To gain insights into the molecular mechanisms triggered in Müller cells following photoreceptor cell death, we performed whole transcriptome sequencing of
rd10 mouse retinas (
Pde6brd10), and used C57Bl6/J WT mice as controls. In
rd10 mice, degeneration is due to a mutation in the β subunit of the rod phosphodiesterase gene (
Pde6b).
41,42 In humans, mutations in the same gene cause retinitis pigmentosa.
43 In
rd10 mice, rod cell death starts around P16, and the vast majority of photoreceptors are lost by P60.
41 We decided to perform RNA-Seq experiments on P30 retina in order to investigate the molecular response of Müller cells. By choosing this time point, we speculated that Müller cell response to injury would still be active since rod photoreceptor cell death is still occurring in contrast to a previous study performed at much later time points when all rods are already lost.
44 Gene level analysis of the sequencing data allowed the identification of 3427 differentially expressed genes (DEGs) out of 24,062 genes using criteria of FC 1.2 with a
P value cutoff of 0.05 and a minimum expression of two FPKMs in at least one sample (
Fig. 1A). Such relaxed parameters allow the identification of several genes with small FC, which may be of importance if they are all part of the same pathway. With this objective in mind, the next step was the enrichment of the dataset with genes mostly expressed in the inner retina, where Müller cells are located. Therefore, we used neural retina leucine zipper (NRL) ChIP-Seq data to filter out photoreceptor-NRL target genes.
33 Indeed, NRL is a rod-specific transcription factor required for rod photoreceptor development and homeostasis and is specifically expressed in photoreceptors.
45 Putative photoreceptor-specific transcripts, as defined by at least one binding site for NRL, were discarded from our dataset, leading to 2643 DEGs named thereafter INL-enriched genes. Although among them, some are photoreceptor-expressed genes without NRL binding sites, such an approach circumvents the identification of a large number of genes expressed in rods and identified as downregulated due to photoreceptor cell death. In addition, based on PANTHER annotation, we kept only extrinsic factors and nuclear factors for further pathway analysis, which represent two of the main categories of signaling pathway regulators (extrinsic cues and effectors, respectively). This approach led to the identification of 293 DEGs that were then used for pathway analysis using KEGG (
Fig. 1A). Among the top pathways, several were already known as deregulated during retinal degeneration such as PI3K-Akt signaling, Jak-STAT, and cytokine-related pathways (
Supplementary Table S3). More interestingly, this analysis of the 293 DEGs belonging to extrinsic factors and nuclear factors categories revealed the Hippo pathway as a new signaling pathway deregulated in degenerative retina. Indeed, KEGG pathway analysis revealed a total of 11 Hippo signaling related genes as significantly differentially expressed in
rd10 versus WT mouse retina (
Fig. 1B) with nine of them upregulated and two downregulated. We then extended our analysis to our entire dataset of 2643 DEGs (regardless of the PANTHER annotation). Among genes identified in the KEGG database as related to the Hippo pathway, 25 were differentially expressed (
Fig. 2A). Chord plot representation illustrates the relationship between those and other signaling pathways such as Wnt or TGFβ (
Fig. 2B).