Active DNA demethylation is upstream of rod photoreceptor fate determination and required for retinal development [Bisulfite-Seq]

The goal of this study is to investigate the role of TET enzyme-mediated DNA demethylation on retinal development and cell fate specification. We utilized an allelic series of TET enzyme retinal conditional mouse mutants using the Chx10-Cre-GFP transgene, removing the TET enzymes from retinal progenitor cells. We observe that inhibition of DNA demethylation results in abnormal retinal development and a loss of visual function. Immunohistological and genomic profiling (RNA-seq, scRNA-seq, WGBS, and bACE-seq) indicate that rod photoreceptor gene regulatory networks are inhibited when DNA demethylation is lost, indicating that DNA demethylation is required for NRL and NR2E3 expression, resulting in a retina with increased specification of cone photoreceptors. Whole Genome Bisulfite Sequencing (WGBS) to profile methylated cytosines (5mC and 5hmC) and Bisulfite-assisted APOBEC-coupled epigenetic sequencing (bACE-seq) to profile hydroxymethylated cytosines (5hmC) in post-natal day 21 (P21) mouse retinas from Cre-negative control and Tet1/Tet2/Tet3 conditional mutant animals. Recombination of floxed TET exons was induced in retinal progenitor cells using the Chx10-Cre-GFP transgene. Experiments were performed on RNA-DNA extractions from retinas, performing biological triplicates for each genotype with paired RNA and DNA samples for each biological replicate being used for RNA-seq, WGBS, and bACE-seq analyses.