Transcriptome Profiling of Embryonic Retinal Pigment Epithelium Reprogramming.

Jared A Tangeman,Chun Liang,Erika Grajales-Esquivel,Panagiotis A Tsonis,Sutharzan Sreeskandarajan,Lin Liu,Katia Del Rio-Tsonis,Agustín Luz-Madrigal

doi:10.3390/genes12060840

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.

Highlights

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and choriocapillaris and constitutes the outer blood-retinal barrier
The observations that human RPE responds to injury by undergoing epithelial-mesenchymal transition (EMT), as well as the capacity for fetal RPE cells to differentiate to retinal neurons, suggest that mature mammalian RPE may retain some capacity for producing neural retina
In the reprogrammed RPE (rRPE), we identified a number of FGF-responsive genes and transcription factors which drive RPE reprogramming, as well as uncover previously unknown roles for EMT and extracellular matrix (ECM) remodeling in facilitating the early transition from RPE to neural retina progenitor

Summary

Introduction

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells located between the neural retina and choriocapillaris and constitutes the outer blood-retinal barrier. Mammalian RPE can generate neuronal or retinal cells under certain conditions. Between embryonic days 12 and 13, rodent RPE can be reprogrammed in vitro to form neural retina when treated with fibroblast growth factor 2 (FGF2) [8,9]. RPE from adult humans can be reprogrammed with small molecules in vitro to form neuron-like cells that synthesize dopamine [11]. The observations that human RPE responds to injury by undergoing EMT, as well as the capacity for fetal RPE cells to differentiate to retinal neurons, suggest that mature mammalian RPE may retain some capacity for producing neural retina. Whether there is a specific combination of factors and conditions that can efficiently reprogram mature human RPE to retinal neurons remains unknown

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