Cell Interactions and Cell Fate Specification

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

0128140 Ettensohn The accumulation of the protein beta-catenin in the nuclei of specific cells is a critical, early step in the development of multicellular animals. In all deuterostomes that have been studied, nuclear accumulation ("nuclearization") of beta-catenin is polarized along one axis of the early embryo. Nuclearization of beta-catenin is required for mesoderm and/or endoderm formation and the establishment of early signaling centers. Despite its fundamental importance in patterning deuterostome embryos, the mechanisms that regulate the differential nuclearization of beta-catenin are unknown. The early sea urchin embryo is an ideal experimental system for the analysis of this problem. The embryo is highly transparent, which facilitates analysis of the dynamics of protein turnover and targeting in vivo using light optical methods. The sea urchin system is also unique in that specific cell types can be isolated from early embryos in large quantities. This critical characteristic means that it is possible to isolate large quantities of early blastomeres that have, or do not have, beta-catenin in their nuclei, and study biochemical differences in the two kinds of cells. Many of the central molecular components of the beta-catenin pathway have been cloned from sea urchin, including several key players that have been cloned recently in our laboratory. This powerful combination of characteristics makes the sea urchin embryo a unique experimental system for the analysis of beta-catenin nuclearization. This proposal tests several specific hypotheses concerning mechanisms that regulate the nuclear accumulation of beta-catenin during early development. To test these hypotheses, a combination of time-lapse, 3-D confocal microscopy, manipulation of embryos by microsurgical and molecular biological methods, and biochemical approaches will be employed. The major questions to be addressed are the following: a) Do interactions between blastomeres play any role in regulating the pattern of beta-catenin nuclearization during early development? b) What is the stability (half-life) of beta-catenin in different cell lineages at different developmental stages? Does the stability of beta-catenin increase or decrease at specific times and in specific cells? c) Is the loss of beta-catenin on the animal side of the embryo mediated through 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-catenin stability, along the A-V axis? e) Is the state of the degradation complex different in animal and vegetal blastomeres? This research project will advance scientific knowledge by elucidating the mechanisms of a fundamental process in animal development. It will also contribute to the development of human resources through the training of undergraduate researchers, graduate students, and postdoctoral fellows.