Axon Guidance Mechanisms in Drosophila

为了找到合适的靶细胞并与之建立突触，发育中的神经元的生长锥会识别 引导他们在环境中的线索，并引导他们进入增长方向的变化。在果蝇中， 生长锥对前连合与后连合（分别为AC与PC）的二元选择 在所有投射到中线的神经元中，由一种非典型酪氨酸受体Derailed（Drl）控制 激酶及其配体Wnt 5，分泌信号分子的Wnt家族的成员。Wnt 5分泌 被PC神经元吸收，并充当化学排斥剂，使表达Drl的AC生长锥远离PC。我们 本项目的目标是了解Wnt 5/Drl指导机制的功能。我们将开发一个 一种新的果蝇神经元体外生长锥转动试验，以测试Wnt 5是否直接作用于果蝇神经元。 DRI表达神经元的生长锥。Wnt 5在体内被蛋白水解切割，但这一事件所起的作用 Wnt 5的功能未知。使用表位标签，我们将确定Wnt 5在何处和何时被切割， 测试切割在Wnt 5功能中的作用。Drl下游的信号传导途径，其转导 生长锥内的排斥信号是未知的。由于Drl是一种新的Wnt受体，我们将在基因上测试 Wnt信号传导的已知组分，如卷曲受体和Disheveled，是否与Drl一起起作用， 轴突导向为了进一步鉴定Drl下游的信号传导组分，我们将确定Drl下游的信号传导组分的身份。 我们已经证明，这两个基因在缺失时强烈抑制Dr 1功能。此外，我们将表达一个 功能性Flag标记形式的Dr 1，以免疫沉淀特异性结合Dr 1的蛋白质 细胞质结构域，并测试这些蛋白质是否结合所需的Drl细胞质结构域的区域。 用于体内信号传导。最后，我们在果蝇中的工作提出了一个问题，即Wnt/Drl轴突有多普遍， 引导机制可能是。我们将测试Ryk（Drl的哺乳动物同源物）的指导作用， 检查Ryk基因敲除小鼠的运动轴突投射。 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。该项目的研究将提供洞察如何 神经细胞会被正确连接

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.