REGULATION OF BETA-CATENIN NUCLEARIZATION

描述（由申请人提供）：β -连环蛋白在特定卵裂球细胞核中的积累是动物发育的关键早期步骤。在所有已被研究过的后齿动物（棘皮动物和脊索动物/脊椎动物）中，卵裂期胚胎沿轴的特定卵裂球细胞核中-连环蛋白的积累是胚胎极性的最初迹象之一。这种β -连环蛋白的差异性“核化”对于适当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-连环蛋白成核的一个强有力的模型系统。胚胎在外部发育，具有光学透明性，促进了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，并且有大量的分子生物学工具可用。基因表达可以通过显性阴性结构的表达和微量注射“morpholino”反义寡核苷酸来操纵。这项工作将确定早期胚胎中β -连环蛋白成核的机制。主要有两个目的：1)使用gfp标记的蛋白质和延时三维共聚焦显微镜，研究体内β -连环蛋白降解关键调控因子的动态和周转。我们将测量β -连环蛋白在早期胚胎特定卵裂球中的半衰期，这将为差异成核的细胞和分子机制提供重要信息。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β -连环蛋白成核中的作用。2)我们将沿着胚胎轴测试三种主要的GSK3调控模型。此外，我们将使用显性负性结构和morpholinos来研究两种GSK3活性调控因子（disheded和Akt/PKB）的发育功能。

DESCRIPTION (provided by applicant): The accumulation of beta-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 beta-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 beta-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 beta-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 beta-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 beta-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 beta-catenin degradation will be studied in vivo. We will measure the half-life of beta-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 beta-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 function of two regulators of GSK3 activity, disheveled and Akt/PKB.