Abstract
Purpose :
RNA sequencing has already identified transcriptional signatures associated with retinal degeneration (RD) in various disease models but provides limited spatial data. In this study, we use spatial sequencing in a well characterized model of early onset degeneration resulting from loss of function of Mouse Frizzled Receptor Protein, to better understand the spatial changes associated with RD.
Methods :
Eyes from 10–12-week-old RD6 mice (n=6) and C57BL/6 (WT)(n=7) mice were fresh frozen in OCT. 10 µm sections were placed on 10X Genomics Visium Spatial slides. Spatial gene expression was performed, creating a cDNA library mapped to specific tissue locations (Fig 1).
Gene set enrichment analysis identified pathways involved in RD. Pathway analysis of the differentially expressed genes was carried out using WebGestalt.org with Kyoto Encyclopedia of Genes and Genomes as a mapping database.
Results :
Uniform Manifold Approximation and Projection clustering identified distinct expression signatures from the ganglion cell layer(GCL), inner nuclear layer(INL), retinal pigment epithelium (RPE)/choroid/sclera, optic nerve, and ciliary body (Fig, 1) but not the outer nuclear layer(ONL) which was contaminated with expression from other layers.
Our findings highlight Clu, C4b, Apoe, and C1qa genes (z-score 3.0, 2.4, 2.3, and 2.2) as potential markers of disease in the RPE.
Gene Set Enrichment analysis between rd6 and WT eyes showed upregulation of glycolysis and carbon metabolism pathways in the GCL and Rap1, Hippo and lysosome pathways in the RPE/Choroid/sclera. The ribosomal pathway was downregulated in these layers. No significant pathways were found in the INL, ciliary body or optic nerve.
Conclusions :
Our study demonstrates that spatial sequencing can be used to identify differentially expressed genes in most layers in mouse retina, although further optimization is required for ONL. Analysis of differentially expressed genes highlighted potential disease associated targets.
Overall gene set enrichment showed that Rap1 and Hippo pathways are perturbed in rd6 mice. These pathways have previously been implicated in RD. However, further validation with RNAscope will likely confirm their association in this model and as potential targets for treatment.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.