Whole transcriptome sequencing in rd10 revealed new factors and pathways involved in stem cells biology and neuroprotection in the inner retina and ciliary body.

Abstract

Purpose : Three main cell types respond to dying photoreceptors: Activated Microglia and Müller glial cells located in the inner retina and secreting neurotrophic factors, and in some species, cells at the retinal periphery located in the ciliary body (CB). These last two can participate in the renewal of lost cells in different species but such properties are very limited in mammals. Thus, we aimed to identify factors involved either in stemness (Müller cells and CB) or in the neurotrophic response originating from the inner retina during photoreceptor cell death.

Methods : Retina and CB from three wild type (WT) and rd10 mice (Pde6B^rd10/rd10) were isolated at P30, when most photoreceptors are lost. Paired-end sequencing on stranded RNA-seq libraries were performed using a Hi-Seq 2500. Pass-filtered reads were mapped using TopHat2 and aligned to mm10. Differentially expressed genes (DEG) were identified with EdgeR. To focus our analysis on inner retina-enriched genes, we removed photoreceptor-expressed genes from adult flow-sorted photoreceptor RNA-seq data. Functional annotation, GO enrichment analysis and pathways analysis were done using DAVID, PANTHER and KEGG, respectively.

Results : Among inner retina-enriched genes, we found clusters of genes highly expressed only in rd10 mice. At the opposite, several unknown genes and miRNA were expressed only in WT. Some nuclear and extrinsic factors exhibiting expression alteration in rd10 context (176 genes) belong to signaling pathways activated by photoreceptor cell death (cytokine, MAPK, PI3K-AKT). Rap1 and the Hippo signaling pathway are among the most interesting pathways with a large number of components induced in Rd10. In addition, several DEG are involved in the regulation of stem cell pluripotency. In contrast to the inner retina, Hippo and Rap1 signaling were unchanged in the CB and a low number of DEG was found overall. However, hierarchical clustering identified sets of genes involved in the maintenance of stem cell pluripotency.

Conclusions : The tremendous amount of data obtained and the promising pathways identified (Hippo in particular) will require further validation. Although no regeneration occurs in the mouse retina, our analysis revealed many genes involved in stem cell biology. Such new cues may help to develop new therapeutic paradigms.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.