Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction

Li, Jin; Ito, Mikako; Ohkawara, Bisei; Masuda, Akio; Ohno, Kinji

doi:10.1038/s41598-018-31949-7

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Published: 11 September 2018

Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction

Scientific Reports volume 8, Article number: 13577 (2018) Cite this article

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Abstract

We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2^−/−) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2^−/− mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.

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Introduction

The release of the neurotransmitter acetylcholine (ACh) from the axon terminal of the spinal motor neuron (SMN) activates the postsynaptic acetylcholine receptor (AChR) and elicits an action potential in the target muscle fiber^1,2,3. To ensure efficient neuromuscular signal transmission, dense AChR clusters must be compactly formed against presynaptic nerve terminals in the proper organization^4,5. Genetic depletion of the embryonic AChR γ-subunit in muscle fibers markedly decreases staining for AChR clusters with progressive accumulation of synaptic vesicles in the presynaptic terminal⁶, suggesting that embryonic AChR clustering is required for further pre- and postsynaptic development. AChR clustering and formation of the neuromuscular junction (NMJ) are mediated by SMN-derived and muscle-derived molecules. Neuronal agrin released from the nerve terminal⁷ binds to the low-density lipoprotein receptor-related protein 4 (LRP4) on the motor endplate and promotes phosphorylation of the muscle-specific kinase (MuSK) to induce AChR clustering^8,9. Other SMN-derived molecules driving AChR clustering and NMJ formation include neuregulin 1 and ACh^{2,10,11,12,13,14,15}. In addition, muscle-derived molecules including laminins, FGFs, collagens, BDNF, GDNF, Wnts, and TGF-β⁴ are implicated in orchestrating pre- and postsynaptic NMJ formation.

Wnts are secreted glycoproteins, and Wnt ligands (Wnt4, Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) are able to promote AChR clustering independent of agrin^16,17. Wnt ligands bind to the Frizzled-like domain of MuSK, and specific deletion of this MuSK domain compromises AChR clustering¹⁸. In addition, β-catenin directly promotes AChR clustering by directly binding to rapsyn and by linking rapsyn with the α-catenin-associated cytoskeleton¹⁹.

R-spondins (Rspo1-4) play essential roles in development through Wnt signaling pathways in vertebrates^20,21,22. As a member of the Rspo family, Rspo2 binds directly to the leucine-rich-repeat-containing G-protein coupled receptors (Lgr) 4 and 5 to enhance stabilization of Wnt receptors on the cell membrane and to activate Wnt signaling pathways^23,24. Rspo2 is implicated in development of the larynx, lung, limb, and trachea in mice^25,26. Rspo2-deficient mice present with an abnormally short left hindlimb and lack of digits on both forelimbs at embryonic day (E) 18.5, and mice die perinatally due to respiratory distress^25,27,28,29. In cultured myogenic C2C12 cells, Rspo2 is able to promote muscle differentiation through Wnt/β-catenin signaling³⁰. We recently reported by laser capture microdissection of mouse SMNs that Rspo2 is highly expressed in SMNs³¹. Rspo2 is likely to be excreted from the axon terminals of SMNs and is accumulated at the NMJ in wild-type mice. In cultured C2C12 myotubes, Rspo2 induces MuSK phosphorylation and AChR clustering, which requires Wnt ligands but not agrin. Lgr5 is associated with MuSK at the NMJ and serves as a receptor for Rspo2. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced with widened synaptic clefts, sparse synaptic vesicles, and markedly reduced frequency of miniature endplate potentials. We assumed that SMN-derived Rspo2 was anchored at the NMJ and promoted AChR clustering as well as NMJ formation, but Rspo2 is also expressed marginally in the diaphragm at E18.5 and in adults³¹. To dissect the differential roles of SMN-derived Rspo2 and muscle-derived Rspo2 on AChR clustering and NMJ formation in mouse embryos, we generated transgenic mice expressing Rspo2 specifically in SMNs or in skeletal muscle on the background of Rspo2^−/−.

Results

Specific expression of Rspo2 in the skeletal muscles and SMNs in Rspo2^−/− mice

To understand the origin of Rspo2 that promotes AChR clustering at the NMJ, transgenic mice expressing Rspo2 specifically in skeletal muscle or SMNs were generated according to the conventional transgenic mice strategy³². Human RSPO2 cDNA with three repeats of FLAG tags at the 3′ end was inserted downstream of either a 3.2-kb mouse muscle creatine kinase (MCK) promoter or a 6.4-kb mouse vesicular acetylcholine transporter (VAChT) promoter (Fig. 1A). The constructs were microinjected into fertilized eggs. PCR genotyping of embryos and adults was carried out with primers targeted to the injected DNA sequence (Supplementary Table S1). Transgenic mice carrying MCK-RSPO2 and VAChT-RSPO2 were morphologically normal, viable, active, and fertile without shortened life spans. To confirm expression of FLAG-tagged Rspo2 protein, frozen sections of the tibialis anterior (TA) muscle and the spinal cord from MCK-RSPO2 and VAChT-RSPO2 mice were stained with anti-FLAG antibody. MCK-RSPO2 mice showed the FLAG signal predominantly in the periphery of muscle fibers of TA but not in the spinal cord (Fig. 1B). In contrast, VAChT-RSPO2 mice showed the FLAG signal in large cells in the ventral horn area of the spinal cord but not in TA (Fig. 1B). Double immunostaining of the spinal cord of VAChT-RSPO2 mice for the FLAG signal and choline acetyltransferase (ChAT), a marker for motor neurons, revealed that most large cells were double-positive for FLAG and ChAT; however, some FLAG-positive cells were not strongly labeled with ChAT, indicating leaky expression of FLAG-RSPO2 in non-motor neurons (Fig. 1C). Thus, Rspo2 was specifically expressed in the skeletal muscle of MCK-RSPO2 mice and in SMNs of VAChT-RSPO2 mice.

