The shifting epigenetic landscape along the developmental trajectory of retinal ganglion cells

Abstract

Purpose : The formation of retinal ganglion cells (RGCs) is a stepwise process subject to tight genetic control. Our purpose in this study is to use single cell ATAC-seq (scATAC-seq) to investigate how the epigenetic landscape changes along the RGC developmental trajectory.

Methods : We used two knock-in mouse lines, Atoh7-zaGreen and Pou4f2-tdTomato, to enrich different cell populations by FACS. scATAC-seq and scRNA-seq libraries were then generated using the 10X Chromium platform and sequenced on an Illumina sequencer. The sequence data were then analyzed using Cell Ranger, Seurat, and archR. We also used CRISPR and RNAscope in situ hybridization to investigate the function of one candidate enhancer of Pou4f2, a key gene for the RGC lineage.

Results : We first performed UMAP clustering and gene score calculation of the scATAC-seq data and were able to cluster the cells into the same groups as achieved by scRNA-seq. We next identified enriched accessible peaks in individual clusters, which represented potential enhancers for each developmental stage/cell types. We further performed DNA motif enrichment and footprinting analysis which revealed stage/cell type-specific motif binding by key transcription factors. Further, we performed peak-to-gene linkage analysis by integrating scATAC-seq data with the scRNA-seq data and assigned the stage/cell type-specific enhancers to the genes they likely regulate, thus generating a global enhancer-gene linkage map for the developing retina at E14.5 and E17.5. We also compared the wild-type and Atoh7-null scATAC-seq data and identified peaks (enhancers) that were dependent on Atoh7. Finally, as a pilot validation, we used CRISPR to delete one of the candidate enhancers of Pou4f2, a key gene involved in RGC genesis, and confirmed by RNAscope in situ hybridization that the enhancer indeed played a critical role in the expression of Pou4f2.

Conclusions : Using scATAC-seq, we identified stage/cell-specific enhancers in the developing retina, assigned them to genes they likely regulate, and identified DNA-motifs in them likely bound by key transcription factors . Our results shed significant new light on the shifting epigenetic landscape along the developmental trajectory of the RGC lineage.

This is a 2021 ARVO Annual Meeting abstract.

 

Figure 1. Enhancers associated with genes expressed in specific stages of the RGC trajectory change along the RGC developmental trajectory, illustrating the shifting epigenetic landscape.