Wnt4 Participates in the Formation of Vertebrate Neuromuscular Junction

Abstract

Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4−/− NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.

Wnts contribute to neuromuscular junction formation through distinct signaling pathways

“…In zebrafish, both Wnt4a and Wnt11r initiate muscle prepatterning, probably by stimulating PCP-dependent MuSK endocytosis in muscle cells (Gordon et al, 2012;Jing et al, 2009). In mice, although recent data have challenged the role of Wnts at the NMJ (Remédio et al, 2016; discussed below), we have demonstrated that Wnt4 contributes to muscle prepatterning (Strochlic et al, 2012). Wnt signaling is also required for later steps of vertebrate NMJ differentiation.…”

“…Growing evidence in several vertebrate species, using both in vitro and in vivo models, suggests that Wnt morphogens act as regulators of NMJ initiation and/or formation (Gordon et al, 2012;Henriquez et al, 2008;Jing et al, 2009;Messéant et al, 2015;Packard et al, 2002;Strochlic et al, 2012;Zhang et al, 2012). Yet, Wnt function and the molecular mechanisms through which Wnts collaborate at the mammalian NMJ remain elusive and controversial.…”

“…At the vertebrate NMJ, Wnt ligands transduce their signals through activation of the receptor complex formed by the muscle-specific tyrosine kinase MuSK and low-density lipoprotein receptor-related protein 4 (Lrp4) and through the activation of classical Frizzled (Fzd) receptors (Avilés et al, 2014;Zhang et al, 2012;Strochlic et al, 2012;Gordon et al, 2012;Messéant et al, 2015). The MuSK-Lrp4 complex constitutes the central scaffold for the formation of the neuromuscular synapse (DeChiara et al, 1996;Weatherbee et al, 2006;Zhang et al, 2008).…”

“…Among the 19 Wnts currently identified in mammals, Wnt2, 3a, 4, 6, 7b, 9a and 11 directly interact with MuSK but only Wnt4, 9a and 11 enhance AChR clustering in muscle cells (Barik et al, 2014;Strochlic et al, 2012;Zhang et al, 2012). In zebrafish, both Wnt4a and Wnt11r initiate muscle prepatterning, probably by stimulating PCP-dependent MuSK endocytosis in muscle cells (Gordon et al, 2012;Jing et al, 2009).…”

β-Catenin gain of function in muscles impairs neuromuscular junction formation

“…In mammalian muscle cells, five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11 and Wnt16), presumably by direct binding to MuSK, are able to stimulate AChR clustering, in a manner dependent on LRP4 . Recently, WNT4 has been implicated in muscle pre-patterning and to stimulate AChR clustering via activation of MuSK (Strochlic et al, 2012). In addition, agrin-induced AChR clustering was enhanced by WNT3, but reduced by WNT3a (Henriquez et al, 2008;.…”