To produce MCK-RSPO2/Rspo2^+/− and VAChT-RSPO2/Rspo2^+/− mice, F1 generations were crossed with heterozygous Rspo2 knockout (Rspo2^+/−) mice. The resulting MCK-RSPO2/Rspo2^+/− and VAChT-RSPO2/Rspo2^+/− mice were morphologically normal, viable, active, and fertile without shortened life spans. Finally, the heterozygous littermates were intercrossed to generate MCK-RSPO2/Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− embryos. Quantitative RT-PCR showed that expression levels of human RSPO2 in the diaphragm of MCK-RSPO2/Rspo2^−/− mice at E18.5 and in the spinal cord of VAChT-RSPO2/Rspo2^−/− mice at E18.5 were 11.6-fold and 1.23-fold higher, respectively, than the expression levels of Rspo2 in wild-type mouse embryos at E18.5 (Fig. 1D). The difference between 11.6- and 1.23-folds may represent the difference between the promoter activities of MCK and VAChT.

Embryonic lethality and morphological defects in the left hindlimb and digits of both forelimbs are not rescued by Rspo2 expression in skeletal muscles or SMNs

Rspo2 is required for normal development of the lungs and limbs²⁵. In accordance with a previous report²⁹, Rspo2^−/− mice had abnormally short left hindlimbs and lacked digits of both forelimbs at E18.5 (Fig. 2A–C), and they died shortly after birth due to respiratory distress. MCK-RSPO2/Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− mice also died perinatally. Similarly, abnormal morphology of the left hindlimb and missing digits in forelimbs (Fig. 2B,C) were also observed in both MCK-RSPO2/Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− embryos at E18.5. The body weights of Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− mice at E18.5 were 15% or more lower than those of wild-type mice (Fig. 2D). However, the body weights of MCK-RSPO2/Rspo2^−/− mice at E18.5 were similar to those of wild-type mice. In contrast, the body lengths of wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− embryos were similar to each other (Fig. 2E).

SMN-derived Rspo2 regulates the bandwidth of AChR clusters and axonal branching in the diaphragm

We next examined AChR clustering at the NMJs of the left hemi-diaphragms at low-magnification in wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice at E18.5. As all mice died shortly after birth, and as immaturity of the NMJs in mouse embryos made it difficult to obtain dependable results, we analyzed the NMJs only at E18.5. First, we observed that innervation patterns were different in these mice. As we previously reported³¹, Rspo2^−/− mice formed broader bands of AChR clusters compared to those in wild-type mice. MCK-RSPO2/Rspo2^−/− mice exhibited similarly broad bands of AChR clusters, whereas VAChT-RSPO2/Rspo2^−/− mice showed markedly narrow bands of AChR clusters (Fig. 3A,B). In contrast, the numbers of AChR were not changed in wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice at E18.5 (Fig. 3C). These findings indicate that SMN-derived Rspo2 ensures proper distribution of AChR clusters.

The axonal branches in the left hemi-diaphragms were observed by immunostaining with anti-peripherin antibody (Fig. 3D,E). The diameters and lengths of the first branches (red arrows in Fig. 3E) were similar in wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice (Fig. 3F,G). In Rspo2^−/− and MCK-RSPO2/Rspo2^−/− mice, the lengths of secondary branches (white arrows in Fig. 3E) were similar to those in wild-type mice, but the numbers of secondary branches were decreased (Fig. 3G,H). VAChT-RSPO2/Rspo2^−/− mice showed more prominent changes in secondary branches; the lengths of secondary branches were increased (Fig. 3G) and their numbers were decreased more than those in Rspo2^−/− mice (Fig. 3H). We also observed that almost all AChR clusters were similarly innervated by motor axons in wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice (Supplementary Fig. S1A,B). This suggests that the lack of Rspo2 or overexpression of Rspo2 is unlikely to have a substantial effect on the gross innervation of AChR clusters. Thus, a comparison of Rspo2^−/− mice against MCK-RSPO2/Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− mice revealed that SMN-derived Rspo2 contributes to branching and outgrowth of SMN axons, whereas muscle-derived Rspo2 has no substantial effect on SMN axons.

SMN-derived Rspo2 regulates the sizes of AChR clusters and nerve terminals

We next investigated AChR clustering at the NMJs of the right hemi-diaphragms at high-magnification in wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice at E18.5. AChRs and nerve terminals were stained by α-bungarotoxin and anti-synaptophysin antibody, respectively (Fig. 4A). The distribution of AChR cluster areas was shifted toward larger sizes in Rspo2^−/− mice (Fig. 4B), as we previously reported³¹. SMN-derived Rspo2 in VAChT-RSPO2/Rspo2^−/− mice corrected the abnormally enlarged AChR cluster areas (Fig. 4B,C) and over-corrected abnormally enlarged presynaptic synaptophysin-positive areas (Fig. 4D). Muscle-derived Rspo2 in MCK-RSPO2/Rspo2^−/− mice had no effect on AChR clusters (Fig. 4B,C), but normalized synaptophysin-positive areas (Fig. 4D). These data support the aforementioned observation at low-magnification that SMN-derived Rspo2 is likely to have more of an effect on AChR clustering and synaptophysin-positive nerve terminals than muscle-derived Rspo2.

