Single-cell RNA sequencing analysis of neurogenic competence in Müller glia in a rabbit retinal injury model

Abstract

Purpose : The capacity for endogenous regeneration of retinal neurons after injury varies greatly among vertebrate species. For example, zebrafish Müller glia can regenerate all major retinal cell types after injury, while those in mice appear to lack this ability. The neurogenic potential of retinal glia in other mammalian model organisms is currently unknown. In this study, we aimed to identify genes and transcriptional networks involved in regulating neurogenic competence in a rabbit model of retinal injury.

Methods : Animal procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Johns Hopkins Animal Care and Use Committee. Unilateral retinal injury was induced in Dutch Belted rabbits by vitrectomy, retinotomy, retinal detachment, platelet-rich plasma injection, and cryotherapy. At different time points after injury, extracted retinas were dissociated and processed to assess the transcriptional profile by single cell RNA sequencing (sc-RNAseq). Fellow eyes were used as non-injury controls.

Results : Retinas were harvested at time points ranging from six hours to 35 days after injury induction. We profiled >70,000 single cells in this study. All major retinal cells including neurons, glia, and retinal pigment epithelium cells were identified in the data sets using selected marker genes. We identified two trajectories of glial cells post-injury: the first expressing classical fibrotic markers (ACTA2 and COL1A1) and the second expressing several genes that are known to promote proliferation (MKI67, MYBL2, UBE2C, TOP2A and HMGA2) and neurogenic competence (ASCL1, GADD45G, KLF6, MSX2, OLIG2, HES6, BHLHE22 and NEUROG2).

Conclusions : Our high throughput transcriptomic analysis identified known fibrotic and neurogenic genes that were differentially expressed between control and injury states in the rabbit model. These findings may facilitate further research in large mammalian models on molecular pathways that could be targeted to induce neurogenesis in human Müller glia following retinal injury.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.