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

Abstract

Neuromuscular junction (NMJ) formation requires proper interaction between motoneurons and muscle cells. β-Catenin is required in muscle cells for NMJ formation. To understand underlying mechanisms, we investigated the effect of β-catenin gain of function (GOF) on NMJ development. In HSA-β-cat(flox(ex3)/+) mice, which express stable β-catenin specifically in muscles, motor nerve terminals became extensively defasciculated and arborized. Ectopic muscles were observed in the diaphragm and were innervated by ectopic phrenic nerve branches. Moreover, extensive outgrowth and branching of spinal axons were evident in the GOF mice. These results indicate that increased β-catenin in muscles alters presynaptic differentiation. Postsynaptically, AChR clusters in HSA-β-cat(flox(ex3)/+) diaphragms were distributed in a wider region, suggesting that muscle β-catenin GOF disrupted the signal that restricts AChR clustering to the middle region of muscle fibers. Expression of stable β-catenin in motoneurons, however, had no effect on NMJ formation. These observations provide additional genetic evidence that pre- and postsynaptic development of the NMJ requires an intricate balance of β-catenin activity in muscles.

Fig. 1.

Increased stability and activity of β-catenin in skeletal muscles in HSA-β-cat^flox(ex3)/+ mice. (A) Schematic of the wild-type and targeted β-catenin allele. E, exon; red triangle, loxP sequence; green arrows, primers for genotyping. (B) Specific expression of exon 3-deleted β-catenin (arrow) in skeletal muscles of HSA-β-cat^flox(ex3)/+ mice, but not in other tissues. (C) Increased stability of exon 3-deleted β-catenin compared with wild type. HEK293 cells were transfected with HA-tagged wild-type (asterisk) and mutant (arrow) β-catenin, and treated with 20 μg/ml CHX for various times. β-Actin was used as a loading control. (D) Quantification of data in C (mean ± SEM, n=3). (E) Schematic of the left hemi-diaphragm. Red dots, AChR clusters; rectangle, area shown in F; D, dorsal; V, ventral; L, lateral; M, medial. (F) Expression of TOP-EGFP in diaphragm muscles by β-catenin GOF mutant. Muscles of control (TOP-EGFP;β-cat^flox(ex3)/+) and TOP-EGFP;HSA-β-cat^flox(ex3)/+ mice were stained with Alexa Fluor 594-conjugated α-BTX (red). Image was taken by confocal fluorescence microscope. Area shown is indicated by rectangle in E. (G) Neonatal mice of indicated genotypes.

Fig. 2.

Aberrant innervation of motor axons in HSA-β-cat^flox(ex3)/+ mice. (A) P0 left hemi-diaphragms of indicated genotypes. Phrenic nerves and terminals were stained with anti-NF/synaptophysin antibodies, which were visualized with Alexa Fluor 488-conjugated goat anti-rabbit antibodies. (B) Decreased number of secondary/intramuscular nerve branches in HSA-β-cat^flox(ex3)/+ muscles (^**P<0.01, n=7, t-test). (C) Increased length of secondary, tertiary, quaternary and 5th branches in HSA-β-cat^flox(ex3)/+ muscles (^**P<0.01, n=6, one-way ANOVA). (D) E13 left hemi-diaphragms of indicated genotypes. (E) Increased secondary branch length in developing HSA-β-cat^flox(ex3)/+ embryos (^*P<0.05, ^**P<0.01, n=5, one-way ANOVA). (F) Developmental changes of secondary branches in HSA-β-cat^flox(ex3)/+ embryos (^**P<0.01, n=5, one-way ANOVA). (G) Increased endplate band width in E16.5 and E18.5 HSA-β-cat^flox(ex3)/+ embryos (^**P<0.01, n=10, one-way ANOVA). In A and D, arrowheads indicate secondary nerve branches; arrow indicates ectopic axon. D, dorsal; V, ventral; L, lateral; M, medial. Error bars indicate s.e.m.

Fig. 3.

Formation and innervation of ectopic muscles in HSA-β-cat^flox(ex3)/+ mice. (A) Schematic of left hemi-diaphragm with ectopic muscles. Square frame indicates parts of diaphragms analyzed in B-D. D, dorsal; V, ventral; M, medial; L, left. (B-D) Staining of diaphragms of different genotypes at indicated ages (NF/synaptophysin, green; AChR, red). White dashed lines indicate the edge of diaphragms. Red dashed lines encircle ectopic muscles. Arrowheads indicate extensive and longer secondary branches. Arrows indicate secondary nerve branches. (E) Enlarged image of an ectopic muscle. Arrow, ectopic axons; arrowheads, AChR clusters.

Fig. 4.