Muscle-derived Rspo2 regulates juxtaposition of the active zones and synaptic folds

The NMJs at E18.5 were ultrastructurally visualized (Fig. 5A) and were morphometrically analyzed in a blinded manner (Fig. 5B–H). Compared to wild-type mice, the areas of nerve terminals (Fig. 5B) and widths of synaptic clefts (Fig. 5C) were increased in Rspo2^−/− mice, and both were normalized in MCK-RSPO2/Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− mice. Similarly, the densities of synaptic vesicles were decreased in Rspo2^−/− mice and were over-corrected in both MCK-RSPO2/Rspo2^−/− and VAChT-RSPO2/Rspo2^−/− mice (Fig. 5D). In contrast, the diameters of synaptic vesicles were increased in Rspo2^−/− mice, and they were normalized in MCK-RSPO2/Rspo2^−/− mice, but not in VAChT-RSPO2/Rspo2^−/− mice (Fig. 5E). Similarly, the numbers of active zones (Fig. 5F) and the numbers of postsynaptic folds (Fig. 5G) were markedly decreased in Rspo2^−/− mice, and they were partially normalized in MCK-RSPO2/Rspo2^−/− mice but not in VAChT-RSPO2/Rspo2^−/− mice. In contrast to the above parameters, the areas of mitochondria at the nerve terminals were similar in wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice, indicating that Rspo2 plays no role in the development of synaptic mitochondria (Fig. 5H). To summarize, muscle-derived Rspo2 (MCK-RSPO2) normalized all six abnormal parameters observed in Rspo2^−/− mice (Fig. 5B–G), whereas SMN-derived Rspo2 (VAChT-RSPO2) was able to normalize only three of them (Fig. 5B–D).

SMN-derived Rspo2 regulates muscle differentiation

Ultrastructural analysis of the diaphragm revealed that the Z-disk length (Fig. 5J), representing the myofibril diameter and the sarcomere length (Fig. 5L) tended to be increased in Rspo2^−/− mice, which were corrected or over-corrected in VAChT-RSPO2/Rspo2^−/− mice, but not in MCK-RSPO2/Rspo2^−/− mice. In contrast, the Z-disk thickness tended to be decreased in Rspo2^−/− mice, which was not mitigated in VAChT-RSPO2/Rspo2^−/− or MCK-RSPO2/Rspo2^−/− mice (Fig. 5K). Thus, SMN-derived Rspo2 is likely to regulate muscle differentiation, which may be secondary to the ameliorated AChR clustering observed with SMN-derived Rspo2.

SMN-derived Rspo2 nearly normalizes abnormally regulated genes in Rspo2 ^−/− mice

To differentiate the effects of muscle-derived Rspo2 and SMN-derived Rspo2 on the NMJ, we examined gene expression levels in the diaphragm and spinal cords of wild-type, Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− mice at E18.5. In Wnt/β-catenin signaling, Wnt protein binds to its receptors (Frizzled and LRP5/6) to inhibit degradation of the co-transcriptional factor β-catenin, encoded by Ctnnb1, which upregulates Myc encoding c-myc, Axin2 encoding Axin2, and others^{33,34,35,36,37}. The target genes of Wnt/β-catenin signaling (Ctnnb1, Myc, and Axin2) were increased in the diaphragms of Rspo2^−/− mice (Fig. 6A). Muscle-derived Rspo2 failed to rescue the abnormal increases, but SMN-derived Rspo2 normalized them. In contrast, these genes were not changed in the spinal cords of Rspo2^−/− mice, and neither muscle-derived Rspo2 nor SMN-derived Rspo2 had any additional effect (Fig. 6B).

We previously reported that Rspo2 enhances MuSK phosphorylation by binding to the Lgr5 receptor expressed at the NMJ³¹. In accordance with our previous report, gene expression levels of Lgr5 and MuSK were increased in Rspo2^−/− diaphragms (Fig. 6A). Muscle-derived Rspo2 failed to normalize the increases, but SMN-derived Rspo2 normalized them. These results suggest that SMN-derived Rspo2 plays a pivotal role in regulation of the Wnt/β-catenin signaling and subsequent AChR clustering at the NMJ.

The expression levels of myogenesis marker genes of Myod and Pax7 were increased in Rspo2^−/− diaphragms (Fig. 6A). Muscle-derived Rspo2 further enhanced the increases, whereas SMN-derived Rspo2 normalized them. Ultrastructurally, the aforementioned increased myofibril sizes in Rspo2^−/− and MCK-RSPO2/Rspo2^−/− mice (Fig. 5J) may be partly accounted for by increased expression levels of myogenesis marker genes. Macroscopically, however, gross measurement of the diaphragm sizes revealed that the diaphragm thickness tended to be rather decreased in Rspo2^−/− and MCK-RSPO2/Rspo2^−/− mice (Supplementary Fig. S1C–G). The increased Myod and Pax7 in these mice were likely a compensation for immature myogenesis due to defective NMJ signal transmission as observed in patients with congenital myasthenic syndromes^38,39, but the compensation was likely to be macroscopically insufficient.

The expression of SMN-specific Chat was markedly elevated in the spinal cords of Rspo2^−/− mice (Fig. 6B). Both muscle-derived Rspo2 and SMN-derived Rspo2 normalized it. The expression of another SMN-specific gene, Agrn, was upregulated in Rspo2^−/− mice (Fig. 6B). Muscle-derived Rspo2 failed to suppress the increase, whereas SMN-derived Rspo2 normalized it.

To summarize, seven out of eight genes (Lgr5, MuSK, Ctnnb1, Myc, Axin2, Myod, and Pax7) were abnormally increased in Rspo2^−/− diaphragms (Fig. 6A). Muscle-derived Rspo2 failed to normalize the increases, whereas SMN-derived Rspo2 normalized them. In contrast, two out of five genes (Chat and Agrn) were increased in the spinal cord of Rspo2^−/− mice (Fig. 6B). SMN-derived Rspo2 normalized both genes, whereas muscle-derived Rspo2 only normalized Chat.

