Single cell genomic analysis to study the retinal cell type specific regulatory landscape of Mfrp-KO mice.

Abstract

Purpose : Our goal is to understand the molecular mechanism underlying retinal degeneration in the Mfrp gene knock out mice (Mfrp-KO).

Methods : We optimized the single nucleus indexing based ATAC-Seq (snATAC-Seq) for use on flash-frozen retina+RPE tissue to analyze one-month-old wildtype and Mfrp-KO mutant mice (n=3). After clustering based on similarity of open chromatin profiles, candidate cis regulatory elements for each cluster were defined and potential master transcriptional regulators were inferred using HOMER. The candidate cis-regulatory regions were assigned to putative target genes using GREAT. Differential accessible regions between cells from wild-type and Mfrp-KO mice were identified using edgeR. Members of AMPK pathway were analyzed by qRT-PCR and western blot analysis to gain insight into this pathway.

Results : Clustering of 53,748 high quality nuclei revealed at least 10 major cell clusters including rods, cones and RPE and >138,000 sites of accessible chromatin of which >35,000 showed cluster specific accessibility (Fig-1). Cell-type resolved comparison between Mfrp-KO and wildtype mice for at this early time point showed 51 peaks with increased and 48 peaks with decreased accessibility for rods; 50 peaks with increased and 26 peaks with decreased accessibility for cones as well as 19 peaks with increased and 10 peaks with decreased accessibility for RPE. Independently, we found a significant decrease in the expression of AdipoR1 and activation of members of AMPK pathway in Mfrp-KO mice when compared to wildtype mice.

Conclusions : We generated a map of candidate cis-regulatory elements in the retina for wildtype and Mfrp-KO mice. These comprehensive maps in conjunction with single nucleus RNA-seq build the basis to understand the gene regulatory logic underlying early onset retinal degeneration. The snATAC-Seq data and additional analysis demonstrated that the loss of Mfrp in mice leads to significant reduction in photoreceptors and AdipoR1. In addition, tissue level based profiling showed decreased activation of AMPK pathway in Mfrp-KO compared to wildtype mice.

This is a 2020 ARVO Annual Meeting abstract.

 

snATAC-Seq analysis of mouse retina from wildtype and Mfrp-KO/KO mice (n=3). (A)UMAP visualization of major cell clusters derived from snATAC-Seq. (B)35,110 peaks showed cluster-specific accessibility patterns. (C)UMAP visualization with labeling based on sample of origin shows overall good admixture between samples.