Genetic Analysis of Mouse Nervous System Development

描述（由申请人提供）：Sonic hedgehog（Shh）信号传导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确定和维持至关重要。然而，尚不清楚Shh信号如何从膜蛋白Smoothened传递到实现该途径的Gli转录因子。已知有四种成分在该途径的这一步起作用：Kif7、蛋白激酶A（PKA）、融合抑制因子（Sufu）和初级纤毛。该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抑制Shh信号。Kif7具有作为Shh通路的核心组分和作为纤毛结构所需的驱动蛋白的双重作用。野生型和突变细胞中的免疫定位和免疫共沉淀实验将确定Kif7如何调节Gli转录因子的活性。高分辨率静态和 活体成像将确定Kif7在纤毛运输中是否具有全局作用或在Gli蛋白运输中是否具有特定作用。PKA是Shh信号传导的强负调节因子，定位于纤毛基部。遗传和细胞生物学实验将测试PKA是否需要定位于纤毛的基部以发挥功能，PKA是否控制纤毛中的运输， Shh是否控制PKA活性。刺猬依赖性疾病和肿瘤的新疗法的开发将取决于对这些信号传导机制的深入理解。纤毛以中心体为模板，破坏中心体的人类遗传疾病会导致小头畸形。该提案的目标3将定义中心体在早期胚胎和发育中的大脑中的发育和细胞功能。Sas4基因（也称为Cenpj或Cpap）是中心粒复制所必需的，Sas4突变体胚胎缺乏中心粒、纤毛和中心体。Sas4突变表型的分析将确定中心体是否调节信号传导、细胞分裂、细胞迁移或细胞存活。数据表明，Sas 4胚胎的早期致死性通过去除p53而得以挽救，并且实验将确定Sas 4突变体中激活的p53依赖性途径。为了确定纤毛和中心体在发育中的大脑中的图案化和细胞行为中的作用，将比较由纤毛形成所需的Sas 4或Ift 88的条件性遗传缺失引起的表型。模式化，增殖，皮质组织和细胞死亡将在突变体中进行分析，这些突变体在发育中的大脑中具有纤毛或中心体。GFP标记的Cre的子宫内电穿孔将用于有条件地删除脑中的Sas 4和Ift 88，然后跟踪缺乏纤毛或中心体的单个细胞的命运。这些实验将明确纤毛和中心体在控制皮层细胞不对称分裂和迁移中的作用。 公共卫生相关性：这个项目将确定初级纤毛的功能，触角样细胞器存在于大多数细胞，在哺乳动物神经系统的发展。实验将测试初级纤毛如何调节Shh信号通路的特定组分，该通路指定神经元细胞类型的模式和增殖。额外 实验将测试初级纤毛和使纤毛成核的中心体的破坏如何导致人类遗传疾病中的小头畸形和其他大脑组织的深刻破坏。

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. PUBLIC HEALTH RELEVANCE: 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.