Discussion

We rescued the phenotypes of Rspo2^−/− mice by MCK-promoter-driven muscle-specific Rspo2 and VAChT-promoter-driven SMN-specific Rspo2. Rspo2 plays critical roles in the development of multiple tissues including the craniofacial structures, kidneys, lungs, and limbs in mice and humans^25,28,40. Rspo2^−/− mice die shortly after birth due to respiratory distress, and they have defects of the left hindlimb and digits of both forearms^25,29. Neither muscle-derived Rspo2 nor SMN-derived Rspo2 rescued the perinatal death or limb anomalies (Fig. 2A–C). Rspo2 promotes myogenic differentiation and hypertrophic myofiber formation in C2C12 myoblasts^20,22,41. Normalization of the reduced body weight in Rspo2^−/− mice by muscle-derived Rspo2 (Fig. 2D) may be partly accounted for by the effect of Rspo2 on myogenesis. Especially, non-physiological overexpression of Rspo2 by the MCK promoter in MCK-RSPO2/Rspo2^−/− mice (Fig. 1D) might have helped normalize the body weight. In contrast, SMN-derived Rspo2 had no effect on the reduced body weight (Fig. 2D). SMN-derived Rspo2 reduced the abnormally increased muscle fiber sizes in Rspo2^−/− mice (Fig. 5J,L), which might have cancelled the effect of Rspo2 on muscle mass. As Rspo2 is a secreted protein, both SMN-derived Rpos2 and muscle-derived Rspo2 can reach the target tissues and exert overlapping effects. SMN-specific and muscle-specific knockdown of Rspo2 would have similar overlapping phenotypes. Identifying the origin of a secreted protein is thus challenging and enigmatic.

The methods of our NMJ analyses can be divided into four categories: (i) low-magnification analysis of the whole-mount diaphragms (Figs 3 and 7), (ii) high-magnification analysis of the NMJ (Figs 4 and 7), (iii) ultrastructural analysis of the NMJ (Figs 5 and 8), and (iv) gene expression analysis of the diaphragm and spinal cord (Fig. 6). In three of the four categories (i, ii, and iv), SMN-derived Rspo2 mitigated aberrant NMJ features in Rspo2^−/− mice more than muscle-derived Rspo2. Ultrastructural features (iii) were normalized by both SMN-derived and muscle-derived Rspo2, but muscle-derived Rspo2 improved more features than SMN-derived Rspo2. Individual NMJ features can also be divided into three categories by the effects of muscle- and SMN-derived Rspo2: (a) features improved by both muscle-derived and SMN-derived Rspo2, (b) features improved by muscle-derived Rspo2 more than SMN-derived Rspo2, and (c) features improved by SMN-derived Rspo2 more than muscle-derived Rspo2. First, features improved by both muscle-derived and SMN-derived Rspo2 were three of the six ultrastructural parameters (Figs 5B–D and 8). Elevated expression of the Chat gene in the spinal cord was also mitigated by both Rspo2’s (Fig. 6B). Second, features improved by muscle-derived Rspo2 were the other three of the six ultrastructural parameters (Figs 5E,G and 8)). Third, features improved by SMN-derived Rspo2 included the bandwidth of AChR clusters (Figs 3A,B and 7) and the area of AChR clusters (Figs 4C and 7). In the diaphragm, elevated expression levels of NMJ-specific genes, target genes of Wnt/β-catenin signaling, and myogenic marker genes were normalized only by SMN-derived Rspo2 (Fig. 6A). In the spinal cord, elevated expression of Agrn was normalized only by SMN-derived Rspo2 (Fig. 6B). Thus, SMN-derived Rspo2 improved the NMJ phenotypes more than muscle-derived Rspo2. Higher expression of Rspo2 in the spinal cord compared to the diaphragm at E18.5 in wild-type mice (Fig. 1D) also supports the notion that SMN-derived Rspo2 plays a pivotal role in NMJ development. Rspo2 is a secreted protein, but the distance that Rspo2 may travel in our body remains unknown. However, we can assume that Rspo2-secreting cells and their proximal cells are exposed to a high concentration of Rspo2. Indeed, only SMN-derived Rspo2 over-corrected the bandwidth of AChR clusters, which was determined by branching of motor axons. The reason why only muscle-derived Rspo2, but not SMN-derived Rspo2, normalized three ultrastructural features remains unknown, but muscle-derived and SMN-derived Rspo2 may have different roles during normal development of the NMJ. Alternatively, as Lgr5 is a receptor for Rspo2, and is highly expressed in motor neurons including SMNs⁴², muscle-derived Rspo2 might have worked on Lgr5 expressed in SMNs. Additionally, as the MCK promoter drove Rspo2 expression in the skeletal muscle 11.6 times more than that in wild-type skeletal muscle (Fig. 1D), the phenotypic rescue observed in MCK-RSPO2/Rspo2^−/− mice might be partly accounted for by over secretion of Rspo2 from the skeletal muscle.

