Abstract
Purpose :
The retinal pigment epithelium (RPE) is a plastic tissue that can regenerate neural retina in embryonic amniotes via cell reprogramming. However, a key question remains unanswered: Why can the RPE be reprogrammed only in the early embryonic stages? In chicken embryos, RPE can reprogram into retina after retinectomy and stimulation of FGF2 at embryonic day 4 (E4). However, this capacity is lost by E5. Based on recent transcriptomic analyses, including bulk and snRNA/ATAC-seq, we hypothesized that signaling pathways and intrinsic cell fate control are coupled mechanisms responsible for restricting RPE neurocompetence. Our data suggest that enhanced activity of the Hippo-YAP pathway and gain in expression of transcription factors, such as NFIA, and NFIB, could restrict RPE neurocompetence.
Methods :
Single-nucleus multimodal profiling was performed on differentiating chicken RPE from E3-E7 to identify transcription factor candidates and signaling pathways for functional perturbation. Using RPE explants from E4 and E5 embryos, we evaluated the capacity of RPE to become retina upon inhibition of the Hippo-YAP pathway by TDI-011536. We also tested the effects of NFIA and NFIB knockdown by using morpholinos. The efficiency of RPE reprogramming was evaluated by gene expression profiling, immuno-detection, and cell proliferation determination.
Results :
Multimodal data analysis pointed to 9 up-regulated and 9 down-regulated transcription factor genes and accompanying changes in motif accessibility that coincide with RPE neurocompetence restriction. Moreover, inhibition of the Hippo-YAP pathway significantly increased cell proliferation in E4 and E5 RPE explants in the absence of FGF2. Hippo-YAP signaling inhibition also up-regulated eye field transcription factors, EMT regulators, and cell cycle-related genes while suppressing RPE identity. This increase in cell proliferation did not coincide with induction of retina. On the other hand, inhibition of NFIA transiently caused an increase in the size of RPE explants only at E4.
Conclusions :
Our data suggest that the neurocompetence of embryonic RPE is not only dependent on the activation of cell proliferation or downregulation of NFIA and NFIB, as they did not promote RPE reprogramming. Instead, these findings indicate that cell proliferation and the regulation of a network of transcription factors may be responsible for controlling RPE reprogramming ability and limiting neural retina regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.