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

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.