Individual NMJ features that were mitigated by either muscle-derived or SMN-derived Rspo2 are further addressed in detail. First, Rspo2^−/− mice showed broadening of the bandwidth of AChR clusters in the diaphragm. Muscle-derived Rspo2 had no effect on this broadening, but SMN-derived Rspo2 made the bandwidth of AChR clusters even narrower than that in wild-type mice. As the number of AChR clusters remained unchanged (Fig. 3C), SMN-derived Rspo2 was likely to have compacted bands of AChR clusters. Since the VAChT promoter drove the expression of Rspo2 1.23-fold higher than wild-type mice in the spinal cord at E18.5 (Fig. 1D), the phenotype in Rspo2^−/− mice was likely to be over-corrected by SMN-derived Rspo2. Broadening of the bandwidth of AChR clusters is observed in the mice knocked out for Agrn^43,44, a gene for a core component of the planar cell polarity (PCP) pathway, Vangl2^17,44,45, Wnt4^44,46, Wnt11^17,44,45, and muscle-specific Ctnnb1 encoding β-catenin^17,44,45. Similar broadening is observed in mice with muscle-specific overexpression of Ctnnb1^17,44,45, in utero co-injection of Wnt4 and Wnt11^17,44,45, and in utero injection of a secreted Wnt antagonist, Dkk1^17,44,45. In contrast, in utero injection of a secreted Wnt antagonist, Sfrp4, narrowed the bandwidth of AChR clusters^17,44,45. As both injection and knockdown of Wnt4 and Wnt11 give rise to similar broadening of AChR clusters, and as injection of Wnt-antagonizing Dkk1 and Sfrp4 have the opposite effects, finely orchestrated Wnt signaling pathways are likely to be critical for normal alignment of AChR clusters. Second, gene expression of Agrn was increased in the spinal cord in Rspo2^−/− mice. Muscle-derived Rspo2 failed to suppress this increase, whereas SMN-derived Rspo2 normalized Agrn expression (Fig. 6B). During the NMJ formation, agrin is also synthesized and released from SMNs to the synaptic clefts and binds to the LRP4/MuSK complex to induce AChR clustering^8,9,47. Although Rspo2 does not require agrin to induce AChR clustering in myotubes³¹, Rspo2 and agrin released from SMNs are likely to cooperate to form AChR clusters in vivo. It is likely that Rspo2^−/− mice overexpress agrin to compensate for the lack of AChR-inducing Rspo2, and SMN-specific rescue of Rspo2 might have made the compensation unnecessary. Third, the reduced number of secondary axonal branches in Rspo2^−/− mice was not affected by muscle-derived Rspo2 and was further reduced by SMN-derived Rspo2 (Fig. 3I). The reduced numbers of secondary branches are observed in mice deficient for Agrn^43,44, Wnt11^17,44,45, and muscle-specific Ctnnb1^17,44,45, as well as in mice overexpressing muscle-specific β-catenin^17,44,45 and in utero injected with Sfrp4^17,44,45. Again, as both up- and down-regulation of Wnt signaling molecules reduces the number of secondary branches, finely tuned activations of Wnt signaling pathways are likely to be crucial for normal terminal arborization of SMN axons. Fourth, muscle-derived Rspo2 rescued six abnormal ultrastructural parameters, whereas SMN-derived Rspo2 rescued only three of them (Fig. 8). The roles of muscle-derived molecules on NMJ formation are demonstrated in mice deficient for Nedd4⁴⁸, muscle-specific Ctnnb1⁴⁴, γ-AChR subunit^6,49, and LRP4^50,51, as well as in mice overexpressing muscle-specific Ctnnb1^17,44,45. Rspo2 may be the fifth muscle-derived molecule that regulates formation of the NMJs. To summarize, we demonstrate that SMN-derived Rspo2 is required for AChR clustering at the motor endplate and for the NMJ formation, and muscle-derived Rspo2 also plays a substantial role in ultrastructural formation of the NMJ.

Materials and Methods

Transgene constructs

Human RSPO2 cDNA (clone ID 100062073) was purchased from Open Biosystems. To generate CMV-RSPO2, the coding region of RSPO2 cDNA was amplified from the clone by PCR (see Supplementary Table S1) and was inserted into the HindIII and Xbal sites of the CMV-driven expression vector, p3XFLAG-CMV-14 (Sigma-Aldrich). A 3198-bp promoter region (positions −3349 to +7 of the ATG translational start site) of the mouse Ckm gene encoding MCK and a 6398-bp promoter region (positions −6358 to +41 of the ATG translational start site) of the mouse Slc18a3 gene encoding VAChT were amplified by PCR (Supplementary Table S1). The amplified promoter regions of MCK and VAChT were inserted into the NotI and HindIII sites of CMV-RSPO2 to substitute for the CMV promoter and to generate MCK-RSPO2 and VAChT-RSPO2, respectively (Fig. 1). Lack of PCR artifacts were confirmed by sequencing the entire inserts.

Generation of transgenic mice

All experiments using mice were approved by the Animal Care and Use Committee of the Nagoya University and were performed in accordance with the relevant guidelines. The MCK- and VAChT-RSPO2 transgenes were excised from the plasmids with NotI and EcoRV (Fig. 1). Then, the fragments were purified from a 1% agarose gel using the Wizard SV Gel and PCR Clean-Up System (Promega) and diluted to a final concentration of 100 ng/μl in 50 μl of Nuclease-Free water (Promega). The purified DNA was microinjected into fertilized eggs of C57BL/6J mice. The microinjection and subsequent transfer to foster mothers were performed as reported elsewhere³². Rspo2-knockout mouse on the C57BL/6J background was kindly provided by Drs. Motoko Aoki and Hitoshi Okamoto at Riken, Japan²⁹. MCK-RSPO2/Rspo2^−/− mice and VAChT-RSPO2/Rspo2^−/− mice were generated by crossing the MCK-RSPO2 and VAChT-RSPO2 transgenic mice with heterozygous Rspo2^+/− mice. The produced heterozygote mice were then intercrossed to generate the homozygotes. The presence of both alleles was identified by PCR genotyping with primers shown in Supplementary Table S1. Genomic DNA was extracted from the mouse tail using 1 mg/ml proteinase K (Sigma-Aldrich) in DirectPCR Lysis Reagent (Mouse Tail) (Viagen). Tails were digested at 55 °C overnight and at 85 °C for 45 min. After centrifugation at 16,000 × g for 5 min, DNA in the supernatant was amplified by PCR for genotyping. Embryos were collected by cesarean sections of pregnant mice because Rspo2-deficient mice died shortly after birth.

