Department of Ophthalmology, University of Alberta, Edmonton, Alberta, Canada.
Invest Ophthalmol Vis Sci. 2013 Jul 10;54(7):4636-47. doi: 10.1167/iovs.13-11674.
Vision is critically dependent on ocular size, which is regulated by environmental and genetic factors. Mutation of human Growth and Differentiation Factor 6 (GDF6) or zebrafish gdf6a results in a spectrum of small eye phenotypes (microphthalmia, anophthalmia, and coloboma). However, current models do not explain their etiology fully. As such, analyses of apoptosis and cell cycle regulation were undertaken in a zebrafish gdf6a mutant.
Microarray analysis was performed at 2 days after fertilization to uncover novel gdf6a-dependent cell cycle regulators. Altered expression of Gdf6a targets was confirmed by in situ hybridization, and resulting changes in cell proliferation were assessed by phosphohistone H3 immunohistochemistry. Analysis of apoptosis was evaluated through activated Caspase 3 immunohistochemistry and chemical inhibitors of cell death.
Reduced numbers of retinal progenitor cells are observed at 24 hours post fertilization (hpf), resulting in microphthalmic eyes in gdf6a(-/-) embryos. At 28 hpf, a wave of apoptosis occurs; however, apoptosis inhibition does not rescue eye size, indicating a limited contribution. Mutants display altered proliferation and expression levels of cell cycle regulators, including members of the forkhead box i (foxi) transcription factor family expressed in the ciliary marginal zone. Notably, inhibition of foxi2 in gdf6a(-/-) embryos further reduces eye size.
These data support a model whereby the gdf6a(-/-)-induced microphthalmia is based on early regulation of retinal progenitor cell number, and later by regulation of proliferation in the ciliary marginal zone. Foxi genes represent downstream effectors of Gdf6a function in the CMZ required for eye size determination.
视觉对眼球大小具有关键依赖性，而眼球大小受环境和遗传因素的调节。人类生长分化因子 6（GDF6）或斑马鱼 gdf6a 的突变会导致一系列小眼球表型（小眼球、无眼球和眼眶裂）。然而，目前的模型并没有完全解释其病因。因此，对斑马鱼 gdf6a 突变体中的细胞凋亡和细胞周期调控进行了分析。
在受精后 2 天进行微阵列分析，以揭示新的依赖于 gdf6a 的细胞周期调控因子。通过原位杂交证实 Gdf6a 靶标的表达改变，并通过磷酸组蛋白 H3 免疫组织化学评估细胞增殖的变化。通过激活 Caspase 3 免疫组织化学和细胞死亡的化学抑制剂分析细胞凋亡。
在受精后 24 小时（hpf）时观察到视网膜祖细胞数量减少，导致 gdf6a(-/-)胚胎出现小眼球。在 28 hpf 时，发生了一波细胞凋亡；然而，细胞凋亡抑制不能挽救眼睛大小，表明其贡献有限。突变体显示细胞增殖和细胞周期调控因子的表达水平发生改变，包括在睫状缘区表达的叉头框 i（foxi）转录因子家族的成员。值得注意的是，在 gdf6a(-/-)胚胎中抑制 foxi2 进一步减小了眼睛大小。
这些数据支持了一种模型，即 gdf6a(-/-)诱导的小眼球是基于对视网膜祖细胞数量的早期调节，以及后来对睫状缘区增殖的调节。Foxi 基因是 Gdf6a 在 CMZ 中发挥作用的下游效应子，是确定眼睛大小所必需的。
