Fig. 7: BMP gain-of-function experiments in limbs and fibroblast cultures.
From: Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning
a–i BMP4/RCAS-producing cells were grafted to the right limbs of E5 chicken embryos (a). Embryos were fixed 5 days later at E10. In situ hybridisation experiments with mBmp4 probe to transverse limb sections indicate the extent of ectopic BMP4 expression in dorsal limb muscles (b). c–f Limb sections were immunostained with MF20 (myosins, green) to visualise the muscle pattern. Asterisks indicate fused muscles in BMP4-grafted limbs (d) versus individual muscles in control limbs (c). e, f High magnifications of dorsal limb muscles immunostained with MF20 (myosins, green), PAX7 (cyan) and TCF4 (red) antibodies in BMP4 (f) and control limbs (e). g, h Representative fields of nuclei (DAPI+) in muscles of control and BMP4 limbs (g) and quantification of the number of nuclei per unit area (h). h Data are presented as mean of values ± SD from 5 BMP4 and 9 control limbs. i Percentages of PAX7+ and TCF4+ nuclei per total number of nuclei in BMP4-grafted limbs versus controls. Data are presented as mean values ± SD from 3 BMP4 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–l Chicken fibroblast cultures transfected with empty/RCAS (control) (j), BMP4/RCAS (k) or BMPR1ca/RCAS (l). Immunostaining with PAX7 antibody (red) and DAPI in BMP4- and BMPR1ca-transfected fibroblast cultures compared to controls. m Quantification of PAX7+ cells versus the total cell number. Data are presented as mean values ± SD from 3 independent experiments.
