Tracking periocular mesenchyme derived anterior segment development using single cell transcriptomic analysis

Purpose: The anterior segment (AS) of the eye, as well as further anterior structures, are formed by a subgroup of neural crest cells, called periocular mesenchyme (POM). Despite the importance for the development of a healthy eye, knowledge about this cell group is limited. Particularly, only very few genetic markers and their respective roles are known. The purpose of this study is to identify formerly unknown markers of POM cells, to further understand their role in eye development and the genetic interactions required for AS formation. To do so, we employed newly available scRNA analysis over the course of AS development in a pseudotime format. Methods: Larval eyes of transgenic zebrafish Tg(Foxc1b:GFP) were collected every 24 hours between 48hpf and 144hpf. GFP+ cells were isolated via FACS cell sorting and processed with the 10x genomics chromium single cell transcriptome kit. The subsequently resulting Illumina sequencing single cell transcriptomes were processed with the Cell Ranger pipeline. Analyses were done with the Cell Loupe Browser as well as with the R based software package Monocle3. Gene expression was studied via in situ hybridization and gene function via Morpholino and CRISPR induced gene knockouts. Results: We collected a total of more than 12.000 Foxc1b+ cells from eyes only, including one biological replicate for each individual time point. Transcriptome analyses with Loupe Cell Browser and Monocle3 showed, that these cells were organized in re-occurring clusters during zebrafish anterior segment development. These clusters are partly localized to different structures within the eye, mainly the cornea and retinal pigment epithelium. Additionally, we found several markers with specific expression patterns within these spatially limited areas with apparent importance for general eye development, as proven by genetic knockout. Conclusions: Our results demonstrate that the combination of an anterior segment labeling transgenic zebrafish line and single cell transcriptome analysis is a powerful tool for studying specification of specific cell types. We were able to identify candidate genes, that are critical for anterior segment development and accordingly, anterior segment dysgenesis. This new knowledge might further enable clinicians to increase their genetic screening for anterior segment related diseases and treatment of ocular diseases such as glaucoma. This is a 2020 ARVO Annual Meeting abstract.