Elucidating the role of pioneer factors in RPC developmental competence

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PROJECT SUMMARY Temporal patterning drives retinal cellular diversity through complex gene regulatory networks (GRNs). These GRNs are headed by transcription factors (TFs) that promote stage-specific cell birth while repressing GRNs associated with other developmental timepoints. This cross play is especially noticeable when comparing the GRNs controlling generation of early and late-born cell types in retinal progenitor cells (RPCs). Notably, over the course of differentiation, mammalian retinal cells lose the regenerative capacity seen in fish and amphibian models. This loss of regenerative capacity permits for the cell death associated with leading causes of blindness such as age-related macular degeneration and glaucoma. Key to therapeutic interventions is an understanding of how to stimulate the birth of specific cell types to allow for successful regrowth of the cell populations damaged in the course of these diseases. This understanding hinges on identifying which TFs within the GRNs drive cell fate specification and the loss of pluripotency for the early and late-stage RPCs. Because the genomic organizational states between the early and late-stage RPCs is so distinct, I hypothesize that the TFs driving the GRNs act in a pioneering capacity to drive the birth of temporally restricted cell types. If this holds true, the TFs associated with early-born cell types will be able to drive the birth of cones, amacrines, and horizontal cells in late-stage RPCs through the opening of genomic regions inaccessible at that temporal window. Likewise, late-stage TFs will induce the production of bipolar cells and Müller glia in the early RPC population through genomic organizational changes. To address these hypotheses, I propose two Aims. Aim 1: Functional analysis of top candidate transcription factors for temporal patterning regulation. This work will help me narrow down the list of candidate regulators of temporal patterning through validation of their impact on cell fate in gain-of and loss-of-function experiments in the developing mouse retina. Aim 2: Determine whether TFs that regulate transition from early to late-stage states have pioneering activity. I will take established temporal patterning regulators and assay how they control the dynamics of epigenetic modulation. Once systems for in vitro and in vivo characterization have been established, I will take candidates identified in Aim 1 and use this pipeline to phenotype their pioneering activity. Through the establishment of a set of PFs that drive early and late-stage cell fate specification in the retina, I will be able to better address current barriers to successful iPSC-derived cell-based therapeutic approaches to glaucoma and age-related macular degeneration.