Cell Interactions and Cell Fate Specification

点击翻译按钮获取中文摘要

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.