Initiation of synapse formation by Wnt-induced MuSK endocytosis

Abstract

In zebrafish, the MuSK receptor initiates neuromuscular synapse formation by restricting presynaptic growth cones and postsynaptic acetylcholine receptors (AChRs) to the center of skeletal muscle cells. Increasing evidence suggests a role for Wnts in this process, yet how muscle cells respond to Wnt signals is unclear. Here, we show that in vivo, wnt11r and wnt4a initiate MuSK translocation from muscle membranes to recycling endosomes and that this transition is crucial for AChR accumulation at future synaptic sites. Moreover, we demonstrate that components of the planar cell polarity pathway colocalize to recycling endosomes and that this localization is MuSK dependent. Knockdown of several core components disrupts MuSK translocation to endosomes, AChR localization and axonal guidance. We propose that Wnt-induced trafficking of the MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis.

Wnts contribute to neuromuscular junction formation through distinct signaling pathways

“…During this step, early nerve-induced signaling is occurring, many AChR clusters are not yet innervated and both aneural and neural AChR clusters can be visualized and quantified, and thus the AChR defects observed are likely to result from initial nerve-independent postsynaptic differentiation. In line with this, Granato and colleagues showed that both Wnt11r and Wnt4a are required for AChR prepatterning in zebrafish and we previously reported that Wnt4 participates in aneural AChR clustering in mice (Gordon et al, 2012;Strochlic et al, 2012). However, only the combined inactivation of wnt11r and wnt4a in zebrafish led to a complete loss of aneural AChR clusters, and loss of Wnt4 function in mice did not fully abolish muscle prepatterning.…”

“…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).…”

“…In view of the complex and still debated role of Wnt4 and Wnt11 in vertebrate NMJ formation (Gordon et al, 2012;Jing et al, 2009;Messéant et al, 2015;Remédio et al, 2016;Strochlic et al, 2012), we investigated events downstream of Wnt4 and Wnt11 and whether they cooperate to induce AChR clustering. Wnt11 alone is known to enhance AChR clustering in vitro; however, conflicting results have been reported regarding the effect of Wnt4 recombinant protein or of Wnt4-conditioned medium treatment of muscle cells on AChR clustering in vitro (Strochlic et al, 2012;Zhang et al, 2012).…”

“…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).…”