Dissecting the roles and molecular mechanisms of Wnt signal transduction at the Drosophila neuromuscular junction

项目摘要 该建议解决了WNT(Wingless/WNT1)信令如何作为细胞间通信的基本机制 通讯，调节神经肌肉接头(NMJ)的发育--通过 中枢神经系统控制肌肉活动。NMJ发育和动态平衡的研究是 非常适合于对突触的形成和功能有全面的了解。此外，它还有可能 为神经肌肉疾病和神经退行性疾病的病因提供深刻的见解- 与NINDS的任务目标一致。 我们研究了果蝇NMJ的发育，其中Wingless(Wg)，这个有机体中的主要Wnt，是 由蛇纹石受体Frizzled型家族的两个功能冗余成员FZ1和FZ2转导。 我们的重点是幼虫的体壁肌肉结构，这是一个成熟的和特别容易驯化的实验 系统。先前的工作导致了流行的教条：(I)运动神经元分泌的Wg控制NMJ 通过作用于肌肉细胞表面的Fz2而发展，以及(Ii)Fz2通过一种新的和 潜在保守的肌肉特异性机制，其中配体结合诱导卵裂和核 其C-末端胞浆结构域的导入。然而，这两个原则都受到了我们初步结果的挑战 而且，如果不正确，将对我们理解突触发生和神经中的Wnt信号构成主要障碍 功能。 在拟议的研究中，我们将对这两个原则进行深入的测试。首先，我们将确认或驳斥卵裂和 确定Fz2的C-末端胞液结构域是否为假定的转导因子的核进口模型 -是肌肉中Wg转导和NMJ发育所必需的。为此，我们将删除其中一个或两个 从肌肉细胞中内源性FZ1和FZ2，并测试Wg信号转导和正常NMJ发育是否可以 通过用部分或完全缺乏C-末端的截短形式的FZ1或FZ2替换它们来恢复 域。我们还将测试肌肉细胞通过经典的“Armadillo/β-”转导Wg的替代可能性。 连环蛋白“途径。其次，我们将明确地确定运动神经元是否表达Wg，如果不是，则确定 相关细胞及其在NMJ中可能的不同作用。为此，我们将使用转基因技术来 对所有WG表达细胞进行荧光标记，然后确定选择性去除WG的后果 基因功能来自运动神经元或任何其他相关的神经元支持细胞(例如，胶质细胞)，它通常位于其中 激活。因此，拟议的研究将测试并可能修改目前对NMJ中WNT信号的看法，如 在突触发生和神经疾病方面也是如此。

Project Summary This proposal addresses how Wnt (Wingless/Wnt1) signaling, a fundamental mechanism of intercellular communication, regulates the development of neuromuscular junctions (NMJs) — the specialized synapses by which the central nervous system controls muscle activity. The study of NMJ development and homeostasis is well suited for gaining general insights into how synapses form and function. In addition, it has the potential to provide profound insights into the etiology of neuromuscular as well as neuro-degenerative disease — concordant with the mission goals of the NINDS. We study NMJ development in Drosophila, where Wingless (Wg), the predominant Wnt in this organism, is transduced by two functionally redundant members of the Frizzled family of serpentine receptors, Fz1 and Fz2. We focus on the body wall musculature of the larva, a well-established and exceptionally tractable experimental system. Prior work has led to the prevailing dogma that (i) Wg secreted by motoneurons controls NMJ development by acting on Fz2 on the surface of muscle cells, and (ii) Fz2 transduces Wg by a novel and potentially conserved muscle-specific mechanism in which ligand binding induces the cleavage and nuclear import of its C-terminal cytosolic domain. However, both these tenets are challenged by our preliminary results and, if incorrect, pose a major barrier to our understanding of Wnt signaling in synaptogenesis and neural function. In the proposed research, we will incisively test both tenets. First, we will confirm or refute the cleavage and nuclear import model by determining if the C-terminal cytosolic domain of Fz2 — the posited transducing factor — is required in muscle for Wg transduction and NMJ development. To do so, we will remove either or both endogenous Fz1 and Fz2 from muscle cells and test if Wg transduction and normal NMJ development can be restored by replacing them with truncated forms of Fz1 or Fz2 that partially or completely lack the C-terminal domain. We will also test the alternative possibility that muscle cells transduce Wg by the canonical “Armadillo/β- Catenin” pathway. Second, we will determine, unequivocally, if motoneurons express Wg, and if not, identify the relevant cells and their potentially distinct roles in NMJs. To do so, we will use transgenic technologies to fluorescently label all Wg expressing cells, and then determine the consequences of selectively removing wg gene function from motoneurons or any other associated neuronal support cells (e.g., glia) in which it is normally active. The proposed research will thus test, and potentially revamp, current views of Wnt signaling in NMJs, as well as in synaptogenesis and neurological disease.