Li PP et al. (2011), Axonal filopodial asymmetry induced by synaptic...

XB-ART-43304

Mol Biol Cell 2011 Jul 15;2214:2480-90. doi: 10.1091/mbc.E11-03-0198.

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Axonal filopodial asymmetry induced by synaptic target.

Li PP , Chen C , Lee CW , Madhavan R , Peng HB .

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During vertebrate neuromuscular junction (NMJ) assembly, motor axons and their muscle targets exchange short-range signals that regulate the subsequent steps of presynaptic and postsynaptic specialization. We report here that this interaction is in part mediated by axonal filopodia extended preferentially by cultured Xenopus spinal neurons toward their muscle targets. Immunoblotting and labeling experiments showed that basic fibroblast growth factor (bFGF) was expressed by muscle and associated with the cell surface, and treatment of cultured spinal neurons with recombinant bFGF nearly doubled the normal density of filopodia in neurites. This effect of bFGF was abolished by SU5402, a selective inhibitor of FGF-receptor 1 (FGFR1), and forced expression of wild-type or dominant-negative FGFR1 in neurons enhanced or suppressed the assembly of filopodia, respectively. Significantly, in nerve-muscle cocultures, knocking down bFGF in muscle decreased both the asymmetric extension of filopodia by axons toward muscle and the assembly of NMJs. In addition, neurons expressing dominant-negative FGFR1 less effectively triggered the aggregation of muscle acetylcholine receptors at innervation sites than did control neurons. These results suggest that bFGF activation of neuronal FGFR1 generates filopodial processes in neurons that promote nerve-muscle interaction and facilitate NMJ establishment.

