Transcriptome Profiling of Embryonic Retinal Pigment Epithelium Reprogramming

Abstract

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

A Stage-Specific OTX2 Regulatory Network and Maturation-Associated Gene Programs Are Inherent Barriers to RPE Neural Competency

“…Reprogrammed RPE forms a neuroepithelium of retinal progenitor cells within 3 days post-retinectomy (PR), and within 7 days, these progenitors will differentiate to produce all major retinal cell types ( Spence et al, 2004 ). Importantly, the incipient transcriptional features of reprogramming are detectable in the RPE as early as 6 h post-retinectomy (6hPR), although the cells remain non-proliferative during this acute window ( Luz-Madrigal et al, 2014 ; Tangeman et al, 2021 ). During this time, RPE cells increase expression of genes associated with the neural retina fate, such as VSX2 and ASCL1 , as well as genes encoding eye field transcription factors, such as SIX6 , RAX , and LHX2 .…”

Pigment Epithelia of the Eye: Cell-Type Conversion in Regeneration and Disease

“…At the stage of the proliferative phase of RPE cells’ conversion in birds, as in other animals, FGF2 activates the cell multiplication and, as a result, the subsequent histogenesis of the NR regenerate. In the layer of amplifying neuroblasts, there is an expression of proneural markers, which are TFs (Pax6 and Chx10) regulated by the FGF2-FGFR/MEK/Erk signaling cascade [ 182 , 183 ]. In chicks, as well as in newts, a down-regulation of RPE65 specific protein and the TF Mitf, which are participants in the RPE specialization of eye development, occurs during the RPE cells’ conversion [ 182 ].…”

“…To characterize the mechanisms underlying the RPE cell conversion in the chicken embryo, RNA was extracted by laser capture microdissection from an intact RPE tissue, tissues at 6 h post retinectomy, and cells converted in the presence of FGF2 [ 183 ]. Using RNA-seq, the authors observed the repression of genes related to cell cycle progression in the early-stage converting RPE, and also the up-regulation of injury-associated genes.…”

“…In contrast, the RPE, converted in a FGF2-dependent manner, was enriched in MAPK-responsive genes and retina development factors, thus, confirming that FGF2 and the downstream MAPK cascade are the main drivers of the embryonic chick RPE reprogramming. Tangeman et al [ 183 ] made an attempt to understand the role that the mechanisms responsible for epithelial–mesenchymal transition (EMT), which is a RPE cell-type conversion underlying retinal pathologies in mammals, can play in the conversion of chicken embryo RPE cells. Since the EMT program is accompanied by an extensive, coordinated regulation of ECM regulators (see below), the major focus is on the regulators of ECM state and composition [ 183 ].…”

“…Tangeman et al [ 183 ] made an attempt to understand the role that the mechanisms responsible for epithelial–mesenchymal transition (EMT), which is a RPE cell-type conversion underlying retinal pathologies in mammals, can play in the conversion of chicken embryo RPE cells. Since the EMT program is accompanied by an extensive, coordinated regulation of ECM regulators (see below), the major focus is on the regulators of ECM state and composition [ 183 ].…”