Sonic hedgehog (Shh) signaling is essential for the organization of the mammalian central nervous system and for determination and maintenance of adult neural stem cells. Nevertheless, it is not known how the Shh signal is transmitted from the membrane protein Smoothened to the Gli transcription factors that implement the pathway. Four components are known to act at this step of the pathway: Kif7, Protein kinase A (PKA), Suppressor of fused (Sufu) and the primary cilium. The first two aims of this proposal will define how Kif7 and PKA function at the primary cilium to transduce the Shh signal. Kif7 has dual roles as a core component of the Shh pathway and as a kinesin required for cilia structure. Immunolocalization and co-immunoprecipitation experiments in wild-type and mutant cells will define how Kif7 regulates the activity of the Gli transcription factors. High-resolution static and live imaging will define whether Kif7 has global roles in ciliary trafficking or has a specific rol in trafficking of Gli proteins. PKA is a strong negative regulator of Shh signaling that is localized o the base of cilia. Genetic and cell biological experiments will test whether PKA needs to be localized to the base of the cilium to function, whether PKA controls trafficking in the cilium and whether Shh controls PKA activity. Development of new therapies for Hedgehog-dependent diseases and tumors will depend on a deep understanding of these signaling mechanisms. Cilia are templated by centrosomes, and human genetic diseases that disrupt the centrosome cause microcephaly.
Aim 3 of the proposal will define the developmental and cellular functions of centrosomes in the early embryo and in the developing brain. The Sas4 gene (also called Cenpj or Cpap) is essential for centriole duplication and Sas4 mutant embryos lack centrioles, cilia and centrosomes. Analysis of the Sas4 mutant phenotype will define whether centrosomes regulate signaling, cell division, cell migration or cell survival. Data indicate that the early lethality o Sas4 embryos is rescued by removal of p53, and experiments will define the p53-dependent pathways activated in Sas4 mutants. To determine the roles of cilia and centrosomes in patterning and cell behavior in the developing brain, the phenotypes caused by conditional genetic deletion of Sas4 or of Ift88, which is required for formation of cilia, will be compared. Patterning, proliferation, cortical organization and cell death will be analyzed in mutants that lak either cilia or centrosomes in the developing brain. In utero electroporation of GFP- tagged Cre will be used to conditionally delete Sas4 and Ift88 in the brain and then follow the fate of individual cells that lack cilia or centrosomes. These experiments will define the roles of cilia ad centrosomes in the control asymmetric cell division and migration in the cortex.
This project will define the function of the primary cilia, antenna-like organelles present on most cells, in the development of the mammalian nervous system. Experiments will test how primary cilia regulate specific components of the Shh signaling pathway, which specifies the pattern and proliferation of neuronal cell types. Additional experiments will test how disruption of primary cilia, and the centrosomes that nucleate cilia, leads to microcephaly and other profound disruptions of the brain organization in human genetic diseases.
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