Regulation of N-cadherin trafficking during cortical development

在哺乳动物新皮层发育过程中，位于脑室区的神经祖细胞产生连续的神经元波，形成皮层板。新生神经元首先在脑室下和中间区呈现多极形态。几个小时后，它们通过延长轴突和前导过程变成双极性。这种从具有多个突起的多极细胞到双极形态的转变（多极到双极的转变）先于双极神经元沿径向胶质细胞的基底突起向皮质板的径向迁移。在多极向双极过渡或径向迁移过程中，这种神经元极化的缺陷是神经发育障碍的主要原因。虽然许多信号通路已经被确定为在原代培养中直接的神经元极化，但对体内调节多极到双极转变的因素知之甚少。对神经元发育中出现缺陷的突变体的分析表明，轴突的形成和导联过程是由不同的、遗传上可分离的途径控制的。在皮质中，一个指导多极到双极转换的中心通路通过小的GTPases Rap1A和Rap1B起作用。我们对Rap1a和Rap1b条件双敲除小鼠的分析表明，它们对皮质发育至关重要。它们冗余地维持放射状胶质细胞的极性，并指导多极到双极的转变。切片培养的活细胞成像显示，Rap1在多极神经元中是细胞自主的极化所必需的。Rap1的一个重要功能是维持n -钙粘蛋白在细胞表面的定位。神经元的极化依赖于细胞内运输的重组，这决定了细胞表面n -钙粘蛋白的表达，这对于与径向胶质细胞的相互作用和向双极形态的转变至关重要。在这个项目中，我们将研究Ral gef和磷脂酰肌醇-4-磷酸5激酶在n -钙粘蛋白调控中的作用，利用细胞片培养的活细胞成像来了解胞外分泌和内吞作用是如何协调调节的，以及Rap1是如何影响这种调节的。

During the development of the mammalian neocortex neural progenitor cells located in the ventricular zone generate successive waves of neurons that give rise to the cortical plate. Newly generated neurons first assume a multipolar morphology in the subventricular and intermediate zone. After several hours, they become bipolar by extending an axon and a leading process. This transition from multipolar cells with several processes to a bipolar morphology (the multi-to-bipolar transition) precedes the radial migration of bipolar neurons along the basal processes of radial glia cells into the cortical plate. Defects in this polarization of neurons during the multi-to-bipolar transition or in their radial migration are a major cause of neurodevelopmental disorders. While many signaling pathways have been identified that direct neuronal polarization in primary cultures much less is known about the factors that regulate the multi-to-bipolar transition in vivo. The analysis of mutants that show defects in neuronal development indicates that the formation of axon and leading process is controlled by different, genetically separable pathways. A central pathway that directs the multi-to-bipolar transition in the cortex acts through the small GTPases Rap1A and Rap1B. Our analysis of conditional double knockout mice for Rap1a and Rap1b showed that they are essential for cortical development. They act redundantly to maintain the polarity of radial glia cells and direct the multi-to-bipolar transition. Live cell imaging of slice cultures showed that Rap1 is required cell-autonomously in multipolar neurons for polarization. An important function of Rap1 is the maintenance of N-cadherin localization at the cell surface. The polarization of neurons depends on a reorganization of intracellular trafficking that determines the cell surface expression of N-cadherin, which is essential for the interaction with radial glia cells and the transition to a bipolar mophology. In this project we will investigate the role of Ral GEFs and Phosphatidylinositol-4-phosphate 5-kinases for the regulation of N-cadherin using live cell imaging of slice cultures to understand how exocytosis and endocytosis are coordinately regulated and how Rap1 impinges on this regulation.