During the development of the vertebrate neuromuscular junction (NMJ), initial contacts between nerve and muscle are established by filopodial processes, which are thought to promote close interactions between the synaptic partners and enable the exchange of molecular cues that regulate subsequent steps in NMJ formation. My thesis research investigated the roles of different growth factors in influencing neuronal growth and filopodial assembly during the early stages of NMJ development....[ Read more ]

During the development of the vertebrate neuromuscular junction (NMJ), initial contacts between nerve and muscle are established by filopodial processes, which are thought to promote close interactions between the synaptic partners and enable the exchange of molecular cues that regulate subsequent steps in NMJ formation. My thesis research investigated the roles of different growth factors in influencing neuronal growth and filopodial assembly during the early stages of NMJ development.

The studies described here were carried on primary cultures of Xenopus embryonic spinal neurons and myotomal muscle cells, an experimental system in which synaptogenesis can be readily visualized and accurately quantified. When spinal neurons were cocultured with muscle cells, the neurons were found to preferentially extend filopodia towards muscle, which suggested that muscle-derived factors induced or stabilized the neuronal filopodia. Results of labeling assays identified basic fibroblast growth factor (bFGF) as a molecule produced by muscle and associated with the cell-surface, and a range of pharmacological, cell biological and molecular assays demonstrated that muscle bFGF enhanced filopodial assembly in spinal neurons and promoted NMJ formation through FGF receptor 1 (FGFR1). This effect of bFGF/FGFR1-signaling was contrasted by that of neurotrophin (NT)-signaling, which, through Trk receptors, promotes neuronal growth and survival but inhibits presynaptic differentiation and NMJ assembly. Characterization here of FGFR1 and Trk signaling in neurons showed that whereas the overexpression of FGFR1 enhanced filopodial formation in neurons, expression of excess TrkB reduced filopodial induction and promote neuronal growth. Intriguingly, FGFR1-dependent increase in filopodial assembly in neurons was blocked by the NT BDNF (brain-derived neurotrophic factor), which was produced by muscle and activated TrkB to enhance neuronal growth. On the other hand, excess bFGF furnished by muscle was able to elicit filopodial extension by TrkB-overexpressing neurons. These results suggested that an FGFR1/TrkB-signaling balance in neurons regulates neuronal growth versus filopodial formation and NMJ establishment. Last, a third muscle-derived molecule – hepatocyte growth factor (HGF) – was found to function through its receptor c-Met in spinal neurons to inhibit filopodial assembly and NMJ formation but promote growth, in a manner akin to NT-signaling.

Taken together, the results of this study revealed distinct and previously unappreciated roles of different growth factor signaling pathways in controlling neuronal growth and the assembly of filopodia which initiate nerve-muscle interactions to NMJ establishment.