Molecular signatures integral to natural reprogramming in the pigment epithelium cells after retinal detachment in Pleurodeles waltl

The phenomenon of retinal pigment epithelium cells (RPE) in vivo natural reprogramming (transdifferentiation) to the neural and glial cells is the basis of the retinal regeneration in some Urodela. The main model in the project is retinal regeneration after experimental detachment in adult newt Pleurodeles waltl. Unlike tailed amphibians, retinal detachment in humans leads to epithelial-mesenchymal transformation of RPE cells and development of neurodegenerative processes (hereditary pathologies, proliferative vitreoretinopathy, rhegmatogenous retinal detachment). The molecular mechanisms of the early RPE response and conversion after injury in vertebrates with different regenerative potencies, remain unexplored.The goal of our project is aimed to identify the key components of endogenous defense cell systems from the stress, that ensure cell reprogramming and full in vivo retina regeneration in P. waltl. The main task is related to the study of RPE transcriptome changes, after disruption interaction with neural retina.In this study, we have sequenced the transcriptome of RPE cells at the early period of cell reprogramming, using the RNA-Seq technology. Total RNA from RPE tissue was extracted using the "Extract RNA" which utilizes guanidine thiocyanate (Cat. #BCO32, Evrogen, RF), and DNA was removed with TURBO DNA-free (Thermo Fisher Scientific, Waltham, MA, USA).De novo transcriptome assembly (obtaining sequences of sequenced transcripts) preceded the assessment of the molecular profile of the RPE, and was carried out using the Trinity package program. We used the transcriptome assembly methodology for RPE P. waltl generally accepted in comparative genomics for animals which are considered as "non-model species", with unstudied or insufficiently studied genomes or transcriptomes - "genome-guided assembly" or "alignment-based assembly". Reads of 250 nucleotides in length were mapped to known homologous genes, including their various splice variants, and further banded into a whole transcript. Blast2GO was used for GO analysis and the functional annotation of contigs. TransDecoder and Trinotate were applied to identify and annotate ORFs.Statistical significance was assigned to terms with a P-value < 0.05. The adjusted p-value based on Bonferroni correction was employed to identify the statistical significance. Genes were clustered into groups according to biological processes in which they participate, as a result of GO annotation.The potential key homologous genes, their GO-cellular and metabolic pathways, involved in regulation of the biological processes that drive and support RPE reprogramming in tailed amphibians have been analyzed. DEGs-associated biological processes and signaling pathway enrichment were estimated by gene ontology and pathway enrichment. The identified molecular signature is associated with signaling pathways, which could ensure the conversion of RPE cells, before their displacement from the layer and formation of the transit cell population as neuroblasts. The identified biological pathways are mainly associated to the early stress-response processes. They include the remodeling extracellular matrix (ECM) and adhesive cell contacts, translation and ribosome biogenesis regulation, components of cell defense system, immune response, proto-oncogenes, oncogene suppressors and suppressors of epithelial mesenchymal transition (EMT).It is assumed, that the features of endogenous defense systems and cellular metabolism in Urodela make RPE cells resistant to stress due to injury and allow them successful reprogramming in neurons and glia. Identification of the key genes are involved in the regulatory signaling and metabolic pathways that make it possible to reprogram newt RPE into retinal cells helps to understand mechanisms of this phenomenon in the newt and to look the approaches to the prevention and treatment of RPE-related degenerative retinal diseases in human.