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Abstract
Primary cilia are present on nearly every cell in the mammalian body, and this microtubule-based organelle can vary greatly in length depending on the cell type producing it. Each cell type tightly regulates the final length of the primary cilium produced, but the mechanisms controlling length regulation are not fully understood. During embryonic development, it is essential for cells to produce a primary cilium to respond to the developmental signaling protein, Hedgehog (Hh). Disruption of cilia production or function can result in dramatic developmental defects, including exencephaly and polydactyly. In this work, I have studied two protein kinases, Cell cycle-related kinase (CCRK) and Intestinal cell kinase (ICK) and their interactions in cilia length regulation and Hh signaling. Single mutant embryos for both genes have defects in Hh signaling and tissue patterning and are embryonic lethal. I analyzed cilia length of CCRK and ICK single mutants in cell culture and in developing embryonic limb buds. Loss of CCRK results in cilia that are both longer and shorter than wild-type cilia. Loss of ICK, also results in embryonic developmental defects related to altered Hh signaling, and ICK mutant cells have cilia that are longer than wild-type. Research has shown that CCRK can phosphorylate ICK in vitro. To understand the biological relevance and genetic interaction of these genes in vivo, I generated Ccrk and Ick double mutant embryos. I found that the double mutant embryos resemble the gross morphology of Ccrk embryos, indicating that Ccrk is indeed epistatic to Ick. However, interestingly, I found that Ccrk and Ick double mutant embryos seem to have earlier lethality than either single mutant and defects in heart looping not seen in Ick single mutants, and only rarely observed in Ccrk single mutants. This defect in heart looping is indicative in defects to left right patterning determination and could potentially stem from defects in the cilia production or length at the embryonic node, the tissue that controls the initial break in symmetry in left-right patterning.