Vsx2 Dependent Regulation of Retinal Progenitor Cell Properties

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A fundamental question in organ formation is how the constituent tissues achieve their correct sizes and cytoarchitectures. Defects in a single tissue can affect the formation of an entire organ and a classic example of this is the eye, where disruptions in retinal histogenesis can cause microphthalmia, a severe ocular anomaly characterized by small, poorly formed eyes and congenital blindness. Mutations in the Visual System Homeobox 2 (Vsx2) gene cause microphthalmia. A definitive marker of retinal specification, Vsx2 functions in retinal progenitor cells to define tissue identity. Concurrently, Vsx2 is required for progenitor proliferation and several aspects of the neurogenic program including the timing of neurogenesis onset (neurogenic timing), and the fate specification of bipolar cells. Two gaps in our understanding are the mechanistic interconnectedness of the progenitor properties regulated by Vsx2 and whether progenitors change in how they utilize Vsx2 over the course of histogenesis. To address these gaps, we generated two new Vsx2 alleles in mice, one with a knock-in reporter/knock-out configuration and the other for conditional gene inactivation. In the first two aims, we will characterize the retinal phenotypes of these mutant alleles and determine how they compare to phenotypes caused by a natural null allele and two missense alleles that correspond to disease-causing mutations in humans. Conditional gene inactivation will be done with tamoxifen-inducible Cre/lox recombination to determine the temporal windows of Vsx2 utilization in progenitors and test the hypothesis that Vsx2’s control of retinal identity, neurogenic timing, and proliferation are separable. We also predict that additional roles in the balanced production of cells in each retinal cell class (neurogenic output) will be unmasked by temporal inactivation after the start of neurogenesis. In the third aim, we test the hypothesis that retinal identity control shifts from a Vsx2-dependent to independent state that is epigenetically defined for some of the earlier targets. In the fourth aim, we will incorporate an ex vivo culture paradigm to test candidate genes identified in the previous aims for their functional significance in promoting or interfering with retinal development. Completion of these studies will provide new insights into how Vsx2 orchestrates retinal progenitor properties and how retinal progenitors drive retinal histogenesis, an essential component of eye organogenesis.