Immunofluorescence

The tibialis anterior (TA) muscle and spinal cord were isolated from wild-type adult mice (C57BL/6J), and they were immediately frozen in liquid nitrogen and kept at −80 °C overnight. The muscle was embedded in OCT compound, frozen on dry ice, and dissected into 20-μm sections using a cryostat (CM3050S, Leica Biosystems). For Rspo2 staining, sections were incubated with rabbit polyclonal anti-FLAG antibody (1:100, PM020, MBL) at 4 °C overnight and were then incubated with FITC-conjugated goat anti-rabbit IgG secondary antibody (1:100, FI1000, Vector Laboratories) at 4 °C for 2 h in a humidified chamber. For ChAT staining, sections were incubated with goat polyclonal anti-Choline Acetyltransferase antibody (1:100, AB144P, Millipore) at 4 °C overnight and were then incubated with biotinylated anti-goat IgG (1:300, BA-9500, Vector Laboratories) for 1 h at room temperature (26 °C), followed by incubation with Alexa 546-conjugated streptavidin (1:500, S11225, Invitrogen) for 2 h at 4 °C in a humidified chamber. Sections were mounted in the VectaShield mounting medium with DAPI (Vector Laboratories) and visualized using the IX71 microscope (Olympus).

For microscopic examination of the whole-mount diaphragms, embryos were sacrificed on E18.5, and the diaphragms were dissected and fixed in 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) at room temperature (26 °C) for 2 h. After the removal of connective tissue, the whole-mount diaphragm was permeabilized with 0.5% Triton X-100 in 0.1 M phosphate buffer (pH 7.4) for 10 min and then incubated in a blocking buffer containing 3% bovine serum albumin (BSA) and 5% normal goat serum (NGS) at 4 °C overnight. After washing with 0.1 M phosphate buffer (pH 7.4) several times, the diaphragms were incubated overnight with anti-peripherin antibody (1:800, AB1530, Millipore) and anti-synaptophysin antibody (1:100, 180130, Thermo Fisher Scientific). After washing the diaphragms, the diaphragms were incubated with α-Bungarotoxin, Alexa Fluor 594 conjugate (1:100, B13423, Invitrogen) at 4 °C overnight. We quantified the whole-mount diaphragms under an Olympus FSX100 fluorescence microscope by two blinded researchers. The NMJ signals were visualized with an A1Rsi microscope (Zeiss). Unsaturated signals were quantified by two blinded researchers using MetaMorph software (Molecular Devices). We defined that the primary branches are two axonal branches originating from the phrenic nerve and extending to the ventral and dorsal sides. The length of the primary branches was defined as the sum of the two primary branches. The secondary branches originate from one of the primary branches and end at the NMJ. The third branches frequently start from the middle of the secondary branch, and we assumed that the longest axonal branch constituted the secondary branch. The length of the secondary branches was defined as the average of all the secondary branches. For quantitative analyses, Alexa Fluor 594 signals for AChR clusters and FITC signals for synaptophysin were manually traced individually. The areas and the perimeters of the traces were automatically measured by MetaMorph software. The lengths of the longest axes of the traces were also measured by MetaMorph.

Quantitative RT-PCR

The diaphragms and spinal cords were harvested from E18.5 embryos. Total RNA was extracted using TRIzol Reagent (Invitrogen). The extracted RNA was reverse-transcribed using ReverTra Ace (Toyobo) with Oligo(dT)₂₀ Primer (Thermo Fisher Scientific). The synthesized cDNA was quantified using with SYBR Premix Ex Taq II (Takara) by real-time PCR on a LightCycler 480 (Roche). The expression level of each gene was normalized for β2-microglobulin (B2m) and also for wild-type mice. The primer pairs are listed in Supplementary Table S2.

Electron microscopy

Ultrastructure of the left diaphragm at E18.5 was analyzed as previously described³¹. Briefly, the diaphragm and thorax of E18.5 embryos were isolated together, and they were fixed in 4% paraformaldehyde for 3 h while applying physiological tension to the diaphragm by the thorax. The middle portion of the diaphragm muscle fibers, where the NMJs were, was isolated and minced into 0.2 to 0.3-mm blocks. The excised blocks were fixed with 2% glutaraldehyde for 2 h, treated with 1% OsO₄, dehydrated in ethanol, and embedded in Epon 812 (TAAB). As the phrenic nerve could not be traced to its nerve terminal even in wild-type embryos, every second block was stained for cholinesterase using the Ellman method to confirm that the excised blocks indeed included the NMJs. Ultrathin sections were made from blocks that were not stained for cholinesterase. Ultrathin sections (60 to 70 nm) were stained with uranyl acetate and lead citrate. We identified the NMJs by inspecting the entire ultrathin sections using a JEM-1400 transmission electron microscope. The following morphometric parameters were measured according to a previous report⁵²: nerve terminal area in μm², synaptic vesicle density in μm² at the nerve terminal area, area of mitochondria/area of nerve terminal (%), the number of active zones, the diameter of synaptic vesicles, and the width of the synaptic cleft. The active zone was defined as the site with a cluster of five or more synaptic vesicles gathered at the presynaptic membrane⁵³. The postsynaptic fold was defined as an indentation in the postsynaptic membrane, where the fold depth was more than 70 nm and the width of the fold aperture was less than a half of the fold depth (see white arrowheads in Supplementary Fig. S2). The numbers of active zones and postsynaptic folds were counted for each nerve terminal. Images were quantified using the ImageJ program (http://imagej.nih.gov/ij/).

Statistical analyses

Data of multiple groups were analyzed by one-way ANOVA and post-hoc Tukey adjustments using Prism 5.0 (GraphPad) software. P values of 0.05 or less were considered statistically significant. Each experiment was conducted three or more times, and the numbers of experiments are indicated in the figure legends.

