Fig. 8: BMP loss-of-function experiments in limbs and myoblast cultures.

a–f NOGGIN/RCAS-producing cells were grafted to right limbs of E5 chicken embryos (a). Embryos were fixed 5 days later at E10. b In situ hybridisation experiments with cNOGGIN probes to transverse limb sections indicate the extent of ectopic NOGGIN expression in dorsal limb muscles. c–f Limb sections were immunostained with MF20 (myosins, green) to visualise muscles. e, f High magnifications of dorsal limb muscles immunostained with PAX7 (cyan) and TCF4 (red) antibodies in NOGGIN and control limbs. g, h Representative fields of nuclei (DAPI+) in muscles of control and NOGGIN limbs (g), and quantification of the number of nuclei per unit area (h). h Data are presented as mean of values ± SD from 4 NOGGIN and 4 control limbs. i Percentages of PAX7+ and TCF4+ nuclei per total number of nuclei in NOGGIN-grafted limbs versus controls. Data are presented as mean values ± SD from 3 NOGGIN and 6 control limbs. The p-value was obtained using the two-tailed Mann–Whitney test. Asterisks indicate the p-values, ****p < 0.0001. j–n Myoblast cultures transfected with empty/RCAS (control) or SMAD6/RCAS were induced to differentiation. j–l Immunostaining with PAX7 (j), MYOG (k), and MF20 (myosins) (l) antibodies (green) in control and SMAD6 cultures. m, n RT-qPCR analyses of the expression levels of muscle genes, PAX7, MYF5, MYOD, MYOG, MYHC, BMP-responsive genes ID1, ID2, ID3 (m) and connective tissue genes, SCX, COL3A1, COL6A1, OSR1, TCF4, PDGFRA (n) in SMAD6-transfected and control cell cultures. Gene mRNA levels were normalised to GAPDH and RPS17. The relative mRNA levels were calculated using the 2^−ΔΔCt method using the control condition as controls. For each gene, the mRNA levels of the control condition were normalised to 1. Graphs show means ± standard deviations of 8 biological samples (m) and 6 biological samples (n). n The exact p-values were obtained using the two-tailed Mann–Whitney test and are noted on the graphs.