Genetic Analysis of Neuromuscular Junction Formation

项目总结 调节乙酰胆碱受体(AChRs)定位的分子过程 神经肌肉接头(NMJ)需要集聚蛋白，这是一种由运动神经元分泌的因子。这一过程包括 各种其他分子，但对集聚蛋白在肌肉中启动的信号传递过程知之甚少。我们 正在研究斑马鱼的这一过程，斑马鱼是脊椎动物，可以进行遗传分析 生物过程中重要基因的鉴定和体内功能的分析 基因。我们产生了斑马鱼Eennui突变，其中AChRs被错误定位，并鉴定了 Enui基因作为编码LRP4的基因，在哺乳动物中是AChRs正确聚集所必需的。我们 建议使用Eennui突变体来更好地了解LRP4在体内对形成 脊椎动物NMJ。 我们分离出了一个活性的ENONUI突变，该突变显示出在 《NMJ》。反应的降低是由于突触AChRs的急剧减少和高水平的 AChRs错误定位于肌末梢。突变的表型是细胞自主的，并且是外源的 集聚蛋白在野生型肌肉中诱导AChR簇，但在疲劳型肌肉中不诱导。这些结果表明， Ennui基因编码了一种肌肉因子，这是agrin诱导AChRs定位到NMJ所必需的。 通过突变的遗传图谱和遗传图谱的结合，将ennui基因鉴定为lrp4。 基因组分析。LRP4是低密度脂蛋白受体家族的成员，由 早期肌肉。尽管最近发现lrp4对于AChRs在 对于LRP4如何介导集聚蛋白信号，以及它可能如何与 其他已被充分研究的集聚蛋白启动的信号通路的组成部分。我们建议探讨这些问题 利用斑马鱼的优势来检测体内基因功能的实验存在问题。 目标1：我们将通过产生抗体和/或表达LRP4来研究LRP4如何在肌肉中分布 荧光标记的LRP4，看看LRP4是否与其他已知的NMJ组分共定位。 目的2：我们将分析LRP4是如何调节神经AChR簇在神经支配前形成的 反义敲除和特定形式的LRP4表达的组合。 目标3：我们将确定LRP4是否是一个聚集因素，并看看LRP4和Musk是如何 集聚蛋白受体复合体的组成成分，在功能上是相关的。 目的：研究LRP4和麝香的相互作用如何影响NMJ的体内发育。

PROJECT SUMMARY The molecular process regulating the localization of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ) requires agrin, a factor secreted by motor neurons. This process involves various other molecules, but the signaling process in muscles initiated by agrin is poorly understood. We are examining this process in zebrafish which are vertebrates amenable to genetic analysis for the identification of genes important for a biological process and analysis of the in vivo function of these genes. We generated the zebrafish ennui mutation in which AChRs are mislocalized and identified the ennui gene as one encoding for LRP4 that in mammals is required for proper clustering of AChRs. We propose to use the ennui mutants to better understand the in vivo role of LRP4 for the formation of the vertebrate NMJ. We isolated the viable ennui mutation that showed a decreased electrophysiological response at the NMJ. The reduced response was due to a dramatic decrease in synaptic AChRs and high levels of AChRs mislocalized to the ends of muscles. The mutant phenotype is cell autonomous, and exogenous agrin induced AChR clusters in wildtype muscles but not in ennui muscles. These results suggested that the ennui gene encoded for a muscle factor required for agrin-induced localization of AChRs to the NMJ. The ennui gene was identified as lrp4 by a combination of genetic mapping of the mutation and genomic analysis. LRP4 is a member of the low-density lipoprotein receptor family and is expressed by early stage muscles. Although lrp4 was recently found to be critical for proper localization of AChRs in mice, there is little known about how LRP4 may mediate agrin signaling and how it might interact with other well studied components of the agrin-initiated signaling pathway. We propose to explore these issues with experiments that utilize the advantages of zebrafish for examining in vivo gene function. Aim 1: We will examine how LRP4 is distributed in muscle by generating antibodies and/or expression of fluorescently labeled LRP4 and see if LRP4 co-localizes with other known NMJ components. Aim 2: We will analyze how LRP4 regulates aneural AChR clusters that form prior to innervation by a combination of antisense knockdowns and expression of specific forms of LRP4. Aim 3: We will establish whether LRP4 is an aggregation factor and see how LRP4 and MuSK, a component of the agrin receptor complex, are functionally related. Aim 4: We will assay how the interaction of LRP4 and MuSK affects the in vivo development of the NMJ.