Wnt Signaling in Synaptic Prepattern formation and Motor Axon Guidance

描述(由申请人提供)：本项目的首要目标是确定神经肌肉连接最初形成的发育机制。神经肌肉接头发展过程中的一个关键分子是跨膜受体unPluted/Musk。非插拔/麝香在肌肉纤维中特异表达，控制神经肌肉连接形成的两个方面：在神经到达之前，肌肉纤维上的“预先形成的”乙酰胆碱受体(AChRs)聚集，以及运动神经引导到其适当的肌肉靶点。在发育后期，它还指导成熟的神经肌肉突触的形成。有了以下三个目标，我建议确定非插拔/马斯克如何在空间上协调“预先设计的”AChR的聚集和生长锥体引导到肌肉中心。首先，我将研究多个Wnt信号是否激活未插入的/麝香受体。我们之前已经确定wnt11r是一个非插拔/麝香配体，但我们怀疑wnt4a也在非插拔/麝香激活中发挥作用。我将使用遗传相互作用和体内结合实验相结合的方法来检查两个WNT之间的合作程度以及它们结合未插拔/马斯克的能力。其次，我将确定哪些分子分子位于UnPlugged/Musk的下游，以及该信号级联是否类似于Wnt平面细胞的极性级联。平面细胞极性样通路的激活可以在肌肉纤维中建立一个中央区，该中心区决定了AChR簇最初形成的位置，以及当它们进入肌肉区域时运动轴突延展到哪里。我将通过扰乱它们在肌肉纤维中的功能来调查各种平面细胞极性成分的参与，并确定这是否会导致未插拔的AChR聚集和轴突引导错误。第三，在包括果蝇翼盘在内的许多系统中，细胞极性最初是通过平面细胞极性途径的各个成员的不对称定位来建立的。我建议用活体共聚焦成像技术来确定这些平面细胞极性成分在肌肉纤维中的定位，并确定Wnt信号的缺失是否会影响这种定位。公共卫生相关性：这项研究与人类疾病的研究直接相关，因为在神经肌肉疾病重症肌无力中，马斯克是抗体介导的自身免疫攻击的目标。此外，麝香通路成分的突变是人类先天性肌无力综合征的原因。

DESCRIPTION (provided by applicant): The overlying goal of this project is to determine the developmental mechanisms by which neuromuscular junctions are initially formed. One key molecular player in the development of the neuromuscular junction is the transmembrane receptor unplugged/MuSK. Unplugged/MuSK is specifically expressed in muscle fibers and controls two aspects of neuromuscular junction formation; the clustering of "prepatterned" acetylcholine receptors (AChRs) on the muscle fiber prior to the arrival of the nerve and the guidance of the motor nerve to its proper muscle target. Later in development it also directs the formation of mature neuromuscular synapses. With the following three aims I propose to determine how unplugged/MuSK spatially coordinates clustering of "prepatterned" AChRs and growth cone guidance to the muscle center. First, I will investigate if multiple Wnt signals activate the unplugged/MuSK receptor. We have previously identified wnt11r as an unplugged/MuSK ligand, but we suspect that wnt4a is also playing a role in unplugged/MuSK activation. I will use a combination of genetic interaction and in-vivo binding experiments to examine the extent of cooperation between the two Wnts and their ability to bind unplugged/MuSK. Second, I will determine which molecular players are downstream of unplugged/MuSK and whether the signaling cascade is similar to the Wnt planar cell polarity cascade. Activation of a planar cell polarity-like pathway could establish a central zone in the muscle fiber that dictates where AChR clusters initially form and where motor axons extend when they enter the muscle territory. I will investigate the involvement of various planar cell polarity components by disrupting their function in muscle fibers and determining whether this results in unplugged- like AChR clustering and axonal guidance errors. Third, in many systems including the Drosophila wing disk, cellular polarity is initially established by asymmetric localization of various members of the planar cell polarity pathway. I propose to determine the localization of these planar cell polarity components in muscle fibers and also determine whether the absence of Wnt signaling affects this localization using in-vivo live confocal imaging techniques. PUBLIC HEALTH RELEVANCE: This research is directly relevant to the study of human diseases in that MuSK is a target of antibody- mediated autoimmune attack in the neuromuscular disease myasthenia gravis. Furthermore, mutations in components of the MuSK pathway are responsible for human congenital myasthenic syndromes.