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Species referenced: Xenopus
Genes referenced: fgf2 fgfr1
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	FIGURE 1:. Axonal filopodia generated by Xenopus spinal neurons. In the absence of muscle target, filopodia were generated on either side of the axon with equal frequency (A). However, when a muscle cell was nearby (cell on the left in B), more filopodia were extended toward the target. (C) This difference was quantified by calculating the AI by scoring filopodia from the muscle-facing side (#Fm) vs. those on muscle-free side (#Ff) along axon segments within 30 μm from muscle. (D–I) Time-lapse recording of filopodia near a muscle cell. Muscle-facing filopodia were more stable (pointed out by arrows) than those on the opposite side (arrowheads pointing to filopodia that disappeared with time). (J) Filopodial density determined by scoring them along the length of the axon. The presence of the muscle target caused a large increase in their total density. Most of this increase was seen asymmetrically along the muscle-facing side of the axon. No asymmetry was seen along axons in pure nerve cultures without muscle target. Fifty-three axons from pure nerve culture and 42 axons from nerve–muscle cocultures were counted. (K) AI calculated from axonal segments in pure nerve cultures and nerve–muscle cocultures (22 axons from pure nerve cultures and 26 from nerve–muscle cocultures). The presence of muscle caused highly asymmetric filopodia disposition, with an AI value of 0.56 ± 0.07. (L) The formation (open bars) and retraction (hatched bars) of axonal filopodia in cocultures. On the muscle-facing side of the axon, more new filopodia were formed than on the muscle-free side. However, the major difference was seen in the stability of filopodia on the muscle-facing side. This is shown by the much lower rate of retraction during a 10-min period. Nineteen nerve–muscle cocultures were scored. In J–L, the mean ± SEM in each case is plotted, *p < 0.05 (Student's t test).
	FIGURE 2:. The expression of bFGF in Xenopus embryos. (A) Western blots of Xenopus embryonic extracts were probed for bFGF. The positive and negative controls used (first two lanes) were extracts of HEK293 cells overexpressing bFGF and GFP, respectively. The other three lanes contained extracts from Xenopus neural tubes, myotomal muscle, and whole embryos. Tubulin was used as protein loading control. Xenopus myotomal muscle expressed a higher level of bFGF than neural tubes. (B–J) Localization of bFGF in Xenopus myotomal muscle cells in culture. Cells were labeled live with anti-bFGF (F, I) or anti-GFP (control, C) and FITC-conjugated secondary antibodies. R-BTX labeling (D, G, J) was used to visualize AChR clusters. Although the muscle cell showed an overall labeling for bFGF, this growth factor was more concentrated at AChR clusters in muscle (E–G) and at developing NMJs in nerve–muscle cocultures (H–J).
	FIGURE 3:. The effect of bFGF/FGFR1 on axonal filopodial density. Spinal neurons were incubated in control medium (A) or in medium containing bFGF (100 ng/ml) (B) or bFGF plus the FGFR1 inhibitor SU5402 (20 μM) (C). Neurite segments were randomly selected, and all the filopodia present in them were counted to calculate filopodial density (number of filopodia/10 μm). More filopodia were found in neurons treated with bFGF than in control neurons, but this change was absent when SU5402 was added with bFGF; by itself SU5402 did not affect the basal filopodial assembly in neurons (J). (D, D′ to I, I′). To test FGFR1'1 role in the induction of neuronal filopodia, spinal neurons were cultured from Xenopus embryos injected with mRNAs encoding GFP (D, D′ and G, G′), WT-FGFR1 and GFP (E, E′ and H, H′), or TR-FGFR1 and GFP (F, F′ and I, I′). Neurons were examined in phase contrast for filopodia, and GFP fluorescence confirmed the expression of exogenous proteins in neurons. In WT-FGFR1 neurons more filopodia were found than in control GFP neurons (E vs. D), but in TR-FGFR1 neurons fewer filopodia were detected than in GFP neurons (F vs. D). The enhancement of filopodial growth in WT-FGFR1 neurons was blocked by the addition of SU5402 (H), but SU5402 did not significantly affect the already low filopodial density in TR-FGFR1 neurons (I) and only slightly reduced filopodia in GFP neurons (G). Filopodial densities in neurons expressing the exogenous proteins are shown in K (mean ± SEM; t test, p < 0.05 and *p < 0.01, compared with control; ^^p < 0.01, compared with no SU5402 treatment).
	FIGURE 4:. Regulation of axonal filopodial asymmetry by neuronal FGFR1 signaling. Xenopus spinal neurons expressing GFP (A, A′) or GFP and exogenous WT-FGFR1 (B, B′), TR-FGFR1 (C, C′), or bFGF (D, D′) were cocultured with normal muscle cells, and filopodia were examined in axons near muscle cells. Nerve–muscle cocultures treated with SU5402 (20 μM) were also compared with control cocultures (E). Relative to control GFP-expressing neurons (A, A′), neurons overexpressing WT-FGFR1 (B, B′) developed more filopodia, and those expressing TR-FGFR1 grew fewer filopodia (C, C′), but in these cases the preferential extension of filopodia toward muscle was less pronounced than it was in bFGF-overexpressing neuron–muscle cocultures (D, D′). (E) Quantification of these results. Axonal filopodial AI values in cocultures involving neurons expressing exogenous bFGF and two kinds of FGFR1 constructs, as well as SU5402 treatment, are shown. Numbers are mean ± SEM; t test, p < 0.05, p < 0.01, and **p < 0.001 for comparisons of bFGF/FGFR1–expressing and control GFP neurons, and ^^p < 0.01 for comparison of untreated and SU5402 treated cocultures.
	FIGURE 5:. Effects of manipulating muscle bFGF expression on axonal filopodial asymmetry. Spinal neurons were cocultured with muscle cells isolated from embryos expressing GFP (A, A′), GFP plus exogenous bFGF (B, B′), control morpholinos (C, C′), or bFGF morpholinos (D, D′). Overexpression of bFGF in muscle led to enhanced filopodial asymmetry in neurons compared with that in cocultures using control GFP-expressing muscle. On the other hand, muscle cells bearing bFGF but not control morpholinos suppressed axonal filopodial asymmetry. (E) Quantification of these results. Values shown are mean ± SEM; t test, *p < 0.05 for comparisons of neurons near GFP- and GFP/bFGF–expressing muscle cells, and ^^^p < 0.001 for comparison of neurons near Ctl-MO– and bFGF-MO–injected muscle cells.
	FIGURE 6:. Regulation of NMJ assembly by FGF signaling. NMJ formation in cocultures with alterations in FGF signaling was assessed by AChR clustering, which was monitored by R-BTX labeling (right). (A–C) AChR clusters (C, arrows) were present along the nerve–muscle contact in the control nerve–muscle coculture. (D–F) Similar to the control, AChR clusters were detected at nerve–muscle contacts in cocultures involving WT-FGFR1–expressing spinal neurons (D–F). (G–I) Suppression of FGFR1 function in neurons through expression of TR-FGFR1–inhibited NMJ formation, as shown by the lack of AChR clusters associated with the nerve–muscle contact. In the absence of nerve-induced AChR clustering, preexistent AChR clusters in the cells (hotspots) persisted (I, h.s.). In these parts of the figure, GFP coexpressed in the neurons was used to mark the expression of exogenous proteins. (J–L) Expressing control morpholinos in muscle (shown by fluorescence in K) did not affect NMJ formation, but the expression of bFGF morpholino (M–O) suppressed NMJ assembly, seen here once again as a lack of AChR clustering at nerve–muscle contacts and the persistence of hotspots. (P) Quantification of data showing the mean ± SEM; t test, **p < 0.01 for comparisons of FGFR1-expressing and control GFP neurons and ^p < 0.05 for comparison of Ctl-MO– and bFGF-MO–injected muscle cells.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase