The vertebrate body plan is established during gastrulation through a series of inductive and morphogenic processes. During the gastrulation movements of convergent extension the entire embryo and most organ rudiments narrow along the mediolateral axis while extending their anteroposterior dimension. The genetic hierarchy regulating convergent extension is only poorly understood. Our cell movement analyses in live zebrafish gastrulae indicate that in the lateral regions directed cell migration drives increasingly faster convergence and extension. Whereas dorsally, intercalation of mediolaterally polarized cells leads to extension with limited convergence.Studies from others and our laboratory indicate that the vertebrate equivalent of the Drosophila planar cell polarity signaling pathway, including Wnt11 and Wnt5, Frizzled7, Dishevelled and Rho kinase 2, mediates convergent extension. We have shown that knypek (kny) and trilobite (tni) mutations impair cell polarization during convergent extension without significantly affecting cell fates. Positional cloning revealed that the kny gene encodes a glypican (heparan sulfate proteoglycan). Epistatic analyses indicate that Knypek promotes Wnt11 signaling during gastrulation. Our central hypothesis is that kny and tn mediate mediolateral polarization of directionally migrating and intercalating cells during convergent extension, acting as components of the Wnt planar cell polarity pathway. To test this hypothesis we will identify cell activities underlying directed cell migration and mediolateral intercalation using in vivo time lapse microscopy, and determine cell activities dependent on kny and tni functions. By genetic and molecular epistasis we will learn whether kny acts in Wnt planar cell polarity pathway. We will employ biochemical assays to test whether Knypek binds Wnt11 ligand. Using embryological methods we will investigate if Knypek mediates propagation of Wnt11 ligand through fields of cells. We will complete molecular characterization of the tri locus and test its involvement in the Wnt planar cell polarity pathway by genetic and molecular epistasis experiments. We will identify additional genes involved in the Wnt planar cell polarity pathway by genetic screens for modifiers of the kny mutant phenotype. Taking advantage of molecular identification of genes involved in convergent extension, methods for in vivo cell movement analyses, and forward genetic approach in zebrafish, this proposal will address the mechanisms of convergent extension at unprecedented molecular genetic and cellular levels. The proposed experiments will provide insights into the vertebrate Wnt planar cell polarity pathway and the mechanisms of cell polarization during vertebrate gastrulation and morphogenesis.
Showing the most recent 10 out of 49 publications
