Nuclear Factor I Regulates Proliferation and Specification of Müller glial and Bipolar Cells

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 to Chx10:Cre-GFP to generate retinal-specific loss of function mutants of these genes. Retinas were dissected and dissociated into single cell suspension. GFP positive cells were obtained by flow sorting and prepared for ATAC sequencing. Cell suspensions of the conditional knockouts and the heterozygous controls were processed on the 10x Genomics Chromium Single Cell system to assess the transcriptional profile at a single cell level across different time points (P2, P14, P60 and P150).

Results : The Nfi conditional knockout mice have altered retinal morphology with a reduction in size of the inner nuclear layer which corresponds to a significant decrease in Müller glial cells and bipolar cells. The loss of the Nfi genes resulted in prolonged progenitor cell proliferation and increased photoreceptor specification. We have profiled >70,000 cells using the 10x Genomics system. ATAC sequencing analysis identified alterations in >14,000 chromatin accessible sites in mutant retinas. Using bioinformatic analyses 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 RNA sequencing and ATAC analyses identified several genes that are differentially expressed in the Nfi mutant retinas. These studies will ultimately reveal candidate genes that are either involved in maintaining progenitor cell state or specifying glial or bipolar cell fate.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.