh7 lineage are required to induce RGC development in Atoh7 mutant mice, suggesting a shared gene regulatory network that connects Atoh7, Pou4f2, and Isl1. What are the downstream targets necessary for proper RGC development in the absence of ATOH7? By exploiting three related but non-contingent aims, we propose to investigate the shared cis-regulatory elements and target genes of Atoh7, Pou4f2, and Isl1 along with identification of potential cofactors influencing Atoh7+ cell fate decisions during RGC genesis. First, we will apply a multi-species approach to test Atoh7, Pou4f2, and Isl1 co-regulated cis-regulatory elements identified by our independent, integrated multi-omic analysis for: A) The spatial-temporal expression pattern (zebrafish) of each transcriptional regulatory element and B) the necessity of each element to activate target gene transcription (mouse) to determine precisely how each component contributes to the dynamic tissue and cellular expression pattern of each target gene. Second, we will investigate the sufficiency of each Atoh7, Pou4f2, and Isl1 target gene to influence RGC development in wild type and atoh7 mutant zebrafish retinae. Third, we will use two transgenic mouse lines in a new experimental paradigm to isolate the proteome of Atoh7+ retinal progenitor cells and evaluate changing Atoh7 protein-protein interactions as they shift dynamically over the course of RGC genesis. These data will define key mechanisms controlling the RGC gene regulatory network: 1) the onset of retinal neurogenesis and RGC fate specification; 2) the changing retinal proteome and Atoh7 protein-protein interactions; and 3) identify new mechanisms for the generation of RGCs in vitro for cell transplantation or new ways to induce RGC regeneration through dedifferentiation of native retinal cells.
