Genetic Analysis of Mouse Nervous System Development

描述（由申请人提供）：Sonic hedgehog （Shh）信号对于哺乳动物中枢神经系统的组织以及成体神经干细胞的决定和维持至关重要。然而，目前尚不清楚Shh信号是如何从膜蛋白Smoothened传递到执行该途径的Gli转录因子的。已知有四种成分在这一过程中起作用：Kif7、蛋白激酶A （PKA）、融合抑制因子（Sufu）和初级纤毛。本提案的前两个目标将定义Kif7和PKA如何在初级纤毛中发挥作用以转导Shh信号。Kif7作为Shh通路的核心组分和纤毛结构所需的驱动蛋白具有双重作用。野生型和突变型细胞的免疫定位和共免疫沉淀实验将确定Kif7如何调节Gli转录因子的活性。高分辨率静态和

DESCRIPTION (provided by applicant): 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.