Synaptic target selection in Drosophila

果蝇的突触目标选择

基本信息

批准号：
8019193
负责人：
KAI G ZINN
金额：
$ 9.07万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8019193
关键词：
Address Affect Antibodies Autistic Disorder Axon Back Basic Science Binding Sites Birth Brain Cell surface Cells Collection Complementary DNA Complex Confocal Microscopy Cues Databases Development Disease Drosophila genus Elements Embryo Event Extracellular Domain Family Genes Genetic Genetic Screening Health Human IGF-1 Signaling Pathway Individual Insulin Insulin Signaling Pathway Insulin-Like Growth-Factor Binding Protein 1 Insulin-Like-Growth Factor I Receptor Invertebrates Knowledge Label Larva Leucine-Rich Repeat Link Maps Methods Motor Motor Neurons Muscle Muscle Fibers Mutation Nature Neuromuscular Junction Neurons Orthologous Gene Paper Pattern Penetrance Phenotype Phosphotransferases Protein Family Proteins RNA Interference Research Personnel Research Project Grants Schizophrenia Signal Pathway Specificity Staining method Stains Synapses System Tissues Work abstracting axon guidance gain of function insulin signaling interest knock-down leucine-rich repeat protein loss of function member muscular structure nerve supply neuromuscular system null mutation overexpression postsynaptic protein function receptor research study selective expression

项目摘要

Synaptic target selection in Drosophila Abstract: 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.

果蝇的突触目标选择抽象的：果蝇中的遗传筛选鉴定了许多细胞表面和分泌（CSS）蛋白如今，该脊椎动物和无脊椎动物系统。该建议描述了CSS蛋白的遗传筛选作为胚胎/幼虫神经肌肉系统中的突触靶标记。该系统是理想的选择检查目标标记机制，因为它仅包含36个运动神经元和30个肌肉目标，具有不变的神经支配模式。每个确定的运动神经元支配特定的肌肉纤维。尽管许多基因调节了轴突指导已经确定了系统，我们对单个肌肉纤维的了解一无所知被电机轴突识别为靶标。为了解决这个问题，我们首先定义了引起轴突误解的CSS蛋白当它们在所有肌肉纤维上过表达时。我们通过构建所有数据库来做到这一点编码CSS蛋白的果蝇中的基因可能参与细胞识别事件。然后，我们搜索了所有现有的UAS（GAL4绑定站点）的集合（'ep-like'）元素线，在这些CSS基因的上游找到插入通过将它们交叉到GAL4“驱动器”线上，用于赋予组织特异性的高级表达。我们获得了类似EP的插入，可以在数据库中驱动979个基因的410个或超过假定的细胞识别库中有40％。我们将每条线越过一个板穆斯勒gal4 通过抗体染色和共聚焦显微镜检查驱动器并检查了F1后代幼虫。我们找到30个基因，其在所有肌肉上的表达都会引起高渗透轴突误解表型，但不会扰动肌肉结构。六个基因在一个特定家族中用包含含亮氨酸重复量的细胞外域（LRR）编码蛋白质，蛋白质相互作用模块。该建议描述了评估功能的实验四种在肌肉中表达并似乎充当突触靶的LRR蛋白标签，并确定LRR家族是否编码其他目标标签。第一个特定目的涉及格子呢（TRN）和反复无常（CAPS）蛋白质。丧失 TRN和CAP的功能表型表明它们以部分冗余的方式起作用在胚胎中。在幼虫中，肌肉12上TRN或帽的选择性表达仅产生定位特异性的改变。我们将确定功能丧失（LOF）幼虫单个肌肉或所有肌肉中TRN和帽子都会产生的表型。我们还将尝试开发一种标记幼虫中单运动轴突的方法，以便我们可以观察遗传扰动如何影响靶向单个已识别轴突的靶向。具体目标2和3涉及两个“新基因”，CG14351/HAF和CG8561。我们有使用的遗传和RNAi分析表明，需要这些基因编码的蛋白质对于腹外侧肌肉的正常神经。我们将在这些基因中进行无效突变并进行遗传相互作用屏幕以查找CG14351/HAF信号的组件路径。我们还将确定CG8561是否是哺乳动物IGF-1结合的直系同源蛋白质是胰岛素/IGF-1信号通路的组成部分。最终的具体目的描述了研究整个LRR家族的实验确定它是否编码其他肌肉目标标签。为此，我们将制作UAS-CDNA 构造并获得或制造41个LRR基因的RNAi线，并评估其表型幼虫。对于所有产生表型的基因，我们将制作其表达的地图轴突产物时期的肌肉纤维模式。这些信息将使我们能够开始结合LRR蛋白扰动，击倒特定肌肉的多个基因，为了检查是否通过表达特异性标记肌肉纤维的靶向 LRR蛋白质的集合。