Biology subjects

Vocking, O.

Publications and source records attributed to Vocking, O..

3 recordsLinked to original sources

bioRxiv2022

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.

developmental biology↗

bioRxiv2022

Zebrafish anterior segment mesenchyme progenitors are defined by function of tfap2a but not sox10.

The anterior segment is a critical component of the visual system. Developing independent of the retina, the AS relies partially on cranial neural crest cells (cNCC) as its earliest progenitors. The cNCCs are thought to first adopt a periocular mesenchyme (POM) fate and subsequently target to the AS upon formation of the rudimentary retina. AS targeted POM is termed anterior segment mesenchyme (ASM). However, it remains unknown when and how the switch from cNCC to POM or POM to ASM takes place. As such, we sought to visualize the timing of these transitions and identify the regulators of this process using the zebrafish embryo model. Using two color fluorescence in situ hybridization, we tracked cNCC and ASM target gene expression from 12-24hpf. In doing so, we identified a tfap2a and foxc1a co-expression at 16hpf, identifying the earliest ASM to arrive at the AS. Interestingly, expression of two other key regulators of NCC, foxD3 and sox10 was not associated with early ASM. Functional analysis of tfap2a, foxd3 and sox10 revealed that tfap2a and foxd3 are both critical regulators of ASM specification and AS formation while sox10 was dispensable for either specification or development of the AS. Using genetic knockout lines, we show that in the absence of tfap2a or foxD3 function ASM cells are not specified, and subsequently the AS is malformed. Conversely, sox10 genetic mutants or CRISPR Cas9 injected embryos displayed no defects in ASM specification, migration or the AS. Lastly, using transcriptomic analysis, we show that GFP+ cNCCs derived from Tg[foxd3:GFP] and Tg[foxc1b:GFP] share expression profiles consistent with ASM development whereas cNCCs isolated from Tg[sox10:GFP] exhibit expression profiles associated with vasculogenesis, muscle function and pigmentation. Taken together, we identify the earliest stage of anterior segment mesenchyme (ASM) specification to be approximately 16hpf and involve tfap2a/foxc1a positive cNCCs.

developmental biology↗

bioRxiv2019

Spatiotemporal characterization of periocular mesenchyme heterogeneity during anterior segment development

Establishment of the ocular anterior segment (AS) is a critical event during development of the vertebrate visual system. Failure in this process leads to Anterior Segment Dysgenesis (ASD), which is characterized by congenital blindness and predisposition to glaucoma. The anterior segment is largely formed via a neural crest-derived population, the Periocular Mesenchyme (POM). In this study, we aimed to characterize POM behaviors and identities during zebrafish AS development. POM distributions and migratory dynamics were analyzed using transgenic zebrafish embryos (Tg[foxC1b:GFP], Tg[foxD3:GFP], Tg[pitx2:GFP], Tg[lmx1b.1:GFP], and Tg[sox10:GFP] throughout the course of early AS development (24-72hpf). In vivo imaging analysis revealed unique AS distribution and migratory behavior among the reporter lines, suggesting AS mesenchyme (ASM) is a heterogenous population. This was confirmed using double in situ hybridization. Furthermore, we generated ASM transcriptomic profiles from our reporter lines and using a four-way comparison analysis uncovered unique ASM subpopulation expression patterns. Taken together, our data reveal for the first time that AS-associated POM is not homogeneous but rather comprised of several unique subpopulations identifiable by their distributions, behaviors, and transcriptomic profiles.

developmental biology↗