Axon Guidance Mechanisms in Drosophila

为了找到合适的靶细胞并与之突触，发育中的神经元的生长锥体识别 在他们的环境中引导线索，并将其转化为生长方向的变化。在果蝇身上， 生长锥对前连合和后连合的二元选择(分别为AC和PC) 在横跨中线投射的所有神经元中，受脱轨(DRL)控制，这是一种非典型的受体酪氨酸 激酶及其配体Wnt5，Wnt5是分泌信号分子Wnt家族的成员。WNT5被分泌出来 通过PC神经元，并起到化学抵抗剂的作用，使表达DRL的AC生长锥体远离PC。我们的 本项目的目标是了解WNT5/DRL指导机制是如何运作的。我们将开发一种 一种新的果蝇神经元体外生长锥体翻转实验，以检测WNT5是否直接作用于 DRL表达神经元的生长锥体。WNT5在体内是蛋白水解性的，但这一事件所起的作用 在WNT5中，函数未知。使用表位标签，我们将确定WNT5在哪里被切割，何时被切割 测试切割在Wnt5功能中的作用。DRL下游的信号转导通路 生长锥体内的排斥信号未知。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，是否与DRL一起在 轴突引导。为了进一步确定DRL下游的信号组件，我们将确定 我们已经证明了两个基因在缺失时会强烈抑制DRL功能。此外，我们还将表示一个 功能标志标记的DRL版本，用于免疫沉淀与DRL特异性结合的蛋白质 并测试这些蛋白是否与DRL胞质结构域所需的区域结合 在活体内发出信号。最后，我们在果蝇身上的研究提出了WNT/DRL轴突的普遍性问题 引导机制可能是。我们将通过以下方式测试DRL的哺乳动物同源物Ryk的指导作用 检测Ryk基因敲除小鼠的运动神经轴突投射。 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。这个项目中的研究将提供对如何 神经细胞会被适当地连接起来。

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.