Wnt Signaling in Synaptic Prepattern formation and Motor Axon Guidance

描述（由申请人提供）：本项目的主要目标是确定神经肌肉接头最初形成的发育机制。神经肌肉接头发育中的一个关键分子是跨膜受体unplugged/MuSK。Unplugged/MuSK在肌纤维中特异性表达，并控制神经肌肉接头形成的两个方面;在神经到达之前肌纤维上的“预图案化”乙酰胆碱受体（AChR）的聚集以及运动神经对其适当肌肉靶的引导。在发育后期，它还指导成熟神经肌肉突触的形成。以下三个目标，我建议，以确定如何不插电/MuSK空间协调集群的“预图案化”AChRs和生长锥的指导肌肉中心。首先，我将研究多个Wnt信号是否激活未插入的/MuSK受体。我们之前已经将wnt 11 r确定为未插入/MuSK配体，但我们怀疑wnt 4a也在未插入/MuSK激活中发挥作用。我将使用遗传相互作用和体内结合实验的组合来检查两个Wnt之间的合作程度和它们结合unplugged/MuSK的能力。其次，我将确定哪些分子参与者是unplugged/MuSK的下游，以及信号级联是否与Wnt平面细胞极性级联相似。平面细胞极性样通路的激活可以在肌纤维中建立一个中心区，该中心区决定AChR簇最初形成的位置以及运动轴突进入肌肉区域时延伸的位置。我将研究各种平面细胞极性成分的参与，通过破坏它们在肌纤维中的功能，并确定这是否会导致不插电样AChR聚集和轴突导向错误。第三，在包括果蝇翅盘在内的许多系统中，细胞极性最初是通过平面细胞极性通路的各种成员的不对称定位建立的。我建议确定这些平面细胞极性组件在肌纤维的本地化，并确定是否Wnt信号的情况下，使用活体共聚焦成像技术影响这种本地化。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MuSK是神经肌肉疾病重症肌无力中抗体介导的自身免疫攻击的靶标。此外，MuSK通路组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.