Slit and RPTP Signaling in Retinal Axon Guidance

描述(申请人提供)：我对分子感兴趣 将轴突引向正确目标的机制。对轴突靶点的洞察 选择机制可能会帮助我们保护或重建神经元连接 因疾病或伤害而受损的。我的实验室用的是果蝇的眼睛 光感受器(R细胞)，研究轴突如何选择正确的目标。这个 不同R细胞亚型的轴突投射到大脑皮质不同层的靶点 大脑。指定R细胞轴突靶向的信号是未知的，以及如何 这些信号使R细胞轴突停止在正确的靶层，这一点尚不清楚。 通过基因筛查，我们发现了两个基因的突变，Sit和 Ptp69，以相关方式扰乱目标选择。狭缝编码一个 可作为信号引导轴突的细胞外蛋白，以及Ptp69d 编码一种受体蛋白酪氨酸磷酸酶。我们将使用Sit和Ptp69d来 探讨决定R细胞轴突靶点选择的机制。我们会： 1.研究Sit在R细胞轴突靶点选择中的作用。狭缝功能损失 使一种亚型的R细胞轴突生长通过其正常的靶层 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 狭缝信号的位置和它在其中发挥作用的细胞 和分子实验。我们将测试缝隙对R细胞轴突行为的影响 无论是在体内，通过异位表达，还是在体外，使用培养的R细胞。 2.鉴定Ptp69d的底物。Ptp69d功能丧失导致R细胞轴突 终止于不合适的目标层。Ptp69d在一个 依赖磷酸酶的方式，但其底物尚不清楚。我们将确定 用Ptp69d生化方法从苍蝇神经系统中提取候选底物 底物陷阱突变体，可以结合但不能使其靶标去磷酸化。我们 将进一步研究在以下情况下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和 与Ptp69d的生化相互作用及其在R细胞轴突靶向中的作用。 3.研究Sit、Ptp69d和R细胞中ROBO家族成员之间的相互作用 轴突目标选择。缝隙受体Robo和Robo2的过表达 导致类似于狭缝的R细胞轴突靶层选择缺陷 功能丧失。我们将检查ROBO家族的功能丧失突变 R细胞轴突靶向缺陷的成员并检查潜在的相互作用 Sit、Ptp69d和ROBO家族成员在R细胞轴突靶点选择中的作用。 我们还将研究潜在的下游信号通路 这些基因与潜在的下游效应器之间的相互作用。

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.