Project Summary This proposal investigates the underlying causes of human ocular diseases using mouse models. Proposed experiments will use complex in vivo conditional (cre-lox) mouse genetics, mouse transgenics, histology, immunohistochemistry, confocal microscopy, in situ hybridization, mouse embryology, single-cell NEXTgen sequencing, bioinformatics, BAC recombineering, qPCR, and PCR technologies to address basic, mechanistic questions about optic stalk-disc development and astrocyte differentiation. The Pax2 transcription factor initiates expression in all optic vesicle cells, but becomes progressively restricted to only the forming optic disc and stalk. Consistent with its role in other embryonic tissues, we will test a hypothesis that Pax2 shuts off neural/retinal progenitor gene programs, via global interactions with cell epigenetic machinery. This activity initially restricts ocular cells to an astrocytic progenitor cell (APC) fate, regulates their rate of cell division, and initiates glial gene expression profiles. In Aim 1, we will test evolutionarily-conserved Pax2 noncoding sequences as long-sought optic disc-nerve enhancer(s) by creating a new Pax2-Cre driver. This tool will be used to conditionally remove Hes1 and assess the consequences to optic stalk development, APC differentiation and mature astrocyte functionality. For Aim 2, we will take advantage of previously characterized Rax-Cre BAC transgenic mouse line, Pax2GFP knock-in and new Pax2 floxed allele to follow the ocular GFP lineages in control and Pax2 conditionally mutant cells. We will also generate and compare the gene expression profiles of Pax2 E11 and E12 heterozygous and homozygous mutant eyes. Here we will use single-cell RNA sequencing and the growing wealth of publicly available information regarding chromatin configurations, and mRNA expression levels during the normal development of mouse ocular cells.