Abstract
Purpose :
Retinal progenitor cells undergo a transition between transcriptionally distinct early- and late-stage specific states during the course of neurogenesis, which control their ability to generate specific postmitotic cell types. Using integrated scRNA- and scATAC-Seq, we identified NR2F1 and NR2F2 as transcription factors that are strong candidates for maintaining early-stage identity in retina progenitors. In this study, we investigate the effects of misexpressing NR2F1 and NR2F2 in late-stage retinal progenitors.
Methods :
We generated single cell RNA sequencing libraries from retinas electroporated ex vivo at P0 with 100ug of either a control GFP-expressing plasmid or individual or a combination of plasmids overexpressing NR2F1 and NR2F2. We then isolated and profiled electroporated cells from 10-15 retinas using scRNA-Seq. We collected between 4500 and 7000 cells per sample and then utilizing the Seurat R package processed the data. We then analyzed expression of known marker genes and conducted differential gene expression analysis to elucidate how NR2F1/2 overexpression impacted cell type population dynamics and gene regulatory networks.
Results :
Within these datasets, we identified cell clusters with marker gene expression representing primary RPC, neurogenic RPC, G2/M phase RPC, microglia, amacrine, starburst amacrine, and photoreceptor cells. NR2F1/2 combined overexpression at P0 led to an increased fraction of RPC and amacrine clusters alongside a decrease in the overall fraction of primary RPCs. In addition, we observed ectopic expression of RGC-enriched markers (Ebf3, Elavl3) within the amacrine and neurogenic RPC populations as well as a loss of RPC markers (Pax6). [SB1] Similar effects of NR2F1/2 overexpression were observed at P2 and P5 individually, with additional RGC-specific markers (Rbpms, Calb2, Snca) detected at this age.
Conclusions :
Our observation that NR2F1/2 overexpression leads to induction of expression of RGC-specific markers implies that these transcription factors may control temporal patterning of retinal progenitors, and render them competent to generate RGCs. These findings also imply that NR2F1/2 promote the transition from primary to neurogenic progenitors and may promote neurogenic competence.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.