Wnt ligands comprise a family of 19 secreted signaling proteins with a wide range of developmental functions including cell fate decisions during early embryogenesis and specific cellular responses such as cell proliferation, differentiation and survival [
36]. Interestingly, Wnt ligands have also emerged as key molecules regulating crucial steps of neurodevelopment, including neuronal fate and differentiation, axonal guidance, dendritic development and synaptogenesis (for reviews, see [
37,
38]). The prevalent idea is that Wnt factors, which act as morphogens during early development, also regulate later, positively and negatively, crucial events resulting in the formation of proper neuronal connections [
37,
38]. Within this section we will focus on the emerging role of Wnt pathways during vertebrate NMJ assembly, taking as a basis what has been better described at the invertebrate neuromuscular synapse.
3.1. Wnt Pathways
Wnt ligands signal through their cognate seven-pass transmembrane G-protein coupled Frizzled (Fz) receptors from which ten members have been described in vertebrates [
39]. Three different Wnt pathways can be activated after Wnt binding to Fz receptors and the subsequent activation of the cytoplasmic protein Dishevelled (Dvl) [
40,
41]. In the so-called “Wnt canonical” pathway, the glycogen synthase kinase-3β (GSK-3β) is inhibited, resulting in the intracellular accumulation of β-catenin which translocates to the nucleus where, along with Tcf/Lef1 transcription factors, activates the expression of specific Wnt target genes [
39,
42,
43]. Two “non-canonical” pathways are also triggered by Wnts. The “Wnt calcium” pathway regulates cell fate decisions and cell movement during development by increasing intracellular Ca
2+ levels that activate specific protein kinases [
44]. In turn, the “planar cell polarity” pathway involves modifications of the cytoskeleton through the small GTPases Rac and Rho and the
N-terminal c-Jun kinase and functions mainly in polarity and morphogenesis [
45].
Along with the three pathways described, Wnt proteins activate several other signaling cascades [
46]. This heterogeneity is due in part to the existence of different membrane proteins, including receptors and regulatory proteins that act as co-receptors, including the LRP5/6 proteins, which are specifically required to activate canonical Wnt signaling [
47–
49]. Also, extracellular molecules, including the secreted Fz-related proteins (Sfrps), act as endogenous antagonists of Wnt signaling [
50]. Therefore, the existence of a large number of Wnt ligands, receptors and modulatory proteins, either cytosolic or extracellular, and, as a consequence, the different signaling pathways they generate, illustrates the broad diversity of functions that Wnt proteins play in different cell types and developmental processes.
3.2. Wnts Play Pro- and Anti-Synaptogenic Roles at the Invertebrate NMJ
At the
Drosophila NMJ, the Wnt orthologue
Wingless (Wg) is secreted by motor terminals whereas its DFz2 receptor is expressed both at pre- and post-synaptic cells [
51]. In agreement with this expression pattern, Wg affects pre- and postsynaptic behavior. Indeed, suppression of Wg at late larval stages results in the formation of aberrant synaptic boutons [
51]. Ultrastructural analysis shows mislocalization of pre and postsynaptic terminals and defective active zones [
51]. A novel mechanism accounts for Wg effects on postsynaptic differentiation, as a Wg/DFz2 complex is endocytosed and translocated to the periphery of the nucleus where a DFz2
C-terminal polypeptide is cleaved and transported into the nucleus to possibly activate transcription of target genes [
52,
53]. This pathway is crucial for NMJ structure, as blocking the internalization of the Wg/DFz2 complex in muscle cells results in severe defects in NMJ synaptic structure [
52,
53].
Wnt signaling also affects presynaptic differentiation at the
Drosophila NMJ. In motor neurons, Wg signaling is transduced through GSK-3β to regulate the formation of synaptic boutons and the recruitment of synaptic components. Indeed, GSK-3β mutants display abnormal synaptic boutons and NMJ growth by affecting presynaptic differentiation through a mechanism involving local β-catenin-independent changes in the dynamics of the microtubule cytoskeleton [
54,
55].
In contrast to the positive effects of nerve-derived Wg on the development of
Drosophila NMJs, studies in
C. elegans have demonstrated that Wnt signaling inhibits neuromuscular synaptogenesis. In this model system, the most proximal segment of the DA9 motor neuron is normally asynaptic whereas the remaining fraction of the axon form functional synapses with muscles along the dorso-ventral axis [
56]. Mutant worms for the Wnt/lin44 ligand and for the Fz/LIN-17 receptor display synaptic puncta in the asynaptic axonal region [
56]. Wnt/lin44 is expressed by few cells located towards the nematode tail, whereas Fz/LIN17 expression is restricted to the axonal asynaptic segment. Interestingly, ectopic expression studies of Wnt/lin44 resulted in mislocalized expression of Fz/LIN17 in areas close to the Wnt/lin44 source [
56]. These findings support a model where a posterior-anterior gradient of Wnt/lin44 positions its Fz/LIN17 receptor at specific areas along the axon to inhibit NMJ formation [
56]. Taken together, data obtained in invertebrates suggest that Wnts exhibit pro- and antisynaptogenic activities to regulate the formation and/or distribution of the neuromuscular synapse.
