Axon Guidance Mechanisms in Drosophila

描述(申请人提供)：为了找到合适的目标细胞并与之突触，发育中的神经元的生长锥体识别环境中的指导信号，并将它们转化为生长方向的变化。在果蝇中，由横跨中线的所有神经元的生长锥体做出的前连合和后连合(AC和PC)的二元选择是由非典型受体酪氨酸激酶(DRL)及其配体Wnt5控制的，Wnt5是Wnt分泌信号分子家族的成员。WNT5是由PC神经元分泌的，作为一种化学抵抗剂将表达DRL的AC生长锥排除在PC之外。我们在这个项目中的目标是了解WNT5/DRL指导机制是如何发挥作用的。我们将建立一种新的果蝇神经元体外生长锥体翻转实验，以测试WNT5是否直接作用于表达DRL的神经元的生长锥体。WNT5在体内被蛋白水解性切割，但该事件在WNT5功能中所起的作用尚不清楚。利用表位标签，我们将确定WNT5在哪里以及何时被切割，并测试切割在WNT5功能中的作用。DRL下游在生长锥内传递排斥信号的信号通路尚不清楚。由于DRL是一种新的Wnt受体，我们将从基因上测试Wnt信号的已知成分，如Frizzleed受体和Disheveled，是否在轴突引导下与DRL一起发挥作用。为了进一步确定DRL下游的信号成分，我们将确定我们已经证明在缺失时强烈抑制DRL功能的两个基因的同一性。此外，我们将表达一个功能性的标记版本的DRL来免疫沉淀与DRL细胞质结构域特异结合的蛋白，并测试这些蛋白是否与体内信号传递所需的DRL细胞质结构域区域结合。最后，我们在果蝇中的工作提出了一个问题，即WNT/DRL轴突引导机制可能有多普遍。我们将通过检测Ryk基因敲除小鼠的运动轴突投射来测试Ryk的指导作用，Ryk是DRL的哺乳动物同源物。相关性：正常运作的大脑依赖于神经细胞之间特定连接的产生，脊髓损伤后的功能恢复也是如此。该项目中的研究将为神经细胞如何正确连接提供洞察力。

DESCRIPTION (provided by applicant): To find and synapse with their appropriate target cells, the growth cones of developing neurons recognize guidance cues in their environment and transduce them into changes in direction of growth. In Drosophila, the binary choice of anterior vs. posterior commissure (AC vs. PC, respectively) made by the growth cones of all neurons that project across the midline, is controlled by Derailed (Drl), an atypical receptor tyrosine kinase, and its ligand Wnt5, a member of the Wnt family of secreted signaling molecules. Wnt5 is secreted by PC neurons and acts as a chemorepellent to keep the Drl-expressing AC growth cones out of the PC. Our goal in this project is to understand how the Wnt5/Drl guidance mechanism functions. We will develop a novel in vitro growth cone turning assay for Drosophila neurons to test whether Wnt5 acts directly on the growth cones of Drl-expressing neurons. Wnt5 is proteolytically cleaved in vivo, but the role this event plays in Wnt5 function is unknown. Using epitope tags, we will determine where and when Wnt5 is cleaved and test the role of cleavage in Wnt5 function. The signaling pathway downstream of Drl that transduces the repulsive signal within the growth cone is unknown. Since Drl is a novel Wnt receptor, we will genetically test whether known components of Wnt signaling, such as Frizzled receptors and Disheveled, function with Drl in axon guidance. To further identify signaling components downstream of Drl, we will determine the identity of two genes that we have shown to strongly suppress Drl function when deleted. In addition, we will express a functional Flag-tagged version of Drl to immunoprecipitate proteins that specifically bind to the Drl cytoplasmic domain and test whether these proteins bind to regions of the Drl cytoplasmic domain required for signaling in vivo. Finally, our work in Drosophila raises the question of how universal the Wnt/Drl axon guidance mechanism might be. We will test the guidance role of Ryk, the mammalian homologue of Drl, by examining motor axon projections in Ryk knockout mice. Relevance: A normal functioning brain relies on the generation of specific connections between nerve cells, as does functional recovery after spinal cord injury. The studies in this project will provide insight into how nerve cells become properly wired.