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 to 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 propose that the earliest stage of anterior segment mesenchyme (ASM) specification in zebrafish is approximately 16hpf and involves tfap2a/foxC1a positive cNCCs.

眼前节是视觉系统的重要组成部分。AS 独立于视网膜发育，部分依赖颅神经嵴细胞 (cNCC) 作为其最早的祖细胞。cNCC 被认为首先采用眼周间充质 (POM) 命运，随后在基本视网膜形成时靶向 AS。AS 靶向 POM 被称为眼前节间充质 (ASM)。然而，从 cNCC 到 POM 或从 POM 到 ASM 的转换何时以及如何发生仍然未知。因此，我们试图将这些转变的时间可视化，并使用斑马鱼胚胎模型识别该过程的监管者。使用双色荧光原位杂交，我们跟踪了 12 至 24hpf 的 cNCC 和 ASM 靶基因表达。在此过程中，我们确定了tfap2a和foxC1a在 16hpf 处共表达，确定最早到达 AS 的 ASM。有趣的是，NCC 的另外两个关键调节因子foxD3和sox10 的表达与早期 ASM 无关。tfap2a、foxD3 和 sox10 的功能分析表明，tfap2a 和 foxD3 都是 ASM 规范和 AS 形成的关键调节因子，而 sox10 对于 AS 的规范或开发都是可有可无的。使用基因敲除线，我们表明在没有 tfap2a 或 foxD3 功能的情况下，ASM 细胞未指定，随后 AS 畸形。相反，sox10 基因突变体或 CRISPR Cas9 注射胚胎在 ASM 规范、迁移或 AS 方面没有显示缺陷。最后，使用转录组学分析，我们表明 GFP + cNCC 源自 Tg [ foxD3:GFP] 和 Tg [ foxC1b :GFP] 共享与 ASM 发育一致的表达谱，而从 Tg [ sox10 :GFP] 分离的 cNCC 表现出与血管发生、肌肉功能和色素沉着相关的表达谱。总之，我们提出斑马鱼前段间充质 (ASM) 规格的最早阶段约为 16hpf，涉及 tfap2a/foxC1a 阳性 cNCC。