References

Hall, Z. W. & Sanes, J. R. Synaptic structure and development: the neuromuscular junction. Cell 72(Suppl), 99–121 (1993).
Article PubMed Google Scholar
Sanes, J. R. & Lichtman, J. W. Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22, 389–442 (1999).
Article PubMed CAS Google Scholar
Burden, S. J. The formation of neuromuscular synapses. Genes Dev 12, 133–148 (1998).
Article PubMed CAS Google Scholar
Wu, H., Xiong, W. C. & Mei, L. To build a synapse: signaling pathways in neuromuscular junction assembly. Development 137, 1017–1033 (2010).
Article PubMed PubMed Central CAS Google Scholar
Hoch, W. Formation of the neuromuscular junction. Agrin and its unusual receptors. Eur J Biochem 265, 1–10 (1999).
Article PubMed CAS Google Scholar
Liu, Y. et al. Essential roles of the acetylcholine receptor gamma-subunit in neuromuscular synaptic patterning. Development 135, 1957–1967 (2008).
Article PubMed PubMed Central CAS Google Scholar
Bezakova, G. & Ruegg, M. A. New insights into the roles of agrin. Nat Rev Mol Cell Biol 4, 295–308 (2003).
Article PubMed CAS Google Scholar
Zhang, B. et al. LRP4 serves as a coreceptor of agrin. Neuron 60, 285–297 (2008).
Article PubMed PubMed Central CAS Google Scholar
Kim, N. et al. Lrp4 is a receptor for Agrin and forms a complex with MuSK. Cell 135, 334–342 (2008).
Article PubMed PubMed Central CAS Google Scholar
Fischbach, G. D. & Rosen, K. M. ARIA: a neuromuscular junction neuregulin. Annu Rev Neurosci 20, 429–458 (1997).
Article PubMed CAS Google Scholar
Carraway, K. L. & Burden, S. J. Neuregulins and Their Receptors. Curr Opin Neurobiol 5, 606–612 (1995).
Article PubMed CAS Google Scholar
Falls, D. L., Rosen, K. M., Corfas, G., Lane, W. S. & Fischbach, G. D. ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family. Cell 72, 801–815 (1993).
Article PubMed CAS Google Scholar
An, M. C. et al. Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms. Proc Natl Acad Sci USA 107, 10702–10707 (2010).
Article ADS PubMed CAS Google Scholar
Lee, Y. I. et al. Neuregulin1 displayed on motor axons regulates terminal Schwann cell-mediated synapse elimination at developing neuromuscular junctions. Proc Natl Acad Sci USA 113, E479–487 (2016).
Article PubMed CAS Google Scholar
Wang, J., Song, F. & Loeb, J. A. Neuregulin1 fine-tunes pre-, post-, and perisynaptic neuromuscular junction development. Dev Dyn 246, 368–380 (2017).
Article PubMed PubMed Central CAS Google Scholar
Zhang, B. et al. Wnt proteins regulate acetylcholine receptor clustering in muscle cells. Mol Brain 5, 7 (2012).
Article ADS PubMed PubMed Central CAS Google Scholar
Messeant, J. et al. Wnt proteins contribute to neuromuscular junction formation through distinct signaling pathways. Development 144, 1712–1724 (2017).
Article PubMed CAS Google Scholar
Messeant, J. et al. MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance. J Neurosci 35, 4926–4941 (2015).
Article PubMed CAS Google Scholar
Zhang, B. et al. Beta-catenin regulates acetylcholine receptor clustering in muscle cells through interaction with rapsyn. J Neurosci 27, 3968–3973 (2007).
Article PubMed CAS Google Scholar
Kim, K. A. et al. R-Spondin family members regulate the Wnt pathway by a common mechanism. Mol Biol Cell 19, 2588–2596 (2008).
Article PubMed PubMed Central CAS Google Scholar
Kamata, T. et al. R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants. Biochim Biophys Acta 1676, 51–62 (2004).
Article PubMed CAS Google Scholar
Kazanskaya, O. et al. R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev Cell 7, 525–534 (2004).
Article PubMed CAS Google Scholar
Ilmer, M. et al. RSPO2 Enhances Canonical Wnt Signaling to Confer Stemness-Associated Traits to Susceptible Pancreatic Cancer Cells. Cancer Res 75, 1883–1896 (2015).
Article PubMed CAS Google Scholar
Wu, C. et al. RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer. Nat Commun 5, 3149 (2014).
Article PubMed CAS Google Scholar
Bell, S. M. et al. R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis. Development 135, 1049–1058 (2008).
Article PubMed CAS Google Scholar
de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).
Article ADS PubMed CAS Google Scholar
Nam, J. S. et al. Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb. Dev Biol 311, 124–135 (2007).
Article PubMed PubMed Central CAS Google Scholar
Neufeld, S. et al. A conditional allele of Rspo3 reveals redundant function of R-spondins during mouse limb development. Genesis 50, 741–749 (2012).
Article PubMed CAS Google Scholar
Aoki, M., Kiyonari, H., Nakamura, H. & Okamoto, H. R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development. Dev Growth Differ 50, 85–95 (2008).
Article PubMed CAS Google Scholar
Han, X. H. et al. Regulation of the follistatin gene by RSPO-LGR4 signaling via activation of the WNT/beta-catenin pathway in skeletal myogenesis. Mol Cell Biol 34, 752–764 (2014).
Article PubMed PubMed Central CAS Google Scholar
Nakashima, H. et al. R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5. Sci Rep 6, 28512 (2016).
Article ADS PubMed PubMed Central CAS Google Scholar
Cho, A., Haruyama, N. & Kulkarni, A. B. Generation of Transgenic Mice. In Curr Protoc Cell Biol (John Wiley & Sons, Inc. 2001).
Kim, W., Kim, M. & Jho, E. H. Wnt/beta-catenin signalling: from plasma membrane to nucleus. Biochem J 450, 9–21 (2013).
Article PubMed CAS Google Scholar
Yu, J. & Virshup, D. M. Updating the Wnt pathways. Biosci Rep 34, (2014).
MacDonald, B. T., Tamai, K. & He, X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17, 9–26 (2009).
Article PubMed PubMed Central CAS Google Scholar
Clevers, H. & Nusse, R. Wnt/beta-catenin signaling and disease. Cell 149, 1192–1205 (2012).
Article PubMed CAS Google Scholar
Gordon, M. D. & Nusse, R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription factors. J Biol Chem 281, 22429–22433 (2006).
Article PubMed CAS Google Scholar
Ohno, K., Ohkawara, B., Ito, M. & Engel, A. G. Molecular Genetics of Congenital Myasthenic Syndromes. In eLS, https://doi.org/10.1002/9780470015902.a9780470024314 (John Wiley & Sons, Inc., 2014).
Ohno, K., Ohkawara, B. & Ito, M. Recent advances in congenital myasthenic syndromes. Clinical and Experimental Neuroimmunology 7, 246–259 (2016).
Article CAS Google Scholar
Yamada, W. et al. Craniofacial malformation in R-spondin2 knockout mice. Biochem Biophys Res Commun 381, 453–458 (2009).
Article PubMed CAS Google Scholar
Han, X. H., Jin, Y. R., Seto, M. & Yoon, J. K. A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis. J Biol Chem 286, 10649–10659 (2011).
Article PubMed PubMed Central CAS Google Scholar
Song, S. J. et al. LGR5/GPR49 is implicated in motor neuron specification in nervous system. Neurosci Lett 584, 135–140 (2015).
Article PubMed CAS Google Scholar
Gautam, M. et al. Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85, 525–535 (1996).
Article PubMed CAS Google Scholar
Li, X. M. et al. Retrograde regulation of motoneuron differentiation by muscle beta-catenin. Nat Neurosci 11, 262–268 (2008).
Article PubMed CAS Google Scholar
Wu, H. et al. beta-Catenin gain of function in muscles impairs neuromuscular junction formation. Development 139, 2392–2404 (2012).
Article PubMed PubMed Central CAS Google Scholar
Strochlic, L. et al. Wnt4 participates in the formation of vertebrate neuromuscular junction. PLoS One 7, e29976 (2012).
Article ADS PubMed PubMed Central CAS Google Scholar
Barik, A., Zhang, B., Sohal, G. S., Xiong, W. C. & Mei, L. Crosstalk between Agrin and Wnt signaling pathways in development of vertebrate neuromuscular junction. Dev Neurobiol 74, 828–838 (2014).
Article PubMed CAS Google Scholar
Liu, Y., Oppenheim, R. W., Sugiura, Y. & Lin, W. Abnormal development of the neuromuscular junction in Nedd4-deficient mice. Dev Biol 330, 153–166 (2009).
Article PubMed PubMed Central CAS Google Scholar
Koenen, M., Peter, C., Villarroel, A., Witzemann, V. & Sakmann, B. Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle. EMBO Rep 6, 570–576 (2005).
Article PubMed PubMed Central CAS Google Scholar
Wu, H. et al. Distinct roles of muscle and motoneuron LRP4 in neuromuscular junction formation. Neuron 75, 94–107 (2012).
Article PubMed PubMed Central CAS Google Scholar
Yumoto, N., Kim, N. & Burden, S. J. Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses. Nature 489, 438–442 (2012).
Article ADS PubMed PubMed Central CAS Google Scholar
Engel, A. G. & Santa, T. Histometric analysis of the ultrastructure of the neuromuscular junction in myasthenia gravis and in the myasthenic syndrome. Ann N Y Acad Sci 183, 46–63 (1971).
Article ADS PubMed CAS Google Scholar
Brandon, E. P. et al. Aberrant patterning of neuromuscular synapses in choline acetyltransferase-deficient mice. J Neurosci 23, 539–549 (2003).
Article PubMed CAS Google Scholar

