Genetic Analysis of Neuromuscular Junction Formation

描述（由申请人提供）：调节神经肌肉接头（NMJ）处乙酰胆碱受体（AChR）定位的分子过程需要聚集蛋白，一种由运动神经元分泌的因子。这一过程涉及各种其他分子，但对聚集蛋白引发的肌肉信号传导过程知之甚少。我们正在研究这一过程中的斑马鱼，这是脊椎动物的遗传分析，以确定重要的基因的生物过程和分析这些基因的体内功能。我们产生了斑马鱼ennui突变，其中AChRs被错误定位，并确定ennui基因编码LRP 4，在哺乳动物中需要适当的AChRs聚类。我们建议使用倦怠突变体，以更好地了解LRP 4的脊椎动物NMJ的形成在体内的作用。我们分离出了在NMJ处显示出降低的电生理反应的可行的倦怠突变。反应降低是由于突触AChRs的急剧减少和高水平的AChRs错误定位到肌肉末端。突变表型是细胞自主的，外源聚集蛋白诱导AChR簇在野生型肌肉，但不是在倦怠肌。这些结果表明，ennui基因编码的肌肉因子所需的聚集蛋白诱导的本地化AChRs的NMJ。结合基因定位和基因组分析，确定该基因为lrp 4。LRP 4是低密度脂蛋白受体家族的成员，由早期肌肉表达。虽然lrp 4最近被发现是在小鼠中的AChRs的适当定位的关键，有很少知道LRP 4如何可能介导聚集蛋白信号，以及它如何可能与聚集蛋白启动的信号通路的其他充分研究的组件相互作用。我们建议探索这些问题的实验，利用斑马鱼的优势，检查体内基因功能。目标1：我们将通过产生抗体和/或荧光标记的LRP 4的表达来检查LRP 4如何在肌肉中分布，并观察LRP 4是否与其他已知的NMJ组分共定位。目标二：我们将分析LRP 4如何调节神经乙酰胆碱受体簇，形成神经支配前的组合反义敲除和表达的特定形式的LRP 4。目标3：我们将确定LRP 4是否是一种聚集因子，并了解LRP 4和MuSK（聚集蛋白受体复合物的一个组成部分）在功能上是如何相关的。目的4：我们将分析LRP 4和MuSK的相互作用如何影响NMJ的体内发育。公共卫生相关性：斑马鱼突变体可以作为人类神经肌肉疾病，如重症肌无力和先天性肌无力综合征的动物模型。倦怠突变体的表型让人联想到患有这些疾病的患者中所见的肌肉无力和AChR缺陷。这使得lrp 4成为病因不明的神经肌肉接头疾病患者的候选基因。无聊突变体是可行的这一事实使它们更类似于人类疾病，并可用于测定治疗剂。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Zebrafish mutants could serve as animal models for human neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndromes. The phenotype of ennui mutants is reminiscent of the muscle weakness and AChR deficiencies seen in patients afflicted with these diseases. This makes lrp4 a candidate gene for neuromuscular junction disorders in patients of unknown etiology. The fact that ennui mutants are viable makes them more analogous to human disorders and useful for assaying therapeutic agents.