Figure 4

BMPs regulate differentiation of primary myoblasts. Myoblasts were treated with Bmp7 (D-F,N) or Noggin (G-I,O) for 3 days under differentiating conditions and double immunolabeled for Desmin (A,D,G) and Myogenin (B,E,H). Nuclei were counterstained with DAPI (C,F,I). Boxed areas in the overlay images (C,F,I) are enlarged in (C',F',I'). Myoblasts of different cell fates are present within cultures: fused (white arrow in C', F' and I'), mononucleated/Myogenin-negative (blue arrow in C',F',I') and mononucleated/Myogenin-positive (yellow arrow in C',F',I') cells. Myotube formation is reduced after Bmp7 stimulation (D-F) compared to control cells (A-C). Enhanced myotube formation was evident after Noggin treatment (G-I). Magnification is 100×. (J) Statistical analysis revealed a significant decrease in the total number of myonuclei upon Noggin treatment (n ≥ 13 individual cultures per treatment from 4 independent experiments). (K) Statistical analysis of the relative number of fused myonuclei (n > 13 individual cultures per treatment from 4 independent experiments) revealed a significant increase in Noggin-treated cultures compared to untreated controls (*p < 0.005; Wilcoxon test), while Bmp7 treatment lead to a significant decrease in the relative number of fused myonuclei (*p = 0.002; Wilcoxon test). (L) Analysis of the relative number of Myogenin-positive myonuclei within the population of mononucleated cells (n > 6 individual cultures per treatment from 2 independent experiments). (M-P) Primary satellite cell descendants do not express alkaline phosphatase in control (M), Bmp7 (N) and Noggin-treated cultures (O). Staining of a positive control is shown in (P). Magnification is 50×.