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Acknowledgements

We thank the staff at Division of Experimental Animals, Nagoya University Graduate School of Medicine for their technical support. We also would like to acknowledge Drs. Motoko Aoki and Hitoshi Okamoto at Riken for providing Rspo2^−/− mice, as well as Drs. Takegami and Nakashima at Nagoya University for valuable discussion. This study was supported by Grants-in-Aids from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (15H04840); the Ministry of Health, Labour, and Welfare of Japan (H29-Nanchi-Ippan-030); the Japan Agency for Medical Research and Development (17ek0109230h0001, 17ek0109281h0001, and 17gm1010002h0002); and the Intramural Research Grant from NCNP (29-4). The first author, JL, obtained a scholarship from the China Scholarship Council (CSC).

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Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
Jin Li, Mikako Ito, Bisei Ohkawara, Akio Masuda & Kinji Ohno

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Jin Li
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Mikako Ito
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Bisei Ohkawara
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Akio Masuda
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Kinji Ohno
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J.L. and K.O. conceived the paper; J.L. and B.O. performed experiments; J.L., M.I. and A.M. analyzed data; J.L., B.O., M.I. and K.O. wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Kinji Ohno.

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Li, J., Ito, M., Ohkawara, B. et al. Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction. Sci Rep 8, 13577 (2018). https://doi.org/10.1038/s41598-018-31949-7

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Received: 05 April 2018
Accepted: 30 August 2018
Published: 11 September 2018
DOI: https://doi.org/10.1038/s41598-018-31949-7

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