Hedgehog signaling and intracellular transport

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DESCRIPTION (provided by applicant): Embryonic cells use the Hedgehog signaling pathway to convey information that controls cell fate, proliferation, and survival. Misregulation of this pathway leads to birth defects and a variety of cancers in humans. Recent data point to key mechanisms controlling the Hedgehog pathway in mammals that do not appear to be evolutionary conserved in invertebrate model organisms. The primary objective of the proposed research is to understand how Hedgehog signal transduction functions at the mechanistic level using the mouse as a model system. Specifically, the roles of 2 novel antagonists of the mammalian pathway, Wdr10 and Tulp3, will be studied using genetic, cellular, and biochemical approaches outlined in 3 specific aims. The Wdr10 protein, as well as its invertebrate homologs, functions in intraflagellar transport, a process used for building cilia and flagella. The experiments proposed in Aim 1 will confirm the requirement for Wdr10 in regulating the Hedgehog pathway through targeted mutagenesis and transgenic rescue approaches. Other experiments will address the subcellular localization of Wdr10 and test whether it acts in retrograde transport using immunocytochemistry and live cell imaging techniques. The mechanism of Wdr10 function in the Hedgehog pathway will be addressed in Aim 2. The hypothesis that Wdr10 acts cell autonomously will be tested through chimera analysis and the step in the Hedgehog pathway at which Wdr10 acts will be clarified through epistasis analysis. In addition, the hypothesis that Wdr10 acts directly or indirectly in controlling nuclear localization of the Gli transcription factors will be pursued using immunohistochemical and biochemical methods. Finally, the experiments in Aim 3 will test 2 hypotheses regarding the function of Tulp3 in Hedgehog signaling. First, the importance of the interaction between Tulp3 and myosin Vb will be functionally tested by blocking myosin Vb activity in vivo. Second, the significance of regulated Tulp3 nuclear translocation in Hedgehog signaling will be examined in tissue culture cells.