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理解取得了很大进展，但几种关键蛋白的作用仍不清楚。拟议的实验将利用斑马鱼遗传学和发育提供的独特优势来确定rapsyn，乙酰胆碱受体，MuSK和β-肌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中提供了超过哺乳动物的巨大优势。使用体外哺乳动物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。体内研究受到了小鼠敲除后不能在突触形成期存活的限制。 例如，在受体敲除的情况下，后果是如此严重，以至于没有研究能够解决无受体发育的后果。相比之下，已经在斑马鱼的突变系中鉴定出乙酰胆碱受体、rapsyn和MuSK的功能性敲除。这些鱼最初是根据游泳异常来识别的，这些游泳异常反映了这些关键突触蛋白中的每一种被敲除的直接后果。这种分析在斑马鱼中是可能的，因为与哺乳动物不同，这些突变动物在突触形成完成后死亡，并且可以评估动物行为。迄今为止，我们的研究结果揭示了乙酰胆碱受体和rapsyn在控制突触发育和功能中最意想不到的作用。特别是，我们发现该受体可能在将rapsyn定位于突触中起关键作用，rapsyn在调节受体功能中起关键作用。此外，我们的研究为人类神经肌肉疾病提供了新的预测，其中一种已在罕见重症肌无力患者身上得到证实。我们相信，这个模型系统将通过其许多独特的优势，解决一些突出的悖论，涉及信号分子在突触形成中的作用。

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.