January 1, 2012;
Reciprocal regulation of axonal Filopodia and outgrowth during neuromuscular junction development.
The assembly of the vertebrate neuromuscular junction (NMJ) is initiated when nerve
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.Whereas 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
cocultures was partly reversed by the overexpression of bFGF
.Our results suggest that the balance between neuronal FGFR1
- and TrkB
-dependent filopodial assembly and axonal outgrowth regulates the establishment of incipient NMJs.
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Figure 2. Influence of BDNF-depletion on the formation of axonal filopodia.Spinal neurons were incubated in (A) control medium or (B) medium containing 1 µg/ml TrkB-FC. Axonal filopodial formation was examined 18 h after plating the neurons. Bath application of TrkB-FC enhanced the formation of axonal filopodia, which is quantified in terms of filopodial densities (C). The density of filopodia (#filopodia/10 µm axon) in TrkB-FC-treated neurons was normalized against that in control neurons. In the presence of TrkB-FC, no significant difference in the speed of axonal outgrowth was observed (D). Mean and SEM shown; t test: **p<0.01, compared with untreated Ctl.
Figure 3. Effect of Trk inhibition on axonal growth.Spinal neurons were incubated in control medium (A–C) or medium containing 500 nM K252a (D–F). The growth speeds of axons were measured from images taken during time-lapse recordings. Treatment with K252a led to a significant reduction in axonal growth speed (G) and also to the elaboration of the distal end of the axon into a web of interconnecting neurites and varicosities within 30 min. Although varicosities were also observed along control axons and were often associated with filopodia (arrow in C), they were much more numerous within the distal neuritic web generated after K252a treatment (arrows in F and quantified in H). Mean and SEM shown; t test: **p<0.01, compared with untreated Ctl.
Figure 4. Opposite effects of bFGF and BDNF signaling on filopodial formation in spinal neurons.Spinal neurons were incubated in control medium (A) or medium containing (B) bFGF or (C) BDNF. Filopodial assembly was enhanced and suppressed by bFGF and BDNF respectively (J). Neurons expressing WT-FGFR1 or TR-FGFR1 were also maintained in control medium (D, G) or medium containing bFGF (E, H) or BDNF (F, I). WT-FGFR1 expression elevated the basal filopodial density in neurons, and this was not increased by bFGF-addition but was suppressed by BDNF-treatment (J). TR-FGFR1 expression blocked both basal and bFGF-induced formation of filopodia in neurons, and BDNF did not further reduce filopodial growth (J). The density of filopodia in control neurons (A) was used for normalizing all other filopodial density values. Mean and SEM shown; t test: *p<0.05 and **p<0.01, compared with untreated Ctl; ??p<0.01, compared to untreated WT-FGFR1-neurons.
Figure 5. Differential control of filopodial assembly and axonal growth by neuronal FGFR1 and TrkB signaling.Compared to control (GFP) neurons (A and A'), WT-TrkB-neurons grew fewer filopodia (B and B') and TR-TrkB-neurons grew more filopodia (C and C'); panels A–C and A'–C' show corresponding phase-contrast and GFP images. In panel D the filopodial densities calculated for these neurons are normalized relative to the density in GFP-neurons and are compared to those obtained for neurons expressing active and inactive FGFR1 proteins (Figure 4). E. Axonal growth speeds of neurons expressing FGFR1 or TrkB proteins were measured by time-lapse imaging and normalized relative to the growth of GFP-neurons. Overexpression of WT-FGFR1 or TR-TrkB slowed axonal growth, whereas expression of TR-FGFR1 or WT-TrkB sped it up. Mean and SEM shown; t test: *p<0.05, compared with Ctl.
Figure 6. The influence of TrkB signaling on filopodial extension by neurons towards muscle.Nerve-muscle cocultures were prepared using neurons expressing GFP (A and A'), WT-TrkB (B and B') or TR-TrkB (C and C'). WT-TrkB-neurons, unlike control neurons, displayed little bias in the extension of filopodia towards muscle, but the TR-TrkB-neurons, which grew more filopodia than GFP-neurons, were able to send out filopodia preferentially in the direction of muscle. (D–F') On muscle cells expressing GFP plus bFGF, neurons expressing GFP (D and D') or TrkB proteins (E–F') were seeded. GFP-neurons extended even more filopodia towards muscle cells overexpressing bFGF (D and D') than towards normal muscle cells (A and A'). WT-TrkB-neurons once again grew fewer filopodia than GFP-neurons, but the bFGF-overexpressing muscle cells induced more filopodia in WT-TrkB-neurons (E and E') than control muscle cells (above). TR-TrkB-neurons also extended more filopodia towards bFGF-expressing muscle cells (F and F') than towards control cells. (G) Calculation of AI values for these cocultures as well as for those cultures in which neurons expressed FGFR1 proteins (pictures not shown). Asymmetric distribution of filopodia was slightly improved in neurons expressing WT-TrkB, TR-TrkB and WT-FGFR1, but not TR-FGFR1. Mean and SEM shown; t test: *p<0.05 and **p<0.01, compared to cocultures between Ctl neurons and normal muscle cells; ?p<0.05 and ???p<0.001, compared to cocultures made between Ctl neurons and bFGF-overexpressing muscle cells.
Figure 7. Reciprocal regulation of NMJ formation by bFGF/NT-signaling.Normal spinal neurons were cocultured with muscle cells expressing GFP (A–C and G–I) or GFP plus bFGF (bFGF O/E; D–F and J–L) and maintained in control medium (A–F) or medium with added BDNF (G–L). Neurons induced AChR clusters equally well in GFP- and bFGF-muscle cells (C, F), and BDNF-treatment inhibited synaptic AChR clustering in both cases (I, L). The inhibitory effect of BDNF, however, was weaker when muscle cells expressed excess bFGF, and in these cases nerve-muscle contacts with AChR were more readily found (L). Muscle cells in which nerves induced new AChR clusters lacked spontaneously occurring AChR aggregates (also called hot-spots); when AChR aggregation at innervation sites was compromised, AChR hot-spots were retained (h.s. in panel I). Synaptogenesis was quantified in terms of the percentages of nerve-muscle contacts with AChR clusters (M). Mean and SEM shown; t test: **p<0.01 and ***p<0.001, compared to cocultures with normal muscle cells; ??p<0.01, compared with BDNF-treated cocultures using bFGF-overexpressing muscle cells. Arrowheads point to nerve tracks, arrows to nerve-induced AChR clusters.
Alsina, Visualizing synapse formation in arborizing optic axons in vivo: dynamics and modulation by BDNF. 2001, Pubmed