A Roadmap to Uncover RPE Plasticity

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Abstract Degenerative retinal diseases represent an enormous public health burden and demand innovative strategies to replace retinal neurons. Ideal solutions will overcome innate barriers associated with terminal differentiation to endogenously regenerate retinal neurons. Retinal pigment epithelium (RPE) cells hold promise for this application, as these cells can reprogram to produce neural retina in embryonic amniotes. For reasons that are not well understood, RPE cells lose neural competence during early amniotic development. The present study proposes to comprehensively map the gene regulatory landscape of RPE at plastic stages of development and to probe the molecular barriers to reprogramming that emerge as these cells differentiate. RPE cells of the chicken are plastic at embryonic day 4 (E4) and can be reprogrammed to neural retina following retinectomy and treatment with FGF2. However, by embryonic day 5 (E5), RPE cell fate becomes restricted and reprogramming capacity is abrogated. Previous studies have demonstrated that E4 RPE cells reprogram through the activation of neural retina transcription factors, such as VSX2, SIX6, and PAX6, and the simultaneous repression of RPE differentiation programs. Concomitantly, an epigenetic reprogramming event resets DNA methylation and poises chromatin into a more active configuration to facilitate the ensuing change in cell identity. However, it is not understood how transcription factor networks interact with a dynamic chromatin landscape to determine RPE neural competence. Our preliminary evidence suggests that pro-neural genes remain comparably inducible in the E5 RPE, but that an incipient RPE differentiation program serves as an inherent barrier to neural identity at this stage. This differentiation program is led by the RPE transcription factors MITF and OTX2, and distinct accessibility footprints from these factors are observable in the chromatin landscape. The current proposal will build on these findings by mapping genome-wide epigenetic patterns associated with RPE competence restriction, including DNA and histone modifications that have been previously demonstrated to facilitate RPE reprogramming (Aim 1). Additionally, RPE differentiation and reprogramming will be profiled at a single cell resolution, enabling the precise identification of transcriptomic features that delineate plastic and fate restricted RPE (Aim 2). In parallel, the DNA binding activity of key effector transcription factors, such as MITF and OTX2, will be profiled across the RPE at different stages of differentiation or reprogramming. These factors, as well as key transcriptional targets, will be perturbed using CRISPR-Cas9 with the intention of recovering RPE neural competence at more advanced stages of differentiation (Aim 3). Together, these findings will provide an expansive view of chromatin states associated with RPE competence restriction, while simultaneously probing the chromatin – transcription factor interactions that drive the observed phenotypes. These results will provide imperative insights toward understanding RPE differentiation and the potentiality of this cell type for use in neuron replacement strategies.