<i>Ascl1</i> Represses Müller Glial and Promotes Rod Photoreceptor Fate Through Repressing Notch Signaling in Late Retinal Progenitor Cells

Purpose: Ascl1 is a transcription factor (TF) that plays key roles in regulating retinal development. Recent studies have further uncovered its intriguing potential to drive retinal neuron regeneration by reprogramming Müller glial cells. However, the efficiency and specificity of Ascl1-mediated regeneration outcomes remain far from optimal, and the molecular mechanisms underlying its dual roles in retinal development and adult Müller reprogramming are not fully elucidated. Given the close developmental relationship between late-stage retinal progenitor cells (RPCs) and Müller glial cells, this study aimed to investigate the functional role and underlying molecular mechanisms of Ascl1 in governing retinal fate specification, using late RPCs as a model system. Methods: In vivo electroporation (IVE) of the mouse retinas was used to deliver Ascl1-overexpressing (Ascl1-OE) plasmids into late RPCs. Immunofluorescence (IF) staining was employed to recognize retinal cells and assess cell proliferation status. RNA sequencing (RNA-seq) and single-cell RNA sequencing (scRNA-seq) were conducted to profile the transcriptomes of RPCs, and the assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted to characterize the chromatin accessibility landscapes of RPCs. Results: Ascl1-OE caused late PRCs to exit the cell cycle prematurely and produce more rods but fewer bipolar and Müller glial cells. Integrative RNA-seq, scRNA-seq, ATAC-seq and chromatin immunoprecipitation (ChIP)-seq analyses of Ascl1-OE late RPCs revealed that Ascl1 promoted the expression of Notch pathway inhibitors and neurogenic genes by binding to and opening up relevant genomic regions. Conclusions: Ascl1 suppresses Müller glial and bipolar fates and promotes rod photoreceptor fate in late RPCs. Mechanistically, Ascl1 achieves the effect by repressing the Notch signaling and activating neurogenic genes through reprogramming the epigenetic landscape of the cells. The findings of this study may help elucidate the molecular mechanisms of Ascl1-driven Müller reprogramming in adult retinas and facilitate the development of novel methods to improve the efficiency and specificity of retinal neuron regeneration outcomes.