Control of Cell Movements During Gastrulation in Xenopus

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0315767MillerThe control of morphogenetic movements during gastrulation is critical for the viability of the organism and, therefore, determining the molecular pathways that regulate cell activities during gastrulation is of fundamental importance. Recent work from Xenopus and zebrafish provide evidence that polarized cell motility and shape changes during gastrulation are regulated by the Wnt/PCP pathway. These studies have defined a critical role for the signaling protein Dishevelled (Dsh) and suggest that Dsh controls cell polarity through regulation of the actin cytoskeleton. Preliminary studies from Dr. Miller's laboratory provide support for the hypothesis that the signaling protein Eps8 interacts with Dsh providing a potential link between extracellular signals and the actin cytoskeleton during gastrulation. The objective of the current research project is to determine the mechanisms by which Eps8 controls morphogenetic movements during gastrulation, using embryos of the clawed frog, Xenopus laevis, as a model system. This proposal will seek to answer the following questions: (1) What is the requirement for Eps8 during development? The function of Eps8 in Xenopus development will be investigated using gene-knockdown strategies and expression of dominant-negative forms of Eps8. The resulting phenotypes will be examined using morphological, histological, and gene expression analyses. (2) What morphogenetic events does Eps8 regulate during gastrulation? Gain- and loss-of-function approaches will be combined with a detailed analysis of (i) cell polarity, shape, and protrusive activity, (ii) actin organization, and (iii) cell-cell adhesion to investigate the mechanism by which Eps8 regulates morphogenesis. They will also characterize the subcellular distribution of Eps8 during gastrulation. (3) What signals regulate Eps8 function during gastrulation? They will utilize biochemical and cell biological assays to examine whether Eps8 function and/or its interaction with Dsh is regulated by PDGF or Wnt signals. Broader impact of the proposed research: By exploiting their expertise in using classical embryological approaches and modern imaging tools for the analysis of cell movements and protein localization in vivo, the Miller laboratory will gain important insights into the mechanisms that control morphogenetic movements during gastrulation in vertebrates. Since convergent extension movements are a fundamental process employed throughout the animal kingdom, their studies in Xenopus will lead to a greater understanding of the how cell movements are coordinated to control tissue patterning in other systems. In addition, a graduate student, and an undergraduate student will work extensively on this project. They will receive extensive training in microscopy, biochemistry, experimental embryology, and cell biology as part of this project.