Cell Interactions and Cell Fate Specification

细胞相互作用和细胞命运规范

• 批准号: 0128140
• 负责人: Charles Ettensohn
• 金额: $ 39万
• 依托单位: Carnegie-Mellon University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0128140&HistoricalAwards=false
• 关键词: Cell; Interactions; Fate; Specification

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.

0128140Ettensohn β-连环蛋白在特定细胞核中的积累是多细胞动物发育中关键的早期步骤。在所有已研究的后口动物中，β-连环蛋白的核积累（“核化”）沿着早期胚胎的一个轴极化。 β-连环蛋白的核化对于中胚层和/或内胚层的形成以及早期信号中心的建立是必需的。尽管β-连环蛋白在后口动物胚胎的模式化中具有根本重要性，但调节β-连环蛋白差异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系统。胚胎高度透明，有利于利用光学方法分析蛋白质周转的动态和体内靶向。海胆系统的独特之处还在于可以从早期胚胎中大量分离特定的细胞类型。这一关键特征意味着可以分离大量细胞核中含有或不含β-连环蛋白的早期卵裂球，并研究这两种细胞的生化差异。β-连环蛋白途径的许多核心分子成分已从海胆中克隆出来，包括我们实验室最近克隆的几个关键分子。这种强大的特征组合使海胆胚胎成为分析 β-连环蛋白核化的独特实验系统。该提案测试了有关早期发育过程中调节 β-连环蛋白核积累的机制的几个具体假设。为了检验这些假设，将采用延时、3D 共聚焦显微镜、显微外科和分子生物学方法以及生化方法对胚胎进行操作的组合。需要解决的主要问题如下：a) 卵裂球之间的相互作用是否在早期发育过程中调节β-连环蛋白核化模式中发挥作用？b) β-连环蛋白在不同发育阶段的不同细胞谱系中的稳定性（半衰期）是多少？ β-连环蛋白的稳定性在特定时间和特定细胞中会增加还是降低？c) 胚胎动物侧 β-连环蛋白的丢失是通过 GSK3 介导的吗？如果是这样，沿 A-V 轴的 GSK3 活性、稳定性或丰度是否存在可测量的差异？d) 轴蛋白（β-连环蛋白稳定性调节剂）沿 A-V 轴的空间分布如何？ e) 动物和植物卵裂球中降解复合体的状态是否不同？该研究项目将通过阐明动物发育基本过程的机制来推进科学知识。它还将通过本科生研究人员、研究生和博士后的培训为人力资源的开发做出贡献。