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

我们的目的是阐明晶状体分化过程作为细胞分化的范例，特别关注转录调控。晶状体细胞在胚胎发生过程中发生终末分化异常早。晶状体分化的开始，除了组织的形态变化外，还以晶体蛋白基因表达的开始为标志。由于delta-crystallin是禽类晶状体中合成最早、数量最多的晶体蛋白，因此我们重点研究了delta-crystallin基因的调控。δ结晶蛋白增强子核心的突变分析确定了两个相互依赖的正调控元件。从这一点和晶状体核提取物的EMSA来看，这两个元素对应于两种不同的激活剂deltaEF2 （Sox-1至3）和deltaEF3的结合位点。抑制因子deltaEF1调节deltaEF3的活性。研究还表明，小鼠γ -结晶蛋白基因的启动子活性需要Sox结合，这提供了证据，证明多种结晶蛋白基因至少在一定程度上受到共同机制的调节。Sox蛋白1至3对晶体蛋白调控至关重要，从其表达的时间和空间分布来看，它们是晶状体分化的关键因素。Pax-6似乎也参与了这些过程，但它的贡献似乎是在更多的步骤上，而不仅仅是引发透镜分化。从整体上看，Pax-6参与了组织的视力赋能，而Sox-1至sox - 3参与了外胚层的晶状度决定。这两个因素似乎是引起晶状体分化的必要因素，但它们本身是不够的，更多的内在和外在因素可能是晶状体分化发生的必要因素。目前正在调查的deltaEF3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.