Abstract
Purpose :
Zebrafish (Zf) have a remarkable capacity to regenerate neurons following retinal injury or disease, making them a suitable model organism for regenerative studies. Such studies have revealed several genetic markers associated with retinal progenitor cells and Muller glial cells that are expressed during retinal regeneration; however, it is unknown how unique is the expression of these markers across all cell types in the retina. We hypothesize that single cell analysis can be used to assess expression of markers of regeneration and the retinal cell types expressing them.
Methods :
Retinas were extracted from 3 randomly selected adult AB Zf. The retinas were pooled, digested and dissociated. Single cell 3’ RNA sequencing analysis was performed using 10x Genomics Chromium platform. Principal component analysis was conducted using Seurat, an R toolkit for single cell analysis. Clusters were then identified using the gene list for each cluster through manual annotation and classified into retinal cell types.
Results :
Initial analysis of data from 4,238 cells from WT Zf retina grouped the cells into 16 clusters. Muller cells, Amacrine cells, bipolar cells, rods, cones and all other retinal cell types including some progenitor cells were identified. Genes found to be associated with retinal regeneration in previous studies were identified in many clusters. Rlbp1a was highly expressed throughout the Muller cell cluster and tailed into amacrine and progenitor cell clusters. Fabp7a was found in the Muller cell cluster, with density highest in cells furthest away from other clusters. It was also found in progenitor cell clusters. Vsx1 and pax6a were found highly expressed in a subset of Muller cells closest to amacrine cells. Amacrine cells showed high density of expression of the pax6a marker while bipolar cells had multiple clusters expressing the Vsx1 marker. Two progenitor cell clusters were identified. One expressed markers found in Muller cells and trailed into the Muller cell cluster. The other had unique markers and trailed into a rod cell cluster.
Conclusions :
The results show that single cell analysis can be used to identify expression levels of these genetic markers as well as the retinal cell type predominantly expressing each marker. Markers previously associated with retinal regeneration were distributed across several cell types in WT Zf retina.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.