Abstract
Purpose :
Embryonic amniotes possess an ephemeral ability to regenerate retinal neurons from cells of the RPE, as well as from progenitors in the ciliary margin. As differentiation proceeds, the neurocompetence of the RPE becomes restricted and the ciliary margin progenitor population is eventually abrogated. In the embryonic chicken, the loss of RPE neurocompetence occurs at embryonic day 5. We sought to examine gene regulatory patterns and environmental factors that emerge during this window which may dispossess the chicken of regenerative competence.
Methods :
Single nucleus RNA-sequencing and ATAC-sequencing were performed on the chicken eye at regeneration competent (day 4) and incompetent (day 5) stages of embryogenesis. Chickens at both stages were subjected to retinectomy and treated with FGF2, a growth factor required for the induction of neural retina regeneration. Gene expression in the RPE was comprehensively examined to delineate features of differentiation and neural reprogramming. These expression patterns were compared to the regenerative sequelae in the ciliary margin. Interplay with accessory cell types, such as the periocular mesenchyme, was evaluated.
Results :
Significant heterogeneity exists amongst RPE cells of the developing chicken, including expression patterns associated with differentiation state and regenerative outcome. We uncover regeneration-associated expression patterns for genes encoding transcription factors (PAX6, SIX6, DACH1, RUNX1), cell adhesion molecules (CDH4, CDH6), RPE maturation factors (OTX2, RLBP1), and cell proliferation regulators. Cell-cell interaction analysis revealed the potential for a dynamic interface between mesenchymal cells and the RPE during this window, engaging activin and BMP signal effectors. Finally, comparison of injured RPE cells to the ciliary margin revealed unique attributes of regeneration-competent progenitors.
Conclusions :
RPE neurocompetence is restricted during early development by an intrinsic program comprised of RPE differentiation factors, as well as by alterations in the regulatory behavior of genes necessary for neurogenesis. A dynamic network of secreted factors from the periocular mesenchyme has the potential to drive RPE lineage restriction. These findings set the basis for understanding emergent barriers to retinal regeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.