3.3. Role of Wnt Ligands in Postsynaptic Differentiation of the Vertebrate NMJ
Important
in vivo evidence supports a role for Wnt signaling during early steps of postsynaptic differentiation at the vertebrate NMJ. For instance,
Dvl1 mutant mice diaphragms display postsynaptic domains organized in a wider end-plate band than control littermates [
57], a phenotype resembling that of mutants of motor neuron proteins such as agrin and ChAT [
10,
12]. In addition, they are consistent with previous
in vitro data obtained in cultured muscle cells that positioned Dvl as a key organizer of postsynaptic differentiation by regulating the function of the agrin receptor MuSK [
58]. Considering that Dvl is a common mediator of Wnt pathways, these results suggest that Wnt signaling could affect the assembly and function of the vertebrate NMJ.
Cell transplantation experiments in the developing chick wing showed that the exposure of muscle cells to the Wnt-binding inhibitor Sfrp1 decreases AChR clustering [
57], revealing that endogenous Wnt ligands could be involved in early steps of postsynaptic differentiation. In this regard, evidence obtained at the developmental stages of mouse NMJ formation showed that Wnt3 is expressed by motor neurons of the lateral motor column [
59]. Consistent with its potential role in postsynaptic assembly, cell transplantation of Wnt3-overexpressing cells in the chick wing increases the clustering of AChRs in developing skeletal muscles [
57]. Experiments in cultured myotubes showed that Wnt3 acts together with agrin to induce postsynaptic differentiation [
57]. It has been described that, depending on the cell context, Wnt3 has the ability to activate either the β-catenin pathway or non-canonical Rho GTPases-dependent signaling [
60,
61]. Experiments using the inhibitors Sfrp1, which precludes Wnt binding to Fz receptors, and Dickkopf-1, which specifically interacts with LRP5/6 and thus blocks canonical signaling, did not to affect the ability of Wnt3 to induce AChR clustering along with agrin [
57]. In turn, these
in vitro studies revealed that Wnt3 activates Rac1 in a more efficient way than agrin does, whereas agrin preferentially increases Rho activity, suggesting that Wnt and agrin signaling pathways cross-talk at the vertebrate NMJ (for a review, see [
62]). Consistent with previous findings addressing a role for small GTPases on AChR clustering [
63,
64], Wnt3-mediated activation of Rac1 resulted in the formation of AChR microclusters, which are converted into full-size aggregates upon agrin-dependent activation of Rho [
57]. Future research will help to elucidate the molecular mechanisms by which Wnt activates Rac1 in this context. Together, these studies point to a positive role of Wnt ligands in postsynaptic differentiation at the vertebrate NMJ via an anterograde mechanism involving the activation of a non-canonical, Rac1-dependent signaling pathway.
In sharp contrast with the effect of Wnt3, the highly identical ligand Wnt3a plays negative roles on AChR clustering [
65]. In support of a physiological role at the NMJ, Wnt3a is expressed by embryonic mouse skeletal muscles during neuromuscular synapse assembly [
65]. Pre-treatment of muscle cells with Wnt3a resulted in impaired AChR clustering; also, agrin-induced aggregates are dispersed by Wnt3a [
65]. As an
in vivo support of these findings, electroporation of Wnt3a in post-natal muscles dispersed stabilized AChR clusters of mature NMJs [
65]. In cultured muscle cells, the dispersal activity of Wnt3a was shown to be mediated by a mechanism involving β-catenin-dependent, but TCF-independent, signaling that results in the down-regulation of rapsyn [
65]. Consistently, mice null for β-catenin in skeletal muscles, but not in neurons, develop bigger AChR clusters distributed in wider end-plate bands than controls [
66], thus supporting the notion that postsynaptic differentiation at the NMJ is negatively affected by a β-catenin-dependent pathway.
More recent evidence demonstrates that Wnt ligands also affect early aneural pre-patterning of AChR clusters at the vertebrate NMJ [
18]. In zebrafish, down-regulation of the Wnt11r ligand induced strong defects in AChR pre-patterning and axonal branching, similar to those of mutants for the agrin receptor MuSK [
67,
68]. Indeed, Wnt11r interacts genetically and biochemically with the ligand-binding domain of MuSK [
18]. These functional studies revealed that Wnt ligands, through MuSK, modulate the pre-patterning of AChR clusters and the guidance of motor axons.
In summary, Wnt ligands affect different features of the vertebrate NMJ formation. Regarding postsynaptic differentiation, they have the ability to modulate the early assembly of aneural AChR clusters, possibly to distribute nascent neuromuscular synapses. In areas of synaptic contact, motor neuron-derived Wnts could act through local pathways as positive inputs for postsynaptic differentiation; in turn, Wnt/β-catenin signaling, possibly activated by non-neuronal Wnt ligands, could negatively regulate postsynaptic differentiation in non-innervated muscle regions [
62] (
Figure 1). Even though a role for Wnt signaling in presynaptic differentiation has been well documented in central neurons [
69–
71], as well as in the
Drosophila NMJ [
54,
55], a potential role for Wnt pathways in the synaptic differentiation of vertebrate motor neurons still remains to be elucidated.