Probing the mechanism of Wnt signal transduction at the Drosophila neuromuscular junction

Intercellular communication is a basic phenomenon of life, essential for cells to know where they are and what to do in order to choose their correct fates and manifest the appropriate morphologies and functional relationships with their neighbors. Cells communicate mainly through secreted and membrane tethered ligands and their receptors, with the signals being transduced by diverse networks of downstream cytosolic effectors that fall into distinct, evolutionarily conserved pathways. These pathways, functioning alone and in combination, govern most if not all aspects of cell, tissue and organismal life - and if impaired, result in diverse diseases, congenital defects and physiological deficits.In this proposal, I focus on Wnt (Wingless/Wnt1) signaling, the prevalence and versatility of which are apparent in all animal systems studied. Research utilizing Drosophila melanogaster has revealed many canonical Wnt pathway components and provided much of what we know about how they normally function in a variety of biological contexts from embryonic patterning to neural plasticity. Conversely, defects in the human homologues of these components contribute to a vast number of medical disorders, including congenital defects, neurological illnesses and cancer.The canonical Wnt signaling pathway has been elucidated in great detail. Wnts that signal by this pathway are received by the Frizzled (Fz) family of 7-pass transmembrane receptors. Fz receptors then regulate the stability and intracellular localization of beta-catenin, a cytosolic protein that functions in adherens junctions between cells and also acts in the nucleus as a transcriptional activator to control target gene expression.A decade ago a novel, non-canonical mechanism of Wingless signal transduction, termed the Frizzled Nuclear Import (FNI) pathway, was proposed to explain how Wnt signals control the development of neuromuscular junctions (NMJs) - the synaptic structures that connect neurons to muscles. This hypothesis is based on genetic and cellular studies of NMJ development in Drosophila larvae deficient for either Wingless (Wg), the founding member of the Wnt superfamily, or Frizzled 2 (dFz2), one of two Drosophila Fz family receptors responsible for transducing Wg. These data have led to the proposal that Wg signal induces a small cytosolic C-terminal fragment of the dFz2 receptor to be cleaved off and transported to the nucleus to directly regulate target gene expression. However, although widely cited and generally accepted, there are published findings that challenge this hypothesis.The central aim of this proposal is to test the validity of the FNI model in light of these challenges, and more generally, to determine the molecular mechanism of Wnt transduction responsible for controlling the development of the NMJ. I’ll largely exploit powerful Drosophila genetics with a combination of various cellular and biochemical approaches to achieve these goals.The outcome of this project will have a significant impact on our understanding of the normal mechanisms for NMJ development and plasticity; it will also influence our understanding of the etiology of neuromuscular diseases.