Lack of Fgf18 causes abnormal clustering of motor nerve terminals at the neuromuscular junction with reduced acetylcholine receptor clusters

Kenyu Ito,Shiro Imagama,Mikito Tsushima,Hiroshi Kiyama,Kinji Ohno,Naoki Ishiguro,Hideki Yagi,Bisei Ohkawara,Akio Masuda,Kyotaro Ota,Hiroyuki Konishi,Hiroaki Nakashima

doi:10.1038/s41598-017-18753-5

Abstract

FGF receptor 2 is involved in the formation of the neuromuscular junction (NMJ), but its in vivo ligand remains to be determined. Laser capture microdissection of the mouse spinal motor neurons (SMNs) revealed that Fgf18 mRNA is highly expressed in SMNs in adults. Expression of Fgf18 mRNA was the highest in the spinal cord at embryonic day (E) 15.5, which gradually decreased to postnatal day 7. FGF18 protein was localized at the NMJs of the tibialis anterior muscle at E18.5 and in adults. Fgf18−/− mice at E18.5 showed decreased expressions of the NMJ-specific Chrne and Colq genes in the diaphragm. In Fgf18−/− diaphragms, the synaptophysin-positive areas at the nerve terminals and the acetylcholine receptor (AChR)-positive areas at the motor endplates were both approximately one-third of those in wild-type embryos. Fgf18−/− diaphragms ultrastructurally showed abnormal aggregation of multiple nerve terminals making a gigantic presynapse with sparse synaptic vesicles, and simplified motor endplates. In Fgf18−/− diaphragms, miniature endplate potentials were low in amplitude with markedly reduced frequency. In C2C12 myotubes, FGF18 enhanced AChR clustering, which was blocked by inhibiting FGFRs or MEK1. We propose that FGF18 plays a pivotal role in AChR clustering and NMJ formation in mouse embryogenesis.

Highlights

The neuromuscular junction (NMJ) is the synapse that is formed between a spinal motor neuron (SMN) and the skeletal muscle
We found that the expression levels of Fgf[1], Fgf[7], Fgf[11], and Fgf[18] were more than 4 times higher in SMNs than in the posterior horn cells (Supplementary Fig. S1A and Supplementary Table 2)
As FGF18 is one of SMN- expressed fibroblast growth factors (FGFs) and is a downstream target for Wnt signaling in cancer cells[26], we analyzed the roles of FGF18 in acetylcholine receptor (AChR) clustering

Summary

Introduction

The neuromuscular junction (NMJ) is the synapse that is formed between a spinal motor neuron (SMN) and the skeletal muscle. Similar requirement of AChR clustering for appropriate NMJ development has been reported in knockout mice lacking Lrp[43] and agrin[4]. Some FGF ligands (FGF4, 6, 7, 9, 10, 17, 18, 22, and 23) facilitate aggregation of synaptophysin, neurite elongation, and/or neurite branching in cultured primary spinal motor neurons (SMNs) isolated from chick embryos[12]. The diaphragm of Fgf18−/− embryos showed small synaptophysin- and acetylcholine receptor (AChR)-positive areas at the NMJs. Ultrastructure of the diaphragm NMJs of Fgf18−/− embryos revealed gigantic presynapse comprised of abnormally aggregated multiple nerve terminals with decreased densities of synaptic vesicles, and simplified postsynaptic folds. FGF18 is likely to be one of the essential regulators at the NMJs in mouse embryogenesis

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Results

Conclusion