Axon Guidance Mechanisms in Drosophila

描述（申请人提供）：发育中的神经元的生长锥识别环境中的引导信号，并将其转化为生长方向的变化，以寻找并与合适的靶细胞发生突触。在果蝇中，通过中线投射的所有神经元的生长锥所做出的前与后连接（分别为AC与PC）的二元选择是由脱轨（Drl）控制的，脱轨（Drl）是一种非典型受体酪氨酸激酶，它的配体Wnt5是分泌信号分子Wnt家族的一员。Wnt5由PC神经元分泌，作为一种化学驱避剂，使表达drl的AC生长锥远离PC。我们在这个项目中的目标是了解Wnt5/Drl引导机制是如何工作的。我们将开发一种新的果蝇神经元体外生长锥转化实验，以测试Wnt5是否直接作用于表达drl的神经元的生长锥。Wnt5在体内发生蛋白水解裂解，但这一事件在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在何时何地被切割，并测试切割在Wnt5功能中的作用。Drl下游在生长锥内转导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，如卷曲受体和Disheveled，是否在轴突引导中与Drl一起起作用。为了进一步确定Drl下游的信号成分，我们将确定两个基因的身份，我们已经证明，当删除Drl功能时，它们会强烈抑制Drl功能。此外，我们将表达一种功能性flag标记的Drl，以免疫沉淀特异性结合Drl细胞质结构域的蛋白质，并测试这些蛋白质是否结合体内信号所需的Drl细胞质结构域区域。最后，我们在果蝇身上的工作提出了Wnt/Drl轴突引导机制的普遍性问题。我们将通过检测Ryk基因敲除小鼠的运动轴突投射，来测试Drl的哺乳动物同源物Ryk的引导作用。相关性：正常的大脑功能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。这个项目的研究将提供对神经细胞如何正确连接的见解。

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.