Cell Interactions and Cell Fate Specification

蛋白质β-连环蛋白在特定细胞核中的聚集是多细胞动物发育的关键早期步骤。在所有已研究的后口动物中，β-连环蛋白的核积累（“核化”）是沿着早期胚胎的单轴极化的。 β-连环蛋白的核化是中胚层和/或内胚层形成和早期信号中心建立所必需的。尽管β-连环蛋白在后口类胚胎发育中具有重要作用，但其调节分化核化的机制尚不清楚。早期海胆胚胎是分析这一问题的理想实验体系。这种胚胎是高度透明的，这有利于使用光学方法分析蛋白质周转和体内靶向的动态。海胆系统的独特之处还在于可以从早期胚胎中大量分离出特定类型的细胞。这一关键特征意味着可以分离出大量在细胞核中含有或不含有β-连环蛋白的早期卵裂球，并研究这两种细胞的生化差异。许多β-连环蛋白通路的核心分子组分已经从海胆中克隆出来，包括我们实验室最近克隆的几个关键分子。这种强大的特性组合使得海胆胚胎成为分析β-连环蛋白核化的独特实验系统。这项提议测试了几个关于在早期发育过程中调节β-连环蛋白核积累机制的具体假设。为了验证这些假设，将采用延时摄影、三维共聚焦显微镜、显微外科和分子生物学方法对胚胎进行操作以及生物化学方法。主要的问题是：a）卵裂球之间的相互作用在早期发育过程中是否对β-连环蛋白核化的模式起调节作用？B）β-连环蛋白在不同发育阶段的不同细胞系中的稳定性（半衰期）是多少？β-连环蛋白的稳定性在特定的时间和特定的细胞中是增加还是减少？c）胚胎动物侧β-连环蛋白的丢失是否通过GSK 3介导？如果是这样的话，沿着A-V轴是否存在GSK 3活性、稳定性或丰度的可测量差异？d）轴蛋白（β-连环不稳定性的调节剂）沿A-V轴的沿着空间分布是什么？e）动物和植物卵裂球中降解复合物的状态是否不同？这个研究项目将通过阐明动物发育中一个基本过程的机制来推进科学知识。它还将通过培养本科研究人员、研究生和博士后研究员，为人力资源的开发做出贡献。

0128140EttensohnThe accumulation of the protein beta-catenin in the nuclei of specificcells is a critical, early step in the development of multicellularanimals. In all deuterostomes that have been studied, nuclearaccumulation ("nuclearization") of beta-catenin is polarized along oneaxis of the early embryo. Nuclearization of beta-catenin is requiredfor mesoderm and/or endoderm formation and the establishment of earlysignaling centers. Despite its fundamental importance in patterningdeuterostome embryos, the mechanisms that regulate the differentialnuclearization of beta-catenin are unknown. The early sea urchin embryois an ideal experimental system for the analysis of this problem. Theembryo is highly transparent, which facilitates analysis of the dynamicsof protein turnover and targeting in vivo using light optical methods.The sea urchin system is also unique in that specific cell types can beisolated from early embryos in large quantities. This criticalcharacteristic means that it is possible to isolate large quantities ofearly blastomeres that have, or do not have, beta-catenin in theirnuclei, and study biochemical differences in the two kinds of cells.Many of the central molecular components of the beta-catenin pathwayhave been cloned from sea urchin, including several key players thathave been cloned recently in our laboratory. This powerful combinationof characteristics makes the sea urchin embryo a unique experimentalsystem for the analysis of beta-catenin nuclearization.This proposal tests several specific hypotheses concerning mechanismsthat regulate the nuclear accumulation of beta-catenin during earlydevelopment. To test these hypotheses, a combination of time-lapse, 3-Dconfocal microscopy, manipulation of embryos by microsurgical andmolecular biological methods, and biochemical approaches will beemployed. The major questions to be addressed are the following:a) Do interactions between blastomeres play any role in regulating thepattern of beta-catenin nuclearization during early development?b) What is the stability (half-life) of beta-catenin in different celllineages at different developmental stages? Does the stability ofbeta-catenin increase or decrease at specific times and in specificcells?c) Is the loss of beta-catenin on the animal side of the embryo mediatedthrough GSK3? If so, are there measurable differences in GSK3 activity,stability, or abundance along the A-V axis?d) What is the spatial distribution of axin, a regulator of beta-cateninstability, along the A-V axis? e) Is the state of the degradation complex different in animal andvegetal blastomeres?This research project will advance scientific knowledge by elucidatingthe mechanisms of a fundamental process in animal development. It willalso contribute to the development of human resources through thetraining of undergraduate researchers, graduate students, andpostdoctoral fellows.