Temporal single cell transcriptome atlas of zebrafish anterior segment development reveals high degree of conservation between the trabecular meshwork and the annular ligament

Anterior segment dysgenesis (ASD), resulting in vision impairment, stems from maldevelopment of anterior segment (AS) tissues. Incidence of ASD has been linked to malfunction of periocular mesenchyme cells (POM). POM cells specify into anterior segment mesenchyme (ASM) cells which colonize and produce AS tissues. In this study we uncover ASM developmental trajectories associated with formation of the AS. Using a transgenic line of zebrafish that fluorescently labels the ASM throughout development, Tg[foxc1b:GFP], we isolated GFP+ ASM cells at several developmental timepoints (48-144hpf) and performed single cell RNA sequencing. Clustering analysis indicates subdifferentiation of ASM as early as 48hpf and subsequent diversification into corneal, epithelium/endothelium/stroma, or annular ligament (AL) lineages. Tracking individual clusters reveals common developmental pathways, up to 72hpf, for the AL and corneal endothelium/stroma, and distinct pathways for corneal epithelium starting at 48hpf. Spatiotemporal validation of over 80 genes found associated AS development demonstrates high degree of conservation with mammalian trabecular meshwork and corneal tissues. In addition, we characterize thirteen novel genes associated with AL and seven with corneal development. Overall, the data provide a molecular verification of the long-standing hypothesis that POM derived ASM give rise to AS tissues and highlight the high degree of conservation between zebrafish and mammals.