Figure 4

AChR clusters and nerve terminals in the diaphragms at high-magnification in wild-type (WT), Rspo2^−/−, MCK-RSPO2/Rspo2^−/−, and VAChT-RSPO2/Rspo2^−/− embryos at E18.5. (A) Representative confocal images of the NMJs of the right hemi-diaphragms at E18.5 stained with anti-synaptophysin antibody (green) and α-bungarotoxin (red) to visualize the nerve terminals and acetylcholine receptors (AChRs), respectively. Scale bar = 25 μm. (B) Size distributions of AChR clusters (n = 32 NMJs for each group) by morphometric analysis. AChR cluster sizes were shifted to the right and were broadly distributed in Rspo2^−/− mice. MCK-RSPO2/Rspo2^−/− narrowed the distribution, but had no effect on the average size [see (C)]. In contrast, VAChT-RSPO2/Rspo2^−/− rescued the right shift. Statistical differences of AChR areas by one-way ANOVA are indicated in (C). (C,D) Morphometric analyses of the area, perimeter, and length of α-bungarotoxin-positive signals for AChR clusters (C) and synaptophysin-positive signals for nerve terminals (D). (B–D) AChR areas and synaptophysin-positive areas were manually traced individually and measured by MetaMorph software. The perimeter is the circumference of the traced area. The length is defined as the longest axes of the traced area. Mean ± SEM (n = 32 NMJs) are indicated. When the p-value by one-way ANOVA is less than 0.05, p-values by post-hoc Tukey test are indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.