During vertebrate gastrulation, convergence and extension (C&E) is a key process, which narrows the mediolateral embryonic axis while extending its anteroposterior dimension. We implicated in this cell polarization process a non-canonical Wnt signaling pathway, similar to the D. melanogaster planar cell oolarity (PCP) pathway, and identified several of its components: Knypek (Glypican4), Trilobite (Van gogh/Strabismus) and Rho kinase. However, the mechanisms this pathway employs to polarize cells during vertebrate gastrulation are not known. Our studies revealed that both knypek (kny/gpc4) and trilobite (tri/vang!2) are essential for a subset of gastrulation cell behaviors, fast directed dorsal migration and mediolateral intercalation. To establish the dynamic properties underlying these polarized cell behaviors that depend on kny and fri function, we will determine formation, polarization and stabilization of protrusions, and the orientation of microtubule cytoskeleton by analyzing cells in kny and tri single, and kny;tri compound mutant gastrulae, and embryos overexpressing dominant negative Dishevelled (dnDsh). We will determine the sequence of cellular events leading to the mediolateral polarization of mesoderm cells. We will test the involvement of kny, tri and dsh in increased cell packing and other aspects of the mediolateral cell polarization process. We will use different types of mosaic analyses to test the hypothesis that kny, and tri are involved in a short-range intercellular communication that mediates cell polarization. We will discover new regulators C&E by analysis of mutant loci identified in our screen for genes that modify the kny gastrulation mutant phenotype. A new mutation, car69 sensitizes embryos to copper levels, and enhances aspects of the kny mutant phenotype. We will characterize patterning and morphogenetic defects caused by calvu69, test its genetic interactions with other C&E mutations, and molecularly characterize the cal locus. Given that aspects of cat phenotype are phenocopied by inhibition of copper uptake, we will test the hypothesis that Wnt signaling via Kny depends on copper homeostasis. We propose to carry out precise phenotypic, genetic and molecular characterization of vu66 mutation, which weakly impairs C&E and acts as homozygous recessive enhancer of the kny gastrulation defect. Recent studies demonstrate that the molecular regulation of tumor growth and metastasis is strikingly similar to that of morphogenetic processes, such as gastrulation. In particular, overexpression of non- canonical Wnts affects cell motility and invasion of melanoma cells. Our work will further elucidate the mechanisms whereby non-canonical Wnt signaling regulates invasive cell behaviors in the context of a developing organism, and will identify additional components of this important pathway.
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