Abstract
Purpose :
In inherited retinal disorders such as retinitis pigmentosa (RP), the initial degeneration of rod photoreceptors is often followed by the secondary death of cones and loss of high-acuity vision. Mechanistic studies of these events are challenging, as cells in different stages of degeneration generally coexist, and the detection of cone responses to rod degeneration is difficult due to the relative paucity of cones in the retina. We utilized droplet-based single-cell RNA sequencing (scRNAseq) to resolve intercellular heterogeneity and elucidate early events in rods and cones during rod photoreceptor degeneration in rd10, an RP mouse model.
Methods :
ScRNAseq was performed on rd10 and C57/BL6 wildtype retinas at postnatal day (P) 21 (N=2, 1 male and 1 female per genotype) using the 10X Genomics platform. Data analysis focused on characterizing the transcriptomes of degenerating rods and cones with differential gene expression and trajectory analysis. Transcriptomics data was validated via immunofluorescence in both rd10 retinas and retinas of BALB/c animals damaged by 1 hour of 13’000 lx white light.
Results :
Trajectory inference revealed two consecutive phases of rod degeneration at P21. The early phase was distinguished by a marked upregulation of Egr1 (avg. fold change (FC): 4.82 over wildtype rods) and the late phase by Cebpd (FC: 4.55 over Egr1+ rods). Egr1 was also the most highly upregulated transcript in rd10 cones (FC: 5.81 over wildtype cones). Notably, EGR1 was the transcription factor most significantly associated with the promoters of differentially regulated genes in Egr1+ rods in silico. Egr1 upregulation preceded transcriptomic changes related to metabolic dysfunction, synapses, and tubulin remodeling in rods. In cones, it accompanied changes in mitochondrial and signaling pathways, including TNFα and TGFβ. Phototransduction genes were downregulated early in both cell types. Immunoreactivity for EGR1 was highly increased in rods and cones of rd10 retinas at P21, with rods losing EGR1 by P28. Cones remained EGR1-positive at least up to P35. EGR1 immunoreactivity also increased 2 hours after light damage, specifically in cones and Müller glia.
Conclusions :
Our results identify early cone responses to rod degeneration in the rd10 model and describe various pathways that are initially affected in degenerating rods. Our data suggest EGR1 as a key regulator of these early events.
This is a 2021 ARVO Annual Meeting abstract.