Figure 5
From: R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5
Apposition of nerve terminals and muscle endplates is compromised in the E18.5 diaphragm of R-spondin 2 (Rspo2)−/− mice.
(A–F) Representative confocal images of the left diaphragm at E18.5 labeled with an anti-synaptophysin antibody and α-bungarotoxin to visualize the nerve terminals and acetylcholine receptors (AChRs), respectively. Endplates of the wild-type muscles were mostly ovoid-shaped (B), whereas the endplates of Rspo2−/− muscles were large, round and heterogeneously stained (E). (G–I) Blinded morphometric analysis of synaptophysin (G) and AChR (H) in the AChR clusters revealed that NMJ areas were markedly enlarged at E18.5. Numbers of synaptophysin-positive clusters (G) and AChR-positive clusters (H) are shown. (I) The ratio is calculated by dividing the synaptophysin-positive area by the AChR-positive area. Note that not all AChR-positive (red) pixels were synaptophysin-positive (green) in each AChR cluster. Mean and standard deviation (SD; n = 6) are indicated. ****p < 0.001, ***p < 0.005 and *p < 0.05 by t-test. n.s., no significant difference. (J,K) Representative electron micrographs of the neuromuscular junctions (NMJs) in the diaphragm of wild-type and Rspo2−/− mice at E18.5. The red two-headed arrow indicates a widened synaptic cleft. The closed arrowhead at wild-type endplate points to a postsynaptic fold. In the Rspo2−/− mice, synaptic vesicles were larger and sparser than those in wild-type mice. SV, synaptic vesicles; TS, terminal Schwann cell. Low magnification images are shown in Supplementary Fig. S6A. Blinded morphometric measurements are shown in Table 1.
