Nuclear Factor I Regulates the Switch from Neurogenesis to Gliogenesis in the Developing Retina

Abstract

Purpose : Retinal progenitor cells undergo a series of temporal transitions during neurogenesis. Many of the mechanisms controlling these processes are still largely unknown. Based on our previous single cell RNA sequencing studies profiling the full course of retinal neurogenesis, the Nuclear Factor I (NFI) transcription factors were found to be enriched in late stage progenitors. In this study, we seek to identify the genes and transcriptional networks regulated by the NFI factors that are involved in cell fate specification.

Methods : All procedures with animals were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Nfia^f/f;Nfib^f/f;Nfix^f/f mice were crossed Chx10:Cre-GFP to generate retinal-specific loss of function mutants of these genes. For explant studies, E14 retinas were dissected and electroporated with either control GFP plasmid or Nfia/Nfib/Nfix over expression plasmid and grown in culture for two days. Retinas were dissected and dissociated into single cell suspension. GFP positive cells were obtained by flow sorting and prepared for ATAC, single cell RNA or single cell ATAC sequencing. Cell suspensions of the conditional knockouts and the heterozygous controls were processed to assess the transcriptional profile at a single cell level across different time points. Retinas from wildtype CD1 mice were used for ChIP-seq studies to identify genomic targets of the NFI factors.

Results : The Nfi conditional knockout mice have altered retinal morphology with prolonged cell proliferation and a significant loss of Müller glial cell markers which recover over time. Nfia/Nfib/Nfix overexpression studies show that cell cycle gene expression is significantly impacted. Using bioinformatic analyses integrating ATAC, ChIP and RNA sequencing, we have identified genes and transcriptional networks that are regulated by Nfia, Nfib and Nfix that correlate with cell-cycle exit and late born retinal cells.

Conclusions : Single cell studies coupled with ATAC and ChIP analysis identified several genes that are regulated by the NFI factors. We have determined that the NFI factors play a critical role in inhibiting cell proliferation. These studies will ultimately reveal candidate factors that are involved in specifying glial cell fate.

This is a 2020 ARVO Annual Meeting abstract.