Investigation of the basic process of cell differentiation utilizing mutant animals of crystallin regulators

我们的目的是阐明透镜分化的过程作为细胞分化的范例，特别注意转录调控。值得注意的是，透镜细胞在胚胎发生中异常早地经历终末分化。透镜分化的开始除了组织的形态学变化外，还以晶状体蛋白基因表达的起始为标志。由于delta-crystallin是禽类晶状体中合成最早、数量最多的晶体蛋白，因此我们对鸡delta-crystallin基因的调控进行了研究，通过对delta-crystallin增强子核心的突变分析，发现了两个相互依赖的正调控元件。根据该结果和透镜核提取物的EMSA，得出结论，这两种元素对应于两种不同激活剂deltaEF 2（Sox-1至3）和deltaEF 3的结合位点。阻遏物deltaEF 1调节deltaEF 3的活性。研究还表明，小鼠γ-晶状体蛋白基因的启动子活性需要Sox结合，这为多种晶状体蛋白基因至少部分地受共同机制的调节提供了证据。Sox蛋白1至3被证明是晶状体蛋白调节所必需的，并且从它们表达的时间和空间分布来看，它们是透镜分化的关键因素的良好候选者。Pax-6似乎也参与了这些过程，但它的贡献似乎比仅仅引起透镜分化要多得多。看起来好像Pax-6参与了整体组织的眼睛赋予，而Sox-1至3则决定了外胚层中的晶状体。这两个因素对于引起透镜分化似乎是必不可少的，但它们本身是不够的，更多的内在和外在因素可能是透镜分化发生所必需的。目前正在研究的deltaEF 3可能是这些重要因素之一。

We have aimed at elucidation of the process of lens differentiation as a paradigm of cell differentiation with special attention to transcriptional regulation. It is remarkable that lens cell undergo terminal differentiation exceptionally early in embryogenesis. Onset of lens differentiation is marked by initiation of crystallin gene expression in addition to morphological change of the tissue. We focused on regulation of delta-crystallin gene of the chicken because delta-crystallin is the earliest and the most abundantly synthesized crystallin in avian lenses.Mutational analysis of the delta-crystallin enhancer core identifed two positive regulatory elements which are interdependent. From this and EMSA of lens nuclear extract, it was concluded these two elements correspond to binding sites of two distinct activators, deltaEF2 (Sox-1 to 3) and deltaEF3. A repressor deltaEF1 modulates the activity of deltaEF3. It was also shown that Sox binding is required for the promoter activity of the mouse gamma-crystallin gene, providing evidence that diversified crystallin genes are regulated, at least partly, by a common mechanism.The Sox proteins 1 to 3 are shown to be essential for crystallin regulation, and from chronology and spatial distribution of their expression they are good candidates as a key factor of lens differentiation. Pax-6 also seems involved in these processes, but its contribution seems to be in larger number of steps than only provoking lens diffentiation. It looks as if Pax-6 is involved in endowment of eyeness to the tissues in global terms, while Sox-1 to 3 are in determination of lensness in the ectoderm. These two factors appear essential for eliciting lens differentiation, but by themselves they are not sufficient, and more intrinsic and extrinsic factors are probably necessary for lens differentiation to occur. deltaEF3, currently under investigation, may be one of these essential factors.