(A) (a–l) Wholemount staining of TS muscles (E18.5) double-labeled with anti-neurofilament antibodies and anti-Syt2 antibodies (nerve, a, d, g, j) and α-bungarotoxin (AChR, b, e, h, k). Merged images are shown in panels c, f, i and l. The inset in each panel shows a high-power view of the image. AChR clusters were markedly reduced both in number and size in Lrp4ECD/ECD;App−/− mice (j–l), compared to wild type (a–c), App−/− (d–f) or Lrp4ECD/ECD (g–i). Furthermore, numerous terminal sprouts (arrowheads in the inset of j and l) were seen in Lrp4ECD/ECD;App−/− mutant mice, whereas the nerve terminals in the wild type (arrowhead in a) juxtaposed with postsynaptic AChR clusters. m–o: 3D reconstruction of confocal images of the NMJs in wholemount diaphragm muscles in wild type (m), Lrp4ECD/ECD (n) and Lrp4ECD/ECD;App−/− (o) illustrate the reduced size of the NMJ in (o). Scale bars: a–l: 100 µm; inset: 20 µm. (B) Wholemount staining of diaphragm muscles (E18.5) double-labeled with anti-neurofilament antibodies and anti-Syt2 antibodies (nerve, a, d, g, j) and α-bungarotoxin (AChR, b, e, h, k). Low-power views of the left dorsal region of the diaphragm muscles. Scale bar, 400 µm. The number of AChR clusters was notably reduced in Lrp4ECD/ECD mice, compared to wild-type and App−/− mice. However both the size and number of AChR clusters were markedly reduced in Lrp4ECD/ECD;App−/− mice, compared to wild-type, App−/− and Lrp4ECD/ECD mice. (C) Quantitative analysis of AChR clusters size and numbers. The number of AChR clusters at the ventral regions of right hemi-diaphragm muscles (E18.5), normalized by muscle area (upper panel). Average size of AChR clusters (lower panel). The numbers of AChR clusters analyzed are: 144 wild type, 132 App−/−, 131 Lrp4ECD/ECD and 96 Lrp4ECD/ECD;App−/− (N = 3 mice for each genotype). Data are shown as average ± S.E.M. Pairwise multiple comparisons were carried out using Tukey’s test and the statistical differences determined by one-way analysis of variance (ANOVA).