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年神经干细胞的确定和维持是必不可少的。然而，Shh信号是如何从膜蛋白平滑到执行该途径的Gli转录因子的还不清楚。已知有四种成分在该途径的这一步骤中起作用：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如何在初级纤毛转导Shh信号。Kif7具有双重作用，既是Shh途径的核心成分，又是纤毛结构所需的激动素。野生型和突变细胞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 实时成像将确定Kif7在纤毛运输中是否具有全球角色，或者在Gli蛋白的运输中具有特定的角色。PKA是一种位于纤毛底部的Shh信号的强负调控因子。遗传学和细胞生物学实验将测试PKA是否需要定位到纤毛底部才能发挥作用，PKA是否控制纤毛的贩运和 Shh是否控制PKA活动。针对Hedgehog依赖性疾病和肿瘤的新疗法的开发将取决于对这些信号机制的深入理解。纤毛是以中心体为模板的，破坏中心体的人类遗传病会导致小头畸形。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突变胚胎缺乏中心粒、纤毛和中心体。对Sas4突变表型的分析将确定中心体是否调节信号、细胞分裂、细胞迁移或细胞生存。数据表明，去除P53可以挽救Sas4胚胎的早期致死性，实验将确定Sas4突变体中激活的P53依赖的通路。为了确定纤毛和中心体在发育中的大脑模式和细胞行为中的作用，我们将比较形成纤毛所需的Sas4或Ift88的条件性遗传缺失所引起的表型。模式、增殖、皮质组织和细胞死亡将在缺乏发育中大脑中纤毛或中心体的突变体中进行分析。在子宫内，GFP标记的Cre的电穿孔将被用来有条件地删除大脑中的Sas4和Ift88，然后跟踪缺乏纤毛或中心体的单个细胞的命运。这些实验将确定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。

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.