Synapse Development in Zebrafish

描述(由申请人提供)：尽管我们对神经肌肉接头处突触发育的了解取得了很大进展，但几个关键蛋白质的作用仍然不清楚。拟议的实验将利用斑马鱼遗传和发育提供的独特优势来确定Rapsyn、乙酰胆碱受体、麝香和β-营养不良聚糖在体内突触形成中的作用。斑马鱼在体外和体内系统上都比哺乳动物具有巨大的优势。使用哺乳动物体外表达系统和培养的肌管的研究都因无法研究真正的突触而受到阻碍。体内研究一直受到小鼠敲除基因无法在突触形成期间存活的限制。例如，在受体敲除的情况下，后果是如此严重，以至于还没有研究能够解决无受体发育的后果。相比之下，在斑马鱼的突变系中，已经发现了乙酰胆碱受体、Rapsyn和麝香的功能性敲除。这些鱼最初是根据游泳异常来识别的，这些异常反映了这些关键突触蛋白中的每一种的敲除的直接后果。这种分析在斑马鱼身上是可能的，因为与它们的哺乳动物不同，这些突变的动物在突触形成完成后会很好地死亡，可以评估动物的行为。到目前为止，我们的发现揭示了乙酰胆碱受体和Rapsyn在控制突触发育和功能方面最意想不到的作用。特别是，我们发现该受体可能在将Rapsyn定位到突触中发挥关键作用，而且Rapsyn在调节受体功能方面发挥着关键作用。此外，我们的研究为人类神经肌肉疾病提供了新的预测，其中一项已在患有罕见形式的重症肌无力的患者上得到证实。我们相信，这个模型系统将通过其许多独特的优势，解决一些涉及信号分子在突触形成中的作用的突出悖论。

DESCRIPTION (provided by applicant): Despite the great advances made in our understanding of synapse development at the neuromuscular junction, the roles of several key proteins remain unclear. The proposed experiments will use the unique advantages offered by zebrafish genetics and development to identify the roles of rapsyn, acetylcholine receptor, MuSK and beta-dystroglycan in synapse formation in vivo. Zebrafish offers tremendous advantages over mammalian in vitro and in vivo systems. Studies using in vitro mammalian expression systems and cultured myotubes have both been hampered by the inability to study bona-fide synapses. The in vivo studies have been limited by the inability of the mouse knock-outs to survive through the period of synapse formation. In the case of receptor knock-out, for example, the consequences are so severe that no studies have been able to address the consequences of receptor-less development. By contrast, functional knock-outs of acetylcholine receptor, rapsyn, and MuSK have been identified in mutant lines of zebrafish. These fish were originally identified on the basis of swimming abnormalities that reflect direct consequences of knock-outs of each of these key synaptic proteins. This analysis is possible in zebrafish because, unlike their mammalian counterparts, these mutant animals die well after synapse formation is completed and the animal behavior can be assessed. To date our findings have revealed most unexpected roles for the acetylcholine receptor and for rapsyn in governing synapse development and function. In particular, we have found that the receptor likely plays a key role in localizing rapsyn to the synapse and rapsyn plays a critical role in regulating receptor function. Additionally, our studies have provided new predictions for human neuromuscular diseases, one of which has been confirmed on patients afflicted with rare forms of myasthenia gravis. We are confident that this model system will, through its many unique advantages, resolve some of the outstanding paradoxes involving the roles of signaling molecules in synapse formation.