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

剖析果蝇神经肌肉接头Wnt信号转导的作用和分子机制

• 批准号: 10527669
• 负责人: Gary Struhl
• 金额: $ 45.24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527669
• 关键词: Achievement; Address; Binding; Brain; C-terminal; Cells; Defect; Development; Drosophila genus; Etiology; Family; Family Dasypodidae; Foundations; Frizzled Domain; Genetic; Goals; Grant; Growth; Homeostasis; Label; Larva; Lead; Life; Ligand Binding; Mediating; Messenger RNA; Mission; Modeling; Molecular; Morphology; Motor Neurons; Muscle; Muscle Cells; National Institute of Neurological Disorders and Stroke; Nervous System control; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neuromuscular Diseases; Neuromuscular Junction; Neurons; Neurophysiology - biologic function; Nuclear; Nuclear Import; Organism; Pathway interactions; Production; Proteins; Research; Role; Route; Secure; Signal Transduction; Source; Supporting Cell; Surface; Synapses; System; Techniques; Technology; Testing; Transgenic Organisms; WNT Signaling Pathway; Work; arm; base; beta catenin; cellular transduction; experimental study; gene function; insight; intercellular communication; knock-down; loss of function; member; nervous system disorder; neuromuscular; novel; receptor; recruit; synaptogenesis; tool; transcription factor

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.

项目摘要 该提案解决了Wnt（Wingless/Wnt 1）信号传导，细胞间的基本机制， 通信，调节神经肌肉接头（NMJs）的发育-通过 中枢神经系统控制肌肉活动。NMJ发育和稳态的研究是 非常适合于了解突触的形成和功能。此外，它还有可能 对神经肌肉和神经退行性疾病的病因学提供了深刻的见解- 与NINDS的使命目标一致。 我们研究了果蝇的NMJ发育，其中无翅（Wg），这种生物体中的主要Wnt， 由蛇形受体Frizzled家族的两个功能冗余成员Fz 1和Fz 2转导。 我们专注于幼虫的体壁肌肉组织，这是一个建立良好且非常易于处理的实验 系统先前的工作已经导致了流行的教条，即（i）运动神经元分泌的Wg控制NMJ 通过作用于肌细胞表面上的Fz 2来促进Wg的发育，以及（ii）Fz 2通过一种新的和 潜在保守的肌肉特异性机制，其中配体结合诱导切割和核 导入其C-末端胞质结构域。然而，这两个原则都受到我们初步结果的挑战 如果不正确的话，这将对我们理解Wnt信号在突触发生和神经元发育中的作用构成重大障碍。 功能 在拟议的研究中，我们将深刻地测试这两个原则。首先，我们将确认或反驳分裂， 通过确定Fz 2的C-末端胞质结构域-假定的转导因子 - 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 内源性Fz 1和Fz 2的肌肉细胞，并测试是否可以Wg转导和正常NMJ发育， 通过用部分或完全缺乏C末端的截短形式的Fz 1或Fz 2替换它们来恢复 域我们还将测试肌肉细胞通过典型的“Armadillo/β- 连环蛋白”通路。第二，我们将明确地确定运动神经元是否表达Wg，如果不表达，则确定运动神经元是否表达Wg。 相关细胞及其在NMJ中的潜在独特作用。为此，我们将利用转基因技术， 荧光标记所有表达Wg的细胞，然后确定选择性去除Wg的结果 来自运动神经元或任何其它相关神经元支持细胞的基因功能（例如，神经胶质）， 活跃因此，拟议的研究将测试并可能修改NMJ中Wnt信号传导的当前观点， 以及突触发生和神经疾病。