Abstract
Purpose :
5hmC is an epigenetic modification required for normal retinogenesis. It is known that tet2-/-tet3-/- (5hmC-null, DMUT) zebrafish show loss of terminal differentiation markers in several retinal cell types, but the extent of developmental impairment and the gene expression events that underly that impairment in each retinal cell type are not well known. The goals of this study are (1) to probe the extent to which developmental phenotypes occur in each cell type in DMUT retinae, and (2) to identify gene expression events required for cell type-specific development that are lost in DMUT retinae.
Methods :
We generated 18 scRNA-seq libraries, including sibling control (CTL) and DMUT cells at four timepoints that span retinogenesis (36, 48,72, 120 hpf). Preprocessing (Bioconductor), batch correction (Batchelor), and cell type identification (AUCell) were done. Computational lineage trajectories were built (URD, Principal Curve), and genes differentially expressed between related CTL and DMUT trajectories were determined (Tradeseq). Genes of interest were tested by injecting target-specific F0 loss-of-function CRISPR complexes into embryos of the Sofa1 transgenic line, in which all major retinal neuron subtypes are labeled. Morphological differences and proportions of different retinal cell types were quantified in F0 crispants.
Results :
146,571 high quality retinal cells were profiled. KS tests performed on lineage-specific pseudotime analyses showed that CTL and DMUT pseudotimes are drawn from significantly different distributions (p<0.05), and 120 hpf CTL cells show significantly older pseudotimes in 7 of 8 retinal lineages (p<2.2e-16, Wilcoxon rank sum). These data suggest significant developmental divergence in timing of CTL and DMUT retinal differentiation, and also suggest that differentiated CTL cells are more mature than DMUT cells of the same lineages. Gene expression alignment of transcriptomic lineage trajectories predicted testable lineage-specific targets that show differential expression between CTL and DMUT retinae.
Conclusions :
Our results suggest that tet2-/-tet3-/- retinae are transcriptionally divergent across all major cell lineages. Tet/5hmC LOF studies that assess divergences in lineage-specific gene expression enable us to further understand the gene expression events that facilitate normal retinogenesis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.