Carrier of Wingless (Cow) Regulation of Neuromuscular Junction Development.

The first Wnt signaling ligand discovered, Wingless [Wg (Wnt1 in mammals)], plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture, and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). Here, we investigate synaptogenic functions of the most recently described HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, we find that Cow secreted from the presynaptic motor neuron acts to limit synaptic architecture and neurotransmission strength. In c null mutants, we find increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. We show null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle fusion frequency based both on GCaMP imaging and electrophysiology recording. We find that membrane-tethered Wg prevents null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It was shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ, and we show here that Cow and Notum work through the same pathway to limit synaptic development. We conclude Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix.