Axon Guidance Mechanisms in Drosophila

描述（由申请人提供）：为了找到合适的靶细胞并与其突触，发育中的神经元的生长锥识别其环境中的指导线索，并将其转化为生长方向的变化。在果蝇中，由投射穿过中线的所有神经元的生长锥做出的前连合与后连合（分别为AC与PC）的二元选择由非典型受体酪氨酸激酶Derailed（Drl）及其配体Wnt 5（分泌信号传导分子的Wnt家族的成员）控制。Wnt 5由PC神经元分泌，并充当化学排斥剂以将表达Drl的AC生长锥保持在PC之外。我们在这个项目中的目标是了解Wnt 5/Drl指导机制如何发挥作用。我们将开发一种新的果蝇神经元体外生长锥翻转试验，以测试Wnt 5是否直接作用于表达Drl的神经元的生长锥。Wnt 5在体内被蛋白水解切割，但该事件在Wnt 5功能中所起的作用尚不清楚。使用表位标签，我们将确定Wnt 5在何处以及何时被切割，并测试切割在Wnt 5功能中的作用。在生长锥内转导排斥信号的Drl下游的信号传导途径是未知的。由于Drl是一种新的Wnt受体，我们将在遗传学上测试Wnt信号传导的已知组分，如Frizzled受体和Disheveled，是否与Drl一起在轴突导向中起作用。为了进一步鉴定Drl下游的信号传导组分，我们将确定我们已经显示在缺失时强烈抑制Drl功能的两个基因的身份。此外，我们将表达一个功能性的Flag标记版本的Drl，以免疫沉淀特异性结合到Drl胞质结构域的蛋白质，并测试这些蛋白质是否结合到体内信号传导所需的Drl胞质结构域的区域。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。我们将通过检查Ryk敲除小鼠中的运动轴突投射来测试Ryk（Drl的哺乳动物同源物）的指导作用。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。该项目的研究将深入了解神经细胞如何正确连接。

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.