Augmented extension and branching of brachial plexuses in HSA-β-cat^flox(ex3)/+ embryos. (A) Schematic lateral view of mouse embryo. Roman numerals indicate cranial nerves. C, cervical; T, thoracic. (B) Embryos at 45-somite stage stained whole-mount with anti-NF antibody, which was visualized with diaminobenzidine (DAB). Arrows indicate brachial plexus. Areas in rectangles in top panels are enlarged in middle panels. Lower panels show camera lucida drawing of axons. an, axillary nerve; rn, radial nerve; un, ulnar nerve. (C) Embryos at 54-somite stage stained as described in B. Areas in rectangles in top panels are enlarged in middle panels. Lower panels show camera lucida drawing of axons. (D) Schematic of axon branches. Color matches data shown in E. (E) Increased numbers of rn tertiary branch points in HSA-β-cat^flox(ex3)/+ embryos shown in C. ^**P<0.01, n=3, one-way ANOVA. (F) Increased length of rn branches in HSA-β-cat^flox(ex3)/+ embryos shown in C. Shown are ratios of rn branch length over limb bud length. ^**P<0.01, n=3, one-way ANOVA. Error bars indicate s.e.m.

Fig. 5.

Postsynaptic deficits of HSA-β-cat^flox(ex3)/+ NMJs. (A) P0, left hemi-diaphragms were stained for AChR. White dashed lines were drawn to include most AChR clusters. (B) Increased endplate band width in HSA-β-cat^flox(ex3)/+ diaphragms (^**P<0.01, n=6, t-test). (C-F) No change in AChR cluster size (C), length (D) or density (E) in HSA-β-cat^flox(ex3)/+ diaphragms (P>0.05, n≥5, t-test). (F) Individual muscle fibers with AChR clusters (red). Nuclei were stained with DAPI (blue). (G) Quantitative analysis of data shown in F (P>0.05, n=22, t-test). (H) Scattered AChE clusters in HSA-β-cat^flox(ex3)/+ diaphragms (P0). V, ventral; L, lateral; M, medial. Error bars indicate s.e.m.

Fig. 6.

Impaired synaptic transmission in HSA-β-cat^flox(ex3)/+ NMJs. (A) Representative superimposed mEPP sample traces. Traces were recorded from neonatal mice at 2 mM Ca²⁺ (1-second × 600 traces). (B) Reduced mEPP frequency in HSA-β-cat^flox(ex3)/+ NMJs (^**P<0.01, n=6, t-test). (C) mEPP amplitude was not changed at mutant NMJs (P>0.05, n=6, t-test). (D) Representative mEPP traces in response to 0.5 M sucrose. Shown are traces 7 minutes after the addition of sucrose. Time scale: 1 second upper panel; 1 minute lower panel. (E) Quantitative analysis of hypertonic evoked mEPP frequency shown in D (P>0.05, n=5, repeat measure). (F-J) No change in synaptic proteins in HSA-β-cat^flox(ex3)/+ muscle lysates. Data are mean ± s.e.m. (P>0.05, n=3 each group, t-test).

Fig. 7.

Reduced number of synaptic vesicles and active zones in muscle β-catenin LOF, but not GOF, NMJs. (A) Representative electron micrographic images of NMJs in β-cat^flox(ex3)/+ control (upper panel) and HSA-β-cat^flox(ex3)/+ (lower panel) mice. N, nerve terminal; M, muscle fiber; SC, Schwann cell; SVs, synaptic vesicles; SBL, synaptic basal lamina; JFs, junctional folds; Asterisks mark active zones. (B-F) Quantitative analysis showed no difference in nerve terminal numbers (B), active zone numbers per nerve terminal (C), synaptic vesicle density (D), synaptic vesicle diameter (E) or synaptic cleft width (F) (P>0.05, n=10, t-test). (G) Representative electron micrographic images of NMJs in β-cat^loxP/loxP control (upper panel) and HSA-β-cat^–/– (lower panel) mice. (H-L) Quantitative data are shown for nerve terminal numbers (H), synaptic vesicle diameter (K), synaptic cleft width (L), active zone numbers per nerve terminal (I) and synaptic vesicle density (J) (^**P<0.01, n=10, t-test). Error bars indicate s.e.m.

Fig. 8.

Different gene expression in HSA-β-cat^flox(ex3)/+ muscles. Total RNA was isolated and subjected to quantitative real-time PCR for indicated genes. mRNA levels were calibrated to Gapdh mRNA levels and normalized to mRNAs from control mice (^**P<0.01, ^*P<0.05, n=3, t-test). Known Wnt/β-catenin target genes are shown in red; morphogens in green; neurotrophic factors in black; and axon guidance molecules in blue. Error bars indicate s.e.m.