Synaptic target selection in Drosophila

描述（由申请人提供）：果蝇中的遗传筛选鉴定了许多细胞表面和分泌（CSS）蛋白，这些蛋白目前作为脊椎动物和无脊椎动物系统中轴突导向的调节剂被深入研究。该提案描述了一种遗传筛选的CSS蛋白质，作为突触的目标标签在胚胎/幼虫神经肌肉系统。该系统是理想的检查目标标记机制，因为它只包含36个运动神经元和30个肌肉目标，并具有不变的神经支配模式。每个被识别的运动神经元支配特定的肌肉纤维。虽然在这个系统中许多调控轴突导向的基因已经被确定，但我们对运动轴突如何识别单个肌肉纤维作为目标知之甚少。为了解决这个问题，我们首先定义了CSS蛋白，当它们在所有肌纤维上过度表达时，它们会导致轴突错误定位。我们通过构建果蝇中编码CSS蛋白的所有基因的数据库来做到这一点，这些基因可能参与细胞识别事件。然后，我们搜索了所有现有的含有UAS（GAL 4结合位点）的（“EP样”）元件系的集合，以找到这些CSS基因的上游插入物，这些CSS基因可用于通过将它们与GAL 4“驱动”系杂交来赋予组织特异性的高水平表达。我们获得了EP样插入，可以驱动数据库中979个基因中的410个，或超过40%的假定细胞识别库。我们将每个品系与泛肌肉GAL 4驱动器杂交，并通过抗体染色和共聚焦显微镜检查F1后代幼虫。我们发现了30个基因，它们在所有肌肉上的表达导致高密度轴突错误定位表型，但不会扰乱肌肉结构。六个基因是在一个特定的家庭编码蛋白质的胞外结构域含有富含亮氨酸的重复序列（LRR），这是蛋白质相互作用模块。该提案描述了评估肌肉中表达的四种LRR蛋白的功能的实验，这些蛋白似乎起到突触靶标签的作用，并确定LRR家族是否编码额外的靶标签。第一个具体目标涉及Tartan（Trn）和Caps（Caps）蛋白。trn和caps的功能丧失表型表明它们在胚胎中以部分冗余的方式发挥作用。在幼虫中，肌肉12上的Trn或Caps的选择性表达仅产生靶向特异性的改变。我们将确定在单个肌肉或所有肌肉中通过敲低Trn和Caps产生的功能丧失（LOF）幼虫表型。我们还将尝试开发一种标记幼虫中单个运动轴突的方法，以便我们可以观察遗传扰动如何影响单个已识别轴突的靶向。具体目标2和3涉及两个“新基因”，即CG 14351/haf和CG 8561。我们已经使用遗传和RNAi分析表明，这些基因编码的蛋白质是腹外侧肌肉正常神经支配所必需的。我们将在这些基因中进行无效突变，并进行遗传相互作用筛选，以找到CG 14351/Haf信号通路的组成部分。我们还将确定是否CG 8561，哺乳动物IGF-1结合蛋白的直系同源物，是胰岛素/IGF-1信号通路的一个组成部分。最后的具体目标描述了检查整个LRR家族的实验，以确定它是否编码其他肌肉靶标签。为此，我们将构建UAS-cDNA构建体，获得或制备41个LRR基因的RNAi株系，并评估其在幼虫中的表型。对于所有产生表型的基因，我们将绘制轴突生长期间肌纤维中它们的表达模式图。这一信息将使我们开始结合联合收割机LRR蛋白扰动，敲低特定肌肉上的多个基因，以检查肌肉纤维是否被标记为通过表达LRR蛋白的特定集合来靶向。

DESCRIPTION (provided by applicant): Genetic screens in Drosophila identified many of the cell-surface and secreted (CSS) proteins that are intensively studied today as regulators of axon guidance in both vertebrate and invertebrate systems. This proposal describes a genetic screen for CSS proteins that function as synaptic target labels in the embryonic/larval neuromuscular system. This system is ideal for examination of target labeling mechanisms, because it contains only 36 motor neurons and 30 muscle targets and has an invariant innervation pattern. Each identified motor neuron innervates a specific muscle fiber. Although many genes that regulate axon guidance in this system have been identified, we know very little about how individual muscle fibers are recognized as targets by motor axons. To address this problem, we first defined CSS proteins that cause axonal mistargeting when they are overexpressed on all muscle fibers. We did this by constructing a database of all genes in Drosophila that encode CSS proteins likely to be involved in cell recognition events. We then searched through all the existing collections of UAS (GAL4 binding site)-containing ('EP-like') element lines to find insertions immediately upstream of these CSS genes that could be used to confer tissue-specific, high-level expression by crossing them to GAL4 "driver" lines. We obtained EP-like insertions that can drive 410 of the 979 genes in the database, or over 40% of the putative cell recognition repertoire. We crossed each line to a pan-muscle GAL4 driver and examined F1 progeny larvae by antibody staining and confocal microscopy. We found 30 genes whose expression on all muscles causes high-penetrance axonal mistargeting phenotypes but does not perturb muscle structure. Six of the genes are in a specific family encoding proteins with extracellular domains containing leucine-rich repeats (LRRs), which are protein interaction modules. This proposal describes experiments to assess the functions of four LRR proteins that are expressed in muscles and appear to function as synaptic target labels, and to determine if the LRR family encodes additional target labels. The first specific aim concerns the Tartan (Trn) and Capricious (Caps) proteins. Loss-of- function phenotypes for trn and caps suggest that they function in a partially redundant manner in the embryo. In larvae, selective expression of Trn or Caps on muscle 12 only produces alterations in targeting specificity. We will determine the loss-of-function (LOF) larval phenotypes generated by knockdown of both Trn and Caps in a single muscle or in all muscles. We will also attempt to develop a method for labeling single motor axons in larvae, so that we can observe how genetic perturbations affect targeting of individual identified axons. Specific aims 2 and 3 concern two "new genes", CG14351/haf and CG8561. We have used genetic and RNAi analysis to show that the proteins encoded by these genes are required for the normal innervation of ventrolateral muscles. We will make null mutations in these genes and conduct a genetic interaction screen to find components of the CG14351/Haf signaling pathway. We will also determine whether CG8561, the ortholog of a mammalian IGF-1 binding protein, is a component of the insulin/IGF-1 signaling pathway. The final specific aim describes experiments to examine the entire LRR family to determine if it encodes other muscle target labels. To do this, we will make UAS-cDNA constructs and obtain or make RNAi lines for 41 LRR genes and assess their phenotypes in larvae. For all genes producing phenotypes, we will then make a map of their expression patterns in muscle fibers during the period of axonal outgrowth. This information will allow us to begin to combine LRR protein perturbations, knocking down multiple genes on specific muscles, in order to examine whether muscle fibers are labeled for targeting by expression of specific ensembles of LRR proteins.