The vertebrate body plan is generated during embryogenesis through a complex sequence of gastrulation cell movements and inductive events. The molecular genetic mechanisms that control these processes are poorly understood. Ga12/13 signaling is implicated in numerous morphogenetic processes including D. melanogaster gastrulation, neutrophil migration, nerve growth cone guidance, and tumor metastasis. However, the roles of these proteins in early vertebrate embryogenesis remain to be elucidated. Ga12/13 proteins constitute the most divergent family of heterotrimeric G protein a subunits. They link G protein coupled receptors (GPCRs) to the actin cytoskeleton and cell motility by regulating small GTPases of the Rho family via guanine nucleotide exchange factors (GEFs). We identified three zebrafish genes encoding Ga12/13 proteins and a pdz rhogefgene, and have implicated them in several distinct gastrulation cell behaviors, including directed migration and intercalation. Our data suggest a novel role of PDZ RhoGEF in primary cilia function during left-right asymmetry specification and kidney physiology. Our central hypothesis is that Ga12/13 and PDZ RhoGEF constitute a regulatory module that regulates cell movements and fates during vertebrate embryogenesis by integrating imputs from several signaling pathways.
In Aim 1, using in vivo cell movement analyses, we will identify aspects of individual gastrulation cell behaviors mediated by Ga12/13 signaling.
In Aim 2, we will determine functions of zebrafish PDZ RhoGEF by overexpression and mutational studies. We will also test whether PDZ RhoGEF and Ga12/13 regulate formation or function of primary cilia jn left-right asymmetry specification, ear and kidney development, and test their functional interactions using cell biological and biochemical approaches.
Our Aim 3 is to test the hypothesis that Ga12/13 integrate inputs from several GPCRs to mediate migration towards different targets in zebrafish gastrula. We propose to identify a subset of GPCRs regulating gastrulation by a combination of bioinformatics with gene expression and interference studies. We will test whether Ga12/13 and PDZ RhoGEF functionally interact with non-canonical Wnt signaling to mediate directed cell migration and intercalation. Given high evolutionary conservation of Ga12/13 and PDZ RhoGEF, our studies will provide new insights into genetic basis of birth defects and tumorigenesis.
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