Wnts contribute to neuromuscular junction formation through distinct signaling pathways

Understanding the developmental steps shaping the formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers, is critical. Wnt morphogens are key players in the formation of this specialized peripheral synapse. Yet, the individual and collaborative functions of Wnts as well as their downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain of function studies in culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. In contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR clustering and mRNA downstream activation of the -catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the Planar Cell Polarity (PCP) pathway, which accumulates at embryonic NMJ. Moreover, mice bearing a Vangl2 loss of function mutation (looptail) exhibit a decreased number of AChR clusters and overgrowth of motor axons bypassing AChR clusters. Taken together, our results provide genetic and biochemical evidences that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways. D ev el o pm en t • A dv an ce a rt ic le Introduction Formation of the vertebrate neuromuscular junction (NMJ), a peripheral cholinergic synapse between motor neurons and skeletal muscle fibers relies on the accurate recognition and apposition of presynaptic motoneurons on postsynaptic muscle targets, a process achieved by a variety of organizing signals from both partners (Tintignac et al., 2015). 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. Wnts are known to activate a canonical signaling pathway that is -catenin (Ctnnb1)dependent as well as several non-canonical ones such as that of the core planar cell polarity (PCP) pathway (Nusse, 2012). At the vertebrate NMJ, Wnt ligands transduce their signals through the activation of the receptor complex formed by the muscle specific tyrosine kinase MuSK and the low-density lipoprotein receptor-related protein4 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; Kim et al., 2008; Weatherbee et al., 2006; Zhang et al., 2008). Activation of this complex is required for i) the early, nerve-independent muscle prepatterning, characterized by AChR aggregation in the prospective synaptic region of the muscle surface that helps to guide growing motor axons towards their final target, and ii) the late, nerve-dependent, differentiation and maturation of the synapse (Tintignac et al., 2015). This later step is orchestrated by the release of a nerve secreted isoform of agrin which binds to muscle Lrp4 D ev el o pm en t • A dv an ce a rt ic le leading to activation of MuSK and AChR clustering in the postsynaptic membrane (Kim et al., 2008; Zhang et al., 2008; Zhang et al., 2011; Zong et al., 2012). 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 likely 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. For example, Dishevelled (Dvl)1, a hub for Wnt signaling, interacts with MuSK and plays several roles during NMJ formation (Henriquez et al., 2008; Jing et al., 2009; Luo et al., 2002; Wang et al., 2014). Wnt3 expressed by motoneurons enhances AChR clustering in developing chicken wings and agrin-induced AChR clustering in cultured myotubes through a non-canonical signaling pathway (Henriquez et al., 2008). In contrast, Wnt3a disperses agrin-induced AChR clusters by down-regulating rapsyn expression in a ß-catenin dependent manner in muscle cell culture (Wang et al., 2008). In addition, muscle ß-catenin gainor loss-of-function in mice, revealed its role in preand postsynaptic differentiation consistent with a critical level of ßcatenin expression required for the proper formation of the NMJ (Li et al., 2008; Liu et al., 2012; Wang and Luo, 2008; Wu et al., 2012a; Wu et al., 2015). Here we have used a set of mutant mice, as well as newly designed in vivo tools and biochemical assays, to identify the signaling pathways activated by Wnt/receptor interaction and their function in pre and postsynaptic differentiation of mammalian NMJs. We show that Wnt11 cooperates with Wnt4 to enhance AChR subunit mRNA levels and aneural AChR clustering in cultured muscle cells in part through activation of -catenin signaling. In D ev el o pm en t • A dv an ce a rt ic le addition, in vivo application of both Wnt4 and Wnt11 before NMJs begin to form, enhances AChR clustering and motor axon outgrowth. In contrast, lack of Wnt11 or inhibition of all Wnt-dependent signaling in vivo decreases AChR clustering and nerve terminal arborization. Specific inhibition of the Wnt canonical pathway similarly affects AChR distribution but not axonal branching suggesting that distinct branches of Wnt signaling regulate nerve terminal arborization. Interestingly a significant number of axons fail to terminate at AChR clusters and grow exuberantly beyond the prepatterned region of the muscle. Finally, we show that: i) both Wnt11 and Wnt4 co-immunoprecipitate with Vangl2, a key component of the core PCP pathway, ii) Vangl2 accumulates at embryonic NMJs and iii) mice bearing the Vangl2 loss of function mutation (Looptail; Vangl2) exhibit disrupted AChR clusters and axons outgrowth that bypasse AChR clusters. Taken together, our results provide compelling evidence that the coordinate action of Wnt4 and Wnt11 regulate NMJ formation through activation of both the canonical and Vangl2-dependant PCP pathways. D ev el o pm en t • A dv an ce a rt ic le