REGULATION OF BETA-CATENIN NUCLEARIZATION

超出提供的空间。-连环蛋白在特定卵裂球的细胞核中的积累是动物发育的关键早期步骤。在所有已研究的后口动物(棘皮动物和脊椎动物/脊椎动物)中，沿着卵裂期胚胎轴线的特定卵裂球的核中-连环素的积累是胚胎极性的最初迹象之一。-连环蛋白的这种不同的“核化”对于正确的胚层形成和诱导中心的建立是必不可少的。海胆胚胎是研究连环蛋白核化的有力模型系统。胚胎在外部发育，是光学透明的，有利于各种光光学技术的应用。该系统的独特之处在于，可以从早期胚胎中大量分离出特定类型的细胞。β-连环蛋白途径的大部分分子已经从海胆中克隆出来，并有大量的分子生物学工具可用。基因表达可通过表达显性负性结构和微量注射“吗啉”反义寡核苷酸来调控。这项拟议的工作将确定早期胚胎中-连环蛋白核化的潜在机制。主要目的有两个：1)利用GFP标记的蛋白质和延时三维共聚焦显微镜，在体内研究β-连环蛋白降解的关键调控因子的动态和周转。我们将测量-连环蛋白在早期胚胎特定的卵裂球中的半衰期，这将为差异核化的细胞和分子机制提供必要的信息。通过卵裂球分离实验，我们将测试细胞-细胞相互作用在调节f_连环蛋白核化中的作用。2)我们将沿着胚轴测试三种主要的GSK3调控模型。此外，利用显性负结构和吗啉，我们将研究GSK3活性的两个调节因子Disheveled和Akt/PKB的发育作用。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. The accumulation of _-catenin in the nuclei of specific blastomeres is a critical early step in animal development. In all deuterostomes (echinoderms and chordates/vertebrates) that have been studied, accumulation of _-catenin in the nuclei of specific blastomeres along the axis of the cleavage stage embryo is one of the first indications of embryonic polarity. This differential "nuclearization" of _-catenin is essential for proper germ layer formation and the establishment of inductive centers. The sea urchin embryo is a powerful model system for studying _-catenin nuclearization. The embryo develops externally and is optically transparent, facilitating the application of a wide variety of light optical technologies. The system is unique in that specific cell types can be isolated from early embryos in large quantities. Most of the molecules of the _-catenin pathway have been cloned from sea urchin and a large number of molecular biological tools are available. Gene expression can be manipulated by expression of dominant negative constructs and by microinjection of "morpholino" antisense oligonucleotides. The proposed work will identify the mechanisms that underlie _-catenin nuclearization in early embryos. There are two major aims: 1) Using GFP-tagged proteins and time-lapse, 3-D confocal microscopy, the dynamics and turnover of key regulators of _-catenin degradation will be studied in vivo. We will measure the half-life of _-catenin in specific blastomeres of the early embryo, which will provide essential information concerning the cellular and molecular mechanisms of differential nuclearization. Through blastomere isolation experiments, we will test the role of cell-cell interactions in regulating f_-catenin nuclearization. 2) We will test three major models of GSK3 regulation along the embryo axis. In addition, using dominant negative constructs and morpholinos, we will examine the developmental ftmction of two regulators of GSK3 activity, dishevelled and Akt/PKB. PERFORMANCE SITE ========================================Section End===========================================