Abstract
Purpose :
Genomic variation across inbred mouse strains underlies variation in gene expression with consequences for retinal development, organization and function, ultimately yielding phenotypic differences across the strains. This study used single cell RNA sequencing (scRNAseq) across four inbred mouse strains, to determine differences in the transcriptional landscape at the level of individual retinal cell types.
Methods :
Adult mouse retinas from four inbred mouse strains (C57BL/6J, A/J, DBA/2J, and 129S1/SvImJ) were dissociated into single cell suspensions, from which rod photoreceptors were depleted using magnetic activated cell sorting. The remaining cells were collected and libraries were prepared for scRNAseq using the Honeycomb HIVE system; libraries were sequenced using an Illumina NextSeq500. The Seurat v4 package in R was used to perform quality control, cluster cells, and search for differentially expressed genes (DEGs). Cell clusters were matched to known cell types due to the presence of both canonical and recently discovered markers. Immunofluorescence was used to validate the expression patterns of DEGs in retinal tissue collected from the different inbred strains.
Results :
Gene expression profiles were obtained for a total of 16,986 single retinal cells across the four strains. Each of the five major classes of retinal neuron were identified, as well as the Müller glia (MG) population. Within those classes, individual clusters of discrete cell types could be distinguished, with conspicuous strain differences in the clusters driven by differential gene expression. Some DEGs were common to multiple cell types within a class or between classes, while others were specific to individual types, differing in relative expression between the strains. For example, HOP homeobox (Hopx) was expressed in rod bipolar cells (RBCs) only of the A/J strain, yet was also expressed in cones and MG of the A/J, C57BL/6J and DBA/2J strains. Immunofluorescent staining of A/J and C57BL/6J retinas confirmed selective expression of HOPX in the RBC population of the A/J strain, as well as common expression in the MG population of both strains, validating the results of the scRNAseq analysis.
Conclusions :
Single cell sequencing reveals cell type specific differences in gene expression across different inbred mouse strains, facilitating the analysis of the polygenic control of various retinal phenotypes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.