Early nerve–muscle interaction that leads to neuromuscular junction formation is mediated by axonal filopodia that are oriented preferentially toward the muscle as a result of target-derived basic fibroblast growth factor.

BackgroundThe assembly of the vertebrate neuromuscular junction (NMJ) is initiated when nerve and muscle first contact each other by filopodial processes which are thought to enable close interactions between the synaptic partners and facilitate synaptogenesis. We recently reported that embryonic Xenopus spinal neurons preferentially extended filopodia towards cocultured muscle cells and that basic fibroblast growth factor (bFGF) produced by muscle activated neuronal FGF receptor 1 (FGFR1) to induce filopodia and favor synaptogenesis. Intriguingly, in an earlier study we found that neurotrophins (NTs), a different set of target-derived factors that act through Trk receptor tyrosine kinases, promoted neuronal growth but hindered presynaptic differentiation and NMJ formation. Thus, here we investigated how bFGF- and NT-signals in neurons jointly elicit presynaptic changes during the earliest stages of NMJ development.Methodology/Principal FindingsWhereas forced expression of wild-type TrkB in neurons reduced filopodial extension and triggered axonal outgrowth, expression of a mutant TrkB lacking the intracellular kinase domain enhanced filopodial growth and slowed axonal advance. Neurons overexpressing wild-type FGFR1 also displayed more filopodia than control neurons, in accord with our previous findings, and, notably, this elevation in filopodial density was suppressed when neurons were chronically treated from the beginning of the culture period with BDNF, the NT that specifically activates TrkB. Conversely, inhibition by BDNF of NMJ formation in nerve-muscle cocultures was partly reversed by the overexpression of bFGF in muscle.ConclusionsOur results suggest that the balance between neuronal FGFR1- and TrkB-dependent filopodial assembly and axonal outgrowth regulates the establishment of incipient NMJs.

Background: During vertebrate neuromuscular junction (NMJ) development, contact between motor axons and muscle fibers is followed by pre-and post-synaptic specialization. Using Xenopus nerve-muscle cocultures, we recently showed that spinal neurons initially contacted muscle cells by means of filopodial processes, and that muscle-derived basic fibroblast growth factor induced axonal filopodia and slowed axonal advance to promote nerve-muscle interaction and NMJ establishment. In contrast, neurotrophins enhanced axonal growth but suppressed the extension of axonal filopodia and blocked NMJ formation. Results: Here we report that hepatocyte growth factor (HGF), which also supports motor neuron survival, was expressed by Xenopus muscle cells, and that forced expression of HGF in Xenopus spinal neurons inhibited the extension of axonal filopodia. Overexpression of the HGF-receptor c-Met in neurons also blocked the formation of axonal filopodia and furthermore sped up axonal growth, but a kinase-dead form of c-Met was unable to effect these changes. Importantly, treatment of nerve-muscle cocultures with recombinant HGF or the expression of HGF or active c-Met in neurons, or that of excess HGF in muscle, inhibited nerve-induced AChR clustering in muscle. Conclusions: Our results suggest that HGF/c-Met signaling in neurons promotes axonal growth but suppresses filopodial assembly in neurons and hinders NMJ establishment. Developmental Dynamics 241:1562-1574,