Single Cell Analysis of Periocular Mesenchyme During Anterior Segment Development - Extension

The anterior segment (AS) of the eye is formed by a subgroup of neural crest cells, called periocular mesenchyme (POM). Despite their importance for a healthy eye, knowledge about these POM cells is limited. Particularly, only very few genetic markers and their respective roles are known. In my initial application, I proposed to study these cells in further detail and identify critical genetic markers. I collected larval eyes of the transgenic line Tg(Foxc1b:GFP) 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 sequenced single cell transcriptomes (scRNA) were processed with the Cell Ranger pipeline. In total, more than 12,000 Foxc1b+ cells were analyzed. By using the Cell Loupe Browser and Monocle3, with focus on pseudotime analyses, I was able to identify reoccurring clusters of cells and assign several candidate genes to these clusters. The purpose of my new proposal is, to a) characterize the new gene candidates, especially but not limited to hgd, si:ch211-251b21.1 and clec14; b) extend my single cell transcriptomic analysis to additional POM cells. Based on pseudotime analyses and initial in situ hybridizations, I hypothesize that the newly identified markers have a critical impact on AS development. As such, I first propose to study their expression patterns over the course of AS development. This analysis will be complemented by SABER multiplex in situ hybridization, enabling examination of expression patterns in native tissue as well as co-expression. Overall, this approach will allow me to make conclusions about potential interactions of the genes in question. Furthermore, I will use the newly developed Alt-R CRISPR/Cas9 method to perform gene knockouts, which will give me a better understanding of the roles these genes play during AS development. Lastly, I propose to extend my POM scRNAseq studies by using zebrafish transgenic lines Tg[foxD3:GFP] and Tg[lmx1b:GFP]. Both of these have GFP signals in the anterior segment throughout early development, up to 9dpf. Thus, I hypothesize that analyzing these two transgenic lines will uncover additional AS specific marker genes. The newly generated data will be analyzed by cell cluster and pseudotime analyses as previously described. Ultimately, the data sets for Foxc1b, FoxD3 and Lmx1b will be combined to give an even broader perspective on anterior segment development. In conclusion, my current analyses have shown, that the combination of transgenic zebrafish lines and single cell RNA sequencing is a powerful approach to study clinically relevant development and specification of cell types within the anterior segment of vertebrate the eye. Summary of plans for extension of project into second year Aim 1: Characterize the role of hgd, si:ch211-251b2.1 and clec14 in anterior segment development -In situ hybridization time course; SABER multiplex in situ hybridization; gene knockouts Aim 2: Uncover developmental signatures of FoxD3 and Lmx1b derived POM cells -Single cell transcriptome generation; pseudotime analysis; in situ hybridization time course; SABER multiplex in situ hybridization; gene knockouts