Department of Ophthalmology and Visual Science, Kumamoto University, Graduate School of Medical Sciences, 1-1-1, Honjo, 860-8556 Kumamoto City, Japan.
Exp Eye Res. 2010 Jan;90(1):81-8. doi: 10.1016/j.exer.2009.09.017. Epub 2009 Sep 24.
The heparan sulfate (HS) is a component of proteoglycans in the extracellular matrix and on cell surfaces, modulating developmental processes. The aim of this study is to investigate whether the defect of HS in the periocular mesenchyme impairs ocular morphogenesis. First, using Protein 0-Cre transgenic mice, we ablated Ext1, which encodes an indispensable enzyme for HS synthesis, in the developing periocular mesenchyme. The expression of Ext1 messenger RNA (mRNA) and HS were observed by RT-PCR and immunohistochemistry, respectively. The phenotypes in the mutant were evaluated by light microscopy and immunohistochemistry for cellular makers. Second, the distribution of the mutant periocular mesenchymal cells was tracked using a Rosa26 Cre-reporter gene. No mutant embryos (Protein 0-Cre;Ext1(flox/flox)) were identified after embryonic day 14.5 (E14.5). RT-PCR showed that an intense band amplified from Ext1 was observed in cDNAs from the control periocular mesenchymal cells at E13.5; however, the band for Ext1 was hardly detectable in cDNA from the mutant embryo, indicating that the mRNA was missing in the mutant periocular mesenchyme at E13.5. The HS expression was disrupted in the periocular mesenchyme of the mutant ocular tissues. The HS deficiency resulted in microphthalmia with reduced axial lengths, lens diameters, and vitreous sizes compared with the littermate eyes. The mutant embryos showed agenesis of the anterior chamber, where cells expressing Cre recombinase were distributed. Moreover, the mutants showed phenotypic alterations in the neural ectoderm including dysgenesis of the presumptive ciliary body and agenesis of the optic nerve head. These findings demonstrate that HS in the periocular mesenchyme plays a critical role in normal ocular morphogenesis, indicating reciprocal interactions between the periocular mesenchyme and the neural ectoderm.
硫酸乙酰肝素（HS）是细胞外基质和细胞表面蛋白聚糖的组成部分，调节发育过程。本研究旨在探讨眼周间质中 HS 缺陷是否会影响眼球形态发生。首先，利用 Protein 0-Cre 转基因小鼠，我们在发育中的眼周间质中敲除了编码 HS 合成必需酶的 Ext1。通过 RT-PCR 和免疫组织化学分别观察 Ext1 信使 RNA（mRNA）和 HS 的表达。通过光镜和细胞标志物免疫组织化学评估突变体的表型。其次，使用 Rosa26 Cre 报告基因跟踪突变的眼周间质细胞的分布。在 E14.5 后未鉴定出突变体胚胎（Protein 0-Cre；Ext1(flox/flox)）。RT-PCR 显示，在 E13.5 时，从对照眼周间质细胞的 cDNA 中观察到强烈的 Ext1 扩增带；然而，在突变体胚胎的 cDNA 中几乎检测不到 Ext1 条带，表明在 E13.5 时突变体眼周间质中缺失了 mRNA。HS 表达在突变体眼组织的眼周间质中被破坏。与同窝对照眼相比，HS 缺乏导致小眼畸形，眼轴长度、晶状体直径和玻璃体体积减小。突变体胚胎的前房发育不全，其中分布着表达 Cre 重组酶的细胞。此外，突变体还表现出神经外胚层的表型改变，包括假定睫状体的发育不良和视神经头的缺失。这些发现表明，眼周间质中的 HS 在正常眼球形态发生中发挥关键作用，表明眼周间质与神经外胚层之间存在相互作用。
