A temporal single cell transcriptome atlas of zebrafish anterior segment development

Abstract

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 (48–144 hpf) and performed single cell RNA sequencing. Clustering analysis indicates subdifferentiation of ASM as early as 48 hpf and subsequent diversification into corneal epithelium/endothelium/stroma, or annular ligament (AL) lineages. Tracking individual clusters reveals common developmental pathways, up to 72 hpf, for the AL and corneal endothelium/stroma and distinct pathways for corneal epithelium starting at 48 hpf. Spatiotemporal validation of over 80 genes found associated with AS development demonstrates a high degree of conservation with mammalian trabecular meshwork and corneal tissues. In addition, we characterize thirteen novel genes associated with annular ligament 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.

Single cell transcriptome analyses of the developing zebrafish eye— perspectives and applications

“…We were able to compare our data to human studies (van Zyl et al, 2020;van Zyl et al, 2022) and found that human marker genes such as krt4, krt5 and myoc are expressed in the zebrafish cornea and AL respectively. Moreover, we found previously unknown markers like hgd and cndp1 expressed in the AL which might also play a role in human TM development (Vöcking and Famulski, 2023). This kind of multiple time point analyses enables the exact determination of the genetic profile of cells during their development and help identify critical moments of their differentiation (Figure 2C) (also see Farnsworth et al, 2020;Farnsworth et al, 2021).…”

“…With the help of fluorescent activated cell sorting (FACS), non-fluorescent cells can simply be sorted out, which allows the use of only the specifically desired cell type for the analysis. This method has already been applied successfully to isolate various neural crest cells including those forming the anterior segment, but also retinal cells, immune cells and cells of the developing gonads among others (see Figure 1) (Carmona et al, 2017;Hernandez et al, 2018;Zhang et al, 2019;Van Der Meulen et al, 2020;Howard et al, 2021;Tatarakis et al, 2021;Liu et al, 2022;Vöcking and Famulski, 2023). Hence, the dissection of the fragile tissue can be avoided altogether.…”

“…The general similarities in ocular development and genetic conservation make zebrafish a powerful model for the study of ocular disease (see Figure 2A). To date, most zebrafish-based studies have focused on retinopathies, but lately zebrafish are also being employed for the study of anterior segment dysgenesis (Gestri et al, 2012;Richardson et al, 2017;Hong and Luo, 2021;Seese et al, 2021;Santhanam et al, 2022;Vöcking and Famulski, 2023). Despite some differences in anatomy, our own group recently suggested that the annular ligament (AL) of zebrafish can be used as a model for the human trabecular meshwork, highlighting the potential use of zebrafish as a glaucoma model (Vöcking and Famulski, 2023).…”

“…To date, most zebrafish-based studies have focused on retinopathies, but lately zebrafish are also being employed for the study of anterior segment dysgenesis (Gestri et al, 2012;Richardson et al, 2017;Hong and Luo, 2021;Seese et al, 2021;Santhanam et al, 2022;Vöcking and Famulski, 2023). Despite some differences in anatomy, our own group recently suggested that the annular ligament (AL) of zebrafish can be used as a model for the human trabecular meshwork, highlighting the potential use of zebrafish as a glaucoma model (Vöcking and Famulski, 2023). In this study, we collected anterior segment (AS) mesenchyme cells from several developmental time via FACS and analyzed expression in AS specific GFP + cells of the anterior segment reporter transgenic line Tg(foxc1b:GFP).…”

“…Since studies of human samples are mostly limited to mature tissues, insights of early timepoints in model organisms such as zebrafish are crucial to understand early development. Compared to other study animals, this approach is most feasible to perform in zebrafish due to ease of tissue collection and their affordability (Farrell et al, 2018;Wagner et al, 2018;Farnsworth et al, 2020;Farnsworth et al, 2021;Vöcking and Famulski, 2023).…”