Regulation of N-cadherin trafficking during cortical development

在哺乳动物新皮层发育过程中，位于脑室区的神经前体细胞产生连续的神经元波，产生皮层板。新产生的神经元首先在室下区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突起而变成双极。这种从具有几个突起的多极细胞到双极形态的转变（多到双极的转变）先于双极神经元沿着放射状胶质细胞的基底突起向皮质板的放射状迁移。在多向双极过渡期间或在其径向迁移期间神经元的这种极化缺陷是神经发育障碍的主要原因。虽然许多信号通路已被确定，在原代培养的直接神经元极化少得多的是已知的因素，在体内调节多到双极转换。对显示神经元发育缺陷的突变体的分析表明，轴突的形成和引导过程是由不同的、遗传上可分离的途径控制的。一个指导皮层中多向双极转换的中枢通路通过小GTP酶Rap 1A和Rap 1B起作用。我们对Rap 1a和Rap 1b的条件性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，并指导多极性到双极性的转变。切片培养的活细胞成像表明，Rap 1是多极神经元极化所需的细胞自主性。Rap 1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，其决定了N-钙粘蛋白的细胞表面表达，这对于与放射状胶质细胞的相互作用和向双极形态学的过渡是必不可少的。在这个项目中，我们将研究的作用，Ral GEFs和磷脂酰肌醇-4-磷酸5-激酶的调节N-钙粘蛋白使用活细胞成像切片培养，以了解如何协同调节胞吐和胞吞作用，以及如何Rap 1冲击这种调节。

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.