Slit and RPTP Signaling in Retinal Axon Guidance

描述（由申请人提供）：我对分子 将轴突导向正确目标的机制对轴突靶点的见解 选择机制可以帮助我们保护或重建神经元连接 因疾病或受伤而受损。我的实验室用果蝇的眼睛 光感受器（R细胞）来研究轴突如何选择正确的目标。的 不同R细胞亚型的轴突投射到不同层的靶点， 个脑袋指定R细胞轴突靶向的信号是未知的，以及如何 这些信号在正确的靶层停止R细胞轴突是不理解的。 通过基因筛选，我们发现两个基因的突变， ptp 69，以相关的方式破坏目标选择。Slit编码一个 细胞外蛋白，可以作为信号引导轴突，和Ptp 69 d 编码受体蛋白酪氨酸磷酸酶。我们将使用slit和Ptp 69 d来 研究决定R细胞轴突靶向选择的机制。我们将： 1.检查狭缝在R细胞轴突目标选择中的作用。狭缝功能丧失 导致一种亚型的R细胞轴突穿过它们的正常靶层生长 并进入其他目标层。我们将检验狭缝是一个信号的假设 控制R细胞轴突的层特异性终止。我们将确定 Slit信号的位置和它在其中发挥作用的细胞使用遗传学方法来确定。 和分子实验。我们将测试狭缝对R细胞轴突行为的影响 通过异位表达在体内和使用培养的R细胞在体外进行。 2.鉴定Ptp 69 d的底物。Ptp 69 d功能丧失导致R细胞轴突 终止于不合适的目标层。ptp 69 d在一个 磷酸酶依赖的方式，但其底物是未知的。我们将确定 候选底物从苍蝇神经系统生化使用Ptp 69 d 能够结合但不能使其靶去磷酸化底物陷阱突变体。我们 将进一步研究那些破坏轴突靶向的候选底物， 用RNAi抑制。这些候选人将接受基因检测， 与Ptp 69 d的生物化学相互作用以及在R细胞轴突靶向中的作用。 3.检查R-cell中slit，Ptp 69 d和robo家族成员之间的相互作用 轴突目标选择。Slit受体Robo和Robo 2的过表达 导致R细胞轴突靶层选择缺陷，类似于狭缝 功能丧失我们将研究机器人家族中的功能缺失突变， 成员的R细胞轴突靶向缺陷，并检查潜在的相互作用 slit、Ptp 69 d和robo家族成员在R细胞轴突靶向选择中的差异。 我们还将研究潜在的下游信号通路， 这些基因和潜在的下游效应子之间的相互作用。

DESCRIPTION (provided by applicant): I am interested in the molecular mechanisms directing axons to their correct targets. Insights into axon target selection mechanisms may help us protect or reestablish neuronal connections compromised by illness or injury. My lab uses the Drosophila melanogaster eye's photoreceptors (R-cells) to study how axons choose their correct targets. The axons of different R-cell subtypes project to targets in distinct layers of the brain. The signals that specify R-cell axon targeting are unknown, and how these signals stop R-cell axons at the correct target layer is not understood. Through genetic screens, we have found that mutations in two genes, slit and Ptp69, disrupt target selection in a related fashion. Slit encodes an extracellular protein that can act as a signal to guide axons, and Ptp69d encodes a receptor protein tyrosine phosphatase. We will use slit and Ptp69d to investigate the mechanisms determining R-cell axon target choice. We will: 1. Examine slit's role in R-cell axon target selection. Slit loss-of-function causes one subtype of R-cell axons to grow through their normal target layer and into other target layers. We will test the hypothesis that Slit is a signal controlling layer-specific termination of R-cell axons. We will identify the location of the Slit signal and the cells in which it functions using genetic and molecular experiments. We will test Slit's effect on R-cell axon behavior both in vivo, through ectopic expression, and in vitro, using cultured R-cells. 2. Identify substrates of Ptp69d. Ptp69d loss-of-function causes R-cell axons to terminate in inappropriate target layers. Ptp69d acts in a phosphatase-dependent fashion, but its substrates are unknown. We will identify candidate substrates from the fly nervous system biochemically using a Ptp69d substrate-trap mutant that can bind but not dephosphorylate its targets. We will further study those candidate substrates that disrupt axon targeting when inhibited with RNAi. These candidates will be tested for genetic and biochemical interactions with Ptp69d and roles in R-cell axon targeting. 3. Examine interactions between slit, Ptp69d, and robo-family members in R-cell axon target selection. Overexpression of the Slit receptors Robo and Robo2 causes R-cell axon target layer selection defects similar to slit loss-of-function. We will examine loss-of-function mutations in robo-family members for R-cell axon targeting defects and examine potential interactions between slit, Ptp69d, and robo-family members in R-cell axon target selection. We will also examine potential downstream signaling pathways by examining interactions between these genes and potential downstream effectors.