Elucidating the gene regulatory networks that drive neural regeneration

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Project Summary / Abstract There are no effective treatments to replace damaged retinal neurons, reflecting a fundamental inability for humans to mount robust regenerative responses within the central nervous system. To address this, it will be critical to understand the diversity of gene regulatory mechanisms that can impede or drive neuron regeneration across vertebrate contexts. Embryonic amniotes have a transitory ability to regenerate retinal neurons from cells of the retinal pigment epithelium (RPE) if supplied with exogenous FGF2 at the time of retinal injury. This mechanism of regeneration can be readily induced at embryonic day 4 (E4) of chicken development, but RPE neural competence is lost by embryonic day 5 (E5). The overarching objective of the proposed research is to profile changes in gene regulation that dispossess RPE cells of their neural competency as they differentiate. Specific aim 1 will interrogate transcription factor regulatory activity within RPE cells across the E4 / E5 developmental window by integrating gene expression analysis, chromatin accessibility profiling, and transcription factor binding assays. Preliminary bulk and single nuclei RNA-seq datasets revealed the acute activation of neural retina transcription factor profiles at both E4 and E5, such as PAX6, ASCL1, and VSX2. In contrast, genes associated with RPE maturity, such as OTX2 and pigmentation genes, were elevated in the E5 RPE independently of retinectomy and FGF2 treatment. Similarly, chromatin accessibility suggested wider dysregulation of OTX2 and related homeobox transcription factor binding sites. During the 1-year F99 phase, OTX2 binding activity will be profiled in intact and FGF2-treated RPE cells at E4 and E5 stages. Additionally, single nuclei RNA-sequencing will capture the heterogeneous transcriptional states of RPE cells during differentiation and FGF2 treatment response at E4 and E5. These results will be integrated to into a model that describes how changes in the RPE gene regulatory landscape culminates in a loss of neural competency. In specific aim 2, the gene regulatory networks present in adult vertebrate models of central nervous system regeneration will be interrogated to inspire novel routes for the induction of mammalian regeneration. This K00 phase will focus on the development of key research skills, including multi-omics data analysis approaches, techniques for spatial transcriptomics and single cell epigenomics, and cross-species genomics / transcriptomics. Up to 4 years will be spent on the K00 phase in an environment directly supportive of these applications. Specific professional development objectives will be concurrently pursued, such as pedagogical development, diversity outreach initiatives, and grant writing. Together, these aims encompass a career development plan that will lead to formation of an independent research program and result in impactful research focused on expanding human central nervous system regenerative capacity.