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

Abstract

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 fluorescencein situhybridization, we tracked cNCC and ASM target gene expression from 12-24hpf. In doing so, we identified atfap2aandfoxc1aco-expression at 16hpf, identifying the earliest ASM to arrive at the AS. Interestingly, expression of two other key regulators of NCC,foxD3andsox10was 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.

A temporal single cell transcriptome atlas of zebrafish anterior segment development

“…Furthermore, based on our data it may be that zebrafish derive all of the aforementioned tissues from a common progenitor pool that arrives early in development. Interestingly, recent work in our lab has observed the earliest ASM cells arriving as early as 16 hpf, while the bulk of ASM are observed to arrive approximately 26-28 hpf 39,87 . The results of our current study suggest that annular ligament, as well as corneal endothelium and the cells in the corneal stroma all share a common origin, a subgroup of ASM that are at part of the foxc1b derived lineage.…”

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

“…Furthermore, based on our data it may be that zebrafish derive all of the aforementioned tissues from a common progenitor pool that arrives early in development. Interestingly, recent work in our lab has observed the earliest ASM cells arriving as early as 16hpf, while the bulk of ASM are observed to arrive approximately 26-28hpf (39, 76). The results of our current study suggest that annular ligament, as well as corneal endothelium and the cells in the corneal stroma all share a common origin, a subgroup of ASM that are at part of the foxc1b derived lineage.…”